Anabolic steroid esters

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The use of anabolic substances for livestock fattening has been prohibited in the European. Union (Directive 96/22/EC). Nevertheless, their uses are still ...

THE POTENTIAL OF ATMOSPHERIC SOLID ANALYSIS PROBE (ASAP) IONISATION MASS SPECTROMETRY FOR RAPID IDENTIFICATION OF ANABOLIC STEROID ESTERS Mickael DOUE, Gaud DERVILLY-PINEL, Audrey GICQUIAU, Laure BEUCHER, Fabrice MONTEAU and Bruno LE BIZEC

LABoratoire d’Étude des Résidus et Contaminants dans les Aliments (LABERCA) USC INRA 1329, Oniris, LUNAM Université, BP 50707, 44307 Nantes Cedex 3, France Fax : +33 (0)2 40 68 78 78 - Tél : +33 (0)2 40 68 78 80 [email protected] - www.laberca.org

NATURAL HORMONES ABUSE IN MEAT PRODUCING ANIMALS INTRODUCTION

OVERVIEW

The use of anabolic substances for livestock fattening has been prohibited in the European Union (Directive 96/22/EC). Nevertheless, their uses are still suspected regarding potential benefits in terms of income. Anabolic steroid esters are the most commonly used due to their higher bioavailability and their longer lasting effect. In this context, the present study aims at providing a new rapid, efficient and multiresidue confirmatory strategy based on Atmospheric Solids Analysis Probe (ASAP) technology.

The potential of ASAP as new, efficient and rapid confirmatory strategy was assessed. Investigation of ionization and fragmentation both in positive and negative mode for 23 steroid esters was carried out. Rapid method to identify large set of steroid esters was developed. Isotopic dilution method in Selected Reaction Monitoring (SRM) mode was validated for the rapid quantification of these compounds.

ATMOSPHERIC SOLIDS ANALYSIS PROBE (ASAP) TARGETED STEROID ESTERS CH3 OR

PRINCIPLE CH3 OR

CH3 OR

CH3 OR

[M+H]+

CH3

CH3

O

6 Testosterone Esters

O

O

'RO

8 Estradiol Esters

. + M

4 Boldenone Esters

Adapted from Nielen et al., TRAC , 30 (2011) 165

5 Nandrolone Esters

Direct Analysis

Ambient Mass Spectrometry

Observed species

R : From C2 to C10 including cyclic esters

ASAP-MS/MS

ANALYTICAL PROCEDURE

MS parameters Mode Polarity Corona Cone Source offset

Probe

XEVO-TQS

Dipping

Introduction

WATERS XEVO TQS®

Acquisition

Values Current Positive 3 µA 45 V 70 V

MS parameters Probe Temperature Gas Desolvation Flow Cone Nebuliser

MS Optimized Tune Conditions O

O H3C

RAPID IDENTIFICATION AND QUANTIFICATION OF ANABOLIC STEROID ESTERS

O CH3

CH3

H3C

O

CH3

O

O

ASAP-MS/MS SCAN of Testosterone Enanthate ASAP POSITIF

ASAP POSITIF

RAPID IDENTIFICATION

O

97.0

H3C

CH3

Observed m/z: 401.3 [M+H]+ 289.2 [T+H]+ 271.2 [T+H-H2O]+ 253.2 [T+H-2H2O]+

O

109.0

85.0 175.1

253.2 271.2 289.2

ASAP NEGATIF

NEUTRAL LOSS SCAN 96.1

O

CH3

271.2 > 175.1 TESTOSTERONE

269.2 > 173.1 BOLDENONE

257.2 > 161.1 NANDROLONE

255.2 > 159.1 ESTRADIOL STEROID IDENTIFICATION

401.3

PRECURSOR ION 271.2 m/z ASAP NEGATIF

Values 450 °C 500 L.H-1 150 L.H-1 4 bars

PRECURSOR ION 269.2 m/z

PRECURSOR ION 257.2 m/z

PRECURSOR ION 255.2 m/z

129.1 O H3C

O CH3

CH3

O

399.3

MASS DETERMINATION

[M+H]+ Observed m/z: 399.3 [M-H]269.2 [T-H-H2O]129.1 [EN]-

PRODUCT ION OF THE CORRESPONDING [M-H]-

269.2

59.1 => Acetate 121.1 => Benzoate 149.1 => Phenyl Propionate

VALIDATION – ASAP SRM POSITIVE MODE Labeled internal standard + Steroid Ester

RSD (1 ppm)

Linearity (R2)

Sensitivity

< 20 %

> 0.95

10 ppb

73.1 => Propionate 129.1 => Enanthate 171.1 => Decanoate 199.1 => Laurate

101.1 = > Valerate 141.1 => Cypionate 183.1 => Undecylenate ESTER IDENTIFICATION

RAPID IDENTIFICATION OF ANABOLIC STEROID ESTERS (3 min)

CONCLUSION TAKE HOME MESSAGE

NEXT STEPS

Rapid, efficient and multiresidue method was developed in order to identify a large set of anabolic steroid esters in less than 5 minutes.

Extend the developed procedure to other anabolic steroid esters.

Thanks to labeled internal standards, quantification of the identified steroid esters was realized in oily commercial solution.

Develop an algorithm to identify both known and also unknown anabolic steroid esters.

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