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.
Wong
T
T
Department of Clinical Biochemistry, Hospital for Sick Children, Toronto, ON, Canada.
Shackleton
C H
CH
Covey
T R
TR
Ellis
G
G
D019326:17-alpha-Hydroxyprogesterone; D000312:Adrenal Hyperplasia, Congenital / Q000097:blood; D002851:Chromatography, High Pressure Liquid; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D006908:Hydroxyprogesterones / Q000097:blood*; D007223:Infant; D007231:Infant, Newborn; D013058:Mass Spectrometry; D011863:Radioimmunoassay; D013256:Steroids / Q000097:blood*
D006908:Hydroxyprogesterones; D013256:Steroids; D019326:17-alpha-Hydroxyprogesterone