Rapid separation of hexabromocyclododecane diastereomers using a novel method combining convergence chromatography and tandem mass spectrometry

Abstract

Analysis of the brominated flame retardant hexabromocyclododecane (HBCDD) is characterized by the separation of its three predominant diastereomers. This analysis is typically performed using reversed phase liquid chromatography (RPLC) coupled with mass spectrometric (MS) detection with analysis times in the order of 10 minutes or greater. Here we describe a rapid method using supercritical CO₂ and methanol to baseline separate the three most abundant HBCDD diastereomers within a three minute run time using a High Strength Silica (HSS) C18 1.8 μm particle size column. A unique elution order of the α-, β- and γ-HBCDD diastereomers using supercritical CO₂ was observed, and can be used as an orthogonal separation for further confidence in diastereomer identification when used in conjuction with RPLC. A tandem quadrupole mass spectrometer with negative mode electrospray ionization was used for detection, operating in multiple reaction monitoring (MRM) mode. Ionization was enhanced by the addition of a make-up flow, which was introduced to the post-column effluent. Method limit of detection (LOD) and limit of quantification (LOQ) for α-, β- and γ-HBCDD were based on peak-to-peak signal to noise ratios of greater than 3 or 10, respectively. The LOD for all HBCDD diastereomers as solvent standards was 100 fg on-column, and LOQs 500 fg on-column for α- and γ-HBCDD and 250 fg on-column for β-HBCDD. In order to test the efficiency of this method, small subsets of complex human serum and whale blubber extracts were analyzed using this method, resulting in positive detections in samples of α-HBCDD.