Method of Steroid Pattern in Urine by Gas Chromatography and its Applications
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Many advances have been made recently in the methods for analysis of steroids by gas liquid chromatography (GLC). These advances comprise the developments of new phases, new reagents and a new high-sensitivity detector (Ni-63). New silylation reagents, BSA (bistrimethylsilylacetamide), TMSI (trimethylsilylimidazole), chloromethyldimethylsilane and others, are very useful for steroid analysis.<BR>Trimethylsilylation of steroids, especially of corticosteroid methyloxime was investigated by using these reagents. TMSI is a stronger silylation reagent than BSA. Trimethylchlorosilame (TMCS) is an effective catalyst for silylation. Hydroxy groups on 3-, 20-, 21-, and other sterically non-hindered groups are trimethylsilylated with BSA for 10-30 minutes at room temperature. Sterically hindered hydroxy groups, such as 11-, 12-, and 17 positions, are trimethylsilylated with BSA-TMCS or TMSI-TMCS. Methyloxime derivatives of adrenocortical steroids and their metabolites can be analyzed by GLC as a single peak after trimethylsilylation of non-hindered hydroxy groups or all of the hydroxy groups. Thus, all neutral urinary steroids, 17-KS, pregnane derivative, 17-OHCS, and others, may be analyzed simultaneously without any preliminary separation by the temperature programmed method.<BR>Correlations between the urinary steroids and diseases may be studied most effectively by using this steroid pattern method. In the urine of a new born infant, as shown in Fig. 1, 16-OH-17-keto, 16-keto-17-OH, 16, 17-diOH-androsten-3β-o1, 21-OH and 16-OH-pregnenolone were found, but not so-called 17-OHCS. In case of adrenocortical cancer, a different urinary steroids profil from that of a normal person was obtained. As shown in Fig. 2, Δ<SUP>5</SUP>-pregnendiol, Δ<SUP>5</SUP>-pregnenetriol, 16-OH and 21-OH pregnenolone were found.<BR>Steroids and steroid derivatives are best characterized in terms of methylene unit (MU) values. These are determined by temperature programmed separations using n-paraffins as reference compounds. MV-Values of about 60 kinds of urinary steroids were determined on OV-1 and OV-17.<BR>Cholesterol and cholesterol ester, C<SUB>16</SUB> and C<SUB>18</SUB> ester, in serum can be determined simultaneously by GLC by using a short column and temperature programmed method from 200° to 300°C. Free and ester forms of cholesterol in the serum patients with various disease were determined by this method. This is useful for the determination of the function of liver, pancreas, and thyroid gland. In the case of some brain tumor and pancreas cancer, no ester form as observed in the serum.
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