Publication Abstract

The objective of this work was to develop a two stage, semi-automated sample extraction/clean up procedure for marine biota and sediments sample containing the brominated flame retardant (BFR), hexabromocyclododecane (HBCD). Tetrabromobisphenol-A (TBBP-A), an environmentally relevant, high volume BFR was also included in this work as little additional effort was required.  The main aims of the method development and refinement exercises were to (1) control interfering sample matrix effects during the electrospray ionisation stage of ion trap mass spectrometric (IT-MS) detection and (2) increase method sensitivity and reduce sample turnaround times using state-of-the-art, ultra performance liquid chromatography (UPLC™) coupled to triple quadrupole, mass spectrometric (MS/MS) detection.

 

The initial stage involved the development of a highly automated normal-phase (NP-) high performance liquid chromatography (HPLC) extract clean up methodology.  For this, standard solutions of HBCD were injected onto a preparative amino-propyl (NH2-) LC column, and eluant fractions were analysed by LC-IT-MS (liquid chromatography coupled to ion trap mass spectrometry).  The optimum conditions for the elution of HBCD were successfully established using an n-hexane:ethyl acetate gradient, with a run time of 34 min/sample. In contrast, TBBP-A could not be recovered.  Additionally, an NH2-column calibration solution was developed to determine accurate HBCD fraction collection times and to assess the performance of the clean up column.

 

A simultaneous sample extraction plus cleanup step using Accelerated Solvent Extraction (ASE®) was subsequently developed.  The suitability of a published ASE® methodology with in-cell clean up (40% w/w sulphuric acid impregnated silica) and n-hexane as the extraction solvent was evaluated using HBCD and TBBP-A standard solutions.  The results were satisfactory for HBCD, but unfortunately TBBP-A was not recovered.  The ASE® methodology was subsequently applied to an in-house laboratory reference material (LRM) using cod muscle spiked with HBCD (20.89 µg/kg) and TBBP-A (19.95 µg/kg).  The absolute recoveries for ΣHBCD were slightly low (60%) although repeatability was acceptable (±13%), while TBBP-A was not recovered.  A second, more polar extraction solvent (n-hexane:ethyl acetate [95:5 v/v]) was also assessed in an attempt to improve TBBP-A recovery. Whilst small recoveries of TBBP-A were observed (34±19%), the recovery of HBCD was significantly impacted and was reduced to 28±3%.  The application of the NP-HPLC clean up after ASE® resulted in improved HBCD recoveries for both n-hexane (66±7%) and n-hexane:ethyl acetate [95:5 v/v] (40±6%) extraction solvents. This was only statistically significant (P<0.05) for the latter.  This suggests that the NP-HPLC step is effective at removing co-extracted sample matrix that cause suppression effects during LC-IT-MS analysis. The combination of ASE® with in-cell clean-up followed by automated NP-HPLC clean-up is promising in terms of significantly shortening analysis time and staff effort, and therefore in reducing analytical costs. The approach has also demonstrated a reduction in potentially inhibiting suppression effects during LC-IT-MS analysis.

 

The LC-IT-MS analysis time was reduced from 40 to 7 min by transferring the method to a ‘fast’, ultra performance liquid chromatography method (UPLC™) with triple quadrupole mass spectrometric detection (MS/MS). This represented an approximately 80% reduction in sample turnaround time and efficiency gains in terms of solvent and gas consumption, and energy costs. After optimization of UPLC and MS instrumental parameters, linearity of detection for HBCDs and TBBP-A were shown to be acceptable and over a range of 0.5 to 200 ng/mL although baseline separation between α- and β-HBCD was not achieved.