Residue analysis of tetracyclines and their metabolites in eggs and in the environment by HPLC coupled with a microbiological assay and tandem mass spectrometry

Tetracyclines are widely used in farm animals. This can cause drug residues in products of animal origin and, after excretion of these substances, in animal slurry and in soil fertilized with that slurry. In this paper, we present a method based on a microbiological assay coupled with HPLC for the detection of oxytetracycline, tetracycline, and chlortetracycline in eggs. After a simple liquid extraction of the samples and HPLC separation, fractions were collected on microtiter plates, and the tetracyclines were analyzed using the Staphylococcus aureus assay. This method was able to identify residues of tetracyclines in eggs at a level set by regulatory agencies (i.e., 200 microg/kg). In addition, it was shown that the described microbiological method can be used as a screening assay for the detection of tetracyclines and possible biologically active metabolites in animal slurry and soil samples. Employing the same extraction procedure, it was demonstrated that LC-MS-MS allowed the quantification of 20-400 microg/kg in eggs with recoveries ranging from 71 to 109% and RSDs of 3-15%.     

Turbidimetric assay of tylosin in animal feeds containing urea

A turbidimetric method is described for determination of tylosin in animal feeds containing urea. This method includes several modified or new steps to existing turbidimetric and AOAC plate assays that improve the extraction of tylosin, remove interferences from feeds, free tylosin activity, concentrate tylosin from low-level feeds, and reduce variability of assay results. A larger analytical sample size has been incorporated into the assay to decrease variability of assay results. A methanol-phosphate buffer extraction solution has replaced the hot buffer and methanol extraction solution. A hydrolysis step, which is not contained in the AOAC plate assay, was developed to free tylosin from the tylosin urea adduct that forms over time in feeds containing urea. A disposable C18 column was used to concentrate tylosin from feeds at levels less than 15 ppm. By increasing the analytical sample size from 25 to 100 g, the coefficient of variation for 12 weighings of cattle feed was reduced from 28.4 to 9.3%. Average recoveries from cattle rations containing tylosin at levels of 8, 10, and 100 ppm were 94, 94, and 91%, respectively.     

Thin-layer chromatographic determination of erythromycin and other macrolide antibiotics in livestock products

A method is described for determination of 4 macrolide antibiotics in livestock products. Erythromycin, tylosin, oleandomycin, and spiramycin were extracted from animal tissues, milk, and egg with acetonitrile at pH 8.5. Cleanup was done by adding sodium chloride and dichloromethane, evaporating the organic layer, and subsequent acid/base partitioning. After the antibiotics were separated by thin-layer chromatography (TLC), they were reacted with xanthydrol and could be detected as purple spots down to 0.02 mg/kg without interference by other commonly used therapeutic drugs (23 were tested). Anisaldehyde-sulfuric acid, cerium sulfate-molybdic acid, phosphomolybdic acid, and Dragendorff's reagent proved to be less sensitive as visualizing agents. For quantitation, TLC plates were scanned at 525 nm. Recoveries were between 71 and 96% for erythromycin and tylosin in liver, muscle, and egg at the 0.1-0.5 mg/kg level and 51% for erythromycin in milk at the 0.02 mg/kg level (coefficient of variation = 10-18%). Bioautography with Bacillus subtilis was used to confirm results, in addition to TLC analysis of derivatized antibiotics and liquid chromatography with electrochemical detection. Various derivatization procedures for erythromycin were investigated for improved ultra-violet or fluorescence detection in liquid chromatography.     

Development and validation of an indirect competitive enzyme-linked immunosorbent assay for the screening of tylosin and tilmicosin in muscle, liver, milk, honey and eggs

Incorrect use of tylosin and tilmicosin could result in allergy and select resistance. To monitor the illegal use of these antibiotics in animals, a monoclonal-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) has been established. Several haptens were synthesized and conjugated to carrier protein. Female Balb/c mice were inoculated with the four different conjugates to produce monoclonal antibodies according to the schemes of immunization. Aftercell fusion and culture several times, nine hybridoma cell lines were isolated. Only one, 3C4 that has isotype IgG2a, was selected for detailed study. The cross-reactivity of the monoclonal antibody 3C4 to tylosin and tilmicosin was 100% and 51% respectively. The standard curves based on the tylosin and tilmicosin matrix calibration ranged from 2.5 to 40 μg L(-1), with an IC(50) value of 6.1 μg L(-1) and 12.1 μg L(-1), respectively. The limits of detection of the ic-ELISA ranged from 5.1 μg kg(-1) to 13.8 μg kg(-1) in edible animal tissues. The recoveries were 74.1% to 120.7% with less than 18.6% of the coefficient of variation when tylosin and tilmicosin were spiked in various biological matrices with the concentrations of 25.0-200.0 μg kg(-1). Good correlations between the results of the ic-ELISA and high performance liquid chromatography were observed in the incurred tissues. These results suggest that the ic-ELISA is a sensitive, accurate and low-cost method that would be a useful tool for the screening of the residues of tylosin and tilmicosin in muscle, liver, milk, honey and eggs.     

Determination and depletion of residues of carbadox, tylosin, and virginiamycin in kidney, liver, and muscle of pigs in feeding experiments

The results of residue determinations of the growth promotors carbadox, tylosin, and virginiamycin in kidney, liver, and muscle from pigs in feeding experiments are described as well as the analytical methods used. Residues of the carbadox metabolite quinoxaline-2-carboxylic acid were found in liver from pigs fed 20 mg/kg in the diet with a withdrawal time of 30 days. No residues were detected in muscle with zero withdrawal time. The limit of determination was 0.01 mg/kg for both tissues. No residues of virginiamycin and tylosin were found in pigs fed 50 and 40 mg/kg, respectively, in the diet, even with zero withdrawal time. Residues of tylosin of 0.06 mg/kg and below were detected in liver and kidney from pigs fed 200 or 400 mg/kg and slaughtered within 3 h after the last feeding.     

Determination and confirmation of melamine residues in catfish, trout, tilapia, salmon, and shrimp by liquid chromatography with tandem mass spectrometry

Pet and food animal (hogs, chicken, and fish) feeds were recently found to be contaminated with melamine (MEL). A quantitative and confirmatory method is presented to determine MEL residues in edible tissues from fish fed this contaminant. Edible tissues were extracted with acidic acetonitrile, defatted with dichloromethane, and cleaned up using mixed-mode cation exchange solid-phase extraction cartridges. Extracts were analyzed by liquid chromatography with tandem mass spectrometry with hydrophilic interaction chromatography and electrospray ionization in positive ion mode. Fish and shrimp tissues were fortified with 10-500 microg/kg (ppb) of MEL with an average recovery of 63.8% (21.5% relative standard deviation, n = 121). Incurred fish tissues were generated by feeding fish up to 400 mg/kg of MEL or a combination of MEL and the related triazine cyanuric acid (CYA). MEL and CYA are known to form an insoluble complex in the kidneys, which may lead to renal failure. Fifty-five treated catfish, trout, tilapia, and salmon were analyzed after withdrawal times of 1-14 days. MEL residues were found in edible tissues from all of the fish with concentrations ranging from 0.011 to 210 mg/kg (ppm). Incurred shrimp and a survey of market seafood products were also analyzed as part of this study.     

Improved turbidimetric assay of tylosin in premix and animal feeds not containing urea

A turbidimetric method is described for the determination of tylosin in premix and animal feeds not containing urea. This method includes several modifications of existing tylosin turbidimetric and AOAC plate assays to remove interferences from the feed, to concentrate low levels of tylosin, and to reduce the variability of the assay results. An acidic alumina column cleanup step has been incorporated into the method to remove interferences from feed ingredients. A disposable C18 column was used to concentrate the tylosin from low-level feeds, and the use of a larger analytical sample size has decreased the variability of the assay results. Average recoveries of tylosin added to chicken and swine rations were 98 and 101%, respectively.     

Multiclass determination and confirmation of antibiotic residues in honey using LC-MS/MS

A multiclass method has been developed for the determination and confirmation in honey of tetracyclines (chlortetracycline, doxycycline, oxytetracycline, and tetracycline), fluoroquinolones (ciprofloxacin, danofloxacin, difloxacin, enrofloxacin, and sarafloxacin), macrolides (tylosin), lincosamides (lincomycin), aminoglycosides (streptomycin), sulfonamides (sulfathiazole), phenicols (chloramphenicol), and fumagillin residues using liquid chromatography tandem mass spectrometry (LC-MS/MS). Erythromycin (a macrolide) and monensin (an ionophore) can be detected and confirmed but not quantitated. Honey samples (approximately 2 g) are dissolved in 10 mL of water and centrifuged. An aliquot of the supernatant is used to determine streptomycin. The remaining supernatant is filtered through a fine-mesh nylon fabric and cleaned up by solid phase extraction. After solvent evaporation and sample reconstitution, 15 antibiotics are assayed by LC-MS/MS using electrospray ionization (ESI) in positive ion mode. Afterward, chloramphenicol is assayed using ESI in negative ion mode. The method has been validated at the low part per billion levels for most of the drugs with accuracies between 65 and 104% and coefficients of variation less than 17%. The evaluation of matrix effects caused by honey of different floral origin is presented.     

Validation of the tetrasensor honey test kit for the screening of tetracyclines in honey

Regarding anti-infectious agents, no maximum residue limits are fixed for honey in the European legislation. Discussions are being conducted in order to set working limits at the European level; for example, for tetracyclines, 20 microg/kg was proposed. The Tetrasensor Honey test kit is a receptor-based assay using dipsticks for a rapid screening (30 min) of honey on the presence of tetracyclines. The test was validated according to Commission Decision 2002/657/EC. The test detects tetracycline, oxytetracycline, chlortetracycline, and doxycycline in honey in a specific and sensitive way. Depending on the type of tetracycline, detection capabilities (CCbeta) between 6 and 12 microg/kg were obtained (4-7 microg/kg for dried dipsticks). The test is rugged and participation with the test in an international ring trial gave compliant results. It can be concluded that the Tetrasensor Honey test kit is a simple and reliable test that can even be used at the production site.     

Determination of tetracycline antibiotic residues in edible swine tissues by liquid chromatography with spectrofluorometric detection and confirmation by mass spectrometry

A sensitive and specific method is described for the simultaneous determination of oxytetracycline, tetracycline (TC), and chlortetracycline residues in edible swine tissues, by combining liquid chromatography with spectrofluorometric and mass spectrometry detection. The procedure involved a preliminary extraction with EDTA-McIlvaine buffer acidified at pH 4.0, followed by solid-phase extraction cleanup using a polymeric sorbent. The liquid chromatography analysis was performed with spectrofluorometric detection after postcolumn derivatization with magnesium ions. The limits of quantification were 50 microg/kg for muscle and 100 microg/kg for kidney tissues. The recovery values were greater than 77.8% for muscle and 65.1% for kidney. The method has been successfully used for the quantification of tetracyclines in swine tissues samples. The selective liquid chromatography mass spectrometric analysis for confirmation of oxytetracycline in one positive swine muscle sample was made by atmospheric pressure chemical ionization (APCI). The APCI mass spectra of the TCs gave the protonated molecular ion and two typical fragment ions, required for their confirmation in single ion monitoring scan mode in animal tissues.     

Use of veterinary drugs in intensive animal production

Background. Veterinary drugs are widely used in animal production and possibly reach soil and water through the excreta. There is only limited information concerning the environmental persistence of these substances. Tetracyclines represent the most frequently used antibiotics and accounted for more than 50% of the total amount of veterinary drugs employed through veterinary purchase orders in the Weser-Ems district in 1997. Objectives. It was therefore the objective of the present study to investigate the excretion patterns of tetracycline hydrochloride in pigs after oral application and to study the stability of tetracycline in pig slurry under different storage conditions. Methods 6 individually kept pigs were orally treated with two doses of tetracycline for 5 days and pooled excreta were regularly sampled. Stability testing was performed using 300 1 tanks which were spiked with 20 and 100 μg ml^-1 tetracycline and stored at different ambient temperatures for seven weeks. 181 random pig slurry samples were obtained from commercial farms. Tetracycline and its epimer 4-epi-tetracycline were detected using HPLC. Results Orally applied tetracyclines were rapidly excreted via feces and urine, and significant amounts (up to 72%) of the active ingredient which had initially been dispensed were recovered until the second day after the end of application. Individual animals continued to excrete tetracyclines over a longer period of time. Tetracycline hydrochloride demonstrated a distinct stability in slurry which was not significantly influenced by the initial concentration of the antibiotic, ambient temperature or aeration through repeated stirring. Half-lives ranged between 55 and 105 days. Conclusion Significant amounts of tetracycline are transferred to soil via manure application. There is considerable need for further research into environmental effects such as sorption and mobility, and microbiological changes. This is also the case for other substances which have not undergone valid environmental risk assessment.     

Investigation of sulfonamide, tetracycline, and quinolone antibiotics in vegetable farmland soil in the Pearl River Delta area, southern China

Thirteen antibiotics in soil from vegetable farmlands of the Pearl River Delta, southern China, were investigated. At least three antibiotics were detected in each sample. Six antibiotics including four quinolones, tetracycline, and sulfamethoxazole were detected in >94% of the samples. The total contents of three tetracyclines, eight sulfonamides, and four quinolones were not detected-242.6, 33.3-321.4, and 27.8-1537.4 μg/kg, respectively. The highest antibiotic concentrations were observed mainly in vegetable farmlands affiliated with livestock farms. Chlortetracycline, sulfameter, and quinolones in some samples exceed the ecotoxic effect trigger value (100 μg/kg) set by the Steering Committee of Veterinary International Committee on Harmonization. The composition and concentration of antibiotics in soil were correlated with vegetable species. This study has revealed an alarming condition of antibiotics in vegetable farmland soil. Further investigation including environmental fate, plant uptake, and human exposure to antibiotics by plant-derived food should be conducted.     

Comparison of liquid chromatographic and bioassay procedures for determining depletion of intramuscularly injected tylosin

Crossbred pigs weighing 80-110 kg were injected intramuscularly in the ham with 8.8 mg/kg tylosin. Animals were slaughtered in groups of 3 at intervals of 4 h, and 1, 2, 4, and 8 days after injection, and samples of blood, injected muscle, uninjected muscle, liver, and kidney were analyzed by liquid chromatography (LC) and by bioassay using Sarcina lutea as the test organism. The LC method was far more sensitive with a detection limit of less than 0.1 ppm, while the detection limit by bioassay was about 0.5 ppm in tissue. Results by bioassay and LC sometimes differed considerably for tissue samples. Residues in all tissues were below the tolerance limit of 0.2 ppm at 24 h, except in the injected muscle in one animal. Residues were not detected in any tissue of any animal at 48 h after treatment.     

土壤及畜禽粪肥中四环素类抗生素固相萃取-高效液相色谱法的优化与初步应用

本研究优化了土壤和有机肥中3 种四环素类抗生素的提取和测定方法。方法经优化后,土壤和有机肥中的抗生素的提取效率达到52%~95%,且分析时间大大缩短。本研究还利用优化方法在天津进行了有机肥和菜地中3 种抗生素残留的初步调查。集约化养殖场的猪、鸡粪便中四环素类抗生素残留状况为: 金霉素（CTC）检出率达到78%,最高残留值达到563.8 mg/kg（干基）; 四环素（TC）和土霉素（OTC）检出率也高达56%,最高值分别达到34.8 mg/kg 和 22.7 mg/kg。在天津销售的几种商品有机肥中同样检测出四环素类抗生素的残留,残留水平与猪粪和鸡粪相当。菜田土壤样品中TCs的总检出率为64%,3.种抗生素中土霉素检出率最低为18%,最高值达到105.6 g/kg（风干基）; 四环素检出率为36%,最高值达到196.7 g/kg; 金霉素检出率为32%,最高值达到477.8 g/kg。在所调查土壤中,温室和大棚土壤TCs的残留水平高于露地土壤。占调查样品 27.3% 的菜田土壤中TCs总量超过欧盟规定的生态安全触发线（100 g/kg）,存在一定的潜在生态风险。     

Simple determination of 40 organophosphate pesticides in raw wool using microwave-assisted extraction and GC-FPD analysis

A validated analytical method for the multiresidue analysis of 40 organophosphate pesticides (OPs) and conversion products in raw wool has been developed. The method is based on the selective microwave-assisted extraction (MAE) of raw wool with acetonitrile and analysis of extracts by gas chromatography-flame photometric detector. The optimum MAE conditions were 20 min duration at 80 °C with 30 mL of acetonitrile per gram of wool. A validation study was performed according to the European SANCO guidelines 10684/2009. Limits of detection and quantification for all pesticides tested were from 0.01 to 0.2 mg/kg and from 0.2 to 1.0 mg/kg, respectively. The average recoveries of pesticides spiked at different levels were in the range of 70-120% with relative standard deviations of ≤ 20%. The extraction performance was compared to the one obtained with a reference Soxhlet extraction. The method was also applied in the analysis of real wool (after field application) samples.     

Residue depletion of ractopamine and its metabolites in swine tissues, urine, and serum

Ractopamine hydrochloride is a beta-adrenergic leanness-enhancing agent approved for use in swine in the United States. Depletion of ractopamine and its metabolites from animal tissues, urine, and serum is of interest for the detection of illegal use. The objectives of this study were to measure the residues of ractopamine in swine incurred samples after treatment with dietary ractopamine for 28 consecutive days. An efficient and sensitive analytical method was developed for the detection of parent ractopamine and its metabolites in swine tissues, urine, and serum by HPLC-FLD. After extraction, enzymatic digestion, and solid-phase cleanup of the samples, ractopamine residues were determined by liquid chromatography (LC) with fluorescence detector. The limits of detection (LOD) for tissues, urine, and serum were 1 ng g(-1), 0.5 ng mL(-1), and 0.5 ng mL(-1), respectively. Recoveries ranged from 70.5 to 94.5% for samples fortified at 1-50 ng g(-1) or ng mL(-1). Sixty pigs were fed twice daily for 28 consecutive days with feeds containing 18 mg kg(-1) ractopamine HCl. The residue concentrations in urine, liver, and kidney were 650.06 ng mL(-1), 46.09 ng g(-1), and 169.27 ng g(-1), respectively, compared with those in muscle, fat, and serum (4.94 ng g(-1), 3.28 ng g(-1), and 7.48 ng mL(-1), respectively) at the feeding period of 7 days. The residue concentrations at withdrawal period of 0 days in all edible tissues were lower than tolerance values established by the FDA and MRL values listed by the JECFA. These data support the withdrawal time of 0 days established by the FDA for ractopamine used as feed additive in swine.     

Matrix solid-phase dispersion (MSPD) isolation and liquid chromatographic determination of oxytetracycline, tetracycline, and chlortetracycline in milk

A multiresidue method for the isolation and liquid chromatographic determination of oxytetracycline (OTC), tetracycline (TC), and chlortetracycline (CTC) antibiotics in milk is presented. Blank and tetracycline (OTC, TC, and CTC) fortified milk samples (0.5 mL) were blended with octadecylsilyl (C18, 40 microns, 18% load, endcapped, 2 g) derivatized silica packing material containing 0.05 g each of oxalic acid and disodium ethylenediaminetetraacetic. A column made from the C18/milk matrix was first washed with hexane (8 mL), following which the tetracyclines were eluted with ethyl acetate-acetonitrile (1 + 3; v/v). The eluate contained tetracycline analytes that were free from interfering compounds when analyzed by liquid chromatography with UV detection (photodiode array, 365 nm). Correlation coefficients of standards curves for individual tetracycline isolated from fortified samples were linear (from 0.982 +/- 0.009 to 0.996 +/- 0.004) with average percentage recoveries from 63.5 to 93.3 for the concentration range (100, 200, 400, 800, 1600, and 3200 ng/mL) examined. The inter-assay variability ranged from 8.5 +/- 2.4% to 20.7 +/- 13.0% with an intra-assay variability of 1.0-9.3%.     

Determination of proteins in refined and nonrefined oils

Five methods using aqueous/organic solvents for the separation of proteins from oils were compared. The extraction with acetone-hexane followed by amino acid analysis was found to be the most suitable method for isolation and quantification of proteins from oils. The detection limit of the method was 0.18 mg protein/kg oil, and the quantification limit was 0.6 mg protein/kg. The relative repeatability limit for samples containing 1-5 mg protein/kg sample was 27%. The protein recovery ranged between 68 and 133%. Using this method, the protein content of 14 refined and nonrefined oils was determined. In none of the refined oils were proteins detected, whereas the protein content of the unrefined oils ranged between undetectable in extra virgin olive oil to 11 mg/kg in rapeseed oil. With sodium dodecyl sulfate-polyacrylamide gel electrophoresis in combination with silver staining, many protein bands were visible in the unrefined soy, olive, peanut, and rapeseed oil samples. Proteins bands were not obtained from the refined fish oil. In the other refined oil samples, a few proteins bands could be visualized. Two protein bands with apparent molecular molecular masses of 58 and 64 kDa were always observed in these oils.     

Development and validation of an optical SPR biosensor assay for tylosin residues in honey

In recent years there has been an increase in the use of tylosin in apiculture as bacterial brood diseases become resistant to oxytetracycline. Confirmatory mass spectrometry based methods have been developed but up until now there has been no complementary screening method available capable of sub 10 microg kg(-1) detection limits. In this paper the development and validation of a screening method using optical biosensor technology is presented. The honey was first dissolved in a phosphate buffer and following solid-phase extraction (SPE) cleanup was analyzed using a Biacore Q instrument. Using the criteria specified in European Commission Decision 2002/657/EC for qualitative screening methods, the detection capability (CCbeta) of the method was determined to be 2.5 microg kg(-)(1). Honey samples containing trace residue levels of tylosin were analyzed by both the biosensor screening method and a LC-MS/MS confirmatory procedure; the results were in good agreement.     

Determination of aflatoxins, ochratoxin a, and zearalenone in mixed feeds, with detection by thin layer chromatography or high performance liquid chromatography

A sensitive, reliable, and economical method for the determination of 6 mycotoxins in mixed feeds is described. The feed is extracted with chloroform-water and the extract is cleaned up by using a disposable Sep-Pak silica cartridge. The procedure requires less time (15 min from sample extraction to extract preparation) and less solvent (approximately one-tenth) compared with conventional methods and is suitable for a fast, economical screen. Additional cleanup procedures, involving dialysis or extraction into base, are described for samples containing high levels of interfering compounds. Thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) with fluorescence detection are described for identification and estimation of mycotoxins. The method has been applied to a wide range of mixed feeds, including laboratory animal diets, and raw materials. The limit of detection is 1 microgram/kg for all mycotoxins measured by HPLC.