Ion-pair reverse-phase high performance liquid chromatography method for determination of Huperzine-A in beagle dog serum.

Huperzine-A (Hup-A), a biologically potent, reversible acetylcholinesterase inhibitor for the treatment of Alzheimer disease (AD) in China, has very low blood concentration. In order to study the pharmacokinetics of newly developed Hup-A transdermal patches in animal, a rapid and sensitive ion-pair reverse-phase high performance liquid chromatography (RP-HPLC) method for the determination of Hup-A in beagle dog serum using mebendazole as internal standard has been developed and validated. The analyte and internal standard were extracted from serum using chloroform-isopropanol (95:5, v/v), analyzed on a C (18) column (5 microm, 150 mm x 4.6 mm i.d.) with a mobile phase consisting of methanol-water-glacial acetic acid (50:48.5:1.5, v/v/v), using sodium dodecylsulfonate as an ion-pair reagent, and detected with UV detector at 313 nm. The chromatographic run time was within 15 min. The assay was linear over the concentration range of 1-12 ng/ml and intra- and inter-day precision over this range was not more than 12.8%. The limit of quantification in serum was 1 ng/ml. The method was successfully applied to characterize the Hup-A concentration-time profiles and study the single and multiple doses phamacokinetics of Hup-A transdermal patches in beagle dogs. The pharmacokinetic study results showed that Hup-A patches has the characteristic of sustained or controlled drug release in vivo.