Keevil B G, Owen L, Thornton S, Kavanagh J
Department of Clinical Biochemistry, Wythenshawe Hospital, South Manchester University Hospitals NHS Trust, Southmoor Road, Manchester M23 9LT, UK.
Ann Clin Biochem. 2005 Sep;42(Pt 5):357-63. doi: 10.1258/0004563054889963.
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.
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.
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.
We have developed a simple LC-MS/MS method for urinary citrate measurement that shows acceptable performance.
尿液柠檬酸盐的测量用于评估进一步形成尿路结石的风险，并评估受影响个体的治疗效果。我们希望开发一种简单快速的液相色谱串联质谱法（LC-MS/MS）来分析尿液中的柠檬酸盐，并将其与我们目前的酶法进行比较。
对于LC-MS/MS分析，在深孔板中制备样品，向400微升水中加入10微升尿液和20微升内标。混合后，将3微升稀释后的样品注入LC-MS/MS系统。使用LC系统以0.4毫升/分钟的流速用含有2毫摩尔/升醋酸铵和0.1%（v/v）甲酸的水等度洗脱C18柱（50×2.1毫米）。使用含有2毫摩尔/升醋酸铵和0.1%（v/v）甲酸的100%甲醇进行梯度洗脱以冲洗柱子。柠檬酸盐的保留时间为1.4分钟，d4-柠檬酸盐的保留时间为1.4分钟。进样间循环时间为4.0分钟。使用串联质谱仪在多反应监测模式下监测分析物，使用以下跃迁，柠檬酸盐m/z 191.0>111.0和d4-柠檬酸盐m/z 195.0>113.0。
在480 - 3800微摩尔/升范围内，批内和批间变异系数均<3%。定量下限为24.0微摩尔/升。回归分析显示LC-MS/MS = 0.8781（酶法）+ 102.5，r = 0.964，n = 73。
我们开发了一种用于尿液柠檬酸盐测量的简单LC-MS/MS方法，其性能可接受。
