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目的:建立UPLC-MS/MS法测定人血浆中DHA,EPA浓度,并用于受试者口服鱼油软胶囊后DHA,EPA血浆浓度测定。方法:以Acquity UPLC BEH C8(50 mm×2.1 mm,1.7μm)为色谱柱,流动相A为0.005%氨水溶液,B相为乙腈,流速为0.4 m L·min-1,柱温为40℃,DHA,EPA和内标他米巴罗汀的检测离子对分别为m/z 327.28/283.09,301.47/257.49和350.20/306.09。游离型DHA,EPA样品经乙腈沉淀蛋白后进样;总浓度DHA,EPA样品经水解后再由乙腈沉淀蛋白后进样。30例健康男性受试者连续口服鱼油软胶囊15 d,并于0 d,8 d和15 d给药前测定血浆中游离型和总DHA,EPA浓度,以及血脂水平变化。结果:DHA,EPA及内标的保留时间分别为1.1,1.0,0.9 min,游离型标准曲线线性范围为2~0.02μg·m L-1,总浓度标准曲线线性范围为50~0.5μg·m L-1。批内及批间RSD均小于10%,方法回收率为91.00%~102.94%,基质效应为3.14%~8.10%。受试者连续服用8 d,15 d后,血浆中DHA,EPA游离型浓度以及总浓度均较基线明显增加(P<0.05);同时血浆TG浓度降低,HDL-C浓度增高(P<0.05);TC和LDL-C与基线比较降低无统计学意义(P>0.05)。结论:本法简单、快速,可用于血浆中DHA,EPA浓度测定。
OBJECTIVE: To establish a method for the determination of DHA and EPA in human plasma by UPLC-MS / MS, and to determine the plasma concentration of DHA and EPA after orally administered fish oil soft capsules. Methods: The mobile phase A was 0.005% aqueous ammonia solution, the phase B was acetonitrile, the flow rate was 0.4 m L · min-1, and the column temperature was 40 ℃ with a column of Acquity UPLC BEH C8 (50 mm × 2.1 mm, 1.7 μm) , DHA, EPA and the internal standard Tamibarotene test ion pairs were m / z 327.28 / 283.09, 301.47 / 257.49 and 350.20 / 306.09 respectively. Free DHA, EPA samples were acetonitrile after precipitation of protein samples; total concentration of DHA, EPA samples after hydrolysis by acetonitrile precipitated protein injection. Thirty healthy male subjects were orally administered with fish oil soft capsules for 15 days. The levels of free plasma and total DHA, EPA, and blood lipid levels were determined before 0, 8 and 15 days. Results: The retention times of DHA, EPA and internal standard were 1.1, 1.0 and 0.9 min, respectively. The linear range of free standard curve was 2 ~ 0.02μg · m L-1, and the linear range of total standard curve was 50 ~ 0.5μg · m L -1. The intra-and inter-batch RSD were less than 10%, the recovery rates were 91.00% ~ 102.94%, and the matrix effects were 3.14% ~ 8.10%. The levels of DHA, EPA free and plasma concentrations in plasma increased significantly from baseline (P <0.05) on the 8th and 15th day after treatment. Plasma TG concentration and HDL-C concentration increased (P <0.05) ; TC and LDL-C decreased compared with the baseline was not statistically significant (P> 0.05). Conclusion: This method is simple and rapid and can be used for the determination of DHA and EPA in plasma.