| The Chinese herb medicine perfusion fluid (patent number:ZL2010 1 0132974.7, China) was composed of 11 kinds of Chinese herbal medicine, such as Herba Polygalae Japonicae, Herba Violae Philippicae, Radix Astragali Mongolici, Herba Scutellariae Barbatae, etc. The Chinese herb medicine perfusion fluid was used for clinical treatment of dairy cow mastitis, which had antibacterial, anti-inflammatory, antipyretic, analgesic and immune enhancement effects. It is very difficult to separate and identify each active ingredient of the Chinese herb medicine prescription in a single way, because of its complicated chemical composition. Through the analysis and study of the prescription, high performance liquid chromatography (HPLC) method for simultaneous determination of 4 kinds of different active ingredients (chlorogenic acid, aesculetin, caffeic acid and rutin) in the Chinese herb medicine perfusion fluid was established in this study. Meanwhile, high-performance liquid chromatography-mass spectrometer (HPLC-MS) was adopted to verify the results, and to study the finger print of the prescription. This may provide a powerful reference to develop the quality standard of the Chinese herb medicine perfusion fluid.The results were as follows:1. Establishment of the method for simultaneous determination of 4 kinds of effective components in Chinese herb medicine perfusion fluid by HPLCHPLC method was established through comparing different chromatographic conditions. Chromatographic column:WatersC18 analytical column (4.6 mm×150 mm,5 μm), Phenomenex guard column. Mobile phase:acetonitrile (B)-5 ml/L glacial acetic acid (C). Elution mode:the gradient elution (0â†'18 min, B5%â†'17%, C95%â†'83%; 18 minâ†'30 min, B17%â†'23%, C83%â†'77%; 30â†'60 min, B23%â†'35%, C77%â†'65%; 60â†'65 min, B35%â†'5%, C65%â†'95%). Flow rate:1.0 mL/min. Detection wavelength: 325 nm. Column temperature:30℃. Standard and test samples can be separated effectively under this method, which could satisfy the simultaneous determination of 4 kinds of effective components (chlorogenic acid, aesculetin, caffeic acid, and rutin) in the perfusion fluid.2. Establishment of the HPLC-MS methodElectrospray ion source (ESI), positive and negative ion detection were used in this experiment. Ion spraying voltage was 4000 V. Capillary temperature was 300℃. Sheath gas velocity was 40 m/s. Auxiliary gas flow velocity was 10 m/s. Capillary voltage was 45 V. The tube lens voltage was 10 V. Sweep range was 100 to 1000 m/z. Full scan of positive and negative mode were used to collect data. MS was analysed through the Roadmap HompPage working software, and the retention time of major peak and the corresponding relative molecular mass were obtained.3. Thin layer identification of Chinese herb medicine perfusion fluidHerba Violae Philippicae, Pericarpium Citri Reticulatae Viride and Rhizoma Cyperi in Chinese herb medicine perfusion fluid were distinguished by thin layer chromatography (TLC). The results showed that the same color of the fluorescent principal spot at the corresponding position in the chromatogram of the test sample and the contrast drugs.4. Determination of effective components of Chinese herb medicine perfusion fluidThe established HPLC method was used to determine the effective components of Chinese herb medicine perfusion. The results showed that the average contents of chlorogenic acid, aesculetin, caffeic acid and rutin were 0.2483,1.1757,0.3071 and 1.8551 mg/g in the compound perfusion fluid, respectively. |
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