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Estimation of chlorzoxazone hydroxylase activity in liver microsomes and of the plasma pharmacokinetics of chlorzoxazone by the same high-performance liquid chromatographic method

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Abstract

We have developed a HPLC method which allows the determination of chlorzoxazone and its hydroxy metabolite in rat liver microsomes and in human plasma. We found that dehalogenated chlorzoxazone or 2-benzoxazolinone was a convenient and stable internal standard. Proteins were precipitated with diluted perchloric acid and the supernatant was extracted with ethyl acetate. Complete resolution of the peaks was achieved within 20 min with a Spherisorb ODS-1 column. The inter-day R.S.D.s were 6.5% at 0.5 μg/ml of hydroxychlorzoxazone and 5.8% at 1 μg/ml of chlorzoxazone in human plasma. The reproducibility of the method has been demonstrated for a large number of samples over a long period.

Introduction

Chlorzoxazone (CZX) has been used for many years as a potent long-acting central muscle relaxant [1]. The drug is rapidly absorbed from the gastro-intestinal tract and 6-hydroxychlorzoxazone (OH-CZX) is the main metabolite present in plasma as a conjugate.

Metabolic investigations of this compound were undertaken after some cases of hepatotoxicity were reported [2]. Human cytochrome P-4502 E1 (CYP2E1) was found to be the primary catalyst of chlorzoxazone 6-hydroxylation in human liver [3].

Considerable variations in the rates for this hydroxylation reaction have been measured in man and a low enzyme activity could be responsible for a hepatotoxic reaction but this remains to be determined 4, 5. Nevertheless, the use of this drug as a non-invasive probe for human CYP2E1 activity is now recognized and widely used 6, 7.

In parallel with these investigations, new high-performance liquid chromatography (HPLC) methods have been developed to measure CZX and OH-CZX concentrations in biological fluids and in microsomes 8, 9, 10, 11, 12, 13. All the published methods are based on the same preparation scheme: deconjugation of OH-CZX and liquid–liquid extraction. In most of these methods, an internal standard has been used and the compounds were separated by isocratic elution and quantified by UV detection. It must be pointed out that most of the compounds used as an internal standard are not chemically related to CZX or are synthesized by the investigators.

We find that dehalogenated CZX or 2-benzoxazolinone which is commercially available could be a convenient and stable internal standard. This paper describes a sensitive HPLC assay for CZX and OH-CZX in rat hepatic microsomes and in human plasma.

Section snippets

Chemicals and standards

CZX and 2-benzoxazolinone (internal standard) were purchased from Aldrich (Milwaukee, WI, USA). OH-CZX was obtained from Ultrafine (Manchester, UK). Acetonitrile HPLC ultra gradient grade, methanol and ethyl acetate HPLC grade were from Labscan (Dublin, Ireland). Helix Pomatia juice and nicotinamide–adenine dinucleotide phosphate, reduced (NADPH) tetrasodium salt were purchased from Boehringer (Mannheim, Germany). All other reagents were of the highest purity available.

Preparation of analytical standards

Stock solutions of CZX (1

Rat liver microsomes

CZX, its metabolite and the internal standard were well separated. Fig. 1 shows typical chromatograms obtained with rat liver microsomes before (A) and 20 min after (B) incubation with CZX. OH-CZX was eluted in 7.5 min, the I.S. in 9.5 min and CZX within 24.5 min.

Discussion

In this paper we present a simplified method, based on the paper of Peter et al. [3], to measure chlorzoxazone hydroxylase activity in rat liver microsomes. The three main modifications introduced are: the use of pure NADPH instead of a generating system, the addition of an internal standard chemically related to CZX and the direct injection of the supernatant, obtained after acid precipitation, onto the HPLC column.

We verify the experimental conditions for what concerns linearity, duration of

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