Development of a capillary electrophoresis method for the determination of allopurinol and its active metabolite oxypurinol

doi:10.1016/j.jchromb.2003.10.002

Journal of Chromatography B

Volume 798, Issue 2, 25 December 2003, Pages 303-308

https://doi.org/10.1016/j.jchromb.2003.10.002 Get rights and content

Abstract

A simple and sensitive capillary zone electrophoresis method with UV absorbance detection is described for the quantitation of allopurinol and its metabolite oxypurinol in aqueous solution. The influence of different parameters on migration times, peak symmetry, efficiency and resolution was systematically investigated; these parameters included the nature and concentration of the separation buffer, pH and applied voltage. A buffer consisting of 15 mM 2-[N-cyclohexylamino]ethanesulfonic acid (CHES) adjusted to pH 8.8 was found to provide a very efficient and stable electrophoretic system for the analysis of these compounds. The optimized method was validated with respect to precision, linearity, limits of detection and quantification, accuracy and robustness. The applicability of the assay was demonstrated by analyzing these compounds in serum and allopurinol in commercial pharmaceutical preparations.

Introduction

Allopurinol (ALP) and its mayor metabolite oxypurinol (OXP) are potent inhibitors of xanthine oxidase, the enzyme that converts hypoxanthine to xanthine, and xanthine to uric acid (Fig. 1). Allopurinol is commonly used in the treatment of chronic gout or of hyperuricaemia associated with leukaemia, radiotherapy, anti-neoplastic agents and treatment with diuretics [1].

Procedures capable of simultaneously detecting the primary drug (ALP) and its metabolite (OXP) in body fluids are of considerable interest for pharmacokinetic and clinical studies. Several assays for the determination of both compounds have been reported using high-performance liquid chromatography (HPLC) with either ultraviolet [2], [3], [4], [5], [6], [7], [8], [9] or electrochemical detection [10], [11], using ion exchange HPLC [2], [3] and reversed phase HPLC [4], [5], [6], [7], [8], [9], [10], [11]. Some of these methods have low limits of detection and quantitation, but others show various shortcomings. For example, the use of organic solvents (acetonitrile or methanol) [3] and excessively long elution times for allopurinol [2].

Capillary electrophoresis (CE) has gained a significant degree of acceptance in the analytical laboratory owing to its many advantageous features such as extremely high efficiency, high resolution, rapid analysis and low consumption of sample and reagents. However, CE methods proposed for the determination of ALP and OXP are scarce. Wang and co-workers [12] have reported a capillary zone electrophoresis (CZE) with end-column amperometric detection method using a running buffer composed of Na₂HPO₄/NaH₂PO₄ at pH 9.55 and detection potential at 1.20 V (versus Ag/AgCl electrode). Hempel and co-workers [13] have developed a CE assay with UV detection for the determination of ALP, OXP and other purine and products in urine with the aid of two running buffer formulations. In a first step, OXP was resolved by CZE using 60 mM sodium tetraborate at pH 8.7 as running buffer and, in a second step, micellar electrokinetic capillary electrophoresis (MEKC) with sodium dodecyl sulphate (80 mM) was used to resolve ALP. Despite the progress in CE separation of these two compounds, there is still room for improvement in the CE separation of allopurinol and oxypurinol. Accordingly, the aim of the present investigation was to optimize the CE conditions for the determination of both analytes. The effects of pH, type of buffer and its concentration and applied voltage on mobility, resolution, sensitivity and speed were carefully evaluated. The assay was validated by determining its accuracy, precision, linearity, specificity and robustness. The method has been successfully applied to the determination of ALP in pharmaceutical formulations and of both analytes in plasma.

Section snippets

Reagents

ALP and OXP were purchased from Sigma (St. Louis, MO, USA). The organic solvents: acetone, acetonitrile, ethanol and methanol were of HPLC grade (Romil, Leoughborough, Leicestershire, UK). Mesityl oxide and sodium dodecyl sulphate were obtained from Sigma; other chemicals were of analytical grade. Ultrapure water from a Milli-Q plus system (Millipore Ibérica, Madrid, Spain) was used throughout. Borate, phosphate, HEPES (N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]), CHES (2-[N

Effect of buffer pH

Structures of allopurinol and oxypurinol are shown in Fig. 1. The ionization of the 1-NH group in the pyrazole moiety leads to the mono-anion form and further ionization of the 5-NH group in the pyridine moiety cause the formation of the di-anion form [15], [16]. Hence, both compounds can exit as neutral, mono-anion and di-anion species, depending on buffer pH.

It is interesting to note that the electrophoretic mobility of allopurinol and oxypurinol continuously decreased with increasing buffer

Conclusion

A simple, rapid, efficient and reliable method for CZE separation and determination of ALP and OXP has been developed using 15 mM CHES buffer adjusted at pH 8.8 as electrophoretic electrolyte. The method was validated with regard to precision, specificity, linearity, limits of detection and quantification and robustness. The usefulness of this method is demonstrated by the excellent results obtained in the determination of ALP and OXP in human serum and ALP in different pharmaceutical

Acknowledgements

We thank Ministerio de Ciencia y Tecnologı́a (Project BJU2000-0262) and Fundación Séneca (Comunidad Autónoma de Murcia, PI62/0556/FS/01) for financial support.

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Being highly sensitive, these methods can be utilized for the analysis of both the analytes in human serum, plasma and urine samples, and hence find applications in pharmacokinetic/bioequivalence studies in clinical practices. Capillary electrophoresis based methods are also reported for the quantitative analysis of ALP and OXY in biological fluids as well as pharmaceutical samples [20–22]. However, there are no reports in the literature reveals which utilize nanoparticles for the concurrent determination of ALP and OXY.
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The useful pH range of CHES is 8.6–10.0. 15 mM of CHES adjusted to pH 8.8 was found to provide a very efficient and stable electrophoretic system for the analysis of allopurinol and its metabolite oxypurinol [2]. Of the various solubilization procedures tried for liberation of diol dehydratase from membranes of C. glycolicum, sonication in CHES buffer released the diol dehydratase in a form that retained enzymic activity.
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Development of a capillary electrophoresis method for the determination of allopurinol and its active metabolite oxypurinol

Abstract

Introduction

Section snippets

Reagents

Effect of buffer pH

Conclusion

Acknowledgements

J. Chromatogr.

Anal. Biochem.

J. Chromatogr.

J. Chromatogr. Biomed. Appl.

Anal. Biochem.

J. Chromatogr. Biomed. Appl.

J. Chromatogr. Biomed. Appl.

Anal. Chim. Acta