CN115508479A - Derivatization analysis method of chloroformic acid n-hexyl ester - Google Patents
Derivatization analysis method of chloroformic acid n-hexyl ester Download PDFInfo
- Publication number
- CN115508479A CN115508479A CN202210731071.3A CN202210731071A CN115508479A CN 115508479 A CN115508479 A CN 115508479A CN 202210731071 A CN202210731071 A CN 202210731071A CN 115508479 A CN115508479 A CN 115508479A
- Authority
- CN
- China
- Prior art keywords
- derivatization
- hexyl chloroformate
- analysis method
- hexyl
- chloroformate
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—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
- G01N30/62—Detectors specially adapted therefor
- G01N30/78—Detectors specially adapted therefor using more than one detector
-
- 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
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The invention provides a derivatization analysis method of n-hexyl chloroformate, which is used for detecting isomers by deriving 4-nitrophenol and n-hexyl chloroformate and detecting related substances by using sodium borohydride ultrasonic reaction to specifically, rapidly and highly selectively reduce the derived n-hexyl chloroformate. The method has good selectivity and simple and convenient operation, can accurately determine the content of isomers and related substances in the n-hexyl chloroformate, can be used for constant analysis and quantitative detection of the n-hexyl chloroformate, and meets the research, development and production requirements.
Description
Technical Field
The invention relates to the field of pharmaceutical analysis, and in particular relates to a derivatization analysis method of n-hexyl chloroformate.
Background
Acyl chloride is an important acylation reagent in drug synthesis, and is an important reagent which can participate in amide coupling and introduction of groups such as ester groups, and the like, and has strong corrosiveness and irritation. Because acid chloride compounds have warning structural groups, genotoxic and carcinogenic impurities may be formed during the course of pharmaceutical production, transport and storage, and patients may face a high risk of genotoxicity even at very low intake doses. Therefore, the regulatory authorities in various countries pay great attention to the detection of residual acyl chloride compounds in drugs, and therefore, the research and development of drugs are required to be fully carried out to establish effective quality control strategies.
Because of the high reactivity of acyl chloride compounds, they are thermally unstable, are easily decomposed when exposed to water, and can cause irreversible damage to instruments and chromatography, and they are usually derivatized and then detected. The n-hexyl chloroformate has the defects of slow reaction rate, low conversion rate, incomplete conversion, easy generation of side reaction products, poor compatibility with instruments and the like in the conventional derivatization method.
An acid chloride derivatization method using 2-nitrophenylhydrazine as the derivatization agent was published by Zheng et al, in the Journal of Pharmaceutical and Biomedical Analysis, and reacted in acetonitrile solvent at room temperature for 30 minutes. However, due to the presence of formic acid in the chloroformate, by-products are very easily produced in practical operation.
Due to the water sensitivity of n-hexyl chloroformate, the product of hydrolysis, hexanol, is an important impurity in the n-hexyl chloroformate starting material. The problems of over-reaction, different derivatization products and the like easily occur when the common hydrolysis derivatization method is applied to chloroformate, so that the conventional derivatization reaction cannot distinguish hydrolyzed chloroformate from unhydrolyzed chloroformate in raw materials, influences the effective determination of hexanol impurities and possibly introduces excessive interference in products. In the process of developing the method, a series of derivatization reactions such as methanol, ammonia water, benzyl alcohol and the like are tried, and similar defects exist. Meanwhile, in the related substance method, the reducing agent, the reaction time and the reaction condition are optimized.
The traditional acyl chloride analysis method in the current medicine research and development can not meet the quality control requirement of the n-hexyl chloroformate, so that a novel high-efficiency n-hexyl chloroformate derivatization analysis method is developed by introducing a new reaction process and simplifying a separation mode aiming at the characteristics of chlorine-containing high-activity organic matters, and is applied to the detection of acyl chloride raw materials and products in the medicine research and development and production. Has important significance for accelerating the research of new process and developing a medicine quality control system with independent intellectual property rights.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: provides a derivatization analysis method of n-hexyl chloroformate, which can accurately determine the content of isomers and related substances in the n-hexyl chloroformate. The method has good selectivity and simple and convenient operation, can be used for conventional analysis and quantitative detection of the n-hexyl chloroformate, and meets the research, development and production requirements.
The technical scheme for solving the technical problems is as follows:
the invention provides a derivatization analysis method of n-hexyl chloroformate, wherein the derivatization comprises derivatization 1 and derivatization 2, and the reaction equation of the derivatization 1 is as follows:
the derivation 2 reaction equation is:
The n-hexyl chloroformate isomer is isohexyl chloroformate, and the structure is as follows (code number DBQM 3-G):
this compound also participates in the reaction, the reaction equation is as follows:
further, the derivatization 1 comprises the following steps:
(1) Adding 4-nitrophenol into a volumetric flask, adding dichloromethane for dispersion, and confirming that no solid exists on the wall;
(2) Measuring triethylamine, directly adding the triethylamine into a volumetric flask, and slightly shaking until the solid is completely dissolved; adding dichloromethane to wash the inner wall from the capacity bottle mouth;
(3) Placing in ice water bath, cooling;
(4) Measuring n-hexyl chloroformate (code number DBQM 3), directly adding into a volumetric flask, slightly shaking, and adding dichloromethane to wash the inner wall from the mouth of the volumetric flask;
(5) Placing in ice water bath, and keeping the temperature;
(6) Adding dichloromethane to constant volume; the solution was taken and detected by liquid chromatography. The product of derivative 1 has the code number DBQM3-1.
Further, the molar ratio of n-hexyl chloroformate, 4-nitrophenol and triethylamine in said derivative 1 is 1: 1.2-1.5: 1.5 to 1.7.
Further, the derivative 1 is cooled by adopting an ice-water bath at the temperature of 0-10 ℃, and the heat preservation time of the ice-water bath is not less than 40 minutes.
Further, the derivation 2 comprises the following steps:
(1) Adding acetonitrile into a volumetric flask, adding n-hexyl chloroformate for dispersion, and slightly shaking;
(2) Measuring sodium borohydride, and adding the sodium borohydride into acetonitrile for dispersion; slowly adding the sodium borohydride suspension into the volumetric flask by using a dropper, adding dichloromethane, washing the inner wall from the mouth of the volumetric flask, and slightly shaking; controlling the reaction;
(3) Adding acetonitrile to constant volume, and filtering with a filter membrane; the filtered solution was taken and detected by gas chromatography.
Further, the molar ratio of n-hexyl chloroformate to sodium borohydride in derivative 2 is 1:1.4 to 1.6.
Further, the derivation 2 control reaction operation is to adopt an ultrasonic mode to carry out reaction, the time is 10 to 20 minutes, and the reaction temperature is controlled to be 0 to 30 ℃.
Further, the filter membrane is a 0.45 μm nylon filter membrane.
Further, the derivatization 1 chromatographic conditions include:
liquid chromatography column: octadecylsilane chemically bonded silica gel column or chromatographic column with equivalent efficiency, the column length is 100-150 mm;
column temperature: 28-32 ℃; flow rate: 0.9-1.1 ml/min; sample introduction amount: 1-5 mul;
UV detector: 252-256 nm;
temperature of the sample pan: 13 to 17 ℃;
mobile phase A: water;
and (3) mobile phase B: acetonitrile;
needle washing liquid: acetonitrile;
mobile phase a and mobile phase B were subjected to gradient elution at different volume ratios.
Further, the gradient elution procedure is as follows: 0-5min,90% A,10% B;5-25min,90% -10% A,10% -90% B;25-30min, 10%; 30-30.1min,10% -90%, A,90% -10%, B;30.1-40min,90% by weight A,10% by weight B.
Further, the gradient elution procedure is: 0-5min,92% A,8% B;5-25min,92% -8% A,8% -92% B;25-30min, 8%; 30-30.1min,8% -92% A,92% -8% B;30.1-40min,92% by weight A,8% by weight B.
Further, the gradient elution procedure is as follows: 0-5min,88% A,12% B;5-25min,88% -12%, A,12% -88%; 25-30min,12% A,88% B;30-30.1min,12% -88% A,88% -12% B;30.1-40min,88% A,12% B.
Further, the derivatization 2 chromatography conditions include: gas chromatographic column: a polar capillary chromatography column or equivalent performance column of 6% cyanopropylbenzene-94% dimethylsiloxane;
carrier gas: nitrogen gas; carrier gas flow rate: 1.9-2.1ml/min; the temperature of the FID detector is 245-255 ℃; hydrogen gas: 35-40ml/min; air: 380-400ml/min; sample injection amount: 1-1.5 mul; the split ratio is as follows: 10:1, temperature programming: the initial column temperature was 38-42 deg.C, ramped up to 220 deg.C at a rate of 10 deg.C per minute, and held for 10 minutes.
The Chinese naming of the compound is in conflict with the structural formula, and the structural formula is taken as the standard; except for a significant error in the formula.
The invention has the beneficial effects that: the invention provides a derivatization analysis method of n-hexyl chloroformate, which is determined by repeatedly optimizing and screening chromatographic conditions and a sample injection program. 4-nitrophenol and n-hexyl chloroformate are used for deriving and detecting isomers, and sodium borohydride is used for carrying out ultrasonic reaction to specifically, rapidly and selectively reduce the derived n-hexyl chloroformate to detect related substances. Aiming at the detection of the n-hexyl chloroformate isomer, the invention utilizes the n-hexyl chloroformate to react with 4-nitrophenol after adding triethylamine as an acid-binding agent into dichloromethane, and the obtained derivative has the characteristic of ultraviolet absorption, and then uses octadecylsilane bonded silica gel column to carry out chromatographic separation, and uses a high performance liquid chromatograph to carry out detection and analysis. Aiming at the decomposition product of the n-hexyl chloroformate and other impurities without ultraviolet absorption, the invention utilizes the reaction of the n-hexyl chloroformate and sodium borohydride to generate the hexyl formate derivative in acetonitrile, then uses a medium polarity capillary chromatographic column of 6 percent cyanopropylbenzene-94 percent dimethyl siloxane for separation, and uses gas chromatography for analysis and detection.
According to the n-hexyl chloroformate derivatization analysis method, the reaction condition is mild, the byproducts are few, the operation is simple and convenient, and the quality of the n-hexyl chloroformate raw material can be comprehensively evaluated and controlled. In the isomer detection method, by controlling the reaction temperature and adding an acid-binding agent, the generation of byproducts is reduced, and the yield of target products is improved; in related substance detection, sodium borohydride is used as a reducing agent and is combined with ultrasonic promotion, so that the reaction rate and the conversion rate are improved, and excessive reaction and generation of byproducts are avoided. The method has the advantages of good selectivity, simplicity, convenience, practicability and high accuracy. The hydrolyzed hexanol impurity and other non-ultraviolet absorbing impurities in the n-hexyl chloroformate can be determined. The two derivatization methods are combined, so that the n-hexyl chloroformate can be better monitored in quality, the quality control in production, the protection of instruments and chromatographic columns and the reduction of cost are of great significance, and the requirements of research and development and production can be met.
The method has the advantages of good reaction selectivity, short derivatization time, mild reaction conditions, simple operation, good specificity and accuracy, high sensitivity and the like. The method is suitable for conventional detection and quantitative analysis of n-hexyl chloroformate in industrial production.
Drawings
FIG. 1 is a liquid phase method of derivatization of n-hexyl chloroformate isomers-solution stability;
FIG. 2 is a liquid phase process for derivatization of n-hexyl chloroformate isomers-durability;
FIG. 3 shows the liquid phase method of derivatization of n-hexyl chloroformate isomers-detection limit, quantitation limit, and precision of sample injection;
FIG. 4 is a liquid phase method-linearity (detection limit to 120% sample concentration) for n-hexyl chloroformate isomer derivatization;
FIG. 5 is a liquid phase method-specific for derivatization of n-hexyl chloroformate isomers;
FIG. 6 is a chromatogram of a sample obtained by a liquid phase method for derivatization of n-hexyl chloroformate isomers;
FIG. 7 is a gas phase method-specific for derivatization of n-hexyl chloroformate-related species;
FIG. 8 is a graph of the 64 hour stability of a derivatization solution of n-hexyl chloroformate related substances in a gas phase derivatization process;
FIG. 9 is a gas phase process for derivatization of n-hexyl chloroformate related species-durability;
FIG. 10 shows the gas phase method of derivatization of n-hexyl chloroformate-detection limit, quantitation limit, and precision of sample injection;
FIG. 11 is a gas phase method-repeatability of derivatization of n-hexyl chloroformate related species;
FIG. 12 gas phase method-linearity for n-hexyl chloroformate related species;
FIG. 13 is a GC/MS chromatogram for identifying the mass spectrum of a derivative product of n-hexyl chloroformate.
Detailed Description
The invention is illustrated but not limited by the following examples. The technical solutions protected by the present invention are all the simple replacements or modifications made by the skilled person in the art.
Example 1:
1. detecting n-hexyl chloroformate isomers:
1.1. equation of reaction
1.2. Reaction charge ratio
| Name of material | Molecular weight | Dosage of | Molar ratio of | Use of |
| Chloroformic acid n-hexyl ester | 164.63 | 50mg/ |
1 | Reactants |
| 4-nitrophenol | 139.11 | 64mg | 1.5 | Reactants |
| Triethylamine | 101.19 | 72.3mg/75ul | 1.7 | Acid-binding agent |
| Methylene dichloride | \ | 5ml | \ | Solvent(s) |
Reaction parameter ranges:
the heat preservation time of the ice-water bath is not less than 40 minutes.
The instrument parameter ranges are shown in the durability section of figure 2.
1.3. Reaction process
(1) To a 20ml volumetric flask was added 4-nitrophenol and 5ml dichloromethane was added to disperse, it being necessary to confirm no apparent solids on the walls. (2) 75ul of triethylamine were measured out and added directly to the flask with gentle shaking until the solid was completely dissolved. And 2ml of DCM were added to wash the inner wall from the vessel neck.
(3) The mixture was cooled for 2min in an ice-water bath at 0 to 10 ℃.
(4) 50ul of DBQM3 was weighed out, added directly to the flask, shaken slightly and 2ml of dichloromethane were added to wash the inner wall from the mouth of the flask.
(5) The temperature is kept for 1h at the temperature of 0 to 10 ℃ in an ice water bath.
(6) Adding dichloromethane to constant volume. The solution was checked by liquid chromatography.
1.4. Parameters of the instrument
1.4.1. Instrument
Liquid chromatography SHIMADZU LC-20AD, UV detector.
Chromatographic conditions are as follows:
1.4.2. chromatographic column
Octadecylsilane bonded silica gel as a filler (Agilent ZORBAX Eclipse Plus C18,4.6 mm. Times.150mm, 3.5 μm, or equivalent performance column)
Wavelength: 254nm; column temperature: 30 ℃; flow rate: 1.0ml/min; sample introduction volume: 5 mul; temperature control of a sample plate: 15 ℃ mobile phase A: water mobile phase B: acetonitrile (ACN)
Needle washing liquid: acetonitrile (ACN)
The linear gradient elution was performed as follows:
1.5 preparation of solution
Diluting liquid: acetonitrile
Blank solution: precisely weighing about 64mg of 4-nitrophenol, placing the 4-nitrophenol into a 20ml measuring flask, adding 5ml of dichloromethane for dispersion, adding 0.075ml of triethylamine, slightly shaking until the solid is completely dissolved, (note: if part of triethylamine is stuck on the wall of the measuring flask, 2ml of dichloromethane is added from the mouth of the measuring flask for washing the inner wall by a dropper), placing the measuring flask into an ice water bath at 0-10 ℃ for 1h, shaking once every 15min, diluting the measuring flask to a scale by using a diluent, and shaking uniformly.
Impurity stock solution: precisely weighing about 64mg of 4-nitrophenol, placing the 4-nitrophenol into a 20ml measuring flask, adding 5ml of dichloromethane for dispersion, adding 0.075ml of triethylamine, slightly shaking until the solid is completely dissolved, (note: if part of triethylamine is stuck on the wall of the flask, a dropper can be used for adding 2ml of dichloromethane from the opening of the measuring flask to wash the inner wall), placing the flask in an ice-water bath at 0-10 ℃ for 2min, precisely weighing about 50mg of DBQM3-G, placing the DBQM3-G into the 20ml measuring flask, and then adding 2ml of dichloromethane to wash the inner wall from the opening of the measuring flask. (Note: this operation is to wash the sample on the bottle wall, dissolve it in the reaction system), put it in ice-water bath at 0-10 deg.C for 1h, shake it once every 15min, dilute it to the scale with diluent, shake it up. Precisely measuring 2.0ml of the solution, placing the solution into a 100ml measuring flask, diluting the solution to a scale with the diluent, and shaking up.
Test solution: precisely weighing about 64mg of 4-nitrophenol, placing the 4-nitrophenol into a 20ml measuring flask, adding 5ml of dichloromethane for dispersion, adding 0.075ml of triethylamine, slightly shaking until the solid is completely dissolved, (note: if part of triethylamine is stuck on the wall of the measuring flask, 2ml of dichloromethane can be added from the opening of the measuring flask to wash the inner wall) and placing the measuring flask in a water bath at 0-10 ℃ for 2min, precisely weighing about 50mg of n-hexyl chloroformate, placing the measuring flask in the 20ml measuring flask, and then adding 2ml of dichloromethane to wash the inner wall from the opening of the measuring flask. (remark: this operation is to wash the sample on the bottle wall, dissolve it in the reaction system), put it in ice-water bath at 0-10 deg.C for 1h, shake it every 15min, dilute it to the scale with diluent, shake it up. Precisely measuring 4ml of the solution, placing the solution into a 20ml measuring flask, diluting the solution to the scale mark with the diluent, and shaking up.
Adding a standard test solution: precisely weighing about 64mg of 4-nitrophenol, placing the 4-nitrophenol into a 20ml measuring flask, adding 5ml of dichloromethane for dispersion, adding 0.075ml of triethylamine, slightly shaking until the solid is completely dissolved, (note: if part of triethylamine is stuck on the wall of the flask, 2ml of dichloromethane can be added from the mouth of the measuring flask for washing the inner wall by a dropper), placing the flask in a water bath at 0-10 ℃ for 2min, precisely weighing about 50mg of n-hexyl chloroformate, placing the flask in the 20ml measuring flask, and then adding 2ml of dichloromethane for washing the inner wall from the mouth of the measuring flask. (remark: this operation is to wash the sample on the bottle wall, dissolve it in the reaction system), put it in ice-water bath at 0-10 deg.C for 1h, shake it every 15min, dilute it to the scale with diluent, shake it up. Precisely measuring 4ml of the solution, placing into a 20ml measuring flask, precisely measuring 0.3ml of impurity stock solution, diluting with diluent to scale, and shaking.
System applicability solution: precisely weighing about 64mg of 4-nitrophenol, placing the 4-nitrophenol into a 20ml measuring flask, adding 5ml of dichloromethane for dispersion, adding 0.075ml of triethylamine, slightly shaking until the solid is completely dissolved, (note: if part of the triethylamine is stuck on the wall of the measuring flask, 2ml of dichloromethane can be added from the opening of the measuring flask for washing the inner wall by a dropper), placing the measuring flask in an ice water bath at 0-10 ℃ for 1min, precisely weighing about 50mg of n-hexyl chloroformate standard substance, placing the measuring flask in the 20ml measuring flask, and then adding 2ml of dichloromethane for washing the inner wall from the opening of the measuring flask. (remark: this operation is to wash the sample on the bottle wall, dissolve it in the reaction system), put it in ice-water bath at 0-10 deg.C for 1h, shake it every 15min, dilute it to the scale with diluent, shake it up. Precisely measuring 4ml of the solution, placing into a 20ml measuring flask, precisely measuring 0.3ml of impurity stock solution, diluting with diluent to scale, and shaking.
DBQM3-G localization solution: the same impurity stock solution.
N-hexyl chloroformate positioning solution: precisely weighing about 64mg of 4-nitrophenol, placing the 4-nitrophenol into a 20ml measuring flask, adding 5ml of dichloromethane for dispersion, adding 0.075ml of triethylamine, slightly shaking until the solid is completely dissolved, (note: if part of the triethylamine is stuck on the wall of the measuring flask, 2ml of dichloromethane can be added from the opening of the measuring flask to wash the inner wall) and placing the measuring flask in a water bath at 0-10 ℃ for 1min, precisely weighing about 50mg of n-hexyl chloroformate reference substance, placing the measuring flask in the 20ml measuring flask, and then adding 2ml of dichloromethane to wash the inner wall from the opening of the measuring flask. (remark: this operation is to wash the sample on the bottle wall, dissolve it in the reaction system), put it in ice-water bath at 0-10 deg.C for 1h, shake it every 15min, dilute it to the scale with diluent, shake it up. Precisely measuring 2ml of the solution, placing the solution in a 100ml measuring flask, diluting the solution to the scale with the diluent, and shaking up.
Quantitative limiting solution: taking n-hexyl chloroformate and the impurity positioning solution to dilute step by step, and detecting that the signal-to-noise ratio is about 10, namely the quantitative limit concentration.
Detection limiting solution: and (3) diluting the 'quantitative limit solution', and detecting that the signal to noise ratio is about 3, namely the detection limit concentration.
Linear solution: the n-hexyl chloroformate and each impurity were selected at 6 points (LOQ, 0.05%, 0.12%, 0.15%, 0.18%, 0.30%, respectively) in the range of LOQ-0.30%.
Sample introduction precision solution: and (6) continuously injecting a quantitative limiting solution into a sample, and recording a chromatogram. Acceptance criteria: the RSD of the peak area should be no more than 10.0%.
Repetitive solution: (1) background test sample solution: the sample solution is prepared in parallel by 6 portions and injected. (2) adding a standard test sample solution: 6 portions of sample injection are prepared in parallel.
The standard specifies:
| impurities in the product | Limit of |
| DBQM3-G | ≤0.15％ |
Example 2:
2. detecting related substances in the chloroformic acid n-hexyl ester:
2.1. equation of reaction
2.2. Reaction charge ratio
| Name of material | Molecular weight | Dosage of | Molar ratio of | Use of |
| Chloroformic acid n-hexyl ester | 164.63 | 200mg/ |
1 | Reactants |
| Sodium borohydride | 37.83 | 65mg | 1.5 | Reactants |
| Acetonitrile | \ | 50ml | \ | Solvent(s) |
The ultrasonic reaction time is 10 to 20 minutes.
The reaction temperature of 0 to 30 ℃ has no obvious influence on the method.
The instrument parameter ranges are shown in the durability section of fig. 9.
2.3. Reaction process
(1) To a 50ml volumetric flask was added 25ml acetonitrile and 200ul n-hexyl chloroformate dispersed and shaken gently.
(2) 65mg NaBH was metered in 4 Then, the mixture was dispersed in 5ml of acetonitrile. NaBH is sprayed by a dropper 4 The suspension was slowly added to the volumetric flask and 2ml of dichloromethane were added to wash the inner wall from the mouth of the volumetric flask, shaking slightly. Sonicate for 15 minutes at room temperature.
(3) Acetonitrile was added to the resulting solution to constant volume, and the mixture was filtered through a 0.45 μm nylon filter. The filtered solution was taken for detection using gas chromatography. The product was hexyl formate as confirmed by mass spectrometry, while there was no hexyl chloroformate residue or hexanol which was a over-reduced product. The mass spectral identification is shown in FIG. 13.
2.4. Conditions of analysis
2.4.1. Instrument for measuring the position of a moving object
Gas chromatography Agilent 7890A, FID detector
2.4.2. Chromatographic column
Gas chromatographic column: a medium polarity capillary column (Agilent DB-624, 30m × 0.320mm,1.8, or equivalent performance column) of 6% cyanopropylbenzene-94% dimethylsiloxane.
Carrier gas: nitrogen gas;
flow rate of carrier gas: 2ml/min;
FID detector temperature: 250 ℃;
sample inlet temperature: 190-210 ℃;
hydrogen gas: 40ml/min;
air: 400ml/min;
sample introduction amount: 1.5 mul;
the split ratio is as follows: 10:1.
temperature programming: the initial column temperature was 40 ℃ and increased to 220 ℃ at a rate of 10 ℃ per minute and held for 10 minutes.
2.5 solution preparation
Blank solution: 25ml acetonitrile was added to a 50ml volumetric flask. 5ml of acetonitrile is added into a 10ml centrifuge tube, and 65mg of NaBH is precisely weighed 4 Adding into a centrifuge tube for dispersion. NaBH is sprayed by a dropper 4 The suspension is slowly added to the volumetric flask and 2ml of acetonitrile are added to wash the inner wall from the volumetric flask mouth, gently shaking. Sonicate for 15 minutes at room temperature. Adding acetonitrile to constant volume, filtering with 0.45 μm nylon filter membrane, and collecting filtrate.
Test solution: 25ml of acetonitrile was added to a 50ml volumetric flask, and about 200mg of n-hexyl chloroformate was precisely weighed and subjected to dispersion by gentle shaking. 5ml of acetonitrile is added into a 10ml centrifuge tube, and 65mg of NaBH is precisely weighed 4 Adding into a centrifuge tube for dispersion. NaBH is added by using a dropper 4 The suspension is slowly added to the volumetric flask and 2ml of acetonitrile are added to wash the inner wall from the volumetric flask mouth, gently shaking. Sonicate for 15 minutes at room temperature. Adding acetonitrile to constant volume, filtering with 0.45 μm nylon filter membrane, and collectingAnd (4) filtering the solution.
N-hexyl chloroformate positioning solution: a50 ml volumetric flask was charged with 25ml of acetonitrile and a control of approximately 200mg of n-hexyl chloroformate was weighed out precisely and dispersed by gentle shaking. 5ml of acetonitrile is added into a 10ml centrifuge tube, and 65mg of NaBH is precisely weighed 4 Adding into a centrifuge tube for dispersing. NaBH is added by using a dropper 4 The suspension is slowly added to the volumetric flask and 2ml of acetonitrile are added to wash the inner wall from the volumetric flask mouth, gently shaking. Sonicate for 15 minutes at room temperature. Adding acetonitrile to constant volume, filtering with 0.45 μm nylon filter membrane, and collecting filtrate.
Quantitative limiting solution: and (3) taking the n-hexyl chloroformate positioning solution to dilute step by step, and detecting that the signal-to-noise ratio is about 10, namely the quantitative limit concentration.
Detection limiting solution: and (3) diluting the 'quantitative limiting solution', and detecting that the signal-to-noise ratio is about 3, namely the detection limit concentration.
The standard specifies:
| impurities in the product | Limit of |
| Total miscellaneous | ≤1.0％ |
Example 3:
3. hydrolytic derivatization of acyl chlorides
3.1. Equation of reaction
3.2. Range of reaction parameters
Reaction temperature (0 ℃,25 ℃,80 ℃), amount of THF/water used (1, 2
3.3. Conditions of analysis
3.3.1. Instrument for measuring the position of a moving object
Gas chromatography Agilent 7890A, FID detector
3.3.2. Chromatographic column
Gas chromatographic column: a medium polarity capillary column (Agilent DB-624, 30m × 0.320mm,1.8, or equivalent performance column) of 6% cyanopropylbenzene-94% dimethylsiloxane.
Carrier gas: nitrogen gas;
carrier gas flow rate: 1.9-2.1ml/min;
FID detector temperature: 245 to 255 ℃;
sample inlet temperature: 190-210 ℃;
hydrogen gas: 40ml/min;
air: 400ml/min;
sample introduction amount: 1.5 mul;
the split ratio is as follows: 10:1.
temperature programming: the initial column temperature was 38-42 deg.C, ramped up to 220 deg.C at a rate of 10 deg.C per minute, and held for 10 minutes.
3.4. Solution preparation
Test solution: to a 50ml volumetric flask was added 25ml of solvent (amount of THF/ water 1,2. The reaction was carried out at a selected temperature (0 ℃,25 ℃,80 ℃) for a suitable time (30min, 1h, 6h). Adding DMSO to fix the volume.
Blank solution: to a 50ml volumetric flask was added 25ml of solvent (amount of THF/ water 1, 9. The reaction is carried out for a suitable time under the selected temperature conditions, and 1.2 times the molar equivalent of the reactants of the base are added. The reaction was carried out at a selected temperature (0 ℃,25 ℃,80 ℃) for a suitable time (30min, 1h, 6h). Adding DMSO to constant volume.
3.5. As a result, the
In the experimental process, DBQM3 remains under different conditions such as reaction temperature, solvent/water dosage, alkali type and the like.
The use of THF was attempted to increase the reaction rate and reduce side reactions. The reaction time is prolonged to 6h, the reflux reaction is carried out, 5V/5V water
THF as a reaction solvent and DMSO as a post-treatment solvent, DBQM3 was completely converted, but an unknown peak was present near the hexanol peak. Example 4:
4. amide derivatization using aqueous ammonia
Acetonitrile was used as solvent and ammonia as derivatizing agent.
4.1. Instrument
Agilent 7890A and FID detector for gas chromatography
4.1.1. Chromatographic column
Gas chromatographic column: a medium polarity capillary column (Agilent DB-624, 30m × 0.320mm,1.8, or equivalent performance column) of 6% cyanopropylbenzene-94% dimethylsiloxane.
Carrier gas: nitrogen gas;
flow rate of carrier gas: 1.9-2.1ml/min;
FID detector temperature: 245 to 255 ℃;
sample inlet temperature: 190-210 ℃;
hydrogen gas: 40ml/min;
air: 400ml/min;
sample introduction amount: 1.5 mul;
the split ratio is as follows: 10:1.
temperature programming: the initial column temperature was 38-42 deg.C, ramped up to 220 deg.C at a rate of 10 deg.C per minute, and held for 10 minutes.
4.2. Solution preparation
Test solution: 10ml of acetonitrile is added into a 50ml volumetric flask, about 200mg of n-hexyl chloroformate is precisely weighed and tested to be dispersed by slight shaking, and 1ml of ammonia water is added. The reaction was carried out at the selected temperature (0 ℃,25 ℃,80 ℃) for the appropriate time (30min, 1h,6 h). Adding acetonitrile to constant volume.
Blank solution: a50 ml volumetric flask was charged with 10ml of acetonitrile and 1ml of ammonia water. The reaction was carried out at a selected temperature (0 ℃,25 ℃,80 ℃) for a suitable time (30min, 1h, 6h). Adding acetonitrile to constant volume.
4.3. Results
The acetonitrile is used as a solvent, ammonia water is used as a derivatization reagent, the obtained reaction liquid is directly sent to a gas phase for detection, and the result shows that an RT 11.5min unknown peak is generated, is large and is not identified.
Example 5:
5. ester derivatization using different alcohols
Methanol, t-butanol, hexanol, benzyl alcohol were tested using gas chromatography except benzyl alcohol.
5.1. Equation of reaction
5.1.1. Methanol
5.1.2. Tert-butyl alcohol
5.1.3. Hexanol
5.1.4. Benzyl alcohol
5.2. Instrument for measuring the position of a moving object
Agilent 7890A and FID detector for gas chromatography
5.2.1. Chromatographic column
Gas chromatographic column: a medium polarity capillary column (Agilent DB-624, 30m × 0.320mm,1.8, or equivalent performance column) of 6% cyanopropylbenzene-94% dimethylsiloxane.
Carrier gas: nitrogen gas;
flow rate of carrier gas: 1.9-2.1ml/min;
FID detector temperature: 245 to 255 ℃;
sample inlet temperature: 190-210 ℃;
hydrogen gas: 40ml/min;
air: 400ml/min;
sample introduction amount: 1.5 mul;
the split ratio is as follows: 10:1.
temperature programming: the initial column temperature was 38-42 deg.C, ramped up to 220 deg.C at a rate of 10 deg.C per minute, and held for 10 minutes.
5.3. Solution preparation
Test solution: see 5.4. Description of results
5.4. Results
Methanol: consider esterification with methanol followed by gas phase detection. Methanol is used as a solvent, potassium carbonate is used as an acid-binding agent, reaction is carried out for 1 hour, and then reaction supernatant is taken for inspection. The gas phase results show that there is no n-hexyl chloroformate, but a small hexanol peak is present. A possible reason is degradation of the methanol derivative product at the gas phase injection port. Therefore, this method is not applicable.
Tert-butyl alcohol: esterification with tert-butanol followed by gas phase detection is contemplated. Through research and study, the product derived from tert-butyl alcohol and n-hexyl chloroformate is more stable than the product derived from methanol, and can be heated for reaction. In the reaction, the reaction is carried out at room temperature, and a large amount of solid is generated after triethylamine is added. The reaction was heated to 70 ℃ for 1 hour. After post-treatment, the gas phase was checked, and the result showed that n-hexyl chloroformate remained, which was not suitable for this method.
Hexanol: the boiling point of dihexyl carbonate is high at 144 ℃ (12 torr). It is therefore inferred that dihexyl carbonate may be stable at high temperatures. Therefore, an attempt to prepare dihexyl carbonate was made. However, after gas phase detection, an unknown peak was found in addition to the main peak of dihexyl carbonate, and this method was not suitable.
Benzyl alcohol: due to the gas phase conditions, partial degradation of the n-hexyl chloroformate to hexanol cannot be avoided. Therefore, introduction of a group having ultraviolet absorption is considered. Benzyl alcohol reacts with n-hexyl chloroformate, which can be detected by a liquid chromatography method. After the reaction, the liquid phase was checked (liquid phase conditions were the same as 1.4. Apparatus parameters in example 1), and the main peak was 35.6min, but an unknown peak at RT 19.6min appeared, which was not applicable.
Therefore, when methanol, ammonia water, benzyl alcohol and the like are used for derivatization, on one hand, the reaction is incomplete, the reaction activity of the n-hexyl chloroformate is not strong, and on the other hand, unknown impurities are generated, so that the derivatization methods are not suitable.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various changes and modifications can be made without departing from the inventive concept of the present invention, and these changes and modifications are all within the scope of the present invention.
Claims (13)
1. A derivatization analysis method of n-hexyl chloroformate is characterized in that the derivatization comprises derivatization 1 and derivatization 2, and the reaction equation of the derivatization 1 is as follows:
the derivation 2 reaction equation is:
2. The n-hexyl chloroformate derivatization analysis method according to claim 1, wherein the derivatization 1 comprises the following steps:
(1) Adding 4-nitrophenol into a volumetric flask, and adding dichloromethane for dispersion;
(2) Measuring triethylamine until the solid is completely dissolved;
(3) Placing in ice water bath, cooling;
(4) Weighing n-hexyl chloroformate, and uniformly mixing;
(5) Placing in ice water bath, and keeping the temperature;
(6) Adding dichloromethane to constant volume; the solution was taken and detected by liquid chromatography.
3. The n-hexyl chloroformate derivatization analysis method according to claim 2, wherein the molar ratio of the n-hexyl chloroformate, 4-nitrophenol, and triethylamine in the derivative 1 is 1: 1.2-1.5: 1.5 to 1.7.
4. The n-hexyl chloroformate derivatization analysis method according to claim 3, wherein the derivatization 1 is cooled in an ice-water bath at 0-10 ℃, and the temperature of the ice-water bath is kept for not less than 40 minutes.
5. The n-hexyl chloroformate derivatization analysis method according to claim 1, wherein the derivatization 2 comprises the following steps:
(1) Adding acetonitrile into a volumetric flask, and adding n-hexyl chloroformate for dispersion;
(2) Measuring sodium borohydride, and adding the sodium borohydride into acetonitrile for dispersion; slowly adding the sodium borohydride suspension into a volumetric flask, and controlling the reaction;
(3) Adding acetonitrile to a constant volume, and filtering by using a filter membrane; the filtered solution was taken and checked by gas chromatography.
6. The n-hexyl chloroformate derivatization analysis method according to claim 5, wherein the molar ratio of n-hexyl chloroformate to sodium borohydride in the derivative 2 is 1:1.4 to 1.6.
7. The method for derivatization analysis of n-hexyl chloroformate according to claim 6, wherein the derivatization 2 is carried out by ultrasonic reaction at 0-30 ℃ for 10-20 minutes.
8. The n-hexyl chloroformate derivatization analysis method according to claim 7, wherein the filter is a 0.45 μm nylon filter.
9. The n-hexyl chloroformate derivatization analysis method according to any one of claims 1-8, wherein the derivatization 1 chromatographic conditions include:
liquid chromatography column: octadecylsilane chemically bonded silica gel column or chromatographic column with equivalent efficiency, the column length is 100-150 mm;
column temperature: 28-32 ℃; flow rate: 0.9-1.1 ml/min; sample introduction amount: 1-5 mul;
UV detector: 252-256 nm;
temperature of sample pan: 13 to 17 ℃;
a mobile phase A: water;
mobile phase B: acetonitrile;
needle washing liquid: acetonitrile;
mobile phase a and mobile phase B were subjected to gradient elution at different volume ratios.
10. The n-hexyl chloroformate derivatization analysis method according to claim 9, wherein the gradient elution procedure is: 0-5min,90% A,10% B;5-25min,90% -10% A,10% -90% B;25-30min,10% A,90% B;30-30.1min,10% -90%, A,90% -10%, B;30.1-40min,90% by weight A,10% by weight B.
11. The n-hexyl chloroformate derivatization analysis method according to claim 9, wherein the gradient elution procedure is: 0-5min,92% A,8% B;5-25min,92% -8% A,8% -92% B;25-30min, 8%; 30-30.1min,8% -92% A,92% -8% B;30.1-40min,92% A,8% B.
12. The n-hexyl chloroformate derivatization analysis method according to claim 9, wherein the gradient elution procedure is: 0-5min,88% A,12% B;5-25min,88% -12% A,12% -88% B;25-30min, 12%; 30-30.1min,12% -88% A,88% -12% B;30.1-40min,88% A,12% B.
13. The n-hexyl chloroformate derivatization analysis method according to any one of claims 1-8, wherein the derivatization 2 chromatographic conditions include: gas chromatographic column: a polar capillary chromatography column or equivalent performance column of 6% cyanopropylbenzene-94% dimethylsiloxane;
carrier gas: nitrogen gas; flow rate of carrier gas: 1.9-2.1ml/min; the temperature of the FID detector is 245-255 ℃; hydrogen gas: 35-40ml/min; air: 380-400ml/min; sample introduction amount: 1-1.5 mul; the split ratio is as follows: 10:1, temperature programming: the initial column temperature was 38-42 deg.C, ramped up to 220 deg.C at a rate of 10 deg.C per minute, and held for 10 minutes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210731071.3A CN115508479B (en) | 2022-06-24 | 2022-06-24 | Derivatization analysis method of n-hexyl chloroformate |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210731071.3A CN115508479B (en) | 2022-06-24 | 2022-06-24 | Derivatization analysis method of n-hexyl chloroformate |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115508479A true CN115508479A (en) | 2022-12-23 |
| CN115508479B CN115508479B (en) | 2023-07-07 |
Family
ID=84501716
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210731071.3A Active CN115508479B (en) | 2022-06-24 | 2022-06-24 | Derivatization analysis method of n-hexyl chloroformate |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115508479B (en) |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130197260A1 (en) * | 2010-08-25 | 2013-08-01 | Cadila Healthcare Limited | Processes for the preparation of fesoterodine |
| CN103293243A (en) * | 2013-06-03 | 2013-09-11 | 福建省产品质量检验研究院 | Detection method and application of carboxy methyl lysine ingredient in food |
| WO2014041559A2 (en) * | 2012-08-27 | 2014-03-20 | Glenmark Pharmaceuticals Limited; Glenmark Generics Limited | Process for the preparation of dabigatran etexilate and intermediates thereof |
| US20140148601A1 (en) * | 2010-12-06 | 2014-05-29 | Msn Laboratories Limited | Process for the preparation of benzimidazole derivatives and its salts |
| CN107311863A (en) * | 2017-07-25 | 2017-11-03 | 利尔化学股份有限公司 | A kind of preparation method and applications of chloroformate derivative |
| CN112707914A (en) * | 2020-12-17 | 2021-04-27 | 中国人民解放军军事科学院防化研究院 | Rhodamine compound based on phenol structure and synthetic method thereof |
| CN113861027A (en) * | 2021-09-17 | 2021-12-31 | 爱斯特（成都）生物制药股份有限公司 | Method for continuous flow synthesis of chloroformate compound |
-
2022
- 2022-06-24 CN CN202210731071.3A patent/CN115508479B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130197260A1 (en) * | 2010-08-25 | 2013-08-01 | Cadila Healthcare Limited | Processes for the preparation of fesoterodine |
| US20140148601A1 (en) * | 2010-12-06 | 2014-05-29 | Msn Laboratories Limited | Process for the preparation of benzimidazole derivatives and its salts |
| WO2014041559A2 (en) * | 2012-08-27 | 2014-03-20 | Glenmark Pharmaceuticals Limited; Glenmark Generics Limited | Process for the preparation of dabigatran etexilate and intermediates thereof |
| CN103293243A (en) * | 2013-06-03 | 2013-09-11 | 福建省产品质量检验研究院 | Detection method and application of carboxy methyl lysine ingredient in food |
| CN107311863A (en) * | 2017-07-25 | 2017-11-03 | 利尔化学股份有限公司 | A kind of preparation method and applications of chloroformate derivative |
| CN112707914A (en) * | 2020-12-17 | 2021-04-27 | 中国人民解放军军事科学院防化研究院 | Rhodamine compound based on phenol structure and synthetic method thereof |
| CN113861027A (en) * | 2021-09-17 | 2021-12-31 | 爱斯特（成都）生物制药股份有限公司 | Method for continuous flow synthesis of chloroformate compound |
Non-Patent Citations (2)
| Title |
|---|
| TOMÁŠ HLOŽEK ET AL.: "Gabapentin, Pregabalin and Vigabatrin Quantification in Human Serum by GC–MS After Hexyl Chloroformate Derivatization", JOURNAL OF ANALYTICAL TOXICOLOGY * |
| 向志祥;李方群;钱春霞;刘敏;: "气相色谱法测定甲磺酸达比加群酯原料药中正己醇的残留量", 中国药房, no. 12 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115508479B (en) | 2023-07-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Montgomery et al. | The modes of decomposition of 1, 3-bis (2-chloroethyl)-1-nitrosourea and related compounds | |
| CN107033101B (en) | A kind of fluorescence probe preparation method and application detecting formaldehyde | |
| MARE | Reaction of Some Aliphatic Amines with tert-Butyl Hydroperoxide. The Fate of the Amine | |
| CN107064350A (en) | Method for detecting suspected genotoxic impurity of tofacitinib citrate | |
| Sweeley et al. | Rearrangement and decarboxylation reactions of N, N-dimethylglycine oxide | |
| Lin et al. | An artifactual solution degradant of pregabalin due to adduct formation with acetonitrile catalyzed by alkaline impurities during HPLC sample preparation | |
| Sakai et al. | Synthesis and DNA binding of [3-[2'-(2-acetamidoethyl)-2, 4'-bithiazole-4-carboxamido] propyl] dimethylsulfonium chloride, a fragment of bleomycin A2 | |
| CN115508479A (en) | Derivatization analysis method of chloroformic acid n-hexyl ester | |
| CN103592377A (en) | Method for determining dimethyl sulfate and diethyl sulfate in electronic and electric products | |
| CN115047093B (en) | Method for detecting dimethyl sulfate in anhydrous caffeine | |
| CN112083084A (en) | Method for detecting bromoacetic acid impurity in cefathiamidine | |
| CN113109473B (en) | Formaldehyde derivatization test method | |
| Staveris et al. | Quantitative analysis of veralipride in plasma and urine by gas chromatography—mass spectrometry and gas chromatography with flame-ionization detection | |
| McDonald et al. | An analytical study of illicit lysergide | |
| CN110376302B (en) | Method for detecting m-fluorobenzaldehyde and m-fluorobenzene cinnamaldehyde | |
| Chattopadhyay et al. | OH radical and chlorine atom kinetics of substituted aromatic compounds: 4-chlorobenzotrifluoride (p-ClC6H4CF3) | |
| Johansson et al. | Determination of tocainide in human plasma by gas chromatography with nitrogen-selective detection after Schiff base formation | |
| CN112903886A (en) | Separation and detection method of 1-butylsulfonyl chloride related substances | |
| CN108414661A (en) | Derivative gas chromatography-mass spectrometry method of ammonia content in a kind of detection biological sample | |
| CN115144480B (en) | Method for detecting morpholine and/or tetramethyl methane diamine from roflumilast intermediate | |
| CN116903541B (en) | Acyl imidazole compound for detecting phenolic pollutants, preparation method and application | |
| Farrar et al. | Intramolecular nucleophilic assistance in the hydrolysis of sulfonate esters: equilibrium constant for sultone formation | |
| Ohya et al. | Analysis of drugs by pyrolysis. I‐selected ion monitoring combined with a pyrolysis method for the determination of carpronium chloride in biological samples | |
| CN116183772B (en) | Method for detecting mutation-causing impurities in bulk drug | |
| CN113252809B (en) | Method for detecting residues of methyl trifluoromethanesulfonate and ethyl trifluoromethanesulfonate |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |