M.P. Hermo

Improved determination of quinolones in milk at their MRL levels using LC–UV, LC–FD, LC–MS and LC–MS/MS and validation in line with regulation 2002/657/EC

This paper reports the multi-residue determination of quinolones included in the European Union regulations (marbofloxacin, ciprofloxacin, danofloxacin, enrofloxacin and flumequine) in bovine milk, using liquid chromatography with ultraviolet (LC-UV), fluorescence (LC-FD), mass spectrometry (LC-MS) and tandem mass spectrometry detection (LC-MS/MS). The methods were validated, in line with EU requirements (Commission Decision 2002/657/EC). Linearity, decision limit (CCalpha), detection capability (CCbeta), detection limit (LOD), quantification limits (LOQ), recoveries, precision, selectivity and stability were determined and adequate results were obtained. Recoveries higher than 80% were obtained for all quinolones. The methods developed were used to quantify enrofloxacin and its main metabolite, ciprofloxacin, in milk from animals treated with enrofloxacin.

Determination of multiresidue quinolones regulated by the European Union in pig liver samples ☆: High-resolution time-of-flight mass spectrometry versus tandem mass spectrometry detection

The use of liquid chromatography coupled to orthogonal acceleration time-of-flight mass spectrometry (LC-ToF-MS) provides an attractive alternative to liquid chromatography coupled to quadrupole (LC-MS) or triple quadrupole mass spectrometry (LC-MS/MS) in multiresidue analysis. ToF-MS provides accurate mass information and a significantly higher mass resolution than quadrupole analyzers. In this work, the influential parameters in time-of-flight detection using an electrospray ionization (ESI) source were studied using a central composite design to obtain the main effects and their two-factor interactions. The method developed uses LC-ESI-ToF-MS to determine and characterize quinolones regulated by the EU in pig liver samples below the maximum residue limits (MRLs). Linearity, decision limit, detection capability, detection and quantification limits, precision and recoveries were determined and adequate results were obtained, with quantification limits between 1.5 and 6 microg kg(-1) and recoveries higher than 60% for all quinolones. Limits of detection are lower than 2 microg kg(-1). Results obtained using LC-ESI-ToF-MS were compared with those obtained using LC coupled to a quadrupole and to triple quadrupole mass spectrometer. The work described in this paper illustrates the suitability and excellent confirmatory potential of LC-ToF-MS for multiresidue analysis in food samples.

Determination of penicillins in milk using LC-UV, LC-MS and LC-MS/MS.

The aim of this work is to establish a method for the simultaneous determination of eight penicillins in milk samples by LC-UV, LC-MS and LC-MS/MS. The procedure involves a step for clean-up and to preconcentrate the analytes by SPE and a subsequent chromatographic analysis. LC-UV, LC-MS and LC-MS/MS have been used for the simultaneous quantification of penicillins in milk. The proposed methods have been validated according to the EU guideline and present LOQ below the maximum limits of residues (MRLs) established by the European Union for penicillins in milk. The developed methods were applied to different milk samples obtained from cows medicated with penicillins.

Multiresidue determination of quinolones regulated by the European Union in bovine and porcine plasma. Application of chromatographic and capillary electrophoretic methodologies

This paper presents the multiresidue determination of the series of quinolones regulated by the European Union (marbofloxacin, ciprofloxacin, danofloxacin, enrofloxacin, sarafloxacin, difloxacin, oxolinic acid and flumequine) in bovine and porcine plasma using capillary electrophoresis and liquid chromatography with ultraviolet detection (CE-UV, LC-UV), liquid chromatography-mass spectrometry and -tandem mass spectrometry (LC-MS, LC-MS/MS) methods. These procedures involve a sample preparation by solid-phase extraction for clean-up and preconcentration of the analytes before their injection into the separation system. All methods give satisfactory results in terms of linearity, precision, accuracy and limits of quantification. The suitability of the methods to determine quinolones was evaluated by determining the concentration of enrofloxacin and ciprofloxacin in real samples from pig plasma and cow plasma.

High-resolution mass spectrometry applied to the study of metabolome modifications in various chicken tissues after amoxicillin administration

The performance of high resolution accurate mass spectrometry (HRMS) operating in full scan MS mode was investigated for the quantitative determination of amoxicillin (AMX) as well as qualitative analysis of metabolomic profiles in tissues of medicated chickens. The metabolomic approach was exploited to compile analytical information on changes in the metabolome of muscle, kidney and liver from chickens subjected to a pharmacological program with AMX. Data consisting of m/z features taken throughout the entire chromatogram were extracted and filtered to be treated by Principal Component Analysis. As a result, it was found that medicated and non-treated animals were clearly clustered in distinct groups. Besides, the multivariate analysis revealed some relevant mass features contributing to this separation. In this context, recognizing those potential markers of each chicken class was a priority research for both metabolite identification and, obviously, evaluation of food quality and health effects associated to food consumption.

Metabolomic assays of amoxicillin, cephapirin and ceftiofur in chicken muscle: application to treated chicken samples by liquid chromatography quadrupole time-of-flight mass spectrometry.

The aim of this study was to identity metabolites and transformation products (TPs) in chicken muscle from amoxicillin (AMX), cephapirin (PIR) and ceftiofur (TIO), which are antibiotics of the β-lactam family. Liquid chromatography coupled to quadrupole time-of-flight (QqTOF) mass spectrometry was utilized due to its high resolution, high mass accuracy and MS/MS capacity for elemental composition determination and structural elucidation. Amoxicilloic acid (AMA) and amoxicillin diketopiperazine (DKP) were found as transformation products from AMX. Desacetylcephapirin (DAC) was detected as a metabolite of PIR. Desfuroylceftiofur (DFC) and its conjugated compound with cysteine (DFC-S-Cys) were detected as a result of TIO in contact with chicken muscle tissue. The metabolites and transformation products were also monitored during the in vivo AMX treatment and slaughtering period. It was found that two days were enough to eliminate AMX and associated metabolites/transformation products after the end of administration.

High-resolution mass spectrometry applied to the identification of transformation products of quinolones from stability studies and new metabolites of enrofloxacin in chicken muscle tissues.

The aim of this work was the identification of new metabolites and transformation products (TPs) in chicken muscle from enrofloxacin (ENR), ciprofloxacin (CIP), difloxacin (DIF) and sarafloxacin (SAR), which are antibiotics that belong to the fluoroquinolones family. The stability of ENR, CIP, DIF and SAR standard solutions versus pH degradation process (from pH 1.5 to 8.0, simulating the pH since the drug is administered until its excretion) and freeze-thawing (F/T) cycles was tested. In addition, chicken muscle samples from medicated animals with ENR were analyzed in order to identify new metabolites and TPs. The identification of the different metabolites and TPs was accomplished by comparison of mass spectral data from samples and blanks, using liquid chromatography coupled to quadrupole time-of-flight (LC-QqToF) and multiple mass defect filter (MMDF) technique as a pre-filter to remove most of the background noise and endogenous components. Confirmation and structure elucidation was performed by liquid chromatography coupled to linear ion trap quadrupole Orbitrap (LC-LTQ-Orbitrap), due to its mass accuracy and MS/MS capacity for elemental composition determination. As a result, 21 TPs from ENR, 6 TPs from CIP, 14 TPs from DIF and 12 TPs from SAR were identified due to the pH shock and F/T cycles. On the other hand, 14 metabolites were identified from the medicated chicken muscle samples. Formation of CIP and SAR, from ENR and DIF, respectively, and the formation of desethylene-quinolone were the most remarkable identified compounds.