Oteef, Mohammed Dhafer Y.
Analysis of the potato sprout inhibitor 1, 4-dimethylnapthalene: HPLC method development and applications.
PhD thesis, University of Glasgow.
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1,4-Dimethylnaphthalene (1,4-DMN) is a pesticide used for inhibiting the sprouting of stored potatoes, and therefore prolonging the storage time. It is registered for commercial use in different parts of the world (e.g. USA and New Zealand) and its registration process in the EU is at an advanced stage. Limited information is available regarding the behaviour and fate of this pesticide in the environment, and therefore, various studies are required in this field. In such studies, analytical methods for the determination of 1,4-DMN in the different environmental samples are a core element.
This work aims to contribute to the knowledge about this pesticide, particularly in the analytical and environmental aspects. Several methods were developed in this work for the determination of 1,4-DMN in environmental samples. HPLC was selected for the final separation and quantification. The development of HPLC separation methods for 1,4-DMN and other related compounds were achieved by the practical step-by-step approach, the use of chromatographic-simulation software, or by a combination of the two approaches. A mixture of seven dimethylnaphthalene isomers and other related naphthalene compounds was used to study the behaviour of these compounds toward the different chromatographic conditions in reversed-phase HPLC with UV detection. This study provides a good understanding of the effect of different chromatographic conditions on the HPLC separation of the compounds studied, and forms a background for the subsequent work in method development. Optimised methods for the separation of this mixture were finally achieved which provide good separation of most of the mixture’s components. In the light of the above study, an HPLC-UV separation method for routine analysis of 1,4-DMN was developed and validated. This method provides good linearity (r2 > 0.999) in the range 0.2 –300 µg/ml, and good precision with %RSD values of 3.45 % and 0.37 % for 0.02 (method LOD) and 50 µg/ml levels. The method was found to be accurate by comparing it statistically to a gas chromatographic method. The two methods were found to produce results which were not significantly different (at the 5% level) by using a regression test.
Several extraction procedures were then compared for their efficiency in extracting 1,4-DMN residues in potato samples, and also for their suitability for routine HPLC analysis. A final HPLC method (TMP/Heat method) for the analysis of 1,4-DMN residues in potato samples was then achieved and validated. This method is based on extracting 1,4-DMN from potato peel with a mixture of ethanol and 2,2,4-trimethylpentane (7:3) by heating at 50 oC. A liquid-liquid extraction is achieved (in the same extraction flask) with the water derived from the fresh peel, to end up with 1,4-DMN concentrated in the 2,2,4-trimethylpentane layer. The evaluation of the volume of this layer, in addition to the correction of any loss of 1,4-DMN during the analysis, was achieved by using 2-methylnaphthalene as an internal standard. 2-methylnaphthalene was selected as a suitable internal standard for 1,4-DMN analysis after comparing it with several other compounds (2-ethylnaphthalene, 1-ethylnaphthalene and n-butylbenzene) for the similarity of their behaviour to 1,4-DMN in the extraction and chromatographic separation processes. An aliquot of the 2,2,4-trimethylpentane layer was then analysed directly by HPLC. This method was validated in the range 0.015 to 3 µg/g of potato fresh weight. It was found to have adequate speed, detection sensitivity (LOQ of 0.015 µg/g of potato fresh weight), accuracy (recovery between 90.3 to 106 %) and precision (%RSD between 1.7 to 10.5 %) for routine analysis of 1,4-DMN residues in treated potatoes.
For the determination of natural 1,4-DMN in potatoes, a new analytical method was developed for the extraction and quantification of 1,4-DMN at trace levels. The method (ACN/PROP method) uses the advantage of injecting large volumes (100 µl) of the extracts containing 1,4-DMN directly to the HPLC as a means of enhancing the detection sensitivity. This advantage was achieved by using a mixture of acetonitrile : 2-propanol (7:3) as the extraction solution, which is compatible with the mobile phase and miscible with the water derived from potato peel. The resulting extracts were ready for direct analysis with HPLC with no further clean up. In addition, a high ratio of sample : solvent (1:1) was used for further enhancement of the detection sensitivity. This method was validated at 7.5 and 15 µg/kg of potato fresh weight. It was found to be adequate for trace analysis of 1,4-DMN in potatoes with a limit of quantification of 4.5 µg/kg of potato fresh weight, recovery values between 86.4 to 87.1 % and a precision expressed by %RSD between 4.0 to 7.9 %.
The ACN/PROP method was used for the determination of the natural levels of 1,4-DMN in potato peel and flesh, in addition to some other plant materials. A small peak was detected in the chromatogram of potato peel extracts, at the right retention time for 1,4-DMN, with an area equivalent to a level of about 4 µg/kg of potato fresh weight. However, it was not possible to confirm the identity of this peak due to its low level and the high background noise. There was no sign of the presence of 1,4-DMN in any of the rest of the plant materials analysed which were potato flesh, apples, orange, celery, spring onion, carrots, rhubarb and poppy seeds. A headspace method and preliminary work using Soxhlet extraction were also examined for the determination of natural 1,4-DMN in potatoes. However, some shortcomings in the development of the methods obstructed the achievement of adequate results.
The ACN/PROP method was also optimised for rapid routine analysis of the residues of 1,4-DMN in treated potatoes. The optimised method was validated in the range of 0.03 to 3 µg/g of potato fresh weight, and found to provide good accuracy (recovery between 89.6 to 93.2 %) and precision (%RSD between 1.6 to 7.3 %). In addition, it is a rapid, easy and straightforward procedure. Because of its advantages, this method was used for different applications regarding the distribution and removal of 1,4-DMN residues in treated potatoes.
To investigate the distribution and removal of 1,4-DMN residues, potato tubers treated and stored for about 18 weeks under commercial storage conditions were analysed for 1,4-DMN residues. 1,4-DMN residues in individual tubers were in the range of 0.63 to 1.16 µg/g fresh weight after 18 weeks of storage with a low variability factor of about 1.5. The residues were found to be concentrated in the peel layer of the tuber and have relatively even distribution across the different parts of the tuber surface. Washing 1,4-DMN-treated potatoes with water and some other solutions was found to remove insignificant amounts of 1,4-DMN residues from potato tubers. In contrast, heating the peel of 1,4-DMN-treated potatoes in an oven at 75 ± 5 oC removed up to 96 % of 1,4-DMN residues.
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