Research article Received: 8 January 2015,
Revised: 22 March 2015,
Accepted: 23 April 2015
Published online in Wiley Online Library: 1 June 2015
(wileyonlinelibrary.com) DOI 10.1002/bmc.3499
Determination and validation of hupehenine in rat plasma by UPLC-MS/MS and its application to pharmacokinetic study Congcong Wena†, Shuanghu Wangb†, Xueli Huangc, Zezheng Liua, Yingying Lina, Suping Yanga, Jianshe Mac, Yunfang Zhoub and Xianqin Wangc* ABSTRACT: In this work, a sensitive and selective ultra-performance liquid chromatography–tandem mass spectrometry (UPLCMS/MS) method for determination of hupehenine in rat plasma was developed and validated. After addition of imperialine as an internal standard (IS), protein precipitation by acetonitrile–methanol (9:1, v/v) was used to prepare samples. Chromatographic separation was achieved on a UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm) with 0.1% formic acid and acetonitrile as the mobile phase with gradient elution. An electrospray ionization source was applied and operated in positive ion mode; multiple reaction monitoring mode was used for quantification using target fragment ions m/z 416.3 → 98.0 for hupehenine, and m/z 430.3 → 138.2 for IS. Calibration plots were linear throughout the range 2–2000 ng/mL for hupehenine in rat plasma. Mean recoveries of hupehenine in rat plasma ranged from 92.5 to 97.3%. Relative standard deviations of intra-day and inter-day precision were both 98%) and imperialine (IS, purity >98%) were purchased from the Chengdu Mansite Pharmaceutical Co. Ltd (Chengdu, China). LC-grade acetonitrile and methanol were purchased from Merck Company (Darmstadt, Germany). Ultra-pure water was prepared by Millipore Milli-Q purification system (Bedford, MA, USA). Rat blank plasma samples were supplied from drug-free rats.
* Correspondence to: X. Wang, Analytical and Testing Center, Wenzhou Medical University, Wenzhou, Zhejiang 325035, China. Email: [email protected] †
These two authors contributed equally to this work
a
Laboratory Animal Centre, Wenzhou Medical University, Wenzhou 325035, China
b
The Laboratory of Clinical Pharmacy, The People’s Hospital of Lishui 323000, Wenzhou Medical University, Lishui, China
c
Analytical and Testing Center, Wenzhou Medical University, Wenzhou 325035, China
Copyright © 2015 John Wiley & Sons, Ltd.
1805
Hubeibeimu is the bulbs of Fritillaria hupehensis Hsiao et K.C.Hsia and has been used as an antitussive and expectorant herb in China for a long time (Xiao, 2002). Many chemical and pharmacological studies have shown that the total alkaloids are responsible for antitussive and expectorant activity (Zhang et al., 2008). Considering that peiminine, hupehenine and peimine have higher contents and hupehenine is the characteristic constituent, hupehenine (Fig. 1) was chosen as the index component (Wu et al., 1986). To date, several analytical methods described in the literature for the determination of hupehenine in biological and other matrices have involved either high-performance liquid chromatography with evaporative light scattering detection (Zhang et al., 2008) or gas chromatography (GC) (Song-Lin et al., 2000), or liquid chromatography–mass spectrometry (LC-MS) (Wu et al., 2010). However, the limit of detection (LOD) of hupehenine using HPLC or GC-MS is not high enough for pharmacokinetic and bioavailability studies, and ultra-performance liquid chromatography– tandem mass spectrometry (UPLC-MS/MS) is known to enable increased throughput, sensitivity and better chromatographic peak resolution (Churchwell et al., 2005). Wu et al. (2010) developed and validated a sensitive LC-MS method for the quantification of verticinone in rat plasma over the concentration range of 0.1– 200 ng/mL. Hupehenine was used as IS their work; the total runtime was 15 min. No current UPLC-MS/MS method exists for the determination of hupehenine to characterize pharmacokinetic properties. In this study, a UPLC-MS/MS method for determination of hupehenine in rat plasma was developed and successfully
applied to pharmacokinetic study of hupehenine after oral and intravenous administration. The UPLC-MS/MS method in this study was validated for selectivity, linearity, accuracy, precision, recovery and stability with a total run time of 3 min.
C. Wen et al.
Figure 1. Mass spectrum of hupehenine (a) and imperialine (IS, b).
Instrumentation and conditions
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A UPLC-MS/MS system with Acquity I-Class UPLC and a XEVO TQD triple quadrupole mass spectrometer (Waters Corp., Milford, MA, USA), equipped with an electrospray ionization interface, was used to analyze the compounds. The UPLC system comprised a Binary Solvent Manager and a Sample Manager with Flow-Through Needle. Masslynx 4.1 software (Waters Corp., Milford, MA, USA) was used for data acquisition and instrument control. Hupehenine and imperialine (IS) were separated on an UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm) maintained at 40 °C. The initial mobile phase consisted of 0.1% formic acid and acetonitrile with gradient elution at a flow rate of 0.4 mL/min and an injection volume of 2 μL. Elution was in a linear gradient, where the acetonitrile increased from 40 to 90% between 0 and 2.0 min, maintained at 90% for 0.5 min, then decreased to 40% within 0.1 min, then maintained at 40% for 0.4 min. The total run time of the analytes was 3 min. After each injection, the sample manager underwent a needle wash process, including both a strong wash (methanol–water, 50/50, v/v) and a weak wash (methanol–water, 10/90, v/v). Nitrogen was used as the desolvation gas (1000 L/h) and cone gas (50 L/ h). Ion monitoring conditions were defined as capillary voltage of 2.5 kV,
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source temperature of 150 °C and desolvation temperature of 500 °C. Multiple reaction monitoring modes of m/z 416.3 → 98.0 for hupehenine and m/z 430.3 → 138.2 for IS were utilized to conduct quantitative analysis.
Calibration standards and quality control samples The stock solutions of hupehenine (1.0 mg/mL) and imperialine (IS) (100 μg/ mL) were prepared in methanol–water (50: 50, v/v). The 0.25 μg/mL working standard solution of the IS was prepared from the IS stock solution by dilution with methanol; working solutions for calibration and controls were prepared from stock solutions in the same manner. All of the solutions were stored at 4 °C and were brought to room temperature before use. Hupehenine calibration standards were prepared by spiking blank rat plasma with appropriate amounts of the working solutions. Calibration plots were offset to range between 2 and 2000 ng/mL for hupehenine in rat plasma (2, 5, 10, 20, 50, 100, 200, 500, 1000 and 2000 ng/mL). Quality-control (QC) samples were prepared in the same manner as the calibration standards, in three different plasma concentrations (4, 800, and 1600 ng/mL). The analytical standards and QC samples were stored at 20 °C.
Copyright © 2015 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2015; 29: 1805–1810
Determination of hupehenine in rat plasma by UPLC-MS/MS Sample preparation Before analysis, the plasma sample was thawed to room temperature. An aliquot of 10 μL of the IS working solution (0.25 μg/mL) was added to 50 μL of the collected plasma sample in a 1.5 mL centrifuge tube, followed by the addition of 150 μL of acetonitrile–methanol (9:1, v/v). The tubes were vortex mixed for 1.0 min. After centrifugation at 14,900 g for 10 min in Allegra 64R High-speed Centrifuge (Beckman Coulter Inc., Los Angeles, California USA), the supernatant (2 μL) was injected into the UPLC-MS/MS system for analysis.
(0.2 mL) were collected from the caudal vein into heparinized 1.5 mL tapered plastic centrifuge tubes at 0.0833, 0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 12 and 24 h after oral (10 mg/kg) or intravenous (5 mg/kg) administration of hupehenine. The caudal vein of the rats was cleaned with 75% alcohol; after that the end of caudal vein was cut with scissors. A 1.5 mL tapered plastic centrifuge tube was used to collect the blood which dropped from the end of caudal vein by squeezing and massaging gently. The samples were immediately centrifuged at 3000 g for 10 min. The plasma (50 μL) was stored as obtained at 20 ° C until UPLC-MS/MS analysis. Plasma hupehenine concentration vs time data for each rat was analyzed using DAS (Drug and Statistics) software (version 2.0, Wenzhou Medical University, China).
Method validation Rigorous tests for selectivity, linearity, accuracy, precision, recovery and stability, according to the guidelines set by the US Food and Drug Administration (2013) and European Medicines Agency (2011), were conducted in order to thoroughly validate the proposed bioanalytical method. Validation runs were conducted on three consecutive days. Each validation run consisted of one set of calibration standards and six replicates of QC plasma samples. The selectivity of the method was evaluated by analyzing blank rat plasma, blank plasma-spiked hupehenine and IS, and a rat plasma sample. Calibration curves were constructed by analyzing spiked calibration samples on three separate days. Peak area ratios of hupehenine-to-IS were plotted against analyte concentrations. Resultant standard curves were well fitted to the equations by linear regression, with a weighting factor of the reciprocal of the concentration (1/x) in the concentration range of 2– 2000 ng/mL. The lower limit of quantitation (LLOQ) was defined as the lowest concentration on the calibration curves. To evaluate the matrix effect, blank rat plasma was extracted and spiked with the analyte at 4, 800 and 1600 ng/mL concentrations. The corresponding peak areas were then compared with those of neat standard solutions at equivalent concentrations; this peak area ratio is defined as the matrix effect. The matrix effect of the IS was evaluated at a concentration of 50 ng/mL in a similar manner. Accuracy and precision were assessed by the determination of QC samples at three concentration levels in six replicates (4, 800 and 1600 ng/mL) over 3 days of validation testing. The precision is expressed as RSD. The recovery of hupehenine was evaluated by comparing the peak area of extracted QC samples with those of reference QC solutions reconstituted in blank plasma extracts (n = 6). The recovery of the IS was determined in the same way. Carry-over was assessed following injection of a blank plasma sample immediately after three repeats of the upper limit of quantification (ULOQ), after which the response was checked for accuracy (Williams et al., 2012). Stability of hupehenine in rat plasma was evaluated by analyzing three replicates of plasma samples at concentrations of 4 or 1600 ng/mL, which were all exposed to different conditions. These results were compared with the freshly prepared plasma samples. Short-term stability was determined after the exposure of the spiked samples to room temperature for 2 h, and the ready-to-inject samples (after protein precipitation) in the HPLC autosampler at room temperature for 24 h. Freeze–thaw stability was evaluated after three complete freeze–thaw cycles ( 20–25 °C) on consecutive days. Long-term stability was assessed after storage of the standard spiked plasma samples at 20 °C for 20 days. The stability of the IS (50 ng/mL) was evaluated similarly (Du et al., 2014a, b; Liu et al., 2014).
Pharmacokinetic study
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Selectivity and matrix effect Figure 2 shows typical chromatograms of a blank plasma sample, a blank plasma sample spiked with hupehenine and IS, and a plasma sample. There were no interfering endogenous substances observed at the retention time of the hupehenine and IS. The matrix effects for hupehenine at concentrations of 4, 800 and 1600 ng/mL were measured to be 96.5, 101.2 and 108.7% (n = 6). The matrix effect for IS (50 ng/mL) was 104.6% (n = 6). As a result, matrix effect from plasma was considered negligible in this method. Calibration curve and sensitivity Linear regressions of the peak area ratios vs concentrations were fitted over the concentration range 2–2000 ng/mL for hupehenine in rat plasma. The equation utilized to express the calibration curve was: y = 0.0111535*x + 0.0240302, r = 0.9973, where y represents the ratios of hupehenine peak area to that of IS, and x represents the plasma concentration. The LLOQ for the determination of hupehenine in plasma was 2 ng/mL. The precision and accuracy at LLOQ were 7.2 and 90.7%, respectively. Precision, accuracy and recovery The precision of the method was determined by calculating RSD for QCs at three concentration levels over 3 days of validation tests. Intra-day precision was 4% or less, and inter-day precision was 6% or less at each QC level. The accuracy of the method ranged from 92.7 to 107.4% at each QC level. Mean recoveries of hupehenine were >92.5%. The recovery of the IS (50 ng/mL) was 95.8%. Assay performance data is presented in Table 1. Carry-over None of the analytes showed any significant peak (≥20% of the LLOQ and 5% of the IS) in blank samples injected after the ULOQ samples. Adding an extra 0.4 min to the end of the gradient elution effectively washed the system between samples, thereby eliminating carry-over (Williams et al., 2012). Stability Results from the autosampler showed that the hupehenine was stable under room temperature, freeze–thaw, and long-term (20 days) conditions, confirmed because the bias in concentrations was within 94.2 and 108.2% of the nominal values (Table 2). To this effect, the established method was suitable for pharmacokinetic study.
Copyright © 2015 John Wiley & Sons, Ltd.
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All 12 Male Sprague–Dawley rats (200–220 g) were obtained from the Laboratory Animal Center of Wenzhou Medical University (Wenzhou, China) to study the pharmacokinetics of hupehenine. The ethical approval number of the experiment animals was wydw2013-0071. All experimental procedures and protocols were reviewed and approved by the Animal Care and Use Committee of Wenzhou Medical University. Diet was prohibited for 12 h before the experiment but water was freely available. Hupehenine (30 mg) was dissolved in 3 mL saline with little 0.1% HCl, about 2 mL for oral admnistration and 1 mL for intravenous admnistration. Blood samples
Results
C. Wen et al.
(a)
(b) Figure 2. Representative UPLC-MS/MS chromatograms of hupehenine and imperialine (IS): (a) blank plasma; (b) blank plasma spiked with hupehenine (50 ng/mL) and IS (50 ng/mL); (c) a rat plasma sample 4 h after intravenous administration of single dosage 5 mg/kg hupehenine.
Application The method was applied to a pharmacokinetic study in rats. The mean plasma concentration–time curve after oral (10 mg/kg) or intravenous (5 mg/kg) administration of hupehenine is shown in Fig. 3. Primary pharmacokinetic parameters, based on noncompartment model analysis, are summarized in Table 3.
Discussion
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Electrospray ionization with positive ion detection resulted in better sensitivity than negative ion mode. Mass detector parameters
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were assessed by infusion of a standard solution directly into the ESI source. In order to optimize MS-MS conditions, the daughter ion spectrum of the [M + H]+ ion was recorded by ramping the capillary voltage and the collision energy. The most prevalent fragment was detected at m/z 98.0 with a capillary voltage of 2500 V, and a collision energy of 54 V. Accordingly, the m/z 416.3 → 98.0 transition was selected for further UPLC-MS/MS analysis in multiple reactions monitoring mode (Fig. 1a). The mobile phase played a critical role in achieving good chromatographic behavior and appropriate ionization (Wang et al., 2013; Wen et al., 2014; Zhang et al., 2014). Acetonitrile was selected
Copyright © 2015 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2015; 29: 1805–1810
Determination of hupehenine in rat plasma by UPLC-MS/MS
(c) Figure 2. (Continued)
Table 1. Precision, accuracy, and recovery for hupehenine of QC samples in rat plasma (n = 6)
4 800 1600
RSD (%)
Accuracy (%)
Intraday
Interday
Intraday
Interday
1.7 3.1 2.7
5.3 4.0 2.4
92.7 101.8 104.0
103.6 107.4 95.4
Recovery (%)
92.5 ± 4.6 95.2 ± 4.3 97.3 ± 2.5
Table 2. Summary of stability of hupehenine and IS under various storage conditions (n = 3) Condition
Concentration (ng/mL) Added
Ambient, 2 h
4 1600 (IS) 50 20 °C, 20 days 4 1600 (IS) 50 Three freeze–thaw cycles 4 1600 (IS) 50 Autosampler ambient, 24 h 4 1600 (IS) 50
RSD Accuracy (%) (%)
Found 3.8 1567.3 52.5 4.3 1550.9 48.7 3.8 1698.8 53.5 3.9 1627.6 51.7
5.4 3.2 3.7 7.6 5.7 8.2 6.7 5.8 4.7 1.7 2.1 1.5
95.3 98.0 105.0 108.2 96.9 97.4 94.2 106.2 107.0 98.5 101.7 103.4
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1200
800
400
0 0
6
12
18
24
Time (h) Figure 3. Mean plasma concentration–time profile after oral (10 mg/kg) or intravenous (5 mg/kg) administration of hupehenine in rats.
because the combination provides proper retention time and peak shape. The total run time for each injection was 3 min. Efficient removal of proteins and other potential interference in the bio-samples prior to LC-MS analysis was a crucial step in the development of this method (Du et al., 2014a, b; Ma et al., 2014a, b). Liquid–liquid extraction by ethyl acetate, ethyl ether and chloroform was tried at first; however, their recovery (between 70.6 and 90.3%) was not very good. Then simple protein precipitation was employed in our work; acetonitrile–methanol (9:1, v/v, 95.2%) was chosen as the protein precipitation solvent because it exhibited better effect than acetonitrile (91.5%), acetonitrile–methanol (1:1, v/v, 74.8%) and methanol (61.6%). An effective and simple protein precipitation is much simpler and faster than liquid–liquid extraction or solid-phase extraction.
Copyright © 2015 John Wiley & Sons, Ltd.
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for the organic phase, as it provides sharper peak shape and lower pump pressure compared with methanol. Acetonitrile and water (containing 0.1% formic acid) were chosen as the mobile phase
Concentration (ng/mL)
Concentration (ng/mL)
Oral administration Intravenous administration
1600
C. Wen et al. Table 3. Primary pharmacokinetic parameters after oral or intravenous administration of hupehenine in rats (n = 6) Parameters Unit
Mean
SD
Mean
Oral, 10 mg/kg AUC(0–t) AUC(0–∞) t1/2 CL V Cmax
ng/mL h ng/mL h h L/h/kg L/kg ng/mL
935.6 1097.4 5.4 11.5 91.5 390.2
Bioavailability, F
SD
Intravenous, 5 mg/kg
441.6 428.6 3.1 7.9 72.5 171.1
3494.9 3514.3 4.6 1.5 9.9 1400.5
723.0 724.5 2.0 0.3 5.4 371.4
13.4%
AUC, Area under the plasma concentration–time curve; t1/2, half-life; CL, clearance; V, volume of distribution.
We tested carbamazepine, diazepam and midazolam first; however, imperialine was more suitable for IS in the present study. Imperialine was selected as the IS because its chemical structure and chromatographic performance are similar to those of hupehenine and the two show similar retention times and extraction efficiencies; and both are suitable for detection in the positive ion electrospray ionization interface. The pharmacokinetic profile of hupehenine in rats was characterized for the first time in this study. This helps to build a better understanding of the pharmacological features of hupehenine. Further, the bioavailability of hupehenine was reported as 13.4% for the first time.
Conclusion The developed and validated UPLC-MS/MS method described here allows accurate and easily reproduced determination of hupehenine in rat plasma, utilizing only 50 μL of plasma with an LLOQ of 2 ng/mL. The UPLC-MS/MS method was successfully applied to a pharmacokinetic study of hupehenine after both oral and intravenous administration. Notably, the pharmacokinetic profile of hupehenine in rats was characterized for the first time. The bioavailability of hupehenine was identified at 13.4% for first time as well.
Acknowledgment This work was supported by fund of the National Natural Science Foundation of China, no. 81401558.
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of UPLC-ES/MS/MS to HPLC-ES/MS/MS. Journal of Chromatography B: Analytical Technology in Biomedicine and Life Sciences 2005; 825: 134–143. Du J, Ma Z, Zhang Y, Wang T, Chen X and Zhong D. Simultaneous determination of ornidazole and its main metabolites in human plasma by LCMS/MS: application to a pharmacokinetic study. Bioanalysis 2014a; 6: 2343–2456. Du J, Zhang Y, Chen Y, Liu D, Chen X and Zhong D. Enantioselective HPLC determination and pharmacokinetic study of secnidazole enantiomers in rats. Journal of Chromatography B: Analytical Technology in Biomedicine and Life Sciences 2014b; 965: 224–230. European Medicines Agency. Guidance on Bioanalytical Method Validation. EMEA/CHMP/EWP/192217/2009 Committee for Medicinal Products for Human Use (CHMP), 2011. Liu J, Wang L, Hu W, Chen X and Zhong D. Development of a UHPLC-MS/MS method for the determination of plasma histamine in various mammalian species. Journal of Chromatography B: Analytical Technology in Biomedicine and Life Sciences 2014; 971: 35–42. Ma J, Ding X, Sun C, Lin C, An X, Lin G, Yang X and Wang X. Development and validation a liquid chromatography mass spectrometry for determination of solasodine in rat plasma and its application to a pharmacokinetic study. Journal of Chromatography B: Analytical Technology in Biomedicine and Life Sciences 2014a; 963: 24–28. Ma J, Lin C, Wen C, Xiang Z, Yang X and Wang X. Determination of bicuculline in rat plasma by liquid chromatography mass spectrometry and its application in a pharmacokinetic study. Journal of Chromatography B: Analytical Technology in Biomedicine and Life Sciences 2014b; 953–954: 143–146. Song-Lin L, Li P, Lin G, Chan SW and Ho YP. Simultaneous determination of seven major isosteroidal alkaloids in bulbs of Fritillaria by gas chromatography. Journal of Chromatography A 2000; 873: 221–228. US Food and Drug Administration. Guidance for Industry on Bioanalytical Method Validation. US Department of Health and Human Services Food and Drug Administration: Rockville, MD, 2013. Wang X, Chen M, Wen C, Zhang Q and Ma J. Determination of chidamide in rat plasma by LC-MS and its application to pharmacokinetics study. Biomedical Chromatography 2013; 27: 1801–1806. Wen C, Lin C, Cai X, Ma J and Wang X. Determination of sec-O-glucosylhamaudol in rat plasma by gradient elution liquid chromatography–mass spectrometry. Journal of Chromatography B: Analytical Technology in Biomedicine and Life Sciences 2014; 944: 35–38. Williams JS, Donahue SH, Gao H and Brummel CL. Universal LC-MS method for minimized carryover in a discovery bioanalytical setting. Bioanalysis 2012; 4: 1025–1037. Wu JZ, Wang YY and Ling DK. Studies on chemical constituents of hubeibeimu (Fritillaria hupehensis Hsiao et K. C. Hsia). V. Isolation and identification of hupehenisine. Yao Xue Xue Bao 1986; 21: 546–550. Wu X, Chen H, Sun J, Peng Y, Liang Y, Wang G, Wu J and Zhang P. Development and validation of a liquid chromatography–mass spectrometry method for the determination of verticinone in rat plasma and its application to pharmacokinetic study. Journal of Chromatography B: Analytical Technology in Biomedicine and Life Sciences 2010; 878: 2067–2071. Xiao PG. A review on the study of hubeibeimu. Zhongguo Zhong Yao Za Zhi 2002; 27: 726–728. Zhang P, Li J, Zhang G, Pi H, Zhang Y, Ruan H and Wu J. Analysis of hupehenine in the total alkaloids from Fritillaria hupehensis by HPLCELSD. Journal of Huazhong University Science and Technology in Medical Science 2008; 28: 118–120. Zhang Q, Wen C, Xiang Z, Ma J and Wang X. Determination of CUDC-101 in rat plasma by liquid chromatography mass spectrometry and its application to a pharmacokinetic study. Journal of Pharmaceutical and Biomedical Analysis 2014; 90: 134–138.
1810 wileyonlinelibrary.com/journal/bmc
Copyright © 2015 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2015; 29: 1805–1810
Revised: 22 March 2015,
Accepted: 23 April 2015
Published online in Wiley Online Library: 1 June 2015
(wileyonlinelibrary.com) DOI 10.1002/bmc.3499
Determination and validation of hupehenine in rat plasma by UPLC-MS/MS and its application to pharmacokinetic study Congcong Wena†, Shuanghu Wangb†, Xueli Huangc, Zezheng Liua, Yingying Lina, Suping Yanga, Jianshe Mac, Yunfang Zhoub and Xianqin Wangc* ABSTRACT: In this work, a sensitive and selective ultra-performance liquid chromatography–tandem mass spectrometry (UPLCMS/MS) method for determination of hupehenine in rat plasma was developed and validated. After addition of imperialine as an internal standard (IS), protein precipitation by acetonitrile–methanol (9:1, v/v) was used to prepare samples. Chromatographic separation was achieved on a UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm) with 0.1% formic acid and acetonitrile as the mobile phase with gradient elution. An electrospray ionization source was applied and operated in positive ion mode; multiple reaction monitoring mode was used for quantification using target fragment ions m/z 416.3 → 98.0 for hupehenine, and m/z 430.3 → 138.2 for IS. Calibration plots were linear throughout the range 2–2000 ng/mL for hupehenine in rat plasma. Mean recoveries of hupehenine in rat plasma ranged from 92.5 to 97.3%. Relative standard deviations of intra-day and inter-day precision were both 98%) and imperialine (IS, purity >98%) were purchased from the Chengdu Mansite Pharmaceutical Co. Ltd (Chengdu, China). LC-grade acetonitrile and methanol were purchased from Merck Company (Darmstadt, Germany). Ultra-pure water was prepared by Millipore Milli-Q purification system (Bedford, MA, USA). Rat blank plasma samples were supplied from drug-free rats.
* Correspondence to: X. Wang, Analytical and Testing Center, Wenzhou Medical University, Wenzhou, Zhejiang 325035, China. Email: [email protected] †
These two authors contributed equally to this work
a
Laboratory Animal Centre, Wenzhou Medical University, Wenzhou 325035, China
b
The Laboratory of Clinical Pharmacy, The People’s Hospital of Lishui 323000, Wenzhou Medical University, Lishui, China
c
Analytical and Testing Center, Wenzhou Medical University, Wenzhou 325035, China
Copyright © 2015 John Wiley & Sons, Ltd.
1805
Hubeibeimu is the bulbs of Fritillaria hupehensis Hsiao et K.C.Hsia and has been used as an antitussive and expectorant herb in China for a long time (Xiao, 2002). Many chemical and pharmacological studies have shown that the total alkaloids are responsible for antitussive and expectorant activity (Zhang et al., 2008). Considering that peiminine, hupehenine and peimine have higher contents and hupehenine is the characteristic constituent, hupehenine (Fig. 1) was chosen as the index component (Wu et al., 1986). To date, several analytical methods described in the literature for the determination of hupehenine in biological and other matrices have involved either high-performance liquid chromatography with evaporative light scattering detection (Zhang et al., 2008) or gas chromatography (GC) (Song-Lin et al., 2000), or liquid chromatography–mass spectrometry (LC-MS) (Wu et al., 2010). However, the limit of detection (LOD) of hupehenine using HPLC or GC-MS is not high enough for pharmacokinetic and bioavailability studies, and ultra-performance liquid chromatography– tandem mass spectrometry (UPLC-MS/MS) is known to enable increased throughput, sensitivity and better chromatographic peak resolution (Churchwell et al., 2005). Wu et al. (2010) developed and validated a sensitive LC-MS method for the quantification of verticinone in rat plasma over the concentration range of 0.1– 200 ng/mL. Hupehenine was used as IS their work; the total runtime was 15 min. No current UPLC-MS/MS method exists for the determination of hupehenine to characterize pharmacokinetic properties. In this study, a UPLC-MS/MS method for determination of hupehenine in rat plasma was developed and successfully
applied to pharmacokinetic study of hupehenine after oral and intravenous administration. The UPLC-MS/MS method in this study was validated for selectivity, linearity, accuracy, precision, recovery and stability with a total run time of 3 min.
C. Wen et al.
Figure 1. Mass spectrum of hupehenine (a) and imperialine (IS, b).
Instrumentation and conditions
1806
A UPLC-MS/MS system with Acquity I-Class UPLC and a XEVO TQD triple quadrupole mass spectrometer (Waters Corp., Milford, MA, USA), equipped with an electrospray ionization interface, was used to analyze the compounds. The UPLC system comprised a Binary Solvent Manager and a Sample Manager with Flow-Through Needle. Masslynx 4.1 software (Waters Corp., Milford, MA, USA) was used for data acquisition and instrument control. Hupehenine and imperialine (IS) were separated on an UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm) maintained at 40 °C. The initial mobile phase consisted of 0.1% formic acid and acetonitrile with gradient elution at a flow rate of 0.4 mL/min and an injection volume of 2 μL. Elution was in a linear gradient, where the acetonitrile increased from 40 to 90% between 0 and 2.0 min, maintained at 90% for 0.5 min, then decreased to 40% within 0.1 min, then maintained at 40% for 0.4 min. The total run time of the analytes was 3 min. After each injection, the sample manager underwent a needle wash process, including both a strong wash (methanol–water, 50/50, v/v) and a weak wash (methanol–water, 10/90, v/v). Nitrogen was used as the desolvation gas (1000 L/h) and cone gas (50 L/ h). Ion monitoring conditions were defined as capillary voltage of 2.5 kV,
wileyonlinelibrary.com/journal/bmc
source temperature of 150 °C and desolvation temperature of 500 °C. Multiple reaction monitoring modes of m/z 416.3 → 98.0 for hupehenine and m/z 430.3 → 138.2 for IS were utilized to conduct quantitative analysis.
Calibration standards and quality control samples The stock solutions of hupehenine (1.0 mg/mL) and imperialine (IS) (100 μg/ mL) were prepared in methanol–water (50: 50, v/v). The 0.25 μg/mL working standard solution of the IS was prepared from the IS stock solution by dilution with methanol; working solutions for calibration and controls were prepared from stock solutions in the same manner. All of the solutions were stored at 4 °C and were brought to room temperature before use. Hupehenine calibration standards were prepared by spiking blank rat plasma with appropriate amounts of the working solutions. Calibration plots were offset to range between 2 and 2000 ng/mL for hupehenine in rat plasma (2, 5, 10, 20, 50, 100, 200, 500, 1000 and 2000 ng/mL). Quality-control (QC) samples were prepared in the same manner as the calibration standards, in three different plasma concentrations (4, 800, and 1600 ng/mL). The analytical standards and QC samples were stored at 20 °C.
Copyright © 2015 John Wiley & Sons, Ltd.
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Determination of hupehenine in rat plasma by UPLC-MS/MS Sample preparation Before analysis, the plasma sample was thawed to room temperature. An aliquot of 10 μL of the IS working solution (0.25 μg/mL) was added to 50 μL of the collected plasma sample in a 1.5 mL centrifuge tube, followed by the addition of 150 μL of acetonitrile–methanol (9:1, v/v). The tubes were vortex mixed for 1.0 min. After centrifugation at 14,900 g for 10 min in Allegra 64R High-speed Centrifuge (Beckman Coulter Inc., Los Angeles, California USA), the supernatant (2 μL) was injected into the UPLC-MS/MS system for analysis.
(0.2 mL) were collected from the caudal vein into heparinized 1.5 mL tapered plastic centrifuge tubes at 0.0833, 0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 12 and 24 h after oral (10 mg/kg) or intravenous (5 mg/kg) administration of hupehenine. The caudal vein of the rats was cleaned with 75% alcohol; after that the end of caudal vein was cut with scissors. A 1.5 mL tapered plastic centrifuge tube was used to collect the blood which dropped from the end of caudal vein by squeezing and massaging gently. The samples were immediately centrifuged at 3000 g for 10 min. The plasma (50 μL) was stored as obtained at 20 ° C until UPLC-MS/MS analysis. Plasma hupehenine concentration vs time data for each rat was analyzed using DAS (Drug and Statistics) software (version 2.0, Wenzhou Medical University, China).
Method validation Rigorous tests for selectivity, linearity, accuracy, precision, recovery and stability, according to the guidelines set by the US Food and Drug Administration (2013) and European Medicines Agency (2011), were conducted in order to thoroughly validate the proposed bioanalytical method. Validation runs were conducted on three consecutive days. Each validation run consisted of one set of calibration standards and six replicates of QC plasma samples. The selectivity of the method was evaluated by analyzing blank rat plasma, blank plasma-spiked hupehenine and IS, and a rat plasma sample. Calibration curves were constructed by analyzing spiked calibration samples on three separate days. Peak area ratios of hupehenine-to-IS were plotted against analyte concentrations. Resultant standard curves were well fitted to the equations by linear regression, with a weighting factor of the reciprocal of the concentration (1/x) in the concentration range of 2– 2000 ng/mL. The lower limit of quantitation (LLOQ) was defined as the lowest concentration on the calibration curves. To evaluate the matrix effect, blank rat plasma was extracted and spiked with the analyte at 4, 800 and 1600 ng/mL concentrations. The corresponding peak areas were then compared with those of neat standard solutions at equivalent concentrations; this peak area ratio is defined as the matrix effect. The matrix effect of the IS was evaluated at a concentration of 50 ng/mL in a similar manner. Accuracy and precision were assessed by the determination of QC samples at three concentration levels in six replicates (4, 800 and 1600 ng/mL) over 3 days of validation testing. The precision is expressed as RSD. The recovery of hupehenine was evaluated by comparing the peak area of extracted QC samples with those of reference QC solutions reconstituted in blank plasma extracts (n = 6). The recovery of the IS was determined in the same way. Carry-over was assessed following injection of a blank plasma sample immediately after three repeats of the upper limit of quantification (ULOQ), after which the response was checked for accuracy (Williams et al., 2012). Stability of hupehenine in rat plasma was evaluated by analyzing three replicates of plasma samples at concentrations of 4 or 1600 ng/mL, which were all exposed to different conditions. These results were compared with the freshly prepared plasma samples. Short-term stability was determined after the exposure of the spiked samples to room temperature for 2 h, and the ready-to-inject samples (after protein precipitation) in the HPLC autosampler at room temperature for 24 h. Freeze–thaw stability was evaluated after three complete freeze–thaw cycles ( 20–25 °C) on consecutive days. Long-term stability was assessed after storage of the standard spiked plasma samples at 20 °C for 20 days. The stability of the IS (50 ng/mL) was evaluated similarly (Du et al., 2014a, b; Liu et al., 2014).
Pharmacokinetic study
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Selectivity and matrix effect Figure 2 shows typical chromatograms of a blank plasma sample, a blank plasma sample spiked with hupehenine and IS, and a plasma sample. There were no interfering endogenous substances observed at the retention time of the hupehenine and IS. The matrix effects for hupehenine at concentrations of 4, 800 and 1600 ng/mL were measured to be 96.5, 101.2 and 108.7% (n = 6). The matrix effect for IS (50 ng/mL) was 104.6% (n = 6). As a result, matrix effect from plasma was considered negligible in this method. Calibration curve and sensitivity Linear regressions of the peak area ratios vs concentrations were fitted over the concentration range 2–2000 ng/mL for hupehenine in rat plasma. The equation utilized to express the calibration curve was: y = 0.0111535*x + 0.0240302, r = 0.9973, where y represents the ratios of hupehenine peak area to that of IS, and x represents the plasma concentration. The LLOQ for the determination of hupehenine in plasma was 2 ng/mL. The precision and accuracy at LLOQ were 7.2 and 90.7%, respectively. Precision, accuracy and recovery The precision of the method was determined by calculating RSD for QCs at three concentration levels over 3 days of validation tests. Intra-day precision was 4% or less, and inter-day precision was 6% or less at each QC level. The accuracy of the method ranged from 92.7 to 107.4% at each QC level. Mean recoveries of hupehenine were >92.5%. The recovery of the IS (50 ng/mL) was 95.8%. Assay performance data is presented in Table 1. Carry-over None of the analytes showed any significant peak (≥20% of the LLOQ and 5% of the IS) in blank samples injected after the ULOQ samples. Adding an extra 0.4 min to the end of the gradient elution effectively washed the system between samples, thereby eliminating carry-over (Williams et al., 2012). Stability Results from the autosampler showed that the hupehenine was stable under room temperature, freeze–thaw, and long-term (20 days) conditions, confirmed because the bias in concentrations was within 94.2 and 108.2% of the nominal values (Table 2). To this effect, the established method was suitable for pharmacokinetic study.
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All 12 Male Sprague–Dawley rats (200–220 g) were obtained from the Laboratory Animal Center of Wenzhou Medical University (Wenzhou, China) to study the pharmacokinetics of hupehenine. The ethical approval number of the experiment animals was wydw2013-0071. All experimental procedures and protocols were reviewed and approved by the Animal Care and Use Committee of Wenzhou Medical University. Diet was prohibited for 12 h before the experiment but water was freely available. Hupehenine (30 mg) was dissolved in 3 mL saline with little 0.1% HCl, about 2 mL for oral admnistration and 1 mL for intravenous admnistration. Blood samples
Results
C. Wen et al.
(a)
(b) Figure 2. Representative UPLC-MS/MS chromatograms of hupehenine and imperialine (IS): (a) blank plasma; (b) blank plasma spiked with hupehenine (50 ng/mL) and IS (50 ng/mL); (c) a rat plasma sample 4 h after intravenous administration of single dosage 5 mg/kg hupehenine.
Application The method was applied to a pharmacokinetic study in rats. The mean plasma concentration–time curve after oral (10 mg/kg) or intravenous (5 mg/kg) administration of hupehenine is shown in Fig. 3. Primary pharmacokinetic parameters, based on noncompartment model analysis, are summarized in Table 3.
Discussion
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Electrospray ionization with positive ion detection resulted in better sensitivity than negative ion mode. Mass detector parameters
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were assessed by infusion of a standard solution directly into the ESI source. In order to optimize MS-MS conditions, the daughter ion spectrum of the [M + H]+ ion was recorded by ramping the capillary voltage and the collision energy. The most prevalent fragment was detected at m/z 98.0 with a capillary voltage of 2500 V, and a collision energy of 54 V. Accordingly, the m/z 416.3 → 98.0 transition was selected for further UPLC-MS/MS analysis in multiple reactions monitoring mode (Fig. 1a). The mobile phase played a critical role in achieving good chromatographic behavior and appropriate ionization (Wang et al., 2013; Wen et al., 2014; Zhang et al., 2014). Acetonitrile was selected
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Determination of hupehenine in rat plasma by UPLC-MS/MS
(c) Figure 2. (Continued)
Table 1. Precision, accuracy, and recovery for hupehenine of QC samples in rat plasma (n = 6)
4 800 1600
RSD (%)
Accuracy (%)
Intraday
Interday
Intraday
Interday
1.7 3.1 2.7
5.3 4.0 2.4
92.7 101.8 104.0
103.6 107.4 95.4
Recovery (%)
92.5 ± 4.6 95.2 ± 4.3 97.3 ± 2.5
Table 2. Summary of stability of hupehenine and IS under various storage conditions (n = 3) Condition
Concentration (ng/mL) Added
Ambient, 2 h
4 1600 (IS) 50 20 °C, 20 days 4 1600 (IS) 50 Three freeze–thaw cycles 4 1600 (IS) 50 Autosampler ambient, 24 h 4 1600 (IS) 50
RSD Accuracy (%) (%)
Found 3.8 1567.3 52.5 4.3 1550.9 48.7 3.8 1698.8 53.5 3.9 1627.6 51.7
5.4 3.2 3.7 7.6 5.7 8.2 6.7 5.8 4.7 1.7 2.1 1.5
95.3 98.0 105.0 108.2 96.9 97.4 94.2 106.2 107.0 98.5 101.7 103.4
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1200
800
400
0 0
6
12
18
24
Time (h) Figure 3. Mean plasma concentration–time profile after oral (10 mg/kg) or intravenous (5 mg/kg) administration of hupehenine in rats.
because the combination provides proper retention time and peak shape. The total run time for each injection was 3 min. Efficient removal of proteins and other potential interference in the bio-samples prior to LC-MS analysis was a crucial step in the development of this method (Du et al., 2014a, b; Ma et al., 2014a, b). Liquid–liquid extraction by ethyl acetate, ethyl ether and chloroform was tried at first; however, their recovery (between 70.6 and 90.3%) was not very good. Then simple protein precipitation was employed in our work; acetonitrile–methanol (9:1, v/v, 95.2%) was chosen as the protein precipitation solvent because it exhibited better effect than acetonitrile (91.5%), acetonitrile–methanol (1:1, v/v, 74.8%) and methanol (61.6%). An effective and simple protein precipitation is much simpler and faster than liquid–liquid extraction or solid-phase extraction.
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for the organic phase, as it provides sharper peak shape and lower pump pressure compared with methanol. Acetonitrile and water (containing 0.1% formic acid) were chosen as the mobile phase
Concentration (ng/mL)
Concentration (ng/mL)
Oral administration Intravenous administration
1600
C. Wen et al. Table 3. Primary pharmacokinetic parameters after oral or intravenous administration of hupehenine in rats (n = 6) Parameters Unit
Mean
SD
Mean
Oral, 10 mg/kg AUC(0–t) AUC(0–∞) t1/2 CL V Cmax
ng/mL h ng/mL h h L/h/kg L/kg ng/mL
935.6 1097.4 5.4 11.5 91.5 390.2
Bioavailability, F
SD
Intravenous, 5 mg/kg
441.6 428.6 3.1 7.9 72.5 171.1
3494.9 3514.3 4.6 1.5 9.9 1400.5
723.0 724.5 2.0 0.3 5.4 371.4
13.4%
AUC, Area under the plasma concentration–time curve; t1/2, half-life; CL, clearance; V, volume of distribution.
We tested carbamazepine, diazepam and midazolam first; however, imperialine was more suitable for IS in the present study. Imperialine was selected as the IS because its chemical structure and chromatographic performance are similar to those of hupehenine and the two show similar retention times and extraction efficiencies; and both are suitable for detection in the positive ion electrospray ionization interface. The pharmacokinetic profile of hupehenine in rats was characterized for the first time in this study. This helps to build a better understanding of the pharmacological features of hupehenine. Further, the bioavailability of hupehenine was reported as 13.4% for the first time.
Conclusion The developed and validated UPLC-MS/MS method described here allows accurate and easily reproduced determination of hupehenine in rat plasma, utilizing only 50 μL of plasma with an LLOQ of 2 ng/mL. The UPLC-MS/MS method was successfully applied to a pharmacokinetic study of hupehenine after both oral and intravenous administration. Notably, the pharmacokinetic profile of hupehenine in rats was characterized for the first time. The bioavailability of hupehenine was identified at 13.4% for first time as well.
Acknowledgment This work was supported by fund of the National Natural Science Foundation of China, no. 81401558.
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