Acidic pesticides, detection

Acetylcholine + H20 AChE> Choline + Acetic acid Choline + Oz + HzO COx >- Betaine + H202 FIGURE 2.3 Bienzymatic reaction for pesticide detection. 

FIGURE 2.4 Acid phosphatase and GOD bioenzymatic reaction for pesticide detection. 

Acidic pesticides and metabolites were concentrated from aqueous solution by the anion procedure of Richard and Fritz (10). The anionic materials in these concentrates were methylated using diazomethane and the derivatized products were separated and detected by gas chromatography. Test results of the recovery efficiencies by this method for several pesticides and suspected metabolites have been reported elsewhere (11). An overall recovery of 93% was achieved for sixteen acidic pesticides and metabolites spiked into water at 200 ppb. 

Earlier work in this field [28] indicated that acetylcholinesterase enzymes would be suitable biomolecules for the purpose of pesticide detection, however, it was found that the sensitivity of the method varied with the type and source of cholinesterase used. Therefore the initial thrust of this work was the development of a range of enzymes via selective mutations of the Drosophila melanogaster acetylcholinesterase Dm. AChE. For example mutations of the (Dm. AChE) were made by site-directed mutagenesis expressed within baculovirus [29]. The acetylcholinesterases were then purified by affinity chromatography [30]. Different strategies were used to obtain these mutants, namely (i) substitution of amino acids at positions found mutated in AChE from insects resistant to insecticide, (ii) mutations of amino acids at positions suggested by 3-D structural analysis of the active site,... 

E. Dijkman, D. Mooibroek, R. Hoogerbmgge, E. Hogendoom, J.-V. Sancho, O. Pozo, F. Hernandez, Study of matrix effects on the direct trace analysis of acidic pesticides in water using various LC modes coupled to MS-MS detection, J. Chromatogr. A, 926 (2001) 113. 

Hogendoom, E. A., Huls, R., Dijkman, E., and Hoogerbmgge, R., Microwave assisted solvent extraction and coupled-column reversed-phase liquid chromatography with UV detection. Use of an anal)4ical restricted-access-medium column for the efficient multi-residue analysis of acidic pesticides in soils, J. Chromatogr. A, 938, 23-33, 2001. 

In many contributions reporting on acidic pesticides in environmental samples ESI applied as ion spray was predominantly performed to analyse these pollutants. APCl, however, was not as effective as ESI as studies with standard solutions of the pesticide mixtures made obvious [325] when phenoxy acid compounds were determined using both types of interface. MSn quantitative results were used for confirmation. Mass detection after CZE-MS interfaced by ESI was successfully apphed to analyse drinking water spiked with chlorinated acid herbicides. Selected-ion elec-... 

Electrochemical biosensors have also been developed for detecting a wide range of other desirable and undesirable components in food such as cholesterol, vitamin C (ascorbic acid), pesticides, and several amino acids, including lactate, malate, glutamate, ° and lysine. They have also been used for detection of alcohols and polyphenols in beverages like wine, beer, and tea. Aside from detection of specific chemicals, electrochemical biosensors have been reported for detecting bacterial contamination of food. In this case, there is no biological catalyst on the electrode. Instead, any bacteria... 

A number of papers describe techniques for the determination of choline esterase activity based on amperometric measurement of the product formed as a result of enzymatic hydrolysis. The oxidation potential of choline is too high to be electro-chemically oxidized directly. In this case, artificial (nonnative) choline esterase substrates are used. Thus, butyiyl or acetyl thiocholine forms thiocholine as a result of choline esterase hydrolysis. When the analyte is not present in the solution, the substrate acetylthiocholine is converted into thiocholine and acetic acid. Thiocholine is oxidized by the appUed voltage. In the presence of an inhibitor, conversion of acetylthiocholine is decreased or even null. Furthermore, the anodic oxidation current is inversely proportional to the concentration of pesticides in samples and the exposed time as well. The procedure of the preparation of an AChE biosensor and pesticide detection is shown in Fig. 13.4. 

An enzymatic assay can also be used for detecting anatoxin-a(s). " This toxin inhibits acetylcholinesterase, which can be measured by a colorimetric reaction, i.e. reaction of the acetyl group, liberated enzymatically from acetylcholine, with dithiobisnitrobenzoic acid. The assay is performed in microtitre plates, and the presence of toxin detected by a reduction in absorbance at 410 nm when read in a plate reader in kinetic mode over a 5 minute period. The assay is not specific for anatoxin-a(s) since it responds to other acetylcholinesterase inhibitors, e.g. organophosphoriis pesticides, and would need to be followed by confirmatory tests for the cyanobacterial toxin. 

The sensitivity of detection is usually between 0.2 and 0.5 pg per chromatogram zone. This is also true for pesticides based on organophosphorus acids [9]. 

Detection limits of 250 ng per chromatogram zone have been reported for THC-11-carboxylic acid [15] and of 500 ng per chromatogram zone for carbamate pesticides [19]. 

Chlorophenoxy acids are relatively polar pesticides which are usually determined by LC because volatile derivatives have to be prepared for GC analysis. This group of herbicides can be detected by multiresidue methods combined with automated procedures for sample clean-up, although selectivity and sensitivity can be enhanced by coupled-column chromatographic techniques (52). The experimental conditions for Such analyses are shown in Table 13.1. 

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