Analysis of Intact Glucosinolates in Beeswax by Liquid Chromatography Tandem Mass Spectrometry

A new method was developed to determine 12 intact glucosinolates (glucoiberin, glucoraphanin, glucoerucin, gluconapin, glucotropaeolin, glucobrassicin, gluconasturtiin, glucoalyssin, 4-hydroxyglucobrassicin, 4-methoxyglucobrassicin, neoglucobrassicin, sinigrin) in beeswax, which is an authorized food additive in the European Union, by means of liquid chromatography tandem mass spectrometry with electrospray ionization. An efficient extraction procedure was proposed (average analyte recoveries were between 88 and 101 %); this involved the dissolution of melted beeswax with hexane, a solvent extraction using a mixture of ethanol and water (80:20, v/v), and a concentration step in a rotary evaporator. Chromatography was performed on a C-18 analytical column with a mobile phase of formic acid in water (0.5 %, v/v) and formic acid in acetonitrile (0.5 %, v/v), at a flow rate of 1 mL/min. The method was fully validated in terms of selectivity, limits of detection and quantification, linearity, precision, and accuracy. The limits of detection and quantification values were below 7 and 23 mu g/kg, respectively. The proposed method was applied to analyze beeswax samples collected from organic apiaries.