Anal. Chem. 1987, 5 9 , 1139-1144
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Effect of Experimental Conditions on the Daughter Ion Spectra Derived from Tandem Mass Spectrometry of Steroid Glucuronides Richard B. Cole,’ Christian R. Guenat,* and Simon J. Gaskel12 Laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709
Tandem mass spectrometry has been used for the direct analysls of stereoisomeric sterold conjugates. Daughter Ion spectra derlved from protonated androsterone and etiocholanolone glucuronides (generated vla fast atom bombardment) were monitored as a function of experlmentai parameters (Le., collision gas pressure, collision energy, Protonating agent concentration, and analyte concentration). Varlatlon with analyte concentration of the daughter ion spectrum of [M 4- HI+ derived from androsterone glucuronide apparently results from an enrichment of analyte at the sample surface. Thls concentrationdependent varlation is attributable to elther a change in the Internal energy or a shift In the distribution of sputtered protonated molecular ion species.
Mass spectrometric analysis has been successfully applied to the investigation of polar and nonvolatile compounds, including steroid glucuronides (1-3), since the introduction of fast atom bombardment (FAB) and related techniques of liquid secondary ion mass spectrometry (SIMS) (4). Typically, information pertaining to molecular weight can be readily derived from these experiments (a characteristic of “soft ionization” particle bombardment techniques (5)),but mass spectra thus obtained may be deficient in structurally diagnostic fragment ions. Furthermore, useful information concerning the analyte may be obscured by interfering ions arising from the matrix, contaminants, and additives used to enhance ion detection. Tandem mass spectrometry can help to overcome these limitations by allowing both increased selectivity and increased fragmentation through collisionally activated decompositions (CAD) of a specified parent ion. This approach has been found to be particularly useful for direct mixture analysis (6), where minimal sample cleanup is required. The success of FAB/MS/MS in novel investigations not previously amenable to conventional mass spectrometric methods, such as the ability to distinguish stereoisomers of biochemical interest, will be determined by the reproducibility of tandem MS data. Experimental parameters, however, may have an important effect on the appearance of a CAD spectrum (7). Thus, isomers differentiable under a particular set of experimental conditions may be difficult to distinguish under another. During the course of a study to assess the ability of FAB and tandem mass spectrometry to distinguish between stereoisomeric steroid glucuronides, we have evaluated the influence of several experimental parameters on the relative abundances observed in the daughter ion spectra. Andro‘Also in the Department of Chemistry, University of North Carolina, Chapel Hill, NC. Present address: Laboratoire de Chimie Organique Structurale, Universit6 Pierre et Marie Curie (Paris VI), Bltiment F, 4, Place Jussieu, 75230 Paris Cedex 05, France. On leave from the Tenovus Institute for Cancer Research, University of Wales College of Medicine, The Heath, Cardiff, Wales, U.K. Present address: Baylor College of Medicine, Department of Medicine, Suite 8263, Texas Medical Center, Houston, TX 77030.
sterone glucuronide (3a-hydroxy-5a-androstan-17-one 3glucuronide) (Figure 1)and etiocholanolone glucuronide (the 5P-isomer) yield abundant [M - HI- ions following FAB, but CAD of these parents gives weak and uninformative spectra. CAD of [M H]+ ions, however, provides diagnostic daughter ion spectra. The experimental parameters varied in this study were collision gas pressure, collision energy, protonating agent concentration, and analyte concentration. Fragmentation pathways of these compounds have also been examined in detail by detecting products of consecutive decompositions occurring in separate field-free regions of the tandem mass spectrometer.
+
EXPERIMENTAL SECTION All experiments were performed on a tandem double-focusing four-sector mass spectrometer (ZAB-4F, VG Analytical, Manchester, U.K.) of BlEl-E2B2 configuration (Figure 2 ) , operated in various modes. The FAB (Xe) gun used in these experiments was an Ion Tech Ltd. (Teddington, Middlesex, U.K.) FABllNF saddle-field source. Androsterone glucuronide and etiocholanolone glucuronide were obtained from Sigma Chemical Co. (St. Louis, MO). These compounds were dissolved in a glycerol matrix at the indicated concentration levels (millimolar in glycerol). Sample introduction was accomplished via application of 2-3 WLof analyte solution to a conventional FAB probe (VG Analytical). Conventional FAB mass spectra were collected at the second detector while B1 was scanned (5 s/decade). Mass calibration was performed by using glycerol matrix ions. The data acquisition system used in all modes of mass spectrometer operation (unless otherwise noted) was an 11-250data system (VG Analytical). All daughter ion spectra displayed represent the accumulation of 10 or more linear scans at 15 s/scan. Daughter Ion Spectra as Functions of Collision Gas Pressure, Collision Energy, Protonating Agent Concentration, and Analyte Concentration. Fragmentations of BIE1selected [M + HIt parent ions, occurring in the third field-free region of the tandem mass spectrometer, were monitored by performing a linked scan of B, and E> When the collision cell was at ground potential, the ratio B2/E2was maintained constant during the daughter ion scan. CAD collision gas (He) pressure in the third field-free region was regulated by observing the attenuation in parent ion intensity resulting from gas introduction; reported values are percentages of parent ion intensity remaining. To probe the effects of varying the collision energy, the cell containing the collision gas was floated at incremental potentials between 0 V and the accelerating potential of the ion source (8 kV in all applications). This approach allowed control of the CAD collision energy between 0 and 8 keV in the laboratory frame of reference. A consequence of floating the collision cell at a positive potential is that daughter ions may no longer be detected by performing a linked scan at constant B2/E2. Instrument electronics, however, provide the capability to perform a daughter ion scan with use of the required complex scan law (8). Temporal Variations in Spectral Characteristics. Temporal changes in either conventional or daughter ion spectra were assessed by repetitive monitoring of appropriate diagnostic signals. When the detection of the respective products was alternated, a correlation in temporal changes associated with the two types of experiments could be established. Changes in the daughter ion profile were monitored by repetitively acquiring daughter ion
0003-2700/87/0359-1139$01.50/00 1987 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 8, APRIL 15, 1987
43 1 I,,
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273
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255
467
559
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500
400
300
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Figure 1. FAB mass spectra of source-formed ions from 6.8 mM androsterone glucuronkle (A) and etiocholanolone glucuronide (B), both in glycerol. MAGNETIC
DETECTOR NO 1
E L E CT R IC SECTOR
FIRST FIELD-FREE REGION
d
*+COLLISION CELLNO 1 SOURCE
REGION
ELECTRIC SECTOR
DETECTOR NO. 3
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82
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ZAB-4F Flgure 2. Schematic diagram of VG Analytical ZAB-4F tandem double-focusing mass spectrometer.
spectra generated in the previously outlined manner using the data system. Fluctuations in the relative signal intensity of the source-formed m/z 559 [M + H + glycerol]’ ion were monitored between daughter ion scans on a strip chart recorder. In order not to disturb the tuning of the first magnet, the mlz 559 ion was transmitted through B,EI (to the second detector) via accelerating voltage switching using the peak matching unit. Consecutive Decompositions in Separate Field-Free Regions. The availability of a second collision cell in the second field-free region enabled the monitoring of consecutive collisionally activated decompositions. This was accomplished by first exposing a B,-selected parent ion to collision gas introduced in the second field-free region. A chosen m / z CAD product ion was then energy-filtered through El, whereupon it was subjected to collision in the third field-free region. Resultant CAD products were detected by a liked scan at constant B2/E2 Since the momentum and kinetic energy of product “granddaughter”ions arising from this two collision cell process were identical with those of daughter ions possessing the same m / z value generated via the more conventional one-cell process, the same B2/E2linked scan calibration ( m / z 460-250) could be conveniently used for both experiments. The collision gas pressure in the second collision cell was adjusted to transmit 30% of the steroid glucuronide [M + H]+ ion. Afterwards, the collision gas pressure in the first collision
cell was adjusted in an identical manner.
RESULTS A N D DISCUSSION Conventional FAB mass spectra for 6.8 mM solutions (in glycerol) of the two steroid glucuronide stereoisomers are shown in parts A and B of Figure 1. Even though subtle differences between the FAB mass spectra of the two isomers may exist, there is precedence (9) to expect that the relative intensities of generated ions can change significantly with conditions (compare to parts A and B of Figure 6). The difficulty encountered in distinguishing between the FAB mass spectra, and the potential interference caused by glycerol matrix ions and/or other sample constituents, underscore the utility of the tandem mass spectrometry experiment. An instrumental feature exploited in this study was the ability to monitor consecutive collisionally activated decompositions occurring in separate field-free regions of the tandem mass spectrometer. The ability to readily induce maximum “granddaughter” ion generation by establishing an optimum collision gas pressure in the third field-free region was impeded somewhat by the fact that when successive E,-selected precursor ions generated by CAD in the second field-free region
ANALYTICAL CHEMISTRY, VOL. 59, NO. 8, APRIL 15, 1987
were chosen, the collisional cross section in the third field-free region for the new ion was no longer the same. The implication, of course, is that a collision gas pressure in the third field-free region transmitting, for example, 30% of a particular El-selected CAD daughter ion to the second mass spectrometer will not necessarily transmit 30% of another El-selected ion. The intent of performing this consecutive collision experiment was to gain insight into decomposition pathways via tracing of multiple collision events. The E,-selected daughter ions arising from collisions of Bl-selected androsterone and etiocholanolone glucuronide m/z 467 [M + H]+ ions were the product ions at m/z 449,431,291, and 273. Among these daughter ions, m / z 449 yielded CAD "granddaughter" ions at m / z 431,291,273, and 255. The m/z 291 daughter ion produced "granddaughter" ions a t m / z 273 and 255, while the m / z 273 daughter ion yielded a CAD product ion at m f z 255. The m / z 431 ion generated by loss of two H 2 0 molecules from the protonated molecular ion was apparently too weak in intensity to allow detection of further fragmentations to cationic products in the monitored range (> m / z 250). This experiment verified that a proportion of the ions formed by loss of two water molecules (with or without previous loss of the glucuronic acid moiety) and a percentage of the ions formed by combined water and glucuronic acid moiety losses (occurring in either order) were the result of consecutive fragmentation reactions. All indicated results were achieved for both stereoisomers. In varying the collision gas pressure in a CAD experiment, one is effectively shifting the statistics governing the number of collision events experienced by each analyte ion. The average energy converted to internal energy of precursor ions via collision can be increased by raising the collision gas pressure. Higher energy decompositions are often required to successfully differentiate between two isomeric species with use of CAD (7). Figure 3 demonstrates the effect of collision gas pressure on the CAD spectra of [M + H]+ (mlz 4671, from androsterone and etiocholanolone glucuronides. As the collision gas pressure was incrementally increased with concomitant loss of parent ion transmission, a steady increase in "aglycone" steroid fragment ion abundance (those incorporating loss of the glucuronic acid moiety, Le., m / z 291, 273, and 255) was observed, reflecting an increase in collision probability. At low collision gas pressures, where transmission of the parent ion was high (100% (metastable decomposition only) and go%),the daughter ion spectra were quite similar. At 60%, 30%, and 15% parent ion transmissions, however, reproducible differences in the relative intensities of CAD products became more evident. The contribution of metastable processes to the observed daughter ion spectra would be expected to diminish with decreasing parent ion transmission since a higher fraction of the parent ions interact with the collision gas. Two characteristics of the CAD spectra obtained a t 30% (or 15%) parent ion transmission can be used as diagnostic criteria to identify the particular stereoisomer in the situation where a unique configuration is present. The first is that for androsterone glucuronide, m / z 291 intensity is always less than m/z 273 intensity. The opposite is true for the etiocholanolone isomer. Secondly, for androsterone glucuronide, the ratio of m / z 273255 is always >2; for the etiocholanolone isomer, this same ratio is always
