Development and validation of a simple HPLC-MS/MS method for the quantification of methylmalonic acid in human serum...

A simple and rapid HPLC-MS/MS analytical method was developed and validated for the determination of methylmalonic acid (MMA) in human serum without a derivatization step. Serum samples (200 μl) were pretreated using a simple method 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 gradient elution using 0.1% (v/v) formic acid in water (mobile phase A) and 0.5% (v/v) formic acid in acetonitrile (mobile phase B) at a flow rate of 0.2 ml min^-1 . The total run time of the analysis was 4.5 min. Negative electrospray ionization and multiple reaction monitoring mode were used. The lower limit of detection and lower limit of quantification for MMA were determined to be 13.6 and 42.3 nmol L^-1 , respectively. The developed method enabled the quantification of MMA in a wide linear range of 42.3-4230 nmol L^-1 with a correlation coefficient of 0.9991.