CN117233309A - Method for detecting impurities of multivitamin injection - Google Patents

Method for detecting impurities of multivitamin injection Download PDF

Info

Publication number
CN117233309A
CN117233309A CN202311173218.2A CN202311173218A CN117233309A CN 117233309 A CN117233309 A CN 117233309A CN 202311173218 A CN202311173218 A CN 202311173218A CN 117233309 A CN117233309 A CN 117233309A
Authority
CN
China
Prior art keywords
solution
vitamin
water
injection
precisely
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.)
Pending
Application number
CN202311173218.2A
Other languages
Chinese (zh)
Inventor
胡军
付国莉
田琳
谢景凤
刘烽
张勇
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Inner Mongolia Baiyi Pharmaceutical Co ltd
Beijing Zangwei Xinkang Medicine Research And Development Co ltd
Original Assignee
Inner Mongolia Baiyi Pharmaceutical Co ltd
Beijing Zangwei Xinkang Medicine Research And Development Co ltd
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Inner Mongolia Baiyi Pharmaceutical Co ltd, Beijing Zangwei Xinkang Medicine Research And Development Co ltd filed Critical Inner Mongolia Baiyi Pharmaceutical Co ltd
Priority to CN202311173218.2A priority Critical patent/CN117233309A/en
Publication of CN117233309A publication Critical patent/CN117233309A/en
Pending legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/8872—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample impurities

Landscapes

  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The application relates to an analysis and detection method of impurities in a multivitamin injection, which belongs to the field of medicine analysis and mainly relates to oxalic acid, 3-aminopropanol, retinol, alpha-tocopherol, menaquinone and vitamin K in the multivitamin injection 1 A method for detecting cis-isomer impurities.

Description

Method for detecting impurities of multivitamin injection
The application is a divisional application of the application application of which the application date is 2018, 7, 12, application number is 201810765320.4 and the application name is a detection method of multi-vitamin injection impurities.
Technical Field
The application relates to an analysis and detection method for multi-vitamin injection impurities, belonging to the field of medicine analysis.
Background
Parenteral nutrition plays an important supporting role in the treatment of diseases and is widely used for the treatment of congenital digestive tract deformity, moderate and severe malnutrition, digestive tract diseases (severe diarrhea, necrotizing enterocolitis, early short bowel syndrome, intestinal obstruction, intestinal fistula, necrotizing pancreatitis), severe-risk neonates who cannot normally ingest, severe gastrointestinal reactions caused by radiotherapy and chemotherapy, severe infection, major surgery, wounds, burns and the like. Vitamins are trace organic substances necessary for maintaining life activities of human bodies, and cannot be generally synthesized in the human body through assimilation of the vitamins, so that normal human bodies can obtain vitamins required by the human bodies through foods, and burns, operations, severe wounds, infections and the like can cause ultrahigh metabolism and the like to cause malnutrition of the human bodies, including deficiency of vitamins, increase of the requirements of patients on vitamins, supplement of extra-intestinal vitamins and benefit of rehabilitation of the patients.
The vitamins can be divided into fat-soluble vitamins and water-soluble vitamins according to solubility, wherein the fat-soluble vitamins mainly comprise vitamin A, vitamin E, vitamin K, vitamin D and analogues thereof, and the water-soluble vitamins mainly comprise vitamin B 1 Vitamin B 2 Vitamin B 6 Vitamin B 12 Folic acid, dexpanthenol, biotin, nicotinamide, vitamin C, and analogs thereof.
According to clinical requirements, the multivitamin injection contains the above water-soluble vitamins and liposoluble vitamins, and related commercial preparations are also marketed, such as injectable water-soluble vitamins containing 9 water-soluble vitamins, vitamin C and vitamin B 1 Compound vitamin (3) for injection and compound vitamin (3) injection containing 3 water-soluble vitamins of riboflavin sodium phosphate, and fat-soluble vitamin (I) for injection and fat-soluble vitamin (II) for injection containing 4 fat-soluble vitaminsThe fat-soluble vitamin injection (II) contains 12 vitamins of multiple vitamins for injection (12), 12 vitamins of multiple vitamins for injection (13), 13 vitamins of multiple vitamins for children injection (13), multiple vitamins for children injection (13) and multiple vitamins for injection (13).
The prior art shows that the physical and chemical properties of the vitamin components are complex, unstable and sensitive to light, heat, oxygen, metal ions, acidity and the like, and the quality control of single vitamin injection is more reported, such as fat-soluble vitamin injection (containing vitamin A palmitate and vitamin D) 2 Vitamin E and vitamin K) and water-soluble vitamins for injection (containing thiamine nitrate, riboflavin sodium phosphate, nicotinamide 40mg, pyridoxine hydrochloride, sodium pantothenate, sodium ascorbate, biotin, folic acid and vitamin B) 12 ) The method has the advantages that the method is carried out in the national drug standard (fifth volume of drug standard), some vitamin raw materials and detection methods of preparations are also available in the literature, as in the invention patent 201510181413.9, the multi-vitamin injection has higher safety requirements due to administration by injection, and the multi-component injection has complex components, different from the single-component injection in detection, has larger mutual interference, has trace vitamin content, especially for degrading impurities, lacks related control measures for the impurities of the multi-component vitamin injection at present, and has the difficulty of quality control detection, especially impurity detection, of the compound vitamin injection. The related technology of the prior literature reports detection of single preparation impurities, such as vitamin C injection and vitamin E injection, but the detection method of single preparation impurities has great interference and is not feasible.
Disclosure of Invention
The invention provides an analysis and detection method for multi-vitamin injection impurities.
The invention establishes oxalic acid, 3-aminopropanol, retinol, alpha-tocopherol, menaquinone and vitamin K in the multivitamin injection 1 Method for detecting cis-isomer and other impurities and method for carrying out related methodThe verification work is carried out to study the specificity, the precision, the reproducibility, the method durability, the linearity, the detection limit and the like, and the verification result shows that the impurity analysis method established by the invention is feasible and can be used for analyzing and detecting the related impurities of the multi-vitamin injection.
The method for detecting oxalic acid impurities of the multivitamin injection comprises the following steps:
(1) Sample solution preparation: taking a multi-vitamin injection solution (freeze-dried powder injection is dissolved by adding water), adding diluted acetic acid and calcium chloride test solution, shaking uniformly, standing, adding water and keeping the volume consistent with that of a control solution to be used as a test sample solution;
(2) Preparation of intermediate solution: taking multiple vitamin injection solution (freeze-dried powder injection is dissolved in water) by the same method, operating by the same method, filtering after standing, and taking filtrate;
(3) Control solution preparation: precisely measuring oxalic acid solution (equivalent to 0.2% -0.5% of the vitamin C), adding into the filtrate of (2), shaking uniformly, and standing to obtain control solution;
(4) And (3) detection: comparing the turbidity generated by the sample solution with the turbidity generated by the control solution.
The method for detecting oxalic acid impurities of the multivitamin injection comprises the following steps:
(1) Taking a multi-vitamin injection solution (equivalent to 200mg of vitamin C, and dissolving a freeze-dried powder injection in water), adding 1ml of dilute acetic acid and 0.5ml of calcium chloride test solution, shaking uniformly, standing for 1 hour, adding 0.5ml of water, and shaking uniformly to obtain a sample solution;
(2) Taking multiple vitamin injection (corresponding to 200mg of vitamin C, dissolving the freeze-dried powder injection in water) by the same method, performing the same method until the vitamin injection is placed for 1 hour, filtering, and taking filtrate;
(3) Precisely measuring 0.5ml of oxalic acid solution (proper amount of oxalic acid is taken, precisely measured, dissolved in water and diluted to prepare a solution containing 1.2mg of oxalic acid in each 1 ml), adding the solution into filtrate, shaking uniformly, and standing for 1 hour to obtain a control solution;
(4) The turbidity produced by the test solution was observed not to be stronger than that of the control solution (0.3%).
The method for detecting the impurity of the 3-aminopropanol of the multivitamin injection comprises the following steps:
(1) Sample solution preparation: taking multiple vitamin injection solution (freeze-dried powder injection is dissolved in water), and diluting with water to obtain sample solution;
(2) Preparing a reference substance solution: taking 3-aminopropanol reference substance, precisely weighing, and dissolving in water to obtain reference substance solution.
(3) And (3) detection: taking a test sample solution and a reference substance solution, measuring according to a high performance liquid chromatography (the fourth edition of China pharmacopoeia 2015, general rule 0512), taking octadecylsilane chemically bonded silica as a filler, taking 0.1% of heptafluorobutyric anhydride aqueous solution as a mobile phase A, taking 0.1% of heptafluorobutyric anhydride acetonitrile solution as a mobile phase B, performing gradient elution according to the following table, taking the detector as an evaporative light scattering detector, respectively precisely measuring different volumes of reference substance solutions, injecting the reference substance solutions into a liquid chromatograph, recording a chromatogram, calculating a regression equation according to the logarithm of the sample injection amount of the reference substance and the corresponding logarithmic value of the peak area, precisely measuring the test sample solution, injecting the reference substance solutions into the liquid chromatograph, and recording the chromatogram. And calculating the content of 3-aminopropanol in the test sample according to a regression equation.
The method for detecting the impurity of the 3-aminopropanol of the multivitamin injection comprises the following steps:
(1) Sample solution preparation: taking multiple vitamin injection solution (freeze-dried powder injection is dissolved in water), and diluting with water to obtain sample solution;
(2) Preparing a reference substance solution: taking 3-aminopropanol reference substance, precisely weighing, and dissolving in water to obtain reference substance solution.
(3) And (3) detection: and (3) taking a sample solution and a reference substance solution, separating and measuring by using an amino acid analyzer, and calculating by using peak area according to an external standard method.
The method for detecting the impurity of the 3-aminopropanol of the multivitamin injection comprises the following steps:
(1) Taking a multi-vitamin injection solution (freeze-dried powder injection is dissolved by adding water), and adding water to dilute the multi-vitamin injection solution to prepare a solution containing about 0.25mg of dexpanthenol in each 1ml serving as a test sample solution;
(2) Taking a proper amount of 3-aminopropanol reference substance, precisely weighing, adding water for dissolving, and preparing a solution containing 2.5 mug of 3-aminopropanol in 1ml as a reference substance solution;
(3) The separation was performed using an amino acid analyzer using SYKNM-Cation Separation Column (LCA K07/Li,4.6 mm. Times.150 mm) as a column; taking buffer solution A, buffer solution C, regeneration solution and ninhydrin derivative as mobile phases, eluting with an elution pump flow rate of 0.45ml per minute and a derivative pump flow rate of 0.25ml per minute, performing gradient elution according to the following table (1), detecting 570nm of wavelength, changing the column temperature according to the gradient of the following table (2), wherein the temperature of the reactor is 130 ℃, the acquisition time is 30 minutes, precisely measuring 50 μl of each of the sample solution and the reference solution, respectively injecting into an amino acid analyzer, recording a chromatogram, and calculating according to an external standard method by peak area to obtain the final product;
Watch (1)
Watch (2)
Buffer a: precisely weighing 5.04g of lithium hydroxide monohydrate and 15.0g of citric acid, adding a proper amount of water for dissolution, precisely adding 50ml of ethanol, 7.8ml of hydrochloric acid and 0.1ml of octanoic acid, diluting to 1000ml with water, uniformly mixing, adjusting the pH value to 2.9 with lithium hydroxide monohydrate or hydrochloric acid, shaking uniformly, filtering and performing ultrasonic treatment;
buffer C: precisely weighing 10.0g of boric acid, 4.2g of lithium chloride, 8.4g of lithium hydroxide monohydrate and 10.0g of citric acid, adding a proper amount of water for dissolution, precisely adding 3.3ml of hydrochloric acid and 0.1ml of octanoic acid, diluting to 1000ml with water, uniformly mixing, adjusting the pH value to 8.0 with lithium hydroxide monohydrate or hydrochloric acid, shaking uniformly, filtering and performing ultrasonic treatment;
regeneration liquid: accurately weighing 21.0g of lithium hydroxide monohydrate and 0.2g of EDTA0, placing into a 1000ml measuring flask, adding water for dissolution, diluting to scale, and shaking uniformly;
ninhydrin derivatizing agent: 600ml of methanol was precisely weighed into a beaker, 20g of ninhydrin crystals and 2g of phenol were added, stirred with a stirrer until the ninhydrin crystals were completely dissolved, filtered, 400ml of filtered potassium-sodium buffer solution (196 g of potassium acetate and 272g of sodium acetate trihydrate were precisely weighed into a beaker, 508ml of water was added to dissolve, 200ml of acetic acid was slowly added into the solution while stirring, mixed uniformly, cooled to room temperature, filtered), mixed uniformly, transferred into a ninhydrin reagent bottle, blown with nitrogen from the bottom for about 3 to 5 minutes, added with 0.2g of ascorbic acid, blown with nitrogen from the bottom for about 3 to 5 minutes, and left to stand for use.
The multivitamin injection of the invention comprises retinol, alpha-tocopherol, menaquinone and vitamin K 1 Method for detecting cis-isomer impurity, operation in dark place, retinol, menaquinone and vitamin K 1 The cis-isomer is detected by liquid chromatography, and the alpha-tocopherol is detected by gas chromatography, and is characterized in that: the preparation method of the test solution comprises dissolving multiple vitamins in water (lyophilized powder for injection), adding water-insoluble organic solvent, shaking for extraction, collecting water-insoluble organic solvent layer, volatilizing, adding organic solvent to dissolve, shaking, and collecting water-insoluble organic solvent selected from n-hexane, cyclohexane, petroleum ether, n-heptane, chloroform, and diethyl ether.
The multivitamin injection of the invention comprises retinol, alpha-tocopherol, menaquinone and vitamin K 1 Method for detecting cis-isomer impurity, operation in dark place, retinol, menaquinone and vitamin K 1 The cis-isomer is detected by liquid chromatography, and the alpha-tocopherol is detected by gas chromatography, and is characterized in that: the preparation method of the test solution comprises dissolving multivitamin injection solution (lyophilized powder for injection in water ) Adding 5% -90% concentration water-miscible organic solvent, adding water-immiscible organic solvent, shaking and extracting, taking water-immiscible organic solvent layer, volatilizing, adding organic solvent to dissolve, shaking uniformly, and taking as sample solution, wherein the water-immiscible organic solvent is selected from n-hexane, cyclohexane, petroleum ether, n-heptane, chloroform and diethyl ether, and the water-miscible organic solvent is selected from methanol, ethanol, absolute ethanol, acetonitrile and isopropanol, and the organic solvent can be selected from the water-immiscible organic solvents and water-miscible organic solvents.
The method for detecting the retinol impurity in the multivitamin injection comprises the following steps:
(1) Sample solution preparation: taking a multi-vitamin injection solution (freeze-dried powder injection is dissolved by adding water), or adding a water-miscible organic solvent with the concentration of 5% -90%, adding a water-immiscible organic solvent, shaking and extracting, taking a water-immiscible organic solvent layer, volatilizing, adding the organic solvent to dissolve, shaking uniformly, and taking the mixture as a sample solution; or taking an organic solvent layer which is not mutually soluble with water as a sample solution;
(2) Preparation of a control solution: precisely weighing retinol reference substance, adding organic solvent to dissolve, shaking to obtain reference substance solution;
(3) And (3) detection: according to high performance liquid chromatography (China pharmacopoeia 2015 edition four general rules 0512), octadecylsilane chemically bonded silica is used as a filler, acetonitrile-methanol (60:40) is used as a mobile phase, the temperature of a column greenhouse is 45 ℃, the detection wavelength is 325+/-3 nm, a sample solution and a reference solution are precisely measured, the sample solution and the reference solution are respectively injected into a liquid chromatograph, a chromatogram is recorded, and the sample solution and the reference solution are calculated according to the external standard method and the peak area.
The method for detecting alpha-tocopherol impurity of the multivitamin injection comprises the following steps:
(1) And (3) preparing an internal standard solution: precisely weighing squalane reference substance, and adding organic solvent to dissolve to obtain internal standard solution;
(2) Sample solution preparation: taking a multi-vitamin injection solution (freeze-dried powder injection is dissolved by adding water), or adding a water-miscible organic solvent with the concentration of 5% -90%, adding a water-immiscible organic solvent, carrying out shaking extraction, taking a water-immiscible organic solvent layer, adding an internal standard solution, and shaking uniformly to obtain a sample solution;
(3) Preparation of a control solution: precisely weighing alpha-tocopherol reference substance, adding organic solvent for dissolving, adding internal standard solution, and shaking to obtain reference substance solution.
(4) And (3) detection: according to the measurement of gas chromatography (China pharmacopoeia 2015 edition four general rules 0521), a capillary chromatographic column using dimethoxy polysiloxane as a fixed liquid is used, the column temperature is 260-310 ℃, the detector temperature is 260-310 ℃, the sample inlet temperature is 270-310 ℃, carrier gas is nitrogen, a sample solution and a reference solution are precisely measured, the sample solution and the reference solution are respectively injected into a gas chromatograph, the chromatogram is recorded, and the peak area is calculated according to an internal standard method, thus obtaining the sample.
The method for detecting the menaquinone impurity of the multi-vitamin injection comprises the following steps:
(1) Sample solution preparation: taking a multi-vitamin injection solution (freeze-dried powder injection is dissolved by adding water), or adding a water-miscible organic solvent with the concentration of 5% -90%, adding a water-immiscible organic solvent, shaking and extracting, taking a water-immiscible organic solvent layer, volatilizing, adding the organic solvent to dissolve, shaking uniformly, and taking the mixture as a sample solution; or taking an organic solvent layer which is not mutually soluble with water as a sample solution;
(2) Preparation of a control solution: taking menadione reference substance, precisely weighing, adding organic solvent to dissolve, shaking uniformly to obtain reference substance solution;
(3) And (3) detection: according to high performance liquid chromatography (China pharmacopoeia 2015 edition four general rules 0512), octadecylsilane chemically bonded silica is used as a filler, methanol-water (65:35) is used as a mobile phase, the temperature of a column greenhouse is 45 ℃, the detection wavelength is 250+/-3 nm, a test sample solution and a reference sample solution are precisely measured, the test sample solution and the reference sample solution are respectively injected into a liquid chromatograph, a chromatogram is recorded, and the test sample solution and the reference sample solution are calculated according to the external standard method and the peak area.
The multivitamin injection vitamin K of the invention 1 A method for detecting cis-isomer impurities, comprising the steps of:
(1) Sample solution preparation: taking a multi-vitamin injection solution (freeze-dried powder injection is dissolved by adding water), or adding a water-miscible organic solvent with the concentration of 5% -90%, adding a water-immiscible organic solvent, shaking and extracting, taking a water-immiscible organic solvent layer, volatilizing, adding the organic solvent to dissolve, shaking uniformly, and taking the mixture as a sample solution; or taking an organic solvent layer which is not mutually soluble with water as a sample solution;
(2) Preparation of a control solution: taking vitamin K 1 Precisely weighing reference substance, adding organic solvent to dissolve, and mixing to obtain reference substance solution;
(3) And (3) detection: according to high performance liquid chromatography (rule 0512 of four edition of China pharmacopoeia 2015), silica gel is used as filler, 1, 4-dioxane-n-octanol-petroleum ether (60-90 ℃) (1:3:96) is used as mobile phase A, n-hexane is used as mobile phase B, gradient elution is carried out according to the following table, the detection wavelength is 264nm, the flow rate is 2.0ml per minute, and the vitamin K is obtained 1 The degree of separation between the cis and trans isomer peaks of (c) should be satisfactory,
measuring reference solution, injecting into liquid chromatograph, recording chromatogram, and vitamin K 1 Sequentially taking the cis isomer and the trans isomer, taking the solution of the sample, injecting the solution into a liquid chromatograph, recording the chromatogram, and calculating according to an area normalization method to obtain the compound.
The main medicine component of the multivitamin injection provided by the invention is selected from vitamin A palmitate and vitamin D 3 Or an analogue thereof, vitamin E or an analogue thereof, vitamin K 1 Vitamin C, its salts and vitamin B 1 Or an analogue thereof, riboflavin sodium phosphate, dexpanthenol or an analogue thereof, nicotinamide, vitamin B 6 Or an analogue thereof, vitamin B 12 2 or more of folic acid and biotin, and pharmaceutically acceptable auxiliary materials.
The multivitamin injection provided by the invention comprises the following components in percentage by weight:
the multivitamin injection provided by the invention comprises the following components in percentage by weight: prescription 1
Or prescription 2
Or prescription 3
The multivitamin injection provided by the invention comprises the following components in percentage by weight: prescription 4
Or prescription 5
Or prescription 6
Vitamin C200.0 mg
Vitamin B 1 10.0mg
Riboflavin sodium phosphate (equivalent to riboflavin 5 mg) 6.355mg or formula 7
Or prescription 8
Or prescription 9
The active ingredients contained in the composition of the invention are:
vitamin D 3 Or its analogue can be vitamin D 2 Vitamin D 3 Alfacalcidol, and the like.
Vitamin E or its analogues are vitamin E, racemic alpha-tocopherol, etc.;
The vitamin C and its salt can be sodium ascorbate and vitamin C.
Vitamin B 1 Or analogs thereof, may be thiamine, thiamine hydrochloride, thiamine nitrate, thiamine phosphate, cocarboxylase tetrahydrate, and the like.
Vitamin B 6 Or its analogues may be pyridoxine hydrochloride, pyridoxine, bipolyaldehyde, bipolyamine, vitamin B 6 Etc.
Dexpanthenol and analogues thereof can be pantothenic acid, sodium pantothenate, calcium pantothenate, panthenol, dexpanthenol, and the like.
The prescriptions 1 to 9 can be in the form of water injection and freeze-dried powder injection.
Detailed Description
The related detection conditions described in the present invention are preferable conditions, such as conditions of high performance liquid chromatography, and the following examples are preferable conditions, and the conditions can be specifically adjusted according to different high performance liquid chromatographs and chromatographic column types, such as adjusting the proportion of eluting solvents, adjusting the time of gradient elution program, adjusting the proportion of gradient elution mobile phases, and defining the adjusted chromatographic proportion under the rule 0512 of four portions of the 2015 edition of Chinese pharmacopoeia to be consistent with the chromatographic conditions described in the present invention.
The detection methods of the invention are verified according to the requirements of the 2015 edition of Chinese pharmacopoeia, and the verification results show that the detection methods of the invention are feasible and reliable.
Example 1
Oxalic acid detection
Prescription 1, prescription 2, prescription 3, prescription 6 sample water needle: taking the product, precisely measuring a solution (about 200mg containing vitamin C), adding 1ml of dilute acetic acid and 0.5ml of calcium chloride test solution, shaking uniformly, standing for 1 hour, adding 0.5ml of water, and shaking uniformly to obtain a sample solution; in addition, the solution (about 200mg containing vitamin C) of the product is precisely measured, the operation is carried out in the same way until the solution is placed for 1 hour, then the solution is filtered, the oxalic acid solution (a proper amount of oxalic acid is taken, precisely measured, water is added for dissolution and dilution to prepare a solution with 1.2mg in each 1 ml) is precisely measured, 0.5ml of the solution is added into the filtrate, and the solution is uniformly shaken and placed for 1 hour to be used as a control solution. The turbidity produced by the test solution must not be greater than that of the control solution (0.3%).
Prescription 1, prescription 2, prescription 3, prescription 4, prescription 5, prescription 6 sample lyophilized powder for injection: dissolving the product in water, quantitatively diluting to obtain solution containing vitamin C40 mg per 1ml, precisely measuring 5ml, adding diluted acetic acid 1ml and calcium chloride 0.5ml, shaking, standing for 1 hr, adding water 0.5ml, shaking, and collecting sample solution; in addition, precisely measuring 5ml of the product solution, carrying out the same operation until the solution is placed for 1 hour, filtering, precisely measuring 0.5ml of oxalic acid solution (a proper amount of oxalic acid is taken, precisely measured, dissolved in water and diluted to prepare a solution containing 1.2mg of oxalic acid in each 1 ml), adding the solution into filtrate, shaking uniformly, and placing for 1 hour to obtain a control solution. The turbidity produced by the test solution must not be greater than that of the control solution (0.3%).
Comparative example: taking a water injection of a prescription 1, a prescription 2, a prescription 3 and a prescription 6 sample and a freeze-dried powder injection (dissolved by adding water) of a prescription 1, a prescription 2, a prescription 3, a prescription 4, a prescription 5 and a prescription 6 sample, and detecting according to a method of injecting two vitamin C or vitamin C injection in China pharmacopoeia 2015, namely precisely measuring a solution (about 200mg containing vitamin C), adding 1ml of dilute acetic acid and 0.5ml of calcium chloride test solution, shaking uniformly, standing for 1 hour, adding 0.5ml of water, shaking uniformly, and taking the solution as a test solution; in addition, 0.5ml of oxalic acid solution (a proper amount of oxalic acid is taken, precisely weighed, dissolved and diluted into a solution containing 1.2mg in each 1 ml) is precisely measured, 1ml of diluted acetic acid and 0.5ml of calcium chloride test solution are added, the volumes of the added water and the test solution are consistent, the mixed solution is uniformly shaken, the mixed solution is placed for 1 hour, and the mixed solution is uniformly shaken to serve as a reference solution, so that the result shows that the reference solution generates white turbidity, but the test solution is yellow and cannot be compared with the reference solution.
Example 2
Retinol assay
Prescription 1, prescription 2, prescription 3, prescription 7, prescription 8, prescription 9 sample water needle: and (5) operating in a dark place. Precisely measuring the solution (about referred to vitamin A6600 IU), placing into a 250ml separating funnel, adding 80% ethanol solution 40ml, shaking, precisely adding n-hexane 25ml, shaking and extracting for 30 min, standing to obtain supernatant, precisely removing 10ml supernatant, placing into a conical flask, blow-drying with nitrogen, precisely adding isopropanol 10ml to dissolve, shaking, and taking as sample solution; and (3) taking a proper amount of retinol reference substance, precisely weighing, adding isopropanol for dissolving and quantitatively diluting to prepare a solution containing about 1.45 mug in each 1ml, and shaking uniformly to serve as a reference substance solution. Determining by high performance liquid chromatography (China pharmacopoeia 2015 edition four general rules 0512), and using octadecylsilane chemically bonded silica gel (4.6 mm×250mm) as filler; acetonitrile-methanol (60:40) as mobile phase; column temperature is 30 ℃; the detection wavelength was 325nm. Precisely measuring 10 μl of each of the sample solution and the reference solution, respectively injecting into a liquid chromatograph, and recording the chromatograms. The chromatographic peak with the retention time consistent with retinol is obtained by calculating the peak area according to the external standard method.
Prescription 1, prescription 2, prescription 3, prescription 4, prescription 7, prescription 8, prescription 9 sample lyophilized powder for injection: and (5) operating in a dark place. Dissolving the product in water, quantitatively diluting to obtain a solution containing 660IU of vitamin A in each 1ml, quantitatively transferring to a 250ml separating funnel, adding 40ml of 80% ethanol solution, shaking uniformly, precisely adding 25ml of n-hexane, shaking and extracting for 30 minutes (shaking frequency is 180 revolutions per minute), standing to obtain supernatant, precisely transferring 10ml of supernatant, placing into an conical flask, drying with nitrogen, precisely adding 10ml of isopropanol to dissolve, shaking uniformly, and taking as a sample solution; and (3) taking a proper amount of retinol reference substance, precisely weighing, adding isopropanol for dissolving and quantitatively diluting to prepare a solution with the concentration of about 1.45 mug in each 1ml serving as the reference substance solution. Determining by high performance liquid chromatography (China pharmacopoeia 2015 edition four general rules 0512), and using octadecylsilane chemically bonded silica gel (4.6 mm×250mm) as filler; acetonitrile-methanol (60:40) as mobile phase; the detection wavelength is 325nm; column temperature was 30 ℃. Precisely measuring 10 μl of each of the sample solution and the reference solution, respectively injecting into a liquid chromatograph, and recording the chromatograms. The chromatographic peak with the retention time consistent with retinol is obtained by calculating the peak area according to the external standard method.
Prescription 3 sample water needle: and (5) operating in a dark place. Precisely weighing the product (about equivalent to vitamin A4600 IU), placing into a 250ml separating funnel, adding 80% ethanol solution 40ml, shaking uniformly, precisely adding n-hexane 25ml, shaking and extracting for 30 minutes (shaking frequency is 180 rpm), placing into an upper layer liquid for clarification, precisely removing 10ml of supernatant, placing into a conical flask, drying with nitrogen, precisely adding isopropanol 10ml to dissolve, shaking uniformly, and taking as a sample solution; and (3) taking a proper amount of retinol reference substance, precisely weighing, adding isopropanol for dissolving and quantitatively diluting to prepare a solution containing about 1.01 mug of each 1ml of retinol reference substance, and shaking uniformly to obtain the reference substance solution. Determining by high performance liquid chromatography (China pharmacopoeia 2015 edition four general rules 0512), and using octadecylsilane chemically bonded silica gel (4.6 mm×250mm) as filler; acetonitrile-methanol (60:40) as mobile phase; column temperature is 30 ℃; the detection wavelength was 325nm. Precisely measuring 10 μl of each of the sample solution and the reference solution, respectively injecting into a liquid chromatograph, and recording the chromatograms. The chromatographic peak with the retention time consistent with retinol is obtained by calculating the peak area according to the external standard method.
Prescription 3 sample lyophilized powder injection: and (5) operating in a dark place. Taking 2 bottles of the product, adding water for dissolution and quantitatively diluting to prepare a solution containing about 575IU of vitamin A in each 1ml, quantitatively transferring to a 250ml separating funnel, adding 40ml of 80% ethanol solution, shaking uniformly, precisely adding 25ml of n-hexane, shaking and extracting for 30 minutes (the shaking frequency of an oscillator is 180 revolutions per minute), standing to obtain supernatant for clarification, precisely transferring 10ml of supernatant, placing into a conical flask, drying by nitrogen, precisely adding 10ml of isopropanol to dissolve, shaking uniformly, and taking the supernatant as a sample solution; and (3) taking a proper amount of retinol reference substance, precisely weighing, adding isopropanol for dissolving and quantitatively diluting to prepare a solution containing about 1.01 mug of each 1ml of retinol reference substance, and shaking uniformly to obtain the reference substance solution. According to high performance liquid chromatography (China pharmacopoeia 2015 edition, ministry of the fourth edition 0512), octadecylsilane chemically bonded silica (4.6 mm×250mm,5 μm) is used as filler; acetonitrile-methanol (60:40) as mobile phase; the detection wavelength is 325nm; column temperature was 30 ℃. Precisely measuring 10 μl of each of the sample solution and the reference solution, respectively injecting into a liquid chromatograph, and recording the chromatograms. The chromatographic peak with the retention time consistent with retinol is obtained by calculating the peak area according to the external standard method. The vitamin A palmitate deficiency prescription 3 solution is prepared into a sample solution by the same method, and chromatographic detection is carried out by the same method, so that no vitamin A palmitate chromatographic peak, no retinol chromatographic peak and no interference to the determination of the product are found.
Comparative test example: taking a water injection of a prescription 3 sample and a freeze-dried powder injection of the prescription 3 sample (adding 4ml of water to dissolve) as a test solution, and taking the retinol reference solution. According to high performance liquid chromatography (China pharmacopoeia 2015 edition, ministry of the fourth edition 0512), octadecylsilane chemically bonded silica (4.6 mm×250mm,5 μm) is used as filler; acetonitrile-methanol (60:40) as mobile phase; the detection wavelength is 325nm; column temperature was 30 ℃. Precisely measuring 10 μl of each of the sample solution and the reference solution, respectively injecting into a liquid chromatograph, and recording the chromatograms, wherein the chromatograms of the sample solution have chromatographic peaks consistent with the retention time of retinol, but the retinol chromatographic peaks are not well separated, are wrapped by adjacent chromatographic peaks, and cannot be well separated and cannot be detected.
Example 3
3-aminopropanol assay
Prescription 1, prescription 2, prescription 3, prescription 4 water injection or freeze-dried powder injection: taking a sample, adding water to prepare a sample solution containing about 3mg of dexpanthenol in each 1 ml; and taking a proper amount of 3-aminopropanol reference substance, precisely weighing, adding water for dissolving, and quantitatively diluting to prepare a solution containing 30 mug of 3-aminopropanol in 1ml serving as the reference substance solution. According to high performance liquid chromatography (China pharmacopoeia 2015 edition, division 0512), octadecylsilane chemically bonded silica is used as filler (250 mm×4.6mm,5 μm); taking 0.1% aqueous solution of heptafluorobutyric anhydride as a mobile phase A and 0.1% acetonitrile solution of heptafluorobutyric anhydride as a mobile phase B; the column temperature is 30 ℃; gradient elution was performed according to the following table; the detector was an evaporative light scattering detector (reference condition: using split mode, drift tube temperature 70 ℃ C., carrier gas flow rate 2.5L per minute). Precisely measuring 5 μl, 10 μl and 20 μl of the reference solution, respectively, injecting into a liquid chromatograph, recording the chromatogram, calculating regression equation with logarithm of the reference sample injection amount and corresponding logarithm of peak area, precisely measuring 20 μl of the sample solution, injecting into the liquid chromatograph, and recording the chromatogram. And calculating the content of 3-aminopropanol in the sample by a regression equation to obtain the product.
Prescription 1, prescription 2, prescription 3 water needle: taking a sample, adding water for dissolving and diluting to prepare a solution containing about 0.75mg of dexpanthenol in each 1ml, and taking the solution as a sample solution; and taking a proper amount of 3-aminopropanol reference substance, precisely weighing, adding water for dissolving and diluting to prepare a solution containing about 7.5 mug of 3-aminopropanol in 1ml serving as a reference substance solution. And (3) measuring by using a proper amino acid analyzer, and calculating by using peak area according to an external standard method.
The reference method comprises the following steps: SYKNM-Cation Separation Column (LCA K07/Li,4.6 mm. Times.150 mm) was used as the column; buffer solution A, buffer solution C, regeneration solution and ninhydrin derivative are taken as mobile phases; the elution pump flow rate was 0.45ml per minute, the derivatization pump flow rate was 0.25ml per minute, and gradient elution was performed as in table (1) below; the detection wavelength is 570nm; the column temperature was varied in gradient according to table (2); the reactor temperature was 130 ℃; the acquisition time is 30 minutes; precisely measuring 50 μl of each of the sample solution and the control solution, respectively injecting into an amino acid analyzer, and recording chromatograms.
Table (1):
table (2):
prescription 1, prescription 2, prescription 3, prescription 4 lyophilized powder injection: dissolving the product in water, and diluting to obtain solution containing about 0.25mg of dexpanthenol in each 1ml as test solution; and taking a proper amount of 3-aminopropanol reference substance, precisely weighing, adding water for dissolving, and preparing a solution containing 2.5 mug of 3-aminopropanol in 1ml serving as a reference substance solution. Respectively carrying out separation and determination by using a proper amino acid analyzer; and calculating according to an external standard method and peak area to obtain the product.
The reference method comprises the following steps: SYKNM-Cation Separation Column (LCA K07/Li,4.6 mm. Times.150 mm) was used as the column; buffer solution A, buffer solution C, regeneration solution and ninhydrin derivative are taken as mobile phases; the elution pump flow rate was 0.45ml per minute, the derivatization pump flow rate was 0.25ml per minute, and gradient elution was performed as in table (1) below; the detection wavelength is 570nm; the column temperature was varied in gradient according to table (2); the reactor temperature was 130 ℃; the acquisition time is 30 minutes; precisely measuring 50 μl of each of the sample solution and the control solution, respectively injecting into an amino acid analyzer, and recording chromatograms.
Table (1):
table (2):
note that: buffer a: precisely weighing 5.04g of lithium hydroxide monohydrate and 15.0g of citric acid, adding a proper amount of water for dissolution, precisely adding 50ml of ethanol, 7.8ml of hydrochloric acid and 0.1ml of octanoic acid, diluting to 1000ml with water, uniformly mixing, adjusting the pH value to 2.90 with lithium hydroxide monohydrate or hydrochloric acid, shaking uniformly, filtering and performing ultrasonic treatment.
Buffer C: precisely weighing 10.0g of boric acid, 4.2g of lithium chloride, 8.4g of lithium hydroxide monohydrate and 10.0g of citric acid, adding a proper amount of water for dissolution, precisely adding 3.3ml of hydrochloric acid and 0.1ml of octanoic acid, diluting to 1000ml with water, uniformly mixing, adjusting the pH value to 8.00 with lithium hydroxide monohydrate or hydrochloric acid, shaking uniformly, filtering and carrying out ultrasonic treatment.
Regeneration liquid: accurately weighing 21.0g of lithium hydroxide monohydrate and 0.2g of EDTA0, placing into a 1000ml measuring flask, adding water for dissolution, diluting to scale, and shaking uniformly.
Ninhydrin derivatizing agent: 600ml of methanol was precisely weighed into a beaker, 20g of ninhydrin crystals and 2g of phenol were added, stirred with a stirrer until the ninhydrin crystals were completely dissolved, filtered, 400ml of filtered potassium-sodium buffer solution (196 g of potassium acetate and 272g of sodium acetate trihydrate were precisely weighed into a beaker, 508ml of water was added to dissolve, 200ml of acetic acid was slowly added into the solution while stirring, mixed uniformly, cooled to room temperature, filtered), mixed uniformly, transferred into a ninhydrin reagent bottle, blown with nitrogen from the bottom for about 3 to 5 minutes, added with 0.2g of ascorbic acid, blown with nitrogen from the bottom for about 3 to 5 minutes, and left to stand for use.
Taking a dexpanthenol-lack prescription 2 solution, preparing a sample solution by the same method, and detecting by the same method, wherein the detection is performed without the chromatographic peaks of dexpanthenol and 3-aminopropanol, i.e. the dexpanthenol-lack prescription does not interfere with the determination of the sample.
Example 4
Alpha-tocopherol assay
Prescription 1, prescription 2, prescription 3, prescription 7, prescription 8, prescription 9 water needle: and (5) operating in a dark place. Precisely weighing a proper amount of squalane reference substance, adding cyclohexane for dissolving and diluting to prepare a solution containing about 2mg of squalane per 1ml as an internal standard solution; precisely measuring the solution (about 50mg corresponding to vitamin E) of the product, placing in a 250ml separating funnel, adding 80% ethanol 100ml, precisely adding cyclohexane 20ml, shaking and extracting for 30 minutes (shaking frequency is 180 rpm), precisely adding internal standard solution 5ml, shaking uniformly, standing for 30 minutes, and taking the supernatant as the solution of the test product; and (3) taking 20mg of alpha-tocopherol reference substance, precisely weighing, placing into a 100ml measuring flask, adding a proper amount of cyclohexane for dissolution and dilution to a scale, precisely weighing 1ml of the solution, placing into a 10ml measuring flask, precisely adding 2ml of internal standard solution, placing into the same measuring flask, diluting to the scale with cyclohexane, and shaking uniformly to obtain the reference substance solution. A capillary chromatographic column using dimethoxy polysiloxane as a fixing solution is measured according to a gas chromatography (second appendix V E of 2010 edition of Chinese pharmacopoeia), and the column temperature is 280 ℃; the detector temperature is 290 ℃; the temperature of the sample inlet is 290 ℃; the carrier gas is nitrogen, the flow rate is 1ml per minute, and the split ratio is 100:1; the theoretical plate number is not less than 5000 according to vitamin E peak, and the separation degree of alpha-tocopherol peak and vitamin E peak and other chromatographic peaks is in accordance with the requirements. Precisely measuring 1 μl of each of the sample solution and the control solution, respectively injecting into gas chromatograph, and recording chromatogram. The chromatogram of the sample solution has chromatographic peaks with the retention time consistent with that of alpha-tocopherol, and the sample solution is obtained by calculating the peak area according to an internal standard method.
Comparative test example: and (5) operating in a dark place. Precisely weighing a proper amount of squalane reference substance, adding ethanol for dissolving and diluting to obtain a solution containing about 2mg per 1ml as an internal standard solution; precisely measuring the water needle solutions (about 50mg equivalent to vitamin E) of the prescription 1, the prescription 2, the prescription 3, the prescription 7, the prescription 8 and the prescription 9 of the product, precisely adding 5ml of an internal standard solution, and shaking uniformly to obtain a sample solution; taking the reference substance solution, and measuring according to a gas chromatography (appendix V E of second edition of Chinese pharmacopoeia 2010), wherein the alpha-tocopherol chromatographic peak of the sample solution is overlapped with other chromatographic peaks, and each chromatographic peak of the reference substance is well separated and cannot be detected.
Prescription 1, prescription 2, prescription 3, prescription 4, prescription 7, prescription 8, prescription 9 lyophilized powder for injection: and (5) operating in a dark place. Precisely weighing a proper amount of squalane reference substance, adding cyclohexane for dissolving and diluting to prepare a solution containing about 2mg of squalane per 1ml as an internal standard solution; dissolving the product in water, quantitatively diluting to obtain a solution containing about 1.75mg of vitamin E per 1ml, taking the solution as a sample stock solution, precisely measuring 40ml of the sample stock solution, placing the sample stock solution into a 250ml separating funnel, adding 100ml of 80% ethanol, precisely adding 20ml of cyclohexane, shaking and extracting for 30 minutes (the shaking frequency of a shaker is 180 revolutions per minute), precisely adding 5ml of an internal standard solution, shaking uniformly, standing for 30 minutes, and taking the supernatant as a sample solution; and 28mg of alpha-tocopherol reference substance is taken, precisely weighed, placed in a 100ml measuring flask, added with a proper amount of cyclohexane for dissolution and dilution to a scale, precisely measured for 1ml of the solution, placed in a 10ml measuring flask, precisely added with 2ml of internal standard solution, placed in the same measuring flask, diluted to the scale by cyclohexane, and uniformly shaken to obtain the reference substance solution. A capillary chromatographic column using dimethoxy polysiloxane as a fixing solution is measured according to a gas chromatography (China pharmacopoeia 2015 edition four general rules of the year 0521); the column temperature is 280 ℃; the detector temperature was 290 ℃; the temperature of the sample inlet is 290 ℃; the carrier gas was nitrogen with a flow rate of 1.0ml per minute. Precisely measuring 1 μl of each of the sample solution and the control solution, respectively injecting into gas chromatograph, and recording chromatogram. The separation degree of alpha-tocopherol peak and vitamin E peak and other impurity peak is required. The chromatogram of the sample solution has chromatographic peaks with the retention time consistent with that of alpha-tocopherol, and the sample solution is obtained by calculating the peak area according to an internal standard method.
Taking a vitamin E deficiency prescription 3 water injection, preparing a test solution by the same method, and detecting by the same method, wherein the chromatogram does not see an alpha-tocopherol chromatographic peak, i.e. the vitamin E deficiency prescription does not interfere with the determination of the product.
Example 5
Menadione detection
Prescription 1, prescription 2, prescription 3, prescription 4, prescription 7, prescription 8, prescription 9 water needle: and (5) operating in a dark place. The solution is taken and precisely measured (about vitamin K is contained) 1 0.3 mg), placing in a 50ml separating funnel, precisely adding 10ml of chloroform, extracting with intense shaking for 30 seconds, standing for 30 minutes, precisely measuring 5ml of lower layer liquid, placing in a 10ml measuring flask, adding methanol for dilution to scale, and shaking to obtain sample solution; taking menadione reference substance about 10mg, precisely weighing, placing into 200ml measuring flask, adding appropriate amount of chloroform to dissolve and dilute to scale, shaking, and precisely measuring3ml, placing in a 50ml measuring flask, adding chloroform for dilution to scale, shaking, taking the mixture as a menadione reference stock solution, precisely measuring 1ml of the reference stock solution, placing in a 100ml measuring flask, adding chloroform for dilution to scale, and shaking to obtain menadione reference solution. Determining by high performance liquid chromatography (China pharmacopoeia 2015 edition four general rules 0512), and using octadecylsilane chemically bonded silica as filler; methanol-water (65:35) was used as mobile phase; the detection wavelength was 250nm. Precisely measuring 20 μl of each of the control solution and the sample solution, respectively injecting into a liquid chromatograph, and recording the chromatograms. The chromatographic peak with the retention time identical to that of the reference solution is obtained by calculating the peak area according to the external standard method.
Prescription 1, prescription 2, prescription 3, prescription 4, prescription 7, prescription 8, prescription 9 lyophilized powder for injection: and (5) operating in a dark place. Dissolving the product in water to obtain a solution containing vitamin K per 1ml 1 0.05mg of solution. Precisely measuring 6ml, placing in a 50ml separating funnel, precisely adding 10ml of chloroform, extracting with intense shaking for 30 seconds, standing for 30 minutes, precisely measuring 5ml of lower layer liquid, placing in a 10ml measuring flask, adding methanol for dilution to scale, and shaking to obtain sample solution; and (3) taking about 10mg of menadione reference substance, precisely weighing, placing into a 200ml measuring flask, adding a proper amount of chloroform to dissolve and dilute to a scale, shaking uniformly, precisely weighing 3ml, placing into a 50ml measuring flask, adding chloroform to dilute to a scale, shaking uniformly, taking 1ml of the reference substance stock solution as menadione reference substance, precisely weighing 1ml of the reference substance stock solution, placing into a 100ml measuring flask, adding chloroform to dilute to a scale, shaking uniformly, and taking menadione reference substance solution. Determining by high performance liquid chromatography (China pharmacopoeia 2015 edition four general rules 0512), and using octadecylsilane chemically bonded silica as filler; methanol-water (65:35) was used as mobile phase; the detection wavelength was 250nm. Precisely measuring 20 μl of each of the control solution and the sample solution, respectively injecting into a liquid chromatograph, and recording the chromatograms. The chromatographic peak with the retention time identical to that of the reference solution is obtained by calculating the peak area according to the external standard method.
Prescription 1, prescription 2, prescription 3, prescription 4, prescription 7, prescription 8, prescription 9 lyophilized powder for injection: and (5) operating in a dark place. Adding water into the product to obtain a product containing vitamins in 1mlElement K 1 0.03mg of the solution, precisely weighing 10ml, placing in a 50ml separating funnel, precisely adding 10ml of chloroform, extracting with vigorous shaking for 30 seconds, and standing for 30 minutes. Precisely measuring 5ml of lower layer liquid, placing in a 10ml measuring flask, adding methanol for dilution to scale, shaking uniformly, and taking as sample solution; and (3) taking about 10mg of menadione reference substance, precisely weighing, placing into a 200ml measuring flask, adding a proper amount of chloroform to dissolve, diluting to a scale, shaking uniformly, precisely weighing 3ml, placing into a 50ml measuring flask, adding chloroform to dilute to a scale, shaking uniformly, taking 1ml of menadione reference substance stock solution, precisely weighing reference substance stock solution, placing into a 100ml measuring flask, adding chloroform to dilute to a scale, shaking uniformly, and taking menadione reference substance solution. Determining by high performance liquid chromatography (China pharmacopoeia 2015 edition four general rules 0512), and using octadecylsilane chemically bonded silica as filler; methanol-water (65:35) was used as mobile phase; the detection wavelength was 250nm. Precisely measuring 20 μl of each of the control solution and the sample solution, respectively injecting into a liquid chromatograph, and recording the chromatograms. The chromatographic peak with the retention time identical to that of the reference solution is obtained by calculating the peak area according to the external standard method.
Taking solutions of a prescription 1, a prescription 2, a prescription 3, a prescription 4 and a prescription 7 as test sample solutions, and taking a storage solution of the menaquinone reference substance, and detecting by the same method, wherein other overlapping peaks exist at the chromatographic peak positions of menaquinone in the chromatographic spectrogram of the test sample solutions, and the detection cannot be performed.
Taking vitamin K 1 Preparing solution of prescription 1, preparing sample solution by the same method, and detecting by the same method without vitamin K 1 And menaquinone chromatographic peaks, i.e. lack of vitamin K 1 The prescription does not interfere with the measurement of the product.
Example 6
Vitamin K1 cis-isomer detection
Prescription 1, prescription 2, prescription 3, prescription 4, prescription 7, prescription 8, prescription 9 water injection or freeze-dried powder injection: and (5) operating in a dark place. Dissolving the extract in water or adding appropriate amount of water, precisely measuring the solution (about corresponding to vitamin K) 1 0.3 mg), and placing into a 250ml separating funnel, adding 80% ethanol solution 40ml, precisely adding n-hexane 25ml, and shaking for 30 min (shaking frequency is equal)180 revolutions per minute), placing the mixture into an upper layer for clarification, and taking supernatant as a sample solution; taking vitamin K 1 Reference substance 10mg, accurately weighing, placing into 50ml measuring flask, adding appropriate amount of n-hexane to dissolve and dilute to scale, mixing, accurately weighing 1ml, placing into 10ml measuring flask, adding n-hexane to dilute to scale, and shaking to obtain reference substance solution. According to high performance liquid chromatography (China pharmacopoeia 2015 edition four general rules 0512), silica gel is used as filler; gradient elution is carried out by taking 1, 4-dioxane-n-octanol-petroleum ether (60-90 ℃) (1:3:96) as a mobile phase A and n-hexane as a mobile phase B according to the following table; the detection wavelength is 264nm; the flow rate is 2.0ml per minute; vitamin K 1 The degree of separation between the cis and trans isomer peaks of (c) should be satisfactory.
Precisely measuring 20 μl of reference solution, injecting into liquid chromatograph, recording chromatogram, and vitamin K 1 The cis-isomer and the trans-isomer of (C) show peaks in sequence. And (3) injecting 20 mu l of the sample solution into a liquid chromatograph, recording a chromatogram, and calculating according to an area normalization method to obtain the liquid chromatograph.
Taking solutions of prescription 1, prescription 2, prescription 3, prescription 4 and prescription 7 as test sample solutions, taking the menaquinone reference sample stock solution, and detecting by the same method to obtain vitamin K in the chromatogram of the test sample solutions 1 The cis-isomer peak and the trans-isomer peak of the kit cannot be well separated, and other edge overlapped chromatographic peaks exist at the positions in the period, so that the detection cannot be performed.
Taking vitamin K 1 Preparing solution of prescription 1, preparing sample solution by the same method, and detecting by the same method without vitamin K 1 Cis-trans isomer peak chromatographic peaks of (B), namely lack of vitamin K 1 The prescription does not interfere with the measurement of the product.
Description of the drawings:
FIG. 1 3-Aminopropanol test sample
FIG. 2 3-amino propanol test control
FIG. 3 Pantoea negative control
FIG. 4 retinol assay test sample
FIG. 5 retinol assay control
FIG. 6 vitamin A deficient palmitate negative control
FIG. 7A-tocopherol assay
FIG. 8A-tocopherol assay control
FIG. 9 alpha-tocopherol assay internal standard chart
FIG. 10 vitamin E deficient negative control
FIG. 11 is a drawing of a test article for menaquinone detection
FIG. 12A-naphthoquinone detection control
FIG. 13 vitamin K deficiency 1 Negative control chart (menadione)
FIG. 14 vitamin K 1 Test sample for cis-isomer detection
FIG. 15 vitamin K 1 Cis-isomer control
FIG. 16 vitamin K deficiency 1 Negative control panels (isoforms).

Claims (7)

1. A detection method of multi-vitamin injection impurities comprises main medicine components and medical acceptable auxiliary materials, wherein the main medicine components are selected from vitamin A palmitate and vitamin D 3 Or an analogue thereof, vitamin E or an analogue thereof, vitamin K 1 Vitamin C, its salts and vitamin B 1 Or an analogue thereof, riboflavin sodium phosphate, dexpanthenol or an analogue thereof, nicotinamide, vitamin B 6 Or an analogue thereof, vitamin B 12 2 or more of folic acid and biotin,
the impurities comprise oxalic acid, 3-aminopropanol, retinol, alpha-tocopherol, menaquinone and vitamin K 1 The cis-isomer of the compound,
wherein, the detection steps of the 3-aminopropanol are as follows:
(1) Sample solution preparation: dissolving multiple vitamins in water, or freeze-dried powder for injection, and diluting with water to obtain sample solution;
(2) Preparing a reference substance solution: taking a 3-aminopropanol reference substance, precisely weighing, and adding water for dissolving to obtain a reference substance solution;
(3) And (3) detection: taking sample solution and reference substance solution, separating and measuring with amino acid analyzer, calculating according to external standard method with peak area,
or is:
(1) Sample solution preparation: dissolving multiple vitamins in water, or freeze-dried powder for injection, and diluting with water to obtain sample solution;
(2) Preparing a reference substance solution: taking a 3-aminopropanol reference substance, precisely weighing, and adding water for dissolving to obtain a reference substance solution;
(3) And (3) detection: taking a test solution and a reference solution, measuring according to high performance liquid chromatography (the fourth edition of China pharmacopoeia 2015, general rule 0512), using octadecylsilane chemically bonded silica as a filler, using 0.1% aqueous solution of heptafluorobutyric anhydride as a mobile phase A, using 0.1% acetonitrile solution of heptafluorobutyric anhydride as a mobile phase B, performing gradient elution according to the following table, wherein the detector is an evaporative light scattering detector,
and respectively precisely measuring the solutions of the reference substances with different volumes, injecting the solutions into a liquid chromatograph, recording the chromatograms, calculating a regression equation according to the logarithm of the sample injection quantity of the reference substances and the logarithm of the corresponding peak areas, precisely measuring the solution of the sample, injecting the solution of the sample into the liquid chromatograph, recording the chromatograms, and calculating the content of 3-aminopropanol in the sample according to the regression equation.
2. The method for detecting impurities in a multivitamin injection according to claim 1, wherein the detecting step of 3-aminopropanol comprises:
(1) Dissolving multiple vitamins in water, or freeze-dried powder for injection, and diluting with water to obtain solution containing dexpanthenol 0.25mg per 1 ml;
(2) Taking a proper amount of 3-aminopropanol reference substance, precisely weighing, adding water for dissolving, and preparing a solution containing 2.5 mug of 3-aminopropanol in 1ml as a reference substance solution;
(3) The separation was performed using an amino acid analyzer using SYKNM-Cation Separation Column (LCA K07/Li,4.6 mm. Times.150 mm) as a column; taking buffer solution A, buffer solution C, regeneration solution and ninhydrin derivative as mobile phases, eluting with an elution pump flow rate of 0.45ml per minute and a derivative pump flow rate of 0.25ml per minute, performing gradient elution according to the following table (1), detecting 570nm of wavelength, changing the column temperature according to the gradient of the following table (2), wherein the temperature of the reactor is 130 ℃, the acquisition time is 30 minutes, precisely measuring 50 μl of each of the sample solution and the reference solution, respectively injecting into an amino acid analyzer, recording a chromatogram, and calculating according to an external standard method by peak area to obtain the final product;
watch (1)
Watch (2)
Buffer a: precisely weighing 5.04g of lithium hydroxide monohydrate and 15.0g of citric acid, adding a proper amount of water for dissolution, precisely adding 50ml of ethanol, 7.8ml of hydrochloric acid and 0.1ml of octanoic acid, diluting to 1000ml with water, uniformly mixing, adjusting the pH value to 2.9 with lithium hydroxide monohydrate or hydrochloric acid, shaking uniformly, filtering and performing ultrasonic treatment;
Buffer C: precisely weighing 10.0g of boric acid, 4.2g of lithium chloride, 8.4g of lithium hydroxide monohydrate and 10.0g of citric acid, adding a proper amount of water for dissolution, precisely adding 3.3ml of hydrochloric acid and 0.1ml of octanoic acid, diluting to 1000ml with water, uniformly mixing, adjusting the pH value to 8.0 with lithium hydroxide monohydrate or hydrochloric acid, shaking uniformly, filtering and performing ultrasonic treatment;
regeneration liquid: accurately weighing 21.0g of lithium hydroxide monohydrate and 0.2g of EDTA0, placing into a 1000ml measuring flask, adding water for dissolution, diluting to scale, and shaking uniformly;
ninhydrin derivatizing agent: 600ml of methanol was precisely weighed into a beaker, 20g of ninhydrin crystals and 2g of phenol were added, stirred with a stirrer until the ninhydrin crystals were completely dissolved, filtered, 400ml of filtered potassium-sodium buffer solution (196 g of potassium acetate and 272g of sodium acetate trihydrate were precisely weighed into a beaker, 508ml of water was added to dissolve, 200ml of acetic acid was slowly added into the solution while stirring, mixed uniformly, cooled to room temperature, filtered), mixed uniformly, transferred into a ninhydrin reagent bottle, blown with nitrogen from the bottom for about 3 to 5 minutes, added with 0.2g of ascorbic acid, blown with nitrogen from the bottom for about 3 to 5 minutes, and left to stand for use.
3. The method for detecting impurities in a multivitamin injection according to claim 1 or 2, wherein the oxalic acid is detected by:
(1) Sample solution preparation: dissolving multiple vitamins in water, or freeze-dried powder for injection, adding diluted acetic acid and calcium chloride solution, shaking, standing, adding water and making the volume of water consistent with that of control solution to obtain sample solution;
(2) Preparation of intermediate solution: dissolving multiple vitamins in water, or lyophilizing, standing, and filtering to obtain filtrate;
(3) Control solution preparation: precisely measuring oxalic acid solution, wherein the amount of the oxalic acid solution is 0.2-0.5% of the amount of vitamin C, adding the oxalic acid solution into the filtrate of the step (2), shaking uniformly, and standing to serve as a control solution;
(4) And (3) detection: comparing the turbidity generated by the sample solution with the turbidity generated by the control solution.
4. The method for detecting impurities in a multivitamin injection according to claim 3, wherein the oxalic acid is detected by:
(1) Taking a multi-vitamin injection solution, wherein the solution quantity can be equal to 200mg of vitamin C, for example, a freeze-dried powder injection can be dissolved by adding water, adding 1ml of dilute acetic acid and 0.5ml of calcium chloride test solution, shaking uniformly, standing for 1 hour, adding 0.5ml of water, shaking uniformly, and taking the mixture as a test solution;
(2) Taking multiple vitamins injection by the same method, operating by the same method until the vitamin injection is placed for 1 hour, filtering, and taking filtrate;
(3) Precisely measuring 0.5ml of oxalic acid solution, which is equivalent to 0.6mg of oxalic acid, adding the oxalic acid solution into the filtrate, shaking uniformly, and standing for 1 hour to obtain a control solution;
(4) The turbidity produced by the test solution was observed not to be stronger than that of the control solution.
5. The method for detecting impurities in a multivitamin injection according to claim 1 or 2, wherein the ratio of each vitamin component in the multivitamin injection is as follows:
6. the method for detecting impurities in a multivitamin injection according to claim 1 or 2, wherein the ratio of each vitamin component in the multivitamin injection is as follows:
or (b)
Or (b)
7. The method for detecting impurities in a multivitamin injection according to claim 1 or 2, wherein the ratio of each vitamin component in the multivitamin injection is as follows:
or (b)
Or (b)
Vitamin C200.0 mg
Vitamin B 1 10.0mg
Riboflavin sodium phosphate (equivalent to riboflavin 5 mg) 6.355mg
Or (b)
Or (b)
Or (b)
CN202311173218.2A 2018-07-12 2018-07-12 Method for detecting impurities of multivitamin injection Pending CN117233309A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202311173218.2A CN117233309A (en) 2018-07-12 2018-07-12 Method for detecting impurities of multivitamin injection

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202311173218.2A CN117233309A (en) 2018-07-12 2018-07-12 Method for detecting impurities of multivitamin injection
CN201810765320.4A CN110715995B (en) 2018-07-12 2018-07-12 Method for detecting impurities of multivitamin injection

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
CN201810765320.4A Division CN110715995B (en) 2018-07-12 2018-07-12 Method for detecting impurities of multivitamin injection

Publications (1)

Publication Number Publication Date
CN117233309A true CN117233309A (en) 2023-12-15

Family

ID=69209147

Family Applications (2)

Application Number Title Priority Date Filing Date
CN202311173218.2A Pending CN117233309A (en) 2018-07-12 2018-07-12 Method for detecting impurities of multivitamin injection
CN201810765320.4A Active CN110715995B (en) 2018-07-12 2018-07-12 Method for detecting impurities of multivitamin injection

Family Applications After (1)

Application Number Title Priority Date Filing Date
CN201810765320.4A Active CN110715995B (en) 2018-07-12 2018-07-12 Method for detecting impurities of multivitamin injection

Country Status (1)

Country Link
CN (2) CN117233309A (en)

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111366657B (en) * 2020-04-10 2022-07-29 江苏海悦康医药科技有限公司 Method for detecting EP impurity and degradation impurity of vitamin B1 by high performance liquid chromatography
CN112763605A (en) * 2020-12-24 2021-05-07 四川欣美加生物医药有限公司 Detection method and application of tocopherol content
CN114437093A (en) * 2021-11-11 2022-05-06 浙江新和成股份有限公司 Identification and extraction method of impurity A in aromatization reaction liquid of vitamin B6 and synthesis method of impurity A
CN114839279B (en) * 2022-02-21 2024-10-29 佛山市南海北沙制药有限公司 Method for measuring impurity content in vinyl diethyl ether
CN115308346A (en) * 2022-09-02 2022-11-08 汤臣倍健股份有限公司 Method for extracting and detecting content of multiple vitamins in blood
CN116990402B (en) * 2023-06-25 2024-06-25 南京汉欣医药科技有限公司 Vitamin K determination1Analysis method of isomer content in injection

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103487518B (en) * 2013-09-02 2015-03-18 成都百裕科技制药有限公司 Impurity detection method and content determination method for clindamycin hydrochloride for injection
CN106153796A (en) * 2015-04-17 2016-11-23 西藏卫信康医药股份有限公司 The content analysis detection method of 12 kinds of compound vitamines of injection
RU2647451C1 (en) * 2017-03-17 2018-03-15 Федеральное Бюджетное Учреждение Науки "Нижегородский Научно-Исследовательский Институт Гигиены И Профпатологии" Федеральной Службы По Надзору В Сфере Защиты Прав Потребителей И Благополучия Человека (Фбун "Нниигп" Роспотребнадзора) Method of the vitamin k1 content determining in products of plant origin
CN107831231A (en) * 2017-11-01 2018-03-23 济南维瑞医药科技开发有限公司 A kind of method for detecting vitamin A impurity in multivitamin preparation
CN108061773B (en) * 2018-02-02 2019-04-12 湖南山水检测有限公司 The method of high-efficient liquid phase chromatogram technology measurement vitamin A

Also Published As

Publication number Publication date
CN110715995A (en) 2020-01-21
CN110715995B (en) 2023-11-03

Similar Documents

Publication Publication Date Title
CN110715995B (en) Method for detecting impurities of multivitamin injection
CN106153796A (en) The content analysis detection method of 12 kinds of compound vitamines of injection
CN108659104B (en) Preparation method of terlipressin and pharmaceutical composition thereof
CN102068453A (en) Stable complex vitamin composite and preparation method thereof
Uddin et al. Simultaneous determination of amoxicillin and chloramphenicol and their drug interaction study by the validated UPLC method
CN106539753A (en) A kind of phloroglucinol injection and preparation method thereof
CN106074422A (en) A kind of succinum love song Ge Lieting oral solid formulation and preparation method thereof
CN109490460B (en) Detection method of L-2-amino-5-guanidino valeric acid related substances
CN109655544B (en) Quality control method of metformin hydrochloride and preparation thereof
CN101623284A (en) Medical composition of compound vitamin C injection
CN101721677B (en) Thymopentin oral microsphere preparation and preparation method thereof
CN115372497B (en) Method for measuring content of 6 alkaloid components in small collateral-activating pills
CN114814060B (en) Detection method of valsartan amlodipine tablet related substances
CN103816126B (en) A kind of pharmaceutical composition containing scopolamine butylbromide
CN102657646B (en) Medicinal composition and preparation thereof
CN104606220A (en) Multivitamin composition and preparation method thereof
CN103565747B (en) Esomeprazole composition and preparation method thereof
CN107184548B (en) A kind of highly-safe L-ornidazole injection liquid and preparation method thereof
CN103860461A (en) Medicinal composition containing active component ambroxol hydrochloride
CN101428020A (en) Freeze-dried powder preparation of kuh-seng native, preparation and detection method thereof
Kozlov et al. HPLC determination of lipoic acid and vitamins A and E in multivitamin compositions.
CN100402030C (en) Pharmaceutical composition containing amoxicillin and preparation method thereof
CN103877579B (en) A kind of pharmaceutical composition and preparation thereof containing famotidine
CN112557558B (en) Method for detecting SCH59566 impurity content in ezetimibe simvastatin tablets
Andhale et al. Development and Validation of Stability Indicating RP-HPLC Method for Estimation of Alogliptin and Metformin HCl Drug from Bulk and Pharmaceutical Dosage Form

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