癌症治疗用光敏剂福大赛因的质量研究及HPLC/DAD/MS法测定藏药手掌参旺拉有效成分的含量
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摘要
本文共分为两部分,第一部为癌症治疗用光敏剂福大赛因的质量研究,第二部分为HPLC/DAD/MS法测定藏药手掌参旺拉有效成分的含量。
第一部分主要阐述了一类新药癌症治疗用光敏剂福大赛因的质量研究,包括了4个酞菁化合物同分异构体的分离纯化、结构鉴定以及含量测定方法学的研究。癌症治疗光敏剂福大赛因,是由4个异构体组成的ZnPcS2P2型取代酞菁化合物的混合物,目前正在进行临床试验。为了更好地对福大赛因进行质量研究和质量控制,需要对这4个结构相似、化学性质相近的同分异构体进行分离纯化,制备FD-1，FD-2,FD-3,FD-4 4个酞菁化合物对照品,并进行FD-1,FD-2,FD-3,FD-4 4种同分异构体HPLC/DAD含量测定方法学的研究,以单一成分来控制福大赛因的质量。
采用反相C18半制备柱(Grace Allsphere ODS-25μ250×10mm),以甲醇-乙腈-离子对缓冲盐溶液为流动相,通过反复进样分离和制备4个同分异构体。由于使用了不挥发性盐类及离子对试剂,故需要脱盐纯化对照品。首先采用SPE C18反相固相萃取小柱,依次以水、甲醇分别洗脱缓冲盐和离子对试剂和样品。除盐不完全部分又经过Sepherdex LH-20凝胶柱进一步进行分离,以甲醇洗脱分离法对4个单体进行脱盐纯化,得到4个异构体的纯品,并进行核磁共振’H谱,gCOSY, NOE1D谱测定。通过已知的酞菁母核结构特征,推断4个异构体的结构,分别为6，5"-二磺酸基-5',5"-二邻苯二甲酰亚胺甲基酞菁锌二钾盐；6,5'"-二磺酸基-6',6"-二邻苯二甲酰亚胺甲基酞菁锌二钾盐；6,5'"-二磺酸基-5',6"-二邻苯二甲酰亚胺甲基酞菁锌二钾盐；6,5'"-二磺酸基-6',5"-二邻苯二甲酰亚胺甲基酞菁锌二钾盐。
比较并优化了不同的色谱柱、流动相类型,以定量分析福大赛因4个不同的同分异构体。确定以反相C18色谱柱(GraceSmart RP18 5μ,4.6x150mm),以乙腈-0.01M十六烷基三甲基溴化铵-0.01MKH2PO4 (用KOH试液调pH值到6.8)为流动相进行梯度洗脱(0min 60%→30min 65%),流速为1.0ml·min-1,在674nm波长处检测,4个同分异构体FD-1,FD-2,FD-3,FD-4得到较好分离,分离度分别为2.5,1.20,1.33,1.8。同时对FD-1,FD-2,FD-3,FD-4四个异构体进行方法学研究,4个异构体在测定范围内0.005-10.0 u g呈良好的线性关系,方法的精密度(以R.S.D.%表示)分别为0.12%,0.66%，0.99%，1.21%,最低检出限分别为15ng，20ng，12ng，25ng。
本课题首次分离制备出了4个异构体的单体,并进行FD-1,FD-2,FD-3,FD-4 4种同分异构体HPLC/DAD含量测定方法学的研究,以单一成分来控制福大赛因的质量,并测定了9份不同批次的样品,完善了福大赛因的质量标准。
第二部分主要阐述了HPLC/DAD/MS法测定藏药手掌参旺拉有效成分的含量,并进行质量研究。兰科手参属植物手参Gymnadenia concpsea R Brown的块根别名旺拉、手掌参、佛手参、手儿参。有填精补髓、增强体质、生津止渴、安神增智的功效,主要有效成分有dactylorhin B, loroglossin, dactylorhin A, militarine。本研究建立了HPLC/DAD/MS方法同时测定手掌参旺拉中4种促智类有效成分的含量,并对不同产地的旺拉进行含量测定,以建立旺拉的质量控制标准。方法采用C18反相色谱柱,以甲醇-水梯度洗脱(0min 15%→10min 20%→20min 45%→35min 70%),流速0.7ml·min-1,在222nm波长处检测4种有效成分的的含量。正交实验优化药材提取方法并测定25个不同产地样品的含量。dactylorhin B, loroglossin, dactylorhin A, militarine线性范围为dactylorhin B 0.2-4.0μg, loroglossin0.01-5.0μg, dactylorhin A 0.08-4.5μg, militarine 0.08-4.5μg;方法的回收率为99.00%-99.52%(RSD＜2%),最低检出限分别为15ng，15ng，10ng，20ng,样品溶液的日内差和日间差测定结果分别为日内精密度0.3%,0.4%，0.4%，0.5%；日间精密度1.2%，1.5%,1.4%,1.5%。建立的HPLC/DAD/MS方法能简便快速的测定旺拉中有效成分的含量,鉴别不同产地药材质量优劣,为全面控制藏药旺拉提供了依据。
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.
第一部分主要阐述了一类新药癌症治疗用光敏剂福大赛因的质量研究,包括了4个酞菁化合物同分异构体的分离纯化、结构鉴定以及含量测定方法学的研究。癌症治疗光敏剂福大赛因,是由4个异构体组成的ZnPcS2P2型取代酞菁化合物的混合物,目前正在进行临床试验。为了更好地对福大赛因进行质量研究和质量控制,需要对这4个结构相似、化学性质相近的同分异构体进行分离纯化,制备FD-1，FD-2,FD-3,FD-4 4个酞菁化合物对照品,并进行FD-1,FD-2,FD-3,FD-4 4种同分异构体HPLC/DAD含量测定方法学的研究,以单一成分来控制福大赛因的质量。
采用反相C18半制备柱(Grace Allsphere ODS-25μ250×10mm),以甲醇-乙腈-离子对缓冲盐溶液为流动相,通过反复进样分离和制备4个同分异构体。由于使用了不挥发性盐类及离子对试剂,故需要脱盐纯化对照品。首先采用SPE C18反相固相萃取小柱,依次以水、甲醇分别洗脱缓冲盐和离子对试剂和样品。除盐不完全部分又经过Sepherdex LH-20凝胶柱进一步进行分离,以甲醇洗脱分离法对4个单体进行脱盐纯化,得到4个异构体的纯品,并进行核磁共振’H谱,gCOSY, NOE1D谱测定。通过已知的酞菁母核结构特征,推断4个异构体的结构,分别为6，5"-二磺酸基-5',5"-二邻苯二甲酰亚胺甲基酞菁锌二钾盐；6,5'"-二磺酸基-6',6"-二邻苯二甲酰亚胺甲基酞菁锌二钾盐；6,5'"-二磺酸基-5',6"-二邻苯二甲酰亚胺甲基酞菁锌二钾盐；6,5'"-二磺酸基-6',5"-二邻苯二甲酰亚胺甲基酞菁锌二钾盐。
比较并优化了不同的色谱柱、流动相类型,以定量分析福大赛因4个不同的同分异构体。确定以反相C18色谱柱(GraceSmart RP18 5μ,4.6x150mm),以乙腈-0.01M十六烷基三甲基溴化铵-0.01MKH2PO4 (用KOH试液调pH值到6.8)为流动相进行梯度洗脱(0min 60%→30min 65%),流速为1.0ml·min-1,在674nm波长处检测,4个同分异构体FD-1,FD-2,FD-3,FD-4得到较好分离,分离度分别为2.5,1.20,1.33,1.8。同时对FD-1,FD-2,FD-3,FD-4四个异构体进行方法学研究,4个异构体在测定范围内0.005-10.0 u g呈良好的线性关系,方法的精密度(以R.S.D.%表示)分别为0.12%,0.66%，0.99%，1.21%,最低检出限分别为15ng，20ng，12ng，25ng。
本课题首次分离制备出了4个异构体的单体,并进行FD-1,FD-2,FD-3,FD-4 4种同分异构体HPLC/DAD含量测定方法学的研究,以单一成分来控制福大赛因的质量,并测定了9份不同批次的样品,完善了福大赛因的质量标准。
第二部分主要阐述了HPLC/DAD/MS法测定藏药手掌参旺拉有效成分的含量,并进行质量研究。兰科手参属植物手参Gymnadenia concpsea R Brown的块根别名旺拉、手掌参、佛手参、手儿参。有填精补髓、增强体质、生津止渴、安神增智的功效,主要有效成分有dactylorhin B, loroglossin, dactylorhin A, militarine。本研究建立了HPLC/DAD/MS方法同时测定手掌参旺拉中4种促智类有效成分的含量,并对不同产地的旺拉进行含量测定,以建立旺拉的质量控制标准。方法采用C18反相色谱柱,以甲醇-水梯度洗脱(0min 15%→10min 20%→20min 45%→35min 70%),流速0.7ml·min-1,在222nm波长处检测4种有效成分的的含量。正交实验优化药材提取方法并测定25个不同产地样品的含量。dactylorhin B, loroglossin, dactylorhin A, militarine线性范围为dactylorhin B 0.2-4.0μg, loroglossin0.01-5.0μg, dactylorhin A 0.08-4.5μg, militarine 0.08-4.5μg;方法的回收率为99.00%-99.52%(RSD＜2%),最低检出限分别为15ng，15ng，10ng，20ng,样品溶液的日内差和日间差测定结果分别为日内精密度0.3%,0.4%，0.4%，0.5%；日间精密度1.2%，1.5%,1.4%,1.5%。建立的HPLC/DAD/MS方法能简便快速的测定旺拉中有效成分的含量,鉴别不同产地药材质量优劣,为全面控制藏药旺拉提供了依据。
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.
引文
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