Research article Received: 4 February 2015,

Revised: 5 April 2015,

Accepted: 18 April 2015

Published online in Wiley Online Library: 1 July 2015

(wileyonlinelibrary.com) DOI 10.1002/bmc.3521

Determination of astragaloside III in rat plasma by liquid chromatography–tandem mass spectrometry and its application to a rat pharmacokinetic study Yongsong Zhaia, Pengyue Lib, Manyuan Wanga, Muxin Gonga and Feng Qiua* ABSTRACT: Astragaloside III (AST III), a naturally occurring saponin compound isolated from Radix Astragali, has been demonstrated to have anti-gastric ulcer, immunomodulatory and antitumor effects. To evaluate its pharmacokinetics in rats, a rapid, sensitive and specific high-performance liquid chromatography–tandem mass spectrometric (HPLC-MS/MS) method has been developed and validated for the quantification of astragaloside III in rat plasma. Samples were pretreated using a simple protein precipitation with methanol–acetonitrile (50:50, v/v) and the chromatographic separation was performed on a C18 column by a gradient elution using a mobile phase consisting of water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid. Astragaloside III and the internal standard (buspirone) were detected using a tandem mass spectrometer in positive multiple reaction monitoring mode. Method validation revealed excellent linearity over the range of 5.00–5000 ng/mL together with satisfactory intra- and inter-day precision, accuracy and recovery. Stability testing showed that astragaloside III spiked into rat plasma was stable for 24 h at 20°C temperature, for up to 30 days at 80°C, and during three freeze–thaw cycles. The method was successfully used to investigate the pharmacokinetic profile of AST III after oral (10 mg/kg) and intravenous (1.0 mg/kg) administration in rats. The oral absolute bioavailability of AST III was calculated to be 4.15 ± 0.67% with an elimination half-life value of 2.13 ± 0.11 h, suggesting its poor absorption and/or strong metabolism in vivo. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: astragaloside III; bioavailability; LC-ESI-MS/MS; rat

Introduction

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* Correspondence to: Feng Qiu, Beijing Key Laboratory of TCM Collateral Disease Theory Research, School of Traditional Chinese Medicine, Capital Medical University, Beijing, 100069, China. Email: [email protected] a

Beijing Key Laboratory of TCM Collateral Disease Theory Research, School of Traditional Chinese Medicine, Capital Medical University, Beijing100069, China

b

Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing100700, China Abbreviations used: AstIII, astragaloside III; DMSO, dimethyl sulfoxide.

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105

Radix Astragali, one of the most popular traditional Chinese medicines in China, is the dried root of Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao or A. membranaceus (Fisch.) Bge. (Pharmacopoeia of the People’s Republic of China, 2010; Sun et al., 2013). It has been widely used in the prevention and treatment of various diseases such as nephritis, diabetes, cancer, etc. (Qu et al., 2014; Cheng et al., 2004; Liu et al., 2011; Xie and Du, 2011). In recent years, saponins in Radix Astragali have attracted much attention for their unique structures and significant biological activities, including anti-inflammatory (Ryu et al., 2008; Chen et al., 2014), hematopoietic functions (Zheng et al., 2011), renoprotection (Meng et al., 2010), anti-hyperglycemic (Yin et al., 2014; Tang et al., 2011), antioxidative (Sun et al., 2014), cardioprotective (Lau et al., 2012; Xu et al., 2007) and immunomodulatory (Wang et al., 2002) effects. Astragaloside III (AST III, chemical structure shown in Fig. 1) is one of these saponins isolated from Radix Astragali, which has shown anti-gastric ulcer (Liu et al., 2014), immunomodulatory (Yang et al., 2008) and significant antitumor (Ionkova et al., 2010) effects. As a result, as one phytosaponin from Radix Astragalus, AST III may become a novel drug candidate that is beneficial for human health. However, based on its relative large molecular structure, the bioavailability of AST III might be of great concern in the period of drug discovery. Therefore, to evaluate the potential of AST III as a drug candidate, it is necessary to develop a rapid and accurate bioanalytical

method to quantify AST III in biological fluids and then apply to the bioavailability study of AST III in animals. Several methods for the separation and analysis of AST III in raw herbs have been reported, including high-performance liquid chromatography coupled with pulsed amperometric detection (Kwon and Park, 2012), high-performance liquid chromatography coupled with evaporative light-scattering detection (Zhang et al., 2014a, b) and ultra-high-performance liquid chromatography– tandem mass spectrometry (Wang et al., 2013; Zhang et al., 2014a, b). However, none of these methods have been used for bioanalysis of AST III in any biological fluids. Thus, the aim of this present work was to develop a rapid, sensitive and specific LC-MS/MS method for the quantification of AST III in rat plasma. The developed method was further validated and then applied to a pharmacokinetic study of AST III after intravenous and oral administration of AST II in rats.

Y. Zhai et al.

+

Figure 1. Chemical structures, full-scan product ion spectra of [M + H] ions and fragmentation schemes for (A) Astragaloside III (AST III) and (B) buspirone (internal standard).

Experimental Materials and reagents Astragaloside III (HPLC purity ≥98.5%, chemical structure shown in Fig. 1) was purchased from Shanghai Jingke Chemicals Co. Ltd (Shanghai, China). Buspirone (chemical structure shown in Fig. 1) was purchased from Sigma (St Louis, MO, USA). Acetonitrile (HPLC grade) was obtained from Merck (Darmstadt, Germany). Hydroxypropyl-β-cyclodextrin was purchased from Beijing Fengli Jingqiu Commerce and Trade Co. Ltd (Beijing, China). Methylcellulose was purchased from Beijing Fengli Jingqiu Commerce and Trade Co. Ltd (Beijing, China). Dimethyl sulfoxide (DMSO) was purchased from Tedia (Fairfield, OH, USA). Ultrapure water was produced by a Milli-Q Reagent Water System (Millipore, MA, USA). All other chemicals were of analytical grade.

buspirone, respectively. The precursor/product ion pairs were monitored at m/z 807.4 → 335.3 for AST III and 386.3 → 122.3 for buspirone. Data were collected and analyzed by the Analyst Data Acquisition and Processing software (version 1.5.2, Applied Biosystems/MDS Sciex, Concord, Ontario, Canada).

Animals Healthy male Sprague–Dawley rats (190–250 g, 8 weeks) were purchased from Beijing Vital River Laboratories Co. Ltd (Beijing, China). Experiments using animals were approved by the Animal Ethics Committee of Capital Medical University (Beijing, China) and conformed to the Guide for Care and Use of Laboratory Animals published by the US National Institutes of Health (1996). Animals were housed in a room at a controlled temperature of 25 ± 2°C and a relative humidity of 40–60%, with access to food and water ad libitum. All animals were acclimated in the laboratory for at least 5 days prior to the experiment and fasted for 12 h but allowed water ad libitum before experiments.

Instrumentation and conditions

106

The HPLC system consisted of an LC-20AD pump, a DGU-20 A3 degasser, an SIL-20AC autosampler and a CTO-20A column oven (Shimadzu, Japan). Samples were separated on an Agilent Zorbax XDB C18 column (50 × 2.1 mm, 3.5 μm, Agilent Co. Ltd, USA) with the column temperature set at 25°C. The mobile phase consisted of a gradient elution by a mobile phase consisting of water containing 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B) with following gradient: 0.00 min 10% B, 0.50 min 10% B, 1.50 min 98% B, 2.50 min 98% B, 2.51 min 5% B, 4.00 min 10% B, with the flow rate of 0.50 mL/min. The injection volume was set at 10 μL and the running time was 4 min. Solvent eluted from chromatography column during the first minute was switched to waste before it entered the ion source. The HPLC system was coupled with an API 4000 Qtrap mass spectrometer (Applied Biosystems/MDS Sciex, Concord, Ontario, Canada) via a Turbo IonSpray ionization interface. Following optimization of the setting parameters, the electrospray ionization source was operated in positive mode with the curtain, nebulizer and turbo-gas (all nitrogen) set at 20, 50 and 50 psi, respectively. The source temperature was 500°C and the ion spray needle voltage was 5500 V. The mass spectrometer was operated at unit resolution for Q1 and low resolution for Q3 in the multiple reaction monitoring mode, with a dwell time of 150 ms per multiple reaction monitoring channel. The collision energy was set at 81 and 44 eV for AST III and buspirone, respectively. The declustering potential was set at 261 and 100 eV for AST III and

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Preparation of standard and QC samples Stock solutions of AST III (1 mg/mL) and buspirone (1 mg/mL) were prepared separately in DMSO. In order to ensure weighing precision, two weighings, as long as their concentrations agreed within 5%, were prepared, then one was used for calibrators and the other for quality control (QC) samples. Primary stock solutions for calibration curve standards and QC samples were prepared using separate weighings. Working standard solutions of AST III for the preparation of calibration curves were obtained by further dilution of the stock solution with methanol to give final concentrations of 50, 100, 200, 500, 2000, 5000, 20,000 and 50,000 ng/mL. QC solutions were prepared at concentrations of 100, 2000 and 40,000 ng/mL by dilution of the primary stock solution with methanol. A solution containing buspirone (10 ng/mL) was also obtained by dilution of buspirone stock solution with methanol:acetonitrile (50:50, v/v). All standard solutions were stored at 4°C. Each drug-free rat plasma sample (50 μL) was spiked with buspirone solution (150 μL) and serial standard solutions of AST III (5 μL) to prepare calibration standards in the concentration range 5.00–5000 ng/mL. QC samples were prepared by spiking control rat plasma in bulk at appropriate concentrations, and then dividing into small aliquots (around 80 μL) in different tubes. These samples were stored under the same conditions as the experimental samples and underwent the same pretreatment procedure (described below).

Copyright © 2015 John Wiley & Sons, Ltd.

Biomed. Chromatogr. 2016; 30: 105–110

LC-MS/MS determination of astragaloside III in rat plasma Sample pretreatment procedure

Results and discussion

After thawing at room temperature for about 30 min and vortexing for 30 s, aliquots of 50 μL plasma were mixed with 5 μL of methanol and 150 μL of IS solution (10 ng/mL buspirone in methanol–acetonitrile, 50:50, v/v). After vortexing for 1 min and then centrifugation at 12,000g for 10 min, aliquots of 100 μL supernatants were transferred to HPLC vials. A volume of 10 μL of this solution was then injected onto the column.

Method validation Method validation for determination of AST III in rat plasma was performed according to the international guidelines (US Food and Drug Administration, 2014; European Medicines Agency, 2011). Selectivity was evaluated by comparing chromatograms of blank plasma samples collected from six different rats with the chromatogram of a plasma sample spiked with AST III and buspirone. The least-squares linear regression 2 method (1/x weighting) was used to determine the slope, intercept 2 and square regression coefficient (r ) of the linear regression equation. Precision and accuracy were calculated by determining QC samples at three concentration levels. Precision is expressed as the relative standard deviation (RSD) and accuracy as the relative error (RE). Precision and accuracy were also assessed at the lowest concentration of the standards (5.00 ng/mL), representing the lower limit of quantification (LLOQ) for the assay. The recovery of analytes was determined by comparing the responses of the analytes from QC samples with analytes spiked in post-extracted blank rat plasma at equivalent concentrations. The matrix effect was measured by comparing the responses obtained from post-extraction blank rat plasma spiked samples with mobile phase spiked with low, middle and high concentrations of analyte. The stability of the analyte in rat plasma was evaluated by analyzing QC samples stored under the following conditions: at 20°C for 24 h and at 80°C for 30 days. The effect of three freeze–thaw cycles on the analyte was also examined.

Pharmacokinetic study

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Operating parameters for MS detection of AST III and buspirone were optimized by flow injection using the standard solutions. The MS2 spectra of AST III and buspirone were recorded and are shown in Fig. 1. In the full-scan Q1 mass spectrum of astragaloside III, the most abundant peak was sodium adduct ion [M + Na]+ found at m/z = 807.4. The abundance of this ion peak ([M + Na]+) was stable enough and sufficient for the accurate quantification. This [M + Na]+ ion was used as the precursor to select product ions formed by collision induced dissociation (CID). The strongest fragment for AST III was the ion at m/z 335.3. The transition m/z 807.4 → 335.3 was then used to optimize ion spray voltage, curtain gas pressure, nebulizer gas pressure, heater gas pressure, source temperature and collision energy for AST III. For buspirone, the most abundant peak was the protonated molecular ion [M + H]+ found at m/z = 386.3. Parameters for buspirone transition m/z 386.3→122.3 were optimized in the same way as those for AST III. Optimization of chromatographic conditions Mixtures of methanol and acetonitrile with water and different buffers/modifiers, such as formic acid, ammonium formate and acetic acid, were investigated as chromatography solvents. Acetonitrile, rather than methanol, was chosen for the quantification of AST III because it produced more symmetrical peaks for AST III and buspirone. The various buffers showed no obvious advantage over water. Finally the mobile phase consisting of acetonitrile with 0.1% formic acid and water with 0.1% formic acid was used on an Agilent Zorbax XDB C18 column (2.1 × 50 mm, 3.5 μm) depending on the analytical experiences in our laboratory. In addition, a sharper gradient elution program was utilized in order to achieve short running time and rapid re-equilibrium. Under the present chromatographic conditions, symmetric peak shapes of AST III and buspirone were obtained. The whole chromatographic running time is only 4.0 min for one sample, and more than 150 samples could be assayed daily, including sample preparation, data acquisition and processing. Choice of internal standard An internal standard is usually required in LC-MS/MS analysis in order to eliminate the effects from matrix and the extraction efficiency. Usually a radio-labeled internal standard is the optimal choose; however, it is not available during the period of drug discovery. In this study, buspirone, a readily available chemical compound, was selected as the IS, which displays similar chromatographic retention behavior (retention time = 2.11 min) with AST III and high extraction efficiency (>80%; Qiu et al., 2015). Most importantly, as a chemically synthesized compound, buspirone will not exist in traditional Chinese medicines and animal feed. Thus, there were no interferences of IS from AST III and solvents. Method validation

Specificity Typical chromatograms (Fig. 2) showed that, under the chromatographic conditions described above, there were no obvious endogenous interferences at the retention time of AST III and buspirone.

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Polyethylene cannulas were implanted in the femoral vein 2 days before the experiment while the rats were anesthetized with pentobarbital (50 mg/kg, intravenous). The cannulas were externalized at the back of the neck and filled with heparinized saline (20 units/mL). The rats were fasted for 16 h before experiments with the exception of free access to water. The intravenous dosing solution with AST III concentration of 1.0 mg/mL was prepared by dissolving appropriate amount of AST III in DMSO–30% hydroxypropyl-β-cyclodextrin (5:95, v/v) and filtering through 0.22 μm Millipore filter prior to use. The oral dosing suspension with AST III concentration of 2.0 mg/mL was prepared by dissolving an appropriate amount of AST III in 0.5% methylcellulose solution. The concentrations of the intravenous and oral dosing solutions will be confirmed using the same LC-MS/MS method. The intravenous and oral doses of AST III were 1.0 and 10 mg/kg, and the intravenous and oral dose volumes were 1.0 and 5.0 mL/kg, respectively. After intravenous administration of 1.0 mg/kg AST III through the tail vein, aliquots of 0.20 mL blood samples were collected in heparinized polyethylene tubes at different time intervals post-dosing (0.033, 0.083, 0.25, 0.50, 1.0, 2.0, 4.0, 6.0 and 8.0 h). After oral administration, aliquots of 0.20 mL blood samples were collected in heparinized polyethylene tubes at different time intervals post-dosing (0.25, 0.50, 1.0, 2.0, 4.0, 6.0 and 8.0 h). Heparinized blood was centrifuged at 12,000g at room temperature for 5 min to obtain plasma, which was stored at 80°C until analysis. Pharmacokinetic parameters including half-life (t1/2z), maximum plasma time (tmax) and concentration (Cmax), area under concentration–time curve (AUC(0–t) and AUC(0–∞)), clearance (CLz), volume of distribution (Vz) and mean residence time (MRT(0–t) and MRT(0–∞)) of AST III were analyzed by noncompartmental method using DAS Version 2.0 (Chinese Pharmacological Society, Beijing, China). All results were expressed as arithmetic mean ± standard deviation (SD).

Optimization of MS conditions

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A

AST III: 807.4/335.3 Da 20

Intensity, cps

10 0 30

IS: 386.3/122.3 Da

20 10 0 0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

2.5

3.0

3.5

4.0

2.5

3.0

3.5

4.0

2.5

3.0

3.5

4.0

Time, min

B

137

2.16 AST III: 807.4/335.3 Da

100

Intensity, cps

50 0 1.25

2.11 IS: 386.3/122.3 Da

8.0e4 4.0e4 0.0 0.0

0.5

1.0

1.5

2.0

Time, min

C

1000

2.16 AST III: 807.4/335.3 Da

500

Intensity, cps

0 9.7e4

2.11 IS: 386.3/122.3 Da

5.0e4

0.0 0.0

0.5

1.0

1.5

2.0

Time, min

D

2.16 600

AST III: 807.4/335.3 Da

400

Intensity, cps

200 0 1.00e5

2.11 IS: 386.3/122.3 Da

5.00e4

0.0 0.0

0.5

1.0

1.5

2.0

Time, min Figure 2. Typical multiple reaction monitoring chromatograms of (A) blank rat plasma; (B) blank rat plasma spiked with AST III (5.00 ng/mL, LLOQ) and IS; (C) an unknown rat plasma sample collected at 2 min after intravenous administration of 1.0 mg/kg AST III (determined concentration 72.5 ng/mL with 100-fold dilution); and (D) an unknown rat plasma sample collected at 30 min after oral administration of 10 mg/kg AST III (determined concentration 37.3 ng/mL).

Linearity, LLOQ and carryover effect

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The calibration curve for AST III exhibited good linearity over the concentration range of 5.00–5000 ng/mL. The typical

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standard curve was described by the equation y = 2.65 × 10 5x + 4.80 × 10 5, where y is the peak area ratio of the component to buspirone and x is the concentration of AST III. The square regression coefficient (r2) was >0.99. The LLOQ

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Biomed. Chromatogr. 2016; 30: 105–110

LC-MS/MS determination of astragaloside III in rat plasma of AST III was 5.00 ng/mL (n = 6, RSD = 9.78%, RE = 8.65%) in this study.

Table 3. Results of stability test for the determination of AST III in rat plasma determined by LC-MS/MS (n = 6) Stability

Spiked Determined Accuracy concentration concentration (%) (ng/mL) (mean ± SD, ng/mL)

Precision, accuracy and dilution effect

Recovery, matrix effect and stability The extraction recoveries of AST III were all >90% (Table 2). The ratio of the peak area resolved in the post-extraction blank sample with that resolved in the mobile phase showed no significant matrix effects. AST III spiked into rat plasma was found to be stable for 24 h at 20°C, for up to 30 days at 80°C, and during three freeze–thaw cycles (Table 3). The stability was thus satisfactory for a routine pharmacokinetic study.

Pharmacokinetic application The mean plasma concentration–time profiles of AST III in rats are illustrated in Fig. 3. The main pharmacokinetic parameters were calculated with DAS software using a noncompartmental model and are presented in Table 4. After intravenous administration of AST III at the dose of 1.0 mg/kg, the elimination half-life (t1/2z) value was estimated to be 1.49 ± 0.10 h, and the mean area under the plasma concentration–time curve from time zero to the last measurable plasma concentration point (AUC(0–t)) and the mean area under the plasma concentration–time curve from time zero to time infinity (AUC(0–∞)) values were 1704 ± 417 and 1722 ± 414 h ng/mL, respectively. Clearance (CLz), mean residence time (MRT(0–∞)) and volume of distribution (Vz) values were estimated to be 0.60 ± 0.13 mL/min/kg, 0.81 ± 0.39 h and 1.28 ± 0.22 L/kg, respectively.

Table 1. Intra- and inter-day precision and accuracy for AST III in rat plasma determined by LC-MS/MS (n = 6) Added concentration (ng/mL) 10.0 200 4000

Precision (RSD, %) Intra-day

Inter-day

7.7 6.0 4.6

7.3 5.1 5.5

Accuracy (RE, %) Intra-day 0.3 1.3 2.9

Inter-day 1.4 0.8 0.7

Table 2. Matrix effects and recoveries for the determination of AST III in rat plasma determined by LC-MS/MS (n = 6) Concentration (ng/mL)

Matrix effect (mean ± SD, %)

92.6 ± 0.75 94.2 ± 2.02 96.3 ± 2.38

87.6 ± 5.24 85.4 ± 6.70 84.6 ± 5.49

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30 days at 80°C Three freeze– thaw cycles

100000

10.0 200 4000 10.0 200 4000 10.0 200 4000

9.30 ± 0.24 188 ± 5.13 3810 ± 62.4 10.3 ± 0.70 182 ± 3.51 3783 ± 25.2 10.7 ± 0.47 189 ± 3.79 3907 ± 41.6

Rat PK Profiles of AST III

93.0 94.0 95.3 103.0 91.0 94.6 107.0 94.5 97.7

IV PO

10000

1000

100 10

1

0

2

4

6

8

Time (hr) Figure 3. Mean plasma concentration–time profiles of AST III after oral (10 mg/kg) and intravenous (1.0 mg/kg) administration in rats (n = 3).

After oral administration of AST III at the dose of 10 mg/kg, AST III was rapidly absorbed, reaching mean Cmax of 189 ± 75.4 ng/mL at tmax of 1.67 ± 0.58 h. The mean AUC(0–t) and AUC(0–∞) values were 650 ± 113 and 714 ± 115 hr ng/mL, respectively. The oral absolute bioavailability (F) of AST III was calculated to be 4.15 ± 0.67% with a t1/2z value of 2.13 ± 0.11 h, suggesting its poor absorption and/or strong metabolism in vivo. Shaw et al. (2012) reported that the plasma concentration–time curve of astragaloside II, another saponin from Radix Astragali, in rats after oral administration of Bu-Yang-Huan-Wu-Tang presented the phenomenon of a double-peak absorption phase in the plasma profile. Likewise, in the present study, the plasma concentration–time curve of astragaloside III in rats after oral administration presents the phenomenon of a double-peak absorption phase in the plasma profile. Several possible factors have been suggested to explain the phenomenon of double-peak behavior: (a) entero-hepatic recycling; (b) the presence of absorption sites along the stomach and different gastrointestinal segments; and (c) variable gastric emptying. In present study, entero-hepatic recycling is the most likely factor to explain the phenomenon of double-peak of AST III (Qiu et al., 2015). Qu et al. (2014) reported a similar pharmacokinetic study in rats after oral and intravenous administration of astragaloside II, which is a structurally similar compound separated from Radix Astragali. The F of AST II was calculated to be 0.79 ± 0.16%, which was similarly low to that of AST III. Overall, the F of AST III is not satisfactory, suggesting that astragaloside III shows poor absorption and/or

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10.0 200 4000

Recovery (mean ± SD, %)

24 h at 20°C

Concentration (ng/mL)

The precision and accuracy of the method were assessed using QC samples (10.0, 200 and 4000 ng/mL). The method was found to be highly precise with intra-day precision ≤7.72% and inter-day precision ≤7.30%, and highly accurate with ≤2.88% deviation from the nominal values at each QC sample concentration (Table 1).

Y. Zhai et al. Table 4. Main pharmacokinetic parameters of AST III in rats after intravenous and oral administration (n = 3, mean ± SD) Parameters AUC(0–t) AUC(0–∞) MRT(0–t) MRT(0–∞) t1/2z Vz CLz C0 tmax Cmax F

Unit

i.v.

p.o.

ng/mL h ng/mL h hr hr hr L/kg L/hr/kg ng/mL hr ng/mL %

1704 ± 417 1722 ± 414 0.7 ± 0.34 0.81 ± 0.39 1.49 ± 0.10 1.28 ± 0.22 0.60 ± 0.13 20,534 ± 3980

650 ± 113 714 ± 115 2.80 ± 0.19 3.56 ± 0.27 2.13 ± 0.11 43.9 ± 8.71 14.3 ±2.33 1.67 ± 0.58 189 ± 75.4 4.15 ± 0.67

AUC(0–t) and AUC(0–∞), area under concentration–time curve; MRT(0–t) and MRT(0–∞), mean residence time; t1/2z, half-life; Vz, volume of distribution; CLz, clearance; C0, plasma concentration at zero time; tmax, maximum plasma time; Cmax, concentration; F, oral absolute bioavailability.

strong metabolism in vivo. As a result, the parenteral administration route is suggested for AST III in order to improve its efficacy.

Conclusion A rapid, sensitive and specific HPLC-MS/MS has been developed and validated for a bioavailability study of AST III in rats. The sample preparation involved a simple protein precipitation procedure with methanol–acetonitrile (50:50, v/v). The LLOQ was 5.00 ng/mL using 50 μL of rat plasma. The assay showed a wide linear dynamic range of 5.00–5000 ng/mL, with acceptable intra- and inter-day accuracy and precision. The developed and validated method was successfully applied in the quantification and pharmacokinetic study of AST III in rats after intravenous and oral administration. The F of AST III was 4.15 ± 0.67% with a t1/2z value of 2.13 ± 0.11 h, suggesting its poor absorption and/or strong metabolism in vivo.

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Biomed. Chromatogr. 2016; 30: 105–110

Determination of astragaloside III in rat plasma by liquid chromatography-tandem mass spectrometry and its application to a rat pharmacokinetic study.

Astragaloside III (AST III), a naturally occurring saponin compound isolated from Radix Astragali, has been demonstrated to have anti-gastric ulcer, i...
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