UPLC-MS/MS法用于人血浆中DHA和EPA浓度测定及应用  

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

作　　者：向瑾[1] 余勤[1] 梁茂植[1] 南峰[1] 秦永平[1] 

机构地区：[1]四川大学华西医院GCP中心,成都610041

出　　处：《中国新药杂志》2017年第22期2726-2730,共5页Chinese Journal of New Drugs

摘　　要：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 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 C8（50 mm × 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 m L·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·m L-1 for free DHA and EPA and 50 ~ 0. 5 μg·m L-1 for total DHA and EPA. The intra-and inter-assay RSDs were all less than10%. 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 andLDL-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.

关 键 词：二十二碳六烯酸(DHA) 二十碳五烯酸(EPA) 超高效液相色谱-质谱联用法 血脂水平 

分 类 号：R969.4[医药卫生—药理学]