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

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 λex  = 340 nm and λem  = 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.