高效液相色谱法测定配合饲料中异绿原酸A、B、C含量的研究

A study on the determination of isochlorogenic acid A,B,and C in feed using high performance liquid chromatography

研究建立了一种高效液相色谱法测定饲料中异绿原酸含量的分析方法。使用C 18色谱柱(250 mm×4.6 mm×5μm),流动相为A:乙腈-水-甲酸(250∶750∶1)和B:乙腈-水-甲酸(100∶900∶1),梯度洗脱,流速1.5 mL/min,柱温25℃,检测波长330 nm,进样量10μL。待测饲料样品经60%甲醇溶液提取,蛋白沉淀处理后经MAX混合阴离子交换柱净化,过有机滤膜后上机检测,外标法定量。结果显示3种异绿原酸分离效果良好,无杂峰干扰,在0.05~5μg/mL范围内线性良好。该方法能减少复杂的饲料基质对结果的影响,准确性好,重复性高,当称样量为1.0 g时,异绿原酸A、B、C的最低检出限均为5.0 mg/kg。研究表明,试验建立的高效液相色谱法可有效适用于饲料中异绿原酸A、B、C的精确检测,为异绿原酸A、B、C在饲料中大规模应用和管理提供了技术支持。

This study aims to establish an analytic approach for determining isochlorogenic acid content in feed using high performance liquid chromatography(HPLC).Related parameters are as follows:C 18 chromatographic column(250 mm×4.6 mm×5μm),mobile phases(A:acetonitrile∶water∶formic acid at 250∶750∶1;and B:acetonitrile∶water∶formic acid at 100∶900∶1),gradient elution,1.5 mL/min flow rate,25℃column temperature,330 nm detection wavelength,and 10μL sampling size.After undergoing extraction with 60%methanol solution and albumen precipitation,the sample feed was then purified through MAX mixed anion-exchange column,tested and ESTD(external standard method)quantified after passing the organic filterable membrane.The results indicate 3 isochlorogenic acids are well separated without miscellaneous peak interference and the linearity appears good within the range of 0.05—5μg/mL.The approach in question could reduce the effect of complicated feed substrate on test results and features high accuracy and repeatability.When sampling size is 1.0 g,the limit of detection is 5.0 mg/kg for all three isochlorogenic acids,A,B and C.The study concludes the applicability of the proposed HPLC(high performance liquid chromatography)approach to accurate detection of isochlorogenic acids A,B and C in feed,which would provide technical support for massive application and management of isochlorogenic acids A,B and C in the feed.