An HPLC assay for sn-1,2-diacylglycerol

A method for the analysis of 1,2-diacylglycerols in biological samples is presented. After tissue extraction and derivatisation with 3,5-dinitrobenzoyl chloride, samples are analysed by normal phase HPLC, using a 3.9 x 300 mm microPorasil column, and ultraviolet detection at 254 nm. The method gives quantitative recovery of 1,2-diacylglycerol, and is of sufficient sensitivity to allow quantitation of 1,2-diacylglycerol in human muscle needle biopsy specimens, from as little as 10 mg muscle. Human skeletal muscle from fasted control subjects was found to have a 1,2-diacylglycerol content of 455 +/- 78 nmol/g wet weight. The method is robust, giving intra- and inter-assay coefficients of variation of 2.9% and 5.9%, respectively, and should prove useful for the analysis of 1,2-diacylglycerol levels in human disease states, such as diabetes, in which no measurements of 1,2-diacylglycerol have yet been undertaken.