Development of a liquid chromatography-tandem mass spectrometry method for the determination of 23 endogenous steroids in small quantities of primate urine

A quantitative method using liquid chromatography-tandem mass spectrometry (LC-MS-MS) was developed for the simultaneous determination of 23 endogenous steroids in primate urine. The introduced method includes estrone, pregnandiol, cortisol, testosterone and several human urinary glucocorticoid and androgen metabolites. As the method is intended for the analysis of steroid hormones in behavioral studies on wild-living primates, it was adapted for a sample volume of 200microL urine. The sample preparation consisted of an enzymatic hydrolysis of steroid glucuronides using beta-glucuronidase from E. coli followed by a solvolytic cleavage of steroid sulfates employing sulfuric acid/ethyl acetate. The extraction of steroids from urine was optimized with respect to pH during extraction, type of ether and the amount of enzyme necessary for complete hydrolysis of glucuronides. The recovery of steroids spiked into urine before hydrolysis was 58.9-103.7% with an intra-day precision of 2.7-14.3% and an inter-day precision of 2.9-14.8%. Detection limits ranged from 0.1-0.5ng/mL. The reproducibility of the whole sample preparation process was also demonstrated for unspiked urine (CV 1.2-16.5%). The proportion of steroid hormone excreted as sulfate was determined for 21 steroids in chimpanzee urine. The solvolysis proved to be essential for all investigated steroids except for pregnandiol, tetrahydrocortisol and tetrahydrocortisone, which were found to be less then 10% in the solvolysis fraction.     

Analysis of prednisone,prednisolone and their 20β-hydroxylated metabolites by high-performance liquid chromatography

A high-performance liquid chromatographic technique primarily developed for use on samples from kidney perfusion studies is presented for simultaneous determination of prednisone, prednisolone and their 20β-hydroxylated metabolites. The technique employs 6β-hydroxycortisol as the internal standard. Samples are extracted with ethyl acetate, washed with sodium hydroxide and water and injected onto a silica gel column with UV detection at 254 nm. Inter- and intraday variability of the assay was determined at two concentrations of each steroid and was less than 10%. Assay steroid recovery ranged from 54.1% for prednisone to 63.2% for 20β-hydroxyprednisone. Sensitivity is 4–10 ng/ml for the steroids measured. The chromatographic conditions may be modified to permit quantitation of these steroids from plasma samples. This method may alternatively be used for quantitation of 6β-hydroxycortisol, an endogenous indicator of enzyme induction. A perfusate concentration-time profile is presented from a kidney perfusion study using prednisolone.     

Capillary electrophoresis with UV detection and mass spectrometry in method development for profiling metabolites of steroid hormone metabolism

The aim of this study was to develop a method for comprehensive profiling of metabolites involved in mammalian steroid metabolism. The study was performed using the partial filling micellar electrokinetic chromatography (PF-MEKC) technique for determination of endogenous low-hydrophilic steroids. The detection techniques in capillary electrophoresis were UV absorption and electrospray mass spectrometry (ESI-MS). Thirteen steroids were included in the method development, and the selected were metabolites involved in major pathways of steroid biosynthesis. Although only eight of them could be separated and detected with UV, they could be identified by ESI-MS using selected ion monitoring (SIM) technique. Tandem MS spectra were also collected. UV detection was more sensitive than MS due to better separation of compounds and the selective signal sensitivity. The lowest limits of detection were 10-100ng/mL for cortisone, corticosterone, hydrocortisone and testosterone. The other steroids could be detected at 500-1000ng/mL. The identification of cortisone, corticosterone, hydrocortisone, estrogen and testosterone were made in patient urine samples and their concentrations were 1-40mug/L.     

Formation of neutral steroids from endogenous precursors in minced human fetal adrenals in vitro

The role of endogenous precursors in steroid biosynthesis in the human fetal adrenals was studied in $\text{in vitro}$ incubations with no added exogenous substrate. The identification and quantitative determination of the steroids was carried out by gas-liquid chromatography and gas chromatography - mass spectrometry. During the incubation a 10–60-fold increase in the concentration of dehydroepiandrosterone sulfate was observed. An increase was also seen in the concentrations of the other two steroid sulfates detected, pregnenolone and 17-hydroxypregnenolone sulfate. The concentrations of the corresponding free steroids were seen to decrease during the incubations. Only traces of free dehydroepiandrosterone and progesterone were detected endogenously or at any stage during the incubations. No corticoids could be found. Endogenous cholesterol was found in high concentrations (1.5 – 3.0 mg/g wet tissue) in the tissue samples studied. A small proportion of it was present as a sulfate conjugate.It is concluded that fetal adrenals can form neutral steroid sulfates of the 3β-hydroxy-5-ene series from endogenous precursors $\text{in vitro.}$ Cholesterol which was detected in high concentrations in the adrenal tissue is a possible precursor of these metabolites. The results obtained would suggest that this endogenous metabolism involves sulfated intermediates principally.     

Simultaneous determination of serum cortisol and cortisone by reversed-phase liquid chromatography with ultraviolet detection

A rapid high-performance liquid chromatographic (HPLC) method for the simultaneous determination of cortisol and cortisone in a single extract of 1 ml of serum is described. The method employs meprednisone as the internal standard. The steroids were analysed isocratically by reversed-phase HPLC with an octadecylsilane-bonded (ODS) column using ultraviolet detection. The matrix effect was reduced by lowering the sample pH by adding glacial acetic acid to the sera. The samples were then filtered through regenerated cellulose membranes at 4°C and extracted with diethyl ether. The dried eluates were redissolved in the mobile phase and injected into the column. The detection limit of the assay for both steroids was 500 ng/l. Cortisol was determined in twenty serum samples by both HPLC and radioimmunoassay (RIA). The results were similar. Interference by other steroids and certain steroid analogue drugs was also studied. The HPLC method yielded no cross-reactivity between the different steroids as may occur with the RIA technique. The HPLC method was technically easy to perform and it allowed us to quantify both cortisol and cortisone in a single serum extract with high specificity.     

Steroid interference in iron-cholesterol reactions: A comparative study

Interference from various physiological and non-physiological steroids in the spectrophotometric determination of cholesterol by the Zak method (ferric chloride) and the method of Parekh and Jung (ferric acetate) was quantitatively measured. Contribution of the steroids at the specific absorption maxima of the cholesterol assays was determined by employing the steroids (40 mg/dl) alone, or added to a serum pool of known cholesterol content. The results show that the method of Parekh and Jung is less influenced by the presence of steroids than the Zak method. Observations on the structural specificity of the iron-cholesterol reaction are discussed.     

Direct interaction between substrates and endogenous steroids in the active site may change the activity of cytochrome P450 3A4

CYP3A4 exhibits unusual kinetic characteristics that result from the metabolism of multiple substrate including endogenous steroids and some drugs that coexist at the active site. To clarify the mechanism of the effect of endogenous steroids on the drug metabolism, the interaction between substrates, nevirapine (NVP) and carbamazepine (CBZ), and endogenous steroids was investigated by theoretical calculations. When the activities of NVP 2-hydroxylation and CBZ 10,11-epoxidation by expressed CYP3A4 were measured in the presence of steroids, NVP 2-hydroxylation was found to be remarkably increased by aldosterone and inhibited by estradiol. CBZ 10,11-epoxidation was increased by androstenedione. Three-dimensional computer modeling has shown that the active site of CYP3A4 is especially large, permitting access of two substrate molecules. The interactions between NVP and aldosterone and between CBZ and androstenedione were estimated by theoretical calculations assuming the substrate and steroids to be present in the active site at the same time. It was shown that NVP or CBZ would be stably fixed close to the oxygen atom at the sixth ligand of heme by interaction with steroids, suggesting that NVP and CBZ may be hydroxylated more easily due to the interaction with steroids. Estradiol was also expected to interact with NVP via a pi/pi interaction between a benzene ring, in which the NVP hydroxylation site is located, and a benzene ring of estradiol, suggested to inhibit the reaction. From these results, interactions between the substrate and endogenous steroids in the active site may change the activity of CYP3A4.     

Gas chromatography-mass spectrometry profiling of steroids in times of molecular biology.

This review's aim is to outline the potential of gas chromatography-mass spectrometry profiling of steroids in the diagnosis of endogenous human steroid disorders. Mass spectrometry currently provides the highest specificity in clinical steroid analysis. The non-invasive and non-selective GC-MS urinary steroid profiling technique enables diagnosis of almost any adrenal enzyme defects in steroid biosynthesis. While enzymatic defects can be diagnosed from spot urine samples in most cases, analysis of 24-hr urinary samples permits determination of hormonal excretion rates or enables diagnostic or therapeutic monitoring of steroid related diseases. Profiling plasma steroids by isotope dilution/GC-MS is particularly suitable where only minimal plasma samples are available and/or the highest specificity is required; therefore, GC-MS steroid profiling presents a complementary analytical technique whenever highest specificity is required. Clinical GC-MS profiling of steroids is also highly recommended as a reasonable initial diagnostic approach--especially in unclear situations--avoiding uncritical and expensive attempts at molecular diagnostic testing.     

Deoxycorticosterone, 18-OH-deoxycorticosterone and corticosterone determination by high performance liquid chromatography in monolayer adrenal cell culture.

Reversed-phase HPLC offers a rapid, qualitative as well as quantitative method for separation and determination of deoxycorticosterone, 18-OH-deoxycorticosterone and corticosterone in primary culture of adrenocortical cells. The resolution is sufficient for the purpose of quantitative determination. The limitation of this method lies in its sensitivity for serum steroid determination, but is perfectly applicable at cell cultures where the concentration of these steroids is highest.     

Quantification of neurosteroids in rat plasma and brain following swim stress and allopregnanolone administration using negative chemical ionization gas chromatography/mass spectrometry

A simplified method for the quantitative analysis of neurosteroids in rat plasma and brain is described. The method uses negative chemical ionization gas chromatography/mass spectrometry and involves the synthesis of pentafluorobenzyloxime/trimethylsilyl ether derivatives with excellent chromatographic and electron-capturing properties. Deuterium-labeled analogs of the steroids of interest were synthesized and used as internal standards. The steroids (allopregnanolone, epiallopregnanolone, pregnenolone, testosterone, and dehydroepiandrosterone) were isolated from the plasma or brain matrix by a rapid and straightforward solid-phase extraction procedure. The mass spectrometer was operated in a selective ion monitoring mode, allowing for picograms of neurosteroids to be quantified from biological extracts. The method was linear (typical R(2) = 0.999) over the concentration range (100 to 8000 pg from 0.3 ml plasma and 250 to 8000 pg from 100 mg brain tissue) with good precision and accuracy. In experimental protocols, the procedure was suitable for measuring concentrations of endogenous neurosteroids in rat plasma and brain. Significant elevations (P < 0.001) were observed in the frontal cortex for allopregnanolone and pregnenolone following a swim stress and for allopregnanolone and epiallopregnanolone following allopregnanolone injection (8 mg/kg, sc). The present method allows accurate determination of neurosteroids and will be helpful in elucidating the role of neurosteroids in health and disease.     

Neuroactive steroids: new biomarkers of cognitive aging

Intensive studies in animals established that neuroactive steroids display neuronal actions and influence behavioral functions. We describe here investigations on the role of neuroactive steroids in learning and memory processes during aging and suggest their role as biomarkers of cognitive aging. Our work demonstrated the role of the steroid pregnenolone (PREG) sulfate as a factor underlying an individual’s age-related cognitive decline in animals. As new perspectives of research we argue that knowing whether neuroactive steroids exist as endogenous neuromodulators and modulate physiologically behavioral functions is essential. To this end, a new approach using the sensitive, specific, and accurate quantitative determination of neuroactive steroids by mass spectrometry seems to have potential for examining the role of each steroid in discrete brain areas in learning and memory alterations, as observed during aging.     

Faecal sexual steroids in sex typing and endocrine status of great bustards

Faecal sexual steroids have been used in field studies evaluating the relationships between gender and the multiple factors influencing endocrine status of individuals. The determination of faecal steroids has been also proposed as an alternative, non-invasive sexing method when other methods were deemed impractical or risky for the health of birds. In this study, we quantified sexual steroid hormones in faeces of the great bustard (Otis tarda), a large and sexually dimorphic polyginic bird species that it is threatened and subjected to intense wildlife management. We evaluated differences between captivity and wild conditions, flocks and sexes, and used faecal steroids to develop sex determination procedures. We found similar steroid levels in captive and wild bustards, no differences between unisexual wild flocks and clear between-sexes differences in testosterone but not estradiol. Faecal steroids accurately discriminated gender in both captive and wild known-sex great bustards. Total testosterone concentration was always higher than estradiol concentration in faecal samples from males, but estradiol was not always higher than testosterone in females. Faecal steroids failed to reveal the presence of young males in female flocks during winter, despite faecal testosterone levels increased with age in a small sample of captive males. Our results show that faecal steroid measurement for both sexing and characterizing the endocrine status of great bustards is feasible, and therefore it should be valuable in wildlife management, especially in combination with additional information obtained from faeces as diet.     

Gas chromatography–combustion–isotope ratio mass spectrometry analysis of 19-norsteroids: application to the detection of a nandrolone metabolite in urine

Determination of whether the major metabolite of nandrolone in urine, 19-norandrosterone (19-NA), is exogenous or endogenous in origin is one of the most exciting challenges for antidoping laboratories. Gas chromatography–combustion–isotope ratio mass spectrometry (GC–C–IRMS) can be used to differentiate these two origins by carbon isotopic ratio analysis. A complete method for purification of 19-NA in urine has been established. Acetylated ketosteroids, and in particular 19-NA, are isolated from the urine matrix before analysis after hydrolysis and purification of urine by reversed-phase and normal solid-phase extraction. The limit of detection for 19-NA was about 60 ng with recoveries of 54–60%. Evidence of exogenous administration of 19-NA may be established from isotope ratio determination from the ¹³C/¹²C ratios of several synthetic 19-norsteroids compared to those obtained for endogenous steroids.     

Repair replication in human cells studied by sodium iodide isopycnic centrifugation of DNA in a fixed-angle rotor

A method for purification of ethynyl steroids from biological fluids has been developed using silver-sulfoethyl cellulose column chromatography. Ethynyl steroids were applied in methanol, and the firm binding allowed the columns to be washed with methanol, ethyl acetate, or diethyl ether to remove endogenous materials. Nonethynylated steroids did not bind to silver-sulfoethyl cellulose. Release of ethynylated steroids was achieved with a saturated NaCl/methanol solution. Dehydration or de-ethynylation of ethynyl estradiol, ethynodiol diacetate, and ethynodiol was not observed. The utility of this technique for purification of ethynyl steroid metabolites from the urine of a beagle metabolizing norethynodrel was demonstrated.     

Urine sex steroid profile in healthy volunteers during isolation in a pressurized compartment

In order to study the time course of the parameters of urine sex steroid profile and its potential boundary changes, the quantitative determination of a number of endogenous steroids and their metabolites in healthy human urine has been carried out by the gas chromatography method using mass-selective detection. The samples were obtained from six volunteers under the conditions of total monitoring of vital activity factors affecting urine steroid profile (diet, water consumption, physical activity, temperature and air composition, day-night rhythm, and the psychoemotional state) in an experimental study using a pressurized compartment. The healthy human profile parameters of urine steroids were found, which were affected by conditions of controlled vital activity in a pressurized compartment. Parameters of the individual and group variability of the parameters of steroid profile and their dependence on experimental factors, salt consumption mode, and autonomous vital activity, were found.     

Stable isotope methodology in the pharmacokinetic studies of androgenic steroids in humans

The use of stable isotopically labeled steroids combined with gas chromatography/mass spectrometry (GC/MS) has found a broad application in pharmacologic studies. Initially, stable isotopically labeled steroids served as the ideal analytic internal standard for GC/MS analysis; however, their in vivo use has expanded and has proven to be a powerful pharmacokinetic tool. We have successfully used stable isotope methodology to study the pharmacokinetic/bioavailability of androgens. The primary advantage of the technique is that endogenous and exogenous steroids with the same basic structure can be differentiated by using stable isotopically labeled analogs. The method was used to examine the pharmacokinetics of testosterone and testosterone propionate, and to clarify the influence of endogenous testosterone. Another advantage of the isotope methods is that steroidal drugs can be administered concomitantly in two formulations (e.g., solution and solid dosage). A single set of blood samples serves to describe the time course of the formulations being compared. This stable isotope coadministration technique was used to estimate the relative bioavailability of 17 alpha-methyltestosterone.     

Ex vivo spontaneous generation of 19-norandrostenedione and nandrolone detected in equine plasma and urine

19-Norandrostenedione (NAED) and nandrolone are anabolic-androgenic steroids (AASs). Nandrolone was regarded solely as a synthetic AAS until the 1980s when trace concentrations of apparently endogenous nandrolone were detected in urine samples obtained from intact male horses (stallions). Since then, its endogenous origin has been reported in boars and bulls; endogenous NAED and nandrolone have been identified in plasma and urine samples collected from stallions. More recently, however, it was suggested that NAED and nandrolone detected in urine samples from stallions are primarily artifacts due to the analytical procedure. The present study was undertaken to determine whether NAED and nandrolone detected in plasma and urine samples collected from stallions are truly endogenous or artifacts from sample processing. To answer this question, fresh plasma and urine samples from ≥8 stallions were analyzed for the two AASs, soon after collection, by liquid chromatography hyphenated to tandem mass spectrometry (LC-MS/MS). NAED and nandrolone were not detected in fresh plasma samples but detected in the same samples post storage. Concentrations of both AASs increased with storage time, and the increases were greater at a higher storage temperature (37°C versus 4°C, and ambient temperature versus 4°C). Although NAED was detected in some fresh stallion urine samples, its concentration (<407 pg/mL) was far lower (<0.4%) than that in the same samples post storage (at ambient temperature for 15 days). Nandrolone was not detected in most of fresh urine samples but detected in the same samples post storage. Based on these results, it is concluded that all NAED and nandrolone detected in stored plasma samples of stallions and most of them in the stored urine samples are not from endogenous origins but spontaneously generated during sample storage, most likely from spontaneous decarboxylation of androstenedione-19-oic acid and testosterone-19-oic acid. To our knowledge, it is the first time that all NAED and nandrolone detected in plasma of stallions and most of them detected in the urine have been shown to be spontaneously generated in vitro during sample storage. This finding would have significant implications with regard to the regulation of the two steroids in horse racing.     

A reversed-phase high-performance liquid chromatographic method for the simultaneous determination of serum concentrations of cyproterone acetate and 15 beta-hydroxycyproterone acetate

A reversed-phase high-performance liquid chromatographic method for the simultaneous determination of cyproterone acetate (CPA), 15 beta-hydroxycyproterone acetate (15 beta-OH-CPA) and cyproterone (CP) was reported. This method was specific, sensitive, precise, easy and rapid for determination of the serum concentrations of these steroids in patients receiving CPA. Although no peak corresponding to CP was observed for serum, peaks corresponding to CPA and 15 beta-OH-CPA were detected and well separated in all subjects undergoing long-term CPA therapy. In these patients, there seemed to be a dose-dependent relationship between the amount of CPA administered and the serum concentrations of these steroids, and the serum concentrations of CPA were either similar or low compared with those of 15 beta-OH-CPA. In conclusion, this simplified method is thought to be very valuable for studies on the pharmacokinetics of CPA and 15 beta-OH-CPA, and on the relationship between the CPA dosage and the therapeutic or side effects on adrenal and gonadal steroid production.     

Steroids and depression.

Patients with endogenous depression (major affective disorder) frequently have high cortisol levels, but the diurnal rhythm is usually maintained and they do not develop the physical signs of Cushing's syndrome. On the other hand, depression is a frequent feature of Cushing's syndrome regardless of etiology, and it is often relieved when the cortisol levels are reduced, by whatever means. The mechanisms of the hypercortisolemia and resistance to dexamethasone suppression commonly found in endogenous depression are poorly understood; contrary to expectations, ACTH levels are not clearly elevated. There is a striking difference in the psychiatric features seen in endogenous hypercorticism compared to those seen after exogenous administration of glucocorticoids or ACTH. This suggests that either there are other stimulating or modifying factors besides ACTH or that the steroids stimulated by ACTH or other peptides differ from those in control subjects, i.e. there may be an alteration in the metabolism of steroids in depression. Little is known about the metabolic changes or the many steroids besides glucocorticoids produced by the hyperactive steroid-producing tissue. Preliminary studies suggest that major depression may be improved by steroid suppression. It is hypothesized that steroids themselves may be important in causing and perpetuating depression.