Anal. Chem. 1907, 59, 1145-1149
1145
Comparison of Desorption Ionization Methods for the Analysis of Neutral Seven-Coordinate Technetium Radiopharmaceuticals Steve E. Unger* and T e r r y J. McCormick The Squibb Institute for Medical Research, P.O.Box 4000, Princeton, New Jersey 08540
Elizabeth N. Treher and A d r i a n D. Nunn The Squibb Institute for Medical Research,
P.O.Box 191, New Brunswick, New Jersey 08903
The maw spectrometry of neutral, seven-coordlnate technetlum radbpharmaceutlcals used as heart-lmaglng agents Is detailed. Fleki desorption, fast atom bombardment, thermorpray Mzatkn, and fast-heatlng chemlcal lonlzatlon were used to effect lonlzatlon and the resultlng spectra are discussed In terms of Information content, detectlon llmlts, and matrix effects. The use of these kniratkn methods wtth mass spectrometry/mass spectrometry and llquld chromatographylmass spectrometry to define fragmentation patterns and resolve mlxtures Is also presented.
Ionization methods in mass spectrometry which affect desorption and ionization from either a solid or liquid phase have proved successful in the characterization of nonvolatile and thermally sensitive biomolecules (I). Such techniques have been applied to the analysis of biologically useful transitionmetal complexes, including platinum anticancer agents (2), gold antiarthritic complexes ( 3 ) ,and technetium radiopharmaceuticals (4-10). Costello and co-workers used positive (4,5) and negative (6)field desorption (FD) mass spectrometry (11)to characterize Tc-containing complexes (12). They performed a chromatographic separation of rat bile and urine folIowed by FD-MS to establish the presence of a ? l ? ccomplex (6). They also used fast atom bombardment (FAB) (13)for the analysis of anionic technetium radiopharmaceuticals (7). Fast atom bombardment mass spectrometry has been used to characterize octahedral cationic Tc(II1) complexes (8) containing phosphorus and arsenic ligands (14). More detailed information including oxidation sites and relative ligand stabilities was apparent from FAB-MS/MS spectra of these cationic complexes (9). Field desorption has also been used to provide mass spectra of cationic Tc complexes and to distinguish these from similar neutral complexes (10). A recent major effort of radiopharmaceutical chemists has been to develop neutral technetium complexes that are passively distributed across biological barriers (15). Volkert and Troutner described a neutral complex of technetium propyleneamine oxime, TcPnAO, which is the first neutral Tc species to enter the brain in quantities sufficient for conventional imaging techniques (16,17). Treher et al. (18) have recently reported the synthesis and characterization (19) of neutral Tc(II1) complexes containing boronic acid adducts of vicinal dioximes for imaging of the heart. One of these neutral, lipid soluble complexes is now in clinical trials as a myocardial imaging agent (20,21). We wish to report here the mass spectral characterization of this and related technetium radiopharmaceuticals. We used numerous ionization methods including field desorption, fast atom bombardment, thermospray ionization, and fast-heating chemical ionization. The results are compared on the basis of detection sensitivity, information content, and the effects 0003-2700/87/0359-1145$01.50/0
of sample matrix upon ionization. This paper also demonstrates the use of thermospray (TSP) LC/MS (22) and MS/MS for the characterization of such neutral technetium complexes. EXPERIMENTAL S E C T I O N Fast atom bombardment and field desorption mass spectra were recorded with a VG-ZAB-2F mass spectrometer (Vacuum Generators, Ltd., Altrincham, Great Britain). FAB mass spectra were obtained by sputtering (8-keV Xe) a thioglycerol or dithiothreitol-dithioerythritolsolution containing approximately 2-3 pg of the analyte. The resulting ions were accelerated to 8 keV and mass spectra were obtained by scanning the magnetic seetor of the reverse-geometry instrument at 20 s/decade. High-resolution analysis under FAB conditions was accomplished using either poly(ethy1ene glycols) or Ethaquad as internal standards (23) for peak matching (1:sooO). MS/MS spectra were obtained by mass analyzing the parent ion, subjecting it to collision (N2; 50% main beam reduction),and analyzing the resulting fragments by scanning the electric sector at 20 s/decade. Approximately 10 such spectra were averaged to enhance detection sensitivity. Field desorption mass spectra were obtained by depositing approximately 0.5 pg of the analyte via syringe onto a carbon emitter. The emitter, prepared by using a Linden ChroMasSpec Activator (ChroMasSpec,Bremen, Germany) and the high-temperature method described by Beckey (24), was composed of carbon dendrites on 10-pm tungsten wire. Manual adjustment of emitter current to 10-15 mA provided the best anode temperature (BAT). The counterelectrode was held at -4 kV while the emitter was maintained at +8 kV. Full mass spectra were acquired by using 20 s/decade scans and spectra over a limited mass range were obtained by averaging 10 scans. A VG-2035 data system was used to record all FAB and FD spectra. Thermospray mass and MS/MS spectra were obtained with a Finnigan TSQ-4600 mass spectrometer (Finnigan Corp., San Jose, CA) and Vestec Thermospray source (Vestec Corp., Houston, TX). The analyte was dissolved in the mobile phase and injected (Rheodyne Model 7125 injector, Cotati, CA) into the interface. A Model 6000A Waters pump (Waters Associates, Milford, MA) was used for some analyses while a Model 400 Kratos pump (Kratos, Inc., Ramsey, NJ) was used for others. The thermospray source was held at 275 OC with the vaporizer adjusted to 180-240 "C, depending upon the analyte and interface nozzle diameter. Direct introduction of 0.2-0.5 pg of analyte sufficed for recording mass spectra of relatively pure samples while prior separation by HPLC using a 10-cm PRP-1 column (Bodman, Inc., Media, PA) and a 0.1 M acetate buffer, pH 5.0acetonitrile 1090 mobile phase was used for the analysis of mixtures. Mass spectra were obtained by scanning the third quadrupole at 2 s/decade while the first and second quadrupoles were set to pass all ions (viz., rf-only mode). MS/MS spectra were acquired by selecting the parent ion in the fiist quadrupole, accelerating the ion to 30 eV energy, and allowing it to undergo collision with argon at 1mtorr pressure. The third quadrupole was scanned under conditions identical with those for recording mass spectra. Fast-heatingchemical ionization (CI) mass and MS/MS spectra were recorded by using instrument parameters identical with those used for thermospray ionization. A commercial Finnigan direct exposure probe was used with approximately0.2-0.3 pg of materialdeposited on the metal loop. Both methane and ammonia were 0 1987 American Chemical Society
1146
ANALYTICAL CHEMISTRY, VOL. 59, NO. 8, APRIL 15, 1987
Table I. Positive Ions in Mass Spectra of Seven-Coordinate Tc(II1) Compounds"
structure
FD-MS
FAB-MS
1
590/592 (100) 2951296 (22)
2
546/548 (100)
3
626/628 (100)
4
582/584 (100)
5
668/670 (100)
590/592 (19) 591/593 (23) 511 (100) 546/548 (15) 547/549 (13) 511 (100) 6261628 (22) 627/629 (28) 547 (100) 5821584 (24) 583/585 (18) 547 (100) 6681670 (23) 669/671 (26) 589 (100)
TSP-MS
CI-MS
591/593 (100) 511 (23)
591/593 (100) 511 (32)
547/549 (100) 511 (33)
547/549 (100) 511 (54)
649/651 (100) Na 547 (22)
627/629 (100) 547 (89)
583/585 (100) 547 (21)
583/585 (100) 547 (44)
691/693 (100) Na 589 (89)
669/671 (100) 589 (55)
"For Br- and C1-containing compounds, intensity is given as the first isotope only. used to record positive chemical ionization mass spectra while a mixture of methane and nitrous oxide was used for negative chemical ionization. The Finnigan TSQ-4600mass spectrometer is interfaced to a Nova 4/C computer and Incos revision 5.4 software was used for data acquisition and processing.
RESULTS AND DISCUSSION Table I illustrates the results of ionizing neutral, sevencoordinate Tc complexes using desorption ionization methods. These techniques include two (field desorption and fastheating chemical ionization) which sample from the solid phase and two (fast atom bombardment and thermospray ionization) in which ionization occurs from solution. Before the introduction of other ionization techniques, field desorption mass spectrometry was used exclusively to characterize involatile transition-metal complexes. The neutral, seven-coordinate Tc complexes examined here gave simple FD spectra, yielding only the molecular ion and limited structurally informative fragmentation. The relatively weak and fluctuating signal precluded the use of high-resolution or MS/MS analysis of the field desorbed ion. The FD spectrum of 1 (Figure 1)showed only the molecular ion, M', at m l z 590/592 (Br isotope), the doubly charged ion, M2+,at mlz 2951296, and a smallquantity of the contaminating chloro compound, 2, a t mlz 5461548. R
Structure
A
Y -
I
Br
bu
2
CI
bu
3
Br
CH3
4
CI
CH3
CH2CH2-
5
Br
bu
CH2CH2-
-
R CH3 CH3
CH2CH2-
These complexes show no ion emission from unactivated wires, whereas previous characterization of cationic octahedral
Figure 1.
Positive field desorption mass spectrum of
1.
technetium complexes had shown intense emission from unactivated wires (IO). The absence of ion emission under low field strength conditions along with their low conductivity (1.1-2.3 Q-' cm2 mol-' in acetonitrile) supports their assignment as neutral compounds (18). The positive FAB mass spectrum of 1 showed both the molecular ion at m l z 5901592 and protonated molecular ion at mlz 5911593 with approximately equal intensities. This is a common feature of these complexes and indicates that under FAB conditions the low ionization potential of the Tc complex directs formation of the M+ ion. Loss of bromide from 1 is responsible for the 511' fragment; the chloro analogue, 2, showed the analogous loss of C1. The MS/MS spectrum of this 511' daughter ion showed several fragments whose identity was confirmed by accurate mass measurement (Chart I, top) of daughter ions observed in the positive FAB mass spectrum of 1. These fragments were also seen in the positive FAB spectrum of 2. In FAB, a significant increase in fragmentation is observed with increased analyte concentration. For less concentrated or impure samples, the use of collisional activation with MS/MS analysis affords structural information absent or obscured in the FAB mass spectrum and resolves parent-daughter relationships in mixtures. The negative FAB mass spectrum of the bromo-containing complex yields an intense (M - H)-parent at mlz 5891591 as well as fragments at m / z 509 and 510 (Brand HBr losses) and 79/81 (Br-). The number of exchangeable hydrogens was indicated by using acidic (0.1 N DCl/D20) deuterated glycerol as the FAB matrix (25,26). For 1 the deprotonated molecular ion at mlz 5891591 was shifted to mlz 5901592 in the presence of acidic deuterated glycerol. As this ion is most likely formed by the loss of an exchanged deuterium, the results are consistent with
ANALYTICAL CHEMISTRY, VOL. 59, NO.
8,APRIL 15, 1987
1147
Chart I. Positive Fragments miz -
ASSIGNMENT
FOUND
CALCD.
51 1
511.1341
51 1.1303
Tc(DMG2-5H) ( 8 - b ~(0) )
410
410.0517
410.0529
TC (DMG2-5H) (6-bu)
394
394.0661
394.0639
313
313.0150
31 3.0128
213-215
214.9647
2 14.9648
213.9564
213.9569
172
171.921 5
171.9226
156
155.9269
155.9276
TC (DMGyPH) (B-bu) + +
+
Tc (DMGpCzH7NO) (8-bu) Tc (DMG2-3H)
338 328
+
TC (DMGz-HzO) + Tc(DMG-yH)+
y = 0-2
Tc (CzH3NO) ( 0 )+ TC (C2H3NO)
+
ASSIGNMENT
miz
Tc (CD03-4H) (6-me) +
547
Tc(CD02-5H) 18-me) (0)
y
m/z
41
365
+
I
r
312
+
Tc (CDO-yH) +
600
404
+
Tc ( C D O ~ - C ~ H S N O ) TC(CD02-HzO)
I 550
500
450
420
+
Tc (CDO2-5H](B-me)
465
0- 2
239-241
Tc (C3H4NO) +
169
547
1
5a9i53'
464
, 450
Figure 3.
400
450
500
550
600
500
400
450
500
550
600
mlz
the presence of two exchangeable hydrogens. Positive FAB mass spectra dowed a similar interpretation showing a 2 m u shift for the less intense M+ parent. IH NMR data of 1 indicate the presence of two intramolecular 0-H-0 bonds, supporting the mass spectral results (18). Complexes containing cyclohexanedione dioxime (CDO) yielded results similar to their dimethylglyoxime (DMG)
( ,
550
,
,
I,
,
,
j 600
m/i
Positive and negative thermospray mass spectra of 1.
counterparts. Under FD conditions the molecular ion was observed; under FAB conditions both the molecular ion and protonated molecular ion were observed with equal intensity. Loss of the halide ligand yielded the base peak in all positive FAB mass spectra (parent:daughter ratio was ca. 1:4);however, the analogous daughter ion was substantially reduced in intensity in the negative FAB spectrum (parent:daughter ratio was ca. 3:l). Portions of the positive (top) and negative (bottom) FAB mass spectra of 4 are shown in Figure 2. Fragments formed from the 547+ daughter common to 3 and 4 are illustrated in the bottom of Chart I. Fragmentation patterns for the (M + H - HX)+ ion (where X = C1 or Br) are similar to those for the DMG-containing complexes (see Chart I, top). Of note is the presence of Tc(DMG - yH) + and Tc(CD0 - yH) fragments (y = 0-2) in which technetium exists in formal oxidation states of I, 11, and I11 in the gas phase. Often FAB and FD mass spectrometry suffer from matrix effects in which ion formation is retarded due to the presence of other impurities. Therefore, alternative ionization methods such as thermospray (which allow chromatographic separation prior to ionization) and fast-heating chemical ionization (which effects desorption before ionization) were investigated. The positive (top) and negative (bottom) thermospray mass spectra of 1 shown in Figure 3 were obtained by using approximately 0.2-0.3 wg of analyte introduced directly into the interface. In general, detection limits using thermospray ionization were comparable to those achieved by using field desorption (10-50 ng) and an order of magnitude lower than those possible using fast atom bombardment. The molecular weight as well as the presence of boron, chlorine, or bromine isotopes are evident from these spectra. The experimentally
+
Figure 2. Portions of the positive and negative FAB mass spectra of 4.
,il
1148
ANALYTICAL CHEMISTRY, VOL. 59, NO. 8, APRIL 15, 1987
1
IONS
420
1
460
440
NEGATIVE
480
500
513
IONS
520
I
Chart 11. Negative Ion Fragments
I
'47
ASSIGNMENT
m/z
Tc (CDOySH) @me)-
546
Tc (CD03-5H - H20) (9-me)-
528
Tc (CD03-3H 2H20)-
486
TC(CD02-3H - H20)-
362
540
41
TC(CDO-2H) ( 0 ) ~ -
271
TC(CDO-2H) (0).
255
TC(CDO-2H)-
239
TcOa-
163
TcO3-
147
ASSIGNMENT
m/z
TC(DMG3-5H) (8-bu)-
510
TC(DMG3-5H-HzO) (6-bu)-
492
TC (DMGz-3H-H20)-
310
Tc (DMG-2H) (0)2.
245
Tc (DMG-2H) (0).
229
Tc ( D M G - 2 H ) ~
213
TcOn-
163
TcO3-
147
C4HgB (OH) (0)-
101
I
1I
545
I
L
I 420
440
460
480
500
520
540
560
mlz
Ammonia positive and methane-nitrous oxide negative chemical ionization mass spectra of 2. Figure 4.
determined isotope pattern for the protonated molecular ion of 1 was m / z 590 (28%), 591 (9670),292 (39%), 593 (loo%), and 594 (16%). This was consistent with the calculated isotope pattern m / z 590 (22%), 591 (9970), 592 (42%), 593 (loo%),and 594 (20%). At high mass, fragmentation paralleled that observed in FAB mass spectra; however, previously described low mass fragments were absent in the thermospray spectrum. The ions at the m / z 571 and 552 in the positive ion spectrum are attributed to the addition of CHBCOzHand CH,CN, respectively, to the complex with loss of bromine. The chloro analogue, 2, showed these ions under identical thermospray conditions, confirming their origin. Methane and ammonia chemical ionization mass spectra of 1 and 2 showed remarkable similarities to the thermospray spectra. Detection limits approached low nanogram levels while full mass spectra could be acquired with as little as 20-50 ng of the analyte. Both the protonated and deprotonated molecular ions were seen along with the common 511' and 510- fragments arising from loss of the halide ligand. For positive ion spectra this fragment represents 50% of the base (parent) ion, while for negative ion spectra it is the base peak (see Figure 4). The deprotonated molecular ion was evident for all Tc complexes examined; however, its intensity is only 10-20% of the base peak which corresponds to the loss of the halide ligand. More detailed fragmentation was apparent from MS/MS spectra of either chemical ionization or thermospray-generated negative ions (see Chart 11). A fragment common to negative ion spectra of CDO-containing complexes (3-5) is the m / z 362 daughter ion whose DMG counterpart (1 and 2) is the 310fragment. Several other similarities are evident including the
'b
404
131
373
I 2 7 9 312 263 240
100
200
I
I
300
400
500
600 m / r
Figure 5 . Reconstructed ion chromatogram of the (M 4- Na)' ion of 4
(a) and its MS/MS spectrum (b).
presence of Tc04- and Tc03- fragments. The fragments Tc(CDO - 2H)- and Tc(CD0 - 2H)(O)- at m / z 239 and 255, respectively, contain Tc in the I and I11 formal oxidation states in the gas phase. Mass and MS/MS spectra of thermospray-generated ions are virtually identical with those formed by fast-heating chemical ionization. Thermospray ionization offers the added advantage of on-line liquid chromatographic analysis as illustrated in Figure 5. A reconstructed ion chromatogram of the (M + Na)+ parent of 4 at m / z 605 is shown in the top portion while the MS/MS spectrum of this ion is shown below. The presence of a small quantity of sodium acetate in the
ANALYTICAL CHEMISTRY, VOL. 59, NO. 8, APRIL 15, 1987
mobile phase is responsible for the sodiated molecular ion. This represents ca. 2 pg of the and@ in 20 pL of mobile phase injected onto a 10-cm PRP-1 column. Losses due to scattering upon introduction of collision gas (analyzer pressure (3-4) X 10" torr) into the second quadrupole account for the increased detection limits. The recent incorporation of an additional cyropump has aided in detection sensitivity for LC/MS/MS. Fragmentation of the sodiated molecular ion of 4 is analogous to that of the protonated molecular ion (see Chart I). Loss of NaCl ( m / z 547), subsequent CDO and H loss ( m / z 404), the common 312' fragment, and the Tc (CDO - yH)+ fragments, y = 0-2, at m / z 239-241 were observed. The sodiated analogue ( m / z 443) of the 420' fragment is also apparent. The fragments at m / z 263 and 279 are unique to the sodiated molecular ion and correspond to NaTc(CD0 H)+ and NaTc(CD0 - H)(O)+. The ion a t m / z 131 is also novel and is assigned to TcOz+. The deliberate introduction of cationizing agents such as Na+ may be useful in thermospray to vary the nature and extent of fragmentation apparent in MS/MS spectra. A mixture of the five technetium complexes has been characterized by use of thermospray LC/MS. Each complex was resolved and yielded structurally informative spectra, suggesting that thermospray LC/MS may be used to characterize more complex reaction mixtures.
CONCLUSIONS Neutral seven-coordinate Tc complexes containing dioxime chelates and alkyl boron groups were analyzed with mass spectrometry. The molecular weight and the presence of C1, Br, and B were evident from both the positive and negative ion spectra. The molecular ion was seen in positive FD and FAB mass spectra, while the protonated molecular ion was observed in positive FAB, TSP, and CI spectra. The deprotonated molecular ion was observed in FAB, TSP, and CI mass spectra. Loss of the halide ligand varied with the charge of the ion formed and the ionization method employed. Additional structural information was gleaned from collisional activation MS/MS spectra of these ions. Parallels in fragmentation between the CDO- and DMG-containing Tc complexes were evident in both their positive and negative ion MS/MS spectra. In general, detection limits showed the following trend: CI < TSP = FD < FAB. As this order also reflects their susceptibility to matrix effects, chemical and thermospray ionization, when applicable, should be considered as ionization techniques of choice.
1149
LITERATURE CITED (1) Busch, K. L.; Cooks, R. 0.Science 1982,218, 247-254. (2) Puzo, G.; Prome, J. C.; Macquet, J. P.; Lewis, I.A. S. Biomed. Mass Spectrom. 1982, 9, 552-556. (3) Rottschaefer, S.; Roberts, G. Thirty-first Annual Conference on Mass Spectrometry and Allied Topics, Boston, MA, May 8-13, 1983; pp 286-287. (4) Wilson, B. W.; Costello, C. E.; Carr, S. E.; Biemann, K.; Orvig, C.; Davison, A,; Jones, A. G. Anal. Lett. 1979, 12, 303-311. (5) Farr, J. P.; Abrams, M. J.; Costello, C. E.; Davison, A,; Lippard, S. J.; Jones, A. G. Organornetalllcs 1985,4 , 139-142. (6) Jones, A. G.; Davison, A.; LaTegola, M. R.; Brodack, J. W.; Orvig, C.; Sohn, M.; Toothaker, A. K.; Lock, C. J. L.; Franklin, K. J.; Costello, C. E.; Carr, S. A.; Biemann, K.; Kaplan, M. L. J. Nucl. Med. 1982,2 3 , 801-809. (7) Costello, C. E.;Brodack, J. W.; Jones, A. G.; Davison, A,; Johnson, D. L.; Kasina, S.; Fritzberg, A. R. J. Nucl. Med. 1983, 24, 353-355. (8) Cohen, A. I.; Glavan, K. A,; Kronauge, J. F. Biomed. Mass Spectrom. 1983, IO, 287-291. (9) Unger, S. E. Anal. Chem. 1984,5 6 , 393-368. (IO) Unger, S. E. Anal. Chem. 1985,5 7 , 776-778. (11) Beckey, H. D. Principles of Field Ionization and Field Desorption Mass Spectrometry; Pergamon: Oxford, 1977. (12) Clarke, M. J.; Fackler, P. H. "The Chemistry of Technetium: Toward Improved Diagnostic Agents" I n Structure and Bonding; SpringerVerlag: New York, 1982; Vol. 50. (13) Barber, M.; Bordoli, R. S.; Sedgwick, R. D.; Tyler, A. N. Nature (London) 1981,293, 270-275. (14) Neirinckx, R. D.; Glavan, K. A.; Kronauge, J. F.; Eakins, M. N. J. Labelled Compd. Radiopharm. 1982, 19, 1575-1577. (15) Kung, H. F.; Molnar, M.; Billings, J.; Wicks, R.; Blau, M. J. Nucl. Med. 1984,25, 326-332. (16) Volkert, W. A.; McKenzie, E. H.; Hoffman, T. J.; Troutner, D. E.; Holmes, R. A. I n t . J. Nucl. Med. Biol. 1984, 11, 243-246. (17) Volkert, W. A.; Hoffman, T. J.; Seger, R. M.; Troutner, D. E.;Holmes, R. A. Nucl. Med. iB8439 , 511-516. (18) Treher, E. N.; Gougoutas, J.; Malley, M.; Nunn, A. D.; Unger, S. E. New Technetium Radlopharmaceuticals: Boronic Acid Adducts of Vicinal Dioxime Complexes"; Sixth International Symposium on Radiopharmaceutical chemistry; Boston, MA, June 29-July 3, 1986; pp 89-9 1, (19) Unger, S. E.; McCormick, T. J.; Nunn, A. D.; Treher, E. N. "Mass Spectra of Cationic and Neutral Technetium Complexes"; Sixth International Symposium on Radiopharmaceutical Chemistry; Boston, MA, June 29-July 3, 1986; pp 92-94. (20) Narra, R, K.; Feld, T.; Wedeking, P.; Matyas, J.; Nunn, A. D. J. Nucl. Med. 1988,27, 1051-1052. (21) Coleman, R . E.; Martin, M.; Nunn, A. D.; Eckelman, W. C.; Juri, P. N.; Cobb, F. R. J. Nucl. Med. 1986,2 7 , 893-894. (22) Blakley, C. R.; Carmody, J. J.; Vestal, M. L. J. Am. Chem. SOC. 1980, 102, 5931-5933. (23) DeStefano. A. J.; Keough, T. "1983 Abstracts," 1983 Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Atlantic City, NJ, March 1983, Abstract 367. (24) Beckey, H. D.; Hilt, E.; Schulten, H. R. J. Phys. E 1973, 6 , 10431044. . .. . . ..
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RECEIVED for review October 28, 1986. Accepted January 20, 1987.
