Chemicals, products and reagents
The analytical standards of enro-, cipro and sarafloxacin (internal standard, IS) were obtained from Sigma-Aldrich (Bornem, Belgium). Water, methanol and acetonitrile (ACN) were of LC-MS grade and obtained from Biosolve (Valkenswaard, The Netherlands). Glacial acetic acid and ethyl acetate were of analytical grade and obtained from VWR (Leuven, Belgium). Phosphate Buffered Saline (PBS) was obtained from Life Technologies (Gent, Belgium).
Preparation of standard solutions
Separate standard stock solutions of enro-, cipro- and sarafloxacin were prepared in 0.01 M acetic acid in water (analyte concentration: ≤ −15°C. A working solution of sarafloxacin and a combined working solution of 0.1 mg/mL of enro- and ciprofloxacin was prepared by transferring 100 μL of each stock solution into an Eppendorf cup, followed by further dilution with water up to a final volume of 1.0 mL. Ten-fold dilutions were obtained by dilution with water. All working solutions were stored at 2-8°C.
Ninety-six three-week-old broiler chickens (Ross 308) of mixed gender were equally divided in 4 groups and allocated to a different treatment with enrofloxacin. Animals had ad libitum access to feed and drinking water throughout the experiment. After a one-week acclimatization period, the animals of the first group were administered the conventional treatment. Enrofloxacin (Baytril® 10 % oral solution, Bayer, Diegem, Belgium) was given as an oral bolus directly in the crop for 5 consecutive days (10 mg enrofloxacin/kg BW). The second group received an elevated dose of enrofloxacin orally (50 mg/kg BW) for five days. Enrofloxacin was given intramuscularly (ad random as several separate injections in both breast muscles) for 5 days at a dose of 10 or 50 mg/kg BW (Baytril® 5%, Bayer) to the birds of the other two groups.
From each group, eight animals were euthanized at each sampling point, namely 2 and 4 h after the first administration and 4 h after the last administration (100 h after first administration). Euthanasia was performed by sodium pentobarbital injection followed by exsanguination. Blood samples (1 mL) from the jugular vein and intestinal content from ileum, cecum, colon and cloaca were collected. Blood samples were centrifugated (2851 × g, 10 min, 4°C). Aliquots (250 μL) of plasma samples were stored at ≤ −15°C until analysis. Intestinal content samples of each treatment group were pooled by segment and stored at ≤ −80°C until analysis. This animal experiment was approved by the Ethical Committee of Ghent University (Case number EC 2013_117).
To 250 μL of plasma were added 12.5 μL of the IS working solution and vortex mixed (15 sec). Three mL of ethyl acetate were added, samples were extracted for 15 min on a roller mixer (Stuart Scientific, Surrey, UK) and centrifugated (2851 × g, 10 min, 4°C). Next, the supernatant was transferred to another tube and evaporated using a gentle nitrogen (N2) stream (45 ± 5°C). The dry residue was reconstituted in 250 μL of water. After vortex mixing (15 sec), the sample was transferred into an autosampler vial and an aliquot (5 μL) was injected onto the LC-MS/MS instrument.
To one gram of intestinal content, pooled per segment, were added 100 μL of the IS working solution followed by a vortex mixing step (15 sec) and addition of 3 mL PBS and 5 mL ethyl acetate. Samples were then treated in the same way as plasma samples. If the detected concentration was out of the linear range, samples were appropriately diluted with PBS, re-extracted and re-analyzed. Samples were analyzed in triplicate (technical replicates).
The LC system consisted of a quaternary, low-pressure mixing pump with vacuum degassing, type Surveyor MSpump Plus and an autosampler with temperature controlled tray and column oven, type Autosampler Plus, both from ThermoFisher Scientific (Breda, The Netherlands). Chromatographic separation was achieved on a Zorbax Eclipse Plus column (100 mm × 3.0 mm i.d., dp: 3.5 μm) in combination with a guard column of the same type (13 mm × 3.0 mm i.d., dp: 3.5 μm), both from Agilent (Diegem, Belgium). The temperatures of the column oven and autosampler tray were set a 45°C and 5°C, respectively. Mobile phase A consisted of 0.1% glacial acetic acid in water whereas mobile phase B was ACN. Following gradient elution program was run: 0–3.5 min (92% A, 8% B), 3.5-4.0 min (linear gradient to 80% A), 4.0-8.0 min (80% A, 20% B), 8.0-8.5 min (linear gradient to 92% A), 8.5-13.0 min (92% A, 8% B). Flow rate was set at 500 μL/min.
Mass spectrometry and method validation
The LC column effluent was interfaced to a TSQ® Quantum Ultra triple quadrupole mass spectrometer, equipped with a heated electrospray ionization (h-ESI) probe operating in the positive ionization mode (all from ThermoFisher Scientific). Instrument parameters were optimized by syringe infusion of working solutions of 1 μg/mL of each compound (flow rate 10 μL/min) in combination with the mobile phases (50% A, 50% B).
The following general MS/MS parameters were used: spray voltage: 3800 V, vaporizer temperature: 300°C, sheath gas pressure: 33 au (arbitrary units), ion sweep gas pressure: 2.0 au, auxilliary gas pressure: 15 au, capillary temperature: 300°C, collision pressure: −1.5 mTorr and quad MS/MS bias: 2.9. The resolution for Q1 and Q3 were set at 0.7 peak width at half-height.
Acquisition was performed in the selected reaction monitoring (SRM) mode. For each compound, the two most intense product ions of the precursor ion were monitored in the SRM mode for quantification and identification, respectively. The SRM transitions for enro-, cipro and sarafloxacin were m/z 360.0 > 316.2*/245.1, 332.0 > 288.1*/314.1 and 386.1 > 299.0*/368.0, respectively. The * indicates the ion used for quantification.
The method was validated for enro- and ciprofloxacin in plasma as well as intestinal content according to a validation protocol previously described by De Baere et al. (2011) . A set of parameters that were in compliance with the recommendations and guidelines defined by the European Community and with criteria described in the literature, were evaluated –.
After determination of normality and homogeneity of variances, one-way ANOVA (SPSS 20.0, IBM, Chicago, IL, USA) was performed on the results from the four different treatments within each matrix and sampling time point. A Scheffé test was performed as post-hoc test. The significance level was set at 0.05.