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 high-performance 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 microgram/mL, and coefficients of variance were 1.14-2.17% suggesting high reproducibility.