Determination of DHA and EPA in human plasma by UPLC-MS/MS

XIANG Jin; YU Qin; LIANG Mao-zhi; NAN Feng; QIN Yong-ping

Abstract：Objective:To establish a UPLC-MS/MS method to determine docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) concentrations in volunteers' plasma after administration of fish oil soft capsules.Methods:The method was established with an Acquity UPLC BEH Cs (50 mm x 2.1 mm,1.7 μm) column.The mobile phase A was 0.005％ ammonia and the mobile phase B was acetonitrile with the flow rate set at 0.4 mL·min-1 The column temperature was maintained at 40 ℃.The MRM detection ions were m/z 327.28/283.09,301.47/257.49 and 350.20/306.09 for DHA,EPA and the internal standard,respectively.Samples for determination of free DHA and EPA were directly injected into the system after protein deposition with acetonitrile.Samples for determination of total DHA and EPA were injected into the system after protein deposition with acetonitrile subsequent to hydrolyzation.Thirty healthy male volunteers were enrolled in the study and orally administered fish oil soft capsules for 15 d.Concentrations of free and total DHA and EPA and serum lipid concentrations were determined before administration on 0,8 and 15 d.Results:The retention time of DHA,EPA and IS were 1.1,1.0 and 0.9 min,respectively.The linear ranges of the calibration curves were within 2 ～ 0.02 μg· mL-1 for free DHA and EPA and 50 ～0.5 μg·mL-1 for total DHA and EPA.The intra-and inter-assay RSDs were all less than 10％.The absolute recovery was 91.00％～ 102.94％ and the matrix effect was 3.14％～ 8.10％.The concentrations of both free and total DHA and EPA significantly increased on d 8 and d 15 (P ＜ 0.05),with decreased plasma TG concentration and increased HDL-C concentration.There was no significant difference between d 8/d 15 TC and LDL-C and baseline TC and LDL-C.Conclusion:The method is simple,accurate,and suitable for detection of DHA and EPA concentration in human plasma.