Analytical strategy for the determination of non-steroidal anti-inflammatory drugs in plasma and improved analytical strategy for the determination of authorised and non-authorised non-steroidal anti-inflammatory drugs in milk by liquid chromatography tandem mass spectrometry Geraldine Dowling, Edward Malone, Tom Harbison, Sheila Martin

To cite this version:

Geraldine Dowling, Edward Malone, Tom Harbison, Sheila Martin. Analytical strategy for the de- termination of non-steroidal anti-inflammatory drugs in plasma and improved analytical strategy for the determination of authorised and non-authorised non-steroidal anti-inflammatory drugs in milk by liquid chromatography tandem mass spectrometry. Food Additives and Contaminants, 2010, 27 (07), pp.962-982. ￿10.1080/19440041003706779￿. ￿hal-00598945￿

HAL Id: hal-00598945 https://hal.archives-ouvertes.fr/hal-00598945 Submitted on 8 Jun 2011

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Food Additives and Contaminants

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Analytical strategy for the determination of non-steroidal anti-inflammatory drugs in plasma and improved analytical strategy for the determination of authorised and non- authorised non-steroidal anti-inflammatory drugs in milk by liquid chromatography tandem mass spectrometry

Journal: Food Additives and Contaminants

Manuscript ID: TFAC-2009-395.R1

Manuscript Type: Original Research Paper

Date Submitted by the 29-Jan-2010 Author:

Complete List of Authors: Dowling, Geraldine; The State Laboratory Malone, Edward; The State Laboratory Harbison, Tom; The State Laboratory Martin, Sheila; The State Laboratory

Methods/Techniques: Chromatography - LC/MS

Additives/Contaminants: Veterinary drug residues

Food Types: Milk, Animal

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1 2 3 4 1 Analytical strategy for the determination of non-steroidal anti- 5 6 2 inflammatory drugs in plasma and improved analytical strategy for 7 8 9 3 the determination of authorised and non-authorised non-steroidal 10 11 12 4 anti-inflammatory drugs in milk by LC-MS/MS 13 14 5 15 16 6 Geraldine DowlingFor * , EdwardPeer Malone Review Tom Harbison Sheila Only Martin 17 18 7 19 20 8 The State Laboratory, Backweston Laboratory Complex, Young’s Cross, Celbridge, 21 22 9 Co. Kildare, Ireland 23 24 10 25 26 27 11 28 12 Abstract 29 30 13 A sensitive and selective method for the determination of 6 non-steroidal anti- 31 32 14 33 inflammatory drugs in bovine plasma was developed. An improved method for the 34 35 15 determination of authorised and non-authorised residues of 10 non-steroidal anti- 36 37 16 inflammatory drugs in milk was developed. Analytes were separated and acquired by 38 39 40 17 HPLC coupled with an electrospray ionisation tandem mass spectrometer (LC-ESI- 41 42 18 MS/MS). Target compounds were acidified in plasma, and plasma and milk samples 43 44 19 were extracted with acetonitrile and both extracts were purified on an improved SPE 45 46 TM 47 20 procedure utilising Evolute ABN SPE cartridges. The recovery of the methods for 48 49 21 milk and plasma was between 73 and 109 %. The precision of the method for 50 51 22 authorised and non-authorised NSAIDs in milk and plasma expressed as % RSD , for 52 53 54 23 the within –laboratory repeatability was less than 16 % and for authorised NSAIDs of 55 56 24 meloxicam, flunixin and tolfenamic acid at their associated MRLs in milk was less 57 58 25 59 than 10 % however for hydroxy flunixin was less than 25%. The methods were 60 26 validated according to Commission Decision 2002/657/EC.

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1 2 3 27 4 5 6 28 . 7 8 29 9 10 30 Keywords: Non-steroidal Anti-Inflammatory Drugs; Plasma; Milk; Liquid Chromatography 11 31 Tandem Mass Spectrometry; Method Validation 12 13 32 14 15 33 16 For Peer Review Only 34 Introduction 17 18 35 Carprofen (CPF), diclofenac (DCF), ibuprofen (IBP), ketoprofen (KPF), mefenamic 19 20 21 36 acid (MFN), phenylbutazone (PBZ), flunixin (FLU), hydroxy-flunixin (FLU-OH), 22 23 37 tolfenamic acid (TLF) and meloxicam (MLX) are non-steroidal anti-inflammatory 24 25 38 drugs (NSAIDs) and their structures are shown in Fig.1. Over the past number of 26 27 28 39 years, residues of NSAIDs in food are a cause for concern. Studies have illustrated 29 30 40 that the second most prescribed class of drugs after microbials is NSAIDs [Sundlof et 31 32 41 al. 1995]. Dairy farmers and veterinarians are using NSAIDs in dairy animals more 33 34 35 42 frequently [US Code, 1988] and studies have shown that their increased use [Kopcha 36 37 43 et al. 1992] poses a threat to human health as permitted residue levels are being 38 39 44 violated [Smith et al. 2008]. In 2007 the EC Rapid Alert System for Food and Feed 40 41 42 45 reported alert notifications in relation to horse meat for these substances. The 43 44 46 European Council recommend rigorous control of NSAIDs in food producing 45 46 47 47 animals [SANCO 2000] because of the health effects in humans such as aplastic 48 49 48 anaemia, gastrointestinal disorders, agranulocytosis [Insel 1990] and changes in renal 50 51 49 function [Goodman et al. 1992]. Long term exposure to PBZ has caused kidney 52 53 54 50 tumors in mice and liver tumors in rats [Kari et al. 1995]. In recent years the COX-II 55 56 51 inhibitor class of NSAIDs has been implicated in cardiovascular harm in humans 57 58 52 [Staa et al. 2008; Debabrata et al. 2008]. According to EU law, all drugs for 59 60 53 veterinary use need to be included in Annexes 1-3 of Regulation 2377/90

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1 2 3 54 [Commission Decision 1990]. This regulation establishes lists of compounds that 4 5 6 55 have a fixed maximum residue limit, MRL (Annex I), that need no MRL (Annex II) 7 8 56 or that have a provisional MRL (III). There are no MRL’s set in plasma as is not an 9 10 57 11 edible matrix. The recommended minimum concentration for NSAIDs in plasma is set 12 -1 13 58 at 5 ng mL [SANCO 2007]. In milk FLU, FLU-OH, TLF and MLX are included in 14 15 59 Annex I. CPF has been included in Annex II of the regulation only for bovine milk 16 For Peer Review Only 17 18 60 [European Commission 2005]. DCF was not authorised for use in animals that 19 20 61 produce milk for human consumption [European Commission 2004] until recently 21 22 62 [EMEA]. KPF is listed in Annex II of the regulation. PBZ, MFN and IBP are 23 24 25 63 considered as prohibited substances and are not included in Annexes 1-3 and have no 26 27 64 maximum residue limit (MRL) established however the minimum recommended 28 29 -1 65 concentration for analysis of NSAIDs with no MRL set in milk is 5 ng mL . The 30 31 32 66 widespread use of NSAIDs presents a risk to the consumer if food containing residues 33 34 67 enter the food chain. In Ireland plasma and milk are some of the target matrices 35 36 37 68 chosen to identify the misuse of NSAIDs in animal production. The advantages of 38 39 69 using plasma in regulatory control are that it is an easy matrix to handle for analysis 40 41 70 and PBZ residues can be found in this matrix for a long time (personal 42 43 44 71 communication with the CRL). Therefore the analytical method developed in this 45 46 72 study in plasma concentrated on the analysis of 6 NSAIDs in bovine species. Methods 47 48 73 have been reported for the analysis of NSAIDs in plasma by LC-UV [De Veau, 1999; 49 50 51 74 Kvaternick et al. 2007; Luo et al. 2004; Hardee et al. 1982; Neto et al. 1996; Grippa et 52 53 75 al. 2000; Jedziniak et al. 2007; Singh et al. 1996; Gowik et al. 1998; Quintana et al. 54 55 76 2004; Fiori et al. 2004], GC-MS [Neto et al. 1996; Singh et al. 1991; Hines et al. 56 57 58 77 2004;Gonzalez et al. 1996; Jaussaud et al. 1992], LC-MS [Luo et al. 2004; Miksa et 59 60 78 al. 2005; Vinci et al. 2006; Quintana et al. 2004; You et al. 2008] and capillary

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1 2 3 79 electrophoresis [Gu et al. 1997]. The majority of methods that have been cited to date 4 5 6 80 have been developed in equine plasma alone or in combination with other matrices 7 8 81 with limits of detection ranging from 0.1 ng mL -1 to 5 ug mL -1 [Miksa et al. 2005; 9 10 82 11 Luo et al. 2004; Hardee et al. 1982; Neto et al. 1996; Grippa et al. 2000; Singh et al. 12 13 83 1991; Hines et al. 2004; Gonzalez et al. 1996; Gowik et al. 1998; Vinci et al. 2006; 14 15 84 Gu et al. 1997; You et al. 2008]. Other methods exist for the determination of 16 For Peer Review Only 17 18 85 NSAIDs in bovine plasma are available but the limits of detection range from 20 ng to 19 20 86 3.4 ug mL -1 [De Veau et al. 1999; Miksa et al. 2005; Jedziniak et al. 2007; Gowik et 21 22 87 al. 1998; Vinci et al. 2006; Quintana et al. 2004; Fiori et al. 2004]. Only two methods 23 24 -1 25 88 are available in equine plasma to date capable of meeting the 5 ng mL requirement. 26 27 89 A method by Luo et al [Luo et al. 2004] for a single residue had a limit of detection of 28 29 -1 90 0.1 ng mL for FLU. A multi-residue method by Gonzalez et al [Gonzalez et al. 30 31 -1 32 91 1996] had a limit of detection of 5 ng mL for IBP, FLU, DCF, TLF but limits of 33 34 92 detection of only 10-25 ng mL -1 could only be achieved for KPF, MFN and PBZ. 35 36 37 93 Therefore no methods are available to date for KPF, MFN and PBZ in plasma that can 38 -1 39 94 meet the target level of 5 ng mL . A disadvantage of the method developed by 40 41 95 Gonzalez et al [Gonzalez et al. 1996] is that the method monitors 3 ions and this is not 42 43 44 96 a confirmatory method according to Commission Decision 2002/657/EC [European 45 46 97 Commission Decision. 2002] and a second analytical technique is required. Overall 47 48 98 there is a paucity of methods in the literature that are available for the analysis of 49 50 51 99 NSAIDs in bovine plasma and of those available, the methods are not sensitive 52 53 100 enough to meet the minimum required concentration of analysis set at 5 ng mL -1. 54 55 101 Milk is the second target matrix analysed in this study and is important in food safety 56 57 58 102 because sampling can often be restricted to sampling of meat, milk, eggs and honey as 59 60 103 in the case of retail import/exports. Milk also allows the detection of drugs in live

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1 2 3 104 animals prior to slaughter. There are few analytical methods for the determination of 4 5 6 105 authorised and non-authorised NSAIDs in milk and usually analyse for only a few 7 8 106 residues. Those that have been described use LC-UV [Martin et al. 1983; Gallo et al. 9 10 107 11 2008; Feely et al. 2002; Rubb et al. 1995; De Veau et al. 1996], LC-MS [Gallo et al. 12 13 108 2008; Boner et al. 2003; Daeseleire et al. 2003; Malone et al. 2009; Dowling et al. 14 15 109 2009] and GC-MS [Dowling et al. 2008; Rubb et al. 1995]. A method by Gallo et al 16 For Peer Review Only 17 18 110 [Gallo et al. 2008] is capable of analysing 16 NSAIDs in milk using two separate 19 20 111 analytical techniques and involves using a screening LC-DAD method with limits of 21 22 112 detection (LOD) of between 2- 15 ng mL -1 and a runtime of 35 min with an 23 24 25 113 equilibration time of 15 min per injection. Confirmation is achieved using an LC ESI- 26 27 114 Iontrap -MS/MS method with an LOD of 5 ng mL -1 except for flurbiprofen with a 28 29 115 runtime of 40 min per injection. The LC-MS method does not meet the requirements 30 31 32 116 for a confirmatory method according to Commission Decision 2002/657/EC and a 33 34 117 third analytical technique is required. A method by Stolker et al [Stolker et al. 2008] is 35 36 37 118 capable of analysing 20 NSAIDs in milk using a quantitative screening method 38 39 119 (UPLC-TOF-MS) with LOD’s for specific NSAIDs such as NAP, PBZ and DCF at 40 41 120 12.5, 25 and 6.3 ng mL -1 and a runtime of 8.5 min per injection. The method cannot 42 43 -1 -1 44 121 meet the 5 ng mL for NAP and PBZ or 0.1 ng mL recently set for DCF and 45 46 122 additionally the analysis by TOF-MS, medium to high resolution of approximately 47 48 123 10,000 FWHM is not included in Commission Decision 2002/657/EC. 49 50 51 124 Other methods for the determination of NSAIDs in milk have limits of detection of 20 52 53 125 ng mL -1 for PBZ [Martin et al. 1983], 0.2 ng mL -1 for FLU and FLU-OH [Boner et al. 54 55 -1 -1 126 2003], 0.5 ug kg for FLU, FLU-OH and 1 ug kg for KPF [Daeseleire et al. 2003], 56 57 -1 58 127 53.05, 15.82, 61.39, 45.04 ng mL for TLF, MLX, 4-MAA and FLU-OH [Malone et 59 60 128 al. 2009], 0.46-2.86 ng mL -1 for CPF, DCF, MFN, niflumic acid (NIFLU), naproxen

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1 2 3 129 (NAP), oxyphenylbutazone (OXYPHEN), PBZ and suxibuzone (SUXI) [Dowling et 4 5 -1 6 130 al. 2009] 0.59, 2.09, 0.90 and 0.70 ng mL for IBP, KPF, DCF and PBZ [Dowling 7 8 131 et al. 2008], 1 ng mL -1 for FLU [Feely et al. 2002], 1.7 ng mL -1 for FLU [Rubb et al. 9 10 132 -1 11 1995] and lowest fortification in matrix was 25 ng mL for PBZ [De Veau et al. 12 13 133 1996]. The objective of this study was to develop an analytical strategy for the 14 15 134 determination of NSAIDs in bovine plasma and for the determination of authorised 16 For Peer Review Only 17 18 135 and non-authorised NSAIDs simultaneously in milk that meet the EU target levels set 19 20 136 and validate according to Commission Decision 2002/657/EC. 21 22 137 In this study an improved purification procedure was developed using Evolute 23 24 TM 25 138 ABN solid phase extraction cartridges for the analysis of a wider range of NSAIDs 26 27 139 including authorised and non-authorised NSAIDs in bovine milk. The developed 28 29 140 procedure was suitable for the purification of 6 NSAIDs in bovine plasma. An 30 31 32 141 improved liquid chromatography tandem mass spectrometry detection technique was 33 34 142 developed to analyse 10 NSAIDs simultaneously with a run-time of 15 min. The 35 36 37 143 methods in each matrix were comprehensively validated according to Commission 38 39 144 Decision 2002/657/EC. The methods were implemented into the National Monitoring 40 41 145 Programme in Ireland for veterinary drug residues and accreditated according to the 42 43 44 146 ISO17025 Standard. The proposed method in milk does not cover the glucuronides. 45 46 147 This is the first time that suitably sensitive methods for the analysis of NSAIDs in 47 48 148 plasma and for the analysis of the selected range of authorised and non-authorised 49 50 TM 51 149 NSAIDs using Evolute ABN solid phase extraction cartridges simultaneously in 52 53 150 bovine milk are available. 54 55 56 57 58 59 60

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1 2 3 151 4 5 152 Materials and Methods 6 7 8 153 Materials and reagents 9 10 154 Water, ethanol, ethyl acetate, methanol, acetonitrile, acetic acid, hydrochloric acid (37 11 12 155 %), n-hexane and iso-octane (HiPerSolv grade) were obtained from BDH (Merck, 13 14 15 156 UK). CPF, DCF, IBP, MFN, FLU, KPF, MLX, TLF and PBZ were purchased from 16 For Peer Review Only 17 157 Sigma (Sigma Aldrich, Ireland). FLU-OH was obtained as a gift from The 18 19 158 Community Reference Laboratory for NSAIDs in the EU in Germany. d -PBZ was 20 10 21 22 159 obtained from Cambridge Isotope Labs (Cambridge Isotope Labs, USA). d3-MLX, d 3- 23 24 160 IBP and d 4-DCF were obtained from CDN Isotopes (CDN Isotopes, Canada). d3-FLU 25 26 161 was obtained from Witega (Witega, Germany). d -TLF was obtained as a gift from 27 4 28 29 162 Stormont, (Stormont, UK). Primary stock standard solutions (stable for 12 months) 30 31 163 were prepared in ethanol at a concentration of 1 mg mL -1. Intermediate single 32 33 34 164 standard solutions (stable for 6 months) were prepared in methanol at a concentration 35 36 165 of 10 g mL -1. CPF, DCF, IBP, MFN, FLU, FLU-OH, KPF, TLF, MLX and PBZ 37 38 166 standard fortification solution for plasma (stable for 6 months) was prepared in 39 40 -1 -1 41 167 methanol at a concentration of 500 ng mL from the 10 g mL intermediate stock 42 43 168 solution. Internal standard fortification solution for milk or plasma containing d 3- 44 45 46 169 MLX, d 4-DCF, d3-IBP d3-FLU d 4-TLF and d 10 -PBZ was prepared at a concentration 47 48 170 of 1.25 ug mL -1. CPF, DCF, IBP, MFN, KPF and PBZ standard fortification solution 49 50 -1 171 for milk (NMRL) was prepared in methanol at a concentration of 500 ng mL from 51 52 -1 53 172 the 10 g mL intermediate stock solution. MLX, FLU, FLU-OH and TLF standard 54 55 173 fortification solution for milk (MRL) was prepared in methanol at a concentration of 56 57 -1 o TM 58 174 1.5, 4, 4 and 5 ug mL . All standards were stored at 4 C in the dark. Isolute 59 60 175 Evolute ABN 50 m solid phase extraction cartridges (10 mL, 100 mg) were obtained

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1 2 3 176 from Biotage (Biotage, UK). Methanol:water (10:90, v/v) and 10 mM ascorbic acid 4 5 6 177 were used as solid phase extraction wash solvents. N-hexane:diethyl 7 8 178 ether:acetonitrile: methanol (45:45:7:3,v/v) was used as the solid phase extraction 9 10 179 11 elution solvent. Injection solvent was water:acetonitrile (90:10, v/v). 12

13 180 14 15 181 LC-MS/MS conditions 16 For Peer Review Only 17 18 182 The LC consisted of an Agilent 1200 Rapid Resolution LC equipped with a G1312B 19 20 183 Binary pump, G1316B-HiPALS SL autosampler and a G1316B-TCCSL column oven 21 22 184 (Agilent Ireland). The NSAIDs were chromatographed on a 1.8 m Agilent Eclipse 23 24 25 185 Plus C 18 column (3.0 × 50 mm) (Agilent, Ireland) and the column temperature was 26 27 186 maintained at 55 o C. A gradient was applied with water containing 0.001 M acetic 28 29 30 187 acid and acetonitrile (90:10, v/v + 0.001 M acetic acid) (A) and acetonitrile (B) (Table 31 32 188 1). The total run time was 15 minutes. The injection volume was 15 L. The mass 33 34 189 spectrometer used was a QTRAP 4000 with a TurboIonSpray source from Applied 35 36 37 190 Biosystems (Applied Biosystems/MDS-Sciex, Canada). The MS was controlled by 38 39 191 version 1.5 of Analyst software. The described LC-MS/MS system was shown to be 40 41 42 192 suitable for the analysis of NSAIDs in plasma (Figure 2-7) and milk (Figure 2-11). 43 44 193 45 46 194 MS/MS parameters 47 48 49 195 The analysis was performed using negative ion electrospray MS/MS in multiple 50 51 196 reaction monitoring (MRM) mode. The collision voltages were optimised as shown 52 53 197 (Table 2). Each transition was performed with a 13 msec dwell time and a pause time 54 55 56 198 of 3 msec. The MS/MS detector conditions were as follows: Ion mode electrospray 57 58 199 negative; curtain gas 45 psi; ion spray voltage 4400 V; temperature 650 oC; ion source 59 60

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1 2 3 200 gas one 70 psi; ions source gas two 70 psi; Interface heater on; entrance potential 10 4 5 6 201 V; Resolution Q1 unit; Resolution Q2 unit; CAD gas =high 7 8 202 9 10 203 11 Plasma/milk samples 12 13 204 Plasma/milk obtained for use as negative controls was separated into 50 mL aliquots 14 15 205 and stored at –20 °C. The plasma/milk was analysed in previous batches and 16 For Peer Review Only 17 18 206 plasma/milk found to contain no detectable residues of NSAIDs were used as negative 19 20 207 controls. 21 22 208 23 24 25 209 Sample extraction and clean-up 26 27 28 210 Plasma Extraction 29 30 31 32 211 Plasma samples (5 mL) were aliquoted into 50 mL polypropylene tubes. The plasma 33 34 212 aliquots (5 mL) were fortified with internal standard at levels corresponding to 15 ng 35 36 -1 -1 213 mL by adding a 60 L portion of a 1.25 ug mL mix solution of d -MLX, d -DCF, 37 3 4 38 39 214 d3-IBP, d 3-FLU , d4-TLF and d 10 -PBZ. Samples were fortified at levels corresponding 40 41 215 to 5, 7.5 and 10 ng mL -1 by adding 50, 75 and 100 L portions of a 500 ng mL -1 42 43 44 216 solution of CPF, DCF, IBP, MFN, KPF and PBZ . After fortification, samples were 45 46 217 held for 15 min prior to extraction. Hydrochloric acid (500 L, 1 M) was added to the 47 48 49 218 plasma samples and they were left to stand at room temperature (10 min). Acetonitrile 50 51 219 (5 mL) was added and the samples were vortexed (30 sec), centrifuged (3568 × g, 10 52 53 o 220 min, 4 C) and the supernatant was transferred to a clean polypropylene tube. 10 mM 54 55 56 221 ascorbic acid ( 15 mL) was added and the samples were vortexed (30 sec) and the 57 58 222 pH of the samples were checked to ensure they were at pH 3 before proceeding to the 59 60 223 solid phase extraction (SPE) stage.

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1 2 3 224 Milk Extraction 4 5 6 7 225 Milk samples (5 mL) were aliquoted into 50 mL polypropylene tubes. The milk 8 9 226 aliquots (5 mL) were fortified with internal standard at levels corresponding to 15 ng 10 11 -1 -1 12 227 mL by adding a 60 L portion of a 1.25 ug mL mix solution of d 3-MLX, d 4-DCF, 13 14 228 d3-IBP, d 3-FLU , d4-TLF and d 10 -PBZ. Samples were fortified at levels corresponding 15 16 For Peer Review Only 229 -1 -1 17 to 5, 7.5 and 10 ng mL by adding 50, 75 and 100 L portions of a 500 ng mL 18 19 230 solution of CPF, DCF, IBP, MFN, KPF and PBZ (NMRL solution) and at 7.5, 15 20 21 231 and 22.5 ng mL -1 with MLX, at 20, 40 and 60 ng mL -1 with FLU and FLU-OH and 22 23 -1 24 232 at 25, 50 and 75 ng mL with TLF by fortifying with 25, 50 and 75 L portions of a 25 26 233 1.5, 4, 4 and 5 ug mL -1 of MLX, FLU, FLU-OH and TLF (MRL solution). After 27 28 29 234 fortification, samples were held for 15 min prior to extraction. Acetonitrile (5 mL) 30 31 235 was added and the samples were vortexed (30 sec), centrifuged (3568 × g, 10 min, 4 32 33 236 oC) and the supernatant was transferred to a clean polypropylene tube. The sample 34 35 36 237 pellet is re-extracted with 5 mL of acetonitrile and the supernatants are combined. 10 37 38 238 mM ascorbic acid (20 mL) and 1 M hydrochloric acid (0.2 mL) were added to the 39 40 239 extracts and the pH of the samples were checked to ensure they were at pH 3 before 41 42 43 240 proceeding to the solid phase extraction (SPE) stage. 44 45 46 241 Solid phase extraction 47 48 49 TM 50 242 The sample extracts were purified by SPE using Evolute ABN SPE cartridges. 51 52 243 Sample extracts were loaded onto the cartridges (preconditioned with 3 mL of n- 53 54 244 hexane:diethyl ether (50:50, v/v) 3 mL of methanol and 5 mL of ascorbic acid. The 55 56 57 245 samples were loaded onto cartridges under gravity. The cartridges were washed with 3 58 59 246 mL of methanol:water (10:90, v/v). The cartridges were dried under vacuum (15 min). 60 247 The cartridges were eluted with 2 × 1.5 mL of n-hexane:diethyl ether: acetonitrile:

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1 2 3 248 methanol (45:45:7:3, v/v). The eluates were reduced to dryness under nitrogen 4 5 6 249 without heat before re-dissolving in 150 L of water:acetonitrile (90:10, v/v) and 7 8 250 vortexed (1 min). An aliquot (15 L) was injected on the LC column. 9 10 11 251 12 13 252 Matrix-Matched Calibration 14 15 16 253 Matrix matchedFor calibration Peer curves wereReview prepared and used Only for quantification. Control 17 18 254 plasma/milk previously tested and shown to contain no residues was prepared as 19 20 255 above (2.4). One control plasma sample and one control milk was used for each 21 22 23 256 calibration standard level. Plasma samples (5 mL) or milk samples (5 mL) were 24 25 257 aliquoted into 50 mL polypropylene tubes. Individual plasma or milk samples were 26 27 -1 258 fortified with internal standard at levels corresponding to 15 ng mL by adding a 60 28 29 -1 30 259 L portion of a 1.25 ug mL mix solution of d 3-MLX, d 4-DCF, d3-IBP, d 3-FLU, d4- 31 32 260 TLF and d 10 -PBZ. Plasma samples were fortified at levels corresponding to 0, 5, 7.5, 33 34 -1 -1 35 261 10 and 20 ng mL by adding 0, 50, 75, 100 and 200 L portions of a 500 ng mL 36 37 262 standard solution of CPF, DCF, IB, KPF, MFN and PBZ. After fortification, plasma 38 39 263 40 samples were held for 15 min prior to the extraction procedure as described above 41 42 264 (2.5). Milk samples were fortified at levels corresponding to 0, 5, 7.5, 10 and 20 ng 43 44 265 mL -1 by adding 0, 50, 75, 100 and 200 L portions of a 500 ng mL -1 standard solution 45 46 47 266 of CPF, DCF, IBP, MFN, KPF and PBZ (NMRL solution) and at levels 48 49 267 corresponding to 0, 7.5, 15, 22.5, 30, 60 ng mL -1 of MLX, 0, 20, 40, 60, 80 and 160 50 51 268 -1 -1 52 ng mL FLU and FLU-OH and 0. 25, 50, 75, 100 and 200 ng mL of TLF. 53 -1 54 269 by adding 0, 25, 50, 75, 100 and 200 L portions of a 1.5, 4, 4, 5 ug mL standard 55 56 270 solution of MLX, FLU, FLU-OH and TLF (MRL solution). After fortification, milk 57 58 59 271 samples were held for 15 min prior to the extraction procedure as described above 60

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1 2 3 272 (2.5). Calibration curves of plasma or milk were prepared by plotting the response 4 5 -1 6 273 factor as a function of analyte concentration (0 to 20 ng mL ) to quantify samples. 7 8 274 9 10 275 11 Method validation 12 13 276 For estimation of accuracy, blank plasma samples were fortified with CPF, DCF, IBP, 14 15 277 MFN, KPF and PBZ at 5, 7.5 and 10 ng mL -1. For estimation of accuracy, blank milk 16 For Peer Review Only 17 18 278 samples were fortified with CPF, DCF, IBP, MFN, KPF and PBZ at 5, 7.5 and 10 ng 19 20 279 mL -1 and at 7.5, 15 and 22.5 ng mL -1 with MLX, at 20, 40 and 60 ng mL-1 with FLU 21 22 280 and FLU-OH and at 25, 50 and 75 ng mL -1 with TLF. Six replicate test portions, at 23 24 25 281 each of the three fortification levels, were analysed. Analysis of the 18 test portions 26 27 282 was carried out on three separate occasions for each matrix. For the estimation of the 28 29 283 precision of the method, repeatability and within-laboratory reproducibility was 30 31 32 284 calculated. For unauthorised substances the decision limit (CC α) of the method was 33 34 285 calculated according to the calibration curve procedure using the intercept (value of 35 36 37 286 the signal, y, where the concentration, x is equal to zero) and 2.33 times the standard 38 39 287 error of the intercept for a set of data with 6 replicates at 3 levels. The detection 40 41 288 42 capability (CC β) was calculated by adding 1.64 times the standard error to the CCα. 43 44 289 For authorised substances the decision limit (CC α) of the method was calculated 45 46 290 according to the ISO 11843 calibration curve procedure by plotting the corresponding 47 48 49 291 concentration at the permitted limit plus 1.64 times the standard deviation of the 50 51 292 within laboratory reproducibility for a set of data with 6 replicates at 3 levels. The 52 53 54 293 detection capability (CC β) was calculated by adding 1.64 times the standard deviation 55 56 294 of the within laboratory reproducibility to the CC α. 57 58 59 295 60 296 Results and Discussion

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1 2 3 297 Development/optimisation experiments 4 5 6 7 298 The ionisation of all NSAIDs was studied in negative and positive mode. Most 8 9 299 NSAIDs can be detected by ESI-MS both in the negative mode and the positive mode, 10 11 12 300 showing different ionisation efficiencies. The optimum parameters (polarity mode, 13 14 301 declustering potential, collision energy, collision cell exit potential) were determined 15 16 302 for each drugFor and the bestPeer diagnostic Review ions for MS/MS analysis Only are shown in Table 2. 17 18 19 303 Negative ion mode was chosen as the required sensitivity was obtained for all 20 21 304 compounds and less baseline noise was obtained. The MS/MS method was developed 22 23 305 to monitor one precursor ion (parent mass) and two daughters (corresponding to 24 25 26 306 strong and weak ion) which is a suitable confirmatory method yielding 4 27 28 307 identification points in accordance with 2002/657/EC. Only one daughter ion could be 29 30 308 obtained for KPF and IBP but two daughter ions could be obtained for all other 31 32 33 309 compounds investigated in the study. A previous method developed at the author’s 34 35 310 laboratory utilising GC-MS/MS was capable of obtaining 2 daughter ions for these 36 37 38 311 substances after derivatisation [Dowling et al. 2008]. The LC method developed in 39 40 312 this study was based on a method developed at the author’s laboratory for the 41 42 313 determination of 8 banned non-steroidal anti-inflammatory drugs but was not suitable 43 44 45 314 for incorporation of the new range of analytes in this study [Dowling et al. 2009]. 46 47 315 Chromatographic tests were carried out to evaluate the suitability of the 1.8 m 48 49 50 316 Agilent Eclipse Plus C 18 column (3.0× 50 mm) and the LC mobile phase utilised in 51 52 317 this study when additional NSAIDs were added. The tests showed that the internal 53 54 318 standards of DCF and FLU overlapped in each internal standard transition when the 55 56 -1 57 319 analytes were eluted with a flow rate of 750 L min and a run time of 6.5 min per 58 59 320 injection. A study was performed using the same composition of mobile phase A and 60 321 B. The times in the gradient were adjusted, the flow rate was reduced and the

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1 2 3 322 chromatographic runtime was extended to separate the internal standard of DCF and 4 5 6 323 FLU. This resulted in the NSAIDs being eluted with good peak shape when using a 7 8 324 mobile phase of water containing 0.001 M acetic acid and acetonitrile (90:10, v/v) (A) 9 10 -1 11 325 and acetonitrile (B) with a flow rate of 0.5 L.min and a runtime of 15 min. The 12 13 326 internal standards d 4-DCF and d 3-FLU were completely separated under these 14 15 327 conditions. As a result a batch of 30 samples can be analysed using the developed 16 For Peer Review Only 17 18 328 LC-MS/MS method for 10 NSAID residues in 7.5 hours allowing the running of up to 19 20 329 3 batches of extracted samples within a 24 hour period. The extraction of the 21 22 330 23 NSAIDs from plasma was based on methods developed by Gowik et al. (Gowik et al. 24 25 331 1998) and by Vinci et al (Vinci et al. 2006) but modified with the addition of 26 27 332 acetonitrile. The extraction of NSAIDs in milk was based on a method previously 28 29 30 333 developed at the author’s laboratory [Dowling et al. 2009]. The extraction procedures 31 32 334 were found to be satisfactory in the extraction of the NSAIDs from milk and plasma 33 34 335 in this study. The purification of NSAIDs from the plasma and milk extracts was 35 36 37 336 investigated initially using a solid phase extraction procedure previously developed at 38 39 337 the authors laboratory using Evolute ABN TM cartridges but the original procedure 40 41 338 was not suitable for the additional range of new analytes in this study. The NSAID 42 43 44 339 FLU-OH was poorly recovered when the method previously developed at our 45 46 340 laboratory was utilised. Elution studies were performed to ascertain where losses were 47 48 341 49 occurring. The cartridges were eluted with different compositions and volumes of 50 51 342 solvents including, 3 mL diethyl ether:hexane:acetonitrile (45:45:10, v/v/v), 3 mL 52 53 343 diethyl ether:hexane, acetonitrile:methanol (45:45:5:5, v/v/v/v), 1.5 mL diethyl ether: 54 55 56 344 hexane (80:20, v/v-elution 1) and 1.5 mL acetonitrile:methanol (90:10, v/v- elution 57 58 345 2), 1.5 mL acetonitrile:methanol 90:10, v/v-elution 1) and 1.5 mL diethyl ether: 59 60 346 hexane 50:50, v/v-elution 2), 3 mL methanol and 3 mL diethyl

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1 2 3 347 ether:hexane:acetonitrile:methanol (37.5:37.5:20:5 v/v/v/v). The results showed that 4 5 6 348 elution of the cartridge with a solvent composition containing n-hexane:diethyl ether: 7 8 349 acetonitrile: methanol (45:45:7:3, v/v-2 × 1.5 mL) gave the best results for all the 9 10 350 11 analytes tested in this study. This is the first time to the best of our knowledge that 12 13 351 CPF, DCF, IBP, KPF, MFN and PBZ residues have been purified from bovine 14 15 352 plasma using Evolute ABN TM solid phase extraction cartridges. The methodology is 16 For Peer Review Only 17 -1 18 353 capable of meeting the minimum concentration of 5 ng mL set for NSAIDs in 19 20 354 plasma. Moreover the values determined for the decision capability (CC α) in this 21 22 355 23 study were lower than those recorded for these substances in the literature in plasma 24 25 356 by LC-MS/MS to date. This is the first time to the best of our knowledge that FLU, 26 27 357 TLF, FLU-OH, IBP, KPF and MLX have been purified from milk using Evolute 28 29 TM 30 358 ABN solid phase extraction cartridges. The method meets the target level of 5 ng 31 32 359 mL -1 for IBP and KPF in milk for the first time. There are no analytical methods that 33 34 360 monitor for authorised and non authorised NSAIDs in milk aswell as FLU and FLU- 35 36 37 361 OH simultaneously in milk that meet the stringent validation requirements according 38 39 362 to Commission Decision 2002/657/EC. The primary advantage of the developed 40 41 363 analytical strategy in this study is that the quantitation and confirmation can be carried 42 43 44 364 out using a single analytical technique according to Commission Decision 45 46 365 2002/657/EC [Commission Decision 2002] except for IBP and KPF. Confirmation of 47 48 366 49 these residues using a second analytical technique is described elsewhere [Dowling et 50 51 367 al. 2008]. After validation of this method the EU changed the legislation for DCF and 52 53 368 a MRL of 0.1 ng mL -1 was set in milk. Preliminary spiking studies at the new MRL 54 55 56 369 for DCF were carried out using the developed analytical strategy in this study. Results 57 58 370 showed that this analytical strategy was sensitive enough to detect DCF at this level. 59 60 371 The same sample extract was also analysed on an Applied Biosystem 5500 triple

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1 2 3 372 quadrupole mass spectrometer and the response using this system gave a better signal- 4 5 6 373 to-noise ratio compared to the 4000 QTRAP. This analytical strategy with detection 7 8 374 using 4000 QTRAP or 5500 Applied Biosystems LC-MS technology shows for the 9 10 375 -1 11 first time during initial studies that the new MRL set for DCF in milk at 0.1 ng mL 12 -1 13 376 can be achieved. A chromatogram of the response for milk fortified at 0.1 ng mL 14 15 377 using Applied Biosystems 5500 is shown in Figure 12. 16 For Peer Review Only 17 18 378 19 20 379 Validation study 21 22 380 Validation of the method in plasma and milk was according to procedures described 23 24 25 381 in Commission Decision 2002/657/EC [Commission Decision 2002] covering 26 27 382 specificity, calibration curve linearity, recovery (accuracy), precision, decision limit 28 29 383 30 (CC α) and detection capability (CC β). 31 32 384 33 34 385 Specificity 35 36 37 386 The technique of LC-MS/MS itself offers a high degree of selectivity and specificity. 38 39 387 To establish the selectivity/specificity of the method, a variety of plasma and milk 40 41 388 samples were fortified with analytes and internal standards and non-fortified samples 42 43 44 389 were also analysed. No interfering peaks were observed at the retention time of the 45 46 390 analytes. To further test specificity in plasma and milk, samples were also fortified 47 48 391 -1 49 with 5.0 ng mL of naproxen (NAP), niflumic acid (NIFLU), oxyphenylbutazone 50 51 392 (OXYPHEN) and suxibuzone (SUXI). No interfering peaks were observed at the 52 53 393 retention window of the analytes. 54 55 56 394 57 58 59 60

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1 2 3 395 Linearity of the response 4 5 6 396 The linearity of the chromatographic response in plasma was tested with matrix 7 8 397 matched curves using 5 calibration points in the concentration range of 0 to 20 ng mL - 9 10 398 1 11 when fortified with CPF, DCF, IBP, KPF, MFN and PBZ. The linearity of the 12 13 399 chromatographic response in milk was tested with matrix matched curves using 5 14 15 400 calibration points in the concentration range of 0 to 20 ng mL -1 when fortified with 16 For Peer Review Only 17 18 401 CPF, DCF, IBP, KPF, MFN and PBZ (NMRL substances). For MRL substances the 19 20 402 linearity of the chromatographic response in milk was tested with matrix matched 21 22 403 curves using 5 calibration points in the concentration range of 0 to 30 ng mL -1 for 23 24 -1 -1 25 404 MLX, 0 to 160 ng mL for FLU and FLU-OH and 0 to 100 ng mL for TLF. Overall 26 2 27 405 the regression coefficients ( r ) were ≥ 0.98 except for FLU-OH. The regression 28 29 30 406 Accuracy 31 32 407 The accuracy was determined using bovine plasma fortified at 5.0, 7.5 and 10.0 ng 33 34 408 mL -1 with CPF, DCF, IBP, KPF, MFN and PBZ. Mean corrected recoveries (n = 18) 35 36 37 409 determined in three separate assays in plasma (Table 3) were between 99 and 109 %. 38 39 410 The accuracy was determined using bovine milk fortified at 5.0, 7.5 and 10.0 ng mL -1 40 41 411 with CPF, DCF, IBP, KPF, MFN and PBZ and the mean corrected recoveries (n = 18) 42 43 44 412 determined in three separate assays in milk (Table 4) were between 74 and 109 %. 45 46 413 The accuracy was determined using bovine milk fortified at MRL levels of 7.5, 15 47 48 414 -1 -1 49 and 22.5 ng mL for MLX, at 20, 40 and 60 ng mL with FLU and FLU-OH and 50 -1 51 415 fortified at 25, 50 and 75 ng mL with TLF and the mean corrected recovery (n = 18) 52 53 416 of the analytes, determined in three separate assays in milk (Table 4) were between 73 54 55 56 417 and 102 %. 57 58 418 59 60 419 Precision

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1 2 3 420 The precision of the method, expressed as RSD values for the within-lab 4 5 6 421 reproducibility of CPF, DCF, IBP, MFN and PBZ in plasma was less than 16 % 7 8 422 (Table 3). No deuterated analogue was available for CPF, MFN, KPF and FLU-OH in 9 10 423 11 our laboratory at the time of carrying out this work. d3-IBP was used as I.S for CPF 12 13 424 and KPF, d 3-TLF was used as I.S for MFN and d 3-FLU was used as I.S for FLU-OH. 14 15 425 The precision of the method, expressed as RSD values for the within-lab 16 For Peer Review Only 17 18 426 reproducibility of CPF, DCF, IBP, KPF, MFN and PBZ when fortified into milk was 19 20 427 less than 16 % (Table 4). The precision of the method, expressed as RSD values for 21 22 428 the within-lab reproducibility of MLX, FLU and TLF when fortified into milk was 23 24 25 429 less than 10 % except for FLU-OH which was less 25 % (Table 4). A one way 26 27 430 analysis of variance was carried out at each of the fortification levels to separate out 28 29 431 estimates for within run, between run and total variance of the method and the results 30 31 32 432 are shown in Tables 3 and 4. Commission Decision 2002/ 657/EC states that the 33 34 433 precision for quantitative methods for mass fractions lower than 100 ng mL -1, the 35 36 37 434 application of the Horwitz Equation gives unacceptable high values. Therefore, the 38 -1 39 435 RSD values for concentrations lower than 100 ng mL shall be as low as possible. 40 41 436 42 43 44 437 CC α and CC β 45 46 438 The decision limit (CC α) is defined as the limit above which it can be concluded with 47 48 49 439 an error probability of α, that a sample contains the analyte. In general, for non-MRL 50 51 440 substances an α equal to 1 % is applied. The detection capability (CC β) is the smallest 52 53 54 441 content of the substance that may be detected, identified and quantified in a sample, 55 56 442 with a statistical certainty of 1-β, were β = 5 %. In the case of non MRL substances 57 58 59 443 CC α is the concentration corresponding to the intercept + 2.33 times the standard 60 444 error of the intercept. CC β is the concentration corresponding to the signal at CC α +

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1 2 3 445 1.64 times the standard error of the intercept (i.e the intercept + 3.97 times the 4 5 6 446 standard error of the intercept). Blank plasma was fortified at 1, 1.5 and 2 times the 7 8 447 minimum required performance level of 5 ng mL -1 set for CPF, DCF, IBP, KPF, 9 10 11 448 MFN, PBZ, FLU, FLU-OH, TLF and MLX. CC α and CC β were calculated in 12 13 449 plasma using the intercept (value of the signal, y, were the concentration, x is equal to 14 15 450 zero) and the standard error of the intercept for a set of data with 6 replicates at 3 16 For Peer Review Only 17 -1 18 451 levels (5.0, 7.5 and 10.0 ng mL ). CC α values of 1.80, 0.58, 0.71, 0.87, 0.70 and 1.19 19 20 452 ng mL -1 were determined for CPF, DCF, IBP, KPF, MFN, PBZ respectively. CC β 21 22 -1 23 453 values of 3.1, 0.99, 1.22 1.49, 1.20, 2.02 ng mL were determined for CPF, DCF, 24 25 454 IBP, KPF, MFN and PBZ respectively. Non authorised substances in milk were 26 27 -1 28 455 fortified at 1, 1.5 and 2 times the minimum required performance level of 5 ng mL 29 30 456 set for CPF, DCF, IBP, KPF, MFN and PBZ. CC α values of 2.11, 0.83, 0.47, 1.63, 31 32 -1 33 457 0.92 and 0.55 ng mL were determined for CPF, DCF, IBP, KPF, MFN and PBZ. 34 35 458 CC β values of 3.59, 1.41, 0.80, 2.77, 1.56 and 0.94 ng mL -1 were determined for CPF, 36 37 459 DCF, IBP, KPF, MFN and PBZ. In the case of substances which have MRLs (MLX, 38 39 40 460 FLU, FLU-OH and TLF) fortification was at 0.5, 1 and 1.5 times the corresponding 41 42 461 MRL. The decision limit (CC α) is calculated by analysing the 18 milk samples 43 44 45 462 fortified at the MRL over three days, and using the concentration at the permitted limit 46 47 463 plus 1.64 times the standard deviation obtained to yield CC α. The detection capability 48 49 464 (CC β) of the proposed method was calculated from the CC α value plus 1.64 times the 50 51 - 52 465 corresponding standard deviation. CC α values of 17.6, 42.89, 55.76 and 54.45 ng mL 53 54 466 1 were determined and CC β values of 20.13, 45.78, 71.50 and 58.9 ng mL -1 were 55 56 57 467 determined for MLX, FLU, FLU-OH and TLF in milk when fortified at their 58 59 468 associated maximum residue limits. 60 469

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1 2 3 470 Measurement Uncertainty 4 5 6 471 According to SANCO/2004/2726 rev 1 the within laboratory reproducibility can be 7 8 472 regarded as a good estimate of the combined measurement uncertainty of individual 9 10 473 11 methods [SANCO 2004]. For the calculation of the extended uncertainty a safety 12 13 474 factor is required. The within laboratory reproducibility should be multiplied by a 14 15 475 value of 2.33 and this should be used when determining the CC α, corresponding to a 16 For Peer Review Only 17 18 476 confidence level of 99 %. As the only source of variation during the validation was 19 20 477 the different days and different plasma or milk sourced from different animals it was 21 22 478 23 decided to use a safety factor of 3.0 instead of 2.33. The measurement uncertainty of 24 25 479 the method in plasma was estimated at 47, 12, 15, 23, 19 and 26 % for 26 27 480 CPF, DCF, IBP, KPF, MFN and PBZ. The measurement uncertainty of the method in 28 29 30 481 milk was estimated at 50, 34, 36, 48, 20, 24, 16, 75, 24 and 30 % for CPF, DCF, IBP, 31 32 482 KPF, MFN, PBZ, FLU, FLU-OH, TLF and MLX. 33 34 483 This was determined by calculating the within laboratory reproducibility of the 35 36 37 484 method, followed by multiplication of the within laboratory reproducibility by the 38 39 485 safety factor of 3.0. 40 41 486 \ 42 43 44 487 Evaluation 45 46 488 The method developed in this study has been used to evaluate the presence of CPF, 47 48 489 49 DCF, IBP, KPF, MFN, PBZ, FLU, FLU-OH, TLF and MLX in bovine milk and CPF, 50 51 490 DCF, IBP, KPF, MFN and PBZ in plasma. In monitoring for these substances in 52 53 491 either matrix at our laboratory it was possible to detect the precursor ion and two 54 55 -1 56 492 daughter ions (at 5 ng mL ) in multiple reaction monitoring mode except for IBP and 57 58 493 KPF. Furthermore the product ion ratio requirement was also met. The method has 59 60 494 been carried out by different analysts under varying environmental conditions and the

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1 2 3 495 method was shown to be robust. To demonstrate the applicability of the method milk 4 5 6 496 samples taken from animals treated with MLX and FLU-OH obtained from the 7 8 497 Community Reference Laboratory in Berlin were tested. These samples had assigned 9 10 498 -1 11 values ranging from 5- 15 ng mL . The samples were analysed by the method 12 -1 13 499 developed in this study and all samples were found to contain 5 ng mL of FLU-OH 14 15 500 and 15 ng mL -1 of MLX. 16 For Peer Review Only 17 501 18 19 20 502 Conclusions 21 22 503 A fast, simple, sensitive and selective LC-MS/MS method for the determination of 23 24 504 CPF, DCF, IBP, KPF, MFN and PBZ in bovine plasma and CPF, DCF, IBP, KPF, 25 26 27 505 MFN, PBZ, FLU, FLU-OH, TLF and MLX in bovine milk has been developed. The 28 29 506 LC-MS/MS method provided quantitative confirmatory data for the analysis of bovine 30 31 507 milk for CPF, DCF, MFN, PBZ, FLU, FLU-OH, TLF and MLX. The method allows 32 33 34 508 the analysis of a wide variety of drugs from different NSAID sub-classes such as CPF, 35 36 509 IBP and KPF (arylpropionic acid derivatives), PBZ (pyrazolidinedione derivatives) 37 38 510 39 DCF, MFN and TLF (anthranilic derivatives) and FLU and FLU-OH (nicotinic acid 40 41 511 derivatives) and MLX (oxicam derivative). There is no published method available to 42 43 512 the best of our knowledge for the simultaneous determination of authorised and non- 44 45 46 513 authorised NSAIDs such as CPF, DCF, IBP, KPF, MFN, PBZ, FLU, FLU-OH, TLF 47 48 514 and MLX in bovine milk that purifies sample extracts using Evolute TM ABN solid 49 50 515 phase extraction cartridge procedure described in this study which is an improvement 51 52 53 516 on previous work carried out utilising this cartridge chemistry. This is the first time 54 55 517 that FLU, TLF, FLU-OH, IBP, KPF and MLX have been purified from milk using 56 57 TM 518 Evolute ABN solid phase extraction cartridges simultaneously with other NSAIDs. 58 59 60 519 This is the first time that a method is available that meets the minimum requirements

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1 2 3 -1 520 of 5 ng mL for IBP and KPF in milk. There is no published method available to the 4 5 6 521 best of our knowledge for the simultaneous determination of CPF, DCF, IBP, KPF, 7 8 522 MFN and PBZ in bovine plasma that purifies sample extracts using Evolute TM ABN 9 10 523 11 solid phase extraction cartridges. This study describes the first such sensitive and 12 13 524 selective methodology. This is also the first time that a rapid multi-residue 14 15 525 methodology for the above authorised and non-authorised NSAIDs has been validated 16 For Peer Review Only 17 18 526 according to Commission Decision 2002/657/EC [Commission Decision 2002] and 19 20 527 the measurement uncertainty of the method has been described. This methodology 21 22 528 shows that suitable sensitivity was obtained and that the method performs very well in 23 24 25 529 terms of accuracy and within-laboratory reproducibility. The developed method was 26 27 530 evaluated by comparison of results when method was performed by different analysts 28 29 531 under different environmental conditions, using different batches of reagents and solid 30 31 32 532 phase extraction cartridges. The results (unpublished data) were highly acceptable 33 34 533 providing evidence of the development of a rugged analytical method in this study. 35 36 37 534 Recently it was proposed by Community Reference Laboratories (CRLs) in Europe 38 -1 39 535 that laboratories should be capable of monitoring for NSAIDS at a level of 5 ng mL 40 41 536 in EU member states where no MRL exists in plasma or milk and this study shows 42 43 44 537 that these limits can be reached using the developed analytical strategy [SANCO 45 46 538 2004]. The objective of the work to anticipate the requirements of the future where 47 48 539 risks could occur due to the administration of NSAIDs by developing a method to 49 50 51 540 monitor for authorised and non-authorised NSAIDs simultaneously has been 52 53 541 achieved. The objective of the work to validate an analytical strategy for these 54 55 542 residues in bovine plasma and milk that meet EU target levels according to the 56 57 58 543 requirements in Commission Decision 2002/657/EC therefore has also been achieved 59 60 544 successfully.

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1 2 3 545 4 5 6 546 Acknowledgements 7 8 547 The authors would like to thank staff at The State Laboratory, Ireland for their 9 10 548 11 practical assistance. 12 13 549 14 15 550 References 16 For Peer Review Only 17 18 551 Boner PL, Liu DD, Feely WF, Wisocky MJ, . Wu J. 2003. Determination and 19 20 552 confirmation of 5-hydroxyflunixin in raw milk using liquid chromatography tandem 21 22 553 mass spectrometry. J. Agric. Food Chem. 51: 3753-3759 23 24 25 26 554 Daeseleire E, Mortier L, De Ruyck H, Geerts N. 2003. Determination of flunixin and 27 28 555 ketoprofen in milk by liquid chromatography –tandem mass spectrometry. Anal. 29 30 556 Chim. Acta. 488:25-34 31 32 33 34 557 De Veau EJ. 1999. Determination of non-protein bound phenylbutazone in bovine 35 36 558 plasma using ultrafiltration and liquid chromatography with ultraviolet detection. J. 37 38 39 559 Chromatogr. B 721:141-145 40 41 42 560 De Veau, EJ. 1996. Determination of phenylbutazone residues in bovine milk by 43 44 561 liquid chromatography with UV detection. J. AOAC Int. 79: 1050-1053 45 46 47 48 562 Debabrata M. 2008. Non-steroidal anti-inflammatory drugs and the heart: what is the 49 50 563 danger? Congest. Heart Failure. 14:75-82 51 52 564 53 54 55 56 565 Dowling G, Gallo P, Fabbrocino S, Serpe L, Regan L. 2008. Determination of 57 58 566 ibuprofen, ketoprofen, diclofenac and phenylbutazone in bovine milk by gas 59 60

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1 2 3 567 chromatography-tandem mass spectrometry. Food Additives and Contaminants 25: 4 5 6 568 1497-1508 7 8 9 569 Dowling G, Gallo P, Malone M, Regan L. 2009. Rapid confirmatory analysis of non- 10 11 12 570 steroidal anti-inflammatory drugs in bovine milk by rapid resolution liquid 13 14 571 chromatography tandem mass spectrometry. J. Chromatogr. A.1216:8117-8131 15 16 For Peer Review Only 17 572 European Commission Decision. 2002. Decision(2002/657/EC) of 12 August 2002 18 19 20 573 implementing Council Directive 96/23/EC concerning the performance of analytical 21 22 574 methods and interpretation of results. Off J Eur Comm L.221:8-36 23 24 25 575 European Commission. 1990. Council Regulation 2377/90/EEC. Off. J. Eur. 26 27 28 576 Communities 1990; L224:1-8 29 30 31 577 European Commission. 2004. Council Regulation 324/2004/EC. Off. J. Eur. 32 33 34 578 Communities 2004; L58: 16- 35 36 37 579 European Commission. 2005. Council Regulation 869/2005/EC. Off. J. Eur. 38 39 580 Communities 2005; L145: 19- 40 41 42 43 581 Feely WF, Chester-Yansen C, Thompson K, Campbell JW, Boner PL, Liu DD, 44 45 582 Crouch LS. 2002. Flunixin residues in milk after intravenous treatment of dairy cattle 46 47 583 with 14C flunixin. J. Agric. Food Chem. 50:7308-7313 48 49 50 51 584 Fiori M, Farne M, Civitareale C, Nasi A, Serpe L, Gallo P. 2004. The use of bovine 52 53 585 serum albumin as a ligand in affinity chromatographic clean-up of non-steroidal anti- 54 55 56 586 inflammatory drugs from bovine plasma. Chromatographia. 60:253-257 57 58 587 Gallo P, Fabbrocino S, Vinci F, Fiori M, Danese V, Serpe L. 2008. Confirmatory 59 60 588 identification of 16 non-steroidal anti-inflammatory drugs in raw milk by liquid

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1 2 3 589 chromatography coupled with ion trap mass spectrometry. Rapid Commun. Mass 4 5 6 590 Spectrom. 22; 841-854 7 8 9 591 Gonzalez G, Ventura R, Smith AK, De la Torre R, Segura J. 1996. Detection of non- 10 11 12 592 steroidal anti-inflammatory drugs in equine plasma and urine by gas chromatography- 13 14 593 mass spectrometry. J. Chromatogr. A. 719:251-264 15 16 594 Goodman A,For Gilman PeerA. 1992, Rall Review TW, Nies AS, Taylor Only P, editors. Goodman and 17 18 19 595 Gilman’s the Pharmacological Basis of Therapeutics. Singapore, McGraw Hill 20 21 22 596 Gowik P. Julicher B, Uhlig S. 1998. Multi-Residue method for non-steroidal anti- 23 24 597 inflammatory drugs in plasma using high performance liquid chromatography- 25 26 27 598 photodiode array detector:Method description and comprehensive in-house validation. 28 29 599 J. Chromatogr B. 716:221-232 30 31 600 32 Grippa E, Santini L, Castellano G, Gatto MT, Leone MG, Saso L. 2000. 33 34 601 Simultaneous determination of hydrocortisone, dexamethasone, indomethacin, 35 36 602 phenylbutazone and oxyphenylbutazone in equine serum by high performance liquid 37 38 39 603 chromatography. J. Chromatogr. B. 738:17-25 40 41 604 Gu X, Meleka-Boules M, Chen CL, Ceska DM. 1997. Determination of flunixin in 42 43 605 equine urine and serum by capillary electrophoresis. J. Chromatogr,B. 692:197-192 44 45 46 606 Hardee GE, Lai JW, Moore JN. 1982. Simultaneous determination of flunixin, 47 48 607 phenylbutazone, oxyphenylbutazone and hydroxyphenylbutazone in equine plasma 49 50 608 by high performance liquid chromatography with application to pharmacokinetics. J. 51 52 53 609 Liq Chromatogr. 5:1991-2003 54 55 610 Hines S, Pearce C, Bright J, Teale P. 2004. Development and validation of a 56 57 611 58 quantitative gas chromatography-mass spectrometry confirmatory method for 59 60 612 phenylbutazone in equine plasma. Chromatographia. 59:S109-S114

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1 2 3 613 http://www.emea.europa.eu/pdfs/vet/mrls/6742109en.pdf 4 5 6 7 614 Insel P.A, 1990. In Goodman A, Gilman A, Rall TW, Nies AS, Taylor P, 8 9 615 editors.Goodman and Gilman’s the Pharmacological Basis of Therapeutics. New 10 11 12 616 York, NY: Pergamon 13 14 15 617 J. Chromatogr. B. 854:313-319 16 For Peer Review Only 17 618 Jaussaud PH, Guieu D, Courtot D, Barbier B, Bonnaire Y. 1992. Identification of 18 19 20 619 tolfenamic acid metabolite in the horse by gas chromatography tandem mass 21 22 620 spectrometry. J. Chromatogr, 573:136-140. 23 24 621 Jedziniak P, Szprengier-Juszkiewicz T, Olejnik M, Jaroszewski J. 2007. 25 26 27 622 Determination of flunixin and 5-hydroxyflunixin in bovine plasma with HPLC-UV 28 29 623 method development, validation and verification. Bull Vet Inst Pulawy. 51:261-266 30 31 624 32 Kari F, Bucher J, Haseman J, Eustis S, Huff H. 1995. Long-term exposure to the anti- 33 34 625 inflammatory agent phenylbutazone induces kidney tumors in rats and liver tumors in 35 36 626 mice. Japanese Journal of Cancer Research. 86:252-263 37 38 39 40 627 Kopcha M, Kaneene JB, Shea ME, Miller R, Alwynelle S, Ahl AS. 1992. Use of non- 41 42 628 steroidal anti-inflammatory drugs in food animal practice. J Am Vet Med Assoc. 43 44 629 201:1868-1872 45 46 47 48 630 Kvaternick V, Malinski T, Wortmann J, Fischer J. 2007. Quantitative HPLC-UV 49 50 631 method for the determination of firocoxib from horse and dog plasma. 51 52 632 Luo Y, Rudy Jeffrey A, Elboh Cornelius E, Soma Laurence R, Gran F, Enright 53 54 55 633 James M, Tsang D. 2004. Quantification and confirmation of flunixin in equine 56 57 634 plasma by liquid chromatography-quadrupole time of flight mass spectrometry. J 58 59 60 635 Chromatogr B Analyt Technive Biomedical Life Science. 801: 2 173-84

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1 2 3 636 Malone E, Dowling G, Elliott CT, Kennedy DG, Regan L,2009. Development of a 4 5 6 637 rapid multi-class method for the confirmatory analysis of anti-inflammatory drugs in 7 8 638 bovine milk using liquid chromatography tandem mass spectrometry. J. Chromatogr. 9 10 639 11 A. 1216:8132-8140 12 13 14 640 Martin K, Stridsberg MI, Wiese BM. 1983. High performance liquid 15 16 641 chromatographicFor method Peer for the Review determination of phenylbutazone Only in milk with 17 18 19 642 special reference to the fat content in milk. J. Chromatogr. 276. 224-229 20 21 22 643 Miksa IR, Cummings MR, Poppenga RH. 2005. Multi-residue determination of anti- 23 24 644 inflammatory analgesics in sera by liquid chromatography-mass spectrometry. J. 25 26 27 645 Anal. Toxicol. 29:95-104 28 29 646 Neto LMR, Andraus MH, Salvadori MC. 1996. Determination of phenylbutazone 30 31 647 32 and oxyphenylbutazone in plasma and urine samples of horses by high performance 33 34 648 liquid chromatography and gas chromatography-mass spectrometry. J Chromatogr B. 35 36 649 678:211-218 37 38 39 650 Quintana MC, Ramos L, Gonzalez MJ, Blanco MH, Hernandez L. 2004. 40 41 651 Development of a solid phase extraction method for simultaneous determination of 42 43 652 corticoids and tranquilizers in serum samples. J. Sep Sci. 27:53-58 44 45 46 653 Rubb HS, Holland DC, Munns RK, Turnipseed SB, Long AR. 1995. Determination 47 48 654 of flunixin in milk by liquid chromatography with confirmation by gas 49 50 655 chromatography/mass spectrometry and selected ion monitoring. J. AOAC Int. 51 52 53 656 78:959-967 54 55 56 657 SANCO. 2000. European Commission-Reference Laboratory for Residues of 57 58 59 658 Veterinary Drugs. Workshop NSAIDs and validation according to SANCO 60 659 1805/2000, Berlin 2001

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1 2 3 660 SANCO. 2007. CRL Guidance Paper CRLs View on state of the art analytical 4 5 6 661 methods for national residue control plans 7 8 9 662 Singh AK, Jang Y, Misra U, Granley K. 1991. Simultaneous analysis of flunixin, 10 11 12 663 naproxen, ethacrynic acid, indomethacin, phenylbutazone, mefenamic acid and 13 14 664 thiosalicylic acid in plasma and urine by high performance liquid chromatography 15 16 665 and gas chromatographyFor Peer mass spectrometry. Review J. Chromatogr. Only 568:351-361 17 18 19 666 Smith GW, Davis JL, Tell LA, Webb AI, Riviere JE. 2008. Extra-label use of non- 20 21 667 steroidal anti-inflammatory drugs in cattle. J. Am. Vet. Med. Assoc. 232: 697-701 22 23 24 668 Staa VP, Smeeth L, Persson I, Parkinson J, Leufkems HGM. 2008. What is the 25 26 27 669 harm-benefit ratio of COX-2 inhibitors. Int. J. Epidemiol. 37:405-413 28 29 670 Stolker AAM, Rutgers P, Oosterink E, Lasaroms JJP, Peters RJP, van Rhijn JA, 30 31 671 32 Nielen MWF. 2008. Comprehensive screening and quantification of veterinary drugs 33 34 672 in milk using UPLC-Tof-MS. Anal Bioanal Chem. 391: 2309-2322 35 36 37 673 Sundlof SF, Kaneene JB, Miller RA. 1995. National survey on veterinarian-initiated 38 39 40 674 drug use in lactating dairy cattle. J. Am. Vet. Med. Assoc. 207:347-352 41 42 43 675 U.S Code of Federal Regulations, Vol 21 (1988), parts 520.1720 and 522.1720. 44 45 46 676 Vinci F, Fabbrocino S, Fiori M, Serpe L, Gallo P. 2006. Determination of fourteen 47 48 49 677 non-steroidal anti-inflammatory drugs in animal serum and plasma by liquid 50 51 678 chromatography/mass spectrometry. Rapid Comm Mass Spectrom. 20:3412-3420 52 53 679 54 You Y, Uboh Cornelius E, Soma Lawrence R, Guan F, Li X, Rudy Jeffrey A, Chen J A. 2009. 55 56 680 J. Anal. Toxicol. 33:41-50 57 58 681 SANCO/2004/2726/Rev 1 Guidelines for implementation of Commission Decision 59 60 682 2002/657EC

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1 2 3 683 4 5 6 7 684 8 9 10 685 Structure a: Carprofen 11 686 12 687 13 14 H 15 16 For Peer ReviewN OnlyO 17 18 19 OH 20 C l 21 22 688 C a rp ro fe n 23 689 24 690 25 691 Structure b: Diclofenac 26 692 27 693 28 29 C l 30 31 32 33 NH 34 35 C l OH 36 37 38 O 39 40 41 42 694 D ic lo fe na c 43 695 44 696 45 697 Structure c: Mefenamic Acid 46 698 47 699 48 700 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 4 5 6 7 8 9 10 NH O 11 12 13 OH 14 15 16 For Peer Review Only 17 18 Mefenamic acid 19 701 702 20 703 21 704 22 705 Structure d: Ibuprofen 23 24 25 26 O 27 28 29 OH 30 31 32 706 Ib up ro fe n 33 707 34 708 35 709 36 710 Structure e: Ketoprofen 37 711 38 712 39 40 O 41 42 O 43 44 45 OH 46 47 48 K e to p ro fe n 49 713 714 50 715 51 716 52 717 53 718 54 719 55 720 56 721 57 722 58 723 59 724 60 725 726

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1 2 3 727 4 728 Structure f: Phenylbutazone 5 729 6 730 7 8 9 10 11 12 13 14 O O 15 16 For Peer Review Only 17 N N 18 19 20 21 22 23 24 731 Phenylbutazone 25 732 26 733 27 734 28 735 29 736 30 737 31 738 32 739 Structure g: Flunixin 33 740 34 741 35 742 36 743 37 744 38 39 40 41 42 43 44 45 46 47 745 48 746 49 747 50 748 51 749 52 750 53 751 54 752 55 753 56 754 57 755 58 756 59 757 758 60 759 760

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1 2 3 761 4 762 Structure h: Hydroxy-Flunixin 5 763 6 764 7 8 9 10 11 12 13 14 15 16 For Peer Review Only 17 18 765 19 766 20 767 21 768 22 769 Structure i: Tolfenamic Acid 23 770 24 771 25 26 27 28 29 30 31 772 773 32 774 33 775 34 776 35 777 36 778 Structure j: Meloxicam 37 779 38 780 39 781 40 782 41 783 42 784 43 44 45 46 47 48 49 50 51 52 53 785 54 786 55 787 56 788 Fig. 1 Structures of the NSAIDs 57 58 59 789 60

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1 2 3 790 4 5 6 7 791 8 9 10 792 11 12 13 14 793 15 16 For Peer Review Only 17 794 18 19 20 795 21 22 23 24 796 25 26 27 797 28 29 30 31 798 32 33 34 799 35 36 37 800 38 39 40 41 801 42 43 44 802 45 803 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3

4 11.91 8.37 6.8e4 5.0e5 5 2 A 6.5e4 4.8e5 4.6e5 6.0e4 4.4e5 4.2e5 6 5.5e4 4.0e5 3.8e5 5.0e4 7 3.6e5 3.4e5 4.5e4 3.2e5 3.0e5 8 Intensity, cps 4.0e4 2.8e5 3.5e4 2.6e5 9 2.4e5 3.0e4 2.2e5 2.0e5 d3 -Ibuprofen 2.5e4 1.8e5 10 1.6e5 2.0e4 1.4e5 1.2e5 1.5e4 11 1.0e5 8.0e4 1.0e4 6.0e4 12.72 12 5.82 4.0e4 5000.0 0.37 0.56 5.29 6.20 13.35 14.49 4.36 4.98 6.38 2.0e4 0.0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 13 Time, min Time, min 14 For Peer Review Only 15

16 7.98 8.36 2 B 1.18e5 1.15e5 5.0e5 17 4.8e5 1.10e5 4.6e5 1.05e5 4.4e5 1.00e5 18 4.2e5 9.50e4 4.0e5 9.00e4 3.8e5 8.50e4 19 3.6e5 8.00e4 3.4e5 7.50e4 3.2e5 20 7.00e4 3.0e5 Intensity, cps 6.50e4 2.8e5 11.97 6.00e4 2.6e5 21 5.50e4 2.4e5 5.00e4 2.2e5 4.50e4 2.0e5 1.8e5 22 4.00e4 1.6e5 3.50e4 1.4e5 3.00e4 1.2e5 23 2.50e4 1.0e5 2.00e4 8.0e4 1.50e4 24 6.0e4 1.00e4 12.60 4.0e4 5000.00 2.0e4 0.00 0.0 25 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 Time, min Time, min 26 27

28 . 2 C 5.70 2.2e6 8.13 1.6e5 29 2.1e6 1.5e5 2.0e6 1.9e6 1.4e5 30 1.8e6 1.3e5 1.7e6 31 1.2e5 1.6e6 1.5e6 1.1e5 1.4e6 1.0e5 32 1.3e6 9.0e4 1.2e6 1.1e6 33 8.0e4 1.0e6 7.0e4 Intensity, cps 9.0e5 34 6.0e4 8.0e5 7.0e5 5.0e4 6.0e5 4.0e4 35 5.0e5 3.0e4 4.0e5 5.2 3.0e5 36 2.0e4 2.7 2.0e5 1.0e4 6.23 1.0e5 0. 0.0 37 1. 2.0 3.0 4.0 5. 6.0 7.0 8.0 9. 10.0 11.0 12. 13.0 14.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 Time, Time, min 38

39 2 D 40

41 1.5e6 7.71 1.9e6 8.13 1.4e6 1.8e6

1.3e6 1.7e6 42 1.6e6 1.2e6 1.5e6 43 1.1e6 1.4e6 Intensity, cps 1.0e6 1.3e6 1.2e6 44 9.0e5 1.1e6 8.0e5 45 1.0e6 7.0e5 9.0e5

8.0e5 6.0e5 46 7.0e5 5.0e5 6.0e5 47 4.0e5 5.0e5 3.0e5 4.0e5 3.0e5 48 2.0e5 5.69 2.0e5 1.0e5 1.0e5

49 0.0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 50 Time, min Time, min 51 52 Fig. 2A. Chromatogram of negative control milk (2A) and plasma (2C) fortified at 15 ng -1 -1 53 mL with internal standard d 3-IBP and fortified with 5 ng mL of CPF in milk (2B) and 54 plasma (2D) 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Page 35 of 52 Food Additives and Contaminants

1 2 3 4

5 8.56 8.19 3400 2.2e5 d4-Diclofenac 6 2.1e5 3200 2.0e5 3000 1.9e5 7 2800 1.8e5 1.7e5 2600 1.6e5 8 2400 1.5e5 7.60 2200 1.4e5

9 2000 1.3e5 Intensity, cps 8.64 1.2e5 1800 9.41 8.21 1.1e5 10 1600 1.0e5 8.13 1400 9.0e4 8.0e4 11 1200 7.55 9.89 3 A 10.33 7.0e4 1000 10.52 6.0e4 12 800 5.0e4 3.97 600 4.0e4 3.0e4 13 400 5.35 6.02 6.37 6.85 1.55 11.00 2.0e4 1.35 3.24 7.24 11.84 12.03 200 1.0e4 14 0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 ForTime, min Peer ReviewTime, min Only 15

16 8.21 8.19 2.3e4 2.3e5 2.2e5 17 2.2e4 2.1e5 3 B 2.1e4 2.0e4 2.0e5 18 1.9e4 1.9e5 1.8e5 1.8e4 1.7e4 1.7e5 19 1.6e4 1.6e5 1.5e4 1.5e5 1.4e5 7.59 20 1.4e4 1.3e4 1.3e5 1.2e4 1.2e5 1.1e5 21 1.1e4 Intensity, cps 1.0e4 1.0e5 9000.0 9.0e4 22 8000.0 8.0e4 7000.0 7.0e4 6000.0 6.0e4 23 5000.0 5.0e4 4000.0 9.32 4.0e4 8.56 3000.0 3.0e4 24 2.0e4 2000.0 8.12 1000.0 1.0e4 0.0 0.0 25 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 Time, min Time, min 26 27

28 .

1650 7.97 2.9e6 7.24 3 C 1600 29 2.8e6 1500 2.6e6 30 1400 2.4e6 1300 31 1200 2.2e6 1100 2.0e6 12.15 1000 0.59 32 1.8e6 900 13.75 1.6e6 800 4.37 8.44 33 1.4e6 700 0.21 2.12 2.46 12.69 13.49 7.97 1.10 3.20 5.61 11.84 Intensity, cps 1.2e6 600 1.66 3.45 4.13 4.76 7.45 9.35 13.96 34 0.88 2.68 6.726.85 13.08 500 4.01 5.54 7.00 8.709.96 11.71 1.0e6 5.83 6.38 9.13 10.54 6.64 35 400 5.31 10.71 8.0e5 300 6.0e5 200 36 4.0e5 100 2.0e5 0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 37 Time, min 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 Time, min 38

39 . . 7.99 7.24 4.4e5 2.9e6 40 4.2e5 2.8e6 3 D 4.0e5 2.6e6 41 3.8e5 3.6e5 2.4e6 3.4e5 2.2e6 42 3.2e5 3.0e5 2.0e6 2.8e5 1.8e6 43 2.6e5 2.4e5 1.6e6 2.2e5 44 1.4e6 Intensity, cps 2.0e5 7.97 1.8e5 1.2e6 1.6e5 45 1.0e6 1.4e5 3.53 6.64 1.2e5 8.0e5 7.80 46 1.0e5 6.0e5 8.0e4

6.0e4 8.74 4.0e5 47 7.04 4.0e4 7.69 5.73 2.0e5 2.0e4 0.0 0.0 48 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 Time, min Time, min 49 50 51 Fig. 3A. Chromatogram of negative control milk (3A) and plasma (3C) fortified at 15 ng -1 -1 52 mL with internal standard d 4-DCF and fortified at 5 ng mL with DCF in milk (3B) and 53 plasma (3D) 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Food Additives and Contaminants Page 36 of 52

1 2 3 4 d3 -Ibuprofen

5 3.46 8.37 9250 5.0e5 9000 6 4.8e5 8500 4.6e5 8000 4.4e5 7 4.2e5 7500 4 A 4.0e5 7000 3.8e5 3.6e5 8 6500 3.4e5 6000 3.2e5 9 5500 3.0e5 2.8e5 5000 Intensity, cps 2.6e5 4500 10 2.4e5 4000 2.2e5 2.0e5 3500 11 1.8e5 3000 1.6e5 2500 1.4e5 12 1.2e5 2000 1.0e5 8.41 1500 12.07 8.0e4 6.73 9.76 11.65 2.83 5.33 5.91 6.29 7.00 7.83 8.29 8.94 9.92 12.26 14.39 6.0e4 0.26 3.93 4.73 9.62 13.14 14.16 13 1000 0.62 4.30 10.97 1.16 1.80 2.64 4.0e4 500 2.0e4 0 0.0 14 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 ForTime, min Peer ReviewTime, min Only 15

16 8.38 8.36 4.4e4 5.0e5 4.2e4 17 4.8e5 4.0e4 4 B 4.6e5 3.8e4 4.4e5 18 3.6e4 4.2e5 3.4e4 4.0e5 3.8e5 3.2e4 19 3.6e5 3.0e4 3.4e5 2.8e4 3.2e5 20 2.6e4 3.0e5 Intensity, cps 2.4e4 2.8e5 2.2e4 2.6e5 2.4e5 21 2.0e4 2.2e5 1.8e4 2.0e5 1.6e4 22 1.8e5 1.4e4 1.6e5 1.2e4 1.4e5 23 1.0e4 1.2e5 1.0e5 8000.0 3.45 8.0e4 6000.0 6.0e4 24 4000.0 4.0e4 2000.0 2.0e4 0.0 0.0 25 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 Time, min Time, min 26

27 ) . 4.8e5 3.69 2.2e6 8.13 28 4.6e5 2.1e6 4.4e5 2.0e6 4.2e5 1.9e6 29 4.0e5 1.8e6 3.8e5 1.7e6 3.6e5 4 C 1.6e6 3.4e5 30 1.5e6 3.2e5 1.4e6 3.0e5 1.3e6 31 2.8e5 2.6e5 1.2e6 2.26 2.4e5 1.1e6 32 2.2e5 1.0e6 2.0e5 9.0e5 Intensity, cps 1.8e5 8.0e5 33 1.6e5 7.0e5 1.4e5 6.0e5 1.2e5 5.0e5 34 1.0e5 4.0e5 8.0e4 2.63 3.0e5 6.0e4 35 4.0e4 2.0e5 6.006.39 2.0e4 1.0e5 0.0 0.0 36 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 Time, min Time, min 37

. . 38 8.14 8.13 4.8e5 1.9e6 39 4.6e5 1.8e6 4.4e5 1.7e6 4.2e5 3.68 1.6e6 40 4.0e5 3.8e5 1.5e6 3.6e5 1.4e6 41 3.4e5 1.3e6 3.2e5 1.2e6 3.0e5 42 4 D 2.8e5 1.1e6 2.6e5 1.0e6 2.4e5 9.0e5 43 2.2e5 2.24 2.0e5 8.0e5 1.8e5 7.0e5

44 1.6e5 6.0e5 Intensity, cps 1.4e5 5.0e5 1.2e5 45 1.0e5 4.0e5 8.0e4 2.62 3.0e5 6.0e4 2.0e5 46 4.0e4 5.99 6.39 2.0e4 1.0e5 0.0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 47 Time, min Time, min

48 49 Fig. 4A. Chromatogram of negative control milk (4A) and plasma (4C) fortified at 15 ng 50 -1 -1 51 mL with internal standard d 3-IBU and fortified at 5 ng mL with IBP in milk (4B) and 52 plasma (4D). 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Page 37 of 52 Food Additives and Contaminants

1 2 3 4 5 5 A 6

11.94 8.37 1.8e4 5.0e5 7 1.7e4 4.8e5 1.6e4 4.6e5 4.4e5 8 1.5e4 4.2e5 1.4e4 d3-Ibuprofen 4.0e5 3.8e5 9 1.3e4 3.6e5 1.2e4 3.4e5 3.2e5 10 Intensity, cps 1.1e4 3.0e5 1.0e4 2.8e5 11 9000.0 2.6e5 2.4e5 8000.0 2.2e5 12 7000.0 2.0e5 1.8e5 6000.0 7.42 1.6e5 5000.0 13 1.4e5 1.2e5 4000.0 7.49 8.37 1.0e5 3000.0 8.0e4 14 6.0e4 9.05 2000.0 7.77 9.91 11.62 4.0e4 For5.90 Peer12.52 Review Only

1000.0 2.0e4 15 0.0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 16 Time, min Time, min 17 18 5 B

7.32 8.36 19 4.3e4 4.2e4 5.0e5 4.8e5 4.0e4 4.6e5 3.8e4 20 4.4e5 3.6e4 4.2e5 3.4e4 4.0e5 21 Intensity, cps 3.2e4 3.8e5 3.0e4 3.6e5 3.4e5 2.8e4 22 3.2e5 2.6e4 3.0e5 2.4e4 2.8e5 23 2.2e4 2.6e5 2.0e4 2.4e5 2.2e5 1.8e4 2.0e5 24 11.96 1.6e4 1.8e5 1.4e4 1.6e5 25 1.2e4 1.4e5 1.0e4 1.2e5 8000.0 1.0e5 7.42 8.0e4 26 6000.0 6.0e4 4000.0 7.49 8.35 4.0e4 2000.0 27 2.0e4 0.0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 28 Time, min Time, min 5 C 29

0.55 2.2e6 8.13 30 440 2.1e6 420 2.0e6 400 1.9e6 31 380 1.8e6 360 1.7e6 340 32 1.6e6 320 1.5e6 300 8.50 33 280 1.4e6 260 1.3e6 Intensity, cps 240 1.2e6 34 220 1.1e6 0.67 1.42 3.90 8.91 10.2310.43 11.81 200 1.0e6 180 9.0e5 35 160 0.07 0.74 2.38 2.65 5.27 5.48 6.64 6.99 7.87 8.14 9.4210.12 12.00 13.95 14.29 8.0e5 140 7.0e5 120 6.0e5 36 100 1.35 2.052.20 3.053.70 4.41 5.00 5.676.02 7.05 9.51 11.07 12.09 12.6813.14 14.41 5.0e5 80 4.0e5 60 37 40 3.0e5 20 2.0e5 0 1.0e5 38 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 Time, min 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 39 Time, min 40 . 5 D 6.98 1.9e6 8.13 41 1.4e6 1.8e6

1.3e6 1.7e6 42 1.6e6 1.2e6 1.5e6 1.1e6 43 1.4e6 1.0e6 1.3e6 44 9.0e5 1.2e6 1.1e6 Intensity, cps 8.0e5 1.0e6 45 7.0e5 9.0e5

6.0e5 8.0e5 46 7.0e5 5.0e5 6.0e5 4.0e5 47 7.08 5.0e5 3.0e5 4.0e5 3.0e5 48 2.0e5 2.0e5 1.0e5 7.40 1.0e5

49 0.0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 50 Time, min Time, min 51 52 Fig. 5A. Chromatogram of negative control milk (5A) and plasma (5C) fortified at 15 ng -1 -1 53 mL with internal standard d 3-IBU and fortified at 5 ng mL with KPF in milk (5B) and 54 plasma (5D). 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Food Additives and Contaminants Page 38 of 52

1 2 3 4

5 d4-Tolfenamic Acid 6 7

8 8.73 440 9 8.89 420 2.0e5 1.18 3.51 8.07 400 1.9e5 6 A 380 10 1.8e5

360 1.7e5 340 11 1.6e5 320 1.5e5 300 12 1.4e5 280 1.3e5 260 Intensity, cps 1.2e5 13 240 1.1e5 220 3.56 6.87 11.74 1.0e5 14 200 9.0e4 180 For Peer Review Only 8.0e4 160 4.03 8.00 8.37 9.39 9.97 13.69 15 7.0e4 140 6.0e4 120 2.98 4.86 4.98 6.94 7.68 9.16 9.67 10.92 12.19 14.34 16 100 5.0e4 80 4.0e4 0.85 2.84 4.31 10.03 11.04 12.26 12.89 13.97 14.64 3.0e4 17 60 0.04 1.91 2.04 4.67 5.91 6.05 6.40 40 2.0e4 18 20 1.0e4 0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 19 Time, min Time, min 20 6 B

8.71 8.88 21 9.9e4 1.7e5 9.5e4 1.6e5 9.0e4 22 1.5e5 8.5e4 1.4e5 8.0e4 23 7.5e4 1.3e5 7.0e4 1.2e5 24 6.5e4 1.1e5 Intensity, cps 6.0e4 1.0e5 5.5e4 25 9.0e4 5.0e4 8.0e4 4.5e4 7.0e4 26 4.0e4 6.0e4 3.5e4 5.0e4 27 3.0e4 2.5e4 4.0e4 28 2.0e4 3.0e4 1.5e4 2.0e4 1.0e4 29 1.0e4 5000.0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 Time, min 30 Time, min 31 6 C

32 750 8.49 6.0e5 8.70 700 5.5e5 33 650 5.0e5 600

34 550 4.5e5 Intensity, cps 500 7.75 35 4.0e5 450 3.5e5 400 36 7.83 3.0e5 350 37 300 2.5e5 250 2.0e5 38 200 0.560.67 4.026.19 7.63 7.89 9.73 11.55 1.5e5 150 0.27 1.371.46 2.3 2.453 2.96 3.78 5.06 6.947.057.40 8.919.31 10.48 10.72 12.01 13.0513.21 14.22

1.09 2.21 2.71 3.46 4.264.93 5.585.67 6.37 9.12 9.80 11.00 12.49 13.39 14.35 39 100 1.0e5 6.26 50 5.0e4 0 40 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 Time, min 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 41 Time, min 42 6 D

1.15e6 8.51 8.70 43 5.2e5 1.10e6 5.0e5 1.05e6 4.8e5 44 1.00e6 4.6e5 9.50e5 4.4e5 9.00e5 4.2e5 4.0e5 45 8.50e5 3.8e5 8.00e5 3.6e5 46 7.50e5 3.4e5 7.00e5 3.2e5 Intensity, cps 6.50e5 3.0e5 6.00e5 2.8e5 47 2.6e5 5.50e5 2.4e5 5.00e5 2.2e5 4.50e5 48 2.0e5 4.00e5 1.8e5 3.50e5 1.6e5 49 3.00e5 1.4e5 2.50e5 1.2e5 1.0e5 2.00e5 8.0e4 6.26 50 1.50e5 6.0e4 1.00e5 4.0e4 51 5.00e4 2.0e4 0.00 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 52 Time, min Time, min 53 54 Fig. 6A. Chromatogram of negative control milk (6A) and plasma (6C) fortified at 15 ng -1 -1 55 mL with internal standard d 4-TLF and fortified at 5 ng mL with MFN 56 in milk (6B) and plasma (6D). 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected]

Page 39 of 52 Food Additives and Contaminants

1 2 3 4 5 6 7 A

7.96 8.53 6.3e4 7 3800 6.0e4 d10 -Phenylbutazone 3600 3400 8 5.5e4 3200 3000 5.0e4 9 2800 4.5e4 2600 2400 10 4.0e4 2200 3.5e4 2000 11 1800 8.76 7.82 3.0e4 1600 12 1400 2.5e4 1200 9.01 9.58 2.0e4 1000 Intensity, cps 7.21 10.15 13 800 6.76 1.5e4 600 7.69 1.0e4 400 5.27 6.61 10.77 6.19 14 4.84 1.77 3.69 4.20 11.48 200 For Peer 5000.0Review Only 0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15 Time, min 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 Time, min 16 17 . 8.57 8.53 6.3e4 18 7 B 4000 3800 6.0e4 3600 5.5e4 19 3400 3200 5.0e4 3000 20 4.5e4 2800

2600 4.0e4 21 2400 Intensity, cps 2200 3.5e4 2000 22 3.0e4 1800 1600 2.5e4 23 1400 2.0e4 1200

1000 1.5e4 24 800 9.03 1.0e4 600 8.31 10.22 9.47 400 10.57 25 7.05 5000.0 200 0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 26 Time, min Time, min 27 28

8.32 8.34 1.25e5 29 5200 1.20e5 5000 1.15e5 4800 1.10e5 4600 30 8.11 1.05 e5 4400 1.00e5 4200 9.50e4 4000 9.00e4 31 7 C 3800 8.50e4 3600 8.00e4 3400 8.99 7.50e4 32 3200 9.92 7.00e4 3000 6.50e4 2800 6.00e4 33 2600 5.50e4 2400 5.00e4 2200 4.50e4 2000 8.71 34 4.00e4 1800 7.65 7.34 3.50e4 1600 3.00e4 1400 8.58 35 2.50e4 1200 10.69 7.15 2.00e4 1000 6.69 9.18 1.50e4 800 36 3.99 6.016.40 10.93 1.00e4 8.20 9.8810.31 600 3.77 5000.00 400 4.17 5.60 11.26 Intensity, cps 1.21 1.68 0.00 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 200 0 0 0 0 0 0 0Time, 0 0 0 0 0 0 0 37 0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 min 38 Time, min 39 Max. 1.1e5 cps. 8.36 8.32 40 1.10e5 9.0e4 1.05e5 8.5e4 1.00e5 8.0e4 41 9.50e4 9.00e4 7.5e4 8.50e4 7.0e4 42 7 D 8.00e4 6.5e4 7.50e4 6.0e4 7.00e4 43 6.50e4 5.5e4 6.00e4 5.0e4 44 5.50e4 4.5e4 5.00e4 4.0e4 4.50e4 8.61 45 4.00e4 3.5e4 9.19 3.50e4 3.0e4 8.58

3.00e4 2.5e4 46 2.50e4 2.0e4 2.00e4 1.50e4 1.5e4 7.39 8.89 8.14 10.33 47 1.00e4 8.06 10.39 1.0e4 8.03 9.85 Intensity, cps 5000.00 5000.0 0.00 48 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 Time, min 49 Time, min 50 51 Fig. 7A. Chromatogram of negative control milk (7A) and plasma (7C) fortified at 15 ng -1 -1 52 mL with internal standard d 10 -PBZ and fortified at 5 ng mL with PBZ in milk (7B) and 53 plasma (7D) 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Food Additives and Contaminants Page 40 of 52

1 2 3 4 8 A 5

6 d3-Meloxicam 7 8 9 10

11 1700 7.33 1600 7.31 12 1500 5.7e5 5.5e5 13 1400 1300 5.0e5

1200 14 Intensity, cps 4.5e5 1100 For Peer Review Only 4.0e5 15 1000 16 900 3.5e5 800 3.0e5 17 700 600 2.5e5 9.25 18 500 8.27 6.20 7.15 8.72 2.0e5 400 7.50 9.71 11.38 6.688.81 10.18 11.00 1.5e5 19 300 3.78 5.88 11.88 14.52 0.20 0.35 4.47 5.48 10.31 12.82 14.25 200 1.442.68 3.43 4.05 5.37 12.27 13.66 20 0.451.34 1.56 2.47 3.30 13.93 1.0e5 100 5.0e4 0 21 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 Time, min 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 22 Time, min 23 8 B 24 25 26 27 28 29

30 7.32 4.4e5 7.31 4.4e5 31 4.2e5 4.2e5 4.0e5 4.0e5 32 Intensity, cps 3.8e5 3.8e5 3.6e5 3.6e5 3.4e5 33 3.4e5 3.2e5 3.2e5 3.0e5 34 3.0e5 2.8e5 2.8e5 2.6e5 2.6e5 35 2.4e5 2.4e5 2.2e5 2.2e5 2.0e5 36 2.0e5 1.8e5 1.8e5 37 1.6e5 1.6e5 1.4e5 1.4e5 38 1.2e5 1.2e5 1.0e5 1.0e5

8.0e4 8.0e4 39 6.0e4 6.0e4 4.0e4 4.0e4 40 2.0e4 2.0e4 0.0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 41 Time, min Time, min 42 43 44 Fig. 8A. Chromatogram of Negative Control milk (8A) fortified at 15 ng mL -1 with 45 -1 46 internal standard d 3-MLX and fortified at 7.5 ng mL with MLX (8B) 47 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Page 41 of 52 Food Additives and Contaminants

1 2 3 4 5 9A 6 7 d3-Flunixin 8 9 10 11 12 13 14 10.32 1.18e4 10.17 1.15e4 For Peer Review Only 8.19 1.10e4 2.2e5 15 9.09 2.1e5 1.05e4 16 1.00e4 9.21 2.0e5 9500.00 1.9e5 Intensity, cps 9000.00 1.8e5 17 8500.00 1.7e5 8000.00 1.6e5 1.5e5 18 7500.00 7.26 7.60 7000.00 1.4e5 6500.00 1.3e5 19 10.03 6000.00 1.2e5

5500.00 1.1e5 20 5000.00 1.0e5 4500.00 8.73 9.0e4 21 4000.00 8.0e4 3500.00 7.0e4 10.52 3000.00 6.0e4 22 2500.00 6.73 5.0e4 2000.00 4.0e4 1500.00 23 8.40 10.80 3.0e4 1000.00 7.65 5.65 10.99 2.0e4 500.00 24 1.0e4 0.00 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 Time, min 25 Time, min 26 27 28 29 9 B 30 31 32 33

34 7.60 4.6e5 35 4.4e5 8.19 2.3e5 4.2e5 2.2e5 36 4.0e5 2.1e5 3.8e5 2.0e5 37 3.6e5 1.9e5 3.4e5 1.8e5

3.2e5 1.7e5 38 3.0e5 1.6e5 2.8e5 1.5e5 39 2.6e5 1.4e5 7.59 1.3e5 2.4e5 Intensity, cps 1.2e5 2.2e5 40 1.1e5 2.0e5 1.0e5 1.8e5 9.0e4 41 1.6e5 8.0e4 1.4e5 7.0e4 42 1.2e5 6.0e4 1.0e5 5.0e4 8.0e4 43 4.0e4 6.0e4 3.0e4 44 4.0e4 10.16 2.0e4 2.0e4 1.0e4 0.0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 45 Time, min Time, min 46 47 Fig. 9A. Chromatogram of negative control milk (9A) fortified at 15 ng mL -1 with 48 -1 49 internal standard d 3-FLU and fortified at 20 ng mL with FLU (9B) 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Food Additives and Contaminants Page 42 of 52

1 2 3 4 5 10 A 6 d3-Flunixin 7 8 9 10 11

7.54 12 300 8.19 280 2.2e5 13 2.1e5 260 8.92 2.0e5 14 240 1.9e5 For Peer Review1.8e5 Only 15 220 1.7e5 1.6e5 200 1.5e5 Intensity, cps 7.60 16 180 1.4e5 1.3e5 160 17 8.83 1.2e5 140 1.1e5

1.0e5 18 120 9.0e4 1.66 2.62 4.09 6.21 6.9 3 9.03 9.71 10.47 100 8.0e4 19 7.0e4 80 6.0e4

20 60 5.0e4 0.48 1.15 2.14 3.15 4.51 4.68 5.20 5.66 6.67 7.02 7.64 8.04 9.18 9.90 10.39 11.05 11.54 12.32 13.76 14.02 4.0e4 21 40 3.0e4 2.0e4 20 22 1.0e4 0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 23 Time, min Time, min 24 25 10 B 26 27

28 7.10 29 9500 9000 8.19 2.3e5 30 Intensity, cps 8500 2.2e5 8000 2.1e5 2.0e5 31 750 1.9e5 700 1.8e5

32 650 1.7e5 1.6e5 600 33 1.5e5 550 1.4e5 7.59 500 1.3e5 34 1.2e5 450 0 1.1e5 400 35 1.0e5 350 9.0e4 0 8.0e4 300 36 0 7.0e4 250 6.0e4 200 5.0e4 37 0 150 4.0e4 0 3.0e4 38 100 0 2.0e4 50 1.0e4 39 0 0.0 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 0 Time, 0 0 0 0 0 Time, min 40 min 41 -1 42 Fig. 10A. Chromatogram of negative control milk (10 A) fortified at 15 ng mL with -1 43 internal standard d 3-FLU and fortified at 20 ng mL with FLU-OH (10B) 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Page 43 of 52 Food Additives and Contaminants

1 2 3 4 5 6 d4-Tolfenamic Acid 7 11 A 8 9

10 . 6200 8.66 11 6000 8.89 12 5500 2.0e5 1.9e5 5000 13 0.94 1.8e5 1.7e5 4500 1.6e5 6.35 14 4000 1.5e5 Intensity, cps For Peer Review1.4e5 Only 3500 15 1.3e5 1.2e5 3000 16 1.1e5 0.43 2500 1.0e5 5.28 17 9.0e4 2000 8.0e4

8.42 7.0e4 7.16 18 1500 9.49 6.0e4 7.44 5.0e4 1000 1.41 4.74 5.98 10.18 12.36 19 2.13 2.33 4.24 5.83 6.74 7.64 12.84 4.0e4 3.08 12.00 13.00 13.73 14.57 0.18 3.84 8.98 500 2.61 3.74 9.65 10.68 3.0e4 20 2.0e4 0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 1.0e4 Time, min 0.0 21 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 Time, min 22 11 B 23 24 25 26 27 28 29 30 31

32 8.90 1.4e6 1.7e5 8.88 1.3e6 33 1.6e5 1.2e6 1.5e5 34 1.1e6 1.4e5 Intensity, cps 1.3e5 1.0e6 35 1.2e5 9.0e5 1.1e5

36 8.0e5 1.0e5 37 7.0e5 9.0e4 8.0e4 6.0e5 7.0e4 38 5.0e5 6.0e4 39 4.0e5 5.0e4 4.0e4 3.0e5 40 3.0e4 2.0e5 2.0e4 1.0e5 41 1.0e4

0.0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 42 Time, min Time, min 43 44 45 -1 46 Fig. 11A. Chromatogram of negative control milk (11A) fortified at 15 ng mL with -1 47 internal standard d 4-TLF and fortified at 25 ng mL with TLF (11B) 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Food Additives and Contaminants Page 44 of 52

1 2 3 4 5 6 . 9.22 7 7.5e5 8 7.0e5 6.5e5 9 6.0e5 5.5e5

10 5.0e5 Intensity, cps 11 4.5e5 4.0e5 12 3.5e5 3.0e5

13 2.5e5 14 2.0e5 8.93 1.5e5 For Peer Review Only 15 1.0e5 5.0e4

16 0.0 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 17 Time, min 18 19 20 21 Fig. 12A. Chromatogram of negative control milk fortified at 0.1 ng mL -1 with DCF and 22 23 analysed by Applied Biosystems 5500 LC-MS 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Page 45 of 52 Food Additives and Contaminants

1 2 3 Table 1 : LC gradient profile for determination of CPF, DCF, IBP, KPF, MFN, PBZ, 4 5 6 FLU, FLU-OH, TLF and MLX 7 8 9 Time Component A Component B 10 11 (min) (%) (%) 12 0.0 90 10 13 1.0 90 10 14 For Peer Review Only 15 3.5 85 15 16 7.5 35 65 17 18 9.5 35 65 19 11.0 90 10 20 15.0 90 10 21 22 23 Component A: Component A: water containing 0.001 M acetic acid + acetonitrile 24 (90 + 10, v/v) 25 26 Component B: Acetonitrile 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Food Additives and Contaminants Page 46 of 52

1 2 3 Table 2 : MS/MS parameters for determination of CPF, DCF, IBP, KPF, MFN, PBZ, 4 5 6 FLU, FLU-OH, TLF and MLX 7 8 9 10 11 Collision 12 Collision Cell Exit 13 Declustering Potential Energy Potential 14 CompoundFor Peer Transition Review[V] Only[eV] [V] 15 16 CPF 271.8>227.9(strong) -60 -16 -13 17 271.8>225.8(weak) -60 -38 -13 18 19 DCF 294.0>250.0(strong) -70 -19 -15 20 294.0>214.0(weak) -70 -28 -15 21 IBP 205.0>161.0(strong) -45 -14 -9 22 23 24 PBZ 306.9>279.0(strong) -70 -30 -16 25 306.9>130.9 (weak) -70 23 -12 26 27 FLU 294.9>250.8(strong) -80 -18 -15 28 294.9>191.0(strong) -80 -25 -17 29 FLU-OH 310.9>266.9(strong) -65 -48 -9 30 31 310.9>226.9(weak) -65 -50 -9 32 MFN 239.8>196 (strong) -60 -34 -11 33 239.8>179.9 (weak) -60 -18 -17 34 35 MLX 349.8>285.9(strong) -50. -19 -18 36 349.8>145.9(strong) -50 -35 -12 37 38 KPF 252.8>209.0(strong) -40. -10 -9 39 40 41 TLF 259.8>215.9(strong) -60. -16 -5 42 259.8>214.0(strong) -60 -30 -5 43 44 d4-DCF 298.0>218.0(strong) -70 -28 -15 45 d3-IBP 208.0>164.0(strong) -45 -14 -9 46 47 d10-PBZ 316.9>289.0(strong) -70 -25 -16 48 d3-FLU 298.0>254.0(strong) -70 -14 -15 49 d3-MLX 353.0>289.0(strong) -50 -15 -18 50 51 d4-TLF 264.0>220(strong) -60 -16 -5 52 Note: Matrix matched curves were used for quantification of all compounds 53 d3-IBP was used as internal standard (I.S) for CPF, IBP and KPF, d 4-DCF was used as 54 I.S for DCF, d -PBZ was used as I.S for PBZ, d - FLU was used as I.S for FLU and 55 10 3 56 FLU-OH. d4- TLF was used as I.S for MFN and TLF. d 3- MLX was used as I.S for 57 MLX. 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Page 47 of 52 Food Additives and Contaminants

1 2 3 Table 3 : Intra- and inter-assay variation for accuracy of CPF, DCF, IBP, KPF, MFN, 4 5 6 PBZ, in plasma 7 8 9 10 11 12 Fortification 13 Analyte level Accuracy Within Run Between Run Total 14 For(ng mLPeer-1) Review CV Only CV CV 15 (%) (%) (%) (%) 16 17 18 CPF 5 104 18.8 9.2 20.9 19 7.5 99 7.1 9.6 12.0 20 10 101 11.0 6.6 12.8 21 22 Combined 5,7.5,10 15.2 23 Variance 24 25 DCF 5 106 4.0 2.1 4.5 26 7.5 102 3.0 2.3 11.9 27 10 101 3.9 1.2 12.8 28 29 30 Combined 5,7.5,10 4.1 31 Variance 32 33 34 IBP 5 104 4.4 0.0 4.4 35 7.5 103 4.3 4.2 6.0 36 10 100 3.8 0.9 3.9 37 38 Combined 5,7.5,10 4.8 39 Variance 40

41 42 KPF 5 108 8.4 3.8 9.2 43 7.5 104 5.4 7.2 9.0 44 10 103 3.2 0.0 3.2 45 46 Combined 5,7.5,10 7.1 47 Variance 48 49 MFN 5 103 7.0 4.3 8.2 50 7.5 103 4.2 4.0 5.8 51 10 99 2.3 3.0 3.8 52 53 Combined 5,7.5,10 5.9 54 55 Variance 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Food Additives and Contaminants Page 48 of 52

1 2 3 4 5 Fortification 6 Analyte level Accuracy Within Run Between Run Total 7 (ng mL -1) CV CV CV 8 (%) (%) (%) (%) 9 10 11 PBZ 5 109 6.8 5.2 8.6 12 7.5 102 10.3 4.9 11.4 13 10 101 4.5 0.8 4.6 14 For Peer Review Only 15 Combined 5,7.5,10 8.2 16 Variance 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Page 49 of 52 Food Additives and Contaminants

1 2 3 4 5 6 Table 4 : Intra- and inter-assay variation for accuracy of CPF, DCF, IBP, KPF, MFN, 7 8 PBZ, FLU, FLU-OH, TLF and MLX in milk 9 10 11 12 Fortification 13 Analyte level Accuracy Within Run Between Run Total 14 For(ng mLPeer-1) Review CV Only CV CV 15 (%) (%) (%) (%) 16 17 18 CPF 5 103 5.2 7.1 8.8 19 7.5 108 11.3 15.4 19.1 20 10 101 12.6 15.0 19.6 21 22 Combined 5,7.5,10 15.8 23 Variance 24 25 DCF 5 92 4.5 13.9 14.6 26 7.5 106 5.8 9.5 11.1 27 10 103 5.3 4.1 6.7 28 29 30 Combined 5,7.5,10 10.8 31 Variance 32 33 34 IBP 5 81 2.7 15.0 15.2 35 7.5 99 3.0 6.4 7.1 36 10 104 3.4 12.0 12.5 37 38 Combined 5,7.5,10 11.6 39 Variance 40

41 42 KPF 5 107 3.6 20.2 20.6 43 7.5 93 8.1 6.3 10.3 44 10 106 13.9 7.3 15.7 45 46 Combined 5,7.5,10 15.5 47 Variance 48 49 MFN 5 74 5.3 3.3 6.2 50 7.5 103 5.9 6.0 5.9 51 10 109 3.1 7.8 8.1 52 53 Combined 5,7.5,10 6.7 54 55 Variance 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Food Additives and Contaminants Page 50 of 52

1 2 3 4 5 6 7 Fortification 8 Analyte level Accuracy Within Run Between Run Total 9 (ng mL -1) CV CV CV 10 11 (%) (%) (%) (%) 12 13 PBZ 5 79 4.5 7.5 8.7 14 For 7.5Peer 99 Review 4.9 Only 4.7 6.8 15 10 104 5.8 5.4 7.9 16 17 Combined 5,7.5,10 7.8 18 Variance 19 20 FLU 20 93 3.0 1.9 3.6 21 22 40 102 3.9 2.1 4.4 23 60 98 4.4 5.8 7.3 24 25 Combined 20,40,60 5.1 26 Variance 27 28 FLU-OH 20 73 9.1 28.3 29.7 29 40 83 12.2 20.7 24.0 30 60 87 9.6 18.2 20.5 31 32 Combined 20,40,60 24.7 33 Variance 34 35 36 TLF 25 97 4.7 7.2 8.6 37 50 101 3.3 4.3 5.4 38 75 92 2.4 8.9 9.2 39 40 Combined 25,50,75 7.7 41 Variance 42 43 44 45 MLX 7.5 88 2.8 6.6 7.2 46 15 92 4.9 9.2 10.4 47 22.5 87 1.9 11.6 11.7 48 49 Combined 7.5,15, 22.5 9.8 50 Variance 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Page 51 of 52 Food Additives and Contaminants

1 2 3 4 Table 5 5 : Calculated CC α and CC β values for plasma 6 7 CC α CC β 8 (ng mL -1) (ng mL -1) 9 10 11 CPF 1.80 3.07 12 13 14 DCFFor Peer 0.58 Review 0.99 Only 15 16 17 IBP 0.71 1.22 18 19 20 KPF 0.87 1.49 21 22 MFN 0.70 1.20 23 24 25 PBZ 1.19 2.02 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected] Food Additives and Contaminants Page 52 of 52

1 2 3 4 5 6 Table 6: Calculated CC α and CC β values for milk 7 8 CC α CC β 9 -1 -1 10 (ng mL ) (ng mL ) 11 12 13 CPF 2.11 3.59 14 For Peer Review Only 15 DCF 0.83 1.41 16 17 18 IBP 0.47 0.80 19 20 21 KPF 1.63 2.77 22 23 MFN 0.92 1.56 24 25 26 PBZ 0.55 0.94 27 28 29 FLU 42.89 45.78 30 31 32 FLU-OH 55.76 71.50 33 34 TLF 54.45 58.90 35 36 MLX 17.57 20.13 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 http://mc.manuscriptcentral.com/tfac Email: [email protected]