Mass spectrometric determination of tert-butyldimethylsilyl derivatized methylmalonic acid

A method for determining the amount of methylmalonic acid in a sample by tandem mass spectrometry, the method comprising:

• (a) adding a deuterated internal standard to a first sample containing methylmalonic acid to form a second sample;
• (b) purifying the second sample by solid phase extraction;
• (c) subjecting the purified sample from step (b) to a derivatizing agent under conditions suitable to generate a derivatized methylmalonic acid;
• (d) subjecting the derivatized methylmalonic acid to liquid chromatography;
• (e) ionizing the derivatized methylmalonic acid by electron impact ionization to generate a precursor ion and fragmenting the precursor ion using collision energy of 3 V or 10 V to generate one or more fragment ions;
• and
• (f) determining the amount of said one or more fragment ions by tandem mass spectrometry;
• wherein the amount of said one or more fragment ions determined in step (f) is related to the amount of methylmalonic acid originally present in the sample, wherein the method is capable of detecting about 9 nMol/L of methylmalonic acid, and wherein the derivatized methylmalonic acid is a tert-butyldimethylsilyl derivatized methylmalonic acid.