Method for Determination of Multi-mycotoxins in Milk: QuEChERS Extraction Modified Followed by HPLC-FL Analysis

The objective was the development and validation of a method for simultaneous mycotoxins determination (aflatoxins B1 - AFB1, G1 - AFG1, G2 - AFG2 and M1 - AFM1, ochratoxin - OTA, and zearalenone - ZEA) in milk by high-performance liquid chromatography with fluorescence detector. A mixture planning was used to define the mobile phase and a fractional factorial design to define a flow rate of 1.4 mL min-1, allowing the subsequent application of a rotational central composite design (CCRD). Through the CCRD, it was possible to obtain the desired resolution of 1.86 and 2.95 between the peaks of AFM1-AFG2 and ZEA-OTA, respectively, using acidification of the mobile phase 0.85%, column temperature 38 degrees C, and injection volume of 40 mu L. In parallel, an extraction method based on the QuEChERS method was developed. The limits of quantification were 0.16, 1.08, 0.01, 0.12, 0.33, and 8.93 mu g center dot kg-1 for AFM1, AFB1, AFG1, AFG2, OTA, and ZEA, respectively. Recovery varied between 73 and 114%. The method was applied to evaluate multi-mycotoxins' occurrence in fluid milk (n = 30). The presence of AFB1, AFG1, AFG2, and AFM1 was detected in 26%, 30%, 93%, and 80% of the samples, respectively; however, the presence of AFM1 was below the maximum limit allowed by Brazilian legislation (0.5 mu g center dot kg-1). OTA and ZEA were below the limit of detection. Thus, the method was capable and efficient in quantifying multi-mycotoxins in fluid milk and can also be applied as a routine method to monitor milk quality.

Automated summary

This study developed and validated a method for the simultaneous determination of multiple mycotoxins in milk using high-performance liquid chromatography. Mixture planning and fractional factorial design were utilized to optimize the mobile phase and flow rate, followed by the application of a rotational central composite design for further optimization. In addition, an extraction method based on the QuEChERS method was developed. The method showed good performance in evaluating the occurrence of mycotoxins in milk samples.