The organophosphorus pesticides are widely utilized in agriculture due to its scarce 
persistence in the environment and great activity.  Its application can cause the 
appearance of residues, whose presence in food should be evaluated regularly due to 
their demonstrated toxicity. Among the most noticeable organophosphorus compounds, 
due to their extensive use, we can found: diazinon, fenthion, malathion and chlorpirifos.  
These insecticides are utilized for agricultural treatments in olive grove cultivation to 
control different plagues. Spain is one of the main producing countries and exporters of 
olive oil, for which to improve its quality and to guarantee food security, is fundamental 
to defend this product.  

The majority of the insecticide residues determinations are carry out in central 
laboratories, using official chromatographic methods. Nevertheless, these techniques 
present certain objections, like long sample preparation procedures with several 
purification steps -and often analyte derivatizations-, besides specialized technical 
personnel and expensive equipments.  These methods have restricted use since the 
number of samples that can be analyzed is limited.  

The objective of these PhD studies is to have fast and sensitive methods that allow the 
screening of residues of organophosphorus insecticides in extra virgin olive oil, with 
minimum sample pre-treatment and low solvent consumption, so that they can be 
applicable in the own field and in the oil mills. In this way, we selected immunoassay 
among the sensitive, specific, simple, cheap and with high throughput screening 
methods.

ELISAs for the pesticides diazinon, fenthion, malathion and chlorpyrifos have been 
developed. Between five and nine different haptens for each analyte have been 
designed. Later, these compounds were conjugated to proteins, being used to immunize 
rabbits. The polyclonal antibodies produced have been characterized by ELISA in two 
formats, immobilized conjugate (indirect) and immobilized antibody (direct). The most 
sensitive  inmunoassays have been obtained in immobilized conjugate format and, after 
the optimization of different variables such as pH, ionic force, surfactant concentration 
and incubation time, they have shown IC50 values of 7.5 ng/mL for diazinon, 0.05 
ng/mL for fenthion, 1.6 ng/mL for malathion and 0.007 ng/mL for chlorpyrifos. The 
developed ELISAs are highly specific, except those for chlorpyrifos since they 
recognized some similar compunds. 

After the organic solvent tolerance studies for the assays, a fast and simple extraction 
method of pesticide residues from extra virgin olive oil has been developed. This 
methodology, based on liquid-liquid extraction and cold cleaning, simplifies the sample 
treatment in comparison with the traditional techniques for this kind of matrix, which 
supposes time analysis reduction, as well as the possibility of accomplishment in oil 
mills. Initially, for the development stage, this technique was applied to the 
determination of atrazine residues in olive oil.
 
It has been verified that this extraction method is also applicable for the ELISA 
determination of the target organophosphorus pesticides, diluting the extract previously 
to ELISA analysis. 

The developed methodology -extraction and determination- has been applied to extra 
virgin olive oil samples from different origins. The obtained results have been validated 
with the reference method (GC-MS), reaching good correlations. The same technique 
has been also applied to other vegetable oils (non-refined olive oil, corn, sunflower, 
soybean) without another change that to do the calibration in the same type of oil to 
analyze. 

Finally, the development of rapid immunoassys based on membranes for residue 
determination of diazinon, fenthion, malathion and chlorpirifos, has been carried out 
using the ELIFA methodology. Combining this methodology and the previously 
developed extraction protocol for olive oil, IC50 values of 22, 4, 10 and 0.4 ng/mL for 
diazinon, fenthion, malathion and chlorpyrifos, respectively, have been obtained in extra 
virgin olive oil samples as a result of the inmunoconcentration step which takes place at 
the membrane. In addition, the direct observation of the membrane allows detecting 
samples with residue concentrations over the MRL for olive oil, allowing the fast and in 
situ screening of a great number of samples.