Advancements and greenification potential of magnetic molecularly imprinted polymers for chromatographic analysis of veterinary drug residues in milk.

Milk, as a widely consumed nutrient-rich food, is crucial for bone health, growth, and overall nutrition. The persistent application of veterinary drugs for controlling diseases and heightening milk yield has imparted substantial repercussions on human health and environmental ecosystems. Due to the high demand, fresh consumption, complex composition of milk, and the potential adverse impacts of drug residues, advanced greener analytical methods are necessitated. Among them, functional materials-based analytical methods attract wide concerns. The magnetic molecularly imprinted polymers (MMIPs), as a kind of typical functional material, possess excellent greenification characteristics and potencies, and they are easily integrated into various detection technologies, which have offered green approaches toward analytes such as veterinary drugs in milk. Despite their increasing applications and great potential, MMIPs' use in dairy matrices remains underexplored, especially regarding ecological sustainability. This work reviews recent advances in MMIPs' synthesis and application as efficient sorbents for veterinary drug extraction in milk followed by chromatographic analysis. The uniqueness and effectiveness of MMIPs in real milk samples are evaluated, current limitations are addressed, and greenification opportunities are proposed. MMIPs show promise in revolutionizing green analytical procedures for veterinary drug detection, aligning with the environmental goals of modern food production systems.

Combining GC-MS/MS with a LLE-SPE sample pretreatment step to simultaneously analyse enrofloxacin and ofloxacin residues in chicken tissues and pork.

A widely applicable original gas chromatography-tandem mass spectrometry (GC-MS/MS) method was explored to qualitatively and quantitatively measure enrofloxacin and ofloxacin residues in chicken tissues and pork. The experimental samples were processed based on liquid-liquid extraction (LLE) and solid-phase extraction (SPE). Trimethylsilyl diazomethane (TMSD) was chosen to react derivatively with enrofloxacin and ofloxacin. In total, 78.25% âˆ¼ 90.56% enrofloxacin and 78.43% âˆ¼ 91.86% ofloxacin was recovered from the blank fortified samples. The limits of detection (LODs) were 0.7-1.0 µg/kg and 0.1-0.2 µg/kg, respectively. The limits of quantitation (LOQs) were 1.6-1.9 µg/kg and 0.3-0.4 µg/kg, respectively. It was verified that various experimental data met the requirements of the FAO & WHO (2014) for the detection of veterinary drug residues. Real samples obtained from local markets were analysed using the established method, and no residues of enrofloxacin or ofloxacin were detected in the samples.

Novel ratiometric electrochemical aptasensor based on broad-spectrum aptamer recognition for simultaneous detection of penicillin antibiotics in milk.

To effectively monitor multi-residues of penicillin antibiotics (PENs) in milk, we developed a novel ratiometric electrochemical aptasensor enabling simultaneous detection of PENs. The aptasensor employed a broad-spectrum aptamer as a recognition element, niobium carbide functionalized with methylene blue (Nb2C-MB) as a reference signal generator, and a ferrocene-labeled aptamer (Fc-Apt) as an output signal. Electrodes were modified with Fe-N-C doped carbon nanotubes (Fe-N-C-CNTs) to amplify detection signals further. During detection, Fc-Apt binding to PENs decreased Fc current intensity (IFc) and increased MB current intensity (IMB). The simultaneous detection of PENs was achieved using IMB/IFc as a quantitative signal. Under optimal conditions, a good linear relationship between IMB/IFc and antibiotic concentration was observed, indicating the aptasensor had a robustness. The limits of detection of aptasensor for four penicillin antibiotics and their mixed targets were 0.093-0.191 nM. This work provides a new approach to multi-residue detection of the same class of antibiotics.

Development and performance evaluation of a microbiological method for screening and LC-MS/MS for conformation of sulfonamides in animal-derived foods.

This study developed a highly sensitive microbiological method utilizing a novel microtiter plate to screen 10 sulfonamides in chicken muscles, eggs, and prawns. This plate was fabricated from agar incorporating trimethoprim and spread with Bacillus megaterium. After residue detection by bioassay, the same test solutions were analyzed by LC-MS/MS for accurate identification and quantification. It also proved eco-friendly compared to using other quantitative methods. The residual drugs were extracted with McIlvaine buffer and purified using an Oasis® MCX cartridge. A triethylamine/methanol/water (0.5:75:24.5, v/v/v) mixture was used as the eluate. The obtained LOD values of the bioassay ranged from 5 to 25 µg kg-1 allowing the detection of the target drugs at the MRLs established in Japan. Adhering to ISO/IEC 17025 standards, the performance of the bioassay was evaluated. Based on the inhibition zone size in bioassay results, quality control yielded a Z score within ±2, indicating reasonable control over the screening process. Proficiency testing of a chicken muscle sample spiked with sulfadimidine demonstrated the inhibition zone detection of the bioassay and quantified value alignment of LC-MS/MS with reference values. In a surveillance study of 91 samples, sulfamethoxazole was detected in one prawn sample.

Hypersensitive electrochemical sensor based on thermally annealed gold-silver alloy nanoporous matrices for the simultaneous determination of sulfathiazole and sulfamethoxazole residues in food samples.

In this study, we have developed a novel, hypersensitive, and ultraselective electrochemical sensor containing thermally annealed gold-silver alloy nanoporous matrices (TA-Au-Ag-ANpM) integrated with f-MWCNTs-CPE and poly(l-serine) nanocomposites for the simultaneous detection of sulfathiazole (SFT) and sulfamethoxazole (SFM) residues in honey, beef, and egg samples. TA-Au-Ag-ANpM/f-MWCNTs-CPE/poly(l-serine) was characterized using an extensive array of analytical (UV-Vis, FT-IR, XRD, SEM, and EDX), and electrochemical (EIS, CV and SWV) techniques. It exhibited outstanding performance over a wide linear range, from 4.0 pM to 490 µM for SFT and 4.0 pM to 520 µM for SFM, with picomolar detection and quantification limits (0.53 pM and 1.75 pM for SFT, 0.41 pM and 1.35 pM for SFM, respectively). The sensor demonstrated exceptional repeatability, reproducibility, and anti-interference capability, with percentage recovery of 95.6-102.4% in food samples and RSD below 5%. Therefore, the developed sensor is an ideal tool to address the current antibiotic residue crisis in food sources.

Residue, distribution and depletion of fluralaner in egg following a single intravenous and transdermal administration in healthy shaver hens: fluralaner residue in egg.

The demand for the use of fluralaner in an extra label manner is increasing due to lack of efficacious treatment to combat mites and bed bugs in the poultry industry in the United States. Fluralaner residue data in eggs is lacking and residues might cause risks to human health. The present study aimed to determine the depletion profiles of fluralaner in eggs and estimate the drug withdrawal interval in whole eggs by adopting the US Food and Drug administration tolerance limit method with single intravenous (0.5 mg/kg) or transdermal administration (average 58.7 mg/kg) in healthy shaver hens. Hens were treated intravenously or trans-dermally with fluralaner. The eggs were collected daily for 28 d for intravenous treated and for 40 d from the transdermal route group. Fluralaner concentrations in yolk and albumen were determined by mass spectrometry. The greater percentage of fluralaner was observed in yolk when compared to the albumen for both administration routes. Noncompartmental analysis was used to calculate the pharmacokinetic parameters in yolk, albumen and whole egg. The longest apparent half-life confirmed in yolk was 3.7 d for intravenous and 14.3 d for the transdermal route. The withdrawal intervals in whole egg for fluralaner following the intravenous and transdermal administration were 7 d and 81 d, respectively, with maximum residue limits (1.3 µg/g) at 13 d and 171 d, respectively, based on the limit of quantification (0.4 µg/g) from the analytical assay reported by EMA and APVMA.

Advances in magnetic analytical extraction techniques for detecting antibiotic residues in edible samples.

The widespread use of antibiotics in agricultural and animal husbandry to treat bacterial illnesses has resulted in a rise in antibiotic-resistant bacteria. These bacteria can grow when antibiotic residues are present in food items, especially in edible animal products. As a result, it is crucial to monitor and regulate the amounts of antibiotics in food. Magnetic analytical extractions (MAEs) have emerged as a potential approach for extracting antibiotic residues from food using magnetic nanoparticles (MNPs). Recent improvements in MAEs have resulted in the emergence of novel MNPs with better selectivity and sensitivity for the extraction of antibiotic residues from food samples. Consequently, this review paper addresses current developments in MAE for extracting antibiotic residues from edible samples. It also provides a critical analysis of contemporary MAE practices. The current issues and potential future developments in this field are also discussed, thereby providing a framework for future study paths.

Stability of thirteen antimicrobials in incurred samples of animal tissues, milk, eggs, and honey after freeze-storage.

Monitoring of antimicrobials residues in food of animal origin is performed by control laboratories to ensure public health, and knowledge of the stability of antimicrobials during storage is essential for the reliability of results. For stability studies, analysis of incurred samples is preferential to fortified samples due to the possible conversion of antimicrobial metabolites back to parent compounds during sample preparation, storage, and analysis of the incurred samples, resulting in an increased concentration of the analyte. We have analyzed the concentrations of 13 antimicrobials from 8 groups (tetracyclines, fluoroquinolones, phenicols, sulfonamides, aminoglycosides, penicillins, macrolides, and nitroimidazoles) at different time points of freeze-storage (1 week; 1, 2, and 3 months) using HPLC-MS/MS. Incurred samples were prepared from muscle tissue, liver, kidneys, eggs, and milk taken from different animals (cows, pigs, poultry, goats, and fish). Incurred and fortified samples of honey were investigated as well. The results have shown that all analytes in all samples were stable during the investigated periods regardless of animal species, matrix, and concentration levels.

Assessment of safety and quality aspects of boiling treatment of quail eggs.

A total of 300 quail eggs were collected randomly from different markets in Cairo and Giza Governorates. Five eggs were represented as one egg sample. Shell and content of each egg were examined for their microbiological contents, sensory evaluation and study of Escherichia coli O157 survival in artificially contaminated eggs. Moreover, qualitative detection of antimicrobial residues by seven plates microbiologically bioassay and confirmed by validated high-performance liquid chromatography (HPLC) methods for positively reacted antimicrobials in raw and boiled samples. There was a significant difference (P < 0·05) between the grading score of eggs after the boiling at 2-, 4-, 5- and 7-min. Based on the survival results, the refrigeration storage and boiling for 5 min of quail eggs was confirmed that such eggs are without E. coli O157. After the boil, the concentrations of oxytetracycline (OTC) and 4-Epi-OTC residues were significantly reduced, and there was no effect on the concentration of sulphadimidine (SDD), amoxicillin (AMO) and Diketo residues. Samples that exceeded the maximum residual limits (MRLs) were 17·0%, 12·0%, 10·0%, 16·0% and 14·0% for SDD, OTC, 4-Epi-OTC, AMO and Diketo, respectively. After boiling, no significant change was noted for SDD, AMO and Diketo, but all OTC and 4-Epi-OTC were completely below MRLs. Therefore, SDD and AMO with their metabolite (Diketo) are heat-stable antimicrobial residues with multiple human health hazards.

Efficient peroxymonosulfate activation by N-rich pyridyl-iron phthalocyanine derivative for the elimination of pharmaceutical contaminants under solar irradiation.

It is of great significance for improving electron transmission performance by changing of the outer ring structure of iron phthalocyanine. Herein, 4 (pyridine-2, 3-yl) iron phthalocyanine (FepyPc), as N-rich pyridyl-iron phthalocyanine derivative, was introduced to degrade pharmaceutical contaminants. The catalytic degradation of organic pollutants with FepyPc was studied by activating peroxymonosulfate (PMS) at room temperature. The results clarified that the removal rate of carbamazepine (CBZ) was close to 100% within 60 min and the calculated apparent rate constant was about 2 times larger than FePc, which proved that FepyPc had superior performance. Four active species were identified for the degradation of CBZ, including superoxide radical (•O2-), singlet oxygen (1O2), sulfate radical (SO4•-) and hydroxyl radical (•OH). In addition, the possible reaction mechanism was inferred in FepyPc/PMS/sunlight system for CBZ removal. Finally, the CBZ degradation pathway was proposed by using ultra-performance liquid chromatography and high definition mass spectrometry (UPLC/HDMS). This research provided a meaningful and efficient method for the elimination of pharmaceutical contaminants.

An ultrasensitive colloidal gold immunosensor to simultaneously detect 12 beta (2)-adrenergic agonists.

In this study, we first prepared a selective monoclonal antibody against 12 beta (2)-adrenergic agonists (Salbutamol, Clenbuterol, Brombuterol, Clenpenterol, Mabuterol, Carbuterol, Cimbuterol, Mapenterol, Pirbuterol, Terbutaline, Cimaterol, and Clenproperol). Then three haptens were designed and derived, among which, haptenS3 used the amino group of the salbutamol analog to derive a carboxyl group containing a spacer, which is unique to this study. The half-maximal inhibitory concentration (IC50) values were 0.35 ng/mL (Salbutamol), 0.42 ng/mL (Clenbuterol), 0.78 ng/mL (Brombuterol), 0.88 ng/mL (Clenpenterol), 1.34 ng/mL (Mabuterol), 1.38 ng/mL (Carbuterol), 1.71 ng/mL (Cimbuterol), 2.24 ng/mL (Mapenterol), 2.25 ng/mL (Pirbuterol), 2.27 ng/mL (Terbutaline), 3.49 ng/mL (Cimaterol), and 4.89 ng/mL (Clenproperol). We further developed a monoclonal antibody-based colloidal gold immunochromatographic test strip for screening and detecting 12 beta (2)-adrenergic agonists in swine urine and lamb samples. The immunochromatographic method developed in this study is a suitable tool for the on-site rapid detection and screening of beta (2)-adrenergic agonists in swine urine and lamb samples.

Comparison of analyte identification criteria and other aspects in triple quadrupole tandem mass spectrometry: Case study using UHPLC-MS/MS for regulatory analysis of veterinary drug residues in liquid and powdered eggs.

Ultrahigh-performance liquid chromatography (UHPLC) coupled with triple quadrupole tandem mass spectrometry (MS/MS) is one of the most powerful tools for the multiclass, multiresidue analysis of veterinary drugs, pesticides, mycotoxins, and other chemical contaminants in foods and other sample types. Until approximately 2010, commercial MS/MS instruments using multiple reaction monitoring (MRM) were generally limited to minimum dwell (and inter-dwell) times of 10 ms per ion transition. To achieve the needed accuracy and detection limits for hundreds of targeted analytes, older UHPLC-MS/MS methods typically acquired only two ion transitions per analyte (yielding only one ion ratio for qualitative identification purposes), which is still the norm despite technological advancements. Newer instruments permit as little as 1 ms (inter-)dwell times to afford monitoring of more MRMs/analyte with minimal sacrifices in accuracy and sensitivity. In this study, quantification and identification were assessed in the validation of 169 veterinary drugs in liquid and powdered eggs. Quantitatively, an "extract-and-inject" sample preparation method yielded acceptable 70-120% recoveries and < 25% RSD for 139-141 (82-83%) of the 169 diverse drug analytes spiked into powdered and liquid eggs, respectively, at three levels of regulatory interest. Qualitatively, rates of false positives and negatives were compared when applying three different regulatory identification criteria in which two or three MRMs/drug were used in each case. Independent of the identification criteria, rates of false positives remained <10% for 95-99% of the drugs whether 2 or 3 ions were monitored, but the percent of drugs with >10% false negatives decreased from 25-45 to 10-12% when using 2 vs. 3 MRMs/analyte, respectively. Use of a concentration threshold at 10% of the regulatory level as an identification criterion was also very useful to reduce rates of false positives independent of ion ratios. Based on these results, monitoring >2 ion transitions per analyte is advised when using MS/MS for analysis, independent of SANTE/12682/2019, FDA/USDA, or 2002/657/EC identification criteria. (Quant)identification results using all three criteria were similar, but the SANTE criteria were advantageous in their greater simplicity and practical ease of use.

A nanocomposite adsorbent of metallic copper, polypyrrole, halloysite nanotubes and magnetite nanoparticles for the extraction and enrichment of sulfonamides in milk.

A composite adsorbent composed of metallic copper (Cu), polypyrrole (PPy), halloysite nanotubes (HNTs) and magnetite nanoparticles (Fe3O4) was developed to extract and enrich sulfonamides by dispersive magnetic solid phase extraction. The composite could adsorb sulfonamides via hydrogen bonding and hydrophobic, π-π and π-electron-metal interactions. The extraction conditions were optimized and the developed composite adsorbent was characterized and provided a large surface area that enhanced extraction efficiency for sulfonamides. Coupled with high performance liquid chromatography, the adsorbent was used to quantitatively determine sulfonamides found in milk samples. The response of the developed method exhibited linearity from 5.0 to 150.0 µg kg-1 for sulfathiazole, and from 2.5 to 100.0 µg kg-1 for sulfamerazine, sulfamonomethoxine and sulfadimethoxine. Limits of detection were between 2.5 and 5.0 µg kg-1. Recoveries of sulfonamides in milk samples ranged from 83.0 to 99.2% with RSDs lower than 6%. The developed composite adsorbent showed good reproducibility and reusability.

Simultaneous determination of 11 quinolone residues in freshwater fish samples by magnetic solid-extraction coupled to liquid chromatography-tandem mass spectrometry.

In this study, well-defined core-shell ethylenediamine-functional magnetic ferroferric oxide polymers were prepared and were fully characterized by transmission electron microscopy, scanning electron microscopy, FTIR spectroscopy, and vibrating sample magnetometry. Then, it was used as a magnetic solid-phase extraction adsorbent for simultaneous determination of 11 trace quinolone residues in freshwater fish samples coupled to liquid chromatography-tandem mass spectrometry. The obtained results revealed that the adsorbent showed good extraction efficiency and the adsorption mechanisms referred to hydrogen bond and π-π stacking interaction. Moreover, the magnetic solid-phase extraction conditions were also carefully optimized. The limits of quantitation of 11 quinolones were in the range of 0.15-0.36 µg/kg, while spiking recoveries were in the range of 80.2-99.5% for the 11 quinolones in freshwater fish samples at four spiked levels including limits of quantitation, 1.0, 40.0, and 80.0 µg/kg with the relative standard deviations ranging from 0.8 to 9.1%. The proposed method was applied to analyze 45 freshwater fish samples, and enrofloxacin was detected in 91.1% samples with concentrations ranging from 0.659 to 333 µg/kg. It could be concluded that the proposed method is fast, simple, sensitive, and accurate for the routine monitor of freshwater fish.

Natural thymol-based ternary deep eutectic solvents: Application in air-bubble assisted-dispersive liquid-liquid microextraction for the analysis of tetracyclines in water.

Four new thymol-based ternary deep eutectic solvents were prepared and evaluated as the extractive phase in air-bubbles assisted dispersive liquid-liquid microextraction for extraction of tetracycline, doxycycline, and oxytetracycline from the water before high-performance liquid chromatography. The maximum extraction efficiencies were obtained using 400 µL of [choline chloride]:[thymol]:[nonanoic acid] in the molar ratio of 1:2:2 at pH = 5. The solvent was characterized by FTIR and NMR spectroscopy. The hydrophobicity of the deep eutectic solvent and its effect on the pH of water samples after mixing was also studied. Besides, the extraction efficiency of the ternary deep eutectic solvent was compared with that of two binary thymol-based deep eutectic solvents, including [choline chloride]:[thymol] and [thymol]:[nonanoic acid] at the same conditions. Under optimal conditions, limits of detection and quantification were 1.2-8.0 and 3.8-26.6 µg/L, respectively. The linear ranges were 18.2-500 µg/L for oxytetracycline, 26.6-500 µg/L for tetracycline, and 3.8-500 µg/L for doxycycline with the determination coefficients > 0.9912. Intra- and inter-day relative standard deviations were 1.2-3.8 and 7.7-11.2%, respectively. The developed method was applied to the analysis of tetracyclines in unspiked and spiked environmental water samples, and the obtained recoveries were 74.5-95.4% with relative standard deviations of 1.2-4.0%.

Effect of food processing (fish burger preparation and frying) on residual levels of enrofloxacin and ciprofloxacin.

The influence of fish burger preparation and frying on residual levels of enrofloxacin (ENR) and ciprofloxacin (CIP) was evaluated. For this purpose, a high-throughput liquid chromatography-mass spectrometry analytical method for the quantitation of ENR and CIP residues in tilapia products (fillet, raw fish burger and fried fish burger) was developed and validated based on European and Brazilian guidelines. Sample preparation was accomplished by extraction with acidified acetonitrile followed by clean-up with hexane. Chromatographic analysis was performed on a C18 column using isocratic elution with 0.1% formic acid and acetonitrile (85:15 v:v). The analytical method showed suitable performance to quantify the residual levels of ENR and CIP in the studied matrices. No reduction in the residual levels of ENR and CIP was observed during fish burger preparation and only a 10% reduction occurred as a consequence of frying, indicating that both compounds were stable to the preparation of the fish burger and to frying conditions.

A single method to analyse residues from five different classes of prohibited pharmacologically active substances in milk.

In the European Union, the use of veterinary drugs belonging to the A6 group is prohibited in food-producing animals according to Commission Regulation (EU) No. 2010/37. The aim of this study was to improve the analytical control strategy by developing a single method to analyse residues of prohibited pharmacologically active substances in milk. For this, a single method was developed to analyse 16 prohibited pharmacologically active substances belonging to five different substance classes at required or recommended levels: nitroimidazoles at 3 µg kg-1, nitrofurans at 0.5 µg kg-1, chloramphenicol at 0.1 µg kg-1, dapsone at 5 µg kg-1 and chlorpromazine at 1 µg kg-1. Milk sample preparation started with an acid hydrolysis combined with a derivatisation. These steps were followed by a clean-up consisting of a dispersive solid-phase extraction and a liquid-liquid extraction. Finally, the sample extracts were analysed by liquid chromatography combined with tandem mass spectrometry, operating alternately in the positive and negative mode. The method was fully validated according to Commission Decision 2002/657/EC for bovine milk and additionally validated for caprine milk. The validation proved that the method is highly effective to detect and confirm all 16 substances in bovine and caprine milk and, additionally to quantify 15 of these substances in bovine milk and 13 of these substances in caprine milk. This study resulted in a new multi-class method to detect, quantify and confirm the identity of 16 prohibited pharmacologically active substances belonging to five different substance classes in two types of milk.

Multi-class analysis of 30 antimicrobial residues in poultry feathers by liquid chromatography tandem mass spectrometry.

Poultry feathers are nowadays partially re-introduced into the animal food chain and the environment. They are valorised by their transformation into feather meal in order to be used as fertilisers in agriculture but also in animal feed (in particular, pet food and fish feed). However, unlike food producing animals for humans, feathers from poultry animals are not subject to a ban or regulatory limits on the presence of antibiotic residue after veterinary treatment. Feathers could therefore be a potential reservoir of antibiotic residues, unintentionally exposing the environment and animals through food, which might contribute to the emergence of antibiotic resistance. To this end, a multi-class liquid chromatographic method coupled with tandem mass spectrometry (LC-MS/MS) was developed for the detection and determination of residues of 30 antibiotics from eight groups of antibacterial (quinolones, lincosamides, macrolides, penicillins, phenicols, tetracyclines, sulphonamides and diaminopyrimidines) in feathers. The extraction of the analytes from the feathers was carried out by the salting out technique. The separation of the analytes employed a Kinetex C18 column. Quantification was made using internal standards. All analytes have been validated according to the performance criteria of Decision 2002/657/EC. Trueness of the method ranged from to 93% to 111% for all analytes and intermediate precision were to 1.2-18.8%. The limits of quantification (LOQ) were from 13 to 150 µg kg-1 depending on the analytes. The method is suitable for the monitoring and quantification of antibiotic residues in feathers over the range 13-600 µg kg-1 depending on the compound.

Depletion of clomiphene residues in eggs and muscle after oral administration to laying hens.

The selective oestrogen receptor modulator (SERM) clomiphene is therapeutically used to induce ovulation. While prohibited as a doping agent in sports, it is frequently detected in sports drug testing urine samples. Few reports exist on clomiphene's (illicit) use in the farming industry to increase the egg production rate of laying hens, which creates a risk that eggs as well as edible tissue of these hens contain residues of clomiphene. To investigate the potential transfer of clomiphene into eggs and muscle tissue, laying hens were orally administered with clomiphene citrate at 10 mg/day for 28 days. To determine clomiphene residues in eggs, chicken breast and chicken thigh, the target analyte was extracted from homogenised material with acetonitrile and subjected to ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis. The test method reached a limit of quantification (LOQ) of 1 µg/kg and was characterised concerning specificity, precision, trueness and linearity. Analyses were performed on whole egg, egg white and yolk separately, and chicken muscle from breast and thigh. Clomiphene was detectable in eggs two days after the beginning of the drug administration period. The drug concentrations increased to 10-20 µg per egg within one week, and after withdrawal of clomiphene, residues decreased after 4 days, but traces of clomiphene were still detectable until the end of the study (14 days after the last administration). In the chicken's muscle tissue, clomiphene levels up to 150 µg/kg (thigh) and 36 µg/kg (breast) were found. Six days after the last dose, tissue clomiphene concentrations fell below the LOQ. Overall, these results underline the concerns that clomiphene may be transferred into animal-derived food and future research will therefore need to focus on assessing and minimising the risk of unintentional adverse analytical findings in doping controls.