Subzero-temperature liquid-liquid extraction of benzodiazepines for high performance liquid chromatography

On the basis of the phenomenon that hydrophilic acetonitrile is separated from the aqueous phase at -20 degrees C, we employed a novel extraction method, "subzero-temperature liquid-liquid extraction", to extract benzodiazepines (estazolam and triazolam) from serum or aqueous solution for liquid chromatography. A 1:1 mixture of acetonitrile and the specimen was cooled at -20 degrees C for 20 min to separate the acetonitrile and aqueous phases. The acetonitrile phase was directly injected into a highperformance liquid chromatograph. Recovery rates of the drugs following the first subzero-temperature liquid-liquid extraction were 50.3 +/- 0.6 - 54.0 +/- 0.9%, which were lower than those (73.9 +/- 3.3 - 80.6 +/- 0.6% and 81.6 +/- 4.7 - 96.1 +/- 2.6%) of the first conventional liquid-liquid extraction using diethyl ether and solid-phase extraction using a Sep-Pak C18 column, respectively. However, three to four repeated subzero-temperature liquid-liquid extractions and conventional liquid-liquid extractions resulted in recovery of almost 100% of the drugs. In the chromatogram of the benzodiazepines recovered from serum by the subzero-temperature extraction, no coextracted component interfered with determination of the drugs. Detection limits of the drugs were 0.02 - 0.08 mu g/mL, land coefficients of variance were 1.14 - 2.17% suggesting high reproducibility.