Rapid and specific extraction of anabolic steroids (A1, A3, A4) and corticosteroids in urine before detection and identification by UPLC-MS/MS

M. Dubois, P.Y.Brasseur, N. Gillard, P. Delahaut CER Groupe, Health Department, rue du point du jour, B-6900 Marloie-Belgium *Corresponding author : [email protected]

I. Introduction III. Results

In the European Union, Council Directive 96/23/EC prohibits the use of Table 1 : Decision limits (CCα) and detection capabilities (CCβ) anabolics as growth promoters for meat production. As the number of CCα CCβ CCα CCβ molecules abused has increased over the years, "anabolic cocktails" (µg L -1) (µg L -1) (µg L -1) (µg L -1) have made their appearance, i.e., mixtures containing several Steroids Steroids Ethyloestranediol ≤2.26 3 Ethynylestradiol ≤1.50 2 different agents at law but still active concentrations. Because their 17β-nortestosterone 0.11 0.125 Methenolone 0.69 0.8 17α-nortestosterone 0.20 0.25 Zearalenone 0.91 1 effects are additive, the resulting mass increases recorded in cattle are 17β-boldenone 0.22 0.25 Zeranol 0.38 0.5 similar to those produced by single compounds at higher 17α-boldenone 0.44 0.5 Taleranol 0.38 0,5 17β-testosterone 0.94 1 Zearalanone 0.92 1 concentration, but since smaller amounts of each agent are eliminated 5α-androst-1-en-17α-ol-3-one 0.45 0.5 α-Zearalenol 0.91 1 Methylboldenone 0.45 0.5 β-Zearalenol 0.87 1 in biological specimens, they are much more difficult to trace. Methyltestosterone 0.22 0.25 Norethandrolone 0.11 0.125 Corticosteroids Detection requires highly sensitive techniques with law background Norgestrel 0.12 0.125 Progesterone 0.93 1 Prednisone 0.88 1 interference. Immunoaffinity purification techniques offer tremendous 4-chloro-androst-4-en-3,17-dione 0.22 0.25 Prednisolone 0.23 0.25 Stanozolol 0.23 0.25 Methylprednisone 0.43 0.5 and decisive advantages over conventional methods used to pre-treat 16β-hydroxy-stanozolol 0.12 0.125 Methylprednisolone 0.47 0.5 biological specimens. In this work, we combined multiresidue Fluoxymesterone 0.22 0.25 Isoflupredone 0.24 0.25 Medroxyprogesterone acetate 0.11 0.125 Dexamethasone 0.10 0.125 immunoaffinity chromatography with efficient liquid chromatographic 17α-trenbolone 0.46 0.5 Flumethasone 0.12 0.125 17α-methyl-5β-androstane-3α,17-β-diol 2.29 3 Clobetasol ≤0.75 1 separation. This efficient method, validated for 44 compounds, is Dienestrol 0.76 1 Triamcinolone acetonide 0.04 0.05 Diethylstilbestrol 0.41 0.5 Fluocinolone acetonide 0.34 0.4 widely applicable to urine samples, i.e., the biological specimens that Hexestrol 0.39 0.5 Fluorometholone 0.33 0.4 veterinary inspectors usually take in slaughter houses and farms. It 17β-estradiol ≤1.50 2 Beclomethasone ≤3.01 4 could provide a basis for a universal procedure for detecting multiple 5.72 6.04 5.98 100 100 6.09 7.39 anabolic agents and their metabolites in such specimens. A 6.13 B 6.81 6.92 % 6.96 303>109 Methyltestosterone (ESI+) % 7.73 0 0 3.00 4.00 5.00 6.00 7.00 8.00 3.00 4.00 5.00 6.00 7.00 8.00

100 6.04 100 5.81 5.88 6.08 6.32 6.93 7.65 7.76

% 303>96.9 Methyltestosterone (ESI+) % 0 0 II. Methods 3.00 4.00 5.00 6.00 7.00 8.00 3.00 4.00 5.00 6.00 7.00 8.00 4.97 5.61 4.75 5.00 100 100 5.63 4.62 6.01 6.08

% 275>108.9 Norstestosterone (ESI+) % 0 0 3.00 4.00 5.00 6.00 7.00 8.00 3.00 4.00 5.00 6.00 7.00 8.00

4.97 4.74 5.14 II.1. Extraction 100 100 4.65 5.23 5.61 5.61 6.02 % 5.70 275>82.9 Norstestosterone (ESI+) % 0 0 3.00 4.00 5.00 6.00 7.00 8.00 3.00 4.00 5.00 6.00 7.00 8.00

3.07 100 3.49 100 7.64 3.41 3.52 3.66 4.36 % % 4.04 295.3>159.2 Ethynyloestradiol (ESI-) 3.28 4.02 6.75 6.83 0 0 3.00 4.00 5.00 6.00 7.00 8.00 3.00 4.00 5.00 6.00 7.00 8.00

100 3.47 100 3.73 3.53 5.31

% 295.3>145.2 Ethynyloestradiol (ESI-) % 5.13 3.19 6.09 0 0 2 mL Urine 3.00 4.00 5.00 6.00 7.00 8.00 3.00 4.00 5.00 6.00 7.00 8.00

100 3.86 100 3.91

% 269>134 Hexestrol (ESI-) 0 % 0 -100 Shake 15 min 3.00 4.00 5.00 6.00 7.00 8.00 3.00 4.00 5.00 6.00 7.00 8.00

3.10 Hydrolysis 100 3.86 100 3.11 3.90 % 269>119 Hexestrol (ESI-) % 3.71 7.44 7.77 0 3.62 0 3.00 4.00 5.00 6.00 7.00 8.00 3.00 4.00 5.00 6.00 7.00 8.00

100 3.72 100 3.03 3.66 3.77 4.02

% 266.9>236.9 DES (ESI-) % 3.39 3.64 0 0 Centrifuge 5 min at 2700 g 3.00 4.00 5.00 6.00 7.00 8.00 3.00 4.00 5.00 6.00 7.00 8.00

100 3.73 100 5.08 3.78 %

266.9>221.9 DES (ESI-) % 0 Time 0 Time 3.00 4.00 5.00 6.00 7.00 8.00 3.00 4.00 5.00 6.00 7.00 8.00

4.17 100 4.39 100 4.74 4.89 Purification on OASIS HLB 4.48 5.36

4.33 435>415 Triamcinolone acetonide (ESI+) % % 0 0 3.00 4.00 5.00 6.00 7.00 8.00 3.00 4.00 5.00 6.00 7.00 8.00

4.39 4.14 4.97 5.16 100 100 4.82 4.12

435>397 Triamcinolone acetonide (ESI+) % % 4.48 4.99 Purification on Multi- 0 0 Injection 1 in ESI – 3.00 4.00 5.00 6.00 7.00 8.00 3.00 4.00 5.00 6.00 7.00 8.00 Immunoaffinity column 100 4.18 100 4.10 4.33 Estrogens 3.56

393>373.3 Dexamethasone (ESI+) % % 0 0 3.00 4.00 5.00 6.00 7.00 8.00 3.00 4.00 5.00 6.00 7.00 8.00

4.26 100 4.19 100 4.06 Analysis by UHPLC-MS/MS 3.50 3.79

4.35 393>355.3 Dexamethasone (ESI+) % % 0 0 Injection 2 in ESI + 3.00 4.00 5.00 6.00 7.00 8.00 3.00 4.00 5.00 6.00 7.00 8.00 100 7.51 100 7.13 7.30 7.80 7.92

387.1>327.1 Medroxyprogesterone acetate (ESI+) % % All others 0 0 compounds 3.00 4.00 5.00 6.00 7.00 8.00 3.00 4.00 5.00 6.00 7.00 8.00 100 7.50 100 7.12 7.20 7.60

387.1>123 Medroxyprogesterone acetate (ESI+) %

% 7.69 7.11 0 0 3.00 4.00 5.00 6.00 7.00 8.00 3.00 4.00 5.00 6.00 7.00 8.00

6.72 6.91 100 100 6.11 7.89 6.28 7.22 7.70

329>121 Stanazolol (ESI+) % % 0 0 II.2. Validation 3.00 4.00 5.00 6.00 7.00 8.00 3.00 4.00 5.00 6.00 7.00 8.00

6.73 6.32 6.91 100 100 6.25 7.667.26 7.94 6.82 329>95 Stanazolol (ESI+) % % 0 0 ÎAccording to EU Decision 2002/657/EC 3.00 4.00 5.00 6.00 7.00 8.00 3.00 4.00 5.00 6.00 7.00 8.00

6.00 β 100 5.77 100 5.65 6.83 ÎSpecificity and CC : analysis of 20 blank and 20 fortified urine samples 6.75 6.98

321.5>143 CLAD (ESI+) % % 0 0 at the decision limit (ppb-level) 3.00 4.00 5.00 6.00 7.00 8.00 3.00 4.00 5.00 6.00 7.00 8.00

5.77 5.935.76 100 100 6.30 6.66 7.00

321.5>131 CLAD (ESI+) % % 0 Time 0 Time II.3. Chromatography 3.00 4.00 5.00 6.00 7.00 8.00 3.00 4.00 5.00 6.00 7.00 8.00 Fig. 1. Selected UHPLC-MS/MS chromatograms in ESI+ and ESI- Mode ÎAcquity UPLC (Waters) (A) a treated sample spiked at CCβ and (B) a blank urine ÎColumn : Acquity HSS T3 (150 x 2.1 mm, 1.8 µm) ÎMobile phase : ESI+(A) H2O+0.1% FA, (B) MeOH+0.1% FA IV. Conclusion ESI- (A) H2O, (B) ACN + post column delivery of NH 4OH The approach presented here, i.e., combining purification by multi- Î Injection volume : 20 µl immunoaffinity chromatography with UPLC-MS/MS detection, has ÎRun time : ESI+ (14 min) ; ESI- (8,5 min) numerous and decisive advantages over conventional purification ÎFlow rate :0.45 ml/min techniques currently used in most accredited laboratories. II.4. Mass spectrometry All products initially present could be clearly detected in all processed samples. Application of the new method should considerably reduce time and labour, thereby reducing the cost of the analytical process Î Sample analysis required two injections per sample ESI-/ESI+ mode and increasing sample throughput. This amply justifies using the Î Xevo TQS (Triple quadrupole mass spetrometer) additional MIAC purification step, which additionally reduces ÎMRM mode : 2 MRM transitions for most compounds background signals urine and increases detection sensitivity. ÎAcquisition method contained 36 MRM for the injection in ESI- and 78 MRM for the injection in ESI+ V. Aknowledgment This work was supported by the Belgian Federal Agency for the Safety of the Food Chain