Method for separating and detecting (R)-1, 2-propylene glycol and (S)-1, 2-propylene glycol
By performing benzoyl chloride derivatization on 1,2-propanediol, combined with liquid chromatography and UV detection, the problem of separating and detecting (R)-1,2-propanediol and (S)-1,2-propanediol was solved, achieving efficient quantitative analysis.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-30
- Publication Date
- 2026-03-31
AI Technical Summary
The lack of effective methods in the existing technology for separating and detecting (R)-1,2-propanediol and (S)-1,2-propanediol affects the quality control of pharmaceutical production.
1,2-propanediol was derivatized using benzoyl chloride reagent, and then separated and detected by liquid chromatography using a polysaccharide derivative-coated normal-phase chiral column or a polysaccharide derivative solvent-resistant chiral column. The mobile phase was a hexane-isopropanol mixture, and quantitative analysis was performed using a UV detector.
Highly sensitive separation and quantitative detection of (R)-1,2-propanediol and (S)-1,2-propanediol were achieved, with good separation and broad application prospects.
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Abstract
Description
Technical Field
[0001] This invention relates to a method for separating and detecting (R)-1,2-propanediol and (S)-1,2-propanediol. Background Technology
[0002] (R)-1,2-propanediol is a key material in the synthesis of the pharmaceutical (R)-(-)-1,2-propanediol (4R)-4-methyl-1,3,2-dioxathiapentane-2,2-dioxide. However, (S)-1,2-propanediol significantly affects the utilization of (R)-1,2-propanediol. Effective separation and detection of (S)-1,2-propanediol content is a crucial step in quality control during pharmaceutical production. The structural formulas are (R)-1,2-propanediol (A) and (S)-1,2-propanediol (B), respectively.
[0003]
[0004] Since 1,2-propanediol has no unsaturated bonds or conjugated groups in its chemical structure, it does not absorb ultraviolet light and cannot be directly separated and determined by high-performance liquid chromatography. Currently, no relevant analytical method has been found to detect it.
[0005] Therefore, it is crucial to develop an effective method for separating and detecting (R)-1,2-propanediol and (S)-1,2-propanediol to achieve quantitative detection of this enantiomer (S)-1,2-propanediol. Given the current limitations in determining the content of the enantiomer (S)-1,2-propanediol, a more accurate and efficient new method is urgently needed. Summary of the Invention
[0006] The technical problem this invention aims to solve is to overcome the shortcomings of existing technologies in separating and detecting (R)-1,2-propanediol and (S)-1,2-propanediol, which suffer from a lack of effective methods. Therefore, this invention provides a method for separating and detecting (R)-1,2-propanediol and (S)-1,2-propanediol. The detection method provided by this invention can effectively separate and quantitatively detect (R)-1,2-propanediol and (S)-1,2-propanediol, and has good application prospects.
[0007] The present invention solves the above-mentioned technical problems through the following technical solutions.
[0008] This invention provides a method for detecting 1,2-propanediol, comprising the following steps:
[0009] Step (1): In an organic solvent, 1,2-propanediol is derivatized with benzoyl chloride reagent;
[0010] Step (2): Separation and detection are performed using liquid chromatography;
[0011] The chromatographic conditions are as follows:
[0012] Chromatographic column: Polysaccharide derivative coated normal-phase chiral chromatographic column or polysaccharide derivative solvent-resistant chiral chromatographic column;
[0013] Mobile phase: a mixture of n-hexane and isopropanol, with n-hexane content of 97%~99%, where the percentage is the volume ratio of n-hexane to the mixture;
[0014] The 1,2-propanediol is (R)-1,2-propanediol, (S)-1,2-propanediol, or a mixture thereof (referring to (R)-1,2-propanediol and (S)-1,2-propanediol).
[0015] In one embodiment, the 1,2-propanediol is a mixture of (R)-1,2-propanediol and (S)-1,2-propanediol.
[0016] In one embodiment, the organic solvent is a non-alcoholic organic solvent, such as a nitrogen-containing aromatic hydrocarbon compound, preferably pyridine.
[0017] In one embodiment, the organic solvent is an alkaline reagent.
[0018] In one embodiment, the derivatization reaction is carried out in the presence of a basic reagent, such as an organic or inorganic base, for example, pyridine.
[0019] In one embodiment, the molar ratio of 1,2-propanediol to benzoyl chloride is 1:(2-4), for example, 1:3.
[0020] In one embodiment, the reaction temperature of the derivatization reaction is 10-30°C, for example, 25°C.
[0021] In one embodiment, the benzoyl chloride is added at a temperature of 0~5℃, for example, 2.5℃.
[0022] In one embodiment, the mass ratio of 1,2-propanediol to the organic solvent is 1:(5-15), for example, 1:10.
[0023] The reaction process is monitored using conventional methods in the art, such as HPLC and TLC. The reaction endpoint is generally defined as the point at which the reactant (e.g., benzoyl chloride) no longer reacts or the product no longer forms. The derivatization reaction time can be 12 hours, for example, overnight.
[0024] In one embodiment, the derivatization reaction further includes the following post-processing steps: after the reaction is completed, separation (e.g., adding an ester solvent (e.g., ethyl acetate) and a water solution), washing (e.g., washing sequentially with hydrochloric acid aqueous solution, sodium bicarbonate aqueous solution and saturated brine), and drying (e.g., rotary evaporation drying).
[0025] In one embodiment, the mass ratio of the ester solvent (e.g., ethyl acetate) to water in the post-processing step can be (5-15):1, for example, 10:1.
[0026] In one embodiment, the mass ratio of ethyl acetate to water in the post-processing step is (8-12):1, for example, 10:1.
[0027] In one embodiment, the reaction preferably includes the following steps: at 10-30°C, 1,2-propanediol and benzoyl chloride reagent undergo the derivatization reaction in the azo aromatic hydrocarbon solvent; after the reaction is completed, the mixture is separated, washed, and dried.
[0028] In one embodiment, the derivatization reaction raw materials are the 1,2-propanediol, the benzoyl chloride reagent, and the solvent.
[0029] In one approach, the product obtained in step (1) is mixed with the mobile phase to obtain a test solution, which is then detected by liquid chromatography. Preferably, the content of the product obtained in step (1) in the test solution is 0.3 μg / mL-0.02 mg / mL, for example, 1 μg / mL-0.02 mg / mL.
[0030] In one embodiment, the particle size of the chromatographic column packing material is 2.5µm to 7.5µm, for example, 5µm.
[0031] In one embodiment, the length of the chromatographic column is 150 mm to 300 mm, for example, 250 mm.
[0032] In one embodiment, the inner diameter of the chromatographic column is 2 mm to 5 mm, for example, 4.6 mm.
[0033] In one embodiment, the liquid chromatography method uses a CHIRALPAK AD-H column, preferably with a size of 250 mm * 4.6 mm and a particle size of 5 μm.
[0034] In one embodiment, the mobile phase contains 98% n-hexane, where the percentage is the volume ratio of n-hexane to the mixture.
[0035] In one embodiment, the flow rate of the mobile phase is 0.4 mL / min to 1.5 mL / min, for example, 0.8 mL / min.
[0036] In one approach, the injection volume of the test solution is 5µL to 20µL, for example, 10µL.
[0037] In one embodiment, the column temperature of the liquid chromatography method is 30℃-40℃, for example, 35℃.
[0038] In one embodiment, the detector for the liquid chromatography method is a UV detector, preferably set to a detection wavelength of 190-400 nm, such as 210 nm.
[0039] In one embodiment, the running time of the liquid chromatography method is 20-40 minutes, for example, 30 minutes.
[0040] The detection method can be performed using a conventional liquid chromatograph, such as an Agilent 1100 liquid chromatograph.
[0041] In one embodiment, the liquid chromatography method is defined under the following conditions: a CHIRALPAK AD-H column with dimensions of 250 mm x 4.6 mm and a diameter of 5 μm; a column temperature of 35°C; and a mobile phase with a volume ratio (V / V). 正己烷 V 异丙醇 The ratio of hexane to isopropanol was 98:2; flow rate: 0.8 mL / min; injection volume: 10 μL; run time: 30 min; detector: UV detector, detection wavelength 210 nm.
[0042] In one embodiment, the method for detecting 1,2-propanediol includes the following steps:
[0043] Step (1): At 0~5℃, in the presence of an alkaline reagent, the 1,2-propanediol and the benzoyl chloride reagent are derivatized. After the reaction is completed, the mixture is separated, washed, and dried.
[0044] Step (2): Separation and detection are performed using liquid chromatography;
[0045] The liquid chromatography method was performed under the following conditions: CHIRALPAK AD-H column, 250 mm * 4.6 mm, 5 μm; column temperature: 35 °C; mobile phase: volume ratio (V / L) 正己烷 V 异丙醇 The sample was prepared as a 98:2 mixture of n-hexane and isopropanol; flow rate: 0.8 mL / min; injection volume: 10 μL; run time: 30 min; detector: UV detector, detection wavelength 210 nm.
[0046] Preferably, the product obtained in step (1) is mixed with the mobile phase to obtain the test solution, and then detected by liquid chromatography.
[0047] In one embodiment, the detection method may be the external standard method, the self-comparison method, or the area normalization method. The external standard method calculates the content of (S)-1,2-propanediol based on peak area. The self-comparison method calculates the content of (S)-1,2-propanediol based on the peak area of (R)-1,2-propanediol. The area normalization method calculates the content of (S)-1,2-propanediol by dividing the peak area of (S)-1,2-propanediol in the chromatogram of the test solution by the sum of the peak areas of (S)-1,2-propanediol and (R)-1,2-propanediol. Both (R)-1,2-propanediol and (S)-1,2-propanediol refer to the substances obtained after reaction (derivation reaction) with benzoyl halide reagent.
[0048] Terminology definition:
[0049] In this invention, the term "active pharmaceutical ingredient" refers to the raw material used in the production of drugs (referring to compounds) and pharmaceutical preparations.
[0050] In this invention, the term "external standard method" refers to a method of quantification by comparing the response signals of (S)-1,2-propanediol in the reference substance and the sample with the pure (S)-1,2-propanediol. Both (R)-1,2-propanediol and (S)-1,2-propanediol refer to the substances obtained after reaction with benzoyl halide reagent (derivation reaction).
[0051] In this invention, the term "self-comparison method" refers to a quantitative method that compares the response signal of the main component in the self-comparison solution with the response signal of (S)-1,2-propanediol in the sample. The (R)-1,2-propanediol and (S)-1,2-propanediol refer to the substances after reacting with benzoyl halide reagent (derivation reaction).
[0052] In this invention, the term "area normalization method" refers to a quantitative method that compares the response signal of (S)-1,2-propanediol in the sample with the sum of the response signals of (S)-1,2-propanediol and (R)-1,2-propanediol in the sample. The (R)-1,2-propanediol and (S)-1,2-propanediol refer to the substances after reacting with benzoyl halide reagents (derivation reaction).
[0053] Without violating common sense in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.
[0054] The reagents and raw materials used in this invention are all commercially available.
[0055] The positive and progressive effects of this invention are as follows: the detection method of this invention realizes the detection and separation of (S)-1,2-propanediol in (R)-1,2-propanediol, with high sensitivity, good separation degree, and good application prospects. Attached Figure Description
[0056] Figure 1 The HPLC chromatograms are of the (R)-1,2-propanediol and (S)-1,2-propanediol positioning chromatograms before derivatization in Example 1.
[0057] Figure 2 The chromatograms of (R)-1,2-propanediol and (S)-1,2-propanediol after derivatization in Example 1 are shown.
[0058] Figure 3 This is the HPLC chromatogram of Example 7.
[0059] Figure 4 This is the HPLC chromatogram of Example 8. Detailed Implementation
[0060] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.
[0061] Example 1
[0062] Sample name: (R)-1,2-propanediol (Source: Bid Pharmaceuticals; Batch number: CPP981; Purity: 99.73% on anhydrous basis)
[0063] Sample name: (S)-1,2-propanediol (Source: Bid Pharmaceuticals; Batch number: DMC044; Purity: 98% on anhydrous basis)
[0064] Sample preparation: Weigh 10 mg each of (R)-1,2-propanediol and (S)-1,2-propanediol, place them in a 10 ml volumetric flask, dilute to the mark with the mobile phase, and shake well to obtain system suitability solution 1.
[0065] Derivatization of 1,2-propanediol: 100 g (10X) of pyridine was added to a 250 mL single-necked flask, followed by 10 g (0.13 mol) of 1,2-propanediol. The mixture was cooled to 0-5 °C, and 55.4 g (0.39 mol) of benzoyl chloride was added dropwise. After the addition was complete, the mixture was stirred overnight at 10-30 °C. 50 g (5X) of ethyl acetate and 5 g (0.5X) of water were added, and the mixture was stirred for 1 hour. The mixture was allowed to stand and separate into layers. The organic phase was washed three times with 50 g (15X) of 1N hydrochloric acid each time, using 50 g (5X) each time. The organic phase was also washed three times with 50 g (15X) of 10% sodium bicarbonate each time, using 50 g (5X) each time. Finally, the organic phase was washed once with 50 g (5X) of saturated brine. The mixture was dried at 40-50 °C and evaporated to dryness to obtain 35 g of the product (molar yield 94.6%).
[0066] Note: Molar yield is calculated based on molar quantity; 10X and 5X represent 10 times and 5 times 10g respectively, referring to the ratio of the amount of feed to the mass of the raw materials.
[0067] The products are: derivatized (R)-1,2-propanediol and derivatized (S)-1,2-propanediol, which are substances (A) and (B) respectively.
[0068]
[0069] Weigh 10 mg each of derivatized (R)-1,2-propanediol and (S)-1,2-propanediol, place them in a 10 mL volumetric flask, add mobile phase to dissolve and dilute to the mark, shake well, and use as system suitability solution 2.
[0070] Chromatographic analysis was performed on system suitability solutions 1 and 2.
[0071] A CHIRALPAK AD-H column (250 mm * 4.6 mm, 5 μm) was used at a column temperature of 35 °C, a detection wavelength of 210 nm, and a UV detector. The chromatograph was an Agilent 1100 HPLC system. The mobile phase was n-hexane-isopropanol (98:2), the flow rate was 0.8 mL / min, the injection volume was 10 μL, and the run time was 30 min.
[0072] Chromatograms of system suitability solutions 1 and 2 (see attached) Figure 1 , Figure 2 As can be seen, (R)-1,2-propanediol and (S)-1,2-propanediol, which have not undergone derivatization, did not elute. After derivatization, (S)-1,2-propanediol and (R)-1,2-propanediol elute sequentially. The retention time of (S)-1,2-propanediol is approximately 15.0 minutes (relative retention time approximately 0.95), and the retention time of (R)-1,2-propanediol is approximately 15.8 minutes. The resolution between the two is not less than 1.5, and the theoretical plate number based on the (S)-1,2-propanediol peak is not less than 10,000.
[0073] Example 1-1
[0074] (1) Limit of quantitation and limit of detection
[0075] 1.1 Solution Preparation
[0076] Accurately measure 1 ml of the system suitability solution 2 into a 10 ml volumetric flask, dilute to the mark with the mobile phase, and mix well. Then accurately measure 1 ml of this solution into a 100 ml volumetric flask, dilute to the mark with the mobile phase, and mix well. This solution is used as the limit of quantitation solution.
[0077] Accurately measure 3 ml of the limit of quantitation solution, place it in a 10 ml volumetric flask, dilute to the mark with the mobile phase, and shake well to obtain the limit of detection solution.
[0078] 1.2 Verification Procedure
[0079] The chromatographic conditions were the same as in Example 1.
[0080] Inject samples in the order shown in the table below:
[0081]
[0082] 1.3 Acceptable Standards
[0083] In the chromatogram of the limit of detection solution, the signal-to-noise ratio of the (S)-1,2-propanediol peak is not less than 3; in the chromatogram of the limit of quantitation solution, the signal-to-noise ratio of the (S)-1,2-propanediol peak is not less than 10, and the relative standard deviation of the peak area of (S)-1,2-propanediol for six consecutive injections is not greater than 10%.
[0084] 1.4 The verification results are shown in the table below:
[0085]
[0086] Note: In the test solution, "-" indicates a repeated batch; for example, "-2" indicates a second injection of the limit of quantitation solution. The unit of peak area in the table above is (mAU*min).
[0087] 1.5 Verification Conclusion
[0088] The limit of quantitation is 1 μg / ml, and the limit of detection is 0.3 μg / ml, which is equivalent to 0.03% of the concentration of the test sample.
[0089] In the chromatogram of the limit of quantitation solution, the signal-to-noise ratio of the (S)-1,2-propanediol peak is 24, and the relative standard deviation of the peak area of (S)-1,2-propanediol for six consecutive injections is 2.3%. In the chromatogram of the limit of detection solution, the signal-to-noise ratio of the (S)-1,2-propanediol peak is 7, which meets the requirements.
[0090] Example 2: Linear Correlation
[0091] 2.1 Solution Preparation
[0092] Accurately measure 1 ml of system suitability solution 2 and place it in a 50 ml volumetric flask. Dilute to the mark with the mobile phase and shake well to obtain linear solution 1.
[0093] Accurately measure 1 ml of system suitability solution 2 and place it in a 100 ml volumetric flask. Dilute to the mark with the mobile phase and shake well to obtain linear solution 2.
[0094] Accurately measure 1.5 ml of the linear solution, place it in a 20 ml volumetric flask, dilute to the mark with the mobile phase, and shake well to obtain linear solution 3.
[0095] Accurately measure 2.5 ml of the linear solution, place it in a 20 ml volumetric flask, dilute to the mark with the mobile phase, and shake well to obtain linear solution 4.
[0096] Accurately measure 1 ml of linear solution 3, place it in a 10 ml volumetric flask, dilute to the mark with the mobile phase, and shake well to obtain linear solution 5.
[0097] 2.2 Verification Procedure
[0098] The chromatographic conditions are the same as in Example 1, and the injection procedure is shown in the table below:
[0099]
[0100] 2.3 Acceptable Standards
[0101] The correlation coefficient r of the linear equation is not less than 0.995, and the ratio of the absolute value of the intercept of the linear equation to the peak area at 100% concentration is not greater than 20%.
[0102] 2.4 The verification results are shown in the table below.
[0103]
[0104] Note: The unit of peak area in the table above is (mAU*min).
[0105] 2.5 Verification Conclusion
[0106] (S)-1,2-propanediol showed a linear correlation between concentration and response signal within the range of 0.0005–0.02 mg / ml. The linear equation was y = 20,186.1957x - 4.5045, with a correlation coefficient r = 0.9996. The absolute value of the intercept relative to the peak area at 100% concentration was 4.67%.
[0107] (R)-1,2-propanediol showed a linear correlation between concentration and response signal within the range of 0.0005–0.02 mg / mL. The linear equation was y = 19,256.8953x - 3.4504, with a correlation coefficient r = 0.9997. The absolute value of the intercept was 3.72% of the peak area at 100% concentration.
[0108] The slope ratio of the linear equations for (R)-1,2-propanediol and (S)-1,2-propanediol is 1.05, and (S)-1,2-propanediol can be determined by self-comparison method.
[0109] Example 3 Repeatability
[0110] 3.1 Solution Preparation
[0111] (S)-1,2-propanediol stock solution: Weigh 10 mg of derivatized (S)-1,2-propanediol, place it in a 200 ml volumetric flask, add mobile phase to dissolve and dilute to the mark, and shake well.
[0112] 100% spiked test solution: Weigh 10 mg of derivatized (R)-1,2-propanediol, place it in a 10 ml volumetric flask, accurately add 1 ml of (S)-1,2-propanediol stock solution, add mobile phase to dissolve and dilute to the mark, shake well, and repeat in 6 parallel applications.
[0113] Self-control solution 1: Accurately measure 1 ml of 100% spiked test solution, place it in a 100 ml volumetric flask, dilute to the mark with the mobile phase, and shake well.
[0114] Unstandardized test solution: Weigh 10 mg of derivatized (R)-1,2-propanediol, place it in a 10 ml volumetric flask, add mobile phase to dissolve and dilute to the mark, shake well, and make 6 parallel portions.
[0115] Self-control solution 2: Accurately measure 1 ml of the unstandardized test solution, place it in a 100 ml volumetric flask, dilute to the mark with the mobile phase, and shake well.
[0116] 3.2 Verification Procedure
[0117] The chromatographic conditions were the same as in Example 1.
[0118] Inject samples in the order shown in the table below:
[0119]
[0120]
[0121] Note: The "-number" in the test solution indicates a repeated batch. For example, "100% spiked test solution-2" means a second injection of the 100% spiked test solution.
[0122] 3.3 Acceptable Standards
[0123] The content of derivatized (S)-1,2-propanediol in the test solution is less than 0.5%, and the relative standard deviation of the content of (S)-1,2-propanediol in 6 samples shall not exceed 10%; the content of derivatized (S)-1,2-propanediol is between 0.5% and 2%, and the relative standard deviation of the content of derivatized (S)-1,2-propanediol in 6 samples shall not exceed 5%.
[0124] 3.4 The verification results are shown in the table below:
[0125]
[0126] Note: The content calculation formula in the table above is: peak area of derivatized (S)-1,2-propanediol or (R)-1,2-propanediol / peak area of the main component of the self-control solution / 100*100%.
[0127] 3.5 Verification Conclusion
[0128] The average content of the derivatized (S)-1,2-propanediol in 6 samples of 100% spiked test specimens was 0.75%, with a relative standard deviation of 0.69%; the average content of the 6 samples of unspecified test specimens was 0.25%, with a relative standard deviation of 3.58%, which met the requirements.
[0129] Example 4 Precision
[0130] 4.1 Solution Preparation
[0131] The test personnel were changed to conduct the test at different times according to the repeatability test requirements.
[0132] 4.2 Verification Procedure
[0133] See item 3.2 under the same repeatability item as in Example 3.
[0134] 4.3 Acceptable Standards
[0135] The content of derivatized (S)-1,2-propanediol in the test solution is less than 0.5%, and the relative standard deviation of the content of derivatized (S)-1,2-propanediol in 12 samples shall not exceed 10%; if the content of derivatized (S)-1,2-propanediol is between 0.5% and 2%, the relative standard deviation of the content of derivatized (S)-1,2-propanediol in 12 samples shall not exceed 5%.
[0136] 4.4 Verification Results
[0137]
[0138] Note: The content calculation formula in the table above is: peak area of derivatized (S)-1,2-propanediol or (R)-1,2-propanediol / peak area of the main component of the self-control solution / 100*100%.
[0139] 4.5 Verification Conclusion
[0140] The relative standard deviation of 12 samples with 100% spiked sample was 0.83%; the relative standard deviation of 12 samples without spiked sample was 3.18%, which meets the requirements.
[0141] The calculation formula is the same as 3.5 in Example 3.
[0142] Example 5 Accuracy
[0143] 5.1 Solution Preparation
[0144] 50% spiked test solution: Weigh 20 mg of derivatized (R)-1,2-propanediol, place it in a 20 ml volumetric flask, accurately add 1 ml of (S)-1,2-propanediol stock solution, add mobile phase to dissolve and dilute to the mark, shake well, and make 3 parallel portions.
[0145] 100% spiked test solution: Weigh 10 mg of derivatized (R)-1,2-propanediol, place it in a 10 ml volumetric flask, accurately add 1 ml of (S)-1,2-propanediol stock solution, add mobile phase to dissolve and dilute to the mark, shake well, and make 3 parallel portions.
[0146] 150% spiked test solution: Weigh 20 mg of derivatized (R)-1,2-propanediol, place it in a 20 ml volumetric flask, accurately add 3 ml of (S)-1,2-propanediol stock solution, add mobile phase to dissolve and dilute to the mark, shake well, and make 3 parallel portions.
[0147] 50% spiked test sample self-control solution: Accurately measure 1 ml of the above 50% spiked test sample solution, place it in a 100 ml volumetric flask, dilute to the mark with the mobile phase, and shake well.
[0148] 100% spiked test sample self-control solution: Accurately measure 1 ml of the above 100% spiked test sample solution, place it in a 100 ml volumetric flask, dilute to the mark with the mobile phase, and shake well.
[0149] 150% spiked test sample self-control solution: Accurately measure 1 ml of the above 150% spiked test sample solution, place it in a 100 ml volumetric flask, dilute to the mark with the mobile phase, and shake well.
[0150] 5.2 Verification Procedure
[0151] The chromatographic conditions were the same as in Example 1.
[0152] Inject samples according to the injection procedure in the table below:
[0153]
[0154] Note: The "-number" in the test solution indicates a repeated batch. For example, "100% spiked test solution-2" means a second injection of the 100% spiked test solution.
[0155] 5.3 Acceptable Standards
[0156] The recovery rate ranges from 92% to 105%, and the relative standard deviation shall not exceed 5%.
[0157] 5.4 Verification Results
[0158]
[0159] 5.5 Verification Conclusion
[0160] The recoveries at all concentrations were in the range of 92% to 105%, with a relative standard deviation of 1.1%, which met the requirements.
[0161] Example 6 Solution Stability
[0162] 6.1 Solution Preparation
[0163] Test solution: Weigh 10 mg of derivatized (R)-1,2-propanediol, place it in a 10 ml volumetric flask, add mobile phase to dissolve and dilute to the mark, and shake well.
[0164] Self-control solution: Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute to the mark with the mobile phase, and shake well.
[0165] 6.2 Verification Procedure
[0166] The chromatographic conditions were the same as in Example 1.
[0167]
[0168] 6.3 Acceptable Standards
[0169] In the chromatograms of the self-reference solution at each time point, the ratio of the peak area of the main component (R)-1,2-propanediol to the peak area at 0 hours should be between 95% and 105%. In the chromatograms of the test solution, the ratio of the peak area of (R)-1,2-propanediol to the peak area at 0 hours should be between 90% and 110%.
[0170] 6.4 Verification Results
[0171]
[0172] Note: The unit of peak area in the table above is (mAU*min).
[0173] 6.5 Verification Conclusion
[0174] The calculation method in the table above is as follows: the ratio of the peak area of the main component (R)-1,2-propanediol at each time point to the peak area at 0 hours in the self-control solution is between 95% and 105%, and the ratio of the peak area of (R)-1,2-propanediol at each time point to 0 hours in the test solution is between 90% and 110%, which meets the requirements and the solution is stable.
[0175] in conclusion:
[0176] The method for detecting derivatized (R)-1,2-propanediol provided by this invention can accurately quantify the content of (S)-1,2-propanediol in active pharmaceutical ingredients. This method has high accuracy and good precision.
[0177] Example 7
[0178] The chromatographic column used was a CHIRALPAK AD-H column (250 mm * 4.6 mm, 5 μm), the column temperature was 35 °C, the detection wavelength was 210 nm, and a UV detector was used.
[0179] The mobile phase was n-hexane-isopropanol (90:10), the flow rate was 0.8 ml / min, the injection volume was 10 μL, and the sample to be tested was system suitability solution 2.
[0180] Conclusion: The samples show a separation trend, with good peak shape and high response, but the resolution is insufficient (see appendix). Figure 3 ).
[0181] Example 8
[0182] The chromatographic column used was a CHIRALPAK AD-H column (250 mm * 4.6 mm, 5 μm), the column temperature was 35 °C, the detection wavelength was 210 nm, and a UV detector was used.
[0183] The mobile phase was n-hexane-isopropanol (95:5), the flow rate was 0.8 ml / min, the injection volume was 10 μL, and the sample to be tested was system suitability solution 2.
[0184] Conclusion: Sample separation increased, peak shape was good, response was high, but resolution was insufficient (see appendix). Figure 4 ).
Claims
1. A method for detecting 1,2-propanediol, characterized by, It comprises the following steps: Step (1): derivatization of 1,2-propanediol with benzoyl chloride in an organic solvent; Step (2): separation and detection by liquid chromatography; The chromatographic column is a polysaccharide derivative-coated normal-phase chiral chromatographic column or a polysaccharide derivative solvent-tolerant chiral chromatographic column; The mobile phase is a mixture of n-hexane and isopropyl alcohol, with the content of n-hexane being 97%-99%, wherein the percentage is the volume ratio of n-hexane to the mixture; The 1,2-propanediol is (R)-1,2-propanediol, (S)-1,2-propanediol, or a mixture thereof.
2. The detection method of claim 1, wherein, It meets one or more of the following conditions: (1) The organic solvent is a non-alcohol organic solvent; (2) The derivatization reaction is carried out in the presence of a basic reagent; (3) The molar ratio of 1,2-propanediol to benzoyl chloride is 1:(2-4); (4) The reaction temperature of the derivatization reaction is 10-30°C; (5) The addition temperature of benzoyl chloride is 0-5°C; (6) The mass ratio of 1,2-propanediol to the organic solvent is 1:(5-15); (7) The product obtained in step (1) is mixed with the mobile phase to obtain a test solution, which is then detected by liquid chromatography; (8) The raw materials for the derivatization reaction are the 1,2-propanediol, the benzoyl chloride reagent, and the solvent; and (9) The 1,2-propanediol is a mixture of (R)-1,2-propanediol and (S)-1,2-propanediol.
3. The detection method of claim 2, wherein, It meets one or more of the following conditions: (1) The organic solvent is an azaheteroaromatic compound, preferably pyridine; (2) The basic reagent is an organic base or an inorganic base, preferably the solvent is a basic reagent; (3) The molar ratio of 1,2-propanediol to benzoyl chloride is 1:3; (4) The reaction temperature of the derivatization reaction is 25°C; (5) The addition temperature of benzoyl chloride is 2.5°C; (6) The mass ratio of 1,2-propanediol to the organic solvent is 1:10; and (7) In the test solution, the content of the product obtained in step (1) is 0.3 µg / mL-0.02 mg / mL, for example, 1 µg / mL-0.02 mg / mL.
4. The detection method as described in claim 1, characterized in that, The derivatization reaction further comprises the following post-treatment steps: adding an ester solvent and water to separate, washing, and drying; Preferably, the post-treatment steps meet one or more of the following conditions: (1) The ester solvent is ethyl acetate; (2) The washing is sequential washing with hydrochloric acid aqueous solution, sodium bicarbonate aqueous solution, and saturated brine; (3) The drying is rotary evaporation drying; and (4) The mass ratio of the ester solvent to water is (5-15):1, for example, 10:
1.
5. The detection method according to claim 1 or 3, wherein The derivatization reaction comprises the following steps: 1,2-propanediol and benzoyl chloride reagent are subjected to the derivatization reaction in an azaheteroaromatic solvent at 10-30°C, after the reaction is completed, separation, washing, and drying are performed.
6. The method of claim 1, wherein, It meets one or more of the following conditions: (1) The particle size of the packing material of the chromatographic column is 2.5 µm-7.5 µm; (2) The length of the chromatographic column is 150 mm-300 mm; (3) the inner diameter of the chromatographic column is 2 mm-5 mm; (4) the liquid chromatography adopts a CHIRALPAK AD-H chromatographic column; (5) the detector of the liquid chromatography is a UV detector; and (6) the liquid chromatography adopts an Agilent 1100 liquid chromatograph.
7. The detection method of claim 6, wherein, One or more of the following conditions are met: (1) the particle size of the packing of the chromatographic column is 5 µm; (2) the length of the chromatographic column is 250 mm; (3) the inner diameter of the chromatographic column is 4.6 mm; and (4) the detection wavelength of the detector is 190-400 nm.
8. The assay of claim 1, 3 or 6 wherein, One or more of the following conditions are met: (1) the flow rate of the mobile phase is 0.4 mL / min-1.5 mL / min; (2) the injection volume of the sample to be measured is 5-20 µL; (3) the column temperature of the chromatographic column is 30℃-40℃; (4) the detection wavelength of the detector is 210 nm; and (5) the running time of the liquid chromatography is 20-40 min.
9. The detection method of claim 8, wherein, One or more of the following conditions are met: (1) the flow rate of the mobile phase is 0.8 mL / min; (2) the injection volume of the sample to be measured is 10 µL; (3) the column temperature of the chromatographic column is 35℃; (4) the liquid chromatography adopts an external standard method, a self-control method or an area normalization method to measure the component content; (5) the running time of the liquid chromatography is 30 min; and (6) in the mobile phase, the content of n-hexane is 98%, wherein the percentage is the volume ratio of n-hexane to the mixed liquid.
10. The method of claim 1, wherein, The liquid chromatography conditions are: a CHIRALPAK AD-H chromatographic column, a chromatographic column specification of 250 mm*4.6 mm, 5 µm; a column temperature of 35℃; a mobile phase of a n-hexane isopropyl alcohol mixed liquid with a volume ratio of 98:2; a flow rate of 0.8 mL / min; an injection volume of 10 µL; and a running time of 30 min; a detector: a UV detector, a detection wavelength of 210 nm; Preferably, the detection method comprises the following steps: Step (1): under 10-30℃, in the presence of an alkaline reagent, the 1,2-propanediol is subjected to a derivatization reaction with the benzoyl chloride reagent, after the reaction is completed, the liquid is separated, washed and dried; Step (2): liquid chromatography is used for separation and detection; The liquid chromatography conditions are: a CHIRALPAK AD-H chromatographic column, a chromatographic column specification of 250 mm*4.6 mm, 5 µm; a column temperature of 35℃; a mobile phase of a n-hexane isopropyl alcohol mixed liquid with a volume ratio of 98:2; a flow rate of 0.8 mL / min; an injection volume of 10 µL; and a running time of 30 min; a detector: a UV detector, a detection wavelength of 210 nm; Further preferably, the product obtained in step (1) is mixed with the mobile phase to obtain a sample to be measured, which is then detected by liquid chromatography.