A simple method for quantification of allopurinol and oxipurinol in human serum by high-performance liquid chromatography with UV-detection

https://doi.org/10.1016/j.jpba.2007.08.002Get rights and content

Abstract

Objectives

Allopurinol is a uric acid lowering drug used in the treatment of gout and the prevention of tumor lysis syndrome. Allopurinol and its active metabolite oxipurinol inhibit xanthine oxidase, which forms uric acid from xanthine and hypoxanthine. Therapeutic drug monitoring is an important option for evaluation and optimization of allopurinol treatment in case of renal impairment, interaction with uricosuric drugs or to verify patient adherence. In this study we developed and validated a simple quantitative assay using reverse phased high-performance liquid chromatography (HPLC) with UV-detection as a method for quantification of allopurinol and oxipurinol in human serum in the presence of different frequently used drugs.

Methods

The HPLC–UV method uses a mobile phase consisting of sodium acetate (0.02 M; pH 4.5), at a flow rate of 1.0 mL/min. Allopurinol and oxipurinol are detected by UV-absorption at 254 nm with a retention time of 9.9 min for oxipurinol and 12.3 min for allopurinol. Aciclovir is used as internal standard.

Results

Validation showed for allopurinol lower and upper limits of quantification of 0.5 and 10 mg/L and for oxipurinol 1 and 40 mg/L, respectively. The assay was linear over the concentration range of 0.5–10 mg/L (allopurinol) and 1–40 mg/L (oxipurinol). Intra- and inter-day precision showed coefficients of variation <15% over the complete concentration range; accuracy was within 5% for allopurinol and oxipurinol. Endogenous purine-like compounds were separated from allopurinol, oxipurinol and aciclovir with a resolution factor >1.5. Exogenous purine-like compounds and co-medication frequently used by gout patients did not hinder the analysis due to the dichloromethane washing step or to low UV-absorpion at 253 nm. Serum levels of 66 patients prescribed allopurinol 300 mg/day were determined using this HPLC–UV method. Measured serum allopurinol and oxipurinol concentrations in clinical practice showed large variability with a range of <0.5–4.3 mg/L for allopurinol and <1.0–39.2 mg/L for oxipurinol, respectively.

Conclusion

We developed an easy-to-operate and validated HPLC–UV method for the quantification of allopurinol and oxipurinol in human serum. This method was proven to be valid for samples of gout patients frequently using concomitant medications.

Introduction

Allopurinol is worldwide the mainstay of modern treatment of gout and prevention of tumor lysis syndrome. Allopurinol, an isomer of hypoxanthine, and its active metabolite oxipurinol (alloxanthine) act by inhibiting xanthine oxidase, an enzyme which forms uric acid (urate) from xanthine and hypoxanthine.

Allopurinol can be administered either orally or intravenously. The oral bioavailability is about 67 to 90% with a peak plasma concentration occurring within one hour; the volume of distribution is approximately 1.6 L/kg [1]. Allopurinol is principally metabolized by aldehyde oxidase to the active compound oxipurinol [2]. The peak plasma concentration of oxipurinol occurs within 3–5 h. Mean elimination plasma half lives ranges between 0.7 and 1.5 h for allopurinol and 18–40 h for oxipurinol [1].

Allopurinol is excreted in urine for less than 10% unchanged and for 70% as oxipurinol; 20% is excreted in feces. In patients with renal impairment (creatinine clearance <80 mL/min [3]), the maintenance dosage of allopurinol must be reduced to prevent toxic effects related to increased oxipurinol serum levels [3], [4]. When renal impairment is present, the initial allopurinol dosage can be calculated based on the estimated creatinine clearance (Table 1) [3]. Optimization of individual allopurinol dosage can be done by targeting of the oxipurinol – steady state – serum concentrations [5], [6], [7], [8], [9] as advised in the product information of allopurinol [5]. Reference serum oxipurinol values which are considered therapeutic, range from 5 to 15 mg/L [9].

The renal excretion of oxipurinol is increased by co-administration of uricosuric drugs (e.g. probenecid and benzbromarone) which are also used to decrease serum urate levels, presumably by interaction at the URAT-1 transporter [10], [11]. Combination of these drugs with allopurinol is frequently used in patients with severe gout, although, optimization of allopurinol dosage by measuring oxipurinol serum levels might be necessary. Another indication for therapeutic drug monitoring (TDM) is to verify a patient's adherence to the use of allopurinol, which in general is reported to be a point of concern [12], [13].

Several methods are described for the analysis of allopurinol and oxipurinol in human serum. However, these published methods, using reversed phase high-performance liquid chromatography, might have several limitations [14], [15], [16], [17], [18], [19], [20], [21], [22]. For example, (1) lack of information on chromatographic interference on detection and quantification of the analytes by concomitant medications frequently used by gout patients; (2) upper limits of quantification not covering the complete concentration range as observed in clinical practice; and (3) absence of stability data of allopurinol and oxipurinol in serum kept under refrigerated conditions.

The objective of the present study was to develop and validate a new analytical method which enables measurement of allopurinol and oxipurinol in representative serum samples obtained from daily clinical practice.

Section snippets

Equipment

The chromatographic system consisted of a Merck-Hitachi L-6200 pump (Merck-Hitachi, Darmstadt, Germany), a Series 200 autosampler (Perkin-Elmer, Wellesley, MA, USA), and a Spectroflow 757 variable absorbance detector (Kratos Analytical, Manchester, UK). Isocratic chromatographic separation was performed on a reversed-phase LiChrospher 100 RP-18 column (5 μm; 250 × 4 mm; Merck, Darmstadt, Germany) connected to a precolumn (LiChroCart Guard column 4–4 packed with Lichrospher 100 RP-18, 5 μm, 15 mm;

Method validation

With the described method, Rs > 1.5 of allopurinol, oxipurinol and the internal standard (aciclovir) was achieved (Fig. 1). The retention times of oxipurinol, allopurinol and aciclovir are 9.9, 12.3 and 17.7 min, respectively. Chromatographic performance depended on column temperature. Because no cooling device and air conditioning was available, we used a column heater and evaluated column temperatures 30–40 °C. Best results were obtained with a column temperature of 32.5 °C. Recovery after

Discussion

Our HPLC–UV method for the quantification of allopurinol and oxipurinol in human serum samples is easy-to-operate, valid, and advantageous over other methods described in literature.

Our method shows acceptable intra- and inter-day accuracy and precision over the allopurinol concentration range 0.5–10 mg/L and oxipurinol concentration range 1–40 mg/L. We did not investigate concentrations allopurinol >10 mg/L and oxipurinol >40 mg/L, because we considered it not clinically relevant. Because

Conclusion

We developed an easy-to-operate and validated HPLC–UV method for the quantification of allopurinol and oxipurinol in human serum for use in clinical practice. The method was shown to be employable for the assay of samples of gout patients frequently using concomitant medications.

Acknowledgements

The authors thank the rheumatologists G.A.W. Bruyn, MD, PhD; E.N. Griep, MD, PhD; P.M. Houtman, MD, PhD; J.P.L. Spoorenberg, MD, PhD (Medical Centre Leeuwarden); K.W. Drossaers-Bakker, MD, PhD; M. Hoekstra MD, PhD; M.W.M. Kruijsen, MD, PhD; H.H. Kuper, MD, PhD (Medical Spectrum Twente) for their work on collecting the patient samples. The authors thank A. Knuif (Pharmaceutical & Toxicological Laboratory Medisch Spectrum Twente) for his work on the operation and validation of the HPLC method.

References (26)

  • S. Reiter et al.

    Clin. Chim. Acta

    (1990)
  • K.R. Hande et al.

    Am. J. Med.

    (1984)
  • R. Boulieu et al.

    J. Chromatogr.

    (1984)
  • P. Nissen

    J. Chromatogr.

    (1982)
  • H. Breithaupt et al.

    J. Chromatogr.

    (1981)
  • W.G. Kramer et al.

    J. Chromatogr.

    (1979)
  • S.G. Tsouli et al.

    Metabolism

    (2006)
  • S.L. Chang et al.

    J. Chrom.

    (1980)
  • F. Pea

    Contrib. Nephrol.

    (2005)
  • F. Perez-Ruiz et al.

    J. Clin. Rheumatol.

    (2005)
  • The Dutch Medicines Evaluation Board. Product Information Zyloric®. Http://www.cbg-meb.nl/nl/prodinfo/index.htm; March...
  • M. Takada et al.

    J. Clin. Pharm. Ther.

    (2005)
  • R.O. Day et al.

    Br. J. Clin. Pharmacol.

    (1988)
  • Cited by (29)

    • Green stability-indicating capillary electrophoretic method for simultaneous determination of Lesinurad and Allopurinol in tablet dosage form: Degradation kinetics investigation

      2020, Microchemical Journal
      Citation Excerpt :

      To the best of our knowledge, very few analytical methods have been published for the analysis of both drugs either alone or simultaneously in their combined dosage form. Recent publications reported the analysis of ALP alone using chromatographic (HPLC and HPTLC) [12,13], spectrophotometric [14] and electrophoretic methods [15]. On the other hand, LES was determined using UHPLC-MS/MS in rat plasma [16].

    • Detection of allopurinol and oxypurinol in canine urine by HPLC/MS-MS: Focus on veterinary clinical pharmacokinetics

      2020, Journal of Pharmaceutical and Biomedical Analysis
      Citation Excerpt :

      The chromatographic technique with UV detector has some limitations mainly regarding the sensitivity for substances that are detectable in the same wavelength. Tada et al. (2003) [18] and Reinders et al. (2007) [19] used this technique for the analysis of allopurinol and oxypurinol in human serum, therefore a matrix with less interference than urine. In their works, Tada et al. (2003) [18] found a linearity for allopurinol at 0.5–50 u g/mL, and for oxypurinol at 0.4–20 u g/mL, while Reinders et al. (2007) [19] found it at 0.5−10 mg/L and 1−40 mg/L, respectively for allopurinol and oxypurinol.

    • Simultaneous analysis of allopurinol and oxypurinol using a validated liquid chromatography–tandem mass spectrometry method in human plasma

      2017, Journal of Pharmaceutical Analysis
      Citation Excerpt :

      AP has also been estimated by micelle-stabilised room temperature phosphorescence in urine samples [11]. However, some of these methods involve lengthy extraction protocols [10], have chromatographic run time more than 10 min [8], are less sensitive [8,9] and employ large sample volume for processing [3]. Few methods report use of liquid chromatography–tandem mass spectrometry (LC–MS/MS) technique for the determination of OP as a single analyte in human urine [12] and for the simultaneous estimation of AP and OP in human plasma [13–15] and urine [14].

    View all citing articles on Scopus
    View full text