Simplified solid-phase extraction method for determination of dihydroergotamine in rabbit and human serum using high-performance liquid chromatography with fluorescence detection

A rapid, selective and sensitive method for the determination of dihydroergotamine (DHE) in serum was developed. Dihydroergocristine (DHEC) was used as an internal standard. Human and rabbit serum samples were extracted using commercial solid-phase cyano (CN) columns. Proteins were washed from these columns with pure acetonitrile, resulting in clean extracts. Extracts were subsequently separated by HPLC in an isocratic way, using a reversed-phase C-18 analytical column. Fluorometric detection was performed at excitation and emission wavelengths of 277 and 348 nm, respectively. Calibration curves with amounts of DHE ranging from 2 to 32 ng, were linear. The limit of detection found for DHE was 0.2 ng, extracted from 0.5 mi rabbit or from 2.5 mi human serum. The limit of quantification in serum of both species was 0.7 ng. The method has been shown to be suitable for monitoring DHE in serum during pharmacokinetic studies in rabbits.