MS and its application in a pharmacokinetic study

Accepted Manuscript Title: Simultaneous Determination of Three Saponins in Human Plasma after Oral Administration of Compound Danshen Dripping Pills by LC-MS/MS and Its Application in a Pharmacokinetic Study Authors: Wei Li, Yi Wu, Mali Wan, Yang Chu, Xiangyang Wang, Shuming Li, Zuhui Liu, Xue Chen, Navaneethakrishnan Polachi, Shuiping Zhou, He Sun PII: DOI: Reference:

S0731-7085(18)32419-1 https://doi.org/10.1016/j.jpba.2019.03.008 PBA 12520

To appear in:

Journal of Pharmaceutical and Biomedical Analysis

Received date: Revised date: Accepted date:

26 October 2018 26 February 2019 4 March 2019

Please cite this article as: Li W, Wu Y, Wan M, Chu Y, Wang X, Li S, Liu Z, Chen X, Polachi N, Zhou S, Sun H, Simultaneous Determination of Three Saponins in Human Plasma after Oral Administration of Compound Danshen Dripping Pills by LC-MS/MS and Its Application in a Pharmacokinetic Study, Journal of Pharmaceutical and Biomedical Analysis (2019), https://doi.org/10.1016/j.jpba.2019.03.008 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.

Simultaneous Determination of Three Saponins in Human Plasma after Oral Administration of Compound Danshen Dripping Pills by LC-MS/MS and Its Application in a Pharmacokinetic Study Wei Li 1,*, Yi Wu 2, #, Mali Wan3,4,* , Yang Chu

4,5,#

1

, Xiangyang Wang 4, Shuming Li 4, Zuhui

1

IP T

Liu 3,4, Xue Chen2,4, Navaneethakrishnan Polachi 4, Shuiping Zhou 4,5, He Sun 4,5,6 School of Pharmacy, North China University of Science and Technology, Tangshan

SC R

063000, China

College of Pharmacy, Jilin University, Changchun 130021, China.

3

China Pharmaceutical University, Nanjing 211198, China

4

Tasly Academy, Tasly Holding Group Co., Ltd., Tianjin 300410, China

5

State Key Laboratory of Core Technology in Innovation Chinese Medicine, Tasly

A

N

U

2

Tasly Pharmaceuticals Inc, Rockville, MD 20850, USA

A

CC

EP

TE D

6

M

Pharmaceutical Group Co., Ltd., Tianjin 300410, China

*These authors contributed equally to this work #Corresponding

author

information:

College

of

Pharmacy,

Jilin

University,

Changchun

Fax:+86-431-85619299; Tel: +86-431-85619299; E-mail address: [email protected] (Y. Wu);

130021,

China.

Division of Pharmacology,

Tasly Academy, Tasly Holding Group Co., Ltd., Tianjin 300410, China. Fax: +86-22-26736376; Tel: +86-22-26735713; E-mail address: [email protected] (Y. Chu). 1

Highlights

SC R



IP T

Graphical abstract

A reliable enough LC-MS/MS method was developed for simultaneous quantification of the notoginsenoside R1, ginsenoside Rg1 and ginsenoside Rb1 in human plasma. A simple precipitating protein method was used to process biological samples.



The method validation was conducted in strict accordance with FDA guidance principle

This was the first report that the PK research of PN has been successfully applied to

M



A

about the bioanalytical method validation.

N

U



ABSTRACT

TE D

Chinese healthy male subjects after oral administration of CDDP.

EP

As one of the main constituents of Compound Danshen Dripping Pills (CDDP), Panax

CC

notoginseng (PN) plays a pivotal role in the treatment of cardiovascular diseases. Numerous researches have proved that the dammarane type saponins including notoginsenoside R1

A

(NR1), ginsenoside Rg1 (GRg1) and ginsenoside Rb1 (GRb1) are the main bioactive components of PN in CDDP. An efficient, realiable and sensitive liquid chromatography tandem-mass spectrometry (LC-MS/MS) analysis method for simultaneously detecting NR1, GRg1 and GRb1 in human plasma was established and applied to the pharmacokinetics study of the three PN saponins after oral administration of CDDP. The human plasma samples were

2

processed using acetonitrile and the target materials were separated on an Eclipse plus C18 column (1004.6 mm, 3.5 m) with a gradient mobile phase consisted of water (containing 0.1% formic acid) and methanol. Within the concentration ranges of 0.25-50 ng/mL, each calibration curve exhibited an excellent linear relationship (r>0.998). The precision deviations of intra-day and inter-day analysis were lower than 9.0%, and accuracy error

IP T

(RE%) ranged between 1.5% and 10.5%. The average recoveries of analytes were >64.0%. The established method was successfully applied to determine the pharmacokinetics of the

SC R

three saponins in human plasma. In addition to providing guidance for clinical safe medication, the experimental results also provided a valuable and reliable basis for further

U

pharmacological studies of PN in the human body after oral administration of CDDP.

N

Keywords: Clinical pharmacokinetics, Compound Danshen Dripping Pills, LC-MS/MS,

1. Introduction

TE D

M

A

Panax notoginseng, Saponins

EP

Compound Danshen Dripping Pills (CDDP), consisting of Radix salvia miltiorrhize

CC

(Danshen, China), Radix Panax notoginseng (Sanqi, China), and Borneolum (Bingpian, China), was developed in accordance with the Traditional Chinese Medicine (TCM) theory

A

and new technologies of modern pharmaceutical development [1]. In 2010, as the first pure Chinese medicine preparation, it completed the FDA Phase II clinical trial (http://clinicaltrials. gov/, NCT00797953) in the USA [2] and it has been demonstrated to have therapeutic effect in the treatment of angina pectoris, coronary arteriosclerosis and improving microcirculation [3,4]. Panax notoginseng (PN), an important tonic and haemostatic Chinese herb medicine [5], 3

plays a crucial role in the clinical treatment using CDDP. Numerous studies have shown that the main bioactive principles of PN are the dammarane type triterpene saponins including 20(S)-protopanaxadiol (PPD) and 20(S)protopanaxatriol (PPT) based on the aglycone moieties [6]. GRb1, as one of the PPD, possesses sugar moieties at the C-3 and C-20 positions but the PPTs, such as NR1 and GRg1,

IP T

have a hydroxyl group at the C-3 position and sugar moieties at the C-6 and (or) C-20 positions [7,8]. Among the total saponin components extracted from the P. notoginseng root,

SC R

the contents of NR1, GRb1 and GRg1 accounted for 2.74%, 29.86% and 20.46%, respectively [9]. Moreover they exhibit important pharmacological effects on improving

U

myocardial ischemia [10], anti-arrhythmia, sedation, anti-diabetic [11], antioxidation [12],

N

and anti-cancer cell proliferation [13]. However, due to the influence of intestinal flora, the

A

plasma concentrations of the three PN saponins are low in the human body [14].

M

For a complex multi-component product, evaluating the pharmacokinetic (PK) properties of the main active constituents may help to better understand the medicinal effects,

TE D

in vivo mechanism as well as clinical application [15,16]. We surveyed a large number of literatures and found that the pharmacokinetics (PK) studies on CDDP were mostly focused on the phenolic acids or diterpenoides from Radix Salvia miltiorrhizae [17-19] and the PK

EP

studies of the three PN saponins from CDDP, especially clinical PK studies, were rare.

CC

Eventhough many PK studies of active saponins from PN have been reported [20,21], the PK results of active ingredients measured after the administration of PN alone is not sufficient to

A

represent the PK results after the administration of the Chinese parent medicines in view of the interactions of different drugs. Previously, Song et al. [22] simultaneously determined the three PN saponins for PK of Compound Danshen tablets, which were in the dog plasma. However, the PK results of PN saponins of CDDP in human plasma have not been reported. In this study, we developed a LC-MS/MS method, which was simple, rapid and reliable

4

for the simultaneous quantifications of the NR1, GRg1 and GRb1 in human plasma. The method was validated in terms of selectivity, linearity, precision, accuracy and stability. This was the first time the PK research of PN was carried out on Chinese healthy male subjects after oral administration of CDDP. The study results provided valuble information for

IP T

pharmacological effects research of the three saponins from PN.

2. Experimental

SC R

2.1. Reagents and materials

NR1, GRg1, GRb1 and Estazolam (internal standard, IS) were all provided by the

U

Chinese National Institute for the Control of Pharmaceutical and Biological Products (Beijing,

N

China). The structural formulas of estazolam and three PN saponins reference substance were

A

shown in Fig.1. CDDP was supplied by Tasly Modern Chinese Medicine Resources Co. Ltd.

M

(Tianjin, China). Formic acid, acetonitrile and methanol (MS grade) were obtained from Merck (Darmstadt, Germany). Ultrapure water was prepared by Milli-Q (Milford, USA)

TE D

water system. Blank plasma was prepared from healthy male volunteers. 2.2. Equipments and chromatographic parameters A LC-MS/MS system equipped with a thermo surveyor four-way infusion pump, an

EP

autosampler, a column oven (Thermo Finnigan, USA) and a TSQ Quantum Discovery MAX

CC

series triple quadrupole tandem mass spectrometer with electrospray ionization (ESI) (Thermo Scientific, San Jose, USA) was used. The chromatographic separation was

A

conducted on an Eclipse plus C18 column (1004.6 mm, 3.5 m) with a C18 pre-column (Phenomenex, USA) using a gradient elution. The temperatures of column and sample room were maintained at 25℃ and 15℃, respectively. The mobile phase consisted of water containing 0.1% formic acid (A) and methanol (B), and the flow rate was set to 0.4 mL/min. Gradient elution was performed according to the following procedure: 0.0-2.0 min (65% B), 5

2.01-5.0 min (65%-90% B), 5.01-6.50 min (90% B), 6.51-9.50 min (90%-65%), 9.51-14.50 min (65% B). The mass parameters of electrospray ionization (ESI) were as follows: positive ion mode; Sheath Gas, 20 psi; Ion Spray voltage, 4000 ev; Temperature (TEM), 350℃; AUX Gas, 10 psi. The quantifications of NR1, GRg1, GRb1 and IS were performed by selective-reaction monitoring (SRM) and the ion collision energies and the ion transitions

IP T

chosen for SRM were shown in Table1. Data analysis was performed by a Xcalibur software. 2.3. Calibration solutions and quality control (QC) samples

SC R

Reference stock solutions of NR1, GRg1 and GRb1 were prepared respectively by accurately weighing a certain amount of standard reference substance, dissolving and diluting

U

to 200 g/mL with methanol. Mixed stock solution containing three analytes were obtained

N

by pipetting the same volume of three reference solutions and continuously diluting to 0.5

A

ng/mL to 100 ng/mL using water-methanol (1:1, v/v). Calibration standards were generated

M

by adding 100 L of the mixed stock solution to blank human plasma and the final concentrations of NR1, GRg1 and GRb1 were 0.25, 0.5, 1, 2, 5, 10, 20 and 50 ng/mL. A 50

TE D

g/mL IS stock solution was obtained by dissolving the reference substance using pure methanol and further diluted to obtain the fresh working solutions of 20 ng/mL. All prepared

EP

solutions were stored at -20℃ and returned to room temperature before analysis. QC samples at three different concentration levels (0.5, 2.0 and 20 ng/mL) were

CC

obtained using similar ways to yield the calibration samples. 2.4. Sample preparation

A

First, the biological samples were thawed at room temperature before treatment. Second,

a 200 L plasma sample was accurately transferred to a 2 mL EP tube, then the IS working solutions (100 L) and acetonitrile (1 mL) were sequentially added to obtain a mixed sample. After vortexing for 1 min, the mixed sample was centrifuged at 12500 rpm for 15 min to obtain a clear supernatant. Next, the supernatant was pipetted and transferred into another EP 6

tube for evaporating at 35℃ to dryness. Finally, the solid residue was redissolved with 50% methanol-water (120 L), centrifuged (12500 rpm for 3 min), and then a 20 L of the supernatant was injected into the instrument for determination. 2.5. Method validation

principle about bioanalytical method validation (draft 2013 version). 2.5.1. Specificity

IP T

The validation of analytical method was conducted in strict accordance to FDA guidance

SC R

To ensure that endogenous substances do not interfere with the determination of the analytes, specificity was evaluated by comparing these chromatograms: blank plasma from

U

six different subjects, blank plasma spiked with NR1, GRg1, GRb1 and IS, and actual plasma

N

samples after oral administeation of CDDP.

A

2.5.2. Linearity and calibration curves

M

The linearity consisting of eight different concentration points was evaluated by correlation coefficient (r>0.99). Calibration curves of NR1, GRg1 and GRb1 were obtained

TE D

by using weighed (1/x2) least squares linear regression baesd on the correlation between the peak area ratio (y) of each analyte/IS and the theoretical concentrations (x) of the corresponding analyte. The acceptable accuracy for each concentration were 85-115% of the

EP

nominal concentrations, except for the lower limit of quantifitation (LLOQ), which was

CC

within 20%. According to the guiding principle, LLOQ was the minimum concentration that the instrument could accurately quantify, where the precision was accepted within 20%.

A

2.5.3. Accuracy and precision The inter-day and intra-day accuracy and precision were investigated through testing

three concentration levels of QC samples on the same day and three independent days, with each concentration measuring six samples. Accuracy, expressed in relative error (RE%), was limited within 15% and precision, represented by relative standard deviation (RSD%), was 7

required to be below 15%. 2.5.4. Recoveries The recoveries of NR1, GRg1, GRb1 and IS were analyzed by comparing the peak area ratio of extracted QC samples (0.5, 2.0 and 20 ng/mL) with those of post-treatment blank plasma samples spiked with analytes of the same concentrations. Each concentration level

IP T

was analyzed with six replicates. 2.5.5. Stability test

SC R

The QC samples of three saponins were prepared to assess stability. Freeze-thaw cycles

stability was investigated after subjected to three freeze-thaw cycles. For processed plasma

U

sample, the stability was assessed by keeping in autosampler (15℃) for 24 h. Room

N

temperature stability was analyzed by testing the QC samples placed at room temperature for

A

24 h. The untreated plasma samples were stored in refrigerator for two weeks at -40℃ and

within the range of 85-115%.

M

then measured to assess long term stability. The stability was accepted when it changed

TE D

2.6. Clinical pharmacokinetic study

In the study, we recruited twelve male volunteers whose ages were from 19 to 45 years

EP

old and BMI was between 19.0 to 25.0 kg/m2. Each volunteer was required to undergo a comprehensive physical examination and all indicators examined were within the normal

CC

range. The entire experimental proposal was permited by the Clinical Medical Ethics Committee and conducted at the Second Affiliated Hospital of Tianjin University of

A

Traditional Chinese Medicine. All volunteers were required to keep a fasting state for 12 h but supplied with water

before giving the CDDP. The CDDP preparation which corresponded to 90 pills was orally administered and blood samples were collected from the human venous blood vessels into heparinized tubes at 0, 0.083, 0.167, 0.333, 0.667, 1, 1.5, 2, 3, 4, 6, 8, 12 and 24 h and 8

centrifuged immediately to obtain plasma samples which were stored at -40℃ until assay. The PK parameters, such as time to reach maximum concentration (Tmax), maximum plasma concentration (Cmax), elimination half-life time (T1/2), time to eliminate 63.2% of the drug and area under plasma concentration and time curve (AUC), were calculated by non-compartmental method using Drug and Statistics 2.0 (Mathematical Pharmacology

IP T

Professional Committee of China, Shanghai, China).

SC R

3. Results and discussion 3.1. Method optimization

U

The analytes and IS were detected respectively in SRM mode to eliminate more noise

N

and background interference. The positive ion mode was adopted by comparing signal peak

A

intensity in both the negative and positive ion modes. In order to obtain the higher signal of

M

selections, we carefully optimized the MS parameters and showed the specific values in Table1. The m/z of extracted ion pairs of NR1, GRg1, GRb1 and IS were chosen at 955775,

TE D

823643, 1131365, 295267, respectively. The precursor and product ion spectrum of [M+Na]+ are shown in Fig.2.

EP

3.2. Method validation 3.2.1. Specificity

CC

Typical chromatogram was displayed in Fig.3 which showed that the retention time of NR1, GRg1, GRb1 and IS were 4.16, 5.07, 8.94 and 6.83 min, respectively. At the same time,

A

we found that no significant endogenous substances or other impurities interfered with the detection of the analytes using the current SRM mode. 3.2.2. Linearity and calibration curves When concentrations were maintained within a range of 0.25-50 ng/mL, the calibration curves of the analytes exhibited better linearity (r>0.998). The accuracy and the precision of 9

LLOQ met the requirement of FDA guideline. The regression equation, correlation coefficients (r) and concentration range were listed in Table2. 3.2.3. Accuracy and precision Accuracy and precision results of intra- and inter-day analysis are displayed in Table3 which indicated that the precision deviations of all concentrations was less than 9.00% and

IP T

the accuracy error was below 11.00% by measuring QC samples at three concentrations. These data suggested that all the results met the critera of the FDA guidelines.

SC R

3.2.4. Recovery and stability

The recoveries of analytes at three QC levels fluctuated between 64.2% and 81.3%. The

U

RSD% values were less than 8.0%. These results indicated that recoveries were stable and the

N

experimental method was feasible for the PK study. The results were shown in Table4.

A

The stabilities of the analytes and IS were measured and the test results showed that the contents of analytes in plasma were not significantly reduced after three freeze-thaw cycles

M

(RE  6.8%, RSD%10.6), storage for 24 h at room temperature (RE  1.2%, RSD%4.0),

8.1%, RSD%6.4).

TE D

in autosampler (4℃) for 24 h (RE  5%, RSD%5.3) and for two weeks at -80℃ (RE 

EP

3.3. Clinical PK application

The validated analytical method was applied to the evaluation of the PK of NR1, GRg1

CC

and GRb1 in human plasma. The mean plasma concentrations versus time curves of the analytes were displayed in Fig.4 and relevant estimated PK parameters were presented in

A

Table5. The elimination half-lifes of NR1, GRg1 and GRb1 were 3.694.91 h, 9.95+10.09 h and 26.2314.89 h, respectively, which indicated that GRb1 had a slower elimination speed than Rg1 and NR1 in human body. The area under the curve of GRg1 was 7 times that of NR1, which is significantly different from the results detected in SD rats [23]. In addition, GRb1 could still be detected at 24h after oral administration of CDDP, which was different 10

from NR1 and GRg1. This was the first clinical data for PK studies of three PN saponins after oral administration of CDDP to Chinese healthy male subjects.

4. Conclusion In this study, a sensitive and reliable LC-MS/MS analytical method was validated for

IP T

simultaneous quantifications of the three PN saponins in human plasma and applied to a clinical PK study of the three PN saponins after oral administration of CDDP. The results of

SC R

PK studies showed that GRb1 had a longer elimination time compared with GRg1 and NR1 in human body, which might be due to different metabolic phathways based on the different

U

structures [24]. The clinical PK results provided a valuable and reliable basis for further

N

pharmacological studies of PN in the human body after oral administration of CDDP.

A

Meanwhile, the results also provided a reference for the formulation of a rational dosage,

TE D

Conflicts of interest

M

frequency of administration and course of treatment in the clinical practice.

All authors involved in this experiment claim that there are no conflicts of interest.

EP

Acknowledgement

CC

The project was supported by the National Project for Standardization of Chinese Materia Medica (ZYBZH-C-TJ-55) and the National Key Special Project of Science and

A

Technology for Innovation Drugs of China (Grant No.2013zx09402202).

References [1] Xin X, Zou HM, Zheng NN, Xu XC, Liu YM, Wang XX, Wu HB et al. Metabonomic Strategy to the Evaluation of Chinese Medicine Compound Danshen Dripping Pills

11

Interfering Myocardial Ischemia in Rats. Evid. Based Complement. Alternat. Med. 2013; 718305. [2] Luo J, Xu H, and Chen KJ. Systematic Review of Compound Danshen Dropping Pills: A Chinese Patent Mwdicine for Acute Myocardial Infarction. Evidence Based Complement. Alternat. Med. 2013; 808076.

IP T

[3] Chu Y, Zhang L, Wang XY, Guo JH, Guo ZX, Ma XH. The effect of Compound Danshen Dripping Pills, a Chinese herb medicine, on the pharmacokinetics and

SC R

pharmacodynamics of warfarin in rats. J. Ethnopharmacol. 2011; 137:1457-1461.

[4] Yang R, Chang L, Guo BY, Wang YW, Wang YL, Jin X, Liu SY and Li YJ. Compound

U

Danshen Drippling Pill Pretreatment to Prevent Contrast-Induced Nephropathy in

N

Patients with Acute Coronary Syndrome Undergoing Percutaneous Coronary

A

Intervention. Evid. Based Complement. Alternat. Med. 2014.

M

[5] Yang TR, Kasai Ryoji, Zhou J, Tanaka Osamu. Dammarane saponins of leaves and seeds of Panax notoginseng. Phytochem. 1983; 22:1473-1478.

TE D

[6] Yang SS, Zhang KZ, Lin X, Miao YQ, Meng LK, Chen W, Tang X. Pharmacokinetic comparisons of single herb extract of Fufang Danshen preparation with different combinations of its constituent herbs in rats. J. Pharm. Biomed. Anal. 2012; 77-85.

EP

[7] Liu HF, Yang JL, Du FF, Gao XM, Ma XT, Huang YH, Xu F, Niu W, Wang FQ, Mao Y,

CC

et al. Absorption and disposition of ginsenosides after oral administration of Panax notoginseng extract to rats. Drug Metab. Dispos. 2009; 37:2290-2298.

A

[8] Zhao J, Qin ZX, Peng B, Liu YG, Liu Y. Fragmentation pathway of ginsenoside in Panax notoginseng using electrospray ionization-quadrupole/time of flight mass spectrometer. J. Chin. Mass Spect. Socie. 2017; 38:97-108. [9] Chen ZH, Li J, Liu J, Zhao Y, Zhang P, Zhang MX and Zhang L. Saponins Isolated from the Root of Panax notoginseng Showed Significant Anti-Diabetic Effects in KK-Ay

12

Mice. Am. J. Chin. Med. 2008; 36:939-951. [10] Yang XC, Xiong XJ, Wang HR, and Wang J. Protective Effects of Panax Notoginseng Saponins on Cardiovascular Diseases: A Comprehensive Overview of Experimental Studies. Evid. Based Complement. Alternat. Med. 2014.

Panax Notoginseng Saponins (PNS). Phytother. Res. 2013.

IP T

[11] Rosette Uzayisenga, Peter Amwoga Ayeka, and Wang Y. Anti-Diabetic Potential of

[12] Zhou NN, Tang Y, Richard F. Keep, Ma XX, Xiang JM. Antioxidative effects of Panax

SC R

notoginseng saponins in brain cells. Phytomed. 2014; 21:1189-1195

[13] He NW, Zhao Y, Guo L, Shang J and Yang XB. Antioxidant, Antiproliferative, and

U

Pro-Apoptotic Activities of a Saponin Extract Derived from the Roots of Panax

N

notoginseng (Burk.) F.H. Chen. J. Med. Food 2012; 15:350-359.

A

[14] Xiao JC, Chen HM, Kang D, Shao YH, Shen BY, Li XN, Yin XX, Zhu ZP, Li HF, Rao T,

M

et al. Qualitatively and Quantitatively Investigating the Regulation of Intestinal Microbiota on the Metabolism of Panax notoginseng saponins. J. Ethnopharmacol. 2016;

TE D

194:324-336.

[15] Zhang ZY, Jia PP, Zhang XX, Zhang QY, Yang HT, Shi H, Zhang LT. LC-MS/MS determination and pharmacokinetic study of seven flavonoids in rat plasma after oral

EP

administration of Cirsium japonicum DC. extract. J. Ethnopharmacol. 2014; 158:66-75.

CC

[16] Lv GY, Lou ZH, Chen SH, Gu H, Shan LT. Pharmacokinetics and tissue distribution of 2,3,5,4’-tetrahydroxystilbene-2-O--D-glucoside from traditional Chinese medicine

A

Polygonum multiflorum following oral administration to rats. J. Ethnopharmacol. 2011; 137: 449-456.

[17] Pei WJ, Zhao XF, Zhu ZM, Lin CZ, Zhao WM, Zheng XH. Study of the determination and pharmacokinetics of Compound Danshen Dripping Pills in human serum by column switching liquid chromatography electrospray ion trap mass spectrometry. J. Chromatogr. 13

B 2004; 809: 237-242. [18] Zheng XH, Zhao XF, Zhao X, Wang SX, Wei YM, Zheng JB. Determination of the main bioactive metabolities of Radix Salvia miltiorrhizae in Compound Danshen Dripping Pills and the tissue distribution of Danshensu in rabbit by SPE-HPLC-MS. J. Sep. Sci. 2007; 30: 851-857.

IP T

[19] Li W, Zhou HJ, Chu Y, Wang XY, Luo RZ, Yang L, et al. Simultaneous determination

and pharmacokinetic of danshensu, protocatechuic aldehyde, 4-hydroxy-3-methyloxy

SC R

phenyl lactic acid and protocatechuic acid in human plasma by LC-MS/MS after oral

administration of Compound Danshen Dripping Pills. J. Pharm. Biomed. Anal. 2017;

U

145: 860-864.

N

[20] Xu QF, Fang XL, Chen DF. Pharmacokinetics and bioavailability of ginsenoside Rb1

A

and Rg1 from Panax notoginseng in rats. J. Ethnopharmacol. 2003; 84:187-192.

M

[21] Li L, Sheng YX, Zhang JL, Wang CS and Guo D. HPLC determination of four active saponins from Panax notoginseng in rat serum and its application to pharmacokinetic

TE D

studies. Biomed. Chromatogr. 2004; 18(10):849-856. [22] Song M, Zhang SY, Xu XY, Hang TJ, Jia L. Simultaneous determination of three Panax notoginseng saponins at sub-nanograms by LC-MS/MS in dog plasma for

CC

3337.

EP

pharmacokinetics of Compound Danshen tablets. J. Chromatogr. B 2010; 878:3331-

[23] You Y, Zhang QY, Zheng Q, Yang M, Xu GL. Pharmacokinetics analysis of multiple

A

active components from Radix salvia miltiorrhizae and Radix notoginseng in rat plasma. Tradit. Chin. Drug Res. Clin. Pharmacol. 2010; 21:614-618.

[24] Ling J, Yu YJ, Long JK, Li Y, Jiang JB, Wang LP, Xu CJ, Duan GL. Indentification of 20 (S)-protopanaxadiol metabolites in human plasma and urine using ultra-performance liquid chromatography coupled with triple quadrupole time-of-flight. J. Ginseng Res.

14

2018; 1-11.

Figure legends Fig.1

Chemical structures of: (A) notoginsenoside R1, ginsenoside Rg1 and Rb1; (B)

estazolam (IS). Production mass spectra of [M+Na]+ ions of: (A) notoginsenoside R1 (m/z

IP T

Fig.2

and (D) estazolam (IS) (m/z 295267) in positive mode. Fig.3

SC R

955775); (B) ginsenoside Rg1 (m/z 823643); (C) ginsenoside Rb1 (m/z 1131365)

Chromatogram of NR1, Rg1, Rb1 and IS in plasma samples: (A) Blank plasma

U

samples; (B) Blank plasma spiked with IS (tR = 6.83 min), ginsenoside Rg1 (tR = 5.07

N

min), notoginsenoside R1 (tR = 4.16 min) and ginsenoside Rb1 (tR = 8.94 min); (C)

Mean plasma concentration-time curves of: (A) notoginsenoside R1, (B) ginsenoside

M

Fig.4

A

Plasma samples obtained from a volunteer at 0.17 h after oral administration of CDDP.

Rg1 and (C) ginsenoside Rb1 in healthy volunteers after oral administration of CDDP

A

CC

EP

TE D

(meanSD, n=12).

15

16

EP

CC

A TE D

IP T

SC R

U

N

A

M

IP T SC R U N A M TE D EP

CC

Table1 The main mass parameters of NR1, Rg1, Rb1 and Estazolam(IS). Table2 The regression equation, concentration range and correlation coefficients(R)

A

Table3 The intra- and inter-day precision and accuracy for the three analytes(n=6) Table4 The extraction recoveries of the analytes and IS(n=6) Table5 The main Pharmacokinetic parameters of NR1, Rg1 and Rb1 in human plasma after oral administration of CDDP (meanSD, n=12)

17

Table1 The main mass parameters of NR1, Rg1, Rb1 and Estazolam(IS). Q1(m/z)

Q3(m/z)

Collision energy

NR1

955

775

40

Rg1

823

643

45

Rb1

1131

365

55

Estazolam(IS)

295

267

22

A

CC

EP

TE D

M

A

N

U

SC R

Note: Q1, parent ion; Q3, product ion.

IP T

Compounds

18

Table2 The regression equation, concentration range and correlation coefficients(r) Concentration range Compounds

Regression equation

r (ng/mL)

Y=0.0014X-0.0008

0.25-50

0.9991

Rg1

Y=0.0012X-0.0012

0.25-50

0.9988

Rb1

Y=0.0014X+0.0005

0.25-50

0.9987

A

CC

EP

TE D

M

A

N

U

SC R

Note: r, the correlation coefficient.

IP T

NR1

19

Table3 The intra- and inter-day precision and accuracy for the three analytes(n=6)

QC-Low

QC-Medium

QC-High

RE (%)

RSD (%)

RE (%)

RSD (%)

RE (%)

RSD (%)

Intra-day

NR1 Rg1 Rb1

2.1 4.5 9.4

4.86 1.96 3.57

5.1 9.2 10.5

4.75 4.54 4.34

6.5 1.5 7.5

5.62 5.48 6.01

Inter-day

NR1 Rg1 Rb1

5.5 3.2 7.1

4.52 2.01 3.52

9.5 8.2 9.1

4.14 8.92 4.54

1.7 0.8 3.6

A

CC

EP

TE D

M

A

N 20

5.46 4.73 5.30

SC R

U

Note: RSD, relative standard deviation; RE, relative error;

IP T

Compounds

Table4 The extraction recoveries of the analytes and IS(n=6) Recovery (%) RSD (%)

Mean  SD

0.5

71.35.52

7.74

2.0

64.21.53

2.38

20

67.72.13

3.15

0.5

79.75.31

6.67

2.0

72.12.45

20

69.31.40

0.5

81.34.15

2.0

72.32.78

Rb1

U

N

Rg1

A

NR1

A

CC

EP

TE D

20

M

20 IS

SC R

(ng/mL)

21

IP T

Nominal Concentration Compounds

3.40 2.02 5.11 3.85

77.42.38

3.08

92.33.23

3.50

Table5 The main Pharmacokinetic parameters of NR1, Rg1 and Rb1 in human plasma after oral administration of CDDP (meanSD, n=12)

Rb1 8.685.63 2.202.10 26.2314.89 39.4422.08

AUC0-t(ngh/mL)

0.690.35

5.102.37

94.0976.73

AUC0-(ngh/mL)

2.001.40

10.776.26

226.38183.45

SC R

IP T

Cmax(ng/mL) Tmax(h) T1/2(h) MRT(h)

NR1 0.670.44 0.700.50 3.694.91 5.546.98

Compounds Rg1 2.511.11 0.390.22 9.9510.09 13.2914.01

Note: Cmax, maximum plasma concentration; Tmax, time to peak plasma concentration; T1/2,

A

CC

EP

TE D

M

A

N

to the last measurable concentration and to infinity.

U

elimination half-life; MRT, mean residence time; AUC0-t and AUC0-, areas under the curve

22