Core Unit Proteomics, Institute of Toxicology, Hannover Medical School, 30625 Hannover, Germany.
Hypertension in Africa Research Team (HART), North-West University, Potchefstroom 2531, South Africa.
Molecules. 2022 Aug 15;27(16):5202. doi: 10.3390/molecules27165202.
Methylmalonic acid (MMA) is a very short dicarboxylic acid (methylpropanedioic acid; CHCH(COOH); K, 3.07; K, 5.76) associated with vitamin B deficiency and many other patho-physiological conditions. In this work, we investigated several carboxylic groups-specific derivatization reactions and tested their utility for the quantitative analysis of MMA in human urine and plasma by gas chromatography-mass spectrometry (GC-MS). The most useful derivatization procedure was the reaction of unlabeled MMA (d-MMA) and trideutero-methyl malonic acid (d-MMA) with 2,3,4,5,6-pentafluorobenzyl bromide (PFB-Br) in acetone. By heating at 80 °C for 60 min, we observed the formation of the dipentafluorobenzyl (PFB) ester of MMA (CHCH(COOPFB)). In the presence of -diisopropylamine, heating at 80 °C for 60 min resulted in the formation of a tripentafluorobenzyl derivative of MMA, i.e., CHCPFB(COOPFB)). The retention time was 5.6 min for CHCH(COOPFB) and 7.3 min for CHCPFB(COOPFB)). The most intense ions in the negative-ion chemical ionization (NICI) GC-MS spectra of CHCH(COOPFB) were mass-to-charge () 233 for d-MMA and 236 for d-MMA. The most intense ions in the NICI GC-MS spectra of CHCPFB(COOPFB) were mass-to-charge () 349 for d-MMA and 352 for d-MMA. These results indicate that the H at C atom at position 2 is C-H acidic and is alkylated by PFB-Br only in the presence of the base -diisopropylamine. Method validation and quantitative analyses in human urine and plasma were performed by selected ion monitoring (SIM) of 349 for d-MMA and 352 for the internal standard d-MMA in the NICI mode. We used the method to measure the urinary excretion rates of MMA in healthy black ( = 39) and white ( = 41) boys of the Arterial Stiffness in Offspring Study (ASOS). The creatinine-corrected excretion rates of MMA were 1.50 [0.85-2.52] µmol/mmol in the black boys and 1.34 [1.02-2.18] µmol/mmol in the white boys ( = 0.85; Mann-Whitney). The derivatization procedure is highly specific and sensitive for MMA and allows its accurate and precise measurement in 10-µl of human urine by GC-MS.
甲基丙二酸（MMA）是一种非常短的二羧酸（甲基丙二酸；CHCH（COOH）；K，3.07；K，5.76），与维生素 B 缺乏和许多其他病理生理状况有关。在这项工作中，我们研究了几种羧酸基团特异性衍生化反应，并测试了它们在气相色谱-质谱法（GC-MS）定量分析人尿和血浆中 MMA 的实用性。最有用的衍生化程序是未标记的 MMA（d-MMA）和氘代甲基丙二酸（d-MMA）与 2,3,4,5,6-五氟苄基溴（PFB-Br）在丙酮中的反应。在 80°C 加热 60 分钟，我们观察到 MMA 的二（五氟苄基）（PFB）酯（CHCH（COOPFB））的形成。在 -二异丙基胺存在下，在 80°C 加热 60 分钟导致 MMA 的三（五氟苄基）衍生物的形成，即 CHCPFB（COOPFB））。CHCH（COOPFB）的保留时间为 5.6 分钟，CHCPFB（COOPFB）的保留时间为 7.3 分钟。CHCH（COOPFB）的负离子化学电离（NICI）GC-MS 谱中最强烈的离子是质量-电荷（m/z）为 233 的 d-MMA 和 m/z 为 236 的 d-MMA。CHCPFB（COOPFB）的 NICI GC-MS 谱中最强烈的离子是 m/z 为 349 的 d-MMA 和 m/z 为 352 的 d-MMA。这些结果表明，C 原子上位置 2 的 H 是 C-H 酸性的，并且仅在存在碱 -二异丙基胺的情况下才被 PFB-Br 烷基化。通过在 NICI 模式下对 349（用于 d-MMA）和 352（用于内部标准 d-MMA）进行选择离子监测（SIM），对人尿和血浆中的方法进行了验证和定量分析。我们使用该方法测量了动脉僵硬后代研究（ASOS）中健康黑（n=39）和白（n=41）男孩的 MMA 尿排泄率。黑人男孩的 MMA 肌酐校正排泄率为 1.50[0.85-2.52]µmol/mmol，白人男孩为 1.34[1.02-2.18]µmol/mmol（=0.85；Mann-Whitney）。衍生化程序对 MMA 具有高度特异性和灵敏度，允许通过 GC-MS 对 10-µl 人尿中的 MMA 进行准确和精确的测量。
