Katia De Wasch

Screening and confirmation of chloramphenicol in shrimp tissue using ELISA in combination with GC-MS2 and LC-MS2

According to the European Commission Decision 2001/699/EC and 2001/705/EC certain fishery and aquaculture products, imported from China, Vietnam or Indonesia and intended for human consumption, must be subjected to a test in order to ensure the absence of chloramphenicol residues. For that reason an analytical method has been developed and validated based on ELISA for screening and gas chromatography–tandem mass spectrometry (GC–MS2) or liquid chromatography–tandem mass spectrometry (LC–MS2) for confirmation. The chloramphenicol ELISA was carried out directly on an aqueous extract of the shrimps or after an extraction with ethyl acetate. Confirmation of suspect samples was performed after extraction with ethyl acetate and defatting with n-hexane. The clean-up was based on solid phase extraction using C18 cartridges or reversed phase HPLC. After derivatisation with N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA), the final extracts were analysed by GC–MS2 in the negative ion chemical ionisation mode. Confirmation of chloramphenicol was also possible with LC–MS2 after the same clean-up. Both selective techniques made it possible to detect chloramphenicol residues at the 0.1 μg kg−1 level starting from 20 g of matrix for enzyme-linked immunosorbent assay (ELISA) with organic solvent extraction, or from 5 g of matrix for ELISA with aqueous extraction.

Detection of residues of tetracycline antibiotics in pork and chicken meat: correlation between results of screening and confirmatory tests†

Residues of the tetracycline group of antibiotics were quantified in pork and chicken muscle tissue that had previously been screened with a microbiological inhibition test and an immunological method. Pieces of frozen pork and chicken meat were screened on a pH 6 culture medium seeded with Bacillus subtilis. An aqueous extract of the inhibitor-positive samples was then screened with a group-specific commercial ELISA kit, able to detect levels of oxytetracycline, chlortetracycline, tetracycline and doxycycline corresponding with the European MRL or lower. The cut-off value of the ELISA was set at a B/B0 value of 75%. Finally, confirmation and quantification were performed using a validated HPLC method with fluorescence detection. The fluorescence was induced by complexation of the tetracyclines with the zirconium cation which is added post-column to the HPLC eluate. This fluorescence makes it possible to quantitate residues below one-half of the MRL. To gain additional qualitative information some samples were also analysed with LC-MS-MS. ELISA analysis demonstrated the presence of residues of tetracyclines in 12 out of 19 inhibitor-positive pork samples and in 19 out of 21 inhibitor-positive chicken samples. Doxycycline was detected with HPLC in 10 of these 12 pork samples and in 18 out of 19 chicken samples. The two other ELISA positive pork samples contained oxytetracycline, while no tetracyclines were found in one ELISA positive chicken meat sample. The correlation between the ELISA B/B0 values and the actual levels determined with the HPLC method was poor, whereas a better correlation was observed between the inhibition zones and the doxycycline levels. Our results indicate that an inhibition test with a medium at pH 6 and B. subtilis as test organism is well suited to screen pork and chicken muscle tissue for residues of tetracycline antibiotics. Since many positive samples contained doxycycline levels below the MRL, a confirmatory method is necessary to quantify the residues.

Trends in the identification of organic residues and contaminants: EC regulations under revision

The use of identification points (IPs) is a new approach to set up quality criteria for the identification of organic residues and contaminants: a laboratory is allowed to use any molecular spectrometric technique or combination of techniques in order to earn a minimum number of points. The system of IPs balances the identification power of the different analytical techniques and has the advantage that new techniques can be introduced very easily.

Rapid and high-performance analysis of thyreostatic drug residues in urine using gas chromatography-mass spectrometry

A more sensitive method was developed using the hyphenated technique of gas chromatography-mass spectrometry (GC-MS) supplementary to the official high-performance thin-layer chromatography (HPTLC) method. Even combined with less efficient extraction and clean-up methods, GC-MS is able to lower the detection limit to less than 50 ppb. The powerful technique of GC-MS-MS is tried out to reduce the detection limit even more, in combination with simplified extraction methods. This time-saving approach combined with the increase in sensitivity is of great importance for a routine technique.

LC-MS-MS to detect and identify four beta-agonists and quantify clenbuterol in liver†

European legislation forbids the use of beta-agonists as growth-promoting substances in cattle raised for human consumption. However, the use of beta-agonists is allowed as a therapeutic treatment of tocolysis for female cattle during calving and of respiratory diseases and tocolysis for horses not raised for human consumption. A maximum residue limit (MRL) of 0.5 microgram kg-1 for clenbuterol in the liver of cattle and horses is proposed by law. Residues of beta-agonists in liver are identified with LC-MS-MS. Using ion trap technology, it was possible to identify each analyte without the need to resolve completely the chromatographic peaks. For each analyte, specific fragment ion spectra were obtained. The coeluting or incompletely resolved peaks were separated mass spectrometrically. For tulobuterol, bromobuterol and mabuterol, qualitative information was obtained. All beta-agonists could be detected up to a concentration of 0.1 microgram kg-1. For clenbuterol, a limited quantitative validation was performed. A working range was defined for which the method was applicable. Quantification was based on the integration of the response of the analytes in spiked blank liver samples. The mean recovery was 15%. The relative standard deviation (RSD) values at different concentrations were below the maximum allowed RSD. The limit of detection of clenbuterol was 0.11 microgram kg-1. The limit of quantification was 0.21 microgram kg-1. It was possible to quantify clenbuterol below one-half of the MRL. The advantage of this method is the ease of use of the mass spectrometric separation to qualify and quantify the presence of four beta-agonists in liver.

Detection of antibiotics in muscle tissue with microbiological inhibition tests: effects of the matrix.

The effects of the tissue matrix on detection limits of antibiotics with microbiological inhibition tests, intended for muscle tissue, were measured. Pieces of frozen meat were laid directly on top of paper disks impregnated with aqueous antibiotic solutions. Inhibition zones were compared with those obtained by the same standard solution without tissue. Only tetracyclines were detected as efficiently with as without muscle tissue. Inhibition zones of the beta-lactam antibiotics ampicillin and penicillin G, and the fluoroquinolone antibiotics enrofloxacin and ciprofloxacin were smaller when muscle tissue was added to low levels of standard solution. At higher levels the differences were not substantial. Inhibition zones of tylosin were smaller and irregular or had disappeared completely, while ceftiofur, sulfadimidine, erythromycin, lincomycin, and streptomycin were not detected in spiked muscle tissue at concentrations fivefold higher than the detection limits without tissue. These results indicate that ceftiofur, sulfonamides, streptomycin and some macrolide antibiotics cannot be detected in intact meat with the plates and bacterial strains prescribed in the European Four Plate Test, a test which was initially intended as a multi-residue method for muscle tissue. Two plates of this system are not suitable for screening purposes; a third one detects tetracyclines and beta-lactam antibiotics in spiked tissue; the fourth one is sensitive for beta-lactam antibiotics and for some but not all macrolides. Samples spiked with the fluoroquinolones enrofloxacin and ciprofloxacin can be detected with an additional plate, not included in the Four Plate Test.

Detection of corticosteroids in injection sites and cocktails by MSn.

In the European Union, the use of growth promoting substances such as thyreostats, anabolics (products with estrogenic, androgenic or gestagenic action) and beta-agonists in animal fattening is forbidden. Corticosteroids, such as dexamethasone, although considered catabolic substances, have been administered to food producing animals in order to achieve mass gains. For the analysis of injection sites and of suspect cocktails (found at the farm), a number of HPTLC and HPLC methods are used. However, in injection sites and also in cocktails found at the farm, sometimes many unknown substances are found. In this investigation, a multiple mass spectrometric (MSn) method was developed. The method is based on rapid extraction of the matrix with methanol and direct infusion of the extract into the interface of the mass spectrometer. Tables that summarise the masses of corticosteroids and their possible esters are presented.

Determination of betamethasone and triamcinolone acetonide by GC-NCI-MS in excreta of treated animals and development of a fast oxidation procedure for derivatisation of corticosteroids†

The use of corticosteroids in combination with other hormonal substances has long been known to result in increased mass gain with bovines. Practice has demonstrated, however, that even the single use of a glucocorticoid may result in growth promoting effects. In addition to the popular dexamethasone, more recently other corticosteroids have also been misused for fattening purposes. The first part of this study deals with the detection of two of them, namely betamethasone and triamcinolone acetonide. Betamethasone was administered orally to a cow at a dose of 50 mg d-1 for 5 d, then later the same cow was injected intramuscularly with a dose of 50 mg of betamethasone dipropionate. Excretion in urine and faeces was followed with both HPLC-enzyme immunoassay and a previously described method based on negative chemical ionization mass spectrometry (NCI-MS) after oxidation. For the triamcinolone acetonide study a cow was treated with 50 mg d-1 of the drug during a 7 d period. Excretion in faeces was followed with GC-NCI-MS. As triamcinolone acetonide is resistant to the previously described oxidation procedure, however, a hydrolysis step had to be introduced prior to oxidation. In addition to this specific modification necessary for triamcinolone acetonide, in a subsequent part of this study the original oxidation procedure with pyridinium chlorochromate was re-investigated especially to shorten the procedure. With the introduction of potassium dichromate the reaction time could be decreased from 3 h to 10 min.

Testosterone and energy metabolism in the estuarine mysid Neomysis integer (Crustacea: Mysidacea) following exposure to endocrine disruptors

A diverse set of reference compounds suspected of having an endocrine-disrupting mode of action (i.e., testosterone, flutamide, ethinylestradiol, precocene, nonylphenol, fenoxycarb, and methoprene) were tested for acute toxicity to the estuarine mysid Neomysis integer (Crustacea: Mysidacea). Neomysis integer was very sensitive to all tested compounds, with 96-h median lethal concentrations in a narrow range between 0.32 and 1.95 mg/L. The pesticides methoprene and fenoxycarb, both synthetic insect juvenile hormone analogs, were most toxic to N. integer. In addition, the short-term sublethal effects of methoprene and nonylphenol (an estrogen agonist) on the energy and steroid metabolism of N. integer were evaluated. Both compounds significantly affected energy and testosterone metabolism of N. integer at concentrations below acute toxicity levels. Energy consumption in methoprene- and nonylphenol-exposed mysids was significantly induced at 100 microg/L, resulting in a lower cellular energy allocation in these animals. Testosterone phase I metabolism was affected at 10 microg/L, whereas glycosylation was the most important phase II pathway affected in mysids exposed to 100 microg/L of both compounds. Methoprene exposure resulted in a concentration-dependent increase in the metabolic androgenization ratio. Mysids exposed to nonylphenol at 10 microg/L had a significantly higher metabolic androgenization ratio. The present study indicates that energy and testosterone metabolism of mysids, as endpoints, are able to detect endocrine-disruptive activity of chemicals after short-term exposure to environmentally realistic levels of endocrine disruptors.

Gas chromatographic-tandem mass spectrometric analysis of clenbuterol residues in faeces

In all EU member states, the use in livestock farming of certain substances having a hormonal action is prohibited. Clenbuterol, the beta-adrenergic agonist, has some growth promoting characteristics. Screening for clenbuterol can be carried out by an immunoassay. Gas chromatography-mass spectrometry (GC-MS) is very valuable for confirmatory purposes. In full scan MS it is impossible to fulfil the EU criteria of four diagnostic ions with one single ionisation mode. Some alternative possibilities are: (1) the use of two different ionisation modes, (2) the use of different derivatization methods or (3) the use of tandem MS. Each derivatisation or ionisation mode on its own did not give a sufficient number of ions. By combining these different possibilities we were able to obtain four ions, fulfilling the EU criteria.