High-throughput liquid chromatography/mass spectrometry method for the quantitation of small molecules using accurate mass technologies in supporting discovery drug screening.

Background: Drug discovery samples are routinely analyzed using liquid chromatography/tandem mass spectrometry (LC/MS/MS) methods on triple quadrupole mass spectrometers employing multiple reaction monitoring (MRM). In order to improve analysis throughput, quantitation of small molecules on a quadrupole time-of-flight (QqTOF) instrument using TOF scan and high-resolution MRM (MRM-HR) modes was evaluated in this study.

Methods: Cassette dosed plasma and brain samples from nine compounds were extracted using a protein precipitation method. Separation was achieved by reversed-phase liquid chromatography. Mass spectrometric analysis was performed using TOF scan and high-resolution MRM approaches on a QqTOF mass spectrometer with turbo-ionspray ionization. Results were compared to those obtained on a triple quadrupole mass spectrometer.

Results: The dynamic range varied depending on compounds and instruments and was similar between the MRM on QqQ and full TOF scan mode on QqTOF. Linear or quadratic regression and 1/x(2) weighting were used. Resolution on the QqTOF instrument was around 32000 and mass accuracy was within 4.4 ppm. The MRM-HR method showed better sensitivity compared to the TOF scan method, and was comparable to the MRM on a QqQ mass spectrometer. Assay accuracy was within ±25%.

Conclusions: A TOF scan method allowed the use of the generic method without compound-specific optimization and was an alternative choice for routine high-throughput quantitation of small molecules. The MRM-HR method on the QqTOF showed good sensitivity which was comparable to that obtained by the MRM method on the triple quadrupole mass spectrometer.