Study On The Separation And Analysis Of Higenamine And Synephrine By Pre-Column Derivatization Hplc

Natural product drug is becoming the significant component of modem drug and the major source of the new drug discovery. Single enantiomer drugs, a chiral Pharmaceuticals of the Single Oriented structure was obtained from racemic drugs by remove the enantiomers which was invalid, inefficient or have some side effects, have been attracted much attention because of the purer active ingredients, the less dosage of the medication in treatment and the faster inducement as a new generation of chiral drug. Therefore, it is very significant to establish a approach for the separating of racemic enantiomers to obtain a single enantiomer drug.Higenamine and synephrine were nature compound which were extracted from natural world with one chiral center. They were have the biological activity which was cardiac and raising the blood pressure respectively."L" is the useful component in the exploration of them as a clinical drug.This dissertation studies on separation and analysis of higenamine and synephrine. It is separated into two major groups:Part1:Establishment of a HPLC method using chiral stationary phases (CsPs) for the separation of Higenamine and Synephrine. To extend the marching degree of the object with solid phase, the analyte was derivated in HPLC with a pre-column before the separation of Higenamine and Synephrine on Chiral PAKR AD-RH CSPS. The derivatization agent was (Boc)2O. Separation and deternation were achieved on a Chiral PAKR AD-RH (150mm×4.6mm,5μm) with the mobile phase of Ethanol/water(50/50) and Ethanol/water(45/55); detection wavelength,284nm and225nm; fluid rate,0.5mL/min. The Rf were2.0and3.42. The methods are simple, rapid and accurate. It is a new way for the separation of the larger quantity of the single enantiomer.Part2:Optimization the HPLC conditon for the Higenamine derivated with the fluorescence derivatization R-(-)/S-(+)-DBD-PyNCS, and the establishment of the method of determinating the Higenamine in rat plasma. The plasma was preprocessed by precipitating the protein with acetonitonitrile/methanol(20/400), then evaporated under the vacuum. The residue was redisolved by the0.1M HC1, and derivated with the fluorescence derivatization reagent S-(+)-DBD-PyNCS. Separation and deternation were achieved on a Diamonsil C18(2)(DiKMA), with the mobile phase of acetonitonitrile/methanol/water (30/20/50) with0.2%Formic acid, the Detecting wavelength, λex=450nm,λem=550nm; Flow rate,0.9mL/min. The retention time of higenamine enantiomers respectively were33.85min and35.07min, the degree of separation is1.54. It indicated that a good linear relationship when Higenamines were in the range of0.02μg/mL～200μg/mL (r=0.9991/0.9992), Lowest limit of quantification was20ng/ml. The extraction recovery of Higenamine in rat plasma was52.4～60.6%, and the method recovery was99.3～104.2%. The intra-and inter-day precisions of the method less than15%repectiely. The freeze-thaw stability is good. The method is sensitive and the condition of the action is mild. It is suitable for determination of Higenamine in rat plasma.