PK and Metabolite Identification
The ability to gather both quantitative and qualitative data in a single run using high-resolution accurate mass spectrometry coupled with UPLC for high-throughput data acquisition can substantially improve efficiency in pharma sample analysis, maintained Suma Ramagiri, Ph.D., application scientist at AB Sciex.
“For example, it’s not necessary to reanalyze the same sample two or three times by separate departments within the same company such as the bioanalytical group for quantitative information and the biotransformation group for qualitative information.”
At “Pittcon”, Dr. Ramagiri presented work demonstrating the ability to gather a buspirone PK quantitation profile in rat plasma along with identification of major metabolites over an eight-hour time period, all in single injection per time point.
The accurate mass high-resolution LC/MS assay was developed on the AB Sciex TripleTOF™ 5600 system using on-the-fly multiple mass defect filter acquisition method. Calibration curve ranging from 0.1 ng/mL–1,000 ng/mL was prepared in rat plasma and evaluated for its linearity, accuracy, and precision. Buspirone-d8 was used as an internal standard. A simple sample protein precipitation was used as sample-extraction method.
The current LC/MS/MS assay showed linearity (R>0.99) with accuracy ranging from 80–120%. Absolute quantification of parent and relative quantification of metabolites was processed by MultiQuant™ 2.0.1 software. From the same sample, both phase I and II metabolites were identified by MetabolitePilot™ 1.0 software.
Dr. Ramagiri believes this approach has wide applicability in drug R&D, including early discovery (in vitro metabolic stability and soft-spot analysis), development (in vivo PK analysis in different animal species like mouse, rat, dog, monkey), and preclinical/early clinical stages (definitive metabolite identification with quantitative capability to address metabolite safety and toxicity issues under MIST guidelines).
One challenge, noted Dr. Ramagiri, “is that more data generation with large file size creates data-storage and data-transfer problems. But with advances in computer technology, this can be controlled.”