免疫亲和净化-液相色谱-串联质谱测定中成药中14个真菌毒素

目的: 建立同时测定中成药中黄曲霉毒素B₁、B₂、G₁、G₂,HT-2毒素,T-2毒素,赭曲霉毒素A,伏马毒素B₁、B₂,玉米赤霉烯酮,α-玉米赤霉烯醇,β-玉米赤霉烯醇,α-玉米赤霉醇以及β-玉米赤霉醇共14个真菌毒素的测定方法。 方法: 采用PBS溶液和70%甲醇-PBS溶液依次提取样品中真菌毒素,提取液经多功能免疫亲和柱净化,0.1%吐温-PBS溶液和水依次淋洗,甲醇洗脱,氮气流下浓缩定容,测定时采用Waters Xterra C₁₈ MS (100 mm×2.1 mm,3.5 μm)分析柱,0.3 mL·min^-1的流速,梯度洗脱,质谱测定采用多反应监测(MRM)模式。 结果: 14个真菌毒素的最小检出浓度(LOQ)为1.0~5.0 μg·kg^-1;4个中成药基质的3个不同水平的添加回收率(n=6)为75.7%~97.9%,RSD为5.3%~13.8%。 结论: 本方法采用了多功能免疫亲和柱净化,质谱定性定量,加快了检测速度,降低了中成药复杂基体的干扰,方法检测限满足了国内外中成药多个真菌毒素限量控制要求。

Objective: To develop a method to determine the content of aflatoxin B₁(AFB₁),aflatoxin B₂(AFB₂),aflatoxin G₁(AFG₁),aflatoxin G₂(AFG₂),HT-2 toxin(HT-2),T-2 toxin(T-2),ochratoxin A(OTA),fumonisin B₁(FB₁),fumonisin B₂(FB₂),zearalenone,α-zearalenol,β-zearalenol,α-zearalanol,β-zearalanol in Chinese patent medicines. Methods: The mycotoxins in with HPLC-MS/MS after twice extracted by PBS and methanol and purified by high selective immunoaffinity column.The mycotoxins were extracted by PBS firstly and by 70% methanol secondly.A certain amount of extraction solution was added to immunoaffinity column.The column was washed by 0.1% twain-PBS and water and eluted by methanol.The elution was dried under nitrogen,dissolved in 1 mL solution of mobile phase.The mycotoxins were separated on a Waters Xterra C₁₈ MS(100 mm×2.1 mm,3.5 μm) and the flow rate of gradient elution was 0.3 mL per minute.The MS/MS was performed with multiple reaction monitoring(MRM) mode. Results: After optimization of extraction,separation of HPLC and conditions of mass spectrometry,the determined limits of the 14 mycotoxins were from 1.0 to 5.0 μg·kg^-1.The average recoveries were from 75.7% to 97.9% when spiked at levels of 1.0 μg·kg^-1,5.0 μg·kg^-1 and 10.0 μg·kg^-1.And the relative standard deviation of 6 repeated spiking experiments ranged from 5.3% to 13.8%. Conclusion: The method is rapid,sensitive and accurate,and suitable for determining these 14 mycotoxins in Chinese patent medicine.The detection limits of aflatoxins can meet the requirement of domestic and foreign limited.