Introduction
Animal-derived products such as meat, milk, eggs, and honey are vital sources of protein and energy in the human food system. The global demand for these products has intensified the need to increase food production, including livestock and aquaculture production (Wilkinson et al., 2017). Veterinary medicines are pharmaceutical compounds used for therapeutic efficiency and as growth stimulants in animal production (Khalifa et al., 2024). The Food and Drug Administration (FDA) defines drug residue as a compound that is present in the edible tissue after the use of a drug or its metabolite, or any other substances formed in food (Pratiwi et al., 2023). The intensification of animal production has led to a consequent increase in the use of veterinary medicines, either to treat infections and prevent diseases, or even illegally, to promote animal growth. Veterinary drugs are essential for maintaining animal health and improving productivity; however, they can enter the environment through the application of manure, animal excreta, treatments against ectoparasites, or through aquaculture practices (Topp et al., 2018). A wide range of compounds are often used, such as antibiotics, anthelmintics, hormones, and anti-inflammatory drugs, among others (Delgado et al., 2023), and their occurrence in food or the environment can have an impact on global health. Small molecules are not biodegradable and may persist in different environmental compartments after excretion. Most medicines are designed to have a prolonged effect for a longer period of time. This stability transforms their occurrence into their persistence in the environment (Berkner & Thierbach, 2014).
The One Health Perspective approach states that human health is closely correlated with the combination of animal and environmental health. Environmental pollutants, such as pharmaceuticals, which are widely used in livestock production and in human medicine, can represent a real threat to wild animals and, more specifically, to natural pollinators such as honeybees. The toxic effects of constant exposure to veterinary drugs and pesticides used in food production can also affect survival (Mahefarisoa et al., 2021). The cumulative exposure to residual levels of pharmaceutical medicines can affect the metabolism of both humans and animals, and the continuous discharges of antibiotics into the environment can promote the development of antimicrobial-resistant microorganisms, which is of global level concern. Antimicrobial resistance (AMR) refers to pathogens, such as bacteria, viruses, and fungi, that no longer respond to antimicrobials. It mainly occurs due to the irrational use of medicines, failure to follow the drug withdrawal period, poor infection prevention and control, pollution, and poor waste and wastewater management. According to global statistics, AMR is directly responsible for 39 million deaths and contributes to more than 169 million deaths between 2025 and 2050 (Larsson & Flach, 2022).
Several studies have shown that veterinary medicines administered to animals are normally excreted at 90 per cent of the dose in urine and feces and thus discharged into sewage plants, which eventually reach surface water. Animal waste is used as manure in crop fields as a natural fertilizer, which leads to the indirect transfer of drug residues to the food chain (Delgado et al., 2023). The release of active pharmaceuticals, mainly due to the excessive use of veterinary medicines in the environment, (Wilkinson et al., 2017), is incomparable to human medicine, which causes the accumulation of pharmaceutical residues in soil, water, plants, and sediments, even though the quantities that are used on animals are much higher, leading to an increase in the impact that it will cause (Gworek et al., 2021).
In the “One Health” approach, and considering the effects that this situation may have in all ecosystems, the persistent contamination of the environment can be one of the main factors causing the decline of pollinators. Bees are assumed to be the most important pollinators in agricultural and natural ecosystems. However, other insects such as beetles, flies, and butterflies are also involved in pollination (Rader et al., 2016). According to the global statistics, bees can pollinate approximately 1500 crops globally, contributing to 15–30% of global food production. (V.S., A., 2024). Honeybees are more prone to environmental contamination because of their natural behavior of gathering nectar and pollen from a diverse range of flowering and nonflowering plants. Bees can encounter multiple unidentified pathways of exposure to chemicals from livestock farms, manure and slurry, animal excreta, urine, surface water, soil, air, and plants (Mahefarisoa et al., 2021).
There is evidence that veterinary medicines can accumulate in hives (Venable et al., 2014) and cause adverse effects on bee health, such as acute toxicity and sublethal effects including weakened immune function, reduced memory and learning capability, compromised reproductive function, and disrupted social behavior.
However, although pharmaceutical pollution of environmental matrices is increasing, its impacts and specific risks on bees are not completely understood. There are knowledge gaps related to the concentration of veterinary drugs on different bee-related matrices (pollen, nectar, and hive matrices), antagonistic or synergetic effects with different chemical contaminants, and chronic effects of low-dose exposure (Di Noi et al., 2021). Identifying these knowledge gaps is essential for the development of strategic risk assessment plans to protect bees and other pollinators. Therefore, this review focuses on a comprehensive overview of environmental contamination by veterinary drugs in animal production and their potential exposure pathway to bee health.
Sources and pathways of contamination
Veterinary drugs can enter the environment through multiple pathways, either directly or indirectly, thereby creating potential risks to the environment, animals, and humans. Several sources should be considered for direct contamination.
Runoff from Livestock Farms: The Waste originating from livestock farms is the major source of direct contamination (Delgado et al., 2023). Most drugs administered to humans and animals after metabolism are transformed into different chemically active structures, which ultimately reach water through agricultural wastes, aquaculture, and sewage treatment plants (Joshua et al., 2022). The concentration of pharmaceuticals in different environmental matrices depends on various factors, such as livestock farm management practices, animal density, and drug administration behaviors in farms (Delgado et al., 2023).
- Use of manure as a natural fertilizer: Manure obtained from livestock farms can comprise a cocktail of different contaminants, such as antimicrobial, antiparasitic, and hormones, among other possible products applied to animals. These pharmaceutical residues eventually accumulate in the soil and affect soil microbes, plant uptake, and groundwater quality (Jaga et al., 2022).
- Direct application of medications to beehives: Depending on the country and purpose of use, medications can be applied to beehives to control the colonies from pests and diseases. Beekeepers use diverse xenobiotics against Varroa mites, which pose a significant threat to their health. Certain insecticides can cause acute toxicity in bees; for example, imidacloprid is a highly toxic insecticide for honeybees, according to the U.S. Environmental Protection Agency (EPA) (Manjon et al., 2018). Therefore, the use of antibiotics is often prohibited.
In terms of indirect contamination also several routes can be expected and here summarized:
- Disposal of pharmaceutical compounds used in human medicine can eventually result in the occurrence of these contaminants in wastewater treatment plans (WWTP). Because WWTP are not well designed to remove pharmaceuticals from water, effluents can contain detectable residues of pharmaceuticals (Delgado et al., 2023);
- Uptake by Plants: Pharmaceuticals and other agrochemicals present in treated wastewater used for irrigation, especially during drought time, can contaminate plants. This assimilation can cause pharmaceutical compounds to accumulate in plant tissues, nectar, and pollen, leading to exposure of bees and other pollinators. Although there are limited data regarding the uptake of pharmaceuticals by plants, some studies have shown that plants can absorb pharmaceuticals from contaminated water and soil (Mastro et al., 2025). The pharmaceutical uptake by plants depends on several factors, such as the specificity of the compounds, soil texture and behavior, and plant species (Carter et al., 2014). Some studies have revealed that medicines are biotransformed into other metabolites by flowering plants, which may be equally or more hazardous. For instance, Obregon et al. (2024) found that anthelmintic medicines administered to bovines undergo biotransformation and can be converted into other compounds by flowering plants.
- Atmospheric deposition of pharmaceutical compounds: There is evidence that volatile and semi-volatile contaminants, such as polycyclic aromatic hydrocarbons (PAHs), originating from incomplete combustion or natural sources, such as volcanic activity and forest fires, exert toxic effects on both the environment and human health (Jiménez et al., 2015). In contrast, the atmospheric deposition of veterinary pharmaceuticals remains a relatively understudied topic despite its potential as a significant pathway for environmental contamination.
Understanding contamination pathways is essential for developing and implementing effective strategies to eradicate environmental contamination caused by veterinary medicines, thus protecting ecosystems and human and animal health, and especially to develop measures to protect essential pollinators such as honeybees. Table 1 shows some examples of previous studies correlating the occurrence of contaminants in hive products with the main impact on bee health, along with possible exposure pathways.
Table 1. Occurrence of contaminants in hive products, probable exposure pathways and possible health effects on bees.
| Contaminants | Matrix | Exposure Pathway | Detected
Concentration | Impact on Bees | Reference |
|---|
| Carbendazim | Honey,
Pollen &
Bees | Atmospheric pollutants,
contaminated food sources
of food such as pollen,
nectar or water | 0.08 to
4.28 mg/kg | Declines foraging
activity, mortality,
impairs reproduction
& Limits pollination
efficiency | Lambert et al. (2013) |
| Amitraz | Honey &
Bees wax | Environmental
contamination & Hive
treatment | ---- | Declines honey
production, diminished
colony growth &
decreased memory and
learning ability | Savarino et al. (2020) |
Streptomycin Sulfanilde
& Tetracyclines | Honey | from livestock farms, water, | ---- | Antimicrobial resistance | Mărgăoan et al. (2024) |
| Chlorpyrifos | Bee pollen,
Honey & Bee
Bread | Surface residues, oral/
contact exposure | Detected in 30%
of samples | Acetyl cholinesterase
inhibition-Affecting
the honeybee brain &
behavioral impairment | Liu et al. (2022) |
Fenpropathrin
(pyrethroid) | Honeybee | Crop raiding | 0.3 to 81.5 ng/g | Decreased homing
ability & survival, acute
and chronic toxicities | Obregon et al. (2024) |
| Avermectin/Ivermecetin | Bee bread | Biotransformation from
ivermectin to abamectin by
flowering plants- (absorbed
animal waste of ivermectin
by cattle) | 9.6–1,856 µg/kg | Adaptive response | Savarino et al. (2020) |
Risks posed by veterinary pharmaceuticals to bees and other pollinators
Veterinary pharmaceutical medicines play a crucial role in maintaining animal health, welfare, and productivity in intensive animal production systems. The presence of veterinary medicines, either by direct application or indirect contamination, can cause potentially harmful consequences, affecting microbial health in the gut. Studies have revealed that honeybees are an important reservoir of antibiotic resistance genes that affect reproduction, immune system, and bee development (Baffoni et al., 2021). For instance, oxytetracycline is a widely employed antibiotic used for the control of bacterial diseases in European Foulbrood (causative agent Streptococcus pluton) and American foulbrood (causative agent Bacillus larvae). Although EU regulations prohibit the use of antibiotics in apiaries, the use of lincomycin, oxytetracycline, and tylosin has been approved for the treatment of honeybees in other countries. Studies conducted by Baffoni et al. (2021) elucidated that tylosin treatment causes a drastic decline in bifidobacteria and lactobacilli found in the rectum but enhances the growth of Frischella spp., Gilliamella, and Snodgrassella in the ileum. The sulfonamides also showed some changes; they increased in Snodgrassella spp. and Bombilactobacillus spp., and declined in Frischella and Bartonella species. Honeybees were found to be a reservoir of tetracycline-resistant genes, as confirmed using qPCR techniques. However, some studies provide evidence that antibiotics, such as sulfonamides, promote the decline of species, such as Frischella and Bartonella, while tylosin decreases Bifidobacteria and Lactobacilli (Baffoni et al., 2021). These imbalances in the gut microbiota due to different factors, such as exposure to chemicals, diet, and season, called dysbiosis, have a negative impact on the bee population (Baffoni et al., 2021). Antibiotic treatment plays a pivotal role in affecting the size and composition of the honey gut microbiome and enhances the risk of infection from widespread opportunistic pathogens (Raymann et al., 2017). Healthy gut microbiota is essential for maintaining proper immune function, nutrition acquisition, and resistance to pathogenic organisms in bees. Dysbiosis can weaken immune function and make bees more prone to attacks by pathogens. (Raymann et al., 2017).
Varroosis (a destructive parasitic disease) is a major threat to honeybees worldwide. To address this problem, insecticides or acaricides, such as amitraz, are often used. Amitraz and its metabolites were found to be potent neurotoxicants, making amitraz more cytotoxic than its metabolites (Giorgini et al., 2023). Bees can also be exposed to fungicides, such as chlorothalonil, and the insecticide chlorpyrifos through contaminated pollen and nectar. These two chemicals are the most commonly found pesticides in bee wax-derived samples. (Walsh et al., 2021).
Even though in some cases there is improper use of chemicals in hives, veterinary drugs should only be administered to honeybee colonies under the guidance of a veterinary practitioner or apiculture specialist. Alternative treatment methods include the growth of bioactive plants containing secondary metabolites such as sesquiterpene lactones or condensed tannins. These metabolites act as antiparasitic agents to avoid the administration of fungicides or herbicides. (Rodríguez-Hernández et al., 2023).
In the following sections, the main classes of medicines that can cause adverse health effects on pollinators are summarized. Apart from these compounds, it may also be considered that possible cross-contamination of the environment can lead to the presence of other groups of compounds used in urbanized regions, mainly human medicines.
Antibiotics
The excessive use of antibiotics and antimicrobial resistance are currently trending topics that compromise both human and animal health. These compounds can be used in apiculture practices to treat and prevent different bee diseases, such as European and American foulbroods, Varroa mites, bee acariasis, and Nosema. This may lead to a rise in concerns about honey contamination (food safety), contribution to the increase in antimicrobial resistance, and other environmental implications from a “One Health” perspective (Moumeni et al., 2025). A recent study revealed the presence of antibiotics such as chloramphenicol and oxytetracycline residues in untreated and treated hives. Situations like this raise concerns about the environmental dissemination of contaminants and/or cross-contamination from sources other than the direct use of medicines. These studies and the detection of residues of medicines in hive products make honeybees act as vectors for transferring antibiotic-resistant genes (Almutairi et al., 2025) and a possible contributor to pollution bioindicators.
Antiparasitic agents
Antiparasitic resistance will be an upcoming threat in the future, elucidated the emergence of resistance from key classes of similar to what is currently being discussed regarding antimicrobial antiparasitic benzimidazoles, which are extensively used by resistance. A recent study from the northern German research group livestock farmers for the treatment and prevention of infections caused by parasites. (Ehnert et al., 2025). The global rise in the population has led to a higher demand for food production and, consequently, to avoid losses due to health problems in animals, there is also an increase in the use of veterinary medicines (Barmpouni et al., 2025), revealing how antiparasitic compounds such as albendazole and ivermectin can persist in animal tissues, leading to drug residues in the food chain and environment (Barmpouni et al., 2025). In Table 2 we can observe Analytical methodologies for detection of different compounds collected from hives products.
Table 2. Analytical methodologies for detection of different compounds collected from hives products.
| Target Compounds | Sample
(g) | Extraction
Method | Appropriate LC
Column | Mobile
Phase | Ionization | MS
Mode | Mass
Analyzer | LOD /
CCβ
(μg/kg) | References |
|---|
| Nitrofurans (Honey) | 1.00 ±
0.02 | Magnetic
SPE | Acclaim 120 C18
(2.1×100 mm,
3 µm) | 0.5% FA–H2O
/ ACN:MeOH
(50:50) | ESI+ and
ESI– | MRM | SCIEX
Triple
Quad
5500 | 0.1–0.3 | Jayasinghe et al. (2021) |
Aminoglycosides
(11) (Honey, milk and
pork) | 2.0 | MIP-SPE | HILIC
(50×2.1 mm,
3.5 µm) | 175 mM
ammonium
formate +
0.3% FA /
MeOH + 0.3%
FA | ESI+ | MRM | Sciex
3200
QTRAP | 2–30 | Ma et al. (2016) |
β-Lactams,
Macrolides,
Sulfonamides, etc.
(Honey) | 1.0 | Oasis HLB
SPE | Synergi Hydro-
RP (150×2 mm,
4 µm) | 0.1% FA /
MeOH | HESI | Full
scan +
DIA | Thermo
Q-
Exactive
Orbitrap | 0.03–
4.80 ng
g⁻¹ | Chen et al. (2021) |
Multi-class antibiotics
(Honey) | 2.0 | Dispersive
SPE | Zorbax SB C18
(100×2.1 mm,
3.5 µm) | H2O + 100
mM HFBA /
ACN | ESI+ | MRM | Agilent
6410
Triple
Quad | 0.73–
3.58 | Zhang et al. (2016) |
Multi-residue
antibiotics (Honey) | 5.0 ±
0.01 | SPE (Strata-
X) | Obelisc R
(2.1×150 mm,
5 µm) | 1% FA / ACN | ESI+ | Full
Scan | Bruker
Q-TOF | 13.4–
47.9 ng
g⁻¹ | Liu et al. (2022) |
Pesticides +
Antibiotics (Honey) | 1.0 | Oasis HLB
SPE | Synergi Hydro-
RP (150×2 mm,
4 µm) | 0.1% FA in
MeOH | HESI | DIA | Thermo
Q-
Exactive
Plus | 0.73–
3.58 | Wang et al. (2023) |
| Perchlorate (Honey) | 2.0 | Freezing-
assisted
LLE | Torus Waters
(2.1×100 mm,
1.7 µm) | 20 mM
ammonium
acetate / ACN | ESI– | MRM | AB Sciex
6500+ | 0.05 | Wang et al. (2023) |
Tylosin A &
Desmycosin (Honey) | 2.00 ±
0.05 | LLE | Kinetex HILIC
(100×2.1 mm,
1.7 µm) | 40 mM
ammonium
formate /
ACN + FA | ESI+ | MRM | Sciex
3500
Triple
Quad | 0.3–0.4 | Li et al. (2019) |
Nitrofurans
Chloramphenicol,
Florfenicol (Honey) | 3.0 | LLE | Zorbax Eclipse
XDB-C18
(50×4.6 mm, 1.8
µm) | 0.1% FA
/ ACN +
ammonium
acetate | APCI+ /
ESI– | MRM | Agilent
1260 +
Sciex
5500 | 0.19–
2.35 | Zhao & Sun (2017) |
Penicillin G,
Dihydrostreptomycin,
Enrofloxacin (Honey) | 10 mL | Micro-LLE | Zorbax SB-Aq /
Eclipse Plus C18 | ACN / 0.1%
FA–H2O | ESI+ | MRM | Waters
Quattro
Triple
Quad | 0.55–
0.79 ng
g⁻¹ | Kim et al. (2021) |
Quinolones &
Nitroimidazoles
(Honey) | 3.0 | QuEChERS | XTerra RP18
(2.1×150 mm,
5 µm) | ACN / 10 mM
ammonium
formate +
0.5% FA | ESI+ | MRM | Waters
Micro
Quattro | 0.83–
1.58 | Alvarenga et al. (2018) |
| Quinolones (Honey) | 5.0 ±
0.01 | QuEChERS | Waters C18
(2.1×50 mm, 1.7
µm) | 0.5 mM
ammonium
formate +
0.005% FA /
MeOH | ESI+ | SRM | Waters
Xevo TQ | 0.61–
0.69 | Patel & Singh (2021) |
Sulfonamides,
Quinolones,
Tetracyclines, etc.
(Honey and royal
jelly) | 2.0 | QuEChERS | Poroshell 120
EC-C18 (100×2.1
mm, 2.7 µm) | 2 mM
ammonium
formate +
0.1% FA /
MeOH | ESI+ | dMRM | Agilent
6460
Triple
Quad | 0.14–
2.91 ng
g⁻¹ | Garcia & Torres (2016) |
Sulfonamides,
Quinolones,
Macrolides (Honey) | 2.0 | QuEChERS | ACQUITY
(100×2.1 mm,
1.7 µm) | MeOH / 0.2%
FA–H2O | ESI+ | MRM | Waters
Xevo TQ-
XS | 0.05–
1.02 | Nguyen & Hoang (2021) |
Sulfonamides
Tetracyclines,
Macrolides (Honey) | 2.0 ±
0.05 | QuEChERS | Acquity
(2.1×150 mm,
1.8 µm) | 0.1% FA /
MeOH | AJS ESI | MRM | Agilent
6470
Triple
Quad | 0.1–2.5 | Silva & Costa (2023) |
Pesticides (96)
(Honey) | 5.0 | QuEChERS | C18 (2.1×150
mm, 1.8 µm) | 5 mM
ammonium
formate +
0.1% FA /
ACN | ESI+ | dMRM | Agilent
6490
Triple
Quad | 0.5–1 | Zhang & Chen (2016) |
Cephalosporins,
Quinolones,
Macrolides, etc.
(Honey) | 2.0 ±
0.05 | QuEChERS | ACQUITY C18
(100×2.1 mm,
1.8 µm) | 0.1% FA–H2O
/ ACN | ESI+ | TOF | Shimadzu
Nexera
UHPLC +
AB Sciex
5600+ | 0.05–
0.64 | Mendes & Oliveira (2025) |
Anti-inflammatory medicines
Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used around the globe in veterinary and human medicine worldwide. NSAIDs are prevalent as parent compounds or metabolites in the environment. However, there are no efficient wastewater treatment methods for the removal of these chemical contaminants from aquatic environments (Izadi et al., 2020) Common NSAIDs such as naproxen, diclofenac and ibuprofen are present in various aquatic resources such as groundwater, sea water, surface water, and sludge (Lakshmi et al., 2024). Coming from direct human and veterinary uses. Bees and other pollinators are directly exposed to contaminated water bodies, flowers, pollen, and soil, and continuous contact with NSAIDs can affect their immune system, foraging ability, and colony vitality. Therefore, a thorough study of NSAID occurrences and their behavior in the environment is vital to elucidate the potential exposure pathways and threats to the bee population and to develop mitigation strategies against pharmaceutical pollution (Izadi et al., 2020).
Analytical methodologies for determination of pharmaceutical residues in honey
Honey is a natural product that is prone to contamination from various sources in the environment (Puścion-Jakubik et al., 2020). Honey comprises 300 distinct compounds produced from Apis mellifera being the main component simple sugars, approximately 80–83%. It is composed of proteins, vitamins, microelements, and organic compounds (Lewiński et al., 2023). Two international regulatory bodies, the Codex Alimentarius (CA) and the European Council Directive, have set standards for honey quality control (Zammit Young & Blundell, 2023). The complex composition and requirements to fulfill the standard regulations pose a significant challenge in developing an analytical method for the determination and quantification of pharmaceuticals in honey. This session aims to provide an overview of different analytical strategies to determine contaminants such as pharmaceuticals in honey, from sample preparation methods to analytical detection techniques to ensure the quality and safety of honey before reaching consumers.
An optimized and targeted sample preparation technique is vital for isolating the target analyte and obtaining results with minimal matrix interference. The new trends in sample preparation include less time, solvent, and sample volume and are more automated, achieving results in less time (Rodrigues et al., 2024).
Regarding the detection techniques, Liquid Chromatography coupled with Mass Spectrometry detection (LC-MS), particularly triple quadruple detectors (LC-MS/MS), (Kivrak et al., 2016), are widely employed for the detection and quantification of pharmaceutical residues from biological matrices including honey. (Gün et al., 2024). This analytical approach offers the possibility of developing high-throughput and fast multidetection screening and confirmatory methods (Er Demirhan & Demirhan, 2022). High resolution Mass Spectrometry (HRMS) allows the possibility to do both targeted and un-targeted analysis (qualitative and quantitative analysis) (Bandini & Spisso, 2021), being the Orbitrap and the time-of-flight mass spectrometer the examples of those technologies (Khalifa et al., 2024). LCMS/MS is often considered the gold standard for the detection and quantification of pharmaceutical residues in honey owing to its high sensitivity and specificity. (Cavaliere et al., 2019). Although other approaches can be used at lower associated costs and speed, the specificity of mass spectrometry and the possibility of multi-detection analysis overcome these characteristics. Despite this, examples in the literature can be found with different methods: a High Fundamental Frequency Quartz Crystal Microbalance with Dissipation (HFF-QCMD) has been developed for detecting pharmaceutical and agrochemical residues in honey (Calero et al., 2022); aptamer-based biosensors have also been employed to isolate and identify streptomycin, an aminoglycoside, in honey samples (Kong et al., 2024); enzyme-linked immunosorbent assays (ELISA) are commonly used for screening tetracycline residues in honey (Yanovych et al., 2023) 43 along with microbiological screening methods for detecting various drug residues.
Sample preparation strategies for honey analysis
Although LC–MS/MS is a powerful analytical technique for unequivocally detecting and quantifying multiple pharmaceutical residues, matrix effects in honey can significantly affect sensitivity and selectivity. Therefore, it is essential to select and optimize the appropriate sample pretreatment and extraction methods. Currently, the focus is on minimizing the sample volume and processing time while maintaining high extraction efficiency (Leite et al., 2020). Different extraction methods are often employed for the extraction of pharmaceutical residues from honey and other hive products, such as solid-phase extraction (SPE), dispersive solid-phase extraction (d-SPE), Liquid-Liquid Extraction (LLE), Quick Easy Cheap Effective Rugged Safe (QuEChERS), and simple dilution (Rodrigues et al., 2024)34. SPE and d-SPE are the most conventional extraction methods, using different solid-phase cartridges, such as C18, Strata X, and Oasis HLB. These methods offer high selectivity by eliminating unwanted analytes and matrices. SPE compensates for many limitations of LLE, such as requiring less solvent volume, more selectivity, and easy operation, and can be automated and integrated with other sample pretreatment techniques (Ścigalski & Kosobucki, 2020). The main limitation of this technique is the relatively long sample preparation time. In addition, there are some variations in SPE, such as the Matrix Solid Phase Dispersion (MSPD), Solid Phase Microextraction (SPME), and Stir Bar Sorptive Extraction (SBSE) (Ścigalski & Kosobucki, 2020).
Liquid-Liquid Extraction (LLE) can also present different possibilities, such as dispersive liquid-liquid microextraction (DLLME), hollow fiber liquid-phase microextraction (HF-LPME), salting-out liquid-liquid extraction (SALLE), aqueous two-phase system extraction (ATPS), single drop microextraction (SDME), liquid-phase microextraction (LPME), and sugaring-out assisted liquid-liquid extraction (SULLE) (Khatibi et al., 2022). Because honey is mainly composed of different sugars, a SULLE strategy is important for eliminating the main sugars and allowing the extraction and purification of diverse target pharmaceutical compounds. Freeze-assisted sugaring-out LLE is another approach developed by Fei et al. (2024) for the extraction of perchlorate from honey. Ultrasonicate-assisted Dispersive Liquid-Liquid Microextraction is a modification made by DLLME to improve extraction efficiency and minimize equilibrium time. A deep-eutectic-solvent-based LLME was developed by Uddin et al. (2025) for the determination of antibiotics in honey.
QuEChERS has been used for residue analysis of pesticides, antibiotics, hormones, mycotoxins, organic pollutants, and polycyclic aromatic hydrocarbons in environmental and food-derived matrices (Kim et al., 2021). Honey analysis is usually carried out using acetonitrile with a modified QuEChERS method (Baša Česnik et al., 2019).
Multi-detection of pharmaceutical veterinary medicines in hive products
Beeswax, pollen, propolis, and royal jelly are the most widely consumed bee-derived products. These substances are produced by bees and collected from the hive; therefore, residues of pharmaceutical medicines can appear in any of them and from multiple sources. To monitor the occurrence of these compounds, either to ensure consumer safety or to understand their effects on the health of bee colonies, it is important to develop efficient analytical methods. The use of multi-detection and multi-class detection methods for pharmaceutical veterinary medicines in hive products offers several significant advantages for both analytical performance and food safety assurance. This approach facilitates the concurrent identification of multiple drug classes, including antibiotics, anti-inflammatory agents, and antiparasitic compounds, in a single analysis. This enhancement in efficiency results in a reduction in the laboratory workload. This approach has been shown to reduce both sample and solvent consumption, leading to a decrease in costs and environmental impact. Furthermore, the use of modern techniques such as LC-MS/MS and GC-MS/MS ensures high levels of sensitivity, selectivity, and accuracy, even when analyzing complex matrices such as honey, wax, or propolis. The utilization of multi-detection methods in conjunction with comprehensive screening facilitates the expeditious identification of contamination sources and augmentation of regulatory control, thereby ensuring that bee-derived products meet the requisite safety standards. Moreover, integration of this analytical approach with emerging technologies, including molecular imprinting, biosensors, and artificial intelligence, has the potential to enhance the speed, automation, and data interpretation of the process. Multidetection has been demonstrated to be a cost-effective, time-efficient, and sustainable solution that supports the principles of One Health by safeguarding human, animal, and environmental health. (Lewiński et al., 2023).
A compilation of recent studies demonstrates that advanced sample preparation and chromatographic techniques are at the core of this strategy. The main advantages of multi-detection techniques for pharmaceutical residues in hive products include their high coverage range, enabling the simultaneous identification of multiple drug classes and contaminants within a single sample preparation and chromatographic analytical run. These methods require only small sample amounts (ranging from 1 to 5 g) and still reach low analytical concentration thresholds; use of advanced extraction sorbents, such as molecularly imprinted polymers (MIP), solid-phase extraction (SPE), and QuEChERS-based methods, which enhance selectivity and method robustness, reduce matrix effects, and improve accuracy. Finally, multi-detection methods provide sufficient data to support both the targeted quantification of known compounds and non-targeted screening for emerging or unknown contaminants. This characteristic makes them powerful and reliable tools for ensuring the safety and quality of hive products.
Emerging technologies for screening pharmaceutical contaminants in honey
Molecular Imprinting Technology is one of the newest innovative advances in sample preparation. Molecularly Imprinted Polymers (MIPs) are synthetic sorbents capable of producing specific binding sites that selectively capture target analytes. These polymers are produced through the interaction of target molecules (such as veterinary drugs) with functional monomers, either by covalent or noncovalent bonding, leading to the formation of polymeric networks that mimic the structural and chemical characteristics of the analyte. MIPs have demonstrated higher selectivity and extraction efficiency than conventional (Yanovych et al., 2023). The application of MIP-based Solid Phase Extraction (SPE) coupled with advanced chromatographic systems, such as LCMS/MS, has proven to be successful for the extraction of veterinary medicines from honey and related matrices. This cutting-edge technology can also be integrated into microchip systems to create miniaturized analytical platforms.
Portable biosensors are another rapid tool for multiresidue detection in environmental and animal-derived products, including honey. In addition to traditional chromatographic, electrochemical, ELISA, and spectroscopic methods, biosensors offer a practical analytical option close to the field. Recent developments have incorporated advanced nanomaterials such as carbon nanotubes, gold nanoparticles, and graphene oxides to enhance their sensitivity and specificity (Elbasiony & Alharthi, 2024). Despite their potential, portable biosensors still face technical limitations, including minimal sample volumes, degradation of biological components, and integration challenges between sample preparation and data-processing systems. Nevertheless, increasing environmental contamination and population growth are driving the demand for portable and miniaturized options such as biosensors capable of detecting residual contaminants in food systems (Jafarzadeh et al., 2024).
Artificial Intelligence (AI) and Machine Learning (ML) are emerging as innovative tools for honey analysis. Honey adulteration and contamination are significant concerns, and the complexity of hive products is a significant challenge. Advanced computational methods have been successfully applied to detect contaminants and ensure product authenticity (Ahmed, 2024). The integration of ML algorithms with spectral and chromatographic techniques, such as infrared (IR), nuclear magnetic resonance (NMR), Fourier-transform infrared (FTIR), Raman spectroscopy, and high-resolution mass spectrometry (HRMS), provides new and unlimited possibilities for multiresidue detection and pattern recognition (Tang et al., 2025). Recent advances by Sánchez et al. combined the least absolute shrinkage and selection operator (LASSO) with support vector regression (SVR) and ultra-fast gas chromatography (UF-GC) to demonstrate how machine learning can enhance analytical performance and interpretation accuracy.
New advanced technologies mark a significant step forward in the monitoring and quality control of honey. By combining high selectivity and sensitivity from molecular imprinting, portability and rapid response of biosensors, and predictive power of artificial intelligence, future analytical systems will be more efficient, sustainable, and accessible. Such progress will not only strengthen food safety and consumer confidence, but also support environmental monitoring and regulatory compliance, ultimately contributing to a more resilient and transparent apicultural sector. The integration of new advanced technologies represents an innovative approach for improving efficient monitoring and quality control of honey. By combining high selectivity and sensitivity from molecular imprinting, portability and rapid response of biosensors, and predictive power of artificial intelligence, future analytical systems will be more efficient and accessible. (Gusarov & Gusarova, 2023). Such progress will not only strengthen food safety and consumer confidence but also support environmental monitoring and regulatory compliance, ultimately contributing to a more resilient apicultural sector.
Conclusion
The overall use of veterinary drugs in animal production and agriculture is essential for managing animal health and productivity, which poses a significant threat to the environment and pollinator health. This review highlights the mapping of different exposure pathways for pharmaceuticals entering the ecosystem through runoff from livestock farms, manure application, direct hive treatments, and atmospheric deposition. Honeybees are key pollinators and are prone to the acute and sublethal effects of diverse pharmaceuticals and agrochemicals, resulting in microbial dysbiosis, immune suppression, reproductive dysfunction, and behavioral impairments. The synergistic effect between veterinary drugs and agrochemicals can contribute to the accelerated increase in AMR, which is a global threat. There are significant knowledge gaps regarding the environmental exposure of bees, particularly to veterinary drugs and agrochemicals that persist in the air after administration and low dose, chronic effects, and interactions among diverse contaminants. This requires interdisciplinary research and the implementation of one health approach to reduce risks. The continuous discovery and studies to archive new methodologies are fundamental, as honey and other hives products are one of the multiple examples in the nature of complex matrixes that are being polluted not only, but mainly by veterinary drugs, creating a general prejudice in the environment and animal and human health. Despite all efforts to avoid the occurrence of contaminants, minimize risks, and avoid undesirable cross-contamination, it is impossible to have a clean system, which makes information, detection, and legislation about these drugs and how to use them even more important, giving farmers, bee keepers, and society all the support they might need to prevent and to take care of this One Health that covers every human being and their quality of life.
Ethics and consent
Ethical approval and consent were not required.
Data availability
No data associated with this article.
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