The determination of pharmaceutically active thiols using hydrophilic interaction chromatography followed postcolumn derivatization with o-phthaldialdehyde and fluorescence detection

Abstract

A rapid, precise and specific hydrophilic interaction chromatography (HILIC) combined with postcolumn derivatization using o-phthaldialdehyde and fluorescence detection was developed and validated for the determination of selected pharmaceutically active thiols. The analysis was carried out on a Diol HILIC column using a mobile phase consisting of acetonitrile and solution of 10 mmol/L citric acid adjusted with 1-propylamine to pH 5.5 in ratio 75:25 (v/v) for separation of cysteine and homocysteine and in ratio 85:15 (v/v) for separation of N-acetyl-l-cysteine and captopril. The postcolumn derivatization reaction was performed at room temperature using reagent (5 mmol/L OPA in 0.05 mol/L 4- (2-hydroxyethyl) piperazine-1-ethanesulfonic acid at pH 7) delivered at the flow rate of 0.3 mL/min. Fluorescence detection was carried out at excitation and emission wavelength of 345 nm and 450 nm, respectively. The effect of chromatographic conditions including acetonitrile content, salt concentration in the mobile phase and mobile phase pH on the retention of tested thiols was investigated. The postcolumn reaction conditions such as reaction temperature, derivatization reagent flow rate, o-phthaldialdehyde concentration and derivatization reagent pH were deeply studied. The developed method was validated in terms of linearity, accuracy, precision and selectivity according to the International Conference on Harmonisation guidelines. The HILIC method was successfully applied for the analysis of commercially available samples of pharmaceutically active thiols such as captopril, N-acetyl-l-cysteine (NAC) and cysteine.

Keywords: Fluorescence detection; HILIC; Pharmaceutically active thiols; Postcolumn derivatization; o-phthaldialdehyde.