液质联用方法对海狗油大鼠药物动力学的初步研究

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,5 μm)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 ⁺、343.30 ⁺、345.30 ⁺、309.20 ⁺。 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 develop a sensitive and specific liquid chromatography-atmosphere pressure chemical ionization quadrupole mass spectrometry(LC-APCI/MS) method for the determination of eicosapentaenoic acid(EPA),docosahexaenoic acid(DHA),and docosapentaenoic acid(DPA) of harp seal oil in rat plasma,and provide a method for pharmacokinetic study. Methods: EPA,DHA,DPA and internal standard methyl nonadecadienoate were extracted from plasma by protein precipitation,extraction with methanol-hexane(1: 1) and derivation then chromatographed by using an Waters C₁₈ column(150 mm×4.6 mm,5 μm).The mobile phase consisted of acetonitrile-water(9: 1) at the flow rate of 0.3 mL·min^-1,the detection wavelength of 210 nm and the column temperature of 40 ℃ were specified.Atmosphere pressure chemical ionization(APCI) source was applied and operated in the positive ion mode.Selected ion monitoring(SIM) mode with m/z 317.30 ⁺,m/z 343.30 ⁺,m/z 345.30 ⁺ and m/z 309.20 ⁺ was used to qualify eicosapentaenoic acid methyl ester(EPAM),docosahexaenoic acid methyl ester(DHAM),docosapentaenoic acid methyl ester(DPAM) and the internal standard,respectively. Results: The linear calibration curve was obtained in the concentration range of 50-10000 ng·mL^-1,the limit of detection was 50 ng·mL^-1,and the RSDs of intra-day and inter-day were less than 6.6%.The results showed that it took ten days to reach the steady state concentration for EPA,DHA and DPA and no correlation was seen between the time and the dosage.While,significant positive correlation was found between the value of steady state concentration and the dosage. Conclusion: The method offers advantages of high sensitivity and accuracy for the determination of three main poly-unsaturated fatty acids of harp seal oil,and is suitable for the primary pharmacokinetic study of harp seal oil in rats.

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