Measurement of citrate in urine using liquid chromatography tandem mass spectrometry: comparison with an enzymatic method

Ann Clin Biochem 2005;42:357-363
© 2005 Association for Clinical Biochemistry


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Original Articles

BG Keevil,
L Owen,
S Thornton and
J Kavanagh

Department of Clinical Biochemistry, Wythenshawe Hospital, South Manchester University Hospitals NHS Trust, Southmoor Road, Manchester M23 9LT, UK;
Department of Urology, Wythenshawe Hospital, South Manchester University Hospitals NHS Trust, Southmoor Road, Manchester M23 9LT, UK

Background: Measurement of urine citrate is used to assess therisk of further urinary stone formation and to assess the benefitof treatment in affected individuals. We wanted to develop asimple and rapid liquid chromatography tandem mass spectrometry(LC-MS/MS) method for the analysis of urinary citrate and tocompare it with our current enzymatic assay.

Methods: For the LC-MS/MS assay, samples were prepared in a deep-well block by adding 10 µL of urine and 20 µL of internal standard to 400 µL of water. After mixing, 3 µL of the diluted sample was injected into the LC-MS/MS system. An LC system was used to isocratically elute a C18 column (50 x 2.1 mm) with 0.4 mL/min water containing 2 mmol/L ammonium acetate and 0.1% (v/v) formic acid. A step gradient of 100% methanol containing 2 mmol/L ammonium acetate and 0.1% (v/v) formic acid was used to wash the column. The retention times were 1.4 min for citrate and 1.4 min for d4-citrate. Cycle time was 4.0 min, injection to injection. The analytes were monitored using a tandem mass spectrometer operated in multiple reaction monitoring mode using the following transitions, citrate m/z 191.0> 111.0 and d4-citrate m/z 195.0> 113.0.

Results: Within and between-batch coefficients of variation were <3% over the range 480-3800 µmol/L. The lower limit of quantification was 24.0 µmol/L. Regression analysis showed LC-MS/MS = 0.8781 (enzymatic assay) + 102.5, r = 0.964, n = 73.

Conclusions: We have developed a simple LC-MS/MS method forurinary citrate measurement that shows acceptable performance.

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