Suma Ramagiri

Structural, bioanalytical, characterization, and quality control studies are critical for successful drug development. These studies must be as accurate, sensitive, and selective as possible, and liquid chromatography coupled to tandem mass spectrometry (LC–MS–MS) has been the technique of choice for many areas of small molecule analysis for the past 30 years. During that time, rapid improvements in analytical technologies have supported the development of more sensitive and robust methods. However, the pharma and biopharma industry continues to need more powerful instruments and more diverse methods, particularly as therapeutics have expanded to include large molecules. This work follows on from an earlier article that explored the limitations of LC–MS–MS for bioanalysis of biologics. This article considers some of the current issues for analysis of small and large molecules, and emerging trends in method development.

Bioanalysis of biologics presents a number of technical challenges. Ligand binding assays (LBA) are the gold standard bioanalytical technique for quantification of biologics in complex matrices such as serum and plasma but selectivity issues and the need for specific capture reagents limit their applicability in the drug discovery and development phase. Liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS) is widely used for highly selective and sensitive bioanalysis of small molecules. However, large molecule bioanalysis presents challenges including the need for extensive and complex sample preparation for LC-MS-MS. This article explores the limitations of LC-MS-MS for bioanalysis of biologics and some of the latest trends for overcoming these in bioanalysis laboratories.

A new time-of-flight mass spectrometer was evaluated for performing simultaneous metabolic stability measurement and metabolite identification with ultrahigh-pressure liquid chromatography. Six representative compounds (clomipramine, diclofenac, imipramine, haloperidol, verapamil, and midazolam) were incubated in rat liver microsomes at a more physiologically relevant substrate concentration (1 ?M). High-resolution full-scan and product-ion spectra were acquired in a single injection using generic methodology. Quantitative clearance of the parent was measured using the full-scan data. Major metabolites were identified using the accurate mass product ion spectra. High scanning speed allowed for a sufficient number of data points to be collected across the chromatographic peak for quantitative analysis. Sensitivity was sufficient for obtaining meaningful kinetics with a 1 ?M initial substrate concentration.