LC-MS Analysis of Antibiotics in Food

By Oliver TrevelyanReviewed by Emily Henderson, B.Sc.

Antimicrobial agents, including antibiotics, are used in large volumes in human and veterinary medicine, and indiscriminate or excessive use is blamed for developing bacterial resistance to these pharmaceuticals. These products and their metabolites are often detected in foodstuffs and environmental samples and can exhibit ecotoxic effects. The action of cells and organisms, physical conditions, or chemical reactions can convert the antibiotics into toxic metabolites, profoundly affecting human health.

Image Credit: Andrzej Rostek/Shutterstock.com

Antimicrobial agents in veterinary medicine are expected to exceed 105,000 tonnes in 2030. Antibiotics are used in healthy animals as metaphylaxis and prophylactics and in unhealthy animals to treat infections. They are used both in agriculture and aquaculture. Aquaculture accounts for over 50% of the world’s food fish.

The intensive nature of animal and fish rearing means that any infection will spread quickly amongst stock. Hence, it is customary to use antibiotics as a preventative measure rather than in response to infection. The application of these antibiotics is permitted in many countries, but the amount and types of antibiotics allowed varies.

Antibiotics in intensive aquaculture are administered in food or directly into the water. This indiscriminate use means that antibiotics and their metabolites end up in fish tissue or are excreted and accumulate in the water or sediment. Intensive agriculture is similar, but the antibiotics and metabolites are present in the soil and runoff and animal tissue and products such as eggs and milk.

Risk to Human Health

The fact that residual traces of antimicrobials in food constitutes a risk to human health is known. It is known that antibiotic contamination can cause allergic reactions, compromise human intestinal and immune systems, and cause neurological disorders. There is not a lot of epidemiological data on the actual effect of these adverse effects. Still, the data indicates that food could be an important factor in the evolution and spread of antibiotic-resistant bacteria.

A sensitive and reliable detection system is required. Even small amounts of antibiotics below minimum inhibitory concentrations (MIC) can lead to bacteria genetic modification, resulting in antibiotic resistance. This resistance can be transferred to bacteria that colonize the human body.

Analytical Methods

There is growing interest in multiclass methods to analyze antimicrobial mixtures in environmental and food samples to detect them in very low concentrations. Liquid chromatography (LC) has become the favorite technique for multiclass analysis, especially when coupled to mass spectrometry (LC-MS) and tandem MS (LC-MS(2)). Due to the complexity of the sample source,  an extraction step for sample clean-up and preconcentration is required in most cases before analysis can be carried out to achieve the necessary sensitivities.

In many Countries, Public health agencies use mass spectrometry (MS) for the detection of contamination and unambiguous identification of residues of antibiotics in animal-derived food products for human consumption. The introduction of liquid chromatography and mass spectrometry (LC)-MS systems which are relatively inexpensive and robust, has boosted the development of methods for confirming antibiotics in foodstuffs. These techniques can analyze biological matrices such as milk, animal tissue, and eggs and give specific results with very few false positives. As many as 40 different antibiotics and 20 classes of antimicrobial agents can be detected using this method with a sample preparation time of around 15 minutes.

Reversed-phase Liquids Chromatography combined with tandem Mass Spectrometry, or triple-quadrupole MS (QqQMS), is currently the preferred technique in most analysis for residual single-class antimicrobials. A recent analytical strategy is to develop methods for detecting various different classes of veterinary drugs and pesticides (multiclass residue analysis).

To do this, generic and straightforward extraction and separation techniques have been developed for a broad range of compounds with differing physical and chemical properties. These methods are still based mainly on LC-QqQMS. Alternative MS detection systems are emerging, such as time-of-flight MS, which provides an accurate mass of the analytes, or Q-linear ion trap (IT) MS, which eliminates some limitations of ITMS(n).

Well-known suppliers such as Perkin Elmer, Agilent Technologies, Shimadzu, Bruker, and many other companies sell LC-MS systems with costs of around US$50,000+ or so for the instrument and around $25+ per sample.

Antibiotics in Milk

Nutritionally milk is considered an essential and beneficial food, being a source of high-quality proteins and a raw material for processed products such as yogurt, cheese, cream, etc. In some places, dry cow therapy and mastitis treatment commonly use an antibiotic in lactating cows, leading to antibiotic traces in milk. In most countries, antibiotics in milk are monitored, and the presence of antibiotics is very low. Still, it is reported that as much as 65% of milk could be contaminated in some areas.

Milk is often analyzed to ensure that the fixed Maximum Residue Limits (MRLs) for antibiotics are not exceeded and the milk is safe for human consumption. Multiclass analytical methods can monitor more drug classes through a single analysis, saving time and money compared to traditional single class analysis methods. These savings are significant for the analysis of perishable foods like milk.

Nevertheless, multiclass methods for determining veterinary drug residues in foodstuffs are real analytical challenges. Some special sample preparation is required to determine antibiotic residues in milk by liquid chromatography coupled with mass spectrometry.

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