DETECTION AND IDENTIFICATION OF CORTICOSTEROIDS IN BIOLOGICAL

MATRICES CONTROL OF THEIR ILLEGAL USE AS GROWTH PROMOTERS IN

CATTLE
Jean-Philippe Antignac, Bruno Le Bizec, Fabrice Monteau, Frdric Poulain and Franois
Andr.
LDH/LNR, Ecole Nationale Vtrinaire de Nantes
Route de Gachet, BP 50707, 44307 Nantes Cedex 03, France
E-mail : ldhlnr@vet-nantes.fr
Abstract
Artificial corticosteroids are used in many veterinary therapeutic drugs for their anti-
inflammatory properties. They are also illegally used as growth promoters in cattle, because
increasing weight gain, reducing feed conversion ratio and having a synergetic effect with -
agonists or anabolic steroids. In order to control their utilisation, a previously described
detection method based on LC-MS/MS (with triple quadrupole analyser and negative
electrospray ionisation) was used. This work presents the developed extraction/purification
method for urine samples, including corticosteroid conjugate hydrolysis, reversed phase SPE
(C18) extraction, liquid/liquid clean up, and normal phase SPE (SiOH) purification. This
method allowed the detection of 11 corticosteroids in bovine urine at the 20-50 pg.mL
-1
(ppt)
level. Results on corticosteroid residues in hair and muscle samples are also presented.
Introduction
Natural corticosteroid anti-inflammatory properties have led to the chemical synthesis of
more active artificial corticosteroids used in many veterinary therapeutic drugs. At the same
time, corticosteroids are illegally used as growth promoters in cattle because increasing
weight gain, reducing feed conversion ratio, and having a synergetic effect with -agonists or
anabolic steroids
1,2
. For dexamethasone, a MRL was established at 2 ppb in liver, 0.75 ppb in
muscle and 0.3 ppb in milk. Many authors have proposed methods based on GC-MS for the
detection of corticosteroids, including chemical oxidation or derivatization
3
. Although
providing good sensitivity, these methods are not routinely applicable and modify the
chemical structure of the molecule, reducing specificity. Several publications have proposed
the detection of corticosteroids in urine or plasma using LC-MS/MS
4-6
. Because of the low
residue levels of corticosteroids or their metabolites in biological matrices, the authors
focused on enhancing specificity and sensitivity. So a previously described detection method
was used, based on triple quadrupole analyser and negative electrospray ionisation
7
. The
objective of the present work was to develop a fast and efficient extraction/purification
method for corticosteroids in urine, including corticosteroid conjugate hydrolysis, reversed
phase SPE (C18) extraction, liquid/liquid clean up, and normal phase SPE (SiOH)
purification. Then, this procedure was adapted for residues in hair and meat samples.
Materials and methods
Apparatus. HPLC separation was achieved on Nucleosil C18AB stationary phase (Macherey-
Nagel, Dren, Germany). An Alliance 2690 pump with gradient system was used (Waters,
Milford, MA, USA). Detection was performed on a QuattroLC

triple quadrupole analyser

with an electrospray interface (Micromass, Manchester, UK).
Chemicals. Reagents, solvents, and SPE columns were provided by SDS (Peypin, France).
Helix Pomatia juice was supplied by Sepracor (Villeneuve La Garenne, France). Reference
corticosteroids (Table 1) were purchased from Sigma (St Louis, MO, USA). Standard
solutions were prepared at 1 mg.mL
-1
in methanol. Working solutions used for spiking were
prepared monthly at concentration from 100 ng.mL
-1
to 100 pg.mL
-1
and stored at -20C.
Extraction procedure. 1 mL acetate buffer (pH adjusted to 5.2) and 50 L Helix pomatia juice
were added to the urine sample (5 mL). Enzymatic hydrolysis was carried out for 15 h at
52C. After centrifugation, supernatant was applied to the C18 SPE cartridge activated with 5
mL methanol and 5 mL water. After a washing step with 5 mL water and 5 mL cyclohexane,
analytes were eluted with 5 mL diethylether. 1 mL Na
2
CO
3
10% was added, and after stirring
and centrifugation, the organic phase was removed. This liquid/liquid cleanup was repeated
one time and the diethylether was dried. The extract was reconstituted in 0.5 mL
cyclohexane/ethyl acetate (50:50, v/v) and applied to the SiOH SPE cartridge activated with
15 mL cyclohexane. After a washing step with 5 mL cyclohexane/ethyl acetate (50:50, v/v),
corticosteroids were eluted with 10 mL ethyl acetate/cyclohexane/acetic acid (90:5:5, v/v/v).
The purified extract was dried and reconstituted in 50 L water/methanol/acetic acid
(60:40:0.5, v/v/v).
LC-MS/MS analysis.10 L of the extract were injected on the HPLC column. Elution solvents
were 0.5% acetic acid in water (A) and methanol (B). The mobile phase gradient (A:B, v/v)
was as follow : 60:40 at 0 min, 10:90 at 10 min, and 60:40 from 20 to 30 min. Detection was
performed in negative electrospray ionisation using the multiple reaction monitoring (MRM)
acquisition mode. Nitrogen was used as nebulisation and desolvation gas, at flow rates of 90
and 600 L/h. Source and desolvation temperatures were 130 and 400 C. Potentials applied on
the capillary (from 2.5 to 4.0 kV) and on the cone (from 15 to 35 V) were adapted to each
molecule. Argon was used as collision gas, at a pressure of 4.0.10
-4
mbar. Collision energy
varied from 2 to 30 V. Diagnostic ions for each corticosteroid are given in Table 1.
Table 1. Structures and diagnostic ions of the investigated corticosteroids.
O
R
11
CH
3
CH
3
CH
2
OH O
R
17
R
16
R
9
R
6
A
Compound Mw A R
6
R
9
R
11
R
16
R
17
Parent
ion
Daughter
ion 1
Daughter
ion 2
Beclomethasone 408.9
1,4
- -Cl -OH -CH
3
-OH 467 377 407
Betamethasone 392.5
1,4
- -F -OH -CH
3
-OH 451 361 391
Cortisol 362.5
4
- - -OH - -OH 421 331 361
Cortisone 360.5
4
- - =O - -OH 419 329 359
Desoxycortisone 346.5
4
- - -OH - -OH 405 315 345
Dexamethasone 392.5
1,4
- -F -OH -CH
3
-OH 451 331 361
Fludrocortisone (IE) 380.5
1,4
- -F -OH - -OH 439 349 379
Flumethasone 410.5
1,4
-F -F -OH -CH
3
-OH 469 379 409
Methylprednisolone 374.5
1,4
-CH
3
- -OH - -OH 433 343 373
Prednisolone 360.5
1,4
- - -OH - -OH 419 329 359
Prednisone 358.4
1,4
- - =O - -OH 417 327 357
Triamcinolone 394.4
1,4
- -F -OH -OH -OH 453 345 393
Results and discussion
Urine samples. Repetability (30 injections of the same spiked urine sample at 0.5 ppb) varied
from 10.0% to 17.9%. Reproductibility (4 injections of 4 different spiked urine samples at 0.5
ppb) varied from 13.1% to 40.5%. The high variability values corresponded to the
triamcinolone and to the endogenous molecules (cortisol, cortisone). Linearity was estimated
by the coefficient of determination, calculated for 4 different urine samples each spiking at
0.05, 0.1, 0.5, 1 and 5 ppb. Results varied from 0.9954 to 0.9990. Detection limits varied from
20 to 50 ppt, quantification limits from 50 to 100 ppt and identification limits from 0.5 to 5
ppb. Recoveries varied from 19% to 36%. Figure 1 shows the MRM ion chromatograms for
spiked samples at 0.1 ppb.
0.00 2.00 4.00 6.00 8.00 10.00
0
100
%
0
100
%
1
100
%
1
100
%
10
100
%
3.26
141
5.25
2106
6.12
1289
5.50
5348
6.37
10239
0.00 2.00 4.00 6.00 8.00 10.00
7
100
%
14
100
%
5
100
%
4
100
%
5
100
%
7.86
497
7.99
449
8.24
401
8.36
98
8.36
302
453>345
Tri
417>327
Prn
439>349
Flu
419>329
Crn/Prl
421>331
Crl
469>379
Flm
451>361
Dex
433>343
Mprl
467>377
Bcl
405>315
Ctx
Figure 1. MRM ion chromatograms of a spiked urine sample at 0.1 ppb (0.5 ppb for
triamcinolone, endogenous cortisol and cortisone). Tri : triamcinolone, Prn : prednisone, Flu :
fludrocortisone, Crn : cortisone, Prl : prednisolone, Crl : cortisol, Flm : flumethasone, Dex :
dexamethasone, MPrl: methylprednisolone, Bcl: beclomethasone, Ctx: desoxycortisone/cor-
texolone.
Hair samples. Samples (100 mg) were incubated 2 h at 52 C with 3 mL HCl 1N and 2 mL
methanol. After centrifugation, supernatants were extracted by C18 SPE and purified by
Na
2
CO
3
liquid/liquid clean-up. Figure 2 shows three MRM ion chromatograms for a bovine
hair sample collected 15 days after injection of dexamethasone and methylprednisolone.
Muscle samples. Fresh samples (15 g) are stirred, freeze-dried, and incubated 15 h at 52C
with 12 mL methanol, 15 mL acetate buffer and 80 L Helix pomatia juice. After
centrifugation, supernatants were removed and the methanol evaporated. The extracts were
then extracted and purified as urine samples. Figure 3 shows the MRM ion chromatograms of
a spiked liver sample at 0.1 ppb.
0.00 2.00 4.00 6.00 8.00 10.00
3
100
%
5
100
%
3
100
%
6.49
478
7.48
256
7.73
426
421>331
Crl
451>361
Dex
433>343
MPrl
Figure 2. MRM ion chromatograms of a bovine hair sample after injection of dexamethasone
(Dex) and methylprednisolone (MPrl). Crl: endogenous cortisol.
Figure 3. MRM ion chromatograms of a spiked liver sample at 0.1 ppb (0.5 ppb for
triamcinolone, endogenous cortisol and cortisone). Tri : triamcinolone, Prn : prednisone, Flu :
fludrocortisone, Crn : cortisone, Prl : prednisolone, Crl : cortisol, Flm : flumethasone, Dex :
dexamethasone, MPrl : methylprednisolone, Bcl : beclomethasone, Ctx : desoxycortisone/cor-
texolone.
0.00 2.00 4.00 6.00 8.00 10.00
8
100
%
15
100
%
5
100
%
5
100
%
4
100
%
8.10
282
8.35
224
8.47
192
8.60
50
8.60
154
0.00 2.00 4.00 6.00 8.00 10.00
0
100
%
1
100
%
0
100
%
10
100
%
10
100
%
3.63
121
5.74
147
6.61
135324
6.11
2206
6.86
9466
453>345
Tri
417>327
Prn
439>349
Flu
419>329
Crn/Prl
421>331
Crl
469>379
Flm
451>361
Dex
433>343
Mprl
467>377
Bcl
405>315
Ctx
Conclusion
The developed method allows the detection of 11 corticosteroids in bovine urine at low
residue levels (20-50 ppt) and their unambiguous identification at very satisfying
concentration (0.5-1 ppb). Only triamcinolone presents lower sensitivity with LOD at 0.5 ppb
(LOI at 1 ppb). Performances of the method are linked to the high specificity of the
[M+acetate]
-
> [M-H-CH
2
O]
-
MRM transition and to the extraction/purification procedure
efficiency. The application to hair and muscle samples appears very promising. In the next
future, the present method will be adapted for a metabolism study. Indeed, focusing on
corticosteroid metabolites could enhance specificity and allow differed controls.
References
1. M. L. J. Rijckaert and H. P. J. Vlemmix, The growth promoting effect of
glucocorticosteroids. Department of chemical engineering, Eindhoven university of
technology (1992).
2. D. Courtheyn, J. Vercammen, M. Logghe, H. Seghers, K. De Wash and H. De
Brabander, Analyst. 123, 2409-2414 (1998).
3. J. Negriolli, PhD Thesis, Faculty of Science and Techniques, University of Nantes,
France (1997).
4. H. M. Dodds, P. J. Taylor, G. R. Cannell and S. M. Pond. Anal. Biochem. 247, 342-
347 (1997).
5. M. Fiori, E. Pierdominici, F. Longo and G. Brambilla, J. Chromatogr. A. 807, 219-227
(1998).
6. S. Rizea Savu, L. Silvestro, A. Haag and F. Srgel, J. Mass Spectrom. 31, 1351-1363
(1996).
7. JP. Antignac, B. Le Bizec, F. Monteau, F. Poulain and F. Andr. Rapid Comun. Mass
Spectrom. 14,33-39 (2000)