Development and validation of an indirect pulsed electrochemical detection method for monitoring the inhibition of Abl1 tyrosine kinase

• It is the first time to report an indirect pulsed electrochemical detection (InPED) for enzyme assay.
• A label free InPED was used to detect peptide, for instance no incorporation of a fluorophore is necessary.
• The measured IC50 values are consistent with the literature.

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. 0.5 M 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 (R2 = 0.99), 32.3 nM (R2 = 0.99) and 1.3 nM (R2 = 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.

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