Abstract

Vitamin B12 is essential for cell function and only accessible in food for mammals. To monitor vitamin B12 deficiency, methylmalonic acid (MMA) is used. Since MMA in serum/plasma is a frequently requested analyte at clinical laboratories the analytical method was improved and validated on a 96 well plate. Using a Tecan robot a working solution of acetonitrile containing MMA-D3 was added to plasma/serum samples. The solution was shaken for 1 min and then centrifuged for 10min. The supernatant was transferred to another plate and evaporated with nitrogen gas. The residual was redissolved with 0.2% formic acid in MilliQ-water and the plate was shaken for 1 min prior to LC-MS/MS analysis. The total analysis time was 3 min, retention time for MMA was 1.1 min and it was well separated from the interfering succinic acid. The calibrator curve was 0.044 – 1.63 μmol/L, which was also the linear range and LLOQ was 0.044 μmol/L. The within- and between-run CV:s were 3-7%. Age dependent clinical cut-offs at 0.28 (age <50 years) and 0.36 μmol/L (age ≥50 years) were applied. In 404 clinical routine samples 10% were >0.28, 7% > 0.4, and only 1% were >0.7 μmol/L. The method has been successfully implemented in the laboratory for routine MMA analysis.

摘要

维生素 B12 对细胞功能至关重要，只能在哺乳动物的食物中获得。为了监测维生素 B12 缺乏，使用甲基丙二酸 (MMA)。由于血清/血浆中的 MMA 是临床实验室经常需要的分析物，因此改进了分析方法并在 96 孔板上进行了验证。使用 Tecan 机器人将含有 MMA-D3 的乙腈工作溶液添加到血浆/血清样品中。将溶液摇动 1 分钟，然后离心 10 分钟。将上清液转移到另一块板上并用氮气蒸发。残留物用 0.2% 甲酸在 MilliQ-水中重新溶解，在 LC-MS/MS 分析之前将板摇动 1 分钟。总分析时间为 3 分钟，MMA 的保留时间为 1.1 分钟，并且与干扰性琥珀酸分离良好。校准曲线为 0。044 – 1.63 μmol/L，这也是线性范围，LLOQ 为 0.044 μmol/L。运行内和运行间的 CV:s 为 3-7%。应用了 0.28（年龄 <50 岁）和 0.36 μmol/L（年龄 ≥50 岁）的年龄依赖性临床临界值。在 404 个临床常规样本中，10% > 0.28，7% > 0.4，只有 1% > 0.7 μmol/L。该方法已在实验室成功实施，用于常规 MMA 分析。