Steroids mixtures

In 1983, Prasad et al.12 first reported the condensation of chloromethyl polystyrene with /V-hydroxyphthalimide to give the ester, hydrazinolysis of which yielded the desired resin-bound hydroxylamine. However, the sole purpose of this reagent was to react with, and hence extract ketones from, a complex steroidal mixture, and its use for the solid-phase synthesis of hydroxamic acids was not explored. Recently, the exploitation of the above solid-phase approach for the synthesis of hydroxamic acids was independently reported by three groups,7-9 all of which differ only in the method for the initial anchoring of TV-hydroxyphtha-limide to an 4-alkoxybenzyl alcohol functionalized polystyrene or trityl chloride polystyrene. Subsequent /V-deprotection was... 

In their studies, Kushnir and co-workers [44, 45] found an increase in sensitivity of ESI detection of testosterone and adrenal steroids when they convert carbonyl groups to oximes. The steroid mixture is dissolved in 300 pi of an aqueous hydroxylamine solution (1.5 mol, pH 10), and following heating for 30 min at 90 C the derivatives are extracted with methyl t-butyl ether (2 ml). 

Using immobilized -glucuronidase reactors, estriol and estradiol glucuronides have been determined in urine by a column-switching technique (270, 271). Both glucuronides were hydrolyzed by the immobilized enzyme at pH 7. The steroid mixture was subsequently separated by gradient elution on a reversed-phase column, to be finally detected by UV absorbance at 280 nm. In this procedure, the activity of enzyme did not alter even after 150 h continuous run and exposure to a mobile phase containing 10% methanol. When a separate reversed-phase precolumn was inserted in the LC system, additional sample purification and shorter analysis time could be attained (272). 

Figure 10.5 shows the separation of the TMSI derivatives of a standard steroid mixture on an XE-60 column using this procedure. Table 10.2 lists the abbreviations used on these chromatograms. Figure 10.6 is the chromatogram obtained from the separation of the TMS derivatives of the steroids extracted from normal urine according to the above procedure. 

Figure 10.5 Gas chromatogram of the TMSI derivatives of standard steroid mixture on a 3% XE-60 column.

Active-Life Boldenone-7-8 day/Methandriol 2-3 days Drug Class Anabolic/Androgenic injectable steroid mixture Average Reported Dosage Men 6-12 ml weekly Women 3-6ml weekly Acne Yes (due to Methandriol)... 

Separations of complex steroid mixtures were achieved recently by Que et al. [76] using both isocratic and gradient elution. Mass spectrometric detection gave femto-mole detection limits while laser-induced fluorescence of dansylated ketosteroids ranged in attomole levels (Fig. 10.16). Monolithic column packings were used with a 35 cm (25 cm packed bed) x 100 pm i.d. capillary packed with a polymer prepared from 5% T (total monomer concentration), 60% C (total crosslinker concentration), 3% polyethylene glycol, 10% vinylsulfonic acid and 15% lauryl acrylate. Details of the monolithic column preparation can be found in refs. 36,76, and 193. Similar monolithic columns can be used for the separation of bile acids [194],... 

Specificity in chemical reactions that form products which have CD Cotton effects at distinctly different wavelength ranges from the parent can be used for the direct analysis of steroids and even steroid mixtures. For instance a simple dilution of the reaction mixture after the enzymatic production of prednisolone from hydrocortisone was used by Bartos [19] in the determination of these two compounds. Hydrocortisone is measured selectively at 326 nm where prednisolone has no dichroism. 

The application of gas chromatography (GC) to steroid analysis seems to have many difficulties due to then-insufficient volatility and thermolability. The development of high-resolution gas chromatography (HRGC) and various derivatization procedures enables the efficient separation of complex steroid mixtures for application in clinical, forensic toxicology, and natural products analysis. The development of low-cost MS detectors, in recent years, has also promoted the application of GC-MS systems for the analysis of the complex mixtures. 

Analysis of steroid mixtures using MIKE/CAD spectra under FI conditions (FI/CAD spectra) [197]... 

Fig. 10. GLC of trimethylsilyl derivatives of synthetic steroid mixture. (A) Procedure using 2% neopentyl glycol succinate on Chromosorb W, acid-washed, dimethyldichlorosilane treated, 80-100 mesh, in 6 foot X 5 mm i.d. glass column at 215° with 10 psi nitrogen. (B) Procedure using 35% XE-60 on Anakrom ABS (90-100 mesh), in 6 foot X 5 mm i.d. glass column, at 232°, with 5 psi nitrogen. Reproduced from Ruchelman and Cole (R4) with permission.

Fast, D.M. Culbreth, P.H. Sampson, E.J. Multivariate and univariate optimization studies of liquid-chromatographic separation of steroid mixtures. Clin.Chem., 1982, 28, 444-448 [also estriol, hydrocortisone, progesterone, testosterone]... 

A HPLC method for the simultaneous determination of prednisolone along other corticosteroids in swine plasma is described. Extraction of the steroid mixture from swine plasma with dexamethasone as internal standard was accomplished by solid-phase (SPE) extraction or by the more traditional... 

Because we know nothing about the potential pheromonal functions of the detected steroid classes (El, E2—3g, Etio) which induce ventilatory responses in Neogobius, the biological significance of the similar discriminatory abilities demonstrated by EOG and behavioural cross-adaptation is not clear. In particular, it is not known whether these be-haviourally active steroids are sex pheromones and, if they are, whether they function as a pheromonal mixture, or whether each steroid class functions as a distinct pheromone. It is also not known what role the ventilation rate response plays in pheromonal function. If the ventilation response serves only as a means to prolong exposure to a putative pheromone, novel individual compounds may evoke a ventilation increase, but a combination of steroidal odours may be necessary to activate a reproductive behaviour or physiological process. However, if encoding of the components of a steroid mixture occurs in and... 

The main field of application of TLC is the rapid characterization of steroid mixtures and checking of the purity of bulk steroid hormones. For the latter purpose the pharmacopoeias widely recommend the semiquantitative estimation of impurities based on visual comparison of their spots with those of reference materials. 

The amount to be applied to the layer in stability testing is governed in particular by the sensitivity of the method of detection and the related question, beyond what degree of decomposition the activity of the preparation becomes really impaired or the decomposition products show an undesireable side effect. An example may be seen in Fig. 168. The resolved steroid mixture can be detected with either dinitrophenyl-hydrazine (Rgt. No. 82) or sulphuric acid (Rgt. No. 241B), followed by inspection in UV light. In this particular case, the sulphuric acid reaction is too sensitive, since it detects impurities and degradation products which are without therapeutic interest [64]. 

Yamamoto et al. (69) have described the combination of TLC with secondary ion mass spectrometry for the determination of acetylcamitine and propionylcamitine in urine. Quantitation was accomplished with a stable isotope dilution method. Kajiura (70) described a TLC/SIMS application to the determination of phospholipid and steroid mixtures, with chromogenic reagents for spot visualization and either triethanolamine or glycerol as the extraction/ionization matrix. A similar application to phospholipids was described the same year by Hayashi et al. (71). Phospholipids as... 

As early as the visual comparison of the chromatograms produced with six different stationary phases, it can be seen that columns nos. 1 and 5 (Synergi POLAR RP andXTerra RP18, respectively) can be considered capable of separating the steroid mixture more effectively than the rest. The primary component is very well separated from the secondary component. The other columns shall no longer be considered for further use in method development due to their inability to separate the mixture. 

Although no simple methods have been developed for these isolations, chromatographic techniques have been used to advantage by many workers. Methods involving combinations of phase extractions and adsorption columns are often applied for the removal of extraneous materials and the concentration of the steroid mixture. There are usually trace... 

As indicated previously, the steroid mixture eluted from the adsorption column (Section VI,2,B) with ethylene chloride-acetone, 16 1, 14 1, 12 1, and 10 1, was now subjected to wide-strip paper chromatography. 

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