MS determination and urinary excretion study of seven alkaloids in healthy Chinese volunteers after oral administration of Shuanghua Baihe tablets

Accepted Manuscript Title: LC-MS/MS determination and urinary excretion study of seven alkaloids in healthy Chinese volunteers after oral administration of Shuanghua Baihe tablets Author: Minlu Cheng Ruijuan Liu Yao Wu Pan Gu Lu Zheng Yujie Liu Pengcheng Ma Li Ding PII: DOI: Reference:

S0731-7085(15)30187-4 http://dx.doi.org/doi:10.1016/j.jpba.2015.10.014 PBA 10290

To appear in:

Journal of Pharmaceutical and Biomedical Analysis

Received date: Revised date: Accepted date:

1-5-2015 7-10-2015 10-10-2015

Please cite this article as: Minlu Cheng, Ruijuan Liu, Yao Wu, Pan Gu, Lu Zheng, Yujie Liu, Pengcheng Ma, Li Ding, LC-MS/MS determination and urinary excretion study of seven alkaloids in healthy Chinese volunteers after oral administration of Shuanghua Baihe tablets, Journal of Pharmaceutical and Biomedical Analysis http://dx.doi.org/10.1016/j.jpba.2015.10.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

LC-MS/MS determination and urinary excretion study of seven alkaloids in healthy Chinese volunteers after oral administration of Shuanghua Baihe tablets

Minlu Chenga, b, 1, Ruijuan Liua, b, 1, Yao Wua, b, Pan Gua, b, Lu Zhengc, Yujie Liuc, Pengcheng Ma d, * *, Li Dinga, b, *

a

Department of pharmaceutical analysis, China Pharmaceutical University, 24 Tongjiaxiang,

Nanjing 210009, P.R. China b

Nanjing Clinical Tech laboratories Inc., 18 Zhilan Road, Jiangning district, Nanjing 211000,

P.R. China c

Yangtze River Pharmaceutical Group, 1 Yangtze River South Road, Gaogang district

Taizhou 225321, P.R. China d

Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical

College, 12 Jiangwangmiao Street, Nanjing 210042, P.R. China

1

Minlu Cheng and Ruijuan Liu are the co-first authors.

*Corresponding author: Prof. Li Ding E-mail: [email protected] Tel.: +86 25 83271485 Fax: +86 25 83271485 **Co-corresponding author: Prof. Pengcheng Ma E-mail: [email protected] Tel.: +86 25 85478929 Fax: +86 25 85471862 1

Graphical abstract

2

Highlights



An LC-MS/MS method for the simultaneous determination of seven alkaloids in human urine



The first urinary excretion study of Shuanghua Baihe tablets in healthy Chinese volunteers



The first excretion profiles of the seven alkaloids except berberine in human urine

3

Abstract An LC-MS/MS method was developed and validated for the simultaneous determination of magnoflorine, berberrubine, jatrorrhizine, coptisine, epiberberine, palmatine and berberine in human urine. The sample preparation procedure involved the four-fold dilution of the urine samples with acetonitrile/water (1:3, v/v). The chromatographic separation was achieved on a Hedera ODS-2 column under gradient elution at a flow rate of 0.4 mL/min with acetonitrile and water containing 0.5% formic acid as the mobile phase. The mass detection was performed in the positive mode. Calibration curves of the seven alkaloids showed good linearity (correlation coefficients >0.9973) over their concentration ranges. To meet the requirements of urinary excretion study for each alkaloid in human, the lower limit of quantification was set at different values from 0.05063 ng/mL to 2.034 ng/mL for the seven alkaloids, respectively. The intra- and inter-batch precision and accuracy were all within ±15%. No matrix effect was observed for the analytes. The validated method was applied to the excretion study for the seven alkaloids in healthy Chinese volunteers after oral administration of Shuanghua Baihe tablets. The average 72 h cumulative urinary excretion of magnoflorine, berberrubine, jatrorrhizine, coptisine, epiberberine, palmatine and berberine accounted for 1.81%, 0.27%, 0.29%, 0.046%, 0.027%, 0.010% and 0.021% of the respective administered dose. Abbreviations: SBT, Shuanghua Baihe tablets; IS, internal standard; LLOQ, lower limit of quantification; LQC, low QC; MQC, medium QC; HQC, high QC;MRM, multiple reaction monitoring; RE, relative error;

Keywords: Berberine; berberrubine; LC-MS/MS; Magnoflorine; Shuanghua Baihe tablets; Urinary excretion

4

1

Introduction Shuanghua Baihe tablets (SBT)[1-3], consisting of Coptidis Rhizoma, Corydalis

Bungeanae Herba, Isatidis Radix, Arnebiae Radix, Lonicerae Japonicae Flos, Lophatheri Herba, Rehmanniae Radix, Lilii Bulbus, Asari Radix et Rhizoma and Snake Bile, is a widely used traditional Chinese medicine formula for treating recurrent oral ulcers and Behcet’s syndrome [4]. It is also reported that SBT could improve clinical symptoms of chemotherapy and radiotherapy induced oral ulcers [5, 6]. Coptidis Rhizoma is the sovereign drug of SBT. Jatrorrhizine, coptisine, epiberberine, palmatine and berberine are the main active alkaloids of Coptidis Rhizoma [7] which have extensive pharmacological effects such as antioxidant [8] and anti-ulcer [9] activities. Other low content alkaloids of Coptidis Rhizoma such as magnoflorine and berberrubine also exhibit a variety of bioactivities [10]. Previous study [11] has reported the characterization of chemical constituents in SBT in rats. Due to the complex mechanisms of action of SBT in human, the principle study on the pharmacokinetics of the SBT active components in human are needed for a better understanding of their absorption, disposition, metabolism and excretion. In this study, the seven active alkaloids, magnoflorine, berberrubine, jatrorrhizine, coptisine, epiberberine, palmatine and berberine, were chosen as the representative components to evaluate their urinary excretion in human. Although studies on urinary excretion profiles of the classical alkaloids such as berberine, palmatine, jatrorrhizine and coptisine have been reported, they were mainly limited to animal experiments [12-16], and few studies focused on magnoflorine and berberrubine. The methods [17-20] for the quantitation of jatrorrhizine, coptisine, epiberberine, palmatine and berberine in traditional Chinese medicine were reported. To our knowledge, the excretion profiles of the seven alkaloids except berberine in human urine have not been reported [21]. There is no report on the simultaneous determination of magnoflorine, berberrubine, jatrorrhizine, coptisine, epiberberine, palmatine and berberine in human urine, and the excretion characteristics of these alkaloids in human urine after oral administration of SBT are also unknown.

5

The purpose of this study was to establish an LC-MS/MS method for the simultaneous determination of magnoflorine, berberrubine, jatrorrhizine, coptisine, epiberberine, palmatine and berberine in human urine, and apply the validated method in urinary excretion study for healthy Chinese volunteers after oral administration of SBT.

2 2.1

Experimental Chemicals and reagents Berberine (86.6% of purity), jatrorrhizine (90.3% of purity), palmatine (86.6% of purity)

and donepezil (100% of purity, internal standard, IS) were purchased from National Institutes for Food and Drug Control (Beijing, China). Coptisine (98% of purity), epiberberine (98% of purity), berberrubine (98% of purity) and magnoflorine (98% of purity) were obtained from Shanghai Youxuan Technology Co., Ltd. (Shanghai, China). Shuanghua Baihe tablets (Batch No. 14032311, 0.6 g/tablet, containing 18.01 mg of berberine, 5.38 mg of epiberberine, 5.11 mg of coptisine, 4.92 mg of palmatine, 2.42 mg of jatrorrhizine, 1.90 mg of magnoflorine and 1.04 mg of berberrubine) were supplied by Yangtze River Pharmaceutical Group (Taizhou, China). Formic acid of analytical grade was provided by Nanjing Chemical Reagent Co., Ltd. (Nanjing, China). Methanol and acetonitrile of HPLC grade were received from Merck KGaA (Darmstadt, Germany). Ultrapure water was obtained from a Milli-Q System (Millipore, Bedford, MA, USA).

2.2

Instrument and LC-MS/MS conditions The chromatographic separation and quantitation of the analytes were performed on

Agilent 1260 Series liquid chromatography (Agilent Technologies, Palo, Alto, CA, USA) coupled with API 4000 tandem mass spectrometer (Applied Biosystems, Foster, USA). Agilent 1260 Series liquid chromatography was equipped with an Agilent 1260 binary pump (model G1312B), a vacuum degasser (model G4225A), an autosampler (model G1367E) and an Agilent 1290 temperature controlled column compartment (model G1330B).

6

The chromatographic separation was achieved on a Hedera ODS-2 column (2.1 mm × 150 mm, 5 μm, Hanbon Sci & Tech, Huai’an, China) with column temperature and flow rate setting as 30°C and 400 μL/min. Gradient elution with the mobile phase consisting of acetonitrile (A) and water containing 0.5% formic acid (B) was utilized to get good chromatographic separation. The gradient elution program was described as follows: (ⅰ) 15% A (0-3.0 min); (ⅱ) 15-20% A (3.0-3.2 min); (ⅲ) 20% A (3.2-14.0 min); (ⅳ) 20-40% A (14.0-15.0 min); (ⅴ) 40% A (15.0-16.0 min); (ⅵ) 40-15% A (16.0-16.1 min); (ⅶ) 15% A (16.1-22.0 min). Quantitation was performed on API 4000 tandem mass spectrometer equipped with a Turbo-V® ionspray source operating in the positive ESI mode. Fig.1 shows the MS/MS spectra of magnoflorine, berberrubine, jatrorrhizine, coptisine, epiberberine, palmatine, berberine and the IS. The optimized multiple reaction monitoring (MRM) parameters for each analyte are displayed in Table 1. The ion spray temperature and ion spray voltage were maintained at 450°C and 5500 V, with nebulizer gas and heater gas set at 40 and 50 psi, respectively. The curtain gas was kept at 30 psi and the collision gas was 12 psi. The system control and data analysis were performed by AB SCIEX Analyst software (version 1.5.2).

2.3

Preparation of calibration standards and quality control samples The stock solutions of magnoflorine, berberrubine, jatrorrhizine, coptisine, epiberberine,

palmatine, berberine and the IS were prepared by respectively dissolving 10 mg of accurately weighed substance in 10 mL methanol. The stock solution (1 mg/mL) of each analyte was further diluted with methanol/water (1:1, v/v) to obtain a series of standard solutions. Then a series of mixed standard working solution were obtained by mixing the standard solution of each analyte at certain concentration. As for the IS, dilute the stock solution with methanol/water (1:1, v/v) to get a working solution at the concentration of 500 ng/mL. All the working solutions were kept at -20°C. Calibration standards and QC samples used to estimate precision and accuracy of the method were prepared from separate stock solutions.

7

The calibration standards and QC samples were prepared by spiking drug-free human urine with the above mixed working solutions. The concentration range and lower limit of quantification (LLOQ) of the seven alkaloids in calibration standards were listed in Table 2. The prepared low QC (LQC), medium QC (MQC) and high QC (HQC) were at the concentrations of 5, 60 and 160 ng/mL for magnoflorine, 0.5, 5 and 40 ng/mL for berberrubine, 0.5, 5 and 40 ng/mL for jatrorrhizine, 0.5, 4 and 16 ng/mL for coptisine, 0.5, 2 and 8 ng/mL for epiberberine, 0.15, 2 and 4 ng/mL for palmatine and 0.5, 4 and 16 ng/mL for berberine, respectively. The dilution QCs at two concentration levels were prepared for magnoflorine (160 and 500 ng/mL), berberrubine (40 and 200 ng/mL), jatrorrhizine (40 and 150 ng/mL), coptisine (16 and 50 ng/mL) and berberine (16 and 80 ng/mL), respectively.

2.4

Urine sample preparation and analysis All of the urine samples were stored at −20°C and allowed to thaw at room temperature

before processing. Aliquot of 200 μL urine sample was spiked with 20 μL of the IS solution (500 ng/mL) and vortex-mixed for 10 s. Then it was vortex-mixed for another 10 s followed by the addition of 600 μL of dilution solvent (acetonitrile-water, 1:3, v/v). The mixture was centrifuged at 15600 rpm for 3 min, then the supernatant was transferred into an autosampler vial. Aliquot of 8 μL supernatant was injected into the LC–MS/MS system for analysis. Urine samples in which the concentration exceeded the upper limit of quantification could be reanalyzed by appropriate dilution with blank human urine.

2.5

Bioanalytical method validation The method was validated for selectivity, carryover effect, linearity, accuracy, precision,

matrix effect, recovery and stability of each analyte in spiked samples according to the guidelines on bioanalytical method validation published by the United States Food and Drug Administration (FDA) and European Medicines Agency (EMA). The results were exhibited in section “Results and discussion”.

8

2.6

Application

2.6.1

Subjects

A total of 12 healthy Chinese volunteers (6 males and 6 females) with age ranging from 19 to 25 (22 ± 2, mean ± SD) and body mass index (BMI) ranging from 19.1 to 23.7 kg/m2 (21.0 ± 1.4 kg/m2, mean ± SD) participated in this urinary excretion study of SBT after signing the informed consent form. All subjects underwent strict physical examinations, electrocardiograms and routine laboratory tests for health assessment. The protocol and statement of informed consent were approved by the Ethics Committee of Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College prior to the start of the study. The study conducted was obedient to the ethical principles established in the Declaration of Helsinki.

2.6.2

Urinary excretion study

This was a randomized and open-label single dose study. Eligible subjects were admitted into the clinic site the day before dosing and fasted for 10 h before drug administration. Each subject was orally administered with SBT (4 tablets, containing 72.04 mg of berberine, 21.52 mg of epiberberine, 20.44 mg of coptisine, 19.68 mg of palmatine, 9.68 mg of jatrorrhizine, 7.60 mg of magnoflorine and 4.16 mg of berberrubine) with 250 mL of water in fast state. No food was permitted until a standardized meal was served 4 h after administration. However, water was allowed as desired 2 h after the drug administration. Neither strenuous exercise nor prolonged bed rest was permitted after dosing. From day 1 to day 4, the urine samples were collected pre-dose and post-dose in the time intervals of 0-2, 2-4, 4-8, 8-12, 12-24, 24-48, 48-60 and 60-72 h. The urine samples in each time interval were well mixed and transferred 20 mL to labeled tubes in duplicates. The volumes of the total collected urine samples for each subject were measured and recorded. All of the urine samples were stored at -20°C until analysis.

2.6.3

Pharmacokinetic analysis

The urine concentrations in all time intervals were directly obtained from the observed data. Parameters for evaluating the urinary excretion characteristics of each alkaloid include 9

excretion amount, excretion rate, cumulative excretion amount and cumulative excretion percentage. The following mathematical equations are utilized for computing the excretion parameters. Excretion amount

Urine concentration

Excretion rate

Excretion amount 2 Time

Cumulative excretion amount Cumulative excretion percentage %

Urine Volume 1

Excretion amount 3

Cumulative excretion amount Dose

100% 4

“Urine concentration” in equation (1) is the observed data in each time interval and “Urine volume” is the recorded data in the same time interval. In equation (2) “Time” refers to the length of the time lasted in the time interval for the urine samples collection. The symbol “n” in equation (3) means the number of the time intervals for collecting the urine samples. “Dose” in equation (4) is the dose amount of each alkaloid in the administered SBT.

3

Results and discussions

3.1

Method development

3.1.1

Mass spectrometer

The mass spectrometer parameters include compound-dependent parameters (DP, EP, CE and CXP) and source-dependent parameters (nebulizer gas, heater gas, curtain gas, collision gas, ion spray temperature and ion spray voltage). The compound-dependent parameters was optimized by injecting the solution of each analyte through a syringe pump. Flow Injection Analysis (FIA) was applied in the optimization of the source-dependent parameters through the automatic function of the AB SCIEX Analyst software. The mass spectrometer parameters set in this study were the optimal ones.

3.1.2

Chromatography

The application of an LC-MS/MS method in the urine analysis is challenging owing to the potential high matrix effect caused by the complex composition of urine. In order to 10

diminish the endogenous interference in the urine, we chose to separate the analyte peaks from the interfering peaks existing in both blank urine and incurred urine samples obtained from the subjects. The composition of mobile phase and the gradient elution programs were optimized to obtain better sensitivity and minimum interference. In this study, acetonitrile rather than methanol was chosen as the organic portion of the mobile phase due to its offered the lower background noise and more appropriate elution strength for the analytes. The use of formic acid in the water portion of the mobile phase was to get better peak shape and mass response to the analytes. The gradient elution program applied in this study was the optimal one.

3.1.3

Sample preparation

Both acetonitrile and methanol were taken into consideration as the dilution solvent. The results proved that the urine samples treated with acetonitrile showed lower background and less interference. To solve the matrix effect from urine components on the analytes and meet the requirements of detection sensitivity for the urine excretion study of the alkaloids, different volume ratios of the dilution solvent to the urine sample (1, 2 and 3) were tested. When the ratio value was set at 3, the urinary matrix effect on the analytes was diminished. In order to improve the peak shape, acetonitrile-water (1:3, v/v) was chosen as the dilution solvent.

3.2

Method validation Blank urine samples obtained from six sources were tested for the interference. The

typical MRM chromatograms of the blank urine sample (A), blank urine sample spiked with the seven alkaloids and the IS (B) and urine sample obtained from a subject after oral administration of SBT (C) were shown in Fig. 2. No endogenous interference in the urine was observed at the retention positions of each analyte. The peaks besides the target analytes present in the chromatograms of the incurred samples but not in the blank urine may be

11

attributed to the complex co-existing compounds in SBT or the multiple metabolites in the incurred urine samples. Carryover effect was assessed by immediately injecting the blank sample after the upper limit of quantification sample, and the response of any interfering peak in the blank sample for each analyte had to be less than 20% of the response of its LLOQ sample. In this study, no carryover effect was observed for each analyte. The typical regression equations of the calibration curves were calculated by least-squares linear regression analysis (weighting factor was 1/x2). The calibration curves of the seven alkaloids showed good linearity over their concentration ranges with weighted correlation coefficients > 0.9973. The results were shown in Table 2. The back calculated concentrations of the calibration standards were within ±15% of the nominal values. Five replicates of LLOQ, LQC, MQC and HQC samples were prepared and analyzed in three analytical batches to evaluate the accuracy (RE, relative error) and the intra- and inter-batch precision (RSD) of the established method for quantification of the alkaloids. The summarized data in Table 3 exhibited good precision and accuracy (RSD<15%, RE within ±15%). The dilution integrity was validated by preparing five replicates of 5-fold diluted QCs at the two concentration levels (low and high). The results of the precision and accuracy of the dilution QCs (RSD<15%, RE within ±15%) showed that urine samples in which the concentration levels exceeded the upper limits of quantification could be analyzed by 5-fold dilution with blank human urine. Matrix effect was assessed by LQC and HQC samples. In this study, IS normalized matrix factors (calculated as the matrix factor ratios of the analytes to the IS) served as the indexes for the assessment of matrix effect. The calculated IS normalized matrix factors were over the range of 94.9% to 102.8%, 86.6% to 94.1%, 95.5% to 98.4%, 104.9% to 108.6%, 107.8% to 111.1%, 87.8% to 99.8% and 96.9% to 97.6% for magnoflorine, berberrubine, jatrorrhizine, coptisine, epiberberine, palmatine and berberine, respectively. The CV of the IS normalized matrix factors calculated from six lots of matrix were less than 15%, suggesting the influence of matrix effect could be ignored. 12

LQC, MQC and HQC samples were involved in the recovery assessment. The mean recoveries of magnoflorine, berberrubine, jatrorrhizine, coptisine, epiberberine, palmatine and berberine ranged from 98.4% to 106.7%, 93% to 110.4%, 99.5% to 110.1%, 101.3% to 103.7%, 100.9% to 106.5%, 100.1% to 107.9% and 98.8% to 107.2%, respectively. The mean recovery of the IS was 101.2%. These data indicated that the recovery of the analytes was consistent, precise and reproducible. The data of spiked samples placed under different storage conditions were compared with that of freshly made ones for three replicates at the low and high concentrations for stability evaluation. Bench-top stability (at room temperature for 8 h), processed sample stability (in autosampler at 4°C for 53 h), freeze and thaw stability (three freeze-thaw cycles) and long-term stability (at -20°C for 41 days) were included in the evaluation. The data of stability samples were all within 15% of the spiked concentrations, which showed that the alkaloids were stable under the above conditions.

3.3

Urinary excretion study The validated method was applied to the determination of the seven alkaloids in the urine

samples collected from the subjects orally administered with SBT. The test results show that the urinary maximum excretion rates were reached at 0-2 h time interval for magnoflorine (9.354 μg/h) and berberrubine (1.820 μg/h), 2-4 h time interval for coptisine (0.5267 μg/h), epiberberine (0.2300 μg/h) and berberine (1.215 μg/h), and 4-8 h time interval for jatrorrhizine (2.059 μg/h) and palmatine (78.28 ng/h). The 72 h cumulative excretion amount of magnoflorine, berberrubine, jatrorrhizine, coptisine, epiberberine, palmatine and berberine were 131.2 μg, 11.16 μg, 27.76 μg, 9.352 μg, 5.870 μg, 1.974 μg and 15.00 μg, respectively, which accounted for 1.81%, 0.27%, 0.29%, 0.046%, 0.027%, 0.010% and 0.021% of the respective administered dose. The mean urinary cumulative excretion percentage-time profiles of the seven alkaloids were shown in Fig. 3. Among the seven isoquinoline alkaloids, magnoflorine was aporphine alkaloid and the other six were protoberberine alkaloids [22]. The 72 h cumulative excretion of aporphine 13

alkaloid was six times more than that of protoberberine alkaloids. In general, the excretion of the unchanged alkaloids in urine accounted for less than 2% of the administered dose for both the aporphine alkaloid and the protoberberine alkaloids. The results indicated that urinary excretion was not the dominant excretion pathway for these prototype alkaloids, and they might be conjugated with glucuronic acid or sulfuric acid to form phase Ⅱ metabolites directly or after biotransformation to the corresponding phase Ⅰ metabolites and then excreted from urine, bile or feces. The poor bioavailability of the quaternary ammonium alkaloids also gives an explanation for the low excretion of the seven alkaloids.

4

Conclusions An LC-MS/MS method for the simultaneous determination of magnoflorine,

berberrubine, jatrorrhizine, coptisine, epiberberine, palmatine and berberine in human urine has been presented and validated for the first time. The method was applied in urinary excretion study of SBT in healthy Chinese volunteers. To our knowledge, there is no report of the excretion profiles of the seven alkaloids except berberine in human urine [21].

The authors have declared no conflicts of interest.

Acknowledgements This study was financially supported by the National Natural Science Foundation of China (No. 81273482) and the Graduate Innovation Fund of Zhejiang Huahai Pharmaceutical Co., Ltd..

14

References [1] P. Gu, R.J. Liu, M.L. Cheng, Y. Wu, L. Zheng, Y.J. Liu, P.C. Ma, L. Ding, Simultaneous quantification and pharmacokinetic study of chlorogenic acid and taurocholic acid by HPLC-MS/MS in human plasma after oral administration of Shuanghua Baihe tablets, Chin J Nat Med, Accepted. [2] Y. Wu, R.J. Liu, P. Gu, M.L. Cheng, L. Zheng, Y.J. Liu, P.C. Ma, L. Ding, Highly sensitive method for simultaneous determination of nine alkaloids of Shuanghua Baihe Tablets in human plasma by LC-MS/MS and its application, J. Chromatogr. B, Accepted. [3] R.J. Liu, P. Gu, L. Wang, M.L. Cheng, Y. Wu, L. Zheng, Y.J. Liu, L. Ding, Study on the pharmacokinetic profiles of corynoline and its potential interaction in traditional Chinese medicine formula Shuanghua Baihe tablets in rats by LC–MS/MS, J. Pharm. Biomed. Anal., 117 (2016) 247-254. [4] J.V. Ghate, J.L. Jorizzo, Behçet’s disease and complex aphthosis, J. Am. Acad. Dermatol., 40 (1999) 1-18. [5] L. Yang, L. Zheng, The clinical value analysis of shuanghua Baihe pill in the prevent of chemotherapy related oral mucositis, Chin. Med. Pharm., (2014) 173-175. [6] L. Zheng, F. zhang, Evaluation of double lily pads in management of head and neck cancer patients after radiotherapy for oral ulcer, Chin. J. Clin. Pharmacol. Oncol. Rehabil., 22 (2015) 230-232. [7] X.C. Qian, L. Zhang, Y. Tao, P. Huang, J.S. Li, C. Chai, W. Li, L.Q. Di, B.C. Cai, Simultaneous determination of ten alkaloids of crude and wine-processed Rhizoma Coptidis aqueous extracts in rat plasma by UHPLC-ESI-MS/MS and its application to a comparative pharmacokinetic study, J. Pharm. Biomed. Anal., 105 (2015) 64-73. [8] T. Yokozawa, A. Ishida, Y. Kashiwada, E.J. Cho, H.Y. Kim, Y. Ikeshiro, Coptidis Rhizoma: protective effects against peroxynitrite-induced oxidative damage and elucidation of its active components, J. Pharm. Pharmacol., 56 (2004) 547-556. [9] J. Jung, J.S. Choi, C.-S. Jeong, Inhibitory Activities of Palmatine from Coptis chinensis Against Helicobactor pylori and Gastric Damage, Toxicol. Res., 30 (2014) 45-48. 15

[10] J.B.I. de la Pena, H.L. Lee, S.Y. Yoon, G.H. Kim, Y.S. Lee, J.H. Cheong, The involvement of magnoflorine in the sedative and anxiolytic effects of Sinomeni Caulis et Rhizoma in mice, J. Nat. Med., 67 (2013) 814-821. [11] L. Zheng, H. Cong, M. Xue, T. Xiang, Z. Yao, B. Wu, W. Lin, Characterization of chemical constituents and rats metabolites of Shuanghua Baihe tablets by HPLC-Q-TOF-MS/MS, Biomed. Chromatogr., 29 (2015) 75-86. [12] B. Tan, Y. Ma, R. Shi, T. Wang, Simultaneous quantification of three alkaloids of Coptidis Rhizoma in rat urine by high-performance liquid chromatography: application to pharmacokinetic study, Biopharm. Drug Dispos., 28 (2007) 511-516. [13] J. Ma, R. Feng, X. Tan, C. Ma, J. Shou, J. Fu, M. Huang, C. He, S. Chen, Z. Zhao, W. He, Y. Wang, J. Jiang, Excretion of Berberine and Its Metabolites in Oral Administration in Rats, J. Pharm. Sci., 102 (2013) 4181-4192. [14] M. Liu, S. Li, S.H. Zhao, H. Wang, H. Wang, P. Tu, Studies on excretion kinetics of ten constituents in rat urine after oral administration of Shensong Yangxin Capsule by UPLC-MS/MS, Biomed. Chromatogr., 28 (2014) 525-533. [15] H. Li, W. Zhang, R. Liu, C. Zhang, T. Han, X. Wang, X. Wang, J. Zhu, C. Chen, Simultaneous determination of four active alkaloids from a traditional chinese medicine Corydalis saxicola Bunting. (Yanhuanglian) in plasma and urine samples by LC-MS-MS, J. Chromatogr. B, 831 (2006) 140-146. [16] H. Li, W. Zhang, C. Zhang, R. Liu, X. Wang, X. Wang, J. Zhu, C. Chen, Bioavailabilty and pharmacokinetics of four active alkaloids of traditional Chinese medicine Yanhuanglian in rats following intravenous and oral administration, J. Pharm. Biomed. Anal., 41 (2006) 1342-1346. [17] P. Ding, L. Chen, Y. Lu, Y. Li, Determination of protoberberine alkaloids in Rhizoma Coptidis by ERETIC H-1 NMR method, J. Pharm. Biomed. Anal., 60 (2012) 44-50. [18] C.Y. Li, S.I. Tsai, A.G. Damu, T.S. Wu, A rapid and simple determination of protoberberine alkaloids in Rhizoma Coptidis by H-1 NMR and its application for

16

quality control of commercial prescriptions, J. Pharm. Biomed. Anal., 49 (2009) 1272-1276. [19] L. Yin, B. Lu, Y. Qi, L. Xu, X. Han, Y. Xu, J. Peng, C. Sun, Simultaneous determination of 11 active components in two well-known traditional Chinese medicines by HPLC coupled with diode array detection for quality control, J. Pharm. Biomed. Anal., 49 (2009) 1101-1108. [20] Y. Wang, R.C. Xu, J. Xiao, J. Zhang, X.H. Wang, R. An, Y.M. Ma, Quantitative analysis of flavonoids, alkaloids and saponins of Banxia Xiexin decoction using ultra-high performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry, J Pharm Biomed Anal, 88 (2014) 525-535. [21] C. Yu, H. Zhang, J. Pan, Y. Hong, J. Ren, D. Zhu, X. Xu, Determination and Preliminary Studies of Metabolism of Berberine in Human Urine After Oral administration, Chin. J. Clin. Pharmacol., 16 (2000) 36-39. [22] Q. Liu, B. Zhou, X. Wang, Y. Ke, Y. Jin, L. Yin, X. Liang, Establishment of a search library about benzylisoquinoline alkaloids based on selective separation on the binaphthyl column and standard analysis on C18 column, J. Sep. Sci., 35 (2012) 3317-3325.

17

Figure captions Fig. 1 MS/MS spectra of magnoflorine, berberrubine, jatrorrhizine, coptisine, epiberberine, palmatine, berberine and the IS.

18

Fig. 2 Typical MRM chromatograms of magnoflorine (Ⅰ), berberrubine (Ⅱ), jatrorrhizine (Ⅲ), coptisine (Ⅳ), epiberberine (Ⅴ), palmatine (Ⅵ), berberine (Ⅶ) and the IS (Ⅷ) in human urine: (A) blank urine sample; (B) blank urine spiked with magnoflorine (2.034 ng/mL), berberrubine (0.3036 ng/mL), jatrorrhizine (0.3036 ng/mL), coptisine (0.2040 ng/mL), epiberberine (0.2043 ng/mL), palmatine (0.05063 ng/mL), berberine (0.2015 ng/mL), and the IS (501.0 ng/mL); (C) urine sample (magnoflorine 48.71 ng/mL, berberrubine 3.943 ng/mL, jatrorrhizine 14.66 ng/mL, coptisine 2.674 ng/mL, epiberberine 1.240 ng/mL, palmatine 0.2410 ng/mL, berberine 2.431 ng/mL) obtained from a subject at 4-8 h after oral administration of SBT.

19

20

Fig. 3 Mean urinary cumulative excretion percentage-time profiles of magnoflorine, berberrubine, jatrorrhizine, coptisine, epiberberine, palmatine and berberine in healthy Chinese volunteers (n = 12) after oral administration of SBT.

21

Table 1 MRM parameters of magnoflorine, berberrubine, jatrorrhizine, coptisine, epiberberine, palmatine, berberine and the IS Precursor ion (Da)

Product ion (Da)

DP (V)

EP (V)

CE (V)

CXP (V)

Magnoflorine

342.2

297.2

37

9

35

36

Berberrubine

322.0

307.2

30

5

37.5

28

Jatrorrhizine

338.1

322.2

60

11.5

38

7

Coptisine

320.1

292.2

19

3

46.5

40

Epiberberine

336.1

320.1

45

13

42

27

Palmatine

352.2

336.2

25

8.5

38

23

Berberine

336.1

292.1

47

3.5

40

18

IS

380.2

243.2

68

7.4

43.5

20

Analytes

a）DP (Declustering Potential); EP (Entrance Potential); CE (Collision Energy); CXP (Collision Cell Exit Potential).

22

Table 2 Linear range, LLOQ, regression equation, correlation coefficient (r) and RSD of the slope of magnoflorine, berberrubine, jatrorrhizine, coptisine, epiberberine, palmatine and berberine

Analytes

Linear range (ng/mL)

LLOQ Regression equation (ng/mL) (w = 1/x2)

RSD of the slope r (%, n=5)

Magnoflorine 2.034-203.4

2.034

y = 0.0418x + 0.0388

0.9999

10.2

Berberrubine

0.3036-50.60

0.3036

y = 0.226x - 0.0118

0.9995

10.8

Jatrorrhizine

0.3036-50.60

0.3036

y = 0.263x - 0.00172

0.9992

3.4

Coptisine

0.2040-20.40

0.2040

y = 0.0342x - 0.000462 0.9983

8.9

Epiberberine

0.2043-10.21

0.2043

y = 0.0845x - 0.00137

0.9973

12.5

Palmatine

0.05063-5.063 0.05063 y = 0.200x + 0.00144

0.9988

2.1

Berberine

0.2015-20.15

0.9998

2.4

0.2015

y = 0.155x + 0.00154

23

Table 3 Precision and accuracy of magnoflorine, berberrubine, jatrorrhizine, coptisine, epiberberine, palmatine and berberine (n = 5) Analytes

Precision (Intra-batch/Inter-batch RSD, %) LLOQ LQC MQC HQC

Accuracy (RE, %) LLOQ LQC MQC HQC

Magnoflorine

5.2/8.6

4.5/5.8

4.8/3.0

7.1/6.9

-5.6

-1.1

-8.4

-10.4

Berberrubine

5.4/2.7

10.5/6.0

5.4/12.9

4.6/9.9

1.8

-9.4

-4.2

-5.4

Jatrorrhizine

3.5/8.4

4.2/7.1

1.8/9.8

1.9/8.0

7.0

4.8

-13.6

-6.1

Coptisine

6.9/11.7

8.2/11.7

2.5/6.8

2.9/5.8

-4.8

-7.1

-8.3

-2.2

Epiberberine

3.7/3.6

5.2/8.1

3.3/4.8

2.5/11.4

10.1

-4.4

-1.8

1.1

Palmatine

10.7/15.4

3.7/7.0

3.5/4.0

3.1/9.4

1.5

5.2

-0.6

-5.3

Berberine

7.3/8.3

6.1/2.2

2.4/0.5

3.1/4.6

3.8

3.7

-2.6

-2.7

24