CN114791469A - Method for detecting content of vitamin C in famotidine injection - Google Patents
Method for detecting content of vitamin C in famotidine injection Download PDFInfo
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- CN114791469A CN114791469A CN202210439412.XA CN202210439412A CN114791469A CN 114791469 A CN114791469 A CN 114791469A CN 202210439412 A CN202210439412 A CN 202210439412A CN 114791469 A CN114791469 A CN 114791469A
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Abstract
The invention discloses a method for detecting the content of vitamin C in famotidine injection, which adopts high performance liquid chromatography for detection and specifically comprises the following steps: a. preparing two parts of test solution; b. preparing a test solution; c. preparing a reference substance solution; d. and respectively sucking the reference solution and the test solution, injecting the reference solution and the test solution into a liquid chromatograph, and recording the chromatogram. The invention can detect the vitamin C in the famotidine injection by specific sample solvent and chromatographic conditions, has accurate result, rapidness and high sensitivity, and provides a new method for the quality control of the famotidine injection containing the vitamin C.
Description
Technical Field
The invention relates to the field of medicine detection, in particular to a method for detecting the content of vitamin C in famotidine injection.
Background
Famotidine injection belongs to gastric acid secretion inhibiting medicine, and famotidine as the effective component is histamine H2 receptor retardant, has obvious gastric acid inhibiting effect, can inhibit pepsin secretion and has protecting effect on experimental gastric ulcer, so that it has curative effect on gastritis caused by gastric acid and related ulcer.
The famotidine as main medicine component in famotidine injection is oxygen sensitive matter and easy to oxidize, and through adding vitamin C as one other kind of sensitive matter, it can compete with famotidine for oxygen to protect the medicine component famotidine. However, since vitamin C is unstable in famotidine injection and is difficult to be effectively separated, the quality of famotidine injection added with vitamin C is not easy to control.
Disclosure of Invention
In order to solve the problems, the invention provides a method for detecting the content of vitamin C in famotidine injection, which comprises the following steps:
a. preparation of control solutions: dissolving vitamin C reference substance in solvent to obtain solution;
b. preparation of a test solution: taking famotidine injection, and adding a solvent to dilute by 3-5 times to obtain the famotidine injection;
c. respectively sucking the reference substance solution and the famotidine injection to be injected into a liquid chromatograph, wherein the chromatographic conditions are as follows:
a chromatographic column: octadecylsilane chemically bonded silica is used as a filling agent; mobile phase: taking a potassium dihydrogen phosphate solution as a mobile phase A and taking an acetonitrile water solution as a mobile phase B; sample injector temperature: 4 ℃;
the gradient elution procedure was:
d. the vitamin C content was calculated as follows:
wherein: a. the For supplying to : the peak area of the main peak in the test solution; v For supplying to : diluting the sample solution by multiple; marking the loading amount: the volume of famotidine injection; marking amount: the famotidine content in the famotidine injection;
the average value of the mass concentration and peak area ratios of a plurality of reference substance solutions;
the solvent is an aqueous solution containing disodium ethylene diamine tetraacetate and monopotassium phosphate;
further, the mass-to-volume ratio of the vitamin C reference substance to the solvent in the step a is 5 mg: 20 ml.
Further, the volume ratio of the famotidine injection in the step b to the solvent is 2.5 ml: 10 ml.
Furthermore, in the chromatographic conditions in the step C, a chromatographic column is GL Inertsutasin C18, 250 multiplied by 4.6mm and 5 mu m, the wavelength is 200-250 nm, the column temperature is 25-40 ℃, the sample injector temperature is 4 ℃, and the sample injection amount is 5-25 mu l.
Further, the column temperature was 30 ℃, the wavelength was 210nm, and the amount of sample was 5. mu.l.
Further, the solvent is an aqueous solution containing 0.3-0.67 mg of disodium ethylenediaminetetraacetate and 1.0-3.0 mg of monopotassium phosphate per 1ml of pH value of 2.0-4.0.
Further, the solvent is an aqueous solution containing 0.56mg of disodium ethylenediaminetetraacetate and 2.0mg of monopotassium phosphate per 1ml at a pH of 3.0.
Further, the mobile phase A is 1-5 mg/mL potassium dihydrogen phosphate solution; the mobile phase B is 70-80% of an acetonitrile water solution of ml/ml.
Further, the mobile phase A is 2.04mg/mL potassium dihydrogen phosphate solution; the mobile phase B is 73 percent of aqueous acetonitrile solution with ml/ml.
Further, step d is as described
The mass concentration and peak area of two reference substance solutionsThe average value of the ratio is specifically represented by the following formula:
wherein A is To pair : peak area of main peak in the reference solution; m is To pair : control weight, mg; v For is to : dilution times of reference solutions; p: content of control.
Furthermore, the famotidine injection comprises the ingredients of famotidine, nicotinamide, vitamin C, D-mannitol, lactic acid, sodium hydroxide and water for injection.
Detection and analysis tests show that the accuracy and precision of the famotidine injection containing vitamin C in the components are poor through high performance liquid chromatography normal temperature sample injection detection, and the content of the vitamin C is lower than the actual addition amount by 2-5%. The method for detecting the content of the vitamin C in the famotidine injection can detect the vitamin C in the famotidine injection through a specific sample solvent and a specific chromatographic condition, has accurate result, high speed and high sensitivity, provides a new method for the quality control of the famotidine injection containing the vitamin C, provides a method basis for improving the quality control standard of the famotidine injection, provides a reference basis for expanding the clinical application of the famotidine injection, and has good application prospect.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1 solvent blank chromatogram
FIG. 2 chromatogram of undegraded blank adjuvant solution
FIG. 3 chromatogram of undegraded test solution
FIG. 4 vitamin control solution chromatogram
FIG. 5 chromatogram of blank adjuvant solution with strong oxidative degradation
FIG. 6 chromatogram of strongly oxidative degradation of test sample solution
FIG. 7 chromatogram of blank adjuvant solution for strong acid degradation
FIG. 8 chromatogram of strongly acidic-degraded test solution
FIG. 9 chromatogram of strong base degradation blank adjuvant solution
FIG. 10 chromatogram of strongly alkaline degradation test solution
FIG. 11 chromatogram of high temperature degradation blank adjuvant solution
FIG. 12 chromatogram of high temperature degradation test solution
FIG. 13 chromatogram of blank adjuvant solution with strong light degradation
FIG. 14 chromatogram of strongly light-degraded test solution
FIG. 15 vitamin C Linearity plot
Detailed Description
EXAMPLE 1 determination of vitamin C content in Famotidine injection
a. Preparation of control solutions: precisely weighing 5mg of vitamin C reference substance, placing the reference substance in a 20ml measuring flask, dissolving the reference substance in an aqueous solution with the pH value of 3.0 and containing 0.56mg of disodium ethylene diamine tetraacetate and 2.0mg of monopotassium phosphate per 1ml, and diluting the solution to a scale to obtain the vitamin C reference substance, wherein 2 reference substance solutions are prepared in parallel.
b. Test solution: 2.5ml of famotidine injection is precisely measured, placed in a 10ml measuring flask, diluted to scale by using an aqueous solution with the pH value of 3.0 and containing 0.56mg of disodium ethylene diamine tetraacetate and 2.0mg of monopotassium phosphate per 1ml, and shaken up.
c. Respectively sucking 5 mul of reference substance solution and famotidine injection, injecting the reference substance solution and the famotidine injection into a liquid chromatograph, recording a chromatogram, and obtaining the following chromatographic conditions:
a chromatographic column: GL Inertsutasin C18, 250X 4.6mm, 5 μm; detection wavelength: 210 nm; column temperature: 30 ℃; mobile phase: taking 2.04mg/mL potassium dihydrogen phosphate solution as a mobile phase A and 73% acetonitrile water solution as a mobile phase B; sample injector temperature: 4 ℃; the gradient elution procedure was:
| time/min | Flow rate/ml/min | Mobile phase/A% | Mobile phase/B% |
| 0 | 0.6 | 100 | 0 |
| 15 | 0.6 | 100 | 0 |
| 19 | 1.5 | 0 | 100 |
| 29 | 1.5 | 0 | 100 |
| 31 | 0.6 | 100 | 0 |
| 38 | 0.6 | 100 | 0 |
d. Vitamin C of famotidine injection was calculated as follows:
wherein: a. the To pair : peak area of main peak in the reference solution; m is a unit of To pair : control weight, mg;
V to pair : control solution dilution factor (20); p: the content of the reference substance; a. the For supplying to : the peak area of the main peak in the test solution; v For supplying to : the dilution ratio of the test solution is 4; marking the loading amount: the volume of famotidine injection; marking amount: the famotidine content in the famotidine injection;
the advantageous effects of the present invention are explained below by way of test examples
Test example 1 methodological Studies
1. Famotidine injection for verification
Famotidine injection prescription (labeling volume 1mL)
The preparation process of the famotidine injection comprises the following steps:
(1) dissolving main medicine and auxiliary materials
Adding water for injection into a 500L thick preparation tank, starting nitrogen, filling nitrogen until the headspace residual oxygen amount of the preparation tank is less than 2.0%, and controlling the temperature of the water for injection to be 40-50 ℃ for later use.
Measuring 40-50 ℃ nitrogen-filled water for injection, placing the water for injection into a diluting preparation tank, setting the stirring frequency to be 30 +/-2 Hz, starting stirring, and filling nitrogen until the headspace residual oxygen amount of the preparation tank is less than 2.0%. Sequentially adding lactic acid, nicotinamide, vitamin C, mannitol and famotidine (operation in dark place), filling nitrogen again until the headspace residual oxygen amount of the preparation tank is less than 2.0%, stirring for 30min until the solution is clarified, and starting small circulation for 5 min.
(2) Adjusting the pH
Weighing water for injection after charging nitrogen, placing in a stainless steel barrel, adding sodium hydroxide, stirring to dissolve completely, charging nitrogen again for 5min to obtain 0.2M sodium hydroxide solution. And adding a sodium hydroxide solution into the preparation tank, and adjusting the pH of the solution to 5.8-6.2.
(3) Constant volume
Charging nitrogen at below 35 deg.C, adding water for injection to desired volume, and circularly stirring for 5 min. And in the process, nitrogen is continuously filled into the tank, and the headspace residual oxygen of the liquid preparation tank is ensured to be less than 2.0%.
2. Authenticating content
Specificity of vitamin C assay, system precision, solution stability, linearity and range, accuracy, precision (repeatability and intermediate precision), durability.
3. Validation acceptability criteria
TABLE 1 validation acceptability criteria
4. Implementation step of verification
4.1. Instrument arrangement
TABLE 2 Instrument configuration
4.2. Verifying the reagents and controls used
TABLE 3 reagent and control
| Name(s) | Specification of | Batch number | Content (wt.) | Suppliers of goods |
| Potassium dihydrogen phosphate | 500 g/bottle | 20180308 | —— | Group of traditional Chinese medicines |
| Acetonitrile | 4L/bottle | 3JH0025 | —— | Spzctaum |
| |
500 ml/bottle | H2020219 | —— | Aladdin |
| Ethylenediaminetetraacetic acid disodium salt | 250 g/bottle | 20200401 | —— | Chongqing Chuandong chemical industry |
| Vitamin |
100 mg/bottle | 100425-201504 | 100％ | Middle school |
| Famotidine injection | 2ml；20mg | 210831 | —— | Sichuan Taiji medicine |
| Purified water | —— | —— | —— | Self-made |
| |
500 ml/bottle | 20180401 | —— | Chongqing Chuandong chemical industry |
| Sodium hydroxide (NaOH) | 500 g/bottle | 20180207 | —— | Wolk & |
| 30 |
500 ml/bottle | 20190501 | —— | Chongqing Chuandong chemical industry |
4.3. Analysis method to be verified
4.3.1. Conditions of the experiment
Detection wavelength: 210 nm; a chromatographic column: GL Inertsutasin C18, 250X 4.6mm, 5 μm; column temperature: 30 ℃; temperature of a sample injection tray: 4 ℃; sample introduction amount: 5 mul; flow rate: 0.6-1.5 ml/min; a mobile phase A: potassium dihydrogen phosphate solution (2.04 g potassium dihydrogen phosphate is dissolved in 1000ml water, and pH is adjusted to 3.0 with phosphoric acid); and (3) fluidity B: 73% acetonitrile; needle washing solution: 0.2% phosphoric acid; gradient elution:
4.3.2. solution preparation
Solvent: disodium ethylenediaminetetraacetate 0.56g and potassium dihydrogen phosphate 2.0g were weighed, dissolved by adding 1000ml of purified water, and the pH was adjusted to 3.0 with phosphoric acid.
Control solution: the vitamin C reference substance of about 12.5mg is precisely weighed and placed in a 50ml measuring flask, dissolved by a solvent and diluted to the scale mark. 2 parts are prepared in parallel.
Test solution: 2.5ml of famotidine injection is precisely measured, placed in a 10ml measuring flask, diluted to scale by a solvent and shaken up. 2 parts are prepared in parallel.
4.3.3. System applicability requirement
In the chromatogram of the test solution, the separation degree of a vitamin C peak and adjacent peaks is more than 1.5, the tailing factor of the vitamin C peak is not more than 1.5, and the theoretical plate number is not less than 5000 according to the vitamin C peak.
4.3.4. Measurement of
The blank (solvent), the reference solution and the sample solution are respectively 5 mul injected into a chromatograph, and the chromatogram is recorded.
TABLE 4 sample introduction needle number setting
4.3.5. Formula for calculation
The vitamin C of the test article is calculated according to the following formula:
wherein: a. the For is to : the peak area of the main peak in the reference solution;
m to pair : control weight, mg;
V to pair : dilution times of reference solutions;
p: the content of the reference substance;
A for supplying to : the peak area of the main peak in the test solution;
V for supplying to : diluting the sample solution by multiple times;
4.3.6. standard provisions
Contains vitamin C (C) 6 H 8 O 6 ) Should be 80.0% -110.0% of the labeled amount, and the RSD should be no more than 2.0%.
5. Method verification
5.1. Specificity test
5.1.1. Interference test
Preparation of a solution:
solvent: 0.56g of disodium ethylenediaminetetraacetate and 2.0g of monopotassium phosphate are weighed, 1000ml of purified water is added for dissolution, and the pH value is adjusted to 3.0 by using phosphoric acid.
Blank solvent: solvent(s)
Blank adjuvant solution: precisely measuring 2.5ml of blank adjuvant solution (prepared from nicotinamide about 500mg, D-mannitol about 200mg, and lactic acid about 35mg, dissolved with water 10 ml), placing in a 10ml measuring flask, diluting with solvent to scale, and shaking.
Vitamin C localization solution: precisely weighing about 12.5mg of vitamin C as reference substance, placing in a 50ml measuring flask, dissolving with solvent, and diluting to scale to obtain reference solution.
Test solution: 2.5ml of famotidine injection is precisely measured, placed in a 10ml measuring flask, diluted to scale by a solvent and shaken up.
Measuring method:
precisely measuring the blank and blank auxiliary material solutions, the reference substance solution and the test solution by 5 μ l respectively, injecting into a liquid chromatograph, and recording chromatogram.
Measuring results:
the chromatogram results are shown in figures 1-4, and the specific chromatogram analysis results are shown in table 5.
TABLE 5 interference test results
Fourthly, conclusion:
the blank solvent and the blank auxiliary materials do not interfere with the determination of the vitamin C; in the chromatogram of the test solution, the separation degree of a vitamin C peak and adjacent peaks is 3.4 and more than 1.5, the tailing factor is 1.1 and less than 1.5, and the theoretical plate number is 15993 and more than 5000 according to the vitamin C peak.
5.1.2. Forced degradation test
Preparation of a solution:
blank: solvent(s)
Blank adjuvant solution: precisely measuring 2.5ml of blank adjuvant solution (prepared from nicotinamide about 500mg, D-mannitol about 200mg, and lactic acid about 35mg, dissolved in water 10 ml), placing in a 10ml measuring flask, diluting with solvent to scale, and shaking.
(1) Strong oxidative degradation
Precisely measuring 2.5ml of famotidine injection, putting the famotidine injection into a 10ml measuring flask, adding 0.03% hydrogen peroxide solution 0.5ml, uniformly mixing, standing for 10min, diluting to a scale with a solvent, and shaking uniformly to obtain a strong oxidative degradation test solution; precisely measuring 2.5ml of blank auxiliary material solution, placing the blank auxiliary material solution into a 10ml measuring flask, and operating the same method to obtain the blank auxiliary material solution for strong oxidative degradation.
(2) Degradation by strong base
Precisely measuring 2.5ml of famotidine injection, putting the famotidine injection into a 10ml measuring flask, adding 0.3ml of 0.1mol/L sodium hydroxide solution, heating in water bath at 40 ℃ for 10 minutes, then adding 0.3ml of 0.1mol/L hydrochloric acid solution, diluting the solution to a scale by using a solvent, shaking up, and degrading a sample solution as strong base; precisely measuring 2.5ml of blank auxiliary material solution, placing the blank auxiliary material solution in a 10ml measuring flask, and performing the same operation to obtain the blank auxiliary material solution for strong alkali degradation.
(3) High temperature degradation
Taking a proper amount of famotidine injection, placing the famotidine injection in a drying oven at 60 ℃ for 4 hours, precisely measuring 2.5ml, placing the famotidine injection in a 10ml measuring flask, diluting the famotidine injection to a scale by using a solvent, and shaking up the famotidine injection to be used as a high-temperature degradation test solution; and taking the blank auxiliary material solution, and performing the same operation as the method to obtain the blank auxiliary material solution for high-temperature degradation.
(4) Degradation by strong acid
Precisely measuring 2.5ml of famotidine injection, placing the famotidine injection into a 10ml measuring flask, adding 0.5ml of 1mol/L hydrochloric acid solution, heating in water bath at 40 ℃ for 10 minutes, then adding 0.5ml of 1mol/L sodium hydroxide solution, diluting the solution to a scale with a solvent, shaking up, and taking the solution as a strong acid degradation sample solution; precisely measuring 2.5ml of blank auxiliary material solution, placing the blank auxiliary material solution in a 10ml measuring flask, and performing the same operation to obtain the blank auxiliary material solution for strong acid degradation.
(5) Degradation under intense illumination
Taking appropriate amount of famotidine injection, and placing in ultraviolet lamp (energy is not less than 200 W.hr/m) 2 ) Placing the sample solution and a cold white fluorescent lamp (the illumination intensity is 4500 +/-500 Lux) for 5 days, precisely measuring 2.5ml, placing the sample solution in a 10ml measuring flask, diluting the sample solution to a scale by using a solvent, shaking up, and using the sample solution as a strong illumination degradation sample solution; taking blank auxiliary material solution, operating the same method, and using the blank auxiliary material solution as blank auxiliary material solution for strong light degradation.
(6) Non-degradation test
2.5ml of famotidine injection is precisely measured, placed in a 10ml measuring flask, diluted to scale by a solvent and shaken up.
Measuring method:
precisely measuring each 5 μ l of the above solutions, respectively injecting into a liquid chromatograph, and recording chromatogram.
TABLE 6 sample needle number setting
Measuring results:
the results of the strong degradation chromatograms are shown in FIGS. 5-14, and the specific chromatogram analysis results are shown in Table 7.
TABLE 7 results of specificity measurement
Fourthly, conclusion:
the main peak purity angle of the test solution under each degradation condition is smaller than the purity threshold, and the separation degree of the main peak and adjacent impurities is larger than 1.5, so that the requirements of the scheme are met.
5.2. System precision test
Preparation of a solution:
blank: solvent(s)
Control solution: taking about 12.5mg of the vitamin C reference substance, accurately weighing, placing in a 50ml measuring flask, dissolving with a solvent, and diluting to scale to obtain a reference substance solution.
Measurement method (v):
and (5) taking blank and reference substance solutions respectively, injecting the blank and the reference substance solutions into a chromatograph, and recording a chromatogram.
TABLE 8 sample needle number settings
Measuring results:
TABLE 9 System precision test results
Fourthly, conclusion:
in a system precision experiment, the retention time RSD of the continuous 6-needle vitamin C is 0.23 percent and less than 1.0 percent, the peak area RSD is 0.61 percent and less than 2.0 percent, and the precision of the instrument meets the requirement.
5.3. Stability of solution
Preparation of a solution:
control solution: precisely weighing about 12.5mg of vitamin C as a reference substance, placing in a 50ml measuring flask, dissolving with solvent, diluting to scale, and shaking.
Test solution: 2.5ml of famotidine injection is precisely measured, placed in a 10ml measuring flask, diluted to the scale by using a solvent and shaken up.
Measuring method:
taking 5 μ l of the reference solution, standing at 2-8 deg.C for 0h, 2h, 4h, 8h, 12h, and 24h, respectively, injecting into a liquid chromatograph, and recording chromatogram; taking 5 mul of the test solution, placing the test solution at the temperature of between 2 and 8 ℃ for 0 hour, 2 hours, 4 hours and 8 hours respectively, injecting the test solution into a liquid chromatograph, and recording a chromatogram;
measuring results:
TABLE 10 results of solution stability measurements
Fourthly, conclusion:
the sample solution is stable within at least 8h and the reference solution is stable within at least 24h when the sample solution is placed at the temperature of 2-8 ℃.
5.4. Linearity
Preparation of a solution:
preparation of a control stock solution: precisely weighing about 50mg of vitamin C as reference substance, placing in a 20ml measuring flask, adding solvent to dilute to scale, shaking, and storing as reference substance.
50% linear solution: precisely measuring 0.5ml of the control stock solution, placing the control stock solution in a 10ml measuring flask, diluting the control stock solution to the scale with a solvent, and shaking up.
80% linear solution: precisely measuring 0.8ml of the control stock solution, placing the control stock solution in a 10ml measuring flask, diluting the control stock solution to the scale with a solvent, and shaking up.
90% linear solution: precisely measuring 0.9ml of the reference stock solution, placing the reference stock solution in a 10ml measuring flask, diluting the reference stock solution to a scale with a solvent, and shaking up.
100% linear solution: precisely measuring 1.0ml of the reference stock solution, placing the reference stock solution in a 10ml measuring flask, diluting the reference stock solution to a scale with a solvent, and shaking up.
110% linear solution: precisely measuring 1.1ml of the reference stock solution, placing the reference stock solution in a 10ml measuring flask, diluting the reference stock solution to a scale with a solvent, and shaking up.
120% linear solution: precisely measuring 1.2ml of the reference stock solution, placing the reference stock solution in a 10ml measuring flask, diluting the reference stock solution to a scale with a solvent, and shaking up.
200% linear solution: precisely measuring 2.0ml of the reference stock solution, placing the reference stock solution in a 10ml measuring flask, diluting the reference stock solution to a scale with a solvent, and shaking up.
Measurement:
and (4) taking 5 mu l of each linear solution, injecting the linear solution into a liquid chromatograph, and recording a chromatogram.
TABLE 11 sample needle number settings
Measuring results:
and (3) performing linear regression by taking the concentration as a horizontal coordinate and the peak area as a vertical coordinate, and calculating the peak area ratio corresponding to 100% of the concentration of the test sample, such as a regression equation, a correlation coefficient and an intercept.
TABLE 12 Linear measurement of vitamin C
Fourthly, conclusion:
when the concentration of the vitamin C is in the range of 124.85 mu g/ml to 499.4 mu g/ml, the linear equation is as follows: y is 4720.9x +14229, the correlation coefficient is 0.9996, which is greater than 0.999, the intercept is 1.21%, which is less than 2.0%, so the peak area and the concentration have a good linear relationship.
5.5. Accuracy of
And determining whether the closeness of the vitamin C determination result and the true value of the test sample at the concentration levels of 50%, 100% and 120% meets the accuracy requirement of the method.
Preparation of a solution:
blank adjuvant solution: precisely weighing about 5000mg of nicotinamide, about 2000mg of D-mannitol and about 350mg of lactic acid, dissolving with 100ml of water, and shaking up to obtain blank adjuvant solution.
Preparation of a reference solution: precisely weighing about 12.5mg of vitamin C as reference substance, placing in a 50ml measuring flask, dissolving with solvent, and diluting to scale to obtain reference solution. 2 parts are prepared in parallel.
Preparation of a reference stock solution: precisely weighing about 50mg of vitamin C as reference substance, placing in a 20ml measuring flask, adding solvent to dilute to scale, shaking, and storing as reference substance.
Preparing a test solution: precisely measuring 2.5ml of famotidine injection blank auxiliary materials, weighing 9 parts in parallel, respectively placing the materials into 10ml measuring bottles, adding 0.5ml of reference substance storage solution into 3 parts of the materials to be used as a test solution with 50% recovery rate, adding 1.0ml of reference substance storage solution into 3 parts of the materials to be used as a test solution with 100% recovery rate, adding 1.2ml of reference substance storage solution into 3 parts of the materials to be used as a test solution with 120% recovery rate, respectively diluting the materials to a scale by using a solvent, and shaking up.
Measuring:
and taking 5 mu l of each solution, injecting the solution into a liquid chromatograph, and recording a chromatogram.
TABLE 13 sample needle number settings
| Serial number | Sample name | Number of sample introduction needles |
| 1 | |
1 |
| 2 | Control solution-1 | 6 |
| 3 | Control solution-2 | 2 |
| 4 | 50% strength recovery solution-1 | 2 |
| 5 | 50% strength recovery solution-2 | 2 |
| 6 | 50% strength recovery solution-3 | 2 |
| 7 | 100% strength recovery solution-1 | 2 |
| 8 | 100% strength recovery solution-2 | 2 |
| 9 | 100% strength recovery solution-3 | 2 |
| 10 | 120% strength recovery solution-1 | 2 |
| 11 | 120% strength recovery solution-2 | 2 |
| 12 | 120% strength recovery solution-3 | 2 |
| 13 | Control solution-1 | 1 |
Measuring results:
the recovery was calculated according to the following formula:
m testing of ＝f×A×V
In the formula: eta: recovery ratio,%;
m add a : addition amount, mg;
m measured in fact : actual measured quantity, mg;
f: a correction factor;
v: diluting the sample solution by volume, ml;
a: main peak area of the test solution.
TABLE 14 results of accuracy measurement
Fourthly, conclusion:
the recovery rates of the 50% limit level, the 100% limit level and the 120% limit level are all between 98% and 102%, the average recovery rate is 100.67%, the RSD is 0.76%, and is less than 2.0%, and the result meets the regulation.
5.6. Precision degree
5.6.1. Repeatability of
Preparation of a solution:
control solution: taking about 12.5mg of the vitamin C reference substance, accurately weighing, placing in a 50ml measuring flask, dissolving with a solvent, and diluting to scale to obtain a reference substance solution.
Test solution: precisely measuring 2.5ml of famotidine injection, putting the famotidine injection into a 10ml measuring flask, diluting the famotidine injection to a scale by using a solvent, and shaking up to obtain the famotidine injection. 6 parts are prepared in parallel.
Measurement:
precisely measuring the sample solution and the control solution by 5 μ l each, respectively injecting into a liquid chromatograph, and recording chromatogram. And calculating the content of the vitamin C according to an external standard method.
TABLE 15 sample needle number setting
| Serial number | Sample name | Number of sample injection needles |
| 1 | |
1 |
| 2 | Control solution-1 | 6 |
| 3 | Control solution-2 | 2 |
| 4 | Repetitive test sample solution-1 (specification 2ml:20mg) | 2 |
| 5 | Repetitive test article solution-2 (specification 2ml:20mg) | 2 |
| 6 | Repetitive test sample solution-3 (specification 2ml:20mg) | 2 |
| 7 | Repetitive test sample solution-4 (specification 2ml:20mg) | 2 |
| 8 | Repetitive test sample solution-5 (specification 2ml:20mg) | 2 |
| 9 | Repeated sample solution-6 (2 ml specification: 20mg) | 2 |
| 10 | Control solution-1 | 1 |
Measuring results:
TABLE 16 repeatability measurements (specification 2ml:20mg)
5.6.2. Intermediate precision
The measurement was performed by the same method as "repeatability" by different persons at different times and using different instruments.
Preparation of a solution:
the procedure was carried out in the same manner as "repeat".
Measuring:
precisely measuring the sample solution and the reference solution by 5 μ l each, injecting into a liquid chromatograph, and recording chromatogram. And calculating the content of the vitamin C according to an external standard method.
TABLE 17 sample needle number setting
| Serial number | Sample name | Number of sample introduction needles |
| 1 | |
1 |
| 2 | Control solution-1 | 6 |
| 3 | Control solution-2 | 2 |
| 4 | Intermediate precision sample solution-1 (specification 2ml:20mg) | 2 |
| 5 | Intermediate precision sample solution-2 (Specification 2)ml:20mg) | 2 |
| 6 | Intermediate precision sample solution-3 (Specification 2ml:20mg) | 2 |
| 7 | Intermediate precision sample solution-4 (specification 2ml:20mg) | 2 |
| 8 | Intermediate precision sample solution-5 (2 ml in specification: 20mg) | 2 |
| 9 | Intermediate precision sample solution-6 (2 ml in specification: 20mg) | 2 |
| 10 | Control solution-1 | 1 |
Measuring results:
TABLE 18 vitamin C content determination results for different persons and different instruments at different times (specification 2ml:20mg)
TABLE 19 measurement of vitamin C content intermediate precision test results (specification 2ml:20mg)
Fourthly, conclusion:
the average content of vitamin C in 6 test sample solutions is 95.94 percent and the RSD is 0.83 percent and less than 2.0 percent through parallel determination, and the repeatability is good. The average content of vitamin C in 12 parts of test solution is 94.81%, RSD is 1.40%, and is less than 2.0%, so that the intermediate precision is good.
5.7 durability
In order to examine the influence of a slight variation in the test conditions on the measurement results, the following typical variation factors were examined: the flow rate, pH value, mobile phase proportion, column temperature, detection wavelength and chromatographic column are examined as follows:
TABLE 20 durability test investigation index
Preparation of a solution:
control solution: precisely weighing about 12.5mg of vitamin C as reference substance, placing in a 50ml measuring flask, dissolving with solvent, and diluting to scale to obtain reference solution. 2 parts are prepared in parallel.
Test solution: precisely measuring 2.5ml of famotidine injection, placing the famotidine injection in a 10ml measuring flask, diluting the famotidine injection to a scale by using a solvent, and shaking up to obtain the famotidine injection. 2 parts are prepared in parallel.
Measuring:
precisely sucking 5 μ l of each of the reference solution and the sample solution, injecting into a liquid chromatograph, and recording the chromatogram.
TABLE 21 sample needle number settings
| Serial number | Sample name | Number of sample injection needles |
| 1 | |
1 |
| 2 | |
6 |
| 3 | Control solution 2 | 2 |
| 4 | |
2 |
| 5 | Test solution 2 | 2 |
| 6 | |
1 |
Measuring results:
TABLE 22 durability measurement results
Fourthly, conclusion:
when the spectrum conditions of each color are slightly changed, the content of the vitamin C is within the range of 80.0-110.0%, and the deviation from the normal condition is less than 2.0%, so the method has good durability.
In conclusion, the invention can detect the vitamin C in the famotidine injection by specific sample solvent and chromatographic conditions, has accurate result, rapidness and high sensitivity, provides a new method for the quality control of the famotidine injection containing the vitamin C, provides a method basis for improving the quality control standard of the famotidine injection, provides a reference basis for expanding the clinical application of the famotidine injection, and has good application prospect.
Claims (10)
1. A method for detecting the content of vitamin C in famotidine injection is characterized by comprising the following steps: it comprises the following steps:
a. preparation of control solutions: dissolving vitamin C reference substance in solvent to obtain solution;
b. preparation of a test solution: diluting famotidine injection by 3-5 times by adding a solvent to obtain the famotidine injection;
c. respectively sucking the reference substance solution and the famotidine injection to be injected into a liquid chromatograph, wherein the chromatographic conditions are as follows:
and (3) chromatographic column: octadecylsilane chemically bonded silica is used as a filling agent; mobile phase: taking a potassium dihydrogen phosphate solution as a mobile phase A and taking an acetonitrile aqueous solution as a mobile phase B; sample injector temperature: 0-4 ℃; the gradient elution procedure was:
d. The vitamin C content is calculated according to the following formula:
wherein: a. the For supplying to : the peak area of the main peak in the test solution; v For supplying to : diluting the sample solution by multiple times; marking the loading amount: famotiVolume of injection; marking amount: the famotidine content in the famotidine injection;
the average value of the mass concentration and peak area ratios of a plurality of reference substance solutions;
the solvent is an aqueous solution containing disodium ethylene diamine tetraacetate and monopotassium phosphate.
2. The detection method according to claim 1, characterized in that: the mass volume ratio of the vitamin C reference substance to the solvent in the step a is 2-10 mg: 20ml, preferably 5 mg: 20 ml.
3. The detection method according to claim 1, characterized in that: the volume ratio of the famotidine injection to the solvent in the step b is 1-5 ml: 10ml, preferably 2.5 ml: 10 ml.
4. The detection method according to claim 1, characterized in that: and C, in the chromatographic conditions, the chromatographic column is GL Inertsutasin C18, the thickness of the chromatographic column is 250 multiplied by 4.6mm, the thickness of the chromatographic column is 5 mu m, the wavelength of the chromatographic column is 200-250 nm, the column temperature is 25-40 ℃, the temperature of a sample injector is 4 ℃, and the sample injection amount is 5-25 mu l.
5. The detection method according to claim 4, characterized in that: the column temperature is 30 ℃, the wavelength is 210nm, and the sample injection amount is 5 mu l.
6. The detection method according to claim 1, characterized in that: the solvent is an aqueous solution containing 0.3-0.67 mg of disodium ethylenediaminetetraacetate and 1.0-3.0 mg of monopotassium phosphate per 1ml of pH value of 2.0-4.0.
7. The detection method according to claim 6, characterized in that: the solvent is an aqueous solution containing 0.56mg of disodium ethylenediaminetetraacetate and 2.0mg of potassium dihydrogen phosphate per 1ml at a pH value of 3.0.
8. The detection method according to claim 1, characterized in that: the mobile phase A is 1-5 mg/mL potassium dihydrogen phosphate solution, preferably 2.04mg/mL potassium dihydrogen phosphate solution; the mobile phase B is 70-80% of an acetonitrile water solution with a concentration of ml/ml, preferably 73% of the acetonitrile water solution with a concentration of ml/ml.
9. The detection method according to claim 1, characterized in that: step d said
The mass concentration of the two reference substance solutions and the average value of the peak area ratio are shown as follows:
wherein A is For is to : the peak area of the main peak in the reference solution; m is To pair : control weight, mg; v For is to : dilution times of reference solutions; p: content of control.
10. The detection method according to any one of claims 1 to 9, characterized in that: the famotidine injection comprises the components of famotidine, nicotinamide, vitamin C, D-mannitol, lactic acid, sodium hydroxide and water for injection.
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