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