Determination of 17-hydroxyprogesterone in plasma by stable isotope dilution/benchtop liquid chromatography-tandem mass spectrometry.
An assay based on stable isotope dilution liquid chromatography-tandem mass spectrometry (ID/LC-MS-MS) was developed for the quantification of 17-hydroxyprogesterone, the most important indicator of 21-hydroxylase deficiency in human plasma. Plasma was extracted using ethyl acetate and Extrelut columns. LC was performed on a reversed-phase C18 column using a water/methanol gradient. A benchtop triple quadrupole mass spectrometer, operating in selected reaction monitoring mode, served as mass detector. The analytical run time was 9 min per sample. The sensitivity was high: 0.06 pmol of 17-hydroxyprogesterone yielded a signal-to-noise ratio of 13. Precision (CV) and accuracy (relative error) derived from the analyses of unspiked and spiked validation samples were 7.4-12.0% and 6.4%, respectively. When analyzing the same samples - median (range), in nanomoles per liter - from neonates and adults independently by ID/LC-MS-MS as well as by ID/gas chromatography (GC)-MS, corresponding results were obtained: neonates (n = 10), ID/LC-MS-MS 3.99 (0.48-16.05), ID/GC-MS 5.39 (1.57-13.02); adults (n = 10), ID/LC-MS-MS 2.66 (1.39-6.15), ID/GC-MS 2.54 (0.51-5.12). The technique permitted reliable detection of classical and nonclassical forms of 21- hydroxylase deficiency. The much simpler sample preparation, the faster analytical run time and the operational ease possible with ID/LC-MS-MS permit a considerable increase of sample testing per day without compromising on analytical sensitivity and specificity. We expect that benchtop tandem mass spectrometry will open new avenues in clinical steroid analysis.
Wudy
S A
SA
Steroid Laboratory, Department of Pediatrics, University of Ulm, Germany. stefan.wudy@medizin.uni-ulm.de
Hartmann
M
M
Svoboda
M
M
D019326:17-alpha-Hydroxyprogesterone / Q000097:blood*; D000312:Adrenal Hyperplasia, Congenital; D000328:Adult; D002853:Chromatography, Liquid / Q000379:methods*; D003903:Deuterium; D005260:Female; D006801:Humans; D007201:Indicator Dilution Techniques; D007231:Infant, Newborn; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D012016:Reference Values; D012680:Sensitivity and Specificity
D019326:17-alpha-Hydroxyprogesterone; D003903:Deuterium