A new way to determine N-acetylcysteine in blood plasma by derivatization and HPLC with UV detection

Abstract

N-acetylcysteine is a medicinal substance used in various medicinal products (mucolytic drugs, cytoprotective agents, drugs for chemotherapy of oncological diseases). There are many methods of HPLC analysis of N-acetylcysteine. Most of these techniques are based on the reaction of the sulfhydryl group of N-acetylcysteine with reagents, resulting in the formation of fluorescent derivatives. These methods are characterized by non-optimal conditions of derivatization reactions, the complexity of chromatographic separation of derivatives and high cost of reagents. Thereby the aim of this work was to develop a simple, quick and affordable way to determine N-acetylcysteine in serum plasma.

Blood plasma of patients taking N-acetylcysteine is analyzed. Blood sampling was performed 1 hour after oral administration or intravenous administration.

A solution of 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl) in acetonitrile was proposed as a derivatization reagent. This reagent is commercially available. In aqueous solutions and organic solvents it reacts quickly with primary and secondary amino groups, and in alkaline environment reacts with phenolic hydroxyls and sulfhydryl groups. Wherein stable chromophores/fluorophores groups with a maximum absorption in the visible range from 425 to 476 nm are formed. 1,4-dithioerythritol was used as the reducing reagent.

The studies were carried out using a high-performance liquid chromatograph which includes a high-pressure pump («Shimadzu» LC-20AT Prominence), a spectrophotometric detector («Shimadzu» SPD-20A Prominence), a manual injector ("Rheodyne" 7725i). Data processing was carried out using software (Ampersand, Multichrome version 3.0).

Chromatography of the standard and the plasma extract was carried out by two ways: 1) on a Chromolith Performance RP-18e column, 100 × 4.6 mm, detection at 425 nm, eluant is acetonitrile-0.05 M phosphate buffer (pH=8.0; v/v=18:82), flow rate is 2000 μl / min, pressure is 40 bars; 2) on a Gemini (Phenomenex) column 50×4.6 mm, C18, 5 μm, detection at 425 nm, eluent is acetonitrile - 0.01 M phosphate buffer (pH=7.0; v/v=20: 80), a flow rate is 1000 μl / min, a pressure is 35 bar.

The results of the chromatographic study showed that the polar aminothiols are eluted by one peak, the less polar derivative of the N-acetylcysteine is completely separated from them, as well as from the degradation product (NBD-OH). After intravenous administration of the drug, its concentration in the plasma is higher and the separation is realized on a short column in this case.

Thus, the developed chromatographic method for determining N-acetylcysteine in blood plasma after derivatization with 4-chloro-7-nitro-2,1,3-benzoxadiazole and subsequent HPLC analysis with UV detection is simple, well reproducible and sufficiently sensitive. The quantification limit (LOQ)  is 0.05 ng per injection.