Development Of New Microemulsion Electrokinetic Chromatography System Using Phospholipids As Biosurfactant And Its Application In Prediction Of Drug Transport Across Blood-brain Barrier

Microemulsion electrokinetic chromatography(MEEKC)is a kind of electrokinetic chromatography technology that uses microemulsion solution as the separation media.It has been paid much attention since the first report due to its special quality including wide applicability,fast speed,less solvent-consuming,and strong solubilization capacity.It is especially applicable for the separation of complicated mixtures.Microemulsions are optically transparent and thermodynamically stable solutions which are formed by the nanometer-sized droplets dispersed in another immiscible liquid.Because microemulsion droplets(oil-in-water type,O/W)have internally hydrophilic,externally hydrophobic and anisotropic structures which are similar to biomembrane,MEEKC also can be used to simulate the passive diffusion process across biomembrane in theory like other biopartitioning chromatography(BPC)such as immobilized artificial membrane(IAM),microemulsion liquid/ electrokinetic chromatography(MELC/ MEEKC),micellar liquid/electrokinetic chromatography(MLC/ MEKC),and liposome electrokinetic chromatography(LEKC).Because the property of microemulsion composed of sodium dodecyl sulfate,which is the surfactant mostly used in MEEKC,is far from that of biomembrane,there is few reports about the applications of MEEKC on membrane permeability prediction.In this paper,a new type of microemulsion system using phospholipids and sodium cholate as biosurfactant was prepared and applied in capillary electrokinetic chromatography.The retention mechanism of solutes in MEEKC was studied by quantitative retention-structure relationship of some representative compounds.One of the developed microemulsion systems was selected to predict the steady-state brain/blood concentration ratio(log BB)of drugs to prove the application value of MEEKC as a high-throughput screening tool of blood-brain barrier penetration.Objective: It is to prepare some new types of microemulsion using phospholipids as the main surfactant which can be applied in electrokinetic chromatography and study the quantitative structure-retention relationships of solutes in these MEEKC systems by solvation parameters model.Then the phospholipids-MEEKC system is designed to predict log BB values of drugs as a high-throughput screening tool for blood-brain barrier penetration of drugs.Methods: Microemulsion systems were prepared using soybean phospholipids(SP),sodium dodecylsulfate(SDS),sodium cholate(SC)or potassium perfluorooctadecyl sulfonate(PFOS-K)as surfactant,n-butyl alcohol as co-surfactant,phosphate buffer as water phase and alkane,octanol,or IPM as oil phase.The particle size and Zeta potential were determined to investigate the stability of microemulsion stored at room temperature.The precisions of retention time and retention factors of three representative compounds including benzamide,o-toluidine,2-naphthol were determined to investigate the stability of MEEKC.Using dynamic coating capillary,dimethyl sulfoxide and dodecyl benzene as the marker of electroosmotic flow and microemulsion droplets,the retention factors(log k)of 26 small neutral compounds were determined in 18 stable MEEKC systems.Linear solvation energy relationship(LSER)equations of these MEEKC systems were developed to explain the contribution of intermolecular forces to solute retention.The vector distance between the coefficients of two LSER equations is used to evaluate the similarity of corresponding MEEKC systems.The log k values of 34 drugs with known log BB values were determined in a selected MEEKC system(ME5)at p H 7.4 using coated capillary.The correlationship between log BB and log k was evaluated and a prediction model of log BB values was constructed by log k values combined with other molecular paramaters.Results: 18 stable microemulsion systems were prepared.The different types and ratios of oil phase had no significant effect on the particle size and Zeta potential of microemulsions.The microemulsion was stable for a month at room temperature.Multiple linear regression equation was developed between log k and the Abraham molecular descriptors of 26 small neutral compounds.LSER characteristics of phospholipids-MEEKC systems were similar to that of other microemulsion systems.The absolute values of coefficients v and b were much larger,and v is positive,b is negative in all equations.The vector distance between any two MEEKC systems was less than 0.25.The different types and concentration of oil phase had no evident influence on these coefficients.Using the microemulsion system of 2.0% SP-3.5% SC-6.0% n-butanol-0.7% octanol-87.0% phosphate buffer(20 mmol·L-1 sodium dihydrogen phosphate buffer,p H 7.4)as the background mobile phase,the log k values of 34 drugs were determined.The correlation between log k and log BB was poor(log BB = 0.7192 log k-0.2713,R2 = 0.487,n = 34).When log k combined with other molecular parameters including polar surface area(PSA),molecular weight(MW),molecular volume(MV),the number of hydrogen bond acceptors/donors(HBA/HBD)and the number of freely rotable bonds(FRB),a good multiple linear regression equation was obtained(log BB = 0.468 + 0.411 log k-0.219 PSA/10 + 0.488 MW/100 + 0.088 HBA-0.007 HBD + 0.050 FRB-0.579 MV/100,R2 = 0.742,n = 34).Conclusions:1 The retention characteristics of phospholipids-MEEKC systems were similar to that of other MEEKC systems for the neutral solutes.That is the volume and HB basicity of solutes have the most influence on the retention and molecular volume is favorable to retention while HB basicity against retention.The change of microemulsion composition including oil phase and surfactant has insignificant effect on the separation selectivity of MEEKC.2 The log k values of drugs determined in the proposed phospholipids-MEEKC microemulsion system can not directly be used to predict the log BB values alone.Only when inputing other molecular parameters including PSA,MW,MV,HBA,HBD and FRB,an improved prediction model for log BB can be developed with log k by multiple linear regression.But the established phospholipids-MEEKC system have advantages of reproducibility,economy and rapidity,so it is still have potential as a high throughput tool of log BB prediction.