1) Blood progesterone:
The early 1950´s saw the beginings of the use of physical chemical methods, such as paper chromatography and, spectrophotometry, in the isolation and identification of progesterone. This has resulted in increaced specificity. Some improvement in sensitivity was made available through the efforts of a number of workers during the past 10-12 years. A slight modification of a method which utilized paper chromatography later allowed measurement in the plasma of nonpregnant females where the level was reported to range from 0.02-0.:05ug/ml. However, these methods still required 10-25m1 of plasma for satisfactory determinations. -in 1963, Wool ever and Gold-fien described a double isotdpe derivative method for, detecting progesterone which required as 1 ml. of peripheral plasma in pregnancy and 5-8m1. in the nonpregnant state. And then, utilizing a combination of thin laver gas chromatography, Neill and his co-workers (1964) were able to measure plasma progesterone to 0. lug/ml.Recently (1967), competitive protein binding assay method was introduced.
2) Urinary pregnanediol:
Because of thedifficulty of working with plasma, most studies of progesterone metabolism in the past have been carried out by measuring the level of excretion of the metabolite of progesterone, pregnanediol. In 1955, !Copper and his co-workers, utilizing the extraction procedures of previous workers, and adding two chromatographic steps involving alumina columns, prose= nted a pregnanediol method that was specific, that resulted in a mean recovery of 90;´0 of added pregnanediol, and that was capable of measuring pregnanediol excretion to 0. 5mg/24hr.
But these physical chemical methods are complicated and time-consuming. The introduction of gas chromatography in steroid analysis has offered the possibility of a more rapid yet still specific and sensitive, approach to the problem. Acid hydrolysis, toluene extraction, and acetylation were still necessary, but the gas chromatography replaced column chromatography and spectrophotometric measurement. According to Sulimovici and his co-workers (1965), for the determination of the relatively low levels of pregnanediol in normal menstrual cycle, thin layer
chromatography alone may be sufficient.
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