Residues of veterinary antibiotics in solid natural and organic fertilizers-method development and sample analysis.

Livestock excrement is used around the world as natural fertilizers or, after processing, as organic fertilizers for crops and grasslands. But due to the presence of veterinary antibiotics in them, they may pose a threat not only to the natural environment, mainly to soil microorganisms, but also to human and animal health. This article describes a method for detecting 21 antibacterial substances in solid natural and organic fertilizers. Antibiotics from fertilizers were extracted with a mixture of acetonitrile and McIlvain-Na2EDTA buffer, twice. The extracts were purified by solid phase extraction technique on Strata-X cartridges and analyzed with the use UHPLC-MS/MS technique. The method was validated in accordance with EU Commission Implementing Regulation 2021/808; the obtained recovery ranged from 93.6 to 116.6% (depending on the analytes), and the linearity ranged from 50 to 1000 µg/kg. The developed method was used to analyze 73 samples of solid natural and organic fertilizers. Our research has shown that over 38% of natural fertilizers were contaminated with antibiotics, mainly doxycycline in concentrations reaching several dozen milligrams per kilogram of fertilizers. In the case of processed organic fertilizers, the presence of antibiotics was found in over 37% of the analyzed samples. The research results showed that the developed and validated analytical method may be useful for assessing the presence and content of antibacterial substances in solid natural and organic fertilizers.

The Identification and Quantification of 21 Antibacterial Substances by LC-MS/MS in Natural and Organic Liquid Fertilizer Samples.

Antibiotics in animal production are widely used around the world for therapeutic and preventive purposes, and in some countries, they still serve as antibiotic growth stimulants. Regardless of the purpose of using antibiotics in livestock, they may be present in animal tissues and organs as well as in body fluids and excretions (feces and urine). Farm animal excrement in unprocessed form (natural fertilizers) or processed form (organic fertilizers) is applied to agricultural fields because it improves soil fertility. Antibiotics present in fertilizers may therefore contaminate the soil, surface, groundwater, and plants, which may pose a threat to the environment, animals, and humans. Therefore, it is important to develop analytical methods that will allow for the control of the presence of antibacterial substances in natural and organic fertilizers. Therefore, in this study, an LC-MS/MS method was developed and validated for the determination of 21 antibacterial substances in natural and organic liquid fertilizers. The developed method was used to analyze 62 samples of natural and organic liquid fertilizers, showing that over 24% of the tested samples were contaminated with antibiotics, mainly from the group of tetracyclines and fluoroquinolones. Studies of post-fermentation sludge from biogas plants have shown that the processes of anaerobic methane fermentation, pH, and temperature changes taking place in bioreactors do not lead to the complete degradation of antibiotics present in the material used for biogas production. For this reason, monitoring studies of natural and organic fertilizers should be undertaken to limit the introduction of antibiotics into the natural environment.

Simultaneous determination of sulfonamides, trimethoprim, amoxicillin and tylosin in medicated feed by high performance liquid chromatography with mass spectrometry.

Introduction: The article presents a rapid and simple analytical procedure for determination of four sulfonamides (sulfadiazine, sulfamerazine, sulfamethazine and sulfamethoxazole), trimethoprim, tylosin and amoxicillin in animal medicated feed. Material and Methods: Eighteen medicated feed samples were analysed for active substances. The analytical protocol used a mixture of acetonitrile and 0.05 M phosphoric buffer, pH 4.5 for the extraction of seven antibacterial substances. After extraction, the samples were diluted in Milli-Q water and analysed by liquid chromatography with mass spectrometry. The developed procedure was subjected to validation in terms of linearity, selectivity, limits of quantification and determination, repeatability, reproducibility and uncertainty. Results: The validation of the method was carried out in accordance with the criteria set out in Commission Implementing Regulation (EU) 2021/808 and ICH guidelines. This method provided average recoveries of 90.8 to 104.5% with coefficients of variation for repeatability and reproducibility in the ranges of 3.2-6.9% and 5.2-8.3%, respectively for all analysed antibacterial substances. The limit of detection and limit of quantification for all seven analytes ranged from 5.4 mg/kg to 48.3 mg/kg and from 10.4 mg/kg to 119.3 mg/kg, respectively. The uncertainty of the method depending on the compound varied from 14.0% to 24.0%. The validated method was successfully applied to the 18 medicated feeds. Conclusion: The developed method can be successfully used to routinely control the content and homogeneity of seven antibacterial substances in medicated feed.

Clostridium perfringens-Opportunistic Foodborne Pathogen, Its Diversity and Epidemiological Significance.

The C. perfringens species is associated with various environments, such as soils, sewage, and food. However, it is also a component of the gastrointestinal (GI) microflora (i.e., microbiota) of sick and healthy humans and animals. C. perfringens is linked with different systemic and enteric diseases in livestock and humans, such as gas gangrene, food poisoning, non-foodborne diarrhoea, and enterocolitis. The strains of this opportunistic pathogen are known to secrete over 20 identified toxins that are considered its principal virulence factors. C. perfringens belongs to the anaerobic bacteria community but can also survive in the presence of oxygen. The short time between generations, the multi-production capability of toxins and heat-resistant spores, the location of many virulence genes on mobile genetic elements, and the inhabitance of this opportunistic pathogen in different ecological niches make C. perfringens a very important microorganism for public health protection. The epidemiological evidence for the association of these strains with C. perfringens-meditated food poisoning and some cases of non-foodborne diseases is very clear and well-documented. However, the genetic diversity and physiology of C. perfringens should still be studied in order to confirm the importance of suspected novel virulence traits. A very significant problem is the growing antibiotic resistance of C. perfringens strains. The aim of this review is to show the current basic information about the toxins, epidemiology, and genetic and molecular diversity of this opportunistic pathogen.

Insect Meals and Insect Antimicrobial Peptides as an Alternative for Antibiotics and Growth Promoters in Livestock Production.

The extensive use of antibiotics in animal production has led to the development of antibiotic-resistant microorganisms and the search for alternative antimicrobial agents in animal production. One such compound may be antimicrobial peptides (AMPs), which are characterized by, among others, a wide range of biocidal activity. According to scientific data, insects produce the largest number of antimicrobial peptides, and the changing EU legislation has allowed processed animal protein derived from insects to be used in feed for farm animals, which, in addition to a protein supplement, may prove to be an alternative to antibiotics and antibiotic growth promoters due to their documented beneficial impact on livestock health. In animals that were fed feeds with the addition of insect meals, changes in their intestinal microbiota, strengthened immunity, and increased antibacterial activity were confirmed to be positive effects obtained thanks to the insect diet. This paper reviews the literature on sources of antibacterial peptides and the mechanism of action of these compounds, with particular emphasis on insect antibacterial peptides and their potential impact on animal health, and legal regulations related to the use of insect meals in animal nutrition.

Prevalence of veterinary antibiotics in natural and organic fertilizers from animal food production and assessment of their potential ecological risk.

BACKGROUND: Veterinary antibiotics are emerging contaminants and enter into soil principally by agricultural application of organic fertilizers. This article presents the results of the research obtained for the analyzed 70 samples of fertilizers (pig and poultry manure and slurry and digestate) for various classes of antibiotics. RESULTS: Doxycycline, oxytetracycline, tetracycline, lincomycin, tiamulin and enrofloxacin were found in tested samples. Doxycycline was found as a dominant compound, and its highest concentration was 175 mg/kg in pig manure. This investigation indicated that fertilization with manure, especially animal feces, might be the primary source of antibiotics. Additionally, a risk assessment based on a risk quotient was carried out, which showed that the determined concentrations of antibiotics in fertilizers may pose a threat to soil microorganisms. CONCLUSIONS: Results suggested that the ecological risk effects of antibiotic contamination on soil bases and their potential adverse risk on human health needs special attention. © 2023 Society of Chemical Industry.

HPLC-FLD-Based Method for the Detection of Sulfonamides in Organic Fertilizers Collected from Poland.

Antibacterial substances such as sulfonamides are widely used in veterinary medicine to treat many bacterial diseases. After their administration to animals, up to 90% of the initial dose of the antibiotic is excreted in the feces and/or urine, which can be applied to farmland as natural or organic fertilizers. In this work, an analytical method was developed with the use of HPLC-FLD for the detection and quantification of five sulfonamides (sulfaguanidine, sulfadiazine, sulfamerazine, sulamethazine and sulfamethoxazol) in poultry and pig feces, slurry and digestates. The method was validated according to EU requirements (Commission Decision 2002/657/EC and VICH GL49). Linearity, decision limit, detection capability, detection and quantification limits, recovery, precision, and selectivity were determined, and adequate results were obtained. Using the HPLC-FLD method for all analyzed matrices, recoveries were satisfactory (77.00-121.16%), with repeatability and reproducibility in the range of 4.36-17.34% to 7.94-18.55%, respectively. Decision limit (CCα) and detection capability (CCß) were 33.87-67.63 and 53.36-92.00 µg/kg, respectively, and limit of detection (LOD) and limit of quantification (LOQ) were 13.53-23.30 and 26.02-40.38 µg/kg, respectively, depending on the analyte. The forty-four samples of natural and organic fertilizers were analyzed, and four samples showed sulfamethoxazole in the amount from range 158 to 11,070 µg/kg. The application of antibiotics including sulfonamides for farming animals is widespread and may lead to the development of antibiotic resistance and other environmental effects.

Identification and Quantification of 29 Active Substances by HPLC-ESI-MS/MS in Lyophilized Swine Manure Samples.

Veterinary drugs are frequently employed to treat and prevent diseases in food-producing animals to improve animal health and to avoid the introduction of microorganisms into the food chain. The analysis of the presence of pharmaceutical residues in animal manure could help to evaluate the legal and illegal practices during food production without harming the animals and to correctly manage manure when it is going to be applied as a fertilizer. This article describes a method for the simultaneous analysis of 29 active substances, mostly antibiotics and antiparasitic agents. Substances were extracted from lyophilized manure with a methanol:McIlvaine solution and analyzed with HPLC-ESI-MS/MS and a C18 HPLC column. The method was validated following European guidelines, the achieved trueness was between 63 and 128% (depending on the analytes), and the linearity was between 100 and 1500 µg/kg. The applicability of the method was demonstrated in 40 manure samples collected from pig farms where tetracycline was quantified in 7.5% of the samples. These results show the viability of this non-invasive method for the control of the legal and illegal administration of pharmaceuticals in food-producing animals.

Application of Micellar Mobile Phase for Quantification of Sulfonamides in Medicated Feeds by HPLC-DAD.

Rapid chromatographic procedure for quantification of five sulfonamides in medicated feeds are proposed. Satisfactory separation of sulfonamides from medicated feeds was achieved using a Zorbax Eclipse XDB C18 column (4.6 × 150 mm, 5 µm particle size) with a micellar mobile phase consisting of 0.05 M sodium dodecyl sulphate, 0.02 M phosphate buffer, and 6% propan-2-ol (pH 3). UV quantitation was set at 260 nm. The proposed procedure allows the determination of sulfaguanidine, sulfadiazine, sulfamerazine, sulfamethazine, and sulfamethoxazole in medicated feeds for pigs and poultry. Application of the proposed method to the analysis of five pharmaceuticals gave recoveries between 72.7% to 94.7% and coefficients of variations for repeatability and reproducibility between 2.9% to 9.8% respectively, in the range of 200 to 2000 mg/kg sulfonamides in feeds. Limit of detection and limit of quantification were 32.7-56.3 and 54.8-98.4 mg/kg, respectively, depending on the analyte. The proposed procedure for the quantification of sulfonamides is simple, rapid, sensitive, free from interferences and suitable for the routine control of feeds. In the world literature, we did not find the described method of quantitative determination of sulfonamides in medicated feeds with the use of micellar liquid chromatography.

Analytical capabilities of micellar liquid chromatography and application to residue and contaminant analysis: A review.

Green chemistry is the use of chemistry to reduce or eliminate the use of generation of feedstocks, products, by-products, solvents, reagents, etc. that are hazardous to human health or the environment. One of the branches of green chemistry is micellar liquid chromatography. Micellar liquid chromatography is a reversed-phase liquid chromatographic mode with mobile phases containing a surfactant above its critical micellar concentration. The applications of micellar liquid chromatography for the determination of numerous compounds in pharmaceutical formulation, biological samples, food, environmental samples, and feeds have been growing rapidly. Micellar liquid chromatography technique has several advantages over other chromatographic techniques. Its main advantage is the small amount of organic modifiers used such as acetonitrile and methanol and the safety and recyclability of the mobile phase. In our work, we discuss the development of "green chemistry" and present what micellar liquid chromatography is. This article presents application methods with the use of micellar liquid chromatography for analysis on antibacterial substances, melamine, biogenic amines, plant protection products, flavonoids, as well as peptides in biological matrices such as milk, eggs, tissues, honey, and feed.

In-house Validation Method for Quantification of Amoxicillin in Medicated Feedingstuffs with the Use of HPLC-DAD Technique.

INTRODUCTION: A high-performance liquid chromatographic-diode array detector (HPLC-DAD) method for the determination of amoxicillin in medicated feedingstuffs was developed and validated. The method was used to investigate the quality requirements of animal feedingstuffs (declared content of active substance and feed homogeneity). MATERIAL AND METHODS: Two-gram samples were extracted by potassium phosphate buffer solution. Extracts were filtered and directly analysed by HPLC-DAD without further clean-up. Amoxicillin was separated by acetonitrile and 0.01M phosphate buffer (pH 5.0) on a Phenomenex Luna C18 column. RESULTS: This method provided average recoveries of 76.1 to 81.6% with coefficients of variation (CV, %) for repeatability and reproducibility in the ranges of 3.7-7.2% and 5.3-7.6%, respectively. The limit of detection was 51.2 mg/kg and limit of quantification was 103.0 mg/kg. CONCLUSION: The method was successfully validated and proved to be efficient, precise, and useful for quantification of amoxicillin in medicated feedingstuffs.

Quantification of Veterinary Antibiotics in Pig and Poultry Feces and Liquid Manure as a Non-Invasive Method to Monitor Antibiotic Usage in Livestock by Liquid Chromatography Mass-Spectrometry.

Antibiotics are active substances frequently used to treat and prevent diseases in animal husbandry, especially in swine and poultry farms. The use of manure as a fertilizer may lead to the dispersion of antibiotic residue into the environment and consequently the development of antibiotic-resistant bacteria. Most pharmaceutical active ingredients are excreted after administration, in some cases up to 90% of the consumed dose can be found in the feces and/or urine as parent compound. Therefore, due to antibiotic metabolism their residues can be easily detected in manure. This article describes a method for simultaneous analysis of ciprofloxacin, chlortetracycline, doxycycline, enrofloxacin, lincomycin, oxytetracycline, tetracycline, tiamulin, trimethoprim and tylosin in feces, liquid manure and digestate. Antibiotics were extracted from the different matrices with McIlvaine-Na2EDTA buffer solution and the extract was purified by the use two techniques: d-SPE and SPE (Strata-X-CW cartridges) and final eluent was analyzed by LC-MS and LC-MS/MS. The European Commission Decision 2002/657/EC was followed to conduct the validation of the method. Recoveries obtained from spiked pig and poultry feces and liquid manures samples ranged from 63% to 93% depending on analytes. The analysis of 70 samples (feces, liquid manure and digestate) revealed that 18 samples were positive for the presence of doxycycline, oxytetracycline, tetracycline, chlortetracycline, enrofloxacin, tiamulin and lincomycin. The results obtained in the presented study demonstrated that animal feces can be used as a non-invasive method detection antibiotic usage in animal production.

Contamination of Animal Feed with Undeclared Tetracyclines-Confirmatory Analysis by Liquid Chromatography-Mass Spectrometry after Microbiological Plate Test.

The presence of tetracycline (TC) antibiotics was determined in animal feed that had been previously screened with a microbiological plate test. Feed samples were screened by a microbiological plate test on a pH 6.0 culture medium seeded with Bacillus cereus ATCC 11778 able to pre-reveal the presence of tetracyclines. Subsequently, confirmation and quantification were performed using a validated HPLC method with mass spectrometric detection. In 2013-2018, 353 feed samples were analysed to detect antibacterial substances, of which 186 (52.7%) were suspected to contain tetracyclines. Forty-two out of 186 (22.6%) samples analysed by the chromatographic method contained undeclared tetracyclines, which were determined at concentrations from 0.3 to 49 mg kg-1. The most frequently identified contaminating tetracyclines were doxycycline and chlortetracycline.

Determination of Florfenicol, Thiamfenicol and Chloramfenicol at Trace Levels in Animal Feed by HPLC⁻MS/MS.

Administration of florfenicol and thiamfenicol through medicated feed is permitted within the European Union, always following veterinary prescription and respecting the withdrawal periods. However, the presence of low levels of florfenicol, thiamfenicol, and chloramfenicol in non-target feed is prohibited. Since cross-contamination can occur during the production of medicated feed and according to Annex II of the European Regulation 2019/4/EC, the control of residue levels of florfenicol and thiamfenicol in non-target feed should be monitored and avoided. Based on all the above, a sensitive and reliable method using liquid chromatography tandem mass spectrometry was developed for the simultaneous detection of chloramfenicol, florfenicol, and thiamfenicol at trace levels in animal feed. Analytes were extracted from minced feed with ethyl acetate. Then, the ethyl acetate was evaporated, the residue was resuspended in Milli-Q water and the extract filtered. The method was in-house validated at carryover levels, with concentration ranging from 100 to 1000 µg/kg. The validation was conducted following the European Commission Decision 2002/657/EC and all performance characteristics were successfully satisfied. The capability of the method to detect amfenicols at lower levels than any prior perspective regulation literature guarantees its applicability in official control activities. The developed method has been applied to non-compliant feed samples with satisfactory results.

HPLC-DAD analysis of florfenicol and thiamphenicol in medicated feedingstuffs.

A simple and reliable method using liquid chromatography with diode array detector was developed for the simultaneous determination of florfenicol and thiamphenicol in medicated feed. The analytes were extracted from the minced feed with methanol and ethyl acetate (1:1, v/v). Next, the extract was further cleaned up by dispersive solid phase extraction using anhydrous magnesium sulfate, PSA and C18 sorbents. Finally, 1 mL of extract was evaporated, the residue resuspended in Milli-Q water, and filtered. The method was validated in-house at medicated levels, in the concentration range 10-300 µg/mL (50-1500 mg/kg). Values of <6.5% and <6.0% were found, respectively, for repeatability and within-laboratory reproducibility. The LODs for the two fenicols were 2.4-5.3 mg/kg, while the LOQs were 3.8-5.6 mg/kg. The expanded uncertainty was estimated to be in the range of 10.0-14.5%, depending on the analyte. Recoveries varied from 81.7% to 97.5%. The methodology was applied to the analysis of animal feedingstuffs collected from poultry and pig farms.

Determination of Sulfonamides in Feeds by High-Performance Liquid Chromatography after Fluorescamine Precolumn Derivatization.

A new multi-residue method for the analysis of sulfonamides (sulfadiazine, sulfamerazine, sulfamethazine, sulfaguanidine and sulfamethoxazole) in non-target feeds using high-performance liquid chromatography-fluorescence detection (HPLC-FLD) and precolumnderivatization was developed and validated. Sulfonamides (SAs) were extracted from feed with an ethyl acetate/methanol/acetonitrile mixture. Clean-up was performed on a Strata-SCX cartridge. The HPLC separation was performed on a Zorbax Eclipse XDB C18 column with a gradient mobile phase system of acetic acid, methanol, and acetonitrile. The method was validated according to EU requirements (Commission Decision 2002/657/EC). Linearity, decision limit, detection capability, detection and quantification limits, recovery, precision, and selectivity were determined, and adequate results were obtained. Using the HPLC-FLD method, recoveries were satisfactory (79.3⁻114.0%), with repeatability and reproducibility in the range of 2.7⁻9.1% to 5.9⁻14.9%, respectively. Decision limit (CCα) and detection capability (CCß) were 197.7⁻274.6 and 263.2⁻337.9 µg/kg, respectively, and limit of detection (LOD) and limit of quantification (LOQ) were 34.5⁻79.5 and 41.3⁻89.9 µg/kg, respectively, depending on the analyte. Results showed that this analytical procedure is simple, rapid, sensitive, and suitable for the routine control of feeds.

Simultaneous analysis of coccidiostats and sulphonamides in non-target feed by HPLC-MS/MS and validation following the Commission Decision 2002/657/EC.

Taking into consideration the maximum level (ML) for coccidiostats included in the European Regulation 574/2011 and the fact that the presence of residues of sulphonamides in non-target feed is forbidden, the aim of this article is to present an analytical method based on HPLC-MS/MS for the identification and quantification of sulphonamides and coccidiostats in non-target feeds. The method was validated following Commission Decision 2002/657/EC, and recovery, repeatability and reproducibility were within the limits established in the Decision. For coccidiostats, the decision limit and detection capability were calculated for the different species taking into account the ML allowed in Regulation 574/2011. The applicability of the method was investigated in 50 feed samples collected from dairy farms, 50 obtained from feed mills and 10 interlaboratory feed samples.

Development and validation of an LC-MS/MS method for the quantification of tiamulin, trimethoprim, tylosin, sulfadiazine and sulfamethazine in medicated feed.

A new multi-compound method for the analysis of veterinary drugs, namely tiamulin, trimethoprim, tylosin, sulfadiazine and sulfamethazine was developed and validated in medicated feeds. After extraction, the samples were centrifuged, diluted in Milli-Q water, filtered and analysed by high performance liquid chromatography coupled to tandem mass spectrometry. The separation of the analytes was performed on a biphenyl column with a gradient of 0.1% formic acid in acetonitrile and 0.1% formic acid in Milli-Q water. Quantitative validation was done in accordance with the guidelines laid down in European Commission Decision 2002/657/EC. Method performances were evaluated by the following parameters: linearity (R2 < 0.99), precision (repeatability <14% and within-laboratory reproducibility <24%), recovery (73.58-115.21%), sensitivity, limit of detection (LOD), limit of quantification (LOQ), selectivity and expanded measurement uncertainty (k. = 2). The validated method was successfully applied to the 2 medicated feeds obtained from the interlaboratory studies and feed manufactures from Spain in August 2017. In these samples, tiamulin, tylosin and sulfamethazine were detected at the concentration levels declared by the manufacturers. The developed method can therefore be successfully used to routinely control the content and homogeneity of these antibacterial substances in medicated feed. Abbreviations AAFCO - Association of American Feed Control Officials; TYL - tylosin; TIAM - tiamulin fumarate; TRIM - trimethoprim; SDZ - sulfadiazine; SMZ - sulfamethazine; UV - ultraviolet detector; FLD - fluorescence detector; HPLC - high performance liquid chromatography; MS/MS - tandem mass spectrometry; LOD - limit of detection; LOQ - limit of quantification; CV - coefficient of variation; SD - standard deviation; U - uncertainty.

Development and validation of multi-residue and multi-class method for antibacterial substances analysis in non-target feed by liquid chromatography - tandem mass spectrometry.

A confirmatory HPLC-MS/MS method for the determination of residues of 11 antibacterial substances from different therapeutic class (ß-lactams, lincosamides, fluoroquinolones, macrolides, pleuromutilins and sulfonamides) in animal feeds has been developed. The sample preparation is based on extraction with 0.1% formic acid in acetonitrile. Separation of the analytes was performed on biphenyl column with a gradient of 0.1% formic acid in acetonitrile and 0.1% formic acid in Milli-Q water. The developed method was validated following the guidelines included in the European Union Commission Decision 2002/657/EC. Limits of detection ranging from 79.22 to 193.60 µg/kg; instrumental and analytical linearity coefficients were above 0.99 for matrix-match calibration; and relative recoveries ranging from 76.04% to 117.39%. Repeatability of the method was in the range of 2.41-19.76% (CV, %), whereas reproducibility ranged from 6.52 to 28.40% (CV %). The method shown to be efficient and precise for quantification of the 11 antibacterial substances in animal feed. The results demonstrate the feasibility of the method for routine use to monitor these substances in feed. The validated method was successfully applied to eight suspect feed samples collected from the Association of American Feed Control Officials (AFFCO) and feed manufactures from Galicia (Spain) in June and July 2017. Of these 8 non-target feeds, 5 were positive for the presence of tiamulin, tylosin and sulfadiazine.

Development and validation of multi-residue analysis for tetracycline antibiotics in feed by high performance liquid chromatography coupled to mass spectrometry.

A new multi-residue method for the analysis of the tetracycline antibiotics oxytetracycline, tetracycline, chlortetracycline, and doxycycline was developed and validated for animal feed. After extraction with 0.1M Na2EDTA-McIlvaine buffer (pH 4), the samples were centrifuged, purified by SPE (Strata-X-CW cartridges) and analysed by HPLC-MS. Validation of the method was performed according to the guidelines indicated in European Commission Decision 2002/657/EC. The procedure was validated by spiking feed samples at three different levels (300, 1000, and 5000 µg/kg). Average recoveries for tetracyclines were in the range 79.70-98.8%, with RSD for repeatability and reproducibility in the ranges of 5.0-9.5% and 6.5-11.0%, respectively. The method was successfully validated and proved to be efficient, precise, and useful for quantification of tetracyclines in animal feed. The validated method was successfully applied to 65 suspect feed samples collected from different regions of Poland in 2015-16 and obtained from farms and feed manufactures. Of these 65 purportedly non-medicated feeds, eight (12.3%) were positive for the presence of doxycycline or chlortetracycline.