Determination of 17alpha-hydroxypregnenolone in human plasma by routine isotope dilution mass spectrometry using benchtop gas chromatography-mass selective detection.
A first assay based on stable isotope dilution/gas chromatography-mass spectrometry has been developed for plasma 17alpha-hydroxypregnenolone, the leading marker of 3beta-hydroxysteroid dehydrogenase deficiency. Equilibration of plasma with internal standard was followed by solid phase extraction. After clean up using Sephadex LH-20 mini columns, heptafluorobutyrates were prepared as derivatives. Quantification was achieved by selected ion monitoring of m/z 467.0 (analyte) and m/z 471.0 (internal standard). 0.030 pmol of 17alpha-hydroxypregnenolone gave a signal to noise ratio of 3.7. Calibration plot was linear. Spiking experiments showed good accuracy with relative errors < 3.7%. Intraassay precision CV was 8.3% and interassay precision CV was 5.6%. Requiring small amounts of plasma, the rapid, convenient work up and the application of bench top GC/MS instrumentation proved our method suitable for routine clinical use in adults and children.
Wudy
S A
SA
Steroid Laboratory, Department of Pediatrics, University of Ulm Prittwitzstr. 43, D-89070, Ulm/Donau, Germany. stefan.wudy@paediat.med.uni-giessen.de
Hartmann
M
M
Solleder
C
C
Homoki
J
J
D006907:17-alpha-Hydroxypregnenolone / Q000097:blood*; D015096:3-Hydroxysteroid Dehydrogenases / Q000172:deficiency; D000293:Adolescent; D000328:Adult; D002648:Child; D002675:Child, Preschool; D005260:Female; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D007553:Isotope Labeling; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*
D006907:17-alpha-Hydroxypregnenolone; D015096:3-Hydroxysteroid Dehydrogenases