Sensitized lanthanide fluorescence detection of steroidal hormones

Several steroidal hormones with an alpha,beta-unsaturated carbonyl group sensitized the fluorescence of terbium(III) ions: medroxyprogesterone, medroxyprogesterone acetate, methyltestosterone, nandrolone, nandrolone decanoate, progesterone, testosterone, testosterone enanthate, and testosterone propionate, but other steroids which have this chromophore did not give any signal: clostebol acetate, dehydrotestosterone, dydrogesterone, methandienone, and methenolone enanthate. Sensitized fluorescence was used to detect the steroids when eluted with micellar mobile phases of sodium dodecyl sulphate (SDS)-acetonitrile and SDS-pentanol containing 0.01 M Tb(III), with 245 nm and 545 nm as excitation and emission wavelengths, respectively. The micelles favour the fluorescence process due to proximity of donor and acceptor, and shielding of the Tb(III) ions from the aqueous medium. Signal intensity increased linearly with the concentration of Tb(III), but decreased with acetonitrile and pentanol. The concentration of organic solvent in the mobile phase should, therefore, be limited. Mobile phases of 0.075 M SDS-12.4% acetonitrile and 0.13 M SDS-4.5% pentanol offered the highest sensitivities, but only pentanol permitted the elution of the most hydrophobic steroids at adequate retention times. With this mobile phase, the limits of detection were in the 0.07-0.2 mu g/mL range. The sensitivities did not change in the 25-60 degrees C range, but the efficiencies of the chromatographic peaks were larger at higher temperature, and consequently, the resolution was improved.