Determination of 17α‐hydroxyprogesterone in serum by liquid chromatography‐tandem mass spectrometry and immunoassay

Abstract

17α‐hydroxyprogesterone (17OHP) is the most important serum marker for congenital adrenal hyperplasia (CAH). 17OHP is usually measured by immunoassay but its detection by mass spectrometry (MS) is a potentially superior method. An LC‐MS (liquid chromatography‐mass spectrometry) method was developed which utilizes 0.5 ml serum spiked with 6‐α‐methylprednisolone (6‐MP) or deuterated 17OHP (d8‐IS) as the internal standard. The samples were extracted with ether/ethylacetate, and the extract was evaporated to dryness and analysed by LC‐MS/MS operating in the positive mode after separation on a reversed‐phase C18 column. The calibration curves for analysis of serum 17OHP exhibited consistent linearity and reproducibility in the range of 5–250 nmol/l. Interassay CVs were 8.5 and 9.2% at mean concentrations of 7.9 and 23 nmol/l, respectively. The detection limit was 1 nmol/l (signal‐to‐noise ratio = 3). The mean recovery of 17OHP added to serum ranged from 76 to 89% and that of internal standards from 75 to 82%. The regression equation for the LC‐MS/MS (x) and in‐house radioimmunoassay (RIA) (y) methods was: y = 0.87x+0.26 (r = 0.97; n = 100) and for a commercial RIA it was: y = 1.32x+0.02 (r = 0.97; n = 26).

• Congenital adrenal hyperplasia
• LC‐MS
• 17‐hydroxyprogesterone
• 21‐hydroxylase deficiency