Resolution and quantitation of diacylglycerol moieties of natural glycerophospholipids by reversed-phase liquid chromatography with direct liquid inlet mass spectrometry

The sn-1,2-diacylglycerol moieties of natural phosphatidylcholines as the tertiary-butyldimethylsilyl ethers were resolved on the basis of their carbon number and degree of unsaturation by high pressure liquid chromatography on reversed-phase C18 columns. Using acetonitrile and propionitrile as eluting solvents and reagent gases, the yields of both quasi molecular and fragment ions were found to vary with the degree of unsaturation and positional distribution of the fatty acids in the diacylglycerol molecules, and appropriate calibration factors were necessary for accurate quantitation. In the absence of pure structural isomer and mixed acid standards, we have determined preliminary calibration factors for total and specific ion current responses, by comparing the peak area ratios obtained by liquid chromatography – mass spectrometry with the weight and mole proportions of molecular species known to be present in the samples from detailed analyses by capillary gas–liquid chromatography on polar liquid phases. It was found that the total chemical ionization current response agreed closely with the weight composition of the molecular species. The relative yields of the (MH – 132)+ ions varied over one- to three-fold, while those of the (MH – RCOOH)+ ions varied over a three- to four-fold range of intensities. After suitable calibration of the relative ion response, it was possible to determine the identity and quantity of all common molecular species in the test diacylglycerophospholipids. Although the derived factors include both chromatographic and mass spectrometrie effects and are obtained with a gradient of reagent gases, they appear to be generally applicable.