Simultaneous Determination of Baicalin, Baicalein, Wogonoside

Simultaneous determination of baicalin, baicalein, wogonoside, wogonin, scutellarin, berberine, coptisine, ginsenoside Rb1 and ginsenoside Re of Banxia xiexin decoction in rat plasma by LC-MS/MS and its application to a pharmacokinetic study

Ying Wanga, Yifan Zhangb, Juan Xiaoa, Ranchi Xua, Qiangli Wangc, Xinhong Wanga* a School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China. b School of Pharmacy, Shanghai University of Medicine & Health Sciences, Shanghai 201318, China c School of Basic Medical Sciences, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China.

Abstract A rapid and high sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for simultaneous determination of nine active constituents, baicalin, baicalein, wogonoside, wogonin, scutellarin, berberine, coptisine, ginsenoside Rb1 and ginsenoside Re in rat plasma after oral administration of Banxia xiexin decoction(BXD). Biological samples were processed wtih acetone-ethyl acetate (4:1, v/v). The mobile phase consisted of methanol and water (containing 0.1% formic acid) with gradient elution at a flow rate of 0.3 mL/min. Detection was performed on a triple quadrupole mass spectrometer using positive ion and negative ESI in the multiple reaction monitoring(MRM) mode. The calibration curves for all analytes had good linearity (r > 0.9933). The mean recovery of all the nine active ingredients was more than 75.2 %, and the intra- and inter-day precisions (RSD) were within 12.0%, the accuracy was between 87.4% and 110.4%. This method was successfully applied to the pharmacokinetic study after administration of BXD. The results of pharmacokinetic study might be helpful for BXD clinical reasonable application and further studies on mechanism.

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/bmc.4083

Key words: Banxia xiexin decoction; Determination; LC-MS/MS; Rat plasma; Pharmacokinetic study

1. Introduction Banxia xiexin decoction (BXD), a classic Chinese herbal formula was originally described in Treatise on Febrile Diseases (Shang Han Lun) compiled by Zhongjing Zhang, composed of seven herbs including Rhizoma Pinelliae, Rhizoma Zingiberis, Radix Scutellariae, Rhizoma Coptidis, Radix et Rhizoma Ginseng, Fructus Jujubae and Radix et Rhizoma Glycyrrhizae Preparata Cum Melle, is commonly used to treat diarrhea, borborygmus, nausea and vomiting in ancient China. The BXD is commonly used to treat diarrhea, borborygmus, nausea and vomiting in ancient China, and it is now being utilized to cure gastroenteritis, gastrointestinal mucositis diarrhea and functional dyspepsia. Moreover, BXD has meritorious performances on treatments of digestive ulcer and ulcerative colitis (UC) (Chen et al, 2015; Wan et al, 2014; Zhang et al, 2007). Flavonoids, alkaloids and saponins are the three main active components of BXD (Wang et al, 2014; Yan et al, 2013). Previous studies demostrated that flavonoids from Radix Scutellariae such as baicalin, baicalein, wogonoside, wogonin and scutellarin exhibits anti-inﬂammatory (Sun et al, 2015), anti-oxidative (Woźniak et al, 2015), anti-allergic (Kim et al, 2010) and anti-viral (Li et al, 2011) activities, which are benefit to UC therapy. Alkaloids including berberine and coptisine that are representative chemicals in Rhizoma Coptidis, have been shown to have anti-bacterial, antispasmodic and antineoplastic activities (Chen et al, 2013; Kong et al, 2012; Wang et al, 2015). Ginsenoside Rb1 and ginsenoside Re derived from Radix et Rhizoma Ginseng, are bioactive saponins and are considered for their remarkable pharmacological effects (Jia et al, 2009; Yu et al, 2017). In addition, their anti-inflammatory property in colitis has attracted more attention recently (Zhang et al, 2015). It has been reported that baicalin, berberine and saponins of ginsenosides are effective against anti-ulcerative colitis in rat (Kawashima et al, 2004). However, only the absorbed compounds are considered to be the potential bioactive constituents. Investigation of the constituents in blood is crucial to elucidate the relationship between chemical components in BXD and the pharmacological effects against UC.

To study the pharmacokinetics of nine compounds including baicalin, baicalein, wogonoside, wogonin, scutellarin, berberine, coptisine, the ginsenoside Rb1 and ginsenoside Re in vivo, several analytical methods have been established by HPLC (Li et al, 2011; Tsai et al, 2002), UPLC (Chen et al, 2015), LC-MS (Xia et al, 2008; Zhu et al, 2010), UPLC-MS (Joo et al, 2010), LC-MS/MS (Feng et al, 2010; Tong et al, 2012; Zhang et al, 2016; Zhao et al, 2012) and UPLC-MS/MS (Cui et al, 2015; Qian et al, 2015). The pharmacokinetic study of the constituents in one of the composition herbs in BXD has been reported previously (Wang et al, 2012). But there are few reports about the pharmacokinetic study of the nine active components after administration of BXD in rats simultaneously. Therefore, the aim of the this study was to develop an efficient, sensitive and selective LC-MS/MS method by optimizing the extraction, separation and analytical conditions for simultaneous determination of baicalin, baicalein, wogonoside, wogonin, scutellarin, berberine, coptisine, ginsenoside Rb1 and ginsenoside Re (Fig.1) in rat plasma. Such methods could be very useful to study the pharmacokinetics of BXD for the clinical applications.

2. Material and methods 2.1. Materials, reagents and animals Rhizoma Pinelliae , Rhizoma Zingiberis, Radix Scutellariae, Rhizoma Coptidis, Radix et Rhizoma Ginseng, Fructus Jujubae and Radix et Rhizoma Glycyrrhizae Preparata Cum Melle were purchased from Kangqiao Medicinal Materials Electuary Co., Ltd. (Shanghai, China).

The analytical standards of baicalin, baicalein, wogonin, scutellarin, berberine, carbamazepine (internal standard (I.S.), used in positive ion mode) and naringin (I.S, used in negative ion mode) with purity over 98.0 % were obtained from the Chinese National Institute for Control of Pharmaceutical and Biological Products (Beijing, China). Coptisine (98.0%) was supplied by Jingke Chemical Technologies Co., Ltd. (Shanghai, China). Wogonoside, ginsenoside Rb1 and ginsenoside Re provided by Ronghe Medical Technology Development Co., Ltd. (Shanghai, China) were over 98.3 % purity. HPLC-grade methanol and formic acid were bought from Merck Company (Darmstadt, Germany). Ultrapure water used in the study has prepared by a Millipore water purification system (Millipore, MA, USA). All other reagents were analytical grade.

Six male Sprague-Dawley (SD) rats (220–240 g) were supplied by Experimental Animal Center of Shanghai University of Traditional Chinese Medicine (certificate NO. SCXK 2008-0016). The rats were bred in an environmentally controlled room (22 ± 2 ℃) with unlimited access to food and water until 12 h before the experiments. The animal study was performed in accordance with the National Research Council’s guidelines.

2.2. Instruments and LC-MS/MS conditions LC-MS/MS system consisted of a LC-20AD Shimadzu liquid chromatography system (Shimadzu Corporation, Kyoto, Japan) and a triple quadruple tandem mass spectrometer Thermo Finnigan TSQ Quantum (Thermo Finnigan, USA) equipped with an electrospray ionization source operated in selected reaction monitoring mode detection. The ion spray voltage was set at 3 kV and 2.5 kV in positive and negative conditions separately, and the heated capillary temperature was maintained at 300 ℃. Sheath gas (nitrogen) and collision gas (argon) pressure were kept at 0.21 MPa and 0.2 Pa respectively. The auxiliary gas flow rate was 8 L/min and the other conditions were fixed as the tuning file. The precursor-to-product ion pairs, tube lens and collision energy (CE) for each analyte are described in Table 1. The operation of the LC-MS/MS and analysis of data were performed using the Xcalibur software version 4.1 (Thermo Electron, Waltham, USA).

Liquid chromatography analyses were performed on an Inertsil ODS-SP analytical column

(2.1 mm×100 mm, 5 µm) coupled with an Agilent C18 guard column at room temperature. The mobile phase consists a composition of a 0.1 % formic acid aqueous solution (A) and methanol solution (B) using a gradient elution. Solution B increased from 30 to 60 % over 0-2.5 min, 60-70 % over 2.5-2.6 min, 70-92 % over 2.6-5.5 min and the composition was maintained at 92 % for 1.5 min, and then returned to the initial condition for 2 min. The samples were kept at 4 ℃ in the auto-sampler. The ﬂow rate was 0.3 mL/min with an operating temperature at 30 ℃.

2.3. Preparation of BXD Seven crude herbal drugs (human daily dose of BXD: Rhizoma Pinelliae 12 g, Rhizoma Zingiberis 9 g, Radix Scutellariae 9 g, Rhizoma Coptidis 3 g, Radix et Rhizoma Ginseng 9 g, Fructus Jujubae 6 g and Radix et Rhizoma Glycyrrhizae Preparata Cum Melle 9 g) were immersed in water for 15 min and decocted with a 10-fold amount of distilled water for 30 min twice. After ﬁltration, two decoctions were mixed and concentrated to 57 mL under reduced pressure for oral administration. In order to calculate the administration dosage, the contents of the nine constituents in BXD were quantitatively determined by internal standardization using the same chromatographic conditions as described above.

2.4. Preparation of calibration standards An appropriate amount of baicalin, baicalein, wogonoside, wogonin and scutellarin were dissolved together in methanol to the final concentrations of 41.5 μg/mL, 19.7 μg/mL, 40.1 μg/mL, 20.4 μg/mL, and 19.7 μg/mL respectively. A series of working solutions at different concentration levels of these flavonoids were obtained by further diluting the stock solutions with the mixture of methanol and water (4:1, v/v). Berberine (10.0 μg/mL), coptisine (11.0 μg/mL), ginsenoside Rb1 (11.1 μg/mL) and ginsenoside Re (10.5 μg/mL) were mixed together and their standard working solutions were prepared in the same way as flavonoids. The I.S. solution containing carbamazepine (2.02 μg/mL) and naringin (1.05 μg/mL) was prepared with methanol. All solutions were stored at 4 ℃. Appropriate 10 μL aliquots of each of the stock solutions were taken separately to prepare a mixed stock solution, which was then diluted with the drug-free rat plasma to a series of concentrations spanning a calibration standard range.

2.5. Preparation of plasma samples and quality control samples 100 μL plasma sample was transferred into a polypropylene tube together with 10 μL I.S. solution and 600 μL methanol, 10 μL 10 % ascorbic acid was added as an antioxidant. After vortex-mixing for 5 min and centrifuging at 10,000 rpm for 8 min at -20 ℃, the organic layer was transferred into a new tube (Portion1). 600 μL acetone-ethyl acetate (4:1, v/v) was added to the residue after a vigorously vortex mixing for 5 min and centrifuging at 10,000 rpm for 8

min at -20 ℃. Then the organic phases were separated to Portion1 and evaporated to dryness under gentle nitrogen stream at 37 ℃. Finally, the residue was reconstituted with 100 μL of mobile phase A/B (1:4, v/v) and centrifuged at 16000 rpm for 8 min after vortex-mixing shortly. 10 μL aliquot was analyzed by LC-MS/MS.

Quality control (QC) samples at low, medium and high concentrations were made by spiking appropriate standard solutions into blank rat plasma with the required plasma concentrations following the same sample preparation and operation method described above.

2.6. Method validation The validation of the method in terms of selectivity, linearity, sensitivity (limits of quantitation and detection), method precision, accuracy and matrix effects was performed according to the guidelines for bioanalytical methods.

Selectivity was assessed by comparing the chromatograms of six different rat blank plasma specimens processed by the LLE method with those spiked with each corresponding standard, in order to detect any peaks that interferes the analytes.

For linearity, three independent six-point calibration curves were evaluated by analyzing the peak area ratios of the analytes to the IS versus the spiked concentration of the calibration standard based on least-squares linear regression with a weighted factor (1/X2). The lower limit of quantiﬁcation (LLOQ) was defined as the concentrations with its signal-to-noise ratio of 10, and the acceptable accuracy and precision were limited within 20 % deviation.

For method precision and accuracy, QC samples at three different concentration levels were measured on the same day and five consecutive validation days with six replicates in order to calculate % of relative standard deviation (RSD) and % of relative error (RE). The variation for the precision and accuracy was less than 15 %.

Extraction recovery was carried out by comparing the peak response of the regularly prepared QC samples pre-extracted from spiked matrix (A) with those post-extracted samples spiked with the analytes (B) at three QC levels with six replicates. The ratio (A/B × 100) % was used to evaluate the extraction recovery. The matrix effect was measured by comparing the peak response of post-extracted samples spiked with analytes (B) with that of standard samples (C) extracted from pure water as QC samples. The ratio (B/C×100) % was calculated to evaluate the matrix effect.

Stability tests of the analytes were also conducted using triplicate of spiked plasma samples at three QC levels under several different storage conditions: at room temperature for 4 h for short-term stability study, at -80 ℃ for at least 28 d for long-term stability test, undergoing three freeze–thaw cycles from -80 ℃ to room temperature and 8 h after extracted at 4 ℃ for auto-sampler storage.

For dilution test, six replicate of high concentration QC samples were diluted ten-fold into calibration range with blank plasma, in order to prove the reliability of the method at concentration levels exceeding the highest concentration of the calibration range.

2.7. Method application Six male SD rats (220–240 g) were fostered overnight with unlimited access to water before the experiment. Each rat was given BXD at a single dose of 20 g/kg (containing baicalin 238.0 mg/kg, baicalein 5.04 mg/kg, wogonoside 40.8 mg/kg, wogonin 3.32 mg/kg, scutellarin

4.48 mg/kg, berberine 11.56 mg/kg, coptisine 11.22 mg/kg, ginsenoside Rb1 4.08 mg/kg and ginsenoside Re 3.12 mg/kg) following intragastric administration. The whole blood sample (about 250 µL) was immediately collected into a heparinized centrifuge tube from the suborbital vein before dosing and at 0.083, 0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 12, 24, 32 and 48 h after dosing. Following the centrifugation at 6000 rpm for 8 min, a ﬁxed volume (100 µL) of each plasma sample was transferred into a clean tube and stored at -20 ℃ until analysis. Pharmacokinetic parameters were calculated by non-compartmental pharmacokinetic analysis with the computer program “Drug and Statistics 2.0” (DAS 2.0) (Shanghai University of Traditional Chinese Medicine, China).

3. Results and Discussion 3.1. Optimization of LC-MS/MS conditions In the full scan of MS spectra, baicalin, baicalein and wogonin obtained the most intensive ions of [M+H]+ in positive ion mode. However, the responses of wogonoside and scutellarin in negative ion mode that generated [M-H]+ demonstrated more sensitive than those in positive ion mode. All ﬂavonoid glucuronides, baicalin, wogonoside and scutellarin produced intense aglycone ions corresponding to the neutral loss of a glucuronic acid moiety from the precursor ion respectively. As for protoberberine alkaloids containing quaternary nitrogen, only the positive mode was suitable in ion monitoring. Both berberine and coptisine produced only single charged molecular ions [M]+ with adjusted tube lens volt (90 V/116 V). Ginsenosides ionized well in positive ion mode in this study. Precursor [M+Na]+ ions were further fragmented at 54 V for ginsenoside Rb1, which was higher than those of the other compounds because it was more difficult to liberate glucose units when ionized and combined by Na+. It was recognized that high-intensity product ions were formed in ﬂavonoids with both ion modes, but sufficient ionization of alkaloids and saponins was only present in positive mode. Therefore, the positive and negative modes were simultaneously applied for maximum sensitivity. Internal standards in both ion modes were selected separately, and other mass detection parameters were seriatim optimized (Table 1).

As mentioned previously, the ionization of baicalin and wogonoside produced baicalein and wogonin ions by neutral loss of a glucuronic acid moiety separately, and berberine has a similar structure and the same fragmentation mechanism as compared with coptisine. Therefore, full chromatographic separation was critically needed to avoid any obvious cross-talk effect. For organic phase, methanol was selected for its better separation and resolution property than acetonitrile in this study. With the addition of formic acid to the aqueous phase, the peak symmetry of all analytes, especially alkaloids, was improved significantly. Thus, formic acid of different concentrations (0.05 %, 0.1 % and 0.2 %) was investigated. Finally, 0.1% formic acid was determined as the right concentrations not only for the peak shape of separation but for abundant ionization of all analytes. In addition, saponins such as ginsenoside Rb1 and ginsenoside Re possess weaker polarity than

flavonoids and alkaloids, so the optimized gradient elution was adopted to separate the nine components in a reasonable time cycle.

3.2. Optimization of sample extraction Protein precipitation (PPT) method with methanol or acetonitrile was initially estimated, and the matrix suppression for the determination of the nine components was too large to be neglected (Table 2). Therefore, liquid–liquid extraction (LLE) method with different organic extraction solvents such as ethyl acetate, methyl tert-butyl ether and n-butyl alcohol was tested. As a result, there was no single solvent that could be adopted with high recovery and low matrix effect (85 ~ 115 %) for all the nine analytes (Table 2). However, methanol performed better in extracting all the nine analytes, and acetone combined with ethyl acetate was also efficient in extracting saponins and flavonoids. Thus, the two-step extraction procedure including PPT by methanol and extraction by acetone-ethyl acetate (4:1, v:v) was optimized and explored.

3.3. Method validation 3.3.1 Selectivity Specificity was conﬁrmed by extracting the blank rat plasma from six different matrices and comparing the MRM chromatographic proﬁles with plasma samples spiked with the nine analytes and the IS, as well as plasma samples at 2 h obtained after oral administration of BXD at a dose of 20 g/kg. As shown in Fig. 2, there was no endogenous interference at retention time of the nine analytes and IS.

3.3.2. Linearity and LLOQ Standard calibration curves ranging from 1.00~250.0 to 4.15 ~ 2075 ng/mL for spiked rat plasma demonstrated a good linearity for the analytes. The coefficients of all were > 0.9933, and the deviation of each point on the calibration curve was less than 15 %. LLOQ of baicalin, baicalein, wogonoside, wogonin, scutellarin, berberine, coptisine, ginsenoside Rb1 and ginsenoside Re in plasma was 4.15, 1.97, 4.01, 2.04, 1.97, 1.00, 1.00, 1.11, 1.05 ng/mL, respectively, which are sensitive enough for pharmacokinetic study of BXD. The results are shown in Table 3.

3.3.3. Precision and accuracy The intra-day accuracy was within 87.4 ~ 110.4 % with RSD less than 11.2 %, and the inter-day accuracy was within 85.6 ~ 109.2 % with RSD less than 12.0 % for the nine analytes in three levels of QCs. All the results of the analytical samples are within the acceptable criteria (RE%: ±15 %; RSD%: ≤15 %), indicating that the present method is precise and accurate (Table 4).

3.3.4. Recovery and matrix effect The mean extraction recovery of the nine analytes from rats plasma was more than 75.19 % with the RSD less than 11.2 % at low, middle and high concentration levels (Table 4), indicating that the recovery of the method is consistent and reproducible. The matrix effect of the analytes ranged from 83.80 % to 117.2 % with the RSD lower than 10.9 % at three QC concentration levels (Table 4), indicating that no signiﬁcant matrix effect was observed for the nine analytes. Therefore, no ion suppression occurred in mass detection.

3.3.5. Stability In terms of stability, the RSD of the concentrations measured for the nine analytes at each QC level was within 15 %, implying that they are stable in biosamples under the following four conditions: (i) at room temperature for 4 h; (ii) at -80 ℃ for at least 28 d; (iii) after three freeze-thaw cycles; and (iv) at 4 ℃ in the autosampler for 8 h after being prepared. The results were presented in Table 5.

3.3.6 Dilution test The accuracy of the dilution test samples ranged from -3.0 % to 5.4 % with the RSD lower than 8.2 %, indicating that the concentrations of the nine analytes in plasma could be accurately determined when the sample was diluted by 10 times. In the present study, only baicalin performed high concentration levels in plasma samples which exceeded the calibration range.

3.4. Pharmacokinetic study The newly developed and optimized LC-ESI-MS/MS method was applied successfully to subsequent pharmacokinetic study. The mean plasma concentration-time proﬁles of the analytes are shown in Fig. 3, and the partial main pharmacokinetic parameters are listed in Table 6.

As shown in Fig. 3, all the five flavonoids exhibited the double-peak phenomenon after BXD administration, which may be attributed to enterohepatic circulation (Xing et al, 2005), intestinal flora biotransformation (Noh et al, 2016), site-specific absorption (Zhang et al, 2011), and transporter-involved mechanisms (Kalapos- Kovács et al, 2015). This finding was consistent with previous studies (Cai et al, 2016; Cui et al, 2015; Tong et al, 2012). Among these studies, the first absorption peak of baicalin, baicalein, wogonoside and scutellarin that occurred at 4 h was significantly longer than that of crude Radix Scutellariae extract as reported before. Additionally, comparing the pharmacokinetic parameters after administration orally of the Radix Scutellariae extract or scutellarine compound (Xing et al, 2011), the Cmax and AUC(0-t) values of baicalin, baicalein, wogonoside, wogonin and scutellarin modified by dose were significantly increased than that of the BXD, which indicated better absorption after dosed BXD. We speculated that mutual influences of multiple constituents of BXD such as ginsenosides, licorice flavonoids and saponins might contribute to the phenomena (Cai et al, 2012; Zhang et al, 2013). It was noteworthy that wogonin in BXD had a good absorption in vivo, with higher exposure level (AUC(0-t) 2.879 ± 1.1 mg/L·h) in plasma than baicalein

(AUC(0-t) 1.210±0.22 mg/L·h), which was different from some previous reports, but agree with one other group’s finding (Cai et al, 2016). Moreover, it was the first time to study the pharmacokinetics of scutellarine after administration of BXD.

In the current study, multiple absorption peaks were observed in plasma concentration-time curves of berberine and coptisine after oral administration BXD to rats, which were once thought to be due to enterohepatic circulation and tissue distribution accompanied by blood re-absorption in rats. However, more and more research findings suggested that the site-specific absorption instead of enterohepatic circulation could be the major reason of the multi-peak phenomenon of berberine and coptisine (Li et al, 2011; Qian et al, 2017; Tan et al,

2010). Comparing dose modified Cmax and AUC(0-t) values (Table 5) of berberine and coptisine to the previous pharmacokinetic studies (Feng et al, 2010; Qian et al, 2015), it was repeatedly found that the absorption of the alkaloids was greatly improved in herbal prescriptions. Certain herbs or compounds of BXD such as baicalin may play a P-glycoprotein inhibitor role in promoting the intestinal absorption of Coptidis Rhizoma alkaloids (Wang et al. 2013). On the other hand, the inhibition effect of compounds detected in BXD on various cytochrome P450 enzymes may decrease the metabolism rate and promote the absorption of berberine and coptisine in vivo (Cai et al. 2012).

Ginsenoside Rb1 was found to have only a single peak at about 6 h in the mean plasma concentration-time profile, which was in agreement with previously reported results (Chen et al, 2017; Kim et al, 2013; Wang et al, 2010). These previous studies have proved the poor bioavailability of ginsenosides after orally administered. In the present study, dose modified

Cmax and AUC(0-t) values of ginsenoside Rb1 exhibited an enhanced absorption by co-administration of Radix et Rhizoma Ginseng with other crude drugs of BXD. This phenomenon might be explained by the weakened metabolism of ginsenoside Rb1 rather than the decreased clearance in vivo (Wang et al, 2010; Xiao et al, 2013; Zhao et al, 2012). Other reasons such as influences of the complex prescription on the membrane permeability of intestinal tract need to be future studied. In addition, the plasma concentrations of ginsenoside Re at most of the time points were lower than those of LLOQ. This may be explained by quick absorption and elimination of ginsenoside Re in the rat body, probably by metabolizing into Rg1 and Rh1 by intestinal microflora before absorting into the blood (Kim et al, 2013; Qi et al, 2011).

4. Conclusions A rapid, sensitive and accurate method for the determination of baicalin, baicalein, wogonoside, wogonin, scutellarin, berberine, coptisine, ginsenoside Rb1 and ginsenoside Re in rat plasma by using LC–MS/MS has been developed. The LC–MS/MS working conditions and sample pretreatment were optimized to obtain reliable experimental results. The unique advantage of this method is its ability of allowing for quantification of nine bioactive compounds simultaneously in rat plasma. Most significantly, this method was successfully

applied to the pharmacokinetic study in rats after the oral administration of BXD, which would provide a methodical application for clinical use and further research on traditional Chinese prescriptions.

Acknowledgements This study was supported by Shanghai municipal construction fund for doctoral program [B201511], Shanghai municipal education commission budget projects [2015YSN04] and the connotation development of the secondary vocational school project [2016-FA1-3903-16-115059]. The authors would like to thank Dr. Yanjun Chen for identifying the medicinal herbs purchased for this study.

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Table 1. Optimal conditions for MRM transitions of the analytes and internal standards

Compound m/z CE(V) Tube Lens (V) Trigger Baicalin 447.1→271.1 26 80 + Baicalein 271.1→123.2 32 45 + Wogonoside 459.1→268.0 33 120 - Wogonin 285.1→270.1 24 60 + Scutellarin 461.1→285.2 25 83 - Berberine 336.1→292.2 29 90 + Coptisine 320.0→204.2 46 116 + Ginsenoside Rb1 1131.5→365.3 54 236 + Ginsenoside Re 969.7→203.1 45 240 + Carbamazepine 237.0→194.3 18 98 + Naringin 579.2→271.0 40 100 -

Table 2. Recovery and Matrix effect of different organic solvents for the extraction of 9 compounds in plasma

Nominal Methanol Acetonitrile Ethyl acetate Methyl tert-butyl ether N-butyl alcohol Analytes concentration Recovery Matrix effect Recovery Matrix effect Recovery Matrix effect Recovery Matrix effect Recovery Matrix effect (ng/mL) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) 10.4 87.72±8.0 72.13±5.2 58.27±6.0 74.39±7.2 47.08±6.2 93.84±7.3 - - 61.43±3.1 98.00±7.7 Baicalin 207.5 81.97±8.4 76.06±3.3 51.41±4.4 71.09±3.6 46.54±6.8 86.25±8.7 57.27±1.2 65.72±3.3 66.50±0.3 116.5±4.3 1038 76.90±6.8 65.13±0.8 47.55±3.8 73.46±1.0 54.37±2.1 102.7±8.2 62.06±3.5 72.99±7.4 60.04±0.8 125.8±6.1 4.93 83.81±9.6 66.40±4.2 75.67±8.2 74.34±8.7 62.76±7.3 85.65±11.2 92.71±8.6 88.50±6.5 80.61±3.6 77.67±8.8 Baicalein 98.5 74.85±2.8 73.08±1.8 86.04±6.7 67.51±1.6 56.88±2.1 98.04±7.3 89.39±1.3 105.8±7.0 84.85±9.6 86.29±6.2 492.5 91.44±8.3 69.44±1.3 81.45±3.1 76.22±2.4 53.37±4.7 119.2±9.5 86.55±8.1 107.1±5.6 93.16±6.4 91.16±7.8 10 67.92±4.2 77.50±10.7 60.26±7.1 64.16±5.0 50.92±6.5 106.3±1.6 - - - - Wogonoside 200.5 59.73±0.6 80.92±2.7 49.67±4.0 78.02±3.5 48.43±5.4 89.26±5.7 - - 55.04±4.2 90.63±3.2 1003 58.51±5.1 80.26±2.5 59.40±3.9 76.14±3.3 42.05±3.7 87.00±4.2 - - 63.92±6.6 79.45±4.6 5.1 59.66±1.0 73.34±3.9 52.41±4.5 65.76±10.7 57.89±4.3 122.7±3.1 79.92±6.5 99.41±8.8 88.90±4.8 91.75±11.5 Wogonin 102 63.85±7.3 67.86±0.3 48.55±4.6 69.42±3.4 54.32±8.2 99.56±7.5 88.40±8.2 108.5±3.5 71.23±2.6 86.78±4.0 510 57.32±1.5 62.65±8.8 53.80±2.6 71.29±0.9 60.75±1.3 117.1±7.0 82.49±6.2 124.8±1.0 68.70±2.1 105.4±8.1 4.93 70.86±1.6 95.18±0.6 65.18±7.2 75.16±2.6 44.82±7.2 85.98±9.5 - - 74.04±7.5 88.05±7.9 Scutellarin 98.5 60.98±7.1 77.42±2.0 78.24±7.4 60.70±2.8 51.42±8.1 104.4±9.8 61.05±2.1 66.27±5.4 69.89±2.3 113.4±6.1 492.5 66.42±4.7 81.76±7.1 70.79±4.9 71.23±3.2 47.31±3.9 82.09±0.4 64.52±7.3 72.49±6.2 77.85±7.0 104.6±8.7 2.5 75.85±7.8 66.68±4.7 75.01±3.0 66.60±6.3 39.71±9.6 88.85±9.2 - - - - Berberine 10 68.57±5.8 74.91±5.0 82.17±5.1 61.03±4.8 40.18±5.2 83.37±1.0 - - - - 100 62.65±4.1 66.08±3.2 83.42±0.7 66.56±1.5 52.75±3.3 87.52±6.2 - - - - 2.5 61.96±2.0 74.25±2.8 85.95±7.3 52.21±9.0 49.10±8.9 88.63±9.8 - - - - Coptisine 10 70.73±5.9 75.67±1.3 93.91±5.5 53.07±2.7 56.09±6.9 98.39±5.8 - - - - 100 70.51±6.3 83.10±3.6 90.33±5.8 55.40±3.2 60.42±5.0 103.4±6.9 - - - - Ginsenoside 2.78 80.23±1.1 69.11±5.6 - - 66.35±8.8 93.03±8.3 - - 114.3±1.2 103.8±5.0 Rb1 27.75 88.07±2.5 74.46±6.3 - - 79.00±2.5 106.3±3.3 - - 94.58±8.5 106.5±3.3

138.8 73.32±3.7 79.84±2.0 - - 80.72±7.0 116.8±10.2 - - 111.5±10.4 90.72±0.6 2.63 56.56±0.3 86.17±4.7 - - 65.38±1.6 98.03±5.2 - - 83.79±6.9 97.40±5.2 Ginsenoside 26.25 44.53±3.1 82.05±7.1 - - 71.35±0.7 107.9±4.8 - - 88.24±0.9 88.09±3.9 Re 131.3 50.97±1.9 75.70±9.5 - - 62.27±7.8 115.4±3.1 - - 91.76±8.2 101.8±2.7

Table 3. Linear range, correlation coefficient (r), and lower limit of quantification (LLOQ) of calibration curve employed in the determination of the 9 analytes in rat plasma

LLOQ (S/N ≥10) Analytes Llinear range(ng/mL) Regression equation r (ng/mL) Baicalin 4.15~2075 Y = 0.0014 X+0.0005 0.9991 4.15 Baicalein 1.97~492.5 Y = 0.0006X-0.0001 0.9974 1.97 Wogonoside 4.01~2005 Y = 0.0027X-0.0009 0.9982 4.01 Wogonin 2.04~510.0 Y = 0.0015 X - 0.0002 0.9982 2.04 Scutellarin 1.97~492.5 Y = 0.0004X-0.0001 0.9979 1.97 Berberine 1.00~250.0 Y =0.0019X-0.0013 0.9983 1.00 Coptisine 1.00~250.0 Y =0.0045X-0.0036 0.9971 1.00 Ginsenoside Rb1 1.11~277.5 Y = 0.0108X-0.0044 0.9940 1.11 Ginsenoside Re 1.05~262.5 Y = 0.0041X-0.0009 0.9933 1.05

Table 4. Precision, accuracy, recovery and matrix effect for 9 analytes in rat plasma

Nominal Intra-day precision Inter-day precision concentration Recovery Matrix effect Analytes measure RSD( measure RSD( (%) (%) (ng/mL) RE(%) RE(%) d %) d %) 10.4 10.9 4.8 7.8 10.6 1.9 6.6 87.97±7.2 93.27±4.7 Baicalin 207.5 195.8 -5.6 5.8 203.5 -1.9 4.8 75.19±1.6 88.89±5.5 1038 965.2 -7.0 4.5 1017 -2.0 7.1 83.55±4.5 91.03±4.2 4.93 5.29 7.3 5.0 4.84 -1.8 8.6 77.61±2.1 86.44±5.2 Baicalein 98.50 95.70 -2.8 4.9 100.2 1.7 6.5 85.61±7.9 84.92±9.0 492.5 536.7 9.0 2.4 482.0 -2.1 9.4 106.7±5.6 98.21±3.7 10.0 9.44 -5.6 9.2 9.68 -3.2 8.7 93.74±4.9 117.2±2.3 Wogonoside 200.5 214.5 7.0 2.6 209.8 4.6 4.4 83.46±2.6 102.6±2.2 1003 1107 10.4 2.6 1064 6.1 5.3 79.77±1.4 90.73±6.4 5.10 5.31 4.1 10.2 5.26 3.1 10.2 76.33±6.7 89.00±8.6 Wogonin 102.0 100.7 -1.3 3.4 96.77 -5.1 5.3 91.86±2.1 112.3±4.3 510.0 526.3 3.2 2.9 521.5 2.3 4.7 87.88±2.6 110.0±5.3 4.93 4.70 -4.7 7.1 5.26 6.7 8.7 108.1±5.4 92.94±4.0 Scutellarin 98.50 103.3 4.9 4.2 107.8 9.4 4.5 92.96±5.7 113.2±4.0 492.5 495.6 0.6 5.7 525.8 6.8 5.9 92.56±2.3 86.84±2.2 2.50 2.43 -2.8 10.2 2.24 -10.4 9.0 92.82±8.6 83.80±8.1 Berberine 10.00 9.09 -9.1 1.9 10.11 1.1 8.1 98.85±3.9 99.25±9.8 100.0 97.04 -3.0 6.0 91.60 -8.4 5.2 91.93±6.6 88.15±5.0 2.50 2.66 6.4 3.5 2.73 9.2 4.8 119.9±8.6 105.9±10.9 Coptisine 10.00 10.22 2.2 11.2 9.93 -0.7 9.5 94.93±5.9 98.26±9.4 100.0 92.68 -7.3 6.1 94.02 -6.0 5.8 90.59±7.3 88.88±8.3 2.78 2.43 -12.6 8.7 2.38 -14.4 12.0 104.5±11.2 108.6±8.5 Ginsenoside 27.75 29.31 5.6 7.6 26.88 -3.1 9.1 93.35±2.7 91.84±6.8 Rb1 138.8 140.3 1.1 5.2 144.6 4.2 7.8 89.58±4.6 108.9±2.4 2.63 2.42 -8.0 7.7 2.54 -3.4 9.8 85.39±5.9 90.81±9.9 Ginsenoside 26.25 24.92 -5.1 6.5 25.35 -3.4 7.1 107.8±8.5 96.17±4.1 Re 131.3 137.6 4.8 3.3 134.3 2.3 4.5 86.61±3.4 93.28±6.2

Table 5. Stability of 9 analytes in rat plasma

Nominal Measured concentration (ng/mL)±SD Analytes concentration Condition (i) Condition (ii) Condition (iii) Condition (iv) (ng/mL) 10.4 10.2±1.1 10.4±0.9 10.7±0.9 10.9±1.1 Baicalin 207.5 194.5±7.7 208.3±8.4 212.3±10.6 195.8±7.1 1038 964.9±42.9 996.6±80.1 971.5±67.0 965.7±30.5 4.93 4.6±0.4 4.7±0.6 4.9±0.7 5.3±0.3 Baicalein 98.50 102.2±6.8 105.5±9.3 105.3±5.0 95.7±8.5 492.5 509.7±13.3 498.9±36.9 494.5±26.8 536.8±26.0 10.0 10.1±0.8 11.0±0.9 9.7±0.7 9.4±0.8 Wogonoside 200.5 204.7±11.1 200.0±15.9 194.4±19.3 214.5±14.7 1003 1029.1±42.2 996.5±54.2 990.8±70.9 1108.3±42.4 5.10 4.9±0.6 5.4±0.4 5.1±0.6 5.2±0.7 Wogonin 102.0 100.8±9.2 105.8±12.4 100.3±4.0 96.6±10.4 510.0 506.1±14.9 518.7±12.1 500.4±27.8 518.2±24.7 4.93 4.9±0.4 5.3±0.6 4.5±0.4 4.7±0.4 Scutellarin 98.50 100.1±3.4 101.6±7.3 95.5±2.3 103.3±7.2 492.5 488.5±26.5 483.1±21.6 509.2±25.4 495.5±25.0 2.50 2.5±0.1 2.5±0.2 2.6±0.3 2.4±0.3 Berberine 10.00 10.0±0.8 9.9±0.6 9.8±0.5 9.1±0.5 100.0 96.4±3.6 104.1±6.5 97.8±8.1 97.0±7.0 2.50 2.6±0.2 2.3±0.2 2.5±0.2 2.7±0.4 Coptisine 10.00 9.2±0.2 9.8±0.6 10.1±0.8 10.2±0.9 100.0 100.8±10.7 98.4±5.5 96.8±7.1 92.7±7.2 2.78 2.9±0.3 3.0±0.2 2.8±0.1 2.4±0.2 Ginsenoside Rb1 27.75 27.1±1.0 28.1±1.1 28.2±1.8 29.3±2.5 138.8 143.1±6.7 143.8±7.4 141.9±7.3 140.3±9.5 2.63 2.4±0.3 2.6±0.2 2.8±0.3 2.4±0.2 Ginsenoside Re 26.25 25.8±1.3 25.5±2.1 25.3±1.5 24.9±1.5 131.3 134.1±8.4 132.6±5.9 132.4±6.4 137.6±5.2

Table 6. Pharmacokinetic parameters of baicalin, baicalein, wogonoside, wogonin, scutellarin, berberine, coptisine, ginsenoside Rb1 after oral administration of BXD (20 g/kg) in rats

Cmax/ AUC(0-t)/ AUC(0-∞)/ CL/F/ Parameter Tmax/h t1/2z/h (mg/L) (mg/L·h) (mg/L·h) (L/h/kg) Baicalin 4.830±0.93 5.3±3.3 76.850±9.1 78.997±8.2 8.7±2.6 3.021±0.36 Baicalein 0.102±0.020 6.7±2.1 1.210±0.22 1.224±0.23 6.4±1.1 3.903±0.85 Wogonoside 2.664±0.74 12.0±0.00 41.698±13.0 41.823±13.0 5.3±1.5 1.048±0.30 Wogonin 0.243±0.074 7.7±0.82 2.879±1.1 2.893±1.1 5.4±1.2 1.301±0.54 Scutellarin 0.051±0.022 3.4±1.4 0.686±0.27 0.726±0.26 11.5±4.2 6.820±2.3 Berberine 0.042±0.014 0.92±0.20 0.675±0.27 0.930±0.36 25.5±4.1 14.318±6.3 Coptisine 0.031±0.012 6.0±0.00 0.527±0.10 0.737±0.31 25.6±9.9 17.168±6.0 Ginsenoside Rb1 0.030±0.013 6.0±0.00 0.508±0.23 0.523±0.23 8.8±2.7 8.685±2.6

Figure 1. Chemical structures of baicalin, baicalein, wogonoside, wogonin, scutellarin, berberine, coptisine, ginsenoside Rb1, ginsenoside Re, carbamazepine (I.S.) and naringin (I.S.)

Figure 2. MRM chromatograms of 9 analytes in (A) blank plasma samples, (B) blank plasma samples spiked with the compounds, (C) plasma sample obtained 2h after oral administration of

BXD

Figure 3. The plasma concentration-time proﬁles of baicalin, baicalein, wogonoside, wogonin, scutellarin, berberine, coptisine, ginsenoside Rb1 after oral administration of BXD (20 g/kg) in rats

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