A Validation and Estimation of Total Eicosapentaenoic and Docosahexaenoic acids Using LC-MS/MS with Rapid Hydrolysis Enzymatic Method for Hydrolysis of Omega Lipids in Human Plasma and its Application in the Pharmacokinetic Study

Background: In this study, we have developed a novel, rapid enzymatic hydrolysis method for conversion of omega lipids (omega fatty acid triglycerides, phospholipids, omega conjugates) in to free fatty acids at room temperature using lipase and esterase enzymes. Objective: To develop simple enzymatic hydrolysis and rapid sample extraction method for quantification of free (un-esterified) and conjugated (esterified) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) to provide the total EPA and DHA lipids present in human plasma. Quantification of total EPA/DHA was performed using liquid chromatography and tandem mass spectrometer instrument. Methods: The plasma sample is digested with lipase and esterase enzymes and extracted by using combined precipitation and liquid-liquid techniques. The LC-MS/MS method was optimized using EPA-D5 and DHA-D5 as labeled internal standards for EPA/DHA respectively. The analytical method is validated, utilized for simultaneous quantification of total EPA and DHA lipids in plasma collected from healthy human volunteers clinical study. Results: The reproducibility of the established enzymatic hydrolysis method was demonstrated by incurred sample reanalysis and the results for total EPA and DHA lipid were 93.33% and 96.67% respectively. The pharmacokinetic and statistical analysis was performed using baseline corrected concentration of total EPA and DHA lipids. Conclusion: The enzymatic hydrolysis method for conversion of omega fatty acid triglycerides, phospholipids, omega conjugates in to free fatty acid was reported first time for the quantitative application. The shorter time for sample workup procedure, simple enzymatic hydrolysis at room temperature and 3 minutes chromatography run time are well suitable for bioavailability/ bioequivalence studies.