Simultaneous quantification six active compounds in ...

Journal of Chromatography B 1061–1062 (2017) 314–321

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Journal of Chromatography B journal homepage: www.elsevier.com/locate/jchromb

Simultaneous quantification six active compounds in rat plasma by UPLC–MS/MS and its application to a pharmacokinetic study of Pien-TzeHuang

MARK

Wen Xua,b,1, Yiping Zhangb,1, Caijie Zhouc,1, Yanni Taia, Xiaoqing Zhanga, Jie Liua, Mei Shaa, ⁎ ⁎ Mingqing Huanga,b, , Yanlin Zhua, Jun Pengd, Jin-Jian Lue, a

College of Pharmacy, Fujian Key Laboratory of Chinese Materia Medica, Fujian University of Traditional Chinese Medicine, Fuzhou 350122, China Third Institute of Oceanography, State Oceanic Administration, Xiamen 361005, China Shenzhen Key Laboratory of ENT, Institute of ENT, Longgang ENT Hospital, Shenzhen 518172, China d Academy of Integrative Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou 350122, China e State Key Laboratory for Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao, China b c

A R T I C L E I N F O

A B S T R A C T

Keywords: Pian-Tze-Huang UPLC–MS/MS Ginsenosides Muscone Pharmacokinetics

Pien-Tze-Huang (PZH) is a popular traditional Chinese medicine (TCM) formula in China, but its pharmacokinetics has not been investigated yet. To better study the pharmacokinetic behaviors of PZH, an optimal ultraperformance liquid chromatography with triple quadrupole mass spectrometry (UPLC–MS/MS) method was developed for rapid quantification of six compounds (notoginsenoside R1, ginsenosides Re, Rg1, Rb1, Rd, and muscone) in rat plasma after oral administration of PZH. All analytes were extracted by protein precipitation with acetonitrile and separated on a Waters Acquity Cortecs C18 column within 3.9 min, and detected by multiple-reaction monitoring in positive ion mode. This proposed method exhibited good linearity (r ≥ 0.9932) with a lower quantification limits of 0.558–1.566 ng/mL for all analytes. The intra- and inter-day precisions were within 8.24%, and the accuracy was within −10.05 to 9.87% for each analyte. The extraction recovery for each analyte ranged from 80.02 to 96.12%. This UPLC–MS/MS method was successfully applied to the pharmacokinetic study for PZH in rats.

1. Introduction Pien-Tze-Huang (PZH, “Pianzaihuang” in Chinese), which consists of Panax Notoginseng, Bovis Calculus, Snake Gall, and Moschus, is a wellknown TCM formula which first prescribed by a royal physician in the Ming Dynasty (around 1555 AD). PZH is officially used in China for treating traumatic injuries and various inflammation-related diseases [1,2]. In addition, PZH is used in folk medicine for treating various cancers in China and other Southeast Asian countries [3,4]. These diseases are possibly caused by extensive inflammation [5–7]. With the increasing attentions on its multiple efficacy, the systemic pharmacokinetics of PZH must be investigated to elucidate the behavior of bioactive compounds and the potential mechanism of PZH in vivo. Thus far, no work has reported the pharmacokinetics of PZH. The four medicinal components of PZH contains three main types of compounds including ginsenosides from Panax Notoginseng, muscone from Moschus, and steroids from Calculus Bovis and Snake Gall [8]. In

⁎

1

Corresponding authors. E-mail addresses: [email protected] (M. Huang), [email protected] (J.-J. Lu). These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.jchromb.2017.07.033 Received 5 February 2017; Received in revised form 17 July 2017; Accepted 18 July 2017 Available online 23 July 2017 1570-0232/ © 2017 Elsevier B.V. All rights reserved.

our previous study, 27 compounds were isolated and identified from PZH; the in vitro anti-inflammatory activities of these compounds were also evaluated. Of these compounds, notoginsenoside R1, ginsenosides Re, Rg1, Rb1, Rd, and muscone showed potent in vitro anti-inflammatory activities [9]. Other studies also reported that these six compounds elicited anti-inflammatory, anti-virus, anti-tumor, cardiovascular protection, and antioxidant effects [10–18]. Several methods have been established to determine one or several of the six compounds both in vitro and in vivo, including gas chromatography-mass spectrometry (GC–MS) [19], high performance liquid chromatography-ultraviolet detection (HPLC-UV) [20], and HPLC-electrospray ionization mass spectrometry (HPLC-ESI–MS) [21]. However, a compatible quantitative method for the five ginsenosides and muscone in rat plasma has not been developed yet. In this study, we developed an optimal method based on UPLC–MS/ MS to simultaneously determine the six active compounds in rat plasma. This proposed method was validated in terms of specificity,


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were purchased from Merck (Darmstadt, Germany). LC–MS grade formic acid was obtained from Macklin (Shanghai, China), and experimental water was purified by a Millipore Alpha-Q system (Bilerica, MA, USA). PZH samples were collected from Zhangzhou Pien Tze Huang Pharmaceutical Co., Ltd. (Zhangzhou, China). A voucher specimen (S1408056) was stored in the College of Pharmacy, Fujian University of Traditional Chinese Medicine. 2.2. UPLC–MS/MS conditions Chromatography separation was conducted on an ACQUITY UPLC IClass system (Waters, Milford, MA, USA) under the following conditions: column, Waters Acquity Cortecs C18 column (2.1 mm × 100 mm, 1.6 μm); mobile phases, 0.1% (v/v) formic acid in water (A) and acetonitrile (B) with gradient elution (0–0.1 min, 30% B; 0.1–0.8 min, 30%–95% B; 0.8–1.0 min, 95%–100% B; 1.0–2.6 min, 100% B; 2.6–3.9 min, 30% B); flow rate, 0.25 mL/min; column temperature, 45 °C; autosampler temperature, 4 °C; injection volume, 5 μL. MS analysis was performed on a Waters Xevo TQ-S tandem quadrupole mass Spectrometer (Waters, Milford, MA, USA) with ESI source in MRM mode. The detection was achieved under the following conditions: positive ion mode; collision gas, argon; nebulizer gas, nitrogen; heater gas, nitrogen; capillary voltage, 2.5 kV; source temperature, 150 °C; dwell time, 20 ms. MS data were analyzed using Mass Lynx version 4.1 data software. The optimized precursor ion, daughter ion, cone voltage, and collision energy of each analyte were listed in Table 1. 2.3. Determination of the concentrations of six compounds in PZH The quantitative analysis of the six compounds in PZH was previously reported [5]. Briefly, 0.050 g of PZH sample powder was precisely weighed, and ultrasonic extracted with 50 mL of methanol for 30 min. The extracted solution was centrifuged (15000 rpm) for 10 min, the filtered supernatant was used for UPLC–MS/MS analysis. The contents of notoginsenoside R1, ginsenosides Re, Rg1, Rb1, Rd, and muscone in PZH were determined as 27.34, 5.223, 26.57, 59.67, 12.56, and 0.829 mg/g, respectively. 2.4. Standard and quality control samples preparation Fig. 1. Chemical structures of the analytes.

A mix stock standard solution including notoginsenoside R1 (139.8 μg/mL), ginsenosides Re (55.8 μg/mL), Rg1 (88.0 μg/mL), Rb1 (156.6 μg/mL), Rd (117.3 μg/mL), and muscone (153.0 μg/mL) was prepared in methanol and diluted to produce a series of working standard solutions. The IS stock solution of astragaloside IV (400 μg/ mL) in methanol was diluted to get IS working solution (4.0 μg/mL). Calibration standards were produced by spiking of standard working solution (8.0 μL) and IS working solution (10.0 μL) into blank plasma (80.0 μL) to obtain the serial concentrations for notoginsenoside R1

linearity, precision, accuracy, recovery, matrix effects, and stability. This approach was subsequently applied to pharmacokinetic study of PZH at a single oral dose of 400 mg/kg body weight to rats. In addition, since steroid compounds including bile acids and conjugated bile acids are both as endogenous substances and exogenous chemicals, so they are were not suitable pharmacokinetic markers of PZH in rats in this study (Supplementary Fig. S1). The present study can provide more useful information to guide the clinical usage of PZH and the developed analytical method can also be applied for further clinical pharmacokinetic study.

Table 1 Retention time, MRM transitions, and MS parameters of the analytes and internal standard (IS).

2. Experimental

Analyte

Retention time (min)

Precursor Ion (m/z)

Daughter ion (m/z)

Cone voltage (v)

Collision energy (eV)

Notoginsenoside R1 Ginsenoside Re Ginsenoside Rg1 Ginsenoside Rb1 Ginsenoside Rd Muscone Astragaloside IV (IS)

1.03 1.13 1.17 1.53 1.63 3.35 1.70

955.5 969.5 823.5 1131.6 969.5 239.4 807.6

775.2 789.6 643.2 365.2 789.6 95.0 627.4

80 80 93 72 80 20 80

45 42 35 64 48 18 48

2.1. Materials and reagents Authentic standards of notoginsenoside R1, ginsenosides Re, Rg1, Rb1, Rd, muscone, and astragaloside IV (internal standard, IS) were obtained from the National Institutes for Food and Drug Control (Beijing, China). These compounds were determined with the purities > 98% by HPLC or GC, and their chemical structures are presented in Fig. 1. Methanol and acetonitrile (chromatographic grade) 315


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Guidelines for the Care and Use of Laboratory Animals, and were approved by the Animal Ethics Committee of Fujian University of Traditional Chinese Medicine (Fuzhou, China). Powdered PZH sample was dissolved in 0.5% carboxymethyl cellulose sodium salt and was orally administered to the fasted rats at a single dose of 400 mg/kg (10.936 mg/kg of notoginsenoside R1, 2.089 mg/kg of ginsenoside Re, 10.628 mg/kg of ginsenoside Rg1, 23.868 mg/kg of ginsenoside Rb1, 5.024 mg/kg of ginsenoside Rd, and 0.3316 mg/kg of muscone) and the dose was according to the clinical application and animal research [22–24]. Blood samples (0.18–0.20 mL) were collected from retro-orbital plexus without hemolysis and subsequently at 0.083, 0.167, 0.33, 0.5, 1, 2, 4, 6, 8, 12, 24, 36, 48 and 72 h after the administration. During the experiment, all rats were given saline solution freely [25]. The blood samples were immediately centrifuged at 4000 rpm (10 min), and the supernatant plasma were immediately stored at −20 °C before analysis. The pharmacokinetic parameters were calculated using DAS software (2.0 version, China Food and Drug Administration) based on non-compartmental method. All data were recorded as mean ± SD.

(1.398–1398 ng/mL), ginsenosides Re (0.558–558 ng/mL), Rg1 (0.88–880 ng/mL), Rb1 (1.566–1566 ng/mL), Rd (1.173–1173 ng/mL) and muscone (1.53–1530 ng/mL). Four concentration levels of the analytes were used as quality control (QC) samples: 2.796, 27.96, 559.2, and 1398 ng/mL for notoginsenoside R1; 1.116, 11.16, 223.2, and 558 ng/mL for ginsenoside Re; 1.76, 17.6, 352, and 880 ng/mL for ginsenoside Rg1; 3.132, 31.32, 626.4, and 1566 ng/mL for ginsenoside Rb1; 2.346, 23.46, 469.2, and 1173 ng/mL for ginsenoside Rd; and 3.06, 30.6, 612, and 1530 ng/mL for muscone. 2.5. Sample preparation The plasma sample (80 μL) was added with 10 μL of the IS working solution and 8 μL of the blank methanol in a 1.5 mL centrifuge tube, followed by adding acetonitrile (800 μL). The tube was vortex mixed for 2 min and centrifuged (15000 rpm) for 10 min. The upper layer was collected and dried under gentle N2 (37 °C). The dried residue was redissolved with 80 μL of the mobile phase and then centrifuged (15000 rpm) for 10 min. The supernatant was injected into UPLC–MS/ MS system for analysis.

3. Results and discussion 2.6. Method validation 3.1. Method development 2.6.1. Specificity To study the specificity of this method, six batches of rat blank plasma from six different rats were analyzed to exclude potential interference at retention time of each analyte.

2.7. Application to a pharmacokinetic study in rats

3.1.1. UPLC and MS/MS conditions optimization In this study, several important chromatographic conditions including analysis time, signal response and peak shape were optimized. First, the solvent composition of mobile phase was tested. Acetonitrilewater was finally used because it could save analysis time and improve chromatographic separation than methanol-water. Second, different buffer solutions including ammonium acetate, formic acid, and acetic acid were tested. Results showed that 0.1% formic acid exhibited the highest elution strength, optimal separation, and strongest signal response in positive ion mode. Third, gradient elution exhibited shorter analysis duration time and higher separation efficiency than isocratic elution. A series of MS/MS conditions were investigated to achieve the optimal ionization efficiency and sensitivity. Each authentic standard solution was directly infused into the MS to select the optimal precursor and product ions for MRM. Similar to our previous report [9], all analytes were abundantly ionized in positive ion mode. Thus, this mode was employed in the present study. Fig. 2 showed the product ion mass spectra of the six analytes and IS. The MS/MS fragmentation behavior was similar among the five saponin compounds because of their identical structural skeleton, the proposed fragmentation pathways of the six analytes and IS were listed at supplementary Table S1. Compared with two reported analysis methods about PZH [9,26], the present method had several improvements over them. Firstly, mobile phase composition (0.1% formic acid in water-acetonitrile) in this method were obviously simplified compared to those of two published methods (0.1% formic acid and 5% methanol in water-acetonitrile; 0.1% formic acid in water-0.1% formic acid in acetonitrile). Secondly, analysis time (3.9 min) was significantly reduced in this method compared to those of two published methods (13 min; 14 min). Thirdly, for better protecting unstable compound, MS conditions including capillary voltage (2.5 kV) and source temperature (150 °C) were optimized in this method compared to those of two published methods (5.5 kV, 550 °C). Fourthly, LLODs of all analytes in this method were significantly increased (0.186–0.522 ng/mL) compared to those of two published methods (0.207–2.726 ng/mL; 1–20 ng/mL).

Six male SD rats (220–260 g, SPF grade) were obtained from the Laboratory Animal Center of Fujian University of Traditional Chinese Medicine (Fuzhou, China) and kept in environmentally controlled conditions (12 h light/12 h night cycle, 25 °C, 55%–60% relative humidity). All animal experiments were strictly conducted according the

3.1.2. Extraction procedure optimization Sample preparation should be optimized to achieve reproducibility, efficiency, and low cost. Therefore, different common extraction methods were investigated in this study. Results showed solid-phase extraction method using HLB column (Milford, MA, USA) was

2.6.2. Calibration curve and lower limits of quantification and detection Calibration curves were established by plotting the ratios of peak area of each analyte to IS versus the plasma concentrations based on weighted linear least-squares regression model (1/x2). The lower limit of detection (LLOD) and quantification (LLOQ) under present UPLC–MS/MS conditions was determined on the basis of response at a signal-to-noise ratio of 3:1 and 10:1, respectively. 2.6.3. Precision and accuracy To evaluated the intra- and inter-day precision (relative standard deviation, RSD) and accuracy (relative error, RE) of this method, different QC samples for each analyte were determined in six replicates for 3 consecutive days. 2.6.4. Recovery and matrix effect Different QC samples (n = 6) were used to evaluate the recovery and matrix effects of the analytes. The recovery of each analyte was calculated by comparing the amount of each analyte in the processed sample with that of the post-processed spiked sample. The matrix effect was evaluated by comparing the amount of each analyte in the postprocessed spiked sample with that of the pure standard solutions. 2.6.5. Stability The stability of each analyte was measured by determining different QC samples under four different conditions (n = 6). Short- and longterm stability were tested with different QC samples at 25 °C for 12 h and at −20 °C for 30 days, respectively. Freeze-thaw stability was determined with different QC samples after 3 times freeze-thaw. Posttreatment stability was investigated with different QC samples stored in the autosampler at 4 °C for 24 h.

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Fig. 2. Product ion mass spectra of notoginsenoside R1 (A), ginsenoside Rg1 (B), ginsenoside Rb1 (C), ginsenoside Rd (D), ginsenoside Re (E), muscone (F), and IS (G).

unsuitable for multi-sample analysis because it was costly, complicated, and time-consuming. We also compared protein precipitation method and liquid–liquid extraction method. The extraction recovery of the six compounds was not significantly different between the two methods. Thus, protein precipitation method was selected as a simple and rapid procedure in this study. In addition, acetonitrile was prove to be the optimal precipitation solution because of its high extraction efficiency and repeatability.

observed at the respective retention time of each analyte.

3.2. Method validation

3.2.3. Precision and accuracy Table 3 showed that the intra- and inter-day precision (RSD) of all analytes was less than 8.24%, and the accuracy (RE) for all analytes ranged from −10.05% to 9.87%, indicating this method was precise and accurate enough to analyze plasma samples.

3.2.2. Linearity and lower limit of quantification and detection Table 2 showed that all calibration curves had good linearity (r, 0.9932–0.9994). The LLOQs and LLODs for all analytes were 0.558–1.566 ng/mL and 0.186–0.522 ng/mL, respectively. Hence, this method was considered sensitive for quantification of the six compounds.

3.2.1. Selectivity Fig. 3 showed the typical MRM chromatograms of the six analytes and IS in rat plasma. It was obvious that no significant interference was 317


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Fig. 3. Typical MRM chromatograms of the six analytes and IS in rat plasma: (A) blank plasma; (B) blank plasma spiked with the six analytes at LLOQ and IS; and (C) plasma sample collected at 0.5 h after oral administration of PZH (400 mg/kg).

Table 2 Calibration curves and linear ranges of the analytes (n = 8). Analyte

Calibration curve

r

Linear range (ng/mL)

LLOD (ng/mL)

LLOQ (ng/mL)

Notoginsenoside R1 Ginsenoside Re. Ginsenoside Rg1 Ginsenoside Rb1 Ginsenoside Rd Muscone

y = 3.584x − 0.005 y = 3.462x − 0.007 y = 2.949x − 0.017 y = 3.158x − 0.027 y = 2.831x − 0.004 y = 0.2895x + 0.002

0.9989 0.9953 0.9959 0.9969 0.9932 0.9994

1.398–1398 0.558–558 0.880–880 1.566–1566 1.173–1173 1.53–1530

0.466 0.186 0.293 0.522 0.391 0.510

1.398 0.558 0.880 1.566 1.173 1.53

3.2.5. Stability As shown in Table 3, after three freeze-thaw cycles, all analytes were stable in plasma (RE: −9.39% to 10.46%) and at 25 °C for 12 h (RE: −7.64% to 6.59%). All analytes were stable when maintained frozen at −20 °C for 30 days (RE: −10.71% to 8.35%). Moreover, no significant change was observed at 4 °C for 24 h (RE: −5.76% to 7.29%).

3.2.4. Extraction recovery and matrix effect The mean extraction recoveries of notoginsenoside R1, ginsenosides Re, Rg1, Rb1, Rd, and muscone were 84.41-93.65%, 89.36-93.07%, 80.02-96.12%, 82.60-88.54%, 89.64-95.28%, and 90.02-94.69%, respectively. In addition, the recovery of IS (500 ng/mL) was 88.32% (n = 6). These results showed that protein precipitation method can achieve precise analytical results. The matrix effect for each analyte was in the range of 85.23-109.27%, indicating no significant matrix effect on the analysis of each analyte in rat plasma (Table 3).

3.3. Application in pharmacokinetic study As shown in Table 4, four ginsenosides (notoginsenoside R1, 318

2.796 27.96 559.2 1398.0 1.116 11.16 223.2 558.0 1.76 17.6 352.0 880.0 3.132 31.32 626.4 1566.0 2.346 23.46 469.2 1173.0 3.06 30.6 612.0 1530.0

Notoginsenoside R1

319

5.83 4.51 2.23 2.46 5.64 3.26 2.98 2.75 4.81 3.77 2.69 2.33 6.54 4.22 2.55 1.89 7.32 5.65 3.02 2.45 5.45 4.89 3.64 2.67

7.84 −6.32 4.63 3.32 7.61 −5.75 −1.43 −2.85 −9.83 4.65 −3.47 −2.62 −9.74 5.86 −2.73 2.04 9.87 −8.44 5.91 3.46 7.77 5.49 4.83 3.52

6.81 4.66 3.57 2.65 7.93 6.22 4.17 3.04 5.87 5.19 4.10 3.56 6.48 5.62 3.23 3.23 8.24 5.69 3.63 3.49 7.13 6.66 4.33 4.53

Precision (RSD)

Precision (RSD)

Accuracy (REa)

Inter-day (%)

Intra-day (%)

Note: RE is expressed as [measured concentration/nominal concentration) − 1] × 100%.

Muscone

Ginsenoside Rd

Ginsenoside Rb1

Ginsenoside Rg1

Ginsenoside Re

Concentration (ng/ mL)

Analyte

Table 3 Precision, accuracy, extraction recovery, matrix effect, and stability of analytes in rat plasma.

8.99 −5.36 4.01 3.84 9.17 −4.65 −3.85 −1.96 8.25 −9.02 6.27 −2.32 −10.05 −6.74 4.16 2.92 −9.71 −7.98 4.76 −2.37 8.63 −7.17 −5.76 −2.49

Accuracy (RE)

88.50 84.41 93.65 92.72 89.36 93.07 91.43 91.69 92.06 96.12 80.02 83.46 88.54 82.60 87.66 88.35 89.64 95.28 90.66 92.93 91.78 94.69 92.38 90.02

Mean (%)

4.97 5.85 5.13 3.69 5.93 9.36 6.23 4.17 5.00 8.37 6.88 3.66 9.25 5.12 5.19 3.05 5.60 6.16 5.26 3.71 4.09 3.61 5.86 3.58

RSD (%)

Extraction recovery

99.97 105.36 109.27 102.32 98.75 94.26 89.23 90.36 96.19 100.80 107.36 104.24 106.21 90.12 99.01 97.58 100.63 92.82 94.84 95.26 85.23 87.38 88.45 89.12

Mean (%)

4.18 3.44 3.06 2.73 6.56 6.26 6.38 3.42 4.60 8.24 7.46 3.27 8.36 8.66 8.36 3.71 5.89 4.61 6.21 2.21 4.72 6.83 3.93 1.94

RSD (%)

Matrix effect –20 °C for 30 days −5.01 −5.05 −4.74 4.41 6.97 −2.4 −2.76 −3.37 −9.8 −10.71 −9.76 −3.01 −8.69 −3.54 4.44 −3.02 5.49 −8.01 8.35 4.88 8.01 −5.05 −3.96 4.18

At 25 °C for 12 h −4.49 −6.55 4.33 1.98 −7.64 −3.55 4.65 2.48 −6.46 6.31 −5.77 −1.95 −4.69 −6.41 −3.63 −1.84 6.59 −3.26 5.34 1.63 −7.49 −6.55 2.33 2.62

Stability (RE, %)

6.46 −8.11 −6.92 3.99 7.69 −9.39 −5.88 −3.25 5.98 −7.98 −4.86 3.83 −4.97 −8.83 −6.03 −4.96 −7.32 −8.98 10.03 3.68 10.46 −8.15 4.92 4.05

Freeze–thaw cycles

7.29 −5.76 3.56 1.87 5.24 −5.67 3.55 1.49 4.68 −3.33 −3.27 1.36 −4.37 94.41 −2.54 91.88 −4.76 3.74 4.83 −1.96 5.12 −2.15 2.74 2.29

Autosampler at 4 °C for 24 h

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Table 4 Pharmacokinetic parameters of analytes in rats after oral administration of PZH (mean ± SD, n = 6). Analyte

Cmax (μg/mL)

Tmax (h)

Notoginsenoside R1 Ginsenoside Re Ginsenoside Rg1 Ginsenoside Rb1 Ginsenoside Rd Muscone

0.387 ± 0.091 0.034 ± 0.006 0.362 ± 0.056 1.256 ± 0.415 0.156 ± 0.050 0.0223 ± 0.002

0.37 0.26 0.27 1.99 0.52 4.01

± ± ± ± ± ±

t1/2 (h) 0.06 0.05 0.09 0.26 0.13 0.98

1.37 4.53 3.27 22.5 7.48 7.95

± ± ± ± ± ±

0.26 1.02 1.01 4.86 2.07 1.90

AUC0–t (μg h/mL)

AUC0–∞ (μg h/mL)

0.77 ± 0.23 0.05 ± 0.01 0.51 ± 0.18 21.10 ± 4.24 1.263 ± 0.31 0.196 ± 0.061

0.775 ± 0.131 0.063 ± 0.008 0.549 ± 0.122 21.967 ± 2.039 1.310 ± 0.412 0.2197 ± 0.0287

Fig. 4. Mean plasma concentration–time curves of the six analytes after oral administration of PZH at a single dose of 400 mg/kg to rats (mean ± SD, n = 6).

longer time for exert improved therapeutic activities, such as anti-inflammatory effect. It may be due to the influences of other constituents in PZH on the pharmacokinetic parameters of muscone in vivo. Overall, the pharmacokinetic behavior of the six active compounds partly revealed the clinical implication of PZH in various inflammation-related diseases, such as acute or chronic hepatitis.

ginsenosides Re, Rg1 and Rd) were rapidly absorbed (Tmax, 0.26–0.52 h) and slowly eliminated because their plasma concentrations were higher than LLOQ after 12 h post-dose. Although ginsenoside Rd is the derivative of ginsenoside Rb1, in which the glycosyl group attached to C-20 is substituted by hydrogen, but their pharmacokinetic behaviors are remarkably different. With Tmax at 1.99 h, ginsenoside Rb1 was absorbed more slowly than ginsenoside Rd (Tmax at 0.26 h). Moreover, A double-peak phenomenon was observed in ginsenoside Rd in contrast to the typical single peak for ginsenoside Rb1 (Fig. 4). The first peak (at 0.5 h post-dose) rose and fell very quickly, and the second peak (at 4 h post-dose) remained at high levels for several hours. This may be ascribed to the biotransformation of ginsenoside Rb1 into ginsenoside Rd by enzymes in vivo. Previous reports have suggested that ginsenoside Rd is one of the major metabolites of ginsenoside Rb1 produced through deglycosylated metabolism [27,28]. In addition, some pharmacokinetic parameters of above five ginsenosides in this study were different from the literature reported [29,30], and it could be explained by the difference of dosage, the administration way and the interactions from complex constituents of this formula [31]. The pharmacokinetics of pure muscone in rat has also been investigated [32], but part of its parameters different from the results observed in the present study. For example, the mean values of Tmax (4.01 h) and t1/ 2 (7.95 h) are higher than those in the literature (Tmax = 0.42 h; t1/ 2 = 1.50 h). A high Tmax allows muscone to remain in the body for a

4. Conclusion A rapid, and accurate UPLC–MS/MS method was developed for simultaneous detection of notoginsenoside R1, ginsenosides Re, Rg1, Rb1, Rd, and muscone in rat plasma. The method features easy sample preparation, short analysis time, and high efficiency. This work is the first pharmacokinetic study of notoginsenoside R1, ginsenosides Re, Rg1, Rb1, Rd, and muscone in vivo following the oral administration of PZH. The results could provide a basis for elucidating the behavior of the bioactive compounds of PZH in vivo and for clinical application of this TCM formula. Acknowledgements The work was supported by the National Natural Science Foundation of China (81503204, 81673561), the Natural Science Foundation of Fujian Province (2017J01838), the Program for New 320


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Century Excellent Talents and Outstanding Young scientific researcher in Fujian Province University ([2015]54, [2016]), the Fujian Province College Students’ Innovative Entrepreneurial Training Plan Project (201610393010, 201410393068), the School Supervision Subject of Fujian University of Traditional Chinese Medicine (X2015015, X2014132) and the Research Fund of University of Macau (MYRG201500091-ICMS-QRCM, MYRG2015-00101-ICMS-QRCM).

[14] [15] [16] [17] [18]

Appendix A. Supplementary data

[19]

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jchromb.2017.07.033.

[20] [21] [22]

References

[23] [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13]

C.P. Commission, Chin. Med. Sci. Tech. Pre. (2015) 670. W.Y. Xu, G.F. Yan, Heilongjiang Med. J. 16 (2003) 542–544. J. Lin, J. Feng, Y. Jin, Z. Yan, Z. Lai, J. Peng, Oncol. Rep. 36 (2016) 3568–3576. Y.Y. Xu, E.X. Yu, Shanghai J. Trad. Chin. Med. 12 (1) (1994) 4–5. J. Lieberthal, N. Sambamurthy, C.R. Scanzello, Osteoarthr. Cartilage 23 (2015) 1825–1834. J. Cheng, W.F. Zhao, Z.Q. Zou, M.X. Zhang, G.Q. Wang, Medicine 94 (2015) e2003. D.R. Powell, A. Huttenlocher, Trends. Immunol. 37 (2016) 41–52. M. Huang, H. Zhao, W. Xu, K. Chu, Z. Hong, J. Peng, L. Chen, J. Sep. Sci. 36 (2013) 3866–3873. M. Huang, W. Xu, Y. Zhang, J. Liu, X. Zhang, J. Lin, J. Peng, J. Lu, J. Chromatogr. B 1027 (2016) 27–39. K.P. Xia, H.M. Ca, C.Z. Shao, Pharmazie 70 (2015) 740–744. X. Zhang, Y. Wang, C. Ma, Y. Yan, Y. Yang, X. Wang, W.D. Rausch, Am. J. Neurodegener. Dis. 5 (2016) 52–61. E.J. Shin, S.W. Shin, T.T. Nguyen, D.H. Park, M.B. Wie, C.G. Jang, S.Y. Nah, B.W. Yang, S.K. Ko, T. Nabeshima, H.C. Kim, Mol. Neurobiol. 49 (2014) 1400–1421. F. Tang, M. Lu, L. Yu, Q. Wang, M. Mei, C. Xu, R. Han, J. Hu, H. Wang, Y. Zhang, J.

[24] [25] [26] [27] [28] [29] [30] [31] [32]

321

Cardiovasc. Pharmacol. (2016), http://dx.doi.org/10.1097/FJC. 0000000000000410. M. Jang, M.J. Lee, J.H. Choi, E.J. Kim, S.Y. Nah, H.J. Kim, S. Lee, S.W. Lee, Y.O. Kim, I.H. Cho, J. Pain 17 (2016) 282–297. L. Yu, N. Wang, Y. Zhang, Y. Wang, J. Li, Q. Wu, Y. Liu, Neurochem. Int. 70 (2014) 10–21. Q.Q. Liang, M. Zhang, Q. Zhou, Q. Shi, Y.J. Wang, Clin. Orthop. Relat. Res. 468 (2010) 1600–1610. Y. Gao, S. Chu, J. Li, Z. Zhang, C. Xia, Y. Heng, M. Zhang, J. Hu, G. Wei, Y. Li, N. Chen, J. Ethnopharmacol. 173 (2015) 231–240. K.M. Joo, J.H. Lee, H.Y. Jeon, C.W. Park, D.K. Hong, H.J. Jeong, S.J. Lee, S.Y. Lee, K.M. Lim, J. Pharm. Biomed. Anal. 51 (2010) 278–283. W. Chang, L. Han, H. Huang, B. Wen, C. Peng, C. Lv, W. Zhang, R. Liu, J Chromatogr B Analyt Technol Biomed Life Sci 963 (2014) 47–53. Q.F. Xu, X.L. Fang, D.F. Chen, J. Ethnopharmacol. 84 (2003) 187–192. J. Zhao, C. Su, C.P. Yang, M.H. Liu, L. Tang, W.W. Su, Z.Q. Liu, J. Pharm. Biomed. Anal. 64–65 (2012) 94–97. L.Y. Deng, F.H. Li, G. Ji, Y.C. Cao, X. Zhang, Chin. J. Exp. Tradit. Med. Form. 21 (2015) 124–128. J. Meng, X.Y. Huang, Q. Li, L.Q. Zhu, Chin. J. Integr. Med. Cardio./Cerebrovasc. Dis. 13 (2015) 754–757. Q. Li, X.Y. Huang, L.Q. Zhu, Chin. J. Neuroanat. 28 (2012) 464–468. K.H. Diehl, R. Hull, D. Morton, R. Pfister, Y. Rabemampianina, D. Smith, J.M. Vidal, C. van de Vorstenbosch, J. Appl. Toxicol. 21 (1) (2001) 15–23. M. Huang, H. Zhao, W. Xu, K. Chu, Z. Hong, J. Peng, L. Chen, J. Sep. Sci. 36 (2013) 3866–3873. T. Qian, Z.H. Jiang, Z. Cai, Anal. Biochem. 352 (2006) 87–96. H. Liu, J. Yang, F. Du, X. Gao, X. Ma, Y. Huang, F. Xu, W. Niu, F. Wang, Y. Mao, Y. Sun, T. Lu, C. Liu, B. Zhang, C. Li, Drug Metab. Dispos. 37 (2009) 2290–2298. G.F. Deng, D.L. Wang, M.X. Meng, F. Hu, T.W. Yao, J. Chromatogr. B 877 (2009) 2113–2122. Q.L. Zhou, D.N. Zhu, Y.F. Yang, W. Xu, X.W. Yang, J. Pharm. Biomed. Anal. 137 (2017) 1–12. G.L. Dai, Z.T. Jiang, L.J. Zhu, Q. Zhang, Y. Zong, S.J. Liu, C.Y. Li, W.Z. Ju, J. Sep. Sci. 39 (2016) 3368–3374. J.F. Li, N.S. Wang, H.W. Liu, T.L. Huang, Tradit. Chin. Drug Res. Clin. Pharmacol. 11 (2000) 208–211.