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Journal of Chromatography A, 1270 (2012) 51– 61
Contents lists available at SciVerse ScienceDirect
Journal of Chromatography A
jou rn al h om epage: www.elsev ier .com/ locat e/chroma
olecularly imprinted polymer applied to the selective isolation of urinaryteroid hormones: An efficient tool in the control of natural steroid hormonesbuse in cattle
ickael Douéa, Emmanuelle Bichona,∗, Gaud Dervilly-Pinela, Valérie Pichonb,lorence Chapuis-Hugonb, Eric Lesellierc, Caroline Westc, Fabrice Monteaua, Bruno Le Bizeca
LUNAM Université, Oniris, Laboratoire d’Etude des Résidus et Contaminants dans les Aliments (LABERCA), Nantes, F-44307, FranceDepartment of Analytical and Bioanalytical Sciences and Miniaturization (LSABM), ESPCI ParisTech, UMR PECSA 7195 (CNRS–UPMC–ESPCI ParisTech), 10 rue Vauquelin, 75231aris Cedex 05, FranceInstitut de Chimie Organique et Analytique (ICOA), Université d’Orléans, CNRS UMR 7311, B.P. 6759, rue de Chartres, 45067 Orléans Cedex 2, France
r t i c l e i n f o
rticle history:eceived 18 July 2012eceived in revised form 22 October 2012ccepted 31 October 2012vailable online 6 November 2012
eywords:hemical food safetyteroid hormonesolecularly imprinted polymer
olid phase extractionupercritical fluid chromatographyas chromatography–combustion–isotope
atio mass spectrometry
a b s t r a c t
The use of anabolic substances to promote growth in livestock is prohibited within the European Unionas laid down in Directive 96/22/EC. Nowadays, efficient methods such as steroid profiling or isotopicdeviation measurements allow to control natural steroid hormones abuse. In both cases, urine is oftenselected as the most relevant matrix and, due to its relatively high content of potential interferents,its preparation before analysis is considered as a key step. In this context, the use of a selective sor-bent such as molecularly imprinted polymer (MIP) was investigated. A MIP was synthesized basedon 17�-estradiol, methacrylic acid and acetonitrile as template, monomer and porogen, respectively.Two approaches were then tested for non-conjugated (aglycons and glucuronides deconjugated) steroidpurification: (i) molecularly imprinted solid phase extraction (MISPE) and (ii) semi-preparative super-critical fluid chromatography with a commercial MIP as stationary phase (SFC–MIP). Parameters for bothapproaches were optimized based on the main bovine metabolites of testosterone, estradiol, nandroloneand boldenone. The MISPE protocol developed for screening purposes allowed satisfactory recoveries(upper 65% for the 12 target steroids) with sufficient purification for gas chromatography–mass spec-trometry (GC–MS) analysis. For confirmatory purposes, the use of isotopic ratio mass spectrometry (IRMS)
requires a higher degree of purity of the target compounds, which can be reached by the SFC–MIP protocolwith three steps less compared to the official and current method. Purity, concentration and absence ofisotopic fractionation of target steroids extracted from urine of treated cattle (treated with testosterone,estradiol, androstenedione, and boldenone) allowed the measurement of 13C/12C isotopic ratios of cor-responding metabolites and endogenous reference compounds (ERC) and proved the relevance of the strategy.. Introduction
Beneficial effects of natural and synthetic steroids related tonimal growth promotion and feed conversion efficiency have led
o a wide use of these compounds in food producing animalsince the 1950s [1]. In the European Union, the use of anabolicubstances in cattle breeding has been prohibited since 1988∗ Corresponding author at: Oniris, Ecole nationale vétérinaire, agroalimentaire ete l’alimentation Nantes-Atlantique, Laboratoire d’Etude des Résidus et Contam-
nants dans les aliments (LABERCA), Atlanpole-La Chantrerie, BP 40706, Nantes,-44307, France. Tel.: +33 2 40 68 78 80; fax: +33 2 40 68 78 78.
E-mail addresses: [email protected] (M. Doué),[email protected] (E. Bichon).
021-9673/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.chroma.2012.10.067
© 2012 Elsevier B.V. All rights reserved.
(Directive 88/146/EC repealed by Directive 96/22/EC) [2]. Never-theless, steroid hormones may still be fraudulently employed andan efficient control is required to monitor such misuse [3–5]. EUlegislation (Directive 2002/657/EC [6]) imposes a two-step strat-egy in laboratories in charge of the control: initial rapid andmultiresidue screening step to sift large numbers of samples forpotential steroids abuse followed by a confirmatory step whichdiscards any doubts on the compliance of the suspicious sam-ples [5,7–9]. Recently and thanks to the advances made in theknowledge of steroid metabolic patterns as well as the associ-ated kinetics of elimination, steroid profiling has been reported as
an efficient screening strategy for natural steroids abuse [10–16].For confirmatory purposes, isotopic deviation measurement bygas chromatography–combustion–isotope ratio mass spectrome-try (GC–C–IRMS) probably remains the most adapted option to
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etermine the endogenous or exogenous origin of steroids [17,18].n both cases, urine is often selected as the most relevant matrixince it contains higher concentration levels of most steroids ofnterest compared to blood [19]. Moreover, urine is available inarge quantities at any time from live animals due to its non invasiveollection. Nevertheless, its preparation before analysis remains aritical step regarding its relatively high content of potential inter-erents [20]. While classical SPE protocols have been described inhe past, more recently several innovative strategies to improvehe purification of urinary steroids have been developed mainlyased on immunoaffinity [21–23], microextraction by packed sor-ent (MEPS) [24], stir bar sorptive extraction (SBSE) [25], and solidhase microextraction (SPME) [26,27]. Immunoaffinity turns to beelatively time-consuming while microextraction techniques, dueo the reduced amount of samples used, leads to an insufficientteroids concentration for subsequent IRMS measurement. In thisontext, molecularly imprinted polymers (MIPs) may appear as aaluable alternative extraction tool due to their specificity and theirigh capacity.
MIPs are synthetic polymers exhibiting specific cavities com-lementary in size, shape and position of the functional groups toarget molecules or families of compounds. They result from theomplexation of template molecules with functional monomers inn appropriate solvent, followed by template molecules removal.IPs are frequently used as selective sorbents for the molecularly
mprinted solid phase extraction (MISPE) of target analytes fromomplex matrices [28–31] due to their numerous advantages suchs selectivity associated to their rapid, easy and cheap use asell as high thermal and chemical stability [29]. The first appli-
ation was carried out by Sellergren in 1994 for the extraction ofentamidine in urine [32]. MIPs specifically designed for steroidxtraction and subsequent analysis have already been developednd successfully applied on water [33–35], milk [36] and urineamples [37]. For the polymerization process, several functionalonomers and initiators have been described, whereas the use
f estradiol as template is by far the most cited in the literature33,34,36,37]. The best results in terms of recovery and selectivityere obtained using methacrylic acid (MAA) as monomer, ethylene
lycol dimethacrylate (EGDMA) as cross-linker and acetonitrile asolvent [38]. Compared to other classical procedures such as SPE oriquid–liquid extraction (LLE), MIPs finally present the advantagesf being a reusable technique allowing a one-step procedure for anmproved extraction, purification and concentration of the targetompounds.
Another sample preparation approach can be based on cou-ling semi-preparative chromatography with MIP as stationaryhase. Indeed, semi-preparative chromatography allows a highurification of compounds in complex matrices. With liquid chro-atography (LC), the main drawback is linked to the large volume
f mobile phase needed. This constraint can be overcome by usingupercritical fluid chromatography (SFC). SFC presents strong eco-omical advantages due to the low percentage of co-solvent needed39] and several advantages linked to the state of supercritical fluidshich exhibit density and dissolving capabilities similar to those
f certain liquids, as well as lower viscosities and better diffu-ion properties [40]. Moreover, and according to literature data,etention rules in SFC mainly depend on the nature of the sta-ionary phase. Indeed, the interactions between compounds andtationary phase are improved in SFC compared to LC. Consider-ng the properties of both techniques, coupling semi-preparativeFC with MIP appeared as an interesting strategy to improve MIPspecificity and therefore selective isolation of steroids. Recently,
pplications using MIP as stationary phase in chromatographiceparation techniques have been reported in literature [41–45].mong these studies, only a limited number reported the use ofIP in chromatographic technique for complex matrices [44,45].. A 1270 (2012) 51– 61
Semi-preparative applications based on MIP have never beendescribed in literature and to the best of our knowledge no studieshave ever focused on steroids.
The aim of the present work was to assess the potential of MISPEand SFC–MIP approaches to purify urinary steroid hormones inorder to propose efficient, cheap and multiresidue sample prepara-tion procedures. Both approaches were optimized using the mainmetabolites of testosterone, estradiol, nandrolone and boldenonein bovine urine which are considered as potential anabolic steroidsused in cattle breeding. A one step MISPE protocol followed bygas chromatography–mass spectrometry (GC–MS) analysis wasdeveloped for screening purposes while the SFC–MIP strategy wasassessed as a highly selective purification strategy prior to IRMSanalysis for confirmatory purposes.
2. Experimental
2.1. Chemicals, reagents, materials
The reference steroids including 5�-androstan-3�-ol-17-one(etiocholanolone), 5�-androstan-3�-ol-17-one (epiandrosterone),androst-4-en-17�-ol-3-one (testosterone), androst-4-en-17�-ol-3-one (epiT), 5-androsten-3�-ol-17-one (DHEA),estra-1,3,5(10)-triene-3,17�-diol (E2), 5�-androstan-3,17-dione(external standard) and estra-1,3,5(10)-triene-3,17�-diol d3(E2-d3) were purchased from Sigma–Aldrich (St. Louis, MO,USA); 5�-androstan-3�,17�-diol (5-aba), 5-androsten-3�,17�-diol (androstenediol), 5�-androst-1-en-17�-ol-3-one (M2),estr-4-en-17�-ol-3-one (17�-nandrolone), estra-1,3,5(10)-triene-3,17�-diol (�-E2) and 5�-estran-3�,17�-diol (E-aba) werepurchased from Steraloids (Newport, RI, USA); whereas 5�-androst-1-en-17�-ol-3-one (M4), 1,4-androstadien-17�-ol-3-one(boldenone), 1,4-androstadien-17�-ol-3-one (epiboldenone),1,4-androstadien-17�-ol-3-one d3 (boldenone-d3) and androst-4-en-17�-ol-3-one d3 (epiT-d3) were purchased from NARL(Pymble, Australia). Each steroid stock solution was preparedat 1 mg mL−1 by dilution in an appropriate volume of ethanol.The working standard solutions were prepared by diluting stocksolutions in ethanol and were stored at −20 ◦C. Derivatisationreagents pyridine and acetic anhydride were purchased fromAldrich (Steinheim, Germany). �-Glucuronidase from Escherichiacoli was obtained from Roche Diagnostics GmbH (Mannheim,Germany). Ethanol, methanol, ethyl acetate, cyclohexane, ace-tonitrile, n-pentane, n-hexane, petrolether and reagents were ofanalytical-grade quality and purchased from Carlo-Erba Reagents(Rodano, Italy). Ultra pure water (UP water) was obtained with aNanopure system from Barnstead (Dubuque, IA, USA). The solidphase extraction (SPE) column (C18: 2000 mg/15 mL) was acquiredfrom UCT (Bristol, PA, USA). For MIP synthesis, methacrylic acid(MAA) and ethylene glycol dimethacrylate (EGDMA) were pur-chased from Sigma–Aldrich. Azo-N,N′-bis-isobutyronitrile (AIBN)was purchased from Acros Organics (Noisy-le-Grand, France).Molecularly imprinted polymer specifically designed for E2 recog-nition (product code: AFFINIMIP Estrogens) was provided byPolyintell (Val de Reuil, France) packed as stationary phase into achromatographic column (250 mm × 4.6 mm, 12–25 �m).
2.2. MIPs synthesis
EGDMA was washed twice with an equal volume of a solu-tion of 10% NaOH in UP water, and then washed twice with an
equal volume of UP water. It was then dried using an equal vol-ume of saturated sodium chloride aqueous solution and next overNa2SO4. AIBN was of a high purity and was therefore used with-out further purification. Washed EGDMA and MAA were distilled
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nder vacuum in order to remove inhibitors and stored at −20 ◦C.or final MIP synthesis, a template/monomer/cross-linker molaratio of 1/8/40 was used. The template (E2, 0.25 mmol) and theonomer (MAA, 2 mmol) were mixed with acetonitrile and left for
0 min in an ice bath. Then, the cross-linker (EGDMA, 10 mmol) andhe initiator (AIBN, 400 �L) were added. The solution was stirred,ransferred into a glass tube and then degassed under nitrogen for5 min. The tube was sealed and transferred into a thermostatedater bath (65 ◦C) during 24 h for thermal polymerization. Afterolymerization, the tube was crushed and the polymer was thenrounded with a ball mill and manually sieved. The particle sizeraction of 25–36 �m was collected and slurried in MeOH/water80:20, v/v) and then dried. Non-imprinted polymer (NIP) wasbtained by performing the same procedure in the absence of theemplate molecules in the polymerization mixture. Two differ-nt rounds of synthesis of MIP/NIP were carried out at differentimes to evaluate the repeatability of the polymerization. Differentmounts of synthesized MIP and NIP were packed between tworits (1/16′, 20 �m, Interchim, Montluc on, France) into 3 mL emptyropylene disposable cartridges (Interchim). In order to eliminatehe remaining reagents from the packed polymers, particularly theemplate molecules in the case of the MIP, a washing step was per-ormed with 20 mL of MeOH. Finally they were conditioned withcetonitrile and kept at 4 ◦C.
.3. Animal experiments
After an acclimation period, one heifer received 17�-estradiol30 mg in sesame oil) once by intramuscular injection (treatment2), one calve received 17�-estradiol benzoate (25 mg) and 17�-androlone laureate once by intramuscular injection (150 mg)treatment E2/NT), one calf received boldione (200 mg) once byral route (treatment B), another heifer received androstenedione250 mg) once by oral route (treatment AED) and another heifereceived testosterone (100 mg) once by oral route (treatment T).nimal experiments (E2, B, AED and T) were conducted in agree-ent with the animal welfare rules currently in force at Onirishile the E2/NT experiment was conducted within the Department
f Veterinary Animal Health of the Faculty of Veterinary Medicinef the Utrecht University (The Netherlands) and approved by thethical committee from Utrecht University. Urine samples wereollected at day one (AED, T, and E2) and day three after treatmentB and E2/NT) and stored at −20 ◦C.
.4. Sample pre-treatment
Five milliliters of urine were thawed at room temperaturend submitted to an enzymatic deconjugation step using �-lucuronidase from E. Coli at 37 ◦C overnight, as described byuisson et al. [17]. Samples were then centrifuged at 1200× g (5 ◦C)
or at least 10 min. The purification was performed directly onhe resulting supernatant for the MISPE method whereas for theFC–MIP method, two additional steps were necessary to preventolumn overload. In a first step, the supernatant was applied onto
C18 SPE column (2000 mg) previously conditioned with 10 mLeOH and 10 mL UP water. Steroids were purified by washingith 10 mL UP water and 10 mL n-hexane and eluted with 5 mLeOH/ethyl acetate (30:70, v/v). The eluted fraction was evapo-
ated to dryness under a gentle stream of nitrogen at 45 ◦C andissolved in 2 mL of acetate buffer (pH 5.2). A LLE was then per-
ormed twice with 5 mL n-pentane. The organic layer containinghe target steroids was kept in a glass tube, evaporated to drynessnder nitrogen, then reconstituted in MeOH (50 �L) and kept at◦C before injection in semi-preparative SFC.. A 1270 (2012) 51– 61 53
2.5. MISPE procedure
For quantification, urine samples were fortified with epiT-d3, E2-d3 and boldenone-d3 at a level of 100 ng mL−1. TheMISPE sorbent was first conditioned with 5 mL acetonitrile and5 mL UP water. Fortified urine samples (pre-treated as previ-ously described in Section 2.4) were applied and then washedwith 5 mL UP water/acetonitrile (90:10, v/v) and 5 mL UPwater/acetonitrile (80:20, v/v). Target steroids were eluted with3 mL UP water/acetonitrile (65:35, v/v). Finally, the sorbent wasrinsed successively with 5 mL MeOH and 5 mL acetonitrile toavoid any carry-over phenomenon and ensure the conditioning ofthe polymer to its original shape. The extracts were evaporatedunder nitrogen, the external standard (5�-androstan-3,17-dione)was added (10 ng �L−1) and acetylation of steroids with 30 �L ofpyridine and 30 �L of acetic anhydride was performed at roomtemperature during 16 h. Finally, the derivatisation reagents wereevaporated to dryness under a nitrogen stream and the residue wasdissolved in 50 �L of cyclohexane.
2.6. SFC–MIP procedure
An Investigator Thar SFC system (Waters, Milford, MA, USA) cou-pled to a photodiode array detector (PDA) was used to perform theseparation and collection of the fractions of interest. The tempera-ture of the column, outlet pressure and flow rate were respectivelyset at 40 ◦C, 15 MPa, and 3 mL min−1. All purified extracts wereinjected in partial injection mode (50 �L). A mixture of acetoni-trile/MeOH (95:5, v/v) (A) was used as co-solvent with CO2(SC) (B)in gradient mode (A:B): 5:95 (3 min), followed by a linear gradientuntil 40:60 at 1% min−1 (5 min). Two fractions were collected as fol-lows: one fraction containing androgen steroids, 17�-nandroloneand E-aba (FA) between 15 and 23 min and another fraction con-taining �-E2 (FE) between 31 and 40 min. Collected fractions andcorresponding time windows were determined by injection of amixture of target steroids (5 �g of each steroid) and by visualiza-tion of the corresponding PDA chromatograms acquired between190 and 400 nm. Collected fractions were evaporated under nitro-gen, external standard was added in each fraction and steroidsacetylation was performed as previously described in Section 2.5.
2.7. Gas chromatography–mass spectrometry (GC–MS)
Quantification of the target compounds and evaluation of thefractions purity obtained after the MISPE or SFC–MIP steps wereachieved by GC–MS. An Agilent 6890 series gas chromatographycoupled to an Agilent 5973N single quadrupole mass analyzer(Agilent Scientific, USA) was used. Chromatographic separationwas achieved using an Optima-17MS column (30 m × 0.25 mmi.d, df: 0.25 �m) (Macherey-Nagel, Duren, Germany). Heliumwas used as carrier gas at a constant flow rate of 1.5 mL min−1.Injections were performed using 4 mm i.d. glass liner contain-ing glass wool (2 �L injected), operating in the pulsed splitlessmode (1.5 min). Inlet temperature was fixed at 250 ◦C. An ovenramp was used to optimize steroid separation. The oven wasconfigured as follows: 1.5 min at 60 ◦C (1.5 min), 20 ◦C min−1 to220 ◦C (0 min), 5 ◦C min−1 to 270 ◦C (1 min), 1 ◦C min−1 to 290 ◦C(0 min), 20 ◦C min−1 to 320 ◦C (3 min). GC–MS transfer line andsource were heated at 320 ◦C and 230 ◦C, respectively. The electronvoltage was set at 70 eV. Mass acquisition was performed in fullscan mode in the 50–500 m/z range. Extracted ion chromatograms(EIC) were used to characterize the response of target compounds,
internal and external standards. Acetylation of target steroidsled to the following diagnostic ions m/z 242 (E-aba), 256 (17�-nandrolone), 268 (epiboldenone), 270 (DHEA and epiT), 272(etiocholanolone, epiandrosterone and boldenone-d3), 273
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epiT-d3), 288 (5�-androstan-3,17-dione and M2), 314androstenediol and �-E2), 316 (5-aba), 317 (E2-d3) and 330 (M4nd testosterone). Chromatograms were recorded and compoundsere quantified using the ChemStation Software (Agilent Scientific,SA).
.8. GC–C–IRMS
GC–C–IRMS measurements were performed on a HP 6890 gashromatograph coupled to an IsoPrime isotope ratio mass spec-rometer via a GC–V Combustion interface (Elementar, Manchester,K). In order to keep the chromatographic separation obtained
n GC–MS, chromatographic conditions used in GC–MS and inC–C–IRMS were identical. The analytes were introduced into
combustion furnace filled with copper oxide wires (Elementalicroanalysis Limited, UK) held at 850 ◦C. The combustion gasesere passed through a liquid nitrogen water removal trap main-
ained at −100 ◦C; the remaining CO2 was introduced in an electrononization source operating at 100 eV. Ions (m/z 44, 45, and 46) wereeparated on a magnet and detected by three Faraday collectors.he calibration of the reference gas was performed with a mixturef three acetylated steroids (DHEA acetate, testosterone acetate and-androstene-3�,17�-diol diacetate) whose �13CVPDB values haveeen previously calibrated and described elsewhere [17,46].
. Results and discussion
.1. MISPE procedure
.1.1. Choice of the conditions of MIP synthesisThe main objective of this work was to develop a MIP for
he selective extraction of steroids from urine samples. A MIPas first synthesized using styrene, divinyl benzene and �-E2 asonomer, cross-linker and template respectively. A polar porogen,
amely MeOH, was selected in order to favor hydrophobic inter-ctions considering the subsequent use of MIP in aqueous media.ith the resulting MIP, a strong retention of �-E2 was obtained
ut the selectivity was poor regarding the very similar recoveriesbtained on the MIP and the corresponding non imprinted poly-er (NIP) (data not shown). As described by Lordel et al. for MIPs
ynthesis with the objective of selective extraction of nitroaro-atic explosives from water [47], a sol–gel approach consisting
n the use of tetraethylorthosilicate (TEOS) as cross-linker and 3-minopropyltriethoxysilane (APTS) as monomer was then tested.PTS was selected for its ability to develop hydrophobic interac-
ions with �-E2 in aqueous media during both the synthesis andhe subsequent use of the MIP. However, a poor selectivity and also
low retention of �-E2 were obtained (results not shown). There-ore, conditions of synthesis similar to those described by Jiang et al.38] were assessed. A template/MAA/EGDMA molar ratio of 1/8/40nstead 1/6/30 was selected in order to increase MIPs capacity. Dueo the poor solubilization of �-E2 in acetonitrile, E2 was finallyhosen as template.
.1.2. Evaluation of the resulting MIPThe resulting MIP performances were evaluated with 3 steroids,
amely 2 androgens (DHEA and epiT) and 1 estrogen (�-E2),onsidered as representative of the panel of steroids of interest.
ater samples (500 �L) spiked at 2500 ng mL−1 with each ana-yte were applied on the MIP and on the NIP in parallel (50 mgf each sorbent). This was followed by a washing step with UP
ater/acetonitrile mixture and an elution step with MeOH. Resultsre shown in Table 1. Recoveries of 85%, 68% and 82% were obtainedor �-E2, epiT and DHEA respectively on the MIP and 62%, 31% and2% on the NIP, thus demonstrating a good selectivity of the MIP
. A 1270 (2012) 51– 61
with a good repeatability (RSD value < 4%, n = 4). Then, matrix influ-ence was assessed by applying the same procedure to calf urinesamples. As shown by the results presented in Table 1, the selec-tivity was maintained and the repeatability was still acceptable(RSD < 10%, n = 4). In order to improve the sensitivity of the methodby increasing the enrichment factor, the sample size was increasedfrom 500 �L to 5 mL which affected neither the recoveries, northe selectivity of the procedure or the repeatability of the results(RSD < 10%, n = 4). Finally, a second MIP, named MIP′, was preparedto test the repeatability of the synthesis, and the retention of thethree analytes was studied with spiked water samples. Very sim-ilar results, reported in Table 1, were obtained compared to thoseof the first MIP (less than 15% of recoveries variation between MIPand MIP′). These results were promising, even if the number of syn-thesized MIPs and the evaluation of analytes retention have to beincreased before proceeding with routine applications. Neverthe-less, previous studies have reported repeatable synthesis using thesame monomer, cross-linker and initiator (MAA, EGDMA and AIBNrespectively) [38,48,49] which support the preliminary promisingperformances in terms of reproducibility of the extraction. To con-clude, synthesized MIPs showed a good selectivity and retentioncapacity toward the 3 selected model compounds.
3.1.3. Optimization of MISPE procedureIn order to improve the MISPE procedure and to extend its appli-
cability to a larger range of steroids, several parameters such aspre-treatment or washing steps were tested and optimized on 12steroids selected for their usefulness in revealing various situationsof anabolic steroid abuse (Fig. 1) [11,14,50,51].
Polymer quantity is a key parameter to optimize, since an exces-sive quantity promotes the development of non-specific bindingsites, whereas a limited one can lead to a low retention capabil-ity [52]. In order to guarantee a maximal recovery of the analytes,100 mg of polymers were used thereafter despite a slight decreasein specificity up to 15% (data not shown).
Preliminary extraction step such as SPE is recommended bymany authors for biological (aqueous) samples in order to trans-fer the target analytes in a solvent close to the porogen resultinginto an optimal selectivity and an increase of the factor enrich-ment. Thus, urinary samples were applied onto C18 cartridges asdescribed in Section 2.4 and the elution fraction was dissolved inacetonitrile. Unfortunately, when this extract was percolated onMISPE, steroids of interest were not retained by the polymer dueto the elution strength of acetonitrile. Therefore, urinary extractswere directly applied into MISPE without preliminary treatment.
In the case of direct percolation of the aqueous sample, analytesretention is mainly due to non-specific hydrophobic or electrostaticinteractions with the polymeric phase. Selective retention mecha-nism resulting from the presence of cavities can be reached by usingan appropriate washing solvent [53]. A combination of acetoni-trile and water was investigated through elution profiles of targetsteroids realized after percolation of 5 mL of fortified urine with the12 selected steroids (100 ng mL−1) and consecutive washing stepswith 3 mL UP water/acetonitrile (from 100:0 to 0:100, v/v by stepof 5%). 17�-nandrolone was the first eluted compound with 25%of acetonitrile and all target compounds were eluted with 35% ofacetonitrile. Therefore, 3 mL UP water/acetonitrile (80:20, v/v) canbe used as washing solvent and 3 mL UP water/acetonitrile (65:35,v/v) as elution solvent in order to obtain all target steroids in thesame fraction. These results show that target analytes were elutedwith a low percentage of acetonitrile. The presence of water seemsto disturb the development of specific recognition between steroids
and MIP and therefore other washing mixtures were tested.Five different protocols were applied on the same urine to com-pare the interest of a non-polar (mixture of hexane/petrolether),an acidic (hydrochloric acid 1 N or acetic acid 1 N/acetonitrile
M. Doué et al. / J. Chromatogr. A 1270 (2012) 51– 61 55
the 12
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Fig. 1. Chemical structures of
80:20, v:v)), a neutral (UP water/acetonitrile (80:20, v:v)) or a basicsodium hydroxide 1 N/acetonitrile (80:20, v:v)) washing step. Asn example, the results obtained for epiT are shown in Table 2xcept for the basic washing step where derivatisation prior tonjection in GC–MS could not be achieved and therefore recover-es for all steroids could not be measured. Results are expressedn ng �L−1 rather than in percentage since the urine samples used
ere from pregnant cows and therefore already contained some ofhe target steroids such as �-E2 and epiT. It should be noted that,hatever the conditions and parameters tested, standard devia-
ion never exceeded 15%. For the hexane/petrolether washing step,
arget steroids were not eluted by such a mixture and no signif-cant improvement of selectivity and purification was observedccording to MIP/NIP comparison and GC–MS chromatograms ofable 1ecoveries and RSD values (n = 4) obtained after the percolation of water and calf urine s50 mg of sorbent). Percolation of 500 �L or 5 mL of samples, washing with a UP water/ac
Water (500 �L) Calf urine (500 �L)
MIP NIP MIP NIP
�-E2 85 ± 4 62 ± 5 83 ± 6 55 ± 13epiT 68 ± 2 31 ± 2 61 ± 10 24 ± 8
DHEA 82 ± 3 52 ± 3 68 ± 8 31 ± 12
able 2ecoveries expressed as results in ng �L−1 obtained for epiT from different MISPE washteroid. Washing step: 3 mL UP water/acetonitrile (80:20, v/v), or 3 mL hexane/petrolethe
N/acetonitrile (80:20, v/v) and or 3 mL acetic acid 1 N/acetonitrile (80:20, v/v). Elution: 3
ere less than 15% of variation (n = 4).
EpiT (ng �L−1) UP water/acetonitrile Hexane/petrolether
MIP NIP MIP
Washing 36.8 8.0 0
Elution 145.9 102.4 137.8
target endogenous steroids.
the eluted fraction (data not shown). Thus, a non-polar washingstep failed to increase specific recognition. For the acidic washingstep, the use of hydrochloric acid led to a decrease of the target ana-lyte recoveries and MIP selectivity whereas the use of acetic acidled to elution of the analytes. The role of electrostatic interactionsin the molecular recognition process seems to play a major part andprevents the use of an acidic washing step [28]. The best results interms of specificity were obtained with the UP water/acetonitrilemixture with recovery differences up to 30% between MIP and NIP.Thus, the neutral washing step was retained for the final protocol.
In order to evaluate the purification power of the developed
protocol, a comparison with two classical preparation steps wasrealized. Three different protocols were applied on the same for-tified urine sample: (i) extraction with SPE C18 followed by LLE aspiked at 2500 ng mL−1 with each compound on MIP and on NIP or on the MIP′/NIP′
etonitrile mixture, elution with 1 mL MeOH.
Calf urine (5 mL) Water (500 �L)
MIP NIP MIP′ NIP′
86 ± 10 65 ± 8 89 ± 4 74 ± 667 ± 7 29 ± 8 59 ± 8 38 ± 9
74 ± 9 44 ± 8 73 ± 9 45 ± 5
ing protocols after percolation of 5 mL of urine spiked with 100 ng �L−1 of eachr several times (from 0:100 to 100:0, v/v by step of 20%), or 3 mL hydrochloric acidmL UP water/acetonitrile (65:35, v/v). Standard deviation values for all experiments
HCl/acetonitrile Acetic acid/acetonitrile
NIP MIP NIP MIP NIP
0 0 0 84.7 72.796.3 128.4 150.2 4.3 8.2
56 M. Doué et al. / J. Chromatogr. A 1270 (2012) 51– 61
14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00
5000000
1e+07
1.5e+07
2e+07
2.5e+07
3e+07
3.5e+07
4e+07
4.5e+07
5e+07
5.5e+07
6e+07
6.5e+07
7e+07
7.5e+07
8e+07
8.5e+07
9e+07
Time-->
SiOH
14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00
5000000
1e+07
1.5e+07
2e+07
2.5e+07
3e+07
3.5e+07
4e+07
4.5e+07
5e+07
Time-->
LLE
12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00
5000000
1e+07
1.5e+07
2e+07
2.5e+07
3e+07
3.5e+07
4e+07
4.5e+07
5e+07
5.5e+07
6e+07
Time-->
MISPE
F �L inja ponse
dtdtsGtSras6eus
3p
deiita[“ocsaru
systematically above 0.99. Results are presented in Table 3 andthe corresponding extracted ion chromatograms (EIC) of the uri-nary extract from E2 and T experiments are featured in Fig. 3.All the main metabolites of testosterone, E2, and boldenone could
Table 3Mean concentrations and corresponding standard deviation (n = 4) of target steroidsobtained with MISPE protocol and GC–MS analysis of urinary samples from treatedbovines.
Treatments Days of collectionafter treatment
Steroids ofinterest
Mean concentration(standard deviations)(�g L−1)
T D1 epiT 68.3 (4.2)Testosterone 28.2 (1.4)
ig. 2. GC–MS chromatograms, total ion current (TIC) acquired in full scan mode: 2
nd MISPE. Chromatograms were normalized according to the external standard res
escribed in Section 2.4 (ii) same procedure with a third extrac-ion on SPE SiOH (SiOH) as described by Buisson et al. [17] and (iii)eveloped MISPE protocol (MISPE). The GC–MS chromatograms ofhe three purified fractions, normalized according to the externaltandard response, are presented in Fig. 2. With respect to theC–MS chromatograms, the baseline value was divided by a fac-
or of ten between the LLE and the MISPE methods, whereas theiOH chromatogram presented less interference but lower steroidecoveries (up to 70% of differences). Thus, MISPE method appeareds a good compromise between both approaches (LLE and SiOH)ince recoveries for all compounds were estimated to be above5% (external calibration) and external standard responses neverxceeded ±20% of variation (measured during a sequence of 10rine samples) showing a sufficient urinary purification for GC–MSingle quadrupole analysis.
.1.4. MISPE protocol applied on urinary steroids for screeningurpose
In the past few years, a lot of research work has been con-ucted to highlight biomarkers of steroids abuse [10,11,13–15]. Forxample, Dervilly-Pinel et al., have shown that some estranediolsomers can be used as biomarkers to indicate nandrolone abusen cattle [11,14]. The unambiguous identification and quantifica-ion of these metabolites required an adapted sample preparationnd the use of sensitive and specific analyzers such as GC–MS/MS14]. Screening may also be based on thresholds in terms of basalendogenous” concentration levels. Despite the high variabilitiesf steroid concentrations in urine [50,54], this approach is alsoonsidered as a method of choice due to its potential multi-
creening application and the use of less specific instruments suchs GC–MS. For boldenone abuse, the current European regulationecommends that epiboldenone levels exceeding 2 �g L−1 in calfrine have to be considered as suspicious [55]. For E2, testosteroneected of the eluted fraction from SPE C18 + LLE (LLE), SPE C18 + LLE + SPE SiOH (SiOH) (indicated by arrows). Squares show the retention time windows of target steroids.
and nandrolone abuse, no official urinary metabolite thresholdshave been published yet. The applicability of the developed MISPEmethod was assessed with urine samples from bovines treated withE2 (E2), boldione (B), testosterone (T) and E2 and 17�-nandrolone(E2/NT) (sampling at day 1 and day 3 after injections). For theE2/NT treatment, the developed sample preparation allowed sub-sequent unambiguous identification of E-aba (previously reportedas a biomarker of interest after such treatment) in urine 3 daysafter treatment, based on its retention time and respective massspectrum. For E2, B and T experimental samples, the quantifica-tion of the main metabolites of the administrated compounds wascarried out by external calibration with an isotopically labeledstandard after GC–MS analysis of the purified extract. Good lin-earity was obtained between 1 and 30 �g L−1 and between 30 and100 �g L−1 for each compound with coefficients of determination
E2 D1 �-E2 261.7 (18.7)E2 4.7 (0.5)
B D3 Epiboldenone 23.1 (0.8)Boldenone 2.9 (0.3)
M. Doué et al. / J. Chromatogr. A 1270 (2012) 51– 61 57
F (epiT( tmen
efwdanNttTpctsTmmp
3
3
pesIpeMA
ig. 3. Extracted ion chromatograms (EIC) m/z 314 (E2 and �-E2), 317 (E2-d3), 2702 �L) of the urinary extracts from E2 (day 1 after treatment) and T (day 1 after trea
asily be detected and measured after MISPE sample preparationollowed by GC–MS analysis. As expected, the epiboldenone levelas found higher than the recommended urinary thresholds atay 3 after treatment (23.1 �g L−1 for a threshold of 2 �g L−1) andllowed to classify the sample as suspicious. As mentioned before,o thresholds have been published yet for testosterone or E2 abuse.evertheless, the developed method allowed the quantification of
estosterone, epiT, E2 and �-E2 at urinary levels useful to monitorheir concentration modifications after anabolic treatment [50,54].hese results showed the applicability of the developed samplereparation for screening purposes. Moreover, compared to otherlassical protocols involving several SPE and LLE steps [56–58],his one-step protocol allowed the analysis of several classes ofteroids (androgens and estrogens) within only one analysis step.hus, the MISPE method can be considered as cheap, efficient andultiresidue, since testosterone, E2, boldenone and nandroloneetabolites can be measured with this one step sample preparation
rocedure.
.2. SFC–MIP procedure
.2.1. Optimization of SFC–MIP procedureSince GC–C–IRMS measurement is very demanding in terms of
eak purity, the sample preparation is a key step which has to be asfficient as possible. The previous MISPE developed method was notufficient (presence of co-eluting interferents with target steroids).ndeed, with biological (aqueous) samples applied on MISPE, the
resence of water seems to disturb specific recognition throughlectrostatic interactions. In order to solve this issue, the use ofIP as stationary phase in semi-preparative SFC was considered.commercial MIP was chosen as stationary phase to guarantee
and testosterone), 273 (epiT-d3) of GC–MS chromatograms obtained by injectionst) experiments obtained after MISPE protocol.
a homogenous particles size and filling of the chromatographiccolumn. The choice of this commercial MIP specifically designedfor E2 recognition was based on the fact that the same retentioncharacteristics and chromatographic profiles of purified extractswere obtained compared to those of the synthesized MIP (data notshown). Unfortunately, the template was not communicated by theprovider.
The nature of the solvent applied on the MIP induces the natureof the interactions that take place during the recognition mech-anism [29]. Thus, two different co-solvents, one protic and oneaprotic i.e. MeOH and acetonitrile respectively were first assessedto optimize the chromatographic separation of the 12 targetsteroids. Gradients from 5 to 30% in 25 min with MeOH and from 5to 40% in 35 min with acetonitrile were applied. UV chromatogramsobtained from a mixture of target compounds are presented inFig. 4. With MeOH, androgens were found to be less retainedcompared with acetonitrile (retention factor kA-MeOH = 13.8 andkA-acetonitrile = 23.8). �-E2 was eluted with 28% of MeOH in CO2(SC)while with acetonitrile this compound was found to be still retainedon the stationary phase despite the highest percentage used (upto 40%). Moreover, chromatographic separation of mono-(MS) anddihydroxylated (DS) steroids could be achieved with acetonitrile.Thus, the use in SFC–MIP of acetonitrile seems to increase specificrecognition. Indeed, the introduction of the porogen, i.e. acetoni-trile, induced the return of the cavities to their original shape andsize and therefore facilitated the specific recognition process. Nev-ertheless, in order to purify all target compounds, the addition of
5% of MeOH in acetonitrile enabled �-E2 to be eluted while pre-serving specificity (Fig. 4). Finally, fractions and corresponding timewindows were determined thanks to the corresponding UV chro-matograms.
58 M. Doué et al. / J. Chromatogr. A 1270 (2012) 51– 61
Fig. 4. PDA chromatograms (from 190 to 400 nm) obtained by injection (5 �g) of a mixture of target compounds with different co-solvents used (MeOH, acetonitrile anda CO2(SC
a d ster
3
oeadirnvt
rpfsicGvu(swfebt
piip
on the expected retention time windows of endogenous steroidspresented in Fig. 5 attest for their satisfactory purity whatever theSFC–MIP methods used. Comparable chromatographic profiles for
Table 4Isotopic deviation (�13CVPDB) values (non-corrected) of target acetylated steroidsdirectly injected (n = 4) in GC–C–IRMS or after SFC–MIP step. Confidence intervals(CI) were given for = 0.01.
Target steroids Introduction mode
Direct injections After SFC–MIP
Mean �13CVPDB Confidence interval�13CVPDB
�13CVPDB
E-aba −37.22 [−36.64 to −37.80] −37.44Etiocholanolone −33.38 [−32.23 to −33.83] −33.74DHEA −37.87 [−37.21 to −38.53] −38.46Androstenediol −37.56 [−36.57 to −38.55] −37.51Epiandrosterone −36.54 [−35.39 to −37.69] −37.135-aba −40.24 [−39.29 to −41.19] −39.81M4 −35.13 [−33.61 to −36.65] −35.97�-Nandrolone −33.36 [−31.98 to −34.74] −34.51
cetonitrile/MeOH (95:5, v:v)). For MeOH, gradient from 5 (3 min) to 30% MeOH incetonitrile in CO2(SC) in 35 min. MS: monohydroxylated steroids, DS: dihydroxylate
.2.2. Isotopic fractionation and purity assessmentSteroid 13C/12C isotopic ratio measurement by GC–C–IRMS is
ne of the methods of choice to determine the exogenous orndogenous origin of steroids for confirmatory purposes. Thedministration of synthetic steroids to bovines leads to a slightepletion in the 13C/12C ratios (expressed as �13CVPDB values) of
ts respective metabolites while the 13C/12C ratios of its precursorsemain unchanged [17,18]. Thus, precursors can be used as endoge-ous reference compounds (ERC) and a difference of �13CVPDBalues between ERC and metabolites proves administration of syn-hetic steroids.
One of the major potential pitfalls in GC–C–IRMS analysis iselated to the isotopic fractionation that can occur during samplereparation [59]. The absence of isotopic fractionation was there-ore assessed for the developed SFC–MIP step. A mixture of targetteroids was first injected in SFC–MIP, collected and then injectedn GC–C–IRMS after derivatisation. Obtained �13CVPDB values wereompared with those of the same steroids directly analyzed inC–C–IRMS after derivatisation. Results (�13CVPDB non-correctedalues) are presented in Table 4. As expected, all �13CVPDB val-es of target steroids were situated in the confidence interval (CI)
= 0.01) of directly injected steroids (except for M2 which pre-ented a slight lower value). These results were in accordanceith those published by Buisson et al., who showed that isotopic
ractionation can occur during chromatographic separation (differ-nces in term of 13C/12C ratio from the start to the end of the peak)ut can be avoided with an appropriate and complete collection ofhe analyte [17].
Another potential issue in GC–C–IRMS analysis is the peak
urity of the target compounds. Chromatographic co-elution ofnterferents with steroids leads to an incorrect estimation of thesotopic composition. Thus, in order to assess the purificationower of the different strategies, two different protocols namely
) in 25 min, for acetonitrile and acetonitrile/MeOH, gradient from 5 (3 min) to 40%oids and E: �-E2.
SFC–MIP with MeOH (SFC–MIP MeOH) and acetonitrile/MeOH(95:5) (SFC–MIP acetonitrile/MeOH) as co-solvent were appliedon fortified pregnant cow urine samples. To prevent column over-load, urinary samples were first applied on SPE C18 and then a LLEwas performed as indicated in Section 2.4 before injection in SFC.Purified extracts were analyzed by GC–MS after derivatisation andtheir chromatograms, as well as the associated mass spectra andthe absence of co-elution with target steroids, allowed us to assesstheir purity. GC–MS chromatograms of the fractions FE focusing
M2 −33.63 [−33.03 to −34.23] −32.69EpiT −38.57 [−36.88 to −40.26] −37.03Testosterone −36.16 [−34.21 to −38.11] −34.78�-E2 −36.92 [−35.12 to −38.72] −37.52
M. Doué et al. / J. Chromatogr. A 1270 (2012) 51– 61 59
F targeS �-E2.
StastMi
3c
w
Fe
ig. 5. GC–MS chromatograms, total ion current (TIC) focused on retention times ofFC–MIP MeOH and SFC–MIP acetonitrile/MeOH methods. 1: external standard, 2:
FC–MIP MeOH and acetonitrile/MeOH methods were obtained forhe fraction FA containing androgens, 17�-nandrolone and E-aband for FE containing �-E2. Nevertheless, according to the externaltandard response, steroid hormone recoveries were higher withhe SFC–MIP MeOH/acetonitrile method compared to the SFC–MIP
eOH one. Thus, this method was then applied to the selectivesolation of urinary steroids in the further scope of IRMS analysis.
.2.3. SFC–MIP protocol applied on urinary steroids foronfirmatory purposes
Sample preparation with SFC–MIP acetonitrile/MeOH methodas applied on urine samples from treated bovines with estradiol
ig. 6. GC–C–IRMS chromatograms (m/z 44) of fractions FA and FE from E2 (day 1 after
ndogenous reference compound.
t steroids acquired in full scan mode: 2 �L injected of the collected fraction FE fromChromatograms were normalized according to the external standard response.
(E2), boldione (B), androstenedione (AED) and testosterone (T).The sample extracts were then injected in GC–MS to confirm ana-lyte identity, assess the peak purity, evaluate the concentration foreventual further dilution/concentration and therefore ensure thatconcentration estimation of target analytes is in the GC–C–IRMSlinearity in the range of 15–70 ng of steroid on column. Sufficientpurity of endogenous steroids was obtained for all urine samples.Urinary extracts were then injected in GC–C–IRMS. IRMS chro-
matograms for E2 experiment are presented in Fig. 6 and �13CVPDBresults in Table 5. Regarding GC–C–IRMS chromatograms, peakpurity is sufficient to allow the measurement of isotopic devia-tion values. All metabolites present a difference in their �13CVPDBtreatment) experiment obtained after SFC–MIP sample preparation method. ERC:
60 M. Doué et al. / J. Chromatogr. A 1270 (2012) 51– 61
Table 5Isotopic deviation (�13CVPDB) values of ERC and metabolites obtained with urinary samples from treated cows.
Experiments/direct metabolites M Days of collection after treatment �13CVPDB values of ERC: DHEA (‰) �13CVPDB values of M: (‰) � (M-ERC) (‰)
T/epiT D1 −26.72 −31.48 −4.7623.9624.9722.75
vctfilccpasasfss
4
appodesmtaerpuaeao
A
(caFtEgn
R
[
[
[
[
[
[
[
[
[
[
[[
[
[
[
[
[[[[[
[
[[[[
[[
[[[[[[[[[
E2/�-E2 D1 −AED/5-aba D1 −B/epiboldenone D3 −
alues with an ERC greater than 3‰ (world anti doping agencyompliant threshold value [60]) confirming the administration ofhe respective steroids under their 17� form. Moreover, for therst time, significant differences in 13C/12C ratio between metabo-
ites compared to their respective ERC were demonstrated inattle after administration of androstenedione and boldione. Toonclude, SFC–MIP method not only showed its multiresidue sam-le preparation application (testosterone, estradiol, boldione, andndrostenedione) but also its relevance to strongly purify urinaryteroids in the further scope of an IRMS analysis. This protocol alsollowed to reduce cost and length of sample preparation since threeteps (1 LLE and 2 semi-preparative HPLC steps) could be removedrom the current method [17]. Indeed, the SFC semi-preparativetep is 90% less expensive than the previous HPLC one and theample preparation time is reduced by 20%.
. Conclusions
The objective of this study was to evaluate the capabilities of selective extraction procedure based on molecularly imprintedolymers in order to propose short, multiresidue and cheap sam-le preparation procedures. For screening purposes, a one stepptimized MISPE protocol with in-house synthesized polymer waseveloped and applied on samples from treated animals. Recov-ries (above 65% for the 12 target steroids) and purities wereufficient for GC-MS analysis and allowed quantification of someetabolites interesting to suspect steroid abuse. MIP used as sta-
ionary phase in SFC in order to improve specific recognition waslso assessed. SFC–MIP procedure was found to be a robust andfficient approach to strongly purify target steroids, with similaresults than the current method but with three steps less (samplereparation time was reduced by 20%). Its application on collectedrines from treated animals allowed the confirmation of steroiddministration. Finally, this sample preparation could be consid-red as multiresidue since confirmation of testosterone, estradiol,ndrostenedione and boldione abuse in producing animals can bebtained.
cknowledgment
We gratefully thank Polyintell (Val de Reuil, France) and WatersMilford, MA, USA) for providing this study with MIP in packedolumn for chromatography and Thar SFC Investigator device. Welso acknowledge Flavia Hanganu for E2 and T animal experiments.or E2/NT animal experiment, the sample has been obtained fromhe 6th Framework Programme “Integrating and strengthening theuropean Research Area within the BioCop project “New technolo-ies to Screen Multiple Chemical Contaminants in Foods”. Contractumber: FOOD-CT-2004-06988.
eferences
[1] P.J. Guiroy, L.O. Tedeschi, D.G. Fox, J.P. Hutcheson, J. Anim. Sci. 80 (2002) 1791.[2] Council Directive 96/22/EC, of 29 April 1996. Off. J. Eur. Commun. (1996) No.
L 125/3. Concerning the prohibition on the use in stockfarming of certain sub-stances having a hormonal or thyrostatic action and on b-agonists and repealingDirectives 81/602/EC, 88/146/EC and 88/299/EC.
[3] D. Courtheyn, B. Le Bizec, G. Brambilla, H.F. De Brabander, E. Cobbaert, M. Vande Wiele, J. Vercammen, K. De Wasch, Anal. Chim. Acta 473 (2002) 71.
[
[
−29.80 −5.84 −33.13 −8.16 −29.16 −6.41
[4] H.F. de Brabander, H. Noppe, K. Verheyden, J. Vanden Bussche, K. Wille, L. Oker-man, L. Vanhaecke, W. Reybroeck, S. Ooghe, S. Croubels, J. Chromatogr. A 1216(2009) 7964.
[5] M.H. Mooney, C.T. Elliott, B. Le Bizec, Trends Anal. Chem. 28 (2009) 665.[6] Commission Decision 2002/657/EC, of 12 August 2002. Off. J. Eur. Commun.
(2002) No. L 221/8. Concerning the performance of analytical methods and theinterpretation of results.
[7] G. Pinel, S. Weigel, J.P. Antignac, M.H. Mooney, C. Elliott, M.W.F. Nielen, B. LeBizec, Trends Anal. Chem. 29 (2010) 1269.
[8] B. Le Bizec, G. Pinel, J.-P. Antignac, J. Chromatogr. A 1216 (2009) 8016.[9] H. Noppe, B. Le Bizec, K. Verheyden, H.F. De Brabander, Anal. Chim. Acta 611
(2008) 1.10] B. Destrez, E. Bichon, L. Rambaud, F. Courant, F. Monteau, G. Pinel, J.-P. Antignac,
B. Le Bizec, Steroids 74 (2008) 803.11] G. Dervilly-Pinel, L. Rambaud, P. Sitthisack, F. Monteau, S.A. Hewitt, D.G.
Kennedy, B. Le Bizec, J. Steroid Biochem. Mol. Biol. 126 (2011) 65.12] S. Anizan, E. Bichon, D. Di Nardo, F. Monteau, N. Cesbron, J.-P. Antignac, B. Le
Bizec, Talanta 86 (2011) 186.13] S. Anizan, E. Bichon, T. Duval, F. Monteau, N. Cesbron, J.-P. Antignac, B. Le Bizec,
J. Mass Spectrom. 47 (2012) 131.14] G. Pinel, L. Rambaud, F. Monteau, C. Elliot, B. Le Bizec, J. Steroid Biochem. Mol.
Biol. 121 (2010) 626.15] B. Le Bizec, F. Courant, I. Gaudin, E. Bichon, B. Destrez, R. Schilt, R. Draisci, F.
Monteau, F. André, Steroids 71 (2006) 1078.16] G. Pinel, L. Rambaud, G. Cacciatore, A. Bergwerff, C. Elliott, M. Nielen, B. Le Bizec,
J. Steroid Biochem. Mol. Biol. 110 (2008) 30.17] C. Buisson, M. Hebestreit, A.P. Weigert, K. Heinrich, H. Fry, U. Flenker, S. Ban-
neke, S. Prevost, F. Andre, W. Schaenzer, E. Houghton, B. Le Bizec, J. Chromatogr.A 1093 (2005) 69.
18] M. Hebestreit, U. Flenker, C. Buisson, F. Andre, B. Le Bizec, H. Fry, M. Lang,A.P. Weigert, K. Heinrich, S. Hird, W. Schänzer, J. Agric. Food Chem. 54 (2006)2850.
19] J. Scarth, C. Akre, L. van Ginkel, B. Le Bizec, H. De Brabander, W. Korth, J. Points,P. Teale, J. Kay, Food Addit. Contam. 26 (2009) 640.
20] D. Ryan, K. Robards, P.D. Prenzler, M. Kendall, Anal. Chim. Acta 684 (2011) 17.21] M. Gasparini, M. Curatolo, W. Assini, E. Bozzoni, N. Tognoli, G. Dusi, J. Chro-
matogr. A 1216 (2009) 8059.22] L. Xu, S. Qiu, C.-J. Sun, Q.-P. Deng, H.-X. Chen, Y.-L. Zhou, X.-X. Zhang, J. Chro-
matogr. B 878 (2010) 1443.23] S. Qiu, L. Xu, Y.-R. Cui, Q.-P. Deng, W. Wang, H.-X. Chen, X.-X. Zhang, Talanta 81
(2010) 819.24] S. Anizan, E. Bichon, F. Monteau, N. Cesbron, J.-P. Antignac, B. Le Bizec, J. Chro-
matogr. A 1217 (2010) 6652.25] W. Liu, L. Zhang, L. Fan, Z. Lin, Y. Cai, Z. Wei, G. Chen, J. Chromatogr. A 1233
(2012) 1.26] Z. Zhang, H. Duan, L. Zhang, X. Chen, W. Liu, G. Chen, Talanta 78 (2009) 1083.27] K. Saito, K. Yagi, A. Ishizaki, H. Kataoka, J. Pharmaceut. Biomed. 52 (2010) 727.28] V. Pichon, J. Chromatogr. A 1152 (2007) 41.29] V. Pichon, F. Chapuis-Hugon, Anal. Chim. Acta 622 (2008) 48.30] A. Beltran, F. Borrull, R.M. Marcé, P.A.G. Cormack, Trends Anal. Chem. 29 (2010)
1363.31] E. Caro, R.M. Marcé, F. Borrull, P.A.G. Cormack, D.C. Sherrington, Trends Anal.
Chem. 25 (2006) 143.32] B. Sellergren, Anal. Chem. 66 (1994) 1578.33] Z. Zhongbo, J. Hu, Water Res. 42 (2008) 4101.34] M.D. Celiz, D.S. Aga, L.A. Colón, J. Microchem. 92 (2009) 174.35] M. Le Noir, A.-S. Lepeuple, B. Guieysse, B. Mattiasson, Water Res. 41 (2007)
2825.36] Q. Zhu, L. Wang, S. Wu, W. Joseph, X. Gu, J. Tang, Food Chem. 113 (2009) 608.37] R. Gadzała-Kopciuch, J. Ricanyová, B. Buszewski, J. Chromatogr. B 877 (2009)
1177.38] T. Jiang, L. Zhao, B. Chu, Q. Feng, W. Yan, J.-M. Lin, Talanta 78 (2009) 442.39] G. Guiochon, A. Tarafder, J. Chromatogr. A 1218 (2011) 1037.40] T.T. Larry, J. Supercrit. Fluids 47 (2009) 566.41] J.A. Richard, Adv. Drug Deliver. Rev. 57 (2005) 1809.42] B.-Y. Huang, Y.-C. Chen, G.-R. Wang, C.-Y. Liu, J. Chromatogr. A 1218 (2011) 849.43] Z. Wang, J. Ouyang, W.R.G. Baeyens, J. Chromatogr. B 862 (2008) 1.44] E. Turiel, J.L. Tadeo, P.A.G. Cormack, A. Martin-Esteban, Analyst 130 (2005) 1601.45] E. Turiel, A. Martín-Esteban, J.L. Tadeo, J. Chromatogr. A 1172 (2007) 97.46] E. Bichon, F. Kieken, N. Cesbron, F. Monteau, S. Prévost, F. André, B. Le Bizec,
Rapid Commun. Mass Spectrom. 21 (2007) 2613.47] S. Lordel, F. Chapuis-Hugon, V. Eudes, V. Pichon, Anal. Bioanal. Chem. 399 (2011)
449.48] F. Chapuis-Hugon, M. Cruz-Vera, R. Savane, W.H. Ali, M. Valcarcel, M. Deveaux,
V. Pichon, J. Sep. Sci. 32 (2009) 3301.
atogr
[
[
[
[[
[
[
[
[
[
M. Doué et al. / J. Chrom
49] F. Hugon-Chapuis, J.U. Mullot, G. Tuffal, M.C. Hennion, V. Pichon, J. Chromatogr.A 1196–1197 (2008) 73.
50] M.W.F. Nielen, J.J.P. Lasaroms, M.L. Essers, M.B. Sanders, H.H. Heskamp,T.F.H. Bovee, J. van Rhijn, M.J. Groot, Anal. Chim. Acta 586 (2007)30.
51] H.F. de Brabander, J. van Hende, P. Batjoens, L. Hendriks, J. Raus, F. Smets, G.Pottie, L. van Ginkel, R.W. Stephany, Analyst 119 (1994) 2581.
52] L.I. Andersson, J. Chromatogr. B 739 (2000) 163.53] F. Chapuis, V. Pichon, F. Lanza, S. Sellergren, M.C. Hennion, J. Chromatogr. A 999
(2003) 23.54] C.J.M. Arts, M.J. Van Baak, J.M.P. Den Hartog, J. Chromatogr. B 564 (1991)
429.
[[
. A 1270 (2012) 51– 61 61
55] European Commission, Health Consumer Protection Directorate. General out-come of the expert meeting on the control of boldenone in veal calves (2003).
56] J. Scarth, A. Clarke, J. Hands, P. Teale, R. Macarthur, J. Kay, Chromatographia 71(2010) 241.
57] S. Biddle, P. Teale, A. Robinson, J. Bowman, E. Houghton, Anal. Chim. Acta 586(2007) 115.
58] P.R. Kootstra, P.W. Zoontjes, E.F. van Tricht, S.S. Sterk, Anal. Chim. Acta 586
(2007) 82.59] A.T. Cawley, U. Flenker, J. Mass Spectrom. 43 (2008) 854.60] TD2004EAAS v1.0. Reporting and evaluation guidance for testosterone,
epitestosterone, T/E ratio and other endogenous steroids. WADA technical doc-ument. 30 May, (2004).
