Quantification of oxalate by novel LC–MS/MS: assay development, validation and application in lumasiran clinical trials: supplementary data

Background: Measurement of plasma oxalate (POx) is challenging, but critical, for management of patients with primary hyperoxaluria type 1. A novel LC–MS/MS assay was developed, validated and used to quantify POx in patients with primary hyperoxaluria type 1. Methods: Samples (100 μl of plasma in K2EDTA) were spiked with internal standard (13C2-labeled oxalic acid), acidified and cleaned by protein precipitation before analysis using anion HPLC–ESI–MS/MS. The assay was validated with a quantitation range of 0.500– 50.0 μg/ml (5.55–555 μmol/l). All parameters successfully met acceptance criteria, including 15% (20% at lower limit of quantification) for accuracy and precision. Conclusion: This assay has advantages over previously published POx quantitation methods, was validated in accordance with regulatory guidelines and accurately determined POx levels in humans.背景：血浆草酸（plasma oxalate, POx）的检测虽颇具挑战，但对于1型原发性高草酸尿症患者的临床管理至关重要。本研究开发、验证并应用了一种新型液相色谱-串联质谱（LC–MS/MS）检测方法，用于定量检测1型原发性高草酸尿症患者体内的POx水平。 方法：取100 μl乙二胺四乙酸二钾（K2EDTA）抗凝血浆样本，加入内标物（13C2标记草酸），经酸化、蛋白沉淀法净化后，采用阴离子交换高效液相色谱-电喷雾电离串联质谱（anion HPLC–ESI–MS/MS）进行分析。该检测方法的定量范围为0.500~50.0 μg/ml（5.55~555 μmol/L），并完成了方法学验证。所有检测参数均符合可接受标准：准确度与精密度要求分别为不超过15%（定量下限处为20%）。 结论：本检测方法相较于已发表的POx定量检测方法具有显著优势，且已按照监管指南完成方法学验证，可准确检测人体血浆中的POx水平。