Synthesis of 21-nitrogen substituted pregna-5,17(20)-dienes from pregnenolone.

The facile synthesis of six [17(20)Z]- and [17(20)E]-isomeric 3ß-hydroxy-pregna-5,17(20)-dien-21-oyl amides and three [17(20)E]-3ß-hydroxy-2-[prergna-5,17(20)-dien-20-yl]-oxazolines from pregnenolone is presented. The synthetic scheme consists of transformation of pregnenolone into the known 17α-bromo-21-iodo-3ß-acetoxypregn-5-en-20-one followed by reaction with ethanolamine, 2-methyl-2-aminopropanol, and (1-aminocyclohexyl)methanol resulted in mixture of [17(20)E]- and [17(20)Z]-pregna-5,17(20)-dien-21-(2-hydroxy)-oyl amides; separation of [17(20)E]- and [17(20)Z]-isomers; their cyclization into [17(20)E]-oxazolines under action of POCl(3) in pyridine, and removal of acetate protecting groups. Significantly different orientation of nitrogen containing substituents in [17(20)Z]- and [17(20)E]-isomers regarding to steroid backbone enables their configuration to be easily identified by NMR spectroscopy. All synthesized compounds did not exhibit marked toxic effects in three cell lines (MCF-7, Hep G2, and LNCaP). In androgen-sensitive LNCaP cells all testing compounds at concentrations of 50 nM potently stimulated proliferation.

Determination of melengestrol acetate in bovine tissues by automated coupled-column normal-phase high-performance liquid chromatography.

A method has been developed for the determination of melengestrol acetate in bovine tissues at lower levels than previously reported. Liquid-liquid extraction of tissue homogenates provided crude clean-up while final isolation, screening, and quantification was done on-line with an automated, normal-phase, coupled-column high-performance liquid chromatographic system. The chromatographic system included phenyl and silica analytical columns for the purposes of isolation and final separation, respectively. These columns provided a large difference in selectivity when operated under normal-phase conditions which allowed for the efficient isolation of melengestrol acetate from the complex tissue extracts. Mobile phases were composed of hexane and dichloromethane modified with methanol and water. Transfer and enrichment of the analyte from the primary phenyl column to the silica column was via a short (12 mm x 4 mm I.D.) silica column. Regeneration and equilibration of the phenyl column was performed after the injection of each tissue extract and was accomplished simultaneously while analytical separation occurred on the final silica column. Routing of the mobile phases and regeneration solvent was performed with automated switching valves. The total time required for each analysis was 12 min. Quantification is demonstrated using external standards with UV detection at 287 nm. The overall recovery of the method was 86% with a coefficient of variation of 9.84% at the 10 ppb [the American billion (10(9] is used in this article] level in bovine liver extracts.

Determination of melengestrol acetate in feedstuffs with liquid chromatographic preparatory column cleanup and quantitative analysis.

Melengestrol acetate (MGA) is determined by liquid chromatography using a fraction from preparatory LC as a means of sample cleanup for feedstuffs, both dry and liquid. Dry ground feed is Soxhlet extracted with hexane and passed through a 2% deactivated alumina column for initial cleanup. The eluate is evaporated, redissolved in methanol, filtered, and injected onto a preparatory LC column. The fraction containing MGA is separated from the remaining matrix, evaporated to dryness, dissolved in methanol, and quantitated by LC analysis. Liquid supplements are extracted in methanol, and the extract is evaporated to near dryness. The residue is diluted with water, extracted with chloroform, passed through sodium sulfate, and evaporated to dryness. The remaining sample is dissolved in methanol prior to preparative LC and quantitative LC. Recoveries for 2 laboratory-fortified commercial feeds, one dry and one liquid, containing 0.39 and 0.40 mg/lb, were 98.3% +/- 4.4 and 95.8% +/- 4.3, respectively. Results compare favorably with existing methods. Up to a 4-fold time savings was realized by this method without automation.

Danazol concentrations in human ovarian follicular fluid and their relationship to simultaneous serum concentrations.

Danazol concentrations in follicular fluid and serum were studied in eight women scheduled for laparoscopy because of suspected endometriosis. In order to obtain some variation in follicular maturity, danazol administration was started 2 to 7 days before the expected day of ovulation. A total of nine doses were given, i.e., 200 mg four times daily for 2 days; the last 200-mg dose was given 3 hours before the laparoscopy during which the follicular fluid from the dominant follicle was aspirated. Peripheral venous blood samples were drawn before, during, and after laparoscopy. Danazol concentrations were assayed by means of a high-performance liquid chromatography method. At the time of follicular aspiration, the mean concentration of danazol was estimated at 96 ng/ml in serum and at 71 ng/ml in follicular fluid, i.e., an average of 73% of the simultaneous serum concentration. The data suggest that even short-term therapy with danazol is likely to produce intrafollicular drug concentrations that have a direct inhibitory effect on follicular steroidogenesis.

Simultaneous quantitative determination of butizide, potassium canrenoate and canrenone in tablets by high-performance liquid chromatography.

A procedure for the simultaneous quantitative determination and selective identification of potassium canrenoate and butizide is proposed. The amount of canrenone, the degradation product of potassium canrenoate, is also determined. Elution of these compounds is investigated on LiChrosorb RP-18 and RP-8 columns. The analysis time was shorter on a RP-8 column while the specific identification was still possible. Therefore, the LiChrosorb RP-8 column was chosen. The eluent consisted of an acetonitrile--0.05 M phosphate buffer, pH 4 (45:55) mixture. Quantitative determination was performed at the absorption maximum of each compound: 286 nm for potassium canrenoate and for canrenone, 271 nm for butizide. This change in wavelength is performed automatically by the computer that controls the spectrophotometric diode array detector.

A new approach for dealing with the stability of drugs in biological fluids.

The purpose of this study was to develop a procedure for investigating the stability of drugs in biological fluids based on sound experimental design and to use a statistical procedure which would allow conclusions to be made concerning stability with an acceptable degree of certainty (95%). The experimental procedure involved replicate analysis of the drug in stored and freshly prepared samples on the same day. The relative difference in response between these two sets of samples and a 90% confidence interval for the true change in response was calculated. This confidence interval enabled us to detect a pharmacokinetically relevant degradation. It is argued that this approach is superior to the stability test procedures based on the t test. The application of the method to the assessment of the stability of three different drugs in biological fluids is described.

Electron impact induced fragmentations of the 1,4-diene analogues of steroid hormones and related steroids.

The electron impact induced fragmentations observed in the mass spectra of eight 1,4-diene-3-ketopregnane steroids are described. The effect of hydroxyl substituents in positions 11 beta, 17 alpha and 21, on the typical fragmentation pattern is analysed; in particular the 'long range' effect of the 17 alpha-hydroxy group on the relative abundance of the ions m/z 121 and 122 is confirmed using specific deuterium labelling.

Determination of canrenone, the major metabolite of spironolactone, in plasma and urine by high-performance liquid chromatography.

An assay procedure for measuring plasma and urine levels of canrenone is described. The drug is extracted with n-hexane-toluene (1:1, v/v) after adding spirorenone as internal standard, and is then separated from plasma constituents and metabolites by high-performance liquid chromatography followed by UV detection at 285 nm. The limit of detection is less than 5 ng/ml. Interference with a series of spironolactone and canrenone metabolites was not observed. Plasma levels and renal excretion of canrenone after oral administration of 200 mg of spironolactone and intravenous injection of 200 mg of potassium canrenoate to a healthy male volunteer were measured.

High pressure liquid chromatographic determination of melengestrol acetate in dry feed supplements.

A high pressure liquid chromatographic (HPLC) method is described for the quantitative determination of melengestrol acetate (MGA) in dry animal feed supplements containing 0.000027-0.000220% MGA (0.125-1.00 mg/lb). Ground feed is Soxhlet-extracted with hexane, and the extract is partitioned from hexane into aqueous methanol and then into methylene chloride, followed by mixed column chromatography. MGA is then quantitated by HPLC. Average recovery of standard MGA through the method at 1.00 ppm (0.454 mg/lb) was 94.8% with a 3.58% standard deviation. Average spike recovery of MGA in fortified feed at 0.100 ppm (0.0454 mg/lb) to 2.00 ppm (0.907 mg/lb) level was 97.8% with a standard deviation of 5.39%. In addition, the method includes a 2-dimensional thin layer chromatographic confirmatory test for MGA.

Gas-liquid chromatographic determination of melengestrol acetate in cattle feed supplements: collaborative study.

The assay for melengestrol acetate (MGA) in cattle feed supplements was the subject of a collaborative study using 6 laboratories. Two feed formulations were each fortified with 0, 0.0625, 0.125, 0.500, 1.00, and 1.50 mg MGA/lb of supplement, and each laboratory assayed 4 samples of each level of each formulation by electron capture gas chromatography after liquid-liquid extraction of each sample followed by solvent partition and column chromatography cleanup procedures. Overall recovery was 83.2% with a pooled within-laboratory sample-to-sample standard deviation of 9.5%. This includes the variability due to formulation, batches, days, levels of MGA, samples, and their interactions. Including the collaborators as a source of variance results in an overall standard deviation of 15.0%. The major sources of variability were associated with collaborator, formulation, and level of MGA. Lower than expected recoveries were attributed to difficulty in recovering MGA from samples containing concentrations of MGA below the range of the method and the inadvertent substitution by some laboratories of ethanol-free chloroform for the ethanol-containing reagent grade chloroform specified in the methodology. On the basis of the collaborative results, the method has been adopted as official first action for MGA in cattle feed supplements at concentrations from 0.125 to 1.00 mg MGA/lb.

21-Hydroxypregna-1,4-diene-3,11,20-trione: a biliary metabolite of a cartilaginous fish, Raja sp.

21-Hydroxypregna-1,4-diene-3,11,20-trione was isolated from skate bile and as an in vivo metabolite of 3H-1alpha-hydroxycorticosterone. Identity was established by chromatography and derivatization to constant 3H/14C ratio and mass spectrometry of the 20,21-acetonide. The new steroid was present in the free form and as the glucuronoside.

A radioimmunoassay for danazol (17alpha-pregna-2,4-dien-20-yno[2,3-d]isoxazol-17-ol).

A method is described for the radioimmunoassay of circulating levels of the pituitary inhibiting agent, danazol. An antigen for danazol was prepared by reacting a 17-carboxy-methyloxime derivative of danazol with bovine serum albumin. By immunizing rabbits with this antigen, antiserum was generated which shows excellent specificity for danazol relative to its known metabolites as well as to many natural steroids. A radioimmunoassay was developed, without using separation or extraction techniques, involving competition for the antiserum between danazol in plasma and 14C-danazol. This assay has been successfully used to measure danazol in a series of normal human subjects receiving the drug at either 100 or 200 mg b.i.d. for 2 weeks. A significant relationship was seen between dosage of danazol and plasma concentrations.

5alpha-Pregna-1,20-dien-3-one and related compounds from a soft coral.

Four unusual pregnane derivatives, 5alpha-pregna-1,20-dien-3-one (1), 1,4,20-pregnatrien-3-one (3), 5alpha-pregn-20-en-3alpha-o1 o-acetate (4), and 5alpha-pregn-20-en-1alpha,3alpha-diol 3-acetate (6) have been isolated from an unidentified soft coral from Canton Island.

Spironolactone: relationship between concentrations of dethioacetylated metabolite in human serum and milk.

The concentration of canrenone, a principal metabolite of spironolactone, was determined in human serum and milk. The milk to serum concentration ratios of this metabolite were 0.72 at 2 hr and 0.51 at 14.5 hr after ingestation of spironolactone. It was estimated that the maximum quantity of canrenone ingested daily by a human infant via its mother's milk would be approximately 0.2% of the daily dose of spironolactone given to the mother.

Validation of plasma and urinary canrenone for determination of bioavailability of spironolactone--comparison of tablets with solution/suspension.

Data showing a linear relationship between spironolactone dose and the levels of canrenone in plasma and urine are presented to support the use of canrenone levels as a measure of the bioavailability of spironolactone. The bioavailability of a selected batch of spironolactone 25 mg tablets was compared to that of an oral aqueous suspension of spironolactone in a separate balanced cross-over study involving 10 healthy subjects. The tablets were equivalent to suspension as regards area under the plasma concentration-time curve and total urinary excretion of canrenone, the prinicipal unconjugated metabolite of spironolactone. Peak plasma concentrations of canrenone were higher, and were attained significantly earlier, after treatment with spironolactone in suspension. The results indicate that spironolactone was absorbed more rapidly from the suspension, but that the total absorption did not differ between the two formulations.

Determination of melengestrol acetate in bovine tissue: collaborative study.

Seven laboratories collaboratively studied a method for the assay of melengestrol acetate at the 0, 10, and 20 ppb levels in bovine fat, liver, muscle, and kidney. The study included fortification of tissue by each laboratory and analysis of fat samples taken from treated heifers which had endogenous levels of 0, 10, and 20 ppb melengestrol acetate. The multistep cleanup procedure used included extraction, solvent partition, column chromatography, and electron capture gas-liquid chromatographic, determination. Results of the study for muscle, liver, kidney, and fat showed that the method gave satisfactory recoveries and accuracy. In fat, the most critical tissue, recovery was greater than 93%. A statistical comparison of the results reported for fat tissue from treated heifers demonstrated that 5 of the 7 laboratories obtained similar results. The results produced by the method can be expected to be repeatable within and among laboratories. On the basis of the collaborative results the method has been adopted as official first action.

High-speed liquid chromatographic determination of canrenone in pharmaceutical dosage forms.

Canrenone can be determined by high-speed liquid chromatography in pharmaceutical dosage forms without interference from common excipients or degradation products. This stability-indicating assay, using o-nitroaniline as the internal standard, is rapid and accurate.