Determination of Butorphanol Using High Performance Liquid Chromatography in Small Volume Plasma Samples

Abstract

A high-performance liquid chromatography (HPLC) method has been developed and validated for the determination of butorphanol in bearded dragon plasma. Following a liquid extraction using ethyl acetate and hexane, samples were separated by gradient reverse phase HPLC on a Symmetry C 18 column and quantified using fluorescence detection at an excitation of 200 nm and an emission of 325 nm. The mobile phase was a mixture of 0.05 M ammonium acetate (pH, 4.1) and acetonitrile, with a flow rate of 1.3 mL/min. A linear curve was produced over the concentration range of 2.5 to 1500 ng/mL. Intra-and inter-assay variability for butorphanol were less than 10% and the average recovery was 90%. The new assay quantifies therapeutic levels of butorphanol in 100 µL samples from pharmacokinetic studies conducted at this institution.