| This present thesis is composed of two parts. One part elucidates the quality research of a cancer therapeutic photosensitizer, Phtotocyanine and the second part explains the quantitative analysis of four active constituents in Tibet herb Gymnadenia conopsea by HPLC/DAD/MS.Part I Medical photonics is a recently emerged interdisciplinary field providing a broad variety of optical technologies and instruments for diagnostic, therapeutic and basic science applications in medicine. Phtotocyanine, consisting of four ZnPcS2P2 metal complex compounds of phthalocyanine, was synthesized by Fuzhou University and had been approved to carry out the clinical trials as a Class I new drug by State Food and Drug Administration of China. As a tumour-localising photosensitiser, it was used as photodynamic therapy (PDT) for solid tumors.Our work focuses on the quality control of Phtotocyanine as a cancer therapeutic photosensitizer. Phtotocyanine is a mixture which contains four ZnPcS2P2 type substituted Phthalocyanine isomers. In order to obtain the single component from Phtotocyanine, the mixture of four isomers possessing the similar structures and chemical property had been isolated and furified. An HPLC method with a mixture of methanol-acetonitrile-ion-pair buffer (0.01M hexadecyl trimethyl ammonium bromide and 0.01 M potassium dihydrogen phosphate, adjusted the pH value to 6.8 by potassium hydroxide solution) as the mobile phase was applied to isolate the four isomers by means of a semi-preparative C18 column (Grace Allsphere ODS-25μ250×10mm). To remove the salts which were mixed in the preparative product, a SPE C18 column was used to separate the salts by elution with water and then the marker component was eluted by methanol. Subsequently, a column of Sepherdex LH-20 gel was applied to elute the crudes with methanol to desalination. The purity of the isolated compound was measured by TLC and four different isomers of phthalocyanine were available. The chemical structures of them were elucidated by NMR spectra of 1H, gCOSY, NOE1D. They were 6, 5'-disulfonato-5',5"-diphthalimidomethyl phthalocyaninato zinc di-potassium salt,6, 5'-disulfonato-6',6"-diphthalimidomethyl phthalocyaninato zinc di-potassium salt,6, 5''-disulfonato-5',6"-diphthalimidomethyl phthalocyaninato zinc di-potassium salt,6, 5"'-disulfonato-6',5"-diphthalimidomethyl phthalocyaninato zinc di-potassium salt, respectively.An HPLC/DAD method was developed for simultaneously determination of four major isomers in Phtotocyanine with a C18 column (Grace Smart,150×4.6 mm i.d.,5 μm) The separation was carried out with a gradient program at a flow rate of 1.0 mL min-1 The mobile phase was a mixture of acetonitrile and ion-pair buffer (0.01M hexadecyl trimethyl ammonium bromide and 0.01 M potassium dihydrogen phosphate, adjusted the pH value to 6.8 by potassium hydroxide solution). The resolution values of four isomers were 2.5,1.20,1.33, and 1.8.Linear regression analysis for four compounds was performed by the external standard method. Four constituents were linear in the concentration range of 0.005 to 10 u g. The values of relative standard deviation (R.S.D.) of intra-day were 0.12%,0.66%, 0.99%, and 1.21%, respectively. The limits of detection for four compounds were 15 ng, 20 ng,12 ng, and 25 ng, respectively. This method was simple, accurate and reproducible. The developed method can be successfully applied to analyze isomers in Phtotocyanine.PartⅡGymnadenia conopsea is a Tibetan medicinal herb commonly used for improving the physique and intelligence which could be a promising nootropic medicine.An HPLC/DAD/MS method has been developed for simultaneously determination of four active constituents, dactylorhin B, loroglossin, dactylorhin A, and militarine present in the Tibetan herb Gymnadenia conopsea. The analysis was achieved on a C18 column (Pravil,4.6 mm x 150 mm,5μm) using a mobile phase of a mixture of methanol (A) and water (B) gradient elution in a total run time of 40 min (0 min:15:85; 10 min: 20:80; 20 min:45:55; 35 min:70:30) and a diode array detector was set at 222 nm. The flow rate was 0.7 mL·min"1. Orthogonal test was adopted to optimize extraction process of four active compounds from Gymnadenia conopsea. Linear regression analysis for four compounds was performed by the external standard method. All the curves were linear within the test concentration range. Four constituents were linear in the concentration range of 0.01 to 5.0μg. The average recoveries (n= 9) were 99.19%,99.16%,99.52% and 99.00% for dactylorhin B, loroglossin, dactylorhin A and militarine respectively (RSD<2%). The relative standard deviation of intra-day were 0.3%,0.4%,0.4%,0.5% and inter-day were 1.2%,1.5%,1.4%,1.5%, respectively. The limits of detection were 15ng,15ng, l0ng,20ng respectively.The method is simple, sensitive, reliable and reproducible which can be used for the quality study of Gymnadenia conopsea.
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