Antibiotics are substances able to kill or inhibit the growth of microorganisms which are produced by living organisms but which can also be produced synthetically (4, 31). Antibiotics have been used on a large scale for several decades in human medicine and as growth promoters in veterinary medicine (26). They can be administered to animals by injection, intrauterine and intramammary infusion, orally in water or feed, and topically (24). Theoretically, all of these routes may lead to the appearance of residues in food of animal origin which are of concern due to their implication in the development of antibiotic resistance of target pathogens, induced allergic reactions in some hypersensitive individuals, potential compromise of the human intestinal and immune systems (24, 26).
According to the Ninth ESVAC Report, sales of veterinary antimicrobial agents, in the European Union ranged from 3.1 to 423.1 milligrams sold per population correction unit (mg/PCU) across 31 countries. Penicillins (1,787 mg/PCU), tetracyclines (2,019 mg/PCU), and sulphonamides (608 mg/PCU) accounted for 67% of the total sales in 2017 (10). These antibacterial substances are used in food production animals and can be administered through medicated feed. These feedstuffs must undergo regular checks, including appropriate laboratory tests of homogeneity by the manufacturers to ensure that they comply with the requirements of Regulation (EU) 2019/4 of the European Parliament and of the Council of 11 December 2018, especially with respect to their homogeneity, declaration of contents, storage conditions, and stability (29).
One of the antibiotics which can be used in medicated feed is amoxicillin (AMO). Amoxicillin is a semi-synthetic penicillin widely used in clinical therapy as a broad-spectrum bactericidal compound. It inhibits peptidoglycan synthesis at the bacterial cell wall and has fast bactericidal action (9). Amoxicillin is used to treat bacterial infections in pigs and poultry. This
group is administered orally either by water or in medicated feeds, the latter appearing to be more suitable than medicated water because the stability of AMO in aqueous solution is poor and medicated water must be freshly prepared every 6–12 h. For this reason, amoxicillin is more often given to pigs as medicated feed (9).
The official methods of the Association of American Feed Control Officials (AAFCO) and the Association of Official Analytical Chemists (AOAC) for the analysis of penicillins in medicated feed samples are based on microbiological plate assays, but these methods are time consuming and not very specific because they do not allow pertinent distinctions,
In Poland amoxicillin is licensed as a veterinary drug for use in feed for pigs and is applied in a dose of 300 or 400 mg/kg feed. AMO is commonly used as an active substance in medicated feedingstuffs so we developed a rapid simple and selective method for quantitative analysis of amoxicillin in medicated feed for pigs based on its extraction from the matrix by phosphate buffer extraction and determination by high-performance liquid chromatography HPLC with a diode array detector (HPLC-DAD).
The LOD is the lowest concentration of analyte that the analytical process can reliably differentiate from background levels, while the LOQ is the lowest concentration of analyte that can be quantified. These were determined using blank feed samples. LOD and LOQ values were calculated from signal-to-noise ratios (S/N) of 3 and 10, respectively. The linearity of the method was evaluated by drawing standard solutions calibration curves with five concentration levels in triplicate. Repeatability was assessed by comparing the results of six replicates prepared on the same day at three different concentrations (150, 300, and 500 mg/kg). The procedure was repeated to determine within-laboratory reproducibility by comparing results from samples prepared and analysed on three different days. Standard deviations (SD) and coefficients of variation (CV, %) were calculated for each level. The recovery was calculated by the formula (measured level/fortified level) × 100%. The overall mean concentration and CV were calculated as reproducibility. The selectivity of the method was found by comparing chromatograms of the blank samples with the chromatograms of the standards and samples with standard additions. Six blank feed samples from different sources were analysed in order to verify the absence of potential interfering endogenous compounds at the target analyte retention times and in this way to test the specificity. The uncertainty (U) was calculated as the ratio of coverage factor (k = 2) and SD of within-laboratory reproducibility, and expressed in percentages.
The developed procedure was designed to obtain a qualitative method of determination of amoxicillin in medicated feedingstuffs. The reversed-phase Phenomenex Luna C18 and gradient of the phosphate buffer and acetonitrile were chosen to separate amoxicillin chromatographically within 18 min. The best separation and peak shape were achieved using 0.01M phosphate buffer adjusted to pH 5.0. A typical chromatogram obtained from a spiked feed sample is presented in Fig. 3. Under selected conditions, AMO displayed high UV absorption at 202 nm, while no interference of the matrix was observed (Figs 2 and 3).
A linearity and regression study was performed for DAD detection. The high correlation coefficient (
Validation parameters of optimised HPLC-DAD method
Validation parameters | Results |
---|---|
Solvent STD regression equations | y = 80.695x + 392.76 |
Coefficient of determination (R2) | 0.99 |
Recovery (%) | |
150 mg/kg | 76.1 |
300 mg/kg | 78.2 |
500 mg/kg | 81.6 |
Repeatability (%) | |
150 mg/kg | 7.2 |
300 mg/kg | 5.8 |
500 mg/kg | 3.7 |
Within-laboratory reproducibility (%) | |
150 mg/kg | 7.6 |
300 mg/kg | 6.8 |
500 mg/kg | 5.3 |
LOD (mg/kg) | 51.2 |
LOQ (mg/kg) | 103.0 |
U (%) | 19.4 |
This investigation aimed to develop a reversed-phase HPLC method for the detection of AMO in medicated feedingstuffs and verification of their declared content.
To ensure the safety of food and feed, efficient methods are required for the simultaneous monitoring of contents of veterinary drugs in food and feed. A large number of available methods have been developed for determining penicillins from biological matrices,
To develop this method for the determination of AMO in medicated feed samples by a solid–liquid extraction technique, the parameters including sample weight, extraction time, extraction solvents, and pH value were studied. For the optimal extraction conditions, we tested 2 and 5 g sample weights, extraction solvents such as water/acetonitrile and phosphate buffer solution at pH ranging through 3, 4, 4.5, 5, and 6, and shaking times between 20 and 60 min. The final protocol for the extraction of amoxicillin was for 2 grams grinded feed samples to be extracted with 0.05M phosphate buffer, pH 4.5, and shaken for 60 min. These parameters allowed good recovery of AMO to be obtained without any additional purification step on SPE cartridges. The biggest advantages of the proposed method are its simplicity and robustness, which make the procedure time-saving and economical.
The mobile phase of the method for the determination of penicillins is usually phosphate buffer or trifluoroacetic acid with acetonitrile for the HPLC-UV or FLD techniques (4, 9, 12, 21, 22) or formic acid in water and acetonitrile or methanol when penicillins are analysed by the LC-MS technique (3, 5, 7, 9, 13, 14, 23, 29, 33). In this method, chromatographic separation was optimised by testing variations of the mobile phase with different concentrations of potassium dihydrogen phosphate (0.01M, 0.02M, and 0.05M) and pH between 4.0 and 6. Experimental results demonstrated that 0.01M phosphate buffer, adjusted to pH 5.0 was the most suitable additive to enhance the peak resolution and sensitivity.
An important part of each chromatographic separation is to choose analytical columns, suitable for the particular group of compound packings. For the separation of amoxicillin from different kinds of matrices researchers commonly used C18 and C8 analytical columns (4, 30, 32) or ODS column (3, 9, 21). Column lengths of 150 mm or 250 mm were equally popular, but a packing of 5 μm and column ID of 4.6 mm were the most common (4, 9, 21, 32). In this study, the best separation results were achieved on a Luna C18 column with 0.01M phosphate buffer in water (pH 5.0) and acetonitrile. The use of gradient elution gave a much better separation of the analysed compound. The developed method gave good results for both samples prepared in-house (spiked with the appropriate levels of AMO) and those delivered by manufacturers. Figs 1, 2, and 3 show chromatograms of standard solution, blank feed, and feed samples spiked with AMO at a concentration of 150 mg/kg.
The proposed HPLC method is simple, economic, accurate, reproducible, and useful for determination of AMO in medicated feedingstufs. The extraction of the antibiotic is based on a simple solid–liquid extraction and no clean-up step is necessary. After filtration, the extracts can be directly injected into the HPLC-DAD system. The presented method has good repeatability and within-laboratory reproducibility, as indicated by the CVs for both being less than 8%. The satisfactory results of validation proved that the method is efficient and precise. Therefore, this method can be applied in official quality control procedures to verify the producers’ declarations of the amount of the active substances in medicated feedingstuffs and their homogeneity.