A column of acetonitrile on diatomaceous earth is used to hold the corticosteroid on the column during the wash of the column with n-heptane to remove decomposition products and interfering substances, after which the corticosteroid and acetonitrile are eluted from the column with chloroform. The recommended procedure significantly reduces or completely eliminates the interference of various substances and certain decomposition products in the blue tetrazolium, phenylhydrazine, isonicotinic acid hydrazide, and UV methods of determination. The column can be readily modified to include acidic, basic, or neutral aqueous-trap layers when necessary. There is no significant difference in precision between the proposed procedure and the normal precision of the determinative methods. Results on typical pharmaceutical preparations, some of which show evidence of extensive decomposition, are given.
Keyphrases 0 Corticosteriod dosage forms-analysis 0 Acetonitrile-diatomaceous earth column-corticosteroid separation 0 Column chromatography-separation 0 Blue tetrazolium, phenylhydrazine, isonicotinic acid hydrazide methods-corticosteroid analysis 0 UV spectrophotometry-analysis
The determination of corticosteroids in many pharmaceutical preparations, other than those official in USP (1) and NF (2), can be difficult due to the interference of certain ingredients and because official methods do not always detect the presence of decomposition products of corticosteroids in the preparation. As a consequence, many "cleanup" procedures have been proposed to remove interferences and/or decomposition products before the actual final determinative step in the analysis of corticosteroids.
Levine (3) has reviewed the column partition chromatography of steroids and listed solka floc (4), silica (5, 6), siliconized diatomaceous earth (7), and diatomaceous earth (8) as the solid supports used. Jakovljevic et al. (9) used magnesium-silica gel' column chromatography with various solvent systems to remove the more polar interferences. Bracey et al. (10) utilized a column of methanol and water on acid-washed diatomaceous earth to remove interfering antibiotics from corticosteroid preparations.
This paper reports a new column partitioning chromatographic procedure which effectively traps the corticosteroid in an acetonitrile layer on a diatomaceous earth column while interferences are removed by washing with n-heptane. The acetonitrile and corticosteroid are then removed from the column with chloroform. The method may be modified readily for the removal of acidic, basic, and/or other water-soluble interferences. EXPERIMENTAL Apparatus-The following were used: Cary model 15 recording spectrophotometer ; glass chromatographic columns, 2.2 X 25 cm. constricted at one end to 0.4 X 5 cm.; and aluminum tamping tool to fit chromatographic column.
1 Florid, Floridin Co., Pittsburgh, PA 15222 Reagents-Solvents-All solvents were spectro, certified, analytical reagent, USP, or distilled-in-glass grade.
Acetonitrile, n-heptane, chloroform, methanol, and alcohol USP were used.
Acetonitrile-n-heptane (mutually saturated): mix 30 ml. of acetonitrile and 400 ml. of n-heptane in a separator. Agitate vigorously for at least 2 min. and separate when both layers are completely clear. These saturated solutions are to be used whenever n-heptane or acetonitrile is called for in these directions.
Chloroform (water saturated): add 50 ml. of water to 400 ml. of chloroform in a separator. Agitate vigorously for 2 min. and separate only when both layers are clear.
Diatomaceous earth,2 acid washed, was used. Standn~ds-Hydrocortisone, hydrocortisone acetate, prednisolone, prednisolone acetate, prednisone (all USP reference standards);