Calf antibiotic and sulfonamide test (CAST) for screening antibiotic and sulfonamide residues in calf carcasses

The Calf Antibiotic and Sulfonamide Test (CAST), a microbial inhibition screening test, was developed for detecting antibiotics and sulfonamides in bob veal calf carcasses. The test uses Bacillus megaterium ATCC 9885 as the indicator organism and Mueller Hinton agar as the growth medium. Compared to Swab Test on Premises (STOP), developed in 1970, this screening test has higher sensitivity and the ability to detect a wider range of veterinary antimicrobial residual drugs, particularly sulfonamides, at lower concentrations. Carcasses that are tested with CAST and suspected of containing chemical residue above tolerance level are retained for confirmation. Disposition of these carcasses are determined upon laboratory result. Routine testing of bob veal calves with CAST allowed the Food Safety and Inspection Service to release most calf carcasses within 24 h post-slaughter, thus conserving shipping and handling resources. However, changes in the regulation in 1990 dictate that disposition of carcasses found to contain violative levels of sulfonamide residues should be based on laboratory findings. The analysis of the data for the years 1990-1994 and 1998 indicate that the use of CAST over the years was significant, and had a direct impact on reduction of residue violations in veal carcasses. With the use of CAST, potentially harmful antimicrobial chemicals entering the human food chain through veal meat have been minimized.     

Monitoring of pesticide residues in Egyptian fruits and vegetables during 1996

Samples of the most common fruits and vegetables were collected from 8 local markets in 6 governorates. These 1,579 samples were analyzed for residues of 53 pesticides, which included organophosphorus and organonitrogen compounds and some synthetic pyrethroids. Samples were also analyzed for residues of organochlorine pesticides, although they had been prohibited from use several years ago. Only 510 of the 1,579 samples were analyzed for dithiocarbamate pesticide residues, which were determined as CS2. Overall, 76.1 % of the total analyzed samples had no detectable residues, 23.9% contained detectable residues, and 2.59% contained residues that exceeded maximum residue limits. For individual crops, contaminated samples ranged from 0 to 96% of the number of samples analyzed. However, the highest violative percentage for samples of individual crops was 12.5. Chlorpyrifos, carbaryl, dimethoate, bromopropylate, and profenofos were the violative pesticides determined in fruit and vegetable samples. The results of the current study demonstrated that no restricted or banned pesticides such as DDT, HCH, and their isomers were found in any of the samples analyzed. Dithiocarbamate residues were detected in 9.4% of the 510 samples analyzed, with a violative percentage of 0.39, representing one grape sample and one peach sample.     

Development and Validation of Multi-Residue Method for Drugs Analysis in Human Feces by Liquid Chromatography–Tandem Mass Spectrometry

The use of veterinary drugs in animal production is a common practice to secure animal and human health. However, residues of administrated drugs could be present in animal food products. Levels of drugs in food of animal origin are regulated within the European Union. In recent years, residues have been detected not only in food, but also in the environmental elements such as water or soil, meaning that humans are involuntarily exposed to these substances. This article presents a multiclass method for the analysis of various therapeutic groups of pharmaceuticals in human feces. Pharmaceuticals are extracted from feces with an acid extraction solvent, and after filtration the extract was analyzed by HPLC–MS/MS. A limit of detection of 10 ng/g was achieved for 9 pharmaceuticals, with linearity over 0.99 and repeatability and reproducibility lower than 20%. The method was satisfactorily applied in 25 feces samples of individuals that had declared not to be under medical treatment for the last two months. Results indicate the presence of six different compounds at concentration between 10 and 456 ng/g. This preliminary study showed the involuntary exposure of human gut microbiota to active substances such as pharmaceuticals     

Chromatographic methods of analysis for penicillins in food-animal tissues and their significance in regulatory programs for residue reduction and avoidance.

Chromatographic methods for penicillin analysis in animal tissues play a significant role in the regulation of the use of these drugs in livestock production. Regulatory agencies rely on data generated from these methods to establish withdrawal times and to determine whether presumptive positive tissue samples from slaughtered animals intended for human consumption contain violative levels of penicillins to necessitate regulatory action. The need to develop sensitive, accurate, and reliable methods to support regulatory programs is examined together with emerging techniques that could be taken advantage of to improve the sensitivity and usefulness of current chromatographic methods for tomorrow's regulatory agency.     

Experiences with an identification and quantification program for inhibitor-positive milk samples

Beta-lactam antibiotics (penicillins, cephalosporins) are still the most commonly used antibiotics for dairy cows in Germany. In routine milk testing, according to the German milk quality regulation, a positive result obtained for bulk tank milk by microbiological inhibitor tests needs no further confirmation, but results in reduced milk payment of 0.05 euros kg(-1) for one month. In some cases, however, further identification of the causative agent can be of interest, either if antimicrobial drugs have not knowingly been used recently, or if improper use of such drugs is denied. As a service for milk producers, our laboratory offers further analyses of violative milk samples, aiming at the identification and quantification of the inhibitor(s). In this program, a panel of microbiological inhibitor tests, receptor tests, and enzyme immunoassays (EIA) is used in a step-by-step analysis, which primarily focusses on beta-lactams, but also includes other compounds such as sulfonamides or tetracyclines, respectively. Here we report results for violative milk samples (n=63) analysed between 2003 and 2005. In most cases (95%), beta-lactam antibiotics could be identified, although not always at levels exceeding the respective MRL values. Penicillin G (mostly together with benzylpenicilloyl metabolites) could be identified in 74.6% of all samples. Other compounds identified were, in decreasing order, ceftiofur (11%), ampicillin/amoxicillin (6.3%), isoxazolyl penicillins (3.2%), and sulfonamides (1.6%). The results indicate that penicillin G is still the predominant antibiotic responsible for violative bulk tank milk samples as detected during regulatory control.     

Validation of the LacTek test applied to spiked extracts of tissue samples: determination of performance characteristics

LacTek tests are competitive enzyme-linked immunosorbent assays intended for rapid detection of antimicrobial residues in bovine milk. In this study, the LacTek test protocol was modified for use with extracts of bovine tissue to detect beta-lactam, tetracycline, and sulfamethazine residues. Test performance characteristics--precision, accuracy, ruggedness, practicability, and analytical specificity and sensitivity--were investigated. Results suggest that LacTek tests can be easily adapted to detect antimicrobial residues in extracts of lean ground beef. However, positive samples may not contain residues at violative concentrations (i.e., Canadian maximum residue limits), and therefore, additional analysis would be required for final confirmation and quantitation (e.g., chromatography).     

Fast simultaneous determination of 23 veterinary drug residues in fish,poultry, and red meat by liquid chromatography/tandem mass spectrometry

The misuses of veterinary drugs can result in the accumulation of residues in food of animal origin that can make its way to the final consumer. Herein we describe a simple method for the accurate determination of beta-lactams, quinolones, sulphonamides, and tetracyclines in fish, poultry, and red meat. No extraction cartridges were used; instead, the extraction process consisted of the addition of an organic solvents, shaking, centrifugation, and dilution. An extensive validation process demonstrated an excellent linearity (R² ≥ 0.99) for 23-drug residues. The recovery of drugs in different matrices at two concentration levels (n = 6) was in the range of 82–119%. The method was proved to be repeatable and reproducible with intra/inter-day measurements (RSDs lower than 20%). The quantification limits of drug residues were in the range of 0.8 to 45.3 ug/kg, which is well below the maximum residue limits set by most regulatory authorities. This method was successfully applied to the routine analysis of 20 fish, poultry, and red meat samples (n = 60).     

Analysis of antibiotics in fish samples

The use of antibiotics in food-producing animals has generated considerable interest because the widespread administration of these drugs may lead to the development of resistant human pathogens. A large increase in the demand for seafood products has occurred in the last century. This has led to a concomitant increase in high-intensity aquaculture methods, characterized by high stock density and volume, and the heavy use of formulated feeds containing antibiotics, among other substances. Therefore, accurate and sensitive determination of antibiotic residues is now a necessity. In order to protect human health, the European Union and other regulatory authorities worldwide have established maximum residue limits (MRL) for antibiotic residues in animal products entering the human food chain. This paper reviews the most recent methods for analysis of antibiotic residues in fish.     

Bioanalytical methods for food contaminant analysis

Foods are complex mixtures of lipids, carbohydrates, proteins, vitamins, organic compounds, and other naturally occurring substances. Sometimes added to this mixture are residues of pesticides, veterinary and human drugs, microbial toxins, preservatives, contaminants from food processing and packaging, and other residues. This milieu of compounds can pose difficulties in the analysis of food contaminants. There is an expanding need for rapid and cost-effective residue methods for difficult food matrixes to safeguard our food supply. Bioanalytical methods are established for many food contaminants such as mycotoxins and are the method of choice for many food allergens. Bioanalytical methods are often more cost-effective and sensitive than instrumental procedures. Recent developments in bioanalytical methods may provide more applications for their use in food analysis.     

Development of an Evidence biochip array kit for the multiplex screening of more than 20 anthelmintic drugs

Anthelmintic drugs are used in clinical and veterinary practice for the treatment of infections caused by parasitic worms. Their extensive use in food-producing animals can cause the presence of residues in food. For consumer protection it is necessary to monitor the levels of anthelmintic residues to ensure that they remain within the legally permitted maximum acceptable concentrations. For this purpose, the use of multiplex screening methods is advantageous. Biochip array technology allows the simultaneous determination of multiple analytes from a single sample at a single point in time. This study reports the development of an Evidence biochip array for the multiplex screening of anthelmintic drugs. Simultaneous competitive chemiluminescent immunoassays are employed. The solid support and vessel is the biochip, which contains an array of discrete test sites. The assays were applied to the semiautomated bench-top analyser Evidence Investigator. The aminobenzimidazoles assay detected aminomebendazole, albendazole 2-aminosulphone and aminoflubendazole, the avermectins assay detected emamectin benzoate, eprinomectin, abamectin, ivermectin and doramectin, the benzimidazoles assay detected albendazole sulphone, albendazole, albendazole sulphoxide, oxibendazole, oxfendazole and flubendazole, the thiabendazole assay detected cambendazole, thiabendazole and 5-hydroxythiabendazole and the triclabendazole assay detected ketotriclabendazole, triclabendazole and triclabendazole sulphoxide. The limits of detection ranged from 0.3 ppb (aminobenzimidazoles) to 2.0 ppb (levamisole) in milk and from 0.15 ppb (aminobenzimidazoles) to 6.5 ppb (levamisole) in tissue. The average recovery range was 71-135 %. This multianalytical approach on a biochip platform is applicable to the screening of more than 20 anthelmintic drugs in different food matrices, leading to consolidation of tests and enhancement of the test result output.     

Immunobiosensor--an alternative to enzyme immunoassay screening for residues of two sulfonamides in pigs.

A rapid immunoassay using an optical biosensor (BIAcore) for determining the presence of sulfamethazine (SMT) residues in pig bile was developed. The assay was used in a routine screening laboratory alongside a previously described biosensor method for sulfadiazine (SDZ). Sulfonamide bile concentrations, determined by enzyme immunoassay (EIA), have already been shown suitable for use in predicting the extent of sulfonamide accumulation in kidney. The ability of immunobiosensor based bile screening to predict violative tissue residues (greater than the maximum residue limit; MRL) was compared with results achieved using two conventional EIAs for two of these drug residues (SMT and SDZ). Analysis of 2081 samples for both sulphonamide residues, over an 8 month period, showed the false positive prediction rate of biosensor analysis to be 0.14% and 0.34% for SMT and SDZ, respectively, compared with false positive rates of 1.54% and 1.44% by EIA. Biosensor analysis showed no false negative predictions for either SMT or SDZ while EIA showed a false negative prediction rate of 0.14% for SMT and 0.24% for SDZ. The present study has clearly demonstrated that immunobiosensor assays can be developed for veterinary drug residue screening programmes. These methods have the potential for generating faster and more reliable results than conventional immunoassay methods.     

Multiresidue method for simultaneous determination of ten quinolone antibacterial residues in multimatrix/multispecies animal tissues by liquid chromatography with fluorescence detection: single laboratory validation study

Quinolone antibacterials are veterinary drugs authorized for use in food animal production. The analysis of residual amounts of drugs in food from animal origin is important for quality control of products for consumers. For this purpose, Maximum Residue Limits (MRLs) have been set up by a European Union Council Regulation on Veterinary Drug Residues (No. 90/2377/EEC and subsequent), and 8 quinolones received MRLs at concentration levels depending on both the matrix and the animal species of interest. A method was developed for screening and confirming 10 quinolone residues (ciprofloxacin, danofloxacin, difloxacin, enrofloxacin, flumequine, marbofloxacin, nalidixic acid, norfloxacin, oxolinic acid, sarafloxacin) in a wide variety of matrixes of different animal species. It involves extraction of the residues from the biological tissues/fluids by acidic aqueous solution, centrifugation and filtration prior to injection on a C18 narrow-bore column, and detection through a 3-step-mode fluorescence detector. The method was validated during a 2-week study for a set of 8 species-matrixes (i.e., bovine raw milk, bovine muscle, porcine muscle, porcine kidney, porcine liver, fish flesh and skin, poultry muscle, whole egg). Residues were quantified down to 15 microg/kg with limits of detection and quantitation ranging from 4 to 11 and 13 to 36 microg/kg, respectively, which are sufficient compared to the wide range of MRLs set for these substances (from 30 microg/kg for danofloxacin in milk to 1900 microg/kg for difloxacin in poultry liver). The limit of performance of the method in terms of CCalpha and CCbeta, the critical concentrations stated in the Decision No. 2002/657/EC and the ISO Standard No. 11843, has been calculated for the authorized (MRL) substances but only estimated in the case of the nonauthorized (non-MRL) substances.     

近红外光谱在品质分析和定量分析中的应用

主要介绍近红外光谱在品质分析和定量分析中的一些应用，作为一种简单、快速、无损的检测手段，近红外光谱在鉴定原料的真伪、原料中有效成分的含量、有毒组分的识别等方面具有独特的效果。因此它在食品、药品、化工产品等领域得到了广泛应用。     

Recent applications of liquid chromatography–mass spectrometry to residue analysis of antimicrobials in food of animal origin

Residual antimicrobials in food constitute a risk to human health. Although epidemiological data on the real magnitude of their adverse effects are very scarce, they indicate that food could be an important vehicle for evolution and dissemination of antimicrobial-resistant bacteria. Public health agencies in many countries rely on detection by mass spectrometry (MS) for unambiguous identification of residues of antimicrobial agents in animal food products for human consumption. The introduction of relatively inexpensive and robust liquid chromatography (LC)–MS systems has given a strong impulse to the development of confirmatory methods for the above medicines in foodstuffs. The initial part of this review, after a brief introduction into the field of antimicrobials, is dedicated to the most important EU regulations and directives for control of residues of these substances in animal products. The main attention in this review is on the sample-treatment and MS detection systems in use today for analysing the most important classes of antimicrobials in various biological matrices (milk, animal tissues, eggs, and honey). As evidenced by this review, reversed-phase LC combined with tandem MS, usually triple-quadrupole MS (QqQMS), is currently the preferred technique in most residue analysis of a single-class of antimicrobials. A recently emerging analytical strategy is that of developing methods for detecting a large variety of veterinary drugs belonging to different classes, including pesticides (multi-class residue analysis). To do this, simple and generic extraction and separation techniques applicable to a broad range of compounds differing in physical and chemical properties have been adopted. Such methods are still based mainly on LC–QqQMS. Emerging alternative MS detection systems are time-of-flight MS, which provides accurate mass of the analyte(s), or Q–linear ion trap (IT) MS that eliminates some limitations of ITMS ⁿ .     

Extension of multi-residue methodology to include the determination of quinolones in food.

The multi-residue procedure for basic drugs described elsewhere by this laboratory has been evaluated for quinolone and fluoroquinolone antibiotics. The fluoroquinolones are a relatively new class of drug and an increasing number of licensed products containing these compounds are becoming available for use in animal husbandry. This, along with the possibility of the development of antibiotic resistant human pathogens, make it an important class of drug for which methodology is required for the monitoring of residues in food. Validation data are presented for a range of compounds including the quinolones; oxolinic acid and nalidixic acid, and the fluoroquinolones; flumequine, ciprofloxacin, danofloxacin, enoxacin, enrofloxacin, lomefloxacin, marbofloxacin, norfloxacin, ofloxacin and sarafloxacin. Foods for which the method was validated included poultry muscle (chicken and turkey), egg, chicken liver, honey, cattle muscle and pig muscle. This application of the multi-residue procedure further demonstrates the importance and wide-ranging usefulness of the technique.     

A quantitative method for the detection of sulfonamide residues in meat and milk samples with a high-performance thin-layer chromatographic method.

Sulfonamides are widely used in veterinary medicine for prophylactic purposes and for the treatment of various infections of food-producing animals. This means that residues of these drugs and their possible metabolites may occur in food of animal origin. In Belgium, a zero tolerance level for sulfonamides in edible animal tissues has been set. In order to check this zero level on a routine basis, a rapid and sensitive method has to be available. For this purpose, a quantitative high-performance thin-layer chromatographic (HPTLC) method for the detection of sulfonamide residues in animal tissue and milk samples has been developed. The sample preparation consists of a liquid extraction followed by a solid phase extraction (SPE) on disposable columns for the meat samples and a matrix solid phase dispersion (MSPD) for the milk samples. A three-multiple development chromatographic system is used for the separation and a derivatization with fluorescamine decreases the minimal detectable quantity per spot from 1.42 to 0.32 ng. The limit of quantification is 4 micrograms/kg for milk and meat samples.     

Antibiotics in food: Legislation and validation of analytical methodologies

In this paper, European food safety legislation is presented, and special attention is devoted to monitoring residues of veterinary drugs in foodstuffs of animal origin. After a short review of the state of the art of analytical methodology for antibiotic residue analysis, the paper focuses on validation of analytical methods, with Decision 657/2002/EC as reference document. Finally, the main issues of the quality control of the analytical data, i.e. analysis of reference materials and participation in proficiency tests, are briefly addressed.     

用于抗生素检测的纳米材料基电化学传感器研究进展

近年来,抗生素作为基本的治疗药物在医学、畜牧业、水产养殖业等方面被广泛应用,但其过量使用造成的抗生素残留问题也给生态环境、食品安全和人类健康造成严重的威胁.鉴于此,纳米电化学传感器在抗生素快速检测方面的研究成为热点,也取得了可观的进展.本文首先对抗生素进行了简单介绍,通过分析抗生素的电化学性质,综述了不同结构和类型的纳...     

LC/MS confirmation of ionophores in animal feeds.

A liquid chromatographic/mass spectrometric (LC/MS) electrospray confirmation method has been developed to confirm 4 ionophores (monensin, lasalocid, salinomycin, and narasin) in a variety of animal feeds using a single quadrupole mass spectrometer. The sodium ions of these compounds are dominant in the electrospray mass spectrum. Using optimized "in-source" collision induced dissociation, characteristic fragment ions seen previously using MS/MS can be observed. The drugs were extracted from the feed matrix using hexane-ethyl acetate and isolated using a silica solid-phase extraction cartridge. These ionophores were confirmed in both medicated feeds and nonmedicated feeds fortified with these drugs at the 1-50 ppm level. In addition, this method was used to confirm residues of monensin in a nonmedicated feed that was collected from a feed mill immediately after the production of a similar feed that was medicated with high levels of monensin.