CN105092724A - Construction method of Pothos chinensis HPLC fingerprint of and HPLC fingerprint - Google Patents
Construction method of Pothos chinensis HPLC fingerprint of and HPLC fingerprint Download PDFInfo
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Abstract
The present invention discloses a construction method of Pothos chinensis HPLC fingerprint of and an HPLC fingerprint based on high-performance liquid chromatography. The fingerprint has 16 common peaks with the following relative retention time tg: 0.178min, 0.268min, 0.309min, 0.354min, 0.411min, 0.488min, 0.507min, 0.633min, 0.730min, 0.922min, 1.000min, 1.318min, 1.326min, 1.525min, 1.544min, and 1.579min. Through the presence or absence of common peaks in the fingerprint, a comprehensive quality evaluation of Pothos chinensis can be carried out, so as to ensure the stability of medicinal quality and effectiveness and safety of clinical medication.
Description
Technical field
The present invention relates to traditional Chinese medicine quality detection technique field, particularly relate to a kind of construction method and HPLC finger-print thereof of Chinese pothos herb HPLC finger-print.
Background technology
Mandarin orange is the dry herb of Araeceae stone mandarin orange platymiscium Chinese pothos herb Pothoschinensis (Raf.) Merr., be precious jade cure old class medicine " five tiger, nine Ns, 18 bore, 72 wind " one of cucurbit bore.Chinese pothos herb is put down, lightly seasoned, have dispel rheumatism, effect of promoting blood circulation to remove blood stasis, food dispersing cough suppressing, be usually used in Yao nationality area treating traumatic injury, treating rheumatic ostealgia, evident in efficacy.Because traditional Chinese medicine ingredients is complicated, only voucher one index is difficult to the quality of accurate evaluation Chinese medicine.Traditional Chinese medicine fingerprint refers to the chromatogram with certain class distinctive or a few constituents that certain (certain place of production) Chinese medicine is common or spectrum atlas.Traditional Chinese medicine fingerprint has globality, ambiguity and quantifiable feature because of it, can react the inherent quality of Chinese medicine all sidedly, has become traditional Chinese medicine quality at present and has controlled one of most effective method.
But there is no the quality standard of Chinese pothos herb medicinal material at present, the control of Chinese pothos herb quality of medicinal material is just seemed very weak, only includes microscopical characters etc. and check routinely, lack perfect quality evaluation system, be difficult to reach the effective control to Chinese pothos herb quality.
Summary of the invention
The object of the present invention is to provide the construction method of Chinese pothos herb HPLC finger-print, the method adopts high performance liquid chromatography (HPLC) to set up its finger-print, and evaluate with similarity software, can evaluate the quality of Chinese pothos herb all sidedly, ensure the stability of quality of medicinal material and the validity of clinical application and security.
Its concrete technical scheme is as follows:
A construction method for Chinese pothos herb HPLC finger-print, comprises the steps:
(1) preparation of need testing solution: get Chinese pothos herb powder (crossing No. four sieves) 2g, accurately weighed, put in tool plug conical flask, precision adds 50% methyl alcohol 25ml, weighed weight, ultrasonic process 30 minutes, let cool, again weigh, supply the weight of less loss with 50% methyl alcohol, filter, accurate absorption subsequent filtrate 10ml, evaporate to dryness, residue adds 50% methyl alcohol and dissolves and be settled to 2ml, cross 0.45 μm of miillpore filter, get subsequent filtrate, to obtain final product;
(2) preparation of reference substance solution: accurately weighed feruloyltyramine, syringic acid, vanillic acid reference substance are appropriate, add methyl alcohol respectively and make the mixed solution of every 1ml containing feruloyltyramine 60.42 μ g, syringic acid 120.14 μ g and vanillic acid 71.25 μ g, cross 0.45 μm of miillpore filter, get subsequent filtrate, to obtain final product;
(3) efficient liquid phase chromatographic analysis:
Accurate absorption reference substance solution and each 10 μ l of need testing solution, inject high performance liquid chromatograph,
Chromatographic condition is as follows: chromatographic column: ThermoODS-2Hypersil chromatographic column, packing material size 5 μm, column length 250mm, column internal diameter 4.6mm;
Mobile phase: take methyl alcohol as mobile phase A, volumn concentration be 0.4% phosphate aqueous solution be Mobile phase B, adopt gradient elution mode: from 0-15mim, the volumn concentration of mobile phase A is increased to 20% by 10%, and the volumn concentration of Mobile phase B is reduced to 80% by 90%; From 15-25mim, the volumn concentration of mobile phase A is 20%, and the volumn concentration of Mobile phase B is 80%; From 25-35mim, the volumn concentration of mobile phase A is increased to 25% by 20%, and the volumn concentration of Mobile phase B is reduced to 75% by 80%; From 35-45mim, the volumn concentration of mobile phase A is increased to 28% by 25%, and the volumn concentration of Mobile phase B is reduced to 72% by 75%; From 45-50mim, the volumn concentration of mobile phase A is 28%, and the volumn concentration of Mobile phase B is 72%; From 50-60mim, the volumn concentration of mobile phase A is increased to 32% by 28%, and the volumn concentration of Mobile phase B is reduced to 68% by 72%; From 60-70mim, the volumn concentration of mobile phase A is increased to 40% by 32%, and the volumn concentration of Mobile phase B is reduced to 60% by 68%; From 70-90mim, the volumn concentration of mobile phase A is increased to 70% by 40%, and the volumn concentration of Mobile phase B is reduced to 30% by 60%; From 90-120mim, the volumn concentration of mobile phase A is 70%, and the volumn concentration of Mobile phase B is 30%; Determined wavelength: 300nm; Column temperature: 30 DEG C; Flow velocity: 1.0ml/min;
(4) measure 24 batches of Chinese pothos herb medicinal materials and carry out com-parison and analysis, obtaining the standard finger-print of the Chinese pothos herb HPLC be made up of its common characteristic peak.
The construction method of described Chinese pothos herb HPLC finger-print, it is further comprising the steps of:
(5) precision of described standard finger-print, reappearance and stability test,
(51) precision test:
Get same need testing solution, continuous sample introduction 6 times, with No. 11 peaks for reference peak, the RSD value of the relative retention time and relative peak area that calculate No. 1-16 total fingerprint peaks is all less than 3%, all be greater than 0.990 by the similarity that similarity evaluation software calculates each chromatographic fingerprinting simultaneously, show instrument stabilizer, precision is good;
(52) reappearance test:
Get with a collection of Chinese pothos herb powder, precision takes 6 parts, by the parallel preparation of test sample preparation method 6 parts of need testing solutions under step (51), sample introduction respectively, with No. 11 peaks for reference peak, the RSD value of the relative retention time and relative peak area that calculate No. 1-16 total fingerprint peaks is all less than 3%, shows that the construction method reappearance of described Chinese pothos herb HPLC finger-print is good;
(53) stability test:
Get with a need testing solution, respectively at 0,2,4,8,16,24h sample detection, with No. 11 peaks for reference peak, the RSD value of the relative retention time and relative peak area that calculate No. 1-16 total fingerprint peaks is all less than 3%, shows that need testing solution is stable in 24 hours.
Another object of the present invention is to provide the standard finger-print that said method builds the Chinese pothos herb HPLC obtained, and the relative retention time tg at 16 total peaks that the standard finger-print of described Chinese pothos herb HPLC has is respectively: 0.178min, 0.268min, 0.309min, 0.354min, 0.411min, 0.488min, 0.507min, 0.633min, 0.730min, 0.922min, 1.000min, 1.318min, 1.326min, 1.525min, 1.544min, 1.579min.
Tool of the present invention has the following advantages and purposes: the standard finger-print being set up Chinese pothos herb HPLC by high performance liquid chromatography (HPLC), and precision, reappearance and the stability by testing confirmed standard finger-print; By the presence or absence at peak total in finger-print, can evaluate the quality of Chinese pothos herb all sidedly, ensure that the stability of quality of medicinal material and clinical application have validity and security.
Below in conjunction with accompanying drawing and embodiment, the present invention is described in more detail.
Accompanying drawing explanation
Fig. 1 is Chinese pothos herb HPLC standard finger-print;
Fig. 2 is the HPLC finger-print stacking diagram of 24 batches of Chinese pothos herbs;
Fig. 3 is the HPLC chromatogram of different flow phase system;
Fig. 4 is the HPLC chromatogram of variable concentrations phosphate aqueous solution;
Fig. 5 is the HPLC chromatogram of different solvents;
Fig. 6 is precision test stacking diagram;
Fig. 7 is replica test stacking diagram;
Fig. 8 is stability test stacking diagram.
Embodiment
See Fig. 1 ~ Fig. 2, below by way of specific embodiment, the present invention is further elaborated.
Select Waterse2695-2489 type high performance liquid chromatograph (U.S.): PAD detecting device, quaternary gradient pump, Empower2 data processing software system; MilliporeAdvantageA10 ultrapure water system (U.S.); MettlerToledoXS205DU type electronic analytical balance (Switzerland); KQ5200DE type numerical control ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.); HH-8 type digital display thermostat water bath (Ke Xi Instrument Ltd. of Jintan City).
Select feruloyltyramine (self-control, purity >=99.5%), syringic acid (self-control: purity >=99.0%), vanillic acid (lot number: X-030-140801, purchased from Rui Fensi bio tech ltd, Chengdu); Liquid phase methyl alcohol is chromatographically pure (Merck); Liquid phase water is ultrapure water; Extraction water is that institute of traditional Chinese medicine of Guangdong Province second Drug Manufacturing Room make purified water by oneself; It is pure that all the other reagent are analysis.
Chromatographic condition: chromatographic column: ThermoODS-2Hypersil chromatographic column (4.6 × 250mm, 5 μm); Mobile phase: take methyl alcohol as mobile phase A, with 0.4% phosphate aqueous solution for Mobile phase B, carry out gradient elution by table 1; Determined wavelength: 300nm; Column temperature: 30 DEG C; Flow velocity: 1.0mL/min.
Table 1 gradient elution program
The preparation of need testing solution: get Chinese pothos herb powder (crossing No. four sieves) about 2g, accurately weighed, put in tool plug conical flask, precision adds 50% methyl alcohol 25ml, weighed weight, ultrasonic process 30 minutes, let cool, again weigh, supply the weight of less loss with 50% methyl alcohol, filter, accurate absorption subsequent filtrate 10ml, evaporate to dryness, residue adds 50% methyl alcohol and dissolves and be settled to 2ml, cross 0.45 μm of miillpore filter, get subsequent filtrate, to obtain final product.
The preparation of reference substance solution: accurately weighed feruloyltyramine, syringic acid, vanillic acid reference substance are appropriate, add methyl alcohol respectively and make the mixed solution of every 1ml containing feruloyltyramine 60.42 μ g, syringic acid 120.14 μ g and vanillic acid 71.25 μ g, cross 0.45 μm of miillpore filter, get subsequent filtrate, to obtain final product.
The foundation of finger-print: measure 24 batches of Chinese pothos herb HPLC-FPSs, and carry out com-parison and analysis, obtain the standard finger-print (as shown in Figure 1, 2) of the Chinese pothos herb HPLC be made up of its common characteristic peak, wherein No. 2 peaks are vanillic acid, No. 3 peaks are syringic acid, and No. 11 peaks are feruloyltyramine.
This 24 batch sample selected, imported " similarity evaluation (2004A version) " that Chinese Pharmacopoeia Commission promulgates, access time, window width was 0.1min, contrast spectrogram is generated with median, Auto-matching after Supplements, generate Chinese pothos herb finger-print common pattern, and calculate the finger-print of 24 batch samples and the similarity of this common pattern.With No. 11 peaks for reference peak, the relative retention time tg calculating 16 total peaks that this standard finger-print has is respectively: 0.178min, 0.268min, 0.309min, 0.354min, 0.411min, 0.488min, 0.507min, 0.633min, 0.730min, 0.922min, 1.000min, 1.318min, 1.326min, 1.525min, 1.544min, 1.579min.
Imported the similarity evaluation (A version) that pharmacopoeia commission is recommended, carry out chromatographic peak coupling, with No. 11 peaks for reference determines that 16 total peaks are for being formed the characteristic peak of Chinese pothos herb finger-print, sample has the relative retention time at peak and relative peak area lists in table 2 respectively, table 3, 24 batches of Chinese pothos herbs and standard finger-print Similarity Measure result are respectively: 0.958, 0.952, 0.916, 0.954, 0.971, 0.919, 0.908, 0.941, 0.926, 0.968, 0.993, 0.952, 0.911, 0.975, 0.998, 0.952, 0.969, 0.948, 0.956, 0.915, 0.992, 0.979, 0.975, 0.907.
Table 224 batch Chinese pothos herb finger-print has peak relative retention time
Table 324 batch Chinese pothos herb finger-print has peak relative peak area
In the present embodiment, see Fig. 3, (in figure, A is acetonitrile-water; B is methanol-water; C is methyl alcohol-formic acid aqueous solution; D is methyl alcohol-phosphate aqueous solution), (in figure, A is 0.05% phosphate aqueous solution to Fig. 4; B is 0.1% phosphate aqueous solution; C is 0.4% phosphate aqueous solution), to being optimized of mobile phase condition, test acetonitrile-water, methanol-water, methyl alcohol-phosphate aqueous solution, methyl alcohol-formic acid aqueous solution 4 kinds of flow phase system respectively, and the phosphate aqueous solution having investigated variable concentrations is on the impact of Chinese pothos herb chromatographic behavior.Result shows, when taking acetonitrile-water as mobile phase, most of composition is eluted prematurely, and chromatographic peak is deposited in first 30 minutes, and degree of separation is poor; When taking methanol-water as mobile phase, spectrogram information is less, cannot reflect the inherent quality of Chinese pothos herb comprehensively; During with methyl alcohol-formic acid aqueous solution for mobile phase, chromatographic peak peak shape is poor.During with methyl alcohol-phosphoric acid for mobile phase, Chromatographic information is comparatively abundant and peak shape is better, sees Fig. 3.Be good with 0.4% phosphate aqueous solution in mobile phase, according to the phosphate aqueous solution of 0.05% or 0.1%, then part chromatographic peak degree of separation is poor, sees Fig. 4.Determine with methyl alcohol-0.4% phosphate aqueous solution for mobile phase carries out gradient elution.
Selecting of Extraction solvent, see Fig. 5, (A is water; B is ethanol; C is methyl alcohol; D is 50% methyl alcohol) sample is extracted using water, ethanol, methyl alcohol, 50% methyl alcohol as Extraction solvent respectively, and gained collection of illustrative plates is compared.Result shows, when adopting water as Extraction solvent, the composition that sample Semi-polarity is less is not fully extracted out, and chromatographic peak mainly concentrates on first 50 minutes; And when adopting ethanol and methyl alcohol as Extraction solvent, the composition that sample Semi-polarity is larger is difficult to extract, the retention time of most of chromatographic peak is all after 60 minutes, and the impurity such as extract Determination of Chlorophyll are more, seriously polluted to chromatographic column; And when adopting 50% methyl alcohol as Extraction solvent, in sample, constituents extraction is complete, and profile information enriches, and can reflect the composition characteristics of medicinal material to greatest extent, and reduce interference and the pollution of the impurity such as chlorophyll, thus adopt 50% methyl alcohol as the Extraction solvent of sample.
Precision test: get same need testing solution (S20), continuous sample introduction 6 times, gained finger-print stacking diagram sees Fig. 6.Adopt chromatographic fingerprints of Chinese materia medica the system of analysis and appraisal 2004A version to analyze its finger-print, compared with the reference fingerprint generated, the similarity of each collection of illustrative plates is all greater than 0.990.With No. 11 peaks for reference peak, the RSD value of the relative retention time and relative peak area that calculate No. 1-16 total fingerprint peaks is all less than 3%, all be greater than 0.990 by the similarity that similarity evaluation software calculates each chromatographic fingerprinting, show instrument stabilizer, precision is good simultaneously.The results are shown in Table 4,5,6.
Table 4 precision test fingerprint similarity analysis result
Table 5 precision test has peak relative retention time
Table 6 precision test has peak relative peak area
Reappearance is tested: get with a collection of Chinese pothos herb powder (S20), precision takes 6 parts, and by the parallel preparation of test sample preparation method 6 parts of need testing solutions, sample introduction respectively, detect with method, gained finger-print stacking diagram sees Fig. 7.Adopt similarity evaluation software to analyze its finger-print, compared with the reference fingerprint generated, the similarity of 6 increment product is all greater than 0.990.With No. 11 peaks for reference peak, the RSD value of the relative retention time and relative peak area that calculate No. 1-16 total fingerprint peaks is all less than 3%, shows that the method reappearance is good.The results are shown in Table 7,8,9.
Table 7 replica test fingerprint similarity analysis result
Table 8 replica test has peak relative retention time
Table 9 replica test has peak relative peak area
Stability test: get with a need testing solution (S20), respectively at 0,2,4,8,16,24h sample detection, gained finger-print stacking diagram sees Fig. 8.Adopt similarity evaluation software to analyze its finger-print, compared with the reference fingerprint generated, the similarity of each collection of illustrative plates is all greater than 0.990.With No. 11 peaks for reference peak, the RSD value of the relative retention time and relative peak area that calculate No. 1-16 total fingerprint peaks is all less than 3%, shows that need testing solution is stable in 24 hours, meets fingerprint pattern technology requirement.The results are shown in Table 10,11,12.
Table 10 stability test fingerprint similarity analysis result
Table 11 stability test has peak relative retention time
Table 12 stability test has peak relative peak area
Similarity evaluation is analyzed: the Similarity Measure of 24 batches of Chinese pothos herb sample finger-prints the results are shown in Table 13, from result, in 24 batch samples, the similarity of 12 batch samples is more than 0.9, the similarity of 8 batch samples is between 0.8 ~ 0.9, and the similarity of all the other 4 batch samples is between 0.7 ~ 0.8.
Analyze 4 batch samples of similarity below 0.8, in No. 11 samples, the part of stem is more and the part of leaf is less, thus causes chemical composition different, and total peak is less; No. 12 sample grown time limits are shorter, and blade is younger tender, and chlorophyll is more, become separating/enriching inadequate; And No. 22 samples have and go mouldy on a small quantity in storage process, fractions may be caused to change.Show that the quality of Chinese pothos herb medicinal material is subject to the impact of the factors such as growth year, pick and process and condition of storage.
Table 1324 batch Chinese pothos herb HPLC fingerprint similarity
The above embodiment only have expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but therefore can not be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some variations and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (3)
1. the construction method of Chinese pothos herb HPLC finger-print, is characterized in that, the method comprises the following steps and condition:
(1) preparation of need testing solution:
Get Chinese pothos herb powder 2g, accurately weighed, put in tool plug conical flask, precision adds 50% methyl alcohol 25ml, weighed weight, ultrasonic process 30 minutes, let cool, again weigh, supply the weight of less loss with 50% methyl alcohol, filter, accurate absorption subsequent filtrate 10ml, evaporate to dryness, residue adds 50% methyl alcohol and dissolves and be settled to 2ml, cross 0.45 μm of miillpore filter, get subsequent filtrate, to obtain final product;
(2) preparation of reference substance solution:
Accurately weighed feruloyltyramine, syringic acid, vanillic acid reference substance are appropriate, add methyl alcohol respectively and make the mixed solution of every 1ml containing feruloyltyramine 60.42 μ g, syringic acid 120.14 μ g and vanillic acid 71.25 μ g, cross 0.45 μm of miillpore filter, get subsequent filtrate, to obtain final product;
(3) efficient liquid phase chromatographic analysis:
Accurate absorption reference substance solution and each 10 μ l of need testing solution, inject high performance liquid chromatograph;
Chromatographic condition is chromatographic column: ThermoODS-2Hypersil chromatographic column, packing material size 5 μm, column length 250mm, column internal diameter 4.6mm;
Mobile phase: take methyl alcohol as mobile phase A, volumn concentration be 0.4% phosphate aqueous solution be Mobile phase B, adopt gradient elution mode: from 0-15mim, the volumn concentration of mobile phase A is increased to 20% by 10%, and the volumn concentration of Mobile phase B is reduced to 80% by 90%; From 15-25mim, the volumn concentration of mobile phase A is 20%, and the volumn concentration of Mobile phase B is 80%; From 25-35mim, the volumn concentration of mobile phase A is increased to 25% by 20%, and the volumn concentration of Mobile phase B is reduced to 75% by 80%; From 35-45mim, the volumn concentration of mobile phase A is increased to 28% by 25%, and the volumn concentration of Mobile phase B is reduced to 72% by 75%; From 45-50mim, the volumn concentration of mobile phase A is 28%, and the volumn concentration of Mobile phase B is 72%; From 50-60mim, the volumn concentration of mobile phase A is increased to 32% by 28%, and the volumn concentration of Mobile phase B is reduced to 68% by 72%; From 60-70mim, the volumn concentration of mobile phase A is increased to 40% by 32%, and the volumn concentration of Mobile phase B is reduced to 60% by 68%; From 70-90mim, the volumn concentration of mobile phase A is increased to 70% by 40%, and the volumn concentration of Mobile phase B is reduced to 30% by 60%; From 90-120mim, the volumn concentration of mobile phase A is 70%, and the volumn concentration of Mobile phase B is 30%; Determined wavelength: 300nm; Column temperature: 30 DEG C; Flow velocity: 1.0ml/min;
(4) measure 24 batches of Chinese pothos herb medicinal materials and carry out com-parison and analysis, obtaining the standard finger-print of the Chinese pothos herb HPLC be made up of its common characteristic peak.
2. the construction method of Chinese pothos herb HPLC finger-print according to claim 1, it is characterized in that, it is further comprising the steps of: the precision of (5) described standard finger-print, reappearance and stability test,
(51) precision test:
Get same need testing solution, continuous sample introduction 6 times, with No. 11 peaks for reference peak, the RSD value of the relative retention time and relative peak area that calculate No. 1-16 total fingerprint peaks is all less than 3%, all be greater than 0.990 by the similarity that similarity evaluation software calculates each chromatographic fingerprinting simultaneously, instrument stabilizer, precision is good;
(52) reappearance test:
Get with a collection of Chinese pothos herb powder, precision takes 6 parts, by the parallel preparation of test sample preparation method 6 parts of need testing solutions under step (51), sample introduction respectively, with No. 11 peaks for reference peak, the RSD value of the relative retention time and relative peak area that calculate No. 1-16 total fingerprint peaks is all less than 3%, and the construction method reappearance of described Chinese pothos herb HPLC finger-print is good;
(53) stability test:
Get with a need testing solution, respectively at 0,2,4,8,16,24h sample detection, with No. 11 peaks for reference peak, the RSD value of the relative retention time and relative peak area that calculate No. 1-16 total fingerprint peaks is all less than 3%, and need testing solution is stable in 24 hours.
3. Chinese pothos herb HPLC finger-print, it is characterized in that, the standard finger-print of Chinese pothos herb HPLC is made up of 16 total fingerprint peakses, and the relative retention time tg at 16 total peaks that the standard finger-print of this Chinese pothos herb HPLC has is respectively: 0.178min, 0.268min, 0.309min, 0.354min, 0.411min, 0.488min, 0.507min, 0.633min, 0.730min, 0.922min, 1.000min, 1.318min, 1.326min, 1.525min, 1.544min, 1.579min.
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| CN110988182A (en) * | 2019-12-19 | 2020-04-10 | 广西壮族自治区药用植物园 | Quality detection method of Zhuang medicine ethnic medicine Huatuo Fengtongbao capsule |
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| CN110988182A (en) * | 2019-12-19 | 2020-04-10 | 广西壮族自治区药用植物园 | Quality detection method of Zhuang medicine ethnic medicine Huatuo Fengtongbao capsule |
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