Research article Received: 6 February 2015,

Revised: 21 April 2015,

Accepted: 18 May 2015

Published online in Wiley Online Library: 6 July 2015

(wileyonlinelibrary.com) DOI 10.1002/bmc.3519

Simultaneous determination of four secoiridoid and iridoid glycosides in rat plasma by ultra performance liquid chromatography–tandem mass spectrometry and its application to a comparative pharmacokinetic study Yun Wua, Yu Aia, Fenrong Wanga, Wen Maa, Qiaoxia Biana, David Y.-W. Leeb and Ronghua Daia* ABSTRACT: A simple, reliable and rapid ultra-performance liquid chromatography–tandem mass spectrometry method was developed and validated for the simultaneous quantification of four secoiridoid ( gentiopicroside, swertiamarin, sweroside) and iridoid glycosides (loganic acid), the bio-active ingredients in rat plasma. After liquid–liquid extraction, chromatographic separation was accomplished on a Shim-pack XR-ODS column with a mobile phase consisting of methanol and 0.1% formic acid in water. A triple quadrupole tandem mass spectrometry equipped with an electrospray ionization source was used as detector operating both in positive and negative ionization mode and operated by multiple-reaction monitoring scanning. The lower limits of quantitation were 0.25–30 ng/mL for all the analytes. Both intra-day and inter-day precision and accuracy of analytes were well within acceptance criteria (±15%). The mean extraction recoveries of analytes and internal standard (amygdalin) from rat plasma were all >71.4%. The validated method was successfully applied to a comparative pharmacokinetic study of four analytes in rat plasma between normal and arthritic rats after oral administration of Huo Luo Xiao Ling Dan and Gentiana macrophylla extract, respectively. Results showed significant differences in pharmacokinetic properties of the analytes among the different groups. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: Huo Luo Xiao Ling Dan; UPLC-MS/MS; comparative pharmacokinetics; arthritis; rat plasma

Introduction

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* Correspondence to: R. Dai, School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China. Email:[email protected]; [email protected] a

School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China

b

Mailman Research Center, McLean Hospital, Harvard Medical School, Boston, MA, United States Abbreviations used: CFA, Complete Freund’s adjuvant; ESI, electrospray ionization; GME, Gentiana macrophylla extract; HLXLD, Huo Luo Xiao Ling Dan; RA, rheumatoid arthritis; TCM, traditional Chinese medicine.

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Rheumatoid arthritis (RA) is a chronic systemic inflammatory autoimmune disease with articular and extra-articular affects. One of the typical clinical features of RA is symmetrical polyarthritis with joint swelling, joint damage and chronic pain, especially of the hands and feet (Davis and Matteson, 2012). These syndromes have a major impact on the quality of life, working productivity and the use of healthcare resources (Lee, 2013; Sarzi-Puttini et al., 2014). The common therapeutic drugs are chemicals with side effects such as serious gastrointestinal disturbances and cardiovascular risk (FitzGerald, 2004). In light of this phenomenon, there is an urgent need for the development of a more effective and safety therapy, and traditional Chinese medicine (TCM) may be a good choice. The Chinese formula is a major clinical application mode of TCM and has been widely used for the prevention and treatment of chronic diseases for many years (Ho and Lai, 2004). Huo Luo Xiao Ling Dan (HLXLD), a modified Chinese herbal compound, has been used in China for the treatment of inflammatory arthritis (Wang et al., 2002; Zhang et al., 2009). HLXLD is composed of 11 crude drugs: Angelica sinensis Diels (12 g), Salvia miltiorrhiza Bge (12 g), Boswellia carterii Birdw (15 g), Angelica pubescens Maxim (12 g), Notopterygium incisum Ting ex H. T. Chang (12 g), Paeonia lactiflora Pall (12 g), Corydalis yanhusuo W. T. Wang (12 g), Ligusticum chuanxiong S.H. Qiu (12 g), Gentiana macrophylla Pall (12 g), Cinnamomum cassia Presl (15 g) and Glycyrrhiza uralensis Fisch

(12 g) (Lao et al., 2006). Among the 11 herbs in HLXLD, G. macrophylla extract (GME) has been widely used in Chinese herbal medicine to treat diverse diseases, such as apoplexy, paralysis and rheumatoid arthritis (Shouzhong, 1998; China, 2010; Jia et al., 2012; Yang et al., 2013). Pharmacological studies have demonstrated that secoiridoid glycosides ( gentiopicroside, swertiamarin, sweroside) and iridoid glycosides (loganic acid) from GME are the major bioactive components, possessing antinociceptive, anti-inflammatory and immunoregulatory activities (Yu et al., 2004; Jia et al., 2012). The four analytes (chemical structures are shown in Fig. 1) in HLXLD have a potential ability to treat RA, so it is necessary to carry out a study on the pharmacokinetics of these compounds in vivo.

Y. Wu et al. Animals

Figure 1. Chemical structures of (A) gentiopicroside, (B) swertiamarin, (C) sweroside, (D) loganic acid and (E) IS.

So far, several analytical methods have been reported for the determination of most secoiridoid glycosides in rat plasma or other biological samples by high-performance liquid chromatography (HPLC) or liquid chromatography–tandem mass spectrometry (LC-MS/MS) (Luo, 2009; Feng et al., 2014; Sheng et al., 2014). However, these assays mainly focused on the quantification of one or two components, and the sensitivity of these methods [lower limits of quantitation (LLOQ) for swertiamarin and sweroside: 2 and 5.84 ng/mL, respectively] is inadequate to meet the requirements of pharmacokinetic and metabolism studies. Further, there is still no method established for estimation of loganic acid in biological samples. In this paper, a simple, sensitive and reliable ultraperformance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) method is developed for the simultaneous quantification of gentiopicroside, swertiamarin, sweroside and loganic acid in rat plasma. The method was applied successfully to a comparative pharmacokinetics investigation between normal and arthritic rats in plasma after oral administration of HLXLD and single-herb GME, respectively. We speculated that both the pathological condition and the compatibility effects of other ingredients present in HLXLD could affect the pharmacokinetics of the analytes. Consequently, this study would be helpful for determining the complexity of the action, understanding the compatibility rationality and facilitating the clinical application of HLXLD.

Experimental Chemicals and materials

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The 11 herbs used in HLXLD, including G. macrophylla, were all purchased from Tong-Ren-Tang TCM store (Shenyang, China) and authenticated by Professor Ying Jia (School of Chinese Material Medica, Shenyang Pharmaceutical University, Shenyang, China). Reference standards material of gentiopicroside was purchased from Chengdu Must Bio-technology Co. Ltd (Chengdu, China). Swertiamarin was obtained from Nanjing Chemlin Bio-technology Co. Ltd (Nangjing, China). Sweroside and loganic acid were bought from Shanghai Winherb Medical Science Co. Ltd (Shanghai, China). The purities of these reference standards were all >98%. The internal standard (IS) of amygdalin was prepared in our laboratory; its structure 1 13 was confirmed by MS, H- and C- NMR spectra (data not shown), and the purity was shown to be >98% by HPLC analysis. Complete Freund’s adjuvant (CFA) was supplied by Sigma-Aldrich Chemical Co. (St Louis, MO, USA). Methanol and acetonitrile (Fisher Technologies Inc., USA) were of HPLC grade. Distilled water was obtained from Wahaha Co. Ltd (Hangzhou, China). Formic acid, ethyl acetate and isopropanol were of HPLC grade and provided by Shandong Yuwang Industrial Co. Ltd (Yucheng, China). Other reagents were of analytical grade.

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Eighteen male Sprague–Dawley rats (220–250 g) were obtained from the Experimental Animal Center of Shenyang Pharmaceutical University. The animals were kept in an air-conditioned animal center (with food and water ad libitum) at a temperature of 22 ± 2°C and a relative humidity of 50 ± 10%, with a 7 a.m. to 7 p.m. natural light–dark cycle for 7 days and then fasted with only access to water for 12 h prior to the experiment. The animal study was carried out following the Guideline of Animal Experimentation of Shenyang Pharmaceutical University, and the protocol was approved by the Animal Ethics Committee of the institution. The arthritis model in rats was induced by CFA as described previously (Lao et al., 2006). The rats in the model group were injected with 100 μL of CFA at the plantar surface of the left hind paw and each rat in normal groups was injected with the same volume of saline once. After the injection of CFA, the inflammation, manifesting as redness, edema and hyperresponsiveness to noxious stimuli, limited to the injected paw, appeared shortly after the injection, peaked around 18 h and lasted about 2 weeks. These phenomena proved the success of the model. The pharmacokinetic study was carried out 18 h after the CFA injection.

Instrumentations and analytical conditions The UPLC-MS/MS system was carried out on a Waters Acquity™ UPLC-MS/ MS system (Waters, Milford, MA, USA), which consisted of an auto-sampler, a quaternary pump, a column oven and a Xevo™ Triple Quadrupole MS/MS system equipped with an electrospray ionization (ESI) source (Waters Corp., Milford, MA, USA). The system was controlled with Unifi™ software for data acquisition and analysis, which was supplied by Waters Technologies. The chromatographic separation of the four analytes and IS were performed on a Shim-pack XR-ODS C18 column (75 × 3.0 mm, 2.2 μm particle size, Shimadzu, Japan) protected by a high-pressure column pre-filter (2 μm; Waters, USA). The mobile phase consisted of methanol–0.1% formic acid aqueous solution (30:70, v/v) at a flow rate of 0.4 mL/min. The column oven temperature and the autosampler temperature were maintained at 30 and 4°C, respectively. The sample injection volume was 10 μL. These analytes were quantified in multiple reaction monitoring mode and detected using electrospray ionization. Among these components, gentiopicroside, swertiamarin, loganic acid and amygdalin (IS) were detected in the negative ionization mode while sweroside was detected in the positive ionization mode in a single run. High-purity nitrogen served as both nebulizing and drying gas. The electrospray source and the mass spectrometer were adjusted to the following parameters: the nebulizer gas pressure, 7.0 bar; capillary voltage, 3.0 kV in positive ionization mode and at 2.0 kV in negative ionization mode; source offset, 50 V; source temperature, 150°C; desolvation temperature, 400°C; cone gas flow, collision gas flow and desolvation gas flow, at 150 L/h, 0.13 mL/min and 700 L/h, respectively. The optimized multiple reaction monitoring parameters of the four analytes and IS are listed in Table 1.

Preparation of HLXLD and GME The dried 11 herbs used in this study were prepared and extracted with 70% aqueous acetone as previously described (Cao et al., 2010). HLXLD was formulated by mixing the 11 extracts and then dissolved in 0.5% CMC-Na aqueous solution. GME was obtained using the same method as described above.

Preparation of standard solution Stock solutions of gentiopicroside (1 mg/mL), swertiamarin (100 μg/mL), sweroside (100 μg/mL), loganic acid (100 μg/mL) and IS (amygdalin) (100 μg/mL) were prepared in methanol. Stock solutions of the analytes were further diluted with methanol to make a series of mixed working solutions at the concentrations of 0.3–150 μg/mL for gentiopicroside, 6–3000 ng/mL for swertiamarin, 2.5–1250 ng/mL for sweroside and 30–15,000 ng/mL for loganic acid. The stock solution of the IS was diluted

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Simultaneous determination of four active compounds by UPLC-MS/MS Table 1. List of selected multiple reaction monitoring parameters, cone voltage, collision energy and retention times for each analyte and IS Analytes Gentiopicroside Swertiamarin Sweroside Loganic acid Amygdalin (IS)

Ionization mode

Q1 mass (Da)

Q3 mass (Da)

Cone voltage (V)

Collision energy (eV)

Retention time (min)

ESI( ) ESI( ) ESI(+) ESI( ) ESI( )

401.1 419.0 359.2 375.0 456.1

179.2 179.1 197.0 213.2 323.1

34 14 17 47 56

10 10 8 18 16

3.03 2.39 3.45 1.85 2.07

to a concentration of 2 μg/mL with methanol as working solution. All the solutions were stored at 4°C and brought to room temperature before use. The calibration standards were prepared by spiking 200 μL aliquots of blank plasma with 20 μL of the corresponding mixed standard solutions to obtain final concentrations in the ranges of 30–15,000 ng/mL for gentiopicroside, 0.6–300 ng/mL for swertiamarin, 0.25–125 ng/mL for sweroside and 3–1500 ng/mL for loganic acid. The low, medium and high quality control (QC) samples were prepared in the same way as the calibration standard samples (60, 900 and 12,000 ng/mL for gentiopicroside; 1.2, 18 and 240 ng/mL for swertiamarin; 0.5, 7.5 and 100 ng/mL for sweroside; and 6, 90 and 1200 ng/mL for loganic acid).

comparing the analyte standard peak areas obtained from extracted samples with post-extracted samples spiked with the analytes. Matrix effect was assessed by comparing the peak areas of the analytes in post-extracted spiked samples with those of the analytes dissolved in initial mobile phase at the same concentration. The stability of analytes was determined by analyzing three replicates of QC samples under different conditions: 8 h at room temperature; after three freeze ( 20°C for 12 h) and thaw cycles (room temperature for 12 h); after storage at 80°C for 2 weeks; and in autosampler vials at 4°C for 8 h, respectively.

Pharmacokinetic studies Sample preparation To a 200 μL aliquot of rat plasma sample in a 5 mL Eppendorf tube, 20 μL of IS solution (2 μg/mL), 20 μL of methanol and 50 μL of 2% formic acid were added and the samples were vortexed for 1 min. Then the mixture was extracted with 2 mL of ethyl acetate–isopropanol (7:3, v/v) by vortex-mixing for 3 min. After centrifugation at 13,000 rpm, 4°C for 10 min, the organic phase was quantitatively transferred into another clean Eppendorf tube and evaporated to dryness under a gentle stream of nitrogen at 35°C. Then the residue was reconstituted with 100 μL of mobile phase and sonicated for 3 min, vortex-mixed for 1 min, then centrifuged at 13,000 rpm for 5 min. Finally, 10 μL of the supernatant was injected into the UPLC-MS/ MS system for analysis.

Method validation

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Results and discussion Optimization of chromatographic and mass conditions The mobile phase is important for improving peak shape and detection sensitivity and shortening run time. Methanol–water and acetonitrile–water systems were investigated. It was found that methanol provided higher responses and lower background noise than acetonitrile. Moreover, the sensitivity and peak symmetry of these analytes were extremely improved with addition of formic acid to the mobile phase. The concentration of formic acid in the water phase was optimized from 0.01 to 0.2%. This showed that the addition of 0.1% formic acid into water phase was optimum. Finally, the optimized analytical method mentioned in the section

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The method was fully validated according to the US Food and Drug Administration and other related guidelines with respect to specificity, LLOQ, linearity, accuracy and precision, recovery, matrix effect and stability (Shah et al., 2000; US Food and Drug Administration, 2001). The specificity was evaluated by comparing chromatograms of blank plasma from six different rats, blank plasma spiked with gentiopicroside, swertiamarin, sweroside, loganic acid and IS, and plasma samples obtained after oral administration of HLXLD. The LLOQ was defined as the lowest concentration on the calibration curve with an acceptable accuracy within ± 20% and precision