Application of different chromatographic techniques and chemometric analysis in authenticity testing of plant protection products containing azoxystrobin as an active substance.

Azoxystrobin (methyl(2E)-2-{2-[6-(2-cyanophenoxy)pyrimidin-4-yloxy] phenyl}-3-methoxyacrylate) is an active ingredient used to protect crops against fungal diseases. The experience of the Polish control laboratory indicates relatively frequent cases of counterfeit plant protection products (PPPs) containing this active substance. The present study aimed to use chemometric methods to model chemical fingerprints obtained by different chromatographic techniques to verify the original formulation of PPPs containing the active substance azoxystrobin. The pesticides used in the study came from different sources (including stores and warehouses), were manufactured at a different time and came from different production batches. The results obtained with the HPLC-DAD and HS-GC-MS techniques were then modeled using principal component analysis (PCA) and soft independent modeling by class analogy (SIMCA) classifier. The proposed approach has been confirmed as useful for verifying the authenticity of PPPs and can be used in the routine control testing of SC pesticides containing azoxystrobin.

Antibacterial potential of the Cistus incanus L. phenolics as studied with use of thin-layer chromatography combined with direct bioautography and in situ hydrolysis.

The main aim of this study was to detect and identify antibacterial components of fraction I derived from eleven commercial C. incanus herbal teas. Fraction I obtained by a well-established phytochemical protocol of a multi-step extraction was expected to contain flavonoid aglycons alone. Antibacterial profile of fraction I was demonstrated by means of thin-layer chromatography - direct bioautography (TLC-DB) using a Gram positive B. subtilis and a Gram negative A. fischeri strain. Six chromatographic zones of fraction I exhibited a well pronounced antibacterial potential. In qualitative terms, a good agreement was observed among chromatographic fingerprints and the corresponding bioautograms of the eleven samples. The compounds isolated from the six zones were analyzed by HPLC- diode array detector (DAD)-electrospray ionization (ESI)-MS. High numerical m/z values valid for certain constituents of these isolates suggested that some selected antibacterial components are, unexpectedly, flavonoid glycosides. In order to confirm this suggestion, three independent HPTLC methods (multi-development on amino phase and two two-dimensional developments on silica gel phase) were devised to in situ hydrolyze flavonoid glycosides and then separate and visualize the liberated glucose and some other building blocks of the zones' components. Additionally, the sensitivity of glucose detection with p-aminobenzoic acid reagent was enhanced by paraffin. In that way, the presence of the kaempferol glycosides (and not only the aglycones alone) in fraction I was confirmed. Beside kaempferol, p-coumaric acid as a building block unit was shown by HPLC-DAD-MS analysis of the hydrolyzed isolates. Results proved apigenin, kaempferide and acylated kaempferol glycosides (cis- and trans-tiliroside and their conjugates with p-coumaric acid) to be antibacterial components of fraction I. Because isomers of the coumaric acid conjugated tiliroside were detected only in fraction I and not in the crude C. incanus extract, they are regarded as artifacts produced through fractionation.