(T1130-01-02) Transforming Quantitative Sensitivity for Small Molecule Analysis Using a High-Resolution Workflow

Purpose: Quantitation of small molecule therapeutics in complex biological matrices requires methods that meet proper selectivity and sensitivity needs. Quantitative analyses of small molecules have commonly been performed on a gold-standard triple quadrupole mass spectrometer. However, more recently, such analyses have been increasingly conducted on a high-resolution mass spectrometer given the improved selectivity, which, consequently, aids in enhancing sensitivity. In this study, quantitation of small molecule therapeutics in extracted rat plasma was performed on a novel QTOF mass spectrometer.

Methods: Rat plasma was prepared using a 1:3 (v/v) plasma/methanol ratio. The samples were vortexed and centrifuged at room temperature. The supernatant was aliquoted and diluted using 1:4 (v/v) supernatant/water (water composition: 2% acetonitrile, 2% acetic acid, and 0.1% formic acid). To this mixture, 6 small molecules were spiked and serially diluted to create a standard curve. Separation was performed using Phenomenex Kinetex XB C18 column (3 x 50 mm, 3 µm, 110 Å) at 40°C with a flow rate of 0.8 mL/min. 0.1% formic acid in water was used as mobile phase A, and 0.1% formic acid in acetonitrile was used as mobile phase B. An injection volume of 5 μL was used for analysis on a novel QTOF mass spectrometer.

Results: In this study, 6 small molecules were analyzed in an extracted rat plasma matrix. Each calibration point was analyzed in triplicate. On average, an 18-fold improvement in LLOQ was achieved across 6 small molecules compared to a previous generation QTOF mass spectrometer using a high-resolution MRM mode. Representative small molecules such as alprazolam, diazepam, and trimethoprim reached an LDR of up to 4.5 orders of magnitude, demonstrating measurement across a wide range of concentrations. Overall, LLOQ accuracy was between 80% to 120% and accuracy for all non-zero calibrators was between 85% and 115%. The overall %CV was < 13% for all small molecules analyzed. Since the analysis was performed on a high-resolution mass spectrometer, compounds such as diazepam showed improved selectivity compared to a traditional triple quadrupole mass spectrometer analysis. In the case of diazepam, a 6.5-fold improvement in signal-to-noise (S/N) was achieved when compared to an MRM analysis on a triple quadrupole mass spectrometer. This was attributed to the capabilities of a high-resolution mass spectrometer, which enabled the differentiation of the target small molecule from matrix components.

Conclusion: Demonstration of a small molecule quantitation method in the biological matrix using high-resolution MRM for improved selectivity and sensitivity.