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Title: Development and validation of an indirect pulsed electrochemical detection method for monitoring the inhibition of Abl1 tyrosine kinase
Authors: Chen, Hui
Wang, Xu
Chopra, Shruti
Adams, Erwin
Van Schepdael, Ann # ×
Issue Date: Mar-2014
Publisher: Pergamon Press
Series Title: Journal of Pharmaceutical and Biomedical Analysis vol:90 pages:52-7
Article number: 10.1016/j.jpba.2013.11.022
Abstract: A new method for monitoring the enzyme inhibition of Abl1 tyrosine kinase by liquid chromatography-indirect pulsed electrochemical detection (LC-InPED) was developed. In this method, adsorption of a peptide analyte at the noble metal electrode suppresses the oxidation of polyols under alkaline condition to elicit an indirect response resulting in a negative peak of the target peptide. Among the reagents tested, d-gluconic acid sodium salt gave the best overall signal to noise (S/N) values for the indirect detection of p-Abltide, the product of Abl1 enzymatic reaction. 50μM d-gluconic acid sodium salt dissolved in a mixture of 78% water-22% acetonitrile-0.03% trifluoroacetic acid (TFA) was used as the mobile phase. Chromatographic separation was achieved on an Alltima C18 (I.D. 5μm; 250mm×4.6mm) column with the mobile phase flow rate of 0.5ml/min. 0.5M sodium hydroxide was added post-column to maintain alkaline conditions in the PED cell. The limit of quantification (LOQ) was 0.2μM for p-Abltide, which was about 50-fold lower than direct PED analysis. The residual plot of the linear calibration curve indicated a good fit with a linear model within the investigated concentration range of p-Abltide. Intra- and inter-day precision was not more than 6.5% and accuracy was from -5.75% to +1.54%. The validated LC-InPED method was successfully applied for monitoring of p-Abltide in Abl1 enzyme reaction and the inhibition study of Abl1. The determined IC50 values of model inhibitors, imatinib, nilotinib and dasatinib, were 601.4nM (R(2)=0.99), 32.3nM (R(2)=0.99) and 1.3nM (R(2)=0.98), respectively. These results were consistent with literature data. To the best of our knowledge this is the first time a LC-InPED method has been used to monitor an enzyme reaction.
URI: 
ISSN: 0731-7085
Publication status: published
KU Leuven publication type: IT
Appears in Collections:Pharmaceutical Analysis
× corresponding author
# (joint) last author

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