Application of simultaneous separation and derivatization for the determination of α-lipoic acid in urine samples by high-performance liquid chromatography with spectrofluorimetric detection

Abstract

To help to clarify therapeutic functions of lipoic acid (LA) in biochemical and clinical practice we have elaborated a fast, simple and accurate HPLC method enabling determination of LA in human urine. The proposed analytical approach includes reduction of LA with tris(2-carboxyethyl)phosphine and simultaneous separation and derivatization of the analyte with butylamine and o-phthaldialdehyde followed by spectrofluorimetric detection at λ_ex = 340 nm and λ_em = 440 nm. The assay was performed using gradient elution and the mobile phase containing 0.0025 mol L^-1 o-phthaldialdehyde in 0.0025 mol L^-1 NaOH and acetonitrile. Linearity of the detector response for LA was observed in the range of 0.3-8 μmol L^-1 . Limits of detection and quantification for LA in urine samples were 0.02 and 0.03 μmol L^-1 , respectively. The total analysis time, including sample work-up, was <20 min. The analytical procedure was successfully applied to analysis of real urine samples delivered from six healthy volunteers who received a single 100 mg dose of LA.

Keywords: o-phthaldialdehyde; simultaneous separation and derivatization; urine sample; α-lipoic acid.