Anal. Chem. 1985, 57, 985-991
ACKNOWLEDGMENT The authors thank Hugo Billiet and Anton Drouen, Laboratory for Instrumental Analysis, Technical University of Delft, The Netherlands, for their technical assistance in measuring the data, and Hans Frank, Department of Biochemistry, Technical University of Delft, The Netherlands, for providing the protein data. This research was supported in part by the providing of equipment by Hewlett-Packard BV, The Netherlands.
LITERATURE CITED Vandeglnste, B. G. M.; de Galan, L. Anal. Chem 2124-2 131.
.
1975, 4 7 ,
985
(2) SpJertvoll, E.; Martens, H.; Volden, R. Technometrlcs 1982, 2 4 , 173- 180. (3) Mallnowskl, E. R.; Howery, 0.G. “Factor Analysis in Chemistry”; Wiley: New York, 1980. (4) Sharaf, M. A.; Kowalskl, B. R. Anal. Chem. 1982, 5 4 , 1291-1296. (5) Lawton, W. H.; Sylvestre, E. A. Technometrlcs 1971, 73, 617-633. (6) Osten, D. W.; Kowalski, B. R. Anal. Chem. 1984, 56, 991-995. (7) Chen, J. H.; Hwang, Lian-Pin Anal. Chlm. Acta 1981, 733, 271-281. (8) Vandeginste, B. G. M. Pure Appl. Chem. 1983, 5 5 , 2007-2016. (9) Zhou, 0.; Chang, Th.; Davies, J. C. Math. Gml. 1983, 15. 581-606. (10) Glllette, P. G.; Lando, J. B.; Koenlg, J. L Anal. Chem. 1983, 55, 630-633.
RECEIVED for review September 24,1984. Accepted January 7, 1985.
On-Line Liquid Chromatography/Fast Atom Bombardment Mass Spectrometry Justin G. Stroh, J. Carter Cook, Richard M. Milberg, Larry Brayton, Tsuyoshi Kihara,’ Zhaogeng Huang,2and Kenneth L. Rinehart, Jr.* School of Chemical Sciences, University of Illinois at Urbana-Champaign, 1209 West California Street, Urbana, Illinois 61801
Ivor A. S. Lewis VG Analytical, Ltd., Wythenshawe, Manchester M23 9LE, United Kingdom
The technique of liquid chromatography/fast atom bombardment mass spectrometry using a modified moving belt interface is described. Compounds studied by this method include antiamoebin I, emerknicins I I A and I IB, digitonin, gramicidin J, and a mixture of tetra- to nonapeptides obtained by partial hydrolysis of antiamoebin I. Partlai structures are assigned to two new antiamoebins identified from the hydrolyzate of crude antiamoebin I.
Recent interest in the coupling of liquid chromatography with mass spectrometry (LC/MS) has been directed toward the design and improvement of two types of interfaces. On the one hand, the direct liquid introduction (DLI) (1) and thermospray (2)techniques involve submitting the LC eluate in the gas phase to the ion source of the mass spectrometer. The DLI method yields chemical ionization (CI) data where the LC solvent becomes the reagent gas. The thermospray method uses ions derived from clusters in the solvent system. As the solvent is vaporized, droplets of solute are formed with salt attached, yielding charged species. On the other hand, the moving belt interface (3)has been employed to obtain both electron ionization (EI) and CI data for samples introduced in the liquid state. Using this interface, the LC eluate is deposited on a moving belt, which is then heated by an infrared heater to remove the solvent. The sample is transported in the condensed phase to the ion source of the mass spectrometer where it is vaporized by means of ‘On leave from The Institute of Physical and Chemical Research,
Saitama, Japan.
*On leave from Wuhan University, Wuhan, China. 0003-2700/85/0357-0985$01.50/0
another infrared heater. Excess sample left on the belt is passed through a clean-up heater. LC has become the method of choice for separating polar and thermally labile compounds. Fast atom bombardment (FAB) MS has proven to be a powerful tool in the analysis of such compounds ( 4 , 5 ) . Therefore, the coupling of these techniques, “LC/FABMS”, would provide a direct method for analysis of mixtures of polar or thermally labile compounds. Surprisingly, only two reports have appeared (6,7) on the use of on-line LC/FABMS. Among polar compounds most amenable to FABMS analysis are peptides. The use of DLI for analysis of mixtures of small underivatized peptides has been reported (8) and thermospray has been used in conjunction with tryptic digest to obtain sequence information on a-melanocyte-stimulating hormone (9). LC/FABMS should be useful in extending the range of peptide mixtures amenable to LC/MS analysis. We report here an on-line LC/FAB system using a moving belt interface to observe pseudomolecular and fragment ions for polar compounds and applications to a series of peptides, including the underivatized hexadecapeptide antiamoebin I (molecular weight 1669) (lo), a mixture of emerimicins (zervamicins) IIA and IIq (molecular weights 1823 and 1837) (IO), and a mixture of tetra- to nonapeptides obtained by partial hydrolysis of antiamoebin I (11).
EXPERIMENTAL SECTION Compounds Studied. The compounds studied were antiamoebin I [Hindustan Antibiotics, complex separated by Pandey a mixture of emerimicins and Meng, University of Illinois (12,13)], IIA and IIB [provided by A. D. Argoudelis, The Upjohn Co., Kalamazoo, MI, and shown earlier (14) to be identical with zervamicins IIA and IIB], gramicidin J hydrochloride (Sigma, St. Louis, MO), digitonin (National Biochemicals Corp., Cleveland, 0 1985 Amerlcan Chemical Societv
986
ANALYTICAL CHEMISTRY, VOL. 57, NO. 6, MAY 1985
I
Heqt/nq Element
I,
LC
-
Cooling
N2
vable Jet Nozzle
Figure 2.
LC interface. 1251
Figure 1.
Jet sprayer.
OH), and a mixture of peptides obtained by partial hydrolysis of antiamoebin I, as previously reported (11). Liquid Chromatography. Two LC pumps were used for these experiments-a Waters M-6000A (Milford, MA) pump for the mixture of peptides obtained from the partial hydrolysis of antiamoebin I, for gramicidin J hydrochloride, and for digitonin, and a Beckman/Altex llOA (Berkeley, CA) pump for the experiments involving intact antiamoebin I and emerimicins IIA and IIB. A Rheodyne 7125 injector (Berkeley, CA) was employed for all experiments. Two columns were used-a Chromanetics Spherisorb ODS column (250 mm X 4.6 mm, P. J. Cobert Assoc., St. Louis, MO) for the antiamoebin I hydrolyzate and an Alltech (2-18 column (250 mm X 2.1 mm, Deerfield, IL) for all the other experiments. Flow rates and solvent systems are given in the Results section. Mass Spectrometer. A VG Analytical ZAB-HF mass spectrometer was used for all experiments. Data were either recorded on a Gould electrostatic recorder or acquired with a VG 11/250 data system. Interface. A commercial moving belt interface (VG Analytical) was modified for operation with FABMS. Samples eluted from the column pass through a jet which deposits the sample onto a Kapton belt as a dry spray. The jet shown in Figure 1is made from a standard VG Analytical E1 solids probe and is based on the design of Hayes et al. (15). The LC line extends approximately 0.5 mm beyond the nozzle. Nitrogen passes through a separate line which extends beyond the vacuum seal of the jet. A nitrogen pressure close to 0.5 psi creates a distribution of sample on the belt and helps vaporization. Heat applied to the LC and nitrogen lines by a heating coil vaporizes the sample and is controlled by the temperature controller on the console of the mass spectrometer. A water line located just above the heating coil prevents overheating of the jet assembly. If additional heat is required, as was the case for the hydrolyzate of antiamoebin I, a custom-built Hotwatt heater (Danvers, MA) (l/g in. i.d.) is used on the nitrogen line. In this case the water line must be disconnected to facilitate the heating. The nozzle of the jet is made from an end nut to which a 3 mm long piece of stainless steel tubing (2 mm i.d.) has been welded with the end beveled to provide a uniform flow of nitrogen. The entire jet assembly is mounted on the housing of the interface so that the angle and distance of deposition can be varied. The optimum angle and distance of deposition have been determined to be ca. 45O and ca. 2 mm, respectively. The entire interface is shown in Figure 2. All infrared heating has been removed from the system and the clean-up heater has been replaced by a clean-up washer. Methanol is passed through the washer in the direction counter to the movement of the belt and then through a liquid nitrogen trap which is attached to a vacuum pump. A mild vacuum of 0.75 atm is applied to the housing to maintain a flow of liquid through the washer. The original belt drive stepping motor has been replaced with a variable ac motor (Japanese Product Co., Elmsford, NY). Calibration of the data system is performed with the normal FAB probe. For easy retraction and insertion of the LC/FAB probe, a probealigning mechanism was built and connected to both the LC/FAB probe and the end plate of the housing. A variable end stop was placed on the mechanism behind the end plate so that when the
1235
I i
1263
z
w
* 5 z
B
I
miz Flgure 3. 1251, M
Molecular ion reglon of LC/FAB spectrum of dlgitonin ( m l z + Na).
probe is moved forward into the ion source, it comes to rest at the correct position. For the analysis of emerimicins IIA and IIB a belt speed of 0.08 in./s was used, and for antiamoebin 10.24 in./s. For all other experiments the speed was 0.13 in./s.
RESULTS Our initial experiments were directed toward determining whether the LC/FAB apparatus using a moving belt would provide spectra. To do this, we injected digitonin and then gramicidin J, in methanol, into the LC injector which was connected directly to the spray depositor. Both gave strong M + Na ions (sodiated molecules) and the molecular ion region of digitonin is shown in Figure 3. The ion intensity varied slightly from scan to scan, and this effect can be magnified by adding water to the solvent system, as will be shown below. Encouraged by these results, we attempted to apply glycerol and thioglycerol separately to the belt as FAB matrices, using as an applicator a self-contained unit with a reservoir filled with the matrix. The applicator tip was designed to spread a thin, even film of the matrix onto the belt, and the unit was
ANALYTICAL CHEMISTRY, VOL. 57, NO. 0, MAY 1985
967
I@. 9s. SI.
6. Rl. 75.
n. 6.
1. 8.
m.
-
0
4
8
12 min.
si lil IS & T1 Figure 4. LC/FABMS total Ion current and LC/UV (inset, X = 220 nm) of crude antiamoebln I hydrolyzate: 80% methanol, 20% water, 0.03% trmuoroacetic acid, 0.05% glycerol, flow rate 0.5 mLlmln, 100 pg in)ected. placed directly after the drive roller. Unfortunately, even when a small amount of matrix was used, the source vacuum could not be maintained due to the large surface area of the belt exposed to high vacuum. Therefore, we have subsequently applied the matrix as part of the LC system a t very low concentrations (0.05% of the solvent employed). With this system, the applicability of LC/FAB to the structural analysis of a mixture of peptides was examined. A mixture of tetra- to nonapeptides produced by partial hydrolysis of antiamoebin I with trifluoroacetic acid (11) was studied by LC/FAB. The presence in this mixture of seven compounds was indicated by LC with UV detection (LC/UV) at 220 nm, and by LC/FAB, as shown in Figure 4, where the compounds are centered around scans 107,120,146,154,172, 195, and 226. LC/FAB spectra for typical scans, 154 and 172, are given in Figures 5 and 6, respectively, and the structures of the peptides, along with the fragment ions of all observed compounds, are given in Table I. It should be noted that the ion signal at m/z 284 is due to the background from the belt. The major cleavage occurs at the peptide bonds with charge retention on the N-terminal portion of the peptide, which greatly facilitates interpretation of the data. Although the spectra differ in quality, they are adequate to identify the peptide and, in some cases, would be adequate to sequence the peptide de novo. Although the antiamoebin I sample was initially assumed to be pure, the LC/FAB data showed at least three other antiamoebins to be present. While scan 172 is for the expected N-terminal nonapeptide (nominal molecular weight 901) from
cleavage of antiamoebin I between Aib and Hyp, scans 146 and 154 indicate the presence of two other nonapeptides with the nominal molecular weight 887, and scan 195 indicates yet another nonapeptide with the nominal molecular weight 915. Scan 154 contains the N-terminal nonapeptide amino acid sequence of antiamoebin I11 (16),indicating the presence of that peptide as an impurity. The N-terminal amino acid sequence contained in scan 146, however, is not attributable to any known antiamoebins and the partial structure of a new antiamoebin, antiamoebin IV, can be assigned from it. Similarly, scan 195 provides the partial (N-terminal) sequence for another new antiamoebin, antiamoebin V. Although ions were observed well above background for the last peptide to elute from the column (appearing at scan 226), none was easily interpretable. However, with this one exception, all the peptides were easily identified from the molecular weight and fragment information, showing the power of LC/FAB as a tool for structure determination. Following this successful LC/FAB analysis of underivatized peptides, we undertook a similar study of larger peptides, including intact antiamoebin I and emerimicins IIA and IIB. Antiamoebin I, a hexadecapeptide of known structure, has been studied by FABMS (10)and is known to give an M + Na ion as well as fragment ions sufficient to define the sequence. By spotting antiamoebin I onto the belt, the M + Na ion is observed as are sequence ions (see Figure 7) corresponding to successive peptide cleavages near the N-terminus. Thus, while the cleavages do not define the complete sequence, they define the N-terminal nonapeptide sequence.
988
ANALYTICAL CHEMISTRY, VOL. 57, NO. 6, MAY 1985
3).
w.2
n. '181.3
Flgure 5. Scan 154.
Table I. LC/FAB Fragmentations of Peptides from Crude Antiamoebin I Hydrolyzate Scan 107 275136014454
Ac-Phe-Aib
Aib
4851
OH
Aib
+
5071
+
Na
523
+
Naz -H
Scan 120
2751360144545441,
Ac-Phe-Aib Scan 146
Aib
Aib
Iva
275$Z80--+431$530++
Ac-Phe-Aib
-H
S621
+
OH
-H
5841
+
H
+
Na
Na
5677
Aib
Ala
Iva
Giy
+
Leu-Aib-Aib-OH
-H Scan 1 5 4
Ac-Phe-Aib
'
~
~
~
~
~
+ ~9N a 7 +~
~
N 93 a z2 1~ -H
Scan 172 2
Ac-Phe-Aib
7
5
~
Aib
3
6
Aib
0
1
4
4
Iva
5
~
S
4
4
~
Leu
~
~
Aib-OH
~
7
1
4
~
+
9N2a4 1
+
9381
~
~
~
+
Nap
Scan 195 275-j-36014454
Ac-Phe-Aib
Aib
Aib
5 4 4 ~ 6 1 5 1
Iva
Ala
Leu-Aib-Aib-OH
Na
9601
-H
~
~
~
~
~
ANALYTICAL CHEMISTRY, VOL. 57, NO. 6, MAY 1985
989
GE W b
5
z
0
L
Flgure 6.
Chart I 2751360l+4451544
Ac-Phe-Aib
Aib
Aib
1601
Iva
7
Gly
714 ++799$8841
Leu
Aib
Aib
Hyp-Gln-Iva-Hyp-Aib-Pro-Phol
+
Hyp-Gln-Aib-Hyp-Aib-Pro-Phol
+
Na
Chart I1 3 4 2 1
Ac-Trp-Ile
S 5 8 ~
Gln-Aib
7691
Ile-Thr
10521
Aib-Leu-Aib
ernerimicin I I A 342i470$569-+
Ac-Trp-Ile
Gln
Iva
10661
783-4
Ile-Thr
Aib-Leu-Aib
Hyp-Gln-
Aib-Hyp-Aib-Pro-Phot
+
ernerimicin I I B
+
The M Na ion (nominal mass 1692) is recognized by the computer as m / z 1693, in accord with its accurate mass (1692.9314) (Chart I). By contrast, the spectra of antiamoebin I and those of emerimicins IIA and IIB obtained by LC/FAB (including a column and interface) are of low intensity (see below). Although M Na ions are present, isotope peaks, as well as the familiar peaks at every mass unit normally present in FABMS, are not observed. In these ,experiments, the resolution was set for 2000 so that isotope peaks should have been observed. The reason for such a low intensity is believed at present to be thermal decomposition of the sample in the jet depositor, resulting in low sample concentrations on the belt. Figure 8 shows the total ion current (TIC) and LC/UV traces of a mixture of emerimicins IIA and IIB. Separation of the peptides is clearly effected. Emerimicin IIA is the first major peptide to be eluted from the column and appears in the TIC trace of those spectra centered around scan 53.
+
Emerimicin IIB is the second major peptide eluted and is centered around scan 93. The solvent system for this LC/FAB run contained 24% water. When the same mixture was injected onto the column with 100% methanol as the solvent system, the TIC trace shown in Figure 9 was obtained. Although the two components are virtually unresolved, the background is much lower and peak breakup is greatly reduced. One possible reason for the peak breakup in the TIC is the material from which the belt is made, the hydrophobic POlyimide, Kapton. As the solvent passes from the heated tip of the jet onto the belt, it may condense onto the belt as very fine droplets, yielding a nonuniform distribution of sample. Since no infrared heating of the belt is used, the only solvent removal is by vacuum pumping. We are currently trying to overcome this problem. The spectra of emerimicins IIA and IIB taken from the LC JFAB run containing 24% water (Figure 8) are shown in
990
ANALYTICAL CHEMISTRY, VOL. 57, NO. 6, MAY 1985
Flgure 7.
LCIFAB spectrum of antiamoebin I.
a
48
€a
88
1H
128
148
1Ea
scan no.
a
38
48
58
68
78
sa
9
im
1
iie
ia
scan no.
Figure 9.
LCIFABMS total Ion current of emerimicins I IA and I IB in
100% methanol, 6 pg injected.
LCIFABMS total ion current and LCIUV (inset, A = 220 nm) of emerimicins I I A and IIB: 7 6 % methanol, 2 4 % water, 0.03% trifluoroacetic acM, 0.05 % glycerol, flow rate 0.3 mLlmin (LCIFABMS), 0.4 mLImin (LCIUV), 13 pg injected. Flgure 8.
Figures 10 and 11. These yielded ion signals corresponding to the sodiated molecules, along with useful sequence ions from cleavage at the peptide bonds as shown in Chart 11. Spectra for this mixture of emerimicins obtained by LC/FAB using 100% methanol (Figure 9) yielded similar results. Although the mixture was not chromatographically resolved using this solvent system, M + Na and fragment ions were observed for both peptides and the relative intensities of the ions for the two sodiated molecules changed over the chromatographic peak. Finally, a preliminary study of the sensitivity of the LC/ FAB technique was undertaken using the peptide Ac-PheAib-Aib-Aib-Iva-OH. Injection of 125 ng of the peptide through the column gave an M Na ion a t m / z 584 with a
+
m,z
Emerimicin IIA. signal-to-background ratio of approximately 3. From this and the foregoing results we conclude that LC/FAB can be a Figure 10.
Anal. Chem. 1985, 57, 991-996
T -
2888 m,z
Figure 11. Emerimicin IIB.
991
(3) McFadden, W. H.; Schwartz, H. L.; Bradford, D. C. J. chromafogf. 1978, 122, 389-396. (4) Rlmhart, K. L., Jr. Science (Washington, D.C.) 1882, 218, 254-260. (5) Barber, M.; Bordoll, R. S.; Elllott, 0. J.; Sedgwlck, R. D.; Tyler, A. N. Anal. Chem. 1982, 5 4 , 645A-657A. (6) Lewis, I. A. S.; Brooks, P. W. Abstracts, 31st Annual Conference on Mass Spectrometry and Allied Toplcs; Boston, MA; May 8-13, 1983; pp 850-85 1. (7) Dobberstein, P.; Korte, E.; Meyerhoff, G.; Pesch, R. I n f . J. Mass Specfrom. Ion Phys. 1883, 46, 185-188. (8) Kenyon, C. N. Blomed. Mass Spectrom. 1883, 10, 535-543. (9) PIIosbf, D.; Kim, H. Y.; Dyckes, D. F.; Vestal, M. L. Anal. Chem. 1884, 56, 1236-1240. (10) Rlnehart, K. L., Jr.; Cook, J. C., Jr.; Pandey, R. C.; Gaudloso, L. A.; Meng, H.; Moore, M. L.; Gloer, J. E.; Wilson, G. R.; Gutowsky, R. E.; Zlerath, P. D.; Shield, L. S.; Li, L. H.; Renls, H. E.; McQovren, J. P.; Canonlco, P. 0. Pure Appl. Chem. 1982, 5 4 , 2409-2424. (11) MmQ, H. Ph.D. Thesis, Unlverslty of Illlnois at Urbana-Chqmpaign, Urbana, IL, 1979. (12) Pandey, R. C.; Meng, H.; Cook, J. C., Jr.; Rinehart, K. L., Jr. J. Am. Chem. SOC. 1977, OQ, 5203-5205. (13) Pandey, R. C.; Cook, J. C., Jr.; Rlnehart, K. L., Jr. J. Antblot. 1978, 3 1 , 241-243. (14) Rinehart, K. L., Jr.; Gaudioso, L. A.; Moore, M. L.; Pandey, R. C.; Cook, J. C., Jr.; Barber, M.; Sedgwick, R. D.; Bordoli, R. S.; Tyler, A. N.; Green, B. N. J. Am. Chem. SOC.1981, 103, 6517-6520. (15) Hayes, M. J.; Lankmayer, E. P.; Vouros, P.; Karger, B. L.; McGuire, J. M. Anal. Chem. 1883, 5 5 , 1745-1752. (16) Rlnehart, K. L., Jr. Trends An81 Chem. 1983, 2 , 10-14.
sensitive and viable technique for the analysis of mixtures of polar and thermally labile compounds. Registry No. Ac-Phe-Aib-Aib-Aib-OH,82527-10-4; Ac-PheAib-Aib-Aib-Iva-OH,95070-15-8;Ac-Phe-Aib-Aib-Aib-Aib-GlyLeu-Aib-Aib-OH,95070-16-9; Ac-Phe-Aib-Aib-Aib-Iva-Gly-LeuAib-Aib-OH,95098-16-1;Ac-Phe-Aib-Aib-Ala-Iva-Gly-Leu-AibAib-OH, 95070-17-0;Ac-Phe-Aib-Aib-Aib-Iva-Ala-Leu-Aib-AibRECEIVED for review September 14,1984. Accepted January OH, 95070-18-1; antiamebin I, 64347-37-1; emerimicin IIA, 18, 1985. Presented in part at the 32nd Annual Conference 79395-86-1; emerimicin IIB, 79395-85-0; digitonin, 11024-24-1. on Mass Spectrometry and Allied Topics, San Antonio, TX, LITERATURE CITED May 27-June 1, 1984, Abstract MPA 20. This work was (1) Baldwin. M. A.; McLafferty, F. W. Org. Mass Specfrom. 1973, 7 , supported in part by grants from the National Institutes of 1111-1 112. Health (GM 27029 and RR 01575) and the National Science (2) Blakley, C. R.; Carmody. J. J.; Vestal, M. L. Anal. Chem. 1880, 5 2 , Foundation (PCM-81-21494). 1636-1641.
Optimization and Application of Thermospray High-Performance Liquid Chromatography/Mass Spectrometry Robert D. Voyksner* Analytical and Chemical Sciences, Research Triangle Institute, P.O. Box 12194, Research Triangle Park, North Carolina 27709
Carol A. Haney North Carolina State University, Department of Research Administration, Box 7003,Raleigh, North Carolina 27695
Thermospray (TSP) HPLC/MS frequently produces molecular weight informatlon and exhibits lower detection limits than other HPLC/MS techniques for certain compounds. Thermospray exhibits the best sensitivlties when used with a 0.1 M ammonium acetate buffer in a high percent water solvent system. The optimal interface temperatures vary with solvent composition. The optimal temperatures can be determined by maximizing the sohrent-buffer Ion Intensities. Thermospray Ionization resembles ammonia C I , which produces protonation, ammonium addition, and proton-bound solvent molecule clusters depending on the basicity of the analyte and existing thermodynamics In the source. The Ionization process is sott; very few fragment ions are Observed. The use of TSP HPLCIMS for the analysis of triazine herbicldes and organophosphorus pesticides shows excellent speclflclty and sensitivity. Thermospray spectra of these compounds were quite simple, consisting of an [M HI+ or [M NH,]' Ion with no fragment ion detected. Detection limits for these compounds Varied from 10 to 300 ng and full scan spectra were obtained ( S I N of about 5 for the base peak).
+
+
Organophosphorus pesticides and triazine herbicides are widely used in agriculture. Methods for the analysis of these compounds and their metabolites are important from both environmental and agricultural points of view. These compounds exhibit persistence in the soil and contamination due to spraying and runoff (1-3).Analytical methods presently used for analyzing these herbicides and pesticides include gas chromatography (GC) (4-lo),GC/mass spectrometry (GC/ MS) (11-161,high-performance liquid chromatography (HPLC) (17-22),and HPLC/MS (23,24).The advantages claimed for HPLC/MS are suitability for high molecular weight and polar compounds or metabolites and thermally labile compounds (not ammenable to GC analysis) coupled with a specific and sensitive means of detection (25-32). The thermospray (TSP) HPLC/MS interface is well suited for combined HPLC/MS since it can handle high quantities of aqueous solvents without shunting a major portion of the effluent and the ionization process is soft, usually resulting in the formation of molecular ions from thermally unstable and involatile molecules. TSP HPLC/MS analysis has been
0003-2700/85/0357-0991$01.50/00 1985 Amerlcan Chemlcal Society
