# Determination of Eleven Veterinary Drugs in Chicken Meat and Liver
## 2 Materials and Methods
### Chicken Samples Preparation and Chromatographic Analysis  
The samples were immediately identified, processed, and submitted to an accredited laboratory, Eurofins, located in Indiaatuba, SP, Brazil. The sample preparation and analyte extraction procedure were as follows:  
1. Internal Method POP-QV028/3 (applied to tianulin, virginamycin, tylosin, spectino-mycin, amoxicillin, trenbolone acetate, halofuginone hydrobromide, dinitolmide + zoalene): The samples were weighed (2.00 \(\pm\) 0.05 g) and internal standard solution was added. The samples were extracted with acetonitrile:water:acetic acid (70:29:1) \(v\)/\(v\) then the mixture was manually stirred or vortexed. In case, if further homogenization was needed, ceramic homogenizer or disposable glass sphere were added to the mixture. Afterward, the mixture was placed on a shaking table for 20 min at 200 rpm and then centrifuged at 4000 rpm for 5 min. Then, 500 \(\mu\)L from the extract and 500 \(\mu\)L deionized water were transferred to 15-mL Falcon tubes. These tubes were vortexed, and the solution was filtered through 0.22 \(\mu\)m membrane directly into vial, which were then submitted to LC-MS/MS analysis;
2. Internal Method POP-QV013/3 (applied to bacitracin and colistin): The samples were weighed (5.00 \(\pm\) 0.05 g) and internal standard solution was added to these samples, as well as to the calibration curve and blank samples. The samples were extracted with MeOH:H20 (25:75) and 1 mol/L sulfuric acid. Afterward, the mixture was stirred to homogenize it properly. Then, the mixture was placed on a shaking table for 20 min at 200 rpm and then centrifuged at 4000 rpm for 5 min. Then, the samples were frozen (\(-\)18 \({}^{\circ}\)C) and allowed to rest for 20 min. After that, samples were centrifuged for another 20 min at 4000 rpm and the supernatant was immediately  
Figure 2: Chicken meat sampling and chemical residues determination by LC-MS/MS.  
separated to another 50-mL polypropylene tube. The samples were submitted to vacuum-controlled SPE extraction, so that the analytes were allowed to interact with the stationary phase. Afterward, clean-up was performed with deionized water and the cartridges were dried under vacuum in this step. The 15-mL polypropylene tubes were placed under the cartridges and the analytes were eluted with MeOH:Formic acid solution (75:25). The mixture was stirred and filtered through 0.22 \(\upmu\)m membrane directly into borosilicate vial, which were then submitted to LC-MS/MS analysis;
3. AOAC Official Method 2013.06 applied to rovarsone extraction and quantification is well established in the literature [4]. Briefly, the sample was submitted to pressure digestion which occurs using nitric acid in a closed vessel with elevated temperature and pressure by conventional or microwave-assisted heating. Determination occurs using inductively coupled plasma (ICP)/MS [4].  
In order to determine the chemical residues by chromatographic analysis, a C8 column (150 mm \(\times\) 3 mm \(\times\) 4 \(\upmu\)m) was used for analytes separation in a system consisting of an HPLC (Sciex Exion LC), coupled to an ABSciex API 5500 mass detector, with a binary pump, a column oven, and an auto-injector. The mobile phase for methods (a) and (b) were the same and consisted of (phase A) an aqueous solution with 0.1% formic acid (_v/v_) + 5 mM ammonium formate and (phase B) methanol with 0.1% formic acid (_v/v_) + 5 mM ammonium formate. The gradient elution ramp was set as follows: 98% A (0 min), 100% B (0.0 to 7.5 min), 100% B (7.5 to 9.5 min), 98% A (9.5 to 9.6 min), and 98% A (9.6 to 17.0 min). The injection volume was 20 \(\upmu\)L, the flow rate was 700 \(\upmu\)L/min, and the column temperature was 40 \({}^{\circ}\)C.