A High-throughput Method For The Determination Of Aflatoxin B₁, B₂,G₁, G₂, Ochratoxin A And Sterigmatocystin In Traditional Chinese Medicines By High Performance Liquid Chromatography-tandem Mass Spectrometry

ObjectiveTo establish a high-throughput method for simultaneous determination of aflatoxin B1, B2, G1, G2, ochratoxin A and sterigmatocystin in traditional Chinese medicines (TCMs) by high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) and estimate the contamination status of real samples of TCMs in Guangzhou, so as to provide important data for the setting of legal maximum limit for mycotoxins in TCMs.MethodsTwo representative TCMs named Semen Armeniacae Amarae and Radix Pseudostellariae were selected to develop the method. To overcome the matrix effect and improve the accuracy of the determination of LC-MS/MS, special focus was placed on some effective processes, including improving the chromatographic separation, the ionization efficiencies of six mycotoxins and reducing the concentration of interferences by dilution procedure. After validation of the method, its applicability to other37TCMs and3foods were investigated by calculating the matrix effect (ME). Moreover, comparison to clean-up procedure using Mycosep226purification columns was also conducted with emphasis on the recovery of aflatoxins. Finally, the established method was used to evaluate the contamination levels of six mycotoxins in365TCM samples and16food samples randomly collected from the markets in Guangzhou.ResultsThe established method is based on a single extraction step using84:16(v/v) acetonitrile-water then analysis of the diluted crude extract without further clean-up procedure. Chromato graphic separation was achieved on a Ci8column with a mobile phase gradient prepared from aqueous4mmol/L ammonium acetate-0.1%formic acid and methanol as the mobile phase. Quantification of the analytes was done by selective reaction monitoring (SRM) on a triple quadrupole mass spectrometer in positive ionization mode (ESI+). The method was validated by determination of linearity (r>0.995), sensitivity (limits of quantification1.6-25.0ng/L), apparent recovery (84.8%-110.6%), extraction recovery (83.6%-106.1%) and precision (relative standard deviation<9.9%) for two representative TCMs. Among the other37TCMs tested,23TCMs were screened with acceptable ME (80.2%-118.6%), while14TCMs were found to have one or two mycotoxins showing obvious matrix effect (lower than80%or above120%). In the comparison experiment, recoveries obtained by using Mycosep226purification column were lower than the novel method reported in this study, especially at medium and low level of aflatoxins (<80%) in Radix Pseudostellariae, which indicated the accuracy, rapid and convenience of the latter method.Finally, the validated method was carried out to evaluate mycotoxin contamination in total365samples of39TCMs collected from local hospitals and TCM pharmacies. Aflatoxin B1and ochratoxin A were detected with contamination rate7.9%and4.4%, respectively. In contrast, sterigmatocystin was the most prevalent mycotoxin contaminant (23.8%) in the samples tested, which however was not stressed in previous reports.Furthermore, the extension of this method to3foods showed that the entire matrix effects were between82.1%and118.3%, indicating the method could be applied to high-throughput determination in these3categories of foods. The analysis of real samples results illustrated that the most prevalent mycotoxins was aflatoxin B1(contamination rate25%) followed by sterigmatocystin, aflatoxin B2and aflatoxin G2(18.8%,12.5%and6.3%), whilst all the food samples were free from aflatoxin G1and OTA.ConclusionA rapid and cost-effective LC-MS/MS method has been developed for simultaneous determination of aflatoxin B1, B2, G1, G2, ochratoxin A and sterigmatocystin in39TCMs and3foods, with a one-step solvent extraction without any clean-up step. This quantitative method has many advantages including simple pretreatment, high-throughput avoiding-cost-consuming and error-prone clean-up procedure proved by comparing to Mycosep226aflatoxin-specific purification column. It could be applied to the detection and quantification of these six mycotoxins in routine analysis of TCMs, in which a large number of samples must be analyzed. The prevalent contamination of sterigmatocystin (a precursor of aflatoxins and a member of group2B of possible carcinogen) implied special attention should be paid to evaluate the potential hazard caused by sterigmatocystin in TCMs in greater depth and breadth, since this contaminant has not been stressed in previous reports.