An Evaluation of Various High-Resolution, Accurate-Mass Scan Modes for In Vitro Drug Discovery Screening

Transcription

1 An Evaluation of Various High-Resolution, Accurate-Mass Scan Modes for In Vitro Drug Discovery Screening Jonathan McNally, 1 Nicholas Duczak Jr., 1 Patrick Bennett, 1 Francois Espourteille, and Maciej Bromirski 3 1 Thermo Fisher Scientific, San Jose, CA, USA; Thermo Fisher Scientific, Franklin, MA, USA; 3 Thermo Fisher Scientific, Bremen, Germany

2 Overview Purpose: To evaluate various scan modes available through high-resolution, accuratemass analysis to determine suitability for in vitro plasma protein binding assay analysis. Methods: An in vitro plasma protein binding assay was analyzed using various scan modes available to a high-resolution, accurate-mass analysis LC-MS system and the results compared to data obtained using a triple quadrupole mass spectrometer. Results: The lower limit of detection was found to be between 5 nm and 5 nm in full scan mode. The 5 nm was detected for a majority of the samples analyzed using full scan mode. The signal response was determined to be linear across 3 orders of magnitude for most test compound calibration curves. The results for the calculated amount of the free fraction remaining (% Free) for the binding assay demonstrated a good correlation between the results for the high-resolution, accurate-mass analysis and the results collected using LC-MS/MS analysis. Sample analysis performed using SIM mode provided a lower limit of detection of 5 nm for all compounds in the assay calibration curve demonstrating an improvement in sensitivity for several compounds in the more targeted scan mode. Introduction High-resolution mass spectrometers are becoming increasingly more powerful and capable of sophisticated scanning experiments that offer new solutions to complex challenges. Additionally, assays that fall into a well defined and routine workspace, such as in vitro screening assay in early drug discovery, will also benefit from the ease of use and high performance of high-resolution mass spectrometric analysis but do not require all available scan capabilities needed for more complex applications. In this evaluation several different full scan and SIM analyses were used to analyze a protein plasma binding assay with an Thermo Scientific Orbitrap mass analyzer and the results compared to previous analysis performed using traditional LC-MS/MS on a triple quadrupole mass spectrometer. Methods Sample Preparation A set of of commercially available drug compounds was selected based on reported binding properties and molecular weight and incubated in an in vitro plasma protein binding assay in triplicate at a concentration of 1 µm. Samples were incubated for 6.5 hours in a dialysis block followed by protein precipitation. Protein precipitation was performed by first adding 15 ml of acetonitrile containing internal standard compound (Alprenolol) to a 96-well 3-mL V-bottomed storage plate followed by addition of 5 ml of each of the assay samples. Calibration curves were also generated for each compound. A working stock solution of 5 mm in DMSO was first made for each compound. A five-point standard curve at concentrations of 5, 5, 5, 1 and nm was prepared for each compound by serial dilution from the working stock solution into a blank mixed matrix using an eight channel pipette 1. Liquid Chromatography Gradient elution was accomplished using water (A) +.1% Formic Acid (v/v) and Acetonitrile (B) +.1% Formic Acid (v/v). The gradient was held at 98% aqueous for.5 minutes, ramped to 98% B over.35 minutes, and held at 98% B for. minutes before returning to the starting conditions at % B for a. minute equilibration time. Chromatographic separation was performed using a C18,.1 x 3 mm, 3µm column with 5uL injections made for each sample. All injections were completed using a Thermo Scientific Accela Open system with DLW (Dynamic Load and Wash) and with Thermo Scientific Accela 15 pumps at a flow rate of 9 µl/min. Mass Spectrometry Samples were analyzed using both a Thermo Scientific Exactive Plus mass spectrometer in Full Scan mode (m/z 9) and a Thermo Scientific Q Exactive mass spectrometer in both Full Scan (m/z 9) and SIM mode with each using a resolution setting of 35, (FWHM) at m/z and a spectral speed of 7 Hz. Generic ion source conditions were used for all sample collection including vaporizer temperature (35 C), capillary temperature (3 C), sheath gas of 5 arbitrary units, and an auxiliary gas of 1 arbitrary units. The instrument was calibrated in positive ion mode before sample acquisition using Thermo Scientific Pierce LTQ Velos ESI Positive Ion Calibration Solution. An Evaluation of Various High-Resolution, Accurate-Mass Scan Modes for In Vitro Drug Discovery Screening

3 Data Analysis Data was acquired using Thermo Scientific Xcalibur. and Exactive Tune.1 software. Chromatographic data review and calibration curve generation was performed and reported using Thermo Scientific QuickCalc software (powered by Gubbs Inc., GMSU Gubbs Mass Spec Utilities, Atlanta, GA). Peak area measurements in the buffer chamber of the dialysis plate were compared to the peak area measurement in the serum chamber of the dialysis plate to calculate the percent of unbound compound (% Free) at assay equilibrium 1. The average % Free for each compound replicate was reported for each analysis scan type and compared to values obtained using a triple quadrupole mass spectrometer. The coefficient of variation of the % Free values for each scan mode was also calculated for each compound analyzed. Results Scan Mode Signal Response Each compound analyzed in the plasma protein binding (PPB) assay was evaluated in a concentration curve to evaluate overall sensitivity and linear dynamic range. All compounds were serially diluted using PPB matrix blank solution with concentrations ranging from 5 nm to nm concentration and analyzed using full scan and SIM analysis. The calibration curves for all compounds were generated using a linear regression and 1/x weighting. Individual calibration points exceeding a % difference of more than % of the regression line fit were excluded from the calibration curve. The majority of the compounds analyzed in full scan and SIM mode analysis exhibit the required sensitivity and linear dynamic range across the full range of the serial dilution and correlate well to the results collected using MS/MS analysis with a triple quadrupole mass spectrometer. Example calibration curves for each evaluated scan mode is displayed below for Fluphenazine (Figure 1). FIGURE 1. Calibration curve of Fluphenazine in each scan mode. (A) MS/MS analysis, (B) Q Exactive SIM analysis, (C) Q Exactive Full Scan Analysis, (D) Exactive Plus Full Scan Analysis (A) Peak Area Ratio (B) Area Ratio (C) Area Ratio (D) Area Ratio R^ =.9936 Fluphenazine Triple Quadrupole R^ = Fluphenazine Q Exactive SIM R^ = Fluphenazine Q Exactive Full Scan R^ = Fluphenazine Exactive Plus Full Scan Thermo Scientifi c Poster Note PN ASMS13_Th66_KMurphy_E 7/13S 3

4 The calibration curves for twenty-three of the twenty-four compounds analyzed using MS/MS analysis were linear across the full range of the calibration curve. One compound calibration curve in the MS/MS analysis required the exclusion of the nm calibration point due to signal saturation. Six of the twenty-four compounds analyzed using full scan and SIM mode analysis required the exclusion of the nm calibration point due to signal saturation (Figure ). High-resolution analysis using an Orbitrap mass analyzer enables a user-definable parameter for the amount of target ions collected for each scan during analysis. An increase in the amount of ions collected during each scan should limit the effects of signal saturation for future analysis. Due to sample volume limitations, optimization of the ion collection target could not be performed for this experiment. Full scan analysis of the compound calibration curves demonstrated adequate sensitivity for the analysis of the calibration curves for twenty of twenty-four compounds or 83%. One compound demonstrated improved sensitivity in full scan mode using the Q Exactive Orbitrap MS, while all other calibration curve signal responses were consistent for full scan analysis across both high-resolution platforms. FIGURE. Heat map display of compound calibration curve points included and excluded for each scan mode used for analysis. Calibration points with a % Difference greater than % were excluded from the linear regression. Excluded calibration points common to 3 scan modes are labeled in yellow. Excluded calibration points in or fewer scan modes are labeled in red. Exactive Plus Full Q Exactive Full Compound 5 nm 5 nm 5 nm 1 nm nm 5 nm 5 nm 5 nm 1 nm nm Propranolol Diltiazem Imipramine Halperidol Carbamazpine Chlorpheniramine Phentolamine Buspirone Verapamil Desipramine Clozapine Acebutolol Retonavir Thioridazine Nefazadone Timolol Minaprine Fluphenazine Metoprolol Ticlopidine Compound A Erythromycin Clomipramin Bendamustine Q Exactive SIM Triple Quadrupole Compound 5 nm 5 nm 5 nm 1 nm nm 5 nm 5 nm 5 nm 1 nm nm Propranolol Diltiazem Imipramine Halperidol Carbamazpine Chlorpheniramine Phentolamine Buspirone Verapamil Desipramine Clozapine Acebutolol Retonavir Thioridazine Nefazadone Timolol Minaprine Fluphenazine Metoprolol Ticlopidine Compound A Erythromycin Clomipramin Bendamustine Incuded in Curve Excluded from curve %Diff > % Excluded from curve %Diff > % and observed in or fewer of the scan modes An Evaluation of Various High-Resolution, Accurate-Mass Scan Modes for In Vitro Drug Discovery Screening

5 Analysis in SIM mode using the Q Exactive MS provided adequate sensitivity for all compounds analyzed and provided a sensitivity improvement for some compounds over full scan analysis (Figure ). PPB % Free Calculation Percent free or unbound amount of compound in the protein binding assay was calculated for each scan mode used for analysis 1. The coefficient of variation of the % Free across each scan mode was calculated for each compound and the results were listed in a table and sorted from lowest to highest by %CV (Table 1). Cells highlighted in red in Table 1 denote a scan mode that did not provide sufficient signal for a specific compound to generate a % Free value and were excluded from the %CV calculation for the respective compound. Table 1. % Free for analyzed compounds in each scan mode and %CV across scan modes. Cells highlighted in red denote scan modes with no results due to lack of analyte signal. Q Exactive SIM QE SIM QE Full E Plus Full Triple Compound % Free % Free % Free % Free Avg(%) StdDev(%) % CV Propranolol Diltiazem Imipramine Halperidol Carbamazpine Chlorpheniramine Phentolamine Buspirone Verapamil Desipramine Clozapine Acebutolol Retonavir Thioridazine Nefazadone Timolol Minaprine Fluphenazine Metoptolol Ticlopidine Compound A Erythromycin Clomipramin Bendamustine The calculated % Free values for each compound were plotted in a bar chart to illustrate differences in the % Free values across each scan mode for the PPB analysis.(figure 3). Figure 3. % Free for individual compounds across each scan mode used for assay analysis. Twenty-two of twenty-four compounds analyzed demonstrate a %CV of less than 5% across the various scan modes while providing adequate sensitivity for assay analysis across all scan modes. PPB % Free Scan Type Comparison 1.% 9.% 8.% 7.% 6.% 5.%.% 3.%.% 1.%.% Propranolol Diltiazem Imipramine Halperidol Carbamazpine Chlorpheniramine Phentolamine Buspirone Verapamil Desipramine Clozapine Acebutolol Retonavir Thioridazine Nefazadone Timolol Minaprine Fluphenazine Metoptolol Ticlopidine Compound A Erythromycin Clomipramin Bendamustine QE SIM QE Full Exactive Plus Triple Thermo Scientifi c Poster Note PN ASMS13_Th66_KMurphy_E 7/13S 5

6 Twenty-two of the twenty-four compounds analyzed in the protein binding assay provide a %CV of less than 5% across the various scan modes while providing adequate sensitivity for analyte analysis in the binding assay. Although four compounds did not provide enough signal in the calibration curve analysis only two did not provide enough signal for % Free calculation in the PPB assay itself. 9% of the compounds analyzed provided sufficient signal in both full scan and SIM mode with a %CV of less than 5%. The two compounds that did not provide enough sensitivity to generate a % Free value in the binding assay were challenging in full scan on the Exactive Plus only and not on the Q Exactive. One explanation for this observation maybe due to the generic mass spec and chromatographic conditions used for data analysis. Although both instruments collected data in full scan mode, the Q Exactive filters all ions outside of the specified full scan mass range. While the Exactive Plus does filter some ions at the s-lens, additional ions outside the specified mass range are also collected and injected into the Orbitrap Mass Analyzer. Further optimization of the ion target amount collected per scan in the mass spec method along with optimized chromatographic clean up of the assay samples in the generic method may improve signal response in full scan mode in the absence of true ion filtering with a quadrupole and will be evaluated in future work. Conclusion 83% of compounds analyzed met the assay calibration curve LOQ of 5nM. 9% of the compounds provided sufficient signal in the assay for calculation of the % Free in all scan modes evaluated. Full scan analysis using high resolution accurate mass provided adequate signal response and linear dynamic range to accurately measure 9% compounds analyzed in the PPB assay. Additional sensitivity and linear dynamic range may be achieved through further method optimization. References 1. Zhang J, Shou WZ, Vath M, Kieltyka K, Maloney J, Elvebak L, Stewart J, Herbst J, Weller HN., Rapid Commun Mass Spectrom. 1 Dec 3;():3593 Acknowledgements We would like to thank Dr. Jun Zhang and Jennifer Maloney of Bristol-Myers Squibb, Wallingford, CT for sample preparation and assistance with data processing. 6 An Evaluation of Various High-Resolution, Accurate-Mass Scan Modes for In Vitro Drug Discovery Screening

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