CN113655133A - Method for detecting N, N-diisopropyl carbodiimide and application - Google Patents
Method for detecting N, N-diisopropyl carbodiimide and application Download PDFInfo
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- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
- G01N2030/324—Control of physical parameters of the fluid carrier of pressure or speed speed, flow rate
Abstract
The invention relates to the technical field of genotoxic impurity detection, and particularly discloses a method for detecting N, N-diisopropyl carbodiimide and application thereof. The method for detecting the N, N-diisopropyl carbodiimide adopts a gas chromatography-mass spectrometry combined mode to detect the N, N-diisopropyl carbodiimide in the object to be detected; the temperature rise procedure of the gas phase part is as follows: the initial temperature is 50-70 ℃, the temperature is kept for 3-7min, the temperature is raised to 300 ℃ at the speed of 20-40 ℃/min, and the temperature is kept for 2-5 min. The invention can directly carry out sample introduction detection, does not need sample pretreatment, and has the advantages of short detection time, strong signal response, high sensitivity and wide applicability.
Description
Technical Field
The invention relates to the technical field of genotoxic impurity detection, in particular to a method for detecting N, N-diisopropyl carbodiimide and application thereof.
Background
In recent years, with the improvement of the research requirements of genotoxic impurities in the raw material medicines, substances such as coupling agent N, N-diisopropyl carbodiimide and the like are determined to be genotoxic impurities, and become necessary research contents in the detection of the raw material medicines. Genotoxic impurities are derived from starting materials, intermediates, reagents and reaction by-products of the drug substance synthesis process, and may also result from degradation of the drug substance during synthesis, storage or formulation. It features that at low concentration, the genetic matter of human body is damaged, resulting in gene mutation and promoting tumor to take place. In the existing drug quality control standard, strict control is required.
The coupling agent N, N-diisopropyl carbodiimide (DIC) is a light yellow transparent liquid and is the most commonly used organic synthetic coupling agent in the process of synthesizing raw material medicaments. Since 1955, the substances are used for polypeptide synthesis, and N, N-diisopropyl carbodiimide is widely applied in many chemical fields. In organic synthesis reactions, the coupling reaction of amino and carboxyl, DIC is the first choice condensing agent, and is widely used in such organic synthesis as polypeptide drugs such as nesiritide, oxytocin, somatostatin and vasopressin, medical excipients such as polyethylene glycol monomethyl ether, and amino and carboxyl condensation reactions.
At present, no mature, reliable and stable method exists for detecting the coupling agent N, N-diisopropyl carbodiimide. In the prior patent, the content of N, N-diisopropyl carbodiimide is detected by adopting liquid chromatography-mass spectrometry, but the sample needs to be subjected to derivatization pretreatment, and the stability and the repeatability of a detection result are poor. If the derivatized product is present in the sample, the N, N-diisopropylcarbodiimide content cannot be accurately quantified. Therefore, further research on the detection mode is needed.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide a method which is simple and quick, has high sensitivity, does not need pretreatment operations such as derivatization and the like, and can realize accurate quality control of N, N-diisopropylcarbodiimide in bulk drugs.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a method for detecting N, N-diisopropyl carbodiimide adopts a gas chromatography-mass spectrometry combined mode to detect the N, N-diisopropyl carbodiimide in a substance to be detected; the temperature rise procedure of the gas phase part is as follows: the initial temperature is 50-70 ℃, the temperature is kept for 3-7min, the temperature is raised to 300 ℃ at the speed of 20-40 ℃/min, and the temperature is kept for 2-5 min.
Preferably, the temperature raising procedure of the gas phase part is as follows: the initial temperature is 60 deg.C, and the temperature is maintained for 5min, and the temperature is increased to 250 deg.C at the rate of 30 deg.C/min, and maintained for 2 min.
The invention fully considers the retention condition of the N, N-diisopropyl carbodiimide in the chromatographic column, the condition after the N, N-diisopropyl carbodiimide is combined with the product to be detected and the condition of an interference peak possibly generated in the solvent, and sets the temperature rise program of the gas phase part, so that the gasified substance of the sample at the sample inlet can completely flow out of the chromatographic column, the interference on the subsequent sample injection can not be generated, the interference peak in the solvent can be effectively eliminated, and the ideal stability, reproducibility and accuracy are realized.
In the invention, the carrier gas of the gas phase part is helium, the flow rate range is 0.9-1.1 ml/min, and the sample injection split ratio is (5-20): 1.
the combination of the split flow ratio and the carrier gas flow rate can meet the requirements of signal response, peak appearance type, tailing factor and the like of the N, N-diisopropyl carbodiimide, so that the N, N-diisopropyl carbodiimide has a good symmetrical Gaussian peak shape, the interference generated by the solvent effect is eliminated, the influence of the flow rate on the retention time can be considered, the peak appearance time of a reference substance is within 10min, the sensitivity of the detection method is effectively improved, the detection of the reference substance at low concentration is facilitated, and the detection limit and the quantification limit are lower.
In the invention, a direct sample injection mode is adopted, and the sample injection amount is 1-5 mul, preferably 1 mul.
In the detection system, the sample injection quantity can ensure the peak type, the sensitivity and the signal response intensity of a chromatographic peak.
In the invention, the concentration of the test sample is 9.0mg/ml-11.0 mg/ml. The solvent is methanol, acetonitrile, dichloromethane, N-dimethylformamide and the like. The solvent selection can be carried out by referring to the solubility of the substance to be detected, and the detection is not obviously influenced.
In the present invention, a weak polarity chromatographic column is used.
Preferably, the chromatographic column is a 100% -dimethylpolysiloxane stationary liquid chromatographic column (nonpolar chromatographic column), or a 5% -diphenyl-95% -dimethyl arylene siloxane copolymer stationary liquid chromatographic column and a chromatographic column (low-polarity chromatographic column) which has similar stationary phase ratio composition with the stationary phase.
More preferably, the column is HP-1, HP-5MS UI or DB-624 UI.
According to the invention, according to the physicochemical property of the N, N-diisopropyl carbodiimide, the retention of the N, N-diisopropyl carbodiimide is considered, the interference peaks of a solvent and a sample are also considered, and the selected chromatographic column can realize the effective detection of the N, N-diisopropyl carbodiimide and can avoid the interference. If the detection sensitivity is reduced after the chromatographic column is replaced, the initial column temperature, the split flow ratio of the sample inlet or the sample volume can be adjusted within the limit of the invention, so that the signal response intensity is improved, and the requirement of the detection limit is met.
In the invention, the injection port temperature of the gas phase part is 160-180 ℃.
The invention fully considers the boiling point of the sample and combines the gasification property of the sample to set the temperature of the sample inlet, thereby ensuring that the sample is completely gasified during detection to be matched with subsequent detection.
In the present invention, the ion source temperature of the mass spectrometry part is 230 ℃; the temperature of the four-level bar is 150 ℃; the electron energy is 70 ev; the monitoring mode is SCAN; the scanning ion range is m/z 30-150.
The ion source selection instrument is configured with ion source types, the electron energy adopts a fixed value, and the temperature adopts a fixed value. And (3) selecting an ion scanning monitoring mode, preferably selecting an ion spectrum of the reference substance, and selecting ions with good response and stable ionization.
In the sample detection and analysis, the coupling agent N, N-diisopropyl carbodiimide in the product (such as a medicine) can be quantitatively detected by adopting a standard curve method and an external standard method. In the standard curve method, the preparation concentration range of the reference substance solution needs to cover the limit of the coupling agent N, N-diisopropylcarbodiimide in the bulk drug. In the external standard method, the concentration of the reference substance solution is the limit concentration of the coupling agent N, N-diisopropyl carbodiimide in the bulk drug.
The invention also provides an application of the method in product quality control.
The product can be a pharmaceutical active ingredient, such as polypeptide, and the like, and can also be a medical auxiliary material, such as polyethylene glycol monomethyl ether, and the like.
The invention has the beneficial effects that:
the invention establishes a simple, stable and reliable detection method based on the chemical property of the coupling agent N, N-diisopropyl carbodiimide and by adopting a gas chromatography-mass spectrometry instrument, and realizes the accurate and quantitative detection method of the coupling agent N, N-diisopropyl carbodiimide residue in the medicine.
According to the method, the coupling agent N, N-diisopropyl carbodiimide is directly injected for detection, sample pretreatment is not needed, the detection time is short, the substance is well reserved in a chromatographic column, the signal response is strong, the sensitivity is high, the applicability is wide, the coupling agent N, N-diisopropyl carbodiimide used in medicines is suitable, and the requirements of different limits of raw material medicines on the substance can be met.
Drawings
FIG. 1 is a total ion flow diagram of the coupling agent N, N-diisopropylcarbodiimide in example 1.
FIG. 2 is a mass spectrum of the coupling agent N, N-diisopropylcarbodiimide in example 1 at a retention time of 5.660 min.
FIG. 3 is a comparison of mass spectra of the instrument with standard library in example 1.
FIG. 4 is a chromatogram of specificity of the detection method for N, N-diisopropylcarbodiimide of the coupling agent in example 1.
FIG. 5 is a linear calibration curve for the coupling agent N, N-diisopropylcarbodiimide used in example 1.
FIG. 6 is a comparison of the total ion flow diagrams of the coupling agent N, N-diisopropylcarbodiimide in example 2 on different chromatographic columns.
FIG. 7 is a linear calibration curve for the coupling agent N, N-diisopropylcarbodiimide in example 3.
FIG. 8 is a comparison of chromatograms of different temperature program in comparative example 1.
Fig. 9 is a comparison of different split ratio chromatograms in comparative example 2.
In each figure, Intensity represents Intensity and Retention time represents Retention time.
Detailed Description
Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The detection instrument adopted in the embodiment of the invention is Agilent7890B +7000D gas chromatography-mass spectrometry combined instrument (Agilent instruments).
Example 1
This example provides a method for detecting the residual content of coupling agent N, N-diisopropylcarbodiimide (residual limit requirement is 10ppm) in polyethylene glycol monomethyl ether polymer.
Acetonitrile is used as a solvent, and direct sample injection is adopted to detect the coupling agent N, N-diisopropyl carbodiimide in the polyethylene glycol monomethyl ether polymer.
(1) Preliminary detection instrument condition of coupling agent N, N-diisopropyl carbodiimide
Solvent: acetonitrile
Coupling agent N, N-diisopropyl carbodiimide stock solution: taking about 10mg of coupling agent N, N-diisopropyl carbodiimide, precisely weighing, placing in a 100ml measuring flask, adding acetonitrile to dissolve and dilute to a scale, and shaking up to obtain 0.1mg/ml stock solution.
10 μ g/ml coupling agent N, N-diisopropylcarbodiimide solution: precisely measuring 1ml of coupling agent N, N-diisopropyl carbodiimide stock solution with the concentration of 0.1mg/ml, placing the coupling agent N, N-diisopropyl carbodiimide stock solution into a 10ml measuring flask, diluting the coupling agent N, N-diisopropyl carbodiimide stock solution to the scale with acetonitrile, and shaking up the coupling agent N, N-diisopropyl carbodiimide stock solution to obtain the coupling agent N, N-diisopropyl carbodiimide. 1ml of the solution is put into a sample injection vial, and is directly injected for analysis.
The initial instrumentation parameter conditions are shown in table 1:
TABLE 1 coupling agent N, N-diisopropyl carbodiimide Detector parameter settings
The total ion flow diagram of the coupling agent N, N-diisopropyl carbodiimide is shown in figure 1, the mass spectrum diagram of the coupling agent N, N-diisopropyl carbodiimide at the retention time of 5.660min is shown in figure 2, and the comparison mass spectrum diagram of the instrument with a standard spectrum library is shown in figure 3.
From this, it was found that the detection effect was the best when the quantitative ion was 69.
(2) Determination of coupling agent N, N-diisopropyl carbodiimide detection instrument condition and methodological verification
The quantitative ion was determined to be 69 and the instrument parameters after adjustment are shown in table 2:
TABLE 2 coupling agent N, N-diisopropyl carbodiimide Detector parameter settings
Preparing a linear solution: 2.0ml, 1.5ml, 1.0ml, 0.5ml and 0.1ml of coupling agent N, N-diisopropyl carbodiimide stock solution with the concentration of 1.0 mu g/ml are precisely measured in sequence and respectively placed in 10ml volumetric flasks, diluted to the scale by acetonitrile and evenly shaken to obtain the solution for the linear standard curve. And precisely measuring 1ml of solution for each linear standard curve, placing the solution in a sample injection vial, directly injecting and analyzing, recording a chromatogram and a peak area, and drawing by taking the concentration as an abscissa and the peak area as an ordinate to obtain the linear standard curve, wherein the concentration range is 0.01-0.2 mu g/ml as shown in figure 5.
Preparing a sample solution: accurately weighing 10mg of polyethylene glycol monomethyl ether, placing the polyethylene glycol monomethyl ether in a 1ml volumetric flask, diluting acetonitrile to a scale, transferring a sample solution into a sample injection vial, and directly injecting a sample for analysis.
Blank solvent: 1ml of acetonitrile is put into a sample injection vial, and sample injection analysis is directly carried out.
Preparation of spiked sample solutions (three horizontal spiked sample solutions of 50%, 100% and 150% were prepared, respectively):
accurately weighing 10mg of a sample, placing the sample in a 1ml measuring flask, diluting the sample to a scale by using a coupling agent N, N-diisopropyl carbodiimide solution with the concentration of 0.05 mu g/ml, shaking up to obtain a 50% labeled sample solution, and preparing 3 parts in parallel; accurately weighing 10mg of a sample, placing the sample in a 1ml measuring flask, diluting the sample to a scale by using a coupling agent N, N-diisopropyl carbodiimide solution with the concentration of 0.1 mu g/ml, shaking up to obtain a 100% labeled sample solution, and preparing 3 parts in parallel; accurately weighing 10mg of a sample, placing the sample in a 1ml measuring flask, diluting the sample to a scale by using a coupling agent N, N-diisopropyl carbodiimide solution with the concentration of 0.15 mu g/ml, shaking up to obtain 150% labeled sample solution, and preparing 3 parts in parallel.
Preparing a reference substance solution: accurately weighing 10mg of a coupling agent N, N-diisopropyl carbodiimide reference substance, placing the reference substance in a 100ml volumetric flask, and adding acetonitrile to dilute to a scale, thus obtaining 0.1mg/ml reference substance mother liquor. Precisely measuring 0.1ml of reference mother liquor, placing the reference mother liquor into a 100ml volumetric flask, adding acetonitrile to dilute the reference mother liquor to a scale, and shaking the reference mother liquor uniformly to obtain a reference solution.
The sample solution, the blank solvent, the standard sample solution (100% standard sample solution) and the reference solution are subjected to sample injection detection, the result is shown in figure 4, and the method is known to have good specificity.
And carrying out sample injection detection by using the prepared standard sample solution, and inspecting the recovery rate. The results are shown in Table 3. From this, the accuracy of the method is good.
TABLE 3 recovery data for 9-pin sample of coupling agent N, N-diisopropylcarbodiimide by spiking
Sample injection precision test solution preparation: precisely measuring 1.0ml of coupling agent N, N-diisopropyl carbodiimide stock solution with the concentration of 1.0 mu g/ml, putting the coupling agent N, N-diisopropyl carbodiimide stock solution into a 10ml volumetric flask, diluting the coupling agent N, N-diisopropyl carbodiimide stock solution to a scale by using acetonitrile, and shaking the solution uniformly to obtain the solution for the sample introduction precision test.
1ml of the sample is taken into a sample introduction bottle, 6 needles are continuously injected by using the instrument parameters in the table 2, the retention time and the relative standard deviation (RSD value) of the peak area are calculated, and the test result is shown in the table 4.
TABLE 4N, N-diisopropylcarbodiimide 6-needle sample introduction precision data
| Sample introduction sequence | Retention time (min) | Peak area (A) |
| Reference 1-1 | 5.667 | 28839 |
| Reference 1-2 | 5.671 | 29213 |
| Reference substances 1-3 | 5.667 | 30034 |
| Reference substances 1-4 | 5.669 | 29707 |
| Reference substances 1-5 | 5.668 | 28029 |
| Reference substances 1-6 | 5.666 | 29678 |
| Mean value | 5.668 | 29250 |
| RSD(％) | 0.03 | 2.50 |
Quantitative limit and detection solution preparation: precisely measuring 1.0ml of a coupling agent N, N-diisopropyl carbodiimide solution with the concentration of 0.1 mu g/ml, putting the coupling agent N, N-diisopropyl carbodiimide solution into a 10ml volumetric flask, diluting the coupling agent N, N-diisopropyl carbodiimide solution to a scale with acetonitrile, and shaking up to obtain a quantitative limiting solution.
1ml of the sample is taken into a sample injection bottle, instrument parameters in the table 2 are adopted, 6 needles of samples are continuously injected, retention time, peak area and signal to noise ratio are obtained, relative standard deviation (RSD value) of the retention time and the peak area is calculated, and test results are shown in the table 5.
Precisely measuring 3.3ml of quantitative limiting solution, placing the quantitative limiting solution in a 10ml volumetric flask, diluting the quantitative limiting solution to a scale with acetonitrile, and shaking up to obtain the detection limiting solution.
1ml of the sample is taken into a sample injection bottle, the sample injection is continuously carried out for 3 needles by adopting the instrument parameters in the table 2, the retention time, the peak area and the signal to noise ratio are obtained, the relative standard deviation (RSD value) of the retention time and the peak area is calculated, and the test result is shown in the table 6.
TABLE 5N, N-diisopropylcarbodiimide 6-pin quantitative limit data
TABLE 6 detection limit data for N, N-diisopropylcarbodiimide 3-pin
Example 2
In this example, the retention times of the coupling agent N, N-diisopropylcarbodiimide were compared by replacing only different chromatography columns with the same instrument set-up parameters as in table 2 above. The specific parameter settings are shown in table 7.
TABLE 7 coupling agent N, N-diisopropyl carbodiimide Detector parameter settings
The coupling agent N, N-diisopropyl carbodiimide with the concentration of 0.1 mug/ml is used for sample injection detection, and the detection result is shown in figure 6.
As can be seen from the figure, under the same instrument parameter setting conditions, different chromatographic columns are used, the retention time of the N, N-diisopropyl carbodiimide is different, and the peak forms are all symmetrical peak forms. And (3) aiming at different samples, for example, when interference peaks appear, different chromatographic columns can be replaced for analysis and detection. The method has wide applicability.
Example 3
The embodiment provides a method for detecting the residual content of a coupling agent N, N-diisopropyl carbodiimide (the residual limit requirement is 1.0ppm) in polypeptide drug bivalirudin.
The instrument parameters are as follows in table 8:
TABLE 8 coupling agent N, N-diisopropyl carbodiimide Detector parameter settings
Preparing a linear solution: 2.0ml, 1.5ml, 1.0ml, 0.5ml and 0.1ml of coupling agent N, N-diisopropyl carbodiimide stock solution with the concentration of 0.1 mu g/ml are precisely measured in sequence and respectively placed in 10ml volumetric flasks, diluted to the scale by methanol and shaken up to obtain the solution for the linear standard curve. And precisely measuring 1ml of solution for each linear standard curve, placing the solution in a sample injection vial, directly injecting and analyzing, recording a chromatogram and a peak area, and drawing by taking the concentration as an abscissa and the peak area as an ordinate to obtain the linear standard curve, wherein the concentration range is 1-20 ng/ml, as shown in figure 7.
Preparation of spiked sample solutions (three horizontal spiked sample solutions of 50%, 100% and 150% were prepared, respectively):
accurately weighing 100mg of a bivalirudin sample (Kunming Longjin pharmaceutical industry Co., Ltd.), putting the bivalirudin sample into a 10ml measuring flask, accurately weighing 0.1 mu g/ml of a coupling agent N, N-diisopropyl carbodiimide solution 0.5ml, putting the coupling agent N, N-diisopropyl carbodiimide solution into the same measuring flask, adding methanol to dilute the solution to a scale, shaking the solution uniformly to obtain 50% labeled sample solution, and preparing 3 parts in parallel; accurately weighing 100mg of a bivalirudin sample, placing the bivalirudin sample in a 10ml measuring flask, accurately weighing 1.0ml of 0.1 mu g/ml coupling agent N, N-diisopropyl carbodiimide solution in the same measuring flask, adding methanol to dilute the solution to a scale, shaking the solution uniformly to obtain 100% labeled sample solution, and preparing 3 parts in parallel; precisely weighing 100mg of a bivalirudin sample, placing the bivalirudin sample in a 10ml measuring flask, precisely weighing 1.5ml of a coupling agent N, N-diisopropylcarbodiimide solution with the concentration of 0.1 mu g/ml, placing the coupling agent N, N-diisopropylcarbodiimide solution in the same measuring flask, adding methanol to dilute the coupling agent N, N-diisopropylcarbodiimide solution to a scale, shaking the solution uniformly to obtain a 150% labeled sample solution, and preparing 3 parts in parallel.
And carrying out sample injection detection by using the prepared standard sample solution, and inspecting the recovery rate. The results are shown in Table 9.
TABLE 9 recovery data for 9-pin sample of coupling agent N, N-diisopropylcarbodiimide by labeling
Comparative example 1
The comparative example provides a method for detecting a coupling agent N, N-diisopropyl carbodiimide in a polyethylene glycol monomethyl ether polymer, which is the same as (2) in example 1, and is different from the following method in that: the temperature rising program (temperature rising program 1 in fig. 8) was set to an initial temperature of 90 ℃ for 2min, the temperature was raised to 250 ℃ at a rate of 30 ℃/min for 5min, and the result of the measurement was that the peak tail was widened in the retention period and quantitative measurement could not be carried out, which was inferior to the method of example 1 (temperature rising program 2 in fig. 8).
Comparative example 2
The comparative example provides a method for detecting a coupling agent N, N-diisopropyl carbodiimide in a polyethylene glycol monomethyl ether polymer, which is the same as (2) in example 1, and is different from the following method in that: the split ratio was 2:1, and the peak pattern was inferior compared to the method of example 1 (split ratio 20: 1). See fig. 9.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. A method for detecting N, N-diisopropyl carbodiimide is characterized in that the detection of the N, N-diisopropyl carbodiimide in a substance to be detected is carried out by adopting a gas chromatography-mass spectrometry combined mode; the temperature rise procedure of the gas phase part is as follows: the initial temperature is 50-70 ℃, the temperature is kept for 3-7min, the temperature is raised to 300 ℃ at the speed of 20-40 ℃/min, and the temperature is kept for 2-5 min.
2. The method according to claim 1, characterized in that the temperature increase of the gas phase fraction is programmed as follows: the initial temperature is 60 deg.C, and the temperature is maintained for 5min, and the temperature is increased to 250 deg.C at the rate of 30 deg.C/min, and maintained for 2 min.
3. The method according to claim 1 or 2, wherein the carrier gas of the gas phase part is helium, the flow rate is in the range of 0.9-1.1 ml/min, and the sample injection split ratio is (5-20): 1.
4. the method according to claim 3, wherein a direct injection mode is adopted, and the injection amount is 1-5 μ l.
5. The method of claim 1, wherein a weakly polar chromatography column is used.
6. The method of claim 5, wherein the chromatographic column is a 100% -dimethylpolysiloxane stationary liquid chromatographic column, or a 5% -diphenyl-95% -dimethylarylenesiloxane copolymer stationary liquid chromatographic column and a chromatographic column having a composition similar to that of the stationary phase.
7. The method of claim 6, wherein the chromatography column is HP-1, HP-5MS UI or DB-624 UI.
8. The method as claimed in claim 1, wherein the injection port temperature of the gas phase portion is 160-180 ℃.
9. The method of any one of claims 1-8, wherein the ion source temperature of the mass spectrometry portion is 230 ℃; the temperature of the four-level bar is 150 ℃; the electron energy is 70 ev; the monitoring mode is SCAN; the scanning ion range is m/z 30-150.
10. Use of the method of any one of claims 1-9 for product quality control.
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| CN (1) | CN113655133B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US20160176918A1 (en) * | 2014-12-19 | 2016-06-23 | Cem Corporation | Coupling method for peptide synthesis at elevated temperatures |
| CN106153748A (en) * | 2015-03-31 | 2016-11-23 | 深圳翰宇药业股份有限公司 | A kind of method of N, N-DIC in detection polypeptide |
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2021
- 2021-06-29 CN CN202110729489.6A patent/CN113655133B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160176918A1 (en) * | 2014-12-19 | 2016-06-23 | Cem Corporation | Coupling method for peptide synthesis at elevated temperatures |
| CN106153748A (en) * | 2015-03-31 | 2016-11-23 | 深圳翰宇药业股份有限公司 | A kind of method of N, N-DIC in detection polypeptide |
Non-Patent Citations (3)
| Title |
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| ANNA KLIMOWSKA 等: "Off-line microextraction by packed sorbent combined with on solid support derivatization and GC-MS: Application for the analysis of five pyrethroid metabolites in urine samples", 《TALANTA》 * |
| 沈菊芳 等: "水中微囊藻毒素的氧化水解产物2-甲基-3-甲氧基-4-苯基丁酸的气相色谱-质谱检测研究", 《环境与职业医学》 * |
| 赵丽华 等: "N,N′-二异丙基碳二亚胺的合成", 《齐鲁药事》 * |
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