Advances in enantioselective analysis of chiral brominated flame retardants. Current status, limitations and future perspectives

STOTEN-20052; No of Pages 11 Science of the Total Environment xxx (2016) xxx–xxx

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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Review

Advances in enantioselective analysis of chiral brominated flame retardants. Current status, limitations and future perspectives Silviu-Laurentiu Badea a,⁎, Violeta Carolina Niculescu a, Roxana-Elena Ionete a, Ethel Eljarrat b a b

National Research and Development Institute for Cryogenics and Isotopic Technologies, Uzinei Street no. 4, 240050 Râmnicu Vâlcea, Romania Water and Soil Quality Research Group, Department of Environmental Chemistry, IDAEA-CSIC, Jordi Girona 18-26, 08034 Barcelona, Spain

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• This paper is an overview of enantioselective methods developed for chiral BFRs. • Enantioselective analysis methods are available only for a limited number of BFRs. • There is a need of further development of chiral GC and LC analytics for BFRs.

a r t i c l e

i n f o

Article history: Received 27 February 2016 Received in revised form 19 May 2016 Accepted 20 May 2016 Available online xxxx Editor: D. Barcelo Keywords: Novel brominated flame retardants (NBFRs) Enantioselective analysis HBCDs CSP

a b s t r a c t Enantioselective analysis is a powerful tool for the discrimination of biotic and abiotic transformation processes of chiral environmental contaminants because their environmental biodegradation is mostly stereospecific. However, it is challenging when applied to new contaminants since enantioselective analysis methods are currently available only for a limited number of compounds. The enantioselective analysis of chiral novel brominated flame retardants (NBFRs) either using gas chromatography (GC) or liquid chromatography (LC) with various chiral stationary phases (CSP) coupled with various mass spectrometric techniques was extensively discussed. The elution order of hexabromocyclododecane (HBCD) enantiomers in chiral LC was reviewed using the experimental LC data combined also with predictions from a multi-mode Hamiltonian dynamics simulation model based on interaction energies of HBCD enantiomers with β-permethylated cyclodextrin. The further development of analytical methodologies for new chiral BFRs using advanced hyphenated analytical techniques, but also the next generation mass spectrometer analyzers (i.e. GC-Qrbitrap MS-MS, LC-Qrbitrap MS-MS), will contribute to a better characterization of the transformation pathways of chiral BFRs. © 2016 Elsevier B.V. All rights reserved.

⁎ Corresponding author. E-mail address: [email protected] (S.-L. Badea).

http://dx.doi.org/10.1016/j.scitotenv.2016.05.148 0048-9697/© 2016 Elsevier B.V. All rights reserved.

Please cite this article as: Badea, S.-L., et al., Advances in enantioselective analysis of chiral brominated flame retardants. Current status, limitations and future perspectives, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.05.148

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Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Development of enantioselective analysis methods for brominated flame retardants . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Challenges in development of chiral gas chromatography–mass spectrometry analytical methods for brominated flame retardants 2.1.1. Sample injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.2. Chiral stationary phase and chiral column dimensions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.3. GC system parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.4. Spectrometric parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Challenges in development of chiral liquid chromatography analytical methods for brominated flame retardants . . . . . . . . 2.2.1. Composition of chiral stationary phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2. Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.3. Mobile phase composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.4. pH and flow rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.5. Spectrometric parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Enantioselective analysis as tool to characterize biodegradation of brominated flame retardants . . . . . . . . . . . . . . . . . . . 3.1. Hexabromocyclododecanes (HBCD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Tetrabromocyclooctane (TBCO) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Tetrabromoethylcyclohexane (TBECH) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Over the last several decades, there has been a rapid growth in flame retardant industry, and there are now N 175 chemicals classified as flame retardants. These chemicals are used in a variety of consumer products, including electronics, textiles, furniture, and toys. Of these, at least 75 are brominated flame retardants (BFRs) (Covaci et al., 2011; Papachlimitzou et al., 2012). Some of the technical flame retardant products contain brominated organic compounds including polybrominated diphenyl ethers (PBDEs), hexabromocyclododecane (HBCD), and tetrabromobisphenol A (TBBPA). The phase out of PBDEs in 2004 (Dodson et al., 2012), has led to an increased market demand for non-regulated flame retardants. These include the less studied polybrominated biphenyls (PBBs) (flame retardants used in plastics), tetrabromocyclooctane (TBCO), tetrabromoethylcyclohexane (TBECH) (Arsenault et al., 2008) and hexachlorocyclopentenyl-dibromocyclooctane (HCDBCO) (Zhu et al., 2007), decabromodiphenylethane (DBDPE) and 1,2-bis(2,4,6tribromophenoxy)ethane (BTBPE), which have been marketed as alternatives to various PBDE formulations. Canadian Environmental Protection Agency listed β-1,2,5,6-TBCO as a non-domestic substance, with an import volume of 10,000 kg/year (Arsenault et al., 2008). Moreover, TBCO was identified in herring gulls from the Great Lakes (Gauthier et al., 2008). TBECH has been shown to bind to and to activate the human androgen receptor in vitro, to bioaccumulate in captive zebrafish and have the potential for long-range atmospheric transport (Tomy et al., 2008a), while 2,3-Dibromopropyl-2,4,6-tribromophenyl ether (DPTE) was detected in the blubber extract of a hooded seal (Cystophora cristata) from the Barents Sea (von der Recke and Vetter, 2007). Nevertheless, the knowledge about the environmental fate and behavior of NBFRs in soils is limited, although some researchers (Nyholm et al., 2010a; Nyholm et al., 2010b) have investigated their degradation in soils and uptake by earthworms. Many of these substances are persistent and lipophilic and have been shown to bioaccumulate (Covaci et al., 2011; de Wit, 2002), therefore being a concern for wildlife biomonitoring. Additionally, it is believed that BFRs are associated with endocrine-, reproductive- and behavioural effects in humans (Lyche et al., 2015). Many brominated flame retardants (i.e. HBCDs, chiral PBBs, etc) have a chiral carbon skeleton and they can be released into the environment as racemate mixtures and can undergo enantiomer specific decomposition during microbial or chemical reactions in the environment. To quantify these enantioselective transformations, the enantiomeric fraction (EF) was commonly used to describe the

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0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

relationship between enantiomers during biodegradation (de Geus et al., 2000; Harner et al., 2000; Wiberg et al., 2001a; Wiberg et al., 2001b). For example, the EF(+) is defined as A+/(A++A−), where A+ and A− correspond to the peak areas of the (+) and (−) enantiomers (Harner et al., 2000), while a racemic mixture has an EF(+) equal to 0.5. Generally, since accumulation of an enantiomer is dependent on the environmental system, it is difficult to predict which enantiomer may be enriched. For example, in the case of enantioselective biodegradation in soil and sediments, the soil parameters (such as pH, organic carbon, nutrients, redox conditions, moisture, and temperature) might have a major impact on soil microbiology, and therefore they influence the enantioselective biodegradation preferences (Buerge et al., 2003). It is believed that enantioselective degradation of a contaminant implies that the enzymes involved in the conversion of such compounds are able to differentiate between the enantiomers (Muller and Kohler, 2004). This means that one enantiomer is degraded faster than the other (Harrison et al., 1998; Zipper et al., 1998a; Zipper et al., 1999; Zipper et al., 1998b), either due to preferential transporter-driven cell uptake or preferential enzymatic reactions (Qiu et al., 2014). Nevertheless, the environmental factors which affect degradation processes of organic contaminants in environmental compartments are far from being fully understood. With the respect of BFRs, most of the enantioselective determinations were focused on HBCD. For example, (Guerra et al., 2008) reported an enantioselective degradation in river sediments for the enantiomers of α and γ HBCD isomers. Regarding enantioselective analysis of NBFRs, an enantioselective biodegradation was recently reported by (Wong et al., 2012) for the enantiomers of TBCO and those of TBECH in spiked soil. The objective of this paper is an overview of enantioselective methods (involving both GC-MS and LC-MS-MS analytical methods) currently developed for organic contaminants, both from analytical and environmental points of view. 2. Development of enantioselective analysis methods for brominated flame retardants The enantioselective analysis of chiral BFRs is usually performed using gas chromatography (GC) and liquid chromatography (LC) (see Table 1) using various modified chiral stationary phases (CSPs). The chromatographic resolution provided by a GC system is typical higher comparing with the one of a LC system and therefore many legacy chiral contaminants were traditionally analyzed by GC using modified cyclodextrin (CD) as a chiral stationary phase (CSP) (Eljarrat et al., 2008;

Please cite this article as: Badea, S.-L., et al., Advances in enantioselective analysis of chiral brominated flame retardants. Current status, limitations and future perspectives, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.05.148

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Table 1 Chiral BFRs and their analysis methods in environmental samples. Chiral BFRs and their metabolites with abbreviations (Bergman et al., 2012)/chemical structure (source)

Origin of compound

No. of enantiomer pairs

Enantioselective analysis methods developed

References

1,2,5,6,9,10-Hexabromocyclododecanes (HBCDs)/ https://pubchem.ncbi.nlm.nih.gov/compound/18529

Parent compounds

6 pairs of enantiomers possible(only 3 (α-, β-, and γ-HBCD) detected in environmental samples)

-chiral LC-MS-MS (QQQ) -chiral LC-(IT)-MS-MS

multiple pairs of enantiomer possible

chiral LC-MS-MS

multiple pairs of enantiomer possible

chiral LC-MS-MS

(Heeb et al., 2014; Heeb et al., 2015; Heeb et al., 2012b; Heeb et al., 2013) (Guerra et al., 2008) (Heeb et al., 2014; Heeb et al., 2015; Heeb et al., 2012b; Heeb et al., 2013) (Heeb et al., 2012b)

multiple pairs of enantiomer possible

chiral LC-MS-MS

(Heeb et al., 2013)

Pentabromocyclododecanols (PBCDOHs)/ Aerobic https://pubchem.ncbi.nlm.nih.gov/compound/102447601 metabolites of HBCDs Tetrabromocyclododecadiols (TBCDDOHs) Aerobic metabolites of HBCDs Pentabromocyclododecenes (PBCDEs)/ Aerobic https://pubchem.ncbi.nlm.nih.gov/compound/102018307 metabolites of HBCDs Tetrabromocyclododecadienes (TBCDs) Aerobic https://pubchem.ncbi.nlm.nih.gov/compound/102018308 metabolites of HBCDs 1,2,5,6-Tetrabromocyclooctanes (TBCOs)/ Parent https://pubchem.ncbi.nlm.nih.gov/compound/296646 compounds 1,2-dibromo-4- (1.2-dibromoethyl) cyclohexane Parent (DBE-DBCH or TBECH)/ compounds https://pubchem.ncbi.nlm.nih.gov/compound/18728

multiple pairs of enantiomer possible

(Abdallah et al., 2014)

1 pair (β-TBCO)

Chiral GC-ECNI/MS

2 pairs (α-TBECH and β-TBECH)

Only α-TBECH (Wong et al., 2012) enantiomers were separated by chiral GC-ECNI/MS. No data reported in literature for β-TBECH enantiomers HPLC sample preparation (Gotsch et al., 2005) followed by chiral GC-MS-MS (QQQ) Chiral HPLC-DAD and (Vetter et al., 2010) chiral GC-ECNI/MS No data reported in literature

Polybrominated biphenyls (PBBs)/ https://pubchem.ncbi.nlm.nih.gov/compound/158629

Parent compounds

19 possible pairs (only 7 enantioseparated)

2,3-Dibromopropyl-2,4,6-tribromophenyl ether (DPTE)/ https://pubchem.ncbi.nlm.nih.gov/compound/118216 Hexachlorocyclopentadienyl-dibromocyclooctane (DBHCTD or HCDBCO)/ https://pubchem.ncbi.nlm.nih.gov/compound/93266

Parent compound Parent compound

1 pair 1 pair

Schurig, 1994) of the chiral column. For commercially available GC columns, the hydroxyl groups at positions 2-, 3- (at the wide end) and 6(at the narrow end) are usually permethylated to form modified cyclodextrins with different selectivity and better thermal stability than pure CD. Unfortunately, the thermal stability of the target analytes (chiral BFRs) is not easy to achieve, since many BFRs can suffer decomposition or thermal rearrangement, thus the chiral GC methods aren't in many cases reliable tools for enantioselective determination of chiral BFRs and chiral LC methods have to be used. 2.1. Challenges in development of chiral gas chromatography–mass spectrometry analytical methods for brominated flame retardants GC/MS is currently the most commonly used technique for enantioselective analysis of BFRs (Abdallah, 2014). Thermal degradation and isomeric interconversion are the main challenges facing analytical chemists with achiral GC/MS analysis of BFRs, while new challenges are facing in enantioselective analysis of chiral BFRs. Therefore, several parameters of the GC/MS system need to be carefully optimized according to the properties of target chiral BFRs. These include injection technique, chiral stationary phase, column dimensions, GC system parameters and mass spectrometer parameters. 2.1.1. Sample injection The chiral BFRs are generally present at much lower concentrations that legacy pollutants, and often require more extensive and rigorous clean-up and processing techniques in order to extract them for reliable determination of enantiomeric fractions from artificial samples and environmental matrices. Because of their relatively low levels in most environmental matrices, the most common injection techniques applied for chiral BFR analysis are split/splitless injection, on-column injection and more recently programmed temperature vaporization (PTV) (Butt et al., 2011). Usually, splitless injection is preferable due to the expected

(Wong et al., 2012)

trace levels of BFRs in most environmental samples, as well to its low cost and availability of split injectors as a standard injection system for most GC/MS instruments. However, thermal degradation and mass discrimination of higher molecular weight compounds (ie. HBCDs, etc) are the main disadvantages of this technique (Abdallah, 2014). To achieve a maximum sensitivity, injector temperature and splitless time are needed to be optimized. On-column injection might appear like an alternative way to minimize thermal degradation in the injector/liner section of the GC since the sample is delivered directly to the entrance of the capillary column which might result in higher precision and less variability of the results (Covaci et al., 2007). Nevertheless, matrix-related interfering substances may cause peak tailing, high noise levels, retention time shifts which can results in low sensitivity and possible in poor separation of enantiomers (Björklund et al., 2004). Additionally, the extensive sample clean-up required makes this injection techniques less desirable in the future. Recently, PTV injection emerged as the method of choice for multiresidue analysis of different classes of legacy and NBFRs with a good potential in enantioselective analysis of chiral BFRs. PTV can provide several advantages including minimal degradation of thermolabile contaminants, reduced thermal discrimination of high molecular weight compounds, and improved response factor of higher molecular weight BFRs. Nevertheless, in the case of PTV injectors, the improvement of sensitivity by larger injection volumes (5–10 μL) might result in peak tailing and possible in poor separation of enantiomers. 2.1.2. Chiral stationary phase and chiral column dimensions With respect of chiral GC analysis of BFRs, the most important consideration for retention and chiral recognition is the proper fit of the analyte into the cyclodextrin cavity. This fit appears to be a function of both molecular size and shape of the analyte, relative to the cyclodextrin cavity, while the choice of chiral GC column depends on the target

Please cite this article as: Badea, S.-L., et al., Advances in enantioselective analysis of chiral brominated flame retardants. Current status, limitations and future perspectives, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.05.148

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chiral contaminants to be investigated. In practice, β-cyclodextrinbased columns were quite successfully in the separation of enantiomers of NBFRs, namely α-TBECH and β-TBCO as shown by (Wong et al., 2012) using a BGB-176MS column (10% 2,3-dimethyl-6-tertbutyldimethylsilyl-β-cyclodextrin in BGB 1, (15 m × 0.25 mm i.d., 0.18 μm film) thickness, BGB Analytik AG, Switzerland). Also (von der Recke and Vetter, 2007) obtained a pioneering chiral GC separation of 2,3-dibromopropyl-2,4,6-tribromophenyl ether (DPTE) (see Fig. 4A) on a β-PMCD (Chirasil-Dex) (25 m × 0.25 mm i.d.) capillary column coated with 0.25 μm permethyl-β-cyclodextrin covalently bonded to dimethyl polysiloxane, nevertheless the quality of separation was lower comparing with enantioselective separation of DPTE by chiral high performance liquid chromatography-diode-array detection (HPLC-DAD) (Fig. 4B). Nevertheless, the amount of cyclodextrin in the stationary phase affects the enantioselectivity and polarity of GC columns. Enantioselectivity (separation factors (α)) increases with higher percentages of CD. Increasing the CD content also increases the polarity of the stationary phase. Thus the GC column with CD content of 20% or higher are likely to be the best in the analysis of more polar chiral BFRs. Decreasing the internal diameter (ID) of chiral columns increases enantiomer resolution, while leaving the separation factors (α values) unaffected. To balance sample loading capacity and enantiomer resolution, the 0.25 mm ID columns appear to be ideal for most separations, while a thin phase (0.1 μm) appear to be the best for separation of enantiomers. Although in the analysis of chiral legacy pollutants, the 30 m length chiral column are preferred, usually the 10–15 m length columns are preferred in the analysis of chiral BFRs, due to a shorter dead time, thus minimizing the possibility of decomposition or isomerization. 2.1.3. GC system parameters Other factors to be considered in GC chiral analysis are the parameters of the GC system. GC method development on chiral stationary phases is quite different from traditional (achiral) methods in achieving and optimizing the selectivity. In chiral chromatography (both GC and LC), the highest enantiomeric selectivity (the higher separation factors (α)) is achieved by maximizing the energy differences in the diastereomeric association complexes formed between each enantiomer and the CSP. These energy differences become smaller with increasing temperature because this will increase the entropy of CSP. Therefore, to optimize a chiral GC separation, low elution temperatures (160 °C or lower) (Badea et al., 2011) in conjunction with relatively high carrier gas linear velocities (about 50 cm/s) are generally the best. Nevertheless, for some chiral BFRs (i.e. TBECH) the decomposition or isomerization can appear at temperature as low as 120 °C (Eljarrat et al., 2008), and in some cases chiral LC measurements are needed for an accurate determination of enantiomers. 2.1.4. Spectrometric parameters Both high resolution (HR) and low resolution (LR) single quadrupole mass spectrometers have been widely applied for detection and quantification of chiral BFRs upon chiral GC separation. The LR/MS instruments could be operated in either electron ionization (EI) or negative chemical ionization (NCI) mode. While EI/MS can provide more selectivity for identification and structural confirmation of target chiral BFRs, LR-EI/ MS is not commonly used for the analysis of higher BFRs (N 6 Br atoms) due to reduced sensitivity. In this respect, chiral GC-LR-EI/MS can be developed on standard solutions of NBFRs, but the amount of analyte required by them makes tricky their applicability to low levels of BFRs from environmental samples. Therefore, NCI, also known as ECNI (electron capture negative ionization), can be used for determination of high molecular weight chiral BFRs (i.e. HBCDs, PBBs). Many BFRs do not produce abundant stable molecular or fragment ions in the ECNI source; hence, only bromide ions (79 and 81) are usually monitored in GC-ECNI/MS (Ali et al., 2011). Nevertheless, other ions were occasionally reported for specific compounds. For example, the chiral

HCDBCO was analyzed via monitoring one molecular fragment at 310 and bromide ion at 79 (Ali et al., 2011), and such selected ion monitoring (SIM) methods can be also applied to chiral methods. As an alternative solution, although not available to most laboratories, gas chromatography–high resolution mass spectrometry in EI mode (GCHR-EI-MS) was applied successfully for detection and quantification of a wide range of NBFRs including chiral molecules: TBCO, HCDBCO, and TBECH (Kolic et al., 2009), although no chiral methods were involved. Recently, advanced MS techniques like tandem mass spectrometry (MS/MS) were applied for multiresidue analysis of legacy BFRs (PBDEs), using atmospheric pressure chemical ionization combined with GC and triple quadrupole mass analysis (GC-APCI-MS/MS) with very low limit of detection (lower than 10 fg on-column) (Portolés et al., 2015). This advancement opens the opportunity for development of chiral GC-APCI-MS/MS methods for NBFRs. Also (Gotsch et al., 2005), enantioseparated 7 PBBs pairs were using HPLC sample preparation followed by chiral GC-EI-MS-MS (QQQ) (see Table 1). Nevertheless, for large molecules like HBCDs and most likely HCDBCO, the chiral GC analysis methods are not at all suitable due to instability and high boiling points of compounds and LC methods must be used. 2.2. Challenges in development of chiral liquid chromatography analytical methods for brominated flame retardants The above mentioned problems encountered with chiral GC/MS analysis, due to the high temperatures applied, resulted to several difficulties in the chiral analysis of some BFRs. Particularly for HBCDs, a racemic isomeric interconversion takes place at temperatures N160 °C rendering enantioselective separation difficult to achieve on GC columns. In this particularly case, a mixture of 78% α-, 13% β-, and 9% γHBCD can form at equilibrium state (Fig. 1) (Peled et al., 1995). Another problem that is facing in chiral GC/MS analysis of BFRs is thermal decomposition of high molecular weight compounds (i.e. metabolites of HBCDs). Furthermore, the increased interest in the relatively polar compounds like hydroxylated HCBB metabolites (Heeb et al., 2012a) it requires the development of new analytical methods involving a derivatization step prior to their chiral GC/MS analysis which may result in significant analyte loss and low recoveries (Covaci et al., 2009; Lupton et al., 2010). Therefore, chiral LC-MS-MS analysis (Fig. 2) is a powerful alternative technique to avoid the above mentioned problems encountered during the analysis of chiral BFRs by GC/MS, nevertheless challenging when applied to chiral BFRs in environmental samples. Regarding the chiral LC separation, the successfulness of the separation is obviously influenced by the chiral stationary phase (CSP) composition (e.g. type and density of chiral selector, column length and internal diameter, particle size). Nevertheless, the LC chiral method development for each column and analyte is more convoluted and complex due to the fact that mobile phase in LC contributes more to the chromatographic separation, in comparison to GC. Beside the composition of CSP, the main parameters which can be manipulated, are temperature, mobile phase composition, pH, and flow rate, as well as mass spectrometric parameters for MS-MS tandem system (Evans and Kasprzyk-Hordern, 2014). 2.2.1. Composition of chiral stationary phase Similarly to the chiral GC analysis, the most common CSP of LC column used in the analysis of chiral BFRs is mainly based on the α-, β,or γ cyclodextrin. In chiral LC, the β-cyclodextrin-based column proved to be the most effective in separation of enantiomers for chiral BFRs. For example, only one chiral stationary phase was reported in literature for efficient separation of HBCD enantiomers. Baseline resolution of the 6 enantiomers from an α-, β-, and γ-HBCD mixture was achieved on βpermethylated cyclodextrin bonded (NUCLEODEX, Macherey-Nagel, GmbH, Düren, Germany) chiral LC column (4 × 200 mm, 5 μm) (Janák et al., 2005) (Fig. 3). It is believed that the interaction of HBCDs enantiomers as hydrophobic molecules with β-permethylated cyclodextrin

Please cite this article as: Badea, S.-L., et al., Advances in enantioselective analysis of chiral brominated flame retardants. Current status, limitations and future perspectives, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.05.148

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Fig. 1. Racemic decomposition of HBCD diasterioisomers in the GC injectors (according to (Peled et al., 1995)) shows the need of chiral analysis of HBCDs enantiomers by LC-MS-MS.

increases in the presence of water from mobile phase. It was observed that (−) α- and (−) β-HBCD eluted before their corresponding (+) α- and (+) β-HBCD enantiomers (fig. 3), followed by the γ-enantiomers with (+) γ- eluting ahead of (−) γ -HBCD (Janák et al., 2005). In order to explain this order of elution, (Durmaz et al., 2012), hypothesized that interaction of HBCD enantiomers with β-permethylated cyclodextrin is more likely in water, while enantiomers elution rate is

higher in more hydrophobic solvents (i.e. acetonitrile, etc). Using a multi-mode Hamiltonian dynamics simulation model, (Durmaz et al., 2012) calculated the interaction energies of HBCD enantiomers with β-permethylated cyclodextrin and they found that the experimental capacity factors k of enantiomers determined by HPLC are increasing with the absolute value of mean interaction energies of the enantiomers (data taken from (Durmaz et al., 2012) were plotted in Fig. 3). However,

Fig. 2. Separation of HBCD enantiomers using LC with an achiral column (i.e. C18) connected to the β-permethylated chiral stationary (Yu et al., 2008) and hyphenated with a triple quadrupole mass analyzer (modified figure from Abdallah, 2014).

Please cite this article as: Badea, S.-L., et al., Advances in enantioselective analysis of chiral brominated flame retardants. Current status, limitations and future perspectives, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.05.148

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using a chiral stationary phase based on cellulose-modified silica gel (cellulose-tris(3,5-dimethylphenyl)-carbamate) packed into NUCLEOCEL DELTA S column (250 mm length × 4.6 mm i.d., particle size 5 μm) purchased from Macherey–Nagel (Düren, Germany). In the same study (Vetter et al., 2010), chiral LC separation was compared with chiral GC separation and only the first one was able to baseline separate the DPTE enantiomers. 2.2.2. Temperature Although not so crucial like in the chiral GC separation, temperature does play a significant role in chiral LC separation; however its variability may be limited by the stability of the chiral selector. Here too, although to a less extend, the lower temperatures increase the chiral selectivity and as the temperature increases, retention times decrease (due to entropy increasing). In the case of chiral LC separation, the optimum temperatures for successful separations are being determined empirically, but usually are between 0 and 50 °C. However, the operating temperature of the LC chiral columns are usually configured to at least 30 °C below the boiling temperature of the eluent, in order to ensure proper detection. 2.2.3. Mobile phase composition Often, the differences in resolution and retention time are the results of relatively small changes and therefore the prediction of order of chiral chromatographic resolution is extremely hard. Functional groups on the stationary phase and analyte might be the bases for method development. For example, the column should be run in ionic organic mode or reversed phase if ionic mechanisms are dominant. On the contrary, if hydrophobic groups are present then reversed phase LC will be preferred. A combination of methanol/acetonitrile/water in the mobile phase is usually a good choice for separation of HBCD enantiomers (Harrad et al., 2009). 2.2.4. pH and flow rate Varying the pH and therefore the charge state on the selector and analyte can play an important role in the chiral separation of molecules with basic functional groups (i.e. hydroxyl), and this it is relevant for the enantioselective separation of BFRs metabolites but less relevant on the parent BFRs. However chiral columns (cyclodextrin-based column) often have relatively strict limits on what pH they can withstand (i.e. 3 to 8). Also, different mobile phase modifiers (i.e. ammonium chloride

Fig. 3. Separation of HBCD stereoisomers on a chiral permethylated β-cyclodextrin (CD) stationary phase column (A). This figure was taken from (Koeppen et al., 2007). HBCDspermethylated β-cyclodextrin Interaction Energy (B) as calculated by (Durmaz et al., 2012).

a quantitative relationship between interaction energy values and liquid chromatographic retention times was difficult to achieve since these depend on many parameters such as flow rate, temperature, column length, density and diameter and pressure and the influence of these factors can explain the elution of (+)-α-HBCD enantiomer before the (+)-β-HBCD enantiomer, beside the smallest difference in the mean interaction energies ΔE mean = 1.2 kJ mol− 1 recorded for them. Furthermore, one strategy to improve the chromatographic resolution of chiral separation is to connect the achiral column (i.e. C18) to the β-permethylated chiral stationary phase in order to separate firstly the HBCD diastereomers (Yu et al., 2008) (in order α, β, γ-HBCD), prior to enantiomeric separation. This provided clear distinction between the respective enantiomers of each HBCD diastereomer in the resulting chromatograms (see Fig. 2). Besides cyclodextrins, also other CSPs were used in the chiral LC determination of BFRs. In one single study (Vetter et al., 2010), the enantiomers of 2,3-dibromopropyl-2,4,6tribromophenyl ether (DPTE) were separated at room temperature

Fig. 4. Separation of DPTE enantiomers by GC-ECNI/MS (A) and HPLC-DAD (B) (modified figures from (von der Recke and Vetter, 2007)).

Please cite this article as: Badea, S.-L., et al., Advances in enantioselective analysis of chiral brominated flame retardants. Current status, limitations and future perspectives, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.05.148

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by (Gomara et al., 2007)) are used in variously concentration, and they slightly modify the pH of the mobile phase, and therefore they can influence the efficiency of enantioselective separation. With the respect of flow rates, lower flow rates are increasing retention times and widening peaks, however, in contrast with chiral GC analysis, they are also increasing resolution which is of crucial importance during chiral chromatography as mass spectrometry cannot usually distinguish between enantiomers. A compromise between resolution and retention time must therefore be made. In the case of cyclodextrin-based column a flow rate of usually 0.5–1.0 mL/min is recommended. 2.2.5. Spectrometric parameters In achiral LC-MS-MS analysis, the tandem mass spectrometric (MSMS) detection conditions must be optimized to the highest analytical signal (relative intensity) and this is performed also by increasing of the temperature of the Turbo-IonSpray source. However, in the particularly case of enantioselective analysis, a compromise between a high signal and a sound accuracy of enantiomeric fractions must be achieved. For example, a higher temperature of Turbo-IonSpray source (i.e. 500 °C) will result in a higher relative intensity (Eljarrat et al., 2008) but a poor quality of a enantiomeric fraction value (i.e. 0.62) of (+) γ-HBCD in an racemic standard, while a lower temperature (i.e. 350 °C) will results in a much better enantiomeric fraction value (0.53). Interestingly, Marvin et al. (2007)) found that both mobile phase composition and column bleed could affect the MS response for different HBCD enantiomers. They formulated the hypothesis that the elution of enantiomers would compete with of co-eluting stationary phase material, during the ion charge process in the ion spray source, especially in the presence of less-polar solvents (i.e. acetonitrile). Dodder et al. (2006)) observed that the MS response changed between the elution of two enantiomers due to the extracted matrix component. In order to avoid such effects on the estimated enantiomeric fractions (EF), Marvin et al. (2007)) introduced a mathematical approach for calculation of corrected enantiomeric fractions (EF values) (Harner et al., 2000). This approach is based on the properties of labelled enantiomeric analogs that are presumable behaving identically to their native counterparts in the MS source (Marvin et al., 2007) and is using deuterium-labelled standards (e.g., d18-HBCDs) but their approach is likely to be applicable also with 13C-labelled standards (e.g., 13C12HBCDs). While electrospray ionization (ESI) source in negative ion mode is the most commonly used interface in analysis of HBCD enantiomers, both atmospheric pressure photoionisation (APPI) (Zhou et al., 2010) and atmospheric pressure chemical ionization (APCI) (Suzuki and Hasegawa, 2006) sources proved also reliable ionization methods for HBCD diastereoisomers/enantiomers detection (Abdallah, 2014). Usually, HBCD molecular ion ([M − H]−, m/z = 640.7) is monitored in all MS techniques. Particularly, the use of tandem mass (MS/MS) detection in triple quadrupole (QpQ) mass spectrometers provided high sensitivity and very low limit of detection (LODs) (≤ 1 pg on column) for all HBCD enantiomers using the ion transition [M − H] − → Br− (m/z = 78.9). In this particulary case, the operating mode of mass spectrometer is named multiple reactions monitoring (MRM). However, while ESI is extremely sensitive for polar compounds, many environmental chiral compounds like HBCDs are non-polar, and the sensivity of ESI in the analysis of chiral of BFRs is not always the best. Therefore, the formation of Cl− and CH3COO– adducts via addition of NH4Cl and CH3COONH4 to the mobile phase was investigated (Galindo-Iranzo et al., 2009) resulting in enhanced sensitivity of LC-ESI-MS/MS analysis of HBCD enantiomers. This methods is based on the specific formation of the chlorine (m/z 676.6) and acetate adduct (m/z 700.6) of the (±) α-, (±) β-, and (±) γ-HBCD enantiomers and their further fragmentation into the more stable [M − H]− molecular ion (m/z 640.6).While both approaches presented a comparable behavior for the analysis of food

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samples, the Cl− method (m/z 676.6 → 640.6) showed higher sensitivity and the limits of detections-LODs (0.2–0.4 pg on column) and limits of quantification-LOQs (0.7–14.0 pg on column) were up to 14 times lower than those obtained applying the acetate method. (Ross and Wong, 2010) compared electrospray ionization (ESI), atmospheric pressure photoionization (APPI), and anion attachment atmospheric pressure photoionization (AA-APPI) for development of enantioselective analysis methods of HBCD enantiomers in environmental samples. Similarly to ESI, the ion transition [M − H]− → Br− (640 → 78.9 and 80.9) was also monitored in the enantioselective analysis of HBCDs by APPI. In AA-APPI analysis, 1,4-dibromobutane in toluene as a bromide source for analysis of HBCDs (Ross and Wong, 2010) were used, while for AA-APPI experiments, the transitions [M + Br]− → Br− (m/z 722.6 → 78.9 and 80.9) were monitored. Comparing with ESI that produced significantly variations in EFs, (Ross and Wong, 2010) found that AA-APPI and APPI produced nearly racemic EFs for all diastereomers. Using APPI, EFs deviated slightly from non-racemic and had larger variation than the EFs found using AA-APPI. Enantiomer fractions, produced using AA-APPI, were racemic for all diastereomers, ranging from 0.492 to 0.507. Although that for HBCDs, ESI produced a 5–25-fold lower LOD than either APPI or AA-APPI, the latter two methods lead to more accurate quantification of the EF and they might be suitable for many chiral NBFRs, for which chiral or otherwise, mass-labelled standards are unavailable. Several mass spectrometric techniques (Eljarrat et al., 2008) (involving both low and high resolution mass analyzers) were reported in literature for enantioselective detection of HBCDs. (Eljarrat et al., 2008) have been developed for the enantiomeric determination of HBCDs. An LCMS-MS method involving a triple quadrupole (QqQ) instrument (Fig. 2) was used frequently in analysis of HBCDs in various environmental samples (Huhnerfuss and Shah, 2009; Tomy et al., 2008b). Trying to solve the problems relating to the low mass cut-off of the ionic trap (IT) and the variable amounts of other adduct peaks in the samples, an LC-ion trap (IT)-MS-MS method was applied successfully by (Gomara et al., 2007) for food samples, while (Guerra et al., 2008) developed an enantiomeric method for HBCDs in sediment samples using a LC-quadrupole linear ion trap (QqLIT)-MS-MS instrument. More recently, (Zacs et al., 2014) developed an ultrahigh performance liquid chromatography (UHPLC) – time-of-flight high resolution mass spectrometry (TOF-HRMS) method for the determination of hexabromocyclododecane (HBCD) diastereomers in fish samples and the method was compared against UHPLC–Orbitrap-HRMS and UHPLC–triplequadrupole (QqQ) tandem MS (MS/MS) techniques. (Zacs et al., 2014) found that UHPLC–TOF-HRMS operated in scan mode over the m/z range of 600–700 was demonstrated to be a good alternative to conventional LC–MS/MS systems for diastereomer analysis of HBCDs and this method could be also applied for enantiomers of HBCDs. Very recently, (Zacs and Bartkevics, 2015) identified and quantified 27 BFRs, including NBFRs ones, by developing an analytical method supported by high performance liquid chromatography (HPLC) coupled to Orbitrap mass spectrometry (Orbitrap-MS) with atmospheric pressure photoionization (APPI) interface operated in negative mode. HPLC-Orbitrap-MS analysis provided a fast separation of selected analytes within 14 min, thus demonstrating a high throughput processing of samples. Beside the molecular ion of (640.6369) for (±) α-, (±) β-, and (±) γ-HBCD diastereoisomers, the hexachlorocyclopentadienyldibromocyclooctane (HCDBCO) was quantified using the adduct ion [M + O2]− with the mass of 571.7285 recorded at a resolution of 17,500. In this study (Zacs and Bartkevics, 2015) proven that Orbitrap-MS is an attractive tool for trace analysis of a wide range of structurally diverse BFRs in a complex matrix like fish tissue, ensuring a selectivity exceeding that provided by commonly used conventional low resolution MS/MS techniques and that HPLCOrbitrap-MS could be also applied for enantiomers of HBCDs and for those of NBFRs (i.e. HCDBCO).

Please cite this article as: Badea, S.-L., et al., Advances in enantioselective analysis of chiral brominated flame retardants. Current status, limitations and future perspectives, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.05.148

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3. Enantioselective analysis as tool to characterize biodegradation of brominated flame retardants 3.1. Hexabromocyclododecanes (HBCD) With the respect of BFRs, most of the enantioselective biodegradation studies were focused on HBCDs (Heeb et al., 2014; Heeb et al., 2015; Heeb et al., 2012b; Heeb et al., 2013) which has already been added in 2009, in the Stockholm Convention for Persistent Organic Pollutants, because of its harmful effect on environment and wildlife, and due to its bioaccumulation potential, process that can be also enantioselective (Eljarrat et al., 2009; Guerra et al., 2008). HBCDs are produced industrially by bromination of cyclododeca-1,5,9-triene (CDT), creating six stereo centers at positions 1, 2, 5, 6, 9, and 10 of the formed reaction products. This bromination reaction can generate a total of 16 possible optical isomers, 6 pairs of enantiomers, and 4 meso forms. To date, only 3 diastereomers—named α-, β-, and γHBCD- were detected in the environmental samples and they can be found in technical grade mixtures with proportions of about 81%, 12%, and 6%, respectively, while two meso forms named δ- and ε-HBCDs account for 0.5% and 0.3% respectively. Like many other halogenated chiral pollutants, HCBCDs follow different degradation pathways in anaerobic conditions comparing with aerobic pathways and the enantioselectivity of the reactions which depends on transporter-driven cell uptake or on enzymatic reactions. (Heeb et al., 2014; Heeb et al., 2015; Heeb et al., 2012b; Heeb et al., 2013) investigated extensively the aerobic metabolism of HBCDs (Fig. 5). In all these studies, pentabromocyclododecanols (PBCDOHs) appear to be the main aerobic metabolites of HBCDs and they can be further transformed into tetrabromocyclododecadiols (TBCDDOHs). Firstly, (Heeb et al., 2012a) suggested that pentabromocyclododecanols (PBCDOHs) are potential environmental transformation products of hexabromocyclododecanes (HBCDs), and the enantioselective separation of 8 diastereoisomers namely α-, β-, γ-, δ-, ε-, ζ-, η-, and θPBCDOHs were separated with a combination of normal (SiO2, F60, 230–400 mesh) and reversed-phase LC (C18-RP, 125 mm × 4 mm, 5 μm, 100 Å, Nucleosil 100–5, Macherey-Nagel, Oensingen,

Fig. 5. Modified figure from (Heeb et al., 2014). Aerobic degradation pathways of to pentabromocyclododecenes (PBCDEs) by LinA2 and to pentabromocyclododecanols (PBCDOHs) by LinB enzymes.

Switzerland), followed by of enantiomeric separation a permethylated-β-cyclodextrin chiral-phase column (PM-β-CP). In the next study, (Heeb et al., 2012b) purified the HCH-converting haloalkane dehalogenases LinB, from Sphigobium indicum B90A and used this enzyme to investigate the enantioselective degradation of HBCD, similarly to the aerobic transformation of α-HCH. It was found that among the enantiomers, (−) α-, (+) β-, and (+) γ-HBCD are preferentially transformed by LinB, while at least seven PBCDOHs and five TBCDDOHs were tentatively identified by LC-MS-MS (triple quadrupole mass analyzer). The recorded enantiomeric enrichment of HBCD (calculated as enantiomeric excess (EE) (Patterson and Schnell, 2014; Shi et al., 2006)) was 8 ± 4% for (+) α-, 27 ± 1% for (−) β-, and 20 ± 2% for (−) γ-HBCD respectively in 32 h comparable to values of 7.1%, 27.0%, and 22.9% as obtained from respective kinetic models. Since this preliminary study (Heeb et al., 2012b) highlighted the fastest transformations of (+) βand (+) γ-HBCDs under given conditions, in the next study (Heeb et al., 2013) focused on LinB-transformation of achiral δ-HBCDs into chiral metabolites, namely to two pentabromocyclododecanols (PBCDOHs) and two tetrabromocyclododecadiols (TBCDDOHs). In these reactions only the stereocenter from C6 is converted to an alcohol with inversion from S- to R-configuration in a nucleophilic, SN2-like substitution reaction, while the other five stereocenters of δ-HBCD remained unchanged. The study suggests that LinB preferentially converted reactive bromine atoms but not those in the so called conserved triple-turn motive of cyclododecane ring conformation. In order to elucidate more these transformation pathways, a successive study (Heeb et al., 2013) investigated the enantioselective aerobic degradation of HBCDs, by LinA2, another dehalogense enzyme from Sphingobium indicum B90A isolated from HCH contaminated soils. In this study, the LinA2 substantially transformed (−) β-HBCD, but all other enantiomers were not biodegraded significantly, so the enantiomeric excess of (+) β-HBCD increased up to 90% in 32 h, while the EE values for α- and γ-HBCD enantiomers did not varied with time. Beside the hydroxylation of HBCDs and formation of PBCDOHs as aerobic metabolites, LinA2 catalyzed also the formation of three pentabromocyclododecenes (PBCDEs) via dehyrobromination (HBr elimination), similarly to the dehydrochlorination of HCHs (Fig. 5). With respect of reaction pathways, in the latest study, (Heeb et al., 2015) investigated more deeply the enantioselective degradation of β-HBCD by LinA2. To elucidate the dehyrobromination of β-HBCD, the crystal structure of its metabolite 1E,5S,6S,9R,10Spentabromocyclododecene (PBCDE) was investigated by X-ray diffraction (XRD) and found out that its enantiomer with the 1E,5R,6R,9S,10R-configuration is the only metabolite formed during LinA2-catalyzed dehydrobromination of (−) β-HBCD, via HBr elimination at C5 and C6, while dehydrobromination of (+) βHBCD gave an yet experimentally unidentified PBCDE, predicted in docking experiments to be 1E,5S,6S,9S,10R-PBCDE. Furthermore, (Abdallah et al., 2014) identified tetrabromocyclododecadienes (TBCDs) as further metabolites of enantioselective biotransformation of hexabromocyclododecane by in vitro rat and trout hepatic sub-cellular fractions. Nevertheless, further studies are needed to elucidate the structures of all PBCDEs and PBCDOHs, and ultimately NMR analysis and even three-dimensional isotope analysis (2H vs. 13C vs 81Br) might be needed for a fully understanding of dehydrobromination of HBCDs. With respect to anaerobic degradation of HBCDs, (Gerecke et al., 2006) investigated the anaerobic degradation of brominated flame retardants in sewage sludge, including the enantioselective one for HBCDs and found out that (±) β-HBCD and (±) γ-HBCD degraded more rapidly than (±) α-HBCD by 1.6 and 1.8 fold, respectively. The enantiomeric fraction (EFs) were measured after 7 h of incubation and the values were 0.47, 0.53, and 0.52 for α-, β-, and γ-HBCD respectively, thus finding no clearly enantioselective degradation of HBCDs, taking into account an estimated standard deviation of ±0.05 EF units. Although, no enantioselective measurements were performed, (Davis et al., 2006) conducted an anaerobic degradation study of HBCDs involving [14C] Hexabromocyclododecane in wastewater sludge and freshwater aquatic sediment and suggested

Please cite this article as: Badea, S.-L., et al., Advances in enantioselective analysis of chiral brominated flame retardants. Current status, limitations and future perspectives, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.05.148

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that HBCD is sequentially debrominated via dihaloelimination where at each step there is the loss of two bromines from vicinal carbons with the subsequent formation of a double bond between the adjacent carbon atoms. This assumption was confirmed by (Peng et al., 2015). In this study, a total of 4 metabolites were detected during the anaerobic biodegradation of HBCDs by process by strain HBCD-1, a pure culture isolated from a continuous anaerobic reactor over 300-days acclimation: Tetrabromocyclododecene (TBCD), Dibromocyclododecadiene (DBCD) 1,5,9-cyclododecatriene (CDT) and 2-Dodecene. Nevertheless, further enantioselective studies are needed to elucidate the anaerobic degradation pathways in different environment compartments. 3.2. Tetrabromocyclooctane (TBCO) (Wong et al., 2012) investigated the degradation of β-TBCO enantiomers in spiked soil. They found out that β-TBCO showed an interesting enantioselective behavior with the EF of the first elution enantiomer (on the BGB-176MS column) increased from 0.502 to 0.537 during the first 90 days of the degradation in soil, followed by a decrease of EF to 0.465 at the end of 360 days the experiment. The switching of the preferential enantiomer degradation may due to a change in the microbial community or other which alters the degradation behavior. 3.3. Tetrabromoethylcyclohexane (TBECH) (Wong et al., 2012) investigated also the degradation of α- and β1,2-dibromo-4-(1,2-dibromoethyl)cyclohexane (TBECH) enantiomers in spiked soil. Nevertheless, the chromatographic resolution of enantiomers was unsuccessful for β-TBECH and only the evolution of EF for αTBECH enantiomers was investigated. Here too, EF of the first elution enantiomer (on the BGB-176MS column) of α-TBECH increased from 0.505 to 0.601 during the first 90 days of the degradation in soil, and afterwards decreased to 0.583 after 360 days, which suggests that the degradation preference might change to favour E1, here too due to unknown factors. Thus, further studies are needed to elucidate the enantioselective transformation of TBECH and other chiral merging BFRs in environment. 4. Conclusions In the last years, analysis and investigation of environmental fate of chiral novel contaminants became a major topic on research in environmental chemistry. The aspects discussed in this review demonstrated that enantioselective analysis is a powerful tool for the discrimination of biotic and abiotic transformation processes of chiral environmental contaminants because their environmental biodegradation is mostly stereospecific, nevertheless challenging and novel when is applied to new contaminants since enantioselective analysis methods are currently only available for a limited number of compounds. Therefore, the understanding of environmental fate and behavior of target BFRs discussed in this review paper will be usefully in understanding the behavior of related compounds (i.e. Octabromotrimethylphenylindane (OBTMPI), DBDPE and BTBPE, etc. (Melymuk et al., 2015)), even for those NBFRs that are achiral since they can form chiral metabolites during their biotransformation. With respect to analytical techniques used in the analysis of chiral NBFRs, there is a need of further development of chiral analytical methodologies that shouldn't be so time consuming and that should have reasonable cost. Beside the lab clean-up, the use of advanced hyphenated analytical techniques, for example, LC-MS/MS, LC-HRMS, two dimensional gas chromatography-time of flight mass spectrometry (GC × GCTOF-MS), two dimensional liquid chromatography-time of flight mass spectrometry (LC × LC-TOF-MS), but also involvement of the next generation mass spectrometer analyzers (i.e. GC-Qrbitrap MS-MS, LCQrbitrap MS-MS) can be the key to such ultimate analytical methodologies. Nevertheless, the validation of these analytical methods is

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challenging since the commercially-available reference standards (labelled and unlabelled) for these compounds are not always available and therefore the identification and even semi-quantification of target analytes can be performed only tentatively using mass spectrometry libraries (i.e. NIST, Willey), while the absolute quantification of the target analytes is not possible in absence of a quantification standard. Regarding the assessment of environmental fate of chiral NBFRs, recently, (Gasser et al., 2012) and (Bashir et al., 2013) proposed to quantitatively describe the fractionation of enantiomers induced by the biotransformation of contaminants using the Rayleigh equation, similarly to the quantification of biodegradation using isotope fractionation. Therefore, using this new concept, the enantiomeric enrichment factors (εe) and the enantiomeric fractions (EFs) of the chiral NBFRs can be used to quantify their biodegradation in environmental samples and even to distinguish between their biotic and abiotic transformation pathways. Nevertheless, since substantial biodegradation (Badea et al., 2011; Padma et al., 2003) can be also non-enantioselective, EF values are not always a reliable tool for assessing the biodegradation of certain contaminants. To overcome this limitation, the enantioselective analysis of NBFRs can be combined with compound specific stable isotope analysis (CSIA) in one single technique namely enantioselective stable isotope analysis (ESIA) (Badea and Danet, 2015), involving either chiral gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) or chiral liquid chromatography-combustion-isotope ratio mass spectrometry (LC-C-IRMS). Also, in the future, it is expected that a combination between enantioselective analysis and a multi-element isotope analysis (Fenner et al., 2013) (i.e. hydrogen vs. carbon vs. bromine) to be developed for a better characterization of transformation pathways of chiral NBFRs, while NMR analysis to be used more extensively to elucidate the structures of their metabolites. References Abdallah, M.A.-E., 2014. Advances in instrumental analysis of brominated flame retardants: current status and future perspectives. Int Sch Res Notices 2014, 21. Abdallah, M.A.-E., Uchea, C., Chipman, J.K., Harrad, S., 2014. Enantioselective biotransformation of hexabromocyclododecane by in vitro rat and trout hepatic sub-cellular fractions. Environ Sci Technol 48, 2732–2740. Ali, N., Harrad, S., Muenhor, D., Neels, H., Covaci, A., 2011. Analytical characteristics and determination of major novel brominated flame retardants (NBFRs) in indoor dust. Anal. Bioanal. Chem. 400, 3073–3083. Arsenault, G., Lough, A., Marvin, C., McAlees, A., McCrindle, R., MacInnis, G., et al., 2008. Structure characterization and thermal stabilities of the isomers of the brominated flame retardant 1,2-dibromo-4-(1,2-dibromoethyl)cyclohexane. Chemosphere 72, 1163–1170. Badea, S.-L., Danet, A.-F., 2015. Enantioselective stable isotope analysis (ESIA) — a new concept to evaluate the environmental fate of chiral organic contaminants. Sci. Total Environ. 514, 459–466. Badea, S.L., Vogt, C., Gehre, M., Fischer, A., Danet, A.F., Richnow, H.H., 2011. Development of an enantiomer-specific stable carbon isotope analysis (ESIA) method for assessing the fate of alpha-hexachlorocyclohexane in the environment. Rapid Commun. Mass Spectrom. 25, 1363–1372. Bashir, S., Fischer, A., Nijenhuis, I., Richnow, H.-H., 2013. Enantioselective carbon stable isotope fractionation of hexachlorocyclohexane during aerobic biodegradation by Sphingobium spp. Environ Sci Technol 47, 11432–11439. Bergman, Å., Rydén, A., Law, R.J., de Boer, J., Covaci, A., Alaee, M., et al., 2012. A novel abbreviation standard for organobromine, organochlorine and organophosphorus flame retardants and some characteristics of the chemicals. Environ. Int. 49, 57–82. Björklund, J., Tollbäck, P., Hiärne, C., Dyremark, E., Östman, C., 2004. Influence of the injection technique and the column system on gas chromatographic determination of polybrominated diphenyl ethers. J Chromatog A 1041, 201–210. Buerge, I.J., Poiger, T., Müller, M.D., Buser, H.-R., 2003. Enantioselective degradation of metalaxyl in soils: chiral preference changes with soil pH. Environ Sci Technol 37, 2668–2674. Butt, C.M., Wang, D., Stapleton, H.M., 2011. Halogenated phenolic contaminants inhibit the in vitro activity of the thyroid-regulating deiodinases in human liver. Toxicol. Sci. 124, 339–347. Covaci, A., Harrad, S., Abdallah, M.A.E., Ali, N., Law, R.J., Herzke, D., et al., 2011. Novel brominated flame retardants: a review of their analysis, environmental fate and behaviour. Environ. Int. 37, 532–556. Covaci, A., Voorspoels, S., Abdallah, M.A.-E., Geens, T., Harrad, S., Law, R.J., 2009. Analytical and environmental aspects of the flame retardant tetrabromobisphenol-A and its derivatives. J Chromatog A 1216, 346–363. Covaci, A., Voorspoels, S., Ramos, L., Neels, H., Blust, R., 2007. Recent developments in the analysis of brominated flame retardants and brominated natural compounds. J Chromatog A 1153, 145–171.

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S.-L. Badea et al. / Science of the Total Environment xxx (2016) xxx–xxx

Davis, J.W., Gonsior, S.J., Markham, D.A., Friederich, U., Hunziker, R.W., Ariano, J.M., 2006. Biodegradation and product identification of C-14 hexabromocyclododecane in wastewater sludge and freshwater aquatic sediment. Environ Sci Technol 40, 5395–5401. de Geus, H.J., Wester, P.G., de Boer, J., Brinkman, U.A.T., 2000. Enantiomer fractions instead of enantiomer ratios. Chemosphere 41, 725–727. de Wit, C.A., 2002. An overview of brominated flame retardants in the environment. Chemosphere 46, 583–624. Dodder, N.G., Peck, A.M., Kucklick, J.R., Sander, L.C., 2006. Analysis of hexabromocyclododecane diastereomers and enantiomers by liquid chromatography/tandem mass spectrometry: chromatographic selectivity and ionization matrix effects. J Chromatog A 1135, 36–42. Dodson, R.E., Perovich, L.J., Covaci, A., Van den Eede, N., Ionas, A.C., Dirtu, A.C., et al., 2012. After the PBDE phase-out: a broad suite of flame retardants in repeat house dust samples from California. Environ Sci Technol 46, 13056–13066. Durmaz, V., Weber, M., Becker, R., 2012. How to simulate affinities for host-guest systems lacking binding mode information: application to the liquid chromatographic separation of hexabromocyclododecane stereoisomers. J. Mol. Model. 18, 2399–2408. Eljarrat, E., Guerra, P., Barcelo, D., 2008. Enantiomeric determination of chiral persistent organic pollutants and their metabolites. Trac-Trend Anal Chem 27, 847–861. Eljarrat, E., Guerra, P., Martínez, E., Farré, M., Alvarez, J.G., López-Teijón, M., et al., 2009. Hexabromocyclododecane in human breast milk: levels and enantiomeric patterns. Environ Sci Technol 43, 1940–1946. Evans, S.E., Kasprzyk-Hordern, B., 2014. Applications of chiral chromatography coupled with mass spectrometry in the analysis of chiral pharmaceuticals in the environment. Trends in Environ Anal Chem 1, e34–e51. Fenner, K., Canonica, S., Wackett, L.P., Elsner, M., 2013. Evaluating pesticide degradation in the environment: blind spots and emerging opportunities. Sci 341, 752–758. Galindo-Iranzo, P., Quintanilla-López, J.E., Lebrón-Aguilar, R., Gómara, B., 2009. Improving the sensitivity of liquid chromatography–tandem mass spectrometry analysis of hexabromocyclododecanes by chlorine adduct generation. J Chromatog A 1216, 3919–3926. Gasser, G., Pankratov, I., Elhanany, S., Werner, P., Gun, J., Gelman, F., et al., 2012. Field and laboratory studies of the fate and enantiomeric enrichment of venlafaxine and Odesmethylvenlafaxine under aerobic and anaerobic conditions. Chemosphere 88, 98–105. Gauthier, L.T., Potter, D., Hebert, C.E., Letcher, R.J., 2008. Temporal trends and spatial distribution of non-polybrominated diphenyl ether flame retardants in the eggs of colonial populations of Great Lakes herring gulls. Environ Sci Technol 43, 312–317. Gerecke, A.C., Giger, W., Hartmann, P.C., Heeb, N.V., Kohler, H.-P.E., Schmid, P., et al., 2006. Anaerobic degradation of brominated flame retardants in sewage sludge. Chemosphere 64, 311–317. Gomara, B., Lebron-Aguilar, R., Quintanilla-Lopez, J.E., Jose, G.M., 2007. Development of a new method for the enantiomer specific determination of HBCD using an ion trap mass spectrometer. Anal. Chim. Acta 605, 53–60. Gotsch, A., Mariussen, E., von der Recke, R., Herzke, D., Berger, U., Vetter, W., 2005. Analytical strategies for successful enantioselective separation of atropisomeric polybrominated biphenyls 132 and 149 in environmental samples. J Chromatog A 1063, 193–199. Guerra, P., Eliarrat, E., Barcelo, D., 2008. Enantiomeric specific determination of hexabromocyclododecane by liquid chromatography-quadrupole linear ion trap mass spectrometry in sediment samples. J Chromatog A 1203, 81–87. Harner, T., Wiberg, K., Norstrom, R., 2000. Enantiomer fractions are preferred to enantiomer ratios for describing chiral signatures in environmental analysis. Environ Sci Technol 34, 218–220. Harrad, S., Abdallah, M.A.E., Covaci, A., 2009. Causes of variability in concentrations and diastereomer patterns of hexabromocyclododecanes in indoor dust. Environ. Int. 35, 573–579. Harrison, I., Leader, R.U., Higgo, J.J.W., Williams, G.M., 1998. A study of the degradation of phenoxy acid herbicides at different sites in a limestone aquifer. Chemosphere 36, 1211–1232. Heeb, N.V., Zindel, D., Bernd Schweizer, W., Lienemann, P., 2012a. 2,5,6,9,10Pentabromocyclododecanols (PBCDOHs): a new class of HBCD transformation products. Chemosphere 88, 655–662. Heeb, N.V., Zindel, D., Geueke, B., Kohler, H.-P.E., Lienemann, P., 2012b. Biotransformation of hexabromocyclododecanes (HBCDs) with LinB—an HCH-converting bacterial enzyme. Environ Sci Technol 46, 6566–6574. Heeb, N.V., Wyss, S.A., Geueke, B., Fleischmann, T., Kohler, H.-P.E., Lienemann, P., 2014. LinA2, a HCH-converting bacterial enzyme that dehydrohalogenates HBCDs. Chemosphere 107, 194–202. Heeb, N.V., Wyss, S.A., Geueke, B., Fleischmann, T., Kohler, H.P.E., Schweizer, W.B., et al., 2015. Stereochemistry of enzymatic transformations of (+)beta- and (−)betaHBCD with LinA2-A HCH-degrading bacterial enzyme of Sphingobium indicum B90A. Chemosphere 122, 70–78. Heeb, N.V., Zindel, D., Graf, H., Azara, V., Bernd Schweizer, W., Geueke, B., et al., 2013. Stereochemistry of LinB-catalyzed biotransformation of δ-HBCD to 1R,2R,5S,6R,9R,10Spentabromocyclododecanol. Chemosphere 90, 1911–1919. Huhnerfuss, H., Shah, M.R., 2009. Enantioselective chromatography-a powerful tool for the discrimination of biotic and abiotic transformation processes of chiral environmental pollutants. J Chromatog A 1216, 481–502. Janák, K., Covaci, A., Voorspoels, S., Becher, G., 2005. Hexabromocyclododecane in marine species from the Western Scheldt Estuary: diastereoisomer- and enantiomer-specific accumulation. Environ Sci Technol 39, 1987–1994. Koeppen, R., Becker, R., Emmerling, F., Jung, C., Nehls, I., 2007. Enantioselective preparative HPLC separation of the HBCD—stereoisomers from the technical product and their absolute structure elucidation using X-ray crystallography. Chirality 19, 214–222.

Kolic, T.M., Shen, L., MacPherson, K., Fayez, L., Gobran, T., Helm, P.A., et al., 2009. The analysis of halogenated flame retardants by GC-HRMS in environmental samples. J Chromatog Sci 47, 83–91. Lupton, S.J., McGarrigle, B.P., Olson, J.R., Wood, T.D., Aga, D.S., 2010. Analysis of hydroxylated polybrominated diphenyl ether metabolites by liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry. Rapid Commun. Mass Spectrom. 24, 2227–2235. Lyche, J.L., Rosseland, C., Berge, G., Polder, A., 2015. Human health risk associated with brominated flame-retardants (BFRs). Environ. Int. 74, 170–180. Marvin, C.H., MacInnis, G., Alaee, M., Arsenault, G., Tomy, G.T., 2007. Factors influencing enantiomeric fractions of hexabromocyclododecane measured using liquid chromatography/tandem mass spectrometry. Rapid Commun. Mass Spectrom. 21, 1925–1930. Melymuk, L., Goosey, E., Riddell, N., Diamond, M., 2015. Interlaboratory study of novel halogenated flame retardants: INTERFLAB. Anal. Bioanal. Chem. 407, 6759–6769. Muller, T.A., Kohler, H.P.E., 2004. Chirality of pollutants - effects on metabolism and fate. Appl. Microbiol. Biotechnol. 64, 300–316. Nyholm, J.R., Asamoah, R.K., van der Wal, L., Danielsson, C., Andersson, P.L., 2010a. Accumulation of polybrominated diphenyl ethers, hexabromobenzene, and 1,2-dibromo4-(1,2-dibromoethyl)cyclohexane in earthworm (Eisenia fetida). Effects of soil type and aging. Environ Sci Technol 44, 9189–9194. Nyholm, J.R., Lundberg, C., Andersson, P.L., 2010b. Biodegradation kinetics of selected brominated flame retardants in aerobic and anaerobic soil. Environ. Pollut. 158, 2235–2240. Padma, T.V., Dickhut, R.M., Ducklow, H., 2003. Variations in α-hexachlorocyclohexane enantiomer ratios in relation to microbial activity in a temperate estuary. Environ. Toxicol. Chem. 22, 1421–1427. Papachlimitzou, A., Barber, J.L., Losada, S., Bersuder, P., Law, R.J., 2012. A review of the analysis of novel brominated flame retardants. J Chromatog A 1219, 15–28. Patterson, D., Schnell, M., 2014. New studies on molecular chirality in the gas phase: enantiomer differentiation and determination of enantiomeric excess. Phys. Chem. Chem. Phys. 16, 11114–11123. Peled, M., Scharia, R., Sondack, D., 1995. Thermal rearrangement of hexabromocyclododecane (HBCD). In: BG, J.-R.D., JG, M. (Eds.), Industrial Chemistry LibraryElsevier 7, 92–99. Peng, X., Huang, X., Jing, F., Zhang, Z., Wei, D., Jia, X., 2015. Study of novel pure culture HBCD-1, effectively degrading hexabromocyclododecane, isolated from an anaerobic reactor. Bioresour. Technol. 185, 218–224. Portolés, T., Sales, C., Gómara, B., Sancho, J.V., Beltrán, J., Herrero, L., et al., 2015. Novel analytical approach for brominated flame retardants based on the use of gas chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry with emphasis in highly brominated congeners. Anal. Chem. 87, 9892–9899. Qiu, S.R., Gozdereliler, E., Weyrauch, P., Lopez, E.C.M., Kohler, H.P.E., Sorensen, S.R., et al., 2014. Small C-13/C-12 fractionation contrasts with large enantiomer fractionation in aerobic biodegradation of phenoxy acids. Environ Sci Technol 48, 5501–5511. Ross, M.S., Wong, C.S., 2010. Comparison of electrospray ionization, atmospheric pressure photoionization, and anion attachment atmospheric pressure photoionization for the analysis of hexabromocyclododecane enantiomers in environmental samples. J Chromatog A 1217, 7855–7863. Schurig, V., 1994. Enantiomer separation by gas-chromatography on chiral stationary phases. J Chromatog A 666, 111–129. Shi, X.Y., Liang, P., Gao, X.W., 2006. Determination enantiomer excess (e.e. %) of chiral sharpless epoxides with beta-cyclodextrin derivatives as chiral stationary phases of capillary gas chromatography. Chin. Chem. Lett. 17, 505–508. Suzuki, S., Hasegawa, A., 2006. Determination of hexabromocyclododecane diastereoisomers and tetrabromobisphenol A in water and sediment by liquid chromatography/mass spectrometry. Anal. Sci. 22, 469–474. Tomy, G.T., Pleskach, K., Arsenault, G., Potter, D., McCrindle, R., Marvin, C.H., et al., 2008a. Identification of the novel cycloaliphatic brominated flame retardant 1,2-dibromo-4(1,2-dibromoethyl)cyclohexane in Canadian Arctic Beluga (Delphinapterus leucas). Environ Sci Technol 42, 543–549. Tomy, G.T., Pleskach, K., Oswald, T., Halldorson, T., Helm, P.A., Macinnis, G., et al., 2008b. Enantioselective bioaccumulation of hexabromocyclododecane and congener-specific accumulation of brominated diphenyl ethers in an eastern Canadian Arctic marine food web. Environ Sci Technol 42, 3634–3639. Vetter, W., von der Recke, R., Ostrowicz, P., Rosenfelder, N., 2010. Liquid chromatographic enantioseparation of the brominated flame retardant 2,3-dibromopropyl-2,4,6tribromophenyl ether (DPTE) and enantiomer fractions in seal blubber. Chemosphere 78, 134–138. von der Recke, R., Vetter, W., 2007. Synthesis and characterization of 2,3-dibromopropyl2,4,6-tribromophenyl ether (DPTE) and structurally related compounds evidenced in seal blubber and brain. Environ Sci Technol 41, 1590–1595. Wiberg, K., Brorstrom-Lunden, E., Wangberg, I., Bidleman, T.F., Haglund, P., 2001a. Concentrations and fluxes of hexachlorocyclohexanes and chiral composition of alphaHCH in environmental samples from the southern Baltic Sea. Environ Sci Technol 35, 4739–4746. Wiberg, K., Harner, T., Wideman, J.L., Bidleman, T.F., 2001b. Chiral analysis of organochlorine pesticides in Alabama soils. Chemosphere 45, 843–848. Wong, F., Kurt-Karakus, P., Bidleman, T.F., 2012. Fate of brominated flame retardants and organochlorine pesticides in urban soil: volatility and degradation. Environ Sci Technol 46, 2668–2674. Yu, Z., Chen, L., Mai, B., Wu, M., Sheng, G., Fu, J., et al., 2008. Diastereoisomer- and enantiomer-specific profiles of hexabromocyclododecane in the atmosphere of an Urban City in South China. Environ Sci Technol 42, 3996–4001. Zacs, D., Bartkevics, V., 2015. Analytical capabilities of high performance liquid chromatography–atmospheric pressure photoionization–orbitrap mass spectrometry

Please cite this article as: Badea, S.-L., et al., Advances in enantioselective analysis of chiral brominated flame retardants. Current status, limitations and future perspectives, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.05.148

S.-L. Badea et al. / Science of the Total Environment xxx (2016) xxx–xxx (HPLC-APPI-Orbitrap-MS) for the trace determination of novel and emerging flame retardants in fish. Anal. Chim. Acta 898, 60–72. Zacs, D., Rjabova, J., Pugajeva, I., Nakurte, I., Viksna, A., Bartkevics, V., 2014. Ultra high performance liquid chromatography–time-of-flight high resolution mass spectrometry in the analysis of hexabromocyclododecane diastereomers: method development and comparative evaluation versus ultra high performance liquid chromatography coupled to Orbitrap high resolution mass spectrometry and triple quadrupole tandem mass spectrometry. J Chromatog A 1366, 73–83. Zhou, S.N., Reiner, E.J., Marvin, C., Kolic, T., Riddell, N., Helm, P., et al., 2010. Liquid chromatography–atmospheric pressure photoionization tandem mass spectrometry for analysis of 36 halogenated flame retardants in fish. J Chromatog A 1217, 633–641. Zhu, J., Hou, Y., Y-l, F., Shoeib, M., Harner, T., 2007. Identification and determination of hexachlorocyclopentadienyl- dibromocyclooctane (HCDBCO) in residential indoor

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air and dust: a previously unreported halogenated flame retardant in the environment. Environ Sci Technol 42, 386–391. Zipper, C., Bunk, M., Zehnder, A.J.B., Kohler, H.P.E., 1998a. Enantioselective uptake and degradation of the chiral herbicide dichlorprop (RS)-2-(2,4dichlorophenoxy)propanoic acid by Sphingomonas herbicidovorans MH. J. Bacteriol. 180, 3368–3374. Zipper, C., Fleischmann, T., Kohler, H.P.E., 1999. Aerobic biodegradation of chiral phenoxyalkanoic acid derivatives during incubations with activated sludge. FEMS Microbiol. Ecol. 29, 197–204. Zipper, C., Suter, M.J.F., Haderlein, S.B., Gruhl, M., Kohler, H.P.E., 1998b. Changes in the enantiomeric ratio of (R)- to (S)-mecoprop indicate in situ biodegradation of this chiral herbicide in a polluted aquifer. Environ Sci Technol 32, 2070–2076.

Please cite this article as: Badea, S.-L., et al., Advances in enantioselective analysis of chiral brominated flame retardants. Current status, limitations and future perspectives, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.05.148