CN113049720A - Method for detecting residual quantity of ethanol in compound fresh bamboo juice - Google Patents
Method for detecting residual quantity of ethanol in compound fresh bamboo juice Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 24
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- 150000001875 compounds Chemical class 0.000 title claims abstract description 17
- 235000011389 fruit/vegetable juice Nutrition 0.000 title claims abstract description 17
- 239000000243 solution Substances 0.000 claims description 37
- 235000019441 ethanol Nutrition 0.000 claims description 33
- 238000012360 testing method Methods 0.000 claims description 20
- 239000013558 reference substance Substances 0.000 claims description 19
- 239000012085 test solution Substances 0.000 claims description 19
- 238000002360 preparation method Methods 0.000 claims description 18
- 238000007789 sealing Methods 0.000 claims description 17
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 16
- 238000001514 detection method Methods 0.000 claims description 13
- 238000005303 weighing Methods 0.000 claims description 13
- 239000000047 product Substances 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- 239000012088 reference solution Substances 0.000 claims description 9
- 239000012159 carrier gas Substances 0.000 claims description 8
- 229910052757 nitrogen Inorganic materials 0.000 claims description 8
- 230000005526 G1 to G0 transition Effects 0.000 claims description 7
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- 239000010453 quartz Substances 0.000 claims description 7
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- UAJTZZNRJCKXJN-UHFFFAOYSA-M sodium;2-dodecoxy-2-oxoethanesulfonate Chemical compound [Na+].CCCCCCCCCCCCOC(=O)CS([O-])(=O)=O UAJTZZNRJCKXJN-UHFFFAOYSA-M 0.000 description 1
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/64—Electrical detectors
- G01N30/68—Flame ionisation detectors
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Abstract
The invention belongs to the field of pharmacy, and relates to a method for detecting residual quantity of ethanol in a medicament. The invention aims to provide a method for detecting the residual quantity of ethanol in compound fresh bamboo juice. The method has the characteristics of high sensitivity, good stability, good specificity, high precision and the like. The invention determines the residual quantity of ethanol in the compound fresh bamboo juice by gas chromatography.
Description
Technical Field
The invention belongs to the field of pharmacy, and relates to a method for detecting residual quantity of ethanol in a medicament.
Background
The compound fresh bamboo juice is prepared from fresh bamboo juice, herba Houttuyniae, rhizoma Pinelliae, rhizoma Zingiberis recens, folium Eriobotryae, radix Platycodi, and peppermint oil. Has the effects of clearing heat, eliminating phlegm and relieving cough. The traditional Chinese medicine composition is clinically used for treating phlegm-heat cough with yellow and viscous phlegm.
In the recipe, fresh bamboo juice is sweet and cold in nature and has the functions of clearing heat and eliminating phlegm, and is the monarch drug. The heartleaf houttuynia herb has the effects of clearing heat and removing toxicity and assisting the fresh bamboo juice in clearing heat and removing toxicity; pinellia ternate, loquat leaves and platycodon grandiflorum are ministerial drugs for eliminating dampness and phlegm, moistening lung and relieving cough. Ginger, rhizoma Zingiberis recens, is used as adjuvant drug for lowering qi and regulating stomach. Mint is pungent and cool, and can dispel heat pathogen. The medicines are used together to play the role of clearing heat, resolving phlegm and relieving cough.
The current standard of the product is collected in one part of the 2015 edition of Chinese pharmacopoeia, ethanol precipitation is introduced under the item of a preparation method in the current standard, but solvent recovery is carried out until no alcohol smell exists, and in order to investigate whether ethanol is completely recovered by different enterprises, ethanol is specified as a third type of solvent in a 0861 residual solvent determination method in the four parts of the 2015 edition of Chinese pharmacopoeia, and the ethanol residual quantity is detected by adopting a gas chromatography.
Therefore, the invention carries out intensive research on the detection method of the residual quantity of the ethanol in the compound fresh bamboo juice.
Disclosure of Invention
The invention aims to provide a method for detecting the residual quantity of ethanol in compound fresh bamboo juice. The method has the characteristics of high sensitivity, good stability, good specificity, high precision and the like.
The compound fresh bamboo juice is the existing product, can be purchased in the market, and can also be prepared by the known method.
The invention determines the residual quantity of ethanol in the compound fresh bamboo juice by gas chromatography.
The method for detecting the residual amount of ethanol in the compound fresh bamboo juice comprises the following steps:
preparation of control solutions:
taking a proper amount of absolute ethyl alcohol reference substance, precisely weighing, adding water to obtain a solution as a reference substance solution,
preparation of a test solution:
precisely measuring compound fresh succus Bambusae, placing in a headspace bottle, sealing the bottle mouth,
the determination method comprises the following steps:
respectively taking a reference solution and a test solution, introducing sample in headspace, recording chromatogram, calculating the residual amount of ethanol in the test solution by peak area according to an external standard method,
the chromatographic condition and system applicability test adopts polyethylene glycol as a stationary phase and adopts an elastic quartz capillary column; column temperature is programmed temperature rise: the initial temperature is 30-50 ℃, the temperature is maintained for 1-3 minutes to 50-80 ℃, and then the temperature is increased to 220 ℃ at 180 ℃; detector (FID) temperature 220 ℃; the carrier gas is nitrogen, and the flow rate is 1-4 ml/min; and (4) carrying out headspace flow splitting and sample injection.
Wherein, the preparation of the reference substance solution:
accurately weighing appropriate amount of anhydrous ethanol reference substance, adding water to obtain solution containing 5mg per 1ml, accurately weighing 2ml as reference substance solution, placing in 20ml headspace bottle, and sealing the bottle mouth.
Wherein, the preparation of the test solution:
precisely measuring 2ml of the product, placing into a 20ml headspace bottle, and sealing the bottle mouth to obtain the final product.
Wherein, the chromatographic condition and the system applicability test take polyethylene glycol as a stationary phase and adopt an elastic quartz capillary column DB-WAXETR; column temperature is programmed temperature rise: the initial temperature is 40 ℃, the temperature is maintained for 2 minutes, the temperature is increased to 65 ℃ at the rate of 3 ℃ per minute, then the temperature is increased to 200 ℃ at the rate of 25 ℃ per minute, and the temperature is maintained for 3 minutes; the temperature of a sample inlet is 200 ℃; the temperature of the detector is 220 ℃; the carrier gas is nitrogen, and the flow rate is 2.0 ml/min; and (4) carrying out headspace flow splitting and sample injection.
Preferably, the detection method of the present invention comprises the following steps:
preparation of control solutions:
accurately weighing appropriate amount of anhydrous ethanol reference substance, adding water to obtain solution containing 5mg per 1ml, accurately weighing 2ml as reference substance solution, placing in 20ml headspace bottle, sealing the bottle mouth,
preparation of a test solution:
precisely measuring 2ml of the product, placing into a 20ml headspace bottle, sealing the bottle mouth to obtain the final product,
the determination method comprises the following steps:
respectively taking a reference solution and a test solution, introducing sample in headspace, recording chromatogram, calculating the residual amount of ethanol in the test solution by peak area according to an external standard method,
the chromatographic condition and system applicability test adopts polyethylene glycol as stationary phase, and adopts elastic quartz capillary column DB-WAXETR (column length is 30m, inner diameter is 0.32mm, and film thickness is 1.0 μm); column temperature is programmed temperature rise: the initial temperature is 40 ℃, the temperature is maintained for 2 minutes, the temperature is increased to 65 ℃ at the rate of 3 ℃ per minute, then the temperature is increased to 200 ℃ at the rate of 25 ℃ per minute, and the temperature is maintained for 3 minutes; the temperature of a sample inlet is 200 ℃; the temperature of the detector is 220 ℃; the carrier gas is nitrogen, and the flow rate is 2.0 ml/min; and (3) injecting the headspace split stream, wherein the split stream ratio is 20:1, the equilibrium temperature of the headspace bottle is 85 ℃, the equilibrium time is 20 minutes, and the number of theoretical plates is not less than 10000 calculated according to the peak of absolute ethyl alcohol.
Compared with two detection methods loaded in 0711 ethanol quantitative determination method of the general rule of the four parts of Chinese pharmacopoeia, the method uses a polar capillary chromatographic column more suitable for aqueous solution determination, adopts a gas chromatography-mass spectrometry combined method for confirmation, does not use an internal standard substance, and has the advantages of better chromatographic peak shape, better separation effect, false positive avoidance, simpler and more convenient operation and the like.
Drawings
FIG. 1, standard curve of absolute ethanol
FIG. 2 GC chromatogram of absolute ethanol control
FIG. 3 GC chromatogram of sample
FIG. 4, negative control GC chromatogram
FIG. 5 Scan Total ion flowgram of ethanol control solution
FIG. 6 shows the peak mass spectrum of the ethanol control solution with a retention time of 2.921min
FIG. 7 is a graph showing Sim (43, 45, 31m/z) of an ethanol control solution
FIG. 8 shows Scan total ion current diagrams of test solutions
FIG. 9 shows the ethanol peak mass spectrum of the sample solution with a retention time of 2.921min
FIG. 10 is a graph showing Sim (43, 45, 31m/z) of a sample solution
FIG. 11 Scan Total ion flowgram of negative control solution
FIG. 12 is a graph showing Sim (43, 45, 31m/z) of the negative control solution
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that the upper and lower limits of the range, and each intervening value therebetween, is specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
Example 1 detection method of the invention
1. Instruments, drugs and reagents
Gas chromatograph: agilent 7890B gas chromatograph, FID detector, ChemStation chromatography workstation; agilent 7890A-5975C gas chromatography-mass spectrometer, Agilent7697 headspace sampling instrument; sartorius BSA 124S-CW electronic balance.
The compound fresh bamboo juice comprises a production unit A and a batch number of: 2005004, 28 lots of samples from production unit B, sample information is shown in Table 5.
Negative control samples: the boiling and cooling ultrapure water was taken as a negative control solution.
Absolute ethanol control: the source is as follows: batch number of Yonghua chemical technology (Jiangsu) Co., Ltd: the content of 64-17-5 is more than or equal to 99.9 percent
Reagent: the water is ultrapure water.
2. Gas chromatography for determining ethanol residue in compound fresh bamboo juice
2.1 chromatographic conditions and System suitability test
Polyethylene glycol is used as a stationary phase, and an elastic quartz capillary column DB-WAXETR (the column length is 30m, the inner diameter is 0.32mm, and the film thickness is 1.0 μm) is adopted; column temperature is programmed temperature rise: the initial temperature is 40 ℃, the temperature is maintained for 2 minutes, the temperature is increased to 65 ℃ at the rate of 3 ℃ per minute, then the temperature is increased to 200 ℃ at the rate of 25 ℃ per minute, and the temperature is maintained for 3 minutes; the temperature of a sample inlet is 200 ℃; detector (FID) temperature 220 ℃; the carrier gas is nitrogen, and the flow rate is 2.0 ml/min; and (3) injecting a headspace split stream, wherein the split stream ratio is 20:1, the equilibrium temperature of a headspace bottle is 85 ℃, and the equilibrium time is 20 minutes. The number of theoretical plates is not less than 10000 calculated according to the absolute ethyl alcohol peak.
2.2 preparation of test solutions
Precisely measuring 2ml of the product, placing into a 20ml headspace bottle, and sealing the bottle mouth to obtain the final product.
2.3 investigation of Linear relationship and minimum detection Limit
Accurately weighing 1.0099g of absolute ethyl alcohol reference substance, placing in a 20ml measuring flask, adding water to dilute to scale, and shaking up; precisely sucking 1ml, 3ml, 5ml, 1ml and 5ml of the above solutions, respectively placing in 100ml, 50ml, 5ml and 10ml measuring bottles, adding water to dilute to scale, and shaking uniformly to obtain the final product. Precisely measuring each 2ml of the above reference substance solution, placing in 20ml headspace bottle, sealing the bottle mouth, introducing headspace sample, measuring peak area according to text chromatogram condition, taking peak area integral value as ordinate, and reference substance sample introduction amount as abscissaCoordinate, drawing standard curve, and regression equation of absolute ethyl alcohol is that y is 849.1211x +120.0482, R2The results are shown in table 1 and fig. 1, and the absolute ethyl alcohol is in good linearity between 1.0089 and 50.4445 mg.
When the signal-to-noise ratio (S/N) is 3, the lowest detection limit of the absolute ethyl alcohol is 0.00504 mg; the lowest detection limit for absolute ethanol was 0.01008mg at a signal-to-noise ratio (S/N) of 10.
TABLE 1 measurement of Linear relationship
2.4 specificity test
Precisely measuring the sample solution, the reference solution and the negative reference solution by 2ml respectively, placing in a 20ml headspace bottle, sealing the bottle mouth, and measuring according to the above conditions. Comparing the chromatogram of the test solution with the chromatogram of the reference sample, the result is negative, other components in the reference sample have no interference to the peak of the reference sample (see fig. 2-4), and the absolute ethyl alcohol has better chromatographic separation.
2.5 precision test
2.5.1 sample introduction precision 2ml of a control solution (5.0495mg/ml) was measured accurately, placed in a 20ml headspace bottle, the mouth of the bottle was sealed, and sample introduction was repeated 6 times under the chromatographic conditions in the text, the results are shown in Table 2.
TABLE 2 control precision test results
2.5.2 repeatability tests six portions of this product (lot 2005004) were collected and tested according to the method under the chromatographic conditions in the text, the results are shown in table 3, the average absolute ethanol content is 1.5752mg/ml, and the RSD is 1.1%.
TABLE 3 results of the repeatability test of absolute ethanol
2.6 recovery test
By sample adding and recovering test, precisely measuring 1ml of product (batch number: 2005004) with known content, placing into 20ml headspace bottle, precisely adding 1ml of reference substance solution (5.0495mg/ml), sealing the bottle mouth, measuring according to chromatographic conditions in text, and calculating recovery rate, the results are shown in Table 4.
TABLE 4 test results on recovery of Anhydrous ethanol
2.7 analysis of results
Referring to the rule that the limit of ethanol residue is 0.5% in a 0861 residual solvent determination method according to the four rules of China pharmacopoeia 2015 edition, the samples tested at this time are determined, and the ethanol residue of 28 batches of samples is found to be serious in standard exceeding (the sample information is shown in table 5), which indicates that the ethanol recovery in the production process of enterprises is incomplete, the production process is problematic and the quality risk exists. To further verify the above test, it was further verified using gas chromatography-mass spectrometry.
TABLE 528 measurement of residual ethanol in samples
Example 2, HS-GCMS confirmation of ethanol in compound fresh bamboo juice
3.1 chromatographic conditions and System suitability test
Using DB-FFAP (30m 250um 0.25um) as chromatographic column, starting temperature of 40 deg.C, holding for 2 min, raising temperature to 65 deg.C for 0.5 min, and raising temperature to 200 deg.C for 2 min; the injection port temperature is 250 ℃, He is used as carrier gas, the column flow rate is 1ml/min, and the split ratio is 300: 1. The equilibrium temperature of the headspace bottle was 85 ℃, the equilibration time was 20 minutes, and the injection volume was 1.0 ml.
3.2 Mass Spectrometry conditions
Electron Impact (EI) source, electron impact energy: 70ev, the ion source temperature is 230 ℃, the quadrupole rod temperature is 150 ℃, and the acquisition mode is as follows: 5-200m/z full scan and 43, 45, 31m/z selective ion monitoring (see Table 6)
TABLE 6 reference quality Spectrum detection parameters
3.3 preparation of the solution:
preparation of control solutions: accurately weighing appropriate amount of anhydrous ethanol, and adding water to obtain solution containing 5mg per 1 ml; precisely measuring the solution in a headspace bottle of 2ml to 20ml, and sealing to obtain the product.
Preparation of a test solution: precisely measuring compound fresh succus Bambusae in 2-20 ml headspace bottle, and sealing.
Preparation of negative control solution: precisely weighing boiled and cooled ultrapure water in a headspace bottle of 2ml to 20ml, and sealing to obtain the product.
3.4 assay: and (3) injecting the reference substance solution and the test solution into a gas chromatography-mass spectrometer for gas-mass spectrometry analysis, and recording full Scan (Scan) and selective ion monitoring (Sim) spectrograms.
3.5 judging the result:
confirmation of ethanol: searching matching (the matching degree of the ethanol peak is more than 95%) and a Sim chart through an NIST spectrum library of three chromatographic peaks of a Scan chart of the ethanol reference solution, sequentially confirming the three peaks in the Scan total ion flow chart of the ethanol reference solution as nitrogen, ethanol and water, and ensuring the retention time of the ethanol peak to be 2.921min, as shown in figures 5-7.
And (5) judging a result: in the ion flow chromatogram of a test sample monitored by three ion selective ions with mass-to-charge ratios of m/z 31, 43 and 45 (ethanol), chromatographic peaks consistent with the retention time of a reference sample and the chromatogram should be presented simultaneously.
And (3) sample determination: and respectively sucking a reference substance solution, a test substance solution and a negative reference solution, and measuring according to the conditions. As a result, in the ion flow chromatogram of the test sample monitored by the ion selective ions with the mass-to-charge ratios of m/z 31, 43 and 45 (ethanol), the solution of the test sample shows chromatographic peaks consistent with the retention time of the chromatogram of the reference sample; and ethanol is not detected in the negative control sample, which indicates that the experiment has certain specificity. FIGS. 8 to 12 show the following.
The 28 samples that were positive for gas chromatography were tested as described above and all ethanol was detected and confirmed as shown in Table 7.
TABLE 7 results of sample measurement
EXAMPLE 3 examination of the column of the invention
| Investigation of chromatographic columns | Head space equilibrium temperature | Sample size | Head space balance time | Split ratio |
| DB-624(30m*0.53mm*3.0μm) | 70℃ | 1ml | 15min | 1:1 |
| DB-WAX(30m*0.32mm*1.0μm) | 80℃ | 2ml | 20min | 10:1 |
| DB-WAXETR(30m*0.32mm*1.0μm) | 85℃ | 3ml | 30min | 20:1 |
Example 4 comparative experiment
The experimental result shows that compared with the existing method, the detection method of the invention has better effect, high separation degree and good chromatographic peak shape.
Claims (5)
1. The method for detecting the residual amount of ethanol in the compound fresh bamboo juice is characterized by comprising the following steps:
preparation of control solutions:
taking a proper amount of absolute ethyl alcohol reference substance, precisely weighing, adding water to obtain a solution as a reference substance solution,
preparation of a test solution:
precisely measuring compound fresh succus Bambusae, placing in a headspace bottle, sealing the bottle mouth,
the determination method comprises the following steps:
respectively taking a reference solution and a test solution, introducing sample in headspace, recording chromatogram, calculating the residual amount of ethanol in the test solution by peak area according to an external standard method,
the chromatographic condition and system applicability test adopts polyethylene glycol as a stationary phase and adopts an elastic quartz capillary column; column temperature is programmed temperature rise: the initial temperature is 30-50 ℃, the temperature is maintained for 1-3 minutes to 50-80 ℃, and then the temperature is increased to 220 ℃ at 180 ℃; detector (FID) temperature 220 ℃; the carrier gas is nitrogen, and the flow rate is 1-4 ml/min; and (4) carrying out headspace flow splitting and sample injection.
2. The detection method according to claim 1,
preparation of control solutions:
accurately weighing appropriate amount of anhydrous ethanol reference substance, adding water to obtain solution containing 5mg per 1ml, accurately weighing 2ml as reference substance solution, placing in 20ml headspace bottle, and sealing the bottle mouth.
3. The detection method according to claim 1,
preparation of a test solution:
precisely measuring 2ml of the product, placing into a 20ml headspace bottle, and sealing the bottle mouth to obtain the final product.
4. The detection method according to claim 1,
the chromatographic condition and system applicability test adopts polyethylene glycol as a stationary phase and adopts an elastic quartz capillary column DB-WAXETR; column temperature is programmed temperature rise: the initial temperature is 40 ℃, the temperature is maintained for 2 minutes, the temperature is increased to 65 ℃ at the rate of 3 ℃ per minute, then the temperature is increased to 200 ℃ at the rate of 25 ℃ per minute, and the temperature is maintained for 3 minutes; the temperature of a sample inlet is 200 ℃; the temperature of the detector is 220 ℃; the carrier gas is nitrogen, and the flow rate is 2.0 ml/min; and (4) carrying out headspace flow splitting and sample injection.
5. The detection method according to claim 1,
preparation of control solutions:
accurately weighing appropriate amount of anhydrous ethanol reference substance, adding water to obtain solution containing 5mg per 1ml, accurately weighing 2ml as reference substance solution, placing in 20ml headspace bottle, sealing the bottle mouth,
preparation of a test solution:
precisely measuring 2ml of the product, placing into a 20ml headspace bottle, sealing the bottle mouth to obtain the final product,
the determination method comprises the following steps:
respectively taking a reference solution and a test solution, introducing sample in headspace, recording chromatogram, calculating the residual amount of ethanol in the test solution by peak area according to an external standard method,
the chromatographic condition and system applicability test adopts polyethylene glycol as stationary phase, and adopts elastic quartz capillary column DB-WAXETR (column length is 30m, inner diameter is 0.32mm, and film thickness is 1.0 μm); column temperature is programmed temperature rise: the initial temperature is 40 ℃, the temperature is maintained for 2 minutes, the temperature is increased to 65 ℃ at the rate of 3 ℃ per minute, then the temperature is increased to 200 ℃ at the rate of 25 ℃ per minute, and the temperature is maintained for 3 minutes; the temperature of a sample inlet is 200 ℃; the temperature of the detector is 220 ℃; the carrier gas is nitrogen, and the flow rate is 2.0 ml/min; and (3) injecting the headspace split stream, wherein the split stream ratio is 20:1, the equilibrium temperature of the headspace bottle is 85 ℃, the equilibrium time is 20 minutes, and the number of theoretical plates is not less than 10000 calculated according to the peak of absolute ethyl alcohol.
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| CN114002341A (en) * | 2021-09-18 | 2022-02-01 | 天地壹号饮料股份有限公司 | Method for determining ethanol content in wine based on gas chromatography |
| CN114441695A (en) * | 2022-01-26 | 2022-05-06 | 武汉九州钰民医药科技有限公司 | Method for detecting N, N-dimethylformamide in ceftazidime residual solvent and application |
| CN114720604A (en) * | 2022-05-19 | 2022-07-08 | 江西省药品检验检测研究院 | Construction method of heat-clearing and phlegm-eliminating traditional Chinese medicine characteristic map and characteristic map thereof |
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