- Email: [email protected]
Simple and sensitive method for determination of nitrated polycyclic aromatic hydrocarbons in diesel exhaust particles by gas chromatography-negative ion chemical ionisation tandem mass spectrometry
Journal of Chromatography A, 1163 (2007) 312–317
Simple and sensitive method for determination of nitrated polycyclic aromatic hydrocarbons in diesel exhaust particles by gas chromatography-negative ion chemical ionisation tandem mass spectrometry Youhei Kawanaka a,∗ , Kazuhiko Sakamoto b , Ning Wang a , Sun-Ja Yun a a
The Institute of Basic Environmental Research, Environmental Control Center Co., Ltd., 323-1 Shimo-ongata, Hachioji, Tokyo 192-0154, Japan b Department of Environment Science and Human Engineering, Graduate School of Science and Engineering, Saitama University, 255 Shimo-ohkubo, Sakura, Saitama 338-8570, Japan Received 18 April 2007; received in revised form 18 June 2007; accepted 21 June 2007 Available online 27 June 2007
Abstract An extremely simple and sensitive method was developed for determination of nitrated polycyclic aromatic hydrocarbons (nitro-PAHs; mono-nitro-PAHs and dinitropyrenes) in diesel exhaust particles (DEPs) by gas chromatography-negative ion chemical ionisation tandem mass spectrometry (GC/NCI/MS/MS). We used two types of column in GC/NCI/MS/MS analysis. A polar column was used for determination of mononitro-PAHs, and a non-polar column was used for determination of dinitropyrenes and mono-nitro-PAHs except nitrofluoranthenes. The proposed method requires no clean-up procedure. The limits of detection ranged from 0.01 to 0.09 pg for all compounds tested. The applicability of the method to DEP samples was validated using diesel particulate standard reference materials (SRMs). Although DEPs contain complex matrices, all compounds could be detected easily in SRM2975 (diesel particulate matter) and SRM1975 (diesel particulate extract) without a clean-up procedure. The RSDs were less than 5% for all compounds examined. The quantitative results for SRMs exhibited good agreement with the available data in the literature. These results indicate that the proposed GC/NCI/MS/MS method is useful for determination of nitro-PAHs in DEP samples. © 2007 Published by Elsevier B.V. Keywords: Nitrated polycyclic aromatic hydrocarbons; Dinitropyrene; Standard reference material; Diesel exhaust particles; Tandem mass spectrometry
1. Introduction Diesel exhaust particles (DEPs) are considered a cause of lung cancer. DEPs contain a wide variety of mutagenic compounds, such as polycyclic aromatic hydrocarbons (PAHs) and their derivatives and show a high degree of mutagenicity. Several studies have indicated that some nitrated polycyclic aromatic hydrocarbons (nitro-PAHs), especially 1-nitropyrene and dinitropyrenes, contribute significantly to the direct-acting mutagenicity of DEPs [1,2]. Various analytical techniques have been used for determination of nitro-PAHs in DEPs: gas chromatography with nitrogen phosphorus detection (GC/NPD) [3], GC with negative ion
∗
Corresponding author. Fax: +81 42 652 0800. E-mail address: [email protected] (Y. Kawanaka).
0021-9673/$ – see front matter © 2007 Published by Elsevier B.V. doi:10.1016/j.chroma.2007.06.038
chemical ionisation mass spectrometry [4–8] (GC/NCI/MS), liquid chromatography with chemiluminescence detection [9,10] (LC/CL) and LC with fluorescence detection (LC/FL) [11]. In particular, GC/NCI/MS and LC/CL have been reported to be highly sensitive methods. The LC/CL method has recently been improved [12] and now requires less complex clean-up procedures. However, the other analytical techniques require several clean-up procedures, such as liquid–liquid extraction, solidphase extraction and normal-phase LC fractionation, because DEPs contain complex matrix components. Tandem mass spectrometry (MS/MS) reduces the interfering background caused by the complex matrix components. In previous studies [13–15], MS/MS has been used in analysis of nitro-PAHs in airborne particulate matter and soil. However, very few MS/MS studies have focused on determination of nitroPAHs in DEPs. Moreover, previous MS/MS studies were not targeted toward dinitropyrenes, which are significant contributors
Y. Kawanaka et al. / J. Chromatogr. A 1163 (2007) 312–317
313
to the mutagenicity of airborne particulate matter, soil and DEPs. In the present study, we developed an extremely simple and sensitive method for the quantitative determination of mono-nitro-PAHs and dinitropyrenes in DEPs using gas chromatography-negative ion chemical ionisation tandem mass spectrometry (GC/NCI/MS/MS). This method requires no clean-up procedure. We also validated the method using diesel particulate standard reference materials (SRMs). 2. Experimental 2.1. Chemicals 9-Nitroanthracene, 6-nitrochrysene, 6-nitrobenzo[a]pyrene and 1,8-dinitropyrene were purchased from AccuStandard (New Haven, CT, USA). 3-Nitrofluoranthene and 1-nitropyrene were obtained from Acros Organics (Geel, Belgium). 1,3Dinitropyrene and 1,6-dinitropyrene were obtained from Aldrich (Milwaukee, WI, USA). 2-Nitrofluoranthene was purchased from Chiron (Trondheim, Norway). [2 H9 ]1-Nitropyrene (1-nitropyrene-d9 ) was obtained from CDN Isotopes (Quebec, Canada). All other chemicals used were pesticide residue analytical grade or special grade. 2.2. DEP samples Diesel particulate matter and diesel particulate extract SRMs (SRM2975 and SRM1975, respectively) from the National Institute of Standards and Technology (NIST, Gaithersburg, MD, USA) were used for validation of the proposed analytical method. The concentrations of nitro-PAHs determined by GC/NCI/MS/MS were compared with those reported in the literature. 2.3. Pretreatment of DEP samples The sample pretreatment is extremely simple. SRM2975 (30 mg) was extracted three times by ultrasonication with 40 mL of dichloromethane. The extract was filtered and evaporated to dryness with a rotary evaporator and under a stream of nitrogen gas. The residue was dissolved in dichloromethane and 1-nitropyrene-d9 was added to the solution as an internal standard.
Fig. 1. Full scan spectrum (a) and product ion scan spectrum (b) in NCI mode of 1,8-dinitropyrene.
SRM1975 (20 L) was diluted with dichloromethane and 1nitropyrene-d9 was added to the solution. Sample pretreatment was performed under darkroom conditions to prevent photochemical reactions. 2.4. GC/NCI/MS/MS systems Analytical conditions were improved based on our previous report [15]. All samples were analysed on a Varian 1200L triple stage quadrupole mass spectrometer coupled to a Varian CP3800 GC system (Walnut Creek, CA, USA). All analyses were carried out in NCI mode. Chromatographic separation was performed on a non-polar DB-1ms capillary column (30 m × 0.25 mm I.D. with film thickness of 0.25 m; Agilent, Santa Clara, CA, USA) or a polar DB-17ms capillary column (30 m × 0.25 mm I.D. with film thickness of 0.25 m; Agilent). The injected volume was 1 L in splitless mode. The column flow rate was 1 mL min−1 . The oven temperature for the DB-1ms analyses was programmed as follows: 1 min at 50 ◦ C, heated at 10 ◦ C min−1 to 220 ◦ C, heated at 5 ◦ C min−1 to 310 ◦ C and held at this temperature for 2 min. The oven temperature for the DB-17ms analyses was programmed as follows: 1 min at 50 ◦ C, heated at 20 ◦ C min−1 to
Table 1 Ions and collision energies used for determination of nitro-PAHs in NCI mode Compound
Molecular mass
Precursor ion (m/z)
Product ion (m/z)
Collision energy (eV)
9-Nitroanthracene 2-Nitrofluoranthene 3-Nitrofluoranthene 1-Nitropyrene 6-Nitrochrysene 6-Nitrobenzo[a]pyrene 1,3-Dinitropyrene 1,6-Dinitropyrene 1,8-Dinitropyrene 1-Nitropyrene-d9
223 247 247 247 273 297 292 292 292 256
223 247 247 247 273 297 292 292 292 256
193 217 217 217 243 267 262 262 262 226
10 10 10 10 10 10 15 15 15 10
314
Y. Kawanaka et al. / J. Chromatogr. A 1163 (2007) 312–317
180 ◦ C, heated at 10 ◦ C min−1 to 240 ◦ C, heated at 5 ◦ C min−1 to 310 ◦ C and held at this temperature for 10 min. The transfer line and the ion source temperature were 300 and 200 ◦ C, respectively. Methane was used as NCI reagent gas at a pressure of 1100 Pa. The filament emission current and the electron energy were 150 A and 150 eV, respectively. 3. Results and discussion 3.1. NCI/MS/MS conditions Optimisation of the MS/MS conditions in NCI mode was performed using nitro-PAH standard solutions. As a typical result,
the full scan spectrum and the product ion spectrum obtained at the optimum collision energy of 1,8-dinitropyrene in NCI mode are shown in Fig. 1a and b, respectively. In the full scan mode, 1,8-dinitropyrene generated the most abundant ion at m/z 292 (Fig. 1a). The ion at m/z 292 is the molecular ion ([M]− ) of 1,8dinitropyrene. Thus, [M]− was used as the precursor ion for the determination of 1,8-dinitropyrene. In product ion scan mode, [M]− of 1,8-dinitropyrene gave fragment ions 262 and 232 (Fig. 1b). The ion at m/z 262, which was formed from the neutral loss of NO from the precursor ion [M]− , was most abundant product ion. Therefore, [M−NO]− was used as the product ion for determination of 1,8-dinitropyrene in MS/MS mode. Similarly, for the other
Fig. 2. MS/MS chromatograms of the target nitro-PAH standard solutions at 2 ng mL−1 : (a) non-polar DB-1ms column separation and (b) polar DB-17ms column separation.
Y. Kawanaka et al. / J. Chromatogr. A 1163 (2007) 312–317
target nitro-PAHs, the most abundant precursor ion and product ion were [M]− and [M−NO]− , respectively. The MS/MS parameters for determination of the nine target nitro-PAHs are summarised in Table 1. 3.2. Calibration curve and limit of detection The linearity of calibration curves and limits of detection (LODs) for the target nitro-PAHs were validated using nitroPAH standard solutions on two different polarity columns, i.e., a non-polar DB-1ms column and a polar DB-17ms column. Fig. 2 shows MS/MS chromatograms of nitro-PAH standard solutions at a concentration of 2 ng mL−1 . As demonstrated in sev-
315
Table 2 Limits of detection for the target nitro-PAHs Compound
9-Nitroanthracene 2-Nitrofluoranthene 3-Nitrofluoranthene 1-Nitropyrene 6-Nitrochrysene 6-Nitrobenzo[a]pyrene 1,3-Dinitropyrene 1,6-Dinitropyrene 1,8-Dinitropyrene
Limit of detection (pg) DB-1ms
DB-17ms
0.01 – 0.02 0.01 0.04 0.01 0.01 0.02 0.03
0.01 0.09 0.02 0.03 0.04 0.03 – – –
Fig. 3. MS/MS chromatograms for the target nitro-PAHs in the extract from SRM2975 (diesel particulate matter): (a) non-polar DB-1ms column separation and (b) polar DB-17ms column separation.
316
Y. Kawanaka et al. / J. Chromatogr. A 1163 (2007) 312–317
Table 3 Nitro-PAH concentrations (g g−1 ) in SRM1975 (diesel particulate extract) Compound
9-Nitroanthracene 2-Nitrofluoranthene 3-Nitrofluoranthene 1-Nitropyrene 6-Nitrochrysene 6-Nitrobenzo[a]pyrene 1,3-Dinitropyrene 1,6-Dinitropyrene 1,8-Dinitropyrene
This study GC/NCI/MS/MS
Bezabeh et al. GC/NCI/MS
DB-1msa
DB-17msb
columnc
Nonpolar
1.58 ± 0.04 – 1.51 ± 0.03f 16.5 ± 0.2 0.689 ± 0.048 0.441 ± 0.024 0.550 ± 0.026 1.41 ± 0.06 1.62 ± 0.08
1.59 ± 0.07 0.091 ± 0.008 1.69 ± 0.05 17.4 ± 0.2 0.718 ± 0.031 0.406 ± 0.039 – – –
1.36 ± 0.03 – 1.47 ± 0.01f 16.4 ± 0.1 0.782 ± 0.007 0.641 ± 0.006 0.603 ± 0.011 1.39 ± 0.04 1.55 ± 0.02
Chiu and Miles GC/NCI/HRMS Polar 1.28 0.094 1.62 16.1 0.900 0.514 0.538 0.934 1.38
columnd ± ± ± ± ± ± ± ± ±
0.02 0.004 0.02 0.6 0.015 0.024 0.039 0.014 0.04
Nonpolar columne 1.19 ± 0.02 – 1.34 ± 0.034f 16.6 ± 0.34 0.873 ± 0.130 0.456 ± 0.012 0.581 ± 0.093 0.640 ± 0.112 0.495 ± 0.143
Values obtained in this study using GC/NCI/MS/MS equipped with a 100% dimethylpolysiloxane column: mean ± 95% confidence intervals, n = 7. Values obtained in this study using GC/NCI/MS/MS equipped with a 50% phenyl methylpolysiloxane column: mean ± 95% confidence intervals, n = 7. c Values based on measurements at NIST by Bezabeh et al. obtained using GC/NCI/MS equipped with a 5% phenyl methylpolysiloxane column: mean ± 95% confidence intervals, n = 4. d Values based on measurements at NIST by Bezabeh et al., using GC/NCI/MS equipped with a 50% phenyl methylpolysiloxane column: mean ± 95% confidence intervals, n = 3. e Values reported by Chiu and Miles obtained using GC/NCI/HRMS (high-resolution mass spectrometry) equipped with a 5% phenyl methylpolysiloxane column: mean ± standard deviation, n = 3. f The concentration of 3-nitrofluoranthene includes 2-nitrofluoranthene. a
b
eral previous studies [4,15], 2-nitrofluoranthene was co-eluted with 3-nitrofluoranthene on the non-polar DB-1ms column. Therefore, in DB-1ms analyses, 2-nitrofluoranthene and 3nitrofluoranthene were determined as the total amount using the calibration curve of 3-nitrofluoranthene. Calibration curves were calculated using linear regression on eight concentrations (0.5, 1, 2, 5, 10, 20, 50 and 100 ng mL−1 ). In DB-1ms analyses, a good linear relationship was obtained for all compounds examined, with correlation coefficients (r2 ) of >0.999. On the other hand, in DB-17ms analyses, good correlation coefficients (r2 > 0.999) were obtained for all target mono-nitro-PAHs; however, calibration curves for dinitropyrenes were not linear (r2 < 0.960). In particular, the linearity of calibration curves for dinitropyrenes using the DB-17ms column were not good over the low concentration range (<5 ng mL−1 ). The DB-17ms column has higher polarity than the DB-1ms col-
umn and dinitropyrenes are more polar than mono-nitro-PAHs. Therefore, we postulated that dinitropyrenes interacted with an active site in the polar column and this polar interaction caused peak tailing and lower sensitivity over the low concentration range. Therefore, we concluded that the use of a DB-17ms column is inadequate for determination of dinitropyrenes. LODs were calculated using a signal to noise ratio of 3 (S/N = 3) and values for each compound examined are shown in Table 2. LODs ranged from 0.01 to 0.09 pg for all compounds examined. These LODs were sufficiently low to allow detection of the target nitro-PAHs at the levels usually present in DEPs. 3.3. Application to DEP samples The applicability of the proposed GC/NCI/MS/MS method to DEP samples was validated using SRM2975 (diesel particulate
Table 4 Nitro-PAH concentrations (g g−1 ) in SRM2975 (diesel particulate matter) Compound
9-Nitroanthracene 2-Nitrofluoranthene 3-Nitrofluoranthene 1-Nitropyrene 6-Nitrochrysene 6-Nitrobenzo[a]pyrene 1,3-Dinitropyrene 1,6-Dinitropyrene 1,8-Dinitropyrene
This study GC/NCI/MS/MS
Bezabeh et al. GC/NCI/MS
DB-1msa
DB-17msb
Nonpolar columnc
Polar columnd
3.21 ± 0.17 – 3.20 ± 0.17e 36.3 ± 0.3 1.33 ± 0.08 1.35 ± 0.12 1.07 ± 0.13 3.04 ± 0.13 3.71 ± 0.15
3.06 ± 0.03 0.22 ± 0.03 3.39 ± 0.21 36.2 ± 1.0 1.49 ± 0.06 1.70 ± 0.20 – – –
3.37 ± 0.19 – 3.32 ± 0.10e 33.1 ± 0.6 1.38 ± 0.07 1.19 ± 0.17 0.961 ± 0.030 1.95 ± 0.06 2.62 ± 0.16
2.93 0.250 4.30 39.6 2.37 1.65 1.15 2.54 3.58
± ± ± ± ± ± ± ± ±
0.06 0.011 0.03 1.7 0.07 0.04 0.06 0.22 0.17
Values obtained in this study using GC/NCI/MS/MS equipped with a 100% dimethylpolysiloxane column: mean ± 95% confidence intervals, n = 3. Values obtained in this study using GC/NCI/MS/MS equipped with a 50% phenyl methylpolysiloxane column: mean ± 95% confidence intervals, n = 3. c Values based on measurements at NIST by Bezabeh et al. obtained using GC/NCI/MS equipped with a 5% phenyl methylpolysiloxane column: mean ± 95% confidence intervals, n = 4. d Values based on measurements at NIST by Bezabeh et al. obtained using GC/NCI/MS equipped with a 50% phenyl methylpolysiloxane column: mean ± 95% confidence intervals, n = 3. e The concentration of 3-nitrofluoranthene includes 2-nitrofluoranthene. a
b
Y. Kawanaka et al. / J. Chromatogr. A 1163 (2007) 312–317
matter) and SRM1975 (diesel particulate extract). Fig. 3 shows MS/MS chromatograms for nitro-PAHs in the extract from SRM2975 on a non-polar DB-1ms column and a polar DB17ms column. All of the target nitro-PAHs could be detected easily in the extract without a clean-up procedure despite the complex matrices in DEPs. Similarly, no matrix interference was observed in the chromatograms for SRM1975 without a clean-up procedure. It was found that no clean-up procedure is required for DEPs in GC/NCI/MS/MS analysis, because the contents of nitro-PAHs in DEPs are higher than those in atmospheric particulate matter and soil. The quantitative results for the target nitro-PAHs in SRM2975 and SRM1975 by the proposed method are shown in Tables 3 and 4, respectively. As described previously, dinitropyrenes were determined using only a non-polar DB-1ms column. The results of previous studies by Bezabeh [4] and Chiu and Miles [6] are also included in Tables 3 and 4 for comparison. In these previous studies, solid-phase extraction and/or normal-phase LC fractionation were used as clean-up procedures for analysis of nitro-PAHs in DEPs. Although the proposed GC/NCI/MS/MS method requires no clean-up procedure, the results obtained using this method showed good agreement (within approximately 30%) with the results of these previous studies. Moreover, the RSDs were less than 5% for all compounds and the results were therefore considered very satisfactory. These results indicate the suitability of the GC/NCI/MS/MS method for analysing nitro-PAHs in DEPs. 4. Conclusions We succeeded in optimisation of a GC/NCI/MS/MS method for determination of mono-nitro-PAHs and dinitropyrenes in DEPs. A polar column was used for determination of mononitro-PAHs, and a non-polar column was used for determination of dinitropyrenes and mono-nitro-PAHs except nitrofluoran-
317
thenes. The LODs were lower than 0.1 pg for all compounds examined. The method was sufficiently sensitive to determine the target nitro-PAHs in DEPs. Although the proposed method requires no clean-up procedure, all the target nitro-PAHs could be detected easily in DEPs. The quantitative results for diesel particulate SRMs showed good agreement with the data in the literature. The technique is useful for determination of nitro-PAHs in DEP samples. References [1] K. Hayakawa, A. Nakamura, N. Terai, R. Kizu, K. Ando, Chem. Pharm. Bull. 45 (1997) 1820. [2] I. Salmeen, A.M. Durisin, T.J. Prater, T. Riley, Mutation Res. 104 (1982) 17. [3] M.C. Paputa-Peck, R.S. Marano, D. Schuetzle, T.L. Riley, C.V. Hampton, T.J. Prater, L.M. Skewes, T.E. Jensen, P.H. Ruehle, L.C. Bosch, W.P. Duncan, Anal. Chem. 55 (1983) 1946. [4] D.Z. Bezabeh, H.A. Bamford, M.M. Schantz, S.A. Wise, Anal. Bioanal. Chem. 375 (2003) 381. [5] H.A. Bamford, D.Z. Bezabeh, M.M. Schantz, S.A. Wise, J.E. Baker, Chemosphere 50 (2003) 575. [6] C. Chiu, W. Miles, Polycyclic Aromat. Compd. 9 (1996) 307. [7] W.M. Draper, Chemosphere 15 (1986) 437. [8] U. Sellstrom, B. Jansson, A. Bergman, T. Alsberg, Chemosphere 16 (1987) 945. [9] K. Hayakawa, R. Kitamura, M. Butoh, N. Imaizumi, M. Miyazaki, Anal. Sci. 7 (1991) 573. [10] K. Hayakawa, M. Butoh, M. Miyazaki, Anal. Chim. Acta 266 (1992) 251. [11] W.A. MacCrehan, W.E. May, S.D. Yang, B.A. Benner Jr., Anal. Chem. 60 (1988) 194. [12] N. Tang, A. Toriba, R. Kizu, K. Hayakawa, Anal. Sci. 19 (2003) 249. [13] M. Vincenti, C. Minero, E. Pelizzetti, M. Fontana, R.D. Maria, J. Am. Soc. Mass Spectrom. 7 (1996) 1255. [14] M. Niederer, Environ. Sci. Pollut. Res. 5 (1998) 209. [15] Y. Kawanaka, K. Sakamoto, N. Wang, S.J. Yun, Bunseki Kagaku 54 (2005) 695 (in Japanese).
Simple and sensitive method for determination of nitrated polycyclic aromatic hydrocarbons in diesel exhaust particles by gas chromatography-negative ion chemical ionisation tandem mass spectrometry Youhei Kawanaka a,∗ , Kazuhiko Sakamoto b , Ning Wang a , Sun-Ja Yun a a
The Institute of Basic Environmental Research, Environmental Control Center Co., Ltd., 323-1 Shimo-ongata, Hachioji, Tokyo 192-0154, Japan b Department of Environment Science and Human Engineering, Graduate School of Science and Engineering, Saitama University, 255 Shimo-ohkubo, Sakura, Saitama 338-8570, Japan Received 18 April 2007; received in revised form 18 June 2007; accepted 21 June 2007 Available online 27 June 2007
Abstract An extremely simple and sensitive method was developed for determination of nitrated polycyclic aromatic hydrocarbons (nitro-PAHs; mono-nitro-PAHs and dinitropyrenes) in diesel exhaust particles (DEPs) by gas chromatography-negative ion chemical ionisation tandem mass spectrometry (GC/NCI/MS/MS). We used two types of column in GC/NCI/MS/MS analysis. A polar column was used for determination of mononitro-PAHs, and a non-polar column was used for determination of dinitropyrenes and mono-nitro-PAHs except nitrofluoranthenes. The proposed method requires no clean-up procedure. The limits of detection ranged from 0.01 to 0.09 pg for all compounds tested. The applicability of the method to DEP samples was validated using diesel particulate standard reference materials (SRMs). Although DEPs contain complex matrices, all compounds could be detected easily in SRM2975 (diesel particulate matter) and SRM1975 (diesel particulate extract) without a clean-up procedure. The RSDs were less than 5% for all compounds examined. The quantitative results for SRMs exhibited good agreement with the available data in the literature. These results indicate that the proposed GC/NCI/MS/MS method is useful for determination of nitro-PAHs in DEP samples. © 2007 Published by Elsevier B.V. Keywords: Nitrated polycyclic aromatic hydrocarbons; Dinitropyrene; Standard reference material; Diesel exhaust particles; Tandem mass spectrometry
1. Introduction Diesel exhaust particles (DEPs) are considered a cause of lung cancer. DEPs contain a wide variety of mutagenic compounds, such as polycyclic aromatic hydrocarbons (PAHs) and their derivatives and show a high degree of mutagenicity. Several studies have indicated that some nitrated polycyclic aromatic hydrocarbons (nitro-PAHs), especially 1-nitropyrene and dinitropyrenes, contribute significantly to the direct-acting mutagenicity of DEPs [1,2]. Various analytical techniques have been used for determination of nitro-PAHs in DEPs: gas chromatography with nitrogen phosphorus detection (GC/NPD) [3], GC with negative ion
∗
Corresponding author. Fax: +81 42 652 0800. E-mail address: [email protected] (Y. Kawanaka).
0021-9673/$ – see front matter © 2007 Published by Elsevier B.V. doi:10.1016/j.chroma.2007.06.038
chemical ionisation mass spectrometry [4–8] (GC/NCI/MS), liquid chromatography with chemiluminescence detection [9,10] (LC/CL) and LC with fluorescence detection (LC/FL) [11]. In particular, GC/NCI/MS and LC/CL have been reported to be highly sensitive methods. The LC/CL method has recently been improved [12] and now requires less complex clean-up procedures. However, the other analytical techniques require several clean-up procedures, such as liquid–liquid extraction, solidphase extraction and normal-phase LC fractionation, because DEPs contain complex matrix components. Tandem mass spectrometry (MS/MS) reduces the interfering background caused by the complex matrix components. In previous studies [13–15], MS/MS has been used in analysis of nitro-PAHs in airborne particulate matter and soil. However, very few MS/MS studies have focused on determination of nitroPAHs in DEPs. Moreover, previous MS/MS studies were not targeted toward dinitropyrenes, which are significant contributors
Y. Kawanaka et al. / J. Chromatogr. A 1163 (2007) 312–317
313
to the mutagenicity of airborne particulate matter, soil and DEPs. In the present study, we developed an extremely simple and sensitive method for the quantitative determination of mono-nitro-PAHs and dinitropyrenes in DEPs using gas chromatography-negative ion chemical ionisation tandem mass spectrometry (GC/NCI/MS/MS). This method requires no clean-up procedure. We also validated the method using diesel particulate standard reference materials (SRMs). 2. Experimental 2.1. Chemicals 9-Nitroanthracene, 6-nitrochrysene, 6-nitrobenzo[a]pyrene and 1,8-dinitropyrene were purchased from AccuStandard (New Haven, CT, USA). 3-Nitrofluoranthene and 1-nitropyrene were obtained from Acros Organics (Geel, Belgium). 1,3Dinitropyrene and 1,6-dinitropyrene were obtained from Aldrich (Milwaukee, WI, USA). 2-Nitrofluoranthene was purchased from Chiron (Trondheim, Norway). [2 H9 ]1-Nitropyrene (1-nitropyrene-d9 ) was obtained from CDN Isotopes (Quebec, Canada). All other chemicals used were pesticide residue analytical grade or special grade. 2.2. DEP samples Diesel particulate matter and diesel particulate extract SRMs (SRM2975 and SRM1975, respectively) from the National Institute of Standards and Technology (NIST, Gaithersburg, MD, USA) were used for validation of the proposed analytical method. The concentrations of nitro-PAHs determined by GC/NCI/MS/MS were compared with those reported in the literature. 2.3. Pretreatment of DEP samples The sample pretreatment is extremely simple. SRM2975 (30 mg) was extracted three times by ultrasonication with 40 mL of dichloromethane. The extract was filtered and evaporated to dryness with a rotary evaporator and under a stream of nitrogen gas. The residue was dissolved in dichloromethane and 1-nitropyrene-d9 was added to the solution as an internal standard.
Fig. 1. Full scan spectrum (a) and product ion scan spectrum (b) in NCI mode of 1,8-dinitropyrene.
SRM1975 (20 L) was diluted with dichloromethane and 1nitropyrene-d9 was added to the solution. Sample pretreatment was performed under darkroom conditions to prevent photochemical reactions. 2.4. GC/NCI/MS/MS systems Analytical conditions were improved based on our previous report [15]. All samples were analysed on a Varian 1200L triple stage quadrupole mass spectrometer coupled to a Varian CP3800 GC system (Walnut Creek, CA, USA). All analyses were carried out in NCI mode. Chromatographic separation was performed on a non-polar DB-1ms capillary column (30 m × 0.25 mm I.D. with film thickness of 0.25 m; Agilent, Santa Clara, CA, USA) or a polar DB-17ms capillary column (30 m × 0.25 mm I.D. with film thickness of 0.25 m; Agilent). The injected volume was 1 L in splitless mode. The column flow rate was 1 mL min−1 . The oven temperature for the DB-1ms analyses was programmed as follows: 1 min at 50 ◦ C, heated at 10 ◦ C min−1 to 220 ◦ C, heated at 5 ◦ C min−1 to 310 ◦ C and held at this temperature for 2 min. The oven temperature for the DB-17ms analyses was programmed as follows: 1 min at 50 ◦ C, heated at 20 ◦ C min−1 to
Table 1 Ions and collision energies used for determination of nitro-PAHs in NCI mode Compound
Molecular mass
Precursor ion (m/z)
Product ion (m/z)
Collision energy (eV)
9-Nitroanthracene 2-Nitrofluoranthene 3-Nitrofluoranthene 1-Nitropyrene 6-Nitrochrysene 6-Nitrobenzo[a]pyrene 1,3-Dinitropyrene 1,6-Dinitropyrene 1,8-Dinitropyrene 1-Nitropyrene-d9
223 247 247 247 273 297 292 292 292 256
223 247 247 247 273 297 292 292 292 256
193 217 217 217 243 267 262 262 262 226
10 10 10 10 10 10 15 15 15 10
314
Y. Kawanaka et al. / J. Chromatogr. A 1163 (2007) 312–317
180 ◦ C, heated at 10 ◦ C min−1 to 240 ◦ C, heated at 5 ◦ C min−1 to 310 ◦ C and held at this temperature for 10 min. The transfer line and the ion source temperature were 300 and 200 ◦ C, respectively. Methane was used as NCI reagent gas at a pressure of 1100 Pa. The filament emission current and the electron energy were 150 A and 150 eV, respectively. 3. Results and discussion 3.1. NCI/MS/MS conditions Optimisation of the MS/MS conditions in NCI mode was performed using nitro-PAH standard solutions. As a typical result,
the full scan spectrum and the product ion spectrum obtained at the optimum collision energy of 1,8-dinitropyrene in NCI mode are shown in Fig. 1a and b, respectively. In the full scan mode, 1,8-dinitropyrene generated the most abundant ion at m/z 292 (Fig. 1a). The ion at m/z 292 is the molecular ion ([M]− ) of 1,8dinitropyrene. Thus, [M]− was used as the precursor ion for the determination of 1,8-dinitropyrene. In product ion scan mode, [M]− of 1,8-dinitropyrene gave fragment ions 262 and 232 (Fig. 1b). The ion at m/z 262, which was formed from the neutral loss of NO from the precursor ion [M]− , was most abundant product ion. Therefore, [M−NO]− was used as the product ion for determination of 1,8-dinitropyrene in MS/MS mode. Similarly, for the other
Fig. 2. MS/MS chromatograms of the target nitro-PAH standard solutions at 2 ng mL−1 : (a) non-polar DB-1ms column separation and (b) polar DB-17ms column separation.
Y. Kawanaka et al. / J. Chromatogr. A 1163 (2007) 312–317
target nitro-PAHs, the most abundant precursor ion and product ion were [M]− and [M−NO]− , respectively. The MS/MS parameters for determination of the nine target nitro-PAHs are summarised in Table 1. 3.2. Calibration curve and limit of detection The linearity of calibration curves and limits of detection (LODs) for the target nitro-PAHs were validated using nitroPAH standard solutions on two different polarity columns, i.e., a non-polar DB-1ms column and a polar DB-17ms column. Fig. 2 shows MS/MS chromatograms of nitro-PAH standard solutions at a concentration of 2 ng mL−1 . As demonstrated in sev-
315
Table 2 Limits of detection for the target nitro-PAHs Compound
9-Nitroanthracene 2-Nitrofluoranthene 3-Nitrofluoranthene 1-Nitropyrene 6-Nitrochrysene 6-Nitrobenzo[a]pyrene 1,3-Dinitropyrene 1,6-Dinitropyrene 1,8-Dinitropyrene
Limit of detection (pg) DB-1ms
DB-17ms
0.01 – 0.02 0.01 0.04 0.01 0.01 0.02 0.03
0.01 0.09 0.02 0.03 0.04 0.03 – – –
Fig. 3. MS/MS chromatograms for the target nitro-PAHs in the extract from SRM2975 (diesel particulate matter): (a) non-polar DB-1ms column separation and (b) polar DB-17ms column separation.
316
Y. Kawanaka et al. / J. Chromatogr. A 1163 (2007) 312–317
Table 3 Nitro-PAH concentrations (g g−1 ) in SRM1975 (diesel particulate extract) Compound
9-Nitroanthracene 2-Nitrofluoranthene 3-Nitrofluoranthene 1-Nitropyrene 6-Nitrochrysene 6-Nitrobenzo[a]pyrene 1,3-Dinitropyrene 1,6-Dinitropyrene 1,8-Dinitropyrene
This study GC/NCI/MS/MS
Bezabeh et al. GC/NCI/MS
DB-1msa
DB-17msb
columnc
Nonpolar
1.58 ± 0.04 – 1.51 ± 0.03f 16.5 ± 0.2 0.689 ± 0.048 0.441 ± 0.024 0.550 ± 0.026 1.41 ± 0.06 1.62 ± 0.08
1.59 ± 0.07 0.091 ± 0.008 1.69 ± 0.05 17.4 ± 0.2 0.718 ± 0.031 0.406 ± 0.039 – – –
1.36 ± 0.03 – 1.47 ± 0.01f 16.4 ± 0.1 0.782 ± 0.007 0.641 ± 0.006 0.603 ± 0.011 1.39 ± 0.04 1.55 ± 0.02
Chiu and Miles GC/NCI/HRMS Polar 1.28 0.094 1.62 16.1 0.900 0.514 0.538 0.934 1.38
columnd ± ± ± ± ± ± ± ± ±
0.02 0.004 0.02 0.6 0.015 0.024 0.039 0.014 0.04
Nonpolar columne 1.19 ± 0.02 – 1.34 ± 0.034f 16.6 ± 0.34 0.873 ± 0.130 0.456 ± 0.012 0.581 ± 0.093 0.640 ± 0.112 0.495 ± 0.143
Values obtained in this study using GC/NCI/MS/MS equipped with a 100% dimethylpolysiloxane column: mean ± 95% confidence intervals, n = 7. Values obtained in this study using GC/NCI/MS/MS equipped with a 50% phenyl methylpolysiloxane column: mean ± 95% confidence intervals, n = 7. c Values based on measurements at NIST by Bezabeh et al. obtained using GC/NCI/MS equipped with a 5% phenyl methylpolysiloxane column: mean ± 95% confidence intervals, n = 4. d Values based on measurements at NIST by Bezabeh et al., using GC/NCI/MS equipped with a 50% phenyl methylpolysiloxane column: mean ± 95% confidence intervals, n = 3. e Values reported by Chiu and Miles obtained using GC/NCI/HRMS (high-resolution mass spectrometry) equipped with a 5% phenyl methylpolysiloxane column: mean ± standard deviation, n = 3. f The concentration of 3-nitrofluoranthene includes 2-nitrofluoranthene. a
b
eral previous studies [4,15], 2-nitrofluoranthene was co-eluted with 3-nitrofluoranthene on the non-polar DB-1ms column. Therefore, in DB-1ms analyses, 2-nitrofluoranthene and 3nitrofluoranthene were determined as the total amount using the calibration curve of 3-nitrofluoranthene. Calibration curves were calculated using linear regression on eight concentrations (0.5, 1, 2, 5, 10, 20, 50 and 100 ng mL−1 ). In DB-1ms analyses, a good linear relationship was obtained for all compounds examined, with correlation coefficients (r2 ) of >0.999. On the other hand, in DB-17ms analyses, good correlation coefficients (r2 > 0.999) were obtained for all target mono-nitro-PAHs; however, calibration curves for dinitropyrenes were not linear (r2 < 0.960). In particular, the linearity of calibration curves for dinitropyrenes using the DB-17ms column were not good over the low concentration range (<5 ng mL−1 ). The DB-17ms column has higher polarity than the DB-1ms col-
umn and dinitropyrenes are more polar than mono-nitro-PAHs. Therefore, we postulated that dinitropyrenes interacted with an active site in the polar column and this polar interaction caused peak tailing and lower sensitivity over the low concentration range. Therefore, we concluded that the use of a DB-17ms column is inadequate for determination of dinitropyrenes. LODs were calculated using a signal to noise ratio of 3 (S/N = 3) and values for each compound examined are shown in Table 2. LODs ranged from 0.01 to 0.09 pg for all compounds examined. These LODs were sufficiently low to allow detection of the target nitro-PAHs at the levels usually present in DEPs. 3.3. Application to DEP samples The applicability of the proposed GC/NCI/MS/MS method to DEP samples was validated using SRM2975 (diesel particulate
Table 4 Nitro-PAH concentrations (g g−1 ) in SRM2975 (diesel particulate matter) Compound
9-Nitroanthracene 2-Nitrofluoranthene 3-Nitrofluoranthene 1-Nitropyrene 6-Nitrochrysene 6-Nitrobenzo[a]pyrene 1,3-Dinitropyrene 1,6-Dinitropyrene 1,8-Dinitropyrene
This study GC/NCI/MS/MS
Bezabeh et al. GC/NCI/MS
DB-1msa
DB-17msb
Nonpolar columnc
Polar columnd
3.21 ± 0.17 – 3.20 ± 0.17e 36.3 ± 0.3 1.33 ± 0.08 1.35 ± 0.12 1.07 ± 0.13 3.04 ± 0.13 3.71 ± 0.15
3.06 ± 0.03 0.22 ± 0.03 3.39 ± 0.21 36.2 ± 1.0 1.49 ± 0.06 1.70 ± 0.20 – – –
3.37 ± 0.19 – 3.32 ± 0.10e 33.1 ± 0.6 1.38 ± 0.07 1.19 ± 0.17 0.961 ± 0.030 1.95 ± 0.06 2.62 ± 0.16
2.93 0.250 4.30 39.6 2.37 1.65 1.15 2.54 3.58
± ± ± ± ± ± ± ± ±
0.06 0.011 0.03 1.7 0.07 0.04 0.06 0.22 0.17
Values obtained in this study using GC/NCI/MS/MS equipped with a 100% dimethylpolysiloxane column: mean ± 95% confidence intervals, n = 3. Values obtained in this study using GC/NCI/MS/MS equipped with a 50% phenyl methylpolysiloxane column: mean ± 95% confidence intervals, n = 3. c Values based on measurements at NIST by Bezabeh et al. obtained using GC/NCI/MS equipped with a 5% phenyl methylpolysiloxane column: mean ± 95% confidence intervals, n = 4. d Values based on measurements at NIST by Bezabeh et al. obtained using GC/NCI/MS equipped with a 50% phenyl methylpolysiloxane column: mean ± 95% confidence intervals, n = 3. e The concentration of 3-nitrofluoranthene includes 2-nitrofluoranthene. a
b
Y. Kawanaka et al. / J. Chromatogr. A 1163 (2007) 312–317
matter) and SRM1975 (diesel particulate extract). Fig. 3 shows MS/MS chromatograms for nitro-PAHs in the extract from SRM2975 on a non-polar DB-1ms column and a polar DB17ms column. All of the target nitro-PAHs could be detected easily in the extract without a clean-up procedure despite the complex matrices in DEPs. Similarly, no matrix interference was observed in the chromatograms for SRM1975 without a clean-up procedure. It was found that no clean-up procedure is required for DEPs in GC/NCI/MS/MS analysis, because the contents of nitro-PAHs in DEPs are higher than those in atmospheric particulate matter and soil. The quantitative results for the target nitro-PAHs in SRM2975 and SRM1975 by the proposed method are shown in Tables 3 and 4, respectively. As described previously, dinitropyrenes were determined using only a non-polar DB-1ms column. The results of previous studies by Bezabeh [4] and Chiu and Miles [6] are also included in Tables 3 and 4 for comparison. In these previous studies, solid-phase extraction and/or normal-phase LC fractionation were used as clean-up procedures for analysis of nitro-PAHs in DEPs. Although the proposed GC/NCI/MS/MS method requires no clean-up procedure, the results obtained using this method showed good agreement (within approximately 30%) with the results of these previous studies. Moreover, the RSDs were less than 5% for all compounds and the results were therefore considered very satisfactory. These results indicate the suitability of the GC/NCI/MS/MS method for analysing nitro-PAHs in DEPs. 4. Conclusions We succeeded in optimisation of a GC/NCI/MS/MS method for determination of mono-nitro-PAHs and dinitropyrenes in DEPs. A polar column was used for determination of mononitro-PAHs, and a non-polar column was used for determination of dinitropyrenes and mono-nitro-PAHs except nitrofluoran-
317
thenes. The LODs were lower than 0.1 pg for all compounds examined. The method was sufficiently sensitive to determine the target nitro-PAHs in DEPs. Although the proposed method requires no clean-up procedure, all the target nitro-PAHs could be detected easily in DEPs. The quantitative results for diesel particulate SRMs showed good agreement with the data in the literature. The technique is useful for determination of nitro-PAHs in DEP samples. References [1] K. Hayakawa, A. Nakamura, N. Terai, R. Kizu, K. Ando, Chem. Pharm. Bull. 45 (1997) 1820. [2] I. Salmeen, A.M. Durisin, T.J. Prater, T. Riley, Mutation Res. 104 (1982) 17. [3] M.C. Paputa-Peck, R.S. Marano, D. Schuetzle, T.L. Riley, C.V. Hampton, T.J. Prater, L.M. Skewes, T.E. Jensen, P.H. Ruehle, L.C. Bosch, W.P. Duncan, Anal. Chem. 55 (1983) 1946. [4] D.Z. Bezabeh, H.A. Bamford, M.M. Schantz, S.A. Wise, Anal. Bioanal. Chem. 375 (2003) 381. [5] H.A. Bamford, D.Z. Bezabeh, M.M. Schantz, S.A. Wise, J.E. Baker, Chemosphere 50 (2003) 575. [6] C. Chiu, W. Miles, Polycyclic Aromat. Compd. 9 (1996) 307. [7] W.M. Draper, Chemosphere 15 (1986) 437. [8] U. Sellstrom, B. Jansson, A. Bergman, T. Alsberg, Chemosphere 16 (1987) 945. [9] K. Hayakawa, R. Kitamura, M. Butoh, N. Imaizumi, M. Miyazaki, Anal. Sci. 7 (1991) 573. [10] K. Hayakawa, M. Butoh, M. Miyazaki, Anal. Chim. Acta 266 (1992) 251. [11] W.A. MacCrehan, W.E. May, S.D. Yang, B.A. Benner Jr., Anal. Chem. 60 (1988) 194. [12] N. Tang, A. Toriba, R. Kizu, K. Hayakawa, Anal. Sci. 19 (2003) 249. [13] M. Vincenti, C. Minero, E. Pelizzetti, M. Fontana, R.D. Maria, J. Am. Soc. Mass Spectrom. 7 (1996) 1255. [14] M. Niederer, Environ. Sci. Pollut. Res. 5 (1998) 209. [15] Y. Kawanaka, K. Sakamoto, N. Wang, S.J. Yun, Bunseki Kagaku 54 (2005) 695 (in Japanese).