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Combined Liquid Chromatography/Electron Paramagnetic Resonance Spectrometry/Electrospray Ionization Mass Spectrometry for Radical Identification Hideo Iwahashi2 Carol E. Parker, Ronald P. Mason, and Kenneth B. Tomer' Laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, North Carolina 27709
Electron paramagnetic resonance (EPR) spectrometry and mass spectrometry (MS) have been coupled together on-line wlth llquld chromatography (LC)/UV detectlon. These combined techniques have been applled to the determlnatlon of spln-trapped radlcal adducts, Including phenyl, 2-, 3-, and 4chlorophenyl, and 2-bromophenyl radlcals trapped wlth CY( loxo-4-pyrldyl)-Ktert-butylnltrone (4-POBN), and phenyl radicals trapped wlth 2-methyl-2-nltrosopropane (MNP), CYphenyl-Kterf-butylnltrone (PBN), and 5,5-dlmethyCl-pyrroline Koxlde (DMPO). Oxldlzed and reduced forms of the radlcal adducts were also detected by the on-llne LC/EPR/ MS system.
INTRODUCTION The possible role of free radicals in cancer, inflammation, and tissue injury has attracted considerableinterest. In order to detect short-lived radicals, the spin-trapping technique was introducedl-3 and has allowed the detection of many free radicals in biological system^.^ With the spin trapping technique, determination of the structures of radical adducts has been based mainly on the hyperfine splitting patterns of the EPR spectra. Hyperfine coupling constants can give detailed information about the radical center but give no molecular weight information. In order to obtain comprehensive knowledge about the structures of the radical adducts, additional information, such as that obtained by mass spectrometry (MS), is necessary. Direct probe MSS9 and GC/MS1+l* have been used to identify the molecular struc+ Permanent address: Department of Chemistry, Wakayama Medical College, 640 Wakayama, Japan. (1) Iwamura, M.; Inamoto, N. Bull. Chem. SOC.Jpn. 1967, 40, 703. Jpn. 1970,43,860-863. (2) Iwamura, M.; Inamoto, N. Bull. Chem. SOC. 1968,90,5909(3) Janzen, E. G.; Blackburn, B. J. J.Am. Chem. SOC.
tures of radical adducts, usually after derivatization. In many cases, however, the radical adducts are only stable in solution and cannot be isolated. For the past few years, we have been developing a technique for the identification of radical adducts in which highperformance liquid chromatography (HPLCVEPR and LC/ thermospray (TSP)/MSor LC/electrospray Ionization (ESI)/ MS are performed under identical HPLC The HPLC/EPR system has an EPR spectrometer as a detector and has allowed us to detect EPR active peaks in the HPLC elution profile.22-26LC/TSP/MSl9vZ0 and LC/ESI/MS,2lboth of which utilize mild ionization techniques, have allowed us to obtain parent ions for these radical species. Identification of radical adducts is thus accomplished by a combination of HPLC/EPR and LC/TSP/MS or LC/ESVMS. Recently, JanZen et al. have also reported the mass spectra of radical adducts using LC/TSP/MS.26 A major difficulty in these experiments is the necessity of identical HPLC conditions in both the LC/EPR and the LC/MS experiments. In this paper we describe the coupling of an HPLC, EPR, and MS where separation,radical adduct detection, and mass spectrometric determination occur on-line. The utility of this instrumentation is illustrated by its application to the determination of the structures of the radical adducta of the spin trapping reagents (~-(1-0~0-4-pyridyl)-N-tert-butylnitrone (CPOBN), a-phenyl-N-tert-butylnitrone(PBN), 2methyl-2-nitrosopropane (MNP), and 5,5-dimethyl-l-pyrroline N-oxide (DMPO) with phenyl and substituted-phenyl radicals. The phenyl and substituted-phenyl radicals are produced by the metal-catalyzed one-electron oxidation of phenyl and substituted-phenyl hydrazines (eqs 1 and Q2' which are then trapped by a spin trapping reagent, e.g. 4POBN, (eq 3). The structures of these radicals are difficult to determine based on their EPR spectra alone because, in general, the spin-trapped radicals of 4-POBN and PBN show
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(4) Mottley, C.; Mason, R. P. Biol. Magn. Reson. 1989,8, 489-546. (5) Hill, H. A. 0.; Thornalley, P. J. FEBS Lett. 1981,125, 235-238. (6) Sinha,B. K.; Motten, A. G. Biochem.Biophys.Res. Commun. 1982, 105, 1044-1051. (7) Ortiz de Montellano, P. R.; Augusto, 0.;Viola, F.; Kunze, K. L. J. Biol. Chem. 1983,258,8623-8628.
(8) Noda, A.; Noda, H.; Ohno, K.; Sendo, T.; Misaka, A.; Kanazawa,
Y.; Isobe, R.; Hirata, M. Biochem. Biophys. Res. Commun. 1985, 133, 1086-1091. (9) Iwahashi, H.; Albro, P. W.; McGown, S. R.; Tomer, K. B.; Mason, R. P. Arch. Biochem. Biophys. 1991,285, 172-180. (10) Suezawa, H.; Abe, K.; Hirota, M.; Ishii, T. Chem. Lett. 1981,10491052. (11) Watanabe, T.; Yoshida, M.; Fujiwara, S.; Abe, K.; Onoe, A.; Hirota, M.; Igarashi, S. Anal. Chem. 1982,54, 2470-2474. (12) Abe, K.; Suezawa, H.; Hirota, M. J.Chem. SOC.,Perkin Trans. 2 1984,29-34. (13) Mikami, N.; Takahashi, N.; Yamada, H.; Miyamoto, J. Pestic. Sci. 1985, 16, 101-112. (14) Janzen, E. G.; Weber, J. R.; Haire, D. L.; Fung, D. M. Anal. Lett. 1985,18, 1749-1757. (15) Janzen,E. G.;Krygsman,P. H.;Towner,R. A.; Haire, D. L.Biomed. Enuiron. Mass Spectrom. 1988, 15, 111-116. This artlcle not subject to U.S. Copyright.
(16) Krygsman, P. H.; Janzen, E. G.; Towner, R. A.; Haire, D. L. A n d . Lett. 1989,22, 1009-1020. (17) Janzen, E. G.; Towner, R. A.; Krygsman, P. H.; Haire, D. L. Free Radical Res. Commun. 1990,9, 343-351. (18) Janzen, E. G.; Towner, R. A.; Krygsman, P. H.;Free Radical Res. .-
Commun. 1990,9, 353-360. (19) Iwahashi, H.; Parker, C. E.; Mason, R. P.; Tomer, K. B. Rapid Commun. Mass Spectrom. 1990,4, 352-354. (20) Iwahashi, H.; Parker, C. E.; Mason, R. P.; Tomer, K. B. Biochem. J. 1991, 276, 447-453. (21) Parker, C. E.: Iwahashi, H.; Tomer, K. B. J.Am. SOC. Mass Spectrom. 1991,2, 413-418. (22) Makino, K.; Hatano, H. Chem. Lett. 1979, 119-122. (23) Makino, K.; Moriyama, F.; Hatano, H. J.Chromatogr. 1985,332, 71-106. (24) Iwahashi, H.; Ikeda, A.; Negoro, Y.; Kido, R. Biochem. J. 1986, 236, 509-514. (25) Sugata, R.; Iwahashi, H.; Ishii, T.; Ryo, K. J. Chromatogr. 1989, 487.9-16. (26) Janzen, E. G.; Lindsay, D. A,; Haire, D. L. J.Am. Chem. SOC.1990, 112,8279-8284. (27) Kalyanaraman, B.; Sinha, B. K. Enuiron. Health Perspect. 1985, 64, 179-184.
Published 1992 by the American Chemlcal Society
ANALYTICAL CHEMISTRY, VOL. 64, NO. 19. OCTOBER 1, 1992
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intermediates during the oxidation of hydrazineen A 0.1-mL aliquot of the reaction mixture was applied directly to the HPLC/ EPR. Reaction of Phenylhydrazinewith PBN. The reaction mixture contained 0.1 M PBN, 14 mg of phenylhydrazine, and 0.5 mM CuC12 in 3.2 mL of 50 mM carbonate buffer (pH 10.0). The reaction mixture without CuCl2 was bubbled with nitrogen gas for 5 min, and then the reaction was started by adding CuC12. The reaction was allowed to proceed for 1h in the absence of light, at which time 6.4 mL of 0.2 M boric acid buffer was added to quench the reaction. The reaction mixture was applied to a Sep-pak Cl8cartridge, washed with 3 mL of water, and was elutad with1 mL of methanol. A 0.05-mL aliquot w& applied to the LC/EPR/MS. cn3 Reaction of Phenylhydrazine with MNP. The reaction mixture contained 0.025 M MNP, 20 mg of phenylhydrazine, and 0.5 mM CuCl2 in 17 mL of 50 mM carbonate buffer (pH 4-POBN PHENYL 4-POBN RADICAL ADDUCl 10.0). The reaction mixture without CuCl2 was bubbled with nitrogen gas for 5 min, and then the reaction was started by only six-line signals typical of carbon-centered radicals, with adding CuCla. The reaction was allowed to proceed for 1h in the only small differences in hyperfine c o ~ p l i n g .On-line ~ couabsence of light, at which time 34 mL of 0.2 M boric acid buffer pling of these three detectors allows the correlation of the was added to quench the reaction. The reaction mixture was UV, LC/EPR, and LC/MS chromatograms, and slight shifts applied to a Sep-pak (218, washed with 3 mL of water, and eluted in elution profiles have allowed the differentiation of overwith 1 mL of methanol. A 0.1-mL aliquot was applied to the lapping species. Our results using LC/EPR/MS are compared LC/EPR/MS. with G U M S for the same radical adducts.lOJ1 Reaction of Phenylhydrazine with DMPO. The reaction mixture contained 0.1 M DMPO, 20 mg of phenylhydrazine, and EXPERIMENTAL SECTION 0.5 mM CuClz in 5 mL of 50 mM carbonate buffer (pH 10.0). The reaction was started by adding CuCla. After a 5-min reaction, Chemicals. 2-Methyl-2-nitrosopropane (MNP), (2-chloro10mLof 0.2 M boricacid buffer was addedtoquench the reaction. pheny1)hydrazine hydrochloride, (3-chloropheny1)hydrazinehyThe reaction mixture was applied to Sep-pak CIS,washed with drochloride, (4-chloropheny1)hydrazinehydrochloride, and (23 mL of water, and eluted with 1mL of methanol. A 0.25-mL bromopheny1)hydrazine hydrochloride were purchased from aliquot was applied to the LC/EPR/MS. Aldrich (Milwaukee, WI). 5,5-Dimethyl-l-pyrrolineN-oxide Instrumentation. HPLC. The HPLC used for the on-line (CPOBN),a(DMPO),a-(l-oxo-4-pyridyl)-N-tert-butylnitrone phenyl-N-tert-butylnitrone(PBN), 2,2,6,6-tetramethyl-l-piper- HPLC/UV/EPR/ESI/MS system consisted of a Waters injector, idine-N-oxy1(TEMPO), 4-hydroxy-2,2,6,6-tetramethyl-l-piper- two Waters 600A pumps (Waters Associates, Milford, MA), a Waters 660 solvent programmer, and a Waters 440 UV detector. idine-N-oxy1 (4-hydroxy-TEMPO), and phenylhydrazine hyThe UV detector was operated at 254 nm, at a sensitivity setting drochloride were obtained from Sigma Chemical Co. (St. Louis, of 2.0 absorbance units full scale. The flow rate was 1mL/min. MO). The DMPO was further purified by passage through a HPLC column conditions were as follows: 1.0 mL/min; gradient charcoal column.28 All other chemicals used were commercial elution [solventA, 10mM ammonium acetate; solvent B, 10mM producta of the highest grade available. ammonium acetate, 80% acetonitrile (v/v)l programmed from Reaction Conditions. Reactions of Phenylhydrazine and 0 to 45% B in 40 rnin for the CPOBN experiment, from 20 to Its Derivatives with 4-POBN. The reaction mixture contained 75% B in 40 min for the PBN and MNP experimente, from 0 to 16 mg of 4-POBN, 7 mg of phenylhydrazine, and 0.5 mM CuCl2 75% B in 40 min for the DMPO experiment, and from 20 to 70 % in 1.6 mL of 50 mM carbonate buffer (pH 10.0). The reaction B in 2Omin for the TEMPO and 4-hydroxy-TEMPOexperiments. mixture without CuCl2 was bubbled with nitrogen gas for 5 min, The column used was a MBondapak C18 [30-cm (L) X 4.6-mm at which time the reactions were started by adding CuClz. The (i.d.)] HPLC column, and the Sep-pak CIScartridges were from reaction was allowed to proceed for 2 h in the absence of light, Waters Associates (Milford, MA). at which time 3.2 mL of 0.2 M boric acid buffer was added to quench the reaction. The reaction mixtures were applied to SepEPR Spectrometer. The EPR spectrometer used in these pak Cl8 cartridges, washed with 3 mL of water, and eluted with experiments was a Varian E-104 EPR (Varian Associates, Palo 2 mL of methanol. A 0.1-mL aliquot of the reaction mixture was Alto, CA) spectrometer of 100-kHz modulation frequency. To appliedto the LC/EPR/MS. To make the concentrated samples, obtain off-line EPR spectra, samples were aspirated into the 3.2 mL of reaction mixture was used and the sample was eluted EPR flow cell which was centered in a microwave cavity. The with 1mL of methanol from a Waters Sep-pak Cl8 (Waters AsEPR spectrometer settings were microwave power, 20 mV; soc., Milford, MA). The other reaction conditions were as modulation amplitude, 0.5 G; time constant, 0.5 a; scan range, described above. 100 G; scan time, 4 min. The spectra were recorded at room Each 3.2-mL volume reaction mixture for (2-chloropheny1)temperature. hydrazine, (3-chlorophenyl)hydrazine,(4-chloropheny1)hydraFor on-line LC/EPR operation, the outlet of the Waters UV zine, and (2-bromopheny1)hydrazine with 4-POBN was eluted detector was connected to the EPR flow cell with 75-cm X 0.010from a Sep-pak Cl8 with 1 mL of methanol. A 0.2-mL aliquot in.4.d. stainless steel HPLC tubing. EPR spectrometry was was applied to the LC/EPR/MS system. The other reaction performed using a Varian E-104 EPR spectrometer. The conditions were as described for the reaction of phenylhydrazine magnetic field of the EPR spectrometer was fiied at each signal with 4-POBN. detected from 4-POBN radical adducts, PBN radical adduct, Reaction of Phenylhydrazine, Horseradish Peroxidase, and MNP radical adduct, and DMPO radical adduct. The EPR Hydrogen Peroxide with 4-POBN. The reaction mixture consettings were microwave power, 20 mW; modulation amplitude, tained 2 mM phenylhydrazine, 50 mM 4-POBN, 1 mg/mL 4 G; modulation frequency, 100 kHz; time constant, 2 s. horseradish peroxidase (294 units/mg), and 1 mM hydrogen Mass Spectrometer. The mass spectrometer used was a VG peroxide in 0.5 mL of 0.12 M potassium phosphate buffer (pH 12-250massspectrometer/datasystem(VGMasslab,Altrincham, 7.4). The reaction was started by adding horseradish peroxidase U.K.). The source used for the ESI experiment was a Veetec and continued for 10minunder aerobicconditions. The hydrogen electrospray source, Model 611B (Vestec Corp., Houston, TX). peroxide/horseradish peroxidase system is known to form radical Typical operating conditions were needle voltage, 3.08 kV; spray current, 0.234 mA; block temperature, 262 "C; chamber tem(28) Buettner,G. R.; Oberley,L. W. Biochern. Biophys. Res. Commun. perature, 51 OC; skimmer voltage, 14 V. The Vestec ESI 1978,83,69-74.
Q
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interfaceB differs from those of other manufacturersNby the absence of a nitrogen curtain gas and the use of a heated block for declustering(similarto Vestec’s thermospray source designa1). In order to have sufficient sensitivity to detect the radicals, the VG peak width parameter was set at 1.1 or 1.2 for most experiments, resulting in a peak width at half-height of approximately 1.5-2.0 Da. The probe used was one developed in this laboratory for CZE/ MS and was of coaxial flow design.s2 Coaxial flow allows the delivery of the make-up solution to the probe tip without loss of chromatographicresolution. The coaxial flow concept was originally developed for postcolumn derivatization with capillary LC columns.33 For the work described here, the probe was operated with
