The Liquid-phase Microextraction / After Extraction

Objectives: The extraction and determination magnolol and honokiol in traditional Chinese medicine (TCM) samples was investigated by liquid phase microextraction with back extraction (LPME/BE) combined with high performance liquid chromatography (HPLC), which is a effective, convenient and environment-friendly method of pre-processing for TCM quality control.Methods: A self-made LPME system was adopted to select and optimize the LPME/BE conditions, such as the solvent carrier-hollow fiber, organic solvent, donor and acceptor phase, stirring rate and extraction time. The analytes were extracted by this self-made LPME system and analyzed by HPLC. The optimal extraction conditions of magnolol and honokiol in TCM samples were as follow, polyacrylonitrile fibre as solvent carrier, n-butanol as organic solvent, 25 % methanol aqueous solution adjusted to pH 5 by HCl as the donor phase, the acceptor solution of pH 12 adjusted by NaOH, a stirring rate of 900 r/min and extraction time of 30 min. The HPLC column was a C₁₈ column. The optimized mobile phase of HPLC was a mixture of methanol and water (82.5:17.5, v/v) flowing at a rate of 0.8 ml/min and the temperature of the column was 28°C. The analytes were analyzed at 294 nm.Results: The linear calibration curves of magnolol and honokiol in TCM samples were obtained in the concentration ranges of 1.56μg/ml～156μg/ml and 1.10μg/ml～110μg/ml, r>0.99, and the detection limits were 0.10μg/ml and 0.07μg/ml, respectively. The relative recoveries were in the range of 98.3 %～105 % and 96.8 %～103 %, respectively. RSD was lower than 2.9 %.Conclusion: LPME/BE was applied to eliminate the interference of matrices and decrease waste of organic solvent, and the results showed the possibility of this method for the determination of bioactive compounds in traditional Chinese medicines. Being friendly to environment, it is a quick and effective technique for TCM pre-processing. Objectives: Liquid phase microextraction with back extraction (LPME/BE) combined with high performance liquid chromatography (HPLC) was investigated for the extraction and determination magnolol and honokiol in biological samples. The method of pre-processing for TCM quality control was established.Methods: A self-made LPME system was adopted to select and optimize the LPME/BE conditions, such as the solvent carrier-hollow fiber, organic solvent, donor and acceptor phase, stirring rate and extraction time. The analytes were extracted by this self-made LPME system and analyzed by HPLC. The optimal extraction conditions of magnolol and honokiol in biological samples were as follow, polyvinylidene difluoride fibre as solvent carrier, n-butanol as organic solvent, 50 % methanol as the donor phase, 100 % methanol as the acceptor phase, 600 r/min of the stirring rate, and 10 min of the extraction time. The HPLC column was a C₁₈ column. The optimized mobile phase of HPLC was a mixture of methanol and water (82:18, v/v) flowing at a rate of 1.0 ml/min and the temperature of the column was 30°C. The analytes were analyzed at 294 nm.Results: The linear calibration curves of magnolol and honokiol in biological samples were obtained in the concentration ranges of 0.150μg/ml～30.0μg/ml and 0.100μg/ml～30.0μg/ml, r>0.90. The average recoveries of magnolol and honokiol were in the ranges of 93.7 %～114 % and 90.5 %～109 %, respectively. RSD were lower than 7.5 %. The detection limits (S/N=3) in plasma of rat were 30 ng/ml and 20 ng/ml, in liver were 25 ng/ml and 15 ng/ml and in kidney were both 10 ng/ml.Conclusion: LPME/BE was applied to eliminate the interference of matrices and decrease waste of organic solvent, and the results showed the possibility of this method for the determination of bioactive compounds in biological samples. Being friendly to environment, it is a quick and effective technique for biological samples pre-processing. Objectives: Some bioactive compounds were selected as they were representative and had definite chemical constitution, such as caffeic acid, ferulaic acid, P-hydroxycinnamic acid, methoxy cinnamic acid and cinnamic acid for explaining the extracted mechanism of LPME/BE. Establishing the relationships of concentration multiple and chemicals'distribution coefficient constitution and distribution coefficient. These relationships will be provide theory evidence to LPME/BE, which was used to separating, depurating and concentrating the TCM samples.Methods: A self-made LPME system was adopted to select and optimize the LPME/BE condition, such as the solvent carrier-hollow fiber, organic solvent, donor and acceptor phase, stirring rate and extraction time. The analytes were extracted by this self-made LPME system and analyzed by HPLC. The optimal extraction conditions of these five phenylpropionic acids in TCM samples as follows: polyvinylidene difluoride fibre as solvent carrier, n-enathol as organic solvent, HCl at pH 3 as donor phase and NaOH at pH 11.7 as acceptor phase, 1800 r/min of the stirring rate, and 60 min of the extraction time. The HPLC column was a C₁₈ column. The optimized mobile phase of HPLC was a mixture of methanol and 0.3 % H3PO4 (53:47, v/v) flowing at a rate of 1.0 ml/min and the temperature of the column was 28°C. The analytes were analyzed at 285 nm.Results:"Charge transfer supramolecule"extraction mechanism of LPME/BE was elucidated initially. The concentration multiple (EF) have good dependability with n-enanthol/water partition coefficient, R2=0.9653, and the regression equation was EF = 113. 99lgP?245.38. The linear calibration curves of phenylpropionic acids as follows: 0.25～2.5×10³ ng/ml of caffeic acid, 1.25 ng/ml～5.0×10³ ng/ml of ferulic acid, 0.10 ng/ml～1.0×10³ ng/ml of P-hydroxycinnamic acid, 0.50 ng/ml～2.0×10³ ng/ml of methoxy cinnamic acid and 0.20 ng/ml～2.0×10³ ng/ml of cinnamic acid, r>0.99. The intra-day precisions were lower than 6.3 % and inter-day precisions were lower than 6.6 %. The detection limit of caffeic acid was 0.025 ng/ml, 0.250 ng/ml of ferulic acid, 0.004 ng/ml of P-hydroxycinnamic acid, 0.100 ng/ml of methoxy cinnamic acid and 0.050 ng/ml of cinnamic acid. The content of caffeic acid in the Shuang huang lian peroral liquid was 0.2528 mg/ml, and average recovery was 100.3 %. The content of ferulic acid in the Dang gui concentrate pellet was 0.2137 mg/g, and average recovery was 99.2 %. The content of cinnamic acid in the Gui zhi fu ling pellet was 0.0128 mg/g, and average recovery was 99.4 %.Conclusion: Extraction mechanism of LPME/BE was elucidated initially. The relationships of concentration multiple and chemicals'distribution coefficient constitution and distribution coefficient were established. These theories provided evidence for LPME/BE as a skill for the pre-processing of TCMs. LPME/BE was applied to eliminate the interference of matrices and decrease waste of organic solvent, and the results showed the possibility of this method for the determination of bioactive compounds in traditional Chinese medicines. Being friendly to environment, it is a quick and effective technique for TCM pre-processing.