CN109959732A - The separation method of Kangfuxin Liquid finger-print and its effective constituents A - Google Patents
The separation method of Kangfuxin Liquid finger-print and its effective constituents A Download PDFInfo
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Abstract
The invention discloses Kangfuxin Liquid finger-print and its separation methods of effective constituents A.The application has carried out analysis and assay to Kangfuxin Liquid effective component by using specific HPLC chromatogram method, while establishing Kangfuxin Liquid finger-print accordingly.This method characterizes the various features substance in Kangfuxin Liquid, is significantly better than the method for just carrying out Quality estimation to Kangfuxin Liquid to a few chemical component only by single wavelength in the prior art, identifies to Kangfuxin Liquid and quality control effect is good.
Description
Technical field
The present invention relates to Kangfuxin Liquid finger-print and its separation methods of effective constituents A.
Background technique
Kangfuxin Liquid standard is the Kangfuxin Liquid quality standard that National Drug Administration on December 11 in 2000 promulgates
Revise official written reply, standard number are as follows: WS3- B-3674-2000 (Z), the every lml of standard requirements Kangfuxin Liquid are containing total amino acid with the third ammonia
Acid (C3H7O2N it) counts, 0.72mg must not be less than.The standard provides only the measuring method and pyrimidine of total amino acid in Kangfuxin Liquid
The qualitative determination method of alkali.It is well known that in Kangfuxin Liquid amino acid many kinds of, such as alanine, tyrosine, bright ammonia
Acid, isoleucine, phenylalanine and tryptophan etc..But the specific type of amino acid in Kangfuxin Liquid is not provided in the standard
And the measuring method of content.In addition, as ucleotides such as another principle active component pyrimidine bases in Kangfuxin Liquid, type
And content assaying method does not also provide in the standard.In face of such situation, generallyd use in this field by amino acid into
The method of row derivatization carries out the assay of amino acid using the method for HPLC, but this method is complicated for operation, and cannot be same
Detection and assay of the Shi Shixian to amino acids and ucleotides substance.
In addition, there is no fingerprints in the standard (WS3-B-3674-2000 (Z)) for the Kangfuxin Liquid that CFDA2001 is promulgated
Atlas analysis has been pointed out only by the finger-print of single wavelength detection Kangfuxin Liquid in the internal standard of most enterprises few
The several shared peaks of number, for example uracil, hypoxanthine, inosine and a small amount of gradient elution, there is no simultaneously to therein
Aminoacid ingredient is detected.
Therefore, simple, conveniently there is an urgent need in the art to establish in order to preferably monitor each component content in Kangfuxin Liquid
And the method that ucleosides and amino acids assay can be measured simultaneously.Meanwhile if to each component content of Kangfuxin Liquid
More preferably understood, then can advanced optimize Kangfuxin Liquid finger-print, to monitoring ucleosides therein and amino acids chemical combination
Object and its quality control are all of great significance.
Summary of the invention
The technical problem to be solved by the present invention is in order to overcome the prior art to the ammonia in Kangfuxin Liquid effective component
Base acid or nucleotide compound are complicated for operation when being measured, or can not accurately determine the wherein defect of specific ingredient or content,
It is separated, analyzed to provide a kind of Kangfuxin Liquid effective constituents A, detection method of content and Kangfuxin Liquid finger-print.The party
Method has carried out analysis and assay to Kangfuxin Liquid effective constituents A by using specific HPLC chromatogram method, while accordingly
Establish Kangfuxin Liquid finger-print.This method is easy to operate, within a short period of time can be phonetic by the urine in Kangfuxin Liquid simultaneously
Pyridine, hypoxanthine, uric acid, adenosine, xanthine, inosine, tyrosine, phenylalanine and tryptophan carry out separation and assay,
And accuracy rate is high.The finger-print for the Kangfuxin Liquid established according to this method has 12 shared peaks, carries out to various features substance
Characterization is significantly better than the side for just carrying out Quality estimation to Kangfuxin Liquid only by a few chemical component in the prior art
Method identifies Kangfuxin Liquid and quality control effect is good.
The present invention is to solve above-mentioned technical problem by the following technical programs.
The present invention provides a kind of separation methods of Kangfuxin Liquid effective constituents A, and it includes following steps: using efficient liquid
Phase chromatography separates Kangfuxin Liquid effective component；
The chromatographic column used in the high performance liquid chromatography is C18Column；
Mobile phase A and Mobile phase B are used in the high performance liquid chromatography；The mobile phase A is 0.06%~0.3%
H3PO4Aqueous solution or 0.06%~0.3% aqueous formic acid, such as: 0.1%~0.3%H3PO4Aqueous solution, the Mobile phase B
For acetonitrile and/or alcohols solvent；Preferably, the mobile phase A is 0.06%~0.1%H3PO4Aqueous solution, the Mobile phase B
For methanol.
The Kangfuxin Liquid effective constituents A be uracil, hypoxanthine, uric acid, adenosine, xanthine, inosine, tyrosine,
Two or more in phenylalanine and tryptophan；
The Kangfuxin Liquid effective component is to include uracil, hypoxanthine, uric acid, adenosine, xanthine, inosine, network ammonia
The second of the dry polypide of the mixture of acid, phenylalanine and tryptophan, for example, American cockroach (Periploneta americana)
Alcohol extracting thing.
Preferably, the Kangfuxin Liquid effective constituents A be uracil, hypoxanthine, uric acid, adenosine, xanthine, inosine,
Tyrosine, phenylalanine and tryptophan.
Preferably, the Kangfuxin Liquid effective component is handled with the miillpore filter that aperture is 0.22 μm.
In the present invention, the chromatographic column can be Boston Luna Clone column, Diamonsail C18 column, Agela
Venusil XBP-C18 column, Acchrom Unitary-C18 column or Ultimate Polar-RP column；Preferably, the chromatography
Column is Boston Luna Clone column
In the present invention, when the mobile phase A is 0.1%~0.3%H3PO4When aqueous solution, the Mobile phase B can be alcohols
Solvent.
In the present invention, the Mobile phase B can be selected from one of methanol, ethyl alcohol and isopropanol or a variety of, such as methanol.
In the present invention, the high performance liquid chromatography can be used isocratic or gradient elution mode, when the gradient elution
The volume ratio of the mobile phase A and the Mobile phase B can be 95:5~30:70；Preferably, the flowing when gradient elution
The volume ratio of phase A and the Mobile phase B can be 95:5~65:35.
In the present invention, in the gradient elution, the mobile phase ratio changes with time range can also be as shown in table 1:
Table 1
In the present invention, the specification of the chromatographic column can be the analytical column of this field routine or prepare column specification:
The filler particles degree of the chromatographic column can be 2.7 μm~5 μm；Such as 3 μm~5 μm；The length of the chromatographic column can
For 100mm~250mm, such as 150mm.
In the present invention, the high performance liquid chromatograph of this field routine can be used in the high performance liquid chromatography, such as
Agilent1260 high performance liquid chromatograph.
In the present invention, the column temperature in the high performance liquid chromatography can be the ordinary temperature of this field generic operation, such as
20~40 DEG C, then such as 30 DEG C.
In the present invention, the flow velocity of the mobile phase can refer to the routine operation in field, such as 0.3~1.2mL/min,
Such as 0.4~0.5mL/min again.
The present invention also provides a kind of analysis methods of Kangfuxin Liquid effective constituents A, and it includes following steps, by institute
High performance liquid chromatography is stated to detect Kangfuxin Liquid effective constituents A with detector combination, the Kangfuxin Liquid is effective
Ingredient A is defined as described above.
The detector is preferably DAD detector.
The Detection wavelength of the DAD detector can be 210nm~254nm；Such as when using gradient elution as shown in Table 1
When mode, when elution time is 0~16min, the Detection wavelength is 254nm；When elution time is 17~26min, the inspection
Survey wavelength is 230nm；When elution time is 27~50min, the Detection wavelength is 214nm.
The present invention also provides a kind of sides for carrying out assay to Kangfuxin Liquid effective constituents A using the analysis method
Method, the Kangfuxin Liquid effective constituents A are defined as described above.
When the progress assay, can refer in this field using high performance liquid chromatography carry out assay method with
Operation, for example, the method for the assay is comprised the steps of: the reference substance of known concentration and Kangfuxin Liquid described in
Analysis method carries out the acquisition of peak area, then using external standard method or internal standard method can be calculated the Kangfuxin Liquid effectively at
Divide the content of each ingredient in A.
The present invention also provides the finger-prints of Kangfuxin Liquid, and it to be phenylpropyl alcohol ammonia referring to peak that it includes 12 shared fingerprint peaks
Sour chromatographic peak, the relative retention time of 12 shared fingerprint peaks are respectively as follows: No. 1 0.36 ± 0.01min of peak, and No. 2 peaks 0.43 ±
0.01min, No. 3 0.47 ± 0.01min of peak, No. 4 0.51 ± 0.01min of peak, No. 5 0.59 ± 0.01min of peak, No. 6 peaks 0.63 ±
0.01min, No. 7 0.86 ± 0.02min of peak, No. 8 1.25 ± 0.02min of peak, No. 9 1.49 ± 0.02min of peak, No. 10 peaks 1.71
± 0.02min and No. 11 1.79 ± 0.02min of peak, wherein the ratio of peak area is peak number 1: peak number 2: peak number 6: peak number 7:
Peak number 8: peak number 9: peak 10=(0.93~1.04): (0.84~1.06): (0.61~0.65): (0.88~0.91):
1.00:(1.02~1.06): (1.017~1.18)；
The Kangfuxin Liquid finger-print is to be measured by using high performance liquid chromatography to Kangfuxin Liquid:
In the high performance liquid chromatography, chromatographic column is Boston Luna Clone, and specification is 4. 6mm × 250mm × 5 μm；Stream
Dynamic phase is with 0.1%H3PO4For mobile phase A, using methanol as Mobile phase B, using gradient elution program, flow velocity: 0.5ml/min is examined
Survey wavelength: 0~16min is 254nm, and 17~26min is 230nm, and 27~50min is 215nm, column temperature: 30 DEG C；The gradient
Elution program is shown in Table 8:
Table 8
Without prejudice to the field on the basis of common sense, above-mentioned each optimum condition, can any combination to get the present invention it is each preferably
Example.
The reagents and materials used in the present invention are commercially available.
The positive effect of the present invention is that:
Method provided by the present application is easy to operate, within a short period of time can simultaneously by uracil, hypoxanthine, uric acid,
Adenosine, xanthine, inosine, tyrosine, phenylalanine and tryptophan carry out separation and assay, and accuracy rate is high；
Kangfuxin Liquid finger-print provided by the present application not only contains 3 uracil, hypoxanthine, inosine ucleosides
Ingredient, has also monitored 3 uric acid, adenosine, xanthine and tyrosine, phenylalanine, tryptophan aminoacid ingredients, and multi objective is more
The monitoring of ingredient carries out quality control to the ingredient in Kangfuxin Liquid more fully hereinafter；
Analysis method provided by the present application detects 28 batches of samples, the map of acquisition with pass through Chinese Pharmacopoeia Commission
The similarity for the control spectrogram that " similarity evaluation " promulgated is formed is high, can reach 0.8 or more,
0.976 can be even up to.
Detailed description of the invention
Fig. 1 is the analysis map of reference substance HPLC in embodiment 1.
Fig. 2 is the analysis map of test sample (S9) HPLC in embodiment 1.
Fig. 3 is the finger-print and standard finger-print of 28 test samples in embodiment 1.
Specific embodiment
The present invention is further illustrated below by the mode of embodiment, but does not therefore limit the present invention to the reality
It applies among a range.In the following examples, the experimental methods for specific conditions are not specified, according to conventional methods and conditions, or according to quotient
The selection of product specification.
Embodiment 1
1.1 instrument Agilent1260 high performance liquid chromatographs (including the online vacuum degassing machine of 1322A type, 1311B type four
First pump, 1367E type autosampler, 1316A column insulating box, 1315C type DAD detector), Agilent company, Switzerland CAMAG
Company；Satorius BSA 124S-CW a ten thousandth electronic analytical balance, Beijing Sai Duolisi instrument system Co., Ltd,
KQ-250DB numerical control ultrasonic cleaner, Kunshan Ultrasonic Instruments Co., Ltd..
1.2 reagent uracils, adenosine, hypoxanthine, inosine reference substance are purchased from SigmaAldrich, phenylalanine, color ammonia
Acid, tyrosine reference substance and NaOH are purchased from Sinopharm Chemical Reagent Co., Ltd., and uric acid, xanthine reference substance are purchased from Shang Haiyuan
Leaf biology Co., Ltd.
Methanol (Honeywell Lot No:R4CG2H) and phosphoric acid are chromatographic grade (Aladdin Lot:J1724005), and water is
Ultrapure water (Merck Mi Libo), other reagents are analysis level.
28 batches of Kangfuxin Liquid information are as follows:
2 methods and result
The preparation of 2.1 reference substance solutions
Precision weighs appropriate uracil, adenosine, hypoxanthine, inosine, phenylalanine, tryptophan product, adds 50% first
It is respectively 1.593,3.016,1.898,2.009,2.633,1.110mgmL that alcohol, which is configured to concentration,-1Reference substance mother liquor, weigh
It is 2.442,0.708,1.090mg that appropriate tyrosine, uric acid, xanthine reference substance, which add 1.5mM NaOH solution to be configured to concentration,
mL-1Reference substance mother liquor.It is accurate respectively to draw uracil, adenosine, hypoxanthine, inosine, phenylalanine, tryptophan product mother
Liquid, adding ultrapure water to be diluted to concentration is 159.3,301.6,189.8,200.9,263.3,111.0 μ gmL-1Reference substance mixing
Liquid, as mixing reference substance stock solution.Absorption tyrosine, uric acid, xanthine reference substance mother liquor add ultrapure water to be diluted to concentration
224.2、70.8、109.0μg·mL-1Reference substance mixed liquor.
The preparation of 2.2 test solutions
Pipette pipettes 1ml Kangfuxin Liquid in 10ml volumetric flask, and appropriate ultrapure water constant volume is added, and crosses 0.22 μm of micropore
Filter membrane (rub fast scientific equipment Co., Ltd in Shanghai) to obtain the final product.
2.3 chromatographic condition
With Boston Luna Clone (4.6mm × 250mm, 5 μm) chromatographic column, with 0.1%H3PO4For mobile phase A, methanol
For Mobile phase B, gradient elution program is shown in Table 9, flow velocity: 0.5ml/min, Detection wavelength: 0~16min is 254nm, 17~26min
It is 215nm, column temperature: 30 DEG C for 230nm, 27~50min.
9 eluent gradient elution program of table
Reference substance HPLC the result is shown in Figure 1；Test sample result is shown in Fig. 2.
In Fig. 1,1 is uracil；2 be hypoxanthine；3 be uric acid；4 be adenosine；5 be xanthine；6 be tyrosine；7 are
Inosine；8 be phenylalanine；11 be tryptophan.
In Fig. 2,1 is uracil；2 be hypoxanthine；3 be adenosine；4 be uric acid；5 be xanthine；6 be tyrosine；7 are
Inosine；8 (S) are phenylalanine；9 be protocatechuic acid -4-O- β-D- glucopyranoside；11: tryptophan
2.4 methodological study
2.4.1 precision
S10 sample is taken, by 22 lower section legal system available test sample solutions.It is descended method continuous sample introduction 6 times on the same day by 2.3, with No. 8
Peak (phenylalanine) is to calculate the relative retention time and relative peak area at each shared peak referring to peak.As a result it is shown in Table 2 and table respectively
3.The relative retention time at each shared peak, the RSD of relative peak area are respectively less than 3% (n=6), illustrate that instrument precision is good.
Table 2
Table 3
2.4.2 repeated
S10 sample is taken, prepares 6 parts of test solutions in parallel by 2.2 lower methods.By 2.3 lower method sample introductions.With No. 8 peak (benzene
Alanine) it is to calculate the relative retention time and relative peak area at each shared peak referring to peak.It the results are shown in Table 4 and table 5.
Table 4
Table 5
Table 4 and table 5 illustrate that the relative retention time at each shared peak, the RSD of relative peak area is respectively less than 3% (n=6), says
Bright method repeatability is good.
2.4.3 stability
S9 sample is taken, by 2.2 lower section legal system available test sample solutions.By 2.3 lower methods respectively at 0,2,4,8,16,24,36h
Sample introduction.It is to calculate the relative retention time and relative peak area at each shared peak referring to peak with No. 8 peaks (phenylalanine).As a result see
Table 6 and table 7.The relative retention time at each shared peak, the RSD of relative peak area are respectively less than 3% (n=6), illustrate sample stability
Well.
Table 6
Table 7
Table 6 and table 7 illustrate that the relative retention time at each shared peak, the RSD of relative peak area is respectively less than 3% (n=6),
Illustrate that sample stability is good.
2.4.4 assay
This experiment prepares sample by 2.2 the methods to above-mentioned 28 batches of Kangfuxin Liquid samples, is surveyed by 2.3 lower methods
The fixed content to get each component in each sample, as follows, the content unit of each component are ug/ml.
2.4.5 similarity analysis
Result is evaluated using similarity software, this experiment is to above-mentioned 28 batches of Kangfuxin Liquid samples, by 2.2 (lower sections
Method prepares sample, is measured the chromatogram for comparing each sample to get each sample content by 2.3 lower methods, while by chromatogram
Data import " similarity evaluation " (2012 editions) software of Chinese Pharmacopoeia Commission's promulgation, first
Chromatography peak match is carried out, control map (map for being R see the number in Fig. 3) is generated, and compares trace analysis, finger-print is shown in
Figure, similarity result are as follows.
The result of similarity can be seen that above-mentioned 28 batches of Kangfuxin Liquid samples with compare the similarity of map in 0.833-
Between 0.976, overall similarity is preferable.
Claims (10)
1. a kind of separation method of Kangfuxin Liquid effective constituents A, is characterized in that comprising the steps of: use high performance liquid chromatography
Method separates Kangfuxin Liquid effective component；
The chromatographic column used in the high performance liquid chromatography is C18Column；
Mobile phase A and Mobile phase B are used in the high performance liquid chromatography；The mobile phase A is 0.06%~0.3%H3PO4Water
Solution and/or 0.06%~0.3% aqueous formic acid；The Mobile phase B is acetonitrile and/or alcohols solvent；
The Kangfuxin Liquid effective constituents A is uracil, hypoxanthine, uric acid, adenosine, xanthine, inosine, tyrosine, phenylpropyl alcohol
Two or more in propylhomoserin and tryptophan；
The Kangfuxin Liquid effective component is to include uracil, hypoxanthine, uric acid, adenosine, xanthine, inosine, tyrosine, benzene
The mixture of alanine and tryptophan.
2. separation method as described in claim 1, which is characterized in that the Kangfuxin Liquid effective constituents A is uracil, secondary Huang
Purine, uric acid, adenosine, xanthine, inosine, tyrosine, phenylalanine and tryptophan；
And/or the Kangfuxin Liquid effective component is handled with the miillpore filter that aperture is 0.22 μm.
3. separation method as described in claim 1, which is characterized in that the Kangfuxin Liquid effective component is American cockroach
The ethanol extract of (Periploneta americana) dry polypide.
4. separation method as described in claim 1, which is characterized in that the chromatographic column be Boston Luna Clone column,
Diamonsail C18 column, Agela Venusil XBP-C18 column, Acchrom Unitary-C18 column or Ultimate
Polar-RP column；
And/or the Mobile phase B is selected from one of methanol, ethyl alcohol and isopropanol or a variety of, such as methanol；
And/or the mobile phase A is 0.1%~0.3%H3PO4Aqueous solution；The Mobile phase B is alcohols solvent；
And/or the high performance liquid chromatography is by the way of isocratic or gradient elution；
And/or the filler particles degree of the chromatographic column is 2.7 μm~5 μm；Such as 3 μm~5 μm；
And/or the length of the chromatographic column is 100mm~250mm, such as 150mm；
And/or the high performance liquid chromatograph in the high performance liquid chromatography is Agilent1260 high performance liquid chromatograph；
And/or the column temperature in the high performance liquid chromatography is 20~40 DEG C, such as 30 DEG C；
And/or the flow velocity of the mobile phase is 0.3~1.2mL/min, such as 0.4~0.5mL/min.
5. separation method as claimed in claim 4, which is characterized in that the mobile phase A and the stream when gradient elution
The volume ratio of dynamic phase B is 95:5~30:70；Preferably, when the gradient elution mobile phase A and the Mobile phase B body
Product is than being 95:5~65:35；For example, the mobile phase ratio changes with time, range is as shown in table 1:
Table 1
6. a kind of analysis method of Kangfuxin Liquid effective constituents A, which is characterized in that comprise the steps of, will as claim 1~
High performance liquid chromatography described in 5 any one detects Kangfuxin Liquid effective component with detector combination, it is described
Kangfuxin Liquid effective constituents A is the Kangfuxin Liquid effective constituents A as described in claims 1 or 2.
7. analysis method as claimed in claim 6, which is characterized in that the detector is DAD detector.
8. analysis method as claimed in claim 7, which is characterized in that the Detection wavelength of the DAD detector be 210nm~
254nm；For example, when using as using gradient elution mode shown in table 1 described in claim 5, elution time 0
When~16min, the Detection wavelength is 254nm；When elution time is 17~26min, the Detection wavelength is 230nm；Elution
When time is 27~50min, the Detection wavelength is 214nm.
9. a kind of side for carrying out assay to Kangfuxin Liquid effective constituents A using analysis method as claimed in claims 6 or 7
Method, which is characterized in that comprise the steps of: and the reference substance of known concentration and Kangfuxin Liquid are subjected to peak by the analysis method
Then the content of each effective component of the Kangfuxin Liquid, institute can be calculated in the acquisition of area using external standard method or internal standard method
Stating Kangfuxin Liquid effective constituents A is the Kangfuxin Liquid effective constituents A as described in claims 1 or 2.
10. a kind of finger-print of Kangfuxin Liquid, which is characterized in that include 12 shared fingerprint peaks, be phenylalanine referring to peak
Chromatographic peak, the relative retention time of 12 shared fingerprint peaks are respectively as follows: No. 1 0.36 ± 0.01min of peak, and No. 2 peaks 0.43 ±
0.01min, No. 3 0.47 ± 0.01min of peak, No. 4 0.51 ± 0.01min of peak, No. 5 0.59 ± 0.01min of peak, No. 6 peaks 0.63 ±
0.01min, No. 7 0.86 ± 0.02min of peak, No. 8 1.25 ± 0.02min of peak, No. 9 1.49 ± 0.02min of peak, No. 10 peaks 1.71 ±
0.02min and No. 11 1.79 ± 0.02min of peak, wherein the ratio of peak area is peak number 1: peak number 2: peak number 6: peak number 7: peak number
8: peak number 9: peak 10=(0.93~1.04): (0.84~1.06): (0.61~0.65): (0.88~0.91): 1.00:
(1.02~1.06): (1.017~1.18)；
The Kangfuxin Liquid finger-print is to be measured by using high performance liquid chromatography to Kangfuxin Liquid: described
In high performance liquid chromatography, chromatographic column is Boston Luna Clone, and specification is 4.6mm × 250mm × 5 μm；Mobile phase is
With 0.1%H3PO4For mobile phase A, using methanol as Mobile phase B, using gradient elution program, flow velocity: 0.5ml/min detects wave
Long: 0~16min is 254nm, and 17~26min is 230nm, and 27~50min is 215nm, column temperature: 30 DEG C；The gradient elution journey
Sequence is shown in Table 8:
Table 8
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