CN114689758B - Method for concentrating, extracting and measuring sorbic acid and benzoic acid in gelatin hollow capsules - Google Patents
Method for concentrating, extracting and measuring sorbic acid and benzoic acid in gelatin hollow capsules Download PDFInfo
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- CN114689758B CN114689758B CN202210384939.7A CN202210384939A CN114689758B CN 114689758 B CN114689758 B CN 114689758B CN 202210384939 A CN202210384939 A CN 202210384939A CN 114689758 B CN114689758 B CN 114689758B
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- benzoic acid
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- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 title claims abstract description 108
- 239000005711 Benzoic acid Substances 0.000 title claims abstract description 54
- 235000010233 benzoic acid Nutrition 0.000 title claims abstract description 54
- 235000010199 sorbic acid Nutrition 0.000 title claims abstract description 54
- BEFDCLMNVWHSGT-UHFFFAOYSA-N ethenylcyclopentane Chemical compound C=CC1CCCC1 BEFDCLMNVWHSGT-UHFFFAOYSA-N 0.000 title claims abstract description 53
- 239000004334 sorbic acid Substances 0.000 title claims abstract description 53
- 229940075582 sorbic acid Drugs 0.000 title claims abstract description 53
- 108010010803 Gelatin Proteins 0.000 title claims abstract description 38
- 229920000159 gelatin Polymers 0.000 title claims abstract description 38
- 239000008273 gelatin Substances 0.000 title claims abstract description 38
- 235000019322 gelatine Nutrition 0.000 title claims abstract description 38
- 235000011852 gelatine desserts Nutrition 0.000 title claims abstract description 38
- 239000002775 capsule Substances 0.000 title claims abstract description 32
- 238000000034 method Methods 0.000 title claims abstract description 28
- 239000000523 sample Substances 0.000 claims abstract description 26
- 239000012488 sample solution Substances 0.000 claims abstract description 25
- 239000013558 reference substance Substances 0.000 claims abstract description 24
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 17
- 239000007788 liquid Substances 0.000 claims abstract description 15
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 14
- 238000005303 weighing Methods 0.000 claims abstract description 9
- 238000009210 therapy by ultrasound Methods 0.000 claims abstract description 7
- CHHHXKFHOYLYRE-UHFFFAOYSA-M 2,4-Hexadienoic acid, potassium salt (1:1), (2E,4E)- Chemical compound [K+].CC=CC=CC([O-])=O CHHHXKFHOYLYRE-UHFFFAOYSA-M 0.000 claims abstract description 5
- 238000005259 measurement Methods 0.000 claims abstract description 5
- 235000010241 potassium sorbate Nutrition 0.000 claims abstract description 5
- 239000004302 potassium sorbate Substances 0.000 claims abstract description 5
- 229940069338 potassium sorbate Drugs 0.000 claims abstract description 5
- WXMKPNITSTVMEF-UHFFFAOYSA-M sodium benzoate Chemical compound [Na+].[O-]C(=O)C1=CC=CC=C1 WXMKPNITSTVMEF-UHFFFAOYSA-M 0.000 claims abstract description 5
- 235000010234 sodium benzoate Nutrition 0.000 claims abstract description 5
- 239000004299 sodium benzoate Substances 0.000 claims abstract description 5
- 239000002904 solvent Substances 0.000 claims abstract description 5
- 238000001914 filtration Methods 0.000 claims abstract description 4
- 239000006228 supernatant Substances 0.000 claims abstract description 4
- 238000005119 centrifugation Methods 0.000 claims abstract 2
- 238000002156 mixing Methods 0.000 claims abstract 2
- 238000012360 testing method Methods 0.000 claims description 22
- 238000000605 extraction Methods 0.000 claims description 12
- 238000001514 detection method Methods 0.000 claims description 10
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 8
- OKKJLVBELUTLKV-UHFFFAOYSA-N methanol Substances OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 7
- 239000002245 particle Substances 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 6
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 5
- 239000005695 Ammonium acetate Substances 0.000 claims description 5
- 235000019257 ammonium acetate Nutrition 0.000 claims description 5
- 229940043376 ammonium acetate Drugs 0.000 claims description 5
- 230000014759 maintenance of location Effects 0.000 claims description 5
- 239000000945 filler Substances 0.000 claims description 4
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 4
- 239000000377 silicon dioxide Substances 0.000 claims description 4
- 238000000862 absorption spectrum Methods 0.000 claims description 3
- 238000007865 diluting Methods 0.000 claims description 3
- 239000000706 filtrate Substances 0.000 claims description 3
- 238000000527 sonication Methods 0.000 claims 1
- 239000000243 solution Substances 0.000 abstract description 19
- 238000011084 recovery Methods 0.000 abstract description 7
- 235000019441 ethanol Nutrition 0.000 abstract description 4
- 238000010790 dilution Methods 0.000 abstract 1
- 239000012895 dilution Substances 0.000 abstract 1
- 238000004090 dissolution Methods 0.000 abstract 1
- 239000003814 drug Substances 0.000 description 11
- 238000004458 analytical method Methods 0.000 description 6
- 229940079593 drug Drugs 0.000 description 6
- 239000012085 test solution Substances 0.000 description 6
- 230000008569 process Effects 0.000 description 5
- 239000003755 preservative agent Substances 0.000 description 4
- 230000002335 preservative effect Effects 0.000 description 4
- 235000013305 food Nutrition 0.000 description 3
- 150000002500 ions Chemical class 0.000 description 3
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 230000001376 precipitating effect Effects 0.000 description 3
- 239000012088 reference solution Substances 0.000 description 3
- WSWCOQWTEOXDQX-UHFFFAOYSA-N 2,4-Hexadienoic acid Chemical compound CC=CC=CC(O)=O WSWCOQWTEOXDQX-UHFFFAOYSA-N 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- HNSDLXPSAYFUHK-UHFFFAOYSA-N 1,4-bis(2-ethylhexyl) sulfosuccinate Chemical compound CCCCC(CC)COC(=O)CC(S(O)(=O)=O)C(=O)OCC(CC)CCCC HNSDLXPSAYFUHK-UHFFFAOYSA-N 0.000 description 1
- IAYPIBMASNFSPL-UHFFFAOYSA-N Ethylene oxide Chemical compound C1CO1 IAYPIBMASNFSPL-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 206010074268 Reproductive toxicity Diseases 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000000022 bacteriostatic agent Substances 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000008468 bone growth Effects 0.000 description 1
- 235000014171 carbonated beverage Nutrition 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 230000003467 diminishing effect Effects 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 230000007674 genetic toxicity Effects 0.000 description 1
- 231100000025 genetic toxicology Toxicity 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 235000021109 kimchi Nutrition 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- LXCFILQKKLGQFO-UHFFFAOYSA-N methylparaben Chemical compound COC(=O)C1=CC=C(O)C=C1 LXCFILQKKLGQFO-UHFFFAOYSA-N 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000007696 reproductive toxicity Effects 0.000 description 1
- 231100000372 reproductive toxicity Toxicity 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000002390 rotary evaporation Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- WSWCOQWTEOXDQX-MQQKCMAXSA-N sorbic acid group Chemical class C(\C=C\C=C\C)(=O)O WSWCOQWTEOXDQX-MQQKCMAXSA-N 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000011003 system suitability test Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000003809 water extraction Methods 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- 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
- G01N30/86—Signal analysis
- G01N30/8624—Detection of slopes or peaks; baseline correction
- G01N30/8631—Peaks
- G01N30/8634—Peak quality criteria
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Pathology (AREA)
- Biochemistry (AREA)
- Immunology (AREA)
- General Physics & Mathematics (AREA)
- Engineering & Computer Science (AREA)
- Quality & Reliability (AREA)
- Investigating Or Analysing Materials By Optical Means (AREA)
- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
Abstract
The invention provides a method for concentrating, extracting and measuring sorbic acid and benzoic acid in a gelatin hollow capsule, which comprises the following steps: precisely weighing gelatin hollow capsules, placing into a centrifuge tube, adding water, immediately vortex oscillating for dissolution, rapidly adding absolute ethyl alcohol after ultrasonic treatment, shaking for uniform mixing to separate out gelatin, ultrasonic treatment and centrifugation, taking supernatant, recovering solvent under reduced pressure, quantitatively transferring into a measuring flask for dilution to scale, shaking for uniform filtration to obtain a sample solution; precisely weighing sodium benzoate and potassium sorbate as reference substances, and adding water to obtain reference substance solution; precisely sucking the reference substance solution and the sample solution respectively, and injecting into a liquid chromatograph for measurement to obtain the sample chromatograph. The invention can extract benzoic acid and sorbic acid in the sample, can rapidly separate gelatin in the system to the maximum extent, can increase the signal intensity of target components, can effectively avoid ethanol interference peaks in the chromatogram, and has the characteristics of simple operation, strong stability, good repeatability and higher recovery rate.
Description
Technical Field
The invention relates to a method for extracting sorbic acid and benzoic acid, in particular to a method for concentrating, extracting and measuring sorbic acid and benzoic acid in a gelatin hollow capsule, belonging to the technical field of food safety.
Background
The gelatin hollow capsule is a two-section capsule shell combined structure refined by medicinal gelatin and auxiliary materials, is mainly used for containing self-made powder, health products, medicaments and other solid medicaments, solves the problems of difficult medicament entrance and poor mouthfeel, has good bioavailability, and can be rapidly, reliably and safely dissolved after being taken.
Benzoic acid, also known as benzoic acid, is a widely used chemical synthetic preservative and has long been used as a preservative for acidic foods such as jams, carbonated beverages, juice beverages, kimchi, and the like. However, in recent years, there is a trend of diminishing the use of benzoic acid in food industry at home and abroad mainly because benzoic acid has a mutation-causing effect, genetic toxicity and reproductive toxicity to male animals according to the research situation in recent years. Sorbic acid is also called 2, 4-hexadienoic acid, and although toxic and side effects are lower than those of benzoic acid, bone growth can be inhibited to a certain extent after long-term administration, and kidney and liver health is endangered, but specific reports about harm of sorbic acid to human bodies are not yet available.
At present, the nipagin is added in the production process of the gelatin hollow capsule as a bacteriostatic agent, or the product is subjected to ethylene oxide sterilization, if other preservative is brought in the processing and manufacturing process of the gelatin raw material or the manufacturing process of the hollow capsule, the medication risk of patients can be increased. The existing gelatin hollow capsule standard does not contain detection items of benzoic acid and sorbic acid components, and the action of illegally adding the preservative does not play a role in supervision and inspection, so that a supplementary inspection method is added to the existing standard of the gelatin hollow capsule, and the detection is carried out on the benzoic acid and sorbic acid components, which is very important for ensuring safe medicines for people.
Disclosure of Invention
In order to further improve the extraction efficiency of illicit substances illegally added in the gelatin hollow capsules and overcome the problem of difficult extraction of the traditional high-protein-content samples, the invention adopts a water extraction and alcohol precipitation method to extract sorbic acid and benzoic acid in the gelatin hollow capsules, and the method is simple and convenient to operate, and the repeatability and the stability meet the requirements of Chinese pharmacopoeia.
The technical solution of the invention is as follows: the method for concentrating, extracting and measuring sorbic acid and benzoic acid in the gelatin hollow capsule specifically comprises the following steps:
1) Preparation of test solution: taking 1g of gelatin hollow capsule, precisely weighing, placing into a 50ml centrifuge tube, adding 10ml of water at 60 ℃, immediately vortex oscillating to dissolve a test sample, ultrasonically treating for 20 minutes at 50 ℃, rapidly adding 40ml of absolute ethyl alcohol, gently shaking to mix uniformly, precipitating gelatin, ultrasonically treating for 1 minute at 50 ℃, centrifuging at 7000RPM for 8 minutes, taking supernatant, recovering the solvent to about 2ml under reduced pressure, quantitatively transferring into a 25ml measuring flask, diluting to a scale with water, shaking uniformly, filtering, and taking subsequent filtrate to obtain the gelatin hollow capsule.
2) Preparation of a control solution: and (3) taking a proper amount of sodium benzoate and potassium sorbate as reference substances, precisely weighing, and adding water to prepare a solution containing 10 mug of benzoic acid and sorbic acid in each 1 ml.
3) Chromatographic conditions and system suitability test: octadecylsilane chemically bonded silica is used as a filler (column length is 250mm, inner diameter is 4.6mm, and particle diameter is 5 μm); taking 0.02mol/L ammonium acetate solution-methanol (93:7) as a mobile phase; the detection wavelength was 229nm. The theoretical plate number is not less than 5000 calculated by sorbic acid peak. The degree of separation of benzoic acid and sorbic acid from adjacent peaks should be in compliance with the regulations.
Assay: respectively precisely sucking 10 μl of the reference solution and the sample solution, and injecting into a liquid chromatograph for measurement.
And (3) judging results: in the sample chromatogram, the chromatogram peak which is consistent with the retention time of the reference substance should not be presented. If the chromatographic peak with the retention time consistent with that of the reference substance appears, a diode array detector is adopted for detection, and if the chromatographic peak is the same as the ultraviolet absorption spectrum of the reference substance, the corresponding component can be judged.
Compared with the prior art, the invention has the advantages that:
1) Adopts an extraction method of firstly dissolving with water and then precipitating with alcohol, not only can extract benzoic acid and sorbic acid in a test sample, but also can maximally and rapidly separate gelatin in a system,
2) The rotary evaporation mode is adopted to concentrate the test solution at low temperature, and then the water is used for volume fixation, so that not only can the signal intensity of the target component be increased, but also the ethanol interference peak in the chromatogram can be effectively avoided;
3) Has the characteristics of simple and convenient operation, strong stability, good repeatability and higher recovery rate.
Drawings
FIG. 1 is a graph of benzoic acid standard calculated from data measured by liquid chromatograph in the examples.
FIG. 2 is a graph of sorbic acid standard calculated from data measured by liquid chromatograph in the examples.
FIG. 3 is a graph showing the results of liquid chromatography of the sample solutions of gelatin hollow capsules in the examples.
Detailed Description
The technical scheme of the invention is further described below with reference to the accompanying drawings.
The instrument model and reagent components adopted in this embodiment are specifically as follows:
instrument: dionex U3000 hplc diode array detector, agilent 1260 hplc diode array detector, waters2695e hplc diode array detector; agilent 1290 high performance liquid chromatograph-AB SCIEX 5500QTRAP mass spectrometer; an Eppendorf 5430 centrifuge; IKA RV10 rotary evaporator; sartorius CPA225D electronic analytical balance.
Ammonium acetate (Ara Ding Shiji, for HPLC, content > 99.0%); absolute ethanol (national pharmaceutical group chemical reagent limited, top purity); methanol (merck, germany, chromatographic purity).
The method for measuring the concentration and extraction of sorbic acid and benzoic acid in the gelatin hollow capsule provided in the embodiment specifically comprises the following steps:
1) Preparation of the solution
(1) Preparation of test solution: taking 1g of gelatin hollow capsule, precisely weighing, placing into a 50ml centrifuge tube, adding 10ml of water at 60 ℃, immediately performing vortex oscillation to dissolve a test sample, performing ultrasonic treatment at 50 ℃ for 20 minutes, rapidly adding 40ml of absolute ethyl alcohol, gently shaking to mix uniformly, precipitating gelatin, performing ultrasonic treatment at 50 ℃ for 1 minute, centrifuging at 7000RPM for 8 minutes, taking supernatant, recovering the solvent under reduced pressure to about 2ml, quantitatively transferring into a 25ml measuring flask, diluting with water to a scale, shaking uniformly, filtering, and taking the subsequent filtrate to obtain a test sample solution; and (3) simultaneously taking 1g of gelatin hollow capsule which does not contain benzoic acid and sorbic acid, precisely weighing, and operating the rest according to the same method as the step (1) to obtain a negative sample solution.
(2) Preparation of a control solution: the sodium benzoate and potassium sorbate are taken as reference substances, the sodium benzoate and potassium sorbate are precisely weighed, water is added to prepare solutions with each 1ml of 1mg, and each solution is diluted with water to prepare serial reference substance solutions with each 1ml of 1 mug, 5 mug, 10 mug, 20 mug and 50 mug of benzoic acid and sorbic acid.
2) Detecting by a liquid chromatograph: respectively precisely sucking 10 μl of each of the series of reference substance solutions and the sample solution, injecting into a liquid chromatograph, measuring corresponding peak areas, drawing a standard curve by taking the mass concentration of the series of reference substance solutions as an abscissa and the peak areas as an ordinate, and then bringing the peak areas of the sample solution into the standard curve to obtain the mass concentrations of benzoic acid and sorbic acid in the sample solution.
Chromatographic conditions: octadecylsilane chemically bonded silica is used as a filler (column length is 250mm, inner diameter is 4.6mm, and particle diameter is 5 μm); taking 0.02mol/L ammonium acetate solution-methanol (93:7) as a mobile phase; the detection wavelength was 229nm.
The linear range, precision, specificity, accuracy, detection limit, quantitative limit, and durability of the method proposed in this example are detected and identified one by one as follows.
1) Linear range
Respectively precisely sucking 10 μl of each concentration of benzoic acid and sorbic acid reference substance solution, measuring peak area, establishing standard curve as shown in figures 1-2, and calculating regression equation as follows: benzoic acid: y=23.693x+1.1295, r=0.9999, linear range: 1.0194-50.9716 mug/ml. Sorbic acid: y=41.984x+1.7136, r=0.9999, linear range: 1.0102-50.5088 mug/ml. The results of liquid chromatography of the gelatin hollow capsule test solution are shown in FIG. 3.
2) Precision of
(1) Repeatability of
6 parts of test solution are prepared in parallel, 10 μl of each sample is introduced, peak areas of benzoic acid and sorbic acid in the 6 parts of test solution are measured, and contents are calculated according to corresponding regression equations, and specific results are shown in tables 1 to 2 below.
TABLE 1
TABLE 2
From the above table, it can be seen that 6 parallel test samples have benzoic acid content RSD of 2.49% and sorbic acid content RSD of 2.73%, and the test sample solutions have good repeatability by referring to the relevant requirements of the guidelines of the quality standard analysis method of the drug in the fourth part of the 2015 year edition of Chinese pharmacopoeia 9101.
(2) Intermediate precision
Three different testers respectively prepare 2 parts of test sample solutions according to different brands of high performance liquid chromatographs and chromatographic columns, simultaneously prepare benzoic acid and sorbic acid series reference substance solutions, respectively establish standard curves, determine the contents of benzoic acid and sorbic acid in the test samples, and calculate RSD.
Standard curve is established by using Agilent 1260 high performance liquid chromatograph, and regression equation is calculatedBenzoic acid: y=32.687x+1.2226, r 2 =0.9999, linear range 1.0050 to 50.2513 μg/ml; sorbic acid: y=55.434x+0.0716, r 2 =0.9999, linear range 0.9978 to 49.8911 μg/ml. Specific results are shown in tables 3 to 4 below.
TABLE 3 Table 3
TABLE 4 Table 4
Standard curves were established using Waters2695e high performance liquid chromatograph, and regression equations were calculated, benzoic acid: y= 40889x-4826.5, r 2 =0.9999, linear range 1.0050 to 50.2513 μg/ml; sorbic acid: y= 71737x-6369.8, r 2 =0.9999, linear range 0.9978 to 49.8911 μg/ml. Specific results are shown in tables 5 to 6 below.
TABLE 5
TABLE 6
And (3) comprehensively using a DionexU3000 high performance liquid chromatograph to establish a standard curve, calculating the content of benzoic acid and sorbic acid in the sample obtained after a regression equation, and calculating RSD of the measured value of the content of the benzoic acid and the sorbic acid among the three. The specific results are shown in Table 7 below.
TABLE 7
From the table, three different testers use different brands of high performance liquid chromatographs and chromatographic columns to measure the contents of benzoic acid and sorbic acid in the test sample, wherein the content of the benzoic acid RSD is 3.11%, the content of the sorbic acid RSD is 1.46%, and the intermediate precision of the test sample solution is good according to the relevant requirements of the guidelines of the drug quality standard analysis method in the fourth edition of the Chinese pharmacopoeia 2015.
3) Specialization of
Comparing chromatograms of the benzoic acid and sorbic acid reference substance solution and the sample solution, wherein chromatographic peaks consistent with retention time of the benzoic acid and sorbic acid reference substance solution appear in the chromatograms of the sample solution. The detection is carried out by adopting a diode array detector, and the ultraviolet absorption spectrum of the corresponding chromatographic peak in the sample solution is basically the same as that of the corresponding chromatographic peak in the reference substance solution, so that the LC-MS method is further adopted for confirmation.
LC-MS method chromatographic conditions: octadecylsilane chemically bonded silica is used as filler (column length is 100mm, inner diameter is 2.1mm, and particle diameter is 3 μm); the column temperature is 35 ℃; taking 0.02mol/L ammonium acetate solution-methanol (93:7) as a mobile phase; the detection wavelength is 229nm; the sample loading was 5. Mu.l. Mass spectrometry conditions: adopting an ESI ion source; the ionization mode is negative ions; the temperature of the ion source is 150 ℃; capillary temperature 375 ℃; the source voltage is 3.5kV; sheath gas flow rate is 30arb; the auxiliary gas flow rate is 5arb; the scanning mode is primary mass spectrum full scanning; scanning range is 50-200 m/z; the m/z of benzoic acid and sorbic acid are 121, 111 respectively.
The LC-MS method confirms that chromatographic peaks in the chromatogram of the sample solution, which are consistent with the retention time of the benzoic acid and sorbic acid reference substance solution, are the benzoic acid and the sorbic acid.
4) Accuracy (recovery rate)
Taking 0.5g of gelatin hollow capsule, precisely weighing, placing into a 50ml centrifuge tube, precisely adding benzoic acid and sorbic acid equivalent to 0.5g of sample, continuously preparing into sample solution, preparing 6 parts, taking 10 μl of sample, and recording chromatogram and corresponding component chromatogram peak area. And calculating the total content of the corresponding components according to the peak area, calculating the content of the corresponding components in the test sample according to the average content of benzoic acid and sorbic acid in 6 parts of the test sample, further calculating the sample adding recovery rate of 6 parts of the test sample, and calculating the RSD. Specific results are shown in tables 8 to 9 below.
TABLE 8
TABLE 9
As can be seen from the table, in the 6 samples to be recovered, the average value of the recovery rate of benzoic acid is 94.98%, and the RSD is 3.76%; the average sorbic acid recovery rate was 92.28% and RSD was 2.56%. The average recovery rate of the two components reaches more than 90%, the RSD is less than 4%, and the accuracy of the method is good by referring to the relevant requirements of the guidelines of the standard analysis method of the quality standard of medicines in the fourth part of the 2015 year edition of Chinese pharmacopoeia 9101.
5) Limit of detection and limit of quantification
Specific results are shown in tables 10 to 11 below.
Table 10
TABLE 11
6) Durability of
(1) Stability of
And (3) sampling and detecting the same sample solution at intervals within 30 hours, recording the peak areas of benzoic acid and sorbic acid in the sample, and calculating the RSD value. The specific results are shown in Table 12 below.
Table 12
From the table, the benzoic acid peak area RSD of the same sample solution is 1.23% and the sorbic acid peak area RSD is 0.22% within 30 hours, and the stability of the sample solution is good according to the relevant requirements of the drug quality standard analysis method guidelines in the fourth edition of the pharmacopoeia 2015, which are referred to in 9101.
(2) Comparison of extraction methods
1g of gelatin hollow capsule is precisely weighed, placed in a 50ml centrifuge tube, added with 10ml of water at 60 ℃, immediately vortex and shake to dissolve the test sample, prepare 5 parts, then respectively ultrasonically process for 20 minutes at 40 ℃, ultrasonically process for 20 minutes at 60 ℃, ultrasonically process for 15 minutes at 50 ℃, ultrasonically process for 20 minutes at 50 ℃, ultrasonically process for 25 minutes at 50 ℃, then prepare test sample solution, and respectively sample injection is carried out to determine peak areas of benzoic acid and sorbic acid in the test sample. The specific results are shown in Table 13 below.
TABLE 13
Referring to the related requirements of the guidelines of the standard analysis method of medicine quality in the four parts of the 2015 edition of Chinese pharmacopoeia, the data in the table are combined, so that the variation of the ultrasonic temperature and the time in a small range during the extraction of the test sample has no obvious influence on the test result.
(3) Comparison of chromatographic conditions
Taking the same sample solution, the same 1 mug/ml benzoic acid reference solution and the same 5 mug/ml sorbic acid reference solution, changing chromatographic conditions, taking the above solutions and sampling once, and recording the chromatogram. The specific results are shown in Table 14 below.
TABLE 14
Referring to the relevant requirements of the guidelines of the standard analytical method of medicine quality in the four parts of the 2015 edition of Chinese pharmacopoeia, the data in the table can be combined, so that the small-range variation of chromatographic conditions such as column temperature, sample injection amount, mobile phase proportion, flow rate and the like has no obvious influence on test results.
Further, a chromatographic column having a column length of 150mm, an inner diameter of 4.6mm and a particle diameter of 5 μm was used for the test, and it was found that the separation degree of sorbic acid from the adjacent impurity peaks was not satisfactory, so that in this example, a chromatographic column having a column length of 250mm, an inner diameter of 4.6mm and a particle diameter of 5 μm was required.
The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art, who is within the scope of the present invention, should make equivalent substitutions or modifications according to the technical scheme of the present invention and the inventive concept thereof, and should be covered by the scope of the present invention.
Claims (7)
1. The method for concentrating, extracting and measuring sorbic acid and benzoic acid in the gelatin hollow capsule is characterized by comprising the following steps:
1) Sample solution preparation: precisely weighing gelatin hollow capsules, placing into a centrifuge tube, adding water, immediately vortex oscillating to dissolve a test sample, rapidly adding absolute ethyl alcohol after ultrasonic treatment, gently shaking and uniformly mixing to precipitate gelatin, performing ultrasonic treatment and centrifugation, taking supernatant, recovering solvent under reduced pressure, quantitatively transferring into a measuring flask, diluting with water to scale, shaking uniformly, filtering, and taking subsequent filtrate to obtain a test sample solution;
2) Preparing a reference substance solution: precisely weighing sodium benzoate and potassium sorbate as reference substances, and adding water to obtain reference substance solution;
3) Liquid chromatograph measurement: respectively precisely sucking 10 μl of the reference substance solution and 10 μl of the sample solution, and injecting into a liquid chromatograph for measurement to obtain sample chromatograph;
the chromatographic conditions of the liquid chromatograph in the step 3) are specifically as follows: the chromatographic column takes octadecylsilane chemically bonded silica as a filler, and takes ammonium acetate solution-methanol with the mass ratio of 0.02mol/L of 93:7 or 94:6 as a mobile phase; the detection wavelength was 229nm or 231nm.
2. The method for concentrated extraction and determination of sorbic acid and benzoic acid in a gelatin hollow capsule according to claim 1 wherein: the gelatin hollow capsule in the step 1) is precisely weighed to 1g, placed in a 50ml centrifuge tube, and then added with water at 60 ℃ for 10ml to dissolve.
3. The method for concentrated extraction and determination of sorbic acid and benzoic acid in a gelatin hollow capsule according to claim 1 wherein: the first ultrasonic treatment in the step 1) is carried out for 20 minutes at 50 ℃, and 40ml of absolute ethyl alcohol is added; the second sonication was performed at 50℃for 1 minute and centrifuged at 7000RPM for 8 minutes.
4. The method for concentrated extraction and determination of sorbic acid and benzoic acid in a gelatin hollow capsule according to claim 1 wherein: the solvent was recovered to 2ml under reduced pressure in step 1), and quantitatively transferred to a 25ml measuring flask and diluted to scale with water.
5. The method for concentrated extraction and determination of sorbic acid and benzoic acid in a gelatin hollow capsule according to claim 1 wherein: the concentration of the reference substance solution in the step 2) is specifically as follows: each 1ml contains 10. Mu.g of benzoic acid and sorbic acid.
6. The method for concentrated extraction and determination of sorbic acid and benzoic acid in a gelatin hollow capsule according to claim 1 wherein: the column length of the chromatographic column is 250mm, the inner diameter is 4.6mm, and the particle size is 5 mu m.
7. The method for concentrated extraction and determination of sorbic acid and benzoic acid in a gelatin hollow capsule according to claim 1 wherein: if the chromatographic peak of the sample obtained by the measurement in the step 3) is consistent with the retention time of the reference substance, detecting the sample by using a diode array detector; if the chromatographic peak is the same as the ultraviolet absorption spectrum of the reference substance, the corresponding component is judged to be contained.
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