A study of the performance characteristics of immunoaffinity solid phase microextraction probes for extraction of a range of benzodiazepines

Immunoaffinity solid phase microextraction (SPME) probes have been developed with antibodies specific for the benzodiazepine class of drugs, covalently immobilized to glass rods. This involved both purification of the polyclonal antibodies to isolate the drug-specific fraction, and optimization of the immobilization procedure. Such probes have been used previously for the extraction of 7-amnoflunitrazepam. This article presents a comprehensive study of their performance and characteristics beyond that described previously, and an evaluation of their application to additional benzodiazepines. The influence of non-specific drug binding (nsb) was determined, with the result that nsb was found to be insignificant for the probes when used in their dynamic range. Immobilized antibodies had specific affinities in the range of 109–1010 M−1. Cross-reactivity was evaluated both for a range of benzodiazepines as well as a structurally unrelated molecule (erythromycin). For analysis of benzodiazepines individually or in the presence of erythromycin, limits of detection were 0.001–0.015 ng/mL depending on the antibody, and the dynamic range (based on 80–90% antigenic site occupancy) extended to 0.2–2 ng/mL.