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Supercritical Fluid Chromatography (SFC) as Orthogonal Technique for Improved Detection of Polar Analytes in Anti-Doping Control

Maria Kristina Parr1), Bernhard Wüst2), Edgar Nägele2), Marica Barbieri2), Francesco Botrè3,4) 1Institute of Pharmacy, Freie Universität Berlin, Germany, 2Agilent Technologies GmbH, Waldbronn, Germany, 3Laboratorio Antidoping FMSI, Rome, Italy, 4Department of Experimental Medicine, ‘Sapienza’ University of Rome, Italy

Introduction

A B C Some highly polar are hardly retained in classical x10 2 … Oc-S Oc-S HPLC-MS/MS due to very low interaction with the reversed phase 1.1 Oc-S Oc-2S Oc Oc-2S Oc 1.0 Oc Oc-2S (RP) columns (Fig.1B&C). Even on more polar RP phases like 0.9 0.8 phenyl-hexyl their analysis remained challenging. 0.7 0.6 Super-/subcritical fluid chromatography (SFC) as orthogonal 0.5

0.4

0.3 separation technique to HPLC was tested to overcome these

0.2

0.1 issues.

0

7 7.5 8 8.5 9 9.5 10 10.5 11 time (min)

Fig 1: Analysis of (Oc), Oc-sulfate (Oc-S) and –disulfate (Oc-2S) obtained by chemical synthesis [1] x10 2 A: SFC-MS/MS as described below B: HPLC-MS/MS on RP18-column C: HPLC-MS/MS: phenylhexyl-column 1.2 1 1

1.1

1

Results and Discussion 0.9 SFC-MS/MS demonstrated its potential for the analysis of polar compounds. A multi-analyte 0.8 dilute-and-inject method was developed for the separation of polar compounds (beta- 0.7 agents, stimulants and narcotics and their metabolites). Method optimization was 0.6

0.5 performed testing supercritical CO2 with different modifiers (methanol, formic acid, ammonia) and several analytical columns of different polarities. 0.4 Screening for 40 analytes (Tab.1 and Fig.2) was possible using conditions as described 0.3 below resulting in total cycle time of 17 min. 0.2

0.1 Post-administration urines of (chromatogram in Fig.3) proved the applicability in 1 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12 12.5 13 real-life samples. Time (min) Fig 2: SFC-MS/MS chromatogram of analyte mixture, conditions as described below

Tab 1: Analytes included in screening procedure (*= unknown concentration of reference) Propranolol Propranolol-OH Propranolol-OH-S

RT Analyte LOD [ng/mL Elution order RT Analyte LOD [ng/mL Elution order in O N O N H H OH OH urine] in HILIC [2] urine] HILIC [2] O N H 1,42 Niketamide 0,25 1 7,89 0,3 22 OH n.r. 29 OSO3H 3,58 Phenazone 0,3 7,89 -d3 1 4,38 0,25 38 7,91 Salbutamol 1 29 OH 4,60 0,4 36 7.94 Propranolol-XOH1 1,2 n.r. 4,74 0,05 6 8,07 0,3 9 4,84 0,6 10 8,17 0,2 44 5,07 MDEA 0,02 21 8,17 0,1 13 5,11 0,1 n.r. 8,54 1 n.r. 5,14 0,02 18 8,42 Propranolol-XOH2 * n.r. 5,46 Esmolol 0,25 20 8,60 1 42 5,63 0,15 24 8,61 Propranolol-4OH 3 n.r. Fig 3: SFC-MS/MS chromatogram of Propranolol post administration urine 5,80 0,6 16 8,64 * 45 (1 tablet Proranolol AL 40, p.o., 3,5-6,75h sampling, diluted 1:5 in THF), 16 46 5,81 Ephedrine-d3 0,6 8,64 Octopamine 2 displayed structures assigned to peaks by reference material, 5,90 Propranolol 0,5 12 8,80 Propranolol-XOH3 * n.r. the others confirmed metabolic results from Oatis et al. [3] 5,95 0,02 35 9,15 0,4 2 6,03 0,5 4 9,52 Propranolol-4OH-Gluc * n.r. Instrumentation 6,09 0,15 15 9,74 0,15 3 Parameters 6,71 Propranolol-Gluc * n.r. 9,95 Fenoterol-sulfate 1 * n.r. System Agilent 1260 Infinity Analytical SFC 7,18 2 23 10,10 -sulfate * n.r. Column Waters BEH 2-EP, 100mm*3.0mm, particle size 1.8 µm 7,30 5 40 10,30 Tyramin-sulfate * n.r. Column temp. 28°C 7,30 4 43 10,47 Phenylephrine-disulfate * n.r. Mobile phase A: CO2, back pressure regulator 150 bar, 60°C 7,47 0,7 37 10,59 Octopamine-sulfate * n.r. B: NH4-OAc (5 mM) in MeOH:H2O (96.5:3.5, v:v) 41 * n.r. 7,61 0,1 10,78 Propranolol-4OH-Sulf Gradient linear 0.0 min 2%B, 3.00 min 5%B, 8.0 min 5%B, 11.0min 50%B 7,67 1 29 10,87 Fenoterol-sulfate 2 * n.r. 12.0 min 65%B, 1.5 min hold, 1.5 min post-time 7,69 Phenylephrine 2 n.r. 10,93 Octopamine-disulfate * n.r. Flow 1.5 mL/min 7,70 0,1 39 11,05 Fenoterol-disulfate * n.r. Injection 5.0 µL, fixed loop

7,82 0,1 33 11,52 Tyramin-disulfate * n.r. Make-up 0.4 mL/min NH4-OAc (5 mM) in MeOH:H2O (96.5:3.5, v:v) 7,82 0,05 n.r. 11,00 Propranolol-XOH-Sulf * n.r. Detector Agilent QQQ 9490, ESI

References [1] Orlovius A, et al. in: Schänzer W, Geyer H, Gotzmann A, Mareck U (Eds.) Recent Advances in Dope Analysis (19), Sportverlag Strauß, Cologne (2011) 34-43 Acknowledgements [2] Mazzarino M, et al. J Chrom A 1218 (2011) 8156-8167 This project is funded by the World Anti-Doping Agency within their research grant [3] Oatis JE, et al. J Med Chem 24 (1981) 309-314 14A03KP.

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