1870
Anal. Chem. 1984, 56, 1870-1876
in Table I for these compounds were obtained in this manner. Linearity of the system 2 peak height with amount derivatized was demonstrated for PGE1, PGE2,8-iso-PGE,, and 16,16dimethyl-PGE, over a range of 150-900 pg. The sensitivity of the method for these prostaglandins was comparable to that observed for the 15-methyl-PGE, epimers. Theoretically, this analysis approach is applicable, after appropriate modification of the mobile phase compositions, to the analysis of other prostaglandins and eicosanoids whose panacyl bromide derivatives elute later (and are generally more polar) than the 15-methyl-PGE, derivatives on HPLC system 1. The analysis of derivatives eluting before 15-methyl-PGE, is complicated by interference from the panacyl bromide derivatizing reagent. Although not designed for prostaglandin profiling applications, this heteromodal column switching technique can be used to analyze a sample for more than one component, provided that the analytes can be forced to elute as a narrow band on HPLC system 1without compromising the sensitivity and specificity of HPLC system 2. This will normally require that the prostaglandin analytes have closely related molecular structures, as was the case for the analysis of (15R)- and (15S)-15-methyl-PGEz and 16,16-dimethylPGE2.
ACKNOWLEDGMENT The authors thank F. A. Fitzpatrick and W. J. Adams for their helpful comments, Dave Gleason for construction of the solid-state and pressure impulse relay devices for switching valve actuation, and Linda Missias for assistance in the preparation of this manuscript. Registry No. PGE1, 745-65-3; PGE2, 363-24-6; (15R)-15methyl-PGE2, 55028-70-1; (15S)-15-methyl-PGE2,35700-27-7; 16,16-dimethyl-PGE2,39746-25-3;8-iso-PGE2,27415-25-4;8-iso[ (15R)-15-methyl-PGE2], 91200-56-5.
LITERATURE CITED (1) Granstrom, E.; Klndahl, H. Adv. Prostaglandin Thromboxane Res. 1978, 5, 119-210, 1 Fischer, C.; Frollch, J. C. Adv. LlpM Res. 1982, 19, 185-202. I Blalr, I. A. Br. M e d . Bull. 1983, 39, 223-226. 1 Waddell, K. A.; Blalr, I . A.; Wellby, J. Blomed. M s s Spectrom. 1983, IO. 03-80. -Fitzpatrick, F. A. Adv. Prostaglandin Thromboxane Res. 1978, 5 , 95-116. FitzpaGick, F. A.; Wynalda, M. A.; Kalser, D. G. Anal. Chem. 1977, 49, 1032-1035. Fltzpatrlck, F. A.; Strlngfellow, D. A.; Maclouf, J.; Rigaud, M. J . Chromatogr. 1979, 177,51-60. Tsuchiya, H.; Hayashi, T.; Naruse, H.; Takagi, M. J . Chromatogr. 1982. 237. 247-254. Hatsuml, M.; Klmata, S. I.; Hlrosawa, K. J . Chromatogr. 1982, 253, 27 1-275. Watkins. W. D.; Peterson, M. B. Anal. Blochem. 1982, 725,30-40. Merrltt, M. V.; Bronson, G. E. J . Am. Chem. SOC. 1978, 100, 1891-1895. Robert, A.; Yankee, E. W. Proc. SOC. Exp. Biol. Med. 1975, 148, 1155-1158. Robert, A.; Magerleln, 8. J. A&. Biosci. 1973, 9 ,247-253. Wickrema Sinha, A. J.; Shaw, S. R.; Thornburgh, B. A. Proceedings of the 33rd National Meeting of the Academy of Pharmaceutlcal Sciences, San Diego, CA, Nov 14-18, 1982; PTOX p-27. Senftleber, F.; Bowling, D.; Stahr, M. S. Anal. &em. 1983, 55, 810-81 2. Freeman, D. H. Anal. Chem. 1981, 53,2-5. Emi, F.; Keller, H. P.; Morln, C.; Schmitt, M. J . Chromatogr. 1981, 204,65-76. Apffel, J. A.; Alfredson, T. V.; Majors, R. E. J . Chromatogr. 1981, 206,43-57. Major, R. E. J . Chromatogr. Sci. 1980, 18,571-579. Snyder, L. R. J . Chromatogr. Scl. 1978, 16,223-234. Stehle, R. G.; Oesterllng, T. 0. J . Pharm. Sci. 1977, 66, 1590-1595. Fenlmore, D. C.; Davis, C. M.; Whitford, J. H.; Harrington, C. A. Anal. Chem. 1976, 48,2289-2290. Turk, J.; Weiss, S. J.; Davis, J. E.;Needleman, P.Prostaglandins 1978, 76, 291-309. Yamada, K.; Onodera, M.; Aizawa, Y. J . Pharmacol. Methods 1983, 9 , 93-100. Savitsky, A.; Golay, J. M. Anal. Chem. 1964, 3 6 , 1627-1639.
~.
RECEIVED for review February 3, 1984. Accepted April 26, 1984.
Direct Comparison of Secondary Ion and Laser Desorption Mass Spectrometry on Bioorganic Molecules in a Moving Belt Liquid Chromatography/Mass Spectrometry System T. P. Fan,E. D. Hardin, and M. L. Vestal* Department of Chemistry, University of Houston, Houston, Texas 77004 The two sofl lonlzatlon/desorptlon technlques, SIMS and LDMS, have been dlrectly compared in our LC/MS system using nonvolatlle blomolecuies as test samples. An ion gun has been Installed in our LC/LDMS Instrument which utlllzes a movlng belt Interface and a thennospray sample deposttlon devlce ( f ). Both secondary Ion mass spectra and laser desorptlon mass spectra can be acqulred under otherwlse Identical condltlons. Some slgniflcant mass spectral differences between SIMS and LDMS have been observed from amino aclds and nucleosides. Surface coverage and dosage effects on sample Ion currents have been studied and are discussed. The advantages and limltatlons of uslng a continuous ion beam vs. a pulsed laser beam In an LC/MS are examlned and evaluated.
Two techniques that have received much interest for molecular weight determinations on nonvolatile, thermally labile biomolecules are secondary ion mass spectrometry (SIMS) and 0003-2700/84/0356-1870$01.50/0
laser desorption mass spectrometry (LDMS). In 1976 Benninghoven and co-workers (2) showed that kiloelectronvolt primary ions at low current densities could be used to desorb intact molecular ions from organic compounds adsorbed on a metal surface with good sensitivity. This technique called “static” SIMS has been used to detect and identify a wide variety of nonvolatile organic samples including amino acids, peptides, vitamins, pharmaceutical compounds, nucleosides, nucleotides, and others ( 3 , 4 ) . Closely related to SIMS is fast atom bombardment (FAB),which uses a neutral primary atom beam and a liquid sample matrix (5, 6). With FAB highperformance magnetic instruments can be used and impressive results have been obtained from a wide variety of difficult samples, including essentially all of the samples determined by SIMS as well as other samples which have not been successfully analyzed before. The important difference between FAB and SIMS appears not to be the charge state of the primary ionizing beam, but rather it is the use of a liquid sample matrix. Laser desorption mass spectrometry (LDMS) of organic 0 1984 Amerlcan Chemical Society
ANAL.YTICAL CHEMISTRY, VOL. 56, NO. 11, SEPTEMBER 1984
SIMS
TO QUADRUPOLE MASS ANALYZER
1
‘I‘
1871
ADENOSINE
I iin I
ION
ION
4“ DIFFUSION PUMP
Figure 1.
Schematic of the ionization source chamber.
solids was investigated earlier (7) but did not achieve widespread interest until 1978 when Kistemaker and co-workers showed that submicrosecond laser pulses could be used to desorb and ionize pseudomolecular ions from many samples including amino acids, peptides, nucleosides, dinucleotides, and oligosaccharides(8). Both bulk solids (8,9)and thin films coated on inert (10) and metal (11)substrates have been used to produce pseudomolecular ions by LDMS. An excellent review of recent LDMS work has been published by Hillenkamp (12). There has also been considerable interest in understanding the ionization mechanisms involved with these two techniques. Although these two techniques use dissimilar energy sources to induce desorption-ionization (ions vs. photons), the spectra they produce are somewhat similar. Both techniques yield even-electron, pseudomolecular ions from nonvolatile samples. A comparison of SIMS and LDMS has been made with data from different labs and different type instruments (13). One of the problems associated with comparing different ionization techniques is that ideritical samples must be used with both techniques if meaningful results are to be obtained (14).In an effort to more clearly understand the differences and similarities between SIMS and LDMS we have installed an ion gun on our laser desorption mass spectrometer (1). Our instrument, with a moving belt interface,liquid chromatograph injection loop, and thermospray sample deposition system has the capability to supply essentially identical samples to the source for either laser desorption or SIMS. SIMS has previously been used as an ionization method with a moving belt LDMS interface (15,16). In this work we have undertaken a systematic study to compare the characteristics of LDMS with SIMS.
INSTRUMENTAL SECTION The instrument used in this work has been described in detail in ref 1. Basically it consists of a quadrupole mass spectrometer, with a moving belt LC/MS interfaceand a Finnigan/INCOS data system. Samples are deposited onto the belt with a thermospray vaporizer to desolvate and spray the sample onto the moving belt under partial vacuum. Samples on the belt are carried to the source through differentially pumped vacuum interlocks. A homebuilt ion gun has been used in this work for obtaining SIMS spectra. Both ion beam and laser beam are aimed at the target surface at a 45’ angle. Figure 1is a schematic of the source chamber showing the mounting arrangement of the laser head and the ion gun for producing comparable spectra. Xenon gas ionized by electron impact is used as the source of primary ions. These ions are focused by an Emel lens and a set of steering plates followed by passing through an aperture to give an oval shaped spot about 5 mm2. The ion beam is impacted onto the belt with a kinetic energy of 3 keV and a measured current of typically 1 pA/cm2. The primary ion dosage to the belt surface can be varied by changing the belt speed or the ion current.
PRIMARY
ION CURRENT ( p / c m * )
Response area of 1 pg of adenosine measured at various primary ion currents: (a) combined ion intensity of protonated adenosine and adenine;(b) protonated adenosine ion intensity after ten times magnification.
Figure 2.
We are now using carbon steel belts (Ebtec Corp.) which have a blackened surface. These blackened &.eel belts apparently allow more efficient absorption of energy from the laser beam than did the stainless steel belts used earlier. When laser power densities are kept low there is no observable degradation of the belt surface. In the SIMS mode there is no observable difference in the spectra obtained from carbon steel compared to stainless steel belts. Details of operation in the laser desorption mode are given in ref 1. There is a major difference in the ion detection methods used with SIMS and LDMS. Since we are using a pulsed laser which results in the desorbed ions coming in pulses, a boxcar integrator is used to process the signal. In the SIMS mode ion production is essentially continuous and the boxcar integrator is set to the continuous mode of operation.
RESULTS AND DISCUSSION In order to obtain sufficient signal level to allow rapid scanning of mass spectra through a wide mass range, we employed primary ion currents of about 1pA/cm2 in the SIMS experiments. The primary ion current was determined by measuring the current collected by a Faraday cup placed at the target position. Primary ion currents in the pA/cm2 range are several orders of magnitude higher than those employed in typical static SIMS studies but with the moving belt system the surface ion dose does not necessarilly exceed the “static” limit. With the moving surface the time that a given surface element is irradiated by a continuous beam is given by the width of the beam in the direction of surface travel divided by the velocity of the surface. In our case the ion beam width is about 0.15 cm and belt speeds in the range from 0.25 to 1.4 cm/s were used in these studies. Thus our total ion doses never exceeded ca. 6 x 10l2 ions/cm2. If the nominal damage cross section is on the order of W4cm2,then at most a few percent of the surface is damaged by ion impact. Measurements of secondary ion intensities and fragmentation patterns give results which are in agreement with this conclusion. For example, Figure 2 shows both the total response and the (M + H)+intensity alone from a series of 1-pg injections of adenosine under various primary ion fluxes. Protonated adenine is the base peak and the major fragment observed in the adenosine spectrum. The protonated molecular ion intensity is maintained at a level 7-87’ of the total ion intensity throughout the primary ion current range applied. Ion intensity as well as background noise level increases or decreases simultaneously with primary ion current; therefore the signal-to-noise ratio is not improved a t high
(In,
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ANALYTICAL CHEMISTRY, VOL. 56, NO. 11, SEPTEMBER 1984
LDMS
(a)
I
lool
BACKGROUND
(b) LDMS
'"1
39 Kt
POSITIVE
ION
5 2
E
L 2
50
w 5 0
>
I-
5
4 W
-I W
K
a
I00
200
300
400
m /z
I"
I A
NEGATIVE
il
IO0
200
ION
4 D
300
m /z
SIMS BACKGROUND
(c)
SIMS
I
BACKGROUND
BACKGROUND I85
POSITIVE ION 43
:1
NEGATIVE ION
100
m/z
200
m/z
Flgure 3. Background mass spectra of the carbon steel belt: (a) LDMS positive ion mode: (b) LDMS negative ion mode: mode; (d) SIMS negative ion mode.
primary ion fluxes. For our work here we have chosen to operate with a primary ion current of 8 X lo-' A/cm2 which produces a strong secondary ion signal without substantial noise interference. In the positive LDMS mode background spectra from the carbon-steel belt under normal operation conditions (power density lo8W/cm2) show intense K+ ion signals together with low intensity Fe+, Na', and salt cluster ion signals corresponding to NaCl.K+, KCl.K+, (CsCl)2-Na+,and (CsC1)z.K+ as shown in Figure 3a. There are no significant low mass hydrocarbon peaks in the background spectrum. In the SIMS mode, the positive background spectrum (Figure 3c) shows intense low mass ions distributed below mass 150. These seem to be typical aliphatic hydrocarbon fragments arising from mechanical pump oil (18). The negative SIMS background spectrum (Figure 3d) shows intense peaks a t mass 93, 185, 277, and 369, all at 92 mass units apart. These ions correspond to fragments from polyphenyl ether diffusion pump oil. LDMS negative ion background spectrum (Figure 3b) also shows low intensity polyphenyl ether fragments. The major ions at mass 43,63, and 79 are presently unidentified but may correspond to C2H30-, POz-, and PO3- ions or to some fragments from pump oil. While our source chamber is maintained under vacuum at 2 x lo4 torr via a 4-in. diffusion pump, the sample introduction chamber is pumped by a mechanical pump and the pressure here is substantially higher, approximately 400 mtorr, when operating the LC at a 1mL/min flow rate. Continuous contamination of the belt by both mechanical and diffusion
(c)SIMS positive ion
pump oil is expected and this is clearly observed from SIMS background spectra. On the other hand, pump oil is essentially transparent in LDMS. Both SIMS and LDMS measurements were made on 14 amino acids and 4 nucleosides. Both positive and negative ion mass spectra were obtained for all compounds. Generally speaking, positive mass spectra obtained from LDMS and SIMS are quite complementary to each other. Figure 4a,b is a set of representative amino acid spectra obtained, in this case, from histidine. In the LD mass spectra there are no significant amounts of fragments and the protonated molecular ion signal is weak compared to mono- or di- Na+ and K+ attached molecular ions. With SIMS, however, protonated molecular ions and characteristic fragment ions such as (M - OH)+ and (M - COOH)+ dominate the mass spectrum. Alkali-attached molecular ion signals are either weak or absent in SIMS spectra of amino acids and nucleosides. In the laser desorption mass spectra of nucleosides, substantial amounts of alkali attached purine or pyrimidine base fragment ions are observed as can be seen in the spectrum of cytidine (Figure 5). The cationized fragments may originate from the cationized molecular ions by loss of a ribose. The SIMS spectrum of cytidine shows a high abundance of protonated cytosine base together with a low intensity of alkali attached molecular ions and a very weak MH+. Adenosine is the only nucleoside tested that yields a substantial (M + H)+ signal by SIMS. The negative ion spectra from LDMS and SIMS are in general very similar. No chloride attached ions are observed.
ANALYTICAL CHEMISTRY, VOL. 56, NO. 11, SEPTEMBER 1984
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(b)
LDMS
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HISTIDINE
194
17s
MK+
MNO+
MH+
(M-COOHT 110
i
160 I50
200
100
2
(c)
LDMS HISTIDINE
250
rn/z (d)
NEG. ION
SIMS
HISTIDINE
100
1
NEG. ION
54
M-H )-
loo
c
200
150
m/z
c
E
E 50
Y
W
5
5 Y
W
50
K
50
100
I50
200
250
Lk 100
2
200
If
0
m /z
m/z
Figure 4. Mass spectra of hlstidlne: (a) LDMS positive ion mode: (b) SIMS positive ion mode; (c) LDMS negative Ion mode; (d) SIMS negative
ion mode.
For amino acids, the (M - H)-ion is the most abundant species observed with both techniques and is often the only ion detected in LDMS. Structually signficant fragment ions with reproducible relative intensities are observed in SIMS. Figure 4c,d shows the negative ion LD and SIMS mass spectra of histidine. With nucleosides, both LDMS and SIMS produced intense deprotonated purine or pyrimidine base ion signals as well as weak deprotonated molecular ion signals as shown by uridine in Figure 6. Loss of a water molecule from the deprotonated base fragment is observed by laser desorption at higher power densities. Our samples are normally introduced into the system using water as the mobile phase, no extra salt is added. Sodium and potassium ions are probably supplied by contaminants in the sample and the belt itself. Therefore the relative ion intensities for different cationized species may very from time to time but the mass spectral pattern remains consistent for the same compound provided the laser power density is maintained constant. Table I summarizes comparisons of relative intensities for major ions produced by SIMS and LDMS for the 14 amino acids and 4 nucleosides studied. It is apparent that the major differences in mass spectra between LDMS and SIMS are stronger cationization signals and less fragmentation by laser desorption. In order to investigate the effects of primary ion beam dosage as well as sample surface coverage, a series of experiments at different belt speeds were performed. Increasing the belt speed results in attenuation of the incident beam dosage by the same factor if all other parameters are kept constant. For these experiments a series of various concen-
Table I. Summary of Major Ion Intensities from 14 Amino Acids and 4 Nucleosides" positive ion amino acids neutrals SIMS LDMS acidic SIMS LDMS basic SIMS LDMS
(M+ alkali)
(M
(M45)
w -
S
W -
-
M
S
-
S
(M-45NFb)
(MH)
(M-HNFb)
s w
M -
S S
W -
M -
S -
S S
-
M-S
M -
S S
-
+ H)
w
negative ion
-
positive ion nucleosides
(M + alkali)
SIMS LDMS
W S
W W
negative ion
(B +
(B +
( M + H)
H)
alkali)
(M-H)
H)
W W
S M
W S
W W
S S
(B-
"S,strong (70-100%) relative intensity; M, moderate (30-70%) relative intensity; W, weak (
