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Increased Analyte Sensitivity through the Utility of Enhanced Mass-Resolution on the FinniganTSQ Quantum Discovery

Chromatography and Mass Spectrometry Application Note

Key Words Sensitivity Quantitation Finnigan TSQ Quantum Discovery Enhanced Mass- Resolution

Xiaoying Xu1, Gary Paul2, Qiao Zhou1, Gregory Tucker1, and Walter Korfmacher1
1 Department of Drug Metabolism and Pharmacokinetics, Schering-Plough Research Institute, Kenilworth, NJ, USA; 2 Thermo Electron Corporation, Somerset, NJ, USA

Overview
Comparison of the limit of quantitation (LOQ) of a drug discovery compound in plasma at unit mass-resolution and enhanced mass-resolution on the Finnigan TSQ Quantum Discovery triple quadrupole mass spectrometer.

Introduction
Recent technological advances in atmospheric pressure ionization (API) techniques and instrumentation are revolutionizing the quantitation of pharmaceutical products.[1] The benchtop Finnigan TSQ Quantum Discovery provides a unique enhanced mass-resolution capability for a triple quadrupole mass spectrometer.[1-6] The utility of enhanced mass-resolution on the Q1 mass analyzer for parent ion selection in the selected reaction monitoring (SRM) experiment has been investigated in previous quantitation studies on the Finnigan TSQ Quantum Discovery. Improved analyte sensitivities were achieved at enhanced mass-resolution, relative to the typical unit mass-resolution mode of a triple quadrupole mass spectrometer.[2-6] These improvements were simply accomplished through the mass separation of the analyte of interest from isobaric matrix/chemical interferences, using the additional resolving power of the quadrupole mass analyzer.

In order to further investigate the benefits of enhanced mass-resolution on the Finnigan TSQ Quantum Discovery, the sensitivity of a drug discovery compound, present in a minimally-treated complex biological matrix, was compared at unit and enhanced mass-resolution. In this manner, an assessment of the improvement in LOQ for this compound at enhanced mass-resolution can be made.

Experimental

Chemicals and Reagents
Rat plasma was purchased from Bioreclamation Inc. (Hicksville,NY, USA). Optima-grade (99.9%) acetonitrile and methanol were obtained from Fisher Scientific Co. (Pittsburgh, PA,USA), ammonium acetate from Sigma Chemical Co.(St.Louis, MO,USA), and glacial acidic acid (99.99+%) from Aldrich Chemical Company (Milwaukee, WI,USA). Water was purified using a compact ultra-pure water system (EASYpure UV, Dubuque, IA,USA).

Sample Preparation
150 μL of internal standard (IS) in acetonitrile solution (conc. 10 ng/mL) was added to 50 μL of drug discovery compound A in rat plasma (concs. 250 ng/mL). After plasma precipitation, the mixture was vortexed for 30 s, and then centrifuged for 10 min. The supernatant was transferred to a clean 96-well plate for LC/MS/MS analysis.

HPLC
HPLC Column: Symmetry C18, 50 4.6 mm, 5 μm (Waters Corporation, Milford, MA,USA)
Mobile Phase A: 0.01 M ammonium acetate in MeOH/H2O (20:80, v/v)
Mobile Phase B: 0.01 M ammonium acetate in MeOH + 0.6 mL/L 10% acetic acid
Flow Rate: 0.8 mL/min Gradient: 5% B for 0.5 min, 5% B to 95%B for 0.5 min, 2.0 min hold at 95% B, return to 5% B at 3.0 min, 3.0 min run time
Divert to MS at 0.8 min Divert to waste at 2.0 min Injection Vol.: 8 μL
Column Temp.: Ambient Temperature
Autosampler Temp: 5C
Retention Time: Compound A - 1.2 min
Internal Standard - 1.5 min

Mass Spectrometer
Mass Spectrometer: Finnigan TSQ Quantum Discovery (ThermoElectron,San Jose,CA,USA)
Source: ESI Mode
Ion Polarity: Positive
Ion Transfer Tube Temperature: 350C
Spray Voltage: 3500 V
Sheath/Auxiliary Gas: Nitrogen
Sheath Gas: 80 arbitrary units
Auxiliary Gas: 20 arbitrary units
Collision Gas Pressure: 1.3 mTorr

The SRM conditions for compound A were as follows:
Parent m/z: 473.28
Product m/z: 456
Scan Width: 1 u
Scan Time: 0.2 s
Collision Energy: 20 eV
Q1 Peak Width (unit mass-resolution): 0.7 u FWHM
Q1 Peak Width (enhanced mass-resolution): 0.2 u FWHM
Q3 Peak Width: 0.7 u FWHM

The internal standard SRM conditions were as follows:
Parent m/z: 544.40
Product m/z: 306
Scan Width: 1 u
Scan Time: 0.2 s
Collision Energy: 28 eV
Q1 Peak Width (unit mass-resolution): 0.7 u FWHM
Q1 Peak Width (enhanced mass-resolution): 0.2 u FWHM
Q3 Peak Width: 0.7 u FWHM

Results and Discussion
The LC/ESI-SRM chromatograms for blank plasma spiked with the internal standard, collected on the Finnigan TSQ Quantum Discovery operating in unit mass-resolution mode (Q1, 0.7 u FWHM), are shown in Figure 1. The arrow shown in Figure 1 indicates the expected retention time of compound A (t = 1.2 min). It can be seen that significant background interference elutes at the same retention time as compound A in the blank plasma (Figure 1). However, when the same blank plasma sample was analyzed by the Finnigan TSQ Quantum Discovery operating at enhanced mass-resolution (Q1, 0.2 u FWHM), the background interference at t =1.2 min was greatly reduced, as shown in Figure 2. The reduction in background noise is a consequence of the added specificity afforded to the SRM experiment by the enhanced mass-resolution feature.

The LC/ESI-SRM chromatograms obtained at unit mass-resolution for compound A in plasma at a concentration of 250 ng/mL, are shown in Figure 3. Background interference to the signal of compound A at this concentration is clearly evident in the chromatographic trace. A calculated signal to noise (S/N) ratio of 8 is determined for this peak (Figure 3). Hence, the LOQ for compound A at unit mass-resolution can be assumed to be of the order of 250 ng/mL.

When the same sample of compound A (conc. 250 ng/mL) was analyzed at enhanced mass-resolution, a much cleaner SRM peak was observed, as shown in Figure 4. This is a consequence of the removal of a considerable amount of isobaric chemical interference from the analyte through the improved mass selectivity at enhanced mass-resolution. In addition to a more uniform SRM peak, an improved S/N ratio of 31 was obtained for compound A at enhanced mass-resolution, relative to unit mass-resolution (Figures 3,4). The dramatic decrease in background noise at the higher mass-resolution setting is responsible for the significant improvement in S/N ratio, despite the loss of a factor of 3-4 in peak height/area for compound A (Figures 3,4). An increase in analyte sensitivity at enhanced mass-resolution, relative to unit, has been reported in previous LC/ESI-SRM and LC/APCI-SRM quantitative studies on the Finnigan TSQ Quantum Discovery.[2-6] Indeed, one of the prior quantitative studies was based on the monitoring of SRM transitions involving small molecule losses,[6] analogous to the transition used in this work (m/z 473456, loss of 17 u). Since small molecule losses through collisionally-induced dissociation processes are common-place and the potential for matrix interference to the analyte SRM is much greater, analysis at enhanced mass-resolution should be an extremely viable method for improved quantitative performance in this situation.

Since the S/N ratio for compound A (conc. 250 ng/mL) at enhanced mass-resolution was above that required for the limit of quantitation (Figure 4), compound A was diluted by a factor of 10 (conc. 25 ng/mL) and reanalyzed. The LC/ESI-SRM chromatograms obtained using enhanced mass-resolution at the lower concentration of compound A are shown in Figure 5. A clearly-defined peak for compound A is observed which is easily quantifiable. Hence, using enhanced mass-resolution on the Finnigan TSQ Quantum Discovery, at least an order of magnitude decrease in LOQ for compound A is achieved, relative to unit mass-resolution operation, without the need for time-consuming chromatographic manipulations. Satisfactory precision and accuracy values for analytes present in plasma have been previously reported for quantitative studies on the Finnigan TSQ Quantum Discovery in enhanced mass-resolution mode.[3,4].

Previous quantitative studies using the Finnigan TSQ Quantum Discovery also found that improvement in analyte sensitivity obtained at enhanced mass-resolution, as shown here, is proportional to an increase in the linear dynamic range for the assay.[3,4,6] An extended linear dynamic range is advantageous in applications such as discovery pharmacokinetic (PK) studies to accommodate the variable concentrations of analytes in the study samples.[3]

References
1. Yang L, Amad M, Winnik WM, Schoen AE, Schweingruber H, Mylechreest I, Rudewiz PJ. Rapid Commun. Mass Spectrom. 2002; 16: 2060.
2. Jemal M, Ouyang, Z. Rapid Commun. Mass Spectrom. 2003; 17: 24.
3. Xu X, Veals J, Korfmacher W. Rapid Commun. Mass Spectrom. 2003; 17: 832.
4. Hughes N, Winnik W, Dunyach JJ, Amad M, Splendore M, Paul G. J. Mass Spectrom, in press.
5. Xu X, Tucker G, Zhou Q, Veals J, Korfmacher, W. 50th ASMS Conf Mass Spectrometry and Allied Topics, Orlando, FL, 2002.
6. Paul G, Winnik W, Schmidt C, Amad M, Splendore M, Lytle C, Hughes JE, Desai B, MacKenzie, KI. 50th ASMS Conf Mass Spectrometry and Allied Topics, Orlando, FL, 2002.


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