Danofloxacin

Chemicals reagents used for this study were of analytical grade, and Ultra-pure water (Direct-Q^® 3UV-R purifier system Millipore, Merck, Darmstadt, Germany) was used throughout the duration of the experiments. Danofloxacin (99.7%) (DANO), enrofloxacin (99.0%) (ENRO), levofloxacin (99%) (LEVO), HPLC grade ethanol, methanol and acetonitrile were used, along with acetic acid, sodium hydroxide, ammonium hydroxide, ferrous chloride, ferric chloride, starch, chitosan, β-cyclodextrin and ortho-phosphoric acid purchased from Sigma-Aldrich (St. Loius, MO, USA). A synthetic sample mixture of the fluoroquinolones (FQs) stock solution was prepared by dissolving appropriate amounts of DANO, ENRO and LEVO in small amounts of methanol. The mixture was then diluted with ultra-pure water to a final volume of 100 mL. The solution were stored in to refrigerator at 4–8 °C.

The following solvents, reagents, and antimicrobial agent analytical standards were purchased from Merck (Darmstadt, Germany):

Methanol and n-hexane (hypergrade for LC-MS LiChrosolv^®);

Trichloroacetic acid (TCA) crystals, disodium hydrogen phosphate dihydrate, citric acid monohydrate, and EDTA (for preparing EDTA-McIlvaine buffer solution, pH 4);

Ampicillin, penicillin G, cloxacillin, amoxicillin, penicillin V, oxacillin, dicloxacillin, nafcillin, enrofloxacin, ciprofloxacin, danofloxacin, marbofloxacin, flumequine, tetracycline, 4-epitetracycline, oxytetracycline, 4-epioxytetracycline, chlortetracycline, 4-epichloretracycline, doxycycline, sulfadiazine, sulfathiazole, sulfadimethoxine, sulfadimidine, and enrofloxacin d5 as the internal standards (IS).

Formic acid (98–100%) was provided from Riedel-de Haën (Sigma-Aldrich, St. Louis, MO, USA).
Water was purified using a Milli-Q system (Millipore, Merck KGaA, Darmstadt, Germany).
The Oasis HLB cartridges (3 mL, 60 mg) were supplied by Waters (Milford, MA, USA).

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.

The following reagents were of analytical standard grade and Sigma-Aldrich (Saint Louis, MO, USA) supplied: danofloxacin, enrofloxacin, ciprofloxacin, norfloxacin, sarafloxacin, bovine serum albumin (BSA), pepsin, α-chymotrypsin and trypsin. SDS (Pepyn, France) supplied methanol (MeOH) and acetonitrile (MeCN) (HPLC grade). PanReac^® (Barcelona, Spain) supplied 2,4-Dinitrophenylhydrazine, potassium phosphate, sodium azide, hydrochloric acid, sodium bicarbonate and calcium chloride. Sodium hydroxide, sodium chloride, potassium chloride, 2-mercaptoethanol, Coomassie blue-brillant G250 and trichloroacetic acid (TCA) (99.5%) were purchased from Merck^® (Darmstadt, Germany). Proteomic or molecular biology grade reagents: sodium dodecyl sulfate, Tris Base and Glycerol were supplied from Amresco^® (West Chester, PA, USA). Orthophosphoric acid, molecular weight marker Spectra Multicolor Broad Range, Rabbit anti-DNP monoclonal and goat anti-Rabbit HRP antibodies were purchased from Scharlab (Barcelona, Spain), Thermo Scientific (Waltham, MA, USA), Invitrogen (Carlsbad, MA, USA) and Bioss Inc. (Woburn, MA, USA), respectively. Water was purified with a Milli-Q system (Millipore, Bedford, MA, USA).

Susceptibility testing was performed using the broth microdilution method with Mueller Hinton II broth (REMEL, LEXENA, KS) according to the CLSI guidelines (CLSI VET01 and CLSI VET08) [12 ,13 ]. Broths were incubated at 37 °C and 5% CO₂ for 72 h. The control strains were Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212 as well as the reference strain D. congolensis ATCC 14637. All isolates were tested in duplicate with the higher concentration taken as the final in cases when variations occurred. Concentrations tested ranged between 0.125 µg/mL and 64 µg/mL for all antimicrobials. Susceptibility was tested against chlortetracycline, ampicillin, florfenicol, tetracycline, sulfadimethoxine, tylosin, novobiocin, neomycin, amoxicillin, danofloxacin, enrofloxacin, ceftiofur, bacitracin, trimethoprim, penicillin and tulathromycin A (all acquired from SIGMA-ALDRICH, Saint Louis, MO, USA). Differentiation between susceptibility and resistance was performed according to the epidemiological cut off (ECOFF) criteria, or otherwise named the microbiological criterium, using visual estimation (“eye-ball” method) [14 (link)].

Standard chemicals, namely, danofloxacin, erythromycin, sulfadimidine, benzylpenicillin potassium, chloramphenicol, lincomycin, tetracycline, spectinomycin, thiamphenicol, dexamethasone, kanamycin, melamine, trimethoprim, zearanol, aflatoxin M₁, flunixin meglumine, and florfenicol, were purchased from Sigma (St. Louis, MO, USA). All the chemicals used were of analytical grade. Lead acetate, potassium oxalate, disodium hydrogen phosphate, trichloroacetic acid, sodium hydroxide, and dichloromethane were obtained from Sinopharm Chemical Reagent Co., Ltd. (China). High-purity water was obtained from a Milli-Q water system (Millipore, Billerica, MA, USA). Pure milk was purchased from a local supermarket. Commercialized semiquantitative ICA strip and an ICA strip reader were supplied by Beijing Meizheng Bio-Tech Co., Ltd. (Beijing, China)