HPLC method for determination of fluorescence derivatives of cortisol, cortisone and their tetrahydro- and allo-tetrahydro-metabolites in biological fluids.

11Beta-hydroxysteroid dehydrogenase isoform 2 (11beta-HSD2) is responsible for conversion of cortisol (F) to inactive cortisone (E). Disturbance of its activity can cause hypertension. To estimate 11beta-HSD2 activity, besides F and E, their tetrahydro- (THF, THE) as well allo-tetrahydro- (allo-THF, allo-THE) metabolites should be determined. This study describes HPLC-FLD method for the quantitative determination of endogenous glucocorticoids (GCs) in plasma and urine (total and free) and their metabolites in urine. Following extraction at pH 7.4 using dichloromethane, GCs (F, E, THF, allo-THF, THE, allo-THE and internal standard--prednisolone) were derivatized with 9-anthroyl nitrile and purified by SPE using C(18) cartridges. The enzymatic hydrolysis of conjugated steroids was provided using beta-glucuronidase. The influence of organic bases on 9-AN derivatization of steroids was investigated. The best yield of the derivatization was obtained in presence of the mixture of 10.0% triethylamine (TEA) and 0.1% quinuclidine (Q). Chromatographic separation was accomplished in the Chromolith RP-18e monolithic column. The elaborated method was validated. Calibration curves were linear in the ranges: for F, E and THF 5.0-1000.0 ng mL(-1), for allo-THF and THE + allo-THE 10.0-1000.0 ng mL(-1). LOD (S/N=3:1) for all analytes amounted 3.0 ng mL(-1). Recoveries of GCs exceeded 90%. The method was precise and accurate, intra- and inter-day precision were 3.0-12.1% and 9.2-14.0%, respectively. Accuracy ranged from 0.2 to 15.1%. The method was applied for estimating endogenous GCs in plasma and urine. Plasma levels of F and E were in the ranges: 133.0-174.5 ng mL(-1) and 17.4-35.9 ng mL(-1), respectively. Free urinary steroids were in the ranges: 12.0-54.1 microg/24 h (UFF) and 37.8-76.2 microg/24 h (UFE). The ratio of (THF + allo-THF)/(THE + allo-THE) amounted from 1.01 to 1.23. The obtained results confirmed utility of the elaborated method in the assessment of 11beta-HSD2 activity in man.

Simultaneous determination of tetrahydrocortisol and tetrahydrocortisone in human plasma and urine by stable isotope dilution mass spectrometry.

A capillary gas chromatographic-mass spectrometric method for the simultaneous determination of tetrahydrocortisol (THF, 3alpha,11beta,17alpha,21-tetrahydroxy-5beta-preg nane-20-one), allo-tetrahydrocortisol (allo-THF, 3alpha,11beta,17alpha,21-tetrahydroxy-5alpha-pre gnane-20-one) and tetrahydrocortisone (THE, 3alpha,17alpha,21-trihydroxy-5beta-pregnane-11,20-dion e) in human plasma and urine is described. [1,2,3,4,5-2H5]THF (THF-d5), allo-[1,2,3,4,5-2H5]THF (allo-THF-d5) and [1,2,3,4,5-2H5]THE (THE-d5) were used as internal standards. A double derivatization (bismethylenedioxypentafluoropropionate, BMD-PFP) made possible the separation of the three tetrahydrocorticoids with good gas chromatographic behavior. Quantitation was carried out by selected-ion monitoring of the characteristic fragment ions ([M-30]+) of the BMD-PFP derivatives of THF, allo-THF and THE. The sensitivity, specificity, precision and accuracy of the method were demonstrated to be satisfactory for measuring low concentrations of THF, allo-THF and THE in human plasma and urine.

A high-performance liquid chromatography-electrospray-tandem mass spectrometry analysis of cortisol and metabolites in placental perfusate.

A reversed-phase high-performance liquid chromatography-electrospray-tandem mass spectrometry assay (HPLC-ESI-MS/MS) was developed to quantitate cortisol, cortisone, 20 alpha- and beta-dihydrocortisol, 20 alpha- and beta-dihydrocortisone, tetrahydrocortisol, and tetrahydrocortisone. The technique was used to analyze perfusate from the isolated human placental lobule for cortisol and its metabolites. Analytes were prepared from the perfusion medium using C18 solid-phase extraction cartridges. The internal standard for the analyses was 6 alpha-methylprednisolone. Chromatography was performed on a Novapak C18 column at ambient temperature using 53% methanol and 47% 10 mM ammonium formate buffer (pH 4.0) as mobile phase, at a flow rate of 80 microL/min. A PE-SCIEX API III triple quadrupole instrument was used for mass spectrometric detection. An ionspray (pneumatically assisted electrospray) interface was used in negative and positive ionization mode. The assay was linear over the range 100-2000 micrograms/L for each analyte. The instrumental limit of detection was 50 pg. Assay imprecision at 400 and 800 micrograms/L was < or = 10% (total coefficient of variation). Accuracy ranged between 83.2% for 20 beta-dihydrocortisone to 102.6% for cortisone. Recovery of 1000 micrograms/L analyte ranged from 91.3% for cortisone to 109.7% for tetrahydrocortisol.

5 alpha-dihydrocortisol in human aqueous humor and metabolism of cortisol by human lenses in vitro.

Glucocorticoids have long been implicated in the etiology of primary open-angle glaucoma (POAG) and cataract. Cortisol metabolites have biologic activity and may affect aqueous humor dynamics. This study was done to determine whether these metabolites are found in human aqueous humor and can be produced by ocular tissues. Radioimmunoassays (RIA) were developed for 5 alpha-dihydrocortisol (5 alpha-DHF) and 5 beta-dihydrocortisol (5 beta-DHF). These assays, as well as a cortisol RIA, were used to quantify these three steroids in 20 surgically derived aqueous humor specimens from patients with and without POAG. The mean concentrations of cortisol and 5 alpha-DHF were 2.5 and 1.3 ng/ml, respectively. In the small group studied, there was no statistically significant difference between the aqueous humor steroid levels in patients with and without POAG. The amount of 5 beta-DHF was at the lower limits of detection of the assay system and could not be uniquivocally shown. Human lenses metabolized cortisol in vitro to 5 alpha-DHF and 3 alpha,5 alpha-tetrahydrocortisol (3 alpha,5 alpha-THF). There was no 5 beta-DHF or cortisone formed. The 5 alpha-DHF and 3 alpha,5 alpha-THF were identified by their positions on thin-layer chromatography, their retention times on high-performance liquid chromatography, and recrystallization with authentic standards to constant specific activity. The data suggest that the lens is the source of 5 alpha-DHF in aqueous humor.

Prenatal diagnosis of 11beta-hydroxylase deficiency congenital adrenal hyperplasia.

To predict 11beta-hydroxylase deficiency congenital adrenal hyperplasia antenatally, studies were performed in urines and amniotic fluids from 2 pregnant women who had previously given birth to affected infants and whose present pregnancies also resulted in infants with the disease. Urinary tetrahydro-11-deoxycortisol [pregnane-3alpha, 17alpha, 21-triol-20-one (THS)] was abnormally elevated in the first, second, and third trimesters (maximal values, 3.5 and 0.9 mg/24 h, respectively) but was undetectable after delivery in these mothers, in 15 normal pregnancies (10--40 weeks of gestation), and in 6 heterozygote parents. Amniotic fluid levels of THS, tetrahydrocortisol [pregnane-3alpha, 11beta, 17alpha, 21-tetra-o1-20-one (THF)], tetrahydrocortisone [pregnane-3alpha, 17alpha, 21-triol-11, 20-dione (THE)] measured by RIA at 18 weeks of gestation in the first mother and at 40 weeks in the second revealed 12.5- and 8.4-fold increases in THS, respectively, but normal THF and THE levels compared to mean levels in normal pregnancies. The THS to THF plus THE ratio, which was constant throughout pregnancy in 125 normal women (mean +/- SD, 0.63 +/- 0.34) despite the variable levels of these metabolites, was significantly elevated in both patients (4.4 and 3.8, respectively). These studies indicate that prenatal diagnosis of 11beta-hydroxylase deficiency congenital adrenal hyperplasia based on hormonal measurements is feasible.