A combined liquid chromatography tandem mass spectrometry assay for the quantification of urinary oxalate and citrate in patients with nephrolithiasis

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 mu L) was combined with mixed internal standard (C-13(2) oxalate/citrate-d(4)) 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% NH3 in methanol, the eluent was dried down and re-constituted with 100 mu L distilled water. Separation was then performed on an HSS T3 2.1 x 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-13(2) oxalate were eluted at 0.29 min; citrate and citrate-d(4) were eluted at 0.52 min. Mean recovery was 100% for oxalate and 103% for citrate; lower limit of quantification of oxalate was 60 mu mol/L and 50 mu mol/L for citrate. Oxalate was linear up to 1388 mu mol/L; citrate was linear up to 4762.5 mu mol/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.