Determination Of Dissociation Constants Of Huperzine A And Its Pharmacokinetics In Human Following Single Oral Administration

Huperzine A, a novel alkaloid isolated from the Chinese herb Huperzia serrata, is a potent, highly specific and reversible acetylcholinesterase (AChE) inhibitor. Huperzine A has a better penetration through the blood-brain barrier, higher oral bioavailability, and longer duration of AChE inhibitory action. Huperzine A has been approved to improve significantly the memory of elderly people and patients with Alzheimer's disease (AD) and vascular dementia (VD) without any notable side effects.Determination of dissociation constants of huperzine A by potentiometric titration method was described in this thesis. And a LC-MS-MS method for the determination of huperzine A in human plasma was developed, validated and successfully applied for the study of pharmacokinetics and bioequivalence.1 Determination of dissociation constants of huperzine AThe bibasic dissociation constants of huperzine A was assayed by potentiometric titration method. The sample solution(C=0.002 mol·L^-1) was titrated with hydrochloric acid volumetric solution (C_HCI=0.01 mol·L^-1). The results of the bibasic dissociation constants were estimated by three methods:(1)Nonlogarithmic titration curves: pK_al = 7.70(2) stepwise linear regression and simultaneous equations algorithm: pK_al = 7.51, pK_a2 = 3.20(3) nH function: pK_al= 7.53, pK_a2= 2.712 Pharmacokinetics of huperzine A in human following single oral administrationA simple, sensitive and selectivity LC-MS-MS method for the quantification of huperzine A in human plasma has been developed and validated and applied for the study of pharmacokinetics and bioequivalence in healthy volunteers.Huperzine A and pseudoephedrine hydrochloride (internal standard, IS) were isolated from human plasma by extraction with ethyl acetate, chromatographed on a Lichrospher C₁₈ column with a mobile phase consisting of 0.2% formic acid and methanol (15:85, v/v), and detected using a tandem mass spectrometer with an electrospray ionization interface. LC-MS-MS (ESI) was performed by using selected reaction monitoring (SRM) with ion transitions of m/z 243 to 226 for Huperzine A, m/z 166 to 148 for pseudoephedrine. The lower limit of quantification (LLOQ) was 0.0508 ng·mL^-1, and the assay exhibited a linear dynamic range of 0.0508-5.08 ng·mL^-1 (r=0.9998). The extraction recovery was more than 70%. And the inter and intra day precision was good with the RSD<15%.The method was successfully applied to investigate the bioequivalence between two kinds of tablets (test versus reference product) in 18 healthy male Chinese volunteers. After a single 0.2 mg dose for the test and reference product, the resulting mean of major pharmacokinetic parameters such as AUC_0-24, AUC_0-∞, C_max, T_max and t_1/2 of huperzine A were (16.35±3.42 ng·h·mL^-1 versus 16.38±3.61 ng·h·mL^-1), (17.53±3.80 ng·h·mL^-1 versus 17.70±3.97 ng·h·mL^-1), (2.47±0.49 ng·mL^-1 versus 2.51±0.51 ng·mL^-1), (1.3±0.4 h versus 1.2±0.3 h) and (5.92±0.75 h versus 6.18±0.66 h), respectively, indicating that these two kinds of tablets were bioequivalent.