Faculty of Pharmaceutical Sciences, University of Iceland, Reykjavik, Iceland; ArcticMass, Reykjavik, Iceland.
Internal Medicine Services, Landspitali-The National University Hospital of Iceland, Reykjavik, Iceland.
J Chromatogr B Analyt Technol Biomed Life Sci. 2024 Mar 1;1235:124041. doi: 10.1016/j.jchromb.2024.124041. Epub 2024 Feb 5.
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 r ≥ 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.
腺嘌呤磷酸核糖基转移酶 (APRT) 缺乏症是一种罕见的遗传性疾病，其特征是肾脏排泄 2,8-二羟腺嘌呤 (DHA)，导致肾结石形成和慢性肾脏病 (CKD)。黄嘌呤氧化还原酶抑制剂别嘌醇或非布司他的治疗可减少尿 DHA 排泄并减缓 CKD 进展。目前用于 APRT 缺乏症治疗监测的方法缺乏特异性，因此需要更可靠的测量技术。在这项研究中，优化和验证了一种超高效液相色谱-串联质谱法，用于同时定量人血浆中的 DHA、腺嘌呤、别嘌醇、氧嘌呤醇和非布司他。使用乙腈进行蛋白沉淀后对血浆样品进行制备，然后蒸发。采用实验设计化学计量学方法优化梯度陡度、有机溶剂用量、流速、柱温、锥电压、蒸发温度和蒸发流量。使用部分因子设计进行实验筛选，并在轴向点添加补充实验，以优化峰面积、峰分辨率和峰宽。该测定法根据美国食品和药物管理局 (FDA) 生物分析方法验证指南进行验证，DHA、别嘌醇和非布司他的浓度范围为 50 至 5000ng/mL，腺嘌呤为 100 至 5000ng/mL，氧嘌呤为 50 至 12000ng/mL，r≥0.99。分析测定法的准确度（-10.8%至 8.3%）和精密度（CV<15%）均达到可接受水平。DHA、腺嘌呤、别嘌醇、氧嘌呤醇和非布司他在-80°C 下经过五次冻融循环和-80°C 储存 12 个月后在血浆样品中稳定。该测定法用于定量分析 6 名 APRT 缺乏症患者的临床血浆样品中的五种分析物，结果证明该测定法既高效又准确。该方法将有助于指导药物治疗，从而为 APRT 缺乏症患者提供更好的、更个性化的治疗。
