Automated extraction and high-performance liquid chromatographic determination of serum clonazepam

We have developed a rapid extraction process using the DuPont Prep I automated extractor/concentrator to prepare serum samples for the high-performance liquid chromatographic (HPLC) determination of clonazepam. The drug, at an alkaline pH, is applied to a styrene divinylbenzene preparatory extraction column. The Prep I is a reversible centrifuge that allows the sample to pass through the preparatory column, followed by a wash solution of deionized water. The rotor then reverses direction and dispenses 20 ml of ethyl acetate, which elutes the adsorbed drug into an aluminum cup that is automatically dried at 68 degrees C. The extract is reconstituted with 100 microliters of mobile-phase, 50-mM sodium acetate (pH 5.4):acetonitrile:methanol (450:235:265, vol/vol). The chromatography is performed on a C-18 radial compression cartridge and detection is by absorbance at 313 nm. A plot of peak height ratio against concentration is linear to at least 160 ng/ml. The recovery of clonazepam with this automated extraction is 97.5%, compared with 90.0% for a manual extraction method. The coefficient of variation is 2.9% with the automated extraction. Only propranolol was found to interfere with clonazepam, whereas clorazepate interferes with the elution of the internal standard, nordiazepam.