Development of a new panel of 9 steroids in amniotic fluid in LC–MSMS

Background Since 1980 (Forest et al. [1] ) steroid analysis in amniotic fluid (AF) by radioimmunoassay were used in our laboratory as a tool for the prenatal diagnosis of 21 hydroxylase deficiency or other disorder of sex development (DSD). LC–MSMS became reference method for several reasons: analytical performance, possibility of studying a panel of several steroids. Objective Establishment of reference values for 21-deoxycortisol (21DF), deoxycorticosterone (DOC), corticosterone (Cortico), 17-hydroxyprogesterone (17OHP), 11-deoxycortisol (CS), dehydroepiandrosterone (DHEA), delta4-androstenedione (D4), testosterone (T) and dihydrotestosterone (DHT) in AF by LC–MS/MS method according to sex and term of pregnancy. Method Liquid chromatography was performed on an Agilent Technologies UPLC 1290 Infinity and selected steroids were analyzed with an Agilent Technologies 6460 triple quadrupole. Internal standard were added in 1 mL of the sample before supported liquid extraction. Materials After informed consent, we included 193 pregnant women AF performed from 14 to 36 weeks of amenorrhea, for increased maternal serum markers. All the samples associated with fetus ultrasound morphological, karyotypic or neonatal abnormalities were excluded. Results We validate the method allowing quantification of the 9 steroids in amniotic fluid with variability coefficient less than 10% intra- and inter-assay. The average concentration of these steroids was similar in both sex for 21DF( p Conclusion LC–MS/MS is a rapid, sensitive method for steroid analysis in AF. References values will be very useful for the management of a mismatch between external genitalia observed by ultrasound and karyotype.