Mineva Ekaterina M, Zhang Mindy, Rabinowitz Daniel J, Phinney Karen W, Pfeiffer Christine M
Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA, 30341, USA.
Anal Bioanal Chem. 2015 Apr;407(11):2955-64. doi: 10.1007/s00216-014-8148-2. Epub 2014 Sep 26.
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.
甲基丙二酸（MMA）是维生素B12缺乏的功能指标，在1999年至2004年的美国国家健康与营养检查调查（NHANES）中，采用气相色谱/质谱（GC/MS）程序对美国人群进行了测量，该程序需要275μL样本且通量较低（每次运行36个样本）。我们的目标是为2011 - 2014年的NHANES引入一种更高效且高度准确的液相色谱 - 串联质谱（LC - MS/MS）方法。我们对已发表的同位素稀释LC - MS/MS程序进行了一些修改来调整样本制备方法。该程序采用液 - 液萃取并生成MMA二丁酯。等度洗脱的反相色谱法可在4.5分钟内实现MMA与其天然存在的结构异构体琥珀酸的基线分离。我们的新方法提高了通量（每次运行≤160个样本），并且仅用75μL样本就能高灵敏度地测量血清MMA（检测限=22.1nmol/L）。添加到血清中的MMA（2天，4个水平，每个水平重复2次）的平均（±标准差）回收率为94%±5.5%。三个血清质量控制样本池的总不精密度（41天，每个样本重复2次）为4.9% - 7.9%（97.1 - 548nmol/L）。与之前的GC/MS方法相比，LC - MS/MS方法显示出极好的相关性（n = 326，r = 0.99）且无偏差（戴明回归，布兰德 - 奥特曼分析）。与美国国家标准与技术研究院的GC/MS方法程序相比，两种方法在一种未来血浆参考物质上产生的平均（±标准差）MMA浓度几乎相同[LC - MS/MS：18.47±0.71ng/mL（n = 17），GC/MS：18.18±0.67ng/mL（n = 11）] [18.41±0.70ng/mL（n = 15）]。比较之前（1999 - 2004年）和未来（2011 - 2014年）的NHANES MMA数据无需进行调整。
