Determination of antibiotic residues in meat samples using analytical methods

Origin of antibiotics

In 1928, Alexander Fleming discovered penicillin, the first antibiotic to be used to treat or prevent bacterial infections in humans. Other antibiotics were then gradually discovered. Antibiotics are antimicrobial agents that are naturally produced by living organisms or by synthetic/semi-synthetic compounds. They kill or inhibit the growth of microorganisms. Today, antibiotics are used to control and prevent diseases caused by Gram-negative and Gram-positive bacteria. Acting in various ways, antibiotics can destroy the cell wall; degrade the structure and function of nucleic acids; and block protein synthesis and important metabolic pathways. Therefore, antibiotic residue analysis becomes extremely important.

Classification of antibiotics

Based on their mechanism of action, antibiotics are divided into 10 main groups: β-lactam, sulfonamide (SAs), aminoglycoside (AGs), glycopeptide, lincosamide, quinolone (Qs), macrolide (MALs), rifamycin, tetracyline (TCs) and amiphenicol (APs).

Chemical structure of some antibiotic groups

Importance of antibiotic residue testing

Antibiotics are widely used in animal husbandry. Nearly 80% of managed animals use antibiotics. Therefore, human consumption of food sources such as meat, milk, eggs and seafood can passively absorb antibiotic residues. Antibiotic residues can cause allergies, cancer, mutations and certain diseases in both humans and animals. However, the most important issue is the development of antimicrobial resistance. For this reason, the World Health Organization (WHO) has established residue limits (MRL) to control antibiotic residues in food (FAO / WHO, 2018). Similarly, the European Union (EU) has amended the technical criteria applied in the screening and analysis of antibiotic residues (2002/657/ EC). This organisation introduces parameters called minimum analytical performance limits (MRPL) to control the amount of antibiotics that can be determined in foodstuffs.

Antibiotic residues in meat samples

Meat is a complex matrix with a diverse composition such as water (72% to 75%), nitrogen compounds (about 21% including protein and non-protein nitrogen compounds) and lipids (2.5% to 5%, or up to 15%). This complex composition makes antibiotic residues in meat very difficult to analyse. Therefore, new, fast, economical, reusable, selective and sensitive processes need to be studied for extracting antibiotic residues. In addition, combining them with other analytical techniques will help determine their composition and detection limits.

Sample preparation

In analytical chemistry, liquid – liquid extraction (LLE) is the first sample preparation technique used. However, LLE requires a lot of solvent and has low reproducibility. Therefore, solid phase extraction (SPE) is used to replace meat sample preparation. SPE has high reproducibility, short time, lower solvent volume and higher sample loading than LLE. The process of preparing meat samples by SPE includes: conditioning, sample loading, washing, analyte elution and sorbent regeneration.

Illustration of the solid phase extraction process in meat sample preparation for antibiotic residue analysis

Silicycle is an American manufacturer of SPE columns and solid phase sorbent materials. It has a full range of materials such as: Silica, modified silica, Florisil, activated carbon,...

Methods for antibiotic residue analysis

Sample preparation is an important step in determining antibiotic residues in meat samples. However, choosing the method for quantifying and identifying antibiotic residues is also very important. Antibiotic residues are usually analysed by liquid chromatography (LC). To separate antibiotics, C18 stationary phase columns, hydrophilic interaction liquid chromatography (HILIC) columns or phenyl-based sorbent materials are used. For detection, spectrometry and MS are the most used. However, spectroscopic detectors (UV detectors) and fluorescence (FLD) sometimes require derivatisation beforehand to obtain coloured or fluorescent compounds. On the other hand, MS is a suitable technique for determining antibiotic residues because of its selectivity, sensitivity and multi-analysis capability.

Macrolide antibiotics

MALs are a group of antibiotics active against Gram-negative bacteria, some Gram-positive bacteria and Mycoplasma. These bacteria are hydrophobic molecules. The structure consists of a lactone ring with 14 – 16 carbon atoms linked to sugar molecules. MALs are widely used in the treatment of infections (respiratory disease) and as growth-promoting additives for animals. MALs antibiotic residues in animal tissues have effects on humans. Examples include gastritis, allergies and liver toxicity. For this reason, the EU has set MRLs for MALs antibiotic residues in tissues of animal origin, such as fat and skin (100 µg/kg for tylosin) or liver (1000 µg/kg).

To separate and determine antibiotic residues of MALs in meat samples, LC coupled with a mass spectrometry detector (LC-MS/MS) is the most used method. However, other methods such as capillary electrophoresis (CE) with a UV detector and UV-Vis spectrometry are also used.

Table 1. Summary of the methods for extracting and determining MALs antibiotic residues in meat samples

Aminoglycoside antibiotics

AGs are a type of antibiotic that is naturally polar and obtained from Actinomycetes bacteria. The chemical structure consists of an amino sugar and an aminocyclitol group linked by glycosidic bonds. Apart from streptomycin and dihydro streptomycin which have a 1 streptidine substitution, other AGs have the same structure. AGs are drugs used in human and veterinary medicine to treat infections such as mastitis, leptospirosis, enteritis and other diseases caused by some Gram-positive and Gram-negative bacteria. The use of these antibiotics as growth promoters in animal feed is banned in the EU. Some other governments (such as China) have also set MRLs for antibiotic residues of AGs. Their accumulation through animal tissues and human consumption through the food chain can cause antimicrobial resistance.
To determine AGs antibiotic residues, LC/MS-MS is one of the most commonly used analytical techniques.

Tetracycline group antibiotics

TCs are a large group of antibiotics semisynthetic in origin and derived from the bacterium Streptomyces aureofaciens. They are used to treat diseases caused by Gram-negative and Gram-positive bacteria in humans and animals. Their chemical structure consists of a ring system with various linking chains. TCs are widely used as growth-promoting feed additives for animals.To determine antibiotic residues of TCs in meat, many techniques are used. LC-MS/MS, LC with a UV detector and LC coupled with a fluorescence detector are the most used in this field. However, other techniques such as Raman spectroscopy and differential pulse voltammetry (DPV) are also used.

See also hormone and growth regulator analysis