液相色谱－串联质谱法测定尿酸及与常规检测方法的比较

摘要

Objective To establish a liquid chromatography-tandem mass spectrometry(LC-MS/MS) for serum uric acid, and to compare LC-MS/MS with clinical routine determination methods.Methods Uric acid-15 N2 was added as internal standard, serum samples were precipitated with acetonitrile and dried with nitrogen flow.A reversed-phase chromatographic separation was performed on Capcell C18 MG Ⅲ analytical column by using 5 mmol/L ammonium acetate plus 0.1% acetate acid in water and methanol(90 ∶10,v/v) as mobile phase.The flow rate was 0.3 mL/min. Uric acid and internal standard were monitored by a negative electrospray ion-tandem mass spectrometry system using ion transitions of 167 /124 amu (quantitation) and 167 /96 amu (qualitation) for uric acid and 169 /125 amu for uric acid-N2 .After being validated, the LC-MS/MS was compared with the uricase ultraviolet ( UV) method and uricase colorimetric (UC) method.Results The LC-MS/MS was validated over a concentration range of 30-952 μmol/L. Within-run and between-run precisions were 2.01%-6.23% and 4.55%-8.08%, respectively.The accuracy was 96.5%-103.4%.The retention time of uric acid was 1.5 min, and total run time was 3 min.The linear correlation formulas among LC-MS/MS, uricase UV method and uricase UC method were YUV =0.898XLC-MS/MS +2.15, r =0.978 and YUC =0.845XLC-MS /MS +22.15, r =0.983.The average biases were 1.56% ±0.65% between LC-MS/MS and the 15 National Institute of Standards and Technology ( NIST) standard reference material and -0.34%-3.05% between LC-MS/MS and correctness verification samples, 2014 from the National Center for Clinical Laboratory.Conclusions Serum uric acid can be simply and accurately measured by LC-MS/MS.There is good comparability between LC-MS/MS, uricase UV method and uricase UC method.%目的：建立液相色谱－串联质谱（LC－MS／MS）检测血清尿酸的方法，并与临床常规生化方法进行比较，为临床实验室不同检测系统尿酸检测结果的一致性提供参考。方法血清添加同位素内标尿酸－15 N2，经乙腈处理吹干重组后采用 LC－MS／MS 测定。以 Capcell C18 MGⅢ为分析柱进行反相色谱分离，以5 mmol／L 乙酸铵＋0．1％乙酸水溶液和甲醇（90∶10，v／v）为流动相，等度洗脱，流速0．3 mL／min，电喷雾离子化串联四极杆质谱，负离子多反应监测，尿酸离子通道为167／124 amu（定量）、167／96 amu（定性）；尿酸－15 N2离子通道为169／125 amu。LC－MS／MS 进行方法学评价后与临床尿酸常规检测方法（酶紫外法和酶比色法）进行比较。结果 LC－MS／MS 检测尿酸的线性范围为30～952μmol／L，批内和批间精密度分别为2．01％～6．23％和4．55％～8．08％，准确度为96．5％～103．4％。尿酸的色谱保留时间为1．5 min，单份样品的分析时间为3 min。酶紫外法、酶比色法与LC－MS／MS 的平均偏差分别为－10．02％、－9．88％；线性相关方程分别为 Y酶紫外法＝0．898XLC－MS／MS ＋2．15，r ＝0．978；Y酶比色法＝0．845XLC－MS／MS ＋22．15，r ＝0．983。 LC－MS／MS 与美国标准技术研究所（NIST）定值参考物质的平均偏差为1．56％±0．65％，与卫生部临床检验中心2014年代谢物正确度验证样品的定值的偏差为－0．34％～3．05％。结论采用 LC－MS／MS 可简便、准确地定量检测尿酸。酶紫外法、酶比色法的血清尿酸测定结果与LC－MS／MS 具有较好的可比性。