Research article Received: 13 September 2013,
Revised: 9 February 2014,
Accepted: 18 February 2014
Published online in Wiley Online Library: 28 March 2014
(wileyonlinelibrary.com) DOI 10.1002/bmc.3184
Determination of swertianolin in rat plasma by LC-MS/MS and its application to a pharmacokinetic study Jun Hea, Chengwang Tianb*, Huizi Ouyangc, Tiwalade A. Adelakuna, Bin Yua, Yanxu Changa, Guixiang Pana, Linghuo Jiangb and Xiumei Gaoa* ABSTRACT: A sensitive and rapid LC-MS/MS method has been developed and validated for quantifying swertianolin in rat plasma using rutin as an internal standard (IS). Following liquid–liquid extraction with ethyl acetate, chromatographic separation for swertianolin was achieved on a C18 column with a gradient elution using 0.1% formic acid as mobile phase A and acetonitrile as mobile phase B at a flow rate of 0.3 mL/min. The detection was performed on a tandem mass spectrometer using multiple reaction monitoring via an electrospray ionization source and operating in the negative ionization mode. The optimized mass transition ion pairs (m/z) for quantitation were 435.1/272.0 for swertianolin and 609.2/300.1 for IS. The lower limit of quantitation was 0.5 ng/mL within a linear range of 0.5–500 ng/mL. Intra-day and inter-day precision was less than 6.8%. The accuracy was in the range of 13.9 to 12.0%. The mean recovery of swertianolin was >66.7%. The proposed method was successfully applied in evaluating the pharmacokinetics of swertianolin after an oral dose of 50 mg/kg Swertia mussotii extract in rats. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: swertianolin; LC-MS/MS; pharmacokinetics
Introduction
1418
Swertia species, which belong to the family Gentianaceae, are widely used in traditional medicine in many Asian countries such as China, India and Japan. Pharmacological research indicated that the plants of the Swertia genus have a variety of biological effects, including hepatoprotective, antidiabetic, antiinflammatory, anticholinesterase, antioxidant and antimicrobial (Brahmachari et al., 2004). Swertianolin, a xanthone glucoside isolated from the Swertia species, is one of the representative constituents of many Swertia species, such as S. chirayita, S. mussotii, S. japonica and S. punctata (Sakamoto et al., 1982; Li et al., 2008; Singh et al., 2012; Menkovic et al., 2002). Swertianolin has been reported to possess hepatoprotective, anticholinesterase, antituberculosis, antioxidant, antiarrhythmic and antidiabetic effects (Brahmachari et al., 2004; Li et al., 2005; Urbain et al., 2004, 2008; Mukherjee et al., 2007; Wani et al., 2013; Wan et al., 2013). Despite its various biological activities, the pharmacokinetic properties of swertianolin have not been reported. It is known that pharmacokinetics plays an important role in drug development and can explain a variety of events related to the efficacy of herbal medicines. However, there are only a few HPLC methods that have been reported to determine swertianolin in herbs (Wan et al., 2013; Li et al., 2007; Bao et al., 2006; Xu et al., 2009), and there is no method that has been developed to study pharmacokinetics of swertianolin. LC-MS/MS has been accepted as a feasible method for pharmacokinetic research on herbal medicines (He et al., 2013). In this study, we developed a sensitive LC-MS/MS method for determination of swertianolin in rat plasma. After validation, this method was successfully applied to pharmacokinetics after administration of
Biomed. Chromatogr. 2014; 28: 1418–1422
Swertia mussotii extract in rats. The results would be useful for evaluating the clinical application of swertianolin.
Experimental Chemicals and reagents Acetonitrile (Fisher, Fair Lawn, USA), methanol (Fisher, Fair Lawn, USA) and formic acid (Tedia, Fairfield, USA) were of HPLC grade. Analytical grade ethyl acetate was purchased from Tianjin Concord Science Co. Ltd (Tianjin, China). Deionized water was prepared using a Milli-Q water purification system (Millipore, Milford, MA, USA). Rutin was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Swertianolin was isolated from Swertia mussotii in our laboratory (>98%). Its structure was confirmed 1 by spectra ( H NMR, IR, and MS), and the purity was confirmed by HPLC and MS analysis.
* Correspondence to: Chengwang Tian and Xiumei Gao, Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin, 300193, China. Email: [email protected]. cn; [email protected] a
Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin, 300193, China
b
School of Pharmaceutical Science and Technology, Tianjin University, Tianjin, 300072, China
c
The First Affiliated Hospital of Tianjin University of Traditional Chinese Medicine, Tianjin, 300193, China Abbreviation used: LLE, liquid–liquid extraction.
Copyright © 2014 John Wiley & Sons, Ltd.
Pharmacokinetic studies of swertianolin in rats Chromatographic and mass spectrometry conditions The LC-MS/MS system was composed of an Agilent 1200 liquid chromatography system coupled to API 3200 triple quadrupole mass spectrometer equipped with electrospray ionization source. Data acquisition and peak integration were performed using Analyst software (version 1.4.2) from Applied Biosystems/MDS Sciex. The chromatographic separation was achieved using an Agilent Eclipse Plus C18 column (2.1 × 150 mm, 3.5 μm). The mobile phase consisted of 0.1% (v/v) formic acid (A) and acetonitrile (B) using a gradient elution of 17–60% B at 0–4 min, 60–60% B at 4–8 min. The reequilibration time of the column was 4 min. The flow rate was kept at 0.3 mL/min, the column temperature was set at 30°C, and the sample injection volume was 5 μL. Mass spectrometer was operated in the negative ion mode with an optimized spray voltage at 4500 V and turbo spray temperature at 450°C. The curtain gas, nebulizer gas (gas 1) and auxiliary gas (gas 2) were at 30, 60 and 60 psi, respectively. For collision-induced dissociation, the collision gas was set at 5 psi. Quantitation was achieved using multiple reaction monitoring of the transitions of m/z 435.1 → 272.0 for swertianolin and 609.2 → 300.1 for the internal standard (IS). The optimized declustering potential, entrance potential, collision energy and collision cell exit potential were 70, 10, 35 and 7 V for swertianolin and 85, 7, 50 and 24 V for the IS.
Preparation of Swertia mussotii extract In the experiment, 200 g of Swertia mussotii was extracted with 90% ethanol (1:12, w/v) for 1.5 h under reflux twice. The extraction solution was concentrated to 200 mL under reduced pressure. Then, the solution was extracted with ligarine to remove the lipophilic constituents. The remaining water fraction was subjected to a HDP-300 macroporous resin column chromatography (water → 30% ethanol → 75% ethanol) to give three fractions. The 75% ethanol fraction was evaporated to dryness for administration. The content of swertianolin in Swertia mussotii extract was 610 mg/g.
Assay specificity. The specificity of the method was investigated by analyzing six blank plasma samples from different rats. Each blank sample was determined for endogenous interference using the proposed preparation procedure and LC-MS/MS conditions. Linearity and sensitivity. The calibration curve was obtained by linear regression of the peak area ratios of swertianolin to IS at eight 2 concentration levels, using 1/x as the weighting factor. Each sample level was prepared and assayed in duplicate on three consecutive days. The sensitivity of the method was represented by the lower limit of quantification (LLOQ), which was determined at a signal-to-noise ratio of about 10 by analyzing the standard plasma samples. Precision and accuracy. The precision and accuracy of this method were assessed by determination of six replicates of QC samples at low, medium and high concentration levels (1, 50 and 500 ng/mL for swertianolin) on three separate days. The intra-day and inter-day precisions were defined as relative standard deviation (RSD) and the accuracy was determined as the relative error (RE), which was assessed by comparing the measured concentration with its true value. The precision for QC samples should not exceed 15%, and accuracy accepted was within ±15%. Extraction recovery and matrix effect. To investigate the recovery and matrix effect, extracted samples, unextracted samples (pure standard solution) and post-extracted spiked samples at three QC levels were assayed using the same method. The recovery was evaluated by comparing the peak area ratios of the analytes in extracted samples with those acquired from post-extracted spiked samples. The matrix effect was obtained by comparing peak areas of analytes in post-extracted spiked samples with those in unextracted samples. Stability. The short-term stability was evaluated by determining QC samples at room temperature for 6 h and in the autosampler after preparation for 12 h. The long-term stability was assessed by storing the QC samples at 20°C for 30 days. The freeze–thaw stability was determined through three complete freeze–thaw cycles on consecutive days. For each condition, three replicates were analyzed at low, medium and high concentrations, respectively.
Preparation of calibration standards and quality control samples Stock solution of swertianolin (1 mg/mL) was prepared in methanol. Then, from the stock solution, working solutions were obtained by sequential dilution with methanol. Calibration standard solutions were prepared by spiking the blank rat plasma (100 μL) with appropriate amount of the working solutions, yielding a final concentration range of 0.5–500 ng/mL. Quality control (QC) plasma samples were prepared in the same manner, and the concentrations were 1, 50 and 500 ng/mL. A quantity of rutin was dissolved in methanol to prepare the IS solution with a concentration of 500 ng/mL.
Sample preparation To each 100 μL of the plasma sample, 25 μL of the IS, 25 μL of methanol (volume of the corresponding working solution for calibration curve and QC samples), 10 μL of formic acid and 1 mL of ethyl acetate were added. The mixture was vortexed for 3 min and then centrifuged for 5 min at 14,000 rpm. The supernatant (900 μL) was transferred to another polypropylene centrifuge tube, and evaporated to dryness under a constant flow of nitrogen gas. The obtained residue was reconstituted in 100 μL methanol and centrifuged at 14,000 rpm for another 10 min. Finally, a 5 μL aliquot of the processed sample was injected into the LC-MS/MS system for analysis.
Method validation
Biomed. Chromatogr. 2014; 28: 1418–1422
Six male Sprague–Dawley rats (210 ± 10 g) were fasted but given free access to water for 12 h prior to experiments. Then, Swertia mussotii extract at a single dose of 50 mg/kg was administered to the rats orally. Blood samples (300 μL) were collected into heparinized centrifuge tubes from the rat fossa orbitalis vein at pre-dose, and 0.03, 0.08, 0.17, 0.25, 0.5, 1, 2, 3, 4, 6, 8, 12 and 24 h post administration. After centrifugation at 6000 rpm for 10 min, plasma samples were finally obtained and stored at 20°C until analysis. Pharmacokinetic parameters were calculated by noncompartmental analysis using the computer program ‘Drug and Statistics 1.0’ (DAS 1.0; Medical College of Wannan, China).
Results and discussion LC-MS/MS method development The standard solutions of swertianolin and IS were delivered directly into the mass spectrometer separately using a syringe pump. Initially, the analytes were scanned in both positive and negative modes, and it was found that ions in negative mode were more sensitive than in positive mode. After mass spectrometric parameters such as declustering potential, entrance potential, collision energy and cell exit potential were optimized, the transitions m/z 435.1/272.0 for swertianolin and 609.2/300.1 for IS were chosen for the quantification studies.
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/bmc
1419
The proposed LC-MS/MS method was validated in accordance with the US Food and Drug Administration guidelines.
Application
J. He et al. Fig. 1 shows the product ion mass spectra and chemical structures of swertianolin and rutin (IS). By testing different mobile phase additives, it was found that 0.1% formic acid in the mobile phase could enhance the sensitivity and improve the peak shape of swertianolin, but ammonium formate had no significant effect. Finally, a mobile phase composed of 0.1% formic acid and acetonitrile was used on Agilent Eclipse Plus C18 column (2.1 × 150 mm, 3.5 μm) in the experiment. As shown in Fig. 2, swertianolin and IS were eluted within 8 min, and no endogenous plasma components interfered with the analytes.
Sample preparation During method development, liquid–liquid extraction (LLE) and a protein precipitation method were compared. Initially, acetonitrile and methanol were added separately to deproteinize
rat plasma sample; however, the recovery of swertianolin was unsatisfactory. Alternatively, the LLE method was used. Several extraction solvent such as ethyl acetate, chloroform, as well as ethyl acetate containing 10 μL of formic acid were tested, after which ethyl acetate containing 10 μL of formic acid was selected as the extraction solvent because of its high extract efficiency.
Method validation Specificity. Specificity was assessed by analyzing six different blank plasma samples. Fig. 2 shows the typical chromatograms for a blank plasma sample, a blank plasma sample spiked with swertianolin and IS, and a plasma sample from a rat at 0.08 h after oral administration of Swertia mussotii extract. The retention times of swertianolin and IS were 6.64 and 5.68 min, respectively. No endogenous interference at the retention time of swertianolin and IS was observed in blank plasma samples.
Figure 1. The product ion mass spectra and chemical structures of swertianolin (A) and rutin (B).
1420
Figure 2. Typical multiple reaction monitoring chromatograms of swertianolin and rutin (IS): (A) blank plasma; (B) blank plasma spiked with swertianolin and IS; (C) 0.08 h plasma sample after a single oral administration of Swertia mussotii extract.
wileyonlinelibrary.com/journal/bmc
Copyright © 2014 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2014; 28: 1418–1422
Pharmacokinetic studies of swertianolin in rats autosampler after preparation, for 30 days at freeze–thaw cycles.
Linearity and sensitivity. The linear regression analysis was created by plotting the peak area ratios of swertianolin to IS vs analyte concentration in spiked plasma samples. The calibration curve ranged from 0.5 to 500 ng/mL, and the regression equation of the curve was y = 0.0133x + 0.00133, with correlation coefficient (r) of 0.9971. The LLOQ of swertianolin was 0.5 ng/mL, the precision (relative standard deviaiton, RSD) and accuracy (relative error, RE) were 6.2 and 11.8%, respectively. This indicated that this method was of great advantage in the measurement of the trace concentrations of swertianolin in rat plasma.
20°C and for three
Application The validated method was applied to investigate the pharmacokinetic parameters of swertianolin in rats after a single oral dose of Swertia mussotii extract at 50 mg/kg. The mean plasma concentration–time profiles (n = 6) of swertianolin are illustrated in Fig. 3, and the major pharmacokinetic parameters are presented in Table 3. As shown in Table 3, the maximum concentration (Cmax) of swertianolin in plasma (135.2 ± 72.3 ng/mL) was attained at 0.15 ± 0.08 h (Tmax), and their elimination half-life (T1/2) was 0.38 ± 0.21 h. The results indicated that swertianolin was rapidly absorbed and eliminated in rat plasma after oral administration of Swertia mussotii extract. A similar phenomenon was also discovered in the pharmacokinetic study of other xanthone glucosides, such as neomangiferin (Cai et al., 2010).
Precision and accuracy. The intra-day and inter-day precision and accuracy were evaluated using QC samples at 1, 50 and 500 ng/mL. The results are summarized in Table 1. At each QC level, the intra-day and inter-day precisions (RSD) of swertianolin were
Revised: 9 February 2014,
Accepted: 18 February 2014
Published online in Wiley Online Library: 28 March 2014
(wileyonlinelibrary.com) DOI 10.1002/bmc.3184
Determination of swertianolin in rat plasma by LC-MS/MS and its application to a pharmacokinetic study Jun Hea, Chengwang Tianb*, Huizi Ouyangc, Tiwalade A. Adelakuna, Bin Yua, Yanxu Changa, Guixiang Pana, Linghuo Jiangb and Xiumei Gaoa* ABSTRACT: A sensitive and rapid LC-MS/MS method has been developed and validated for quantifying swertianolin in rat plasma using rutin as an internal standard (IS). Following liquid–liquid extraction with ethyl acetate, chromatographic separation for swertianolin was achieved on a C18 column with a gradient elution using 0.1% formic acid as mobile phase A and acetonitrile as mobile phase B at a flow rate of 0.3 mL/min. The detection was performed on a tandem mass spectrometer using multiple reaction monitoring via an electrospray ionization source and operating in the negative ionization mode. The optimized mass transition ion pairs (m/z) for quantitation were 435.1/272.0 for swertianolin and 609.2/300.1 for IS. The lower limit of quantitation was 0.5 ng/mL within a linear range of 0.5–500 ng/mL. Intra-day and inter-day precision was less than 6.8%. The accuracy was in the range of 13.9 to 12.0%. The mean recovery of swertianolin was >66.7%. The proposed method was successfully applied in evaluating the pharmacokinetics of swertianolin after an oral dose of 50 mg/kg Swertia mussotii extract in rats. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: swertianolin; LC-MS/MS; pharmacokinetics
Introduction
1418
Swertia species, which belong to the family Gentianaceae, are widely used in traditional medicine in many Asian countries such as China, India and Japan. Pharmacological research indicated that the plants of the Swertia genus have a variety of biological effects, including hepatoprotective, antidiabetic, antiinflammatory, anticholinesterase, antioxidant and antimicrobial (Brahmachari et al., 2004). Swertianolin, a xanthone glucoside isolated from the Swertia species, is one of the representative constituents of many Swertia species, such as S. chirayita, S. mussotii, S. japonica and S. punctata (Sakamoto et al., 1982; Li et al., 2008; Singh et al., 2012; Menkovic et al., 2002). Swertianolin has been reported to possess hepatoprotective, anticholinesterase, antituberculosis, antioxidant, antiarrhythmic and antidiabetic effects (Brahmachari et al., 2004; Li et al., 2005; Urbain et al., 2004, 2008; Mukherjee et al., 2007; Wani et al., 2013; Wan et al., 2013). Despite its various biological activities, the pharmacokinetic properties of swertianolin have not been reported. It is known that pharmacokinetics plays an important role in drug development and can explain a variety of events related to the efficacy of herbal medicines. However, there are only a few HPLC methods that have been reported to determine swertianolin in herbs (Wan et al., 2013; Li et al., 2007; Bao et al., 2006; Xu et al., 2009), and there is no method that has been developed to study pharmacokinetics of swertianolin. LC-MS/MS has been accepted as a feasible method for pharmacokinetic research on herbal medicines (He et al., 2013). In this study, we developed a sensitive LC-MS/MS method for determination of swertianolin in rat plasma. After validation, this method was successfully applied to pharmacokinetics after administration of
Biomed. Chromatogr. 2014; 28: 1418–1422
Swertia mussotii extract in rats. The results would be useful for evaluating the clinical application of swertianolin.
Experimental Chemicals and reagents Acetonitrile (Fisher, Fair Lawn, USA), methanol (Fisher, Fair Lawn, USA) and formic acid (Tedia, Fairfield, USA) were of HPLC grade. Analytical grade ethyl acetate was purchased from Tianjin Concord Science Co. Ltd (Tianjin, China). Deionized water was prepared using a Milli-Q water purification system (Millipore, Milford, MA, USA). Rutin was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Swertianolin was isolated from Swertia mussotii in our laboratory (>98%). Its structure was confirmed 1 by spectra ( H NMR, IR, and MS), and the purity was confirmed by HPLC and MS analysis.
* Correspondence to: Chengwang Tian and Xiumei Gao, Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin, 300193, China. Email: [email protected]. cn; [email protected] a
Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin, 300193, China
b
School of Pharmaceutical Science and Technology, Tianjin University, Tianjin, 300072, China
c
The First Affiliated Hospital of Tianjin University of Traditional Chinese Medicine, Tianjin, 300193, China Abbreviation used: LLE, liquid–liquid extraction.
Copyright © 2014 John Wiley & Sons, Ltd.
Pharmacokinetic studies of swertianolin in rats Chromatographic and mass spectrometry conditions The LC-MS/MS system was composed of an Agilent 1200 liquid chromatography system coupled to API 3200 triple quadrupole mass spectrometer equipped with electrospray ionization source. Data acquisition and peak integration were performed using Analyst software (version 1.4.2) from Applied Biosystems/MDS Sciex. The chromatographic separation was achieved using an Agilent Eclipse Plus C18 column (2.1 × 150 mm, 3.5 μm). The mobile phase consisted of 0.1% (v/v) formic acid (A) and acetonitrile (B) using a gradient elution of 17–60% B at 0–4 min, 60–60% B at 4–8 min. The reequilibration time of the column was 4 min. The flow rate was kept at 0.3 mL/min, the column temperature was set at 30°C, and the sample injection volume was 5 μL. Mass spectrometer was operated in the negative ion mode with an optimized spray voltage at 4500 V and turbo spray temperature at 450°C. The curtain gas, nebulizer gas (gas 1) and auxiliary gas (gas 2) were at 30, 60 and 60 psi, respectively. For collision-induced dissociation, the collision gas was set at 5 psi. Quantitation was achieved using multiple reaction monitoring of the transitions of m/z 435.1 → 272.0 for swertianolin and 609.2 → 300.1 for the internal standard (IS). The optimized declustering potential, entrance potential, collision energy and collision cell exit potential were 70, 10, 35 and 7 V for swertianolin and 85, 7, 50 and 24 V for the IS.
Preparation of Swertia mussotii extract In the experiment, 200 g of Swertia mussotii was extracted with 90% ethanol (1:12, w/v) for 1.5 h under reflux twice. The extraction solution was concentrated to 200 mL under reduced pressure. Then, the solution was extracted with ligarine to remove the lipophilic constituents. The remaining water fraction was subjected to a HDP-300 macroporous resin column chromatography (water → 30% ethanol → 75% ethanol) to give three fractions. The 75% ethanol fraction was evaporated to dryness for administration. The content of swertianolin in Swertia mussotii extract was 610 mg/g.
Assay specificity. The specificity of the method was investigated by analyzing six blank plasma samples from different rats. Each blank sample was determined for endogenous interference using the proposed preparation procedure and LC-MS/MS conditions. Linearity and sensitivity. The calibration curve was obtained by linear regression of the peak area ratios of swertianolin to IS at eight 2 concentration levels, using 1/x as the weighting factor. Each sample level was prepared and assayed in duplicate on three consecutive days. The sensitivity of the method was represented by the lower limit of quantification (LLOQ), which was determined at a signal-to-noise ratio of about 10 by analyzing the standard plasma samples. Precision and accuracy. The precision and accuracy of this method were assessed by determination of six replicates of QC samples at low, medium and high concentration levels (1, 50 and 500 ng/mL for swertianolin) on three separate days. The intra-day and inter-day precisions were defined as relative standard deviation (RSD) and the accuracy was determined as the relative error (RE), which was assessed by comparing the measured concentration with its true value. The precision for QC samples should not exceed 15%, and accuracy accepted was within ±15%. Extraction recovery and matrix effect. To investigate the recovery and matrix effect, extracted samples, unextracted samples (pure standard solution) and post-extracted spiked samples at three QC levels were assayed using the same method. The recovery was evaluated by comparing the peak area ratios of the analytes in extracted samples with those acquired from post-extracted spiked samples. The matrix effect was obtained by comparing peak areas of analytes in post-extracted spiked samples with those in unextracted samples. Stability. The short-term stability was evaluated by determining QC samples at room temperature for 6 h and in the autosampler after preparation for 12 h. The long-term stability was assessed by storing the QC samples at 20°C for 30 days. The freeze–thaw stability was determined through three complete freeze–thaw cycles on consecutive days. For each condition, three replicates were analyzed at low, medium and high concentrations, respectively.
Preparation of calibration standards and quality control samples Stock solution of swertianolin (1 mg/mL) was prepared in methanol. Then, from the stock solution, working solutions were obtained by sequential dilution with methanol. Calibration standard solutions were prepared by spiking the blank rat plasma (100 μL) with appropriate amount of the working solutions, yielding a final concentration range of 0.5–500 ng/mL. Quality control (QC) plasma samples were prepared in the same manner, and the concentrations were 1, 50 and 500 ng/mL. A quantity of rutin was dissolved in methanol to prepare the IS solution with a concentration of 500 ng/mL.
Sample preparation To each 100 μL of the plasma sample, 25 μL of the IS, 25 μL of methanol (volume of the corresponding working solution for calibration curve and QC samples), 10 μL of formic acid and 1 mL of ethyl acetate were added. The mixture was vortexed for 3 min and then centrifuged for 5 min at 14,000 rpm. The supernatant (900 μL) was transferred to another polypropylene centrifuge tube, and evaporated to dryness under a constant flow of nitrogen gas. The obtained residue was reconstituted in 100 μL methanol and centrifuged at 14,000 rpm for another 10 min. Finally, a 5 μL aliquot of the processed sample was injected into the LC-MS/MS system for analysis.
Method validation
Biomed. Chromatogr. 2014; 28: 1418–1422
Six male Sprague–Dawley rats (210 ± 10 g) were fasted but given free access to water for 12 h prior to experiments. Then, Swertia mussotii extract at a single dose of 50 mg/kg was administered to the rats orally. Blood samples (300 μL) were collected into heparinized centrifuge tubes from the rat fossa orbitalis vein at pre-dose, and 0.03, 0.08, 0.17, 0.25, 0.5, 1, 2, 3, 4, 6, 8, 12 and 24 h post administration. After centrifugation at 6000 rpm for 10 min, plasma samples were finally obtained and stored at 20°C until analysis. Pharmacokinetic parameters were calculated by noncompartmental analysis using the computer program ‘Drug and Statistics 1.0’ (DAS 1.0; Medical College of Wannan, China).
Results and discussion LC-MS/MS method development The standard solutions of swertianolin and IS were delivered directly into the mass spectrometer separately using a syringe pump. Initially, the analytes were scanned in both positive and negative modes, and it was found that ions in negative mode were more sensitive than in positive mode. After mass spectrometric parameters such as declustering potential, entrance potential, collision energy and cell exit potential were optimized, the transitions m/z 435.1/272.0 for swertianolin and 609.2/300.1 for IS were chosen for the quantification studies.
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/bmc
1419
The proposed LC-MS/MS method was validated in accordance with the US Food and Drug Administration guidelines.
Application
J. He et al. Fig. 1 shows the product ion mass spectra and chemical structures of swertianolin and rutin (IS). By testing different mobile phase additives, it was found that 0.1% formic acid in the mobile phase could enhance the sensitivity and improve the peak shape of swertianolin, but ammonium formate had no significant effect. Finally, a mobile phase composed of 0.1% formic acid and acetonitrile was used on Agilent Eclipse Plus C18 column (2.1 × 150 mm, 3.5 μm) in the experiment. As shown in Fig. 2, swertianolin and IS were eluted within 8 min, and no endogenous plasma components interfered with the analytes.
Sample preparation During method development, liquid–liquid extraction (LLE) and a protein precipitation method were compared. Initially, acetonitrile and methanol were added separately to deproteinize
rat plasma sample; however, the recovery of swertianolin was unsatisfactory. Alternatively, the LLE method was used. Several extraction solvent such as ethyl acetate, chloroform, as well as ethyl acetate containing 10 μL of formic acid were tested, after which ethyl acetate containing 10 μL of formic acid was selected as the extraction solvent because of its high extract efficiency.
Method validation Specificity. Specificity was assessed by analyzing six different blank plasma samples. Fig. 2 shows the typical chromatograms for a blank plasma sample, a blank plasma sample spiked with swertianolin and IS, and a plasma sample from a rat at 0.08 h after oral administration of Swertia mussotii extract. The retention times of swertianolin and IS were 6.64 and 5.68 min, respectively. No endogenous interference at the retention time of swertianolin and IS was observed in blank plasma samples.
Figure 1. The product ion mass spectra and chemical structures of swertianolin (A) and rutin (B).
1420
Figure 2. Typical multiple reaction monitoring chromatograms of swertianolin and rutin (IS): (A) blank plasma; (B) blank plasma spiked with swertianolin and IS; (C) 0.08 h plasma sample after a single oral administration of Swertia mussotii extract.
wileyonlinelibrary.com/journal/bmc
Copyright © 2014 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2014; 28: 1418–1422
Pharmacokinetic studies of swertianolin in rats autosampler after preparation, for 30 days at freeze–thaw cycles.
Linearity and sensitivity. The linear regression analysis was created by plotting the peak area ratios of swertianolin to IS vs analyte concentration in spiked plasma samples. The calibration curve ranged from 0.5 to 500 ng/mL, and the regression equation of the curve was y = 0.0133x + 0.00133, with correlation coefficient (r) of 0.9971. The LLOQ of swertianolin was 0.5 ng/mL, the precision (relative standard deviaiton, RSD) and accuracy (relative error, RE) were 6.2 and 11.8%, respectively. This indicated that this method was of great advantage in the measurement of the trace concentrations of swertianolin in rat plasma.
20°C and for three
Application The validated method was applied to investigate the pharmacokinetic parameters of swertianolin in rats after a single oral dose of Swertia mussotii extract at 50 mg/kg. The mean plasma concentration–time profiles (n = 6) of swertianolin are illustrated in Fig. 3, and the major pharmacokinetic parameters are presented in Table 3. As shown in Table 3, the maximum concentration (Cmax) of swertianolin in plasma (135.2 ± 72.3 ng/mL) was attained at 0.15 ± 0.08 h (Tmax), and their elimination half-life (T1/2) was 0.38 ± 0.21 h. The results indicated that swertianolin was rapidly absorbed and eliminated in rat plasma after oral administration of Swertia mussotii extract. A similar phenomenon was also discovered in the pharmacokinetic study of other xanthone glucosides, such as neomangiferin (Cai et al., 2010).
Precision and accuracy. The intra-day and inter-day precision and accuracy were evaluated using QC samples at 1, 50 and 500 ng/mL. The results are summarized in Table 1. At each QC level, the intra-day and inter-day precisions (RSD) of swertianolin were
