药材中黄曲霉毒素的免疫亲和层析净化高效液相色谱法

Determination of Aflatoxins Content in Traditional Chinese Medicine Using Immunoaffinity Column Cleanup and by High Performance Liquid Chromatography

目的建立高效液相色谱法测定中药材砂仁中黄曲霉毒素B1、B2、G1、G2的方法。方法样品经免疫亲和层析净化,色谱柱为Zorbax SB C18柱(4.6mm×150mm,5μm),流动相为甲醇-0.01mol/L的KH2PO(44+6),流量为0.5ml/min,柱温为35℃,激发波长为360nm,发射波长为425nm,柱后衍生反应器温度为70℃,进行HPLC检测。结果黄曲霉毒素B1在12.00～300.00μg/L范围内、黄曲霉毒素B2、G1、G2在24.00～600.00μg/L范围内时,回归方程呈良好的线性关系,检出限分别为0.025、0.085、0.060、0.055μg/L,r≥0.996。该方法的平均回收率为81.7%～101.2%,RSD为0.7%～4.9%。结论该方法灵敏度高,选择性好,方法稳定,可用于中药材中黄曲霉毒素的检验。

Objective To develop a method for the determination of aflatoxins B1, B2, G2 and G： in Chinese herbal medicine. Methods The herbal medicine samples were cleaned up on immnnoaffinity columns and analyzed by high performance liquid chromatography （HPLC） with fluorescence detection. For chromatographic separation, a Zorbax SB Cis （4.6 mm×150 mm,5 μm） column was employed. The separation was carried out as the mobile phase of methanol-0.01 mol/L KH2PO4（ 4＋6 ） with a flow rate of 0.5 ml/min at column temperature of 35 ℃. The reaction tube temperature of posteolumn derivatization system was 70 ℃. The detection was observed by fluorescence with excitation at 360 nm and emission wavelength at 425 nm. Results The calibration curve showed good linearity in the range of 12.00-300.00 μg/L for aflatoxin Bt, and 24.00-600.00 μg/L for aflatoxin B2,Gt and G2. The lowest limits of detection for aflatoxins in Chinese herbal medicine were 0.025 μg/L for AFBI, 0.085 μg/L for AFB2,0.060 μg/L for AFGl,and 0.055 μg/L for AFG2, and the correlation coefficients were ≥0.996. Recoveries were 81.7%-101.2% for aflatoxin B1, B2, G1 and G2 spiked to Fructus amomi, Radix angelicae sinensis, Rhizoma polygonati, Rhizorrti, atractylodis macrocephalae and Rhizorna dioscoreae at the level of 5μg/L, and the relative standard deviations were 0.7%-4.9% in all instances. Conclusion The method has good stability,high sensitivity and high selectivity,and it is applicable to the determination of aflatoxins in Chinese herbal medicine.