The bioanalysis of the major Echinacea purpurea constituents dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamides in human plasma using LC–MS/MS

► An LC–MS/MS assay for the most abundant alkylamides (DTAI) in E. purpurea has been validated for quantification in human plasma. ► Most sensitive FDA-validated bioanalytical assay for DTAI with a quantification limit of 0.01ng/mL. ► Only 300μL plasma underwent simple liquid–liquid extraction. Run time was 7.5min. ► DTAI was successfully quantified in patients after ingestion of E. purpurea extract. Alkylamides are a group of active components of the widely used herb Echinacea purpurea (E. purpurea), which have immunostimulatory and anti-inflammatory effects. For the most abundant alkylamides, dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamides (DTAI), an LC–MS/MS assay has been developed and validated for quantification in human plasma. This assay will be used to support a clinical interaction study with E. purpurea. A 300μL plasma aliquot underwent liquid–liquid extraction with diethylether-n-hexane (50:50, v/v). After evaporization and reconstitution in 100μL of acetonitrile–water (50:50, v/v) 20μL of sample were injected into the HPLC system. Chromatographic separation was achieved with a Polaris 3 C18-A column (50mm×2mm ID, particle size 3μm), a flow rate of 0.3mL/min and isocratic elution with acetonitrile–water (50:50, v/v) containing 0.1% formic acid during the first 5min. Hereafter, gradient elution was applied for 0.5min, followed by restoration of the initial isocratic conditions. The total run time was 7.5min. The assay was validated over a concentration range from 0.01 to 50ng/mL for DTAI, with a lower limit of quantification of 0.01ng/mL. Validation results show that DTAI can be accurately and precisely quantified in human plasma. DTAI also demonstrated to be chemically stable under relevant conditions. Finally, the applicability of this assay has been successfully demonstrated by measuring the plasma concentration of DTAI in patients after ingestion of a commercial extract of E. purpurea.