CN110082448A - A kind of detection method of D-VB5 calcium impurities - Google Patents
A kind of detection method of D-VB5 calcium impurities Download PDFInfo
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- CN110082448A CN110082448A CN201910394767.XA CN201910394767A CN110082448A CN 110082448 A CN110082448 A CN 110082448A CN 201910394767 A CN201910394767 A CN 201910394767A CN 110082448 A CN110082448 A CN 110082448A
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The invention belongs to chromatographic technology fields, and in particular to a kind of detection method of D-VB5 calcium impurities, impurity include Beta-alanine, D-VB5 lactone and pantoic acid sodium；Detection method is high performance liquid chromatography, the mixed solution that mobile phase is phosphate buffer and acetonitrile volume ratio is 8.5~9.5: 1.Above-mentioned impurity is the critical impurities for influencing D-VB5 calcium finished product stability, high performance liquid chromatography selected by this method can make to realize separation well between D-VB5 calcium chromatographic peak and each impurity peaks, and Accurate Determining goes out the content of above-mentioned each impurity in sample to be tested simultaneously, detection sensitivity is high, detection limit is low, convenient for detecting in the production process of D-VB5 calcium to above-mentioned impurity.
Description
Technical field
The invention belongs to chromatographic technology fields, and in particular to a kind of detection method of D-VB5 calcium impurities.
Background technique
D-VB5 calcium is mainly used for pantothenic acid calcium deficiency in clinic and (is mainly shown as malabsorption syndrome, tropical stomatitis
Property diarrhea, coeliac disease, regional enteritis etc.) prevention and treatment or when application calcium pantothenate agonist drug.It can also be used in vitamin B
Deficiency disease, peripheral neuritis, postoperative intestine colic pain and other diseases (such as streptomycin poisoning, postoperative ileus, rheumatoid)
Adjuvant treatment.
Currently, need to be sampled inspection to in-between product, finished product in the actual production of D-VB5 calcium, it is miscellaneous to understand
Matter situation.Thin-layered chromatography is mostly used to detect greatly the detection method of impurity in D-VB5 calcium in the prior art, it is sensitive
Low, detection time length (generally at least needing 3~4 hours) is spent, and specific a certain impurity can not be carried out qualitative and quantitative
Analysis；High performance liquid chromatography for the detection of D-VB5 calcium carrys out the total content of checked for impurities mainly with external standard method, can not learn
The content of specific a certain kind impurity, and the rate of recovery is low, accuracy needs to be further increased.And accuracy, the essence of defects inspecting
The problems such as density, detection limit for judging that intermediate products or final product quality are of great significance, especially Beta-alanine, D-VB5
Lactone and pantoic acid sodium etc. produce the intermediate of D-VB5 calcium, and as finished product impurity, its content should be in D-VB5 calcium production process
Strict control, but there is no the detection methods to above each impurity in the prior art.
Summary of the invention
For in the prior art can not specific impurity content the technical issues of, the present invention provides impurity in a kind of D-VB5 calcium
Detection method.
To achieve the above object of the invention, the embodiment of the present invention uses the following technical solution:
The detection method of impurity in a kind of D-VB5 calcium, the impurity includes Beta-alanine, D-VB5 lactone and pantoic acid
Sodium, the detection method are high performance liquid chromatography, and mobile phase is the mixed solution of phosphate buffer and acetonitrile；The phosphoric acid
The volume ratio of salt buffer and the acetonitrile is 8.5~9.5: 1；The pH of the phosphate buffer is 2~4.
Impurity provided by this method is the intermediate for synthesizing D-VB5 calcium, is to influence finished product in D-VB5 calcium finished product to stablize
Property critical impurities, it is carried out accurately detect can aid in control finished product in impurity content, to improve final product quality.
Mobile phase in high performance liquid chromatography selected by this method can make real between D-VB5 calcium chromatographic peak and each impurity peaks
Now separation well can go out the content of plurality of impurities in sample to be tested by Accurate Determining simultaneously, and detection sensitivity is high, detection limit
It is low, convenient for carrying out plurality of impurities detection to intermediate products and finished product in the production process of D-VB5 calcium.And the present invention is used
High performance liquid chromatography detection time it is short, the working efficiency of detection can be significantly improved.
Preferably, the phosphate buffer is the potassium phosphate buffer of 0.02mol/L.The solution is mixed with acetonitrile
Share in mobile phase, can make detect baseline it is steady, and D-VB5 calcium chromatographic peak and above-mentioned impurity chromatographic peak can efficiently separate,
Peak shape is good.
Preferably, the volume ratio of the phosphate buffer and acetonitrile is 9: 1.It can be obtained under the ratio more perfect
Peak shape.
Preferably, the pH of the phosphate buffer is 3.0.
Preferably, the Detection wavelength of the high performance liquid chromatography is 205~215nm.
Preferably, the column temperature of the high performance liquid chromatography is 25~35 DEG C.The column temperature range is easy to control close to room temperature
Temperature makes its stabilization, and sample separates well within this temperature range.
Preferably, the flow velocity of the mobile phase is 0.7~1.0mL/min.
Preferably, the chromatographic column of the high performance liquid chromatography is octadecylsilane chemically bonded silica chromatographic column.
Preferably, above-mentioned detection method includes the following steps:
Step a, the reference substance solution of the impurity is prepared；
Step b, the reference substance solution is detected with the high performance liquid chromatography, is drawn according to gained peak area
The standard curve of the impurity obtains the concentration-peak area equation of linear regression；
Step c, sample to be tested is provided, the sample to be tested is detected with the high performance liquid chromatography, will be obtained
Peak area substitute into the concentration-peak area equation of linear regression, calculate the content of impurity described in the sample to be tested.
In terms of existing technologies, the present invention uses in the operating procedure of high performance liquid chromatography, the processing side of sample
Method is simple, operation is convenient, and working efficiency is higher；The present invention is detected using high performance liquid chromatography, is not needed using valuableness
Instrument.
Specifically, the reference substance solution of impurity described in above-mentioned steps a is at least 5 parts, and each impurity concentration range is 4
~40mg/L, solvent are the mobile phase.It may insure that the reference substance of the impurity sufficiently dissolves in preferred concentration range,
And baseline will not be had an impact using mobile phase as solvent, keep baseline in detection process more steady, testing result is accurate
Du Genggao.
Detailed description of the invention
Present invention will be further explained below with reference to the attached drawings and examples, in attached drawing:
Fig. 1 is the high-efficient liquid phase chromatogram of Beta-alanine reference substance solution in the embodiment of the present invention 1；
Fig. 2 is the high-efficient liquid phase chromatogram of D-VB5 lactone reference substance solution in the embodiment of the present invention 1；
Fig. 3 is the high-efficient liquid phase chromatogram of pantoic acid sodium reference substance solution in the embodiment of the present invention 1；
Fig. 4 is the product to be tested solution high-efficient liquid phase chromatogram of the embodiment of the present invention 1.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to the accompanying drawings and embodiments, right
The present invention is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, and
It is not used in the restriction present invention.
Embodiment 1
The embodiment of the invention provides a kind of detection method of D-VB5 calcium impurities, the high performance liquid chromatograph used is peace
Prompt human relations 1100, chromatographic column are octadecylsilane chemically bonded silica column (5 μm, 4.6 × 250mm), and sampling volume is 20 μ l (quantitative
Ring).Include the following steps:
1, standard solution is prepared:
4mg/L solution is prepared: precision measures impurity 1 (Beta-alanine), impurity 2 (D-VB5 lactone), 3 (pantoic acid of impurity
Sodium) each 40mg until adding flowing phased soln in 100ml volumetric flask, being settled to scale takes 1ml into 100ml volumetric flask, add flowing
Mutually be settled to scale to get.
10mg/L solution is prepared: precision measures impurity 1 (Beta-alanine), impurity 2 (D-VB5 lactone), 3 (pantoic acid of impurity
Sodium) each 40mg until adding flowing phased soln in 100ml volumetric flask, being settled to scale takes 2.5ml into 100ml volumetric flask, add stream
Dynamic phase be settled to scale to get.
20mg/L solution is prepared: precision measures impurity 1 (Beta-alanine), impurity 2 (D-VB5 lactone), 3 (pantoic acid of impurity
Sodium) each 40mg until adding flowing phased soln in 100ml volumetric flask, being settled to scale takes 5ml into 100ml volumetric flask, add flowing
Mutually be settled to scale to get.
30mg/L solution is prepared: precision measures impurity 1 (Beta-alanine), impurity 2 (D-VB5 lactone), 3 (pantoic acid of impurity
Sodium) each 40mg until adding flowing phased soln in 100ml volumetric flask, being settled to scale takes 7.5ml into 100ml volumetric flask, add stream
Dynamic phase be settled to scale to get.
40mg/L solution is prepared: precision measures impurity 1 (Beta-alanine), impurity 2 (D-VB5 lactone), 3 (pantoic acid of impurity
Sodium) each 40mg until adding flowing phased soln in 100ml volumetric flask, being settled to scale takes 10ml into 100ml volumetric flask, add stream
Dynamic phase be settled to scale to get.
2, the reference substance solution is detected with high performance liquid chromatography, chromatographic condition are as follows:
Chromatographic column: octadecylsilane chemically bonded silica chromatographic column；
Mobile phase: with buffer (0.02mol/L potassium phosphate buffer, with phosphoric acid tune pH value to 3.0): acetonitrile=9:
1 is mobile phase；
Detection wavelength: 210nm；
Column temperature: 30 DEG C；
Flow rate of mobile phase: 1.0mL/min.
20 μ l of reference substance solution injection high performance liquid chromatograph is taken to be detected, impurity reference substance solution concentration and peak area
The results are shown in Table 1~3.The theoretical cam curve of each chromatogram is greater than 5000, and good with impurity peaks separating degree, and wherein concentration is
The chromatogram of the pantoic acid sodium of the Beta-alanine of 20.270 μ g/ml, the D-VB5 lactone of 119.5 μ g/ml and 19.483 μ g/ml point
Not not as shown in Figures 1 to 3.
1 Beta-alanine reference substance solution concentration of table and peak area
| 1 concentration of impurity (μ g/ml) | 1 peak area of impurity |
| 4.054 | 18.9 |
| 10.162 | 49.6 |
| 20.270 | 100.1 |
| 30.540 | 149.4 |
| 40.810 | 198.9 |
Table 2D- pantolactone reference substance solution concentration and peak area
| 2 concentration of impurity (μ g/ml) | 2 peak area of impurity |
| 4.204 | 23.3 |
| 10.812 | 61.6 |
| 20.021 | 119.5 |
| 30.042 | 178.3 |
| 40.062 | 238.5 |
3 pantoic acid sodium reference substance solution concentration of table and peak area
| 3 concentration of impurity (μ g/ml) | 3 peak area of impurity |
| 3.897 | 74.1 |
| 10.690 | 246.9 |
| 19.483 | 469.8 |
| 30.966 | 745.1 |
| 40.449 | 985.9 |
The standard curve that each impurity reference substance is drawn according to each impurity reference substance concentration and corresponding peak area is with peak area
Abscissa show that concentration-peak area equation of linear regression of each impurity reference substance is as shown in table 4 using concentration as ordinate.
Concentration-peak area equation of linear regression of 4 impurity reference substance of table
| Impurity title | Standard curve | Related coefficient |
| Beta-alanine | Y=4.894x-0.2116 | R=0.9999 |
| D-VB5 lactone | Y=6.0195x-2.2629 | R=0.9999 |
| Pantoic acid sodium | Y=24.845x-19.801 | R=0.9999 |
3, sample to be tested is provided, the solution to be measured of 4mg/ml is configured to mobile phase；Separately take Beta-alanine reference substance, D- general
Acid lactone reference substance and pantoic acid sodium reference substance is appropriate and appropriate test solution, is placed in same measuring bottle, is dissolved with solvent
And dilute be made in every 1ml containing Beta-alanine, D-VB5 lactone and pantoic acid sodium respectively be about 20 μ g solution.With octadecyl silicon
Alkane bonded silica gel is filler (4.6 × 250mm, 5 μm), and with buffer, (0.02mol/L potassium phosphate buffer, uses phosphoric acid
Adjust pH value to 3.0): acetonitrile=9:1 is mobile phase, is operated, is measured according to the examination criteria program of high performance liquid chromatograph
Beta-alanine peak area is 21.3, and D-VB5 lactone peak area is 50.1, and pantoic acid sodium peak area is 100.1.According to above-mentioned reason
It is 4.4 μ g/ml, the concentration of D-VB5 lactone by the concentration that linear equation calculates Beta-alanine in D-VB5 calcium in sample to be tested
For 8.7 μ g/ml, the concentration of pantoic acid sodium is 4.8 μ g/ml, therefore the content of Beta-alanine is that 0.11%, D- is general in sample to be tested
The content of acid lactone is 0.22%, and the content of pantoic acid sodium is 0.12%.
4, Intermediate precision is investigated
Reference substance solution is prepared, the Intermediate precision of this method is investigated by above-mentioned chromatographic condition, the results are shown in Table 5.
5 Intermediate precision of table investigates result
5, the rate of recovery is investigated
Reference substance solution is prepared, the rate of recovery of this method is investigated by above-mentioned chromatographic condition, the results are shown in Table 6.
6 rate of recovery of table investigates result
6, quantitative limit
Quantitative limit detection is carried out to each impurity with above-mentioned chromatographic condition, quantifying for Beta-alanine is measured and is limited to 2 μ g/ml, RSD
It is 3.7%；Quantifying for D-VB5 lactone is limited to 3.5 μ g/ml, RSD 2.6%；Quantifying for pantoic acid sodium is limited to 3 μ g/ml, RSD
It is 4.1%.The RSD of three impurity < 10.0%, meets methodology requirement.
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
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (10)
1. the detection method of impurity in a kind of D-VB5 calcium, which is characterized in that the impurity includes Beta-alanine, D-VB5 lactone
With pantoic acid sodium；The detection method is high performance liquid chromatography, and mobile phase is the mixed solution of phosphate buffer and acetonitrile；
The volume ratio of the phosphate buffer and the acetonitrile is 8.5~9.5: 1；The pH of the phosphate buffer be 2.0~
4.0。
2. the detection method of impurity in D-VB5 calcium according to claim 1, which is characterized in that the phosphate buffer
For the potassium phosphate buffer of 0.02mol/L.
3. the detection method of impurity in D-VB5 calcium according to claim 2, which is characterized in that the phosphate buffer
Volume ratio with acetonitrile is 9: 1.
4. the detection method of impurity in D-VB5 calcium according to claim 3, which is characterized in that the phosphate buffer
PH be 3.0.
5. the detection method of impurity in D-VB5 calcium according to claim 1, which is characterized in that the high performance liquid chromatography
The Detection wavelength of method is 205~215nm.
6. the detection method of impurity in D-VB5 calcium according to claim 1, which is characterized in that the high performance liquid chromatography
The column temperature of method is 25~35 DEG C.
7. the detection method of impurity in D-VB5 calcium according to claim 1, which is characterized in that the flow velocity of the mobile phase
For 0.7~1.0mL/min.
8. the detection method of impurity in D-VB5 calcium according to claim 1, which is characterized in that the high performance liquid chromatography
The chromatographic column of method is octadecylsilane chemically bonded silica chromatographic column.
9. according to claim 1~8 in described in any item D-VB5 calcium impurity detection method, which is characterized in that above-mentioned height
Effect liquid phase chromatogram method includes the following steps:
Step a, the reference substance solution of the impurity is prepared；
Step b, the reference substance solution is detected with the high performance liquid chromatography, according to the drafting of gained peak area
The standard curve of impurity obtains the concentration-peak area equation of linear regression；
Step c, sample to be tested is provided, the sample to be tested is detected with the high performance liquid chromatography, the peak that will be obtained
Area substitutes into the concentration-peak area equation of linear regression, calculates the content of impurity described in the sample to be tested.
10. the detection method of impurity in D-VB5 calcium according to claim 9, which is characterized in that described in above-mentioned steps a
The reference substance solution of impurity is at least 5 parts, and each impurity concentration range is 4~40mg/L, and solvent is the mobile phase.
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Cited By (2)
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| CN112379007A (en) * | 2020-09-27 | 2021-02-19 | 安徽泰格生物科技有限公司 | Method for detecting content of DL-pantoic acid lactone |
| CN115078585A (en) * | 2022-06-27 | 2022-09-20 | 辰欣药业股份有限公司 | Method for measuring content of pantothenic acid in total-nutrient formula food with special medical application |
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