Dye residues in aquaculture products: Targeted and metabolomics mass spectrometric approaches to track their abuse.

The aim of the study was to initiate an exhaustive strategy of control by implementing both targeted and non-targeted metabolomics approaches. A LC-MS/MS method including an oxidative step for most of dyes was developed and validated to target the analysis of 14 residues belonging to different families of dyes. The method was suitable for the quantitative confirmation of 13 dyes at low ppb levels. The metabolomics approach objective was to compare fingerprints between farmed fish treated with malachite green and farmed fish treated with victoria pure blue bo. Analytical information on modifications in the metabolome of muscle, liver and plasma was exploited by HRMS following by multivariate statistics and revealed some direct or endogenous metabolites among relevant mass features contributing to the constructed models. These two approaches, either appropriate biomarkers either enlarged targeted dyes are explored concomitantly to help improving the strategy for tracking new illegal practices in aquaculture.

Evaluation and validation of two microbiological tests for screening antibiotic residues in honey according to the European guideline for the validation of screening methods.

Two microbiological kits based on Bacillus stearothermophilus (Eclipse 50® and Premi®Test) have been evaluated and validated according to the European guideline for the validation of screening methods (January 2010) and in relation to the concentrations recommended by the EU-RL in 2007. Both tests are robust, a fast method and easy to implement. Both tests are applicable to a very large variety of honeys from different floral and geographical origins (rosemary, lavender, scrub, heath, alder, forest, lemon, acacia, chestnut, raspberry, mountain and flowers) as well as honey of different colours (from blank honey to brown honey, including yellow and orange honey). A satisfactory false-positive rate of 5% was obtained for the Eclipse 50® test. The observed detection capabilities CCß of the Eclipse 50® kit were: chlortetracycline (>75 µg kg(-1)), oxytetracycline (≤200 µg kg(-1)), tetracycline (>100 µg kg(-1)), cloxacillin (≤40 µg kg(-1)), tylosin (≤200 µg kg(-1)), desmycosin (>400 µg kg(-1)), sulfadiazine (≤300 µg kg(-1)), sulfadimethoxine (≤250 µg kg(-1)), sulfamerazine (>300 µg kg(-1)), sulfamethazine (>1000 µg kg(-1)), sulfamethizole (>75 µg kg(-1)), sulfamethoxazole (≤25 µg kg(-1)), sulfanilamide (>>1000 µg kg(-1)), sulfaquinoxaline (>75 µg kg(-1)), sulfathiazole (≤250 µg kg(-1)) and lincomycin (>1500 µg kg(-1)). These levels were all higher than the recommended concentrations where they exist. Due to its lack of sensitivity, it cannot be recommended for reliable routine use. The observed CCß of the Premi®Test kit were: chlortetracycline (10 µg kg(-1)), oxytetracycline (>10 µg kg(-1)), tetracycline (≤10 µg kg(-1)), cloxacillin (≤5 µg kg(-1)), tylosin (≤10 µg kg(-1)), desmycosin (≤15 µg kg(-1)), sulfadiazine (≤25 µg kg(-1)), sulfadimethoxine (≤25 µg kg(-1)), sulfamerazine (≤25 µg kg(-1)), sulfamethazine (≤25 µg kg(-1)), sulfamethizole (≤25 µg kg(-1)), sulfamethoxazole (≤10 µg kg(-1)), sulfanilamide (≤25 µg kg(-1)), sulfaquinoxaline (≤10 µg kg(-1)), sulfathiazole (25 µg kg(-1)) and lincomycin (≤25 µg kg(-1)). The Premi®Test kit could be recommended for reliable use in routine control due to its low detection capabilities (except for aminoglycosides), but the disadvantage is a high false-positive rate of 14%.

Validation of a commercial receptor kit Sulfasensor Honey for the screening of sulfonamides in honey according to Commission Decision 2002/657/EC.

The Sulfasensor Honey kit is a receptor test dedicated to the screening of sulphonamide residues respectively in different matrices. The aim of this project was to evaluate and validate this kit according to the Community Reference Laboratory (CRL) guideline for the validation of screening methods to achieve the French control plan for honey. The test is robust, quick (90 min for 40 samples), easy to perform and easy to read. The false-positive rate was estimated to be 12.5%. The detection capabilities CCß of the kit were lower than or equal to 25 µg kg(-1) for sulfamethazine, sulfamerazine, sulfathiazole and sulfapyridine, and between 25 and 50 µg kg(-1) for sulfadiazine and sulfadimethoxine, 150 µg kg(-1) for sulfaquinoxaline, and 1000 µg kg(-1) for sulfamethoxazole and sulfamethizole. Sulfanilamide was not detected by the kit. The kit was applicable to a wide variety of honeys (different floral and geographical origins, liquid or solid). This kit was used to implement the French control plan for the detection of antibiotic residues in honey in 2010 in parallel with an HPLC method. However, in 2011 the kit was replaced by an LC-MS/MS method for the screening and confirmation of sulfonamide residues in honey, which detects all the sulfonamides of interest.

Development of a new screening method for the detection of antibiotic residues in muscle tissues using liquid chromatography and high resolution mass spectrometry with a LC-LTQ-Orbitrap instrument.

A liquid chromatography-high resolution mass spectrometry (LC-HRMS) method was developed for screening meat for a wide range of antibiotics used in veterinary medicine. Full-scan mode under high resolution mass spectral conditions using an LTQ-Orbitrap mass spectrometer with resolving power 60,000 full width at half maximum (FWHM) was applied for analysis of the samples. Samples were prepared using two extraction protocols prior to LC-HRMS analysis. The scope of the method focuses on screening the following main families of antibacterial veterinary drugs: penicillins, cephalosporins, sulfonamides, macrolides, tetracyclines, aminoglucosides and quinolones. Compounds were successfully identified in spiked samples from their accurate mass and LC retention times from the acquired full-scan chromatogram. Automated data processing using ToxId software allowed rapid treatment of the data. Analyses of muscle tissues from real samples collected from antibiotic-treated animals was carried out using the above methodology and antibiotic residues were identified unambiguously. Further analysis of the data for real samples allowed the identification of the targeted antibiotic residues but also non-targeted compounds, such as some of their metabolites.

Liquid chromatography-tandem mass spectrometry method for the determination of dye residues in aquaculture products: development and validation.

A method is described for the identification and the quantitative determination of the triphenylmethane dyes, malachite green (MG), crystal violet (CV), brilliant green (BG) and leuco malachite green (LMG) and leuco crystal violet (LCV). The analytes were isolated from the matrix by liquid-liquid extraction with acetonitrile. Determination was performed using LC-MS/MS with positive electrospray ionisation. 4 different deuterated internal standards were introduced to improve the quantitative performance of the method. The method has been validated in line with the EU criteria of Commission Decision 2002/657/EC in accordance with the minimum required performance limit (MRPL) set at 2 µgkg(-1) for the sum of MG and LMG. For all the monitored compounds, accuracy, intra-day and inter-day precision were determined at each level of fortification (0.5, 0.75, 1.0 and 2.0 µgkg(-1)). Decision limits CCα and detection capabilities CCß were calculated according to the standard ISO 11843-2. A study on the applicability of the method was conducted on various aquacultured species with the aim to assess the matrix effects. The presence of residues of leuco brilliant green in fish has also been confirmed from experimental study performed on trout treated with brilliant green, using LTQ-Orbitrap mass spectrometer.

Validation of a Five Plate Test, the STAR protocol, for the screening of antibiotic residues in muscle from different animal species according to European Decision 2002/657/EC.

The STAR protocol is a Five Plate Test (FPT) developed several years ago at the Community Reference Laboratory (CRL) for the screening of antimicrobial residues in milk and muscle. This paper presents the validation of this method according to European Decision 2002/657/EC and to an internal guideline for validation. A validation protocol based on 'simulated tissues' and on a list of 16 representative antimicrobials to be validated was implemented in our laboratory during several months for the STAR protocol. The performance characteristics of the method were determined (specificity, detection capabilities CCbeta, applicability, ruggedness). In conclusion, the STAR protocol is applicable to the broad-spectrum detection of antibiotic residues in muscles of different animal species (pig, cattle, sheep, poultry). The method has good specificity (false-positive rate = 4%). The detection capabilities were determined for 16 antibiotics from different families in relation to their respective maximum residue limit (MRL): beta-lactams (penicillins and cephalosporins < or = MRL), tetracyclines (< or = MRL and < or = 2.5 MRL), macrolides (2 MRL), quinolones (< or = 2 MRL), some sulphonamides (< or = 3 MRL), and trimethoprim (2 MRL). However, the sensitivity of the STAR protocol towards aminoglycosides (> 8 MRL) and florfenicol (< or = 10 MRL) was unsatisfactory (>>MRL). The two objectives of this study were met: firstly, to validate the STAR protocol according to European Decision 2002/657/EC, then to demonstrate that the validation guideline developed to implement this decision is applicable to microbiological plate tests even for muscle. The use of simulated tissue appeared a good compromise between spiked discs with antibiotic solutions and incurred tissues. In addition, the choice of a list of representative antibiotics allowed the reduction of the scope of the validation, which was already costly in time and effort.

Comparative study of three screening tests, two microbiological tube tests, and a multi-sulphonamide ELISA kit for the detection of antimicrobial and sulphonamide residues in eggs.

The screening of antimicrobial residues in eggs is an especially important subject. Three different commercial kits for the screening of sulphonamides and other antimicrobials in eggs were validated in accordance with Decision 2002/657/EC: one enzyme-linked immunoabsorbant assay (ELISA) kit multi-sulphonamides (from RAISIO Diagnostics) and two microbiological tests (a Premi test from DSM and an Explorer kit from Zeu-Inmunotec). The false-positive rates were lower than 2% for all kits. The detection capabilities (CCbeta) have to be as low as possible for banned substances and lower than the maximum residue limit (MRL) when MRLs have been set. The sensitivity of the Premi test was better than that of the Explorer test, probably because of the dilution of the eggs before the Explorer test was used. The CCbeta values towards most of the tested sulphonamides were satisfactory with the Premi test (< or = 100 microg kg(-1)). Performance in a proficiency test for the detection of sulphonamides in eggs with the Premi test confirmed these results. The detection capabilities of tetracycline and doxycycline were at the level of the MRL or twice the MRL maximum. The detection capabilities for chlortetracycline and oxytetracycline were higher (four to six times the MRL). The detection capabilities for amoxicillin, neomycin, tylosin and erythromycin were lower than their respective MRLs. Detection capabilities for sulphonamides were much lower for the ELISA kit than for microbiological tests. The ELISA kit could be recommended for the targeted screening of sulphonamides in eggs. On the other hand, the Explorer and Premi tests could be used as wide screening tests allowing the detection of most of the antimicrobial families.

Validation of a liquid chromatography-tandem mass spectrometry screening method to monitor 58 antibiotics in milk: a qualitative approach.

A multi-residue method was developed for monitoring antibiotic residues in milk using liquid chromatography coupled to a tandem quadrupole mass spectrometer (LC/MS-MS). Two very short extractions followed by two LC/MS-MS acquisitions allowed the screening of 58 antibiotics belonging to eight different families (penicillins, cephalosporins, sulfonamides, macrolides, lincosamides, aminoglycosides, tetracyclines, and quinolones). This method is currently implemented in the laboratory in a qualitative way, i.e. monitoring the presence or absence of residue in a sample and identification of the analyte before the confirmation step. In order to assess the performance of this method, a validation strategy described in an internal guideline for the validation of screening methods was applied. The aim of the validation was to prove sufficient sensitivity of the method to detect all the targeted antibiotics at the level of interest (maximum residue limit, MRL) at least. According to European Commission Decision 2002/657/EC, the suitable sensitivity of a screening method can be demonstrated when the CCbeta is below or equal to the MRL and so the false-compliant rate below or equal to 5% at the MRL level. The validation scheme was established in order to take into account various variability factors: the apparatus response, the interday repeatability, the matrix effect, etc. The results of the validation clearly demonstrate the suitability of this method for the detection and identification of more than 50 antibiotics and they are in agreement with the results obtained in routine analysis.

Proficiency study for the determination of nitrofuran metabolites in shrimps.

A proficiency test for the determination of nitrofuran metabolites in shrimp tissue was organized in the first half of 2003. This test was intended to allow the participants to use their routine method and to assess their competence on this specific analysis. The participation in this proficiency test was offered to all the National Reference Laboratories (NRLs) of the European Union (EU) in charge of the analysis of nitrofurans, to Official Laboratories of the then 10 Candidate Countries for entry in EU and to some countries exporting food to the EU. The participants (20) analysed nitrofuran metabolites in eight randomly coded frozen samples including three blank samples. All participants performed a confirmatory method using liquid chromatography/mass spectrometry to detect total nitrofuran metabolite residues. Both qualitative and quantitative analyses of the results were investigated. Qualitatively, 16 laboratories out of 20 gave the correct interpretation of the results in term of compliant/non-compliant sample. Quantitatively, laboratory performance was evaluated by calculating the z-scores.

Separation of basic compounds by capillary electrochromatography on an X-Terra RP18 stationary phase.

In this work we demonstrate that the X-Terra RP18 stationary phase, specially designed for the analysis of basic compounds in liquid chromatography, may be successfully used in capillary electrochromatography. Although this packing material does not afford a sufficient electroosmotic flow with classical hydro-organic mobile phases, the addition of a surfactant that adsorbs onto the stationary phase allows to generate a sustainable electroosmosis flow (EOF), the direction of which depends on the charge of the surfactant. The way of manipulating the electroosmotic flow is described (nature and concentration of the added surfactant, proportion of the organic modifier in the mobile phase, pH). It is then demonstrated that high efficiencies can be reached with this packing material (up to 220,000 plates/m with a mean diameter particles of 3.5 microm) when it is operated at high linear velocities. Then the separations of different classes of compounds such as amphenicol antibiotics, macrolide antibiotics or basic test solutes with mobile phases with pH up to 10.8 are described. The influence of the addition of sodium dodcylsulfate (SDS) to the mobile phase on the retention is described and the selectivity of the X-Terra RP18 stationary phase is compared to that of a more traditional phase, i.e. Hypersil C18 stationary phase with SDS added to the mobile phase. However, it is shown that a good repeatability of the retention factors can only be obtained when the ionization of the compounds is totally suppressed since electrolysis of the buffered hydro-organic mobile phase occurs in the buffer reservoirs leading to a variation of the mobile phase pH and consequently to a modification of the ionization degree of the solutes having their pKa close to the mobile phase pH.

Stability of penicillin antibiotic residues in meat during storage. Ampicillin.

Incurred samples from a pig treated with ampicillin, one of the most important penicillin antibiotic drugs used in food-producing animal treatments, were analyzed at the residue level of the drug in muscle tissue (approximately 100 microg kg(-1)) during their freezing storage and using three different techniques: quantitative microbiological assay, HPLC-UV and LC-MS. Two parameters have been specifically monitored: storage temperature (-20 and -75 degrees C) and storage packaging (ground meat or bulk meat). No significant decrease was observed during the first 3 months of storage monitoring at -20 and -75 degrees C. On the contrary, the sample preparation significantly affected the drug concentration in muscle from the very beginning of the storage. Grinding the meat before storage allowed to keep the drug near the higher level of concentration (approximately 100 microg kg(-1)) when bulk meat stored frozen systematically led to a decreased value (approximately 75 microg kg(-1)). After 8 months of storage at -20 degrees C, a significant decrease arose and was never observed at -75 degrees C. All the results were similarly obtained with the three different techniques used simultaneously, which allows to indicate a good correlation between the techniques.

Multiresidue analytical method for the determination of eight penicillin antibiotics in muscle tissue by ion-pair reversed-phase HPLC after precolumn derivatization.

A high-performance liquid chromatographic multiresidue method was developed for the determination of 8 penicillin compounds (benzylpenicillin, phenoxymethylpenicillin, ampicillin, amoxicillin, nafcillin, oxacillin, cloxacillin, and dicloxacillin) at trace levels in muscle tissue. This method involves extraction of the penicillins with phosphate buffer pH 9 followed by cleanup and concentration on a C18 solid-phase extraction column and reaction with benzoic anhydride at 50 degrees C for 5 min and with 1,2,4-triazole and mercury(II) chloride solution pH 9 at 65 degrees C for 10 min. The derivatized compounds are eluted on a C8 column with a mobile phase containing acetonitrile and phosphate buffer (pH 6; 0.1 mol/L) loaded with sodium thiosulfate and ion-pairing tetrabutylammonium hydrogenosulphate. The method detection limit is approximately 3-11 micrograms/kg and the limit of determination was evaluated down to 25 micrograms/kg in line with the criteria of the EU decision No. 93/256/EEC.

Determination of isoxazolylpenicillins residues in milk by ion-pair reversed-phase high-performance liquid chromatography after precolumn derivatization.

A high-performance liquid chromatographic method has been developed for the determination of isoxazolylpenicillins (oxacillin, cloxacillin and dicloxacillin) residues in milk. This method involves extraction of the penicillins from milk with phosphate buffer pH 8, deproteinization by acidification with sulfuric acid followed by cleanup and concentration on a C18 solid-phase extraction column and reaction with 1,2,4-triazole and mercury(II) chloride solution pH 9.0 at 65 degrees C. The derivatized compound is eluted on a C8 column with a mobile phase containing acetonitrile, methanol and phosphate buffer (pH 6.5, 0.1 mol l(-1)) loaded with sodium thiosulfate and ion-pairing tetrabutylammonium hydrogenosulphate. The detection limit of the method is 2 microg l(-1) for oxacillin, 3 microg l(-1) for cloxacillin and 5 microg l(-1) for dicloxacillin in milk and the three penicillins have been quantified down to 15 microg l(-1) in line with the EU criteria of the directive No. 93/256/EEC.

Determination of ampicillin residues in milk by ion-pair reversed phase high performance liquid chromatography after precolumn derivatization.

A high performance liquid chromatography method has been developed for the determination of ampicillin residues in milk. The method involves extraction of ampicillin from milk with trichloroacetic acid solution followed by concentration on a conditioned C18 solid phase extraction column, acetylation with acetic anhydride in aqueous solution (pH 8.0) at ambient temperature for 3 min followed by reaction with 2 M 1, 2, 4-triazole and 10(-2) M mercury (II) chloride solution (pH 9.0) at 65 degrees C for 10 min. The resulting product is eluted on a C18 column with a mobile phase containing phosphate buffer (pH 6.5; 0.1 M), the ion-pairing agent tetrabutylammonium hydrogenosulphate, acetonitrile and methanol. The detection limit of the method is 3 ng ml-1 in milk.

Atherosclerotic lesions of the aorta and renal arteries: results of simultaneous surgical treatment.

Between 1975 and 1985, 43 patients underwent simultaneous aortic and renal artery reconstruction. Twenty-two patients had infrarenal abdominal aortic aneurysms and 21 had aortoiliac occlusive disease. In addition, 40 patients had severe lesions of one or both renal arteries and three patients had a lesion in an accessory renal artery. Hypertension was present in 29 patients, 15 of whom had impaired renal function. Four patients had chronic renal insufficiency without hypertension. Ten patients underwent prophylactic renal artery reconstruction. Infrarenal aortic repair was carried out simultaneously with thromboendarterectomy of one or both renal arteries, or reimplantation of a renal artery into the aorta, in two cases with contralateral nephrectomy. In one patient, the celiac and superior mesenteric arteries were also bypassed. Three patients (7%) died in the immediate postoperative period, two of these from myocardial infarction. Long-term survival was studied in 37 patients. Sixty-seven percent of patients with preoperative hypertension and less than 50% of those with preoperative renal insufficiency had good results.