Simultaneous determinations of 17 marker compounds in Xiao–Chai–Hu–Tang by LC–MS/MS: Application to its pharmacokinetic studies in mice

Rongjin Sun, Min Zeng, Ting Du, Li Li, Guangyi Yang, Ming Hu, Song Gao
2015 Journal of chromatography. B  
The purpose of this study is to develop and validate an UPLC-MS/MS method to quantify different marker compounds from Xiao-Chai-Hu-Tang (XCHT, a Chinese traditional herbal) in biological samples and apply the method to pharmacokinetic study. A Waters BEH C 18 UPLC column was used with acetonitrile/0.1% formic acid mobile phases. The mass analysis was performed in a triple quadrupole mass spectrometer using multiple reaction monitoring (MRM) with positive scan mode. A one-step protein
more » ... on by methanol was used to extract the analytes from blood. Seventeen commercially available compounds from the different compositing herbals were selected as markers. The results revealed that all of the calibration curves showed good linear regression (r 2 > 0.9918). The intra-day and inter-day precisions (RSD) of all of these markers at three different levels were less than 15.0% and the bias of the accuracies ranged from −13.5% to 16.6%. The extraction recoveries of all of these 17 markers were from 70.8% to 113.7% and the matrix effects ranged from 71.8% to 114.8%. The stabilities of these compounds in blood were evaluated by analyzing three replicates of QC samples at three different concentrations following storage at 25°C for 6 h, 4°C for 24 h, and −80°C for 30 days. All the samples displayed 85-15% precision and accuracy after various stability tests. The validated method was successfully applied to pharmacokinetic study in A/J mouse with oral administration of XCHT. All of these markers were detected and the pharmacokinetic parameters of 8 compounds were able to be calculated. This method is sensitive and reproducible that can be used for XCHT's in vivo study. The herbals were bought from Taihe Hospital (Shiyan, Hubei Province, China). Liquiritin, glycyrrhetinic acid, wogonoside, wogonin, baicalin, baicalein, saikosaponin a (SSa), saikosaponin d (SSd), zingerone, 6-gingernol, rutin and quecertin were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Ginsenoside Rd (Rd), ginsenoside Rg1 (Rg1), ginsenoside Rg3 (Rg3), ginsenoside Re (Re), formononetin, and daidzein were purchased from LKT Laboratories (St. Paul, MN). The chemical structures of these standard were shown in Fig. 1 . The purity of all standards was 95.0% or above. Acetonitrile and methanol were purchased from Merck (Darmstadt, Germany). Other chemicals (analytical grade or better) were used as received. Preparation of XCHT The powdered herbal materials, including Bupleuri Radix (12 g), Scutellariae Radix (9 g), Ginseng Radix (9 g), Glycyrrhizae Radix (6 g), Pinelliae Tuber (9 g), Zingiberis Rhizoma (9 g), and Jujubae Fructus (12 g), were extract twice with 8-fold volumes of water under reflux for 1 h. The combined filtrate was concentrated and abrown sticky extract (2 g/mL) was afforded. The sample for quality control was prepared by diluting the original extract for 1000-folds in 50% methanol. Before injection, the quality control samples were centrifuged for 15 min at 15,000 rpm, then, 100 μL of the supernatant was filtered through a 0.22 μm membrane and 20 μL of internal standard (daidzein 0.5 μM) was added for LC-MS analysis. Instruments and conditions 2.3.1 UPLC-The UPLC conditions were: Waters Acquity™ with diode array detector (DAD); column, Acquity UPLC BEH C 18 column (50 mm × 2.1 mm I.D., 1.7 μm, Waters, Milford, MA, USA); mobile phase A (MPA), 0.1% formic acid in water; mobile phase B (MPB), acetonitrile; gradient, 0-2 min, 5% B; 2.0-3.0 min, 10% B; 3.0-5.0 min, 10-15% B; 5.0-9.0 min, 15-20% B; 9.0-11.0 min, 20-40% B; 11.0-14.0 min, 40-45% B; 14.0-14.5 min, 45-60% B; 14.5-15.0 min, 60-100% B; 15.0-15.2 min, 100%; 15.2-16.0 min, 100-5% B; 16.0-17.0 5% B; column temperature, 45°C; sample temperature, 20°C; and injection volume, 10 μL. MS- The MS analysis was performed on an API 5500 Qtrap triple quadrupole mass spectrometer (Applied Biosystem/MDS SCIEX, Foster City, CA, USA) equipped with a TurboIonSpray™ source. The compounds were determined by using MRM (multiple reaction monitoring) scan type in positive mode. The instrument dependent parameters for mass spectrum were set as follows: ion-spray voltage, 5.5 kV; ion source temperature, 500°C; nebulizer gas (gas 1), nitrogen, 40 psi; turbo gas (gas 2), nitrogen 40 psi; curtain gas, nitrogen 10 psi. Unit mass resolution was set in both mass-resolving quadruples Q1 and Q3. Compound-dependent parameters were listed in Table 1 . Sun et al.
doi:10.1016/j.jchromb.2015.09.004 pmid:26397748 pmcid:PMC4608253 fatcat:qhazfxju7za5demhcoycfn5ffe