Results and Discussion The screening method was set up for the identification and confirmation of more than 120 molecules, including anabolic agents, steroids, anesthetics, anti-inflammatory agents, and diuretics, as listed in Table 1. Table 1 : List of compounds detected in the screening. High Resolution and High Mass Accuracy: A New Approach for Screening in Doping Control Analysis Yves Moulard, Yves Bonnaire, Laboratoire des Courses Hippiques, Verrières-Le Buisson, France Bénédicte Duretz, Sarah Robinson, Samuele Scurati, Thermo Fisher Scientific, Les Ulis, France Dennis Nagtalon, Thermo Fisher Scientific, San Jose, CA, USA

Introduction Results and Discussion The injected concentrations were 50 pg/ml for dexamethasone Liquid chromatography–mass spectrometry (LC-MS/MS) The screening method was set up for the identification and and flumethasone, and 1 ng/ml for triamcinolone and technology has revolutionized the types of detection assays used confirmation of more than 120 molecules, including anabolic triamcinolone acetonide. In the positive mode, the analytes were in doping control analysis over the last decade. Triple quadrupole agents, steroids, anesthetics, anti-inflammatory agents, and identified as protonated species and in the negative mode, as or tandem mass spectrometers have been used most frequently diuretics, as listed in Table 1. formate adducts. in this area and provide accurate identification, confirmation, and Figure 2 shows an example of a real sample that has been quantification of prohibited compounds in a single analysis. In analyzed using this method. addition, ion trap and quadrupole time-of-flight mass Table 1 : List of compounds detected in the screening. spectrometers have been useful for screening and confirming 20 Beta dihydrocortisol Diazoxide Naftidrofuryl results. However, these technologies cannot address all of the Figure 2: Dexamethasone identified in a real sample in 4 Methylamino antypirine Dichlorisone Niketamide main requirements of doping control analysis such as: positive and negative mode. 5' Hydroxy Omeprazole Diphenydramine Nimesulide • Data re-interrogation Acepromazine Diphylline Nordazepam RT: 0.00 - 30.01 2.05 • Unlimited number of compounds scanned during the Acide ethacrynic Etamiphylline Omeprazole 100 analysis Althiazide Etophylline ( Etofylline) Oxazepam 80 • Fast and easy method development Ambroxol Fenspiride Oxyphenbutazone • High resolution to efficiently separate analytes from Amcinonide Fludrocortisone Paramethasone 60

interferences present in the matrix Amitryptylline Flufenamic acid Pentoxyphylline + 40 MH C22H28FO5 m/z 393.20718 • High mass accuracy to identify compounds by exact mass Antipyrine (phenazone) Flumethasone Petidine (meperidine) Relative Abundance Relative 20 15.92 Here we present a screening approach that uses high mass Beclomethasone Flunisolid Phenobarbital 22.03 0 Bendroflumethiazide Flunixin Phenylbutazone 15.93 accuracy and high resolution (R = 50,000) in positive and 100 negative polarities for the accurate screenThermo Scientific Benzocaine Fluocinolone acetonide Phenytoin Exactive mass spectrometer. More than 120 analytes are ing of Benzoylecgonine Fluocinonide Piroxicam 80 illicit substances in urine matrix using the screened using this Benzydamine Fluorometholone Prednisolone 60 2.79 Betamethasone Fluoroprednisolone Prednisone method. Unequivocal compound identification and confirmation 40 Formiate adduct C23H31F107 are made using the exact mass of the analytes in positive and Budesonide Flurandrenolide Probenicid m/z 437.19701 20 3.17 4.69 negative mode (if available) and the retention time. Buflomedil Fluticasone propionate Procaine 1.98 8.22 9.60 Bumetanide Furosemide Prolintane 0 Bupivacaine Guaifenesin Promazine 0 5 10 15 20 25 3 Goal Time (min) Butorphanol Halcinonide Pyrilamine To demonstrate a new approach using high mass accuracy and Caffeine Hydrochlorothiazide Ranitidine high resolution (> 50,000) for the accurate screening of illicit Capsaicine Hydroflumethiazide Sildenafil substances in a urine matrix using the Exactive™ mass Carbetapentane Hydroxy Lidocaine Sildenafil hydroxy spectrometer, a new benchtop instrument equipped with All data have been processed using Thermo Scientific ToxID Chlorothiazide Hydroxy Meloxicam Sulindac OrbiTrap™ technology. automated compound screening software. ToxID™ for Exactive Chlorpheniramine Hydroxy Piroxicam Tenoxicam processes data and identifies the compounds using the mass Chlorpromazine Hydroxy Tenoxicam Tetracaine Experimental accuracy and retention time of the analytes. An example of the Chlorthalidone OH-Triamcinolone Aceto. Tetrahydrogestrinone automatically-generated report can be seen in Figure 3. Sample preparation Cimetidine Imipramine Tetramisole Solid phase extraction (SPE) was used for sample pre-treatment Clenbuterol Indapamide Theobromine and clean up. The details of the procedure are described below. Clobetasol Isoflupredone Theophylline • To 5 mL of urine add 25 µL of hydrocortisone-d3 at 10 Cortisol Ketamine Timolol Figure 3: ToxID report – short summary style µg/mL Cortisol d3 ketoprofen Tixocortol pivalate • Add 1 mL of phosphate buffer Cortivazol Ketorolac Tramadol • Add 50 µL of β glucuronidase and 50 µL of protease Cyclothiazide Lidocaine Triamcinolone • Incubate for 1 hour at 55°C Dantrolene Meloxicam Triamcinolone acetonide • Centrifuge at 4000 rpm for 30 minutes Dantrolene hydroxy Mepivacaine hexacetonide • Transfer the supernatant to a tube Desonide Meprednisone Trichlormethiazide • Add 5 mL of water Desoximethasone Methyl phenidate Tripelennamine • Condition the C18-HF cartridge (Varian, Les Ulis, France) Dexamethasone Metocarbamol Xipamide with 3 mL of methanol and 3 mL of water Diazepam Morphine Xylazine • Load the sample and wash the cartridge with 3 mL of water and 3 mL of hexane • Elute with 3 mL of a mixture containing dichloromethane and Acquisition was performed in the full scan mode, in both ethanol positive and negative mode, using external calibration. All data • Evaporate to dryness were reprocessed using 5 ppm mass accuracy. Figure 1 shows • Reconstitute with 100 µL of a mixture containing water and the sensitivity obtained for a urine sample spiked with 4 acetonitrile (80/20) molecules: dexamethasone, flumethasone, triamcinolone acetonide, and triamcinolone. Instrumentation Method HPLC conditions Chromatographic analyses were performed using Shimadzu Figure 1 : Extracted ion chromatograms for dexamethasone, binary pumps LC-20ADxr (Champs sur Marne, France). The flumethasone, triamcinolone acetonide, and triamcinolone in chromatographic conditions were as follows: the positive and negative modes using 5 ppm mass accuracy

• Column: Reversed-phase, silica-based C18 (3.5 µm, 150 x + 11.41 15.95 MH C22H28FO5 100 Dexamethasone m/z 393.20718 2.1 mm) column 10.49 18.45 12.54 • Flow rate: 0.3 mL/min 0 15.94 100 Formiate adduct C23H31F107 • Injection volume: 10 µL Relative Abundance m/z 437.19701 3.31 5.57 6.45 8.44 9.36 10.48 16.13 23.50 • Mobile phase: 0 • A: Water containing 0.1% formic acid 4 6 8 10 12 14 16 18 20 22 24 26 28 Time (min) • B: Acetonitrile containing 0.1% formic acid 16.19 • Gradient Mode 100 + Flumethasone MH C22H28F2O5 Conclusion 3.52 4.28 m/z 411.19776 Mass Spectrometry conditions 0 16.17 The Exactive benchtop mass spectrometer is an ideal system for 100 Formiate adduct C H F O m/z Relative Abundance 23 30 2 7 MS analysis was carried out on a Thermo Scientific Exactive 455.18759 screening illicit substances in the doping control laboratory. 19.51 4.42 6.16 13.47 benchtop mass spectrometer with an electrospray ionization 0 Equipped with ToxID software, it demonstrates unequivocal 4 6 8 10 12 14 16 18 20 22 24 26 28 (ESI) source. The MS conditions were as follows: Time (min) analyte identification and confirmation in real urine samples using high resolving power, high mass accuracy, and retention • Ion Polarity: Polarity switching scan dependent 17.13 100 + Triamcinolone MH C24H31FO6 m/z time. Method development is fast and simple with the ability to experiment 435.21774 Acetonide configure screening for an unlimited number of compounds 0 • Spray Voltage: 4500 V in positive mode and -3900 V in 17.14 100 Formiate adduct C25H33FO8 during the analysis. As the analysis is done in full scan mode, negative mode Relative Abundance m/z 479.20757 4.95 6.27 8.97 16.89 19.19 21.53 22.92 25.33 data re-interrogation can easily be done without the need for • Sheath gas pressure (N2): 45 (arbitrary units) 0 4 6 8 10 12 14 16 18 20 22 24 26 28 sample re-injection. • Auxiliary gas pressure (N2):3 (arbitrary units) Time (min) • Capillary temperature: 300 °C MH+ C H FO 23.58 Currently, over 120 compounds are screened routinely and • Resolution: 50,000 100 Triamcinolone 21 27 6 9.57 395.18644 12.09 24.82 succesfully on thousands of real urine laboratory samples to • Automatic Gain Control: Target value 500,000 5.90 6.40 6.57 14.02 17.13 19.74 0 23.57 date with this approach. 100 Formiate adduct Relative Abundance 9.60 C22H27FO8 439.17627 23.84 17.85 21.05 22.81 25.01 0 All trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. 4 6 8 10 12 14 16 18 20 22 24 26 28 Time (min)