Measurement of citrate in urine using liquid chromatography tandem mass spectrometry: comparison with an enzymatic method.
BACKGROUND
Measurement of urine citrate is used to assess the risk of further urinary stone formation and to assess the benefit of treatment in affected individuals. We wanted to develop a simple and rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the analysis of urinary citrate and to compare it with our current enzymatic assay.


METHODS
For the LC-MS/MS assay, samples were prepared in a deep-well block by adding 10 microL of urine and 20 microL of internal standard to 400 microL of water. After mixing, 3 microL 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 micromol/L. The lower limit of quantification was 24.0 micromol/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 for urinary citrate measurement that shows acceptable performance.
Keevil
B G
BG
Department of Clinical Biochemistry, Wythenshawe Hospital, South Manchester University Hospitals NHS Trust, Southmoor Road, Manchester M23 9LT, UK. bkeevil@smuht.nwest.nhs.uk
Owen
L
L
Thornton
S
S
Kavanagh
J
J
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D019343:Citric Acid / Q000652:urine*; D006801:Humans; D007669:Kidney Calculi / Q000175:diagnosis / Q000652:urine; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D019343:Citric Acid