Research article Received: 9 September 2013,

Revised: 17 October 2013,

Accepted: 29 October 2013

Published online in Wiley Online Library: 11 December 2013

(wileyonlinelibrary.com) DOI 10.1002/bmc.3095

Quantification of carbamazepine and its 10,11epoxide metabolite in rat plasma by UPLC-UV and application to pharmacokinetic study Avital Beig and Arik Dahan* ABSTRACT: A rapid, selective and sensitive UPLC-UV method was developed and validated for the quantitative analysis of carbamazepine and its epoxide metabolite in rat plasma. A relatively small volume of plasma sample (200 μL) is required for the described analytical method. The method includes simple protein precipitation, liquid–liquid extraction, evaporation, and reconstitution steps. Samples were separated on a Waters Acquity UPLC BEH C18 column (1.7 μm, 2.1 × 100 mm) with a gradient mobile phase consisted of 60:40 going to 40:60 (v/v) water–acetonitrile at a flow rate of 0.5 mL/min. The total run time was as low as 6 min, representing a significant improvement in comparison to existing methods. Excellent linearity (r2 > 0.999) was achieved over a wide concentration range. Close to complete recovery, short analysis time, high stability, accuracy, precision and reproducibility, and low limit of quantitation were demonstrated. Finally, we successfully applied this analytical method to a pre-clinical oral pharmacokinetic study, revealing the plasma profiles of both carbamazepine and carbamazepine-10,11-epoxide following oral administration of carbamazepine to rats. The advantages demonstrated in this work make this analytical method both time- and cost-efficient approach for drug and metabolite monitoring in the pre-clinical/clinical laboratory. Copyright © 2013 John Wiley & Sons, Ltd. Keywords: carbamazepine; carbamazepine-10,11-epoxide; UPLC-UV; pharmacokinetics; pre-clinical study

Introduction

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Carbamazepine (CBZ) is a widely used antiepileptic drug. It is indicated for partial, generalized tonic–clonic and mixed seizures. It is the first-choice antiepileptic drug for a wide range of seizure disorders in both adults and children owing to its efficacy and acceptable safety profile (Simhandl and Meszaros, 1992; Bourin and Thibaut, 2013). Like other classic antiepileptic drugs, the therapeutic and most of the toxic effects of carbamazepine are better correlated with plasma concentrations than with dose. Monitoring the concentration of CBZ in plasma is of great importance to clinical analysis and disease treatment (Johannessen and Landmark, 2008; Patsalos et al., 2008). Likewise, the active metabolite of carbamazepine, carbamazepine-10,11-epoxide, is equipotent to carbamazepine and contributes significantly to therapeutic benefits of carbamazepine therapy; therefore quantifying its blood concentrations is of great importance (Lertratanangkoon and Horning, 1982). Various analytical methods for determining CBZ concentrations in the plasma have been reported, including high-performance liquid chromatography in conjunction with various detectors, such as UV (Yoshida et al., 2006; Ateş et al., 2007; Beig et al., 2012), electrochemical (Messiha, 1986) and mass spectroscopy (MS; Kim et al., 2011; Zhu et al., 2005), and are universally considered accepted methods. Chromatography-based techniques allow simultaneous detection and quantification of multiple drugs from one sample; however, these methods have high quantification limits and long chromatographic run times, which are not preferable for a large number of samples (McMillin et al., 2010; Fortuna et al., 2011). Ultra-performance liquid chromatography (UPLC) is a method with both high sensitivity and high throughput, which bears

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significant advantages, including greater efficiency in preclinical pharmacokinetics, toxicokinetic and clinical studies (Bhatt et al., 2011; Marin et al., 2012; Shibata et al., 2012). UPLC has gained a considerable attention in recent years and has emerged as the preeminent analytical tool for pharmaceutical and biomedical analysis because of its high speed, low solvent usage, low time consumption, better resolution and better sensitivity. The present study describes the development and validation of a UPLC method for the quantification of carbamazepine and carbamazepine-10,11-epoxide (CBZ-E) in rat plasma. The method was found to be simple, sensitive, and reproducible. Then, we examined the application of this analytical method in a pharmacokinetic study of CBZ and its active metabolite, CBZ-E, after oral administration of CBZ in a rat model.

Experimental Chemicals and reagents Carbamazepine, carbamazepine-10,11-epoxide, phenacetin, MES buffer, ethyl acetate, polyethylene glycol 400, and trifluoroacetic acid (TFA) were

* Correspondence to: Arik Dahan, Department of Clinical Pharmacology, School of Pharmacy, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel. Email: [email protected] Department of Clinical Pharmacology, School of Pharmacy, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel Abbreviations used: ACN, acetonitrile; CBZ, carbamazepine; CBZ-E, carbamazepine-10,11-epoxide; TFA, trifluoroacetic acid.

Copyright © 2013 John Wiley & Sons, Ltd.

Quantification of CBZ and its metabolite in plasma using UPLC-UV purchased from Sigma Chemical Co. (St Louis, MO, USA). Acetonitrile (ACN) and water (Merck KGaA, Darmstadt, Germany) were UPLC grade. All other chemicals were of analytical reagent grade.

Stocks and working solutions Stock solutions of carbamazepine and phenacetin (internal standard) at 0.5 mg/mL in ACN were prepared. Working solutions containing both compounds at different concentrations (50 and 5 μg/mL respectively) were obtained by dilution of the stock solutions with ACN. Blank plasma samples were spiked with the corresponding working solution in an appropriate volume to prepare independently the calibration and the validation standard samples at the desired concentrations (0.1–10 μg/mL) and volume (200 μL).

Equipment UPLC experiments were performed on a Waters (Milford, MA, USA) Acquity UPLC H-Class system equipped with photodiode array detector and Empower software. Carbamazepine was assayed using a Waters (Milford, MA, USA) Acquity UPLC BEH C18 1.7 μm, 2.1 × 100 mm column. The detection wavelengths were 285 nm for CBZ and 210 nm for CBZ-E and phenacetin. The retention times were 4.75, 3.95 and 4.45 min for CBZ, CBZ-E and phenacetin, respectively. A gradient mobile phase was used, consisting of 60:40 going to 40:60 (v/v) 0.1% TFA in water–0.1% TFA in acetonitrile over 6 min, pumped at a flow rate of 0.5 mL/min. Injection volumes for all UPLC analyses ranged from 25 to 75 μL.

Calibration curves Rat plasma samples, spiked with CBZ and CBZ-E, to produce various concentrations (0–10 μg/mL) and the appropriate volume (200 μL) were prepared to obtain the calibration curve. The internal standard was prepared by dissolving known amounts of phenacetin in acetonitrile. An equivalent volume of internal standard solution (5 μg/mL) was added to each 200 μL plasma sample, mixed for 1 min and centrifuged at 13,000g for 10 min. Ethyl acetate (3 mL) was added to the plasma, vortex-mixed for 3 min and centrifuged at 5000g for 5 min. Following centrifugation, the clear supernatant was transferred to another glass test tube and evaporated in a multiple-sample evaporator (Labconco RapidVap, Kansas City, MO, USA) at 37°C with vortex motion (30%) and a vacuum level of 100 mbar. Complete dryness was achieved within approximately 20 min. The dried residue was reconstituted in 150 μL of mobile phase, filtered, and a 75 μL aliquot was injected into the UPLC system.

by assaying six replicates of QC samples (0.25, 2.5 and 10 μg/mL) on three different days. Extraction recovery was determined by comparing the peak areas of CBZ or CBZ-E from QC samples (0.25, 2.5 and 10 μg/mL) with those obtained from the analyte spiked into an equivalent volume of postextraction supernatant. Matrix effect was measured by comparing the peak area of analyte spiked after extraction with that of an equivalent concentration of the pure standard solution. Stability was investigated at low- and high-concentration QC samples (0.25 and 10 μg/mL) under different conditions, including short-term stability of CBZ in rat plasma for 4 h at room temperature, for 24 h at 4°C and freeze–thaw stability through three repeated freeze–thaw cycles.

Oral administration of CBZ to rats All animal experiments were conducted using protocols approved by the Ben-Gurion University of the Negev Animal Use and Care Committee (protocol IL-60-11-2010). Animals were housed and handled according to the Ben-Gurion University of the Negev Unit for Laboratory Animal Medicine Guidelines. Male Wistar rats (Harlan, Israel) weighing 250–280 g were used for all studies. Prior to each experiment, the rats were fasted overnight (12 h) with free access to water. On the day prior to the pharmacokinetic (PK) study, a cannula was placed in the right jugular vein of the rats to allow blood withdrawal. The animals were anesthetized for the period of surgery by intramuscular injection of 1 mL/kg of ketamine:xylazine solution (9:1%, respectively) and placed on a heated surface maintained at 37°C (Harvard Apparatus Inc., Holliston, MA, USA). An indwelling cannula was placed in the right jugular vein of each animal for systemic blood sampling, by a previously described method (Dahan and Hoffman, 2005, 2006, 2007; Dahan et al., 2007). The cannula was tunneled beneath the skin and exteriorized at the dorsal part of the neck. After completion of the surgical procedure, the animals were transferred to metabolic cages to recover overnight (12 h). During this recovery period and throughout the experiment, food, but not water, was withheld. CBZ was dissolved in polyethylene glycol 400 (PEG-400) for the oral PK study. The dose was 25 mg/kg. Blood samples (400 μL) were collected via the jugular vein cannula at 0, 30, 60, 90, 120, 150, 180, 240, 360, and 480 min post-dosing. Blood samples were immediately centrifuged and 200 μL of plasma were collected and stored at 70°C until UPLC analysis. Plasma concentrations vs time curves for CBZ and CBZ-E in individual rats were analyzed using WinNonlin® Professional software (Certara, St Louis, MO, USA) by means of the noncompartmental analysis model. The systemic bioavailability of CBZ was calculated from the ratio of the AUCs normalized by dose after oral and intravenous administration.

Method validation

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Results and discussion Chromatography Chromatographic conditions, especially the composition of mobile phase, were optimized through several trials to achieve good resolution and increase the signal to noise, as well as short run time. A gradient mobile phase consisting of 60:40 going to 40:60 (v/v) of 0.1% TFA in water–0.1% TFA in acetonitrile was finally adopted. The maximum UV absorbance of CBZ was determined at the wavelength 285 nm, while CBZ-E and phenacetin were determined at 210 nm. A representative chromatogram of rat plasma sample obtained using the analytical method described in this paper is presented in Fig. 1. It can be seen that a good separation of CBZ, CBZ-E and phenacetin was obtained. The limits of detection (LOD) and limits of quantification (LOQ) were 20 and 40 ng/mL, and 80 and 100 ng/mL, for CBZ and CBZ-E, respectively. The total run time was as low as 6 min for each sample. This short run time represents a significant

Copyright © 2013 John Wiley & Sons, Ltd.

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The method was validated according to the US Food and Drug Administration guidelines for selectivity, linearity, precision, accuracy, matrix effects, recovery and stability (www.fda.gov). Briefly, selectivity was assessed by comparing blank plasma from six individual rats with the corresponding spiked plasma samples. Calibration curves were constructed by the peak area ratio of analyte (CBZ or CBZ-E) to internal standard (phenacetin) vs analyte concentration, which was fitted via a 2 1/x weighted linear least-squares regression model, where x represents the concentration of analyte in spiked samples. The limit of quantitation (LOQ) was defined as the lowest drug concentration that could be determined with an accuracy and precision of

Quantification of carbamazepine and its 10,11-epoxide metabolite in rat plasma by UPLC-UV and application to pharmacokinetic study.

A rapid, selective and sensitive UPLC-UV method was developed and validated for the quantitative analysis of carbamazepine and its epoxide metabolite ...
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