Quantification of ochratoxin A in red wines by conventional HPLC–FLD using a column packed with core–shell particles

Abstract

Ochratoxin A (OTA) is a mycotoxin commonly present in red wine and other foodstuffs. It is widely studied due to its nephrotoxic, immunotoxic, teratogenic and carcinogenic effects. Nowadays, liquid chromatography is the main method for OTA analysis. With the recent development on the column technology, core–shell columns were commercialized in recent years. It is with high performance while maintain low back pressure thus could be used on conventional HPLC instrument. However, as current conventional benchmark high-performance liquid chromatographs were not initially designed for the recently developed high-efficiency packed columns, the HPLC–FLD parameter should be carefully investigated. In this work the performance of chromatography column packed with 2.6 μm core–shell particles on conventional benchmark HPLC were investigated. Parameters including flow rate, mobile phase composition (volume fraction of ACN in water), temperature, response time, sampling frequency, and injection volume for quantitative analysis of OTA in red wines were investigated in detail.

The results show that core–shell column is more effective and faster to be operated on conventional HPLC than other total porous particle packed column. Optimized conditions provided a method for the separation of OTA in less than 5 min, with the limit of detection (LOD) 0.0025 μg/L and the limit of quantification (LOQ) 0.0083 μg/L in the sample, respectively. The developed method was validated with red wine samples with OTA concentrations ranging from 0.0028 to 0.0437 μg/L. The use of a core–shell column allows highly efficient, sensitive, and accurate determination of OTA with an outstanding sample throughout.

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.