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APPLICATION OF EXACT MASS MS IN BIOANALYSIS DISCOVERY QUANTITATION WITH UPLC AND XEVO QTOF MS


Robert S. Plumb and Marian Twohig, Waters Corporation, Milford, MA, U.S. INTRODUCTION


The quantification of candidate pharmaceuticals and/or their metabolites in biological fluids plays a key role in drug discovery.


The information generated is used to determine key pharmacokinetics parameters such as clearance, half-life, Tmax, and bioavailability. During discovery ADME studies the metabolic fate of the molecule is determined as well as its pharmacokinetics. Currently this involves using two analytical instruments: one to provide the quantitative information and one for the qualitative analysis. Quantitative information is normally derived from a tandem quadrupole instrument due to sensitivity; qualitative data is gathered either from an ion trap or quadrupole time-of-flight MS (QTof) instrument. This need for multiple instruments and analytical runs results in reduced productivity and increased instrument capital costs.


QTof technology is well-recognized as the platform of choice for exact mass MS/MS structural elucidation.1-3 note, we present the use of a high-sensitivity QTof mass spectrometer coupled with UltraPerformance LC® at the levels of sensitivity required for drug discovery.


(UPLC®


However, its use in quantitative DMPK studies has yet to be fully exploited. In this application ) separation technology for the quantitative analysis of a model candidate pharmaceutical


APPLICATION NOTE


Here we can see that using a narrower mass range of 50 mDa simplifies the chromatogram and reduces the detected chemical noise.


Linear dynamic range


The assay displayed sensitivity down to 50 pg/mL, which is more than adequate for use in discovery projects for quantification. The Xevo QTof MS provided linearity in excess of three orders of magnitude, as shown in Figure 4. The narrow peak widths produced by the ACQUITY UPLC System of 4 to 6 sec at the base required a data collection rate of 100 to 50 mSec per data point in order to correctly define the peaks for quantification.


Figure 1. Xevo QTof MS with ACQUITY UPLC. EXPERIMENTAL


A calibration line for a model drug candidate molecule was prepared in blank rat plasma at the concentration level of 50 pg/mL to 50 ng/mL. The samples were prepared by the protein precipitation of 50 µL of plasma with cold acetonitrile (2:1). The supernatant was evaporated to dryness and reconstituted in 50 µL of water for injection. An aliquot of the sample was injected onto the LC/MS system for analysis.


LC conditions LC system: Waters®


Column:


Column temp.: 40°C Flow rate:


ACQUITY UPLC® System


ACQUITY UPLC BEH C18 Column 2.1 x 100 mm, 1.7 µm


600 µL/min


Mobile phase A: Aqueous formic acid (0.1%) Mobile phase B: Methanol Gradient:


5 to 95% B/10 min MS conditions


MS system: Waters Xevo™ QTof MS Mass Spectrometer


Ionisation mode: ESI positive Acquisition range: 100 to 800 m/s


RESULTS


The popularity of tandem quadrupole mass spectrometers for use in quantitative analysis stems from the specificity and sensitivity derived from the multiple reaction monitoring (MRM) process. These instruments still provide the most sensitive mode of analysis, especially with the complex matrices encountered in bioanalysis.


Accurate mass instrumentation generates full spectrum MS and MS/MS data that provides information regarding analytes in the sample. These accurate mass instruments, although less sensitive than tandem quadrupole instruments, can provide a similar level of specificity by using accurate mass chromatograms with small mass window ranges. The data displayed in Figure 2 shows the effect of changing the mass error range from 1 Da to 30 mDa for propranolol with a M+H mass 260.1651.


Figure 4. Propranolol calibration line from 50 pg/mL to 50 ng/mL.


Figure 2. Exact mass chromatogram of propranolol protein- precipitated plasma sample with 1 Da and 50 mDa mass windows.


The reduction in chemical noise produced by using a smaller mass window significantly increases the signal-to- noise (S/N) ratio from 3:1 to 13:1, as shown in Figure 3. In this expanded figure we can see that when using the 30 mDa window, the propranolol peak at 3.94 minuntes is now the biggest peak in the chromatogram and well- defined above the noise compared to the 1 Da window.


The Xevo QTof MS is capable of acquiring data at acquisition speeds of 50 mSec per spectra without reduction in mass accuracy or spectral quality. This mass stability allows the use of a narrow mass window for data processing, improving the specificity and signal-to-noise obtained for the analysis. Using a data capture rate of 50 spectra/sec, a calibration line was generated over four orders of magnitude, from 50 pg/mL to 50 ng/mL.


Data were processed using TargetLynx™ Application Manager for MassLynx™ Software. TargetLynx automates sample data acquisition, processing, and reporting for quantitative results. It incorporates a range of confirmatory checks that identify samples that fall outside user-specified or regulatory thresholds. The TargetLynx method editor allows the mass window to be selected for the quantification of the analyte(s) of interest. This allows very narrow mass windows to be selected, improving the mass selectivity. The example shown in Figure 5 illustrates the selection of the mass window. Here we can see that a mass window of 30 mDa has been employed for integration. The lower the mass window that can be employed, the more specific the analysis; however, the ability to use very low mass windows relies on the mass stability of the mass spectrometer. The Xevo QTof MS is equipped with LockSpray™ Technology, which allows for stable, long-term operation with low mass drift.


Figure 3. Expanded chromatogram window focused on propranolol peak at 3.9 minutes.


The Xevo QTof MS’s exact mass MS and MS/MS capability also makes it ideal for de novo identification of small molecules such as drug metabolites and impurities. The Xevo QTof MS has been shown to provide excellent sensitivity and spectral quality in both MS and MS/MS modes for metabolite identification,[1-3] especially when combined with the data processing power of the MetaboLynx XS Application Manager.


LAB ASIA - NOVEMBER/DECEMBER 2009 - MASS SPECTROSCOPY FOCUS


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