Loxapine represents an interesting example of old "new" drug and is recently drawing attention for its novel inhalation formulation for the treatment of both psychiatric and non-psychiatric disorders. It is extensively metabolized to several active metabolites with diverging pharmacological properties. To further pursue the contribution of metabolites to the overall outcome after loxapine administration, quantification of both loxapine and its active metabolites is essential. The current study developed a rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of loxapine and its five metabolites (amoxapine, 7-hydroxy-loxapine, 8-hydroxy-loxapine, 7-hydroxy-amoxapine and 8-hydroxy-amoxapine) in rat brain tissues, plasma and cerebrospinal fluid (CSF). By evaluating the effects of perchloric acid and methanol on analyte recovery, the extraction methods were optimized and only small amounts of sample (100 μl for plasma and less than 100mg for brain tissue) were required. The lower limits of quantification (LLOQs) in brain tissue were 3 ng/g for loxapine and amoxapine and 5 ng/g for the four hydroxylated metabolites of loxapine. The LLOQs were 1 ng/ml for loxapine and amoxapine and 2 ng/ml for the four hydroxylated metabolites in plasma, and 10 ng/ml for all analytes in CSF. The developed method was applied to a pharmacokinetic study on rats treated with a low-dose loxapine by oral administration. Four hours after loxapine dosing, high levels of 7-hydroxy-loxapine were found throughout the ten brain regions examined (68-124 ng/g), while only trace amount of loxapine was measured in brain (<5 ng/g) and plasma (<3 ng/ml). The method provides a useful tool for both preclinical and clinical investigations on the dispositions of loxapine and its metabolites, which would help to elucidate their roles in neurotherapeutics.
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