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.
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 uit 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 x 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 lambda ex 355 nm and lambda em greater than 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 min-1.
Bryan
P D
PD
Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Georgia, Athens 30602.
Capomacchia
A C
AC
D002851:Chromatography, High Pressure Liquid; D008163:Luminescent Measurements; D010070:Oxalates / Q000737:chemistry*; D012176:Retinoids / Q000032:analysis*; D012996:Solutions
D010070:Oxalates; D012176:Retinoids; D012996:Solutions