Dummy-template molecularly imprinted solid phase extraction for selective analysis of ractopamine in pork

Food Chemistry 139 (2013) 24–30

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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 b

School of Medicine, Xi’an Jiaotong University, Xi’an 710061, PR China Xi’an Institute for Food and Drug Control, Xi’an 710061, PR China

a r t i c l e

i n f o

Article history: Received 2 July 2012 Received in revised form 20 December 2012 Accepted 28 January 2013 Available online 10 February 2013 Keywords: Ractopamine Dummy-template Molecularly imprinting polymers In situ polymerisation Solid phase extraction Liquid chromatography- mass spectrometry

a b s t r a c t Molecularly imprinted polymers (MIPs) for selective adsorption of ractopamine hydrochloride (RAC) were synthesised by an in situ method, in which salbutamol (SAL) was used as the dummy-template to avoid the template leakage. Scanning electron microscopy (SEM), mercury porosimerty and Fourier transform infrared spectroscopy (FTIR) were used to investigate the physical and morphological 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 dummy-template MIPs was 90.9 lg g1. Based on the dummy-template polymers, a liquid chromatography–mass spectrometry (LC–MS) method was developed for the selective analysis of RAC in real pork samples. The averages of intra- and inter-day accuracy ranged from 78.9% to 92.2% and from 90.7% to 93.1%, respectively. The RSD% of repeatability ranged from 1.9% to 6.3%, and the RSD% of intermediate precision ranged from 3.5% to 9.2%, while the limit of detection (LOD) was 0.02 lg kg1. Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved.

1. Introduction Ractopamine (RAC) is a synthetic phenethanolamine b2-adrenoceptor agonist, which can be used as clinical medicine for the treatment of asthma. It, however, can be illegally used as a growth promoter for meat-producing animals with high dosage. Therefore, RAC is strictly banned as a feed additive in many countries (Commission of the European Communities, 1996; The Ministry of Agriculture, 2002) due to the potential risk to human beings who consume products made from RAC-treated animals (Brambilla et al., 2000; Smith, Ehrenfried, Dalidowicz, & Turberg, 2002; Xiao, Xu, & Chen, 1999). Nevertheless, the use of RAC remains attractive to swine producers because it can improve feed efficiency. This makes it essential to establish sensitive and selective analytical methods to monitor the residual RAC in food samples. Several analytical methods have been developed for the determination of RAC in animal tissues, urine and feed, such as highperformance liquid chromatography (HPLC) (Shelver & Smith, 2003), gas chromatography–mass spectrometry (He, Su, Zeng, Liu, & Huang, 2007; Wang, Li, & Zhang, 2006), liquid chromatography–mass spectrometry (LC–MS) (Antignac, Marchand, Le, & Andre, 2002; Blanca et al., 2005; Dong et al., 2011; Kootstra et al., 2005), ultra-performance liquid chromatography–tandem mass spectrometry (Shao et al., 2009; Zheng et al., 2010), capillary electrophoresis (Wang, Zhang, Wang, Shi, & Ye, 2010), and surface ⇑ Corresponding author. Tel.: +86 29 82655382. E-mail address: [email protected] (Q. Fu).

plasmon resonance-based biosensor inhibition immunoassays (Lu et al., 2012). However, these methods usually require sample pretreatment processes, such as solid-phase extraction (SPE) (Dong et al., 2011; Qu et al., 2011; Shao et al., 2009; Wang et al., 2010), and the routine SPE suffered from the disadvantage of low selectivity and poor recovery. Recently, molecularly imprinted polymers (MIPs) have attracted much attention in different areas, attributed to their high affinity and pre-determined selectivity for target analytes and other structural analogues (Tamayo, Turiel, & Martin-esteban, 2007). Molecularly imprinted solid-phase extraction (MISPE), as a relatively new concept in the clean-up of biological samples, has proved to be an efficient and selective approach for purification and pre-concentration of RAC from complex matrices (Hu, Li, Liu, Tan, & Li, 2011; Tang, Fang, Wang, & Li, 2011; Wang, Liu, Fang, Zhang, & He, 2009; Widstrand et al., 2004). However, the drawback of the MIPs is the unavoidable template leaking, which may influence the accuracy of identification and quantitation of the analytes (Tamayo et al., 2007). A strategy to avoid template leaking is the utilisation of a dummy template, structural analogue of target analyte itself, during the preparation of MIPs (Yin et al., 2012). To our best knowledge, there is still no developed method concerning the template leaking of MISPE during the analysis of RAC residue. In this study, the MISPE for selective analysis of RAC in pork was prepared by an in situ method, using the analogue, salbutamol (SAL), as the dummy-template. The chemical structures of RAC and SAL are shown in Fig. 1. The adsorption characteristics of the obtained dummy-template MIPs were investigated. The adsorption

0308-8146/$ - see front matter Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.01.109

W. Du et al. / Food Chemistry 139 (2013) 24–30

Fig. 1. Chemical structures: (a) ractopamine and (b) salbutamol.

isotherms were modelled using Langmuir and Freundlich models. The adsorption rate was determined based on Lagergren’s pseudo first and second order kinetic equations. The application of the dummy-template MISPE, coupled with LC–MS method, was developed for selective analysis of RAC in pork. 2. Materials and methods 2.1. Reagents and solutions RAC was purchased from Sigma–Aldrich (New Jersey, USA). SAL was purchased from Cunyi Chemical Co. (Jiangsu, China). Clenbuterol hydrochloride was obtained from Jinhe Pharmaceutical Co. (Wuhan, China). Terbutaline sulphate was purchased from Gangzheng Pharmaceutical Co. (Wuhan, China). Adrenaline hydrochloride was obtained from Hefeng Pharmaceutical Co. (Shanghai, China). Methacrylic acid (MAA) was purchased from Tianjin Chemical Reagent Plant (Tianjin, China). 4-Vinylpyridine (4-VPY), 2-vinylpyridine (2-VPY) and trifluoromethacrylic acid (TFMAA) were obtained from Sigma–Aldrich (New Jersey, USA). MAA, 4VPY and 2-VPY were distilled under vacuum to remove inhibitors prior to use. Ethylene glycol dimethacrylate (EDMA) was obtained from Sigma–Aldrich (New Jersey, USA). 2,20 -Azobisisobutyronitrile (AIBN) was purchased from Shanghai No. 4 Reagent Factory (Shanghai, China) and recrystallised in methanol before use. Methanol and acetonitrile were of HPLC grade, purchased from Kemite Co. (Tianjin, China). Water was purified with Molement 1805b (Shanghai, China). All other chemicals were of analytical grade and obtained from local suppliers. Empty SPE cartridges (10 ml) were purchased from Shenzhen Doudian Co. (Shenzhen, China). Blank pork sample was supplied by a local farmer. Real pork samples were obtained from local markets and stored at 20 °C prior to use. Standard stock solutions of RAC, clenbuterol, terbutaline and adrenaline were prepared separately in water at the concentration of 500 lg ml1. Working solutions of RAC (0.5–500 lg ml1) were prepared by independently diluting stock solutions with acetonitrile. 2.2. Instrument and analytical conditions The adsorption selectivity of the dummy-template MIPs was analysed by HPLC. The dummy-template MIPs and non-imprinted polymers (NIPs) were directly synthesised in a stainless-steel column (100 mm  4.6 mm, id.) according to the procedures in Section 2.4, respectively. Then this MIPs or NIPs column was directly attached to an HPLC pump. The HPLC analysis was performed with a Shimadzu HPLC system, equipped with an LC-20A pump, an SPD-20A UV detector and CS-Light Real Time Analysis Chromatographic Software. The mobile phase was acetonitrile– phosphate buffer (20 mM, pH 5.0) (60:40, v/v). The detection wavelength for RAC, clenbuterol, terbutaline and adrenaline were

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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 workstation. The analysis was performed in the positive electrospray ionisation mode (ESI) at m/z of 302. The column was a VP-ODS column (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 were as follows: nebulizer gas (N2, purity > 99.999%), flow rate of 1.5 l min1, drying gas (N2, purity > 99.999%), pressure of 0.1 MPa, interface temperature of 300 °C, heat block temperature of 220 °C, and detector voltage of 1.25 kV. The injected volume was 10 ll. 2.3. Preparation of dummy-template MISPE The dummy-template MISPE was prepared by an in situ polymerisation in the SPE cartridge according to the method reported previously (Fu et al., 2011). Briefly, the dummy-template (SAL), toluene, MAA, EDMA, dodecanol, and AIBN were sequentially added to a 10 ml test tube. The mixture was thoroughly mixed before use and then degassed for 15 min. After purging with a nitrogen stream for 15 min, the pre-polymerising solution was transferred into a 10 ml empty SPE cartridge. The cartridge was then sealed and set up vertically. The polymerisation was allowed to proceed at 50 °C for 20 h. The obtained MISPE cartridge was washed by a mixture of methanol–acetic acid (90:10, v/v) with a flow rate of 0.5 ml min1 to remove the template molecule and residual porogenic solvents. Finally, the MISPE cartridge was washed with methanol at a flow rate of 0.5 ml min1 to remove the residual acetic acid. A similar procedure, without dummy-template, was used to prepare the non-imprinted solid-phase extraction (NISPE) cartridge. 2.4. Physical and morphological characterisation The morphologies of the dummy-template MIPs and NIPs were observed by a JSM-6390A Scanning Microscope (Jeol, Japan). The porosity, total pore volume, and average pore diameter were measured by mercury porosimerty with an Auto Pore IV 9510 porosimeter (Micromeritics, USA). Fourier transform infrared spectra (FTIR) were recorded on an FTIR-8400S spectrometer (Shimadzu, Japan) with a scanning range from 400 to 4000 cm1. 2.5. Selectively test The adsorption selectivities of the dummy-template MIPs and NIPs were analysed in a stainless-steel column (100 mm  4.6 mm, id.). The retention factor (K) was determined by the following formula:

K¼

tR  to to

ð1Þ

where tR is the retention time of a solute and t0 is void time of the column (measured by injecting acetone). The selectivity factor (S) was expressed by the following formula:

S¼

K MlPs K NlPs

ð2Þ

where kMIPs and kNIPs are the retention factors of a compound on MIPs and NIPs, respectively.

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W. Du et al. / Food Chemistry 139 (2013) 24–30

2.6. Adsorption test 2.6.1. Adsorption isotherm To investigate the adsorption ability of dummy-template MIPs for RAC, 50 mg of the dummy-template MIPs particles were added to 10 ml of RAC working solution at different concentrations (5–500 lg ml1). The suspensions were placed on a SHZ-82 Vapour-bathing Constant Temperature shaker (Jintan, China) for 300 min at 25 °C and then centrifuged at 8000 rpm for 15 min. The supernatant was measured for free RAC by HPLC. A similar procedure was performed for NIPs particles. The adsorption amount (Qe, lg g1) was calculated by the following formula:

Qe ¼

ðC o  C e Þv m

ð3Þ

where C0 (lg ml1) is the initial concentration of RAC, Ce (lg ml1) is equilibrium concentration of RAC in solution, V (ml) is sample volume and m (mg) is the mass of the polymer. The adsorption isotherms were described by the Langmuir equation (Eq. (4)) and Freundlich equation (Eq. (5)) (Singh & Mishra, 2010). The linearised forms of the two isotherms are:

Ce 1 Ce ¼ þ Q e qm K L qm ln Q e ¼

ð4Þ

1 ln C e þ ln K F n

ð5Þ

where Ce (lg ml1) and Qe (lg g1) are the equilibrium concentration and the amount of RAC adsorbed at equilibrium, respectively, and qm (lg g1) and KL (l g1) are theoretical maximum adsorption capacity and Langmuir equilibrium constant, respectively. KF and n are the Freundlich constants, which are indicators of adsorption capacity and adsorption intensity. According to the Freundlich theory, n can be used to determine whether the adsorption is favourable. When n > 1, it is favourable adsorption; when n = 1, it is linear adsorption; when n < 1, it is unfavourable adsorption. 2.6.2. Adsorption kinetics The uptake kinetic study was performed with 150 lg ml1 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 capacity was determined by HPLC. The Lagergren’s pseudo first order (Eq. (6)) and pseudo second order (Eq. (7)) (Singh & Mishra, 2010) models were used to describe the adsorption kinetic mechanism of dummy-template MIPs. Both the first and second order rate equations were commonly employed in parallel, and one was often claimed to be better than another according to a marginal difference in correlation coefficient.

logðQ e  Q t Þ log Q e  t 1 t ¼ þ Q t k2 Q 2e Q e

k1 t 2:303

ð6Þ

ð7Þ

where Qe (lg g1) and Qt (lg g1) are the adsorption amount of RAC at equilibrium and at time t (min), respectively; k1 (min1) and k2 (g lg1 min1) are the pseudo first order and pseudo second order adsorption rate constants, respectively.

eluents were evaporated to dryness under a nitrogen stream and the residues were dissolved in 500 ll of acetonitrile for LC–MS analysis. 2.8. Sample preparation Two grams, of real pork sample were accurately weighed into a 10 ml glass beaker. After adding 4 ml of acetonitrile–water (80:20, v/v), the sample was vortex-mixed for 2 min and centrifuged for 10 min. Subsequently, 3 ml of supernatant were loaded onto the MISPE cartridge. 2.9. Method validation and real sample analysis The method validation was performed for specificity, linearity, range, limit of detection (LOD), limit of quantification (LOQ), accuracy and precision, following the recommendations of the International Conference on Harmonization Q2(R1) (ICH-International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use, 2005). The calibration was established by measuring different concentrations (0.05– 100 lg kg1) of RAC in spiked pork samples after the MISPE procedure. In order to avoid undue bias, the calibration curve was split into two ranges: 0.05–2 lg kg1 and 2–100 lg kg1. Least squares linear regression analysis was used to determine the slope, intercept and correlation coefficient. The LOD and LOQ were calculated from injection of the spiked sample, providing signal to noise ratios of 3 and 10, respectively. The accuracy was expressed as a percentage of recovery. The precision was tested by studying the repeatability and intermediate precision. The precision was expressed by relative standard deviation (R.S.D%), with acceptable values for the RSD% being less than 15% (Bressolle, Bromet-Petit, & Audran, 1996). In order to evaluate the stability of the MISPE cartridge, the same MISPE cartridge was reused 12 times for the measurement of RAC in a spiked pork sample. Twenty real pork samples, obtained from local markets, were analysed by MISPE, coupled with LC–MS. 3. Results and discussion 3.1. Preparation conditions of dummy-template MIPs In the dummy-template MIPs preparation, commonly used acidic monomers, MAA and TFMAA, and basic monomers 2-VPY and 4-VPY, were tested for the validity. It was observed that, with 2-VPY, 4-VPY and TFMAA, the pre-polymerisation mixtures were difficult to polymerise successfully. Therefore, MAA was employed as the functional monomer in this study, which was the same as published elsewhere (Fu et al., 2011). Several factors affecting the adsorption properties of the MIPs were optimised, including the mole ratio of the template (SAL) to functional monomer (MAA), the content of cross-linker (EDMA) and different porogenic solvents. As shown in Table 1, the MIPs10 showed the highest selectivity factor. Hence, its preparation conditions were selected to be the optimum ones. 3.2. Physical and morphological observation

2.7. MISPE procedure The MISPE cartridge was first washed with 2 ml of water and semi-dried at a low positive pressure of 0.05 MPa. Then 3 ml of the pre-treated sample was loaded onto the MISPE cartridge. The cartridge was washed with 5 ml of acetonitrile–water (50:50, v/ v), and eluted with 8 ml of methanol–acetic acid (90:10, v/v). The

SEM images of the dummy-template MIPs and NIPs are shown in Fig. 2A. The dummy-template MIPs and NIPs showed appreciable differences in morphology. The NIPs possessed crosslinked microglobules which yielded to large clusters, whereas the dummy-template MIPs exhibited more rough and porous structures than did NIPs, indicating that the presence of recognition sites in

W. Du et al. / Food Chemistry 139 (2013) 24–30 Table 1 Optimisation for preparation and separation performance of dummy-template MIPs and NIPs. Polymers

Molar ratio of SAL/MAA

Content of cross linkera (v%)

Toluene in porogenb (v%)

kMIPs

kNIPs

S

MIPs1 MIPs2 MIPs3 MIPs4 MIPs5 MIPs6 MIPs7 MIPs8 MIPs9 MIPs10 MIPs11

1:2 1:3 1:4 1:5 1:6 1:4 1:4 1:4 1:4 1:4 1:4

85 85 85 85 85 80 83 87 90 85 85

18 18 18 18 18 18 18 18 18 15 20

0.5 1.9 8.7 1.3 4.5 7.0 7.1 5.7 4.9 14.8 8.4

/ 0.7 1.9 0.6 1.4 1.9 1.6 1.1 – 2.5 2.3

/ 2.6 4.6 2.2 3.2 3.6 4.4 5.2 – 5.9 3.6

HPLC conditions: mobile phase, acetonitrile – phosphate buffer (20 mM, pH 5.0) (60:40, v/v); flow rate, 1.0 ml min1; column temperature, 25 °C. ‘‘/’’ Indicated that the polymers were too flexible to be evaluated. ‘‘–’’ Denoted that the polymers were too rigid to allow the mobile phase to flow through. kMIPs: retention factor for RAC on MIPs. kNIPs: retention factor for RAC on NIPs. S: selectivity factor for RAC. 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 dodecanol.

the dummy-template MIPs could be ascribed to the removal of template molecules. According to the measurements, the total pore volumes of the dummy-template MIPs and NIPs were 1.9 cm3 g1 and 1.6 cm3 g1,

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respectively. The porosity of the dummy-template MIPs (60.0%) was higher than that of NIPs (50.3%), which was of benefit to the adsorption of analytes from complex matrices. Besides, the average pore diameter of the dummy-template MIPs (134.0 nm) was smaller than that of NIPs (283.3 nm). The results were consistent with the description of the SEM images. FTIR spectra of dummy-template (SAL), NIPs, MIPs after and before removal of SAL, are shown in Fig. 2B. For SAL, the bands at 3409 cm1, 3193 cm1, and 1234 cm1 were the characteristic vibrations of AOH, NAH and CAN. The bands at 2970.17 cm1 and 1612 cm1 were attributed to the CAH antisymmetic stretching vibration and C@C stretching vibration in benzene, respectively. For NIPs and MIPs after removal of SAL, the FTIR spectra almost had the same characteristic bands. The bands at 3448 cm1, 2950 cm1, 1720 cm1 and 1157 cm1 were attributed to the stretching vibrations of AOH, ACH3, C@O and CAOAC for MAA and EDMA, respectively, indicating that the NIPs and MIPs were synthesised by the polymerisation of MAA and EDMA. For MIPs before removal of SAL, the presence of SAL in MIPs led to a reduction of the C@O stretching intensity, and the bands of CAN stretching shift to 1265 cm1, revealing the existence of the interactions between MAA and SAL. 3.3. Selectivity of dummy-template MIPs The selectivities of dummy-template MIPs and NIPs for SAL, RAC, clenbuterol, terbutaline, and adrenaline were evaluated by the parameters K and S. The values of S for SAL, RAC, clenbuterol, terbutaline, and adrenaline were 6.5, 5.5, 3.6, 3.3 and 2.9, respectively, 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.

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for SAL and good cross-recognition for RAC; meanwhile, the MIPs had moderate affinity for other analogues. The possible reason was that the molecular recognition of MIPs mainly depends on the molecular dimension of the template and matching degree of the bonding sites in the three-dimensional network polymers. Although the RAC molecule had a larger molecular dimension than had SAL, there may be similar molecular interactions and structures between RAC and SAL. Here, the dummy-template MIPs were prepared because of its good cross-recognition for RAC.

tion time. At 200 min, the adsorption gradually reached equilibrium. The pseudo first order and pseudo second order kinetic models were used to evaluate the adsorption kinetics of RAC on dummy-template MIPs. log (Qe  Qt) versus t and t/Qe versus t were plotted, using the pseudo first order equation and pseudo second order, respectively. The correlation coefficient (R2 = 0.9788) for the pseudo second order was higher than that (R2 = 0.9417) for the pseudo first order, indicating that the pseudo second order kinetic 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 NIPs were investigated at 25 °C. As shown in Fig. 3A, the adsorption capacity of dummy-template MIPs for RAC increased with the increment of RAC concentration in the initial solution. Meanwhile, the adsorption capacity for dummy-template MIPs was apparently higher than that of NIPs at the same RAC concentration, suggesting that the resultant dummy-template MIPs showed a higher affinity for RAC than NIPs. The equilibrium data were modelled with the Langmuir equation and Freundlich equation, respectively. The plot Ce/Qe versus Ce was used to validate the linearised Langmuir isotherm. The equation for dummy-template MIPs can be described as: y = 0.0085x + 7.7318, with the correlation coefficient R2 = 0.7808. The plot log Qe versus log Ce was used to validate the linearised Freundlich isotherm, and the equation for dummy-template MIPs can be described as: y = 0.8053x  0.1862, with the correlation coefficient R2 = 0.9935, suggesting that the Freundlich isotherm model was more suitable for the experimental data than the Langmuir isotherm model because of the higher correlation coefficient. According to the Freundlich theory, the value of n was calculated to be 1.2418 for dummy-template MIPs, indicating that the dummy-template MIPs adsorption for RAC is favourable. 3.5. Adsorption kinetics The adsorption kinetic curve is shown in Fig. 3B. The absorption capacity of dummy-template MIPs for RAC increased with adsorp-

Fig. 3. (A) Adsorption isotherm curves of RAC on MIPs and NIPs and (B) adsorption kinetic curve of RAC on MIPs.

3.6. Optimisation of MISPE procedure In this study, the MISPE procedure was investigated using a 0.5 lg ml1 RAC standard solution. The washing step was optimised to reduce the matrix interference and maximise the special interactions between RAC and MISPE. The washing solutions, such as methanol, water, acetonitrile, and different ratios of acetonitrile–water (20:80, 30:70, 50:50, and 80:20, v/v), were also investigated. The results showed that, when 5 ml of acetonitrile–water (50:50, v/v) was used, the hydrophilic impurities in samples could be mostly cleaned up; meanwhile, 8.7% of RAC was washed out from MISPE, but 42.6% of that from NISPE, suggesting that different ratios of acetonitrile–water (v/v) could influence the specific and nonspecific interactions between RAC molecule and the MIPs, which led to a significant loss on the MISPE column. Then different volumes (1 ml, 3 ml and 5 ml) of acetonitrile–water (50:50, v/v) were optimised. It was observed that the recoveries of RAC had no obvious change. Therefore, 5 ml of acetonitrile–water (50:50, v/v) was selected as the washing solution. With the concern about the strong eluting effect of acetic acid (Song et al., 2008), different types of solvents, including methanol, methanol–acetic acid (90:10, v/v), acetonitrile–acetic acid (90:10, v/v) and dichloromethane–acetic acid (90:10, v/v), with different volumes, were investigated in the eluting step. As shown in Fig. 4, methanol–acetic acid (90:10, v/v) offered the highest recovery 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/ v) and different ratios of methanol–acetic acid (10:90, 30:70, 50:50 and 90:10, v/v) with different volumes in the eluting step, respectively.

W. Du et al. / Food Chemistry 139 (2013) 24–30

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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 3.7.1. Specificity The representative chromatograms of a RAC standard solution, blank and spiked pork samples were compared. Good separation was achieved between RAC and endogenous compounds, indicating that this method could detect RAC selectively in pork samples. 3.7.2. Linearity, LOD and LOQ Under the optimised LC–MS conditions, the linear regression analysis was y = 5239.9x + 1907.3 for the low concentration range (0.05–2 lg kg1) with a correlation coefficient of 0.9966 and y = 5025.5x + 10962 for the high concentration range (2–100 lg kg1) with a correlation coefficient of 0.9985, where y is the peak area and x is the analyte concentration. The LOD was 0.02 lg kg1 and the LOQ was 0.05 lg kg1. 3.7.3. Precision, accuracy and matrix effect The repeatability (intra-day) and intermediate (inter-day) precision of this method were assessed using spiked pork samples at three concentrations (0.05, 1 and 20 lg kg1). The repeatability and intermediate precision were conducted with five replicates for each concentration level on the same day and on three consecutive days, respectively. The results showed that the RSD% of repeatability ranged from 1.9% to 6.3%, and the RSD% of intermediate precision ranged from 3.5% to 9.2%. The averages of intra- and inter-day accuracy ranged from 78.9% to 92.2% and from 90.7% to 93.1%, respectively. The results in this study were similar to those previously reported (Hu et al., 2011; Tang et al., 2011). The matrix effect was in the range of 86.3–94.6%. Moreover, the developed MISPE coupled with LC–MS method was applied to analyse spiked pork samples with 0.05 lg kg1. As shown in Fig. 5, RAC could be selectively extracted on MISPE, and the chromatogram of eluate collected from the MISPE was much cleaner than that before extraction, which indicated that the dummy-template MISPE is an efficient sample pretreatment tool. 3.8. Stability and carryover 12 successive measurements of RAC in spiked pork samples, using the same MISPE cartridge, yielded the R.S.D of 6.2%, indicating that the obtained MISPE column was stable between the cycles. The MISPE cartridge needed to be washed with 5 ml of methanol– acetic acid (90:10, v/v), 5 ml of methanol and 5 ml of water

between extractions. RAC was not detected in blank pork sample extracts from reused MISPE cartridge, indicating that no carryover effect was observed. 3.9. Application to real pork sample In order to verify the applicability of the validated method, the developed MISPE coupled with the LC–MS method was applied to analyse twenty pork samples obtained from different markets. The results showed that RAC was not detected in all pork samples, which demonstrated that the use of RAC was effectively controlled in local food-producing animals. 4. Conclusions In this study, MISPE for selective adsorption of RAC was prepared by an in situ method, using salbutamol as the dummytemplate. The test of adsorption selectivity indicated that the dummy-template MIPs displayed high selectivity to RAC. The mechanism for adsorption of RAC on dummy-template MIPs was found to be a Freundlich isotherm and pseudo second order model. The proposed dummy-template MISPE coupled with LC–MS method was successfully applied to the selective analysis of RAC in pork. Acknowledgements This work was financially supported by the National Natural Science Foundations of China (Nos. 30873193 and 81173024). The authors also express their gratitude to professor Jun Haginaka from Mukogawa Women’s University for his great help in the polymer preparation. References Antignac, J. P., Marchand, P., Le, B. B., & Andre, F. (2002). Identification of ractopamine residues in tissue and urine samples at ultra-trace level using liquid chromatography–positive electrospray tandem mass spectrometry. 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 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). Clinical and pharmacological profile in a clenbuterol epidemic poisoning of contaminated beef meat in Italy. Toxicology Letters, 114, 47–53. Bressolle, F., Bromet-Petit, M., & Audran, M. (1996). Validation of liquid chromatographic and gas chromatographic methods, applications to pharmacokinetics. Journal of Chromatography B: Biomedical Science and Application, 686, 3–10.

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