Anal. Chem. 1986, 58,345-348
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Pentafluorobenzylation of 04-Ethylthymidineand Analogues by Phase-Transfer Catalysis for Determination by Gas Chromatography with Electron Capture Detection Jeanette Adams,l Mordechai David, a n d Roger W.Giese* Department of Medicinal Chemistry, College of Pharmacy and Allied Health Professions, and Barnett Institute of Chemical Analysis a n d Materials Science, Northeastern University, 360 Huntington Avenue, Boston, Massachusetts 02115
O4-EthyIthymidlne,$methylthymidine, thymidine, and thymlne are derivatlzed with pentafluorobenzyl bromide under condltlons uslng phase-transfer catalysis. All active hydrogens, whether on the base or the sugar, are alkylated. The products exhibit good chromatographlc peak shapes and have strong molar responses when determined by gas chromatography with electron capture detectlon. A detection iimtl of 27 fg Is observed for 3',5'-bls( O-pentafluorobenzyl)-04-ethylthymidine, lowerlng the detection limit for nucleoside GC by 103.
Exposing DNA to ethylating agents may result in the formation of 04-ethylthymidine (I). As a lesion on DNA, this ethyl-adduct causes misincorporation of deoxyguanosine during in vitro DNA replication and transcription (2). In vivo, this adduct has a relatively long half-life and may be important in the initiation of carcinogenesis and mutagenesis (3-5). Although the quantitative relationship between such an adduct and carcinogenesis has been studied (3-5), its minimum threshold for carcinogenesis is not known. Additionally, routine physiological samples generally provide only small amounts of DNA. Thus, there is interest in quantitating 04-ethylthymidine in DNA at ultratrace levels. The methodology currently available for quantitating 04-ethylthymidine with high sensitivity is incomplete. A promising immunoradiometric assay is reported to detect 0.09 fmol of 04-ethylthymidinein DNA (6);however, the sample of DNA was ethylated in vitro to give an initial ratio of 3 X O4-ethy1thymidine/thymidine and then diluted as a standard with unmodified DNA. Also, immunoassays only provide an indirect measurement. As an alternative to radioimmunoassay, the determination of @-ethylthymidine from DNA by gas chromatography could provide a highly sensitive and specific method of quantitation. The sensitivity and specificity could be maximized by forming highly electrophoric derivatives detectable by electron capture detection (GC-ECD) and negative ion chemical ionization mass spectrometry (GC-NCI-MS) (7,8). Previous methods for derivatizing 2'-deoxyribonucleosides for determination by GC and GC-MS have involved trialkylsilanization (9, IO),acetylation and trifluoroacetylation (11,221, and permethylation (13)of the 3'- and 5'-deoxyribose hydroxyls. Of these, peralkylation should provide the most stable derivatives amenable to postderivatization sample cleanup prior to quantitation by GC-ECD and GC-NCI-MS. Typical reqctions for permethylation of nucleosides use methyl iodide and a strong nucleophile such as the methylsulfinyl or silver oxide (15). A pyrolysis technique carbanion (13,14) using trimethylanilinium hydroxide also has been used to 'Present address: Midwest Center for Mass Spectrometry, Deartment of Chemistry, University of Nebraska-Lincoln, Lincoln, E 68588-0362.
Kr
permethylate ribonucleosides and thymidine, giving, for example, a 55-60% preparative yield of permethylthymidine (16). No yields were cited for the other permethylation methods. Toward the goal of determining DNA adducts at a low level by GC-ECD and GC-NCI-MS, we report here the preparation of pentafluorobenzyl derivatives of 04-ethylthymidine and some related compounds with a relatively mild phase-transfer alkylation reaction. Although guanosine has been permethylated previously using a crown ether catalyst (17), this appears to be the first alkylation of a nucleoside by phasetransfer catalysis. The pentafluorobenzyl derivatives described here exhibit good chromatographic properties, including high molar responses by GC-ECD. EXPERIMENTAL SECTION Apparatus. High-Pressure Liquid Chromatography (HPLC). A Series 4 liquid chromatograph (Perkin-Elmer, Norwalk, CT) was used for preparative HPLC. lt was equipped with a Model 7125 injector and a 2-mL sample loop (Rheodyne, Cotati, CA) and a 20 cm long, 1.2 cm o.d., 10 pm particle size column of DavisiLC8 (Applied Science, State College, PA). Detection was at 254 nm with a Spectroflow 773 absorbance detector (Kratos, Ramsey, NJ). Liquid chromatograms were recorded with a SP4270 integrator (Spectra-Physics, San Jose, CA). For the purification of 3',5'-bis(O-pentafluorobenzy1)-3methylthymidine, the following conditions were used: (1) the column was equilibrated for 5 min at a flow of 6 mL min-' with 75:25 CH,CN/water; (2) upon injection, the solvent composition was kept the same for 10 min; (3) over a period of 1 min, the solvent composition was linearly changed to 100% CH&N and maintained for 10 min. A similar program beginning with 8020 CH,CN/water was used to purify 03',05'-bis(0-pentafluorobenzyl)-04-ethylthymidine. Gas Chromatography. A Vista 6000 gas chromatograph equipped with a @Niconstant-current,variable frequency electron capture detector, a Model 11095 nonvaporizing on-column capillary injector, and mass-flow controlled pneumatics (Varian, Sunnyvale, CA) was used for gas chromatography with electron capture detection (GC-ECD). On-column injections were made with a 5-pL syringe equipped with a stainless steel needle (Scientific Glass Engineering, Austin, TX). The analytical column with a 15 m, 0.31 mm i.d., 0.17-pm film thickness, ULTRA cross-linked 5 % phenylmethyl silicone fused silica capillary column (Hewlett-Packard, Palo Alto, CA). Gas chromatograms were recorded with a SP4270 integrator (Spectra-Physics, San Jose, CA). To determine the molar responses of the derivatized compounds relative to lindane, the following conditions were used: upon injection, the injector was programmed from 30 OC to 200 "C at a setting of 180 "C min-l with a 14-min final hold; the column was held for 1.5 min at 90 "C and then programmed at 20 O C min-l to 280 "C with a 4-min final hold. For the determination of derivatized 04-ethylthymidine individually, the following conditions were used upon injection, the injector was programmed from 30 "C to 200 "C at a setting of 180 "C min-l with a 9 min final hold; the column was held at 180 "C for 1.5 min and then programmed at 30 "C rnin-l to 260 OC with a 5.84 min final hold. The carrier gas was UHP nitrogen (Granite State Oxygen, Nashua,
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NH) at a flow of 6 mL min-' (purge is normally open but this was measured with purge closed); make-up gas was UHP helium (Granite State Oxygen) at a flow of 24 mL min-'; detector temperature was 340 "C. Carrier and make-up gases were equipped with OxiClear gas purifiers (Labclear, Oakland, CA). Reagents. For the organic syntheses, thymidine and thymine were Sigma grade (Sigma Chemical, St. Louis, MO); K2C03and KOH were ACS Grade (Baker Chemical, Phillipsburg, NJ); pentafluorobenzyl bromide, petroleum ether, CH2C12,CH31,THF, and CH3CN were 99% pure, ethanol was anhydrous and denatured, (Bu)4NHS04 was 97% pure, and HMDS (1,1,1,3,3,3hexamethyldisilazane) was 98% pure (Aldrich Chemical, Milwaukee, WI); and ethyl acetate and hexane (Eastman Kodak, Rochester, NY) and acetone (VWR Scientific, San Francisco, CA) were ACS grade. Silica gel for column chromatography was Kieselgel 60, 230-400 mesh (EM Science, Gibbstown, NJ); preparative TLC plates were silica gel GF, 1000 pm thickness (Analtech, Newark, DE). Sodium ethoxide was prepared by adding 45 mg (1.96 mmol) of sodium metal (ACS grade, MCB, Cincinnati, OH) to 5 mL of anhydrous ethanol. For HPLC, CH3CN was glass-distilled (Burdick & Jackson, Muskegon, MI); water was HPLC grade (Baker Chemical, Phillipsburg, NJ). For GC-ECD, stock solutions of analytical standards were prepared in toluene and diluted for use in either toluene, acetonitrile,methanol, or isooctane (Burdick & Jackson). Preparation of Glassware. For GC-ECD, glassware was cleaned by soaking it in a hot (70 "C) solution of 10% H2S04in water for approximately 16 h. It was rinsed with tap water and soaked in a hot (70 "C) solution of 2% Micro (International Products, Trenton, NJ) in water for approximately 4 h. After being rinsed with tap water and drained, the glassware was heated at 280 "C in the GC oven for approximately 16 h. Vapor-phase silanization was performed by transferring the clean glassware to a vacuum oven (VWR Scientific, San Francisco, CA) and heating it at 200 "C under high vacuum for approximately 2 h. After the vacuum was turned off, 1-2 mL of HMDS was added through a valve, and the glassware was silanized for 2 h. Vacuum was again applied, and the glassware was heated for an additional 2 h. The glassware was covered with aluminum foil, transferred to the GC oven, and heated at 280 "C for approximately 16 h. A series of pieces were then serially rinsed with hexane, and the hexane was analyzed by GC-ECD to confirm that the glassware was clean. The liners, made of Teflon, for the vial caps (Arthur H. Thomas, Philadelphia, PA) were boiled for approximately 10 min each in methanol, acetone, hexane, and pentane. They were then heated at 280 OC for 16 h. Synthesis. Unless noted otherwise, the structure of each product was determined by NMR, IR, and mass spectral analysis. 3-Methylthymidine.Thymidine (1.2 g, 4.9 mmol), K2CO3 (3.4 g, 25 mmol), and CH,I (3.1 mL, 49 mmol) were suspended in acetone (50 mL) and stirred at room temperature for 24 h. The solids were removed by filtration, and the acetone was concentrated. The product was dissolved in ethyl acetate, and the solids that separated were removed by filtration. The product was recrystallized from ethyl acetate/hexane to give a 37% yield (mp 94 "C). 04-Ethylthymidine.04-Ethylthymidinewas prepared from thymidine (2.4 g) according to Singer et al. (2) by using ethanol and sodium ethoxide instead of methanol and sodium methoxide in the second step. The product was isolated by preparative TLC using 2 1 acetone/benzene (mp 182-184 "C). The UV spectrum was identical with that published by Kusmierek and Singer (18). 3-Pentafluorobenzylthymidine. Thymidine (0.48 g, 2.0 mmol), K,C03 (0.69 g, 5.0 mmol), and pentafluorobenzyl bromide (0.3 mL, 2 mmol) in acetone (20 mL) were stirred at room temperature for 30 h. After the solids were removed by filtration, the acetone was evaporated to dryness. The residual oil was dissolved in ethyl acetate, and the solids that separated were removed by filtration. The solution was then applied to a column of silica gel, and the product was eluted wiih ethyl acetate. The oily product was resuspended in petroleum ether and cooled overnight at 4 "C. The crystals that formed were isolated to give a 78% yield (mp 134-136
"C). 1,3-Bis@entafluorobenzyl)thymine. Thymine (0.12 g, 0.95 mmol), pentafluorobenzyl bromide (1.0 g, 3.9 mmol), and K2CO3
(0.70 g, 5.1 mmol) in acetone (8 mL) were stirred at room temperature for 15 h. After the residual solids were removed by filtration, the acetone was evaporated to dryness. The residue was dissolved in a small amount of ether and applied to preparative TLC plates. After development with 80:20 hexane/ethyl acetate, the product was isolated and recrystallized from acetone/hexane (mp 113-115 "C)giving a 46% yield. 3',5'-Bis(O-pentafluorobenzyl)-3-methylthymidine. 3Methylthymidine (0.25 g, 1.0 mmol) was suspended in a solution of pentafluorobenzyl bromide (1.5 mL, 9.8 mmol) in CH2C12(17 mL) and CH3CN (8 mL). A solution of KOH (1.1 g) and (Bu),NHS04 (0.84 g, 2.5 mmol) in water (40 mL) was added, and the mixture was stirred at room temperature for 4 h. The organic layer was isolated was washed three times with 20-mL portions of water. The solution was concentrated to a small volume, and the product was isolated by preparative TLC using 7:3 petroleum ether/ethyl acetate. The oily product was isolated in a 43% yield. Further purification by preparative HPLC gave a solid (mp 105-107 "C). O3',O5',3-Tris(pentaf2uorobenzyl)thymidine. 3-Pentafluorobenzylthymidine (0.18 g, 0.42 mmol) was dissolved in a solution of pentafluorobenzyl bromide (0.64 mL, 4.2 mmol) in CH2C12(7 mL). An aqueous solution (17 mL) containing KOH (0.5 g) and (Bu)~NHSO,(0.36 g, 1.1mmol) was then added, and the mixture was stirred vigorously at room temperature for 1.5 h. The organic layer was isolated and washed 3 times with 30-mL portions of water. The CH2C12was evaporated to a small volume, and the solution was applied to preparative TLC plates. After development with 4 1 petroleum ether/ethyl acetate, the desired product was isolated as an oil to give a 50% yield. 3',5'-Bis(0-pentafluorobenzyl)-O4-ethylthymidine. 04Ethylthymidine (0.042 g, 0.16 mmol) was suspended in a solution of CH3CN (2 mL), CH2Clz(4 mL), and THF (0.5 mL) containing pentafluorobenzyl bromide (0.46 mL, 3.1 mmol). An aqueous solution containing 0.5 N KOH (6.5 mL) and (Bu)~NHSO,(0.26 g, 0.78 mmol) was added, and the mixture was stirred at room temperature for 6 h. The aqueous layer was removed and washed twice with CHzClz(5 mL). The organic layers were combined, evaporated to a small volume, and applied to preparative TLC plates. After development with 2:3 ethyl acetate/petroleum ether, the desired product was isolated as an oil to give a 55% yield. Further purification by preparative reverse-phase HPLC resulted in a 45% yield. Product identity was supported by NMR and mass spectral analysis.
RESULTS AND DISCUSSION Derivatization with pentafluorobenzyl bromide (PFBzBr) can facilitate the determination of polar compounds by gas chromatography with electron capture detection (GC-ECD) and GC with detection by negative ion chemical ionization mass spectrometry (GC-NCI-MS) (1+21). However, attempts here to react the sugar hydroxyls of thymidine with PFBzBr under conventional conditions were unsuccessful: using PFBzBr with K2C03and acetone gave only the N-alkylated product. Similarly, PFBzBr and Ag,O (15),gave three product spots by TLC along with a major spot of unreacted thymidine. Pettit et al. (17)used crown ether catalysis with methyl iodide and Ag,O to give a 19% yield of permethylguanosine. Davis, using 18-crown-6, KzC03, and CH3CN successfully alkylated phenols with PFBzBr (22). However, the latter conditions here gave no alkylation of the deoxyribose hydroxyls of thymidine. As an alternative to crown ether catalysis, reactions using phase-transfer catalysis (23,24) were investigated. Phasetransfer Catalysis has been used for the N-, s-,and 0-alkylation of thiouracil (25),the N-alkylation of purines (26)and pyrimidines (27), and the 0-alkylation of carbohydrates (28). Carboxylic acids also have been esterified with PFBzBr under phase-transfer catalysis conditions (29, 30). Here it was found that 3-methylthymidine, 3-pentafluorobenzylthymidine, and 04-ethylthymidine could be converted in good yields to their corresponding 3',5'-bis( 0-pentafluorobenzyl) ether derivatives by phase-transfer catalysis.
ANALYTICAL CHEMISTRY, VOL. 58, NO. 2, FEBRUARY 1986 CH O, ,
CH
2
1
3
A
Flgure 1. Structure of 3‘,5’-bis(0gentafluorobenzyl~04-ethy~ymidine (compound 4 in Table I and Figure 2).
347
c
B
T
41
6
Table I. GC-ECD Characteristics of the Pentafluorobenzyl (PFBz)Derivatives compound 1,3-bis(PFBz)thymine 3’,5’-bis-(O-PFBz)3-methylthymidine 04-ethylthymidine 3-(PFBz)thymidine
no.@
mol w t
re1 molar responseb
2
486
1.6 f 0.11
3
616 630
1.1f 0.062 0.60 f 0.068 1.5 f 0.092
4 5
782
“Refersto peak number in Figure 2. *Area units/mol relative to lindane; represents mean f standard deviation from 1 to 2 injections of duplicate sets of dilutions at each concentration level containing all four compounds and lindane covering the linear range: for compound 2, n = 18; for 3, n = 26; for 4, n = 42; for 5, n = 26. (n = total number of data Doints throughout the linear range.) The structure of the derivative of P-ethylthymidine is shown in Figure 1. Thymidine itself gives no reaction without prior N-alkylation for instance with methyl iodide/K2C03 or PFBzBr/K2C03;N-alkylation presumably increases the solubility of the thymidinelquaternary amine ion pair in the organic phase. Equivalent results are obtained with tetrabutylammonium hydrogen sulfate, tetrahexylammonium bromide, or Adogen 464 [trademark of Ashland Chemical Co. for methyltrialkyl(C8-C,o)ammoniumchloride]. At a point where no starting nucleoside remains, based on monitoring the phase-transfer reaction by TLC, two minor side product spots are observed. The pentafluorobenzyl derivatives exhibit good peak shapes and elicit high molar responses when determined by GC-ECD as shown in Figure 2 and Table I. Thus, 27 fg (0.045 fmol) of 3‘,5’-bis(O-pentafluorobenzyl)-@-ethylthyrnidine can be detected at a signal/noise of about 2, as shown in Figure 2C. The additional peaks seen in chromatograms B and C arise, a t least in part, from the polyimide coating on the column extending into the on-column injector (31). Trace amounts of the derivatives diluted in either toluene, acetonitrile, or methanol give the same response by GC-ECD. However, the compounds diluted in isooctane are adsorbed onto the walls of silanized glass vials. This adsorption causes a reduction in solution concentration of approximately 40% for derivatized thymine, 80-90% for thymidine and 3methylthymidine, and virtually 100% for derivatized 04ethylthymidine at the 10 fmol bL-l level. Similar losses are seen at the 50 fmol pL-l level. An adsorption mechanism is indicated since the derivatives are recovered when the above isooctane solution is diluted 1:l with toluene. The appearance of a plot of peak area vs. solute amount varies with each analyte using a GC fitted with a conventional ECD. Derivatized thymine gives the least linear range, approximately 102-fold(correlation coefficient r = 0.997 for the analyte range of 0.11-16 fmol). For the other compounds, the linear ranges (r 1 0.997) are 0.24-41 fmol for 3,0.44-610 fmol for 4, and 0.25-39 fmol for 5. A plot of response factor (area per solute amount) vs. solute amount shows a “roller coaster” type of nonlinearity analogous to that observed previously for lindane with a conventional ECD (32). This nonlinear “roller coaster” behavior is slightly less pronounced for derivatized
0 9 2 7J
--
b
&
lb
lk
b
5
IO time imin)
I
15
-
O
5
IO
Figure 2. Gas chromatograms of lindane (peak 1) and pentafluorobenzyl derivatives. Peaks 2-5 refer to derivatives listed in Table I. One
microliter of analytical standards containing a mixture of all five compounds in toluene was inJected: chromatogram A, 1 = 1.1, 2 = 4.0, 3 = 5.0, 4 = 5.0, and 5 = 6.0 pg; attenuation, 64 X 1; chromatogram B, (a) 1 = 0.11, 2 = 0.53, 3 = 0.15, 4 = 0.28, and 5 = 0.20 pg; (b) blank; attenuation, 16 X 1; chromatogram C , (a) 57 fg and (b) 27 fg (0.045 fmol) of compound 4, (c) = blank; attenuatlon, 4 X 1. thymidine and 3-methylthymidine, and it is absent for derivatized 04-ethylthymidine. As suggested in another study, this type of nonlinearity and its dependence on solute structure may be due to surface effects in the ECD and perhaps can be improved, as before, by using a more inert version of this detector (32).
CONCLUSION Alkylation using phase-transfer catalysis with pentafluorobenzyl bromide is a promising method for derivatizing nucleoside DNA adducts such as 04-ethylthymidine prior to their determination by GC-ECD and GC-NCI-MS. The reaction involves relatively mild conditions, gives a good yield, and affords derivatives that elicit strong molar responses by GC-ECD. This procedure should be applicable to determiniig 04-ethylthymidine at trace levels in biological samples. It should also be applicable to determining many other covalent adducts of DNA potentially relevant to carcinogenesis and mutagenesis. Registry No. 04-Ethylthymidine, 59495-22-6; 3-methylthymidine, 958-74-7;thymidine, 50-89-5;thymine, 65-71-4;pentafluorobenzyl bromide, 1765-40-8;3-pentafluorobenzylthymidine, 99268-58-3; 1,3-bis(pentafluorobenzyl)thymine,99268-59-4; 3’,~bis(O-pentafluorobenzyl)-3-methylthymidine, 99268-60-7; O3,Os-3-tris(pentafluorobenzyl)thymidine,99280-64-5;3’,5’-bis(O-pentafluorobenzyl)-04-ethylthymidine, 99268-61-8.
LITERATURE CITED (1) Singer, B. Nature (London) 1978, 264, 333-339. (2) Singer, 6.; Sagi, J.; Kusmierek, J. T. R o c . Nafl. Acad. Sci. U . S . A . 1983. 80, 4884-4888. (3) Slnger, 8.; Spengier, S.; Bodell, W. J. Carcinogenesis 1981, 2 , 1069-1073. (4) Swenberg, J. A.; Dyroff, M. C.; Bedell, M. A.; Popp. J. A.; Huh, N.; Klrstein, U.; Rajewsky, M. F. R o c . Nafl. Acad. Sci. U . S . A . 1984. 81, 1692-1895. ( 5 ) Scherer, E.; Timmer, A. P.; Emmeiot, P. Cancer Lett. 1880, 10, 1-6. (6) Nehls, P.; Adamklewicz. J.; Rajewsky, M. F. J. Cancer Res. Cih. OnCOI. 1984, 108, 23-29. (7) Nazareth, A.; Joppich, M.; Abdel-Baky, S.;O’Connell, K.; Sentlssi, A.; Glese, R. W. J . Chromatogr. 1984, 314, 201-210. (8) Mohamed, G. E.; Nazareth, A.; Hayes, M. J.; Giese, R. W.; Vouros. P. J. Chromatogr. 1984, 314, 211-217. (9) Quilliam, M. A.; Westmore, J. B. Anal. Chem. 1978, 50, 59-68. (10) Gehrke, C. W.; Patel, A. 8. J . Chromatogr. 1978, 123, 335-345. (11) Quilliam, M. A.; Ogilvle, K. K.; Sadana, K. L.; Westmore, J. B. J. Chromatogr. 1880, 196, 367-378.
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(12) Boutagy, J.; Harvey, D. J. J . Chromatogr. 1978, 156, 153-166. (13) De Leenheer, A. P.; Gelljkens, C. F. Anal. Chem. 1976, 48, 2203-2206. (14) von Mlnden, D. L.; McCloskey, J. A. J . Am. Chem. SOC. 1973, 9 5 , 7480-7490. (15) Dolhun, J. J.; Wiebers, J. L. Org. Mass Spectrom. 1970, 3 , 669-681. (16) Einck, J. J.; Pettit, G. R.; Brown, P.; Yamauchi, K. J . Carbohydr., Nucleosides, Nucleotides 1980, 7, 1-20. (17) Pettit, G. R.; Blazer, R. M.; Einck, J. J.; Yamauchi, K. J . Org. Chem. 1980, 45, 4073-4076. (18) Kusmierek, J. T.; Singer, B. Nucleic Acids Res. 1976, 3 , 989-1000. (19) Roberts, J. L.; Oates, J. A. Anal. Blochem. 1984, 136, 258-263. (20) Waddeli, K. A.; Blair, I. A.; Wellby, J. Biomed. Mass Spectrom. 1983, 10,83-88. (21) Epstein, E.; Cohen, J. D. J. Chromatogr. 1981, 209, 413-420. (22) Davis, D. Anal. Chem. 1977, 49, 832-834. (23) Brandstrom, A. J . Mol. Catal. 1983, 2 0 , 93-103. (24) Jones, R. A. Aldrichimica Acta 1976, 9 (3), 35-45. (25) Hassanaly, P.; Dou, H.; Ludwlkow, M. Bull. SOC. Chlm. Be@. 1982, 9 1 , 661-662.
Hedayatullah, M. J . Heterocycl. Chem. 1982, 19, 249-251. Hedayatullah, M. J . Heterocycl. Chem. 1981, 18, 339-342. Szeja, W. Pol. J . Chem. 1981, 5 5 , 1503-1509. Rosenfeld, J. M.; Crocco, J. L. Anal. Chem. 1978, 5 0 , 701-704. Gyllenhaai, 0.;Broteil, H.; Hartvig, P. J . Chromstogr. 1976, 129, 295-302. (31) Adams, J.; Giese, R. W., unpublished results. (32) Nazareth, A.; O'Connell, K.: Sentissi, A,; Giese, R. W. J. Chromatogr. 1984, 314, 219-232.
RECEIVED for review June 3, 1985. Accepted October 1, 1985. Financial support for this research was provided by National Cancer Institute Grant CA35843 and Oak Ridge Subcontract 19X4335C from the Reproductive Effects Assessment Group, U.S. Environmental Protection Agency. This is Contribution No. 253 from the Barnett Institute of Chemical Analysis and Materials Science.
Neutralization Agents for Neutralization-Reionization Mass Spectrometry Paul 0. Danis, Bong Feng, and Fred W. McLafferty* Chemistry Department, Cornell Uniuersity, Ithaca, New York 14853
Vaporlzed metals provide the most efficient targets for charge-exchange neutrailratlon of fast ions; wlth Hg or Na, at least 25% of the 10-keV acetone enol Ions can be converted Into collectable neutrals. These targets are superior for measuring neutralization-relonlration (NR) mass spectra, producing 5 times the abundance of neutrals from acetone Ions as acetone itself, for which resonant charge exchange Is possible, or as xenon, In contrast to a recent recommendation. Metal vapor targets produce approximately an order of magnitude more neutrals by charge exchange than by colllslonally activated dlssoclatlon, while the proportions are reversed wlth hellum as the target. Targets such as Na of much lower ioniratlon energy produce neutrals of higher internal energy, which can fragment differently than the orlginal ions, provlding addltlonal sructural Infformatlon. Experimental procedures are detalled for producing routine NR mass spectra, for which mercury Is a particularly convenient target.
Several years ago it was shown (1,2) that the fast neutral products from the dissociation of mass-selected organic ions of relatively high kinetic energy could be characterized by reionization and subsequent mass analysis. An extension of this method, termed neutralization-reionization mass spectrometry (NRMS) (3, 4) incorporates the methodology of Gellene and Porter ( 5 ) for the alternative preparation of the fast neutrals by charge exchange of mass-selected precursor ions. Similar experimental techniques have studied electron transfer and energy states of simple mono- to triatomic species such as H, (5-10). NRMS makes possible the preparation of neutral species whose reference reionization mass spectra can be directly compared to those of neutral dissociation products. For example, the neutrals formed with CSH6+-from dissociation of metastable aniline ions must be HNC, not HCN, as their reionization spectrum matches the NR spectrum from HCN'., not that from HCN'. (3). The neutralization capability also makes possible the preparation and study of hypervalent and other unstable species such as H2CC1H, CH3-
C(OH)=CH2, CH30H2 ( 3 ) , .(CH2),0H2 (111, and RNH3 (12-14). Subsequent dissociation of the neutral can involve reaction pathways quite different than those of its precursor ion (15), as shown for protonated alkylbenzenes (3),C2H,+ (16),and distonic radical ions such as .CH2CH2CH2N+H3(11). Thus NR should complement collisionally activated dissociation (CAD) as a method for primary ion characterization in tandem mass spectrometry (17,18). Other important work, particularly by Holmes and co-workers (19-21), has used reionization to identify neutral products of unimolecular ion dissociations, such as HNC from aniline+. (3,19)and C H 2 0 H from CH3COOCH3+-(20). These neutral beams are prepared from the fast ions exiting MS-I of a tandem mass spectrometer by charge-exchange (CE) collision (3-10) with a target gas. These collisions can also produce neutrals by CAD, for which optimum conditions have been extensively studied (17, 22, 23). Extending previous studies (1-13, 19-21), we report here the effect of neutralization agents, pressure, and kinetic energy on the CE and CAD efficiency of neutral targets interacting with fast ions. These results lead to recommendations on optimal experimental conditions for NRMS. As neutralization targets, metal vapors are substantially superior to xenon, in contrast to a recent report (24).
EXPERIMENTAL SECTION The tandem mass spectrometer utilizes a Hitachi RMH-2 double-focusinginstrument as MS-I and an electrostatic sector as MS-11, as described previously (1-3,25). Ions were prepared by 70-eV electron ionization and accelerated at 10 kV unless noted otherwise. Figure 1shows MS-I1 and the region between MS-I and MS-I1 incorporating the metal vapor furnace or gas inlet (Cls-I) for neutralization, the ion lens for deflection (Dfl-I) of unneutralized ions (in experiments to be reported a collision gas is introduced here separately for neutral dissociation) (26), the molecular beam for reionization (Cls-III), a second electrostatic ion deflector (Dfl-11), and a retractable channel electron multiplier (Mlt-I) for direct measurement of the neutral and ion beams. Oxygen was used as the reionization target (9, 27) at a pressure giving 50% transmittance of
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