Modern Extraction Techniques for Pesticide Residues Determination (Part 1)

Due to intensive use of pesticides, their residues have become an unavoidable part of the environment, and they are often detected in all environmental segments and therefore their monitoring has been frequently performed throughout the world. As the presence of trace amounts of both pesticide residues and their degradation products could be potential health hazards, UN organization has formed specialized groups: World Health Organization (WHO) and Food Agriculture Organization (FAO), with the aim to establish restrictive measures to protect the environment against pollution. These organizations and their experts’ groups on annual meetings summarize international achievements in pesticides domain, establish legislation and make recommendations obligating member states to act in accordance with international standards.

As pesticides are a very heterogeneous group of compounds with different biological and physicochemical properties, the current trend in pesticide residues analysis is developing multi-residual methods that not only provide simultaneous determination of large number of pesticides, but also can be applicable to large numbers of samples of different origin.

Although separation chemical analysis involves several stages (sample preparation, analyte separation i.e. quantification and data analysis), sample preparation step can be marked as “the most critical” one. Traditional sample preparation methods (liquid-liquid extraction, Soxhlet extraction, etc.) are laborious, time consuming, expensive, requires large amounts of

organic solvents and usually involve many steps, leading to loss of some analyte quantity. Additionally, consequences of hydrocarbon solvents use, such as ozone depletion and generation of considerable cancer waste, lead to reduction of not only their use but also their manufacture. As a result, modern sample preparation procedures, such as accelerated solvent extraction (ASE), supercritical fluid extraction (SFE), microwave assisted extraction (MAE), solid phase extraction (SPE), solid phase microextraction (SPME), matrix solid phase dispersion (MSPD) extraction and QuEChERS (quick, easy, cheap, effective, rugged and safe), have been developed to overcome the drawbacks of the traditional approaches.

Overall, a comprehensive analytical procedure is carried out in the way that obtained results can be found within prior established concentration range. Whether the measurement will be performed at the desired concentration range is determined by the instrument sensitivity and the choice of sample preparation method. Multiple factors, related to the physical properties of not only tested active ingredients (volatility, solubility in water and organic solvents, stability, acid-base properties, etc.), but also sample matrix (water, lipids, pigments content etc.), must be considered during an experiment planning. The choice of sample treatment depends directly on mentioned factors, but also on analysis purpose i.e. on required detection method sensitivity (limits of detection) and quantification accuracy (whether the aim is to establish the exact concentration value regardless maximum residues limits (MRLs), or just to establish if result is above or under MRLs).

Considering the complexity of these issues, analytical procedure for each specific case should be chosen in order to minimize problems relating to analysis duration, consumption of solvents and other necessary reagents, and also to reduce number of involved analytical steps, which would minimize potential sources of errors. Additionally, it is desirable that chosen analytical procedure allows the simultaneous determination of large number of pesticides. This paper summarizes the basic principles of modern extraction techniques, comparing their advantages and drawbacks, and their ability and applicability for pesticide residues determination.

1. Supercritical fluid extraction (SFE)

This technique uses supercritical fluid (SF)1 as an extraction tool for “drawing out” the organic compounds from solid matrices. Commonly used for this purpose is CO2, as it has relatively low critical temperature (310 C) and low critical pressure (73 kPa). It is not reactive and is accessible in a high degree of purity at low cost. Changes in temperature and pressure at which the supercritical CO2 is held will increase or decrease the “strength” of solvent and thus the selectivity of extraction performed. At constant temperature which exceeds critical temperature, the supercritical CO2 will be able to extract analytes of low polarity at low pressure, and high polarity analytes at high pressure. SFE with CO2 is usually performed at pressures that are not high enough to achieve efficient extraction of polar compounds. In such conditions, the supercritical CO2 is a good extraction medium for non-polar compounds and moderately polar ones, such as PAHs, PCBs, organochlorine (OCPs) and organophosphorus (OPPs) pesticides, etc. The efficiency of supercritical CO2 can be improved by adding small amounts of modifiers, which identity is often more important than their concentration, since the major role of a modifier is to interact with the sample matrix to promote desorption into the fluid. Some of the common solvents such as acetone and methanol are now mostly used as modifiers. Besides CO2, supercritical N2O has been much in use as well, and it could be used both with and without modifiers.

The basic principles and possibilities of applying the SFE technique for determining pesticide residues in samples of different origin have been documented in several reports.  In general, SFE usually lasts less than two hours, and the further analysis can be accomplished in several ways. According to one, SF with analytes is passed through a capillary that is immersed in an appropriate solvent. While in the capillary, SF exists, but after leaving the capillary it becomes a gas (the pressure falls below the critical pressure). The largest part of this gas passes through the solvent, while the extracted analytes are retained in the solvent (the degree of retention depends on the solvent, i.e. the solubility of the analyte in it). Also, the flow of SF can be directed to a solid sorbent, which will then bind analytes, and its elution by appropriate solvent, analysts translate into a solution suitable for further analysis. Also, the flow of SF could be directed directly to capillary column of the gas chromatograph (GC), thus obtaining the “on-line” SFE. This approach enables analytical scheme with the highest sensitivity for a limited amount of sample available for analysis.

Recent studies have shown that SFE methods, followed by additional purification of the obtained extracts, meet the strict criteria of the pesticide residues analysis.  One study showed that SFE method combined with purification on the SPE columns (C18 and Envicarb/NH2), could be used for determination of 242 pesticides in spinach, 245 in green beans and 263 in orange, while another showed that the SFE preparation of potato, tomato, lettuce and apple samples, combined with the extracts clean-up on amino propylene columns, could be used for determination of 37 pesticides.

 2. Solid phase extraction (SPE)

SPE is one of the most commonly used sorbent techniques in analyzing pesticide residues. This method is based on the omission of extracts containing target analytes through a column filled with the appropriate sorbent (which was previously conditioned by an appropriate solvent or solvent mixture), or passing of an appropriate solvent through the SPE column to which a suitable amount of sample was previously added. Using selective solvents, first the co-extractants from the SPE column can be successfully eluted, and then the target analytes or the elution of analytes can be direct, where undesirable co-extractants derived from the sample matrix remain in the SPE column. Compared with the traditional methods, SPE has many attractive features. It is easy to operate, costs less, it has been automated and uses small amounts of solvent. SPE is a multifunctional technique, since the purification and the concentration occur in the same step. Unfortunately, SPE has certain limitations, primarily related to lower yields (recovery), i.e. slightly lower sensitivity, in situations where there is “clogging” of the SPE column (blocking of the sorption centres by solid and oily components originating from the sample).

Fig. 1. The basic principle of SPE technique

The most commonly used SPE sorbents in pesticide residues determination are: reverse phase octadecyl (C18), normal-phase aminopropyl (-NH2) and primary-secondary amine (PSA), anion-exchanger three-methyl ammonium (SAX) and adsorbents such as graphitized carbon black (GCB). Normal-phase sorbents such as florisil (MgSiO3), aluminum oxide (Al2O3) and silica (SiO2) are usually used in combination with the previously mentioned sorbents. The SPE cartridge should be chosen depending on the physicochemical properties of pesticides that are searched for in a particular sample, and the nature of the sample matrix. SPE has been used for the determination of 446 pesticides in cabbage, tomato, cucumber, spinach, cauliflower, celery, peas, carrots, potatoes, lettuce, onion and leek, and for the determination of 180 pesticides in tomato, lettuce, pepper, broccoli, spinach, orange, apple and banana.


Reference :

Rada Ðurović and Tijana Ðorđević : Modern Extraction Techniques for Pesticide Residues Determination in Plant and Soil Samples; Institute of Pesticides and Environmental Protection, Belgrade Serbia

Posted by Muyiwa Adebola

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