Isotope dilution analysis using chromatographic separation of isotopic forms of the compound to be measured.

Using progesterone, testosterone, androstenedione, 11-oxoprogesterone and 11 beta-hydroxyprogesterone as models, a new form of isotope dilution assay has been developed. A known mass of deuterium-labelled steroid is added to the serum sample. High-performance liquid chromatography is used to separate endogenous steroid from its deuterium-labelled form. After separation, the two forms of the analyte are quantitated using conventional methods: radioimmunoassay, enzyme-linked immunoassay and, where the concentrations are high enough, ultraviolet light absorption. The ratio of the amounts of the two forms of the analyte is used to calculate the amount of unlabelled material in the original sample. The assay principle is quite general. A variety of high resolution methods are available to separate isotopic analogues of the same compound. A number of detection methods can be used to quantitate the separated isotopic forms. Extension of this principle to other fields of interest in bio-medicine is discussed briefly.