Determination of Rhynchophylline in Rat Plasma by Liquid Chromatography Mass Spectrometry and Its Application

Journal of Chromatographic Science 2014;52:661– 665 doi:10.1093/chromsci/bmt096 Advance Access publication July 9, 2013 Article Determination of Rhynchophylline in Rat Plasma by Liquid Chromatography Mass Spectrometry and Its Application Jinzhang Cai1, Chongliang Lin2, Jianshe Ma3, Lufeng Hu2, Guanyang Lin2 and Xianqin Wang4* 1 The Second Affiliated Hospital of Wenzhou Medical College, Wenzhou 325000, China, 2The First Affiliated Hospital of Wenzhou Medical College, Wenzhou 325000, China, 3Function Experiment Teaching Center of Wenzhou Medical College, Wenzhou 325035, China, and 4School of Pharmacy of Wenzhou Medical College, Wenzhou 325035, China *Author to whom correspondence should be addressed. Email: A sensitive and selective liquid chromatography mass spectrometry method was developed for the determination of rhynchophylline in rat plasma. After the addition of estazolam as the internal standard (IS), protein precipitation by acetonitrile was used for sample preparation. Chromatographic separation was achieved on a Zorbax SB-C18 column (2.1 3 150 mm, 5 mm) with acetonitrile –0.1% formic acid as the mobile phase with gradient elution. The electrospray ionization source was applied and operated in positive ion mode; selective ion monitoring mode was used for quantification by using target fragment ions m/z 385 for rhynchophylline and m/z 295 for the IS. Calibration plots were linear over the range of 5–500 ng/mL for rhynchophylline in plasma. The lower limit of quantification for rhynchophylline was 5 ng/mL. The mean recovery of rhynchophylline from plasma was in the range of 87.7–92.6%. The coefficients of variation of intra-day and inter-day precision were both less than 11%. This method is sensitive and selective enough to be used in pharmacokinetic research for the determination of rhynchophylline in rat plasma. Introduction China has an abundant resource of Uncaria rhynchophylla (Miq.) jacks. The hooks on this plant are used in the treatment of infantile convulsion, headache, dizziness, hypertension and stroke in traditional Chinese medicine and Japanese Kampo medicine (1). Oxindole alkaloids such as rhynchophylline (R) and isorhynchophylline (IR) are major components of U. rhynchophylla (2), responsible for the cardiocerebral vascular effects, including hypotension, vasodilation (3), anti-platelet aggregation (4) and protective effects against neuronal damage (5). One analytical liquid chromatography mass spectrometry (LC– MS) method has been published for the determination of R in rat plasma (6). However, the analysis method was not fully verified; the selectivity, matrix effect and stability were missing. LC– tandem mass spectrometry (MS-MS) does not guarantee the effective elimination of interferences from endogenous impurities, but an easy and effective way to make this adjustment is to modify gradient conditions. In this paper, a sensitive and selective LC–MS method was developed and validated for the determination of R in rat plasma, using one-step protein precipitation with gradient elution. The LC–MS method was successfully applied to a pharmacokinetic study of R after oral administration to rats. Experimental Chemicals and reagents R ( purity . 98%) was purchased from Chengdu Mansite Pharmaceutical Co. (Chengdu, China). Estazolam (purity . 98%) was purchased from the National Institute for Control of Pharmaceutical and Biological Products (Beijing, China). Highperformance liquid chromatography (HPLC) grade acetonitrile and methanol were purchased from Merck (Darmstadt, Germany). Ultra-pure water was prepared by a Millipore Milli-Q purification system (Bedford, MA). Instrumentation and conditions All analyses were performed with a 1200 Series liquid chromatograph (Agilent Technologies, Waldbronn, Germany) equipped with a quaternary pump, a degasser, an autosampler, a thermostatted column compartment and a Bruker Esquire HCT ion-trap mass spectrometer (Bruker Technologies, Bremen, Germany) equipped with an electrospray ion source and controlled by ChemStation software [Version B.01.03 (204); Agilent Technologies]. Chromatographic separation was achieved on an Agilent Zorbax SB-C18 (2.1  150 mm, 5 mm) column at 258C, with acetonitrile – 0.1% formic acid as the mobile phase. The flow rate was 0.4 mL/min. A gradient elution program was conducted for chromatographic separation with mobile phase A (0.1% formic acid) and mobile phase B (acetonitrile) as follows: 0 –4.0 min (10–80% B), 4.0 –7.0 min (80–80% B), 7.0 –8.0 min (80–10% B), 8.0 –12.0 min (10 –10% B). Drying gas flow and nebulizer pressure were set at 7 L/min and 25 psi, respectively. Drying gas temperature and capillary voltage of the system were adjusted at 3508C and 3,500 V, respectively. LC–MS was performed with selected ion monitoring (SIM) mode using target ions at m/z 385 for R (Figure 1A) and m/z 295 for estazolam [internal standard (IS); Figure 1B], in the positive ion electrospray ionization (ESI) interface. Calibration standards and quality control samples The stock solution of R (1.0 mg/mL) was prepared in methanol –trichloromethane (70:30) and the stock solution of the IS (100 mg/mL) was prepared in methanol. Working solutions for calibration and controls were prepared from the stock solution by dilution using methanol. The 2.0 mg/mL working standard solution of the IS was prepared by dilution of the IS stock solution with methanol. All of the solutions were stored at 48C and brought to room temperature before use. R calibration standards were prepared by spiking blank rat plasma with appropriate amounts of the working solutions. Calibration plots were constructed in the range of 5 –500 ng/mL for R in rat plasma (concentrations of 5, 10, 20, 50, 100, 200 and 500 ng/mL). Quality control (QC) samples were prepared by the # The Author [2013]. Published by Oxford University Press. All rights reserved. For Permissions, please email: Received 2 May 2012; revised 28 May 2013 same method as the calibration standards at three different plasma concentrations (10, 100 and 500 ng/mL). The analytical standards and QC samples were stored at –208C. Sample preparation Before analysis, the plasma sample was thawed to room temperature. In a 1.5 mL centrifuge tube, an aliquot of 10 mL of the IS working solution (2.0 mg/mL) was added to 100 mL of the collected plasma sample, followed by the addition of 300 mL of acetonitrile. The tubes were vortex-mixed for 0.5 min. After centrifugation at 15,000 rpm for 10 min, the supernatant was collected, transferred into clean tubes and evaporated to dryness with a gentle stream of nitrogen gas at 408C. The residues were dissolved in 150 mL of acetonitrile –water (50:50) and the supernatant (5 mL) was injected into the LC–MS system for analysis. Method validation Validation of the method was conducted following Food and Drug Administration (FDA) guidelines with respect to specificity, recovery, intra-day and inter-day precision, lower limit of detection (LOD) (...truncated)