CN113176371B - Method for determining free polyethylene glycol in fatty alcohol-polyoxyethylene ether - Google Patents
Method for determining free polyethylene glycol in fatty alcohol-polyoxyethylene ether Download PDFInfo
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Abstract
The invention discloses a method for detecting the content of polyethylene glycol in fatty alcohol-polyoxyethylene ether by using a reversed-phase high-performance liquid chromatography method, wherein a reversed-phase chromatography system is used for a stationary phase, an evaporative light scattering detector is used as the detector, and a logarithmic regression equation is used for quantification. The method is suitable for a series of products, and can be applied to analysis and detection of the content of free polyethylene glycol in fatty alcohol-polyoxyethylene ether in the fields of medicines, cosmetics and the like.
Description
Technical Field
The invention belongs to the field of analytical chemistry, and particularly relates to a method for determining free polyethylene glycol in fatty alcohol-polyoxyethylene ether.
Background
The fatty alcohol-polyoxyethylene ether is a product formed by the addition of fatty alcohol and ethylene oxide, and can form a certain amount of free polyethylene glycol in the reaction process. The invention discloses a method for determining the content of free polyethylene glycol in fatty alcohol-polyoxyethylene ether by reversed-phase high-performance chromatography, which comprises the following steps: and (4) calculating a result by using an evaporative light scattering detector and adopting a logarithmic regression equation. The analysis method provided by the invention has the advantages of high sensitivity, good repeatability, rapidness, high efficiency and good precision, and is applicable to similar products in the market. The method for determining free PEG generally adopts gel chromatography, the principle is to separate according to molecular weight, and the method has the defect that for free PEG with similar molecular weight, the separation degree cannot meet the requirement, so that the integral is unreasonable, and the accuracy of the final detection result is influenced.
Polyethylene glycol is a reaction byproduct in the reaction process of fatty alcohol-polyoxyethylene ether, is a product formed by condensation polymerization of ethylene oxide and water, has a molecular formula consisting of a plurality of repeated oxyethyl structural units, and is represented by HO (CH 2CH 2O) nH, wherein n represents the average number of oxyethylene groups.
The fatty alcohol-polyoxyethylene ether series products take polidocanol as an example, and are widely applied to medicines. The polidocanol injection is suitable for hardening treatment of central veins, reticular veins and venules varicose veins of spider veins. In order to control the raw material residue in the crude drug polidocanol, the content of free polyethylene glycol in the raw material polidocanol needs to be controlled.
Disclosure of Invention
In order to overcome the blank of the existing detection method, the invention aims to provide a simple, convenient and accurate method for detecting the content of free polyethylene glycol in fatty alcohol-polyoxyethylene ether such as polidocanol, thereby ensuring the medication safety.
In order to achieve the above object, the present invention provides the following technical solutions:
a method for determining free polyethylene glycol in fatty alcohol-polyoxyethylene ether, the method comprising:
(1) Preparation of standard curve solutions: accurately weighing 0.2g of polyethylene glycol standard substance, placing the polyethylene glycol standard substance in a 20ml measuring flask, dissolving with tetrahydrofuran, fixing the volume to scale, shaking up to be used as standard stock solutions, respectively taking a proper amount of the standard stock solutions, and diluting with tetrahydrofuran to obtain diluent solutions with the concentrations of 0.08mg/ml, 0.2mg/ml, 0.5mg/ml, 0.9mg/ml, 1.2mg/ml and 1.6mg/ml respectively as standard curve solutions;
(2) Preparing a test solution: taking a corresponding fatty alcohol-polyoxyethylene ether sample to be detected, precisely weighing, placing the sample in a 10ml measuring flask, dissolving and diluting the sample to a scale by using tetrahydrofuran, and shaking up the sample to be used as a sample solution;
(3) And (3) chromatographic measurement: respectively and precisely measuring a standard curve solution and a test sample solution, and injecting the standard curve solution and the test sample solution into a liquid chromatograph for measurement;
(4) And (3) calculating the concentration: performing linear regression on the logarithm value of the peak area of the evaporation light signal in the standard curve solution and the logarithm value of the concentration to obtain a linear equation, calculating the content of free polyethylene glycol by using the equation and the peak area of the fatty alcohol-polyoxyethylene ether signal to be detected,
the stationary phase in the chromatograph uses a reversed phase chromatographic system, the detector is an evaporative light scattering detector, and the quantification is carried out by using a logistic regression equation.
Preferably, the reversed-phase chromatographic column of the reversed-phase chromatographic system uses reversed-phase carbooctadecylsilane chemically bonded silica as a filler, acetonitrile-water 1 and tetrahydrofuran as mobile phases, and gradient elution is carried out; the temperature of a drift tube of the evaporative light scattering detector is 80 to 85 ℃, the carrier gas flow is 2.3L/min, and the measurement is carried out in a shunting mode.
Further, the procedure of gradient elution is that the flow rate is 1.0ml/min and the column temperature is 35 to 38 ℃ in terms of min/b% of time and 0/20, 20/60, 25/100, 30/100, 35/20 and 40/20.
In the method, the standard substance is polyethylene glycol with the molecular weight of 1000 to 4000.
In the above method, the fatty alcohol-polyoxyethylene ether to be measured is polidocanol.
Has the beneficial effects that: the analysis method provided by the invention has the advantages of high sensitivity, good repeatability and precision, rapidness, high efficiency, simplicity and convenience, is suitable for measuring the content of free polyethylene glycol in the same series of products, can effectively supervise production, and ensures the stability of product quality.
Drawings
FIG. 1 is a spectrum of 0.9mg/ml polyethylene glycol 1000;
FIG. 2 is a spectrum of a polidocanol test solution.
Detailed Description
The invention provides a method for detecting the content of polyethylene glycol in fatty alcohol-polyoxyethylene ether by using a reversed-phase high performance liquid chromatography method, which is suitable for a series of products. The method can be applied to the analysis and detection of the content of free polyethylene glycol in fatty alcohol-polyoxyethylene ether in the fields of medicines, cosmetics and the like.
In the method, a reversed phase chromatographic system is used as a stationary phase, an evaporative light scattering detector is used as a detector, and the quantification is carried out by using a logistic regression equation, wherein the chromatographic conditions are as follows:
a. octadecylsilane chemically bonded silica is used as a packed column;
b. gradient elution is carried out by taking acetonitrile-water (1);
c. the gradient elution procedure (time min/b%) was: 0/20, 20/60, 25/100, 30/100, 35/20, 40/20;
d. the flow rate is 1.0ml/min;
e. the column temperature is 35 ℃;
f. the temperature of a drift tube of the evaporative light scattering detector is 83 ℃, the carrier gas flow is 2.3L/min, and the flow distribution mode is adopted.
The standard curve solution was prepared as follows: 0.2g of polyethylene glycol 1000 is precisely weighed, placed in a 20ml measuring flask, dissolved by tetrahydrofuran, and shaken up to a constant volume to be used as a standard stock solution. Taking appropriate amount of standard stock solution respectively, diluting with tetrahydrofuran to obtain the following concentration: 0.08mg/ml, 0.2mg/ml, 0.5mg/ml, 0.9mg/ml, 1.2mg/ml, 1.6mg/ml as standard curve solutions.
The test solution was prepared as follows: taking polidocanol as an example, 0.3g of polidocanol is precisely weighed, placed in a 10ml measuring flask, dissolved and diluted to the scale by tetrahydrofuran, and shaken up to be used as a test solution.
The determination method comprises the following steps: respectively and precisely measuring the standard curve solution and the sample solution, and injecting into a liquid chromatograph for measurement.
The concentration calculation method comprises the following steps: and performing linear regression on the logarithm value of the peak area and the logarithm value of the concentration of the evaporation light signal in the solution with the standard curve to obtain a linear equation. And calculating the content of free polyethylene glycol by using the equation and the peak area of the polyethylene glycol signal in the polidocanol.
The present invention is described in further detail below with reference to specific examples, but the embodiments of the present invention are not limited to the scope of the description. In addition, various modifications may occur to those skilled in the art upon reading the present disclosure, and such equivalent variations are within the scope of the present invention as defined in the appended claims.
The liquid chromatography is carried out according to the liquid chromatography of the four parts of China pharmacopoeia 2020 edition.
[ examples ] A method for producing a compound
Standard curve solution preparation: taking 0.2g of PEG1000 as a reference, accurately weighing, placing in a 20ml measuring flask, dissolving with tetrahydrofuran, metering to a certain volume, and shaking uniformly to obtain a standard stock solution. Taking appropriate amount of standard stock solution respectively, diluting with tetrahydrofuran to obtain the following concentrations: 0.08mg/ml, 0.20mg/ml, 0.50mg/ml, 0.90mg/ml, 1.2mg/ml, 1.6mg/ml were used as standard curve solutions.
Preparing a test solution: adding tetrahydrofuran into polidocanol to dissolve, and preparing a solution of 30mg/ml to serve as a test solution.
The chromatographic conditions were as follows: performing gradient elution by using an octadecylsilane chemically bonded silica gel column as a chromatographic column, acetonitrile-water (50) as a mobile phase A and tetrahydrofuran as a mobile phase B, wherein the flow rate is 1.0ml/min, the column temperature is 35 ℃, the temperature of an evaporative light scattering detector drift tube is 83 ℃, the carrier gas flow rate is 2.3L/min, and the flow distribution mode is adopted. 10 μ l of the test solution and 10 μ l of the standard curve solution were injected into a liquid chromatograph to be measured.
Gradient elution procedure (time min/b%): 0/20, 20/60, 25/100, 30/100, 35/20 and 40/20.
According to the above detection method, the following specificity test was performed.
1. Specificity
The operation method comprises the following steps: and (3) carrying out acid, alkali, oxidation, high temperature, high humidity and illumination damage on the sample, and then injecting the sample.
Acceptance criteria: tetrahydrofuran does not interfere with the position of free PEG, each forced degradation peak has good separation degree with the free PEG, and the measurement is not interfered.
The results show that: acid, alkali and 105 ℃ degradation obviously increase the content of free PEG, and other degradation effects are not large, and no other degradation impurity peak exists.
2. Detection limit amount limit
The operation method comprises the following steps: taking PEG1000 solution to gradually dilute until the S/N is about 3 as a detection limit. Taking PEG1000 solution to dilute step by step until S/N is about 10 as a quantitative limit, and continuously injecting samples for 6 times.
The results showed that the sample could be detected at 8. Mu.g/ml and quantified at 20. Mu.l/ml. The RSD for the six needle quantification limit was 12, which is within the acceptable range.
3. Linear range of
The operation method comprises the following steps: PEG1000 control was diluted to concentrations of: 0.08mg/ml, 0.20mg/ml, 0.50mg/ml, 0.90mg/ml, 1.2mg/ml, 1.6mg/ml were used as standard curve solutions. 10 mul of the sample was injected into a liquid chromatograph, and linear regression was performed using the concentration C (mg/ml) as the abscissa and the peak area A as the ordinate.
Acceptance criteria: taking the concentration as the abscissa X, and taking the peak area as the ordinate Y: the correlation coefficient (R) should not be less than 0.999, and the Y-axis intercept should be within 25% of the 100% response value
The result shows that when the PEG1000 concentration is between 78.3 and 1566.6 mug/ml (0.26 to 5.2 percent), the linear relation between the peak area logarithm value and the concentration logarithm value is good, and the correlation coefficient R value is 0.999.
4. Precision experiment
The operation method comprises the following steps: taking 0.9mg/ml of PEG1000 standard curve solution, and taking 6 needles for sample injection precision; taking a polidocanol test solution (6 parts of each 1 needle) as repeatability; repeat experiments were performed again at different times by different experimenters as intermediate precision.
The sample injection precision and the repeatability RSD of the invention are not more than 2, the intermediate precision RSD is not more than 4, and the result shows that the precision of the invention is better.
5. Recovery rate
The operation method comprises the following steps: taking a proper amount of polidocanol, adding tetrahydrofuran to dissolve, adding a certain amount of PEG1000 control solution according to 50%, 100% and 150% of limits, operating 3 parts in parallel for each level, and calculating the recovery amount and the recovery rate.
Acceptance criteria: the recovery rate is between 92 and 105 percent, and the RSD is not more than 5 percent.
The result shows that the recovery rate of the invention is 92 to 105 percent, the RSD is not more than 5 percent, and the accuracy of the invention is good.
6. Durability
a. Solution stability: taking 100% sample-added sample solution, standing at room temperature, and injecting into liquid chromatograph at different times.
The results show that the solution is stable within 10 hours.
b. Modified chromatography column
The chromatographic columns with the same packing of different batches are replaced for measurement.
The result shows that the content of the replaced chromatographic column only changes slightly, and the influence on the invention is small.
c. Modifying flow rate
The flow rate was changed to 1.1ml/min for measurement.
The result shows that the content of free polyethylene glycol at the change flow rate is only slightly changed, and the influence on the invention is small.
d. Changing the column temperature
The column temperature was changed to 38 ℃ for measurement.
The result shows that only slight change exists in the content of the free polyethylene glycol when the column temperature is changed, and the influence on the method is small.
e. Fine adjustment of mobile phase ratio
The results show that fine adjustment of the mobile phase ratio has little effect on the results.
The above results show that the present invention has better durability.
The method can conveniently, quickly and efficiently detect the content of the free polyethylene glycol in the fatty alcohol-polyoxyethylene ether series products.
While the embodiments of the present invention have been described in detail with reference to the drawings and the specific examples, the present invention is not limited to the embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the spirit of the present invention.
Claims (4)
1. A method for determining free polyethylene glycol in fatty alcohol-polyoxyethylene ether is characterized by comprising the following steps:
(1) Preparation of standard curve solutions: accurately weighing 0.2g of polyethylene glycol standard substance, placing the polyethylene glycol standard substance in a 20ml measuring flask, dissolving with tetrahydrofuran, fixing the volume to scale, shaking up to be used as standard stock solutions, respectively taking a proper amount of the standard stock solutions, and diluting with tetrahydrofuran to obtain diluent solutions with the concentrations of 0.08mg/ml, 0.2mg/ml, 0.5mg/ml, 0.9mg/ml, 1.2mg/ml and 1.6mg/ml respectively as standard curve solutions;
(2) Preparing a test solution: taking a corresponding fatty alcohol-polyoxyethylene ether sample to be detected, precisely weighing, placing the sample in a 10ml measuring flask, dissolving and diluting the sample to a scale by using tetrahydrofuran, and shaking up the sample to be used as a sample solution;
(3) And (3) chromatographic measurement: respectively precisely measuring a standard curve solution and a test sample solution, and injecting the standard curve solution and the test sample solution into a liquid chromatograph for measurement;
(4) And (3) calculating the concentration: performing linear regression on the logarithm value of the peak area of the evaporation light signal in the standard curve solution and the logarithm value of the concentration to obtain a linear equation, calculating the content of free polyethylene glycol by using the equation and the peak area of the fatty alcohol-polyoxyethylene ether signal to be detected,
wherein, the stationary phase in the chromatograph uses a reversed phase chromatographic system, the detector is an evaporative light scattering detector, and the quantification is carried out by using a logarithmic regression equation;
the reverse phase chromatographic column of the reverse phase chromatographic system uses reverse phase octadecylsilane chemically bonded silica as a filler, acetonitrile-water 1 as a mobile phase A and tetrahydrofuran as a mobile phase B to perform gradient elution, wherein the gradient elution is performed by the following procedures of 0/20, 20/60, 25/100, 30/100, 35/20 and 40/20 in terms of time min/B%; the temperature of a drift tube of the evaporative light scattering detector is 80-85 ℃, the flow rate of carrier gas is 2.3L/min, and the measurement is carried out in a split-flow mode.
2. The method for determining free polyethylene glycol in fatty alcohol-polyoxyethylene ether according to claim 1, wherein in the gradient elution process, the flow rate is 1.0ml/min, and the column temperature is 35-38 ℃.
3. The method for determining free polyethylene glycol in fatty alcohol-polyoxyethylene ether according to claim 1, wherein the standard substance is polyethylene glycol with molecular weight of 1000 to 4000.
4. The method for determining free polyethylene glycol in fatty alcohol-polyoxyethylene ether according to claim 1, wherein the fatty alcohol-polyoxyethylene ether to be determined is polidocanol.
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