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

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Most veterinary antibiotics are excreted after administration, in some cases up to 90% of the consumed dose can be found in the faeces and/or urine as parent compound. Livestock excrement is used around the world as natural fertilizers or, after processing, as organic fertilizers for crops and grasslands. Natural and organic fertilizers are an excellent source of nutrients for plants, but due to the presence of veterinary antibiotics in them, they may pose a threat to the natural environment, mainly to soil microorganisms, but also to human and animal health. Therefore, the work aimed to develop and validate an analytical method using the UHPLC-MS/MS technique and to analyze real samples of natural and organic fertilizers. Our research has shown that over 38% of natural fertilizers are contaminated with antibiotics, mainly doxycycline in concentrations reaching up to several dozen milligrams per kilogram of fertilizers. In the case of organic fertilizers that undergo processing, the presence of antibiotics was found in over 37% of the analyzed samples. The presented analytical method can be a tool for assessing the presence of veterinary antibiotics in solid natural and organic fertilizers. veterinary antibiotics natural fertilizer organic fertilizer UHPL-MS/MS analysis validation Figures Figure 1 Figure 2 Introduction Veterinary antibiotics are widely used in animal husbandry around the world to prevent and treat disease and are still used in some countries to promote animal growth as feed additives (Hou et al., 2015 ; Zhou et al., 2013 ; Zhi et al., 2020 ). Antibiotics in farm animals can be administered in the form of injections (treatment of individual animals, especially ruminants and horses) or orally, by administering them in medicated feed or after dissolving in water. The second method of drug application is used to treat entire groups of animals (mainly pigs and poultry). The popularity of this method of administering antibacterial substances is due to the fact that the drug is administered to an entire group of animals at the same time, which reduces time and work expenditure (Osiński and Patyra 2022 ). Regardless of the route of administration of antibacterial substances to farm animals, these compounds are excreted from the body in amounts ranging from 30–90% of the administered initial dose, in unmetabolized form or in the form of active and/or inactive metabolites with faeces or urine, which are then used as natural fertilizers for fertilizing arable lands and grassland (Berendsen et al., 2015 ) or processed (granulation, composting, fermentation) and available on the market as organic fertilizers. In accordance with the Act of July 10, 2007 in force in Poland on fertilizers and fertilization, for natural fertilizers include manure and slurry well as excrements from farm animals without the addition of other substances, as well as guano, which are intended for agricultural use. Manure or slurry are very good natural fertilizers used in plant cultivation. In accordance with the applicable Directive 91/676/EEC on natural fertilizers, it contains guidelines on the timing of their use on permanent grasslands, permanent crops and arable land, as well as specifies the requirements for their storage and the permissible amount of nitrogen introduced per hectare of crops. In addition to natural fertilizers, organic fertilizers are also used for growing plants, which, in accordance with Regulation (EC) No. 1069/2009 of the European Parliament and of the Council of 21 October 2009, includes manure, which is defined as faeces or urine farm animals, other than farmed fish, with or without litter. In addition, the Regulation defines the concepts of "organic fertilizer" and "soil improver", which mean materials of animal origin used separately or in combination to maintain or improve plant nutrition, as well as the physical and chemical properties and biological activity of soils. They may contain manure, digestive tract contents, non-mineralized guano, compost and post-fermentation residues. Manure, organic fertilizers and soil improvers are Category 2 materials. This means that they may be by-products containing residues of approved substances or contaminants at levels above permitted levels, including antibiotics, sulfonamides and quinolones, which should be monitored in live animals and products animal origin (Regulation (EC) No. 1069/2009). Natural and organic fertilizers are not subject to any control for the presence of antibacterial substances, and the agricultural use of these fertilizers may cause contamination of the natural environment with these compounds. Antibiotics are detected unchanged in manure, slurry and poultry litter from farms in concentrations ranging from several µg/kg to several hundred mg/kg. Differences in the levels of antibacterial substances in the above-mentioned fertilizers depend on the animal species, the class of antibiotic and the geographical location and type of breeding farm. Their content depends on the antibiotic used because some substances, such as amoxicillin or tetracycline, are metabolized only by 10–20%, while others, e.g. sulfamethoxazole, by approximately 85% (Hirsh et al. 1999). Active substances can be metabolized into inactive conjugates, which, after being excreted from the animal's body, can be converted to the active form of the drug. Moreover, some excreted compounds may be similar or even more harmful than the parent compounds (Hirsch et al. 1999 ). Large-scale farms have significantly higher concentrations of antibiotics in fertilizers compared to small family farms (Zhi et al. 2020 ) (Zhi et al. 2020 ). Moreover, higher concentrations of antibiotics are detected in fertilizers from pigs than in fertilizers from poultry and cattle. This is mainly due to the fact that antibiotics are administered in higher doses and more frequently to pigs than to other farm animals (Xin et al. 2016 ). The available scientific literature contains information that antibiotics are present in high concentrations in feces obtained from slaughtered animals. Wolters et al. ( 2016 ) examined derived manure from eight fattening farms and six breeding pigs farms. In the material examined, the authors found eleven different antibiotics belonging to up to six classes. Antibiotic residue analysis showed a maximum tetracycline concentration reaching up to 300 mg/kg dry matter (DM) in pig manure (Walters et al. 2016). Martinez-Carballo et al. examined pig faeces in Austria, in which they found the presence of antibiotics from the tetracycline group in amounts of several dozen milligrams per kilogram of faeces (oxytetracycline—29 mg/kg, chlorotetracycline – 46 mg/kg and tetracycline—23 mg/kg), (Martinez-Carballo et al. 2007 ). Researchers from China were analyzing fertilizers from chicken, in which they found high concentrations of enrofloxacin – 1420 mg/kg and norfloxacin – 225 mg/kg (Zhao et al. 2010 ). Due to the lack of data on the presence and content of veterinary antibiotics in solid natural fertilizers and commercial organic fertilizers, this work was undertaken to develop a multi-residue analytical method using UHPLC-MS/MS. The study compared various solvents for antibiotic extraction and various solid phase extraction cartridges. The developed method was validated and used to analyze real samples of solid natural fertilizers from pigs, poultry and cattle as well as solid organic fertilizers on the market produced with animal by-products. Materials and methods Chemicals and Reagents HPLC-grade acetonitrile and methanol were obtained from J.T. Baker (Deventer, The Netherlands). Formic acid and citric acid were purchased from Acros Organics (Geel, Belgium). Disodium ethylenediaminetetraacetate (Na 2 EDTA) was from Sigma Aldrich (California, MO, USA), and disodium hydrogen phosphate was from Chempur (Piekary Śląskie, Poland). Water was purified using a Milli-Q water system from Millipore (Billerica, Ma, USA). All target veterinary antibiotics (VAs) and internal standards (IS) were purchsed from Dr. Ehrenstorfer Gmbh (Augsburg, Germany). The 21 target antibiotics belonged to 6 classes: tetracyclines (TCs), oxytetracycline (OXT), epi-oxytetracycline (epi-OXT), tetracycline (TC), chlortetracycline (CTC), epi-chlotetracycline (epi-CTC), doxycycline (DC) and demeclocycline (DMC; IS), sulfonamides (SAs): sulfamethoxazole (SMX), sulfaguanidine (SGD), sulfamethazine (SMZ), sulfadiazine (SDZ), sulfamerazine (SMR) and sulfadiazine- 13 C 6 (SDZ- 13 C 6 ; IS); fluoroquinolones (FQs): sarafloxacin (SAR), enrofloxacin (ENR), ciprofloxacin (CIP), flumequine (FLU) and norfloxacin (NOR; IS), macrolides (MAs) tylosin (TYL), spiramycin (SPIR) and erythromycin (ERM; IS), pleuromutilin (PLM): tiamulin (TIAM) and valnelmulin (VAL) and lincosamides: lincomycin (LINCO) and lincomycin-d 3 (LINCO-d3;IS). McIlvaine-Na 2 EDTA buffer was prepared by mixing 115.65 mL of 0.2 M phosphate buffer and 184.65 mL of 0.1 M citric acid, then 11.406 g of Na 2 EDTA was added and mixed. The pH was adjusted to 4.0. Four SPE cartridges were tested: Strata-XCW (3 ml, 300 mg), Strata-X (6 ml, 200 mg) and Strata-SAX (12 ml, 500 mg) from Phenomenex (Torrance, CA, USA) and Oasis HLB ( 3 ml, 60 mg), from Waters (Milliford, MA, USA). An SPE manifold (J.T. Baker, PA, USA) and a pump as a vacuum source were used. Preparation of standard solutions Stock standard solutions 1 mg/mL of OTC, epi-OTC, TC, CTC, epi-CTC, DC, SGD, SMR, SMZ, SXZ, TRIM, ENR, SAR, LICO, TIAM, TYL, SPIR, VAL and internal standards of ERT, DMC and LINCO-d 3 were prepared by dissolving 5 mg of individual compounds in 5 mL of methanol. SDZ, SDZ- 13 C 6, FLU and NOR were dissolved in acetonitrile and CIP was dissolving in mixture of methanol and 1M sodium hydroxide (99:1; v/v). All standard solutions were stored in volumetric flasks at − 18°C for 6 months. All VAs and IS working solutions of 10 µg/mL were prepared by diluting the stock solutions in methanol and stored in amber volumetric flask at -18°C for less than one month. Instrument analysis Quantitative evaluation of antibiotics was performed using the UHPLC-MS/MS method. A liquid chromatograph consisting of an Exion LC with a SCIEX Triple Quad 5500 + system (SCIEX, Framingham, MA, USA) was used. A Kinetex C18 column (2.1 mm x 75 mm; 2.6 µm) was used to separate the target compounds. The mobile phase consisted of 0.1% formic acid in milli-Q water (A) and 0.1% formic acid in acetonitrile (B), and the gradient elution was as follows: 0–2 min 5% B; 2–10 minutes 5 − 15% B, 10–12 min. 15–20%B, 12–15 min. 20–50% B, 15–16 min. 50–70% B, 16–17 min. 70–100%, 17–18 100–5% B and 18–21 min. 5% B. The mobile phase flow rate was set to 0.25 mL/min, the injection volume was 10 µl, and the column thermostat temperature was set to 35°C. Antibiotics and their active metabolites selected for testing were identified by detecting the masses of precursor ions and their fragments. Positive electrospray ionization (ESI+) and multiple reaction monitoring (MRM) mode were used for all antibiotics. Depending on the analyzed compound, different operating parameters of the mass detector were used. The operating parameters of the MS/MS detector used are presented in Tables 1 and 2 . Chromatographic integration of samples was performed using Sciex OS MQ version 2.1.6 software (SCIEX, Framingham, MA, USA). Table 1 Operating parameters of the MS/MS detector Parameters Analytical conditions Curtain gas 30 psi Collision gas 10 psi Ion spray voltage 4500 V Temperature 400°C Ion source gas 1 40 psi Ion source gas 2 40 psi Entrance potential 10 Table 2 Transitions and optimal conditions used for MS/MS analysis. No. Analyte Precursor ion Q1 Product ion Q3 DP CE CXP EP Dwell time [msec] TETRACYCLINES 1. Oxytetracycline 1 461.0 426.2 116 27 22 10 150 Oxytetracycline 2 461.0 444.2 116 19 12 10 150 2. Tetracycline 1 445.0 410.0 50 27 15 10 150 Tetracycyline 2 445.0 427.0 60 20 15 10 150 3. Chlorotetracycline 1 479.0 444.2 126 31 22 10 150 Chlorotetracycline 2 479.0 462.2 126 25 20 10 150 4. Doxycycline 1 445.0 428.1 70 27 12 10 150 Doxycycline 2 445.0 410.0 70 33 12 10 150 5. epi-oxytetarcycline 1 461.0 426.0 27 116 22 10 150 epi-oxytetracycline 2 461.0 444.2 19 116 12 10 150 6. epi-chlorotetracyklina1 479.0 444.2 31 126 22 10 150 epi-chlortetracycline 2 479.0 462.2 25 126 20 10 150 IS Demeclocycline 465.0 448.1 124 25 16 10 150 FLUOROQUINOLONE 7. Ciprofloxacin 1 332.0 314.0 79 29 13 10 100 Ciprofloxacin 2 332.0 245.0 61 35 13 10 100 8. Enrofloxacin 1 360.0 342.0 80 30 24 10 100 Enrofloxacin 2 360.0 286.0 80 36 24 10 100 9. Sarafloxacin 1 386.0 368.0 29 31 13 10 100 Sarafloxacin 2 386.0 299.0 39 45 13 10 100 10. Flumequine 1 262.0 202.0 63 45 14 10 150 Fumequine 2 262.0 174.0 60 51 14 10 150 IS Norfloxacin 319.1 151.1 104 17 18 10 150 SULFONAMIDES 11. Sulfaguandine 1 215.0 156.0 86 21 18 10 150 Sulfaguanidine 2 215.0 92.0 86 33 12 10 150 12. Sulfadiazine 1 251.0 156.0 31 23 18 10 150 Sulfadiazine2 251.0 108.0 55 32 15 10 150 13. Sulfamerazine1 265.0 156.0 35 25 12 10 150 Sulfamerazine 2 265.0 92.0 44 40 12 10 150 14. Sulfamethazine 1 279.0 186.0 14 25 12 10 150 Sulfamethazine 2 279.0 124.0 13 26 12 10 150 15. Sulfamethoxazole 1 254.0 156.0 56 23 18 10 150 Sulfamethoxazole 2 254.0 108.0 53 33 18 10 150 16. Trimethoprim 1 291.0 230.1 181 33 12 10 150 Terimethoprim 2 291.0 261.1 181 35 14 10 150 IS Sulfadiazine- 13 C 6 257.0 162.0 89 23 16 10 150 LINCOSAMIDES 17. Lincomycin 1 407.0 126.0 65 39 13 10 150 Lincomycin 2 407.0 359.0 81 27 13 10 150 IS Lincomycin-d 3 410.1 129.2 129 37 14 10 150 MACROLIDES 18. Spiramycin 1 422.0 174.0 126 29 16 10 150 Spiramycin 2 422.0 101.0 126 25 12 10 150 19. Tylosin 1 916.0 174.1 139 51 22 10 150 Tylosin 2 916.0 772.4 125 43 20 10 150 IS Erytrhomycin 734.4 576.4 157 41 12 10 150 PLEUROMUTILIN 20. Tiamulin 1 494.0 192.0 114 29 10 10 150 Tiamulin 2 494.0 119.0 135 55 12 10 150 21. Valnemulin 1 565.2 263.1 141 25 16 10 150 Valnemulin 2 565.2 164. 1 141 25 10 10 150 Method development Due to the fact that the analytical matrices selected for research, such as natural and organic fertilizers, are complex analytical matrices, developing an appropriate extraction method for all antibacterial substances listed in Table 2 turned out to be a time-consuming step. The extraction method was optimized for samples fortified with analytes at a level of 100 µg/kg for solid fertilizers. Extraction experiments Twenty one compounds were selected from six different antibiotics classes: the tetracyclines OXT, epi-OXT, TC, CTC, epi-CTC and DC; fluoroquinolones CIP, ENR, SAR and FLU, the sulfonamides SGD, SDZ, SMR, SMZ, SXZ and TRIM, the macrolides TYL and SPIR, pleuromutilin TIAM and VAL, and lincosamide LINCO. The mentioned antibiotics are characterized by different physico-chemical properties, therefore experiments had to be carried out using various extraction mixtures that would allow for good extraction of all the mentioned analytes from natural and organic fertilizers. In addition, due to the complexity of the analytical matrix, it was necessary to select an appropriate technique for purifying the obtained extracts to ensure detection and good recovery of all analyzed antibiotics. Experiment I : Two- and five-gram samples of solid fertilizer were extracted with 25 mL of McIlvaine-Na 2 EDTA buffer at pH = 7 and acetonitrile mixture in a ratio of 23:2 v/v. The samples were shaken and centrifuged, then the extract was filtered through a cellulose filter and acidified with 85% orthophosphoric acid, using 50 µL of orthophosphoric acid for each 5 mL of extract. After this stage of work, the extract was clean-up using "tandem" SPE according to the scheme below (Fig. 1 ). The obtained eluate was evaporated in a stream of nitrogen, and the precipitate was dissolved in 500 µL of 0.1% formic acid in water and additionally filtered through a PVDF syringe filter (13 mm, 0.22 µm). Experiment II Two- and five-gram solid fertilizer samples were extracted with 25 mL of McIlvaine-Na 2 EDTA buffer with pH = 4. The samples were shaken and centrifuged. The extract was purified by SPE using Strata-XCW cartridges (3 mL, 100 mg). The cartridges were conditioned with 5 mL of methanol, 5 mL of water, then 9 mL of the extract was placed on the cartridge. The impurities were washed out with 6 mL of water, 6 mL of methanol and 3 mL of acetonitrile. The cartridges were dried under vacuum for 5 min, and the antibacterial substances were eluted from the columns with 3 mL of 2% formic acid in methanol. The eluate was evaporated in a stream of nitrogen, and the obtained precipitate was dissolved in 0.1% formic acid in water and additionally filtered through a PVDF syringe filter (13 mm, 0.22 µm). Experiment III 2 mL of acetonitrile were added to a two- and five-gram sample of fertilizer and mixed using a vortex mixer for 30 s. Then, 18 mL of McIlvaine-Na2EDTA buffer with pH = 4 was added and shaken for 45 min. The samples were centrifuged at 4500 rpm, 15 min, 20ºC, and then the supernatant was transferred to a new 50 mL centrifuge tube. 5 mL of McIlvaine-Na2EDTA buffer with pH = 4 was added to the sediment obtained after centrifugation and shaken again for 30 min. The samples were then centrifuged and the obtained extracts combined. 5 mL of n-hexane was added to the extract and shaken manually for about 1 min. The samples were then centrifuged again. 18 mL of the extract was purified and concentrated using solid phase extraction on Strata-X cartridges (200 mg, 6 mL). The cartridges were conditioned by washing with 6 mL of methanol and 6 mL of water. Dispense 18 mL of sample onto the cartridges. Subsequently, the cartridges were washed with 12 mL of water, and then the cartridges were dried under vacuum for 10 min. Antibacterial substances were eluted from the cartridges with 3 mL of methanol and then evaporated to dryness in a stream of nitrogen at a temperature of 45°C ± 5°C. The residue was dissolved in 500 µL of 0.1% formic acid in water and filtered through a PVDF filter (13 mm, 0.22 µm) into a chromatography vial. Experiment IV : A two-gram solid fertilizer sample was extracted with a mixture of McIlvaine-Na 2 EDTA buffer at pH = 4, acetonitrile and methanol in the proportion of 3:3.75:1.25; v/v/v or for five-gram samples in the ratio 6:7.5:2.5; v/v/v. The samples were shaken and centrifuged. To reduce the content of organic reagents such as acetonitrile and methanol, the obtained extract was diluted in water by adding 6 mL of extract to 25 mL of water. The extracts were purified by SPE using Strata-X cartridges (6 mL, 200 mg). The cartridges were conditioned with 6 mL of methanol and 6 mL of water. After conditioning, 18 mL of extract was dosed onto the cartridges, the impurities were washed out with 12 mL of water, and then the cartridges were dried for 10 min. under vacuum. Antibacterial substances were eluted from the cartridges with 3 mL of methanol. The eluate was evaporated under a stream of nitrogen, and the precipitate was dissolved in 500 µL of 0.1% formic acid in water and filtered through a PVDF syringe filter (13 mm, 0.22 µm). Method validation Validation of the method was carried out following the guidelines set out in the EU Commission Implementing Regulation 2021/808 of 22 March 2021 on the performance of analytical methods for residues of pharmacologically active substances used in food-producing animals and the interpretation of the results, as well as on the methods used for sampling and repealing Decisions 2002/657/EC and 98/179/EC. Linearity, selectivity, LOD and LOQ The working range of the method was determined by preparing calibration curves for fortified samples. For this purpose, blank samples were fortified with antibacterial substances at seven adopted concentration levels (0, 50, 100, 250, 500, 750 and 1000 µg/kg). The regression coefficient (R 2 ), slope (a) and shift (b) were calculated for the linear regression equation of the type y = ax + b. Then, curves were determined for fortified samples based on the relationship between concentration and signal size or the ratio of the analyte signal to the corresponding signal of the internal standard. It was assumed that the value of the coefficient of determination (R 2 ) of the calibration curve for each analyte should be higher than 0.98 for quantification in order to consider the linearity as satisfactory. The limit of detection and limit of quantification were assessed by the signal-to-noise ratio (3 for LOD and 10 for LOQ). The presence of interferences resulting from the composition of the endogenous matrix in the retention times of the monitored antibiotics was checked to determine the selectivity of the method. Selectivity tests were performed on 20 animal faeces samples. Recovery, repeatability, and within-laboratory reproducibility To determine the recovery and repeatability of the analyses, blank samples were fortified at three concentration levels: 50, 500 and 1000 µg/kg (six samples for each levels). The recovery was calculated based on the results obtained when determining repeatability. Recovery is the percentage of the actual concentration of a substance contained in a sample determined during the analytical process, according to the following equation: % recovery = 100 × measured content/fortification level. Intralaboratory reproducibility is the precision under conditions of intralaboratory reproducibility in which independent analytical results for the same quantities are obtained by the same method by different operators, in the same laboratory and with the same equipment over long time intervals. In order to determine intra-laboratory reproducibility, data from the determination of repeatability are used and additionally the same analyzes were performed - blank samples were fortified at 3 concentration levels, in two repetitions, at specified time intervals, by different operators. The mean value (x), standard deviation (SD) and coefficient of variation (CV) were calculated for each fortification level. Decision limit and detection capability The decision limit (CCα) is the concentration of an analyte in a sample above which the analytical result is considered inconsistent with the error probability α. The α value means the probability that the tested sample will be considered falsely non-compliant and in the case of tested antibiotics it has a value of 0.01. The detection capacity (CCβ) is the concentration of an analyte that can be detected, identified and determined in a sample with probability 1 – β. The β value means the probability that the tested sample will be considered a false match and in the case of tested drugs it has a value of 0.05. The CCα and CCβ parameters were determined based on the analysis of the calibration curve from the matrix (y = ax + b; y – signal; x – concentration; b – curve slope coefficient; a curve slope coefficient) prepared in accordance with the ISO 11843-4:2007 standard. The calibration curve was obtained from the results of the fortified samples. Uncertainty The uncertainty components are expressed as standard uncertainty, which is measured by the standard deviation. The total uncertainty was expressed as the combined standard uncertainty (uc), and the expanded uncertainty (U) was assumed as the product of the combined standard uncertainty and the coverage factor k = 2 for the adopted significance level α = 0.05 according to the formula: U = k*u c (y). Antibiotics monitoring in solid manures and commercial organic fertilizers Samples of solid natural and organic fertilizers were collected from pig, poultry and cattle farms and organic fertilizer producers in Poland. In general, the developed method analyzed 24 samples of commercial organic fertilizers produced with animal by-products and 49 samples of solid natural fertilizers, including 41 samples from pigs, 7 samples from poultry and 1 sample from beef cattle. After delivery to the laboratory, the samples were stored in plastic containers at a temperature of − 18°C to avoid degradation of antibiotics. Before the analysis day, the samples were slowly thawed in a refrigerator at temperatures of + 2–8°C. Results and discussion Instrumental conditions The selected compounds were detected with a mass spectrometer (MS). All the compounds in the study were sensitive in electrospray ionization (ESI) positive mode. The protonated ion was present as the base peak of the MS spectrum and selected as the precursor ion, and two transitions of the precursor ion were selected for quantification and confirmation by optimizing the collision energy. Daughter ion with higher response and better shaps were selected as quantitative ions. Additionally, standard solutions of all antibacterial substances were infused at 1 µg/L to ensure correct identification of analytes and optimization of precursor and product ions. For the purposes of proper optimization and validation of the method, Commission Implementing Regulation (EU) 2021/808 was used. During the development of the method, the operating conditions of the liquid chromatograph were optimized to obtain the best separation of the analyzed antibiotics and satisfactory selectivity and sensitivity for each compound. Optimization of the mobile phase was carried out by checking its individual components (formic acid and ammonium formate in water and comparison of separation conditions using acetonitrile or methanol and acetonitrile or methanol with the addition of formic acid or ammonium formate). Optimization of the analyte separation conditions also involved the selection of an appropriate gradient elution for the mobile phase for the tested antibiotics in complex analytical matrices such as solid natural and organic fertilizers. In the available scientific literature relating to the analysis of antibacterial substances in natural fertilizers (animal faeces) and soil, researchers use formic acid, ammonium formate or ammonium acetate in water in combination with methanol or acetonitrile with/or without ammonium formate, ammonium acetate or formic acid as a component of the mobile phase. (Berendsen et al., 2015 ; Jensen et al., 2019; Ho et al., 2014 ; Li et al., 2015 ; Haller et al. 2002 ; Wallace and Aga 2016 ; Wu et al. 2014 ). In the case of our research, the most favorable mobile phase for separation was a mixture of 0.1% formic acid in water combined with 0.1% formic acid in acetonitrile. The separation of pharmaceuticals belonging to different chemical groups requires the appropriate selection of chromatographic columns in order to obtain the appropriate shape, separation and peak area. For this purpose, researchers used chromatographic columns such as: such as Genesis C18, Nucleosil C18 HD, Kinetex C18, and ACQUITY UPLC BEH C18 (Berendsen et al., 2015 ; Jensen et al., 2019; Martinez-Carballo et al. 2007 ; Blackwell et al. 2004 ; Li et al. 2015 ; Wu et al. 2014 ; Hu et al. 2010 ). However, all chromatographic columns used were filled with octadecyl (C18). In our work, we tested two Kinetex C18 chromatographic columns (both from Phenomenex) differing in length and grain diameter of the filling: 75 mm x 2.1 mm, 2.6 um and 100 x 4.6 mm, 5 um. Ultimately, the shorter Kinetex C18 column 75 x 2.1 mm, 2.6 um was chosen to develop the method. Extraction experiments Analyzing pharmaceuticals in animal faeces, manure and organic fertilizers can be difficult because it is a complex matrix with a high organic matter content. In addition, the desire to analyze many antibiotics belonging to different chemical groups and exhibiting different physico-chemical properties requires a lot of effort from the analyst to optimize the extraction mixture and select the appropriate purification method. Therefore, as part of the presented work, we checked four different extraction mixtures (described in other scientific studies), different fertilizer sample weights, and solid-phase extraction cartridges with different sorbents and from different manufacturers. Based on the tested extraction mixtures, sample weights and SPE cartridges described in the "Extraction experiments" section, it was shown that the use of "tandem" solid phase extraction (Experiment I) using combined Strata-SAX and Oasis HLB cartridges may be useful for the analysis of tiamulin, tylosin, trimethoprim, oxytetracycline, epi-oxytetarcycline, tetracycline, chlortetracycline, epi-chlortetarcycline, doxycycline, ciprofloxacin, enrofloxacin, sulfamerazine and sulfamethazine - a total of 11 compounds out of 21 antibacterial substances selected for testing. In addition, the purification technique used, combining two SPE cartridges, did not allow for the detection of 10 analytes and increased the limit of detection of the analytes selected for testing. The presented method was described by Blackwell et al. ( 2004 ) and is used for the quantitative analysis of oxtetracycline, sulfachloropyridazine and tylosin in soil and slurry samples in the concentration range from 0.2 to 5 mg/kg. In the described extraction and purification procedure for Experiment II, the method described by Patyra and Kwiatek ( 2017 ) was used, which concerns the analysis of tetracyclines in feed using the LC-MS technique. For this extraction method, McIlvaine-Na 2 EDTA buffer with pH = 4 was used. McIlvaine-Na 2 EDTA buffer is often used alone or in combination with methanol or acetonitrile for the extraction of tetracycline antibiotics from biological matrices and feeds. Better results for the analyzed antibiotics were obtained for 5 g of fertilizer samples compared to 2 g of samples. Strata-XCW cartridges were used to purify the extract, and satisfactory results were obtained for trimethoprim, tiamulin, tylosin and ciprofloxacin, enrofloxacin, sarafloxacin and oxytetracycline, tetracycline, chlortetracycline, doxycycline and their epimeric forms: epi-oxytetracycline, epi-chloroteracycline. The tested method was unsuitable for the sulfonamides selected for testing, lincomycin, spiramycin, valnemulin, and flumequine. The best results were obtained for the described Experiments III and IV of extraction and purification of antibacterial substances from solid fertilizers because all analyzed antibacterial substances were observed in the chromatograms. However, both presented methods still had some shortcomings that needed to be improved to obtain the best possible parameters for the developed method. Preparation of fertilizer samples according to the extraction procedure described as “Experiment IV” required additional dilution of the extract in water in order to be able to clean-up and concentrate the obtained extract on reverse-phase polymer cartridges, Strata-X. This was due to the extraction mixture used, which included McIlvaine-NA 2 EDTA buffer and acetonitrile and methanol in the proportions of 3 mL/3.75 mL/1.25 mL (or 6 mL/ 7.5 mL/ 2.5 mL for a sample weighing 5 g). Direct application of an extract containing approximately 62.5% of the organic mixture to a Strata-X cartridge would cause the analytes to pass through the cartridge along with the dosed extract. In the case of extraction of antibacterial substances from solid natural fertilizers, better results were obtained for a two-gram sample and a mixture of McIlvaine-Na 2 EDTA buffer with pH = 4 and acetonitrile in the proportion of 23:2; v/v. Samples were extracted for 45 min, then centrifuged at 4500 rpm, 20°C, 20 min. The obtained extract was transferred to a new centrifuge tube, and 5 mL of McIlvaine-Na 2 EDTA buffer with pH = 4 was added to the resulting sediment and the samples were shaken again for 30 min. Subsequently, the samples were centrifuged at 4500 rpm, 20°C, 20 min, and the obtained extracts were combined. Due to the fact that fertilizer is a very complex analytical matrix containing many organic compounds such as undigested food remains (fiber, proteins, fats), pigments (bilirubin and biliverdin), significant amounts of nitrogen and phosphorus compounds, enzymes, bacteria and dead lining cells intestinal walls, 5 mL of n-hexane was added to the samples for degreasing. The samples were shaken manually for approximately 1 min. and centrifuged. Samples were purified using Strata-X cartridges (6 ml, 200 mg). The extraction and purification method used allowed for the detection and quantitative determination of all 21 antibiotics belonging to 6 chemical classes. MRM chromatograms for all analysing antibiotics at a concentration 50 µg/kg are shown in Fig. 2 . Method validation Validation of the developed method was carried out in accordance with with Commission Implementing Regulation (EU) 2021/808. The linearity and working range of the method were checked in the concentration range from 50 µg/kg to 1000 µg/kg for all tested antibiotics in solid fertilizers. A calibration curve showing the ratio of the analyte signal to the corresponding internal standard signal or the dependence of the peak area on the analyte concentration was plotted at seven points, taking into account the blank sample. This curve was used to calculate the concentration of antibiotics in fortified samples. The test results of three series of samples fortified to concentrations of 50, 500 and 1000 µg/kg for solid fertilizers were the basis for determining such validation parameters of the procedure as: recovery, repeatability, intra-laboratory reproducibility, CCα, CCβ, LOD and LOQ and uncertainty. The results of the experiments performed are presented in Table 3 . Table 3 Validation results of the LC-MS/MS method for determining antibacterial substances in solid natural fertilizers (pig faeces) Validation parameters Analyte OTC epi-OTC TC CTC epi-CTC DC CIP ENR SAR FLU Selectivity no interference LOD (µg/kg) 15.4 25.1 22.9 20.1 22.6 31.1 20.9 29.0 21.7 24.0 LOQ (µg/kg) 24.3 43.2 40.0 31.0 37.4 45.5 43.6 62.0 41.2 45.1 CCα (µg/kg) 62.7 63.7 61.0 56.8 67.6 68.9 60.8 66.5 58.3 51.2 CCβ (µg/kg) 74.3 76.8 74.1 68.3 84.0 90.1 76.3 97.0 72.1 62.7 Repeatability (CV %) 50 µg/kg 8.7 15.0 5.4 12.6 15.0 12.9 13.9 17.4 17.1 14.9 500 µg/kg 8.7 11.3 9.2 12.1 11.3 6.1 14.0 15.7 10.0 15.8 1000 µg/kg 6.6 6.3 6.7 10.0 6.3 9.4 12.5 15.9 13.0 14.4 Reproducibility (CV %) 50 µg/kg 10.1 16.1 10.7 14.5 16.1 17.6 11.7 18.4 13.6 12.0 500 µg/kg 14.0 11.4 15.5 14.5 11.4 15.0 14.3 21.4 13.7 15.5 1000 µg/kg 8.4 9.9 9.0 8.9 6.3 10.3 11.9 18.8 16.8 14.0 Recovery (%) 50 µg/kg 105.4 110.4 102.6 108.4 103.4 101.7 112.4 103.9 112.4 114.2 500 µg/kg 93.6 102.5 94.2 104.0 99.4 99.6 97.5 97.5 93.9 98.9 1000 µg/kg 98.9 103.6 99.8 107.2 106.8 110.7 105.1 100.7 103.2 98.6 Uncertainty (U %) 50 µg/kg 33.0 35.0 25.0 30.0 33.5 35.2 28.0 37.0 34.0 30.1 500 µg/kg 29.0 27.3 33.0 29.2 31.0 32.0 28.4 35.0 31.0 28.7 1000 µg/kg 27.8 24.0 24.0 18.0 28.4 23.1 26.5 35.7 30.5 25.3 Table 3 continued Validation parameters Analyte SGD SDZ SMR SMZ SMK TRIM LINKO TIAM TYL SPIR VAL Selectivity no interference LOD (µg/kg) 36.6 14.0 16.5 19.3 21.0 9.3 13.4 13.4 12.4 15.5 19.7 LOQ (µg/kg) 48.9 27.5 39.0 46.5 49.0 18.8 27.5 24.8 22.3 27.0 33.1 CCα (µg/kg) 68.9 58.4 52.2 57.7 54.3 55.1 64.8 63.3 74.5 60.0 65.0 CCβ (µg/kg) 82.3 64.4 59.1 64.3 71.2 62.7 82.3 76.2 94.2 81.1 84.2 Repeatability (CV %) 50 µg/kg 18.9 10.3 7.7 10.8 10.8 18.2 8.2 14.2 17.0 15.7 16.1 500 µg/kg 15.3 11.4 11.3 11.3 8.7 11.5 14.4 8.7 12.1 10.8 13.8 1000 µg/kg 12.3 5.7 6.8 8.3 10.6 5.8 10.3 8.1 6.0 16.1 8.6 Reproducibility (CV %) 50 µg/kg 19.1 10.8 14.3 10.1 10.6 14.4 14.3 9.8 17.5 13.4 16.4 500 µg/kg 13.5 17.8 11.1 10.8 18.3 12.5 13.0 12.6 10.9 18.7 14.4 1000 µg/kg 10.3 12.6 10.5 11.5 9.8 6.9 12.5 12.6 8.8 12.7 12.0 Recovery (%) 50 µg/kg 110.0 103.6 113.8 116.6 11.8 98.7 108.0 110.5 107.9 105.1 104.4 500µg/kg 99.6 102.4 101.3 98.7 101.3 99.4 9.8 95.5 95.4 100.7 97.7 1000 µg/kg 106.7 103.3 104.0 107.1 102.0 94.8 96.2 102.5 96.0 101.0 105.5 Uncertainty(U %) 50 µg/kg 36.2 28.6 28.5 24.5 22.0 30.0 25.0 27.6 35.0 26.7 33.0 500 µg/kg 30.4 25.1 23.0 22.3 27.7 24.8 22.1 25.0 30.0 34.1 27.1 1000 µg/kg 29.5 24.4 18.8 22.0 21.9 18.5 15.6 24.3 28.8 26.6 22.9 Table 4. Results for positive samples of solid natural and organic fertilizers Real sample analysis Seventy-three samples of solid natural fertilizers (pig and cattle manure and poultry droppings) and organic fertilizers produced using animal by-products (such as cattle, horse, sheep and chicken manure) were analyzed in the study. Including 49 samples of natural fertilizers and 24 samples of commercial organic fertilizers. The results showed that in the case of samples from farmed poultry, only one sample contained the presence of antibacterial substances - sulfamethoxazole at a concentration below 2 mg/kg. Manure from beef cattle analyzed for antibacterial substances were free of antibiotics, sulfonamides and quinolones. The most contaminated with antibiotics was solid manure from pigs from large-scale farms. The most frequently detected antibiotics were tetracyclines, mainly doxycycline. The presence of doxycycline was confirmed in fifteen samples of natural fertilizers. Doxycycline has been found at concentrations ranging from 103.0 µg/kg to over 57000.0 µg/kg. The tested solid manure from pigs also contained the presence of sulfamethoxazole (in 4 samples), oxytetracycline and epi-oxytetracycline (in 3 samples), and tiamulin in 2 samples. Moreover, the data obtained indicate that two or even three antibiotics were used one after another in the animals from which the material was collected to treat the entire group of animals with feed or water, which indicates their intensive use on large-scale farms. In two samples of manure from pigs, the presence of three different antibiotics was also found: doxycycline, oxytetracycline, and tiamulin. Antibiotics were detected in the manure of piglets at 5 and 8 weeks of age. This indicates the intensive, perhaps irrational use of antibiotics in young animals to prevent the development of bacterial infections. Based on the results obtained, it can be concluded that over 38.77% of the tested samples of natural fertilizers, mainly from pigs, were contaminated with antibiotics. The results were also confirmed by data on the sale of antibiotics in European Union countries, which show that antibiotics from the tetracycline group are among the most frequently prescribed antibacterial drugs for farm animals by veterinarians. The results of the analysis of natural fertilizers showed that tetracycline antibiotics, including oxytetracycline and doxycycline, accounted for 75% of all antibacterial substances determined in natural fertilizers. Laboratory analysis of commercial organic fertilizers also confirmed the presence of antibacterial substances. The determined levels of antibacterial substances in organic fertilizers were lower in relation to the concentrations of antibacterial substances determined in natural fertilizers and ranged from 47.0 µg/kg to 757.9 µg/kg, but the results obtained may indicate that the applied processing processes such as increase the temperature during the processing or composting does not lead to the complete degradation of antibacterial substances present in the material used. The tests carried out showed the presence of antibacterial substances in 9 out of 24 analyzed samples of commercial organic fertilizers made from manure (cattle, horse or sheep), which constitutes over 37.5% of the positive results obtained. In solid commercial organic fertilizers, the most frequently found antibiotics were oxytetracycline and epi-oxytetracycline, but also tiamulin, flumequine, sulfamethazine, sulfamerazine, sulfadiazine, lincomycin and trimethoprim. Moreover, due to the fact that commercial organic fertilizers are produced from animal excrement from different farms, the presence of up to five different antibiotics was found in one fertilizer sample. Antibiotics are detected unchanged in manure, slurry and poultry droppings from farms in concentrations ranging from several µg/kg to several hundred mg/kg. Differences in the levels of antibacterial substances in the fertilizers mentioned above depend on the animal species, the class of antibiotic and the geographical location and type of breeding farm. The highest concentrations of antibiotics are detected in fertilizer from large-scale farms compared to small family farms (Zhi et al. 2020 ). In addition, detection rates and antibiotic concentrations are usually higher in pigs' fertilizers than poultry and cattle fertilizers. This is mainly due to the fact that antibiotics are administered in higher doses and more frequently to pigs than to other farm animals (Xin et al. 2016 ). In a study conducted in the Netherlands on 680 faeces samples from 20 pig farms and 20 cattle farms, the presence of antibiotics was found respectively: in 55% and 75% of samples (Berendsen et al. 2015 ). Moreover, more than one antibiotic was detected in as many as 34% of the tested samples. The most frequently detected compounds were: oxytetracycline, doxycycline and sulfadiazine, tetracycline, flumequine, lincomycin and tylosin. The antibacterial substances in the samples ranged from 1 µg/kg to 95 mg/kg of faeces. All antibiotics detected in the faeces of pigs and/or calves are registered for use in these animal species. Moreover, in animal feces Berendsen et al. in 34% of samples they found the presence of two or more antibiotics, in one sample they determined as many as eight different antibiotics. In the case of some compounds, their concentrations indicated for use for therapeutic purposes. It is possible that antibiotics were used illegally and/or for purposes other than those intended. Another reason for the presence of antibacterial substances may be contamination of feed or water on the farm (Berendsen et al. 2015 ). Similar results were obtained in the present study conducted on samples of poultry droppings, cattle and pig solid manure. The most frequently found antibiotics in the fertilizers mentioned were tetracyclines, mainly doxycycline, and the determined levels of antibacterial substances were to over 57 mg/kg of fertilizers and similar to those mentioned by Berendsen et al. ( 2015 ). In the research conducted by Zhao et al. ( 2010 ), 143 samples of faeces from eight Chinese provinces were analyzed, in which the presence of ciprofloxacin, enrofloxacin, oxytetracycline and chlortetracycline in pig and cattle faeces at concentrations ranging from 21 to over 59 mg/kg. No significant concentrations of sulfonamides (below 10 mg/kg) were found in any of the analyzed faeces samples and only sulfadimidine was observed in chicken droppings at a maximum concentration of 6.04 mg/kg. The residues found by the authors for most antibiotics showed significant statistical differences between the provinces from which they were obtained samples collected and animal species (Zhao et al. 2010 ). In the case of detection and quantification of antibiotics in solid commercial organic fertilizers produced with animal by-products, there is practically no literature data on their analysis and the presence of antibiotics in them. Based on the results obtained in this work and those of other researchers, it should be concluded that antibiotics are often present in natural and organic fertilizers that are used on agricultural fields and grasslands, which may pose a threat to the natural environment. The presence of veterinary antibiotics in natural and organic fertilizers should be monitored to ensure the safety of the environment, but also of animals and consumers consuming agricultural produce and food of animal origin. Moreover, the European Parliament Resolution of 1 June 2023 on EU action to combat antimicrobial resistance adopted by the European Union states that the use of sewage sludge and manure as fertilizers on agricultural soil may lead to the development of antimicrobial resistance through the spread of antimicrobial-resistant bacteria and antimicrobial resistance genes in the environment, which causes further contamination of the food chain and it is necessary to introduce prudent manure management practices. The results for positive samples of solid natural and organic fertilizers are summarized in the Table 4. Conclusion An UHPLC-MS/MS method for antibiotic residues was developed and validated in the present study in solid natural and organic fertilizers. The study focused on optimization of the sample weight, sample clean-up steps, including the use of different extraction solutions and SPE cartridges. In this work 73 samples of natural and organic solid ferilizers were analysed. Tweenty eight samples were positive for the presence of antibacterial substances. Antibiotics conatmination was more severe in the natural fertilizers than in the organic fertilizers in general, natural fertilizers from pigs exhibited the highest concentration of antibiotics in which the most frequently found antibiotic was doxycycline. High residue of antibiotics in natural and organic fertilizers could probably cause risks to the soil ecosystem after they were applied on the arable fields and grassland. Therefore, it is necessary to limit the amount of natural and organic fertilizers application based to containing toxic substances in order to reduce the associated risk to ecosystems, human and animal health. Declarations Author Contributions Statement All authors contributed to the study conception and design. Ewelina Patyra: conceptualization, methodology, supervision, administration, formal analysis, investigation, writing – original draft, writing – review & editing. Zbigniew Osiński: sampling, review – original draft. Krzysztof Kwiatek: supervision, writing – original draft. All authors read and approved the final manuscript. Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors. Funding The study was financially supported by the „Science for Society” programm, decision of Ministry of Science and Higher Education No. NdS/545025/2022/2022 Consent for publication Not applicable. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Consent to Publish Not applicable. Consent to participate Not applicable. References Berendsen BJA, Wegh RS, Memelink J, Zuidema T, Stolker LAM (2015). The analysis of animal faeces as a tool to monitor antibiotic usage. Talanta 132: 258-268 Blackwell PA, Lützhøft H-CHH, Ma H-P, Halling-Sørensen B, Boxall ABA, Kay P (2004) Ultrasonic extraction of veterinary antibiotics from soils and pig slurry with SPE clean-up and LC-UV and fluorescence detection. Talanta 64:1058–1064 Commission Implementing Regulation (EU) 2021/808 of 22 March 2021 on the performance of analytical methods for residues of pharmacologically active substances used in food-producing animals and on the interpretation of results as well as on the methods to be used for sampling and repealing Decisions 2002/657/EC and 98/179/EC. Council Directive 91/676/EEC of 12 December 1991 concerning the protection of waters against pollution caused by nitrates from agricultural sources. 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Environ Sci Pollut Res Int 22:4545-4554 Hu X, Luo Y, Zhou Q (2010) Simultaneous analysis of selected typical antibiotics in manure by microwave-assisted extraction and LC–MS. Chromatographia 1:217–223. Jansen LJM, van de Schans MGM, de Boer D, Bongers IEA, Schmitt H, Hoeksma P, Berendsen BJA (2019) A new extraction procedure to abate the burden of non-extractable antibiotic residues in manure. Chemosphere 224: 544–553. Li Ch, Chen J, Wang J, Ma Z, Ha P, Luan Y, Lu A (2015) Occurrence of antibiotics in soils and manures from greenhouse vegetable production bases of Beijing, China and associated risk assessment. Sci Total Environ 521-522:101-107 Martinez-Carballo E, Gonzalez-Barreiro C, Scharf S, Gans O (2007) Environmental monitoring study of selected veterinary antibiotics in animal manure and soils in Austria. Environ Pollut 162:56–62 Osiński Z., Patyra E., 2022. Nawozy naturalne i organiczne jako źródło zanieczyszczenia środowiska substancjami przeciwbakteryjnymi. Veterinary Medicine. Sciene and Practice. dx.doi.org/10.21521/mw.6638 Patyra E, Kwiatek K (2017) Development and validation of multi-residue analysis for tetracycline antibiotics in feed by high performance liquid chromatography coupled to mass spectrometry. Food Addit Contam 9:1553–1561 Regulation (EC) No 1069/2009 of the European Parliament and of the Council of 21 October 2009 laying down health rules as regards animal by-products and derived products not intended for human consumption and repealing Regulation (EC) No 1774/2002 (Animal by-products Regulation) Ustawa z dnia 10 lipca 2007 r. o nawozach i nawożeniu. Dz. U. 2007, nr 147, poz. 1033. Wallace JS, Aga D (2016) Enhancing Extraction and Detection of Veterinary Antibiotics in solid and Liquid Fractions Manure. J Environ Qual 45: 471–479. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3984638","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":277000450,"identity":"73192f6c-84b4-4a77-99c4-6495e7c7f193","order_by":0,"name":"Ewelina Patyra","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-3827-2314","institution":"Panstwowy Instytut Weterynaryjny - Panstwowy Instytut Badawczy w Pulawach","correspondingAuthor":true,"prefix":"","firstName":"Ewelina","middleName":"","lastName":"Patyra","suffix":""},{"id":277000451,"identity":"bf392f88-673c-4330-afce-1c6d8cf3d015","order_by":1,"name":"Zbigniew Osiński","email":"","orcid":"","institution":"Panstwowy Instytut Weterynaryjny - Panstwowy Instytut Badawczy","correspondingAuthor":false,"prefix":"","firstName":"Zbigniew","middleName":"","lastName":"Osiński","suffix":""},{"id":277000452,"identity":"c3f46049-1d61-4752-92d8-c0c36151b0ae","order_by":2,"name":"Krzysztof Kwiatek","email":"","orcid":"","institution":"Panstwowy Instytut Weterynaryjny - Panstwowy Instytut Badawczy","correspondingAuthor":false,"prefix":"","firstName":"Krzysztof","middleName":"","lastName":"Kwiatek","suffix":""}],"badges":[],"createdAt":"2024-02-24 10:23:48","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3984638/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3984638/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11356-024-33956-w","type":"published","date":"2024-06-18T15:42:52+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":52452825,"identity":"013ceb8c-7826-4928-a979-e62bf45834b1","added_by":"auto","created_at":"2024-03-11 19:17:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":79335,"visible":true,"origin":"","legend":"\u003cp\u003ePurification of the extract using \"tandem\" solid phase extraction\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3984638/v1/21831f1cdaca9c730cefdd27.png"},{"id":52452813,"identity":"0ba4732e-15a4-43c3-98b7-e468f8e94477","added_by":"auto","created_at":"2024-03-11 19:17:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":635275,"visible":true,"origin":"","legend":"\u003cp\u003eMRM chromatograms for all analysing antibiotics at a concentration 50 µg/kg\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3984638/v1/f7a037d6177705234d50c0d0.png"},{"id":58823559,"identity":"5fd17e49-3307-46b0-b0d9-c7e80443286c","added_by":"auto","created_at":"2024-06-21 17:02:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1638806,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3984638/v1/5a40bafd-4cf7-4d7d-b02f-08ad5bdbc7ac.pdf"}],"financialInterests":"","formattedTitle":"Residues of veterinary antibiotics in solid natural and organic fertilizers – method development and sample analysis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eVeterinary antibiotics are widely used in animal husbandry around the world to prevent and treat disease and are still used in some countries to promote animal growth as feed additives (Hou et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Zhou et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Zhi et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Antibiotics in farm animals can be administered in the form of injections (treatment of individual animals, especially ruminants and horses) or orally, by administering them in medicated feed or after dissolving in water. The second method of drug application is used to treat entire groups of animals (mainly pigs and poultry). The popularity of this method of administering antibacterial substances is due to the fact that the drug is administered to an entire group of animals at the same time, which reduces time and work expenditure (Osiński and Patyra \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Regardless of the route of administration of antibacterial substances to farm animals, these compounds are excreted from the body in amounts ranging from 30\u0026ndash;90% of the administered initial dose, in unmetabolized form or in the form of active and/or inactive metabolites with faeces or urine, which are then used as natural fertilizers for fertilizing arable lands and grassland (Berendsen et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) or processed (granulation, composting, fermentation) and available on the market as organic fertilizers. In accordance with the Act of July 10, 2007 in force in Poland on fertilizers and fertilization, for natural fertilizers include manure and slurry well as excrements from farm animals without the addition of other substances, as well as guano, which are intended for agricultural use. Manure or slurry are very good natural fertilizers used in plant cultivation. In accordance with the applicable Directive 91/676/EEC on natural fertilizers, it contains guidelines on the timing of their use on permanent grasslands, permanent crops and arable land, as well as specifies the requirements for their storage and the permissible amount of nitrogen introduced per hectare of crops. In addition to natural fertilizers, organic fertilizers are also used for growing plants, which, in accordance with Regulation (EC) No. 1069/2009 of the European Parliament and of the Council of 21 October 2009, includes manure, which is defined as faeces or urine farm animals, other than farmed fish, with or without litter. In addition, the Regulation defines the concepts of \"organic fertilizer\" and \"soil improver\", which mean materials of animal origin used separately or in combination to maintain or improve plant nutrition, as well as the physical and chemical properties and biological activity of soils. They may contain manure, digestive tract contents, non-mineralized guano, compost and post-fermentation residues. Manure, organic fertilizers and soil improvers are Category 2 materials. This means that they may be by-products containing residues of approved substances or contaminants at levels above permitted levels, including antibiotics, sulfonamides and quinolones, which should be monitored in live animals and products animal origin (Regulation (EC) No. 1069/2009). Natural and organic fertilizers are not subject to any control for the presence of antibacterial substances, and the agricultural use of these fertilizers may cause contamination of the natural environment with these compounds.\u003c/p\u003e \u003cp\u003eAntibiotics are detected unchanged in manure, slurry and poultry litter from farms in concentrations ranging from several \u0026micro;g/kg to several hundred mg/kg. Differences in the levels of antibacterial substances in the above-mentioned fertilizers depend on the animal species, the class of antibiotic and the geographical location and type of breeding farm. Their content depends on the antibiotic used because some substances, such as amoxicillin or tetracycline, are metabolized only by 10\u0026ndash;20%, while others, e.g. sulfamethoxazole, by approximately 85% (Hirsh et al. 1999). Active substances can be metabolized into inactive conjugates, which, after being excreted from the animal's body, can be converted to the active form of the drug. Moreover, some excreted compounds may be similar or even more harmful than the parent compounds (Hirsch et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). Large-scale farms have significantly higher concentrations of antibiotics in fertilizers compared to small family farms (Zhi et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) (Zhi et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Moreover, higher concentrations of antibiotics are detected in fertilizers from pigs than in fertilizers from poultry and cattle. This is mainly due to the fact that antibiotics are administered in higher doses and more frequently to pigs than to other farm animals (Xin et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The available scientific literature contains information that antibiotics are present in high concentrations in feces obtained from slaughtered animals. Wolters et al. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) examined derived manure from eight fattening farms and six breeding pigs farms. In the material examined, the authors found eleven different antibiotics belonging to up to six classes. Antibiotic residue analysis showed a maximum tetracycline concentration reaching up to 300 mg/kg dry matter (DM) in pig manure (Walters et al. 2016). Martinez-Carballo et al. examined pig faeces in Austria, in which they found the presence of antibiotics from the tetracycline group in amounts of several dozen milligrams per kilogram of faeces (oxytetracycline\u0026mdash;29 mg/kg, chlorotetracycline \u0026ndash; 46 mg/kg and tetracycline\u0026mdash;23 mg/kg), (Martinez-Carballo et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Researchers from China were analyzing fertilizers from chicken, in which they found high concentrations of enrofloxacin \u0026ndash; 1420 mg/kg and norfloxacin \u0026ndash; 225 mg/kg (Zhao et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDue to the lack of data on the presence and content of veterinary antibiotics in solid natural fertilizers and commercial organic fertilizers, this work was undertaken to develop a multi-residue analytical method using UHPLC-MS/MS. The study compared various solvents for antibiotic extraction and various solid phase extraction cartridges. The developed method was validated and used to analyze real samples of solid natural fertilizers from pigs, poultry and cattle as well as solid organic fertilizers on the market produced with animal by-products.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eChemicals and Reagents\u003c/h2\u003e \u003cp\u003eHPLC-grade acetonitrile and methanol were obtained from J.T. Baker (Deventer, The Netherlands). Formic acid and citric acid were purchased from Acros Organics (Geel, Belgium). Disodium ethylenediaminetetraacetate (Na\u003csub\u003e2\u003c/sub\u003eEDTA) was from Sigma Aldrich (California, MO, USA), and disodium hydrogen phosphate was from Chempur (Piekary Śląskie, Poland). Water was purified using a Milli-Q water system from Millipore (Billerica, Ma, USA). All target veterinary antibiotics (VAs) and internal standards (IS) were purchsed from Dr. Ehrenstorfer Gmbh (Augsburg, Germany). The 21 target antibiotics belonged to 6 classes: tetracyclines (TCs), oxytetracycline (OXT), epi-oxytetracycline (epi-OXT), tetracycline (TC), chlortetracycline (CTC), epi-chlotetracycline (epi-CTC), doxycycline (DC) and demeclocycline (DMC; IS), sulfonamides (SAs): sulfamethoxazole (SMX), sulfaguanidine (SGD), sulfamethazine (SMZ), sulfadiazine (SDZ), sulfamerazine (SMR) and sulfadiazine-\u003csup\u003e13\u003c/sup\u003eC\u003csub\u003e6\u003c/sub\u003e (SDZ-\u003csup\u003e13\u003c/sup\u003eC\u003csub\u003e6\u003c/sub\u003e; IS); fluoroquinolones (FQs): sarafloxacin (SAR), enrofloxacin (ENR), ciprofloxacin (CIP), flumequine (FLU) and norfloxacin (NOR; IS), macrolides (MAs) tylosin (TYL), spiramycin (SPIR) and erythromycin (ERM; IS), pleuromutilin (PLM): tiamulin (TIAM) and valnelmulin (VAL) and lincosamides: lincomycin (LINCO) and lincomycin-d\u003csub\u003e3\u003c/sub\u003e (LINCO-d3;IS).\u003c/p\u003e \u003cp\u003eMcIlvaine-Na\u003csub\u003e2\u003c/sub\u003eEDTA buffer was prepared by mixing 115.65 mL of 0.2 M phosphate buffer and 184.65 mL of 0.1 M citric acid, then 11.406 g of Na\u003csub\u003e2\u003c/sub\u003eEDTA was added and mixed. The pH was adjusted to 4.0. Four SPE cartridges were tested: Strata-XCW (3 ml, 300 mg), Strata-X (6 ml, 200 mg) and Strata-SAX (12 ml, 500 mg) from Phenomenex (Torrance, CA, USA) and Oasis HLB ( 3 ml, 60 mg), from Waters (Milliford, MA, USA).\u003c/p\u003e \u003cp\u003eAn SPE manifold (J.T. Baker, PA, USA) and a pump as a vacuum source were used.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of standard solutions\u003c/h2\u003e \u003cp\u003eStock standard solutions 1 mg/mL of OTC, epi-OTC, TC, CTC, epi-CTC, DC, SGD, SMR, SMZ, SXZ, TRIM, ENR, SAR, LICO, TIAM, TYL, SPIR, VAL and internal standards of ERT, DMC and LINCO-d\u003csub\u003e3\u003c/sub\u003e were prepared by dissolving 5 mg of individual compounds in 5 mL of methanol. SDZ, SDZ-\u003csup\u003e13\u003c/sup\u003eC\u003csub\u003e6,\u003c/sub\u003e FLU and NOR were dissolved in acetonitrile and CIP was dissolving in mixture of methanol and 1M sodium hydroxide (99:1; v/v). All standard solutions were stored in volumetric flasks at \u0026minus;\u0026thinsp;18\u0026deg;C for 6 months. All VAs and IS working solutions of 10 \u0026micro;g/mL were prepared by diluting the stock solutions in methanol and stored in amber volumetric flask at -18\u0026deg;C for less than one month.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eInstrument analysis\u003c/h2\u003e \u003cp\u003eQuantitative evaluation of antibiotics was performed using the UHPLC-MS/MS method. A liquid chromatograph consisting of an Exion LC with a SCIEX Triple Quad 5500\u0026thinsp;+\u0026thinsp;system (SCIEX, Framingham, MA, USA) was used. A Kinetex C18 column (2.1 mm x 75 mm; 2.6 \u0026micro;m) was used to separate the target compounds. The mobile phase consisted of 0.1% formic acid in milli-Q water (A) and 0.1% formic acid in acetonitrile (B), and the gradient elution was as follows: 0\u0026ndash;2 min 5% B; 2\u0026ndash;10 minutes 5 \u0026minus;\u0026thinsp;15% B, 10\u0026ndash;12 min. 15\u0026ndash;20%B, 12\u0026ndash;15 min. 20\u0026ndash;50% B, 15\u0026ndash;16 min. 50\u0026ndash;70% B, 16\u0026ndash;17 min. 70\u0026ndash;100%, 17\u0026ndash;18 100\u0026ndash;5% B and 18\u0026ndash;21 min. 5% B. The mobile phase flow rate was set to 0.25 mL/min, the injection volume was 10 \u0026micro;l, and the column thermostat temperature was set to 35\u0026deg;C. Antibiotics and their active metabolites selected for testing were identified by detecting the masses of precursor ions and their fragments. Positive electrospray ionization (ESI+) and multiple reaction monitoring (MRM) mode were used for all antibiotics. Depending on the analyzed compound, different operating parameters of the mass detector were used. The operating parameters of the MS/MS detector used are presented in Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Chromatographic integration of samples was performed using Sciex OS MQ version 2.1.6 software (SCIEX, Framingham, MA, USA).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eOperating parameters of the MS/MS detector\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAnalytical conditions\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCurtain gas\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30 psi\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCollision gas\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10 psi\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIon spray voltage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4500 V\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTemperature\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e400\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIon source gas 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40 psi\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIon source gas 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40 psi\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEntrance potential\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTransitions and optimal conditions used for MS/MS analysis.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAnalyte\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePrecursor ion Q1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eProduct ion\u003c/p\u003e \u003cp\u003eQ3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCE\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCXP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eEP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eDwell time\u003c/p\u003e \u003cp\u003e[msec]\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"9\" nameend=\"c9\" namest=\"c1\"\u003e \u003cp\u003eTETRACYCLINES\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e1.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOxytetracycline 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e461.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e426.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e116\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOxytetracycline 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e461.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e444.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e116\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e2.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTetracycline 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e445.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e410.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTetracycyline 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e445.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e427.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e3.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChlorotetracycline 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e479.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e444.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e126\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChlorotetracycline 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e479.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e462.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e126\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e4.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDoxycycline 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e445.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e428.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDoxycycline 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e445.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e410.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e5.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eepi-oxytetarcycline 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e461.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e426.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e116\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eepi-oxytetracycline 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e461.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e444.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e116\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e6.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eepi-chlorotetracyklina1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e479.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e444.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e126\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eepi-chlortetracycline 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e479.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e462.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e126\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDemeclocycline\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e465.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e448.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e124\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"9\" nameend=\"c9\" namest=\"c1\"\u003e \u003cp\u003eFLUOROQUINOLONE\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e7.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCiprofloxacin 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e332.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e314.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCiprofloxacin 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e332.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e245.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e8.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEnrofloxacin 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e360.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e342.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEnrofloxacin 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e360.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e286.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e9.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSarafloxacin 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e386.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e368.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSarafloxacin 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e386.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e299.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e10.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFlumequine 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e262.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e202.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFumequine 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e262.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e174.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNorfloxacin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e319.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e151.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e104\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"9\" nameend=\"c9\" namest=\"c1\"\u003e \u003cp\u003eSULFONAMIDES\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e11.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSulfaguandine 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e215.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e156.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSulfaguanidine 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e215.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e92.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e12.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSulfadiazine 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e251.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e156.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSulfadiazine2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e251.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e108.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e13.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSulfamerazine1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e265.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e156.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSulfamerazine 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e265.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e92.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e14.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSulfamethazine 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e279.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e186.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSulfamethazine 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e279.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e124.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e15.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSulfamethoxazole 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e254.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e156.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSulfamethoxazole 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e254.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e108.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e16.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTrimethoprim 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e291.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e230.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e181\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTerimethoprim 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e291.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e261.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e181\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSulfadiazine-\u003csup\u003e13\u003c/sup\u003eC\u003csub\u003e6\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e257.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e162.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"9\" nameend=\"c9\" namest=\"c1\"\u003e \u003cp\u003eLINCOSAMIDES\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e17.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLincomycin 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e407.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e126.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLincomycin 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e407.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e359.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLincomycin-d\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e410.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e129.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e129\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"9\" nameend=\"c9\" namest=\"c1\"\u003e \u003cp\u003eMACROLIDES\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e18.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSpiramycin 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e422.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e174.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e126\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSpiramycin 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e422.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e101.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e126\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e19.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTylosin 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e916.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e174.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e139\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTylosin 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e916.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e772.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e125\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eErytrhomycin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e734.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e576.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e157\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"9\" nameend=\"c9\" namest=\"c1\"\u003e \u003cp\u003ePLEUROMUTILIN\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e20.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTiamulin 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e494.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e192.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e114\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTiamulin 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e494.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e119.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e135\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e21.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eValnemulin 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e565.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e263.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e141\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eValnemulin 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e565.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e164. 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e141\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eMethod development\u003c/h2\u003e \u003cp\u003eDue to the fact that the analytical matrices selected for research, such as natural and organic fertilizers, are complex analytical matrices, developing an appropriate extraction method for all antibacterial substances listed in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e turned out to be a time-consuming step. The extraction method was optimized for samples fortified with analytes at a level of 100 \u0026micro;g/kg for solid fertilizers.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eExtraction experiments\u003c/h2\u003e \u003cp\u003eTwenty one compounds were selected from six different antibiotics classes: the tetracyclines OXT, epi-OXT, TC, CTC, epi-CTC and DC; fluoroquinolones CIP, ENR, SAR and FLU, the sulfonamides SGD, SDZ, SMR, SMZ, SXZ and TRIM, the macrolides TYL and SPIR, pleuromutilin TIAM and VAL, and lincosamide LINCO. The mentioned antibiotics are characterized by different physico-chemical properties, therefore experiments had to be carried out using various extraction mixtures that would allow for good extraction of all the mentioned analytes from natural and organic fertilizers. In addition, due to the complexity of the analytical matrix, it was necessary to select an appropriate technique for purifying the obtained extracts to ensure detection and good recovery of all analyzed antibiotics.\u003c/p\u003e \u003cp\u003e \u003cb\u003eExperiment I\u003c/b\u003e: Two- and five-gram samples of solid fertilizer were extracted with 25 mL of McIlvaine-Na\u003csub\u003e2\u003c/sub\u003eEDTA buffer at pH\u0026thinsp;=\u0026thinsp;7 and acetonitrile mixture in a ratio of 23:2 v/v. The samples were shaken and centrifuged, then the extract was filtered through a cellulose filter and acidified with 85% orthophosphoric acid, using 50 \u0026micro;L of orthophosphoric acid for each 5 mL of extract. After this stage of work, the extract was clean-up using \"tandem\" SPE according to the scheme below (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The obtained eluate was evaporated in a stream of nitrogen, and the precipitate was dissolved in 500 \u0026micro;L of 0.1% formic acid in water and additionally filtered through a PVDF syringe filter (13 mm, 0.22 \u0026micro;m).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eExperiment II\u003c/strong\u003e \u003cp\u003eTwo- and five-gram solid fertilizer samples were extracted with 25 mL of McIlvaine-Na\u003csub\u003e2\u003c/sub\u003eEDTA buffer with pH\u0026thinsp;=\u0026thinsp;4. The samples were shaken and centrifuged. The extract was purified by SPE using Strata-XCW cartridges (3 mL, 100 mg). The cartridges were conditioned with 5 mL of methanol, 5 mL of water, then 9 mL of the extract was placed on the cartridge. The impurities were washed out with 6 mL of water, 6 mL of methanol and 3 mL of acetonitrile. The cartridges were dried under vacuum for 5 min, and the antibacterial substances were eluted from the columns with 3 mL of 2% formic acid in methanol. The eluate was evaporated in a stream of nitrogen, and the obtained precipitate was dissolved in 0.1% formic acid in water and additionally filtered through a PVDF syringe filter (13 mm, 0.22 \u0026micro;m).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eExperiment III\u003c/strong\u003e \u003cp\u003e2 mL of acetonitrile were added to a two- and five-gram sample of fertilizer and mixed using a vortex mixer for 30 s. Then, 18 mL of McIlvaine-Na2EDTA buffer with pH\u0026thinsp;=\u0026thinsp;4 was added and shaken for 45 min. The samples were centrifuged at 4500 rpm, 15 min, 20\u0026ordm;C, and then the supernatant was transferred to a new 50 mL centrifuge tube. 5 mL of McIlvaine-Na2EDTA buffer with pH\u0026thinsp;=\u0026thinsp;4 was added to the sediment obtained after centrifugation and shaken again for 30 min. The samples were then centrifuged and the obtained extracts combined. 5 mL of n-hexane was added to the extract and shaken manually for about 1 min. The samples were then centrifuged again. 18 mL of the extract was purified and concentrated using solid phase extraction on Strata-X cartridges (200 mg, 6 mL). The cartridges were conditioned by washing with 6 mL of methanol and 6 mL of water. Dispense 18 mL of sample onto the cartridges. Subsequently, the cartridges were washed with 12 mL of water, and then the cartridges were dried under vacuum for 10 min. Antibacterial substances were eluted from the cartridges with 3 mL of methanol and then evaporated to dryness in a stream of nitrogen at a temperature of 45\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u0026deg;C. The residue was dissolved in 500 \u0026micro;L of 0.1% formic acid in water and filtered through a PVDF filter (13 mm, 0.22 \u0026micro;m) into a chromatography vial.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eExperiment IV\u003c/b\u003e: A two-gram solid fertilizer sample was extracted with a mixture of McIlvaine-Na\u003csub\u003e2\u003c/sub\u003eEDTA buffer at pH\u0026thinsp;=\u0026thinsp;4, acetonitrile and methanol in the proportion of 3:3.75:1.25; v/v/v or for five-gram samples in the ratio 6:7.5:2.5; v/v/v. The samples were shaken and centrifuged. To reduce the content of organic reagents such as acetonitrile and methanol, the obtained extract was diluted in water by adding 6 mL of extract to 25 mL of water. The extracts were purified by SPE using Strata-X cartridges (6 mL, 200 mg). The cartridges were conditioned with 6 mL of methanol and 6 mL of water. After conditioning, 18 mL of extract was dosed onto the cartridges, the impurities were washed out with 12 mL of water, and then the cartridges were dried for 10 min. under vacuum. Antibacterial substances were eluted from the cartridges with 3 mL of methanol. The eluate was evaporated under a stream of nitrogen, and the precipitate was dissolved in 500 \u0026micro;L of 0.1% formic acid in water and filtered through a PVDF syringe filter (13 mm, 0.22 \u0026micro;m).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMethod validation\u003c/h2\u003e \u003cp\u003eValidation of the method was carried out following the guidelines set out in the EU Commission Implementing Regulation 2021/808 of 22 March 2021 on the performance of analytical methods for residues of pharmacologically active substances used in food-producing animals and the interpretation of the results, as well as on the methods used for sampling and repealing Decisions 2002/657/EC and 98/179/EC.\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003eLinearity, selectivity, LOD and LOQ\u003c/h2\u003e \u003cp\u003eThe working range of the method was determined by preparing calibration curves for fortified samples. For this purpose, blank samples were fortified with antibacterial substances at seven adopted concentration levels (0, 50, 100, 250, 500, 750 and 1000 \u0026micro;g/kg). The regression coefficient (R\u003csup\u003e2\u003c/sup\u003e), slope (a) and shift (b) were calculated for the linear regression equation of the type y\u0026thinsp;=\u0026thinsp;ax\u0026thinsp;+\u0026thinsp;b. Then, curves were determined for fortified samples based on the relationship between concentration and signal size or the ratio of the analyte signal to the corresponding signal of the internal standard. It was assumed that the value of the coefficient of determination (R\u003csup\u003e2\u003c/sup\u003e) of the calibration curve for each analyte should be higher than 0.98 for quantification in order to consider the linearity as satisfactory. The limit of detection and limit of quantification were assessed by the signal-to-noise ratio (3 for LOD and 10 for LOQ). The presence of interferences resulting from the composition of the endogenous matrix in the retention times of the monitored antibiotics was checked to determine the selectivity of the method. Selectivity tests were performed on 20 animal faeces samples.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003eRecovery, repeatability, and within-laboratory reproducibility\u003c/h2\u003e \u003cp\u003eTo determine the recovery and repeatability of the analyses, blank samples were fortified at three concentration levels: 50, 500 and 1000 \u0026micro;g/kg (six samples for each levels). The recovery was calculated based on the results obtained when determining repeatability. Recovery is the percentage of the actual concentration of a substance contained in a sample determined during the analytical process, according to the following equation: % recovery\u0026thinsp;=\u0026thinsp;100 \u0026times; measured content/fortification level. Intralaboratory reproducibility is the precision under conditions of intralaboratory reproducibility in which independent analytical results for the same quantities are obtained by the same method by different operators, in the same laboratory and with the same equipment over long time intervals. In order to determine intra-laboratory reproducibility, data from the determination of repeatability are used and additionally the same analyzes were performed - blank samples were fortified at 3 concentration levels, in two repetitions, at specified time intervals, by different operators. The mean value (x), standard deviation (SD) and coefficient of variation (CV) were calculated for each fortification level.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDecision limit and detection capability\u003c/h2\u003e \u003cp\u003eThe decision limit (CCα) is the concentration of an analyte in a sample above which the analytical result is considered inconsistent with the error probability α. The α value means the probability that the tested sample will be considered falsely non-compliant and in the case of tested antibiotics it has a value of 0.01. The detection capacity (CCβ) is the concentration of an analyte that can be detected, identified and determined in a sample with probability 1 \u0026ndash; β. The β value means the probability that the tested sample will be considered a false match and in the case of tested drugs it has a value of 0.05.\u003c/p\u003e \u003cp\u003eThe CCα and CCβ parameters were determined based on the analysis of the calibration curve from the matrix (y\u0026thinsp;=\u0026thinsp;ax\u0026thinsp;+\u0026thinsp;b; y \u0026ndash; signal; x \u0026ndash; concentration; b \u0026ndash; curve slope coefficient; a curve slope coefficient) prepared in accordance with the ISO 11843-4:2007 standard. The calibration curve was obtained from the results of the fortified samples.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eUncertainty\u003c/h2\u003e \u003cp\u003eThe uncertainty components are expressed as standard uncertainty, which is measured by the standard deviation. The total uncertainty was expressed as the combined standard uncertainty (uc), and the expanded uncertainty (U) was assumed as the product of the combined standard uncertainty and the coverage factor k\u0026thinsp;=\u0026thinsp;2 for the adopted significance level α\u0026thinsp;=\u0026thinsp;0.05 according to the formula: \u003cem\u003eU\u0026thinsp;=\u0026thinsp;k*u\u003c/em\u003e\u003csub\u003e\u003cem\u003ec\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e(y).\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eAntibiotics monitoring in solid manures and commercial organic fertilizers\u003c/h2\u003e \u003cp\u003eSamples of solid natural and organic fertilizers were collected from pig, poultry and cattle farms and organic fertilizer producers in Poland. In general, the developed method analyzed 24 samples of commercial organic fertilizers produced with animal by-products and 49 samples of solid natural fertilizers, including 41 samples from pigs, 7 samples from poultry and 1 sample from beef cattle. After delivery to the laboratory, the samples were stored in plastic containers at a temperature of \u0026minus;\u0026thinsp;18\u0026deg;C to avoid degradation of antibiotics. Before the analysis day, the samples were slowly thawed in a refrigerator at temperatures of +\u0026thinsp;2\u0026ndash;8\u0026deg;C.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eInstrumental conditions\u003c/h2\u003e\n \u003cp\u003eThe selected compounds were detected with a mass spectrometer (MS). All the compounds in the study were sensitive in electrospray ionization (ESI) positive mode. The protonated ion was present as the base peak of the MS spectrum and selected as the precursor ion, and two transitions of the precursor ion were selected for quantification and confirmation by optimizing the collision energy. Daughter ion with higher response and better shaps were selected as quantitative ions. Additionally, standard solutions of all antibacterial substances were infused at 1 \u0026micro;g/L to ensure correct identification of analytes and optimization of precursor and product ions. For the purposes of proper optimization and validation of the method, Commission Implementing Regulation (EU) 2021/808 was used.\u003c/p\u003e\n \u003cp\u003eDuring the development of the method, the operating conditions of the liquid chromatograph were optimized to obtain the best separation of the analyzed antibiotics and satisfactory selectivity and sensitivity for each compound. Optimization of the mobile phase was carried out by checking its individual components (formic acid and ammonium formate in water and comparison of separation conditions using acetonitrile or methanol and acetonitrile or methanol with the addition of formic acid or ammonium formate). Optimization of the analyte separation conditions also involved the selection of an appropriate gradient elution for the mobile phase for the tested antibiotics in complex analytical matrices such as solid natural and organic fertilizers. In the available scientific literature relating to the analysis of antibacterial substances in natural fertilizers (animal faeces) and soil, researchers use formic acid, ammonium formate or ammonium acetate in water in combination with methanol or acetonitrile with/or without ammonium formate, ammonium acetate or formic acid as a component of the mobile phase. (Berendsen et al., \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e; Jensen et al., 2019; Ho et al., \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e; Li et al., \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e; Haller et al. \u003cspan class=\"CitationRef\"\u003e2002\u003c/span\u003e; Wallace and Aga \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e; Wu et al. \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e). In the case of our research, the most favorable mobile phase for separation was a mixture of 0.1% formic acid in water combined with 0.1% formic acid in acetonitrile. The separation of pharmaceuticals belonging to different chemical groups requires the appropriate selection of chromatographic columns in order to obtain the appropriate shape, separation and peak area. For this purpose, researchers used chromatographic columns such as: such as Genesis C18, Nucleosil C18 HD, Kinetex C18, and ACQUITY UPLC BEH C18 (Berendsen et al., \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e; Jensen et al., 2019; Martinez-Carballo et al. \u003cspan class=\"CitationRef\"\u003e2007\u003c/span\u003e; Blackwell et al. \u003cspan class=\"CitationRef\"\u003e2004\u003c/span\u003e; Li et al. \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e; Wu et al. \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e; Hu et al. \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e). However, all chromatographic columns used were filled with octadecyl (C18). In our work, we tested two Kinetex C18 chromatographic columns (both from Phenomenex) differing in length and grain diameter of the filling: 75 mm x 2.1 mm, 2.6 um and 100 x 4.6 mm, 5 um. Ultimately, the shorter Kinetex C18 column 75 x 2.1 mm, 2.6 um was chosen to develop the method.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003eExtraction experiments\u003c/h2\u003e\n \u003cp\u003eAnalyzing pharmaceuticals in animal faeces, manure and organic fertilizers can be difficult because it is a complex matrix with a high organic matter content. In addition, the desire to analyze many antibiotics belonging to different chemical groups and exhibiting different physico-chemical properties requires a lot of effort from the analyst to optimize the extraction mixture and select the appropriate purification method. Therefore, as part of the presented work, we checked four different extraction mixtures (described in other scientific studies), different fertilizer sample weights, and solid-phase extraction cartridges with different sorbents and from different manufacturers.\u003c/p\u003e\n \u003cp\u003eBased on the tested extraction mixtures, sample weights and SPE cartridges described in the \u0026quot;Extraction experiments\u0026quot; section, it was shown that the use of \u0026quot;tandem\u0026quot; solid phase extraction (Experiment I) using combined Strata-SAX and Oasis HLB cartridges may be useful for the analysis of tiamulin, tylosin, trimethoprim, oxytetracycline, epi-oxytetarcycline, tetracycline, chlortetracycline, epi-chlortetarcycline, doxycycline, ciprofloxacin, enrofloxacin, sulfamerazine and sulfamethazine - a total of 11 compounds out of 21 antibacterial substances selected for testing. In addition, the purification technique used, combining two SPE cartridges, did not allow for the detection of 10 analytes and increased the limit of detection of the analytes selected for testing. The presented method was described by Blackwell et al. (\u003cspan class=\"CitationRef\"\u003e2004\u003c/span\u003e) and is used for the quantitative analysis of oxtetracycline, sulfachloropyridazine and tylosin in soil and slurry samples in the concentration range from 0.2 to 5 mg/kg.\u003c/p\u003e\n \u003cp\u003eIn the described extraction and purification procedure for Experiment II, the method described by Patyra and Kwiatek (\u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e) was used, which concerns the analysis of tetracyclines in feed using the LC-MS technique. For this extraction method, McIlvaine-Na\u003csub\u003e2\u003c/sub\u003eEDTA buffer with pH\u0026thinsp;=\u0026thinsp;4 was used. McIlvaine-Na\u003csub\u003e2\u003c/sub\u003eEDTA buffer is often used alone or in combination with methanol or acetonitrile for the extraction of tetracycline antibiotics from biological matrices and feeds. Better results for the analyzed antibiotics were obtained for 5 g of fertilizer samples compared to 2 g of samples. Strata-XCW cartridges were used to purify the extract, and satisfactory results were obtained for trimethoprim, tiamulin, tylosin and ciprofloxacin, enrofloxacin, sarafloxacin and oxytetracycline, tetracycline, chlortetracycline, doxycycline and their epimeric forms: epi-oxytetracycline, epi-chloroteracycline. The tested method was unsuitable for the sulfonamides selected for testing, lincomycin, spiramycin, valnemulin, and flumequine. The best results were obtained for the described Experiments III and IV of extraction and purification of antibacterial substances from solid fertilizers because all analyzed antibacterial substances were observed in the chromatograms. However, both presented methods still had some shortcomings that needed to be improved to obtain the best possible parameters for the developed method. Preparation of fertilizer samples according to the extraction procedure described as \u0026ldquo;Experiment IV\u0026rdquo; required additional dilution of the extract in water in order to be able to clean-up and concentrate the obtained extract on reverse-phase polymer cartridges, Strata-X. This was due to the extraction mixture used, which included McIlvaine-NA\u003csub\u003e2\u003c/sub\u003eEDTA buffer and acetonitrile and methanol in the proportions of 3 mL/3.75 mL/1.25 mL (or 6 mL/ 7.5 mL/ 2.5 mL for a sample weighing 5 g). Direct application of an extract containing approximately 62.5% of the organic mixture to a Strata-X cartridge would cause the analytes to pass through the cartridge along with the dosed extract. In the case of extraction of antibacterial substances from solid natural fertilizers, better results were obtained for a two-gram sample and a mixture of McIlvaine-Na\u003csub\u003e2\u003c/sub\u003eEDTA buffer with pH\u0026thinsp;=\u0026thinsp;4 and acetonitrile in the proportion of 23:2; v/v. Samples were extracted for 45 min, then centrifuged at 4500 rpm, 20\u0026deg;C, 20 min. The obtained extract was transferred to a new centrifuge tube, and 5 mL of McIlvaine-Na\u003csub\u003e2\u003c/sub\u003eEDTA buffer with pH\u0026thinsp;=\u0026thinsp;4 was added to the resulting sediment and the samples were shaken again for 30 min. Subsequently, the samples were centrifuged at 4500 rpm, 20\u0026deg;C, 20 min, and the obtained extracts were combined. Due to the fact that fertilizer is a very complex analytical matrix containing many organic compounds such as undigested food remains (fiber, proteins, fats), pigments (bilirubin and biliverdin), significant amounts of nitrogen and phosphorus compounds, enzymes, bacteria and dead lining cells intestinal walls, 5 mL of n-hexane was added to the samples for degreasing. The samples were shaken manually for approximately 1 min. and centrifuged. Samples were purified using Strata-X cartridges (6 ml, 200 mg). The extraction and purification method used allowed for the detection and quantitative determination of all 21 antibiotics belonging to 6 chemical classes. MRM chromatograms for all analysing antibiotics at a concentration 50 \u0026micro;g/kg are shown in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003eMethod validation\u003c/h2\u003e\n \u003cp\u003eValidation of the developed method was carried out in accordance with with Commission Implementing Regulation (EU) 2021/808. The linearity and working range of the method were checked in the concentration range from 50 \u0026micro;g/kg to 1000 \u0026micro;g/kg for all tested antibiotics in solid fertilizers. A calibration curve showing the ratio of the analyte signal to the corresponding internal standard signal or the dependence of the peak area on the analyte concentration was plotted at seven points, taking into account the blank sample. This curve was used to calculate the concentration of antibiotics in fortified samples. The test results of three series of samples fortified to concentrations of 50, 500 and 1000 \u0026micro;g/kg for solid fertilizers were the basis for determining such validation parameters of the procedure as: recovery, repeatability, intra-laboratory reproducibility, CC\u0026alpha;, CC\u0026beta;, LOD and LOQ and uncertainty. The results of the experiments performed are presented in Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e.\u0026nbsp;\u003c/p\u003e\u0026nbsp;\u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eValidation results of the LC-MS/MS method for determining antibacterial substances in solid natural fertilizers (pig faeces)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colspan=\"3\" rowspan=\"2\"\u003e\n \u003cp\u003eValidation parameters\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"20\"\u003e\n \u003cp\u003eAnalyte\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eOTC\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eepi-OTC\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eTC\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eCTC\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eepi-CTC\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eDC\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eCIP\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eENR\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eSAR\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eFLU\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eSelectivity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"20\"\u003e\n \u003cp\u003eno interference\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eLOD (\u0026micro;g/kg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e15.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e25.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e22.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e20.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e22.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e31.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e20.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e29.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e21.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e24.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eLOQ (\u0026micro;g/kg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e24.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e43.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e40.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e31.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e37.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e45.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e43.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e62.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e41.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e45.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eCC\u0026alpha; (\u0026micro;g/kg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e62.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e63.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e61.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e56.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e67.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e68.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e60.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e66.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e58.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e51.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eCC\u0026beta; (\u0026micro;g/kg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e74.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e76.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e74.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e68.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e84.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e90.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e76.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e97.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e72.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e62.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eRepeatability\u003c/p\u003e\n \u003cp\u003e(CV %)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50 \u0026micro;g/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e8.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e15.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e5.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e12.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e15.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e12.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e13.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e17.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e17.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e14.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e500 \u0026micro;g/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e8.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e11.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e9.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e12.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e11.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e6.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e14.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e15.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e10.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e15.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1000 \u0026micro;g/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e6.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e6.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e6.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e10.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e6.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e9.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e12.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e15.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e13.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e14.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eReproducibility (CV %)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50 \u0026micro;g/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e10.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e16.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e10.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e14.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e16.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e17.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e11.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e18.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e13.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e12.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e500 \u0026micro;g/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e14.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e11.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e15.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e14.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e11.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e15.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e14.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e21.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e13.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e15.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1000 \u0026micro;g/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e8.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e9.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e9.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e8.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e6.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e10.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e11.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e18.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e16.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e14.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eRecovery (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50 \u0026micro;g/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e105.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e110.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e102.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e108.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e103.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e101.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e112.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e103.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e112.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e114.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e500 \u0026micro;g/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e93.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e102.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e94.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e104.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e99.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e99.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e97.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e97.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e93.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e98.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1000 \u0026micro;g/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e98.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e103.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e99.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e107.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e106.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e110.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e105.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e100.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e103.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e98.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eUncertainty (U %)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50 \u0026micro;g/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e33.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e35.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e25.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e30.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e33.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e35.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e28.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e37.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e34.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e30.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e500 \u0026micro;g/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e29.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e27.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e33.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e29.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e31.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e32.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e28.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e35.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e31.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e28.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1000 \u0026micro;g/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e27.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e24.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e24.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e18.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e28.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e23.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e26.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e35.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e30.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e25.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003econtinued\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colspan=\"4\" rowspan=\"2\"\u003e\n \u003cp\u003eValidation parameters\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"20\"\u003e\n \u003cp\u003eAnalyte\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"1\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSGD\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSDZ\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eSMR\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eSMZ\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eSMK\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eTRIM\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eLINKO\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eTIAM\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eTYL\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eSPIR\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eVAL\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"1\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eSelectivity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"20\"\u003e\n \u003cp\u003eno interference\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eLOD (\u0026micro;g/kg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e36.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e16.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e19.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e21.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e9.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e13.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e13.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e12.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e15.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eLOQ (\u0026micro;g/kg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e48.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e39.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e46.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e49.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e18.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e27.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e24.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e22.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e27.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eCC\u0026alpha; (\u0026micro;g/kg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e68.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e58.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e52.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e57.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e54.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e55.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e64.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e63.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e74.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e60.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e65.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003eCC\u0026beta; (\u0026micro;g/kg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e82.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e64.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e59.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e64.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e71.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e62.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e82.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e76.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e94.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e81.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e84.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\" rowspan=\"3\"\u003e\n \u003cp\u003eRepeatability\u003c/p\u003e\n \u003cp\u003e(CV %)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e50 \u0026micro;g/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e7.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e10.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e10.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e18.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e8.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e14.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e17.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e15.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e500 \u0026micro;g/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e11.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e11.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e8.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e11.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e14.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e8.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e12.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e10.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e1000 \u0026micro;g/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e6.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e8.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e10.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e5.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e10.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e8.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e6.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e16.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\" rowspan=\"3\"\u003e\n \u003cp\u003eReproducibility (CV %)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e50 \u0026micro;g/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e14.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e10.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e10.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e14.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e14.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e9.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e17.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e13.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e500 \u0026micro;g/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e11.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e10.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e18.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e12.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e13.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e12.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e10.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e18.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e1000 \u0026micro;g/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e10.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e11.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e9.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e6.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e12.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e12.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e8.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e12.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\" rowspan=\"3\"\u003e\n \u003cp\u003eRecovery (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e50 \u0026micro;g/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e110.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e103.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e113.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e116.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e11.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e98.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e108.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e110.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e107.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e105.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e104.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e500\u0026micro;g/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e99.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e102.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e101.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e98.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e101.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e99.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e9.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e95.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e95.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e100.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e97.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e1000 \u0026micro;g/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e106.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e103.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e104.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e107.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e102.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e94.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e96.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e102.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e96.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e101.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e105.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\" rowspan=\"3\"\u003e\n \u003cp\u003eUncertainty(U %)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e50 \u0026micro;g/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e36.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e28.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e24.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e22.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e30.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e25.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e27.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e35.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e26.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e500 \u0026micro;g/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e23.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e22.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e27.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e24.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e22.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e25.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e30.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e34.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e1000 \u0026micro;g/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e18.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e22.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e21.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e18.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e15.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e24.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e28.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e26.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eTable 4.\u003c/strong\u003e Results for positive samples of solid natural and organic fertilizers\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cimg src=\"https://myfiles.space/user_files/122228_c8a1650c59388082/122228_custom_files/img1710158670.png\"\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003eReal sample analysis\u003c/h2\u003e\n \u003cp\u003eSeventy-three samples of solid natural fertilizers (pig and cattle manure and poultry droppings) and organic fertilizers produced using animal by-products (such as cattle, horse, sheep and chicken manure) were analyzed in the study. Including 49 samples of natural fertilizers and 24 samples of commercial organic fertilizers. The results showed that in the case of samples from farmed poultry, only one sample contained the presence of antibacterial substances - sulfamethoxazole at a concentration below 2 mg/kg. Manure from beef cattle analyzed for antibacterial substances were free of antibiotics, sulfonamides and quinolones. The most contaminated with antibiotics was solid manure from pigs from large-scale farms. The most frequently detected antibiotics were tetracyclines, mainly doxycycline. The presence of doxycycline was confirmed in fifteen samples of natural fertilizers. Doxycycline has been found at concentrations ranging from 103.0 \u0026micro;g/kg to over 57000.0 \u0026micro;g/kg. The tested solid manure from pigs also contained the presence of sulfamethoxazole (in 4 samples), oxytetracycline and epi-oxytetracycline (in 3 samples), and tiamulin in 2 samples. Moreover, the data obtained indicate that two or even three antibiotics were used one after another in the animals from which the material was collected to treat the entire group of animals with feed or water, which indicates their intensive use on large-scale farms. In two samples of manure from pigs, the presence of three different antibiotics was also found: doxycycline, oxytetracycline, and tiamulin. Antibiotics were detected in the manure of piglets at 5 and 8 weeks of age. This indicates the intensive, perhaps irrational use of antibiotics in young animals to prevent the development of bacterial infections. Based on the results obtained, it can be concluded that over 38.77% of the tested samples of natural fertilizers, mainly from pigs, were contaminated with antibiotics. The results were also confirmed by data on the sale of antibiotics in European Union countries, which show that antibiotics from the tetracycline group are among the most frequently prescribed antibacterial drugs for farm animals by veterinarians. The results of the analysis of natural fertilizers showed that tetracycline antibiotics, including oxytetracycline and doxycycline, accounted for 75% of all antibacterial substances determined in natural fertilizers.\u003c/p\u003e\n \u003cp\u003eLaboratory analysis of commercial organic fertilizers also confirmed the presence of antibacterial substances. The determined levels of antibacterial substances in organic fertilizers were lower in relation to the concentrations of antibacterial substances determined in natural fertilizers and ranged from 47.0 \u0026micro;g/kg to 757.9 \u0026micro;g/kg, but the results obtained may indicate that the applied processing processes such as increase the temperature during the processing or composting does not lead to the complete degradation of antibacterial substances present in the material used. The tests carried out showed the presence of antibacterial substances in 9 out of 24 analyzed samples of commercial organic fertilizers made from manure (cattle, horse or sheep), which constitutes over 37.5% of the positive results obtained. In solid commercial organic fertilizers, the most frequently found antibiotics were oxytetracycline and epi-oxytetracycline, but also tiamulin, flumequine, sulfamethazine, sulfamerazine, sulfadiazine, lincomycin and trimethoprim. Moreover, due to the fact that commercial organic fertilizers are produced from animal excrement from different farms, the presence of up to five different antibiotics was found in one fertilizer sample. Antibiotics are detected unchanged in manure, slurry and poultry droppings from farms in concentrations ranging from several \u0026micro;g/kg to several hundred mg/kg. Differences in the levels of antibacterial substances in the fertilizers mentioned above depend on the animal species, the class of antibiotic and the geographical location and type of breeding farm. The highest concentrations of antibiotics are detected in fertilizer from large-scale farms compared to small family farms (Zhi et al. \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). In addition, detection rates and antibiotic concentrations are usually higher in pigs\u0026apos; fertilizers than poultry and cattle fertilizers. This is mainly due to the fact that antibiotics are administered in higher doses and more frequently to pigs than to other farm animals (Xin et al. \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). In a study conducted in the Netherlands on 680 faeces samples from 20 pig farms and 20 cattle farms, the presence of antibiotics was found respectively: in 55% and 75% of samples (Berendsen et al. \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e). Moreover, more than one antibiotic was detected in as many as 34% of the tested samples. The most frequently detected compounds were: oxytetracycline, doxycycline and sulfadiazine, tetracycline, flumequine, lincomycin and tylosin. The antibacterial substances in the samples ranged from 1 \u0026micro;g/kg to 95 mg/kg of faeces. All antibiotics detected in the faeces of pigs and/or calves are registered for use in these animal species. Moreover, in animal feces Berendsen et al. in 34% of samples they found the presence of two or more antibiotics, in one sample they determined as many as eight different antibiotics. In the case of some compounds, their concentrations indicated for use for therapeutic purposes. It is possible that antibiotics were used illegally and/or for purposes other than those intended. Another reason for the presence of antibacterial substances may be contamination of feed or water on the farm (Berendsen et al. \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e). Similar results were obtained in the present study conducted on samples of poultry droppings, cattle and pig solid manure. The most frequently found antibiotics in the fertilizers mentioned were tetracyclines, mainly doxycycline, and the determined levels of antibacterial substances were to over 57 mg/kg of fertilizers and similar to those mentioned by Berendsen et al. (\u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e). In the research conducted by Zhao et al. (\u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e), 143 samples of faeces from eight Chinese provinces were analyzed, in which the presence of ciprofloxacin, enrofloxacin, oxytetracycline and chlortetracycline in pig and cattle faeces at concentrations ranging from 21 to over 59 mg/kg. No significant concentrations of sulfonamides (below 10 mg/kg) were found in any of the analyzed faeces samples and only sulfadimidine was observed in chicken droppings at a maximum concentration of 6.04 mg/kg. The residues found by the authors for most antibiotics showed significant statistical differences between the provinces from which they were obtained samples collected and animal species (Zhao et al. \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e). In the case of detection and quantification of antibiotics in solid commercial organic fertilizers produced with animal by-products, there is practically no literature data on their analysis and the presence of antibiotics in them.\u003c/p\u003e\n \u003cp\u003eBased on the results obtained in this work and those of other researchers, it should be concluded that antibiotics are often present in natural and organic fertilizers that are used on agricultural fields and grasslands, which may pose a threat to the natural environment. The presence of veterinary antibiotics in natural and organic fertilizers should be monitored to ensure the safety of the environment, but also of animals and consumers consuming agricultural produce and food of animal origin. Moreover, the European Parliament Resolution of 1 June 2023 on EU action to combat antimicrobial resistance adopted by the European Union states that the use of sewage sludge and manure as fertilizers on agricultural soil may lead to the development of antimicrobial resistance through the spread of antimicrobial-resistant bacteria and antimicrobial resistance genes in the environment, which causes further contamination of the food chain and it is necessary to introduce prudent manure management practices. The results for positive samples of solid natural and organic fertilizers are summarized in the Table\u0026nbsp;4.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eAn UHPLC-MS/MS method for antibiotic residues was developed and validated in the present study in solid natural and organic fertilizers. The study focused on optimization of the sample weight, sample clean-up steps, including the use of different extraction solutions and SPE cartridges. In this work 73 samples of natural and organic solid ferilizers were analysed. Tweenty eight samples were positive for the presence of antibacterial substances. Antibiotics conatmination was more severe in the natural fertilizers than in the organic fertilizers in general, natural fertilizers from pigs exhibited the highest concentration of antibiotics in which the most frequently found antibiotic was doxycycline. High residue of antibiotics in natural and organic fertilizers could probably cause risks to the soil ecosystem after they were applied on the arable fields and grassland. Therefore, it is necessary to limit the amount of natural and organic fertilizers application based to containing toxic substances in order to reduce the associated risk to ecosystems, human and animal health.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Ewelina\u003cstrong\u003e\u0026nbsp;Patyra:\u003c/strong\u003e conceptualization, methodology, supervision, administration, formal analysis, investigation, writing \u0026ndash; original draft, writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eZbigniew Osiński:\u003c/strong\u003e sampling, review \u0026ndash; original draft. \u003cstrong\u003eKrzysztof Kwiatek:\u0026nbsp;\u003c/strong\u003esupervision, writing \u0026ndash; original draft. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis article does not contain any studies with human participants or animals performed by any of the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was financially supported by the \u0026bdquo;Science for Society\u0026rdquo; programm, decision of Ministry of Science and Higher Education No. NdS/545025/2022/2022\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eConsent to Publish \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBerendsen BJA, Wegh RS, Memelink J, Zuidema T, Stolker LAM (2015). The analysis of animal faeces as a tool to monitor antibiotic usage. Talanta 132: 258-268\u003c/li\u003e\n\u003cli\u003eBlackwell PA, L\u0026uuml;tzh\u0026oslash;ft H-CHH, Ma H-P, Halling-S\u0026oslash;rensen B, Boxall ABA, Kay P (2004) Ultrasonic extraction of veterinary antibiotics from soils and pig slurry with SPE clean-up and LC-UV and fluorescence detection. Talanta 64:1058\u0026ndash;1064 \u003c/li\u003e\n\u003cli\u003eCommission Implementing Regulation (EU) 2021/808 of 22 March 2021 on the performance of analytical methods for residues of pharmacologically active substances used in food-producing animals and on the interpretation of results as well as on the methods to be used for sampling and repealing Decisions 2002/657/EC and 98/179/EC.\u003c/li\u003e\n\u003cli\u003eCouncil Directive 91/676/EEC of 12 December 1991 concerning the protection of waters against pollution caused by nitrates from agricultural sources.\u003c/li\u003e\n\u003cli\u003eEuropean Parliament resolution of 1 June 2023 on EU action to combat antimicrobial resistance (2023/2703(RSP).\u003c/li\u003e\n\u003cli\u003eHaller MY, Muller SR, McArdell CS, Alder AC, Suter MJF (2002) Quantification of veterinary antibiotics (sulfonamides and trimethoprim) in animal manure by liquid chromatography mass spectrometry. J Chromatogr A 952:111-120\u003c/li\u003e\n\u003cli\u003eHirsch R, Ternes T, Haberer K, Krat KL (1999) Occurrence of antibiotics in the aquatic environment. Sci Total Environ 225:109-118\u003c/li\u003e\n\u003cli\u003eHo YB, Zakaria MP, Latif PA, Saari N (2014) Occurrence of vaterinary antibiotics and progesterone in broiler manure and agricultural soil in Malaysia. Sci Total Environ 261\u0026ndash;267\u003c/li\u003e\n\u003cli\u003eHou JW., Wan D, Mao C, Wang Q, Mu S, Qin Y (2015) Occurrence and distribution of sulfonamides, tetracyclines, quinolones, macrolides, and nitrofurans in livestock manure and amended soils of Northern China. Environ Sci Pollut Res Int 22:4545-4554\u003c/li\u003e\n\u003cli\u003eHu X, Luo Y, Zhou Q (2010) Simultaneous analysis of selected typical antibiotics in manure by microwave-assisted extraction and LC\u0026ndash;MS. Chromatographia 1:217\u0026ndash;223.\u003c/li\u003e\n\u003cli\u003eJansen LJM, van de Schans MGM, de Boer D, Bongers IEA, Schmitt H, Hoeksma P, Berendsen BJA (2019) A new extraction procedure to abate the burden of non-extractable antibiotic residues in manure. Chemosphere 224: 544\u0026ndash;553.\u003c/li\u003e\n\u003cli\u003eLi Ch, Chen J, Wang J, Ma Z, Ha P, Luan Y, Lu A (2015) Occurrence of antibiotics in soils and manures from greenhouse vegetable production bases of Beijing, China and associated risk assessment. Sci Total Environ 521-522:101-107\u003c/li\u003e\n\u003cli\u003eMartinez-Carballo E, Gonzalez-Barreiro C, Scharf S, Gans O (2007) Environmental monitoring study of selected veterinary antibiotics in animal manure and soils in Austria. Environ Pollut 162:56\u0026ndash;62\u003c/li\u003e\n\u003cli\u003eOsiński Z., Patyra E., 2022. Nawozy naturalne i organiczne jako źr\u0026oacute;dło zanieczyszczenia środowiska substancjami przeciwbakteryjnymi. Veterinary Medicine. Sciene and Practice. dx.doi.org/10.21521/mw.6638\u003c/li\u003e\n\u003cli\u003ePatyra E, Kwiatek K (2017) Development and validation of multi-residue analysis for tetracycline antibiotics in feed by high performance liquid chromatography coupled to mass spectrometry. Food Addit Contam 9:1553\u0026ndash;1561\u003c/li\u003e\n\u003cli\u003eRegulation (EC) No 1069/2009 of the European Parliament and of the Council of 21 October 2009 laying down health rules as regards animal by-products and derived products not intended for human consumption and repealing Regulation (EC) No 1774/2002 (Animal by-products Regulation)\u003c/li\u003e\n\u003cli\u003eUstawa z dnia 10 lipca 2007 r. o nawozach i nawożeniu. Dz. U. 2007, nr 147, poz. 1033.\u003c/li\u003e\n\u003cli\u003eWallace JS, Aga D (2016) Enhancing Extraction and Detection of Veterinary Antibiotics in solid and Liquid Fractions Manure. J Environ Qual 45: 471\u0026ndash;479. \u003c/li\u003e\n\u003cli\u003eWolters B, Widyasari-Mehta A, Kreuzing R, Smalla K (2016) Contaminations of organic fertilizers with antibiotic residues, resistance genes, and mobile genetic elements mirroring antibiotic use in livestock? Applied Microbiol Biotech 100:9343-9353\u003c/li\u003e\n\u003cli\u003eWu XL, Xiang L, Jiang YN, Li YW, Huang XP, Li H, Cai QY, Mo CH (2014) Distribution and risk assessment of quinolone antibiotics in the soils from organic vegetable farms of a subtropical city, Southern China. Sci Total Environ 487:399\u0026ndash;406\u003c/li\u003e\n\u003cli\u003eXin CY, Li GJ, Qiu HPZ, Chen R, Xu J, Kong XJ, Shan ZJ, Wang N (2016) Pollution characteristics of 23 veterinary antibiotics in livestock manure and manure-amended soils in Jiangsu province. J Environ Sci Health 51:383-392\u003c/li\u003e\n\u003cli\u003eZhao L, Dang YH, Wang H (2010) Residues of veterinary antibiotics in manures from feedlots livestock in eight provinces of China. Sci Total Environ 408:1069-1075\u003c/li\u003e\n\u003cli\u003eZhi S, Shen S, Zhou J, Ding G, Zhang K (2020) Systematic analysis of occurrence, density and ecological risks of 45 veterinary antibiotics: Focused on family livestock farms in Erhai Lake basin, Yunnan, China. Environ Pollution 115539, doi: 10.1016/j.envpol.2020.115539.\u003c/li\u003e\n\u003cli\u003eZhi S, Jing Z, Haixue L, Huihui W, Zulin Z, Yongzhen D, Keqiang Z (2020) Simultaneous extraction and determination of 45 veterinary antibiotics in swine manure by liquid chromatography-tandem mass spectrometry. J Chromatogr B 1154:122286. https://doi.org/10.1016/j.jchromb.2020.122286\u003c/li\u003e\n\u003cli\u003eZhou L-J, Ying G-G, Zhang R-Q, Liu S, Lai H-J, Chen Z-F, Yang B, Zhao J-L (2013) Use patterns, excretion masses and contamination profiles of antibiotics in a typical swine farm, south China. Environ Sci 15: 802-813.\u003cstrong\u003e\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"environmental-science-and-pollution-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"espr","sideBox":"Learn more about [Environmental Science and Pollution Research](https://www.springer.com/journal/11356)","snPcode":"11356","submissionUrl":"https://submission.nature.com/new-submission/11356/3","title":"Environmental Science and Pollution Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"veterinary antibiotics, natural fertilizer, organic fertilizer, UHPL-MS/MS analysis, validation","lastPublishedDoi":"10.21203/rs.3.rs-3984638/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3984638/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAntibiotics are active substances frequently used to treat and prevent diseases in animal husbandry. Most veterinary antibiotics are excreted after administration, in some cases up to 90% of the consumed dose can be found in the faeces and/or urine as parent compound. Livestock excrement is used around the world as natural fertilizers or, after processing, as organic fertilizers for crops and grasslands. Natural and organic fertilizers are an excellent source of nutrients for plants, but due to the presence of veterinary antibiotics in them, they may pose a threat to the natural environment, mainly to soil microorganisms, but also to human and animal health. Therefore, the work aimed to develop and validate an analytical method using the UHPLC-MS/MS technique and to analyze real samples of natural and organic fertilizers. Our research has shown that over 38% of natural fertilizers are contaminated with antibiotics, mainly doxycycline in concentrations reaching up to several dozen milligrams per kilogram of fertilizers. In the case of organic fertilizers that undergo processing, the presence of antibiotics was found in over 37% of the analyzed samples. The presented analytical method can be a tool for assessing the presence of veterinary antibiotics in solid natural and organic fertilizers.\u003c/p\u003e","manuscriptTitle":"Residues of veterinary antibiotics in solid natural and organic fertilizers – method development and sample analysis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-11 19:17:38","doi":"10.21203/rs.3.rs-3984638/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revision","date":"2024-04-19T02:48:29+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-03-13T08:58:48+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-03-07T13:55:36+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Environmental Science and Pollution Research","date":"2024-03-06T22:37:31+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-02-29T05:08:21+00:00","index":"","fulltext":""},{"type":"submitted","content":"Environmental Science and Pollution Research","date":"2024-02-23T04:12:04+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"environmental-science-and-pollution-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"espr","sideBox":"Learn more about [Environmental Science and Pollution Research](https://www.springer.com/journal/11356)","snPcode":"11356","submissionUrl":"https://submission.nature.com/new-submission/11356/3","title":"Environmental Science and Pollution Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f0f17240-fa68-44f5-a12c-beabcf929057","owner":[],"postedDate":"March 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-06-21T15:42:52+00:00","versionOfRecord":{"articleIdentity":"rs-3984638","link":"https://doi.org/10.1007/s11356-024-33956-w","journal":{"identity":"environmental-science-and-pollution-research","isVorOnly":false,"title":"Environmental Science and Pollution Research"},"publishedOn":"2024-06-18 15:42:52","publishedOnDateReadable":"June 18th, 2024"},"versionCreatedAt":"2024-03-11 19:17:38","video":"","vorDoi":"10.1007/s11356-024-33956-w","vorDoiUrl":"https://doi.org/10.1007/s11356-024-33956-w","workflowStages":[]},"version":"v1","identity":"rs-3984638","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3984638","identity":"rs-3984638","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}