Determination of sub-nanogram amounts of dihydroergotamine in plasma and urine using liquid chromatography and fluorimetric detection with off-line and on-line solid-phase drug enrichment

A highly sensitive and selective high-performance liquid chromatographic method, involving sample pre-treatment, column switching and fluorimetric detection, is described for the determination of dihydroergotamine in plasma and urine samples. The pre-chromatographic sample treatment consists of extraction by means of an Extrelut column for plasma samples, and pre-separation with enrichment steps on a Sep-Pak column for urine samples. The samples are then injected onto a pre-separation column (Aquapore), and the fraction containing dihydroergotamine are automatically diverted onto an analytical column (ODS reversed phase). An acetonitrile-ammonium carbamate gradient is used as the mobile phase. High recovery of dihydroergotamine from both plasma (87%) and urine (100%) and a detection limit as low as 100 pg/ml were achieved, with a linear response up to 5 ng/ml. The assay demonstrated a high degree of selectivity with regard to the extensive metabolism of dihydroergotamine especially to the main metabolite 8'-hydroxydihydroergotamine. The assay was successfully applied for more than one year to the determination of plasma and urine concentrations of dihydroergotamine after parenteral administration.