Ammonia Concentration in the Eluent Influences Fragmentation Pattern of Triacylglycerols in Mass Spectrometry Analysis.

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 (NHOH) is the preferred choice for the solvent additive for the formation of the ammonium adduct ([M + NH]). In this study, the influence of different NHOH concentrations in the eluents on TAG adduct formation and fragmentation under LC-MS analysis was assessed. Increasing NHOH 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 [NHOH], 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.