牛血浆中乙酰氨基阿维菌素HPLC-FLD检测方法的建立

Establishment of HPLC-FLD determination method for eprinomectin in bovine plasma

为了研究乙酰氨基阿维菌素(eprinomectin, EPR)缓释注射液在牛体内的药代动力学情况,试验建立了检测牛血浆中EPR浓度的高效液相色谱荧光(HPLC-FLD)法,并进行了系统的方法学验证,向空白牛血浆样品中加入内标阿维菌素(AVM),用乙腈沉淀蛋白质,再用固相萃取柱进行净化,洗脱液浓缩至干后以N-甲基咪唑和三氟乙酸酐对EPR及AVM进行衍生化,采用HPLC-FLD法进行检测。该检测方法所用色谱柱为Elite Hypersil ODS(4.6 mm×250 mm, 5μm);流动相为甲醇∶乙腈∶水=64∶32∶4;流速为1 mL/min;柱温为35℃;荧光检测器激发波长为365 nm,发射波长为463 nm。结果表明:建立的HPLC-FLD检测方法中EPR的保留时间为8.076 min, AVM保留时间为12.922 min。检测限为0.5 ng/mL,定量限为1 ng/mL。血浆样品中EPR浓度在1~100 ng/mL之间时,血浆药物浓度与峰面积之比呈良好的线性关系,线性方程为y=0.912x+0.924,相关系数(R2)为0.994 7。准确度为80.79%~119.20%,批内相对标准偏差为2.02%~11.87%,批间相对标准偏差为4.16%~10.83%。不同储存条件下血浆样品中EPR稳定性良好,符合生物样品分析的具体要求。说明试验建立的HPLC-FLD法操作简便,灵敏度高,干扰少,适用于牛血浆中EPR的检测。

In order to perform pharmacokinetic studies of eprinomectin(EPR) extended-release injection in bovine, a high performance liquid chromatography fluorescence method(HPLC-FLD) for the detection of EPR in bovine plasma was established, and its methodological verification was carried out. The internal standard avermectin(AVM) was added to the plasma samples, and the proteins were precipitated by acetonitrile and purified by solid phase extraction column. EPR and AVM were derivated with N-methylimidazole and trifluoroacetic anhydride after the eluent was concentrated up to dryness, and were detected by HPLC-FLD method. The chromatographic column was an Elite Hypersil ODS(4.6 mm×250 mm, 5 μm) with the mobile phase of methanol∶acetonitrile∶water=64∶32∶4 at the flow rate of 1 mL/min and the column temperature was kept at 35 ℃, and the fluorescence detector was at an excitation wavelength of 365 nm and an emission wavelength of 463 nm. The results showed that the retention time of EPR and AVM was 8.076 min and 12.922 min, respectively. The limit of detection was 0.5 ng/mL, and the limit of quantification was 1 ng/mL. When the EPR concentration in plasma samples ranged from 1 ng/mL to 100 ng/mL, the ratio of plasma drug concentration to peak area showed a good linear relationship. The linear equation was y=0.912x+0.924, and the correlation coefficient(R~2) was 0.994 7. The accuracy was 80.79%-119.20%;the intra-assay RSD was 2.02%-11.87%, and the inter-assay RSD was 4.16%-10.83%. The stability of EPR in plasma under different storage conditions was good, which met the specific requirements of biological sample analysis. The results indicated that the established HPLC-FLD method was simple, sensitive with less interference, and suitable for the detection of EPR in bovine plasma.