Food safety is an important consideration for everyone in the food chain, from producers, suppliers and vendors right through to the consumer. Contaminants may be introduced at any stage, so it is important that monitoring is performed at multiple points through the food production and supply chain. Common contaminants that are introduced prior to harvest or processing include pesticides, fungicides, antibiotics and other veterinary drugs, aflatoxins, heavy metals and other environmental contaminants. During processing substances associated with the processing machinery can pose a risk, and even after production, chemicals such as per- and polyfluoroalkyl substances (PFASs) can leach from packaging into the products – termed food contact contaminants.
With so many potential routes for contaminants, both natural and otherwise, having the tools to detect them, determine the source of the problem and prevent them from reaching the consumer is therefore vitally important. However, it is not only enough to identify a contaminant in a particular sample, it important to then be able to trace where it has come from to enable other potentially affected items to be isolated.
Challenges of food analysis
One major challenge in food analysis, especially once multiple ingredients are combined, is the sheer complexity of samples. Not only can unwanted interference from the sample matrices pose an analytical headache (think oils and lumpy sauces), but the dynamic range of the compounds within the sample can be very broad. Consequently, sample preparation may not be straightforward with traditional analytical techniques. This is where two-dimensional liquid chromatography (2DLC) comes into its own.
2DLC as a tool for food safety and traceability testing
With 2DLC, samples for separation may be injected directly or with a reduced number of pre-preparation steps depending on the sample type. This reduces time, cost and the risk of cross-contamination or sample loss, helping to improve result accuracy. When the substance an analyst is looking to detect poses a risk to human health, such as aflatoxins, this also has the added benefit of reducing the opportunity for exposure of personnel too.
Who can solve a problem like pH?
Pyrrolizidine alkaloids (PAs) and their N-oxide derivatives (PAs-ox) are an interesting case. Produced by some plants and particularly linked to teas, herbal infusions and some food supplements, they are considered a health risk and have been linked to liver damage and are potentially carcinogenic. PAs and PAs-ox come in many forms with widely varying toxicities, so it is important to be able to identify which form is present. However, successful separation of these compounds varies widely depending on the pH of the separation conditions used, with some separating well in alkaline conditions whilst others will not separate at all at that pH. This poses a headache for analysts trying to determine which forms are present in a sample. With 2DLC, this problem is alleviated as differing pH conditions can be used in each dimension of the separation, enabling a much wider spectrum of compounds to be isolated.
Wide polarity range? No worries!
In food production animals, antibiotic administration may be necessary to keep them healthy. Low doses of antibiotics supplemented in feed are sometimes also used indiscriminately for growth promotion, despite the issues this can cause with the spread of antimicrobial resistance. The use of some antibiotics (those of particular importance to human health) for this practice is banned in some countries, but that by no means includes all antibiotics and not in all countries. Irrespective of how or why antibiotics have been administered, there is however the danger that drug residues may persist in foods derived from animals, for example in dairy, egg or meat products, and reach the consumer. Consequently, there are regulations governing the presence of many antibiotics in foodstuffs, for which analysts need to be able to identify which antibiotics are there and at what level.
Antibiotics were first detected in milk in the 1960s , since when there has been a sharp increase in studies to detect antibiotics in foodstuffs as awareness and concern about their presence has grown. However, antibiotics cover a diverse spectrum, from aminoglycosides, amphenicols and β-lactams to tetracyclines, and have an equally wide polarity range. Consequently, the flexibility offered by being able to combine two chromatography chemistries into a single analysis expands the range of antibiotics that may be separated, differentiated and quantified. In one study, researchers wanted to identify the presence of 20 different antibiotics from seven subtypes (aminoglycosides, amphenicols, β-lactams, macrolides, quinolones, sulphonamides and tetracyclines). For this purpose, they chose to use 2DLC for sample separation to exploit their polarity range. Five antibiotics were not retained in the first dimension where hydrophilic interaction chromatography (HILIC) was used, eight antibiotics were not retained in the second dimension where reverse phase (RP) chromatography was used, and the remaining seven antibiotics were retained in both dimensions. This improved subdivision of the antibiotics present therefore greatly simplified the challenges for downstream analysis.
Embrace the added separation power of going multidimensional
Detection of compounds with broad structural diversity and multiple isomers can be challenging. Therefore, multidimensional chromatography can provide the enhanced separation power required to sift out these compounds. Polycyclic aromatic hydrocarbons (PAHs) are one such group whose detection is benefitting from 2DLC. Known carcinogens and mutagens, PAHs are produced by burning carbon-based materials. Cooking techniques such as char-grilling and smoking therefore introduce PAHs, but they can also be present in foods such as shellfish who accumulate PAHs from the environment, and foods that have been improperly dried. Edible oils are another source of concern, with PAHs generated during frying for example.
Their structure can vary greatly depending on how they were generated and factors such as fat content, the duration of heat exposure and oxygen level to name a few. PAHs of low molecular weight are volatile, whilst those of higher molecular weights are more chemically inert, factors that impact their environmental persistence and therefore likelihood of becoming a problem for bio accumulation. Determining which PAHs are present in food samples is therefore important to determining the risk to health.
Samples rich in polyunsaturates, like oils, or with complex matrices can be tricky to analyze, but using 2DLC enables multiple cleanup processes during separation to be combined. Heart-cutting 2DLC is also consequently a useful tool for detection of PAHs in a range of foods.
In conclusion, the flexibility that 2DLC offers to combine the differing attributes of multiple chromatographic strategies in the separation of complex samples means that food safety analysts are better able to detect even trace levels of contaminants. The improved clarity and range of data mean that they have more pieces of the puzzle with which to be able to effectively trace the sources of contaminants in a timely fashion and prevent further dissemination of unsafe products.
1. Siddique IH, Loken KI, Hoyt HH. Antibiotic residues in milk transferred from treated to untreated quarters in dairy cattle. J Am Vet Med Assoc 1965; 146:589–93.