(1) 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 laberca@oniris-nantes.fr - www.laberca.org

Poster presented at the EuroResidue VIIth Conference, Egmond aan Zee, The Nertherlands, 14th-16th May 2012.

Introduction Experimental

Results and discussion

Conclusion

(2) Banat’s University of Agriculture and Veterinary Medecine, Timisoara, Romania

(3) LUNAM Université, Oniris, Centre Hospitalier Vétérinaire, Atlanpole - La Chantrerie, BP 50707, Nantes F-44307, France

Sandrine Rochereau1, Emmanuelle Bichon1, Frédérique Courant1, Fabrice Monteau1, Stéphanie Prévost1, Flavia Hanganu1,2, Nora Cesbron3, Gaud Dervilly-Pinel1, Bruno Le Bizec1

QUANTIFICATION OF ESTROGENS AT PPT LEVELS IN BOVINE PLASMA BY

MOLECULARLY IMPRINTED SOLID PHASE EXTRACTION AND GC-MS/MS ANALYSIS

The use of anabolic agents in food producing animals has been prohibited

within the EU since 1988 (96/22/EC directive). In particular, the control of

natural steroid hormones administration in cattle still represent a difficult

challenge because of the high variability observed for endogenous levels of

such hormones, in relation with inter-individual polymorphisms originating

from genetic, environmental, age... Nevertheless, in 1989, the EU has

established a threshold for 17-estradiol at 40 pg mL-1 in plasma or serum of

bovine animals below 2 years old, above which a sample has to be considered

as suspect. Therefore, a screening strategy allowing pointing out estradiol

administration has been investigated through monitoring low ppt levels of

estrogens in plasma. The purification is based on molecular imprinted

polymer (MIP) is proposed here for the analysis of 17-estradiol (E2), 17-

estradiol (E2) and estrone (E1) by GC-EI-MS/MS after TMS derivatisation.

The efficiency of the developed method has been successfully assessed on samples

collected on incurred samples. The MIP selectivity was assessed and adapted

according to the expected purification quality of the biological extract. This

method will be implemented for on an experimental plan in 2012 to validate its

robustness and to adjust the compliant threshold for a future screening method of

estradiol in bovine serum. This strategy was then implemented on urine to assess

steroid purification efficiency on MIP-SPE before gas chromatography-combustion-

isotope ratio mass spectrometry (GC-C-IRMS) analysis to confirm the origin of

target natural steroids. A new protocol was then based on an efficient combination

between MIP packed in chromatographic column and preparative SFC to obtain an

excellent purification (E. Bichon, 2012).

Animal experiment

The animal experiment was conducted in agreement with animal welfare rules. After an

acclimation period, each two calves (6327 and 7669) received one intramuscular

injection of 30 mg of estradiol diluted in sesame oil at day 0. Blood samples were

collected from 3 days before administration (Day-3) and 5 hours, 1, 2, 6 and 7 days after

administration respectively called D0+5h, D1, D2, D6 and D7. The sera were obtained

after blood centrifugation at 3000 rpm for 10 min and were stored at -18°C.

Chemicals

Standard reference steroids were purchased from Steraloids (Wilton, NY, USA) and

Cambridge Isotope Laboratories (Andover, MA, USA). MIPs (AFFINIMIP-estradiol) were

from Polyintell (Val de Reuil, France). β-glucuronidase was provided by Roche (Rosny

sous Bois, France). N,O-bis(trimethylsilyl)-trifluoroacetamide (BSTFA) was from Fluka

(Buchs, Switzerland). All solvents and reagents were of analytical grade quality and

provided by Carlo Erba (Val-de-Reuil, France).

Sample preparation

Serum samples (2 mL) were spiked with 40 pg 17β-estradiol-d3 (internal standard). Two

milliliters of 0.8 M acetate buffer (pH 6.8) and 100 µL β-glucuronidase were added.

Hydrolysis was performed overnight at 37°C and samples were centrifuged at 4000 rpm

for 10 min. The upper layer was loaded onto an AFFINIMIP-estradiol previously

conditioned with 4 mL methanol, 4 mL acetonitrile and 4 mL water. Two washing steps

with 5 mL water and 5 mL water / acetonitrile (60:40,v/v) preceded elution. The

molecules of interest were eluted with 3 mL of methanol. Extracts were evaporated to

dryness and estrogens were derivatised 40 min at 60°C with BSTFA before to be injected

onto a GC-MS/MS (2 µL).

GC-MS/MS instrument

Estrogens’ determination was carried out by GC-MS/MS in electronic impact. An Agilent

7890 gas chromatograph was coupled to a triple quadripole mass analyser Agilent 7000

(Agilent Technologies, Santa Clara, USA). GC column was a 15 m × 0.25 mm × 0.10 µm,

RTX-1614 (Restek, Lisses, France). The gradient of temperature was set from 80°C to

320°C (15°C.min-1). Two transitions were monitored per compound.

GC-MS/MS

Preparative HPLC (1)

PFB Derivatisation(1)

LLE Pentane (1)

copolymeric SPE (1)

Enzymatic hydrolysis

LLE ether (1)

SiOH SPE (1)

MIP: estradiol

(AFFINIMIP®, Polyintell)

2 mL bovine plasma

TMS Derivatisation

Fig 1: New sample preparation with MIP purification to replace the approach of Courant et al (2010)

Analytical results

The quantification can be performed at concentration level as low as 10 pg mL-1 with two

diagnostic signals (MRM transition) monitored per analyte. Consequently, the target

threshold at 40 pg mL-1 can now be monitored with a rapid and efficient sample

preparation and a classical ionisation in electronic impact with TMS derivatisation (Fig 2).

Concentrations in the incurred samples

The quantification of αE2, βE2 and E1 was performed in all plasma samples collected

from the animal experimentation. The results are presented in Figure 3. The apex of the

kinetic corresponds to samples collected 5 hours after administration, when the

concentrations reaches 1 ppb. In addition, the plasma samples, taken between day 0+5h

and day 2 after administration, present βE2 concentrations above the proposed threshold

(i.e. 40 ppt). They can then directly been concluded as suspicious.

Moreover, the sensitivity of this method allows the detection, identification and

quantification of estrogens during all the experimentation procedure, i.e. during the

seven days after administration. During this week after treatment, the return to the basal

values is achieved only at D7. In conclusion, with this very efficient method, we may

consider suspicious samples until 7 days after administration. This strategy has to be

confirmed with additional plasma samples (coming from untreated animals with ages,

different sexes and races). All these results show the potentiality of this method to be

used for the screening of estrogens in bovine plasma.

Blank plasma 10 ppt 40 ppt 100 ppt

17-estradiol-d3

419>285

17/-estradiol

416>129

17/-estradiol

416>285

Fig 2: MRM chromatograms obtained from fortified calves’ plasma samples at 0, 10, 40 and 100 pg.mL-1 with 17α-estradiol, 17β-estradiol and estrone.

0

500

1000

1500

2000

2500

0 2 4 6 8

Co

nce

ntr

atio

n (

pg.

mL-1

)

Days

αE2

βE2

E1

0

20

40

60

80

100

120

0 2 4 6 8

Co

nce

ntr

atio

n (

pg.

mL-1

)

Days

αE2

βE2

E1

Fig 3: Plasmatic concentration of 17-, 17-estradiol and estrone after estradiol one IM injection (30mg).

References

E. Bichon, S. Anizan, S. Prevost, L. Sérée, M. Doué, P. Sitthisack, D. Di Nardo, T. Duval, F. Monteau, J.-P. Antignac and B. Le Bizec. (2012) Strategies todetect natural steroids misuse in cattle : the steroidomic powerful screening and GC-C-IRMS for confirmation. Proceeding EuroResidue VII Conference.

Council Directive 96/22/EC concerning the prohibition on the use in stockfarming of certain substances having a hormonal or thyrostatic action and of beta-agonists, and repealing Directives 81/602/EEC, 88/146/EEC and 88/299/EEC. Off. J. Eur. Commun. 1996;L125:3.

F. Courant, L. Aksglaede, J.-P. Antignac, F. Monteau, K. Sorensen, A.-M. Andersson, N. E. Skakkebaek, A. Juul, B. Le Bizec. (2010) Assessment of Circulating Sex Steroid Levels in Prepubertal and Pubertal Boys and Girls by a Novel Ultrasensitive Gas Chromatography-Tandem Mass Spectrometry Method. J Clin Endocrinol Metab, 95(1):82–92