7-[125I]iodoclonazepam: purification by high-performance liquid chromatography for use in a very sensitive benzodiazepine radioimmunoassay of broad specificity

The purification of 7-125I]iodoclonazepam by high-performance liquid chromatography (HPLC) for use in a very sensitive benzodiazepine radioimmunoassay is described. A silica column is used with a non-aqueous eluent and sequential ultra-violet and gamma-ray detection. A commercially available antiserum is used at a dilution of 1:1000. Blood samples are diluted 10-fold with buffer before analysis and only 25 microliters of diluted sample are required per assay tube. Benzodiazepines, but not the radiolabel, appear to be bound by blood proteins in competition with the antiserum and so, if undiluted blood is assayed, erroneously low results are obtained. The minimal sample requirement and the high sensitivity of the assay described here largely avoid this problem while maintaining acceptable detection limits. For diazepam, the detection limit is 2.5 ng/ml in blood or urine (after correction for the initial 10-fold dilution) and therapeutic or sub-therapeutic levels of many other benzodiazepines can be detected. In practice, the assay is reliable, simple to perform and extremely economical.