Ammonia Concentration in the Eluent Influences Fragmentation Pattern of Triacylglycerols in Mass Spectrometry Analysis
2022
Marta Velasco | David Balgoma | Olimpio Montero
Correct assessment of the fatty acyl at the glycerol <i>sn</i>-2 position in triacylglycerol (TAG) analysis by liquid chromatography and mass spectrometry (LC-MS) is challenging. Ammonium hydroxide (NH<sub>4</sub>OH) is the preferred choice for the solvent additive for the formation of the ammonium adduct ([M + NH<sub>4</sub>]<sup>+</sup>). In this study, the influence of different NH<sub>4</sub>OH concentrations in the eluents on TAG adduct formation and fragmentation under LC-MS analysis was assessed. Increasing NH<sub>4</sub>OH concentrations delayed the chromatographic elution time according to a power function. The [M + NH<sub>4</sub>]<sup>+</sup> and [M + ACN + NH<sub>4</sub>]<sup>+</sup> adducts (where ACN means acetonitrile) were formed at all ammonium concentrations assayed. [M + ACN + NH<sub>4</sub>]<sup>+</sup> predominated above 18.26 mM [NH<sub>4</sub>OH], and the intensity of [M + NH<sub>4</sub>]<sup>+</sup> dropped. TAG fragmentation for fatty acyl release in the MS<sup>E</sup> was reduced with increasing [M + ACN + NH<sub>4</sub>]<sup>+</sup> 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<sub>sn-I</sub> = a × H<sub>[M+NH4]+</sub>, where <i>sn</i>-I refers to the <i>sn</i> 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<sub>4</sub>]<sup>+</sup> intensity in the full scan. This equation had a slope mean value of 0.369 ± 0.058 for the <i>sn</i>-1 and <i>sn</i>-3 positions, and of 0.188 ± 0.007 for the <i>sn</i>-2 position.
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