Journal of Chromatography B, 941 (2013) 10–16
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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Determination of allopurinol and oxypurinol in human plasma and urine by liquid chromatography-tandem mass spectrometry Xia Liu a , Xiao-Jia Ni a , De-Wei Shang a , Ming Zhang a , Jin-Qing Hu a , Chang Qiu a , Fu-Tian Luo b , Yu-Guan Wen a,∗ a b
Department of Pharmacy, Guangzhou Brain Hospital, Guangzhou Medical University, 36 MingXin Road, Guangzhou 510370, China School of Public Health, Sun Yat-sen University, Guangzhou, China
a r t i c l e
i n f o
Article history: Received 11 June 2013 Accepted 19 September 2013 Available online 1 October 2013 Keywords: Allopurinol Metabolite HPLC-MS/MS Human plasma Pharmacokinetics
a b s t r a c t Allopurinol is used widely for the treatment of gout, but its pharmacokinetics is complex and some patients show hypersensitivity, necessitating careful monitoring and improved detection methods. In this study, a sensitive and reliable liquid chromatography-tandem mass spectrometry method was developed to determine the concentrations of allopurinol and its active metabolite oxypurinol in human plasma and urine using 2,6-dichloropurine as the internal standard (IS). Analytes and the IS were extracted from 0.5 ml aliquots of plasma or urine using ethyl acetate and separated on an Agilent Eclipse Plus C18 column using methanol and ammonium formate–formic acid buffer containing 5 mM ammonium formate and 0.1% formic acid (95:5, v/v) as the mobile phase (A) for allopurinol or methanol plus 5 mM ammonium formate aqueous solution (95:5, v/v) as the mobile phase (B) for oxypurinol. Allopurinol was detected in positive ion mode and the analysis time was about 7 min. The calibration curve was linear from 0.05 to 5 g/mL allopurinol in plasma and 0.5–30 g/mL in urine. The lower limit of quantification (LLOQ) was 0.05 g/mL in plasma and 0.5 g/mL in urine. The intra- and inter-day precision and relative errors of quality control (QC) samples were ≤11.1% for plasma and ≤ 8.7% for urine. Oxypurinol was detected in negative mode with an analysis time of about 4 min. The calibration curve was linear from 0.05 to 5 g/mL in plasma (LLOQ, 0.05 g/mL) and from 1 to 50 g/mL in urine (LLOQ, 1 g/mL). The intra- and inter-day precision and relative errors were ≤7.0% for plasma and ≤9.6% for urine. This method was then successfully applied to investigate the pharmacokinetics of allopurinol and oxypurinol in humans. © 2013 Elsevier B.V. All rights reserved.
1. Introduction The xanthine oxidase inhibitor allopurinol (Fig. 1A) is the most frequently prescribed medication for the treatment of gout and hyperuricaemia [1,2]. Allopurinol is rapidly absorbed from the gastrointestinal tract and metabolized by xanthine oxidase to oxypurinol (Fig. 1B), the main metabolite responsible for the pharmacological activity [3,4]. Oxypurinol is also a potent inhibitor of xanthine oxidase, the enzyme that converts hypoxanthine to xanthine and xanthine to uric acid. Like allopurinol, it is excreted via the kidney. However, oxypurinol exhibits a prolonged serum half-life [5,6], complicating allopurinol dosing and increasing the risk of side effects in some patients. A new allopurinol intravenous preparation is in development, necessitating a reliable and practical analytical method for high-throughput detection in pharmacokinetic and therapeutic drug monitoring studies.
∗ Corresponding author. Tel.: +86 20 81268052; fax: +86 20 81891391. E-mail addresses: [email protected], [email protected] (Y.-G. Wen). 1570-0232/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jchromb.2013.09.028
Several methods have been developed for the measurement of allopurinol and oxypurinol in plasma and urine, including HPLC with ultra-violet detection [7–11] or electrochemical detection [12–14]. Brown and Bye [7] described the HPLC method for allopurinol and oxipurinol assay within human plasma and urine with linear calibration curves for allopurinol over the range 0.068–1.36 g/ml in plasma and 0.68–136 g/ml in urine and for oxipurinol 0.076–15.2 g/ml in plasma and 15.2–304 g/ml in urine. However, this method is based on complex ion-exchange chromatography. Moreover, the selectivity of UV detection is reduced by the interferences of residual protein [8–10], detection of single sample extends as long as 20 min [10], preparation method is time-consuming [11], and sensitivity is still limited [8–11]. Electrochemical detection involves complex sample preparation using enzymatic reactions [13] or solid phase extraction [14]. Other methods such as capillary electrophoresis detection [15,16] and micelle-stabilised room temperature phosphorescence detection [17] require specific modifications for plasma and urine sample detection. HPLC-MS method [18] has been adopted to determine allopurinol and oxypurinol in plasma by protein precipitation and liquid–liquid extraction following the lower limit of quantification
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Fig. 1. Chemical structures and mass spectra of (A) allopurinol (M + H)+ , (B) 2,6-dichloropurine (M + H)+ , (C) oxypurinol (M − H)− , and (D) 2,6-dichloropurine (M − H)− .
of the assay was 50 ng/mL for allopurinol and 100 ng/mL for oxypurinol, yet the paper did not present the concrete assay method. Modern HPLC-MS/MS method [19] for the determination of oxypurinol in urine is suitable for the quantification of oxypurinol in the urine of patients (n = 34) participating in a clinical trial to optimize therapy of gout with allopurinol but the selectivity and sensitivity are still not sufficient for general application. Because allopurinol and its metabolite oxypurinol are main for the treatment of gout and hyperuricaemia so a validated analytical methods for urine matrix to explore relationships with plasma urate concentrations is also needed. As a result, we have developed novel HPLC-MS/MS method to systemically measure allopurinol and oxypurinol in both human urine and plasma for the first time. This simple, sensitive, and practical method was fully validated and then successfully applied to a pharmacokinetic study of an injectable allopurinol formulation in healthy Chinese volunteers. 2. Materials and methods 2.1. Chemicals and reagents Intravenous Preparations of Allopurinol Sodium (0.5 g, Batch No. 120601) were prepared for a clinical study by Hainan Generalsanyang Pharmaceutical Co., Ltd. (China). Allopurinol standard reference (purity: 99.5%, lot. C10118000) was provided by Dr. Ehrenstorfer GmbH Company. Oxypurinol standard (purity: 99.5%, lot. D454550) was supplied by Toronto Research Chemicals Inc. The internal standard 2,6-dichloropurine (purity: 99.5%, lot. 100603200401) was provided by Wuhan Ariel Chemical Technology Co., Ltd. (China). Chromatographic pure grade methanol was purchased from Merck, chromatographic pure grade formic acid and ethyl acetate from Dikma Company (USA), mass spectrum pure grade ammonium formate from Sigma (USA), and analytical grade HCl from Guangzhou Chemical Agent Factory (China). Deionized water was produced using a Milli-Q academic reagent grade water purification system (Millipore, USA) and distilled before using.
and internal standard were detected using an Agilent 6410 triple quadrupole mass spectrometer (Agilent Technologies, Inc., USA) with electrospray ionization. Ultra-high purity (UHP) nitrogen was used as the drying gas. Raw data were processed using the Agilent MassHunter Chemstation (B.01.03). A Salvis Lab vacuum drying apparatus (Vacucenter VC20, CH-6343 Rotkreuz, Switzerland) was used to evaporate the supernatant extracted from plasma or urine samples. Pharmacokinetic parameters were calculated using DAS 2.1.1 software (Mathematical Pharmacology Professional Committee of China). 2.3. LC-ESI-MS/MS Chromatographic separation was performed at 40 ◦ C on an Agilent Eclipse Plus-C18 column (4.6 mm × 150 mm, 3.5 m). The mobile phase consisting of methanol −5 mmol/l ammonium formate aqueous solution (95:5, v/v) with or without 0.1% formic acid was employed for allopurinol and oxypurinol, separately. The flow rate for detection of both analytes was 0.5 mL/min. The mass spectrometer was operated in positive ion multiple reaction monitoring (MRM) mode for allopurinol detection and negative ion MRM mode for oxypurinol detection. The optimized condition consisted of a nebulizer set at 45 psi, drying gas flow of 10 L/min at 350 ◦ C, and a HV capillary set at 4000 V. Impact energy and split voltage of allopurinol were 30 units and 125 V separately and these were 20 units and 135 V for 2,6dichloropurine. The dwell time for each transition was 0.2 s and the electron multiplier set at 300 V. The injection volumes were 5 L plasma and 2 L urine. The impact energy of oxypurinol and IS were separately 18 units and 22 units with a split voltage of 115 V and 135 V, separately. The dwell time for each transition was 0.2 s and the electron multiplier set to 400 V. The injection volumes were 5 L plasma and 1 L urine. 2.4. Preparation of stock solutions, calibration curves, and quality control (QC) samples
2.2. Apparatus The chromatographic analysis system (Agilent 1100 series LC system, Agilent Technologies, Inc., USA) included a quaternary pump, autosampler, column oven, and degasser. The analytes
Stock solution of allopurinol in mobile phase A, oxypurinol in mobile phase B, and 2,6-dichloropurine in corresponding mobile phase A or B were prepared individually at a concentration of 1 g/L. Stocks were then serially diluted with the mobile phase
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to respectively prepare working solutions of 0.5, 1, 5, 10, 20, 40, and 50 g/ml analytes and either 10 g/mL IS for allopurinol or 20 g/mL IS for oxypurinol to construct plasma calibration curves. For urine calibration curves, stocks were respectively diluted to 5, 10, 20, 50, 100, 200, and 300 g/mL allopurinol and 40 g/mL IS or 10, 20, 50, 100, 200, 400, and 500 g/ml oxypurinol and 100 g/mL IS. All the solutions were kept at 4 ◦ C and warmed to room temperature before use. For preparation of calibration samples, 50 L of vacuum-dehydrated working analyte solution (all concentrations as above) and 50 L IS solution were added to 0.5 ml blank plasma or urine. The final concentrations in plasma were 0.05, 0.1, 0.5, 1, 2, 4, and 5 g/mL allopurinol plus 1 g/mL IS or 0.05, 0.1, 0.5, 1, 2, 4, and 5 g/mL oxypurinol plus 2 g/mL IS. The final concentrations in urine calibration samples were 0.5, 1, 2, 5, 10, 20, and 30 g/mL allopurinol plus 4 g/mL IS and 1, 2, 5, 10, 20, 40, and 50 g/mL oxypurinol plus 10 g/mL IS. Quality control samples for plasma analysis were prepared at 0.1, 1, and 4 g/mL allopurinol or oxypurinol in blank plasma. Quality control samples for urine analysis were prepared at 1, 10, and 20 g/mL for allopurinol or 2, 10, and 40 g/mL for oxypurinol in blank urine. 2.5. Sample preparation Standard (calibration curve) samples, quality control samples and real plasma or urine samples from human subjects were all processed by liquid–liquid extraction. In each tube, 0.5 mL plasma or urine, 50 L IS stock, and 0.2 M of 100 L hydrochloric acid were combined and vortexed at high speed for 15 s. Then, 3 mL of ethyl acetate was added and the mixture vortexed for 30 s. Samples were then centrifuged at 3000 r/min for 10 min. The upper organic phase was transferred into a 5 ml glass centrifuge tube and vacuum dried at 42 ◦ C. Samples were reconstituted with 200 L of respective mobile phase (A for allopurinol and B for oxypurinol) for analysis. 2.6. Method validation 2.6.1. Selectivity and lower limit of quantification To test the selectivity of the method, blank plasma or urine samples from six healthy volunteers were pretreated and analyzed. The absence of interfering peaks at the analyte retention times was considered acceptable selectivity. The lower limit of quantification (LLOQ) was defined as the lowest detectable analyte concentration for which the values of precision (RSD) and accuracy (RE) were ≤20% and the signal-to-noise (S/N) ≥5. 2.6.2. Calibration curves Calibration equations were derived using a linear weighted least-squares regression analysis of the peak area ratios of analyte to IS versus nominal analyte concentration. The regression parameters of slope, intercept, and correlation coefficient were calculated by the weighting factor 1/x2 . Linearity was assessed for calibration curves of human plasma or urine in three separate duplicates by correlation coefficient (r2 ) and by determining the residues and errors between nominal and measured concentrations for each point of the calibration curve. Residues were evaluated by alternating injections of the highest calibration concentration with blank samples. The area of blank sample was no more than 20% of LLOQ. The error values of precision (RSD) and accuracy (RE) were ≤20% for calibration curve standard concentrations at the LLOQ and 15% for other standard concentrations. 2.6.3. Precision and accuracy To evaluate precision and accuracy, five sets of spiked plasma or urine QC samples at the same three concentrations as above (Section 2.4) were analyzed on the same day and over three consecutive
days. Samples for a standard curve were prepared and analyzed on the same day to calculate the concentration of each QC sample. Relative standard deviation (RSD, %) and relative error (RE, %) were calculated to estimate precision and accuracy, where RE (%) = ((measured conc. − nominal conc.)/nominal conc.) × 100. Dilution factors were determined in plasma or urine to measure the sample concentrations out of analytical range. Quality control samples with 16 g/mL allopurinol or oxypurinol were diluted 1/4 with blank plasma while QC samples containing 50 g/mL and 100 g/mL allopurinol were diluted 1/5 with blank urine. These dilution factors were accurate and precise, as measured concentrations multiplied by 4 or 5 were 85–115% of the nominal concentrations. 2.6.4. Extraction recovery Recovery experiments were performed at the three QC concentrations with five replicates for allopurinol and oxypurinol and at one concentration for the IS. Analyte peak areas obtained from QC samples after extraction were compared to unextracted standard solutions at the same concentrations. 2.6.5. Matrix effects To evaluate the effect of co-eluting matrix components following ion suppression or enhancement, three different batches of blank plasma or urine were extracted and then spiked with the analyte at three QC levels. The peak areas of the analyte in spiked plasma or urine (B) were then compared with those of corresponding standard solutions in mobile phase (A) at equivalent concentrations. The ratio (B/A × 100) is defined as the matrix effect. A matrix effect value of 100% indicates that the response in the mobile phase and in the plasma or urine extracts was the same and no absolute matrix effect was observed. Usually RSD < 15% is satisfactory. Matrix effects of IS were investigated in a similar way at one concentration. 2.6.6. Stability The stability of the stock solutions and working solutions was examined by measuring allopurinol, oxypurinol, and IS stock concentrations after 3 days at 4 ◦ C and after 3 h at room temperature. The stability of analytes in plasma or urine QC samples was tested after 24 h at RT (25 ◦ C), after three complete freeze/thaw cycles from −20 to 25 ◦ C, and after long-term storage at −20 ◦ C (15 days for allopurinol, 30 day for oxypurinol). Placement stability of the ready-to-inject samples was also investigated after 24 h at 25 ◦ C in the autosampler rack. A sample was considered stable in the biological matrix when the calculated concentration was 85–115% of the freshly prepared samples. 2.7. Pharmacokinetic study Thirty-six healthy Chinese volunteers (19–28 years of age, 19–24 of BMI) participated in a pharmacokinetic study on an injectable allopurinol formulation. Health status was confirmed by review of medical histories and complete physical and biochemical tests prior to enrollment. The study was approved by the ethics committee of Guangzhou Brain Hospital. The volunteers fasted for 10 h and were then divided into three dose groups (6 males and 6 females per group) receiving a singledose of either 100 mg/m2 (L), 200 mg/m2 (M), or 300 mg/m2 (H) allopurinol in physiological saline by iv gtt administration. Body surface area was calculated using formulae for ethnic Chinese [20]: Smale = 0.0057 × height + 0.0121 × body weight + 0.0882, Sfemale = 0.0073 × height + 0.0127 × body weight − 0.2106. Blood samples were collected into heparin tubes before drug administration and at 0.17, 0.33, 0.5, 0.58, 0.75, 1, 1.5, 2, 2.5, 3, 4, and 5 h for allopurinol measurements and at these same times plus 8,
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12, 24, 36, 48, 72, and 96 h for oxypurinol. Blood samples were immediately centrifuged at 3000 r/min for 10 min to obtain plasma samples. Samples were stored at −20 ◦ C for later analysis. Volunteers also evacuated urine before drug administration and then drank water freely 2 h later. Urine samples from volunteers in the 200 mg/m2 (M) group were collected at 0, 0–2, 2–4, 4–6, 6–8, 8–12, and 12–24 h after injection. Volumes of urine were recorded and 10.0 mL samples stored at −20 ◦ C for later analysis. 3. Results 3.1. HPLC–ESI-MS/MS In positive ion mode, the most intense product ions observed in the MS/MS spectra were m/z 137.1 → 54.3 for allopurinol (Fig. 1A) and m/z 188.8 → 153.0 for the IS 2,6-dichloropurine (Fig. 1B). In negative ion mode, the most intense product ions were m/z 150.8 → 42.1 for oxypurinol (Fig. 1C) and m/z 186.8 → 150.8 for the IS (Fig. 1D). No interfering signals were observed in plasma or urine (Figs. 2–5). The allopurinol retention time was 5.8 min (Fig. 2B and C, left panels) and that for the IS was 6.5 min (Fig. 2B and C, right panels). The oxypurinol retention time was 2.9 min (Fig. 3B and C, left panels) and that for the IS was 3.1 min (Fig. 3B and C, right panels). 3.2. Method validation 3.2.1. Selectivity and LLOQ Typical chromatograms for allopurinol and oxypurinol in human plasma or urine are shown in Figs. 2–5. Figs. 2A and 3A are blank plasma samples and Figs. 4A and 5A are blank urine samples. Figs. 2B and 3B are plasma samples and Figs. 4B and 5B are urine samples all spiked with allopurinol, oxypurinol, or IS at the LLOQ to show drug, metabolite, or IS at their respective retention times free from endogenous matrix interference. Plasma and urine samples from a volunteer after a single-dose of 100 mg/m2 allopurinol by iv gtt are presented in Figs. 2C and 3C (plasma) and in Figs. 4C and 5C (urine).
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The LLOQ value for both allopurinol and oxypurinol in plasma was 0.05 g/mL (S/N ≥ 5). For allopurinol, precision was 11.6% and accuracy 3.8%. For oxypurinol, precision was 10.4% and accuracy −4.5%. The LLOQ for allopurinol in urine was 0.5 g/mL, with precision of 9.2% and accuracy of 4.2%. The LLOQ for oxypurinol in urine was 1 g/mL (S/N ≥ 5), with precision and accuracy of 4.2% and 5.8%, respectively. 3.2.2. Linearity Both the allopurinol and oxypurinol in plasma were linear in the range 0.05–5.0 g/mL. The regression equations (n = 7) were y = 0.3971x + 0.0141 (r2 = 0.997) for allopurinol and y = 0.6169x + 0.0036 (r2 = 0.996) for oxypurinol, where y is the peak area ratio of analyte to IS and x is the corresponding plasma concentration. In urine, good linear responses were measured over 0.5–30 g/mL allopurinol and 1–50 g/mL oxypurinol. The regression equations were y = 0.1607x + 0.0026 (r2 = 0.995) for allopurinol and y = 0.3499x + 0.0655 (r2 = 0.996). The residues and error values for each point of the calibration curve were satisfactory. 3.2.3. Precision and accuracy Precision and accuracy are summarized in Table 1. The intraand inter-day precisions in plasma were within 8.6% for allopurinol and within 6.9% for oxypurinol at all QC concentrations. In urine, corresponding values were 11.1% and 7.0% at all QC concentrations. Relative errors in plasma ranged from −2.2% to 5.1% for all allopurinol QC concentrations and from −9.6% to −1.3% for all oxypurinol QC concentrations. In urine, relative errors ranged from −2.9% to 8.7% for allopurinol and from −2.5% to 4.6% for oxypurinol at all QC concentrations. Thus, the assay was precise and accurate. 3.2.4. Extraction recovery The extraction recoveries from plasma were 51.1% of 0.1 g/mL allopurinol, 55.8% of 1.0 g/mL, and 53.0% of 4 g/mL. Corresponding extraction recovery values for oxypurinol in plasma were 72.4%, 75.6%, and 76.7%. Recoveries of IS were 46.7% and 50.8% in
Fig. 2. MRM chromatograms of (A) drug free plasma, (B) drug free plasma spiked with allopurinol (0.05 g/mL) and IS (1 g/mL), and (C) plasma sample 2 h after a single injection at 100 mg/m2 allopurinol with concentration 1.00 g/mL.
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Fig. 3. MRM chromatograms of (A) drug free plasma, (B) drug free plasma spiked with oxypurinol (0.05 g/mL) and IS (2 g/mL), and (C) plasma sample 96 h after a single injection at 100 mg/m2 allopurinol with concentration 0.06 g/mL.
plasma, respectively. In urine, 79.3% was extracted from 1 g/mL allopurinol, 80.1% from 10 g/mL, and 83.8% from 20 g/mL. Corresponding values for oxypurinol were 75.0% of 2 g/mL, 75.4% of 10 g/mL, and 78.0% of 40 g/mL. Accordant recoveries of IS were 78.7% and 62.0%. 3.2.5. Matrix effects The matrix effects of allopurinol at three QC concentrations were 103.6%, 94.3%, and 97.2% in plasma and 97.1%, 96.8%, and 99.1% in urine, respectively. The matrix effects of IS were 95.7% in plasma and 94.9% in urine. For oxypurinol, matrix effects at three QC levels were 99.4%, 107.0%, and 100.4% in plasma, and 107.2%, 96.8%, and 104.2% in urine, respectively. Matrix effects of IS were 96.3%
in plasma and 95.9% in urine. These matrix effects values were all near 100%, indicating no significant matrix effects. 3.2.6. Stability Measured concentrations of allopurinol, oxypurinol, and IS were between 90.1% and 109.7% of the initial concentrations when stocks or working solutions were stored for 3 days at 4 ◦ C or for 3 h at 25 ◦ C. All stocks were remade every 3 days to obviate any possible effect of analyte or IS degradation. There was no obvious degradation of allopurinol or oxypurinol following short-term storage, 3 freeze-thaw cycles, or long-term storage (Table 2), so it is unlikely that the delay in testing affected the pharmacokinetic results presented below.
Fig. 4. MRM chromatograms of (A) drug free urine, (B) drug free urine spiked with allopurinol (0.5 g/mL) and IS (4 g/mL), and (C) urine sample 4–6 h after a single injection at 200 mg/m2 allopurinol with concentration 18.35 g/mL.
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Fig. 5. MRM chromatograms of (A) drug free urine, (B) drug free urine spiked with oxypurinol (1 g/mL) and IS (10 g/mL), and (C) urine sample 12–24 h after a single injection at 200 mg/m2 allopurinol with concentration 37.09 g/mL.
Table 1 The precision and accuracy of determination method for allopurinol and oxypurinol in human plasma and urine. Matrix
Measurement interval
Plasma
Allopurinol
Nominal conc. (g ml)
Measured conc. (g ml) (mean ± SD)
Intra-day (n = 5)
0.1 1 4 16 1 10 20 50 100
0.11 1.02 3.92 3.91 0.97 10.07 21.74 10.06 19.84
± ± ± ± ± ± ± ± ±
Inter-day (n = 15)
0.1 1 4 16 1 10 20 50 100
0.10 1.03 3.98 4.06 1.00 9.80 20.96 10.06 19.56
± ± ± ± ± ± ± ± ±
Urine
Plasma
Oxypurinol
Urine
RSD (%)
RE (%)
Nominal conc. (g ml)
Measured conc. (g ml) (mean ± SD)
RSD (%)
RE (%)
0.01 0.05 0.20 0.17 0.07 0.67 0.53 0.98 0.58
7.82 4.53 5.05 4.47 7.62 6.63 2.44 9.71 2.94
5.12 2.36 −2.05 −2.22 −2.90 0.69 8.68 0.58 −0.78
0.1 1 4 16 2 10 40
0.10 0.96 3.64 3.95 2.03 10.10 41.84
± ± ± ± ± ± ±
0.00 0.06 0.08 0.22 0.10 0.17 2.34
4.56 6.11 2.15 5.63 4.79 1.72 5.60
−4.47 −4.21 −9.05 −1.30 1.60 0.98 4.60
0.01 0.04 0.20 0.09 0.07 0.64 0.76 1.12 0.57
8.56 3.36 5.16 2.24 6.68 6.50 3.63 11.09 2.92
1.82 3.17 −0.63 1.43 −0.38 −2.04 4.81 0.64 −2.18
0.1 1 4 16 2 10 40
0.09 0.95 3.62 3.74 1.96 9.75 40.11
± ± ± ± ± ± ±
0.01 0.06 0.06 0.12 0.14 0.38 2.58
6.86 6.31 1.58 3.12 7.03 3.92 6.42
−6.12 −5.06 −9.56 −6.58 −2.23 −2.52 0.26
Table 2 The stability of allopurinol and oxypurinol in human plasma and urine under tested conditions (mean ± S.D., n = 5). Matrix
Plasma
Urine
Allopurinol
Oxypurinol
Nominal (g ml)
Short-term (24 h, 25 ◦ C)
0.1 1 4 1 10 20
0.09 0.98 3.49 1.01 9.88 19.58
± ± ± ± ± ±
0.01 0.04 0.09 0.02 0.07 0.08
Freeze-thaw (3 cycles, −20 ◦ C to 25 ◦ C) 0.11 1.03 4.11 1.06 9.98 22.42
± ± ± ± ± ±
0.01 0.04 0.09 0.01 0.73 0.23
Long-term (−20 ◦ C) 0.10 0.92 3.63 1.01 9.62 22.21
± ± ± ± ± ±
0.01 0.07 0.15 0.12 0.69 0.54
Autosampler (24 h, 4 ◦ C) 0.10 0.93 3.96 1.01 10.15 19.80
± ± ± ± ± ±
0.01 0.05 0.38 0.06 0.10 0.09
Nominal (g ml)
Short-term (24 h, 25 ◦ C)
0.1 1 4 2 10 40
0.10 0.94 3.70 2.10 10.07 42.54
± ± ± ± ± ±
0.00 0.02 0.04 0.18 0.06 2.20
Freeze-thaw (3 cycles, −20 ◦ C to 25◦ C) 0.10 1.01 3.76 2.00 9.46 39.15
± ± ± ± ± ±
0.00 0.02 0.04 0.10 0.06 0.26
Long-term (−20 ◦ C) 0.10 1.02 3.59 2.00 9.68 39.79
± ± ± ± ± ±
0.00 0.09 0.08 0.06 0.25 0.78
Autosampler (24 h, 4 ◦ C) 0.09 0.95 3.56 2.05 10.51 43.16
± ± ± ± ± ±
0.00 0.01 0.05 0.07 0.23 2.14
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Fig. 6. Mean plasma concentration-time curves of allopurinol (A) and oxypurinol (B) in 36 volunteers after single injections at 100 mg/m2 (L), 200 mg/m2 (M), and 300 mg/m2 (H) allopurinol.
interrupted. We optimized the analysis time by using acidified ethyl acetate to extract the samples and adjusted the mobile phase with ammonium formate. As a result, there are no interfering peaks and analysis time was reduced by about 2 min per sample. Simple and rapid liquid–liquid extraction was adopted and ethyl acetate was used as the extraction agent owing to its optimal polarity for efficient recovery. 2,6-Dichloropurine is an appropriate choice of IS due to the similarity of structure to both allopurinol and oxypurinol (Fig. 1). Indeed, recovery was similar and no significant matrix effect was observed. We have developed and validated the first systematically highsensitivity LC-MS/MS assay for determination of allopurinol and oxypurinol in human plasma urine. We improved the LC-MS/MS determination of oxypurinol in urine by increasing the selectivity and sensitivity while reducing assay time. Our method was successfully applied to a human pharmacokinetic study of an injectable allopurinol preparation. The simple and rapid liquid–liquid extraction protocol, combined with fast analytic times, makes this technique amenable to high-throughput detection of allopurinol and oxypurinol in plasma and urine for preclinical experiments and clinical trials.
3.3. Pharmacokinetic study Acknowledgments Fig. 6 depicts the mean plasma concentration-time curves for allopurinol (Fig. 6A) and oxypurinol (Fig. 6B) following single injections at 100 mg/m2 (low dose, L), 200 mg/m2 (mid dose, M), and 300 mg/m2 (high dose, H) in 36 healthy volunteers. Allopurinol reach a peak within 30 min and was almost completely metabolized within 4–5 h. In contrast, oxypurinol peaked at 4 h, consistent with the kinetics of allopurinol metabolism, and remained in the circulation and urine for more than 96 h. Allopurinol was metabolized and converted to oxypurinol and partially eliminated via the urine in healthy volunteers. In our study, the volumes of urine (V0−t ) were recorded and 1 mL of each sample was reserved to determine analyte concentration (C0−t ), excretion (S0−t = C0−t × V0−t ), and urine excretory rate (S0−t /Sdose × 100%). Urine excretory rate was 13.9% for allopurinol (S0−24 h = 47.2 mg) and 13.8% for oxypurinol (S0−24 h = 46.9 mg). The excretion of oxypurinol was lower because the T1/2 was too long to collect enough urine.
This work was supported by a grant from National Science and Technology Major Project for investigational new drug (Grant No. 2008ZX09312-002) and Guangdong Science and Technology Major Project (Grant No. 2011A080300003). References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11]
4. Discussion and conclusion We initially assumed that negative ion mode detection would be appropriate for both allopurinol and oxypurinol because they are both acidic. However, detection of allopurinol was poor in negative ion mode. To produce a high [M + H]+ precursor ion of allopurinol and IS, we used formic acid in the mobile phase to provide an acidic environment and ammonium formate to adjust the retention on the column. We also added hydrochloric acid to plasma and urine samples to accelerate analyte extraction and to increase the ionization response. Stocker et al. [19] developed a HPLC-MS/MS protocol for detection of oxypurinol in urine, but late eluting peaks were observed in some samples, which may be problematic when the analysis is
[12] [13] [14] [15] [16] [17] [18] [19] [20]
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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Determination of allopurinol and oxypurinol in human plasma and urine by liquid chromatography-tandem mass spectrometry Xia Liu a , Xiao-Jia Ni a , De-Wei Shang a , Ming Zhang a , Jin-Qing Hu a , Chang Qiu a , Fu-Tian Luo b , Yu-Guan Wen a,∗ a b
Department of Pharmacy, Guangzhou Brain Hospital, Guangzhou Medical University, 36 MingXin Road, Guangzhou 510370, China School of Public Health, Sun Yat-sen University, Guangzhou, China
a r t i c l e
i n f o
Article history: Received 11 June 2013 Accepted 19 September 2013 Available online 1 October 2013 Keywords: Allopurinol Metabolite HPLC-MS/MS Human plasma Pharmacokinetics
a b s t r a c t Allopurinol is used widely for the treatment of gout, but its pharmacokinetics is complex and some patients show hypersensitivity, necessitating careful monitoring and improved detection methods. In this study, a sensitive and reliable liquid chromatography-tandem mass spectrometry method was developed to determine the concentrations of allopurinol and its active metabolite oxypurinol in human plasma and urine using 2,6-dichloropurine as the internal standard (IS). Analytes and the IS were extracted from 0.5 ml aliquots of plasma or urine using ethyl acetate and separated on an Agilent Eclipse Plus C18 column using methanol and ammonium formate–formic acid buffer containing 5 mM ammonium formate and 0.1% formic acid (95:5, v/v) as the mobile phase (A) for allopurinol or methanol plus 5 mM ammonium formate aqueous solution (95:5, v/v) as the mobile phase (B) for oxypurinol. Allopurinol was detected in positive ion mode and the analysis time was about 7 min. The calibration curve was linear from 0.05 to 5 g/mL allopurinol in plasma and 0.5–30 g/mL in urine. The lower limit of quantification (LLOQ) was 0.05 g/mL in plasma and 0.5 g/mL in urine. The intra- and inter-day precision and relative errors of quality control (QC) samples were ≤11.1% for plasma and ≤ 8.7% for urine. Oxypurinol was detected in negative mode with an analysis time of about 4 min. The calibration curve was linear from 0.05 to 5 g/mL in plasma (LLOQ, 0.05 g/mL) and from 1 to 50 g/mL in urine (LLOQ, 1 g/mL). The intra- and inter-day precision and relative errors were ≤7.0% for plasma and ≤9.6% for urine. This method was then successfully applied to investigate the pharmacokinetics of allopurinol and oxypurinol in humans. © 2013 Elsevier B.V. All rights reserved.
1. Introduction The xanthine oxidase inhibitor allopurinol (Fig. 1A) is the most frequently prescribed medication for the treatment of gout and hyperuricaemia [1,2]. Allopurinol is rapidly absorbed from the gastrointestinal tract and metabolized by xanthine oxidase to oxypurinol (Fig. 1B), the main metabolite responsible for the pharmacological activity [3,4]. Oxypurinol is also a potent inhibitor of xanthine oxidase, the enzyme that converts hypoxanthine to xanthine and xanthine to uric acid. Like allopurinol, it is excreted via the kidney. However, oxypurinol exhibits a prolonged serum half-life [5,6], complicating allopurinol dosing and increasing the risk of side effects in some patients. A new allopurinol intravenous preparation is in development, necessitating a reliable and practical analytical method for high-throughput detection in pharmacokinetic and therapeutic drug monitoring studies.
∗ Corresponding author. Tel.: +86 20 81268052; fax: +86 20 81891391. E-mail addresses: [email protected], [email protected] (Y.-G. Wen). 1570-0232/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jchromb.2013.09.028
Several methods have been developed for the measurement of allopurinol and oxypurinol in plasma and urine, including HPLC with ultra-violet detection [7–11] or electrochemical detection [12–14]. Brown and Bye [7] described the HPLC method for allopurinol and oxipurinol assay within human plasma and urine with linear calibration curves for allopurinol over the range 0.068–1.36 g/ml in plasma and 0.68–136 g/ml in urine and for oxipurinol 0.076–15.2 g/ml in plasma and 15.2–304 g/ml in urine. However, this method is based on complex ion-exchange chromatography. Moreover, the selectivity of UV detection is reduced by the interferences of residual protein [8–10], detection of single sample extends as long as 20 min [10], preparation method is time-consuming [11], and sensitivity is still limited [8–11]. Electrochemical detection involves complex sample preparation using enzymatic reactions [13] or solid phase extraction [14]. Other methods such as capillary electrophoresis detection [15,16] and micelle-stabilised room temperature phosphorescence detection [17] require specific modifications for plasma and urine sample detection. HPLC-MS method [18] has been adopted to determine allopurinol and oxypurinol in plasma by protein precipitation and liquid–liquid extraction following the lower limit of quantification
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Fig. 1. Chemical structures and mass spectra of (A) allopurinol (M + H)+ , (B) 2,6-dichloropurine (M + H)+ , (C) oxypurinol (M − H)− , and (D) 2,6-dichloropurine (M − H)− .
of the assay was 50 ng/mL for allopurinol and 100 ng/mL for oxypurinol, yet the paper did not present the concrete assay method. Modern HPLC-MS/MS method [19] for the determination of oxypurinol in urine is suitable for the quantification of oxypurinol in the urine of patients (n = 34) participating in a clinical trial to optimize therapy of gout with allopurinol but the selectivity and sensitivity are still not sufficient for general application. Because allopurinol and its metabolite oxypurinol are main for the treatment of gout and hyperuricaemia so a validated analytical methods for urine matrix to explore relationships with plasma urate concentrations is also needed. As a result, we have developed novel HPLC-MS/MS method to systemically measure allopurinol and oxypurinol in both human urine and plasma for the first time. This simple, sensitive, and practical method was fully validated and then successfully applied to a pharmacokinetic study of an injectable allopurinol formulation in healthy Chinese volunteers. 2. Materials and methods 2.1. Chemicals and reagents Intravenous Preparations of Allopurinol Sodium (0.5 g, Batch No. 120601) were prepared for a clinical study by Hainan Generalsanyang Pharmaceutical Co., Ltd. (China). Allopurinol standard reference (purity: 99.5%, lot. C10118000) was provided by Dr. Ehrenstorfer GmbH Company. Oxypurinol standard (purity: 99.5%, lot. D454550) was supplied by Toronto Research Chemicals Inc. The internal standard 2,6-dichloropurine (purity: 99.5%, lot. 100603200401) was provided by Wuhan Ariel Chemical Technology Co., Ltd. (China). Chromatographic pure grade methanol was purchased from Merck, chromatographic pure grade formic acid and ethyl acetate from Dikma Company (USA), mass spectrum pure grade ammonium formate from Sigma (USA), and analytical grade HCl from Guangzhou Chemical Agent Factory (China). Deionized water was produced using a Milli-Q academic reagent grade water purification system (Millipore, USA) and distilled before using.
and internal standard were detected using an Agilent 6410 triple quadrupole mass spectrometer (Agilent Technologies, Inc., USA) with electrospray ionization. Ultra-high purity (UHP) nitrogen was used as the drying gas. Raw data were processed using the Agilent MassHunter Chemstation (B.01.03). A Salvis Lab vacuum drying apparatus (Vacucenter VC20, CH-6343 Rotkreuz, Switzerland) was used to evaporate the supernatant extracted from plasma or urine samples. Pharmacokinetic parameters were calculated using DAS 2.1.1 software (Mathematical Pharmacology Professional Committee of China). 2.3. LC-ESI-MS/MS Chromatographic separation was performed at 40 ◦ C on an Agilent Eclipse Plus-C18 column (4.6 mm × 150 mm, 3.5 m). The mobile phase consisting of methanol −5 mmol/l ammonium formate aqueous solution (95:5, v/v) with or without 0.1% formic acid was employed for allopurinol and oxypurinol, separately. The flow rate for detection of both analytes was 0.5 mL/min. The mass spectrometer was operated in positive ion multiple reaction monitoring (MRM) mode for allopurinol detection and negative ion MRM mode for oxypurinol detection. The optimized condition consisted of a nebulizer set at 45 psi, drying gas flow of 10 L/min at 350 ◦ C, and a HV capillary set at 4000 V. Impact energy and split voltage of allopurinol were 30 units and 125 V separately and these were 20 units and 135 V for 2,6dichloropurine. The dwell time for each transition was 0.2 s and the electron multiplier set at 300 V. The injection volumes were 5 L plasma and 2 L urine. The impact energy of oxypurinol and IS were separately 18 units and 22 units with a split voltage of 115 V and 135 V, separately. The dwell time for each transition was 0.2 s and the electron multiplier set to 400 V. The injection volumes were 5 L plasma and 1 L urine. 2.4. Preparation of stock solutions, calibration curves, and quality control (QC) samples
2.2. Apparatus The chromatographic analysis system (Agilent 1100 series LC system, Agilent Technologies, Inc., USA) included a quaternary pump, autosampler, column oven, and degasser. The analytes
Stock solution of allopurinol in mobile phase A, oxypurinol in mobile phase B, and 2,6-dichloropurine in corresponding mobile phase A or B were prepared individually at a concentration of 1 g/L. Stocks were then serially diluted with the mobile phase
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to respectively prepare working solutions of 0.5, 1, 5, 10, 20, 40, and 50 g/ml analytes and either 10 g/mL IS for allopurinol or 20 g/mL IS for oxypurinol to construct plasma calibration curves. For urine calibration curves, stocks were respectively diluted to 5, 10, 20, 50, 100, 200, and 300 g/mL allopurinol and 40 g/mL IS or 10, 20, 50, 100, 200, 400, and 500 g/ml oxypurinol and 100 g/mL IS. All the solutions were kept at 4 ◦ C and warmed to room temperature before use. For preparation of calibration samples, 50 L of vacuum-dehydrated working analyte solution (all concentrations as above) and 50 L IS solution were added to 0.5 ml blank plasma or urine. The final concentrations in plasma were 0.05, 0.1, 0.5, 1, 2, 4, and 5 g/mL allopurinol plus 1 g/mL IS or 0.05, 0.1, 0.5, 1, 2, 4, and 5 g/mL oxypurinol plus 2 g/mL IS. The final concentrations in urine calibration samples were 0.5, 1, 2, 5, 10, 20, and 30 g/mL allopurinol plus 4 g/mL IS and 1, 2, 5, 10, 20, 40, and 50 g/mL oxypurinol plus 10 g/mL IS. Quality control samples for plasma analysis were prepared at 0.1, 1, and 4 g/mL allopurinol or oxypurinol in blank plasma. Quality control samples for urine analysis were prepared at 1, 10, and 20 g/mL for allopurinol or 2, 10, and 40 g/mL for oxypurinol in blank urine. 2.5. Sample preparation Standard (calibration curve) samples, quality control samples and real plasma or urine samples from human subjects were all processed by liquid–liquid extraction. In each tube, 0.5 mL plasma or urine, 50 L IS stock, and 0.2 M of 100 L hydrochloric acid were combined and vortexed at high speed for 15 s. Then, 3 mL of ethyl acetate was added and the mixture vortexed for 30 s. Samples were then centrifuged at 3000 r/min for 10 min. The upper organic phase was transferred into a 5 ml glass centrifuge tube and vacuum dried at 42 ◦ C. Samples were reconstituted with 200 L of respective mobile phase (A for allopurinol and B for oxypurinol) for analysis. 2.6. Method validation 2.6.1. Selectivity and lower limit of quantification To test the selectivity of the method, blank plasma or urine samples from six healthy volunteers were pretreated and analyzed. The absence of interfering peaks at the analyte retention times was considered acceptable selectivity. The lower limit of quantification (LLOQ) was defined as the lowest detectable analyte concentration for which the values of precision (RSD) and accuracy (RE) were ≤20% and the signal-to-noise (S/N) ≥5. 2.6.2. Calibration curves Calibration equations were derived using a linear weighted least-squares regression analysis of the peak area ratios of analyte to IS versus nominal analyte concentration. The regression parameters of slope, intercept, and correlation coefficient were calculated by the weighting factor 1/x2 . Linearity was assessed for calibration curves of human plasma or urine in three separate duplicates by correlation coefficient (r2 ) and by determining the residues and errors between nominal and measured concentrations for each point of the calibration curve. Residues were evaluated by alternating injections of the highest calibration concentration with blank samples. The area of blank sample was no more than 20% of LLOQ. The error values of precision (RSD) and accuracy (RE) were ≤20% for calibration curve standard concentrations at the LLOQ and 15% for other standard concentrations. 2.6.3. Precision and accuracy To evaluate precision and accuracy, five sets of spiked plasma or urine QC samples at the same three concentrations as above (Section 2.4) were analyzed on the same day and over three consecutive
days. Samples for a standard curve were prepared and analyzed on the same day to calculate the concentration of each QC sample. Relative standard deviation (RSD, %) and relative error (RE, %) were calculated to estimate precision and accuracy, where RE (%) = ((measured conc. − nominal conc.)/nominal conc.) × 100. Dilution factors were determined in plasma or urine to measure the sample concentrations out of analytical range. Quality control samples with 16 g/mL allopurinol or oxypurinol were diluted 1/4 with blank plasma while QC samples containing 50 g/mL and 100 g/mL allopurinol were diluted 1/5 with blank urine. These dilution factors were accurate and precise, as measured concentrations multiplied by 4 or 5 were 85–115% of the nominal concentrations. 2.6.4. Extraction recovery Recovery experiments were performed at the three QC concentrations with five replicates for allopurinol and oxypurinol and at one concentration for the IS. Analyte peak areas obtained from QC samples after extraction were compared to unextracted standard solutions at the same concentrations. 2.6.5. Matrix effects To evaluate the effect of co-eluting matrix components following ion suppression or enhancement, three different batches of blank plasma or urine were extracted and then spiked with the analyte at three QC levels. The peak areas of the analyte in spiked plasma or urine (B) were then compared with those of corresponding standard solutions in mobile phase (A) at equivalent concentrations. The ratio (B/A × 100) is defined as the matrix effect. A matrix effect value of 100% indicates that the response in the mobile phase and in the plasma or urine extracts was the same and no absolute matrix effect was observed. Usually RSD < 15% is satisfactory. Matrix effects of IS were investigated in a similar way at one concentration. 2.6.6. Stability The stability of the stock solutions and working solutions was examined by measuring allopurinol, oxypurinol, and IS stock concentrations after 3 days at 4 ◦ C and after 3 h at room temperature. The stability of analytes in plasma or urine QC samples was tested after 24 h at RT (25 ◦ C), after three complete freeze/thaw cycles from −20 to 25 ◦ C, and after long-term storage at −20 ◦ C (15 days for allopurinol, 30 day for oxypurinol). Placement stability of the ready-to-inject samples was also investigated after 24 h at 25 ◦ C in the autosampler rack. A sample was considered stable in the biological matrix when the calculated concentration was 85–115% of the freshly prepared samples. 2.7. Pharmacokinetic study Thirty-six healthy Chinese volunteers (19–28 years of age, 19–24 of BMI) participated in a pharmacokinetic study on an injectable allopurinol formulation. Health status was confirmed by review of medical histories and complete physical and biochemical tests prior to enrollment. The study was approved by the ethics committee of Guangzhou Brain Hospital. The volunteers fasted for 10 h and were then divided into three dose groups (6 males and 6 females per group) receiving a singledose of either 100 mg/m2 (L), 200 mg/m2 (M), or 300 mg/m2 (H) allopurinol in physiological saline by iv gtt administration. Body surface area was calculated using formulae for ethnic Chinese [20]: Smale = 0.0057 × height + 0.0121 × body weight + 0.0882, Sfemale = 0.0073 × height + 0.0127 × body weight − 0.2106. Blood samples were collected into heparin tubes before drug administration and at 0.17, 0.33, 0.5, 0.58, 0.75, 1, 1.5, 2, 2.5, 3, 4, and 5 h for allopurinol measurements and at these same times plus 8,
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12, 24, 36, 48, 72, and 96 h for oxypurinol. Blood samples were immediately centrifuged at 3000 r/min for 10 min to obtain plasma samples. Samples were stored at −20 ◦ C for later analysis. Volunteers also evacuated urine before drug administration and then drank water freely 2 h later. Urine samples from volunteers in the 200 mg/m2 (M) group were collected at 0, 0–2, 2–4, 4–6, 6–8, 8–12, and 12–24 h after injection. Volumes of urine were recorded and 10.0 mL samples stored at −20 ◦ C for later analysis. 3. Results 3.1. HPLC–ESI-MS/MS In positive ion mode, the most intense product ions observed in the MS/MS spectra were m/z 137.1 → 54.3 for allopurinol (Fig. 1A) and m/z 188.8 → 153.0 for the IS 2,6-dichloropurine (Fig. 1B). In negative ion mode, the most intense product ions were m/z 150.8 → 42.1 for oxypurinol (Fig. 1C) and m/z 186.8 → 150.8 for the IS (Fig. 1D). No interfering signals were observed in plasma or urine (Figs. 2–5). The allopurinol retention time was 5.8 min (Fig. 2B and C, left panels) and that for the IS was 6.5 min (Fig. 2B and C, right panels). The oxypurinol retention time was 2.9 min (Fig. 3B and C, left panels) and that for the IS was 3.1 min (Fig. 3B and C, right panels). 3.2. Method validation 3.2.1. Selectivity and LLOQ Typical chromatograms for allopurinol and oxypurinol in human plasma or urine are shown in Figs. 2–5. Figs. 2A and 3A are blank plasma samples and Figs. 4A and 5A are blank urine samples. Figs. 2B and 3B are plasma samples and Figs. 4B and 5B are urine samples all spiked with allopurinol, oxypurinol, or IS at the LLOQ to show drug, metabolite, or IS at their respective retention times free from endogenous matrix interference. Plasma and urine samples from a volunteer after a single-dose of 100 mg/m2 allopurinol by iv gtt are presented in Figs. 2C and 3C (plasma) and in Figs. 4C and 5C (urine).
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The LLOQ value for both allopurinol and oxypurinol in plasma was 0.05 g/mL (S/N ≥ 5). For allopurinol, precision was 11.6% and accuracy 3.8%. For oxypurinol, precision was 10.4% and accuracy −4.5%. The LLOQ for allopurinol in urine was 0.5 g/mL, with precision of 9.2% and accuracy of 4.2%. The LLOQ for oxypurinol in urine was 1 g/mL (S/N ≥ 5), with precision and accuracy of 4.2% and 5.8%, respectively. 3.2.2. Linearity Both the allopurinol and oxypurinol in plasma were linear in the range 0.05–5.0 g/mL. The regression equations (n = 7) were y = 0.3971x + 0.0141 (r2 = 0.997) for allopurinol and y = 0.6169x + 0.0036 (r2 = 0.996) for oxypurinol, where y is the peak area ratio of analyte to IS and x is the corresponding plasma concentration. In urine, good linear responses were measured over 0.5–30 g/mL allopurinol and 1–50 g/mL oxypurinol. The regression equations were y = 0.1607x + 0.0026 (r2 = 0.995) for allopurinol and y = 0.3499x + 0.0655 (r2 = 0.996). The residues and error values for each point of the calibration curve were satisfactory. 3.2.3. Precision and accuracy Precision and accuracy are summarized in Table 1. The intraand inter-day precisions in plasma were within 8.6% for allopurinol and within 6.9% for oxypurinol at all QC concentrations. In urine, corresponding values were 11.1% and 7.0% at all QC concentrations. Relative errors in plasma ranged from −2.2% to 5.1% for all allopurinol QC concentrations and from −9.6% to −1.3% for all oxypurinol QC concentrations. In urine, relative errors ranged from −2.9% to 8.7% for allopurinol and from −2.5% to 4.6% for oxypurinol at all QC concentrations. Thus, the assay was precise and accurate. 3.2.4. Extraction recovery The extraction recoveries from plasma were 51.1% of 0.1 g/mL allopurinol, 55.8% of 1.0 g/mL, and 53.0% of 4 g/mL. Corresponding extraction recovery values for oxypurinol in plasma were 72.4%, 75.6%, and 76.7%. Recoveries of IS were 46.7% and 50.8% in
Fig. 2. MRM chromatograms of (A) drug free plasma, (B) drug free plasma spiked with allopurinol (0.05 g/mL) and IS (1 g/mL), and (C) plasma sample 2 h after a single injection at 100 mg/m2 allopurinol with concentration 1.00 g/mL.
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Fig. 3. MRM chromatograms of (A) drug free plasma, (B) drug free plasma spiked with oxypurinol (0.05 g/mL) and IS (2 g/mL), and (C) plasma sample 96 h after a single injection at 100 mg/m2 allopurinol with concentration 0.06 g/mL.
plasma, respectively. In urine, 79.3% was extracted from 1 g/mL allopurinol, 80.1% from 10 g/mL, and 83.8% from 20 g/mL. Corresponding values for oxypurinol were 75.0% of 2 g/mL, 75.4% of 10 g/mL, and 78.0% of 40 g/mL. Accordant recoveries of IS were 78.7% and 62.0%. 3.2.5. Matrix effects The matrix effects of allopurinol at three QC concentrations were 103.6%, 94.3%, and 97.2% in plasma and 97.1%, 96.8%, and 99.1% in urine, respectively. The matrix effects of IS were 95.7% in plasma and 94.9% in urine. For oxypurinol, matrix effects at three QC levels were 99.4%, 107.0%, and 100.4% in plasma, and 107.2%, 96.8%, and 104.2% in urine, respectively. Matrix effects of IS were 96.3%
in plasma and 95.9% in urine. These matrix effects values were all near 100%, indicating no significant matrix effects. 3.2.6. Stability Measured concentrations of allopurinol, oxypurinol, and IS were between 90.1% and 109.7% of the initial concentrations when stocks or working solutions were stored for 3 days at 4 ◦ C or for 3 h at 25 ◦ C. All stocks were remade every 3 days to obviate any possible effect of analyte or IS degradation. There was no obvious degradation of allopurinol or oxypurinol following short-term storage, 3 freeze-thaw cycles, or long-term storage (Table 2), so it is unlikely that the delay in testing affected the pharmacokinetic results presented below.
Fig. 4. MRM chromatograms of (A) drug free urine, (B) drug free urine spiked with allopurinol (0.5 g/mL) and IS (4 g/mL), and (C) urine sample 4–6 h after a single injection at 200 mg/m2 allopurinol with concentration 18.35 g/mL.
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Fig. 5. MRM chromatograms of (A) drug free urine, (B) drug free urine spiked with oxypurinol (1 g/mL) and IS (10 g/mL), and (C) urine sample 12–24 h after a single injection at 200 mg/m2 allopurinol with concentration 37.09 g/mL.
Table 1 The precision and accuracy of determination method for allopurinol and oxypurinol in human plasma and urine. Matrix
Measurement interval
Plasma
Allopurinol
Nominal conc. (g ml)
Measured conc. (g ml) (mean ± SD)
Intra-day (n = 5)
0.1 1 4 16 1 10 20 50 100
0.11 1.02 3.92 3.91 0.97 10.07 21.74 10.06 19.84
± ± ± ± ± ± ± ± ±
Inter-day (n = 15)
0.1 1 4 16 1 10 20 50 100
0.10 1.03 3.98 4.06 1.00 9.80 20.96 10.06 19.56
± ± ± ± ± ± ± ± ±
Urine
Plasma
Oxypurinol
Urine
RSD (%)
RE (%)
Nominal conc. (g ml)
Measured conc. (g ml) (mean ± SD)
RSD (%)
RE (%)
0.01 0.05 0.20 0.17 0.07 0.67 0.53 0.98 0.58
7.82 4.53 5.05 4.47 7.62 6.63 2.44 9.71 2.94
5.12 2.36 −2.05 −2.22 −2.90 0.69 8.68 0.58 −0.78
0.1 1 4 16 2 10 40
0.10 0.96 3.64 3.95 2.03 10.10 41.84
± ± ± ± ± ± ±
0.00 0.06 0.08 0.22 0.10 0.17 2.34
4.56 6.11 2.15 5.63 4.79 1.72 5.60
−4.47 −4.21 −9.05 −1.30 1.60 0.98 4.60
0.01 0.04 0.20 0.09 0.07 0.64 0.76 1.12 0.57
8.56 3.36 5.16 2.24 6.68 6.50 3.63 11.09 2.92
1.82 3.17 −0.63 1.43 −0.38 −2.04 4.81 0.64 −2.18
0.1 1 4 16 2 10 40
0.09 0.95 3.62 3.74 1.96 9.75 40.11
± ± ± ± ± ± ±
0.01 0.06 0.06 0.12 0.14 0.38 2.58
6.86 6.31 1.58 3.12 7.03 3.92 6.42
−6.12 −5.06 −9.56 −6.58 −2.23 −2.52 0.26
Table 2 The stability of allopurinol and oxypurinol in human plasma and urine under tested conditions (mean ± S.D., n = 5). Matrix
Plasma
Urine
Allopurinol
Oxypurinol
Nominal (g ml)
Short-term (24 h, 25 ◦ C)
0.1 1 4 1 10 20
0.09 0.98 3.49 1.01 9.88 19.58
± ± ± ± ± ±
0.01 0.04 0.09 0.02 0.07 0.08
Freeze-thaw (3 cycles, −20 ◦ C to 25 ◦ C) 0.11 1.03 4.11 1.06 9.98 22.42
± ± ± ± ± ±
0.01 0.04 0.09 0.01 0.73 0.23
Long-term (−20 ◦ C) 0.10 0.92 3.63 1.01 9.62 22.21
± ± ± ± ± ±
0.01 0.07 0.15 0.12 0.69 0.54
Autosampler (24 h, 4 ◦ C) 0.10 0.93 3.96 1.01 10.15 19.80
± ± ± ± ± ±
0.01 0.05 0.38 0.06 0.10 0.09
Nominal (g ml)
Short-term (24 h, 25 ◦ C)
0.1 1 4 2 10 40
0.10 0.94 3.70 2.10 10.07 42.54
± ± ± ± ± ±
0.00 0.02 0.04 0.18 0.06 2.20
Freeze-thaw (3 cycles, −20 ◦ C to 25◦ C) 0.10 1.01 3.76 2.00 9.46 39.15
± ± ± ± ± ±
0.00 0.02 0.04 0.10 0.06 0.26
Long-term (−20 ◦ C) 0.10 1.02 3.59 2.00 9.68 39.79
± ± ± ± ± ±
0.00 0.09 0.08 0.06 0.25 0.78
Autosampler (24 h, 4 ◦ C) 0.09 0.95 3.56 2.05 10.51 43.16
± ± ± ± ± ±
0.00 0.01 0.05 0.07 0.23 2.14
16
X. Liu et al. / J. Chromatogr. B 941 (2013) 10–16
Fig. 6. Mean plasma concentration-time curves of allopurinol (A) and oxypurinol (B) in 36 volunteers after single injections at 100 mg/m2 (L), 200 mg/m2 (M), and 300 mg/m2 (H) allopurinol.
interrupted. We optimized the analysis time by using acidified ethyl acetate to extract the samples and adjusted the mobile phase with ammonium formate. As a result, there are no interfering peaks and analysis time was reduced by about 2 min per sample. Simple and rapid liquid–liquid extraction was adopted and ethyl acetate was used as the extraction agent owing to its optimal polarity for efficient recovery. 2,6-Dichloropurine is an appropriate choice of IS due to the similarity of structure to both allopurinol and oxypurinol (Fig. 1). Indeed, recovery was similar and no significant matrix effect was observed. We have developed and validated the first systematically highsensitivity LC-MS/MS assay for determination of allopurinol and oxypurinol in human plasma urine. We improved the LC-MS/MS determination of oxypurinol in urine by increasing the selectivity and sensitivity while reducing assay time. Our method was successfully applied to a human pharmacokinetic study of an injectable allopurinol preparation. The simple and rapid liquid–liquid extraction protocol, combined with fast analytic times, makes this technique amenable to high-throughput detection of allopurinol and oxypurinol in plasma and urine for preclinical experiments and clinical trials.
3.3. Pharmacokinetic study Acknowledgments Fig. 6 depicts the mean plasma concentration-time curves for allopurinol (Fig. 6A) and oxypurinol (Fig. 6B) following single injections at 100 mg/m2 (low dose, L), 200 mg/m2 (mid dose, M), and 300 mg/m2 (high dose, H) in 36 healthy volunteers. Allopurinol reach a peak within 30 min and was almost completely metabolized within 4–5 h. In contrast, oxypurinol peaked at 4 h, consistent with the kinetics of allopurinol metabolism, and remained in the circulation and urine for more than 96 h. Allopurinol was metabolized and converted to oxypurinol and partially eliminated via the urine in healthy volunteers. In our study, the volumes of urine (V0−t ) were recorded and 1 mL of each sample was reserved to determine analyte concentration (C0−t ), excretion (S0−t = C0−t × V0−t ), and urine excretory rate (S0−t /Sdose × 100%). Urine excretory rate was 13.9% for allopurinol (S0−24 h = 47.2 mg) and 13.8% for oxypurinol (S0−24 h = 46.9 mg). The excretion of oxypurinol was lower because the T1/2 was too long to collect enough urine.
This work was supported by a grant from National Science and Technology Major Project for investigational new drug (Grant No. 2008ZX09312-002) and Guangdong Science and Technology Major Project (Grant No. 2011A080300003). References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11]
4. Discussion and conclusion We initially assumed that negative ion mode detection would be appropriate for both allopurinol and oxypurinol because they are both acidic. However, detection of allopurinol was poor in negative ion mode. To produce a high [M + H]+ precursor ion of allopurinol and IS, we used formic acid in the mobile phase to provide an acidic environment and ammonium formate to adjust the retention on the column. We also added hydrochloric acid to plasma and urine samples to accelerate analyte extraction and to increase the ionization response. Stocker et al. [19] developed a HPLC-MS/MS protocol for detection of oxypurinol in urine, but late eluting peaks were observed in some samples, which may be problematic when the analysis is
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