Authors: Ekaterina M Mineva Mindy Zhang Daniel J Rabinowitz Karen W Phinney Christine M Pfeiffer
Publish Date: 2014/09/26
Volume: 407, Issue: 11, Pages: 2955-2964
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 LCMS/MS method for NHANES 2011–2014 We adapted the sample preparation with some modifications from a published isotopedilution LCMS/MS procedure The procedure utilized liquid–liquid extraction and generation of MMA dibutyl ester Reversedphase chromatography with isocratic elution allowed baseline resolution of MMA from its naturally occurring structural isomer succinic acid within 45 min Our new method afforded an increased throughput ≤160 samples/run and measured serum MMA with high sensitivity LOD = 221 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 ± 55 Total imprecision 41 d 2 replicates each for three serum quality control pools was 49 –79 971–548 nmol/L The LCMS/MS method showed excellent correlation n = 326 r = 099 and no bias Deming regression BlandAltman analysis compared to the previous GC/MS method Both methods produced virtually identical mean ±SD MMA concentrations LCMS/MS 1847 ± 071 ng/mL n = 17 GC/MS 1818 ± 067 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 1841 ± 070 ng/mL n = 15 No adjustment will be necessary to compare previous 1999–2004 to future 2011–2014 NHANES MMA dataNo specific sources of financial support The findings and conclusions in this report are those of the authors and do not necessarily represent the official views or positions of the Centers for Disease Control and Prevention/Agency for Toxic Substances and Disease Registry Certain commercial equipment instruments or materials are identified in this paper to specify adequately the experimental procedure Such identification does not imply recommendation or endorsement by the National Institute of Standards and Technology nor does it imply that the materials or equipment identified are necessarily the best available for the purposeThe Division of Laboratory Sciences at the National Centers for Environmental Health contributed to this project The authors thank Michael Welch and Lorna Sniegoski from NIST for their involvement in MMA measurements in SRM 1950 They thank Hui Ping Chen and Bridgette Toombs members of Nutritional Biomarkers group at the CDC for their contribution in sample supply for stability and collection tube studies
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