Steroid Derivatization

Note The derivatized steroids can be extracted from the blue chromatogram zones with alcohol and quantitatively determined by means of the Zimmermann reaction, which is not interfered with by the presence of phosphoric acid and phosphomolybdic acid. A yellow background can be bleached by exposure to anunonia vapor [2]. 

Using a series of PASSflow reactors, with typical dimensions of 5 mm (i.d.) x 10 cm (long), Kirschning et al. (Kirschning and Gas 2003 Kirschning et al., 2001, 2006) demonstrated the ability to combine a series of immobilized reagents in order to perform a sequence of reaction steps. In one example, the group demonstrated an oxidation, followed by a silyl deprotection and reductive amination steps to afford a derivatized steroid 147, as illustrated in Scheme 38, in excellent yield and purity. 

One of the major advantages of LC-MS/MS over GC-MS or GC-MS/MS is that steroid hormones may be analyzed directly by LC-MS/MS without derivatization procedures, which are time-consuming and tedious [22,50-53], However, a number of studies demonstrated that the chemically derivatized steroid hormones were significantly more sensitive to LC-MS/MS detection than the underivatized hormones, because the neutral molecules of estrogens and metabolites might not be effectively ionized under electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) modes [4, 21, 25, 54, 55], In order to enhance the steroid hormone molecules sensitivity for LC-MS/MS analysis at pg/mL level, chemical derivatization is an effective technique for analysis of steroid hormones and metabolites. A list of derivatization reagents and application examples for steroid hormone analyses by LC-MS/MS and GC-MS are presented in Table 3. 

Hala D, Overturf MD, Petersen LH, Huggett DB (2011) Quantification of 2-hydrazinopyri-dine derivatized steroid hormones in fathead minnow (pimephales promelas) blood plasma using LC-ESI+/MS/MS. J Chromatogr B 879 591-598... 

Antagonists from Natural Sources. Merck performed a screen ( I-CXCLIO) on a library consisting of extracts from microbial, plant and marine sources [86]. A diverse series of hits was found, including sugar-derivatized steroids and dipyridinium salts. 

The literature indicates that t-butyldimethylsilylimida-zole is probably the most popular reagent for derivatizing steroids, and it is often used in conjunction with t-butyldi-methylchlorosilane. These reagents form derivatives under relatively mild conditions (room temperature, reaction... 

Gas-Liquid Chromatography of Un-derivatized Steroids. Simultaneous Determination of Urinary 17-Keto Steroids, Pregnanediol, Preg-nanetriol, and Pregnanetriolone Clin. Chim. Acta 56(1) 49-57 (1974) CA 82 1735y... 

Chromatography Steroids (54). The methyloxime-trimethylsilyl ether derivative has become the method of choice for derivatizing steroids. The success of this derivatization procedure is due to the ease by which both carbonyl and hydroxyl groups can be protected. 

One anabohc steroid, the presence of which has proven difficult to analyze, is stanozolol [10418-03-8] C2 H22N20. A metaboUte of the parent dmg, hydroxy stanozolol, detected in equine urine eight hours after ingestion of the parent dmg is actually identified, usually at very low levels. Analysis was done by Ic/ms/ms which had a shortened analysis time advantage over gc/ms procedures because of the elimination of the need for a derivatization step (33). 

In Older to improve the poor oral absorption of carbenicillin [4697-36-3] a bpophilic rndanyl ester has been formulated, Geocillin [33331-88-3] (5). Prednisolone [30-24-8] a steroid, is derivatized to its C-21 hemisuccinate sodium salt (6) to make it extremely water-soluble (108). 

Other examples of irreversible derivatization on treatment with iodine have been described for phenohc steroids (estrone derivatives [256]), morphine [257] and 23 other pharmaceuticals [258]. These reactions are probably favored by the presence of silica gel as stationary phase and by the influence of light. 

Pentafluorobenzyl bromide has been used in the derivatization of mercaptans [55] and phenols [36], m the analysis of prostaglandins [37], and in quantitative GC-MS [5S] 1,3 Dichlorotetrafluoroacetone is used for the derivatization of amino acids to the corresponding cyclic oxazolidinones and allows the rapid analysis of all 20 protein ammo acids [d] Pentafluorophenyldialkylchlorosilane derivatives have facilitated the gas chromatographic analysis of a wide range of functionally substituted organic compounds, including steroids, alcohols, phenols, amines, carboxylic acids, and chlorohydrms [4]... 

A. Molecular Ion Because of low volatility, most steriods are derivatized before analysis by GC/MS. Molecular ions are usually observed for steriods sufficiently volatile to be analyzed underivatized by GC/MS (see Figure 31.1) Some important steroids in urine include estrone, estradiol, estriol, pregnanediol, and 17-ketosteroids. which can be analyzed by GC/MS as the TMS or the MO-TMS derivatives. The plant steroids, such as camposterol, ergosterol. stigmasterol, cholestanol, and sitosterol, are generally analyzed as the TMS derivatives. 

Cyclic diethylsilylene and di-tert.-butylsilylene derivatives have been used to protect diol groups in steroids and prostanoids [539-541]. N,0-bis(diethylhydrogensilyl)trifluoro-methylacetamide simultaneously converts Isolated hydroxyl groups to the diethyllu ogensilyl ether allowing a single step derivatization toVp used for the analysis of corticosteriods. 

Membranes offer a format for interaction of an analyte with a stationary phase alternative to the familiar column. For certain kinds of separations, particularly preparative separations involving strong adsorption, the membrane format is extremely useful. A 5 x 4 mm hollow-fiber membrane layered with the protein bovine serum albumin was used for the chiral separation of the amino acid tryptophan, with a separation factor of up to 6.6.62 Diethey-laminoethyl-derivatized membrane disks were used for high-speed ion exchange separations of oligonucleotides.63 Sulfonated membranes were used for peptide separations, and reversed-phase separations of peptides, steroids, and aromatic hydrocarbons were accomplished on C18-derivatized membranes. 

The combination of a Stille and a Diels-Alder reaction has also been used for a derivatization of steroids, for example, in the synthesis of pentacydic compounds such as 6/1-189, as described by Kollar and coworkers [97]. Reaction of 17-iodoan-drosta-16-ene 6/1-187 with vinyltributyltin and various dienophiles such as diethyl... 

Derivatized compounds with fluorophores (amino acids, steroids, aliphatic amines, carboxylic acids, catecholamines, etc.)... 

Historically, the target analytes in clinical mass spectrometric applications were small, volatile compounds that could be analyzed by GC-MS (see Chapter 4). With time, new chemical preparation techniques and derivatization schemes broadened the scope of these metabolites to include fatty acids, amino acids, intermediates of glucose oxidation, phospholipids, steroids, neurogenic amines, nucleic acids, etc. The molecular weights (molar masses) after derivatization were less than 1000 Da, a mass range easily within the limits of most conventional mass spectrometers. 

Endogenous and exogenous androgens can be derivatized with trimethylsilyl (TMS) for hydroxy functions and by 0-methylation for ketones, and analyzed with GC-FID or GC-MS (Shimada et al., 2001). MS is more prevalent due to unequivocal identification and greatly increased sensitivity but FID is still used in laboratories for some steroids. Sterols have typically been analyzed by GC-FID and GC-MS with derivatization to optimize peak shape (Shimada et al., 2001), and bile acids can be derivatized with M-butyl ester-TMS ether and analyzed by GC-FID from plasma samples (Batta et al., 1998). Juricskay and Telegdy (2000) reported an assay capable of analyzing 28 steroids in urine samples using GC-FID. 

A prevailing problem with neuroactive steroid analysis by HPLC is the lack of sufficient chromaphores or fluorophores within their chemical structures to allow suitable spectrophotometric end-point detection such as with UV/VIS or fluorescence with adequate sensitivity. The multitude of structural isomers of the metabolites also decreases the applicability of RP-HPLC since the chromatographic profiles become very complex with co-eluting peaks. Due to these inherent problems, it is often necessary to derivatize this group of compounds prior to chromatographic separation and suitable end-point detection to allow their direct determination at physiological concentrations. 

Others have reported the use of fluorescent dansyl derivatives for the identification of 17a-oestradiol from serum samples (O Figure 2-2) (Nozaki et al., 1988). The derivatization of the keto functional group of particular steroids (ketosteroids) can also be achieved by hydrazone formation (O Figure 2-3) the most... 

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