Analysis of steroidal lipids by gas and liquid chromatography.

This article describes the most commonly used procedures and recent laboratory methodologies using gas and liquid chromatography developed for separation and quantitation of non-saponifiable steroidal lipids from clinical (human) studies, edible fats and oils or fatty foods.

Reversed-phase high-performance liquid chromatographic screening method for serum steroids using retention index and diode-array detection.

A multisteroid screening method has been developed based on the use of 1-[4-(2,3-dihydroxypropoxy)phenyl]-1-alkanones as retention index standards and UV absorbance spectra recorded on-line with a diode-array detector using reversed-phase high-performance liquid chromatographic gradient elution with acetonitrile and water. The effect of chromatographic conditions on retention indices of steroids were studied. The method was tentatively applied to profiling of steroids in serum samples.

Structure-retention relationships of steroid hormones in reversed-phase liquid chromatography and micellar electrokinetic capillary chromatography.

Quantitative structure-retention relationship (QSRR) studies are useful in retention prediction, finding the relevant structural descriptors for analytes and estimating the relative biological activities of a series of analytes. Most of the studies have been conducted by RP-HPLC and a few by micellar electrokinetic capillary chromatography (MECC). The aim of this work was to find structural parameters and characteristics related to the RP retention and electrophoretic migration of steroids in order to predict the retention/migration of steroid hormones on the basis of their molecular structure. Retention data were obtained with an ODS column using a mobile phases aqueous methanol-acetonitrile (mobile phase A) and methanol-tetrahydrofuran (mobile phase B). MECC was conducted with a sodium dodecyl sulfate (SDS)-borate system and with a mixed micellar solution of SDS and sodium cholate. Several topological indices, such as the connectivity indices, chi, were used as structural parameters. Steric factors seem to have a great effect on the retention of steroid hormones, especially with the MeCN-containing mobile phase. Retention in mobile phase B could be predicted more accurately. Topological indices can be used in the modelling and prediction of the retention/migration of steroid hormones when the solutes form a congeneric series and stereochemical properties do not govern the separation process.

High-performance liquid chromatographic analysis of corticosteroids.

This review presents recent developments in high-performance liquid chromatographic (HPLC) analysis of corticosteroids for the determination of clinically important steroids in biological specimens. Various sample preparation techniques are described.

Simple and specific reversed-phase liquid chromatographic method with diode-array detection for simultaneous determination of serum hydroxychloroquine, chloroquine and some corticosteroids.

This paper describes a simple, specific, and sensitive high-performance liquid chromatographic (HPLC) method using ion-pair reversed-phase chromatography with photodiode-array detection for the simultaneous determination of hydroxychloroquine (HCQ) and chloroquine (CQ) in serum samples from rheumatoid arthritis patients receiving either HCQ sulphate or CQ diphosphate. The assay is also applicable to the simultaneous determination of corticosteroids. The method consisted of two diethyl ether extractions of 1.0 ml of serum, to which two internal standards (2,3-diaminonaphthalene and 18-hydroxy-11-deoxycorticosterone) and 1.0 ml of 0.25 M sodium hydroxide had been added. After the organic phase was evaporated to dryness at 30-40 degrees C under a stream of nitrogen, the extract was reconstituted with a 1:1 mixture of 0.1 M perchloric acid and methanol, an aliquot of which was injected on to the system. Peak-height ratios at different wavelengths (A245/343, A245/256, A245/265 and A245/275) were utilized as a method of assessing peak homogeneity. Some anti-inflammatory drugs which may be used for rheumatic disorders were shown not to interfere with the assay. The method provides selectivity by using diode-array detection at several wavelengths. The use of two internal standards not only compensates for losses during the sample manipulation but also prevents erroneous results in case of interference.

Determination of free urinary catecholamines by high-performance liquid chromatography with electrochemical detection.

The present paper compares a manual high-performance liquid chromatographic (HPLC) method with electrochemical detection (ED) with an on-line HPLC-ED method for the determination of free catecholamines (CAs) (norepinephrine, epinephrine, and dopamine) in urine. The manual method involves a two-stage extraction procedure with home-packed columns filled with weakly acidic cation-exchange resin followed by adsorption onto alumina while the on-line method uses a strong cation-exchange polymer as precolumn material which allows the simultaneous determination of free CAs and metanephrines (MNs) in urine. Reference control urine samples analysed with both methods gave levels of CAs within the acceptable reference ranges. Dihydroxybenzylamine was used as an internal standard in both methods and the separation was performed by ion-pairing reversed-phase HPLC.

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 degrees 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.

Determination of urinary normetanephrine, metanephrine and 3-methoxytyramine by high-performance liquid chromatography with electrochemical detection: comparison between automated column-switching and manual dual-column sample purification methods.

This report describes a high-performance liquid chromatographic method with electrochemical detection for the simultaneous quantitation of urinary metanephrine, normetanephrine and 3-methoxytyramine. This method, which involves manual dual-column purification steps for the routine determination of urinary metanephrines, is compared with the previously used spectrophotometric Pisano method and an on-line sample preparation procedure, where the automated sequential trace enrichment (ASTED) apparatus is used for the column-switching procedure. In order to automate the metanephrine assay, the enrichment technique was evaluated against the reference chromatographic method. Bio-Rad urine controls gave coefficients of variation of less than 9% at all levels for the reference method. Values of less than 19% were found in the reference range with the enrichment method, and the recovery of 3-methoxytyramine was also too poor to be measured in normal concentrations. The linearity of both methods is sufficient to determine pathological levels of these biogenic amines. Future developments should be focused on decreasing the variation of between-day assays in an on-line, automated procedure.

Some endocrine responses to sauna, shower and ice water immersion.

Nine winter swimmer men were exposed to: (A) sauna and ice water immersion; (B) sauna and 15 degrees C shower; (C) sauna and room temperature; (D) head-out ice-water immersion and room temperature. The exposures were repeated and ended with a recumbent recovery. The initial, post-exposure and post-recovery concentrations of plasma ACTH, serum cortisol, serum melatonin, plasma norepinephrine and plasma epinephrine were determined. ACTH and cortisol indicated a slightly increased post-exposure level. Melatonin concentration did not change. Post-exposure norepinephrine levels increased (P less than 0.05) from the initial. Post-exposure epinephrine indicated a tendency to elevated levels with a nearly doubled (P less than 0.05) concentration in experiment A. The tendency toward enhanced ACTH and cortisol secretion and sympathetic activity shown by increased catecholamine secretion suggest that the winter swimming practice may raise the pain threshold and develop a potential for improved cold tolerance, possibly by nonshivering thermogenesis.