title: Development and validation of an indirect pulsed electrochemical detection method for monitoring the inhibition of Abl1 tyrosine kinase.
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. 50 μM 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. 5 μm; 250 mm × 4.6 mm) column with the mobile phase flow rate of 0.5 ml/min. 0.5M sodium hydroxide was added post-column to maintain alkaline conditions in the PED cell. The limit of quantification (LOQ) was 0.2 μM 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 601.4 nM (R(2)=0.99), 32.3 nM (R(2)=0.99) and 1.3 nM (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.
authors: Pharmaceutical Analysis, Faculty of Pharmaceutical Sciences, KU Leuven, O&N 2, PB 923, Herestraat 49, B-3000 Leuven, Belgium.
authors: Hui
authors: H
authors: Chen
authors: Pharmaceutical Analysis, Faculty of Pharmaceutical Sciences, KU Leuven, O&N 2, PB 923, Herestraat 49, B-3000 Leuven, Belgium.
authors: Xu
authors: X
authors: Wang
authors: Pharmaceutical Analysis, Faculty of Pharmaceutical Sciences, KU Leuven, O&N 2, PB 923, Herestraat 49, B-3000 Leuven, Belgium.
authors: Shruti
authors: S
authors: Chopra
authors: Pharmaceutical Analysis, Faculty of Pharmaceutical Sciences, KU Leuven, O&N 2, PB 923, Herestraat 49, B-3000 Leuven, Belgium.
authors: Erwin
authors: E
authors: Adams
authors: Pharmaceutical Analysis, Faculty of Pharmaceutical Sciences, KU Leuven, O&N 2, PB 923, Herestraat 49, B-3000 Leuven, Belgium. Electronic address: Ann.VanSchepdael@pharm.kuleuven.be.
authors: Ann
authors: A
authors: Van Schepdael
mesh_terms: D001549:Benzamides / Q000008:administration & dosage / Q000494:pharmacology; D002138:Calibration; D002853:Chromatography, Liquid / Q000379:methods*; D000069439:Dasatinib; D055664:Electrochemical Techniques / Q000379:methods*; D016044:Fusion Proteins, bcr-abl / Q000037:antagonists & inhibitors*; D005942:Gluconates / Q000737:chemistry; D000068877:Imatinib Mesylate; D020128:Inhibitory Concentration 50; D057230:Limit of Detection; D016014:Linear Models; D010879:Piperazines / Q000008:administration & dosage / Q000494:pharmacology; D047428:Protein Kinase Inhibitors / Q000008:administration & dosage / Q000494:pharmacology*; D011743:Pyrimidines / Q000008:administration & dosage / Q000494:pharmacology; D013844:Thiazoles / Q000008:administration & dosage / Q000494:pharmacology
chemical_list: C581634:BCR-ABL1 fusion protein, human; D001549:Benzamides; D005942:Gluconates; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; D013844:Thiazoles; D000068877:Imatinib Mesylate; D016044:Fusion Proteins, bcr-abl; C498826:nilotinib; C030691:gluconic acid; D000069439:Dasatinib
keywords: Abl1 tyrosine kinase; Abltide; Inhibition assay; LC–InPED