ACETAZOLAMIDE

Jagdish Parasrampuria

Abbott Laboratories

Pharmaceutical R&D

Abbott Park, Illinois 60064

ANALYTICAL PROFILES OF DRUG SUBSTANCES Copyright 0 1993 by Academic Press, Inc.

AND EXCIPIENTS-VOLUME 22 1 All rights of reproduction in any form reS0Ned.
2 JAGDISH PARASRAMPURIA

1. I NTRODUCTION
1.1 History
1.2 Therapeutic Category
1.3 Chemistry and Structure-Activity
Re1 ati onshi p
2. DESCRIPTION
2.1 Name, Formula, and Molecular Weight
2.2 Appearance, Color, Odor, and Taste
2.3 Pharmaceutical Dosage Forms
3. PHYSICAL PROPERTIES
3.1 Dissociation Constant (pKa)
3.2 U1 traviolet Spectra
3.3 Solubility
3.3.1 pH-Sol ubil i ty Profi 1 e
3.3.2 Solubility in Various Commonly Used
Pharmaceutical Sol vents
3 -3-3 Sol ubi 1 i zation through Cosol vency
3.4 Polymorphism
3.5 Melting Point
3.6 Differential Scanning Calorimetry
3.7 Infrared Spectra
3.8 X-Ray Diffraction
3.9 Nuclear Magnetic Resonance Spectra
4. METHODS OF ANALYSIS
4.1 Identification Tests
4.2 U1 traviolet Spectroscopy
4.3 Polarography
4.4 Nuclear Magnetic Resonance Spectroscopy
4.5 Chromatography
4.5.1 High-Performance Liquid
Chromatography
4.5.2 Gas Liquid Chromatography
5. STABILITY
5.1 pH-Rate Profile
5.2 Stability in Intravenous Admixtures
5.3 Effect of Temperature
5.4 Effect of Buffer Species, Buffer
Concentration, and Ionic Strength
5.5 Stability in Various Solvents
6. BIOPHARMACEUTICS
6.1 Pharmacokinetics and Metabolism
6.2 Bi oavai 1 abi 1 i ty
7. REFERENCES
 ACETAZOLAMIDE 3

1. INTRODUCTION

1.1 History
I n t h e e a r l y 1930s, Roughton (1) discovered t h e
existence i n erythrocytes of an enzyme which promotes t h e
hydration o f carbon d i o x i d e and dehydration o f carbonic
acid. The enzyme was found t o be carbonic anhydrase. Since
then, t h e presence of carbonic anhydrase has been
demonstrated i n p r a c t i c a l l y every physiological b a r r i e r
where i o n exchange occurs, e.g., t h e kidney, sweat glands,
s a l i v a r y glands, b r a i n and spinal cord t i s s u e , choroid
plexus, p a r i e t a l t i s s u e o f g a s t r i c mucosa, pancreatic
tissue, and t h e eye. I n the eye, carbonic anhydrase has
been found i n t h e lens, r e t i n a , c i l i a r y body, i r i s , and
v i t r e o u s body.
According t o the c l assf cal concept developed between
1930 and 1950, carbonic anhydrase was thought t o have a
s i n g l e chemical r o l e , t h a t i s , c a t a l y s i s of t h e r a t e o f
a t t a i n i n g e q u i l i b r i u m i n t h e primary r e v e r s i b l e r e a c t i o n :

d
H20 + CO2 CARBONIC ANHYDRASE

A secondary, p r a c t c a l l y instantaneous e q u i l i b r i u m

L
H2CO3 H+ + ~ ~ 0 3 -
7
i s n o t c a t a l wed. Thus t h e enzyme-sensitive r e a c t i o n i s t h e
r a t e - c o n t r o l i i n g step i n t h e o v e r a l l process (2).
I n 1950, a series o f unsubstituted h e t e r o c y c l i c
sulfonamides were synthesized. Among these was
acetazol ami de (3 *4), a compound found t o possess speci f ic
carbonic anhydrase-inhibiting a c t i v i t y and a low incidence
o f acute and chronic t o x i c i t y . Thus, acetazolamide was
f i r s t used by physicians i n 1953 as a d i u r e t i c .

1.2 Therapeutic Category

Acetazolamide i s a potent and r e v e r s i b l e carbonic
anhydrase i n h i b i t o r , e f f e c t i v e i n t h e c o n t r o l o f f l u i d
secretion, e.g., glaucomas ( 5 ) , i n t h e treatment of c e r t a i n
convulsive disorders, e.g., epilepsies (6), and i n t h e
promotion o f d i u r e s i s i n instances o f abnormal f l u i d
r e t e n t i o n (7).
4 JAGDISH PARASRAMPURIA

Acetazolamide i s indicated f o r centrencephal i c

e p i l e p s i e s ( p e t i t malt unlocalized seizures), chronic simple
(open angl e) glaucoma, secondary glaucoma, and
preoperatively i n acute angle closure glaucoma where delay
o f surgery i s desired i n order t o lower i n t r a o c u l a r pressure
( 8 ) . Acetazolamide i s used as an adjuvant i n t h e treatment
o f c e r t a i n dysfunctions of t h e central nervous system i n
which cerebral spinal f l u i d pressure i s increased, e.g.,
hydrocephalus (9-13). Acetazolamide i s a l s o used f o r t h e
f o l l o w i n g i n d i c a t i o n s n o t included i n USA product l a b e l i n g :
i n t h e treatment o f hypokalemic and hyperkalemic forms o f
f a m i l i a l p e r i o d i c p a r a l y s i s (14,15); as prophylaxis f o r and
treatment o f a1t i t u d e (mountain) sickness (16-19); t o f o r c e
a l k a l i n e d i u r e s i s t o increase e l i m i n a t i o n o f weakly a c i d i c
medications (20); and as a a n t i u r o l i t h i c t o a l k a l i n i z e t h e
u r i n e t o prevent c y s t i n e and u r i c a c i d renal stone formation
(21).
Acetazolamide has a l s o been used t o prevent metabolic
a l k a l o s i s (22) and as a d i u r e t i c i n t h e treatment o f edema
due t o congestive heart f a i l u r e o r due t o drugs. However,
i t has been replaced by newer d i u r e t i c s f o r these
indications.

1.3 Chemistry and Structure-Activity Relationship

Acetazolamide i s n o t a mercurial d i u r e t i c . Rather, i t
i s a nonbacteriostatic sulfonamide possessing a chemical
s t r u c t u r e and pharmacol og ical a c t i v i t y d ist in c t l y d if f erent
from t h e b a c t e r i o s t a t i c sulfonamides. Among t h e enormous
number o f sulfonamides t h a t have been synthesized and
tested, acetazolamide has been studied most extensively as
an i n h i b i t o r o f carboni c anhydrase.
The most s t r i k i n g s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p i s
t h a t aromatic sulfonamides, unsubstituted on -S02NH2
nitrogen, are speci f ic carbonic anhyqrase i n h i b i t o r s . Thi s
i n h i b i t o r y e f f e c t i s l o s t when t h e N -(sulfonamide) n i t r o g e n
i s s u b s t i t u t e d (23).

2. DESCRIPTION

2.1 Name, Formula, and Molecular Weight

Acetazolamide i s 2-acetylamido-1,3,4-thiadiazole-5-
sul fonamide; N-( 5-Sul famoyl-l,3,4-thiadiazol e-Z-yl )
 ACETAZOLAMIDE S

acetamide with a molecular formula of CqH6Nq03S2 and

molecular weight o f 222.24. The structure of acetazolamide
is shown in Figure 1.

Figure 1: Structure o f Acetazolamide

2.2 Appearance, Color, Odor, and Taste

A fine, white-to-faintly-yellowish-white, odorless,
bitter crystalline powder. A 3.85% solution of
acetazolamide is iso-osmotic with serum (24).

2.3 Pharmaceutical Dosage Forms

Acetazolamide Tablets, USP: 125 and 250 mg; Diamox by
Lederle Laboratory, Division of American Cyanamid Co.;
Generic version by Bolar Pharmaceuticals, Danbury
Pharmaceuticals, Lannett Co., and Mutual Pharmaceutical Co.
(25) -
Acetazol ami de Extended-Re1ease Capsul es : 500 mg ; D i amox
Sequel s by Lederl e Laboratory, Division of Ameri can Cyanamid
co.
Sterile Acetazolamide Sodium, USP: 500 mg; Diamox by
Lederle Laboratory, Division of American Cyanamid Co.,
Generic version by Quad Pharmaceuticals (25).
6 JAGDISH PARASRAMF'URIA

3. PHYSICAL PROPERTIES

3.1 D i s s o c i a t i o n Constant

Acetazolamide i s a weak acid w i t h a d i s s o c i a t i o n

constant (pKa) value o f 7.2.

3.2 U l t r a v i o l e t Spectroscopy

The u l t r a v i o l e t absorbance o f acetazolamide scanned

from 200 t o 400 nm i s presented i n F i g u r e 2. The wavelength
o f maximum absorbance i s a t 265 nm (26).

3.3 Solubility

Acetazolamide i s very s l i g h t l y s o l u b l e i n water:

s l i g h t l y s o l u b l e i n alcohol and acetone; p r a c t i c a l l y
i n s o l u b l e i n carbon t e t r a c h l o r i d e , chloroform, and ether.
Dissolves i n s o l u t i o n o f a l k a l i hydroxides; s p a r i n g l y
s o l u b l e i n p r a c t i c a l l y b o i l i n g water (27).

3.3.1 pH-Sol ubi 1it y prof ile

The concentration o f acetazolamide i n saturated

s o l u t i o n s o f various pH values i s shown i n F i g u r e 3 (28).
The s o l u b i l i t y ranges from 0.8 t o 2.8 mg/mL between pH
values o f 1.7 and 8.2. The p H - s o l u b i l i t y p r o f i l e i n d i c a t e s
t h e s o l u b i l i t y between pH 4 and 7 i s approximately t h e same
(0.8-1 mg/mL). S o l u b i l i t y i s higher on t h e basic s i d e
because o f sodium s a l t formation. However, degradation
increases manyfold on t h e basic side, which precludes
measurement o f e q u i l i b r i u m s o l u b i l i t y above pH 8.2.

3.3.2 S o l u b i l i t y i n Various Commonly Used Pharmaceutical

Sol vents:
Table 1 shows t h e s o l u b i l i t y o f acetazolamide i n
various common1y used pharmaceutical solvents. The maximum
s o l u b i l i t y i s i n polyethylene g l y c o l 400 (PEG 400), w i t h a
value o f about 87.8 mg/mL. The s o l u b i l i t y i n propylene
g l y c o l , alcohol, g l y c e r i n , and water i s 7.4, 3.9, 3.6, and
0.7 mg/ml, r e s p e c t i v e l y (28).
 ACETAZOLAMIDE

Wavelength (nrn)

Figure 2: UV Spectra o f Acetazolamide

 JAGDISH PARASRAMPURIA

I I I I I i I I 1
1 2 3 4 5 6 7 8 9
PH

Figure 3: pH-Sol ubi 1 ity Prof i1 e of Acetazolamide

 ACETAZOLAMIDE 9

Table 1: S o l u b i l i t y o f Acetazolamide i n Various Solvents

a t 25'C

Sol vent Sol ubi 1 it y (mg/mL)

Pol y e t h y l ene G1y c o l 87.81 (3.45)*

Propyl ene G1ycol 7.44 (1.20)

Ethanol 3.93 (0.11)

Glycerin 3.65 (0.09)

Water 0.72 (0.05)

*Mean
~

( + std. dev.)

3.3.3 S o l u b i l i z a t i o n through Cosolvency

F i g u r e 4 ( A and 6) i l l u s t r a t e s t h e e f f e c t o f g l y c e r i n ,
1,Z-propylene g l y c o l , PEG 300, PEG 400, polypropylene g l y c o l
420, e t h y l alcohol, dimethylacetamide, and dimethylsulpoxide
a l l mixed w i t h water a t d i f f e r e n t r a t i o s . There i s an
increase i n aqueous s o l ubi 1it y o f acetazol ami de upon t h e
a d d i t i o n o f cosolvents. A s o l u b i l i t y - e n h a n c i n g e f f e c t i s
dependent on t h e t y p e and volume f r a c t i o n o f cosolvent used.
G l y c e r i n showed l e a s t e f f e c t , w h i l e dimethylsulphoxide i s
t h e most e f f i c i e n t cosolvent f o r i n c r e a s i n g t h e s o l u b i l i t y
(29).
According t o t h e equation derived by Yalkowski e t al.,
(30) l o g a r i t h m s o l u b i l i t y i s p l o t t e d against volume f r a c t i o n
o f t h e cosolvent. As depicted i n F i g u r e 5, t h e r e i s a
1 inear r e 1a t i onshi p between 1og sol ubi 1 it y o f acetazol amide
and volume f r a c t i o n o f g l y c e r i n , 1,2-propylene g l y c o l ,
methyl alcohol, e t h y l alcohol, PEG 300, PEG 400, and
dioxane. The r e l a t i v e s o l u b i l i z i n g power o f these s o l v e n t s
can be obtained from t h e slope o f t h e l i n e as l i s t e d i n
Table 2 (29).
10 JAGDISH PARASRAMPURIA

0' I

lo 20 30 40 50
Concentration (% v/v)

Figure 4A: Sol ubi 1 i t y o f Acetazolamide in Some Mixed

Solvent Systems. a--17 Polypropylene Glycol
420; A-A PEG 400; m.- PEG 300; 04 1,2
Propyl ene G1 ycol ; O-. G1 ycerol ;
 ACETAZOLAMIDE

.---.
Figure 46: Solubility o f Acetazolamide in Some Mixed
Solvent Systems. 0---0Dimethyl Sulfoxide;
A---A Dimethyl Acetamide; 0---o Dioxane;
Methanol ; A---A Ethanol
12 JAGDISH PARASRAMPURIA

0
2.4

2.2 -
2.0 -

1.8 -

.-
-
5
.- 1.4-
a
3

*
0.0 0.1 0.2 0.3 0.4 0.5 Fc

F i g u r e 5: Log S o l u b i l i t y versus Volume F r a c t i o n o f

Cosolvent f o r Acetazolamide. w
Polypropylene Glycol 420; A-A
PEG 300; 04
PEG 400;
1,2 Propylene Glycol ; .-. m- .m

Glycerol ; (7---o Oimethyl Sulfoxide; A---A

Oimethyl Acetamide; 0---o Oioxane;
Methanol ; A---A Ethanol
.---.
 ACETAZOLAMIDE

Table 2

Solubilizing Power of Some Binary Solvents

Toward Acetazolamide

Cosol vent Solubilizing Power

G1 ycerol 5.294 x

1,P-Propylene Glyco? 8.437 x

Methyl A1 coho1 10.00 x 10-3
Ethyl Alcohol 12.00 x 10-3
PEG 300 16.50 x
PEG 400 20.60 x
Oioxane 26.70 x
14 JAGDISH PARASRAMPURIA

3.4 Polymorphism
Acetazolamide has two polymorphic forms (Forms A and
B). The solubility and dissolution rate of Form B is about
1.1 times higher those of Form A (31). The transition
temperature obtained by solubility measurement is 78"C, and
the heats of transition (AHtrans) calculated by solubility
measurement and by differential scanning calorimetry are 2.6
and 1.7 kJ.mol-l, respectively. The free energy change
( ~ G 2 5 0 ~between
) the two polymorphic forms is 357 J. mol-l,
which is a relatively small value. It is therefore
presumed, following Aguiar and Zelmer, that acetazolamide
polymorphic forms do not significantly affect
bioavailability. The kinetics of isothermal transition from
Form A to Form B at high temperature follows the mechanism
of random nucleation with first-order kinetics. The
activation energy for this tran ition as derived from
Arrhenius plots is 246 kJ. mol-I. The results from the
scanning electron microscope indicate that the crystal shape
of acetazolamide during isothermal transition from Form A to
Form B does not change significantly (31).

3.4 Melting Point

The me1 ting point range of acetazol amide is 258-263OC,
accompanied by decomposition.

3.5 Differential Scanning Calorimetry

Differential Scanning Calorimetry (DSC) curves of
acetazolamide's two polymorphic forms are shown in Figure 6
(31). The DSC curve of Form A exhibits two endothermic
peaks: one at 205OC corresponding to the transition from
Form A to Form 3, and the other at 263OC attributable to the
melting point accompanied by decomposition. Form B gives
only one endothermic peak at 263OC corresponding to the
melting point which is accompanied by decomposition as
i ndi cated by the exothermic peak.

3.6 Infrared Spectra

Infrared spectra of acetazol amide's two polymorphic
forms are shown in Figure 7 (31). The spectrum of Form A is
different from that o f Form B. In particular, Form A shows
 ACETAZOLAMIDE IS

3.4 Melting Point

The me1t i ng p o i n t range o f acetazol ami de is 258-263OC,
accompanied by decomposition.

3.5 Differential Scanning Calorimetry

D i f f e r e n t i a l Scanning Calorimetry (DSC) curves o f
acetazolamide's two polymorphic forms a r e shown i n F i g u r e 6
(31). The DSC curve o f Form A e x h i b i t s two endothermic
peaks: one a t 205OC corresponding t o t h e t r a n s i t i o n from
Form A t o Form B, and t h e o t h e r a t 263OC a t t r i b u t a b l e t o t h e
m e l t i n g p o i n t accompanied by decomposition. Form B g i v e s
o n l y one endothermic peak a t 263OC corresponding t o t h e
m e l t i n g p o i n t which i s accompanied by decomposition as
i n d i c a t e d by t h e exothermic peak.

form A

100 150 200 250

Temperature ("c

F i g u r e 6: OSC-TG Curves o f Acetazolamide Polymorphic

Forms. -, DSC Curves; --- TG Curves;
S e n s i t i v ' t y Range 41.8 mJ.s-l; Heating Rate,
5"C.min -1
 16 JAGDISH PARASRAMPURIA

3.6 Infrared Spectra

Infrared spectra of acetazolamide's two polymorphic
forms are shown i n Figure 7 (31). The spectrum of Form A is
different from that of Form 6. In particular,
characteristic absorption peaks in the 1100-900
and Form B gives a specific peak at about 940 cm-

Form A
n

1 1 I I I I
4000 3000 2000 1500 1000 650

Wave number (cm-l)

Figure 7: Infrared spectra of Acetazolamide Polymorphic

Forms (in N u j o l )
 ACETAZOLAMIDE 17

3.7 X-Ray Diffraction

The X-ray powder diffraction patterns of the two

polymorphic forms a r e shown i n Figure 8 (31). The
d i f f r a c t i o n pattern of Form A is different from t h a t of Form
B. Very h i g h peaks a t 9.9', 24.8' and 29.4" were observed
i n the diffraction pattern o f Form A ; these were not found
i n the diffraction pattern of Form B. On the other hand,
Form B gave the h i g h e s t diffraction pattern a t 13.7" w i t h
c h a r a c t e r i s t i c peaks a t 19.6" 22.3" 26.0' and 26.9".

Form A

5 10 '0

-
Form B

5 10
28 degrees

Figure 8: X-Ray Diffraction Pattern of Two Polymorphic

Forms o f Acetazol ami de
18 JAGDISH PARASRAMPURIA

3.8 Nuclear Uagnetic Resonance Spectra

The NMR spectra of acetazolamide contains broad peaks
centered a t 790 cps ( 6 13.17, 1 H) and 514 cps ( 6 8.57, 2H)
a r i s i n g from the N-protons of the carboxamide and
sulfonamides, respectively (32).
The 15N NMR spectra of acetazolamide was measured in
hexadeuteriodimethyl sufoxide. I n i t i a l l y protondecoupled ad
protoncoupled spectra of the nitrogens bearing hydrogens
were measured. Then, tris-
(acetylacetonate)Cr(111) [Cr(acac) was added t o sol ution
1
because the Cr(acac)3 considerab y shortens the relaxation
tim and thus enables f a s t e r pulse r e p e t i t i o n . The changes
of f S N chemical s h i f t s induced by the addition of t h i s
laxation reagent i s small (usually 4 ppm able 3 l i s t s
“N chemical s h i f t s and coupling constants ;(’ 15NH) of
acetazol ami de.

Table 3: 1 5 N hemical s h i f t s and coupling constants

J (“NH) in acetazol amide

~ ~~~

6 15N Chemical S h i f t ‘J(15NH) Coupling Constant

-241.2 (CONH)
[-241.4b (CONH)]
-282.4 (S02NH2)
-282.6b (SOzNHz)
-15. 7b [ N=C ( S ) -SO21
-58.3b [N=C(S)-NH]

aCoupl ing constants were observed e i t h e r f o r reasons of

small intermolecular proton exchange or small sol ubi 1 i t y of
sampl es
bmeasured a f t e r the addition o f 10 rng/mL Cr(acac)3, as
re1 axati on agent
 ACETAZOLAMIDE 19

4. UEMODS OF ANALYSIS

Several assay methods f o r acetazolamide have been

reported, such as u l t r a v i o l e t absorption spectroscopy (33-
35), p o l oragraphy (34), e l e c t r o n capture gas-1 iq u i d
chromatography (36), high-performance 1i q u i d chromatography
(37-44), amperometric determination using a s e s s i l e mercury-
drop d e t e c t o r (45), and nuclear magnetic resonance (32).

4.1 Identification Tests

A) The i n f r a r e d absorption spectrum of a potassium
bromide d i s p e r s i o n e x h i b i t s maxima o n l y a t t h e same
wavelength as t h a t o f a s i m i l a r p r e p a r a t i o n o f USP
acetazolamide reference standard. I f a d i f f e r e n c e appears,
p o r t i o n s o f both t h e t e s t specimen and t h e Reference
Standard should be dissolved i n methanol, t h e s o l u t i o n s
evaporated t o dryness, and t h e t e s t on residues repeated.

B ) When d i ssol ved acetazol ami de is m i xed w i t h

hydroxylarnine hydrochloride and c u p r i c s u l f a t e and heated i n
a steam bath f o r 5 minutes, a c l e a r b r i g h t y e l l o w s o l u t i o n
i s produced. No heavy p r e c i p i t a t e o r dark brown c o l o r
r e s u l t s a f t e r t h e mixing o r heating.

4.2 Ultraviolet Spectroscopy

The USP-NF methods f o r t h e q u a n t i t a t i o n o f
acetazolamide sodium i n i n j e c t i o n i s based on UV
spectroscopy i n HC1 solution (34). Since t h e h y d r o l y s i s
product 5-arnino-1,3,4-thiadiazole-2-sulfonamide a l s o absorbs
l i g h t a t same wavelength (265 nrn), t h e USP-NF method
t h e r e f o r e cannot be s t a b i l i t y - i n d i c a t i n g .

I n t e r f e r i n g absorbance due t o e x c i p i e n t s i n
pharmaceutical formulations a f f e c t s t h e accuracy o f t h e
spectrophotometric method. A p p l i c a t i o n o f pH-induced
s p e c t r a l changes f o r acetazol ami de, however, nu1 1 i f ies t h e
i r r e l e v a n t absorbance i f we assume t h a t t h e e x c i p i e n t i s n o t
a f f e c t e d by these pH changes. As seen i n F i g u r e 9, i n 0.1 N
NaOH acetazol ami de e x h i b i t s a bathochromi c s h i f t together
w i t h a hyperchromic e f f e c t . The bathochromic s h i f t i s
a t t r i b u t e d t o t h e formation o f SQ.NH-, which i s conjugated
with the t h i a d i a z o l e r i n g (33).
20 JAGDISH PARASRAMPURIA

fi''\\,,
I-
,t-\
I \.
0 5 . I '\\

Ob .
0 3 .

a
a2
0Ob
a2 '
.-
13 '. ;
0
' L' '. u \\

a1

t
b
211 250 270
1

290 30
\

330 A

Figure 9: UV Spectrum of Acetazolamide i n 0.1 N H2SO4 -,

and 0.1 N NaOH ---

4.3 Poloragraphy
Acetazolamide can be assayed i n a polarographic c e l l
t h a t i s immersed i n a water bath regulated a t 25 k 0.5"C,
and de-aerated by b u b b l i n g nitrogen through the solution f o r
10 minutes. A mercury-drop electrode should b e immersed i n
a s u i t a b l e polarograph, and the polarogram recorded from -
0.20 volts t o -0.75 volts, using a saturated calomel
electrode (SCE) as the reference electrode i n 0.1 M HC1.
Diffusion current i s recorded a t -0.70 volts.
A method of determining acetazolamide by reductive
amperometry w i t h flow injection using a s e s s i l e mercury-drop
electrode i s reported ( 4 5 4 Acetazolamide was determined i n
the range of 10-70 mcg m l a t -0.85 V vs. SCE i n 0.1 M HC1.
4.4 Nuclear Magnetic Resonance Spectroscopy
Acetazolamide can be assayed using an NMR spectrometer
equipped w i t h a variable temperature probe having a six-turn
insert. Spectra were scanned a t a probe temperature of
42'C. A 25% ammonia solution i s selected as the solvent
and t-butanol is used as the internal standard (32).
 ACETAZOLAMLDE ?I

4.5 Chromatography
4.5.1 High-performance Liquid Chromatography
Several systems have been developed for acetazolamide.
Table 4 describes assays published in the literature.

Table 4: HPLC Conditions for Acetazolamide Assay

Internal Std. Col umn Conc. Mobile Phase Ref.

( PLghL)

Sul famerazine C18 15 12% MeoH, 2% ACN, 37 t

0.02 M Phosphate 3a
Chl orothi azide C18 1-20 6% ACN, 0.05 M 39
Acetate, pH 4.5
Sul fadiazine C 18 1-50 3% ACN, 2% MeOH, 40
pH 4.0
Propazolamide Porasil 1-50 9.7% Ethanol , 41
79.4% Di chloro-
methane, 1%HAC
Chl oroth azide Silica Gel 1-30 65% Hexane, 25% 42
Chloroform, 10%
MeOH, 0.25% HAC
Chl oroth azide U1 trasphere 0.05 10% ACN, 0.05 M 43
-20 Acetate, pH 4.5
2-Acetamido-4- cia 1-25 23.8% MeOH, 44
methyl-5-thiadiazole 0.02 M Ammonium
sul fonamide Phosphate
22 JAGDISH PARASRAMPURIA

5, STABILITY

5.1 pH-Rate Profile

The decomposition o f acetazolamide follows a first-
order kinetics (Figure 10). The pH-rate profile curve
(Figure 11) is V-shaped, which indicates specific acid-base
catalysis. The slopes o f the pH-rate profile curve for the
acidic and alkaline solutions is -1.72 and 1.15,
respectively. The pH of maximum stability i s 4 (46, 47).

Time (days)

Figure 10: First-order plots o f acetazolamide at different

pH values. (0)pH 5.46; ( 0 ) pH 6.06; (A) , pH
6.86; (*) pH 1.68; (A) pH 8.17
 ACETAZOLAMIDE 23

-3 -
-4 -

-5 -

-6 -
Y
C
- -7 -

-8 -

d
0
-1-g/ 1 2 3 4 5 6 7 8 9

PH

Figure 11: The pH-rate p r o f i l e curve of acetazolamide

5.2 Stabi 1 ity in Intravenous Admixtures

Acetazolamide sodium s o l u t i o n s i n 5% dextrose and 0.9%
sodium c h l o r i d e i n j e c t i o n s a r e s t a b l e f o r 5 days a t 25°C
w i t h a l o s s o f potency of l e s s than 7.2%. A t 5 ' C t h e l o s s
i n potency i n both t h e s o l u t i o n s i s 6% a f t e r 44 days o f
storage. A t -10°C t h e l o s s i n potency i s l e s s than 3% i n
both s o l u t i o n s ( 4 8 ) . The s o l u t i o n s remain c l e a r throughout
but a s l i g h t change i n pH occurs. Results of frozen samples
thawed i n a microwave oven a r e s i m i l a r t o those samples
thawed using tap water ( 4 8 ) . Thawing i n a microwave can be
completed i n l e s s than two minutes.

5.3 Effect of Temperature

The decomposi t i on of acetazol ami de f o l 1ows f ir s t - o r d e r
k i n e t i c s a t higher temperature. The higher-temperature data
24 JAGDISH PARASRAMPURIA

f o l l o w t h e Arrhenius equation a t a l l three d i f f e r e n t pH

values o f 3.1, 5.85, and 6.64. The energy o f a c t i v a t i o n , Ea
values, are d i r e c t l y r e l a t e d t o pH values and t h e f o l l o w i n g
equation i s reported (49) using t h e experimental data
I n Ea = I n K + 0.083 pH Eq. 1

where k i s a constant w i t h a value o f 11.94 kcal/mole and

0.083 i s t h e slope o f t h e s t r a i g h t l i n e . Using Equation 1,
t h e Ea value a t pH 4 (pH value o f maximum s t a b i l i t y ) i s
calculated t o be 16.6 kcal/mole. The other a c t i v a t i o n
parameters a r e l i s t e d i n Table 5. The p o s i t i v e enthalpy
value determined using Equation 1 i n d i c a t e s endothermic
r e a c t i o n . The increase i n enthalpy as pH values a r e
increased i n d i c a t e s higher heat contents o f t h e s o l u t i o n a t
higher pH values. The change i n t h e entropy values from
-46.77 cal/mole/deg a t pH 3.10 t o -20.75 cal/mole/deg a t pH
6.64 i n d i c a t e s t h a t l e s s energy i s a v a i l a b l e f o r work due t o
random motion and t h a t t h e r e i s greater d i s o r d e r l i n e s s o f
t h e molecules a t pH 3.10.

Table 5: A c t i v a t i o n Parameters f o r the Degradation o f

Acetazolamide i n Aqueous Solutions a t D i f f e r e n t
pH Values

PH Ea In A AG AH As
kcal /mol e kcal /mol e kcal /mol e cal /mol /deg

3.10 15.43 29.24 28.77 14.84 -46.77

5.85 19.35 26.04 28.03 18.76 -31.11
6.64 20.69 18.27 26.28 20.10 -20.75

5.4 Effect of Buffer Species, Buffer Concentration, and

Ionic Strength
The phosphate b u f f e r has very l i t t l e e f f e c t on t h e
kobs values o f acetazolamide a t pH 7.5 w i t h b u f f e r
concentrations between 0.05 M and 0.15 M. S i m i l a r r e s u l t s
 ACETAZOLAMIDE 25

a r e reported w i t h a c i t r a t e buffer a t pH 6.25. The kobs

values a r e very similar w i t h different ionic strengths.
T h i s indicates t h a t it is the unionized acetazolamide which
reacts w i t h e i t h e r H or OH-. Furthermore, the pH-rate
p r o f i l e i s a l s o a typical plot of a s p e c i f i c acid-base
c a t a l y s i s (47).
The hydrolysis of acetazolamide may b e represented as
f 01 1ows :

kobs = ko + kH (H+) + k0H (OH-) Eq. 2

where kobs i s the overall observed r a t e constant, and ko,
kH, k0H a r e r a t e constants f o r hydrolysis due t o solvent,
hydrogen ion concentration ( H ), and hydroxyl ion
concentration (OH-), respectively. Assuming the k o t o be
0.0001 per day (k value a t pH 4 where hydrolysis is a t i t s
m i n i m u m ) , and neglecting the effect of OH- a t pH 1.68, the
kH value i s estimated t o be 0.23 per day. Using the kobs
valuf of 0.0495 per day a t pH 12.5 and neglecting the e f f e c t
of H , the k0H value i s estimated t o be 1.56 per day.

5.5 Stability in Various Solvents

The stabi 1 i t y of acetazolamide i n pure unbuffered
sol vents 1 i ke propyl ene glycol , pol yethyl ene glycol 400, and
water i s not optimum, probably because of the higher pH
values of these solutions a t which acetazolamide i s not
stab1e. Progressive rep1 acement of water w i t h propyl ene
glycol improves the s t a b i l i t y of acetazolamide because of
solution pH values approaching 4 , which i s the pH value for
maximum s t a b i l i t y (26, 50).
T h e s t a b i l i t y of acetazolamide i n an extemporaneous
suspension compounded from t a b l e t s w i t h a predicated shelf-
l i f e of 371 days i s also reported (51).

6 BIOPHARIUICEUTICS

6.1 Phariacokinetics and Metabolism

Acetazolamide i s rapidly and almost completely
absorbed from the gastrointestinal t r a c t . Food intake does
not appear t o influence absorption (52). Peak
concentrations i n plasma occur w i t h i n two hours (53). Usual
26 JAGDISH PARASRAMPURIA

therapeutic serum acetazol amide concentration range is 15-20

mcg/mL, with variations in response from patient to patient
(54). Acetazol ami de is 70-90% protei n-bound (55). The
apparent volume of distribution i s about 0.2 L/kg (56).
Acetazolamide is not metabolized (56) and 90% of the
administered dose is excreted unchanged in the urine within
the first 24 hours. This process involves both active
tubular secretion and passive reabsorption. Plasma
concentrations of acetazolamide are proportional to dose,
fall in the therapeutic range, and can be detected for six
to 12 hours after administration (57). The saliva
concentration is about 1% o f the plasma levels, elimination
half-life about 4-8 hours, and the therapeutic index 2.7.
Renal clearance is approximately two-thirds of
simultaneously administered creatinine. Acetazolamide is
widely distributed throughout the body, including the CNS
(58). No tissue, with the exception of red blood cells, has
special affinity for acetazolamide. In red blood cells the
drug remains for several days after a single dose and
appears to reach a fixed range of concentration, independent
of dose or duration of administration, due to the apparent
saturation and delay in elimination. Only a small
proportion of the carbonic anhydrase in circulating red
cells is apparently inhibited by acetazolamide (59). Peak
acetazolamide levels in erythrocyte are 45% higher in the
elderly group (60). It is unknown if acetazolamide is
excreted into human breast milk; however, no harmful effects
have been reported in breast-fed infants whose mothers were
taking acetazolamide (61).

6.2 Bioavailability
Bi oequi Val ent comparisons of two sustained re1 eases
and an immediate-release acetazolamide dosage form performed
in normal human volunteers (n = 18) demonstrates a large
statistical difference between the preparations. The
sustained-re1 ease dosage forms are 40-70% 1 ess avai lab1 e
than the immediate-release dosage form, based on the AUC
data (62).
Comparisons were made between the ocular hypotensi ve
effects and blood levels achieved with the single-dose
administration of either generic acetazolamide or brand-name
acetazolamide (Diamox). The generic and brand-name
acetazolamide were equivalent in their effects on
intraocul ar pressure. Comparable bl ood 1 eve1 s of
acetazolamide were obtained with the two products.
 ACETAZOLAMIDE 21

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28 JAGDISH PARASRAMPURIA

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 ACETAZOLAMIDE 29

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