Keevil Brian G, Thornton Susan
Department of Clinical Biochemistry, Wythenshawe Hospital, South Manchester University Hospitals National Health Service Trust, Manchester, United Kingdom.
Clin Chem. 2006 Dec;52(12):2296-9. doi: 10.1373/clinchem.2006.075275. Epub 2006 Oct 13.
Urinary oxalate is commonly measured with an enzymatic assay that is specific but requires a manual clean-up step to reduce ascorbic acid interference. We developed a urinary oxalate assay that uses liquid chromatography-tandem mass spectrometry (LC-MS/MS) with anion exchange chromatography and simple sample preparation.
We added calibrator or urine sample (10 microL) to 10 microL of (13)C2 oxalate and 400 microL of water and performed separation on a Waters OASIS WAX column, flow rate 0.6 mL/min, and then elution for 0.3 min with water containing 2 mmol/L ammonium acetate and 1 mL/L formic acid and for 1.0 min with 750 mL/L methanol containing 20 mL/L ammonia. We detected multiple reaction monitoring transitions m/z 88.6 > 60.5 and m/z 90.5 > 61.5 for oxalic acid and 13C2-oxalate, respectively, with a Quattro micro tandem mass spectrometer in electrospray-negative mode.
Oxalate and 13C2-oxalate eluted at 1.2 min. Mean recovery was 95%, limit of detection 3.0 micromol/L, lower limit of quantification 100.0 micromol/L, linearity to 2212 micromol/L, imprecision <6%, and bias <3% at 166, 880, and 1720 micromol/L. Oxalate eluted after the main area of ion suppression. Mean response ratios for urine and aqueous samples, enriched at 200 and 1000 micromol/L, were 3.7% and 5.4%, respectively. No interference was observed from other organic acids. Passing and Bablock regression analysis comparing the Trinity Biotech enzymatic reagent set and LC-MS/MS showed LC-MS/MS = 1.06 (enzymatic assay) -21.2, r = 0.964, n = 110. Bland Altman analysis showed general agreement, with a mean bias of -1.9 mumol/L.
This LC-MS/MS assay is applicable for quantifying urinary oxalate excretion.
尿草酸通常采用一种特异性的酶法测定，但需要一个手工净化步骤以减少抗坏血酸的干扰。我们开发了一种尿草酸测定法，该方法采用液相色谱 - 串联质谱法（LC-MS/MS），结合阴离子交换色谱和简单的样品制备方法。
我们将校准物或尿液样本（10微升）加入到10微升的（13）C2草酸和400微升水中，在Waters OASIS WAX柱上进行分离，流速为0.6毫升/分钟，然后先用含2毫摩尔/升醋酸铵和1毫升/升甲酸的水洗脱0.3分钟，再用含20毫升/升氨水的750毫升/升甲醇洗脱1.0分钟。我们使用Quattro micro串联质谱仪在电喷雾负离子模式下分别检测草酸和13C2 - 草酸的多反应监测转换m/z 88.6 > 60.5和m/z 90.5 > 61.5。
草酸和13C2 - 草酸在1.2分钟时洗脱。平均回收率为95%，检测限为3.0微摩尔/升，定量下限为100.0微摩尔/升，线性范围至2212微摩尔/升，在166、880和1720微摩尔/升时不精密度<6%，偏差<3%。草酸在离子抑制的主要区域之后洗脱。在200和1000微摩尔/升富集的尿液和水性样品的平均响应比分别为3.7%和5.4%。未观察到其他有机酸的干扰。通过Passing和Bablock回归分析比较Trinity Biotech酶试剂组和LC-MS/MS，结果显示LC-MS/MS = 1.06（酶法测定） - 21.2，r = 0.964，n = 1 | 0。Bland Altman分析显示总体一致性，平均偏差为 - 1.9微摩尔/升。
这种LC-MS/MS测定法适用于定量尿草酸排泄。
