CN110967421A - Method for detecting related substances of flunarizine hydrochloride preparation - Google Patents
Method for detecting related substances of flunarizine hydrochloride preparation Download PDFInfo
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Abstract
The invention discloses a method for detecting related substances of a flunarizine hydrochloride preparation, which comprises the steps of respectively taking a test sample solution, a reference solution and an impurity mixed solution to carry out high performance liquid chromatography detection, and respectively detecting the content of each impurity in the test sample at 210nm and 253 nm; wherein, the peaks of the impurity A and the 4, 4-difluoro benzhydryl alcohol are detected at 210nm, and the content of the impurity is calculated according to an external standard method; and detecting other impurity peaks at 253nm, and calculating the impurity content according to a self-contrast method. The method disclosed by the invention is easy to operate, short in analysis time, strong in specificity and high in sensitivity, and can effectively separate 7 impurities, wherein 2 process impurities and 5 degraded impurities exist, the separation degree of the most difficult-to-separate impurity B from a main peak reaches more than 1.2, and the separation degrees of the other impurities and adjacent peaks can reach more than 1.5.
Description
(I) technical field
The invention relates to a method for detecting impurities in a medicament, in particular to a method for detecting related substances of a flunarizine hydrochloride preparation.
(II) background of the invention
Flunarizine is a selective calcium ion antagonist, has the main action mechanism of selectively blocking T and L type calcium channels, reducing the intracellular calcium level, inhibiting the contraction of vascular smooth muscle caused by calcium inflow and relieving vasospasm, can prevent cell damage caused by intracellular pathological calcium overload caused by ischemia and other reasons, and has wide effect on the nervous system. The composition is mainly used for treating cerebral ischemia and hypoxic diseases clinically, and has obvious curative effect on preventing and treating injury of peripheral vascular endothelial cells caused by ischemia and hypoxia. In addition, it is also used for reducing cell fragility caused by ischemia and acidosis, so as to improve deformability and reduce blood viscosity.
The chemical name of the flunarizine hydrochloride is (E) -1- [ bis (4-fluorophenyl) methyl]-4-2 (2-propenyl-3-phenyl) -piperazine dihydrochloride of formula: c26H26F2N22HCl, molecular weight 477.42, having the formula:
at present, a plurality of flunarizine hydrochloride dosage forms are on the market at home, such as flunarizine hydrochloride capsules, flunarizine hydrochloride tablets, flunarizine hydrochloride dispersible tablets and other dosage forms.
The method for detecting related substances of flunarizine hydrochloride raw materials is recorded in the Chinese pharmacopoeia 2015 edition and the European pharmacopoeia, wherein the method for detecting related impurities in the flunarizine hydrochloride raw materials in the pharmacopoeia 2015 edition adopts isocratic elution, cannot effectively separate related impurities, and is low in sensitivity. The European pharmacopoeia method only monitors impurities possibly introduced in the synthesis of raw materials, has high requirements on a system, and has overhigh concentration of a test sample, but the specifications of various formulations of flunarizine hydrochloride are smaller, so the concentration is not suitable for the formulations. The existing literature, Meidan and other quality evaluation of the commercially available flunarizine hydrochloride capsules has the defects of long detection time, more time consumption, poor impurity separation effect and small reflected information amount, and cannot achieve the aim of effectively controlling the quality of the flunarizine hydrochloride. The existing literature, namely 'improvement of related substances and content determination methods of flunarizine hydrochloride' such as Lijie, can not effectively separate related impurities, and the method only aims at the raw material of the flunarizine hydrochloride and does not aim at a preparation. Therefore, the research of a related substance detection method of the flunarizine hydrochloride preparation, which has the advantages of strong specificity, high separation degree, more comprehensive control and mild detection condition, is very important.
Disclosure of the invention
The invention aims to provide a method for detecting related substances of a flunarizine hydrochloride preparation, which is simple, convenient and quick, has strong specificity, accurate detection and high separation degree of impurity compounds, has more comprehensive detection and control on the related substances of the flunarizine hydrochloride preparation, mild detection conditions and wide application to detection equipment and instruments.
The technical scheme adopted by the invention is as follows:
the invention provides a method for detecting related substances of a flunarizine hydrochloride preparation, which comprises the following steps:
(1) solution preparation:
test solution: dissolving flunarizine hydrochloride sample in diluent, diluting to obtain solution containing flunarizine, filtering (preferably with 0.45 μm filter membrane), and collecting filtrate as sample solution; the diluent is methanol and phosphate buffer solution with the pH value of 3.5 in a volume ratio of 75: 25;
control solution: precisely measuring a test solution, and diluting the test solution by 100 times by using a diluent to serve as a reference solution;
impurity a and 4, 4-difluorobenzhydrol mixed solution: accurately weighing an appropriate amount of impurity A and 4, 4-difluorobenzhydrol, and preparing a mixed solution of impurity A and 4, 4-difluorobenzhydrol by using a diluent, wherein each 1mL of the mixed solution contains 3 mu g of impurity A and 1.2 mu g of 4, 4-difluorobenzhydrol; the impurity A is 1- [ bis- (4-fluorophenyl) methyl ] piperazine;
(2) and (3) high performance liquid chromatography detection: respectively taking a test solution, a reference solution and a mixed solution of impurities A and 4, 4-difluorobenzhydrol for high performance liquid chromatography detection at 210nm and 253nm, and calculating the content of each impurity in the test solution according to high performance liquid chromatography (general rule 0512) of Chinese pharmacopoeia; wherein, the peaks of the impurity A and the 4, 4-difluoro benzhydryl alcohol are detected at 210nm, and the content of the impurity is calculated according to an external standard method; detecting other impurity peaks at 253nm, and calculating the impurity content according to a self-contrast method; the related substances of the flunarizine hydrochloride preparation are impurity A, impurity B, impurity D, trans-1-cinnamyl piperazine, 4, 4-difluorobenzophenone, 4, 4-difluorobenzhydrol and nitrogen oxide, wherein the impurity A is 1- [ bis- (4-fluorophenyl) methyl ] piperazine, the impurity B is 1- [ (4-fluorophenyl) phenylethyl ] -4- [ (2E) -3-phenylpropenyl ] piperazine, and the impurity D is 1- [ bis- (4-fluorophenyl) methyl ] -4- [ (2Z) -3-phenylpropenyl ] piperazine;
the detection conditions of the high performance liquid chromatography are as follows: using a chromatographic column C18, and using 0.01 mol.L of triethylamine with the volume concentration of 0.4% and the pH value of 3.3-3.7-1Performing gradient elution with phosphate buffer solution as mobile phase A and methanol or acetonitrile as mobile phase B at column temperature of 30 deg.C and flow rate of 0.8-1.2 mL/min; the gradient elution method comprises the following steps: at 0min, the mobile phase A is 70-80%, and the mobile phase B is 20-30%; at 14-20min, the mobile phase A is 20-30%, and the mobile phase B is 70-80%; at 22-25min, the mobile phase A is 20-30%, and the mobile phase B is 70-80%.
Preferably, the gradient elution method is: at 0min, the mobile phase A is 70%, and the mobile phase B is 30%; at 18min, the mobile phase A is 20% and the mobile phase B is 80%; at 25min, mobile phase a was 20% and mobile phase B was 80%.
Preferably, the specification of the C18 chromatographic column is that the length is 100mm, the inner diameter is 4.6mm, and the grain diameter of a packing material is 3.0 mu m.
Preferably, the flow rate is 1.0 mL/min.
Preferably, the pH of the phosphate buffer solution is 3.5, and the mobile phase a is prepared by: dissolving potassium dihydrogen phosphate in water, diluting, adding triethylamine, and adjusting pH to 3.5 with phosphoric acid; the mass concentration of the potassium dihydrogen phosphate is 1.36g/L calculated by the volume of water, and the adding volume of the triethylamine is 4ml/L calculated by the volume of water.
Preferably, the mobile phase B is methanol.
Preferably, the concentration of flunarizine in the test solution is 0.1-2.0 mg/ml.
If the content of impurities in the test sample is low, adding an impurity standard substance into the test sample to prepare a mixed solution of the test sample and each impurity for detecting the separation degree condition of the impurities, wherein each 1mL of the mixed solution contains 0.6mg of flunarizine hydrochloride, 3 mug of impurity A, 3 mug of impurity B, 3 mug of 4, 4-difluorobenzophenone, 1.2 mug of impurity D, 1.2 mug of trans-1-cinnamyl piperazine, 1.2 mug of 4, 4-difluorobenzhydryl and 1.2 mug of nitric oxide.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a method for detecting related substances of a flunarizine hydrochloride preparation, which is a high performance liquid chromatography method and can be used for detecting related substances of the flunarizine hydrochloride preparation, particularly related substances of a flunarizine hydrochloride capsule. The detection method adopts gradient elution, can effectively separate 7 impurities, wherein 2 process impurities and 5 degraded impurities exist, the separation degree of the most difficult-to-separate impurity B from a main peak reaches more than 1.2, the separation degrees of the rest impurities from adjacent peaks can reach more than 1.5, and can effectively monitor each impurity, especially the degraded impurities. In addition, the dual-wavelength detection of 210nm and 253nm is adopted, the peaks of the impurity A and the 4, 4-difluorobenzhydrol are detected at 210nm, the quantification limits are 0.6044ug/ml and 0.613ug/ml respectively, the average recovery rates are 96.80 percent and 101.01 percent respectively, and the effective detection and accurate quantification of the impurity A and the 4, 4-difluorobenzhydrol can be realized; the peak of other impurities is detected at 253nm, the quantitative limit of the main component is 0.1219ug/ml, the quantitative limit of the impurity B is 0.3004ug/ml, the recovery rate is 98.76%, the quantitative limit of the impurity D is 0.30225ug/ml, the recovery rate is 102.56%, the quantitative limit of the trans-1-cinnamyl piperazine is 0.1214ug/ml, the recovery rate is 101.63%, the quantitative limit of the 4, 4-difluorobenzophenone is 0.2982ug/ml, the recovery rate is 103.82%, the quantitative limit of the nitrogen oxide is 0.2388ug/ml, the recovery rate is 103.50%, and the effective detection and accurate quantification of the other impurities can be realized. The method adopts the small-particle-size chromatographic column, improves the detection efficiency and simultaneously ensures the separation effect of each impurity.
In conclusion, the method for detecting related substances of the flunarizine hydrochloride preparation provided by the invention is easy to operate, short in analysis time, good in separation effect, high in sensitivity, more comprehensive in detection and control of related substances of the flunarizine hydrochloride, mild in detection conditions, and wide in application to detection equipment and instruments. Fills the blank of the related substance detection field of the flunarizine hydrochloride preparation, and provides reliable guarantee for improving and better controlling the quality of the flunarizine hydrochloride product.
(IV) description of the drawings
FIG. 1: the method detects an HPLC (high performance liquid chromatography) diagram of a mixed solution of a test sample and each impurity standard substance, wherein A is a 253nm spectrogram, B is a 210nm spectrogram, wherein a peak 1 is flunarizine, a peak 2 is impurity A, a peak 3 is 4, 4-difluorobenzophenone, a peak 4 is 4, 4-difluorobenzhydrol, a peak 5 is trans-1-cinnamyl piperazine, a peak 6 is impurity B, a peak 7 is impurity D, and a peak 8 is nitrogen oxide.
FIG. 2: the method detects an HPLC (high performance liquid chromatography) diagram of a mixed solution of a flunarizine hydrochloride standard substance and each impurity, wherein A is a 253nm spectrogram and B is a 210nm spectrogram, wherein a peak 1 is flunarizine, a peak 2 is impurity A, a peak 3 is 4, 4-difluorobenzophenone, a peak 4 is 4, 4-difluorobenzhydrol, a peak 5 is trans-1-cinnamyl piperazine, a peak 6 is impurity B, a peak 7 is impurity D, and a peak 8 is nitrogen oxide.
FIG. 3: example 2 HPLC profile of the sensitive solution, a 253nm profile and B210 nm profile, with peak 1 flunarizine.
FIG. 4: example 2 HPLC profile of resolution solution, a 253nm profile, B210 nm profile, peak 1 is flunarizine, peak 6 is impurity B, and peak 7 is impurity D.
FIG. 5: example 2 HPLC profile of the test solution, a is 253nm profile, B is 210nm profile, peak 1 is flunarizine, peak 2 is impurity a, peak 3 is 4, 4-difluorobenzophenone, peak 5 is trans-1-cinnamyl piperazine, and peak 6 is impurity B.
FIG. 6: example 2 HPLC profile of control solution, A253 nm spectrum, B210 nm spectrum, peak 1 flunarizine.
FIG. 7: example 2 HPLC profile of a mixed solution of impurity a and 4, 4-difluorobenzhydrol, a being 253nm spectrum, B being 210nm spectrum, peak 2 being impurity a, peak 4 being 4, 4-difluorobenzhydrol.
FIG. 8: example 3 HPLC plot of the sensitive solution, A is 253nm spectrum, B is 210nm spectrum, peak 1 is flunarizine.
FIG. 9: example 3 HPLC profile of the split solution, a 253nm profile and B210 nm profile, with peak 1 being flunarizine, peak 6 being impurity B and peak 7 being impurity D.
FIG. 10: example 3 HPLC profile of the mixed solution of the test sample and each impurity standard, a is 253nm profile, B is 210nm profile, wherein peak 1 is flunarizine, peak 2 is impurity a, peak 3 is 4, 4-difluorobenzophenone, peak 4 is 4, 4-difluorobenzhydrol, peak 5 is trans-1-cinnamylpiperazine, peak 6 is impurity B, peak 7 is impurity D, peak 8 is nitrogen oxide.
FIG. 11: example 3 HPLC profile of control solution, a 253nm profile and B210 nm profile, with peak 1 flunarizine.
FIG. 12: example 3 HPLC profile of a mixed solution of impurity a and 4, 4-difluorobenzhydrol, a being 253nm spectrum, B being 210nm spectrum, peak 2 being impurity a, peak 4 being 4, 4-difluorobenzhydrol.
FIG. 13: example 4 HPLC profile of the sensitive solution, a 253nm profile and B210 nm profile, with peak 1 flunarizine.
FIG. 14: example 4 HPLC profile of the split solution, a 253nm profile and B210 nm profile, with peak 1 being flunarizine, peak 6 being impurity B and peak 7 being impurity D.
FIG. 15: example 4 HPLC profile of the mixed solution of the test sample and each impurity standard, a is 253nm profile, B is 210nm profile, wherein peak 1 is flunarizine, peak 2 is impurity a, peak 3 is 4, 4-difluorobenzophenone, peak 4 is 4, 4-difluorobenzhydrol, peak 5 is trans-1-cinnamylpiperazine, peak 6 is impurity B, peak 7 is impurity D, peak 8 is nitrogen oxide.
FIG. 16: example 4 HPLC profile of control solution, a 253nm profile and B210 nm profile, with peak 1 flunarizine.
FIG. 17: example 4 HPLC profile of a mixed solution of impurity a and 4, 4-difluorobenzhydrol, a being 253nm spectrum, B being 210nm spectrum, peak 2 being impurity a, peak 4 being 4, 4-difluorobenzhydrol.
FIG. 18: example 5 HPLC profile of a mixed solution of a test sample and each impurity standard, a is 253nm profile, B is 210nm profile, wherein peak 1 is flunarizine, peak 2 is impurity a, peak 3 is 4, 4-difluorobenzophenone, peak 4 is 4, 4-difluorobenzhydrol, peak 5 is trans-1-cinnamylpiperazine, peak 6 is impurity B, peak 7 is impurity D, peak 8 is nitrogen oxide.
FIG. 19: example 6 HPLC profile of the mixed solution of the test sample and each impurity standard, a is 253nm profile, B is 210nm profile, wherein peak 1 is flunarizine, peak 2 is impurity a, peak 3 is 4, 4-difluorobenzophenone, peak 4 is 4, 4-difluorobenzhydrol, peak 5 is trans-1-cinnamylpiperazine, peak 6 is impurity B, peak 7 is impurity D, peak 8 is nitrogen oxide.
FIG. 20: example 7 HPLC profile of the mixed solution of the test sample and each impurity standard, a is 253nm profile, B is 210nm profile, wherein peak 1 is flunarizine, peak 2 is impurity a, peak 3 is 4, 4-difluorobenzophenone, peak 4 is 4, 4-difluorobenzhydrol, peak 5 is trans-1-cinnamylpiperazine, peak 6 is impurity B, peak 7 is impurity D, peak 8 is nitrogen oxide.
FIG. 21 shows a standard curve, where A is the impurity A standard curve, B is the 4, 4-difluorobenzhydrol standard curve, and C is the flunarizine standard curve.
(V) detailed description of the preferred embodiments
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
example 1 Standard Curve, quantitation Limit and recovery
1. Standard curve
Diluent agent: methanol: the ph3.5 phosphate buffer was 75:25, v/v.
Accurately weighing 12mg of impurities A15mg, 4-difluorobenzhydrol and 35.4mg of flunarizine hydrochloride (equivalent to 30mg of flunarizine) in a 100ml measuring flask respectively, dissolving and diluting the impurities in methanol to scale, shaking up, and preparing the impurities A stock solution, the 4, 4-difluorobenzhydrol stock solution and the flunarizine hydrochloride stock solution respectively.
The appropriate amount of each stock solution is precisely measured, the stock solutions are diluted into an impurity A solution, a 4, 4-difluorobenzhydrol solution and a flunarizine hydrochloride solution with different concentration gradients by a diluent, high performance liquid chromatography detection is carried out respectively (the same as example 2), linear regression is carried out by taking the linear solution concentration (mu g/ml) as an X axis and the peak area A as a Y axis, the linearity is good, and the results are shown in tables 1, 2 and 3.
Table 1 impurity a linearity and range experimental results
| Concentration (ug/ml) | 0.6044 | 1.2088 | 1.511 | 3.022 | 6.044 | Correlation coefficient |
| Peak area average | 17.58344 | 36.3659 | 43.75229 | 86.51976 | 174.03 | 0.99994 |
TABLE 24, 4-Difluorodiphenylmethanol Linear and Range results
| Concentration (ug/ml) | 0.613 | 0.9195 | 1.226 | 1.839 | 2.452 | Correlation coefficient |
| Peak area average | 24.90 | 35.31 | 46.89 | 69.17 | 97.87293 | 0.99805 |
TABLE 3 Flunarizine Linearity and Range test results
| Concentration (ug/ml) | 0.1219 | 0.6097 | 3.0487 | 6.0975 | 12.1949 | Correlation coefficient |
| Peak area average | 6.15343 | 21.60776 | 93.68465 | 184.7155 | 367.251 | 1 |
2. Limit of quantification
And (3) diluting each impurity standard substance stock solution step by step, wherein the mass concentration when the signal-to-noise ratio S/N is approximately equal to 10 is taken as a quantification Limit (LOQ), and the mass concentration when the signal-to-noise ratio S/N is approximately equal to 3 is taken as a detection Limit (LOD). LOQ solution was continuously fed into 6 needles and LOD solution was continuously fed into 3 needles, and the results are shown in Table 4.
TABLE 4 detection and quantitation limits for various impurity standards
3. Recovery rate
12 parts of blank auxiliary materials (mixture composed of the auxiliary materials: 80 parts of lactose, 13 parts of corn starch, 6 parts of talcum powder and 1 part of magnesium stearate) 0.92g are respectively weighed in a 50ml measuring flask, and each single impurity stock solution (samples are prepared according to four concentrations of 50%, 100% and 200% of LOQ of each impurity and the limit of mass content of each impurity) is precisely transferred and taken, wherein the results are shown in tables 5 to 11.
TABLE 5 results for recovery of impurity A
TABLE 64, 4-Difluorodiphenylmethanol recovery results (LOQ and Limit 50% identical)
TABLE 7 Nitrogen oxide recovery results (correction factor 1.2)
TABLE 8 impurity B recovery results (correction factor 1.1)
TABLE 94, 4-Difluorobenzophenone recovery results (correction factor 0.70)
TABLE 10 results for recovery of trans-1-cinnamylpiperazine (correction factor 0.60)
TABLE 11 impurity D recovery results (correction factor 2.3)
Example 2:
1. the instrument comprises the following steps: an analytical balance is adopted, namely a high performance liquid chromatograph, an Agilent 1260/1100 high performance liquid chromatograph, a Chem Station chromatographic workstation, a Mettler FE20pH meter and a Mettler ME204/XS205 analytical balance.
2. Reagent: the test sample is the content of a commercial flunarizine hydrochloride capsule, and the standard sample is a flunarizine hydrochloride standard sample; 7 impurity standards: impurity a (1- [ bis- (4-fluorophenyl) methyl ] piperazine), impurity B (1- [ (4-fluorophenyl) phenylethyl ] -4- [ (2E) -3-phenylpropenyl ] piperazine), impurity D (1- [ bis- (4-fluorophenyl) methyl ] -4- [ (2Z) -3-phenylpropenyl ] piperazine), trans-1-cinnamyl piperazine, 4, 4-difluorobenzophenone, 4, 4-difluorobenzhydrol, nitrogen oxide; the methanol and the acetonitrile are chromatographically pure, and the other reagents are analytically pure.
Diluent agent: methanol: the ph3.5 phosphate buffer was 75:25, v/v.
3. Solution preparation
Test solution: weighing the content of a commercial flunarizine hydrochloride capsule by 0.96g precisely, placing the content in a 50mL volumetric flask, adding a diluent, carrying out ultrasonic dissolution at 40kHz for 10min, diluting the content to the scale with the diluent, filtering the content with a 0.45-micron filter membrane, and taking the filtrate as a test solution, wherein the concentration of the flunarizine is 0.6 mg/mL.
Control solution: precisely measuring 1ml of test solution, placing the test solution in a 100ml volumetric flask, diluting the test solution to a scale with a diluent, and taking the test solution as a control solution, wherein the concentration of the fluorine-containing cinnarizine is 0.006 mg/ml.
Sensitivity solution: precisely measuring 1ml of the control solution, putting the control solution into a 10ml volumetric flask, diluting the control solution to a scale with a diluent to obtain a sensitivity solution, wherein the concentration of the fluorine-containing cinnarizine is 0.0006 mg/ml.
Resolution solution: and dissolving a proper amount of the flunarizine hydrochloride standard substance and the impurity B standard substance in methanol, and diluting to prepare a solution with the separation degree of 0.6mg of flunarizine and 3 mu g of impurity B per 1ml as a separation degree solution.
A standard flunarizine hydrochloride stock solution: dissolving the standard substance flunarizine hydrochloride with methanol, and diluting to obtain standard substance stock solution containing 6mg of flunarizine per 1 ml.
Stock solutions of various impurity standards: dissolving impurity A, impurity B, impurity D, trans-1-cinnamyl piperazine, 4-difluorobenzophenone, 4-difluorobenzophenone and a nitrogen oxide standard substance with methanol to respectively prepare impurity A with the concentration of 151.1 mu g/mL-1The concentration of impurity B was 150.2. mu.g/mL-1The concentration of impurity D was 120.9. mu.g/mL-1Concentration of trans-1-cinnamyl piperazine 121.4. mu.g.mL-1Concentration of 4, 4-difluorobenzophenone was 149.1. mu.g.mL-1Concentration of 4, 4-difluorobenzhydrol 122.6. mu.g.mL-1And nitric oxide concentration 119.4. mu.g.mL-1A stock solution of each impurity standard of (a);
mixed solution of the test sample and each impurity standard substance: precisely weighing 0.96g of flunarizine hydrochloride capsule content, placing the content into a 50mL measuring flask, precisely weighing a proper amount of each impurity standard substance stock solution into the same measuring flask, and performing volume metering by using a diluent to prepare a mixed solution of a test article and each impurity, wherein each 1mL of the mixed solution contains flunarizine hydrochloride 0.6mg, impurity A3 mu g, impurity B3 mu g, 4-difluorobenzophenone 3 mu g, impurity D1.2 mu g, trans-1-cinnamyl piperazine 1.2 mu g, 4-difluorobenzyl alcohol 1.2 mu g and nitric oxide 1.2 mu g.
The mixed solution of the flunarizine hydrochloride standard substance and each impurity standard substance is as follows: taking a proper amount of a standard substance stock solution of flunarizine hydrochloride, placing the appropriate amount of the standard substance stock solution of the flunarizine hydrochloride into a 50mL measuring flask, respectively and precisely measuring a proper amount of each standard substance stock solution of impurities into the same measuring flask, and carrying out volume metering by using a diluent to prepare a mixed solution of the standard substance and each impurity, wherein each 1mL of the mixed solution contains 0.6mg of the flunarizine hydrochloride, 3 mug of the impurity A, 3 mug of the impurity B, 3 mug of 4, 4-difluorobenzophenone, 1.2 mug of the impurity D, 1.2 mug of trans-1-cinnamyl piperazine, 1.2 mug of 4, 4-difluorobenzhydrol and 1.2 mug of nitric oxide.
Mixed solution of impurity A and 4, 4-difluorobenzhydrol: precisely measuring an appropriate amount of impurity A and 4, 4-difluorobenzhydrol stock solution in the same measuring flask, and diluting to constant volume by using a diluent to prepare a mixed solution containing 3 mu g of impurity A and 1.2 mu g of 4, 4-difluorobenzhydrol per 1 mL.
4. Chromatographic conditions are as follows:
a chromatographic column: pursuit 3C 18(4.6mm 100mm, 3.0 μm);
mobile phase A: 1.36g of monopotassium phosphate is taken, dissolved in water and diluted into 1000ml, 4ml of triethylamine is added, and the pH value is adjusted to 3.5 by phosphoric acid.
Mobile phase B: methanol.
Column temperature: at 30 ℃. The flow rate was 1.0 ml/min.
TABLE 12 gradient elution conditions
5. Chromatographic detection
Because the content of impurities in the test sample is low, the impurities are added into the test sample, so that the method is convenient to verify the separation degree of the impurities. Respectively and precisely measuring a mixed solution (figure 1) of a test sample and each impurity standard substance, a mixed solution (figure 2) of a flunarizine hydrochloride standard substance and each impurity, a sensitivity solution (figure 3), a separation degree solution (figure 4), a test sample solution (figure 5), a reference solution (figure 6), a mixed solution (figure 7) of impurities A and 4, 4-difluorobenzhydrol, respectively injecting the mixed solution into a liquid chromatograph, taking 253nm and 210nm as detection wavelengths, recording a chromatogram, and calculating the separation degree of the impurities by referring to a high performance liquid chromatography (general rule 0512) of Chinese pharmacopoeia, wherein the calculation formula of the separation degree is as follows:
R＝2(tR2-tR1)/(W1+W2)
wherein R is the degree of separation, tR2The retention time of the latter peak in two adjacent chromatographic peaks; t is tR1The retention time of the former peak in two adjacent chromatographic peaks is shown; w1、W2The peak width of two adjacent chromatographic peaks.
The results of the degrees of separation are shown in Table 16. The embodiment can effectively separate 7 impurities, the separation degree of the impurity B and the main peak reaches more than 1.2, the separation degrees of the rest impurities and the adjacent peaks can reach more than 2.0, and the signal-to-noise ratio of the main peak of the sensitivity solution is more than 10. The results of calculating the contents of the respective impurities are shown in Table 17 based on the standard curve of example 1.
Example 3:
test solution: the preparation method comprises the steps of grinding commercially available flunarizine hydrochloride tablets, precisely weighing the tablets to be equal to 30mg of flunarizine, placing the tablets into a 50mL volumetric flask, adding a diluent (same as example 2), carrying out ultrasonic treatment at 40kHz for 10min, dissolving and diluting the tablets to a scale, filtering the tablets through a 0.45-micron filter membrane, taking the filtrate as a sample solution, wherein the concentration of the flunarizine is 0.6 mg/mL.
Mixed solution of the test sample and each impurity standard substance: taking a commercially available flunarizine hydrochloride tablet, finely grinding the tablet, precisely weighing the tablet to be equal to 30mg of flunarizine hydrochloride, placing the tablet in a 50mL volumetric flask, precisely weighing a proper amount of each impurity standard substance stock solution in the same volumetric flask, adding a diluent (same as example 2), dissolving the tablet by 40kHz ultrasonic for 10min, diluting the solution to a scale, filtering the solution by a 0.45 mu m filter membrane, and taking a filtrate as a solution of a test article and each impurity standard substance, wherein the concentration of the flunarizine is 0.6mg/mL, 3 mu g of impurity A, 3 mu g of impurity B, 3 mu g of 4, 4-difluorobenzophenone, 1.2 mu g of impurity D, 1.2 mu g of trans-1-cinnamyl piperazine, 1.2 mu g of 4, 4-difluorobenzhydryl alcohol and 1.2 mu g of nitrogen oxide.
Control solution: 1ml of the test solution was measured precisely and diluted to the scale with a diluent (same as in example 2) in a 100ml volumetric flask as a control solution.
Sensitivity solution: 1ml of the control solution was measured precisely, and diluted to the scale with a diluent (same as example 2) in a 10ml volumetric flask to obtain a sensitive solution.
Resolution solution: the same as in example 2.
Mixed solution of impurity A and 4, 4-difluorobenzhydrol: the same as in example 2.
Chromatographic conditions are as follows: xbridge C18(4.6mm 100mm, 3.5 μm);
mobile phase A: the preparation is as in example 2, the pH is adjusted to 3.3.
Mobile phase B: methanol. The column temperature was 30 ℃. The flow rate was 0.8 ml/min.
Gradient elution is as in example 2 Table 12, and other conditions and operations are as in example 2.
Precisely measuring a sensitivity solution (figure 8), a resolution solution (figure 9), a mixed solution of a test sample and each impurity standard (figure 10), a reference solution (figure 11), and a mixed solution of impurity A and 4, 4-difluorobenzhydrol (figure 12), respectively measuring 10 μ l, injecting into a liquid chromatograph, and recording chromatograms with 253nm and 210nm as detection wavelengths, wherein the impurity resolution is shown in table 16. The embodiment can separate 7 impurities, the separation degree of the impurity B and the main peak reaches more than 1.0, and the separation degrees of the rest impurities and the adjacent peaks can reach more than 2.0. The results of calculating the contents of the respective impurities are shown in Table 17 based on the standard curve of example 1.
Example 4:
test solution: taking the content of a commercial flunarizine hydrochloride capsule, precisely weighing 30mg equivalent to flunarizine, placing the content in a 50mL volumetric flask, adding a diluent (same as the example 1) to dissolve and dilute the content to a scale, filtering the content by a 0.45 mu m filter membrane, taking the filtrate as a test solution, wherein the concentration of the flunarizine is 0.6 mg/mL.
Mixed solution of the test sample and each impurity standard substance: taking the content of a commercially available flunarizine hydrochloride capsule, precisely weighing 30mg of flunarizine hydrochloride, placing the content in a 50mL volumetric flask, precisely weighing a proper amount of each impurity standard substance stock solution in the same volumetric flask, adding a diluent (same as the example 2) to dissolve and dilute the stock solution to a scale, filtering with a 0.45 mu m filter membrane, taking the filtrate as a mixed solution of a test sample and each impurity standard substance, wherein the concentration of the flunarizine is 0.6mg/mL, 3 mu g of impurity A, 3 mu g of impurity B, 3 mu g of 4, 4-difluorobenzophenone, 1.2 mu g of impurity D, 1.2 mu g of trans-1-cinnamyl piperazine, 1.2 mu g of 4, 4-difluorobenzophenone and 1.2 mu g of nitrogen oxide.
Control solution: 1ml of the test solution was measured precisely and diluted to the scale with a diluent (same as in example 2) in a 100ml volumetric flask as a control solution.
Sensitivity solution: 1ml of the control solution was measured precisely, and diluted to the scale with a diluent (same as example 2) in a 10ml volumetric flask to obtain a sensitive solution.
Resolution solution: the same as in example 2.
Mixed solution of impurity A and 4, 4-difluorobenzhydrol: the same as in example 2.
Chromatographic conditions are as follows: poroshell 120C 18(4.6mm 100mm, 2.7 μm).
Mobile phase A: the preparation is as in example 2, the pH is adjusted to 3.7.
Mobile phase B: methanol. The column temperature was 30 ℃ and the flow rate was 1.2 ml/min.
Gradient elution is as in example 2 Table 12, and other operations and conditions are as in example 2.
A sensitive solution (FIG. 13), a resolution solution (FIG. 14), a mixed solution of a sample and each impurity standard (FIG. 15), a control solution (FIG. 16), and a mixed solution of impurity A and 4, 4-difluorobenzhydrol (FIG. 17) each 10. mu.l were precisely measured and injected into a liquid chromatograph, and chromatograms were recorded with 253nm and 210nm as detection wavelengths, and the resolution of impurities is shown in Table 16. The embodiment can effectively separate 7 impurities, the separation degree of the impurity B and the main peak reaches more than 1.5, the separation degree of the impurity nitrogen oxide and the main peak reaches more than 1.2, and the separation degrees of the rest impurities and the adjacent peaks can reach more than 2.0. The respective impurity contents were calculated based on the standard curve of example 1, and the results are shown in Table 17.
Example 5:
impurity stock solution: respectively taking a proper amount of 7 impurity standard substances in example 2, precisely weighing, and dissolving with methanolDissolving, fixing the volume, shaking up, and respectively preparing into 151.1 mug. mL-1150.2. mu.g.mL of the impurity A standard stock solution of (1)-1120.9. mu.g.mL of the impurity B standard stock solution of (1)-1121.4. mu.g.mL of the impurity D standard stock solution of (1)-1149.1. mu.g.mL of trans-1-cinnamyl piperazine stock solution-1122.6. mu.g.mL of the stock solution of 4, 4-difluorobenzophenone-1Stock solution of 4, 4-difluorobenzhydrol standard and 119.4. mu.g.mL-1The nitrogen oxide standard substance stock solution is used for standby.
Mixed solution of the test sample and each impurity standard substance: taking 0.96g of content of a commercially available flunarizine hydrochloride capsule, precisely weighing, placing in a 50mL measuring flask, precisely weighing a proper amount of each impurity stock solution in the same measuring flask, and preparing into a mixed solution of a sample and each impurity (the concentration of the flunarizine is 0.6mg/mL, the content of the impurity A, B and 4, 4-difluorobenzophenone is 3 mu g per 1mL, and the content of the other 4 impurities is 1.2 mu g per 1 mL).
Chromatographic conditions are as follows: hypersil Gold Thermo (4.6mm 100mm, 3.0 μm);
mobile phase A: as in example 2, the mobile phase B was methanol and the column temperature was 30 ℃ with a flow rate of 1.0 ml/min.
TABLE 13 gradient elution conditions
Other operations and conditions were the same as in example 2, 10. mu.l of a mixed solution of the sample and each impurity standard was precisely measured, and the mixed solution was injected into a liquid chromatograph, and a chromatogram was recorded with 253nm and 210nm as detection wavelengths, as shown in FIG. 18, and the impurity separation degree is shown in Table 16. The embodiment can effectively separate 7 impurities, the separation degree of the impurity B and the main peak reaches more than 1.5, and the separation degrees of the rest impurities and the adjacent peaks can reach more than 2.0.
Example 6:
mixed solution of the test sample and each impurity standard substance: the same as in example 5.
Chromatographic conditions are as follows: pursuit 3C 18(4.6mm 100mm, 3.0 μm);
mobile phase A: as in example 2, the mobile phase B was methanol and the column temperature was 30 ℃ with a flow rate of 1.0 ml/min.
TABLE 14 gradient elution conditions
Gradient elution was performed according to Table 14, and other operations and conditions were the same as in example 2, wherein 10. mu.l of a mixed solution of the sample and each impurity standard was precisely measured, and the mixed solution was injected into a liquid chromatograph, and 253nm and 210nm were used as detection wavelengths, and a chromatogram, as shown in FIG. 19, was recorded, and the degree of separation of impurities is shown in Table 16. The embodiment can effectively separate 7 impurities, the separation degree of the impurity B and the main peak reaches more than 1.5, and the separation degrees of the rest impurities and the adjacent peaks can reach more than 2.0.
Example 7:
mixed solution of the test sample and each impurity standard substance: the same as in example 4.
Chromatographic conditions are as follows: pursuit 3C 18(4.6mm 100mm, 3.0 μm);
mobile phase A: as in example 2, mobile phase B was acetonitrile and the column temperature was 30 ℃ and the flow rate was 1.0 ml/min.
TABLE 15 gradient elution conditions
Gradient elution was performed as shown in Table 15, otherwise the same procedure as in example 2 was carried out, and 10. mu.l of the mixed solution was precisely measured and injected into a liquid chromatograph, and the chromatogram was recorded with 253nm and 210nm as detection wavelengths, as shown in FIG. 20, and the degree of impurity separation as shown in Table 16. The embodiment can effectively separate 7 impurities, the separation degree of the impurity B and the main peak reaches more than 1.2, and the separation degrees of the rest impurities and the adjacent peaks can reach more than 1.5.
TABLE 16 degrees of separation of the impurities
TABLE 17 respective impurity contents
| Impurities | Example 2 | Example 3 | Example 4 |
| Trans-1-cinnamyl piperazine | 0.061％ | 0.19％ | 0.19％ |
| Impurity A | 0.073％ | 0.62％ | 0.61％ |
| 4, 4-Difluorobenzyl alcohol | Not detected out | 0.24％ | 0.23％ |
| 4, 4-difluorobenzophenone | 0.130％ | 0.47％ | 0.49％ |
| Impurity B | 0.135％ | 0.24％ | 0.46％ |
| Impurity D | Not detected out | 0.16％ | 0.18％ |
| Nitrogen oxides | Not detected out | 0.18％ | 0.18％ |
| Total miscellaneous | 0.40％ | 2.10％ | 2.34％ |
Claims (9)
1. A method for detecting related substances of a flunarizine hydrochloride preparation is characterized by comprising the following steps:
(1) solution preparation:
test solution: dissolving flunarizine hydrochloride sample in diluent, diluting to obtain solution containing flunarizine, filtering, and collecting filtrate as sample solution; the diluent is methanol and phosphate buffer solution with the pH value of 3.5 in a volume ratio of 75: 25;
control solution: precisely measuring a test solution, and diluting the test solution by 100 times by using a diluent to serve as a reference solution;
impurity a and 4, 4-difluorobenzhydrol mixed solution: accurately weighing an appropriate amount of impurity A and 4, 4-difluorobenzhydrol, and preparing a mixed solution of impurity A and 4, 4-difluorobenzhydrol by using a diluent, wherein each 1mL of the mixed solution contains 3 mu g of impurity A and 1.2 mu g of 4, 4-difluorobenzhydrol;
(2) and (3) high performance liquid chromatography detection: respectively taking a test solution, a reference solution and a mixed solution of impurities A and 4, 4-difluorobenzhydrol for high performance liquid chromatography detection, and respectively detecting the content of each impurity in the test solution at 210nm and 253 nm; wherein, the peaks of the impurity A and the 4, 4-difluoro benzhydryl alcohol are detected at 210nm, and the content of the impurity is calculated according to an external standard method; detecting other impurity peaks at 253nm, and calculating the impurity content according to a self-contrast method; the related substances of the flunarizine hydrochloride preparation are impurity A, impurity B, impurity D, trans-1-cinnamyl piperazine, 4, 4-difluorobenzophenone, 4, 4-difluorobenzhydrol and nitrogen oxide, wherein the impurity A is 1- [ bis- (4-fluorophenyl) methyl ] piperazine, the impurity B is 1- [ (4-fluorophenyl) phenylethyl ] -4- [ (2E) -3-phenylpropenyl ] piperazine, and the impurity D is 1- [ bis- (4-fluorophenyl) methyl ] -4- [ (2Z) -3-phenylpropenyl ] piperazine.
2. The method of claim 1, wherein the high performance liquid chromatography detection conditions are: using a chromatographic column C18, and using 0.01 mol.L of triethylamine with the volume concentration of 0.4% and the pH value of 3.3-3.7-1The phosphate buffer solution is used as a mobile phase A, methanol or acetonitrile is used as a mobile phase B, and gradient elution is carried out under the conditions that the column temperature is 30 ℃ and the flow rate is 0.8-1.2 mL/min.
3. The method of claim 2, wherein the gradient elution method is: at 0min, the mobile phase A is 70-80%, and the mobile phase B is 20-30%; at 14-20min, the mobile phase A is 20-30%, and the mobile phase B is 70-80%; at 22-25min, the mobile phase A is 20-30%, and the mobile phase B is 70-80%.
4. The method of claim 3, wherein the gradient elution method is: at 0min, the mobile phase A is 70%, and the mobile phase B is 30%; at 18min, the mobile phase A is 20% and the mobile phase B is 80%; at 25min, mobile phase a was 20% and mobile phase B was 80%.
5. The method of claim 2, wherein the C18 column is sized to have a length of 100mm, an internal diameter of 4.6mm, and a packing particle size of 3.0 μm.
6. The method of claim 2, wherein the flow rate is 1.0 mL/min.
7. The method of claim 2, wherein the phosphate buffered solution has a pH of 3.5.
8. The method of claim 2, wherein said mobile phase B is methanol.
9. The method of claim 1, wherein the concentration of flunarizine in the test solution is between 0.1 and 2.0 mg/ml.
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