Determination of olanzapine in rat brain by means of HPLC with coulometric detection

Olanzapine (Zyprexa®, 2-methyl-4-(4-methyl-1-piperazinyl)-10H-thieno[2,3b][1,5]-benzodiazepine, OLA) is an atypical antipsychotic drug which demonstrates high interaction with serotonin 5-HT2A, 5-HT2C, 5-HT3, dopamine D1, D2, D4, adrenergic, histamine and muscarinic receptors. It has chemical structure and many therapeutic properties (activity against both negative and positive symptoms of schizophrenia) and side effects similar to those of clozapine, yet with the advantage that it seems to not cause agranulocytosis. Brain olanzapine levels are an important index which can verify if pharmacological effects are due to direct receptor occupancy or to secondary modifications due to the therapeutically effective treatment. Aim of the present study is the implementation of a sensitive and selective analytical method for the determination of brain olanzapine levels, based on the use of HPLC with electrochemical coulometric detection. Analyte separation was achieved on a C8 column (150×4.6 mm I.D., 5 µm) using a mobile phase composed of methanol and a pH 3.5 phosphate buffer containing triethylamine (21:79). Analyte detection was carried out using a Coulochem III coulometric detector (working electrode: porous graphite; reference electrode: alpha-hydrogen/palladium, auxiliary electrode: stainless steel). The guard cell was set at 0.000 V; the analytical cell was set at: detector 1, + 0.350 V; detector 2, -0.200 V. Sample pre-treatment was carried out by solid-phase extraction (SPE) on Oasis HLB cartridges, loading 500 µL of brain homogenate, eluting with methanol and obtaining a final sample volume of 125 µL. The extraction yield of this procedure is very good, always higher than 95%. The preliminary results of the method are very promising, with good sensitivity and repeatability. Assays are in progress to fully validate the method.