黑茶茶汤中赭曲霉毒素A的提取与检测方法

日常饮茶中，茶叶若因加工或贮藏不当污染了一定量霉菌毒素，同时对于冲泡过程中的浸出率及实际的摄入量，仍欠缺深入了解。为建立茶汤中赭曲霉毒素A（OTA）的提取方法，为饮茶所摄入生物毒素的安全评估提供更合理的分析方法，研究使用乙腈作为提取溶剂提取茶汤中的OTA，然后加入盐离子使乙腈和水相分层，去除杂质后，以高效液相色谱法（HPLC）分析检测，通过对比4种不同方法的准确度、线性、精密度、灵敏度等指标，选择最合适于茶汤的检测方法。结果表明，方法M4通过用含0.1%甲酸酸化乙腈提取，用氯化钠和硫酸镁使乙腈和茶汤分层，在准确度、精密度、灵敏度等指标上，均优于其他3种方法。方法M4在茶汤中OTA添加浓度为1～45 µg·L^-1时回收率为82.5%～99.8%，标准偏差为1.4%～7.3%；该方法添加回收的回归方程相关系数为0.9971。在日间和日内验证试验中，高浓度毒素组日内回收率为90.9%，相对标准偏差为2.2%，日间回收率为87.5%，相对标准偏差为3.2%；低浓度毒素组的日内回收率为91.9%，相对标准偏差为5.2%，日间回收率为92.4%，相对标准偏差为6.7%；该方法的定量限为1.1 µg·L^-1，检出限为0.33 µg·L^-1。不同类型黑茶茶汤验证实验发现，该方法的回收率均高于88.65%。对比这4种方法，方法M4在分析结果上均好于其他3种方法，适合用于茶汤中OTA的检测，该方法对于OTA在茶水冲泡过程中浸出情况及安全风险评估有重要的参考意义。

Extraction and detection of ochratoxin A in dark tea infusions

In daily tea drinking, the tea leaves may be contaminated with a certain amount of mycotoxins due to improper processing or storage, meanwhile, we still lack of understanding of the mycotoxin migration rate and actual intake during the tea brewing process. Therefore, we aim to establish a method for the extraction and detection of ochratoxin A (OTA) in tea infusions, so as to make an appropriate safety assessment of biotoxins ingested through tea infusion. In this study, acetonitrile was used as major solvent to extract OTA in tea infusions, and different salt ions were added to separate acetonitrile and aqueous phase. The supernatant was cleaned and analyzed by HPLC (high performance liquid chromatography), four extraction methods were established and compared, and the most appropriate method was selected by evaluation of the accuracy, linearity, precision, sensitivity and other indicators. As results, method 4, which was extracted with acetonitrile containing 0.1% formic acid and separated the acetonitrile and aqueous phase with sodium chloride and magnesium sulfate, obtained the best results. Method validation of method 4 showed that recovery was between 82.5% and 99.8%, the standard deviation was 1.4% - 7.3%, and the correlation coefficient of the determination method was 0.997 1. In high-concentration fortified group, the intra-day recovery was 90.9%, and the relative standard deviation (RSD) was 2.2%; the inter-day recovery was 87.5% and the RSD was 3.2%. In low-concentration fortified group, the intra-day recovery was 91.9% and the RSD was 5.2%; the inter-day recovery was 92.4% and the RSD was 6.7%. The limit of quantification of the method was 1.1 µg·L^-1, and the detection limit was 0.33 µg·L^-1. Evaluation results of different types of dark tea infusions showed that the recovery rates of the method were all higher than 88.65%. In conclusions, by comparison of the four