Journal of Chromatography B, 953–954 (2014) 143–146

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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb

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Determination of bicuculline in rat plasma by liquid chromatography mass spectrometry and its application in a pharmacokinetic study Jianshe Ma a , Chongliang Lin b , Congcong Wen c , Zheng Xiang c , Xuezhi Yang b , Xianqin Wang c,∗ a

Laboratory Animal Centre of Wenzhou Medical University, Wenzhou 325035, China The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, China c Analytical and Testing Center of Wenzhou Medical University, Wenzhou 325035, China b

a r t i c l e

i n f o

Article history: Received 15 November 2013 Accepted 10 February 2014 Available online 18 February 2014 Keywords: Bicuculline LC–MS Pharmacokinetics Rat plasma

a b s t r a c t Bicuculline, a phthalide isoquinoline alkaloid is of current interest as an antagonist of gammaaminobutyric acid (GABA). A simple and sensitive liquid chromatography mass spectrometry method for determination of bicuculline in rat plasma was developed over the range of 5–500 ng/mL. After addition of midazolam as internal standard, protein precipitation with acetonitrile–methanol (9:1, v/v) was used as sample preparation. Chromatographic separation was achieved on a Zorbax SB–C18 (2.1 mm × 150 mm, 5 ␮m) column with acetonitrile −0.1% formic acid in water as mobile phase with gradient elution. Electrospray ionization (ESI) source was applied and operated in positive ion mode; selective ion monitoring (SIM) mode was used for quantification using target fragment ions m/z 368 for bicuculline and m/z 326 for the IS. Linear calibration was obtained with correlation coefficients r > 0.99. The CV of the precision measurements was less than 13%. The accuracy of the method ranged from 93.6% to 100.5%. Mean recoveries of bicuculline in plasma were in the range of 80.5–91.8%. The method was successfully applied to the pharmacokinetic study after gavage administration of 15 mg/kg bicuculline in rats. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Bicuculline is a plant alkaloid of the phthalide isoquinoline alkaloid [1]. It could be isolated from Dicentra cucullaria, Adlumia fungosa, Fumariaceae and several Corydalis species [2]. Bicuculline is a relatively selective and competitive antagonist [2] of GABA particularly of GABAA receptors [3] at various sites of the nervous system. Thus bicuculline has pharmacological activity, which proved to be very useful in drug discovery and design [4]. There have been several literatures reported for analysis of bicuculline, such as high performance liquid chromatography (HPLC) analysis [5,6] and mass spectrometric analysis [7–10]. There only one literature [7] reported for determination of bicuculline in biological fluid. Due to the unique selectivity and sensitivity, liquid chromatography–mass spectrometry become widely used technique for pharmacokinetic study. In this paper a simple and sensitive LC–MS method for determination of bicuculline in rat plasma using one-step protein precipitation was

∗ Corresponding author. Tel.: +86 57786699156; fax: +86 57786699156. E-mail address: [email protected] (X. Wang). http://dx.doi.org/10.1016/j.jchromb.2014.02.006 1570-0232/© 2014 Elsevier B.V. All rights reserved.

developed and validated. This method was successfully applied to the pharmacokinetic study of bicuculline in rats. 2. Experimental 2.1. Chemicals and reagents Bicuculline (purity >98%) was purchased from the Chengdu Mansite Pharmaceutical Company Limited. (Chengdu, China). Midazolam (IS, purity >98%) was purchased from the Institute of Forensic Science, Ministry of Justice (Shanghai, 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). 2.2. Instrumentation and conditions The chromatographic workstation was a Bruker Esquire HCT ion-trap mass spectrometer (Bruker Technologies, Bremen, Germany) equipped with 1200 Series liquid chromatograph (Agilent Technologies, Waldbronn, Germany) controlled by ChemStation (Version B.01.03 [204], Agilent Technologies, Waldbronn, Germany).

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J. Ma et al. / J. Chromatogr. B 953–954 (2014) 143–146

2.4. Sample preparation Before sample treatment, the plasma samples were thawed to room temperature. In a 1.5 mL centrifuge tube, an aliquot of 10 ␮L of the IS working solution (2.0 ␮g/mL) was added to 100 ␮L of collected plasma followed by the addition of 200 ␮L acetonitrile–methanol (9:1, v/v). The tubes were vortex mixed for 1.0 min. After centrifugation at 14,900g for 10 min, the supernatant (2 ␮L) was injected into the LC–ESI–MS for analysis. 2.5. Method validation

Fig. 1. Mass spectrum of of bicuculline (a) and midazolam (IS, (b)).

Chromatographic separation was achieved on an Agilent Zorbax SB-C18 (2.1 mm × 150 mm, 5 ␮m) column at 40 ◦ C. The flow rate was set at 0.4 mL/min. A gradient elution program was conducted for chromatographic separation with the mobile phase A (0.1% formic acid in water) and mobile phase B (acetonitrile) as follows: the gradient elution method consisted of an opening condition of 10% solvent B, with a linear increase to 80% solvent B over 4.0 min, then 4.0 min at 80% solvent B, and then a return to the opening condition (10% solvent B) via a linear gradient over 1.0 min, followed by 4.0 min re-equilibration at opening conditions. The total run time was 13 min for each sample. Drying gas (nitrogen) flow and nebulizer pressure were set at 6 L/min and 20 psi. Dry gas temperature and capillary voltage of the system were adjusted at 350 ◦ C and 3500 V. LC–MS was performed with SIM mode using target ions at m/z 368 for bicuculline (Fig. 1a) and m/z 326 for midazolam (IS, Fig. 1b), in positive ion ESI interface, respectively.

2.3. Calibration standards and quality control samples The stock solution of bicuculline (1.0 mg/mL) and midazolam (IS, 100 ␮g/mL) were prepared in methanol–water (50:50), repectively. Working solutions for calibration and controls were prepared from the stock solution by dilution with methanol–water (50:50). The 2.0 ␮g/mL working standard solution of IS was prepared from the IS stock solution by dilution with methanol–water (50:50). All the solutions were stored at 4 ◦ C and brought to room temperature before use. Bicuculline 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 bicuculline in rat plasma (5, 10, 20, 50, 100, 200 and 500 ng/mL). Quality-control (QC) samples (10, 50 and 400 ng/mL) were prepared by the similar way as the calibration standards. The calibration standards and QC samples were stored at −20 ◦ C.

Calibration curves were constructed by analyzing spiked calibration samples on three separate days. Peak area ratios of bicuculline to IS were plotted against analyte concentrations, and 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 5–500 ng/mL. The lower limit of quantification (LLOQ) is the lowest concentration of analyte in a sample which can be quantified reliably, with an acceptable accuracy (80–120%) and precision (

Determination of bicuculline in rat plasma by liquid chromatography mass spectrometry and its application in a pharmacokinetic study.

Bicuculline, a phthalide isoquinoline alkaloid is of current interest as an antagonist of gamma-aminobutyric acid (GABA). A simple and sensitive liqui...
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