Sustainable Chemistry and Pharmacy 18 (2020) 100345

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Sustainable Chemistry and Pharmacy

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Microwave assisted solid phase microextraction for extraction and selective

enrichment of four alkaloids in lotus leaf
Shuhan Zou a, b, Chenhong Wang a, b, Jin Li a, **, Jun He a, b, Xiumei Gao a, Yan-xu Chang a, b, *
a
State Key Laboratory of Component-based Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin, 301617, China
b
Tianjin Key Laboratory of Phytochemistry and Pharmaceutical Analysis, Tianjin University of Traditional Chinese Medicine, Tianjin, 301617, China

A R T I C L E I N F O A B S T R A C T

Keywords: A novel method was developed for extraction and enrichment of the four alkaloids (nuciferine, O-nornuciferine,
Lotus leaf armepavine and N-nornuciferine) from lotus leaf by coupling microwave-assisted extraction (MAE) with solid
Microwave-assisted extraction (MAE) phase microextraction (SPME) before ultra-high-performance liquid chromatography (UHPLC) analysis. The
Solid phase microextraction (SPME)
influence parameters of MAE and SPME process including solvent-to-materials ratio, extraction solvent, irradi­
ation power, extraction time were investigated. The method exhibited a good linearity within the range of
0.4–100 μg mL− 1 for nuciferine and O-nornuciferine, 0.1–25 μg mL− 1 for armepavine and N-nornuciferine (r2 >
0.9990), respectively. Compared with the other extraction method, this method obviously shortens analytical
time and enhances extraction efficiency. In addition, the concentration of the four alkaloids (nuciferine, O-
nornuciferine, N-nornuciferine and armepavine) included two trace alkaloids were increased by 13, 10, 5 and 12
times after concentration, respectively. It was concluded that the newly MAE-SPME was an efficient method for
the extraction and enrichment for alkaloids from herbs.

1. Introduce (HPLC-QTOF/MS) (Guo et al., 2016) and high-performance liquid

chromatography (HPLC) (Ma et al., 2010). Due to limited sensitivity, the
Lotus, Nelumbo nucifera Gaertn., is an aquatic plant that is widespread common analytical technique such as HPLC was hardly used to detect
distributed in Asia. Multiple parts of the lotus including the seed, root, the trace components. Therefore, some high resolution and sensitivity
fruits and leaf have been used as food for thousands of years (Lin et al., techniques included ultra-high-performance liquid
2009). Lotus leaf, dry leaf of the lotus (Nelumbo nucifera Gaertn), is one of chromatography-mass (UHPLC-MS) and IC-ESI-MS/MS were applied to
traditional Chinese herbal medicines compiled in Chinese Pharmaco­ analyze the trace components in waste waters and food (Kisiala et al.,
poeia (The State of Commission Chinese Pharmacopoeia, 2015). It was 2019; Armbruster et al., 2019). Moreover, capillary electrophoresis with
also served as tea in China. Chemical studies demonstrated that the main mass spectrometric (CE-MS) was used to determine the major alkaloids
chemical components were flavonoids, alkaloids and polysaccharides included some trace alkaloids in lotus leaf (Do et al., 2013). However,
and so on (Chen et al., 2019). Alkaloids have been proved to be the these techniques were not widely applied in the determination of trace
major bioactivity compounds, which had the remarkable antimicrobial components due to high cost and tedious operation. Therefore, a simple,
(Zhao et al., 2016), anti-HIV (Kashiwada et al., 2005), anti-obesity ac­ efficient and economic method should be established for determining
tivity (Guo et al., 2013). In recent years, the increasing attentions were the trace alkaloid.
drawn to the trace alkaloid compounds in the lotus leaf. Some trace al­ Due to the low contents of active components, it is difficult to analyze
kaloids such as armepavine and N-nornuciferine possess the remarkable the trace components with a common analytical technique in TCM.
antimicrobial and antioxidation activity (Ma et al., 2015; Costa et al., Therefore, there are two key points to solve this problem such as
2010; Weng et al., 2012). Many researchers have reported the quanti­ increasing extraction efficiency and concentrationof active components
fication of major alkaloids in lotus leaf by using high-performance liquid in sample solution. In terms of increasement of the extraction efficiency,
chromatography-quadrupole time-of-flight mass spectrometry the major extraction methods for lotus leaf included the heat reflux

* Corresponding author. State Key Laboratory of Component-based Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin, 301617, China.
** Corresponding author.
E-mail addresses: [email protected] (J. Li), [email protected] (Y.-x. Chang).

https://doi.org/10.1016/j.scp.2020.100345
Received 6 July 2020; Received in revised form 30 October 2020; Accepted 31 October 2020
Available online 13 November 2020
2352-5541/© 2020 Elsevier B.V. All rights reserved.
S. Zou et al. Sustainable Chemistry and Pharmacy 18 (2020) 100345

extraction (HRE) and ultrasonic extraction (Wu et al., 2018; Jiang et al.,
2004; Zhao et al., 2003). These general extraction techniques were not
only low-effective but also cumbersome, time-consuming and
solvent-consuming (Wu et al., 2014). Recently, microwave assisted
extraction (MAE) with some merits such as high-effective, less solvent
and energy consumption have been widely applied for extracting the
chemical components from TCMs (Zhang et al., 2015; Chan et al., 2011).
For concentrating active components in sample solution, there are
some conventional concentrating techniques such as liquid-liquid
extraction (LLE), liquid-phase extraction (LPE), aqueous two-phase
extraction (ATPE) (Cheng et al., 2017) and dispersive liquid-liquid
extraction (DLLME) (Wang et al., 2018). However, these techniques
possess some drawbacks such as fussy operation, lengthy extraction
time, large solvent consumption and high cost. Recently, solid phase
microextraction (SPME) was widely applied to concentrate various
components in the field of environment, medicine and food (Wang et al.,
2019; Cui et al., 2019; Hafezian et al., 2019), especially for the con­
centration of trace components (Jinadasa et al., 2020; Li et al., 2020;
Mehdinia et al., 2015) because of its simplicity, lower cost and less
consumption of reagent. The little amount of adsorbent with greater
selectivity was used to enrich the trace components in SPME process.
Various sorbents have been reported for separation and concentration of
trace components (Wang et al., 2016; Mroczek et al., 2006; Luo et al.,
2019).
In current research, a MAE assisted SPME method was established for Fig. 1. The structure of four alkaloids and internal standard.
enhancing the extraction efficiency and sensitivity of four alkaloids from
lotus leaf. In brief, the MAE procedure was established by optimizing 2.3. UHPLC condition
some key parameters. Then, the SPME procedure was investigated by
optimizing some key parameters. The MAE-SPME-UHPLC method was The chromatographic analysis was performed on a Waters ACQUITY
successfully applied to extract, enrich and determine the four alkaloids UHPLC system equipped with a photo-diode array (PDA) detector. The
(nuciferine, O-nornuciferine, N-nornuciferine and armepavine) from mobile phase consisted of acetonitrile (A) and 10 mM ammonium ace­
lotus leaf. Two of them (Armepavine and N-nornuciferine) were trace tate solution (B) were used to separate the analytes on an ACQUITY
alkaloids. The developed method was successfully applied to determine UPLC BEHC18 (1.7 μm, 2.1 × 100 mm) with 35 ◦ C column temperature.
the contents of four alkaloids and would be helpful to the further sep­ The gradient elution was as follows: 0–4 min, 5–45% (A); 4–8 min,
aration of trace alkaloid and be useful to quality evaluation of lotus leaf. 45–70% (A); 8–9 min, 70–95% (A); 9–10 min, 95-5% (A), then post-run
time was 5min. The flow rate was 0.3 mL min− 1 and injection volume
2. Experimental was 2 μL. The peaks of analytes possess a good chromatographic sepa­
ration with above chromatographic conditions.
2.1. Chemical and material

Four standard references including nuciferine, O-nornuciferine, 2.4. Microwave-assisted extraction

armepavine, N-nornuciferine (Fig. 1) were obtained from Chengdu
DeSiTe Biological Technology Co., Ltd. (Chengdu, China). The purities In order to get a better extraction efficiency of all analytes, four
of these four standard references were more than 98%. Eight kinds of parameters (extraction solvent, material-solvent ratio, microwave
absorbent such as ODS C18 (50 μm, 60A), COOH (50 μm, 60A), PCX power and extraction time) were investigated in this study. The dried
(40–60 μm), SAX (50 μm, 60A), PAX (40–60 μm), PWCX (40–60 μm), sample powder (0.250 g) was mixed and extracted with 10 mL of
HP-20 (0.3–1.25 mm) and SCX (50 μm, 60A) were achieved from Agela extraction solvent (methanol:1% formic acid = 75:25, v/v) by Multi­
Tec. Co. Ltd (Tianjin, China). The chromatographic grade reagents were wave PRO SOLV (Anton Paar Co. Ltd., Graz, Austria). The extracting
bought from Fisher (USA). The analysis grade ammonium acetate was solutions were irradiated, then they were cooled down to the room
obtained from Tianjin Kemiou Chemical Reagent Co. The deionize water temperature and centrifuged at 10,000 rpm for 10 min. Then, the su­
wasprovided by the Mili-Q system (Millipore Corp., MA, USA). pernatant (1 mL) was added in 25 mL volumetric flask, then diluted to
The sample of lotus leaf were bought from Tongrentang medicine calibration with extraction solvent and shook well. The final solution
store of Tianjin and authenticated by Dr. Yan-xu Chang (Tianjin Uni­ was used for SPE procedure.
versity of Traditional Chinese Medicine). The sample was pulverized,
then sifted through 60-mesh sieve. 2.5. SPE procedure

2.2. Preparation of standard solutions To enrich and enhance the sensitivity of the analytes, some param­
eters including the type and amount of sorbent, adsorption time, type of
Two mg⋅mL− 1 nuciferine, O-nornuciferine, N-nornuciferine and eluent, elution time and eluent volume were optimized by univariate
armepavine were dissolved in methanol, respectively. The mixed stock investigation. Each test was repeated in triplicate. One microliter sample
solution containing 100 μg mL− 1 nuciferine, 100 μg mL− 1 O-nornuci­ solution was mixed with 25 mg sorbent (cation exchange polymer PCX)
ferine, 25 μg mL− 1 armepavine and 25 μg mL− 1 N-nornuciferine was and vortexed for 2 min. Then, the mixed solution was centrifuged at
accurately performed and attenuated with methanol to get nine different 7000 rpm for 10min. The supernatant was discarded. Moreover, the
concentrations for calibration curves. acetonitrile solution (contain 1% ammonia) was added to the sorbent
adsorbed with analytes and vortexed for 3 min. Then, the mixed solution
was centrifuged at 10,000 rpm for 10min. Moreover, the supernatant

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was diverted into the 1.5 mL tube and evaporated to dryness using ni­ extraction efficiency, but also decreases the consumption of sample and
trogen gas. Finally, the dried residue was reconstituted by adding 100 μL organic solvent. Thus, the material-solvent ratio (1:40, 1:30, 1:20, 1:10
methanol and vortexed for 2 min. The final solutions were centrifuged m/V) with the amounts of sample varying from 0.250 g to 1 g was raised.
twice at 14,000 rpm for 10 min prior to UHPLC analysis. The schematic The result demonstrates that the extraction efficiency of analytes grad­
diagram of was described in Fig. 2. ually increased when the material-solvent ratio varied from 1:10 to 1:40
(Fig. 3C). Therefore, the optimal material-solvent ratio was 1:40 (g/mL).
3. Results and discussion
3.1.4. Optimization of extraction time
3.1. Optimization of MAE procedure Appropriate extraction time can ensure more complete extraction.
When the time during under microwave powder was change from 0.5
3.1.1. Selection of extraction solvent ratio min to 4 min, the extraction efficiency first increased (0.5 min–2 min)
Alkaloids with a certain alkalinity often exist in the form of salt in and then decreased (2 min–4 min) (Fig. 3D). Thus, the optimal extrac­
plants. They are usually extracted in acid water. The advantages of this tion time was set at 2 min.
extraction method as follows: the solubility was increased by converting
macromolecular organic salt of alkaloid into micromolecular inorganic 3.2. Optimization of SPE procedure
salt. Therefore, methanol solution with 1% formic acid was selected as
extraction solvent. To obtain a better extraction efficiency and suitable 3.2.1. Type of sorbent
acidity, the different ratios of extraction solvent (methanol:1% formic It is crucial to choose an appropriate sorbent in the SPE procedure.
acid) were evaluated The result showed that the extraction efficiency The sorbent is not only used to adsorb the target compounds, but also to
was highest, when the ratio of extraction solvent (methanol: 1% formic remove the interfering substance. Silica ODS C18, microporous resin HP-
acid) was 75:25 (Fig. 3A). Thus, the optimal extraction solvent ratio was 20, three kinds of cation exchange polymer including SCX, PCX, PWCX
75:25 (methanol:1% formic acid). and three kinds of cation exchange polymer including COOH, SAX, PAX
were examined. These results demonstrated that the peak areas of the
3.1.2. Optimization of microwave power four alkaloids in supernatant were least when PCX was selected as sor­
The microwave power has been regarded as a parameter which bent (Fig. 4A). It was obviously indicated that the four alkaloids had
affected the interaction and equilibrium rate of analytes between sample strong retention in PCX. The reason was that alkaloid cation from
and extraction solvent. The optimal microwave power was achieved by alkaloid salt were exchanged with the cation on the strong acid cation
comparing the extraction efficiency at the condition of 150, 300, 450, exchange in acid water. PCX is a water-wetting polymer with cation
600 and 750 W. The result showed that microwave power had no exchange mechanism. It was stable in pH (0–14) and possess a larger
obvious influence on extraction efficiency of four components (Fig. 3B). binding capacity. Thus, cation exchange polymer PCX was selected as
There is a little higher extraction efficiency for the four alkaloids when optimal sorbent for next step.
microwave power was 300 W. Therefore, microwave power at 300 W
was used for further experiments. 3.2.2. Ratio of sample solution to sorbent
To ensure that the targeted alkaloids in sample solution were
3.1.3. Optimization of material-solvent ratio absorbed as completely as possible, the different ratios of sample solu­
Material-solvent ratio was an important parameter that influenced tion to sorbent (v/m) were explored. These results demonstrated that the
extraction yields of analytes from sample. In MAE procedure, the peak areas of the four alkaloids in supernatant were gradually decreased
appropriate material-solvent ratio not only achieves the higher when the ratio of sample solution to sorbent increased from 1:5 (1 mL/5

Fig. 2. The schematic diagram of microwave assisted (MAE) and solid phase microextraction (SPME) method.

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Fig. 3. Effects of parameters on peak area of four alkaloids: (A) Ratio of extract solution. (B) Microwave power. (C) Ratio of material to solvent. (D) Extract time in
microwave extraction process.

Fig. 4. Effects of parameters on peak area or content of four alkaloids: (A) Type of sorbent; (B) amount of sorbent; (C) adsorption time; (D) type of elution solvent; (E)
elution time; (F) elution volume in enrichment process.

mg) to 1:25 (1 mL/25 mg) (Fig. 4B). When the ratio reached 1:30, the as completely as possible in a short time. The result shows that the peak
peak areas of the four alkaloids were not increased any more. Therefore, areas of the two alkaloids (nuciferine and N-nornuciferine) has a slightly
the sample solution to sorbent ratio of 1:25 (1 mL/25 mg) was chosen in changes with the increase of the adsorption time from 0.5 min to 4 min)
this experiment. (Fig. 4C). However, the peak areas of the two alkaloids (O-nornuciferine
and armepavine) in supernatant were least when the adsorption time
3.2.3. Adsorption time was 2 min. Therefore, the optimal adsorption time was 2 min.
The appropriate adsorption time is a critical factor for the extraction
efficiency. It can ensure that targeted analytes were absorbed on sorbent

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3.2.4. Type of eluent ammonia) from 0.2 mL to 1 mL (Fig. 4F). It was found that the more
The suitable eluent is crucial for extraction efficiency. Using the way eluent was used, the higher extraction efficiency was obtained. Thus, 1
of acid extraction and alkaline precipitation, the adsorbed alkaloids in mL of acetonitrile solution (contain 1% ammonia) was applied in the
sorbent were eluted by alkaline solution. The alkaloid cation from follow experiment.
alkaloid salt was exchanged with the cation on the strong acid cation
exchange in acid water in the above adsorption process. Then, the 3.3. Method validation
alkaloid cations in resin were replaced by the cation from alkaline so­
lution in the desorption process. Finally, the free alkaloid was achieved. The specificity of the established method was investigated by
Thus, three kinds of alkaline eluents including methanol solution analyzing the chromatogram of the blank solvent and real sample of
(contain 1% ammonia), ethyl alcohol solution (contain 1% ammonia), lotus leaf (Fig. 5). It was shown that no interference was tested at the
acetonitrile solution (contain 1% ammonia) were evaluated in this same time of the four target alkaloids.
study. The result shows that the peak areas of the four alkaloids in eluent The calibration curves of four alkaloids were built by performing the
was maximum when acetonitrile solution (contain 1% ammonia) was peak areas versus the concentration of analytes. The linear range was
used as the eluent (Fig. 4D). Therefore, the optimal eluent was aceto­ 0.4–100 μg mL− 1 for nuciferine, O-nornuciferine and 0.1–25 μg mL− 1 for
nitrile solution (contain 1% ammonia) in this study. N-nornuciferine, armepavine. The correlation coefficients (r2) of all
analytes were not lower than 0.9990 (Table 1). The repeatability was
3.2.5. Elution time investigated by using the developed method parallelly six times. The
The appropriate elution time was achieved by evaluating the influ­ RSDs of all analytes were no more than 1.6% (Table 1). It was indicated
ence of five different elution times including 1, 2, 3, 4 and 5 min on that the established method possessed a good reproducibility. The intra-
extraction efficiency of four target alkaloids. The result demonstrates day and inter-day accuracies of four alkaloids ranged from 92.6% to
that the peak areas of those alkaloids increased when the elution time 102% (≤4.9%) and 91.4 %–103% (≤3.9%), respectively (Table 2).
increased from 1 to 3 min (Fig. 4E). It was likely that the longer time, the The stabilities were assessed by determining the accuracies of four
more alkaloid cations in resin were replaced by the cation from alkaline analytes on three concentration levels at the condition of temperature
solution. However, the peak areas were decreased from 3 to 5 min. The over 24 h. The accuracies of four alkaloids were in the range of 92.7 %–
possible reason was that the replacement reached saturation, when the 103% (≤5.0%), which indicated that the four alkaloids were stable
elution time was 3 min. Thus, 3 min was taken as the optimum elution within 24 h (Table 2).
time. To evaluate the accuracy of the developed method, recoveries of
analyte were investigated. The mean recoveries of four target alkaloids
3.2.6. Eluent volume were in the range of 97.7%–99.7% and the RSDs were less than 3.6%
To achieve the higher extraction effect with the less eluent, different (Table 3).
volumes were tested. The result shows that the contents of targeted al­
kaloids increased with the volume of acetonitrile solution (contain 1%

Fig. 5. Chromatogram of (A) real sample, (B) reference substance and (C) blank before enrichment; chromatogram of (D) real sample, (E) reference substance and (F)
blank after enrichment. (1 = armepavine; 2 = N-nornuciferine; 3 = O-nornuciferine; 4 = nuciferine.)

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Table 1
The calibration curves, linearity range, limit of detection (LOD), limit of quantification (LOQ) and repeatability of the developed method (n = 6).
Compounds Regression equation r2 Linearity range (μg⋅mL− 1) LOD (μg⋅mL− 1) LOQ (μg⋅mL− 1) Repeatability RSD (%)

nuciferine Y = 22954X-211.6 0.9998 0.4–100 0.01 0.04 1.6

N-nornuciferine Y = 12696X-263.5 0.9992 0.1–25 0.01 0.04 1.4
O-nornuciferine Y = 22834X-11095 0.9990 0.4–100 0.02 0.05 1.1
armepavine Y = 3427.6X+767.32 0.9999 0.1–25 0.05 0.1 1.1

Table 2
Intra-day, inter-day precision and stability of four compounds.
Compounds Concentration (μg⋅mL− 1) Intra-day Inter-day Stability

Accuracy (%) RSD (%) Accuracy (%) RSD (%) Accuracy (%) RSD (%)

nuciferine 2.0 92.6 0.7 91.4 1.3 95.2 2.0

10 100 4.5 97.7 2.5 98.3 5.0
50 95.9 4.6 95.0 3.3 95.2 3.7
N-nornuciferine 0.5 96.3 3.8 96.7 3.1 94.4 0.8
2.5 95.5 2.6 95.5 0.4 92.7 4.0
12.5 93.1 1.6 92.9 0.3 93.1 2.2
O-nornuciferine 2.0 102 0.7 103 0.6 102 3.5
10 100 0.5 102 1.9 103 0.7
50 102 0.3 102 1.2 98.8 2.7
armepavine 0.5 97.6 1.9 94.6 3.7 95.5 4.1
2.5 100 3.1 101 1.0 101 3.6
12.5 101 4.9 96.5 3.9 100 1.0

Table 3
The results of recovery test (n = 6).
Compounds Original (μg) Spike (μg) Detected (μg) Average recovery (%) RSD (%)

nuciferine 2.23 1.12 1.12 ± 0.03 99.7 3.1

N-nornuciferine 0.38 0.27 0.27 ± 0.01 99.5 3.1
O-nornuciferine 2.49 1.25 1.22 ± 0.04 97.7 3.6
armepavine 0.65 0.33 0.33 ± 0.01 97.9 1.6

3.4. Application 3.5. Comparison with other methods

The established MAE-SPME method was applied to extraction and To assess the advantage and availability of developed MAE-SPME
concentration of the four alkaloids from lotus leaf. Before enrichment, method, the features of reflux extraction with acid solvent, reflux
the concentrations of nuciferine, O-nornuciferine, N-nornuciferine and extraction with organic solvent and microwave extraction with ILs were
armepavine were 1.5, 1.7, 0.5 and 0.4 μg mL− 1, respectively. Moreover, reported (Ma et al., 2010) (Table 4). Firstly, compared with extraction
the concentrations of those after enrichment were 19.9, 16.9, 2.6 and time in this work, more extraction time were used in this reported
4.7 μg mL− 1, individually. These results obviously demonstrated that the method (Ma et al., 2010). Secondly, the consumptions of organic reagent
concentrations of the four alkaloids were remarkable increased after in this study were more than that in the reported research (Ma et al.,
enrichment. The concentration of nuciferine, O-nornuciferine, N-nor­ 2010). Finally, the detection time in this work were more than that in
nuciferine and armepavine was increased the times of 13, 10, 5 and 12, the reported studies. Moreover, the numbers of target compounds in this
respectively after enrichment. Moreover, the peak of armepavine and N- study were more than those of target compounds in these reported
nornuciferine is too small to be clear enough before concentrating studies (Ma et al., 2010). The two of four target alkaloids of lotus leaf,
(Fig. 5). However, the response of armepavine and N-nornuciferine were including N-nornuciferine and armepavine, were analyzed by combing
obviously enhanced after enrichment so that the accuracy of quantifi­ MAE-SPME with UHPLC method in current work. It was demonstrated
cation was improved (Fig. 5). In addition, the analytical time was that the established method was satisfactory and efficient for extraction,
shortened from 45 min to 10 min. Moreover, the chromatographic peaks enrichment and determination of target alkaloids of lotus leaf, especially
of each compound were separated excellently. some trace alkaloids.

Table 4
Comparison of the MAE-SPE method with other methods.
Sample Analytes Type of solvent Solvent Extraction Extraction Detection Detection Reference
amounts volumn method time method time

0.50 g nuciferine, O-nornuciferine, N- ILs 10 mL MAE 2 min HPLC 20 min Ma et al.,

nornuciferine, 2010
0.50 g nuciferine, O-nornuciferine, N- Hydrochloride acid 15 mL HRE 2h HPLC 20 min Ma et al.,
nornuciferine, aqueous solution 2010
0.50 g nuciferine, O-nornuciferine, N- 95% ethanol 15 mL HRE 2h HPLC 20 min Ma et al.,
nornuciferine, 2010
0.25 g nuciferine, O-nornuciferine, 75:25 (methanol:1% 10 mL MAE-SPME 7 min UHPLC 10 min This work
armepavine, N-nornuciferine formic acid)

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Declaration of competing interest solid-phase microextraction in drinking water. Environ. Technol. 21, 1–8. https://
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Luo, Z.M., Chen, G.N., Li, X., Wang, L., Cui, X., Chang, C., Zeng, A.G., Fu, Q., 2019.
The authors declare that they have no known competing financial Molecularly imprinted polymer solid-phase microextraction coupled with ultra high
interests or personal relationships that could have appeared to influence performance liquid chromatography and tandem mass spectrometry for rapid
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Acknowledgments nuciferine inhibit lipogenesis in 3T3-L1 adipocytes by activating the AMPK signaling
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This research was supported by Tianjin Research Program of Appli­ liquids based microwave-assisted extraction of three alkaloids N-nornuciferine, O-
cation Foundation and Advanced Technology (18JCYBJC95000), Na­ nornuciferine, and nuciferine from lotus leaf. Talanta 80, 1292–1297. https://doi.
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