Corticosteroids methods

Let us start with a classic example. We had a dataset of 31 steroids. The spatial autocorrelation vector (more about autocorrelation vectors can be found in Chapter 8) stood as the set of molecular descriptors. The task was to model the Corticosteroid Ringing Globulin (CBG) affinity of the steroids. A feed-forward multilayer neural network trained with the back-propagation learning rule was employed as the learning method. The dataset itself was available in electronic form. More details can be found in Ref. [2]. 

This structure encoding method has been applied both for the classification of a data set comprising 31 corticosteroids, for which affinity data were available in the literature, binding to the corticosteroid-binding globulin (CBG) receptor, and for the simulation of infrared spectra [28, 29). 

Efforts toward producing synthetic steroids, particularly cortisol, expanded during World War II to enable researchers to explore the possibiUty of medicinal appHcations of corticosteroids. In 1948, the discovery that cortisone dramatically alleviates the symptoms of arthritis led to intensive research on the antiinflammatory properties of corticosteroids. The development of partial and total syntheses for the commercial preparation of cortisone, alternative methods for producing cortisone, and the search for more potent antiinflammatory analogues gready stimulated both academic and industrial steroid research. 

Systemic corticosteroids (Table 80-4) are indicated in all patients with acute severe asthma not responding completely to initial inhaled /J2-agonist administration (every 20 minutes for three to four doses). Prednisone, 1 to 2 mg/kg/day (up to 40 to 60 mg/day), is administered orally in two divided doses for 3 to 10 days. Because short-term (1 to 2 weeks), high-dose systemic steroids do not produce serious toxicities, the ideal method is to use a short burst and then maintain the patient on appropriate long-term control therapy with inhaled corticosteroids. 

Immunoaffinity procedures have also been developed to selectively extract corticosteroids from different sample matrices. Thus, Seymour et al. demonstrated the higher efficiency of the immunoaffinity methods compared with the conventional extraction procedures using organic solvents [177]. Immunosorbents have also been used for online procedures followed by HLPC-UV [178, 179], HPLC-APCI-MS [179,180], GC-MS [176,181], or capillary electrophoresis [182]. Poly(hydroxyethyl methacrylate) (HEMA) was evaluated as a support material for the anti-dexamethasone antibodies used in IAC. The online IAC-HPLC-MS allowed determination of dexamethasone and flumethasone in equine urine with LODs in the range 3-4 ng mL-1 [180]. The cross-reactivity values obtained in the ELISA and the recoveries of an IAC-HPLC procedure are presented in Table 7. Bagnati et al. developed an immunoaffinity extraction... 

A mixture of six corticosteroids, namely deoxycortisone, hydrocortisone acetate, cortisone, prednisone, hydrocortisone and prednisolone can be assayed by HPLC method. The chromatogrpahic parameters for the assay are as follows ... 

L. Gagliardi, D. De Orsi, M.R. Del Giudice, F. Gatta, R. Porra, P. Chimenti and D. Tonelli, Development of a tandem thin-layer high-performance liquid chromatography method for the identification and determination of corticosteroids in cosmetic products. Anal. Chim. Acta 457 (2002) 187-198. 

The administration of spironolactone (Aldactone) interferes in the determination of 11-hydroxy corticosteroid by methods that depend on formation of fluorescence in strong sulfuric acid (W15). In 5 patients, the administration of the drug produced as much as a 5-fold increase in the apparent plasma cortisol levels. Aspirin interferes in the determinations of homovanillic acid (HVA) by a fluorometric method. The HVA fluorophore occurs at 320 nm and 420 nm, and acetylsalicylic acid produces fluorescence at 305 to 405 nm (H12). 

Ishii, D., Hibi, K., Asai, K., Nagaya, M., Mochizuki, K., and Mochida, Y., Studies of micro high-performance liquid-chromatography. 4. Application of micro pre-column method to analysis of corticosteroids in serum. Journal of Chromatography 156(1), 173-180,1978. 

De Beilis MD, Baum AS, Birmaher B, Keshavan MS, Eccard CH, Boring AM, Jenkins FJ, Ryan ND (1999) A.E. Bennett Research Award. Developmental traumatology. Part 1 Biological stress systems [see comments]. Biol Psychiatry 45 1259-1270 de Kloet ER, Joels M, Oitzl M, Sutanto W (1991) Implication of brain corticosteroid receptor diversity for the adaptation syndrome concept. Methods Achiev Exp Pathol 14 104-132 Delahanty DL, Raimonde AJ, Spoonster E (2000) Initial posttraumatic urinary cortisol levels predict subsequent PTSD symptoms in motor vehicle accident victims. Biol Psychiatry 48 940-947... 

The isoniazid method was applied to the determination of corticosteroids in urine [74] and to the determination of some steroids in pharmaceutical formulations [75]. 

The 2,6-di-tertbutyl-p-cresol reaction also produces fluorescent products with certain corticosteroids, which allows the development of fluorimetric methods for their determination. These will be discussed in a subsequent section. 

Reactions based on the presence of the 21-hydroxy-20-keto functionality (ketol group) are much more specifie for corticosteroids, and these methods have been used for the determination of corticosteroids in pharmaceutical formulations or in biological fluids [72]. For instance, only 21-unesterified corticosteroids react with sodium molybdate in acetic acid medium [81]. The blue color obtained by reducing the ketol group allows the determination of 40-200 pg of these steroids. 

The tetrazolium blue method was first recommended by Chen et al. [90], and was modified by several authors [91-94]. It has been the most widely used procedure in the routine determinations of corticosteroids. The following procedure has been recommended [88]. 

This method is specific for the 17,21-dihydroxy-20-keto corticosteroids (such as cortisone and hydrocortisone). The method depends upon the action of sulfuric acid with the 17,21-dihydroxy-20-keto groups to give rise to a glyoxal side chain. Subsequent condensation with phenylhydrazine yields a stable yellow color. This reaction was developed by Porter and Silber [98], and its mechanism was elucidated by Lewbart and Mattox [99] as follows ... 

The fluorimetric determination of corticosteroids in sulfuric acid-ethanol mixtures has given rise to numerous studies, particularly with the aim of estimating these compounds in urine or plasma. These all require prior methods of fractionation to permit a more or less selective separation of the various corticosteroids. The purified sample solution is then evaporated to dryness, and the residue treated with the reagent to yield fluorescence which can be measured [72]. 

Clarke described a chromatographic system for the identification of corticosteroids including cortisone [2]. The method uses Whatman paper 1, impregnated with a 40% v/v solution of formamide in methanol, which must be prepared immediately before use. A saturated solution of formamide in chloroform was used as the solvent system [125]. The paper was equilibrated for 2 1/2 hours in a tank that had been saturated with the solvent for 24 hours. Development was continued in a descending manner until the solvent front had advanced about 35 cm. To locate the analytes,... 

The system just described has been modified to identify corticosteroidal salts, including cortisone acetate. The solvent is a saturated solution of formamide in a 1 1 mixture of benzene and chloroform. Development is descending, until the solvent front has advanced about 22 cm [2,125], and the spot location is the same as described for the preceding method. The reported Rf values were cortisone acetate = 0.80, hydrocortisone acetate = 0.59, prednisone acetate = 0.74, and prednisolone acetate = 0.49. 

System (2) has been described for the assay of corticosteroids (cortisone, hydrocortisone, prednisone, and prednisolone) in urine [141]. Prior to introduction into the GC system, the sample was eluted with 2 1 ethyl acetate-methanol, the extracts evaporated to dryness, and then oxidized with sodium bismuthate. Used in the method was a silanized column (132 cm X 5 mm) containing 2,2-dimethylpropane-l,3-diol adipate (0.65%) supported on celite, and operated at 230°C. The carrier gas was argon, and the detector used strontium 90-ionization. The standard deviation was 3.5 % (based on 47 determinations). 

Corticosteroids have been determined by several GC-MS methods in various body fluids [179-181]. The following represent some selected examples of this methodology. 

System (1) has been described for the analysis of corticosteroids in urine, and uses HPLC and thermospray LC-MS technology. The method can be... 

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