Use of stop-flow oxalate ester chemiluminescence as a means to determine conditions for high-performance liquid chromatography chemiluminescence detection of retinoids using normal-phase chromatography

Abstract

Stop-flow chemiluminescence (CL) has been used to determine the conditions necessary for the oxalate ester CL detection of selected retinoids after separation by normal-phase HPLC. Also, the detection of the selected retinoids by bis(2,4,6-trichlorophenyl) oxalate (TCPO)-hydrogen peroxide (H2O2) chemiluminescence and fluorescence (FL) are compared. Stop-flow CL was performed on a prototype unit from High-Tech Scientific, Ltd (Salisbury, UK). Detection limits were determined for retinyl palmitate, retinyl acetate, retinol, etretinate, acitretin and tretinoin, after separation on a YMC PVA-sil, 25 × cm 4.6 mm column using hexane-tetrahydrofuran-acetic acid (75:25:0.01, v/v/v) as eluent at 1.5 ml min−1. A Schoeffel 970 detector was used for both CL and FL detection. Detection by FL was determined with γex 355 nm and λem > 418 nm. CL detection was performed with the deuterium lamp off and no emission filter. CL was induced by mixing the reagents post-column. Pump A — hexane-THF-AcOH (75:25:0.01, v/v/v) at 1.5 ml min−1 pump B — 70 mM TCPO in THF at 0.5 ml min−1 pump C — THF-50% aq H2O2 (75:25, v/v) 1.0 mg ml−1 imidazole at 1.0 ml−1 imidazole at 1.0 ml min−1.