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Development and validation of an LC‐ESI‐MS/MS quantification method for a potential γ ‐aminobutyric acid transporter 3 (GAT3) marker and its application in preliminary MS binding assays
Author(s) -
Polley Maria,
Höfner Georg,
Wanner Klaus Theodor
Publication year - 2013
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.2841
Subject(s) - chemistry , chromatography , bioanalysis
Binding assays for the γ ‐aminobutyric acid (GABA) transporter GAT3 can be assumed to significantly facilitate screening for respective inhibitors. As appropriate labeled ligands for this promising drug target are not available so far, we started efforts to set up mass spectrometry‐based binding assays (MS binding assays), for which labeled markers are not required. Therefore, we developed a sensitive and rapid LC‐ESI‐MS/MS quantification method for DDPM‐1007 {( RS )‐1‐[4,4,4‐Tris(4‐methoxyphenyl)but‐2‐en‐1‐yl]piperidine‐3‐carboxylic acid}, one of the most potent GAT3 inhibitors yet known, as a potential GAT3 marker. Using a 50 × 2 mm C 8 column in combination with a mobile phase composed of 10 m m ammonium bicarbonate buffer pH 8.0 and acetonitrile (60:40, v/v) at a flow rate of 450 μL/min DDPM‐1007 could be analyzed in the positive multiple reaction monitoring mode [( m / z ) 502.5 → 265.4] within a chromatographic cycle time of 3 min. Deuterated DDPM‐1007 [( 2 H 9 )DDPM‐1007] was synthesized and employed as internal standard. This way DDPM‐1007 could be quantified in a range from 100 p m to10 n m in the matrix resulting from respective binding experiments without any sample preparation. The established quantification method met the requirements of the FDA guidance for bioanalytical method validation concerning linearity and intra‐ and inter‐batch accuracy. Based on this LC‐ESI‐MS/MS quantification preliminary MS binding assays employing membrane preparations obtained from a stably GAT3 expressing HEK293 cell line and DDPM‐1007 as nonlabeled GAT3 marker could be performed. In these experiments specific binding of DDPM‐1007 at GAT3 could be unambiguously detected. Additionally, the established LC‐MS method provides a suitable analytical tool for further pharmacokinetic characterization of DDPM‐1007, as exemplified for its log D determination. Copyright © 2012 John Wiley & Sons, Ltd.