Identification of the steroids in neonatal plasma that interfere with 17 alpha-hydroxyprogesterone radioimmunoassays

Neonatal plasma contains interferents that increase the apparent 17 alpha-hydroxyprogesterone (17-OHP) content measured by direct (no-extraction) radioimmunoassay. We fractionated extracts from neonatal plasma pools by liquid chromatography with a Sephadex LH-20 column and measured 17-OHP immunoreactivity by a direct test kit. We found immunoreactivity in the free steroid and glucuronide fraction and also in the monosulfate fraction. We analyzed these two fractions by reversed-phase high-performance liquid chromatography (HPLC) and HPLC-mass spectrometry. We collected fractions and assayed for 17-OHP immunoreactivity. The HPLC fractions containing the interfering steroid monosulfates were analyzed by ion-spray mass spectrometry and, after solvolysis, by gas chromatography-mass spectrometry. Several monosulfates were identified, including those of 17 alpha-hydroxy-pregnenolone, 16 alpha-hydroxypregnenolone, pregnenolone, and several pregnenetriols. 17 alpha-Hydroxypregnenolone sulfate was the most significant interferent. Other commercially available 17-OHP assays showed similar interference when used without an extraction step. Kit manufacturers should select antibodies and protocols to minimize cross-reaction with sulfates, especially 17 alpha-hydroxypregnenolone sulfate.