Correct assessment of the fatty acyl at the glycerol sn-2 position in triacylglycerol (TAG) analysis by liquid chromatography and mass spectrometry (LC-MS) is challenging. Ammonium hydroxide (NH4OH) is the preferred choice for the solvent additive for the formation of the ammonium adduct ([M + NH4]+). In this study, the influence of different NH4OH concentrations in the eluents on TAG adduct formation and fragmentation under LC-MS analysis was assessed. Increasing NH4OH concentrations delayed the chromatographic elution time according to a power function. The [M + NH4]+ and [M + ACN + NH4]+ adducts (where ACN means acetonitrile) were formed at all ammonium concentrations assayed. [M + ACN + NH4]+ predominated above 18.26 mM [NH4OH], and the intensity of [M + NH4]+ dropped. TAG fragmentation for fatty acyl release in the MSE was reduced with increasing [M + ACN + NH4]+ adduct, which suggests that ACN stabilizes the adduct in a way that inhibits the rupture of the ester bonds in TAGs. A linear equation (Hsn-I = a × H[M+NH4]+, where sn-I refers to the sn position of the glycerol (I = 1, 2, or 3) and H is the peak height) was deduced to quantify the dehydroxydiacylglycerol fragment intensity in relation to [M + NH4]+ intensity in the full scan. This equation had a slope mean value of 0.369 ± 0.058 for the sn-1 and sn-3 positions, and of 0.188 ± 0.007 for the sn-2 position.
Keywords: mass spectrometry; regioisomery; triacylglycerols.