CN110455944A - Method that is a kind of while detecting apo- Changchun amino acid and Changchun amino acid in vinpocetine - Google Patents
Method that is a kind of while detecting apo- Changchun amino acid and Changchun amino acid in vinpocetine Download PDFInfo
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Abstract
The present invention provides a kind of method for detecting apo- Changchun amino acid and Changchun amino acid in vinpocetine simultaneously, this method includes that (1) takes vinpocetine raw material or Vinpocetine injection to prepare test solution；(2) test solution is diluted 1000 times and is used as contrast solution；(3) by the test solution, contrast solution, chromatographic isolation is carried out with high performance liquid chromatography, and record chromatogram, chromatographic condition are as follows: Detection wavelength is 262nm~266nm；Column temperature is 25 DEG C~35 DEG C；Mobile phase A is phosphate buffer, and Mobile phase B is methanol；The flow velocity of elution is 0.8~1.5ml/min；(3) content of apo- Changchun amino acid and Changchun amino acid in test solution is calculated by the principal component Self-control method of the correction up factor.Changchun amino acid can be made to reach with apo- Changchun amino acid, vinpocetine peak preferably to separate, there is higher system suitability, detection limit is low, and precision is high.
Description
Technical field
The present invention relates to technical field of analytical chemistry, more particularly to one kind to detect apo- Changchun amino acid in vinpocetine simultaneously
With the method for Changchun amino acid.
Background technique
The impurity of drug refers to present in drug without therapeutic effect or influences the stability of drug, curative effect or even right
The harmful substance of health of human body.The research of drug, production, storage and in terms of, it is necessary to keep the pure of drug
Degree, reduces the impurity of drug, just can guarantee the validity and safety of drug in this way.
Vinpocetine is a kind of derivative of the alkaloid extracted from Apocynaceae periwinkle, and effective component is Ah
Plain Vincaminic acid ethyl ester.Vinpocetine has a variety of effects, can improve cerebral metabolism, blood flow and hemorheology properties.
Vinpocetine raw material impurity used in this product is more, is mainly derived from reaction raw materials introduced in synthesis process, synthetic intermediate, anti-
Answer by-product and degradation impurity.And Changchun amino acid is vinpocetine raw material synthetic intermediate, by first eliminate afterwards be esterified and will produce
Raw apo- Changchun amino acid.Apo- Changchun amino acid be also vinpocetine enter in vivo after active metabolite, pharmacological action with
Original shape medicine is similar, but activity is relatively low.Vinpocetine can rapidly be converted to apo- Changchun amino acid through liver metabolism, and pass through
Glomerular filtration is drained；It is harmless to liver kidney in vivo without accumulation.Apo- Changchun amino acid is without apparent toxic side effect, safety
Property is preferable.But it is safety, the validity for guaranteeing product clinical use, Changchun amino acid and apo- Changchun amino acid need to be subject to strictly
Control.Currently, Changchun amino acid and apo- pervone acid content control method are not embodied in the domestic Extra Pharmacopoeia Martindale such as CP, EP, USP.
Documents 1: document " C-MS/MS measures vinpocetine cylinder metabolism-ure apo- Changchun amino acid in human plasma " (in
State's pharmaceutical journal, 2010) it describes and is disclosed in the detection method using the content of C-MS/MS detection apo- Changchun amino acid
The mobile phase used is 2mmol/L ammonium acetate-methanol (8:92) and 2mmol/L ammonium acetate-methanol (13:87), but in the condition
Lower apo- Changchun amino acid chromatographic peak number of theoretical plate is very low, and peak shape symmetry is poor；Methanol ratio is reduced, retention time is slightly postponed,
But peak shape is without improvement.
Documents 2: " ultra performance liquid chromatography-mass spectrum/Mass Spectrometry measures the Changchun in rat plasma to document simultaneously
Xi Ting and metabolin apo- Changchun amino acid " (Chinese Pharmaceutical Journal, 2013) also describes and detects apo- Changchun using C-MS/MS
The content of amino acid.In the detection method, the mobile phase used is disclosed as water-methanol (13:87), is equally deposited under this condition
In the low problem with peak shape difference of theoretical cam curve.Consider that there is ionizable carboxyl in apo- pervone acid molecule, in mobile phase
It is middle be added 0.1%~0.3% phosphoric acid, while adjust methanol ratio be respectively 0.1% phosphoric acid solution-methanol (13:87),
0.1% phosphoric acid solution-methanol (28:72), 0.3% phosphoric acid solution-methanol (28:72), 0.3% phosphoric acid solution-methanol (90:
10), but amino acid peak number of theoretical plate in apo- Changchun is not improved, also by the interference of solvent peak under partial condition.It is above two
Method reported in the literature is not applicable while detecting apo- Changchun amino acid and pervone acid content in vinpocetine.Therefore, it is necessary to
A kind of method of simple and effective is established effectively to control the content of apo- Changchun amino acid and Changchun amino acid in vinpocetine, to ensure
The safety of drug.
Summary of the invention
It is an object of the invention to overcome the defect of the prior art, provides while detecting apo- pervone in vinpocetine
Acid and the method for Changchun amino acid, can be such that Changchun amino acid reaches with apo- Changchun amino acid peak and preferably separate, with vinpocetine and other
Also it can reach preferable separation between chromatographic peak, and peak type symmetry is higher, conducive to the detection in relation to substance, there is higher system
Adaptability, while unrivaled advantage is all shown in specificity, quantitative limit, detection limit, the range of linearity and repeatability,
Precision with higher.
The present invention is implemented as follows:
Apo- Changchun amino acid and Changchun amino acid in vinpocetine are detected simultaneously the object of the present invention is to provide a kind of
Method, described method includes following steps:
The preparation of S1, test solution: vinpocetine raw material or Vinpocetine injection is taken to prepare test solution；
The preparation of S2, contrast solution: the test solution is diluted 1000 times and is used as contrast solution；
S3, measurement: precision measures test solution, appropriate contrast solution, and sample introduction carries out color using high performance liquid chromatography
Spectrum separation, and record chromatogram；The test condition of the high performance liquid chromatography are as follows: chromatographic column used is bonded with octadecylsilane
Silica gel is filler, Detection wavelength 264nm；Column temperature is 25 DEG C~35 DEG C；Mobile phase A is phosphate buffer, and Mobile phase B is
Methanol；The flow velocity of elution is 0.8~1.5ml/min.
S4, calculating: by the correction up factor principal component Self-control method calculate test solution in apo- Changchun amino acid with
The content of Changchun amino acid.
Preferably, the concentration of vinpocetine is 2mg/mL in test solution in the S1；Specifically: taking vinpocetine former
Expect (or Vinpocetine injection) in right amount, adds mobile phase A-methanol (60:40) to dissolve and quantify dilution and be made in every 1ml containing about length
The solution of Chun Xiting 2mg, shakes up, as test solution.
Preferably, the concentration of vinpocetine is 2 μ g/mL in contrast solution in the S2.Specifically: precision measures appropriate institute
Test solution is stated, quantitatively dilution is made in every 1ml containing about the solution of 2 μ g with mobile phase A-methanol (60:40), molten as compareing
Liquid.Mobile phase A-methanol (60:40) in the present invention is volume ratio.
Preferably, phosphate buffer in the S3 are as follows: after sodium dihydrogen phosphate or sodium dihydrogen phosphate dihydrate are dissolved in water,
The phosphoric acid that volume ratio is 0.05% is added, mixes to obtain the final product.The present invention selects sodium dihydrogen phosphate to prepare the specific of phosphate buffer
Method are as follows: take sodium dihydrogen phosphate 1.40g, add water 1000ml to dissolve, add phosphoric acid 0.5ml, mix to obtain the final product.
Preferably, most preferably 30 DEG C of column temperature in the S3；Detection wavelength most preferably 264nm；The velocity optimization of elution is
1ml/min。
Preferably, the elution program that HPLC is detected in the S3 are as follows:
0-10min, mobile phase A: Mobile phase B volume ratio is 60:40, carries out isocratic elution, 10-20min, mobile phase A: stream
Dynamic phase B volume ratio is 55:45 by linear gradient, carries out linear gradient elution；20-40min, mobile phase A: Mobile phase B volume ratio
For 55:45, isocratic elution is carried out；40-50min, mobile phase A: Mobile phase B volume ratio is 60:40 by linear gradient, is carried out linear
Gradient elution；After 50min, mobile phase A: Mobile phase B volume ratio is that 60:40 is eluted to all appearance of the peak in relation to substance.
Preferably, according to the principal component Self-control method calculation formula of the correction up factor in the S4: related substance contains
Amount=F × (ATest sample/AControl× extension rate) × 100%, it calculates and obtains the content in relation to substance；Specifically:
Content=F of apo- Changchun amino acidApo- Changchun amino acid×(AFor/AIt is right× 1000) apo- Changchun amino acid × 100%, is calculated
Content, FApo- Changchun amino acidFor the relative correction factor of apo- Changchun amino acid, specially 0.69；
Content=F of Changchun amino acidChangchun amino acid×(AFor/AIt is right× 1000) content of Changchun amino acid, F × 100%, are calculatedChangchun amino acid
For the relative correction factor of Changchun amino acid, specially 1.6；
Wherein AForFor the peak area of test solution, AIt is rightFor the main peak peak area of contrast solution used in S2.
Relative correction factor (the FApo- Changchun amino acidAnd FChangchun amino acid) it is the oblique of principal component (vinpocetine) equation of linear regression
The ratio of the slope of rate and the equation of linear regression in relation to substance (apo- Changchun amino acid, Changchun amino acid).The relative correction because
Son is relative value, will not theoretically be become, but because of the presence of experimental error, has minor change, on final result without influence.Institute
The specific calculating process for stating relative correction factor is shown in embodiment 1.
The invention has the advantages that:
A kind of method detecting apo- Changchun amino acid and Changchun amino acid in vinpocetine simultaneously provided by the invention, can make to grow
Spring amino acid reaches with apo- Changchun amino acid peak preferably to be separated, and preferable point can be also reached between vinpocetine and other chromatographic peaks
From and peak type symmetry is higher, conducive to the detection in relation to substance, has a higher system suitability, while in specificity, quantitative
Unrivaled advantage, precision with higher are all shown in limit, detection limit, the range of linearity and repeatability.
Detailed description of the invention
Fig. 1 is the chromatogram for the system suitability solution that experimental example 1 provides；
Fig. 2 is the apo- Changchun amino acid reference substance solution UV scanning figure that experimental example 7 provides；
Fig. 3 is the chromatogram for the blank solution that experimental example 1 provides；
Fig. 4 is the chromatogram for the vinpocetine test solution that embodiment 1 provides；
Fig. 5 is the chromatogram for the vinpocetine contrast solution that embodiment 1 provides；
Fig. 6 is the chromatogram for the Vinpocetine injection test solution that embodiment 1 provides；
Fig. 7 is the chromatogram for the Vinpocetine injection contrast solution that embodiment 1 provides；
Fig. 8 is the blank auxiliary solution chromatogram for the Vinpocetine injection that experimental example 1 provides；
Fig. 9 is that the Vinpocetine injection that experimental example 2 provides does not destroy sample chromatogram figure；
Figure 10 is that the Vinpocetine injection illumination that experimental example 2 provides destroys sample chromatogram figure；
Figure 11 is the Vinpocetine injection high temperature sample chromatogram figure that experimental example 2 provides；
Figure 12 is the Vinpocetine injection Oxidative demage sample chromatogram figure that experimental example 2 provides；
Figure 13 is that the Vinpocetine injection alkali that experimental example 2 provides destroys sample chromatogram figure；
Figure 14 is that the Vinpocetine injection acid that experimental example 2 provides destroys sample chromatogram figure；
Figure 15 is that the detection limit that experimental example 6 provides and quantitative limit test 1 chromatogram of solution；
Figure 16 is that the detection limit that experimental example 6 provides and quantitative limit test 2 chromatogram of solution；
Figure 17 is that the detection limit that experimental example 6 provides and quantitative limit test 3 chromatogram of solution.
Specific embodiment
Embodiment 1
1, instrument and reagent
(1) LC-20AD type high performance liquid chromatograph (Shimadzu, Japan), including LC-20AD pump, SPD-M20A type
Diode array detector (190nm~800nm), SIL-20A type autosampler, Labsolution work station；E2695 type
High performance liquid chromatograph (Waters, US), including 2998 type diode array detector (190nm~800nm),
Empower3 work station；AUW 220D type double-range assay balance (sensibility reciprocal 0.1mg/0.01mg, Shimadzu, Japan)；
SHH-100GD-2 type drug strong illumination chamber (immortality laboratory apparatus factory, Chongqing City)；GXZ-9240 MBE type forced air drying
Case (Medical Equipment Plant, Shanghai Boxun Industrial Co., Ltd.)
(2) reagent: other than methanol is chromatographic grade, other reagents are that analysis is pure.
(3) reference substance: vinpocetine reference substance (CAS:42971-09-5), lot number: 100947-201203 is eaten by China
Product drug assay research institute provides.Apo- Changchun amino acid reference substance (CAS:27773-65-5), lot number: C4X-18434-1606,
It is provided by CATO company, the U.S..Changchun amino acid reference substance (CAS:59413-21-7), lot number: B9-12-6, by CATO company, the U.S.
It provides.Vinpocetine is provided by northeast Pharmacy stock Co., Ltd；Vinpocetine injection is applicant according to conventional existing skill
Art self-control.
2, test solution preparation
Reference substance solution: first prepare reference substance stock solution I, II, III: precision weighs vinpocetine reference substance 25.62mg, sets
In 100ml measuring bottle, adds mobile phase A-methanol (60:40) to dissolve and be diluted to scale, shake up, as reference substance stock solution I；Respectively
Changchun amino acid reference substance 6.42mg, apo- Changchun amino acid reference substance 6.38mg are weighed, sets in 100ml measuring bottle, adds mobile phase A-first
Alcohol (60:40) dissolves and is diluted to scale, shakes up, as reference substance stock solution II, III.The storage of vinpocetine reference substance is weighed respectively
Standby liquid I, Changchun amino acid reference substance stock solution II, apo- Changchun amino acid reference substance stock solution III, respectively plus mobile phase A-methanol is molten
It solves and dilutes, the solution containing vinpocetine, Changchun amino acid and each 2 μ g of apo- Changchun amino acid in every 1ml is made, respectively as Changchun
Xi Ting, Changchun amino acid, apo- Changchun amino acid reference substance solution.
Test solution: taking vinpocetine raw material (or Vinpocetine injection) in right amount, adds mobile phase A-methanol (60:40)
It dissolves and quantifies dilution and be made in every 1ml containing about the solution of vinpocetine 2mg, shake up, as test solution；
Contrast solution: precision measures in right amount, and with mobile phase A-methanol (60:40), quantitatively dilution is made in every 1ml containing about 2 μ g
Solution, as contrast solution.
3, accurate to measure test solution, each 10 μ l of contrast solution, it is injected separately into liquid chromatograph, using high-efficient liquid phase color
Spectrometry carries out chromatographic isolation, and records chromatogram；Chromatographic condition are as follows:
Chromatographic column: using octadecylsilane chemically bonded silica as filler, Detection wavelength: 264nm；Column temperature: 30 DEG C of mobile phases:
With phosphate buffer (take sodium dihydrogen phosphate 1.40g, water 1000ml added to make to dissolve, add phosphoric acid 0.5ml, shake up) for mobile phase A,
Methanol is Mobile phase B, carries out gradient elution, Gradient program such as table 1.Flow velocity: 1ml/min；Runing time: 60 minutes.Note: because examining
Considering has ionizable carboxyl in apo- pervone acid molecule, therefore 0.05% phosphoric acid is added in mobile phase A, with elimination solvent
The interference at peak improves the theoretical cam curve of apo- Changchun amino acid and Changchun amino acid peak.
1 gradient elution table of table
4, correction factor is measured by the principal component Self-control method of the correction up factor
According to the requirement of " Chinese Pharmacopoeia " guideline, the range of linearity of this method is investigated, and calculates pervone
The correction factor of acid and apo- Changchun amino acid.6 concentration points are taken in the range of 0~150% limit concentration of quantitative limit concentration, point
Not Xuan Yong two high performance liquid chromatographs of LC-20AD type and Waters e2695 type simultaneously determine each component the range of linearity (want
Ask r >=0.999), the correction factor of Changchun amino acid and apo- Changchun amino acid is calculated using the slope (k) of linear equation, formula is such as
Under:
Changchun propylhomoserin correction factor=kVinpocetine/kChangchun propylhomoserin；Apo- Changchun propylhomoserin correction factor=kVinpocetine/kApo- Changchun propylhomoserin；
Using the average value of the correction factor of two Instrument measurings as this product Changchun amino acid with the correction of apo- Changchun amino acid
The factor.
(1) range of linearity
The lower reference substance stock solution I~III of accurate measurement embodiment 1 is configured to mixing reference substance stock solution in right amount respectively, uses
Linear series test solution is made in flowing phase dilution.Standard solution is prepared as follows: gradient 1: stock solution I
II 0.4ml+ stock solution of 0.2ml+ stock solution, III 0.4ml；Gradient 2: I 0.5ml+ stock solution of stock solution, II 1ml+ stock solution, III 1ml；
Gradient 3: I 1ml+ stock solution of stock solution, II 2ml+ stock solution, III 2ml；Gradient 4: I 2ml+ stock solution of stock solution, II 4ml+ stock solution
Ⅲ4ml；Gradient 5: I 2.5ml+ stock solution of stock solution, II 5ml+ stock solution, III 5ml；Gradient 6: I 3ml+ stock solution of stock solution, II 6ml
III 6ml of+stock solution；
6 gradients are respectively placed in 100ml measuring bottle, adds mobile phase to be diluted to scale, shakes up, be configured to contain respectively
0.5094 μ g/ml of vinpocetine, 1.2735 μ g/ml, 2.5470 μ g/ml, 5.0940 μ g/ml, 6.3675 μ g/ml, 7.6410 μ g/
Ml, 0.2547 μ g/ml of Changchun amino acid, 0.6369 μ g/ml, 1.2737 μ g/ml, 2.5474 μ g/ml, 3.1843 μ g/ml, 3.8212
μ g/ml, 0.2536 μ g/ml of apo- Changchun amino acid, 0.6340 μ g/ml, 1.2680 μ g/ml, 2.5360 μ g/ml, 3.1700 μ g/
The linear series test solution of ml, 3.8040 μ g/ml.
Precision measures 10 μ l of linear series test solution, is injected separately into two liquid phases of LC-20AD and Waters e2695
Chromatograph records chromatogram.With reference substance solution concentration (C, μ g/ml) for abscissa, peak area (A) is ordinate, is calculated back
Return equation, concrete outcome see the table below 2:
2 equation of linear regression of table and correction factor calculated result
As shown in Table 2:
1. Changchun amino acid linear relationship: within the scope of 0.2547 μ of μ g/ml~3.8212 g/ml, the concentration (C) of Changchun amino acid
Good with peak area (A) linear relationship, correlation coefficient r is respectively 0.9995 and 0.9999.
2. apo- Changchun amino acid linear relationship: within the scope of 0.2536 μ of μ g/ml~3.8040 g/ml, apo- Changchun amino acid
Concentration (C) and peak area (A) linear relationship it is good, correlation coefficient r is respectively 0.9993 and 0.9999.
3. vinpocetine linear relationship: within the scope of 0.5094 μ of μ g/ml~7.6410 g/ml, the concentration (C) of vinpocetine
Good with peak area (A) linear relationship, correlation coefficient r is respectively 0.9994 and 0.9996.
(2) correction factor
According to the regression equation that above-mentioned two different high performance liquid chromatographs measure, calculate vinpocetine and Changchun amino acid,
The ratio of apo- pervone acid slope of standard curve, acquires the correction factor and average value of Changchun amino acid and apo- Changchun amino acid,
I.e. the correction factor of apo- Changchun amino acid is 0.69, and the correction factor of Changchun amino acid is 1.6.
5, it calculates: calculating the content of apo- Changchun amino acid and Changchun amino acid in test solution.
(1) Vinpocetine injection sample is taken, test solution and contrast solution is made, experimental group uses the correction up factor
Principal component Self-control method, control group uses external standard method, checks respectively for containing for Changchun amino acid and apo- Changchun amino acid in sample
Amount.Actually detected the results are shown in Table 3, and principal component Self-control method and the external standard method result of the correction up factor are not much different, preceding
Person Changchun amino acid detected level higher 0.004%~0.007%, apo- Changchun amino acid detected level higher 0~0.001%, more favorably
The control of Changchun amino acid and apo- Changchun amino acid impurity in strict control Vinpocetine injection.Accordingly, it is considered to method can
By the accessibility of property, inspection cost and reference substance, determine that Vinpocetine injection Changchun amino acid is adopted with the amino acid inspection of apo- Changchun
It is calculated with the principal component Self-control method of the correction up factor.As shown in Table 3, long in the specification 9 batches of Vinpocetine injection 3 sample
Spring amino acid content is 0.035%~0.042%, apo- pervone acid content is 0.007%~0.010%.
(2) take Vinpocetione crude drug test solution and its contrast solution, Vinpocetine injection test sample molten respectively
Liquid and its each 10 μ l of contrast solution inject liquid chromatograph, record chromatogram.
Fig. 4 is Vinpocetione crude drug test solution chromatogram；Fig. 5 is Vinpocetione crude drug contrast solution chromatography
Figure；Fig. 6 is Vinpocetine injection test solution chromatogram；Fig. 7 is Vinpocetine injection contrast solution chromatogram.Fig. 4-
In 7, each ingredient peak sequence is followed successively by Changchun amino acid, apo- Changchun amino acid, vinpocetine, and retention time is at 7 minutes or more.
3 Vinpocetine injection sample measurement result of table
1 system suitability of experimental example and interference test
1, it measures 10 μ l of system suitability solution and injects liquid chromatograph, record chromatogram, the system suitability solution:
It takes vinpocetine, Changchun amino acid and apo- Changchun amino acid reference substance each appropriate, adds mobile phase A-methanol (60:40) to dissolve and dilute
It is made in every 1ml containing about the mixed solution of 1 μ g of vinpocetine, Changchun amino acid and each 0.5 μ g of apo- Changchun amino acid, it is suitable as system
With property solution.
4 system suitability result of table
| Chemical combination name | Concentration | Retention time (min) | Separating degree |
| Vinpocetine | 1μg/ml | 39.146 | 43.9 |
| Changchun amino acid | 0.5μg/ml | 7.179 | / |
| Apo- Changchun amino acid | 0.5μg/ml | 8.690 | 4.2 |
As a result as shown in Figure 1, peak sequence is followed successively by Changchun amino acid, apo- Changchun amino acid, vinpocetine.Vinpocetine master
Peak retention time is 39.146 minutes, and Changchun amino acid and apo- Changchun amino acid peak retention time are respectively 7.179 minutes and 8.690
Minute, Changchun amino acid number of theoretical plate 7279 is 4.2 with apo- Changchun amino acid separating degree, and each chromatographic peak theoretical cam curve is not low
In 7000, for separating degree 4 or more, separating degree is good.
2, blank solution, 10 μ l of blank auxiliary solution injection liquid chromatograph are taken respectively, are recorded chromatogram, are investigated this method
To the selectivity of Changchun amino acid and apo- Changchun amino acid.
The blank solution: the blank solution is mobile phase A: Mobile phase B volume ratio is the mixed solution of 60:40；Institute
It states blank auxiliary solution: being reference with Vinpocetine injection (specification 2ml:20mg) prescription, weigh L-TARTARIC ACID respectively
600mg, vitamin C 20mg, sorb ester 600mg, sodium pyrosulfite 100mg add a small amount of water for injection (or molten with the blank
Liquid dilution) dissolution, it is settled to 100ml, as Vinpocetine injection blank auxiliary solution.Fig. 3 is blank solution chromatogram, figure
8 be blank auxiliary solution chromatogram, the results showed that blank auxiliary solution is to the measurement of Changchun amino acid and apo- Changchun amino acid without dry
It disturbs.
The test of 2 forced degradation of experimental example
In order to further investigate in sample impurity or catabolite that may be present to Changchun amino acid and apo- Changchun amino acid
The influence of measurement, this method have carried out the research of forced degradation test.Pass through the side such as strong illumination, high temperature, acid and alkali hydrolysis, oxidation
Method, acceleration destroy vinpocetine and Vinpocetine injection, investigate catabolite and unknown impuritie and the Changchun of sample
The separation situation of amino acid and apo- Changchun amino acid, the efficiency and applicability of evaluation method.
1, vinpocetine raw material forced degradation is tested
Under the conditions of illumination and high temperature, vinpocetine destroys in sample not without degradation, suspension and powder substantially
Changchun amino acid and apo- Changchun amino acid are detected, is had good stability.Under oxidation, alkali, sour failure condition, vinpocetine is degraded
20% or so, apo- Changchun amino acid is produced, wherein acid, which destroys, also creates Changchun amino acid, other unknown impurities are also different
The increase of degree.But the separating degree of Changchun amino acid, apo- Changchun amino acid and other impurities is not less than 3.8, it can be completely separable,
Therefore in this method, unknown impuritie and potential catabolite do not influence Changchun amino acid and apo- Changchun in Vinpocetione crude drug
The inspection of amino acid.
Table 5
2, Vinpocetine injection forced degradation is tested
Fig. 9-Figure 14, which is respectively Vinpocetine injection, not to be destroyed and illumination, high temperature, oxidation, alkali, sour failure condition
Chromatogram；As a result as a result basic with bulk pharmaceutical chemicals it is found that Vinpocetine injection is under illumination, oxidation, alkali, sour failure condition
Unanimously, but in high temperature test, the extent of the destruction of preparation is much higher than raw material under equality strength.Under illumination condition, in preparation
The stability of vinpocetine is still good, and substantially without degradation, the variation of apo- pervone acid content is little.High temperature, oxidation, alkali, acid
Under the conditions of, vinpocetine degrades 4%~18% in injection, and Changchun amino acid is dramatically increased with apo- pervone acid content,
Increase both under the conditions of high temperature most；It is still that alkali destroys vinpocetine hydrolysis degree maximum, apo- pervone in sample
Acid content far surpasses other samples.But the separating degree of Changchun amino acid, apo- Changchun amino acid and other impurities is all larger than 1.5, can be complete
It is complete to separate, therefore in this method, in Vinpocetine injection unknown impuritie and potential catabolite do not influence Changchun amino acid with
The inspection of apo- Changchun amino acid.
Table 6
In conclusion vinpocetine raw material and injection carry out under illumination, high temperature, Strong oxdiative, highly basic and strong acid condition
Forced degradation test, under degree of the main peak degradation no more than 25%, each known impurity level increases in various degree, new degradation
Product assay is no more than 3%, and does not interfere to Changchun amino acid and the inspection of apo- Changchun amino acid, the validity of this method with
Applicability is met the requirements, and specificity is preferable.
3 precision test of experimental example
1, repeated
It takes Vinpocetine injection appropriate, prepares 6 parts of test solutions and contrast solution in parallel by the lower method of embodiment 1,
It measures in accordance with the law, respectively by external standard method, the principal component Self-control method of the correction up factor and the principal component itself that correction factor is not added
The percentage composition of counter point calculating Changchun amino acid and apo- Changchun amino acid.
Table 7
As shown in Table 7, external standard method, the principal component Self-control method of the correction up factor and be not added the principal component of correction factor from
Pervone acid content is respectively 0.045%, 0.048% and in the Vinpocetine injection that three kinds of calculation methods of body counter point measure
0.029%, RSD in 2.36%~3.08% range (n=6)；Apo- pervone acid content is respectively 0.008%, 0.007%
With 0.010%, RSD in 6.74%~11.02% range (n=6).RSD is respectively less than 15%, meets the requirements, and illustrates this method
It is repeated good.Compared with external standard method calculated result, the principal component Self-control method of the correction up factor calculates pervone acid content
Higher 0.003%, apo- pervone acid content relatively low 0.001%, the two result is not much different.Without the correction up factor it is main at
Self-control method is divided to calculate pervone acid content relatively low 0.016%, apo- pervone acid content higher 0.002%, error is up to
36% and 25%, the quality for not being suitable for this product Changchun amino acid and apo- Changchun amino acid controls.
2, Intermediate precision
Waters e2695 type high performance liquid chromatograph is optionally used, in different time by 2 different analysis personnel by " weight
Method separately prepares 6 parts of Vinpocetine injection test solutions and contrast solution under renaturation " item, measures peak by above-mentioned chromatographic condition
Area.Respectively by external standard method, the principal component Self-control method of the correction up factor and the principal component own control that correction factor is not added
The percentage composition of method calculating Changchun amino acid and apo- Changchun amino acid.
Changchun amino acid that analysis personnel II measure and apo- pervone acid content by three kinds of methods calculate RSD no more than
15%, the difference of different analysis personnel measurements is no more than 0.004%, and 12 parts of data RSD are no more than 20%, meets Intermediate precision
It is required that illustrating that this method Intermediate precision is good.
4 accuracy test of experimental example
1, the accuracy test of Vinpocetione crude drug
Precision weighs Vinpocetione crude drug about 40mg, sets in 20ml measuring bottle, accurate respectively that poly-doped impurity reference substance is added
Solution (6.1135 μ g/ml of Changchun amino acid, 6.3400 μ g/ml of apo- Changchun amino acid) 1ml, 3ml, 5ml, again with methanol are settled to quarter
Degree, shakes up, and is configured to Changchun amino acid and apo- pervone acid concentration is approximately equivalent to Vinpocetione crude drug test solution concentration
The mixed solution of (2mg/ml) 0.015%, 0.046% and 0.076%, as sample recovery rate test solution, each concentration
It is each to prepare 3 parts.Precision measures 10 μ l of series of concentrations test solution, is injected separately into liquid chromatograph, records chromatogram, and measurement is each
The peak area of impurity calculates measured amount by external standard method to get the rate of recovery.
The rate of recovery of Changchun amino acid and apo- Changchun amino acid in table 8- Vinpocetione crude drug
Pervone acid recovering rate is between 81.58%~90.32% in Vinpocetione crude drug, apo- pervone acid recovery
Rate is between 110.27%~116.30%, in 80.0%~120.0% claimed range；RSD is respectively 0.68%~
2.08%, 0.22%~1.58%, it no more than 10%, meets the requirements, it was demonstrated that this method is for long in Vinpocetione crude drug
Spring amino acid and the acid quantitative check of apo- pervone have good accuracy.
2, Vinpocetine injection accuracy test
Vinpocetine injection (specification 2ml:20mg) 2ml for taking known content, sets in 10ml measuring bottle, accurate respectively to be added
Poly-doped impurity reference substance solution (6.1135 μ g/ml of Changchun amino acid, 6.3400 μ g/ml of apo- Changchun amino acid) 0.5ml, 3ml, 5ml,
Again with methanol is settled to scale, shakes up, and is configured to Changchun amino acid and apo- pervone acid concentration is approximately equivalent to vinpocetine injection
The mixed solution of liquid test solution concentration (2mg/ml) 0.015%, 0.092% and 0.15%, as sample recovery rate for examination
Product solution, each concentration respectively prepare 3 parts.Precision measures 10 μ l of series of concentrations test solution, is injected separately into liquid chromatograph, remembers
Chromatogram is recorded, the peak area of each impurity is measured, calculates measured amount by external standard method to get the rate of recovery.
The rate of recovery of Changchun amino acid and apo- Changchun amino acid in table 9- vinpocetine REFRACTIVE LIQUID
As shown in Table 9, for pervone acid recovering rate between 85.15%~96.04%, apo- is long in Vinpocetine injection
The spring amino acid rate of recovery is between 112.05%~119.10%, in 80.0%~120.0% claimed range；RSD difference
It is 1.01%~4.68%, 0.20%~1.95%, no more than 10%, meets the requirements, it was demonstrated that this method is used for vinpocetine
Changchun amino acid and the acid quantitative check of apo- pervone in injection, have good accuracy.
5 stability of solution of experimental example
Changchun amino acid, the stability of apo- Changchun amino acid and vinpocetine in the solution are investigated, with determination measurement appropriate
Time.Since Changchun amino acid and apo- Changchun amino acid being not detected in Vinpocetione crude drug, vinpocetine raw material is not investigated
The stability of medicine test solution.Reference substance solution is (by vinpocetine, Changchun amino acid, apo- Changchun amino acid point in Example 1
Not plus methanol dissolution is configured to each reference substance solution), Vinpocetine injection test solution and its reference substance solution be in room temperature
It is placed under (20~25 DEG C), it is accurate respectively at 0,3,6,9 hour to measure 10 μ l, it measures in accordance with the law, records chromatogram, more each chromatography
Peak area.
Reference substance solution is placed at room temperature for 9 hours, and Changchun amino acid, apo- Changchun amino acid and vinpocetine peak area do not decline
Trend, RSD are respectively 1.96%, 3.21%, 0.95%, no more than 8%, are had good stability.In test solution, Changchun
Amino acid and apo- Changchun amino acid are being placed at room temperature for 9 hours peak areas also without downward trend, RSD is respectively 3.88%,
5.56%, show that Changchun amino acid and apo- pervone absolute acid stability are good in Vinpocetine injection.Its reference substance solution places 9
Vinpocetine peak area RSD is 0.76% after hour, and stability is good, can be used for principal component Self-control method and calculates test solution
Middle Changchun amino acid and apo- pervone acid content.To sum up the result shows that: Changchun amino acid and apo- in reference substance and test solution
The concentration of Changchun amino acid is no more than 0.5 μ g/ml, by " Chinese Pharmacopoeia " 2015 editions guideline regulations, the essence of measurement result
Density (RSD) tolerance interval is 8%.
The detection of experimental example 6 limit and quantitative limit
One, detection limit
Reference substance stock solution I~III under Example 1 is appropriate, is diluted to mobile phase A-methanol (60:40) different dense
The quantitative limit of degree tests solution 1-3, and 10 μ l is taken to inject liquid chromatograph, records chromatogram, is determined according to signal-to-noise ratio (S/N) each
The detection limit and quantitative limit of impurity.Sample concentration when S/N is about 3 is that the detection of the substance limits, and sample when S/N is about 10 is dense
Degree is the quantitative limit of the substance.
10 quantitative limit of table and detection limit result
When signal-to-noise ratio is about 10, Changchun amino acid, apo- Changchun amino acid, vinpocetine concentration be respectively 0.2547 μ g/
ml,0.1268μg/ml,0.5094μg/ml；When signal-to-noise ratio is about 3, concentration be respectively 0.0764 μ g/ml, 0.0380 μ g/ml,
0.1528μg/ml.It is thus determined that each be quantitatively limited to pervone acid: 2.55ng；Apo- Changchun amino acid: 1.27ng；Vinpocetine:
5.09ng；Lowest detection is limited to pervone acid: 0.76ng；Apo- Changchun amino acid: 0.38ng；Vinpocetine: 1.53ng.Pervone
The quantitative limit of acid and apo- Changchun amino acid is respectively equivalent to 0.013% of vinpocetine sample volume in test solution (2mg/ml)
With 0.006%, i.e., this method can accurately detect that content is 0.013% Changchun amino acid and 0.006% apo- pervone
Acid, sensitivity are met the requirements.It is specifically shown in attached drawing 15-17.
Two, quantitative limit precision
1, the 10 μ l of Changchun amino acid and apo- Changchun amino acid reference substance solution for taking quantitative limit concentration, the weight under this chromatographic condition
Multiple sample introduction 6 times records chromatogram, investigates quantitative limit precision according to each chromatographic peak area, and to the number of theoretical plate of chromatographic system,
The parameters such as separating degree, sensitivity, tailing factor and repeatability are verified.
11 quantitative limit precision of table and system suitability repeatability result
2, result is as shown in table 11, and the RSD of Changchun amino acid peak area is 5.6%, and the RSD of apo- Changchun amino acid peak area is
7.7%, the RSD of 6 needle peak area of quantitative limit solution continuous sample introduction is no more than 10%, and the Changchun amino acid and apo- that this method determines are long
Spring amino acid quantitative limit is all satisfied requirement.In the continuous 6 needle chromatogram of the reference substance solution of quantitative limit concentration, Changchun amino acid, apo- are long
The number of theoretical plate of spring amino acid and vinpocetine is 7000 or more, and separating degree is not less than 4.5, and signal-to-noise ratio is respectively 11.0,10.0
With 10.6, it is able to satisfy the requirement of this method system suitability, is 0.013% Changchun amino acid and 0.006% apo- to content
Changchun amino acid energy accurate quantitative analysis.The RSD of Changchun amino acid and apo- Changchun amino acid retention time is respectively 0.09% and 0.76%, small
In 1.0%；Tailing factor is respectively 1.20 and 1.25, less than 2.0；The RSD of Changchun amino acid and apo- Changchun amino acid peak area points
Not Wei 5.6% and 7.7%, less than 2.0%；Each RSD value is all satisfied requirement.Show that the precision of instrument and sample introduction is good, chromatography
The parameters such as number of theoretical plate, separating degree, sensitivity, tailing factor and the repeatability of system close requirement.
7 durability of experimental example
Since Changchun amino acid and apo- Changchun amino acid being not detected in Vinpocetione crude drug, only under Example 1
Vinpocetine injection test solution and its reference substance solution carry out the durable Journal of Sex Research of this method.
1, the selection (attached drawing 2) of Detection wavelength
(1) take vinpocetine reference substance, apo- pervone reference substance and vinpocetine impurity A, B, C, D reference substance appropriate,
Accurately weighed, respectively plus mobile phase A-methanol (60:40) dilution is made in every 1ml containing about the solution of 25 μ g, and 10 μ l is taken to inject liquid
Chromatography records the UV-Vis abosrption spectrogram at 200nm to 800nm wavelength, and apo- Changchun amino acid is at 264nm, 311nm
There is absorption maximum.In conjunction with vinpocetine content and the Detection wavelength in relation to substance selects 264nm as the inspection of apo- Changchun amino acid
Survey wavelength.It is specifically shown in attached drawing 2.
(2) comparison of different Detection wavelengths
Investigate influence of the change of Detection wavelength to Changchun amino acid and apo- Changchun amino acid inspection result.Use different inspections
It surveys wavelength (± 2nm), other chromatographic conditions are constant, and precision measures system suitability solution, test solution and control solution
Each 10 μ l is injected separately into liquid chromatograph, records chromatogram, investigates the durability of method.Respectively by external standard method and correction up because
The principal component Self-control method of son calculates Changchun amino acid and apo- pervone acid content.
Under three Detection wavelengths (262nm, 264nm, 266nm), the reason of vinpocetine chromatographic peak in system suitability solution
It is not less than 3000 by plate number, Changchun amino acid and separating degree, sensitivity, the tailing factor of apo- Changchun amino acid chromatographic peak etc. meet
The requirement of this method system suitability.Changchun amino acid and apo- Changchun amino acid chromatographic peak and other compositions peak energy base in test solution
Line separation, retention time, peak area do not change substantially, meet durability requirements.
Pervone acid content is 0.045%~0.052% under different Detection wavelengths, and apo- pervone acid content is
0.008%~0.009%, the absolute value of changes of contents caused by Detection wavelength is no more than 0.004%, different calculation methods gap
No more than 0.007%, RSD no more than 8%, therefore Detection wavelength is that 262nm~266nm influences less this method, each to examine
Changchun amino acid is measured under survey wavelength and apo- pervone acid is almost the same.And Detection wavelength be 264nm when, effect is best.
3, the comparison of different column temperatures
Investigate influence of the change of column temperature to apo- Changchun amino acid inspection result.Using different column temperatures (± 5 DEG C), other
Chromatographic condition is constant, and precision measures system suitability solution, each 10 μ l of test solution and control solution, is injected separately into liquid phase
Chromatograph records chromatogram, investigates the durability of method.The principal component Self-control method of external standard method and the correction up factor is pressed respectively
Calculate Changchun amino acid and apo- pervone acid content.
Under different column temperatures (25 DEG C, 30 DEG C, 35 DEG C), the number of theoretical plate of vinpocetine chromatographic peak is equal in system suitability solution
Not less than 3000, separating degree, sensitivity, the tailing factor etc. of Changchun amino acid and apo- Changchun amino acid chromatographic peak meet the our genealogy of law
Applicability of uniting requirement.Changchun amino acid and apo- Changchun amino acid chromatographic peak and other compositions peak energy baseline separation in test solution,
Retention time change is larger, but peak area does not change substantially, and pervone acid content is 0.038%~0.051%, apo- Changchun
Amino acid content is 0.006%~0.009%, and the absolute value of changes of contents caused by column temperature is no more than 0.008%, different calculating sides
Method gap is no more than 0.010%, RSD no more than 20%, and durability is preferable.Therefore column temperature is 25 DEG C~35 DEG C to this method shadow
It rings less, Changchun amino acid is measured under each column temperature and apo- pervone acid is almost the same；And column temperature be 30 DEG C when effect it is best.
4, the comparison of different chromatographic columns
Investigate influence of the different chromatographic columns to apo- Changchun amino acid inspection result.Using different chromatographic column (chromatographic column 1:
Shimadzu Inertsil ODS-4 (5 μm, 4.6 × 250mm), chromatographic column 2: Shimadzu Shim-pack VP-ODS (5 μm, 4.6 ×
250mm), chromatographic column 3: moon rising sun Ultimate XB-C18 (5 μm, 4.6 × 250mm)), precision measures system suitability solution, supplies
Test sample solution and each 10 μ l of reference substance solution are injected separately into liquid chromatograph, record chromatogram, investigate the durability of method.Point
Not An the principal component Self-control method of external standard method and the correction up factor calculate Changchun amino acid and apo- pervone acid content.
Under different chromatographic columns, the number of theoretical plate of vinpocetine chromatographic peak is not less than 3000 in system suitability solution, length
Separating degree, sensitivity, the tailing factor etc. of spring amino acid and apo- Changchun amino acid chromatographic peak meet this method system suitability requirement.
Changchun amino acid and apo- Changchun amino acid chromatographic peak and other compositions peak energy baseline separation in test solution, retention time change compared with
Greatly, but peak area variation is little, and pervone acid content is 0.039%~0.051%, and apo- pervone acid content is 0.008%
~0.010%, the absolute value of changes of contents caused by chromatographic column changes is no more than 0.012%, and different calculation methods gap does not surpass
0.005%, RSD is crossed no more than 15%, good tolerance.Therefore changing chromatographic column influences less this method, each chromatographic column
It measures Changchun amino acid and apo- pervone acid is almost the same.
Comprehensive durability result can determine that Detection wavelength is 262nm~266nm, column temperature is 25 DEG C~35 DEG C and chromatographic column
Etc. the minor changes of conditions the measurement result of Changchun amino acid and apo- Changchun amino acid is influenced less, the durability of method is preferable.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (10)
1. a kind of method for detecting apo- Changchun amino acid and Changchun amino acid in vinpocetine simultaneously, which is characterized in that the method
Include the following steps:
The preparation of S1, test solution: vinpocetine raw material or Vinpocetine injection is taken to prepare test solution；
The preparation of S2, contrast solution: the test solution is diluted 1000 times and is used as contrast solution；
S3, measurement: precision measures the test solution, appropriate contrast solution, and sample introduction carries out color using high performance liquid chromatography
Spectrum separation, and record chromatogram；The test condition of the high performance liquid chromatography are as follows: chromatographic column used is bonded with octadecylsilane
Silica gel is filler, and Detection wavelength is 262nm~266nm；Column temperature is 25 DEG C~35 DEG C；Mobile phase A is phosphate buffer, stream
Dynamic phase B is methanol；The flow velocity of elution is 0.8~1.5ml/min；
S4, calculating: apo- Changchun amino acid and Changchun in test solution are calculated by the principal component Self-control method of the correction up factor
The content of amino acid.
2. the method as described in claim 1, which is characterized in that test solution is to be matched using mobile phase A-methanol in the S1
The test solution that concentration is 2mg/mL is made.
3. the method as described in claim 1, which is characterized in that contrast solution is by test solution using flowing in the S2
Phase A- methanol dilution is configured to the contrast solution for the vinpocetine that concentration is 2 μ g/mL.
4. the method as described in claim 1, which is characterized in that phosphate buffer in the S3 are as follows: sodium dihydrogen phosphate or two
After hypophosphite monohydrate sodium dihydrogen is dissolved in water, the phosphoric acid that volume ratio is 0.05% is added, mixes to obtain the final product.
5. method as claimed in claim 4, which is characterized in that take sodium dihydrogen phosphate 1.40g, add water 1000ml to dissolve, add phosphorus
Sour 0.5ml, mixes to obtain the final product.
6. the method as described in claim 1, which is characterized in that column temperature is 30 DEG C in the S3；Detection wavelength is 264nm；It washes
De- flow velocity is 1ml/min.
7. the method as described in claim 1, which is characterized in that the elution program that HPLC is detected in the S3 are as follows:
0-10min, mobile phase A: Mobile phase B volume ratio is 60:40, carries out isocratic elution, 10-20min, mobile phase A: mobile phase
B volume ratio is 55:45 by linear gradient, carries out linear gradient elution；20-40min, mobile phase A: Mobile phase B volume ratio is 55:
45, carry out isocratic elution；40-50min, mobile phase A: Mobile phase B volume ratio is 60:40 by linear gradient, carries out linear gradient
Elution；After 50min, mobile phase A: Mobile phase B volume ratio is that 60:40 is eluted to all appearance of the peak in relation to substance.
8. the method as described in claim 1, which is characterized in that according to the principal component own control of the correction up factor in the S4
Method calculation formula:
Content=F of apo- Changchun amino acidApo- Changchun amino acid×(AFor/AIt is right× 1000) content of apo- Changchun amino acid × 100%, is calculated,
FApo- Changchun amino acidFor the relative correction factor of apo- Changchun amino acid；
Content=F of Changchun amino acidChangchun amino acid×(AFor/AIt is right× 1000) content of Changchun amino acid, F × 100%, are calculatedChangchun amino acidFor length
The relative correction factor of spring amino acid；Wherein AForFor the peak area of test solution in S1, AIt is rightFor the master of contrast solution used in S2
Peak peak area.
9. the method as described in claim 1, which is characterized in that the relative correction factor FApo- Changchun amino acidFor principal component Chang Chunxi
The ratio of the slope of the equation of linear regression of the slope and apo- Changchun amino acid of spit of fland equation of linear regression；The relative correction factor
FChangchun amino acidFor the ratio of the slope of the equation of linear regression of the slope and Changchun amino acid of principal component vinpocetine equation of linear regression.
10. method as claimed in claim 9, which is characterized in that calculate reference substance solution used in correction factor in the S4
Preparation process are as follows: weigh vinpocetine reference substance, Changchun amino acid reference substance, apo- Changchun amino acid reference substance respectively, respectively plus
Mobile phase A-methanol is dissolved and is diluted, and is made in every 1ml containing the molten of vinpocetine, Changchun amino acid and each 2 μ g of apo- Changchun amino acid
Liquid, respectively as vinpocetine, Changchun amino acid, apo- Changchun amino acid reference substance solution.
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