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