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).

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.

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.

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).

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.

Analytical strategies to determine antibiotic residues in fish.

The accelerated growth of aquaculture has resulted in a series of harmful effects to human health. The widespread and unrestricted use of antibiotics in this industry, to prevent bacterial infections, leads to remaining amounts in the aquatic environment. This has resulted in the emergence of antibiotic-resistant bacteria in aquaculture environments, in the increase in antibiotic resistance in fish pathogens as well as in the transfer of these resistance determinants to human pathogens. Moreover, the use of large amounts of antibiotics may lead to the presence of residual antibiotics in fish tissue and fish products. Fluoroquinolones, tetracyclines, penicillins, sulphonamides and other antibiotics, exhibiting activity against both Gram-positive and Gram-negative bacteria, are widely used for the treatment and prevention of diseases in fish. An extended and comprehensive review on the recent analytical methodologies concerning antibiotic residues in fish reported in the literature is provided in the present article. Emphasis is given on sample preparation regarding isolation and purification, chromatographic conditions and method validation according to legislation. Results of published assays are comparatively presented and criticised.

Chromatographic analysis of penicillins in pharmaceutical formulations and biological fluids.

Natural penicillin (benzylpenicillin) is the oldest antibiotic observed by Alexander Fleming in 1928. To broaden its spectrum of activity, natural penicillin was modified, giving rise to a group of antibiotics under the name 'penicillins'. Although an increasing number of bacteria appear to be resistant to them, penicillins are used to treat a variety of bacterial infections including Gram-positive, Gram-negative aerobic and anaerobic bacteria. Consequently, they are widely used in human and veterinary medicine to prevent and treat diseases. This review covers the analytical methodologies, mainly chromatographic, employed to the penicillins determination in pharmaceutical formulations, biological fluids and in production-scale fermentations reported in the literature. Results of published assays are comparatively presented focusing on sample preparation regarding isolation and purification, chromatographic conditions and method validation. Information on chemical structure, spectrum of activity and action mechanism of common penicillins has also been given.

Development of a validated HPLC method for the determination of four penicillin antibiotics in pharmaceuticals and human biological fluids.

A quantitative method for the determination of four penicillin antibiotics, amoxicillin (AMO), oxacillin (OXA), cloxacillin (CLO), and dicloxacillin (DICLO), has been developed. Separation was achieved on an Inertsil ODS-3 (250 x 4 mm, 5 microm) column after selective extraction of penicillin drugs from biological matrices by means of SPE. Gradient elution with a mobile phase consisting of 0.1% TFA (pH 1) and ACN, and PDA detection with monitoring at 240 nm was applied. Salicylic acid (5 ng/microL) was used as the internal standard. RP-8 Adsorbex Merck cartridges provided high absolute recoveries (98-101%). The developed method was fully validated in terms of selectivity, linearity, accuracy, precision, stability, and sensitivity. Repeatability (n = 8) and between-day precision (n = 8) revealed RSD <10%. Recoveries from biological samples ranged from 91 to 103%. The detection limits were estimated as 3.3 ng for AMO, OXA, and CLO, and 6.6 for DICLO in blood plasma. LOD in whole blood and urine was 6.6 ng. Injection volume was 20 microL. The method was applied to commercially available AMO containing pharmaceuticals and spiked biological matrices. The method was also applied to biological samples after AMO oral administration, where the drug was successfully identified and quantified.

Simultaneous determination of quinine and chloroquine anti-malarial agents in pharmaceuticals and biological fluids by HPLC and fluorescence detection.

Even nowadays millions of people suffer and even die each year from malaria and hundreds of millions of people especially in tropical countries. Quinine (Q) a natural occurring alkaloid and chloroquine (CQ) a synthetic drug are widely used as anti-malarial agents. Herein an isocratic reversed-phase high performance liquid chromatographic (RP-HPLC) method is described for the simultaneous determination of quinine and chloroquine, at low concentrations, in pharmaceuticals and biological fluids. The present method is characterized by higher sensitivity and analytes are separated in less time than the already published methods. The analytical column, an MZ Kromasil, C18, 5 microm, 250 x 4mm, was operated at ambient temperature with backpressure values of 230 kg/cm(2). Mobile phase consisted of methanol-acetonitrile-0.1 mol/L ammonium acetate, (45:15:40 v/v) at a flow rate of 1.0 mL/min. Fluorescence detection was performed at excitation 325 nm and emission 375 nm, respectively. Salicylic acid was used as internal standard at a concentration of 0.5 ng/microL, resulting in a detection limit of 0.3 ng, while upper limit of linear range was 0.7 ng/microL for quinine and 0.5 ng/microL for chloroquine. Separation was completed within 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. Solid phase extraction provided high relative extraction recoveries from biological matrices: 92.1% for quinine and 105.4% for chloroquine from blood serum and 101.8% for quinine and 90.7% for chloroquine from urine.