Einar Pontén

Derivatization of Steroids with Dansylhydrazine Using Trifluoromethanesulfonic Acid as Catalyst

A new dansylation reaction, where trifluoromethanesulfonic acid is used as catalyst, has been characterized for six ketosteroids by employing experimental design followed by multivariate data analysis. The molar ratio between the steroid and the derivatization reagent was found to be the factor most strongly affecting the reaction. Faster reaction kinetics was achieved when the molar ratio between dansylhydrazine and the steroid was increased. Mass spectroscopic analysis showed that the dual peaks observed when derivatized progesterone was separated on an octadecyl silica stationary phase were due to the syn and anti hydrazones formed. We furthermore conclude that the dansylation reaction is subject to alkyl catalysis rather than acid catalysis, since methyl trifluoromethanesulfonate showed a strong catalytic action, while the catalytic action of trifluoromethanesulfonic acid was lower when diluted in other alcohols and disappeared in aprotic solvents. A sensitivity to water in the reaction mixture strengthens the evidence for alkyl catalysis. When optimal experimental conditions were used, derivatization of picomole amounts of ketosteroids could be accomplished in 25 min. Analysis of spiked plasma containing 0.2-2.0 nmol each of progesterone and 3α-hydroxy-5β-pregnan-20-one showed overall recoveries of 69-76% and 40-55%, respectively. The corresponding 3σ detection limits estimated from calibration curve data were 12 and 15 pmol (n = 4, 500 μL injected).

Trifluoromethanesulfonic acid as a catalyst for the formation of dansylhydrazone derivatives

Abstract Trifluoromethanesulfonic acid (TFMSA) is presented as a new, efficient catalyst in the pre-column fluorescent derivatization of the 3-ketosteroid budesonide with dansylhydrazine and compared to the commonly used catalyst trifluoroacetic acid. With TFMSA the derivatization reaction may be carried out at room temperature, with a considerably higher reaction rate compared to previously used acids. The chromatograms also show that TMFSA results in less formation of spurious peaks from the reagent. Derivatization of steroid solutions ranging from 0.5 to 64 μM could be performed using identical reaction conditions.

Non-porous sphérical polymér particlés for luminéscént solid phase detection reactors

Abstract Support materials for analytical solid phase detection applications have been prepared. A method for synthesis and functionalization of non-porous particles based on suspension co-polymerization of ethylene dimethacrylate (EDMA) and 2-hydroxyethyl methacrylate (HEMA) is reported. Attachment of the functionalities 5-amino-8-hydroxyquinoline, and the fluorescent 3-aminofluoranthene, as well as the chemiluminescent isoluminol were accomplished by reeaction of the HEMA hydroxyl groups with epichlorhydrin and subsequent attachment of the amino groups to thee oxirane group thus introduced. Calcein and Calcein Blue were immobilized through esterification to the HEMA hydroxyl group of the carrier bead. The solid phase reeagents were verified qualitatively by colour changes, fluorescence for the fluorophores, and photon emission for the isoluminol deerivative in presence of chlorine dioxide. The particles are intendeed for analytical solid phase deetection and mechanistic studies of luminesceence phenomena.