A Novel Use of RIA and HPLC in the Identification of Plasma Metabolites of HR 158
The benzodiazepine HR 158 is active at dose levels of 1 mg, resulting in peak plasma levels of 5–6 ng/ml. Although radio-labelled metabolism studies in animals had indicated the nature of the main metabolites, these could not be confirmed in man due to the obligatory low doses. Moreover, conventional modes of analysis such as HPLC or GC were too insensitive for metabolite detection and identification in human plasma. The high sensitivity of radioimmunoassay (RIA) was used to overcome this problem. Of the two antisera available, one was highly specific for HR 158 furnishing plasma levels similar to those found by a specific HPLC method, and one was non-specific yielding much higher values and thus indicating the presence of cross-reacting metabolites. The latter was used on eluate fractions in an HPLC system capable of separating the known and putative metabolites in human plasma. The resulting RIA-chromatogram allowed identification at ng/ml level.
KeywordsPeak Plasma Level Plasma Metabolite Ammonium Carbonate Solvent Flow Rate Eluate Fraction
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