A Zwitterionic Hydrophilic Interaction Liquid Chromatographic Photo Diode Array Method as a Tool to Investigate Oxalic Acid in Bees: Comparison with Mass Spectrometric Methods

Background: Oxalic acid constitutes beekeepers′ leading choice for the battle of the parasitic mite Varroa destructor considering its efficacy, low cost, and easy application. Nevertheless, its broad use and reported synergistic effects prompted us to explore analytical methodologies for its determination in honeybees, especially after death incidents. Methods: The extraction of oxalic acid from bees was conducted by applying a simplified water extraction protocol. Oxalic acid′s content in honeybees was investigated through a novel zwiterionic hydrophilic interaction liquid chromatographic coupled to photo diode array (ZIC-HILIC-PDA) method and paralleled to gas/liquid chromatographic mass spectrometric methods. Results: The analytical method was validated, exhibiting a sufficient limit of quantification (LOQ) of 1.46 μg/g bee bw and precision and accuracy within the acceptable statistical limits as expressed by the relative standard deviation (RSD%) obtained from repeatability-reproducibility and recovery studies <12.5%. The application of the analytical method to 45 real honeybee samples demonstrated a fluctuation of oxalic acid’s concentrations from 1.6 to 1202.4 μg/g bee bw, verifying its frequent use in apiculture, exemplified by an overall 19% of positive samples. Conclusions: ZIC-HILIC-PDA proves a pivotal and alternative method to mass spectrometry tools in the determination of oxalic acid and other organic acids in honeybees and pertinent commodities.