Quantification of aflatoxin and ochratoxin contamination in animal milk using UHPLC-MS/SRM method: a small-scale study

Abstract

Mycotoxin contamination in animal milk is an emerging concern around the globe. Here we developed and validated an ultrahigh-performance liquid chromatography and mass spectrometry-selected reaction monitoring (UHPLC/MS-SRM) method to quantify low concentrations of aflatoxins (AFs) and ochratoxins (OTs) in routinely consumed animal milk samples collected from southern India. Stable isotope dilution methodology was applied to quantify AFB₁, AFB₂, AFG₁, AFG₂, AFM₁, AFM₂ and OTA, OTB in n = 38 different milk samples, using 1 mL of milk. Bioanalytical parameters including method accuracy, precision, recovery, regression analysis and stability were assessed. Dynamic ranges for quantification were between 15.6–1000 pg/mL for AFB₁, AFB₂, AFG₁, and OTA; 7.8–500 pg/mL for AFM₁, AFM₂ and OTB; 78.6–5000 pg/mL for AFG₂. Method accuracy ranged between 80–120%, with ± 15% precision. Recoveries for spiked standards were > 88% in water and 75% in milk, with limits of quantification (LOQ) ranging between 31.3 pg/mL for AFB₁, AFB₂, AFG₁ and OTA, 15.6 pg/mL for AFM₁, AFM₂ and OTB and 156 pg/mL for AFG₂. R² values for regression analyses ranged between 0.9991–0.9999. AFB₂ [mean: 38 pg/mL (0.038 µg/kg)] was quantified in goat milk, AFM₁ was quantified in cow, goat, pasteurized milk [mean: 331 pg/mL (0.331 µg/kg), 406 pg/mL (0.406 µg/kg), 164 pg/mL (0.164 µg/kg)]. Additionally, 90% of cow, goat and pasteurized milk samples were above European Union (EU) limits of 50 pg/mL (0.05 µg/kg) and 40% of cow and goat milk samples were above the Food Safety Standards Authority of India (FSSAI) limit of 500 pg/mL (0.5 µg/kg). AFM₂ was also quantified in cow, goat, and pasteurized milk samples [mean: 249 pg/mL (0.249 µg/kg), 375 pg/mL (0.375 µg/kg), 81 pg/mL (0.081 µg/kg)]. Our dynamic ranges for quantification are lower than other published methods, with need for a smaller volume of milk. This validated method can be applied for routine quantification of mycotoxins in milk.