Bioanalysis, the study of the concentration of components of interest in biological matrices, covers a range of applications from small molecule discovery PK quantitation to regulated bioanalysis in toxicological and human clinical studies of large and small molecule therapeutics.
Workflow Overview for Bioanalysis
Pharmacokinetics (PK) studies the distribution of an externally administered compound or drug candidate inside a living animal. The primary endpoint is the calculation of various important pharmacokinetic parameters such as bioavailability (F), exposure (as area under the curve, AUC), and clearance (CL). Although in vitro models are used to predict potential pharmacokinetic performance prior to performing the in vivo study, the PK assay is a key step in determining the quality of a potential drug candidate as well as validating in vitro/in vivo correlation (IVIVC) efforts. PK samples often consist of plasma and, therefore, require a sensitive and robust method of analysis. Data acquisition using high-resolution accurate mass analysis by full-scan MS, SIM (selected ion monitoring) or PRM (parallel reaction monitoring, see refs (1) & (2)) offers a simple alternative to traditional SRM analysis on triple quadrupoles without compromising robustness or sensitivity. In addition, added selectivity can be achieved through high resolution accurate mass selection of the desired mass. The Thermo Scientific Q Exactive™ MS provides unparalleled specificity, sensitivity, and robustness without the need for SRM optimization generally required for triple quadrupole analysis. Additionally Q Exactive MS experiments can be designed to collect supplementary information regarding qualitative information for both known and unknown sample components without compromising primary quantitative requirements.
Sample Preparation Workflow for Bioanalysis
For discovery PK studies, plasma samples are crashed with an organic solvent such as ACN containing the internal standard for the LC-MS analysis. Samples are then centrifuged for several minutes, and the supernatant is transferred to 96-well plate format for analysis. Typical injection volumes range between 2 and 10 µL per sample.
Mass Spectrometry Workflow for Bioanalysis
The Thermo Scientific Q Exactive™ benchtop mass spectrometer combines high-performance quadrupole precursor selection with high-resolution, accurate-mass (HR/AM) Orbitrap detection to deliver high performance and tremendous versatility. With fast scanning, the Q Exactive system ensures exact mass measurement for UHPLC applications, and additionally allows for metabolic profile data to be obtained without compromising the primary quantitative purpose of PK studies.
Data Analysis Workflow for Bioanalysis
Thermo Scientific QuickCalc™ software can be utilized for peak detection, integration, and reporting of pharmacokinetic results. The software integrates LC/MS data review with PK calculations for results in a single view, allowing for instantaneous updates of calculated results during chromatographic data interrogation. The PK module allows for quick calculations of T1/2, AUC, Cmax, and clearance for rapid determination of the PK properties of the compound of interest. After review, results are stored in the QuickCalc database and custom reports can be generated.
Orbitrap of Choice for Bioanalysis
A Thermo Scientific Q Exactive™ MS with a HESI source, operated at a resolution of 70,000 FWHM, provides a level of sensitivity for pharmacokinetic studies comparable to that of triple quadrupole technology. The capability to combine parent compound quantitation and preliminary metabolite identification in a single LC/MS experiment provides the potential for cost-effective and streamlined workflow. The simple-to-use interface enables Q Exactive MS operators the ability to easily obtain results similar or better than seen with their QqQ, without the need to dedicate limited resources and time for compound optimization normally required for QqQ-based SRM experiments.