High-performance liquid chromatography with light-scattering detection and desorption chemical-ionization tandem mass spectrometry of milk fat triacylglycerols

The utility of reverse-phase high-performance liquid chromatography (HPLC), desorption chemical-ionization mass spectrometry (DCI-MS) and tandem mass spectrometry (MS/MS) for the characterization of triacylglycerols in complex mixtures has been further explored. Triacylglycerols of anhydrous bovine milk fat were separated by using two reversephase C₁₈ HPLC columns, and eluents were monitored with an evaporative light-scattering detector. Fifty-eight fractions were resolved and analyzed by positive ion isobutane DCI-MS. The formation of protonated molecules and of major fragments corresponding to the random loss of any one of the constituent fatty acids readily identified acyl carbon numbers and the number of double bonds within each fatty acid. MS/MS was only required when the original mass spectra indicated the presence of more than one triacylglycerol or of impurities in a fraction. Protonated molecules produced by DCI were fragmented using high energy collisional activation, and the resulting ions were detected by MS/MS. Odd-chain triacylglycerols were also readily distinguished using this methodology. The positive ion DCI and MS/MS techniques described here demonstrate the usefulness of this approach for the characterization of triacylglycerols in complex mixtures.