Quantification of methylmalonic acid in serum measured by capillary gas chromatography—mass spectrometry as tert.-butyldimethylsilyl derivatives

Abstract

Quantification of methylmalonic acid in serum is considered one of the most sensitive parameters for diagnosis of cobalamin deficiency. Methylmalonic acid was measured as tert.-butyldimethylsilyl derivatives formed in the presence of dimethylformamide. Under these conditions the derivative was quantified using gas chromatography—mass spectrometry with selected-ion monitoring at 403 and 406 for methylmalonic acid and methyl-d 3-malonic acid, respectively. The characteristics of the method were: linearity from 0.04 to 1.7 μmol/l with linear regression equation y = −0.0199 + 0.727 x ( r = 0.999); recovery 95.5 ± 6.8%; detection threshold 17 fmol injected; within-day variation coefficient ranging from 3.10% to 5.6% according to sample concentration; and day-to-day coefficient of variation of 6.8% and 5.3% for methylmalonic acid concentrations of 0.103 μmol/l and 0.360 μmol/l, respectively. The normal range after log transformation was estimated at 0.03–0.26 μmol/l, 0.06–0.33 μmol/l and 0.02–0.40 μmol/l in children of 4–14 years ( n = 39), in healthy subjects at 20–40 years ( n = 70) and in healthy elderly persons older than 60 years ( n = 14), respectively. The normal range in children was significantly lower than that in adults and, in contrast, normal values in the elderly were significantly higher.