Perfluorosulfonated ionomer-modified polyethylene. A material for simultaneous solid-phase enrichment and enhanced precolumn dansylation of C-21 ketosteroids in human serum

A new derivatization procedure has been developed where solid-phase catalys is is utilized to facilitate the formation of hydrazones in precolumn label ing of keto-containing compounds. This procedure has been implemented on a solid-phase enrichment and enhanced derivatization (SPEED) device, prepared from porous polyethylene that has been coated with Nafion and dansylhydraz ine. The SPEED devices have been optimized using experimental design and ch aracterized for dansylation of C-21 ketosteroids by multivariate data analy sis, using progesterone as the model compound. The reaction temperature and the molar ratio between the steroid and the derivatization reagent were fo und to be the factors most strongly affecting the reaction. Faster reaction kinetics were achieved when the molar ratio between dansylhydrazine and th e steroid was increased. Mass spectroscopic analysis showed that the four d erivative peaks eluting when derivatized progesterone was separated on an o ctadecyl silica stationary phase were due to the syn and anti mono- and bis - (hydrazones) formed in the reaction. Using optimal reaction conditions, t he derivatives mainly constitute the syn and anti conformers of bis-derivat ives. In contrast to solution-based acid catalysis, the SPEED device was re markably insensitive to water in the reaction mixture. A sample volume of 4 00 muL was found to be the maximum, enabling sample enrichment prior deriva tization. Using optimal experimental conditions, picomole amounts of ketost eroids could be derivatized in 10 min at room temperature. Analysis of spik ed serum samples containing 0.4-2.0 nmol of progesterone showed overall rec overies of 52-63%. The corresponding 3 sigma detection limit was 1.3 pmol ( n = 4, 100 muL injected), as estimated from calibration curve data.