An automated high throughput liquid chromatography-mass spectrometry process to assess the metabolic stability of drug candidates.
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.
Drexler
Dieter M
DM
Discovery Analytical Sciences, Pharmaceutical Candidate Optimization, Pharmaceutical Research Institute, Bristol-Myers Squibb Company, Wallingford, CT 06492, USA. dieter.drexler@bms.com
Belcastro
James V
JV
Dickinson
Kenneth E
KE
Edinger
Kurt J
KJ
Hnatyshyn
Serhiy Y
SY
Josephs
Jonathan L
JL
Langish
Robert A
RA
McNaney
Colleen A
CA
Santone
Kenneth S
KS
Shipkova
Petia A
PA
Tymiak
Adrienne A
AA
Zvyaga
Tatyana A
TA
Sanders
Mark
M
D002853:Chromatography, Liquid; D003627:Data Interpretation, Statistical; D004353:Drug Evaluation, Preclinical; D007202:Indicators and Reagents; D013058:Mass Spectrometry; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D011786:Quality Control; D015203:Reproducibility of Results; D012371:Robotics; D012984:Software; D012997:Solvents; D013056:Spectrophotometry, Ultraviolet
D007202:Indicators and Reagents; D004364:Pharmaceutical Preparations; D012997:Solvents; D009251:NADPH-Ferrihemoprotein Reductase