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Determination of C-21 ketosteroids in serum using trifluoromethanesulfonic acid catalyzed precolumn dansylation and 1,1’-oxalyldiimidazole postcolumn peroxyoxalate chemiluminescence detection
Umeå University, Faculty of Medicine, Department of Clinical Sciences. Umeå University, Faculty of Science and Technology, Department of Chemistry.
Umeå University, Faculty of Science and Technology, Department of Chemistry.
Umeå University, Faculty of Medicine, Department of Clinical Sciences, Obstetrics and Gynaecology.
Umeå University, Faculty of Science and Technology, Department of Chemistry.
1998 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 70, no 23, 5002-5009 p.Article in journal (Refereed) Published
Abstract [en]

A new procedure for the quantitation of C-21 ketosteroids using trifluoromethanesulfonic acid-catalyzed precolumn dansylation and coupled column liquid chromatographic separation, followed by postcolumn 1,1‘-oxalyldiimidazole peroxyoxalate chemiluminescence detection is presented. In the simultaneous optimization of chromatographic resolution and chemiluminescence intensity, a coupled column chromatographic system and a stopped-flow system were used. An eluent containing 20 mM phosphate buffer at pH 6.7 accomplished an efficient separation of 3α-hydroxy-5β-pregnan-20-one from a mixture containing 10 C-21 ketosteroids. Phosphate buffer also proved to be the most advantageous, among the six buffers tested, for sensitive detection. Experimental design and multivariate data analysis were used to characterize and optimize the postcolumn reaction chemistry in the chromatographic system. A valid full factorial design with excellent predictability showed that the flow rates for both 1,1‘-oxalyldiimidazole and hydrogen peroxide were the factors most strongly affecting the sensitivity of the system. The theoretical plate numbers were above 11 000 for all 10 dansylated ketosteroids. The 3σ detection limit estimated from 3α-hydroxy-5β-pregnan-20-one calibration curve data was 1.6 pmol (n = 4, 125 μL injected) and spiked serum containing 0−74 pmol of this compound showed overall recoveries of 73 ± 9% (n = 12). Quantitation of 3α-hydroxy-5β-pregnan-20-one was finally carried out on 45 serum samples and the results compared to those from a radioimmunoassay (RIA) method. The data acquired with the procedure described in this work compare well with the results from RIA, which confirms the reliability of the new analytical procedure.

Place, publisher, year, edition, pages
Washington: American Chemical Society (ACS), 1998. Vol. 70, no 23, 5002-5009 p.
Keyword [en]
performance liquid chromatography, bile-acids, steroids, reagent, system
National Category
Analytical Chemistry
URN: urn:nbn:se:umu:diva-3055DOI: 10.1021/ac980511sISI: 000077278700031OAI: diva2:141500
Available from: 2003-01-24 Created: 2003-01-24 Last updated: 2016-08-25Bibliographically approved
In thesis
1. Peroxyoxalate Chemiluminescence for Miniaturized Analytical Flow Systems
Open this publication in new window or tab >>Peroxyoxalate Chemiluminescence for Miniaturized Analytical Flow Systems
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis deals with the peroxyoxalate chemiluminescence (POCL) reaction and its application as a detection technique in flow systems for chemical analysis. Particularly, miniaturized flow systems aimed for separation of molecules. In such systems, a high light intensity and a rapid development of the emission are the desired reaction characteristics, for reasons discussed in this text. The work tries to develop an understanding of the chemical processes involved in POCL, with special emphasis to the species favoring or hindering a rapid light evolution. Hence, is the focus placed on the nature of catalysis and the desired properties of substances acting as catalysts in this reaction. Consequently, the scientific papers on which this work is founded includes both systematic stopped-flow studies of catalyst candidates and of the causes for diminished light emission. In addition, multivariate strategies for reaction optimization in practical analysis situations are treated, and the application of the POCL technique to detection of serum-extracted neuroactive steroids, derivatized with fluorescent moieties, is presented.

From the experiments in this thesis it is clear nucleophilic catalysts are the most efficient enhancing compounds, which means that they must possess a carefully balanced characteristics of nucleophilicity, leaving group ability, and basicity. The investigations also conclude that the feature of basicity efficiently can be delegated to a non-nucleophilic co-catalyst, which allow the use of nucleophilic catalysts that need to be deprotonated to be active. This thesis also shows the importance of minimizing the amount of competing nucleophiles at the site of reaction to maintain the emission. This implies that also solvents and buffer substances should be carefully chosen not to interfere with the emission process.

The most promising combination of catalysts found in this work was 4,5- dichloroimidazole together with 1,2,2,6,6-pentamethylpiperidne. This arrangement was capable of speeding the reaction more than tenfold while increasing the maximum emission intensity by about the same factor.

37 p.
peroxyoxalate, chemiluminescence, nucleophilic catalysis, non-nucleophilic base, imidazole, triazole, aminopyridine, miniaturized, flow system, capillary electrophoresis, multivariate
Research subject
Analytical Chemistry
urn:nbn:se:umu:diva-16 (URN)91-7305-377-5 (ISBN)
Public defence
2003-01-24, 00:00
Available from: 2003-01-24 Created: 2003-01-24 Last updated: 2012-06-05Bibliographically approved

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Appelblad, PatrikJonsson, TobiasBäckström, TorbjörnIrgum, Knut
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