MS Method for Determination of Natural

Anal. Chem. 2008, 80, 9748–9755

Negative APCI-LC/MS/MS Method for Determination of Natural Persistent Halogenated Products in Marine Biota Koichi Haraguchi,*,† Yoshihisa Kato,‡ Kazutaka Atobe,‡ Syohei Okada,‡ Tetsuya Endo,§ Futoshi Matsubara,† and Takayoshi Oguma‡ Daiichi College of Pharmaceutical Sciences, 22-1 Tamagawa-cho, Minami-ku, Fukuoka 815-8511, Japan, Kagawa School of Pharmaceutical Sciences, Tokushima Bunri University, 1314-1 Sanuki-shi, Kagawa 769-2193, Japan, and Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, 1757, Ishikari-Tobetsu, Hokkaido 061-0293, Japan A sensitive and selective method utilizing high performance liquid chromatography coupled to negative atmospheric pressure chemical ionization tandem mass spectrometry (APCI-LC/MS/MS) was developed to enable analysis of selected natural persistent organohalogens accumulated in marine biota. The analytes were three methoxylated tetrabromodiphenyl ethers (6-MeO-BDE47, 2′-MeO-BDE68, and 2′,6-diMeO-BDE68), a dimethoxylated tetrabromobiphenyl (2,2′-diMeO-BB80), and two halogenated methyl bipyrroles (Cl7-MBP and Br4Cl2DBP). These products were well resolved on a 150 mm reversed-phase column with methanol as the mobile phase. The fragmentation pathways of the Cl7-MBP and Br4Cl2-DBP produced characteristic multiple reaction monitoring (MRM) transitions. Determination was performed in the MRM mode using phenoxide ion [M-Br+O]- and product Br- ions for MeO-BDE analogues, or the precursor [M-Cl+O]- to Br- ion for Br4Cl2DBP, and to C4NCl4- ion for Cl7-MBP. The APCI-LC/MS/ MS method is acceptable for calibration of the linearity and repeatability of all products studied in the low ng/g (lipid weight) level and with similar sensitivity to the electron ionization (EI)-GC/MS method. The proposed method was applied for quantification of natural organohalogens accumulated in melon-headed whale (Peponocephala electra) blubber (N ) 15) in the Asia-Pacific Ocean. The concentration was positively correlated between different groups of compounds except for 2′-MeOBDE68. The use of the analytical method based on negative ion APCI-LC/MS/MS would provide a new way for rapid monitoring of halogenated natural products from marine biota, such as sponges or algae. Many brominated flame retardants (BFRs) are mainly represented by polybrominated diphenyl ethers (PBDEs), tetrabromobisphenol A (TBBPA), and hexabromocyclododecane (HBCD). * To whom correspondence should be addressed. Phone: +81-92-541-0161. Fax: +81-92-553-5698. E-mail: [email protected]. † Daiichi College of Pharmaceutical Sciences. ‡ Tokushima Bunri University. § Health Sciences University of Hokkaido.

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Analytical Chemistry, Vol. 80, No. 24, December 15, 2008

They are persistent and lipophilic and they show bioaccumulation in the environment, animals, and humans, similarly to other persistent organic pollutants (POPs).1 In recent years, lipophilic POPs of natural origin have been detected in the marine food chain from different regions. Some are methoxylated tetrabromodiphenyl ethers (MeO-BDEs), dimethoxylated tetrabromobiphenyl (diMeO-BB), hexachlorinated 1-methyl 1′,2-bipyrrole (Cl7-MBP, referred to Q1), and hexahalogenated 1,1′-dimethyl 2,2′-bipyrroles (HDBPs). MeO-BDEs have been found in red algae, blue mussels, marine sponges, fish, and mammals,2-5 whereas diMeO-BB has been found in whale products from Japanese markets6 and in mammals from Australia.7 Cl7-MBP has been detected in mammals and fish from the southern hemisphere,5,8 whereas HDBPs have been shown to be distributed in mammals from the North Pacific.9,10 These components possess POP-like properties and have been proposed to be of biogenic origin,5,11 although their geographical sources and toxicological effects are still unclear. Powerful analytical methods for BFRs, MeO-BDEs, and related halogenated POPs have been developed using gas chromatography coupled to mass spectrometry (GC/MS). Hydroxylated PBDEs have also been identified in the Baltic Sea salmon,12 red algae, and blue mussels4 by GC/MS using electron ionization (EI) or electron capture negative ionization (ECNI) with diazomethane as the derivatizing agent. Currently, ECNI based on monitoring (1) de Wit, C. A. Chemosphere 2002, 46, 583–624. (2) Bowden, B. F.; Towerzey, L.; Junk, P. C. Aust. J. Chem. 2000, 53, 299– 301. (3) Agrawal, M. S.; Bowden, B. F. Mar. Drugs 2005, 3, 9–14. (4) Malmva¨rn, A.; Marsh, G.; Kautsky, L.; Athanasiadou, M.; Bergman, Å.; Asplund, L Environ. Sci. Technol. 2005, 39, 2990–2997. (5) Vetter, W. Rev. Environ. Contam. Toxicol. 2006, 188, 1–57. (6) Marsh, G.; Athanasiadou, M.; Athanassiadis, I.; Bergman, Å.; Endo, T.; Haraguchi, K. Environ. Sci. Technol. 2005, 39, 8684–8690. (7) Vetter, W. Anal. Chem. 2001, 73, 4951–4957. (8) Vetter, W.; Scholz, E.; Gaus, C.; Müller, J. F.; Haynes, D. Arch. Environ. Contam. Toxicol. 2001, 41, 221–231. (9) Tittlemier, S.; Borrell, A.; Duffe, J.; Duignan, P. J.; Fair, P.; Hall, A.; Hoekstra, P.; Kovacs, K. M.; Krahn, M. M.; Lebeuf, M.; Lydersen, C.; Muir, D.; O’Hara, T.; Olsson, M.; Pranschke, J.; Ross, P.; Siebert, U.; Stern, G.; Tanabe, S.; Norstrom, R. Arch. Environ. Contam. Toxicol. 2002, 43, 244–255. (10) Haraguchi, K.; Hisamichi, Y.; Endo, T. Arch. Environ. Contam. Toxicol. 2006, 51, 135–141. (11) Teuten, E. L.; Reddy, C. M. Environ. Pollut. 2007, 145, 668–671. (12) Marsh, G.; Athanasiadou, M.; Bergman, Å.; Asplund, L Environ. Sci. Technol. 2004, 38, 10–18. 10.1021/ac801824f CCC: $40.75  2008 American Chemical Society Published on Web 11/14/2008

Chart 1. Abbreviations According to IUPAC Nomenclature and Structures of the Analyzed Compounds and Internal Standard

of the m/z 79 and 81 ions for the bromine trace is the most widely used method for the analysis of BFRs and natural halogenated POPs.13-15 However, the analysis of highly brominated congeners, such as HBCD, is known to be difficult because of thermal degradation problems and the relative lack of sensitivity of EI- or ECNI-GC/MS. Indeed, GC/MS may be less suited in the context of degradation or metabolism studies because of its limitations for the analysis of some hydroxylated derivatives, and even more inappropriate for glucuronic acid or glutathione conjugates, all of which are encountered as usual xenobiotic metabolites. Instead of GC/MS, liquid chromatography/mass spectrometric (LC/MS) techniques using electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) have been developed.16,17 More recently, atmospheric pressure photoioniza¨ stman, C. J. Mass Spectrom. 2003, 38, 394– (13) Bjo ¨rklund, J.; Tollba¨ck, P.; O 400. (14) Covaci, A.; Voorspoels, S.; Ramos, L.; Neels, H.; Blust, R. J. Chromatogr. A. 2007, 1153, 145–171. (15) Gómara, B.; Herrero, L.; González, M. J. Anal. Chim. Acta. 2007, 597, 121–128. (16) Thomson, B. A.; Davidson, W. R.; Lovett, A. M. Environ. Health Perspect. 1980, 36, 77–84.

tion (APPI) has been developed as a complementary ionization technique, allowing the ionization of a wide range of hydrophobic compounds, and broadening the applicability of LC/MS.18 This method was applied for analysis of BFRs, such as TBBPA,19 higher brominated PBDEs,20,21 and HBCD.22 Mas et al.23 proposed the negative ion spray ionization (ISP)-LC/MS/MS method for eight OH-PBDEs, which was shown to be an efficient, robust, sensitive, and selective tool, although the simultaneous determination of (17) Polo, M.; Gómez-Noya, G.; Quintana, J. B.; Liompart, M.; Garcia-Jares, C.; Cela, R. Anal. Chem. 2004, 76, 1054–1062. (18) Kauppila, T. J.; Kotiaho, T.; Kostiainen, R. J. Am. Soc. Mass Spectrom. 2004, 15, 203–211. (19) Debrauwer, L.; Riu, A.; Jouahri, M.; Rathahao, E.; Jouanin, I.; Antignac, J-P.; Cariou, R.; le Bizec, B.; Zalko, D. J Chromatogr. A. 2005, 1082, 98– 109. (20) Cariou, R.; Antignac, J-P.; Debrauwer, L.; Maume, D.; Monteau, F.; Zalko, D.; le Bizec, B.; Andre, F. J. Chromatogr. Sci. 2006, 44, 489–497. (21) Riu, A.; Zalko, D.; Debrauwer, L. Rapid Commun. Mass Spectrom. 2006, 20, 2133–2142. (22) Budakowski, W.; Tomy, G. Rapid Commun. Mass Spectrom. 2003, 17, 1399– 1404. (23) Mas, S.; Ja´uregui, O.; Rubio, F.; de Juan, A.; Tauler, R.; Lacorte, S. J. Mass Spectrom. 2007, 42, 890–899.


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Table 1. Compounds Studied, Chemical Formulas, Molecular Weights, LC Retention Times, Product Ions, and MRM Acquisition Parameters Used for Determination

compounds

chemical formula

[M-H](m/z)

LC retention time (min)

6-MeO-BDE47

C13H8O2Br4

510.7

6.16

2′-MeO-BDE68

C13H8O2Br4

510.7

7.74

2′,6-diMeO-BDE68

C14H10O3Br4

540.7

6.63

2,2′-diMeO-BB80

C14H10O2Br4

524.7

7.71

Cl7-MBP

C9H3N2Cl7

382.8

6.12

Br4Cl2-DBP

C10H6N2Br4Cl2

538.7

5.32

4′-MeO-BDE121 (IS)

C13H7O2Br5

588.7

9.04

a

major precursor and product ions [M-Br+O][M-CH3][M-Br+CH3O]b Bra [M-Br+O][M-CH3][M-Br+CH3O]b Bra [M-Br+O][M-CH3][M-Br+CH3O]b Bra [M-Br+O][M-CH3][M-Br+CH3O]b [M′-CH3]a [M-Cl+O][M-NCH3+O]b C4N2Cl4a [M-Cl+O][M-NCH3+O]b Bra [M-Br+O][M-Br+CH3O]b [M′-CH3]a

MRM transition (m/z)

DP (V)

CE (V)

450.7f78.8

-35

-54

450.7f78.8

-35

-62

480.8f78.8

-40

-66

464.8f449.6

-45

-22

366.8f203.8

-25

-32

526.5f81.0

-30

-46

530.6f515.6

-55

-22

Precursor ion. b Product ion used in this study. M′) [M-Br+O]-.

natural POPs, such as MeO-BDEs and Br4Cl2-DBP, has not been investigated. Our recent study indicated that APCI may be a promising technique for LC/MS analysis of natural POPs or related degradation products, because halogenated POPs are efficiently ionized to [M-X+O]- ions (X ) halogen) in a negative ion mode. The initial objective was to develop a powerful methodology for the quantification of these natural POPs, which was validated in comparison to EI-GC/MS. This paper reports a novel approach for LC/MS/MS quantification of natural halogenated POPs using negative ion APCI as the ionization technique. The analytes investigated were 2′-MeO-BDE68, 6-MeO-BDE47, 2′,6-diMeOBDE68, 2,2′-diMeO-BB80, Cl7-MBP, and Br4Cl2-DBP (Chart 1), all of which are frequently accumulated at higher concentrations than PBDEs in marine biota from the Pacific.24 Thus, the proposed method was applied to monitor the natural halogenated POPs accumulated in blubber of melon-headed whales (Peponocephala electra) mass-stranded in Japan. EXPERIMENTAL SECTION Chemicals and Reagents. Chart 1 lists the chemical names (according to the IUPAC nomenclature) and chemical structures of the analytes, together with the internal standard (IS). 6-MeOBDE47, 2′-MeO-BDE68, 2′,6-diMeO-BDE68, 2,2′-diMeO-BB80, and 4′-MeO-BDE121 were synthesized by Dr. Go¨ran Marsh of Stockholm University, Sweden.6 Cl7-MBP and Br4Cl2-DBP were synthesized according to Wu et al.25 and Gribble et al.,26 respectively. (24) Hisamichi, Y.; Endo, T.; Nishimura, E.; Haraguchi, K. Organohalogen Compd. 2007, 69, 1709–1712. (25) Wu, J.; Vetter, W.; Gribble, G. W.; Schneekloth, J.S., Jr.; Blank, D. H.; Görls, H. Angew. Chem., Int. Ed. 2002, 41, 1740–1742.

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A solution containing all standards was prepared in methanol at a concentration of 4 to 2000 ng/mL. LC/MS-grade methanol was purchased from Wako Pure Chemical Industries Ltd. Sampling and Extraction. On February, 2006, melon-headed whales (P. electra) mass-stranded on the coast of Boso Peninsula, Ichinomiya, Chiba Prefecture, Japan. Despite rescue efforts by volunteers, about seventy whales died. Samples (N ) 15) of blubber from the stranded whales (male, N ) 10; female, N ) 5; body length, 2.5-2.7 m) were collected on March, 2006.27 These samples were stored at -20 °C until analysis. Accurately weighed samples (1-2 g) were cut into small species and mixed with 10 volumes of anhydrous sodium sulfate. The mixtures were wetpacked with acetone/n-hexane (1:3, v/v) into a glass column (2 cm, id.). The filtered extracts were concentrated and the lipid contents were determined gravimetrically. Portions of the lipids were spiked with an internal standard (4′-MeO-BDE121) and then purified by gel permeation chromatography (GPC) (Bio-Beads S-X3, Bio-Rad Laboratories, CA), with elution with dichloromethane (DCM)/n-hexane (1:1 v/v) for organohalogen residues. The eluate containing organohalogens was concentrated to dryness and dissolved in methanol (1 mL). Half of the solution (0.5 mL) was directly subjected to LC/MS/MS. The rest (0.5 mL) was concentrated and further purified by passage over 1 g of activated silica gel column (5 mm i.d.,Wako gel S-1, Wako Pure Chemical Industries Ltd., Osaka), with elution with DCM/nhexane (12:88 v/v, 15 mL). The eluate containing natural POPs was reduced to 0.5 mL and subjected to GC/MS for comparison. (26) Gribble, G. W.; Blank, D. H.; Jasinski, J. P. Chem. Commun. 1999, 2195– 2196. (27) Endo, T.; Hisamichi, Y.; Kimura, O.; Haraguchi, K.; Baker, C. S. Sci. Total Environ. 2008, 401, 73–80.

Figure 1. Mass spectra (Q1 scan) of four natural POPs: (a) 6-MeOBDE47, (b) 2′-MeO-BDE68, (c) 2′,6-diMeO-BDE68, (d) 2,2′-diMeOBB80, and internal standard (e) 4′-MeO-BDE121.

GC/MS Identification and Quantification. Analyses of natural POPs were first performed using a GC (Agilent 6980N) equipped with a mass-selective detector (5973i) in EI and selected ion monitoring (SIM) mode (EI-SIM). An HP-5MS column (30 m × 0.25 mm, i.d., stationary phase 0.25 µm in thickness; J&W Scientific Inc., Folsom, CA) was installed in the GC. In the full scan EI mode, m/z 50 to 650 were recorded. Helium was used as a carrier gas at a constant flow rate of 1.0 mL/min. The injector and transfer line temperatures were 250 and 280 °C, respectively.

The GC oven program was as follows. After injection at 70 °C (1.5 min), the temperature was increased at 20 °C/min to 230 °C (2 min), then at 4 °C/min to 280 °C (20 min). Total run time was 35 min. Target analytes were quantified by the SIM mode, using m/z 386 for Cl7-MBP, m/z 546 for Br4Cl2-DBP and 2′,6-diMeOBDE68, m/z 516 for 2′-MeO-BDE68 and 6-MeO-BDE47, m/z 530 for 2,2′-diMeO-BB80 and m/z 594 for 4′-MeO-BDE121 (IS), together with m/z 362 for 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB153), m/z 486 for 2,2′,4,4′-tetrabromodiphenyl ether (BDE47). Procedural blanks were analyzed simultaneously with each batch of five samples to check interference or contamination from solvents and glassware. LC/MS/MS Identification and Quantification. Analyses were carried out using a liquid chromatograph (Prominence 20A; Shimadzu Co., Japan) coupled to a tandem mass spectrometer (API 3200Q Trap triple-quadrupole MS/MS system; Applied Biosystems Inc.). A reversed phase Shim-pack FC-ODS column (150 mm × 4.6 mm, i.d., 3.1 µm particle size; Shimadzu Co.) was used. The mobile phase composition was optimized with 100% methanol at a flow rate of 0.5 mL/min. The column conditions were all programmed at room temperature, and the column was equilibrated for more than 10 min between runs. The samples were kept at 15 °C in an autosampler, and a volume of 20 µL of each sample was injected into the HPLC column. The data were acquired and processed using the Analyst 1.4.2 software package. MS/MS parameters were optimized in infusion experiments using individual standard solutions of six natural POPs and an internal standard at a concentration of 2 µg/mL in methanol. Each solution was pumped into the APCI source through a syringe pump at a constant flow rate of 0.2 mL. The best conditions for the selected natural POPs were attained with the APCI source in the negative ion mode with the following settings. First, the precursor ions were chosen from Q1 scan mode. Next, for each stable selected precursor ion, the declustering potential (DP), entrance potential (EP), and collision cell entrance potential (CEP) were all adjusted to optimize the signal intensity using the “Quantitative Optimization” software tool. Then, a product ion scan of the precursor ion was performed to identify useful fragment ions. The collision energy (CE) and the collision cell exit potential (CXP) were optimized manually at the selected MRM transition. Finally, the APCI source parameters, that is, the collision-activated dissociation (CAD) gas (N2, 5 psig, arbitrary units) in Q2, curtain gas (CUR ) 10 psig, arbitrary units), ion source gas (GS1 ) 30 psig, arbitrary units), heated nebulizer temperature (TEM ) 400°), and the discharge needle current (NC ) -2 µA), were all optimized manually during flow injection until the highest signal was achieved. The optimized parameters, DP and CE for MS/MS of the natural POPs, are summarized in Table 1 (Capillary voltage -4000 V, drying gas (N2) was introduced at a flow rate of 5000 cm3/min). Full-scan data acquisition was performed by scanning from m/z 300 to 600 (Q1 scan range) in the profile mode, using a scan time of 1 s with a step size of 0.1 amu and a pause between each scan of 5 ms. To choose the fragmentation patterns of m/z (Q1) f m/z (Q3) ions for the MRM transitions, product ion scan (PIS) mass spectra were recorded by CAD of selected precursor ions in the collision cell of the triple-quadrupole mass spectrometer and analyzed using the second analyzer of the instrument. MRM Analytical Chemistry, Vol. 80, No. 24, December 15, 2008

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Figure 2. Mass spectra (Q1 scan) of (a) Cl7-MBP and (b) Br4Cl2-DBP and product ion scan (PIS, Q3 scan) of [M-Cl+O]- ions for (c) Cl7-MBP and (d) Br4Cl2-DBP. Table 2. Comparison of Concentrations of Natural POPs in Melon-Headed Whale Blubber between APCI-LC/MS/MS and EI-GC/MS Methodsa concentration (µg/g lipid weight) APCI-LC/MS/MS mean ± SD 6-MeO-BDE47 2′-MeO-BDE68 2′,6-MeO-diBDE68c 2,2′-MeO-diBB80 Cl7-MBP Br4Cl2-DBP BDE47 PCB153

0.46 ± 0.15 ± 0.07 ± 0.32 ± 4.25 ± 23.4 ± NAd NA

0.21 0.05 0.01 0.18 2.43 13.7

EI-GC/MS range

0.07-0.72 0.05-0.23 0.05-0.08 0.05-0.55 0.53-7.62 2.86-42.3

mean ± SD

range

CV (%)b

± ± ± ± ± ± ± ±

0.07-0.80 0.06-0.24 0.05-0.08 0.04-0.54 0.44-9.19 1.85-51.7 0.04-0.54 0.62-12.1

2.8 1.7 4.6 6.3 3.0 3.9

0.47 0.14 0.07 0.35 4.13 24.3 0.31 5.53

0.22 0.05 0.01 0.20 2.65 16.1 0.18 3.99

a Concentrations are expressed as mean ± standard deviation (SD) in micrograms per gram of lipid weight for 15 samples. b Coefficients of variance (%) between two methods calculated from mean concentrations. c Concentrations are mean ± SD (range) for 12 samples. d Not analyzed.

experiments were performed using a dwell time of 150 ms. The MRM transition with higher selectivity was used to avoid decrease in sensitivity. The retention time shift was lower than 1.1%. Quality Assurance and Quality Control. A seven-point calibration curve in the concentration range of 4-2000 ng/mL was used for quantification of natural POPs to determine the linear concentration range. Limits of quantification (LOQ) were determined using a signal-to-noise ratio (S/N) of 20, which ranged from 90 to 570 pg on the column (Supporting Information, Table S1), whereas the LOQ using EI-GC/MS was determined using S/N of 10, and ranged from 1 to 50 pg on the column for all analytes. Method detection limit (MDL) was calculated from the standard deviation of the response at lower levels of analytes (ranged from 0.1 to 1.0 ng/g lipid), correcting for blank contamination. Levels of natural POPs in laboratory blanks were all below MDLs (0.05 and 0.2 ng/mL for MeO-BDEs and Cl7-MBP, respectively). Solvent blanks did not contain any of the analytes investigated, indicating no carryover effect between LC/MS/MS runs. Repeatability (%RSD) was evaluated by interassay variations, which were assessed by three consecutive injections of 200 pg/ µL standard solution and by measuring the same standard solution 9752


on different days. Replicate extractions were performed on two of the blubber samples, and values for the replicates were within 8% of each other for all analytes. Recoveries of natural POPs from blubber were assessed by spiking with 10-200 ng of each compound through the entire extraction method. Six target compounds were recovered between 85% and 102% (N ) 7, RSD)6-11%) with no statistical difference to each other. All reported concentrations were calculated by peak area relative to internal standard (4′-MeO-BDE121). PCB153 and BDE47 concentrations were determined by GC/MS using PCB205 as an internal standard. Statistical Analysis. Linear regression analyses between concentrations of organohalogens using APCI-LC/MS/MS and GC/MS were assessed using the SPSS software (ver. 14.0, SPSS Inc.). The existence of a correlation between concentrations of natural POPs was verified by applying the Spearman correlation coefficient (r). The Mann-Whitney U test was used to evaluate the significance of differences between males and females after verifying that size distributions were not significantly different. Statistical significance was set at P < 0.01.

Figure 3. Total ion chromatogram (TIC) in MRM mode at 1000 ng/mL and characteristic transitions of a standard mixture of six natural products and the internal standard. (a) Br4Cl2-DBP, (b) 6-MeO-BDE47, (c) Cl7MBP, (d) 2′,6-diMeO-BDE68, (e) 2′-MeO-BDE68, (f) 2,2′-diMeO-BB80, (g) 4′-MeO-BDE121 (IS), analyzed on a 150 × 4.6 mm, i.d., Shim-pack FC-ODS column (injection volume, 20 µL).

RESULTS AND DISCUSSION Structural Information by LC/MS/MS. Using a Q1 scan in infusion experiments, mass spectra were acquired for 2′-MeOBDE68 and 6-MeO-BDE47, both of which yielded a fragment [M-CH3]- ion, as well as two substitution products [M-Br+O]and [M-Br+CH3O]- ions (Figure 1). The [M-Br+O]- phenoxide ion was probably formed by the reaction with O2-. As the ion source is operated at atmospheric pressure, the most likely source of O2- was the ambient air. As reported previously, even 1 ppm of oxygen as an impurity is sufficient for substitution reactions to take place.28 In addition, another [M-Br+CH3O]- ion emerged in the spectra, where the bromine atom in the molecule was substituted with CH3O-, probably formed by deprotonation of the solvent methanol (Figure 1). The CH3O- anion was assumed to originate from solvent molecules that break down in the ion source, possibly catalyzed by the hot metal source block. This could be explained by the observation that higher proportions of CH3O- ions were observed in methanol solvent but not in acetonitrile or hexane solvent in APCI.18 Similarly, mass spectra of 2′,6-diMeO-BDE68 and 2,2′-diMeOBB80 exhibited three characteristic ions, [M-Br+O]-, [M-CH3]-, and [M-Br+CH3O]- (Figure 1c,d). In contrast, the mass spectrum of 4′-MeO-BDE121 exhibited no [M-CH3]- ion (28) Dzidic, I.; Carroll, D. I.; Stillwell, R. N.; Horning, E. C. Anal. Chem. 1975, 47, 1308–1312.

Figure 4. Sex-related differences in concentrations of natural and anthropogenic POPs. *The difference between male (N ) 10) and female (N ) 5) was significant at P < 0.01. Concentrations of BDE47 and PCB153 were measured by EI-GC/MS.

(Figure 1e). The intensities of the phenoxide ion and the [M-CH3]- ion in the negative ion spectra of MeO-BDEs may aid in the assignment of ring substitution patterns. On the other hand, the spectra of Cl7-MBP and Br4Cl2-DBP indicated the formation of phenoxide [M-Cl+O]- ions (Figure 2a,b), probably by substitution of the chlorine atom in the molecule with O2-. Reactions between M and O2- or M- and O2 may yield [M-Cl+O]- and OCl, which have been observed for hexachlorobenzene (HCB) or polychlorinated biphenyl (PCB) in APPI and APCI.28,29 The [M-Cl+O]- ion has also been observed in negative ion chemical ionization GC/MS for PCB metabolites.30 The reaction can be utilized to enhance the ionization efficiency when this type of compound is analyzed in negative ion mode.31 In Br4Cl2-DBP, the [M-Cl+O]- was formed preferentially instead of [M-Br+O]- ions, indicating that the phenoxide ions are formed by replacement of a chlorine at the 5 or 5′-position of Br4Cl2-DBP. (29) Horning, E. C.; Carroll, D. I.; Dzidic, I.; Lin, S-N.; Stillwell, R. N.; Thenot, J. P. J. Chromatogr. 1977, 142, 481–495. (30) Haraguchi, K.; Bergman, Å.; Jakobsson, E.; Masuda, Y. Fresenius’ J. Anal. Chem. 1993, 347, 441–449. (31) Wolkers, H.; Lydersen, C.; Kovacs, K. M. Sci. Total Environ. 2004, 319, 137–146.


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Table 3. Pearson Correlation Coefficients between Different Groups of Natural and Anthropogenic POPs 2′-MeO-BDE68 2′,6-diMeO-BDE68 2,2′-diMeO-BB80 6-MeO-BDE47 0.509 (P ) 0.05) 2′-MeO-BDE68 2′,6-diMeO-BDE68 2,2′-diMeO-BB80 Cl7-MBP Br4Cl2-DBP BDE47 a

0.000 (P ) 0.99) 0.729a

Cl7- MBP

0.934b 0.925b 0.313 (P ) 0.26) 0.256 (P ) 0.36) -0.374 (P ) 0.20) -0.396 (P ) 0.20) 0.989b

Br4Cl2-DBP

BDE47

PCB153

0.924b 0.261 (P ) 0.35) -0.352 (P ) 0.26) 0.985b 0.993b

0.900b 0.262 (P ) 0.35) -0.344 (P ) 0.27) 0.985b 0.978b 0.971b

0.790b 0.133 (P ) 0.64) -0.371 (P ) 0.23) 0.949b 0.960b 0.944b 0.950b

P < 0.05, b P < 0.001.

For another product ion, the [M-NCH3+O]- ion was observed in both Cl7-MBP and Br4Cl2-DBP spectra (Figure 2). This ion was probably formed by substitution reaction of CH3N with O2- at greater intensity than [M-Cl+O]- ion in the spectra of Cl7-MBP, but was not optimized for MRM transition in this study. [M-NCH3-CCl]- and [M-NCH3-CBr]- ions were observed in the GC/MS fragmentation patterns of Cl7-MBP and Br4Cl2-DBP, respectively.5 MRM Transition. The formation of specific fragment ions produced by the different congeners allowed the development of efficient analytical methods based on the MRM mode. The most intense MRM transition for each compound was used for quantification. The precursor ion, the product ions, and their proposed formulas and the MRM transitions with their optimized DPs and CEs for each compound are listed in Table 1. The [M-Br+O]- ions of the different congeners behaved similarly toward CAD, but they could be distinguished because of the differences observed in the fragment ions. For 2′-MeO-BDE68, 6-MeO-BDE47, and 2′,6-diMeO-BDE68, the most intense MRM transition took place at ([M-Br+O]- f Br- ion (m/z 79 or 81)), whereas the MRM transition for 2,2′-diMeO-BB80 and 4′-MeOBDE121 was most abundant at ([M-Br+O]- f [MCH3Br+O]-) because of the loss of CH3 from phenoxide ions. While Br4Cl2-DBP exhibited MRM transition including ([M-Cl+O]- f Br–), Cl7-MBP exhibited MRM transition with ([M+Cl-O]- f [C4NCl4]-) probably because of cleavage of the interannular C-N bond, producing C4NCl4- (tetrachloropyrrolate anion) from the phenoxide ion. For 2′-MeO-BDE68 and 6-MeOBDE47, only the same MRM transition was best selected, although both compounds could be completely separated from each other on the ODS column condition. Cl7-MBP and 2′,6-diMeO-BDE68 also had the same retention time, but it was highly specific because of the relatively high masses of the MRM transition and different voltages. Optimization of HPLC Condition. Figure 3 shows the total ion chromatogram (TIC) in the MRM mode, and the characteristic transitions for a standard mixture of the six natural POPs and the internal standard. Good resolution values for all adjacent chromatographic peaks were achieved except between 6-MeOBDE47 and Cl7-MBP and between 2′-MeO-BDE68 and 2,2′-diMeOBB80. However, LC overlap of all materials was resolved by the selective MRM transition. ODS column and methanol mobile phase provided the best resolution and shorter retention times, although the separation may be further optimized to resolve the other brominated POPs. Quantification and Quality Parameters. To evaluate the performance of the optimized APCI-LC/MS/MS approach for the selected compounds, (1) linearity, (2) repeatability, and (3) 9754


sensitivity were examined (Table 2). (1) Calibration curves were linear by both external and internal standard calibration. The calibration range studied was at least 2 orders of magnitude, ranging from 4 to 1000 ng/mL, with good values of the correlation factor (r2 > 0.992), indicating excellent linearity over the environmentally relevant concentration range. (2) At the level of 0.2 µg/mL, precision, as measured by relative standard deviation (%RSD), was less than 7% for all compounds, independent of the quantification procedure. The %RSD values obtained using internal standard calibration were better (

MS Method for Determination of Natural

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