Sampling detection method for phenacetin content

Abstract

The present invention discloses a phenacetin content sampling detection method, and relates to the technical field of drug analysis and detection, the method comprises the following steps: 1, preparing a test sample and an internal standard substance into a phenacetin solution, carrying out chromatographic separation through a high performance liquid chromatograph, and using solvents: 50% methanol, the test sample solution and a proper amount of the product; 2, sample preparation: precisely measuring a test sample, precisely weighing a reference substance preparation solution, and preparing an internal standard solution, wherein an impurity positioning solution is prepared by taking a proper amount of p-aminophenetole and a proper amount of p-chlorophenylacetamide reference substances; and 3, precisely measuring 1ml of the test solution, placing the test solution in a 50ml measuring flask, diluting the test solution to a scale by using a solvent, taking 1ml of the test solution and 1ml of the impurity positioning solution, diluting the test solution and the impurity positioning solution to 20ml by using a mobile phase, and the like. The method has high precision, accuracy and linear range, the concentration of phenacetin can be detected, and quantitative analysis and monitoring of phenacetin can be realized through proper sample preparation, chromatographic conditions and standard curve preparation.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis and detection, and in particular to a sampling and detection method for phenacetin content. Background Art

[0002] Phenacetin, chemically known as acetaminophenethyl ether, is an antipyretic and analgesic API with good efficacy in treating headaches, neuralgia, joint pain and fever. It has antipyretic and analgesic effects, but no anti-inflammatory or anti-rheumatic effects. This product is mainly metabolized to acetaminophen in the liver, and a small part is deacetylated to form acetaminophenethyl ether, which is further metabolized to iminoquinone, which can oxidize hemoglobin into methemoglobin. When the amount is large, cyanosis may occur, and even severe hypoxia symptoms may occur, which is especially common in children.

[0003] Phenacetin is a widely used drug in clinical practice. It is used to treat epilepsy, anticonvulsant diseases, etc. Accurate monitoring and detection of phenacetin concentration is very important for determining efficacy and adjusting drug dosage. In the pharmaceutical field, painkillers are compound preparations made of aminopyrine, phenacetin, caffeine and phenobarbital. They have been used clinically for toothache, joint pain and migraine. Long-term and large-scale use can cause kidney poisoning. In order to control the quality of drugs, ensure the safety and effectiveness of clinical drugs, simplify the inspection steps, and reduce experimental errors, it is necessary to determine the content of phenacetin in painkillers.

[0004] After searching the prior art, we did not find any patent website that describes the determination method of phenacetin. We need a new analytical method for the determination of phenacetin content. In the existing literature, only the ministerial standard WS1-55(B)-89 involves the quality standard of phenacetin, and there is no related substance detection item. Its impurity detection is carried out by hydrolyzing the sample under catalytic conditions and using the chloride test method to control p-chlorophenylacetamide; and by using the colorimetric method to control p-ethoxyaniline. The above two methods are time-consuming and insensitive, and the accurate content of impurities cannot be obtained.

[0005] In view of the above problems, there is a need in the art to provide a method for detecting phenacetin-related substances with strong specificity, high accuracy, and simple and rapid detection. Summary of the invention

[0006] The object of the present invention is to provide a sampling and detection method for the content of phenacetin. The device is used to work, thereby solving the problem that the traditional method for detecting the content of phenacetin in the above background is that most of the existing documents only have the quality standard of phenacetin, but no related substance detection items, and the impurity detection is carried out by hydrolyzing the sample under catalyst conditions, using the chloride inspection method to control p-chlorophenylacetamide, and using the colorimetric method to control p-ethoxyaniline. The above two methods are time-consuming and insensitive to detection, and the problem that the accurate content of impurities cannot be obtained.

[0007] To achieve the above object, the present invention provides the following technical solution: a sampling and detection method for phenacetin content, comprising the following main steps;

[0008] Step 1: Prepare the test sample and internal standard into phenacetin solution, and perform chromatographic separation by high performance liquid chromatography, using solvent: 50% methanol, test sample solution and appropriate amount of this product, accurately weigh, add solvent to dissolve and quantitatively dilute to make a solution containing about 1 mg per 1 ml;

[0009] Step 2: The sample preparation step includes accurately measuring the test sample, accurately weighing the reference sample to prepare the solution, and preparing the internal standard solution. The impurity location solution is to take appropriate amounts of p-aminophenyl ether and p-chlorophenyl acetamide reference samples, accurately weigh them, place them in different volumetric bottles, dissolve them with solvents and dilute them to make a solution containing about 10 μg per 1 ml;

[0010] Step 3: The control solution is to accurately measure 1 ml of the test solution, place it in a 50 ml volumetric flask, dilute it to the mark with solvent, then take 1 ml of this solution and 1 ml of each impurity location solution, and dilute it to 20 ml with mobile phase;

[0011] Step 4: The operation procedure is to accurately aspirate 5 μL of the test sample and reference sample solutions, inject them into the high performance liquid chromatograph, measure the absorbance value at a wavelength of 254 nm, and calculate the phenacetin content according to the internal standard method.

[0012] Preferably, octadecylsilane bonded silica gel is used as a filler, and the separation degree between phenacetin and the internal standard substance peak should meet the requirements. The chromatographic conditions in the determination method use octadecylsilane bonded silica gel as a filler; 0.01 mol / l potassium dihydrogen phosphate solution-methanol (6:4) is used as the mobile phase; the detection wavelength is 245nm; the column temperature is 40°C, the flow rate is 1.0ml / min, and the injection volume is 10μl.

[0013] Preferably, the instrument includes a high performance liquid chromatograph equipped with an octadecylsilane bonded silica gel chromatographic column, the peak separation meets the standard, and an ultraviolet absorption detector is also required.

[0014] Preferably, the system suitability requirement is that in the reference solution chromatogram, the number of theoretical plates calculated based on the phenacetin peak is not less than 5000, and the resolution between the phenacetin peak and the p-chlorophenylacetamide peak should be greater than 2.5.

[0015] Preferably, the assay method comprises accurately measuring the test solution and the control solution, injecting them into a liquid chromatograph respectively, and recording the chromatogram to 3 times the retention time of the main component peak.

[0016] Preferably, if there are impurity peaks in the chromatogram of the limit test solution that are consistent with the p-aminophenyl ether peak and p-chlorophenylacetamide peak in the control solution, calculated by peak area using the external standard method, they shall not exceed 0.05%, the peak area of ​​other individual impurity peaks shall not be greater than the peak area of ​​phenacetin in the control solution, the sum of all impurities shall not exceed 1.0%, and the peaks in the chromatogram of the test solution that are less than 0.1 times the main peak area of ​​the control solution shall be ignored.

[0017] Preferably, the inner wall of the pipette is washed with ether several times for the test sample, the washing solution is added to a volumetric flask, and ether is added to dilute to the scale; shake well and accurately measure 5 mL and place it in a stoppered centrifuge tube, and the ether is evaporated in a warm water bath; shake and extract with methanol 4 times, shake for 10 minutes each time, centrifuge for 15 minutes, and transfer the methanol to a 25 mL volumetric flask with a dropper, combine the extracts, accurately add 5 mL of the internal standard solution, dilute to the scale with methanol, shake well, and obtain the test sample solution.

[0018] Preferably, the high performance liquid phase analysis method of phenacetin is used in the preparation of drugs for detecting high-risk groups of liver cancer, and the phenacetin is the qualitative and quantitative analysis of phenacetin in oral preparations, injections or tablets.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] Reversed-phase high performance liquid chromatography can accurately and reliably detect the concentration of phenacetin in medicines. This method has high precision, accuracy and linear range, and can detect low concentrations of phenacetin. Through appropriate sample preparation, chromatographic condition setting and standard curve preparation, quantitative analysis and monitoring of phenacetin can be achieved, providing effective support for clinical drug use. This method fills the gap in the detection of phenacetin by liquid chromatography. This detection method can be used for the detection of phenacetin content and related substances, and can be used to guide the inspection of stability samples. When taking related drugs in the later stage, it is convenient for medical staff to understand the content of phenacetin, which is convenient for controlling the amount of drugs and improving the safety of phenacetin use. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is an illustration of the chromatograms of the blank solvent, the impurity localization solution and the control solution of the present invention;

[0022] Figure 2 It is a diagram illustrating the specific test results of the present invention;

[0023] Figure 3 It is an illustration of the chromatographic condition detection according to an embodiment of the present invention;

[0024] Figure 4 It is a diagram illustrating the precision of sample injection under the chromatographic conditions of the present invention;

[0025] Figure 5 It is a diagram illustrating the repeatability of the chromatographic condition detection of the present invention;

[0026] Figure 6 is a graph illustrating the stability of the control solution of the present invention;

[0027] Figure 7 It is a diagram illustrating the stability of the test solution of the present invention;

[0028] Figure 8 is an illustration of the linear range of phenacetin of the present invention;

[0029] Fig. 9 It is an illustration of the linear range of p-phenylamino ether of the present invention;

[0030] Fig.10 It is an illustration of the linear range of p-chlorophenylacetamide of the present invention. DETAILED DESCRIPTION

[0031] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] In order to further understand the content of the present invention, the present invention is described in detail in conjunction with the accompanying drawings.

[0033] The present invention provides a technical solution: a sampling and detection method for phenacetin content, comprising the following main steps:

[0034] Step 1: Prepare the test sample and internal standard into phenacetin solution, and perform chromatographic separation by high performance liquid chromatography, using solvent: 50% methanol, test sample solution and appropriate amount of this product, accurately weigh, add solvent to dissolve and quantitatively dilute to make a solution containing about 1 mg per 1 ml;

[0035] Step 2: The sample preparation step includes accurately measuring the test sample, accurately weighing the reference sample to prepare the solution, and preparing the internal standard solution. The impurity location solution is to take appropriate amounts of p-aminophenyl ether and p-chlorophenyl acetamide reference samples, accurately weigh them, place them in different volumetric bottles, dissolve them with solvents and dilute them to make a solution containing about 10 μg per 1 ml;

[0036] Step 3: The control solution is to accurately measure 1 ml of the test solution, place it in a 50 ml volumetric flask, dilute it to the mark with solvent, then take 1 ml of this solution and 1 ml of each impurity location solution, and dilute it to 20 ml with mobile phase;

[0037] Step 4: The operation procedure is to accurately aspirate 5 μL of the test sample and reference sample solutions, inject them into the high performance liquid chromatograph, measure the absorbance value at a wavelength of 254 nm, and calculate the phenacetin content according to the internal standard method.

[0038] Octadecylsilane bonded silica gel is used as filler, and the separation degree between phenacetin and internal standard substance peaks should meet the requirements. The chromatographic conditions in the determination method use octadecylsilane bonded silica gel as filler; 0.01 mol / l potassium dihydrogen phosphate solution-methanol (6:4) is used as the mobile phase; the detection wavelength is 245nm; the column temperature is 40℃, the flow rate is 1.0ml / min, and the injection volume is 10μl.

[0039] The instruments and equipment include a high performance liquid chromatograph equipped with an octadecylsilane bonded silica gel column with peak separation meeting the standards. An ultraviolet absorption detector is also required.

[0040] The system suitability requirement is that in the reference solution chromatogram, the theoretical plate number calculated based on the phenacetin peak should be no less than 5000, and the separation between the phenacetin peak and the p-chlorophenylacetamide peak should be greater than 2.5.

[0041] Determination method: Accurately measure the test solution and the reference solution, inject them into the liquid chromatograph respectively, and record the chromatogram to 3 times the retention time of the main component peak.

[0042] If there are impurity peaks in the chromatogram of the test solution that are consistent with the peaks of p-aminophenylethyl ether and p-chlorophenylacetamide in the control solution, the peak areas calculated by the external standard method shall not exceed 0.05%. The peak areas of other individual impurity peaks shall not be greater than the peak area of ​​phenacetin in the control solution. The sum of all impurities shall not exceed 1.0%. Peaks in the chromatogram of the test solution that are less than 0.1 times the main peak area of ​​the control solution shall be ignored.

[0043] Example 1

[0044] Sample preparation

[0045] Phenacetin in the sample can be firstly extracted from an oral preparation, an injection or a tablet to obtain a sufficient phenacetin sample.

[0046] Chromatographic conditions

[0047] The reverse phase high performance liquid chromatography method was used to detect phenacetin under the following conditions:

[0048] Chromatographic column: C18 column (length, diameter, filling material, etc. are selected according to needs)

[0049] Mobile phase: prepared by mixing methanol and 0.01M phosphoric acid solution (pH 2.5)

[0050] Flow rate: Determined by column and detection conditions (typical range is 0.5-2.0 mL / min)

[0051] Detector: UV detector (typical wavelength is 254nm)

[0052] Standard curve preparation

[0053] In order to quantitatively analyze the concentration of phenacetin, a series of standard solutions were prepared. The specific preparation steps are as follows:

[0054] 1. Prepare concentration: Determine the concentration range of the standard based on the concentration range and the requirements of the sample to be tested.

[0055] 2. Dilute the standard: dilute the high concentration standard in sequence to prepare a series of standards with different concentrations (usually including 3-5 concentrations)

[0056] 3. Color development reaction: Appropriate color development reaction can be used to increase the sensitivity and selectivity of the detection.

[0057] Method validation

[0058] In order to verify the reliability and accuracy of reversed-phase HPLC, a series of method validations are required. The following are common validation parameters:

[0059] 1. Exclusivity

[0060] Take blank solvent, impurity localization solution and control solution, respectively, and detect according to the chromatographic conditions of the embodiment, and record the chromatogram. The chromatograms of blank solvent, impurity localization solution and control solution are shown in the attached manual. Figure 1 .

[0061] The test results show that the blank solvent does not interfere with the detection of the main component peak and impurity peak; the peak order of the control solution is p-aminophenyl ether peak, phenacetin peak and p-chlorophenyl acetamide peak, and the separation degree between each peak is greater than 2.5, indicating that the method has good specificity. The specificity test results are shown in the appendix of the manual. Figure 2 .

[0062] 2. Limit of quantification and limit of detection

[0063] Take the impurity location solution and the test solution, dilute them gradually with solvent until the main peak S / N is ≈ 10, and use it as the quantitative limit solution. Detect according to the chromatographic conditions of the example. The results are shown in the attached manual. Figure 3 shown.

[0064] The results showed that the method has high sensitivity and can produce responses at extremely low limits.

[0065] 3. Injection precision

[0066] Take the control solution and repeat the injection 6 times according to the chromatographic conditions of the example. Figure 4 shown.

[0067] The RSD of the peak area of ​​each impurity and the main component is ≤2.0%, and the RSD of the retention time is ≤2.0%, indicating good precision.

[0068] 4. Repeatability

[0069] Take the same test sample and prepare 6 copies in parallel, and test them according to the chromatographic conditions in the example. Figure 5 shown.

[0070] The extreme differences of the impurity contents did not exceed 0.01%, proving that the method had good repeatability.

[0071] 5. Solution stability

[0072] Take the control solution and the test solution, and select no less than 3 time points for testing according to the chromatographic conditions of the embodiment. The results are attached to the specification. Figure 6 as well as Figure 7 shown.

[0073] Description: Instructions attached Figure 6 For the control solution, the instruction manual is attached. Figure 7 For the test solution

[0074] The ratio of the peak area measured at each time point to the peak area measured at 0 h was between 98% and 102%, indicating that the control solution and the test solution were stable at room temperature for at least 24 h.

[0075] 7. Linear range

[0076] Take the impurity location solution and the test solution, dissolve and dilute them with a solvent to form a series of reference solutions (equivalent to the limit concentration of 20%, 50%, 100%, 150%, 200%), take each reference solution and test it according to the chromatographic conditions of the embodiment, record the peak area, and draw a linear regression curve with the peak area as the ordinate and the concentration of the reference solution (μg / ml) as the abscissa. The results are shown in the attached manual. Figure 8-Figure 10 .

[0077] The results showed that the linear relationship between the concentration and the peak area of ​​phenacetin, p-aminophenyl ether and p-chlorophenylacetamide was good within the limit concentration range of 20% to 200%.

[0078] Data processing

[0079] The data obtained from the experiment is processed and analyzed through the data acquisition software of the chromatograph or other data processing software. Common data processing methods include:

[0080] 1. Peak area determination: The concentration of phenacetin is quantified by measuring the peak area.

[0081] 2. Standard curve: Use the standard curve to convert peak areas to concentrations.

[0082] In summary, the present invention proposes a method for sampling and detecting the content of phenacetin, and the detection conclusion is:

[0083] Reversed-phase high performance liquid chromatography can accurately and reliably detect the concentration of phenacetin in medicines. This method has high precision, accuracy and linear range, and can detect low concentrations of phenacetin. Through appropriate sample preparation, chromatographic condition setting and standard curve preparation, quantitative analysis and monitoring of phenacetin can be achieved, providing effective support for clinical drug use.

[0084] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0085] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A sampling and detection method for phenacetin content, characterized in that: The main steps include: Step 1: Prepare the test sample and internal standard into phenacetin solution, and perform chromatographic separation by high performance liquid chromatography, using solvent: 50% methanol, test sample solution and appropriate amount of this product, accurately weigh, add solvent to dissolve and quantitatively dilute to make a solution containing about 1 mg per 1 ml; Step 2: The sample preparation step includes accurately measuring the test sample, accurately weighing the reference sample to prepare the solution, and preparing the internal standard solution. The impurity location solution is to take appropriate amounts of p-aminophenyl ether and p-chlorophenyl acetamide reference samples, accurately weigh them, place them in different volumetric bottles, dissolve them with solvents and dilute them to make a solution containing about 10 μg per 1 ml; Step 3: The control solution is to accurately measure 1 ml of the test solution, place it in a 50 ml volumetric flask, dilute it to the mark with solvent, then take 1 ml of this solution and 1 ml of each impurity location solution, and dilute it to 20 ml with mobile phase; Step 4: The operation procedure is to accurately aspirate 5 μL of the test sample and reference sample solutions, inject them into the high performance liquid chromatograph, measure the absorbance value at a wavelength of 254 nm, and calculate the phenacetin content according to the internal standard method.

2. The method for sampling and detecting phenacetin content according to claim 1, characterized in that: Octadecylsilane bonded silica gel is used as a filler, and the separation degree between phenacetin and the internal standard substance peak should meet the requirements. The chromatographic conditions in the determination method use octadecylsilane bonded silica gel as a filler; 0.01 mol / l potassium dihydrogen phosphate solution-methanol (6:4) as a mobile phase; the detection wavelength is 245 nm; The column temperature was 40°C, the flow rate was 1.0 ml / min, and the injection volume was 10 μl.

3. A method for sampling and detecting phenacetin content according to claim 2, characterized in that: The instruments and equipment include a high performance liquid chromatograph equipped with an octadecylsilane bonded silica gel column with peak separation meeting the standards. An ultraviolet absorption detector is also required.

4. The method for sampling and detecting phenacetin content according to claim 3, characterized in that: The system suitability requirement is that in the reference solution chromatogram, the theoretical plate number calculated based on the phenacetin peak should be no less than 5000, and the separation between the phenacetin peak and the p-chlorophenylacetamide peak should be greater than 2.

5.

5. The method for sampling and detecting phenacetin content according to claim 4, characterized in that: Determination method: Accurately measure the test solution and the reference solution, inject them into the liquid chromatograph respectively, and record the chromatogram to 3 times the retention time of the main component peak.

6. A method for sampling and detecting phenacetin content according to claim 5, characterized in that: If there are impurity peaks in the chromatogram of the test solution that are consistent with the peaks of p-aminophenylethyl ether and p-chlorophenylacetamide in the control solution, the peak areas calculated by the external standard method shall not exceed 0.05%. The peak areas of other individual impurity peaks shall not be greater than the peak area of ​​phenacetin in the control solution. The sum of all impurities shall not exceed 1.0%. Peaks in the chromatogram of the test solution that are less than 0.1 times the main peak area of ​​the control solution shall be ignored.

7. A method for sampling and detecting phenacetin content according to claim 6, characterized in that: Preparation of test solution: Wash the inner wall of the pipette with ether several times, add the washing solution into the volumetric flask, and dilute to the scale with ether; shake well and accurately measure 5mL and place it in a stoppered centrifuge tube, and evaporate the ether in a warm water bath; shake and extract with methanol 4 times, shake for 10 minutes each time, centrifuge for 15 minutes, and use a dropper to transfer the methanol to a 25mL volumetric flask, combine the extracts, accurately add 5mL of internal standard solution, dilute to the scale with methanol, shake well, and the test solution is obtained.

8. The method for sampling and detecting phenacetin content according to claim 7, characterized in that: The high performance liquid phase analysis method of phenacetin is used in the preparation of drugs for detecting high-risk groups of liver cancer, and the phenacetin is the qualitative and quantitative analysis of phenacetin in oral preparations, injections or tablets.

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