Colour Reactions for the in Situ Characterisation of Steroid Oestrogens on Thin-Layer Chromatograms

Lisboa, B. P.; Diczfalusy, E.

doi:10.1530/acta.0.0430545

Cited by 38 publications

(7 citation statements)

References 9 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…cinnamic aldehyde and p-dimethylaminobenzaldehyde; acids with salts, e.g. ammonium molybdate and perchloric acid (Witter and Stone, 1957); acids in combination with phenol reagents (Monder, 1964); metal salts (Lisboa and Diczfalusy, 1963) and iodine vapour (Thamm et al, 1963). A mixture of aromatic aldehydes with acids was often used as a derivatization reagent for steroids (SanniC et d., 1951).…”

Section: Discussionmentioning

confidence: 99%

Thin‐layer chromatography—the forgotten alternative for the quantitative determination of steroids

Golf

Graef

Schiller

et al. 1987

Biomedical Chromatography

View full text Add to dashboard Cite

We have developed a high performance thin layer chromatography (HPTLC) system for quantitative determination of androgens, corticosteroids, mineralocorticoids and gestagens on silicagel KG-60 HPTLC-plates with different solvent systems. A complete separation of androgens, gestagens and metabolites was achieved with dichlormethane/cyclohexane/acetone (70:25:5). Corticosteroids, mineralocorticoids and their derivatives were completely separated with diethylether/isooctane/isopropanol (70:25:5). The quantitative in situ fluorescence determination was carried out after post-chromatographic derivatization with cinnamic aldehyde, 4-dimethylaminobenzaldehyde and sulfuric acid. The sensitivity of detection was found between 500 pg and 1 ng per spot. The steroid metabolism as catalysed by rat liver microsomal oxidoreductases was measured by these procedures, and was compared with determination of steroids by gas chromatography (GC). According to HPTLC, steroids were reduced by NADPH-5 alpha-reductase (EC 1.3.1.4) in the order progesterone greater than testosterone greater than aldosterone greater than cortisol greater than corticosterone. The enzyme activities as measured by HPTLC agree well with those obtained by GC (r = 0.94). When turnover of enzyme assays, speed of determination, detection limit, application to labile steroids and costs of steroid determination are considered, all points speak in favour of HPTLC.

show abstract

Section: Discussionmentioning

confidence: 99%

Thin‐layer chromatography—the forgotten alternative for the quantitative determination of steroids

Golf

Graef

Schiller

et al. 1987

Biomedical Chromatography

View full text Add to dashboard Cite

show abstract

“…For the detection and characterization of the oestrogen sulphates on the chromatograms Folin-Ciocalteu reagent (Barton, Evans & Gardner, 1952), anisaldehyde (Lisboa & R. HAHNEL AND M. GHAZALI BIN ABDUL RAHAN Diczfalusy, 1963) and rhodizonic acid combined with hydrolysis in situ were used (Schneider & Lewbart, 1956 Characterization offree oestrogens. The free oestrogens in the hydrolysates were characterized by thin-layer chromatography in the following systems: (6) ethyl acetate-watersaturated n-hexane-ethanol (16:3:1, by vol.)…”

Section: Methodsmentioning

confidence: 99%

Separation of urinary oestrogen sulphates from oestrogen glucosiduronates on diethylaminoethyl-Sephadex columns

Hähnel

Rahman

1967

Biochemical Journal

View full text Add to dashboard Cite

1 Gradient elution (sodium chloride concentration gradient) on DEAE-Sephadex columns is used to separate urinary oestrogen conjugates of pregnancy urine. Changes in the shape of the gradient alter the chromatograms in a predictable manner so that the dispersion of peaks can be modified at will. 2. In a previous paper (Hahnel, 1965) on the separation of conjugated urinary oestrogens on columns of the anion-exchanger DEAE-Sephadex it was reported that oestrogen sulphates partly overlap with the oestriol 16-glucosiduronate peak. Preliminary experiments at that time had demonstrated that a satisfactory separation can in principle be achieved by suitable adjustment of the sodium chloride gradient used for the elution. Altering the shape of the gradient to fit the requirements indicated by previous chromatograms has already proved successful for separating the overlapping peaks of pregnanediol glucosiduronate and oestrogen 3-glucosiduronates (Hahnel & Ghazali bin Abdul Rahman, 1966 Rahman, 1966). A 10-30ml. sample of diluted pregnancy urine (urine-water, 2:1, v/v) was put on top of a DEAE-Sephadex column (50ml. burette, l1mm. internal diam., height of DEAE-Sephadex filling approx. 500mm. at the start of the experiment) prepared in water. When the urine had percolated into the bed material gradient elution was started. The new gradient for elution was obtained by filling into each of the nine identical chambers of a Buchler Varigrad 95 ml. of the following aqueous solutions: 2M-NaCl in chambers 8 and 9, 0 3M-NaCl in chamber 3 and 0-05M-NaCl in chambers 1, 2, 4, 5, 6 and 7. After completion of this gradient the DEAE-Sephadex column was further eluted with a gradient resulting from 50ml. of 2m-NaCl in chambers 1 and 2 and 50ml. of 4m-NaCl in chamber 3 (alternatively, the column was further eluted with 150ml. of 2m-NaCl). The column effluent was collected in fractions of 100 drops each. Samples of the effluent fractions were used for quantitative estimation of individual oestrogens by the method of Brown (1960) or of total oestrogens (Ittrich, 1958; Bradshaw, 1961).Chromatography of oestrogen sulphates. The fractions of DEAE-Sephadex chromatograms containing oestrogen sulphates were extracted with diethyl ether-ethanol (3: 1,

show abstract

“…The Chromatographie plate was sprayed with fluorescein and then inspected in u.v. light (Lisboa & Diczfalusy, 1963). Spots corresponding to the reference substances were eluted with methanol and an aliquot was taken for the determination of oestrogens according to Mitchell & Davies (1954).…”

Section: Methodsmentioning

confidence: 99%

“…(Kushinsky & Demetriou, 1963). The oestrogens were detected by their reactions with ferrichloride : ferricyanide,^-toluene sulphonic acid, and anisaldehyde (Lisboa & Diczfalusy, 1963).…”

Section: Methodsmentioning

confidence: 99%

AROMATIZATION OF 7α-Hydroxydehydroepiandrosterone AND OF ITS 3-Sulphate BY OVARIAN AND PLACENTAL TISSUE CULTURES

Stárka¹,

Janata²,

Novak³

1966

Journal of Endocrinology

View full text Add to dashboard Cite

show abstract

Colour Reactions for the in Situ Characterisation of Steroid Oestrogens on Thin-Layer Chromatograms

Cited by 38 publications

References 9 publications

Thin‐layer chromatography—the forgotten alternative for the quantitative determination of steroids

Thin‐layer chromatography—the forgotten alternative for the quantitative determination of steroids

Separation of urinary oestrogen sulphates from oestrogen glucosiduronates on diethylaminoethyl-Sephadex columns

AROMATIZATION OF 7α-Hydroxydehydroepiandrosterone AND OF ITS 3-Sulphate BY OVARIAN AND PLACENTAL TISSUE CULTURES

Contact Info

Product

Resources

About