Carvalho Valdemir Melechco, Kok Fernando
Fleury Medicine and Health, São Paulo 04344-070, Brazil.
Anal Biochem. 2008 Oct 1;381(1):67-73. doi: 10.1016/j.ab.2008.06.023. Epub 2008 Jun 20.
Despite the new advances in bioanalytical techniques, the analysis of low-molecular-weight organic acids in complex matrices is still a challenge. Although new strategies applying liquid chromatography-tandem mass spectrometry (LC-MS/MS) seem to be promising, sample preparation methodologies hamper its application in most clinical laboratories. The quantitation of methylmalonic acid (MMA) in biological matrices is an emblematic example due to its low concentration, the need for derivatization to increase its molecular weight, and the presence of the physiologically more abundant isomer succinic acid. Here we present a new strategy for rapid and sensitive MMA quantitation by combining alkylative extraction and LC-MS/MS. Alkylative extraction conditions were optimized to allow endogenous detection of MMA using only 50 microL of serum with a short sample preparation procedure. The formation of a unique ion from the MMA dipentafluorobenzyl derivative in negative atmospheric pressure chemical ionization (APCI) allowed its detection with high sensitivity and with no interference from succinic acid, a more abundant physiologically present isomer.
尽管生物分析技术有了新进展，但在复杂基质中分析低分子量有机酸仍是一项挑战。虽然应用液相色谱 - 串联质谱法（LC-MS/MS）的新策略似乎很有前景，但样品制备方法阻碍了其在大多数临床实验室中的应用。生物基质中甲基丙二酸（MMA）的定量分析就是一个典型例子，这是因为其浓度低、需要衍生化以增加分子量，以及存在生理上含量更高的异构体琥珀酸。在此，我们提出一种通过结合烷基化萃取和LC-MS/MS进行快速灵敏的MMA定量分析的新策略。优化了烷基化萃取条件，仅使用50微升血清并通过简短的样品制备程序就能实现MMA的内源检测。在负大气压化学电离（APCI）中，MMA二五氟苄基衍生物形成独特离子，从而实现了高灵敏度检测，且不受生理上含量更高的异构体琥珀酸的干扰。
