The direct determination of double bond positions in lipid mixtures by liquid chromatography/in-line ozonolysis/mass spectrometry

The direct determination of double bond positions in unsaturated lipids using in-line ozonolysis-mass spectrometry (O₃-MS) is described. In this experiment, ozone penetrates through the semi-permeable Teflon AF-2400 tubing containing a flow of a solution of fatty acid methyl esters (FAME). Unsaturated FAME are thus oxidized by the ozone and cleaved at the double bond positions. The ozonolysis products then flow directly into the atmospheric pressure photoionization (APPI) source of a mass spectrometer for analysis. Aldehyde products retaining the methyl ester group are indicative of the double bond positions in unsaturated FAME. For the first time, O₃-MS is able to couple directly to high performance liquid chromatography (HPLC), making the double bond localization in lipid mixtures possible. The application of LC/O₃-MS has been demonstrated for a fat sample from bovine adipose tissue. A total of 9 unsaturated FAME including 6 positional isomers were identified unambiguously, without comparison to standards. The in-line ozonolysis reaction apparatus is applicable to most mass spectrometers without instrumental modification; it is also directly compatible with various LC columns. The LC/O₃-MS method described here is thus a practical, versatile and easy to use new approach to the direct determination of double bond positions in lipids, even in complex mixtures.