High Performance Liquid Chromatography Applied To Herbal Medicine Analysis

High performance liquid chromatography (HPLC) as an important modern measurement of separation and analysis is developed on the basis of classical Liquid Chromatography. Due to its convenience, high-speed, sensitivity and accuracy, it has been widely used in the areas of biology engineering, pharmacy, foodstuff, environment and petroleum so far.This thesis establishes several detection methods by Reversed-phase High-performance Liquid Chromatography (RP-HPLC). It includes review and research report. In the review section, information about high performance liquid chromatography such as characteristic, the development of chromatography technology, application in herbal medicine analysis and technology of herbal medicine fingerprint was summarized. In the next research report section, analysis methods of several herbal medicines were reported. That is: Simultaneous determination of flavonoids and triterpenoids in the leaves of kaki and fructus forsythiae by high performance liquid chromatography; Determination of three alkaloids in different parts of Coptis om eiensis; Study on fingerprints of rhizoma alpiniae officinarum by HPLC; Study on fingerprints of rhizoma alpiniae officinarum by HPLC.1. Simultaneous determination of flavonoids and triterpenoids in the leaves of kaki and fructus forsythiae by high performance liquid chromatographyA reversed-phase high performance liquid chromatography method for the separation and determination of three flavonoids (rutin, hyperoside and quercetin) and two triterpenoids (oleanolic acid and ursolic acid) was developed. The column was Eclipse XDB-C₁₈ (150 mm×4.6 mm, 5μm). The mobile phase was a mixture of methanol-acetonitrile-0.05 % H₃PO₄ (adjust to pH 3.0 with triethylamine ), column temperature was 30℃, DAD detector was performed at 360 nm within 0-24 min, 210 nm after 24 min. External standard was used and the calibration curves showed good linearity over the range of 1.632-489.6 mg/L (r=0.9995), 1.045-522.5 mg/L(r=0.9997), 1.349-134.9 mg/L (r=0.9996), 5.290-529.0 mg/L (r=0.9996) and 5.075-507.5 mg/L (r= 0.9996) for rutin, hyperoside, quercetin, oleanolic acid and ursolic acid, respectively. This method was sensitive, accurate, rapid and reproducible. It can be applied to separate and determine five compounds in the leaves of kaki and fructus forsythia.2. Determination of three alkaloids in different parts of Coptis om eiensisTo establish a HPLC method for the determination of the contents of berberine, pa(?)matine and jatrorrhizine in rhizome, fibre, lamina and petiole parts from Coptis om eiensis. A good chromatographic separation among the compounds was achieved using a ZORBAX Eclipse XDB-C₈ column (150×4.6 mm; 5μm) and a mobile phase consisting of acetonitrile-0.025 mol/L KH₂PO₄ (adjust to pH 3.0 with H₃PO₄ ) (25:75) at the flow rate of 1.0 mL/min. Column temperature was 30℃. DAD detector was performed at 342 nm. External standard was used and the calibration curves showed good linearity over the range of 0.0256-160μg/mL (r=0.9999), 0.0147-92.0μg/mL (r=0.9999) and 0.0371-116μg/mL (r=0.9999) for jatrorrhizine, palmatine and berberine, respectively. A detection limit of 0.005-0.010μg/mL was reached. This method was sensitive, accurate, rapid and reproducible. It can be applied to separate and determine three alkaloids in different parts of Coptis om eiensis.3. Study on fingerprints of rhizoma alpiniae officinarum by HPLCSet up the HPLC fingerprints of rhizoma alpiniae officinarum. The HPLC separation was performed on a Eclipse XDB-C₁₅(150 mm×4.6 mm, 5μm) analytical column gradient eluted with methanol-acetonitrile-water (adjust to pH 3.0 with acetic acid) at the flow rate of 1.0 mL/min. The column temperature was controlled at 30℃, and the detection wavelength was at 254 nm. 13 common peaks were found in the fingerprints of 12 batches rhizoma alpiniae officinarum, and good similarities with correlation coefficients higher than 0.9 were found in fingerprints between the herbs from different sources and the standard chromatographic fingerprint, which can be utilized for the identification of rhizoma alpiniae officinarum. The method is accurate and reproducible, providing a new reference for quality control of rhizoma alpiniae officinarum.4. Study on fingerprints of Folium Pyrrosiae by HPLCBy using the orthogonal design experiment to optimize the extraction condition for Folium Pyrrosiae, and the fingerprints of the extracts was set up by high-performance liquid chromatography. The HPLC separation was performed on a Eclipse XDB-C₁₈(150 mm×4.6 mm, 5μm) analytical column gradient eluted with acetonitrile-water (adjust to pH 3.0 with acetic acid) at the flow rate of 0.8 mL/min. The column temperature was controlled at 35℃, DAD detector was performed at 254 nm within 0-9.5 min, 285 nm within 9.5-35 min, 254 nm after 35 min. 22 common peaks were found in the fingerprints of 8 batches Folium Pyrrosiae, and good similarities with correlation coefficients higher than 0.9 were found in fingerprints between the herbs from different sources and the standard chromatographic fingerprint, which can be utilized for the identification of Folium Pyrrosiae. It is simple and rapid to identify and differentiate Folium Pyrrosiae collected from different sources with the method which providing a new reference for quality control of Folium Pyrrosiae.