Talanta 138 (2015) 40–45

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Talanta
journal homepage: www.elsevier.com/locate/talanta

Rapid analysis of ractopamine in pig tissues by dummy-template

imprinted solid-phase extraction coupling with surface-enhanced
Raman spectroscopy
Xiaohua Xiao n, Kuanglin Yan, Xianfang Xu, Gongke Li n
School of Chemistry and Chemical Engineering, Sun Yat-sen University, Guangzhou 510275, China

art ic l e i nf o a b s t r a c t

Article history: Ritodrine has similar skeleton structure to ractopamine and it was selected as the dummy-template
Received 12 December 2014 molecule to synthesize the molecular imprinted polymers (MIPs). The MIPs exhibited better selectivity
Received in revised form to ractopamine than to the dummy-template molecule: the imprint factor for ractopamine was 8.9,
27 January 2015
while 7.6 for ritodrine. The MIPs were used as sorbents in solid-phase extraction for selective enrichment
Accepted 1 February 2015
Available online 9 February 2015
of ractopamine, and some key parameters were optimized. After that, a rapid surface-enhanced Raman
spectroscopy method was developed for analysis of ractopamine and isoxuprine in pig tissue samples.
Keywords: Under the optimal conditions, good linearity was achieved in the range of 20.0–200.0 μg/L for
Dummy-template ractopamine and isoxsuprine at 842 cm
1 and 993 cm
1, respectively. The limits of detection were
Molecularly imprinted polymers
3.1–4.3 μg/L, which were lower than the maximum allowed by U. S. Food and Drug Administration. The
Solid-phase extraction
recoveries of ractopamine and isoxsuprine were 72.4–79.7% and 71.0–78.2% for the spiked pork and pig
Surface-enhanced Raman spectroscopy
Ractopamine liver, respectively, while the relative standard deviations ranging from 7.4% to 13.0%. The results suggest
Pig tissues that the proposed method is sensitive and selective, and it has good potential on the quantitative
analysis of trace amounts of β-agonists in complex samples.
& 2015 Elsevier B.V. All rights reserved.

1. Introduction silver [3]. The magnitude of Raman signals in SERS can be enhanced
to 104–107 due to the effects of electromagnetic field and chemical
The demand for faster, more sensitive and cost-effective analytical enhancement [4]. Hence, the ultrasensitive SERS has been used to
methods is rapidly increasing during the last decades, especially on characterize and detect organic chemicals and microorganisms such
the determination of trace analytes in complex matrix such as foods, as melamine [5–7], malachite green [8–10], pesticides [11–13], anti-
environmental or biological samples [1]. Normally, an analytical biotics [14–16], β-agonists [17,18], etc. Although the limits of detection
process has several steps including sample preparation, sampling, (LODs) of these methods were acceptable, their further application to
separation, determination, data handling and treatment, etc. Of these detect specific analytes in complex matrices, especially for the quanti-
steps, selective sample preparation and sensitive analytical methods tative analysis of trace analytes, were greatly limited by the serious
might be the most important. Chromatographic methods such as matrix interferences and poor selectivity. Therefore, sample pretreat-
liquid chromatography–mass spectroscopy, gas chromatography– ment to reduce or remove undesirable interferences is required before
mass spectroscopy and immunoassay such as enzyme-linked immu- SERS detection.
nosorbent assay (ELISA) were typically used in the most cases [2]. Conventional liquid–liquid extraction method is considered the
However, the chromatographic methods are relatively expensive and most time-consuming and error-prone part of the analytical scheme.
complex operation while ELISA is likely to be influenced by matrix Some new extraction techniques such as solid-phase extraction (SPE),
interferences and so lead false-positive results. Surface-enhanced solid-phase microextraction (SPME) [19] or liquid-phase microextrac-
Raman spectroscopy (SERS) is a special surface-enhanced optical tion (LPME) [20], were developed to reduce the initial sample sizes,
phenomenon on the nano-scale metal surfaces, which has overcome and to minimize the amount of hazardous organic solvents. Among
the low sensitivity of normal Raman spectroscopy via adsorption of them, both SPE and SPME were well established in analytical labora-
analytes on rough nano-scale surfaces typically made from gold and tories. However, the main drawback associated to them is the lack of
selectivity of the sorbents. Molecularly imprinted polymers (MIPs), the
synthetic materials with artificially generated recognition sites which
n
Corresponding authors. Tel.: þ 86 20 84110922; fax: þ 86 20 84115107.
are able to specifically rebind target molecule, have attracted more and
E-mail addresses: [email protected] (X. Xiao), more attention for their high affinity and selectivity for target analyte
[email protected] (G. Li). and its analogues. Molecular imprinted solid-phase extraction (MISPE)

http://dx.doi.org/10.1016/j.talanta.2015.02.003
0039-9140/& 2015 Elsevier B.V. All rights reserved.
 X. Xiao et al. / Talanta 138 (2015) 40–45 41

was also developed as a relatively new concept in the pretreatment (MeOH) and acetonitrile (ACN) of HPLC grade were purchased from
of biological sample, it had been successfully used for enrichment Merck (Darmstadt, Germany). Deionized water was used thoroughly.
of ractopamine from complex matrices [21]. MISPE had also been All other reagents were of analytical grade.
combined with SERS for the detection of chemical compounds from The individual stock solutions of standards were prepared at
complicated samples. Feng [18] reported a MISPE-SERS biosensor the concentration of 100.0 mg/L in acetonitrile, and further solu-
system for the detection of α-tocopherol from four different types of tions of lower concentration were prepared by serial dilution of
vegetable oils based on using a dendritic silver nanostructure as SERS the stock solutions. The mixed standard solution was prepared
substrate. This biosensing system demonstrated good sensitivity and with iso-proportional mass of each solution.
selectivity for the quantitative detection of different spiking levels of Au nanoparticles (Au NPs) was prepared according to the
α-tocopherol in vegetable oils. Meanwhile, the further applications method reported previously [3]. Briefly, 200 mL deionized water
of traditional MIPs were greatly limited owing to the unavoidable consist of 5.0 mL 10 mmol/L HAuCl4 was heated to boiling under
template leaking, which may influence the accuracy of identification vigorous stirring, then 1.2 mL 1% (w/v) trisodium citrate was
and quantization of the analytes. The structural analogues of the injected rapidly and kept boiling for 40 min. The red solution
analyte itself, namely dummy-template, can avoid the leakage of the was detected by Cary-100 UV–vis spectrophotometer (Varian,
template. To guarantee the selectivity and capacity of the sorbents, American) and its absorption wavelength was 530 nm, the average
the selection of the dummy template is a key factor, while the available diameter of Au NPs was about 55 nm.
dummy template molecules are limited especially for simultaneous
determination of homologue compounds. In most case, the imprinting 2.2. Instruments
factor or selectivity for dummy template was significantly higher than
that for the cross-recognition targets in these molecular imprinted An S-4300 scanning electron microscope (Hitachi, Japan) was
polymers [22]. used to investigate the surface of the MIP polymer. A Nicolet
Ractopamine was a typical β-agonists, which can promote Avatar 330 Fourier transform infrared (FT-IR) spectrometer with a
bronchodilation, vasodilatation and increase heart rate. It is also scanning range from 400 to 4000 cm
1 was applied to investigate
used in animal feeding, effects seen in the skeletal muscle include the composition of the polymer. An LC-20A HPLC system (Shi-
speed up feeding efficiency and carcass leanness. However, the madzu, Japan) consists of a RF-10AXL fluorescence detector and a
residual of ractopamine in pork products may pose health risks, diode-array detector was utilized to analyze ractopamine and its
particularly to those with asthma or cardiovascular disease. The analogues. Aspirator A-3S circulating pump was applied to pro-
use of ractopamine in swine has been banned by the majority mote the solid-phase extraction. The solid-phase extraction tube
of countries and areas in the world, such as China, Japan, and and frits were purchased from Anpel company (Shanghai, China).
European Union, the maximum residue limit of ractopamine in A battery-powered Raman spectrometer with 785 nm laser
swine liver is strictly limited to 0.15 μg/g or lower in the United excitation wavelength (Delta Nu Inspector Raman, Laramie, WY)
States. In recent years, monitoring the residues of β-agonists in was used to perform SERS detection. This system consists of a CCD
swine liver or pork products had been attracted great interest to detector (ModelSpec-10:400B, Roper Scientific, Trenton, NJ) with a
the government regulatory agencies and the food industry [18]. spectral resolution of 8 cm
1, and a data acquisition system
Since the residue of ractopamine in complex samples is lower than (Photometrics, Tucson, AZ).
μg/g, efficient sample pretreatments as well as sensitive analytical
methods are significantly important. Solid-phase extraction (SPE) 2.3. Synthesis of the dummy-template MIPs
[23] or solid-phase microextraction (SPME) [24] with chromato-
graphic methods were commonly used to analysis β-agonists in The dummy-template MIPs were prepared according to our
different matrices. Recently, Du et al. [25] reported a dummy- reported work [26] with some modifications. Briefly, 0.33 g RTD,
template MISPE method for selective analysis of ractopamine in 347 μL MAA and 3.1 mL EGDMA were dissolved in 6.8 mL methanol
pork, in which salbutamol was used as the dummy-template, the to prepare the pre-polymer solution. This solution was stirred for
selectivity factors of dummy-template MIPs for salbutamol was 12 h at room temperature, and then 30 mg AIBN was added and
significantly higher than that for ractopamine. dissolved adequately. After purging with nitrogen stream for 5 min,
In the present work, ritodrine was selected as the dummy- the bottle was sealed immediately and allowed to perform poly-
template molecule to synthesize the molecular imprinted poly- merization at 60 1C for 24 h. Non-imprinted polymers (NIPs) were
mers due to its highly similar structure skeleton to ractopamine. prepared by the same way with the imprinted polymers except
The MIPs were used as sorbents of SPE for the selective extraction without the addition of RTD.
of β-agonists, then a rapid SERS method for analysis of ractopa- The polymers were ground and sieved with a mesh gauge. The
mine in pig tissue samples was developed. template molecule was removed by soxhlet extraction with
methanol-acetic acid (9:1, v/v) until no template molecule was
detected by HPLC. The polymers were heated at 120 1C for 12 h
2. Experimental before use.

2.1. Chemicals and materials 2.4. Optimization of molecular imprinted solid-phase extraction
procedures
Ritodrine hydrochloride (RTD) was purchased from Ruihe Chemi-
cal Plant (Taiyuan, China); Ractopamine hydrochloride (RAT), iso- 200 mg of MIP particles were packed into a 3.0 mL empty
xsuprine hydrochloride (ISP) and clenbuterol hydrochloride (CLB) polypropylene cartridge with glasswool frit at each end. The
were purchased from Sigma (Shanghai, China); 4-nitrophenol (4- cartridge was activated with 3.0 mL of methanol and equilibrated
NP) and trisodium citrate was purchased from Guangzhou Reagent with 3.0 mL of acetonitrile. Then, the loading solutions in different
Plant (Guangzhou, China). Methacrylic acid (MAA) and azo-bis(iso- solvent including deionized water, methanol, acetonitrile and their
butyronitrile) (AIBN) were purchased from Damao Reagent Plant corresponding aqueous solutions were investigated. 5.0 mL of the
(Tianjin, China). Ethylene glycol dimethacrylate (EGDMA) was pur- pre-treated sample was added into the cartridge at 0.5 mL/min. After
chased from Rohm-Haas AG (USA). Chloroauric acid was purchased that, the cartridge was washed with methanol and eluted with
from Guangfu Chemical Research Institute (Tianjin, China). Methanol methanol containing 20% (v/v) acetic acid. The eluent was
42 X. Xiao et al. / Talanta 138 (2015) 40–45

evaporated to dryness and the residue was re-dissolved with 100 μL ture and time were optimized. Non-imprinted polymers (NIPs) were
of methanol for further analysis. prepared by the same way with the imprinted polymers without the
addition of RTD.
2.5. Sample preparation FT-IR spectra of the MIPs before and after removal of the template
molecule, as well as that of the NIPs and the template molecule of
Five grams of pork and liver samples were accurately weighed into RTD are illustrated in Fig. 2. When RTD was removed from the MIPs,
a 50 mL beaker flask. After adding 5.0 mL of acetonitrile, the sample the MIPs (curve b) had the similar characteristic bands with that of
was ultrasound for 15 min and centrifuged for 10 min at 3000 rpm, the NIPs (curve c). The bands at 3440 cm
1, 2970 cm
1, 1728 cm
1
repeated twice and combined the extracts. The extracts were re- and 1156 cm
1 were corresponding to the stretching vibration of –
extracted with 10 mL hexane, 6 mL acetonitrile, 4 mL methylene OH, –CH3, –C¼ O and –C–O–C, respectively. The absorption peak at
chloride and 20 mL water in a separator funnel. Then, the supernatant 1515 cm
1, which was corresponding to C¼ C stretching vibration in
was evaporated to dryness and the residue was re-dissolved with the benzene ring of RTD (curve a), was not shown in MIPs and NIPs,
5.0 mL of acetonitrile before loading onto the MISPE cartridge. indicating the leakage of the template can be neglected.
The SEM images of the MIPs are shown in Fig. 3. Rough and
2.6. SERS and HPLC analysis porous structure was obtained, indicating that the presence of
recognition sites in the dummy-template MIPs, which could be
In a typical experiment for detecting RAT measured by SERS, 1.5 mL ascribed to the removal of template molecules.
of the prepared Au NPs was concentrated to 30 μL, then 2.0 μL of the After removing the template and other substances, 200 mg of
concentrated Au NPs was mixed with 2.0 μL of the sample solution in a MIPs particles were packed into a 3.0 mL empty polypropylene
silicon substrate (0.5  0.5 cm2) treated in Piranha solution (98% H2SO4/ cartridge with glasswool frit at each end. The NISPE was prepared
30% H2O2, 3:1, v/v) and rinsed thoroughly with ultrapure water. A with the same procedure.
portable Raman spectrometer with laser wavelength of 785 nm, and a
power of 60 mW was used for Raman detection. The typical exposure
time was 1 s with three accumulations.
3.2. Evaluation of the dummy-template MIP as SPE sorbents
HPLC measurement was performed on a Shimadzu LC-20A system
and the analytical column was a C18 column (250 mm  4.6 mm id,
The MISPE requires the same processes used in a common SPE
5 mm, Dikma). RF-10AxL detector was used to analyze RAT, RTD and
procedure, including conditioning, sample loading, washing, and elut-
ISP. The excitation wavelength was 226 nm while the emission
ing. Factors that probably influenced the extraction process, such as
wavelength was 305 nm. The mobile phase was acetonitrile and
loading solvent, washing solvent, eluting solvent, amount of the
0.1% acetic acid solution. The ratio of acetonitrile was changed from
washing and eluting solvent, should be evaluated to achieve the
10% (v/v) to 48% (v/v) within 8 min at a flow rate of 1.0 mL/min.
highest extraction efficiency. In this study, 5.0 mL 100 μg/L RAT

3. Results and discussion

3.1. Preparation of dummy-template MISPE

To avoid the undesirable RAT template interferences in the MIPs,

salbutamol was used as the dummy-template, but the selectivity
factor of salbutamol was significantly higher than that ractopamine
[25]. In this work, RTD was used as the dummy-template because the
chemical structure of RTD is highly similar to RAT except the position
of methyl group and the carbon number of the main chain in the
molecule. The MIPs were prepared according to reference [26] with
some modification (Fig. 1). The conditions such as the amount of
monomer (MAA) and cross linker (EGDMA), the reaction tempera- Fig. 2. The infrared spectra of the MIPs and NIPs. (a) RTD; (b) MIPs; (c) NIPs.

Fig. 1. The preparation procedure of dummy-template MISPE.

 X. Xiao et al. / Talanta 138 (2015) 40–45 43

Fig. 3. The SEM images of the MIPs (A)  500; (B)  3000.

Fig. 4. The binding isotherm of MIPs and NIPs for RTD (A), the Scatchard plot of MIPs and NIPs toward RTD (B). The binding isotherm of MIPs and NIPs for RAT (C), the
Scatchard plot of MIPs and NIPs toward RAT (D). Chemical structure of the studied target compounds (E) and extraction amount of target compounds with MISPE and
NISPE (F).

standard solution was employed to optimize the loading, washing and solutions were investigated. The results showed that the recovery of
elution steps of MISPE. RAT was more than 90% when using acetonitrile as loading solution,
First, the loading solutions in different solvent including demo- suggesting that acetonitrile effectively promotes the rebinding of
nized water, methanol, acetonitrile and their corresponding aqueous analytes to the specific sites. Generally, the MIPs exhibit best extraction
44 X. Xiao et al. / Talanta 138 (2015) 40–45

performance and molecular recognition ability when the polarity 8.6 μg/g, respectively. There are 5.4 and 5.3 times of MISPE over
of extraction solvent is similar to the polymerization solvent. [26] the NISPE. The results revealed that the saturated adsorption
Therefore the synthesized MIPs were more compatible with polar capacity of MISPE was significantly higher than NISPE, suggesting
environment, especially applying acetonitrile as loading solution. the resultant MISPE showed a higher affinity for RTD and its
The washing step was optimized to reduce the matrix inter- analogues than NISPE for the specific adsorption. Notably, the
ference and maximize the special interactions between RAT and saturated adsorption amount of RAT in MISPE was slightly higher
MIP sorbents. The washing solutions, such as methanol, acetonitrile, than RTD-the dummy-template molecule.
water, 50% methanol/water (v/v), 50% acetonitrile/water (v/v), 50% Moreover, Scatchard relationship was usually determined to assess
methanol/acetonitrile (v/v) were investigated. When 4.0 mL metha- the affinity of MIPs and NIPs. Here, we constructed a Scatchard plot
nol was used, the impurities in samples could be mostly cleaned up using the expression Q/C¼
Q/Kd þ Qmax/Kd, where Q is the adsorp-
and the recovery of RAT reached a maximum of 92.2% in MISPE. tion capacity at adsorption equilibrium, C is the initial concentration
Therefore 4.0 mL methanol was selected as the optimal washing of RTD, Kd is the distribution coefficient, and Qmax is the saturated
solution for the following experiments. adsorption capacity [27]. Both of the adsorption isotherm of MIPs for
In eluting step, different ratios of acetic acid/methanol (10%, 20%, RTD and RAT (Fig. 4) showed a good linear regression (R2 ¼0.9759
v/v) and acetic acid/acetonitrile (10%, 20%, v/v) were investigated. and 0.9761, respectively). The Qmax of MIPs were 86.3 μg/g for RTD
Acetic acid/methanol was better than acetic acid/acetonitrile accord- and 87.7 for RAT, which was in keeping with that obtained from the
ing to the recovery of RAT and 20% acetic acid/methanol (v/v) offered saturated adsorption capacity experiments. By contrast, NIPs showed
the highest recovery. Additionally, 2.0 mL of 20% acetic acid/metha- nonlinearity, indicating no selective adsorption sites are present for
nol (v/v) provided the best elution efficiency. RTD and RAT. Since the hydrogen bond was formed between the
Overall, the optimized MISPE procedures included acetonitrile template and monomers and an ordered arrangement was kept by
as loading solution, 4.0 mL methanol as washing solution and the polymerization of MIPs, those binding sites came from the
2.0 mL 20% acetic acid/methanol (v/v) as the eluting solution. template could recognize some compounds with similar chemical
structure to the template. Although the characterization of FT-IR
3.3. Evaluation of the adsorption properties for the dummy-template indicated the MISPE and NISPE had the same chemical composition,
MIPs their extraction capabilities were significantly different, which would
be due to the imprinting effect of the template.
3.3.1. Extraction capability of the MISPE
The saturated adsorption capacity of RTD and RAT on MISPE 3.3.2. Selectivity of the MISPE
and NISPE were investigated with 100.0 μg/L individual standard The selectivity of the MISPE and the NISPE for RAT, RTD, ISP,
solution. The saturated adsorption capacities of MISPE for RAT, CLB and 4-NP were investigated with 100 μg/L of mixed standard
RTD were 47.5 and 45.9 μg/g, while that of NISPE were 8.8 and solution and the molecular structures of the analytes are shown in

Fig. 5. SERS spectra of different concentrations of RAT (A), ISP (B) and their linear curves. (The concentrations from bottom to top were 0, 20.0, 50.0, 80.0, 100.0, 150.0 and
200.0 μg/L, respectively).

Fig. 6. SERS spectra of the pig tissue samples spiked with different amount of RAT (A) and ISP (B). ((a) 0 μg/kg; (b) 20.0 μg/kg; (c) 50.0 μg/kg.).
 X. Xiao et al. / Talanta 138 (2015) 40–45 45

Table 1 4. Conclusion
Recoveries of RAT and ISP in spiked pork and pig liver samples (n¼ 3).
In the present work, a selective and sensitive MIP-SERS method
Samples Compounds Spiked level (μg/kg)
was developed for ractopamine in complex samples by combining
50.0 100.0 150.0 the selective enrichments of molecular imprinting polymers (MIPs),
the higher capacity of solid phase extraction and the sensitive
Recovery RSD Recovery RSD Recovery RSD detection of SERS. The quantitative detection limits of 10.0 μg/L
(%) (%) (%) (%) (%) (%)
was less than the maximum allowed limits of FDA for ractopamine
Pork RAT 73.4 11.6 76.0 9.7 79.7 11.0 in pig samples. These results suggested that the MISPE and SERS
ISP 72.8 10.5 74.5 7.9 78.2 7.4 method is sensitive and accurate, it is of great significance to
Pig liver RAT 72.4 13.0 74.1 9.7 78.6 9.4 improve the selectivity and sensitivity for detecting trace amounts
of β-agonists in complex samples.
ISP 71.0 10.3 74.0 9.0 78.0 7.6

Fig. 4E. Fig. 4F illustrates the binding amounts of these targets on

Acknowledgments
the MISPE and the NISPE. The extraction amounts of RAT and RTD
in MISPE was 1.51 and 1.17 nmol, which was 8.9 and 7.8 times over
This work was supported by the Major National Scientific Instru-
that in NISPE, suggesting satisfactory selectivity of the MISPE for
ment and Equipment Development Project (2011YQ03012409), the
RTD and cross-recognition for RAT. Notably, best selectivity for RAT
Special Funds of the National Natural Science Foundation of China
was also obtained than that for the dummy-template of RTD. The
(Grant no. 21127008), the National Natural Science Foundation of
reason could be the relatively higher steric hindrance of RTD since
China (Grant nos. 91232703 and 21375155) and Specialized Research
its methyl group was closed to the hydroxyl group in the alkyl
Fund for the Doctoral Program of Higher Education (20120171110001),
chain. And then, better recognition for RAT in MISPE was obtained
respectively.
for the molecular interaction [28] and molecular structure differ-
ence [29]. On the other hand, the MISPE had moderate selectivity
for another analogue of CLB, its extraction amount was 0.69 nmol,
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