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

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 x 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.

Automated summary

This study assessed the influence of different ammonium hydroxide concentrations on triacylglycerol adduct formation and fragmentation under liquid chromatography mass spectrometry analysis. The results showed that increasing ammonium hydroxide concentrations delayed the chromatographic elution time and affected the formation and fragmentation of different adducts. Additionally, a linear equation was derived to quantify the intensity of dehydroxydiacylglycerol fragments relative to the intensity of the ammonium adduct.