Determination of Nitrated Pyrenes and Their Derivatives by High Performance Liquid Chromatography with Chemiluminescence Detection after Online Electrochemical Reduction Noriko Imaizumit, Kazuichi HayakawaS, Yasuo Suzuki, Motoichi Miyazaki Faculty of Pharmaceutical Sciences, Kanazawa University, Takara-machi 13-1, Kanazawa, 920, Japan
A sensitive and selective high performance liquid chromatographic method for the simultaneous determination of nitrated polycyclic aromatic hydrocarbons and their reduced compounds has been developed. As the model compounds for the proposed method, pyrene, aminopyrene, nitrosopyrene, and several nitrated pyrenes were used. After separation on a reversed phase column, both nitropyrenes and nitrosopyrene which were not fluorescent, were electrochemically reduced to strongly fluorescent aminopyrenes, and detected chemilumigenically by using bis(2,4,6-trichloropheny1)oxalate and hydrogen peroxide as post column reagents. Their detection limits were all at the fmol levels, which were one or two orders lower than those by fluorescence detection. By the proposed method, nitropyrene, 1,3-, and l,8-dinitropyrenes were detected in sooty emissions of cars after simple purifications.
INTRODUCTION Polycyclic aromatic hydrocarbons (PAH) and nitrated polycyclic aromatic hydrocarbons( N PAH) have attracted much attention because of their potent mutagenicity (Rosenkranz et al., 1980; Arey et al., 1988) and carcinogenicity (Hirose et al., 1984; Howard et al., i965). They are formed as undesirable by-products through burning processes of various materials, such as petroleum, and are found in various environmental samples (Davis et al., 1987; Kames et al., 1988). PAH has been determined mainly by gas chromatography ( G C ) with flame ionization and mass spectrometric (MS) detectors, and NPAH has been determined by GC with MS, electron capture, nitrogen-phosphorus and chemiluminescence detectors. MS and nitrogenphosphorus detection in particular were used in sensitive determination methods for environmental samples. However, these methods required very complex purification of samples to remove interfering compounds (Sellstrom et al., 1987) and concentration of trace levels of PAH and NPAH. On the other hand, high performance liquid chromatographic (HPLC) methods with UV absorbance, fluorescence (Tanabe et aZ., 1986; Tejada et al., 1986; Arashidani et aZ., 1987) and amperometric (MacCrehan et al., 1988) detectors have been reported. In these methods, fluorescence detection has been used for sensitive determination of several fluorescent PAH’s and amino-PAH’s since it was so selective that purification procedures could be simpler than those for GC. Recently, chemiluminescence detection has been developed as a highly sensitive detection method for HPLC. Such aryl oxalates as bis(2,4,6-trichloro-
pheny1)oxalate (TCPO) and hydrogen peroxide have been mainly used as post column chemilumigenic reagents (Imai, 1988; Sigvardson and Birks, 1983, 1984a,b; Hayakawa et al., 1989). The sensitivities of some fluorescent compounds by chemiluminescence detection increased 10-100 times higher than those given by fluorescence detection (Sigvardson and Birks, 1984b; Hayakawa et al., 1989). The method has been already applied to the determination of fluorescent PAH’s (Sigvardson and Birks, 1983; Imaizumi et al., 1989b) and amino-PAH’s (Sigvardson and Birks, 1984a, b). For the determination of non-fluorescent NPAH’s, several reducing columns packed with zinc powder (Sigvardson and Birks, 1984b) or Pt/Rh-coated alumina (Tejada et al., 1986) have been developed to reduce NPAH to strongly fluorescent amino-PAH. But they have not been favorable for a routine analysis of NPAH because of their short life-time. In this paper, we have established a new HPLC determination method for NPAH using chemiluminescence detection after online electrochemical reduction. Online reduction has such an advantage that it does not require any particular reagents which might affect chromatographic separation and chemiluminescence detection of NPAH. We used 1-nitropyrene(NP), l-nitrosopyrene(NSP), 1-aminopyrene(AP), 1,3-, 1,6-, and 1,8dinitropyrenes( D N P s ) and pyrene as analytes in this work, because NP, DNP’s and pyrene are found in many environmental samples and NSP and AP were the feasible metabolites of NP in biological systems. By the proposed method, simultaneous and sensitive determination of PAH’s and NPAH’s was made possible.
EXPERIMENTAL Current Address: Niigata College of Pharmacy, Kamishin’ eicho 5-13-2, Niigata, 950-21, Japan. * Author to whom correspondence should be addressed.
Chemicals. TCPO of analytical reagent grade (Tokyo Kasei, Tokyo, Japan) and hydrogen peroxide of electrochemical
CCC-O269-3879/90/OlOS-O112 $2.50 108 BIOMEDICAL CHROMATOGRAPHY, VOL. 4, NO. 3, 1990
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NITRATED PYRENE DETERMINATION BY HPLC WITH CL DETECTION
grade (Kanto Chemicals, Tokyo, Japan) were used as received. NP was synthesized by the method of Paputa-peck et al. (1983) using pyrene (chemical grade, Tokyo Kasei) and fuming nitric acid (Katayama Chemical, Osaka, Japan). NSP was synthesized by the method of Howard et a/. (1985) using AP (Aldrich, Milwaukee, WI, USA) and m-chloroperoxybenzoic acid (Wako Pure Chemical, Osaka, Japan). 1,3-, 1,6-, and 1,sDNPs were purchased from Aldrich. All other reagents used were analytical reagent grade and were commercially purchased. Apparatus. The HPLC system consisted of two Shimadzu (Kyoto, Japan) LC-6A pumps, a Rheodyne (Cotati, CA, USA) model 7125 loop injector (loop volume of 20pL), an Atto (Tokyo, Japan) AC-2220 chemiluminescence detector (spinal type cell of 60 pL), a Jasco (Hachioji, Tokyo, Japan) 820-FP fluorescence detector, an ESA (Bedford, CA, USA) Coulochem model 5100A electrochemical detector with two glassy-carbon working electrodes, a Kyowa (Mitaka, Tokyo, Japan) KZS-1 mixing device, two Shimadzu C-R1B integrators and a 0.5 mm ID x 300 mm stainless steel tubing as a reaction coil. The separation and guard columns were Nagel (Duran, West Germany) Nucleosil ODS (4.6 mm ID x 250 mm) and a Brownlee (Berkley, CA, USA) RP-18 (4.6 mm ID x 30 mm), respectively. Figure 1 shows a schematic diagram of the present system. Other details of the system were the same as those reported in our previous work (Imaizumi et a/., 1989a) except assembly for electrochemical reduction. Preparation of solutions. The mixture of acetonitrile and aqueous buffers was used as a mobile phase at a flow rate of 1.0 mL/min. The mobile phase was continuously purged with nitrogen gas (purity was over 99.99%) to remove oxygen which disturbed the electrochemical reduction of nitropyrenes remarkably. The acetronitrile content in the mobile phase was SO%, except in the case for the simultaneous determination of AP, NP, NSP, D N P s and pyrene, it was 65%. As aqueous buffers, imidazole(2 mM) perchloric acid, imidazole(2 mM) + acetic acid and sodium acetate+ acetic acid (2 mM as the total acetate concentration) were tested. The chemilumigenic reagent solution was prepared by the addition of hydrogen peroxide to acetonitrile solution of TCPO just before use. The final concentrations of TCPO and hydrogen peroxide were 0.5 mM and 150 mM, respectively. The solution was kept in a polyethylene bottle cooled in an icewater bath. The flow rate of the solution was l.OmL/min. These conditions were fixed according to our previous investigations on one-pump post column chemiluminescence detection method for PAH (Imaizumi el al., 1989a). The stock solutions of pyrene, AP, NP, NSP and DNPs were prepared by dissolving in benzene and stored in a refrigerator. The sample solutions were prepared by diluting
+
N~
gas
Preparation of practical samples. Sooty emissions (about 25 mg) obtained from car-exhaust pipes were extracted with 50 mL of benzene ethanol (3 : 1) under ultrasonication and filtered with a Millipore (Bedford, CA, USA) column guard FH-13. The extracts were evaporated to dryness and redissolved in 10mL of n-hexane. The solution was applied to a Waters (Bedford, CA, USA) Sep-Pak alumina cartridge. After washing with benzene, the analytes were eluted with 10 mL of chloroform. The eluate was evaporated, dissolved in acetonitrile, and the sample solution was injected into the present HPLC system.
+
RESULTS AND DISCUSSION Chemiluminescence generation with hydrogen peroxide and TCPO has been applied to a post column reaction system as a highly sensitive detection method for several fluorigenic compounds. The chemiluminescence intensity was strongly enhanced by the addition of such basic compounds as imidazole ( H o n d a et af., 1985; Imai et af., 1989; Imaizumi et af., 1989b; Hanaoka, 1989). We reported that the acetonitrile solution of TCPO and hydrogen peroxide was stable in a polyethylene or a borosilicate glass bottle cooled in a n ice-water bath (Imaizumi et aL, 1989a). In the determination of PAH's, we used the combination of acetonitrile imidazole buffer a n d acetonitrile solution of TCPO + hydrogen peroxide as mobile phase and chemilumigenic reagent solution, respectively. The same combination has been also used in the determination of dansyl derivatives of amphetamine related compounds (Hayakawa et al., 1989). Also, electrochemical reactions are enhanced by the addition of some electrolytes. In a n HPLC system with amperometric detector, perchlorate a n d acetate (Jin and Rappaport, 1983; Ang et al., 1987; MacCrehan et af., 1988) were popular as electrolytes added in the mobile phases. Buffers added in the mobile phase in the present HPLC system should have two important functions: catalyst for chemiluminescence generation a n d supporting electrolyte for electrochemical reduction, in view of the above reports. At first, we investigated the electrochemical reducing efficiencies of N P and NSP with several buffers to find the most effective mobile phase. Table 1 shows relative peak areas of AP, NP, a n d N S P by chemiluminescence detection after online electrochemical reduction. The largest peak areas were observed with imidazole perchloric acid in the three buffers for both analytes, N P and NSP. The interesting facts were that the relative peak area of AP, which is itself fluorescent, was reduced by the addition of acetate and that the relative values of both N P a n d NSP were smaller than that of AP with the buffers containing acetate. As the reason, inhibition of acetate on chemiluminescence generation and electrochemical reduction should be considered. The inhibition of acetate on chemiluminescence generation was explained by the comparison of the relative values of A P with the three buffers, since AP was not affected by
+
+
Eluent
CL reagent
I
Waste
Figure 1. Schematic diagram of the HPLC system for chemiluminescence detection after online electrochemical reduction. P1, pump for mobile phase; P2, pump for chemilumigenic reagent; I, injector; El,E2, working electrodes; G, guard column; C, separation column; M, mixing device; RC, reaction coil; FL, fluorescence detector; CL. chemiluminescence detector; R1 ,R2, integrators.
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stock solutions with acetonitrile adequately just before use and were kept in the dark during the experiments. These samples were used only for on-the-day analysis.
BIOMEDICAL CHROMATOGRAPHY, VOL. 4, NO. 3. 1990 109
N. IMAlZUMl E T A L .
Table 1. Effect of buffers on the chsmiluminescence intensities of AP, NP, and NSP after online electrochemical reduction' Bufferb
Relative peak areaC
Sodium acetate + acetic acid lmidazole + acetic acid lmidazole i perchloric acid a
AP
NP
NSP
0.09
0.03
0.04
0.73
N D~
0.58
1.oo
1.oo
1.oo
Applied potential - 1.6 V. pH adjusted to 4.75. By chemiluminescence detection. ND, not detected.
electrochemical reduction. The values decreased with increase in the concentration of acetate. To explain the other effect of acetate on electrochemical reduction of NP and NSP, relative peak areas by fluorescence detection at the wavelengths of 360 nm for excitation and 430nm for emission, which are the optimum wavelengths for AP, were compared in Table 2. Relative peak areas of both NP and NSP, which are not fluorescent, in the two buffers containing acetate, were smaller than those in imidazole + perchloric acid buffer. Based on these results, imidazole + perchloric acid was selected as the most suitable buffer system in the proposed method. Fluorescence (emission) spectra of electrochemically reduced products of NP and NSP showed the same spectrum of AP in imidazole+ perchloric acid bufferby the stopped flow method. This fact suggested strongly that the electrochemical reduction product of NP and NSP in the proposed system might be AP. The pH of imidazole + perchloric acid buffer affects both chemiluminescence generation and electrochemical reduction. The chemiluminescence intensity of AP with the TCPO+hydrogen peroxide system increased with the pH up to 7 as shown in Fig. 2. This was in accordance with the reports that the chemiluminescence intensities of several fluorophores with TCPO + hydrogen peroxide were highest at pH 6 - 7 (Honda el al., 1985; Imai et aL, 1989; Imaizumi et aL, 1989b; Hanoaka, 1989). But the maximum chemiluminescence intensities of both NP and NSP after
O Li
5
PH
6
7
Figure 2. Effect of pH on chemiluminescence intensity of AP. Amount of AP injected 0.2 prnol. Mobile phase 80% acetonitrile solution containing 2 mM imidazole adjusted at each pH with perchloric acid.
online electrochemical reduction were observed at pH 4.75. The reason was explained by the effect of pH on electrochemical reduction efficiencies of two analytes. The effects of pH on peak areas of both NP and NSP by fluorescence detection after online electrochemical reduction were shown in Fig. 3. The maximum values were obtained at pH4.75 for both NP and NSP. Although the chemiluminescence intensity of AP at pH 7 was stronger than at 4.75, the change of effluent pH was not easy in the present system, since an extra pump is necessary. From these results, the following mobile phase was used in the work: 80% acetonitrile solution containing 2 mM imidazole buffer with pH adjusted to 4.75 with perchloric acid. Figure 4 shows the hydrodynamic voltamograms of NP, NSP and 1,8-DNP plotted as relative peak areas by fluorescence detection. The peak of NSP was detected at - 0.3 V, and the area increased with decrease in potential. The area was constant at potentials below - 1.2 V. Both NP and I,8-DNP showed similar patterns without any plateau. The lower potential provided the more increased peak areas of the two compounds. However, too high a potential might result in impairment of the working electrodes, so that the applied potential for the reduction of NP, NSP and DNP's was fixed at - 1.6 V. Under the conditions described above, six analytes, AP, NP, NSP, 1,3-,1,6-, and 1,8-DNPs were chromatographed with both fluorescence and chemiluminescence detection after online electrochemical reduction. A typical chromatogram is shown in Fig. 5. Contrary to chemiluminescence detection, determination of NP, NSP and DNP's by electrochemical detection was
0
1.ot
Table 2. Effect of buffers on the electrochemical reducing efficiency" of NP and NSP Bufferb
Relative peak areac
+
Sodium acetate acetic acid lmidazole + acetic acid lrnidazole + perchloric acid
AP
NP
NSP
0.97
0.34
0.38
1.01
N D ~
0.37
1 .oo
1 .oo
1.oo 1
4 a
Applied potential - 1.6 V. pH adjusted to 4.75. By fluorescence detection. ND, not detected.
110 BIOMEDICAL CHROMATOGRAPHY, VOL. 4, NO. 3, 1990
5
6
7
PH Figure 3. Effect of pH on reducing efficiencies of NP and NSP: 0, NP (20 pmol); m. NSP (10 pmol). Detection by fluorescence after online electrochemical reduction at an applied potential of - 1.6V.
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NITRATED PYRENE DETERMINATION BY HPLC WITH CL DETECTION
Table 3. Detection limits for AP, NP, NSP, 1,3-, 1,6-, and I,8-DNP9s and pyrene by fluorescence and chemiluminescence detections with or without electrochemical reduction Detection limit (fmolr Fluorescenceb Chemiluminescence With Without With Without
Compound
a4
0.8
AP NP NSP 1.3-DNP 1.6 DNP 1.8-DNP
l.6
1.2
Applied potential ( - V )
Figure 4. Hydrodynamic voltamograms of pyrene derivatives: A, NSP (10 pmol); 0. NP (20 pmol); 0, 1.8-DNP (2 pmol). Mobile phase 80% acetonitrile solution containing 2 mM imidazole adjusted at each pH with perchloric acid. Detection by fluorescence after online electrochemical reduction at an applied potential of - 1.6 V.
difficult, because of the large base line noise using the buffer system proposed here. Their detection limits were summarized in Table 3. The detection limits by chemiluminescence detection for these compounds except pyrene were significantly lower than those by fluorescence detection. In particular, high sensitivities of chemiluminescence detection might be favorable in the determination of such D N P s as 1,6-and 1,8-DNP's. Since D N P s are very strong direct-acting mutagens in spite of their very low concentrations in the environment. Nevertheless, these values are not lower than those reported by Sigvardson and Birks (1984b) using chemiluminescence detection after online reduction with a Zn column. The significantly large value for NP compared with AP in Table 3 suggested that the reduction of NP to AP might not be complete. Peak areas of both NP and NSP by the present system were increased by decreasing the flow rate of the mobile phase. Calibration curves for these compounds were linear over three orders. The reproducibilities for AP, NP, and NSP by fluorescence and chemiluminescence detection are shown in
0
10
20
30
40
Pyrene
2 40 17 10 2 2 1000
20 NDC ND ND ND ND 150
20 380 170 500 140 140 1500
2 NDC ND ND ND ND 1000
Each value represented with signal to-noise ratio 3 by peak height method Excitation and emission wavelengths for pyrene were 332 nm and 392 nm, respectively Excitation and emission for other compounds were 360 nm and 430 nm, respectively N D , not detected a
Table 4. Accuracies for the determination of AP, N P and NSP by fluorescence and chemiluminescence detection after online electrochemical reduction Coefficient of variation (%)" Fluorescence Chemiluminescence Peak area Peak height Peak area Peak height
Compound
3.6 5.4 6.3
AP NP NSP a
3.3 11.4 3.1
3.9 6.7 6.1
4.4 55 10.0
Values based on ten iniections.
Table 4. These three analytes could be determined with coefficients of variation below 7% by the peak area method. To explain why the values of NP and NSP were larger than that of AP, the variations of reducing efficiencies of NP and NSP should be considered. The proposed method was applied to the determination of NP and DNP's in sooty emissions obtained from car-exhaust pipes as an example. A typical chromatogram is shown in Fig. 6 . With electrochemical
L with
I
without
Time ( m i n )
Figure 5. Typical chromatograms of pyrene derivatives with chemiluminescence(a) and fluorescence(b) detection after online electrochemical reduction. Peak assignment: 1, AP (1 pmol); 2, 1.8DNP (1 pmol); 3, 1.6-DNP (1 pmol); 4, 1.3-DNP (1 pmol); 5, NP (5 pmol); 6, NSP (5 prnol).
@ Heyden & Son Limited, 1990
0
20
40
60
Time(mln)
Figure 6. Chromatograms of car emission particles by chemiluminescence detection with or without online electrochemical reduction. Peak assignment: 1, 1,8-DNP; 2, 1,J-DNP (1 pmol); 3, NP.
BIOMEDICAL CHROMATOGRAPHY, VOL. 4,
NO. 3, 1990 111
N. I M A I Z U M I ET AL.
reduction, many peaks were detected by chemiluminescence detection, whereas any peaks except the solvent front were not observed without electrochemical reduction. Contents of NP, 1,3-, and 1,8-DNP's were estimated at about 50, 3 and 2.5 ng per g sooty emission, respectively. The important thing to be emphasized was that purification procedures used here were simpler than those usually used for GC/MS because of the higher selectivity of the present method. Simple purification is desirable to reduce the loss of analytes of low concentration and to prevent the chemical changes of unstable analytes. Not only nitrated pyrenes but also other reducible compounds can be detected by the proposed
method. It is known that nitrofluoranthenes, nitrobenzo[ alpyrenes and other nitrated PAH's are generally to be found in environmental samples. Moreover, the existence of several of these oxygenated PAH's in environmnental samples has also been reported ( Levsen, 1988). The identification of the other peaks observed in Fig. 6 is now under examination.
Acknowledgement The authors wish to thank Dr. Kazuhiro Imai of the University of Tokyo for his helpful advice.
REFERENCES Ang, K. P.. Tay, B. T. and Guwasingharn. H. (1987).lnt. J. Environ. Studies 29, 163. Arashidani, K.,Yoshikawa, M. and Kodarna, K. (1987). J. UOEH,9,19. Arey, J., Zielinska, B., Harger, W. P., Atkinson, R. and Winer, A. M. (1988).Mutation Res. 207,45. Davis, I. L., Raynor, M. K., Williams, R. T., Andrews, G. E. and Bartle, K. D. (1987).Anal. Chem. 59,2579. Hanaoka. N. (1989).Anal. Chem. 61,1300. Hayakawa, K., Hasegawa, K., Irnaizurni. N., Wong, 0. S. and Miyazaki, M. (1989).J. Chromatogr. 464,343. Hirose, M., Lee, M-S., Wang, C. Y. and King C. M. (1984).Cancer Res. 44,1 1 58. Honda, K., Miyaguchi. K. and Imai, K. (1985).Anal. Chim. Acta 177,
103. Howard, P. C., Flamrnang, T. J. and Beland, F. A. (1985).Carcinogenesis 6,243. Irnai. K. (1988).In Methods in Enzymology, ed by M. DeLuca and W. D. McElroy, Vol. 133,p. 435,Academic Press, New York. Irnai, K., Nishitani, A.. Tsukarnoto, Y. and Akirnoto, H. (1989). Xenobiotic Metabolism and Disposition, ed by R. Kato, R. W. Estabrook and M. N. Cayen, p. 325.Taylor and Francis, London. Irnaizumi, N., Hayakawa, K., Miyazaki, M. and Imai, K. (1989a). Analyst 114, 161. Irnaizumi, N., Hayakawa, K. and Miyazaki, M. (1989b).Eisei Kagaku,
35,4.
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Jin, 2. and Rappaport, S . M. (1983).Anal. Chem. 55, 1788. Kames, R. M.,Guo, Z., Fulcher, J. H. and Ball, D. A. (1988).Environ. Sci. Techno/. 22, 103. Levsen, K. (1988).Fresenius Z.Anal. Chem. 331,467. MacCrehan, W . A., May, W . E.. Yang, S. D. and Benner, B. A. Jr., (1988).Anal. Chem. 60,194. Paputa-peck, M. C., Marano. R. S., Schuetzle, D.. Rilly. T. L., Harnpton, C. V., Prater, T. J., Skewes, L. M., Jensen, T. E., Ruehle, P. H., Bosch, L. C. and Duncan, W. P. (1983).Anal. Chem. 55,1940. Rosenkranz, H. S., McCoy, E. C., Sanders, D. R., Butler, M., Kiriazides, D. K. and Mermelstein R. (1980).Science 209, 1039. Sellstrorn, U., Jansson, N., Bergrnan, A. and Alsberg, T. (1987). Chemosphere 16,945. Sigvardson, K. W. and Birks, J. W. (1983).Anal. Chem. 55,432. Sigvardson, K. W. and Birks, J. W. (1984a). Anal. Chem. 56, 1096. Sigvardson, K. W . and Birks, J. W. (1984b).J. Chromatogr. 316,507. Tanabe, K., Matsushita, H., Kuo, C-T. and Irnamiya, S. (1986).Taiki Osen Gakkaishi 21,535. Tejada, S. B., Zweidinger, R. B. and Sigsby, J. E. Jr. (1986).Anal. Chem. 58, 1827.
Received 30 July 1989,accepted 22 August 1989.
0 Heyden & Son Limited,
1990
A sensitive and selective high performance liquid chromatographic method for the simultaneous determination of nitrated polycyclic aromatic hydrocarbons and their reduced compounds has been developed. As the model compounds for the proposed method, pyrene, aminopyrene, nitrosopyrene, and several nitrated pyrenes were used. After separation on a reversed phase column, both nitropyrenes and nitrosopyrene which were not fluorescent, were electrochemically reduced to strongly fluorescent aminopyrenes, and detected chemilumigenically by using bis(2,4,6-trichloropheny1)oxalate and hydrogen peroxide as post column reagents. Their detection limits were all at the fmol levels, which were one or two orders lower than those by fluorescence detection. By the proposed method, nitropyrene, 1,3-, and l,8-dinitropyrenes were detected in sooty emissions of cars after simple purifications.
INTRODUCTION Polycyclic aromatic hydrocarbons (PAH) and nitrated polycyclic aromatic hydrocarbons( N PAH) have attracted much attention because of their potent mutagenicity (Rosenkranz et al., 1980; Arey et al., 1988) and carcinogenicity (Hirose et al., 1984; Howard et al., i965). They are formed as undesirable by-products through burning processes of various materials, such as petroleum, and are found in various environmental samples (Davis et al., 1987; Kames et al., 1988). PAH has been determined mainly by gas chromatography ( G C ) with flame ionization and mass spectrometric (MS) detectors, and NPAH has been determined by GC with MS, electron capture, nitrogen-phosphorus and chemiluminescence detectors. MS and nitrogenphosphorus detection in particular were used in sensitive determination methods for environmental samples. However, these methods required very complex purification of samples to remove interfering compounds (Sellstrom et al., 1987) and concentration of trace levels of PAH and NPAH. On the other hand, high performance liquid chromatographic (HPLC) methods with UV absorbance, fluorescence (Tanabe et aZ., 1986; Tejada et al., 1986; Arashidani et aZ., 1987) and amperometric (MacCrehan et al., 1988) detectors have been reported. In these methods, fluorescence detection has been used for sensitive determination of several fluorescent PAH’s and amino-PAH’s since it was so selective that purification procedures could be simpler than those for GC. Recently, chemiluminescence detection has been developed as a highly sensitive detection method for HPLC. Such aryl oxalates as bis(2,4,6-trichloro-
pheny1)oxalate (TCPO) and hydrogen peroxide have been mainly used as post column chemilumigenic reagents (Imai, 1988; Sigvardson and Birks, 1983, 1984a,b; Hayakawa et al., 1989). The sensitivities of some fluorescent compounds by chemiluminescence detection increased 10-100 times higher than those given by fluorescence detection (Sigvardson and Birks, 1984b; Hayakawa et al., 1989). The method has been already applied to the determination of fluorescent PAH’s (Sigvardson and Birks, 1983; Imaizumi et al., 1989b) and amino-PAH’s (Sigvardson and Birks, 1984a, b). For the determination of non-fluorescent NPAH’s, several reducing columns packed with zinc powder (Sigvardson and Birks, 1984b) or Pt/Rh-coated alumina (Tejada et al., 1986) have been developed to reduce NPAH to strongly fluorescent amino-PAH. But they have not been favorable for a routine analysis of NPAH because of their short life-time. In this paper, we have established a new HPLC determination method for NPAH using chemiluminescence detection after online electrochemical reduction. Online reduction has such an advantage that it does not require any particular reagents which might affect chromatographic separation and chemiluminescence detection of NPAH. We used 1-nitropyrene(NP), l-nitrosopyrene(NSP), 1-aminopyrene(AP), 1,3-, 1,6-, and 1,8dinitropyrenes( D N P s ) and pyrene as analytes in this work, because NP, DNP’s and pyrene are found in many environmental samples and NSP and AP were the feasible metabolites of NP in biological systems. By the proposed method, simultaneous and sensitive determination of PAH’s and NPAH’s was made possible.
EXPERIMENTAL Current Address: Niigata College of Pharmacy, Kamishin’ eicho 5-13-2, Niigata, 950-21, Japan. * Author to whom correspondence should be addressed.
Chemicals. TCPO of analytical reagent grade (Tokyo Kasei, Tokyo, Japan) and hydrogen peroxide of electrochemical
CCC-O269-3879/90/OlOS-O112 $2.50 108 BIOMEDICAL CHROMATOGRAPHY, VOL. 4, NO. 3, 1990
@ Heyden & Son Limited, 1990
NITRATED PYRENE DETERMINATION BY HPLC WITH CL DETECTION
grade (Kanto Chemicals, Tokyo, Japan) were used as received. NP was synthesized by the method of Paputa-peck et al. (1983) using pyrene (chemical grade, Tokyo Kasei) and fuming nitric acid (Katayama Chemical, Osaka, Japan). NSP was synthesized by the method of Howard et a/. (1985) using AP (Aldrich, Milwaukee, WI, USA) and m-chloroperoxybenzoic acid (Wako Pure Chemical, Osaka, Japan). 1,3-, 1,6-, and 1,sDNPs were purchased from Aldrich. All other reagents used were analytical reagent grade and were commercially purchased. Apparatus. The HPLC system consisted of two Shimadzu (Kyoto, Japan) LC-6A pumps, a Rheodyne (Cotati, CA, USA) model 7125 loop injector (loop volume of 20pL), an Atto (Tokyo, Japan) AC-2220 chemiluminescence detector (spinal type cell of 60 pL), a Jasco (Hachioji, Tokyo, Japan) 820-FP fluorescence detector, an ESA (Bedford, CA, USA) Coulochem model 5100A electrochemical detector with two glassy-carbon working electrodes, a Kyowa (Mitaka, Tokyo, Japan) KZS-1 mixing device, two Shimadzu C-R1B integrators and a 0.5 mm ID x 300 mm stainless steel tubing as a reaction coil. The separation and guard columns were Nagel (Duran, West Germany) Nucleosil ODS (4.6 mm ID x 250 mm) and a Brownlee (Berkley, CA, USA) RP-18 (4.6 mm ID x 30 mm), respectively. Figure 1 shows a schematic diagram of the present system. Other details of the system were the same as those reported in our previous work (Imaizumi et a/., 1989a) except assembly for electrochemical reduction. Preparation of solutions. The mixture of acetonitrile and aqueous buffers was used as a mobile phase at a flow rate of 1.0 mL/min. The mobile phase was continuously purged with nitrogen gas (purity was over 99.99%) to remove oxygen which disturbed the electrochemical reduction of nitropyrenes remarkably. The acetronitrile content in the mobile phase was SO%, except in the case for the simultaneous determination of AP, NP, NSP, D N P s and pyrene, it was 65%. As aqueous buffers, imidazole(2 mM) perchloric acid, imidazole(2 mM) + acetic acid and sodium acetate+ acetic acid (2 mM as the total acetate concentration) were tested. The chemilumigenic reagent solution was prepared by the addition of hydrogen peroxide to acetonitrile solution of TCPO just before use. The final concentrations of TCPO and hydrogen peroxide were 0.5 mM and 150 mM, respectively. The solution was kept in a polyethylene bottle cooled in an icewater bath. The flow rate of the solution was l.OmL/min. These conditions were fixed according to our previous investigations on one-pump post column chemiluminescence detection method for PAH (Imaizumi el al., 1989a). The stock solutions of pyrene, AP, NP, NSP and DNPs were prepared by dissolving in benzene and stored in a refrigerator. The sample solutions were prepared by diluting
+
N~
gas
Preparation of practical samples. Sooty emissions (about 25 mg) obtained from car-exhaust pipes were extracted with 50 mL of benzene ethanol (3 : 1) under ultrasonication and filtered with a Millipore (Bedford, CA, USA) column guard FH-13. The extracts were evaporated to dryness and redissolved in 10mL of n-hexane. The solution was applied to a Waters (Bedford, CA, USA) Sep-Pak alumina cartridge. After washing with benzene, the analytes were eluted with 10 mL of chloroform. The eluate was evaporated, dissolved in acetonitrile, and the sample solution was injected into the present HPLC system.
+
RESULTS AND DISCUSSION Chemiluminescence generation with hydrogen peroxide and TCPO has been applied to a post column reaction system as a highly sensitive detection method for several fluorigenic compounds. The chemiluminescence intensity was strongly enhanced by the addition of such basic compounds as imidazole ( H o n d a et af., 1985; Imai et af., 1989; Imaizumi et af., 1989b; Hanaoka, 1989). We reported that the acetonitrile solution of TCPO and hydrogen peroxide was stable in a polyethylene or a borosilicate glass bottle cooled in a n ice-water bath (Imaizumi et aL, 1989a). In the determination of PAH's, we used the combination of acetonitrile imidazole buffer a n d acetonitrile solution of TCPO + hydrogen peroxide as mobile phase and chemilumigenic reagent solution, respectively. The same combination has been also used in the determination of dansyl derivatives of amphetamine related compounds (Hayakawa et al., 1989). Also, electrochemical reactions are enhanced by the addition of some electrolytes. In a n HPLC system with amperometric detector, perchlorate a n d acetate (Jin and Rappaport, 1983; Ang et al., 1987; MacCrehan et af., 1988) were popular as electrolytes added in the mobile phases. Buffers added in the mobile phase in the present HPLC system should have two important functions: catalyst for chemiluminescence generation a n d supporting electrolyte for electrochemical reduction, in view of the above reports. At first, we investigated the electrochemical reducing efficiencies of N P and NSP with several buffers to find the most effective mobile phase. Table 1 shows relative peak areas of AP, NP, a n d N S P by chemiluminescence detection after online electrochemical reduction. The largest peak areas were observed with imidazole perchloric acid in the three buffers for both analytes, N P and NSP. The interesting facts were that the relative peak area of AP, which is itself fluorescent, was reduced by the addition of acetate and that the relative values of both N P a n d NSP were smaller than that of AP with the buffers containing acetate. As the reason, inhibition of acetate on chemiluminescence generation and electrochemical reduction should be considered. The inhibition of acetate on chemiluminescence generation was explained by the comparison of the relative values of A P with the three buffers, since AP was not affected by
+
+
Eluent
CL reagent
I
Waste
Figure 1. Schematic diagram of the HPLC system for chemiluminescence detection after online electrochemical reduction. P1, pump for mobile phase; P2, pump for chemilumigenic reagent; I, injector; El,E2, working electrodes; G, guard column; C, separation column; M, mixing device; RC, reaction coil; FL, fluorescence detector; CL. chemiluminescence detector; R1 ,R2, integrators.
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stock solutions with acetonitrile adequately just before use and were kept in the dark during the experiments. These samples were used only for on-the-day analysis.
BIOMEDICAL CHROMATOGRAPHY, VOL. 4, NO. 3. 1990 109
N. IMAlZUMl E T A L .
Table 1. Effect of buffers on the chsmiluminescence intensities of AP, NP, and NSP after online electrochemical reduction' Bufferb
Relative peak areaC
Sodium acetate + acetic acid lmidazole + acetic acid lmidazole i perchloric acid a
AP
NP
NSP
0.09
0.03
0.04
0.73
N D~
0.58
1.oo
1.oo
1.oo
Applied potential - 1.6 V. pH adjusted to 4.75. By chemiluminescence detection. ND, not detected.
electrochemical reduction. The values decreased with increase in the concentration of acetate. To explain the other effect of acetate on electrochemical reduction of NP and NSP, relative peak areas by fluorescence detection at the wavelengths of 360 nm for excitation and 430nm for emission, which are the optimum wavelengths for AP, were compared in Table 2. Relative peak areas of both NP and NSP, which are not fluorescent, in the two buffers containing acetate, were smaller than those in imidazole + perchloric acid buffer. Based on these results, imidazole + perchloric acid was selected as the most suitable buffer system in the proposed method. Fluorescence (emission) spectra of electrochemically reduced products of NP and NSP showed the same spectrum of AP in imidazole+ perchloric acid bufferby the stopped flow method. This fact suggested strongly that the electrochemical reduction product of NP and NSP in the proposed system might be AP. The pH of imidazole + perchloric acid buffer affects both chemiluminescence generation and electrochemical reduction. The chemiluminescence intensity of AP with the TCPO+hydrogen peroxide system increased with the pH up to 7 as shown in Fig. 2. This was in accordance with the reports that the chemiluminescence intensities of several fluorophores with TCPO + hydrogen peroxide were highest at pH 6 - 7 (Honda el al., 1985; Imai et aL, 1989; Imaizumi et aL, 1989b; Hanoaka, 1989). But the maximum chemiluminescence intensities of both NP and NSP after
O Li
5
PH
6
7
Figure 2. Effect of pH on chemiluminescence intensity of AP. Amount of AP injected 0.2 prnol. Mobile phase 80% acetonitrile solution containing 2 mM imidazole adjusted at each pH with perchloric acid.
online electrochemical reduction were observed at pH 4.75. The reason was explained by the effect of pH on electrochemical reduction efficiencies of two analytes. The effects of pH on peak areas of both NP and NSP by fluorescence detection after online electrochemical reduction were shown in Fig. 3. The maximum values were obtained at pH4.75 for both NP and NSP. Although the chemiluminescence intensity of AP at pH 7 was stronger than at 4.75, the change of effluent pH was not easy in the present system, since an extra pump is necessary. From these results, the following mobile phase was used in the work: 80% acetonitrile solution containing 2 mM imidazole buffer with pH adjusted to 4.75 with perchloric acid. Figure 4 shows the hydrodynamic voltamograms of NP, NSP and 1,8-DNP plotted as relative peak areas by fluorescence detection. The peak of NSP was detected at - 0.3 V, and the area increased with decrease in potential. The area was constant at potentials below - 1.2 V. Both NP and I,8-DNP showed similar patterns without any plateau. The lower potential provided the more increased peak areas of the two compounds. However, too high a potential might result in impairment of the working electrodes, so that the applied potential for the reduction of NP, NSP and DNP's was fixed at - 1.6 V. Under the conditions described above, six analytes, AP, NP, NSP, 1,3-,1,6-, and 1,8-DNPs were chromatographed with both fluorescence and chemiluminescence detection after online electrochemical reduction. A typical chromatogram is shown in Fig. 5. Contrary to chemiluminescence detection, determination of NP, NSP and DNP's by electrochemical detection was
0
1.ot
Table 2. Effect of buffers on the electrochemical reducing efficiency" of NP and NSP Bufferb
Relative peak areac
+
Sodium acetate acetic acid lmidazole + acetic acid lrnidazole + perchloric acid
AP
NP
NSP
0.97
0.34
0.38
1.01
N D ~
0.37
1 .oo
1 .oo
1.oo 1
4 a
Applied potential - 1.6 V. pH adjusted to 4.75. By fluorescence detection. ND, not detected.
110 BIOMEDICAL CHROMATOGRAPHY, VOL. 4, NO. 3, 1990
5
6
7
PH Figure 3. Effect of pH on reducing efficiencies of NP and NSP: 0, NP (20 pmol); m. NSP (10 pmol). Detection by fluorescence after online electrochemical reduction at an applied potential of - 1.6V.
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NITRATED PYRENE DETERMINATION BY HPLC WITH CL DETECTION
Table 3. Detection limits for AP, NP, NSP, 1,3-, 1,6-, and I,8-DNP9s and pyrene by fluorescence and chemiluminescence detections with or without electrochemical reduction Detection limit (fmolr Fluorescenceb Chemiluminescence With Without With Without
Compound
a4
0.8
AP NP NSP 1.3-DNP 1.6 DNP 1.8-DNP
l.6
1.2
Applied potential ( - V )
Figure 4. Hydrodynamic voltamograms of pyrene derivatives: A, NSP (10 pmol); 0. NP (20 pmol); 0, 1.8-DNP (2 pmol). Mobile phase 80% acetonitrile solution containing 2 mM imidazole adjusted at each pH with perchloric acid. Detection by fluorescence after online electrochemical reduction at an applied potential of - 1.6 V.
difficult, because of the large base line noise using the buffer system proposed here. Their detection limits were summarized in Table 3. The detection limits by chemiluminescence detection for these compounds except pyrene were significantly lower than those by fluorescence detection. In particular, high sensitivities of chemiluminescence detection might be favorable in the determination of such D N P s as 1,6-and 1,8-DNP's. Since D N P s are very strong direct-acting mutagens in spite of their very low concentrations in the environment. Nevertheless, these values are not lower than those reported by Sigvardson and Birks (1984b) using chemiluminescence detection after online reduction with a Zn column. The significantly large value for NP compared with AP in Table 3 suggested that the reduction of NP to AP might not be complete. Peak areas of both NP and NSP by the present system were increased by decreasing the flow rate of the mobile phase. Calibration curves for these compounds were linear over three orders. The reproducibilities for AP, NP, and NSP by fluorescence and chemiluminescence detection are shown in
0
10
20
30
40
Pyrene
2 40 17 10 2 2 1000
20 NDC ND ND ND ND 150
20 380 170 500 140 140 1500
2 NDC ND ND ND ND 1000
Each value represented with signal to-noise ratio 3 by peak height method Excitation and emission wavelengths for pyrene were 332 nm and 392 nm, respectively Excitation and emission for other compounds were 360 nm and 430 nm, respectively N D , not detected a
Table 4. Accuracies for the determination of AP, N P and NSP by fluorescence and chemiluminescence detection after online electrochemical reduction Coefficient of variation (%)" Fluorescence Chemiluminescence Peak area Peak height Peak area Peak height
Compound
3.6 5.4 6.3
AP NP NSP a
3.3 11.4 3.1
3.9 6.7 6.1
4.4 55 10.0
Values based on ten iniections.
Table 4. These three analytes could be determined with coefficients of variation below 7% by the peak area method. To explain why the values of NP and NSP were larger than that of AP, the variations of reducing efficiencies of NP and NSP should be considered. The proposed method was applied to the determination of NP and DNP's in sooty emissions obtained from car-exhaust pipes as an example. A typical chromatogram is shown in Fig. 6 . With electrochemical
L with
I
without
Time ( m i n )
Figure 5. Typical chromatograms of pyrene derivatives with chemiluminescence(a) and fluorescence(b) detection after online electrochemical reduction. Peak assignment: 1, AP (1 pmol); 2, 1.8DNP (1 pmol); 3, 1.6-DNP (1 pmol); 4, 1.3-DNP (1 pmol); 5, NP (5 pmol); 6, NSP (5 prnol).
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0
20
40
60
Time(mln)
Figure 6. Chromatograms of car emission particles by chemiluminescence detection with or without online electrochemical reduction. Peak assignment: 1, 1,8-DNP; 2, 1,J-DNP (1 pmol); 3, NP.
BIOMEDICAL CHROMATOGRAPHY, VOL. 4,
NO. 3, 1990 111
N. I M A I Z U M I ET AL.
reduction, many peaks were detected by chemiluminescence detection, whereas any peaks except the solvent front were not observed without electrochemical reduction. Contents of NP, 1,3-, and 1,8-DNP's were estimated at about 50, 3 and 2.5 ng per g sooty emission, respectively. The important thing to be emphasized was that purification procedures used here were simpler than those usually used for GC/MS because of the higher selectivity of the present method. Simple purification is desirable to reduce the loss of analytes of low concentration and to prevent the chemical changes of unstable analytes. Not only nitrated pyrenes but also other reducible compounds can be detected by the proposed
method. It is known that nitrofluoranthenes, nitrobenzo[ alpyrenes and other nitrated PAH's are generally to be found in environmental samples. Moreover, the existence of several of these oxygenated PAH's in environmnental samples has also been reported ( Levsen, 1988). The identification of the other peaks observed in Fig. 6 is now under examination.
Acknowledgement The authors wish to thank Dr. Kazuhiro Imai of the University of Tokyo for his helpful advice.
REFERENCES Ang, K. P.. Tay, B. T. and Guwasingharn. H. (1987).lnt. J. Environ. Studies 29, 163. Arashidani, K.,Yoshikawa, M. and Kodarna, K. (1987). J. UOEH,9,19. Arey, J., Zielinska, B., Harger, W. P., Atkinson, R. and Winer, A. M. (1988).Mutation Res. 207,45. Davis, I. L., Raynor, M. K., Williams, R. T., Andrews, G. E. and Bartle, K. D. (1987).Anal. Chem. 59,2579. Hanaoka. N. (1989).Anal. Chem. 61,1300. Hayakawa, K., Hasegawa, K., Irnaizurni. N., Wong, 0. S. and Miyazaki, M. (1989).J. Chromatogr. 464,343. Hirose, M., Lee, M-S., Wang, C. Y. and King C. M. (1984).Cancer Res. 44,1 1 58. Honda, K., Miyaguchi. K. and Imai, K. (1985).Anal. Chim. Acta 177,
103. Howard, P. C., Flamrnang, T. J. and Beland, F. A. (1985).Carcinogenesis 6,243. Irnai. K. (1988).In Methods in Enzymology, ed by M. DeLuca and W. D. McElroy, Vol. 133,p. 435,Academic Press, New York. Irnai, K., Nishitani, A.. Tsukarnoto, Y. and Akirnoto, H. (1989). Xenobiotic Metabolism and Disposition, ed by R. Kato, R. W. Estabrook and M. N. Cayen, p. 325.Taylor and Francis, London. Irnaizumi, N., Hayakawa, K., Miyazaki, M. and Imai, K. (1989a). Analyst 114, 161. Irnaizumi, N., Hayakawa, K. and Miyazaki, M. (1989b).Eisei Kagaku,
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Jin, 2. and Rappaport, S . M. (1983).Anal. Chem. 55, 1788. Kames, R. M.,Guo, Z., Fulcher, J. H. and Ball, D. A. (1988).Environ. Sci. Techno/. 22, 103. Levsen, K. (1988).Fresenius Z.Anal. Chem. 331,467. MacCrehan, W . A., May, W . E.. Yang, S. D. and Benner, B. A. Jr., (1988).Anal. Chem. 60,194. Paputa-peck, M. C., Marano. R. S., Schuetzle, D.. Rilly. T. L., Harnpton, C. V., Prater, T. J., Skewes, L. M., Jensen, T. E., Ruehle, P. H., Bosch, L. C. and Duncan, W. P. (1983).Anal. Chem. 55,1940. Rosenkranz, H. S., McCoy, E. C., Sanders, D. R., Butler, M., Kiriazides, D. K. and Mermelstein R. (1980).Science 209, 1039. Sellstrorn, U., Jansson, N., Bergrnan, A. and Alsberg, T. (1987). Chemosphere 16,945. Sigvardson, K. W. and Birks, J. W. (1983).Anal. Chem. 55,432. Sigvardson, K. W. and Birks, J. W. (1984a). Anal. Chem. 56, 1096. Sigvardson, K. W . and Birks, J. W. (1984b).J. Chromatogr. 316,507. Tanabe, K., Matsushita, H., Kuo, C-T. and Irnamiya, S. (1986).Taiki Osen Gakkaishi 21,535. Tejada, S. B., Zweidinger, R. B. and Sigsby, J. E. Jr. (1986).Anal. Chem. 58, 1827.
Received 30 July 1989,accepted 22 August 1989.
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1990
