Optimization of Triacylglycerol‐estolide Analysis by Matrix‐Assisted Laser Desorption/Ionization‐Mass Spectrometry

Acylglycerols containing more than three acyl groups (TAG‐estolides) have been reported in plant seed oils and oil from ergot fungus. These TAG‐estolides have considerable potential for industrial use, however, costs of producing synthetic TAG‐estolides limits their use in large‐scale applications. Identification and structural characterization of additional natural sources of TAG‐estolides has been restricted by their complexity and limitations of current analytical techniques. In this work, detection and characterization of TAG‐estolides was optimized for use with MALDI‐TOF‐MS. Eight commonly used matrices were compared; 2,5‐dihydroxybenzoic acid (DHB) and 2,4,6‐trihydroxyacetophenone (THAP) gave good quality mass spectra. Matrix additives were examined and lithium was the most suitable, since MS/MS spectra of lithiated TAG‐estolides provided the most informative fragmentation using an optimized method. The matrix solution pH was examined, and for THAP, replacing LiCl with 10–40 mM LiOH resulted in a slightly basic pH and significantly more intense TAG‐estolide signals (up to eightfold higher). Since DHB is acidic, a larger amount of LiOH (>150 mM) was required for the matrix solution to become basic, leading to ion suppression and reduced signal intensities. Thus, for TAG‐estolide analysis, THAP with ~20 to 30 mM LiOH gives the highest quality spectra and the most informative MS/MS fragmentation.