Quantification of Urinary Oxalate by Liquid Chromatography–Tandem Mass Spectrometry with Online Weak Anion Exchange Chromatography

Abstract Background: 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. Methods: We added calibrator or urine sample (10 μL) to 10 μL of 13C2 oxalate and 400 μL 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. Results: Oxalate and 13C2-oxalate eluted at 1.2 min. Mean recovery was 95%, limit of detection 3.0 μmol/L, lower limit of quantification 100.0 μmol/L, linearity to 2212 μmol/L, imprecision <6%, and bias <3% at 166, 880, and 1720 μmol/L. Oxalate eluted after the main area of ion suppression. Mean response ratios for urine and aqueous samples, enriched at 200 and 1000 μmol/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 μmol/L. Conclusion: This LC-MS/MS assay is applicable for quantifying urinary oxalate excretion.