An LC-MS/MS method for serum methylmalonic acid suitable for monitoring vitamin B12 status in population surveys

Methylmalonic acid (MMA), a functional indicator of vitamin B12 insufficiency, was measured in the US population in the National Health and Nutrition Examination Survey (NHANES) from 1999 to 2004 using a GC/MS procedure that required 275 μL of sample and had a low throughput (36 samples/run). Our objective was to introduce a more efficient yet highly accurate LC-MS/MS method for NHANES 2011–2014. We adapted the sample preparation with some modifications from a published isotope-dilution LC-MS/MS procedure. The procedure utilized liquid–liquid extraction and generation of MMA dibutyl ester. Reversed-phase chromatography with isocratic elution allowed baseline resolution of MMA from its naturally occurring structural isomer succinic acid within 4.5 min. Our new method afforded an increased throughput (≤160 samples/run) and measured serum MMA with high sensitivity (LOD = 22.1 nmol/L) in only 75 μL of sample. Mean (±SD) recovery of MMA spiked into serum (2 d, 4 levels, 2 replicates each) was 94 % ± 5.5 %. Total imprecision (41 d, 2 replicates each) for three serum quality control pools was 4.9 %–7.9 % (97.1–548 nmol/L). The LC-MS/MS method showed excellent correlation (n = 326, r = 0.99) and no bias (Deming regression, Bland-Altman analysis) compared to the previous GC/MS method. Both methods produced virtually identical mean (±SD) MMA concentrations [LC-MS/MS: 18.47 ± 0.71 ng/mL (n = 17), GC/MS: 18.18 ± 0.67 ng/mL (n = 11)] on a future plasma reference material compared with a GC/MS method procedure from the National Institute of Standards and Technology [18.41 ± 0.70 ng/mL (n = 15)]. No adjustment will be necessary to compare previous (1999–2004) to future (2011–2014) NHANES MMA data.