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.

Routine method for the simultaneous quantification of 17alpha-hydroxyprogesterone, testosterone, dehydroepiandrosterone, androstenedione, cortisol, and pregnenolone in human serum of neonates using gas chromatography-mass spectrometry.

Steroid determination by immunoassays results in significant interferences and inaccurate results. This study describes the development and validation of a new gas chromatographic-mass spectrometric method for the simultaneous quantification of 17alpha-hydroxyprogesterone (17alphaOHP), testosterone (T), dehydroepiandrosterone (DHEA), androstenedione (Delta4-A), cortisol (F) and pregnenolone (Preg) in serum of neonates. Steroids were extracted and purified from 0.5 mL serum using diethyl ether and Extrelut mini NT1 column. The extracts were derivatized with N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA)/trimethylsilyl iodide (TMSI)/dithioerythritol (DTE) and the resulting trimethylsilyl derivatives were quantified by gas chromatography-selected ion monitoring-mass spectrometry (GC-SIM-MS). The detection limit for all steroids was lower than 0.1 ng/mL. The limit of quantification was 0.1 ng/mL for all steroids except cortisol which was at 0.25 ng/mL. d3-Testosterone and methyltestosterone served as internal standards. Precision for all compounds at the concentrations of 0.5, 1, 5 and 10 ng/mL (n = 10) in fortified steroid-free serum samples ranged from 0.8% to 16.6%. Accuracy was calculated at the concentrations of 0.5, 1, 5 and 10 ng/mL and ranged from -9.2% to 10.6% (n = 10). Linear calibration equations were obtained for all five steroids (0.125-31.25 ng/mL) and for cortisol (0.125-200 ng/mL). Relative recoveries at concentrations 1.0 and 12.5 ng/mL ranged from 70.5% to 97.5%. Absolute recoveries at the same concentrations ranged from 73.2% to 96.6%. Reference intervals were estimated for infants aged from 9 to 40 days. The proposed steroid profile is suitable for routine analysis and provides meaningful data for samples within normal range as well as those with elevated levels.

A study of the expression of the xenobiotic-metabolising cytochrome P450 proteins and of testosterone metabolism in bovine liver.

The expression of xenobiotic-metabolising cytochrome P450 proteins in the liver of cattle was determined using substrate probes and immunologically by Western blot analysis. Compared to the rat, cattle displayed much higher coumarin 7-hydroxylase (CYP2A) and ethoxyresorufin O-deethylase (CYP1) activity but, in contrast, it exhibited much lower debrisoquine 4-hydroxylase (CYP2D) and lauric acid hydroxylase activities (CYP4A). The ethoxyresorufin O-deethylase activity was markedly inhibited by furafylline and a-naphthoflavone, and coumarin 7-hydroxylase by 8-methoxypsoralen. Immunoblot analysis employing antibodies to rat CYP1A1 recognised two immunorelated proteins in bovine liver whose expression appeared to be higher compared with rat. Kinetic studies indicated that a single enzyme is likely to be responsible for the O-deethylation of 7-ethoxyresorufin in bovine liver. When bovine microsomes were probed with antibodies to rat CYP2A2, a single protein was detected in cattle liver. Kinetic analysis followed by construction of Eadie-Hofstee plots indicated that more than one enzyme contributes to the 7-hydroxylation of coumarin. Immunoblot analysis employing antibodies to human CYP2D6 and rat CYP4A1 revealed in both cases a single, poorly expressed immunoreacting band in bovine microsomes. Similar immunoblot studies detected proteins in cattle liver immunorelated to the CYP2B, CYP2C, CYP2E, and CYP3A subfamilies. Bovine microsomes metabolised testosterone but, in contrast to the rat, failed to produce 2alpha- and 16alpha-hydroxytestosterone. On the other hand, bovine microsomes produced levels of another hydroxylated metabolite, possibly 12-hydroxytestosterone. In conclusion, results emanating from this study indicate the presence of proteins in the cattle liver belonging to all the xenobiotic-metabolising families of cytochrome P450.