- Email: [email protected]
Determination of ochratoxin A in wines by capillary liquid chromatography with laser induced fluorescence detection using dispersive liquid–liquid microextraction
Food Chemistry 135 (2012) 368–372
Contents lists available at SciVerse ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Analytical Methods
Determination of ochratoxin A in wines by capillary liquid chromatography with laser induced fluorescence detection using dispersive liquid–liquid microextraction Natalia Arroyo-Manzanares, Laura Gámiz-Gracia, Ana M. García-Campaña ⇑ Department of Analytical Chemistry, Faculty of Sciences, University of Granada, Campus Fuentenueva s/n, E-18071 Granada, Spain
a r t i c l e
i n f o
Article history: Received 11 July 2011 Received in revised form 24 April 2012 Accepted 2 May 2012 Available online 11 May 2012 Keywords: Ochratoxin A Capillary-HPLC Laser induced fluorescence detection Dispersive liquid–liquid microextraction Wine
a b s t r a c t A method based on reverse phase capillary high performance liquid chromatography (capillary HPLC) coupled to laser-induced fluorescence detection (LIF) has been proposed for the determination of ochratoxin A (OTA) in wine samples. An anionic micellar medium was added to the mobile phase for increasing the fluorescence intensity and peak efficiency. Dispersive liquid–liquid microextraction (DLLME) has been used as a simple and efficient sample pretreatment method for the analysis of OTA in wines, being optimised by means of experimental design. The limit of detection was 5.5 ng L 1 (3 S/N) and recoveries for different wines ranged from 91.7 to 98.1%. The proposed methodology could be classified as a green analytical chemistry alternative, combining the low organic solvent volumes required in the DLLME with the reduced consumption of mobile phase in capillary HPLC. The use of LIF as detector provided an extremely sensitive method for the determination of OTA in wines. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Ochratoxin A is a mycotoxin naturally found in various foods, including cereals, wine or coffee. It is produced by several species of Aspergillus (A. carbonarius) and Penicillium (P. verrucosum) moulds. As it has potent nephrotoxic, carcinogenic and teratogenic effects, the International Agency for Research of Cancer (IARC) has classified OTA in group 2B, as a possible human carcinogen (IARC, 1993). OTA has been widely detected in cereals (e.g. wheat, barley and maize), cereal-derived products (Duarte, Pena, & Lino, 2010), dried fruits (Bircan, 2009), spices (Santos, Marín, Sanchis, & Ramos, 2010), beer and wine (Mateo, Medina, Mateo, Mateo, & Jiménez, 2007). After cereals, wine is considered the major source of daily OTA intake. Thus, European Union (EU) has set maximum levels for this compound at 2 lg kg 1 in wine samples (European Commission, 2006a). As a consequence, very sensitive analytical methods for OTA detection, with performance characteristics fulfilling the established legislation are needed (European Commission, 2006b). The most common analytical method for OTA determination in foodstuff is HPLC coupled to fluorescence detection (FL) (González-Osnaya, Soriano, Moltó, & Mañes, 2008; Hernández, García-Moreno, Durán, Guillén, & Barroso, 2006; Sáez, Medina, ⇑ Corresponding author. Tel.: +34 958 242385; fax: +34 958 249510. E-mail address: [email protected] (A.M. García-Campaña). 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.05.009
Gimeno-Adelantado, Mateo, & Jiménez, 2004; Tessini et al., 2010), including the reference method described by the European Standard EN 14133 for the determination of OTA in wine (European Committee for Standardization, 2003). Alternative detection methods, such as photodiode array detection (LC–DAD) (Soleas, Yan, & Goldberg, 2001) or mass spectrometry (LC–MS) and tandem MS (LC–MS/MS) (Zöllner & Mayer-Helm, 2006) have also been proposed. Others applied techniques are thin layer chromatography (TLC) (Welke, Hoeltz, Dottori, & Noll, 2010), gas chromatography coupled with MS (GC–MS) (Olsson, Borjesson, Lundstedt, & Schnurer, 2002), enzyme immunoassay (EIA) (Saha, Acharya, Roy, Shrestha, & Dhar, 2007) and fluorescence polarisation immunoassay (Zezza, Longobardi, Pascale, Eremin, & Visconti, 2009). Also, capillary electrophoresis (CE) with UV/Vis detection has also been proposed for the determination of OTA in wine (Almeda, Arce, & Valcárcel, 2008; González-Peñas, Leache, López de Cerain, & Lizarraga, 2006). The use of laser induced fluorescence (LIF) as a more sensitive detection system has been proposed for the determination of OTA in different food samples (roasted coffee, corn, and sorghum) using CE (Corneli & Maragós, 1998). Concerning the sample treatment, different extraction and cleanup methods for the determination of OTA have been reported. Most of them are based on immunoaffinity columns (IACs), which contain specific antibodies to the analyte of interest, allowing higher cleanup and sample purification. However, this methodology is high-cost and tedious. Liquid–liquid extraction (LLE) in milk, wine, beer and must (Sáez et al., 2004; González-Osnaya et al., 2008),
N. Arroyo-Manzanares et al. / Food Chemistry 135 (2012) 368–372
solid-phase extraction (SPE) in wine, beer and must (Sáez et al., 2004; Hernández et al., 2006; Tessini et al., 2010), SPE with molecular imprinted polymers (MISPE) in different matrices (Yu & Lai, 2010) or pressurised liquid extraction (PLE) in rice (Juan, González, Soriano, Moltó, & Mañés, 2005), have been also used for the determination of OTA. The stability of OTA during its extraction using microwave-assisted extraction (MAE), PLE, ultrasound-assisted and magnetic stirring-assisted extraction, has also been evaluated (Liazid, Palma, Brigui, & Barroso, 2007). Some reviews devoted to the analysis of OTA and mycotoxins compile most of these analytical and extraction methodologies (Turner, Subrahmanyam, & Piletsky, 2009; Cigic´ & Prosen, 2009). Recently, dispersive liquid–liquid microextraction (DLLME) has been introduced for treatment of liquid samples (Rezaee, Yamini, & Faraji, 2010; Zang, WU, Zhang, Xi, & Wang, 2009; Herrera-Herrera, Asensio-Ramos, HernándezBorges, & Rodríguez-Delgado, 2010), and has been recently used in the determination of OTA in wine by LC-MS (Campone, Piccinelli, & Rastrelli, 2011). It is based on the use of a ternary component solvent system, where an appropriate mixture of a few microlitres of a organic extraction solvent (usually with a density higher than water), and a small volume of a disperser solvent (miscible with the extraction solvent and with water), is injected rapidly into an aqueous sample, resulting in the formation of a stable emulsion. The organic analytes present in the aqueous sample are rapidly extracted into the extraction solvent as a result of the large contact surface between the organic and the aqueous phases. Phase separation is performed by centrifugation and an organic phase with the analytes of interest is settled in the bottom of a conical tube and subsequently analysed by an appropriate technique. In this paper, we propose a method based on DLLME followed by capillary HPLC–LIF for the very sensitive determination of OTA in wine samples, combining the advantages of capillary HPLC (better resolution, lower detection limits and lower solvent consumption) with the low detection limits provided by LIF detection. This methodology could be classified among the alternatives proposed by the recent trends of green analytical chemistry (Tobiszewski, Mechlinska, Zymunt, & Namiesnik, 2009; Welch et al., 2010), as it achieves a reduction of organic solvent per analysis, thanks to both the applied extraction technique and the capillary HPLC system.
2. Material and method 2.1. Chemicals All the reagents were analytical reagent grade, solvents were HPLC grade and OTA was analytical standard grade. Methanol (MeOH), ethanol (EtOH), acetonitrile (ACN), sodium chloride (NaCl) and sodium dodecyl sulphate (SDS) were supplied by Panreac (Madrid, Spain); acetic acid, chloroform and tetrachloroethylene were purchased from VWR BDH Prolabo (West Chester, Pensilvania, USA); carbon disulphide was obtained from Carlo Erba (Rodano, MI, Italy); tetrahydrofuran (THF) and acetone (ACO) was supplied by Merck (Darmstadt, Germany); chlorobenzene was obtained from Alfa Aesar (Karlsruhe, Germany) and b-cyclodextrin was purchased from Sigma–Aldrich (St. Louis, MO, USA). Ultrapure water (18.2 MO cm 1, Milli–Q Plus system, Millipore Bedford, MA, USA) was used throughout the work. Acrodisc 13 mm syringe filters with 0.2 lm nylon membrane (Pall Corp., MI, USA) were used for filtration of samples prior to the injection in the chromatographic system. A 0.2 lm nylon membrane filter (Supelco, Bellefonte, PA, USA) was used for filtration of mobile phase. A stock standard solution of 10 lg mL 1 of OTA in ACN was obtained from Supelco. Working solutions were prepared by evaporation of the stock standard solution to near dryness using a gentle
369
stream of N2, and diluting with the appropriate amount in MeOH and H2O. These solutions were stored at 20 °C. 2.2. Instruments and equipments All experiments were carried out using an Agilent HP-1200 series capillary HPLC (Agilent Technologies, Waldbron, Germany) equipped with a binary pump (20 lL min 1 maximum flow-rate), online degasser, autosampler (8 lL loop), and a column thermostat. This system was coupled with a LIF detector (Zetalif Evolution model LIF UV-01, Picometrics S.A., Ramonville, France) equipped with a 325 nm HeCd laser. A fused-silica capillary (75 lm I.D.) from Polymicro Technologies (Phoenix, AZ, USA) was used to coupled the HPLC and LIF detector. ChemStation software (A.10.20 [1757] version) was used for data acquisition and processing. A Luna C18 (150 0.5 mm, 5 lm) from Phenomenex (Torrance, CA, USA) was used as chromatographic column. A centrifuge Model Universal 320R (Hettich, Tuttlingen, Germany), a vortex-2 Genie (Scientific Industries, Bohemia, NY, USA), a mechanical shaker (model 384 from Vibromatic, Noblesville, USA) and a Evaporator EVA EC-S/EVA LS-S (VLM GmbH, Bielefeld, Germany) were also used for sample treatment. The statistic software STATGRAPHICS Centurion XV.II was used for data treatment. 2.3. Optimum chromatographic conditions The analyses were performed on a Luna C18 column (150 0.5 mm, 5 lm, 100 Å). An isocratic mobile phase of water (2% acetic acid, 0.2 M SDS): MeOH (30:70, v/v) at a flow rate of 14 lL min 1 was used; the column temperature was kept at 40° C and the injection volume was 1.20 lL. Under optimum conditions, the analysis took around 3.5 min. 2.4. Sample preparation White, rose and red wine samples were purchased from a local market. An aliquot of 5.0 mL of sample was placed into a 10 mL screw cap test tube with conical bottom and 0.25 g of NaCl (5%; w/v) were added. The mixture of the disperser solvent (940 lL of ACN) and the extraction solvent (660 lL of chloroform) was rapidly injected into the test tube with a 2.0 mL syringe. The solution was shaken and a cloudy solution was formed in the tube. In this step, the OTA was extracted into the fine droplet of chloroform. Later, the mixture was centrifuged at 5000 rpm for 1 min and the fine particles were sedimented at the bottom of the tube. The sedimented phase (approximately 700 lL) was removed using a 1 mL syringe, evaporated to near dryness using a gentle stream of N2 and reconstituted with 1 mL of MeOH:H2O (v/v). The solution was filtered and injected into the capillary HPLC–LIF system for analysis. 3. Results and discussion 3.1. Optimisation of capillary HPLC–LIF method Peak area, efficiency and analysis time were taken into account to select the adequate determination procedure. Different natures of mobile phase (water with acetic acid from 1% to 5% as solvent A, and ACN, MeOH or a mixture of both of them as solvent B) were tested. The best results were obtained with an isocratic phase consisting on water (2% acetic acid): MeOH (30:70, v/v). The use of cyclodextrins has been previously reported as modifiers of native fluorescence of mycotoxins (Maragos et al., 2008). Thus, the addition of 6 mM b-cyclodextrin to solvent A was considered. However,
370
N. Arroyo-Manzanares et al. / Food Chemistry 135 (2012) 368–372
the improvement in the sensitivity was not significant and a higher noise in the baseline was observed. As an alternative, we proposed the addition of a micellar anionic media (SDS) to solvent A. The addition of this anionic surfactant at a concentration above its critical micellar concentration not only provided an increase on fluorescence quantum yield of the analyte, as could be expected as effect of this organised media on fluorophore solutions (McIntire, 1990), but also improved the chromatographic efficiency, giving a sharper peak. Thus, concentrations of 0.1, 0.2 and 0.3 M of SDS were tested, obtaining the best efficiency and the highest signal in a shorter time with 0.2 M of SDS in the aqueous phase. Higher concentrations did not improve the analysis; so 0.2 M was selected for the rest of experimental work. The flow rate was also tested between 10–15 lL min 1, selecting a final optimum value of 14 lL min 1, as higher flows involved higher pressure without a significant improvement in the analysis. The effect of temperature was examined in the range of 30–50 °C; an optimum value of 40 °C was chosen, as a compromise between analysis time and of column life. Finally, the injection volume was increased from 0.6 to 1.4 lL, selecting 1.2 lL as optimum. At these final conditions, the analysis of OTA took less than 3.5 min. 3.2. Optimisation of DLLME The optimisation of DLLME involved the study of the following parameters: nature and volume of extraction solvent, nature and volume of disperser solvent, percentage of NaCl, extraction time and shake mode. All the experiments were performed using samples of 5 mL of white wine spiked with 400 ng L 1 of OTA, and the goal of this optimisation was to obtain the highest absolute recovery of OTA. First, it was necessary to select the extraction and disperser solvents. Chloroform, carbon disulphide, chlorobenzene and tetrachloroethylene were tested as extraction solvents, and
MeOH, EtOH, ACO, ACN and THF were tested as disperser solvents. The best results were obtained with a combination of chloroform and ACN. Once the extraction and disperser solvents have been selected, their volumes and the percentage of NaCl were optimised by a multivariate approach using an experimental design, which takes into account possible interactions between the variables. A central composite design (23 + star, face centered), with three spaced central points, involving 17 runs, was used an as approach to generate the response surface, using the recovery percentage as analytical response. The different factors were studied in the following ranges: ACN volume (800–1000 lL); chloroform volume (500–700 lL); and percentage of NaCl (0–5%). A Pareto chart (Fig. 1A) was obtained from the screening experimental design, showing what variables and/or interactions between them have significant effects when their value is changed inside the selected experimental domain. The extraction solvent volume and the quadratic terms of extraction solvent volume, disperser solvent volume and percentage of salt (AA, BB and CC, respectively) had a significant effect on the recovery of OTA; so finally the three variables were simultaneously optimised. The obtained response surface (Fig. 1B) gave the optimum conditions for DLLME, being as follows: extraction solvent = 660 lL; disperser solvent = 940 lL; percentage of salt = 5%. The P-value for the lack of fit test for the model was 38.0% and the determination coefficient (R2) was 93.9%, showing the suitability of this design. Under the previous optimum conditions, the effects of extraction time (defined as the interval between the injection of disperser and extraction solvents, and centrifugation) and shake mode (mechanical shake, vortex, and manual shake) were tested in the range of 0–10 min. It was concluded that these variables had no influence in the extraction efficiency. The reason is that extraction solvent can be evenly dispersed after the formation of the cloudy solution, the transition of analyte from the sample to
Fig. 1. (a) DLLME screening design: Pareto chart showing the effects of the studied variables on the recovery percentage: (+) Positive effects on the response; ( ) negative effects on the response. Blue line shows the limit of decision to consider the significance of the factors (based on the effect = estimated effect/standard error, P-value = 0.05 at 95% of confidence); (b) Estimated response surface obtained in the optimisation procedure using a central composite design (23 + star, face centered), with three spaced central points.
371
N. Arroyo-Manzanares et al. / Food Chemistry 135 (2012) 368–372
the extraction phase can be very fast, and the equilibrium state can be subsequently achieved very quickly, resulting in a very short extraction time (Zang et al., 2009). Thus, the extraction was carried out by shaking the sample manually for a few seconds. Finally, under the optimum conditions, an enrichment factor of 6.6 (calculated as the ratio of analyte concentration in the sediment, and the initial concentration of analyte in the aqueous sample) and a final preconcentration factor of 5 was achieved. 3.3. Validation of the method In order to check the suitability of the proposed method of DLLME-capillary HPLC–LIF for the determination of OTA in wine samples, it was characterised in terms of linear dynamic range, limit of detection and quantification, precision and trueness. Table 1 Precision study of the whole method. The results are expressed as RSD of peak areas.
Level 1 (0.2 lg L Level 2 (1.0 lg L Level 3 (2.0 lg L
1
) 1 ) 1 )
Repeatability (n = 9)
Intermediate precision (n = 15)
1.9 2.6 1.3
4.9 2.9 5.2
1
Level 2 (1.0 lg L
1
Level 3 (2.0 lg L
1
) ) )
R (%) RSD (%) R (%) RSD (%) R (%) RSD (%)
The matrix-matched calibration curve was obtained using white wine samples spiked with the following concentrations of OTA: 0.02, 0.1, 0.2, 1, 2 and 4 lg L 1. These concentrations, after the sample treatment by DLLME, gave concentrations of OTA in the final extracts of 0.1, 0.5, 1, 5, 10 and 20 lg L 1, respectively. Each concentration level was prepared by duplicate and injected by triplicate. A blank sample was also analysed and no interferences from the matrix were found co-migrating with the analyte. The statistical parameters were calculated by least-square regression, and the resulting calibration curve was: Area = 27790.0 Concentration + 711.3 (R2 = 0.998). The standard deviations of the slope and intercept were 200.7 and 334.2, respectively. The limit of detection (LOD) and the limit of quantification (LOQ) in the sample were 5.5 ng L 1 (3 S/N) and 18.4 ng L 1 (10 S/N), respectively. As can be seen, the sensitivity of the method allows the quantification of OTA at concentrations much lower than the maximum level established for wine samples (European Commission, 2006a). 3.5. Precision study
Table 2 Data about the recovery assay to the whole method for white, rose and red wine. Each level was prepared by triplicate and injected by triplicate (n = 9).
Level 1 (0.2 lg L
3.4. Calibration curve and performance characteristics of the method
White wine
Rose wine
Red wine
94.9 1.9 94.8 2.9 91.7 1.4
94.4 3.9 92.7 2.2 95.8 1.7
98.3 4.1 96.3 1.7 98.1 1.8
The precision of the whole method was evaluated in terms of repeatability (intraday precision) and intermediate precision (interday precision). Repeatability was assessed by application of the whole procedure to three white wine samples (experimental replicates) spiked at three concentration levels of OTA (0.2, 1.0 and 2.0 lg L 1), analysed on the same day and injected by triplicate (instrumental replicates). Intermediate precision was evaluated with a similar procedure, with five samples analysed in five different days. The results, expressed as RSD of peak areas are shown in Table 1. As can be observed, in all cases good precision was
Fig. 2. Chromatogram of a white wine sample analysed using DLLME with capillary HPLC–LIF: (a) blank sample; (b) sample spiked at 1 lg L conditions as indicated in the text.
1
of OTA. Optimum separation
372
N. Arroyo-Manzanares et al. / Food Chemistry 135 (2012) 368–372
obtained, being in agreement with current legislation (European Commission, 2006b). 3.6. Recovery studies In order to check the trueness of the proposed methodology, recovery experiments were carried out in white, rose and red wine samples. Samples were spiked with OTA at three levels, similar to those used in the precision study, and submitted to the whole method. Each level was prepared by triplicate and injected by triplicate. Blank samples were also analysed, and none of them gave a positive result for OTA. The results are shown in Table 2; as can be seen, very good absolute recoveries were obtained, according with current legislation, which establishes that the recoveries must be between 70% and 110 % (European Commission, 2006b). Typical chromatograms of a blank and a spiked white wine sample are shown in Fig. 2. Similar chromatograms were obtained for rose and red wine. As can be seen, a clean extract was obtained using this DLLME, showing its suitability as extraction method. 4. Concluding remarks A method based on a high sensitive and miniaturised technique, capillary HPLC with LIF detection, has been developed for the determination of OTA in different kinds of wine (white, rose and red). The addition of a micellar medium to the mobile phase has been used as an efficient way to improve sensitivity and efficiency. In addition, DLLME has been proposed as an alternative to the most frequent extraction methods for mycotoxins, based on SPE or immunoaffinity columns. DLLME has the advantages of simplicity of operation, rapidity, low-cost, and high-recovery, being also environmentally friendly. The proposed method DLLE-capillary HPLCLIF could be classified as a ‘‘green approach’’ for the fast, simple and sensitive analysis of OTA. It provided satisfactory results in terms of accuracy, so its suitability for the analysis of OTA in wine samples has been demonstrated. It can be concluded that the method fulfill the requirements established by the legislation for the determination of mycotoxins in foodstuff (European Commission, 2006b) in terms of both recovery and limit of detection, which could even be decreased by increasing the preconcentration factor, if requires. Acknowledgements The Andalusia Government (Junta de Andalucía) supported this work (Project Ref: P07-AGR-03178). Natalia Arroyo Manzanares thanks the ‘‘Junta de Andalucía’’ for a predoctoral grant. References Almeda, S., Arce, L., & Valcárcel, M. (2008). Combined use of supported liquid membrane and solid-phase extraction to enhance selectivity and sensitivity in capillary electrophoresis for the determination of ochratoxin A in wine. Electrophoresis, 29, 1573–1581. Bircan, C. (2009). Incidence of ochratoxin A in dried fruits and co-occurrence with aflatoxins in dried figs. Food and Chemical Toxicology, 47, 1996–2001. Campone, L., Piccinelli, A. L., & Rastrelli, L. (2011). Dispersive liquid–liquid microextraction combined with high-performance liquid chromatographytandem mass spectrometry for the identification and the accurate quantification by isotope dilution assay of ochratoxin A in wine samples. Analytical and Bioanalytical Chemistry, 399, 1279–1286. Cigic´, I. K., & Prosen, H. (2009). An overview of conventional and emerging analytical methods for the determination of mycotoxins. International Journal of Molecular Sciences, 10, 62–115. Corneli, S., & Maragós, C. M. (1998). Capillary electrophoresis with laser-induced fluorescence. method for the mycotoxin ochratoxin A. Journal of Agricultural and Food Chemistry, 46, 3162–3165. Duarte, S. C., Pena, A., & Lino, C. M. (2010). A review on ochratoxin A occurrence and effects of processing of cereal and cereal derived food products. Food Microbiology, 27, 187–198.
European Commission. (2006a). Commission Regulation (EC) No. 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs. Official Journal of the European Union L, 364, 5–24. European Commission. (2006b). Commission Regulation (EC) No. 401/2006 of 23 February 2006. Laying down the methods of sampling and analysis for the official control of the levels of mycotoxins in foodstuffs. Official Journal of the European Union L, 70, 12–34. European Committee for Standardization (CEN), (2003). Foodstuffs – determination of ochratoxin A in wine and beer – HPLC method with immunoaffinity column clean-up, EN 14133:2003. Brussels, Belgium. González-Osnaya, L., Soriano, J. M., Moltó, J. C., & Mañes, J. (2008). Simple liquid chromatography assay for analyzing ochratoxin A in bovine milk. Food Chemistry, 108, 272–276. González-Peñas, E., Leache, C., López de Cerain, A., & Lizarraga, E. (2006). Comparison between capillary electrophoresis and HPLC-FL for ochratoxin A quantification in wine. Food Chemistry, 97, 349–354. Hernández, M. J., García-Moreno, M. V., Durán, E., Guillén, D., & Barroso, C. G. (2006). Validation of two analytical methods for the determination of ochratoxin A by reversed-phased high-performance liquid chromatography coupled to fluorescence detection in musts and sweet wines from Andalusia. Analytica Chimica Acta, 566, 117–121. Herrera-Herrera, A. V., Asensio-Ramos, M., Hernández-Borges, J., & RodríguezDelgado, M. A. (2010). Dispersive liquid-liquid microextraction for determination of organic analytes. Trends in Analytical Chemistry, 29, 728–751. IARC, 1993. (1993). Some naturally occurring substances: items and constituents heterocyclic aromatic amines and mycotoxins. Monographs on the evaluation of carcinogenic risks to humans (Vol. 56). Lyon, France: International Agency for Research on Cancer. Juan, C., González, L., Soriano, J. M., Moltó, J. C., & Mañés, J. (2005). Accelerated solvent extraction of ochratoxin A from rice samples. Journal of Agricultural and Food Chemistry, 53, 9348–9351. Liazid, A., Palma, M., Brigui, J., & Barroso, C. G. (2007). Investigation on Ochratoxin A stability using different extraction techniques. Talanta, 71, 976–980. Mateo, R., Medina, A., Mateo, E. M., Mateo, F., & Jiménez, M. (2007). An overview of ochratoxin A in beer and wine. International Journal of Food Microbiology, 119, 79–83. Maragos, C. M., Appell, M., Lippolis, V., Visconti, Z., Catucci, L., & Pascale, M. (2008). Use of cyclodextrins as modifiers of fluorescence in the detection of mycotoxins. Food Additives & Contaminants, 25, 164–171. McIntire, G. L. (1990). Micelles in analytical chemistry. Critical Reviews in Analytical Chemistry, 21, 257–278. Olsson, J., Borjesson, T., Lundstedt, T., & Schnurer, J. (2002). Detection and quantification of ochratoxin A and deoxynivalenol in barley grains by GC–MS and electronic nose. International Journal of Food Microbiology, 72, 203–214. Rezaee, M., Yamini, Y., & Faraji, M. (2010). Evolution of dispersive liquid–liquid microextraction method. Journal of Chromatography A, 1217, 2342–2357. Sáez, J. M., Medina, A., Gimeno-Adelantado, J. V., Mateo, R., & Jiménez, M. (2004). Comparison of different sample treatments for the analysis of ochratoxin A in must, wine and beer by liquid chromatography. Journal of Chromatography, A, 1029, 125–133. Saha, D., Acharya, D., Roy, D., Shrestha, D., & Dhar, T. K. (2007). Simultaneous enzyme immunoassay for the screening of aflatoxin B1 and ochratoxin A in chili samples. Analytica Chimica Acta, 584, 343–349. Santos, L., Marín, S., Sanchis, V., & Ramos, A. J. (2010). Co-occurrence of aflatoxins, ochratoxin A and zearalenone in capsicum powder samples available on the Spanish market. Food Chemistry, 122, 826–830. Soleas, G. J., Yan, J., & Goldberg, D. M. (2001). Assay of ochratoxin A in wine and beer by high-pressure liquid chromatography photodiode array and gas chromatography mass selective detection. Journal of Agricultural and Food Chemistry, 49, 2733–2740. Tessini, C., Mardones, C., von Baer, D., Vega, M., Herlitz, E., Saelzer, R., et al. (2010). Alternatives for sample pre-treatment and HPLC determination of Ochratoxin A in red wine using fluorescence detection. Analytica Chimica Acta, 660, 119–126. Tobiszewski, M., Mechlinska, A., Zymunt, B., & Namiesnik, J. (2009). Green analytical chemistry in sample preparation for determination of trace organic pollutants. TrAC, Trends in Analytical Chemistry, 28, 943–951. Turner, N. W., Subrahmanyam, S., & Piletsky, S. A. (2009). Analytical methods for determination of mycotoxins: A review. Analytica Chimica Acta, 632, 168–180. Welch, C. J., Wu, N., Biba, M., Hartman, R., Brkovic, T., Gong, X., et al. (2010). Greening analytical chromatography. TrAC, Trends in Analytical Chemistry, 28, 667–680. Welke, J. E., Hoeltz, M., Dottori, H. A., & Noll, I. B. (2010). Determination of ochratoxin A in wine by high-performance thin-layer chromatography using charged coupled device. Journal of the Brazilian Chemical Society, 21, 441–446. Yu, J. C. C., & Lai, E. P. C. (2010). Molecularly imprinted polymers for ochratoxin A extraction and analysis. Toxins, 2, 1536–1553. Zang, X. H., WU, Q. H., Zhang, M. Y., Xi, G. H., & Wang, Z. (2009). Developments of dispersive liquid–liquid microextraction technique. Chinese Journal of Analytical Chemistry, 37, 161–168. Zezza, F., Longobardi, F., Pascale, M., Eremin, S. A., & Visconti, A. (2009). Fluorescence polarization immunoassay for rapid screening of ochratoxin A in red wine. Analytical and Bioanalytical Chemistry, 395, 1317–1323. Zöllner, P., & Mayer-Helm, B. (2006). Trace mycotoxin analysis in complex biological and food matrices by liquid chromatography–atmospheric pressure ionisation mass spectrometry. Journal of Chromatography, A, 1136, 123–169.
Contents lists available at SciVerse ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Analytical Methods
Determination of ochratoxin A in wines by capillary liquid chromatography with laser induced fluorescence detection using dispersive liquid–liquid microextraction Natalia Arroyo-Manzanares, Laura Gámiz-Gracia, Ana M. García-Campaña ⇑ Department of Analytical Chemistry, Faculty of Sciences, University of Granada, Campus Fuentenueva s/n, E-18071 Granada, Spain
a r t i c l e
i n f o
Article history: Received 11 July 2011 Received in revised form 24 April 2012 Accepted 2 May 2012 Available online 11 May 2012 Keywords: Ochratoxin A Capillary-HPLC Laser induced fluorescence detection Dispersive liquid–liquid microextraction Wine
a b s t r a c t A method based on reverse phase capillary high performance liquid chromatography (capillary HPLC) coupled to laser-induced fluorescence detection (LIF) has been proposed for the determination of ochratoxin A (OTA) in wine samples. An anionic micellar medium was added to the mobile phase for increasing the fluorescence intensity and peak efficiency. Dispersive liquid–liquid microextraction (DLLME) has been used as a simple and efficient sample pretreatment method for the analysis of OTA in wines, being optimised by means of experimental design. The limit of detection was 5.5 ng L 1 (3 S/N) and recoveries for different wines ranged from 91.7 to 98.1%. The proposed methodology could be classified as a green analytical chemistry alternative, combining the low organic solvent volumes required in the DLLME with the reduced consumption of mobile phase in capillary HPLC. The use of LIF as detector provided an extremely sensitive method for the determination of OTA in wines. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Ochratoxin A is a mycotoxin naturally found in various foods, including cereals, wine or coffee. It is produced by several species of Aspergillus (A. carbonarius) and Penicillium (P. verrucosum) moulds. As it has potent nephrotoxic, carcinogenic and teratogenic effects, the International Agency for Research of Cancer (IARC) has classified OTA in group 2B, as a possible human carcinogen (IARC, 1993). OTA has been widely detected in cereals (e.g. wheat, barley and maize), cereal-derived products (Duarte, Pena, & Lino, 2010), dried fruits (Bircan, 2009), spices (Santos, Marín, Sanchis, & Ramos, 2010), beer and wine (Mateo, Medina, Mateo, Mateo, & Jiménez, 2007). After cereals, wine is considered the major source of daily OTA intake. Thus, European Union (EU) has set maximum levels for this compound at 2 lg kg 1 in wine samples (European Commission, 2006a). As a consequence, very sensitive analytical methods for OTA detection, with performance characteristics fulfilling the established legislation are needed (European Commission, 2006b). The most common analytical method for OTA determination in foodstuff is HPLC coupled to fluorescence detection (FL) (González-Osnaya, Soriano, Moltó, & Mañes, 2008; Hernández, García-Moreno, Durán, Guillén, & Barroso, 2006; Sáez, Medina, ⇑ Corresponding author. Tel.: +34 958 242385; fax: +34 958 249510. E-mail address: [email protected] (A.M. García-Campaña). 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.05.009
Gimeno-Adelantado, Mateo, & Jiménez, 2004; Tessini et al., 2010), including the reference method described by the European Standard EN 14133 for the determination of OTA in wine (European Committee for Standardization, 2003). Alternative detection methods, such as photodiode array detection (LC–DAD) (Soleas, Yan, & Goldberg, 2001) or mass spectrometry (LC–MS) and tandem MS (LC–MS/MS) (Zöllner & Mayer-Helm, 2006) have also been proposed. Others applied techniques are thin layer chromatography (TLC) (Welke, Hoeltz, Dottori, & Noll, 2010), gas chromatography coupled with MS (GC–MS) (Olsson, Borjesson, Lundstedt, & Schnurer, 2002), enzyme immunoassay (EIA) (Saha, Acharya, Roy, Shrestha, & Dhar, 2007) and fluorescence polarisation immunoassay (Zezza, Longobardi, Pascale, Eremin, & Visconti, 2009). Also, capillary electrophoresis (CE) with UV/Vis detection has also been proposed for the determination of OTA in wine (Almeda, Arce, & Valcárcel, 2008; González-Peñas, Leache, López de Cerain, & Lizarraga, 2006). The use of laser induced fluorescence (LIF) as a more sensitive detection system has been proposed for the determination of OTA in different food samples (roasted coffee, corn, and sorghum) using CE (Corneli & Maragós, 1998). Concerning the sample treatment, different extraction and cleanup methods for the determination of OTA have been reported. Most of them are based on immunoaffinity columns (IACs), which contain specific antibodies to the analyte of interest, allowing higher cleanup and sample purification. However, this methodology is high-cost and tedious. Liquid–liquid extraction (LLE) in milk, wine, beer and must (Sáez et al., 2004; González-Osnaya et al., 2008),
N. Arroyo-Manzanares et al. / Food Chemistry 135 (2012) 368–372
solid-phase extraction (SPE) in wine, beer and must (Sáez et al., 2004; Hernández et al., 2006; Tessini et al., 2010), SPE with molecular imprinted polymers (MISPE) in different matrices (Yu & Lai, 2010) or pressurised liquid extraction (PLE) in rice (Juan, González, Soriano, Moltó, & Mañés, 2005), have been also used for the determination of OTA. The stability of OTA during its extraction using microwave-assisted extraction (MAE), PLE, ultrasound-assisted and magnetic stirring-assisted extraction, has also been evaluated (Liazid, Palma, Brigui, & Barroso, 2007). Some reviews devoted to the analysis of OTA and mycotoxins compile most of these analytical and extraction methodologies (Turner, Subrahmanyam, & Piletsky, 2009; Cigic´ & Prosen, 2009). Recently, dispersive liquid–liquid microextraction (DLLME) has been introduced for treatment of liquid samples (Rezaee, Yamini, & Faraji, 2010; Zang, WU, Zhang, Xi, & Wang, 2009; Herrera-Herrera, Asensio-Ramos, HernándezBorges, & Rodríguez-Delgado, 2010), and has been recently used in the determination of OTA in wine by LC-MS (Campone, Piccinelli, & Rastrelli, 2011). It is based on the use of a ternary component solvent system, where an appropriate mixture of a few microlitres of a organic extraction solvent (usually with a density higher than water), and a small volume of a disperser solvent (miscible with the extraction solvent and with water), is injected rapidly into an aqueous sample, resulting in the formation of a stable emulsion. The organic analytes present in the aqueous sample are rapidly extracted into the extraction solvent as a result of the large contact surface between the organic and the aqueous phases. Phase separation is performed by centrifugation and an organic phase with the analytes of interest is settled in the bottom of a conical tube and subsequently analysed by an appropriate technique. In this paper, we propose a method based on DLLME followed by capillary HPLC–LIF for the very sensitive determination of OTA in wine samples, combining the advantages of capillary HPLC (better resolution, lower detection limits and lower solvent consumption) with the low detection limits provided by LIF detection. This methodology could be classified among the alternatives proposed by the recent trends of green analytical chemistry (Tobiszewski, Mechlinska, Zymunt, & Namiesnik, 2009; Welch et al., 2010), as it achieves a reduction of organic solvent per analysis, thanks to both the applied extraction technique and the capillary HPLC system.
2. Material and method 2.1. Chemicals All the reagents were analytical reagent grade, solvents were HPLC grade and OTA was analytical standard grade. Methanol (MeOH), ethanol (EtOH), acetonitrile (ACN), sodium chloride (NaCl) and sodium dodecyl sulphate (SDS) were supplied by Panreac (Madrid, Spain); acetic acid, chloroform and tetrachloroethylene were purchased from VWR BDH Prolabo (West Chester, Pensilvania, USA); carbon disulphide was obtained from Carlo Erba (Rodano, MI, Italy); tetrahydrofuran (THF) and acetone (ACO) was supplied by Merck (Darmstadt, Germany); chlorobenzene was obtained from Alfa Aesar (Karlsruhe, Germany) and b-cyclodextrin was purchased from Sigma–Aldrich (St. Louis, MO, USA). Ultrapure water (18.2 MO cm 1, Milli–Q Plus system, Millipore Bedford, MA, USA) was used throughout the work. Acrodisc 13 mm syringe filters with 0.2 lm nylon membrane (Pall Corp., MI, USA) were used for filtration of samples prior to the injection in the chromatographic system. A 0.2 lm nylon membrane filter (Supelco, Bellefonte, PA, USA) was used for filtration of mobile phase. A stock standard solution of 10 lg mL 1 of OTA in ACN was obtained from Supelco. Working solutions were prepared by evaporation of the stock standard solution to near dryness using a gentle
369
stream of N2, and diluting with the appropriate amount in MeOH and H2O. These solutions were stored at 20 °C. 2.2. Instruments and equipments All experiments were carried out using an Agilent HP-1200 series capillary HPLC (Agilent Technologies, Waldbron, Germany) equipped with a binary pump (20 lL min 1 maximum flow-rate), online degasser, autosampler (8 lL loop), and a column thermostat. This system was coupled with a LIF detector (Zetalif Evolution model LIF UV-01, Picometrics S.A., Ramonville, France) equipped with a 325 nm HeCd laser. A fused-silica capillary (75 lm I.D.) from Polymicro Technologies (Phoenix, AZ, USA) was used to coupled the HPLC and LIF detector. ChemStation software (A.10.20 [1757] version) was used for data acquisition and processing. A Luna C18 (150 0.5 mm, 5 lm) from Phenomenex (Torrance, CA, USA) was used as chromatographic column. A centrifuge Model Universal 320R (Hettich, Tuttlingen, Germany), a vortex-2 Genie (Scientific Industries, Bohemia, NY, USA), a mechanical shaker (model 384 from Vibromatic, Noblesville, USA) and a Evaporator EVA EC-S/EVA LS-S (VLM GmbH, Bielefeld, Germany) were also used for sample treatment. The statistic software STATGRAPHICS Centurion XV.II was used for data treatment. 2.3. Optimum chromatographic conditions The analyses were performed on a Luna C18 column (150 0.5 mm, 5 lm, 100 Å). An isocratic mobile phase of water (2% acetic acid, 0.2 M SDS): MeOH (30:70, v/v) at a flow rate of 14 lL min 1 was used; the column temperature was kept at 40° C and the injection volume was 1.20 lL. Under optimum conditions, the analysis took around 3.5 min. 2.4. Sample preparation White, rose and red wine samples were purchased from a local market. An aliquot of 5.0 mL of sample was placed into a 10 mL screw cap test tube with conical bottom and 0.25 g of NaCl (5%; w/v) were added. The mixture of the disperser solvent (940 lL of ACN) and the extraction solvent (660 lL of chloroform) was rapidly injected into the test tube with a 2.0 mL syringe. The solution was shaken and a cloudy solution was formed in the tube. In this step, the OTA was extracted into the fine droplet of chloroform. Later, the mixture was centrifuged at 5000 rpm for 1 min and the fine particles were sedimented at the bottom of the tube. The sedimented phase (approximately 700 lL) was removed using a 1 mL syringe, evaporated to near dryness using a gentle stream of N2 and reconstituted with 1 mL of MeOH:H2O (v/v). The solution was filtered and injected into the capillary HPLC–LIF system for analysis. 3. Results and discussion 3.1. Optimisation of capillary HPLC–LIF method Peak area, efficiency and analysis time were taken into account to select the adequate determination procedure. Different natures of mobile phase (water with acetic acid from 1% to 5% as solvent A, and ACN, MeOH or a mixture of both of them as solvent B) were tested. The best results were obtained with an isocratic phase consisting on water (2% acetic acid): MeOH (30:70, v/v). The use of cyclodextrins has been previously reported as modifiers of native fluorescence of mycotoxins (Maragos et al., 2008). Thus, the addition of 6 mM b-cyclodextrin to solvent A was considered. However,
370
N. Arroyo-Manzanares et al. / Food Chemistry 135 (2012) 368–372
the improvement in the sensitivity was not significant and a higher noise in the baseline was observed. As an alternative, we proposed the addition of a micellar anionic media (SDS) to solvent A. The addition of this anionic surfactant at a concentration above its critical micellar concentration not only provided an increase on fluorescence quantum yield of the analyte, as could be expected as effect of this organised media on fluorophore solutions (McIntire, 1990), but also improved the chromatographic efficiency, giving a sharper peak. Thus, concentrations of 0.1, 0.2 and 0.3 M of SDS were tested, obtaining the best efficiency and the highest signal in a shorter time with 0.2 M of SDS in the aqueous phase. Higher concentrations did not improve the analysis; so 0.2 M was selected for the rest of experimental work. The flow rate was also tested between 10–15 lL min 1, selecting a final optimum value of 14 lL min 1, as higher flows involved higher pressure without a significant improvement in the analysis. The effect of temperature was examined in the range of 30–50 °C; an optimum value of 40 °C was chosen, as a compromise between analysis time and of column life. Finally, the injection volume was increased from 0.6 to 1.4 lL, selecting 1.2 lL as optimum. At these final conditions, the analysis of OTA took less than 3.5 min. 3.2. Optimisation of DLLME The optimisation of DLLME involved the study of the following parameters: nature and volume of extraction solvent, nature and volume of disperser solvent, percentage of NaCl, extraction time and shake mode. All the experiments were performed using samples of 5 mL of white wine spiked with 400 ng L 1 of OTA, and the goal of this optimisation was to obtain the highest absolute recovery of OTA. First, it was necessary to select the extraction and disperser solvents. Chloroform, carbon disulphide, chlorobenzene and tetrachloroethylene were tested as extraction solvents, and
MeOH, EtOH, ACO, ACN and THF were tested as disperser solvents. The best results were obtained with a combination of chloroform and ACN. Once the extraction and disperser solvents have been selected, their volumes and the percentage of NaCl were optimised by a multivariate approach using an experimental design, which takes into account possible interactions between the variables. A central composite design (23 + star, face centered), with three spaced central points, involving 17 runs, was used an as approach to generate the response surface, using the recovery percentage as analytical response. The different factors were studied in the following ranges: ACN volume (800–1000 lL); chloroform volume (500–700 lL); and percentage of NaCl (0–5%). A Pareto chart (Fig. 1A) was obtained from the screening experimental design, showing what variables and/or interactions between them have significant effects when their value is changed inside the selected experimental domain. The extraction solvent volume and the quadratic terms of extraction solvent volume, disperser solvent volume and percentage of salt (AA, BB and CC, respectively) had a significant effect on the recovery of OTA; so finally the three variables were simultaneously optimised. The obtained response surface (Fig. 1B) gave the optimum conditions for DLLME, being as follows: extraction solvent = 660 lL; disperser solvent = 940 lL; percentage of salt = 5%. The P-value for the lack of fit test for the model was 38.0% and the determination coefficient (R2) was 93.9%, showing the suitability of this design. Under the previous optimum conditions, the effects of extraction time (defined as the interval between the injection of disperser and extraction solvents, and centrifugation) and shake mode (mechanical shake, vortex, and manual shake) were tested in the range of 0–10 min. It was concluded that these variables had no influence in the extraction efficiency. The reason is that extraction solvent can be evenly dispersed after the formation of the cloudy solution, the transition of analyte from the sample to
Fig. 1. (a) DLLME screening design: Pareto chart showing the effects of the studied variables on the recovery percentage: (+) Positive effects on the response; ( ) negative effects on the response. Blue line shows the limit of decision to consider the significance of the factors (based on the effect = estimated effect/standard error, P-value = 0.05 at 95% of confidence); (b) Estimated response surface obtained in the optimisation procedure using a central composite design (23 + star, face centered), with three spaced central points.
371
N. Arroyo-Manzanares et al. / Food Chemistry 135 (2012) 368–372
the extraction phase can be very fast, and the equilibrium state can be subsequently achieved very quickly, resulting in a very short extraction time (Zang et al., 2009). Thus, the extraction was carried out by shaking the sample manually for a few seconds. Finally, under the optimum conditions, an enrichment factor of 6.6 (calculated as the ratio of analyte concentration in the sediment, and the initial concentration of analyte in the aqueous sample) and a final preconcentration factor of 5 was achieved. 3.3. Validation of the method In order to check the suitability of the proposed method of DLLME-capillary HPLC–LIF for the determination of OTA in wine samples, it was characterised in terms of linear dynamic range, limit of detection and quantification, precision and trueness. Table 1 Precision study of the whole method. The results are expressed as RSD of peak areas.
Level 1 (0.2 lg L Level 2 (1.0 lg L Level 3 (2.0 lg L
1
) 1 ) 1 )
Repeatability (n = 9)
Intermediate precision (n = 15)
1.9 2.6 1.3
4.9 2.9 5.2
1
Level 2 (1.0 lg L
1
Level 3 (2.0 lg L
1
) ) )
R (%) RSD (%) R (%) RSD (%) R (%) RSD (%)
The matrix-matched calibration curve was obtained using white wine samples spiked with the following concentrations of OTA: 0.02, 0.1, 0.2, 1, 2 and 4 lg L 1. These concentrations, after the sample treatment by DLLME, gave concentrations of OTA in the final extracts of 0.1, 0.5, 1, 5, 10 and 20 lg L 1, respectively. Each concentration level was prepared by duplicate and injected by triplicate. A blank sample was also analysed and no interferences from the matrix were found co-migrating with the analyte. The statistical parameters were calculated by least-square regression, and the resulting calibration curve was: Area = 27790.0 Concentration + 711.3 (R2 = 0.998). The standard deviations of the slope and intercept were 200.7 and 334.2, respectively. The limit of detection (LOD) and the limit of quantification (LOQ) in the sample were 5.5 ng L 1 (3 S/N) and 18.4 ng L 1 (10 S/N), respectively. As can be seen, the sensitivity of the method allows the quantification of OTA at concentrations much lower than the maximum level established for wine samples (European Commission, 2006a). 3.5. Precision study
Table 2 Data about the recovery assay to the whole method for white, rose and red wine. Each level was prepared by triplicate and injected by triplicate (n = 9).
Level 1 (0.2 lg L
3.4. Calibration curve and performance characteristics of the method
White wine
Rose wine
Red wine
94.9 1.9 94.8 2.9 91.7 1.4
94.4 3.9 92.7 2.2 95.8 1.7
98.3 4.1 96.3 1.7 98.1 1.8
The precision of the whole method was evaluated in terms of repeatability (intraday precision) and intermediate precision (interday precision). Repeatability was assessed by application of the whole procedure to three white wine samples (experimental replicates) spiked at three concentration levels of OTA (0.2, 1.0 and 2.0 lg L 1), analysed on the same day and injected by triplicate (instrumental replicates). Intermediate precision was evaluated with a similar procedure, with five samples analysed in five different days. The results, expressed as RSD of peak areas are shown in Table 1. As can be observed, in all cases good precision was
Fig. 2. Chromatogram of a white wine sample analysed using DLLME with capillary HPLC–LIF: (a) blank sample; (b) sample spiked at 1 lg L conditions as indicated in the text.
1
of OTA. Optimum separation
372
N. Arroyo-Manzanares et al. / Food Chemistry 135 (2012) 368–372
obtained, being in agreement with current legislation (European Commission, 2006b). 3.6. Recovery studies In order to check the trueness of the proposed methodology, recovery experiments were carried out in white, rose and red wine samples. Samples were spiked with OTA at three levels, similar to those used in the precision study, and submitted to the whole method. Each level was prepared by triplicate and injected by triplicate. Blank samples were also analysed, and none of them gave a positive result for OTA. The results are shown in Table 2; as can be seen, very good absolute recoveries were obtained, according with current legislation, which establishes that the recoveries must be between 70% and 110 % (European Commission, 2006b). Typical chromatograms of a blank and a spiked white wine sample are shown in Fig. 2. Similar chromatograms were obtained for rose and red wine. As can be seen, a clean extract was obtained using this DLLME, showing its suitability as extraction method. 4. Concluding remarks A method based on a high sensitive and miniaturised technique, capillary HPLC with LIF detection, has been developed for the determination of OTA in different kinds of wine (white, rose and red). The addition of a micellar medium to the mobile phase has been used as an efficient way to improve sensitivity and efficiency. In addition, DLLME has been proposed as an alternative to the most frequent extraction methods for mycotoxins, based on SPE or immunoaffinity columns. DLLME has the advantages of simplicity of operation, rapidity, low-cost, and high-recovery, being also environmentally friendly. The proposed method DLLE-capillary HPLCLIF could be classified as a ‘‘green approach’’ for the fast, simple and sensitive analysis of OTA. It provided satisfactory results in terms of accuracy, so its suitability for the analysis of OTA in wine samples has been demonstrated. It can be concluded that the method fulfill the requirements established by the legislation for the determination of mycotoxins in foodstuff (European Commission, 2006b) in terms of both recovery and limit of detection, which could even be decreased by increasing the preconcentration factor, if requires. Acknowledgements The Andalusia Government (Junta de Andalucía) supported this work (Project Ref: P07-AGR-03178). Natalia Arroyo Manzanares thanks the ‘‘Junta de Andalucía’’ for a predoctoral grant. References Almeda, S., Arce, L., & Valcárcel, M. (2008). Combined use of supported liquid membrane and solid-phase extraction to enhance selectivity and sensitivity in capillary electrophoresis for the determination of ochratoxin A in wine. Electrophoresis, 29, 1573–1581. Bircan, C. (2009). Incidence of ochratoxin A in dried fruits and co-occurrence with aflatoxins in dried figs. Food and Chemical Toxicology, 47, 1996–2001. Campone, L., Piccinelli, A. L., & Rastrelli, L. (2011). Dispersive liquid–liquid microextraction combined with high-performance liquid chromatographytandem mass spectrometry for the identification and the accurate quantification by isotope dilution assay of ochratoxin A in wine samples. Analytical and Bioanalytical Chemistry, 399, 1279–1286. Cigic´, I. K., & Prosen, H. (2009). An overview of conventional and emerging analytical methods for the determination of mycotoxins. International Journal of Molecular Sciences, 10, 62–115. Corneli, S., & Maragós, C. M. (1998). Capillary electrophoresis with laser-induced fluorescence. method for the mycotoxin ochratoxin A. Journal of Agricultural and Food Chemistry, 46, 3162–3165. Duarte, S. C., Pena, A., & Lino, C. M. (2010). A review on ochratoxin A occurrence and effects of processing of cereal and cereal derived food products. Food Microbiology, 27, 187–198.
European Commission. (2006a). Commission Regulation (EC) No. 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs. Official Journal of the European Union L, 364, 5–24. European Commission. (2006b). Commission Regulation (EC) No. 401/2006 of 23 February 2006. Laying down the methods of sampling and analysis for the official control of the levels of mycotoxins in foodstuffs. Official Journal of the European Union L, 70, 12–34. European Committee for Standardization (CEN), (2003). Foodstuffs – determination of ochratoxin A in wine and beer – HPLC method with immunoaffinity column clean-up, EN 14133:2003. Brussels, Belgium. González-Osnaya, L., Soriano, J. M., Moltó, J. C., & Mañes, J. (2008). Simple liquid chromatography assay for analyzing ochratoxin A in bovine milk. Food Chemistry, 108, 272–276. González-Peñas, E., Leache, C., López de Cerain, A., & Lizarraga, E. (2006). Comparison between capillary electrophoresis and HPLC-FL for ochratoxin A quantification in wine. Food Chemistry, 97, 349–354. Hernández, M. J., García-Moreno, M. V., Durán, E., Guillén, D., & Barroso, C. G. (2006). Validation of two analytical methods for the determination of ochratoxin A by reversed-phased high-performance liquid chromatography coupled to fluorescence detection in musts and sweet wines from Andalusia. Analytica Chimica Acta, 566, 117–121. Herrera-Herrera, A. V., Asensio-Ramos, M., Hernández-Borges, J., & RodríguezDelgado, M. A. (2010). Dispersive liquid-liquid microextraction for determination of organic analytes. Trends in Analytical Chemistry, 29, 728–751. IARC, 1993. (1993). Some naturally occurring substances: items and constituents heterocyclic aromatic amines and mycotoxins. Monographs on the evaluation of carcinogenic risks to humans (Vol. 56). Lyon, France: International Agency for Research on Cancer. Juan, C., González, L., Soriano, J. M., Moltó, J. C., & Mañés, J. (2005). Accelerated solvent extraction of ochratoxin A from rice samples. Journal of Agricultural and Food Chemistry, 53, 9348–9351. Liazid, A., Palma, M., Brigui, J., & Barroso, C. G. (2007). Investigation on Ochratoxin A stability using different extraction techniques. Talanta, 71, 976–980. Mateo, R., Medina, A., Mateo, E. M., Mateo, F., & Jiménez, M. (2007). An overview of ochratoxin A in beer and wine. International Journal of Food Microbiology, 119, 79–83. Maragos, C. M., Appell, M., Lippolis, V., Visconti, Z., Catucci, L., & Pascale, M. (2008). Use of cyclodextrins as modifiers of fluorescence in the detection of mycotoxins. Food Additives & Contaminants, 25, 164–171. McIntire, G. L. (1990). Micelles in analytical chemistry. Critical Reviews in Analytical Chemistry, 21, 257–278. Olsson, J., Borjesson, T., Lundstedt, T., & Schnurer, J. (2002). Detection and quantification of ochratoxin A and deoxynivalenol in barley grains by GC–MS and electronic nose. International Journal of Food Microbiology, 72, 203–214. Rezaee, M., Yamini, Y., & Faraji, M. (2010). Evolution of dispersive liquid–liquid microextraction method. Journal of Chromatography A, 1217, 2342–2357. Sáez, J. M., Medina, A., Gimeno-Adelantado, J. V., Mateo, R., & Jiménez, M. (2004). Comparison of different sample treatments for the analysis of ochratoxin A in must, wine and beer by liquid chromatography. Journal of Chromatography, A, 1029, 125–133. Saha, D., Acharya, D., Roy, D., Shrestha, D., & Dhar, T. K. (2007). Simultaneous enzyme immunoassay for the screening of aflatoxin B1 and ochratoxin A in chili samples. Analytica Chimica Acta, 584, 343–349. Santos, L., Marín, S., Sanchis, V., & Ramos, A. J. (2010). Co-occurrence of aflatoxins, ochratoxin A and zearalenone in capsicum powder samples available on the Spanish market. Food Chemistry, 122, 826–830. Soleas, G. J., Yan, J., & Goldberg, D. M. (2001). Assay of ochratoxin A in wine and beer by high-pressure liquid chromatography photodiode array and gas chromatography mass selective detection. Journal of Agricultural and Food Chemistry, 49, 2733–2740. Tessini, C., Mardones, C., von Baer, D., Vega, M., Herlitz, E., Saelzer, R., et al. (2010). Alternatives for sample pre-treatment and HPLC determination of Ochratoxin A in red wine using fluorescence detection. Analytica Chimica Acta, 660, 119–126. Tobiszewski, M., Mechlinska, A., Zymunt, B., & Namiesnik, J. (2009). Green analytical chemistry in sample preparation for determination of trace organic pollutants. TrAC, Trends in Analytical Chemistry, 28, 943–951. Turner, N. W., Subrahmanyam, S., & Piletsky, S. A. (2009). Analytical methods for determination of mycotoxins: A review. Analytica Chimica Acta, 632, 168–180. Welch, C. J., Wu, N., Biba, M., Hartman, R., Brkovic, T., Gong, X., et al. (2010). Greening analytical chromatography. TrAC, Trends in Analytical Chemistry, 28, 667–680. Welke, J. E., Hoeltz, M., Dottori, H. A., & Noll, I. B. (2010). Determination of ochratoxin A in wine by high-performance thin-layer chromatography using charged coupled device. Journal of the Brazilian Chemical Society, 21, 441–446. Yu, J. C. C., & Lai, E. P. C. (2010). Molecularly imprinted polymers for ochratoxin A extraction and analysis. Toxins, 2, 1536–1553. Zang, X. H., WU, Q. H., Zhang, M. Y., Xi, G. H., & Wang, Z. (2009). Developments of dispersive liquid–liquid microextraction technique. Chinese Journal of Analytical Chemistry, 37, 161–168. Zezza, F., Longobardi, F., Pascale, M., Eremin, S. A., & Visconti, A. (2009). Fluorescence polarization immunoassay for rapid screening of ochratoxin A in red wine. Analytical and Bioanalytical Chemistry, 395, 1317–1323. Zöllner, P., & Mayer-Helm, B. (2006). Trace mycotoxin analysis in complex biological and food matrices by liquid chromatography–atmospheric pressure ionisation mass spectrometry. Journal of Chromatography, A, 1136, 123–169.