Validation of GC-MS method for determination of varroacide residues in propolis

Propolis is a resinous material collected by honeybees (Apis mellifera L.) from various plants and enriched with bee salivary gland secretions and wax. Number of factors, such as beekeeping practice, can affect the quality and consequently the therapeutic value of propolis and its preparations. The most important source of propolis contamination is improper administration of varroacides that have to be used for long-term control of honeybee mite Varroa destructor. Therefore, the analysis of these noxious substances is essential in quality control of propolis.

In this study the method based on the extraction with n-hexane followed by purifying of extract on florisil column was used prior to sample analysis. Purified extracts were analyzed by gas chromatography-mass spectrometry hyphenated technique. The procedure was optimized and validated for the determination of bromopropylate, amitraz and coumaphos residues in propolis.

Investigated validation parameters of applied procedure were selectivity, linearity, precision, accuracy, robustness and limits of detection (LOD) and quantification (LOQ). All these parameters satisfied the ICH criteria [1] in the test samples of standards mixture for all three examined compounds. The applied method for varroacide analysis in validation sample of propolis was suitable for determination of bromopropylate and coumaphos. Recoveries were 100% for bromopropylate and 63% for coumaphos with LOQ of 0.08 and 1.5µg/g, respectively. On the other hand, the procedure was inappropriate for determination of amitraz, probably due to its instability in complex matrix of propolis as was previously described for honey and beeswax [2].

References: [1] International Conference on Harmonisation (ICH) of Technical Requirements for the Registration of Pharmaceuticals for Human Use (2005) Validation of Analytical Procedures: Text and Methodology Q2(R1).

[2] Korta, E. et al. (2001)J. Agric. Food Chem. 49:5835–5842.