Fatty Acid Sentinels as Covalently Bound Randomization Standards for Triacylglycerol (TAG) Quantitative Analysis

RATIONALE. Quantitative analysis of triacylglycerols (TAG) is impeded by a lack of standards and the huge number of potential TAG molecular species that may be present due to the combinatorial nature of glycerolipids. Randomization of acyl groups yields TAG mixtures with profiles predictable from fatty acid profiles, however their use as calibration mixtures has been limited. METHODS. We introduce here the principle of fatty acid isotopic sentinels that are quantitatively added prior to randomization to enable verification that randomization is complete, and that can be used as internal standards. A mixture of two or moreisotopically labeled fatty acid methyl esters (FAME) are prepared in quantitative proportions and randomized covalently into the acyl groups of TAG mixtures.RESULTS. Reaction with catalytic amounts of NaOCH3 yields complete randomization, such that the product FAME and TAG have the same fatty acid profile. TAG mixtureanalysis reveals that the isotopic sentinels have been covalently incorporated into TAG molecular species and <1% of the expected proportions thus verifying randomization within experimental error.CONCLUSIONS. The sentinel principle demonstrated here as covalently incorporated internal standards verifies that randomization chemistry went to completion. It applies ingeneral to use of combinatorial chemistry for quantitative standards.