Fructus Aurantii Immaturus (枳实).pptVIP

* Agenda 8: Assay 1. Fructus Aurantii Immaturus (枳實) Determination of Synephrine in Fructus Aurantii Immaturus by HPLC Synephrine are used as the markers for the quality control of Fructus Aurantii Immaturus in C.P. (2005) Same markers are used as TLC and fingerprint Synephrine Molecular weight: 167 Chemical formula: C9 H13NO2 Selection of detection wavelength UV 224 nm (Synephrine) are chosen as detection wavelengths UV spectrum of Synephrine Selection of mobile phase (224 nm) Water – ACN (75:25) Isocratic Synephrine 30 32 68 32 68 0 ACN Water Time Same as fingerprint condition Water(0.1%SDS, 0.75%H3PO3) – ACN (68:32) Isocratic Synephrine The retention times of Synephrine are very different due to their polarity and structures. Synephrine + SDS = stability SDS-SO3- + Selection of extraction solvent 50% Methanol is chosen as the extraction solvent. ZS sample (0.5 g) + 25 mL solvent sonicate 30 min x 2 HPLC analysis 75% MeOH MeOH 2132940 50%MeOH Synephrine 272807 CH2Cl2 627431 EtOH 680923 H2O 1634510 25%MeOH 1975483 75%MeOH 1519732 MeOH Peak area Solvent One-way ANOVA (n=3) 50% MeOH CH2Cl2 EtOH H2O 25% MeOH 2025781 2025781 Selection of extraction method Sonicate 30 min twice is chosen as the extraction method. Peak area Extraction method Synephrine 1852777 Soxhlet extraction 2079484 Reflux 2118892 Sonicate One-way ANOVA (n=3) ZS sample (0.5 g) + MeOH HPLC analysis Reflux 2 hr Soxhlet Extraction 4 hr Sonicate 30 min x 2 Selection of extraction time Andrographolide: PA 1% 224 nm 3rd extraction 1st + 2nd extraction 224 nm Synephrine Synephrine HPLC condition for determination of andrographolide and dehydroandrographolide in Herba Andrographidis Column : Injection volume: Detector: Detector wavelength: Flow rate: Mobile phase: C18 HPLC column, Alltima, 4.6 x 250 mm, 5 mm or equivalent 10 μL PDA 224 nm 1.0 mL/min 0.075%phosphoric acid with 0.1%SDS Acetonitrile Gradient of mobile phase: Acetonitrile (%) Time 0.075%phosphoric acid