Analytical Sciences Advance Publication by J-STAGE Received January 28, 2020; Accepted April 22, 2020; Published online on May 1, 2020 DOI: 10.2116/analsci.20P025 1 Original Papers 2 3 Quantification of Brominated Polycyclic Aromatic Hydrocarbons in 4 Environmental Samples by Liquid Chromatography Tandem Mass 5 Spectrometry with Atmospheric Pressure Photoionization and 6 Post-Column Infusion of Dopant (LC-DA-APPI-MS/MS) 7 8 Misato Masuda, Qi Wang, Masahiro Tokumura, Yuichi Miyake†, and Takashi Amagai† 9 10 Graduate Division of Nutritional and Environmental Sciences, University of Shizuoka, 52-1 11 Yada, Suruga, Shizuoka 422-8526, Japan 12 † To whom correspondence should be addressed. 13 E-mail: [email protected] (Miyake), [email protected] (Amagai) 14 1 Analytical Sciences Advance Publication by J-STAGE Received January 28, 2020; Accepted April 22, 2020; Published online on May 1, 2020 DOI: 10.2116/analsci.20P025 15 Abstract 16 A sensitive method for the quantification of brominated polycyclic aromatic hydrocarbons 17 (BrPAHs) in environmental samples is yet to be developed. Here, we optimized the analytical 18 conditions for liquid chromatography tandem mass spectrometry with atmospheric pressure 19 photoionization and post-column infusion of dopant (LC-DA-APPI-MS/MS). We then 20 compared the sensitivity of our developed method with that of conventional gas 21 chromatography high-resolution MS (GC-HRMS) by comparing the limits of quantification 22 (LOQs) for a range of BrPAHs. Finally, to evaluate our developed method, 12 BrPAHs in 23 sediments and fish collected from Tokyo Bay, Japan, were analyzed; 9 common PAHs were also 24 targeted. The LOQs of the developed analytical method were 14−160 times lower than those of 25 GC-HRMS for the targeted BrPAHs. The developed analytical method is a sensitive approach 26 for determining the concentrations of BrPAHs in sediment and fish samples. 27 28 Keywords: polycyclic aromatic hydrocarbons, brominated polycyclic aromatic hydrocarbons, 29 LC-MS/MS, APPI, dopant. 30 2 Analytical Sciences Advance Publication by J-STAGE Received January 28, 2020; Accepted April 22, 2020; Published online on May 1, 2020 DOI: 10.2116/analsci.20P025 31 Introduction 32 Brominated or chlorinated polycyclic aromatic hydrocarbons (Br/ClPAHs) are compounds in 33 which one or more hydrogen atoms have been substituted with bromine or chlorine atoms. 34 These halogenated PAHs are structurally similar to dioxins (e.g., polyhalogenated 35 dibenzo-p-dioxins, dibenzofurans, and polyhalogenated biphenyls), and some have higher 36 carcinogenicity and mutagenicity than their parent PAHs.1 Like PAHs,2-5 halogenated PAHs 37 have been ubiquitously detected in environmental samples.6-12 Ohura et al.13 reported that the 38 total concentration (sum of individual concentrations) of 11 BrPAHs and 20 ClPAHs in surface 39 sediment collected from the Yellow Sea was in the range 5.5–250 and 290–1200 ng g−1, 40 respectively. Jin et al.14 reported that the total concentration of 19 BrPAHs and 19 ClPAHs in 41 haze in China was 24 and 260 pg m−3, respectively. The total toxic equivalency concentration, 42 calculated based on toxicity relative to benzo[a]pyrene (BaP), of 20 ClPAHs (36–1210 pg g−1) 43 was higher than that of polychlorinated dibenzo-p-dioxins (0.039–29 ng g−1) and dibenzofurans 44 (0.034–5.5 ng g−1) in sediment cores collected from Tokyo Bay, Japan.15,16 Together, these 45 findings suggest that halogenated PAHs are emerging pollutants. 46 Currently, the prevalences of BrPAHs in the environment are much less understood than 47 those of ClPAHs. One reason for this is the lack of a suitable analytical method for determining 48 the concentrations of BrPAHs in environmental samples. Although gas chromatography-mass 49 spectrometry (GC-MS) is traditionally used for the analysis of PAHs, and of halogenated PAHs 50 in general, high-molecular-weight compounds undergoing GC analysis can thermally 51 decompose and adsorb onto the GC inlet and column.17 In addition, the high molecular weight 52 of bromine and the weakness of the carbon–bromine bond could result in higher limits of 53 quantification (LOQ) for BrPAHs compared with ClPAHs. An alternative approach for the 54 analysis of polar organic compounds is liquid chromatography-mass spectrometry (LC-MS)18-21 55 with electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) for 3 Analytical Sciences Advance Publication by J-STAGE Received January 28, 2020; Accepted April 22, 2020; Published online on May 1, 2020 DOI: 10.2116/analsci.20P025 56 ionization of the target compounds; however, the low polarity of PAHs means they cannot be 57 ionized by ESI and APCI.22 Thus, a sensitive method for determination of BrPAH 58 concentrations in environmental samples is needed. 59 Atmospheric pressure photoionization (APPI) is a soft ionization technique that can ionize 60 molecules that cannot be ionized by ESI or APCI.23 In APPI, ultraviolet light emitted from a 61 krypton lamp photochemically ionizes target compounds or dopants added to indirectly improve 62 the ionization of the target compounds via subsequent gas-phase reactions.24 For the analysis of 63 PAHs, toluene and anisole are suitable dopants that can be introduced into the sample stream to 64 create a source of charge carriers that then react with neutral target molecules via proton transfer 65 and charge exchange reactions.25 Itoh et al.25 have reported that a 99.5:0.5 (v/v) toluene/anisole 66 mixture reduces the limit of detection for PAHs by 3.8–40 times. When coupled with liquid 67 chromatography tandem mass spectrometry (LC-MS/MS), APPI provides increased sensitivity 68 for high-molecular-weight PAHs compared with conventional ionization methods.26 69 Furthermore, the mass spectra obtained using APPI are much simpler to interpret, both for 70 compound identification and quantification, compared with the spectra obtained using 71 conventional ionization methods.27 Although APPI has the potential to become the standard 72 ionization method for the analysis of PAHs, the optimal APPI conditions for the analysis of 73 BrPAHs are currently unknown. 74 Here, we optimized the APPI conditions for analysis of BrPAHs by LC-MS/MS with 75 post-column dopant infusion (LC-DA-APPI-MS/MS). We then compared the LOQs obtained 76 using our approach with those obtained by GC–high-resolution MS (GC-HRMS), which is the 77 current standard method for the analysis of halogenated PAHs. Finally, we used our approach to 78 evaluate the concentrations of 12 BrPAHs in real-world marine environmental samples. 79 4 Analytical Sciences Advance Publication by J-STAGE Received January 28, 2020; Accepted April 22, 2020; Published online on May 1, 2020 DOI: 10.2116/analsci.20P025 80 Experimental 81 82 Chemicals and materials 83 In the present study, 17 BrPAHs and 9 PAHs were targeted (Table 1). Analytical standards for 84 the target compounds were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan) 85 or Sigma-Aldrich (St. Louis, MO, USA), or were prepared by organic synthesis in our 13 13 13 86 laboratory. Isotope-labeled phenanthrene- C6, fluoranthene- C6, chrysene- C6, and 13 87 bromobenz[a]anthracene- C6 as recovery standards and fluoranthene-d10 as an internal standard 88 were obtained from Cambridge Isotope Laboratories (Andover, MA, USA). Hexane, and 89 dichloromethane (residual-pesticide-analysis grade) for extraction and clean-up were obtained 90 from Wako Pure Chemical (Osaka, Japan). A 50-mL KOH silica gel column filled with 12 g of 91 2% KOH silica gel (dioxin-analysis grade) was obtained from Wako Pure Chemical. An 92 activated carbon cartridge (Carboxen 1016, 200 mg) was obtained from Supelco (St. Louis, MO, 93 USA). Reduced copper was obtained from Wako Pure Chemical. 94 To evaluate our approach for the analysis of BrPAHs in real-world environmental samples, 95 sediment and fish samples (Lateolabrax japonicus and Pseudopleuronectes yokohamae) were 96 collected from 10 stations in Tokyo Bay, Japan, in 2016–2017; the samples were stored in the 97 dark at −18 °C until analysis. 98 99 Clean-up procedure 100 The BrPAHs and PAHs in the samples were extracted by Soxhlet extraction for 16 h using 250 101 mL of dichloromethane spiked with the recovery standards. The determination of BrPAHs and 102 PAHs in the extracts was performed according to an established method.28,29 The silica gel 103 column with reduced copper (5 g) was washed with 20 mL of 10% dichloromethane/hexane, 104 and the activated carbon cartridge was washed with 120 mL of toluene. The column and 5 Analytical Sciences Advance Publication by J-STAGE Received January 28, 2020; Accepted April 22, 2020; Published online on May 1, 2020 DOI: 10.2116/analsci.20P025 105 cartridge were connected in series, and the sample extract was loaded onto the column. After 106 loading the extract, the column was washed with 20 mL of 10% dichloromethane/hexane. After 107 washing, the cartridge was removed and back-flushed with 120 mL of toluene to extract the 108 target compounds. The collected toluene fraction containing the target compounds was spiked 109 with the internal standard and concentrated to 100 µL for further analysis. 110 111 Analytical methods for LC-MS/MS 112 A liquid chromatograph (Thermo Fisher Scientific Vanquish, Thermo Scientific Inc., Waltham, 113 MA, USA) coupled to a tandem mass spectrometer (TSQ Quantiva, Thermo Scientific Inc.) was 114 used to determine the concentrations of PAHs. A Fusion 101 syringe pump (Chemyx, Stafford, 115 TX, USA) was integrated into the system for post-column dopant infusion. A Fusion 101 116 syringe pump was also used for injection of standard solutions used to optimize the MS 117 parameters, such as quantitative ion, reference ion, collision energy, and lens settings, using the 118 Xcalibur data acquisition (version 1.3, Thermo Scientific Inc.) and interpretation software 119 (version 4.2, TraceFinder, Thermo Scientific Inc.).