CN101979052A - Method for establishing high-performance liquid chromatography (HPLC) fingerprint of Guangxi dendrobium candidum wall. ex lindl and standard fingerprint of Guangxi dendrobium candidum wall. ex lindl - Google Patents
Method for establishing high-performance liquid chromatography (HPLC) fingerprint of Guangxi dendrobium candidum wall. ex lindl and standard fingerprint of Guangxi dendrobium candidum wall. ex lindl Download PDFInfo
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Abstract
The invention discloses a method for establishing a high-performance liquid chromatography (HPLC) fingerprint of Guangxi dendrobium candidum wall. ex lindl, which comprises the following steps of: moisturizing dendrobium candidum wall. ex lindl powder with 10 percent ammonia water; performing reflux extraction for 4 h in a methanol water-bath at the temperature of 85 DEG C; filtering the extract and drying the filtered product by distillation; fixing a constant volume with methanol; passing solution through a microporous filter membrane; and performing chromatographic assay on filtrate to obtain the HPLC fingerprint of the dendrobium candidum wall. ex lindl, wherein the HPLC assay conditions are that: a chromatographic column is Alltima C18 (with an area of 4.6mm*250mm wide and a length of 5 mu m long), acetonitrile-0.1 percent water solution of phosphoric acid serving as a flow phase is used for gradient eluting at a flow speed of 1.2mL/min, a detection wavelength is 300nm, and a column temperature is 30 DEG C. The invention also discloses a standard fingerprint prepared by the method, and the standard fingerprint has 13 common chromatographic peaks. The method in the invention is precise, stable and high in repeatability, and the obtained fingerprint can provide scientific reference for manual rational collection and the quality control of the dendrobium candidum wall. ex lindl.
Description
Technical field
The present invention relates to the TCD identification method, especially relate to the method for building up and the standard finger-print thereof of Guangxi Herba Dendrobii HPLC finger printing.
Background technology
Herba Dendrobii (Dendrobium candidum Wall.ex Lindl.) is the perennial herbaceous plant that grows nonparasitically upon another plant of the orchid family Dendrobium, is a kind of famous and precious Chinese crude drug, and Guangxi is one of main distributed areas of Herba Dendrobii.Have pharmacological effects such as enhancing immunity, antioxidation, blood sugar lowering and inhibition cancer.Because artificial long-term uncontrolled excavating and the irrational utilization Herba Dendrobii, its wild resource reduces day by day, has occurred in the industry that some are mixed the spurious with the genuine, shoddy trouble waters.At present, some researcheres carry out the artificial culture Herba Dendrobii, perhaps attempt substituting crude drug with tissue culture.Therefore, be necessary utilizing chromatographic fingerprinting that the quality of medicinal material of Herba Dendrobii, tissue culture and the crude drug of cultivation is estimated.
Chromatographic fingerprinting is a kind of comprehensive, quantifiable discriminating means, can judge medical material the true and false, estimate good and bad, can be wild and the medical material of plantation or tissue culture's material, the medical material in the different places of production of same kind, the same place of production of same kind different year in medical material, the same kind of whole and local differentiation different cultivars.Up to now, the patent of relevant Herba Dendrobii HPLC finger printing has 1, the paper of having delivered has 1 piece, wherein, Cheng Jinle etc. have carried out HPLC fingerprint map analyzing, (Cheng Jinle to phenols in the Herba Dendrobii (comprising Herba Dendrobii) and flavones ingredient, Li Xianxia, Xu Jiyin, etc. dendrobe chromatogram finger print measuring method, CN200810218494.5).The method is suitable for the quality control of phenols and this two constituents of flavonoid.Yin Fangzhou etc. have set up the finger printing of 10 batches of Herba Dendrobii medical materials that pick up from Simao, Yunnan Gypsum Fibrosum Jing Zhen, have obtained 15 total fingerprint peakses (Yin Fangzhou, land rabbit woods, Cai Baochang, etc. the research of Herba Dendrobii medical material HPLC finger printing, Chinese herbal medicine, 2008,39 (3): 433-435).
Because Herba Dendrobii contains the multiclass chemical constituent, control the quality of Herba Dendrobii medical material, will detect a plurality of compositions as far as possible it is carried out quality control.In addition, because different places of production Herba Dendrobii quality of medicinal material difference is bigger, sample collecting will have enough representativenesses.
Summary of the invention
The technical problem to be solved in the present invention provides the method for building up and the standard finger-print thereof of a kind of Guangxi Herba Dendrobii HPLC finger printing, for manually rationally gather Herba Dendrobii and quality control provide the science reference.
Adopt following technical scheme for solving the problems of the technologies described above: the method for building up of Guangxi Herba Dendrobii HPLC finger printing comprises the steps:
(1) preparation of need testing solution: precision took by weighing the Herba Dendrobii powder 1.0g of 60 mesh sieves in the ground triangular flask, an amount of 10% ammonia moistening, add methanol 50mL and the airtight 30min that leaves standstill, under 85 ℃, carry out 4h water-bath reflux, extract,, filtered while hot, filtrate in 65 ℃ down rotation be evaporated to do after, with 5mL dissolve with methanol and standardize solution, the back is got filtrate and is carried out chromatographic determination with 0.45 μ m filtering with microporous membrane;
(2) HPLC chromatographiccondition: chromatographic column is the Alltima C18 of 4.6mm * 250mm 5 μ m, is that mobile phase is carried out gradient elution with acetonitrile-0.1% phosphate aqueous solution, and the detection wavelength is 310nm, and flow velocity is 1.2mL/min, and column temperature is 30 ℃;
(3) measure: accurately draw need testing solution 20 μ L and inject chromatograph of liquid, the conditional operation of (2) set by step, the chromatogram of record 60min.
The program of gradient elution is made as time gradient 0,10,20,25,40,55,60min, the Concentraton gradient 15%, 22%, 25%, 27.5%, 30%, 30%, 15% of acetonitrile correspondence.
The total chromatographic peak of the standard finger-print of the method for building up gained of Guangxi Herba Dendrobii HPLC finger printing has 13, and its retention time is respectively 6.387min, 7.595min, 8.091min, 9.104min, 10.494min, 11.743min, 13.133min, 14.418min, 16.777min, 25.759min, 28.555min, 31.284min, 32.533min.
Methodological study is the result show, the method for building up favorable reproducibility of Guangxi of the present invention Herba Dendrobii HPLC finger printing, RSD＜3% of each total peak relative peak area, the RSD of relative retention time＜1%; RSD＜3% of each total peak relative peak area during precision is investigated, the RSD of relative retention time＜1%, need testing solution is better at the 24h internal stability, meets the requirement of finger printing research.With respect to existing method, have more following advantage and effect:
(1) the present invention is the HPLC finger printing of setting up with the Herba Dendrobii (comprising tame Herba Dendrobii) in the different places of production, Guangxi, and total peak is many, and is representative strong, can objectively reflect the inherent quality of Herba Dendrobii;
(2), can control the reasonable collection period of artificial culture Herba Dendrobii effectively by the size of total peak-to-peak area in the finger printing;
(3) the inventive method accurately, stable, favorable reproducibility, easy and simple to handle, be easy to grasp.
Therefore, the HPLC finger printing of the different places of production, the Guangxi that the present invention sets up Herba Dendrobii all has great importance to Guangxi Herba Dendrobii standardization plantation and quality of medicinal material control.
Description of drawings
Fig. 1 is the standard finger-print of the method for building up gained of Guangxi Herba Dendrobii HPLC finger printing.
The specific embodiment
The present invention is described in further detail below in conjunction with embodiment and accompanying drawing.
The foundation of embodiment Guangxi Herba Dendrobii HPLC finger printing
＜1〉key instrument and reagent
U.S. waters2695 high performance liquid chromatograph, U.S. Waters 2996 PAD diode array detector, chromatographic column: Alltima C18 (4.6mm * 250mm, 5 μ m).
Acetonitrile (Merck KGaA company, chromatographically pure), other reagent are analytical pure.
＜2〉material
Shared 13 batch of materials, wherein 5 batch of materials are from Rong County, Guangxi, Guangxi Hechi, Guiping, Guangxi, Guilin and other places, and 8 batch of materials are the artificial culture Herba Dendrobii of different growing.
＜3〉preparation of need testing solution
Precision takes by weighing Herba Dendrobii powder (crossing 60 mesh sieves) 1.0g in the ground triangular flask, an amount of 10% ammonia moistening, add methanol 50mL and the airtight 30min that leaves standstill, under 85 ℃, carry out 4h water-bath reflux, extract,, filtered while hot, filtrate in 65 ℃ down rotation be evaporated to do after, with 5mL dissolve with methanol and standardize solution, the back is got filtrate and is carried out chromatographic determination with 0.45 μ m filtering with microporous membrane.
＜4〉finger printing is set up and is analyzed
Chromatographic condition
Chromatographic column: Alltima C18 (4.6mm * 250mm, 5 μ m);
Mobile phase: acetonitrile-0.1% phosphate aqueous solution gradient elution;
Elution program: time gradient 0 → 10 → 20 → 25 → 40 → 55 → 60min, the Concentraton gradient 15% → 22% → 25% → 27.5% → 30% → 30% → 15% of acetonitrile correspondence;
Detect wavelength: 310nm;
Flow velocity: 1.2mL/min;
Column temperature: 30 ℃
Accurately draw machine on the need testing solution 20 μ L, obtain writing down the chromatogram of 60min.According to 13 batches of Guangxi Herba Dendrobii material HPLC fingerprint map analyzings, find out its total chromatographic peak (13 peaks).Because the separating degree at No. 6 peaks is better, and peak area ratio more greatly, more stable, therefore selecting No. 6 peaks is the confidential reference items peak.As shown in Figure 1, the retention time of these 13 total chromatographic peaks is respectively 6.387min, 7.595min, 8.091min, 9.104min, 10.494min, 11.743min, 13.133min, 14.418min, 16.777min, 25.759min, 28.555min, 31.284min, 32.533min.
＜5〉methodological study
＜5.1〉precision is measured
With same need testing solution, go up the machine sample introduction continuously 6 times, the record chromatogram.The result shows, the RSD of each total chromatographic peak relative retention time＜1.0%, and the RSD of relative peak area＜3.0% shows that the precision of instrument system is good.
＜5.2〉stability is measured
With same need testing solution, go up machine respectively at 0,2,4,8,12, in the 24h and measure the record chromatogram.The result shows, the RSD of each total chromatographic peak relative retention time＜1.0%, and the RSD of relative peak area＜3.0% shows that need testing solution is stable in 24h.
＜5.3〉repeatability is measured
Get 6 parts of same batch of materials, prepare need testing solution according to preparation method, chromatogram is measured and write down to the machine of going up respectively.The result shows, the RSD of each total chromatographic peak relative retention time＜1.0%, and the RSD of relative peak area＜3.0% shows that the method repeatability is good.
Claims (3)
1. the method for building up of a Guangxi Herba Dendrobii HPLC finger printing is characterized in that this method for building up comprises the steps:
(1) preparation of need testing solution: precision took by weighing the Herba Dendrobii powder 1.0g of 60 mesh sieves in the ground triangular flask, an amount of 10% ammonia moistening, add methanol 50mL and the airtight 30min that leaves standstill, under 85 ℃, carry out 4h water-bath reflux, extract,, filtered while hot, filtrate in 65 ℃ down rotation be evaporated to do after, with 5mL dissolve with methanol and standardize solution, the back is got filtrate and is carried out chromatographic determination with 0.45 μ m filtering with microporous membrane;
(2) HPLC chromatographiccondition: chromatographic column is the Alltima C18 of 4.6mm * 250mm 5 μ m, is that mobile phase is carried out gradient elution with acetonitrile-0.1% phosphate aqueous solution, and the detection wavelength is 310nm, and flow velocity is 1.2mL/min, and column temperature is 30 ℃;
(3) measure: accurately draw need testing solution 20 μ L and inject chromatograph of liquid, the conditional operation of (2) set by step, the chromatogram of record 60min.
2. the method for building up of Guangxi according to claim 1 Herba Dendrobii HPLC finger printing, it is characterized in that: the program of described gradient elution is made as time gradient 0,10,20,25,40,55,60min, the Concentraton gradient 15%, 22%, 25%, 27.5%, 30%, 30%, 15% of acetonitrile correspondence.
3. the standard finger-print of the method for building up gained of Guangxi according to claim 1 Herba Dendrobii HPLC finger printing, it is characterized in that: the total chromatographic peak of this finger printing has 13, and its retention time is respectively 6.387min, 7.595min, 8.091min, 9.104min, 10.494min, 11.743min, 13.133min, 14.418min, 16.777min, 25.759min, 28.555min, 31.284min, 32.533min.
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Application publication date: 20110223 |