1975
Anal. Chem. 1984, 56, 1975-1977 (3) (4) (5) (6) (7)
(8)
(9) (10) (1 1) (12) (13) (14) (15)
Jolley, R. L. J . Am. Water 1875, 6 7 , 601. Glaze, W. H.; Henderson, J. E. J . WaferP. C . 1977, 8 , 1818. Peieg, M. Water Res. 1878. 70, 361. Zeilig, N. M. J . Am. Wafer 1883, 75, 34. Symons, J. M. "Ozone, Chlorine Dioxide, and Chloramines as Alternatives to Chlorine for Dlsinfectlon of Drinking Water"; presented at the Second Conference on Water Chlorination: Envlronmental Impact and Health Effects, Gatllnburg, TN, Oct 31-Nov 4, 1977. "A Comparison of Analytical Methods for Residual Ozone"; a paper presented by 0. Gordon and J. Grunwell at the 6th Ozone World Congress, Washlngton, DC, May 1983. Bader, H.; Holgne', J. Ozone: Sb. Eng. 1982, 4 , 169. Hoigne', J.; Bader, H. Vom Wasser 1880, 55, 261. Ruzlcka, J.: Hansen, E. "Flow Injection Analysis"; Wiley: New York, 1981. Bahnemann, D.; Hart, E. J. J . Phys. Ch8m. 1882, 86, 252. Nicolson, N. J. Analyst(London) 1985, 9 0 , 188. "Standard Methods for the Examlnatlon of Water and Wastewater", 14th ed.; American Public Health Association: Washlngton, DC, 1975. Cohen, H. Thesis, Miami University, Oxford, OH, 1981.
(16) (17) (18) (19)
Benga, J. Dissertation, Miami Universlty, Oxford, OH, 1980. Bader, H.; Holgne', J. Water Res. 1981, 15, 449. Gilbert, E.: Hoigne', J. GWF, Wasser Abwasser 1983, 124, 527. Gordon, G. "Improved Methods of Analysis for Chlorate, Chlorite and Hypochlorite Ions at the Sub-mg/L Level"; Proceedings of the American Water Works Association, The 1982 Water Quality Technology Conference, 1982, Vol. 175, p 189.
Michael R. S t r a k a Gilbert E.Pacey* Gilbert Gordon Department of Chemistry Miami University Oxford, Ohio 45056
RECEIVED for review February 8,1984.Accepted May 7,1984.
Dehalogenation Reactions in Fast Atom Bombardment Mass Spectrometry Sir: Fast atom bombardment (FAB) mass spectrometry is a method of considerable importance for the structural analysis of highly polar or labile organic compounds (1, 2). The technique is generally regarded as a "soft"-ionization method in which low-energy processes predominate, with fragment ions formed by unimolecular dissociation of protonated molecules (MH+) and similar species. The general appearance of FAB mass spectra can often be predicted by analogy to the corresponding chemical ionization or field desorption spectra ( 3 , 4 )with which FAB shares some characteristics. We report here that halogenated nucleosides, a class of compound typically well suited for FAB mass spectrometry, undergo extensive dehalogenation during FAB in glycerol matrix. These processes, in which halogen bound to the heterocyclic base is replaced by H in the net reaction, have no analogy as major reactions of ions produced by other desorption methods or electron ionization and cannot represent low-energy processes in the usual context of gaseous ion chemistry. The FAB mass spectra of nucleosides 1-8 (Chart I) have been studied to determine whether chemical selectivity resulta from the identity of the halogen (I, Br, C1, F) or position of halogen substitution and to determine whether structural selectivity exists for the source of hydrogen incorporated after dehalogenation, by using selectively deuterated glycerol as matrix. EXPERIMENTAL SECTION All compounds were of reagent grade, obtained from the following sources: 1, 5-iodouridine, P-L Biochemicals, Inc., Milwaukee, WI; 2, 5-bromouridine, 3, 5-chlorouridine, and 4, 5fluorouridine, Calbiochem, San Diego, CA; 5,2-chloroadenosine, 8, 8-bromoinosine, thyroxine, and 5-(4,6-dichlorotriazin-2-yl)aminofluorescein, Sigma Chemical Co., St. Louis, MO; 6, 8bromoadenosine, Cancer Chemotherapy National Service Center, National Cancer Institute, Bethesda, MD; 7,8-bromoguanosine, Nutritional Chemicals Corp., Cleveland, OH; glycerol, Fisher ScientificCo., Fair Lawn, NJ; [hydr0xy-~H,]glycer01(98atom % 2H), 1,1,2,3,3-[2H51glycerol(C-perdeuterioglycerol) (98 atom % 'H), and 2Hz0(100 atom % 2H),MSD isotopes, St. Louis, MO. Compounds 1-8 were examined by electron ionization mass spectrometry in free form or as trimethylsilyl derivatives (5) and were judged to be greater than 98% pure. In addition, 1-8 were analyzed by high-performance liquid chromatography [0.1 M NH40Ac, 4 % CH30H, 0.5% tetrahydrofuran, 2% CH,CN, pH 5.1, RSil C,, LL-DA (Alltech Assoc., Deerfield, IL)] for presence of dehalogented nucleosides which could be present as impurities.
Chart I
0
O
AN
Y9
HOCH2
HO
OH
x= 3, x =c1
1, I 2, X = Br
Hocw HO
OH
5, XI = C1, X2 = H 6, X' = H, X1 = Br
4,X=F
0
0
CY HoctY
HOCH2
OH
HO
7
HO
OH
8
In the pyrimidine series 1-4, uridine was absent above the limit of detection, approximately 1 %. Also, samples of 5-8 were found to be over 99% pure, and no dehalogented nucleosides were observed above the limit of detection. Samples were mixed in glycerol-H20 (1:l)at a concentration of 1, 2, and 10 pg/pL with 2 fiL of the solution applied to the stainless steel probe tip. After exposure to vacuum for 3 min in the probe vacuum lock, only glycerol of approximately 1-pL volume remained. Conditions for use of deuterated glycerol-2Hz0 matrix were previously published (6). Mass spectra were acquired on a MAT 731 instrument operated at 8-keV accelerating potential, utilizing an Ion Tech FAB 11N saddle field-type ion source, closely following the configuration described by Biemann and co-workers (7). A neutral Xe beam of 6-keV energy and neutral current of approximately 5 X
0003-2700/84/0356-1975$01.50/00 1984 Amerlcan Chemical Society
1976
ANALYTICAL CHEMISTRY, VOL. 56, NO. 11, SEPTEMBER 1984
Table I. Dehalogenation of Halonucleosides during Fast Atom Bombardment compd 5-iodouridine (1) 5-bromouridine (2) 5-chlorouridine (3) 5-fluorouridine (4) 2-chloroadenosine (5) 8-bromoadenosine (6) 8-bromoguanosine (7) 8-bromoinosine (8)
%
exchangea 33 21 12
5.3 17 30 33 32
MxH"
m/z (% re1 intens)* MHH+
245 (49) 245 (53) 245 (19) 245 (5.6)
371 (100)
323 (100) 279 (100) 263 (100) 302 (100) 346 (100) 362 (100) 347 (100)
268 (29) 268 (73) 284 (95) 269 (82)
Sum of 35Cl and 37Cl or 79Brand 81Brvalues were used to calculate percent exchange. V l and 81Brisotopic species not listed.
A were employed. Spectra were recorded at resolution 600 (1-pug samples) or 10000 (10-pg samples for 1-4,2-pg samples for 5-8) by scanning the magnetic field. Mass spectra, at both resolution levels and all concentration levels, were qualitatively the same; data in Table I were taken from the high-resolution experiments in order to exclude the possiblity of interference from the same nominal mass ions from glycerol. The negative ion mass spectrum of 2 was recorded at resolution 600 from a 2-pg sample;the positive ion spectrum of thyroxine was recorded at resolution 900 from approximately 10 pg of material. The Xe beam was turned on 10 s after insertion of the sample probe. Data in the table are from scans initiated 30 s after the beam was turned on; additional scans were taken at 30-s or 1-min intervals up to 5 min.
RESULTS AND DISCUSSION Results from positive ion FAB mass spectra showing abundances of the principal molecular species and extent of dehalogenation from compounds 1-8 are given in Table I. Halogen-containing molecules are denoted as Mx and those which have undergone dehalogenation with addition of H as MH. Percent dehalogenation is defined in terms of relative ion abundances: M"+/ (M"+ + MxH'). The order of percent dehalogenation in the 5-halouridine series (1-4) was found to be I > Br > C1> F. The extent of reaction in each case is also qualitatively reflected in the fragment ions representing the protonated free base (8) (e.g., protonated 5-iodouracil and uracil from 1). Glycerol adducts with MxHf and MHH+ were observed (data not shown), a common characteristic of molecular species. The spectra were otherwise devoid of other major or unassigned ions. The extent of dehalogenation was monitored a t 30-s and I-min intervals up to 5 min. Small fluctuations, generally within the 1-3% range, were observed; after 5 min, dehalogenation levels were within several percent of the initial values and generally within the limits of scan-to-scan fluctuations. A negative ion FAB mass spectrum of 2 was recorded, which showed ions representing (8)5-bromouridine, uridine, and Bras shown in Table 11. The extent of dehalogenation on the basis of (Mx - H)- and (MH - H)- abundances was 11%. This lower value, from negative ion mass spectra, compared with 21% measured from positive ion mass spectra (Table I) may result from several factors, including higher ionization efficiency for the halogen-containing (Mx - H)- ion relative to (MH - H)-, compared with the analogous MH' ions in the positive ion spectrum. The positive ion FAB spectrum of 1 dissolved in C-perdeuterioglycerol [(HOCD2)2CDOH]showed no shift for MxH' ( m / z 371) compared with use of unlabeled glycerol, as expected (6). However, MHH' showed partial incorporation of deuterium, with the abundance ratio mlz 2451246 = 0.72. After correction for natural heavy isotopes and the extent of deuterium enrichment in glycerol, it is calculated that 57% of hydrogen (deuterium) introduced upon dehalogenation originates from glycerol carbon. This value compares with a theoretical value of 62.5% for statistical utilization of all eight hydrogens of glycerol. When 0-perdeuterioglycerol
Table 11. Negative Ion Mass Spectrum from Fast Atom Bombardment of 5-Bromouridine (2) in Glycerol m/za
ionb
% re1 intens
321
(Mx - H)(MH - HIBXBH-
50 14 96 33 100
243 189 111
79
79Br-
a81Brisotopic species not listed. *B,pyrimidine base fragment. [ (DOCHJ&HOD] was used as matrix, and following correction for natural isotopes and the extent of active hydrogen-deuterium exchange (6), 88%, it was found that active hydrogens from glycerol contribute approximately 34-37% toward incorporation after dehalogenation, compared with the statistical theoretical value of 37.5%. These results demonstrate that dehalogenation, with concomitant incorporation of hydrogen, occurs as major processes during FAB. These reactions were not a priori predicted and have not otherwise been observed in gas-phase ionic reactions associated with particle desorption or electron ionization (9, 10) mass spectrometry. Analogous reactions in which halogen is replaced by hydrogen overall have been reported by Harrison and co-workers, who studied the chemical ionization mass spectra of halogenated benzenes and toluenes (11,12). The mechanism which they proposed differs from the one discussed below, in proceeding through a cationic intermediate which reacts with reagent gas (13). That low-energy gas-phase ionic reactions are not involved in the present case is suggested by the random distribution of hydrogen abstracted from glycerol and the fact that the net reaction is bimolecular. However, chemical selectivity is evident in the extent of dehalogenation in 1-4 and 5 vs. 6, 7, and 8, which inversely follows the order of carbon-halogen bond strengths: C-F > C-C1> C-Br > C-I. From the positive ion FAB mass spectra, the extent of dehalogenation fluctuates slightly over the period of bombardment, typically in the range 1-3% over the first 5 min. These results are entirely consistent with the reaction represented in eq 1,resulting from particle bombardment at the glycerol surface. Ionization of MX and MH in the vicinity of particle collision then leads to a distribution of products observed in either the postive or negative ion FAB mass spectrum. The observation that product Mn does not undergo appreciable accumulation during FAB suggests that a large proportion of MH species are continuously lost from the region of particle impact as either neutrals or ions. Reaction 1has direct analogy in both UV-photolysis (14) and radiolysis experiments in solution using y-rays or energetic electrons (15). The dehalogenation of 5-bromopyrimidines, following UV irradiation, has been extensively studied in relation to photolytic damage to DNA (16). The extent of photolysis follows the order I > Br > C1 (14, 16). The ura-
Anal. Chem. 1984, 56, 1977-1979
. HO
. OH
C1 during FAB in glycerol (J. A. Kelley, National Cancer Institute, personal communication), indicating the possibility of dehalogenation from nonaromatic carbon.
0
0
HO
OH
HO
ACKNOWLEDGMENT We are grateful to D. L. Smith for conversion of the MAT 731 mass spectrometer for negative ion measurments and to J. Franks for helpful discussions. Registry NO.1,1024-99-3;2,957-75-5;3,2880-89-9;4,316-46-1; 5,146-77-0;6, 2946-39-6; 7,4016-63-1;8, 55627-73-1; thyroxine, 51-48-9; glycerol, 56-81-5.
OH
MH
Mx
1, x =I 2, X = Br
3, x =
1977
c1
4,X=F
LITERATURE CITED (1)
cil-5-yl radical is highly reactive and rapidly extracts hydrogen from a structural variety of compounds (16)a t rates which are sufficiently similar to imply little selectivity in the abstraction step. For example, the relative rate constant for H abstraction by the uracil-5-yl radical a t pH 7 for benzene is 16 f 2 and is 12 f 2 for ethanol (17). The photochemical analogy is of added interest because of the finding by White et al. that bombardment of surfaces by neutral or ion beams in the 30 eV-100 keV energy range induces optical emission in the visible, ultraviolet, and infrared regions (18,19). Of perhaps greater significance are radiolysis studies (20) of reactions induced by the solvated electron, generated by y-irradiation, or direct electron bombardment of aqueous solutions. Upon irradiation, 5-bromouracil thus reacts by dissociative electron capture to form the uracil-by1 radical and Br- (15,21, 22)) the former then producing uracil by hyrogen abstraction from the medium. When the reaction with 5-bromouridine is initiated by FAB, both uridine and unreacted nucleoside are observed in the positive ion mass spectrum, while Br- is additionally observed in the negative ion spectrum. The generality of reactions of this type in FAB mass spectrometry has not yet been determined, so two additional polar halogenated compounds were examined for evidence of dehalogenation: thyroxine and 5-(4,6-dichlorotriazin-2-y1)aminofluorescein. The fluorescein derivative (MxH+ = m / z 495) showed no observable dechlorination (MHH+ = m/z 461) measured at resolution 10000 to avoid overlap from m / z 461 of glycerol. On the other hand, thyroxine showed extensive dehalogenation by replacement of both one and two iodine atoms: MxH+, m/z 778,100% relative intensity; M"+, m/z 652,88%; m / z 526, 34%. The overall significance of these studies is twofold. First, the interpretation of FAB mass spectra must take into account the possiblity of processes which are unexpected on the basis of conventional gaseous ion chemistry, as earlier suggested by Field (23).For example, under most circumstances, the finding of ions representing M H from eq 1would lead to the conclusion that the original sample contains uridine. Second, extensive radiation damage to glycerol occrs during FAB (23)) with consequences that are largely unexplored at the present time. Note Added i n Proof. 5'-Chloro-5'-deoxy-5,6-dihydro-5azacytidine was observed to undergo 20% exchange of H for
(1) Rinehart, K. L., Jr. Science(Washington, D.C.) 1982,218,254-260. (2) Burlingame, A. L.; Whitney, J. 0.; Russell, D. H. Anal. Chem. 1984, 56,417R-467R. (3) Harrison, A. G. "Chemical Ionlzatlon Mass Spectrometry"; CRC Press: Boca Raton, FL, 1983. (4) Morris, H. R. "Soft Ionization Biological Mass Spectrometry"; Heyden: Philadelphia, PA, 1981. (5) Pang, H.; Schram, K. H.; Smith, D. L.; Gupta, S. P.; Townsend, L. B.; McCloskey, J. A. J . Org. Chem. 1982,4 7 , 3923-3932. (6) Sethi, S. K.; Smlth, D. L.; McCloskey, J. A. Biochem. Biophys. Res. Commun. 1983, 112, 126-131. (7) Martln, S. A.; Costello, C. E.; Blemann, K. Anal. Chem. 1982, 5 4 ,
2362-2368. Crow, F. W.; Tomer, K. 8.; Gross, M. L.; McCloskey, J. A.; Bergstrom, D. E. Anal. Biochem. 1984, 139, 243-262. Reiser, R. W. Org. M s s Spectfom. 1969,2,467-479. McCloskey, J. A. I n "Basic Principles in Nucleic Acid Chemistry"; Ts'o, P. 0. P., Ed.; Academic Press: New York, 1974; Voi. I,pp
209-309. Harrison, A. G.; Lin, P.-H. Can. J . Chem. 1975,53, 1314-1318. Leung, H.-W.; Harrlson, A. G. Can. J . Chem. 1976,5 4 , 3439-3452. Reference 3,pp 102-106. Wang, S. Y. I n "Photochemistry and Photobiology of Nucleic Acids"; Academic Press: New York. 1976:Vol. I.DO 296-31 1. (15) Bhatia, K.; Schuler, R. H. J.'Phys.'Chem.'fS73, 7 7 , 1888-1896. (16) Hutchinson, F. 0.Rev. Biophys. 1973,6 , 201-246. (17) Ebel, R.; Kraljic, 1. Eur. Biophys. Congr., Proc., Ist, 1971, 1971,2,
109-1 14. (18) White, C. W.; Simms, D. L.: Tolk, N. H. Science (Washinaton, D . C . ) 1972, 177, 481-486. (19) White, C. W.; Thomas, E. W.; Van der Weg, W. F.; Tolk, N. H. I n
"Inelastic Ion-Surface Collisions"; Tolk, N. H.; Tuliy, J. C.; Heiland, W.; White, C. W., Eds.; Academic Press: New York, 1977; pp 201-252 (20) S c h o k r G . I n "Effects of Ionizing Radiation on DNA"; Huttermann, J.; Kohnlein, W.; Teoule, R.; Bertinchamps, A. J., Eds.; Springer-Verlag: New York, 1978;pp 153-170. (21) Bansal. K. M.; Patterson, L. K.; Schuier, R. H. J . Phys. Chem. 1972, 7 6 , 2386-2392. (22) Rivera, E.; Schuler, R. H. J . Phys. Chem. 1983,8 7 , 3966-3971 and references therein. (23) Field, F. H. J . Phys. Chem. 1962,8 6 , 5115-5123.
'
Current address: Travenol Laboratories, Inc., 6301 Lincoln Avenue, Morton Grove, I L 60053.
Satinder K. Sethi' Chad C. Nelson James A. McCloskey* Departments of Medicinal Chemistry and Biochemistry University of Utah Salt Lake City, Utah 84112
RECEIVED for review March 16,1984. Accepted May 3,1984. S.K.S. gratefully acknowledges support from National Cancer Institute Training Grant CA 09038. This work was supported by Grant GM 21584 from the National Institute of General Medical Sciences.
Microcolumn Gel Permeation Chromatography with Inductively Coupled Plasma Emission Spectrometric Detection Sir: The increasing need to characterize the components of complex mixtures separated by high-performance liquid
chromatography (HPLC) has encouraged the development of hyphenated techniques such as HPLC-MS (1-9), HPLC-
0003-2700/84/0356-1977$01.50/00 1984 American Chemical Society
