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

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-d3-malonic acid, respectively. The characteristics of the method were: linearity from 0.04 to 1.7 mumol/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 mumol/l and 0.360 mumol/l, respectively. The normal range after log transformation was estimated at 0.03-0.26 mumol/l, 0.06-0.33 mumol/l and 0.02-0.40 mumol/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.