Pyrene sulphonyl chloride as a reagent for quantitation of oestrogens in human serum using HPLC with conventional and...

A highly sensitive HPLC method with conventional and laser-induced fluorescence detection for the analyses of oestrogens is described. beta-oestradiol (beta-ES) was chosen as a model and derivatized with pyrenesulphonyl chloride (PSCL), a novel derivatizing reagent, using a two-phase system with tetrapentylammonium bromide (TPABR) as a phase transfer catalyst. Derivatization was complete in 30 s at room temperature and concentrations as low as 5 x 10(-10) M could be derivatized and detected. The concentration detection limit PSCL derivatized beta-ES was 2 x 10(-11) M which corresponds to an on-column detection limit of one femtomole using conventional chromatography and detection. The relative standard deviation (n = 6) of the derivatization carried out at 2.5 x 10(-9) M was 4%. No significant loss of peak-height of the derivative was observed at room temperature over 24 h period. Baseline resolution of PSCL derivatized oestrone, equilin and beta-oestradiol was achieved with ACN:H2O (75:25 v/v) using a Bondclone C-18 column. The method described here is sufficiently sensitive for analyses of beta-oestradiol at low pg/mL concentrations in 1 mL of human serum. The PSCL derivatives of oestrogens demonstrate excellent potential for excitation by laser-induced fluorescence using the 351 nm line of an Ar ion laser or the 325 nm line of He-Cd laser. A concentration limit of detection of 4 x 10(-12) M for beta-oestradiol was achieved using the 325 nm line of an He-Cd laser. This corresponds to 0.2 fmol of derivatized beta-oestradiol on-column.