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Anal. Chem. 1985, 57, 2425-2427 (7) Meyer, R. F.; Hartwick, R. A. Anal. Chem. 1984, 56, 2211-2214. (8) Lin, P. Y. T.; Bulawa, M. C.; Wong, P.;Lin, L.; Scott, J.; Blarlk, C. L. J . Liq. Chromafogr. 1984 7 (3). 509-538. (9) Welling, P.; Poppe. H.; Kraak, J. C. J . Chromatogr. 1985, 327, 450-457. (10) Scott, R. P. W. I n “Advances in Chromatography”; Giddings, J. C., Grushka, E., Cazes, J., Brown, P. R., E&.; Marcel Dekker: New York, 1983; Vol. 22. pp 247-294.
(11) DiCesare, J. L.; Dong, M. W.; Ettre, L. S. Chromafographia 1981, 74 (5).257-268.
RECEIVED for review February 19, 1985. Accepted June, 11, 1985. This ~ ~ owas r k supported by a grant from the Scottish Rite Schizophrenia Research Foundation.
Utility of Silver Ion Attachment in Fast Atom Bombardment Mass Spectrometry Brian D. Musselman,* John Allison, and J. Throck Watson
Departments of Biochemistry and Chemistry, Michigan State University, East Lansing, Michigan 48824 The addition of metal salts to the glycerol (G) solvent utilized in fast atom bombardment (FAB) mass spectrometry (MS) results in the generation of adducts between the cation and molecules of both the analyte and solvent. Several investigators have outlined procedures for the addition of a salt such as LiCl to the solvent in order to produce adduct ions such as (M Li)+ (where M = analyte) (1). The presence of these salts leads to additional unwanted ionic species in the mass spectrum. These include adduct ions arising from the combination of the cation with the solvent to form ion clusters Li)+ and ions which may be the product of such as (G, unimolecular dissociation of these clusters (2). Additional problems are apparent when samples of biological origin are analyzed. The presence of sodium and potassium ions in these samples results in the formation of both cationated solvent ions (G, + Na+),cationated analyte (M Na)+, and cluster ions containing one or more cations such as (G, + Na + K - H)+, which further complicate interpretation of the mass spectrum. These experiments detail the use of silver salts dissolved in the FAB solvent for the generation of unique cation-analyte clusters which can be used to identify the molecular weight of unknown components while minimizing the generation of new cluster species. For purposes of illustration, consider the FAB mass spectrum of cholic acid (mol wt 408) in Figure la as an unknown. The three major peaks could represent protonated molecules of three different analytes having even molecular weights or they could represent cluster ions and fragment ions of a single analyte. An alternate interpretation of the spectrum suggests that the peak a t m / z 373 could while the peak represent a protonated molecule (M H)+, at m / z 355 could represent the fragment ion (M H - H20)+ arising from the loss of water from the protonated molecule, a fragmentation frequently observed in mass spectra obtained by FAB ionization of carbohydrates. Both of these interpretations are reasonable, but incorrect in this case, as revealed during subsequent analysis of the analyte in glycerol containing 0.14 M AgN03 which resulted in the generation of an adduct, (M + Ag)+. Figure l b illustrates the results of this analysis. Abundant ionic species which were not apparent in the original mass spectrum were obtained a t m / z 515 and 517. Additional analyses of the sample a t different concentrations indicated that the abundance of these ions could be correlated to sample concentration. The silver ion adducts are easily recognized by virtue of the characteristic peak intensity pattern (51%:49%) of the 1mAg:109Agisotopes. The appearance of (Aganalyte)+adducts is evident even when the protonated analyte molecule is not observed in mass spectra obtained using pure glycerol as a solvent (Figure la). This analysis of cholic acid in two different FAB matrices illustrates the potential of using silver salts to produce (Aganalyte)+ adducts which can be used to
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0003-2700/85/0357-2425$01.50/0
determine molecular weight information for unknown components. The molecular weight of the analyte in this case is determined to be 408 as obtained by subtracting 107 from the nominal mass of the (Aganalyte)+adduct a t m / z 515. With this verification of the molecular weight by intentional formation of the silver ion adduct, major peaks in the original FAB mass spectrum, Figure la, can be interpreted as labeled in Figure lb. Note that the peak at m / z 409 representing the protonated molecule is not readily distinguishable in either spectrum (Figure l), a feature that would surely contribute to misinterpretation of the FAB spectrum in Figure la, if it were an unknown. Protonated cholic acid dimer (mlz 817) is also present, but not shown in the partial mass spectra in Figure 1.
EXPERIMENTAL SECTION Solutions of silver salts in glycerol were prepared by first dissolving the salt in water to make a 5% w/w solution; aliquots of this solution were subsequently added to known volumes of glycerol to obtain the desired concentration of silver salt. Mixing was completed with a magnetic stirring apparatus. All FAB analyses were completed using 1pL of a glycerol solution containing the silver salt as delivered from a 0-5-pL micropipet onto the aluminum FAB probe tip. A series of experiments were conducted to determine the relative utility of silver salts (AgC10, and AgN03) dissolved in glycerol. The utility of granulated silver metal (-222, +325 mesh size Cerac, Inc., Milwaukee, WI) suspended in the solvent, was also examined. Optimum concentrations of each silver salt required for generation of silver adducts were determined. Silver metal suspended in the glycerol did not result in the formation of silver adducts with the analyte or the solvent. Solutions of sucrose, acetaminophen sulfate, arachidonic acid, the tripeptide Leu-Gly-Phe, and cholic acid were prepared by dissolving each in H,O/methanol (75:25) to the desired concentration; 1-pL aliquots of this solution were applied to the FAB probe tip upon which a 1-pL aliquot of the silver salt in glycerol solution had been applied previously. Excess water/methanol was evaporated from the probe tip by cautious use of a heat gun. Aliquots of a solution of 1.8 g of sucrose in 10 mL of water were g of AgN03, pipetted into 0.5 g of glycerol containing 9 X mixed by agitation, and analyzed after 5-min intervals. The relative abundances of the protonated molecule and the silver ion adduct of the analyte were monitored as a function of sucrose concentration. A similar experiment was completed using aliquots of 1.0 X lo-’ g of tripeptide dissolved in 5 mL of water/methanol (4;l). Determination of the minimum concentration of silver salt necessary to provide useful analytical data was completed as follows: glycerol (0.5 i 0.03 g) was added to each of ten l/*-dram vials. A stock solution of 0.45 g of AgN03 in 1 mL of distilled water was prepared. Volumes of 5, 10, 15, 20, 25, 30, 40, 50, 7 5 , and 100 pL of the stock solution were pipetted into each of the ten vials. Distilled water was added to each of the first nine vials in order to make a final volume of 100 FL in each vial. Analysis of the tripeptide, Leu-Gly-Phe, was completed by measuring the 0 1985 American Chemical Society
a-
rnh 355 m l z 373
l
m
7x10'
E 0
m
0
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9 a -
4
mi2
501
5x103
ABSOLUTE ION INTENSITY 3x10
I I
(S+ H+)
I
I
I
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I
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3
4
[~ucrose], pg/pr
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Figure 1. FA6 mass spectra of cholic acid (mol wt 408)dissolved in (a)glycerol, and (b) glycerol containing 0.14M silver nitrate. Promlnent fragment ions at m l z 355 and 373 indicate that even under "soft"
ionization conditions the molecule is unstable. Silver adduct ions at m l z 515 and 517 demonstrate that even when the protonated molecule is not present under normal FAB conditions, it is possible to obtain molecular weight information using the silver salt in the glycerol solvent. abundance of the protonated molecule and that of the silver ion adduct generated upon addition of a constant concentration of analyte to each of the ten solutions. It should be noted that all FAB mass spectra are time dependent; as the sample and solvent on the FAB probe tip are depleted, relative intensities of ions vary. In these experiments, this is also the case. The results discussed represent spectra obtained early in the experiment- at times when consecutive spectra do not change and no significant depletion of the sample has occurred. It should also be noted that solutions of glycerol/analyte/silver salt should not stand for long periods of time before the FAB analysis is performed. Experiments were carried out on a modified Varian MAT CH5 double focusing mass spectrometer equipped with an Ion Tech FAB gun (Teddington, UK). Details of the instrument configuration have been published previously (3). Ultra-high-purity xenon was used to generate a beam of fast atoms (8 kV) in all experiments.
RESULTS AND DISCUSSION Cation attachment has been exploited in numerous experiments involving desorption ionization (DI) techniques. The addition of alkali metal salts to samples in field desorption ( 4 ) and molecular secondary ion mass spectrometry (SIMS) (5) was found to enhance the ionization of polar molecules being analyzed. A common problem in these and other DI techniques is the appearance of ions in the mass spectrum arising from the association of multiple cations with analyte and/or solvent. Thus, the addition of LiCl to the sample results in the formation of ionic species such as (M + 2Li H)+and (2M Li)+which can complicate attempts to identify the analyte (M). Mass spectra obtained from SIMS experiments of silver surfaces demonstrate the utility of silver adduct ion formation in producing distinguishable molecular ion containing species; however, the presence of ionic surface contaminants reduces the effectiveness of these experiments in unequivocable identification of peaks which can be used
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Flgure 2. Absolute ion Intensity of molecular Ion obtained from analysis of sucrose (S)in glycerol ( 0 )and in glycerol containing 0.14M AgNO, (0, 0).Absolute ion intensity of (S 3. Ag)' in glycerol containing AgNO, is greater than that of (S H)' In glycerol alone. A decrease in the absolute ion intensity of (S 4- H)' is apparent upon additon of AgN0, to the glycerol.
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to calculate or determine molecular weights (6). Sucrose has been used as a model nonvolatile compound in numerous studies of DI techniques (7-9); for this reason our initial investigations of cationization in FAB were conducted with this analyte. The addition of successive aliquots of sucrose (S), to a matrix of 0.14 M AgN03 in glycerol was completed in order to determine the concentration of analyte necessary for the formation of detectable ion currents for the protonated sucrose and the silver ion adduct of sucrose. A plot of ion intensity for both the protonated sucrose, (S H)+, and the silver ion adduct, (S Ag)+, vs. the concentration of sucrose is shown in Figure 2. The graph indicates that the silver ion adduct species appears at concentrations as low as 0.4 X pg of sucrose/pL while the protonated molecules are not apparent until a concentration of 2.5 X ,ug/pL sucrose is present on the probe tip. The absolute abundance of the adduct ion was found to increase rapidly as sucrose concentration increased. These data suggest that the addition of silver nitrate to glycerol lowers the detection limit for this analyte by as much as a factor of 5. Experiments similar to those described above were completed using the tripeptide, Leu-Gly-Phe, and cholic acid with somewhat different results. The results of the tripeptide analyses are plotted in Figure 3. The formation of the protonated tripeptide was suppressed in the presence of a silver salt in the glycerol. Also, the silver ion adduct did not appear until ions from the protonated tripeptide were already apparent in the mass spectrum. These data are in direct contrast to the earlier results from the analysis of sucrose and suggest that the formation of ionic adduct species varies from molecule to molecule. A third type of behavior is seen for the analyte cholic acid. Cholic acid does not form an (M + 1)' ion, while the addition of AgN03 does produce an (M + Ag)' adduct, the abundance of which is concentration dependent. Compounds whose behavior in a glycerol/AgN03 matrix under FAB conditions have been examined include the dipeptide Ser-Ala, arachidonic acid, methyl linoleadic acid ester, and the Acetaminophen (APAP) metabolites APAP sulfate, APAP-cystine, and 3-0-methyl-APAP. These compounds are representative of a wide range of functional groups frequently
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ANALYTICAL CHEMISTRY, VOL. 57, NO. 12, OCTOBER 1985
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L I
6~10~Absolute Ton Titensity 4 x IO3
Absolute
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Intensity
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P
Ion
2 x lo3
1 2
tI
P
2 x io4
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IO
Ixi04
P
[Leu-Gly-Phe] x IO'M Figure 3. Absolute ion intensity of molecular ion obtained from the analysis of the tripeptlde (Leu-Gly-We) in glycerol (U)and glycerol containing 0.14 M AgNO, (0, 0). A decrease in absolute ion Intensity Is observed upon addition of silver ,saR to the glycerol.
encountered in FAB-MS analysis. Silver ion adduct formation with each analyte (M) was limited consistently to ions corresponding to m/z of (M 107) and (M 109) even in instances where FAB of the anal@ from pure glycerol generated the most prominent ion containing the analyte molecule as (M - H)+ or (M 2K - H)+, which would complicate interpretation of the original mass spectrum. The use of other salts such as silver perchlorate as a source of silver ions for the formation of silver adducts was also investigated. The results of these investigations were that, although silver ion containing adducts were readily formed with analyte, the spectrum background was excessive and background ions containing C1 were formed. Addition of silver nitrate salt to the glycerol did not increase contributions to the spectrum background other than to produce those ions due to the combination of silver with glycerol at m/z values corresponding to (G, Ag - H)+ which can be readily identified. Studies of different concentrations of silver nitrate salt in glycerol indicate that 0.43 M AgN03 in glycerol yields intense adduct ion concentrations; however, the intensity of these ions was not reproducible for solutions analyzed immediately after preparation. The use of 0.14 M AgNO, in glycerol resulted in the formation of stable relative intensities of the silver adducts of glycerol over a 24-h period. The formation of more complex ions such as those containing multiple silver atoms is also minimized by utilizing the lower concentration of silver salt. Experiments directed at determining the optimum concentration of silver salt for generating adducts were carried out using the tripeptide Leu-Gly-Phe as analyte. A plot of intensity of protonated molecule ion current and tripeptidesilver adduct ion current vs. silver ion concentration in glycerol is shown in Figure 4. The plot indicates that as the concentration of the silver salt increases, the abundance of protonated molecules initially increases and then decreases. The silver-tripeptide adduct ion abundance increases slowly as silver salt concentration rises until a plateau is reached a t 2 X M AgNOB in glycerol. This experiment indicates that the presence of excess silver salt results in decreased abundance of protonated molecules of analyte, thus use of a 1.2 x M AgNOB in glycerol matrix is recommended for the
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Flgure 4. Absolute Ion intensity of protonated trlpeptide (M H)' and silver-tripeptide adduct (M Ag)' as a function of silver salt concentration in the glycerol solvent used for FAB-MS. A 30 nmol sample of Leu-Gly-Phe was used for each analysis.
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formation of the maximum intensity of silver adducts while minimizing the loss of the protonated molecular species. That is, we feel the ideal situation for analysis is to obtain a spectrum which contains the (M Ag)+ doublet and the confirming (M H)+ ion. The presence of ions containing multiple silver cations, e.g., (M 2Ag)+, was not evident in the mass spectra acquired using this technique, owing to the use of low concentrations of silver salts in the glycerol. Use of glycerol containing more than 0.14 M AgN03 results in the formation of (G 2Ag H)+ ions which contribute additional ionic species to the background in the mass spectrum. Finally, an additional advantage of the use of this tehcnique is that trace quantities of contaminating cations (e.g., Na+ and K+) need not be eliminated for obtaining adequate silver ion adducts. Formation of ions containing both silver and sodium was not evident in any of the mass spectra obtained by using this methodology. Registry No. Leu-Gly-Phe, 17608-53-6;Ag, 7440-22-4;cholic acid, 81-25-4; sucrose, 57-50-1.
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LITERATURE CITED Dell, A.; Baiiou, C. E. Biomed. Mass Spectrom. 1983, 10, 50. Cooks, R. G.; Busch, K. L. I n t . J . Mass Spectrom. Ion Phys. 1983,
53,111. Ackermann, E. L.; Watson, J. T.; Newton, J. F., Jr.; Hook, J. E.; Braselton, W. E., Jr. Biomed. Mass Spectrom. 1984, 1 1 , 502. Vieth, H. J. Angew, Chem. 1976, 88, 762. Liu, L. K.; Busch, K. L.; Cooks, R. G. Anal. Chem. 1981, 53, 109. Grade, H.; Winograd, N.; Cooks, R. G. J . Am. Chem. SOC.1977, 99, 23. Slmons, D. S.; Colby, E. N.; Evans, C. A., Jr. I n t . J . Mass Spectrom. Ion Phys. 1974, 15,291. Moor, J.; Waight, E. S. Org. Mass Spectrom. 1974, 9 , 903. Posthumus, M. A.; Kistemaker, P. G.; Meuzelaar, H. L. C.; Ten Noever de Brauw, M. C. Anal. Chem. 1970, 50, 985.
RECEIVED for review March 8,1985. Accepted June 14,1985. This work was made possible through the financial support of the Biotechnology Resources Program of the Division of Research Resources of the National Institutes of Health (NIH Grant RR00480-16).
