Evaggelia N. Evaggelopoulou

Multi-residue determination of seven quinolones antibiotics in gilthead seabream using liquid chromatography–tandem mass spectrometry

A sensitive and selective confirmatory analytical method for the multi-residue determination of seven quinolones (ciprofloxacin, enrofloxacin, sarafloxacin, danofloxacin, oxolinic acid, nalidixic acid and flumequine) in gilthead seabream (Sparus aurata) was developed. The sample pre-treatment involves extraction with 0.1 M NaOH and purification by solid-phase extraction (SPE) on Waters Oasis HLB cartridges followed by the determination of all compounds in a single LC-electrospray ionization MS/MS run. Separation was achieved on a Perfectsil ODS-2, 5 microm, 250 mm x 4 mm, analytical column (MZ Analysentechnik) by gradient elution using a mixture of 0.2% (v/v) formic acid, methanol and acetonitrile within 30 min. Multiple reaction monitoring (MRM) was used for selective detection of each quinolone. Accuracy was evaluated through recovery studies at three different fortification levels. The mean recoveries are between 90 and 132% for the selected levels with RSD values lower than 20%. The method presents satisfactory results for linearity, precision and limits of quantification. The latter are much lower than the maximum residue limits (MRLs) established by the European Union for quinolones in fish tissues (6-8 microg/kg).

Development and validation of an HPLC method for the determination of six penicillin and three amphenicol antibiotics in gilthead seabream (Sparus Aurata) tissue according to the European Union Decision 2002/657/EC.

A confirmatory high performance liquid chromatography method for the determination of six penicillin antibiotics and three amphenicol antibiotics in gilthead seabream (Sparus Aurata) tissue was developed. Ampicillin (AMP), penicillin G (PG), penicillin V (PV), oxacillin (OXA), cloxacillin (CLO), dicloxacillin (DICLO), thiamphenicol (TAP), florfenicol (FFC) and chloramphenicol (CAP) were separated on an Inertsil, C(8) (250×4 mm, 5 μm) column by gradient elution with a mobile phase consisting of ammonium acetate 0.05 M and acetonitrile at 25°C. Diode array detection with monitoring at 225 nm (for the determination of AMP, PG, PV, TAP and FFC), 240 nm (for OXA, CLO and DICLO) and 278 nm (for CAP) was applied. Examined antibiotics were isolated from gilthead seabream tissue by liquid-liquid extraction and further clean-up was performed by solid phase extraction using Oasis HLB (200 mg/6 mL) cartridges. The developed method was fully validated in terms of selectivity, linearity, accuracy, precision, stability and sensitivity according to the European Union Decision 2002/657/EC.

HPLC confirmatory method development for the determination of seven quinolones in salmon tissue (Salmo salar L.) validated according to the European Union Decision 2002/657/EC.

A confirmatory high pressure liquid chromatographic method for the determination of seven quinolone antibiotics in tissue of Atlantic salmon (Salmo salar L.) was developed. Ciprofloxacin (CIP), danofloxacin (DAN), enrofloxacin (ENR), sarafloxacin (SAR), oxolinic acid (OXO), nalidixic acid (NAL) and flumequine (FLU) were separated on a Perfectsil ODS-2 120 (250 mm × 4 mm, 5 μm) column by gradient elution with a mobile phase consisting of 0.1% trifluoroacetic acid (pH=1), acetonitrile and methanol at 25°C within 22 min. Analytes were monitored at 255 nm (for the determination of OXO, NAL and FLU) and 275 nm (for CIP, DAN, ENR and SAR) by means of photodiode array detector. Examined quinolones were isolated from salmon tissue by extraction with citrate buffer solution (pH=4.7) and purified by solid phase extraction using Oasis HLB (200mg/6 mL) cartridges. The developed method was fully validated in terms of selectivity, linearity, accuracy, precision, stability and sensitivity according to the European Union Decision 2002/657/EC. The accuracy of the method was additionally proved by its application to certified reference material of salmon tissue (BCR® 725).

Chromatographic analysis of banned antibacterial growth promoters in animal feed

The issue of antimicrobial use in animals used as food is of global concern. Antimicrobials are used in animal agriculture to improve health and welfare of animals, meat quality, the economic efficiency of growth and production and public health by decreasing shedding of zoonotic pathogens. However, large quantities are often used without professional supervision. The growth-promotant (now reclassified as zootechnical feed additives) effect of low levels of antibiotics in animal feeds was first described in the late 1940s. Already in 1969 the Swann Committee recommended that use of antibiotics as a supplement in animal feedstuff should be restricted to those with little or no application as therapeutic agents for humans and animals, which would not impair the efficacy of therapeutic antibiotics through the development of resistant strains of organisms. Antimicrobials like avoparcin, ardacin, zinc bacitracin, virginiamycin, tylosin, spriramycin, carbadox and olaquindox were withdrawn within the period 1997-1999. Four others (monensin sodium, salinomycin sodium, avilamycin and flavophospholipol) were still permitted for use as growth promoters in animal feed to animals marketed in the European Union (EU). Since January 2006, they have been banned as well. This review focuses on the analytical methods developed to be an effective tool for monitoring compliance with the ban.

Simple and Rapid HPLC Method for the Determination of Quinine in Soft Drinks Using Fluorescence Detection

Abstract A simple and rapid reversed‐phase high performance liquid chromatographic (RP‐HPLC) method was developed for the routine determination of quinine in soft drinks, which is added when a bitter taste is required. The analytical column, an MZ Kromasil, C18, 5 µm, 250 × 4 mm2, was operated at ambient temperature with backpressure of 230 kg/cm2. The mobile phase consisted of CH3OH–CH3CN–CH3COONH4 0.1 M, (45:15:40 %v/v/v) and was delivered at a flow rate of 1.0 mL/min. Fluorescence detection was performed at 325 nm (excitation) and 375 nm (emission). For the quantitative determination of quinine, salicylic acid was used as internal standard at a concentration of 0.5 ng/µL, resulting in a detection limit (signal‐to‐noise ratio 3:1) of 0.3 ng, while the upper limit of linear range was 0.7 ng/µL. Analysis time was less than 5 min. The statistical evaluation of the method was examined performing intra‐day (n = 8) and inter‐day calibration (n = 8) and was found to be satisfactory, with high accuracy and precision results. The method was applied to the analysis of soft drinks containing quinine, such as tonic water and bitter lemon. No interferences from other food additives were observed.

DEVELOPMENT AND VALIDATION OF AN LC-DAD METHOD FOR THE ROUTINE ANALYSIS OF RESIDUAL QUINOLONES IN FISH EDIBLE TISSUE AND FISH FEED. APPLICATION TO FARMED GILTHEAD SEA BREAM FOLLOWING DIETARY ADMINISTRATION

The authors in a previous work had reported on the determination of seven quinolone antibiotics in gilthead sea bream using liquid chromatography–tandem mass spectrometry, which is a rather expensive and not always available analytical technique. In this work, photodiode array detection is used, so that no sophisticated instrumentation is necessary, following the same sample preparation procedure. A high performance liquid chromatography method for the determination of seven quinolone antibiotics in fish feed and gilthead sea bream (Sparus aurata L.) tissue was developed and validated in terms of selectivity, linearity, accuracy, precision, sensitivity, robustness, and stability, according to European Decision 2002/657/EC. Ciprofloxacin, danofloxacin, enrofloxacin, sarafloxacin, oxolinic acid, nalidixic acid, and flumequine were separated on a Perfectsil ODS-3 (250 mm × 4 mm, 5 µm) column by gradient elution, using a mobile phase consisting of 0.1% trifluoroacetic acid (pH = 1), acetonitrile, and methanol at 25°C. Analytes were monitored by diode array detector at 255 nm for acidic quinolones (oxolinic acid, nalidix acid, and flumequine) and 275 nm for ciprofloxacin, danofloxacin, enrofloxacin, and sarafloxacin. The method was successfully applied to gilthead sea bream from aquaculture after dietary administration of flumequine and oxolinic acid.

Confirmatory development and validation of HPLC-DADmethod for the determination of tetracyclines in gilthead seabream (Sparus aurata) muscle tissue.

A confirmatory high-pressure liquid chromatographic method for the determination of nine tetracyclines in Sparus aurata (gilthead seabream) muscle tissue is developed and presented herein. Tetracycline, 4-epi-tetracycline, oxytetracycline, 4-epi-oxytetracycline, chlortetracycline, 4-epi-chlortetracycline, doxycycline, methacycline and demeclocycline were separated on a Kromasil, C18 (250 mm × 4 mm, 5 μm) analytical column by gradient elution with a mobile phase consisting of 0.001 M ethylenediaminetetraacetic acid/sodium salt and acetonitrile at 25°C. Diode array detection with monitoring at 280 nm (for the determination of chlortetracycline, 4-epi-chlortetracycline, methacycline and demeclocycline) and 355 nm (for tetracycline, 4-epi-tetracycline, oxytetracycline, 4-epi-oxytetracycline and demeclocycline) was applied for peak identification and quantification of analytes. Examined antibiotics were isolated from gilthead seabream tissue by leaching using a citrate buffer (pH 4.0) and purified by solid phase extraction using Oasis HLB(200 mg/6 mL) cartridges. The developed method was fully validated in terms of selectivity, linearity, accuracy, precision, stability and sensitivity according to the European Union Decision 2002/657/EC.