RohanA.Thakur, AndrewW. Guzzetta, and Julie
Ion Trap Analysis
The data presented here can be acquired using
a Thermo Finnigan LCQ Advantage, LCQ Deca XP, and Deca XP Plus ion
trap mass spectrometer.
Two important considerations in many LC/MS analyses
are ruggedness and sensitivity. These two factors are important because
analytes of interest are often present in complex matrices that can interfere
with, suppress, or overwhelm the analyte signal.
In assays such as metabolite identification or peptide mapping, the full
MS scan plays a pivotal role in setting up Data-Dependant MS/MS analyses.
Information derived from the full MS scan triggers the sequence of events
driven by the data-dependant set of parameters. Therefore, improvements
in the quality of the full MS scan, such as increased signal-to-noise (S/N),
make the resultant Data-Dependent acquisition more useful. Reducing API
solvent ion noise, especially in the m/z 100-250 mass range, will enable
Data-Dependent acquisitions for small molecules to be performed more efficiently
on a per scan basis by lowering the number of nonsense full-scan MS/MS
spectra. This reduction in the amount of MS/MS spectra acquired decreases
data analysis time and increases the chances of finding elusive components
in baseline noise.
Furthermore, metabolism and peptide samples inherently have matrices such
as plasma or serum. Therefore, any analytical method used must have the
ruggedness to withstand repeated analyses in these types of matrices.
Ion Sweep technology in combination with orthogonal electrospray ionization
(ESI) and atmospheric pressure chemical ionization (APCI) probes has resulted
in a dramatic reduction in API solvent ion noise and overall increased source
robustness. Examples of increased robustness and solvent noise reduction
in the full-scan MS mode when using Ion Sweep gas clearly illustrate the
power of Ion Sweep technology that is now standard on both LCQ Deca XP Plus
and LCQ Advantage platforms.
This report demonstrates the improvements in
robustness and full-scan MS sensitivity observed when Ion Sweep technology
is utilized. Both APCI and ESI techniques are examined. Several small molecules
ranging from m/z 190900 were selected for APCI and ESI LC/MS assays to
illustrate the improvement in full-scan performance of the API source using
Ion Sweep technology. Repeated injections of alprazolam in plasma were performed
to emphasize the robustness of the source when Ion Sweep is used.
Surveyor MS Pump: 400 L/min
Thermo Finnigan LCQ Deca XP Plus mass spectrometer with Ion Sweep Cap
Ion transfer tube temperature: 275-300C
APCI corona discharge current: 4.8 A (probe position: 2C)
ESI spray voltage: 5kV
(probe position: 3C)
Sheath gas: 45-65 au
Ion Sweep Gas: 10 au (using auxiliary gas line)
HPLC conditions: variable; see figure legends for details
Figure 1: Picture of the Ion Sweep cap
The Ion Sweep cap is shown in Figure 1. Nitrogen gas from the auxiliary
gas line is plumbed through the API flange and exits the Ion Sweep cap through
the openings of the three tubes positioned in front of the ion transfer
tube orifice. The flow of nitrogen gas over the opening of the ion transfer
tube ensures that the ions pierce this gentle stream of nitrogen gas as
they travel into the ion transfer tube. Solvent clusters formed in the probe
region interact with the nitrogen gas and dissociate before they enter the
ion transfer tube. Furthermore, a small amount of the Ion Sweep nitrogen
gas is drawn into the ion source, where the gas plays an important role
in eliminating adduct formation during the free jet expansion.
Influence on ion source robustness
The physical presence of the Ion Sweep cap, combined with the flowing nitrogen
gas, increases the overall ion source robustness by gently sweeping the
front face of the ion transfer tube, thereby protecting it from contaminant
build-up. Figure 2 shows over 200 injections of alprazolam in crashed bovine
plasma analyzed over a 24-hour period. No significant decrease in signal
intensity was observed, testifying to the increased ruggedness due to Ion
Improving full-scan MS S/N
Figures 3 through 6 illustrate the power of Ion Sweep technology on eliminating
solvent ion noise in the low m/z region (100-250) during APCI full-scan
MS flow injection analysis (FIA). Figures 3 and 4 show five injections of
caffeine (m/z 195.3) with and without the use of Ion Sweep gas, respectively.
Figure 2: Multiple injections of alprazolam
in crashed bovine plasma. No loss in signal intensity was observed over
200 injections within a 24-hour time period.
Without Ion Sweep gas, the caffeine signal is
lost in the base peak chromatogram, as observed in Figure 3a. The signal
due to caffeine (m/z 195) is overwhelmed by signal due to solvent clusters
at m/z 150 and m/z 160, as evident from the mass spectrum (Figure 3b).
Figure 3: Flow injection analysis of caffeine
without use of Ion Sweep gas. The presence of solvent adduct ions (m/z 150
and m/z 160) overwhelm the caffeine signal at m/z 195 in both a) the base
peak chromatogram and b) mass spectrum.
When Ion Sweep gas is used, the background noise
is drastically reduced and each injection is clearly visible in the base
peak chromatogram, as illustrated in Figure 4a. Furthermore, m/z 195 is
the base peak in the mass spectrum (Figure 4b).
Figure 4: Flow injection analysis of caffeine
using Ion Sweep gas at a setting of 10 arbitrary units. Reducing the background
solvent adduct ions yields significantly greater analyte signal-to-noise
in the base peak chromatogram (a). The mass spectrum (b) also shows a base
peak of the caffeine (m/z 195) and no significant noise due to solvent adduct
ions at m/z 150 and m/z 160, as seen in Figure 3b.
Similar improvements are observed in analysis
of buspirone (m/z 386.4), as illustrated in Figures 5 and 6. Again, the
solvent ion clusters at m/z 150 and m/z 160 elevate the background in the
base peak ion trace, as well as the mass spectrum, when the Ion Sweep gas
is turned off (see Figure 5). The remarkable increase in S/N when the Ion
Sweep gas is turned on at 10 arbitrary units (au), as presented in Figure
6, illustrates the advantage of using Ion Sweep gas in full-scan MS mode,
especially when the analysis requires scanning from m/z 150 and higher.
Figure 5: Flow injection analysis of buspirone
without use of Ion Sweep gas. The presence of solvent adduct ions at m/z
160 overwhelm the buspirone signal at m/z 386.4 in both a) the base peak
chromatogram and b) mass spectrum.
Figure 6: Flow injection analysis of buspirone
using Ion Sweep gas at a setting of 10 arbitrary units. The base peak chromatogram
(a) clearly shows dramatic improvement in S/N when compared with analysis
without use of Ion Sweep gas (Figure 5). Solvent adduct ions are absent
from the mass spectrum (b) which shows a base peak of 386.4, the molecular
ion for buspirone.
Figures 7 and 8 illustrate the utility of Ion
Sweep gas during ESI full-scan LC-MS positive ion analysis for paclitaxel
(Taxol) (m/z 854). Taxol is present in a nine component test mixture
and elutes at 30.1 min. Figure 7 is a chromatogram and mass spectrum without
the use of Ion Sweep gas. Taxol is barely visible in the baseline of the
chromatogram. As can be seen from the mass spectrum, the ammonium adduct,
(m/z 871), is the base peak. Ion Sweep gas is useful
in reducing the formation of the ammonium adduct and allows for unambiguous
determination of molecular weight, as observed in Figure 8. The background
is reduced, allowing better detection of the Taxol peak in the base peak
chromatogram. Furthermore, a clear advantage of Ion Sweep is the ability
to trigger Data-Dependent full-scan MS/MS on the basis of the molecular
ion rather than the ammonium adduct, as m/z 854 is the base peak in the
mass spectrum (Figure 8b).
Figure 7: Positive full-scan ESI LC/MS analysis
of a nine component test mixture without Ion Sweep gas. The target analyte,
paclitaxel [Taxol (m/z 854)] elutes at 30.1 min. The base peak chromatogram
(a) shows a high background that increases with increasing organics in the
content of the mobile phase. This high background is due to solvent adducts.
The base peak in the mass spectrum (b) is the ammonium adduct (m/z 871)
instead of the molecular ion (m/z 854).
Figure 8: Positive full-scan ESI LC/MS analysis
a nine component test mixture using Ion Sweep gas. Taxol (m/z 854) elutes
at 30.1 min. Use of Ion Sweep gas reduces the background level in the base
peak chromatogram (a), as seen by comparing Figure 8a with Figure 7a. Also,
the gas reduces ammonium adducts resulting in the molecular ion (m/z 854)
as the major ion present in the mass spectrum (b).
Figure 9 is the ESI negative ion full MS scan
base peak chromatogram of imazethapyr ([M-H]-=m/z 288) without use of Ion
The negatively charged solvent-ammonium adduct at m/z 136 [2(M-H) -17)
where M is the ammonium acetate ion] dominates the ion current, elevating
the base peak ion trace and completely overwhelming any analyte signal.
The high background from the solvent-ammonium adduct analyte would make
identification in cases of unknown or Data-Dependent analysis difficult,
at best. The mobile phase modifier, ammonium acetate, is always present
in solution phase and, therefore, clusters are always a potential problem;
Ion Sweep technology can be used to de-cluster the solvent-adducts.
Figure 9: Negative ESI base peak chromatogram
of imazethapyr (RT 9.6 min, m/z 288) without Ion Sweep gas. a) Solvent-adduct
ions overwhelm the signal in the base peak chromatogram making detection
of the analyte difficult, at best. b) The mass spectrum has a base peak
of 136.6, corresponding to ammonium acetate clusters/adducts from the solvent.
Elimination of the solventadduct ions cleans
up the full-scan MS trace, as shown in Figure 10. Imazethapyr can now be
detected at 9.7 min in the base peak chromatogram (Figure 10a), allowing
for accurate and efficient Data-Dependant analysis. Figure 10b also shows
the drastic reduction of solvent adducts by the use of Ion Sweep gas, as
the base peak is the molecular ion at m/z 288.
Figure 10: Negative ESI base peak chromatogram
of imazethapyr (m/z 288) using Ion Sweep gas. a) Solvent-adduct ions are
reduced compared with analysis without Ion Sweep gas (Figure 9a) and imazethapyr
is detected in the base peak chromatogram at 9.7 min. b) The mass spectrum
shows a base peak at m/z 288, the molecular ion for imazethapyr; the presence
of adduct ions (m/z 136.7) is greatly reduced compared to analysis without
Ion Sweep gas (Figure 9b).
The new Thermo Finnigan API source with Ion
Sweep technology resulted in a dramatic improvement in S/N in full-scan
MS mode. The examples presented illustrate the decrease in chemical noise
when Ion Sweep gas is used for full-scan MS analyses, thereby resulting
in increased S/N. The performance enhancement for APCI in full-scan MS mode
was remarkable, while solvent background noise was greatly reduced for ESI
full-scan analysis in negative ion mode. This decrease in background/chemical
noise and correlating increase in S/N significantly enhances the information
content of the Data-Dependent experiment by reducing the number of nonsense
spectra collected, a consequence of chemical noise triggering an MS/MS
acquisition. The ion source was also subjected to over 200 injections of
alprazolam in crashed bovine plasma without significant signal roll-off
over a 24-hour period. The use of new Ion Sweep technology results in a
more sensitive ion trap mass spectrometer with the added benefit of enhanced
ion source robustness.
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