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

Reducing the amount of antibiotics used in animal production is one of the priority actions of the European Green Deal, which aims to achieve a sustainable and ecological transformation of the economy. In the context of reducing antibiotics in the food chain, the European Green Deal sets an ambitious target for their reduction in animal husbandry by 2050. As part of this goal, the EU plans to introduce strict regulations on the use of these drugs, especially of antibiotics critical for the treatment of human infections. These activities are aimed at reducing the risk of bacterial resistance and limiting the presence of antibiotics in food products. Public health and agricultural organisations such as the World Health Organization (WHO) and the World Organisation for Animal Health (WOAH) are developing guidelines and recommendations to control the use of antibiotics in the food chain. EU Member States, including Poland, have implemented control programmes for the presence of antibiotics, sulfonamides and quinolones in drinking water for animals, medicated and non-target feed, and food and products of animal origin, which are constantly being improved and adapted to current EU and national legislation. But according to the data published in the European Surveillance of Veterinary Antimicrobial Consumption (ESVAC) European Medicines Agency (EMA) report for 2021, in Poland in 2011–2021 there was a 35% increase in the total sales of veterinary antimicrobials (mg/population correction unit (PCU)). In 2021, the most frequently sold groups of antibacterial drugs in Poland were penicillins, tetracyclines, macrolides and sulfonamides. In the same year, Poland ranked second only to Spain in terms of sales of antibacterial substances in livestock (6). According to Regulation (EU) 2019/4 of the European Parliament and of the Council of 11 December 2018 (24), one of the routes for the oral administration of veterinary medicinal products is medicated feed, which is a homogeneous mixture of feed and such products. Medicated feed should be manufactured only with veterinary medicinal products authorised for incorporation into medicated feed during manufacture, and the compatibility of all compounds used should be ensured for safety and efficacy of the product. Medicated feed should only be manufactured with approved medicinal premixes. The uniform incorporation of the veterinary medicinal product into the feed is essential for safe production and effective medicated feed to ensure food safety throughout the food chain.

In Poland, medicated feeds are subject to official control by the Veterinary Inspection and quality control by manufacturers, which ascertains that the actual content of the active substance added to the feed is the declared content of that substance and tests the homogeneity of the feed. The first methods used for this purpose were microbiological methods. However, with the development and availability of other analytical methods, such as chromatographic methods with various types of detectors, and the impossibility of using microbiological methods for some groups of antibacterial substances, such as sulfonamides or phenicols, other methods began to be developed and implemented in laboratory practice.

In scientific publications, methods can be found of quantitative analysis of single substances in medicated feed (e.g. sulfaguanidine, tylosin, tiamulin, amoxicillin, florfenicol and sulfamethazine) (5, 10, 11, 18, 20, 23, 25, 26), and of analysis of several compounds belonging to the same chemical group (sulfonamides, phenicols, β-lactams and tetracyclines) (1, 2, 4, 8, 12–17, 21, 22). However, few developed analytical methods exist for several antibacterial substances belonging to different chemical groups (19). For the analysis of antibacterial substances in medicated feeds, the technique of liquid chromatography with various types of detectors is used: diode array detector, fluorescence detector, single mass spectrometer or tandem mass spectrometer (1, 2, 4, 5, 7, 8, 10–23, 25, 26).

In this publication, an analytical method for the analysis of seven antibacterial substances such as: sulfadiazine (SDZ), sulfamerazine (SMR), sulfamethazine (SMZ), sulfamethoxazole (SMX), trimethoprim (TRIM), amoxicillin (AMO) and tylosin (TYL) in one analytical procedure in medicated feeds using only liquid extraction and liquid chromatography with mass spectrometry is presented. The method was „in-house” validated, verified by analysis of real samples, and successfully applied in laboratory practice. The development of multi-analyte protocols is practical for laboratories monitoring the content and homogeneity of antimicrobial substances in medicated feeds, as it allows for much greater analysis of feed samples without changing the extraction or analytical method.

There are few chromatographic methods described in the scientific literature that allow the analysis of several antibacterial substances in medicated feeds in one analytical procedure (19). This may be due to the long-standing application of cheap and simple microbiological methods that have been used for years to control the content of active substances declared by the manufacturer or to test the homogeneity of manufactured medicated feeds. Unfortunately, these methods are not useful for ascertaining the content of all antimicrobial substances approved for use in medicated feeds, including sulfonamides and phenicols. Therefore, work has been undertaken on the optimisation and validation of a new, multicomponent analytical method involving seven antibacterial substances: sulfadiazine, sulfamerazine, sulfamethazine, sulfamethoxazole, trimethoprim, tylosin and amoxicillin. Trimethoprim is the drug that is most commonly administered in medicated feed in combination with sulfadiazine or sulfamethoxazole. So far, no chemical or microbiological analytical methods have been implemented in Poland that would allow for quantitative determination of the combination of these two antibacterial substances in the feed mixture. In the case of methods available for the quantitative determination of amoxicillin, microbiological and chromatographic methods are available, but the chemical methods developed for this compound most often allow only the analysis of amoxicillin or amoxicillin and penicillin V or G or ampicillin in one analytical procedure (1, 7). For this purpose, liquid chromatography with ultraviolet-visible spectroscopy (UV/Vis) and diode array detection (DAD) is also most often used, which may hinder the quantitative analysis of these compounds because the wavelength of UV light that amoxicillin absorbs is about 200–210 nm. In the UV wavelength range similar to that of amoxicillin, other endogenous compounds present in the analysed extract also show significant absorption, which may significantly hinder the quantitative analysis of this antibiotic. The presence of interfering substances can be partly eliminated by purifying the sample, but in this case the development of the method requires more work and increases the cost of the analysis. Patyra and Kwiatek (18) described a method of analysing amoxicillin in medicated feed using the HPLC-DAD technique. They used 0.01 M of phosphate buffer at pH 5.0 and acetonitrile as the mobile phase and gradient elution. Tylosin is an antibiotic from the group of macrolides, which is quite often used in the production of medicated feed. The content of this compound in medicated feed is 50 or 100 mg/kg. The content of this drug is most often determined using the microbiological method, but chromatographic analysis methods for this compound in medicated feed are available in scientific publications. The author of such a method is Pietroń et al. (20), who used liquid chromatography with UV/Vis detection to quantify tylosin in medicated feed. They used a mobile phase consisting of methanol and acetonitrile (50:50, v/v), and solution triethylamine 70 mM adjusted to pH 2.5 with orthophosphoric acid. The wavelength of UV light for tylosin was set at 282 nm.

However, there are few described chromatographic methods that concern the analysis of several antibacterial substances from different chemical groups in one course of analytical procedure. One method which can analyse several antibacterial substances in one performance of a procedure is the method described by a Polish–Spanish team of scientists (19). The method allows for the analysis of five antibacterial substances in medicated feeds (sulfadiazine, sulfamethazine, trimethoprim, tylosin and tiamulin) using liquid chromatography with tandem mass spectrometry (LC-MS/MS). In the developed method, a combination of 0.1% formic acid in water and acetonitrile with 0.1% formic acid was used as the mobile phase, and chromatography was in a biphenyl column. In our work, we developed an analytical method using liquid chromatography with single mass spectrometry for the analysis of seven antibacterial substances belonging to 3 chemical groups. The most important and labour-intensive stage of work was the optimisation of the stage of extraction and purification of analytes from the feed matrix. The physical and chemical properties of the antibiotics in the optimised method were different because the drugs were from three antibiotic classes; for this reason, solvents that would allow the extraction of all analytes were challenging to select. Sulfonamides and trimethoprim are most often extracted with organic solvents such as methanol, acetonitrile and ethyl acetate (2, 17, 21). For tylosin, methanol in citrate buffer (20), sodium bicarbonate solution with acetonitrile (10) or a mixture of water and acetonitrile with the addition of formic acid are used (20). However, antibiotics from the penicillin (amoxicillin, ampicillin and penicillin) class are most often extracted from biological matrices using acetonitrile, an acetonitrile and water mixture or phosphate buffer at different pH ranges (1, 5, 12, 18). In order to optimise the extraction process, we had to find the all-round best, which meant selecting the solvents which could obtain the highest possible recoveries for all the analysed antibacterial substances. Various extraction mixtures were tested for this purpose, such as a combination of methanol and acetonitrile, a mixture of acetonitrile and water with the addition of formic acid, and a combination of acetonitrile with a phosphate buffer at a range of pH (3, 3.5, 4, 4.5 and 5). The best extraction mixture was a combination of acetonitrile and phosphate buffer at pH 4.5 (1 : 1; v/v). The high concentrations of antibacterial substances in medicated feed and their wide range from several dozen to several hundred milligrams per kilogram of feed necessitate the obtained extracts’ dilution for analyses using the LC-MS technique. When optimising the method, we used 0.1% formic acid in water and pure water to dilute the extract. Different dilutions of the extract were also tested: 100-fold and 50-fold. It was found that the best results were obtained by diluting the extract 100-fold in water. The developed method was validated, giving satisfactory results for all seven analysed compounds.

The conducted research on market samples of medicated feed showed that in Poland there is no problem with the concentration of active substances in the feed declared by the manufacturer, because the antibiotic contents are consistent with the manufacturers’ declarations. Moreover, medicinal feeds produced in Poland are homogeneous, which was confirmed by our research.