Oxolinic acid

Antibiotics and pesticide residues were analyzed using analytic grade reagents. Standards of high purity grades (>99%) for 26 antibiotics belonging to four families: Sulfonamides (sulfacetamide, sulfisomidine, SMT, sulfapyridine, sulfamerazine, sulfameter, sulfachloropyridazine, sulfamethoxazole, sulfabenzamide, sulfadimethoxine, sulfaquinoxaline, sulfamethoxypyridazine, and sulfadoxine), quinolones (nalidixic acid, oxolinic acid, CIP, norfloxacin, ofloxacin, flumequine, danofloxacin, enrofloxacin, lomefloxacin, and sarafloxacin), tetracyclines (tetracycline and oxytetracycline), and β-lactams (ampicillin) in addition to a hundred pesticide standards were procured from Sigma-Aldrich. Each pesticide stock solution was accurately prepared in acetonitrile at a concentration of 1 g/L. The working standards were freshly prepared by diluting the stock solution in acetonitrile to the desired concentration. Both stock and working solutions were stored at −20°C.
High-performance liquid chromatography (HPLC)-grade water, HPLC-grade acetonitrile, disodium ethylenediaminetetraacetate dihydrate (Na₂ EDTA), and magnesium sulfate (MgSO₄) were also supplied by Sigma-Aldrich.

The Prestwick library (Prestwick Chemical) contains 1120 approved drugs dissolved in DMSO at a stock concentration of 10 mM. Other compounds were purchased from the following sources: from Cayman Chemical: flumequine, oxolinic acid, etoposide, mitoxantrone (hydrochloride), ellipticine, bleomycin (sulfate), hydroxyurea, actinomycin D, triptolide, and UNC1999; from Sigma-Aldrich: lomefloxacin hydrochloride, ofloxacin, and sodium salicylate; from ApexBio: amsacrine; from Santa Cruz Biotechnology: ICRF-193, sobuzoxane, and aclarubicin (aclacinomycin A); from Tocris: UNC2400. All individually purchased compounds were dissolved in DMSO or water. Chemical structures were generated using PubChem Sketcher.

The antimicrobial agents used to perform the susceptibility assays of F. psychrophilum isolates were those currently licensed for treatment of Gram-negative bacterial pathogens in the Chilean salmonid farm industry. In addition, due to its worldwide use in fish aquaculture, enrofloxacin was included in the study. The antimicrobials used were amoxicillin, florfenicol, oxytetracycline, oxolinic acid, flumequine and enrofloxacin, and were commercially obtained from Sigma (Sigma, Poole, UK). Antimicrobial solutions were prepared according to CLSI guidelines (CLSI, 2006 ) and solvents used were deionized distilled water for oxytetracycline-HCl, 95% ethanol for amoxicillin and florfenicol and 0.1 M NaOH for oxolinic acid, flumequine, and enrofloxacin. All solutions were filter-sterilized (0.22 μm) and used immediately.

All reagents and solvents were of analytical reagent grade and were used without further purification. Oxolinic acid, YCl₃·6H₂O, LaCl₃, EuCl₃·6H₂O, GdCl₃·6H₂O, SmCl₃·6H₂O, TbCl₃·6H₂O, human transferrin, human serum albumin and double-stranded calf-thymus DNA were purchased from Sigma Aldrich Chemical Co. (Schnelldorf, Germany). The following abbreviations were used to designate signal intensity: b = broad, w = weak, m = medium, s = sharp.

All reagents were of analytical grade. Antibiotic standards of 4 different families, sulfonamides (sulfacetamide, sulfapyridine, sulfamerazine, sulfamethazine, sulfadimidine, sulfameter; sulfamethoxypyridazine, sulfachloropyridazine, sulfadimethoxine, sulfadoxine, sulfamethoxazole, sulfabenzamide, and sulfaquinoxaline), quinolones (nalidixic acid, oxolinic acid, ciprofloxacin, and norfloxacin, ofloxacin, flumequine, danofloxacin, enrofloxacin, lomefloxacin, and sarafloxacin), tetracyclines (tetracycline, oxytetracycline, and doxycycline), and beta-lactam antibiotics (amoxicillin, ampicillin, cloxacillin, and penicillin G), were purchased from (Sigma-Aldrich, St louis, MO, United States). All the standards were of high purity grades (>99%). Individual stock solutions were prepared at 1 g/L in acetonitrile and stored at −18 °C.
The working standard solutions containing all analytes except the lactams group with variable concentrations, according to their MRLs, was prepared as dilution of the stock solution in water /acetonitrile ratio of 70:30 (v/v), and the working standard solutions were kept at −20 °C in brown glass and were used for 1 week.
HPLC-grade water, HPLC-grade acetonitrile, trifluoroacetic acid (TFA; 0.2%), disodium ethylenediaminetetraacetate dihydrate (Na₂EDTA), and magnesium sulfate (MgSO₄) were also supplied by Sigma-Aldrich.