A validated and optimized method for separation and quantification of total fatty acids by gas chromatography–ion trap mass spectrometry in human plasma

•A GC–IT/MS has been validated to identify and quantify 19 fatty acids simultaneously in plasma.•The method is reliable, sensitive, robust, precise, and accurate.•The method applied to quantify fatty acids in T2DM patients with or without ACS. Fatty acids (FAs) are associated with many physiological functions of tissues, and their alteration has been linked with tissue-specific or systemic diseases. The current situation warrants us to have a sensitive and specific method for analysis of total FAs simultaneously from the biological fluid so that the risk prediction, diagnosis or prognosis of the disease can be made effectively. Because of greater sensitivity and resolution, a method of gas chromatography-ion trap mass spectrometry (GC–IT/MS) has been optimized and validated to quantify simultaneously 19 total FAs levels in plasma and compared with GC–triple quadrupole mass spectrometry. FAs have been transesterified by methanolic acetyl chloride to fatty acid methyl esters (FAMEs). A 65 min GC method separated all 19 FAMEs. The calibration curve had good linearity up to 313–922 μM with a correlation coefficient between 0.9882 and 0.9998. The LODs and LOQs of FAMEs were in the range of 0.63 to 9.55 and 2.12 to 31.8 μM, respectively. The method has recovery up to 144 %, stability at 4 °C for 48 h and one freeze–thaw cycle, and good intra-day and inter-day precision. The optimized method has been used to quantify plasma total FAs in type 2 diabetes mellitus patients with and without acute coronary syndrome. Though a significant difference has been found between IT/MS and triple quadrupole mass spectrometry, the GC–IT/MS can help to quantify total FAs in the clinical setting.