Simultaneous analysis of aflatoxins B1, B2, G1, G2, M1 and ochratoxin A in breast milk by high-performance liquid chromatography/fluorescence after liquid–liquid extraction with low temperature purification (LLE–LTP)

•A HPLC/FLD multi-mycotoxin method in breast milk was developed and validated.•Liquid–liquid extraction with low temperature purification was used.•LOQs ranged from 0.005 to 0.03 ng/mL.•224 breast milk samples were analyzed.•Two samples showed the presence of aflatoxin B2 at the LOQ level (0.005 ng/mL).

The aims of this study were to optimize and validate a methodology for the simultaneous analysis of aflatoxins B1, B2, G1, G2, M1 (AFB1, AFB2, AFG1, AFG2, AFM1) and ochratoxin A (OTA) in breast milk, and to analyze these mycotoxins in samples obtained from human milk banks in the Federal District, Brazil. The optimized analytical method was based on liquid–liquid extraction with low temperature purification (3.25 mL of acidified acetonitrile + 0.75 mL of ethyl acetate), followed by analysis by high-performance liquid chromatography with fluorescence detector (HPLC/FLD) and a photochemical post-column reactor. Limits of quantification (LOQ) ranged from 0.005 to 0.03 ng/mL, recoveries from 73 to 99.5%, and relative standard deviations (RSD) from 1.8 to 17.3%. The LLE–LTP extraction method was shown to be simple and cost-effective, since no columns were needed for clean-up. Only 2 of the 224 breast milk samples analyzed were positive for the mycotoxins, both samples containing AFB2 at the LOQ level (0.005 ng/mL). The identity of the mycotoxin detected was confirmed by liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). This result indicates that infants who are fed with breast milk from the milk banks are not at risk from aflatoxin and ochratoxin exposure.