Dummy-Template Molecularly Imprinted Solid Phase Extraction For

Food Chemistry 139 (2013) 24–30

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Food Chemistry

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Analytical Methods Dummy-template molecularly imprinted solid phase extraction for selective analysis of ractopamine in pork ⇑ Wei Du a, Qiang Fu a, , Gang Zhao a, Ping Huang b, Yuanyuan Jiao a, Hao Wu a, Zhimin Luo a, Chun Chang a a School of Medicine, Xi’an Jiaotong University, Xi’an 710061, PR China b Xi’an Institute for Food and Drug Control, Xi’an 710061, PR China article info abstract

Article history: Molecularly imprinted polymers (MIPs) for selective adsorption of ractopamine hydrochloride (RAC) Received 2 July 2012 were synthesised by an in situ method, in which salbutamol (SAL) was used as the dummy-template Received in revised form 20 December 2012 to avoid the template leakage. Scanning electron microscopy (SEM), mercury porosimerty and Fourier Accepted 28 January 2013 transform infrared spectroscopy (FTIR) were used to investigate the physical and morphological Available online 10 February 2013 characteristics of the dummy-template MIPs. The test of adsorption selectivity indicated that the dummy-template MIPs exhibited high selectivity to RAC. The saturated adsorption capacity for RAC on Keywords: dummy-template MIPs was 90.9 lgg1. Based on the dummy-template polymers, a liquid chromatogra- Ractopamine phy–mass spectrometry (LC–MS) method was developed for the selective analysis of RAC in real pork Dummy-template Molecularly imprinting polymers samples. The averages of intra- and inter-day accuracy ranged from 78.9% to 92.2% and from 90.7% to In situ polymerisation 93.1%, respectively. The RSD% of repeatability ranged from 1.9% to 6.3%, and the RSD% of intermediate 1 Solid phase extraction precision ranged from 3.5% to 9.2%, while the limit of detection (LOD) was 0.02 lgkg . Liquid chromatography- mass spectrometry Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved.

1. Introduction plasmon resonance-based biosensor inhibition immunoassays (Lu et al., 2012). However, these methods usually require sample pre-

Ractopamine (RAC) is a synthetic phenethanolamine b2-adreno- treatment processes, such as solid-phase extraction (SPE) (Dong ceptor agonist, which can be used as clinical medicine for the treat- et al., 2011; Qu et al., 2011; Shao et al., 2009; Wang et al., 2010), ment of asthma. It, however, can be illegally used as a growth and the routine SPE suffered from the disadvantage of low selectiv- promoter for meat-producing animals with high dosage. Therefore, ity and poor recovery. RAC is strictly banned as a feed additive in many countries Recently, molecularly imprinted polymers (MIPs) have at- (Commission of the European Communities, 1996; The Ministry tracted much attention in different areas, attributed to their high of Agriculture, 2002) due to the potential risk to human beings affinity and pre-determined selectivity for target analytes and who consume products made from RAC-treated animals (Brambilla other structural analogues (Tamayo, Turiel, & Martin-esteban, et al., 2000; Smith, Ehrenfried, Dalidowicz, & Turberg, 2002; Xiao, 2007). Molecularly imprinted solid-phase extraction (MISPE), as a Xu, & Chen, 1999). Nevertheless, the use of RAC remains attractive relatively new concept in the clean-up of biological samples, has to swine producers because it can improve feed efficiency. This proved to be an efficient and selective approach for purification makes it essential to establish sensitive and selective analytical and pre-concentration of RAC from complex matrices (Hu, Li, Liu, methods to monitor the residual RAC in food samples. Tan, & Li, 2011; Tang, Fang, Wang, & Li, 2011; Wang, Liu, Fang, Several analytical methods have been developed for the deter- Zhang, & He, 2009; Widstrand et al., 2004). However, the drawback mination of RAC in animal tissues, urine and feed, such as high- of the MIPs is the unavoidable template leaking, which may influ- performance liquid chromatography (HPLC) (Shelver & Smith, ence the accuracy of identification and quantitation of the analytes 2003), gas chromatography–mass spectrometry (He, Su, Zeng, (Tamayo et al., 2007). A strategy to avoid template leaking is the Liu, & Huang, 2007; Wang, Li, & Zhang, 2006), liquid chromatogra- utilisation of a dummy template, structural analogue of target ana- phy–mass spectrometry (LC–MS) (Antignac, Marchand, Le, & lyte itself, during the preparation of MIPs (Yin et al., 2012). To our Andre, 2002; Blanca et al., 2005; Dong et al., 2011; Kootstra best knowledge, there is still no developed method concerning the et al., 2005), ultra-performance liquid chromatography–tandem template leaking of MISPE during the analysis of RAC residue. mass spectrometry (Shao et al., 2009; Zheng et al., 2010), capillary In this study, the MISPE for selective analysis of RAC in pork was electrophoresis (Wang, Zhang, Wang, Shi, & Ye, 2010), and surface prepared by an in situ method, using the analogue, salbutamol (SAL), as the dummy-template. The chemical structures of RAC ⇑ Corresponding author. Tel.: +86 29 82655382. and SAL are shown in Fig. 1. The adsorption characteristics of the E-mail address: [email protected] (Q. Fu). obtained dummy-template MIPs were investigated. The adsorption

225 nm, 246 nm, 223 nm and 256 nm, respectively. The injection volume was 5 ll, and the column temperature was maintained at 25 °C by an Automatic AT-330 column heater (Tianjin, China). LC–MS equipment consisted of a Shimadzu mass spectrometry system (Kyoto, Japan), which included three LC-20AD pumps, a DGU-20A3 degasser, a SIL-20A autosampler, a CTO-20A column oven, a SPD-20A UV/VIS detector, a SPD-M20A diode array detector, a LCMS2010EV mass spectrometer, and a LCMS solution work- station. The analysis was performed in the positive electrospray ionisation mode (ESI) at m/z of 302. The column was a VP-ODS col- Fig. 1. Chemical structures: (a) ractopamine and (b) salbutamol. umn (150 2.0 mm I.D., 5 lm), and the mobile phase was acetonitrile–0.2% formic acid solution (12:88, v/v) at a flow rate of 0.2 ml min1 with a column temperature of 37 °C. MS conditions isotherms were modelled using Langmuir and Freundlich models. were as follows: nebulizer gas (N2, purity > 99.999%), flow rate of 1 The adsorption rate was determined based on Lagergren’s pseudo 1.5 l min , drying gas (N2, purity > 99.999%), pressure of 0.1 first and second order kinetic equations. The application of the MPa, interface temperature of 300 °C, heat block temperature of dummy-template MISPE, coupled with LC–MS method, was devel- 220 °C, and detector voltage of 1.25 kV. The injected volume was oped for selective analysis of RAC in pork. 10 ll.

2.3. Preparation of dummy-template MISPE 2. Materials and methods The dummy-template MISPE was prepared by an in situ poly- 2.1. Reagents and solutions merisation in the SPE cartridge according to the method reported previously (Fu et al., 2011). Briefly, the dummy-template (SAL), tol- RAC was purchased from Sigma–Aldrich (New Jersey, USA). SAL uene, MAA, EDMA, dodecanol, and AIBN were sequentially added was purchased from Cunyi Chemical Co. (Jiangsu, China). Clenbu- to a 10 ml test tube. The mixture was thoroughly mixed before terol hydrochloride was obtained from Jinhe Pharmaceutical Co. use and then degassed for 15 min. After purging with a nitrogen (Wuhan, China). Terbutaline sulphate was purchased from Gang- stream for 15 min, the pre-polymerising solution was transferred zheng Pharmaceutical Co. (Wuhan, China). Adrenaline hydrochlo- into a 10 ml empty SPE cartridge. The cartridge was then sealed ride was obtained from Hefeng Pharmaceutical Co. (Shanghai, and set up vertically. The polymerisation was allowed to proceed China). Methacrylic acid (MAA) was purchased from Tianjin at 50 °C for 20 h. The obtained MISPE cartridge was washed by a Chemical Reagent Plant (Tianjin, China). 4-Vinylpyridine (4-VPY), mixture of methanol–acetic acid (90:10, v/v) with a flow rate of 2-vinylpyridine (2-VPY) and trifluoromethacrylic acid (TFMAA) 0.5 ml min1 to remove the template molecule and residual poro- were obtained from Sigma–Aldrich (New Jersey, USA). MAA, 4- genic solvents. Finally, the MISPE cartridge was washed with meth- VPY and 2-VPY were distilled under vacuum to remove inhibitors anol at a flow rate of 0.5 ml min1 to remove the residual acetic prior to use. Ethylene glycol dimethacrylate (EDMA) was obtained acid. A similar procedure, without dummy-template, was used to 0 from Sigma–Aldrich (New Jersey, USA). 2,2 -Azobisisobutyronitrile prepare the non-imprinted solid-phase extraction (NISPE) (AIBN) was purchased from Shanghai No. 4 Reagent Factory cartridge. (Shanghai, China) and recrystallised in methanol before use. Meth- anol and acetonitrile were of HPLC grade, purchased from Kemite 2.4. Physical and morphological characterisation Co. (Tianjin, China). Water was purified with Molement 1805b (Shanghai, China). All other chemicals were of analytical grade The morphologies of the dummy-template MIPs and NIPs were and obtained from local suppliers. Empty SPE cartridges (10 ml) observed by a JSM-6390A Scanning Microscope (Jeol, Japan). The were purchased from Shenzhen Doudian Co. (Shenzhen, China). porosity, total pore volume, and average pore diameter were mea- Blank pork sample was supplied by a local farmer. Real pork sured by mercury porosimerty with an Auto Pore IV 9510 porosi- samples were obtained from local markets and stored at 20 °C meter (Micromeritics, USA). Fourier transform infrared spectra prior to use. (FTIR) were recorded on an FTIR-8400S spectrometer (Shimadzu, Standard stock solutions of RAC, clenbuterol, terbutaline and Japan) with a scanning range from 400 to 4000 cm1. adrenaline were prepared separately in water at the concentration of 500 gml1. Working solutions of RAC (0.5–500 gml1) l l 2.5. Selectively test were prepared by independently diluting stock solutions with acetonitrile. The adsorption selectivities of the dummy-template MIPs and NIPs were analysed in a stainless-steel column (100 mm 4.6 mm, 2.2. Instrument and analytical conditions id.). The retention factor (K) was determined by the following formula: The adsorption selectivity of the dummy-template MIPs was t t analysed by HPLC. The dummy-template MIPs and non-imprinted K ¼ R o ð1Þ t polymers (NIPs) were directly synthesised in a stainless-steel o column (100 mm 4.6 mm, id.) according to the procedures in where tR is the retention time of a solute and t0 is void time of the Section 2.4, respectively. Then this MIPs or NIPs column was column (measured by injecting acetone). The selectivity factor (S) directly attached to an HPLC pump. The HPLC analysis was per- was expressed by the following formula: formed with a Shimadzu HPLC system, equipped with an LC-20A K pump, an SPD-20A UV detector and CS-Light Real Time Analysis S ¼ MlPs ð2Þ K Chromatographic Software. The mobile phase was acetonitrile– NlPs phosphate buffer (20 mM, pH 5.0) (60:40, v/v). The detection wave- where kMIPs and kNIPs are the retention factors of a compound on length for RAC, clenbuterol, terbutaline and adrenaline were MIPs and NIPs, respectively. 26 W. Du et al. / Food Chemistry 139 (2013) 24–30

2.6. Adsorption test eluents were evaporated to dryness under a nitrogen stream and the residues were dissolved in 500 ll of acetonitrile for LC–MS 2.6.1. Adsorption isotherm analysis. To investigate the adsorption ability of dummy-template MIPs for RAC, 50 mg of the dummy-template MIPs particles were added 2.8. Sample preparation to 10 ml of RAC working solution at different concentrations 1 (5–500 lgml ). The suspensions were placed on a SHZ-82 Two grams, of real pork sample were accurately weighed into a Vapour-bathing Constant Temperature shaker (Jintan, China) for 10 ml glass beaker. After adding 4 ml of acetonitrile–water (80:20, 300 min at 25 °C and then centrifuged at 8000 rpm for 15 min. v/v), the sample was vortex-mixed for 2 min and centrifuged for The supernatant was measured for free RAC by HPLC. A similar pro- 10 min. Subsequently, 3 ml of supernatant were loaded onto the cedure was performed for NIPs particles. The adsorption amount MISPE cartridge. 1 (Qe, lgg ) was calculated by the following formula:

ðCo CeÞv 2.9. Method validation and real sample analysis Q ¼ ð3Þ e m The method validation was performed for specificity, linearity, where C (lgml1) is the initial concentration of RAC, C (lgml1) 0 e range, limit of detection (LOD), limit of quantification (LOQ), accu- is equilibrium concentration of RAC in solution, V (ml) is sample racy and precision, following the recommendations of the Interna- volume and m (mg) is the mass of the polymer. tional Conference on Harmonization Q2(R1) (ICH-International The adsorption isotherms were described by the Langmuir Conference on Harmonisation of Technical Requirements for Reg- equation (Eq. (4)) and Freundlich equation (Eq. (5)) (Singh & Mish- istration of Pharmaceuticals for Human Use, 2005). The calibration ra, 2010). The linearised forms of the two isotherms are: was established by measuring different concentrations (0.05– 1 Ce 1 Ce 100 lgkg ) of RAC in spiked pork samples after the MISPE proce- ¼ þ ð4Þ Q e qmKL qm dure. In order to avoid undue bias, the calibration curve was split into two ranges: 0.05–2 lgkg1 and 2–100 lgkg1. Least squares 1 linear regression analysis was used to determine the slope, inter- ln Q ¼ ln C þ ln K ð5Þ e n e F cept and correlation coefficient. The LOD and LOQ were calculated 1 1 from injection of the spiked sample, providing signal to noise ra- where Ce (lgml ) and Qe (lgg ) are the equilibrium concentra- tios of 3 and 10, respectively. The accuracy was expressed as a per- tion and the amount of RAC adsorbed at equilibrium, respectively, 1 1 centage of recovery. The precision was tested by studying the and qm (lgg ) and KL (l g ) are theoretical maximum adsorption repeatability and intermediate precision. The precision was ex- capacity and Langmuir equilibrium constant, respectively. KF and n pressed by relative standard deviation (R.S.D%), with acceptable are the Freundlich constants, which are indicators of adsorption values for the RSD% being less than 15% (Bressolle, Bromet-Petit, capacity and adsorption intensity. According to the Freundlich the- & Audran, 1996). ory, n can be used to determine whether the adsorption is favour- In order to evaluate the stability of the MISPE cartridge, the able. When n > 1, it is favourable adsorption; when n =1, it is same MISPE cartridge was reused 12 times for the measurement linear adsorption; when n < 1, it is unfavourable adsorption. of RAC in a spiked pork sample. Twenty real pork samples, obtained from local markets, were analysed by MISPE, coupled with 2.6.2. Adsorption kinetics LC–MS. The uptake kinetic study was performed with 150 lgml1 RAC standard solutions and 50 mg of MIPs for different periods of time (10–300 min). The mixture was shaken at 25 °C and the adsorption 3. Results and discussion capacity was determined by HPLC. The Lagergren’s pseudo first order (Eq. (6)) and pseudo second order (Eq. (7))(Singh & Mishra, 3.1. Preparation conditions of dummy-template MIPs 2010) models were used to describe the adsorption kinetic mechanism of dummy-template MIPs. Both the first and second order In the dummy-template MIPs preparation, commonly used rate equations were commonly employed in parallel, and one acidic monomers, MAA and TFMAA, and basic monomers 2-VPY was often claimed to be better than another according to a mar- and 4-VPY, were tested for the validity. It was observed that, with ginal difference in correlation coefficient. 2-VPY, 4-VPY and TFMAA, the pre-polymerisation mixtures were difficult to polymerise successfully. Therefore, MAA was employed k1t logðQ e Q tÞ log Q e ð6Þ as the functional monomer in this study, which was the same as 2:303 published elsewhere (Fu et al., 2011). Several factors affecting t 1 t the adsorption properties of the MIPs were optimised, including ¼ 2 þ ð7Þ the mole ratio of the template (SAL) to functional monomer Q t k Q Q e 2 e (MAA), the content of cross-linker (EDMA) and different porogenic 1 1 solvents. As shown in Table 1, the MIPs showed the highest selec- where Qe (lgg ) and Qt (lgg ) are the adsorption amount of RAC 10 1 at equilibrium and at time t (min), respectively; k1 (min ) and k2 tivity factor. Hence, its preparation conditions were selected to be (g lg1 min1) are the pseudo first order and pseudo second order the optimum ones. adsorption rate constants, respectively. 3.2. Physical and morphological observation 2.7. MISPE procedure SEM images of the dummy-template MIPs and NIPs are shown The MISPE cartridge was first washed with 2 ml of water and in Fig. 2A. The dummy-template MIPs and NIPs showed apprecia- semi-dried at a low positive pressure of 0.05 MPa. Then 3 ml of ble differences in morphology. The NIPs possessed crosslinked the pre-treated sample was loaded onto the MISPE cartridge. The microglobules which yielded to large clusters, whereas the dum- cartridge was washed with 5 ml of acetonitrile–water (50:50, v/ my-template MIPs exhibited more rough and porous structures v), and eluted with 8 ml of methanol–acetic acid (90:10, v/v). The than did NIPs, indicating that the presence of recognition sites in W. Du et al. / Food Chemistry 139 (2013) 24–30 27

Table 1 respectively. The porosity of the dummy-template MIPs (60.0%) Optimisation for preparation and separation performance of dummy-template MIPs was higher than that of NIPs (50.3%), which was of beneﬁt to the and NIPs. adsorption of analytes from complex matrices. Besides, the average

Polymers Molar ratio Content of Toluene in kMIPs kNIPs S pore diameter of the dummy-template MIPs (134.0 nm) was smal- a b of SAL/MAA cross linker porogen ler than that of NIPs (283.3 nm). The results were consistent with (v%) (v%) the description of the SEM images. MIPs1 1:2 85 18 0.5 / / FTIR spectra of dummy-template (SAL), NIPs, MIPs after and be- MIPs 1:3 85 18 1.9 0.7 2.6 2 fore removal of SAL, are shown in Fig. 2B. For SAL, the bands at MIPs3 1:4 85 18 8.7 1.9 4.6 3409 cm1, 3193 cm1, and 1234 cm1 were the characteristic MIPs4 1:5 85 18 1.3 0.6 2.2 A A A 1 MIPs5 1:6 85 18 4.5 1.4 3.2 vibrations of OH, N H and C N. The bands at 2970.17 cm 1 MIPs6 1:4 80 18 7.0 1.9 3.6 and 1612 cm were attributed to the CAH antisymmetic stretch- MIPs7 1:4 83 18 7.1 1.6 4.4 ing vibration and C@C stretching vibration in benzene, respec- MIPs 1:4 87 18 5.7 1.1 5.2 8 tively. For NIPs and MIPs after removal of SAL, the FTIR spectra MIPs9 1:4 90 18 4.9 – –

MIPs10 1:4 85 15 14.8 2.5 5.9 almost had the same characteristic bands. The bands at 1 1 1 1 MIPs11 1:4 85 20 8.4 2.3 3.6 3448 cm , 2950 cm , 1720 cm and 1157 cm were attributed A A @ A A HPLC conditions: mobile phase, acetonitrile – phosphate buffer (20 mM, pH 5.0) to the stretching vibrations of OH, CH3,C O and C O C for (60:40, v/v); flow rate, 1.0 ml min1; column temperature, 25 °C. MAA and EDMA, respectively, indicating that the NIPs and MIPs ‘‘/’’ Indicated that the polymers were too flexible to be evaluated. were synthesised by the polymerisation of MAA and EDMA. For ‘‘–’’ Denoted that the polymers were too rigid to allow the mobile phase to flow MIPs before removal of SAL, the presence of SAL in MIPs led to a through. reduction of the C@O stretching intensity, and the bands of CAN k : retention factor for RAC on MIPs. MIPs stretching shift to 1265 cm1, revealing the existence of the inter- kNIPs: retention factor for RAC on NIPs. S: selectivity factor for RAC. actions between MAA and SAL. a The volume content of EDMA in the total volume of monomer and EDMA. b The volume content of EDMA in the total porogen volume of toluene and 3.3. Selectivity of dummy-template MIPs dodecanol.

The selectivities of dummy-template MIPs and NIPs for SAL, the dummy-template MIPs could be ascribed to the removal of RAC, clenbuterol, terbutaline, and adrenaline were evaluated by template molecules. the parameters K and S. The values of S for SAL, RAC, clenbuterol, According to the measurements, the total pore volumes of the terbutaline, and adrenaline were 6.5, 5.5, 3.6, 3.3 and 2.9, respec- dummy-template MIPs and NIPs were 1.9 cm3 g1 and 1.6 cm3 g1, tively, which indicated that the obtained MIPs had high selectivity

Fig. 2. (A) SEM images of NIPs and MIPs: (a) NIPs and (b) dummy-template MIPs. (B) The FTIR spectra of (a) SAL, (b) NIPs, (c) dummy-template MIPs after removal of SAL, and (d) dummy-template MIPs before removal of SAL. 28 W. Du et al. / Food Chemistry 139 (2013) 24–30 for SAL and good cross-recognition for RAC; meanwhile, the MIPs tion time. At 200 min, the adsorption gradually reached equilib- had moderate affinity for other analogues. The possible reason rium. The pseudo first order and pseudo second order kinetic was that the molecular recognition of MIPs mainly depends on models were used to evaluate the adsorption kinetics of RAC on the molecular dimension of the template and matching degree of dummy-template MIPs. log (Qe Qt) versus t and t/Qe versus t were the bonding sites in the three-dimensional network polymers. plotted, using the pseudo first order equation and pseudo second Although the RAC molecule had a larger molecular dimension than order, respectively. The correlation coefficient (R2 = 0.9788) for had SAL, there may be similar molecular interactions and struc- the pseudo second order was higher than that (R2 = 0.9417) for tures between RAC and SAL. Here, the dummy-template MIPs were the pseudo first order, indicating that the pseudo second order ki- prepared because of its good cross-recognition for RAC. netic model provided better correlation for the adsorption of RAC on dummy-template MIPs. 3.4. Adsorption isotherm

The adsorption isotherms of RAC on dummy-template MIPs and 3.6. Optimisation of MISPE procedure NIPs were investigated at 25 °C. As shown in Fig. 3A, the adsorption capacity of dummy-template MIPs for RAC increased with the In this study, the MISPE procedure was investigated using a 1 increment of RAC concentration in the initial solution. Meanwhile, 0.5 lgml RAC standard solution. The washing step was opti- the adsorption capacity for dummy-template MIPs was apparently mised to reduce the matrix interference and maximise the special higher than that of NIPs at the same RAC concentration, suggesting interactions between RAC and MISPE. The washing solutions, such that the resultant dummy-template MIPs showed a higher affinity as methanol, water, acetonitrile, and different ratios of acetoni- for RAC than NIPs. The equilibrium data were modelled with the trile–water (20:80, 30:70, 50:50, and 80:20, v/v), were also inves- Langmuir equation and Freundlich equation, respectively. The plot tigated. The results showed that, when 5 ml of acetonitrile–water (50:50, v/v) was used, the hydrophilic impurities in samples could Ce/Qe versus Ce was used to validate the linearised Langmuir isotherm. The equation for dummy-template MIPs can be described be mostly cleaned up; meanwhile, 8.7% of RAC was washed out as: y = 0.0085x + 7.7318, with the correlation coefficient R2 = from MISPE, but 42.6% of that from NISPE, suggesting that different ratios of acetonitrile–water (v/v) could influence the specific and 0.7808. The plot logQe versus logCe was used to validate the linearised Freundlich isotherm, and the equation for dummy-template nonspecific interactions between RAC molecule and the MIPs, MIPs can be described as: y = 0.8053x 0.1862, with the correlation which led to a significant loss on the MISPE column. Then different coefficient R2 = 0.9935, suggesting that the Freundlich isotherm volumes (1 ml, 3 ml and 5 ml) of acetonitrile–water (50:50, v/v) model was more suitable for the experimental data than the were optimised. It was observed that the recoveries of RAC had Langmuir isotherm model because of the higher correlation coeffi- no obvious change. Therefore, 5 ml of acetonitrile–water (50:50, cient. According to the Freundlich theory, the value of n was calcu- v/v) was selected as the washing solution. lated to be 1.2418 for dummy-template MIPs, indicating that the With the concern about the strong eluting effect of acetic acid dummy-template MIPs adsorption for RAC is favourable. (Song et al., 2008), different types of solvents, including methanol, methanol–acetic acid (90:10, v/v), acetonitrile–acetic acid (90:10, 3.5. Adsorption kinetics v/v) and dichloromethane–acetic acid (90:10, v/v), with different volumes, were investigated in the eluting step. As shown in The adsorption kinetic curve is shown in Fig. 3B. The absorption Fig. 4, methanol–acetic acid (90:10, v/v) offered the highest recov- capacity of dummy-template MIPs for RAC increased with adsorp- ery of RAC compared to other solvents, and the recovery reached a maximum of 90.3%. Additionally, 8 ml of methanol–acetic acid (90:10, v/v) provided the best elution efficiency. Therefore, the optimised MISPE procedure included washing with 5 ml of acetonitrile–water (50:50, v/v), and elution with 8 ml of methanol– acetic acid (90:10, v/v).

Fig. 4. Recoveries of RAC on MISPE with methanol, methanol–acetic acid (90:10, v/ v), acetonitrile–acetic acid (90:10, v/v) and dichloromethane–acetic acid (90:10, v/ Fig. 3. (A) Adsorption isotherm curves of RAC on MIPs and NIPs and (B) adsorption v) and different ratios of methanol–acetic acid (10:90, 30:70, 50:50 and 90:10, v/v) kinetic curve of RAC on MIPs. with different volumes in the eluting step, respectively. W. Du et al. / Food Chemistry 139 (2013) 24–30 29

Fig. 5. (A) Chromatograms of RAC in pork sample: (a) spiked with RAC, (b) after washing step of MISPE, and (c) after eluting step of MISPE. (B) Mass spectra of RAC.

3.7. Method validation between extractions. RAC was not detected in blank pork sample extracts from reused MISPE cartridge, indicating that no carryover 3.7.1. Specificity effect was observed. The representative chromatograms of a RAC standard solution, blank and spiked pork samples were compared. Good separation 3.9. Application to real pork sample was achieved between RAC and endogenous compounds, indicating that this method could detect RAC selectively in pork samples. In order to verify the applicability of the validated method, the developed MISPE coupled with the LC–MS method was applied to 3.7.2. Linearity, LOD and LOQ analyse twenty pork samples obtained from different markets. The Under the optimised LC–MS conditions, the linear regression results showed that RAC was not detected in all pork samples, analysis was y = 5239.9x + 1907.3 for the low concentration range which demonstrated that the use of RAC was effectively controlled (0.05–2 lgkg1) with a correlation coefficient of 0.9966 and in local food-producing animals. y = 5025.5x + 10962 for the high concentration range (2–100 lgkg1) with a correlation coefficient of 0.9985, where y is the 4. Conclusions peak area and x is the analyte concentration. The LOD was 0.02 lgkg1 and the LOQ was 0.05 lgkg1. In this study, MISPE for selective adsorption of RAC was prepared by an in situ method, using salbutamol as the dummy- 3.7.3. Precision, accuracy and matrix effect template. The test of adsorption selectivity indicated that the The repeatability (intra-day) and intermediate (inter-day) pre- dummy-template MIPs displayed high selectivity to RAC. The cision of this method were assessed using spiked pork samples at mechanism for adsorption of RAC on dummy-template MIPs was three concentrations (0.05, 1 and 20 lgkg1). The repeatability found to be a Freundlich isotherm and pseudo second order model. and intermediate precision were conducted with five replicates The proposed dummy-template MISPE coupled with LC–MS meth- for each concentration level on the same day and on three consec- od was successfully applied to the selective analysis of RAC in pork. utive days, respectively. The results showed that the RSD% of repeatability ranged from 1.9% to 6.3%, and the RSD% of intermedi- Acknowledgements ate precision ranged from 3.5% to 9.2%. The averages of intra- and inter-day accuracy ranged from This work was financially supported by the National Natural 78.9% to 92.2% and from 90.7% to 93.1%, respectively. The results Science Foundations of China (Nos. 30873193 and 81173024). in this study were similar to those previously reported (Hu et al., The authors also express their gratitude to professor Jun Haginaka 2011; Tang et al., 2011). The matrix effect was in the range of from Mukogawa Women’s University for his great help in the poly- 86.3–94.6%. Moreover, the developed MISPE coupled with LC–MS mer preparation. method was applied to analyse spiked pork samples with 0.05 lgkg1. As shown in Fig. 5, RAC could be selectively extracted References on MISPE, and the chromatogram of eluate collected from the Antignac, J. P., Marchand, P., Le, B. B., & Andre, F. (2002). Identification of MISPE was much cleaner than that before extraction, which indi- ractopamine residues in tissue and urine samples at ultra-trace level using cated that the dummy-template MISPE is an efficient sample pre- liquid chromatography–positive electrospray tandem mass spectrometry. treatment tool. Journal of Chromatography B, 774, 59–66. Blanca, J., Munoz, P., Morgado, M., Mendez, N., Aranda, A., Reuvers, T., et al. (2005). Determination of clenbuterol, ractopamine and zilpaterol in liver and urine by 3.8. Stability and carryover liquid chromatography tandem mass spectrometry. Analytica Chimica Acta, 529, 199–205. Brambilla, G., Cenci, T., Franconi, F., Galarini, R., Macr, A., Rondoni, F., et al. (2000). 12 successive measurements of RAC in spiked pork samples, Clinical and pharmacological profile in a clenbuterol epidemic poisoning of using the same MISPE cartridge, yielded the R.S.D of 6.2%, indicat- contaminated beef meat in Italy. Toxicology Letters, 114, 47–53. ing that the obtained MISPE column was stable between the cycles. Bressolle, F., Bromet-Petit, M., & Audran, M. (1996). Validation of liquid chromatographic and gas chromatographic methods, applications to The MISPE cartridge needed to be washed with 5 ml of methanol– pharmacokinetics. Journal of Chromatography B: Biomedical Science and acetic acid (90:10, v/v), 5 ml of methanol and 5 ml of water Application, 686, 3–10. 30 W. Du et al. / Food Chemistry 139 (2013) 24–30

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