Journal of Pharmaceutical and Biomedical Analysis 117 (2016) 1–10
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Journal of Pharmaceutical and Biomedical Analysis
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imultaneous determination of four phenolic acids and sevenlkaloids in rat plasma after oral administration of traditional Chineseedicinal preparation Jinqi Jiangtang Tablet by LC-ESI–MS/MS
an-xu Chang a,b,∗, Ai-hua Ge a,b, Xie-an Yu a,b, Xiu-cheng Jiao a,b, Jin Li a, Jun He a,b,i Tian a,b, Wei Liu a,b, John Teye Azietaku a,b, Bo-li Zhang a, Xiu-mei Gao a
Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, ChinaTianjin Key Laboratory of Phytochemistry and Pharmaceutical Analysis, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China
r t i c l e i n f o
rticle history:eceived 9 May 2015eceived in revised form 18 August 2015ccepted 19 August 2015vailable online 24 August 2015
eywords:henolic acidserberine
a b s t r a c t
A rapid, sensitive and selective high performance liquid chromatography–tandem mass spectrometry(LC–MS/MS) method was developed and validated for the simultaneous determination of four phenolicacids (neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid and ferulic acid) and seven alka-loids (berberine, epiberberine, coptisine, magnoflorine, berberubine, palmatine and jatrorrhizine) in ratplasma. After mixing with the internal standards tetrahydropalmatine (IS1) and rosmarinic acid (IS2),plasma samples were pretreated by protein precipitation using acetonitrile. The HPLC analysis was per-formed on an Agilent Eclipse plus C18 (4.6 mm × 100 mm, 1.8 �m) column with mobile phase consistingof 0.1% formic acid aqueous solution and acetonitrile at a flow rate of 0.3 mL min−1. The detection was
hlorogenic acidinqi Jiangtang tabletC–MS
accomplished for the analytes and internal standards using positive electrospray ionization for the alka-loids and negative electrospray ionization for the phenolic acids in multiple-reaction monitoring mode.The method showed a good linearity over a wide concentration range (r2 > 0.99). The lower limit ofquantification of seven alkaloids was lower than 2 ng mL−1 and that of four phenolic acids was less than20 ng mL−1. The developed method was applied to the pharmacokinetic study of 11 components afteroral administration of traditional Chinese medicinal preparation Jinqi Jiangtang Tablet in rats.
. Introduction
Traditional Chinese medicines (TCMs) have been used as anmportant resource for the prevention and treatment of various dis-ases for more than 1000 years [1]. Typically, TCMs have differentinds of components. It is well known that multiple componentsct on multiple targets and exert synergistic therapeutic efficacies,hich are the unique mechanism of action of TCMs [2]. Nowadays,ore and more attention has been focused on the active compo-
ents from TCMs. The pharmacokinetic study on active components
n TCM could have a great effect on evaluating the mechanism ofction to better elucidate the efficacy of TCMs.
Traditional Chinese medicinal preparation Jinqi Jiangtang (JJT)ablet consists of three herbal plants: Coptis chinensis (rhizome
∗ Corresponding author at: Tianjin State Key Laboratory of Modern Chi-ese Medicine, Tianjin University of Traditional Chinese Medicine, China.ax: +86 25 59596163.
of C. chinensis Franch.), Astragalus membranaceus (root of A.membranaceus Moench) and Lonicera japonica (flower buds of L.japonica Thunb.). It has been used for the treatment of diabetesand its complications clinically [3–4]. Modern pharmacologicalresearches demonstrated that JJT had various bioactivities suchas anti-inflammation, gastrointestinal tract and radical scaveng-ing effects [5]. Phenolic acids like neochlorogenic acid, chlorogenicacid, cryptochlorogenic acid and ferulic acid are the main bioac-tive compounds in L. japonica [6], while the alkaloids berberine,epiberberine, coptisine, magnoflorine, berberubine, palmatine andjatrorrhizine are the main bioactive components in Rhizome Cop-tidis [7]. In previous studies, it was found out that the total alkaloidsof C. chinensis had hypoglycemic effect and the phenolic acids hadfree radical scavenging activity [8]. It was these components thatgave JJT tablet its pharmacological activity.
There are many studies on the simultaneous determination ofalkaloids in TCMs and their application in rat plasma using HPLC-UV [9], HPLC-ED [10], LC–MS/MS [11] and UHPLC–MS/MS as theanalytical methods [12]. However, these methods only focused onpharmacokinetics of the normal alkaloids with high concentrations
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n TCMs and their preparations. Moreover, a UPLC–MS/MS methodas reported to simultaneously determine phenolic acids after oral
dministration of Flos Lonicerae prescriptions [13]. A method waseveloped and validated to evaluate the pharmacokinetic prop-rties of ferulic acid in normal and blood deficiency rats [14].owever, there is currently no analytical method to assess theharmacokinetic study of JJT tablet, of which both alkaloids andhenolic acids are the main components. The physicochemical dif-
erences between alkaloids and phenolic acids, the low contentsf the components and the low absorption of alkaloids after oraldministration may be the reasons why no method has been estab-ished for the simultaneous determination of these components inat plasma. Obviously, it is important to make a comprehensivenvestigation into the pharmacokinetic study of the active com-onents, which could help to determine the rational dose, avoidndesirable interactions and minimize side effects in clinic. There-
ore, it is necessary to develop a rapid and sensitive method forhe simultaneous determination of the alkaloids and the phenoliccids which will help in effectively evaluating the pharmacokineticroperty of JJT tablet.
In this study, a simple, rapid and sensitive LC-ESI–MS/MSethod was firstly developed and validated for the simultane-
us determination of the four phenolic acids (neochlorogeniccid,chlorogenic acid, cryptochlorogenic acid and ferulic acid) andeven alkaloids (berberine, epiberberine, coptisine, magnoflorine,erberubine, palmatine and jatrorrhizine) after oral administrationf JJT tablet in rat plasma. The chemical structures of the 11 com-onents and 2 internal standards are shown in Fig. 1. The methodas successfully applied to the pharmacokinetic study after oral
dministration of the JJT tablet. To the best of our knowledge,his is the first report of a simultaneous determination of berber-ne, epiberberine, coptisine, magnoflorine, berberubine, palmatine,atrorrhizine, chlorogenic acid, cryptochlorogenic acid, neochloro-enic acid and ferulic acid after oral administration of JJT tablet.his is also the first study of the pharmacokinetic property of JJTablet and berberubine after oral administration.
. Experimental
.1. Chemicals and reagents
Acetonitrile of MS-grade was purchased from Merck (Darm-tadt, Germany) and methanol (Tianjin Concord Science Co. Ltd.,ianjin, China) were of HPLC grade. HPLC-grade formic acid wasurchased from Tedia Company Inc. (Tedia, Fairfield, OH, USA).eionized water was purified with a Milli-Q Academic ultra-pureater system (Millipore, Milford, MA, USA). Reference Standards
f chlorogenic acid, cryptochlorogenic acid, neochlorogenic acid,erulic acid, magnoflorine, coptisine, epiberberine, jatrorrhizinydrochloride, berberine hydrochloride, berberubine and palma-ine hydrochloride (purity > 98%) were purchased from Chengdu
ust Biotechnology Co. Ltd. (Chengdu,China). traditional Chi-ese medicinal preparation Jinqi Jiangtang Tablet was obtained
rom Tianjin pharmacy (Tianjin, China). Voucher specimens wereeposited at the Institute of Traditional Chinese Medicine (Tian-
in university of Traditional Chinese Medicine). All other reagentsere of analytical grade and obtained commercially.
.2. Equipment and LC–MS/MS conditions
The LC–MS/MS system was made up of an Agilent 1200eries HPLC system (Agilent Technologies, USA), consisting of a1312A binary pump, a vacuum degasser unit (G1322A), a Hip-LS auto-sampler (G13678) and an API3200 triple quadruple mass
d Biomedical Analysis 117 (2016) 1–10
spectrometer with an ESI source (Concord, Ontario, Canada). Datawere analyzed by Analyst 1.4.2 software (AB MDS Sciex).
The separation was achieved on an Agilent Eclipse plus C18(4.6 mm × 100 mm, 1.8 �m) column. The mobile phases consistedof acetonitrile (A) and 0.1% formic acid (B) using a gradient elutionof 10–20% (v/v) A at 0–10 min; 20–20% A at 10–14 min; 20–27% Aat 14–17 min; 27–50% A at 17–20 min; 50–70% A at 20–25 min;70–80% A at 25–30 min. The flow rate was set at 0.3 mL min−1.The injection volume was 5 �L. The column temperature was setat 30 ◦C. The ESI–MS data was obtained in positive electrosprayionization for alkaloids (berberine, epiberberine, coptisine, mag-noflorine, berberubine, palmatine and jatrorrhizine) and negativeelectrospray ionization for the phenolic compounds (neochloro-genic acid, chlorogenic acid, cryptochlorogenic acid and ferulicacid) using multiple-reaction monitoring mode. The source param-eters were optimized to obtain the best sensitivity and response ofevery component. The results are listed in Table 1.
2.3. Preparation of standards and quality control (QC) samples
Stock solutions at 1.0 mg mL−1 for each compound wereprepared in methanol. The mixture of IS stock solutions of tetrahy-dropalmatine (1.0 �g mL−1) and rosmarinic acid (1.0 �g mL−1)were also prepared in methanol and kept at 50 and 100 ng mL−1
level in working solutions and samples. Appropriate volumes ofeach compound stock solution were mixed together. Then, themixture was diluted serially by methanol to achieve the standardworking solutions. All the solutions were kept at 4 ◦C until use.Quality control (QC) samples of each compound were preparedby spiking 10 �L of the standard working solutions into 100 �L ofblank rat plasma to provide the final concentration in the ranges of0.04, 0.12, 1.2, 12 ng mL−1 for berberine and palmatine; 0.08, 0.24,2.4, 24 ng mL−1 for epiberberine and berberubine; 0.16, 0.48, 4.8,48 ng mL−1 for jatrorrhizin; 0.2, 0.6, 6, 60 ng mL−1 for coptisine; 2,6, 60, 600 ng mL−1 for magnoflorine; 0.4, 1.2, 12, 120 ng mL−1 forchlorogenic acid; 4, 12, 120, 1200 ng mL−1 for ferulic acid and 20, 60,600, 6000 ng mL−1 for cryptochlorogenic acid and neochlorogenicacid.
2.4. Content of the 11 components in JJT tablet
The contents of berberine, epiberberine, coptisine, mag-noflorine, berberubine, palmatine, jatrorrhizine, cryptochlorogenicacid, chlorogenic acid, cryptochlorogenic acid, neochlorogenic acidand ferulic acid in JJT tablet for oral administration were deter-mined by the established LC–MS/MS method. The powder of JJTtablet (0.10 g) was extracted with 10 mL 70% methanol and ultra-sonicated for 40 min according to previous research [15]. Aftercentrifugation at 14,000 rpm for 10 min, the supernatant was fil-tered through a 0.22 �m membrane filter. At last, an aliquot of2 �L of supernatant was injected into the LC-ESI–MS/MS system.Finally, the contents of berberine, epiberberine, coptisine, mag-noflorine, berberubine, palmatine, jatrorrhizine, chlorogenic acid,cryptochlorogenic acid, neochlorogenic acid and ferulic acid were20.8, 5.5, 8.33, 0.22, 0.10, 3.48, 8.70, 27.4, 1.49, 1.17 and 0.19 mg g−1
in traditional Chinese medicine JJT tablet, respectively.
2.5. Plasma sample preparation
Plasma samples were pretreated employing a simple proteinprecipitation with acetonitrile. After thawing to room temperature,
100 �L of the rat plasma was mixed with 10 �L of the IS workingsolutions containing tetrahydropalmatine (50 ng mL−1) and ros-marinic acid (100 ng mL−1) and 10 �L of formic acid. The mixturewas vortex-mixed for 1 min and then 400 �L acetonitrile was addedand centrifuged for 10 min at 14,000 rpm. Supernatant was trans-
Y.-x. Chang et al. / Journal of Pharmaceutical and Biomedical Analysis 117 (2016) 1–10 3
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Fig. 1. Chemical structures of 11
erred and condensed to dryness by nitrogen gas. The dried residueas reconstituted by 100 �L methanol by vortex-mixing for 1 min
nd centrifuged at 14,000 rpm for 10 min. Then, 5 �L of the solutionas injected into the LC–MS/MS system for analysis.
.6. Method validation
A full validation according to the industrial guidelines forioanalytical method validation from the US Food and Drug Admin-
stration (FDA) was carried out for the assay in rat plasma (US Foodnd Drug Administration, 2001) [16].
.6.1. SelectivityThe selectivity was assessed by comparing the chromatograms
f six different batches of blank rat plasma samples with corre-ponding spiked plasma samples and obtained plasma after oraldministration of JJT tablet.
.6.2. Linearity and lower limit of quantification (LLOQ)For the calibration curves, the diluted standard working solu-
ions were spiked into blank rat plasma to obtain the finaloncentrations in the ranges of 0.04–15 ng mL−1 for berberinend palmatine; 0.08–30 ng mL−1 for epiberberine and berberubine;
.16–60 ng mL−1 for jatrorrhizin; 0.2–75 ng mL−1 for coptisine;–750 ng mL−1 for magnoflorine; 0.4–150 ng mL−1 for chlorogeniccid; 4–1200 ng mL−1 for ferulic acid and 20–7500 ng mL−1 forryptochlorogenic acid and neochlorogenic acid. All the standardorking solutions were kept at 4 ◦C before use. The calibration
able 1ource and SRM parametersof 11 compounds and two internal standards (IS).
Compounds CUR CAD IS TEM GS1
Neochlorogenic acid 30 4 −4500 500 40
Chlorogenic acid 30 4 −4500 500 40
Cryptochlorogenic acid 30 4 −4500 500 40
Ferulic acid 30 4 −4500 500 40
Magnoflorine 40 12 5000 600 40
Coptisine 40 12 5000 600 40
Epiberberine 40 12 5000 600 40
Jatrorrhizin 40 12 5000 600 40
Berberine 40 12 5000 600 40
Palmatine 40 12 5000 600 40
Berberubine 40 12 5000 600 40
Tetrahydropalmatine (IS1) 40 12 5000 600 40
Rosmarinic acid (IS2) 30 4 −4500 500 40
onents and 2 internal standards.
curve of each compound was constructed by plotting the ratio ofthe peaks area of analytes and internal standard (IS) (y) against thecorresponding concentration (x, ng mL−1) using a 1/X2 weightedlinear least-squares regression model.
The lower limit of quantification (LLOQ) is the lowest concen-tration of analyte in a sample which can be quantified reliably, withan acceptable accuracy and precision. In addition, the analyte sig-nal of the LLOQ sample should be at least 5 times the signal of ablank sample. The relative standard deviation (RSD) of the LLOQsample within 20% and the accuracy was in the range of 80% to120% according to the USFDA guidelines.
2.6.3. Precision and accuracyThe accuracy and precision were determined by using six repli-
cates of QC samples at four levels on the same day (intra-day)and between three different days (inter-day) and finally assessedusing the calibration curve constructed on the same testing day.The precision of intra-day and inter-day were expressed as RSDand accuracy was evaluated by comparing the calculated concen-tration with the spiked concentration. The RSD values should notbe more than 15% and accuracy should be in the range of 85% to115%.
2.6.4. The recovery and matrix effectThe recovery and matrix effects at four QC levels were deter-
mined in sets of six replicates. The recovery was assayed bycomparing the peak areas obtained from plasma samples spiked
4 Y.-x. Chang et al. / Journal of Pharmaceutical and Biomedical Analysis 117 (2016) 1–10
F plasma g (C).
be
oostTTm
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ig. 2. The chromatograms of the analytes in rat plasma: blank plasma (A), blank ratfter oral administration of traditional Chinese medicinal preparation Jinqi Jiangtan
efore extraction to those of analytes from solutions spiked in post-xtracted blank plasma at equivalent concentration.
The matrix effect is defined as the ion suppression/enhancementn the ionization of analytes, which was measured via comparisonf the peak response from post extraction blank plasma samplespiked with standard solutions at three levels in three replicates tohose of the corresponding concentration neat standard solutions.he same procedure was applied for IS at a single concentration.
he matrix effect is not negligible if the ratio is less than 85% orore than 115%..
able 2he calibration curves,linearity range,LLOQs of the assay(n = 6).
Compounds Regression equation
Neochlorogenic acid Y = 0.00566X + 0.236
Chlorogenic acid Y = 0.173X + 0.492
Cryptochlorogenic acid Y = 0.0229X + 0.0414
Ferulic acid Y = 0.0803X + 0.129
Magnoflorine Y = 0.00706X − 0.000419
Coptisine Y = 0.0154X + 0.0154
Epiberberine Y = 0.0206X + 0.00792
Jatrorrhizin Y = 0.019X + 0.000988
Berberine Y = 0.0201X + 0.0201
Palmatine Y = 0.021X + 0.000668
Berberubine Y = 0.0447X + 0.00429
a spiked with standard compounds (B) and plasma samples taken from rats 30 min
2.6.5. StabilityThe stability of analytes in plasma was investigated by analyzing
QC samples at four concentrations. The autosampler 24 h stabilitywas assayed after keeping the treated sample under autosam-pler conditions (room temperature) for 24 h. The QC samples weresubjected to three freeze (at about −20 ◦C) to thaw (at room tem-perature) cycles for freeze and thaw stability. The stability wasexpressed as RSD and accuracy. The long-term stability was exam-
ined by analyzing samples stored at about −80 ◦C for 1 month.For all stability testing of QC samples, the concentration wascompared with those of freshly prepared QC samples and the per-
entage concentration deviations were calculated to evaluate thetability.
.6.6. Cross-talkThe cross-talk was evaluated by comparing the observed peak
rea of the single analyte with that of the corresponding mix stan-ards QC samples at high level and this was to make sure that eachnalyte could not be influenced by others during a simultaneousetermination.
.7. Applications to pharmacokinetic studies
The PK study was carried out in accordance with the guidelinesor the care and use of laboratory animals and approved by the Ani-
al Ethics Committee of Tianjin University of Traditional Chineseedicine. Seven male Sprague-Dawley rats weighing 180–220 gere kept at the animal center of Tianjin University of Tradi-
ional Chinese Medicine (Tianjin, China) with controlled conditionstemperature at 25 ± 2 ◦C and relative humidity of 55 ± 5%). Theyere allowed free access to food and water and acclimatized for
everal days until 12 h before the experiment. According to thelinical dose used in human for each day which is 0.21 g/kg, the
11.3 95.0 14.214.4 104 5.44
4.46 100 2.377.89 98.7 4.63
dose in rat is 1.31 g/kg. Therefore, the JJT extract at 1.31 g kg−1
(suspended in 0.5% carboxymethyl cellulose sodium salt aque-ous solution) was administrated orally to the rats. Blood samples(about 200 �L) were collected before dosing and at 5, 10, 15, 30,45 min, 1, 1.5, 2, 4, 6, 8, 12, 24 h after administration from thefossa orbitalis of rats. After centrifugation at 7000 rpm for 10 minat 4 ◦C, the samples were immediately transferred into heparinizedtubes. The plasma was stored frozen at about −80 ◦C until analy-sis.
2.8. Data analysis
All the pharmacokinetic parameters were calculated using theDAS 1.0 software (Drug and Statistics 1.0, Medical College ofWannan, China) [17]. The appropriate pharmacokinetic modelwas chosen to fit the concentration–time curve in plasma. The
maximum drug concentration in plasma (Cmax) and the time toreach maximum drug concentration (Tmax) were obtained directlyfrom concentration–time data. Other pharmacokinetic parame-ters like the area under the plasma concentration–time curve(AUC), distribution half-life (t1/2�), elimination half-life (t1/2�), and
6 Y.-x. Chang et al. / Journal of Pharmaceutical and Biomedical Analysis 117 (2016) 1–10
Table 4Recoveries and matrix effects of 11compounds (n = 6).
ean residence time (MRT) were also calculated. The values werexpressed as mean ± SD.
. Results and discussion
.1. Optimization of LC–MS/MS conditions
The HPLC conditions were optimized to obtain better separa-ion in a short time. Different mobile phases (acetonitrile-water,
ethanol-water), concentrations of additive (0.05%, 0.1%, and 0.2%ormic acid), column temperatures (20, 30 and 40 ◦C) and flow rates0.2, 0.3 and 0.4 mL min−1) were optimized. After continuous andppropriate adjustment of the chromatographic separation condi-ions, the best results were obtained when the HPLC conditionsere set as follows: acetonitrile-0.1% formic acid as mobile phases,
ow rate at 0.3 mL min−1 and column temperature at 30 ◦C.
To optimize ESI conditions, the analytes and ISs were character-zed by MS scan and MS/MS products in SRM mode. The ESI–MSata were obtained in both negative and positive mode. The par-nt ions and product ions of the analytes, source and parameters
14.4 79.3 12.36.76 82.2 8.15
were optimized to obtain the maximum sensitivity and responseof every component. The results are listed in Table 1.
Other parameters such as drying gas flow rate, drying gastemperature, nebulizing gas pressure, and capillary voltage wereoptimized to obtain the highest intensity of the charged moleculesof analytes.
3.2. Optimization of sample preparation
Sample preparation is a critical step for developing an accu-rate and reliable LC-ESI MS/MS method to eliminate interferencefrom the sample matrix and achieve satisfactory recovery. In orderto extract the target analytes from biological fluids more effec-tively, we used the liquid–liquid extraction (LLE) with ethyl acetatemethod. However, matrix effects of some analytes were lowerthan 50% which could not meet the analysis requirements. Com-
pared with above methods, protein precipitation with acetonitrile(PPA) was found to offer satisfactory recovery and desired extractefficiency in determining the concentrations of the analytes. Inaddition, PPA was much simpler and less time-consuming than LLEwith ethyl acetate. In order to improve the extraction efficiency,
Y.-x. Chang et al. / Journal of Pharmaceutical and Biomedical Analysis 117 (2016) 1–10 7
Table 5Stability of the 11 analytes (n = 6).
Compounds Concentration(ng mL−1)
Freeze-thaw cycles At −80 ◦C for 1 month Autosampler for 24 h
he volumes of formic acid (5, 10, and 20 �L) and the precipitationeagents (methanol and acetonitrile) were optimized. Finally, theighest extraction recoveries of all analytes were obtained when0 �L formic acid (v/v) and 400 �L acetonitrile were employed forhe sample pre-preparation.
.3. Method validation
.3.1. SpecificityUnder the optimized LC–MS/MS conditions, the specificity was
valuated by analyzing six blank plasma samples, blank plasmadded to 11 analytes and 2 ISs, and the obtained plasma after oraldministration of JJT tablet The results demonstrated that thereere no interfering peaks in the peaks region of analytes (Fig. 2).
.3.2. Linearity and LLOQ
The calibration curves were constructed with a series of diluted
oncentrations in blank plasma by plotting the peak area ratio ofhe analytes to IS against the concentration of each analyte. Theesults are illustrated in Table 2. The results demonstrated that cal-bration curves showed good linearity in the linear range of the
85.2 15.4 109 12.587.9 9.36 102 9.98
analytes with the correlation coefficient r more than 0.99. The LLOQsamples of six rat plasma samples independent of the calibrationcurves were determined. The results are listed in Table 3. It wasobserved that the LLOQs of seven alkaloids were less than 2 ng mL−1
and that of four phenolic acids were less than 20 ng mL−1.TheirRSDs of inter-day and intra-day were less than 15% and theaccuracies ranged from 80% to 118%, respectively. These resultsdemonstrated that the limits were sufficient enough for the phar-macokinetic study of 11 analytes after oral administration of JJTtablet.
3.3.3. Precision and accuracyThe intra-day and inter-day precision and accuracy were calcu-
lated by preparing and analyzing six replicates of the analytes atspiked QC samples at LLOQ low, medium and high concentrationlevels on the same day and continuously for 3 days, respectively.
The results are shown in Table 3. The RSD and accuracy at three lev-els QC samples was less than 15% and more than 93.6.The RSD atLLOQ was less than 20% with accuracies in the range of 80–118%. Itwas concluded that the precision and accuracy of the method wereacceptable.
8 Y.-x. Chang et al. / Journal of Pharmaceutical an
.3.4. Recovery and matrix effectThe results of recovery and matrix effect of the analytes are listed
n Table 4. The mean recoveries of the analytes of the QC samplest four concentrations were more than 80.0% and the matrix effectere higher than 76.7% and less than 115%, indicating that theethod was precise, consistent and reproducible and the assess-ent of the matrix effect showed no effect.
.3.5. StabilityThe freeze–thaw stability, auto-sampler for 24 h stability and
ong-term stability were evaluated by the mean concentrations ofhe analytes of the QC samples at four levels. The results are listedn Table 5, indicating that all the analytes were stable in rat plasmaor three freeze/thaw cycles, auto-sampler for 24 h at room tem-erature. The long term stability for 1 month was relatively stable
or all the target analytes with the accuracy for all the compoundsnalysed passing FDA approved range.
.3.6. Cross-talk validationThe accuracy of each compound is shown in Table 6. There
ere two types of isomers including neochlorogenic acid, chloro-enic acid, and cryptochlorogenic acid isomer and berberine andpiberberine isomer. The extracted ion pair results was obtainedimultaneously with others, however, it could not be infulencedwing to retention time. For examples, the retention time ofeochlorogenic acid, chlorogenic acid and cryptochlorogenic acidere different, and the three peaks were separated well. The Q3
f jatrorrhizin was the same with the Q1 of the berberubine. Therere two peaks which were separated well (rententian times were4.76 min and 25.12 min) in the chromatographic figures when the
on pair of berberubine in the mix compound was extracted. Whenntegrated, the area of berberubine, the peak with retention time
5.12 min was calculated. The results showed that jatrorrhizin didot influence the accuracy of berberubine. Based on above results, itas concluded that there was no cross-talk influence in the newly
stablished LC–MS/MS methods.
able 7harmacokinetic parameters of the nine components in JinqiJiangtang tablet (n = 7, mean
In the study, the selection of the internal standard was investi-gated. Usually, the internal standard should have similar chemicalstructures and physicochemical property and should not interferewith the analytes. Based on this, two types of chemical compo-nents, tetrahydropalmatine (IS1) and rosmarinic acid (IS2) wereselected for the alkaloids and phenolic acids, respectively. Theresults showed that the internal standards had no interference withthe endogenous matrix and suitable retention time for separationwas obtained for the analytes. As a result, the tetrahydropalmatine(IS1) and rosmarinic acid (IS2) were chosen to evaluate the concen-tration of alkaloids and phenolic acids respectively in rat plasmaafter orally administrated the JJT tablet.
3.5. Pharmacokinetic study
The developed method was employed to determine the con-centration of seven alkaloids and four phenolic acids in ratplasma after oral administration of JJT tablet at a dose of1.31 g kg−1 (27.3 mg kg−1 berberine, 7.22 mg kg−1 epiberberine,10.9 mg kg−1 coptisine, 0.29 mg kg−1 magnoflorine, 0.13 mg kg−1
berberubine, 4.57 mg kg−1 palmatine, 11.4 mg kg−1 jatrorrhizine,36.0 mg kg−1 chlorogenic acid, 1.96 mg kg−1 cryptochlorogenicacid, 1.54 mg kg−1 neochlorogenic acid and 0.25 mg kg−1 fer-ulic acid). The results showed that cryptochlorogenic acid andneochlorogenic acid were not detected in rat plasma after orallyadministrated JJT tablet. The possible reasons might be that theconcentration of these two compounds in plasma was below theLLOQ, or they were not absorbed after oral administration, orthey were metabolized rapidly and their metabolites were notdetected in plasma. Further experiments are in progress to validatethe reason. The mean plasma concentration–time curve profilesof berberine, epiberberine, coptisine, magnoflorine, berberubine,palmatine, jatrorrhizine, chlorogenic acid and ferulic acid areshown in Fig. 3. The compartmental pharmacokinetic analysis ofconcentration–time data was performed by DAS 1.0 software. Theresults showed that all the compounds except palmatine werefitted best to the one-compartment model and palmatine was con-sistent with two-compartment model. The mean pharmacokineticparameters are presented in Table 7.
As shown in Fig 3, multiple plasma concentration peaks wereobserved in the mean plasma concentration curves profiles ofberberine, epiberberine, coptisine, palmatine and jatrorrhizine,which was consistent with results from previous studies [18–20].
Previous studies reported multiple blood concentration peaks inthe alkaloid PK and attributed it to the distribution, reabsorption,
a complex prescription [21–23]. Other studies have demonstratedthat TCMs and natural components from TCMs can be identified asinhibitors, substrates, and/or inducers of a variety of CYP enzymes,and TCMs-CYP interactions may occur and affect the pharmacoki-
Y.-x. Chang et al. / Journal of Pharmaceutical and Biomedical Analysis 117 (2016) 1–10 9
minis
nsT
Tif2acrtloTfpf
cdpetoTdtato
as15wr
Fig. 3. Mean plasma concentration–time profiles in rats after oral ad
etics [23]. The results also indicated that the absorption of theeven alkaloids and two phenolic acids might be rapid with themax values within 2 h.
However, the Cmax of seven alkaloids was less than 20 ng mL−1.he AUC0−24 h values were 80.7 ± 9.89 ng mL−1 h−1 for berber-
ne, 126 ± 19.6 ng mL−1 h−1 for coptisine, 48.2 ± 16.0 ng mL−1 h−1
or epiberberine, 37.9 ± 9.38 ng mL−1 h−1 for palmatine and2.9 ± 6.24 ng mL−1 h−1 for jatrorrhizine. These showed that theselkaloids shared low plasma concentration. Previous studies con-luded that the poor absorption and extensive metabolism mightesult in low plasma concentration of berberine after oral adminis-ration [23]. Because of their similar structures with berberine andower contents for administration, the plasma concentration of thether four protoberberine-type alkaloids was lower. The values of1/2 were in the range of 0.28–12.4 h, which were greatly differentrom previous study [23]. This was probably due to the differentroportions of the components in JJT tablet and the individual dif-
erences in rats.As we can see from Fig 3, the mean plasma concentration–time
urve profiles of magnoflorine and berberubine was remarkablyifferent from the above alkaloids. They only have a typical single-eak in the concentration–time curve. The Tmax value exceededpiberberine and jatrorrhizine and the AUC0 − 24 h value was higherhan epiberberine, jatrorrhizine and palmatine, though the contentf magnoflorine was much lower than them. However, the Tmax and1/2 values were similar to them. The results might be presumablyue to the different physicochemical properties of compounds orhe PK interaction of the prescribed chemical constituents. The Tmax
nd AUC0 – 24 h values of berberubine were low for its low concen-ration in JJT tablet. It is the first time that pharmacokinetic studyf berberubine after oral administration has been reported.
It was found that values of Tmax and T1/2 of chlorogenic acidnd ferulic acid were much closer, demonstrating that they have
imilar absorption and elimination rate. The Cmax values were61 ± 55.2 and 40.1 ± 34.3 ng mL−1 and values of AUC0 – 24 h were21 ± 155 and 46.2 ± 21.7 ng mL−1 h−1 respectively. The resultsere not correspondent with their dosages administered to the
tration of traditional Chinese medicinal preparation Jinqi Jiangtang.
The possible reason may be that some of the ferulic acid detectedin rat plasma was the metabolites of the components in JJT tablet.
3.6. Method comparison with existing reports
There are several studies on evaluating the alkaloids in Rhi-zome Coptidis and its preparations [9–12]. In general, it is essentialto simultaneously determine different kinds of components fromTCMs and preparations in biological samples in clinical practice. Butno analytical methods for simultaneous determination of chloro-genic acid, cryptochlorogenic acid, neochlorogenic acid, ferulicacid, magnoflorine, coptisine, epiberberine, jatrorrhizin, berberine,berberubine and palmatine in biological samples were availablein these studies. These studies mainly focused on the pharma-cokinetics of alkaloids with high contents. There was a report onthe simultaneous determination of wogonin, coptisine, berber-ine, palmatine, jatrorrhizine, phellodendrine, magnoflorine andwogonoside from Huanglian Jiedu Decoction in rat plasma [24].It was the first study on the pharmacokinetics of magnoflorine(low content). However, the LLOQs for coptisine, berberine, jatr-orrhizine, magnoflorine and palmatine were 0.20, 0.48, 0.10, 0.32,and 0.30 ng mL−1, respectively. The berberubine was not detectedin plasma. In our study, the LLOQs were 0.04 ng mL−1 for berberineand palmatine, 0.16 ng mL−1for jatrorrhizine, 0.2 ng mL−1for copti-sine, 2 ng mL−1 for magnoflorine. Though the LLOQ of magnoflorinewas a little higher than the above study, the method described inour study is more suitable for clinical applications. To the best of ourknowledge, this is the first time chlorogenic acid, cryptochlorogenicacid, neochlorogenic acid, ferulic acid, magnoflorine, coptisine,epiberberine, jatrorrhizin, berberine, berberubine and palmatinehas been simultaneously determined using an LC–MS–MS method.
4. Conclusion
For the first time, a rapid, sensitive, and convenient LC-ESI–MS/MS method for the simultaneous determination of fourphenolic acids (neochlorogenic acid, chlorogenic acid, cryp-tochlorogenic acid and ferulic acid) and seven alkaloids (berberine,
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piberberine, coptisine, magnoflorine, berberubine, palmatine andatrorrhizine) in rat plasma has been developed and validated. The
ethod offers the advantages of high sensitivity and simple plasmaample preparation. It was successfully applied to simultaneouslyvaluate the PK properties of the bioactive components after oraldministration of JJT tablet. The PK parameters obtained from thistudy and the validated method would be useful in clinical appli-ations of JJT tablet and other related TCM preparations.
cknowledgments
This research was supported National Natural Science Foun-ation of China (81001632 and 81374050), National Science andechnology Support Program Projects (2014BA105B01), Programor Innovative Research Team in Universities of Tianjin (TD12-033), PCSIRT (IRT-14R41) and State the Science & Technologyommission of MOST of China (2014ZX09304307001). The authorsave declared no conflict of interest.
eferences
[1] J.J. Lee, W.H. Hsu, T.L. Yen, N.C. Chang, Y.J. Luo, G. Hsiao, J.R. Sheu, TraditionalChinese medicine, Xue-Fu-Zhu-Yu decoction, potentiates tissue plasminogenactivator against thromboembolic stroke in rats, J. Ethnopharmacol. 134(2011) 824–830.
[2] L. Wang, G.B. Zhou, P. Liu, J.H. Song, Y. Liang, X.J. Yan, F. Xu, B.S. Wang, J.H.Mao, Z.X. Shen, S.J. Chen, Z. Chen, Dissection of mechanisms of Chinesemedicinal formula Realgar-Indigo naturalis as an effective treatment forpromyelocytic leukemia, Proc. Natl. Acad. Sci. U. S. A. 105 (2008) 4826–4831.
[3] M. Chao, D. Zou, Y. Zhang, Y. Chen, M. Wang, H. Wu, G. Ning, W. Wang,Improving insulin resistance with traditional Chinese medicine in type 2diabetic patients, Endocrine 36 (2009) 268–274.
[4] D.P. Lin, Effects of Jinqi Jiangtang tablet on blood glucose and pancreatic betacell function in 60 patients with type 2 diabetics, Chin. J. New Drugs Clin.Rem. 25 (2006) 5–7.
[5] L.H. Gao, Q. Liu, S.N. Liu, Z.Y. Chen, C.N. Li, L. Lei, S.S. Sun, L.Y. Li, J.L. Liu, Z.F.Shen, A refined-JinQi-JiangTang tablet ameliorates prediabetes by reducinginsulin resistance and improving beta cell function in mice, J.Ethnopharmacol. 151 (2014) 675–685.
[6] J.H. Xiong, S.C. Li, W.J. Wang, Y.P. Hong, K.J. Tang, Q.S. Luo, Screening andidentification of the antibacterial bioactive compounds from Lonicera japonicaThunb. Leaves, Food Chem. 138 (2013) 327–333.
[7] C.L. Kuo, C.W. Chi, T.Y. Liu, The anti-inflammatory potential of berberinein vitro and in vivo, Cancer Lett. 203 (2004) 127–137.
[8] T. Schmeller, B. Latz-Bruning, M. Wink, Biochemical activities of berberine,palmatine and sanguinarine mediating chemical defence againstmicroorganisms and herbivores, Phytochemistry 44 (1997)257–266.
[9] S.J. Lin, H.H. Tseng, K.C. Wen, T.T. Suen, Determination of gentiopicroside,
mangiferin, palmatine, berberine, baicalin, wogonin and glycyrrhizin in thetraditional Chinese medicinal preparation Sann-Joong-Kuey-Jian-Tang byhigh-performance liquid chromatography, J. Chromatogr. A 730 (1996) 17–23.
10] L. Liu, Z. Chen, Analysis of four alkaloids of Coptis chinensis in rat plasma byhigh performance liquid chromatography with electro chemical detection,Anal. Chim. Acta 737 (2012) 99–104.
[
d Biomedical Analysis 117 (2016) 1–10
11] T. Lu, J. ong, F. Huang, Y. Deng, L. Xie, G. Wang, X. Liu, Comparativepharmacokinetics of baicalin after oral administration of purebaicalin, radixscutellariae extract and Huang-Lian-Jie-Du-Tang to rats, J. Ethnopharmacol.110 (2007) 412–418.
12] J. Yuan, Y. Wang, R. An, S. Wang, S.J. Li, J.Y. Jia, S.W. Bligh, X.H. Wang, Y.M. Ma,Simultaneous determination of six alkaloids and one monoterpene in ratplasma by liquid chromatography–tandem mass spectrometry andpharmacokinetic study after oral administration of a Chinese medicineWujiPill, J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 895–896 (2012)154–161.
13] W. Zhou, S.J. Liu, W.Z. Ju, J.J. Shan, M.X. Meng, B.C. Cai, L.Q. Di, Simultaneousdetermination of phenolic acids by UPLC–MS/MS in rat plasma and itsapplication in pharmacokinetic study after oral administration of FlosLonicerae preparations, J. Pharm. Biomed. Anal. 86 (2013) 189–197.
14] W. Li, J. Guo, Y. Tang, H. Wang, M. Huang, D. Qian, J.A. Duan, Pharmacokineticcomparison of ferulic acid in normal and blood deficiency rats after oraladministration of angelica sinensis, Ligusticum chuan xiong and theircombination, Int. J. Mol. Sci. 13 (2012) 3583–3597.
15] Y.X. Chang, A.H. Ge, S. Donnapee, J. Li, Y. Bai, J. Liu, J. He, X. Yang, L.J. Song, B.L.Zhang, X.M. Gao, The multi-targets integrated fingerprinting for screeninganti-diabetic compounds from a Chinese medicine Jinqi Jiangtang Tablet, J.Ethnopharmacol. 164 (2015) 210–222.
16] US Department of Health and Human Services, Food and Drug Administration,Center for Drug Evaluation and Research (CDER), Guidance for Industry,Bioanalytical Method Validation, May 2001.
17] Y.X. Chang, Z.W. Zhu, J. Li, Q.H. Zhang, Y.R. Deng, L.Y. Kang, B.L. Zhang, X.M.Gao, Quantitative determination of anti-inflammatory columbianetin in ratplasma by LC–ESI–MS/MS for pharmacokinetic studies after oraladministration of duhuo extract, Chromatography 74 (2011)639–643.
18] W. He, G.H. Liu, H. Cai, X.M. Sun, W. Hou, P.T. Zhang, Z.Y. Xie, Q.F. Liao,Integrated pharmacokinetics of five protoberberine-type alkaloids in normaland in somnic rats after single and multiple oral administration ofJiao-Tai-Wan, J. Ethnopharmacol. 154 (2014) 635–644.
19] J. Feng, W. Xu, X. Tao, H. Wei, F. Cai, B. Jiang, W.S. Chen, Simultaneousdetermination of baicalin, baicalein, wogonin, berberine, palmatine andjatrorrhizine in rat plasma by liquid chromatography—tandem massspectrometry and application in pharmacokinetic studies after oraladministration of traditional Chinese medicinal preparations containingscutellaria-coptis herb couple, J. Pharm. Biomed. Anal. 53 (2010)591–598.
20] C.C. Peng, S.P. Wang, H.Z. Jin, J.F.G. Jin, W. Wang, P.L. Wei, X.F. Zhang, L. Li, R.H.Liu, W.D. Zhang, Bioanalysis and pharmacokinetics of eight activecomponents from Huanglian Jiedu decoction in rat plasma by LC-ESI–MS/MSMethod, Chin. Herbal Med. 6 (2014) 198–210.
21] Y.T. Deng, Q.F. Liao, S.H. Li, K.S. Bi, B.Y. Pan, Z.Y. Xie, Simultaneousdetermination of berberine, palmatine and jatrorrhizine by liquidchromatography–tandem mass spectrometry in rat plasma and itsapplication in a pharmacokinetic study after oral administration ofcoptis-evodia herb couple, J. Chromatogr. B 863 (2008) 195–205.
22] B. Zan, R. Shi, T.M. Wang, J.S. Wu, Y.M. Ma, N.N. Cheng, Simultaneousquantification of multiple active components from Xiexin decoction in ratplasma by LC-ESI–MS/MS: application in pharmacokinetics, Biomed.Chromatogr. 25 (2011) 816–826.
23] Y. Li, J.P. Gao, X. Xu, L.X. Dai, Simultaneous determination of baicalin, rheinand berberine in rat plasma by column-switching high-performance liquid
chromatography, J. Chromatogr. B 838 (2006) 50–55.
24] T. Lu, Y. Liang, J. Song, L. Xie, G.J. Wang, X.D. Liu, Simultaneous determinationof berberine and palmatine in rat plasma by HPLC-ESI–MS after oraladministration of traditional Chinese medicinal preparationHuang-Lian-Jie-Du decoction and the pharmacokinetic application of themethod, J. Pharm. Biomed. Anal. 40 (2006) 1218–1224.
