RP-HPLC/MS-APCI Analysis of Branched Chain TAG Prepared by Precursor-Directed Biosynthesis with Rhodococcus erythropolis

Reversed phase liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (RP-HPLC/MS-APCI) was used to analyze both synthetic triacylglycerols (TAG) having 1-3 branched fatty acids (FA) in the molecule, and natural TAG prepared by precursor directed biosynthesis from valine, leucine and isoleucine and the corresponding branched short-chain acids in cultivations of Rhodococcus erythropolis. The technique made it possible to identify and quantify TAG differing in a single branched-chain FA. Altogether 11 TAG were synthesized, out of which 8 were synthesized stereospecifically. Branched- and straight-chain-TAG were separated and identified while TAG differing only in iso or anteiso FA could not be separated. The APCI mass spectra of iso-, anteiso- and straight-chain TAG were completely identical. The natural material was found to contain 19 TAG having at least one branched FA. Cultivation on six different substrates showed, apart from the presumed and common incorporation of precursors to iso-even, iso-odd and anteiso FA, also some unusual features such as an increase in the content of odd-FA after the addition of Val (attributed to catabolism of Val to propionate) or the appearance of branched monounsaturated FA. The two-sample paired t test, when applied to the TAG, showed that only the pair Val and isobutyrate differ in incorporation into FA-see, e.g. proportions of M/M/O and brM/brM/O (1.2:1.2 and 1.9:1.2, respectively). Also, incorporation of Val (isobutyrate) yielded only TAG having two branched FA in the molecule, whereas Leu and Ile (isovalerate and 2-methyl-butyrate) gave only TAG with a single branched FA in the molecule.