Analytical aspects of veterinary drug residues

A survey is given with respect to recent developments in the analysis of veterinary drug residues in food of animal origin. The application of solidphase extraction, immunoaffinity and matrix solid-phase dispersion as rapid clean-up and concentration techniques is discussed. Attention is paid as well to automated methods such as FAST-LC and immunoassays.     

Development of fast screening methods for the analysis of veterinary drug residues in milk by liquid chromatography-triple quadrupole mass spectrometry

Two rapid multi-residue screening methods for the determination of 21 veterinary drugs in milk by ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) have been developed and compared. For both methods, veterinary drugs were extracted from milk samples using a rapid extraction procedure based on the modification of the well-known QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) method, and no further clean-up steps were necessary. One screening method was based on the selection of a characteristic neutral loss or product ion of the various families of compounds, whereas another one was based on the choice of a selected reaction monitoring (SRM) for each compound. These methods were compared with regards to false negatives, cut-off values and the unreliability region. The total run time for both methods was 3 min, allowing quick selection of samples that contained veterinary drugs. Non-negative samples were re-analyzed by the UHPLC-MS/MS confirmation/quantification method, which consisted in the monitoring of two SRM for each compound. The methods were validated according to international guides. The proposed analytical methods allow for the identification and confirmation of the target veterinary drugs at trace levels employing quick analysis time.     

Immunochemical screening for antimicrobial drug residues in commercial honey.

Honey samples (n = 100; origin: various countries from Eurasia, Oceania, and the Americas) were analysed by enzyme immunoassays (EIA) for tetracyclines, streptomycin, and sulfathiazole. Considering antibody specificity, these EIAs are either quantitative (streptomycin) or qualitative (tetracyclines, sulfathiazole) tests. Honey extract purification was achieved by liquid-liquid partition (tetracyclines), and by solid phase extraction-immunoaffinity chromatography (streptomycin, sulfathiazole). Detection limits were 20 micrograms kg-1 (tetracycline equivalents), 10 micrograms kg-1 (streptomycin), and 50 micrograms kg-1 (sulfathiazole equivalents), with mean recoveries of 100-117%. A total of 42% of the samples was found positive by EIA; 25% were positive in one assay, 13% in two, and 3% were positive in all three tests. In the EIA for tetracyclines, 26% were positive, with 12 samples exceeding a level of 50 micrograms kg-1 (tetracycline equivalents). In the EIA for streptomycin, 19% were positive, with a mean concentration of 19 +/- 12 micrograms kg-1. In the sulfathiazole EIA, 16% of the samples were positive, with 13 samples exceeding a level of 100 micrograms kg-1 (sulfathiazole equivalents). However, when samples which were positive in the sulfathiazole EIA were reanalysed for sulfonamides by HPLC, no sulfa drugs could be detected. Experimental heating (40 degrees C) of honey spiked with sulfathiazole indicated that the sulfa drug(s) responsible for positive EIA results could be present a sugar derivatives.     

Different approaches to legal requirements on validation of test methods for official control of foodstuffs and of veterinary drug residues and element contaminants in the EC

 In order to ensure food consumer protection as well as to avoid barriers to trade and unnecessary duplications of laboratory tests and to gain mutual recognition of results of analyses, the quality of laboratories and test results has to be guaranteed. For this purpose, the EC Council and the Commission have introducedprovisions – on measures for quality assurance for official laboratories concerning the analyses of foodstuffs on the one hand and animals and fresh meat on the other, – on the validation of test methods to obtain results of sufficient accuracy. This article deals with legal requirements in the European Union on basic principles of laboratory quality assurance for official notification to the EC Commission and on method validation concerning official laboratories. Widespread discussions and activities on measurement uncertainty are in progress, and the European validation standards for official purposes may serve as a basis for world-wide efforts on quality harmonization of analytical results. Although much time has already been spent, definitions and requirements have to be revised and further additions have to be made.     

High-throughput screening for multi-class veterinary drug residues in animal muscle using liquid chromatography/tandem mass spectrometry with on-line solid-phase extraction

A rapid qualitative method using on-line column-switching liquid chromatography/tandem mass spectrometry (LC/MS/MS) was developed and validated for screening 13 target veterinary drugs: four macrolides - erythromycin A, josamycin (leucomycin A3), kitasamycin (leucomycin A5), and tylosin A; six (fluoro)quinolones - ciprofloxacin, danofloxacin, enrofloxacin, flumequine, oxolinic acid, and sarafloxacin; and lincomycin, virginiamycin M1, and trimethoprim in different animal muscles. Clindamycin, norfloxacin, nalidixic acid, oleandomycin, ormetoprim, and roxithromycin were used as the internal standards. After simple deproteination and analyte extraction of muscle samples using acetonitrile, the supernatant was subjected to on-line cleanup and direct analysis by LC/MS/MS. On-line cleanup with an extraction cartridge packed with hydrophilic-hydrophobic polymer sorbent followed by fast LC using a short C18 column resulted in a total analysis cycle of 6 min for 19 drugs. This screening method considerably reduced the time and the cost for the quantitative and confirmatory analyses. The application of a control point approach was also introduced and explained.     

固相萃取高效液相色谱法测定乳粉中苯并咪唑类药物残留

研究了乳粉中丙硫咪唑、噻苯咪唑、苯硫咪唑、噻苯咪唑酯、苯亚砜苯咪唑等五种苯并咪唑类药物液相色谱测定方法。乳粉中苯并咪唑类药物残留经碱性乙酸乙酯提取,Waters Oasis HLB固相萃取柱净化,以Cloversil C18柱为固定相,甲醇和0．02mol／LKH2P04为流动相,紫外检测器下测定。该法检出限为0．020mg／kg,添加标准0．10mg／kg的回收率为71％-90％。     

A review on the determination of veterinary drug residues in food products

In this paper, we discuss veterinary medicine and its applications in the food industry as well as the risk to the health of humans and animals caused by these residues. We review how the veterinary residues enter and cause some detrimental effects. We also mention two techniques to determine the residue of veterinary medications that exist in food originating from animals, including classic and advanced techniques. Finally, we discuss the potential of various developed methods and compare them with some traditional techniques.     

Validation study on avermectine residues in foodstuffs

Avermectines are antiparasitic agents widely used as veterinary drugs for food producing animals. The European Community, due to their side effects, limited the use of these molecules establishing maximum residue limits (MRLs) in some foods. A validated qualitative and quantitative high performance liquid chromatography method with fluorescence detection (HPLC-FL) is presented for the simultaneous determination of ivermectin (IVM), abemectin (ABA), moxidectin (MOX), eprinomectin (EPR), doramectin (DOR) and emamectin (EMA) in foodstuffs (muscle, eggs and milk). Samples were extracted with acetonitrile, purified with liquid-liquid extraction (LLE), and analysed by HLPC-FL previous derivatization with trifluoroacetic anhydride (TFAA) in presence of 1-methyl-imidazole (MI) and acetic acid. To date, the presented method is the first validated for the matrix eggs, and in accordance with the requirements set by Commission Decision 2002/657/EC. Recoveries of the methods, calculated spiking the samples in the range 5.0-100.0 μg kg(-1), were 64-83% for muscle, 65-89% for milk and 63-84% for eggs. The precision (CV) ranged between 9.2 and 17.1% for muscle, 9.9 and 16.6% for milk and from 9.4 to 17.4% for eggs. Linearity for the six analytes was calculated from 5.0 to 200.0 μg kg(-1). The main advantages of the presented method are its rapidity, the specificity, the good precision and recovery that make it very suitable to the detection and determination of avermectines.     

免疫亲和柱在兽药残留检测应用中的研究进展

免疫亲和柱(IAC)是一种有效的兽药残留检测净化技术,可以简化样品净化过程并且提高待测物的提取效率,近几年在兽药残留检测中得到广泛应用,并表现出良好的发展前景。本文简要叙述了IAC的原理、制备过程以及在各种抗微生物类兽药检测中的应用,就IAC对目前已研制出的抗微生物药的残留检测及净化效果进行综述,并展望了IAC在未来兽药残留检测应用中的发展趋势,为研究者提供参考和研发思路。     

Effect of cooking on veterinary drug residues in food. Part 9. Nitroimidazoles

Most food containing drug residues is consumed after cooking or processing, yet surveillance for these residues is almost always conducted on raw tissue. This investigation was to establish the effect of cooking on residues of the nitroimidazole drugs dimetridazole and ronidazole in chicken muscle and egg, to enable dietary intake calculations based on surveillance results. In model aqueous and lipid solutions, dimetridazole and its 2-hydroxy metabolite, which is usually found when residues are present, were relatively stable for times and temperatures normally encountered during standard cooking methods. Ronidazole in hot aqueous solutions, except at acidic pH, was converted into the 2-hydroxy metabolite. Chicken meat and eggs from birds treated with dimetridazole or ronidazole were used to investigate the effects of cooking on food containing these residues. It was apparent that these residues were not destroyed by cooking chicken meat although some of the residue leached from the sample with juices which exuded as it cooked. Residue concentration in egg reduced during cooking by between 14% and 32% of the original concentration.     

Determination of veterinary drug residues in chicken meat using corona discharge ion mobility spectrometry

A positive corona discharge ion mobility spectrometry (CD-IMS) has been evaluated for the determination of three residual veterinary drugs including furazolidone (FUR), chloramphenicol (CAP), and enrofloxacin (ENR) in poultry for the first time. Pretreatment included extraction of the drugs from samples and further treatment of the extracts by solid phase extraction (SPE) using C₁₈ sorbents. The limits of quantification (LOQs) were less than 20 μg kg⁻¹ for all compounds. The calibration plots for these compounds were linear to about three orders of magnitude. The validity of the method was demonstrated by the analysis of spiked and real samples.     

Regulatory problems caused by contamination, a frequently overlooked cause of veterinary drug residues

The occurrence of violative residues of veterinary medicines and other, unauthorised, drugs in food of animal origin is an issue of popular concern within the European Union. Violations can occur as a result of improper use of a licensed product or through the illegal use of an unlicensed substance. However, a "violative" analytical result does not necessarily mean that abuse has occurred. Contamination of animal feedingstuffs, environmental contamination and animal-to-animal transfer of drugs can also cause residue violations. This paper reviews these inadvertent causes of residues violations in food, and includes data generated using chromatographic and non-chromatographic methods of analysis.     

Development of a multi-class method for the quantification of veterinary drug residues in feedingstuffs by liquid chromatography-tandem mass spectrometry

A simple multi-residue method was developed for detecting and quantifying 33 analytes from 13 classes of antibiotics (tetracyclines (3), quinolones (7), penicillins (3), ionophore coccidiostats (7), macrolides (3), sulfonamides (1), quinoxalines (2), phenicols (2), lincosamides (1), diaminopyrimidines (1), polypeptides (1), streptogramins (1) and pleuromutilins (1)) in animal feeds. Extraction and clean-up procedures were optimized with spiked piglet feed. Samples were extracted by ultrasonic-assisted extraction with a mixture of methanol/acetonitrile/McIlvaine buffer at pH 4.6 (37.5/37.5/25, v/v/v) containing 0.3% of EDTA-Na₂, followed by a clean up using a dispersive solid-phase extraction (d-SPE) with PSA (primary secondary amine). Detection of antibiotics was achieved by liquid chromatography-electrospray tandem mass spectrometry (LC–ESI-MS/MS) within 28 min using both positive and negative ESI mode. Average recoveries ranged from 51% (oxytetracycline) to 116% (tilmicosin) with associated relative standard deviations of 7.3% and 9.0% and an overall mean of 87%. Limits of quantification ranged from 3.8 ng g⁻¹ (lincomycin) to 65.0 ng g⁻¹ (bacitracin). Following optimization, the method was further verified for bovine and lamb feedingstuffs; negative matrix effects were evaluated and overcome by a standard addition method.     

Determination of pesticides and veterinary drug residues in food by liquid chromatography-mass spectrometry: A review

Monitoring of pesticides and veterinary drug residues is required to enforce legislation and guarantee food safety. Liquid chromatography-mass spectrometry (LC-MS) is the prevailing technique for assessing both types of residues because LC offers a versatile and universal separation mechanism suitable for non-gas chromatography (GC) amenable and the majority of GC-amenable compounds. This characteristic becomes more relevant when LC is coupled to MS because the high sensitivity and specificity of the detector allows to apply generic sample preparation procedures, which simultaneously extract a wide variety of residues with different physico-chemical properties. Determination of metabolites and degradation products, non-target suspected screening of an increasing number of residues, and even unknowns identification are also becoming inherent LC-MS advantages thanks to the latest advances. For routine analysis and, in particular, for official surveillance purposes in food control, analytical methods properly validated following strict guidelines are needed. After a brief introduction and an outline of the legislation applicable around the world, aspects such as improvement of specificity of high-throughput methods, resolution and mass accuracy of identification strategies and quantitative accuracy are critically reviewed in this article. In them, extraction, separation and determination are emphasized. The main objective is to offer an assessment of the state of the art and identify research needs and future trends in determining pesticide and veterinary drug residues in food by LC-MS.     

Biosensor assay of sulfadiazine and sulfamethazine residues in pork

Biosensor-based immunochemical screening assays for the detection of sulfadiazine (SDZ) and sulfamethazine (SMT) in muscle extract from pigs were developed. Samples were extracted with aqueous buffer and then centrifuged. This simple and straightforward preparation allowed up to 40 samples to be processed and analysed in 1 d. The limits of detection for the assays were found to be 5.6 ng g-1 for SDZ and 7.4 ng g-1 for SMT. These figures were well below the European and US legal limits for sulfonamides (100 ng g-1). The precision (RSD) between runs was < 8% and the recovery was between 91 and 98%. The validation proved the assays to be accurate and the analysis of routine field samples showed good correlation with an established TLC screening procedure. No false negative or positive results were obtained with blank and spiked samples. The influence of cross-reacting metabolites on immunoassays was demonstrated by testing incurred tissue samples, collected from sulfonamide treated pigs after only a short withdrawal period. The quantitative results obtained by biosensor analysis were a combination of parent sulfonamide plus N4-acetyl metabolite while the HPLC method used for confirmatory analysis detected only the parent sulfonamide. This gave rise to some false positive results and highlighted the need to use real samples in evaluating any assay thoroughly. False negative results were not obtained.     

Evaluation of an immunobiosensor for the on-site testing of veterinary drug residues at an abattoir. Screening for sulfamethazine in pigs

A study was conducted to determine the feasibility of performing "on-site" screening for sulfamethazine (SMT), at an abattoir, using a rapid immunobiosensor method. This involved transfer of the biosensor technology and an assay developed in the laboratory, to the cold, humid conditions of a modern pig-processing factory. A pre-determined threshold limit of 0.4 microgram ml-1 SMT in bile was used to identify the likelihood that corresponding tissue samples contained SMT concentrations in excess of the European maximum permissible residue limit of 0.1 mg kg-1. Bile samples containing SMT concentrations above the threshold limit were deemed positive and the corresponding kidney and muscle samples were sent to the laboratory for HPLC analysis. The robustness of the biosensor instrumentation in the harsh operating conditions was monitored throughout the project. The performance of the assay, on-site, was assessed by the regular inclusion of QA samples and by the submission of control 'SMT-positive' pigs to the abattoir. Sampling procedures, identification and traceability were also under scrutiny. During the project, 337 (9.35%) of the total kill were tested for SMT residues, representing 75% of all producers submitting pigs for slaughter. Twelve animals, including the ten controls, gave positive bile results. HPLC analysis confirmed SMT residues in all 12 kidneys (11 in excess of the permissible level). Ten muscle samples also contained violative SMT levels. Throughout the project, the biosensor performed reliably, with no adverse reaction of any mechanical or electrical components. The SMT assay also performed reliably. This is the first report of a biosensor being used for 'on-site' drug screening.     

超高效液相色谱-四极杆飞行时间质谱法测定克氏螯虾中39种兽药残留

建立了超高效液相色谱-四极杆飞行时间质谱（UHPLC-Q-TOF-MS）快速筛查克氏螯虾中39种兽药残留的分析方法。样品经酸化乙腈提取,增强型脂质去除净化剂（EMR-Lipid）结合石墨化多壁碳纳米管（GMWNTs）净化,采用UHPLC-Q-TOF-MS分析检测。通过比对待测物准分子离子的精确质量数、同位素丰度比、保留时间等特征信息进行定性初筛,通过二级谱图谱库检索比对进行确证,通过一级提取离子色谱图峰面积进行定量分析。39种化合物在各自的浓度范围内均呈良好的线性关系,线性相关系数均大于0.99,定量限为3~15 μg/kg,添加回收率为62.4%~105.8%,相对标准偏差（RSD）为2.5%~13.5%,方法简便、快速、准确,可应用于克氏螯虾中喹诺酮类、磺胺类、三苯甲烷类39种药物残留的快速筛查定量。