HPLC assay for albendazole and metabolites in human plasma for clinical pharmacokinetic studies

Abstract

A sensitive and selective HPLC chromatography method using UV detection (295 nm) was developed for the determination of albendazole, albendazole sulfoxide (ABZSO), and albendazole sulfone (ABZSO2) in human plasma. Albendazole, ABZSO, ABZSO2, and the internal standard, oxibendazole, were extracted from human plasma by loading onto a conditioned C₁₈ SPE cartridge, rinsing with 15% methanol, and eluting with 90% methanol. Samples were evaporated under a stream of nitrogen, reconstituted with mobile phase, 1.25% triethylamine in water–methanol–acetonitrile (72:15:13, v/v/v) (pH* 3.1), and injected onto a Waters μBondapak™ Phenyl 3.9×300 mm HPLC column. Mobile phase flow rate was 1.0 ml/min. The retention times of albendazole, ABZSO, ABZSO2, and the internal standard were approximately 24.4, 7.9, 13.4, and 11.3 min, respectively. Total run time was 30 min. The assay was linear for concentration ranges in human plasma of 20–600 ng/ml for albendazole, 20–1000 ng/ml for ABZSO, and 20–300 ng/ml for ABZSO2. The analysis of quality control samples demonstrated excellent precision. Coefficients of variation for albendazole (20, 400, 600 ng/ml) were 6.7, 8.1 and 7.0%; ABZSO (20, 400, 800 ng/ml) were 6.0, 8.5 and 5.9%; ABZSO2 (20, 150, 300 ng/ml) were 3.1, 3.9 and 2.3%, respectively. The method appears to be robust and has been applied to a pharmacokinetic study of albendazole in healthy volunteers.

Introduction

Albendazole is a benzimidazole derivative used for treatment of intestinal helminthiasis and echinococcosis. Albendazole is an effective drug for treatment of these diseases but the therapeutic response in echinococcosis is unpredictable due to poor bioavailability [1], [2]. A single dose of albendazole in combination with diethylcarbamazine has been found safe and efficacious for the treatment of Brugia malayi infection and is used in treatment programs for the elimination of lymphatic filariasis [3], [4]. Albendazole undergoes first-pass metabolism and during the absorption process it is likely that the albendazole is metabolized by intestinal mucosal cells and the liver into the active metabolite ABZSO, a mixture of R(+) and S(−) enantiomers. In animals, formation of R(+) ABZSO is catalyzed by microsomal flavin monooxidase (FMO) and of S(−) ABZSO by cytochrome P450 enzymes (CYP3A) [5], [6]. The R(+) enantiomer seems to predominant, but the contribution of both enzyme systems to the metabolic process is variable and not well characterized in humans. This first-pass metabolism is extensive and thus, albendazole plasma levels are relatively low after a normal therapeutic dose (400 mg). Subsequently, ABZSO is metabolized by another cytochrome P450 enzyme (CYP2C) to the inactive metabolite, ABZSO2 [7].

Bogan and Marriner reported an HPLC assay of albendazole for plasma and gastrointestinal (GI) fluid [8]. The procedure involved liquid–liquid extraction and had a recovery of 83–100%, and had a sensitivity of 20 ng/ml with a 4 ml sample of plasma or GI fluid. The assay only quantitated albendazole and not the sulfoxide or sulfone metabolites. Another HPLC assay method for albendazole and its principal metabolites in sheep plasma has been reported by Alvinerie and Galtier [9]. The method has been reported to be sensitive, specific and reproducible using normal phase chromatography with mebendazole as the internal standard and UV detection at 225 nm. Only 100 μl of the sample was used and extracted using ethyl acetate as extraction solvent. The standard curves in plasma were linear for albendazole and its metabolites at concentrations of 0.1–10 μg/ml. The extraction recovery of albendazole, ABZSO and ABZSO2 were reported to be 78.2, 84.2 and 81.2%, respectively.

Hoaksey et al. reported a sensitive and selective reversed-phase HPLC method for the determination of albendazole and its active metabolite, ABZSO in human plasma [10]. They carried out a single step extraction of plasma with dichloromethane and separation on a μBondapak phenyl column. The assay was characterized down to 50 ng/ml for albendazole and 20 ng/ml for ABZSO. Hurtado et al., have described a rapid and sensitive assay for albendazole and ABZSO determination in plasma and CSF using an analytical wavelength of 295 nm, mebendazole as internal standard, and a ODS C₁₈ column for chromatographic separation [11]. The calibration curve was validated over a concentration range of 30–1000 ng/ml. Extractions were made using Sep-Pak C₁₈ cartridges. Recovery of albendazole and ABZSO from plasma extracts ranged from 95 to 100%. Valois et al., reported a method for the determination of ABZSO and ABZSO2. They use a liquid extraction with chloroform-isopropanol, separation on an RP-18 column and detection at 290 nm [12]. Garcia and coworkers have reported chromatographic conditions for quantitative determination of albendazole and another for ABZSO and ABZSO2 by using a reversed phase HPLC method with an ODS2 column and two different mobile phases [13]. Lanchote developed a HPLC method for the simultaneous determination of ABZSO enantiomers and ABZSO2 in human plasma [14]. The compounds were extracted from plasma with ethyl acetate, separated on a Chiralpak AD column and detected by fluorescence. The limits of quantification were 5 ng/ml for both ABZSO enantiomers and 1 ng/ml for ABZSO2. Chiap et al. reported a method for albendazole, ABZSO and ABZSO2 in ovine plasma by liquid chromatography [15]. The method uses dialysis as a purification step, followed by enrichment of the dialysate on a precolumn and liquid chromatography. A gradient elution was applied for separation of the analytes. UV detection at 295 nm was used and the limits of quantification for albendazole and metabolites were 10 and 7.5 ng/ml, respectively.

A nonaqueous capillary electrophoresis method for the determination of plasma albendazole, ABZSO and ABZSO2 was described by Prochazkova and coworkers [16]. The assay uses liquid–liquid extraction using dichloromethane with recovery between 63 and 98%. The limit of detection for the three compounds was 8×10⁻⁷ M, which is less sensitive than most HPLC methods. Albendazole was undetectable in all patient samples and ABZSO2 was below or close to the limit of detection.

An inexpensive, robust method for the quantitization of plasma levels of albendazole and ABZSO after 400 mg doses was needed to study pharmacokinetic effects when used in combination with diethylcarbamazine for lymphatic filariasis elimination. Separation of ABZSO enantiomers was deemed unnecessary and determination of albendazole bioavailability was the primary objective of this project. For the analysis of albendazole and its metabolites in body fluids, most methods are unable to efficiently determine albendazole and its metabolites simultaneously. Other methods lack the sensitivity required for clinical pharmacokinetic studies [15]. Some of the published liquid–liquid extraction procedures do not adequately remove plasma interference from the chromatograms to yield reproducible results. Initially, in the development of this methodology, only albendazole and ABZSO were validated. When actual subject samples were analyzed it became evident that the ABZSO2 metabolite was clearly present in some specimens and this methodology was also capable of quantitating ABZSO2. At this point validation work was done for ABZSO2 and it was added to the assay. This study reports a sensitive, selective, accurate, and reproducible HPLC assay using UV detection for the simultaneous determination of albendazole, ABZSO and ABZSO2 in human plasma using oxibendazole as an internal standard. With the use of an autoinjector, this method is suitable for processing the large number of albendazole plasma samples withdrawn during clinical pharmacokinetic studies.