| Phospholipids are natural biological surface active agents, widely exist in plants and animals. In addition to phosphatidyl choline (PC) as a principal component, phospholipids contain a small amount of phosphatidyl ethanolamine (PE), phosphatidyl inositol (PI), lysophosphatidyl ethanolamine (LPE), sphingomyelin (SM), lysophosphatidyl choline (LPC), glyceryl tripalmitate (TG), cholesterol (CH) and other components. They are not only the basic composition of animals and plants cell, nuclear and plasmid membrane, but also one of the basic materials in life. It has an important nutritional and medicinal value, which is widely used in medicine, food, chemical industry and so on. Due to the complexity of components and the lack of ultraviolet absorption, quality control of phospholipids drugs and excipients has been the research hotspot and difficulty in pharmaceutical analysis. This paper adopts respectively high performance liquid chromatography (HPLC)-evaporative light -scattering detector and supercritical fluid chromatography to separate and determinate phospholipid components of Poractant Alfa Injections(trade name:Curosurf) as the representative of phospholipid drugs and egg yolk lecithin, soybean lecithin as the representative of phospholipid drugs phospholipid drug excipients. It provides a technical means and reference basis for its quality control. The thesis is divided into two parts as follows:(1) A HPLC-ELSD method was established for the firstl time to separate and determinate nine phospholipid components in Curosurf. Compared with the analysis method of Curosurf with two-way thin layer chromatography only controling the PC content in registered standard, the method is simple, stable and has good reproducibility. It can provide a variety of datas for phospholipids determinatio n and is more suitable for the control of batch-to-batch consistency of the products among enterprise. In the optimization process of chromatographic conditions, diol, amino and different brand silica gel columns were investigated for effect on separation of phospholipid components. Chloroform-methanol-water-ammonia was choosed as the elution system. The experimental conditions including mobile phase ratio, flow rate and column temperature were investigated for effect on separation of phospholipid components in Poractant Alfa Injection. Finally, a silica column (4.6 mm×250 mm,5 ?m) was used as stationary phase. The mobile phase A was chloroform: methanol:water:ammonia water (78:21:0.2:0.4) and the mobile phase B was chloroform: methanol:water:ammonia water (59:35:5:0.4) by gradient elution. The column temperature was 25?. The flow rate was 1.0 ml/min and samples were detected by evaporative light scattering detector. Five batches of samples which involved two specifications in a manufacturer were determinated by the established method. Nine known phospholipids and an unknown component in five batchs of Poractant Alfa Injections were separated and determined. Nine known phospholipids were quantitatively analysed according to respective standards and an unknown component was quantitatively analysed according to SM standard. All kinds of method validation were investigated, which included reproducibility, linearity and range, limit of detection and recovery rate. The result showed phospholipid components were separated completely. Linear regression is made by logarithm value (lgc) of standard solution concentrations with logarithm value (1gA) of the corresponding peak areas. The linear relationships of nine phospholipids were good. The correlation coefficient were 0.9984 ?0.9995.Within-day and intra-day precision of the method were 0.81%?1.83% and 0.74% ?2.15%, respectively. Limit of detection were 4.1?106.5 ng and limit of quantification were 5.5?221.3 ng. The recovery rates were 98.5%?101.3%. The results of phospholipid contents in five batchs of Poractant Alfa Injections were as follows:CH 0.10-0.13mg/ml, PE 2.62?2.93mg/ml, PI 3.51?3.62 mg/ml, PS 0.50?0.56 mg/ml, LPE 0.05?0.07 mg/ml, PC 68.51?72.42 mg/ml, SM 1.10?1.15 mg/ml, LPC 0.1?0.34 mg/ml, an unknown component 1.07?1.26 mg/ml.(2) A SFC (Supercritical Fluid Chromatography) method was established for the first time to separate and determinate eight phospholipid components in egg yolk lecithin and soybean lecithin. The proposed method is proved to be simple, rapid, selective, accurate and reliable with good reproducibility by method validation. Compared with existed analysis method of phospholipids, organic solvent is used much less in this SFC method and the damage to human body can be considerably reduced in. It provides a new testing technology and idea of quality control for phospholipids. Combinating the supercritical fluid chromatography with evaporative light-scattering detector, a series of columns were investigated for effect on separation of phospholipid components, which involved a Zorbax Rx-SIL column (3.0 mm×100mm,1.8?m), a Zorbax Silica column (4.6mm×250mm,5?m), a Zorbax CN column (3.0 mm×100mm,1.8?m),a Zorbax NH2 column (4.6 mm×250mm,5?m), a Pursuit PFP column (4.6 mmx 150mm,5?m) and a LiChrospher 100 Diol column (4mm×250mm,5?m).. A Zorbax Silica column (4.6mm×250mm,5?m) and a LiChrospher 100 Diol (4mmx250mm,5?m) in series were choosed. The type and addition amount of additives in mobile phase B, flow rate, column temperature and parameter of evaporation light detector were investigated for effect on separation of phospholipid components. Finally the mobile phase A was carbon dioxide and the mobile phase B was methanol-ammonia solution (100:0.5) by gradient elution. The flow rate was 2.3 ml/min. The column temperature was 30? and samples were detected by evaporative light scattering detector. Eleven batchs of egg yolk lecithins in four manufacturer and four batchs of soybean lecithins in three manufacturers were determinated by the established method. Eight phospholipid components in samples were separated and determined quantitatively according to respective standards. All kinds of method validation were investigated, which included reproducibility, linearity and range, limit of detection and recovery rate. The result showed that the resolutions of phospholipid components were 1.90?11.92 and the theoretical plate numbers of phospholipid components were 3575?18786. Linear regression is made by logarithm value (lgc) of standard solution concentrations with logarithm value (1gA) of the corresponding peak areas. The linear relationships of eight phospholipids were good. The correlation coefficient were 0.9967?0.9998. Within-day and intra-day precision of the method were 0.2%?2.0% and 0.5%?2.0%, respectively. Limit of detection were 137?584 ng and limit of quantification were 273?1169 ng. The recovery rate were 97.1%?100.6%. The results of phospholipid contents in eleven batchs of egg yolk lecithins were as follows:TG 0.2%?1.8%, CH 0.4%?2.0%, PE 7.8%?15.0%, LPE 0.7%?1.0%, PI 0.7%?1.8%,PC 68.0%?89.4%, SM 1.0%?1.9%, LPC 0.6%?1.7%. The results of phospholipid contents in four batchs of soybean lecithins were as follows:TG 0.1%?0.8%, CH 0.2%?0.3%,PE 5.0%?17.8%, LPE 0.0%, PI 0.9%?1.6%,PC 53.6%?72.4%,SM 0.0%,LPC 1.0%?2.9%. |
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