Identification of Novel Brominated Compounds in Flame Retarded

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Identification of novel brominated compounds in flame retarded plastics containing TBBPA by combining isotope pattern and mass defect cluster analysis Ana Ballesteros-Gómez, Joaquin Ballesteros, Xavier Ortiz, Willem Jonker, Rick Helmus, Karl J. Jobst, John Robert Parsons, and Eric J Reiner Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b03294 • Publication Date (Web): 22 Dec 2016 Downloaded from http://pubs.acs.org on December 27, 2016

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Environmental Science & Technology

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Identification of novel brominated compounds in flame retarded plastics

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containing TBBPA by combining isotope pattern and mass defect cluster analysis

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Ana Ballesteros-Gómez1*, Joaquín Ballesteros2, Xavier Ortiz3, Willem Jonker4, Rick Helmus5, Karl J. Jobst3, John R. Parsons5, Eric J. Reiner3 1

6 7 8 9 10 11 12 13 14 15

VU University Amsterdam, Institute for Environmental Studies, De Boelelaan 1087, 1081 HV

Amsterdam, the Netherlands. 2

University of Málaga, Department of Electronic Technology, Bulevar Louis Pasteur 35, 29010 Málaga, Spain 3

Ontario Ministry of the Environment and Climate Change, 125 Resources Road, M9P 3V6, Toronto (ON), Canada 4

VU University Amsterdam, Division of Bioanalytical Chemistry, De Boelelaan 1108, 1081 HZ Amsterdam, the Netherlands 5

University of Amsterdam, Institute for Biodiversity and Ecosystem Dynamics, Science Park 904, 1098 XH Amsterdam, The Netherlands

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*Corresponding author

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E-mail: [email protected]; [email protected]

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Tel: + 31205983193

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Fax +31205989553

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1 ACS Paragon Plus Environment


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ABSTRACT

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The study of not only main flame retardants but also of related degradation products or

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impurities has gained attention in the last years and is relevant to assess the safety of our

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consumer products and the emission of potential contaminants into the environment. In this

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study, we show that plastics casings of electric/electronic devices containing TBBPA contain

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also a complex mixture of related brominated chemicals. These compounds were most

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probably coming from impurities, byproducts or degradation products of TBBPA and TBBPA

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derivatives. A total of 14 brominated compounds were identified based on accurate mass

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measurements (formulas and tentative structures proposed). The formulas (or number of

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bromine elements) for other 19 brominated compounds of minor intensity are also provided. A

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new script for the recognition of halogenated compounds based on combining simplified

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isotope pattern and mass defect cluster analysis was developed in R for the screening. The

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identified compounds could be relevant from an environmental and industrial point of view

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Keywords: flame retardants, mass defect plots, non-target screening, TBBPA, mass spectrometry

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TOC (graphical abstract)

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INTRODUCTION

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Brominated flame retardants (BFRs) are chemicals of concern due to their persistence and

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ubiquity in the environment and their potential toxicity.1,2 In recent years, new halogenated

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flame retardants have been identified for the first time in products or environmental samples.

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For example, two chlorinated organophosphate flame retardants were identified in

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polyurethane foam from baby care products, namely 2,2-bis(chloromethyl)propane-1,3-diyl-

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tetrakis(2-chloroethyl)bis(phosphate), known commercially as “V6”, and the analogue 2,2-

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bis(chloromethyl)propane-1,3-diyl tetrakis(1-chloropropan-2-yl) bis(phosphate), known as “U-

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OPFR”.3 “V6” was later reported in dust samples collected in houses and cars.4 Subsequently, a

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triazine-based flame retardant [2,4,6-tris(2,4,6-tribromophenoxy)-1,3,5-triazine, TTBP-TAZ] was

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identified by our research group in plastics of items usually found at homes and in house dust.5

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Special attention has also been given to tetrabromobisphenol A (TBBPA) derivatives, since

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TBBPA and TBBPA-based flame retardants are widely used in electrical/electronic equipment.

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Tetrabromobisphenol A is indeed considered the most widely used BFR nowadays and it has

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raised environmental concern, as occurring with other halogenated flame retardants, due to its

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ubiquity and potential toxicity.6 The global market demand for the major brominated flame

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retardants was 203,790 metric tonnes in 2001 and TBBPA accounted for 58.7% of this total

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market.7

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reported in dust in 2008.8 TBBPA-DBDPE together with other derivatives [tetrabromobisphenol-

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A-bis(allyl ether) (TBBPA-AE) were also reported in 2010 in Great Lakes herring gull eggs9 and

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later in 2013 in soil, sediment, rice hull, and earthworm samples collected near a BFR

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manufacturing plant.10 Not only BFRs, but also new byproducts or impurities derived from

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TBBPA, or from its alternative tetrabromobisphenol-S (TBBPS), have been very recently

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reported in environmental samples.11,12 The persistence and toxicity of these related

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compounds as well as their presence in the environment is still unknown.

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In this study, we analyzed plastics of casings of electronic/electrical products commonly found

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in homes (televisions, computers, etc.) that contained a high concentration of TBBPA for further

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detection of brominated impurities or byproducts coming from the synthesis or of degradation

Tetrabromobisphenol-A-bis-(2,3-dibromopropylether)

(TBBPA-BDBPE)

was

first



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products formed during the processing or the aging of the product. Polyhalogenated molecules

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have a unique negative mass defect (difference between exact and nominal mass) and

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particular isotopic patterns, which readily distinguishes them from non-halogenated

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compounds in a complex mass spectrum. These differences can be visualized for example by

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constructing a mass defect (MD) plot, where mass defect is represented against exact mass. 13-

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15

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been reported in the literature.16-18 Recently, precursor isolation based on characteristic

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fragments (e.g. Br) has been also proposed as a suitable method for the non-target screening of

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halogenated compounds.19

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In this study, we identify a variety of unreported brominated compounds, most probably

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degradation products, byproducts or impurities of TBBPA or TBBPA derivatives in plastic casings

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of consumer electronics. These compounds could be relevant with respect to the environment

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or for industrial quality control purposes. For simplifying the screening, a new script was

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developed in R to automate the construction of MD plots and the recognition of potential

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brominated compounds. The script is based on recognition of characteristic halogenated

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isotope patterns and specific mass defects. To the best of our knowledge the recognition of

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halogenated peaks by construction of mass defect plots is done by a visual and time consuming

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step. The automation of this process has not been up to date reported and is in this study

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described for the first time.

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proposed based on mass accuracy, isotopic patterns and MS/MS spectra.

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MATERIALS AND METHODS

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Chemical and reagents

Another common strategy is the recognition of typical halogenated isotopic patterns in the

Possible molecular formulas and tentative structures were

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Methanol (MeOH) was obtained from J.T. Baker® (Center Valley, USA). Tetrahydrofuran

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(THF) was acquired from Biosolve (Valkenswaard, The Netherlands). Ultrapure water was

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obtained from an “PURELAB Ultra Mk2” system (ELGA, High Wycombe, United Kingdom). All

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solvents and reagents were of analytical grade and used as supplied. For sample treatment,

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micro-centrifuge filters (0.2 μm, nylon) from Costar Spin-X obtained from Sigma-Aldrich were

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used for removing micro-particles from sample extracts when necessary. The compounds 2,4,6-


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tribromophenol (2,4,6-TBP) and TBBPA were obtained from Wellington Laboratories (Guelph,

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Ontario, Canada).

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Instruments

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A high resolution QTOF instrument (maXis 4G upgraded with HD collision cell, Bruker

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Daltonics, Bremen, Germany) equipped with an electrospray ionization (ESI)-ion booster source

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operating in negative mode was used for analysis (resolving power up to 80,000 FWHM). The

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ion booster ESI source employs an additional soft voltage and a vaporizer temperature, with

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respect to a standard ESI source, in order to improve ionization efficiency. Mainly [M-H]- anions

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were observed for brominated compounds with ion booster ESI.

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The source parameters were as follows: capillary, 1 kV; end plate offset, 400V; charging

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voltage, 500 V; nebulizer gas, 4.1 bar; dry gas, 3.0 L/min; dry temperature, 200°C and vaporizer

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temperature, 320°C. The mass analyzer settings were: funnel 1 RF, 400 Vpp (peak-to-peak

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voltage); multipole RF, 400 Vpp; quadrupole ion energy, 3.0 eV; collision RF, 750 and 1000 Vpp

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for m/z below and above 560, respectively; transfer time, 50 and 60 μs for m/z below and

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above 560, respectively, and pre-pulse storage, 10 μs. Collision energies for MS/MS

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experiments were 40 eV for every compound except for m/z of 792.5 and of 928.5 for which 50

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and 70 eV were used instead, respectively. Mass calibration was performed using a 2mM

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sodium acetate solution in water:isopropanol 1:1 v/v.

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Liquid chromatography was performed using an UHPLC system (Nexera, Shimadzu, Den Bosch,

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The Netherlands) equipped with a binary pump, autosampler and column oven. An InertSustain

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C18 (3 µm particle size, 10 mm length) precolumn and an InertSustain C18 (3 µm particle size,

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2.1 mm i.d., 100 mm length) column were used as stationary phase (GL Sciences, Eindhoven,

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The Netherlands). For the mobile phase, ultrapure water and MeOH were used in the following

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gradient: 60% MeOH for 0.5 min, a linear gradient to 98% MeOH in 15 min followed by 98%

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MeOH for 10 min. The flow was 0.3 mL/min, the column temperature was set at 35 ºC and the

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injection volume was 5 µL.

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Sample collection and preparation



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Four plastic samples from casings of electrical/electronic devices that were bought in

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electronic stores in the Netherlands in 2014 and 2015 and that contained a high concentration

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of TBBPA (0.2%-6.7% w/w) were included in the study with the aim of identifying related

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compounds.20 The samples were pre-selected by using a simple screening technique based on

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direct probe ambient mass spectrometry.20 The samples were hard plastics coming from casings

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of an electrical adaptor (5.2% w/w TBBPA, sample 1), a television (0.2% w/w TBBPA, sample 2),

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a decorative item (0.2 % w/w TBBPA, sample 3) and a router (6.7 % w/w TBBPA, sample 4). The

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extraction method was adapted from Ballesteros-Gómez et al.20 Plastic samples (around 50 mg)

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were taken from each device (casing) by using a surgical cutter and a Stanley knife and

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extracted with 10 mL of a mixture THF:MeOH (70:30, v/v) by sonicating (60min) and stirring

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(200 rpm) for 12 h. Extracts were ultracentrifuged (10,.000 rpm, 5min) to precipitate solids and

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evaporated to around ~0.5 mL (N2, 40 ◦C). After the addition of 0.5 mL of methanol:THF (70:30,

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v/v) to each extract, samples were ultracentrifuged (10.000 rpm, 5min) again and further

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filtrated if required (with 0.2 μm microcentrifuge filters). Procedural blanks were prepared in

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the same way (solvent extraction, evaporation, filtration) but without containing any sample.

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Finally, aliquots of 2-5 µL were analyzed by LC-QTOF. In order to prevent losses of compounds

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during sample treatment, no clean-up was performed before analysis.

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Data processing for untargeted screening of brominated compounds

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The software Data Analysis and Metabolic Detect from Bruker Daltonics (Bremen, Germany)

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was used for data processing. Metabolic detect (originally intended for the identification of

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metabolites) was used to subtract the background noise of the total ion chromatogram by using

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the eXpose algorithm provided by the software. The eXpose algorithm performs a background

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subtraction in a chromatogram from a reference chromatogram (in this study coming from a

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procedural blank) by setting certain tolerance values for retention time and mass position.

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Blank subtraction was very useful to provide cleaner spectra and improve in this way the

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isotope pattern recognition by the developed script.

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The mass spectra within the retention time window of interest (1.5-17 min) was summed and a

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list of m/z and corresponding intensities generated and exported in .xls format. The use of LC


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instead of direct injection made easier a simultaneous confirmation of the identified

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compounds by later inspection of the chromatographic peaks corresponding to the extracted

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ion chromatograms of the target ions. In this sense we could avoid false positives due to in-

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source fragmentation, relate expected retention times with the calculated logP of the

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structures20 and perform further MS/MS experiments. The.xls file was directly imported in the

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in-house script (written in R language, www.r-project.org) for the generation of the mass defect

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(MD) plot and for the recognition of potential halogenated compounds. As an output of the

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script, we obtained a MD plot highlighting in a distinctive color (green) those m/z values

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corresponding to potential halogenated compounds and a list of the m/z of interest. The scale

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factor −H/+Br (78/77.91051) was used for the construction of the MD plot to visually identify

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structures correlated by subsequent addition of bromine atoms.

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Finally, suitable formulas were generated with the smart formula tool from Data Analysis that is

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based on generating the formulas that match both mass accuracy and mSigma values settings

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(match factor between the measured isotopic pattern and the theoretical pattern for a given

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formula). Values of less than 5 ppm of mass error and less than 100 of mSigma were considered

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acceptable for positive confirmation (mSigma

Identification of Novel Brominated Compounds in Flame Retarded

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