868
Anal. Chem. 1990, 62,868-870
Laser Desorption Electron Impact: Application to a Study of the Mechanism of Conjugation of Glutathione and Cyclophosphamide Zhi-min Yuan, Catherine Fenselau,* D. M. Dulik, William Martin, W. Bart Emary, R, B. Brundrett, 0. M. Colvin, and R. J. Cotter Department of Chemistry a n d Biochemistry, University of Maryland Baltimore County, Baltimore, Maryland 21228, a n d Department of Pharmacology and Molecular Sciences, Johns Hopkins University, Baltimore, Maryland 21205
Toward the objective of producing ion radical species from involatile and thermally labile samples, we have combined laser desorption of neutral molecules with electron impact ionization on a time-of-flight mass analyzer with a delayed draw-out pulse. The analytical capabilities of this method are tested in the analysis of lsotope labels in the lnvolatlle product in a mechanistic study of both the chemical and the enzyme catalyzed reactions of cyclophosphamide with glutathione.
INTRODUCTION Isotope analysis and other applications of mass spectrometry require cleavage of carbon-carbon bonds within a molecule. Such bond scissions are prevalent in the ion radical species produced by electron impact ionization, as for example, cleavage of bonds CY t o a heteroatom and the McLafferty rearrangement ( I ) . Ion radicals and their fragmentation pathways are not usually initiated by the newer desorption methods and have thus far been confined to samples sufficiently volatile for electron impact ionization. Fast atom bombardment, for example, produces even electron ions in most cases, which do not usually undergo homolytic or other carbon-carbon bond scission unimolecularly ( 2 ) . Thus, we have combined laser desorption of neutral molecules with electron impact ionization ( 3 , 4 )as a means for producing ion radical species from involatile samples. As part of continuing studies of the formation of glutathione conjugates as deactivated metabolites of anticancer alkylating mustards (5),we have designed an isotope labeling experiment to distinguish whether glutathione conjugation with cyclophosphamide takes place via direct displacement of chloride (reaction 1 in Figure l), or by opening a cyclic atiridinium ion (reaction 2 in Figure 1). Direct halide displacement is the accepted mechanism for glutathione conjugation of halocarbons (6);however, kinetic (7) and isotope labeling evidence (8) indicate that many reactions of sulfur and nitrogen mustards take place subsequent to internal displacement and formation of the cyclic aziridinium intermediate. As indicated in Figure 1, cyclophosphamide labeled with deuterium isotopes in the terminal methylene group of each mustard arm would bond with glutathione in the two reaction schemes to produce products in which the isotope labels have different positional distributions. The fragmentation pattern observed for the cyclophosphamide family in classical electron impact spectra (9) features bond scission between the two methylene groups in question (Figure 1) and would permit distinction of the patterns of labels in the two products (8). Nonselective opening of the aziridinium ring (path 2 in Figure 1) would be revealed by a 5 0 5 0 split in the peaks, i.e., m/z 213 and 215 for the 35Clspecies and 215 and 217 for the 37Cl species. Glutathione conjugates are involatile and thermally labile, and they have not been successfully analyzed by
electron impact, even derivatized. T h e monoglutathione conjugate derived from cyclophosphamide by both chemical and enzyme-catalyzed reactions, and reported here for the first time, was no exception, and this isotope labeling study was selected as a n excellent candidate for the laser desorption electron impact method.
EXPERIMENTAL SECTION Materials. Cyclophosphamide and 2’,2’,2’’,2’’-tetradeuterocyclophosphamide had been synthesized previously ( I O ) . Glutathione was purchased from Sigma Chemical Co. (St. Louis, MO). All other chemicals were reagent grade and used without further purification. Conjugation Reactions. The chemical reaction was carried out by mixing cyclophosphamide (0.1 M) and glutathione (1.0 M) in aqueous solution adjusted to pH 10 with ammonium hydroxide, for 24 h at room temperature. The conjugation reaction was also carried out under catalysis by immobilized rabbit liver microsomal glutathione S-transferases (11). The incubation mixture contained cyclophosphamide (1.0 mM), glutathione (5.0 mM), and 15 mL of microsomal protein immobilized to beaded Sepharose in phosphate buffer (0.1 M, pH 7.4). The total volume was 20 mL. This incubation was carried out for 24 h a t 37 “C and terminated by addition of enough trifluoroacetic acid to bring the pH to 3.0. Conjugation was not observed a t pH 7.4 in a control incubation run without immobilized protein. The monoglutathione products from both the chemical and the enzymatic reactions were purified by high-pressure liquid chromatography on a Beckman/ Altex (Fullerton, CA) liquid chromatograph using a RP-C18 Brownlee Labs (Santa Clara, CA) column (250 X 4.6 mm) and a gradient mobile phase (A, 0.1% trifluoroacetic acid (TFA); B, 60/40 acetonitrile-0.1% TFA, B from 10% to 30% in 25 min) a t a flow rate of 2.0 mL/min. Ultraviolet detection was used at 215 nm. Molecular weights of the conjugates were obtained by fast atom bombardment mass spectrometry on a Kratos (Manchester, U.K.) MS-50 mass spectrometer operating at 3000 resolution. Laser Desorption Electron Impact Mass Spectrometry. LDEI mass spectra were obtained on a modified CVC Products (Rochester, NY) Model 2000 time-of-flight mass spectrometer fitted with a Tachisto (Needham, MA) Model 215G pulsed carbon dioxide laser (3). The laser wavelength was 10.6 pm with a pulse width of 40 ns and energy of 100 mJ. This instrumental configuration has been described in detail (12) and has been used for the recording of ions produced directly by the laser (LD) as well as those produced by electron impact ionization of desorbed neutrals (3).In the LDEI method, desorption of neutral species was initiated with the instantaneous power density of the pulsed laser reduced below ca. lo7 W/cm2. When the laser power is reduced, few ions are evident unless the electron beam is turned on. Thus, after a 90-ps delay, each laser pulse was then followed by an 0.5 gs 30-eV electron beam pulse, a second delay of approximately 8 ps, and a drawout pulse. The second time delay provides time lag focusing (13)for optimizing the mass resolution. In this case, resolution was about lj200. The drawout pulse extracts the ions from the ion source and initiates their acceleration into the 1-m drift region. The drawout pulse also provides the trigger pulse for a LeCroy (Spring Valley, NY) Model 3500SA
0003-2700/90/0362-0868$02 50/0 IC 1990 American Chemical Society
0
ANALYTICAL CHEMISTRY, VOL. 62, NO. 8, APRIL 15, 1990
mn
215
/&H
869
1
-
Figure 1. Alternative reactions for conjugation of glutathione (GSH) and 2r,2r,2”,2rr-tetradeuterocyclophosphamide.
100
d.
C
I-
Q
00 (L
-:
delay time 0-100
30v ov
ov
ov
-e
60
JJS
i
-
LASER TRIGGER
40 I
20
ION ORAWOUT PULSE
: I_
: -150~
1 time lag focus
s us I -
Flgure 2. Timing sequence of the laser desorption electron ionization instrument used for this study.
100
a.
‘i’
(MIK)+ 171
W
40
= l 0
3
20
m
a W
> F a
100
1 W LL
219
219
MASS/ CHAR G E
Flgure 4. Partial laser desorption electron impact spectra of (a) chemically formed Cyclophosphamide glutathione, (b) chemically formed 2’,2’,2”,2”-tetradeuterocycbphosphamide glutathione, (c) enzymatically formed cyclophosphamide glutathione, and (d) enzymatically formed 2’,2’,2rr,2rr-tetradeuterocyclophosphamideglutathione.
RESULTS AND DISCUSSION A schematic representation of the timing sequence is shown
60
z
A
I
are obtained by repeating this cycle.
80
v
I .
so 6C
4c
2c
190
252
323
403
492
590
MASS/CHARGE
Figure 3. Laser desorption (a) and laser desorption electron impact (b) spectra of cyclophosphamide glutathione produced by chemical reaction.
transient recorder and signal averager, which records the mass spectrum from each laser pulse. Signal-averaged mass spectra
in Figure 2. The time intervals between desorption and ionization and between ionization and drawout can be regarded as experimental variables ( 4 ) ,which in this case were adjusted t o optimize the abundances and resolution (respectively) of the ions of interest. The laser desorption (LD) mass spectrum of the cyclophosphamide glutathione conjugate is shown in Figure 3a. Consistent with previous studies using the C 0 2 laser ( 3 ) , molecular ions are formed as their sodium or potassium adducts (555 and 571 amu). Signals for the chloride isotopes in the heavier ions are not resolved in this spectrum, and the mass values indicated are average masses. Fragment ions are formed by the loss of HCl from molecular ion adducts, mass 535 for the kalinated species. The spectra of the tetradeutero analogues (not shown) indicate that ions of mass 312 are derived from kalinated glutathione, apparently by elimination of RSH, R being the cyclophosphamide moiety. The base peak a t m / z 261 corresponds to a kalinated ion resulting from both of these processes and is shown in Chart I. This ion and its natriated and protonated analogues (245 and 223 m u ) appear at mass values increased by four in the laser desorption spectrum of the tetradeuterated conjugate. The electron impact spectrum (Figure 3b) of cyclophosphamide glutathione ablated by the laser beam has quite a different appearance. Potassium and sodium adduct ions are not observed. Consistent with previously published
870
Anal. Chem. 1990, 62, 870-875
Chart I. L a s e r D e s o r p t i o n F r a g m e n t Ions
tribution in both cases is consistent with conjugation by direct displacement of one chloride.
LITERATURE CITED
X -
m/z -
K Na H
261 245 223
electron impact spectra of cyclophosphamide (9),molecular ion radicals (masses 531 and 533) are also not detected in the laser desorption electron impact spectrum. Facile cleavage between the methylene groups in the mustard arm (Figure 1) leads to the base peak a t m / z 211 and its 37Clsatellite a t 213, illustrated more clearly in Figure 4. Additional ions occur at masses 209,222, and 223, which appear to be formed in part by pyrolytic processes, since their relative intensities vary from spectrum to spectrum. The fragment ions of major interest in this study are those of mass 211 and 213 amu. In the LDEI spectra of the tetradeutero analogues formed either chemically or enzymatically (Figure 4b,d), this pair of peaks occurs a t m / z 213 and 215, recorded with approximately the 3:l intensity ratio characteristic of chlorine isotopes. The absence of signals a t m / z 217 confirms the retention of only two deuterium labels in the ions. This in turn excludes the symmetrical aziridinium ion as a reaction intermediate in either the chemical reaction (pH 10) or the enzyme catalyzed reaction. The isotope dis-
( I ) McLafferty, F. W. Interpretation of Mass Spectra; University Science Books: Mill Valley, VA, 1980. (2) Fenselau, C.; Cotter, R. J. Chem. Rev. 1987, 87,501-512. (3) VanBreeman, R. 8 . ; Snow, M.; Cotter, R. J. Int. J . Mass Spechom. Ion Phys. 1983, 4 9 , 35-50. (4) Fenselau, C.; Emary, W. 8.; Cotter, R. J. Org. Mass Spectrom. 1989, 2 4 , 694-698. (5) Colvin, M.; Russo, J. E.; Hilton, J.; Dulik, D. M.: Fenselau, C. Advances in Enzyme Regulation 1988, 27, 211-221. (6) Mannervik, 8.; Danielson, U. H. CRC, Crit. Rev. Biochem. 1988, 23, 283-337. (7) Peterson, L. A,; Harris, T. M.; Guengerich, F. P. J . Am. Chem. SOC. 1988, 110, 3284-3291. (8) Colvin, M.; Brundrett, R. 8.; Kan, M. N.; Jardine, I.; Fenselau. C. CancerRes. 1978, 36, 1121-1126. (9) Fenselau, C. I n Physical Methods in Modern Chemical Analysis; Kuwana, T., Ed.; John Wiley and Son: New York, 1978, Vol. I, pp 123- 125. (10) Jardine, I.; Fenselau, C.; Appler, M.; Kan, M. N.; Brundrett, R. 6.: Colvin, M. Cancer Res. 1978. 38,408-415. (11) Pallante, S.; Lisek, C. A.; Dulik, D. M.; Fenselau, C. Drug Metab. Disp. 1986, 14, 313-318. (12) Oithoff. J. K.; Lys, I.; Demirev, P.; Cotter, R. J. Anal. Instrum. 1978, 16, 93-1 15. (13) Wiley, W. C.; McLaren, I . H. Rev. Sci. Instrum. 1955, 26. 1150.
RECEIVED for review November 10, 1989. Accepted January 24, 1990. This research was supported by grants from the National Institutes of Health, GM 21248 (to C.F.) and CA 16783 (to O.M.C.), and from the National Science Foundation, BBS 85-15390 (to R.J.C.). This work was presented in part a t the Thirty-Seventh Annual Conference on Mass Spectrometry and Allied Topics, Miami Beach, FL, 1989
Quantitative Static Secondary Ion Mass Spectrometry of (L-DOPA) Molecular Ions from 1-~-3,4=Dihydroxyphenylalanine and Indolic Derivatives Michael B. Clark, Jr.,l and Joseph A. Gardella, Jr.*P2
Department of Chemistry and Surface Science Center, State University of New York at Buffalo, Buffalo, New York 14214
TradltbnaUy, statlc secondary ion mass spectrometry (SIMS) Is believed to yield qualltatlve information and very little quantltatlve information. A method to obtain quantitative molecular Ion data from organlc static SIMS analysis of LDOPA and rdated compounds is presented. Linear callbration curves have been constructed by integrating the protonated molecular Ion to sliver Ion peak area ratios over a known ion dosage and plotting versus the orlginai sample concentration.
INTRODUCTION Secondary ion mass spectrometry (SIMS) is a simple and very powerful surface analytical technique. Typically, a primary ion beam of 0.5-30 keV energy bombards a solid
*
To w h o m correspondence should b e addressed. ‘ C u r r e n t address: R o h m a n d Haas Co., N o r r i s t o w n Rd., Spring House. PA 19477. * C u r r e n t address: Analytica! and Surface C h e m i s t r y Program, Chemistry Divison, N a t i o n a l Science Foundation, Washington, DC 20550.
surface, and the sputtered secondary ions are mass analyzed ( I ) . The interested reader is referred to ref 2 for an excellent description of the basic concepts and applications of the SIMS technique. As a surface analytical technique, SIMS has several advantages over ESCA (electron spectroscopy for chemical analysis) and Auger electron spectroscopy (2,3). These advantages include detection of all elements and their isotopic distributions, and a low detection limit, microgram per gram and nanogram per gram for elements and attomolar concentrations for molecular species (2). Unfortunately, SIMS suffers from so-called matrix effects, producing vastly different ion yields from different chemical structures and preparations. The SIMS experiment can be performed with several different types of experimental conditions, divided loosely into “dynamic” or “static” modes. There are several review articles (2-4) comparing and contrasting these two modes of SIMS. High current densities, greater than 1pA/cm2, can be used to gain elemental information as a function of sputter depth into the bulk. In addition, elemental maps or imaging can be obtained by focusing the ion beam to small (1-3 pm) spot sizes (“ion microprobe”) or by illuminating the sample with a large-diameter ion beam and using ion optics to preserve
0003-2700/90/0362-0870$02.50/0 1990 American Chemical Society
