Ανάπτυξη και εφαρμογές της αναλυτικής τεχνικής μικροεκχύλισης δια της στερεάς φάσης (SPME) για τον προσδιορισμό υπολειμμάτων φυτοφαρμάκων σε φυτικά προϊόντα

Abstract

Pesticide residue analysis in food is nowadays a priority objective in pesticide research in order to get an extensive evaluation of food quality and avoid possible risks to human health. Organophosphorus insecticides (OPs) constitute a class of pesticides widely used in agriculture to combat a high number of pests in a great variety of crops. The utilization of this class of pesticides is favoured over their more persistent organochlorine counterparts because of their ability to degrade more readily in the environment. OPs insecticides demonstrate rather low environmental persistence but high toxicity. As a consequence, the determination of OPs insecticide residues in crops has been strictly regulated by governments in all countries, with two basic aims, namely, to detect the presence of forbidden pesticides on a particular commodity and to determine whether the concentrations of the pesticides used exceed their maximum residue limits (MRLs). To achieve a practical and reliable method for the determination of pesticide residues in complex matrices such as food samples, several sample preparation methods have been developed including liquid-liquid extraction (LLE), supercritical fluid extraction (SFE), solid-phase extraction (SPE) and solid phase microextraction (SPME). However, in LLE and SPE large quantities of solvent waste are generated, multiple operation steps are needed, preconcentration of the extract prior to analysis is required, and interfering compounds are more likely to be coextracted. On the other hand, headspace sampling and purge and trap methods are simpler, less laborious, faster and solvent-free techniques. Nevertheless, these methods have some disadvantages, such as the risk of cross-contamination and leaks, and the use of high flow rates that can sometimes be incompatible with on-line operation. SPME constitutes a convenient alternative to other commonly used extraction methods because it integrates sampling, extraction, concentration and sample introduction into a single step without the use of solvents. This technique is of increasing interest in the field of pesticide residue analysis and has been applied for the determination of several classes of pesticides in aqueous media, or in other sample matrices. Recently, headspace SPME (HS-SPME) has also been used to determine pesticides in water and biological fluid samples. Compared with direct SPME, headspace SPME can shorten the time of extraction significantly because of the faster diffusion rate of the analytes in gaseous phase than in liquid phase. Because the fiber is not in contact with the sample, matrix effects can be reduced, enhancing the life expectancy of the fiber. Furthermore, HS-SPME eliminates the possibility of introducing trace-level water caused by the wick effect in direct SPME. SPME is an equilibrium process that involves the partitioning of analytes between the sample and the extraction phase. Therefore, sampling conditions must be systematically optimised in order to increase the partitioning of analytes toward the coated fiber. Besides sampling conditions and analyte properties, the type of fiber coating is one of the most important aspects to optimisation. At present, the Supelco Company has made available a number of SPME fiber coatings for the extraction of non-polar and more polar compounds from liquid, solid or gaseous samples. The commercially available SPME fibers can be divided (depending on the coating phase) into absorbent and adsorbent type fibers. Absorbent type fibers extract the analytes by partitioning of analytes into a 'liquid - like' phase. While adsorbent type fibers extract the analytes by physical interacting with the analytes. In the present study both types of coatings have been selected in order to examine their extraction efficiencies for organophosphorus (OPs) insecticides and their metabolites (omethoate, dimethoate, diazinon, fenitrothion, malathion, fenthion, ethyl parathion, chlorpyrifos, methyl bromophos, methidathion, ethyl bromophos, ethion, fenthion sulfoxide, and fenthion sulfone) in olive oil, olives' pulp, tomatoes and cucumbers samples. Specifically, an in depth study on the optimum extraction efficiency of four commercially available SPME coatings (PDMS, PA, CW-DVB, PDMS-DVB) for the quantitave determination of selected pesticides in olive oil, and vegetables samples has been done. The parameters that affect the SPME procedure were evaluated and optimised by analysing spiked samples using all the tested fibers. Furthermore, the analytical characteristics of the method such as, precision, linearity, detection limits and recoveries were evaluated for each fiber. The obtained results have shown that HS-SPME procedure is a sensitive, reproducible, simple rapid and economical technique to rule out the presence of the selected pesticides in olive oil and vegetables samples at trace levels. Recoveries were in relatively high levels over 70% in the selected samples and the calibration curves were reproducible and linear (R²> 0.98) for all analytes with all of the tested fibers. The limits of detection ranged from 0.005 to 0.01 μg/Kg for olives, from 0.005 to 0.01 mg/L for olive oil and from 0.04 to 0.2 μg/Kg for tomatoes and cucumbers samples. Optimization of the parameters affecting the method sensitivity should be carefully developed in order to enable substantial increase in the amount extracted of most analytes and to improve the limit of detection with each fiber. All fibers displayed efficient analytical characteristics for the extraction of the target analytes in the tested samples. PDMS 100μm fiber proved to be more suitable for the analysis of the selected pesticides due to its higher sensitivity in the selected samples. The last part of the work presents the results of the occurrence of organophosphorus pesticides (OPs) and metabolite residues in 167 samples of Greek virgin olive oil during a two-year (2004-2005) sampling campaign. The purpose of this study was to apply the HS-SPME method for the determination of OPs in virgin olive oil samples, within the context of a pesticide-monitoring program for the Mediterranean diet in order to estimate the dietary risk exposure to OPs in the Greek population which was not reported so far in previous studies. Both chronic and acute exposure were considered for the risk assessment that was made based on individual pesticides as well as on cumulative approaches since OPs have a common toxicological way of acting. Two deterministic approaches, the Hazard Index (HI) and the Toxicity Equivalence Factor (TEF), were used for the cumulative risk assessment of the intake of OPs and to assess potential health concerns. A total of 30.5% of samples contained detectable residues while only one sample contained dimethoate residues higher than the maximum residue limits. Among the seven detected OPs, fenthion and fenthion sulfoxide residues were detected in 10.8% and 14.4% of the samples respectively at 0.003 to 0.6118 mg kgˉ¹ followed by dimethoate that was detected in 10.2% of the samples, at 0.003 to 0.0573 mg kgˉ¹. The acute dietary risk assessment was evaluated by determining the National Estimated Short-Term Intake (NESTI) while for the chronic dietary risk assessment the National Theoretical Maximum Daily Intake (NTMDI) and National Estimated Daily Intake (NEDI) were calculated. The estimated intakes (NESTI and NEDI) of each pesticide were <7% and <0.86%, much lower than the corresponding acute reference doses (ARfDs) and acceptable daily intakes (ADIs), respectively. Furthermore, a cumulative risk assessment was performed using the Hazard Index (HI) and the Toxicity Equivalence Factor (TEF) approaches taking into account that OPs share the same toxicological mechanism. The determined HI and TEFs were found to represent only a small portion of the respective ADIs or ARfDs. These results indicate that neither acute nor chronic risk derives for the Greek population through olive oil consumption.