CN109884224B - Carbazochrome sodium sulfonate and method for measuring content of semicarbazide hydrochloride in preparation thereof - Google Patents
Carbazochrome sodium sulfonate and method for measuring content of semicarbazide hydrochloride in preparation thereof Download PDFInfo
- Publication number
- CN109884224B CN109884224B CN201910169423.9A CN201910169423A CN109884224B CN 109884224 B CN109884224 B CN 109884224B CN 201910169423 A CN201910169423 A CN 201910169423A CN 109884224 B CN109884224 B CN 109884224B
- Authority
- CN
- China
- Prior art keywords
- solution
- semicarbazide hydrochloride
- sodium sulfonate
- blank
- sample
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
Abstract
The invention provides a method for preparing detection samples of carbazochrome sodium sulfonate and a preparation thereof, which comprises the following steps: respectively taking a sample to be detected, a semicarbazide hydrochloride reference substance and a blank, adding water and methanol to dissolve, adjusting the pH value to be 2.0-8.0, shaking up, adding a 2-naphthaldehyde solution, adding water to a constant volume, shaking up, reacting at room temperature for 1-5 h, filtering, adding n-hexane into a subsequent filtrate for extraction, centrifuging, taking a bottom layer aqueous solution for repeated extraction, and centrifuging to obtain the composition. The invention also provides a method for measuring the content of semicarbazide hydrochloride in the carbazochrome sodium sulfonate and the preparation thereof, which considers the measurement of the content of semicarbazide hydrochloride in the carbazochrome sodium sulfonate preparation with different dosage forms, has better universality and specificity, high sensitivity, convenient operation and moderate analysis time, provides effective guarantee for the quality control of the content of semicarbazide hydrochloride in the carbazochrome sodium sulfonate and the preparation thereof, ensures the medication safety and has good application prospect.
Description
Technical Field
The invention belongs to the field of drug analysis, and particularly relates to carbazochrome sodium sulfonate and a content determination method of semicarbazide hydrochloride in a preparation thereof.
Background
Carbazochrome sodium sulfonate, chemical name is 1-methyl-6-oxo-2, 3,5, 6-tetrahydroindole-5-semicarbazone-2-sodium sulfonate, has the structural formula shown in formula I:
carbazochrome sodium sulfonate is originally developed by Mitsubishi pharmaceutical corporation of Ribes-Hedi (Mitsubishi TANBE Co., Ltd.) under the trade name Adona Arona, and is suitable for bleeding tendency (e.g., purpura, etc.) caused by decrease in capillary resistance and increase in permeability; fundus, renal, and uterine bleeding that exude from the skin or mucosa and endometrium due to attenuation of capillary resistance; abnormal bleeding during and after surgery caused by a decrease in capillary resistance.
Semicarbazide hydrochloride having the formula shown in formula II:
the semicarbazide hydrochloride belongs to hydrazine series chemical substances, early researches show that the semicarbazide hydrochloride has mutagenicity and potential carcinogenicity, and recent researches show that the semicarbazide hydrochloride not only can cause various tissue morphology changes on the tissue morphology level, but also can affect the functions of a nervous system and an endocrine system. The semicarbazide hydrochloride of 75-225 mg/L can cause the nucleic acid level in the lung and liver organ of a rat fetus to be obviously reduced, and the incidence rate of lung tumor of a young mouse is increased; in addition, the micronucleus rate of mouse bone marrow pleochromocyte can be obviously increased, and weak genetic toxicity is shown. Semicarbazide hydrochloride is an inhibitor of GABA synthase GAD (glutamate decarboxylase) and has an antagonistic effect on NMDAR (N-methyl-D-aspartate receptor), resulting in neurotoxicity. The semicarbazide hydrochloride can cause the morphological structure change of a plurality of target tissues and organs of rats and has a certain sex difference, wherein, the histological change of thymus and adrenal gland is only shown in female, the change of thyroid gland tissue structure is only shown in male, and the toxicity to reproductive organs is shown in that the semicarbazide hydrochloride can change the microstructure of female ovary and male testis. Female rats orally administered 60, 110, 210mg/kg semicarbazide hydrochloride 28d were able to reduce their plasma levels of E2 (estradiol) and showed dose-response relationship, showing antiestrogenic effect.
Semicarbazide hydrochloride, which is a starting material for the synthesis of carbazochrome sodium sulfonate, is a synthetic reagent for adrenal color hydrazone (also called carbazochrome) and may remain during the synthesis process. In addition, because semicarbazone groups exist in the structure of the carbazochrome sodium sulfonate, the semicarbazone groups are broken due to the influences of high temperature, oxidation, illumination and the like in the storage process of the carbazochrome sodium sulfonate and the preparation thereof, so that semicarbazone hydrochloride is generated. Based on the potential toxicity of the semicarbazide hydrochloride, in order to control the content of the carbazochrome sodium sulfonate and the semicarbazide hydrochloride in the preparation thereof and ensure the safety of the medicine, the development of a method for measuring the content of the carbazochrome sodium sulfonate and the semicarbazide hydrochloride in the preparation thereof is a problem to be solved urgently.
By searching relevant standards and documents at home and abroad, the legal standard for measuring the content of the semicarbazide hydrochloride in the carbazochrome sodium sulfonate and the preparation thereof at home and abroad is not available, and only a method for detecting the content of the semicarbazide hydrochloride in the carbazochrome sodium sulfonate and the carbazochrome sodium sulfonate is found in the document published by Kossana Canavesi in Chromolographia (2017)80: 1535-154. However, the method has the disadvantages of long reaction equilibrium time, poor specificity, high recovery rate, influence on result accuracy, slow pre-column derivatization of the carbazochrome sodium sulfonate preparation containing auxiliary materials, and influence on detection efficiency and accuracy, so the method is not suitable for detection of carbazochrome sodium sulfonate and the preparation thereof.
Disclosure of Invention
In order to solve the problems, the invention provides a method for measuring the content of carbazochrome sodium sulfonate and a preparation thereof, namely semicarbazide hydrochloride, which is characterized by comprising the following steps: the detection is carried out by adopting a high performance liquid chromatography, and the method comprises the following steps:
(1) sample preparation and derivatization
a. Blank solution: taking methanol and water, adjusting the pH value, shaking up, adding a 2-naphthaldehyde solution, fixing the volume with water, and reacting at room temperature to obtain the product;
b. control solution: dissolving semicarbazide hydrochloride in water, diluting with methanol and water, adjusting pH, shaking, adding 2-naphthaldehyde solution, adding water to desired volume, shaking, and reacting at room temperature to obtain the final product;
c. test solution: taking a sample to be detected, adding methanol and water for dissolving, adjusting the pH value, shaking up, then adding a 2-naphthaldehyde solution, adding water for constant volume, shaking up, and reacting at room temperature to obtain the product;
(2) liquid-liquid extraction
Filtering blank solution, reference solution and sample solution, extracting with n-hexane, centrifuging, extracting the bottom layer water solution repeatedly, and centrifuging;
(3) high performance liquid chromatography detection
Injecting the blank test solution, the reference test solution and the test solution to be tested into a chromatograph respectively, wherein the chromatographic conditions are as follows:
a chromatographic column: octadecylsilane chemically bonded silica is used as a filling agent; mobile phase: methanol is taken as a mobile phase A, and water is taken as a mobile phase B; isocratic elution procedure: phase A: phase B is 65: 35;
(4) calculated content
Calculating the content of semicarbazide hydrochloride in the sample according to an external standard method, wherein the specific calculation formula is as follows:
a is describedTo pairThe peak area of 2-naphthalene formaldehyde semicarbazone in the control solution is shown; a. theSample (A)Is the peak area corresponding to the 2-naphthalene formaldehyde semicarbazone in the test solution; cTo pairThe concentration (mu g/ml) of semicarbazide hydrochloride in the control solution; cSample (A)The concentration (mg/ml) of carbazochrome sodium sulfonate in the test solution is shown.
Further, the volume ratio of the methanol to the 2-naphthaldehyde solution in the step a is 2: 1-5: 1, preferably 4: 1; the water is used for fixing the volume until the methanol concentration is 0.2 ml/ml-0.5 ml/ml, and preferably 0.4 ml/ml.
Further, the mass-to-volume ratio of the semicarbazide hydrochloride reference substance in the step b to the methanol and 2-naphthaldehyde solution is (1-10) mug to 1ml, preferably (1-10) mug to 4ml to 1 ml; and (3) diluting with water until the semicarbazide hydrochloride concentration is (0.1-1) mu g/ml.
Further, the mass-to-volume ratio of the sample to be detected in the step c to the methanol and 2-naphthaldehyde solution is (1-10) mg: (2-5) ml:1ml, preferably (1-10) mg:4ml:1 ml; the water is used for fixing the volume until the concentration of the sample to be detected is (0.1-1) mg/ml; the dissolving is ultrasonic dissolving, and the time is 5-40 min, preferably 20 min.
Further, in the step (1), the reagent for adjusting the pH value is hydrochloric acid, phosphoric acid, sulfuric acid or glacial acetic acid, preferably hydrochloric acid solution, and the concentration is (3 → 1000); the pH value is adjusted to be 2.0-8.0, preferably 3.0-6.0, and more preferably 3.5-5.5.
Further, the concentration of the solution indicated by "(3 → 1000)" means that 3.0ml of hydrochloric acid as a liquid solute is added with water to make 1000ml of the solution.
Further, in the step (1), the 2-naphthaldehyde solution is prepared by dissolving 2-naphthaldehyde in 95% ethanol to obtain a solution with a concentration of 10 mg/ml.
Further, in the step (1), the reaction time is 1 to 5 hours, preferably 2 hours.
Further, in the step (2), the volume ratio of the subsequent filtrate to the n-hexane is 1: 2; the rotation speed of the centrifugation is 4000rpm, and the time is 3 min.
Further, in the step (2), other chromatographic peaks in the blank solution chromatogram after repeated extraction have no interference with the chromatographic peak of the 2-naphthalene formaldehyde semicarbazone.
Further, in the step (3), the flow rate in the chromatographic condition is 1.0ml/min, the column temperature is room temperature, the detection wavelength is 272nm +/-10 nm, and the injection volume is 20 mul.
According to the invention, deep research is carried out on different derivatization temperatures, different derivatization pH environments and addition of a derivatization catalytic reagent, the derivatization temperatures and the derivatization catalytic reagent have no obvious influence on the reaction rate, the pH environment of pre-column derivatization reaction has obvious improvement on the reaction rate, and when the derivatization pH environment reaches 3.5-5.5, the derivatization reaction rates of semicarbazide hydrochloride reference substances, carbazochrome sodium sulfonate and carbazochrome sodium sulfonate related preparations are consistent. The test data shows that: by the method, samples of different preparation types can react with the 2-naphthaldehyde for 1 hour to achieve reaction balance, and the problems of slow pre-column derivatization reaction rate and poor detection accuracy in the prior art are solved.
According to the invention, the derivatization reagent 2-naphthaldehyde and the extracting agent used in liquid-liquid extraction are analyzed and researched, and through screening, the n-hexane serving as the extracting agent can not interfere with the chromatographic peak of the 2-naphthaldehyde semicarbazide, but other impurity peaks which are consistent with the retention time of the chromatographic peak of the 2-naphthaldehyde semicarbazide exist in the 2-naphthaldehyde, and further research shows that the impurities can be eliminated by increasing the n-hexane extraction times and dissolving the impurities in the n-hexane. And then, carrying out comparison tests on the 2-naphthaldehyde of different brands, and finding that the impurities contained in the 2-naphthaldehyde of different brands are different, and the elution times of normal hexane are different, for example, the 2-naphthaldehyde produced by SIGMA needs liquid-liquid extraction for 4 times, while the 2-naphthaldehyde produced by Aladdin needs liquid-liquid extraction for 6 times, and because the 2-naphthaldehyde used for the derivatization reaction is difficult to keep the same practical brand during detection, the liquid-liquid extraction times are determined by the extraction times used when other chromatographic peaks in a blank solution chromatogram have no interference with the chromatographic peak of the 2-naphthaldehyde semicarbazone. The key technology of the invention solves the problems existing in the prior art that: the chromatographic peak in the blank solution interferes with the chromatographic peak of the 2-naphthalene formaldehyde semicarbazone, and the specificity of the method is influenced.
Aiming at the problems in the prior art, the method is further improved, the pH value of the pre-column derivatization reaction is determined, and the step of liquid-liquid extraction is reasonably limited, so that the method can quickly and accurately detect the content of semicarbazide hydrochloride in the carbazochrome sodium sulfonate and the preparation thereof.
The content determination method of carbazochrome sodium sulfonate and the semicarbazide hydrochloride in the preparation thereof considers the content determination of the semicarbazide hydrochloride in the carbazochrome sodium sulfonate preparation with different formulations, has better universality and specificity, high sensitivity, convenient operation and moderate analysis time, provides effective guarantee for the quality control of the content of the semicarbazide hydrochloride in the carbazochrome sodium sulfonate and the preparation thereof, ensures the medication safety and has good application prospect. The present invention can satisfy more features of novelty, creativity and practicality.
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 chromatogram of semicarbazide hydrochloride control solution.
FIG. 2 is a comparison of the blank solution with the control solution (1, semicarbazide hydrochloride control solution 2, blank solution).
FIG. 3 is a comparison of sample and control solutions (1, carbazochrome sodium sulfonate 2, semicarbazide hydrochloride control solution).
FIG. 4 shows the comparison of the sample solution and the control solution (1, carbazochrome sodium sulfonate injection 2, semicarbazide hydrochloride control solution).
FIG. 5 is a comparison of sample and control solutions (1, carbazochrome sodium sulfonate-sodium chloride injection 2, semicarbazide hydrochloride control solution).
FIG. 6 is a comparison of sample and control solutions (1, carbazochrome sodium sulfonate for injection 2, semicarbazide hydrochloride control solution).
FIG. 7 is a comparison of sample and control solutions (1, carbazochrome sodium sulfonate tablet 2, semicarbazide hydrochloride control solution).
Detailed Description
Example 1 measurement of semicarbazide hydrochloride content in carbazochrome sodium sulfonate
Carbazochrome sodium sulfonate sample to be tested
(1) Sample preparation and derivatization
a. Blank solution: taking 4ml of methanol and a proper amount of water, placing the methanol and the proper amount of water in a 10ml volumetric flask, adjusting the pH to 3.5-5.5 by using a hydrochloric acid solution (3 → 1000), shaking up, then adding 1.0ml of a 2-Naphthaldehyde (NCA) solution, fixing the volume by using the water, and shaking up; the solution was reacted at room temperature for 2 hours.
b. Control solution: taking 10mg of semicarbazide hydrochloride, precisely weighing, placing in a 100ml volumetric flask, adding a proper amount of water to dissolve, diluting to a scale, and shaking up; precisely measuring 5ml to 50ml volumetric flasks, adding water for dilution until scales are uniformly shaken, and taking the diluted solution as a reference stock solution; precisely measuring 1.0ml, putting the solution into a 10ml volumetric flask, adding 4ml of methanol and a proper amount of water, adjusting the pH to 3.5-5.5 by using a hydrochloric acid solution (3 → 1000), shaking up, then adding 1.0ml of NCA solution, fixing the volume by using water, and shaking up; the solution was reacted at room temperature for 2 hours.
c. Test solution: taking a carbazochrome sodium sulfonate sample of 10mg, placing the carbazochrome sodium sulfonate sample in a 10ml volumetric flask, adding 4ml of methanol and a proper amount of water, carrying out ultrasonic treatment for 20min to dissolve the carbazochrome sodium sulfonate, adjusting the pH to 3.5-5.5 by using a hydrochloric acid solution (3 → 1000), shaking up, then adding 1.0ml of NCA solution, adding water to a constant volume, and shaking up; the solution was reacted at room temperature for 2 hours.
(2) Liquid-liquid extraction
Filtering the blank solution, the reference solution and the sample solution, respectively, adding 1ml of the subsequent filtrate into a centrifuge tube, adding 2ml of n-hexane, fully mixing, centrifuging at 4000rpm for 3min, removing the upper organic layer, repeating the operation for several times, and taking the aqueous solution as a test solution. Respectively injecting 20 mu l of blank solution and 20 mu l of reference solution into a liquid chromatograph, determining that other chromatographic peaks in the chromatogram of the blank solution do not interfere with the chromatographic peak of the 2-naphthalene formaldehyde semicarbazone, and continuously increasing the extraction centrifugation times if interference exists until other chromatographic peaks in the chromatogram of the blank solution do not interfere with the chromatographic peak of the 2-naphthalene formaldehyde semicarbazone.
(3) Detection of
Injecting the blank test solution, the reference test solution and the test solution to be tested into a chromatograph respectively, wherein the chromatographic conditions are as follows:
the instrument comprises the following steps: high performance liquid chromatograph-ultraviolet detector
A chromatographic column: a chromatographic column using octadecylsilane chemically bonded silica as a filler;
mobile phase: methanol: water (65:35)
Flow rate 1.0ml/min, column temperature room temperature, detection wavelength: 272nm +/-10 nm and injection volume of 20 mu l
(4) Calculation of semicarbazide hydrochloride
If a chromatogram of the test solution has a chromatogram peak which is consistent with the retention time of the 2-naphthalene formaldehyde semicarbazone peak in the reference solution, calculating the content of semicarbazone hydrochloride according to the peak area of the 2-naphthalene formaldehyde semicarbazone in the reference solution by an external standard method, and specifically calculating the content of semicarbazone hydrochloride according to the following formula:
Ato pair: peak area of 2-naphthalene formaldehyde semicarbazone in the control solution;
Asample (A): peak area corresponding to 2-naphthalene formaldehyde semicarbazone in the test solution;
Cto pair: concentration of semicarbazide hydrochloride in control solution (μ g/ml);
Csample (A): the concentration (mg/ml) of carbazochrome sodium sulfonate in the test solution.
Example 2 measurement of the content of semicarbazide hydrochloride in carbazochrome sodium sulfonate injection
The specification of the carbazochrome sodium sulfonate injection to be detected is 20 ml: 100mg of
(1) Process for preparing and derivatizing test and reference substances
a. Blank solution: taking 4ml of methanol and a proper amount of water, placing the methanol and the proper amount of water in a 10ml volumetric flask, adjusting the pH to 3.5-5.5 by using a hydrochloric acid solution (3 → 1000), shaking up, then adding 1.0ml of an NCA solution, fixing the volume by using water, and shaking up; the solution was reacted at room temperature for 2 hours.
b. Control solution: taking 10mg of semicarbazide hydrochloride, precisely weighing, placing in a 100ml volumetric flask, adding a proper amount of water to dissolve, diluting to a scale, and shaking up; precisely measuring into a volumetric flask of 5ml to 50ml, adding water to dilute until scales are uniformly shaken, and taking the diluted solution as a reference stock solution. Precisely measuring 1.0ml, putting the solution into a 10ml volumetric flask, adding 4ml of methanol and a proper amount of water, adjusting the pH to 3.5-5.5 by using a hydrochloric acid solution (3 → 1000), shaking up, then adding 1.0ml of NCA solution, fixing the volume by using water, and shaking up; the solution was reacted at room temperature for 2 hours.
c. Test solution: precisely measuring 2.0ml of the product, putting the product into a 10ml volumetric flask, adding 4ml of methanol and a proper amount of water, shaking up, adjusting the pH to 3.5-5.5 by using a hydrochloric acid solution (3 → 1000), shaking up, then adding 1.0ml of NCA solution, fixing the volume by using water, and shaking up; the solution was reacted at room temperature for 2 hours.
(2) Liquid-liquid extraction process
Filtering the blank solution, the reference solution and the sample solution, respectively, adding 1ml of the subsequent filtrate into a centrifuge tube, adding 2ml of n-hexane, mixing completely, centrifuging at 4000rpm for 3min, and removing the upper organic layer. Repeating the operation for several times, taking the aqueous solution as a test solution, respectively taking 20 mu l of each of the blank solution and the reference solution, injecting into a liquid chromatograph, determining that other chromatographic peaks in the chromatogram of the blank solution have no interference to the chromatographic peak of the 2-naphthaldehyde semicarbazide, and continuously increasing the extraction centrifugation times if the interference exists until other chromatographic peaks in the chromatogram of the blank solution have no interference to the chromatographic peak of the 2-naphthaldehyde semicarbazide.
(3) Detection of
Injecting the blank test solution, the reference test solution and the test solution to be tested into a chromatograph respectively, wherein the chromatographic conditions are as follows:
the instrument comprises the following steps: high performance liquid chromatograph-ultraviolet detector
A chromatographic column: a chromatographic column using octadecylsilane chemically bonded silica as a filler;
mobile phase: methanol: water (65:35)
Flow rate 1.0ml/min, column temperature room temperature, detection wavelength: 272nm +/-10 nm and injection volume of 20 mu l
(4) Calculation of semicarbazide hydrochloride
If a chromatogram of the test solution has a chromatogram peak which is consistent with the retention time of the 2-naphthalene formaldehyde semicarbazone peak in the reference solution, calculating the content of semicarbazone hydrochloride according to the peak area of the 2-naphthalene formaldehyde semicarbazone in the reference solution by an external standard method, and specifically calculating the content of semicarbazone hydrochloride according to the following formula:
Ato pair: peak area of 2-naphthalene formaldehyde semicarbazone in the control solution;
Asample (A): peak area corresponding to 2-naphthalene formaldehyde semicarbazone in the test solution;
Cto pair: concentration of semicarbazide hydrochloride in control solution (μ g/ml);
Csample (A): the concentration (mg/ml) of carbazochrome sodium sulfonate in the test solution.
Example 3 content determination of semicarbazide hydrochloride in Carbazochrome sodium sulfonate sodium chloride injection
The specification of the carbazochrome sodium sulfonate and sodium chloride injection to be tested is 100 ml: 80mg of
(1) Process for preparing and derivatizing test and reference substances
a. Blank solution: taking 4ml of methanol and a proper amount of water, placing the methanol and the proper amount of water in a 10ml volumetric flask, adjusting the pH to 3.5-5.5 by using a hydrochloric acid solution (3 → 1000), shaking up, then adding 1.0ml of an NCA solution, fixing the volume by using water, and shaking up; the solution was reacted at room temperature for 2 hours.
b. Control solution: taking 10mg of semicarbazide hydrochloride, precisely weighing, placing in a 100ml volumetric flask, adding a proper amount of water to dissolve, diluting to a scale, and shaking up; precisely measuring into a volumetric flask of 1ml to 50ml, adding water to dilute until scales are uniformly shaken, and taking the diluted solution as a reference stock solution. Precisely measuring 1.0ml, putting the solution into a 10ml volumetric flask, adding 4ml of methanol and a proper amount of water, adjusting the pH to 3.5-5.5 by using a hydrochloric acid solution (3 → 1000), shaking up, then adding 1.0ml of NCA solution, fixing the volume by using water, and shaking up; the solution was reacted at room temperature for 2 hours.
c. Test solution: precisely measuring 2.0ml of the product, putting the product into a 10ml volumetric flask, adding 4ml of methanol and a proper amount of water, shaking up, adjusting the pH to 3.5-5.5 by using a hydrochloric acid solution (3 → 1000), shaking up, then adding 1.0ml of NCA solution, fixing the volume by using water, and shaking up; the solution was reacted at room temperature for 2 hours.
(2) Liquid-liquid extraction process
Filtering the blank solution, the reference solution and the sample solution, respectively, adding 1ml of the subsequent filtrate into a centrifuge tube, adding 2ml of n-hexane, mixing completely, centrifuging at 4000rpm for 3min, and removing the upper organic layer. Repeating the operation for several times, taking the aqueous solution as a test solution, respectively taking 20 mu l of each of the blank solution and the reference solution, injecting into a liquid chromatograph, determining that other chromatographic peaks in the chromatogram of the blank solution have no interference to the chromatographic peak of the 2-naphthaldehyde semicarbazide, and continuously increasing the extraction centrifugation times if the interference exists until other chromatographic peaks in the chromatogram of the blank solution have no interference to the chromatographic peak of the 2-naphthaldehyde semicarbazide.
(3) Detection of
Injecting the blank test solution, the reference test solution and the test solution to be tested into a chromatograph respectively, wherein the chromatographic conditions are as follows:
the instrument comprises the following steps: high performance liquid chromatograph-ultraviolet detector
A chromatographic column: a chromatographic column using octadecylsilane chemically bonded silica as a filler;
mobile phase: methanol: water (65:35)
Flow rate 1.0ml/min, column temperature room temperature, detection wavelength: 272nm +/-10 nm and injection volume of 20 mu l
(4) Calculation of semicarbazide hydrochloride
If a chromatogram of the test solution has a chromatogram peak which is consistent with the retention time of the 2-naphthalene formaldehyde semicarbazone peak in the reference solution, calculating the content of semicarbazone hydrochloride according to the peak area of the 2-naphthalene formaldehyde semicarbazone in the reference solution by an external standard method, and specifically calculating the content of semicarbazone hydrochloride according to the following formula:
Ato pair: peak area of 2-naphthalene formaldehyde semicarbazone in the control solution;
Asample (A): peak area corresponding to 2-naphthalene formaldehyde semicarbazone in the test solution;
Cto pair: concentration of semicarbazide hydrochloride in control solution (μ g/ml);
Csample (A): the concentration (mg/ml) of carbazochrome sodium sulfonate in the test solution.
Example 4 measurement of semicarbazide hydrochloride content in carbazochrome sodium sulfonate for injection
The standard of the carbazochrome sodium sulfonate for injection of the sample to be tested is 20mg
(1) Process for preparing and derivatizing test and reference substances
a. Blank solution: adding 4ml of methanol and a proper amount of water into a 10ml volumetric flask, adjusting the pH to 3.5-5.5 by using a hydrochloric acid solution (3 → 1000), shaking up, then adding 1.0ml of NCA solution, fixing the volume by using water, and shaking up; the solution was reacted at room temperature for 2 hours.
b. Control solution: taking 10mg of semicarbazide hydrochloride, precisely weighing, placing in a 100ml volumetric flask, adding a proper amount of water to dissolve, diluting to a scale, and shaking up; precisely measuring into a volumetric flask of 5ml to 50ml, adding water to dilute until scales are uniformly shaken, and taking the diluted solution as a reference stock solution. Precisely measuring 1.0ml, putting the solution into a 10ml volumetric flask, adding 4ml of methanol and a proper amount of water, adjusting the pH to 3.5-5.5 by using a hydrochloric acid solution (3 → 1000), shaking up, then adding 1.0ml of NCA solution, fixing the volume by using water, and shaking up; the solution was reacted at room temperature for 2 hours.
c. Test solution: taking 1 bottle (20mg) of the product, transferring the bottle into a 20ml volumetric flask, adding a proper amount of water into the residual content in the bottle for dissolving, transferring the bottle into the volumetric flask together, then rinsing the empty bottle with a small amount of water for 2 times, transferring the empty bottle into the volumetric flask together, adding 8ml of methanol into the volumetric flask, performing ultrasonic treatment for 20min to dissolve carbazochrome sodium sulfonate, adjusting the pH to 3.5-5.5 by using a hydrochloric acid solution (3 → 1000), shaking up, then adding 2.0ml of an NCA solution, adding water for fixing the volume, and shaking up; the solution was reacted at room temperature for 2 hours.
(2) Liquid-liquid extraction process
Filtering the blank solution, the reference solution and the sample solution, respectively, adding 1ml of the subsequent filtrate into a centrifuge tube, adding 2ml of n-hexane, mixing completely, centrifuging at 4000rpm for 3min, and removing the upper organic layer. Repeating the operation for several times, taking the aqueous solution as a test solution, respectively taking 20 mu l of each of the blank solution and the reference solution, injecting into a liquid chromatograph, determining that other chromatographic peaks in the chromatogram of the blank solution have no interference to the chromatographic peak of the 2-naphthaldehyde semicarbazide, and continuously increasing the centrifugal extraction times if the interference exists until other chromatographic peaks in the chromatogram of the blank solution have no interference to the chromatographic peak of the 2-naphthaldehyde semicarbazide.
(3) Detection of
Injecting the blank test solution, the reference test solution and the test solution to be tested into a chromatograph respectively, wherein the chromatographic conditions are as follows:
the instrument comprises the following steps: high performance liquid chromatograph-ultraviolet detector
A chromatographic column: a chromatographic column using octadecylsilane chemically bonded silica as a filler;
mobile phase: methanol: water (65:35)
Flow rate 1.0ml/min, column temperature room temperature, detection wavelength: 272nm +/-10 nm and injection volume of 20 mu l
(4) Calculation of semicarbazide hydrochloride
If a chromatogram of the test solution has a chromatogram peak which is consistent with the retention time of the 2-naphthalene formaldehyde semicarbazone peak in the reference solution, calculating the content of semicarbazone hydrochloride according to the peak area of the 2-naphthalene formaldehyde semicarbazone in the reference solution by an external standard method, and specifically calculating the content of semicarbazone hydrochloride according to the following formula:
Ato pair: peak area of 2-naphthalene formaldehyde semicarbazone in the control solution;
Asample (A): peak area corresponding to 2-naphthalene formaldehyde semicarbazone in the test solution;
Cto pair: concentration of semicarbazide hydrochloride in control solution (μ g/ml);
Csample (A): the concentration (mg/ml) of carbazochrome sodium sulfonate in the test solution.
Example 5 measurement of semicarbazide hydrochloride content in carbazochrome sodium sulfonate tablets
The standard of the sample carbazochrome sodium sulfonate tablet to be detected is 10mg
(1) Process for preparing and derivatizing test and reference substances
a. Blank solution: taking 4ml of methanol and a proper amount of water, placing the methanol and the proper amount of water in a 10ml volumetric flask, adjusting the pH to 3.5-5.5 by using a hydrochloric acid solution (3 → 1000), shaking up, then adding 1.0ml of an NCA solution, fixing the volume by using water, and shaking up; the solution was reacted at room temperature for 2 hours.
b. Control solution: taking 10mg of semicarbazide hydrochloride, precisely weighing, placing in a 100ml volumetric flask, adding a proper amount of water to dissolve, diluting to a scale, and shaking up; precisely measuring into a volumetric flask of 5ml to 50ml, adding water to dilute until scales are uniformly shaken, and taking the diluted solution as a reference stock solution. Precisely measuring 1.0ml, putting the solution into a 10ml volumetric flask, adding 4ml of methanol and a proper amount of water, adjusting the pH to 3.5-5.5 by using a hydrochloric acid solution (3 → 1000), shaking up, then adding 1.0ml of NCA solution, fixing the volume by using water, and shaking up; the solution was reacted at room temperature for 2 hours.
c. Test solution: taking 1 tablet (10mg) of the product, putting the product into a 10ml volumetric flask, adding 4ml of methanol and a proper amount of water, carrying out ultrasonic treatment for 20min to dissolve carbazochrome sodium sulfonate, adjusting the pH to 3.5-5.5 by using a hydrochloric acid solution (3 → 1000), shaking up, then adding 1.0ml of NCA solution, adding water to a constant volume, and shaking up; the solution was reacted at room temperature for 2 hours.
(2) Liquid-liquid extraction process
Filtering the blank solution, the reference solution and the sample solution, respectively, adding 1ml of the subsequent filtrate into a centrifuge tube, adding 2ml of n-hexane, mixing completely, centrifuging at 4000rpm for 3min, and removing the upper organic layer. Repeating the operation for several times, taking the aqueous solution as a test solution, respectively injecting 20 mu l of blank solution reference solution into a liquid chromatograph, determining that other chromatographic peaks in the chromatogram of the blank solution have no interference to the chromatographic peak of the 2-naphthaldehyde semicarbazone, and continuously increasing the extraction centrifugation times if the interference exists until other chromatographic peaks in the chromatogram of the blank solution have no interference to the chromatographic peak of the 2-naphthaldehyde semicarbazone.
(3) Detection of
Injecting the blank test solution, the reference test solution and the test solution to be tested into a chromatograph respectively, wherein the chromatographic conditions are as follows:
the instrument comprises the following steps: high performance liquid chromatograph-ultraviolet detector
A chromatographic column: a chromatographic column using octadecylsilane chemically bonded silica as a filler;
mobile phase: methanol: water (65:35)
Flow rate 1.0ml/min, column temperature room temperature, detection wavelength: 272nm +/-10 nm and injection volume of 20 mu l
(4) Calculation of semicarbazide hydrochloride
If a chromatogram of the test solution has a chromatogram peak which is consistent with the retention time of the 2-naphthalene formaldehyde semicarbazone peak in the reference solution, calculating the content of semicarbazone hydrochloride according to the peak area of the 2-naphthalene formaldehyde semicarbazone in the reference solution by an external standard method, and specifically calculating the content of semicarbazone hydrochloride according to the following formula:
Ato pair: peak area of 2-naphthalene formaldehyde semicarbazone in the control solution;
Asample (A): peak area corresponding to 2-naphthalene formaldehyde semicarbazone in the test solution;
Cto pair: concentration of semicarbazide hydrochloride in control solution (μ g/ml);
Csample (A): the concentration (mg/ml) of carbazochrome sodium sulfonate in the test solution.
The beneficial effects of the invention are further illustrated by way of test examples as follows:
test example 1 verification of semicarbazide hydrochloride detection analysis method in carbazochrome sodium sulfonate
1. Analytical method
In the same manner as in example 1, chromatograms were obtained (see FIGS. 1 to 3).
2. Specificity
The blank solution obtained in the step a of the embodiment 1 is analyzed according to the chromatographic conditions obtained in the step (3) of the embodiment 1, and the result shows that the blank solution has no interference on the chromatographic peak of the 2-naphthalene formaldehyde semicarbazone and has good specificity.
3. Limit of quantification
When a control solution was prepared by the method described in step b of example 1, and the test solution subjected to liquid-liquid extraction was diluted and subjected to the detection under the chromatographic conditions described in step (3) of example 1, the results showed that the semicarbazide hydrochloride concentration was 4.86ng/ml, corresponding to 0.0005% of the carbazochrome sodium sulfonate concentration, at an S/N of about 10.
4. Linearity
And (3) respectively putting control solutions corresponding to 48.6ng, 809.3ng, 1618.6ng, 4046.5ng, 6474.4ng, 8093.0ng, 9711.6ng and 12139.5ng of semicarbazide hydrochloride into a 10ml volumetric flask, carrying out derivatization reaction according to the method in the step (1) of the example 1, and detecting a linear series of solutions subjected to liquid-liquid extraction according to the chromatographic conditions in the step (3) of the example 1, wherein the measured concentration of the semicarbazide hydrochloride is in the range of 4.86ng/ml to 1213.95ng/ml, the linear equation is 4972.4x +4535.9, the correlation coefficient r is 0.9998, the correlation coefficient is more than 0.99, and the linearity meets the requirement.
5. Recovery rate
Precisely weighing about 10mg of carbazochrome sodium sulfonate and 9 parts of carbazochrome sodium sulfonate respectively, placing the carbazochrome sodium sulfonate into a 10ml volumetric flask, adding 0.5ml, 1.0ml and 1.5ml of the reference substance stock solution of the method respectively and 3 parts of the reference substance stock solution respectively, carrying out derivatization reaction according to the method in the step (1) of the embodiment 1, carrying out high, medium and low recovery solutions after liquid-liquid extraction, and detecting according to the chromatographic conditions in the step (3) of the embodiment 1, wherein the result shows that the recovery rate of semicarbazide hydrochloride is between 97% and 101%, RSD is less than 1.0%, and the recovery rate meets the requirement.
6. Repeatability of
Respectively and precisely weighing about 10mg and 6 parts of carbazochrome sodium sulfonate, placing the carbazochrome sodium sulfonate into a 10ml volumetric flask, precisely adding 0.5ml of a reference substance stock solution according to the method described in the step (1) of the embodiment 1, carrying out a derivatization reaction according to the method described in the step (1) of the embodiment 1, and detecting a repetitive series solution after liquid-liquid extraction according to the chromatographic conditions described in the step (3) of the embodiment 1, wherein the result shows that the content result RSD of semicarbazide hydrochloride is less than 1.0%, and the repeatability meets the requirement.
7. Stability of solution
The sample solution is placed at room temperature, the peak area RSD of the chromatographic peak of the 2-naphthalene formaldehyde semicarbazone in the sample solution is 1.3% within 24 hours, and the result shows that the sample solution is stable within 24 hours at room temperature.
Test example 2 verification of carbazochrome sodium sulfonate injection analysis method
Methodology verified that the used carbazochrome sodium sulfonate injection specification was 20 ml: 100 mg.
1. Analytical method
In the same manner as in example 2, a chromatogram was obtained (see FIG. 4)
2. Specificity
A blank preparation solution is prepared according to a prescription of a carbazochrome sodium sulfonate injection specification, a sample solution is prepared according to the method in the embodiment 2, a blank auxiliary material solution is subjected to liquid-liquid extraction, and the analysis is performed according to the chromatographic conditions in the step (3) in the embodiment 2, so that the blank auxiliary material solution has no interference on the chromatographic peak of the 2-naphthaldehyde semicarbazone and has good specificity.
3. Limit of quantification
A control solution was prepared in accordance with the method of example 2, and the test solution subjected to liquid-liquid extraction was diluted and examined under the chromatographic conditions of the step (3) of example 2, and as a result, it was found that when S/N was about 10, the concentration of semicarbazide hydrochloride was 4.86ng/ml, which corresponds to 0.0005% of the concentration of carbazochrome sodium sulfonate.
4. Linearity
And (3) putting control solutions corresponding to 48.6ng, 809.3ng, 1618.6ng, 4046.5ng, 6474.4ng, 8093.0ng, 9711.6ng and 12139.5ng of semicarbazide hydrochloride into a 10ml volumetric flask, performing derivatization reaction according to the method in the step (1) of the example 2, and detecting a linear series of solutions subjected to liquid-liquid extraction according to the chromatographic conditions in the step (3) of the example 2, wherein the measured concentration of the semicarbazide hydrochloride is in the range of 4.86ng/ml to 1213.95ng/ml, the linear equation is 4972.4x +4535.9, the correlation coefficient r is 0.9998, the correlation coefficient is more than 0.99, and the linearity meets the requirements.
5. Recovery rate
2.0ml and 9 parts of carbazochrome sodium sulfonate injection are precisely measured respectively, and are placed into a 10ml volumetric flask, 3 parts of carbazochrome sodium sulfonate injection are respectively added with 0.5ml, 1.0ml and 1.5ml of reference substance stock solution of the method of the step b of the embodiment 2, the derivatization reaction is carried out according to the method of the step (1) of the embodiment 2, high, medium and low recovery solution after liquid-liquid extraction is carried out, and the detection is carried out according to the chromatographic conditions of the step (3) of the embodiment 2, and the results show that the recovery rate of semicarbazide hydrochloride is between 99% and 102%, and the RSD is less than 1.0%, so that the recovery rate meets the requirements.
6. Repeatability of
2.0ml and 6 parts of carbazochrome sodium sulfonate injection are precisely measured respectively, the carbazochrome sodium sulfonate injection is placed in a 10ml volumetric flask, derivatization is carried out according to the derivatization method in the step (1) of the embodiment 2, a repetitive series solution after liquid-liquid extraction is detected according to the chromatographic conditions in the step (3) of the embodiment 2, and the result shows that the content result RSD of semicarbazide hydrochloride is less than 1.0 percent and the repeatability meets the requirement.
7. Stability of solution
The sample solution is placed at room temperature, the peak area RSD of the chromatographic peak of the 2-naphthalene formaldehyde semicarbazone in the sample solution is 1.4% within 24 hours, and the result shows that the sample solution is stable within 24 hours at room temperature.
Test example 3 verification of analysis method of carbazochrome sodium sulfonate and sodium chloride injection
Methodology verifies that the specification of the carbazochrome sodium sulfonate and sodium chloride injection is 100 ml: 80 mg.
1. Analytical method
In the same manner as in example 3, a chromatogram was obtained (see FIG. 5)
2. Specificity
A blank preparation solution is prepared according to the prescription of the carbazochrome sodium sulfonate and sodium chloride injection specification, a sample solution is prepared according to the method in the embodiment 3, a blank auxiliary material solution is subjected to liquid-liquid extraction, and the analysis is carried out according to the chromatographic conditions in the embodiment 3, so that the blank auxiliary material solution has no interference on the chromatographic peak of the 2-naphthaldehyde semicarbazone and has good specificity.
3. Limit of quantification
A control solution was prepared as described in example 3, and the test solution subjected to liquid-liquid extraction was diluted and examined under the chromatography conditions of example 3, and the results showed that when S/N was about 10, the semicarbazide hydrochloride concentration was 4.86ng/ml, which corresponds to 0.003% of the carbazochrome sodium sulfonate concentration.
4. Linearity
And (3) respectively putting reference substance solutions corresponding to 48.6ng, 809.3ng, 1618.6ng, 4046.5ng, 6474.4ng, 8093.0ng, 9711.6ng and 12139.5ng of semicarbazide hydrochloride into a 10ml volumetric flask, carrying out derivatization reaction according to the method in the step (1) of the example 3, detecting a linear series of solutions after liquid-liquid extraction according to the chromatographic condition of the example 3, wherein the result shows that the measured concentration of the semicarbazide hydrochloride is in the range of 4.86ng/ml to 1213.95ng/ml, the linear equation is 4972.4x +4535.9, the correlation coefficient r is 0.9998, the correlation coefficient is more than 0.99, and the linearity meets the requirement.
5. Recovery rate
2.0ml of carbazochrome sodium sulfonate and sodium chloride injection, 9 parts in total, are precisely measured respectively, and are placed into a 10ml volumetric flask, 0.5ml, 1.0ml and 1.5ml of reference substance storage solution of the method described in the step b of the embodiment 3 are respectively added, 3 parts of the reference substance storage solution are respectively added, the derivatization reaction is carried out according to the derivatization method described in the step (1) of the embodiment 3, high, medium and low recovery solutions after liquid-liquid extraction are detected according to the chromatographic conditions described in the embodiment 3, and the result shows that the semicarbazide hydrochloride recovery rate is between 97% and 101%, the RSD is less than 1.0%, and the recovery rate meets the requirement.
6. Repeatability of
2.0ml and 6 parts of carbazochrome sodium sulfonate and sodium chloride injection are precisely measured respectively, the carbazochrome sodium sulfonate and sodium chloride injection is placed in a 10ml volumetric flask, derivatization is carried out according to the derivatization method in the step (1) of the embodiment 3, and the result shows that the content result RSD of semicarbazide hydrochloride is less than 1.0% and the repeatability meets the requirement by detecting the repeatability series solution after liquid-liquid extraction according to the chromatographic conditions.
7. Stability of solution
The sample solution is placed at room temperature, the peak area RSD of the chromatographic peak of the 2-naphthalene formaldehyde semicarbazone in the sample solution is 1.5% within 24 hours, and the result shows that the sample solution is stable within 24 hours at room temperature.
Test example 4 test of carbazochrome sodium sulfonate for injection
Methodology verified that the specification of carbazochrome sodium sulfonate for injection used was 20 mg.
1. Analytical method
In the same manner as in example 4, a chromatogram was obtained (see FIG. 6)
2. Specificity
A blank preparation solution is prepared according to a prescription of a carbazochrome sodium sulfonate instruction for injection, a test sample solution is prepared according to the method in the embodiment 4, a blank auxiliary material solution is subjected to liquid-liquid extraction, and the analysis is performed according to the chromatographic conditions in the embodiment 4, so that the blank auxiliary material solution has no interference on a chromatographic peak of 2-naphthaldehyde semicarbazone and has good specificity.
3. Limit of quantification
When a control solution was prepared as described in example 4, and the test solution subjected to liquid-liquid extraction was diluted and subjected to the chromatography conditions as described in example 4, the results showed that the semicarbazide hydrochloride concentration was 4.86ng/ml, corresponding to 0.0005% of the carbazochrome sodium sulfonate concentration, at an S/N of about 10.
4. Linearity
And (3) respectively putting control solutions corresponding to 48.6ng, 809.3ng, 1618.6ng, 4046.5ng, 6474.4ng, 8093.0ng, 9711.6ng and 12139.5ng of semicarbazide hydrochloride into a 10ml volumetric flask, carrying out derivatization reaction according to the derivatization method in the step (1) of the example 4, and detecting a linear series of solutions subjected to liquid-liquid extraction according to the chromatographic conditions in the step (3) of the example 4, wherein the measured concentration of the semicarbazide hydrochloride is in the range of 4.86ng/ml to 1213.95ng/ml, the linear equation is 4972.4x +4535.9, the correlation coefficient r is 0.9998, and the correlation coefficient is more than 0.99, so that the linearity meets the requirements.
5. Recovery rate
Taking 1 bottle (20mg) of the product, placing the bottle in a 20ml volumetric flask (9 parts in total), respectively adding 1.0ml, 2.0ml and 3.0ml of the reference substance stock solution in the step b of the embodiment 4, and 3 parts of the reference substance stock solution respectively, performing derivatization according to the derivatization method described in the step (1) of the embodiment 4, performing liquid-liquid extraction on high, medium and low recovery solutions, and detecting according to chromatographic conditions described by the invention, wherein the results show that the recovery rate of semicarbazide hydrochloride is between 98% and 101%, RSD is less than 1.0%, and the recovery rate meets the requirements.
6. Repeatability of
Taking 1 bottle (20mg) of the product, putting 6 parts in a 20ml volumetric flask, performing derivatization according to the derivatization method in the step (1) of the embodiment 4, detecting repetitive series solutions after liquid-liquid extraction according to the chromatographic conditions in the step (3) of the embodiment 4, and indicating that the content result RSD of semicarbazide hydrochloride is less than 1.0 percent and the repeatability meets the requirements.
7. Stability of solution
The sample solution is placed at room temperature, the peak area RSD of the chromatographic peak of the 2-naphthalene formaldehyde semicarbazone in the sample solution is 1.0% within 24 hours, and the result shows that the sample solution is stable within 24 hours at room temperature.
Test example 5 verification of carbazochrome sodium sulfonate tablet analysis method
Methodology verified that the carbazochrome sodium sulfonate tablet size used was 10 mg.
1. Analytical method
In the same manner as in example 5, a chromatogram was obtained (see FIG. 7)
2. Specificity
A blank preparation solution is prepared according to the prescription of the carbazochrome sodium sulfonate tablet specification, a test solution is prepared according to the method in the embodiment 5, a blank auxiliary material solution is subjected to liquid-liquid extraction, and the analysis is performed according to the chromatographic conditions in the embodiment 5, so that the blank auxiliary material solution has no interference on the chromatographic peak of the 2-naphthalene formaldehyde semicarbazone and has good specificity.
3. Limit of quantification
When a control solution was prepared as described in example 5, and the test solution subjected to liquid-liquid extraction was diluted and subjected to the chromatography conditions of example 5, it was found that the semicarbazide hydrochloride concentration was 4.86ng/ml, which corresponds to 0.0005% of the carbazochrome sodium sulfonate concentration, at an S/N of about 10.
4. Linearity
And (3) respectively putting control solutions corresponding to 48.6ng, 809.3ng, 1618.6ng, 4046.5ng, 6474.4ng, 8093.0ng, 9711.6ng and 12139.5ng of semicarbazide hydrochloride into a 10ml volumetric flask, performing derivatization according to the derivatization method in the step (1) of the example 5, and detecting a linear series of solutions subjected to liquid-liquid extraction according to the chromatographic conditions in the step (3) of the example 5, wherein the measured concentration of the semicarbazide hydrochloride is in the range of 4.86ng/ml to 1213.95ng/ml, the linear equation is 4972.4x +4535.9, the correlation coefficient r is 0.9998, the correlation coefficient is more than 0.99, and the linearity meets the requirement.
5. Recovery rate
Taking 1 tablet (10mg) of the product, putting the tablet into a 10ml volumetric flask (9 parts in total), respectively adding 0.5ml, 1.0ml and 1.5ml of the method reference product stock solution, 3 parts respectively, performing derivatization according to the derivatization method described in the step (1) of the example 5, performing detection on high, medium and low recovery solutions obtained after liquid-liquid extraction according to the chromatographic conditions described in the example 5, and showing that the semicarbazide hydrochloride recovery rate is between 98% and 101%, the RSD is less than 1.0%, and the recovery rate meets the requirements.
6. Repeatability of
Taking 1 tablet (10mg) of the product, putting 6 parts in a 10ml volumetric flask, performing derivatization according to the derivatization method in the step (1) of the example 5, detecting repetitive series solutions after liquid-liquid extraction according to the chromatographic conditions in the step (3) of the example 5, and indicating that the content result RSD of semicarbazide hydrochloride is less than 1.0 percent and the repeatability meets the requirements.
7. Stability of solution
The sample solution is placed at room temperature, the peak area RSD of the chromatographic peak of the 2-naphthalene formaldehyde semicarbazone in the sample solution is 0.9% within 24 hours, and the result shows that the sample solution is stable within 24 hours at room temperature.
The test proves that: the content determination method of the carbazochrome sodium sulfonate and the semicarbazide hydrochloride in the preparation thereof can determine the semicarbazide hydrochloride content of carbazochrome sodium sulfonate preparations of different formulations, has good universality and specificity, high sensitivity, convenient operation and moderate analysis time, provides effective guarantee for quality control of the carbazochrome sodium sulfonate and the semicarbazide hydrochloride content of the preparation thereof, and has good application prospect.
Claims (14)
1. A method for preparing detection samples of carbazochrome sodium sulfonate and a preparation thereof is characterized in that: it comprises the following steps:
respectively taking a sample to be detected, a semicarbazide hydrochloride reference substance and a blank, adding water and methanol to dissolve, adjusting the pH value to 3.5-5.5, shaking up, adding a 2-naphthaldehyde solution, adding water to a constant volume, shaking up, reacting at room temperature for 1-5 h, filtering, adding n-hexane into a subsequent filtrate for extraction, centrifuging, taking a bottom layer aqueous solution for repeated extraction, and centrifuging to obtain the composition.
2. The method for arranging a test sample according to claim 1, wherein: the mass-volume ratio of the sample to be detected or the semicarbazide hydrochloride reference substance to the methanol and 2-naphthaldehyde solution is 1-10 mg: 2-5 ml:1 ml; the volume is determined by water until the concentration of the sample to be detected or the semicarbazide hydrochloride reference substance is 0.1-1 mg/ml; the dissolving is ultrasonic dissolving, and the time is 5-40 min.
3. The method for arranging a test sample according to claim 2, wherein: the mass volume ratio of the sample to be detected or the semicarbazide hydrochloride reference substance to the methanol and 2-naphthaldehyde solution is 1-10 mg:4ml:1 ml.
4. The method for arranging a test sample according to claim 2, wherein: the ultrasonic dissolution time is 20 min.
5. The method for arranging a test sample according to claim 1, wherein: the volume ratio of methanol to the 2-naphthalene formaldehyde solution in the blank is 2: 1-5: 1; and (3) diluting the blank with water until the methanol concentration is 0.2-0.5 ml/ml.
6. The method of claim 5, wherein: the volume ratio of methanol to the 2-naphthaldehyde solution added into the blank is 4: 1; and (4) adding water into the blank to make the methanol concentration be 0.4 ml/ml.
7. The method for arranging a test sample according to any one of claims 1 to 6, comprising: the 2-naphthaldehyde solution is prepared by dissolving 2-naphthaldehyde in 95% ethanol to prepare a solution with the concentration of 10 mg/ml.
8. The method for arranging a test sample according to claim 1, wherein: the reagent for adjusting the pH value is hydrochloric acid, phosphoric acid, sulfuric acid or glacial acetic acid.
9. The method for arranging a test sample according to claim 8, wherein: the reagent for adjusting the pH value is hydrochloric acid solution, and the concentration of the hydrochloric acid solution is 3 → 1000.
10. The method for arranging a test sample according to claim 1, wherein: the reaction was carried out at room temperature for 2 h.
11. The method for arranging a test sample according to claim 1, wherein: the volume ratio of the secondary filtrate to the normal hexane is 1: 2; the rotation speed of the centrifugation is 4000rpm, and the time is 3 min.
12. A method for measuring the content of semicarbazide hydrochloride in carbazochrome sodium sulfonate and a preparation thereof is characterized in that: it comprises the following steps:
(1) taking a sample solution to be tested, a reference solution and a blank solution obtained in the claim 1;
(2) respectively injecting the solution obtained in the step (1) into a high performance liquid chromatograph to obtain a chromatogram; the chromatographic conditions were as follows:
a chromatographic column: octadecylsilane chemically bonded silica is used as a filling agent; mobile phase: methanol is taken as a mobile phase A, and water is taken as a mobile phase B; isocratic elution procedure: phase A and phase B are 65: 35;
(3) calculated content
Calculating the content of semicarbazide hydrochloride in the sample according to an external standard method, wherein the specific calculation formula is as follows:
a is describedTo pairThe peak area of 2-naphthalene formaldehyde semicarbazone in the control solution is shown; a. theSample (A)Is the peak area corresponding to the 2-naphthalene formaldehyde semicarbazone in the test solution; cTo pairIs the concentration mu g/ml of semicarbazide hydrochloride in the reference solution; cSample (A)Is the concentration mg/ml of carbazochrome sodium sulfonate in the test solution.
13. The content measurement method according to claim 12, characterized in that: and the chromatographic peak in the blank solution chromatogram should not interfere with the chromatographic peak of the 2-naphthalene formaldehyde semicarbazone in the chromatogram of the sample solution to be detected and/or the control solution, if interference exists, the sample solution to be detected, the control solution and the blank solution should be extracted and centrifuged again.
14. The content measurement method according to claim 12, characterized in that: and (3) under the chromatographic conditions in the step (2), the flow rate is 1.0ml/min, the column temperature is room temperature, the detection wavelength is 272nm +/-10 nm, and the sample injection volume is 20 mu l.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910169423.9A CN109884224B (en) | 2019-03-06 | 2019-03-06 | Carbazochrome sodium sulfonate and method for measuring content of semicarbazide hydrochloride in preparation thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910169423.9A CN109884224B (en) | 2019-03-06 | 2019-03-06 | Carbazochrome sodium sulfonate and method for measuring content of semicarbazide hydrochloride in preparation thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109884224A CN109884224A (en) | 2019-06-14 |
| CN109884224B true CN109884224B (en) | 2020-06-16 |
Family
ID=66931068
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910169423.9A Active CN109884224B (en) | 2019-03-06 | 2019-03-06 | Carbazochrome sodium sulfonate and method for measuring content of semicarbazide hydrochloride in preparation thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109884224B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4642286A (en) * | 1984-05-07 | 1987-02-10 | Moldowan Mervin J | Composition and method for ethanol determination |
| CN108938626A (en) * | 2018-07-27 | 2018-12-07 | 四川联成迅康医药股份有限公司 | A kind of good and highly-safe Carbazochrome sodium sulfonate pharmaceutical composition and its preparation method and application of stability |
-
2019
- 2019-03-06 CN CN201910169423.9A patent/CN109884224B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4642286A (en) * | 1984-05-07 | 1987-02-10 | Moldowan Mervin J | Composition and method for ethanol determination |
| CN108938626A (en) * | 2018-07-27 | 2018-12-07 | 四川联成迅康医药股份有限公司 | A kind of good and highly-safe Carbazochrome sodium sulfonate pharmaceutical composition and its preparation method and application of stability |
Non-Patent Citations (4)
| Title |
|---|
| In vitro biotransformation of 3-nitropropanol (miserotoxin aglycon) by horse liver alcohol dehydrogenase;M. H. Benn 等;《Toxicology Letters》;19890531;第47卷(第2期);第165-172页 * |
| Isolation, Structural Characterization, and Validation of a New Compound Present in Non‐Carbonyl Curcuma longa (NCCL): A Potential Lead for Stroke;Swati Gupta 等;《Journal of Heterocyclic Chemistry》;20180704;第55卷(第8期);第1926-1934页 * |
| Mechanism of semicarbazone formation in methanol solution. Evidence of a side reaction and origin of the optimum pH;H. P. Figeys 等;《Bulletin des Societes Chimiques Belges》;19660101;第75卷(第9-10期);第601-619页 * |
| Semicarbazide Hydrochloride as Impurity in Drug Substances: a Validated LC-DAD-UV Method for Its Determination in Carbazochrome and Carbazochrome Sodium Sulfonate;Rossana Canavesi 等;《Chromatographia》;20170802;第80卷(第10期);第1535-1544页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109884224A (en) | 2019-06-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103983725B (en) | The rapid assay methods of cumarin and safrole in a kind of essence and flavoring agent | |
| CN111693633B (en) | Method for detecting 3,4-dimethoxy benzoyl chloride in itopride hydrochloride | |
| CN109580821B (en) | Method for detecting impurity succinic acid in S-benzylsuccinic acid | |
| CN113125611B (en) | Method for detecting content of impurity 6-formyl pterin folic acid | |
| CN108152425B (en) | Method for detecting lignanoids in sesame oil by high performance liquid chromatography | |
| CN108445098B (en) | Analysis method for detecting impurities in vitamin A palmitate | |
| CN109884224B (en) | Carbazochrome sodium sulfonate and method for measuring content of semicarbazide hydrochloride in preparation thereof | |
| CN107688065B (en) | Gas chromatography-mass spectrometry combined detection method for chloromethyl methyl ether residue in bulk drug | |
| CN111983054B (en) | Method for separating and measuring related substances of empagliflozin intermediate by using HPLC (high performance liquid chromatography) | |
| CN115616133A (en) | Method for detecting cysteine in compound amino acid injection and application thereof | |
| CN115774061A (en) | Method for detecting acetic acid in 1-cyclohexyl piperazine | |
| CN110231416B (en) | Method for measuring 2-iodoxybenzoic acid related substances by using HPLC (high performance liquid chromatography) | |
| CN110187021B (en) | Method for simultaneously determining contents of two main drugs in closantel sodium ivermectin injection | |
| Rivai et al. | Development and validation of bisacodyl analysis method in tablet with absorbance method and area under curves method in ultraviolet spectrophotometry | |
| CN110907541B (en) | Method for simultaneously measuring R-epoxypropanol and R-3-chlorine-1,2-propylene glycol residues in L-alpha-glycerophosphorylcholine | |
| CN109374778B (en) | Method for determining organic impurities in 2-mercaptobenzimidazole | |
| CN114264765A (en) | Analysis method for determining related substances in glimepiride intermediate by using HPLC | |
| Gujral et al. | A validated method without derivatization for the determination of gabapentin in bulk, pharmaceutical formulation and human urine samples | |
| Sajedi-Amin et al. | Development and validation of ultrasound assisted and dispersive liquid-liquid microextractions combined with HPLC-UV method for determination of bosentan in human plasma and urine | |
| CN114113402B (en) | Method for measuring pinanediol content in bortezomib by adopting high performance liquid chromatography | |
| CN111351886A (en) | Method for determining impurities contained in etamsylate and content of main drug thereof | |
| CN115327006B (en) | Method for detecting clopidogrel isomer | |
| CN117471001B (en) | Method for measuring content of N-bromosuccinimide in starting material of Rate Lu Geli | |
| CN111443141B (en) | Method for detecting content of effective components in health wine | |
| CN117054579B (en) | Method for treating a sample, method for determining the content of a substance of interest in a sample |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |