Search

Clenbuterol is a steroid-type drug used in respiratory treatments in both humans and animals. However, it has a secondary effect related to the hypertrophy process in muscle and fat reduction. The illegal or bad use of clenbuterol has been reported in several countries, but there is scarce information in South America, where the production and consumption of meat are considerable. In this sense, the present study aimed at evaluating the occurrence of clenbuterol in bovine muscle and liver samples from a high cattle production area of Ecuador in 2015 and 2018. For this purpose, 57-58 samples were evaluated in 2015 and 20 samples in 2018 using the Enzyme-Linked Inmuno Sorbent Assay and ultrahigh-performance liquid chromatography-tandem mass spectrometry. The results showed complained results for clenbuterol in meat samples from both years and 23% (2015) and 85% (2018) of the samples of meat complied the maximum residue level defined by CODEX.

Coccidiostats are authorized in the European Union (EU) to be used as poultry feed additives. Maximum (residue) levels (M(R)Ls) have been set within the EU for consumer and animal protection against unintended carry-over, and monitoring is compulsory. This paper describes the single-laboratory validation of a previously developed multiplex flow cytometric immunoassay (FCIA) as screening method for coccidiostats in eggs and feed and provides and compares different approaches for the calculation of the cut-off levels which are not described in detail within Commission Decision 2002/657/EC. Comparable results were obtained between the statistical (reference) approach and the rapid approaches. With the most rapid approach, the cut-off levels for narasin/salinomycin, lasalocid, diclazuril, nicarbazin (DNC) and monensin in egg, calculated as percentages of inhibition (%B/B0), were 60, 32, 76, 80 and 84, respectively. In feed, the cut-off levels for narasin/salinomycin, lasalocid, nicarbazin (DNC) and monensin were 70, 64, 72 and 78, respectively, and could not be determined for diclazuril. For all analytes, except for diclazuril in feed, the rate of false positives (false non-compliant) in blank samples was lower than 1 %, and the rate of false negatives (false compliant) at the M(R)Ls was below 5 %. Additionally, very good correlations (r ranging from 0.994 to 0.9994) were observed between two different analysers, a sophisticated flow cytometer (FlexMAP 3D(®)) and a more cost-efficient and transportable planar imaging detector (MAGPIX(®)), hence demonstrating adequate transferability.

The influence of natural organic matter on the screening of pharmaceuticals in water was determined by using high resolution liquid chromatography (HRLC) combined with full scan mass spectrometry (MS) techniques like time of flight (ToF) or Orbitrap MS. Water samples containing different amount of natural organic matter (NOM) and residues of a set of 11 pharmaceuticals were analyzed by using Exactive Orbitrap (TM) LC-MS. The samples were screened for residues of pharmaceuticals belonging to different classes like benzimidazoles, macrolides, penicillins, quinolones, sulfonamides, tetracyclines, tranquillizers, non-steroidal anti-inflammatory drugs (NSAIDs), anti-epileptics and lipid regulators. The method characteristics were established over a concentration range of 0.1-500 mu g L(-1). The 11 pharmaceuticals were added to two effluent and two influent water samples. The NOM concentration within the samples ranged from 2 to 8 mg L(-1) of dissolved organic carbon. The HRLC-Exactive Orbitrap (TM) LC-MS system was set at a resolution of 50,000 (FWHM) and this selection was found sufficient for the detection of the list of pharmaceuticals. With this resolution setting, accurate mass measurements with errors below 2 ppm were found, despite of the NOM concentration of the different types of water samples. The linearities were acceptable with correlation coefficients greater than 0.95 for 30 of the 51 measured linearities. The limit of detection varies between 0.1 mu g L(-1) and 100 mu g L(-1). It was demonstrated that sensitivity could be affected by matrix constituents in both directions of signal reduction or enhancement. Finally it was concluded that with direct shoot method used (no sample pretreatment) all compounds, were detected but LODs depend on matrix-analyte-concentration combination. No direct relation was observed between NOM concentration and method characteristics. For accurate quantification the use of internal standards and/or sample clean-up is necessary. The direct shoot method is only applicable for qualitative screening purposes. The use of full scan MS makes it possible to search for unknown contaminants. With the use of adequate software and a database containing more than 50,000 entries a tool is available to search for unknowns.

This paper describes the discrimination of eight different isomers of chloramphenicol (CAP), an antibiotic banned for use in food producing animals, by reversed phase and chiral liquid chromatography in combination with tandem mass spectrometric detection. Previously, by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) the presence of CAP was confirmed in some grass and herb samples collected on Mongolian pastures up to concentrations of 450 μg kg −1 . It was not possible to establish the cause of CAP residues which has initiated research on the natural occurrence of this drug. CAP occurs in the para -configuration and in the meta -configuration and contains two chiral centers thus eight different isomeric configurations exist, namely four (RR, SS, RS, SR) meta -stereoisomers and four para -stereoisomers. It is known that only RR- p -CAP has antimicrobial properties. To find out if the CAP detected in the plant material samples is the active configuration, a high resolution reversed phase LC-MS/MS system was tested for its ability to separate the different isomers. This system proved to be able to discriminate between some isomers, but not between RR- p -CAP and SS- p -CAP, also called dextramycin. Despite a detailed elucidation of the product ions and the fragmentation patterns of all isomers, MS/MS did not add sufficient specificity for full discrimination of the isomers. Therefore a chiral liquid chromatographic separation with MS/MS detection that is able to distinguish all isomers was developed and finally the isomeric ratio of non-compliant plant material samples and some CAP formulations was determined using this system. This showed that Mongolian grass and herb samples only contain the biological active isomer of CAP as do the obtained formulations. Therefore the CAP present in the plant material might origin from the production by soil organisms or from a manufactured source.

A proficiency test including the screening analysis of antibiotics in beef using cryogenicly minced materials was organized by RIKILT in 2009. The test included blank beef samples and beef samples spiked with either flumequine or a combination of lincomycin and spectinomycin around the maximum residue limits [1]. The suitability of the materials was demonstrated with a homogeneity and a stability study. This study showed that cryogenically minced spiked muscle material is suited for proficiency tests aiming at the screening and the confirmatory analysis. Of the 26 participants, 23 carried out their in-house screening approach involving microbial, biochemical or instrumental methods, or a combination of these to cover the broad range of antibiotic groups. The false negative rate was 73% for microbial methods, 50% for biochemical and 22% for instrumental methods. These results indicate that substantial effort is needed to improve screening approaches and that more regular proficiency tests are needed to reveal the shortcomings in the currently applied screening methods.

The analysis of antibiotics in animal faeces is important to obtain more insight in the possible formation of bacterial resistance in the animals׳ gut, to learn about the dissemination of antibiotics to the environment, to monitor trends in antibiotic usage and to detect the illegal and off-label use of antibiotics. To facilitate these studies a comprehensive method for the analysis of trace levels of 44 antibiotic compounds including tetracyclines, quinolones, macrolides and sulfonamides in animal faeces by liquid chromatography in combination with tandem mass spectrometric (LC–MS/MS) detection is reported. The method is fully validated according to European regulation and showed satisfactory quantitative performance according to the stringent criteria adopted, with the exception of some of the macrolide compounds, which can be analysed with somewhat high measurement uncertainty. A large survey was carried out monitoring swine and cattle faeces and the outcomes were striking. In 55% of the swines, originating from 80% of the swine farms and in 75% of the calves, originating from 95% of the cattle farms, antibiotics were detected. Oxytetracycline, doxycycline and sulfadiazine were the most detected antibiotics, followed by tetracycline, flumequine, lincomycin and tylosin. Over 34% of the faeces samples contained two or more different antibiotics with a maximum of eight. Possible explanations for these findings are given and the effects are discussed.

We have investigated the potential of capillary-column-switching liquid chromatography coupled to tandem mass spectrometry (cLC-MS-MS) for the quantitative on-line trace analysis of target compounds in aqueous solutions. The technical design of the nano-scale cLC system developed at our Institute for peptide and protein identification has been tested and evaluated for the direct trace analysis of drugs in water samples. Sulphametoxazole, bezafibrate, metoprolol, carbamazepine and bisoprolol occurring frequently in Dutch waters, were selected as test compounds. Adequate conditions for trapping, elution and MS-MS detection were investigated by employing laboratory made 200 microm i.d. capillary columns packed with 5 microm aqua C18 material. In the final cLC-MS-MS conditions, a 1 cm length trapping column and a 4 cm length analytical column were selected. Under these conditions, the target compounds could be directly determined in water down to a level of around 50 ng/l employing only 25 microl of water sample. Validation was done by recovery experiments in ground-, surface- and drinking-water matrices as well as by the analysis of water samples with incurred residues and previously analyzed with a conventional procedure involving off-line solid-phase extraction and narrow-bore LC with MS-MS detection. The new methodology provided recoveries (50-500 ng/l level) between 50 and 114% with RSDs (n = 3, each level) below 20% for most of the compounds. Despite the somewhat less analytical performance in comparison to the conventional procedure, the on-line approach of the new methodology is very suitable for screening of drugs in aqueous samples.

Due to the unexpected findings of the banned antibiotic chloramphenicol in products of animal origin, feed, and straw, the hypothesis was studied that the drug is naturally present in soil, through production by soil bacteria, and subsequently can be transferred to crops. First, the stability of chloramphenicol in soil was studied. The fate of chloramphenicol highly depends on soil type and showed a half-life of approximately one day in nonsterile topsoil. It was found to be more stable in subsoil and sterile soils. Second, the production of chloramphenicol in soil was studied, and it was confirmed that Streptomyces venezuelae can produce chloramphenicol at appreciable amounts in nonsterile soil. Third, a transfer study was carried out using wheat and maize grown on three different soils that were weekly exposed to aqueous chloramphenicol solutions at different levels. Chloramphenicol was taken up by crops as determined by chiral liquid chromatography coupled to tandem mass spectrometric analysis, and the levels in crops were found to be bioavailability related. It was concluded that chloramphenicol residues can occur naturally in crops as a result of the production of chloramphenicol by soil bacteria in their natural environment and subsequent uptake by crops.

Commission Decision (CD) 2002/657/EC describes detailed rules for method validation within the framework of residue monitoring programmes. The approach described in this CD is based on criteria. For (qualitative) screening methods, the most important criteria is that the CCβ has to be below any regulatory limit. Especially when microbiological or immunochemical methods are involved, the approach described in the CD is not easily applied. For example, by those methods, a large number of analytes (all antibiotics) within several different matrices (meat, milk, fish, eggs, etc.) are detected. It is not completely clear whether all those analytes and all matrices have to be taken into account during method validation. To clarify this, a working group - from EU Reference Laboratories - came up with a practical approach to validate multi-analyte multi-matrix screening methods. It describes how many analyte/matrix combinations have to be tested and how these combinations are selected. Furthermore it describes how to determine CCβ for screening methods in relation to a large list of compounds and maximum residue limits (MRLs). First for each analyte/matrix combination the 'cut-off' level - i.e. the level at which the method separates blanks from contaminated samples - is established. The validation is preferably at the concentration of 50% of the regulatory limit. A minimum set of 20 different samples has to be tested. From the experiences with applying these guidelines it was concluded that the validation approach is very 'practical'; however, there are some remarks. One has to be careful with selecting 'representative' analytes and matrices and it is strongly recommended to collect additional validation data during the routine application of the method.

We developed a procedure to determine the "identification power" of an LC-MS/MS method operated in the MRM acquisition mode, which is related to its selectivity. The probability of any compound showing the same precursor ion, product ions, and retention time as the compound of interest is used as a measure of selectivity. This is calculated based upon empirical models constructed from three very large compound databases. Based upon the final probability estimation, additional measures to assure unambiguous identification can be taken, like the selection of different or additional product ions. The reported procedure in combination with criteria for relative ion abundances results in a powerful technique to determine the (un)certainty of the selectivity of any LC-MS/MS analysis and thus the risk of false positive results. Furthermore, the procedure is very useful as a tool to validate method selectivity. Figure

The use of cephalosporin antibiotics in veterinary practice is likely to play an important role in the development of ß-lactam-resistant bacteria. To detect off-label cephalosporin antibiotic usage, an analytical method is needed that, besides the native compound, also detects their active metabolites. In this paper, the applicability of three approaches for the quantitative analysis of ceftiofur using LC–MS/MS is assessed, viz. (A) analysis of ceftiofur, desfuroylceftiofur and/or desfuroylceftiofur cystein disulfide, (B) derivatisation of ceftiofur metabolites to desfuroylceftiofur acetamide and (C) chemical hydrolysis using ammonia, to produce a marker compound for ceftiofur. We found that approach A was not suited for quantitative analysis of total ceftiofur concentration or for effectively detecting off-label use of ceftiofur. Approach B resulted in adequate quantitative results, but was considered a single compound method because it depends on cleavage of a thioester group, which is present in only a limited number of cephalosporin antibiotics. Approach C showed adequate quantitative results but, in contrast to approach B, it is applicable to a range of cephalosporin antibiotics. Therefore, it is applicable as a broad quantitative screening of cephalosporin compounds in poultry tissue samples to indicate off-label use of cephalosporins in poultry breeding. Based on this study, it was concluded that approach C is the most suitable to detect off-label use of a range of cephalosporin antibiotics.

Chloramphenicol is a broad-spectrum antibiotic with, apart from its human medicinal use, veterinary abuse in all major food-producing animals. Chloramphenicol occurs in four stereoisomers (all para-nitro substituted) and furthermore four meta-nitro analogs of chloramphenicol exist. In this paper these are referred to as eight chloramphenicol isomers. According to EU regulations an analytical method should be able to discriminate the analyte from interfering substances that might be present in the sample, including isomers. For the first time a quantitative method for the analysis of trace levels of eight chloramphenicol isomers in urine by chiral liquid chromatography in combination with tandem mass spectrometric detection is reported. The separation of the isomers on the analytical column, the clean-up of urine and the selectivity of the monitored product ions turned out to be critical parameters. To obtain reproducible retention isocratic elution on a chiral AGP column was applied. For urine samples matrix compounds present in the final extract caused decreased retention of the isomers on the chiral stationary phase and a lack of chromatographic resolution. Therefore an extended clean-up procedure that combines solid phase extraction and liquid–liquid extraction had to be developed. The final method was fully validated and showed satisfactory performance for all isomers with decision limits (CCa) ranging from 0.005 to 0.03 µg L-1 and within-laboratory reproducibility of all isomers below 20% at the minimum required performance limit level of 0.3 µg L-1.

This study reports the findings of a supplement marketed on the Internet for prostate problems. The supplement was orally taken by a 60-year-old man with divergent hormonal levels and who was surgically treated for gynaecomastia: development of abnormally large mammary glands in males. The supplement showed a strong effect in a yeast oestrogen bioassay, expressing a yeast-enhanced green fluorescent protein (yEGFP) upon exposure to oestrogens. Using both nuclear magnetic resonance (NMR) and a gradient liquid chromatographic time-of-flight mass spectrometric (LC/TOF-MS) method, the response was shown to be caused by very high levels of diethylstilbestrol, known for causing gynaecomastia. The gynaecomastia was most probably caused by this orally taken 'natural' herbal supplement, as the patient's hormonal levels also returned to normal again when stopping the use of it. This case demonstrates that physicians need to be aware of the use of supplements with illegal components that may be responsible for unwanted side-effects.

We have investigated the potential of capillary-column-switching liquid chromatography coupled to tandem mass spectrometry (cLC-MS-MS) for the quantitative on-line trace analysis of target compounds in aqueous solutions. The technical design of the nano-scale cLC system developed at our Institute for peptide and protein identification has been tested and evaluated for the direct trace analysis of drugs in water samples. Sulphametoxazole, bezafibrate, metoprolol, carbamazepine and bisoprolol occurring frequently in Dutch waters, were selected as test compounds. Adequate conditions for trapping, elution and MS-MS detection were investigated by employing laboratory made 200 microm i.d. capillary columns packed with 5 microm aqua C18 material. In the final cLC-MS-MS conditions, a 1 cm length trapping column and a 4 cm length analytical column were selected. Under these conditions, the target compounds could be directly determined in water down to a level of around 50 ng/l employing only 25 microl of water sample. Validation was done by recovery experiments in ground-, surface- and drinking-water matrices as well as by the analysis of water samples with incurred residues and previously analyzed with a conventional procedure involving off-line solid-phase extraction and narrow-bore LC with MS-MS detection. The new methodology provided recoveries (50-500 ng/l level) between 50 and 114% with RSDs (n = 3, each level) below 20% for most of the compounds. Despite the somewhat less analytical performance in comparison to the conventional procedure, the on-line approach of the new methodology is very suitable for screening of drugs in aqueous samples.

Chloramphenicol (CAP), a broad-spectrum antibiotic, was detected in several herb and grass samples from different geographic origins. Due to its suspected carcinogenicity and linkages with the development of aplastic anemia in humans, CAP is banned for use in food-producing animals in the European Union (EU) and many other countries. However, products of animal origin originating from Asian countries entering the European market are still found noncompliant (containing CAP) on a regular basis, even when there is no history of chloramphenicol use in these countries. A possible explanation for the continued detection of these residues is the natural occurrence of CAP in plant material which is used as animal feed, with the consequent transfer of the substance to the animal tissues. Approximately 110 samples were analyzed using liquid chromatography coupled with mass spectrometric detection. In 26 samples, the presence of CAP was confirmed using the criteria for banned substances defined by the EU. Among other plant materials, samples of the Artemisia family retrieved from Mongolia and from Utah, USA, and a therapeutic herb mixture obtained from local stores in the Netherlands proved to contain CAP at levels ranging from 0.1 to 450 microg/kg. These findings may have a major impact in relation to international trade and safety to the consumer. The results of this study demonstrate that noncompliant findings in animal-derived food products may in part be due to the natural occurrence of chloramphenicol in plant material. This has implications for the application of current EU, USA, and other legislation and the interpretation of analytical results with respect to the consideration of CAP as a xenobiotic veterinary drug residue and the regulatory actions taken upon its detection in food.

In residue analysis of antibiotics quantitative and qualitative aspects are involved in declaring a sample non-compliant. The quantitative aspect regards the determination of the amount of the compound present in the sample. Validation procedures are available to determine the uncertainty of this result, which is taken into account in the decision making process. The qualitative aspect regards the confirmation of the identity of the compound present. In this, selectivity is the main parameter which is defined as the ability of a method to discriminate the analyte being measured from other substances. A trend observed in residue analysis is towards more generic methods for the detection of a broad range of compounds in a single run. As a result, by definition, selectivity is compromised. Procedures to determine the uncertainty of the qualitative aspect are lacking and, as a result, whether or not a method is adequately selective is a matter of experts’ judgment. In this thesis a method is presented for grading selectivity of methods using liquid chromatography coupled to tandem mass spectrometry. Based on the outcome it can be stated if selectivity is adequate and thus if a confirmatory result stands strong when challenged in a court case. If selectivity is found inadequate, additional measures can be taken like the selection of another product ion or the use of a third product ion to obtain adequate selectivity. Furthermore, two examples of analyses are presented in which selectivity plays an important role. First, the analysis of the banned antibiotic chloramphenicol (CAP). CAP contains two chiral centers and the nitro-group can either be para- or meta-substituted. Therefore, eight different isomers of CAP occur of which only RR-p-CAP is antimicrobially active. In the analysis of CAP, extreme selectivity is needed to distinguish the antimicrobially active compound from its inactive isomers. A method applying chiral liquid chromatography with tandem mass spectrometry was developed to discriminate antimicrobially active CAP for its inactive isomers. Also the research for the possible natural occurrence of this drug is presented. It is shown that CAP can be produced in unamended soil by Streptomyces venezuelaein appreciable amounts and that crops can take up CAP from soils. Therefore, it is concluded that CAP can occur in crops and animal feed due to its natural production by soil bacteria. Second, the development of a multi-ß-lactam method is presented. In this method a derivatization is applied to be able to effectively detect off-label ceftiofur use. In this selectivity is intentionally compromised and no unequivocal confirmation can be carried out using this method. The developed method is applicable to a wide range of ß-lactam antibiotics including penicillins, cephalosporins and carbapenems and is the best method available today for effective monitoring of off-label ß-lactam usage in poultry breeding.