An Automated High Throughput Liquid Chromatography–Mass Spectrometry Process to Assess the Metabolic Stability of Drug Candidates

Abstract

An automated high throughput process, termed the MetFast assay, is described to assess in vitro the general microsomal cytochrome P450 beta-nicotinamide adenine dinucleotide phosphate-mediated first-pass metabolic stability of potential drug candidates as a utility for pharmaceutical profiling. Utilizing robotic protocols with a multiprobe liquid handler, compounds are incubated with liver microsomes from different species. Samples are then analyzed by in-line liquid chromatography (LC)-mass spectrometry (MS) to determine the amount of compound remaining after a certain time, which allows calculation of metabolism rates. To quantitatively assess large numbers of structurally diverse compounds by LC-MS, a strategy based on an iterative two-step process was devised. Initially compounds are qualitatively analyzed by LC-ultraviolet (UV)/MS (step 1) to determine purity (UV detection) and structural integrity (MS detection). This step ensures that only correct and verified compounds with sufficient purity are being assayed to obtain reproducible high data quality. In addition, all necessary information is gathered to automatically generate specific quantitative methods for the subsequent bioanalytical analysis of metabolic stability samples by LC-UV/MS (step 2). In-house-developed, highly flexible and sophisticated data management software, termed SmartReport, is utilized for automated qualitative and quantitative LC-MS analysis set-up, data processing, and results reporting. The integration of key aspects, inherent "universal" collision-induced dissociation settings of ion trap mass spectrometers for tandem mass spectrometric scan functions utilized for compound-specific and sensitive quantitative MS methods, generic fast-LC conditions, generic MS instrument settings, and the functionality of SmartReport software resulted in an analytical process that routinely provides reproducible high-quality metabolic stability data on structurally diverse compounds. Described here is the setup of the MetFast assay, and metabolic stability data from assay validation compounds are given.

Quantitative-qualitative data acquisition using a benchtop orbitrap mass spectrometer

“…This no-metabolism point is arbitrarily set to 100% parent remaining and all subsequent time points are calculated relative to this sample. As such, standard curves are not used and the instrument response is assumed to be linear across the range of concentrations detected (Յ1 M) [19,20]. When using a full-scan approach, no prior method developed is required to determine multiple reaction monitoring (MRM) transitions for the compounds of interest.…”

A review of LC–MS techniques and high-throughput approaches used to investigate drug metabolism by cytochrome P450s

“…In this regard, the speed and sensitivity associated with liquid chromatography/tandem mass spectrometry (LC-MS/MS) lends itself to supporting high-throughput analyses from drug-metabolism studies. However, recent advances in ion-trap mass analysers make these systems a suitable alternative [24].…”

“…Drexler et al [24] recently described an iterative two-step process addressing some of these processes involved in a screening strategy. Compounds were initially analysed by LC-ultraviolet (UV)/MS for purity (UV detection) and identity confirmation (MS detection) in the structural integrity (SI) assay, ensuring that the correct and verified compound with sufficient purity is used in an assay.…”

“…These have included obtaining SRM MS/MS parameters under a 'universal' collision-induced dissociation condition on ion-trap mass spectrometers for MS/MS [24] and single quadrupole mass spectrometers analyzing samples without MS method development [31]. Support of in vitro screens by LC-MS quantitation without the need for MS/MS method development has also been demonstrated using TOF analysers with accurate mass measurement [32].…”

Acoustic Sample Deposition MALDI-MS (ASD-MALDI-MS): A Novel Process Flow for Quality Control Screening of Compound Libraries

“…2 However, compound consumption, sample preparation, online sample manipulation by the LC autosampler, and analysis time are concerns when dealing with library collections within CM. Matrix-assisted laser desorption/ionization (MALDI-MS) is an alternative MS technique that can be employed for molecular analysis.…”