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Food Control 33 (2013) 337e343

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Food Control

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Molecularly imprinted polymer-based solid phase clean-up foranalysis of ochratoxin A in ginger and LC-MS/MS confirmation

Jiliang Cao a,b,1, Shujun Zhou a,c,1, Weijun Kong a, Meihua Yang a,d,*, Li Wan b, Shihai Yang c

aKey Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education, Institute of Medicinal PlantDevelopment, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, 100193, Chinab School of Pharmacy, Chengdu University of TCM, Chengdu, 611137, Chinac Jilin Agricultural University, Changchun, 130118, ChinadHainan Branch Institute of Medicinal Plant (Hainan Provincial Key Laboratory of Resources Conservation and Development of Southern Medicine), ChineseAcademy of Medical Sciences & Peking Union Medical College, Wanning, 571533, China

a r t i c l e i n f o

Article history:Received 22 January 2013Received in revised form11 March 2013Accepted 16 March 2013

Keywords:GingerOchratoxin AMolecularly imprinted polymerSolid-phase extractionUPLC-FLRLC-ESI-MS/MS

* Corresponding author. Key Laboratory of BioactivUtilization of Chinese Herbal Medicine, Ministry of EduPlant Development, Chinese Academy of Medical SciCollege, Beijing 100193, China. Tel./fax: þ86 10 57833

E-mail address: yangmeihua15@hotmail.com (M. Y1 These authors contributed equally to this work.

0956-7135/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.foodcont.2013.03.023

a b s t r a c t

Ginger, one of the most commonly used spices and additives, is also widely used to prevent and treathuman disease as a traditional medicine. However, ginger is prone to contamination by ochratoxin A(OTA), which is toxic, carcinogenic and thermostable. Here, a simple, reliable and low-cost method basedon molecularly imprinted polymer (MIP) as selective sorbent of solid-phase extraction (SPE) was pro-posed for the determination of OTA in ginger by ultra-performance liquid chromatography coupled withfluorescence detection (UPLC-FLR). The samples were firstly extracted and then cleaned up with anAFFINIMIP� SPE OTA column for UPLC-FLR analysis. Under the optimized conditions, the limit ofdetection (LOD) and limit of quantification (LOQ) for OTA were 0.09 and 0.30 ng mL�1, respectively. Therecoveries of OTA from ginger spiked at 5, 15 and 25 mg kg�1 ranged from 87.6 to 94.5%. In addition, aftera simple regenerated procedure, the MIP-based SPE column could be reused at least forty-one times toobtain more than 80% recoveries of OTA for ginger samples. The developed method was applied to thedetection of twenty batches of ginger and six samples were contaminated by OTA with levels below thenewest legal limits.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Ginger (Zingiber officinale Rosc.) is one of the most widely usedfreshherbs and spices all over theworld.Meanwhile gingerwas alsoused as a commondietary supplement for its therapeutic benefits totreat nausea (Kawai, Kinoshita, Koyama, & Takahashi, 1994),pregnancy-related nausea (Boone & Shields, 2005; Keating & Chez,2002) and motion sickness (Chrubasik, Pittler, & Roufogalis, 2005)in China and India. It has a long and well-documented history ofmedicinal uses in traditional Chinesemonographs and also recordedas a traditional Chinese medicine in Chinese Pharmacopoeia(Chinese Pharmacopoeia, 2010, p. 273). However, ginger is prone tocontamination by mycotoxins which greatly threatened human

e Substances and Resourcescation, Institute of Medicinalences, Peking Union Medical277.ang).

All rights reserved.

health and several publications have reported that ochratoxin A(OTA, Fig. 1) has been found in ginger with various contaminationlevels (Thirumala-Devi et al., 2001; Whitaker, Trucksess, Weaver, &Slate, 2009).

Ochratoxin A is awidely distributedmycotoxinmainly producedby several fungal species of the genera Aspergillus and Penicillium(Frisvad, Thrane, & Samson, 2007). OTA exhibits multi-facettedtoxicity, including hepatotoxic, nephrotoxic, carcinogenic andimmunotoxic effects, and has been classified as a possible carcin-ogen to humans (Group 2B) by the International Agency forResearch on Cancer (IARC) (IARC, 1993). Moreover, OTA possessesthe longest half-life for its elimination of any of the mycotoxinsexamined (Iha & Trucksess, 2010). The health risk posed by OTA hasbeen assessed in various studies, and a provisional tolerable weeklyintake level of 100 ng kg�1 bodyweight has been established by theJoint Food and Agriculture Organization/World Health OrganizationExpert Committee on Food Additives (JECFA) (JECFA, 2001).Depending on both environmental and processing conditions, OTAis commonly found in a variety of food commodities such as cereals,dried fruits, oleaginous seeds, beans, wine, green coffee, cocoa,

O

OH

Cl

NH

O OCO OH

H

CH3

Fig. 1. Chemical structure of ochratoxin A (OTA).

J. Cao et al. / Food Control 33 (2013) 337e343338

spices, etc (Blesa, Berrada, Soriano, Moltó, &Mañes, 2004; Lee, Saad,Ng, & Salleh, 2012; Romani, Sacchetti, Chaves López, Pinnavaia, &Dalla Rosa, 2000; Sánchez-Hervás, Gil, Bisbal, Ramón, & Martínez-Culebras, 2008; Zinedine et al., 2007). Due to the occurrence ofOTA and the consumer safety purpose, maximum levels, rangingfrom0.5 to80mgkg�1, are set fordifferent foodstuffs in theEuropeanUnion. The newest Commission Regulation (EU) No. 594/2012,amending Regulation 1881/2006, has further reduced maximumlevels of 15 mg kg�1 instead of 30 mg kg�1 for ginger just recently(Commission regulation, 2012).

To date, many different analytical methods have been developedfor the determination of OTA, such as thin-layer chromatography(TLC) (Pittet & Royer, 2002), capillary electrophoresis (CE) (Almeda,Arce, & Valcárcel, 2008), gas chromatographyemass spectrometry(GCeMS) (Olsson, Börjesson, Lundstedt, & Schnürer, 2002; Soleas,Yan, & Goldberg, 2001), liquid chromatography (LC) (Yang et al.,2010) and liquid chromatography-mass spectrometry (LC-MS)(Goryacheva et al., 2007). However, these methods have severaldisadvantages such as long analysis time (Yang et al., 2010), envi-ronmental unfriendly solvents (Olsson et al., 2002) or expensiveinstrumentation (Goryacheva et al., 2007). Ultra-performanceliquid chromatography coupled with fluorescence detection(UPLC-FLR) makes it possible to achieve not only very high-resolution separations in short periods of time with little organicsolvent consumption (Swartz, 2005) but also good sensitivity(Nováková, Matysová, & Solich, 2006) for mycotoxins, the nativefluorescence of OTA favoring the development of a very sensitivemethod (Turner, Subrahmanyam, & Piletsky, 2009).

On the other hand, since the determination of mycotoxins be-longs to trace analysis, sample clean-up and pre-concentrationsteps are necessary to remove matrix components and enhancesensitivity prior to chromatographic analysis. Generally, samplepretreatment for the determination of OTA mainly involves liquideliquid extraction (LLE) (Monaci, Tantillo, & Palmisano, 2004) andsolid-phase extraction (SPE), the latter being preferred due tosimplicity in operation and saving solvent requirements. Home-made and commercially available SPE cartridges with differentkinds of bonding phases such as silica-kieselguhr (Han, Zheng,Luan, Ren, & Wu, 2010), C18 (Romero-González, Martínez Vidal,Aguilera-Luiz, & Garrido Frenich, 2009), OASIS HLB (Varelis,Leong, Hocking, & Giannikopoulos, 2006), ionexchange (Reinsch,Töpfer, Lehmann, & Nehls, 2005) and immunoaffinity (Pascale &Visconti, 2000), have already been applied to the pretreatmentfor decades. Particularly the immunoaffinity columns (IACs),composed of anti-OTA antibodies covalently immobilized on asolid-sorbent, are widely used for the clean-up procedure due totheir high specificity and selectivity toward selected mycotoxins.However, they suffer from some problems such as the relativelyhigh cost, limited capacity, single used and limited shelf-life.

Molecular imprinted technique (MIT) has been investigated tobe an efficient and powerful technique for sample clean-up and

pre-concentration of mycotoxins in applications to differentmatrices successfully (De Smet, Dubruel, Van Peteghem, Schacht, &De Saeger, 2009; De Smet et al., 2010; Weiss, Freudenschuss, Krska,& Mizaikoff, 2003). Molecularly imprinted polymer (MIP) is asynthetic material with an artificially generated three-dimensionalnetwork that is able to specifically rebind a target molecule (Lucci,Derrien, Alix, Pérollier, & Bayoudh, 2010). MIP has the advantages tobe inexpensive, chemically, thermally stable and compatible withall solvents. MIP specifically designed for OTA has already beengenerated using either OTA or a structural analog as template(Jodlbauer, Maier, & Lindner, 2002; Yu & Lai, 2010) and successfullyapplied as SPE sorbent (MIP-SPE) for sample pretreatment (Maier,Buttinger, Welhartizki, Gavioli, & Lindner, 2004). In comparisonwith IAC, MIP-SPE exhibits reusability, simple operation and longerstorage time and is further considered as an alternative to IAC.

The aim of this studywas to develop a simple, sensitive and low-cost method based on MIP as selective SPE sorbents for the deter-mination of OTA in ginger by ultra-performance liquid chroma-tography with fluorescence detection to better control thecontamination of OTA. In addition, the reusability of the MIP-SPEcolumn was also evaluated for the purpose of reducing the sam-ple testing costs compared with IAC. The method developed herewas applied to twenty ginger samples to investigate the contami-nation of OTA and the positive samples were analyzed by liquidchromatography tandem electrospray ionization mass spectrom-etry (LC-ESI-MS/MS) in the multiple reaction monitoring (MRM)mode for confirmation.

2. Experimental

2.1. Chemicals and reagents

A standard solution of ochratoxinA (1mgmL�1 inmethanol)waspurchased from ALEXIS (Lausen, Switzerland) and stored at �18 �C.Molecularly imprinted solid-phase extraction columns for OTA(AFFINIMIP� SPE Ochratoxin A) were provided by Polyintell (Val deReuil, France). Immunoaffinity columns for OTA (OchraTest�) andglass microfiber filters (MG 550-HA, 110 mm) were obtained fromVicam (Watertown,MA,USA).HPLC-grademethanolwas purchasedfrom Honeywell (Burdick & Jackson, USA). Other reagents andchemicals were of analytical grade and water was Wahaha purifiedwater (Wahaha, Hangzhou, China).

Phosphate-buffered saline (PBS, pH 7.0) was prepared by dis-solving 0.20 g potassium chloride, 0.20 g monobasic potassiumphosphate, 8.00 g sodium chloride and 1.2 g dibasic sodiumphosphate in 1 L of double-distilled water.

2.2. Samples

Twenty batches of ginger samples were purchased from severaldifferent local markets (Beijing, China). The samples were stored inplastic bags at 4e5 �C in a fridge until analysis.

2.3. Extraction and clean-up based on MIP-SPE

Twenty-grams of ginger samples were extracted by sonicationfor 20 minwith 40 mL of acetonitrile/water (60/40, v/v). The extractwas filtered and an aliquot of 20 mL of filtrate were diluted with20 mL of water and then adjusted to pH 1.0 with 1.0 M hydrochloricacid (HCl). After filtrated through a glass microfiber filter, 4 mL (1 gequivalent) of the filtratewere then passed through the AFFINIMIP�

SPE Ochratoxin A cartridge at a flow rate of 1 drop per second,previously conditioned with 5 mL of acetonitrile and equilibratedwith 5 mL of water. The cartridge was washed with 5 mL of 0.1 MHCl/acetonitrile (60/40, v/v) at theflowrateof 1e2drops per second.

Table 1Recovery data of ochratoxin A (OTA) from spiked ginger samples, n ¼ 3.

Spiking level (mg kg�1) Recovery (%) Mean (%) RSD (%)

1 2 3

5.0 89.4 92.9 94.5 92.3 2.815.0 93.1 87.6 92.5 91.1 3.325.0 89.8 92.7 93.0 91.9 1.9

Fig. 2. Optimization of MIP-SPE conditions. *The optimal condition; Wash 1: HCl0.1 M/acetonitrile (60/40, v/v), Wash 2: HCl 0.1 M/acetonitrile (50/50, v/v), Wash 3: HCl0.1 M/methanol (60/40, v/v) and Wash 4: HCl 0.1 M/methanol (50/50, v/v).

J. Cao et al. / Food Control 33 (2013) 337e343 339

At last, OTAwas eluted with 2 mL of methanol containing 2% aceticacid at a flow rate of 1 drop per second and collected in a clean vial.

The eluates were evaporated to dryness under a nitrogen streamat 50 �C and the residues were dissolved in 1 mL of mobile phaseprior to injection for UPLC analysis. The cartridge can be reusedafter cleaning with 10 mL of methanol.

2.4. Extraction and clean-up using immunoaffinity column (IAC)

Ten-grams of ginger samples with 2 g NaCl were extracted with50 mL of extraction solution methanol/water (80/20, v/v) by soni-cation for 20 min. The extract were filtered through a filter paperand 10 mL of extract were diluted with 40 mL of PBS solution in a100 mL conical flask. After filtration through a glass microfiberfilter, 25 mL of the filtrate were passed through an OchraTest�immunoaffinity column at the flow rate of approximately 1 dropper second. Wash the immunoaffinity column with 20 mL of waterat a flow rate of 1e2 drops per second until 2e3 mL of air passedthrough the column. Then, the analytes were eluted into the vialwith 2 mL of methanol at a flow rate of 1 drop per second. Theeluates were evaporated to dryness under a stream of nitrogen at50 �C. The residues were reconstituted in 1 mL of the mobile phaseand 1 mL was analyzed in UPLC.

2.5. Apparatus and UPLC conditions

UPLC was carried out on a Waters Acquity UPLC H-Class system(Waters, MA, USA) equipped with quaternary solvent deliverypump, an auto sampler and fluorescence detector, connected toWaters Empower data software. The chromatographic separationwas performed on a Waters Acquity UPLC HSS T3 column(50 mm � 2.1 mm, 1.8 mm) operated at 30 �C.

OTA was detected at lex 333 nm and lem 460 nm. The mobilephase consisted of methanol/0.5% aqueous acetic acid (65/35, v/v)at a flow rate of 0.2 mL min�1 and the sample injection volumewas1 mL. Under optimum conditions, the analysis took around 4.0 min.

2.6. Reusability of MIP-SPE cartridges

In order to assess the reusability of MIP-SPE columns, 50consecutive clean-up cycles were performed on a single MIP-SPEcolumn for spiked ginger samples (15 mg kg�1). After each cycle,the column was washed with 10 mL of methanol for the regener-ation of polymers, which could avoid carry-over effects betweenthe individual cycles.

2.7. LC-MS/MS conditions

A Shiseido Nanospace SI-2 HPLC system (Shiseido, Japan)coupled to an API 5500 triple-quadrupole mass spectrometerequipped with an electrospray ionization (ESI) source (AB SCIEX,Foster City, CA, USA) was used for the LC-MS/MS analysis. AppliedBiosystems Analyst software (version 1.5.1) was used to control theLC-MS/MS system and for data acquisition and processing. OTAwasseparated on a Capcell Core C18 column (50 mm � 2.1 mm, 2.7 mm)(Shiseido, Japan) operated at 30 �C. The mobile phase consisted ofmethanol-0.5% aqueous acetic acid (65:35, v/v) at a flow rate of0.3 mL/min and the sample injection volume was 2 ml. The massspectrometer was operated in the positive ESI mode with MRM atunit resolution. Two precursor-to-product ion transitions weresimultaneously monitored (in parentheses: collision energy, CE;collision cell exit potential, CXP; declustering potential, DP): m/z404/ 358 (CE 19 V, CXP 15 V, DP 120 V), m/z 404/ 239 (CE 31 V,CXP 15 V, DP 120 V). The operating parameters used for ionizationsource were set as follows: curtain gas (CUR), 33 psi; nebulizer gas

(GS 1), 50 psi; auxiliary gas (GS 2), 50 psi; ion spray voltage (IS),4500 V; and source temperature, 500 �C.

3. Results and discussion

3.1. UPLC method development

In order to achieve optimal UPLC parameters for the determi-nation of OTA, the peak area, efficiency and analysis time weretaken into account.

As OTA is a weak acid, the mobile phase must be acidic to avoidstrong tailing and unspecific adsorption to the column (Valenta,1998). Therefore, different compositions of mobile phase (waterwith acetic acid from 0.1% to 1.0% as solvent A and acetonitrile,methanol or a mixture of both of them as solvent B) were investi-gated andfinallymethanol/0.5% aqueous acetic acid (65/35, v/v)wasselected as a preferred mobile phase due to its better separationefficiency, shorter analysis time (w3.3 min) and lower toxicity.Furthermore, the effect of column temperatures, flow rates and in-jection volumes had also been evaluated. As a comparison betweenseparation speed and sensitivity, the following conditions wereadopted: mobile phase composition, methanol/0.5% aqueous aceticacid (65/35, v/v); flow rate, 0.2 min mL�1; column temperature,30 �C; and injection volume, 1 mL. Under the optimized chromato-graphic conditions, OTA was well separated in less than 4.0 min,which was shorter than most of the published methods (González-Osnaya, Soriano, Moltó, & Manes, 2008; Reiter et al., 2011).

The excitation and emission wavelengths are the most impor-tant parameters to be optimized in fluorescence detection, whichdepend not only on the absorption of the target analytes but also onthe operational conditions (Kong et al., 2012). To obtain the optimal

J. Cao et al. / Food Control 33 (2013) 337e343340

wavelengths, an OTA standard solution of 100 ng mL�1 wasanalyzed under isocratic conditions. According to the results ofrepeated preliminary experiments, the optimal excitation andemission wavelengths were 333 and 460 nm, respectively.

3.2. Optimization of MIP-SPE conditions

The influence of several parameters such as the pH value ofloading solution, the composition of washing solution, volume ofwashing and eluting solution was evaluated to identify key pa-rameters that affected MIP-SPE efficiency (Fig. 2). Optimizationwascarried out by triplicate analysis with non-contaminated gingersamples spiked with OTA at 15 mg kg�1.

As is known, OTA is a weak acid, with pKa values of 4.4 for thecarboxylic and 7.5 for the phenolic groups (Aresta, Palmisano,Vatinno, & Zambonin, 2006). Therefore, the acidic environment ofthe loading solution is extremely necessary for the absorption ofOTA as completely as possible. In this research, the pH values of theloading solutions were adjusted to 0.5, 1.0 and 1.5 with the additionof 1.0 M HCl and finally the results showed that the optimum pHvalue was 1.0 (Fig. 2). Therefore, the loading solution was carriedout at pH 1.0 for the rest of the study.

Different solvent mixtures and volumes were assessed in orderto select a suitable solvent combination to eliminate interferents forwashing step. Mixtures of 0.1 M HCl/acetonitrile (60/40, v/v), 0.1 MHCl/acetonitrile (50/50, v/v), 0.1 M HCl/methanol (60/40, v/v) and0.1 M HCl/methanol (50/50, v/v) were tested. Better washing effi-ciency with high recoveries was obtained when using the mixtureof 0.1 M HCl/acetonitrile (60/40, v/v), so it was selected as thewashing solution. And then, the volumes of washing solution (3, 5,7 and 9 mL) were investigated and it was found that 5 mL couldafford not only the similarly high recovery compared to 3 mL(Fig. 2) but also better chromatograms with low response ofinterferents. Hence the volume of 5 mL was chosen as the washingvolume for the subsequent experiment.

When optimizing the elution conditions, the addition of aceticacid in the eluting fraction appears absolutely necessary for acomplete elution (Ali et al., 2010). Six desorption solvents includingmethanol, methanol/water (90/10, v/v), methanol/acetic acid (98/2,v/v), acetonitrile, acetonitrile/water (90/10, v/v) and acetonitrile/acetic acid (98/2, v/v) as reported by Lee, Saad, Khayoon, and Salleh(2012) were tested. As a result, methanol/acetic acid (98/2, v/v) waspreferred as the eluting solution with the largest peak area. For thenext study, different volumes (1, 2, 3 and 4 mL) of methanol/aceticacid (98/2, v/v) were used for the test of eluting efficiency. The re-coveries had no significant increase when using more than 2 mL ofeluting solutions (Fig. 2). Therefore, 2 mL of methanol/acetic acid(98/2, v/v) was selected as the eluting volume.

3.3. Method validation

The established UPLC method was assessed for linearity, limitof detection (LOD), limit of quantification (LOQ), recovery, andprecision.

3.3.1. Linearity, LOD and LOQCalibration curve for OTA standards was prepared by diluting

appropriate volumes of the standard solutionwith methanol/water(50/50, v/v) into nine different concentrations (0.5e100 ng mL�1)with triplicate injections. The calibration curve was constructed byplotting the peak areas (y) versus the concentrations (x) of OTA

Fig. 3. Typical chromatograms of (a) OTA standard solution (15 ng mL�1); (b) blankginger sample after clean-up with MIP-SPE; ginger sample spiked with OTA at the levelof 15 mg kg�1 after clean-up with (c) MIP-SPE and (d) IAC.

Table 2Precision study of the established method.

Spiking level(mg kg�1)

Intra-dayprecision (RSD, %)

Inter-dayprecision (RSD, %)

5.0 2.5 3.715.0 1.8 2.225.0 1.6 2.4

Table 3OTA concentrations in ginger samples.

Sample no. OTA (mg kg�1) Sample no. OTA (mg kg�1)

1 NDa 11 0.702 ND 12 7.123 0.82 13 ND4 ND 14 <LOQ5 1.65 15 <LOQ6 1.34 16 7.567 ND 17 ND8 ND 18 ND9 <LOQ 19 ND10 ND 20 <LOQ

a ND: no detected.

J. Cao et al. / Food Control 33 (2013) 337e343 341

obtained from UPLC analysis. The regression equation wasexpressed as y ¼ 3999x þ 1773 with correlation coefficients (R2) of0.9999. The LOD and LOQ were determined by injecting serialdiluted standard solutions based on the signal to noise (S/N) ratio of3:1 for LOD and 10:1 for LOQ. The LOD and LOQ for OTA standardwere 0.09 and 0.30 ng mL�1, respectively.

3.3.2. Recovery, intra-day and inter-day precisionIn order to test the reliability of the proposed method, recovery

studies were carried out by spiking OTA of three different con-centrations to the non-contaminated ginger samples. Each fortifiedsample mixture was extracted and analyzed in triplicate pursuantto themethod described above. As reported in Table 1, all recoveriesranged from87.6% to 94.5% and the corresponding relative standarddeviation (RSD) values were between 1.9% and 3.3%, indicating thatthe developed method was considered appropriate for detection ofOTA in ginger. As can be seen in typical chromatograms of a blankand a spiked ginger sample presented in Fig. 3b and c, no relevantinterferences were observed in the chromatogram of blank sampleat the retention time of OTA, due to the high specificity of theproposed method.

The precision of the established method was estimated in termsof measuring intra- and inter-day precision by application of thewhole procedure to non-contaminated ginger samples spiked atthree concentration levels of OTA and the data are summarized inTable 2. For intra-day precision test, the samples were determinedon six replicates within one day, while the samples were examinedduring five consequent days for inter-day precision test. The rangesof intra-day and inter-day precision, expressed as the RSD of peakareas of OTA, were 1.6e2.5% and 2.2e3.7%, respectively.

3.4. Reusability of MIP-SPE cartridges

Although reusability has been claimed for immunoaffinity col-umns, the complete regeneration of the selective binding properties

Fig. 4. Recovery of OTA quantified in spiked ginger sample (15 mg kg�1) after multipleSPE cycles on a single MIP column.

was found to be challenging and was reported to require extendedincubation (>10 h) in special buffers at low temperatures (Zimmerli& Dick, 1996). Compared with IACs, polymers in MIP-SPE columnscould be cleaned and reactivated under relatively harsh conditionsfor repeated uses owing to their high chemical robustness.

The result showed that though the regenerate polymers showedindications of irreversible adsorptions of some dye componentsfrom the matrix, the performance of the MIP-SPE column remaineduncompromised for the sample clean-up and the data were pre-sented in Fig. 4. From Fig. 4, it can be observed that the recoverywas still more than 80% even after forty-one reuse cycles high-lighting the good reusability and high efficiency for the singlecolumn. Then it dropped to less than 80% ranged from 79.6% to75.8% for the next consecutive nine cycles which seemed to be alsoacceptable. However, it was worth noting that, due to many irre-versible adsorptions of impurities on the sorbents, the loading andwashing solutions were increasingly difficult to pass through thecolumn even using a vacuum and the total time spent on the clean-up procedure became longer than before which lacked of simplicityand rapidity. Therefore, a single MIP-SPE column could afford highrecoveries of OTA in at least forty-one reuse-times with respect tothe clean-up of ginger.

3.5. Comparison of MIP-SPE with immunoaffinity column

The potential of MIP was evaluated through comparing chro-matograms obtained by analyzing the elution part resulting fromMIP and IAC purification of spiked ginger samples. These chro-matograms are presented in Fig. 3c and d. Comparedwith IAC, more

Fig. 5. Typical chromatograms of naturally contaminated ginger sample after clean-upwith MIP-SPE.

a b

Fig. 6. Typical chromatograms of XIC chromatograms of positive ginger sample with (a) MRM mass transitions 404e358 and (b) MRM mass transitions 404e239.

J. Cao et al. / Food Control 33 (2013) 337e343342

responses of interferents were observed in the chromatogram ofginger sample spiked with OTA at the level of 15 mg kg�1 afterclean-up with MIP-SPE, whereas these had no effect on the sepa-ration of OTA under the optimal chromatographic conditions.Moreover, similar recoverieswere obtainedwith 91.1% and 91.5% onMIP and IAC, respectively. Although IACs offer high selectivity andreproducibility, they are relatively expensive and single use. On thecontrast, as previously mentioned, the MIP-SPE column could bereused up to forty-one times with high recoveries of more than 80%which greatly reduced the cost of sample pretreatment in com-parison with IACs.

3.6. Real sample analysis

The established method was applied for the determination ofOTA in twenty ginger samples collected from local markets inBeijing, China. OTAwas detected in six (30%) ginger samples rangedfrom 0.70 to 7.56 mg kg�1 (Table 3). The typical chromatogram ofcontaminated ginger samples at 7.12 mg kg�1 was shown in Fig. 5.

As a result, the OTA levels of the contaminated ginger samplesdid not exceed the legal limits newly established by the EuropeanUnion (15 mg kg�1 for ginger). However, considered both OTAtoxicity and the large consumption of ginger all over the world,more attention should be paid to not only the development of moresensitive detection methods but also the prevention strategies tomonitor and manage the OTA contamination of ginger duringstorage and processing.

3.7. Confirmatory results by LC-ESI-MS/MS

The occurrence of OTA in all positive samples was furtherconfirmed by LC-ESI-MS/MS. Retention time and fragment ions ofOTA (m/z 404/ 358 and m/z 404/ 239) from LC-ESI-MS/MSanalysis were used to confirm the positive samples. As a result,there were no false positive samples, which indicated that theproposed method was sensitive and reliable. Typical MRM chro-matograms of OTA in positive ginger samples were shown in Fig. 6.

4. Conclusion

A simple and rapid method based on molecularly imprintedpolymer as selective SPE sorbents has been established for thedetermination of OTA in ginger by UPLC coupled with fluorescence

detection, and the validated method shows satisfactory linearity,precision and accuracy. Moreover the developed method was suc-cessfully applied for the analysis of 20 ginger samples which werewidely used as medicinal and edible substances in China.

Additionally, the reusability of the MIP-SPE columns was eval-uated and the column showed an excellent reusability that morethan 80% recoveries of OTA could be achieved in at least forty-onetimes of reuse for ginger samples, which could greatly reduce thetesting costs for large numbers of ginger samples in comparisonwith IACs. MIP-SPE exhibits reusability, simplicity and longerstorage time and can be considered as an alternative to thecommonly used IAC technique for the sample clean-up and pre-concentration. In a future study, we would focus on the detectionof OTA in more complex medicinal and edible matrices based onMIP-SPE clean up to prevent the healthy threat of mycotoxins toconsumers.

Acknowledgment

We are grateful to Sami Bayoudh and Johann Travers from Pol-yintell (Val de Reuil, France) for their expert technical assistance.The research was supported from National Science Foundation ofChina (No. 81274072 and 81173539) and the Program for Chang-jiang Scholars and Innovative Research Team in University ofMinistry of Education of China (Grant no. IRT1150).

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