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

RATIONALE: 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.

Comparison of triple quadrupole and high-resolution TOF-MS for quantification of peptides.

BACKGROUND: With an increased interest in peptides and proteins as potential new drug candidates, new approaches for sensitive and selective quantitative analysis are required. LC-MS/MS analysis provides a good alternative to immunoassays with reduced method development times and increased specificity. RESULTS: We have evaluated two state-of-the-art triple quadrupole and high-resolution TOF mass spectrometers with respect to their performance for quantification of six peptides (glufibrinopeptide B, somatostatin, enfuvirtide, TRI1144, C34 and exenatide). The peptides were spiked into protein-precipitated plasma supernatant. Triple quadrupole quantification was performed in SRM mode, and in high-resolution, MS narrow-width extracted chromatograms were generated for quantification. Specificity, accuracy, reproducibility and robustness were found to be comparable between the two instruments. The triple quadrupole instrument is still the most sensitive instrument for quantification of peptides with a median factor of about four-times higher sensitivity (based on LLOQ evaluation). CONCLUSION: Based on sensitivity, the newest generation triple quadrupole MS systems are still the preferred technology for quantification of peptides. Since the sensitivity difference between triple quadrupole instruments and the new-generation high-resolution TOF-MS instruments is minor, the latter offer a useful alternative whenever additional selectivity is preferred or the use of a generic approach not requiring method optimization is advantageous.