Determination of Antibiotic Residues in Honey

LUPU Daniela1, HANCU Gabriel1, BLEBEA Nicoleta Mirela2

1 University of Medicine, Pharmacy, Science and Technology “G.E. Palade” of Târgu Mureș, Faculty of Pharmacy, Department of Pharmaceutical Chemistry

2 Pharmacology and Clinic Pharmacy Department, Faculty of Pharmacy, Ovidius University from Constanta, Romania



Honey is a complex natural sweet food, composed mainly of sugars and minor components like aminoacids, minerals, organic acid, vitamins, and aromatic substances. The presence of xenobiotics in honey can be detrimental to its quality and represents a risk to consumer health. Antibiotics are commonly used by beekeepers to prevent and eradicate bacterial infections among bees. Widespread antibiotic administration may cause bacteria to become resistant to certain drugs. Sample preparation and determination of extremely low concentration of antibiotic from honey are true challenges for an analyst. The aim of this study is to provide an insight into the use of antibiotic treatment in beekeeping and to evaluate possible contamination of honey with antibiotic residues.

Keywords: honey, antibiotic residue, analytical methods, sample preparation.


Honey is a natural product used for nutritional and medicinal purposes composed mainly of sugars and water together with other minor components like minerals, vitamins, flavonoids, aminoacids, organic acids, phenols, and aromatic substances. Its composition is variable, depending on its botanical and geographical source. Its composition, colour, aroma and taste are strongly influenced by the flowers, climate, bee species, weather conditions, manufacturing and storage time [1].

The use of antibiotics to fight bacterial infections in bee colonies is known since their large-scale introduction in human therapy, starting with the 1950s. The presence of antibiotic residues in food products can be correlated with the proliferation of antibiotic resistant bacterial strains. Taking in consideration this danger, legislation in many countries restricted antibiotic use in farming activities [2].

Currently in the EU the maximum residue limits (MRLs) for antibiotics in food are listed in EU Regulation No 37/2010, however in EU antibiotic treatment of bees is not permitted and consequently there are no official MRL data for the presence of antibiotic in honey [3].

The lack of harmonization between legislative stipulations and appropriate control methods, detection limits leads to differences in interpretation among EU member states. In some states, limits of tolerance are established and recommended concentrations, for the analysis of antibiotics are set [4].

Environment pollution and improper beekeeping practices represents the main source of antibiotic residues in honey.

The development of new analytical techniques for the detection of antibiotic residues in honey samples is a difficult but fundamental task to verify product quality and safety.

Antibiotics in beekeeping

Among antibacterials detected in honey, different studies mention the following: aminoglycosides (streptomycin), macrolides (erythromycin), sulphonamides (sulfathiazole, sulfamethoxazole, sulphanilamide, sulfadiazine), tetracyclines (tetracycline, oxytetracycline, doxycycline), chloramphenicol [5].

Antibiotics/Chimioterapics detected in honey can be classified according to Annex I of Council Directive 96/23/EC in group A (banned substances) (chloramphenicol, nitrofurans, nitroimidazoles) and group B (admitted substances) (aminoglycosides, macrolides, quinolones, sulphonamides, tetracyclines) [6].

Determination of antibiotic residues in honey

Beginning with the 1990s, the first methods dealing with detection of antibiotics in honey, were developed at trace levels; the initial methods were based on liquid chromatography coupled with UV (LC-UV) or fluorescence (LC-LIF) detection. However, the limits of detection (LOD) of these initial methods are considered today unsatisfactory. Everything changed in the 2000s, with the emergence of LC coupled to mass spectrometry (LC-MS) detector, which allowed the development of sensitive and selective analytical methods [7].

For the banned antibiotic substances (limits of 0.1 – 1.0 µg/kg), the use of LC-MS method for their detection is mandatory, while for the permitted antibiotic substances (higher accepted limits), the more traditional LC-UV or LC-LIF are acceptable in some situations, too [7].

Since antibiotics are present in honey in extremely low concentration, a high priority should be awarded to sample preparation and extraction before the determination.

To determine antibiotics in honey, in the sample preparation stage, sugars and other substances which may interfere with the analysis should be separated from the sample. Methods typically begin with acidic hydrolysis, followed by liquid-liquid extraction (LLE) to release sugarbound substances, followed by sample clean-up and a solid phase extraction (SPE) [8].

Two strategies are usually implemented for antibiotics detection in honey: screening tests and multistage analytical methods. When using the conventional testing approach, a single target analyte or a group of analytes can be detected and quantified. Modern analytical techniques are developed usually to detect simultaneous determination of several analytes from different groups in a single analytical procedure [9].

Screening tests can detect the presence of an antibiotic or a group of antibiotics, and typically provide quantitative/semi-quantitative results. These tests are rapid, easy to operate and relatively cheap, however they often give false results. For this purpose, microbiological and immune-enzyme tests are usually used [10].

The samples found positive with screening techniques have to be confirmed with more specific analytical methods, however according to EU regulations ‘‘methods based only on chromatographic analysis without the use of molecular spectrometric detection are not suitable for use as confirmatory methods” [11].

LC-MS in combination with electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) are the most frequently used technique in the analysis of antibiotics from honey. Due to the high selectivity in multiple reaction mode (MRM), a triple-quadrupole analyser is widely used for quantitative analysis of antibiotics [12].

Studies showed that honey can be contaminated by more than one class of xenobiotics, consequently multiscreen approaches are suitable to detect residues.


Xenobiotic contamination of the environment is related to intense industrialisation and intensive farming [13]. Bees products like honey, can be used as a strong biological indicator based on the antibiotic residues found in its composition.

Since in EU countries antibiotics are prohibited in beekeeping, there are no MRL values for antibacterial substances in honey.

The development of new analytical methods for the detection of antimicrobials residues in honey at trace levels is a difficult task; advanced sample preparation techniques have to be applied to allow the detection of trace quantities of antibiotics, due to the high complexity of the honey matrix.

The performance of an analytical method are mainly determined by the applied sample preparation and instrumental technique.

The latest trends in honey analysis are directed towards the development of multi-class analytical procedures and microextraction techniques.


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