Sample Preparation Techniques

Sample preparation is a very important part of the analytical method because the sample must initially be cleaned up before the final analysis. Due to the low levels of pesticides that may be found in samples, the analyte concentration must be enriched before its instrumental determination [43].

Pesticides overall are compounds from different organic groups, having different chemical properties. As a result, there are two type of methods:

Multiresidue methods: cover a large number of pesticides

Single methods and common moiety methods: for a pesticide residue or a group of pesticides than cannot be determined using a multi-residue method Single methods using derivatisation: For the analysis of some compounds derivatisation may be required. These derivatives may be prepared prior to chromatographic analysis or as part of the chromatographic procedure, either pre- or post-column [44].

Nowadays, the high number of possible pesticide residues in foodstuff indicates the need to develop multiresidue methods to cover compounds of different polarities and to allow routine analysis . Below, there is a description of some of the most used sample preparation techniques for pesticide residues determination.

60 2.2.1 Solvent Extraction

Solvent extraction is the most widely used technique, mainly because of its ease of use and wide ranging applicability. The extraction process varies slightly, depending on whether the sample is liquid or solid. Analysis of liquid samples has an advantage over analysis of solid samples that one fewer pretreatment step is usually required, because of their liquid state. The latter are usually repeatedly extracted with an immiscible organic solvent. Solid samples are usually homogenized before extraction, by mechanical grinding, mixing, rolling, agitating, stirring, chopping, crushing, macerating, mincing, pressing, pulverizing, or any other reasonable means of comminuting the sample. A portion is then blended or stirred with an organic solvent which is then homogenized with sodium sulfate to bind water present in the sample.

The dried powder is then centrifuged and the supernatant is either concentrated or injected directly in the chromatographic system. Sample clean-up is usually performed before final chromatographic analysis.

Solvent extraction procedures have such as, they are laborious, time- consuming, expensive, and subject to problems arising from evaporation of large volumes of solvent and the disposal of toxic or inflammable solvents [45].

2.2.2 Solid Phase Extraction (SPE)

SPE (Solid Phase Extraction) is one of the most frequently used procedures for cleanup, extraction, and preconcentration of pesticide residues from different samples. Disposable cartridges for SPE were introduced more than 30 years ago. In addition to cartridges, SPE can be done in disc, pipette tips, and 96-well plates and is recognized as beneficial alternative to LLE, because it overcomes many drawbacks. The advantages of this technique are related to low solvent consumption, low costs, and reduction of processing time. The SPE can be performed in off-line or online mode (automated process). During SPE sample preparation, the steps are (Figure 2.2):

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Conditioning of solid phase for activation of sorbents with the appropriate solvent

Loading the sample in order of retention the target compounds existing in sample

Washing with solvent for removal of interferences

Eluting with appropriate solvent in order to elute the target compounds

More frequently, the interferences are retained in the cartridge and the analytes pass through and are collected for analysis. In this occasion the third step is not done [22].

Figure 2.2 : Steps of SPE

2.2.3 Quechers

Quechers acronym stands for quick, easy, cheap, effective, rugged, and safe.

QuEChERS furnishes high-quality results in a minimum number of steps and with low consumption of solvent and glassware. The original procedure consists in extraction of the homogenized sample by hand-shaking or vortex mixing with the same amount of acetonitrile to furnish a final extract sufficiently concentrated to remove the need for solvent evaporation. Gram quantities of salts, like sodium chloride and magnesium sulfate, are then added to the sample, with mixing, to drive partitioning of the analytes between the aqueous residue and the solvent. After simple vortex mixing and centrifugation, which results in perfect physical separation of the phases, clean-up and removal of residual water is performed simultaneously by use of a rapid procedure, called dispersive solid-phase extraction (dSPE), in which a

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primary–secondary amine (PSA) adsorbent and more anhydrous magnesium sulfate are mixed with the sample extract. Dispersive SPE is based on SPE methodology, but the adsorbent is added directly to the extract without conditioning and the clean-up is easily performed by shaking and centrifugation. The latest procedure requires less time than traditional SPE and simultaneously removes residual water and many polar matrix components, for example organic acids, some polar pigments, and sugars [45].

The original unbuffered version of Quechers evolved into two official methods using citrate buffering at a relatively low buffering capacity, CEN Standard Method EN 15662, or acetate buffering at higher concentration to give a greater buffering strength, AOAC Official Method 2007). Both versions lead to a pH around 5, which corresponds to a compromise to extract satisfactorily those pesticides that are sensitive under acidic or basic regardless of the matrix. These versions have been widely evaluated and adopted as routine methods in many laboratories around the world [45].

The QuEChERS method has the advantages of high recovery, accurate results, high sample throughput, low solvent and glassware usage (no chlorinated solvents), less labor and bench space, lower reagent costs, and ruggedness. Organic acids and other potential contaminants are removed during clean-up. The main disadvantage of QuEChERS is that for one grammar of sample per milliliter of final extract the concentration of the extract is lower than for the concentrated extracts obtained by use of most traditional procedures [45][46].