Reference standards
Amoxicillin (99.6 %), ampicillin (99.8 %), benzylpenicillin (99.3 %), cloxacillin (98.7 %), oxacillin (98.4 %), clenbuterol hcl (99.1), isoxsuprine hcl (100 %), salbutamol (99.4 %), zilpaterol hcl (96.0), ractopamine hcl (95.5 %), terbutaline hemisulfate salt (100.0 %), taleranol (99.5 %), 19 nortestosterone (99.8 %), clostebol (99.1 %), boldenone (99.1 %), methyltestosterone (99.5 %), testosterone (100.0 %), carbofuran (99.9 %), carbaryl (99.9 %), parathion (99.7 %), malathion (99.2 %), diazinon (98.3 %), dimethoate (99.8 %), atrazine (99.5 %), cypermethrin (98.4 %), permethrin (98.1 %), deltamethrin (99.9 %), coumaphos (99.7 %), dicholphos (99.8 %), chlorpyrifos (99.8 %), fenvalerate (99.4 %) were purchased Sigma-Aldrich (St. Louis, MO, USA). Brombuterol (98.0 %), mabuterol hcl (98.0 %), cimbuterol (98.0 %), clenpenterol hcl (98.0 %) were obtained from Witega (Berlin, Germany). Zeranol (99.9 %), stanozolol (99.8 %), ceftioflur (98.01), cephalexin (96.6 %), oxytetracycline (96.5%), enrofloxacin (99.74 %), ciprofloxacin (98.0 %), sulfadimidine (99.6 %), sulfamethoxazole (99.7 %), sulfadiazine (99.8 5), sulfachloropiridazine (99.1 %) and sulfadimethoxine (99.7 %) were obtained from Dr. Ehrenstorfer GmbH (Augsburg, Germany); ochratoxin a (≥ 98.0 %) and zearalenon (99.0 %) were obtained from Trylogy Analytical Laboratory, Inc. (Washington, USA).
Isotopic labelled internal standards
Clenbuterol-d6 HCl (98.0 %), brombuterol-d9 hcl (98.0 %), mabuterol-d9 hcl (98.0 %), clenpenterol-d5 hcl (98.0 %), cimbuterol-d9 (98.0 %), were obtained from Witega (Berlin, Germany); isoxsuprine-d5 hemifumarate (≥ 98.0 %) and ractopamine-d6 hcl (≥ 98.0 %) were obtained from the European reference laboratory (EURL) at RIKILT, The Netherlands, salbutamol (albuterol)-d9 (≥ 98.0 %) was obtained from Dr. Ehrenstorfer GmbH (Augsburg, Germany); zilpaterol–d7 (≥98.0 %) and β-zearalenol-d4 (≥98.0 %) were obtained from Toronto Research Chemicals Inc. (Toronto, Canada), while terbutaline-d9 acetate hemihydrate (99.3 %), flunixin–d3 (100.0 %) and penicillin G-d7 N-ethylpiperidinium (98.1 %) salt were obtained from Sigma-Aldrich (St.Louis, MO, USA).
Preparation of standard solutions
Individual stock standard solutions and internal standards were prepared in methanol (MeOH) at concentration from 0.528 to 3.610 mg/mL, while the concentration of ochratoxin A was 50 μg/ml and for zearalenone was 100 μg/ml after reconstruction in methanol. After preparation of individual stock standard solutions and internal standards, the standards were divided in groups in accordance with the maximul recommended residue levels (MRL) and Minimum Required Performance Limits (MRPL) values according to the acceptance criteria of European Commision 2002, European Commision 2006, European Commision 2010, SANCO 2007 and EU pesticide database.
The initial mixed working standards with concentration at 10 µg/ml were prepared in MeOH and kept at–18°C. The groups of standards were: clenbuterol, brombuterol, mabuterol in group 1; cimbuterol, clenpenterol, isoxsuprine, ractopamine in group 2; salbutamol, terbutaline, zilpaterol in group 3; testosterone, methyltestosterone, boldenone, zeranol, 19 nortestosterone, stanozolol, clostebol, taleranol in group 4; amoxicillin, ampicillin, benzylpenicillin, cloxacillin, carbaryl, parathion, dimethoate, atrazine, permethrin, dicholphos, zearalenon, ochratoxin a in group 5; enrofloxacin, ciprofloxacin, oxytetracycline, sulfadimidine, sulfamethoxazole, sulfadimethoxine, sulfadiazine, sulfachloropiridazine in group 6; carbofuran, chlorpyrifos in group 7; malathion, diazinone, coumaphos in group 8. Working standards for oxacillin, cephalexin, deltamethrin and fenvalerate were prepared at 10 µg/ml and ceftioflur and cypermetrin at 100 µg/ml.
Mixed internal standard working solutions including brombuterol-d9 hcl, cimbuterol-d9, clenbuterol-d6 hcl, clenpenterol-d5 hcl, isoxsuprine-d5 hemifumarate, mabuterol-d9 hcl, ractopamine-d6 hcl, salbutamol (albuterol)-d9, terbutaline-d9 acetate hemihydrate, zilpaterol-d7) at 100 ng/ml and (β-zearalenol – d4, flunixin –d3 and penicillin g-d7) at 10 μg/mlwere prepared and kept at –18°C.
Chemicals and reagents
LC-MS/MS grade MeCN, water and MeOH, HPLC grade ethylacetate (EtOAc), dichloromethane, ammonium hydroxide, n-hexane, acetic acid, ammonium acetate were obtainedfrom Carlo Erba Reagent S.A.S (Val de Reuil, France); LC-MS/MS grade formic acid was from Merck (Darmstad, Germany and Oasis HLB cartridge (500mg/6ml) from Waters (Milford, MA, USA).
Sample preparation
Meat samples were collected from local markets of North Macedonia and transported to the laboratory at 4 °C. After homogenization, 10 g of meat sample was fortified with the analytes and the internal standards and let to stand for 20 min. Then, 20 ml of extraction mixture (MeCN:EtOAc:acetic acid, 49.5:49.5:1, v/v/v) was added and shaken vigorously for 1 min ona vortex mixer. The mixture was then shaken for 30 min with an automated shaker and centrifuged at 8,000 rpm for 10 min, at 0°C. The extraction step was repeated and the combination of the supernatants was transferred to a 50 mL tube and kept at −80°C, for 20 min. The solution was filtered through filter paper ( > 95% α-cellulose content, dia. 240 mm) and evaporated to nearly dryness under stream of nitrogenin a water bath at 35°C. The extract was dissolved in 10 mL of MeOH:water (10:90, v/v) and the solution shaken for 1 min on avortex mixer followed by cleanup by solid phase extraction using Oasis HLB cartridges. The cartridge was activated and conditioned by passing through 5 ml of MeOH and 5 mL water before supernatant was loaded and cartridges washed with 5mL of water, and then vacuum-dried for 10 min. The residues were eluted into a test tube using 4 mL MeOH:MeCN:ammonium hydroxide (47.5:47.5:5, v/v/v) followed by 4 mL MeOH:dichloromethane (30:70, v/v). The samples were evaporated to dryness under stream of nitrogen at 35°C and the residue reconstituted with 1 mL of MeCN:water (10:90,v/v). Defatting was attained by adding 3 mL of n-hexane. The lower layer was centrifuged at 10,000 rpm for 3 min and the extract was filtered through a 0.45 µm membrane filter into autosampler vials prior to LC–MS/MS analysis.
LC-MS/MS analysis
The analysis was performed with a Waters (Milford, MA, USA) quadrupole LC–MS/MS equipped with abinary pump, vacuum degasser, thermostated autosampler and thermostated column manager. MassLynx software (Waters, Milford, MA, USA) version 4.1 was used for instrument control, data acquisition and calculation of results. Chromatographic separation was carried out using an Kinetex C18 column (50 x 2.1 mm, 2.6 μm, Phenomenex, Torrance, CA, USA).
A gradient mobile phase was used where mobile phase A consisted of 0.1% formic acid solution in water containing 5 mM ammonium acetate and mobile phase B consisted of 0.1% formic acid solution in MeCN. The elution program was as follows: 0–1 min, 95-80 % A; 1–4 min, 80-60 % A; 4–8 min, 60-95 % A; 8-12 min, 95 % A while the flow rate was 0.2 mL/min. The column temperature was 40°C and injection volume was 10 µL.
The MS/MS acquisition was carried out using electrospray ionization positive and negative mode (ESI+ and ESI-). The main MS conditions were optimized and finally set as follows: capillary voltage of 3.0 kV; source temperature of 150°C; desolvation temperature of 400°C; cone gas at 100 L/h; and desolvation gas at 300 L/h. Three multiple reaction monitoring (MRM) transitions of banned analytes were chosen, while for analytes with permitted limits, 2 MRM transitions were chosen. Analysis of internal standards involved one MRM transition. The optimized MRM conditions for each analyte are given in Table 1.
Method validation
The analytical method was validated according to the European Commission decision 2002/657 and validation approach of the SANTE/11813/2017 guidelines. For the method validation were elaluated various parameters: linearity, accuracy, precision, limit of detection (LOD), limit of quantification (LOQ), Decision limit (CCα) and Detection capability (CCβ). The linearity of the method was evaluatedon the basis of matrix-match calibration. Instead of trueness the recovery parameter was determined because certified reference materials (CRMs) were not available.