2006
Anal. Chem. 1982, 5 4 , 2006-2008
Characterization of Anthracycline Antibiotics by Desorption Chemical Ionization Mass Spectrometry Ronald G. Smith Department of Developmental Therapeutics, The University of Texas
Desorption chemical ionization mass spectrometry is used to obtain positive and negative ion spectra of 10 anthracyciine antibiotics. These compounds provide relatively few, but intense, negative ions due to resonance electron capture of thermal electrons by the anthraquinone nucleus. The spectra exhibit molecular anions and ions derived from the aglycon, aglycon H,O, aglycon - 2 H,O, and aglycon 0 these can be obtained from as little as 1.0 ng of the compound. By comparison, a greater variety of positive ions are observed, including several derived from the giycosldlc moiety.
-
-
Anthracycline antibiotics are clinically important in the treatment of cancer. This is due, in part, to the success of doxorubicin, which has demonstrated a broad range of clinical activity and is one of the most effective anticancer drugs in use. This success has stimulated a great deal of interest in new anthracyclines, obtained from both natural sources and semisynthetic methods, in the hope of finding analogues with even greater clinical efficacy. Structure elucidations of naturally occurring anthracyclines are tedious, involving chemical degradation and derivatization coupled with IR, UV, IH NMR, 13CNMR, circular dichroism, and mass spectrometry (1-3). The glycosidic components frequently can be identified by cochromatography of hydrolysis products with authentic samples ( 4 ) . Other than providing the molecular weight of the aglycone after hydrolysis, the role of mass spectrometry has been minor. Recent developments in producing ions from thermally labile and nonvolatile compounds ( 5 , 6 )should enable molecular weight measurements of the intact anthracyclines and carbohydrate moieties as well as other structural characteristics. In addition to aiding the identification of new anthracycline natural products, mass spectrometry should be invaluable for characterizing minute samples of metabolites in pharmacologic studies. Anthracycline metabolites previously have been characterized by electron ionization mass spectra of the underivatized aglycons (7,8).Another method was developed to characterize the per(trimethylsily1)methoxime derivatives of aglycons by electron ionization (9, 10). A similar method was unable to provide molecular weight information from the intact compound (11). Field desorption mass spectrometry gives molecular ions for several anthracyclines (12,13) but is not always unambiguous (14) and involves instrumentation and techniques not available to many researchers. Desorption chemical ionization (DCI)has been useful for characterizing carbohydrate conjugates (15,16)and offers the advantages of simplicity and relative low cost. We have obtained positive and negative DCI spectra for a series of anthracycline antibiotics. The negative ion spectra are particularly useful, providing intense anions characteristic of these compounds and their aglycons.
EXPERIMENTAL SECTION Mass spectra were obtained with a Finnigan 3300F mass spectrometer equipped with a chemical ionization source, positive-negative ion detection capability with a conversion dynode
M. D. Anderson
Hospital and Tumor Institute, Houston, Texas 77030
electron multiplier, and interfaced with an Incos 2300 data system. The DCI probe (Vacumetrics,Ventura, CA) is a modification of that described by Bruins (17) and maintains the potential of the probe tip to that applied to the ion source. The probe current was linearly programmed from 0 to 2 A at 2 A/min. Sample ion currents were greatest with 1.0-1.5 A probe current. The source temperature was held at 120 "C and the electron multiplier was set at 1300 V. Methane (0.5 torr) was the reagent gas for both positive and negative ion spectra. The instrument was calibrated with perfluorotributylamine (FC-43) in the positive mode, and mass assignments above m/z 614 were checked by using a perfluorinated polyether (Fomblin-L)in the negative mode. Spectra were obtained every 3 s, scanning m / z ranges of 100 to 50-60 beyond the molecular weight of each compound. Anthracyclines 1-10 (Figure 1) were provided by the Division of Cancer Treatment, National Cancer Institute, National Institutes of Health. Methanolic solutions of these compounds were applied to the DCI probe tip with a syringe. The sensitivities of positive and negative DCI were compared with samples obtained by serial dilutions of daunorubicin (4) and N-trifluoroacetyldoxorubicinvalerate (8) in methanol. The mass ranges scanned for these experiments were m/z 350-550 for 4 and 450-750 for 8. The platinum probe tip was cleaned by heating in a Bunsen flame before the application of each sample. A second component of the dibenzyldaunorubicin sample (4) was observed and isolated by preparative thin layer chromatography using silica gel plates with fluorescent indicator. The developing solvent, ethyl acetate, moved 4 with the solvent front, while the impurity remained at the origin. The impurity was eluted from the silica gel with methanol.
RESULTS AND DISCUSSION Anthracyclines 1-10 represent a variety of naturally occurring (1-5) and semisynthetic (6-10) compounds of interest for their antitumor activity. Although both positive and negative ion mass spectra of the anthracyclines provide structural information, the greater negative ion currents make this mode particularly useful. The anthracyclines possess the anthraquinone nucleus and are thus capable of undergoing one- and two-electron reductions (It?), change transfer complex formation (19),and other chemical reactions indicative of their high electron affinities. Indeed, anthracyclines are believed to undergo an autocatalytic reduction-oxidation in the presence of quinone-reducing enzyme systems and molecular oxygen, which may account for lipid peroxidation in biological membranes (20), a possible cause of the dose-limiting cardiotoxicity associated with these drugs. The electron affinities of these compounds suggest they should produce stable anions by resonance capture of thermal electrons generated under chemical ionization conditions. This is supported by the negative ion spectra reported for anthraquinone (using IO-eV electrons)(21) and for two anthraquinones related to aflatoxins (22). The negative ion spectra of anthracyclines (Table I) have relatively few ions; these possess the anthraquinone moiety, with and without the carbohydrate group. The molecular anions are usually very intense and constitute the base ion in six of the ten compounds studied. Three anthracyclines (7,9, 10) with significantly less intense molecular anions have synthetically modified side chains at the 9-position. The increased bulk of these side chains may contribute to their
0003-2700/82/0354-2006$01.25/00 1982 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 54, NO. 12, OCTOBER 1982
2007
Table I. Methane Negative Ion Chemical Ionization Spectra of Anthracyclines % re1 abundance compd no.
(aglycon -
mD1
wt
M-,
1
787
100
2
811
97
3 4 5
513 527 543
100 100 42
6 7
707 67 3
8 9 10
723 683 645
a
(aglycon)-.
H,O)-.
(aglycon - (aglycon 2H,O)-.
O)-.
other, m / z ( % re1 abundance) 773 (3), 769 (3), 729 ( 7 ) , 535 (3) 797 (si, $93 (21), 699 (31, 569 ( 2 ) , 215 (2) 527 (13), 482 ( 4 ) 543 (3), 336 ( 2 ) 507 ( 8 ) , 505 (lo), 489 (12), 483 (6), 336 (92) 617 (14)a 655 (2), 414 (5), 378 (3), 336 ( 4 ) 621 ( 3 ) 354 (3), 336 (53) 527 (4), 376 (3), 362 (1)
1
82
12
5
2
100
8
100
100
2 2 18
10 4 62
12 38
100 7
100
2
4 20
9
100 1 6
2 14 8
9 100 100
28 27 38
4 23 21
Monobenzyldaunoru bicin contaminant ion.
Table 11. Methane Positive Ion Chemical Ionization Spectra of Anthracyclines % re1 abundance
(glycoside (aglycon compd no.
( M + H)+
+ H)+
1
100
2
41
3
9
3
4
16
5 6
3 100
7
2
8 9
2
10 a
(aglycon
-
(aglycon
H,O
+ H)+ 12
(glycoside - 2H,O (glycoside - H,O t H)+ + H)+ + H)' 70
-
H,O
14
98
78
14
4
47
7
100
31
13
2u
35
18
49
100
5 1
26 2
7 9
8 3
21 27
100 8
6
7
7
16
38
26 2
100 52
17
12
21 32
42 24
3
20
17
2
11
61
18
-
NR,R, + H)+
other, m / z ( % re1 abundance) 770 ( 1 3 ) , 185 (15), 1 7 1 (13), 113 (46) 794 (12). 570 ( 7 ) , 550 (17), 176 (69), 158 (82), 113 (100) 478 (9), 460 ( 8 ) , 377 (lo), 369 ( l l ) ,307 (6), 224 (ll), 206 (17) 510 (4), 427 (14), 321 (41), 224 (7), 206 (10) 337 (7), 224 (lo), 206 ( 9 ) 618 (12),a 220 (20),a 132 (19) 529 (6), 463 (25), 379 ( 3 ) , 224 (4), 206 ( 4 ) , 103 (100) 639 (1).483 (20) 337 (5jI 172 (isj, 159 (171, 146 (52), 144 (100) 224 (3), 206 (6), 191 (6), 137 (38), 115 (100)
Monobenzvldaunoru bicin contaminant ion.
thermal instability. The dissociative electron capture process, which leads to (M - H)- ions in some hydroxylated compounds (23), is not observed, presumably due to the dominant influence of the anthraquinone electron affinity. Four different types of #anionsapbear in the aglycone region of these spectra; these correspond to the aglycon, aglycon H,O, aglycon - 2Hz0, anld aglycon - 0. The losses of water are clearly from the nonaromatic D ring t~ form double bonds, presumably in conjugation with the dnthraquinone, and to form a fully aromatic nalphthacenequinone. The aglycon 0 ions arise from the reductive loss of the carbohydrate group. Although this would seem an unusual mass spectrometric process, it is readily explained in light of similar reductive cleavages of anthracyclines in chemical (24) and enzymatic reactions (4). The reducing agent ih this case is thermal electrons. The major positive ionis in the methane CI spectra of anthracyclines 1-10 are listed in Table 11. The ion current is generally lower than that observed in the negative ion mode, and the relative intensities of the M + H+ ions are less than the corresponding molecular gnions. The positive ion spectra
can be useful for confirmation of the molecular weights of the intact aiithracycline as well as the individual aglycon and carbohydrate groups. The molecular weights are usually supported by the presence of M + H+, M + C2H5+,and M + C3HS+ions. Aglycon ions are present as the protonated form of the aglycon, aglycon - HzO, and aglycon - 2H20. Some of these are accompanied by ions at 29 and 41 mass units up from the aglycon molecular weights. Several prominent ions are derived from the carbohydrate group. Anthracyclines with daunosamine exhibit ions a t mlz 148 (protonated daunosamine), mlz 130 (148 - H,O), and m / z 113 (148 - H 2 0 - NH3). Anthracyclines with N and 0 substituents on daunosamine (2,8,8) exhibit ions at mlz values that reflect these changes; the ion at m / z 113 is not shifted. The positive ion spectra frequently exhibit a number of additional ions not readily explained. This study was conducted essentially without regard to quantitative sample measurements. Most spectra were obtained from sample sizes of 100 ng to a few micrograms. Despite this, it became obvious that useful negative ion spectra could be obtained from considerably smaller sample sizes than
ANALYTICAL CHEMISTRY, VOL. 54, NO. 12, OCTOBER 1982
2008
OH
I
CH,O
I,
nogalamycln
2,
aclacinomycin,
A
consistent with monobenzyldaunorubicin with a molecular anion at mlz 617 and aglycon ions at mlz 362,382, and 398. Because dealkylation has been reported as a photolytic process of aclacinomycin A (4), it is uncertain whether this impurity was present in the original sample or formed during the present study. Anthracyclines are an important class of compounds that can be characterized by DCI mass spectrometry,which affords the molecular weights of these compounds and their aglycons. Particularly well suited for this purpose is the detection of negative ions, owing to the high electron affinity of the anthraquinone nucleus. We are currently using this technique in the characterization of anthracycline metabolites isolated in clinical studies.
ACKNOWLEDGMENT The author thanks John D. Douros, Natural Products Branch, Division of Cancer Treatment, NCI, and Ven L. Narayanan, Drug Synthesis & Chemistry Branch, Division of Cancer Treatment, NCI, for samples of the anthracyclines used in this study.
HO R, 3-10
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Rz
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M
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LITERATURE CITED Wiley, P. F.; Kelley, R. B.; Caron, E. L.; Wiley, W. H.; Johnson, J. H.; MacKellar, F. A.; Mizsak, S. A. J. Am. Chem. SOC. 1977, 9 9 ,
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