CN103115973B - Method for judging consistency of traditional Chinese medicine batches - Google Patents
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Abstract
The invention discloses a method for judging consistency of traditional Chinese medicine batches. The method comprises the following steps: establishing a reference map on the basis of finger-print similarity analysis, and calculating the mahalanobis distance between a high performance liquid chromatography (HPLC) finger-print in traditional Chinese medicine samples in various batches and the reference map; converting the mahalanobis distance into F check so as to judge whether obvious difference exists between the HPLC finger-print in traditional Chinese medicine samples in different batches and the reference map under the level at a certain probability; and determining whether obvious difference exists in the content of components corresponding to chromatographic peaks in the HPLC finger-print in traditional Chinese medicine samples in various batches by employing a t checking method if the batch samples with obvious difference are determined according to the mahalanobis distance. The method is based on the chromatographic fingerprint and is simple, feasible and practical, the consistency of various batch samples can be judged from the probability according to the difference of the finger-print in traditional Chinese medicine samples in different batches, and method basis is provided for judging the consistency of the batches of the traditional Chinese medicine.
Description
Technical field
The present invention relates to a kind of for Chinese medicine batch conforming determination methods.
Background technology
Chinese medicine is used for preventing and treating and treating the history of the existing more than one thousand years of disease.Different from synthetic drug, the curative effect performance of Chinese medicine and preparation thereof is mainly the synergy of Multiple components.Recently decades, due to its hypotoxicity and high curative effect, Chinese medicine is at global demand increase year after year, especially in Asia, Europe and North America.The increase of demand also just means the higher requirement of quality standard proposition.The Chinese medicine of each batch all should meet the code requirement of specific products, as date of manufacture and shelf-life etc.But even if only there is the simple formulations that several herbal medicine are made just may comprise hundreds of uniqueness, narrow spectrum composition, its quality control is many more than synthetic drug difficulty.If it is the huge challenge that traditional Chinese medicine quality control and evaluation face that contained Chinese medicine whole compositions are characterized
[1].In addition, only screening one or more compositions carries out traditional Chinese medicine quality control and does not meet traditional tcm theory
[2].In the last few years, show by a large amount of research, chromatographic fingerprinting has become a kind of traditional Chinese medicine quality control and evaluation method of accepting extensively.
1991, WHO promulgated the guilding principle based on traditional Chinese medicine quality, safety, effectiveness and desired use assessment.At that time, chromatographic fingerprinting is applied as a kind of acceptable assay method
[3].After more than ten years, U.S. food Drug Administration (FDA), EMEA (EMES) and State Food and Drug Administration (SFDA) all accept the quality control of chromatographic fingerprinting for herbal drug, and have promoted its development
[4～6].As a kind of quality evaluating method, chromatographic fingerprinting can reflect the number of chemical composition information comprising in complex analyses sample (as Chinese medicine), can provide more comprehensively quality assessment.In order to advance fingerprint spectrum method to be effective to traditional Chinese medicine quality control, SFDA suggestion chromatographic fingerprints of Chinese materia medica should carry out similarity evaluation and (comprise two kinds of computing method of related coefficient and included angle cosine
[7]).Nowadays, similarity has been widely used in the similarity of evaluating finger-print.But similarity is higher means that the difference of finger-print is just less? in recent years, high performance liquid chromatography (HPLC) finger-print has been set up in conjunction with the content assaying method of some characteristic chemical constituent and for the quality control of Chinese medicine preparation
[8,9].In these research work, satisfactory although the fingerprint similarity of sample is evaluated, in different samples, the content of some composition still presents larger difference
[10～12].This explanation fingerprint similarity evaluation can not fully reflect the similarity between finger-print, can not absolutely prove the similarity of sample ingredient content.The similarity (or difference) of how evaluating between finger-print is a problem that is worth further investigation.Have good stability and consistance in order to ensure each batch of sample quality of Chinese medicine, the finger-print difference of each batch of sample is the smaller the better.But, in present stage, for the such complex analyses system of Chinese medicine, realize quality control unrealistic by its contained all the components being carried out to qualitative and quantitative analysis.Therefore, the application has proposed a kind of for traditional Chinese medicine quality batch conforming determination methods based on chromatographic fingerprinting, the method can judge that whether the difference between different batches sample finger-print is remarkable from the angle of probability, and can provide which chromatographic peak tie element in finger-print and on content, have larger difference, to providing a kind of effective, practical method mode for traditional Chinese medicine quality control and evaluation.
Summary of the invention
For above-mentioned prior art, the present invention is based on chromatographic fingerprinting, a kind of determination methods for each batch of unanimity of samples of Chinese medicine has been proposed, the method is easy, Yi Hang, practicality, can realize the each batch of conforming judgement of sample by judging the difference of each batch of sample finger-print of Chinese medicine from the angle of probability.The consistance of each batch of sample of Chinese medicine can be by judging that the each batch of difference between sample fingerprint collection of illustrative plates embodies: if the difference of different batches sample chromatogram finger-print on certain probability level without conspicuousness, the consistance there was no significant difference of each batch of sample; Otherwise consistance has significant difference; There is batch sample of significant difference for consistance, can analyze which chromatographic peak in finger-print and differ greatly.
The present invention is achieved by the following technical solutions:
For a determination methods for each batch of unanimity of samples of Chinese medicine, step is as follows:
(1) select the traditional Chinese medicine sample of multiple different batches to be judged, after grinding, adopt the solvent extraction that is applicable to extracting this Effective Component of Chinese Medicine, obtain extract;
(2) adopt high performance liquid chromatograph to gather original high efficiency liquid chromatography (HPLC) finger-print of the extract of the traditional Chinese medicine sample of above-mentioned different batches at the detection wavelength place that is applicable to this Chinese medicine;
(3) the HPLC finger-print of above-mentioned gained is carried out to methodology checking, (that is: precision test, reappearance test and stability test meet the requirements to make measured HPLC finger-print meet fingerprint pattern technology code requirement.According to precision test and reappearance testing requirements, investigate the relative retention time of chromatographic peak, the consistance of peak area ratio.Each chromatographic peak of the total peak area ratio of regulation in finger-print, the relative standard deviation (RSD) of its peak area ratio must not be greater than 3%; According to stability test requirement, investigate the relative retention time of chromatographic peak, the consistance of peak area ratio, determine detection time); Then calculate the average collection of illustrative plates collection of illustrative plates in contrast of the HPLC finger-print of the traditional Chinese medicine sample of each batch; Calculate respectively the HPLC finger-print and the similarity that contrasts collection of illustrative plates of the traditional Chinese medicine sample of each batch; If similarity (r) is more than or equal to 0.900, retain, if r<0.900, rejected, then recalculated the contrast collection of illustrative plates of traditional Chinese medicine sample of residue batch, and remained the HPLC finger-print of traditional Chinese medicine sample and the similarity that contrasts collection of illustrative plates of the traditional Chinese medicine sample remaining batch of each batch, and then adopt criterion similarity same as described above (if that is: r is more than or equal to 0.900, retain, if r<0.900 is rejected); Repeat this process, until it meets the requirements (that is: similarity r all >=0.900);
(4) from above-mentioned selection, in remaining batch, select the sample that active constituent content is high (can select a collection of, also can select some batches) HPLC finger-print as a reference, select the traditional Chinese medicine sample of multiple batch nearest with the HPLC finger-print Euclidean distance of reference sample, then build with reference to classification with reference sample, and the average finger-print of computing reference classification sample, with reference to collection of illustrative plates;
(5) based on above-mentioned with reference to classification, calculate each batch traditional Chinese medicine sample HPLC finger-print with reference to the mahalanobis distance of collection of illustrative plates, then mahalanobis distance is converted into F inspection, thus judge different batches traditional Chinese medicine sample HPLC finger-print and with reference between collection of illustrative plates, under probability level necessarily, whether there were significant differences;
(6) find out mahalanobis distance and be judged to be the traditional Chinese medicine sample of those batches of significant difference, adopt t inspection to be tested in the total peak of the HPLC finger-print of the traditional Chinese medicine sample of these batches and the total peak of the HPLC finger-print of the traditional Chinese medicine sample with reference to each batch of classification, in the total peak of judgement, whether each chromatographic peak area has significant difference under certain probability level; Be judged as the chromatographic peak of significant difference, show in the traditional Chinese medicine sample of this batch, on the represented active constituent content of this chromatographic peak, be starkly lower than or higher than the average content of the corresponding composition of other traditional Chinese medicine sample of reference class.
Preferably, described step (4) is specific as follows: select the HPLC finger-print of batch sample (reference sample) that active constituent content is high as a reference, calculate the Euclidean distance of the HPLC finger-print of other traditional Chinese medicine sample of each batch and the HPLC finger-print of reference sample, according to Euclidean distance is ascending, other traditional Chinese medicine sample of each batch is arranged, select the traditional Chinese medicine sample of multiple (being no less than 10) nearest with the HPLC finger-print Euclidean distance of reference sample batch, and building with reference to classification with reference sample, and computing reference collection of illustrative plates.
Preferably, in described step (5), the consistance quality that judges the traditional Chinese medicine sample of each batch is that the probable value providing based on mahalanobis distance is determined; Before calculating mahalanobis distance, the HPLC finger-print data of the first traditional Chinese medicine sample with reference to classification are carried out principal component analysis (PCA) (Principal component analysis, PCA), number of principal components is that the principle that is greater than All Eigenvalues mean value according to character pair value is determined.Determine score vector according to number of principal components, and then calculate mahalanobis distance; Then mahalanobis distance is checked and provided corresponding probable value (P) by F, if P<0.05(α=0.05 of the traditional Chinese medicine sample of certain batch), judge this batch traditional Chinese medicine sample HPLC finger-print with reference to collection of illustrative plates, there were significant differences, represent that other traditional Chinese medicine sample consistance of this batch of sample and reference class exists significant difference.If without significant difference, judge that the traditional Chinese medicine sample of this batch and other traditional Chinese medicine sample of reference class are other batch of sample of same class, consistance there was no significant difference.
In described step (6), adopt t inspection to carry out peak area significance test, in theory, chromatographic peak area and the proportional relation of tie element content, the t assay based on peak area is consistent with the t assay based on tie element content; In other words, adopt t to upcheck and judge whether peak area has significant difference, can judge whether tie element content has significant difference, without the quantitative test of chromatographic peak tie element.
Determination methods for each batch of unanimity of samples of Chinese medicine of the present invention, method is easy, Yi Hang, practicality, is suitable for, accurately judgement batch conforming fast to Chinese medicine, for a Chinese medicine batch conforming judgement provides method foundation.
Brief description of the drawings
Fig. 1 is the efficient liquid-phase chromatograph finger print atlas that adopts U.S. Agilent1200series high performance liquid chromatograph to obtain.
Fig. 2 is the each batch of sample HPLC finger-print that adopts that " chromatographic fingerprints of Chinese materia medica similarity evaluation software " use that related coefficient or Cosin method calculate and the similarity evaluation result that contrasts collection of illustrative plates.
Fig. 3 is the probability that different batches sample calculates according to mahalanobis distance with respect to other traditional Chinese medicine sample of reference class.The dotted line that is parallel to horizontal ordinate in figure represents α=0.05 level of significance.
Fig. 4 is the z value that mahalanobis distance is judged to be the total peak of 19 batch samples of significant difference.Wherein z
critaccording to t inspection t under the level of α=0.05
critcalculate by formula (2).
Fig. 5 is the frequency number percent that has the total peak of significant difference.
Fig. 6 is the representative HPLC finger-print of Chinese medicine Xin Ke Shu ' tablet for treating coronary heart disease.Wherein the chromatographic peak of mark is the chromatographic peak of frequency large percentage shown in Fig. 5 (>60%).
Embodiment
Below in conjunction with embodiment, the present invention is further illustrated.
The preparation of 1.1 need testing solutions
First get the Xin Ke Shu ' tablet for treating coronary heart disease sample of appropriate different batches (this example is 71 batches), remove outer coatings film, grind.Precision takes ground sample fine powder 100mg, adds 70% ethanol (percent by volume) 5mL, and ultrasonic extraction 20min under room temperature, gets 4mL at the centrifugal 10min of 3000r/min.Then get supernatant after 0.45 μ m membrane filtration, sample introduction, adopts efficient liquid phase chromatographic analysis instrument to record can the relax original HPLC finger-print of ethanol extract composition of these different batches hearts, as shown in Figure 1.
1.2 instruments and chromatographic condition
Agilent1200 liquid chromatograph (Agilent Corp., Santa Clara, CA, USA), DAD detecting device, PhenomexC
18(250mm × 4.6mm, 4 μ are m); Mobile phase: acetonitrile (A) and 0.1%(percent by volume) formic acid (B) aqueous solution; Gradient elution: 0-21min, 8-12% (A); 21-31min, 12-17% (A); 31-55min, 17-38% (A); 55-65min, 38-90% (A); 65-75min, 90% (A), flow velocity is 1.0ml/min, and sample size is 10 μ L, and column temperature is 30 DEG C.Detection wavelength is 278nm.
The methodology checking of 1.3 finger-prints
By same batch of sample extracting solution continuous sample introduction 6 times, the total relative retention time at peak of the finger-print of mensuration and the relative standard deviation of peak area ratio (RSD) are respectively and are less than 1.2% and 3.0%, show that the precision of method is better.Core 5 parts, Xin Ke Shu' tablet sample, obtain 5 duplicate samples extracts by 1.1 described methods, measure HPLC finger-print in accordance with the law, the total relative retention time at peak and the RSD of peak area ratio are less than respectively 1.4% and 1.8%, the repeatability that shows method is better, meets the technical requirement of finger-print.Same batch of sample extracting solution carried out to 6 times in 3 days and measure, obtain the total relative retention time at peak of HPLC finger-print and the RSD of peak area ratio and be less than respectively 2.3% and 3.0%, show that sample extracting solution is stable in 3 days.
1.4 similarity analysis
Adopt " chromatographic fingerprints of Chinese materia medica similarity evaluation software " (2004A version, pharmacopoeia commission's recommendation) the HPLC finger-print data of obtaining are carried out to Supplements, coupling, generates contrast collection of illustrative plates, obtains the data matrix of each batch of sample HPLC finger-print.In this matrix, " OK " represents sample, and " row " represent variable (namely chromatographic peak).Calculate respectively each batch of sample HPLC finger-print and the similarity that contrasts collection of illustrative plates by related coefficient and Cosin method, result is as Fig. 2.The similarity value of the each batch of sample HPLC finger-print that visible two kinds of methods are calculated is very high, is all greater than 0.900.
Other structure of 1.5 reference classes
Select the high batch sample of active constituent content as with reference to sample.It is reference sample that this experiment adopts batch sample 5, because of its puerarin content maximum in each batch of sample.Based on reference sample HPLC finger-print, adopt the Euclidean distance of each batch of sample HPLC finger-print of Matlab software calculating and reference sample HPLC finger-print, each batch of sample pressed to the ascending arrangement of size of Euclidean distance.Select batch sample that some and reference sample HPLC finger-print Euclidean distance are less to build with reference to classification.Other sample size of reference class can be determined by user.This experiment is according to the technical requirement of finger-print research, and finger-print sample batch quantity is no less than 10 batches for foundation, determines that reference class other style product batch quantity is 11, comprises reference sample (being sample 5).
1.6 each batches of sample consistance judgements
The average finger-print (with reference to collection of illustrative plates) that adopts Matlab software computing reference classification sample HPLC finger-print, then calculates all batches of sample HPLC finger-prints and the mahalanobis distance (Mahalanobis distance) with reference to collection of illustrative plates.Before calculating mahalanobis distance, need first to carry out principal component analysis (PCA) (Principalcomponent analysis, PCA) with reference to classification sample HPLC finger-print data.Because the reference class very variable number (being chromatogram peak number) of product HPLC finger-print data is greater than sample number, such matrix cannot directly carry out mahalanobis distance calculating.In PCA process, number of principal components is that the principle that is greater than the mean value of All Eigenvalues according to character pair value is determined.In this example, the eigenwert of front 2 major components is greater than the average of All Eigenvalues, and therefore number of principal components is defined as 2.Reference class very product HPLC finger-print data obtains load vector (being proper vector) through PCA, then calculate the score of all batches of sample HPLC finger-print data according to load vector, and then calculate again each batch of sample HPLC finger-print and the mahalanobis distance with reference to collection of illustrative plates.The mahalanobis distance calculating is checked and calculated corresponding probable value (P) by F, judge each batch of sample HPLC finger-print and whether have significant difference with reference to collection of illustrative plates (α=0.05) under certain level of significance with this.If P<0.05(α=0.05), corresponding batch sample HPLC finger-print, with there were significant differences with reference to collection of illustrative plates, illustrates this batch of sample and has significant difference with reference to a classification batch sample consistance.If P >=0.05(α=0.05), corresponding batch sample HPLC finger-print with reference to collection of illustrative plates without significant difference, show this batch of sample with reference to classification batch sample consistance there was no significant difference.Different batches sample is shown in Fig. 3 with respect to the reference class probability that very product calculate by mahalanobis distance, and wherein front 11 samples are very product of reference class.As shown in Figure 3, under the level of α=0.05, very product HPLC finger-print and there is no significant difference with reference to collection of illustrative plates of reference class.In other batch of sample, there are 19 batches of samples (being Fig. 3 the right) with respect to there is significant difference (the namely other population of samples of reference class) with reference to collection of illustrative plates, these batch of sample finger-print is described and may has larger difference with reference to collection of illustrative plates.
Mahalanobis distance is used for judging each batch of sample HPLC finger-print and with reference to whether there being significant difference between collection of illustrative plates, has significant difference but be difficult to find out which finger-print chromatographic peak area in there were significant differences batch sample.In this example, adopt the t method of inspection can judge which chromatographic peak area has significant difference.Because the total peak of finger-print is the common characteristic of each batch of sample, the area discrepancy of these chromatographic peaks is particularly important for batch sample consistance.For this reason, the total peak area of batch sample HPLC finger-print that adopts the t method of inspection to be judged to be significant difference to mahalanobis distance is tested, and judges which total peak area and has significant difference (α=0.05) with reference to the total peak area of classification batch sample HPLC finger-print.In t checkout procedure, chromatographic peak area need to be transformed by formula (1),
In formula (1), x
irefer to the peak area of certain batch of sample HPLC finger-print chromatographic peak i.
and S
ibe respectively peak area mean value and standard deviation with reference to each batch of sample HPLC finger-print chromatographic peak i in classification.By z
ivalue is calculated t by formula (2)
i:
t
i＝z
i[n/(n+1)]
1/2 （2）
In formula (2), n represents the other sample size of reference class.T
ikey value (t with student t distribution
crit, i.e. α=0.05 o'clock corresponding t value) compare, to determine x
iwith
whether there is significant difference.From formula (1) and (2), although t
ibe to calculate based on the area of finger-print chromatographic peak i, but it is consistent with the result of calculation based on chromatographic peak i tie element content.Proof procedure is as follows:
In chromatographic quantitative analysis, the area of a certain chromatographic peak i and the following relation of the content of tie element, as the formula (3),
x
i＝f
ic
i+b
i （3）
Wherein c
irepresent the concentration of chromatographic peak i tie element, f
iand b
ibe constant in chromatographic condition one timing.Formula (3) substitution formula (1) is had
From formula (4), based on the z of calculated by peak area
iwith the z based on the cubage of chromatographic peak tie element
iidentical, therefore t
i(seeing formula (2)) is also identical, and card is finished.
From above-mentioned proof, the t assay based on chromatographic peak area is consistent with the t assay based on chromatographic peak tie element content.Therefore, we can just can be judged in certain batch of sample HPLC finger-print chromatographic peak i tie element content and whether be had significant difference with reference to the average content of classification batch sample composition i by the t assay based on chromatographic peak area, without in advance component i being carried out to quantitative test.Whether this result has significant difference very convenient for investigating which chromatographic peak tie element content in certain batch of sample HPLC finger-print, make to adopt other method (as mass spectrophotometry) to do further to identify that Objective is stronger to composition, for determining that affecting a Chinese medicine batch conforming factor has important directive significance.
In this example, while adopting t inspection to carry out significance test to total peak, by the t of α=0.05 o'clock
critthrough type (2) is converted into z
crit, then directly compare to judge with the z value that adopts formula (1) to calculate based on peak area.The total peak z value that is judged as 19 batch samples of significant difference by mahalanobis distance is shown in Fig. 4.As shown in Figure 4, as long as z
i>z
crit, illustrate that chromatographic peak i is judged to be the total peak of significant difference through t inspection.In Fig. 6, major part has the total peak area of significant difference all relatively little, illustrates that the content of tie element may be lower than the very average content of the corresponding composition of product of reference class.Also inconsistent (as shown in Figure 5) of frequency number percent that has the total peak of significant difference to occur in 19 batch samples.Wherein, the ratio of No. 13 and No. 19 total peak appearance is the highest, is secondly No. 16 and No. 20 total peaks.There is the retention time of the total peak (>60%) of large percentage in HPLC finger-print as shown in Figure 6.
As above embodiment shows, on the basis that the present invention analyzes at fingerprint similarity, can judge a Chinese medicine batch consistance by mahalanobis distance, whether significantly can provide the difference of batch sample room from the angle of probability.Be judged to be for mahalanobis distance batch sample that there were significant differences, adopt the t method of inspection can judge in these batch of sample HPLC finger-print, which chromatographic peak tie element content whether have significant difference.
Embodiment recited above limits design of the present invention and protection domain, and various modification and improvement that those skilled in the art make technical scheme of the present invention, all should fall into protection scope of the present invention.
List of references
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[2]P.S.Xie,S.B.Chen,Y.Z.Liang,X.H.Wang,R.T.Tian,R.Upton,J.Chromatogr.A?1112(2006)171.
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[4]FDA?Guidance?for?Industry–Botanical?Drug?Products(Draft?Guidance),US?Food?and?Drug?Administration,Rockville,2000,pp.4.
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[6]State?Food?and?Drug?Administration?of?China,Note?for?Studying?Fingerprint?of?TraditionalChinese?Medicine?Injections(Draft),Chinese?Traditional?Patent?Medicine,22(2000)671.
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Claims (3)
1. for a determination methods for each batch of unanimity of samples of Chinese medicine, it is characterized in that: step is as follows:
(1) select the traditional Chinese medicine sample of multiple different batches to be judged, after grinding, adopt the solvent extraction that is applicable to extracting this Effective Component of Chinese Medicine, obtain extract;
(2) adopt high performance liquid chromatograph to gather the original high efficiency liquid-phase chromatograph finger print atlas of the extract of the traditional Chinese medicine sample of above-mentioned different batches at the detection wavelength place that is applicable to this Chinese medicine;
(3) the HPLC finger-print of above-mentioned gained is carried out to methodology checking, make measured HPLC finger-print meet fingerprint pattern technology code requirement; Then calculate the average collection of illustrative plates collection of illustrative plates in contrast of the HPLC finger-print of the traditional Chinese medicine sample of each batch; Calculate respectively the HPLC finger-print and the similarity that contrasts collection of illustrative plates of the traditional Chinese medicine sample of each batch; If similarity r is more than or equal to 0.900, retain, if r<0.900, by its rejecting, then recalculate the contrast collection of illustrative plates of traditional Chinese medicine sample of residue batch, and remain the similarity that contrasts collection of illustrative plates of traditional Chinese medicine sample of HPLC finger-print and the residue batch of the traditional Chinese medicine sample of each batch, and then adopt criterion similarity same as described above; Repeat this process, until it meets the requirements;
(4) the HPLC finger-print of selecting the sample that active constituent content is high in remaining batch from above-mentioned selection as a reference, select the traditional Chinese medicine sample of multiple batch nearest with the HPLC finger-print Euclidean distance of reference sample, then build with reference to classification with reference sample, and the average finger-print of computing reference classification sample, with reference to collection of illustrative plates;
(5) based on above-mentioned with reference to classification, calculate each batch traditional Chinese medicine sample HPLC finger-print with reference to the mahalanobis distance of collection of illustrative plates, then mahalanobis distance is converted into F inspection, thus judge different batches traditional Chinese medicine sample HPLC finger-print and with reference between collection of illustrative plates, under probability level necessarily, whether there were significant differences;
(6) find out mahalanobis distance and be judged to be the traditional Chinese medicine sample of those batches of significant difference, adopt t inspection to be tested in the total peak of the HPLC finger-print of the traditional Chinese medicine sample of these batches and the total peak of the HPLC finger-print of the traditional Chinese medicine sample with reference to each batch of classification, in the total peak of judgement, whether each chromatographic peak area has significant difference under certain probability level; Be judged as the chromatographic peak of significant difference, show in the traditional Chinese medicine sample of this batch, on the represented active constituent content of this chromatographic peak, be starkly lower than or higher than the average content of the corresponding composition of other traditional Chinese medicine sample of reference class;
In t checkout procedure, chromatographic peak area need to be transformed by formula (1),
In formula (1), x
irefer to the peak area of certain batch of sample HPLC finger-print chromatographic peak i;
and S
ibe respectively peak area mean value and standard deviation with reference to each batch of sample HPLC finger-print chromatographic peak i in classification; By z
ivalue is calculated t by formula (2)
i:
t
i＝z
i[n/(n+1)]
1/2（2）
In formula (2), n represents the other sample size of reference class; t
ikey value t with student t distribution
critcompare, to determine x
iwith
whether there is significant difference.
2. a kind of determination methods for each batch of unanimity of samples of Chinese medicine according to claim 1, it is characterized in that: described step (4) is specific as follows: select the HPLC finger-print of batch sample that active constituent content is high as a reference, calculate the Euclidean distance of the HPLC finger-print of other traditional Chinese medicine sample of each batch and the HPLC finger-print of reference sample, according to Euclidean distance is ascending, other traditional Chinese medicine sample of each batch is arranged, select the traditional Chinese medicine sample of multiple batch nearest with the HPLC finger-print Euclidean distance of reference sample, and build with reference to classification with reference sample, and computing reference collection of illustrative plates.
3. a kind of determination methods for each batch of unanimity of samples of Chinese medicine according to claim 1, is characterized in that: in described step (5), the consistance quality that judges the traditional Chinese medicine sample of each batch is that the probable value providing based on mahalanobis distance is determined; Before calculating mahalanobis distance, the HPLC finger-print data of the first traditional Chinese medicine sample with reference to classification are carried out principal component analysis (PCA); Determine score vector according to number of principal components, and then calculate mahalanobis distance; Then mahalanobis distance is checked and provided corresponding probable value P by F, if the P<0.05 of the traditional Chinese medicine sample of certain batch, α=0.05, judge this batch traditional Chinese medicine sample HPLC finger-print with reference to collection of illustrative plates, there were significant differences, represent that other traditional Chinese medicine sample consistance of this batch of sample and reference class exists significant difference; If without significant difference, judge that the traditional Chinese medicine sample of this batch and other traditional Chinese medicine sample of reference class are other batch of sample of same class, consistance there was no significant difference.
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