Quantification of ochratoxin A in red wines by conventional HPLC–FLD using a column packed with core–shell particles
Highlights
► Performance of core–shell column on HPLC was optimized for OTA analysis in red wines. ► Parameters were optimized with special attention different with totally porous columns. ► The using of core–shell column allows highly efficient quantification of OTA.
Introduction
Ochratoxin A (OTA) is a toxic metabolite produced by several fungi of the genera Aspergillus and Penicillium and commonly found in red wine and other foodstuffs (Amézqueta, González-Peñas, Murillo-Arbizu, & López de Cerain, 2009; Bhat, Rai, & Karim, 2010; Mateo, Medina, Mateo, Mateo, & Jiménez, 2007). OTA is considered as teratogenic, embryotoxic, genotoxic, immunosuppressive, carcinogenic (IARC group 2B), and nephrotoxic (JECFA, 2001). As it can contaminate a wide variety of foodstuffs and often presents in red wines, maximum permitted levels have been established by the EU, with a maximum level of 2 μg/L for wine samples (European Commission, 2006). Nowadays, OTA is routinely analyzed by high performance liquid chromatography (HPLC) coupled with a variety of detectors. FLD is still the method of choice because of the popularity, stability, and sensitivity (Rahmani, Jinap, & Soleimany, 2009; Turner, Subrahmanyam, & Piletsky, 2009).
Fast separations of OTA with very high efficiency and sufficient resolution to perform analysis within few minutes have always been of great interest and have become increasingly important in recent years mainly driven by the challenges of either more complex samples or increasing numbers of samples (Bhat et al., 2010). Higher separation efficiency and faster speed have been achieved with the introduction of very efficient packing materials such as sub-2 μm fully porous particles, core–shell particles and silica monolithic rods (Carr, Stoll, & Wang, 2011; Gritti & Guiochon, 2012a; Núñez, Gallart-Ayala, Martins, & Lucci, 2012). The efficiency of the monolithic columns that are now available is low, due to their radial heterogeneity (Gritti & Guiochon, 2010b). The current leaders are columns packed with sub-2 μm totally porous and sub-3 μm core–shell particles, which are increasingly widespread nowadays to conduct fast and efficient separations (Fekete, Oláh, & Fekete, 2012). Columns packed with sub-2 μm totally porous particles require heavy costs of ultra-high pressure instrument (cable of withstanding up to 1200 bar of inlet pressures), and require to switch from conventional HPLC systems we have been accustomed to for many years to chromatographs of the new generation (Carr et al., 2011; Gritti & Guiochon, 2012b). Core–shell particles are different from the totally porous particles in that they have a solid core surrounded by a thin porous shell. They are also referred to as fused-core, shell or superficially porous particles (Carr et al., 2011; Fekete et al., 2012). Columns packed with core–shell particles at approximately 2.6 μm can provide speed and efficiency similar to columns packed with sub-2 μm totally porous particles while maintain low back pressure thus could be used on conventional HPLC instrument, which usually could only operate at a maximum inlet pressure of 400 bar (Fekete et al., 2012; Guiochon & Gritti, 2011; Gritti & Guiochon, 2010b; Gritti, Leonardis, et al., 2010; Gritti, Sanchez, Farkas, & Guiochon, 2010; Manchón et al., 2010). However, the theoretical increase in performance from the use of high efficiency columns with conventional HPLC equipment is generally limited due to the design limitations of such equipment, particularly with respect to extra-column volume (ECV) and extra-column dispersion (ECD) (Alexander, Waeghe, Himes, Tomasella, & Hooker, 2011). These contributions that have negligible influence on the effective efficiency of conventional columns cause an obviously decrease of the intrinsic performance of highly efficient columns (Alexander et al., 2011).
Herein, a commercially available 4.6 mm ID × 100 mm Kinetex columns packed with core–shell particles was used to quantify OTA concentrations in red wines on a benchmark Agilent 1200 HPLC–FLD system. Parameters including flow rate, mobile phase composition (percentages of acetonitrile in water), temperature, response time, sampling frequency, and injection volume were investigated in detail. Optimized conditions provided a method for the separation of OTA in less than 5 min. The developed method was validated with 28 red wine samples from China with OTA concentrations ranging from 0.0028 to 0.044 μg/L. The ability of using the core–shell column on conventional instruments permits large savings without hardware update or modification and the transfer of analytical methods from the old to the new instrument while allows highly efficient, sensitive, and accurate quantification of OTA with an outstanding sample throughout.
Section snippets
Materials
Twenty eight commercial red wine samples were purchased in supermarkets (Chengdu, Sichuan Province, China). OTA standard 5 μg/mL in a benzene/acetic acid solution (99:1; v/v) was purchased from Supelco (Bellefonte, PA, USA). Stock OTA standard solution 500 μg/L was obtained by diluting 1 mL standard OTA solution to 10 mL with acetonitrile. Working standard solutions of 0.5, 5, 10, 25, 50 μg/L of OTA are prepared by diluting stock standard with acetonitrile: water 50/50. Immunoaffinity columns
Optimization of the HPLC condition
Columns packed with core–shell particles show higher efficiencies for the smaller diffusion distance and improved mass transfer, lower internal porosity (smaller B term of van Deemter/Knox type equations, −20 to −30%) and narrower particle size distribution particles and better packing (smaller eddy diffusion or A term, −40%). The C term of shell particles is also more favorable than that of the fully porous particles however the benefits of core–shell particles mostly lie in the A and B term (
Conclusions
An HPLC–FLD method based on core–shell column and IAC cleanup, has been developed for the determination of OTA in red wine. Under optimized condition with injection volume 20 μL, flow rate 0.66 mL/min, temperature 40 °C, response time 1 s, sampling rate 9.25 Hz, OTA is separated in less than 5 min. The observed efficiencies of columns is 72% of the intrinsic theoretical column efficiency and 85% resolution power of the column was maintained. The LOD and LOQ of the proposed method are
Acknowledgments
This work was supported by the Ministry of Agriculture of the People's Republic of China, within the project of “Risk assessment of mycotoxins in black teas”.
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