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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 &lt;50 years) and 0.36 μmol/L (age ≥50 years) were applied. In 404 clinical routine samples 10% were &gt;0.28, 7% &gt; 0.4, and only 1% were &gt;0.7 μmol/L. The method has been successfully implemented in the laboratory for routine MMA analysis.

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