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Enhancing Ruggedness and Full-Scan MS Sensitivity Using Ion Sweep ,,, Technology

RohanA.Thakur, AndrewW. Guzzetta, and Julie A.Horner

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. Introduction 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. Goal 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. Experimental Conditions Surveyor MS Pump: 400 L/min
Surveyor Autosampler
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 Discussion Theory
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 Sweep technology.

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+17]+ (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 Sweep gas.

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). Conclusions 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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