Validation of an automated UPLC-MS/MS method for methylmalonic acid in serum/plasma and its application on clinical samples.

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.