PhD: Development and optimizaton of HPLC methods for determination of permethrin and its residues in different matrices

Maja Shishovska
Institute of Chemistry, Faculty of Natural Sciences and Mathematics, Ss. Cyril and Methodius University, Skopje, Macedonia
March, 2012


The research presented in this work is contribution in the field of the new analytical methods for identification and quantification of permethrin [3-(2,2-dichloro-ethenyl)-2,2-dimethylcyclopropanecarboxylic acid-(3-phen-oxyphenyl) methylester] and its residues in various matrices. The proposed HPLC methods are for determination of its geometrical isomer forms (cis and trans) and its enantiomers (R-cis, R-trans, S-cis and S-trans). For the separations, chromatographic columns with various dimensions, stationary phases and particle size (LiChrosorb® RP18 (250 mm x 4 mm i.d., 5 µm), Chromolith® Performance RP18e (100 mm x 4.6 mm i.d., monolithic rod), Zorbax SB-C18 (50 mm x 4.6 mm i.d., 1.8 µm), Supelcosil Si 60 (150 mm x 4,6 mm i. d., 5 µm)) were used, and one column with chiral stationary phase on base of ß-cyclodextrin (ChiraDex® (250 mm x 4 mm, 5 µm)), among them. n-Hexane or mixtures of acetonitrile and water or methanol and water were used as mobile phases, in isocratic or gradient mode, with various volumetric ratio of the components, at various flow rates, by controlled temperature and detection at 215 nm or 220 nm.

The monolithic rod and 1.8 µm particle sized columns applicability was evaluated and their chromatographic parameters data were compared with data obtained for other column types. It was shown that for analysis, using monolithic column, less time is needed without loss in chromatographic performance and when using 1.8 µm particle size column the runtime is 4.5 min in normal and 1.6 min in rapid mode, using only 3.6 mL mobile phase.

All proposed methods are validated with regards to: retention and separation factors, resolution, linearity, limits of detection and quantification, accuracy and precision. The validation data show that the proposed methods are selective, sensitive, precise and accurate, and simple and rapid, as well.
When developing the method for permethrin residues analysis in wine, the quantity of the solvent (ethanol) needed for sample preparation has been optimized and the matrix effect studied, which is important when testing various types of wine.

As for the method for separation and quantification of permethrin enantiomers, the whole procedure is shorter and the separation of the trans-pair is achieved with better analytical performance compared to reported methods. The correlation between the temperature (288 - 318 K) and chromatographic parameters was studied. The obtained vant Hoff’s correlations are presented (ln k x 1/T) and (ln α x 1/T), for cis and trans-enatiomers and enantiomeric pairs. It was found that the first correlation is linear, and the second is linear in short range (318 - 303 K), only. At higher temperatures, cis-enantiomers are more stable compared to the trans-pair, but at lower temperatures the two enantiomeric pairs act similarly. The values for: ΔGº, ΔHº and ΔSº were calculated for every enantiomer, separately. The obtained values of free Gibbs energy (ΔGº) for the cis-pair are higher than the values obtained for the trans-pair, which results in better chiral separation. For the trans-pair, the ΔHº-values are very similar, but for the cis-pair they differ more, leading to their better separation. Also, it has been calculated the free Gibbs energy difference (Δ(ΔG)) for the cis and trans pairs, for the temperature range 288 - 318 K. According to the results, both the entropy and the enthalpy have part in the separation, which is affected by many processes acting simultaneously in the separation of enantiomers. This method was applied for permethrin enantiomers testing in correlation with a temperature stability study in a tomato juice matrix.

Proposed methods have been applied for permethrin determination in various formulations (medical shampoo, veterinary powder, gel and powder for household use, mattresses cleaner) and raw material samples as well as for permethrin and residues determination in wine samples. It was proved that the same methods might be used for analysis of various formulations and matrixes.