Shotgun Lipidomics Approach to Stabilize the Regiospecificity of Monoglycerides Using a Facile Low-Temperature Derivatization Enabling Their Definitive Identification and Quantitation.

Monoglycerides play a central role in lipid metabolism and are important signaling metabolites. Quantitative analysis of monoglyceride molecular species has remained challenging due to rapid isomerization via α-hydroxy acyl migration. Herein, we describe a shotgun lipidomics approach that utilizes a single-phase methyl tert-butyl ether extraction to minimize acyl migration, a facile low temperature diacetyl derivatization to stabilize regiospecificity, and tandem mass spectrometric analysis to identify and quantify regioisomers of monoglycerides in biological samples. The rapid and robust diacetyl derivatization at low temperatures (e.g., -20 °C, 30 min) prevents postextraction acyl migration and preserves regiospecificity of monoglyceride structural isomers. Furthermore, ionization of ammonium adducts of diacetyl monoglyceride derivatives in positive-ion mode markedly increases analytic sensitivity (low fmol/μL). Critically, diacetyl derivatization enables the differentiation of discrete monoglyceride regioisomers without chromatography through their distinct signature fragmentation patterns during collision induced dissociation. The application of this approach in the analysis of monoglycerides in multiple biologic tissues demonstrated diverse profiles of molecular species. Remarkably, the regiospecificity of individual monoglyceride molecular species is also diverse from tissue to tissue. Collectively, this developed approach enables the profiling, identification and quantitation of monoglyceride regioisomers directly from tissue extracts.