Phase Microextraction Concentrated Trace Phenylalanine Active Ingredients Of Traditional Chinese Medicine, Enrichment And Chromatographic Analysis

Objectives Time-resolved binary-solvent synergy liquid-phase microextraction (TRBSS-LPME) combined with HPLC was developed for the determination of chlorogenic acid and its metabolites:caffeic acid, p-hydroxycinnamic acid, and ferulic acid, from biological specimens. chlorogenic acid and metabolites of transformations in the biosystem had been revealed, at the same time this experiment explained the TRBSS-LPME mechanism. The effect of biological matrix and emulsification phenomenon on the extraction efficiency was discussed.Methods The optimal extraction conditions of the target drugs were as follow:porous polyvinylidene difluoride fiber was used as solvent carrier, previously diluted and acidification with HCl aqueous solution as donor solutions (pH 2.1-2.4), and NaOH (pH12) aqueous solution as the acceptor solutions. Binary-solvent (n-pentanol:n-heptanol,7:3, v/v) as extraction solvent. In 1200 rpm, an extraction time for chlorogenic acid determination was set at 180 min in plasma and 60 min in urine, and an extraction time for metabolite determination was set at 40 min in plasma and 20 min in urine. The HPLC were carried out on a C18 column, the column was kept at 37℃during the whole sequence. The injection volume was 20μL and the detection wavelength was set at 320nm. Optimum separation in a short amount of time was achieved with a binary mobile phase using gradient conditions and a flow rate of 1.OmL/min. Solvent A was methanol and solvent B was 0.2% aqueous phosphoric acid. The gradient elution program was:0→6 min 22% A; 6→15 min,28% A, then returning to initial conditions.Results In plasma samples, the calibration curve of chlorogenic acid was linear in the concentration range of 1.0×10-3-13.5μg·mL-1, and the detection limit (LOD) was 0.1ng·mL-1; the calibration curve of caffeic acid was linear in the concentration range of 2.0×10-3-10.8μg·mL-1, and the LOD was 0.8ng·mL-1; the calibration curve of ferulic acid was linear in the concentration range of 8.0×10-3-13.5μg·mL-1, and the LOD was 5.0ng·mL-1; the calibration curve of p-hydroxycinnamic acid was linear in the concentration range of 2.5×10-3-5.5μg·mL-1, and the LOD was 2.0ng·mL-1, all correlation coefficient (r>0.99). The mean recoveries in plasma were 90.8%-119.8% for all enantiomers evaluated. The values of relative standard deviations (RSD) for intra-day and inter-day precision were lower than 5.0% and 8.4% in plasma, respectively. In urine samples, the calibration curve of chlorogenic acid was linear in the concentration range of 0.5×10-2-13.5μg·mL-1, and the LOD was 1.0ng·mL-1; the calibration curve of caffeic acid was linear in the concentration range of 10.0×10-2-13.5μg·mL-1, and the LOD was 50.0ng·mL-1; the calibration curve of ferulic acid was linear in the concentration range of 50.0×10-2-10.8μg·mL-1, and the LOD was 50.0ng·mL-1; the calibration curve of p-hydroxycinnamic acid was linear in the concentration range of 2.5×10-2-6.8μg·mL-1, and the LOD was 10.0ng·mL-1, all correlation coefficient (r>0.97). The mean recoveries of caffeic acid and p-hydroxycinnamic acid in urine were 81.6%-111.6% and the RSD for intra-day precision was lower than 6.3% in urine. These data suggested that chlorogenic acid and its metabolites were likely biologically transformations after the oral administration. Most of the chlorogenic acid was rapidly absorbed by microbial metabolism in the colon and only a small percentage of the parent compound was absorbed by the small intestine. Chlorogenic acid was hydrolyzed into caffeic acid by the microflora in the lower part of the gut, and then, caffeic acid was absorbed into the blood and hepato-enteral circulation occurred. Caffeic acid was metabolized in the liver and ferulic acid in the methyl fixation reaction was generated by O-methyltransferase activity. In the microsome metabolism, the oxidation reaction was catalyzed by CYP450 and the reaction converted ferulic acid into p-hydroxycinnamic acid. We hypothesize that chlorogenic acid is mainly metabolized by the kidney and excreted in the urine.Conclusion TRBSS-LPME was successfully used for the determination of target drugs in biological specimens. It not only extended the linear range of chlorogenic acid determination in biological samples and improved the sensitivity, but also eliminated interference from complex constituents in the biological specimens and reduced the LOD. Objective To study the concentration and enrichment of low abundance active ingredients such as cinnamic acid,p-hydroxycinnamic acid and methoxy cinnamic acid in traditional Chinese medicine Gui zhi fu ling pellet and Giant Typhonium Rhizome by liquid phase microextraction with back extraction (LPME/BE) and to determine cinnamic acid and its derivatives combined with high performance liquid chromatography. The relationships were provided between analytes concentration, donor phase volume and the enrichment factor.Methods Polyvinylidene difluoride fibre was used as solvent carrier,2.0×10-4mol·L-1HCl as the donor phase, 0.008mol·L-1 NaOH as the acceptor phase, n-heptanol 1 as organic solvent. In 1800rpm extraction time was 60min. The compounds were determined by HPLC after extraction. The HPLC were carried out on a C18 column, the column was kept at 28℃during the whole sequence. The injection volume was 20μL and the detection wavelength was set at 290nm. Optimum separation in a short amount of time was achieved with a binary mobile phase using gradient conditions and a flow rate of 1.0mL·min-1. Solvent A was methanol and solvent B was 0.2% H3PO4. The gradient elution program was:0→5min 40% A; 5→20min,42% A, then returning to initial conditions.Results The result showed that the linear range of cinnamic acid,p-hydroxycinnamic acid and methoxycinnamic acid were up to six orders of magnitude. The linear calibration curves of cinnamic acid, p-hydroxycinnamic acid and methoxycinnamic acid were obtained in the concentration ranges of 2.0×10-6-2.0μg·mL-1,2.0×10-7-2.0μg·mL-1,2.0×10-6-2.0μg·mL-1, correlation coefficient> 0.998. The limits of detection (LODs) were 0.2,0.08,0.2pg·mL-1, respectively. The intra RSD were less than 10.6% and inter RSD were less than 11.9%. The mean recoveries of Gui zhi fu ling pellet were in the range of 94.7%-101.3% and Giant Typhonium Rhizome were in the range of 92.1%-105.9%.Conclusion The method was simple and cinnamic acid, p-hydroxycinnamic acid and methoxy cinnamic acid were enriched to nearly 100 times in the best extraction conditions. It had a great significance on the analysis of low-abundance trace element which were difficult to treat with conventional pre-treatment method.