title: Measurement of citrate in urine using liquid chromatography tandem mass spectrometry: comparison with an enzymatic method.
abstract: 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.
authors.affiliation: Department of Clinical Biochemistry, Wythenshawe Hospital, South Manchester University Hospitals NHS Trust, Southmoor Road, Manchester M23 9LT, UK. bkeevil@smuht.nwest.nhs.uk
authors.forename: B G
authors.initials: BG
authors.lastname: Keevil
authors.forename: L
authors.initials: L
authors.lastname: Owen
authors.forename: S
authors.initials: S
authors.lastname: Thornton
authors.forename: J
authors.initials: J
authors.lastname: Kavanagh
mesh_terms: 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*
chemical_list: D019343:Citric Acid