| Objective:Four kind of furocoumarin compounds have been selected as extraction object to explaining the extracted mechanism of hollow fiber liquid phase microextraction (HFLPME) and dispersive liquid liquid microextraction (DLLME). Two sample preparation methods, in combination with high performance liquid chromatography (HPLC) have been critically compared for analytical performance in this paper.Method:The experimental conditions including hollow fiber, extraction solvent, solvent volume, extraction time, stirring rate in both mocroextraction techniques have been examined, and comparison through extraction efficiency, extraction time, purity, accuracy and operation were carried out.Results:Under the optimal experimental conditions, two methods have been developed to determine four furocoumarin compounds. No obvious difference was observed except extraction time. The enrichment factors ranged from 12.2 to 18.5 and 11.6 to 21.6 folds for HFLPME and DLLME, respectively, and the limits of detection for four compounds (psoralen, oxypeucedanin, imperatorin, isoimperatorin) by HFLPME/HPLC and DLLME/HPLC were ranged from 1.2 to 5.0 ng.L-1 and 1.0 to 3.0 ng.L-1, respectively. RSD<6.2%.Conclusion:Both methods were found to be simple, highly accuracy and precision, applied to extract weak polarit compounds. Compared with DLLME, HFLPME was easier to operate and more stable. While DLLME was much shorter time required to reach extraction equilibrium and no more special materials required. DLLME technique was less extraction time, and suitable for batches of samples pretreatment simultaneousl. HFLPME was stronger purification for multiplicity material by the selectivity of chemical structure properties of HF. For analysis of four compounds, the linear ranges of psoralen and oxypeucedanin were wider for HFLPME, but isoimperatorin was narrower than DLLME. The average recovery of 90.8%-106.5%and 97.2.1%-106.1%were obtained for HFLPME and DLLME, respectively. Because of both methods were higher extraction effficiency, psoralen as trace constitute in traditional Chinese medicine Radix angelicae dahurica was detected. Objectives:Four furocoumarin drugs were investigated as a model system:psoralen, oxypeucedanin, imperatorin and isoimperatorin. A hollow fiber liquid-liquid phase microextraction (HFLLPME) method, in combination with high performance liquid chromatography (HPLC), was applied to measure the free drug concentration in protein solution. A Klotz plot was employed for characterizing drug binding to bovine serum albumin (BSA).Methods:In the microextraction setup, the donor phase (protein or blank) was placed in a 2 mL vial into which was immersed the hollow fiber with the acceptor phase (n-heptitol) situated in the lumen. The microextraction was optimized for 60 min with 1800 rpm stirring. The analytes were extracted by LPME system and analyzed by HPLC.Results:The percents of drug-protein binding of four furocoumarin compounds were determined directly by hollow fiber liquid phase microextraction. The results suggested that the percent of drug-protein binding did not completely dependent on the total concentrations of drug and protein. The four furocoumarin drugs did not compete for binding sites of protein, and the percent of binding for each drug could be determined simultaneously by HFLLPME. The percents of binding for psoralen, oxypeucedanin, imperatorin and isoimperatorin to protein were 58%-62%,18%-21%,27%-33%and 40%-48%, respectively. The parameters of binding were estimated by Klotz linear regression analyses. The numbers of binding sites for psoralen, oxypeucedanin, imperatorin and isoimperatorin were 1.2,1.6,0.6 and 0.3, respectively. The binding constants were 1801,212,937.3 and 4025 M-1, respectively.Conclusion:This developed method presents a new, simple, improved and effective way for the determination of free drug concentration in protein solution, and it extends to the determination of drug-protein binding parameters, as well. |
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