Harnessing the Benefits of HPLC with Fluorescence Detection for the Analysis of Aflatoxins in Raw Milk

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Wilhad M. Reuter and Nicola Vosloo, PerkinElmer, Inc., Shelton, Connecticut, USA

This article highlights the analytical advantages of analyzing raw milk for aflatoxins, focusing on M1, using high performance liquid chromatography (HPLC) with fluorescence detection - without derivatization. Leveraging M1’s native fluorescence, this is particularly welcome since this approach affords the requisite analytical sensitivity with minimal method complexity.

Aflatoxins, a type of mycotoxin, are the most carcinogenic natural compounds known to man. They were first identified in the 1960s following the death of over 100,000 turkeys in the UK (1). Produced by the fungus Aspergillus flavis and Aspergillus parasiticus, they consist of about 20 similar compounds, however, only four strains are naturally found in food - B1, B2, G1, and G2 - with B1 as the most common and the most toxic.

Aflatoxins can occur in foods such as groundnuts, maize, rice, corn, nuts, and spices, to name just a few. Pre- and post‑harvest conditions, exposure to moisture, and warm temperatures all impact on the risk and rate of mould formation. When aflatoxin B1 is ingested by animals from contaminated feed, it is converted to aflatoxin M1, which is the predominant aflatoxin found in milk. Although less potent than B1, M1 has been shown to cause liver cancer in certain animals (2). While a concern to all milk consumers, this is a particular red flag to the dairy industry.

Although it is primarily food commodities that become contaminated with aflatoxins, these toxins are very stable during processing, including pasteurization, and, consequently, are also a risk in processed foods, such as peanut butter. If present in raw milk, there is a high degree of certainty that M1 will persist in any final products intended for human consumption. The toxicity and risk to human health has led to over 100 countries setting out regulations relating to the testing and confirmation of aflatoxins. Prevention is preferred to maintain the health safety of the food supply chain and early detection provides a key role in ensuring that this strategy is successful.

Both the Food and Drug Administration (FDA) and the European Union (EU) have set out clear guidelines on the allowable limits of primary aflatoxin in milk, M1. The EU has established a stringent control limit for M1, set at 0.05 ppb in milk (3). This is currently the strictest global control limit, setting a significantly lower level than the FDA’s limit of 0.5 ppb (2).

There are a number of different tests to monitor the levels of aflatoxin M1 in milk, ranging from ELISA to high performance liquid chromatography (HPLC) analyses. HPLC offers exceptional analyte separation because of the LC column’s high selectivity. By coupling HPLC with fluorescence (FL) detection, one is provided with quantitative analyte determination down to very low detection limits as a result of the inherent sensitivity of fluorescence detectors. Fluorescence also affords great analyte specificity because only certain compounds natively fluoresce, and they will only do so under specific excitation and emission wavelength conditions. Considering that many analytes do not fluoresce, this can be a disadvantage. However, as aflatoxin M1 natively fluoresces, the use of HPLC coupled to FL allows this to be leveraged.

The following article demonstrates the effectiveness of using this analytical technique for monitoring aflatoxin M1 in raw milk at ppb or ppt levels. Though the primary focus was on aflatoxin M1, aflatoxins B1, B2, G1, and G2 were also included as part of the standards and spikes to confirm the chromatographic separation of M1 from other aflatoxins, and allow for accurate and reliable quantification.

Samples of raw milk were obtained from a local health food store for analysis. Samples were initially spiked with aflatoxin calibrant, diluted, and then passed through immunoaffinity solid-phase extraction (SPE) columns to extract the aflatoxins.

Initial chromatographic results of the 0.382 ppb aflatoxin calibrant, shown in Figure 1, shows all five aflatoxins were readily separated isocraticly in under 6 min. The expanded view highlights the presence of M1 relative to the other aflatoxins.

Based upon a signal-to-noise (S/N) ratio of >10/1, the limit of quantitation (LOQ) for aflatoxin M1 was calculated to be 0.011 ppb, making M1 easily quantifiable down to the 0.05 ppb level established by the EU. Having convincingly demonstrated that the achievable detection limit is well below that required by the regulations, we can now turn our attention to actual milk samples.

Figure 2 shows the expanded chromatographic views of a 0.0765-ppb spiked raw milk sample. The run time had to be extended to 15 min to account for the large unidentified matrix peak eluted at 11 min. The calculated LOQ of 0.014 ppb was again considerably below the EU control limit, clearly demonstrating the applicability of this method in effectively monitoring for regulatory compliance.

As the dairy industry must continuously monitor milk batches sent for processing, it is important that any applied method is both robust and reproducible. Figure 3 highlights the chromatographic reproducibility that can be expected, showing the overlay of three replicate injections of a 0.0765-ppb spiked raw milk sample.

Whilst there is always a risk of the presence of aflatoxins in feed and grain, the possibility of contamination in milk is here to stay, and all concerned must stay vigilant. A key focus needs to be in rapid detection, without delaying dairy processing or distribution. With aflatoxins having a high carryover rate and with no overt signs of contamination, the routine screening of M1 in particular is critical, especially in areas of elevated risk of grain or feed contamination. This will help to ensure the safety of all dairy products.

To this end, as this work has shown, a reliable chromatographic method exists for the quantitation of aflatoxin M1, affording quantitation down to <0.02 ppb, well below the 0.05 ppb acceptable limit in milk, as established by the EU. The analytical approach was accomplished via FL detection, by taking advantage of aflatoxin M1’s native fluorescence, obviating the need for precolumn derivatization to enhance detection. This was particularly fortuitous, since most analytes do not inherently fluoresce, significantly reducing their sensitivity unless first being derivatized - the latter being a procedure that most lab scientists try to avoid.

References

1. Food Safety Watch, Aflatoxins, 1 February 2013.

1. US Food and Drug Administration, Inspections, Compliance, Enforcement, and Criminal Investigations; CPG Section 527.400 Whole Milk, Lowfat Milk, Skim Milk - Aflatoxin M1 (FDA, Rockville, Maryland, USA, 2005).

1. Commission Regulation (EU) No 165/2010, “Amending Regulation (EC) No 1881/2006 Setting Maximum Levels for Certain Contaminants in Foodstuffs as Regards Aflatoxins”, Official Journal of the European Union, 26 February 2010, pp. L 50/8–L 50/12.

Wilhad M. Reuter has worked at PerkinElmer for the past 32 years, the first 23 of which he was engaged as a product specialist, covering HPLC applications, method development, data handling and validation. He later served as LC Technology/Applications Specialist and now serves as Strategic LC and LC–MS Applications Specialist for PerkinElmer.

Nicola Vosloo completed her degree and Ph.D. in organic chemistry at the University of Exeter, utilizing electrospray and MALDI–MS. Nicola joined a chromatography company where she undertook a variety of roles including technical support, application and method development for LC and LC–MS. A move to the product inspection division of Mettler Toledo gave her experience of the food sector. Nicola joined PerkinElmer’s OneSource business and then moved to lead and develop the European marketing team. Following a period in the mass spectrometry business, managing sales support across Europe and the Middle East, Nicola now leads food and material characterization applications strategy and collaborations globally.

E-mail:wilhad.reuter@perkinelmer.com

Website: www.perkinelmer.com