ELECTROSPRAY AND TANDEM MASS-SPECTROMETRIC CHARACTERIZATION OF ACYLGLYCEROL MIXTURES THAT ARE DISSOLVED IN NONPOLAR-SOLVENTS

This paper presents electrospray mass spectrometric analysis of mixtures containing monoglycerides, diglycerides, and triglycerides. Sample compounds were dissolved in concentrations of 1-50 pmol/mu-L in chloroform:methanol (70:30, v:v), which was modified by the addition of alkali-metal or ammonlum salts or by addition of formic acid to favor the addition of a cationic species to the sample molecules. Electrospray mass spectrometric analysis of acylglycerol standards yielded positive-ion current signals for (M + Na)+ or (M + NH4)+ of all the species that were present at low picomole per microliter concentrations with no fragmentation. For equimolar concentrations of these sample compounds, there was a general decrease in ion current response as the analyte polarity decreased. Therefore, acylglycerols that contained unsaturated fatty acid chains were observed to exhibit a response in the mass spectrum greater than those with saturated chains, and ion signals resulting from the molecular adduct ions of monoglycerides were more abundant than those of diglycerides, which were more abundant than those of triglycerides in the mass spectrum. Electrospray mass spectrometric analysis of an unknown lipid material recovered from a mammalian cell culture reactor revealed a mixture of triglycerides containing mostly C-14, C-16, and C18 fatty acids with varying degrees of unsaturation. The results obtained by electrospray mass spectrometry compared favorably to those obtained by gas chromatography after saponification and methylation of fatty acid components of the triglycerides. MS/MS fragmentation of sodiated acylglycerols required a dissociation energy significantly greater than that required for fragmentation of ammoniated acylglycerols, so MS/MS characterization of acylglycerols was generally performed on the ammonlated compounds. The most abundant product ions that were formed from the ammoniated acylglycerols resulted from loss of fatty acids, and the acylium ions of the fatty acids were also present in the MS/MS spectra. If the MS/MS collision energy was set at greater than 100 eV, fragmentation of the carbon-carbon bonds of the fatty acid chains was evident in the low-mass region of the mass spectrum, but locations of double bonds could not be distinguished because the site of unsaturation apparently migrated during the collisions. Application of nonpolar solvents to the dissolution and electrospray mass spectrometric analysis of other nonpolar compound classes should follow from the methodology presented in this paper.