High Performance Liquid Chromatography earthworm granules in the content of hypoxanthine_678

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High Performance Liquid Chromatography earthworm granules in the content of hypoxanthine_678

    High Performance Liquid Chromatography earthworm granules in the content of hypoxanthine

     Abstract Objective To determine the earthworm granules in the content of hypoxanthine. Methods RP-HPLC method. Column:

    Inertsil ODS-EP column, mobile phase: water - methanol -

    tetrahydrofuran (100:0.5:0.5), flow rate: 1. 0 ml * min-1,

    detection wavelength: 254 nm. Results hypoxanthine at 0.040 6 ~ 0.406 0 μg between the peak area and showed good linear relationship, r = 0.999 9; the average recovery was 97.6%, RSD

    of 0.63% (n = 9). Conclusion The method is simple, accurate, and can be used as a method for the determination of the preparation.

     Key words Earthworm granules hypoxanthine RP-HPLC

     Abstract: ObjectiveTo determine the content of

    Hypoxanthine in Pheretima Compounding Granules.MethodsAn RP-

    HPLC was established.The chromatographic column was Inertsil ODS-EP.The mobile phase was H2O-CH3OH-C4H8O (100:0.5:0.5). The

    flow rate was 1.0 ml * min-1, and the detection wavelength was 254 nm. ResultsHgpoxanthine showed good linear relationship in the range of 0.040 6 ~ 0.406 0μg. The average recovery for

    Hypoxanthine was 97.6% and RSD was 0.63% (n = 9). ConclusionThis method is simple, accurate and can be used for content determination of Pheretima Compounding Graunles.

     Key words: Pheretima Compounding Granules; Hypoxanthine; RP-HPLC

     In Long Branch for the giant earthworm Pheretima animal reference or Lumbricidae earthworm and other animals, dorsal

    dark lips full parasites. Salty taste of cold, there is heat, Pinggan, Dingchuan, Tongluo the efficacy, attending high fever frenzy, convulsion convulsion, wind-heat headache, Mu Chi,

    stroke hemiplegia, wheezing, sore throat, joint pain, teeth bleed at the nose, urine unreasonable, M. scrofulaceum, mumps,

    sores and other diseases [1]. It is reported that hypoxanthine is the expansion of bronchoalveolar Dragon of ingredients, from the dragon in the isolated hypoxanthine has anti-

    histamine, reducing the role of blood pressure [2]. Earthworm

    granules inherent quality, this paper seq hypoxanthine as an index component, the establishment of earthworm granules in hypoxanthine determination of high-performance liquid

    chromatography-based indicators of quality control, provide reference materials.

     1 Equipment

     1.1 Instrument Shimadzu high-performance liquid

    chromatography (LC-10ATVP solvent delivery pump, SPD-10AVP UV

    detector); HW-2000 chromatography workstation (Nanjing 1000 Spectrum Software Co., Ltd.).

     1.2 Examination of drugs and reagents hypoxanthine reference substance (Sinopharm Chemical Reagent Ltd., batch number F20040219, purity 99.8%); and earthworm granules samples (batch of 030598,0407136,0409959,0504632,0507026) and the lack of land dragon herbs negative reference substance

    (Guangdong Yifang Pharmaceutical Co., Ltd. to provide specifications for the bag 1 g, equivalent to 10 g Pieces per bag); methanol, tetrahydrofuran-specific reagents for

    chromatography, the remainder AR; water for pairs distilled water.

     2 Methods and Results

     2.1 Chromatographic conditions and system suitability test column Inertsil ODS-EP column (250 mm × 4.6 mm, 5 μm,

    Dumas companies); mobile phase for the water - methanol -

    tetrahydrofuran (100:0.5:0.5); flow rate of 1. 0 ml * min-1;

    detection wavelength of 254 nm. Column temperature: room

    temperature. Reference substance solution were taken for the test product solution and negative sample solution into the chromatograph 20 μl, according to the above-mentioned

    chromatographic conditions, recording chromatograms (Figure 1

    ~ 3). Hypoxanthine in the sample separation of adjacent peaks well (R> 1.5), theoretical plate number seq xanthine peak calculation should not be less than 2 600, the retention time of about 5.5 min.

     Figure 1 hypoxanthine reference substance HPLC diagram


     Figure 2 Sample Earthworm granules HPLC diagram (omitted)

     Figure 3 negative control samples of HPLC diagram (omitted)

     2.2 Preparation of solution for the test materials obtained in this study fine powder product of about 0.075 g,

    precision that will placed in 25 ml flask, add water, dissolve and dilute to the scale, shake, filtration, take added filtrate, as for the test product solution.

     2.3 Preparation of negative reference substance solution for the test items with the same preparation of the solution.

     2.4 Preparation of reference substance solution, said precision reference substance obtained hypoxanthine 20.30 mg, placed in 100 ml flask, add water, dissolved and diluted to the ultrasound treatment scale, shake, so as 0.203 mg * ml-1

    reserve liquid, and then precision Lessons reserve liquid 10 ml placed in 100 ml flask, add water, diluted to scale, shake, may hypoxanthine concentration of 0.020 3 mg * ml-1 of the

    reference substance reserve liquid, respectively, absorb a

    certain amount of reference substance precision 0.020 3 mg * ml-1 reserve liquid water diluted into a concentration of 2.03,4.06,8.12,10.15,12.18,16.24,20.30 μg * ml-1 reference

    substance solution, shake, that is, too.

     2.5 linear relationship between the study were drawn precision of the reference substance solutions of different concentrations of 20μl, injecting liquid chromatography to

    measure in order to peak area (A) of the reference substance concentration (C) regression, the regression equation was A = 41 342C 6 140.3, r = 0.999 9. Experimental results show that hypoxanthine at 0.040 6 ~ 0.406 0 μg peak area within the

    range of a good linear relationship. Reposted elsewhere in the paper for free download http://

     Taking a concentration of 2.6 precision experiments 8.12 μg * ml-1 of the reference substance solution hypoxanthine 20 μl, injected into liquid chromatography determination of 5 consecutive times, recording the peak area and calculate the result, hypoxanthine average peak area 338 681, RSD is 1.01% (n = 5), showed good precision instruments.

     2.7 stability test taken to test products for the Long Granule solution (lot number 0409959), and 0,2,3,4,6,8 h injection 20 μl, recorded peak area, the results of the

    average peak area of hypoxanthine to 194 873, RSD 1.46%, indicating that the tested materials was stable solution at 8h.

     2.8 to reproduce the experiment to take the same batch number (batch number 0409959) Sample 6 copies, according to the sample, "2.10" entry method of measuring the results of the average level of Hypoxanthine 1.561 4 mg * g-1, RSD 1.96%,

    indicating that analysis a good precision.

     2.9 The average recovery obtained experimental precision that is known content (batch number 0409959) and Dragon of

    granules sample of about 0.0750g, a total of nine copies, respectively, by adding precise concentration of 0.203 mg * ml-1 of the reference substance Hypoxanthine reserve liquid , 3.8,3.8,3.8,7.6,7.6,7.6,11.4,11.4,11.4 ml, according to "2.2"

    entry method for the test materials were prepared solution in the above chromatographic conditions, 20 μl sample for

    analysis. The results in Table 1.

     Table 1 The average recovery determination results (omitted)

     2.10 sample measured in different batches obtained Dragon of the sample granules 5 batches, according to "2.2" entry method for the test materials were prepared solution, and the other precision drawing concentration of 8.12 μg * ml-1 of

    the hypoxanthine reference substance solution and prepared

    test items for the solution of 20 μl, into the liquid

    chromatograph to measure, calculate the content of hypoxanthine. The results in Table 2.

     Table 2 Determination of Sample Results (abbreviated)

     3 Discussion

     3.1 Preparation for the test solution, the choice of materials were investigated in water and different concentrations of ethanol (10% ~ 95%) solution dissolved samples were determined hypoxanthine content of the results of the highest content of water-soluble extract and ethanol with

    increasing concentration Ci Huang purine content was decreased. Note hypoxanthine in aqueous solution is greater than the solubility of alcohol solution.

     3.2 Determination of the wavelength you choose to hypoxanthine 8.12 μg * ml-1 reference substance solution and

    the batch number 009,959 for the test product solution with water to do the reference, and placing UV-VIS

    spectrophotometer equipment, in the 200 ~ 400 nm wavelength scanning, results of hypoxanthine solution, reference

    substance and for the test items were part has a maximum absorption at 254 nm wavelength, and therefore selected as detection wavelength of 254 nm.

     3.3 mobile phase choice we made reference to the literature the use of water - methanol - tetrahydrofuran

    (100:0.12:0.04) as mobile phase conditions [3], the results of separation of less than ideal, we tried a different proportion of water - methanol - tetrahydrofuran flow relative separation the impact results show that water - methanol -

    tetrahydrofuran (100:0.5:0.5) as mobile phase conditions, earthworm granules in hypoxanthine, and other components could be baseline separated, and the good peak shape.

     Experimental results show that earthworm granules of different batches of preparation are very different

    hypoxanthine levels, so in their preparations for quality control, particularly important. Formula used as the hypoxanthine content of the active ingredients of preparations to establish appropriate quality control standards, can

    accurately reflect the quality of the preparation. This method is a good separation effect, simple, accurate, and can serve as a measure of earthworm granules formulations based on the quality of good and bad.


     [1] Long Yanhua. Dilong Overview of chemical and

    pharmacological studies [J]. Journal of Mathematical Medicine, 2005,18 (4): 379.

     [2] ZHANG Li-ping, LI Ru-hui. Earthworm pharmacological

    study and clinical application of [J]. Fujian Chinese medicine, 1990,21 (6): 52.

     [3] Hu Xin, Wang Qian. HPLC determination of hypoxanthine Dragon of Lu [J]. Chinese herbal medicine, 2005,36 (6): 928. Reposted elsewhere in the paper for free download http://www.hi138. com

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