SummaryWe report a stable isotope dilution method for the accurate measurement of methylmalonic acid (MMA) in amniotic fluid and its successful application to the prenatal diagnosis of methylmalonic acidemia. The stable isotopically-labeled analogue of MMA, (2-[2H3]methyl)malonic acid, was synthesized and used as an internal standard. Samples were extracted, methylated, and analyzed by chemical ionization gas chromatography-mass spectrometry (CI-GC-MS) operated in the selected ion monitoring (SIM) mode. The analytical method is rapid (8 h), ultrasensitive (lower limit ~10 ng/ml), linear over three orders of magnitude, and reproducible. The concentration of MMA in amniotic fluids from normal pregnant women was 38 ± 15 ng/ml (mean ± 1 S.D.). This value is 20-fold less than our previous estimate by gas chromatographic analysis, indicating that interference by co-eluting materials was eliminated by use of SIM in the present method. Amniotic fluids from eight at-risk pregnancies were examined. MMA concentration in six of the pregnancies was in the normal range whereas the concentration in the remaining two pregnancies was elevated by greater than two orders of magnitude (125–250-fold). Analysis of urine from ten at-risk pregnancies showed a smaller (31/2–9-fold) but significant increase in urinary MMA excretion in two women carrying affected fetuses. A control group of normal pregnant women had a mean urinary MMA excretion of 1.7 ± 0.5 μg/mg creatinine, in excellent agreement with previous estimates. There was concordance in prenatal diagnosis of fetal status in all cases between amniotic fluid cell studies and MMA measurements in amniotic fluid and/or maternal urine. Amniotic fluid MMA determination by the stable isotope dilution technique provides a highly accurate technique for prenatal diagnosis of methylmalonic acidemia that is complementary to amniotic fluid cell studies, and may supplant such studies in selected instances.Speculation The stable isotope dilution method may be extended to prenatal diagnosis of other inborn errors of metabolism in which low molecular weight metabolites accumulate in amniotic fluid. The concentrations of metabolites, which accumulate in some of these disorders, are below the sensitivity limits of currently available analytical techniques and it is thus impossible to distinguish differences that may be present in pregnancies carrying affected and unaffected fetuses. The enhanced sensitivity of the stable isotope dilution method may permit the desired discrimination.