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www.rsc.org/methods Page 1 of 24 Analytical Methods
1 2 3 1 A multi residue analysis of sulphonamides in edible animal tissues 4 5 2 6 using QuEChERS extraction and HPLC – MS/MS 7 8 3 Hiba Abdallah,ab Carine Arnaudguilhem,b Ryszard Lobinski bc & Farouk Jaber *ad 9
10 a 11 4 CNRSL, Lebanese Atomic Energy Commission (LAEC), Laboratory for Analysis of Organic Compound 12 5 (LAOC), Beirut, Lebanon 13 14 6 b CNRS/UPPA, Laboratory of Bio Inorganic Analytical and Environmental Chemistry (LCABIE), UMR5254, 15 7 16 Hélioparc, 2, Av. President Angot, 64053 Pau, France 17 c 18 8 Department of Chemistry, Warsaw University of Technology, ul. Noakowskiego 3, 00 664 Warsaw, Poland 19 20 9 d Laboratory of Analysis of Organic Compounds (509), Faculty of Sciences I, Lebanese University, Hadath, 21 10 Beirut, Lebanon Manuscript 22 23 24 11 Abstract 25 26 12 A HPLC double reaction monitoring MS/MS method was developed for the determination of 27 28 13 a wide range (>20) sulphonamide residues in several edible animal (sheep, pork, beef, 29 30 14 chicken and dromedary) tissues. Sample preparation was based on the simultaneous
31 15 extraction into acetonitrile solution followed by a clean up using primary secondary amine Accepted 32 33 16 beads. Quantification was carried out using matrix matched calibration curves. The limit of 34 1 35 17 detection (LODs) and limit of quantification (LOQs) ranged from 0.5 to 14.5 µg.kg and 36 18 from 1.8 to 48.4 µg.kg 1, respectively. Decision limit (CCα) and decision capability (CCβ) 37 38 19 obtained were below 100 µg.kg 1 for sulphonamides and below 5 µg.kg 1 for dapsone. The 39 20 method was validated in terms of recoveries and inter and intra day precision by reference
40 Methods 41 21 analyses of meat samples using LC Orbitrap MS and by the analysis of a reference material. 42 43 22 The method was applied to the analysis of several animal tissue samples collected in 44 45 23 Lebanon. The highest values were observed for sulfamethazine and sulfadimethoxine at 70.2 46 24 and 62.5 µg.kg 1 in sheep tissues. 47 48 49 * Corresponding author: Farouk Jaber 50 Analytical 51 Address : Laboratory for Analysis of Organic Compound Lebanese Atomic Energy Commission, B. P. 11 8281, 52 53 Riad El Solh 1107 2260 Beirut, Lebanon 54 55 Tel : 00 96 11 45 08 11 Fax : 00 96 11 45 08 10 56 E mail address: [email protected] 57 58 59 60 1 | P a g e
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1 2 3 25 Keywords: QuEChERS, mass spectrometry, sulphonamides residues, edible animal tissues. 4 5 6 26 7 8 27 1. Introduction 9 10 28 11 12 29 Sulphonamides (SAs) are the most common (after tetracyclines) veterinary antibiotics used in 13 14 30 the EU. They are relatively cheap and efficient to combat many common bacterial 15 1,2 16 31 infections. SAs are N substituted derivatives of the p aminobenzenesulfonic acid with 17 32 amphoteric properties. They can be metabolized in the animal body to produce N1 18 19 33 (oxidation) and N4 (acetylation) derivatives. Glucuronide conjugation and aromatic 20 3,4 21 34 hydroxylation can also take place leading to sulfinamide, AZO SAs or nitro SAs (Fig. 1). Manuscript 22 35 As a consequence of the extensive usage of SAs, their residues (parent compounds or 23 24 36 metabolites) can persist in edible tissues of farm animals.4 6 The exposure of consumers to 25 7,8 26 37 SAs can lead to allergies and hematological, gastrointestinal and neurological diseases. The 27 38 use of SAs in animals is regulated; according to the EU regulation 37/2010, SAs are 28 29 39 authorized substances whereas dapsone is a prohibited one. The maximum residue limit 30 40 (MRL) for the total amount of SAs in edible tissues, such as muscle, liver, kidney and milk, 31 Accepted 32 41 is 100 µg.kg 1 9 which requires the development of relevant monitoring analytical methods. 33 34 35 42 High performance liquid chromatography (HPLC) coupled to triple quadrupole mass 36 43 spectrometry (MS),10 13 operated in “Multiple Reaction Monitoring” (MRM) or “Selected 37 38 44 Reaction Monitoring” (SRM),10,12,14 16 mode, is a common technique of choice for a wide 39 45 range of chemical residues. The analytes are usually detected by monitoring the ions 40 Methods 41 46 corresponding to at least two mass transitions which, in combination with their 42 43 47 chromatographic retention time, offer sufficient analytical selectivity. The high throughput of 44 45 48 HPLC MS/MS analysis is dependent on the simultaneous multispecies efficient extraction 46 49 method. These criteria are fulfilled by leaching with aqueous acetonitrile solution followed 47 48 50 by the extract cleanup. This principle, referred to as QuEChERS (Quick, Easy, Cheap, 49 17 50 51 Effective, Rugged, Safe) was first developed for the extraction of pesticides but has been 51 Analytical 52 increasingly used for the recovery of veterinary drugs from various types of matrices, 52 53 53 offering an increased sample throughput and reducing the cost of analysis. 54 55 56 54 The literature concerning the simultaneous HPLC triple quad MS/MS analysis of 57 55 SAs residues in edible animal tissues is relatively scarce and limited to few tissue varieties 58 59 60 2 | P a g e
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1 2 3 56 (poultry and fish 16,18 20 ) and to a limited number of compounds (6 16 , 7 19 and 16 compounds 4 18 5 57 ). The objective of this work was to develop a wide scope method in terms of the number of 6 58 compounds determined (23 – the most complete list reported recently by non targeted high 7 8 59 resolution MS 21 ) and in terms of the variety of matrices analysed (sheep, chicken, beef, pork, 9 10 60 and dromedary kidney, liver and muscle). 11 12 61 13 14 62 2. Materials and methods 15 16 63 17 64 2.1. Reagents and samples 18 19 20 65 The structures of the studied SAs are summarized in Fig. 1 . SAs were obtained from 21 66 Ehrenstorfer (Augsburg, Germany) (Sulfaguanidine (SGN), Sulfadiazine (SD), Sulfathiazole Manuscript 22 23 67 (STZ), Sulfamerazine (SM), Sulfamethoxypyridazine (SMP), Sulfamonomethoxine (SMM), 24 25 68 Sulfadoxine (SDO), Sulfaphenazole (SNZ), Sulfadimethoxine (SDM) and Sulfaquinoxaline 26 69 (SQX)) and Sigma Aldrich (China) (Sulfacetamide (SAA), Sulfisomidine (SIM), 27 28 70 Sulfapyridine (SP), Sulfameter (SME), Sulfamethizole (SMT), Sulfamethazine (SMZ), 29 30 71 Sulfachloropyridazine (SCP), Sulfamethoxazole (SMX), Sulfisoxazole (SIX),
31 72 Sulfabenzamide (SB), Sulfanitrane (SNT), Sulfaclozine (SCL) and Dapsone (Da)). The Accepted 32 33 73 internal standard SMX D4 was obtained from C/D/N Isotopes (Pointe Claire, QC, Canada). 34 35 74 All the standards were of high purity grade (>95 %). 36 37 75 LC MS grade methanol (MeOH), acetonitrile (MeCN), acetic acid (AA) and formic 38 39 76 acid 98% (FA) were purchased from Honeywell (Germany), Fluka (Germany), Sharlu
40 TM Methods 41 77 (Spain) and BDH AnulaR (England), respectively. Water was purified using Easypure II 42 78 (Thermo Scientific, USA). For the “QuEChERS” extraction sodium citrate, sodium 43 44 79 hydrogencitrate sesquihydrate, magnesium sulfate and primary secondary amine were 45 46 80 purchased from Sigma Aldrich. Sodium chloride was purchased from Riedel de Haen. 47 81 Purified extracts were filtered through a 0.2 µm Ultrafree CL Centrifugal filter with a low 48 49 82 binding Durapore PVDF membrane (Millipore, France). 50 Analytical 51 83 Edible beef, sheep, chicken, pig and dromedary tissues (liver, kidney, muscle) were 52 53 84 collected from slaughterhouses and farms in Lebanon. A reference material (FAPAS pig 54 55 85 kidney N°02227) was obtained from the Food and Environment Research Agency (United 56 86 Kingdom). 57 58 59 60 3 | P a g e
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1 2 3 87 2.2. Standards solutions 4 5 88 Individual standard stock solutions (ca 1000 mg.l 1) were prepared by dissolving an 6 7 89 appropriate amount of each compound in MeCN and MeOH depending on their solubility. A 8 1 9 90 mixed standard working solution (10 mg.l ) used for the spiking of the control samples was 10 91 prepared by appropriate dilutions with MeCN. Another mixed standard working solution (1 11 12 92 mg.l 1) was prepared by dilution of the 10 mg.l 1 mixed standard working solution with the 13 14 93 initial mobile phase (water/MeOH 0.01% formic acid (95:5, v/v )). A working internal 15 94 standard solution (10 mg.l 1) of SMX D was prepared by dilution of the stock solution (ca. 16 4 17 95 550 mg.l 1) in MeCN. All stock and working solutions were stored in dark at 20 0C. 18 19 20 96 2.3. Extraction 21 Manuscript 22 97 Extraction and cleanup were based on QuEChERS extraction as described elsewhere.21 23 24 98 Extraction efficiency was evaluated using samples spiked with appropriate amounts of 25 99 solutions of SAs and SMX D (IS) at 100 µg.kg 1. A 5 g finely ground sample of meat was 26 4 27 100 weighed. Then, 5 mL of water and 10 mL 1% acetic acid in MeCN (v/v) were added to the 28 29 101 sample. After agitation for 1 min, 0.5 g of sodium hydrogencitrate sesquihydrate, 1.0 g 30 102 sodium citrate, 4.0 g of anhydrous magnesium sulfate and 1g of sodium chloride were added.
31 Accepted 32 103 The mixture was vigorously shaken, vortexed for 1 min and centrifuged at 3500 rpm for 5 33 34 104 min. 6 mL of the supernatant was purified with 150 mg of primary secondary amine and 900 35 105 mg of anhydrous magnesium sulfate followed by shaking and centrifugation in the conditions 36 0 37 106 as above. 4 mL of the supernatant was evaporated to dryness with N 2 (35 C), reconstituted 38 39 107 with 500 µl 0.01% (v/v) formic acid in 95% (v/v) MeOH and then filtered through a 0.2µm
40 108 PVDF, low binding Durapore (Millipore) filter. Methods 41 42 2 43 109 2.4. Liquid chromatography–mass spectrometry (LC–MS ) 44 45 110 Chromatographic analysis was performed using an Agilent 1200 HPLC system (Agilent, 46 47 111 USA). Separations were achieved with a Zorbax Eclipse XDB C8 column (3.5µm, 2.1 100 48 49 112 mm, Agilent). The column was kept at 30 0C. The flow rate and injection volume were 0.2 50 51 113 ml/min and 5 L, respectively. The mobile phases used were: (A) 0.01% formic acid and (B) Analytical 52 114 0.01% formic acid in MeOH. The gradient elution program was: 0 10 min (5% 10%) B, 10 53 54 115 12 min (10% 50%) B, 12 15 min (50% 100%) B, 15 17 min (100%) B. Then, the elution 55 56 116 gradient was linearly ramped down to 5 % B for 2 min and maintained for 11 min to allow 57 117 the conditioning of the column prior to next injection. 58 59 60 4 | P a g e
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1 2 3 118 Mass spectrometry analysis was carried out using an Agilent 6410 electrospray triple 4 5 119 quadrupole mass spectrometer operated in positive mode. All the SAs were measured in the 6 120 same chromatographic run by tandem MS carried out in the MRM acquisition mode. Two 7 8 121 precursor to product ion transitions were monitored of each analyte (Table 1) . The most 9 10 122 intense transition was used for quantification (“quantification transition”) and the second 11 123 transition for confirmation of the presence of the analyte (“confirmation transition”). 12 13 14 124 The optimization of MS parameters (precursor ions, skimmer voltage, collision 15 125 energy, and quantification and confirmation transitions) was performed by flow injection 16 17 126 analysis for each compound dissolved in the mobile phase. Data acquisition was carried out 18 19 127 using MassHunter software (Agilent). 20 21 128 2.5. Validation Manuscript 22 23 24 129 Linearity, accuracy, intra day and inter day precision, limits of detection (LOD) and 25 130 quantification (LOQ), decision limit (CC ), detection capability (CC ) and stability were 26 α β 27 131 studied to validate the whole procedure according to the European Commission 28 22 29 132 2002/657/EEC recommendations . SAs quantification was performed using matrix matched 30 133 calibration. Linearity was verified by spiking meat samples with the target compounds at 5
31 Accepted 32 134 levels (blank, 50, 100, 150, 200 µg.kg 1) for SAs and (blank, 1.25, 2.5, 3, 5 µg.kg 1) for 33 1 34 135 dapsone and a fixed concentration of SMX D4 (100 µg.kg ). Calibration curves were 35 136 obtained by least squares linear regression analysis of the peak area versus concentration 36 37 137 corrected with a deuterated internal standard SMX D4. 38 39 138 Accuracy of the method was assessed by determining the concentration of 3
40 Methods 1 1 41 139 uncontaminated meat samples spiked with 100 g.kg and 5 g.kg of SAs and dapsone 42 43 140 respectively, using matrix matched calibration, and comparing the calculated concentration 44 45 141 with the theoretical concentration. Precision (intra and inter day) was investigated at the 46 142 same concentration level. The values of CC α and CC β were calculated for all analytes using a 47 48 143 matrix matched calibration curve. CCα was calculated at the statistical certainty of 1−α (α = 49 50 144 0.05 for authorized compounds and 0.01 for unauthorized compounds) and CC β for 1−β (β= Analytical 51 145 0.05 for both authorized and unauthorized compounds) to detect the concentration at the 52 1 23,24 53 146 spiked levels 100 and 5 µg.kg for SAs and dapsone, respectively. LOD and LOQ were 54 55 147 determined as the lowest amount of analyte which could be detected and quantified, 56 148 respectively. The LOD and LOQ were estimated at 3 and 10 times the standard deviation of 57 1 58 149 the response obtained for 10 samples spiked at 25 and 5 µg.kg for SAs and dapsone 59 60 5 | P a g e
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1 2 3 150 respectively, divided by the slope of the calibration curve. All the experiments were repeated 4 5 151 for each concentration level on 3 different days. The stability was assessed by spiking beef 6 152 samples at different concentration (50 (1.25), 100 (2.5), 150 (3) and 200 (5) µg.kg 1 for SAs 7 8 153 (and dapsone) compounds, respectively, stored at 18 0C for 12 weeks. 9 10 154 11 12 13 155 3. Results and Discussion 14 15 156 21 16 The choice of the analytes was made to match the most complete list reported so far. 17 18 157 3.1. Extraction procedure 19 20 158 The extraction procedure was developed for beef muscle in a previous study.21 In order to 21 Manuscript 22 159 evaluate the efficiency of this procedure, samples of liver, muscle and kidney derived from 23 1 1 24 160 beef and pork were spiked at 100 µg.kg of SAs and 5 µg.kg of dapsone. 25 26 161 For pork matrices (Fig.2a) recoveries of 70 120% for 19 SAs and of 4 SAs 50 70% 27 28 162 were achieved for muscle and kidney samples. Most SAs were extracted from liver with 29 30 163 recoveries higher than 50%. For beef matrices (Fig.2b), QuEChERS allowed the extraction
31 164 recoveries of 70 120% for 21 SAs from kidney and for 16 SAs in muscle tissues. In liver, Accepted 32 33 165 most of the tested SAs yielded recoveries of 50 70%. Similar results were obtained for sheep 34 35 166 (Fig.2c ) ( 70 100% from muscle and kidney and 54 80% from liver samples) and dromedary 36 167 (Fig.2d) (70 120% from muscle and kidney and 60 90% from liver samples), and chicken 37 38 168 (70 90% and 55 70%, respectively) (Fig.2e). In general, the recoveries decreased in the order 39 169 kidneys > muscle > liver for beef, pork, sheep, and in the order: muscle > kidneys > liver for
40 Methods 41 170 dromedary. 42 43 44 171 3.2. LC MS/MS determination 45 46 172 ESI source and positive ionization mode were selected due to the presence of primary or 47 48 173 secondary amino groups in the SAs. MRM mode was applied; two transitions per analyte 49 50 174 were selected. The more sensitive one was used for quantitation whereas the other one for the Analytical 51 175 identity confirmation. A typical Total Ion Count (TIC) chromatogram for a beef muscle 52 53 176 spiked with 23 SAs at the fixed levels (100 and 5 µg.kg 1 for SAs and dapsone respectively) 54 55 177 is shown in Fig. 3. 56 57 58 59 60 6 | P a g e
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1 2 3 178 Typical fragment ions were observed for most SAs at m/z 156 (cleavage of the S N 4 + 5 179 bond [M RNH 2] ), m/z 108 (elimination of the RNH 2SO group) and at m/z 92 (cleavage of 6 + 180 the M RNH 2 SO 2 group] . Another number of specific transitions were detected for some 7 8 181 compounds due to the variable amine substituent, such as, e.g., ions of m/z 124 and 186 for 9 10 182 SIM and SMZ, m/z 215 for SME and SMM, m/z 184, 126, 113 and 130 for SP, SMP, SIX and 11 183 SCL, respectively. For the di substituted SAs, SNT was detected at m/z 134 and 156. The 12 13 184 values of the parameters optimized and the MRM transitions selected are given in Table 1 . 14 15 185 3.3. Figures of merit 16 17 18 186 The monitoring of SAs in the animal tissues was performed by Multiple Reaction Monitoring 19 20 187 (MRM). The identity confirmation was accomplished by comparing the retention time and 21 188 the ion ratio of the 2 transitions within 2% and 20%, respectively. The quantification was Manuscript 22 23 189 performed with the most intense transition by matrix matched calibration. The method was 24 25 190 validated following the criteria defined in the Decision 2002/657/EC for quantitative 26 191 confirmatory methods.22 Method detection limit (LOD), quantitation limit (LOQ), precision 27 28 192 (intra day and inter day), accuracy, decision limit (CC α), detection capability (CC β) and 29 30 193 stability were evaluated for all compounds using spiked beef tissue. No SAs compound was
31 194 detected in any of the blank beef tissue samples. Accepted 32 33 34 195 Linearity was that of a matrix matched calibration curve obtained by spiking a beef 35 196 tissue with the selected antibiotics in the range from 50 to 200 and 1 to 5 µg.kg 1 for SAs and 36 37 197 dapsone, respectively. A correlation coefficient (R 2) higher than 0.990 was obtained for all 38 39 198 the compounds, except for SNT (Table 2 ). Accuracy (expressed as A (%) = mean measured
40 199 concentration * 100 / theoretical concentration), intra day and inter day precision (expressed Methods 41 42 200 as Relative Standard Deviation, RSD ) of the analytical method were assessed by the analysis 43 1 44 201 of 3 different samples spiked at 100 and 5 µg.kg levels for SAs and dapsone, respectively. 45 202 The analysis was performed by the same operator on three separate days (3 experiments per 46 47 203 day) (Table 2 ). The A% value varied from 71% to 117%. The inter day precision ( RSD R ) 48 49 204 values were below 23% except for SGN and SNT and the intra day precision ( RSD r ) below 50 205 15% for all SAs except for SNT. These results obtained for A% , RSD R and RSD r are Analytical 51 52 206 consistent for all the analytes with the requirements of the 2002/657/EC decision.22 53 54 55 207 The decision limit (CC α) was defined as “the limit at and above which it can be 56 208 concluded with an error probability of α that a sample is non compliant”, and the detection 57 58 209 capability (CC β) as “the smallest content of the substance that may be detected, identified 59 60 7 | P a g e
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1 2 3 210 and/or quantified in a sample with an error probability of β”. In the case of SAs, α and β 4 5 211 errors were set at 5% (authorized antibiotics) and 1% in the case of dapsone (unauthorized 6 212 antibiotic). The decision limit (CC α) and the detection capability (CC β) were calculated from 7 8 213 the matrix matched calibration curve using the ISO 11843 method by using the following 9 24 10 214 equations: 11 12 13 14 15 215 16 17 18 19 216 20 21 217 Manuscript 22 23 218 where a is the slope of the regression line which equals the recovery of the analyte, 24 25 219 CMRL is the MRL value of the analyte, tv,α the associated t value, σ is an estimation of the 26 220 residual standard deviation of the regression function, I the number of replicates per 27 28 221 concentration, J the number of concentrations of the spiked samples, xMRL is the referenced 29 30 222 MRL value of the analyte, x is the mean of the xij values (Eq.1) and δ v,α,β is a statistical
31 Accepted 223 function that can be fairly approximated by 2 tv,α (Eq.2). 32 33 34 224 The results reported for CC α and CC β values in Table 2 ranged from 101 to 118 35 225 µg.kg 1 which is similar to the SAs MRL level. For dapsone, CC and CC values were 0.5 36 α β 37 226 and 0.6 µg.kg 1 respectively, which is less than the lowest spiked concentration. We can thus 38 39 227 conclude that the developed method is applicable for the detection of SAs and dapsone with a
40 228 statistical certainty of 95 and 99%, respectively. In comparison with the values reported in Methods 41 10,15,18 42 229 literature for SAs, the calculated CC α and CC β values from this study are equal (in most 43 44 230 cases) indicating a high sensitivity of the reported methodology. 45 46 231 The limit of detection (LOD) is the smallest value of the concentration of an analyte 47 48 232 which can be detected and the limit of quantification (LOQ) is the smallest value of the 49 233 50 concentration of an analyte which can be quantified. These limits were calculated as the Analytical 51 234 standard deviation (SD) of the intensity obtained for tissues spiked at levels close to the LOD 52 53 235 and LOQ divided by the slope (a) of the calibration curve according to the formulae: LOD = 54 236 55 3.3 (SD/a) and LOQ = 10 (SD/a). In each case, LOD was found to be lower than the MRL 56 237 and ranged from 1.7 to 15 µg.kg 1; LOQ ranged from 5.8 to 49.7 µg.kg 1for SGN and SQX, 57 58 59 60 8 | P a g e
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1 2 3 238 respectively. For dapsone, LOD and LOQ values of 0.5 and 1.8 µg.kg 1, respectively, were 4 5 239 found (Table 2 ). 6 7 240 In order to evaluate the stability of 23 SAs in meat samples, different beef muscles 8 9 241 were spiked with the analytes at 4 concentration levels (50 (1.25), 100 (2.5), 150 (3) and 200 10 242 (5) µg.kg 1 for SAs (and dapsone) respectively, stored at 18 0C for 1, 2, 6 and 12 weeks and 11 12 243 extracted as described in section 2.3. All SAs were found to be stable for at least 12 weeks at 13 0 14 244 18 C (Appendix A, Fig. A1 ). 15 16 245 3.4. Analysis of marketed samples 17 18 19 246 The developed method was tested on different matrices (kidney, muscle, liver) collected from 20 247 beef, pig, sheep, chicken and dromedary. Forty samples were analyzed: 12 beef (6 muscle, 3 21 Manuscript 22 248 liver and 3 kidney), 12 sheep (4 muscle, 4 liver and 4 kidney), 8 pig (4 muscle, 2 liver and 2 23 24 249 kidney), 4 chicken (2 muscle and 2 liver) and 4 dromedary (2 muscle, 1 liver and 1 kidney). 25 250 The concentrations of the detected compounds are summarized in Table 4. LC/MS/MS 26 27 251 chromatograms obtained for samples S5 and S20 are shown in Appendix B, Fig. B1 . 28 29 30 252 Seventeen samples showed the presence of SDM, SMZ, SD and SQX with some 1 31 253 detected at MRL/2 40 Methods 41 259 retention time was successful in all cases. 42 43 260 44 261 3.5. Quality assurance 45 46 262 47 263 The samples with the detectable concentrations of SAs were analysed using HPLC Orbitrap 48 49 264 MS according to the procedure reported elsewhere.21 Note that in terms of precision, 50 51 265 linearity, accuracy, CC α and CC β, HR Orbitrap MS matches the LC MS/MS performance for Analytical 52 266 the most compounds but shows lower sensitivity. The results presented in Fig. C1 show good 53 54 267 (R 2 > 0.992) correlation between LC Orbitrap MS and LC MS/MS. 55 56 268 57 58 59 60 9 | P a g e Analytical Methods Page 10 of 24 1 2 3 269 A FAPAS (Food Analysis Performance Assessment Scheme) test material 02227 of 4 1 5 270 pig kidney containing SP, SMZ and SDO at concentrations of 120, 68.7 and 63.4 µg.kg was 6 271 analysed. The concentrations found for SP, SMZ and SDO were 106, 44 and 62 µg.kg 1 with 7 8 272 Z scores 0.52, 1.6 and 0.53, respectively. The fitness for purpose of the presented method 9 10 273 was thus demonstrated with all 2 ≤ Z scores ≤ 2 (Table 3 ). 11 12 274 13 275 4. Conclusion 14 15 16 276 HPLC – double reaction monitoring MS/MS preceded by HPLC allows a rapid, sensitive, 17 277 18 precise and accurate determination of 23 sulfonamides in different edible animal tissues 19 278 required to be monitored by the European Commission 2002/657/EEC decision. The method 20 21 279 offers lower detection limits (1 3 times) in comparison with the use of Orbitrap mass analyser Manuscript 22 280 23 making triple Quad MS better for quantitative analysis. However, unlike MS/MS, the 24 281 Orbitrap MS methods using both “full scan” and “MS n” mode may offer the possibility to 25 26 282 explore sets of data retrospectively. 27 28 283 Acknowledgements 29 30 284 We thank the Lebanese National Council for Scientific Research (CNRSL) and the Lebanese 31 Accepted 32 285 Atomic Energy Commission (LAEC) for financial support. 33 34 35 286 Caption to the tables 36 37 287 Table 1: SAs retention time (RT) and MS/MS parameters 38 39 288 Table 2: Extraction recovery, AR (%) and validation parameters of analysis of SAs in beef 40 289 muscle using QuEChERS LC MSMS Methods 41 42 290 Table 3: Results for analysis of CRM (pig kidney) using QuEChERS extraction method, LC 43 291 MSMS and Orbitrap MS 44 45 292 Table 4: Concentration of SAs in positively analysed samples 46 293 47 48 294 Caption to the figures 49 50 295 Fig.1: Structures of SAs antibiotics Analytical 51 52 296 Fig.2: Recovery values (%) obtained from a) pork samples, b) beef, c) sheep, d) dromedary 53 54 297 and e) chicken with selected buffered QuEChERS method 55 56 298 Fig.3: Total Ion Counts (TIC) of a beef muscle spiked with 23 sulphonamides at the fixed 57 299 levels: 100 µg.kg 1 and 5 µg.kg 1 for SAs and dapsone, respectively. a) SGN, b) SAA, c) SD, 58 59 60 10 | P a g e Page 11 of 24 Analytical Methods 1 2 3 300 d) SIM, e) SMZ, f) STZ, g) SP, h) SM, i) SME, j) SMP, k) SMM, l) SMT, m) Da, n) SCP, o) 4 301 SCL, p) SMX, q) SDO, r) SDM, s) SIX, t) SB, u) SNZ, v) SQX, w) SNT and x) SMX D4 5 6 302 Captions to supplementary data 7 8 9 303 Appendix A 10 11 304 Fig.A1: Stability of SAs in spiked beef muscle at different concentrations a) 50 (1.25), b) 100 12 305 (2.5), c) 150 (3) and d) 200 (5) µg.kg 1 for SAs and dapsone, respectively . 13 14 306 Appendix B 15 16 17 307 Fig.B1: LC MS/MS chromatograms of some analysed samples (a) S5 containing SDM (59.1 18 308 µg.kg 1) and (b) S20 containing SMZ (70.2 µg.kg 1) 19 20 309 Appendix C 21 Manuscript 22 310 Fig.C1: Correlation graph of concentrations (µg.kg 1) determined of studied compounds with 23 311 triple quadrupole mass spectrometry (QqQ) and Orbitrap analyzers in meat samples 24 312 25 26 313 27 314 28 315 29 316 30 317 31 318 Accepted 32 319 33 320 34 321 35 36 322 37 323 38 324 39 325 40 326 Methods 41 327 42 328 43 329 44 330 45 46 331 47 332 48 333 49 334 50 335 Analytical 51 336 52 337 53 338 54 339 55 56 340 57 341 58 342 59 60 11 | P a g e Analytical Methods Page 12 of 24 1 2 3 343 References 4 5 6 344 1 L. Kantiani; M. Llorca; J. Sanchís; M. Farré; D. Barceló, Emerging food contaminants: a 7 345 review, Anal. Bioanal. Chem. , 2010, 398 , 2413. 8 9 346 2 S. A. E. Kools; J. F. Moltmann; T. Knacker, Estimating the use of veterinary medicines in 10 347 11 the European union, Regul. Toxicol. Pharm. , 2008, 50 , 59. 12 13 348 3 M. J. García Galán; M. Silvia Díaz Cruz; D. Barceló, Identification and determination of 14 349 metabolites and degradation products of sulfonamide antibiotics, TrAC Trends Anal. Chem. , 15 350 2008, 27 , 1008. 16 17 351 4 C. Huang; B. Guo; X. Wang; J. Li; W. Zhu; B. Chen; S. Ouyang; S. Yao, A generic 18 352 approach for expanding homolog targeted residue screening of sulfonamides using a fast 19 353 matrix separation and class specific fragmentation dependent acquisition with a hybrid 20 21 354 quadrupole linear ion trap mass spectrometer, Anal. Chim. Acta , 2012, 737 , 83. Manuscript 22 23 355 5 M. M. Aguilera Luiz; J. L. Martínez Vidal; R. Romero González; A. Garrido Frenich, 24 356 Multiclass method for fast determination of veterinary drug residues in baby food by ultra 25 357 high performance liquid chromatography–tandem mass spectrometry, Food Chem. , 2012, 26 358 132 , 2171. 27 28 359 6 D. Hurtaud Pessel; T. Jagadeshwar Reddy; E. Verdon, Development of a new screening 29 360 method for the detection of antibiotic residues in muscle tissues using liquid chromatography 30 361 and high resolution mass spectrometry with a LC LTQ Orbitrap instrument, Food Addit. 31 Accepted 32 362 Contam. Part A , 2011, 28 , 1340. 33 34 363 7 W. Baran; E. Adamek; J. Ziemiańska; A. Sobczak, Effects of the presence of sulfonamides 35 364 in the environment and their influence on human health, J. Hazard. Mater. , 2011, 196 , 1. 36 37 365 8 R. S. Gruchalla, 10. Drug allergy, J. Allergy Clin. Immunol. , 2003, 111 , S548. 38 39 366 9 Commission Regulation (EC) No. 37/2010 of 22 December 2009 on pharmacologically 40 Methods 41 367 active substances and their classification regarding maximum residue limits in foodstuffs of 42 368 animal origin. Off. J. Eur. Commun. , 2010, 15 , 1. 43 44 369 10 H. Yu; Y. Tao; D. Chen; Y. Wang; L. Huang; D. Peng; M. Dai; Z. Liu; X. Wang; Z. 45 370 Yuan, Development of a high performance liquid chromatography method and a liquid 46 371 chromatography–tandem mass spectrometry method with the pressurized liquid extraction for 47 372 the quantification and confirmation of sulfonamides in the foods of animal origin, J. 48 373 Chromatogr. B , 2011, 879 , 2653. 49 50 51 374 11 C. Chiaochan; U. Koesukwiwat; S. Yudthavorasit; N. Leepipatpiboon, Efficient Analytical 52 375 hydrophilic interaction liquid chromatography–tandem mass spectrometry for the multiclass 53 376 analysis of veterinary drugs in chicken muscle, Anal. Chim. Acta, 2010, 682 , 117. 54 55 377 12 P. A. Martos; F. Jayasundara; J. Dolbeer; W. Jin; L. Spilsbury; M. Mitchell; C. Varilla; B. 56 378 Shurmer, Multiclass, multiresidue drug analysis, including aminoglycosides, in animal tissue 57 58 59 60 12 | P a g e Page 13 of 24 Analytical Methods 1 2 3 379 using liquid chromatography coupled to tandem mass spectrometry, J. Agric. Food Chem. , 4 380 2010, 58 , 5932. 5 6 381 13 H. Li; H. Sun; J. Zhang; K. Pang Highly, Sensitive and simultaneous determination of 7 382 sixteen sulphonamide antibiotics, four acetyled metabolites and trimethoprim in meat by 8 9 383 rapid resolution liquid chromatography tandem mass spectrometry, Food Control , 2013, 31 , 10 384 359. 11 12 385 14 L. Kantiani; M. Farré; J. M. G. i. Freixiedas; D. Barceló, Development and validation of a 13 386 pressurised liquid extraction liquid chromatography–electrospray–tandem mass spectrometry 14 387 method for β lactams and sulfonamides in animal feed, J. Chromatogr. A, 2010, 1217 , 4247. 15 388 16 389 15 M. McDonald; C. Mannion; P. Rafter, A confirmatory method for the simultaneous 17 390 extraction, separation, identification and quantification of Tetracycline, Sulphonamide, 18 19 391 Trimethoprim and Dapsone residues in muscle by ultra high performance liquid 20 392 chromatography–tandem mass spectrometry according to Commission Decision 21 393 2002/657/EC, J. Chromatogr. A , 2009, 1216 , 8110. Manuscript 22 23 394 16 R. P. Lopes; R. C. Reyes; R. Romero González; A. G. Frenich; J. L. M. Vidal, 24 395 Development and validation of a multiclass method for the determination of veterinary drug 25 396 residues in chicken by ultra high performance liquid chromatography–tandem mass 26 397 spectrometry, Talanta , 2012, 89 , 201. 27 28 29 398 17 I. S. Jeong; B. M. Kwak; J. H. Ahn; S. H. Jeong, Determination of pesticide residues in 30 399 milk using a QuEChERS based method developed by response surface methodology, Food 400 31 Chem. , 2012, 133 , 473. Accepted 32 33 401 18 G. Stubbings; T. Bigwood, The development and validation of a multiclass liquid 34 402 chromatography tandem mass spectrometry (LC–MS/MS) procedure for the determination of 35 403 veterinary drug residues in animal tissue using a QuEChERS (QUick, Easy, CHeap, 36 404 Effective, Rugged and Safe) approach, Anal. Chim. Acta , 2009, 637 , 68. 37 38 39 405 19 M. Villar Pulido; B. Gilbert López; J. F. García Reyes; N. R. Martos; A. Molina Díaz, 406 Multiclass detection and quantitation of antibiotics and veterinary drugs in shrimps by fast 40 Methods 41 407 liquid chromatography time of flight mass spectrometry, Talanta , 2011, 85 , 1419. 42 43 408 20 R. P. Lopes; R. C. Reyes; R. Romero González; J. L. M. Vidal; A. G. Frenich, 44 409 Multiresidue determination of veterinary drugs in aquaculture fish samples by ultra high 45 410 performance liquid chromatography coupled to tandem mass spectrometry, J. Chromatogr. B , 46 411 2012, 895–896 , 39. 47 48 49 412 21 H. Abdallah; C. Arnaudguilhem; F. Jaber; R. Lobinski, Multiresidue analysis of 22 50 413 sulfonamides and their metabolites in animal tissues using quick, easy, cheap, effective, 51 414 rugged, and safe extraction and high resolution mass spectrometry (hybrid linear ion trap Analytical 52 415 Orbitrap), J. Chromatogr. A ., 2014, 61 72 , 1355. 53 54 416 22 Commission Decision 2002/657/EEC. Implementing Council Directive 96/ 23/EC 55 417 concerning the performance of analytical methods and the interpretation of results, Off. J. 56 418 Eur. Commun ., 2002, L 221 , 8. 57 58 59 60 13 | P a g e Analytical Methods Page 14 of 24 1 2 3 419 23 R. J. B. Peters; Y. J. C. Bolck; P. Rutgers; A. A. M. Stolker; M. W. F. Nielen, Multi 4 420 residue screening of veterinary drugs in egg, fish and meat using high resolution liquid 5 421 chromatography accurate mass time of flight mass spectrometry, J. Chromatogr. A , 2009, 6 422 1216 , 8206. 7 8 9 423 24 E.Verdon; D. Hurtaud Pessel; P. Sanders, Evaluation of the limit of performance of an 10 424 analytical method based on a statistical calculation of its critical concentrations according to 11 425 ISO standard 11843: Application to routine control of banned veterinary drug residues in 12 426 food according to European Decision 657/2002/EC, Accred. Qual. Assur. , 2006, 11 , 58. 13 14 427 15 16 428 17 18 19 20 21 Manuscript 22 23 24 25 26 27 28 29 30 31 Accepted 32 33 34 35 36 37 38 39 40 Methods 41 42 43 44 45 46 47 48 49 50 51 Analytical 52 53 54 55 56 57 58 59 60 14 | P a g e Page 15 of 24 Analytical Methods 1 2 3 4 5 429 6 Table 1:SAs retention time (RT) and MS/MS parameters. 7 8 Analyte RT(min) Quantification transition Cone Confirmation transition Ion ratio * (%) 9 m/z (collision energy V) voltage (V) m/z (collision energy V) 10 11 12 SGN 1.9 214.9 > 156 (10) 60 214.9 > 108 (20) 55.1 13 SAA 4.94 215 > 156 (5) 60 214.9 > 107.9 (15) 58.9 14 SD 8.8 251.2 > 92 (25) 60 251.2 > 108 (20) 92.5 15 SIM 9.8 279.1 > 124 (20) 60 279.1 > 186 (15) 48.0 16 STZ 12.9 256 > 156 (10) 60 256 > 108 (20) 85.0 Manuscript 17 SP 13.8 250.2 > 156 (10) 80 250.2 > 184.1 (15) 55.0 18 SM 15.3 265 > 156 (10) 60 265 > 172 (1 0) 84.4 19 20 SME 17.8 281 > 155.9 (15) 60 281 > 215.1 (15) 28.8 21 SMZ 18 279 > 186 (15) 60 279 > 124 (15) 79.9 22 SMT 18 271 > 155.9 (10) 60 271 > 107.9 (25) 62.5 23 Da 18.1 249 > 108 (20) 120 249 > 156 (10) 80.7 Accepted 24 SMP 18.3 281.6 > 108 (25) 60 281.6 > 126.5 (20) 65.2 25 SCP 18.5 285 > 156 (10) 100 285 > 107.9 (25) 71.2 26 SMX 18.7 254.1 > 108 (25) 100 254.1 > 156 (10) 79.5 27 SMM 18.8 281 > 155.9 (15) 60 281 > 215 (15) 65.4 28 SDO 18.8 311.2 > 156 (15) 60 311.2 > 107.9 (25) 73.4 29 SIX 18.9 268.2 > 156 (10) 60 268.2 > 112.9 (10) 94.6 30 Methods 31 SB 19.17 277.2 > 156 (5) 60 277.2 > 108 (20) 65.0 32 SCL 19.4 285 > 155.7 (10) 60 285 > 130 (25) 24.2 33 SNZ 19.3 315.5 > 92 (35) 60 315.5 > 156 (30) 77.4 34 SDM 19.4 311.2 > 156 (20) 60 311.2 > 107.9 (30) 58.7 35 SQX 19.5 301.1 > 156 (15) 60 301.1 > 108 (25) 94.1 36 SNT 20.1 336.1 > 133.9 (25) 120 336.1 > 156 (5) 76.6 37 18.6 258.1 > 96 (25) 70 258.1 > 160 (10) 62.5 SMX D4 Analytical 38 430 *Relative intensity of the analytical response of the confirmation transition / quantification transition 39 431 40 41 42 15 | P a g e 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Analytical Methods Page 16 of 24 1 2 3 4 5 432 Table 2: Extraction recovery, AR (%) and validation parameters of analysis of SAs in beef muscle using QuEChERS LC MSMS 6 433 7 (1) 2(3) (6) (7) (8) (9) SAs AR % Accuracy R RSD RSD LOD LOQ CC α CC β 8 A (2) (%) r(4) % R(5) % (µg.kg 1) (µg.kg 1) (µg.kg 1) (µg.kg 1) 9 10 SGN 53 111 0.995 11.7 34.3 1.7 5.8 109 118 11 SAA 70 105 0.996 4.7 18.4 11.7 39.2 106 113 12 SD 84 88 0.996 2.6 7.5 6.5 21.8 103 106 13 SIM 70 95 0.997 2.9 12.7 14.4 48.0 103 106 14 STZ 73 100 0.996 4.3 9.4 13.7 45.8 102 104 15 SP 81 99 0.998 2.3 2.4 14.2 47.5 103 106 16 SM 83 95 0.998 3.8 4.7 13.4 44.7 103 105 Manuscript 17 SME 82 92 0.997 2.5 8.7 14.5 48.4 103 107 18 19 SMZ 82 96 0.997 2.5 6.3 5.5 18.5 103 105 20 SMT 70 101 0.993 4.3 16.7 10.8 35.9 106 112 21 Da 54 97 0.992 8.7 9 0.5 1.8 0.5 0.6 22 SMP 75 98 0.998 3.9 7.1 10.6 35.3 103 105.6 23 SCP 75 99 0.999 4.1 5.8 5.1 17.0 105.5 111 Accepted 24 SMX 81 97 0.999 2.5 4.9 2.4 8 102 105 25 SMM 78 98 0.999 2.6 9.5 5.3 17.6 102 104 26 SDO 84 91 0.997 2.6 6.2 10.4 34.8 101 103 27 SIX 77 100 0.998 2.3 4.0 12 40 103 106 28 29 SB 64 104 0.995 2.8 6.3 8.7 29.2 105 111 30 SCL 65 98 0.998 5.2 9.9 11.8 39.2 105 109 Methods 31 SNZ 73 94 0.997 5.6 6.9 7.1 24 105 110 32 SDM 70 99 0.998 6.6 7.6 13.4 44.6 102 104 33 SQX 60 96 0.997 9.6 11.7 15 49.7 105 111 34 SNT 42 71 0.960 17 47.8 103.5 107 35 434 (1) Absolute recovery (AR); (2) Accuracy (A); (3) Squared regression coefficient (R 2); (4) Relative standard deviation of intra day precision (RSD r); (5) Relative standard deviation of inter day precision (RSD R); (6) Limit 36 (7) (8) (9) 435 of detection (LOD); Limit of quantification (LOQ); Decision limit (CC α); Detection capability (CC β). 37 38 Analytical 39 40 41 42 16 | P a g e 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 17 of 24 Analytical Methods 1 2 3 436 Table 3: Results for the analyses of CRM (pig kidney) using QuEChERS extraction method, 4 5 437 LC MSMS and Orbitrap MS 6 7 (1) (2) Analyte C target (µg.kg 1) C (µg.kg 1) Z scores 8 9 SDO 63.4 MS/MS 62 0.10 10 Orbitrap 73 0.68 11 68.7 MS/MS 44 1.63 12 SMZ 13 Orbitrap 14 SP 120 MS/MS 106 0.53 15 Orbitrap 98 0.81 (1) (2) 16 438 Calculated concentration obtained by FAPAS; Calculated concentration obtained by our method; 17 439 18 440 19 441 20 442 21 443 Manuscript 22 444 23 445 24 446 25 26 447 27 448 28 449 29 30 31 Accepted 32 33 34 35 36 37 38 39 40 Methods 41 42 43 44 45 46 47 48 49 50 51 Analytical 52 53 54 55 56 57 58 59 60 17 | P a g e Analytical Methods Page 18 of 24 1 2 3 4 5 450 6 451 7 452 Table 4: Concentration of SAs in positive analysed samples 8 9 Sample Sample SDM SMZ SD SQX 10 number type Concentration Ion Concentration Ion Concentration Ion Concentration Ion 1 1 1 1 11 (µg.kg ) Ratio (µg.kg ) Ratio % (µg.kg ) Ratio % (µg.kg ) Ratio % 12 % 13 S4 BM MS/MS 70.4 58.1 14 Orbitrap 62.5 15 S5 BM MS/MS 63.0 58.2 LOD 23 S17 SL LOD 30 S22 SK 23.2 80.6 Methods 31 S25 SK LOD 454 Analytical 38 Kidney 39 40 41 42 18 | P a g e 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 19 of 24 Analytical Methods 1 2 3 4 5 6 7 Sulfamethoxazole D4 (SMX D4) Sulfaguanidine (SGN) Sulfacetamide (SAA) Dapsone (DA) 8 9 10 11 12 Sulfadiazine (SD) Sulfisomidine (SIM) Sulfathiazole (STZ) Sulfapyridine (SP) 13 14 15 16 17 18 Sulfamerazine (SM) Sulfameter (SME) Sulfamethizole (SMT) Sulfamethazine (SMZ) 19 20 21 Manuscript 22 23 24 Sulfamethoxypyridazine (SMP) Sulfachloropyridazine(SCP) Sulfamethoxazole(SMX) Sulfamonomethoxine (SMM) 25 26 27 28 29 30 Sulfadoxine (SDO) Sulfisoxazole (SIX) Sulfabenzamide (SB) Sulfaphenazole (SNZ) 31 Accepted 32 33 34 35 36 37 Sulfadimethoxine (SDM) Sulfaquinoxaline (SQX) Sulfaclozine (SCL) Sulfanitrane (SNT) 38 455 39 456 Fig.1: Structures of SAs antibiotics. 40 Methods 41 42 457 43 44 45 46 47 48 49 50 51 Analytical 52 53 54 55 56 57 58 59 60 19 | P a g e Analytical Methods Page 20 of 24 1 2 3 120 a) Pork 4 liver muscle kidney 5 90 6 7 60 8 30 9 Recovery% 10 0 11 12 13 Name of compounds 14 120 b) Beef 15 Liver Muscle Kidney 16 90 17 18 60 19 30 20 Recovery% 21 Manuscript 22 0 23 24 Name of compounds 25 26 120 c) Sheep liver muscle kidney 27 28 90 29 60 30 31 Accepted Recovery% 30 32 33 0 34 35 Name of compounds 36 37 120 d) Dromedary 38 liver muscle kidney 39 90 40 Methods 41 60 42 30 43 % Recovery 44 0 45 46 47 Name of compounds 48 120 Liver muscle 49 e) Chicken 50 90 51 Analytical 52 60 53 54 Recovery% 30 55 0 56 57 58 458 Name of compounds 59 60 20 | P a g e Page 21 of 24 Analytical Methods 1 2 3 459 Fig.2: Recovery values (%) obtained from a) pork samples, b) beef, c) sheep, d) dromedary 4 460 and e) chicken with selected buffered QuEChERS method. 5 6 a c d 7 10 30 b 6 e 8 7.5 20 4 9 5 2 10 10 2.5 0 0 0 11 2 4 6 8 10 12 0 2 4 6 8 3 8 13 18 23 13 8 f g h 14 6 3 3 15 4 2 2 16 2 1 1 17 0 0 0 8 10 12 14 16 18 6 8 10 12 14 10 12 14 16 18 2 19 60 j 15 l 1.5 20 40 k 10 m 21 i 1 20 5 Manuscript 22 0.5 0 0 0 23 18 20 22 24 26 18 20 22 24 26 24 18 20 22 24 26 25 20 n 16 p 80 r 26 15 12 60 q 10 o 8 40 27 5 4 20 28 0 0 0 29 18 20 22 24 26 18 20 22 24 26 18 20 22 24 26 30 10 u 24 s 25 t Accepted 31 20 7.5 32 16 15 5 8 10 33 5 2.5 34 0 0 0 35 18 20 22 24 26 18 20 22 24 26 18 20 22 24 26 36 2 16 37 30 v 1.5 w 12 x 20 38 1 8 10 39 0.5 4 0 0 40 0 Methods 41 18 20 22 24 26 20 22 24 26 28 18 20 22 24 26 42 3 43 461 Counts (*10 ) vs. acquisition time (min) 44 462 Fig.3: Total Ion Counts (TIC) of a beef muscle spiked with 23 sulphonamides at the fixed 45 1 1 46 463 levels: 100 µg.kg and 5 µg.kg for SAs and dapsone, respectively. a) SGN, b) SAA, c) SD, 47 464 d) SIM, e) SMZ, f) STZ, g) SP, h) SM, i) SME, j) SMP, k) SMM, l) SMT, m) Da, n) SCP, o) 48 49 465 SCL, p) SMX, q) SDO, r) SDM, s) SIX, t) SB, u) SNZ, v) SQX, w) SNT and x) SMX d4. 50 51 Analytical 52 53 54 55 56 57 58 59 60 21 | P a g e Analytical Methods Page 22 of 24 1 2 3 4 5 6 466 Supplementary data 7 8 467 Appendix A 9 10 a) Da SAA b) Da SAA 1.4 1.6 11 SB SCL SB SCL 1.4 12 1.2 SCP SD SCP SD 13 1 SDM SDO 1.2 SDM SDO SGN SIM 1 0.8 SGN SIM 14 SIX SME 0.8 SIX SME 0.6 15 SMM SMP 0.6 SMM SMP 0.4 SM SMT Manuscript 16 Relativeabundance 0.4 SM SMT Relativeabundance SMX SMZ 17 0.2 0.2 SMX SMZ SNT SNZ SNT SNZ 0 0 18 SP SQX SP SQX 0 2 4 6 8 10 12 STZ 0 2 4 6 8 10 12 19 Weeks Weeks STZ 3 20 2.5 c) Da SAA d) Da SAA 21 SB SCL 2.5 SB SCL 22 2 SCP SD SCP SD SDM SDO 2 SDM SDO 23 1.5 Accepted SGN SIM SGN SIM 1.5 24 SIX SME SIX SME 1 25 SMM SMP 1 SMM SMP 26 SM SMT Relativeabundance SM SMT Relativeabundance 0.5 27 SMX SMZ 0.5 SMX SMZ SNT SNZ SNT SNZ 28 0 0 SP SQX SP SQX 0 2 4 6 8 10 12 0 2 4 6 8 10 12 29 STZ STZ 468 Weeks Weeks 30 Methods 31 469 Fig.A1: Stability of SAs in spiked beef muscle at different concentrations a) 50 (1.25), b) 100 (2.5), c) 150 (3) and d) 200 (5) µg.kg 1 for 32 33 470 respectively SAs and dapsone compounds. 34 35 36 37 38 Analytical 39 40 41 42 22 | P a g e 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 23 of 24 Analytical Methods 1 2 3 471 Appendix B 4 5 6 SDM > 156 a) 7 8 9 10 SDM > 108 11 12 13 14 SMZ > 186 15 b) 16 17 18 19 SMZ > 156 20 21 Manuscript 22 23 472 24 473 Fig. B1: LC MS/MS chromatograms of some analysed samples (a) S5 containing SDM (59.1 25 474 µg.kg 1) and (b) S20 containing SMZ (70.2 µg.kg 1). 26 27 475 28 29 476 30 31 477 Accepted 32 33 478 34 35 479 36 37 38 480 39 481 40 Methods 41 42 482 43 44 483 45 46 484 47 48 485 49 50 486 51 Analytical 52 487 53 54 488 55 56 489 57 58 59 60 23 | P a g e Analytical Methods Page 24 of 24 1 2 3 490 Appendix C 4 5 120 6 y = 0.925x 7 100 8 R² = 0.992 9 80 ) 10 -1 11 60 12 13 (µg.kg 40 14 15 20 16 17 Orbitrap Concentration 0 18 0 20 40 60 80 100 120 19 20 Concentration MS/MS (µg.kg -1) 21 Manuscript 22 491 23 1 492 Fig. C1: Correlation graph of concentrations (µg.kg ) determined of studied compounds 24 25 493 with triple quadrupole mass spectrometry (QqQ) and Orbitrap analyzers in meat samples. 26 494 27 28 29 30 31 Accepted 32 33 34 35 36 37 38 39 40 Methods 41 42 43 44 45 46 47 48 49 50 51 Analytical 52 53 54 55 56 57 58 59 60 24 | P a g e