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 isotopically labeled fatty acid methyl esters (FAME) is prepared at a ratio of 2:1 and the exact ratio was carefully measured by GC-FID 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 mixture analysis reveals that the isotopic sentinels have been covalently incorporated into the TAG molecular species at <1% deviation from 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 in general to use of combinatorial chemistry for quantitative standards.