Liquid chromatography-tandem mass spectrometry method for the analysis of N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine, a biocidal disinfectant, in dairy products.

A novel and reliable method to quantify residual levels of N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine in dairy products using ion-pairing reversed-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and fully validated. Sample extraction was done with salting-out technique using acetonitrile and sodium chloride. For LC-MS/MS, the analyte was detected using positive electrospray ionization (ESI+) and two multiple reaction monitoring (MRM) transitions were monitored. The method was validated in the 5-150 µg kg-1 range using total error approach. Thus, performance criteria of the method were evaluated. Relative standard deviations for trueness and precision were lower than 10%; with the exception of hard pressed cheese at 5 µg kg-1 for precision. The limit of quantification (LOQ) was around 5-7 µg kg-1 depending on the matrix of interest. The method was successfully applied to accurately quantify N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine in 146 various dairy products with a maximum contamination level of 225 µg kg-1 in cheese.

Liquid chromatography-tandem mass spectrometry multiresidue method for the analysis of quaternary ammonium compounds in cheese and milk products: Development and validation using the total error approach.

Quaternary ammonium compounds (QACs) are both cationic surfactants and biocidal substances widely used as disinfectants in the food industry. A sensitive and reliable method for the analysis of benzalkonium chlorides (BACs) and dialkyldimethylammonium chlorides (DDACs) has been developed that enables the simultaneous quantitative determination of ten quaternary ammonium residues in dairy products below the provisional maximum residue level (MRL), set at 0.1mgkg-1. To the best of our knowledge, this method could be the one applicable to milk and to three major processed milk products selected, namely processed or hard pressed cheeses, and whole milk powder. The method comprises solvent extraction using a mixture of acetonitrile and ethyl acetate, without any further clean-up. Analyses were performed by liquid chromatography coupled with electrospray tandem mass spectrometry detection (LC-ESI-MS/MS) operating in positive mode. A C18 analytical column was used for chromatographic separation, with a mobile phase composed of acetonitrile and water both containing 0.3% formic acid; and methanol in the gradient mode. Five deuterated internal standards were added to obtain the most accurate quantification. Extraction recoveries were satisfactory and no matrix effects were observed. The method was validated using the total error approach in accordance with the NF V03-110 standard in order to characterize the trueness, repeatability, intermediate precision and analytical limits within the range of 5-150µgkg-1 for all matrices. These performance criteria, calculated by e.noval® 3.0 software, were satisfactory and in full accordance with the proposed provisional MRL and with the recommendations in the European Union SANTE/11945/2015 regulatory guidelines. The limit of detection (LOD) was low (<1.9µgkg-1) and the limit of quantification (LOQ) ranged from 5µgkg-1 to 35µgkg-1 for all matrices depending on the analytes. The validation results proved that the method is suitable for quantifying quaternary ammoniums in foodstuffs from dairy industries at residue levels, and could be used for biocide residues monitoring plans and to measure the exposition consumer to biocides products.

Validation of a liquid chromatography-high-resolution mass spectrometry method for the analysis of ceftiofur in poultry muscle, kidneys and plasma: A unique accuracy profile for each and every matrix.

A robust, selective and specific liquid chromatography-high resolution mass spectrometry (LC-HRMS) method was developed for the quantification of total residues of ceftiofur, an antibiotic belonging to the 3rd generation cephalosporins in plasma, muscle and kidney of poultry. Ceftiofur and conjugates in samples were firstly hydrolyzed with dithioerythritol into desfuroylceftiofur, which was then stabilized by derivatization with iodoacetamide into desfuroylceftiofur acetamide. Sample were then submitted to a solid phase extraction followed the accurate mass analysis of desfuroylceftiofur acetamide by LC-HRMS in full scan mode using a linear trap quadrupole (LTQ)-Orbitrap mass spectrometer with a resolving power 60,000 full width at half maximum (FWHM). The method was fully validated over a dosing range between 100 and 2000 µg kg(-1) (or µg L(-1)) using the total error approach. Accuracy profiles a graphical decision-making tool were built by computing results of validation procedure with acceptance limits set at ±60%, and ß-expectation tolerance intervals, i.e. the interval assuming to contain a ß % of future measurements (ß=90% in this study). Total measurement error including trueness, repeatability and intermediate precision were evaluated. Relative bias of trueness was never exceeding the threshold of 6% in all matrices at all level of concentration. The mean relative standard deviation for repeatability was lower than 16% at all levels of concentration for all matrices; the mean relative standard deviation for intermediate precision was lower than 25% at all levels of concentration for all matrices. This validation approach proved that the method is reliable for the quantification in each and every matrix (i.e. plasma, kidneys and muscle of chicken) thanks to only one single regression model (i.e. linear) obtained from external calibration standards (without matrix) with deuterated labelled internal standard. The developed method was applied during a depletion study of ceftiofur in chicken tissues and plasma.

6-Iso-chlortetracycline or keto form of chlortetracycline? Need for clarification for relevant monitoring of chlortetracycline residues in food.

Chlortetracycline (CTC) is a broad-spectrum antibiotic used in veterinary medicine for pulmonary or digestive infections and having a regulatory maximum residue limit (MRL) necessitating an official analytical control method. The purpose of this study was to clarify the identification of different forms of CTC observed in standard solution, in spiked muscle samples and in naturally incurred muscle samples of pigs analysed by LC-MS/MS and to demonstrate the in vivo formation of 6-iso-chlortetracycline and 4-epi-6-iso-CTC as a metabolite of CTC and 4-epi-CTC in muscle. The six following forms were identified, all being isobaric with a protonated molecule at m/z 479 (precursor ion): the keto-enol forms of CTC and the keto-enol forms of 4-epi-chlortetracycline (4-epi-CTC), 6-iso-chlortetracycline (6-iso-CTC) and 4-epi-6-iso-chlortetracycline (4-epi-6-iso-CTC). The 6-iso-CTC and 4-epi-6-iso-CTC were observed only in incurred pig samples so were identified for the first time as metabolites of CTC and 4-epi-CTC. Identification of the different forms was obtained by comparing incurred muscle samples with standard solutions and with spiked samples. Then the differences between the features of the chromatograms obtained by LC-TQ-MS and the fragmentation study of the different forms of CTC obtained by LC-Q-TOF-MS helped us to support this identification. The extraction steps and the LC-MS/MS conditions developed to analyse muscle tissue samples are described. This clarification concerning the rigorous identification of chromatographic peaks allowed us to evaluate the relevance of our monitoring method with regard to the regulations in place in the European Union and could be of help to laboratories involved in official control of antibiotic residues in food of animal origin. Additional results are also presented highlighting the transformation of the CTC when prepared in a mixture with other antibiotics.

Liquid chromatography-electrospray tandem mass spectrometric method for quantification of monensin in plasma and edible tissues of chicken used in pharmacokinetic studies: applying a total error approach.

A liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for use in pharmacokinetic studies in order to determine the concentrations of monensin in plasma and edible tissues of chicken. Two sample preparations were performed, one for determining monensin concentrations in plasma using acetonitrile for protein precipitation and another one for determining monensin concentrations in muscle, liver, and fat using methanol-water followed by a clean up on a solid-phase extraction cartridge. Sample extracts were injected into the LC-MS/MS system, and a gradient elution was performed on a C18 column. Narasin was used as internal standard. The LC-MS/MS method was validated using an approach based on accuracy profiles, and applicability of the method was demonstrated for the determination of monensin in chicken plasma, muscle, liver, and fat in a pharmacokinetic study.