The aim of the study was to compare efficiency of various extraction methods of benzodiazepine derivatives: diazepam, estazolam,
flunitrazepam and nitrazepam. The study compared the recovery of benzodiazepines isolated from biological material (blood and
human blood serum) using liquid-liquid extraction and solid-phase extraction. The efficiency of each extraction was evaluated using
high-performance liquid chromatography with diode array detector. In addition, benzodiazepines immunoassay reactivity was estimated.
The following methods of extraction were used: liquid-liquid extraction (a classical liquid-liquid extraction and microextraction), solid-
-phase extraction (Baker’s columns and United Chemical Technologies’ (UTC columns). The reactivity was evaluated using V-Twin System
with EMIT technology by Siemens.
The results showed that the lowest recovery (nitrazepam – 16%, diazepam – 23%, flunitrazepam – 28%, estazolam – 37%) was obtained
using liquid-liquid microextraction of whole blood and the highest recovery was obtained in solid-phase extraction of whole blood
using United Chemical Technologies’ columns (nitrazepam – 86%, diazepam – 89%, estazolam – 91%, flunitrazepam – 94%). The lowest
recovery in classical liquid-liquid extraction was obtained for diazepam isolated from whole blood (36%), and the highest – for flunitrazepam
isolated from serum (74%). Solid-phase extraction with Baker’s columns was successful only in case of drugs isolation from serum and the recovery range from 57% to 89% for flunitrazepam. The results indicated higher efficiency of solid-phase extraction,
especially with use of columns specific for the extraction of benzodiazepines. The immunoassay analysis showed a decreased reactivity
of the tested benzodiazepine derivatives on the reagent used for the EMIT assay.
Comparative analysis of the recovery efficiency of selected benzodiazepine derivatives led to the conclusion that use of solid-phase
extraction should be considered more often in routine toxicological analysis. The knowledge of benzodiazepine derivatives cross-reactivity
in immunoassay method is essential for correct interpretation of obtained results.
