Surface chemical ionization mass spectrometry - Analytical Chemistry

A microcomputer current programmer for mass spectrometer direct exposure probes. Robert J. Cotter. Biological Mass Spectrometry 1979 6 (11), 508-513 ...
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ANALYTICAL CHEMISTRY, VOL. 50, NO. 8, JULY 1978

(155.,,,,,'"

30 0 1 0 3 Torr

%TIC 2 0

'0 40

60

83

803

20

E T H Y L BUTYRATE 1'0DC 129 Vlcm Torr

%TIC

30

2oL__.d 10

43

60 "ROPYL

80

103

120

PRCP OluA-E

Flgure 3. Water CI spectra of ethyl butyrate and propyl propionate obtained in the drift-tube CI source with water reagent gas at 110 OC and 0.103 Torr. The drift field strength was 129 Vkm-Torr, sufficient to produce substantial amounts of fragmentation characteristic of these two structural isomers

\

in propyl propionate are RCO+ ( m / e 57), RCOOH2' ( m / e 75), and RCOOHzf.0H2 ( m / e 93). The differences in the spectra are so great that these two structural isomers are readily distinguished. There is one important difference between methane (6,8) and water CI spectra of esters which should be noted, and that is the complete absence of M+ and (M - 1)+ions in the water CI spectra. The absence of these peaks is a great advantage when attempting to assess the isotopic or chemical purity of a compound from its water CI spectrum at low E / P .

preferential solvation of the product ion favors formation of RCOOH2+-OH2from (RCOOR')H+.OH, over formation of RCOOH2+ from (RCOOH')H+. Inasmuch as the origins and analytical utility of all of the various ions observed in the CI spectra of esters have been discussed extensively in previous work ( 6 , 8 , 1 4 ) ,there is no need to repeat that information here. As an example of the ease of identifying esters from their water CI spectra a t high E / P in the drift tube source, Figure 3 compares the spectra of ethyl butyrate and propyl propionate a t 129 V/cm-Torr. The MH+ ions at m / e 117 are present for both esters, but none of the other peaks are identical. The fragment ions in ethyl butyrate are R+ ( m / e 43), RCO' ( m / e 711, RCOOH2+ ( m / e 89), and RCOOH2+.0H2( m / e 107), while the fragment ions

LITERATURE CITED (1) P. Price, H. S. Swofford, Jr., and S. E. Buttrill, Jr., Anal. Chem., 49, 1497

(1977). (2) For recent reviews, see B. Munson, Anal. Chem., 49, 772A (1977)and F. H. Field in "Ion Molecule Reactions", J. L. Franklin, Ed., Plenum Press, New York, N.Y., 1972,pp 261-313. (3) E. W. McDaniel, "Collision Phenomena in Ionized Gases", John Wiley and Sons, New York, N.Y., 1964,pp 426-440. (4) W. Lindinger, M. McFarland, F. C. Fehsenfeld, D. L. Albritton, A. L. Schmeltekopf, and E. E. Ferguson, J. Chem. fhys., 63, 2175 (1975). (5) H. E. Rivercomb and E. A. Mason, Anal. Chem., 47, 970 (1975). (6) M. S.B. Munson and F. H. Field, J. Am. Chem. Soc., 68, 4337 (1966). (7) F. H. Field, J . Am. Chem. SOC., 91,2827 (1969). (8) C. V. Pesheck and S. E. Buttrill, Jr., J. Am. Chem. Soc., 96, 6027 (1974). (9) P. Price, H. S. Swofford, and S. E. Buttrill, Jr., Anal. Chem., 48, 494

(1976).

(IO) P. C. Price, D. P. Martinsen, R. P. Upham, H. S. Swofford, Jr., and S. E. Buttrill, Jr., Anal. Chem., 47, 190 (1975). (11) H. M. Rosenstock, K. Draxl, B. W. Steiner, and J. T. Herron, J. fhys. Chem. Ref. Data, 6,Suppl. 1 (1977). (12) R. Yamdagni and P. Kebarle, J . Am. Chem. SOC.,98, 1320 (1976). (13) W. A. Chupka and J. Berkowitz, J . Chem. f h y s . , 54, 4256 (1971). (14) C. W. Tsang and A. G. Harrison, J. Chem. Soc., ferkln Trans. 2 , 1718 (1975). (15) W. T. Huntress, Jr., R. F. Pinizzotto, Jr., and J. B. Laudenslager, J . Am. Chem. SOC.,95,4107 (1973). (16) Unpublished data from this laboratory.

RECEIVED for review January 27, 1978. Accepted April 10, 1978. This work was supported by NSF Grants GP-38764X, MPS-7510940, and CHE 76-20096.

Surface Chemical Ionization Mass Spectrometry Gordon Hansen and Burnaby Munson" Department of Chemistry, University of Delaware, Newark, Delaware

The use of "in-beam" sample introduction to obtain chemical ionization (CI)mass spectra of thermally labile compounds and salts of low volatilHy is descrlbed. Samples are introduced on the surface of Teflon tubing and the effects of changing the sample position in the source are shown. The time and temperature dependence of some CI mass spectra are shown. The results are interpreted in terms of a model of sample ion production whlch involves surface ionization by ion-molecule reactlons followed by thermal desorption.

A major limitation in the use of mass spectrometry for the analysis of thermally labile compounds is the requirement that a sample must first be vaporized before it can undergo ionization. For many polar compounds, the energy required to disrupt the bonds between molecules may be greater than the energy required to break intramolecular bonds; therefore,

1971 I

decomposition of the molecules may occur a t temperatures lower than those required for volatilization of the intact molecules. Field desorption mass spectrometry has been used successfully for the analysis of thermally labile compounds and of salts of low volatility. The spectra produced by this technique contain few ions for structure determination but generally give an indication of the molecular weight of the compounds. Some complications arise from the presence of ions of masses higher than the molecular weight of the analyte ( I , 2). Recently, the mechanism of ion production in field desorption has been challenged ( 3 ) . Another recent and highly successful technique in obtaining mass spectra of thermally labile compounds is that developed by MacFarlane and Torgerson, known as Fission Fragment Desorption or Plasma Desorption ( 4 ) . This technique utilizes energetic fission fragments from 252Cfto volatilize and ionize solid samples deposited on thin Ni-foils. Ions characteristic

0003-2700/78/0350-1130$01.00/0 0 1978 American Chemical Society

ANALYTICAL CHEMISTRY, VOL. 50, NO. 8, JULY 1978

of molecular weight have been recorded for high molecular weight nonvolatile compounds such as Vitamin Blz (mol wt 1327) and xanthine-tyrosine (mol wt 331) using this technique. The instrumental modifications needed for the implementation of plasma desorption are substantial and its acceptance for routine analysis does not appear to be likely in the near future. Two techniques, developed over the past five years, do stand out in their effectiveness and feasibility for possible widespread use with mass spectrometers used for electron impact (EI) and chemical ionization (CI) mass spectrometry. First is the technique of Baldwin and McLafferty known as direct source insertion or in-beam chemical ionization (5). This technique consists of depositing a sample on the surface of an extended tip of a conventional sample probe. The probe is introduced into the source chamber so that the tip is exposed to the ion plasma. The source temperature is then raised until spectra characteristic of the sample are obtained. The authors reported that spectra of many relatively nonvolatile compounds could be obtained a t least 150 "C below temperatures usually required for vaporization from a conventional solid sample probe. As an example, the CH4 CI spectrum of the LeuLeuValTyr contains an abundant (M + H)+ peak with in beam chemical ionization, but no ions were observed, even a t 340 "C using conventional introduction methods. Although the initial work of Baldwin and McLafferty showed promising results using a seemingly simple technique, many of those attempting to use the technique obtained erratic results and little has been published concerning the details of the technique. Field and co-workers have reported the use of in-beam CI in obtaining spectra of biologically significant quaternary amines. In no case were ions characteristic of the quaternary amines observed. The highest masses observed for the compounds studied were the ions resulting from protonation of the amine formed by the loss of CH3X (6). Recently Ohashi and co-workers have reported the successful use of in-beam electron impact ionization to produce useful spectra of several amino acids. The authors report the observation of MH+ as well as M+ ions for the amino acids studied. Phenylalanine, whose E1 spectrum contains an M+ ion with an abundance of 3% of the base peak with conventional sample introduction, exhibits an MH+ ion with an abundance of 31% of the base peak in the in-beam E1 mass spectrum (7). An even more recent technique for obtaining CI spectra of thermally labile compounds was reported by Hunt and coworkers (8). Their work involved the use of activated field desorption emitters as direct source insertion probes under chemical ionization conditions, FD/CI. The technique consists of depositing the sample from dilute solution on an activated field desorption emitter which is located in the ion beam within a high pressure source. The sample is heated by passing a current through the wire until ions are produced. As examples, creatine, arginine, and guanosine give abundant ions characteristic of molecular weight using this technique, FD/CI, whereas conventional introduction techniques under CI conditions give no molecular weight information for these compounds. Another research group which has been active in mass spectral studies of thermally labile compounds is that of Friedman and co-workers a t the Brookhaven National Laboratories (9, I O ) . Although their specific instrument, a tandem mass spectrometer, is not readily adaptable for general use, the underlying principles of their work may be generally applicable. These workers have studied the effects of surfaces on the spectra of Thyrotropin Releasing Hormone, as well as the effects of rapid heating on the spectra of this and other thermally labile compounds. They have reported a 300%

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increase in the relative intensity of the (M t H)+ ion for TRH with a Teflon-lined collision chamber and glass probe tip. The technique of rapid heating was used to minimize the effects of decomposition during sample vaporization. Samples are applied as dilute solutions to a Teflon probe which is inserted into a Teflon-lined collision chamber of a tandem mass spectrometer. The samples are heated at a rate of 5-12 "C/s and single collision proton transfer spectra from NH4+ are obtained. The NH4+spectrum of arginine, for example, shows an (M + H)' ion with an intensity of 20% of the base peak (M - 16)'. The techniques discussed above have similarities and it may be that they owe some of their success to similar phenomena. We wish to report the relatively simple operating and optimization procedures for obtaining in-beam/CI spectra which are comparable to spectra obtained by FD/CI.

EXPERIMENTAL The experiments were performed with a duPont 21-llOB mass spectrometer which has been modified for high pressure operation (11). In addition, a data acquisition system has been added to the instrument t o allow rapid scanning (4.5 s for the mass range of 28 to 600) of spectra, every 6.5 s. The design of our chemical ionization source is such that the electron entrance aperture and the ion exit slit are on the same axis. Sample introduction is perpendicular to this axis. The distance between the sample introduction axis and the ion exit plate is fixed at 3 mm. With this configuration, the only parameter which can be varied is the distance between the tip of the sample holder and the electron entrance/ion exit axis. The direct insertion probe was a glass probe made by Mass Spectrometer Associates, College Station, Texas. The temperature of the capillary well of the probe could be varied and measured independently from the temperature of the metal ionization chamber or source block of the mass spectrometer. After these experiments were completed, it was discovered that the temperature measurements were in error, but the relative values should be reliable. Isopentane (99+%), methane (99.97%), and nitrogen (high purity, dry) were used as reagent gases at pressures of approximately 0.5 Torr. The samples were obtained from different commercial sources without further purification. RESULTS AND DISCUSSION Fales et al. ( I ) have suggested that creatine undergoes dehydration to the cyclic lactone, creatinine, at temperatures lower than those required for volatilization into the source of a mass spectrometer. As a result, conventional vaporization techniques of the sample into the source chamber (EI, CI, FI) show no ions characteristic of sample molecular weight. Only FD (where (M + H)+ is the base peak) and FD/CI (where (M + H)+ is 92% of the base peak) give significant abundances of ions characteristic of the molecular weight for this sample ( I , 8 ) . Experiments in our laboratory showed that in-beam/CI using isopentane as the reagent gas could also produce ions characteristic of molecular weight from creatine. The appearance of the (M + H)+ ion using direct source insertion CI showed a strong dependence on experimental conditions. For this reason, creatine was chosen for use in optimizing experimental parameters. Baldwin and McLafferty ( 5 ) ,in their description of direct source insertion chemical ionization, reported that the tip of the direct insertion probe must be placed within a few millimeters of the electron beam. Since the detailed effects of variation of the insertion position were not known to us, the insertion distance was the first parameter investigated. The experiments to investigate the effects of the insertion distance were conducted in the following manner: the samples were lightly dusted (-50 wg) on approximately 1mm of the tips of varying lengths of Teflon capillary tubing. The tubing was placed in the well of the unheated glass probe and the probe introduced into the source a t a constant source temperature.

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ANALYTICAL CHEMISTRY, VOL. 50, NO. 8. JULY 1978

TEYPEilrTU4E

('C)

Figure 2. Temperature dependence of the relative abundance of characteristic ions for creatine (0)m l r 1321mlr 114, arginine (A) m l r 174/rnlz 157, choline chloride (0)m l r 1041mlr 90 i

Figure 1. Variation of (M 4- l)+/(M - 17)' ratio with distance from the ion exit-electron entrance axis. (V) Creatine, 190 "C; (0)creatine, 210 "C;(0)aspartyl glycine, 175 "C The results of these experiments for creatine at two temperatures (Figure 1) indicate that fractional abundance of m / e 132, (M H)', is strongly dependent on insertion distance. The maximum value for the ratio of (M + H)+/(M - OH)+, (M - OH)+ = base peak, was obtained when the tip of the probe was 2.5 mm from the ion exit/electron entrance axis. It was also observed that the total sample ion current was independent of insertion distance. Similar data for the i-C5H12CI spectra of aspartylglycine are also shown in Figure 1. With conventional probe introduction, the isopentane CI mass spectrum of aspartylglycine contains no (M + HI+. The effects of sample insertion distance of the in-beam/CI technique with aspartylglycine are the same as those obtained with creatine. The same observations have been made with other thermally labile compounds and routine use of this technique has been achieved for several months in our laboratory. These variations in ionic abundance are inconsistent with a model of vaporization followed by gas phase reactions with t-C$ll+. The irreproducibility of our early experiments led us to investigate the effects of temperature on i-CsHlz spectra obtained by this technique. Heating the glass probe from 25 to 300 "C in 20 min had no effect on the spectra. If the source temperature was low, no sample ions were observed. If the source temperature was high enough so that sample ions were observed, no increase in sample ionization or change in ionic ratios was noted with increasing temperature of the glass probe. We attribute these observations to the low thermal conductivity of Teflon; consequently, the temperature of the sample on the surface of the Teflon capillary is determined by the gas temperature rather than the temperature of the probe well. Changing the temperature of the gas by heating the source block is relatively slow with our instrument. Increasing the source temperature for a single sample in this manner gave only minor effects on the CI spectra. The compounds being studied in these experiments appeared to decompose during this slow heating process. In order to obtain spectra at different temperatures, the source block temperature, and therefore the gas temperature, was raised in approximately 10 "C steps and a different sample was inserted at each temperature. Spectra were recorded as a function of time after each insertion and about 15 s elapsed before the first spectrum could be taken. During this time the pressure in the CI source was increasing to its equilibrium value. The ratio, (M + H)+/(M - OH)+,for creatine varied with both time and gas temperature. Figure 2 shows the temperature variation of this ratio. The values chosen for this

+

25

50

75

100

TlrlE (SEC)

Figure 3. Time dependence of the major ions in the in-beam spectrum of arginine. Source temp = 260 "C. (M) m l z 139, (0) m l z 157, (0) m l z 158, (A)m l z 175

plot were the maxima at each temperature. Similar temperature effects are shown in Figure 2 for arginine, (M + H)+/(M - OH)', and for choline chloride, 1(104)/1(90). This increase in the ratio of ion currents, (M + H)+/(M OH)', is inconsistent with observed temperature effects on CI spectra with conventional introduction of thermally stable compounds. These observations, however, are consistent with the results of the rapid heating experiments, in which abundant (M + H)' ions, 20% the most abundant ion, were observed in the spectra of arginine if the heating rate of the sample was about 5 "C/s. Figure 3 illustrates the time dependence of the major ions in the isopentane CI spectra of arginine at a constant source and gas temperature of -260 "C. The total sample ion current increases rapidly to a maximum in about 45 s and then decreases more slowly with time. If we assume that the sample on the surface of the Teflon capillary has attained the temperature of the gas by the time the sample ion current has reached its maximum value, then the sample is being heated at a rate of approximately 4-5 "C/s. It is also apparent from Figure 3 that the relative abundances of the major fragment ions in the isopentane CI spectra of arginine obtained by this technique are strongly time dependent. There may be some thermal decomposition of the samples on the Teflon surface. In the spectrum of choline chloride that has been reported previously from the in-beamlchemical ionization technique with isobutane as the reagent gas, only the protonated amine, (M - CHBX + H)+ at m / z = 90 and not the quaternary ammonium cation, MC at m / z = 104, was observed for slowly heated samples. No sample ions are observed in our ex-

ANALYTICAL CHEMISTRY, VOL. 50, NO. 8 , JULY 1978

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Table I. Creatine, M = 131 NHCH, I1 I H,N-C-N-CH,-COOH i-CSH,*,Source temp = 2 4 0 C

cI

O

0.05

TIME

Flgure 4. Time dependence of (M

(0)220 "C, ( 0 ) 240 "C

% Base peak

114 132 227 245 263

100.0% (MH - H,O)' 69.5%MH+ 56.3% (M,H - 2H,O)* 32.2% fM,H - H,O)' 12.9% M,H' .

periments for temperatures below 250 "C. The (M - CH8X + HI+ ions, at m/z = 90, are produced when the samples are rapidly heated to temperatures above 250 "C. The quaternary ammonium cation, m / z = 104, is produced for samples rapidly heated to temperatures above 290 "C and the relative abundance of m / z = 104 increased with increasing temperature. A spectrum similar to the previously reported FD/CI spectrum (8) is obtained at 300 "C. In many of these experiments, we noted that although the ion current of the (M H)+ions increased with increasing temperature, the time during which (M + H)+ ions could be detected decreased. In addition, the time of this maximum ion current of (M H)+ ions decreased. Consequently, there was only a relatively narrow window of time and temperature in which (M + H)+ ions could be detected. Figure 4 illustrates this phenomenon with a plot of relative intensity of (M H)+ ions vs. time for creatine at three different source temperatures. This strong variation of spectra with experimental conditions is an obvious potential problem for the identification or quantitation of compounds. Additional experiments are planned to study these effects further; however, we suggest that our observations and the FD/CI experiments of Hunt and co-workers (8)involve some surface ionization by the CI reagent ions. Our results are consistent with a model in which gas phase ionization of molecules by reaction with CsHI1+ions gives primarily (M X)+. This ionization is substantially independent of the position of the sample in the source because of rapid mixing of gaseous sample and increases with increasing temperature because of the increasing vapor pressure of the samples. Some of the (M - X)+ ions may also be formed by proton transfer to (M - HX) molecules formed by thermal decomposition of the sample as well as from decomposition of gaseous (M + H)+ ions. The observed (M H)+ ions may be formed by ion-molecule reactions of the reactant ions with the solid sample molecules on the tip of the Teflon probe. The excess energy of reaction which causes the decomposition of the gas phase ions is transferred to the surface. Only those ions formed in a small volume of the reactant ion plume near the ion exit slit can be collected and detected. Vaporization of the positive ions is aided by the interionic repulsion on the surface and an increase in temperature should increase the rate of desorption of the sample ions. Protonated sample ions, (M + H)+, are produced from the surface under conditions of rapid heating when nonequilibrium vaporization can occur. The extent to which the charge density in the gas phase enhances ionic volatilization has not been established. No striking variations in sample ion currents have been observed with changes of approximately a factor of two in electron current. Smaller samples (-5 pg) have been used, which produced the same spectra under the same conditions as the much larger samples; however, useful spectra can be obtained only for very

+

+

+

I

---

(SEC)

+ H)+ for creatlne at (0)200 "C,

+

m lz

short times. The smaller samples are deposited on the Teflon tubing by evaporation of a drop of dilute aqueous solutions. Microscopic examination of the Teflon surfaces after deposition of powder or evaporation of the dilute solution indicated that samples were present in both cases as small crystals. A few experiments indicate that Teflon is not essential to the technique, but systematic studies of the effects of different surfaces on the isopentane CI spectra have not yet been completed. The spectra are dependent upon the nature of the reactant ions since different spectra are obtained with a weak proton, hydride, or X-, transfer reactant ion like C5Hll and a relatively high energy charge exchange ion like Nz+. Changing the reagent gas in this in-beam technique produces spectral changes similar to those observed under conventional chemical ionization conditions. When isopentane is used as the reagent gas under in-beam conditions at a source temperature of 280 "C, the (M + H)+/(M - OH)+ ratio is 0.57. Nitrogen, under the same conditions does not produce molecular weight ions. The charge exchange reaction of N2+and N4+with neutral arginine is quite exothermic, and the excited molecular ions produced by charge exchange dissociate rapidly. Similarly, differences can also be observed for in-beam spectra obtained using reagent gases of different proton affinities. A comparison of in-beam methane and isopentane spectra obtained at a source temperature of 310 "C shows that isopentane, a weak proton transfer or X- abstraction reagent, produces a greater relative abundance of the (M H)+ ion than does methane, a strong proton and X- transfer reagent. For isopentane, the (M + H)+/(M - OH)+ ratio is 1.1 and for methane, the (M + H)+/(M- OH)+ ratio is 0.36 under comparable conditions of time and temperature. This simple sample introduction technique has been generally useful in our laboratory to produce CI mass spectra of samples for which spectra could not be obtained with conventional techniques. Tables I-IV show typical isopentane in-beam/CI spectra obtained in our laboratory. Table I shows the in-beam/CI spectrum of creatine obtained at a source temperature of 260 "C. The presence of ions at m / e 227, 245, and 263 indicates that some sample ion/sample molecule interactions are occurring and is probably due to a high vapor phase concentration of creatine and perhaps its neutral thermal decomposition product creatinine. These higher mass ions decreased in relative abundance with increasing repeller voltage; so we consider that they result in part from gaseous ion-molecule reactions. Table I1 shows the in-beam/CI spectrum of arginine obtained at 260 "C. As mentioned earlier, it is quite similar to the rapid heating spectrum obtained using a heating rate of 5 "C/s (9, 10). The technique of in-beam/CI has also been useful for obtaining spectra of thermally stable compounds of vapor pressure too low for conventional E1 or CI introduction, e.g., phosphonium salts. For some phosphonium salts, the field desorption spectra are ambiguous and positive molecular weight determination is not always possible. The in-beam

+

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ANALYTICAL CHEMISTRY, VOL. 50, NO. 8 , JULY 1978

Table 11. Arginine, M = 174 NHH II

COOH

I

Table IV. Aspartylglycine, M = 190 H O H I

N H,-C-N-GH,-CH,-CH,-C-NH, I H

II

I

NH,-C-C-N-CH,-C I

CH,

/ \

OH

i-C,H, ,, Source temp = 260 C

m lz

% Base peak

60 61 112

27.5% 3.3% 1.6% 35.8% (MH - NH,CONH,)' 5.4% 30.6% 5.3% 44.1% (MH - 2 H 2 0 ) + 2.8% 2.5% 100.0% (MH - H,O)+ 23.3% (MH - NH,)+ 20.2% MH'

115 116

133 137 139

140 154 157 158 175

Table 111. Phosphonium Salt CH3 CH2-N

I

P' @)Br-

H

H

1

C H2-CH 2

MW = 4371439, RPPh,' = 358, i-C5H,,, Source temp = 300 "C rn lz

% Base peak

98 263 357 358 359

4.2% 37.9% Ph,PH+ 19.2% 100.0% RPPh,+ 6.8%

isopentane CI spectra of these compounds are quite simple, and in some cases superior to the field desorption spectra and frequently contain essentially only RPPh3+ ions. An inbeam/CI spectrum of a typical phosphonium salt is shown in Table 111. The protonated triphenylphosphine ion at 263 may result from either a gas phase decomposition of the cation, RPPh3+,or from protonation of neutral triphenylphosphine present as an impurity or decomposition product. Table IV shows the in-beam isopentane CI spectrum of aspartylglycine. As mentioned earlier, normal sample introduction using isobutane as reagent gas gives no (M + H)+ ions for aspartylglycine; however, in-beam/CI using isopentane has produced MH' ions with a relative intensity of -30% of the base peak, (M - OH)', for aspartylglycine. The sample was obtained commercially and used without further puri-

/\

OH 550 p i-C5H12,Source temp = 180 C m iz % Base peak 0

76 134 173 191 248 263 a

34.8% (Gly + H)+a 8.3% (Asp + H)'a 100.0% (MH - H20)+ 28.9% MH' 17.6% (( Asp-Gly-Gly )H)" 28.9% (M,H - H,O)+

Probably due to an impurity.

fication. The ions observed at m / e 76,134, and 248 may be due to protonation of impurities: free glycine, aspartic acid, and tripeptide asp-gly-gly. The ion at m / e = 263 may result from loss of water from a protonated dimer or from addition of the most abundant fragment ion, 173, to the neutral aspartylglycine. This observation is similar to those of sample ion/sample molecule product ions noted previously with creatine. In conclusion, analytically useful spectra of thermally labile and involatile compounds can be obtained using in-beam/CI. The spectra obtained are similar to those obtained using FD/CI and those obtained with moderately rapid heating techniques. One difficulty with this introduction technique is the narrow window of time and temperature over which (M H)+ions can be detected and the sensitivity of the relative ionic abundances to experimental conditions.

+

LITERATURE CITED (1) H. M. Fales, G. W. A. Milne, H. V. Winkler, H. D. Beckey, J. N. Damico,

and R . Barrow, Anal. Chem., 47, 207 (1975). (2) C. N. McEwen, S. F. Layton, and S. K.Taylor, Anal. Chem., 49,922 (1976). (3) J. F. Holland, B. Soltmann, and C. C. Sweeley, B/Omed. Mass Spectrom., 3, 340 (1976). (4) R. D. MacFarlane and D. F. Tagerson, Science, 191, 920 (1976). (5) M. A. Baklwin and F. W. McLafferty, Org. Mas Spechom.,7, 1353 (1973). (6) J Shabanowitz, P. Brynes, A . Maelike, D. V. Bowen, and F. H. Field, Biomed. Mass Spectrom., 2 , 164 (1975). (7) M. Ohashl, K.Tsujinioto and A. Yasuda, Chem. Lett., 1976, 439-440. ( 8 ) D. F. Hunt, J. Shabanowitz, F. K.Botz, and D. Brent, Anal. Chem., 49,

1160 (1977). (9) R. J. Beuhler, E. Flanigan, L. J. Greene, and L. Friedman, Biochemistry, 13, 5060 (1974)

(IO) R. J. Beuhler, E. Flanigan, L. J. Greene, and L. Friedman, J. Am. Chem. Soc., 96, 3990 (1974). (1 1) F. Hatch and B. Munson, Anal. Chem., 49, 169 (1977).

RECEIVED for review January 18,1978. Accepted March 21, 1978.