Perfluorosulfonated Ionomer-Modified Polyethylene. A Material for Simultaneous Solid-Phase Enrichment and Enhanced Precolumn Dansylation of C-21 Ketosteroids in Human Serum

Abstract

A new derivatization procedure has been developed where solid-phase catalysis is utilized to facilitate the formation of hydrazones in precolumn labeling 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 dansylhydrazine. The SPEED devices have been optimized using experimental design and characterized for dansylation of C-21 ketosteroids by multivariate data analysis, using progesterone as the model compound. The reaction temperature and the molar ratio between the steroid and the derivatization reagent were found to be the factors most strongly affecting the reaction. Faster reaction kinetics were achieved when the molar ratio between dansylhydrazine and the steroid was increased. Mass spectroscopic analysis showed that the four derivative peaks eluting when derivatized progesterone was separated on an octadecyl silica stationary phase were due to the syn and anti mono- and bis(hydrazones) formed in the reaction. Using optimal reaction conditions, the derivatives mainly constitute the syn and anti conformers of bis-derivatives. In contrast to solution-based acid catalysis, the SPEED device was remarkably insensitive to water in the reaction mixture. A sample volume of 400 microL was found to be the maximum, enabling sample enrichment prior derivatization. Using optimal experimental conditions, picomole amounts of ketosteroids could be derivatized in 10 min at room temperature. Analysis of spiked serum samples containing 0.4-2.0 nmol of progesterone showed overall recoveries of 52-63%. The corresponding 3delta detection limit was 1.3 pmol (n = 4, 100 microL injected), as estimated from calibration curve data.

Development of High-Performance Chemical Isotope Labeling LC–MS for Profiling the Carbonyl Submetabolome

“…Metabolites containing carbonyl group (ketones and aldehydes) include several important classes of molecules that often have low ionization efficiency (e.g., sugars) and/or are present at very low concentrations (e.g., steroids in plasma) . GC/MS methods have been used for analyzing ketones and aldehydes, − while a number of derivatization chemistries − including dansylhydrazine labeling − have been reported for labeling ketones/aldehydes to improve MS detectability; however, these methods were often used for the analysis of a limited number of analytes. In this report, we present a method using 12 C-/ 13 C-dansylhydrazine labeling for analyzing the carbonyl submetabolome with high coverage and high accuracy and precision of relative quantification.…”