Marshall David J, Adaway Joanne E, Keevil Brian G
Department of Clinical Biochemistry, Manchester University NHS Foundation Trust, Manchester, UK.
Ann Clin Biochem. 2018 Jul;55(4):461-468. doi: 10.1177/0004563217739035. Epub 2017 Nov 20.
Background Analysis of citrate and oxalate in a 24-h urine sample is important in the screening and monitoring of patients with nephrolithiasis. To streamline the analytical process, it was decided to combine oxalate and citrate and analyse them simultaneously in the same assay. Objective A highly sensitive and specific assay for analysis of urine citrate and oxalate was developed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with a simple weak anion exchange solid phase extraction (WAX SPE) clean-up procedure. Method Premixed calibrator/acidified urine (50 µL) was combined with mixed internal standard (C oxalate/citrate-d) and 5% v/v formic acid in water and passed through a Waters WAX SPE plate. After clean-up steps, the plate was eluted with 5% NH in methanol, the eluent was dried down and re-constituted with 100 µL distilled water. Separation was then performed on an HSS T3 2.1 × 50 mm column (Waters, Manchester, UK), flow rate of 0.5 mL/min using a gradient of aqueous and organic mobile phases. We detected multiple reaction monitoring transitions m/z citrate 191.1>110.9, citrate IS 195.1>112.9, oxalate 88.9>60.85, oxalate IS 90.9>61.9 using a Waters TQD in electrospray-negative mode. Results Oxalate and C oxalate were eluted at 0.29 min; citrate and citrate-d were eluted at 0.52 min. Mean recovery was 100% for oxalate and 103% for citrate; lower limit of quantification of oxalate was 60 µmol/L and 50 µmol/L for citrate. Oxalate was linear up to 1388 µmol/L; citrate was linear up to 4762.5 µmol/L. Oxalate was found to be affected by ion suppression (matrix effect: -23 to +65%) but was compensated for by the internal standard used in all cases. The coefficient of variation of the assay in urine for oxalate was <7% for oxalate and 5% for citrate. Discussion We have developed a rapid assay for LC-MS/MS measurement of urinary oxalate and citrate in a routine clinical laboratory. It is simple, reproducible and easy to perform.
对24小时尿液样本中的柠檬酸盐和草酸盐进行分析，对于肾结石患者的筛查和监测具有重要意义。为简化分析过程，决定将草酸盐和柠檬酸盐合并，在同一项检测中同时进行分析。目的：采用液相色谱 - 串联质谱法（LC-MS/MS）结合简单的弱阴离子交换固相萃取（WAX SPE）净化程序，开发一种高灵敏度和特异性的尿液柠檬酸盐和草酸盐分析方法。方法：将预混合的校准物/酸化尿液（50 μL）与混合内标（草酸盐/柠檬酸盐 - d）以及5% v/v甲酸水溶液混合，通过Waters WAX SPE板。净化步骤完成后，用5%氨甲醇溶液洗脱该板，将洗脱液吹干，再用100 μL蒸馏水复溶。然后在HSS T3 2.1×50 mm柱（Waters，英国曼彻斯特）上进行分离，使用水相和有机流动相的梯度，流速为0.5 mL/min。使用Waters TQD在电喷雾负离子模式下检测多反应监测转换m/z柠檬酸盐191.1>110.9、柠檬酸盐内标195.1>112.9、草酸盐88.9>60.85、草酸盐内标90.9>61.9。结果：草酸盐和草酸盐 - d在0.29分钟洗脱；柠檬酸盐和柠檬酸盐 - d在0.52分钟洗脱。草酸盐的平均回收率为100%，柠檬酸盐为103%；草酸盐的定量下限为60 μmol/L，柠檬酸盐为50 μmol/L。草酸盐在高达1388 μmol/L范围内呈线性；柠檬酸盐在高达4762.5 μmol/L范围内呈线性。发现草酸盐受离子抑制影响（基质效应：-23%至+65%），但在所有情况下均通过使用内标进行了补偿。该检测方法在尿液中草酸盐的变异系数<7%，柠檬酸盐为5%。讨论：我们开发了一种在常规临床实验室中通过LC-MS/MS测量尿草酸和柠檬酸的快速检测方法。它简单、可重复且易于操作。
