Development and validation of a high-performance liquid chromatography method for the simultaneous determination of selected varroacidal active substances in bee health products

The presence of substandard and counterfeit veterinary medicinal products represents a significant concern from a One Health perspective, given the potential consequences for human and animal health, as well as the environment. In this study, an isocratic flow liquid chromatography method was developed for the simultaneous determination of thymol, coumaphos, amitraz, tau-fluvalinate, and flumethrin in bee health products used for varroosis control. The chromatographic separation was conducted at a wavelength of 276 nm, utilizing an RP18e column and an acetonitrile:water mobile phase ratio of 75:25 (v/v), with a flow rate of 0.75 mL/min. The validation of the method was conducted using wood and plastic strips. Good linearity (R2 > 0.995) and accuracy values in the concentration range of 8–12 μg/mL were obtained for wood and plastic strips, respectively. The method performed well in terms of intraday and interday repeatability (%RSD ≤ 2) and robustness (%RSD ≤ 2). The detection limits for thymol, coumaphos, amitraz, tau-fluvalinate, and flumethrin were calculated to be 0.029, 0.033, 0.007, 0.041, and 0.016 μg/mL for plastic strips and 0.233, 0.071, 0.016, 0.108, and 0.067 μg/mL for wood strips, respectively. Using the developed method, the contents of 39 products identified as being sold outside the legal supply chain were analyzed for five active substances. The analysis revealed that 20 products contained at least one active substance, while 19 products had no detectable level of any active substance. It is imperative to adhere to technological advancements in the domain of medicinal products quality control, whilst concurrently cultivating chromatographic techniques and methodologies to combat the proliferation of illicit substances. This study proposes a methodology that has the potential to contribute to the enhancement of quality control measures and the suppression of the illicit trade in bee health products.