| Objectives To investigate the hollow fiber liquid-phase microextraction (HFLPME) mechanism of free anthraquinones, to compare their behaviors via two-phase HFLPME (2p-HFLPME) and three-phase HFLPME(3p-HFLPME) and to determine these compounds in Radix Polygoni Multiflori Preparata by HFLPME coupling high performance liquid chromatography (HPLC), especially microcrystalline and trace anthraquinones which could not be determined with rout sample processing methods.Methods With a self-made HFLPME system, experimental parameters of 2p-HFLPME and 3p-HFLPME were optimized, including the organic solvent supportor-hollow fiber, organic solvent, donor and acceptor phase, stirring rate and extraction time. For 2p-HFLPME, the analytes were extracted under 1800 rpm agitation for 60 min with MOF503 polyvinylidene difluoride hollow fiber as the organic solvent supportor, n-octanol as the organic phase and acceptor phase, 2 mmol/L HCl and 50 % methanol in the donor phase. After extraction, the acceptor phase was dried on 80°C water-bath, dissolved with 60μL methanol and then analyzed by HPLC; For 3p-HFLPME, the analytes were extracted at 1500 rpm agitation for 50 min with MOF503 polyvinylidene difluoride hollow fiber as the organic solvent supportor, n-hexanol as the organic phase, 100 % methanol solution as the donor phase and 1 mmol/L NaOH as the acceptor phase. After extraction, the acceptor phase was directly analyzed by HPLC. The optimal mobile phase was methanol-0.6% phosphoric acid with a composition of 90:10 (v/v) flowing at 1.0 ml/min and the temperature was 30°C. The HPLC column was a Kromasil C18 column.Results Under the optimal conditions, relationship between enrichment factor (EF) and concentration of anthraquinones (c) was EF = aLnc +b and EF at 1μg/mL (EF1μg/mL) was introduced for enrichment comparison of analytes; logPAPP (apparent n-octanol/water partition coefficient) of the five analytes were determined by 2p-HFLPME and shake flask method and logPAPP obtained by 2p-HFLPME was positively correlated with EF1μg/mL. Detection limits and presicion (RSD) of the analytes by 2p-HFLPME and 3p-HFLPME were 0.25~0.35 ng/mL and 2.9~20 ng/mL; 8.1%~14.1% and 2.5%~5.2%. Recoveries of the analytes in Radix Polygoni Multiflori Preparata by 2p-HFLPME were 75.4%~111.5%; 2p-HFLPME coupling HPLC was successfully applied to determine emodin, physcione, trace rhein and chrysophanol in Radix Polygoni Multiflori Preparata.Concusion 2p-HFLPME could be employed to quickly determine apparent n-octanol/water partition coefficient of free anthraquinones, providing trails for active constituents screening for traditional Chinese medicines; EF1μg/mL obtained by HFLPME of free anthraquinones under the optimal conditions could be applied to compare and estimate enrichment of homoplastic compounds, seting up LPME optimized models; 2p-HFLPME significantly surpassed 3p-HFLPME for analysis of microcrystalline and trace anthraquinones in Radix Polygoni Multiflori Preparata. Objectives To deeply penetrate the extraction mechanism of three-phase hollow fiber liquid-phase microextraction (3p-HFLPME) for hydroxybenzoic acids (HBAs) and investigate the correlativities between enrichment factor (EF) and structural and physicochemical parameters of HBAs; to determine HBAs in Calyx Kaki by 3p-HFLPME coupling HPLC, especially microcrystalline and trace HBAs which couldn't be analyzed with rout sample processing methods.Methods 3p-HFLPME behaviors of six HBAs (i.e.3,4,5-trihydroxybenzoic acid: 3,4,5-THBA, 3,5-dihydroxybenzoic acid: 3,5-DHBA, 3,4-dihydroxybenzoic acid: 3,4-DHBA, 4-hydroxybenzcic acid: 4-HBA, 2,5-dihydroxybenzoic acid: 2,5-DHBA and 2,4-dihydroxybenzoic acid: 2,4-DHBA) were worked-over with a self-made HFLPME system. Under the optimal 3p-HFLPME conditions, HBAs were extracted at 1200 rpm agitation for 35 min with MOF503 polyvinylidene difluoride hollw fiber as the organic solvent supporter, n-heptanol as the organic phase, 5 mmol/L HCl in the donor phase and 80 mmol/L NH3.H2O as the acceptor phase. After extraction, the extract was directly analyzed by HPLC at 246 nm. The optimal mobile phase was acetonitrile-0.15 % phosphoric acid (12:88) flowing at 1.0 ml/min and the Kromasil C18 column temperature was 35°C.Results Under the optimal 3p-HFLPME conditions, EF of HBAs showed good linearity with their conditional n-heptanol/water partition coefficients, pKa and sum of–OH (N); polyvinylidene difluoride hollow fiber contributed to promoting the extraction efficiency by forming charge transfer compound with HBAs and n-alkanols contributed to promoting the extraction efficiency by forming hydrogen bond with HBAs. Linear range, detection limits and EF of 3,4,5-THBA, 3,5-DHBA, 3,4-DHBA, 4-HBA, 2,5-DHBA and 2,4-DHBA were: 4.50×10-2~18.00μg/mL, 9.00×10-3~4.50μg/mL, 9.00×10-2~9.00μg/mL, 4.50×10-4~4.50μg/mL, 4.50×10-2~18.00μg/mL and 9.00×10-3~9.00μg/mL; 9, 1.8, 30, 0.09, 15 and 1.8 ng/mL; 14.9, 28.2, 31.9, 53.7, 91.2 and 107.6, the RSD was lower than 5.5 % and the average recovries of the model compounds in Calyx Kaki were 93.3%~107.1%. The optimized 3p-HFLPME coupled with HPLC was employed to determine HBAs in a traditional Chinese medicine Calyx Kaki.Conclusions Structure and acidity of HBAs, chemical constitution of the hollow fiber, acidity of the donor and the organic phase were critical factors deciding their extraction efficiency obtained by 3p-HFLPME. Correlativities between these parameters and EF could be employed to build optimal model for other HBAs, estimating the best 3p-HFLPME conditions and enrichment. For analysis of HBAs in Calyx Kaki, 3p-HFLPME could provide good enrichment factor, high sensibility and efficiently clean up the sample. 3p-HFLPME could be utilized to extraction, sample clean-up and concentration of microcrystalline and trace HBAs. |
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