Dominique Hurtaud-Pessel

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 CCβ 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.

Determination of four nitroimidazole residues in poultry meat by liquid chromatography-mass spectrometry

A multi-residue liquid chromatography-mass spectrometry (LC-MS) assay method is described for the determination of four nitroimidazoles in poultry muscle. The extraction procedure is based on liquid-liquid extraction with ethyl acetate followed by an evaporation step. A deuterated internal standard is used. The LC separation was made on a C18 bonded silica column with an aqueous formic acid (0.2%) solution-methanol-acetonitrile (81:13:6) mobile phase. Following electrospray ionization, the protonated molecular ion [M+H]+ is obtained for each compound. Monitoring several ions for each nitroimidazole provides the specificity required for confirmatory assay. Validation of the method was performed to estimate linearity, intra-day and inter-day repeatability, accuracy and detection limit. The present method is capable of identifying nitroimidazole residues in muscle at levels below 5 microg/kg.

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 CCβ 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.

Confirmation of 13 sulfonamides in honey by liquid chromatography-tandem mass spectrometry for monitoring plans: Validation according to European Union Decision 2002/657/EC

A rapid and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous confirmation of 13 sulfonamides in honey was developed and fully validated in accordance with the European Commission Decision No 2002/657/EC. The validation scheme was built in accordance with the target level of 50μgkg(-1) for all analytes. The sulfonamides investigated were as follows: sulfaguanidine (SGN), sulfanilamide (SNL), sulfadiazine (SDZ), sulfathiazole (STZ), sulfamerazine (SMR), sulfamethizole (SMZ), sulfadimerazine (SDM), sulfamonomethoxine (SMNM), sulfamethoxypiridazine (SMP), sulfadoxine (SDX), sulfamethoxazole (SMX), sulfaquinoxaline (SQX) and sulfadimethoxine (SDT). Several extraction procedures were investigated during the development phase. Finally, the best results were obtained with a procedure using acidic hydrolysis and cation exchange purification. Chromatographic separation was achieved on a C18 analytical column. Matrix effects were also investigated. Data acquisition implemented for the confirmatory purpose was performed by monitoring 2 MRM transitions per analyte under the positive electrospray mode. Mean relative recoveries ranged from 85.8% to 110.2% and relative standard deviations lying between 2.6% and 19.8% in intra-laboratory reproducibility conditions. The decision limits (CCα) ranged from 1.8 to 15.5μgkg(-1). High resolution mass spectrometry was used to investigate the possible formation of sulfonamide metabolites in honey. The validation results proved that the method is suitable for the screening and confirmatory steps as implemented for the French monitoring residue plan for sulfonamides residue control in honeybees.

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.

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.

CYP3A4 activity reduces the cytotoxic effects of okadaic acid in HepaRG cells

The biotoxin okadaic acid (OA), produced by dinoflagellates in marine environment, can accumulate in sponges and shellfish. Consumption of contaminated shellfish induces acute toxic effects such as diarrhea, nausea, vomiting, and abdominal pain. CYP3A4, one of the most important human xenobiotic metabolizing enzymes, is supposed to be involved in the metabolism of OA. Aim of our study was to evaluate the role of CYP3A4 in OA in vitro metabolism as well as in cell cytotoxicity in parallel. Therefore, a metabolic competent HepaRG cell line was exposed to OA with and without addition of the CYP3A4 inhibitor ketoconazole. Without the inhibitor, two mono-hydroxylated metabolites could be identified, whereas in its presence, no metabolites could be detected. Confirmation of the formed metabolites was accomplished by measuring the exact masses and investigating the fragmentation pattern. Data obtained from cytotoxicity assays showed that OA cytotoxicity is reduced when CYP3A4 is active. Thus, hydroxylation appears to be a crucial step for metabolic OA detoxification.

In vitro metabolism of the cyanotoxin cylindrospermopsin in HepaRG cells and liver tissue fractions.

No evidence for phase I metabolites of the cyanotoxin cylindrospermopsin (CYN) was given using HepaRG cells and different liver tissue fractions when studying metabolic conversion. Although the application of ketoconazole, a CYP3A4 inhibitor, led to a decreased cytotoxicity of CYN, no metabolites were detected applying high resolution mass spectrometry. Quantification of non-modified CYN led to recovery rates of almost 100%. Consequently, reduction of CYN toxicity in the presence of metabolism inhibiting agents must be attributed to alternative pathways.

The Monitoring of Triphenylmethane Dyes in Aquaculture Products Through the European Union Network of Official Control Laboratories.

Aquaculture has been the fastest growing animal production industry for the past four decades, and almost half of the fish eaten in the world are now farmed fish. To prevent diseases in this more intensive aquaculture farming, use of therapeutic chemicals has become a basic choice. The monitoring of malachite green, a triphenylmethane dye and one of the oldest and widely used chemicals in fish production, has gained more interest since the mid 1990s when this substance was finally proven to be toxic enough to be prohibited in seafood products destined for human consumption. The enforcement of the European Union (EU) regulation of this banned substance along with some other triphenylmethane dye congeners and their metabolites in its domestic production and in seafood imports was undertaken through the National Residue Monitoring Plans implemented in nearly all of the 28 EU member states. The reliability of the overall European monitoring of this dye contamination in aquaculture products was assessed by using the results of proficiency testing (PT) studies provided by the EU Reference Laboratory (EU-RL) in charge of the network of the EU National Reference Laboratories (NRLs). The proficiency of each NRL providing analytical support services for regulating dye residues was carefully checked during three PT rounds. In the process, the analytical methods developed and validated for this purpose have gradually been improved and extended over the last two decades.

Comprehensive validation of a liquid chromatography–tandem mass spectrometry method for the confirmation of chloramphenicol in urine including stability of the glucuronide conjugate and efficiency of deconjugation

This paper describes a method to reveal the illegal use of chloramphenicol (CAP) in animals intended for human consumption based on the detection of free CAP and chloramphenicol-glucuronide (CAP-glu) in urine. It details the different steps of the method, including hydrolysis of CAP-glu, extraction and cleanup with molecularly imprinted polymers and detection by LC-MS/MS, as well as the validation design. The efficiency of chloramphenicol release during the hydrolysis step and the stability of CAP-glu in urine samples stored at -20°C were also investigated. These verifications were important to ensure the methods suitability for checking CAP misuse in veterinary medicine. Validation results were fully compliant with the qualitative and quantitative criteria required by European regulations. Intraday relative standard deviations were all below 7.5%, while interday relative standard deviations were below 6.9%. Recoveries lay between 93.3 and 104.6%. Purification appears very effective since no matrix effect was demonstrated. CAP-glu was found to be stable for at least 3 months, and the mean recovery following deconjugation was assessed to be 79.4%. The decision limits (CCa) were all found to be lower than 0.1μg/kg.