Rapid Quantification of Low-Viscosity Acetyl-Triacylglycerols Using Electrospray Ionization Mass Spectrometry

Acetyl‐triacylglycerols (acetyl‐TAG) possess an sn‐3 acetate group, which confers useful chemical and physical properties to these unusual triacylglycerols (TAG). Current methods for quantification of acetyl‐TAG are time consuming and do not provide any information on the molecular species profile. Electrospray ionization mass spectrometry (ESI–MS)‐based methods can overcome these drawbacks. However, the ESI–MS signal intensity for TAG depends on the aliphatic chain length and unsaturation index of the molecule. Therefore response factors for different molecular species need to be determined before any quantification. The effects of the chain length and the number of double‐bonds of the sn‐1/2 acyl groups on the signal intensity for the neutral loss of short chain length sn‐3 groups were quantified using a series of synthesized sn‐3 specific structured TAG. The signal intensity for the neutral loss of the sn‐3 acyl group was found to negatively correlated with the aliphatic chain length and unsaturation index of the sn‐1/2 acyl groups. The signal intensity of the neutral loss of the sn‐3 acyl group was also negatively correlated with the size of that chain. Further, the position of the group undergoing neutral loss was also important, with the signal from an sn‐2 acyl group much lower than that from one located at sn‐3. Response factors obtained from these analyses were used to develop a method for the absolute quantification of acetyl‐TAG. The increased sensitivity of this ESI–MS‐based approach allowed successful quantification of acetyl‐TAG in various biological settings, including the products of in vitro enzyme activity assays.