Efficient GC/MS analysis of hydroxy lipid compounds from geochemical samples using tertiary-butyldimethylsilyl etherification

In order to improve GC/MS analysis of hydroxy compounds from various sources, such as alkanols, sterols, diols, keto-ols and hydroxy fatty acids (FAs) with complex compositions, we evaluated their column-chromatographic separation after silylation. N-tertiary-butyldimethylsilylimidazole (t-BDMS) was expected to be the most effective silylating reagent to make –OH groups more inert and hence to promote separation according to the chemical and structural differences in their individual alkyl groups. Results indicate that the t-BDMS etherification of hydroxy compounds remarkably improved their purification and separation by strongly constraining or enhancing the chromatographically different behaviour possibly caused by chemical and structural differences in the alkyl groups. Thus, the complex mixtures of hydroxy compounds from lacustrine and marine sediments were clearly fractionated into each major compound group, i.e., alkanols, sterols, diols, hydroxy FAs and keto-ols. Moreover, the individual sterols, diols and hydroxy FAs were separated into four, two and five specific sub-groups, respectively. It is worth noting that, by using only one column, this analytical improvement was achieved with excellent reproducibility. Such fine fractionations resulted in a substantial decrease in the complexity of the gas chromatograms of all major hydroxy compounds. The improvement enabled us to simply and accurately identify and quantify major as well as trace homologues and isomers of various hydroxy compounds with a high level of sensitivity using GC/MS. Consequently, we found some series of hitherto unknown compounds to serve as information sources for organisms and environments. The chromatographic fractionation following t-BDMS etherification of lipids from various geochemical samples provides not only a greater understanding of their molecular distributions and stable carbon isotopic ratios, but also substantially upgrades lipid analytical efficiency as a whole.