Optimization and validation of a UPLC-MS/MS assay for simultaneous quantification of 2,8-dihydroxyadenine, adenine, allopurinol, oxypurinol and febuxostat in human plasma.

Adenine phosphoribosyltransferase (APRT) deficiency is a rare , hereditary disorder characterized by renal excretion of 2,8-dihydroxyadenine (DHA), leading to kidney stone formation and chronic kidney disease (CKD). Treatment with a xanthine oxidoreductase inhibitor, allopurinol or febuxostat, reduces urinary DHA excretion and slows the progression of CKD. The method currently used for therapeutic monitoring of APRT deficiency lacks specificity and thus, a more reliable measurement technique is needed. In this study, an ultra-performance liquid chromatography-tandem mass spectrometry method for simultaneous quantification of DHA, adenine, allopurinol, oxypurinol and febuxostat in human plasma was optimized and validated. Plasma samples were prepared with protein precipitation using acetonitrile followed by evaporation. The chemometric approach design of experiments was implemented to optimize gradient steepness, amount of organic solvent, flow rate, column temperature, cone voltage, desolvation temperature and desolvation flow rate. Experimental screening was conducted using fractional factorial design with addition of complementary experiments at the axial points for optimization of peak area, peak resolution and peak width. The assay was validated according to the US Food and Drug Administration guidelines for bioanalytical method validation over the concentration range of 50 to 5000 ng/mL for DHA, allopurinol and febuxostat, 100 to 5000 ng/mL for adenine and 50 to 12,000 ng/mL for oxypurinol, with r2 ≥ 0.99. The analytical assay achieved acceptable performance of accuracy (-10.8 to 8.3 %) and precision (CV < 15 %). DHA, adenine, allopurinol, oxypurinol and febuxostat were stable in plasma samples after five freeze-thaw cycles at -80 °C and after storage at -80 °C for 12 months. The assay was evaluated for quantification of the five analytes in clinical plasma samples from six APRT deficiency patients and proved to be both efficient and accurate. The proposed assay will be valuable for guiding pharmacotherapy and thereby contribute to improved and more personalized care for patients with APRT deficiency.

Urinary 2,8-dihydroxyadenine excretion in patients with adenine phosphoribosyltransferase deficiency, carriers and healthy control subjects.

BACKGROUND: Adenine phosphoribosyltransferase (APRT) deficiency is a rare autosomal recessive disorder of adenine metabolism that results in excessive urinary excretion of the poorly soluble 2,8-dihydroxyadenine (DHA), leading to kidney stones and chronic kidney disease. The purpose of this study was to assess urinary DHA excretion in patients with APRT deficiency, heterozygotes and healthy controls, using a recently developed ultra-performance liquid chromatography - tandem mass spectrometry (UPLC-MS/MS) assay. METHODS: Patients enrolled in the APRT Deficiency Registry and Biobank of the Rare Kidney Stone Consortium (http://www.rarekidneystones.org/) who had provided 24-h and first-morning void urine samples for DHA measurement were eligible for the study. Heterozygotes and healthy individuals served as controls. Wilcoxon-Mann-Whitney test was used to compare 24-h urinary DHA excretion between groups. Associations were examined using Spearman's correlation coefficient (rs). RESULTS: The median (range) 24-h urinary DHA excretion was 138 (64-292) mg/24 h and the DHA-to-creatinine (DHA/Cr) ratio in the first-morning void samples was 13 (4-37) mg/mmol in APRT deficiency patients who were not receiving xanthine oxidoreductase inhibitor therapy. The 24-h DHA excretion was highly correlated with the DHA/Cr ratio in first-morning void urine samples (rs = 0.84, p < .001). DHA was detected in all urine samples from untreated patients but not in any specimens from heterozygotes and healthy controls. CONCLUSIONS: High urinary DHA excretion was observed in patients with APRT deficiency, while urine DHA was undetectable in heterozygotes and healthy controls. Our results suggest that the UPLC-MS/MS assay can be used for diagnosis of APRT deficiency.

Comparison of the effect of allopurinol and febuxostat on urinary 2,8-dihydroxyadenine excretion in patients with Adenine phosphoribosyltransferase deficiency (APRTd): A clinical trial.

INTRODUCTION: Adenine phosphoribosyltransferase (APRT) deficiency is a rare, but significant, cause of kidney stones and progressive chronic kidney disease. The optimal treatment has not been established. The purpose of this pilot study was to compare the effect of the xanthine oxidoreductase inhibitors allopurinol and febuxostat on urinary 2,8-dihydroxyadenine (DHA) excretion in APRT deficiency patients. MATERIALS AND METHODS: Patients listed in the APRT Deficiency Registry of the Rare Kidney Stone Consortium, currently receiving allopurinol therapy, were invited to participate. The trial endpoint was the 24-h urinary DHA excretion following treatment with allopurinol (400mg/day) and febuxostat (80mg/day). Urinary DHA was measured using a novel ultra-performance liquid chromatography - electrospray tandem mass spectrometry assay. RESULTS: Eight of the 10 patients invited completed the study. The median (range) 24-h urinary DHA excretion was 116 (75-289) mg at baseline, and 45 (13-112) mg after 14days of allopurinol therapy (P=0.036). At the end of the febuxostat treatment period, 4 patients had urinary DHA below detectable limits (<20ng/mL) compared with none of the participants following allopurinol treatment (P=0.036). The other 4 participants had a median 24-h urinary DHA excretion of 13.2 (10.0-13.4) mg at the completion of febuxostat therapy (P=0.036). CONCLUSION: Urinary DHA excretion in APRT deficiency patients decreased with conventional doses of both allopurinol and febuxostat. Febuxostat was, however, significantly more efficacious than allopurinol in reducing DHA excretion in the prescribed doses. This finding, which may translate into improved outcomes of patients with APRT deficiency, should be confirmed in a larger sample.

Quantitative UPLC-MS/MS assay of urinary 2,8-dihydroxyadenine for diagnosis and management of adenine phosphoribosyltransferase deficiency.

Adenine phosphoribosyltransferase (APRT) deficiency is a hereditary disorder that leads to excessive urinary excretion of 2,8-dihydroxyadenine (DHA), causing nephrolithiasis and chronic kidney disease. Treatment with allopurinol or febuxostat reduces DHA production and attenuates the renal manifestations. Assessment of DHA crystalluria by urine microscopy is used for therapeutic monitoring, but lacks sensitivity. We report a high-throughput assay based on ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) for quantification of urinary DHA. The UPLC-MS/MS assay was optimized by a chemometric approach for absolute quantification of DHA, utilizing isotopically labeled DHA as an internal standard. Experimental screening was conducted with D-optimal design and optimization of the DHA response was performed with central composite face design and related to the peak area of DHA using partial least square regression. Acceptable precision and accuracy of the DHA concentration were obtained over a calibration range of 100 to 5000ng/mL on three different days. The intra- and inter-day accuracy and precision coefficients of variation were well within ±15% for quality control samples analyzed in replicates of six at three concentration levels. Absolute quantification of DHA in urine samples from patients with APRT deficiency was achieved wihtin 6.5min. Measurement of DHA in 24h urine samples from three patients with APRT deficiency, diluted 1:15 (v/v) with 10mM ammonium hydroxide (NH4OH), yielded a concentration of 3021, 5860 and 10563ng/mL and 24h excretion of 816, 1327 and 1649mg, respectively. A rapid and robust UPLC-MS/MS assay for absolute quantification of DHA in urine was successfully developed. We believe this method will greatly facilitate diagnosis and management of patients with APRT deficiency.