Reversed-phase Liquid Chromatography-atmospheric Pressure Research Articles

Comparative analysis of triacylglycerols from different Stichococcus strains by RP-HPLC/APCI-MS and chiral HPLC

The main analytical benefit of this study is the development of methods enabling a rapid determination of triacylglycerols (TAGs) in different strains of the green alga Stichococcus bacillaris and detailed identification and quan- tification of a complex mixture of natural TAGs by reversed phase liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (RP-HPLC/APCI-MS) and chi- ral HPLC. Neutral ion scan can readily identify, both qualita- tively and semiquantitatively, triacylglycerols containing 16:2 and16:3fattyacids(FAs)inthemolecule.Sincemoredetailed studies in this respect have been stymied by the shortage of 16:2 and 16:3 FAs, we decided to study the alga Stichococcus as a potential new biotechnological source of C16 polyunsat- urated fatty acids (PUFAs). We conclude that the genus Stichococcus is a major producer of C16 PUFAs mostly contained in TAGs. Further, the ratio of regioisomers and enantiomers of TAGs depends on the content of fatty acids; with lowering temperature, the proportion of the achiral TAG increases, and so does the enantiomer ratio from 1:1. Dienoic FAs preferentially esterify position sn-3, whereas in trienoic acids, it is position sn-1.

Regioisomer separation and identification of triacylglycerols containing vaccenic and oleic acids, and α- and γ-linolenic acids, in thermophilic cyanobacteria Mastigocladus laminosus and Tolypothrix sp.

Reversed phase liquid chromatography–atmospheric pressure chemical ionization mass spectrometry (RP-HPLC/APCI-MS) was used for direct analysis of triacylglycerols (TAGs) from different strains of the cyanobacteria Mastigocladus laminosus, Tolypothrix cf. tenuis and Tolypothrix distorta. This technique enabled us to identify and quantify the specific molecular species of TAGs directly from lipid extracts of the cyanobacteria. The regioisomeric series of TAGs having α-linolenic and γ-linolenic and also oleic and cis-vaccenic acids were separated by RP-HPLC and identified by APCI-MS. M. laminosus produced only a few molecular species of TAGs, including both isomers of octadecenoic (oleic and vaccenic) acid, while T. distorta contained tens of molecular species of TAGs having FAs with up to four double bonds (stearidonic acid and including also its positional isomer, i.e. 3,6,9,12-octadecatetraenoic acid) and both positional isomers (α and γ) of linolenic acids. Individual strains of both cyanobacteria exhibited different contents of polyunsaturated fatty acids (Tolypothrix sp.) and different distribution of positional isomers of monoenoic fatty acids in TAGs (M. laminosus).

The genus Dracunculus – A source of triacylglycerols containing odd-numbered ω-phenyl fatty acids

Reversed phase liquid chromatography–atmospheric pressure chemical ionization mass spectrometry (RP-HPLC/MS-APCI) was used to identify and quantify triacylglycerols (TAGs) having odd-numbered ω-phenylalkanoic acids from seeds of the flower plant Dracunculus vulgaris, and TAGs from the bacterium Rhodococcus erythropolis prepared by precursor directed biosynthesis from phenylalanine and having the corresponding even-numbered ω-phenylalkanoic acids. Model compounds, which are not commercially available, were prepared by organic synthesis and this allowed us to extend the number of identified natural TAGs to nearly 140 molecular species. Both synthetic and natural compounds containing ω-phenylalkanoic acids were found to have antioxidant and free radical scavenging properties.

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.

RP-HPLC/MS-APCI analysis of odd-chain TAGs from Rhodococcus erythropolis including some regioisomers

We used reversed phase liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (RP-HPLC/MS-APCI) for direct analysis of triacylglycerols (TAGs) from four different cultivations of Rhodococcus erythropolis CCM 2595. This technique enabled us to identify and quantify the specific molecular species of TAGs directly from lipid extracts of the bacterium, including the determination of their basic characteristics such as retention time and mass spectra. A total of 17 TAGs having at least one odd-numbered-chain FA (fatty acid) were found. This is the first time when odd-chain TAGs, predominantly with pentadecanoic, margaric, and margaroleic acids, were identified. Multiple linear regression analyses were used for predicting retention times of molecular species, predominantly of odd-chain TAGs. Cultivations on two different substrates at two different temperatures (20 and 30 degrees C) showed that only temperature had any effect on the content of TAGs. In cultivations with succinate as a carbon source the content of these TAGs increased by half (i.e., from 23.5 to 34.2%) when the cultivation temperature was lowered from 30 to 20 degrees C. The content of the PoPoMo, PoMoO and PPoMo (see Table 1) TAGs containing the odd-numbered-chain margaroleic acid rose from 0.0 to 7.1% while in cultivation on phenol as a carbon source the lowering of cultivation temperature caused an increase of these TAGs from 0.5 to 6.7%.