A method for simultaneously detecting p-hydroxyphenylhydantoin and impurities
By using a ShimNex HE C18-Ph column and a methanol-perchloric acid aqueous solution gradient elution system, the problem of separating and quantitatively detecting the target product and various impurities in the synthesis reaction solution of p-hydroxyphenylhydantoin was solved, and the monitoring of the reaction process and the purification separation were improved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- QINGDAO UNIV OF SCI & TECH
- Filing Date
- 2026-05-07
- Publication Date
- 2026-07-17
AI Technical Summary
Existing technologies make it difficult to simultaneously and effectively separate and quantitatively detect the target product and multiple structurally similar impurities in the reaction solution for the synthesis of p-hydroxyphenylhydantoin, leading to difficulties in monitoring the reaction process and an increased risk of impurities being introduced into downstream products.
A method for the simultaneous detection of p-hydroxyphenylhydantoin and its impurities 1-6 was developed using a ShimNex HE C18-Ph column and a methanol-perchloric acid aqueous solution gradient elution system combined with high performance liquid chromatography. The gradient elution program and appropriate detection wavelength were used to achieve effective separation and quantitative analysis of each component.
This method enables the simultaneous separation and quantification of the target product and major impurities in the reaction solution for the synthesis of p-hydroxyphenylhydantoin, improving the accuracy of reaction process monitoring and the efficiency of subsequent purification and separation, and reducing the impact of impurities on the quality of downstream products.
Smart Images
Figure CN122409930A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of analytical testing technology, and specifically to a method for simultaneously detecting p-hydroxyphenylhydantoin and impurities. Background Technology
[0002] p-Hydroxyphenylhydantoin is an important intermediate in the synthesis of D-p-hydroxyphenylglycine, which is an important pharmaceutical intermediate widely used in the synthesis of the side chain of β-lactam semi-synthetic antibiotics such as amoxicillin and cephalosporins. Currently, p-hydroxyphenylhydantoin is mainly prepared by condensation and further cyclization of glyoxylic acid, phenol, and urea under acidic conditions. In this reaction, glyoxylic acid first forms an intermediate with urea, which then undergoes a substitution reaction with phenol to generate the corresponding precursor and finally cyclize to obtain p-hydroxyphenylhydantoin. However, since multiple active components exist in the reaction system, and the ratio of raw materials, acidity, temperature, and crystallization process all affect the reaction pathway, in addition to the target product, ortho-substituted byproducts, polymeric impurities formed by further condensation of phenol or intermediates, and byproducts formed by backmixing of the target product and intermediates are easily generated, resulting in a complex reaction solution composition and increasing the difficulty of subsequent separation and purification. Therefore, in the process development and production control, simultaneously detecting the content of p-hydroxyphenylhydantoin and various major impurities in the reaction solution not only helps to accurately evaluate the reaction yield and selectivity, but also enables timely judgment of the degree of side reactions, analysis of process progress, and provides important basis for optimization of reaction conditions, central control analysis, and subsequent purification and separation.
[0003] Currently, analytical methods for p-hydroxyphenylhydantoin include spectrophotometry and high-performance liquid chromatography (HPLC). The literature "Study on the Determination of p-hydroxyphenylhydantoin by Reversed-Phase HPLC" (Zhu Zhijia, Vol. 41, 2005, Physical and Chemical Testing - Chemistry Section) reports a reversed-phase HPLC method for the determination of p-hydroxyphenylhydantoin. This method is mainly for the determination of the content of p-hydroxyphenylhydantoin itself and can achieve the analysis of p-hydroxyphenylhydantoin in product samples. However, this literature does not cover the simultaneous detection of the target product and various impurities in the p-hydroxyphenylhydantoin synthesis reaction solution, thus failing to meet the needs of reaction process monitoring, impurity formation law research, and process control analysis. In particular, byproducts with similar structures and polarities contained in the p-hydroxyphenylhydantoin synthesis reaction solution are difficult to separate effectively under existing chromatographic conditions, easily causing peak overlap or insufficient resolution, thus making it difficult to accurately measure the content of p-hydroxyphenylhydantoin. Furthermore, impurities may be carried downstream with the product during subsequent purification, thereby affecting the quality and performance of downstream products.
[0004] Currently, there is a lack of analytical methods for the synthesis reaction solution of p-hydroxybenzylhydantoin that can simultaneously and accurately separate and quantify the target product from multiple major impurities. Given the complexity of the reaction system and the variable composition of impurities, developing a detection method suitable for this reaction system is crucial for accurately monitoring the reaction process, revealing the patterns of impurity formation, guiding process optimization, and improving subsequent separation and purification efficiency. Therefore, establishing an analytical method suitable for the p-hydroxybenzylhydantoin reaction solution that can simultaneously determine the content of the target product and major impurities has significant practical application value. Summary of the Invention
[0005] To address the aforementioned limitations of existing technologies, the purpose of this invention is to provide a method for the simultaneous detection of p-hydroxyphenylhydantoin and impurities. This invention develops a high-performance liquid chromatography (HPLC) method suitable for the synthesis reaction solution of p-hydroxyphenylhydantoin. Impurities with similar structures and polarities to p-hydroxyphenylhydantoin exhibit overlapping and mutual interference in ultraviolet absorption, thus establishing an analytical method capable of simultaneously detecting the product and multiple major impurities in the reaction solution. This method is of great significance for accurately monitoring the reaction process, revealing the patterns of impurity formation, guiding process optimization, and improving the efficiency of subsequent separation and purification.
[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for simultaneously detecting p-hydroxyphenylhydantoin and impurities, the method comprising: A mixed standard solution was prepared by taking the standard of p-hydroxyphenylhydantoin and the standards of impurities 1, 2, 3, 4, 5, and 6. A sample solution was prepared by taking the p-hydroxyphenylhydantoin synthesis reaction solution. The mixed standard solution and the sample solution were detected by high performance liquid chromatography using a gradient elution program. The peak areas of the standard and the sample were obtained, and the contents of p-hydroxyphenylhydantoin and its impurities 1 to 6 in the p-hydroxyphenylhydantoin synthesis reaction solution were calculated. The structural formula of impurity 1 is: ; The structural formula of impurity 2 is: ; The structural formula of impurity 3 is: ; The structural formula of impurity 4 is: ; The structural formula of impurity 5 is: ; The structural formula of impurity 6 is: ; The high-performance liquid chromatography column is a ShimNex HE C18-Ph column; the mobile phase is perchloric acid aqueous solution and methanol; the concentration of the perchloric acid aqueous solution is 0.04M~0.10M.
[0007] Preferably, the mixed standard solution is obtained by dissolving the standard of p-hydroxyphenylhydantoin and the standards of impurities 1 to 6 in a solvent and filtering them through a membrane; the solvent is a mixture of methanol and water at a volume ratio of 20:80; and the filter membrane is a 0.45 μm filter membrane.
[0008] Preferably, the sample solution is obtained by dissolving the p-hydroxyphenylhydantoin synthesis reaction solution in a solvent and filtering it through a membrane; the solvent is a mixture of methanol and water at a volume ratio of 20:80; and the filter membrane is a 0.45 μm filter membrane.
[0009] Preferably, the standard of impurity 1 is prepared by the following method: p-Hydroxybenzane, urea, and potassium carbonate were added to a solvent, stirred thoroughly, and heated. Glyoxylic acid was added dropwise, and the reaction was continued at this temperature. After the reaction was completed, the solvent was removed by vacuum concentration, pure water was added, and the mixture was stirred at room temperature. The mixture was then filtered, and the filter cake was washed three times with water. The filter cake was collected and dried. The dried filter cake was purified to obtain the standard of impurity 1.
[0010] Preferably, the solvent is DMF or DMSO; the molar ratio of p-hydroxyphenylhydantoin, urea, potassium carbonate, glyoxylic acid and solvent is 1:1:(0.5-0.7):(1-3):(5-35).
[0011] Preferably, the heating temperature is 90–120°C; the heat preservation reaction time is 2 hours; and the glyoxylic acid is added dropwise over a period of 0.5–2 hours.
[0012] Preferably, the crude product purified to impurity 1 is subjected to gradient elution with dichloromethane / methanol / glacial acetic acid, each eluent fraction is collected, the fraction containing impurity 1 is combined, and the fraction is concentrated under reduced pressure to obtain pure impurity 1.
[0013] Preferably, the calculation formula is as follows: Where: wi - mass fraction of p-hydroxyphenylhydantoin and impurities 1-6 in the sample, in %; Ai - peak area of the sample; As - peak area of the standard; Ci - mass of the sample, in g; Cs - mass of the standard, in g; Ps - purity of the standard, in %.
[0014] Preferably, the concentration of p-hydroxyphenylhydantoin standard in the mixed standard solution is 1.0 mg / mL to 4.0 mg / mL, the concentration of impurity 1 standard is 0.001 mg / mL to 0.01 mg / mL, the concentration of impurity 2 standard is 0.001 mg / mL to 0.01 mg / mL, the concentration of impurity 3 standard is 0.2 mg / mL to 0.6 mg / mL, the concentration of impurity 4 standard is 0.001 mg / mL to 0.01 mg / mL, the concentration of impurity 5 standard is 0.001 mg / mL to 0.01 mg / mL, and the concentration of impurity 6 standard is 0.001 mg / mL to 0.01 mg / mL.
[0015] Preferably, the detection conditions for the high-performance liquid chromatography include: Mobile phase A is a 0.04M~0.10M aqueous perchloric acid solution, and mobile phase B is methanol; During gradient elution, from 0 to 5 min, A was 87%–95% and B was 5%–13%; Over 5-15 minutes, A gradually decreased to 35%-45%, while B increased to 55%-65%. Over 15-18 minutes, the percentage of A decreased from 35%-45% to 20%-25%, while the percentage of B increased from 55%-65% to 75%-85%. 18~23 min, A is 20%~25%, B is 75%~85%; 23.01~30 min to restore initial ratio and achieve equilibrium; Flow rate: 0.8–1.2 mL / min; Detection wavelength: 254~275 nm; Column temperature 30~40℃; Injection volume: 5~20 μL.
[0016] A second aspect of the present invention provides the application of the above-described method in monitoring the reaction process of p-hydroxyphenylhydantoin or improving the detection accuracy of p-hydroxyphenylhydantoin.
[0017] The beneficial effects of this invention are: (1) The present invention provides a method for simultaneous detection of p-hydroxyphenylhydantoin synthesis reaction solution, which can simultaneously separate and determine p-hydroxyphenylhydantoin and impurities 1 to 6 in the reaction solution, thus overcoming the shortcomings of the prior art which can only analyze p-hydroxyphenylhydantoin finished product and is difficult to simultaneously detect target product and multiple impurities in reaction solution.
[0018] (2) The present invention uses a ShimNex HE C18-Ph column and combines it with a methanol-perchloric acid aqueous solution gradient elution system, which can effectively improve the separation of each component, especially the separation between p-hydroxyphenylhydantoin and impurity 1.
[0019] (3) The detection method of the present invention has good practicality and application value. On the one hand, it can be used to track the reaction process of hydroxyphenylhydantoin synthesis, evaluate the yield and selectivity, and optimize the process parameters. On the other hand, it can accurately monitor impurities, especially impurities 1 that are easily carried downstream with the product, to provide a basis for subsequent purification and separation, and reduce the adverse effects of impurities on the quality and performance of downstream products.
[0020] (4) The present invention establishes a method for preparing impurity 1 standard, which provides a standard material basis for the qualitative and quantitative analysis of impurities and the verification of method accuracy, and further improves the reliability of the detection method. Attached Figure Description
[0021] Figure 1 Chromatogram of the standard solution in Example 2; Figure 2 Chromatogram of the standard solution in Example 3; Figure 3 Chromatogram of the standard solution in Example 4; Figure 4 Comparative Example 1: Chromatogram of the standard. Figure 5 Chromatogram of the standard in Comparative Example 2; Figure 6 Impurity 1 HPLC chromatogram.
[0022] Figure 7 : NMR spectrum of impurity 1 hydrogen. Detailed Implementation
[0023] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.
[0024] As described in the background section, the byproducts in the p-hydroxyphenylhydantoin synthesis reaction solution, which have similar structures and polarities, are difficult to separate effectively under existing chromatographic conditions. This can easily lead to peak overlap or insufficient resolution, making it difficult to accurately measure the p-hydroxyphenylhydantoin content. Furthermore, these impurities may be carried downstream with the product during subsequent purification, thus affecting the quality and performance of downstream products.
[0025] Based on this, the purpose of this invention is to provide a method for the simultaneous detection of p-hydroxyphenylhydantoin and its impurities. Through research, this invention has found that using methanol and water as solvents, and employing high-performance liquid chromatography (HPLC) with a ShimNex HE C18-Ph column and acidic elution, can effectively detect p-hydroxyphenylhydantoin and its impurities 1-6 (structural formulas shown in Table 1).
[0026] Table 1 Components of p-hydroxyphenylhydantoin reaction solution Compared with conventional chromatographic columns, this method offers superior separation and peak shape. When using the single-point external standard method, it effectively controls the consistency of analyte content in the standard and sample solutions, reducing integration errors and resulting in better spike recovery and higher method accuracy. Furthermore, the method of this invention has a shorter analysis time and can be applied to the controlled analysis of the p-hydroxyphenylhydantoin synthesis process, achieving rapid and accurate analysis.
[0027] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.
[0028] The test materials used in the embodiments of this invention are all conventional test materials in the art and can be purchased through commercial channels.
[0029] Example 1: Preparation of Impurity 1 Standard In a reactor equipped with a stirrer, thermometer, and dropping device, 192 g of p-hydroxyphenylhydantoin, 60 g of urea, 76 g of potassium carbonate, and 570 ml of dimethyl sulfoxide were added. Stirring was initiated to ensure thorough mixing of all components. The temperature was then raised to 110 °C. While maintaining stirring, 296.16 g of a 50 wt% glyoxylic acid aqueous solution was added dropwise to the reaction system at a constant rate over a period of 1.5 h. After the addition was complete, the reaction was maintained at 110 °C for another 2 h. After the reaction was complete, the solvent was removed by vacuum concentration. An appropriate amount of purified water was added to the concentrated system, and the mixture was stirred at room temperature for 30 min. The mixture was then filtered, and the resulting filter cake was washed three times with purified water. The filter cake was collected and dried under vacuum to obtain the crude product.
[0030] Dissolve 5 g of the crude product in 50 mL of a dichloromethane / methanol mixed solvent (dichloromethane to methanol volume ratio 95:5), add an appropriate amount of silica gel, stir, remove the solvent under reduced pressure, and then load the sample dry. Pack a 200–300 mesh silica gel column and perform gradient elution with dichloromethane / methanol / glacial acetic acid as eluents. The eluent volume ratios are 100:3:0.2, 100:5:0.5, and 100:8:0.5, used for the initial, intermediate, and final elution phases, respectively (the volume ratio of eluent used in the initial, intermediate, and final phases is 7:9:4). Collect each eluent fraction and analyze by thin-layer chromatography. Combine the fractions containing impurity 1, concentrate under reduced pressure, and obtain the white impurity 1 standard. 1 H NMR (400MHz, DMSO-d6)δ10.74 (s, 1H), 9.75 (s, 1H), 8.59 (s, 1H), 8.23 (s, 1H), 7.15-7.13 (m, 2H), 6.84-6.77 (m, 2H), 5.44 (s, 1H). MS (ESI - m / z: 289 [MH] - The system name is: 5-(4-hydroxyphenyl)-[1,4'-biimidazolidine]-2,2',4,5'-tetraone, with a molecular weight of 290. In the HPLC elution method, impurity 1 had a retention time of 4.62 min and an area-normalized purity of 99.32%. The largest single impurity had a retention time of 5.16 min and an area-normalized purity of 0.59%. The detection wavelength was 275 nm.
[0031] Example 2: Detection of p-hydroxyphenylhydantoin and its impurities A Shimadzu LC 20AT high-performance liquid chromatograph and a Shimadzu ShimNex HE C18-Ph column (250mm×4.6mm, 5μm) were used.
[0032] Mobile phase A: water (with 3 mL of 12 mol / L perchloric acid added to every 500 mL of ultrapure water); mobile phase B: methanol. The gradient elution program is shown in Table 2.
[0033] Table 2 Gradient elution program Flow rate: 1.0 mL / min; Elution mode: gradient elution; Detector: Ultraviolet detector; Detection wavelength: 275 nm; Column temperature: 35℃; Injection volume: 10 μL.
[0034] Weigh the corresponding standards of p-hydroxyphenylhydantoin and impurity 3 into a 100 mL volumetric flask according to Table 3. Weigh the corresponding standards of impurities 1, 2, 4, 5 and 6 into another 100 mL volumetric flask. Dissolve and dilute to volume with a solvent (methanol and water at a volume ratio of 20:80) to prepare a mixed impurity stock solution. Transfer 1 mL of the mixed impurity stock solution to the volumetric flask containing p-hydroxyphenylhydantoin and impurity 3. Dissolve and dilute to volume with a solvent (methanol and water at a volume ratio of 20:80). Shake well and filter through a 0.45 μm filter membrane to prepare the mixed standard solution in Table 3.
[0035] Table 3 Sample weighing and dilution concentration Weigh 0.2165 g of the p-hydroxyphenylhydantoin synthesis reaction solution, dissolve it in solvent and bring the volume to 100 mL, shake well, filter through a 0.45 μm filter membrane, and prepare the sample solution.
[0036] Under the chromatographic conditions described above, after the instrument baseline stabilized, inject 10 μL of the mixed standard solution and the sample solution into the chromatograph, and record the chromatograms of the seven components. The standard chromatogram is shown below. Figure 1 .
[0037] Figure 1 The retention times of impurities 1, p-hydroxyphenylhydantoin, 2, 3, 4, 5, and 6 were 4.62 min, 5.16 min, 6.23 min, 7.58 min, 8.52 min, 10.91 min, and 18.92 min, respectively, with resolutions greater than 2, meeting the baseline separation requirements.
[0038] According to the calculation formula, the content of p-hydroxyphenylhydantoin in the p-hydroxyphenylhydantoin synthesis reaction solution is 80.23%, the content of impurity 1 is 0.30%, the content of impurity 2 is 0.34%, the content of impurity 3 is 16.13%, the content of impurity 4 is 0.32%, the content of impurity 5 is 0.36%, and the content of impurity 6 is 0.30%.
[0039] Example 3 A Shimadzu LC 20AT high-performance liquid chromatograph and a Shimadzu ShimNex HE C18-Ph column (250mm×4.6mm, 5μm) were used.
[0040] Mobile phase A: water (with 2 mL of 12 mol / L perchloric acid added to every 500 mL of ultrapure water), mobile phase B: methanol. The gradient elution program is shown in Table 4.
[0041] Table 4 Gradient elution program Flow rate: 1.2 mL / min; Elution mode: gradient elution; Detector: Ultraviolet detector; Detection wavelength: 254 nm; Column temperature: 30℃; Injection volume: 5 μL.
[0042] According to Table 5, weigh the standard corresponding to p-hydroxyphenylhydantoin and impurity 3 into a 100 mL volumetric flask. Weigh the standard corresponding to impurity 1, impurity 2, impurity 4, impurity 5 and impurity 6 into another 100 mL volumetric flask. Dissolve and dilute to volume with solvent (methanol and water at a volume ratio of 20:80) to prepare a mixed impurity stock solution. Then transfer 1 mL of the mixed impurity stock solution into the volumetric flask containing p-hydroxyphenylhydantoin and impurity 3. Dissolve and dilute to volume with solvent (methanol and water at a volume ratio of 20:80), shake well, and filter through a 0.45 μm filter membrane to prepare the mixed standard solution in Table 5.
[0043] Table 5 Sample weighing and dilution concentration Weigh 0.4257 g of p-hydroxyphenylhydantoin synthesis reaction solution (prepared at a different time from the p-hydroxyphenylhydantoin synthesis reaction solution in Example 2), dissolve in solvent and bring to a final volume of 100 mL, shake well, filter through a 0.45 μm filter membrane, and prepare the sample solution.
[0044] Figure 2 The retention times of impurities 1, p-hydroxyphenylhydantoin, 2, 3, 4, 5 and 6 were 4.33 min, 4.86 min, 6.05 min, 6.76 min, 7.72 min, 8.56 min and 17.81 min, respectively, with resolution greater than 2, meeting the baseline separation requirements.
[0045] According to the calculation formula, the content of p-hydroxyphenylhydantoin in the p-hydroxyphenylhydantoin synthesis reaction solution is 71.42%, the content of impurity 1 is 0.11%, the content of impurity 2 is 0.13%, the content of impurity 3 is 11.98%, the content of impurity 4 is 0.15%, the content of impurity 5 is 0.10%, and the content of impurity 6 is 0.13%.
[0046] Example 4 A Shimadzu LC 20AT high-performance liquid chromatograph and a Shimadzu ShimNex HE C18-Ph column (250mm×4.6mm, 5μm) were used.
[0047] Mobile phase A: water (with 4 mL of 12 mol / L perchloric acid added to every 500 mL of ultrapure water); mobile phase B: methanol. The gradient elution program is shown in Table 6.
[0048] Table 6 Gradient elution program Flow rate: 0.8 mL / min; Elution mode: gradient elution; Detector: Ultraviolet detector Detection wavelength: 260 nm; Column temperature: 40℃; Injection volume: 20 μL.
[0049] Weigh the corresponding standards of p-hydroxyphenylhydantoin and impurity 3 into a 100 mL volumetric flask according to Table 7. Weigh the corresponding standards of impurities 1, 2, 4, 5 and 6 into another 100 mL volumetric flask. Dissolve and dilute to volume with a solvent (methanol and water at a volume ratio of 20:80) to prepare a mixed impurity stock solution. Transfer 1 mL of the mixed impurity stock solution to the volumetric flask containing p-hydroxyphenylhydantoin and impurity 3. Dissolve and dilute to volume with a solvent (methanol and water at a volume ratio of 20:80). Shake well and filter through a 0.45 μm filter membrane to prepare the mixed standard solution in Table 7.
[0050] Table 7 Sample weighing and dilution concentration Figure 3 The retention times of impurities 1, p-hydroxyphenylhydantoin, 2, 3, 4, 5 and 6 were 5.61 min, 6.87 min, 7.81 min, 8.75 min, 9.83 min, 11.83 min and 19.62 min, respectively, with a resolution greater than 2, meeting the baseline separation requirements.
[0051] Weigh out 0.2216 g, 0.2196 g, 0.2279 g, 0.2244 g, 0.2303 g, 0.2268 g and 0.2215 g of the p-hydroxyphenylhydantoin synthesis reaction solution (from the same batch), dissolve and dilute to 100 mL with solvent, shake well, filter through a 0.45 μm filter membrane, and prepare sample solutions.
[0052] Table 8 Content of p-hydroxyphenylhydantoin and impurities 1-6 in the reaction solution Assuming the content of p-hydroxyphenylhydantoin in the reaction solution is 80.09%, the content of impurity 1 is 0.30%, the content of impurity 2 is 0.32%, the content of impurity 3 is 15.01%, the content of impurity 4 is 0.25%, the content of impurity 5 is 0.28%, and the content of impurity 6 is 0.36%, approximately 0.15 g of the reaction solution was weighed. The standard solutions of p-hydroxyphenylhydantoin and impurities 1-6 were added to the weighed reaction solution at 80%, 100%, and 120% of the content of the seven components in the sample, respectively. The spiked recovery rate was determined, and the results are shown in Table 9.
[0053] Table 9 Spike Recovery Rate As shown in the table above, the spiked recoveries of p-hydroxyphenylhydantoin ranged from 98.67% to 101.32%, impurity 1 from 96.03% to 102.14%, impurity 2 from 96.50% to 105.89%, impurity 3 from 98.28% to 103.78%, impurity 4 from 95.16% to 104.51%, impurity 5 from 95.57% to 104.25%, and impurity 6 from 96.04% to 104.65%. These recoveries meet the general sample determination range of 90% to 110%, indicating that the method yields accurate and reliable results.
[0054] Comparative Example 1 The difference from Example 2 is that the Shimadzu LC 20AT high-performance liquid chromatograph and Shimadzu ShimNexHE C18-Ph column (250mm × 4.6mm, 5μm) were replaced with a Shimadzu LC 20AT high-performance liquid chromatograph and a Midspect Red C18 (250mm × 4.6mm, 5μm) column. The chromatogram of the standard is shown below. Figure 4 .
[0055] Figure 4 In the case of impurity 1 and p-hydroxyphenylhydantoin, the C18 column cannot separate the seven components simultaneously, and under the same chromatographic conditions, the impurity 1 cannot be separated from the p-hydroxyphenylhydantoin at the baseline.
[0056] Comparative Example 2 The difference from Example 2 is that a Shimadzu LC 20AT high-performance liquid chromatograph and a Shimadzu ShimNexHE C18-Ph column (250mm × 4.6mm, 5μm) will be used; the mobile phase will be acetonitrile-water-tetrahydrofuran-glacial acetic acid = 70:30:0.5:0.5 (volume ratio). The chromatogram of the standard is shown below. Figure 5 .
[0057] Figure 5 In the chromatogram, due to the high polarity and poor separation of impurity 1 and p-hydroxyphenylhydantoin, the retention times of the two chromatographic peaks completely overlapped. Except for impurity 6, the retention times of other impurities were relatively early and the separation was poor.
[0058] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.
Claims
1. A method for simultaneously detecting p-hydroxyphenylhydantoin and impurities, characterized in that, The method is as follows: A mixed standard solution was prepared by taking the standard of p-hydroxyphenylhydantoin and the standards of impurities 1, 2, 3, 4, 5, and 6. A sample solution was prepared by taking the p-hydroxyphenylhydantoin synthesis reaction solution. The mixed standard solution and the sample solution were detected by high performance liquid chromatography using a gradient elution program. The peak areas of the standard and the sample were obtained, and the contents of p-hydroxyphenylhydantoin and its impurities 1 to 6 in the p-hydroxyphenylhydantoin synthesis reaction solution were calculated. The structural formula of impurity 1 is: ; The structural formula of impurity 2 is: The structural formula of impurity 3 is: ; The structural formula of impurity 4 is: ; The structural formula of impurity 5 is: ; The structural formula of impurity 6 is: ; The high-performance liquid chromatography column used is a ShimNex HE C18-Ph column; The mobile phase is an aqueous solution of perchloric acid and methanol; the concentration of the aqueous solution of perchloric acid is 0.04M~0.10M.
2. The method according to claim 1, characterized in that, The mixed standard solution is obtained by dissolving the standard of p-hydroxyphenylhydantoin and the standards of impurities 1 to 6 in a solvent and filtering them through a membrane; the solvent is a mixture of methanol and water at a volume ratio of 20:80; the filter membrane is a 0.45 μm filter membrane.
3. The method according to claim 1, characterized in that, The sample solution was obtained by dissolving the p-hydroxyphenylhydantoin synthesis reaction solution in a solvent and filtering it through a membrane; the solvent was a mixture of methanol and water at a volume ratio of 20:80; and the filter membrane was a 0.45 μm filter membrane.
4. The method according to claim 1, characterized in that, The standard for impurity 1 is prepared by the following method: p-Hydroxybenzane, urea, and potassium carbonate were added to a solvent, stirred thoroughly, and heated. Glyoxylic acid was added dropwise, and the reaction was continued at this temperature. After the reaction was completed, the solvent was removed by vacuum concentration, pure water was added, and the mixture was stirred at room temperature. The mixture was then filtered, and the filter cake was washed three times with water. The filter cake was collected and dried. The dried filter cake was purified to obtain the standard of impurity 1.
5. The method according to claim 4, characterized in that, The solvent is DMF or DMSO; the molar ratio of p-hydroxyphenylhydantoin, urea, potassium carbonate, glyoxylic acid and solvent is 1:1:(0.5-0.7):(1-3):(5-35).
6. The method according to claim 4, characterized in that, The heating temperature is 90–120°C; the heat preservation reaction time is 2 hours; and the glyoxylic acid is added dropwise over a period of 0.5–2 hours.
7. The method according to claim 4, characterized in that, The crude product purified to impurity 1 was subjected to gradient elution with dichloromethane / methanol / glacial acetic acid. The eluents were collected, and the fractions containing impurity 1 were combined and concentrated under reduced pressure to obtain pure impurity 1.
8. The method according to claim 1, characterized in that, The formula for the calculation is as follows: Where: wi - mass fraction of p-hydroxyphenylhydantoin and impurities 1-6 in the sample, in %; Ai - peak area of the sample; As - peak area of the standard; Ci - mass of the sample, in g; Cs - mass of the standard, in g; Ps - purity of the standard, in %.
9. The method according to claim 1, characterized in that, The detection conditions for the high-performance liquid chromatography include: Mobile phase A is a 0.04M~0.10M aqueous perchloric acid solution, and mobile phase B is methanol; During gradient elution, from 0 to 5 min, A was 87%–95% and B was 5%–13%; Over 5-15 minutes, A gradually decreased to 35%-45%, while B increased to 55%-65%. Over 15-18 minutes, the percentage of A decreased from 35%-45% to 20%-25%, while the percentage of B increased from 55%-65% to 75%-85%. 18~23 min, A is 20%~25%, B is 75%~85%; 23.01~30 min to restore initial ratio and achieve equilibrium; Flow rate: 0.8–1.2 mL / min; Detection wavelength: 254~275 nm; Column temperature 30~40℃; Injection volume: 5~20 μL.
10. The application of the method according to any one of claims 1 to 9 in monitoring the reaction process of p-hydroxyphenylhydantoin or improving the accuracy of p-hydroxyphenylhydantoin detection.