Liquid chromatographic determination of oestradiol in serum by pre-column derivatization with dansyl chloride or laryl chloride and peroxyoxalate chemiluminescence detection.

Column liquid chromatography with chemiluminescence detection is used to determine oestradiol in serum. Oestradiol is labelled by means of a two-phase derivatization procedure with either dansyl chloride or laryl chloride. After derivatization the excess reagent is removed on a short amino-bonded column: the sulphonyl chloride functional group reacts with the amino groups causing removal of the label; the oestradiol derivatives are not retained. Chromatography of oestradiol derivatives is performed with methanol-water mixtures as eluent. Chemical excitation is carried out by adding bis(2-nitrophenyl)oxalate and hydrogen peroxide dissolved in acetonitrile to the column eluate. Linearity is observed (r = 0.9998; n = 7) over at least three decades (10(-6)-10(-9) M) for both dansyl chloride and laryl chloride. A detection limit of 5 x 10(-10) M (50 fmol injected) has been achieved and the dansyl derivatization method has been applied to the determination of oestradiol in spiked serum.