Trimethoprim Residues Research Articles

Comparative study of the plasma pharmacokinetics and tissue residues of trimethoprim in silky fowls and 817 broilers after single oral administration

A comparative study was performed to investigate the differences in plasma pharmacokinetics (PKs) and tissue residues of trimethoprim (TMP) between silky fowls and 817 broilers. The 2 breeds of chickens received compound sulfadiazine suspension by gavage at 20 mg/kg (measured as TMP). Blood and tissue samples were collected at predetermined time points. The concentrations of TMP in plasma and tissue samples were determined by a validated high-performance liquid chromatography (HPLC) method. The plasma concentration-time data were subjected to noncompartment analysis by WinNonlin program (Pharsight Co., Mountain View, CA). The mean plasma concentrations of TMP in silky fowls were significantly lower than those in 817 broilers at all time-points. Significant differences were also observed between silky fowls and 817 broilers in maximum concentration (Cmax), area under the curve from time 0 to 24 h (AUC0→24 h), apparent volume of distribution (Vd), and total body clearance (ClB). Silky fowls had significantly higher muscle TMP concentrations and longer tissue residual time than 817 broilers. The tissue concentration of TMP followed the order of leg muscle > breast muscle > liver, which was obviously different from that of 817 broilers. The half-lives of TMP in the leg muscle, breast muscle, and liver of silky fowls were 31.42, 10.78, and 0.38 d, respectively. The current withdrawal time (WDT) was not sufficient to prevent violative residues of TMP in the edible tissues of silky fowls, and a WDT much longer than 8 d might be required.

THE DEVELOPMENT AND THE VALIDATION OF THE METHOD FOR TRIMETOPRIM RESIDUES DETERMINATION IN TISSUES SAMPLES USING THE METHOD OF ENZYME-LINKED IMMUNOSORBENT ASSAY

The article presents the results of the development of sample preparation method of muscle and parenchymal tissues for their further testing on trimethoprim residues by enzyme-linked immunosorbent assay using Trimethoprim test kits from Kwinbon Biotechnology (China), designed to analyze another target matrix, viz. honey. The results of the comparison of sample preparation methods with the use of different techniques of analyte extraction from tissues, with the established percentage of extraction are presented. The most effective conditions are proposed: acetonitrile extraction using ultrasonic intensification, subsequent concentration of the analyte by evaporation, reconstitution of dried residue and degreasing. This approach makes it possible to determine trimethoprim residues in poultry tissues at the level of 5-10 μg/kg with an absolute recovery of about 75%. In accordance with the Decision of the European Commission 2002/657/EC, the validation of the proposed method was carried out taking into account the maximum permissible levels (MRL) of trimethoprim in the tissues of productive animals. The suitability of the method was confirmed on the basis of establishing of the main validation parameters for screening methods (technical threshold and cut-off factor) using control (pure) and spiked with a standard solution of trimethoprim at the level of ½ MRL samples of muscle and parenchymal tissues of broiler chickens. The main advantages of the developed technique are simplicity of performance, rapidity and cost effectiveness. The certainty of the results obtained using the developed method was verified using the confirmatory UPLC-MS/MS method. The article presents the results of a comparative study of pure samples of broiler chickens muscles and parenchymal tissues and spiked at the level of ½ MRL with a standard solution of trimethoprim. The developed screening technique of enzyme-linked immunosorbent assay is recommended for determination of residual amounts of trimethoprim in tissue samples and can be used for routine laboratory tests, as well as to study the withdrawal procedure of veterinary drugs based on this drug.

Multi-Residue Determination of Sulfonamides, Dapsone, Ormethoprim, and Trimethoprim in Fish and Shrimp Using Dispersive Solid Phase Extraction with LC–MS/MS

This study reports an analytical method to simultaneously determine the residues of sulfonamides, dapsone, ormethoprim, and trimethoprim in fish and shrimp. The samples were extracted with acetonitrile, magnesium sulfate, and sodium acetate, followed by dispersive solid-phase extraction (C18) for cleanup. The target analytes were confirmed and quantified by liquid chromatography–tandem mass spectrometry. The developed method was validated according to the Codex guidelines (CAC/GL 71-2009). As a result, linearity was expressed between 0.25 and 200 μg kg−1 with a correlation coefficient > 0.98. The limits of quantitation were 0.01–2.8 μg kg−1 and the accuracy (expressed as average recovery) was 76.1–115%. The precision (expressed as the coefficient of variation) was <20%. The decision limits of sulfonamides, dapsone, ormethoprim, and trimethoprim were 94.2–114, 1.3–1.5, 105–111, and 53.3–54 μg kg−1 and detection capabilities of 102–129, 1.4–1.6, 109–120, and 55.6–58.2 μg kg−1, respectively. The proposed method was applied to the analysis of real samples (n = 54) obtained from domestic markets in Korea, resulting in a detection rate of 13% (seven samples). This method is applicable for the efficient determination of sulfonamides, dapsone, ormethoprim, and trimethoprim residues in fish and shrimp according to Korean MRL.

THE DEVELOPMENT OF THE METHOD FOR THE DETERMINATION OF SULFONAMIDES AND TRIMETOPRIM RESIDUAL QUANTITIES IN MILK SAMPLES BY UPLC-MS/MS METHOD AND ITS VERIFICATION THROUGH INTERLABORATORY TESTING

This manuscript presents the results of development and adoption of the confirmatory UPLC- MS/MS method for determination of sulfonamides and trimethoprim residues in whole and powdered cow's milk samples. Sample preparation includes a recovery with water (in the case of milk powder), separation of milk fat by centrifugation at 4 °C, proteins precipitation with acetonitrile using ultrasonic intensification, extraction of analytes with a two-component mixture of organic solvents from weak acidic phosphate buffer solution, concentration of obtained extracts, degreasing and reconstitution with the mobile phase. The use of tandem-mass spectrometric detector in multiple reaction monitoring mode provides high specificity for all ten sulfonamides, which molecules contain the same structural elements, and trimethoprim, as three ion transitions were used for each of the target analytes. The approach of matrix matched calibration on the milk matrix was applied, and it allowed us to disregard taking into consideration the absolute extraction of analytes during sample preparation, as well as the influence of sample matrix components on analytes ionization. The main advantages of the developed technique are simplicity, rapidity and cost effective sample preparation, fast procedure of sample analysis (the time of chromatographic separation is 2.0 min), high selectivity, and sensitivity at the level of ≤ 0.5 μg/kg (as compared with the maximum residues level of sulfonamides in dairy products, viz. 100 μg/kg). According to the European Commission Decision 2002/657/EC, the values of CCα and CCβ for sulfonamides and trimethoprim in milk were calculated. The capability of the method was confirmed on the basis of the established validation parameters, using spiked milk samples and "added – found" approach. The method was tested during the analysis of spiked samples of whole and powdered cow's milk (CV ~ 20%). The developed technique was verified by interlaboratory comparative testing of milk powder samples for sulfonamides content. The UPLC-MS/MS method for the determination of sulfonamides and trimethoprim residues in whole and milk powder samples can be used both for routine laboratory analysis of dairy products and for the examination of suspicious or questionable samples.

Detection of antibacterial residues in milk by HPLC-DAD and microbial inhibitor tests

The objective of this study was to assess the detection sensitivity of four commercial microbial inhibition assays (MIAs) in comparison with the results by High Pressure Liquid Chromatography with Diode Array Detection (HPLC-DAD) for residues of three tetracyclines, trimethoprim and sulfadiazine in fortified raw milk samples. MIAs showed fairly good results at detecting sulfadiazine residues, whereas concerning tetracyclines and trimethoprim residues, most of the kits showed insufficient results. HPLC analysis revealed high recoveries of examined compounds with detection limits below their respective maximum residue levels (MRLs). The HPLC method was validated determining linearity, accuracy, precision, detection capability and decision limit. Subsequently, both methods were used to monitor several cow and sheep milk samples for the presence of antibacterial agents. Results obtained showed that MIAs could be successfully used for the analysis of different milk types. However, milk spoilage which was observed in some cases could decrease the sensitivity of the kits, a fact that does not seem to influence the effectiveness of HPLC-DAD method.&lt;br /&gt;&lt;br /&gt;

Gold immunochromatographic assay for trimethoprim in milk and honey samples based on a heterogenous monoclonal antibody

ABSTRACTThree heterogenous haptens (3,4,5-Trimethoxybenzoic acid [H1], 3,4,5-Trimethoxyphenylacetic acid [H2], 3-(3,4,5-Trimethoxyphenyl) propanoic acid [H3]) were used to synthesize immunogens, with the aim of screening a more sensitive monoclonal antibody (mAb) for trimethoprim (TMP). H2 proved to be the best heterogenous hapten. The mAb was specific with no cross-reactivity with other structurally related chemicals. The 50% maximal inhibitory concentration of the mAb against TMP was 1.98 µg L−1, which is more sensitive compared with the mAb reported in our previous work. A mAb-based gold immunochromatographic assay (GICA) was established to analyze TMP residues in milk and honey samples. The concentration of coating antigen in test line and concentration of mAb conjugated with gold nanoparticles was 0.3 µg mL−1 and 0.25 µg mL−1, respectively. Under optimized conditions, the visual limit of detection concentration of TMP spiked in milk and honey samples was 10 µg L−1 and 15 µg kg−1, respectively. The proposed GICA provides rapid and simple determination of TMP residues in milk and honey samples.

Establishment of a monoclonal antibody-based indirect enzyme-linked immunosorbent assay for the detection of trimethoprim residues in milk, honey, and fish samples

ABSTRACTA specific monoclonal antibody (mAb) against trimethoprim (TMP) was produced and a mAb-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was established to detect TMP residues in milk, honey, and fish samples. The working range of ic-ELISA and IC50 was 1.83–9.36 and 4.14 µg L−1. The average recoveries of TMP from TMP-spiked milk, honey, and fish sample were 103.5–118.0%, 100.3–107.1%, and 91.5–108.1%, respectively. The results revealed that our developed mAb-based ic-ELISA can be effectively applied to detect TMP residues in these food products.

Determination of sulfadiazine, trimethoprim, and N(4) -acetyl-sulfadiazine in fish muscle plus skin by Liquid Chromatography-Mass Spectrometry. Withdrawal-time calculation after in-feed administration in gilthead sea bream (Sparus aurata L.) fed two different diets.

This study presents a depletion study for sulfadiazine and trimethoprim in muscle plus skin of gilthead sea bream (Sparus aurata L.). N(4) -acetyl-sulfadiazine, the main metabolite of sulfadiazine (SDZ), was also examined. The fish were held in seawater at a temperature of 24-26°C. SDZ and trimethoprim (TMP) were administered orally with medicated feed for five consecutive days at daily doses of 25mg SDZ and 5mg TMP per kg of fish body weight per day. Two different diets, fish oil- and plant oil-based diets, were investigated. Ten fish were sampled at each of the days 1, 3, 5, 6, 8, 9, 10, and 12 after the start of veterinary medicine administration. However for the calculation of the withdrawal periods, sampling day 1 was set as 24 h after the last dose of the treatment. Fish samples were analyzed for SDZ, TMP, and acetyl-sulfadiazine (AcSDZ) residues by liquid chromatography-mass spectrometry. SDZ and TMP concentrations declined rapidly from muscle plus skin. Considering a maximum residue limit of 100μg/kg for the total of sulfonamides and 50μg/kg for TMP residues in fish muscle plus skin, the withdrawal periods of the premix trimethoprim-sulfadiazine 50% were calculated as 5 and 6days, at 24-26 °C, in fish oil (FO) and plant oil (PO) groups, respectively. The investigation of this work is important to protect consumers by controlling the undesirable residues in fish.

Monitoring of trimethoprim antibiotic residue in livestock and marine products commercialized in Korea

A survey for trimethoprim (TMP) was performed using 369 livestock and marine products distributed in Korea. Product samples included beef, pork, milk, egg, chicken, flatfish, jacopever, common eel, and shrimp. TMP residues were analyzed using ultra performance LC with electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS). The analytical method was validated according to FDA (U.S. Food and Drug Administration) guidelines and all results fully complied with FDA recommendations. TMP was detected in 7 product samples (1.9% incidence), including 5 jacopever, 1 flatfish, and 1 common eel. Residue levels were 1.17 to 16.43 μg/kg in jacopever, 40.0 μg/kg in flatfish, and 13.3 μg/kg in common eel. All positive product samples were below the legal residue limit of 20- 50 μg/kg in the Korean food code.

Distribution of sulfamonomethoxine and trimethoprim in egg yolk and white

The distribution of sulfamonomethoxine (SMM) and trimethoprim (TMP) in egg yolk and white was measured during and after administration of a SMM/TMP combination in laying hens in doses of 8gl−1 and 12gl−1 in drinking water for 7days. The SMM concentration reached maximal levels on day 2 of the post-treatment period for both doses (μgkg−1): 5920 and 9453 in yolk; 4831 and 6050 in white, in doses 1 and 2, respectively. Significant differences in the SMM and TMP concentrations between yolk and white in post treatment period were found. SMM dropped below the LOD (1.9μgkg−1) in yolk after day 16 and 19 for doses 1 and 2. TMP reached maximal levels on day 3 after drug administration for doses 1 and 2 (μgkg−1): 6521 and 7329 in yolk, 1370 and 1539 in white. TMP residues were measured above LOD (0.3μgkg−1) in yolk for both doses on day 37 post-treatment.

Evaluation and validation of a biochip multi-array technology for the screening of 14 sulphonamide and trimethoprim residues in honey according to the European guideline for the validation of screening methods for veterinary medicines

Honey could be contaminated by antibiotic residues. There is still a great need for a cheap and single multi-residue method. The Evidence Investigator™ system is a biochip and semi-automated system. The microarray kit I (AM I) for the detection of sulphonamide and trimethoprim (TMP) residues in honey was evaluated, and then validated according to the European decision EC/2002/657 and to the European guideline for the validation of screening methods for veterinary medicines (2010). The method was found to be rapid and able to screen simultaneously 14 sulphonamides and TMP in honey with a very easy sample preparation procedure. The false–positive rate of 4% maximum is very satisfactory. All detection capabilities (CCβ) were well below the recommended concentration (RC) of 50 µg kg−1. The applicability of AM I kit with different types of honey (monofloral or multifloral, liquid or solid, different floral origins, etc.) has been proved.

ANALYSIS OF SULFONAMIDE RESIDUES IN REAL HONEY SAMPLES USING LIQUID CHROMATOGRAPHY WITH FLUORESCENCE AND TANDEM MASS SPECTROMETRY DETECTION

This paper presents new reversed phase liquid chromatographic methods (HPLC-FLD and LC-MS/MS) for the quantification of sulfonamides in spiked and incurred honey samples. The sample preparation was optimized using Oasis HLB (hydrophilic–lipophilic balance) solid-phase extraction (SPE) cartridge. Elutions of sulfonamides were carried out under acidic, neutral, and basic conditions using methanol. Recoveries under acid condition were in the range from 66.8–90%, which were approximately 10% higher than those obtained under other conditions. The sample clean-up was also tested using Strata-XL cartridges. The HPLC-FLD separation was performed using a Varian C18 column and a ternary (methanol-acetonitrile-phosphate buffer, pH 5) mobile phase resulting good selectivity for the determination. The robustness of the ternary gradient method was evaluated by computer simulation (DryLab). LC-MS/MS separation was carried out on a Kinetex XB core-shell type HPLC column that enabled a low limit of detection (0.01–0.5 µg/kg) and faster separation (6 min). The developed methods were validated in accordance with the European Union Commission Decision 2002/657/EC and were applied successfully for more than four hundred honey samples (under a national monitoring program). The concentrations of sulfadimethoxine, sulfachloropyridazine, and trimethoprim residues in samples were found in a concentration range from 0.03 up to 686 µg/kg.

Simultaneous determination of 13 kinds of sulfonamides and trimethoprim residues of aquatic products by ASE-GPC-HPLC method

Simultaneous determination of 13 kinds of sulfonamides and trimethoprim residues of aquatic products by ASE-GPC-HPLC method

Effect of roasting, boiling and microwaving cooking method on sulfadiazine + trimethoprim residues in edible tissues of broiler by microbial inhibition method

The purpose of this study was to determine the effects of different cooking processes like boiling, roasting and microwaving on sulfadiazine + trimethoprim residues in edible tissues of broiler chickens. Each of chicks was fed by water and food with 0.05% of sulfadiazine + trimethoprim in their drinking water for consecutive 5 days. After the 5th day of the drug administration, they were killed and breast muscle; liver and gizzard were sampled aseptically from each carcass. The residue was analyzed using microbial inhibition test by plates seeded with Bacillus subtilis. The positive raw samples cooked by various cooking procedures (roasting, boiling and microwaving). Then, we surveyed cooked samples for the present of residue. The results show the reduction in concentration of sulfadiazine + trimethoprim residue after different cooking processes. The most reduction of sulfadiazine + trimethoprim residues in cooked muscle samples related to microwaving process and a part of residue excreted from tissue to cooking fluid in boiling process. Muscle samples had the most resistance to residue reduction rather than other tissues in boiling and roasting processes. The reduction effect of all cooking processes on liver and gizzard samples was greater than muscle samples and the inhibitory zone around all cooked liver and gizzard samples were not detectable. Regarding to the results of this study, we can concluded that cooking processes do not guarantee a full break-down of these drugs present in condemned animals. Between the various agents affecting antibiotics residue after cooking process, cooking time and temperature can play major role about antibiotic residue reduction.

Bis[2,4-diamino-5-(3,4,5-trimethoxybenzyl)pyrimidin-1-ium]DL-malate

Racemic malic acid and trimethoprim [5-(3,4,5-trimethoxybenzyl)pyrimidine-2,4-diamine] form a 1:2 salt (monoclinic, P2(1)/c), 2C(14)H(19)N(4)O(3)(+).C(4)H(4)O(5)(2-), in which the malate component is disordered across a centre of inversion. The crystal structure of the salt consists of protonated trimethoprim residues and a malate dianion. The carboxylate group of the malate ion interacts with the trimethoprim cation in a linear fashion through pairs of N-H...O hydrogen bonds to form a cyclic hydrogen-bonded motif. This is similar to the carboxylate-trimethoprim cation interaction observed earlier in the complex of dihydrofolate reductase with trimethoprim. The structure of the salt of trimethoprim with racemic DL-malic acid reported here is the first of its kind. The present study investigates the conformations and the hydrogen-bonding interactions, which are very important for biological functions. The pyrimidine plane makes a dihedral angle of 78.08 (7) degrees with the benzene ring of the trimethoprim cation. The cyclic hydrogen-bonded motif observed in this structure is self-organized, leading to novel types of hydrogen-bonding motifs in supramolecular patterns.

Analytical methods for multiresidue determination of sulfonamides and trimethoprim in meat and ground water samples by CE‐MS and CE‐MS/MS

This paper presents two methods based on CZE-MS detection and CZE-MS/MS detection developed for the multiresidue determination of ten sulfonamides (sulfapyridine, sulfadoxin, sulfamethazine, sulfadimethoxine, sulfameter, sulfamerazine, sulfachlorpyridazine, sulfadiazine, sulfamethoxazole, and sulfamethizole) and a potentiator, trimethoprim (TMP), whose contents are regulated by the EU Council Regulation no. 2377/90 in animal edible tissues. Experimental designs were employed to optimize the electrospray conditions. MS/MS experiments using an IT as analyzer operating in multiple reaction monitoring (MRM) mode were carried out to achieve the minimum number of points according to the 2002/657/EC European Decision for unambiguous identification. The proposed procedures have been compared in terms of the performance characteristics and trueness. The limits of detection and quantification were in all cases lower than the maximum residue limits legislated for these compounds and the recoveries were satisfactory, being possible the application for their monitoring in foodstuff of animal origin and in environmental samples, allowing the determination of sulfonamides and TMP residues in meat and in superficial water in the low microg/L range.

Residues of selected antibiotics in water and mud from shrimp ponds in mangrove areas in Viet Nam

In recent years, antibiotics have been used widely in intensive shrimp culture and this may lead to their contamination of the environment. Surveys on residues of trimethoprim (TMP), sulfamethoxazole (SMX), norfloxacin (NFXC) and oxolinic acid (OXLA) in water and mud in shrimp ponds in mangrove areas were conducted in the north as well as in south of Viet Nam in July and August, 2002. The results show that these antibiotics are found in all samples in both shrimp ponds and surrounding canals. The highest concentrations of TMP, SMX, NFXC and OXLA are 1.04, 2.39, 6.06, and 2.50 ppm in water samples; and 734.61, 820.49, 2615.96, 426.31 ppm (based on wet mud weight), respectively. The comparison of antibiotics residues between study sites and types of shrimp ponds will be discussed in this paper. The results also suggest that antibiotics residues may cause harmful effect on ecosystems in the study sites.

Detection Limits of Antimicrobials in Ewe Milk by Delvotest Photometric Measurements

The Delvotest method detection limits per manufacturer's instructions at a fixed reading time of 3h for 24 antimicrobial agents were determined in ewe milk by photometric measurement. For each drug, eight concentrations were tested on 20 ewe milk samples from individual ewes. Detection limits, determined by means of logistic regression models, were (μg/kg): 3, amoxycillin; 2, ampicillin; 18, cloxacillin; 1, penicillin “G”; 34, cefadroxil; 430, cephalosporin “C”; 40, cephalexin; 20, cefoperazone; 33, Ceftiofur; 18, cefuroxime; 6100, streptomycin; 1200, gentamycin; 2600, neomycin; 830, erythromycin; 100, tylosin; 180, doxycycline; 320, oxytetracycline; 590, tetracycline; 88, sulfadiazine; 44, sulfamethoxazole; 140, sulfametoxypyridazine; 48, sulfaquin-oxaline; 12,000, chloramphenicol; and 290, trimethoprim. Whereas the beta-lactam antibiotics, sulphonamides, and tylosin were detected by Delvotest method at levels equal to those of maximum residue limits, its sensitivity needs to be enhanced to detect aminoglycosides, tetracyclines, streptomycin, chloramphenicol, and trimethoprim residues in ewe milk or to develop an integrated residue detection system for ewe milk with different sensitive microorganisms for each group of antiinfectious agents.

HPLC determination of trimethoprim in meat and milk with an SPE preseparation procedure

An HPLC method in combination with solid-phase extraction and dual wavelength detection has been developed in order to monitor trimethoprim residues in meat and milk. The extraction recoveries for the maximal residual limit concentration in food (50 ng/g) were 86±5% for meat and 73±6 % for milk samples. The limits of determination of trimethoprim were 5 ng/g at 229 nm and about 15 ng/g at 280 nm. The method has been developed in response to specific needs of food control laboratories. This publication introduces the assay for milk and meat samples.

High performance liquid chromatographic determination of trimethoprim residues in egg yolk and albumen in a feeding experiment

In a feeding experiment, the residues of trimethoprim (TMP) in egg yolk and albumen were determined by high performance liquid chromatography (HPLC). Laying hens were divided into four groups. The first group was designated as control and fed with TMP free diet during the experimental period. The second, third and fourth groups were administered with the feeds containing 4 p.p.m., 16 p.p.m. and 56 p.p.m. TMP, respectively, for 19 days and, thereafter, fed with TMP free diet. TMP was mainly found in yolk in all the three administered levels. In the medication period, the average concentration of TMP in the yolk of the second, third and fourth groups was 0.05, 0.25, and 0.90 p.p.m., respectively. After withdrawal of medication, the residues of TMP in yolk in the second, third and fourth groups decreased below the detection limit at 0.02 p.p.m. on days 4, 9, and 11, respectively.