Simultaneous quantification of 17α-OH progesterone, 11-deoxycortisol, Δ4-androstenedione, cortisol and cortisone in newborn blood spots using liquid chromatography-tandem mass spectrometry.

Adrenal steroid profiling, including 17α-OH progesterone (17OHP), 11-deoxycortisol (S), Δ4-androstenedione (Δ4-A) and cortisol (F) in blood spots by tandem mass spectrometry, is used for newborn screening to detect congenital adrenal hyperplasia (CAH). Pre-analytical sample processing is critical for assay specificity and accuracy; however, it is laborious and time-consuming. This study describes the development and validation of a new Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) method for the simultaneous quantification of five steroids: 17OHP, S, Δ4-A, F and cortisone (E) in blood spots from newborns. Whole blood was eluted from a 5.00 mm dried blood spot by an aqueous solution containing the deuterium-labeled internal standards d8-17OHP and d4-cortisol. The steroids extracted from blood spot into aqueous solution were subsequently purified via Extelut mini NT1 column using diethylether. The extracts were evaporated and quantified using LC-MS/MS. The detection limit was 0.25 ng/mL for 17OHP and S, 0.4 ng/mL for Δ4-A and 0.5 ng/mL for F and E. The limit of quantification was 0.5 ng/mL for 17OHP, S and Δ4-A and 1 ng/mL for F and E. Precision for 17OHP, S, Δ4-A at concentrations of 0.5, 2, and 8 ng/mL (n=5) in fortified steroid free serum samples was 1.3-3.5% (intra-assay CV) and 7-14.8% (inter-assay CV). Precision for F and E at concentrations of 5 and 20 ng/mL was 1.5-4.8% (intra-assay, CV%) and 6-15% (inter-assay, CV%). Accuracy was calculated at concentrations of 0.5, 2, and 8 ng/mL for 17OHP, S and Δ4-A and ranged from -0.3 to 0.2%, while for F and E it ranged from -3.2 to 0.2%. Relative recoveries at concentration 2 ng/mL and 8 ng/mL for 17OHP, S, Δ4-A and at 5 ng/mL and 20 ng/mL for F and E ranged from 55% to 80%. Reference intervals were estimated for all steroids in newborns (on day 3). The steroid profile assay herein described is sensitive, specific and accurate and involves a simple pre-analytical sample manipulation; it is therefore suitable for routine analysis and provides data for samples within normal range as well as those with elevated levels. For the first time to our knowledge, cortisone levels are reported in dried blood spots from newborns.

Isolation of a novel maturation-inducing steroid produced in vitro by ovaries of Atlantic croaker.

All the steroids produced by Atlantic croaker ovaries during final oocyte maturation (FOM) were assayed for their ability to induce germinal vesicle breakdown (GVBD) of croaker oocytes in vitro. Ovarian tissue in the process of FOM was removed from Atlantic croaker (Micropogonias undulatus) and incubated with human chorionic gonadotropin (hCG) and pregnenolone in tissue culture medium for 8 hr. Steroids were extracted from the medium and fractionated by HPLC and TLC. Fractions were bioassayed for their potency to induce GVBD of Atlantic croaker oocytes in vitro. 17 alpha, 20 beta,21-Trihydroxy-4-pregnen-3-one (20 beta-dihydro-11-deoxycortisol, 20 beta-S) was identified as the predominant steroid product and the major maturation-inducing steroid produced by the ovary of Atlantic croaker in vitro. A small amount of another steroid with equal potency to induce GVBD was tentatively identified as 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-P4); however, over ten times more 20 beta-S than 17 alpha,20 beta-P4 accumulated in the incubation medium. Trace amounts of testosterone and estradiol-17 beta were also isolated. Ovarian tissue incubated under more physiological conditions, without the addition of excess pregnenolone, failed to produce 17 alpha,20 beta-P4. These results provide further evidence that 20 beta-S is a major maturation-inducing steroid in Atlantic croaker.

Polarization fluoroimmunoassay of 11-deoxycortisol in serum and saliva.

We developed polarization fluoroimmunoassays for 11-deoxycortisol in serum and saliva. To avoid interfering factors, the steroid was initially extracted from the biological fluids with dichloromethane. Assays could then be completed without any further separation procedures or need to correct for blank signals. The serum assay was suitable for following the response to the metyrapone test and results correlated acceptably with those by an established, specific, direct 125I-radioimmunoassay. The method is not sufficiently sensitive to detect 11-deoxycortisol in normal saliva, but greatly increased concentrations were found in post-metyrapone saliva and results agreed well with those by the radioimmunoassay as modified for salivary assay. Salivary 11-deoxycortisol assay would provide a convenient means of monitoring results of the metyrapone test.

A convenient and efficient method for the extraction and fractionation of steroid hormones from serum or urine.

A convenient solid phase method for extracting and fractionating steroid hormones from serum or urine is presented. The influence of the solvent used for extraction, of the body fluid to be extracted, and of the degree of sample dilution on the efficiency of extraction was studied for progesterone and cortisol. The potency of fractionation was demonstrated by the separation of the steroid pairs, deoxycortisol--cortisol and oestradiol--oestriol, from a urine sample. Influence of ionic strength, pH and temperature on the reproducibility was assessed for deoxycortisol in undiluted urine. With respect to practicability and efficiency, this method has proved to be considerably superior to conventional liquid-liquid techniques.

Potential limitations of recrystallization for the definitive identification of radioactive steroids.

The usefulness of recrystallization in establishing the radiochemical purity of steroids is widely recognized, but the potential limitations of the technique have received little attention. The current study reports the failure of standard recrystallization procedures using methanol/water as the solvent pair to separate contaminating 14C-17-hydroxyprogesterone (17-hydroxy-4-pregnene-3, 20-dione) from 3H- and 14C-labeled 11-deoxycortisol (17,21-dihydroxy-4-pregnene-3,20-dione) despite ten serial crystallizations. The standard criteria of radiochemical purity were met despite gross impurity of the crystals as evidenced by thin layer chromatography. Thus, recrystallization may, under certain conditions, yield misleading results when employed as the only method for identifying radioactive steroids. These observations illustrate the importance of an optimal choice of solvent and crystallization conditions, and emphasize the need for confirmation by derivative formation and chromatography.

Preparation of 1,2-3H-21-deoxycortisol by incubation of 1,2-3H-17-hydroxyprogesterone with rat adrenal mitochondria.

A method is described for preparation of 1,2-3H-21-deoxycortisol by incubation of 1,2-3H-17-hydroxyprogesterone with rat adrenal mitoochondria. The radiochemical purity of the product was established by two subsequent chromatographies, reversed isotopic dilution and oxidation to 21-deoxycortisone. The final yield of 1,2-3H-21-deoxycortisol was approximately 15%.