CN115078572B - Method for determining folic acid impurity by ultra-high performance liquid chromatography - Google Patents
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Abstract
The invention belongs to the technical field of medicine analysis, and particularly relates to a method for detecting folic acid impurities by ultra-high performance liquid chromatography, which adopts a Waters ultra-high performance liquid chromatograph, adopts a chromatographic column with octadecylsilane chemically bonded silica gel as a filler, and adopts a volume ratio of a buffer solution to methanol as a mobile phase of 90:10 is mobile phase A, the volume ratio of buffer solution and methanol is 70:30, carrying out gradient elution, the method not only can completely separate and detect 9 known impurities carried in each pharmacopoeia of folic acid at the same time, but also can separate and detect other impurities of folic acid, especially 6-formylpterin and pterin-6 carboxylic acid, and can more simply, conveniently, efficiently and accurately ensure controllable quality of folic acid and finally determine the safety of products.
Description
Technical Field
The invention belongs to the technical field of medicine analysis, and particularly relates to a method for detecting folic acid impurities by ultra-high performance liquid chromatography separation.
Background
Folic Acid (Folic Acid) is a water-soluble cellulose, known under the chemical name N- [4- [ (2-amino-4-oxo-1, 4-dihydro-6-pteridine) methylamino]Benzoyl group]L-glutamic acid of formula C 19 H 19 N 7 O 6 The molecular weight is 441.40, the CAS number is 59-30-3, and the structural formula of the compound is shown as the formula (a).
Folic acid is easy to damage in acid solution, is unstable to heat, is easy to lose at room temperature, and is easy to damage in visible light. Folic acid plays an important role in protein synthesis, cell division and growth, has promotion effect on normal erythrocyte formation, and can reduce hemoglobin production in erythrocytes and prevent cell maturation in erythrocytes when lacking, so that megaloblastic anemia is caused, and the folic acid is especially suitable for pregnant women and infants. In the folic acid preparation process, various related process impurities or degradation impurity impurities can appear, the impurities can influence the purity and quality of folic acid due to incomplete removal, 9 processes and degradation impurities are involved in the pharmacopoeia quality standard of folic acid at present, 2 larger degradation impurities are found in the experimental process, so that the 11 impurities are required to be subjected to quality control in folic acid and preparations, therefore, the separation and measurement of the impurities in folic acid and preparations have important significance for the production and storage of folic acid and preparations, and the structural formula of 10 impurities is as follows:
impurity I: the EP standard is noted as an unknown structure.
At present, the analysis and detection methods of folic acid related substances are carried out in Chinese pharmacopoeia, united states pharmacopoeia, european pharmacopoeia and British pharmacopoeia, and high performance liquid chromatography is adopted. Wherein, octadecylsilane chemically bonded silica is adopted as a filler, phosphate buffer (pH5.0) is adopted (2.0 g of monopotassium phosphate is taken, about 650mL of water is added for dissolution, 15mL of 0.5mol/L tetrabutylammonium hydroxide methanol solution, 7mL of 1mol/L phosphoric acid solution and 270mL of methanol are added, the mixture is cooled, 1mol/L phosphoric acid solution or ammonia test solution is used for adjusting the pH value to 5.0, water is used for diluting to 1000 mL) is adopted as a mobile phase for isocratic elution, the flow rate is 1.2mL/min, the detection wavelength is 280nm, the sample injection volume is 10 mu L, the detection time is 3 times of the main peak retention time, the detection of impurities is carried out by adopting a self-contrast method, and the 6-formylpterin can be detected by the Chinese pharmacopoeia. The detection method of the United states pharmacopoeia is similar to that of Chinese pharmacopoeia. The European pharmacopoeia is the same as the British pharmacopoeia, and uses octyl silane bonded silica gel as filler and methanol: phosphate (11.16 g/L monobasic potassium phosphate and 5.50g/L dibasic potassium phosphate) =12: 88 is the mobile phase, the flow rate is 0.6ml/min, the detection wavelength is 280nm, the sample injection volume is 5 μl, the detection time is 3.3 times of the retention time of the main peak, the impurity A, D is detected by an external standard method, other impurities are detected by a self-contrast method, and the European pharmacopoeia can not detect the 6-formylpterin. None of the above methods can simultaneously separate and detect the 11 impurities described above. In the method for measuring the content of 6-formylpterin as an impurity in folic acid (application number: 202110435473.4), the simultaneous detection of 6-formylpterin and pterin-6 carboxylic acid was carried out by HPLC, but it was not mentioned whether other impurities could be detected simultaneously.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for determining folic acid impurities by ultra-high performance liquid chromatography, which can detect and degrade impurities 6-formylpterin and pterin-6 carboxylic acid besides 9 impurities mentioned in European pharmacopoeia and impurity D mentioned in Chinese pharmacopoeia simultaneously, thereby greatly improving working efficiency and saving time and cost.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
a method for determining folic acid impurity by ultra-high performance liquid chromatography, wherein a chromatographic column adopted in the analysis process of the ultra-high performance liquid chromatography uses octadecylsilane chemically bonded silica as a filler, and a mobile phase uses a buffer solution and methanol with the volume ratio of 90:10 is mobile phase A, the volume ratio of buffer solution and methanol is 70:30 is mobile phase B, the gradient elution method is as follows:
from 0 minutes to 15 minutes, mobile phase A being 100% and mobile phase B being 0%;15 minutes to 20 minutes, mobile phase a decreased linearly to 0% and mobile phase B increased linearly to 100%;20 minutes to 25 minutes, mobile phase a maintained 0%, mobile phase B maintained 100%;25 to 28 minutes, mobile phase A increased linearly to 100% and mobile phase B decreased linearly to 0%;28 minutes to 35 minutes, mobile phase A remained 100% and mobile phase B remained 0%.
The method is suitable for separating and detecting 11 impurities, can be used for independently detecting one impurity, and can also be used for simultaneously separating and detecting 11 impurities.
The ultra-high performance liquid chromatography is reversed phase liquid chromatography, the adopted chromatographic column takes octadecylsilane chemically bonded silica as a filler, the particle size of the octadecylsilane chemically bonded silica particles is 1.7 mu m, the length of the chromatographic column is 150mm, the inner diameter is 2.1mm, and the Waters ACQUITY is preferredCSH C18 column. When the chromatographic column is used for separation, the temperature range of the chromatographic column is 20-30 ℃, preferably 25 ℃.
In the above method, the buffer solution is ammonium acetate, and glacial acetic acid is used for adjusting the pH to 4.6-5.0, preferably 4.8.
In the above method, the ammonium salt is one or more of ammonium acetate, monoammonium phosphate, and diammonium phosphate, preferably ammonium acetate.
In the above method, the concentration of the phosphate in the mixed buffer solution is 0.018 to 0.022mol/L, preferably 0.02mol/L.
In the method, when the ultra-high performance liquid chromatography is adopted and the mobile phase is eluted, the flow rate of the mobile phase is 0.28-0.32ml/min, preferably 0.3ml/min.
In the above method, the amount of the sample is 0.5 to 2. Mu.l, preferably 1. Mu.l.
After elution, detection is carried out by means of a DAD detector, an ultraviolet detector is also suitable, the detection wavelength being 278-282nm, preferably 280nm.
The method comprises the following specific steps: preparing a system applicable solution, a control solution and a folic acid sample solution respectively, injecting samples, and calculating the content of each impurity in the sample by a self-control method.
The system applicable liquid comprises: about 5mg of the 10 impurity reference substances are respectively taken, precisely weighed, placed in a 25ml measuring flask, added with a proper amount of solvent A to dissolve, fixed to a scale with solvent B, shaken uniformly to serve as impurity stock solutions, respectively weighing 0.5 ml-1.0 ml of each impurity stock solution, placed in a 10ml measuring flask, added with 2ml of folic acid test sample solution, diluted to the scale with solvent B, and shaken uniformly to serve as a system applicable solution.
The impurity I is as follows: EP impurity I2mg is taken, precisely weighed, placed in a 10ml measuring flask, dissolved by adding a proper amount of solvent A, and uniformly shaken by using the solvent B to fix the volume to the scale, thus being taken as an impurity I identification solution.
The solvent is as follows: 2.86% sodium carbonate as solvent A, and 0.02mol/L ammonium acetate (pH adjusted to 4.8 by glacial acetic acid) and methanol at a volume ratio of 90:10 as solvent B.
The folic acid test sample solution is as follows: about 50mg of folic acid test sample is taken, precisely weighed, placed in a 50ml measuring flask, added with 2.5ml of solvent A for dissolution, and diluted to a scale with solvent B.
The control solution is: precisely measuring 1ml of folic acid test sample solution, placing in a 200ml measuring flask, adding the solvent B, diluting to scale, shaking uniformly, and taking as a control solution.
Drawings
FIG. 1 is a liquid chromatogram for a system
FIG. 2 is a chromatogram of an impurity I identification solution
FIG. 3 is a chromatogram of a raw material sample solution
FIG. 4 is a chromatogram of a control solution
FIG. 5 is a solvent chromatogram
Detailed Description
The examples are presented for better illustration of the present invention, but are not intended to limit the scope of the present invention to the examples. Those skilled in the art will appreciate that various modifications and adaptations of the embodiments described above are possible in light of the above teachings and are intended to be within the scope of the invention.
Example 1
The instrument and chromatographic conditions used were as follows:
(1) Ultra-high performance liquid chromatograph: waters UPLC H-class
A detector: PDA
Chromatography workstation: empower
(2) Chromatographic column: waters ACQUITYCSH C18 column (1.7 μm,2.1 mm. Times.150 mm)
(3) Mobile phase a phase: the volume ratio of 0.02mol/L ammonium acetate (glacial acetic acid to adjust the pH to 4.8) to methanol is 90:10. mobile phase B phase: the volume ratio of 0.02mol/L ammonium acetate (glacial acetic acid to adjust the pH to 4.8) to methanol is 70:30.
(4) Detection conditions
Eluting the mobile phase A and the mobile phase B according to the following gradient proportion;
mobile phase flow rate: 0.3ml/min;
chromatographic column temperature: 25 DEG C
Detection wavelength: 280nm of
Sample injection amount: 1 μl
Gradient table: from 0 minutes to 15 minutes, mobile phase A being 100% and mobile phase B being 0%;15 minutes to 20 minutes, mobile phase a decreased linearly to 0% and mobile phase B increased linearly to 100%;20 minutes to 25 minutes, mobile phase a maintained 0%, mobile phase B maintained 100%;25 to 28 minutes, mobile phase A increased linearly to 100% and mobile phase B decreased linearly to 0%;28 minutes to 35 minutes, mobile phase A remained 100% and mobile phase B remained 0%.
Preparation of test solution
Solvent: 2.86% sodium carbonate as solvent A, and 0.02mol/L ammonium acetate (pH adjusted to 4.8 by glacial acetic acid) and methanol at a volume ratio of 90:10 as solvent B.
About 5mg of the 10 impurity reference substances are respectively taken, precisely weighed, placed in a 25ml measuring flask, added with a proper amount of solvent A to dissolve, fixed to a scale with solvent B, shaken uniformly to serve as impurity stock solutions, respectively weighing 0.5 ml-1.0 ml of each impurity stock solution, placed in a 10ml measuring flask, added with 2ml of folic acid test sample solution, diluted to the scale with solvent B, and shaken uniformly to serve as a system applicable solution.
And (3) taking 2mg of the EP impurity I, precisely weighing, placing into a 10ml measuring flask, adding a proper amount of the solvent A to dissolve, fixing the volume to a scale, and shaking uniformly to obtain the impurity I identification solution.
About 50mg of folic acid test sample is taken, precisely weighed, placed in a 50ml measuring flask, added with 2.5ml of solvent A for dissolution, and diluted to a scale with solvent B.
Precisely measuring 1ml of folic acid test sample solution, placing in a 200ml measuring flask, adding the solvent B, diluting to scale, shaking uniformly, and taking as a control solution.
Operating procedure
Respectively taking solvent, system applicable solution, impurity I identification solution, folic acid test sample solution, control solution, and liquid chromatograph, measuring under the above chromatographic conditions, and recording chromatogram, with the results shown in figures 1-4. The content of each impurity in the test sample is calculated according to the self-control, and the peak smaller than the main peak area of the control solution is ignored.
The calculation formula is as follows:
impurity content (%) =ax/as×100%
Wherein: ax: peak area of impurities in test sample solution
As: peak area of control solution
Typical patterns are shown in FIGS. 1-5.
Example 2
The instrument and chromatographic conditions used were as follows:
(1) Ultra-high performance liquid chromatograph: waters UPLC H-class
A detector: PDA
Chromatography workstation: empower
(2) Chromatographic column: waters ACQUITYCSH C18 column (1.7 μm,2.1 mm. Times.150 mm)
(3) Mobile phase a phase: 0.018mol/L ammonium acetate (glacial acetic acid to adjust pH to 4.6) and methanol at a volume ratio of 90:10. mobile phase B phase: 0.018mol/L ammonium acetate (glacial acetic acid to adjust pH to 4.6) and methanol at a volume ratio of 70:30.
(4) Detection conditions
Eluting the mobile phase A and the mobile phase B according to the following gradient proportion;
mobile phase flow rate: 0.28ml/min;
chromatographic column temperature: 20 DEG C
Detection wavelength: 278nm
Sample injection amount: 0.5 μl
Gradient table: from 0 minutes to 15 minutes, mobile phase A being 100% and mobile phase B being 0%;15 minutes to 20 minutes, mobile phase a decreased linearly to 0% and mobile phase B increased linearly to 100%;20 minutes to 25 minutes, mobile phase a maintained 0%, mobile phase B maintained 100%;25 to 28 minutes, mobile phase A increased linearly to 100% and mobile phase B decreased linearly to 0%;28 minutes to 35 minutes, mobile phase A remained 100% and mobile phase B remained 0%.
The preparation of the test solution was the same as in example 1
Procedure same as in example 1
The calculation formula is the same as that of example 1
Example 3
The instrument and chromatographic conditions used were as follows:
(1) Ultra-high performance liquid chromatograph: waters UPLC H-class
A detector: PDA
Chromatography workstation: empower
(2) Chromatographic column: waters ACQUITYCSH C18 column (1.7 μm,2.1 mm. Times.150 mm)
(3) Mobile phase a phase: 0.022mol/L ammonium acetate (glacial acetic acid to adjust pH to 5.0) and methanol in a volume ratio of 90:10. mobile phase B phase: 0.022mol/L ammonium acetate (glacial acetic acid to adjust pH to 5.0) and methanol in a volume ratio of 70:30.
(4) Detection conditions
Eluting the mobile phase A and the mobile phase B according to the following gradient proportion;
mobile phase flow rate: 0.32ml/min;
chromatographic column temperature: 30 DEG C
Detection wavelength: 282nm
Sample injection amount: 2 μl
Gradient table: from 0 minutes to 15 minutes, mobile phase A being 100% and mobile phase B being 0%;15 minutes to 20 minutes, mobile phase a decreased linearly to 0% and mobile phase B increased linearly to 100%;20 minutes to 25 minutes, mobile phase a maintained 0%, mobile phase B maintained 100%;25 to 28 minutes, mobile phase A increased linearly to 100% and mobile phase B decreased linearly to 0%;28 minutes to 35 minutes, mobile phase A remained 100% and mobile phase B remained 0%.
The preparation of the test solution was the same as in example 1
Procedure same as in example 1
Example 4
The invention was studied with a blank solvent used in the test procedure and found that the blank solvent did not interfere with the invention.
The linearity, detection limit and quantitative limit of folic acid and each impurity are detected, and the result shows that the correlation coefficient of the linear equation of folic acid and each impurity is more than or equal to 0.999.
TABLE 1 detection limits for folic acid and various impurities, quantitative limit test results
As can be seen from Table 1, the folic acid and each impurity of the present invention have high detection sensitivity.
Preparing a raw material sample solution, respectively injecting samples within 24 hours after preparation, recording a map, counting and calculating the content of each impurity, calculating to obtain the pterin-6-carboxylic acid, impurity A, 6-formylpterin, impurity C, impurity E, impurity G, impurity H in the sample, wherein the relative standard deviation RSD of the content of unknown impurities and the content of impurity D is less than 5%, and counting the number of newly-generated impurities to be 0.
The results showed that the test solution was stable over 24 h.
The folic acid impurity is subjected to repeatability and intermediate precision tests respectively, and the results show that the folic acid repeatability and intermediate precision meet the requirements, and the folic acid repeatability and intermediate precision are good.
The system is suitable for liquid sample injection and recording of chromatograms, and the separation degree between folic acid and each impurity can meet the requirements.
Taking the impurity I identification solution for sample injection and recording a chromatogram, wherein folic acid and each impurity do not influence the detection of the impurity I.
Taking folic acid test sample solution, sampling and recording a chromatogram, and calculating the content of 11 impurities in the test sample according to a self-contrast method, wherein the result is shown in Table 2.
TABLE 2 determination of the content of impurities in folic acid
| First batch of | Second batch | Third batch | ||
| Pterin-6-carboxylic acid | 0.03％ | 0.01％ | 0.02％ | |
| Impurity A | 0.38％ | 0.19％ | 0.37％ | |
| 6-formylpterin | 0.17％ | 0.09％ | 0.18％ | |
| Impurity C | 0.09％ | 0.09％ | 0.08％ | |
| Impurity E | 0.13％ | 0.14％ | 0.11％ | |
| Impurity G | 0.12％ | 0.12％ | 0.11％ | |
| Impurity H | 0.13％ | 0.11％ | 0.12％ | |
| Unknown impurities | 0.09％ | 0.05％ | 0.08％ | |
| Impurity D | 0.08％ | 0.07％ | 0.05％ | |
| Total impurities | 1.22％ | 0.87％ | 1.01％ |
As can be seen from the results of Table 2, the folic acid test sample contains pterin-6-carboxylic acid, A, 6-formylpterin, C, E, G, H, D, which is a known impurity, and contains an unknown impurity.
The invention can rapidly, effectively and accurately monitor the impurities in folic acid; the invention has good specificity, the folic acid peak and the adjacent impurities can be separated from each other, and the theoretical plate number of the folic acid peak is more than 10000; the detection limit of folic acid and 11 impurities is smaller, and the quantitative limit is smaller; the invention has high sensitivity; the repeatability and intermediate precision of the invention are good; in the invention, folic acid and 11 impurities have good linear relationship in a wider concentration range.
Claims (7)
1. The method for determining folic acid impurity by using ultra-high performance liquid chromatography is characterized in that ultra-high performance liquid chromatography, DAD detector or ultraviolet detector is adopted, octadecylsilane chemically bonded silica is used as filler in a chromatographic column, and the volume ratio of a buffer solution to methanol is 90:10 is mobile phase A, the volume ratio of buffer solution and methanol is 70:30 is mobile phase B, the detection wavelength is 278-282nm, the column temperature is 20-30 ℃, the flow rate is 0.28-0.32ml/min, the sample injection amount is 0.5-2 μl, and gradient elution is carried out;
the buffer solution is ammonium acetate aqueous solution with the concentration of 0.018-0.022mol/L, the pH value of glacial acetic acid is adjusted to 4.6-5.0, and the gradient elution method comprises the following steps: from 0 minutes to 15 minutes, mobile phase A being 100% and mobile phase B being 0%;15 minutes to 20 minutes, mobile phase a decreased linearly to 0% and mobile phase B increased linearly to 100%;20 minutes to 25 minutes, mobile phase a maintained 0%, mobile phase B maintained 100%;25 to 28 minutes, mobile phase A increased linearly to 100% and mobile phase B decreased linearly to 0%;28 minutes to 35 minutes, mobile phase A maintained 100% and mobile phase B maintained 0%;
the impurities are as follows:
2. the method for determining folic acid impurities of claim 1, wherein the particle size of the filler for chromatography column is 1.7 μm, the length of the chromatography column is 150mm, and the inner diameter is 2.1mm.
3. The method for determining folic acid impurities by ultra-high performance liquid chromatography according to any one of claims 1 to 2, characterized in that the chromatographic column is a Waters ACQUITYCSH C18 column.
4. The method for determining folic acid impurities of claim 2, wherein the aqueous solution of ammonium acetate and glacial acetic acid are adjusted to pH 4.8.
5. The method for determining folic acid impurities by ultra performance liquid chromatography according to claim 4, comprising the steps of:
step 1, preparing a solvent: 2.86% sodium carbonate is taken as a solvent A, and a mixed solution of 0.02mol/L ammonium acetate aqueous solution and methanol with the volume ratio of 90:10 is taken as a solvent B;
step 2, preparing a system applicable liquid: respectively taking about 5mg of the 10 impurity reference substances, precisely weighing, putting into a 25ml measuring flask, adding a proper amount of solvent A to dissolve, fixing the volume to a scale by using the solvent B, shaking uniformly, taking 0.5 ml-1.0 ml of each impurity stock solution as an impurity stock solution, putting into a 10ml measuring flask, adding 2ml of folic acid test sample solution, diluting to the scale by using the solvent B, shaking uniformly, and taking as a system applicable solution;
step 3, preparing an impurity I identification solution: taking 2mg of EP impurity I, precisely weighing, placing into a 10ml measuring flask, adding a proper amount of solvent A to dissolve, and using solvent B to fix the volume to the scale;
step 4, preparing a sample solution: taking about 50mg of folic acid test sample, precisely weighing, placing into a 50ml measuring flask, adding 2.5ml of solvent A for dissolution, and diluting to scale with solvent B;
and 5, precisely measuring 1ml of folic acid test sample solution, placing the folic acid test sample solution into a 200ml measuring flask, adding the solvent B for dilution to the scale, and shaking the folic acid test sample solution to be used as a control solution.
6. The method for determining folic acid impurities of claim 5, wherein the flow rate of the mobile phase is 0.3ml/min.
7. The method for determining folic acid impurities of claim 6, wherein the detection wavelength is 280nm.
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| CN114324618A (en) * | 2020-09-27 | 2022-04-12 | 重庆华邦制药有限公司 | Method for separating and detecting calcium levofolinate intermediate and related substances |
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