Molecularly imprinted polymer applied to the selective isolation of urinary steroid hormones: An efficient tool in the control of natural steroid hormones abuse in cattle

Journal of Chromatography A, 1270 (2012) 51–61

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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma

Molecularly imprinted polymer applied to the selective isolation of urinary steroid hormones: An efficient tool in the control of natural steroid hormones abuse in cattle Mickael Doué a , Emmanuelle Bichon a,∗ , Gaud Dervilly-Pinel a , Valérie Pichon b , Florence Chapuis-Hugon b , Eric Lesellier c , Caroline West c , Fabrice Monteau a , Bruno Le Bizec a a

LUNAM Université, Oniris, Laboratoire d’Etude des Résidus et Contaminants dans les Aliments (LABERCA), Nantes, F-44307, France Department of Analytical and Bioanalytical Sciences and Miniaturization (LSABM), ESPCI ParisTech, UMR PECSA 7195 (CNRS–UPMC–ESPCI ParisTech), 10 rue Vauquelin, 75231 Paris Cedex 05, France c Institut de Chimie Organique et Analytique (ICOA), Université d’Orléans, CNRS UMR 7311, B.P. 6759, rue de Chartres, 45067 Orléans Cedex 2, France b

a r t i c l e

i n f o

Article history: Received 18 July 2012 Received in revised form 22 October 2012 Accepted 31 October 2012 Available online 6 November 2012 Keywords: Chemical food safety Steroid hormones Molecularly imprinted polymer Solid phase extraction Supercritical fluid chromatography Gas chromatography–combustion–isotope ratio 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 Union as laid down in Directive 96/22/EC. Nowadays, efficient methods such as steroid profiling or isotopic deviation measurements allow to control natural steroid hormones abuse. In both cases, urine is often selected 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 sorbent such as molecularly imprinted polymer (MIP) was investigated. A MIP was synthesized based on 17␤-estradiol, methacrylic acid and acetonitrile as template, monomer and porogen, respectively. Two approaches were then tested for non-conjugated (aglycons and glucuronides deconjugated) steroid purification: (i) molecularly imprinted solid phase extraction (MISPE) and (ii) semi-preparative supercritical fluid chromatography with a commercial MIP as stationary phase (SFC–MIP). Parameters for both approaches were optimized based on the main bovine metabolites of testosterone, estradiol, nandrolone and 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 spectrometry (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 protocol with three steps less compared to the official and current method. Purity, concentration and absence of isotopic fractionation of target steroids extracted from urine of treated cattle (treated with testosterone, estradiol, androstenedione, and boldenone) allowed the measurement of 13 C/12 C isotopic ratios of corresponding metabolites and endogenous reference compounds (ERC) and proved the relevance of the strategy. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Beneficial effects of natural and synthetic steroids related to animal growth promotion and feed conversion efficiency have led to a wide use of these compounds in food producing animals since the 1950s [1]. In the European Union, the use of anabolic substances in cattle breeding has been prohibited since 1988

∗ Corresponding author at: Oniris, Ecole nationale vétérinaire, agroalimentaire et de l’alimentation Nantes-Atlantique, Laboratoire d’Etude des Résidus et Contaminants dans les aliments (LABERCA), Atlanpole-La Chantrerie, BP 40706, Nantes, F-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). 0021-9673/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chroma.2012.10.067

(Directive 88/146/EC repealed by Directive 96/22/EC) [2]. Nevertheless, steroid hormones may still be fraudulently employed and an efficient control is required to monitor such misuse [3–5]. EU legislation (Directive 2002/657/EC [6]) imposes a two-step strategy in laboratories in charge of the control: initial rapid and multiresidue screening step to sift large numbers of samples for potential steroids abuse followed by a confirmatory step which discards any doubts on the compliance of the suspicious samples [5,7–9]. Recently and thanks to the advances made in the knowledge of steroid metabolic patterns as well as the associated 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 by gas chromatography–combustion–isotope ratio mass spectrometry (GC–C–IRMS) probably remains the most adapted option to

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M. Doué et al. / J. Chromatogr. A 1270 (2012) 51–61

determine the endogenous or exogenous origin of steroids [17,18]. In both cases, urine is often selected as the most relevant matrix since it contains higher concentration levels of most steroids of interest compared to blood [19]. Moreover, urine is available in large quantities at any time from live animals due to its non invasive collection. Nevertheless, its preparation before analysis remains a critical step regarding its relatively high content of potential interferents [20]. While classical SPE protocols have been described in the past, more recently several innovative strategies to improve the purification of urinary steroids have been developed mainly based on immunoaffinity [21–23], microextraction by packed sorbent (MEPS) [24], stir bar sorptive extraction (SBSE) [25], and solid phase microextraction (SPME) [26,27]. Immunoaffinity turns to be relatively time-consuming while microextraction techniques, due to the reduced amount of samples used, leads to an insufficient steroids concentration for subsequent IRMS measurement. In this context, molecularly imprinted polymers (MIPs) may appear as a valuable alternative extraction tool due to their specificity and their high capacity. MIPs are synthetic polymers exhibiting specific cavities complementary in size, shape and position of the functional groups to target molecules or families of compounds. They result from the complexation of template molecules with functional monomers in an appropriate solvent, followed by template molecules removal. MIPs are frequently used as selective sorbents for the molecularly imprinted solid phase extraction (MISPE) of target analytes from complex matrices [28–31] due to their numerous advantages such as selectivity associated to their rapid, easy and cheap use as well as high thermal and chemical stability [29]. The first application was carried out by Sellergren in 1994 for the extraction of pentamidine in urine [32]. MIPs specifically designed for steroid extraction and subsequent analysis have already been developed and successfully applied on water [33–35], milk [36] and urine samples [37]. For the polymerization process, several functional monomers and initiators have been described, whereas the use of estradiol as template is by far the most cited in the literature [33,34,36,37]. The best results in terms of recovery and selectivity were obtained using methacrylic acid (MAA) as monomer, ethylene glycol dimethacrylate (EGDMA) as cross-linker and acetonitrile as solvent [38]. Compared to other classical procedures such as SPE or liquid–liquid extraction (LLE), MIPs finally present the advantages of being a reusable technique allowing a one-step procedure for an improved extraction, purification and concentration of the target compounds. Another sample preparation approach can be based on coupling semi-preparative chromatography with MIP as stationary phase. Indeed, semi-preparative chromatography allows a high purification of compounds in complex matrices. With liquid chromatography (LC), the main drawback is linked to the large volume of mobile phase needed. This constraint can be overcome by using supercritical fluid chromatography (SFC). SFC presents strong economical advantages due to the low percentage of co-solvent needed [39] and several advantages linked to the state of supercritical fluids which exhibit density and dissolving capabilities similar to those of certain liquids, as well as lower viscosities and better diffusion properties [40]. Moreover, and according to literature data, retention rules in SFC mainly depend on the nature of the stationary phase. Indeed, the interactions between compounds and stationary phase are improved in SFC compared to LC. Considering the properties of both techniques, coupling semi-preparative SFC with MIP appeared as an interesting strategy to improve MIPs specificity and therefore selective isolation of steroids. Recently, applications using MIP as stationary phase in chromatographic separation techniques have been reported in literature [41–45]. Among these studies, only a limited number reported the use of MIP in chromatographic technique for complex matrices [44,45].

Semi-preparative applications based on MIP have never been described in literature and to the best of our knowledge no studies have ever focused on steroids. The aim of the present work was to assess the potential of MISPE and SFC–MIP approaches to purify urinary steroid hormones in order to propose efficient, cheap and multiresidue sample preparation procedures. Both approaches were optimized using the main metabolites of testosterone, estradiol, nandrolone and boldenone in bovine urine which are considered as potential anabolic steroids used in cattle breeding. A one step MISPE protocol followed by gas chromatography–mass spectrometry (GC–MS) analysis was developed for screening purposes while the SFC–MIP strategy was assessed as a highly selective purification strategy prior to IRMS analysis 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-en17␣-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)-triene3,17␣-diol (␣-E2) and 5␣-estran-3␤,17␣-diol (E-aba) were purchased 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 androst4-en-17␣-ol-3-one d3 (epiT-d3 ) were purchased from NARL (Pymble, Australia). Each steroid stock solution was prepared at 1 mg mL−1 by dilution in an appropriate volume of ethanol. The working standard solutions were prepared by diluting stock solutions in ethanol and were stored at −20 ◦ C. Derivatisation reagents pyridine and acetic anhydride were purchased from Aldrich (Steinheim, Germany). ␤-Glucuronidase from Escherichia coli was obtained from Roche Diagnostics GmbH (Mannheim, Germany). Ethanol, methanol, ethyl acetate, cyclohexane, acetonitrile, n-pentane, n-hexane, petrolether and reagents were of analytical-grade quality and purchased from Carlo-Erba Reagents (Rodano, Italy). Ultra pure water (UP water) was obtained with a Nanopure system from Barnstead (Dubuque, IA, USA). The solid phase extraction (SPE) column (C18 : 2000 mg/15 mL) was acquired from UCT (Bristol, PA, USA). For MIP synthesis, methacrylic acid (MAA) and ethylene glycol dimethacrylate (EGDMA) were purchased 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 recognition (product code: AFFINIMIP Estrogens) was provided by Polyintell (Val de Reuil, France) packed as stationary phase into a chromatographic column (250 mm × 4.6 mm, 12–25 ␮m). 2.2. MIPs synthesis EGDMA was washed twice with an equal volume of a solution of 10% NaOH in UP water, and then washed twice with an equal volume of UP water. It was then dried using an equal volume of saturated sodium chloride aqueous solution and next over Na2 SO4 . AIBN was of a high purity and was therefore used without further purification. Washed EGDMA and MAA were distilled

M. Doué et al. / J. Chromatogr. A 1270 (2012) 51–61

under vacuum in order to remove inhibitors and stored at −20 ◦ C. For final MIP synthesis, a template/monomer/cross-linker molar ratio of 1/8/40 was used. The template (E2, 0.25 mmol) and the monomer (MAA, 2 mmol) were mixed with acetonitrile and left for 30 min in an ice bath. Then, the cross-linker (EGDMA, 10 mmol) and the initiator (AIBN, 400 ␮L) were added. The solution was stirred, transferred into a glass tube and then degassed under nitrogen for 15 min. The tube was sealed and transferred into a thermostated water bath (65 ◦ C) during 24 h for thermal polymerization. After polymerization, the tube was crushed and the polymer was then grounded with a ball mill and manually sieved. The particle size fraction of 25–36 ␮m was collected and slurried in MeOH/water (80:20, v/v) and then dried. Non-imprinted polymer (NIP) was obtained by performing the same procedure in the absence of the template molecules in the polymerization mixture. Two different rounds of synthesis of MIP/NIP were carried out at different times to evaluate the repeatability of the polymerization. Different amounts of synthesized MIP and NIP were packed between two frits (1/16 , 20 ␮m, Interchim, Montluc¸on, France) into 3 mL empty propylene disposable cartridges (Interchim). In order to eliminate the remaining reagents from the packed polymers, particularly the template molecules in the case of the MIP, a washing step was performed with 20 mL of MeOH. Finally they were conditioned with acetonitrile and kept at 4 ◦ C.

2.3. Animal experiments After an acclimation period, one heifer received 17␤-estradiol (30 mg in sesame oil) once by intramuscular injection (treatment E2), one calve received 17␤-estradiol benzoate (25 mg) and 17␤nandrolone laureate once by intramuscular injection (150 mg) (treatment E2/NT), one calf received boldione (200 mg) once by oral route (treatment B), another heifer received androstenedione (250 mg) once by oral route (treatment AED) and another heifer received testosterone (100 mg) once by oral route (treatment T). Animal experiments (E2, B, AED and T) were conducted in agreement with the animal welfare rules currently in force at Oniris while the E2/NT experiment was conducted within the Department of Veterinary Animal Health of the Faculty of Veterinary Medicine of the Utrecht University (The Netherlands) and approved by the ethical committee from Utrecht University. Urine samples were collected at day one (AED, T, and E2) and day three after treatment (B and E2/NT) and stored at −20 ◦ C.

2.4. Sample pre-treatment Five milliliters of urine were thawed at room temperature and submitted to an enzymatic deconjugation step using ␤glucuronidase from E. Coli at 37 ◦ C overnight, as described by Buisson et al. [17]. Samples were then centrifuged at 1200× g (5 ◦ C) for at least 10 min. The purification was performed directly on the resulting supernatant for the MISPE method whereas for the SFC–MIP method, two additional steps were necessary to prevent column overload. In a first step, the supernatant was applied onto a C18 SPE column (2000 mg) previously conditioned with 10 mL MeOH and 10 mL UP water. Steroids were purified by washing with 10 mL UP water and 10 mL n-hexane and eluted with 5 mL MeOH/ethyl acetate (30:70, v/v). The eluted fraction was evaporated to dryness under a gentle stream of nitrogen at 45 ◦ C and dissolved in 2 mL of acetate buffer (pH 5.2). A LLE was then performed twice with 5 mL n-pentane. The organic layer containing the target steroids was kept in a glass tube, evaporated to dryness under nitrogen, then reconstituted in MeOH (50 ␮L) and kept at 4 ◦ C before injection in semi-preparative SFC.

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2.5. MISPE procedure For quantification, urine samples were fortified with epiTd3 , E2-d3 and boldenone-d3 at a level of 100 ng mL−1 . The MISPE sorbent was first conditioned with 5 mL acetonitrile and 5 mL UP water. Fortified urine samples (pre-treated as previously described in Section 2.4) were applied and then washed with 5 mL UP water/acetonitrile (90:10, v/v) and 5 mL UP water/acetonitrile (80:20, v/v). Target steroids were eluted with 3 mL UP water/acetonitrile (65:35, v/v). Finally, the sorbent was rinsed successively with 5 mL MeOH and 5 mL acetonitrile to avoid any carry-over phenomenon and ensure the conditioning of the polymer to its original shape. The extracts were evaporated under nitrogen, the external standard (5␤-androstan-3,17-dione) was added (10 ng ␮L−1 ) and acetylation of steroids with 30 ␮L of pyridine and 30 ␮L of acetic anhydride was performed at room temperature during 16 h. Finally, the derivatisation reagents were evaporated to dryness under a nitrogen stream and the residue was dissolved in 50 ␮L of cyclohexane. 2.6. SFC–MIP procedure An Investigator Thar SFC system (Waters, Milford, MA, USA) coupled to a photodiode array detector (PDA) was used to perform the separation and collection of the fractions of interest. The temperature of the column, outlet pressure and flow rate were respectively set at 40 ◦ C, 15 MPa, and 3 mL min−1 . All purified extracts were injected in partial injection mode (50 ␮L). A mixture of acetonitrile/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 gradient until 40:60 at 1% min−1 (5 min). Two fractions were collected as follows: one fraction containing androgen steroids, 17␣-nandrolone and E-aba (FA ) between 15 and 23 min and another fraction containing ␣-E2 (FE ) between 31 and 40 min. Collected fractions and corresponding time windows were determined by injection of a mixture of target steroids (5 ␮g of each steroid) and by visualization of the corresponding PDA chromatograms acquired between 190 and 400 nm. Collected fractions were evaporated under nitrogen, external standard was added in each fraction and steroids acetylation 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 the fractions purity obtained after the MISPE or SFC–MIP steps were achieved by GC–MS. An Agilent 6890 series gas chromatography coupled to an Agilent 5973N single quadrupole mass analyzer (Agilent Scientific, USA) was used. Chromatographic separation was achieved using an Optima-17MS column (30 m × 0.25 mm i.d, df : 0.25 ␮m) (Macherey-Nagel, Duren, Germany). Helium was 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 containing glass wool (2 ␮L injected), operating in the pulsed splitless mode (1.5 min). Inlet temperature was fixed at 250 ◦ C. An oven ramp was used to optimize steroid separation. The oven was configured as follows: 1.5 min at 60 ◦ C (1.5 min), 20 ◦ C min−1 to 220 ◦ 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 and source were heated at 320 ◦ C and 230 ◦ C, respectively. The electron voltage was set at 70 eV. Mass acquisition was performed in full scan 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 steroids led 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), 314 (androstenediol and ␣-E2), 316 (5-aba), 317 (E2-d3 ) and 330 (M4 and testosterone). Chromatograms were recorded and compounds were quantified using the ChemStation Software (Agilent Scientific, USA). 2.8. GC–C–IRMS GC–C–IRMS measurements were performed on a HP 6890 gas chromatograph coupled to an IsoPrime isotope ratio mass spectrometer via a GC–V Combustion interface (Elementar, Manchester, UK). In order to keep the chromatographic separation obtained in GC–MS, chromatographic conditions used in GC–MS and in GC–C–IRMS were identical. The analytes were introduced into a combustion furnace filled with copper oxide wires (Elemental Microanalysis Limited, UK) held at 850 ◦ C. The combustion gases were passed through a liquid nitrogen water removal trap maintained at −100 ◦ C; the remaining CO2 was introduced in an electron ionization source operating at 100 eV. Ions (m/z 44, 45, and 46) were separated on a magnet and detected by three Faraday collectors. The calibration of the reference gas was performed with a mixture of three acetylated steroids (DHEA acetate, testosterone acetate and 5-androstene-3␤,17␤-diol diacetate) whose ␦13 CVPDB values have been previously calibrated and described elsewhere [17,46]. 3. Results and discussion 3.1. MISPE procedure 3.1.1. Choice of the conditions of MIP synthesis The main objective of this work was to develop a MIP for the selective extraction of steroids from urine samples. A MIP was first synthesized using styrene, divinyl benzene and ␣-E2 as monomer, cross-linker and template respectively. A polar porogen, namely MeOH, was selected in order to favor hydrophobic interactions considering the subsequent use of MIP in aqueous media. With the resulting MIP, a strong retention of ␣-E2 was obtained but the selectivity was poor regarding the very similar recoveries obtained on the MIP and the corresponding non imprinted polymer (NIP) (data not shown). As described by Lordel et al. for MIPs synthesis with the objective of selective extraction of nitroaromatic explosives from water [47], a sol–gel approach consisting in the use of tetraethylorthosilicate (TEOS) as cross-linker and 3aminopropyltriethoxysilane (APTS) as monomer was then tested. APTS was selected for its ability to develop hydrophobic interactions with ␣-E2 in aqueous media during both the synthesis and the subsequent use of the MIP. However, a poor selectivity and also a low retention of ␣-E2 were obtained (results not shown). Therefore, conditions of synthesis similar to those described by Jiang et al. [38] were assessed. A template/MAA/EGDMA molar ratio of 1/8/40 instead 1/6/30 was selected in order to increase MIPs capacity. Due to the poor solubilization of ␣-E2 in acetonitrile, E2 was finally chosen as template. 3.1.2. Evaluation of the resulting MIP The resulting MIP performances were evaluated with 3 steroids, namely 2 androgens (DHEA and epiT) and 1 estrogen (␣-E2), considered as representative of the panel of steroids of interest. Water samples (500 ␮L) spiked at 2500 ng mL−1 with each analyte were applied on the MIP and on the NIP in parallel (50 mg of each sorbent). This was followed by a washing step with UP water/acetonitrile mixture and an elution step with MeOH. Results are shown in Table 1. Recoveries of 85%, 68% and 82% were obtained for ␣-E2, epiT and DHEA respectively on the MIP and 62%, 31% and 52% on the NIP, thus demonstrating a good selectivity of the MIP

with a good repeatability (RSD value < 4%, n = 4). Then, matrix influence was assessed by applying the same procedure to calf urine samples. As shown by the results presented in Table 1, the selectivity was maintained and the repeatability was still acceptable (RSD < 10%, n = 4). In order to improve the sensitivity of the method by increasing the enrichment factor, the sample size was increased from 500 ␮L to 5 mL which affected neither the recoveries, nor the selectivity of the procedure or the repeatability of the results (RSD < 10%, n = 4). Finally, a second MIP, named MIP , was prepared to test the repeatability of the synthesis, and the retention of the three analytes was studied with spiked water samples. Very similar results, reported in Table 1, were obtained compared to those of the first MIP (less than 15% of recoveries variation between MIP and MIP ). These results were promising, even if the number of synthesized MIPs and the evaluation of analytes retention have to be increased before proceeding with routine applications. Nevertheless, previous studies have reported repeatable synthesis using the same monomer, cross-linker and initiator (MAA, EGDMA and AIBN respectively) [38,48,49] which support the preliminary promising performances in terms of reproducibility of the extraction. To conclude, synthesized MIPs showed a good selectivity and retention capacity toward the 3 selected model compounds. 3.1.3. Optimization of MISPE procedure In order to improve the MISPE procedure and to extend its applicability to a larger range of steroids, several parameters such as pre-treatment or washing steps were tested and optimized on 12 steroids selected for their usefulness in revealing various situations of anabolic steroid abuse (Fig. 1) [11,14,50,51]. Polymer quantity is a key parameter to optimize, since an excessive quantity promotes the development of non-specific binding sites, whereas a limited one can lead to a low retention capability [52]. In order to guarantee a maximal recovery of the analytes, 100 mg of polymers were used thereafter despite a slight decrease in specificity up to 15% (data not shown). Preliminary extraction step such as SPE is recommended by many authors for biological (aqueous) samples in order to transfer the target analytes in a solvent close to the porogen resulting into an optimal selectivity and an increase of the factor enrichment. Thus, urinary samples were applied onto C18 cartridges as described in Section 2.4 and the elution fraction was dissolved in acetonitrile. Unfortunately, when this extract was percolated on MISPE, steroids of interest were not retained by the polymer due to the elution strength of acetonitrile. Therefore, urinary extracts were directly applied into MISPE without preliminary treatment. In the case of direct percolation of the aqueous sample, analytes retention is mainly due to non-specific hydrophobic or electrostatic interactions with the polymeric phase. Selective retention mechanism resulting from the presence of cavities can be reached by using an appropriate washing solvent [53]. A combination of acetonitrile and water was investigated through elution profiles of target steroids realized after percolation of 5 mL of fortified urine with the 12 selected steroids (100 ng mL−1 ) and consecutive washing steps with 3 mL UP water/acetonitrile (from 100:0 to 0:100, v/v by step of 5%). 17␣-nandrolone was the first eluted compound with 25% of acetonitrile and all target compounds were eluted with 35% of acetonitrile. Therefore, 3 mL UP water/acetonitrile (80:20, v/v) can be 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 the same fraction. These results show that target analytes were eluted with a low percentage of acetonitrile. The presence of water seems to 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 compare the interest of a non-polar (mixture of hexane/petrolether), an acidic (hydrochloric acid 1 N or acetic acid 1 N/acetonitrile

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Fig. 1. Chemical structures of the 12 target endogenous steroids.

(80:20, v:v)), a neutral (UP water/acetonitrile (80:20, v:v)) or a basic (sodium hydroxide 1 N/acetonitrile (80:20, v:v)) washing step. As an example, the results obtained for epiT are shown in Table 2 except for the basic washing step where derivatisation prior to injection in GC–MS could not be achieved and therefore recoveries for all steroids could not be measured. Results are expressed in ng ␮L−1 rather than in percentage since the urine samples used were from pregnant cows and therefore already contained some of the target steroids such as ␣-E2 and epiT. It should be noted that, whatever the conditions and parameters tested, standard deviation never exceeded 15%. For the hexane/petrolether washing step, target steroids were not eluted by such a mixture and no significant improvement of selectivity and purification was observed according to MIP/NIP comparison and GC–MS chromatograms of

the eluted fraction (data not shown). Thus, a non-polar washing step failed to increase specific recognition. For the acidic washing step, the use of hydrochloric acid led to a decrease of the target analyte recoveries and MIP selectivity whereas the use of acetic acid led to elution of the analytes. The role of electrostatic interactions in the molecular recognition process seems to play a major part and prevents the use of an acidic washing step [28]. The best results in terms of specificity were obtained with the UP water/acetonitrile mixture 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 was realized. Three different protocols were applied on the same fortified urine sample: (i) extraction with SPE C18 followed by LLE as

Table 1 Recoveries and RSD values (n = 4) obtained after the percolation of water and calf urine spiked at 2500 ng mL−1 with each compound on MIP and on NIP or on the MIP /NIP (50 mg of sorbent). Percolation of 500 ␮L or 5 mL of samples, washing with a UP water/acetonitrile mixture, elution with 1 mL MeOH. Water (500 ␮L)

␣-E2 epiT DHEA

Calf urine (500 ␮L)

Water (500 ␮L)

Calf urine (5 mL)

MIP

NIP

MIP

NIP

MIP

NIP

MIP

NIP

85 ± 4 68 ± 2 82 ± 3

62 ± 5 31 ± 2 52 ± 3

83 ± 6 61 ± 10 68 ± 8

55 ± 13 24 ± 8 31 ± 12

86 ± 10 67 ± 7 74 ± 9

65 ± 8 29 ± 8 44 ± 8

89 ± 4 59 ± 8 73 ± 9

74 ± 6 38 ± 9 45 ± 5

Table 2 Recoveries expressed as results in ng ␮L−1 obtained for epiT from different MISPE washing protocols after percolation of 5 mL of urine spiked with 100 ng ␮L−1 of each steroid. Washing step: 3 mL UP water/acetonitrile (80:20, v/v), or 3 mL hexane/petrolether several times (from 0:100 to 100:0, v/v by step of 20%), or 3 mL hydrochloric acid 1 N/acetonitrile (80:20, v/v) and or 3 mL acetic acid 1 N/acetonitrile (80:20, v/v). Elution: 3 mL UP water/acetonitrile (65:35, v/v). Standard deviation values for all experiments were less than 15% of variation (n = 4). EpiT (ng ␮L−1 )

Washing Elution

UP water/acetonitrile

Hexane/petrolether

HCl/acetonitrile

MIP

NIP

MIP

NIP

MIP

NIP

Acetic acid/acetonitrile MIP

NIP

36.8 145.9

8.0 102.4

0 137.8

0 96.3

0 128.4

0 150.2

84.7 4.3

72.7 8.2

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5e+07

9e+07 8.5e+07 8e+07 7.5e+07 7e+07 6.5e+07 6e+07 5.5e+07 5e+07 4.5e+07 4e+07 3.5e+07 3e+07 2.5e+07 2e+07 1.5e+07 1e+07 5000000

LLE

4.5e+07 4e+07 3.5e+07 3e+07 2.5e+07 2e+07 1.5e+07 1e+07 5000000

Time-->

14.00

16.00

18.00

20.00

22.00

24.00

26.00

28.00

30.00

32.00

SiOH

Time-->

14.00

16.00

18.00

28.00

30.00

32.00

20.00

22.00

24.00

26.00

28.00

30.00

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6e+07 5.5e+07

MISPE

5e+07 4.5e+07 4e+07 3.5e+07 3e+07 2.5e+07 2e+07 1.5e+07 1e+07 5000000 12.00 Time-->

14.00

16.00

18.00

20.00

22.00

24.00

26.00

Fig. 2. GC–MS chromatograms, total ion current (TIC) acquired in full scan mode: 2 ␮L injected of the eluted fraction from SPE C18 + LLE (LLE), SPE C18 + LLE + SPE SiOH (SiOH) and MISPE. Chromatograms were normalized according to the external standard response (indicated by arrows). Squares show the retention time windows of target steroids.

described in Section 2.4 (ii) same procedure with a third extraction on SPE SiOH (SiOH) as described by Buisson et al. [17] and (iii) developed MISPE protocol (MISPE). The GC–MS chromatograms of the three purified fractions, normalized according to the external standard response, are presented in Fig. 2. With respect to the GC–MS chromatograms, the baseline value was divided by a factor of ten between the LLE and the MISPE methods, whereas the SiOH chromatogram presented less interference but lower steroid recoveries (up to 70% of differences). Thus, MISPE method appeared as a good compromise between both approaches (LLE and SiOH) since recoveries for all compounds were estimated to be above 65% (external calibration) and external standard responses never exceeded ±20% of variation (measured during a sequence of 10 urine samples) showing a sufficient urinary purification for GC–MS single quadrupole analysis. 3.1.4. MISPE protocol applied on urinary steroids for screening purpose In the past few years, a lot of research work has been conducted to highlight biomarkers of steroids abuse [10,11,13–15]. For example, Dervilly-Pinel et al., have shown that some estranediol isomers can be used as biomarkers to indicate nandrolone abuse in cattle [11,14]. The unambiguous identification and quantification of these metabolites required an adapted sample preparation and the use of sensitive and specific analyzers such as GC–MS/MS [14]. Screening may also be based on thresholds in terms of basal “endogenous” concentration levels. Despite the high variabilities of steroid concentrations in urine [50,54], this approach is also considered as a method of choice due to its potential multiscreening application and the use of less specific instruments such as GC–MS. For boldenone abuse, the current European regulation recommends that epiboldenone levels exceeding 2 ␮g L−1 in calf urine have to be considered as suspicious [55]. For E2, testosterone

and nandrolone abuse, no official urinary metabolite thresholds have been published yet. The applicability of the developed MISPE method was assessed with urine samples from bovines treated with E2 (E2), boldione (B), testosterone (T) and E2 and 17␤-nandrolone (E2/NT) (sampling at day 1 and day 3 after injections). For the E2/NT treatment, the developed sample preparation allowed subsequent unambiguous identification of E-aba (previously reported as a biomarker of interest after such treatment) in urine 3 days after treatment, based on its retention time and respective mass spectrum. For E2, B and T experimental samples, the quantification of the main metabolites of the administrated compounds was carried out by external calibration with an isotopically labeled standard after GC–MS analysis of the purified extract. Good linearity was obtained between 1 and 30 ␮g L−1 and between 30 and 100 ␮g L−1 for each compound with coefficients of determination systematically above 0.99. Results are presented in Table 3 and the corresponding extracted ion chromatograms (EIC) of the urinary extract from E2 and T experiments are featured in Fig. 3. All the main metabolites of testosterone, E2, and boldenone could

Table 3 Mean concentrations and corresponding standard deviation (n = 4) of target steroids obtained with MISPE protocol and GC–MS analysis of urinary samples from treated bovines. Treatments

Days of collection after treatment

Steroids of interest

Mean concentration (standard deviations) (␮g L−1 )

T

D1

E2

D1

B

D3

epiT Testosterone ␣-E2 E2 Epiboldenone Boldenone

68.3 (4.2) 28.2 (1.4) 261.7 (18.7) 4.7 (0.5) 23.1 (0.8) 2.9 (0.3)

M. Doué et al. / J. Chromatogr. A 1270 (2012) 51–61

57

Fig. 3. Extracted ion chromatograms (EIC) m/z 314 (E2 and ␣-E2), 317 (E2-d3), 270 (epiT and testosterone), 273 (epiT-d3) of GC–MS chromatograms obtained by injections (2 ␮L) of the urinary extracts from E2 (day 1 after treatment) and T (day 1 after treatment) experiments obtained after MISPE protocol.

easily be detected and measured after MISPE sample preparation followed by GC–MS analysis. As expected, the epiboldenone level was found higher than the recommended urinary thresholds at day 3 after treatment (23.1 ␮g L−1 for a threshold of 2 ␮g L−1 ) and allowed to classify the sample as suspicious. As mentioned before, no thresholds have been published yet for testosterone or E2 abuse. Nevertheless, the developed method allowed the quantification of testosterone, epiT, E2 and ␣-E2 at urinary levels useful to monitor their concentration modifications after anabolic treatment [50,54]. These results showed the applicability of the developed sample preparation for screening purposes. Moreover, compared to other classical protocols involving several SPE and LLE steps [56–58], this one-step protocol allowed the analysis of several classes of steroids (androgens and estrogens) within only one analysis step. Thus, the MISPE method can be considered as cheap, efficient and multiresidue, since testosterone, E2, boldenone and nandrolone metabolites can be measured with this one step sample preparation procedure. 3.2. SFC–MIP procedure 3.2.1. Optimization of SFC–MIP procedure Since GC–C–IRMS measurement is very demanding in terms of peak purity, the sample preparation is a key step which has to be as efficient as possible. The previous MISPE developed method was not sufficient (presence of co-eluting interferents with target steroids). Indeed, with biological (aqueous) samples applied on MISPE, the presence of water seems to disturb specific recognition through electrostatic interactions. In order to solve this issue, the use of MIP as stationary phase in semi-preparative SFC was considered. A commercial MIP was chosen as stationary phase to guarantee

a homogenous particles size and filling of the chromatographic column. The choice of this commercial MIP specifically designed for E2 recognition was based on the fact that the same retention characteristics and chromatographic profiles of purified extracts were obtained compared to those of the synthesized MIP (data not shown). Unfortunately, the template was not communicated by the provider. The nature of the solvent applied on the MIP induces the nature of the interactions that take place during the recognition mechanism [29]. Thus, two different co-solvents, one protic and one aprotic i.e. MeOH and acetonitrile respectively were first assessed to optimize the chromatographic separation of the 12 target steroids. Gradients from 5 to 30% in 25 min with MeOH and from 5 to 40% in 35 min with acetonitrile were applied. UV chromatograms obtained from a mixture of target compounds are presented in Fig. 4. With MeOH, androgens were found to be less retained compared with acetonitrile (retention factor kA-MeOH = 13.8 and kA-acetonitrile = 23.8). ␣-E2 was eluted with 28% of MeOH in CO2(SC) while with acetonitrile this compound was found to be still retained on the stationary phase despite the highest percentage used (up to 40%). Moreover, chromatographic separation of mono-(MS) and dihydroxylated (DS) steroids could be achieved with acetonitrile. Thus, the use in SFC–MIP of acetonitrile seems to increase specific recognition. Indeed, the introduction of the porogen, i.e. acetonitrile, induced the return of the cavities to their original shape and size and therefore facilitated the specific recognition process. Nevertheless, in order to purify all target compounds, the addition of 5% of MeOH in acetonitrile enabled ␣-E2 to be eluted while preserving specificity (Fig. 4). Finally, fractions and corresponding time windows were determined thanks to the corresponding UV chromatograms.

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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 and acetonitrile/MeOH (95:5, v:v)). For MeOH, gradient from 5 (3 min) to 30% MeOH in CO2(SC) in 25 min, for acetonitrile and acetonitrile/MeOH, gradient from 5 (3 min) to 40% acetonitrile in CO2(SC) in 35 min. MS: monohydroxylated steroids, DS: dihydroxylated steroids and E: ␣-E2.

3.2.2. Isotopic fractionation and purity assessment Steroid 13 C/12 C isotopic ratio measurement by GC–C–IRMS is one of the methods of choice to determine the exogenous or endogenous origin of steroids for confirmatory purposes. The administration of synthetic steroids to bovines leads to a slight depletion in the 13 C/12 C ratios (expressed as ␦13 CVPDB values) of its respective metabolites while the 13 C/12 C ratios of its precursors remain unchanged [17,18]. Thus, precursors can be used as endogenous reference compounds (ERC) and a difference of ␦13 CVPDB values between ERC and metabolites proves administration of synthetic steroids. One of the major potential pitfalls in GC–C–IRMS analysis is related to the isotopic fractionation that can occur during sample preparation [59]. The absence of isotopic fractionation was therefore assessed for the developed SFC–MIP step. A mixture of target steroids was first injected in SFC–MIP, collected and then injected in GC–C–IRMS after derivatisation. Obtained ␦13 CVPDB values were compared with those of the same steroids directly analyzed in GC–C–IRMS after derivatisation. Results (␦13 CVPDB non-corrected values) are presented in Table 4. As expected, all ␦13 CVPDB values of target steroids were situated in the confidence interval (CI) (˛ = 0.01) of directly injected steroids (except for M2 which presented a slight lower value). These results were in accordance with those published by Buisson et al., who showed that isotopic fractionation can occur during chromatographic separation (differences in term of 13 C/12 C ratio from the start to the end of the peak) but can be avoided with an appropriate and complete collection of the analyte [17]. Another potential issue in GC–C–IRMS analysis is the peak purity of the target compounds. Chromatographic co-elution of interferents with steroids leads to an incorrect estimation of the isotopic composition. Thus, in order to assess the purification power of the different strategies, two different protocols namely

SFC–MIP with MeOH (SFC–MIP MeOH) and acetonitrile/MeOH (95:5) (SFC–MIP acetonitrile/MeOH) as co-solvent were applied on fortified pregnant cow urine samples. To prevent column overload, urinary samples were first applied on SPE C18 and then a LLE was performed as indicated in Section 2.4 before injection in SFC. Purified extracts were analyzed by GC–MS after derivatisation and their chromatograms, as well as the associated mass spectra and the absence of co-elution with target steroids, allowed us to assess their purity. GC–MS chromatograms of the fractions FE focusing on the expected retention time windows of endogenous steroids presented in Fig. 5 attest for their satisfactory purity whatever the SFC–MIP methods used. Comparable chromatographic profiles for

Table 4 Isotopic deviation (␦13 CVPDB ) values (non-corrected) of target acetylated steroids directly 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

E-aba Etiocholanolone DHEA Androstenediol Epiandrosterone 5-aba M4 ␣-Nandrolone M2 EpiT Testosterone ␣-E2

After SFC–MIP

Mean ␦13 CVPDB

Confidence interval ␦13 CVPDB

␦13 CVPDB

−37.22 −33.38 −37.87 −37.56 −36.54 −40.24 −35.13 −33.36 −33.63 −38.57 −36.16 −36.92

[−36.64 to −37.80] [−32.23 to −33.83] [−37.21 to −38.53] [−36.57 to −38.55] [−35.39 to −37.69] [−39.29 to −41.19] [−33.61 to −36.65] [−31.98 to −34.74] [−33.03 to −34.23] [−36.88 to −40.26] [−34.21 to −38.11] [−35.12 to −38.72]

−37.44 −33.74 −38.46 −37.51 −37.13 −39.81 −35.97 −34.51 −32.69 −37.03 −34.78 −37.52

M. Doué et al. / J. Chromatogr. A 1270 (2012) 51–61

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Fig. 5. GC–MS chromatograms, total ion current (TIC) focused on retention times of target steroids acquired in full scan mode: 2 ␮L injected of the collected fraction FE from SFC–MIP MeOH and SFC–MIP acetonitrile/MeOH methods. 1: external standard, 2: ␣-E2. Chromatograms were normalized according to the external standard response.

SFC–MIP MeOH and acetonitrile/MeOH methods were obtained for the fraction FA containing androgens, 17␣-nandrolone and E-aba and for FE containing ␣-E2. Nevertheless, according to the external standard response, steroid hormone recoveries were higher with the SFC–MIP MeOH/acetonitrile method compared to the SFC–MIP MeOH one. Thus, this method was then applied to the selective isolation of urinary steroids in the further scope of IRMS analysis. 3.2.3. SFC–MIP protocol applied on urinary steroids for confirmatory purposes Sample preparation with SFC–MIP acetonitrile/MeOH method was applied on urine samples from treated bovines with estradiol

(E2), boldione (B), androstenedione (AED) and testosterone (T). The sample extracts were then injected in GC–MS to confirm analyte identity, assess the peak purity, evaluate the concentration for eventual further dilution/concentration and therefore ensure that concentration estimation of target analytes is in the GC–C–IRMS linearity in the range of 15–70 ng of steroid on column. Sufficient purity of endogenous steroids was obtained for all urine samples. Urinary extracts were then injected in GC–C–IRMS. IRMS chromatograms for E2 experiment are presented in Fig. 6 and ␦13 CVPDB results in Table 5. Regarding GC–C–IRMS chromatograms, peak purity is sufficient to allow the measurement of isotopic deviation values. All metabolites present a difference in their ␦13 CVPDB

Fig. 6. GC–C–IRMS chromatograms (m/z 44) of fractions FA and FE from E2 (day 1 after treatment) experiment obtained after SFC–MIP sample preparation method. ERC: endogenous reference compound.

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Table 5 Isotopic deviation (␦13 CVPDB ) values of ERC and metabolites obtained with urinary samples from treated cows. Experiments/direct metabolites M

Days of collection after treatment

␦13 CVPDB values of ERC: DHEA (‰)

␦13 CVPDB values of M: (‰)

 (M-ERC) (‰)

T/epiT E2/␣-E2 AED/5-aba B/epiboldenone

D1 D1 D1 D3

−26.72 −23.96 −24.97 −22.75

−31.48 −29.80 −33.13 −29.16

−4.76 −5.84 −8.16 −6.41

values with an ERC greater than 3‰ (world anti doping agency compliant threshold value [60]) confirming the administration of the respective steroids under their 17␤ form. Moreover, for the first time, significant differences in 13 C/12 C ratio between metabolites compared to their respective ERC were demonstrated in cattle after administration of androstenedione and boldione. To conclude, SFC–MIP method not only showed its multiresidue sample preparation application (testosterone, estradiol, boldione, and androstenedione) but also its relevance to strongly purify urinary steroids in the further scope of an IRMS analysis. This protocol also allowed to reduce cost and length of sample preparation since three steps (1 LLE and 2 semi-preparative HPLC steps) could be removed from the current method [17]. Indeed, the SFC semi-preparative step is 90% less expensive than the previous HPLC one and the sample preparation time is reduced by 20%. 4. Conclusions The objective of this study was to evaluate the capabilities of a selective extraction procedure based on molecularly imprinted polymers in order to propose short, multiresidue and cheap sample preparation procedures. For screening purposes, a one step optimized MISPE protocol with in-house synthesized polymer was developed and applied on samples from treated animals. Recoveries (above 65% for the 12 target steroids) and purities were sufficient for GC-MS analysis and allowed quantification of some metabolites interesting to suspect steroid abuse. MIP used as stationary phase in SFC in order to improve specific recognition was also assessed. SFC–MIP procedure was found to be a robust and efficient approach to strongly purify target steroids, with similar results than the current method but with three steps less (sample preparation time was reduced by 20%). Its application on collected urines from treated animals allowed the confirmation of steroid administration. Finally, this sample preparation could be considered as multiresidue since confirmation of testosterone, estradiol, androstenedione and boldione abuse in producing animals can be obtained. Acknowledgment We gratefully thank Polyintell (Val de Reuil, France) and Waters (Milford, MA, USA) for providing this study with MIP in packed column for chromatography and Thar SFC Investigator device. We also acknowledge Flavia Hanganu for E2 and T animal experiments. For E2/NT animal experiment, the sample has been obtained from the 6th Framework Programme “Integrating and strengthening the European Research Area within the BioCop project “New technologies to Screen Multiple Chemical Contaminants in Foods”. Contract number: FOOD-CT-2004-06988. References [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 substances having a hormonal or thyrostatic action and on b-agonists and repealing Directives 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. Van de Wiele, J. Vercammen, K. De Wasch, Anal. Chim. Acta 473 (2002) 71.

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