title: An LC-MS/MS method for serum methylmalonic acid suitable for monitoring vitamin B12 status in population surveys.
abstract: 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.
authors.affiliation: Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA, 30341, USA.
authors.forename: Ekaterina M
authors.initials: EM
authors.lastname: Mineva
authors.forename: Mindy
authors.initials: M
authors.lastname: Zhang
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Rabinowitz
authors.forename: Karen W
authors.initials: KW
authors.lastname: Phinney
authors.forename: Christine M
authors.initials: CM
authors.lastname: Pfeiffer
mesh_terms: D000925:Anticoagulants / Q000097:blood / Q000494:pharmacology; D002138:Calibration; D002853:Chromatography, Liquid / Q000379:methods* / Q000592:standards; D056148:Chromatography, Reverse-Phase / Q000379:methods; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D059625:Liquid-Liquid Extraction; D008764:Methylmalonic Acid / Q000097:blood*; D009749:Nutrition Surveys; D011786:Quality Control; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019802:Succinic Acid / Q000097:blood; D053719:Tandem Mass Spectrometry / Q000379:methods* / Q000592:standards; D014805:Vitamin B 12 / Q000032:analysis*
chemical_list: D000925:Anticoagulants; D008764:Methylmalonic Acid; D019802:Succinic Acid; D014805:Vitamin B 12