CN114518413A - Method for measuring content of proline in captopril raw material medicine - Google Patents
Method for measuring content of proline in captopril raw material medicine Download PDFInfo
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- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 title claims abstract description 94
- FAKRSMQSSFJEIM-RQJHMYQMSA-N captopril Chemical compound SC[C@@H](C)C(=O)N1CCC[C@H]1C(O)=O FAKRSMQSSFJEIM-RQJHMYQMSA-N 0.000 title claims abstract description 64
- 229960000830 captopril Drugs 0.000 title claims abstract description 64
- 239000003814 drug Substances 0.000 title claims abstract description 59
- 239000002994 raw material Substances 0.000 title claims abstract description 56
- 238000000034 method Methods 0.000 title claims abstract description 30
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 claims abstract description 122
- 239000000243 solution Substances 0.000 claims abstract description 110
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 77
- 239000013558 reference substance Substances 0.000 claims abstract description 37
- 238000005303 weighing Methods 0.000 claims abstract description 37
- 239000012085 test solution Substances 0.000 claims abstract description 34
- 238000007865 diluting Methods 0.000 claims abstract description 20
- 229940079593 drug Drugs 0.000 claims abstract description 16
- 239000007788 liquid Substances 0.000 claims abstract description 16
- 239000012088 reference solution Substances 0.000 claims abstract description 11
- 238000003556 assay Methods 0.000 claims abstract description 10
- 238000002360 preparation method Methods 0.000 claims abstract description 10
- 238000010812 external standard method Methods 0.000 claims abstract description 9
- 229960002429 proline Drugs 0.000 claims description 119
- 229930182821 L-proline Natural products 0.000 claims description 29
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 26
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 24
- ONIBWKKTOPOVIA-SCSAIBSYSA-N D-Proline Chemical compound OC(=O)[C@H]1CCCN1 ONIBWKKTOPOVIA-SCSAIBSYSA-N 0.000 claims description 18
- 229930182820 D-proline Natural products 0.000 claims description 18
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 13
- 235000019253 formic acid Nutrition 0.000 claims description 13
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 11
- 239000012535 impurity Substances 0.000 claims description 10
- 238000010828 elution Methods 0.000 claims description 9
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 9
- 238000000889 atomisation Methods 0.000 claims description 8
- 239000000945 filler Substances 0.000 claims description 8
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical group CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 8
- 239000012488 sample solution Substances 0.000 claims description 8
- 239000000741 silica gel Substances 0.000 claims description 5
- 229910002027 silica gel Inorganic materials 0.000 claims description 5
- 239000007921 spray Substances 0.000 claims description 5
- 239000000377 silicon dioxide Substances 0.000 claims description 3
- 239000011550 stock solution Substances 0.000 description 24
- 239000000523 sample Substances 0.000 description 19
- 239000012071 phase Substances 0.000 description 18
- 238000011084 recovery Methods 0.000 description 14
- 238000012360 testing method Methods 0.000 description 14
- 239000012086 standard solution Substances 0.000 description 12
- 238000009210 therapy by ultrasound Methods 0.000 description 10
- 238000001514 detection method Methods 0.000 description 8
- 230000014759 maintenance of location Effects 0.000 description 8
- 238000005259 measurement Methods 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 5
- 238000001212 derivatisation Methods 0.000 description 5
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 4
- 239000002253 acid Substances 0.000 description 4
- 229910052500 inorganic mineral Inorganic materials 0.000 description 4
- 230000004807 localization Effects 0.000 description 4
- 239000011707 mineral Substances 0.000 description 4
- 238000011002 quantification Methods 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 238000012795 verification Methods 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 2
- 150000007513 acids Chemical class 0.000 description 2
- 239000000443 aerosol Substances 0.000 description 2
- 239000012490 blank solution Substances 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 206010020772 Hypertension Diseases 0.000 description 1
- 239000008186 active pharmaceutical agent Substances 0.000 description 1
- 150000001408 amides Chemical group 0.000 description 1
- 239000002220 antihypertensive agent Substances 0.000 description 1
- 229940127088 antihypertensive drug Drugs 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 229940088679 drug related substance Drugs 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 239000003595 mist Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 206010038464 renal hypertension Diseases 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 150000003335 secondary amines Chemical group 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N2030/042—Standards
- G01N2030/047—Standards external
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Abstract
The invention relates to a method for measuring the content of proline in captopril raw material medicines, which comprises the following steps: step 1, preparation of a test solution: taking a proper amount of captopril raw material medicine, precisely weighing, adding a proper amount of water, dissolving by ultrasonic waves, and quantitatively diluting by adding water to prepare a solution containing about 12mg of the captopril raw material medicine in each 1ml of the solution to be used as a test solution; step 2, preparing a reference substance solution: taking a proper amount of proline reference substance, precisely weighing, adding water to dissolve, and quantitatively diluting to obtain a solution containing about 12 μ g of proline in 1ml as reference substance solution; step 3, assay: and respectively injecting the reference solution and the test solution into a high performance liquid chromatograph by 20 mu l, recording a chromatogram, and calculating the proline content by peak area according to the chromatogram and an external standard method.
Description
The technical field is as follows:
the invention relates to an analysis and detection method of impurity components in chemical drugs, in particular to a content determination method of impurity component proline in antihypertensive drug captopril raw material drug.
Background art:
captopril was first developed and marketed by the company centurie meishi, precious in 1981. It is suitable for treating various types of hypertension, but not for renal hypertension. The chemical name of the L-proline is 1- [ (2S) -2-methyl-3-mercaptooxopropyl ] -L-proline, and the structural formula is as follows:
the preparation method of captopril comprises the following steps:
the captopril raw material medicine contains proline as an impurity component, and the source of the captopril raw material medicine can be analyzed as follows:
among them, L-proline is a starting material in the preparation of captopril as a raw material, and there is a possibility that the captopril formed may be hydrolyzed at the amide position to produce proline. In order to ensure the quality of the captopril raw material medicine product, the impurity component proline contained in the captopril raw material medicine is required to be detected.
Because the ultraviolet absorption of proline is very weak and the polarity is large, the proline cannot be directly and quantitatively determined, a derivatization method is generally adopted for indirect determination. During the derivatization reaction, the sulfhydryl group in captopril molecules has stronger nucleophilicity than secondary amine group in proline, and has great interference on the proline derivatization reaction, so the content of proline in captopril raw material medicine cannot be determined by a derivatization method.
The invention content is as follows:
the invention provides a method for detecting proline as an impurity component contained in captopril raw material medicines, which comprises the following steps:
step 1, preparation of a test solution:
taking a proper amount of raw materials of captopril, precisely weighing, adding a proper amount of water, dissolving by ultrasonic waves, and quantitatively diluting by adding water to prepare a solution containing 10-14mg of the raw materials in 1ml as a test solution;
step 2, preparing a reference substance solution:
taking a proper amount of proline reference substance, precisely weighing, adding water to dissolve, and quantitatively diluting to obtain a solution containing about 10-14 μ g of proline in 1ml as reference substance solution;
step 3, assay:
respectively injecting 10-30 μ l of the reference solution and the sample solution into a high performance liquid chromatograph, recording a chromatogram, and calculating the proline content according to the chromatogram and the peak area by an external standard method;
wherein, the chromatographic conditions of the high performance liquid chromatography are as follows: octadecylsilane bonded silica gel as a filler (Welch Ultimate XB-C18, 4.6 mm. times.150 mm, 5 μm); the column temperature is 25-35 ℃; the flow rate is 0.8-1.2ml per minute; the detector is a charged-aerosol detector (CAD); the atomization temperature is 30-40 ℃; eluting with 0.04-0.06% formic acid solution as mobile phase A and acetonitrile as mobile phase B in the following gradient elution manner;
preferably, the method of the invention is as follows:
step 1, preparation of a test solution:
taking a proper amount of captopril raw material medicine, precisely weighing, adding a proper amount of water, dissolving by ultrasonic waves, and quantitatively diluting by adding water to prepare a solution containing about 12mg of the captopril raw material medicine in each 1ml of the solution to be used as a test solution;
step 2, preparing a reference substance solution:
taking a proper amount of proline reference substance, precisely weighing, adding water to dissolve, and quantitatively diluting to obtain a solution containing about 12 μ g of proline in 1ml as a reference substance solution;
step 3, assay:
respectively injecting the reference solution and the test solution into a high performance liquid chromatograph by 20 mu l, recording a chromatogram, and calculating the proline content by peak area according to the chromatogram and an external standard method;
wherein the chromatographic conditions of the high performance liquid chromatography are as follows: octadecylsilane bonded silica gel as a filler (Welch Ultimate XB-C18, 4.6 mm. times.150 mm, 5 μm); the column temperature is 30 ℃; the flow rate was 1.0ml per minute; the detector is a charged-aerosol detector (CAD); the atomization temperature is 35 ℃; taking 0.05% formic acid solution as a mobile phase A and acetonitrile as a mobile phase B, and eluting in the following gradient elution mode;
wherein the proline comprises L-proline and D-proline, and the content of the proline is the sum of the L-proline and the D-proline.
The detection method is obtained by screening, and the screening process is as follows:
the proline in captopril bulk drug is directly measured by HPLC-CAD (high performance liquid chromatograph equipped with CAD detector, a novel universal detector, the detection of which is independent of the molecular structure of the compound and has been widely applied to the analysis of ultraviolet non-absorbing or weakly absorbing compounds). Using a conventional C18 chromatography column; since proline is weakly retained on a conventional C18 column, it was proposed to use an aqueous phase containing mineral acids (trifluoroacetic acid and formic acid were chosen) for 0-5min to increase the retention of proline on the column; other chromatographic conditions have less effect on proline retention and are therefore unchanged.
The results of optimizing the aqueous mobile phase are as follows:
TABLE 1 screening of the aqueous mobile phase
CAD detectors are easily contaminated with high concentrations of mineral acids, so low concentrations of formic acid, i.e., 0.05% formic acid in water, are used with satisfactory resolution.
Therefore, the method for determining and analyzing the proline content in the captopril raw material medicine comprises the following steps:
taking a proper amount of the product, precisely weighing, adding a proper amount of water, dissolving by ultrasonic treatment, and quantitatively diluting with water to obtain a solution containing about 12mg per 1ml as a test solution.
And taking a proper amount of proline reference substance, precisely weighing, adding water to dissolve, and quantitatively diluting to obtain a solution containing about 12 mu g of proline in 1ml as a reference substance solution.
Performing high performance liquid chromatography (China pharmacopoeia 2015 edition four-part general regulation 0512) test with octadecylsilane chemically bonded silica as filler (Welch Ultimate XB-C18, 4.6mm × 150mm, 5 μm); taking 0.05% formic acid solution as a mobile phase A and acetonitrile as a mobile phase B, and carrying out gradient elution according to the following table; the column temperature is 30 ℃; the flow rate was 1.0ml per minute; the detector is a charged-mist detector (CAD) and the atomization temperature is 35 ℃. Precisely measuring 20 μ l of each of the reference solution and the sample solution, respectively injecting into a liquid chromatograph, and recording chromatogram. If a peak consistent with the retention time of proline exists in the chromatogram of the test solution, the peak area is calculated according to an external standard method, and the peak area is not more than 0.10%.
TABLE 2 gradient elution conditions
| Time (minutes) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 100 | 0 |
| 5 | 100 | 0 |
| 6 | 60 | 40 |
| 10 | 60 | 40 |
| 11 | 100 | 0 |
| 15 | 100 | 0 |
TABLE 3 Instrument for test, column and reagent
And (3) verification of methodology:
1. specificity
(1) Blank solution: and (3) water.
(2) L-proline localization solution: taking about 12mg of L-proline, precisely weighing, placing in a 100ml measuring flask, adding water to dissolve and dilute to a scale, and shaking up to obtain an L-proline stock solution; precisely measuring 1ml, placing into a 10ml measuring flask, adding water to dilute to scale, and shaking up to obtain L-proline positioning solution.
(3) D-proline localization solution: taking about 12mg of D-proline, accurately weighing, placing in a 100ml measuring flask, adding water for dissolving, diluting to scale, and shaking up to obtain D-proline stock solution; precisely measuring 1ml, placing into a 10ml measuring flask, adding water to dilute to scale, and shaking up to obtain the D-proline positioning solution.
(4) Test solution: taking about 120mg of the captopril raw material medicine (batch number: 1810081), precisely weighing, placing in a 10ml measuring flask, adding a proper amount of water, performing ultrasonic treatment to dissolve, adding water to dilute to a scale, and shaking up to obtain a test solution.
(5) Adding a standard solution into a test sample: taking about 120mg of captopril raw material medicine (batch number: 1810081), precisely weighing, placing in a 10ml measuring flask, precisely adding 1ml of L-proline stock solution, adding a proper amount of water, performing ultrasonic dissolution, adding water to dilute to a scale, shaking up, and taking the scale added solution as a sample.
Precisely measuring the above solutions at a volume of 20 μ l each, injecting into a liquid chromatograph, and recording chromatogram.
TABLE 4 proline peak retention time and separation degree of each positioning solution and sample added standard solution
| Solutions of | Retention time/min | Degree of separation from adjacent peaks |
| L-proline localization solution | 2.032 | 3.98 |
| D-proline localization solution | 2.033 | 4.01 |
| Sample adding solution | 2.032 | 3.85 |
The results show that: the retention time of the L-proline peak is consistent with that of the D-proline peak, and the content of proline (the sum of L-proline and D-proline) in captopril raw material medicines is researched by taking the L-proline as a reference substance; the separation degree of the proline peak and the adjacent impurity peak is good; the blank solution and the test solution have no interference on proline determination; the method for determining and analyzing the proline content in the captopril raw material medicine has good specificity.
2. Linearity and range
Taking about 12mg of L-proline, accurately weighing, placing in a 100ml measuring flask, adding water to dissolve and dilute to a scale, shaking up, and using as a reference substance stock solution.
Precisely measuring the reference stock solutions 0.2ml, 0.5ml, 0.8ml, 1.0ml, 1.2ml and 1.5ml, respectively placing in 10ml measuring bottles, adding water to dilute to scale, shaking up to obtain 20%, 50%, 80%, 100%, 120% and 150% linear solutions respectively.
Each 20. mu.l of the linear solutions was measured precisely, and the solutions were injected into a liquid chromatograph, and the chromatogram was recorded, and the results are shown in Table 5.
TABLE 5 proline Linear assay results
| / | Concentration (μ g/ml) | Peak area |
| LOQ | 0.7999 | 1816612 |
| 20％ | 2.3998 | 7804764 |
| 50％ | 5.9994 | 19107575 |
| 80％ | 9.5990 | 29394869 |
| 100％ | 11.9988 | 36099353 |
| 120％ | 14.3986 | 41245502 |
| 150％ | 17.9982 | 49032825 |
The results show that proline is in the range of 0.7999. mu.g/ml to 17.9982. mu.g/ml, the regression equation: y 2762143x +1421643, correlation index: r is2The linear relationship is good at 0.9927.
3. Detection limit and quantification limit
(1) Limit of quantification
The limit of quantitation was determined by the signal-to-noise ratio method, with the corresponding concentration at a signal-to-noise ratio of about 10: 1. Precisely measuring 1ml of 100% linear solution under the linear term, placing the solution into a 15ml measuring flask, adding water to dilute the solution to a scale, and shaking up the solution to be used as a quantitative limiting solution. The concentration of the proline quantification limit solution was 0.7999. mu.g/ml, which is 6.67% of the limit.
Precisely measuring 20 mu l of limiting solution, repeatedly introducing sample for 6 times, recording chromatogram, and inspecting peak area RSD, wherein the results are shown in Table 6.
TABLE 6 measurement results of proline quantitation limit solution
The results showed that the proline quantitation limit was accurate.
(2) Detection limit
The detection limit is determined by a signal-to-noise ratio method, and is determined by the corresponding concentration when the signal-to-noise ratio is about 3: 1. Precisely measuring 3ml of proline quantitative limiting solution, placing in a 10ml measuring flask, diluting with water to scale, shaking up, and using as proline detection limiting solution (chromatogram shown in figure 3).
The concentration of the proline detection limit solution was 0.2400. mu.g/ml, which is 2.00% of the limit.
4. Precision degree
(1) Precision of the instrument
Taking about 12mg of L-proline, precisely weighing, placing in a 100ml measuring flask, adding water to dissolve and dilute to a scale, shaking up, and taking as a reference stock solution; precisely measuring 1ml, placing into a 10ml measuring flask, adding water to dilute to scale, and shaking to obtain a control solution.
Precisely measuring 20 μ l of the control solution, repeating the sample injection for 6 times, and examining the peak area of proline and the RSD of the retention time, wherein the results are shown in Table 7.
TABLE 7 results of instrumental precision tests
| Control solution | Retention time/min | Peak area |
| 1 | 2.033 | 36540462 |
| 2 | 2.033 | 36779409 |
| 3 | 2.033 | 36707081 |
| 4 | 2.033 | 35687852 |
| 5 | 2.032 | 35258853 |
| 6 | 2.032 | 35796583 |
| Mean value of | 2.033 | 36128373 |
| SD | 0.0005 | 630678 |
| RSD％ | 0.03 | 1.75 |
The result shows that the method for measuring and analyzing the proline content in the captopril raw material medicine has good instrument precision.
(2) Repeatability of
Control solution: taking about 12mg of L-proline, accurately weighing, placing in a 100ml measuring flask, adding water to dissolve and dilute to scale, shaking up, and taking as a reference substance stock solution; precisely measuring 1ml, placing into a 10ml measuring flask, adding water to dilute to scale, and shaking to obtain a control solution.
Test solution: taking about 120mg of the captopril raw material medicine (batch number: 1810081), precisely weighing, placing in a 10ml measuring flask, adding a proper amount of water, performing ultrasonic treatment to dissolve, adding water to dilute to a scale, and shaking up to obtain a test solution.
Adding a standard solution into a test sample: 120mg of the raw material medicine of captopril (batch number: 1810081) is precisely weighed and placed in a 10ml measuring flask, 1ml of the stock solution of the reference substance is precisely added, a proper amount of water is added, ultrasonic treatment is carried out to dissolve the raw material medicine, the raw material medicine is diluted to a scale by adding water and shaken up to be used as a test sample and added with a standard solution (6 parts are prepared in parallel).
Precisely measuring 20 μ l of each solution, injecting into a liquid chromatograph, recording chromatogram, and observing RSD of proline content in the sample solution, wherein the results are shown in Table 8.
TABLE 8 results of reproducibility measurement
| Sample adding solution | Proline content% |
| 1 | 0.0998 |
| 2 | 0.0991 |
| 3 | 0.0998 |
| 4 | 0.0978 |
| 5 | 0.0973 |
| 6 | 0.0982 |
| Mean value of | 0.0987 |
| SD | 0.001 |
| RSD％ | 1.08 |
The result shows that the method for measuring and analyzing the proline content in the captopril raw material medicine has good repeatability.
(3) Intermediate precision
Control solution: taking about 12mg of L-proline, precisely weighing, placing in a 100ml measuring flask, adding water to dissolve and dilute to a scale, shaking up, and taking as a reference stock solution; precisely measuring 1ml, placing into a 10ml measuring flask, adding water to dilute to scale, and shaking to obtain a control solution.
Test solution: taking about 120mg of the captopril raw material medicine (batch number: 1810081), precisely weighing, placing in a 10ml measuring flask, adding a proper amount of water, performing ultrasonic treatment to dissolve, adding water to dilute to a scale, and shaking up to obtain a test solution.
Adding a standard solution into a test sample: 120mg of the raw material medicine of captopril (batch number: 1810081) is precisely weighed and placed in a 10ml measuring flask, 1ml of the stock solution of the reference substance is precisely added, a proper amount of water is added, ultrasonic treatment is carried out to dissolve the raw material medicine, the raw material medicine is diluted to a scale by adding water and shaken up to be used as a test sample and added with a standard solution (6 parts are prepared in parallel).
Different personnel precisely measure 20 mul of the solution on different dates, inject the solution into a liquid chromatograph, and record chromatogram. The RSD of the proline content of 12 sample solutions (including 6 sample solutions under the repeatability test) was examined. The results are shown in Table 9.
TABLE 9 results of intermediate precision measurement
The result shows that the intermediate precision of the determination and analysis method for the proline content in the captopril raw material medicine is good.
5. Accuracy of
Reference stock solution: taking about 12mg of L-proline, accurately weighing, placing in a 100ml measuring flask, adding water to dissolve and dilute to scale, shaking up, and using as a reference stock solution.
Control solution: precisely measuring 1ml of the reference stock solution, placing in a 10ml measuring flask, adding water to dilute to scale, and shaking to obtain the reference solution.
Test solution: test solution: taking about 120mg of the captopril raw material medicine (batch number: 1810081), precisely weighing, placing in a 10ml measuring flask, adding a proper amount of water, performing ultrasonic treatment to dissolve, adding water to dilute to a scale, and shaking up to obtain a test solution.
10% recovery from spiked solution: taking about 120mg of captopril bulk drug (batch number: 1810081), accurately weighing, placing in a 10ml measuring flask, accurately adding 0.1ml of reference substance stock solution, adding a proper amount of water, dissolving by ultrasonic, adding water to dilute to a scale, shaking up, and preparing three parts in parallel as 10% standard recovery rate solution.
Recovery of 80% spiked solution: taking about 120mg of captopril bulk drug (batch number: 1810081), accurately weighing, placing in a 10ml measuring flask, accurately adding 0.8ml of reference substance stock solution, adding a proper amount of water, dissolving by ultrasonic, adding water to dilute to a scale, shaking up, and preparing three parts in parallel as 80% solution with standard recovery rate.
100% recovery solutions with standard addition: 120mg of raw captopril (batch number: 1810081) is precisely weighed and placed in a 10ml measuring flask, 1.0ml of reference stock solution is precisely added, a proper amount of water is added, ultrasonic treatment is carried out to dissolve the raw captopril, the mixture is diluted to a scale by adding water and shaken up to be used as a 100 percent solution with standard recovery rate, and three parts are prepared in parallel.
Recovery of 120% spiked solution: 120mg of the raw material medicine of captopril (batch number: 1810081) is precisely weighed and placed in a 10ml measuring flask, 1.2ml of the stock solution of the reference substance is precisely added, a proper amount of water is added, ultrasonic treatment is carried out to dissolve the stock solution, water is added to dilute the stock solution to a scale, the stock solution is shaken up to be used as a 120 percent solution with the standard recovery rate, and three parts are prepared in parallel.
Precisely measuring the reference substance solution, the test substance solution, the 10% standard adding recovery rate solution, the 80% standard adding recovery rate solution, the 100% standard adding recovery rate solution and the 120% standard adding recovery rate solution by 20 μ l respectively, injecting into a liquid chromatograph, recording chromatogram, and inspecting the proline recovery rate average value and RSD of each standard adding recovery rate solution. The results are shown in Table 10.
TABLE 10 accuracy measurement results
The result shows that the method for determining and analyzing the proline content in the captopril raw material medicine has good accuracy.
6. Durability
Control solution: taking about 12mg of L-proline, precisely weighing, placing in a 100ml measuring flask, adding water to dissolve and dilute to a scale, shaking up, and taking as a reference stock solution; precisely measuring 1ml, placing into a 10ml measuring flask, adding water to dilute to scale, and shaking to obtain a control solution.
Test solution: taking about 120mg of the captopril raw material medicine (batch number: 1810081), precisely weighing, placing in a 10ml measuring flask, adding a proper amount of water, performing ultrasonic treatment to dissolve, adding water to dilute to a scale, and shaking up to obtain a test solution.
Adding a standard solution into a test sample: taking about 120mg of captopril bulk drug (batch number: 1810081), precisely weighing, placing in a 10ml measuring flask, precisely adding 1ml of reference substance stock solution, adding appropriate amount of water, dissolving by ultrasonic, adding water to dilute to scale, shaking up, and using as a sample and adding standard solution.
The results of examining chromatographic columns of different flow rates of 1.0 +/-0.1 ml/min, different column temperatures of 35 +/-2 ℃ and different batches are shown in Table 11, wherein the RSD% of the proline content of the test sample added with the standard solution is obtained.
TABLE 11 durability measurement results
The result shows that the durability of the method for measuring and analyzing the proline content in the captopril raw material medicine is good.
7. Stability of solution
Control solution: taking about 12mg of L-proline, precisely weighing, placing in a 100ml measuring flask, adding water to dissolve and dilute to a scale, shaking up, and taking as a reference stock solution; precisely measuring 1ml, placing into a 10ml measuring flask, adding water to dilute to scale, and shaking to obtain a control solution.
Adding a standard solution into a test sample: taking about 120mg of captopril bulk drug (batch number: 1810081), precisely weighing, placing in a 10ml measuring flask, precisely adding 1ml of reference substance stock solution, adding appropriate amount of water, dissolving by ultrasonic, adding water to dilute to scale, shaking up, and using as a sample and adding standard solution.
Precisely measuring the reference solution and the sample standard solution by 20 μ l each, injecting into a liquid chromatograph, recording chromatogram, and observing the peak area RSD of proline for 24h, the results are shown in Table 12.
TABLE 12 results of solution stability measurements
The result shows that the solution has good stability for 24 hours by the method for measuring and analyzing the proline content in the captopril raw material medicine.
8. Verification summary of method for determining and analyzing proline content in captopril raw material medicine
TABLE 13 verification summary of proline content determination analysis method in captopril drug substance
9. Determination of proline content in captopril raw material medicine
The proline content in 8 batches of captopril raw material medicines was determined according to the method for determining and analyzing the proline content in captopril raw material medicines, and the results are shown in table 14.
TABLE 14 proline assay results
The results show that proline (the sum of L-proline and D-proline) in 8 batches of captopril raw material medicines is not detected.
The method provided by the invention has the following advantages:
1. based on that ultraviolet absorption of proline is very weak and polarity is large, the invention adopts a derivatization method to briefly determine proline, wherein:
depending on the sensitivity, the mineral acid was chosen, and to avoid contamination of the CAD detector with high concentrations of mineral acid, a 0.05% aqueous formic acid solution was finally used.
2. The method provided by the invention has good specificity, linear relation, quantitative limit precision, analytical method instrument precision, repeatability, intermediate precision, accuracy, durability, solution 24h stability and the like.
Description of the drawings:
FIG. 1: chromatogram with 0.5% trifluoroacetic acid as mobile phase;
FIG. 2: chromatogram with 0.05% formic acid as mobile phase;
FIG. 3: a linear plot of proline;
FIG. 4 is a schematic view of: a control chromatogram;
FIG. 5: chromatogram of the test sample.
The specific implementation mode is as follows:
the invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto.
Example 1: method for detecting impurity component proline contained in captopril raw material medicine
Step 1, preparation of a test solution:
taking a proper amount of captopril raw material medicine, precisely weighing, adding a proper amount of water, dissolving by ultrasonic waves, and quantitatively diluting by adding water to prepare a solution containing about 12mg of the captopril raw material medicine in each 1ml of the solution to be used as a test solution;
step 2, preparing a reference substance solution:
taking a proper amount of proline reference substance, precisely weighing, adding water to dissolve, and quantitatively diluting to obtain a solution containing about 12 μ g of proline in 1ml as a reference substance solution;
step 3, assay:
injecting 20 μ l of each of the reference solution and the sample solution into a high performance liquid chromatograph, recording chromatograms (the chromatogram of the reference and the chromatogram of the sample are respectively shown in fig. 4 and fig. 5), and calculating the proline content according to the chromatograms by peak area according to an external standard method;
wherein, the chromatographic conditions of the high performance liquid chromatography are as follows: octadecylsilane bonded silica gel as a filler (Welch Ultimate XB-C18, 4.6 mm. times.150 mm, 5 μm); the column temperature is 30 ℃; the flow rate was 1.0ml per minute; the detector is a spray detector (CAD); the atomization temperature is 35 ℃; taking 0.05% formic acid solution as a mobile phase A and acetonitrile as a mobile phase B, and eluting in the following gradient elution mode;
wherein the proline comprises L-proline and D-proline, and the content of the proline is the sum of the L-proline and the D-proline.
Example 2: method for detecting proline serving as impurity component in captopril bulk drug
Step 1, preparation of a test solution:
taking a proper amount of captopril raw material medicine, precisely weighing, adding a proper amount of water, dissolving by ultrasonic waves, and quantitatively diluting by adding water to prepare a solution containing 14mg of the captopril raw material medicine per 1ml, wherein the solution is used as a test solution;
step 2, preparing a reference substance solution:
taking a proper amount of proline reference substance, precisely weighing, adding water to dissolve, and quantitatively diluting to obtain a solution containing about 14 μ g of proline in 1ml as a reference substance solution;
step 3, assay:
respectively injecting 30 mu l of reference solution and 30 mu l of test solution into a high performance liquid chromatograph, recording a chromatogram, and calculating the proline content by peak area according to the chromatogram and an external standard method;
wherein the chromatographic conditions of the high performance liquid chromatography are as follows: octadecylsilane chemically bonded silica was used as a filler (Welch Ultimate XB-C18, 4.6 mm. times.150 mm, 5 μm); the column temperature was 35 ℃; the flow rate was 1.2ml per minute; the detector is a spray detector (CAD); the atomization temperature is 40 ℃; taking 0.06% formic acid solution as a mobile phase A and acetonitrile as a mobile phase B, and eluting in the following gradient elution mode;
wherein the proline comprises L-proline and D-proline, and the content of the proline is the sum of the L-proline and the D-proline.
Example 3: method for detecting proline serving as impurity component in captopril bulk drug
Step 1, preparation of a test solution:
taking a proper amount of captopril raw material medicine, precisely weighing, adding a proper amount of water, dissolving by ultrasonic waves, and quantitatively diluting by adding water to prepare a solution containing about 10mg of the captopril raw material medicine per 1ml, wherein the solution is used as a test solution;
step 2, preparing a reference substance solution:
taking a proper amount of proline reference substance, precisely weighing, adding water to dissolve, and quantitatively diluting to obtain a solution containing about 10 μ g of proline in 1ml as a reference substance solution;
step 3, assay:
respectively injecting 10 mu l of reference solution and test solution into a high performance liquid chromatograph, recording a chromatogram, and calculating the proline content by peak area according to the chromatogram and an external standard method;
wherein, the chromatographic conditions of the high performance liquid chromatography are as follows: octadecylsilane bonded silica gel as a filler (Welch Ultimate XB-C18, 4.6 mm. times.150 mm, 5 μm); the column temperature was 25 ℃; the flow rate was 0.8ml per minute; the detector is a spray detector (CAD); the atomization temperature is 30 ℃; taking 0.04% formic acid solution as a mobile phase A and acetonitrile as a mobile phase B, and eluting in the following gradient elution mode;
wherein the proline comprises L-proline and D-proline, and the content of the proline is the sum of the L-proline and the D-proline.
Claims (3)
1. A method for measuring the content of proline as an impurity component in captopril raw material medicines is a high performance liquid chromatography and specifically comprises the following steps:
step 1, preparation of a test solution:
taking a proper amount of raw materials of captopril, precisely weighing, adding a proper amount of water, dissolving by ultrasonic waves, and quantitatively diluting by adding water to prepare a solution containing 10-14mg of the raw materials in 1ml as a test solution;
step 2, preparing a reference substance solution:
taking a proper amount of proline reference substance, precisely weighing, adding water to dissolve, and quantitatively diluting to obtain a solution containing 10-14 μ g of proline in 1ml as reference substance solution;
step 3, assay:
respectively injecting 10-30 μ l of the reference solution and the sample solution into a high performance liquid chromatograph, recording a chromatogram, and calculating the proline content according to the chromatogram and the peak area by an external standard method;
wherein, the chromatographic conditions of the high performance liquid chromatography are as follows: octadecylsilane bonded silica gel as a filler (Welch Ultimate XB-C18, 4.6 mm. times.150 mm, 5 μm); the column temperature is 25-35 ℃; the flow rate is 0.8-1.2ml per minute; the detector is a spray detector (CAD); the atomization temperature is 30-40 ℃; eluting with 0.04-0.06% formic acid solution as mobile phase A and acetonitrile as mobile phase B in the following gradient elution manner;
2. the method according to claim 1, characterized in that it comprises the following steps:
step 1, preparation of a test solution:
taking a proper amount of captopril raw material medicine, precisely weighing, adding a proper amount of water, dissolving by ultrasonic waves, and quantitatively diluting by adding water to prepare a solution containing 12mg of the captopril raw material medicine in each 1ml of the solution to serve as a test solution;
step 2, preparing a reference substance solution:
taking a proper amount of proline reference substance, precisely weighing, adding water to dissolve, and quantitatively diluting to obtain a solution containing 12 μ g of proline in 1ml as a reference substance solution;
step 3, assay:
respectively injecting the reference solution and the sample solution into a high performance liquid chromatograph by 20 mu l, recording a chromatogram, and calculating the proline content by peak area according to the chromatogram and an external standard method;
wherein, the chromatographic conditions of the high performance liquid chromatography are as follows: octadecylsilane chemically bonded silica was used as a filler (Welch Ultimate XB-C18, 4.6 mm. times.150 mm, 5 μm); the column temperature is 30 ℃; the flow rate was 1.0ml per minute; the detector is a spray detector (CAD); the atomization temperature is 35 ℃; taking 0.05% formic acid solution as a mobile phase A and acetonitrile as a mobile phase B, and eluting in the following gradient elution mode;
3. the method of claim 1, wherein the proline comprises L-proline and D-proline in the total amount of L-proline and D-proline.
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