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目的:应用LC/APCI-MS法检测大鼠血浆中海狗油的二十碳五烯酸(EPA)、二十二碳六烯酸(DHA)、二十二碳五烯酸(DPA)3个主要成分,为其药动学研究提供灵敏、准确和实用的方法。方法:取血浆样品0.1 mL经甲醇-正己烷(1∶1)沉淀蛋白并萃取后,氮气吹干后进行柱前转酯化衍生,采用Waters C18色谱柱(150 mm×4.6 mm,5μm)分离,以乙腈-水(9∶1)为流动相,流速0.3 mL·min-1,检测波长210 nm,柱温40℃。通过大气压化学电离四极杆质谱,以选择离子监测(SIM)方式进行检测。用于定量分析的EPA甲酯(EPAM)、DHA甲酯(DHAM)、DPA甲酯(DPAM)、十九碳二烯酸甲酯(内标物)的m/z分别为317.30[M+H]+、343.30[M+H]+、345.30[M+H]+、309.20[M+H]+。结果:EPAM、DHAM和DPAM的线性范围均为50～10000 ng.mL-1,定量限为50 ng.mL-1,日内、日间RSD均小于6.6%。对海狗油的大鼠药物动力学初步研究结果表明,EPA、DHA和DPA在连续给药第10 d达到稳态浓度,且时间与给药剂量之间没有关联性;大鼠血浆中EPA、DHA和DPA稳态浓度的高低与给药剂量正相关。结论:该法快速灵敏,准确度高,适用于大鼠体内EPA、DHA、DPA的药动学研究。
OBJECTIVE: To detect the levels of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and docosapentaenoic acid (DPA) in seal oil of rat by LC / APCI-MS method The main components for its pharmacokinetic study to provide a sensitive, accurate and practical method. Methods: 0.1 mL plasma samples were precipitated by methanol-n-hexane (1: 1) and extracted. After drying with nitrogen and pre-column derivatization, the compounds were separated on a Waters C18 column (150 mm × 4.6 mm, 5 μm) , The mobile phase was acetonitrile-water (9:1), the flow rate was 0.3 mL · min-1, the detection wavelength was 210 nm and the column temperature was 40 ℃. Atmospheric pressure chemical ionization quadrupole mass spectrometry was used to perform ion selective ion monitoring (SIM). The m / z of EPA methyl ester (EPAM), DHA methyl ester (DHAM), DPA methyl ester (DPAM), and nonadecyl methyl ester (internal standard) for quantitative analysis were 317.30 [M + H ] +, 343.30 [M + H] +, 345.30 [M + H] +, 309.20 [M + H] +. Results: The linear range of EPAM, DHAM and DPAM was 50 ~ 10000 ng.mL-1, and the limit of quantification was 50 ng.mL-1. The intra-day and inter-day RSD were less than 6.6%. The pharmacokinetics of fur seal oil in rats showed that EPA, DHA and DPA reached steady-state concentration on the 10th day of continuous administration, and there was no correlation between time and dosage. The contents of EPA, DHA And DPA steady-state concentration level and the dose is positively correlated. Conclusion: The method is rapid, sensitive and accurate and suitable for pharmacokinetic studies of EPA, DHA and DPA in rats.