| Microemulsion electrokinetic chromatography(MEEKC) is a kind of electrophoresis technology using microemulsion as the separation media. It has applied in the filed of the separation science extensively due to its special quality, such as wide applicability, fast speed, less solvent-consuming, et al. In addition, because microemulsion droplets exhibit ordered hydrophilic and hydrophobic microstructure, which resembles that of cell membrane in a certain extent, so the partition of drugs into microemulsion droplets can be simulated to the passive diffusion process across biomembrane. Then microemulsion chromatography can be used to study drug permeability across cell membrane. But the microemulsion system applied in electrokinetic chromatography mainly use sodium dodecyl sulfate as surfactant, whose composition and property are far from those of cell membrane. In this paper, a new type of microemulsion system was prepared using phospholipids as biosurfactant and applied to capillary electrokinetic chromatography. The retention mechanism of solutes in MEEKC was studied by quantitative retention-structure relationship of some representative compounds. The optimal microemulsion system was selected to simulate skin permeation process of drugs and establish quantitative retention-permeability relationship. The results were used to prove the application value of MEEKC as a high-throughput screening tool of drug membrane permeability. Part one Preparation of microemulsion systems used in MEEKC andthe repeatability determination of retention factors inMEEKCObjective: It is to prepare stable microemulsion systems using soybean phospholipid as the main surfactant and apply it to capillary electrokinetic chromatography. Then the inter-batch and inter-day precisions of solute retention factors in MEEKC will be studied.Methods: 1 Pseudo-ternary phase diagram method was used to determine the compositions of microemulsion with soybean phospholipids(SP), sodium dodecylsulfate(SDS), sodium cholate(SC) as the surfactant, n-butyl alcohol as co-surfactant, phosphate buffer as water phase and alkane, octanol, IPM as oil phase. The clear appearance which indicated the formation of microemulsion, and particle size and Zeta potential which revealed the microstructure properties of microemulsion droplets were used to observe the stability of microemulsion at room temperature and among the p H range of 3 to 10. 2 The inter-batch and inter-day precisions of retention time and retention factors of solutes in MEEKC were determined by three representative compounds including thiourea, o-toluidine, 2-naphthol. DMSO was used as the marker of electroosmotic flow and dodecyl benzene was used as microemulsion marker.Results: 1 Seven microemulsion systems were prepared. Particle size range was between 4.14 to 15.9 nm, Zeta range between-12 to-40.5 mv. The types of oil phase had no significant effect on the particle size and Zeta potential of microemulsions. The microemulsion was stable within the p H range of 3 to 10 at room temperature for a month. 2 Using the microemulsion system of 1.56% SP-1.5% SDS-7.5% butanol-0.85% heptane-0.16% acetonitrile-88.43% phosphate buffer(20m M sodium dihydrogen phosphate buffer, microemulsion system p H 9.2) as the background mobile phase, the inter-batch precisions of retention time and retention factors in MEEKC for three batches of microemulsions were 0.36% to 0.81% and 3.50% to 5.8%. The inter-day precisions of solute retention time and retention factors in MEEKC for the same batch of microemulsion were 1.0% to 1.6% and 0.7% to 3.0%.Conclusion: 1 Seven stable microemulsion system using soybean phospholipid as the main surfactant were prepared and applied successfully in MEEKC. 2 The inter-batch precision of retention time in MEEKC was good, but the inter-day precision for the same batch of microemulsion was not very ideal. Because DMSO and dodecyl benzene were injected with each sample, the repeatability of retention factors was better. Part two Study on the solute retention mechanism in MEEKC andselection of the optimal microemulsion system to predict drugskin permeabilityObjective: It is to elucidate the solute retention mechanism in MEEKC using phospholipid microemulsion system by linear solvation energy relationship(LSER) model and then choose the optimal microemulsion system to simulate skin penetration process according to the similarity of LSER equation coefficients for logk and skin permeability coefficient(log Kp).Methods: Enough compounds with diverse chemical property were collected and determined in MEEKC to obtain the logk values. Multiple linear regression equation was made between logk and the molecular descriptors of these compounds to clarify the intermolecular forces contributed to the partition of solutes into microemulsion. The normalized coefficients of LSER equations were used to estimate the similarity among various partition systems.Results: 26 small aromatic compounds and their Abraham descriptors were collected. The logk values and corresponding LSER equations were determined under the studied seven microemulsion systems. The volume(V) and hydrogen bond(HB) basicity of solutes had the maximum influence on the retention. The main changes of phospholipid-microemulsion system compared to SDS-microemulsion system were the basicity(a) increased and the cohesive energy(v) and its interaction with n or π electrons of solutes(e) decreased. The ME1, ME5 and ME7 systems are most close to skin penetration process according to the coefficients of LSER equations. ME7 system was selected to simulate skin permeability of drugs because the baseline and peak response were better.Conclusion: LSER equation coefficients showed that the influence of volume and HB basicity on partition was similar to other chromatographic systems, but the extent was different. The phospholipid-microemulsion system was different from SDS-microemulsion. Compared to the reported LSER characteristics of the skin permeation, the microemulsion composed of 2% SP, 3.5% SC, 6% n-butyl alcohol, 0.7% octanol and 87.8% phosphate buffer(20 m M sodium dihydrogen phosphate buffer) was suitable to simulate skin penetration process of drugs. Part three Prediction of log Kp and log P values by phospholipids-MEEKC systemObjective: With skin permeability coefficient(log Kp) and octanol-water partition coefficient(log P) as the objective functions, it is to establish the correlation equations between logk and log Kp or log P and evaluate the application value of MEEKC in predicting drug skin permeability and octanol- water partition coefficient.Methods: 34 drugs with known log Kp and log P values were collected. The logk values of 34 drugs were determined in ME7 system under p H 7.4 using coated capillary. The correlationships between logk and log Kp of log P values were studied for all 34 drugs and for the divided subgroups composed of acid, neutral and basic drug sets, respectively.Results: The logk values of 34 drugs had good linear correlation with log P(y=1.9124x+1.2263, R2 =0.7635, n=34), but no significant correlation with log Kp. The logk values of acid, neutral and basic drug sets were also correlated well with log P and the best result was for neutral drug set(y=1.6595x+1.0705, R2 =0.9393). While only the logk values of basic drug set showed a relative good correlation with log Kp values(y=2.1377x-6.7621 R2=0.7692, n=16). When introducing other physical and chemical parameters into multivariate linear regression equation, the correlation coefficient was improved(log Kp=-6.329-0.184MW/100+2.376logk-0.006PSA+0.186HBA-0.285HBD-0.043 FRB R2=0.882, F=111.745, SE=0.287, n=16). The established correlation equations between logk and log P for neutral drugs was applied to predict the log P values of eight natural products. The predicted log P values were all within ±0.3 log unit of experimental values.Conclusion: The proposed phospholipids-MEEKC microemulsion system can be used to predict the log Kp values of basic drugs, and log P values of drugs with diverse structures and properties. |
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