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Laboratory Manual of Biopharmaceutics and

Pharmacokinetics
Chapter · January 2010
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LABORATORY MANUAL OF
BIOPHARMACEUTICS
AND PHARMACOKINETICS
Dr. S. B. Bhise
M. Pharm PhD
Principal,
Govt College of Pharmacy, Karad
Dr. R. J. Dias
M. Pharm PhD MBA
Professor,
Satara College of Pharmacy, Satara
Dr. S. C. Dhawale
M. Pharm PhD
Professor,
Govt College of Pharmacy, Karad
Shri. K. K. Mali
M. Pharm (Biopharm)
Associate Professor,
Satara College of Pharmacy, Satara
INNOVATE
P
TRINITY PUBLISHING HOUSE
PUBLISH
Serving Pharmacy Profession
Laboratory Manual of Bipharmaceutics & Pharmacokinetics
Published by
Mrs. Anita R. Dias

For Trinity Publishing House,
475/8, F-3, Suryanandan Apartments,
Near Hotel Suruban, Sadar Bazaar,
Satara - 415 001. India.
Tel: (02162) 236229.
E. mail: [email protected]
© 2010 Trinity Publishing House

All rights reserved. No part and style of this book be reproduced or transmitted, in any form, or by any
means- electronic, mechanical, photocopying, recording or otherwise, without prior permission of the
publishers and authors.
Disclaimer: As new information becomes available, changes become necessary. The

editors/authors/contributors and the publishers have, as far as it is possible, taken care to ensure that
the information given in this book is accurate and up-to-date. In view of the possibility of human error or
advances in medical science neither the editor nor the publisher nor any other party who has been
involved in the preparation or publication of this work warrants that the information contained herein is in
every respect accurate or complete. Readers are strongly advised to confirm. This book is for sale in
India only and cannot be exported without the permission of the publisher in writing. Any disputes and
legal matters to be settled under Mumbai jurisdiction only.
Rs. 225/-
Printed at
Print Om Offset, Satara.
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Phone- (02162)234049.
Designed by
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[email protected].
PREFACE
Right kind of laboratory manuals suitable to Indian conditions is a dire necessity for promoting professional
sciences. Teaching in Pharmacy lacks it. The present laboratory manual is expected to fill up the yawning gap.
Good quality articles in Journal of Chemical Education and American Journal of Pharmaceutical Education is a
rich source of ready experiments to be implemented in laboratories; however all these publications have
American perspectives. In recent years few research papers on laboratory experiments are being published in
Indian Journal of Pharmaceutical Education and Research.
We have included the simple experiments which are feasible in laboratory of undergraduate courses instead of
experiments requiring sophisticated instruments and costly setup. This book is divided into seven sections
comprising 32 experiments as per their feasibility in laboratories and we are in process of designing more such
experiments for the benefits of students.
We acknowledge the help and co-operation extended by various persons in bringing out this book. We are
highly indebted to the authors of the various books and articles mentioned in bibliography which became a
major source of information for writing this book. We also thank the publishers and designers who graciously
worked hard to publish this book in time.
Our request to all users of this book is to provide constructive criticism in improving further editions of the
book. We sincerely hope that readers will certainly welcome the book.
Satara
Authors
August 31, 2010.
CONTENTS
Experiment No. Title of the experiment Page No.
Section 1 Physicochemical properties of drugs and dosage forms
1 Determination of partition coefficient and dissociation constant......................... 01
2 Verification of Noyes Whitney law of dissolution............................................... 07
3 Kinetic study of dissolution of drug......................................................................13
4 In vitro dissolution of compressed tablet..............................................................18
5 In vitro dissolution of fast dissolving tablet......................................................... 22
6 In vitro dissolution of sustained release tablet......................................................25
7 Effect of pH on dissolution of Benzoic acid sticks...............................................31
8 Effect of pH on dissolution behavior of drug....................................................... 36
Section 2 Absorption of drugs
9 Intestinal permeability using chicken intestine.................................................... 41
10 Effect of permeation enhancers on intestinal permeability of drug..................... 46
11 Percutaneous absorption of drug from various ointment bases............................ 50
12 Percutaneous absorption of drug through different membranes...........................56
13 In vitro permeation study using Franz diffusion cell............................................ 62
Section 3 Protein binding of drugs
14 Protein binding study using equilibrium dialysis method................................... 66
15 Protein binding study using dynamic dialysis method......................................... 71
16 Determination of binding sites using bovine serum albumin............................... 75
Section 4 Metabolism of drugs
17 Metabolism of drug using in vitro method............................................................79
18 Effect of food on metabolism of drug...................................................................84
Section 5 Excretion of drugs
19 Urinary excretion of drug..................................................................................... 87
20 Influence of urinary pH on excretion of drug.......................................................91
Section 6 Pharmacokinetics
21 Simulation of plasma elimination and urine excretion after an IV bolus dose.....95
22 Calculation of various pharmacokinetic parameters after an IV bolus injection.105
23 Calculation of urinary excretion rate constant and elimination rate constant......109
24 Simulation of plasma elimination after an IV Infusion........................................114
25 Calculation of various pharmacokinetic parameters after an IV infusion............119
26 Calculation of area under curve by Trapezoidal rule...........................................122
27 Calculation of absorption rate constant by method of residuals..........................125
28 Calculation of absorption rate constant by of Wagner-Nelson method................129
29 Calculation of various pharmacokinetic parameters after extravascular
administration.......................................................................................................133
30 Pharmacokinetic study of drug using plasma and urinary data............................140
31 Pharmacokinetic study of drug using salivary drug concentration......................146
Section 7 Bioavailability and Bioequivalence
32 Bioequivalence testing of drug using salivary samples....................................... 151
Bibliography....................................................................................................... 159
ABBREVIATIONS
A Amount of drug in body at time t

A0 Amount of drug at zero time
API Active pharmaceutical ingredient
ARA Amount of drug remaining to be absorbed
Au Amount of drug excreted in urine
AUC Area under the plasma drug concentration-time curve
BCS Biopharmaceutical classification system
BSA Bovine serum albumin
C Plasma concentration at time t
Cmax Maximum plasma concentration
C0 Initial plasma concentration
Css Steady state plasma concentration
Cv Initial concentration of drug in donar compartment
CADD Cumulative amount of drug diffused
CDR Cumulative drug release
Cl Clearance
CYP Cytochrome
ER Excretion rate
F Absolute bioavailability
Fr Relative bioavailability
FDTs Fast dissolving tablets
HSA Human serum albumin
IAEC Institutional Animal Ethics Committee
IEC Institutional Ethics Committee
Jss Steady state flux
K Elimination rate constant (Pharmacokinetic studies experiment )
K Dissolution rate constant (Dissolution studies experiment)
Ka Absorption rate constant
Ka Acid constant
Kp Permeability coefficient
Ku Excretion rate constant
Papp Apparent permeability
PC Partition coefficient
PC Apparent partition coefficient
pKa Dissociation rate constant
Ro Constant infusion rate
t1/2 Biological half life
tmax Time required to achieve maximum plasma concentration
V Volume of distribution
SECTION 1
Physicochemical properties of drugs and dosage forms
Experiment 1
Determination of partition coefficient and dissociation constant
Aim
To determine Ka, pKa, and partition coefficient (PC) of Salicylic acid and study their relationship.
Learning objectives
1. To study partition coefficient (PC) and dissociation constant (pKa) of Salicylic acid.
2. To measure the extraction of Salicylic acid from aqueous buffer into organic solvent.
3. To understand the relationship between pKa and pH.
Theory
pH-partition hypothesis was put forth by Brodie et al., which states that drugs are absorbed from the
gastrointestinal tract by passive diffusion depending on the fraction of undissociated drug at the pH of the
intestine. Thus, the process of absorption is governed by:
1. the dissociation constant (pKa) of the drug
2. the lipid solubility of the unionized drug ( PC) and
3. the pH at the absorption site.
Drug pKa and gastrointestinal pH

pKa is a measure of the strength of an acid or a base. pKa is defined as the negative logarithm of the
equilibrium coefficient of the neutral and charged forms of a compound. Calculation of pKa allows the
proportion of neutral and charged species at any pH to be estimated, as well as the basic or acidic properties of
the compound to be defined.
Lower the pKa of an acidic drug, stronger is the acid i.e. greater the proportion of ionized form at a
particular pH. Higher the pKa of a basic drug, the stronger is the base. Thus, from the knowledge of pKa of drug
and pH at the absorption site, the relative amount of ionized and unionized drug in solution at a particular pH and
the percent of drug ionized at this pH can be determined by Henderson-Hasselbalch equation:
For weak acids,
ionized drug concentration
pH = pK a + log …1
unionized drug concentration
10 pH - pKa
% drug ionized = ´100 …2
1 + 10 pH - pKa
For weak bases,
unionized drug concentration
pH = pK a + log ...3
ionized drug concentration
1
2 Laboratory Manual of Biopharmaceutics and Pharmacokinetics
10 pKa - pH
% drug ionized = ´100 …4
1 + 10 pKa - pH
Lipophilicity and drug absorption
PC of a drug is a measure of how well a substance partitions between a lipid (oil) and water. It is
defined as the ratio of concentration of compound in aqueous phase to the concentration in an immiscible
solvent, as the neutral molecule.
Partition Coefficient, PC = [Conc in organic phase] / [Conc in aqueous phase]
If the drug exists predominantly in the unionized form, it will be poorly absorbed if it has poor lipid
solubility. Ideally, for optimum absorption, a drug should have sufficient aqueous solubility to dissolve in the
fluids at the absorption site and lipid solubility high enough to facilitate partitioning of the drug in the lipoidal
biomembrane and into systemic circulation.
The lipid solubility of a drug is determined from its oil/water PC value. PC is a measure of the degree of
distribution of drug between one of the several organic, water immiscible, lipophilic solvent such as n-octanol,
chloroform, n-heptane, etc. and an aqueous phase (water or a suitable buffer). In general, the octanol/pH 7.4
buffer partition coefficient value in the range of 1 to 2 of a drug is sufficient to predict passive absorption across
lipoidal membranes.
Principle
Salicylic acid is a relatively polar, poorly aqueous soluble material. The salt form however is quite
water soluble. By changing pH of the aqueous buffer, you are able to alter the ratio between the ionized and
unionized form of the acid. Since the unionized form is extracted into the organic phase, the fraction extracted
will vary with pH of the aqueous solution. The definition of ionization constant (Ka) can be useful.
In aqueous buffer H+
[H + ] ×[ A- ] K a ×[H A]
Ka = or [ A- ] = ...5
[H A] [H + ]
The partition between organic and aqueous buffer can be described by a 'true' partition coefficient, PC, as
[ HA - organic ]
PC =
[ HA - aqueous]
or
[HA- organic] = PC [HA- aqueous] …6
In laboratory an apparent partition coefficient, PC' is measured, which will vary with pH or [H+]. This
apparent partition coefficient is given by:
[ HA - organic ]
PC ¢ =
[ HA] + [ A- ] …7
Substituting for [HA-organic] from equation 6 and [A-] from equation 5 gives
Section 1 Physicochemical properties of drugs and dosage forms 3
[PC ]´ [H A]
PC ¢ =
é Ka ù
êë 1 + H + úû ´ [ H A ] …8
PC
PC ¢ =
é K a ù
êë 1 + H + úû …9
This gives us an equation with PC' as function of [H+]with two unknown parameters, PC and Ka.
This can be converted into a straight line equation by taking the reciprocal of both sides of the
equation. Thus,
1 [H + ] Ka
= +
PC ¢ PC ´ [H + ] PC ´ [H + ] … 10
1 1 Ka
= + … 11
PC ¢ PC PC ´ [H + ]
Therefore plotting 1/PC' versus 1/[H+] gives a straight line with a slope of Ka/PC and an intercept of 1/PC.
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
0 2000 4000 6000 8000
+
1/[H ]
PC' can be estimated by determining concentration of Salicylic acid partitioned in organic phase.
Salicylic acid present in organic phase is estimated by adding ferric nitrate solution. The reaction of Salicylic
acid with ferric nitrate produces an intensely colored complex, whose maximum absorbance can be detected at
540 nm spectrophotometrically.
Prerequisite
1. Concept of pH and pKa.
2. pH partition hypothesis.
3. Concept of drug absorption.
Requirements
1. Glasswares: Volumetric flasks, pipettes, etc.
2. Chemicals: Salicylic acid, sodium hydroxide, hydrochloric acid, etc.
3. Instruments: Balance, spectrophotometer.
Procedure
1. Prepare 100 ml of buffer at pH 2.5.
2. Weigh accurately 20 mg of Salicylic acid and transfer to 100 ml volumetric flask and adjust volume by buffer
of pH 2.5. Similarly prepare 0.02 % solution of Salicylic acid with buffers of pH 2.8, 3.0, 3.5, 3.8, and 4.0.
3. Take 4 ml of the buffer pH of 2.5 containing Salicylic acid stock solution and add 1 ml of the ferric nitrate
solution. Allow the color to form and measure the absorbance at 540 nm using spectrophotometer. This is
absorbance ONE (ABS I).
4. Next, take 5 ml of the buffer pH 2.5 containing Salicylic acid stock solution and add 5 ml of the organic
solvent hexane/ethyl acetate. Stopper and shake the test tube for 5 minutes to complete the extraction. Allow the
two phases to settle, remove 4 ml of the aqueous phase, add 1 ml of ferric nitrate solution (0.55% ferric nitrate in
0.4 M nitric acid), allow the color to form, and measure the absorbance at 540 nm. This is absorbance two (ABS
II). Using the two absorbance readings, calculate the apparent partition coefficient, PC', as
[total in organic]
PC ¢ = …12
[total in aqueous]
Where, [total in organic] = First absorbance - Second absorbance and [total in aqueous] = Second
absorbance. Thus,
(ABS I - ABS II)
PC ¢ = …13
ABS I
5. Repeat the experiment with the buffers of pH 2.8, 3.0, 3.5, 3.8, and 4.0. Plot 1/PC' versus 1/[H+] and calculate
PC, Ka, and pKa.
6. Calculate H+ ion concentration with the help of equation, pH = -log10 [H+].
Observations
Table 1.Apparent partition coefficient (PC') of Salicylic acid
Absorbance Absorbance Extraction of SA
Sr. No. pH PC' = (A -B) / B
I (A) II (B) (A - B)
1 2.5
2 2.8
3 3
4 3.5
5 3.8
6 4
PC'-Apparent partition coefficient
+
Table 2. Values of [H ] and PC' of Salicylic acid
Sr. No. pH [H+] 1/[H+] PC' 1/PC'
1 2.5
2 2.8
3 3
4 3.5
5 3.8
6 4
Calculations
+
1. Concentration of H at given pH
+
pH = - log10 [H ]
[H+] = -Antilog (pH)
2.Apparent partition coefficient (PC')
PC' = (ABS I -ABS II)/ABS II
3. Partition Coefficient (PC)
+
Plot the graph of 1/PC' versus 1/H
You will get straight line with a slope, m = Ka/PC and an intercept, c = 1/PC.
Calculate intercept c, and determine PC = 1/c.
4.Acid Constant, Ka
Substitute the values of slope (m) and PC in the formula Ka= m x PC
5. Dissociation Constant, pKa
pKa = -log10 (Ka)
Results
1. Extraction of Salicylic acid in organic layer at pH 2.5 was_______.
2. The partition coefficient (PC) of Salicylic acid was found to be ______.
3. pKa of Salicylic acid was found to be _____.
Conclusion
Dissociation constant, pKa value gives idea regarding the ionization of the drug at given pH while
partition coefficient, PC gives idea regarding its lipid solubility at given pH. Thus, the drug existing in
unionized form with higher lipophilicity at given pH ensures its absorption. As the pH goes on increasing,
extraction of Salicylic acid in organic layer goes on increasing.
Applications
1. Determination of pKa: We can calculate the relative amount of unionized (absorbable) and ionized
(unabsorbable) forms of the drug and predict the extent of absorption at a given pH of gastro intestinal tract, if
pKa of drug is known.
2. Determination of partition coefficient: We can predict the extent of absorption by knowing the lipid solubility
because high lipid solubility facilitates the partitioning of the drug in the lipoidal biomembrane and into the
systemic circulation.
3. The knowledge of value of pKa and PC of particular drug will be useful for designing appropriate dosage
form for optimum bioavailability.
Questions
1. Define PC, pKa, and pH.
2. How will you utilize the values of pKa and PC of drug while designing of drug deliveries?
3.An acid has a pKa of 5.2. What percentage of the acid will be ionized at pH 6.0?
4. Consider following compounds:
Compound Type pKa
Toluene-4-sulphonic acid Acid -1.3
Benzoic acid Acid 4.2
Thiopental Acid 7.6
Codeine Base 8.2
Atropine Base 10
Which will be best absorbed from the stomach ( pH = 2)?

Which will be best absorbed from the small intestine (pH = 4.2)?
(Assume partition coefficients of compounds are same.)
Exercise
Determine Ka, pKa and PC of Caffeine or any other weak base.
Experiment 2
Verification of Noyes Whitney law of dissolution
Aim
To verify Noyes Whitney law of dissolution using Benzoic acid sticks.
Learning objectives
1. To study and understand the concept of Noyes Whitney law of dissolution.
2. To verify Noyes Whitney law of dissolution using Benzoic acid sticks.
Theory
When a drug is given orally in the form of a tablet, capsule or suspension the rate of absorption is
controlled by how fast the drug dissolves in the fluids at the absorption site. Therefore, dissolution rate is often
rate limiting step in the following sequence.
Disintegration
Permeability
Dissolution
Fraction absorbed
Drug in systemic circulation

Drug in solution
Degradation Complexation Gut wall metabolism Liver metabolism
Absorption
Bioavailability
Figure 1. Dissolution and absorption process of tablet

When dissolution is the rate controlling step in the over all process, absorption is said to be dissolution
rate limited.Absorption from the solution proceeds more rapidly than from a solid dosage form. In case of freely
soluble drugs absorption is independent of dissolution while sparingly soluble drugs tend to result in dissolution
limited absorption.
Dissolution of a solid in a liquid involves transfer of mass from a solid to a liquid phase. The overall
transfer process may be regarded as being composed of two consecutive steps. The first involving an interfacial
reaction that results in the liberation of solute molecules from the solid phase, followed by the transport of solute
molecules away from the interfacial boundary under the influence of diffusion or convection. Like any complex
reaction that involves consecutive stages, the overall rate of mass transfer in dissolution will be determined by
the slowest stage. If rates of two consecutive stages are comparable in magnitude, then both stages will
influence the overall rate of transfer.
In 1897, Noyes and Whitney described the quantitative analysis that correlated the amount of time it
took to dissolve a drug from solid particles. Noyes and Whitney law states that the rate at which a solid
substance dissolves in its own solution is proportional to the difference between the concentration of that
solution and the concentration of the saturated solution. Mathematical expression of Noyes and Whitney law is
as follows
dc
= K (C s - C)
dt ...1
Where, dc/dt= rate of dissolution, Cs= saturation solubility of the substance, C= concentration at the
expiration of the time t, and K= dissolution constant.
Drug particle Diffusion layer Bulk solution
Cs
Ch
h
Figure 2. Dissolution of a drug according to diffusion layer model
The current version of the equation is slightly modified from the original but remains based on a
diffusion layer model of dissolution (figure 2) of drug from a particle into a large excess bulk medium.
The rate of dissolution depends upon the surface area of the solid, which in turns depends upon how
finely the drug is subdivided. It also depends upon energy and energy states within the crystals of drug. A
general relationship descending the dissolution process was first observed by Mayer and Whitney. The
modified Noyes Whitney equation states that
dc
= K S (C s - C )
dt …2
Where, S = surface area of dissolving solid, Cs = saturation solubility of drug (concentration of
diffusion layer), C = concentration of drug in the dissolution medium at time t.
K, the dissolution rate constant, is equal to the diffusion layer (D/h), like the unstirred water layer in the
intestine and is a thin, stationary film of solution adjacent to the surface of solid. The layer is saturated with the
drug. Thus the drug concentration in the layer is equal to Cs. The term Cs-C represents the concentration
gradient between the diffusion layer and the bulk solution. In dissolution rate limited absorption, C is negligible.
Then equation 2 is reduced to
dc D S C s
=
dt h …3
Noyes and Whitney equation assumes that the rate of mass transfer depends on the rate at which the
solute diffuses from the thin boundary layer into the bulk of solution. Therefore K, will depend on the diffusion
coefficient of the solute and the thickness of the diffusion pathway and it will be influenced by factors that
influences the diffusion coefficient and film thickness.
If surface area is kept constant, then
KS = K' …4
Therefore, equation 2 can be reduced to
dc
= K ¢(C s - C)
dt …5
On integrating above equation,
Cs K¢t
log = …6
( C s - C) 2.303
Dissolution rate for a particular drug in a particular solvent can be calculated since
K ¢ 2.303 Cs
K= = ´ log
S St (C s - C ) …7
Principle
The principle of this experiment is based on the fact that the dissolution rate constant remains same
despite variation in surface area of Benzoic acid sticks. The dissolution rate constant of Benzoic acid is
calculated by using equation 7. The saturation solubility (Cs) of Benzoic acid can be calculated by dissolving its
excess amount in water while the concentration of Benzoic acid (C) in the solution at given time (t) can also be
estimated. The dissolution rate constant for Benzoic acid stick A and stick B remains same even though the
surface areas of both these sticks are different. Thus, Noyes and Whitney law can be verified using Benzoic acid
sticks.
Prerequisite
1. Concept of dissolution and factors affecting dissolution process.
2. Saturation solubility.
3. Logarithmic calculations.
Requirements
1. Glasswares: Beakers, Nesseler's cylinder, test tubes, glass rods, burette and conical flask.
2. Chemicals: Benzoic acid, sodium hydroxide, oxalic acid and phenolphthalein.
3. Equipments: Vernier caliper.
Procedure
1. Standardization of 0.05 N sodium hydroxide solution
Take 10 ml of 0.05 N oxalic acid (dissolve 315 mg of oxalic acid in 100 ml of distilled water) solution
into a conical flask and add 2 drops of phenolphthalein indicator. Titrate contents of the flask against sodium
hydroxide solution until permanent pink color is obtained. Repeat the titration to get concordant values.
2. Preparation of saturated solution of Benzoic acid (BA)
Prepare saturated solution of Benzoic acid in water by putting excess of Benzoic acid in 100 ml of
water. Stir resulting solution using magnetic stirrer for 2 hours and filter. Withdraw 10 ml of filtered saturated
solution and determine the quantity of Benzoic acid by titrating with 0.05 N NaOH (this will give value of Cs)
3. Preparation of Benzoic acid sticks
1. Place required quantity of pure crystals of Benzoic acid in a beaker and heat till it melts (heating should not be
so vigorous that Benzoic acid gets discolored).
2. Take a glass rod and test tube. Flatten one end of glass rod to fit into the test tube and place this test tube on a
test tube stand vertically.
3. Hold glass rod in the center of the test tube and pour molten Benzoic acid carefully. Fill it to 9-10 cm height.
Hold the rod in the center till acid begins to solidify.
4. After cooling at room temperature, place test tube on ice-bath for 10 to 15 minutes. Due to further cooling
Benzoic acid will shrink and dislodge from surface of the test tube.
5. After thorough cooling, pull out the glass rod along with the Benzoic acid cylinder. Cut with a blade to a
length of 6 to 8 cm. Measure the exact length with scale and also the diameter with the help of a vernier caliper.
6. Smear some soft paraffin on two opposite surfaces of stick (cylinder). The idea is to prevent the surfaces for
dissolution. Excess of soft paraffin should be avoided as it diffuses to the Benzoic acid stick and spoils the
circular surface.
7. Prepare Benzoic acid sticks of varying length and mark as StickAand Stick B.
4. Dissolution study of Benzoic acid sticks
1. Fill a pair of Nesseler's cylinder/measuring cylinder to 100 ml with distilled water. Then dip Benzoic acid
sticks into cylinder and note initial time. Move sticks up and down for 10 minutes. Then remove 10 ml of
solution and titrate with 0.05N NaOH using phenolphthalein as an indicator for analysis of Benzoic acid salt.
Maintain sink conditions with water.
2. Similarly determine the concentration of Benzoic acid after 20, 30 and 40 minutes.
3. Perform the blank titration omitting the sample and substract the readings from the above said titrations and
report the results.
5. Plotting of graph
1. Plot the graph of log (Cs/Cs-C) versus time (t) on graph paper.
Observations
Table 1. Standardization of 0.05 N NaOH
Burette Burette Vol. of
Sr. Volume of 0.05N Oxalic acid
reading reading NaOH Normality
No. solution (ml)
initial (ml) final (ml) used (ml)
1 10
2 10
3 10
Normality of NaOH
Table 2. Parameters of Benzoic acid stick
Sr. Diameter (D) Length (L) Surface area (S)

Stick
No. (cm) (cm) (cm2)
1 Stick A
2 Stick B
Saturation solubility of Benzoic acid in water (Cs)= ___________g/l.
Table 3. Observation table for stickA
Cs/(Cs-C)
Cs/(Cs-C)
Volume of
Time NaOH Normality Concentration K' =
log
Slope K=K'/S
(min) used (a-b)* of NaOH of BA (A) slope x 2.303
(ml)
10
20
30
40
Average K
* a is volume of NaOH used for Benzoic acid titration, b is blank titration i.e. without Benzoic acid.
Table 3. Observation table for stick B
Cs/(Cs-C)
Cs/(Cs-C)
Volume of
log
Slope K=K'/S
(min) used (a-b)* of NaOH of BA (B) slope x 2.303
(ml)
10
20
30
40
Average K
Calculations
1. Calculation for normality of NaOH
N1V1 = N2V2
N1= Normality of NaOH, V1= Volume of NaOH used, N2= Normality of Benzoic acid, V2= Volume of
Benzoic acid used
2. Calculation for concentration of saturated solution of Benzoic acid (Cs)
N1V1 = N2V2
NaOH Benzoic acid
Concentration of BAsolution = N2 x Molecular weight of BA
= N2 x 122.1
Saturation solubility = N2 x 122.1
3. Calculation for surface area of BAstick (S)
S = p DL
Where, D- diameter, L - length
4. Calculation for concentration of Benzoic acid sticks at time t (C)
N1V1 = N2V2
NaOH Benzoic acid
Concentration of BA solution = N2 x Molecular weight of BA
= N2 x 122.1
5. Calculation of dissolution rate constant (K)

0.4
Plot the graph of log (CS/CS-C) 0.35
versus time (t) on graph paper. 0.3
log (Cs/Cs-C)
Graphically calculate K' 0.25
K' = Slope x 2.303 0.2
K=D/h= K'/S 0.15
0.1
0.05
0
0 10 20 30 40
Time (min)
Result
Dissolution rate constant for Benzoic acid stick was found to be ________.
Conclusion
It can be concluded from this experiment that the dissolution rate constant 'K' remains constant for
given drug at given conditions and hence Noyes and Whitney law of dissolution is obeyed.
Applications
1. Dissolution tests are used in the pharmaceutical industry for quality control and to assist with the
determination of bioequivalence.
2. Dissolution test provides useful information at several stages of drug development.
3. Dissolution test can be used as a prognostic tool of oral drug absorption.
4. Dissolution can also be an essential tool for the development and evaluation of sustained release
formulations.
5. Dissolution test can be a tool for in vitro- in vivo correlation of drug.
Questions
1. Define solubility. How will you determine saturation solubility?
2. Describe factors affecting dissolution rate of drug.
3. Describe the sequential events during the transfer of a drug from a solid dosage form in the gastrointestinal
tract to the systemic circulation.
4. Define dissolution and describe film theory for dissolution.
Exercise
Verify Noyes Whitney law of dissolution using any weakly basic drug.
Experiment 3
Kinetic study of dissolution of drug
Aim
To study the kinetics of dissolution ofAspirin and report dissolution rate constant.
Learning objectives
1. To study the kinetics of dissolution of solid substances by Guggenheim's method.
2. To correlate the obtained data for determination of dissolution rate constant.
Theory
Dissolution of solid substances is one of the heterogeneous processes occurring at the boundary
between two phases, which is called as phase interface. Obviously one of the phase is solid, so it is reaction on
the solid surface and it can be divided into following phases.
1. Diffusion of interacting substances to the surface,
2.Adsorption on the surface,
3. Reaction on the surface,
4. Desorption from the surface,
5. Diffusion of product from the surface,
The total reaction rate of heterogeneous process is controlled by the rate of the slowest step and in the
case of solid/liquid systems, the rate determining stage is sub processes involving diffusion.
In biological systems, water represents the most frequent liquid environment-solvent. In the process of
dissolution of crystalline solid compounds into aqueous solution, the above steps are supplemented with
hydration of the surface, and the products of dissolution. Dissolution of solid substance is controlled by the
slowest reaction stage, which is the diffusion of dissolved and hydrated compound from the solid surface. The
diffusion transports the dissolved substance across a thin diffusion layer h, where the concentration of dissolved
substance continuously decreases from the concentration of saturated solution (Cs) at the solid surface to the
concentration level (C) in the bulk solution. The driving force of diffusion is the spatial concentration gradient
in according to First Fick's Law:
dn dc
= - DS …1
dt dx
Where, dn = amount of the dissolved substance within time interval dt, D = diffusion coefficient, S =
total surface (phase interface) of the dissolved solid and finally dc/dx = concentration gradient. When the
mixing is efficient, the diffusion layer is very thin (0.02-0.05 nm) and the concentration gradient may be
replaced by a single linear approximation
dc (C - C S )
= …2
dx h
For calculating the amount of dissolved substance, ‘dn’ we can then write
dn = Vdc …3
Where, V = total volume of solution and dc = concentration increment.

The final shape of the Nernst equation is
dc DS dc
= (C S - C ) or = K (C S - C ) …4
dt Vh dt
where, K = rate constant of dissolution.
After separation of variables and integration we obtain following equation:
C = C S (1 - e - kt ) …5
which is formally equivalent to the equation for the first order reaction kinetics.
Chemical kinetics can be defined as a quantitative study on concentration (or pressure) changes with
time brought about by a chemical reaction. In other words, the chemical kinetics investigates velocities of
various chemical reactions. Reaction rate is decrease of the concentration per unit time of one of the reactants.
The rate constant is a measure of the rate of a given chemical reaction under specified conditions (pressure,
temperature). It may be defined as the rate of changes in concentration of reactant or product with time for a
reaction in which all the reactants are at unit concentration. The order of reaction is usually a small whole
number, but in special case it may have a fractional value or be zero. It is formally defined as sum of the powers
of the concentration terms that occur in the differential form of the rate law. If the chemical reaction proceeds in
a series of sequential stages, then the rate of reaction is limited by the slowest stage. This stage is referred to as
the rate determining stage.
Principle
Kinetic measurements are usually performed for determination of reaction rate (or rate constant) or
reaction order at given conditions. Dissolution process of ionic substances can be observed by measuring the
conductivity changes with time:
G ( t ) = G s (1 - e - k t ) . . .6
where G(t)= conductivity at time t, Gs = saturated conductivity
Assume the surface changes of the dissolved substance are negligible. In case of less stable organic
compounds, precise determination of the conductivity of saturated solution is impossible because of side
reactions (e.g. acetylsalicylic acid hydrolyses to acetic and salicylic acid). Thus the saturated conductivity Gs is
handled as unknown quantity and must be determined, too.
The Guggenheim's method of evaluation of the rate constant will be used, because the final
concentration is unknown. The essence of the method may be characterized as follows: there are some
measurements series constructed within the experiment, where the time shift between the series remains
constant. The concentration data are recorded at time t and t+dt (dt is the appropriate time shift). Using the
Guggenheim's method the mentioned kinetic equation for the conductivity dependence may be written as:
In [G(t + dt ) - G(t )] = A - e - kt
. . .7
Where
A = In [GS (1 - e - kd )] . . .8
The last equation is used for determination of the unknown saturated conductivity.
The series are not to be taken from the start and the end of measurement process, because the
experimental errors are bigger at these points.
Prerequisite
1. Theory of dissolution.
2. Chemical kinetics.
Requirements
Glasswares: Electrode holder, 400 ml beaker, perforated test tube.
Chemicals: 4 tablets ofAcetylsalicylic acid (maximum solubility 2.5 g/l at 150C).
Instruments: Conductometer, electromagnetic stirrers, stop watch.
Procedure
1. Place the beaker with 200 ml of double distilled water on the electromagnetic stirrer.
2. Using the laboratory stand, fix the perforated test tube and conductivity cell into beaker.
3. Set the mixing rate at 800 rpm, which is constant for the experiment (note that no heating is needed).
4. Determine conductivity of pure redistilled water (it must be less than 2mS). Now carefully put the tablets into
the test tube and start the stop-watch. Write down the conductivity data every minute and from the 5th minute
every 5 minutes. Repeat the described conductivity determinations for 85 minutes.
5. After finishing the measurements switch off the stirrer and conductometer and rinse the conductivity cell with
distilled water.
6. Plot the graph of function G=f(t) to characterize the overall development of the dissolution in time. Select two
series from the measured conductivity data, which differ in reaction times by a constant time shift (t=40 min).
Then calculate the appropriate quantity: log [G(t+dt) G(t)].
7. The data set is given by conductivities at reaction time: t=5, 10, 15, 20, 25, 30, 35 min. The second data set is
defined sequentially: (t+dt) =45, 50, 55, 60, 70, 75.
8. Using of least squares method determine parameters for the following linear function:
In[G(t+dt) - G(t)] =A-kt where, slope = -K

5.00
Where, the slope is negative value of the
ln[G(t+dt)-G(t)]
dissolution rate constant at a given 4.00

temperature. 3.00
or alternately
2.00
log [G(t+dt) - G(t)] = logA- Kt/2.303
Slope = -K/2.303 1.00
0.00
5 10 15 20 25 30 35
Time (min)
Figure 1. Design of apparatus

A- Electrode, B- Perforated tube,
C- Beaker, D- Magnetic stirrer
Observations
Table1. Table of conductivity dependence on time
Time (t/min) Conductivity G(t)/mS

1
2
3
4
5
10
Table 2. Guggenheim's method of determination of the rate constant
t (min) G (t) (mS) t+dt /min G(t+dt) (mS) G(t+dt)-G(t) In [G(t+dt)-G(t)]
5 45
10 50
15 55
20 60
25 65
Result
Dissolution rate constant forAspirin tablet by Guggenheim's method was found to be _______ .
Conclusion
It can be concluded that dissolution rate constant can be determined by Guggenheim's method using
conductometer.
Applications
The Guggenheim method for the evaluation of rate constants is shown to be applicable to a wide range
of problems that are of pharmaceutical interest. These include:
1. Reaction kinetics in which more than one product is produced from a common reactant.
2. Consecutive first-order reactions.
3. Dissolution followed by partitioning into a lipid phase.

4. The use of dissolution kinetics to obtain drug solubility and
5. The analysis of drugs through kinetics.
Question
Enlist the applications of conductance property of drug in the field of pharmacy.
Exercise
Study dissolution kinetics of any weakly basic drug.
Experiment 4
In vitro dissolution of compressed tablet
Aim
To study in vitro drug release of the given compressed tablet of Paracetamol.
Learning objectives
1. To understand the working of dissolution test apparatus.
2. To understand the dissolution process of an uncoated tablet.
Theory
Dissolution is the process by which a solid solute enters a solution. In the pharmaceutical industry, it
may be defined as the amount of drug substance that goes into solution per unit time under standardized
conditions of liquid/solid interface, temperature and solvent composition.
Dissolution is considered as one of the most important quality control tests performed on
pharmaceutical dosage forms and is now developing into a tool for predicting bioavailability, and in some cases,
replacing clinical studies to determine bioequivalence. Dissolution behaviour of drugs has a significant effect
on their pharmacological activity. In fact, a direct relationship between in vitro dissolution rate of many drugs
and their bioavailability has been demonstrated and is generally referred to as in vitro-in vivo correlation,
IVIVC.
Solid dosage forms may or may not disintegrate when they interact with gastrointestinal fluid
following oral administration depending on their design (Figure 1).
Tablet
Tablet Non disintegrating
D
isi
nt
eg
ra Blood
tio
n
Dissolution
Granules
Figure 1: Schematic diagram of the dissolution process

Dissolution kinetics is important in determining the bioavailability of a drug. The dissolution rate
controls rate of build up of certain drugs in the blood stream. It was thus recognized that in-vitro dissolution
kinetics provides useful information on the availability of drugs and their subsequent therapeutic effects in-
vivo. This led to the inclusion of dissolution tests in the United States NF XIII (1970) and USP XVIII (1970)
monographs for one capsule and twelve tablet preparations. In 1975, dissolution tests were included in the
British Pharmacopoeia (amendment to BP 1973) for Digoxin tablets. Various pharmacopoeias contain
specifications on the dissolution requirements of various drugs. A variety of designs of apparatus for dissolution
testing have been proposed and tested, varying from simple beaker with stirrer to complex systems with lipid
phases and lipid barrier where an attempt is made to mimic the biological milieu. The choice of the apparatus to
be used depends largely on the physicochemical properties of the dosage form.
Compressed tablets are the standard uncoated tablets made by either direct compression or wet
granulation or dry granulation or double compaction. They may be used for local action in gastro-intestinal tract
or systemic action. When tablet exert local action, they are formulated as more water insoluble by means of
selecting slow dissolving excipients and thus provides local action for long time period. e.g., antacids and
adsorbents. The drugs that produce systemic action have some aqueous solubility and are designed to
disintegrate and dissolve quickly so that the drug can be quickly absorbed and produce systemic action.
Generally, an active pharmaceutical ingredient (API) exhibits bioavailability depending upon
biopharmaceutical class, which is based on water solubility and gastro-intestinal membrane permeability
criteria. But, it can be altered by appropriate selection of excipients and processing technology.
Requirements
a) Glasswares: Beaker, volumetric flasks, pipettes, test tubes, funnel.
b) Equipments: Dissolution test apparatus, UV spectrophotometer.
c) Chemicals: Pure Paracetamol, Paracetamol tablets, sodium hydroxide, potassium dihydrogen phosphate.
Procedure
A. Preparation of solutions
1. Preparation of 0.2 M potassium dihydrogen phosphate: Dissolve 27.22 gm of potassium dihydrogen
phosphate in sufficient quantity of water to produce 1000 ml.
2. Preparation of 0.2 M sodium hydroxide: Dissolve 8 gm of sodium hydroxide in sufficient quantity of water to
produce 1000 ml.
3. Preparation of phosphate buffer of pH 5.8: Place 50 ml of 0.2 M potassium dihydrogen phosphate in a 200 ml
volumetric flask, add 3.6 ml of 0.2 M sodium hydroxide and then add water to make up the volume.
B. Calibration curve of Paracetamol
1. Preparation of standard stock solution: Weigh accurately 100 mg of pure Paracetamol and transfer it into 100
ml volumetric flask and adjust volume (Stock I, 1 mg/ml). Transfer 10 ml stock I to another 100 ml volumetric
flask and adjust volume (Stock II, 100 mg/ml).
2. Preparation of working solution: From stock solution II, pipette out 0.2, 0.4, 0.6, 0.8 and 1 ml into 10 ml
volumetric flask and adjust volume to get concentration in the range of 2-10 mg/ml.
3. Measurement of absorbance: Measure absorbance of the respective dilutions at l max 243 nm using UV-
Visible spectrophotometer. Plot the graph of absorbance of Paracetamol against concentration in MS Excel and
determine slope and intercept.
C. Procedure for dissolution
1. Remove one of the dissolution vessels and fill acrylic tank with distilled water up to the level mark. Place
dissolution vessel back.
2. Fix six stirring shafts with blades to the spindles projecting out of stirrer platform with the help of screw.
3. Fill the dissolution vessel with dissolution media up to mark, 900 ml.
4. Connect the mains cord to the mains supply (230V). Put on mains switch.
5. Press the platform down key to lower down the platform. Place the acrylic covers on each vessel.
6. Set the parameters such as RPM, test temperature, test time and alarm time. Monitor the temperature of
vessel by inserting the thermometer.
0
7. Once the temperature of vessels reaches to 37 C, insert three tablets of Paracetamol in first row of 3 vessels
and press enter key to start test.
8. Withdraw samples at predetermined time interval. Filter and dilute sample if required and analyze
spectrophotometrically at 243 nm.
9. From the absorbance values determine concentration of drug (mg/ml) and percent drug release.
10. With help of MS Excel, plot the graph of percent drug dissolved versus time.
A. Parameters set for plotting of calibration curve

1) Beer's & Lambert range: 2-10 mg/ml
2) Solvent: Phosphate buffer pH 5.8
3) l max for Paracetamol: 243 nm
B. Parameters set for dissolution studies

1) Tablet: Paracetamol
2) Strength of tablet: 500 mg
3) Apparatus: IPType I dissolution apparatus (Paddle).
4) Rotation Speed: 50 rpm
5) Test time: 30 min
6) Dissolution medium: Phosphate buffer (pH 5.8)
7) Volume of dissolution medium: 900 ml
Observations
Table 1. Calibration curve of Paracetamol
Concentration Absorbance
(mg/ml)
2
4
6
8
10
Slope
Intercept
Table 2. In-vitro dissolution of Paracetamol
Time Percent cumulative drug release

(min) I II III Mean ± SD
10
20
30
Calculations
1. Determination of concentration of dissolved drug (mg/ml)
Y= m X + c
Where, Y= absorbance, m= slope, X= concentration (mg/ml), c= intercept.
2.Amount of drug released (mg)
Amount of drug released =
[Concentration (mg/ml) x (volume of dissolution medium) x (dilution factor)]/1000
3. Dilution factor
Dilution factor = volume of diluted sample (ml)/ volume of sample removed (ml)
4. Percent cumulative drug release
Percent cumulative drug release = (amount of drug released) x 100 /strength of tablet
Result
From the release data it was found that _______ % of drug was released at 30 min.
Conclusion
Paracetamol tablet was investigated for in vitro drug release study and it passes/fails IP standards.
Application
As given in previous experiment (Expt No. 2).
Questions
1. State difference between Type I and Type II dissolution apparatus.
2. Enlist the major parts and functions of Type I dissolution apparatus.
Exercise
Perform in vitro dissolution studies of an uncoated tablet of any weakly basic drug.
Experiment 5
In vitro dissolution of fast dissolving tablet
Aim
To study in vitro drug release of the fast release tablets of Domperidone.
Learning objective
To understand the dissolution process of fast release tablet of Domperidone.
Theory
Domperidone shows significant first pass metabolism and poor bioavailability. Thus, a strategy to
improve bioavailability should aim at improving its aqueous solubility and overcoming first pass metabolism.
Innovative drug delivery systems known as melt in mouth or mouth dissolving tablets are novel types of tablets
that disintegrate/disperse/dissolve in saliva. Advantage of mouth dissolving tablets allowing administration
without water anywhere anytime, leads to their suitability for geriatric and pediatric patients. They are also most
suitable for drugs that undergo extensive first pass metabolism. The benefits, in terms of patient compliance,
rapid onset of action as the drug goes directly into systemic circulation and good stability, make these tablets
popular as a dosage form of choice in the current market.
Fast dissolving tablets (FDTs) of Domperidone contains superdisintegrants, which accelerate the
disintegration of tablets by virtue of their ability to absorb large amount of water when exposed to aqueous
environment. This rapid disintegration of FDTs is due to penetration of saliva into the pores, which leads to
swelling of superdisintegrants to create enough hydrodynamic pressure for quick and complete disintegration
of the tablets. This increases bioavailability / rapid absorption through pre-gastric absorption of drugs from
mouth, pharynx and esophagus as saliva passes down.
Requirements
b) Equipments: Dissolution test apparatus, UV spectrophotometer.
c) Chemicals: Domperidone tablets, pure Domperidone , methanol, hydrochloric acid, potassium dihydrogen
phosphate.
Procedure
A. Calibration curve of Domperidone
1. Preparation of standard stock solution: Transfer 100 mg of Domperidone to 100 ml volumetric flask. Add 30
ml of methanolic HCl (0.1 N) so as to dissolve the drug. Make up the volume to 100 ml (Stock I). Withdraw 10
ml of solution and transfer to another 100 ml volumetric flask and make up volume (Stock II).
2. Preparation of working solutions: Transfer from Stock II, 0.2, 0.4, 0.8, 1.2, 1.6, and 2 ml solutions to 10 ml
volumetric flasks (ten flasks) and make volume up to 10 ml with 0.1 N HCl in each flask so as to get
concentration in the range of 2 to 20 µg/ml.
3. Measurement of absorbance: Measure absorbance of the respective dilutions at l max 284 nm using UV-
Visible spectrophotometer. Plot the graph of absorbance of Domperidone against concentration in MS Excel
and determine slope and intercept.
B. Procedure for dissolution

1. Remove one of the dissolution vessels and fill acrylic tank with distilled water up to the level mark. Place
dissolution vessel back.
2. Fix six stirring shafts with blades to the spindles projecting out of stirrer platform with the help of a screw.
3. Fill the dissolution vessel with dissolution media up to mark, 900 ml.
4. Connect the mains cord to the mains supply (230V). Put on main switch, heater switch & motor switch.
5. Press the platform down key to lower down the platform. Place the acrylic covers on each vessel.
6. Set the parameters such as RPM, test temperature, test time and alarm time. Monitor the temperature of vessel
by inserting the thermometer.
0
7. Once the temperature of vessels reaches to 37 C, insert three tablets of Domperidone in first row of 3 vessels
and press key to start test.
8. Withdraw samples at predetermined time interval (5 min). Filter and dilute sample if required and analyze
9. From the absorbance values determine concentration of the drug (mg/ml) and percent drug release.
10. With the help of MS Excel, plot the graph of percent drug dissolved versus time.

1) Beer's and Lambert range : 2-20 µg/ml
2) Solvent: Phosphate buffer pH 6.8
3) l max for Domperidone: 284 nm
1) Tablet: Domperidone tablet
3)Apparatus: IPType I dissolution apparatus (Paddle).
6) Dissolution medium: 0.1N HCl
Observations
Table 1. Calibration curve of Domperidone
Concentration (mg /ml) Absorbance
2
4
8
12
16
20
Slope
Intercept
Table 2. In vitro dissolution of Domperidone

(min) I II III Mean ± SD
10
20
30
Calculations
1. Determination of concentration of Domperidone (mg/ml)
Y= m X + c
Where, Y= absorbance, m= slope, X= concentration (mg /ml), c = intercept.
[Concentration (mg /ml) x (volume of dissolution medium) x (dilution factor)]/ 1000
3. Dilution factor
Percent cumulative drug release = Amount of drug released x 100 /strength of tablet
Result
It was found that Domperidone fast dissolving tablet shows 100 % drug release at _____ min.
Conclusion
Domperidone fast release tablet was investigated for in vitro drug release study.
Applications
1. Fast dissolving tablets can improve the bioavailability of drug by virtue of overcoming first pass metabolism.
2. The comparison of various superdisintegrants can be done.
3. The drugs inactivated by gastric juice can be formulated as FDTs.
Questions
1. Give the fast dissolving tablets available in market.
2. Enlist various superdisintegrants with their mechanisms.
Exercise
Study in vitro release of marketed fast dissolving tablet of Diclofenac sodium.
Experiment 6
In vitro dissolution of sustained release tablet
Aim
To study in vitro dissolution of the given sustained release Diclofenac sodium tablet.
Learning objective
To understand dissolution of a sustained release tablet of Diclofenac sodium.
Theory
Modified release tablet
The main aim behind formulation of this dosage form is to release the medicament slowly for a long
duration after administration of a single tablet.
A widespread use of this type of tablet is mainly because of improvement in patient's compliance. As
the dosage frequency is reduced, patient can take an undisturbed sleep at night. It's also beneficial for
psychiatric patients who forget to take their tablets regularly and the dose related side effects and toxicities are
reduced. Any adjuvant that can alter water uptake rate, swelling and gelling characteristics of matrixing agents
can alter the release rate of API e.g., electrolytes in hydroxy propyl methyl cellulose (HPMC) matrix tablet. It's
also possible to achieve pulsed drug release. Weakly basic drugs exhibit good solubility at low pH while less
soluble drugs show at high pH conditions, which can result in incomplete drug release for sustained release
formulations. The drug release can be modified by providing suitable micro environmental pH in the tablet e.g.,
acidic polymer, succinic acid, etc. Similarly, inclusion of alkaline polymers results in desirable drug release of
acidic drugs. Classic approaches are usually based on adaptation of either film coated or multiparticulate
technologies or those involving slow release matrices, which are discussed below:
1. Coating technology
It combines semi permeable coatings and osmotic tablet cores to produce “zero order release”
technology. Attention is also focused to trigger drug release at critical time point e.g., to achieve drug release 1 -
2 hours before the patient awakens. Alza's prolific research activities have yielded a technology called
“Ringcap” which is based on a tablet, preferentially film coated, partially coated with a series of rings whose
respective thickness provides the means of moderating the rate at which the drug is released from final dosage
form.
2. Matrix technology
Classically matrix products exhibit first order (or perhaps square-root-of-time) drug release
characteristics. In order to achieve zero order release characteristics, it's necessary to employ specially designed
materials or strategies that seek to manipulate tablet structure or geometry. Combination of conventional
HPMC matrix technology with upper and lower layer helps to moderate drug release by increase in surface area
with concomitant reduction in drug concentration within the device.
Release of drug can follow various mechanisms
1. Diffusion is rate limiting
Diffusion is driving force where the movement of drug molecules occurs from high concentration in
the tablet to lower concentration in gastro intestinal fluids. This movement depends on surface area exposed to
gastric fluid, diffusion pathway, drug concentration gradient and diffusion coefficient of the system.
In practice, we can follow either of the two methods,
1. The drug is formulated in an insoluble matrix; the gastric fluid penetrates the dosage form and dissolves the
drug and release it through diffusion.
2. The drug particles are coated with polymer of defined thickness so as the portion of drug slowly diffuse
through the polymer to maintain constant drug level in blood.
2. Dissolution is rate limiting
The drugs with poor water solubility (BCS class 2 and 4) are inherently sustained release forms. While
for water soluble drugs, it's possible to incorporate a water insoluble carrier to reduce dissolution of the drug
particles. They are coated with this type of materials e.g. Polyethylene glycol. One may skip the use of
disintegrating agent to promote delayed release.
3. Osmotic pressure is rate limiting
Osmosis is a phenomenon in which the flow of liquid occurs from lower concentration to higher
concentration through a semi permeable membrane which allows transfer of liquid only. The whole drug is
coated with a semi permeable membrane with a hole on one end of tablet made by a laser beam. The gastric fluid
penetrates through the membrane, solubilizes the drug and increases the internal pressure which pumps the drug
solution out of the aperture and releases the drug in gastric environment. The delivery rate is constant provided
that the excess of drug is present inside the tablet. But, it declines to zero once the concentration drops below
saturation.
4. Release is controlled by ion exchange
Ion exchangers are water insoluble resinous materials containing salt forming anionic or cationic
groups. While manufacturing, the drug solution is mixed with resin and dried to form beads which are tableted.
The drug release depends upon high concentration of charged ions in gastro intestinal tract where, the drug
molecules are exchanged and diffused out of the resin into the surrounding fluid. This mechanism relies upon
the ionic environment of resin and not pH or an enzyme on absorption site.
3. Delayed action tablet
Enteric coated tablet is such an example of delayed action tablet. This formulation is preferred when,
1. TheAPI irritates gastric mucosa e.g.,Aspirin or strong electrolytes.
2. Drugs that produce nausea and vomiting.
3.API is sensitive to low pH e.g., Erythromycin.
4. When it's necessary to release the drug undiluted e.g., intestinal antibacterial, antiseptic agents, etc.
The commonly used coating agents are: Cellulose acetate phthalate, Hydroxy methyl propyl
phthalate, polyvinyl acetate phthalate, Eudragit®, etc. This dosage form is intended to hydrate and begin to
dissolve in duodenum (pH 4 to 6) or in small intestine where pH increases to 7 to 8. The presence of esterases or
bile salts like surface active agents plays a role in drug release.
Requirements
b) Chemicals: NaCl, NaOH, HCl, KH2PO4, Diclofenac sodium, methanol.
c) Equipments: USP dissolution apparatus type II, Balance and UV spectrophotometer.

Procedure
A. Preparation of solutions
1.Acidic buffer of pH 1.2: Dissolve 2 gm of sodium chloride and 7 ml of concentrated HCl in sufficient quantity
of water to produce 1000 ml.
2. Preparation of phosphate buffer (pH 6.8): Place 50 ml of 0.2 M potassium dihydrogen phosphate in 200 ml
volumetric flask, add 22.4 ml of 0.2 M sodium hydroxide and make up the volume.
B. Plotting of calibration curve
1. Preparation of standard stock solution: Transfer 100 mg of Diclofenac to 100 ml volumetric flask. Add 30 ml
of methanol to it so as to dissolve the drug. Make up the volume to 100 ml (Stock I). Withdraw 10 ml of solution
and transfer to another 100 ml volumetric flask and make up the volume to 100 ml with methanol (Stock II).
2. Preparation of working solutions: From Stock II, pipette out 0.2, 0.4, 0.6, 0.8, 1, 1.2 and 1.4 ml into seven 10
ml volumetric flasks and adjust the volume to 10 ml with respective buffer (acidic buffer (pH 1.2) or phosphate
buffer (pH 6.8)) to get concentration in the range of 2 to 14 µg/ml.
3. Measurement of absorbance: Measure the absorbance of respective dilutions at 276 nm using UV-Visible
spectrophotometer. Plot the graph of absorbance of Diclofenac versus concentration in MS Excel and determine
slope and intercept.
C. Procedure for dissolution
1. Remove one of the dissolution vessels and fill acrylic tank with distilled water up to the level mark. Replace
back the dissolution vessel.
2. Fit six stirring shafts with blades in the spindles projecting out of stirrer platform with the help of screw.
3. Fill the dissolution vessel with dissolution medium (acidic buffer solution) up to mark, 900 ml.
4. Connect the mains cord to the mains supply (230V). Put on mains switch, heater switch and motor switch.
5. Lower down the platform and fit covers on each vessel.
6. Set parameters such as temperature, RPM, test time and alarm time according to experimental conditions.
0
7. Once the temperature of vessels reaches 37 C insert three Diclofenac SR tablets (75 mg) in first row of 3
vessels and start the dissolution.
8. Withdraw 2 ml sample at regular time interval, filter, dilute and analyze by UV spectrophotometer at 276 nm.
Replace same quantity of fresh dissolution medium.
9.After 2 h, replace dissolution medium with phosphate buffer and continue dissolution for further 10 h.
10. From the absorbance values, determine concentration of drug (mg/ml) and percent drug release along with
standard deviation.

2) Solvent: Acidic buffer/phosphate buffer pH 6.8
3) l max for Diclofenac: 276 nm
1) Tablet: Diclofenac sodium SR
3)Apparatus: IPType I dissolution apparatus (Paddle)
5) Test time: 12 h
6) Dissolution medium: Acidic buffer/ phosphate buffer pH 6.8
Observations
Table 1. Calibration curve of Diclofenac sodium (acidic buffer)
Concentration
Absorbance
(mg/ml)
2
4
6
8
10
12
14
Slope
Intercept
Coefficient of correlation
Table 2. Calibration curve of Diclofenac sodium (phosphate buffer)
Concentration
Absorbance
(mg/ml)
2
4
6
8
10
12
14
Slope
Intercept
Coefficient of correlation
Table 3. Drug release profile of Diclofenac sodium tablet

Time Percent drug release
% CDR
(h) 1 2 3 Mean
Acidic buffer
0.5
1
1.5
2
Phosphate buffer
3
4
5
6
7
8
9
10
11
12
% CDR- Percent cumulative drug release
Calculations
1. Determination of concentration of Diclofenac sodium (mg/ml)
Y= m X + c
Where, Y= absorbance, m= slope, X= concentration (mg /ml), c = intercept.
[Concentration (mg /ml) x (volume of dissolution medium) x (dilution factor)]/ 1000
3. Dilution factor
Percent cumulative drug release =Amount of drug released x 100 /strength of tablet
Result
In vitro drug release from Diclofenac sodium sustained release tablet was found to be______% at 12 h.
Conclusion
Diclofenac SR tablet was investigated for in vitro drug release study.
Applications
1. Dissolution of sustained release tablet can be studied.
2. Sustained release dosage form reduces frequent dosing thereby improving patient compliance.
3. The behaviour of tablets in fluids of varying pH can be studied.
Questions
1. Why enteric coating of tablets is done?
2. Give various approaches of modified release tablets.
Exercise
Study the dissolution of Theophylline SR tablets.
Experiment 7
Effect of pH on dissolution of Benzoic acid sticks
Aim
To study the effect of pH on dissolution of Benzoic acid sticks.
Learning objectives
1. To study and understand the concept of Noyes and Whitney law of dissolution at different pH.
2. To study its applications.
3. To study the effect of pH on dissolution of Benzoic acid sticks.
Theory
The solubility of a weak electrolyte usually varies considerably as a function of pH. Hence, differences
are expected in the dissolution rate of a weak acid or weak base in different regions of the GIT, which has large
differences in surface areas as well as a notable difference in H+ ion concentration. The pH range of the fluids in
various portions of the GIT varies between 1-8.
The degree of acidity or alkalinity of biological fluids at the absorption site is one of the most critical
factors in the absorption of many drugs. Majority of drugs used in therapeutics are weak acids or bases. The
organic electrolytes tend to exists in solution as unionized or ionized species. The relative fraction of each
species present in solution depends on the pH of the fluid in which the drug is dissolved.
Since, the GIT barrier is selectively permeable to unchanged, lipid soluble solutes, a drug may be well
absorbed from one portion of the tract where a favorable pH exists and poorly absorbed from another portion
where a much less favorable pH is found. The absorption of the weakly basic drug is favored in intestine, where
they exist in essentially unionized form. Conversely, the acidic gastric fluids tend to retard absorption of weakly
basic drugs, but promote absorption of weakly acidic drugs.
The transition from stomach to duodenum usually involves an abrupt and marked change in acidity.
Beyond the pylorus, the pH of intestinal fluids gradually increases to the maximum of pH 8 in the colon.
The total solubility (CS) of a weak acid is given by
Cs = [HA] + [A-] …1
Where [HA] is the intrinsic solubility of the nonionized acid (denoted as C0) and [A-] is the
concentration of its anion, which is infinitely soluble. The concentration of the anion can be expressed in terms
of the dissociation constant, Ka and C0; that is
KaC 0
Cs = C0 +
[H + ] …2
In a similar manner, the solubility of a weak base is given by:
C 0 [H + ]
Cs = C0 +
Ka …3
Substituting equations 2 and 3 into modified Noyes Whitney law, the following dissolution rate
equations are obtained,
For weak acids

dc é C + K aC 0 ù
= K' ê 0 ú
dt êë [H + ] úû …4
dc é Ka ù
= K'C0 ê1 + ú
dt êë [ H + ] úû …5
For weak bases
dc é [H + ] ù
= K'C 0 ê1 + ú
dt êë K a úû …6
Where, K'=DS/h
Equations 5 and 6 indicate that the dissolution rate of weak acids increases with increasing pH,
whereas the dissolution rate of weak bases decreases with increasing pH. The dissolution rate of weak bases is at
a maximum in gastric fluids but that of weak acids is at a minimum. The dissolution rate of weak acid increases
as the undissolved drug particles are transported to the more alkaline regions of the GIT.
According to equation 5, a linear relationship should exist between dissolution rate (dc/dt) of a weak
acid and the reciprocal of [H+] concentration (i.e. 1/H+). In practice, however a plot of dc/dt versus 1/H+ shows
linearity only at low pH.
As, [H+] decreases, negative deviation from linearity is observed. The reason for this deviation is that
[H+] concentration of the bulk solution is not equal to [H+] concentration of the diffusion layer except at low pH
values. So, in case of weak acid
[H+]d >[H+]
Where, [H+]d is the H+ concentration of the diffusion layer
Since the diffusion layer is saturated with respect to drug, it is reasonable to accept that in solutions
with a pH greater than pKa of the drug, the relatively large acid drug concentration in the layer may overcome
the buffer capacity of the solution. In this case, the pH of the diffusion layer would be lower than the pH of the
bulk solution.
Principle
Dissolution rate of weak acids increases with increasing pH, whereas dissolution rate of weak bases
decreases with increasing pH. The dissolution rate of weak bases is at maximum in gastric fluids but that of
weak acid is at a minimum. The dissolution rate of weak acid increases as the undissolved drug particles are
transported to the more alkaline regions of the gastrointestinal tract. As Benzoic acid is a weak acid, its
ionization is less in low pH and hence the dissolution rate is low. However, as the pH increases the ionization of
Benzoic acid increases thereby increasing dissolution rate.
Prerequisite
1. Understanding of concept of dissolution.
2. Understanding of Noyes Whitney law of dissolution.
3. Understanding of the concept of pH and Henderson Hasselbalch equation.
Requirements
1. Glasswares: Beakers, test tubes, glass rods, burette and conical flask.
2. Chemicals: Benzoic acid, sodium hydroxide, phenolphthalein, potassium dihydrogen orthophosphate.
3. Equipments: Vernier caliper, pH meter.
Procedure
A. Preparations of solutions
1. Preparation of 0.05 N sodium hydroxide
Dissolve 2 gm of sodium hydroxide in 150 ml of carbon dioxide free water, cool the solution to room
temperature, and filter through filter paper. Dilute clear filtrate to 1000 ml.
2. Preparation of phosphate buffer of pH 4.0
Dissolve 5.04 g of disodium hydrogen phosphate and 3.01 g of potassium dihydrogen phosphate in
sufficient water to produce 1000 ml.Adjust the pH with glacial acetic acid.
Solution I- Dissolve 13.61g of potassium dihydrogen phosphate in sufficient water to produce 1000
ml. Solution II- Dissolve 35.81 g of disodium hydrogen phosphate in sufficient water to produce 1000 ml. Mix
96.4 ml of solution I with 3.6 ml of solution II.
Dissolve 28.8 g of disodium hydrogen phosphate and 11.45 g of potassium dihydrogen phosphate in
sufficient water to produce 1000 ml.
B. Dissolution of Benzoic acid sticks at different pH
1. Prepare three Benzoic acid sticks as per previous experiment (Expt No. 2). The prepared stick should have
same length and diameter.
2. Prepare three buffers of pH 4.0, 5.5 and 6.8 and check their pH with pH meter. If required adjust their pH.
3. Prepare the saturated solution of Benzoic acid at various pH (4.0, 5.5 and 6.8) as per previous experiment
(Expt No. 2) and calculate saturation solubility of Benzoic acid at various pH.
4. Perform the dissolution of Benzoic acid sticks as per previous experiment (Expt No. 2) at different pH values
e.g. stickAat pH 4.0, stick B at pH 5.5 and stick C at pH 6.8.
5. Calculate the dissolution rate constant, for buffers of pH 4.0, 5.5 and 6.8.
6. Plot a graph between pH and dissolution rate constant.
Observations
Table 1. Parameters of Benzoic acid stick
Diameter (D) Length (L) Surface area (S)
Sr. No. Stick
(cm) (cm) (cm2)
1 Stick A 8
2 Stick B 8
3 Stick C 8
1. Saturation solubility of Benzoic acid at pH 4.0 = ____________g/l.


B. Observation table for Benzoic acid stickAat pH 4.0
Cs/(Cs-C)
Cs/(Cs-C)
Volume of
log
Slope K=K'/S
(min) used (a-b)* of NaOH of BA (A) slope x 2.303
(ml)
10
20
30
40
Average K
C. Observation table for Benzoic acid stick B at pH 5.5
Cs/(Cs-C)
Cs/(Cs-C)
Volume of
log
Slope K=K'/S
(min) used (a-b)* of NaOH of BA (B) slope x 2.303
(ml)
10
20
30
40
Average K
D. Observation table for Benzoic acid stick C at pH 6.8
Cs/(Cs-C)
Cs/(Cs-C)
Volume of
log
Slope K=K'/S
(min) used (a-b)* of NaOH of BA (C) slope x 2.303
(ml)
10
20
30
40
Average K
Calculations
1. Calculation for concentration of saturated solution of Benzoic acid (Cs)
N 1V 1 = N2V2
NaOH Benzoic acid
N1= Normality of NaOH, V1= Volume of NaOH used, N2= Normality of Benzoic acid, V2= Volume of
Benzoic acid used
Concentration of BAsolution (g/l) = N2 x Molecular weight of BA
= N2 x 122.1
2. Calculation for surface area of BAstick (S)

S = p DL
where, D- diameter, L - length.
3. Calculation for concentration of Benzoic acid sticks at time t (C)
N1V1 = N2V2
NaOH Benzoic acid
Concentration of BA solution = N2 x Molecular weight of BA
= N2 x 122.1
4. Calculation of dissolution rate constant (K)
Graphically calculate K'
K = Slope x 2.303
K'=D/h= K/S
Result
The dissolution rate constant (K) of Benzoic acid at pH 4.0, pH 5.5 and pH 6.8 are __________,
__________ and ____________ respectively.
Conclusion
It can be concluded from this experiment that, the dissolution rate of Benzoic acid (weak acid)
increases, as the pH of the dissolution medium increases.
Application
Thorough understanding of effect of pH on dissolution rate of weakly acidic or basic drug will help in
designing various dosage forms.
Questions
1. Describe effect of pH on weak acid and weak base.
2. Correlate your results with theoretical results of Benzoic acid.
3. Why Benzoic acid is a weak acid? Describe effect of pH on Benzoic acid solubility.
Exercise
Study the effect of pH on dissolution rate of a weakly basic drug.
Experiment 8
Effect of pH on dissolution behavior of drug
Aim
To study effect of pH on dissolution behavior of commercially available enteric coated Diclofenac
sodium tablets.
Learning objectives
1. To understand the effect of pH on dissolution of a drug.
2. To utilize Hixon and Crowell cube root law for determining dissolution rate constant.
3. To compare dissolution of Diclofenac sodium at different pH.
4. To compare dissolution of different brands of Diclofenac sodium at same pH.
Theory
Enteric-coated dosage forms are those that remain intact in stomach but dissolve and release their
contents on arriving in small intestine. Factors responsible for this include the difference in pH of gastric and
intestinal fluids; where the coatings that are acid functionally or acid ester functionally remain unionized and
remain intact in the low pH (pH 1-4) gastric environment, they ionize and thus disintegrate in the intestinal
fluids, pH may vary from 5 in the duodenum to around 7.4 further down the intestinal tract. Other factors
responsible for loss of film integrity include hydration and the presence of esterase in the intestinal fluid that are
responsible for cleavage of ester linkage present in some kinds of enteric films.
Dissolution kinetics may be influenced by the physicochemical characteristics of the drug and the
formulation factors and in this case the most probable variants may be the kind of coating materials used, the
coating material thickness, the amount of plasticizer used and the age of the coating film.
To make the comparisons of test formulations easier, dissolution rate constants for every brand at
different pH can be calculated by using Hixon and Crowell cube root law. Hixon and Crowell equation takes
into account the changing surface area of the dissolving molecule. Mathematical form of Hixon and Crowell
cube root law is given as follows:
1/3 1/3
[W0] - [W] =kt …1
where, W0 = initial amount of drug in grams, W = amount of drug remaining to dissolve, k = cube root
dissolution rate constant.
For the calculation of “k”, percent dissolution of Diclofenac sodium is subtracted from 100% to get the
percentage of the drug that remains undissolved. The percentage of undissolved Diclofenac sodium is then
converted into grams (W) and used to compute “k” by using equation (2)
1 - [W]1/3 = k t …2
Based on the dissolution rate constants of various brands, it can be predicted that the brand with lowest
dissolution rate constant is least bioavailable.
Principle
As the pH in GIT goes on increasing the enteric coat gets dissolved and the dissolution rate of
Diclofenac goes on increasing. The release of drugs from enteric-coated tablets is highly dependent on pH of
the buffer solution.
At any particular pH, the difference in dissolution rates among different brands may be due to the use
of different coating materials by different manufacturers. Commonly used enteric coating materials are:
cellulose acetate derivatives such as cellulose acetate phthalate (CAP), hydroxyl propyl methyl cellulose
(HPMC) and the two grades of hydroxy propyl methylcellulose phthalate (HP-50 and HP-55),
polymethacrylate polymers (Eudragit L and Eudragit S), polyvinyl acetate phthalate (PVAP). All the enteric
coatings in current use possess ionizable acid groups, usually a free carboxylic acid. The equilibrium between
unionized insoluble polymer and ionized soluble polymer will be determined by pH of the medium and pKa of
the polymer.
As pH of the dissolution medium increases, the coating materials ionize and start breaking and
dissolving which cause release of the drug. At lower pH, however, the polymer remains unionized and the
integrity of the film is maintained. pKa of the film former and nature of the polymer backbone are the two most
important factors that cause variation in the dissolution behaviour of film formers. The enteric coating
materials with respect to their resistance to the gastric dissolution media are as follows: CAP > PVAP > HP55 >
HP50.
Numerous other factors can play their part in causing the observed variation in drug release behaviour.
These factors include film thickness, plasticizers added to the coating material and the amount of diluent used.
Requirements
1. Glasswares: Funnel, beakers, pipettes, test tubes, etc.
2. Chemicals: Two compressed oral formulations of enteric-coated Diclofenac sodium tablets (same
strength 50 mg), pure Diclofenac sodium used as reference standard, analytical grade sodium hydroxide,
potassium dihydrogen phosphate, 37 % HCl, distilled water.
3. Instruments: Balance, dissolution test apparatus, UV spectrophotometer.
Procedure
1. Plot a standard curve of Diclofenac sodium by making standard dilutions of pure Diclofenac sodium in 0.1N
HCl in the range of 1-10 mg/ml and note their absorbance at 276 nm. Similarly prepare the dilutions of
Diclofenac sodium at phosphate buffer pH 4.0, 4.5, 5.5, 6.0, 6.5 and 7.0 and measure the absorbance at 276 nm
and plot the calibration curves at different pH.
2. Conduct the dissolution test for all brands of Diclofenac sodium tablets as specified in USP XXII for
Diclofenac sodium.
3. Use USP dissolution apparatus type II for dissolution study.
4. Place 900 ml of simulated gastric (0.1N HCl adjusted to pH 1.2) dissolution medium in hemispherical bottom
flask.
5. Maintain the temperature of dissolution medium at 370C and rotation speed at 50 rpm. Place three tablets of
brandAand three tablets of brand B in dissolution vessels.
6. Withdraw 10 ml samples from the dissolution vessel at predetermined time intervals (0, 15, 30, 60, 90, and
120 min) and add same volume of fresh dissolution medium to maintain sink conditions.
7. Analyze samples spectrophotometrically at 276 nm and determine percent cumulative drug release of
Diclofenac sodium using calibration curve.
8. Repeat the experiment in simulated intestinal media (mixed phosphate buffer) at pH 4.0, 4.5, 5.5, 6.0, 6.5 and
7.0 for 240 minutes.
9. Determine dissolution rate constants for every brand at different pH by using Hixon and Crowell cube root
1/3 1/3
law. Plot the graph of (W0 -W ) versus time, t. Slope of line is the dissolution rate constant.
10. Plot the graph of dissolution rate constant versus pH of dissolution medium.
11. Report the effect of pH of dissolution medium on dissolution rate constant.
2) Solvent: 0.1 N HCl or phosphate buffer (pH 4.0, 4.5, 5.5, 6, 6.5, 7.0)
3) l max for Diclofenac sodium: 276 nm
1) Tablet: Diclofenac sodium
2) Strength of tablet: 50/75/100 mg
3) Apparatus: IPType I dissolution test apparatus
6) Dissolution medium: 0.1N HCl, phosphate buffer (pH 4.0, 4.5, 5.5, 6, 6.5, 7.0)
Observations
Table 1. Calibration curve of Diclofenac sodium
pH Slope Intercept
0.1 N HCl
PB pH 4.0
PB pH 4.5
PB pH 5.5
PB pH 6.0
PB pH 6.5
PB pH 7.0
PB- phosphate buffer
Table 2. Percent cumulative drug release of brandA
Time
pH 1.2 pH 4 pH 4.5 pH 5.5 pH 6 pH 6.5 pH 7.0
(min)
0
15
30
60
90
120
150
180
210
240
Table 3. Cumulative percent drug release of Brand B

Time
pH 1.2 pH 4 pH 4.5 pH 5.5 pH 6 pH 6.5 pH 7.0
(min)
0
15
30
60
90
120
150
180
210
240
Calculations
1. Concentration of dissolved drug (mg/ml)
Plot the graph of absorbance versus concentration and determine slope and intercept.
Y= m X + c
Where, Y= absorbance, m= slope, X= concentration (mg/ml), c = intercept.
3. Dilution factor
Percent cumulative drug release = (amount of drug released) x 100 /strength of tablet
5. Determination of dissolution rate constant
1/3 1/3
[W0] - [Wt] = k t
Where, W0- Initial amount of drug in grams,
Wt - Amount of drug released at time t in grams.
1/3 1/3
Plot the graph of [W0] - [Wt] versus time
Calculate the slope of the line as a dissolution rate constant.
Result
1. The dissolution of the brandAvaries from ____ % to ____ % as the pH rises from 1.2 to 7.5.
2. The dissolution of the brand B varies from ____ % to ____ % as the pH rises from 1.2 to 7.5.
3. Dissolution rate constant of Brand A is low/high as compared to brand B at 7.0. Hence Brand A is more/less
dissolved with given time.
Conclusion
It can be concluded that the enteric coating is greatly affected by changes in pH of dissolution medium.
As the pH increases the dissolution becomes rapid and as the pH of the dissolution medium decreases, the
dissolution becomes slow.
It can also be concluded that variation in dissolution behavior of the brands tested may be due to any of
the factors like coating material used, film thickness, diluent used etc.
Applications
1. Comparison of various brands of Diclofenac sodium in terms of bioavailability.
2. Studying the effect of pH of dissolution medium on various parameters like coating material, film thickness,
plasticizers or diluents used in coating of tablet.
3. Studying drug release of enteric coated tablet throughout GIT.
4. Estimation of dissolution rate constant.
Questions
1. Why enteric coating is required for Diclofenac sodium?
3. Enlist drugs requiring enteric coating.
Exercise
Similarly students can study effect of pH on dissolution behavior of commercially available
Domperidone DT tablet. Dissolution medium for the study are 0.1N HCl and phosphate buffer pH 6.8.
Correlate pH of medium and dissolution of Domperidone.
SECTION 2
Absorption of drugs
Experiment 9
Intestinal permeability using chicken intestine
Aim
To perform in vitro absorption-permeation study of marketedAcyclovir tablet.
Learning objectives
1. To study dissolution and in vitro absorption ofAcyclovir tablet.
2. To study and understand continuous dissolution absorption system using isolated everted intestine.
Theory
Good oral bioavailability occurs when the drug has maximum permeability and maximum solubility at
the site of absorption. The extent of absorption of drug in vivo, thus, could be predicted based on permeability
and solubility measurements. Hence, the intestinal permeability represents one essential part in the prediction
of oral bioavailability. The intestinal permeability data can be used in preformulation studies to evaluate the
effects of various pharmaceutical excipients on drug absorption. A number of in vitro methods for assessing the
intestinal permeability of a given drug have been developed. In vitro absorption (permeability) studies based on
isolated intestinal sacs are routinely performed. The advantages of this model are that it contains all the types of
cells and mucus layer; it is relatively fast and inexpensive; and it can be used for preformulation studies. This
kind of model is suitable for measuring kinetic parameters with high reliability and reproducibility. Several
animal species including rat, rabbit, pig, dog, and monkey have been used in permeability studies based on
isolated intestinal sacs. The chicken small intestine could be a useful model for intestinal absorption based on
the assumption that membrane permeability of drugs is not species-dependant, since the composition of plasma
membrane of intestinal epithelial cells is similar across the species. Thus the permeability across the chicken
intestinal segment could be expected to be the same. Furthermore, the model would have advantages as less
labor intensive, less time consuming, lower cost per assay and, since slaughtered chicken is used, no special
permission from animal ethical committees would be required.
Design of continuous dissolution-absorption system using everted intestine segment
The in vitro continuous dissolution absorption system design is illustrated in Figure 1. The system
consists of USP dissolution apparatus 1 and a side-by-side perfusion apparatus holding isolated everted
intestine segment. In this system, drug dissolution from the slow release tablet and permeation across everted
intestine occurs simultaneously. Use 1000 ml of distilled water as dissolution medium maintained at 37 ± 0.5
°C. The perfusion apparatus is consisted of two glass tubes, A and B, connected together (Figure 1). Tube B is a
bent cannula at its lower end, and tube A, a straight cannula at its lower end. The distance between two cannula
is kept constant. The isolated everted intestinal segment is fixed between the ends of tubes A and B as shown in
the figure 1. The ends of the intestine are tied in position with a thread. The apparatus is immersed completely
into the dissolution vessel.
41
Principle
Acyclovir [9-(2-hydroxyethoxymethyl) guanine], a synthetic purine nucleoside analogue derived
form guanine is the most widely used antiviral agent. According to BCS classification, Acyclovir is categorized
as class III drug i.e. having high solubility and less permeability. Its absorption in GIT is slow, variable and
incomplete. The bioavailability of Acyclovir after oral administration ranges from 10-30%. Approximately 80
% of an oral dose is never absorbed and excreted through feces. The continuous-absorption system using
everted intestine segment can be used to study the dissolution of the Acyclovir tablet and at the same time the
absorption of Acyclovir through intestinal membrane can be studied. High solubility and low permeability of
Acyclovir can be substantiated by this experiment as both solubility and permeability can be simultaneously
studied. The absorption ofAcyclovir is permeation rate limited.
Figure 1. Design of in vitro continuous dissolution-absorption system.

Labels:1. Dissolution flask, 2. Rotating shaft, 3. Dissolution medium, 4. Basket, 5. Tablet, 6. Oxygen tube, 7.
Tube B, 8. TubeA, 9. Everted intestine, I. Dissolution absorption system, II.Absorption (perfusion) apparatus.
Prerequisite
1. Intestinal absorption of drugs.
2. Ex vivo absorption models.
Requirements
Glasswares: Beaker, test tube, funnel, volumetric flask, etc.
Chemicals: Sodium lauryl sulphate, phosphate buffer pH 5, distilled water, Krebs ringer solution pH 7.4,
Acyclovir tablet 200 mg.
Instruments: Weighing balance, dissolution apparatus, UV spectrophotometer.
Procedure
1. Preparation of solutions
Krebs ringer solution: Prepare the Krebs ringer solution by combining 6.3 g NaCl, 0.35 g KCl, 0.14 g CaCl2,
0.16 g KH2PO4, 0.15 g MgSO4·7 H2O, 2.1 g NaHCO3, and 5 g glucose in one liter of distilled water.
Phosphate buffer pH 5.0: Dissolve 6.8 g of potassium dihydrogen phosphate in 1000 ml water and adjust the pH
Section 2 Absorption of drugs 43
to 5.0 with 10 M potassium hydroxide.

2. Plotting of calibration curve
Transfer accurately weighed 100 mg of Acyclovir in to 100 ml volumetric flask containing sufficient
quantity of phosphate buffer pH 5.0. Finally adjust volume to get stock solution containing 100 µg/ml
Acyclovir. Withdraw adequate quantities of aliquots from standard stock solution in 10 ml volumetric flask and
dilute with phosphate buffer pH 5.0 to get the concentration of 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 µg/ml of
Acyclovir. Measure the absorbance of these dilute solutions at a l max of 254 nm by using double beam UV
spectrophotometer against a blank of phosphate buffer pH 5.0. Plot the graph of absorbance versus
concentration and determine slope, intercept and coefficient of correlation.
3. Isolation of everted intestine
Bring male white Leghorn chicks weighing between 500 and 600 g from the local market. For isolation
of everted intestine, slaughter the chicks, perform a median incision of the abdomen and free the small intestine.
Clean the lumen carefully from mucus by rinsing with a pH 7.4 buffer solutions (Krebs ringer solution).
Remove an intestinal segment of the first 6 cm length and transfer to oxygenated Krebs ringer solution. Wash it
thoroughly with warm Krebs ringer solution. Turn back the proximal extremity of the intestine and ligate on a
glass rod to form an everted bag.
Alternatively, chick intestine can be obtained from slaughter house by taking proper care of providing
oxygenated Krebs ringer solution to intestinal segment removed. This will eliminate the requirement of taking
permission of Ethics Committee for performing this experiment.
4. Dissolution absorption studies
1. Maintain the dissolution medium consisting of 900 ml of phosphate buffer pH 5.0 at 37 ± 0.5 °C. Clamp a
fresh intestinal segment to perfusion apparatus, as shown in the figure 1.
2. Fill Krebs ringer solution in perfusion apparatus and record total volume of absorption compartment (tube A
and tube B of perfusion apparatus).
3. Place the perfusion apparatus into dissolution medium and aerate it.
4. Place marketedAcyclovir tablet into basket and rotate it at 50 rpm.
5. Withdraw 2 ml sample at interval of 10 min up to 120 min and determine released Acyclovir
6. Withdraw the transported drug from absorption compartment with replacement of Krebs ringer solution at 13
min to 123 min at the interval of 10 min and analyze it spectrophotometrically for transported Acyclovir at 254
nm. To allow time for drug to circulate from the dissolution vessel to the everted intestine surface, absorption
samples should be collected 3 min later than their corresponding dissolution samples.
7. Repeat whole experiment in triplicate (n=3) using fresh dissolution medium as well as fresh intestinal
segment each time.
8. Plot the graph of percent drug released versus time.
9. Plot the graph of cumulative amount of drug diffused versus time and determine slope of linear portion as
steady state appearance rate (mg/min) namely the amount of a compound traversing the tissue in time t (min).
Observations
Table 1. Percent cumulative drug release (% CDR)
Time Concentration Amount of drug released

Absorbance % CDR
(min) (mg/ml) (mg)
0
10
20
30
40
50
60
70
80
90
100
110
120
Table 2.Amount ofAcyclovir diffused

Amount
Time Concentration
Absorbance diffused CADD
(min) (mg/ml)
(mg)
0
13
23
33
43
53
63
73
83
93
103
113
123
CADD= Cumulative amount of drug diffused in mg
Calculations
1. Concentration of diffused drug (mg/ml)
Y= mX + c
Where, Y= absorbance, m= slope, X= concentration (mg/ml), c = intercept.
2. Cumulative amount of drug diffused (mg)
Cumulative amount of drug diffused (CADD) =
[Concentration (mg/ml) x (volume of diffusion medium) x (dilution factor)]/1000
3. Surface area (A) of chicken intestine (cm2)

A= 2prl
Where, r = internal radius of everted intestine, l = length of everted intestine.
4. Dilution factor
5.Amount of drug released in dissolution medium (mg)
Percent cumulative drug release = (Amount of drug released) x (100 /strength of tablet)
7.Apparent permeability (cm/s)
dQ 1
Papp = ´
dt C0 ´ A ´ 60
Where, dQ/dt = steady state appearance rate, namely the amount of a compound traversing the tissue in
2
time t (min), A = exposed area of the tissue (cm ), C0 = initial concentration of the drug in the donor
compartment.
Results
1. Percent drug release of marketedAcyclovir tablet in phosphate buffer is _________ % in 2 h.
2.Amount ofAcyclovir diffused through intestinal membrane is _________ in 2 h.
Conclusion
It can be concluded from this experiment that the in vitro continuous dissolution-absorption system
design can be successfully used for simultaneous determination of dissolution and apparent permeability of
Acyclovir.
Applications
1. Simultaneous determination of dissolution and apparent permeability is possible using this model.
2. In vitro absorption studies can be done by using this model.
Question
Give various models used for in vitro absorption studies.
Exercise
Perform ex vivo absorption-permeation study of marketed Metformin HCl tablet.
Experiment 10
Effect of permeation enhancers on intestinal permeability of drug
Aim
To study the effect of permeation enhancer on absorption of Acyclovir by using in vitro dissolution-
absorption system.
Learning objectives
1. To study dissolution and in vitro absorption ofAcyclovir tablet.
2. To study the effect of permeation enhancer on absorption ofAcyclovir.
Theory
As per previous experiment (Expt No. 9).
Principle
One of the approaches to improve the permeability of poorly absorbed drug from GIT is co
administration of absorbance enhancers including surfactants, bile salts, fatty acids and some mucoadhesive
polymers.
Surfactants are often included in the oral solid dosage forms in order to improve their wetting or
dissolution properties of drug. It is important to know whether these surfactants at concentrations that are
achieved in the intestinal lumen enhances the permeability of the active ingredients. Sodium lauryl sulphate
(SLS) is a commonly used surfactant to study the permeability at various concentrations. Increase in
permeability is due to the ability of permeation enhancer (SLS) to promote permeability in the absorptive
direction by opening the tight junctions and/or by inhibiting the active efflux system. Thus, by use of SLS the
problem of less permeability can be solved which may lead to increased absorption of Acyclovir which in turn
may increase its bioavailability. Increasing concentrations of SLS in the dissolution medium increases the
permeability coefficient and enhancement ratio.
Prerequisite
1. Knowledge about permeation enhancers.
2. In vitro absorption methods.
3. Concept of dissolution.
Requirements
Glasswares: Beaker, test tube, funnel, volumetric flask, etc.
Chemicals: Sodium lauryl sulphate, phosphate buffer pH 5, distilled water, Krebs ringer solution pH 7.4,
Acyclovir tablet 200 mg.
Instruments: Weighing balance, dissolution apparatus, UV spectrophotometer.
Procedure
1. Preparation of solutions
Krebs ringer solution: Prepare the Krebs ringer solution by combining 6.3 g NaCl, 0.35 g KCl, 0.14 g CaCl2,
0.16 g KH2PO4, 0.15 g MgSO4·7 H2O, 2.1 g NaHCO3, and 5 g glucose in one liter of distilled water.
Phosphate buffer of pH 5.0: Dissolve 6.8 g of potassium dihydrogen phosphate in 1000 ml water and adjust the
pH to 5.0 with 10 M potassium hydroxide.
Transfer accurately weighed 100 mg of Acyclovir in a 100 ml volumetric flask containing sufficient
quantity of phosphate buffer pH 5.0. Finally adjust volume to get stock solution containing 100 µg/ml
Acyclovir. Withdraw adequate quantities of aliquots from standard stock solution in ten, 10 ml, volumetric
flasks and dilute with phosphate buffer pH 5.0 to get the concentration of 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20
µg/ml of Acyclovir. Measure absorbance of these dilute solutions at a lmax of 254 nm by using double beam
UV spectrophotometer against a blank of phosphate buffer pH 5.0. Plot the graph of absorbance versus
concentration and determine slope, intercept and coefficient of correlation.
3. Isolation of everted intestine
Bring male white Leghorn chicks weighing between 500 and 600 g from the local market. For isolation
of everted intestine, slaughter the chicks, perform a median incision of the abdomen and free the small intestine.
Clean the lumen carefully from mucus by rinsing with a pH 7.4 buffer solutions (Krebs ringer solution).
Remove an intestinal segment of the first 6 cm length and transfer to oxygenated Krebs ringer solution. Wash it
thoroughly with warm Krebs ringer solution. Turn back the proximal extremity of the intestine and ligate on a
glass rod to form an everted bag.
Alternatively, chick intestine can be obtained from slaughter house by taking proper care of providing
oxygenated Krebs ringer solution to intestinal segment removed. This will eliminate the requirement of taking
permission of Ethics Committee for performing this experiment.
4. Dissolution absorption studies
1. Maintain the dissolution medium consisting of 900 ml of phosphate buffer pH 5.0 at 37 ± 0.5 °C. Clamp a
fresh intestinal segment to perfusion apparatus.
2. Fill Krebs ringer solution in perfusion apparatus and record total volume of absorption compartment (tube A
and tube B of perfusion apparatus).
3. Place the perfusion apparatus into dissolution medium and aerate it.
4. Place marketedAcyclovir tablet into basket and rotate it at 50 rpm.
5. Withdraw 2 ml sample at interval of 10 min up to 120 min and determine released Acyclovir
6. Withdraw the transported drug from absorption compartment with replacement of Krebs ringer solution at 13
min to 123 min at the interval of 10 min and analyze it spectrophotometrically for transported Acyclovir at 254
nm.
7. Repeat whole experiment in triplicate (n=3) using fresh dissolution medium as well as fresh intestinal
segment each time.
8. Repeat the same procedure for other tablets except that, add SLS at three varying concentrations of 1%, 2%
and 3% in the dissolution medium.
9. Plot the graph of percent drug released versus time.
10. Plot the graph of cumulative amount of drug diffused versus time and determine slope of linear portion as
steady state appearance rate (mg/min) namely the amount of a compound traversing the tissue in time t (min).
Observations
Table 1. Percent cumulative drug release
(min) 0% SLS 1% SLS 2% SLS 3% SLS
10
20
30
40
50
60
70
80
90
100
110
120
Table 2. Amount of drug diffused
Time Percent cumulative amount of drug diffused
(min) 0% SLS 1% SLS 2% SLS 3% SLS
13
23
33
43
53
63
73
83
93
103
113
123
Calculations
Y= mX + c
2
3. Surface area (A) of chicken intestine (cm )
A= 2prl
Where, r = internal radius of everted intestine, l = length of everted intestine.
4. Dilution factor
5.Amount of drug released in dissolution medium (mg)
[concentration (mg/ml) x (volume of dissolution medium) x (dilution factor)]/1000
Percent cumulative drug release = (amount of drug released) x (100 /strength of tablet)
7.Apparent permeability (cm/s)
dQ 1
Papp = ´
dt C0 ´ A ´ 60
Where, dQ/dt = steady state appearance rate, namely the amount of a compound traversing the tissue
2
in time t, A = exposed area of the tissue (cm ), C0 is the initial concentration of the drug in the donor
compartment.
6. Enhancement ratio (ER)
Papp of drug with enhancer
ER =
Papp of drug without enhancer
Results
1. Percent drug release of marketedAcyclovir tablet in phosphate buffer is ________ % in 2 h.
2.Amount ofAcyclovir diffused through intestinal membrane is ________ in 2 h.
3. The apparent permeability and enhancement ratio of Acyclovir at different concentrations of SLS is given
below: 0% SLS 1% SLS 2% SLS 3% SLS
Apparent permeability
Enhancement Ratio
Conclusion
It can be concluded from this experiment that as the concentration of SLS is increased permeability of
Acyclovir is also increased.
Applications
1. Permeability of Class III drugs with low permeability can be improved by using permeation enhancers.
2. The effect of various absorption enhancers on absorption of drugs can be studied.
Questions
1. Give list of various permeation enhancers used in oral dosage forms.
2. Give the mechanisms by which permeation enhancers increase the permeability of drugs.
Exercise
Study the effect of bile salts on permeability ofAcyclovir.
Experiment 11
Percutaneous absorption of drug from various ointment bases
Aim
To study the ex vivo percutaneous absorption of Metronidazole from water soluble and oleaginous
vehicles.
Learning objectives
1. To study the ex vivo percutaneous absorption method.
2. To understand the concept of permeability coefficient and its estimation.
3. To study the effect of ointment bases on percutaneous absorption of Metronidazole.
Theory
Stratum corneum is the principle barrier for cutaneous penetration allowing slow absorption for
majority of drugs. In any case, the permeability of the stratum corneum is increased by using appropriate
vehicle. It is generally assumed that nature of the selected vehicle strongly influences the rate and extent of
drug release. Release may be improved by selecting the appropriate vehicle. The best vehicle for topical use has
been described as the one which contributes to reversible decrease in the stratum corneum resistance and allows
diffusion of molecules into the vehicle itself.
Most in vitro experimental designs aim to mimic as closely as in vivo situation. The most common in
vitro design is one where a membrane (usually the epidermis) separates two compartments. One compartment
contains the drug in a vehicle, possibly a simple aqueous or buffer solution (termed the donor solution), and the
other compartment contains a receptor (or receiver) solution that provides sink conditions (i.e. essentially zero
concentration). After sufficient time, steady-state permeation across the membrane is achieved when
concentration gradient of the drug across the membrane is constant. Under these conditions following equation
can be used.
dM DC0
=
dt h …1
Where M = cumulative mass of the drug that passes through per unit area of the membrane in time t,
C0 = concentration of the drug in the first layer of the membrane (at the skin surface, in contact with the donor
solution) and h = membrane thickness.
In practical terms, it is very difficult to measure C0 , concentration of the drug in first layer of the
membrane; removal of the outer layer for assay is problematic and contamination from the applied donor
solution is almost inevitable. However, concentration of the drug in the vehicle (donor solution) bathing the skin
membrane (Cv) is usually known or can be determined relatively easily. Since C0 and CV are simply related by:
C0 C0 = PCV
P= so …2
CV
Where P is the partition coefficient of the drug between the membrane and the vehicle. Substitution of
equation 2 into equation 1 gives:
dM DPCV
=
dt h …3
This is most widely applied equation in examining transdermal drug delivery data. A plot of M,
cumulative amount of the drug passing through a unit area of membrane (mg/cm ) against time gives the typical
2
permeation profile.
The permeability coefficient (KP), rate of drug transport per unit concentration of a drug through a
membrane can be defined by equation,
DP
KP = …4
h
Which can be substituted into equation 3 to give:
dM
= J SS = K P CV
dt
…5
The steady state flux (JSS) is simply obtained as the gradient of the linear portion of the permeation
profile (by plotting the graph of cumulative amount of drug diffused per unit area versus time) and if
concentration of the drug in the applied vehicle is known, then the permeability coefficient can be determined.
Higher the value of KP and JSS obtained for the formulation, higher will be permeation.
Principle
Release of the drug from the dosage forms depend directly on the physicochemical properties of the
vehicle and the drug employed. Solubility of the drug is one of the most important physical properties that affect
release in both base and its surrounding medium.
Metronidazole (MTD) is soluble in phosphate buffer. Hence the solubility does not constitute a
limiting factor in the absorption process. Lipophilicity is a very useful physicochemical parameter reflecting
transfer properties of a compound. The partition coefficient (aqueous phase:n-octanol) is commonly used in the
pharmaceutical industry to reflect lipophilicity of a drug. The partition coefficient of MTD is 0.56 and 0.69 in
water and phosphate buffer (pH 7.4) respectively. So, MTD has more affinity to phosphate buffer than water.
Release of MTD is dependent on the solubilizing effect of the vehicle. MTD will be released fast and high from
water-soluble bases than oleaginous bases due to favorable partitioning of MTD toward the aqueous phase. Free
concentration of MTD will be greater in water soluble vehicles. In contrast, for the oleaginous vehicles,
partitioning towards the internal aqueous phase would render the drug almost unavailable in the external oil
phase.
It is possible to assume based on solubility of the drug in the vehicle, that formulation containing water
soluble base will be good candidates for the topical delivery of MTD.
Prerequisite
1. Permeation through skin.
2. Ointment Bases.
Requirements
1. Glasswares: Beaker, open diffusion cell, beakers, test tubes.
2. Chemicals: Metronidazole, potassium dihydrogen orthophosphate, etc.
3. Equipments: UV spectrophotometer.
4. Membrane: Rat membrane.
Procedure
1. Preparation of phosphate buffer 0.05M, pH 7.4:
Dissolve required quantity of potassium dihydrogen phosphate in sufficient quantity of water to produce 1000
ml and adjust the pH to 7.4.
2. Calibration curve:
1. Preparation of standard stock solution: Weigh accurately 100 mg of pure MTD and transfer it into 100 ml
volumetric flask and adjust volume (Stock I, 1mg/ml). Transfer 10 ml stock I to another 100 ml volumetric flask
and adjust volume (Stock II, 100 mg/ml).
2. Preparation of working solution: From stock solution II, pipette out 0.5, 1, 1.5, 2, 2.5 and 3 ml into 10 ml
volumetric flasks and adjust volume to get concentration in the range of 0-30 mg/ml.
3. Measurement of absorbance: Measure absorbance of the respective dilutions at lmax 320 nm using UV-
Visible spectrophotometer. Plot the graph of absorbance of MTD against concentration in MS Excel and
4. Preparation of formulations
0
1. Heat separately aqueous and oil phases at 70 C, and mix together with continuous stirring.
0
2. After cooling to (40-50 C), add MTD (1%) and mix to obtain a homogeneous and uniform mixture as per
formula given below
Ingredients F1 F2
Beeswax 5g -
White Vaseline 95 g -
Stearic acid - 20 g
Cetyl alcohol - 1g
Spermaceti wax - 1g
Glycerin - 5g
Potassium hydroxide - 1.05 g
Distilled water - 71.95 g
3. In vitro release and permeation

1. Use dialysis cell method to determine the amount of the drug diffused from different ointment formulations.
2. Shave abdominal hair of the rat and cut the skin samples in full thickness. Remove rat skin and wash with
water. Carefully remove fat and connective tissues and keep the skin in contact with receptor liquid (0.05 M
phosphate buffer; pH 7.4) for 1 h.
Hollow tube
Beaker
Membrane
Magnetic Stirrer
Figure 1. Design of diffusion assembly

3. Cover the tubes with a rat skin. Immerse the tubes into a 100 ml beaker containing 50 ml of the receptor phase
(0.05 M phosphate buffer; pH 7.4).
4. Stir the receptor phase continuously with a small magnetic bar at a speed of 100 rpm during the experiments
0
to ensure homogeneity and maintain at 37 C.
5. Apply known quantity of the formulation F1 (oleagineous base) on membrane. At predetermined time
interval remove samples and measure absorbance spectrophotometrically at 320 nm.
6. Repeat the experiment for formulation F2 (water soluble base).
7. Use the calibration curve for determination of the amount of MTD diffused.
8. Plot graph of amount of the drug diffused per unit area versus time and determine slope of linear portion of
graph.
9. Calculate permeability coefficients by using following formula
Kp = JSS/CV
2
Where, KP = permeability coefficient (cm/h), JSS = flux (mg/cm /hr), A = area of the diffusion
2
membrane (cm ); CV = initial concentration of the drug in the formulations (mg).

1. Beer's & Lambert range: 5-30 mg/ml
2. Solvent: 0.05 M phosphate buffer, pH 7.4
3. l max for Metronidazole: 320 nm
B. Parameters set for diffusion studies
1. Formulation: Ointment
2. Strength: 1%
3.Apparatus: Open tube dialysis diffusion cell
4. Rotation Speed: 100 rpm
5. Test time: 8h
6. Diffusion medium: 0.05 M phosphate buffer, pH 7.4.
7. Volume of diffusion medium: 50 ml
Observation
Table 1. Calibration curve of Metronidazole
Concentration
Absorbance
(mg/ml)
5
10
15
20
25
30
Slope
Intercept
Table 2. Ex vivo absorption of formulation F1 through rat skin

Time Concentration Total amount CADD/unit
Absorbance
(h) (mg/ml) diffused (mg) area
0.5
1
2
3
4
6
8
Table 3. Ex vivo absorption of formulation F2 through rat skin

Absorbance
0.5
1
2
3
4
6
8
CADD- cumulative amount of drug diffused
Calculations
Y= m X + c
Where, Y = absorbance, m = slope, X = concentration (mg/ml), c = intercept.

3. Surface area (A) of rat skin
A= pr
2
Where, r = radius of rat skin.

2
4. Cumulative amount of drug diffused per unit area (CADD/cm )
2
CADD/cm = CADD/Area of rat skin used
5. Dilution factor
6. Flux (JSS)
Slope (JSS) of linear portion of plot of amount of drug diffused per unit area versus time
7. Permeability coefficient (KP)
J ss
KP =
Cv
Results
1. Permeability coefficient and flux for formulation F1 were found to be ___cm/h and ___mg/cm /h respectively.
2
2. Permeability coefficient and flux for formulation F2 were found to be ___cm/h and ___mg/cm /h respectively.
2
Conclusion
It can be concluded from this study that the MTD shows higher permeability in water soluble base as
vehicle than oleaginous base.
Applications
1. Determination of suitable vehicle for the topical delivery of drug.
2. Estimation of flux and permeation coefficient of the drug.
3. Comparison of various brands available in the market by estimating flux and permeation coefficient.
Questions
1. What is permeation coefficient?
2. Define flux.
3. Discuss different types of vehicle bases.
Exercise
Study the effect of various bases on percutaneous absorption of Diclofenac gel.
Experiment 12
Percutaneous absorption of drug through different membranes
Aim
To study ex vivo percutaneous absorption of marketed Metronidazole ointment/gel through different
membranes.
Learning objectives
1. To understand the concept of permeability coefficient and its estimation.
2. To study the percutaneous absorption of Metronidazole through different membranes.
Theory
An ideal way to determine percutaneous absorption of a compound in human is to do the actual study in
humans. However, many compounds are potentially toxic to be tested in vivo in humans. Besides this,
evaluation of these systems in vivo using human beings is difficult from the viewpoint of cost, time
consumption, and ethical restrictions. Therefore the studies must be conducted in vitro using excised skin
(human cadaver, animals). Membranes from rats, mice, pigs, guinea pigs, snakes, rabbits and humans as well as
synthetic membranes have been used for these drug diffusion studies. The animal skins differ significantly from
human skin (HS) due to differences in thickness, nature of stratum cornea, density of hair follicles and sweat
glands. In this respect, in vitro studies are generally conducted using a diffusion cell system with either static or a
flow-through cell. The difficulty in obtaining excised skin and the variation in their permeability due to race,
age, sex, anatomical site and concern for restricted use of animals has led the workers to use simulated skin or an
artificial membrane.
Permeation of drugs through various artificial membranes, such as collagen membrane and oil
saturated membrane filter have been investigated. In addition, many studies have been reported on drug release
from ointments using silicon rubber membrane, cellulose membrane and membrane filter. However, results
obtained with these artificial membranes do not always reflect percutaneous absorption.
Natural membranes such as peach and tomato skin, inter-lamellar layer of the onion, and inner layer of
the egg could be applied instead of HS, synthetic, simulated or artificial skin in in vitro drug permeation studies.
Natural membranes can be good alternatives for permeability studies because of their easy availability, very low
cost and no ethical issues involved in it. Various artificial and natural membranes are compared with animal skin
for permeation studies, in this experiment.
Principle
Metronidazole (MTD), being intermediate lipophilic with low molecular weight (171.2), shows
highest permeability through cellophane membrane than all other membranes. Therefore for permeation study
of low molecular weight drugs, cellophane membranes cannot be the membranes of choice for in vitro studies,
instead of human skin. Some of the natural membranes are better models for such drugs. Hydrophilic drugs will
permeate well through onion membrane while lipophilic drugs permeate well through egg membrane. Natural
membranes have pores and channels with hydrophilic properties which permeate small to middle size
hydrophilic drugs to diffuse in a manner similar to human skin, and because of their availability thus can be used
for in vitro diffusion studies.
Prerequisite
1. Permeation through various membranes.
2. Permeability coefficient and flux.
Requirements
1. Glasswares: Beaker, open diffusion cell, beakers, test tubes etc.
2. Chemicals: Metronidazole, marketed Metronidazole gel or ointment, potassium dihydrogen orthophosphate,
etc.
3. Membranes: Rat skin, cellophane membrane, egg membrane and onion membrane.
Procedure
1. Preparation of phosphate buffer 0.05 M, pH 7.4
Dissolve required quantity of potassium dihydrogen phosphate in sufficient quantity of water to produce 1000
ml and adjust the pH to 7.4.
1. Preparation of standard stock solution: Weigh accurately 100 mg of pure Metronidazole and transfer it into
100 ml volumetric flask and adjust volume (Stock I, 1mg/ml). Transfer 10 ml stock I to another 100 ml
volumetric flasks and adjust volume (Stock II, 100 mg/ml).
2. Preparation of working solution: From stock solution II, pipette out 0.5, 1, 1.5, 2, 2.5 and 3 ml into 10 ml
volumetric flask and adjust volume to get concentration in the range of 0-30 mg/ml.
Visible spectrophotometer. Plot the graph of absorbance of MTD against concentration in MS Excel and
3. Preparation of membranes
1. Shave abdominal hair of the rat and cut the skin samples in full thickness. Remove rat skin and wash with
water. Carefully remove fat and connective tissues and leave the skin in contact with receptor liquid (0.05 M
phosphate buffer; pH 7.4) for 1 hour.
2. Peal the middle membrane of the Allium cepa L. (onion) with caution to prepare at least 10 cm2 uniform
membrane without any crack or orifice.
3. The outer shell membrane of the egg of Gallus domesticus that is just located inside the shell exactly under the
hard calcified layer is taken. This membrane is prepared by immersing the egg in 0.01N HCl solution for 6 h to
dissolve the calcified layer without any further process. Cut the membrane cautiously to expel the content of the
egg and wash it with normal saline solution.
4. Inspect the membrane by a microscope to assure about its integrity and uniformity.
1. Use the dialysis cell method to determine the amount of the drug diffused from MTD ointment using different
membranes prepared as given above.
2. Cover the tube with a rat skin. Immerse the tube into a 100 ml beaker containing 50 ml of the receptor phase
(0.05 M phosphate buffer; pH 7.4).
3. Stir the receptor phase continuously with a small magnetic bar at a speed of 100 rpm during the experiments to
0
ensure homogeneity and maintain at 37 C.
4. Apply known quantity of the formulation on membrane. At predetermined time intervals, remove sample and
measure absorbance spectrophotometrically at 320 nm.
5. Repeat the same experiment by using cellophane, onion and egg membrane.
6. Use the calibration curve for the determination of the amount of MTD diffused.
7. Plot the graph of amount of drug diffused per unit area versus time and determine slope of linear portion of
graph.
8.Calculate permeability coefficients by using following formula
KP = JSS/CV
2
Where, KP = permeability coefficient (cm/h), JSS = flux (mg/cm /hr), A = area of the diffusion
membrane (cm2); CV = initial concentration of the drug in the formulation (mg).

1. Beer's & Lambert range for MTD: 5-30 mg/ml
2. Solvent: 0.05 M phosphate buffer, pH 7.4
3.l max for MTD: 320 nm
4. Instrument: UV spectrophotometer
B. Parameters set for diffusion studies
1. Formulation: Ointment
2. Strength: 1%
3. Apparatus: Open tube dialysis diffusion cell
4. Rotation Speed: 100 rpm
5. Test time: 8h
6. Diffusion medium: 0.05 M phosphate buffer, pH 7.4
7. Volume of diffusion medium: 50 ml
Observations
Table 1. Calibration curve of Metronidazole
Concentration
Absorbance
mg/ml
0
5
10
15
20
25
30
Slope
Intercept
Table 2. Ex vivo absorption of drug through rat skin

Time Absorbance Concentration Total amount CADD/unit
0.5
1
2
3
4
6
8
CADD = cumulative amount of drug diffused
Table 3. Ex vivo absorption of drug through cellophane membrane
Absorbance
0.5
1
2
3
4
6
8
Table 4. Ex vivo absorption of drug through onion membrane
Time Absorbance Concentration Total amount CADD/unit
0.5
1
2
3
4
6
8
Table 5. Ex vivo absorption of drug through egg membrane
Absorbance
0.5
1
2
3
4
6
8
Calculations
1. Determination of concentration of diffused drug (mg/ml)
Plot the graph of absorbance versus concentration and determine slope and intercept. Calculate
unknown concentration using formula
Y= m X + c
Where, Y = absorbance, m = slope, X= concentration (mg /ml), c = intercept.
[Concentration (mg /ml) x (volume of diffusion medium) x (dilution factor)]/1000
3. Surface area (A) of membrane used
A=p r
2
Where, r = radius of rat skin / cellophane membrane/ onion membrane / egg membrane.
2
2
CADD/cm = CADD/Area of membrane
5. Dilution factor
6. Flux (JSS)
Plot the graph of amount of drug diffused per unit area versus time. The slope of linear portion of plot
will give flux, JSS.
Permeability coefficient calculated by using formula
J
K P = ss
Cv
Where CV is the initial concentration of the drug in donor compartment (mg).
Results
1. Permeability coefficient and flux of MTD through rat membrane is ____cm/h and ____mg/cm /h respectively.
2
2. Permeability coefficient and flux of MTD through cellophane membrane is ____cm/h and ____mg/cm /h 2
respectively.
3. Permeability coefficient and flux of MTD through onion membrane is ___cm/h and ___mg/cm /h respectively.
2
4. Permeability coefficient and flux of MTD through egg membrane is ____cm/h and ____mg/cm /h respectively.
2
5. The permeability of MTD is in the order _____> _____> _______ > _______.
Conclusion
It can be concluded from this study that the formulation of MTD shows highest permeability through
cellophane membrane while lowest permeability through rat membrane as the drug is hydrophilic in nature.
Applications
1. Determination of suitable membrane for the permeability studies.
2. Estimation of flux and permeation coefficient of the drug.
3. Comparison of various brands of drugs available in the market can be done by estimating flux and permeation
coefficient.
Questions
1. Discuss various in vitro and ex vivo models of percutaneous absorption studies.
2. Which membrane is ideal membrane for percutaneous absorption studies and why?
3. How will you calculate the flux of absorption through membrane?
4. Describe permeability coefficient of drug.
Exercise
Perform this experiment using tomato, peach and capsicum membrane.
Experiment 13
In vitro permeation study using Franz diffusion cell
Aim
To study the in vitro permeation of Diclofenac gel using Franz diffusion cell.
Learning objectives
1. To understand construction of Franz diffusion cell.
2. To study the percutaneous absorption of Diclofenac using Franz diffusion cell.
Theory
Principle
The in vitro permeation of drug can be studied by Franz diffusion cells. The Franz diffusion cells are
made of glass with a contact area of 4.7 cm2 and pretreated with a silanizing agent. The Franz diffusion cell
consists of a donor compartment (A) and a receptor compartment (B). The membrane is mounted between two
cell compartments. The two cell compartments are held together with a clamp. The receptor compartment has a
volume of 37 ml and is filled with diffusion medium. It is kept at 37°C by circulating water through an external
water jacket. After 30 min of equilibration of the membrane with the receptor solution, specific quantity of the
drug/ formulation is applied in the donor compartment. The donor compartment is then covered with parafilm to
prevent evaporation of the solvent. The receptor solution is continuously stirred by means of a spinning bar
magnet, at 400 rpm. Receptor solution samples, 2.0 ml aliquots, are withdrawn through the sampling port of the
receptor compartment at various time intervals. The cells are refilled with receptor solution to keep the volume
of receptor solution constant during the experiment.
Sample holder A
Sample port
Membrane
Figure 1. Diffusion study cell assembly

B
Stir bar
Magnetic stirrer
Prerequisite
Requirements
1. Glasswares: Franz diffusion cell, test tubes, pipettes, etc.
2. Chemicals: Pure Diclofenac sodium, marketed Diclofenac gel, potassium dihydrogen orthophosphate, etc.
3. Membrane: Cellophane membrane.
4. Equipments: UV Spectrophotometer, magnetic stirrer, thermostatic water bath, pump.
Procedure
1. Preparation of Sorensen's phosphate buffer, pH 7.4
Solution A: Take 3.5 g of disodium hydrogen phosphate and dissolve it in 100 ml distilled water. Solution B:
Take 2.76 g of sodium dihydrogen phosphate and dissolve it in 100 ml distilled water. Mix 40.5 ml of solution A
with 9.5 ml of solution B and make volume to 100 ml.
2. Plotting of calibration curve of Diclofenac sodium
1. Preparation of standard stock solution: Weigh accurately 100 mg of pure Diclofenac sodium and transfer it
into 100 ml volumetric flask and adjust volume with water (Stock I, 1mg/ml). Transfer 10 ml stock I to another
100 ml volumetric flask and adjust volume (Stock II, 100 mg/ml).
2. Preparation of working solution: From stock solution II, pipette out 0.4, 0.8, 1.2, 1.6, 2, and 2.4 ml into 10 ml
volumetric flasks and adjust volume to get concentration in the range of 4-24 mg/ml.
Visible spectrophotometer. Plot the graph of absorbance of Diclofenac sodium versus concentration in MS
Excel and determine slope and intercept.
1. For the permeation study use Franz diffusion cells with a diffusion area of 4.7 cm2 and 37 ml capacity.
2. Place cellophane membrane between the donor and receptor compartments of the cells.
3. Fill receptor compartment with approximately 37 ml of Sorensen's phosphate buffer (pH 7.4). Maintain
temperature of cell at 370C by means of circulating contents of a thermostatic water bath by a pump through the
surrounding layer of the cell. Stir content of receptor compartment at 600 rpm with Teflon-coated magnetic bar
placed inside cell throughout experiment.
4. Ensure that membrane should be in contact with the receptor medium.
5. Place 1 gm Diclofenac gel in a donor compartment and cover donor cell with aluminum foil to avoid
evaporation.
6. Remove 2 ml sample from receptor compartment at predetermined time intervals and immediately replace
with 2 ml of the receptor solution, at the same temperature.
7. Dilute withdrawn samples if required and analyze spectrophotometrically at 270 nm.
8. Use the calibration curve for the determination of the amount of Diclofenac sodium diffused.
9. Plot the graph of amount of drug diffused per unit area versus time and determine slope of linear portion of
graph.
10. Calculate permeability coefficients by using following formula:
KP = JSS/CV
Where, KP = permeability coefficient (cm/h), JSS = flux (mg/cm2/hr), A = area of the diffusion
membrane (cm2); CV = initial concentration of the drug in donor compartment (mg).
Obervations
Table 1. Calibration curve of Diclofenac sodium
Concentration
Absorbance
(mg/ml)
0
4
8
12
16
20
24
Slope
Intercept
Table 2. In vitro permeation of Diclofenac sodium through cellophane membrane
Time Absorbance Concentration Cumulative CADD/unit

(h) (mg/ml) amount of drug area
diffused (mg)
0.5
1
1.5
2
Calculations
1. Determination of concentration of diffused drug (mg/ml)
Y= m X + c
3. Surface area (A) of cellophane membrane
A= pr
2
Where, r = radius of cellophane membrane exposed to diffusion medium.

2
2
CADD/cm = CADD/Area of cellophane membrane
5. Dilution factor
6. Flux (JSS)
Slope (JSS) of linear portion of plot of amount of drug diffused per unit area versus time.
J ss
KP =
Cv
Where, CV = initial concentration of the drug in donor compartment (mg).
Result
Permeability coefficient and flux of Diclofenac gel through cellophane membrane is __________cm/h
and ________mg/cm /h respectively.
2
Conclusion
It can be concluded from this study that in vitro permeation of Diclofenac gel through cellophane can
be studied using Franz diffusion cell.
Applications
1. This experiment allows us to study in vitro permeation of various transdermal formulations.
2. This model can be used to study effect of formulation variables on permeation of drugs.
3. Estimation of flux and permeation coefficient is possible.
4. This model can be utilised for studing permeation of drug through nasal and buccal route.
Question
Give various in vitro permeation models
Exercise
Perform in vitro permeation study of Ketoconazole gel using Franz diffusion cell.
SECTION 3
Protein binding of drugs
Experiment 14
Protein binding study using equilibrium dialysis method
Aim
To study the protein binding of Salicylic acid by method of equilibrium dialysis.
Learning objectives
1. To understand the protein binding process of a drug.
2. To confirm protein binding of Salicylic acid by equilibrium dialysis method.
Theory
The desired therapeutic response of a drug is elicited as a result of its interaction with the specific
receptor site. On its way to that receptor, the drug may bind to other constituents of biological system, such as
plasma proteins like albumin, high density lipoproteins and globulins. It is the free drug that can diffuse to and
from the extra vascular fluid across the endothelium and other biological barriers. The unbound fraction of drug
in plasma/serum is available for binding to a specific site. The nature and magnitude of drug protein interaction
significantly influence biologic activity of a drug by affecting its pharmacokinetic fate and pharmacodynamic
performance.
A drug in the body can interact with several tissue components of which two major categories are
blood and extra vascular tissues. The interacting molecules are generally macromolecules such as proteins,
DNA, etc. Proteins are particularly responsible for such an interaction. The phenomenon of complex formation
with proteins is called as protein binding of a drug. Importance of such a binding derives from the fact that the
bound drug is both pharmacokinetically as well as pharmacodynamically inert. Binding of drugs generally
involves weak chemical bonds such as hydrogen bonds, ionic bonds or van der Waal's forces and, therefore, is a
reversible process.
Binding of a drug to protein contained in the body influence their action in a number of ways.
1. Protein may facilitate the distribution of a drug throughout the body.
2. It may inactivate the drug by not enabling a sufficient concentration of free drug to develop at the receptor
site.
3. It may retard excretion of a drug.
4. The interaction of a drug with protein may cause
a) The displacement of body hormones or a co administered agent.
b) Aconfigurational change in the protein.
c) Formation of a drug protein complex that itself is biological active.
Methods to study protein binding of drug
Number of methods are available for studying protein binding of drugs. Apart from satisfying the
general criteria for any analytical technique, they must not upset the equilibrium between free and bound drug.
66
Section 3 Protein binding of drugs 67
Equilibrium dialysis, dynamic dialysis, ultra filtration and electrophoresis are the classic techniques widely
used to study protein binding. In recent years other methods such as gel filtration and nuclear magnetic
resonance have been used with satisfactory results, all of them having their own inherent advantages and
disadvantages.
Spectral changes
Most drugs have distinct UV spectra because of the conjugated chromophores in the molecule. When a
drug interacts with a protein the UV or visible spectrum may be changed because of alterations in the electronic
configuration. These alterations can be quantitated and used to determine the extent of binding. Changes in
fluorescence spectra can be used in the same way.
Gel filtration
This involves the use of porous gels that are molecular sieves. They separate components on the basis
of size. Low molecular weight drugs are held on the gel whereas bound drugs and proteins are washed through.
Equilibrium dialysis Membrane
Protein
Buffer
Drug Free drug
Drug
Buffer
Figure 1. Equilibrium across a semi-permeable membrane

The protein solution (e.g. plasma) containing a drug and a buffer solution are placed on opposite sides
of a dialysis membrane.
After a sufficient time (may be 12- 24 h), free drug concentration will be same on either side of the
membrane. Protein binding can be determined by measuring the concentration of a drug on either side of the
membrane. On left the concentration will involve free and bound drug, whereas on the right there is no binding
and the concentration will be equal to the free drug concentration.
Ultrafiltration
Drug and protein solution
Membrane
Free drug filtrate
Figure 2. Ultrafiltration as a method of measuring protein binding

A quicker method of separating free and bound drug is the ultrafiltration method. Drug and protein
solutions are placed in a filter membrane and liquid containing free drug is forced through the membrane by
centrifugation.
Dynamic dialysis
One of the methods widely used to study in vitro protein binding of drug is dynamic dialysis. Dynamic
dialysis uses flow dynamics to increase both the rate and efficiency of dialysis. Circulating the sample and/or
the dialysis creates the highest possible concentration gradient to significantly decrease dialysis time. Other
benefit of the sweeping action is that it prevents membrane fouling and in some situation generates a pressure
differential. This supplemental driving force increases the hypo-osmotic mass transfer rate across the semi-
permeable membrane and allow for sample concentration during the dialysis process.
Principle
TubeAcontains only Salicylic acid with no protein in it. The equilibrium of Salicylic acid in tubeAand
beaker is achieved through semipermeable membrane and the concentration of Salicylic acid in beaker can be
measured. However, Tube B contains Salicylic acid in presence of protein (0.5 ml egg albumin) and hence the
protein binding of Salicylic acid takes place. This allows only free Salicylic acid left after protein binding to
equilibrate through semipermeable membrane, which reduces the concentration of Salicylic acid in beaker.
Further, reduction in concentration of Salicylic acid takes place in tube C because of increased presence of
protein (1 ml egg albumin). This increases protein binding of salicylic acid, thereby reducing free Salicylic acid
in the tube and in beaker too.
Salicylic acid present in beaker (non protein compartment) is estimated by adding ferric nitrate
solution. The reaction of Salicylic acid with ferric nitrate produces an intensely colored complex (deep wine),
whose maximum absorbance can be detected at 540 nm spectrophotometrically.
Prerequisite
1. Concept of protein binding.
2. Significance of protein binding.
Requirements
Chemicals: Salicylic acid, egg albumin, ferric nitrate, hydrochloric acid, mercuric chloride, cellophane
membrane, and distilled water.
Instruments: UV spectrophotometer, magnetic stirrer and equilibrium dialysis unit.
Procedure
1. Calibration curve of Salicylic acid
1. Preparation of coloring agent: Dissolve mercuric chloride (4 g) in about one-fifth volumes of water.
Separately dissolve ferric nitrate (4 g) in 0.12 N HCl (12 ml). Mix these two solutions and adjust volume to 100
ml, filter and use the filtrate as a coloring agent.
2. Preparation of standard stock solution: Weigh accurately 100 mg of Salicylic acid. Dissolve in 100 ml of
distilled water using 5 ml alcohol. Take 10 ml of this solution and dilute to 100 ml with water.
3. Preparation of working solution: From stock solution, pipette out 0.2, 0.4, 0.6, 0.8 and 1 ml into 10ml
volumetric flask and add 4 ml coloring agent. Dilute resulting solution to 10 ml with water to get concentration
in range of 2-10 µg/ml.
4. Measurement of absorbance: Record absorbance of the working solution at lmax of 540 nm using UV-
Visible spectrophotometer against water as a blank. Plot a graph of absorbance versus concentration and
2. Protein binding study of drug

1. Take three hollow tubes and mark them asA, B and C.
2. To one end, tie a semipermeable membrane in such a way that a sack is formed.
3. Place 1ml of 1% Salicylic acid and 1 ml of distilled water in tubeA.
4. Place 1ml of 1% Salicylic acid and 0.5 ml of egg albumin and 0.5 ml distilled water in tube B.
5. Place 1ml of 1% Salicylic acid and 1 ml of egg albumin in tube C.
6. Place the tubes in a 50 ml of distilled water and pipette out 5 ml of sample from beaker at the intervals of 15,
30, 45, 60, 75 and 90 min.
7. Add 4 ml of coloring agent and 1 ml of distilled water and determine absorbance at 540 nm. Replace the fluid
with 5 ml of distilled water in the beaker.
8. Plot comparative curves of amount of Salicylic acid present in non protein compartment versus time.
Figure 3. Equilibrium dialysis unit
Observations
Table 1. Calibration curve of Salicylic acid
Concentration of SA Absorbance
mg/ml
2
4
6
8
10
Slope
Intercept
Table 2. Percent cumulative drug release with and without protein through cellophane membrane

(min) Tube A Tube B Tube C
15
30
45
60
75
90
Calculations
A. Determination of unknown concentration of Salicylic acid
Determine unknown concentration of Salicylic acid from receptor compartment by using following
equation
Y = mX +c
Where, Y = absorbance, m = slope, X = concentration, c = the intercept.
B.Amount of drug diffused (mg)
Amount of drug diffused =
C. Dilution factor
Dilution factor = volume of diluted sample (ml) / volume of sample removed (ml)
D. Percent cumulative drug release
Percent cumulative drug release = (amount of drug diffused x 100) /total amount of dose
Result
The percent release of Salicylic acid without any proteins was found to be _______, with 0.5 ml of egg
albumin was______, and with 1 ml of egg albumin was_______.
Conclusion
The protein binding of Salicylic acid is confirmed and the increase in protein binding is found to be
proportional to the amount of protein available.
Applications
1. It can be used for studying protein binding of various other drugs.
2. Equilibrium dialysis method provides the non invasive in vitro method for studying protein binding.
Questions
1. Discuss about protein binding of Salicylic acid.
2. Give clinical significance of protein binding.
Exercise
Study the protein binding of Ranitidine hydrochloride using equilibrium dialysis method.
Experiment 15
Protein binding study using dynamic dialysis method
Aim
To study the effect of protein binding on drug diffusion by dynamic dialysis method.
Learning objective
To compare protein binding of human serum and plasma with a model drug Tetracycline.
Theory
Theory as per previous experiment (Expt No. 14).
Principle
Percentage of Tetracycline release is estimated by knowing its concentration in non protein
compartment at various time intervals. The release of a drug is very less in presence of serum and plasma than
that of the release of a dug without any proteins. This represents well that plasma proteins, serum proteins have
great affinity towards the drug and bind with it.
While comparing the drug release in presence of serum or plasma, the percentage of drug release in
presence of plasma is less than that in presence of serum, since the serum lacks the clotting factors that are
normally present in plasma but have been consumed during the process of coagulation. It indicates that the
extent of protein binding is directly proportional to the amount of protein.
Due to the affinity of plasma proteins towards drugs they bind with the drug and release the free drug
slowly to maintain the equilibrium between free drug and bound drug. This may help to prevent the quick
elimination of drug and since the therapeutic action of a drug depends upon the concentration of free drug alone,
protein binding may also help to prolong action of the drug.
Prerequisite
Requirements
Chemicals: Tetracycline, human serum, human plasma, hydrochloric acid, egg membrane and distilled water.
Instruments: UV spectrophotometer, magnetic stirrers, dynamic dialysis setup.
Procedure
1. Calibration curve of Tetracycline
a) Preparation of standard stock solution: Weigh accurately 100 mg of Tetracycline and dissolve it in 100 ml of
distilled water. Take 10 ml of this solution and dilute to 100 ml with water.
b) Preparation of working solution: From stock solution, pipette out 0.2, 0.4, 0.6, 0.8, 1 and1.5 ml into 10 ml
volumetric flask and adjust the volume to get concentration in range of 2-15 µg/ml.
c) Measurement of absorbance: Record absorbance of working solution at lmax of 360 nm using UV-Visible
spectrophotometer against water as a blank. Plot a graph of absorbance versus concentration and determine
2. Preparation of egg shell membrane

Soak a whole chicken egg in 0.5 N HCl solution. The outer calcareous shell gets dissolved, then cutoff
a part of the egg shell membrane and remove the inner contents. Wash obtained membrane thoroughly in
distilled water and store it in a refrigerator and use within a week.
3. Protein binding study of Tetracycline
1. Tie egg membrane to one end of an open-ended glass cylinder and use it as a protein compartment (donor).
2. Use a glass beaker capacity of 25 ml as a non-protein compartment (receptor) and fill it with 20 ml of distilled
water.
3. Place drug solution (1 mg/ml) of 2 ml to the inner tube and immerse into the beaker. Take care to maintain the
level of the drug solution coincide with the water level in outside compartment and fix it with a stand.
4. Keep this whole set-up on a magnetic stirrer and stirr the outer compartment continuously with an optimal
0
speed (see figure 1). Maintain the temperature at 35 ± 2 C for the whole experiment.
5. At predetermined time intervals (5, 10, 15, 30, 60, 90 min), remove 1 ml of the sample from beaker and
replace it with same volume of fresh distilled water.
6. Determine the concentration of Tetracycline spectrophotometrically.
7. Repeat the experiment by using 1 ml of human blood serum and 1 ml of drug solution (2 mg/ml) in the protein
compartment and determine the percentage of drug released from the protein compartment into the non-protein
compartment in the same time period as above.
8. Also repeat this experiment using 1 ml of human blood plasma and 1 ml of drug solution (2 mg/ml) in the
protein compartment and determine the percentage of the drug released.
9. Plot the graph of percent cumulative drug release versus time.
Figure 1. Dynamic dialysis unit

Observations
Table 1. Calibration curve of Tetracycline
Concentration
Absorbance
(mg/ml)
2
4
6
8
10
15
Slope
Intercept
Table 2. Percentage cumulative drug release with and without protein through egg membrane

(min) Without any protein In presence of serum In presence of plasma
5
10
15
30
60
90
Calculations
1. Determination of unknown concentration of Tetracycline
For the determination of unknown concentration of Tetracycline from receptor compartment
following equation is used.
Y = mX + c
Where, Y = absorbance, m = slope, X = concentration, c = intercept.
2.Amount of drug diffused (mg)
Amount of drug diffused =
3. Dilution factor
Percent cumulative drug release = amount of drug diffused x 100 /drug dose
Result
The percent release of Tetracycline without any proteins was found to be _______, with plasma was
_______, and with serum was ______.
Conclusion
The protein binding of Tetracycline is confirmed and is dependant on the amount of proteins available.
Therefore, the extent of protein binding in plasma is more giving lesser drug release.
Applications
1. Protein binding of drug can be studied.
2. The displacement of one drug from protein binding site in presence of another drug can be studied.
3.Affinity of various drugs towards protein can be studied.
4. Protein binding study is very important for calculation of dose of the drug.
Questions
1. Give significance of protein binding of a drug.
2. Give five examples of displacement of drugs from protein binding site and their pharmacological effects.
Exercise
Study the protein binding of Phenylbutazone using dynamic dialysis method.
Experiment 16
Determination of binding sites using bovine serum albumin
Aim
To determine binding site of Propranolol using Warfarin sodium/Diazepam as a site-I/II specific
probe.
Learning objectives
1. To understand and study binding of drugs to albumin.
2. To determine binding site of Propranolol HCl.
Theory
Bovine serum albumin (BSA) is a large multi-domain protein folded into three domains, each of which
is built of three loops. On the basis of probe displacement method, there lie at least three relatively high specific
binding sites on the BSA molecule. These sites are generally called the warfarin-binding site, the
benzodiazepine-binding site and digoxin-binding site and are denoted as site-I, II and III, respectively. Site-II is
more specific than site-I whereas site-III is an independent binding site. Serum albumin, the most abundant
protein in the blood, plays a very important role in the binding phenomenon and serves as a depot protein and
transport protein for numerous endogenous compounds. Displacement of a drug is defined as reduction in the
extent of binding of a drug to other agent, the displacer. This type of interaction may occur when two drugs or
agents, capable of binding to proteins, are administered concurrently. Competitive displacement is more
significant, when two drugs or agents are capable of binding to the same sites on the protein. From different
investigations, it has been suggested that human serum albumin (HSA) has limited number of binding sites.
Since the number of protein binding sites is limited, competition will exist between drugs or drugs with metals
or other agents and the agent with higher affinity will displace the other causing increased free drug
concentration leading to higher toxicity or short duration of action. Ability of one drug to inhibit the other is a
function of their relative concentration, binding affinities and specificity of binding.
Principle
Interactions of Propranolol HCl and its binding characteristics on BSA can be studied by equilibrium
dialysis method. It provides the possible way of in vitro estimates of protein binding to Propranolol HCl. The
relative strength and specificity of binding to BSA is determined by its ability to displace the probes (warfarin as
site-I specific probe and diazepam as site-II specific probe) specific for particular sites (site-I or site-II) on the
BSA molecule. By measuring the free concentration of the site specific probe it is inferred with regard to the
binding of Propranolol HCl to BSA. BSA and HSA have structural similarity. In this study BSA, in lieu of
human serum albumin (HSA), can be used because of its low cost and easy availability.
Propranolol HCl is known to increase the free concentration of Warfarin to a greater extent than that of
Diazepam, showing its high affinity binding to site-I and low affinity to site-II in the BSA. So, in patients
suffering from hypertension, if they take drugs having high affinity for site-I, it may result in rapid action or
rapid excretion from the body or even may cause toxicity at the normal doses.
Prerequisite
Requirements
1. Drugs: Warfarin sodium, Diazepam, Propranolol HCl.
2. Reagents: Disodium hydrogen phosphate (Na2HPO4), potassium dihydrogen phosphate (KH2PO4), borax
(NaB4O7.10H2O), cellulose membrane (M.W. 1200 Daltons), bovine serum albumin (fatty acid free, fraction V,
96-98%, M.W. 66500).
3. Instruments: UV/VIS spectrophotometer, pH Meter, metabolic shaking incubator, micro syringes.
4. Method: Equilibrium dialysis method.
Procedure
Calibration curve of Warfarin
1. Preparation of standard stock solution: Weigh accurately 100 mg of Warfarin. Dissolve in 100 ml of
phosphate buffer pH 7.4. Take 10 ml of this solution and dilute to 100 ml with buffer (100 mg/ml).
2. Preparation of working solution: From stock solution pipette out 0.2, 0.4, 0.6, 0.8 and 1 ml into 10 ml
volumetric flasks and make volume to 10 ml with buffer to get concentration in range of 2-10 µg/ml.
3. Measurement of absorbance: Record absorbance of working solution at l max of 308 nm using UV-Visible
Calibration curve of Diazepam
1. Preparation of standard stock solution: Weigh accurately 100 mg of Diazepam. Dissolve in 100 ml of
phosphate buffer pH 7.4. Take 10 ml of this solution and dilute to 100 ml with buffer (100 mg/ml).
2. Preparation of working solution: From stock solution, pipette out 0.2, 0.4, 0.6, 0.8 and 1 ml into 10 ml
volumetric flask and make volume to 10 ml with buffer to get concentration in range of 2-10 µg/ml.
3. Measurement of absorbance: Record absorbance of working solution at max of 235 nm using UV-visible
Protein binding study
Perform the experiment in the following successive steps:
-5
1. Prepare 2×10 M BSAsolution and transfer 3 ml in each of fifteen clean and dry test tubes.
-3
2.Add 1×10 M Warfarin solution to seven test tubes so that the final ratio of protein and Warfarin is maintained
-5 -5 -3
1:1 (2×10 M: 2×10 M) in each of first seven test tubes. Now, add 1×10 M Diazepam solution to next seven
-5 -5
test tubes so that the final ratio of protein and Diazepam is maintained 1:1 (2×10 M: 2×10 M) in each of next
seven (8-14) test tubes. Mark the fifteenth test tube containing only BSAsolution as "Blank" or "Control".
3. Allow these mixtures to stand for 10 minutes in order to allow binding of the Warfarin to its particular binding
site (to site-I) and that of Diazepam to binding site II.
-5
4. Add Propranolol HCl solution with increasing concentrations (0-12 x 10 ) into six out of seven test tubes
containing 1:1 mixture of protein and Warfarin and also into six out of seven test tubes containing 1:1 mixture,
protein and Diazepam. The final ratios of protein: Warfarin: Propranolol HCl or, protein: Diazepam:
Propranolol HCl are 1:1:0, 1:1:1, 1:1:2, 1:1:3, 1:1:4, 1:1:5 and 1:1:6. The test tube No. 7 will contain only
protein-Warfarin mixture and test tube No. 14 will contain only, protein-Diazepam mixture in the ratio 1:1.
5. Mix the solution properly and allow to stand for 10 minutes to ensure maximum binding of Propranolol HCl
to site-I and site-II and thereby displacing the probes from site-I and site-II on BSA.
6. Take 2 ml of solution from each test tube into fourteen different semi-permeable membrane tubes. Clip the
two ends of the membrane tube and ensure that there is no leakage.
7. Immerse the membrane tubes in fourteen separate 50 ml conical flasks containing 30 ml of phosphate buffer
solution pH 7.4.
0
8. Place the conical flasks in a metabolic shaker for dialysis at 25 C and shake at 20 rpm. Continue the shaking
for 10 hours.
9. At the end of dialysis, collect samples from each flask. Measure the free concentrations of Warfarin and
Diazepam by a UV spectrophotometer at lmax 308 and 235 nm respectively.
10. Plot the graph of free concentration of Warfarin sodium and free concentration of Diazepam as percent of
initial concentration versus ratio of Propranolol to BSAconcentration.
Observations
Table I. Calibration curve of Warfarin
mg/ml
Slope
Intercept
Table 2. Calibration curve of Diazepam
mg/ml
Slope
Intercept
Table 3. Displacement of Warfarin/ Diazepam by Propranolol HCl
Propranolol to Warfarin Free concentration of Diazepam Free concentration of

BSA ratio concentration Warfarin as percent Concentration Diazepam as percent
of initial of initial
0
1
2
3
4
5
6
Results
1. The free fraction of Warfarin (site-I specific probe) increased from 100% (as % of initial) to
_______ with an increasing concentration from 0-12×10-5 M of Propranolol HCl.
2. The free fraction of Diazepam (site-II specific probe) increased from 100% (as % of initial) to _______ with
the same increment range of Propranolol HCl.
Conclusion
As % increment of the free fraction Warfarin is more than that of Diazepam, it can be concluded that
Propranolol shows high affinity binding to site-I (Warfarin binding site) than to site-II (Diazepam binding site).
Applications
Questions
1. Give various drug binding sites on human serum albumin.
2. Give various factors affecting protein-drug binding.
Exercise
Study displacement of Warfarin and Diazepam by Tetracycline.
SECTION 4
Metabolism of drugs
Experiment 17
Metabolism of drug using in vitro method
Aim
To study metabolism of Hexobarbitone sodium by enzymes of the liver microsomal fraction.
Learning objectives
1.To study the metabolism of Hexobarbitone sodium.
2.To understand the activity of enzymes most concerned in metabolism of exogenous compounds.
Theory
Metabolism is the process of transformation of water insoluble, lipophilic, non polar drugs into polar
and water soluble products that can be easily excreted by the kidneys. Hence metabolism is a detoxification
process. The enzymes that biotransform drugs are broadly divided into two categories: microsomal and non
microsomal. The microsomes are isolated from endoplasmic reticulum of liver homogenate. The large varieties
of microsomal enzymes catalyse a number of oxidative, reductive, hydrolytic and glucuronidation reactions.
The non microsomal enzymes are those that are present in soluble form in the cytoplasm, that catalyse few
oxidative reactions, a number of reductive and hydrolytic reactions and conjugation reactions other than
glucuronidation.
Principle
The preparation of enzymes first involves separation of the supernatant fraction of the liver
homogenate by centrifugation at 10,000 g. The supernatant fraction contains soluble enzymes and the
microsomes. Metabolism reactions are first performed with the supernatant fraction. Conversion of
hexobarbitone takes place due to oxidation by several routes given below:
CH3
H CH3
O N OH
O N OH O N OH H3C
H 3C N
H 3C
N N
O
O O
OH
Desmethylhexobarbitone Hexobarbitone 3 -Hydroxyhexobarbitone
Microsomes are then prepared and same reactions are performed.
The enzymes concerned require NADPH and oxygen. NADPH can be added directly but it is more
satisfactory to use a generating system. The soluble fraction can be used to generate NADPH
Mg++
Glucose-6-phosphate + NADP 6-phosphogluconolactone + NADPH
glucose-6phosphate dehydrogenase
79
When the 10,000 g supernatant is used it is necessary to reinforce endogenous glucose-6 phosphate
and NADP. When microsomes are used, it is necessary to add glucose-6 phosphate dehydrogenase as well. In
addition, nicotinamide is added to prevent destruction of NAD by tissue nucleosides.
It is necessary also to add Mg++ ions when hydroxylation is desired, but demethylation apparently
proceeds satisfactorily with no addition of magnesium.
The enzymes involved are most commonly named cytochrome P-450 and b5, and these enzymes are
characterized by their spectral properties. Their concentrations are related to pre-treatment of the animals and
microsomal protein. Metabolism of the test compound can be related to amounts of the microsomal enzymes.
Requirements
Apparatus: Refrigerated centrifuge, tissue homogenizer, spectrophotometer, water bath, surgical instruments.
Chemicals: KCl (1.15%) in phosphate buffer (0.01M, pH 7.6), phosphate buffer (0.5M, pH 7.6), phosphate
buffer (0.05M, pH 7.6), hexobarbitone sodium, NADP, glucose-6-phosphate, nicotinamide, magnesium
chloride, glucose-6-phosphate dehydrogenase, citrate buffer (0.5M, pH 5.5) saturated with NaCl, n-heptane,
amyl alcohol.
Animal: Rats (150-250g, fasted overnight before the experiment).
Procedure
1. Preparation of the 10,000 g supernatant fraction
1.Take the approval of the study from institutional animal ethics committee.
2. Kill the rat with a blow on the head.
3. Remove the liver(s) immediately (25 g of liver is required) into the beaker containing ice and KCl/0.01M
buffer. Weigh the liver and add more KCl/0.01 M buffer to form a 25% suspension of liver in water (volume
approximately 100 ml).
4. Chop or mince the liver to form small pieces. This can be done on a cold tile or under the buffer in the tared
beaker.
5. Homogenize in the tissue homogenizer taking small quantity at a time subjecting each fraction to six or eight
vertical excursions.Also keep everything cold by using ice bucket.
6. Centrifuge the suspension at 10,000 g for 20 min at 40C. The tubes should be as full as possible and balanced,
and generally 2 x 50 ml will be sufficient.
7. Remove the supernatant (designated 10,000 g supernatant) and store on ice to be used on the day of
preparation. One milliliter is equivalent to 250 mg of liver.
2. Preparation of microsomes
1. Centrifuge 4 x 14 ml of the 10,000 g supernatant at 100,000 g for 60 min.
2. Decant the supernatants and discard.
3. Re-suspend the pellets in 10 ml of KCl/0.01M buffer in the centrifuge tubes and fill the tube after re-
suspension is complete.
4. Re-centrifuge as before for 30 min and decant the supernatant.
5. Overlay pellets with 1 ml (or more if needed) of 0.05 M phosphate buffer pH 7.6 for storage. Store at -180 for
further study.
Section 4 Metabolism of drugs 81
3. Preparation of a cofactor solution for metabolic reactions using the 10,000 g supernatant and
microsomes
Table 1. Formula for preparation of a cofactor solution
Quantity for each Formula for
Code Constituent
incubation flask experiment
a NADP 0.65 mmol 5 mg
b Glucose-6-phosphate 10 mmol 28.2 mg
c Nicotinamide 50 mmol 61.05 mg
d Magnesium chloride 25 mmol 50.8 mg
e Glucose-6-phosphate dehydrogenase 2 units 20 units
f Phosphate buffer pH 7.6 (0.5M) to 3 ml to 30 ml
4. Metabolism of Hexobarbitone sodium in liver homogenate
1. Prepare an aqueous solution of Hexobarbitone sodium at 1mg/ml. Salt is freely soluble.
2. Using 25 ml Erlenmeyer flasks (wide mouthed conicals) prepare a series of solutions as given in table 2.
Prepare these solutions from ice cold reagents and keep the mixtures ice cold until starting the experiment, as
preliminary incubation can inactivate microsomal enzymes. The cofactor solution must contain constituents of
table 1 except (e).
Table 2. Solutions required to demonstrate Hexobarbitone metabolism
Flask Hexobarbitone solution Distilled Cofactor KCl/buffer 10,000g
No (1mg/ml) (ml) water (ml) solution (ml) (ml) supernatant
1 - 1 3 - 2
2 - 1 3 - 2
3 1 - 3 - 2
4 1 - 3 - 2
5 1 - 3 2 -
6 1 - 3 2 -
7 1 - 3 - 2
8 1 - 3 - 2
0
3. Incubate flasks 1 to 6 with gentle shaking for 30 min at 37 C; retain flasks 7 and 8 on ice.
4.At the end of 30 minutes cool flasks 1 to 6 upto 40C. Store all eight flasks if necessary at -180C.
5.Assay of Hexobarbitone
1. Prepare distilled water blanks and hexobarbitone sodium standards in water at 10, 20, 50, 100 and 200 mg/ml
from a 1mg/ml solution prepared.
2. Add 0.5 ml aliquots of these preparations to screw capped tubes containing 1 ml of 0.5 M citrate buffer pH 5.5
saturated with NaCl and 10 ml of n-heptane containing 1.5 % amyl alcohol.
3. Shake for 10 min and centrifuge.
4. Remove the aqueous layer completely with pipettes.
5.Add 1.5 ml of the citrate buffer to the same tubes and once again shake for 10 minutes and centrifuge.
6. Transfer 8 ml of the heptane layer to a fresh tube containing 4 ml of 0.8 M phosphate buffer pH 11.
7. Shake and centrifuge.
8. Record the spectrum of the aqueous layer over the range 200-400 nm with water in the reference position.
9. Determine values of (OD245-OD280) and compare experimental unknowns with standard curves.
Hexobarbitone sodium is relatively unstable at pH 11 and delays in reading the OD values should be avoided.
6. Metabolism of Hexobarbitone by microsomes
Follow the procedures in the previous sections except
0
1. Resuspend one microsomal pellet in 14 ml of 0.05 M phosphate buffer pH 7.6 at 4 C (use the Teflon pestle
after decanting and discarding the storage overlay buffer).
2. Substitute cofactor solutions containing constituents (e) in table 1 in addition to other materials.
3. Use 2 ml microsomal suspension prepared in (1) above in place of the 10,000 g supernatant.
Observations
A. Hexobarbitone sodium metabolism in liver homogenates
1. Hexobarbitone sodium concentration at start _______ mg/ml
2.Absorbance differences for standards
Concentration (mg/ml) Absorbance difference

10
20
50
100
200
3.Absorbance differences for flasks 1 to 8

Flask No Absorbance difference
1
2
3
4
5
6
7
8
B. Hexobarbitone sodium metabolism in microsomes

1. Hexobarbitone sodium concentration at start ______ mg/ml
2.Absorbance differences for standards
Concentration (mg/ml) Absorbance difference
10
20
50
100
200
3.Absorbance differences for flasks 1 to 8

Flask No Absorbance difference
1
2
3
4
5
6
7
8
Calculations
1. Metabolism of Hexobarbitone sodium after incubation =
Initial concentration -Average concentration obtained for flask 3 & 4.
2. Metabolism of Hexobarbitone sodium without incubation =
Initial concentration -Average concentration obtained for flask 7 & 8.
Results
1. Metabolism of Hexobarbitone sodium with and without incubation was found to be ______mg/ml and
______ mg/ml respectively.
2. Metabolism of Hexobarbitone sodium in liver microsomes with and without incubation was found to be
______mg/ml and ______ mg/ml respectively.
Conclusion
It can be concluded that metabolism of Hexobarbitone sodium takes place by oxidation in both liver
homogenate and microsomes.
Applications
1. This experiment allow us to study in vitro metabolism of drugs.
2. The activity of enzymes most concerned in metabolism of particular drug can be studied.
3. Metabolism of drug by liver homogenate and microsomes can be separately studied.
4. Various metabolic processes viz. oxidation, reduction etc. can be studied.
5. The enzyme induction and inhibition due to drugs can be studied in vitro.
Questions
1. Give the various enzymes involved in metabolism of drugs.
2. Give the significance of metabolism.
3. Explain the process of enzyme induction and inhibition.
Exercise
Study the metabolism of thiopental sodium by this method.
Experiment 18
Effect of food on metabolism of drug
Aim
To study the effect of food products on metabolism of drugs.
Learning objectives
1. To understand the metabolism of drug.
2. To understand the effect of food on metabolism of Thiopental.
Theory
Majority of drugs undergo metabolic change. Metabolism usually results in increased polarity, and
this reduces the degree of tissue penetration and increase the rate of urinary excretion. Metabolism commonly
results in reduction of pharmacological potency, but this is not inevitable, and there are cases of metabolism
leading to enhancement or induction of activity.
Metabolism often occurs in two phases. Phase I is usually an oxidation, a reduction, or a hydrolysis.
Phase II is always a synthetic reaction, such as a conjugation. Phase I often leads to introduction of a substituent,
which can then combine with conjugation group. Phase II is the major mechanism for increasing polarity, as
Phase I is often an intermediate step making increase in polarity possible. The commonest combination is
aromatic hydroxylation followed by conjugation with glucuronic acid.
Major site of metabolism of exogenous drugs is liver. A wide variety of compounds is oxidized by a
non specific mixed function oxidase system found in the microsomal fraction of the liver homogenate.
Endogenous drugs tend to depend on enzymes of higher specificity for their metabolism, located more
specifically at places relevant to their sites of action.
Principle
Biotransformation of drugs is primarily carried out by mixed-function oxidase system localized in the
endoplasmic reticulum of the liver. The relationship between the nutritional status of an animal and the ability to
metabolize drugs is well established.
The protein rich diet induces cytochrome-p-450 (CYP), a metabolizing enzyme leading to increased
metabolism of drugs. Deficiency of various nutrients such as calcium, zinc, iron, vitamin C, thiamin, or lipid
have been shown to alter the rate of drug metabolism. The rate of metabolism in liver from rats fed with gluten
diet (normal diet) is significantly lower than that rats fed with casein diet. There is a direct relation between
metabolism of thiopental and sleeping time. As drug metabolism is reduced by feeding gluten diet, more
thiopental is available for action and hence sleeping time is increased while rats fed with casein diet show
increased metabolism of thiopental sodium thus decreasing seeping time.
Drug concentration is relatively lower in the animals fed with gluten than in the corresponding groups
pair-fed with casein. The effective dietary variable is probably the imbalance and/or deficiency of amino acids
in gluten. Either amino acid imbalance or deficiency or both might be hindering the de novo synthesis of
microsomal enzymes or of some cofactors or components of the mixed function oxidase system for drug
metabolism.
Thiopental is an ultra short acting barbiturate which produces sleep in mice within 15-20 seconds and
it regenerates within 10-20 mins. Elimination half life is 7-12 h and is metabolized by hepatic metabolism.
Requirements
Apparatus: Righting reflex chamber, tuberculin syringe.
Chemicals: Food products (gluten diet, casein diet), thiopental injection, water for injection I.P.
Animal: Mice.
Procedure
1. Select twelve mice weighing around 30-50 gm for the study after approval fromAnimal Ethics Committee.
2. Keep the animals in controlled laboratory condition for 2 days.
3. Divide animals into two groups, six each in a group.
4. Group I is fed with gluten diet and group II with casein diet for 5 days.
5.After 5 days, weigh the animals and note their weights.
6. Thiopental in saline is injected intraperitoneally at a dose of 3-5 mg/kg body weight to the mice of both
groups.
7. Record the time between the loss and the recovery of righting reflex, after Thiopental administration.
Observations
Table 1. Weight gain in group I and II
Animal Group I Group II
No th
Initial Wt Wt on 5 day Gain in Wt Initial Wt Wt on 5th day Gain in Wt
1
2
3
4
5
6
Mean
SD
Table 2. Sleeping time observed after Thiopental injection

Sleeping time
Animal No
Group I Group II
1
2
3
4
5
6
Mean
SD
Results
1. Mean weight of mice with casein diet is __________g while mean weight of mice fed with glutein diet is
_____ g.
2. Mean Thiopental sleeping time found for mice fed with casein diet is ______min while mean sleeping time
for mice fed with gluten diet is ______min.
The mean Thiopental sleeping time for mice fed with gluten is significantly longer than for mice with
casein diet.
Conclusion
It can be concluded that mice fed with casein diet (protein diet) show significantly more body weight
and increased metabolism of Thiopental than the mice fed with gluten diet (normal diet).
Applications
1. The effect of food products on metabolism of various drugs can be studied.
2. Metabolism of drug in vivo can be studied.
Questions
1. Give various steps involved in process of metabolism.
2. Why protein rich diet increases the metabolism of drug?
Exercise
Study the effect of deficiency of calcium, zinc, iron and lipid on metabolism of Thiopental sodium
SECTION 5
Excretion of drugs
Experiment 19
Urinary excretion of drug
Aim
To study the urinary excretion of Acetylsalicylic acid (Aspirin) in healthy, male volunteers after its
oral administration.
Learning objective
To determine urinary excretion of aspirin in free and conjugated forms.
Theory
Acetylsalicylic acid, most commonly known asAspirin is a member of salicylate group of compounds.
It is a non steroidal anti-inflammatory drug that possesses analgesic, anti- inflammatory and antipyretic
properties.Aspirin is hydrolyzed in stomach and in blood to salicylic acid and acetic acid.
Salicylates are excreted mainly by kidney as salicyluric acid (75%), free salicylic acid (10%), salicylic
phenol (10%) and acyl (5%) glucuronides, and gentisic acid (<1%). When small doses (less than 250 mg in an
adult) are ingested, all the pathways proceed by first order kinetics with an elimination half life of about 2-3 h.
Aspirin is rapidly deacetylated to salicylic acid which is pharmacologically active metabolite.
Salicylic acid is then oxidized to gentisic acid and conjugated with glycine to form salicyluric acid and with
glucuronide to form ester and ether conjugates.
Urinary excretion is a process by which drug or its metabolites are eliminated from the body without
chemical change. Aspirin is excreted as free salicylic acid or its conjugates in urine and these can be estimated
spectrophotometrically.
Principle
When the urine is added with 1 % ferric nitrate solution, then the salicylate ions formed will give an
intensely colored complex with the ferric ion in acidic solution. When treated with basic solution the
acetylsalicylic acid hydrolyzes quickly to salicylic acid and acetate ions.
O
OCCH3 OH
2-
+ 2OH- + CH3CO + H2O
-
COH CO2
O
-
acetylsalicylic acid salicylate C7H5O3
The salicylate ions will form an intensely colored complex with the ferric ion in acidic solution.
3C7H5O-3 + Fe2+ (C7H5O3)3Fe
(colorless) (faint yellow) (deep red wine)
The wavelength of maximum absorption for this complex is about 525 nm. Thus visible spectroscopy
87
can be used to measure the amount ofAspirin present in the urine.
Prerequisite
Concept of urinary excretion of drug.
Requirements
Glassware: Test tubes, volumetric flasks, conical flasks, measuring cylinder, etc.
Chemicals: Ferric nitrate, nitric acid, salicylic acid, etc.
Instrument: Spectrophotometer.
Procedure
1. Plotting of calibration curve in blank urine:
1. Preparation of standard stock solution: Weigh accurately 100 mg of pure salicylic acid and transfer it into 100
ml volumetric flask and adjust volume with urine (Stock I, 1 mg/ml). Transfer 10 ml stock I to another 100 ml
volumetric flask and adjust volume with urine (Stock II, 100 mg/ml).
2. Preparation of working solution: From stock solution II, pipette out 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 and 5 ml
into 10 ml volumetric flask and adjust volume with urine to get concentration in the range of 0-50 mg/ml.
3. Color formation: Take 5 ml of working solution in test tube and add 1 ml coloring reagent (1% ferric nitrate in
the ratio of 1:99 nitric acid: distilled water) to produce violet color and centrifuge.
4. Measurement of absorbance: Measure the absorbance at lmax 525 nm using UV-Visible spectrophotometer.
Plot the graph of absorbance of Salicylic acid against concentration in MS Excel and determine slope and
intercept.
2. Study protocol
1. Prepare study protocol and take approval from IEC.
2. Select one healthy volunteer and obtain a written informed consent.
3. No other medications should be permitted to be taken by the volunteer one week prior to and during the study.
4.After an overnight fasting, water loading should be done by taking 400 ml water.
5.After 1 hour from water loading collect drug free urine sample as blank.
6.Administer 600 mg ofAspirin (soluble aspirin 300 mg x 2) tablets with 200 ml water.
7. Every hour give 200 ml of water to get sufficient urine sample.
8. Instruct volunteers to completely empty their bladder while collecting urine sample.
9. Collect the urine samples at 30, 60, 90, 120, 180, 240, 360, and 600 minutes after an oral administration of
drug.
10. Record the total volume of each urine sample voided during period. Also record the pH of urine samples and
0
keep in freezer at -20 C till analysis.
3. Urine analysis
1.Analyze urine sample in triplicate by spectrophotometric method.
2. Take 5 ml urine sample and 1 ml coloring agent (1% ferric nitrate in the ratio of 1:99 nitric acid: distilled
water). The violet color will be developed, centrifuge and note the absorbance (absorbance I) at 525 nm. This
will give free salicylic acid.
Section 5 Excretion of drugs 89
3. Estimate total salicylic acid after hydrolyzing 5 ml urine sample by 1ml of 0.25 M hydrochloric acid and then
boiling it for 1 h. Add coloring agent (1% ferric nitrate in the ratio of 1:99 nitric acid: distilled water) and record
the absorbance (absorbance II).
4. The conjugated salicylic acid can be determined from the difference of total salicylic acid and free salicylic
acid.
Observations
Table 1. Plotting of calibration curve
mg/ml
0
10
20
30
40
50
Slope
Intercept
Table 2. Estimation of free, conjugated and total salicylic acid (SA)
Conjugated
Free SA Absorbance Total SA
Time Absorbance I SA
A II B
(B-A)
30
60
90
120
180
240
360
600
Table 3. Urinary excretion data after oral administration of 600 mg aspirin (460 mg of salicylic acid)
Total SA Amount excreted A u
Time of urine collection Urine volume Cumulative A u
Conc (mg)
(min) (ml) (mg)
(mg/ml)
30
60
90
120
180
240
360
600
Calculations
1. Concentration of excreted drug in urine (mg/ml)
Y= m X + c
2.Amount of drug excreted in urine,Au (mg/ml)
Au = Conc. of drug in urine (mg) x Total volume of urine voided (ml)
3. Total Salicylic acid in urine
Total Salicylic acid in urine =Au =Absorbance II
Free Salicylic acid in urine =Absorbance I
Conjugated Salicylic acid= Total SA– Free SA
Results
The cumulative amount of total Salicylic acid excreted in urine was found to be __________mg.
Conclusion
From this experiment it can be concluded that the cumulative amount of total Salicylic acid excreted in
free and conjugated forms can be estimated by this method.
Applications
1. This experiment allow us to estimate free, cojugated and total concentration of Aspirin excreted through
urine.
2. The process of elimination of drug can be studied.
3. Urinary excretion of certain drugs can help us to determine the functioning of kidney.
Questions
1. Give the various routes of excretion of drugs.
2. Give the principle involved in estimation of Salicylic acid in urine.
Exercise
Estimate the concentration of Paracetamol and its metabolites in urine.
Experiment 20
Influence of urinary pH on excretion of drug
Aim
To study the influence of urinary pH on excretion of Acetylsalicylic acid (Aspirin) in healthy male
volunteers.
Learning objective
To study the effect of urinary pH on salicylate excretion.
Theory
The most studied excreting organ is the kidney, although excretion of both unmetabolized drugs and
their metabolites in bile is of considerable importance. Drug molecules, dissolved in plasma, are delivered to the
kidney by the renal artery. Filtration of plasma occurs at the glomerulus, and compounds with molecular
weights below approximately 66,000 dalton pass through into the renal tubule. However, molecules with
sufficient lipid solubility are reabsorbed through the membranes of the renal tubule into plasma, so that the net
loss may be quite low. This degree of loss varies from compound to compound. In particular, metabolites of
exogenous compounds, because of their greater polarity, are less likely to be absorbed and so are more likely to
remain in tubular urine. In case of weak electrolytes, the ionized forms fail to be reabsorbed while the non
ionized forms pass the tubular membranes relatively easily. Therefore, an influence of urinary pH on renal
excretion of drugs is very considerable.
The relative amount of ionized and unionized drug in the urine at a particular pH and the percent of
drug ionized at this pH can be computed from the Henderson-Hasselbalch equations:
For weak acids,
ionized
pH = pK a + log ... 1
unionized
10 pH - pKa ... 2
% drug ionized = ´100
1 + 10 pH - pKa
For weak bases,
unionized ... 3
pH = pK a + log
ionized
10 pKa - pH
% drug ionized = ´100
1 + 10 pKa - pH ... 4
The concentration ratio R of the drug in urine to that in plasma (U:P) can be given by equations:
For weak acids,
U 1 + 10 pH urine - pKa
Ra = = ... 5
P 1 + 10 pH plasma - pKa
For weak bases,

U 1 + 10 pKa -pH urine ... 6
Ra = =
P 1 + 10 pKa -pH plasma
Principle
As the pH of urine changes from acidic to basic, the renal clearance of Salicylic acid goes on increasing
due to increased ionization of Salicylic acid preventing its reabsorption from tubules. Thus, the amount of
Salicylic acid will be more in basic urine while less in acidic urine.
Prerequisite
1. pH partition hypothesis.
2. Effect of pH on excretion of drugs.
Requirements
Glassware: Test tubes, volumetric flasks, conical flasks, measuring cylinder, etc.
Chemicals: Ferric nitrate, nitric acid, Salicylic acid, aspirin tablets, sodium bicarbonate, ammonium chloride.
Instruments: Spectrophotometer, pH meter, etc.
Procedure
1. Plotting of calibration curve in blank urine
1. Preparation of standard stock solution: Weigh accurately 100 mg of pure salicylic acid and transfer it into 100
ml volumetric flask and adjust volume with urine (Stock I, 1 mg/ml). Transfer 10 ml stock I to another 100 ml
volumetric flask and adjust volume with urine (Stock II, 100 mg/ml).
2. Preparation of working solution: From stock solution II, pipette out 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 and 5 into
10 ml volumetric flask and adjust volume with urine to get concentration in the range of 0-50 mg/ml.
3. Color formation: Take 5 ml of working solution in test tube and add 1 ml coloring reagent (1% ferric nitrate in
the ratio of 1:99 nitric acid: distilled water) to produce color and centrifuge.
4. Measurement of absorbance: Measure the absorbance at lmax 525 nm using UV-Visible spectrophotometer.
Plot the graph of absorbance of Salicylic acid against concentration in MS Excel and determine slope and
intercept.
2. Study Protocol
1. Prepare study protocol and take approval from IEC.
2. Select nine healthy volunteers and obtain a written informed consent. Make three groups comprising 3
volunteers and designate as Group I (receive only Aspirin), Group II (receive Aspirin + ammonium chloride)
and Group III (receiveAspirin + sodium bicarbonate).
3. No other medications should be permitted to be taken by volunteers one week prior to and during the study.
4.After an overnight fasting, water loading should be done by taking 400 ml water.
5. After 1 hour from water loading collect drug free urine sample as blank and administer ammonium chloride to
Group II and sodium bicarbonate to Group III.
6. After 30 min, administer 600 mg of Aspirin (soluble Aspirin 300 mg x 2) tablets with 200 ml water to each
group.
7. Every hour give 200 ml of water to get sufficient urine sample.
8. Instruct volunteers to completely empty their bladder while collecting urine sample.
9. Collect the urine samples at 30, 60, 90, 120, 180, 240, 360, and 600 minutes after an oral administration of
drug.
10. Record the total volume of each urine sample voided during period. Also record the pH of urine samples and
0
keep in freezer at -20 C till analysis.
3. Urine analysis
1.Analyze urine sample in triplicate by spectrophotometric method.
2. Take 5 ml urine sample and 1 ml coloring agent (1% ferric nitrate in the ratio of 1:99 nitric acid: distilled
water). The violet color will be developed, centrifuge and note the absorbance (absorbance I) at 525 nm.
3. Plot the graph of percent cumulative amount of Salicylic acid excreted versus time.
4. Plot the graph of cumulative excretion rate versus time.
Observations
Table 1. Calibration curve of Salicylic acid
mg/ml
0
10
20
30
40
50
Slope
Intercept
Table 2. Urinary excretion data after oral administration of 600 mg aspirin for group I
Time of urine Subjects Urine SA Amount Cumulative
pH of
collection volume Conc excreted A u Au
urine
(min) (ml) (mg/ml) (mg) (mg)
1
30 2
3
Mean
1
60 2
3
Mean
1
90 2
3
Mean
1
120 2
3
Mean
1
180 2
3
Mean
1
240 2
3
Mean
1
360 2
3
Mean
1
600 2
3
Mean
Construct similar tables for Group II and Group III.
Calculations
Y= m X + c Where, Y = absorbance, m = slope, X = concentration (mg/ml), c = intercept.
2.Amount of drug excreted in urine,Au (mg/ml)

Au = Concentration of drug in urine (mg) x Total volume of urine voided (ml)
Results
The amount of Salicylic acid excreted in urine by various groups is given below:
Group No. pH of urine Cumulative amount of Salicylic acid
excreted
I
II
III
Conclusion
It can be concluded that the excretion ofAspirin goes on increasing as the urinary pH increases.
Applications
1. The influence of urinary pH on excretion of drug can be studied.
2. The toxicity of drug can be reduced during overdosage by increasing excretion through changing the pH of
urine.
3. The excretion of drug can be predicted based on pKa and lipophilicity of drug and pH of urine.
Questions
1. Give the relationship between pKa and lipophilicity of drug and pH of urine.
2. Why the excretion of Salicylic acid increases with increasing pH?
Exercise
Study the influence of pH of urine on weakly basic drug.
SECTION 6
Pharmacokinetics
Experiment 21
Simulation of plasma elimination and urinary excretion after an IV bolus dose
Aim
To investigate a first-order process by simulating plasma elimination and urine excretion after an
IV bolus dose.
Learning objectives
1. To understand various pharmacokinetic parameters viz., apparent volume of distribution, elimination half
life, elimination rate constant and clearance.
2. To determine pharmacokinetic parameters from either plasma or urinary data.
3. To calculate pharmacokinetic parameters after an IV bolus dose administration.
Theory
When a drug is administered as an injection of a sterile solution formulation, several important points
are to be considered. They are
1. IV route of administration ensures entire administered dose to be reaching to the general circulation,
2. the desired drug concentration in the blood is promptly attained and
3. the doses should be calculated very carefully because of the danger of adverse or toxic effects.
One compartment open pharmacokinetic model depicts the body as single kinetically homogenous
unit that has no barrier to the movement of drug. Also, the final distribution equilibrium between the drug in
plasma and other body fluids is attained instantaneously and maintained at all time. This model applies only to
those drugs that distribute rapidly. The plasma drug concentration is representative of all body tissues
concentration. The term open indicates the input and output are unidirectional.
Intravenous bolus administration

When a drug that distributes rapidly in the body is given in the form of rapid IV bolus or slug, it takes
about 1 to 3 minutes for complete circulation, therefore the absorption rate is not considered in calculation.
Following intravenous bolus administration of a drug, by considering following assumptions the
fundamental pharmacokinetic parameters of a drug can be obtained.
one-compartment model, first-order process and passive diffusion are operative
no metabolism takes place (elimination is 100% via renal excretion)
the drug is being monitored in blood (plasma/serum) and urine.
IV Injection K (Elimination)
Dose Blood and body tissue
Central compartment
95
The differential equation for a first-order process can be given by equation 1

dX
= - KX
dt …1
where dX/dt = rate of change of a substance over time.
Applying this equation to the elimination of drug (amount) in the body, gives
dA
= - KA
dt …2
The integrated form of equation 2 is given by
A = A0 e - Kt …3
ln A = ln A0 - Kt …4
Kt
log A = log A0 -
2.303 …5
where A = amount of unchanged drug in the body at time zero (t=0). Please note that A is the
0 0
administered intravenous bolus dose of the drug.

When drugs are monitored in plasma or serum, it is concentration (not mass or amount) that is measured.
amount of drug (A), (mg, mg) A
Concentration (C) = =
Unit volume (V), (ml, L) V …6
Dividing equation 3 by the volume term, V, yields
A A0 - Kt
= e
V V …7
Substituting equation 6 (A/V)=C, equation 7 can be written as
C = C0 e - Kt …8
ln C = ln C0 - Kt …9
Kt
log C = log C0 - …10
2.303
Equation 10 can be plotted as shown in Figure 1 by taking the concentration versus time data by the use
of semilogarithmic co-ordinates (Figure 1).
Figure 1. A semilog plot of plasma concentration of a drug versus time, following IV bolus dose
Section 6 Pharmacokinetics 97
Important Pharmacokinetic Parameters

1. Elimination half life (t1/2)
The elimination half life is sometimes called ''biological half-life'' of a drug. At a time after
administering a dose when equilibrium has been established, the elimination half life may be defined as the time
at which the amount of unchanged drug becomes half (or 50%) of the initial amount of drug. Elimination half
life can be calculated from following equation
0.693
t1/ 2 = …11
K
Alternatively, the elimination half life may be obtained from the semilogarithmic plot of plasma
concentration versus time data, as described in Figure 2. Choose any two concentration values (read off the y-
axis of the concentration versus time plot) that are one half of each other (i.e. 200 and 100 mg/mL) and the
corresponding time values (from the x-axis of the plot). The difference between the two time values represents
the elimination half life of the drug.
Figure 2. Semilog plot of plasma concentration versus time

2. Elimination rate constant (K)
Speed at which drug eliminates from body is known as elimination rate constant. The elimination or
removal of drug from the body is the sum of urinary excretion, metabolism, biliary excretion, pulmonary
excretion etc. Thus, the elimination rate constant is an additive property of all rate constants. Total elimination
rate constant can be calculated from following equations
-K log C 2 - log C1 …12

Slope = =
2.303 t 2 - t1
or
0.693
K=
t1 / 2 …13
3.Apparent volume of distribution (V)
Concentrations (amount per unit volume) and not masses (mg), are usually measured in plasma or
serum. Therefore, a term is needed to relate the measured concentration (C) at a time to the mass of drug (A) at
that time. This term is defined as the apparent volume of distribution (V). It is the measure of the extent of
distribution of drug in compartment. It is defined as the hypothetical volume of body fluid into which a drug is
dissolved or distributed. Please note that the apparent volume of distribution (V) is simply a proportionality
constant whose sole purpose is to relate the plasma concentration (C) and the mass of a drug (A) in the body at a
given time. It is not a physiological volume.
In order to determine the apparent volume of distribution of a drug, it is necessary to have
plasma/serum concentration versus time data.
Now, from equation 6, C =A/V
A …14
V= t
Ct
where (At) = amount of drug; V = apparent volume of distribution; and (Ct) = plasma concentration at
time, t.
At t = 0, the equation can be written as
A Dose
V= 0 = …15
C0 C0
whereA0 = administered dose of a drug, (C0) = plasma concentration at time t=0.
Equation 15 permits the determination of the apparent volume of distribution of a drug from the
knowledge of the initial plasma or serum concentration [i.e. (C0)] and the administered dose. It is important to
note that the apparent volume of distribution is a constant for a given drug and is independent of the
administered dose and route of drug administration.
4. Clearance (Cl)
Clearance is the most important parameter in clinical drug applications and is useful in evaluating the
mechanism by which a drug is eliminated by the whole organism or by a particular organ. It is defined as
theoretical volume of body fluid containing drug from which the drug is completely removed in a given period
of time. Clearance for a drug is constant if the drug is eliminated by first-order kinetics. Mathematically,
clearance (L/hr) is the product of the first-order elimination rate constant (K) and the apparent volume of
distribution (V). Thus
Rate of elimination dA / dt
Clearance (Cl) = =
Plasma drug concentration C …16
Substituting dA/dt =KAin above equation, we get
KA
Cl = …17
C
SinceA/C = V, the equation 17 can be written as
Cl = KV …18
5. Total area under curve (AUC)

AUC represents the total integrated area under the plasma level-time profile and expresses the total
amount of drug that comes into the systemic circulation after its administration. When a drug is given as an IV
bolus and the decline in plasma concentration is monoexponential, total AUC can be calculated by dividing the
extrapolated zero time concentration (C0) by the elimination rate constant.
6. Calculation of K from urinary excretion data

The elimination rate constant K can also be calculated from urinary excretion data by using excretion
rate method. In this calculation the excretion rate of the drug is assumed to be first order. The term Ku is the renal
excretion rate constant, andAu is the amount of drug excreted in urine.
Following differential equation describes the process
dAu
= Ku A …19
dt
where dAu/dt = rate of excretion;A = amount of drug in the body; and Ku = excretion rate constant.
SubstitutingAfrom equation 3 in the equation 19, gives
dAu
= K u A0 e - Kt …20
dt
The logarithmic form of the equation becomes

dAu Kt
log = log( K u A0 ) - …21
dt 2.303
If the rate of excretion is plotted (Fig. 3) against the average time (t*), on semilogarithmic paper, the
slope will permit the determination of the elimination rate constant (K); and the intercept will represent the
initial rate of excretion (KuA0). Please note that from the knowledge of the intercept value (mg/h) and the
administered dose (mg), one can determine the excretion rate constant (Ku).
Figure 3. A typical semilogarithmic plot of rate of excretion against average time t*

In practice, equation 22 becomes
dAut* Kt *
log = log( K u A0 ) -
dt 2.303 …22
where dAut*/dt = average rate of excretion; t* = average time between urine collection; Ku = excretion
rate constant; andA0 = dose.
Both equations 21 and 22 suggest that the rate of excretion of a drug declines monoexponentially with
time, as shown in Fig. 3. The elimination half life (t1/2), elimination (K) and excretion rate constant (Ku) can be
determined from semilogarithmic plot of the rate of excretion versus average time t*.
Principle
By an arrangement of beakers and a constant head water reservoir, it is possible to simulate plasma
concentrations and drug amounts in urine after IV bolus administration. The constant flow of water through the
system, causes a first order dilution of the marker, potassium permanganate. The marker concentrations can be
determined spectrophotometrically at 540 nm, by sampling the water from plasma compartment (P) and urine
compartment (U) periodically. The pharmacokinetic parameters of the system can be determined by analyzing
the obtained data.
The plasma concentration versus time is plotted on semilog paper and the parameters; elimination rate
constant, elimination half life, plasma concentration at zero time (C0), volume of distribution, clearance and
area under curve can be determined.
The rate of excretion versus time is plotted on semilog paper and parameters elimination rate constant,
excretion rate constant and elimination half life can be determined.
Figure 4. Design of apparatus to simulate one compartment open model IV bolus dose
Prerequisite
1. Knowledge of various pharmacokinetic parameters.
2. Knowledge of first order process.
Requirements
Potassium permanganate, beaker, measuring cylinder, test tubes, spectrophotometer, regular graph
paper, semilog graph paper, calculator, pencil.
Procedure
1. Prepare 100 mg/ml of potassium permanganate in distilled water. Further prepare 10 ml volumes of working
solutions 10, 20, 40, 60, 80, and 100 mg/ml from the stock solution. Measure the absorbance of each solution at
540 nm. Use distilled water as the blank. Plot calibration curve by using MS Excel and record intercept and
slope.
2. Identify and mark the plasma and urine compartment to the apparatus. Make sure that the water reservoir (W)
is full. Turn on the stirrer in the plasma beaker and adjust for gentle mixing. Establish a flow rate of 20 (any
where 15-25) ml/min.
3. Collect plasma samples at 2.5, 7.5, 12.5, 17.5, 22.5, 27.5, 32.5, 37.5, 45, and 55 minutes and urine samples at
5, 10, 15, 20, 25, 30, 35, 40, 50, and 60 minutes after the dose. Arrange and label test-tubes to accommodate the
samples.
4. 'Inject' the plasma beaker with the 'dose' of potassium permanganate (15 drops of saturated solution as bolus),
place an empty beaker in position to collect the urine sample. Use distilled water as the blank. Take 5 ml sample
using a Pasteur pipette. The dose injected can be determined by taking the same volume as injected into the
beaker and diluting it to 250 ml and measuring the absorbance. A dose of about 15 drops (saturated potassium
permanganate solution) should be considered to equal 250 mg. Thus the absorbance measured is that for a 1
mg/ml solution.
5. Collect 5 ml plasma sample at the times designated quickly by pipette. Don't rinse the pipette between
samples.
6. At each time point for urine collection, replace the beaker with an empty beaker. Measure the volume
collected, mix the contents of the beaker to obtain a uniform solution, and take 5 ml sample for analysis.
7. Analyze each sample spectrophotometrically at 540 nm. Early samples may need to be diluted to give
absorbance readings below 1. Remember to apply this dilution factor in the analysis of the results.
8. Plot plasma concentration (C) versus time on semi-log graph paper. Calculate K from slope and t1/2 from K.
Calculate apparent volume of distribution by using C0 and dose.
9. Plot rate of excretion (dAu/dt) versus time (t*) on semi log graph paper and calculate K.
10. Construct a 'clearance' plot by plotting the rate of excretion (dAu/dt) versus C (plasma concentration at the
midpoint of the urine collection time). Measure the clearance as the slope of this line.
Observations
Table 1. Calibration curve
(mg/ml)
10
20
40
60
80
100
Slope
Intercept
Table 2. Concentration of potassium permangnate in plasma

Plasma Data
Time (min) Dilution Factor Absorbance Concentration
2.5
7.5
12.5
17.5
22.5
27.5
32.5
37.5
45
55
Table 3. Concentration of potassium permanganate in urine
Urinary Data
Time Time Time Conc Volume of Amt of Cumulative amount Au/dt t* C at
Interval t interval in Urine drug in excreted (mg/hr) midpoint midpoint
of urine dt Urine collected urine Au Au (mg)
collection mg/ml (ml) mg
0 5 5 5 2.5
5 - 10 10 5 7.5
10 - 15 15 5 12.5
15 - 20 20 5 27.5
20 - 25 25 5 22.5
25 - 30 30 5 27.5
30 - 35 35 5 32.5
35 - 40 40 5 37.5
40 - 50 50 10 45.5
50 - 60 60 10 55
Calculations
1. Concentration of potassium permanganate in plasma and urine
Y= m X + c
Where, Y= absorbance, m= slope, X= concentration (microgram/ml), c= intercept.
2. Dilution factor
3. Elimination half life (t1/2)
Plot graph of plasma concentration (C) versus time (t) on semilog paper and graphically determine half
life. Alternatively, also plot the graph of rate of excretion (dAu/dt) versus time (t*) on semi-log graph paper and
determine half life.
4. Elimination rate constant (K)

Elimination rate constant can be determined using following formula for plasma and urinary data.
0.693
K=
t1 / 2
Alternatively for both plasma and urinary data plot the graphs of plasma concentration C versus time
(t) and rate of excretion (dAu/dt) versus time (t*) on semilog paper and determine slope of line.
-K log C 2 - log C1
Slope = = For plasma data
2.303 t 2 - t1
-K log( Au / t ) 2 - log( Au / t )1 For urinary data

Slope = =
2.303 t 2* - t1*
5. Plasma concentration at 0 time (C0)

· Plasma concentration at zero time can be determined by back extrapolating (i.e. y intercept) the
decline in plasma concentration.
6. Volume of distribution (V)
Volume of distribution can be determined using following formula
A0 Dose
V= =
C0 C0
7. Clearance (Cl)
Clearance can be determined using following formula
Cl = KV
· Plot the graph of the rate of excretion (dAu/dt) versus C (plasma concentration at the midpoint of the
urine collection time). Determine the slope of line as clearance.
8.AUC
DetermineAUC by using following formula
C0
AUC =
K
9. Excretion rate constant (Ku)
Determine the y intercept of semilog plot of rate of excretion (dAu/dt) versus time (t*).
y intercept = K u A0
K u = y intercept x dose
Result
The various pharmacokinetic parameters of given plasma data and urinary excretion data of potassium
permanganate are found as given below:
Sr. No. Parameter Plasma data Urine data

1 Elimination half life (t1/2)
2 Elimination rate constant (K) -
3 Excretion rate constant (Ku) -
3 Area under curve (AUC) -
5 Volume of Distribution (V) -
6 Clearance (Cl) -
Conclusion
Plasma concentrations and drug amounts in urine after IV bolus administration can be simulated by an
arrangement of beakers and a constant head water reservoir.
Applications
1. Non invasive method of determination of pharmacokinetic parameters if the drugs follow first order process.
2. The pharmacokinetic study can be understood well by simulating first order process using simple
arrangement of beakers.
3. All the pharmacokinetic parameters can be estimated by generating the plasma and urine data from this
experiment.
Questions
1. Give significance of volume of distribution.
2. How half life and elimination rate constant can be determined graphically?
3.Are elimination rate constant and excretion rate constant same? How?
Exercise
Plasma and urine data obtained after a bolus dose of 50 mg of drug is given in following table
Plasma data Urine data
Time Concentration Time interval of Volume of urine Concentration of unchanged drug in
(h) (mg/l) collection (h) (ml) urine (mg/l)
1 2 0-2 120 133
3 1.13 2-4 180 50
5 0.70 4-6 89 63
7 0.43 6-8 340 10
10 0.20 8-12 178 18
18 0.025 12-24 950 2
Determine AUC, elimination half life, elimination rate constant, volume of distribution and excretion
rate constant.
Experiment 22
Calculation of various pharmacokinetic parameters after IV bolus injection
Aim
To calculate various pharmacokinetic parameters from given blood data of IV bolus injection (one
compartment model).
Learning objective
To understand calculation of various pharmacokinetic parameters after one compartment IV bolus
administration following first order elimination.
Requirements
Regular graph paper, semilog graph paper, calculator and pencil.
Given data
Plasma data obtained after a bolus dose of 184 mg of ceftriaxone, a semi synthetic cephalosporin
antibiotic, in a newborn infant is summarized below:
Time (h) 1 6 12 24 48 72 96 144
Conc 137 120 103 76 42 23 12 3.7
(mg/l)
a. Prepare a semilogarithmic plot and estimate the half-life of drug

b. Estimate the totalAUC
c. Calculate volume of distribution
d. Calculate total clearance.
Solution
Note: If the graph of concentration versus time is plotted on regular graph paper, the curvilinear plot is obtained
which makes it difficult to calculate slope and hence the semilog plot is used which gives the linearity and makes
it easy and accurate to calculate the pharmacokinetic parameters (Refer figure 1).
1. Semilog Plot
a. Plot the graph on semi logarithmic graph paper as the elimination data follows first order kinetics.
b.According to the given data select number of cycles on semilog paper.
c. Plot concentration on Y axis and time on X axis.
d. Mark concentration for respective time on the graph paper.
e. Draw a straight line in such a way that it covers maximum elimination phase points up to Y axis.
f. Y intercept is the C0 concentration.
g. Determine slope of the line and calculate elimination rate constant.
2. Elimination half life
a. On Y axis near to elimination phase select initial concentration as (C, 20). From that point draw a
straight line which intersects plotted line (refer figure 2). This point would be the first point of intersection.
From intersected point draw perpendicular straight line on X axis which gives the time (t1).
b. On Y axis draw a second straight line from half concentration of initial (C/2, 10) which intersects
plotted line. This will be the second point of intersection. From that point draw perpendicular on X axis which
gives the time (t2).
c. Calculate half life by subtracting t2 form t1.
Figure 1. Regular and semilog plot of plasma concentrations versus time showing y intercept and slope
Figure 2. Semilog plot of plasma concentration versus time showing determination of half life
1. Elimination rate constant

Calculate elimination rate constant by using slope of line.
log C 2 - log C1
Slope =
t 2 - t1
K = -( Slope ´ 2.303)
Calculate elimination half life by using following formula
0.693
t1/ 2 = K
Alternatively, half life can be determined graphically.
3.Area Under Curve (AUC)

By using concentration at zero time AUC can be calculated from following equation (Y intercept is C0
concentration).
C0
AUC0-¥ =
K
4. Clearance
Calculate clearance by using following equation
Cl = KV
Result
The various pharmacokinetic parameters calculated from given plasma data of ceftriaxone are given in
table below:
Sr. No. Parameter Result
1 Biological half life
2 Elimination rate constant
3 Total AUC
4 Volume of Distribution
5 Clearance
Applications
1. Various pharmacokinetic parameters can be estimated.
2. Pharmacokinetic parameters are very useful in calculating the dose of new drug.
3. Bioequivalence study can be undertaken based on plasma data of various brands.
Questions
1. Why semilog plot is for estimation of pharmacokinetic parameters?
2. HowAUC can be calculated when IV bolus dose is given?
Excercise
1. Plasma data obtained after a bolus dose of 500 mg phenylethylmalonide (PEMA), a metabolite of
antiepileptic drug primidone is summarized in table below.
Time (h) 0.33 0.5 0.67 1.5 2 4 6 10 16 24 32 48

Conc 14.7 12.6 11 9 8.2 7.9 6.6 6.2 4.6 3.2 2.3 1.2
(mg/l)

2. The data given in table below are the plasma concentrations of cocaine as a function of time after IV
administration of 33 mg cocaine hydrochloride to a subject. (Molecular weight of cocaine hydrochloride = 340
g/mol; molecular weight of cocaine = 303 g/mol)
Time (h) 0.16 0.5 1 1.5 2 2.5 3

Conc 170 122 74 45 28 17 10
(mg/l)
3. Plasma concentration profile after a single 600 mg intravenous dose of ampicillin to an adult is given below
Time (h) 1 2 3 5
Conc (mg/l) 37 21.5 12.5 4.5

Experiment 23
Calculation of urinary excretion rate constant and elimination rate constant
Aim
To calculate the urinary excretion rate constant Ku and elimination rate constant K from the given data.
Learning objective
1. To understand excretion rate method and sigma minus method for calculating elimination rate constant.
2. To calculate Ku and K from given data.
Theory
A urinary excretion study for assessing bioavailability is based on the principle that the urinary
excretion of unchanged drug is directly proportional to the plasma concentration of drug. Thus, even if a drug is
excreted to some extent in the urine, bioavailability can be determined. The study is particularly useful for drugs
extensively excreted unchanged in the urine. For example, certain thiazide diuretics and sulphonamides and
drugs that have urine as the site of action e.g. urinary antiseptics such as Nitrofurantoin and Hexamine.
There are two methods that permit us to compute some pharmacokinetic parameters from urinary
excretion data.
the rate of excretion method and
the ''amount remaining to be excreted'' (ARE) method, also known as the sigma-minus method.
Excretion rate method

This method is already discussed before (Expt No. 21, Page No. 99 ).
If the rate of excretion is plotted (Fig. 3) against the average time (mid point of collection period) (t*), on
semilogarithmic paper, the slope will permit the determination of the elimination rate constant (K); and the
intercept will represent the initial rate of excretion (KuA0). Please note that from the knowledge of the intercept
value (mg/h) and the administered dose (mg), one can determine the excretion rate constant (Ku).
Figure 1.Atypical semilogarithmic plot of rate of excretion against average time t*

The method tends to give overestimate of intercept. The overestimation can be minimized by
collecting urine samples more frequently.

Sigma-minus method/ amount remaining to be excreted (ARE) method
The amount of unchanged drug in urine can be expressed as a function of time through the following
equation
AK
Au = 0 u (1 - e - Kt )
K …1
Where Au = cumulative amount of drug excreted into urine at time t; A0 = administered dose of drug; K
= elimination rate constant; and Ku = excretion rate constant.
The amount of unchanged drug excreted in the urine, A¥u , can be determined by making time t equal t¥.
Thus, the term e-Kt progresses to zero and equation 4 is reduced to
A0 K u
Au¥ = …2
K
¥
SubstitutingA u for the term (KuA0)/K in equation 1 gives
Au = Au¥ (1 - e - Kt ) …3
Au = Au¥ - Au¥ e - Kt …4
Au¥ - Au = Au¥ e - Kt
…5
Equation 6 can be written in logarithmic form to obtain a linear equation
Kt
log ( Au¥ - Au ) = log Au¥ -
2.303 …6
¥
A plot of [Au -Au], that is the amount of drug remaining to be excreted (ARE) against time (t*) (mid
point of collection period) on semilogarithmic co-ordinates gives a straight line. The slope of the line permits
the determination of the elimination rate constant (K) and the intercept represents the cumulative amount of
¥
drug excreted in the urine at time infinity, (Au ), which in this case is not equal to the administered dose. Figure 1
¥
represents the semilogarithmic plot of [Au -Au] against time.
Figure 1. Semilogarithmic plot ofARE against average time t*

Limitations of the ''amount remaining to be excreted'' (ARE) method

1. Urine samples must be collected until such time that, practically, no additional drug appears in the urine.
2. No urine samples can be lost, or urine from any samples used in the determination of Au (the exact volume of
urine at each time interval must be known).
3. This is a time-consuming method for a drug with a long elimination half life (t1/2).
4. There is a cumulative build up of error.
Given Data
Table 1. A single IV dose of an antibiotic was given to 50-kg woman at a dose level of 20 mg/kg. Urine samples
were removed periodically and assayed for parent drug. The following data were obtained. (50 x 20 = 1000 mg
dose)
Time (t)
0.25 0.5 1 2 4 6
h
Au (mg) 160 140 200 250 188 46
Calculate urinary excretion rate constant Ku and elimination rate constant K.
1. Solution: Rate of Excretion Method
1. Construct the table for the determination of excretion rate.
2. Plot the semilog graph of excretion rate versus mid point of urine collection period.
3. Determine slope of line.
4. Determine elimination half life form graph.
5. Determine total elimination rate constant (K) from elimination half life.
6. Determine urinary excretion rate (Ku) from Y intercept = log Ku A0
2. Solution: Sigma-minus Method
1. Construct table for calculation of cumulative drug excreted and amount remaining to be excreted.
2. Plot the graph of amount remaining to be excreted on semi logarithmic scale versus mid point of urine
collection period (t*).
4. Determine elimination half life from graph.
5. Determine total elimination rate constant (K) from elimination half life.
Table 2. Observation table for rate of excretion method

Mid point of Urine Excretion rate
Time of urine Time interval(dt)for dAu(amount
collection period t* Au/dt
collection (h) urine sample collection excreted) (mg)
(h) (mg/h)
0.25 0.125 0.25 160
0.5 0.375 0.25 140
1 0.750 0.5 200
2 1.50 1 250
4 3 2 188
6 5 2 46
Table 3. Observation table for Sigma-minus method

Au (amount Cumulative
Time of urine Mid point of Urine collection Au¥ -
excreted) Au
collection (h) period t* Au
mg mg
0.25 160 0.125
0.5 140 0.375
1 200 0.750
2 250 1.50
4 188 3
5 46 5
Calculations
1. Elimination rate constant (Rate of excretion method & Sigma-minus method)
Determine elimination rate constant from half life or by using slope of line.
-K
Slope =
2.303
Calculate slope of line either from graph or select two points from decline phase of given data.
Also calculate elimination rate from t1/2 obtained from graph.
0.693
t 1/ 2
=
K
2. Excretion rate constant (Ku) by using rate of excretion method
Plot the graph of excretion rate versus mid point of urine collection period on semilog paper.
Determine the intercept (A0Ku) by extrapolating the line to y axis. Use following formula to determine excretion
rate constant
Intercept Intercept
K u
=
A0
=
Dose
Result
The various pharmacokinetic parameters calculated from given urinary data are as given below:
Sr. No. Parameter Rate excretion method Sigma-minus method

2 Excretion rate constant -
3 Elimination half life
Conclusion
It can be concluded from this experiment that the pharmacokinetic parameters of the drug can be
determined from urinary data, without doing the invasive method of estimation of plasma concentration.
Applications
1. Excretion rate constant can be determined.
2. Provides alternative to invasive method.
Questions
1. What is excretion rate constant?
2. Which two methods are used for computing pharmacokinetic parameters from urinary excretion data?
Exercise
1. Urinary excretion data following IV bolus dose of 80 gm of drug is given in following table.
Time of urine collection (h) 1 2 3 4 5 6 12
Amount excreted in mg 40 20 10 5 2.5 1.25 1.25
Calculate elimination rate constant, excretion rate constant and elimination half life.
2. Urinary excretion data following IV bolus dose of 50 mg of drug is given in following table.
Time of urine
2 4 6 8 12 24
collection (h)
Amount excreted in
16 9 5.6 3.4 3.2 1.9
mg
3. Urinary excretion data following IV bolus dose of 1000 mg of drug is given in following table.
Time of urine
3 6 9 12 15 24 48
collection (h)
Amount excreted in
534 436 181 139 110 202 195
mg
Experiment 24
Simulation of plasma elimination after an IV infusion
Aim
To investigate a first-order elimination process by simulating data during and after an IV infusion.
Theory
When a single intravenous bolus dose of a drug is given, the desired therapeutic concentration is
achieved immediately. However, this mode of administration is unsuitable when it is necessary to maintain
plasma or tissue concentrations for prolonged duration. Here the aim is to reach the therapeutic range and then
maintaining drug concentration within the therapeutic range for a longer duration. It is common practice, in the
hospital setting to infuse a drug at a constant rate (constant rate input or zero-order input). This method permits
precise and readily controlled drug administration.
The infusion rate of a drug is controlled by flow rate and concentration of the drug in solution. Flow
rate is controlled by adjusting the height of an infusion bottle or by regulating the aperture size of the tube that
connects the bottle to the needle. When greater precision and control of drug administration is desired, an
infusion pump is used. Drug is monitored in blood under two conditions:
during infusion (while the drug is being infused)
In the post-infusion period (following the cessation of infusion).
Figure 1 shows the scheme and set up for drug changes in the body or blood under constant infusion. Following
text will consider first the issues of sampling during infusions and then those of monitoring drugs after an
infusion is stopped.
Constant infusion rate R0 K (Elimination)

Zero order process Central Compartment First order process
Since drug is being monitored in the blood, the change in the amount of drug in the blood/body (dX/dt)
for above set up will be
dA
= R0 - KA
dt …1
where dA/dt = rate of change in the mass or amount of drug in the body; R0 = zero order input or
constant infusion rate; and KA= first-order elimination rate.
Upon integration, equation 1 yields
R
A = 0 (1 - e - Kt )
K …2
Equation 2 indicates that the amount of drug in the body rises asymptotically with time. Since drug
concentration and not the amount of drug is measured, we can substitute equation C=A/V (or A=VC), in
equation 2 to give
R
C = 0 (1 - e - Kt )
KV …3
Where C = plasma (or serum) drug concentration at time t; V = apparent volume of distribution; K =
elimination rate constant; and KV is the systemic clearance (Cl).
R0
C= (1 - e - Kt )
Cl …4
The first order elimination rate constant and elimination half life can be calculated from a
semilogarithmic plot of post infusion concentration time data as given in figure 1.
At the start of constant rate infusion, the amount of drug in the body is zero and hence, there is no elimination.As
time passes, the amount of drug in the body rises gradually until a point after which the rate of elimination equals
the rate of infusion i.e. the concentration of drug in plasma approaches a constant value called as steady-state.
Semilog plot
12
Regular plot 10
Css
10 Infusion stopped
P l a s m a d r u g c o n c e n tr a ti o n
8
When plotted on a
Infusion
semilog graph yields
6 rate = R0
a straight line with
slope = -K/2.303 1
4
2
Infusion time T
0
0 5 10 15 20 0.1
Time (h) 0 5 10 Time 15 20 25
Figure 1. Regular and semilog plot of plasma concentration versus time profile by constant rate infusion.
At steady-state, the change of amount of drug in the body is zero, hence, the equation 1 becomes:
Zero = R0 - KASS
R0 = KASS …5
Transforming to concentration terms and rearranging the equation:
R R
CSS = 0 = 0
KV Cl …6
Where, Ass and Css are amount of drug in the body and concentration of drug in plasma at steady-state
respectively. The value of K can be obtained from the slope of straight line obtained after a semilogarithmic plot
(log C versus t) of the plasma concentration time data gathered from the time when infusion is stopped.
Principle
By an arrangement of beakers and a constant head water reservoir, it is possible to simulate plasma
concentrations after constant rate IV infusion (See figure 2). The constant flow of water through the system
causes a first order dilution of the marker, potassium permanganate while the infusion of potassium
permanganate at constant rate in plasma compartment gives a zero order process. The samples from the plasma
compartment can be withdrawn at predetermined intervals and the concentration of samples can be determined
spectrophotometrically. The semilogarithmic plot of post infusion concentration time data will give the
elimination rate constant (K) and half life (t1/2). The concentration of sample at the time at which infusion is
stopped is considered as steady state concentration (Css). Various parameters can be calculated using various
formulae discussed in theory.
Figure 2. Design to simulate constant rate IV infusion
Procedure
1. Prepare 100 mg/ml of potassium permanganate in distilled water. Further prepare 10 ml volumes of working
solutions 10, 20, 40, 60, 80, and 100 mg/ml from the stock solution. Measure the absorbance of each solution at
540 nm. Use distilled water as the blank. Plot calibration curve and record intercept and slope.
2. Identify and mark the plasma compartment (P) to the apparatus. Make sure that the water reservoir is full.
Turn on the stirrer in the plasma beaker and adjust for gentle mixing. Establish a flow rate of 20 ml/min.
Between 15 and 25 ml/min is OK.
3. Collect plasma samples from plasma compartment at 0, 5, 10, 15, 20, 25, 30, 45, and 60 minutes after starting
the infusion.Arrange and label test-tubes to accommodate the samples.
4. One person in the group should be given the responsibility of performing activity of injection pump. This
person should take 15 drops of the saturated potassium permanganate solution and 'inject' one drop per minute
into the plasma compartment until the full dose has been injected. Record this time, T min. The infusion rate is
then 250/T mg/min (16.66 mg/min).
5. Collect 5 ml plasma sample from plasma compartment at the times designated quickly by pipette. Don't rinse
the pipette between samples.
6. Each sample will be analyzed spectrophotometrically at 540 nm. Early samples may need to be diluted to give
absorbance readings below 1. Remember to apply this dilution factor in your analysis of the results.
7. Plot the post infusion data of plasma concentration (C) versus time (t) on semi-log graph paper and determine
slope.
8. Determine steady state concentration (Css).
9. Calculate the apparent volume of distribution (V).
Observations
1. Infusion rate R0 = 16.66 mg/ml
2. C15 = Css = ______
Table 1 Concentration of potassium permangnate in plasma compartment
Time (min) Dilution Factor Absorbance Concentration

0
5
10
15
20
25
30
45
60
Calculations
Determine the slope of plot of post infusion data of concentration (C) versus time (t). Calculate
elimination rate constant by using slope of line.
-K log C 2 - log C1
Slope = =
2.303 t 2 - t1
Elimination half life can be calculated by using following formula
0.693
t1/ 2 =
K
3. Steady state concentration (Css)
Consider concentration of potassium permanganate in plasma compartment at the time of stopping of
infusion as Css (here Css = concentration at 15 min, as infusion was stopped at 15 min).
4. Volume of distribution
Volume of distribution can be determined by using following formula
R
CSS = 0
KV
Therefore,
R0
V=
KC SS
5. Clearance
Clearance can be determined by using following formula
R0
Cl =
CSS
Result
The various pharmacokinetic parameters obtained from plasma data of potassium permanganate are as
given below
Sr. No. Parameter Plasma data
1 Concentration at steady state
5 Clearance
Conclusion
Plasma concentrations after IV infusion can be simulated by an arrangement of beakers and a constant
head water reservoir.
Applications
1. Non invasive method of determination of pharmacokinetic parameters.
2. Simulation of first order elimination process during and after an IV infusion is possible.
Questions
1. What is steady state concentration?
2. What is zero order and first order process?
Exercise
Plasma concentration of a drug during and after a constant infusion (40 mg/h) for 12 h is given in
following table:
Time (h) 1 2 4 6 8 10 12 14 16 18 20 22
Concentration
3.3 5.4 7.6 8.7 9.3 9.6 9.5 4.1 1.8 0.76 0.33 0.14
(mg/l)
Calculate concentration at steady state, elimination rate constant, elimination half life, volume of
distribution and clearance.
Experiment 25
Calculation of various pharmacokinetic parameters after IV Infusion
Aim
To calculate various pharmacokinetic parameters from the given blood data of IV infusion (one
compartment model).
Learning objective
To understand calculations of various pharmacokinetic parameters after one compartment IV infusion
following first order elimination.
Prerequisite
Knowledge of pharmacokinetics of IV infusion.
Requirements
Regular graph paper, semilog graph paper, calculator, pencil.
Given data
Estimate the volume of distribution, elimination rate constant, half life and clearance from the data in
following table obtained on infusing a drug at the rate of 50 mg/hr for 7.5 h.
Time (h) 0 2 4 6 7.5 9 12 15
Conc 0 3.4 5.4 6.5 7 4.6 2 0.9
(mg/l)
b. Calculate elimination rate constant
Solution
Note: If the graph of post infusion concentration versus time is plotted on regular graph paper, the curvilinear
plot is obtained which makes it difficult to calculate slope and hence the semilog plot is used which gives the
linearity and makes it easy and accurate to calculate the pharmacokinetic parameters (Refer figure 1).
1. Semilog plot
a. Plot the graph on semi logarithmic graph paper as the elimination data follows first order kinetics.
b.According to the given data select number of cycles on semilog paper.
c. Plot concentration on Y axis and time on X axis.
d. Mark concentration for respective time on the graph paper.
e. Draw a straight line in such a way that it covers maximum elimination phase points.
f. Determine slope of the line and calculate elimination rate constant.
a. On Y axis near to elimination phase select initial concentration (C1) and from that point, draw a
straight line which intersects plotted line. This point would be the first point of intersection. From intersected
point, draw perpendicular straight line on X axis which gives the time (t1).
b. On Y axis draw a second straight line from half concentration of initial (C2) which intersects plotted line. This
will be the second point of intersection. From that point draw perpendicular on X axis which gives the time (t2).
c. Calculate half life by subtracting t2 form t1.
8 Regular plot Se milog plot of post infusion da ta
10
7
Concentration (mg/l)
Concentration (mg/l)
6
5
4
1
3
2
1
0 0.1
0 5 10 15 20 0 5 10 15 20
Time (h) Time (h)
Figure 1. Regular and semilog plot
Calculations
Calculate elimination rate constant by using slope of line.
log C 2 - log C1
Slope =
t 2 - t1
K = - ( Slope ´ 2 .303)
This can be calculated graphically as discussed above or by using formula given below:
0.693
t 1/ 2
=
K
3. Volume of distribution
Calculate volume of distribution by using following equation
R0
V=
KC SS
Where, R0 = infusion rate = 50 mg/h, Css = 7 mg/l.
· Consider concentration of drug in plasma compartment at the time of stopping of infusion as Css (here
Css = concentration at 7.5 h, as infusion was stopped at 7.5 h).
4. Clearance
Calculate clearance by using following equation
Cl = KV
Also it can be calculated by using following formula

R0
Cl =
CSS
Results
The various pharmacokinetic parameters calculated from given plasma data of drug are given as
follows:
1 Biological half life
4 Clearance
Applications
1. Pharmacokinetic parameters during and after IV infusion can be estimated.
2. Steady state concentration of drug can be estimated.
Questions
1. How elimination rate constant can be determined graphically?
2. How volume of distribution can be calculated in case of IV infusion data?
Exercise
1. Estimate the pharmacokinetic parameters from the data given in following table obtained on infusing
droperidol at the rate of 0.125 mg/hr for 7.5 h.
Time (h) 0 0.5 2 4 6 8 10 14 18 24 26 28 30

Conc 0 0.9 1.8 2.6 2.5 2.5 2.7 2.7 2.9 3.10 1.4 0.67 0.36
(mg/l)
2. What is the plasma drug concentration 4 h after an IV dose 1 mg/kg followed by a constant infusion of 0.2
mg/kg per hour?
Time (h) 0 2 4 6 8 10 12 18 24
IV dose (mg/l) 10 6.7 4.5 3.0 2.0 1.35
IV infusion
0 3.3 5.5 7.0 8.0 8.6 9.1 9.7 9.9
(mg/l)
Experiment 26
Calculation of area under curve by Trapezoidal rule
Aim
To calculate the area under curve (AUC) for given set of data by Trapezoidal rule.
Learning objective
1. To understand the calculations of area under curve using trapezoidal rule.
2. To understand the importance ofAUC for various studies.
Theory
AUC represents the total integrated area under the plasma level-time profile and expresses the total
amount of drug that comes into the systemic circulation after its administration. AUC is expressed in mg.hr/ml
and is useful in estimating rate of absorption. The parameter is of particular importance in assessing the efficacy
of drugs used to treat acute conditions like pain and insomnia which can be treated by a single dose. Several
methods are used for measuring the area under the plasma concentration-time curve like counting square,
cutting and weighing, use of planimeter and trapezoidal rule.
Trapezoidal rule is the simple numeric method of estimation of area. In this method, the plasma
concentration versus time is plotted and the curve is divided by a series of vertical lines into a number of
trapezoids, the area of each trapezoid is calculated separately and addition of area of all trapezoids gives AUC.
When concentration at time zero is not indicated in data then in determination of AUC from a concentration
versus time plot obtained following extravascular administration of drug, concentration at zero time is taken as
zero. However, when concentration versus time is plotted after IV administration of drug, concentration at zero
time should not to be taken as zero because concentration at zero time is maximum. So, concentration at zero
time must be determined graphically in order to draw first segment for trapezoidal rule. The advantage of this
method is that it only requires a simple extension of a table of experimental data. Other methods involve either
greater numeric complexity or fitting of an equation to the observation and then calculating the area by
integrating the fitted equation.
AUC for data plotted in graph is obtained by adding area of each segment represented by geometric
figure. The area of each segment is calculated using formula for geometric figure of that particular segment.
50
Regular graph
45
40
35
30
25
20
15
10
5
A B C D E F G H I J
0
0 1 2 3 4 5
Time (h)
Figure 1. Plot of plasma concentration versus time data after extravascular administration
Given Data
Table. 1 The plasma concentration as a function of time following oral administration of a single 500 mg dose
of a drug is shown below
Time
0.5 1 1.5 2 2.5 3 3.5 4 4.5 5
h
Concentration
1.5 16 35 45 32 20 14 10 8 5
mg/ml
Calculate area under curve from 0-5 h.
Requirement
Graph paper, scale, pencil, etc
Solution
a. Plot given data of plasma concentration versus time profile by using regular graph paper.
b. Generate total ten segments (trapezoids) on the graph paper and denote asA, B, …. to J.
c. Calculate the area of each segment using formula for geometric figure of segment.
d. The area bounded by trapezoids approximates the area under the curve.
e. The sum of area of all these segments yields area under curve from time zero to time of last data point plotted
on graph.
f. Put the values of concentration and corresponding time in the following formula
C1 + C 0
AUC0-1 = ´ (t1 - t 0 )
2 …1
AUC0-¥ = AUC0-0.5 + AUC0.5-1 + ..... + AUC4- 4.5 + AUC4.5-5

…2
Table 2. Calculation of totalAUC using the Trapezoidal rule
Time Concentration Cn -1 + Cn
Segment tn-tn-1 AUC
(h) (mg/ml) 2
0.5 1.5 A
1 16 B
1.5 35 C
2 45 D
2.5 32 E
3 20 F
3.5 14 G
4 10 H
4.5 8 I
5 5 J
AUC0-5 =
Result
By using Trapezoidal rule method, theAUC from 0-5 hrs was ________ mg.hr/ml.
Conclusion
It can be concluded that Trapezoidal rule can be used for the determination of the area under the plasma
concentration versus time curve.
Applications
1. In absence of the knowledge of the intercept of the plasma concentration versus time plot and rate constants
accompanying the data, this method permits the determination of the area under plasma concentration time
curve (AUC).
2. One can determine systemic, renal and metabolic clearance of a drug using this method.
3.AUC is one of methods the to determine the extent of bioavailability of drug.
4.AUC is very important parameter in the bioequivalence testing.
Questions
1. What isAUC? Give its importance.
2. What is Trapezoidal rule of estimatingAUC?
3. Enlist various methods for estimatingAUC.
Exercise
1. The plasma concentration as a function of time following oral administration of a single 50 mg dose of a drug
is given below:
Time
1 2 3 4 5 6 7 8
h
Concentration
7 10 5 2.5 1.25 0.6 0.2 0
mg/l
Calculate area under curve from 0-8.
2. The plasma concentration as a function of time following oral administration of a single 50 mg dose of a drug
is given below:
Time
0 0.5 1 1.5 2 3 4 6 8 12
h
Concentration
0 0.38 0.6 0.73 0.85 0.95 0.94 0.87 0.66 0.37
mg/l
Calculate area under curve from 0-12.
Experiment 27
Calculation of absorption rate constant by method of residuals
Aim
To calculate the absorption half life and absorption rate constant (Ka) for given set of data by using
method of residuals.
Learning objective
1. To understand method of residuals for calculating absorption rate constant.
2. To calculate Ka from given data.
Theory
Absorption rate constant can be calculated by method of residuals. The technique is also known as
feathering, peeling and stripping. It is commonly used in pharmacokinetics to resolve a multi-exponential curve
into its individual components.
For drugs that follows one-compartment kinetics and are administered extravascularly, the
concentration of drug in plasma is expressed by a bi-exponential equation
K a FA 0
C = [ e - Kt - e - K a t ]
V (K a - K ) …1
Where F = fraction of drug absorbed systemically after extra vascular administration; Ka = first order
absorption rate constant; K = first order elimination rate constant;A0 = amount of drug at zero time;
If KaFA0/V(Ka-K) = X, a hybrid constant, then:
C = Xe - Kt - Ae - K at
…2
During the elimination phase, when absorption is almost over, Ka >> K and the value of second
-Kat
exponential e retains some finite value.At this time, the equation 2 reduces to:
s -K t
C = Xe …3
Converting equation 3 into logarithmic form
s Kt
log C = log X -
2.303
…4
s
Where C represents the back extrapolated plasma concentration values.
A plot of log C verses t yields a biexponential curve with a terminal linear phase having slope K/2.303.
Back extrapolation of this straight line to time zero yields y intercept equal to log (X).
Substraction of true plasma concentration values i.e. equation 2 from the extrapolated plasma
concentration values i.e. equation 3 yields a series of residual concentration values Cr:
s
(C - C ) = Cr = Xe - K t a
…5
In log form, the equation is
K at
log Cr = log A -
2.303 …6
Aplot of log Cr versus t yields a straight line with slope Ka/2.303 and y intercept log X.Absorption half-
life can then be computed from Ka using the relation 0.693/Ka. Thus, the method of residuals enables resolution
of the biexponential plasma level-time curve into its two exponential components.
Ideally, the extrapolated and the residual lines intersect each other on y axis i.e. at time t = zero and
there is no lag in absorption. If an intersection occurs at a time greater than zero, it indicates time lag.
The method is best suited for drugs which are rapidly and completely absorbed and follow one compartment
kinetics.
Figure 1. Semilog plot of plasma concentration versus time profile after oral administration of a drug
When using the method of residuals, a minimum of three points should be used to define the straight
line. Data points occurring shortly after tmax may not be accurate, because drug absorption is still continuing at
that time. Because this portion of the curve represents the post-absorption phase, only data points from the
elimination phase should be used to define the rate of drug absorption as a first order process.
Prerequisite
1. Concept of one compartment extravascular administration.
Requirements
Regular graph paper, semilog graph paper, calculator, pencil.
Given data
The following data were obtained when a 500 mg of an antibiotic was given orally. Assume 100% of
the administered dose was absorbed.
Table 1. Plasma data obtained after oral administration of 500 mg dose of drug
Time
1 2 3 4 5 6 8 16 18 20
h
Conc.
26.501 36.091 37.512 36.055 32.924 29.413 22.784 7.571 5.734 4.343
mg/ml
Calculate absorption rate constant.
Solution
1. Plot the drug concentration (C) versus time on semilog paper with the concentration values on logarithmic
axis.
2. Extrapolate elimination phase back upto Y axis.
3. Denote concentration from extrapolated line as C .
4. Obtain C concentrations for time (t1, t2, ….tn) versus concentration (C1, C2,….Cn) from back extrapolated line
( C1, C 2,......, Cn ).
5. Obtain residual concentrations (Cr) by subtracting C from C with respect to time.
6. Plot a residual concentration (Cr) versus time on same semilog paper.
7. Determine slope of residual line.
Table 2. Estimation of residual concentrations

Time PDC Extrapolated Residual PDC
(h) (mg/ml) C PDC C Cr = C - C
1 26.501
2 36.091
3 37.512
4 36.055
5 32.924
6 29.413
8 22.784
16 7.571
18 5.734
20 4.343
PDC: Plasma drug concentration
Calculations
1.Absorption rate constant (Ka)
Calculate absorption rate constant by using slope of residual line.
s s
log C 2 - log C1
Slope =
t 2 - t1
K a = -(Slope ´ 2.303)
2.Absorption half life

Calculate absorption half life by using following formula
0.693
t 1/ 2
=
Ka

Results
By using method of residuals absorption rate constant (Ka) and absorption half life (t1/2) for given data
set were found to be ________ h-1 and __________ h respectively.
Conclusion
It can be concluded that absorption rate constant and absorption half life (t1/2) can be estimated using
method of residuals.
Applications
1. Determination of absorption rate constant from oral absorption data is possible.
2. Method of residuals is easy method of calculating absorption rate constant.
Questions
1. What is absorption rate constant?
2. How absorption rate constant can be determined by method of residuals?
Exercise
1. The following data were obtained when a 500 mg of an antibiotic was given orally. Assume 100% of the
administered dose was absorbed.
Time
0.25 0.5 0.75 1 1.5 2 3 4 5 6 7
h
Conc.
1.91 2.98 3.54 3.80 3.84 3.62 3.04 2.49 2.04 1.67 1.37
mg/l
Calculate absorption rate constant and absorption half life.

2. Plasma samples from a patient were collected after an oral bolus dose of 10 mg of a new benzodiazepine
solution and the following data was obtained.
Time
0.25 0.50 0.75 1 2 4 6 10 14 20
h
Conc.
2.85 5.43 7.75 9.84 16.20 22.15 23.01 19.09 13.90 7.97
ng/ml

Experiment 28
Calculation of absorption rate constant by Wagner-Nelson method
Aim
To calculate absorption rate constant (Ka) using Wagner-Nelson method.
Learning objective
To understand Wagner-Nelson method for estimating absorption rate constant (Ka).
Theory
Estimation of absorption rate constant: Wagner-Nelson method
After a single oral dose of a drug, at any time, the amount of drug absorbed into the systemic circulation
Aa, is the sum of amount of drug in the bodyAand the amount of drug eliminated from the bodyAe. Thus
Aa = A + Ae …1
The amount of drug in the body is A= VC while the amount of drug eliminated at any time t can be
calculated as follows
Ae = KV [ AUC ]t0 …2
Substituting the values ofAandAe in equation 1 gives
Aa = VC + KV [ AUC ]t0 …3
¥
The total amount of drug absorbed into the systemic circulation from time zero to infinity Aa can be
given as
Aa¥ = VC ¥ + KV [ AUC ]¥0 …4
Since at t = ¥ , C= 0, the above equation can be reduced to
Aa¥ = KV [ AUC ]¥0

…5
The fraction of drug absorbed at any time t is given as
Aa VC + KV [ AUC ]t0
=
Aa¥ KV [ AUC ]¥0 …6
Aa C + K [ AUC ]t0
=
Aa¥ K [ AUC ]¥0 …7
Percent drug unabsorbed at any time is therefore
é A ù é C + K [ AUC ]t0 ù
% ARA = ê1 - ¥a ú100 = ê ¥ ú100 …8
ë Aa û ë K [ AUC ]0 û
The method requires collection of blood samples after a single oral dose at regular intervals of time till
the entire amount of drug is eliminated from the body. K is obtained from semilog plot of C versus t. A semilog
plot of percent unabsorbed (i.e percent ARA) versus t yields a straight line whose slope is Ka/2.303. If a regular
plot of the same is a straight line, then absorption is zero order.
Ka can also be similarly estimated from urinary excretion data. The biggest disadvantage of Wagner-
Nelson method is that is applies only to drugs with one compartment characteristics.
Given data
Bioavailability of phenylpropanolamine hydrochloride was studied in 24 adult male subjects. The
following data represent the mean blood phenylpropanolamine hydrochloride concentrations (ng/ml) after the
oral administration of a single 25 mg dose of phenylpropanolamine hydrochloride solution.
Time
0.25 0.5 0.75 1 1.5 2 3 4 6 8 12 18 24
(h)
Conc
51.33 74.05 82.91 85.11 81.76 75.51 62.98 52.32 36.08 24.88 11.83 3.88 1.27
(ng/ml)
Determine absorption rate constant and absorption half life by using Wagner-Nelson method.
Solution
Wagner-Nelson Method
1. Plot the semilog graph of plasma concentration versus time.
2. Determine elimination rate constant from the slope of terminal part of line.
3. Plot the regular graph of plasma concentration versus time and determine [ AUC ]t0 by trapezoidal rule.
t
Determine cumulative [ AUC ]0.
t
4. Determine K [ AUC ]0 by multiplying each [ AUC ]0 by K.
t
¥
5. Find [ AUC ]0 by adding up all theAUC pieces from zero to infinity.
6. Determine the fraction unabsorbed value corresponding to each time point t by using observation table.
7. Plot fraction unabsorbed versus time on semilog paper.
Observation table for determination of fraction of drug unabsorbed

C + K x Cum Ab/Ab¥ 1 ( Ab/A b¥ )
Time C [ AUC ]t0 Cumulative K x Cum
[ AUC ]t0 (Fraction (Fraction
[ AUC ]t0 [ AUC ]t0 (amount absorbed) absorbed) unabsorbed)
0.25 51.33
0.5 74.05
0.75 82.91
1 85.11
1.5 81.76
2 75.51
3 62.98
4 52.32
6 36.08
8 24.88
12 11.83
18 3.88
24 1.27
¥
Consider (C + K x Cum [ AUC ]024 ) value as amount absorbed at infinity (Ab )
Calculations
1. Determination of absorption rate constant
Determine totalAUC by the trapezoidal rule.
C n -1 + C n
( A U C ) tt nn -1 = ( t n - t n -1 )
2
C la s t
( A U C ) t¥ =
K
Determine absorption rate constant.
Determine slope of line and calculate absorption half life by using formula
0.693
t1/ 2 = K a
Result
By using Wagner-Nelson absorption rate constant (Ka) and absorption half life (t1/2) for given data set
-1
was found to be ______h and ________h respectively.
Conclusion
It can be concluded that absorption rate constant and absorption half life (t1/2) can be estimated using
Wagner-Nelson method.
Application
This method allows calculation of absorption rate constant (Ka).
Questions
1. How absorption rate constant can be determined by Wagner Nelson method?
2. What are the disadvantages of this method?
Exercise
The following data were obtained when a 100 mg of an antibiotic was given orally. Assume 100% of
the administered dose was absorbed.
Time
0.25 0.5 1 2 3 4 6 8 10 12
h
Conc.
1.6 2.7 3.7 3.5 2.7 2 1.02 0.49 0.26 0.12
mg/ml
Experiment 29
Calculation of various pharmacokinetic parameters after extravascular administration
Aim
To calculate various pharmacokinetic parameters after extravascular administration of drug (one
compartment model).
Theory
Administration of drug dose by an extravascular route involves passage of the drug by absorption
through a biological membrane. The plasma profile obtained following extravascular administration of a drug is
different from plasma profile of same drug obtained after the drug administered as a rapid intravenous bolus
injection because the entire dose of administered drug is not absorbed all at once.
Absorption phase
Elimination phase
Time (h)
For a drug that enters the body by a first order absorption process, gets distributed in the body
according to one-compartment kinetics and is eliminated by a first order process, the model can be depicted as
follows
Central Compartment
Ka (absorption) K (Elimination)
First order First order
After extravascular administration, the rate of change in the amount of drug in the body dA/dt is the
difference between the rate of input (absorption), dAa/dt and rate of output (elimination), dAe/dt.
Amount of drug in the body = Rate of absorption - Rate of elimination
dA dAa dAe
= - …1
dt dt dt
The differential equation that follows relates changes in drug concentration in the blood with time to the
absorption and the elimination rates
dA …2
= K a Aa - KA
dt
where dA/dt = rate of change of amount of drug in the blood; A = amount of drug in the blood at time t;
Aa = amount of absorbable drug at the absorption site at time t; Ka = absorption rate constant; K elimination
rate constant, respectively, KaAa = first order rate of absorption and KA= first-order rate of elimination.
Integration of equation 2 yields
FK a A0
A= (e - Kt - e - Kat )
(K a - K ) …3
Where A = amount of drug in the body at time t; A0 = amount of drug at the site of administration at
t=0 (the administered dose), F = fraction of drug absorbed.
Equation 3 shows that the amount of drug in the body or blood follows a biexponential profile, first
rising and then declining. For orally or extravascularly administered drugs, generally Ka>>K; therefore, the
rising portion of the graph denotes the absorption phase. If K>>Ka (perhaps indicating a dissolution rate-limited
absorption) the exact opposite will hold true.
Converting equation 3 into concentration form, as V=A/C
FK a A0
C= (e - Kt - e - Kat )
V (K a - K ) …4
FAoKa
Intercept=
V (Ka- K)
-K
Slope=
2.303
t=0 Time (h)

Determination of elimination rate constant (K)
This parameter can be estimated from elimination phase of the plasma level time curve profile. For
most drugs administered extravascularly, absorption rate is significantly greater than the elimination rate i.e.
Kat>>Kt. Hence, one can say that e approaches to zero much faster than does e-Kt . At this stage, when
-Kat
absorption is complete, the change in plasma concentration is dependent only on elimination rate and equation 4
becomes
FK a A0
C= e - Kt
V (Ka - K ) ...5
Transforming into log form, the equation 5 becomes:
FK a A0 Kt
log C = log - …6
V ( K a - K ) 2.303
Aplot of C versus t yields a straight line with slope K/2.303.
Elimination half life

One can estimate elimination half life form elimination rate constant.
0.693
t1/ 2 = …7
K
Apparent volume of distribution
For a drug administered by oral, or any other extravascular route of administration, the apparent
volume of distribution cannot be calculated from plasma drug concentration data alone. The reason is that the
value of F (the fraction of administered dose that reaches the general circulation) is not known. From equation 6
we get,
FK a A0
Intercept =
V (Ka - K ) …8
In the absence of data for the fraction of administered dose that reaches the general circulation, the best
one can do is to obtain the ratio of V/F:
V K a A0 1
= ( ) …9
F ( K a - K ) intercept
Time of maximum drug concentration, peak time (tmax)

The peak time (tmax) is the time at which the body displays the maximum plasma concentration, (Cmax).
It occurs when the rate of absorption is equal to the rate of elimination. At the peak time, therefore, KaAa = KA.
The rate of change in plasma drug concentration dc/dt = zero. This rate can be obtained by differentiating
following equation 4.
dC FK a A0
= ( - Ke - Kt + K a e - Kat ) …10
dt V ( K a - K )
On simplifying, above equation becomes:
Ke - Kt = K a e - Kat
Converting into logarithmic form,
Kt Ka
log K - = log K a -
2.303 2.303
When t is tmax, rearrangement of above equation yields

2.303 log( K a / K )
t max =
Ka - K ...11
Equation 11 indicates that peak time depends on, or is influenced by, only the absorption and
elimination rate constants; therefore, any factor that influences the absorption and the elimination rate constants
will influence the peak time value; however, the peak time is always independent of the administered dose of a
drug.
Maximum (peak) plasma concentration (Cmax)
There are three methods available for determining peak plasma concentration Cmax. Two are given here:
Method 1. Peak plasma concentration obtained from the graph of plasma concentration versus time.
Method 2. Peak plasma concentration can also be obtained by using an equation 4,
FK a A0
C= (e - Kt - e - Kat )
V (K a - K )
If tmax is substituted for t then

FK a A0
Cmax = (e -Kt max - e -Kat max )
V (K a - K ) …12
Where,
FA 0 K a
= Intercept
V (Ka - K )
Obtain intercept by plotting plasma concentration versus time profile on semilog paper.
Given Data
The bioavailability of phenylpropanolamine hydrochloride was studied in 24 adult male subjects. The
following data represent the mean blood phenylpropanolamine hydrochloride concentrations (ng/ml) after the
oral administration of a single 25 mg dose of phenylpropanolamine hydrochloride solution. Plasma
concentration time profile of phenylpropanolamine hydrochloride is given below:
Time
0.25 0.5 0.75 1 1.5 2 3 4 6 8 12 18 24
h
Conc
51.33 74.05 82.91 85.11 81.76 75.51 62.98 52.32 36.08 24.88 11.83 3.88 1.27
ng/ml
1. Determine elimination rate constant and elimination half life.

2. Determine totalAUC.
3. Determine Cmax and Tmax.
Solution
Table 1. Determination ofAUC
Time C Segment Cn-1 + Cn tn-tn-1 AUC
h mg/ml 2
0.25 51.33 A
0.5 74.05 B
0.75 82.91 C
1 85.11 D
1.5 81.76 E
2 75.51 F
3 62.98 G
4 52.32 H
6 36.08 I
8 24.88 J
12 11.83 K
18 3.88 L
24 1.27 M
AUC0-24
Clast/K
Total AUC= AUC 0-24 + Clast/K
Table 2. Determination of residual concentrations

Time Conentration Extrapolated Residual Conc.
h (mg/ml) C Cr = C - C
0.25 51.33
0.5 74.05
0.75 82.91
1 85.11
1.5 81.76
2 75.51
3 62.98
4 52.32
6 36.08
8 24.88
12 11.83
18 3.88
24 1.27
Calculations
1. Elimination rate constant and elimination half life
Plot a graph of decline in plasma concentration versus time on semilog paper and determine slope of line.
K = - ( Slope ´ 2 . 303 )
2.Area under curve

Plot a graph of plasma concentration versus corresponding time on simple graph paper and prepare
segmentsAto M.
C1 + C 0
AUC0-1 = ´ (t1 - t 0 )
2
C24
AUC0-¥ = AUC0-0.25 + AUC0.25-0.5 + ..... + AUC18- 24 +
K
3.Absorption rate constant (Ka)
Calculate absorption rate constant by using slope of line.
s s
log C 2 - log C1
Slope =
t 2 - t1
K a = -( Slope ´ 2.303)

Calculate absorption half life by using following formula
0.693
t 1/ 2
=
Ka
5. Determination of tmax and Cmax
FAoKa
I=
V (Ka- K)
Cmax
Elimination
phase
-K
Slope=
2.303
Absorption tmax
phase
Time (h)
2.303 log( K a / K )
t max =
Ka - K
FA 0 K a
Cmax = (e - Kt max - e - Kat max )
V (Ka - K )
Where (FA0Ka)/V(Ka-K) is the y intercept of semilog plot of plasma concentration versus time curve.
Results
The various pharmacokinetic parameters calculated from given plasma data of phenylpropanolamine
hydrochloride are given as follows:
3 Cmax
4 t max
5 Total AUC
6 Absorption rate constant
7 Absorption half life
Conclusion
It can be concluded that various pharmacokinetic parameters can be calculated after extravascular
administration of drug.
Applications
1.Various pharmacokinetic parameters can be estimated from given plasma data after extravascular
administration of drug.
2.Bioequivalence testing between various brands can be done.
3.Bioavailability of drug can be studied.
Exercise
After an oral administration of 500 mg drug, the following data was obtained
Time (h) 0.5 1 1.5 2 4 6 10 16 24 32 48

Plama Conc (mg/l) 2.4 3.8 4.2 4.6 8.1 5.8 5.1 4.1 3.0 2.3 1.3
Calculate elimination half, elimination rate constant, Cmax, tmax, AUC, absorption rate constant and
absorption half life.
Experiment 30
Pharmacokinetic study of drug using plasma and urinary data
Aim
To study the pharmacokinetics of amoxicillin capsules by using plasma and urinary data.
Learning objectives
1. To study the pharmacokinetics of amoxicillin in humans.
2. To estimate various pharmacokinetic parameters using plasma and urinary data.
Theory
1. Calculations of pharmacokinetics ofAmoxicillin using plasma data
As per previous experiment ( Expt No. 29 ).
2. Calculations of pharmacokinetic parameters using urinary data
When plasma drug level-time data is not available, one can estimate pharmacokinetics of drug by
using urinary excretory data. It is possible to estimate first-order elimination, excretion and absorption rate
constant, fraction excreted unchanged and renal clearance of drug. If volume of distribution is known then total
clearance and non renal clearance can also be calculated.
Urinary drug excretion data can be used for calculation of the first order elimination rate constant. The
rate of drug excretion after a single oral dose of drug is given by
dAu FK a K u A0
= - (e - Kt - e - Kat )
dt Ka - K …1
Where dAu/dt = rate of urinary drug excretion, Ku = excretion rate constant, K = elimination rate
constant, Ka = absorption rate constant and F = fraction of drug absorbed. A graph of rate of urinary drug
excretion (dAu/dt) versus time yield a curve identical in appearance to the plasma level-time curve for the drug.
After complete drug absorption -e-kat approaches to zero and equation is reduced to
dAu FK a K u A0 - Kt
= e …2
dt Ka - K
We can rewrite equation 2 in logarithmic form as follows
dAu FK a K u A0 Kt
log = log - …3
dt Ka - K 2.303
When a graph of rate of urinary drug excretion versus time (t* is midpoint of the collection period) is
plotted on semilog paper yields straight line with a slope of –K/2.303.
The first order elimination rate constant can be calculated from urine data by using sigma-minus
method. Following equation will be utilized to determine rate constant.
Au¥
ARE = ( Au¥ - Au ) = ( K a e- Kt - Ke- Kat )
Ka - K …4
Where, ARE = amount remaining to be excreted, K = first-order elimination rate constant, A¥ is u
amount excreted at infinity, Au is amount excreted at time t, Ka is first order absorption rate constant and t is the
time.
A plot of ARE versus t results in a biexponential curve and if (Ka>K), the slope of the terminal linear
portion of the curve will define K of the drug. Intercept of equation 4 is equal to Au /(Ka-K) so it is possible to
determine absorption rate constant. The absorption rate constant Ka can be estimated by method of residuals
using the same data.
Urinary excretion data after oral administration can also be treated according to Wagner-Nelson
method to calculate Ka by construction of percent amount remaining to be absorbed (ARA) plots. The method
requires urine collection for sufficiently long time to ensure accurate estimation of K but need not be collected
to time infinity. The equation derived to relate %ARAwith urinary excretion rate is:
A é dA / dt + KAu ù
% ARA = (1 - ¥a )100 = ê1 - u ú100
Aa ë KAu¥ û …5
Where ARA = amount remaining to be absorbed, Aa = amount of drug absorbed in to systemic
circulation at time t, Aa ¥= total amount of drug absorbed into the systemic circulation from time zero to infinity,
dAu/dt = rate of urinary drug excretion (proportional to the amount of drug in body) and K = elimination rate
constant.
A semilog plot of %ARAversus t yields a straight line with slope -Ka/2.303.
Requirements
Glassware: Petri plates, micropipettes, test tubes etc.
Chemicals: Pure amoxicillin, nutrient agar etc.
Instrument:Autoclave, UV Spectrophotometer, centrifuge etc.
Procedure
Assay of amoxicillin
1. Maintain Streptococcus pneumoniae cultures in nutrient broth. Maintain stock cultures of Streptococcus
0
pneumoniae from isolates on nutrient agar plates and store at 4 C. Before each assay, run subculture into
nutrient broth. Give at least two transfers in nutrient broth before the test assay.
0
2.Keep the nutrient broth cultures at 37 C for 24 h. Measure absorbance of broth cultures at 630 nm in a
spectrophotometer on the day of the assay. Select the culture showing an optical density ranging from 0.275 to
0.325.
3. Dissolve 11.5 gm of nutrient agar in 500 ml of distilled water and adjust pH to 7.0 using 0.4 N sodium
hydroxide solution. Autoclave nutrient agar for 20 min at 15 lbs pressure. Then, bring temperature of agar
0
solution to 37 to 40 C and then seed the organism kept in nutrient broth (0.4 ml culture/500 ml solution). Then
pour this into sterile petri plates and wait till it gets solidify. Finally with the help of sterile borer cut the wells of
2 mm.
4. Prepare 1 mg/ml solution ofAmoxicillin by dissolving it in minimal amount of suitable sterile solvent.
5. Collect blank plasma and urine samples from volunteers, centrifuge and remove supernatant. Dilute
supernatant with distilled water and prepare standard samples of Amoxicillin in concentration range of 0-12
mg/ml in quadruplicate.
0
6. Load ten micro liters of the standard solutions of drug into the wells and keep the petri plates at 4-10 C for 1-2
h and incubate the plates at 370C for 24 h.
7. Measure the zone of inhibition and its diameter. Plot the graph of log concentration of Amoxicillin versus
diameter of zone of inhibition and determine unknown concentration of drug from regression line.
Study participants
1. Take approval of the study protocol from Institutional Ethics Committee and obtain written informed consent
from all the volunteers.
2. Select six healthy male volunteers with age ranging between 20-30 years and a mean body weight ranging
between 45-69 kg.
3. Assess the volunteers as healthy on the basis of medical history, hepatic and renal function tests. All subjects
should be non-smokers and should not ingest any medicine two weeks before or during the study for any
ailment.
4. Exclude volunteers with history of penicillin allergy or who takes alcohol.
5. Instruct subjects to avoid antibiotics and beverages for 14 days before the study.
Study design
1. After an overnight fast, administer amoxicillin 500 mg on an empty stomach after emptying the bladder with
180 ml drinking water.
2.Allow food, 4 h after ingestion of amoxicillin.
Sample collection and processing
1. Collect blood samples (5 ml) from a peripheral vein before drug administration and then at 0.25, 0.5, 0.75, 1,
1.5, 2, 4, 8, and 12 hours after dosing into heparinised tubes.
2. Centrifuge collected samples within 10 minutes at 3000 rpm for 15 min and separate plasma. Store plasma
samples at -400C prior to analysis.
3. Collect urine samples prior to drug administration considered as blank sample and then in block samples at
0-2, 2-4, 4-6, 6-8, 8-10 and 10-12 h post dosing.
4. Measure the volume of urine, collected during each period from each volunteer. Collect the urine samples in
coded eppendorf and store at -400C until further analysis.
Pharmacokinetic analysis: Plasma data
Assume that Amoxicillin follows one compartment open model with first order absorption and
calculate pharmacokinetic parameters as given in calculations.
Pharmacokinetic analysis: Urinary excretion data
1. Plot the graph of percent cumulative amount ofAmoxicillin excreted versus time.
2. Plot the graph of excretion rate ofAmoxicillin versus mid point of urine collection period on semilog paper.
3. Plot the graph of amount ofAmoxicillin to be excreted ( Au¥ - Au ) versus mid point of urine collection period on
semilog paper.
4. Calculate pharmacokinetic parameters as given in calculations.
Observations
Table 1. Calibration curve ofAmoxicillin
Concentration Log C Zone of Inhibition

(mg/ml) (mm)
1
2
4
6
8
10
12
Slope
Intercept
Table 2. Plasma data obtained after oral administration of 500 mgAmoxicillin

Time Plasma Concentration Average
(h) mg/ml (SD)
S1 S2 S3 S4 S5 S6
0.25
0.5
0.75
1
1.5
2
4
8
12
S-Subject, SD-standard deviation
Table 3. Urinary excretion data obtained after oral administration of 500 mgAmoxicillin
Time Amount
Time of Mid point ARE
interval Urine excreted Au/t Cumulative
urine of urine Conc (A¥u
of urine volume Au ER Au
collection collection (mg/ml) - A u)
collection (ml) (mg) (mg/min) (mg)
(h) period t*
t (h)
0-2 2 1
2-4 2 3
4-6 2 5
6-8 2 7
8-10 2 9
10-12 2 11
¥
ER- excretion rate;Au- amount of drug excreted in mg;Au CumulativeAu at time 12h.
Calculations
Plasma data
1. Plasma concentration ofAmoxicillin (mg/ml)
Plot the graph of absorbance versus log concentration and determine slope and intercept.
Y= m X + c
Where, Y = absorbance, m= slope, X = log concentration (mg/ml), c = intercept.
2. Peak amoxicillin concentration (Cmax, mg/ml) in plasma and time to peak concentration (tmax, h)
Plot the graph of plasma concentration versus time and determine peak concentration (Cmax) and time to
peak (tmax) from the graph.
3. Elimination half life (t½, h) and Elimination rate constant (K, /h)
Plot the graph of plasma concentration of Amoxicillin versus time on semilog paper and determine
slope of decline in plasma concentration.
Elimination rate constant can be determined using following equation
K = -2.303 (slope)
Elimination half life can be determined using following formula
t1/2 = 0.693/K
4. Area under curve (AUC, mg/h/ml)
DetermineAUC from 0-t by using trapezoidal rule.
C1 + C 0
AUC0-1 = + (t1 - t 0 )
2
AUC0-t = AUC0- 25 + .......... + AUC8-12
Determine totalAUC by using following formula

C12
AUC12-¥ =
K
TotalAUC =AUC0-12 +AUC12- ¥

Urinary data
Plot the graph of absorbance versus log concentration and determine slope and intercept using formula
Y= m X + c
Where, Y= absorbance, m= slope, X= log concentration (mg/ml), c= intercept.
2. Amount of drug excreted in urine,Au (mg/ml):
Au = Conc. of drug in urine (mg) x Total volume of urine voided (ml)
3. Excretion rate
Excretion rate =Amount excreted (Au) / Time interval of urine collection
4. Elimination rate constant (sigma-minus method)

Plot the graph of amount remaining to be excreted versus mid point of urine collection period on
semilog paper. Determine slope of terminal linear portion (last three to four points) of the curve and determine
elimination rate constant.
Slope=(-K)/2.303
5. Excretion rate
Excretion rate =Amount excreted (Au) / Time interval of urine collection
Determine elimination half life using following formula
t1/2 = 0.693/K
Result
1. The concentration ofAmoxicillin at 1.5 h was found to be ________mg/ml.
2. The pharmacokinetic parameters ofAmoxicillin were found as follows:
Parameter Plasma data Urinary data
Elimination rate constant
Elimination half life
Total AUC -
Volume of distribution -
Conclusion
It can be concluded that the pharmacokinetic parameters can be calculated from both plasma and
urinary excretion data.
Applications
1. Estimation of pharmacokinetic parameters using urinary excretion data is possible.
2. Comparison of pharmacokinetic parameters obtained by plasma and urinary data is possible.
3. Bioequivalence testing of different brands can be done.
Questions
1. Give two methods for calculating various pharmacokinetic parameters using urinary excretion data.
2. What is tmax and Cmax?
Exercise
Study pharmacokinetics of Ofloxacin by both plasma and urinary excretion data.
Experiment 31
Pharmacokinetic study of drug using salivary drug concentration
Aim
To study the pharmacokinetics of ofloxacin tablets using salivary drug concentration.
Learning objectives
1. To study the salivary pharmacokinetics of ofloxacin in humans.
2. To estimate various pharmacokinetic parameters using salivary drug concentration.
Theory
Determination of drug concentrations in the saliva has gained widespread acceptance in a variety of
settings. Estimation of drugs in the saliva has been employed for therapeutic drug monitoring and for
calculation of pharmacokinetic variables. The rational use of such determinations can provide knowledge of
patient-specific pharmacokinetic parameters leading to improved therapy. Saliva can serve as an alternative
body fluid for pharmacokinetic investigations. It can be collected with minimal patient discomfort and can be
easily obtained on multiple occasions. It is particularly suitable for investigations in geriatrics and pediatrics.
Pharmacokinetic studies have shown that Ofloxacin penetrates into saliva and its concentration correlates well
with serum levels.
Therefore, once the salivary concentration of Ofloxacin is determined, the pharmacokinetic data can
be generated by the following equations
1. Elimination rate constant (K) can be determined from the slope of concentration time plot
Slope=(-K)/2.303
2. Elimination half life (t1/2) = 0.693/K
3. TotalAUC
C
AUC0-¥ = AUC0-0.5 + AUC0.5-1 + ..... + AUC5-6 + AUC6-8 + last
K
Principle
Ofloxacin is a synthetic fluorinated carboxy quinolone which is reported to have a broad antimicrobial
spectrum. Pharmacological studies of ofloxacin require sensitive and specific methods for the estimation of the
drug in biological fluids such as blood, saliva and urine. HPLC methods using fluorescence detector are
available but they involve a series of steps for preparation of the sample before analysis. More over, all the
laboratories cannot afford to have such sophisticated equipment. Since E.coli is sensitive to Ofloxacin, it can be
used as test organism for the microbiological assay. The zone of inhibition exhibited by Ofloxacin against E.coli
is proportional to the concentration of the drug present. Hence the estimation of Ofloxacin in saliva can be done
by microbial method. Here, simple spectrophotometric method is also discussed for estimation of Ofloxacin in
saliva.
Prerequisite
1. Basic pharmacokinetics.
2. Plotting of graphs
Requirements
Glassware: Petri plates, pipettes, test tubes etc.
Chemicals: Pure Ofloxacin, nutrient broth, nutrient agar etc.
Instrument:Autoclave, UV Spectrophotometer.
Procedure
Assay of Ofloxacin
Method I (Spectrophotometric estimation)
1. Collect blank saliva samples from volunteers, centrifuge and remove supernatant. Dilute supernatant with
distilled water and prepare standard samples of Ofloxacin in concentration range of 0-16 mg/ml.
2. Determine the absorbance with UV spectrophotometer at 283 nm. Determine the unknown concentration of
Ofloxacin in saliva from the calibration curve.
Method II (Microbiological assay)
1. Maintain E. coli cultures in nutrient broth. Maintain stock cultures of E. coli from isolates on nutrient agar
0
plates and store at 4 C. Before each assay, run subculture onto nutrient broth. Give at least two transfers in
nutrient broth before the test assay.
0
2. Keep the nutrient broth cultures at 37 C for 24 h. Measure the absorbance of broth cultures at 630 nm in a
spectrophotometer on the day of the assay. Select the culture showing an optical density ranging from 0.275 to
0.325.
3. Dissolve 11.5 gm of nutrient agar in 500 ml of distilled water and adjust pH to 7.0 using 0.4 N sodium
hydroxide solution. Autoclave nutrient agar for 20 min at 15 lbs pressure. Then, bring temperature of agar
0
solution to 37 - 40 C and then seed the organism kept in nutrient broth (0.4 ml culture/500 ml solution). Pour this
solution into sterile petri plates and wait till it gets solidify. Finally with the help of sterile borer cut the wells of 2
mm.
4. Prepare 1 mg/ml solution of Ofloxacin by dissolving it in minimal amount of sterile 0.1 N hydrochloric acid
solution.
5. Collect blank saliva samples from volunteers, centrifuge and remove supernatant. Dilute supernatant with
distilled water and prepare standard samples of Ofloxacin in concentration range of 0-16 mg/ml in
quadruplicate.
6. Load ten micro liters of the standard solutions of drug onto the wells and keep the petri plates at 4-100C for 1-2
h and incubate the plates at 370C for 24 h.
7. Measure the zone of inhibition and its diameter. Plot the graph of log concentration of Ofloxacin versus
diameter of zone of inhibition and determine unknown concentration of drug from regression line.
Study design
1. Take approval of the study protocol from Institutional Ethics Committee and obtain written informed consent
from all the volunteers.
2. Select six healthy male volunteers with age ranging between 20-30 years and a mean body weight ranging
between 45-69 kg.
3. Assess the volunteers as healthy on the basis of medical history, hepatic and renal function tests. All subjects
should be non-smokers and should not ingest any medicine one week before or during the study for any ailment.
4. After an overnight fast, administer Ofloxacin 200 mg on an empty stomach after emptying the bladder with
180 ml drinking water.
5. On each occasion, collect 2-3 ml of saliva samples in clean test tubes at 0, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0 and
8.0 h period after oral administration of the drug.
6. Instruct the volunteers participated in the study to wash their oral cavity and use a piece of unsweetened,
unflavored chewing gum to stimulate salivary secretion.
0
7. Collect saliva sample from volunteers, centrifuge and separate the supernatant and store the samples at -20 C
till assayed.
Pharmacokinetic analysis
1.Assume that Ofloxacin follows one compartment open model with first order absorption.
2. Plot the regular graph of salivary concentration of drug versus time. Fit data into bell shape curve and obtain
peak Ofloxacin concentration (Cmax) in saliva and time to peak (tmax) concentration from the graph.
3. Calculate various pharmacokinetic parameters as per given in calculations section.
Observations
Table 1. Calibration curve of Ofloxacin
Spectrophotometric method Microbial assay
Concentration (C) Absorbance Log C Zone of Inhibition
(mg/ml) (mm)
0.5
1
2
4
6
16
Slope
Intercept
Table 2. Salivary concentrations and pharmacokinetic parameters calculated for individuals

Time Salivary Concentration Average
(h) mg/ml (SD)
S1 S2 S3 S4 S5 S6
0
0.5
1.0
2.0
3.0
4.0
5.0
6.0
8.0
S-Subject, SD-standard deviation
Calculations
1. Salivary concentration of Ofloxacin (mg/ml)
Y= m X + c
Where, Y= absorbance, m = slope, X= concentration (mg/ml), c = intercept.
or
Plot the graph of log concentration versus diameter of zone of inhibition.
Y= m X + c
Where, Y = absorbance, m = slope, X = log concentration (mg/ml), c = intercept.
2. Peak Ofloxacin concentration (C , mg/ml) in saliva and time to peak concentration (t , h)
max max
Plot the graph of salivary concentration versus time and determine peak concentration (C ) and time
max
to peak (tmax) from the graph.

-1
3. Elimination half life (t½, h) and Elimination rate constant (K, h )
Plot the graph of salivary concentration of Ofloxacin versus time on semilog paper and determine
slope of decline in salivary concentration.
Elimination rate constant can be determined using following equation
K = -2.303 (slope)
Elimination half life can be determined using following formula
t1/2 = 0.693/K
4.Area under curve (AUC, mg/h/ml)
DetermineAUC from 0-t by using trapezoidal rule.
C1 + C 0
AUC0-1 = + (t1 - t 0 )
2
AUC0-8 = AUC0-0.5 + AUC0.5-1 + ........ + AUC6-8
Determine totalAUC by using following formula

C
AUC8-¥ = 8
K
TotalAUC =AUC0-8+AUC8- ¥
Results
1. The mean salivary concentration of Ofloxacin at 8 h is found to be_______(mg/ml) .
2. The pharmacokinetic parameters of Ofloxacin found as follows:
Cmax:
tmax:
Elimination rate constant:
Absorption rate constant:
Elimination half-life:
TotalAUC:
Conclusion
It can be concluded that the pharmacokinetic parameters can be calculated from salivary concentration
(noninvasive method) provided if the correlation between plasma to saliva concentration exists.
Applications
1. Non invasive method determining drug concentration.
2. Various pharmacokinetic parameters can be calculated from salivary concentrations of drug at various time
points.
Questions
1. Why estimation of drug concentration has gained popularity?
2. Give the principle of microbiological assay of Ofloxacin.
Exercise
Estimate the salivary concentration of Theophylline.
SECTION 7
Bioavailability and Bioequivalence
Experiment 32
Bioequivalence testing of drug using salivary samples
Aim
To study bioequivalence of Paracetamol tablets by means of salivary samples.
Learning objectives
1.To understand the concepts of bioavailability and bioequivalence.
2.To study the use of saliva in estimating bioavailability of different brands of Paracetamol.
3.To estimate various pharmacokinetic parameters of Paracetamol through salivary samples.
Theory
The term “bioavailability” refers to the extent to which a drug/nutrient reaches its site of action or a
biological fluid such as blood that has access to its site of action. “Relative bioavailability” is assessed using a
reference product and “absolute bioavailability” is determined using an IV as 100%.
The term “bioequivalence” refers to pharmaceutically equivalent drug products where the
rates/extents of bioavailability of the actives are not significantly different under suitable test conditions. In
other words, this is a comparison of two or more products with respect to their bioavailability.
Bio-equivalent means that one brand or dosage form of a drug or supplement is equivalent to a
reference brand or dosage form of the same drug or supplement in terms of various bioavailability parameters
measured via in vivo testing in human subjects.
Bio-equivalence cannot be claimed based on in vitro testing only or on the basis of animal studies only.
Bio-equivalence of human drugs must be determined in humans via established measures of bioavailability.
Likewise, animal drugs must be tested for bio-equivalence in the animal species for which the drug in intended.
Once bio-equivalence has been established via bioavailability testing in a statistically significant
manner subsequent batches of the same product are deemed bio-equivalent based on in vitro measures such as
drug dissolution.
There are two types of bioavailability; absolute and relative.
Absolute bioavailability
Absolute bioavailability is assessed by comparing the values of (AUC)¥0 and/or cumulative mass of
drug excreted in the urine (Au), obtained following the administration of a drug in an extravascular dosage form
and an equal dose of the same drug intravenously (intravenous bolus).
From plasma data, it can be calculated by following equation
( AUC0¥ ) oral DoseIV
F= ´
( AUC0¥ ) IV Doseoral
...1
151
From urinary data, it can be calculated by following equation

( Au¥ ) oral DoseIV
F= ´
( Au¥ ) IV Doseoral ...2
Relative bioavailability
The ratio comparative (relative) bioavailability is assessed by comparing the bioavailability
parameters derived from plasma drug concentration–time plot data and/or urinary excretion data following the
administration of a drug in two different dosage forms (i.e. tablet and syrup, capsule and suspension, etc.) and/or
two different extravascular routes of administration. It also compares a generic formulation with a standard
formulation of the same dosage form of the same drug.
From plasma data, it can be calculated by following equation
( AUC0¥ ) test Dosestd
Fr = ´
( AUC0¥ ) std Dosetest ...3
From urinary data, it can be calculated by following equation
( A¥ ) Dosestd
Fr = u¥ test ´
( Au ) std Dosetest ...4
Paracetamol (N-acetyl-p-aminophenol) has been in use as analgesic and antipyretic drug over 50
years. It is used for an effective medication for the relief of pain and fever in adults and children. It is rapidly
absorbed and has a elimination half life of around 2 h. Serum concentrations between 10 to 20 mg/ml are
generally considered to be therapeutically effective, while >150 mg/ml may produce hepatic necrosis.
According to current Biopharmaceutical Classification System (BCS) criteria, Paracetamol is a BCS
class III compound. Differences in the composition of Paracetamol formulation show differences in the rate of
absorption, as in the case of tablets containing high amount of sodium bicarbonate which increases absorption
of Paracetamol by an effect on gastric emptying. It is suggested that these differences are due to differences in
disintegration and/or gastric emptying rates.
Pharmacokinetics of Paracetamol in human is found to be affected by formulation. The present
experiment examines the relative bioavailability of one generic formulation of Paracetamol tablets in
comparison to the standard brand. Assessment of the bioequivalence of one locally manufactured tablet brand is
undertaken with reference to standard tablet brand by generating in vivo data from saliva concentration of
twelve volunteers. The use of saliva concentration data for bioavailability assessment of Paracetamol is
considered feasible since the ease of collection and analysis of saliva samples besides the good correlation
between saliva and plasma concentration of Paracetamol. The spectrophotometric method is applied for the
determination and assessment of the bioavailability of Paracetamol using saliva samples.
Principle
The spectrophotometric method is based on the reduction of iron (III) by p-aminophenol which is
formed due to the hydrolysis of Paracetamol. A Prussian blue color is formed due to the reaction of iron (II) with
potassium ferricyanide, whose intensity is proportional to the concentration of Paracetamol. P-aminophenol is a
reducing agent due to the presence of phenolic hydroxyl and aromatic amine groups.
Absorbance of colored complex can be estimated at lmax 700 nm and hence the estimation of
Section 7 Bioavailability and Bioequivalence 153
Paracetamol in saliva can be done.

Figure 1: Oxidation and hydrolysis of Paracetamol in spectrophotometric method
NHCOCH3 NH2 O
heat -3H+
+ 3Fe(III) + 3Fe(II)
Hcl -3e
OH OH O
Paracetamol p-aminophenol Benzoquinone
Requirements
Glassware: volumetric flasks, test tubes, micropipettes, etc.
Chemicals: Pure Paracetamol pure drug, different brands of Paracetamol (one standard & one generic), Na2SO4,
diethyl ether, ferric sulfate, potassium ferricyanide, HCl and distilled water.
Instruments: UV spectrophotometer.
Procedure
1. Preparation of standard stock solution: Prepare standard stock solution of Paracetamol (100 m g/ml, Stock I).
From stock solution I, pipette out 2.5 ml into 100 ml volumetric flask and adjust volume with water to get
concentration of 2.5 mg/ml.
2.Preparation of working solution: Transfer 0.25 ml of drug free saliva into 10 test tubes using micropipette.
From stock solution II, pipette out 0, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 1.2, 1.6 and 2 ml into test tubes. Adjust volume
with water to 2.5 ml so as to get concentration in the range of 0-2 µg/ml. The final concentrations of Paracetamol
in each saliva sample are 0, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.6 and 2 µg/ml.
3. Extraction of samples: Transfer 0.25 ml of working solution into test tube. Add 1.0 g of Na2SO4 and 10 ml of
diethyl ether into it and mix thoroughly. Separate diethyl ether layer and evaporate to dryness. Reconstitute
residue in 2.5 ml distilled water.
4. Color formation: Take 2.5 ml of working solution in test tube and add 0.5 ml of 1.0 M HCl solution followed
by 1ml of ferric sulfate. Heat the mixture on boiling water bath for 10 min and cool it. Add 1 ml of potassium
ferricyanide and if required dilute with water to make volume 5 ml. Keep solutions for 24 min and measure
absorbance.
5. Measurement of absorbance: Measure absorbance at lmax 700 nm using UV Visible spectrophotometer. Plot
the graph of absorbance of Paracetamol against concentration in MS Excel and determine slope and intercept.
2. Study design
1.Prepare study protocol and take approval from IEC.
2. Take informed consent from all participants involved in study.
3. Select 12 healthy volunteers and make two groups each comprising six subjects.
4. After an overnight fast, give 2 tablets (500 mg x 2) of Paracetamol brand A (standard) orally along with 150
ml of water to group I. Similarly give brand B (generic) of Paracetamol to group II.

5. Rinse the mouth promptly with another 100 ml of water and swallow it.
6. Withheld food for a further period of 4 h to ensure complete absorption of the drug.
7. Collect 3 ml saliva samples at 0, 0.25, 0.5, 0.75, 1, 1, 1.25, 1.5 1.75, 2, 4, 6 and 8 h into a centrifuge tube.
Instruct volunteers to drink water regularly during study to keep saliva flow.
3. Salivary analysis of drug
1. Centrifuge the samples at about 5000 rpm for 5 min to remove mucous and particulate matter from saliva.
0
Separate the salivary supernatant and keep in freezer at 20 C till analysis.
2. Transfer 0.25 ml of working solution into test tube. Add 1.0 g of Na2SO4 and 10 ml of diethyl ether into it and
mix thoroughly. Separate diethyl ether layer and evaporate to dryness. Reconstitute residue in 2.5 ml distilled
water.
3. Take 2.5 ml of working solution in test tube and add 0.5 ml of 1.0 M HCl solution followed by 1ml of ferric
sulfate. Heat the mixture on boiling water bath for 10 min and cool it. Add 1 ml of potassium ferricyanide and if
required dilute with water to make volume 5 ml. Keep solutions for 24 min and measure absorbance at lmax
700 nm using UV-Visible spectrophotometer.
4. Pharmacokinetic analysis:
The estimation of pharmacokinetic parameters is done as per given in calculations.
Observations
Table 1. Plotting of calibration curve
mg/ml
0.1
0.2
0.4
0.6
0.8
1
1.2
1.6
2
Slope
Intercept
Table 2. Salivary data for Group I, who have taken standard brand (A) of Paracetamol
Time Salivary concentration (mg/ml) Mean Conc SD
(h) A1 A2 A3 A4 A5 A6 (mg/ml)
0.25
0.5
0.75
1
1.25
1.5
1.75
2
4
6
8
Table 3. Salivary data for Group II, who have taken generic brand (B) of Paracetamol
Time Salivary concentration (mg/ml) Mean Conc SD
(h) A1 A2 A3 A4 A5 A6 (mg/ml)
0.25
0.5
0.75
1
1.25
1.5
1.75
2
4
6
8
Table 4. TotalAUC using the Trapezoidal rule for standard brand of Paracetamol (A)
Time Mean Conc. Cn -1 + Cn
Segment tn-tn-1 AUC
(h) (mg /ml) 2
0 - - - - -
0.25 A 0.25
0.5 B 0.25
0.75 C 0.25
1 D 0.25
1.25 E 0.25
1.5 F 0.25
1.75 G 0.25
2 H 0.25
4 I 2
6 J 2
8 K 2
Table 5. TotalAUC using the Trapezoidal rule for generic brand of Paracetamol (B)
Time Mean Conc. Cn -1 + Cn
Segment tn-tn-1 AUC
(h) (mg /ml) 2
0 - - - - -
0.25 A 0.25
0.5 B 0.25
0.75 C 0.25
1 D 0.25
1.25 E 0.25
1.5 F 0.25
1.75 G 0.25
2 H 0.25
4 I 2
6 J 2
8 K 2
Calculations
1. Concentration of drug in saliva (mg/ml)
Y= m X + c
· Plot graph of salivary concentrations of Paracetamol versus time on a semi-logarithmic graph paper.
Calculate elimination rate constant by using slope of terminal portion of line.
log C 2 - log C1
Slope =
t 2 - t1
K = -( Slope ´ 2.303)

Calculate elimination half life by using following formula
0.693
t 1/ 2
=
K
4. Peak concentration and time to peak
Determine mean peak salivary concentration (Cmax) and mean time to peak salivary concentration
(tmax) by plotting salivary concentrations versus time on regular graph paper.
5.Area under curve (AUC)

DetermineAUC from 0 to 8 h by using trapezoidal rule.
DetermineAUC from 8 to infinity by using following equation:
Clast
AUC8-¥ =
K
TotalAUC is sum ofAUC from 0 to 8 h andAUC from 8 to infinity.
6. Relative bioavailability (Fr)
Determine relative bioavailability by using following formula:
AUCTest
% Fr = ´100
AUCStd
Results
1. The pharmacokinetic parameters estimated for both brands are summarized below:
Pharmacokinetic parameter Standard Brand (A) Generic Brand
(B)
Elimination rate constant (h-1)
Elimination half life (h)
Mean maximum salivary concentration (mg/ml)
Mean time to peak saliva concentration (h)
AUC0-8 mg h/ml
Total AUC
2. Relative bioavailability for generic Paracetamol tablet is found to be ________.
Conclusion
It can be concluded from this experiment that the generic brand (B) is bioequivalent / not bioequivalent
to standard brand (A) of Paracetamol.
Applications
Bioavailability and bioequivalence studies can provide useful information regarding the drug such as:
1.Bioavailability studies provide an estimate of the fraction of the orally administered dose that is absorbed into
the systemic circulation when compared to the bioavailability for a solution, suspension, or intravenous dosage
form that is completely available.
2.Bioavailability studies provide other useful information that is important to establish dosage regimens and to
support drug labeling, such as distribution and elimination characteristics of the drug.
3.Bioavailability studies provides indirect information regarding the presystemic and systemic metabolism of
the drug and the role of transporters such as p-glycoproteins.
4.Bioavailability studies are designed to study the effect of food and other nutrients on the absorption of the
drug substance.
5.Bioavailability studies provide information regarding the performance of the formulation and subsequently
are a means to document product quality.

6. Bioequivalence studies provide a link between the pivotal and early clinical trial formulation, a link between
formulation used in the pivotal clinical trial, and the stability studies, the pivotal clinical trial and the to-be-
marketed drug product, and other comparisons as appropriate.
7. Bioequivalence studies are the basis for determination of the therapeutic equivalence between a
pharmaceutically equivalent generic drug product and a corresponding reference listed drug.
8. Bioequivalence studies provide information on product quality and performance when there are changes in
components, compositions, and method of manufacture after approval of the drug product.
Questions
1. Give the significance of bioequivalence studies.
2. What is the difference between relative and absolute bioavailability.
3.Comparison of plasma concentrations (mg/ml) of antibiotic taken from 10 humans (average weight 70 kg), as
related to dosage form and time are given below:
Time IV solution Oral solution Oral tablet Oral capsule

(h) (2 mg/kg) (10 mg/kg) (10 mg/kg) (10 mg/kg)
0.5 5.94 23.4 13.2 18.7
1.0 5.30 26.6 18.0 21.3
1.5 4.72 25.2 19.0 20.1
2.0 4.21 22.8 18.3 18.2
3.0 3.34 18.2 15.4 14.6
4.0 2.66 14.5 12.5 11.6
6.0 1.68 9.14 7.92 7.31
8.0 1.06 5.77 5.00 4.61
10.0 0.67 3.64 3.16 2.91
12.0 0.42 2.30 1.99 1.83
AUC 29 145 116 116
a. Which of the four drug products would be preferred as a reference standard for the determination of relative
bioavailability? Why?
b. From which oral drug product is absorbed more rapidly?
c. What is the absolute bioavailability of the drug from the oral solution?
d. What is the relative bioavailability of the drug from the oral tablet compared to the reference standard?
e. From the data determine volume of distribution, elimination half life, elimination rate constant and total
clearance.
Exercise
Perform the bioequivalence testing of various brands of Ofloxacin by salivary means.
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Laboratory Manual of Biopharmaceutics and

Chapter · January 2010

Remeth Dias Kailas K Mali

SEE PROFILE SEE PROFILE

Available from: Kailas K Mali

Mrs. Anita R. Dias

© 2010 Trinity Publishing House

Disclaimer: As new information becomes available, changes become necessary. The

A Amount of drug in body at time t

Physicochemical properties of drugs and dosage forms

Drug pKa and gastrointestinal pH

[HA- organic] = PC [HA- aqueous] …6

Which will be best absorbed from the stomach ( pH = 2)?

Drug in systemic circulation

Degradation Complexation Gut wall metabolism Liver metabolism

Figure 1. Dissolution and absorption process of tablet

Table 2. Parameters of Benzoic acid stick

Sr. Diameter (D) Length (L) Surface area (S)

Table 3. Observation table for stickA

5. Calculation of dissolution rate constant (K)

versus time (t) on graph paper. 0.3

K' = Slope x 2.303 0.2

K=D/h= K'/S 0.15

Where, V = total volume of solution and dc = concentration increment.

In[G(t+dt) - G(t)] =A-kt where, slope = -K

dissolution rate constant at a given 4.00

Figure 1. Design of apparatus

Time (t/min) Conductivity G(t)/mS

3. Dissolution followed by partitioning into a lipid phase.

Tablet Non disintegrating

Figure 1: Schematic diagram of the dissolution process

A. Parameters set for plotting of calibration curve

B. Parameters set for dissolution studies

Table 2. In-vitro dissolution of Paracetamol

Time Percent cumulative drug release

B. Procedure for dissolution

A. Parameters set for plotting of calibration curve

Table 2. In vitro dissolution of Domperidone

Time Percent cumulative drug release

c) Equipments: USP dissolution apparatus type II, Balance and UV spectrophotometer.

A. Parameters set for plotting of calibration curve

Table 2. Calibration curve of Diclofenac sodium (phosphate buffer)

Table 3. Drug release profile of Diclofenac sodium tablet

In a similar manner, the solubility of a weak base is given by:

For weak acids

1. Saturation solubility of Benzoic acid at pH 4.0 = ____________g/l.

2. Saturation solubility of Benzoic acid at pH 5.5 = ____________g/l.

2. Calculation for surface area of BAstick (S)

Table 3. Cumulative percent drug release of Brand B

Figure 1. Design of in vitro continuous dissolution-absorption system.

to 5.0 with 10 M potassium hydroxide.

Time Concentration Amount of drug released

Table 2.Amount ofAcyclovir diffused

3. Surface area (A) of chicken intestine (cm2)

3. In vitro release and permeation

Figure 1. Design of diffusion assembly

A. Parameters set for plotting of calibration curve

Table 2. Ex vivo absorption of formulation F1 through rat skin