Development and validation of a simple HPLC-MS/MS method for the quantification of methylmalonic acid without a derivatization step in human serum.

Abstract

A simple and rapid HPLC-MS/MS analytical method was developed and validated for the determination of methylmalonic acid without a derivatization step in human serum. Serum samples with a volume of 200 μL were pre-treated in a simple way based on ultrafiltration using a VIVASPIN 500 ultrafiltration column. Chromatographic separation was achieved on a Luna Omega C18 column with a PS C18 precolumn guard by a gradient elution using 0.1% (v/v) formic acid in water (component A of the mobile phase) and 0.5% (v/v) formic acid in acetonitrile (component B of the mobile phase) with flow rate 0.2 ml.min-1 . The total run time of the analysis was 4.5 minutes. Negative electrospray ionization and multiple reaction monitoring mode were used. The lower limit of detection (LLOD) and lower limit of quantification (LLOQ) for methylmalonic acid were determined to be 13.6 nmol. L-1 and 42.3 nmol. L-1 , respectively. The developed method allows the quantification of methylmalonic acid in a wide linear range of 42.3-4230 nmol. L-1 with a correlation coefficient of 0.9991.