Determination of Aflatoxin M1 in Raw Milk Using an HPLC-FL Method in Comparison with Commercial ELISA Kits—Application in Raw Milk Samples from Various Regions of Greece

Abstract

The highly toxic Aflatoxin M1 (AFM1) is most often detected in milk using an Enzyme-Linked-Immunosorbent Assay (ELISA) for screening purposes, while High-Performance Liquid Chromatography with Fluorescence Detector (HPLC-FL) is the reference method used for confirmation. The aim of the present study was the comparison between three commercially available ELISA kits and a newly developed HPLC-FL method for the determination of the AFM1 in milk samples. The developed HPLC-FL method was validated for the AFM1 and Aflatoxin M2 (AFM2), determining the accuracy, precision, linearity, decision limit, and detection capability with fairly good results. All three ELISA kits were also validated and showed equally good performance with high recovery rates. Moreover, the Limit Of Detection (LOD) and Limit Of Quantification (LOQ) values were found to be significantly lower than the Maximum Residue Limit (MRL) (50 ng kg−1). After the evaluation of all three commercial kits, the ELISA kit with the optimum performance along with the HPLC method was used for the determination of AFM1 in raw cow’s, goat’s, and sheep’s milk samples (396) obtained from producers in different regions of Greece. The evaluation of both methods showed that this ELISA kit could be considered as a faster and equally reliable alternative method to HPLC in routine analysis for the determination of AFM1 in milk.

Assessment of aflatoxin M1 and B1 in some dairy products with referring to the analytical performances of enzyme-linked immunosorbent assay in comparison to high-performance liquid chromatography

“…The most commonly used analytical methods for the quantification of AFM 1 in milk include TLC, HPLC with a fluorescence detector (HPLC-FL), and the ELISA [50]. Notwithstanding its extensive and time-consuming sample preparation that necessitates the use of numerous chemical solvents, HPLC-FL is currently the most accurate method [51][52][53]; however, ELISA gives quick and sensitive results, cost-effective, and requires small sample volumes and fewer preparation procedures. Therefore, ELISA can be a reliable alternative to HPLC-FL and a preferred method at the routine level and in research studies [53].…”

“…Notwithstanding its extensive and time-consuming sample preparation that necessitates the use of numerous chemical solvents, HPLC-FL is currently the most accurate method [51][52][53]; however, ELISA gives quick and sensitive results, cost-effective, and requires small sample volumes and fewer preparation procedures. Therefore, ELISA can be a reliable alternative to HPLC-FL and a preferred method at the routine level and in research studies [53]. HPLC-FL as a reference method is used for confirming the obtained ELISA results.…”

“…HPLC-FL as a reference method is used for confirming the obtained ELISA results. Especially due to cross-reaction interferences, particularly at concentrations <50 ng/L, the ELISA method may not be completely reliable as it is resulting in false-positive or false-negative results [53,54].…”

Aflatoxin M1 in traditional and industrial pasteurized milk samples from Tiran County, Isfahan Province

“…AFM1 stock solution (50 mg/L) was obtained by solubilizing the AFM1 standard powder (Sigma Aldrich, Germany) in a chloroform/methanol solution with a volume ratio (v/v) of 19:81 and kept at a temperature of -20°C. Before analysis, by chloroform:methanol at a 1:1 v/v ratio, the stock solution to obtain different standard solution concentrations was diluted (Fallah, 2010;Maggira et al, 2021;Tajik et al, 2016). AFM1 analysis was carried out according to the instructions given in the kit.…”

Validation of New ELISA Technique for Detection of Aflatoxin B1 Contamination in Food Products versus HPLC and VICAM

“…VICAM was less sensitive than HPLC analysis for aflatoxin AFB1 detection in several samples-peanut 2, flours 2 and 3, and milk-powder 3. HPLC is widely used for the analysis of aflatoxins for sensitivity and accuracy [13] (Table 1). HPLC is an excellent quantitative method in detection of aflatoxins [14], although it requires skilled operators, extensive sample preparation, and is a high-cost equipment [15].…”