Determination of C-21 Ketosteroids in Serum Using Trifluoromethanesulfonic Acid Catalyzed Precolumn Dansylation and 1,1‘-Oxalyldiimidazole Postcolumn Peroxyoxalate Chemiluminescence Detection

Abstract

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 3 alpha-hydroxy-5 beta-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 3 sigma detection limit estimated from 3 alpha-hydroxy-5 beta-pregnan-20-one calibration curve data was 1.6 pmol (n = 4, 125 microL injected) and spiked serum containing 0-74 pmol of this compound showed overall recoveries of 73 +/- 9% (n = 12). Quantitation of 3 alpha-hydroxy-5 beta-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.

Kinetic studies on the peroxyoxalate chemiluminescence reaction: determination of the cyclization rate constant

“…The increasing interest in the peroxyoxalate system is due to its high analytical applicability as a sensitive tool for quantifying numerous analytes (19)(20)(21)(22). In order to optimize analytical conditions and sensitivity, a better understanding of the mechanism of the steps before chemiexcitation is crucial.…”

Detection of synthetic corticosteroids in bovine urine by chemiluminescence high‐performance liquid chromatography

“…Chemiluminiscence (CL) has been proposed by other workers (10)(11)(12)(13) for the detection of corticosteroids, but in most of those cases prior derivatization with different compounds is mandatory. In previous works, the CL reaction produced by natural and synthetic corticosteroids when reacting with luminol in the presence of a catalyst [hexacyanoferrate (III)] in alkaline solution was studied under both static (6) and dynamic (7,14) conditions.…”

Chemiluminescence detection of nine corticosteroids in liver

“…17 Chemiluminescence has been proposed by other workers. [14][15][16][17][18] for the detection of corticosteroids, but in those cases the corticosteroids were previously derivatized with different compounds. In other work, 16 the chemiluminescence detection system used luminol in the presence of a catalyst [hexacyanoferrate(III) in alkaline solution].…”

“…Several methods have been developed for the determination of corticosteroids in different matrices e.g., enzyme immunoassay, 5,6 thin-layer chromatography, [3][4][5][6][7] HPLC-diode array detection, 8,9 HPLC-atmospheric pressure chemical ionization mass spectrometry, 10,11 gas chromatography-mass spectrometry, 12 tandem mass spectrometry (MS). 13 Chemiluminescence methods 14,15 for sensitive and selective detection are available for liquid chromatography, in which the light is formed through a chemical reaction and emitted against an ideally dark background; such methods have been applied to corticosteroid detection. However, these methods use derivatization procedures for each corticosteroid, whereas in the method proposed here no derivatization procedure is needed.…”