Anal. Chem. 1982, 5 4 , 2219-2224 (14) McCluskey, G. b.; Kondrat, R. W.; Cooks, R. 12. J . Am. Chem. SOC. 1978, 100,6045. (15) Wilson, B. W.; Pelroy, R.; Cresto. J. T. Mutal. Res. 1980, 79, 193. (16) Paudler, W. W.; Cheplen, M. Fuel 1979, 5 8 , 775. (17) Novotny, M.; Kump, R.; Merli, F.; Todd, L. J. Anal. Chem. 1980, 52, 401. (18) Later, D. W.; Lee, M. L.; Bartle, K. D.; Kong, R. C.; Vassllaros, D. L. Anal. Chem. 1981, 5 3 , 1612. (19) Later, D. W.; Li?e, M. L.; Wilson, B. W. Anal. Chem. 1982, 5 4 , 117. (20) Wilson, B. W.; Peiroy, R. A.; Lee, M. L.; Later, D. W. Presented at the 29th Annual Conference of the American Society for Mass Spectrometry, Minneapolis, MN, May 1981; Abstr. MPMOB5. (21) Zakett, D.; Shaddock, V. W.; Cooks, R. G. Anal. Chem. 1979, 51, 1849. (22) Beynon, J. H.; Cooks, R. G.; Amy, J. W.; Baitinger, W. E.; Ridley, T. Y. Anal. Chem. 1973, 4 5 , 1023A. (23) Wood, K. V.; Cooks, R. G., Purdue University, unpubllshed results, 1981. (24) Latven, R. K.; Enke, C. G. Presented at the 29th Annual Conference of the American Eioclety for Mass Spectrometry, Minneapolis, MN, May 1981; Abstr. FAMOA3.
2219
(25) McLuckey, S. A.; Glish, G. L.; Cooks, R. G. Int. J . Mass Spectrom. Ion Phys. 1981, 3 9 , 219. (26) Douglas, D. J. J . Phys. Chem. 1982, 8 6 , 185. (27) Wrlght, G. L.; McLuckey, S. A.; Cooks, R. G.; Wood, K. V. Int. J . Mass Spectrom. Ion Phys 1982, 42, 1 15. (28) Ast, T.; Beynon, J. H.; Cooks, R. G. Org. Mass Spectrom. 1976, 1 1 , 857. (29) Zakett, D. Ph.R. Thesis, Purdue Unlverslty, 1981. (30) Yost, R. A.; Enke, C. G.; McGllvery, D. C.; Smith, D.; Morrison, J. D. I n t . J . Mass Spectrom. Ion Phys. 1979, 3 0 , 127. (31) Richter, W. J.; Schwarz, H. Angew. Chem., I n t . Ed. Engl. 1978, 77, 424.
.
RECEIVED for review December 23, 1981. Accepted July 8, 1982. This research was supported with funds from the Department of Energy (DE-FG22-81PC40780), the Department of Transportation, and Conoco. J.D.C. thanks International Harvester for a fellowship in support of this research.
Liquid Ion Evaporation/Mass Spectrometry/Mass Spectrometry for the Detection of Polar and Labile Molecules B. A. Thomson" S C I f X , 55 Glen Cameron Road, Thornhill, Oniario, Canada L3T l P 2
J. V. Irlbarne and P. J. Dzledzic McLennan Physical Laboratories, University of Toronto, Toronto, Ontario, Canada M5S l A 7
The new technique termed llquld Ion evaporatlon has been coupled to a trlple quadrupole mass spectrometer system In order to demonstrate the detection of labile compounds. Quasl-molecular parent Ions are generated, whlch can be fragmented In the CID region of the Instrument to provide structural Informatlon. Prelonlzed specks Including acids, salts, drugs, amino aclds, peptides, nucleosides, and nucleotides can be detected, and thelr CID spectra are reported. The coupllng of i~gentle Ionization technlque to a tandem mass spectrometer system Is shown to provide an attractive comblnatlon of molecular weight and fragmentatlon Information for blochemlcal compounds.
The inability to thermally vaporize many compounds without inducing decomposition has in the ]past been a limiting factor in the appliication of mass spectrometry to the detection of involatile and polar compounds. Identification usually requires the presence of molecular weight information in the mass spectrum in the form of molecular (M+, M-) or quasimolecular (MH+, [M - HI-) ion peaks, and uncontrolled decomposition often results in a spectrum which is representative of pyrolysis products of the molecule. Therefore, beginning in the late 1960s with field desorption ( I ) ,there has been a steadily increasing effort, directed a t developing more gentle vaporization/ionization techniques which can provide ions Characteristic of the molecular weight. The past several years has seen, for example, the development of laser desorption (2), plasma desorption (33,direct exposure {chemicalionization ( 4 ) ,electrohydrodynamic ionization (5), fast atom bombardment (6),secondary ion mass spectrometry (9, and ion 0003-2700/82/0354-2219$0 1.25/0
thermospray (8). Each technique has its own more or less special requirement for sample preparation, and no one technique has proven to be a universal solution to the problem, either in the types of compounds to which they are suited or in their ability to provide both ions which are characteristic of the molecular weight, and fragment ions characteristic of the structure. A partial resolution of this latter problem may be provided by the relatively recent developments in the technique of tandem mass spectrometry or MS/MS (9-11), wherein ions from the source region may be individually selected according to their mass, and fragmented in either low- or high-energy collisions with a neutral gas target. The resulting fragment ions are then analyzed by either their mass or energy, to produce a daughter ion spectrum for each parent ion extracted from the source. The use of this technique thus relaxes the requirements of producing both molecular ions and fragment ions in the source. In fact the overall signal to noise can be improved by using a very gentle ionization technique to concentrate all of the ion signal from the source in the parent ion and electronically tuning the MS/MS for the desired degree of fragmentation and maximum transmission. The full potential of combining a soft ionization source with MS/MS has not yet been explored but appears to be promising (12). This paper presents results of coupling one such technique-liquid ion evaporation-with a triple quadrupole MS/MS instrument, to show both the range of compounds for which the ionization technique is suitable and to show the daughter ion spectra of some of the quasi-molecular parent ions. The examples which are discussed have been chosen as examples of relatively polar or labile compounds of biological or pharmaceutical interest. In all cases, the quasi0 1982 American Chemical Society
2220
ANALYTICAL CHEMISTRY, VOL. 54, NO. 13, NOVEMBER 1982
molecular parent ion dominates the spectrum when the collision gas is turned off. Each parent ion can be selected (from compounds which are individually present in the source, or present in a mixture) and fragmented in order to elucidate the structure of the compound. The process of liquid ion evaporation has recently been described from both the theoretical and practical point of view (13, 14). Briefly, ions which are present in a liquid solution, such as water, can be emitted into the air by the presence of a strong electric field at the surface, in a manner which is analogous to that of field desorption. No heat is employed. This process differs from field desorption in that the ions are emitted into an atmospheric pressure gas-generally airrather than into a vacuum. The extremely high electric fields required are generated at the surfaces of tiny charged solvent droplets containing the dissolved ions. The ions are ejected from the liquid in the final stages of droplet evaporation and are sampled from the high-pressure (760 torr) source through a small orifice into the mass spectrometer. A variety of compounds have been tested, including organic and inorganic salts, acids, alkaloids, amino acids, catecholamines, nucleosides and nucleotides. All were dissolved in aqueous solution, with the pH adjusted where necessary to ensure a high degree of ionization in solution, and the solution was sprayed into the air. All preionized compounds were detected as either MHf or (M - H)-, in accordance with their form in solution, and with virtually no fragmentation attributable to the evaporation process. In addition, sugars and other less polar compounds have been observed (although with reduced sensitivity) by either alkali or halide ion attachment in solution (14). In this paper, we discuss results from some of the more labile compounds which have been tested.
EXPERIMENTAL SECTION Compounds to be ionized are dissolved in aqueous solution at to mol/L to provide a suitable ion signal. the level of Between 10 and 60 s are required to record a full scan depending upon the signal strength. At a liquid flow rate of 1mL/min and a solution concentration of M, approximately 16-100 nmol of compound is required for analysis at the present time. Salts, hydrochlorides, and compounds which are strongly acidic or basic can be analyzed with no further treatment. Other compounds which may not be fully dissociated or ionized in solution are treated by adding an acid (HCl and H2S04have been used) or a base (NaOH or NH40H) to provide the fullest degree of preionization possible. In general, equimolecular mixtures of the compound with the base or acid have been used in the experiments to date. For compounds which are only weakly basic or acidic, this addition increases the ion signal. The solution is nebulized into the air with a simple air-blast sprayer consisting of fine stainless steel hypodermic needles, and a charge is induced on the sprayed droplets by means of a small electrode placed close to the sprayer. This electrode, maintained at 2-3 kV, is of the opposite polarity from the ions to be analyzed. The plume of charged mist is carried by the air stream from the sprayer past the orifice leading into the mass spectrometer, and the ions which evaporate from the droplets are repelled toward the orifice by a large repeller electrode at 3.5 kV. The mass spectrometer is a TAGA 6000 triple quadrupole system. It has been designed to sample ions from an atmospheric pressure chemical ionization (APCI) source and so is eminently suitable for sampling ions from the atmospheric pressure spray source. The APCI source is simply unclipped from the core instrument and replaced with this source. Ions, which in the moist gas are surrounded by a sheath of several water molecules (12), are sampled by drawing them through a curtain of dry nitrogen in front of the orifice. The dry curtain gas acts to keep droplets and particles out of the orifice and also helps to remove solvent molecules from the ions by shifting the equilibrium of the cluster toward a lower number of molecules. As the ions pass through the orifice into the vacuum, a first collision induced dissociation region is traversed in which further declustering (removal of
attached solvent molecules) can be performed to leave only the core ion or in which fragmentation can be induced if desired. The ions enter the first quadrupole, and those of the correct massto-charge ratio are selected and transmitted into the central quadrupole, operated in the rf-only mode to act as a high pass filter and confinement region. Here a neutral gas jet (usually nitrogen or argon) can be added across the axis of the quadrupole, to provide a well-defined region in the center of the quadrupole in which ion-neutral collisions can occur. The mean energy of the collisions can be varied from 0 to at least 150 eV with an energy spread of a few electronvolts acquired during the expansion of the ions into vacuum (15), and the gas target thickness can be varied over 2 orders of magnitude. Together these provide overall control over the average number of collisions per ion, and the energy of each collision (15). This range of collision energy and gas target thickness is advantageous in optimizing conditions for the degree of fragmentation desired. The fragment ions, or "daughters" of each parent, are then mass analyzed in the third quadrupole, operated in the resolving mode. The entire system is under digital control, with a data system and a pulse counting detector. It can be operated as a single mass spectrometer, by switching either the third quadrupole or the first quadrupole to the total ion mode (rf only), in order to examine the ions from the source in the absence of the CID fragmentation process. The application of this type of instrument for direct mixture analysis has been well documented (16, 17). The results which are presented here were obtained by spraying the solutions and acquiring a full mass spectrum of the ions emitted from the droplets. Each parent ion was then selected and fragmented to obtain a daughter ion spectrum. Argon was used as a collision gas in all cases reported here, with collision energies which ranged, depending on the compound, from 30 V up to 100 V (lab energy). Most of the compounds were prepared from pure analytical standards, with some being obtained from commercial pharmaceutical formulations. Laboratory deionized water was used as the solvent. Each spectrum was acquired in a single scan and is presented without background subtraction or data smoothing.
RESULTS AND DISCUSSION Amino Acids. Creatine and arginine are examples of thermally labile amino acids which are difficult to characterize by E1 or CI using conventional direct probe techniques (18, 19). Thermal dehydration occurs at temperatures below that required to vaporize these compounds, unless special care is taken to deactivate the support surface. For example, Cotter (19) reports that with normal direct probe techniques, no molecular or quasi-molecular ions could be generated from either compound. However, by using an exposed Vespel probe, spectra containing approximately 5% intensity of MH+ for arginine hydrochloride and 11% intensity of MH' for creatine hydrochloride could be obtained, using isobutane CI. Similarly, Hansen and Munson (20) used a Teflon surface to obtain results in which MH+ ions were observed from both compounds, but in which the loss of water (MH' - 18) still dominated. An activated FD emitter used as a solid probe (18) showed a significant improvement, giving MH' peaks which were comparable in intensity to the (MH' - 18) peak. The technique of FD has been shown to generate abundant MHf from both compounds (21, 22),probably as a result of much lower thermal energy transfer to the sample molecule. The spectra shown in Figures 1and 2 are the positive and negative spectra of creatine and the positive spectrum of arginine, respectively, obtained with the technique of liquid ion evaporation. In each case, the sample was dissolved at M in water, and mixed 1:lwith a concentration of M HC1 (for the positive specta) and M NaOH (for the negative spectra) before being nebulized. The spectra are dominated by the quasi-molecular MH+ or (M - H)- ions which preexist in the aqueous solution and reveal few or no decomposition products which are indicative of the structure of the sample molecules except for m / z 45 (formate) and m / z 59 (acetate) in Figure lb. The small, lower mass peaks ob-
ANALYTICAL CHEMISTRY, VOL. 54, NO. 13, NOVEMBER 1982 (Mtll’
2221
loot (=j
(Mfl)’
5
r -
200
Figure 3. Collision-induced-dissociation (CID) spectrum of MH+ of arginine.
Flgure 1. (a) Positive ion spectrum of M creatine in M HCI aqueous solution. (12) Negative ion spectrum of lo4 M creatine in M NaOH aqueous !solution.
lo4
l iN tigN
/
CNHCHZCH2CH2CH[NH2)COOH
(Mtl)+ 1 75
oL--k0 0
m 100 /z
Flgure 2. Positive ion spectrum of M HCI aqueous solution.
M solution of arginine in
served in the spectra appear to be due to impurities in either the sample or the water, being in no clear way related to the parent ion or molecule. In particular, there is no visible evidence of ions corresponding to MH+ - 18 or to (M - H)- 44, the two mOEk likely decomposition products. These two examples therefore provide an indication of the relative absence of mechanisms which transfer energy to the ion during either the emission process (from the liquid drop) or during the sampling into the vacuum chamber. The molecular weight information obtained in the previous spectra by operating the triple quadrupole as a single mass spectrometer and deliberately minimizing the extent of fragmentation in the ion source (the term “ion source” will be taken to refer to the nebulizer and vacuum interface region, even though in this process the ions are already existing in solution) can now be complemented by aldmitting a collision gas and operating the instrument in the MS/MS mode to produce a CID sipectrum of the parent ion. Figure 3 shows
the CID spectrum of the MH+ (mlz 175) ion from arginine. Conditions were such that multicollision processes dominated, so that the spectrum (termed “daughter ion spectrum”) shows several relatively low mass (and structurally significant) fragments. In this mode, there is no interference a t the low mass where reagent ions normally occur. Major fragmentation pathways appear to correspond to the loss of guanidine and the loss of guanidine plus formic acid from the MH+, giving ions at mlz 116 and m/z 70, respectively, and to the formation of the stable protonated guanidine (mlz 60) and protonated cyanamide (mlz 43). It is interesting to compare these products with those observed by Hunt et al. (18) in the CI experiments with an FD probe as the sample support, where even the gentle heat applied to the sample undoubtedly played a major role. In their case, products corresponding to the loss of water and the loss of NH3 were apparently formed in the vaporization process, since adducts were observed. Fragments from these ions in the source then were observed, as well as fragments corresponding to the loss of formic acid and guanidine together, and guanidine alone, from the free arginine MH+. Thus, if thermal decomposition products were eliminated, fragments of the parent MH+ in the CI source and in the CID region of the triple quadrupole appear to be similar. Quaternary Ammonium Salts. Several quarternary ammonium salts (NR4C1) have tested, and all are readily observed with this technique as the NR4+ion. These ions are surface active in aqueous solution (23), a property which promotes their concentration at the surface of the liquid and appears to result in greater ease of desorption or evaporation from the liquid surface. Sensitivity toward these salts is therefore somewhat better than for other more polar organic ions. The mass spectra show little or no evidence of fragments or decomposition products. Figure 4a shows the spectrum of tetrabutylammonium chloride, in which N(C4H9)4+at m/z 242 is the base peak with a fragment a t m / z 142 of only 2% intensity. Figure 4b shows the full CID spectrum obtained by selecting the parent cation and fragmenting it with an argon target gas a t an energy of approximately 60 V. Although a small fragment at m/z 184 reveals the loss of butane indicative of the butyl substituent, the dominant daughter ions at m / z 142, 100, and 57 correspond to the loss of groups which are probably the neutral species C7H16and CloHzzand to the formation of the C4Hg+ion, respectively. Other significant daughters are a t m / z 58 (loss of C13HZs), m / z 44 (loss of C&30), m / z 41 (C3H5+),and m / z 29 (CzH6+). Several of the same ions (at m / z 100,142,184,and 57) have been observed in CW laser desorption spectra of N(C4Hg),I (24). The authors ascribed these fragments to thermal process (except for m/z 57); however Figure 4b shows that collisional
2222
ANALYTICAL CHEMISTRY, VOL. 54, NO. 13, NOVEMBER 1982
0-
0
: ?
14
,
1
)/'I
, '1, mh
,
1
2w
153
50
0
1,
100
,i
,,,,, 100 mh
200
Flgure 5. (a) Positive ion spectrum of M dopamine in M HCI aqueous solution. (b) Positive Ion spectrum of lo4 M serotonin in M HCI aqueous solution. (c) Positive ion spectrum of M norepinephrine in 10"' M HCI aqueous solution. Figure 4. (a) Posltlve ion spectrum of (b) C I D spectrum of N(C4Hs)4+.
M solution of N(C4Hg)&I.
excitation can produce these same ions. Organic Acids. Several organic acids and compounds with acidic functional groups have been examined. Acetylsalicylic acid (ASA) produced a dominant (M - H)- ion a t m / z 179, with minor fragments of mlz 137, 59, and 45. Collision-induced dissociation of the m/z 179 gave daughter ions of m / z 137 (the (M - H)- of salicylic acid), m / z 93 (the (M - H)-of phenol), and m / z 59 (the acetate ion). Picric acid (trinitrophenol) gave exclusively the (M - H)- ion at m / z 228 from the spray which under CID conditions showed daughter ions of mlz 182 (loss of NOz), mlz 63 and 78 (unexplained), and m / z 46 (NO,-). Ascorbic acid (obtained from a commercial vitamin C tablet) showed a base peak of (M - H)-at m/z 175, with minor fragments at m/z 115,87,59, and 57 which were probably formed by CID processes in the cluster breaking region. Collision-induceddissociation of the parent (M - H)gave the same ions but with greater relative intensity as well as daughter ions of mlz 43 and 71. The daughter ions can each be ascribed to the loss of stable neutral species from the parent, or to the formation of a stable negative ion. For example, m / z 115 corresponds to the loss of acetic acid, mlz 71 to the loss of COPand acetic acid, and m/z 87 to the loss of CO and acetic acid (as well as to the formation of the (M - H)- of pyruvic acid), while m / z 59 (acetate ion) and m / z 43 (CH,CO-) are stable negative ions. The daughter at m / z 57 remains unexplained. In addition to these compounds, carboxylic acids up to C5 have been tested and detected, as well as citric acid (with a dominant (M - H)- at m / z 191 and daughters at m / z 111,87, 85, and 43). Alkaloids. Several alkaloids have been examined, and all give MH+ ions with few or no fragments. Those tested include methaqualone, antipyrine, caffeine, morphine, amphetamine, cocaine, codeine, quinine, lidocaine, methadone, meth-
amphetamine, imiprimine, and sparteine. All have basic functional groups and so are readily ionizable in aqueous solution. In most cases, optimum sensitivity is ensured by the addition of an equimolecular amount of HCl (or another strong acid) to the compound of interest. Catecholamines. The catecholamines (dopamine, epinephrine, and norepinephrine) and one related biogenic amine (serotonin) have been detected with this technique. Figure 5 shows the mass spectra of dopamine, serotonin, and norepinephrine. The first two spectra are dominated by the expected MH'; however, norepinephrine is characterized by a fragment corresponding to the loss of ammonia ( m / z 17) from the parent as the base peak (with the abundance of the parent being 70%). Our opinion (not yet fully confirmed) is that this fragment was in fact produced in the region of the free jet expansion from the atmospheric pressure ion source region into the vacuum, where electric draw-out and focusing fields can impart energy to the ion in a region where there is still a significant gas density. This region is normally a declustering region and can also be useful as a preliminary or first CID region in the instrument. The appearance of significant fragmentation in the spectra of epinephrine and norepinephrine, and not in any of the other spectra, remains to be further explored. However, the presence of a strong (MH)+is still sufficient for molecular weight determination and for the selection and fragmentation to generate a daughter ion spectra. All four of the biogenic amines show loss of ammonia MH+ - 17 from the parent ion as the dominant CID process. Glutathione. Glutathione was selected as an example of a tripeptide in order to explore the possible utility of the technique for polypeptide analysis and sequencing. This peptide has been examined by FD (25) which gave a strong MH+ ion, as well as by electrohydrodynamic ionization (26). In the latter case, the compound was dissolved in a glycerol solution with NaI as an electrolyte. The quasi-molecular MH'
ANALYTICAL CHEMISTRY, VOL. 54, NO. 13, NOVEMBER 1982 HzNCHCH~CHZCONHCHCONHCH~ COCH
I CH2SH
I COOH
2223
U
308 !MS1)+
E
A
VI
0
200 m/ z
Figure 8. Posltive ion spectrum of
7 300
M guanosine in
M HCI
solution.
L 0 -4 100 -
200
300
m/z
M in Flgure 8. (a) Positive ion spectrum of glutathione at M HCI solution. (b) Negatlvu Ion spectrum of lom4M glutathione in MNaOH. (M+l)'30 8
0
Flgure 7. C I D spectrum of MH+ mlz 308 of glutathione.
a t mlz 308 was readily observed, but ions related to several other species interpreted as breakdown products were also observed. The positive and negative ion evaporation spectra of glutathione (Figure 6) show the quasi-molecular MH+ and (M - H)- as the dominant ions. The presence of several minor peaks in the range of 100 to 200 amu in the positive spectrum is attributed to impurities in the solution rather than to fragments or products of the compound itself. Complementary information can be obtained from the CID spectra of the MH+ and (M - H)- ions. Sequence information is more readily obtained from the former (see Figure 7), which shows daughter ions at mlz 179 (MH+ of cysteinylglycinle), m / z 161 (loss of water from 179),,mlz 1.30 (loss of water from the MH+ of glutamic acid), ntlz 83 (unexplained), arid m / z 76 (MH+ of glycine). The negative CID spectrum shows daughters at mlz 143 (unexplained), m/z 128 (from the glutmyl residue), m / z 99 (unexplained), and mlz 74 ((M - H)- of glycine). No attempt has been made to optimize the CID conditions and it may be that a careful study of the target thickness and collision energy conditions might reveal conditions which provide better sequence information (missing in these spectra is a fragment showing the glutamyl-cysteine residue). However, the ability to generate the quasi-molecular ion from an
underivatized tripeptide, and obtain significant information on the amino acids and their sequence, could be a very promising field of application for this technique, particularly if linked on-line at an LC system. Nucleosides. Adenosine and guanosine are two nucleosides which thermally decompose at temperatures required for vaporizing from a probe, so that both E1 and CI spectra lack the presence of molecular ions. Guanosine has been successfully detected as MH+ when volatilized from a field desorption probe under CI conditions (18). In addition, quasi-molecular (MH+, MNa+) ions have been observed by laser desorption (27),by electrohydrodynamic ionization ( 5 ) ,and by SIMS (28). The LD spectrum was dominated by alkali attachment ions, while the EHD spectrum was dominated by clusters of glycerol with the various species. The SIMS spectra in general showed the protonated base as the dominant peak. In contrast, the spectrum of guanosine obtained with this technique shows an MH+ ion at mlz 284 which is of the same intensity as the mlz 152 peak due to the protonated base (Figure 8). The spectrum of adenosine likewise shows an MH+ peak which is the base peak in the spectrum. Both compounds were analyzed in acidic solution M guanosine and adenosine in MHCl solution). The spectrum of guanosine in Figure 8 is only shown for mlz > 100. The low range of the spectrum contains a number of mass peaks which appear to be due to impurities in the aqueous solution. Nucleotides. Free underivatized nucleotides cannot be analyzed by direct probe E1 or CIJMS. Molecular ions have been obtained, however, by 252Cf plasma desorption ( 3 ) , CI/MS with field desorption emitters (18),FAB (29),EHD ( 5 ) , SIMS (28), and ion thermospray (8). The liquid ion evaporation technique being tested here was successful in generating both MH+ from adenosine monophosphate and adenosine triphosphate as well as M- ions from the sodium salt of adenosine monophosphate (MNa) and M2- from the disodium salt of adenosine triphosphate (MNaJ. The spectrum of the latter is shown in Figure 9. The base peak at m / z 252.5 is the doubly charged anion of molecular weight 505, while the other peaks all correspond to various states of dissociation or ionization of the salt. They are identified as m / z 528 (MNa-), mlz 506 (MH-), and mlz 448 and 426, the corresponding ions of adenosine diphosphate. Figure 10 shows the CID spectrum of the M2- parent ion, which gives daughters of m / z 79 (PO,) and 426 [(M - PO,)-]. Thus, an ion of apparent mass 252.5 gives a daughter of lower mass and one of higher apparent mass.
2224
ANALYTICAL CHEMISTRY, VOL. 54, NO. 13, NOVEMBER 1982 (M-'2)'-
252 5
100-
OH ER A
T vI
I
T N E
__
OH
I P-0-
NoO-
l
8 A-
/I
---
ONa
l P-
0- P
50-
OH
-.
__
OH
4?6
m/z
Flgure 9. Negative ion spectrum of lo4 M disodium salt of adenosine trlphosphate.
0
0
I
,
#!
LITERATURE CITED
(M-2)2252.5
'7
I
,
,
1
I
260
,I,, , ,
Beckey, H. D. I n f . J . Mass Spectrom. Ion Phys. 1969, 2 , 500. Cotter, R. J. Anal. Chem. 1980, 52, 1767-1780. Macfarlane, R. D.; Torgerson, D. F. Science 1976, 191, 920-925. Cotter, R. J. Anal. Chem. 1980, 52, 1589A-1606A. Lal, S. T. F.; Evans, C. A,, Jr. Biomed. Mass Spectrom. 1979, 6 , 10-14 .- . .. (6) Barber, M.; Bordoll, R. S.;Sedgwick, R. D.; Tetler, L. W. Org. Mass. Specfrom. 1981, 16, 256-260. I Day, R. J.; Unger, S.E.; Cooks, R. G. Anal. Chem. 1980, 52,557A572A. Blakley, C. R.; Carmody, J. J.; Vestal, M. L. J . Am. Chem. SOC. 1980, 102,5933-5934. Beynon, J. H.; Cooks, R. G.; Amy, J. W.; Baitinger, W. E.; Ridley, T. Y. Anal. Chem. 1973, 45, 1023A-1031A. Kondrat, R. W.; McClusky, G. A,; Cooks, R. G. Anal. Chem. 1978, 50, 1222-1 223. McLafferty, F. W. Science 1981, 209, 675-677. Weber, R.; Levsen, K. Biomed. Mass Spectrom. 1980, 7 , 314-316. Iribarne, J. V.; Thomson, B. A. J . Chem. Phys. 1976, 6 4 , 2287-2293. Thomson, B. A.; Iribarne, J. V. J . Chem. Phys. 1979, 7 1 , 4451-4463. Dawson, P. H.; French, J. 8.; Buckley, J. A,; Douglas, D. J.; Simmons, D., submitted to Org Mass Spectrom. Rees, G. A. V.; Tosine, H.; Sakuma, T.; Davidson, W. R.; Thomson, B. A.; Danylewych, L. M.; Shushan, B.;Reid, N. M. Presented at the 29th Annual Conference on Mass Spectrometry and Allied Topics, Mlnneapolis, MN, May 24-29, 1981. Paper number RPB18. Hunt, D. F.; Shabanowitz, J. Anal. Chem. 1982, 54, 574-576. Hunt, D. F.; Shabanowitr, J.; Botz, F. R.; Brent, D. A. Anal. Chem. 1977, 4 9 , 1160-1163. Cotter, R. J. Anal. Chem. 1979, 51, 317-316. Hansen, G.; Munson, B. Anal. Chem. 1980, 52,245-248. Fales, H. M.; Milne, G. W. A,; Winkler, H. U.; Beckey, H. D.; Damico, J. N.; Barron, R. Anal. Chem. 1975, 4 7 , 207-219. Wlnkler, H. U.; Beckey, H. D. Org. Mass Spectrom. 1972, 6 , 655-660. Tamaki, K. Bull. Chem. SOC.Jpn. 1974, 4 7 , 2764-2767. Stoll, R.; Rollgen, F. W. Org. Mass Specfrom. 1979, 14, 642-645. Anbar, M.; St. John, G. A. Anal. Chem. 1976, 48, 198-203. Stimpson, B. P.; Evans, C. A., Jr. Biomed. Mass Spectrom. 1978, 5 , 52-63. Hardln, E. D.; Vestal, M. L. Anal. Chem. 1981, 53, 1492-1497. Elcke, A,; Sichtermann, W.; Benninghoven, A. Org. Mass Specfrom. 1980, 15, 289-294. Wllliams, D. H.; Bradley, C.; Bojesen, G.; Santikarn, S.; Taylor, L. C. E. J . Am. Chem. SOC.1981, 103, 5700-5704. (1) (2) (3) (4) (5)
I
I
pected that enough compounds either are ionic themselves or can be derivatized to make them ionic that the technique can be of some general utility for many applications. The results of coupling the technique to an MS/MS instrument have also demonstrated that an ion source which produces almost exclusively quasi-molecular ions linked to a controllable fragmentation process provides an attractive combination for both molecular weight determination and structural elucidation. A full scan of the mass spectrum shows the parent ion peak or peaks with little or no interference from fragments. Each one can then be selected for fragmentation by the CID process. While much work remains to be done to show the utility of CID process in structural elucidation of large biochemical molecules, results so far (7)indicate that, in almost every case, some structurally significant fragment is generated.
I
,
,
,I,
A60
f
,
, ,
6b0
m/z
Figure 10. CIb spectrum of the m l z 252.5 doubly charged parent ion of adenosine triphosphate.
In summary, the preliminary results reported here suggest that the technique of ion evaporation may be suitable for the direct introduction of labile and polar compounds into a mass spectrometer system. Salts, acids, and bases have all been tested, and all of the compounds which have given positive results have been characterized by the dominance of the quasi-molecular parent ion in the spectrum. It appears to be the case from our experience that most compounds which can be made ionic in solution can be observed with this technique, as long as the ion is monovalent. (The only true exceptions which have been found so far are the monovalent transition metal ions, Cu+, T1+, and Au+, which have not been observed.) Adenosine triphosphate is the only polyvalent ion observed to d a h Compounds which have been tested so far have been selected on the basis of being sufficiently polar or ionic in solution, and so all which would be expected to produce ions have actually been observed. Sugars (sucrose and raffinose) as an example did not produce ions a t any pH but, in the M Na+, did exhibit weak signals due to sodium presence of ion attachment. The number of successes to date therefore greatly exceed the number of failures due to careful and deliberate selection of trial compounds. Nevertheless, it is ex-
.
RECEIVED for review May 28,1982. Accepted August 2,1982. This work was carried out, in part, under contract through the IRAP and PRAI programs of the National Research Council of Canada.
