Analysis of methylcitric acid by enantioselective multidimensional gas chromatography-mass spectrometry

Methylcitric acid (2-hydroxybutane-1,2,3-tricarboxylic acid-MCA) is a struc tural analogue of citric acid, but due to an additional methyl group it is a chiral molecule with two stereogenic centers and thus four stereoisomers are conceivable, MCA occurs naturally as prominent metabolite in body fluid s of patients with inherited metabolic diseases such as propionic acidemia, methylmalonic aciduria, or holocarboxylase synthetase deficiency, Therefor e methylcitric acid is considered to be an important diagnostic marker for these diseases. MCA is most likely produced from accumulated propionyl-CoA in these diseases by the enzyme si-citrate synthase from the citric acid cy cle; however, there are other enzymes known which could catalyze the same r eaction with different stereoselectivity, such as re-citrate synthase or th e more specific enzyme methylcitrate synthase, found in microorganisms. Almost all methods dealing with MCA in the literature are non-enantioselect ive, For that reason there is no information about occurrence of MCA enanti omers in healthy people, patients with propionic acidemia, methylmalonic ac iduria, or holocarboxylase synthetase deficiency and about value of enantio meric distribution for diagnosis and long-term treatment. The enantioselective analysis of MCA as corresponding trimethyl ester was a chieved by enantioselective multidimensional gas chromatography coupled wit h mass spectrometry using heptakis-(2,3-di-O-methyl-6-O-tert-butyl-dimethyl silyl)-beta-cyclodextrin as chiral stationary phase. The described method allows a reliable screening of MCA in complex matrices like urine without time consuming sample preparation and with mass selecti ve detection. During this investigation urine samples from various patients and healthy controls were analyzed, As concluded, MCA is a good diagnostic marker and can be easily measured by the method presented. Only the two st ereoisomers (2S,3R) and (2S,3S) were detectable in patients and healthy con trols. The varying ratios of these stereoisomers cannot presently be correl ated with the health status of patients, although there are some indication s that this might be possible. However, the quantitative levels of MCA, det ermined as the ratio of MCA absolute peak area divided by 1,000 to the crea tinine contents of urine samples in this investigation, showed a dependence on the state of health and MCA would thus also be a possible marker for lo ng-term treatment. Such a substance is of major interest nowadays since the re are different studies searching for such a long-term marker in propionic acidemia or methylmalonic aciduria.