| To prepare safe, effective, stable, easy to use of pharmaceutical preparations has always been our goal. Nowadays, there are a number of pharmaceutical preparations with complicated base. How to evaluate the quality of drugs needs high-efficiency methods as eyes to judge.In pharmaceutical preparation, except for active ingridents, there are still some other additives, especially macromolecular substances, which sometimes could affect the determination of analytes. The presence of coexisting components or macromolecules will result in a decrease of tolerance of analytical method; even have adverse effect on accuracy and precision. Therefore, it is necessary to establish a high-efficiency analytical pretreatment method to eliminate the matrix interference, further improve analytical sensivity, and guarantee to get a correct analytical result.Traditional pharmaceuticals, such tablets, granules, injection, etc, have relatively simple matrix with less interference. The active compounds can be separated from matrix by commonly used methods, such as solvent extraction, ultrasonic extraction, protein precipitation, etc. However, the analysis of some modern pharmaceuticals with complex matrix, such as liposomes, is relatively difficult. In liposome, employing phospholipid bilayer as membrance to enclose drugs does raise specific-targeting, controlled release in vivo, but, the instability of membrance increases the difficulty of characterization the qulity of liposome to some extent. How to maintain the original stable state of liposme, separate unentrapped drug from liposme, and accurately character the quality of liposome, is always a challenge faced by analytical workers. The Entrapment efficiency(EE) is of great significant parameter to control the quality of the liposome, which is known as a regulatory requirement. For liposomes containing lipophilic drug, the presence of microparticle or molecular aggregates, which are produced by water-insoluble unentrapped drugs in water, may affect the accuracy of entrapment efficiency. In this paper, a comprehensive evaluation of entrapment efficiency(EE) of liposomes containing lipophilic drug was carried out for four pretreatment methods(size-exclusion chromatography(SEC), solid-phase extraction(SPE), centrifugation ultrafiltration(CF-UF) and hollow fiber centrifugal ultrafiltration(HF-CF-UF)), which are used for characterization of EE of liposome. Each method has its own feature in characterization. This paper can provide a reasonable guideline for choosing methods for characterization of EE of liposome containing lipophilic drug.The quality of drug involves not only to guaranteeing the quality and quantity of active ingridents, but also to monitoring toxical ingredients, which is an important issue for safety of drug. Proprietary Chinese medicines(PCM), are made up of different kinds of traditional Chinese medicine or herbs extracts, and have heavier color, strong smell and complicated chemical components. They prone to mildew in the processes of manufacture, storage and circulation, and produce harmful fungal toxics, especially aflatoxins, which can not be ignored.In this topic, a novel multi-pretreatment technology for separation trace aflatoxins from complex, commercial, traditional proprietary Chinese medicines was established. Get rid of impurity interference and purity and enrich AFB1, AFB2, AFG1 and AFG2 from PCM. Children cough oral liquid was representative for evaluating the extraction efficiency of established pretreatment method. The method is simple, rapid, low cost, good reproducibility and higher purification efficiency. It provides a novel pretreatment method for purification, enrichment and detection of trace aflatoxins from PCM. Part 1 Theoretical study of characterization of liposomes containing lipophilic drugObjective: A comprehensive evaluation of entrapment efficiency(EE) of liposomes containing lipophilic drug was carried out for 4 pretreatment methods(size-exclusion chromatography(SEC), solid-phase extraction(SPE), centrifugation ultrafiltration(CF-UF) and hollow fiber centrifugal ultrafiltration(HF-CF-UF)), which are used for characterization of EE of liposome. This paper provides a theoretical guideline for choosing methods for characterization of EE of liposome containing lipophilic drug.Method: With Am Bisome as model, the entrapment efficiency was determined with SEC, SPE, CF-UF and HF-CF-UF pretreatment mthods. Analyze the difference of results by the above mthods. For SPE, some uncertainty reasons, which caused large difference in result, were studied and further analyzed through experiments. For CF-UF and HF-CF-UF methods, the reasons, which leaded to a deviation result, were analyzed by comparing the recoveries of different concentrations of reference solution. The chromatographic condition would be as follows, a Diamonsil C18 column(250×4.6mm, 5μm); mobile phase: acetonitrile-0.02 mol·L-1 ethylenediamine tetraacetic acid disodium salt(38:62,v/v) with the flow rate of 1.0 m L·min-1; the UV wavelength was set at 405 nm.Results: The EE of liposome by SEC was about 93%. It was only 6~13% with using C18 or HLB columns, which was caused by the interaction between stationary phase C18 or HLB and stabilizer in liposome. In CF-UF, there was no detection of unentrapped drug, which could be explained by concentration polarization. The experimental results showed that before and after ultrafiltration the concentrations of standard solution in sample reservoir were no change, however, with an increase of the concentration of standard solution, the recoveries decreased gradually. The EE of HF-CF-UF reached up to nearly 99%. When increasing the concentration of Am B from 0.67 μg·m L-1 to 21.4 μg·m L-1, the recoveries keep in constant. Continuously increasing the concentration up to 25.0 μg·m L-1, a dramatic result was achieved with the recoveries lower than 85%. This dramatic result was caused by the presence of microparticle or molecular aggregates.Conclusion: The HF-CF-UF process could maintain the original stable state of liposme without drug leakage, and effectively separate unentrapped drug from liposomes containing lipophilic drug, which seemed more suitable for characterization of the EE of liposome with a lower concentration of unentrapped drug. For SEC, it is more suitable for characterization of the EE of liposomes containing hydrophilic drug on the premise that the SEC process could accurately separate unentrapped drug from liposomes. As to SPE, C18 and HLB columns may be not suitable for characterization of liposomes composed of cholesterol or sodium deoxycholate as stabilizer owing to the interaction between the stationary phase and membrane. For CF-UF, it was also not suitable for characterization of the EE of liposome containing lipophilic drug. Part 2 Determination of Aflatoxin B1, B2, G1 and G2 in proprietary Chinese medicines with a novel multi-pretreatment methodObjective: A novle multi-pretreatment method was established for getting rid of impurity interference or purity, enriching AFB1, AFB2, AFG1 and AFG2 from complex, commercial, traditional proprietary Chinese medicines(PCM), and providing a reference approach for purifying and enrichment of aflatoxins from PCM.Method: The study of sample pretreatment method was as follows:(1) Purifying process: The ratio of methanol and sample and extraction time, the type and addition amont of dehydrant, as well as the type and addition amount of adsorbant were all optimized for improving purification efficiency.(2) Extraction process: The extraction conditions of AFG1 including methanol concentration, extraction time and GO amount were simultaneously optimized with Box-Behnken(BBD). A second-order polynomial regression model was used to predict the experimental results. At the same time, the addition amount of Na Cl was also investigated. Detection: Elution and separation were conducted on a phenomenexc® C18 column(250 mm×4.6 mm, 5 μm). Me OH was used as mobile phase A, Me CN was mobile phase B, and Phosphoric acid aqueous solution(p H=3.5) was used as mobile phase C. HPLC separation was achieved by an isocratic elution with a mobile phase consisting of A,B,C(3:3:5,v/v/v) at 0.8 m L·min-1. The post-column derivatization reagent was prepared by dissolving 500 mg of iodine in 100 m L of methanol and diluted to 1L with ultrapure water, and delivered at a flow rate of 0.3 m L·min-1. The reaction coil was maintained at 70℃. The eluate was monitored by a fluorescence detector set at excitation wavelength of 360 nm and emission wavelength of 450 nm. Method validation: Linear, accuracy, precision and LOD, LOQ were all determined.Results: The most optimization conditions were achieved.(1) Purifying process: The ratio of methanol and sample was 9:1 with extraction time 10 min, anhydrous magnesium sulfate 1.0 g, and neutral alumina 0.5g.(2) Extraction process: The most optimization condintions were that methanol concentration was 12.5%, extraction time was 15 min, and GO amount was 3.0 mg. Method validation:Good linear relationships have been achieved with the linear of AFB1 and AFG1 obtained from 0.125 to 2.50 ng·m L-1, and AFB2 and AFG2 from 0.0625 to 0.625 ng·m L-1.The recoveries were more than 83% with RSD less than 3.7%. The LODs of AFB1, AFB2, AFG1 and AFG2 were 0.027, 0.020, 0.041, and 0.021 ng·m L-1, respectively.Conclusion: The method is simple, good quantitative, strong tolerance,good reproducibility, as well as higher purification efficiency, which could be used as a reference method for purifying, extracting and enriching aflatoxins from PCM. |
PDF Full Text Request