Development of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of glucocorticoid residues in edible tissues of swine,cattle, sheep,and chicken

A confirmatory and quantitative method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to determine the presence of eight glucocorticoids (prednisone, prednisolone, hydrocortisone, methylprednisolone, dexamethasone, betamethasone, beclomethasone, and fludrocortisone) in the muscles and livers of swine, cattle, and sheep and the muscle of chicken is described. After deconjugation in alkali media, samples were extracted with ethyl acetate for glucocorticoids followed by solid-phase extraction clean-up and reconstitution in the LC mobile phase. The hydrolysis procedure with sodium hydroxide was used to reduce handling time. A single-step solid-phase extraction method was optimized which is suitable for the clean-up of the compounds of interest in many diverse tissue matrices. LC separations were performed on a C₁₈ column with gradient elution using acetonitrile and water (containing 0.2% formic acid) and the two epimers betamethasone and dexamethasone were successfully separated. LC-electrospray ionization (ESI)-MS/MS in negative mode with selected reaction monitoring (SRM) mode was performed to improve method sensitivity and reduce matrix interference. Two SRM transitions were used for each compound. The recovery of glucocorticoids spiked at levels of 0.5–16 µg kg^?1 ranged from 55% to 107%; the between-day relative standard deviations were no more than 15%. The limits of quantification were 0.5–2.0 µg kg^?1 in muscle and 1–4 µg kg^?1 in liver. The optimized procedure was successfully applied to monitor the food at the 2008 Summer Olympics Games in Beijing, China, demonstrating the method to be simple, fast, robust, and suitable for identification and quantification of glucocorticoids residues in foods of animal origin.     

同时检测畜禽肉中呋喃唑酮、地塞米松残留高效液相色谱法的建立

为建立畜禽肉中呋喃唑酮、地塞米松HPLC检测方法。经试验,得到的色谱条件为流动相:乙腈-水(40+60);检测波长:240nm;流速:1.0mL/min。采用该方法检测了市售畜禽肉中呋喃唑酮、地塞米松的残留量,结果表明:该方法分离效果良好,平均回收率范围85.41%～95.2%,相对标准偏差为2.0%～4.5%,最低检出限达1.0～2.0μg/mL,说明该方法灵敏、准确、精密、无杂质干扰,可以用于肉品中呋喃唑酮、地塞米松残留的检测。     

Concentrations of antibiotic residues vary between different edible muscle tissues in poultry

Antibiotics are used by veterinarians and producers to treat disease and improve animal production. The federal government, to ensure the safety of the food supply, establishes antibiotic residue tolerances in edible animal tissues and determines the target tissues (e.g., muscle) for residue monitoring. However, when muscle is selected as the target tissue, the federal government does not specify which type of muscle tissue is used for monitoring (e.g., breast versus thigh). If specific muscle tissues incorporate residues at higher concentrations, these tissues should be selected for residue monitoring. To evaluate this possibility in poultry, chickens were divided into four groups and at 33 days of age were dosed with enrofloxacin (Baytril), as per label directions, at either 25 ppm for 3 days, 25 ppm for 7 days, 50 ppm for 3 days, or 50 ppm for 7 days. Breast and thigh muscle tissues were collected from each bird (n = 5 birds per day per group) during the dosing and withdrawal period, and fluoroquinolone concentrations were determined. The results indicate higher overall enrofloxacin concentrations in breast versus thigh muscle for each treatment group (P < 0.05). These data indicate, at least for enrofloxacin, that not all muscle tissues incorporate antibiotics at the same concentrations. These results may be helpful to regulatory agencies as they determine what tissues are to be monitored to ensure that the established residue safety tolerance levels are not exceeded.     

Long-term residues arising from 14CO2 fixation in swine tissues

Pigs injected intramuscularly with ¹⁴C sodium earbonate solution were killed after 9 and 21 days. The protein hydrolysate from liver tissues were treated with ninhydrin to liberate CO₂ which was used for determination of residual radioactivity. Most of the ¹⁴C from the fixed CO₂ was liberated by this treatment, which was considered to be a suitable method for estimating long-term residues of ¹⁴C in tissues arising from a ¹⁴CO₂ fixation process. The proportion of ¹⁴C in various amino acids remained almost constant over the period of the test.     

Determination method for ractopamine in swine and cattle tissues using LC/MS

Simple and reliable methods using LC/MS have been developed for the determination of the beta-agonist ractopamine in swine and cattle tissues. Ractopamine was extracted with ethyl acetate from muscle and liver, and the ethyl acetate layer was evaporated to dryness. The residue was purified by partition with acetonitrile/n-hexane. In the case of fat, ractopamine was extracted and purified by partition with acetonitrile/n-hexane. The resulting acetonitrile solutions were evaporated to dryness. The residue was dissolved in methanol, and subjected to LC/MS. The LC separation was performed on a Wakosil-II 3C18HG column (150 x 3 mm i.d.) in isocratic mode with 0.05% trifluoroacetic acid-acetonitrile (80:20) as a mobile phase at a flow rate of 0.4 mL/min. The MS detection was performed in the selected ion recording (SIR) mode, with detection of the M + H+ ion of ractopamine (m/z 302) produced by electrospray ionization (ESI). The mean recoveries of the drug from swine muscle (0.01 microg fortified), fat (0.01 microg fortified) and liver (0.04 microg/g fortified) were 99.7%, 99.5% and 100.8%, and those from cattle samples were 108.3%, 97.0% and 109.4%, respectively. The relative standard deviations (RSDs) ranged from 0.1% to 9.5%. The limit of quantification (LOQ) of the drug was 1 ng/g.     

Oxytetracycline and oxolinic acid residues in kuruma prawn (Penaeus japonicus) and the effect of cooking procedures on the residues

Tissue distribution and residue depletion of oxytetracycline (OTC) and oxolinic acid (OA) were studied in the kuruma prawn (Penaeus japonicus). The prawn were kept in tanks with recirculated artificial seawater at a salinity of 22-23@1000. The water temperature was maintained at 25 degrees C. The average body weight was 22.9 +/- 4.9 g for OTC and 22.5 +/- 3.6 g for OA. The drug was mixed with the diet and orally administered through a catheter to the prawn. The doses of OTC and OA, respectively, were 50 mg/kg body weight. At each sample time, four prawns were sacrificed and tissues were sampled. OTC and OA levels were determined by high-performance liquid chromatography. At the highest levels, the concentrations of OTC were in the other: shell (13.57 micrograms/g) > hemolymph (12.20 micrograms/mL) > muscle (8.30 micrograms/g). For OA, the order was: shell (20.74 micrograms/g) > hemolymph (7.06 micrograms/mL) > muscle (2.05 micrograms/g). The elimination half-lives of hemolymph and muscle were 44.7 and 46.8 hours for OTC and 55.0 and 107.9 hours for OA, respectively. Residual OTC could not be detected in hemolymph and muscle at 20 days after dosing. Residual OA disappeared from hemolymph and muscle at 25 days after dosing. A 25-day period for OTC and 30-day period for OA could be regarded as the proper withdrawal time established for kuruma prawn by the Pharmaceutical Law in Japan. However, the elimination half-lives of shell for OTC and OA could not be calculated because both drug residues persisted in shell tissues, and the elimination phase was not completed during the experimental period. Residual OTC (14.10 +/- 2.26 micrograms/g, n = 6) and OA (0.32 +/- 0.06 microgram/g, n = 7) were detected in exuviae at 3 days and 4 days after dosing, respectively. Residual OTC was reduced to 50-70% in muscle by the usual methods of cooking (boiling, baking at 200 degrees C and frying at 180 degrees C), whereas reduction levels in shell were only 20-30%. Residual OA was reduced to 20-30% in muscle and shell by the cooking. These results confirm that the cooking procedures could only reduce but not completely eliminate these drug residues in prawn.     

Antibiotic residues in edible tissues and antibiotic resistance of faecal Escherichia coli in pigs from Portugal.

The aims were to provide data on antibiotic residues in pig tissues and the trends in the occurrence of antibiotic resistance in Escherichia coli (a commensal bacteria that can colonize the intestinal tract of humans and animals) isolated from the animals studied. Muscle and kidney samples collected from freshly slaughtered pigs were tested for the presence of antibiotic residues using rapid screening tests. Ninety muscle and 90 kidney samples from the same animals, collected in the slaughterhouse of the central region of Portugal, were screened by microbial tests and only six samples did not fulfil the European Community regulations concerning maximum residue limits. When a more sensitive method was used for the detection of sulphamethazine, 36% of the animals contained residues of the antibiotic. This indication of a larger antibiotic occurrence than anticipated was confirmed by studying the susceptibility of their enteric E. coli. In all the animal faeces studied (n = 60), E. coli isolates resistant to amoxycillin, tetracyclines and sulphonamides were found. The majority of the faecal samples demonstrated a high degree of E. coli resistant to sulphonamides and tetracyclines, respectively 88% and 85%. These data raise important questions about the relevance of the detection of antibiotic residues as the only method to establish the quality and safety of animal products for human consumption.     

Drug residues in dairy cattle industry: epidemiological evaluation of factors influencing their occurrence

A random study of 3000 dairy farmers was designed to determine 1) management factors that may be associated with the occurrence of drug residues; 2) the dairy farmers' attitudes and knowledge about residues; 3) how these variables influence the occurrence of residues in dairy cattle. Management factors perceived as having the greatest influence on drug residues in milk were insufficient knowledge about withdrawal periods, errors due to hired help, insufficient identification and record of animals treated for mastitis, metritis treatment, dry cow treatment for mastitis, and not reading the label. Forty-one percent of farms with residue problems used medicated feeds compared with 38.9% of control farms. Factors significantly associated with the occurrence of residues were herd size, increased number of hired persons, increased frequency of use of medicated feeds, category of medicated feed, and producer's attitude toward the public health significance of residues. Farmers needed more information in five areas: likelihood of persistent residues from various types of chemicals (26.8%), consequences of chemicals occurring in livestock products (22.4%), withholding times for specific chemicals (20.4%), disposal of surplus farm chemicals (17%), preparation of products for use in livestock (13.4%).     

Rapid and sensitive enzyme-linked immunosorbent assay and immunochromatographic assay for the detection of chlortetracycline residues in edible animal tissues

Two rapid and sensitive enzyme-linked immunosorbent assays (ELISA) and an immunochromatographic assay (ICA) for the detection of chlortetracycline (CTC) residues in edible animal tissues were developed based on a monoclonal antibody (MAb) produced by using the chlortetracycline-bovine serum albumin (CTC-BSA) conjugate as the immunogen. A total of 50% inhibiting concentration (IC(50)) of the modified ELISA was 0.66 ng ml(-1) and the recoveries from spiked chicken muscle and liver were 78.8-92.2% and 80.3-90.2%, respectively. The corresponding coefficient variations (CVs) were 3.2-9.5% and 6.5-10.2%. The detection limit was 0.06 ng g(-1) in chicken muscle and 0.07 ng g(-1) in liver. However, the detection limit of ICA was 0.12 ng ml(-1), and the recoveries in negative samples spiked at concentrations of 10, 50 and 100 ng g(-1) ranged from 79.0% to 88.6% for muscle samples and from 75.2% to 87.0% for liver samples. The cut-off values for the test lines were 80 ng g(-1) and the analysis can be completed within 5-10 min. Comparisons with an HPLC method were performed by testing 200 swine muscle samples and chicken muscle samples from local markets, and an agreement rate of 99.5% was obtained between the three methods.     

A simple procedure for the enzymic digestion of edible tissues prior to detection of drug residues

Summary A simple procedure for the enzymic digestion of edible tissues is described and compared with other procedures. The samples are digested overnight in an enzyme suspension containing subtilisin A at 60° C and pH 9. The resulting digest contains only a few small tissue fragments. This method is suitable for routine analysis, since the manipulation of the samples is very limited.

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Eine einfache enzymatische Zersetzung eßbarer Gewebe zur Auffindung von medikamentösen Rückständen

Zusammenfassung Es wird ein einfaches Verfahren für die enzymatische Auflösung eßbarer Gewebe beschrieben und mit anderen Verfahren verglichen. Die Proben werden über Nacht bei 60° C und pH 9 in einer subtilisinhaltigen Enzymsuspension zersetzt. Die erhaltenen Lösungen enthalten lediglich kleine Gewebefragmente. Diese Methode eignet sich für die Routineanalyse, weil die Handhabung der Proben sehr einfach ist.

     

The effect of cooking on veterinary drug residues in food: nicarbazin (dinitrocarbanilide component).

The change of concentration of residues of the marker compound for the anti-coccidial drug nicarbazin, N,N'-bis(4-nitrophenyl)urea (dinitrocarbanilide, DNC), was investigated in model oil and aqueous solutions and in chicken muscle and egg. In model aqueous solutions, DNC decreased rapidly in concentration upon heating followed by a much more gradual decomposition. The curves produced when this information was plotted were not typical of exponential decay. In model cooking oil solutions, DNC generally showed a slower decrease in concentration over time when compared with aqueous solutions. DNC residues in egg were stable to microwave cooking and residues in chicken muscle were stable to stewing and microwaving. Other cooking procedures led to a decrease in amount of DNC by 22% to 48% of the total amount of analyte present. Only a small amount (<2%) of residue leached with juices which exuded as the food was cooked.     

Evaluation of three different microbial inhibition tests for the detection of sulphamethazine residues in the edible tissues of rabbit

The aim of the present study was to evaluate three microbial inhibition tests (MIT) based on inhibition of growth of the test organisms: (a) four plate test (FPT) containing Bacillus subtilis BGA, (b) screening test for antibiotic residues (STAR) containing Bacillus stearothermophilus var. calidolactis_ATCC 10149 and (c) the Premi®Test containing Bacillus stearothermophilus var. calidolactis. The tests were used to determine sulphamethazine (SMZ) residues in edible tissues of rabbit after oral administration up to day 15 of the withdrawal period (WP). A solvent extraction procedure was used to enhance the capability of the tests to detect SMZ residues at or below the maximum residue limit (MRL). Para-aminobenzoic acid (PABA) was employed to previously identify SMZ residues in the first stage of the residue screening. The presence of SMZ residues in the samples was confirmed and quantified by a validated HPLC method. The Premi®Test detected SMZ residues in the muscle and heart tissue up to day 9 of the WP, and in the liver, lungs and kidneys up to day 10 of the WP. The STAR detected SMZ residues in the edible organs of rabbits up to day 8 of the WP. The kidneys were positive up to day 5 of the WP, the liver until day 4 of the WP and the lungs until day 3 of the WP. No SMZ residues were detected in the muscle and heart. By using the solvent extraction procedure, SMZ residues were detected in the muscle extract up to day 10 of the WP and the muscle was positive until day 6 of the WP. No detection sensitivity was observed using the FPT. After solvent extraction, SMZ residues were detected in the muscle extract until day 8 of the WP and the muscle was positive until day 3 of the WP. No positive results were detected after the addition of PABA into/onto the agar medium. PABA at a concentration of 10 µg ml^?1 completely reversed the inhibitory activity of SMZ and enabled reliable identification of SMZ in the examined samples. Using HPLC, SMZ was detected in the muscle samples until day 10 of WP (0.02 mg kg^?1) and in the liver until day 12 of the WP (0.09 mg kg^?1). The results obtained by the HPLC method and the limit of detection (LOD) of screening tests for SMZ (FPT 0.4 µg ml^?1, STAR 0.2 µg ml^?1, Premi® Test 0.05 µg ml^?1) allowed us to state that the most suitable screening tests for the detection of SMZ residues in the edible tissues of rabbits at level corresponding to the MRL of 0.1 mg kg^?1, established for sulphonamides, are the Premi®Test and STAR in conjunction with the solvent-extraction procedure.     

Assessing the depletion of lincomycin in feathers from treated broiler chickens: a comparison with the concentration of its residues in edible tissues

ABSTRACT

Lincomycin is the first antimicrobial agent described for the lincosamide class and it is commonly used for the treatment of infectious enteric and respiratory diseases in poultry. Maximum residue limits (MRLs) in edible tissues have been established for this antimicrobial, however, no regulation has been proposed yet for by-products that are not intended for direct human consumption. Feathers are a by-product from poultry farming that might be used as an ingredient for diets fed to other farm animal species. The presence of antimicrobial residues in them is not monitored in spite of the fact that several studies have proved that they can persist in feathers. Currently though, no evidence has been presented regarding the behaviour of lincomycin in this matrix. Hence, this work intended to assess the depletion of lincomycin residues in feathers of birds treated with therapeutic doses and compare them with those detected in muscle and liver samples. Samples were collected for several days after ceasing treatment from a group of broiler chickens treated with a 25% lincomycin formulation. Methanol and Florisil® columns were used to extract and retain the analyte, and samples were analysed using a triple quadrupole mass spectrometer (API 5500, AB SCIEX?). On day 1 after ceasing treatment, average concentrations of lincomycin detected in feather samples reached up to 8582 μg kg^?1 and by day 16, these had only declined by 63%, to an average of 3138 μg kg^?1. Lincomycin residues were detected in feathers at every sampling point, even after they were not detectable in edible tissues. Depletion time was 98 days for feathers, considering the LOQ established for the methodology as cut-off value for the calculations. Data showed that lincomycin is highly persistent in feathers, which may result in this matrix becoming a re-entry route for its residues into the food chain.     

A simple procedure for the enzymic digestion of edible tissues prior to detection of drug residues.

A simple procedure for the enzymic digestion of edible tissues is described and compared with other procedures. The samples are digested overnight in an enzyme suspension containing subtilisin A at 60 degrees C and pH 9. The resulting digest contains only a few small tissue fragments. This method is suitable for routine analysis, since the manipulation of the samples is very limited.     

气相色谱法测量植物油中六六六和滴滴涕残留量的不确定度评定

介绍了气相色谱法测定植物油中六六六、滴滴涕残留量的不确定度评定方法。按测量程序完整地确定了不确定度的来源,并对分析结果的已识别来源的不确定度影响进行评价。     

液相色谱—串联质谱法测定猪可食用组织中氨茶碱残留

目的:研究猪可食用组织中氨茶碱药物残留的定性定量测定方法。方法:建立固相萃取净化结合高效液相色谱—串联质谱法,试样中的氨茶碱药物残留被甲醇溶液提取后,经PRiME HLB固相萃取柱一步净化,随后用C₁₈色谱柱分离,以0.1%甲酸水溶液和乙腈分别作为水相和有机相对氨茶碱进行色谱柱梯度洗脱,利用电喷雾正电压离子化并采用多反应监测模式进行化合物检测,空白基质匹配标准曲线外标法定量。结果:氨茶碱药物在3.5 min内完成仪器分析,不同基质的匹配曲线在0.005～0.1μg/mL质量浓度范围内呈良好的线性关系(R>0.998 0),猪肉、猪肝、猪肾的方法加标平均回收率为87.2%～101.3%,相对标准偏差为0.7%～6.2%(n=6),方法检出限为2μg/kg,定量限为5μg/kg。结论:该方法简便高效、重复性好、灵敏度高,适用于猪的可食用组织中氨茶碱药物残留的定性定量分析。     

An evaluation of edible red seaweed (Chondrus crispus) components and their modification during the cooking process

In this study, phycobiliproteins, carotenoids (β-carotene and lutein), volatile compounds and antioxidant activity were determined in dried, hydrated, boiled and steamed Chondrus crispus seaweed.     

Tylosin depletion in edible tissues of turkeys

The depletion of tylosin residues in edible turkey tissues was followed after 3 days of administration of tylosin tartrate at 500 mgl^-1 in drinking water, to 30 turkeys. Immediately after the end of the treatment (day 0) and at day 1, 3, 5 and 10 of withdrawal, six turkeys (three males and three females) per time were sacrificed and samples of edible tissues were collected. Tissue homogenates were extracted, purified and analysed by HPL C according to a method previously published for the analysis of tylosin residues in pig tissues. In all tissues, tylosin residues were already below the detection limits of 50 μg kg^-1 at time zero. However, in several samples of tissues (skin ₊ fat, liver, kidney, muscle), from the six turkeys sacrificed at that time, one peak corresponding to an unknown tylosin equivalent was detected at measurable concentrations. The identification of this unknown compound was performed by L C-MS/MS analysis of the extracts from incurred samples. The mass fragmentation of the compound was consistent with the structure of tylosin D (the alcoholic derivative of tylosin A), the major metabolite of tylosin previously recovered and identified in tissues and/or excreta from treated chickens, cattle and pigs.     

Analysis of sulphonamide residues in edible animal products: A review

The methods of analysis for sulphonamide residues in edible animal products are reviewed. Sulphonamides are widely used for therapeutic and prophylactic purposes in both humans and animals, sometimes as growth promoters as additives in animal feed. As a result of their widespread use, there is concern about whether the levels used of these drugs can generate serious problems in human health, e.g., allergic or toxic reactions. Several methods for the determination of sulphonamides have been reported in the literature and this review considers high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC/MS), gas chromatography (GC), thin-layer chromatography (TLC), high-performance capillary electrophoresis (HPCE), enzyme-linked immunosorbant assay (ELISA), biosensor immunoassay (BIA) and microbiological methods. Specific aspects of analysing sulphonamides, such as sample handling, chromatographic conditions and detection methods are discussed. Methods for drug residue monitoring should be accurate, simple, economical in both time and cost, and capable of detecting residues below the maximum residue limits (MRL). The current sulphonamide detection technologies are based on chromatographic methods or bacteriological growth inhibition. The instrumental methods such as HPLC and GC are both sensitive and specific, but are laborious and expensive. Because of the labour-intensive processes, only a few cases of GC methods applied to residue analysis have been published. These methods are suitable for confirmation but not for screening of large numbers of samples. Microbiological methods do not require highly specialized and expensive equipment. They also use highly homogeneous cell populations for testing and thus result in better assay precision. Although HPCE has powerful separation ability, the precision is poor and the instrument still needs to be improved. To date, this technique has not been widely applied to routine analysis. Currently, TLC has been almost replaced by other instrumental analysis. A rapid, sensitive and specific assay is required to detect positive samples in routine analysis, which can then be confirmed for the presence of sulphonamides by HPLC. Immunochemical methods such as ELISA can be simple, rapid and cost-effective, with enough sensitivity and specificity to detect small molecules. This review can be considered as a basis for further research aimed at identifying the most efficient approaches.     

Effect of cooking on enrofloxacin residues in chicken tissue

The aim of this study was to determine the effect of different cooking processes (microwaving, roasting, boiling, grilling and frying) on naturally incurred enrofloxacin residues in chicken muscle. Enrofloxacin and its metabolite, ciprofloxacin, were analysed using a validated LC-MS method with limits of detection (LOD) and quantification (LOQ), respectively, of 2 and 5 ng g^-1 quinolones in muscle samples. The method was shown to be linear over the range 5-500 ng g^-1. Mean intra-day relative standard deviation (RSD) at a concentration of 50 ng g^-1 (n = 6) was 6%; inter-day RSD was 12%. A recovery study demonstrated that 65-101%, of the drug and metabolite could be recovered from the tissue. The RSD with naturally incurred roasted chicken breast was 9.18% at a concentration of 11 ± 1.01 ng g^-1 (n = 6). In water, enrofloxacin remained stable for 3 h when heated at 100°C. It was concluded that residue data from raw tissue are valid for estimation of consumer exposure to this drug, as well as the ADI calculations because cooking procedures did not affect enrofloxacin residues, which remained stable during heating. However, there was an apparent decrease in quinolone concentration in tissue because some was lost by exudation into the liquid used for cooking. Conversely, for a cooking procedure with water loss, there was an apparent increase in residue concentration.