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Quantification of Brominated Polycyclic Aromatic Hydrocarbons in Environmental Samples by Liquid Chromatography Tandem Mass Spectrometry with Atmospheric Pressure Photoionization and Post-column Infusion of Dopant

A sensitive method for the quantification of brominated polycyclic aromatic hydrocarbons (BrPAHs) in environmental samples is yet to be developed. Here, we optimized the analytical conditions for liquid chromatography tandem mass spectrometry with atmospheric pressure photoionization and post-column infusion of dopant (LC-DA-APPI-MS/MS). We then compared the sensitivity of our developed method with that of conventional gas chromatography high-resolution MS (GC-HRMS) by comparing the limits of quantification (LOQs) for a range of BrPAHs. Finally, to evaluate our developed method, 12 BrPAHs in sediments and fish collected from Tokyo Bay, Japan, were analyzed; 9 common PAHs were also targeted. The LOQs of the developed analytical method were 14 – 160 times lower than those of GC-HRMS for the targeted BrPAHs. The developed analytical method is a sensitive approach for determining the concentrations of BrPAHs in sediment and fish samples.


Introduction
Brominated or chlorinated polycyclic aromatic hydrocarbons (Br/ClPAHs) are compounds in which one or more hydrogen atoms have been substituted with bromine or chlorine atoms. These halogenated PAHs are structurally similar to dioxins (e.g., polyhalogenated dibenzo-p-dioxins, dibenzofurans, and polyhalogenated biphenyls), and some have higher carcinogenicity and mutagenicity than their parent PAHs. 1 Like PAHs, 2-5 halogenated PAHs have been ubiquitously detected in environmental samples. [6][7][8][9][10][11][12] Ohura et al. 13 reported that the total concentration (sum of individual concentrations) of 11 BrPAHs and 20 ClPAHs in surface sediment collected from the Yellow Sea was in the range of 5.5 -250 and 290 -1200 ng g -1 , respectively. Jin et al. 14 reported that the total concentration of 19 BrPAHs and 19 ClPAHs in haze in China was 24 and 260 pg m -3 , respectively. The total toxic equivalency concentration, calculated based on the toxicity relative to benzo[a]pyrene (BaP), of 20 ClPAHs (36 -1210 pg g -1 ) was higher than that of polychlorinated dibenzo-p-dioxins (0.039 -29 ng g -1 ) and dibenzofurans (0.034 -5.5 ng g -1 ) in sediment cores collected from Tokyo Bay, Japan. 15,16 Together, these findings suggest that halogenated PAHs are emerging pollutants.
Currently, the prevalences of BrPAHs in the environment are much less understood than those of ClPAHs. One reason for this is the lack of a suitable analytical method for determining the concentrations of BrPAHs in environmental samples. Although gas chromatography-mass spectrometry (GC-MS) is traditionally used for the analysis of PAHs, and of halogenated PAHs in general, high-molecular-weight compounds undergoing GC analysis can thermally decompose and adsorb onto the GC inlet and column. 17 In addition, the high molecular weight of bromine and the weakness of the carbon-bromine bond could result in higher limits of quantification (LOQ) for BrPAHs compared with ClPAHs. An alternative approach for the analysis of polar organic compounds is liquid chromatographymass spectrometry (LC-MS) [18][19][20][21] with electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) for ionization of the target compounds; however, the low polarity of PAHs means that they cannot be ionized by ESI and APCI. 22 Thus, a sensitive method for the determination of BrPAH concentrations in environmental samples is needed.
Atmospheric pressure photoionization (APPI) is a soft ionization technique that can ionize molecules that cannot be ionized by ESI or APCI. 23 In APPI, ultraviolet light emitted from a krypton lamp photochemically ionizes target compounds or dopants added to indirectly improve the ionization of the target compounds via subsequent gas-phase reactions. 24 For the analysis of PAHs, toluene and anisole are suitable dopants that can be introduced into the sample stream to create a source of charge carriers that then react with neutral target molecules via proton transfer and charge-exchange reactions. 25 Itoh et al. 25 have reported that a 99.5:0.5 (v/v) toluene/anisole mixture reduces the limit of detection for PAHs by 3.8 -40 times. When coupled with liquid chromatography tandem mass spectrometry (LC-MS/MS), APPI provides increased sensitivity for highmolecular-weight PAHs compared with conventional ionization methods. 26 Furthermore, the mass spectra obtained using APPI are much simpler to interpret, both for compound identification and quantification, compared with the spectra obtained using conventional ionization methods. 27 Although APPI has the potential to become the standard ionization method for the analysis of PAHs, the optimal APPI conditions for the analysis of BrPAHs are currently unknown.
Here, we optimized the APPI conditions for the analysis of BrPAHs by LC-MS/MS with post-column dopant infusion (LC-DA-APPI-MS/MS). We then compared the LOQs obtained using our approach with those obtained by GC-high-resolution MS (GC-HRMS), which is the current standard method for the analysis of halogenated PAHs. Finally, we used our approach to evaluate the concentrations of 12 BrPAHs in real-world marine environmental samples.

Chemicals and materials
In the present study, 17 BrPAHs and 9 PAHs were targeted (Table 1). Analytical standards for the target compounds were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan) or Sigma-Aldrich (St. Louis, MO, USA), or were prepared by organic synthesis in our laboratory. Isotope-labeled phenanthrene-13 C6, fluoranthene-13 C6, chrysene-13 C6, and bromobenz[a]anthracene-13 C6 as recovery standards and fluoranthene-d10 as an internal standard were obtained from Cambridge Isotope Laboratories (Andover, MA, USA). Hexane, and dichloromethane (residual-pesticide-analysis grade) for extraction and clean-up were obtained from Wako Pure Chemical (Osaka, Japan). A 50-mL KOH silica gel column filled with 12 g of 2% KOH silica gel (dioxin-analysis grade) was obtained from Wako Pure Chemical. An activated-carbon cartridge (Carboxen 1016, 200 mg) was obtained from Supelco (St. Louis, MO, USA). Reduced copper was obtained from Wako Pure Chemical.
To evaluate our approach for the analysis of BrPAHs in realworld environmental samples, sediment and fish samples (Lateolabrax japonicus and Pseudopleuronectes yokohamae) were collected from 10 stations in Tokyo Bay, Japan, in 2016 -2017; the samples were stored in the dark at -18 C until analysis.

Clean-up procedure
The BrPAHs and PAHs in the samples were extracted by Soxhlet extraction for 16 h using 250 mL of dichloromethane spiked with the recovery standards. The determinations of BrPAHs and PAHs in the extracts were performed according to an established method. 28,29 The silica-gel column with reduced copper (5 g) was washed with 20 mL of 10% dichloromethane/ hexane, and the activated carbon cartridge was washed with 120 mL of toluene. The column and cartridge were connected in series, and the sample extract was loaded onto the column. After loading the extract, the column was washed with 20 mL of 10% dichloromethane/hexane. After washing, the cartridge was removed and back-flushed with 120 mL of toluene to extract the target compounds. The collected toluene fraction containing the target compounds was spiked with the internal standard and concentrated to 100 μL for further analysis.

Analytical methods for LC-MS/MS
A liquid chromatograph (Thermo Fisher Scientific Vanquish, Thermo Scientific Inc., Waltham, MA, USA) coupled to a tandem mass spectrometer (TSQ Quantiva, Thermo Scientific Inc.) was used to determine the concentrations of PAHs. A Fusion 101 syringe pump (Chemyx, Stafford, TX, USA) was integrated into the system for post-column dopant infusion. A Fusion 101 syringe pump was also used for the injection of standard solutions used to optimize the MS parameters, such as quantitative ion, reference ion, collision energy, and lens settings, using the Xcalibur data acquisition (Ver. 1.3, Thermo Scientific Inc.) and interpretation software (Ver. 4.2, TraceFinder, Thermo Scientific Inc.). Standard solutions were diluted to 1.0 μg mL -1 with a solvent mixture containing methanol, water, toluene, and anisole (80:10:5:5, v/v) and injected into the MS system at 10 -20 μL min -1 . The positive ion mode was used. The Q1 and Q3 resolutions were both 0.7 Da. The optimal MS parameters for each compound were determined individually. Sheath gas (arbitrary unit), auxiliary gas (arbitrary unit), and sweep gas (arbitrary unit) were optimized by manual adjustment to 50, 15, and 0, respectively. The temperature of the ion transfer tube and vaporizer was optimized in the present study. Toluene, anisole, and acetone were used as dopants. The dopant injection volume was 2 μL min -1 .
A Poroshell 120 SB-C18 column (2.1 mm i.d. × 150 mm length, 2.7 μm, Agilent Technologies Japan, Ltd., Tokyo, Japan) was used for the separation in the LC. The flow rate was 0.25 mL min -1 . Water (Eluent A), and methanol (Eluent B) were used as the mobile phases. The gradient program was as follows: isocratic at 50% Eluent B for 3 min, 50 to 100% Eluent B in 10 min, isocratic at 100% Eluent B for 4 min, 100 to 50% Eluent B in 0.1 min, and then isocratic at 50% Eluent B for 3 min. The column temperature was kept at 50 C.
The MS/MS was operated under the selected reaction monitoring (SRM) mode. Peaks were identified by comparing the retention times of samples with those of the standards if the signal-to-noise ratio was >3, and they were quantified if the target/qualifier ion ratio was within 15% of the theoretical value.

Analytical methods for GC-HRMS
A GC-HRMS (JMS-700, JEOL, Tokyo, Japan) was used to provide data for comparing with our developed approach. Gas chromatographic separation was accomplished with a 60-m BPX-DXN fused silica capillary column (0.25 mm i.d., Kanto Chemical Co., Inc., Tokyo, Japan). A 2-μL aliquot of the sample was injected to the system in the splitless mode at 280 C. The temperature of the column oven was kept at 130 C for 1 min, raised at a rate of 5 C min -1 to 250 C, raised at a rate of 10 C min -1 to 320 C, and then held for 18 min. The MS was operated in the electron-impact selected ion monitoring mode at a resolution >10000 (10% valley). Peaks were identified by comparing the retention times of samples to those of standards if the signal-to-noise radio was >3, and they were quantified if the target/qualifier ion ratio was within 15% of the theoretical value.

Results and Discussion
Analytical method optimization Table 2 shows the quantitative and reference ion transitions and collision energies determined in the present study. The most abundant ion transition was selected for quantification.   31 The present results are consistent with the report by Ohura et al. 1 that certain BrPAHs can be photodegraded and debrominated. The ion transitions of the BrPAHs were similar between GC-MS/MS and LC-DA-APPI-MS/MS.
The effect of the ion source temperature (range, 240 -320 C) on the BrPAH peak intensity is shown in Fig. 1. The signal intensities of three-and four-ring compounds with low molecular weight, such as 2-BrFle/9-BrFle, 2,7-Br2Fle, 2-BrAnt, and 3-BrFlu, were 2 -3 times larger at the lowest ion source temperature (240 C) than at the highest ion source temperature (320 C).
We also examined three commonly used dopants (toluene, anisole, and acetone) for their ability to increase the sensitivity of LC-APPI-MS/MS for the analysis of BrPAHs. 22,24,25 The effects of the dopant type on the signal intensity are shown in Fig. 2. The relative peak intensity was calculated as a value with respect to the signal of 100% anisole dopant. Although using toluene or acetone alone as the dopant did little to increase the signal intensity (data not shown), using a 1% or 50% mixture of anisole in toluene or using 100% anisole increased the signal intensity. Itoh et al. 25 and Smith et al. 32 have also reported that anisole is suitable for the analysis of PAHs using LC-DA-APPI-MS. These results are in reasonably good agreement with the present results.
Comparing the LOQs of our approach for BrPAHs and their parent PAHs, the LOQs of BrAnt (150 -190 times lower), BrFlu (210 times), and BrPyr (220 -240 times) were significantly lower than those of the parent compounds. These results suggest that BrPAHs are more easily ionized than their parent compounds. This may be attributed to the fact that BrPAHs are more polar than PAHs. In addition, the fact that the dibrominated PAHs (i.e., Br2Fle, Br2Phe, and Br2Ant) had lower LOQs than the corresponding monobrominated PAHs (i.e., BrFle, BrPhe, and BrAnt) suggested that BrPAHs containing a larger number of bromine atoms are more easily ionized. Adding bromine atoms to a PAH likely improves absorption of the light energy emitted by the krypton lamp of APPI and thereby improves the ionization of the compound.

BrPAHs and PAHs in sediment and fish samples
To evaluate the use of our LC-DA-APPI-MS/MS approach for determining the concentrations of BrPAHs and PAHs in realworld samples, samples of sediment and fish collected from Tokyo Bay, Japan, were examined. Fig. 1 Effect of the ion source temperature on the peak intensities for brominated polycyclic aromatic hydrocarbons. Fig. 2 Effects of the dopant on the peak intensities for brominated polycyclic aromatic hydrocarbons.
In the present study, the concentrations of Pyr were in the range of 130 -240 ng g -1 , respectively, and the concentrations of 1-BrPyr and 1,6-/1,8-Br2Pyr were in the range of 0.012 -0.042 and 0.043 -0.068 ng g -1 , respectively. The concentrations of Pyr were about 3100 -10000 and 2400 -4300 times higher than that of BrPyr and Br2Pyr. Table 4 shows the concentrations of BrPAHs and PAHs in fish from Tokyo Bay. The total concentration of PAHs in Lateolabrax japonicus (Japanese sea bass) and Pseudopleuronectes yokohamae (aka Limanda yokohamae; marbled flounder) was 21 and 1.9 ng g -1 , respectively. Among the PAHs, Fle (P. yokohamae: 1.5 ng g -1 ; L. japonicus: <LOQ), Phe (17 and 1.2 ng g -1 ), Ant (1.5 and 0.73 ng g -1 ), Flu (0.80 ng g -1 and <LOQ), and Pyr (0.067 ng g -1 and <LOQ) were detected in the fish. Takeuchi et al. 33 reported that the concentrations of Phe, Ant, Flu, and Pyr in Acanthogobius flavimanus (yellowfin goby) collected from Tokyo Bay were 0.77, 0.11, 0.17, and 0.12 ng g -1 , respectively.  Liang et al. 34 reported that the concentrations of Phe, Ant, Flu, and Pyr in the muscle tissues of tilapia (Sarotherodon mossambicus), a freshwater fish, collected from Mai Po Marshes (Hong Kong) were in the range of 16 -23, 2.7 -3.7, 9.9 -13, and 8.2 -11 ng g -1 , respectively. These previous studies show that PAH concentrations vary among different species. The concentrations of PAHs in the present study were most comparable with those reported by Takeuchi et al. in A. flavimanus. 33 The concentrations of the target BrPAHs in the fish samples studied were lower than the LOQs. Wickrama-Arachchige et al. 35 reported that the concentrations of PAHs and BrPAHs in tuna fish species (three individuals of Thunnus albacares and three individuals of Katsuwonus pelamis) collected from the Indian Ocean near Sri Lanka were in the ranges of 238 -2023 and 4.73 -776 ng g -1 , respectively. These previously reported PAH concentrations are 100 times higher than those obtained in the present study, and the BrPAH concentrations are higher than the LOQs of our present analytical method. These inconsistent findings may be a result of differences in the concentrations and the accumulation pattern of PAHs in different fish species. The concentrations of BrPAHs in the environment should be lower than those of PAHs according to the formation mechanism of halogenated PAHs. 36,37 Bioconcentration of BrPAHs could be different in different fish species, and that in tuna could be higher than that in L. japonicus and P. yokohamae. The low LOQs of the present method for the detection of BrPAHs in fish samples suggests that the concentrations of BrPAHs in fish collected from Tokyo Bay are low. However, to obtain bioconcentration factors for BrPAHs, a more sensitive analytical method is required.

Conclusion
Here, we developed a sensitive analytical method for the quantification of BrPAHs using LC-DA-APPI-MS/MS. Our method was 100 times more sensitive at detecting BrPAHs compared with the corresponding PAHs, and was successfully used to determine the concentrations of BrPAHs and PAHs in sediment and fish samples.