A specific immunoassay for the detection of flunitrazepam.

The development of an immunochemical procedure for the determination of flunitrazepam in whole blood is described. Flunitrazepam was derivatized in position 3 of the benzodiazepine ring to a hapten which was coupled to a carrier protein. To obtain antibodies, rabbits were immunized with these immunogens and the collected antisera were tested in a heterogeneous, competitive RIA. The antibodies showed a very specific reaction with flunitrazepam and hardly any cross-reactivity with related 1,4-benzodiazepines. Because of its high specificity the antiserum has the advantage of a definite determination of low levels of flunitrazepam without the risk of false-negative results obtained by using the commercially available group-specific test systems. The drug was extracted from whole blood in a simple batch process with a polystyrene suspension and the extracts were measured by RIA. The advantages of an immunochemical system, such as short analysis time and simple sample preparation, and the exactness of a drug-specific method are combined in this procedure, which allowed the specific and very sensitive determination of flunitrazepam in the low therapeutic range.