Abstract：
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.
摘要：
腺嘌呤磷酸核糖基转移酶（APRT）缺乏是一种罕见的，以2,8-二羟基腺嘌呤（DHA）的肾脏排泄为特征的遗传性疾病，导致肾结石形成和慢性肾病（CKD）。用黄嘌呤氧化还原酶抑制剂治疗，别嘌呤醇或非布索坦，减少尿DHA排泄并减缓CKD的进展。目前用于治疗性监测APRT缺乏症的方法缺乏特异性，因此，需要更可靠的测量技术。在这项研究中,用于同时定量DHA的超高效液相色谱-串联质谱方法，腺嘌呤,别嘌呤醇,优化并验证了人血浆中的氧代嘌呤醇和非布索坦。使用乙腈用蛋白质沉淀制备血浆样品,随后蒸发。实施了化学计量学方法的实验设计，以优化梯度陡度，有机溶剂的量，流量,柱温,锥电压，去溶剂化温度和去溶剂化流速。使用分数阶乘设计进行实验筛选，在轴向点添加互补实验以优化峰面积，峰分辨率和峰宽。根据美国食品和药物管理局在50至5000ng/mL的DHA浓度范围内的生物分析方法验证指南，别嘌呤醇和非布索坦，腺嘌呤为100至5000ng/mL，氧嘌呤醇为50至12,000ng/mL，r2≥0.99。分析测定实现了准确度(〜10.8至8.3%)和精密度(CV<15%)的可接受性能。DHA,腺嘌呤,别嘌呤醇,在-80°C下进行五次冻融循环并在-80°C下储存12个月后，血浆样品中的氧代嘌呤醇和非布索坦是稳定的。评估了该测定以定量来自六名APRT缺乏症患者的临床血浆样品中的五种分析物，并证明是有效且准确的。所提出的测定对于指导药物治疗将是有价值的，从而有助于为APRT缺乏症患者提供改进的和更个性化的护理。
