A simple and sensitive methodology for quantification of aflatoxins in milk and butter by HPLC: Method development, validation and application to real samples

Abstract

Aflatoxins (AFTB1, AFTB2, AFTG1, AFTG2 and AFTM1) are toxic and carcinogenic compounds produced by Aspergillus species. They can accumulate in animal-derived foods such as milk and dairy products, posing a serious threat to public health. In this study, sensitive high-performance liquid chromatography (HPLC) and a modified QuEChERS extraction method with simple process steps and using smaller sample and chemical volumes were developed and validated for the detection of aflatoxins (AFTB1, AFTB2, AFTG1, AFTG2 and AFTM1) in cow milk and butter samples. Simple sample preparation was carried out, including only protein precipitation with acetonitrile, followed by phase separation induced by adding potassium chloride. HPLC separation was carried out at 60◦C using a C18 analytical column (Zorbax Eclipse Plus, 250 mm × 4.6 mm, 5 µm). The mobile phase consisted of ultra-pure water (H₂O) with potassium bromide (4 M nitric acid, pH: 3.40) and acetonitrile:methanol (50:50), delivered in a gradient mode at a flow rate of 1 ml/min. The fluorescence detector (FLD) was set at an excitation wavelength of 360 nm and an emission wavelength of 435 nm. The elution of all five analytes was completed within 21 min. The method provides values for the linearity (R2 > 0.998) and recovery rates (89.15 % to 95.14 % for milk and 50.81 % to 86.08 % for butter), the limits of detections (LOD, 0.004–0.008 ng/g for milk and butter), the limits of quantifications (LOQ, 0.012–0.025 ng/g for milk and butter), precision (RSD < 10 %), repeatability (RSD values 1.30–6.08 % for milk and 2.32–6.39 % for butter) and reproducibility (RSD values 5.03–11.09 % for milk and 6.38–11.99 % for butter). This developed and validated method offers an alternative option for the simultaneous analysis of multiple aflatoxin residues in milk and butter samples intended for human consumption, through its fewer processing steps, lower volume and use of smaller variety of chemicals. Unlike other methods in the literature, this technique uses very small sample amounts, such as 2 g. Furthermore, this method was applied to real milk (106 raw and 32 pasteurised milk) and 103 butter samples. While these findings demonstrate that the technique can be applied to the detection of aflatoxin residues in milk and butter, the limitations of the butter matrix must be considered.