Evaluación de la microextracción líquido-líquido dispersiva para la determinación de patulina en zumos de manzana mediante electroforesis capilar

Abstract

Patulin (PAT) is a mycotoxin produced in fruits by several Penicillium and Aspergillus species, especially Penicillium expansum. Its occurrence as a natural contaminant of fruit juices is an indicative of fruit quality in production [1]. The maximum content of patulin has been established by the EU by the Directive (CE) Nº 1881/2006 in 50 μg·kg-1 for fruit juices. In this work, dispersive liquid–liquid microextraction (DLLME) has been proposed for the extraction and preconcentration of PAT in apple juice samples, followed by its determination by micellar electrokinetic chromatography (MEKC) with diode-array detection. DLLME is a novel environmentally friendly sample preparation technique, showing important advantages because it is fast, inexpensive, easy to operate with a high enrichment factor and consumes low volume of organic solvents. This method is based on a ternary component solvent system in which the extraction solvent and disperser solvent are rapidly injected into the aqueous sample containing the analyte, using a syringe. The mixture is then gently shaken and a cloudy solution (water/disperser solvent/extraction solvent) is formed in the test tube. After centrifugation, the fine particles of extraction solvent are sedimented in the bottom of a conical test tube and the analyte can be directly analyzed or this phase can be evaporated and reconstituted in the appropriate solvent before analysis [2]. In this work PAT has been analyzed in the presence of 5-hydroxymethylfurfural (HMF) (main interference in apple juice PAT analysis) using an extended light path fused-silica capillary (64.5 cm × 75 μm I.D., 56 cm effective length) and an electrophoretic buffer consisting of borate/HCl 35 mM at pH 9.0, sodium dodecyl sulphate (SDS) 65 mM and acetonitrile 5%. Samples were introduced by hydrodynamic injection (50 mbar, 17 s), dissolved in water at pH 4.0, acidified with an aqueous acetic acid solution [3]. Variables affecting DLLME efficiency were optimized, selecting: 5 mL of sample solution, 1000 μL of chloroform as extraction solvent, 1000 μL of 2-propanol as disperser solvent and 20% (w/v) of NaCl. The sedimented phase was evaporated to near dryness using a stream of nitrogen and reconstituted with 0.5 mL of water at pH 4. The solution was filtered and injected into the electrophoretic system. Under these conditions, a very clean extract was obtained. Following this treatment, sample throughput was approximately 10 samples per hour, obtaining a preconcentration factor of 10. Calibration curve was established for PAT in apple juice samples, applying the DLLME-MECK procedure. LOD (obtained as 3xS/N) was 0.64 μg·L-1 and LOQ (obtained as 10xS/N) was 2.14 μg·L-1. The precision of the method was evaluated in terms of repeatability and intermediate precision and the result was acceptable (RSD < 10%). Recoveries obtained for spiked apple juice samples at four different concentration levels (5, 20, 50 and 75 μg·L-1), were above 75% with RSD lower than 9%. From the results, DLLME has shown to be a very easy, fast, efficient and green extraction procedure, in comparison with conventional alternatives for PAT analysis. Combined with the advantages of capillary electrophoresis, it could provide a very useful method for the analysis of PAT in fruit juices in routine laboratories.

[1] E. M.S.M. Gaspar, A. F.F. Lucena. Food Chem. 114 (2009) 1576-1582 [2] C. Bosch Ojeda, F. Sánchez Rojas. Food Control 18 (2007) 530–534. [3] M. Murillo, E. González-Peñas, S. Amézqueta, Food Chem. Toxicol. 46 (2008) 57–64.