Anal. Chem. 1988, 60,774-780
774
Characteristics of a Liquid Chromatograph/Atmospheric Pressure Ionization Mass Spectrometer Minoru Sakairi* and Hideki Kambara Advanced Research Laboratory, Hitachi, Ltd., Kokubunji, Tokyo 185, J a p a n
The wide appiicabiUty and high sensitivity of a liquid chromatograph/atmospheric pressure ionization mass spectrometer (LWAPI-MS) using an interface with direct heating nebulizer and vaporizer are reported. This system can produce molecular ions of many nonvolatile compounds comprislns amlnes, amino acids, peptides, antlblotlcs, nudeoskles, nucleotide, steroids, sugars, lipids, vitamins, and alkaloids, which are dlfflcult of Impossible to handle by gas chromatography/mass spectrometry. The detection limit of this system by selected ion detection (SID) mode is 5 pg for some compounds such as theophyiilne and caffelne. I n the API method, reduced fragmentation for some types of compounds provides lnsufficlent structural informath. However, a simple CID method operated at a relatlvely high pressure proves to be useful for producing fragment ions which give structural information.
Recently, analysis of mixtures of nonvolatile compounds has become increasingly important. For this reason, a liquid chromatograph/atmospheric pressure ionization mass spectrometer (LC/MS) shows promise as a new analysis method in various fields. Many groups have reported various kinds of LC/MS systems including a thermospray method ( I d ) , a moving belt method (6, 7), a DLI method (8,9),a vacuum nebulizing method (10, I I ) , an atmospheric pressure ionization (API) method (12-20), and so on. Basically, an API method has a moderately high sensitivity. However, the sensitivities of the reported liquid chromatograph/atmospheric pressure ionization mass spectrometer (LC/API-MS) systems are not so high (12, 16-18). In a LC/API-MS system, the sensitivity strongly depends on the size of gas clusters produced by an interface. T o obtain very small gas clusters, a nebulizer producing very small droplets is required in the interface. Previously, the authors used a nebulizer with an ultrasonic vibrator for this purpose (14,15). However, the lifetime of the vibrator was not long enough. Consequently, a direct heating nebulizer like a thermospray interface probe (1-3) is used here. This nebulizer is very simple and has a long life. Other problems in LC/API-MS system are formation of cluster ions (19,21,22)and reduced fragmentation resulting from mild ionization (19). These difficulties can be overcome by a collision-induced dissociation (CID) method in a relatively high pressure region of a mass spectrometer (21,22). This paper reports the detail of this system and demonstrates the high performance and wide applicability of the system.
EXPERIMENTAL SECTION Apparatus. A schematicdiagram of the interface and the API ion source of the LC/API-MS system is shown in Figure 1. In this system, effluents from a LC are vaporized by the interface. The vaporized sample and solvent molecules are then ionized by corona discharge followed by ion/molecule reactions. The resulting ions are introduced into mass analyzing region of a sector type mass spectrometer through two apertures. LC. A Hitachi 655A high-performance liquid chromatograph (HPLC) with a loop injector and a Hitachi L-5000 LC controller was used. A Hitachi 655A-UV detector was used for obtaining
UV chromatograms. A column (4 mm X 150 mm) packed with Hitachi gel 3056 (ODS)for reversed-phase chromatography (RPC) mode was used for separating mixtures. The LC was connected with the interface through a Teflon tube (1.6 mm o.d., 0.25 mm i.d., 1 m long). Interface. The interface consisted of the nebulizer and the vaporizer. The nebulizer consisted of a stainless pipe (1.6 mm o.d., 0.1 mm i.d.) brazed to a copper block, which could be heated to 400 "C with cartridge heaters and could be temperature controlled. The nebulizer structure was almost the same as that of a thermospray vaporizer probe (1-3). The small droplets produced were introduced into the vaporizer for further heating and subsequent vaporization. The vaporizer consisted of a bored stainless block (50 mm X 70 mm) which could be uniformly heated to 400 "C by cartridge heaters and could be temperature controlled. In this interface, independent control of nebulizer and vaporizer temperatures was possible. Therefore, the optimum conditions for each temperature could be obtained by carefully investigating variations of molecular ion intensities for several nonvolatile compounds with nebulizer and vaporizer temperatures. Careful attention was also paid to heating the vaporizer to high temperature as uniformly as possible. Consequently,tailing of mass chromatogram peaks was always neglibible. The nebulizer, vaporizer axes, and first electrode axis were joined in alignment in order to efficiently introduce vaporized sample molecules to the API ion source. Vaporized sample and solvent molecules were introduced into the ion source of the API-MS system. Atmospheric Pressure Ionization Mass Spectrometer. Introduced sample and solvent molecules were ionized by corona discharge followed by ion/molecule reactions. Corona discharge current was 5 X lo4 A. The ion source including the first and second electrodeswas heated to 120 "C with two ceramic heaters. Heating of the first and second electrodes was very important to reduce contamination with solvents and samples. The ions produced at atmospheric pressure were introduced into the ion source housing through the intermediate region a t about 60 Pa, which was located between the first electrode with a 250-wm aperture and the second electrode with a 400-wm aperture. The ions were focused by an electrostatic lens. The total ion currents monitored at the intermediate region, at the 5 ion source housing, and a t the collector were about 5 X x and 5 X lo-" A, respectively. A drift voltage was applied to the intermediate region to increase transmission efficiency of ions into the ion source housing and to dissociate cluster ions into quasi-molecular ions (21,22). When a voltage was applied to the intermediate region, cluster ions were accelerated and collided with neutral molecules in the intermediate region. As a result, kinetic energy of cluster ions was partly converted into internal energy of cluster ions such as vibration and rotation. Dissociation of cluster bonds occurred if internal energy of cluster ions was sufficient. Since the kinetic energy of ions was easily controlled by the drift voltage, covalent bonds could be also dissociated by increasing the drift voltage. Increasing fragmentation using this CID method was very easy compared with a m w spectrometry/mw spectrometry (MS/MS) method. Normally, the drift voltage was set to 40-50 V. The intermediate region was evacuated by a 1500 L/min mechanical booster pump and a 500 L/min rotary pump to about 60 Pa. A discharge suppressor was installed inside a polymer hose connecting the intermediate region with the mechanical booster pump. The ion source housing and the mass analyzing region were evacuated with two 700 L/s diffusion pumps to about 1 X lo-* and 1 X lo-* Pa, respectively. Acceleration energy was determined to be 3 keV by the second electrode potential. A Hitachi
0003-2700/88/0360-0774$01.50/0 0 1988 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 60, NO. 8, APRIL 15, 1988 COMPOUNDS
1 4.6
MS
11
4 1 0 PUMP
ou
1.13 14.16
7 . 6 11
> 236-238 7 186-188
2 INOSINE
> 218
4
GUANOSINE
> 236
6
6'-AMP
182
E
PROLINE
228-222
7
LYSINE
224-226
8
SERINE
223-228
a
8
ARGININE
238
P
10 THREONINE
263
1 1 GLUTAHIC ACID
247-249
l2
288
13 LEUCINE
283-296
14 VALINE
316
16 TYROSINE
342 34 4
4
0 6
E:
n
p
3
{
2.9 18
12
N
Flgurs 1. Schematlc dlagram of Interface and API Ion source: (1) Teflon tube; (2) stalnless pipe; (3)copper block, (4) cartridge heater; (5) stalnless Modc; (6) needle electrode; (7) first electrode with aperture; (8) second electrode with aperture: (9) lens; (10) intermediate region:
DECOMPOSI TI ON TEMPERATURES ('C
3
/ (I--
PUMP
ADENOSINE
300 30 0
400
NEEULI ZER TEMPERATURE (OC1
(11) ceramic heater. >
775
-
+ L
(8)
250
300
350
uv
400
L
W
L
c 0
2-
TEMPERATURE OF NEBULIZER ( ' C )
Flgus 2. Variation of molecular ion intensify for inosine with nebulizer and vaporizer temperatures.
M-80B double focusing maes spectrometer was used, and a Hitachi 0101 data acquisition system was used for obtaining LC/MS data. The scan range was 1-1200 amu and scan speed was 1200 amu/6 s under the typical measurement condition. Operation. Nebulizer and vaporizer temperatures were determined to be 330 and 380 "C, respectively, as the quasi-molecular ion intensity of inosine, which was very thermolabile, came to maximum. Quasi-molecular ions for various kinds of nonvolatile compounds could be observed under this condition. In the subsequently described experiment, temperatures were set at these levels. PEG 200,400,600, and 1000 were used for mass markers. API mass spectra of nonvolatile compounds were measured without a column using a sample amount of 5 r g and mobile phase of methanol/water (1:l)at a flow rate of 1 mL/min. Analysis of a spiramycin mixture was carried out with the RPC type column, and mobile phase of 0.1 M ammonium acetate solution/acetonitrile/methanol (55/40/5) at a flow rate of 1mL/min. Reagents. Amines, amino acids, phenylthiohydantoin (PTH) amino acids, nucleosides, steroids, sugars, vitamins, and alkaloids were purchased from Wako Pure Chemical Industry (Tokyo, Japan). Peptides were purchased from the Institute of Peptides (Osaka, Japan). Lipids were purchased from Kanto Chemical Industry (Tokyo, Japan). Antibiotics were kindly supplied from the Institute of Microbial Chemistry (Tokyo, Japan) and Meijyo University (Nagoya, Japan). Distilled water, methanol, and acetonitrile were purchased from Wako Pure Chemical Industry (Tokyo, Japan).
RESULTS A N D D I S C U S S I O N Variation of Molecular I o n Intensity with Nebulizer and Vaporizer Temperatures. It is very important to study optimum conditions of nebulizer and vaporizer temperatures to obtain intense molecular ions of nonvolatile compounds in the LC/API-MS system. Therefore, variation of molecular ion intensities for several nonvolatile compounds with nebulizer and vaporizer temperatures were carefully investigated. Figure 2 shows variation of molecular ion intensity for inosine with nebulizer and vaporizer temperatures. This result reveals the importance of controlling both the nebulizer and vaporizer temperatures. Figure 3 shows the optimum nebulizer and
II
T
---
0
1
(mi
2
n)
3 0
1
(mi
2
n)
3 0
1
2
3
(ai n )
Figure 4. Full width at half maximum comparlson of mass chromatogram peaks of molecular ion and fragment ion and UV chromatogram peak obtained by a 1 kg adenosine injection.
vaporizer temperatures for several nonvolatile compounds with different decomposition temperatures. From this result, it becomes apparent that optimum nebulizer and vaporizer temperatures are different for each compound and lower nebulizer and vaporizer temperatures are preferable for more thermolabile compounds. For normal operation, however, setting the vaporizer temperature to 380 "C and optimizing only the nebulizer temperature was enough for intense molecular ions of many nonvolatile compounds because optimum vaporizer temperature ranged from 380 to 400 "C and molecular ion intensities did not vary much when the vaporizer temperature ranged from 380 to 400 "C, as shown in Figures 2 and 3. Consequently, optimizing parameters in the interface for the LC/API-MS system was very easy. T o reduce the tailing effect of mass chromatogram peaks, the vaporizer was designed for being heated up to high temperature as uniformly as possible. This resulted in minimal tailing of mass chromatogram peaks. Figure 4 shows the mass chromatogram peaks of adenosine molecular ion, MH+ ( m / z 268), and base moiety fragment ion, BHz+ (m/z 136), and UV chromatogram peak (A = 250 nm), obtained from a 1-kg adenosine injection without a column. Both full width at half maximum (FWHM) ratios of MH' and BHz+ chromatogram peaks to the UV chromatogram peak are 1.02. This result shows that tailing of the mass chromatogram peaks is almost negligible. Reproducibility of M a s s Chromatogram Peaks. The mass chromatogram ( m / z 268) obtained by injecting 1 pg of adenosine repeatedly is shown in Figure 5. The mobile phase was water/methanol (1:l)a t a flow rate of 1 mL/min. The relative standard deviation of the ion intensities was about
778
ANALYTICAL CHEMISTRY, VOL. 60, NO. 8, APRIL 15, 1988 Kananyci n A(n.v.484)
? .
w -x >* -c I
