Liquid-phase Microextraction Analysis Of Protein Binding Parameters In The Determination Of Flavonoids Ingredient In Traditional Chinese Medicine

Objective: To establish a rapid sample pretreatment technology-solidification of floating organic drop liquid-phase microextraction (SFODLPME)combined with HPLC determination of eight kinds of low-abundance flavonoids ingredients dihydromyricetin, myricetin, fisetin, quercetin, naringenin, kaempferide, isorhamnetin and chrysin from traditional Chinese medicines(TCMs). In this method, the relations between the enrichment factor and concentration of flavonoids compounds were revealed, EF_1μg/mL was defined and expounded; solidification phase volume of floating organic drop and apparent partition coefficient of flavonoids compounds were calculated by the relations between the enrichment factor and the donor phase volume of flavonoids compounds.Method: The main parameters affecting the performance of SFODLPME were optimized. The optimal conditions of SFODLPME: 40μL of 1-dodecanol as extraction organic solvent was delivered to the surface of 5.00mL donor phase in vial and the solution was stirred under 600r/min for microextraction 40min at room temperature. The sample vial was standed to laminate, and then placed into a refrigerator at -20℃. 1-dodecanol was solidificated after 6 min. The solidified solvent was transferred into a pan small beaker and immediately melted, then volatilized solvent to dryness by hair dryer and cooled. The extraction was diluted to 50μL with methanol, 20μL was injected into a high performance liquid chromatography (HPLC) for analysis. Chromatographic conditions: the HPLC column was a Eclipse XDB-C₁₈ column(4.6 mm×150 mm,5μm). The mobile phase was acetonitrile, methanol and 0.3% phosphoric acid solution with gradient elution. The flow rate was 1 mL/min and 0.8 mL/min with gradient elution and the temperature was 35°C. In the wavelength of 300 nm, the content of eight flavonoids ingredients was determined in TCMs.Results: The linear range of SFODLPME-HPLC determination of dihydromyricetin, myricetin, isetin, quercetin, naringenin, kaempferide, isorhamnetin and chrysin were 0.01¹0.00μg/mL, the limits of detection (LODs) were 1.0⁰.01ng/mL(S/N=3). The intra RSD were less than 6.0% and inter RSD were less than 7.0%. The average recoveries of analytes in TCMs ranged from 96.0% to 104.2%. Solidification phase volume of floating organic drop was (52±14)μL when extraction solvent volume is 40μL; apparent partition coefficients of eight kinds of flavonoids ingredients ranged from 44.8 to 243.9.Conclusion: The method was simple and the enrichment factor was high under the optimal conditions. It had a great significance on the analysis of low-abundance trace elements which were difficult to treat with conventional pre-treatment method. Objective: Eight kinds of flavonoids compounds have been selected as model compounds to compare differences of extractive behaviors via two-phase hollow fiber liquid phase microextraction (2p-HFLPME) and three-phase hollow fiber liquid phase microextraction (3p-HFLPME), and to determine the content of flavonoids active ingredients in combination with high performance liquid chromatography (HPLC).Method: The main parameters affecting the performance of 2p-HFLPME and 3p-HFLPME were optimized by a self-made HFLPME system. The optimal conditions of 2p-HFLPME: the analytes were extracted under 1500 r/min agitation for 45 min with MOF503 polyvinylidene difluoride hollow fiber as the organic solvent supportor, 10^-5mol/L HCl in the donor phase, n-hexanol as the acceptor phase. After extraction, the acceptor phase was collected into a pan small beaker, dried by hair dryer, dissolved with 50μL methanol and then 20μL injected into a high performance liquid chromatography (HPLC) for analysis. For 3p-HFLPME, the analytes were extracted at 600 r/min agitation for 30 min with MOF503 polyvinylidene difluoride hollow fiber as the organic solvent supportor, 0.1mol/L HCl in the donor phase, n-hexanol as the organic phase, 10^-6mol/L NH₃·H₂O as the acceptor phase. After extraction, the acceptor phase was directly analyzed by HPLC. Chromatographic conditions: the HPLC column was a Eclipse XDB-C18 column(4.6 mm×150 mm,5μm). The mobile phase was acetonitrile, methanol and 0.3% phosphoric acid solution with gradient elution. The flow rate was 1 mL/min and 0.8 mL/min with gradient elution and the temperature was 35°C. In the wavelength of 300 nm, the content of eight flavonoids ingredients was determined in TCMs.Results: The linear range of 2p-HFLPME-HPLC determination of dihydromyricetin, myricetin, isetin, quercetin, naringenin, kaempferide, isorhamnetin and chrysin were 0.01¹0.00μg/mL, the limits of detection (LODs) were 0.01¹.0ng/mL(S/N=3). The intra RSD were less than 8.0% and inter RSD were less than 10.0%. The average recoveries of analytes in TCMs ranged from 95.0% to 101.2%. The linear range of 3p-HFLPME-HPLC determination of dihydromyricetin, myricetin, isetin, quercetin, naringenin, kaempferide, isorhamnetin and chrysin were 0.5²5.00μg/mL, the limits of detection (LODs) were 1.0¹00ng/mL(S/N=3). The intra RSD were less than 7.0% and inter RSD were less than 8.0%. The average recoveries of analytes in TCMs ranged from 94.7% to 99.3%. Conclusion: The enrichment factor of 2p-HFLPME was higher than 3p-HFLPME. 2p-HFLPME was obviously better than 3p-HFLPME for analysis of the content of trace flavonoids compounds from Geranium wilfordii, Ginkgo, Pagodatree flower bud and Platycladus orientalis in TCMs. 2p-HFLPME was more suitable for the analysis of the content of trace flavonoids compounds in TCMs. Objectives: Hollow fiber-liquid phase microextraction–high performance liquid chromatography(HF-LPME-HPLC) combined with Bjerrum and Scatchard methods was applied to simultaneous and fast research of protein binding rates, protein binding constants and binding sites of flavonoids compounds. It was discussed that six kinds of flavonoids compounds when coexisted competitively reacted on the protein binding. The protein binding rates of flavonoids compounds whether depended on concentrations were analyzed.Methods: Polyvinylidene fluoride (PVDF) was used as organic solvent carrier, n-heptylalcohol as extraction phase, pH 7.4 phosphate buffer solution (PBS) of sample solution as donor phase, 900 r/min of stirring rate and 2 h of extraction time, respectively. The free drug concentration after drugs and protein binding was determined by HPLC and accordingly calculated the binding rates of drug and protein. Bjerrum and Scatchard methods were employed for obtaining binding constants and binding sites of drugs and bovine serum albumin (BSA).Results: The protein binding rates of six flavonoids compounds were 29.3%, 56.8%, 12.2%, 25.7%, 25.2% and 12.8% respectively; the binding constants with BSA were 17900, 18300, 5900, 8900, 9900, 18100 respectively; the number of binding sites were 1.2, 2.8, 0.6, 1.3, 1.1, 0.5 respectively. Six kinds of flavonoids compounds when coexisted did not competitively combine with protein. The protein binding rates of flavonoids compounds did not depend on protein concentrations, but part of them depended on drug concentrations.Conclusion: This method was simple and quick, organic solvent less. The protein binding rates of many kinds of flavonoids compounds can be determined simultaneously and fast by the method.