RAPID COMMUNICATIONS IN MASS SPECTROMETRY, VOL. 5, 354-356 (1991)
Four Interfaces for Liquid Chromatography/ Atmospheric-pressure-ionization Mass Spectrometry Minoru Sakairi* Central Research Laboratory, Hitachi Ltd., Kokubunji, Tokyo 185, Japan
Alfred L. Yergey National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA
Mixture analyses are demonstrated using the liquid chromatographlatmospheric-pressure-ionizationmass spectrometric system with four modes. These modes are atmospheric-pressurespray with electron ionization, atmospheric-pressurechemical ionization, atmospheric-pressurespray ionization, and electrospray ionization modes. This system can deal with a wide variety of compounds from hydrocarbons with low polarity to proteins with high polarity.
We have already developed atmospheric-pressure chemical ionization (APCI) and atmospheric-pressure spray ionization (APSI) modes suitable for liquid chromatography/mass spectrometry (LC/MS), and have applied these modes to the analyses of various kinds of compounds.'-3 Recently, Fenn and his coworkers have reported that the combination of electrospray ionization (ESPI) with a quadrupole mass spectrometer is very powerful for the analyses of large peptides and proteins because of highly sensitive detection of multiply charged ions of these corn pound^.^.^ These three interface structures resemble one another. If we could develop an interface for an electron ionization (EI) mode by using a similar interface structure to those of the three other modes, we could measure a great variety of compounds from hydrocarbons with low polarity to proteins with high polarity in one LC/MS system by using the four modes. This paper reports mixture analyses using the liquid chromatograph/atmospheric-pressure-ionization mass spectrometer with the four modes, in order to demonstrate the wide applicability of the system.
\
OUADRUPOLE MASS SPECTROMETER
NOZZLE SUIMMER
1
PIPE
* Author to whom correspondence should be addressed. 0951-4 198/91/080354-03 $05.00 01991 by John Wiley & Sons, Ltd.
\
I
NOZZLE
+
SKIMMER
I
LENS
\
(c) APSI mode
OUADRUPOLE MASS SPECTROMETER
\
\
IMPACT IONIZATION SOURCE
(a) APE1 mode
sK1?uER LENS
\
NOZZLE
1
SKIMMER
OUADRUPOLE YASS SPECTROMETER
NEBULIZER
VAPORIZER PUMP
EXPERIMENTAL APEZ mode. Ions are produced under atmospheric pressure in the APCI, APSI and ESPI modes, but the resultant ions must be introduced into a mass-analyzing region under low pressure to detect them. For this purpose, we have used a differential pumping system combined with a collision-induced dissociation techRecently, this nique for APSI and APCI technique has also been applied to electrospray mass spectrometry by Smith et al. If sample solutions can be effectively vaporized under atmospheric pressure and the sample molecules produced can be efficiently introduced into an electron ionization source through the differential pumping region used for the other ionization modes, even the interface for the EI mode becomes similar to the other interfaces. The interface for the EI mode to achieve this, is shown in Fig. l(a).
LENS
QUADRUPOLE MASS SPECTROMETER
NOZZLE
PUMP
NEEDLE ELECTRWE
(b) APCI mode
COUNTERCURRENT GASOUTLET
(d) ESP1 mode
Figurel. Schematic diagrams of (a) the APEI interface, (b) the APCI interface, (c) the APSI interface and (d) the ESPI interface.
Here, we temporarily call this EI mode the atmospheric pressure spray with electron ionization (APEI) mode. Sample solutions were nebulized by the vaporizer consisting of a stainless-steel capillary tube brazed to a stainless-steel block. The fine droplets produced were introduced directly into a low-pressure region through a differential pumping region located between a nozzle and a skimmer. This differential pumping region was evacuated to about 1Torr by a mechanical pump (1500 L/min). The block temperature was usually set to 420 "C for obtaining high ion currents of molecular-ion species. Even under such conditions there were no problems with clogging of the stainless-steel capillary Receioed I 7 May 1991 Accepted 2 June 1991
355
FOUR INTERFACES FOR LCIAPI-MS
tube. Vaporized sample molecules were introduced into an electron ionization source through a heated glass guide-pipe (OD 4 mm X ID 2 mm). This region was evacuated to about Torr by a diffusion pump (1200 L/s). The guide-pipe and the electron ionization source were heated to 150 and 250 "C.Intense molecular ion species could not be seen without heating the guide-pipe. The edge of the guide-pipe was supported by the einzel lens used for focusing ions in the other ionization modes. A quadrupole mass spectrometer was operated at a pressure of Torr. This analyzing region was evacuated by a diffusion pump (700 L/s). The analysis of a mixture of naphthalene (MW 128) and anthracene (MW 178) was carried out by using a reverse-phase chromatography mode (column: 4 X 150 rnm, CIS).Water acetonitrile (30/70) was used as a mobile phase at a flow rate of lmL/min. The electron beam energy was set to 70 eV. APCZ mode. The detail of the APCI interface shown in Fig. l(b) has been already described elsewhere.' The analysis of a mixture of glycocholic acid (MW 465), cholic acid (MW 408), glycodeoxycholic acid (MW 449) and deoxycholic acid (MW392) was carried out by using a reverse-phase chromatography mode (column: 4 x 150 mm, CIS).Acetonitrile + 0.1 M ammonium acetate (35/65) solution was used as a mobile phase at a flow rate of 1mL/min. APSZ mode. The detail of the APSI interface shown in Fig. l(c) has already been described elsewhere.2 The analysis of a mixture of glucose (MW 180), sucrose (MW 342) and raffinose (MW 504) was carried out by using a reverse-phase chromatography mode (Column: 4 x 150 mm, CIS). 100% water was used as a mobile phase at a flow rate of 1mL/min. ESPZ mode. The schematic diagram of the interface for ESPI mode is shown in Fig. l(d). In this mode, a pneumatically assisted electrospray (ion spray) interface was used for dealing with higher flow rates, as Bruins has already reported .' The probe used consisted of an approximately 25 mm stainless-steel tube (100 pm ID) attached by epoxy glue to a 100mm long and 1.5mm O D stainless-steel tube. The probe tip was
+
l . . . . . . . . .10. . . . .15. . . . .20. . . . .25. . . . .30I 5
0
RETENTION TIME (min)
100.
483 (M+NH4 f
t v 2
C ;H Z ;
l
"
412
.
w
466
+
z
350
m/z
Figure3. The analysis of bile acids by APCI mode. (a) Total-ion intensity of glycocholic acid (A), cholic acid (B), glycodeoxycholic acid (C) and deoxycholic acid (D); (b) mass spectrum of glycocholic acid.
positioned approximately 15 mm from the sample aperture and was typically biased to 3 kV with respect to ground. Countercurrent nitrogen gas flow was used for promoting vaporization of droplets produced by electrospray . The analysis of a mixture of bovine insulin (average molecular weight (AMW) 5734), bovine glucagon (AMW 3483) and myoglobin (AMW 16951) was performed by using a reverse-phase chromatography mode (column: 1 X 100mm, C,,). A linear gradient of 20/80 acetonitrile water (0.05% formic acid) to 100% acetonitrile (0.05% formic acid) in 30min was utilized. The flow rate was set to 40 pL/min.
+
RESULTS A N D DISCUSSION APEZ mode. Figure 2 shows the total-ion chromatogram (mlz 50-250) of naphthalene and anthracene, and the mass spectrum of anthracene. It is noteworthy
RETENTION TIME(min)
100-
527
fb)
>
245
(M+Na)+
t vl 2
w
.
+ 5
0
100
543 (M+K)+
I 200
Figure2. The analysis of aromatic compounds by APEI mode. (a) Total-ion chromatogram of naphthalene (A) and anthracene (B); (b) mass spectrum of anthracene.
FOUR INTERFACES FOR LC/API-MS
356
I . . . . . . . . . . . . . . . . . . . . . . . .25' . . 5
0
10
20
15
.do
RETENTION TIME(min)
100-
t v) Z
w
848
(b)
>
*
(M+H)
+
679
I-
z 1131
600
1 . .I , ,. h , ,
800
1000
,
1200 (C)
m/z
Figure 5. The analysis of proteins by ESP1 mode. (a) Total-ion chromatogram of insulin (A), glucagon (B) and myoglobin (C); (b) mass spectrum of myoglobin; (c) structure of myoglobin.
that the chromatogram tailing is almost negligible. Intense molecular ions are observed at mlz 178 in the anthracene mass spectrum which is almost the same as that obtained by GUMS. Although an analysis using 1nmol of each sample is demonstrated here, 0.1 nmol or less is enough for obtaining mass spectra. The sensitivity of this technique is lower than that of G U M S by two or more orders of magnitude, but comparable to that of the method reported by Browner et ~ 1 This . ~ technique may be very useful for the analyses of hydrocarbons, aromatic compounds and other compounds with high volatility and thermal stability. APCZ mode. Figure 3 shows the total ion chromatogram (mlz 250-550) of glycocholic acid (MW 465), cholic acid (MW 408), glycodeoxycholic acid (MW 449) and deoxycholic acid (MW 392), and the mass spectrum of glycocholic acid as a representative example. A stable chromatogram is obtained and intense ammonium adduct ions [M NH,]' are observed at mlz 483 in the mass spectrum of glycodeoxycholic acid. Although the APCI mode generally produces protonated molecules, ammonium adduct ions are easily obtained in the case of bile acids using ammonium acetate solution as a mobile phase. APSZmode. Figure 4 shows the total ion chromatogram (mlz 100-600) of glucose, sucrose and raffinose, and the mass spectrum of raffinose. We can observe the
+
fragment ions due to glycosidic bond cleavages, which are very useful for estimating saccharide sequences, in addition to cationized molecules. In the case of raffinose, the fragment ions corresponding to disaccharide (mlz 365) and monosaccharide structures (mlz 203) are obtained with the cationized molecules at mlz 504. From these ions, we can easily estimate that a raffinose molecule has the structure hexose-hexose-hexose. ESP1 mode. Figure 5 shows the stable total ion chromatogram (mlz 200-1200) of bovine insulin, bovine glucagon and myoglobin, and the mass spectrum of myoglobin. The mass spectrum exhibits the distribution of multiply charged ion peaks resulting from the attachment of 15-27 protons to myoglobin molecules.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.
M. Sakairi and H. Kambara, Anal. Chem. 60, 774 (1988). M. Sakairi and H. Kambara, Anal. Chem. 61, 1159 (1989). M. Sakairi, Doctoral Thesis (The University of Tokyo) (1989) M. Yamashita and J. B . Fenn, J . Phys. Chem. 88, 4451 (1984). C. M. Whitehouse, R. N. Dreyer, M. Yamashita and J. B. Fenn, Anal. Chem. 57, 675 (1985). H. Kambara, Anal. Chem. 54, 143 (1982). R. D. Smith et al, Anal. Chem. 62, 882 (1990). A. P. Bruins, T. R. Covey and J. D. Henion, Anal. Chem. 59, 2647 (1987). . R. C: Willoughby and R. F. Browner, Anal. Chem. 56, 2626 (1984).
Four Interfaces for Liquid Chromatography/ Atmospheric-pressure-ionization Mass Spectrometry Minoru Sakairi* Central Research Laboratory, Hitachi Ltd., Kokubunji, Tokyo 185, Japan
Alfred L. Yergey National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA
Mixture analyses are demonstrated using the liquid chromatographlatmospheric-pressure-ionizationmass spectrometric system with four modes. These modes are atmospheric-pressurespray with electron ionization, atmospheric-pressurechemical ionization, atmospheric-pressurespray ionization, and electrospray ionization modes. This system can deal with a wide variety of compounds from hydrocarbons with low polarity to proteins with high polarity.
We have already developed atmospheric-pressure chemical ionization (APCI) and atmospheric-pressure spray ionization (APSI) modes suitable for liquid chromatography/mass spectrometry (LC/MS), and have applied these modes to the analyses of various kinds of compounds.'-3 Recently, Fenn and his coworkers have reported that the combination of electrospray ionization (ESPI) with a quadrupole mass spectrometer is very powerful for the analyses of large peptides and proteins because of highly sensitive detection of multiply charged ions of these corn pound^.^.^ These three interface structures resemble one another. If we could develop an interface for an electron ionization (EI) mode by using a similar interface structure to those of the three other modes, we could measure a great variety of compounds from hydrocarbons with low polarity to proteins with high polarity in one LC/MS system by using the four modes. This paper reports mixture analyses using the liquid chromatograph/atmospheric-pressure-ionization mass spectrometer with the four modes, in order to demonstrate the wide applicability of the system.
\
OUADRUPOLE MASS SPECTROMETER
NOZZLE SUIMMER
1
PIPE
* Author to whom correspondence should be addressed. 0951-4 198/91/080354-03 $05.00 01991 by John Wiley & Sons, Ltd.
\
I
NOZZLE
+
SKIMMER
I
LENS
\
(c) APSI mode
OUADRUPOLE MASS SPECTROMETER
\
\
IMPACT IONIZATION SOURCE
(a) APE1 mode
sK1?uER LENS
\
NOZZLE
1
SKIMMER
OUADRUPOLE YASS SPECTROMETER
NEBULIZER
VAPORIZER PUMP
EXPERIMENTAL APEZ mode. Ions are produced under atmospheric pressure in the APCI, APSI and ESPI modes, but the resultant ions must be introduced into a mass-analyzing region under low pressure to detect them. For this purpose, we have used a differential pumping system combined with a collision-induced dissociation techRecently, this nique for APSI and APCI technique has also been applied to electrospray mass spectrometry by Smith et al. If sample solutions can be effectively vaporized under atmospheric pressure and the sample molecules produced can be efficiently introduced into an electron ionization source through the differential pumping region used for the other ionization modes, even the interface for the EI mode becomes similar to the other interfaces. The interface for the EI mode to achieve this, is shown in Fig. l(a).
LENS
QUADRUPOLE MASS SPECTROMETER
NOZZLE
PUMP
NEEDLE ELECTRWE
(b) APCI mode
COUNTERCURRENT GASOUTLET
(d) ESP1 mode
Figurel. Schematic diagrams of (a) the APEI interface, (b) the APCI interface, (c) the APSI interface and (d) the ESPI interface.
Here, we temporarily call this EI mode the atmospheric pressure spray with electron ionization (APEI) mode. Sample solutions were nebulized by the vaporizer consisting of a stainless-steel capillary tube brazed to a stainless-steel block. The fine droplets produced were introduced directly into a low-pressure region through a differential pumping region located between a nozzle and a skimmer. This differential pumping region was evacuated to about 1Torr by a mechanical pump (1500 L/min). The block temperature was usually set to 420 "C for obtaining high ion currents of molecular-ion species. Even under such conditions there were no problems with clogging of the stainless-steel capillary Receioed I 7 May 1991 Accepted 2 June 1991
355
FOUR INTERFACES FOR LCIAPI-MS
tube. Vaporized sample molecules were introduced into an electron ionization source through a heated glass guide-pipe (OD 4 mm X ID 2 mm). This region was evacuated to about Torr by a diffusion pump (1200 L/s). The guide-pipe and the electron ionization source were heated to 150 and 250 "C.Intense molecular ion species could not be seen without heating the guide-pipe. The edge of the guide-pipe was supported by the einzel lens used for focusing ions in the other ionization modes. A quadrupole mass spectrometer was operated at a pressure of Torr. This analyzing region was evacuated by a diffusion pump (700 L/s). The analysis of a mixture of naphthalene (MW 128) and anthracene (MW 178) was carried out by using a reverse-phase chromatography mode (column: 4 X 150 rnm, CIS).Water acetonitrile (30/70) was used as a mobile phase at a flow rate of lmL/min. The electron beam energy was set to 70 eV. APCZ mode. The detail of the APCI interface shown in Fig. l(b) has been already described elsewhere.' The analysis of a mixture of glycocholic acid (MW 465), cholic acid (MW 408), glycodeoxycholic acid (MW 449) and deoxycholic acid (MW392) was carried out by using a reverse-phase chromatography mode (column: 4 x 150 mm, CIS).Acetonitrile + 0.1 M ammonium acetate (35/65) solution was used as a mobile phase at a flow rate of 1mL/min. APSZ mode. The detail of the APSI interface shown in Fig. l(c) has already been described elsewhere.2 The analysis of a mixture of glucose (MW 180), sucrose (MW 342) and raffinose (MW 504) was carried out by using a reverse-phase chromatography mode (Column: 4 x 150 mm, CIS). 100% water was used as a mobile phase at a flow rate of 1mL/min. ESPZ mode. The schematic diagram of the interface for ESPI mode is shown in Fig. l(d). In this mode, a pneumatically assisted electrospray (ion spray) interface was used for dealing with higher flow rates, as Bruins has already reported .' The probe used consisted of an approximately 25 mm stainless-steel tube (100 pm ID) attached by epoxy glue to a 100mm long and 1.5mm O D stainless-steel tube. The probe tip was
+
l . . . . . . . . .10. . . . .15. . . . .20. . . . .25. . . . .30I 5
0
RETENTION TIME (min)
100.
483 (M+NH4 f
t v 2
C ;H Z ;
l
"
412
.
w
466
+
z
350
m/z
Figure3. The analysis of bile acids by APCI mode. (a) Total-ion intensity of glycocholic acid (A), cholic acid (B), glycodeoxycholic acid (C) and deoxycholic acid (D); (b) mass spectrum of glycocholic acid.
positioned approximately 15 mm from the sample aperture and was typically biased to 3 kV with respect to ground. Countercurrent nitrogen gas flow was used for promoting vaporization of droplets produced by electrospray . The analysis of a mixture of bovine insulin (average molecular weight (AMW) 5734), bovine glucagon (AMW 3483) and myoglobin (AMW 16951) was performed by using a reverse-phase chromatography mode (column: 1 X 100mm, C,,). A linear gradient of 20/80 acetonitrile water (0.05% formic acid) to 100% acetonitrile (0.05% formic acid) in 30min was utilized. The flow rate was set to 40 pL/min.
+
RESULTS A N D DISCUSSION APEZ mode. Figure 2 shows the total-ion chromatogram (mlz 50-250) of naphthalene and anthracene, and the mass spectrum of anthracene. It is noteworthy
RETENTION TIME(min)
100-
527
fb)
>
245
(M+Na)+
t vl 2
w
.
+ 5
0
100
543 (M+K)+
I 200
Figure2. The analysis of aromatic compounds by APEI mode. (a) Total-ion chromatogram of naphthalene (A) and anthracene (B); (b) mass spectrum of anthracene.
FOUR INTERFACES FOR LC/API-MS
356
I . . . . . . . . . . . . . . . . . . . . . . . .25' . . 5
0
10
20
15
.do
RETENTION TIME(min)
100-
t v) Z
w
848
(b)
>
*
(M+H)
+
679
I-
z 1131
600
1 . .I , ,. h , ,
800
1000
,
1200 (C)
m/z
Figure 5. The analysis of proteins by ESP1 mode. (a) Total-ion chromatogram of insulin (A), glucagon (B) and myoglobin (C); (b) mass spectrum of myoglobin; (c) structure of myoglobin.
that the chromatogram tailing is almost negligible. Intense molecular ions are observed at mlz 178 in the anthracene mass spectrum which is almost the same as that obtained by GUMS. Although an analysis using 1nmol of each sample is demonstrated here, 0.1 nmol or less is enough for obtaining mass spectra. The sensitivity of this technique is lower than that of G U M S by two or more orders of magnitude, but comparable to that of the method reported by Browner et ~ 1 This . ~ technique may be very useful for the analyses of hydrocarbons, aromatic compounds and other compounds with high volatility and thermal stability. APCZ mode. Figure 3 shows the total ion chromatogram (mlz 250-550) of glycocholic acid (MW 465), cholic acid (MW 408), glycodeoxycholic acid (MW 449) and deoxycholic acid (MW 392), and the mass spectrum of glycocholic acid as a representative example. A stable chromatogram is obtained and intense ammonium adduct ions [M NH,]' are observed at mlz 483 in the mass spectrum of glycodeoxycholic acid. Although the APCI mode generally produces protonated molecules, ammonium adduct ions are easily obtained in the case of bile acids using ammonium acetate solution as a mobile phase. APSZmode. Figure 4 shows the total ion chromatogram (mlz 100-600) of glucose, sucrose and raffinose, and the mass spectrum of raffinose. We can observe the
+
fragment ions due to glycosidic bond cleavages, which are very useful for estimating saccharide sequences, in addition to cationized molecules. In the case of raffinose, the fragment ions corresponding to disaccharide (mlz 365) and monosaccharide structures (mlz 203) are obtained with the cationized molecules at mlz 504. From these ions, we can easily estimate that a raffinose molecule has the structure hexose-hexose-hexose. ESP1 mode. Figure 5 shows the stable total ion chromatogram (mlz 200-1200) of bovine insulin, bovine glucagon and myoglobin, and the mass spectrum of myoglobin. The mass spectrum exhibits the distribution of multiply charged ion peaks resulting from the attachment of 15-27 protons to myoglobin molecules.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.
M. Sakairi and H. Kambara, Anal. Chem. 60, 774 (1988). M. Sakairi and H. Kambara, Anal. Chem. 61, 1159 (1989). M. Sakairi, Doctoral Thesis (The University of Tokyo) (1989) M. Yamashita and J. B . Fenn, J . Phys. Chem. 88, 4451 (1984). C. M. Whitehouse, R. N. Dreyer, M. Yamashita and J. B. Fenn, Anal. Chem. 57, 675 (1985). H. Kambara, Anal. Chem. 54, 143 (1982). R. D. Smith et al, Anal. Chem. 62, 882 (1990). A. P. Bruins, T. R. Covey and J. D. Henion, Anal. Chem. 59, 2647 (1987). . R. C: Willoughby and R. F. Browner, Anal. Chem. 56, 2626 (1984).
