Navigation Links
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

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.

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 min imally-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.


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 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,US A)
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]

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.



Page: All 1 2 3 4 5 6

Related biology technology :

1. Increased Sensitivity in Microarray Analysis
2. Increased Transfer Efficiency Using a Discontinuous Buffer System With the Trans-Blot SD Cell
3. Improved Sensitivity for Staining RNA with SYBR Gold Stain
4. Enhancing Ruggedness and Full-Scan MS Sensitivity Using Ion Sweep Technology
5. Increase the Power and Sensitivity for Your cDNA Synthesis with the New Transcriptor Reverse Transcriptase
6. Femtogram Sensitivity of the Antipsychotic Drugs Raclopride and Ritanserin Using a Varian 1200L LC/MS/MS
7. Maintaining High Sensitivity when Working with Small-Volume Samples
8. High Sensitivity, Wide Dynamic Range, Horseradish Peroxidase (HRP) ELISA Using the LMax Microplate Luminometer (MaxLine Application Note #41)
9. Sensitivity and Quantitation using a Finnigan LCQ Deca XP Plus Ion Trap Mass Spectrometer
10. Trace Analysis Optimizing Sensitivity Finnigan Surveyor PDA Detector
11. A Screen of shRNAs Targeting Tumor Suppressor Genes to Identify Factors Involved in Paclitaxel Sensitivity
Post Your Comments:

(Date:10/8/2015)... Celemics, a global pioneer of personalized medicine ... market with the establishment of Celemics America, Inc. ... Europe , has secured itself in the global ... sample preparation for DNA sequencing and genetic testing.  Next ... genetic testing more accurately, quickly, and cost effectively than ...
(Date:10/8/2015)... 8, 2015  The ALS Association, in partnership with ... Challenge to revolutionize communication technology solutions for people living ... ALS (amyotrophic lateral sclerosis) is a progressive neurodegenerative ... and the spinal cord. Eventually, people with ALS lose ... often leads to total paralysis and death within two ...
(Date:10/8/2015)... ... October 08, 2015 , ... ProMIS ... license agreement to develop and commercialize intellectual property rights belonging to The University ... February 2009, and affords exclusive worldwide rights to intellectual property based on use ...
(Date:10/7/2015)... Dallas-Fort Worth, Texas (PRWEB) , ... October 07, ... ... Stem Cell Institute ( ) will present a public educational seminar on ... diseases on Saturday, October 10th from 1:00 pm – 3:00 pm at the ...
Breaking Biology Technology:
... Nov. 28 On Monday, December 1, 2008, at,8:30 ... Therapeutics, Inc.,s (CTI) (Nasdaq and MTA: CTIC) management team ... Spectrum Pharmaceuticals,of Irvine, California to jointly market and develop ... Call Numbers, Monday, December 1, ...
... today that it is refocusing all projects and priorities around ... be seen as a,consequence of the negative recommendation of the ... 2008 regarding the,approval of Iclaprim in the U.S. , ... FDA advisory board,s recommendation the Arpida,board intends to put the ...
... house always looking good, without cleaning or giving it ... with paint that can maintain itself and get rid ... process for producing photo-catalytic, self-cleaning coatings has been developed ... and Research), and licensed to Haruna (S) Pte Ltd. ...
Cached Biology Technology:
(Date:9/24/2015)... YORK , Sept. 24, 2015  EyeLock LLC, ... be showcasing its award winning and latest technology in ... Anaheim, California . EyeLock,s iris ... security with unmatched biometric accuracy, making it the most ... EyeLock,s platform uses video technology to deliver a fast ...
(Date:9/10/2015)... 10, 2015 Report Details ... Selling Opportunities and Revenue Prospects to Help You ... biologics, especially new drug classes? Get the latest ... with exclusive market data and industry knowledge, benefitting ... results, opportunities and sales predictions. Visiongain,s new ...
(Date:9/10/2015)... -- NXT-ID, Inc. (NASDAQ: NXTD ) ("NXT-ID" or the ... mobile commerce market, announces that the San ... will feature the Wocket® smart wallet for the first ... Touch Of Modern is an online retailer ... its 7.2 million members. Customers are affluent, urban influencers ...
Breaking Biology News(10 mins):
... and their colleagues in Japan are conducting research that may ... necessary step if man someday hopes to build molecular machines ... Dr. Eric Blough, a member of the research ... Biological Sciences, said his group has shown how bionanomotors can ...
... Biologists have known for long that ecology, the interaction ... in forming new species and in modifying living ones. ... The environment defines a template and the process of ... template. Some specialized theory, a few ...
... plant virus lures aphids to infected plants by making the ... they quickly leave for tastier, healthier ones. In the process, ... entomologists. "The virus improves the cues that insects use to ... is already in the plant," said Mark C. Mescher, assistant ...
Cached Biology News: