Determination of N-Acetyl-l-cysteine Ethyl Ester (NACET) by Flow Injection Analysis and Spectrophotometric Detection Using Different Thiol-Sensitive Ligands.

Lea Kukoc-Modun,Njegomir Radić,Dimitrios Tsikas,Tomislav Kraljević,Darko Modun

doi:10.3390/molecules26226826

Abstract

A new flow injection spectrophotometric method for the determination of N-acetyl-l-cysteine ethyl ester (NACET) was developed and validated. The method is based on the reduction of Cu(II)-ligand complexes to chromophoric Cu(I)-ligand complexes with the analyte. The studied ligands were neocuproine (NCN), bicinchoninic acid (BCA) and bathocuproine disulfonic acid (BCS). The absorbance of the Cu(I)-ligand complex was measured at 458, 562 and 483 nm for the reactions of NACET with NCN, BCA and BCS, respectively. The method was validated in terms of linear dynamic range, limit of detection and quantitation, accuracy, selectivity, and precision. Experimental conditions were optimized by a univariate method, yielding linear calibration curves in a concentration range from 2.0 × 10−6 mol L−1 to 2.0 × 10−4 mol L−1 using NCN; 2.0 × 10−6 mol L−1 to 1.0 × 10−4 mol L−1 using BCA and 6.0 × 10−7 mol L−1 to 1.2 × 10−4 mol L−1 using BCS. The achieved analytical frequency was 90 h−1 for all three ligands. The method was successfully employed for NACET determination in pharmaceutical preparations, indicating that this FIA method fulfilled all the essential demands for the determination of NACET in quality control laboratories, as it combined low instrument and reagent costs with a high sampling rate.

Highlights

N-Acetylcysteine ethyl ester (NACET) is a lipophilic, charge-free, cell-permeable cysteine derivative with highly improved pharmacological properties in comparison with its congener N-acetyl-L-cysteine (NAC)
The method was successfully employed for N-acetyl-Lcysteine ethyl ester (NACET) determination in pharmaceutical preparations, indicating that this flow injection analysis (FIA) method fulfilled all the essential demands for the determination of NACET in quality control laboratories, as it combined low instrument and reagent costs with a high sampling rate
The reactions involved in the present study are based on the one-step redox reaction (Equations (1)–(3)), in which NACET (RSH compound) reduces the Cu(II)-ligand complex to the Cu(I)-ligand complex, which exhibits absorption maxima at 458 nm [19], 562 nm [20] and 483 nm [21] for NCN, bicinchoninic acid (BCA) and bathocuproine disulfonic acid (BCS), respectively: 2RSH + 2[Cu(NCN)2]2+ RSSR + 2[Cu(NCN)2]+ + 2H+

Summary

Introduction

N-Acetylcysteine ethyl ester (NACET) is a lipophilic, charge-free, cell-permeable cysteine derivative with highly improved pharmacological properties in comparison with its congener N-acetyl-L-cysteine (NAC). At physiological pH values, the free carboxylic group of NAC is almost entirely negatively charged. The esterification of the carboxyl group of NAC increases the lipophilicity of NAC and improves its pharmacokinetics. A recent report showed that NAC did not act as a GSH enhancer in human endothelial cells in a dose-dependent manner. The treatment with NACET had an evident ceiling effect. The levels reached by intracellular GSH after treatments with NACET resulted in a balance between the induction of its synthesis (by increased cysteine) and enzyme inhibition (by increased intracellular NAC) [3].

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Conclusion