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

摘要

Methylmalonic acid (MMA), a functional indicator of vitamin B-12 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 mu L 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 (a parts per thousand currency sign160 samples/run) and measured serum MMA with high sensitivity (LOD = 22.1 nmol/L) in only 75 mu L 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.