Premium
Development of a high‐throughput mass spectrometry based analytical method to support an in vitro OATP1B1 inhibition screening assay
Author(s) -
Wagner Andrew D.,
Elkin Lisa,
Mosure Kathy,
Gallagher Lizbeth,
Stavola Lindsey K.,
Soars Matthew G.,
Shou Wilson
Publication year - 2016
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.7655
Subject(s) - chemistry , pitavastatin , chromatography , mass spectrometry , tandem mass spectrometry , detection limit , selected reaction monitoring , reproducibility , electrospray ionization , ion suppression in liquid chromatography–mass spectrometry , analyte , biochemistry , statin
Rationale It is well known that the organic anion transporting polypeptide 1B1 (OATP1B1) plays a major role in the hepatic uptake of a range of drugs. To this end, it is pivotal that the potential for new molecular entities (NMEs) to inhibit OATP1B1 activity be assessed during early drug discovery. The work reported herein describes the development of a high‐throughput analytical method to measure the clinically relevant probe substrate, pitavastatin, for the in vitro assessment of OATP1B1 inhibition. Methods Development of an analytical method capable of very fast throughput was crucial for the success of this assay and was accomplished using a system which combines direct, on‐line solid‐phase extraction (SPE) with highly sensitive, label‐free tandem mass spectrometry (MS/MS)‐based detection. Mass spectrometry analysis of pitavastatin, along with the stable isotopically labeled internal standard d5‐pitavastatin, was conducted using positive electrospray ionization (ESI) in selected reaction monitoring (SRM) mode. Results The on‐line SPE‐MS/MS platform demonstrated similar sensitivity, selectivity, reproducibility, linearity and robustness to existing methodologies while achieving analytical cycle times of 10.4 seconds per well. Sensitivity exceeded what was necessary for our assay conditions, with a determined lower limit of quantification (LLOQ) for pitavastatin of 10 pM (picomolar) in assay matrix. Furthermore, the potency of multiple reference compounds was shown to be within 2‐fold of IC 50 values generated from liquid chromatography (LC)/MS/MS‐based literature values. Conclusions A very fast and robust analytical method was successfully developed for the measurement of the clinically relevant OATP1B1 substrate, pitavastatin. The successful development and implementation of this very important early liability screen has helped to facilitate judicious lead candidate progression and will ultimately help build a greater understanding of OATP1B1‐NME interactions, in general. Copyright © 2016 John Wiley & Sons, Ltd.