A method for the simultaneous determination of 4,4'-dichloro-2-hydroxydiphenyl ether and various intermediates and impurities thereof

Abstract

This invention relates to the field of analytical detection technology and discloses a method for simultaneously determining 4,4'-dichloro-2-hydroxydiphenyl ether and its various intermediates and impurities. The method includes the following steps: S1, preparing a series of mixed standard solutions containing standards of the target analytes, wherein the target analytes are 4,4'-dichloro-2-hydroxydiphenyl ether, 4,4'-dichloro-2-nitrodiphenyl ether, 4,4'-dichloro-2-aminodiphenyl ether, p-chlorophenol, 2,5-dichloronitrobenzene, toluene, and 2,8-dichlorodibenzofuran; S2, preparing a test solution; and S3, performing detection using high-performance liquid chromatography (HPLC). This invention enables simultaneous determination of the purity of the main component, monitoring of residual synthetic intermediates, and limit checks of impurities in raw materials and by-products in a single detection, covering seven components with highly polar and structurally different properties, providing a comprehensive related substance detection solution for industrial pharmaceutical raw materials.

Description

Technical Field

[0001] This invention relates to the field of analytical detection technology, and in particular to a method for simultaneously determining 4,4'-dichloro-2-hydroxydiphenyl ether and its various intermediates and impurities. Background Technology

[0002] 4,4'-Dichloro-2-hydroxydiphenyl ether (also known as diclosan) is a broad-spectrum antibacterial agent widely used in daily chemicals, medical, textiles, and building materials. Currently, the mainstream industrial production process for diclosan is the nitro etherification-diazo hydrolysis route. For example, the preparation methods described in CN116444353A and CN107353185A both belong to this route: using p-chlorophenol and 2,5-dichloronitrobenzene as starting materials, etherification condensation is performed to generate nitro ethers, followed by nitro reduction, diazotization, and hydrolysis to obtain the diclosan product. This route is relatively mature, with an overall yield of 65%–72%. In addition, although alternative routes such as one-step chlorination exist, their industrial application is limited due to complex processes, numerous byproducts, and unsuitability for large-scale production.

[0003] In the synthesis of diclosan, various process-related impurities inevitably remain, including unreacted raw materials p-chlorophenol and 2,5-dichloronitrobenzene, toluene (a solvent used in the synthesis), intermediates 4,4'-dichloro-2-aminodiphenyl ether and 4,4'-dichloro-2-nitrodiphenyl ether, and the reaction byproduct 2,8-dichlorodibenzofuran. These substances pose multiple risks to the quality and safety of the final product: p-chlorophenol is skin irritant and potentially carcinogenic; 2,5-dichloronitrobenzene and 4,4'-dichloro-2-nitrodiphenyl ether are genotoxic structural impurities, and even trace amounts pose unacceptable risks of genotoxicity; residual solvent toluene is neurotoxic, and its residue limits are strictly regulated by international guidelines such as ICH Q3C; and 2,8-dichlorodibenzofuran belongs to the dibenzofuran class of compounds and has the potential characteristics of a persistent organic pollutant. Furthermore, the presence of residual intermediates not only poses safety risks but also signifies incomplete synthesis and transformation, directly impacting the purity and antibacterial activity of the product. Therefore, establishing a method for the simultaneous and accurate detection of diclosan and its various related substances is crucial for ensuring product quality and safe use.

[0004] Several existing methods exist for detecting diclosan, but most focus on determining the content of diclosan as the main component in daily chemical products. For example, CN109541069A discloses a method for detecting diclosan content in detergents, using acetonitrile-water (80:20) isocratic elution with a detection wavelength of 278 nm; CN115078583A discloses a method for detecting triclosan (a structural analog) content in antibacterial detergents; CN120352543A discloses a method for detecting diclosan in laundry liquids, using acetic acid aqueous solution-acetonitrile as the mobile phase with a detection wavelength of 278 nm. However, the above-mentioned existing methods have at least the following drawbacks: First, these methods only determine the content of the main diclosan component and do not consider the peak elution and quantification of synthetic intermediates and other process impurities. When these methods are applied to the detection of related substances in diclosan industrial products, significant overlap occurs between key impurity peaks and the main component peaks, or between impurity peaks themselves. For example, in an acetonitrile-water system, the chromatographic peaks of toluene and 2,5-dichloronitrobenzene completely overlap, and baseline separation cannot be achieved between the main diclosan component and the intermediate 4,4'-dichloro-2-aminodiphenyl ether. This makes simultaneous quantification of multiple related substances impossible.

[0005] Secondly, all the methods described above use 278 nm as the detection wavelength. However, toluene has no UV absorption at 278 nm, which means that under single-wavelength conditions of 278 nm, toluene as a residual solvent cannot be detected at all, resulting in a blind zone in its content determination.

[0006] The technical root of the above defects lies in the fact that the seven target substances of diclosan (diclosan, two intermediates, and four impurities) have vastly different chemical properties. p-Chlorophenol is a strongly polar compound (containing phenolic hydroxyl groups), toluene is a non-polar aromatic hydrocarbon, and 2,8-dichlorodibenzofuran is a weakly polar fused-ring compound. Their polarity range far exceeds the separation difficulty faced by the detection of a single target substance in conventional daily chemical products.

[0007] Simultaneously separating so many target analytes with vastly different polarities under the same chromatographic conditions places extremely high demands on the selectivity of the mobile phase system and the precision of the gradient elution procedure. However, existing methods were originally designed for the rapid determination of diclosan content in daily chemical products, falling under the category of component presence verification. Their technical focus is on speed, simplicity, and low cost, rather than the comprehensive quality control requirements for pharmaceuticals / APIs that demand the simultaneous and accurate quantification of multiple related substances. These two application scenarios have fundamentally different technical requirements for the detection methods. The former only needs to confirm whether the main component content meets the standard, while the latter requires a comprehensive screening and accurate quantification of all related substances that may affect safety.

[0008] Therefore, there is an urgent need to develop a method for the simultaneous determination of 4,4'-dichloro-2-hydroxydiphenyl ether and its various intermediates and impurities, so as to simultaneously separate and quantify seven target compounds with large polarity differences. Summary of the Invention

[0009] One of the objectives of this invention is to provide a method for simultaneously determining 4,4'-dichloro-2-hydroxydiphenyl ether and its various intermediates and impurities, so as to simultaneously separate and quantify seven target substances with large polarity differences, thereby ensuring product quality and safety of use.

[0010] To achieve the above-mentioned objectives, the specific technical solution is as follows: A method for simultaneously determining 4,4'-dichloro-2-hydroxydiphenyl ether and its various intermediates and impurities includes the following steps: S1 prepares a series of mixed standard solutions containing the standard of the detection target, wherein the detection target is 4,4'-dichloro-2-hydroxydiphenyl ether, 4,4'-dichloro-2-nitrodiphenyl ether, 4,4'-dichloro-2-aminodiphenyl ether, p-chlorophenol, 2,5-dichloronitrobenzene, toluene and 2,8-dichlorodibenzofuran; S2 is used to prepare the test solution; S3 was detected using high-performance liquid chromatography (HPLC) under the following chromatographic conditions: Column: Packed with octadecylsilane-bonded silica gel; Mobile phase A: Ammonium acetate-phosphoric acid aqueous solution; Mobile phase B: A mixed solution of methanol and acetonitrile; Gradient elution is employed, and the gradient elution procedure is as follows: within a time range of 0 to 40 minutes, the volume fraction of mobile phase A is switched from a first ratio to a second ratio and then restored to the first ratio, wherein the first ratio is 38% to 42% and the second ratio is 13% to 17%. Detection wavelengths: dual wavelengths of 220nm and 278nm.

[0011] According to embodiments of the present invention, one of the technical solutions has at least one of the following advantages or beneficial effects: Existing technologies can only solve the technical problem of quantifying the main components in daily chemical matrices, but cannot take into account the separation of multiple types of impurities, and are completely unsuitable for the quality control scenarios of industrial pharmaceutical raw materials.

[0012] The method of this invention can simultaneously complete the determination of the purity of the main component, the monitoring of residual synthetic intermediates, and the limit inspection of impurities in raw materials and by-products in a single test, covering seven components with extremely different polarities and structures, providing a comprehensive related substance detection solution for industrial pharmaceutical raw materials.

[0013] More specifically: Existing technologies either employ a single organic phase system of acetonitrile-water, resulting in insufficient separation selectivity and an inability to differentiate the retention differences of components with similar polarities; or they utilize a single long-wavelength detection method, leading to the undetectability of components such as toluene. This creates a persistent dilemma: improving separation at the expense of stability, or increasing sensitivity at the expense of increased baseline interference. The solution presented in this invention is not a simple parameter adjustment, but rather a systematic matching of multiple technical characteristics: First, the present invention uses an ammonium acetate-phosphate buffer solution phase to ensure consistent retention behavior of ionizable target substances (such as p-chlorophenol); Secondly, the use of a methanol-acetonitrile mixed organic phase, compared to acetonitrile or methanol alone, can precisely control the difference in partition coefficients of different structural components. Combined with step-gradient elution, weakly retained polar impurities are separated first under a high aqueous phase ratio, and then the organic phase ratio is gradually increased to elute the strongly retained components. This allows gradient elution to elute target substances with large polarity differences from the chromatographic column in order of polarity. Furthermore, a specific gradient elution method is employed. Polar targets, such as p-chlorophenol, require a higher aqueous phase ratio to retain on the column, while weakly polar targets, such as 2,8-dichlorodibenzofuran, require a lower aqueous phase ratio for elution. This gradient framework first maintains the retention of p-chlorophenol with 38–42% mobile phase A, then reduces the mobile phase A to 13–17% to elute the weakly polar components, and finally restores equilibrium. This maintenance-rapid reduction and reequilibration pattern is a necessary means to simultaneously separate targets with a wide polarity range. Finally, this invention employs a dual-wavelength approach of 220nm + 278nm. The 220nm wavelength ensures sufficient UV response for all components (including toluene), resolving the issue of toluene's lack of absorption at longer wavelengths. The 278nm wavelength serves as a reference wavelength for auxiliary qualitative analysis, while the buffer salt system suppresses baseline noise at low wavelengths. Furthermore, conventional understanding suggests that low-wavelength detection at 220nm is accompanied by significant baseline noise and drift. However, the ammonium acetate-phosphate aqueous solution system in this invention stabilizes the background absorption of the mobile phase, significantly reducing baseline fluctuations at 220nm. Simultaneously, the 278nm wavelength in the dual-wavelength approach serves as a reference to subtract background interference. Together, these two approaches address the technical problem of high sensitivity but high noise at low wavelengths, achieving a balance between high sensitivity and low baseline noise.

[0014] According to one embodiment of the present invention, in the ammonium acetate-phosphoric acid aqueous solution of the mobile phase A, the concentration of ammonium acetate is 18~22 mmol / L and the volume fraction of phosphoric acid aqueous solution is 0.09%~0.11%.

[0015] According to one embodiment of the present invention, in the mobile phase B, the volume ratio of methanol to acetonitrile is 49~51:51~49.

[0016] Acetonitrile alone exhibits strong elution of weakly polar targets but poor selectivity for polar targets, while methanol alone shows the opposite. When methanol and acetonitrile are mixed at a volume ratio of 49–51:51–49, the resulting mixed solvent effect is significant, simultaneously optimizing the separation of polar and non-polar targets. Experiments demonstrate that baseline separation was achieved for all seven targets at this ratio.

[0017] According to one embodiment of the present invention, the mobile phase A is a 20 mmol / L ammonium acetate-0.10% vol phosphoric acid solution; the mobile phase B is a 50% vol methanol-50% vol acetonitrile solution.

[0018] Under this preferred flow phase ratio, the retention factor change curves of each target are steepest when the gradient changes during the gradient program, and the separation is maximized at the inflection points (15 min, 20 min).

[0019] According to one embodiment of the present invention, the gradient elution procedure is shown in Table 1 below: Table 1 Gradient Elution Procedure

[0020] The precise procedure ensures that each target analyte is eluted within its optimal elution window. Specifically, the 15-minute inflection point allows for sufficient retention of polar analytes, the 20-minute inflection point allows for the separation of toluene and 2,5-dichloronitrobenzene within a limited time, the 30-minute inflection point allows for complete elution of all weakly polar components, and the 31-minute rapid equilibration recovery shortens the total analysis time.

[0021] More specifically: The polarity order of the seven target analytes is: p-chlorophenol > toluene > 2,5-dichloronitrobenzene > 4,4'-dichloro-2-hydroxydiphenyl ether > 4,4'-dichloro-2-aminodiphenyl ether > 4,4'-dichloro-2-nitrodiphenyl ether > 2,8-dichlorodibenzofuran. The gradient elution program is designed to accommodate this order. 0-15 min (40% mobile phase A), maintain high aqueous phase to ensure sufficient retention time of p-chlorophenol on the column and avoid overlap with solvent peaks; 15-20 min (40% mobile phase A → 15% mobile phase A): Rapidly reduce the proportion of aqueous phase, the elution intensity jumps, and toluene and 2,5-dichloronitrobenzene elute sequentially during this time period. The two peaks completely overlap under isocratic conditions, but separation is achieved in this jump segment due to the amplification of the difference in retention factors. 20-30 min (maintained with 15% mobile phase A): Under constant strong elution conditions, diclosan, 4,4'-dichloro-2-aminodiphenyl ether, 4,4'-dichloro-2-nitrodiphenyl ether and 2,8-dichlorodibenzofuran were eluted in descending order of polarity, with diclosan and 4,4'-dichloro-2-aminodiphenyl ether being separated selectively under stationary phase conditions; 30-40 min (restore 40% of mobile phase A): Equilibrate the column in preparation for the next injection.

[0022] According to one embodiment of the present invention, in the series of mixed standard solutions described in S1, the concentration range of each target standard is 9~81 μg / mL.

[0023] In pharmaceutical quality control, it is necessary to accurately determine trace impurities (detection limits as low as 0.1 μg / mL) and the content of the main component. This concentration range covers the typical content of impurities (usually 0.01% to 1%) and the content of the main component in the test sample, ensuring that the correction factor method and external standard method are effective within this range.

[0024] According to one embodiment of the present invention, the series of mixed standard solutions are prepared by the following method: dissolving the standards in acetonitrile and making up to volume to prepare a single target analyte stock solution, and then mixing and diluting the stock solutions to obtain a series of mixed standard solutions.

[0025] The seven target analytes have significantly different solubilities. If they are directly mixed and weighed, the sparingly soluble substances may not dissolve completely, leading to inaccurate concentrations in the standard solution. Therefore, dissolving them separately before mixing ensures that each target analyte dissolves completely.

[0026] According to one embodiment of the present invention, the test solution is prepared by taking 4,4'-dichloro-2-hydroxydiphenyl ether as a test sample, dissolving it in acetonitrile and making up to volume to prepare a test solution with a concentration of 2.5~5 mg / mL.

[0027] The optimal concentration is 2.5–5 mg / mL. When the concentration of the test sample is too low, the peak of impurities will be below the detection limit. When the concentration is too high, the main component peak will be overloaded and broadened, which may easily mask the adjacent impurity peaks, especially the adjacent 4,4'-dichloro-2-aminodiphenyl ether.

[0028] According to one embodiment of the present invention, the chromatographic conditions in S3 further include: a flow rate of 0.9~1.1 mL / min, a column temperature of 25~35 ℃, and an injection volume of 5~15 μL.

[0029] According to one embodiment of the present invention, the flow rate is 1 mL / min, the column temperature is 30 °C, and the injection volume is 10 μL. This combination results in symmetrical peak shapes, optimal separation, and good repeatability for each target analyte.

[0030] According to one embodiment of the present invention, the chromatographic column in S3 has a specification of 250 mm × 4.6 mm and a packing particle size of 5 μm. Under this column specification, the linear velocity generated by the flow rate (1 mL / min) in the gradient program is optimal, allowing sufficient mass transfer time for the target analyte within the column to achieve efficient separation.

[0031] According to one embodiment of the present invention, in the dual-wavelength detection spectrum of 220nm and 278nm, qualitative analysis is performed based on the retention time of each detection target, and quantitative analysis is performed at 220nm based on the peak area of ​​each detection target; during the quantitative analysis, the content of toluene is calculated using the external standard method, and the content of the other detection targets is calculated using the area normalization method with a correction factor.

[0032] Since toluene has no absorption at 278 nm, quantification must be performed at 220 nm; however, other target compounds all exhibit strong absorption at 220 nm. This invention uses 278 nm as an auxiliary method to verify the attribution of the main component and impurity peaks, avoiding misjudgment, because diclosan has characteristic absorption at 278 nm, while toluene does not.

[0033] Furthermore, experiments conducted in this invention revealed that the correction factor for toluene at 220 nm is 17.8, which is greater than 5.0. If the correction factor area normalization method is used, the toluene content will be severely underestimated. The correction factors for other target analytes are between 0.6 and 1.8, making the correction factor method suitable. This strategy ensures the accuracy of toluene quantification while avoiding the cumbersome nature of the full external standard method.

[0034] According to one embodiment of the present invention, when calculating using the area normalization method with added correction factors, the correction factors for each detected target are as follows: p-chlorophenol 1.8, 2,5-dichloronitrobenzene 1.1, 4,4'-dichloro-2-hydroxydiphenyl ether 1.0, 4,4'-dichloro-2-aminodiphenyl ether 1.3, 4,4'-dichloro-2-nitrodiphenyl ether 1.3, and 2,8-dichlorodibenzofuran 0.6.

[0035] A second objective of this invention is to provide an application of the method for simultaneously determining 4,4'-dichloro-2-hydroxydiphenyl ether and its various intermediates and impurities.

[0036] The specific technical solution is as follows: The method for simultaneously determining 4,4'-dichloro-2-hydroxydiphenyl ether and its various intermediates and impurities is applied in the safety and quality control of 4,4'-dichloro-2-hydroxydiphenyl ether raw material.

[0037] The method of this invention can simultaneously determine the main component, two intermediates, and four impurities (including genotoxicity warning structures and residual solvents), providing comprehensive data support for safety evaluation, which is something that existing detection methods cannot achieve.

[0038] In addition to the objectives, features, and advantages described above, the present invention has other objectives, features, and advantages. The invention will now be described in further detail with reference to the figures. Attached Figure Description

[0039] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which: Figure 1 This is a chromatogram of the method in Example 1 for detecting related substances in the test sample solution.

[0040] Figure 2 This is the chromatogram of Comparative Example 1 for detecting related substances in the test sample solution.

[0041] Figure 3 This is the chromatogram of Comparative Example 2 for detecting related substances in the test sample solution.

[0042] Figure 4 The chromatograms obtained by the method in Comparative Example 3 for the detection of toluene and 2,5-dichloronitrobenzene standards are shown.

[0043] Figure 5 The chromatograms obtained by the method for detecting 4,4'-dichloro-2-hydroxydiphenyl ether and 4,4'-dichloro-2-aminodiphenyl ether standards are shown in Comparative Example 3.

[0044] Figure 6 The detection chromatograms are for the methods used in Comparative Example 4 to detect 4,4'-dichloro-2-hydroxydiphenyl ether and 4,4'-dichloro-2-aminodiphenyl ether standards.

[0045] Figure 7 The detection chromatogram is shown in Comparative Example 4, obtained by detecting a mixed solution containing all the target analytes.

[0046] Figure 8 This is the chromatogram of the method used in Comparative Example 5 for detecting related substances in the test solution.

[0047] Figure 9 This is the chromatogram of the method used in Comparative Example 6 for detecting related substances in the test solution.

[0048] Figure 10 This is a diagram showing the repeatability test of the test sample solution in Experiment 1 of this invention.

[0049] Figure 11 This is a diagram showing the repeatability test of the mixed solution in Experiment 2 of this invention.

[0050] Figure 12 This is a linear graph of p-chlorophenol at 220 nm in Experiment 3 of this invention.

[0051] Figure 13 This is a linear graph of toluene at 220 nm in Experiment 3 of this invention.

[0052] Figure 14 This is a linear graph of 2,5-dichloronitrobenzene at 220 nm in Experiment 3 of this invention.

[0053] Figure 15 This is a linear graph of 4,4'-dichloro-2-hydroxydiphenyl ether at 220 nm in Experiment 3 of this invention.

[0054] Figure 16 This is a linear graph of 4,4'-dichloro-2-aminodiphenyl ether at 220 nm in Experiment 3 of this invention.

[0055] Figure 17 This is a linear graph of 4,4'-dichloro-2-nitrodiphenyl ether at 220 nm in Experiment 3 of this invention.

[0056] Figure 18 This is a linear graph of 2,8-dichlorodibenzofuran at 220 nm in Experiment 3 of this invention.

[0057] Figure 19 This is a test diagram for the detection limit experiment in Experiment 4 of this invention.

[0058] Figure 20 The chromatogram for detecting related substances in the test sample solution is shown in Example 2.

[0059] Figure 21 The chromatogram for detecting related substances in the test sample solution is shown in Example 3.

[0060] Figure 22 The chromatogram for detecting related substances in the test sample solution is shown in Example 4.

[0061] Figure 23 The chromatogram for detecting related substances in the test sample solution is shown in Example 5.

[0062] Figure 24 The chromatogram for detecting related substances in the test sample solution is shown in Example 6.

[0063] Figure 25 The chromatogram for detecting related substances in the test sample solution is shown in Example 7.

[0064] Figure 26 The chromatogram is of commercially available test sample 1 produced by Dixin Chemical in Experiment 6.

[0065] Figure 27 The chromatogram is of commercially available test sample 2 produced by Dido Pharmaceutical Chemical in Experiment 6.

[0066] Figure 28 The chromatogram is of commercially available test sample 3 produced by Xinjiecheng Chemical in Experiment 6. Detailed Implementation

[0067] The terms "preferred," "more preferred," etc., used in this invention refer to embodiments of the invention that provide certain beneficial effects under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the description of one or more preferred embodiments does not imply that other embodiments are unavailable, nor is it intended to exclude other embodiments from the scope of this invention.

[0068] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0069] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of the present invention.

[0070] Unless otherwise specified, the reagents, methods and equipment used in this invention are all conventional reagents, methods and equipment in this technical field.

[0071] The target main component detected in this invention is 4,4'-dichloro-2-hydroxydiphenyl ether (also known as dichlorosan), with the English name 4,4'-Dichloro-2-hydroxydiphenyl ether and the molecular formula C. 12 H8Cl2O2, CAS number 3380-30-1.

[0072] The 4,4'-dichloro-2-hydroxydiphenyl ether test samples involved in this invention include self-made samples and commercially available samples from multiple manufacturers. All samples were prepared using the currently mainstream industrial production process of nitro etherification-diazo hydrolysis. The specific sources of each test sample are as follows: Self-made test sample: Prepared through independent synthesis, with process reference CN 107353185 B. The process route is as follows: using 2,5-dichloronitrobenzene and p-chlorophenol as starting materials, the intermediate 4,4'-dichloro-2-nitrodiphenyl ether is obtained through nucleophilic substitution (etherification) reaction; the nitro group is converted to amino group through reduction reaction to obtain the intermediate 4,4'-dichloro-2-aminodiphenyl ether; finally, the amino group is converted to hydroxyl group through diazotization-hydrolysis reaction to obtain crude 4,4'-dichloro-2-hydroxydiphenyl ether, which is then purified to obtain the 4,4'-dichloro-2-hydroxydiphenyl ether test sample. Commercially available test sample 1: Dichloroform produced by Zhongshan Dixin Chemical Co., Ltd., batch number DX20260411; Commercially available test sample 2: 4,4-dichloro-2-hydroxydiphenyl ether produced by Shaanxi Didu Pharmaceutical Chemical Co., Ltd., batch number 2604190119; Commercially available test sample 3: Dichloroform produced by Hubei Xinjiecheng Chemical Technology Co., Ltd., batch number 20260415-01.

[0073] Since all the above samples were produced using the same mainstream nitro etherification-diazo hydrolysis process, and the synthesis reaction route and purification process were consistent, the types of related substances that may remain in the products are highly common: they all contain starting material impurities such as p-chlorophenol and 2,5-dichloronitrobenzene, process intermediates such as 4,4'-dichloro-2-nitrodiphenyl ether and 4,4'-dichloro-2-aminodiphenyl ether, production solvent toluene, and 2,8-dichlorodibenzofuran generated by high-temperature side reactions.

[0074] In the examples, comparative examples, and performance tests, the preparation method of the standard solution includes the following steps: Accurately weigh appropriate amounts of standards for the target analytes, including 4,4'-dichloro-2-hydroxydiphenyl ether, 4,4'-dichloro-2-nitrodiphenyl ether, 4,4'-dichloro-2-aminodiphenyl ether, p-chlorophenol, 2,5-dichloronitrobenzene, toluene, and 2,8-dichlorodibenzofuran. Dissolve and quantitatively dilute each standard in acetonitrile to prepare a single target analyte stock solution with a concentration of 2.5 mg / mL. Accurately measure appropriate amounts of each stock solution and dilute it with acetonitrile to prepare a series of mixed standard solutions containing each target analyte.

[0075] In the examples, comparative examples, and performance tests, the preparation method of the test solution includes the following steps: Accurately weigh 25 mg of the test sample and place it in a 10 mL volumetric flask. Dissolve and dilute to the mark with acetonitrile, and shake well to obtain a test sample solution with a concentration of 2.5 mg / mL.

[0076] Example 1 A method for simultaneously determining 4,4'-dichloro-2-hydroxydiphenyl ether and its various intermediates and impurities includes the following steps: S1 prepares a series of mixed standard solutions containing standards for the detection target, wherein the detection target includes 4,4'-dichloro-2-hydroxydiphenyl ether, 4,4'-dichloro-2-nitrodiphenyl ether, 4,4'-dichloro-2-aminodiphenyl ether, p-chlorophenol, 2,5-dichloronitrobenzene, toluene, and 2,8-dichlorodibenzofuran; S2 is used to prepare the test solution; S3 was detected using high-performance liquid chromatography (HPLC) under the following chromatographic conditions: Instrument: Agilent 1260 high performance liquid chromatograph, equipped with VWD detector (variable wavelength detector). Column: Agilent ZORBAX SB-C18 (250 mm × 4.6 mm, 5 μm); Mobile phase A: 20 mmol / L ammonium acetate - 0.10% vol phosphoric acid solution; Mobile phase B: 50% vol methanol - 50% vol acetonitrile; The gradient elution procedure is as follows: Table 1: Table 1 Gradient elution program

[0077] Flow rate: 1.0 mL / min; Column temperature: 30 ℃; Detection wavelengths: 220 nm and 278 nm; Injection volume: 10 μL.

[0078] Accurately pipette 10 μL each of the series of mixed standard solutions and the test solution into the liquid chromatograph, record the chromatograms, and qualitatively determine the content by the retention time in the chromatograms at dual wavelengths of 220 nm and 278 nm. Quantitatively determine the content by calculating the peak area of ​​each target analyte at 220 nm. The content of toluene is calculated by peak area using the external standard method, and the content of other target analytes is calculated by area normalization with correction factors. The contents of 4,4'-dichloro-2-hydroxydiphenyl ether, 4,4'-dichloro-2-nitrodiphenyl ether, 4,4'-dichloro-2-aminodiphenyl ether, p-chlorophenol, 2,5-dichloronitrobenzene, toluene, and 2,8-dichlorodibenzofuran in the test solution are then calculated.

[0079] When using the external standard method, a standard curve of the content-peak area of ​​the target analyte is plotted using a series of concentration solutions of the target analyte (the linear result of Experiment 3 is the standard curve). Then, the peak area of ​​the target analyte at 220 nm is calculated. By comparing the standard curve of the content-peak area of ​​the target analyte, the content of the target analyte in the test sample can be obtained.

[0080] When using the area normalization method with added correction factors, the correction factor data are shown in Table 17.

[0081] Selection of mobile phase: Because strong acids and bases are used in the synthesis process, if there is no buffer salt system in the mobile phase, the retention time of the target analyte peaks will drift severely and the chromatographic column will be easily damaged. Therefore, a special buffer salt is added to the mobile phase. That is, mobile phase A is 20 mmol / L ammonium acetate-0.10% vol phosphoric acid solution. The target analytes are complex and have a large polarity range. In order to optimize the separation selectivity, mobile phase B is set to 50% vol methanol-50% vol acetonitrile.

[0082] The detection spectrum of the test solution in Example 1 is shown below. Figure 1 ,Depend on Figure 1 As can be seen, the method of the present invention can simultaneously and accurately determine 4,4'-dichloro-2-hydroxydiphenyl ether, 4,4'-dichloro-2-nitrodiphenyl ether, 4,4'-dichloro-2-aminodiphenyl ether, p-chlorophenol, 2,5-dichloronitrobenzene, toluene, and 2,8-dichlorodibenzofuran, and all achieve baseline separation, symmetrical peak shape, and strong specificity.

[0083] Example 2 The only difference between Example 2 and Example 1 is that the flow rate in Example 2 is 0.9 mL / min.

[0084] Example 3 The only difference between Example 3 and Example 1 is that the flow rate in Example 3 was 1.1 mL / min.

[0085] Example 4 The only difference between Example 4 and Example 1 is that the column temperature in Example 4 is 25°C.

[0086] Example 5 The only difference between Example 5 and Example 1 is that the column temperature in Example 5 is 35°C.

[0087] Example 6 The only difference between Example 6 and Example 1 is that the initial volume fraction of the mobile phase B in the gradient elution program of Example 6 is 58%. Example 6 aims to verify whether the separation degree of each target analyte can still meet the requirements when the initial organic phase ratio fluctuates slightly (±2%).

[0088] The gradient elution procedure for Example 6 is shown in Table 2: Table 2 Gradient elution program

[0089] Example 7 The only difference between Example 7 and Example 1 is that the initial volume fraction of the mobile phase B in the gradient elution program of Example 7 is 62%. Example 7 aims to verify whether the separation degree of each target analyte can still meet the requirements when the initial organic phase ratio fluctuates slightly (±2%).

[0090] The gradient elution procedure for Example 7 is shown in Table 3: Table 3 Gradient elution program

[0091] Comparative Example 1 The only difference between Comparative Example 1 and Example 1 is that Comparative Example 1 used a different gradient elution procedure.

[0092] The gradient elution procedure for Comparative Example 1 is shown in Table 4: Table 4 Gradient elution procedure for Comparative Example 1

[0093] The chromatogram of Comparative Example 1 for detecting related substances in the test solution is as follows: Figure 2 As shown, under this elution gradient, 4,4'-dichloro-2-hydroxydiphenyl ether and 4,4'-dichloro-2-aminodiphenyl ether can be effectively separated, but toluene and 2,5-dichloronitrobenzene cannot achieve baseline separation and almost overlap.

[0094] Comparative Example 2 The only difference between Comparative Example 2 and Example 1 is that Comparative Example 2 used a different gradient elution procedure.

[0095] The gradient elution procedure for Comparative Example 2 is shown in Table 5: Table 5 Gradient elution procedure for Comparative Example 2

[0096] Comparative Example 2: Chromatogram for detecting related substances in the test solution (as shown below) Figure 3 As shown, under this gradient, 4,4'-dichloro-2-hydroxydiphenyl ether and 4,4'-dichloro-2-aminodiphenyl ether can be effectively separated, but toluene and 2,5-dichloronitrobenzene cannot achieve baseline separation.

[0097] Comparative Example 3 The difference between Comparative Example 3 and Example 1 lies in the different chromatographic conditions, as shown in Table 6.

[0098] Table 6 Chromatographic conditions for Comparative Example 3

[0099] The test results for Comparative Example 3 are shown below. Figure 4 and Figure 5 .in, Figure 4 The chromatograms obtained by the method in Comparative Example 3 for the detection of toluene and 2,5-dichloronitrobenzene standards are shown. Figure 5 The chromatograms obtained by the method for detecting 4,4'-dichloro-2-hydroxydiphenyl ether and 4,4'-dichloro-2-aminodiphenyl ether standards are shown in Comparative Example 3. Figure 4 The peaks of toluene and 2,5-dichloronitrobenzene impurities overlap. Figure 5 The peak of the 4,4-dichloro-2-aminodiphenyl ether impurity overlaps with the peak of the 4,4'-dichloro-2-hydroxydiphenyl ether.

[0100] Comparative Example 4 The only difference between Comparative Example 4 and Example 1 is the chromatographic conditions, as shown in Table 7.

[0101] Table 7 Chromatographic conditions for Comparative Example 4

[0102] The test results for Comparative Example 4 are shown below. Figure 6 and Figure 7 . Figure 6 The detection chromatograms are for the methods used in Comparative Example 4 to detect 4,4'-dichloro-2-hydroxydiphenyl ether and 4,4'-dichloro-2-aminodiphenyl ether standards. Figure 7 The detection chromatogram obtained by the method of Comparative Example 4 is the same as the mixed solution used in the "Repeatability Experiment of Mixed Solution Containing All Detection Targets".

[0103] Figure 6 The peaks of 4,4'-dichloro-2-hydroxydiphenyl ether and 4,4'-dichloro-2-aminodiphenyl ether were poorly separated and easily confused. Figure 7 The above is a comparison spectrum obtained from the chromatographic detection of a mixed solution containing all the target analytes under the chromatographic conditions of Comparative Example 4 (the same mixed solution was used in the repeatability experiment of the mixed solution containing all the target analytes). Figure 7 The theoretical plate number of p-chlorophenol is 970, and that of toluene is 3083. The difference in peak shape directly affects the detection sensitivity and the separation of unknown impurities.

[0104] Comparative Example 5 The only difference between Comparative Example 5 and Example 1 is that in Comparative Example 5, each target analyte was detected and scanned using ultraviolet light at dual wavelengths of 237 nm and 220 nm.

[0105] Figure 8 This is the chromatogram of the method used in Comparative Example 5 for detecting related substances in the test solution. Figure 8 The results show that at a wavelength of 237 nm, the ultraviolet absorption of each detected target is significantly weaker than that at 220 nm, and toluene has no absorption at this wavelength.

[0106] Comparative Example 6 The only difference between Comparative Example 6 and Example 1 is that in Comparative Example 6, each target analyte was detected and scanned using ultraviolet light at a single wavelength of 220 nm.

[0107] Figure 9 This is the chromatogram of the method used in Comparative Example 6 for detecting related substances in the test solution. Figure 9 The results show that the baseline noise is higher at a single wavelength of 220 nm than at 278 nm, which may cause some unknown impurities to be overlooked.

[0108] By comparing Example 1 and Comparative Examples 5-6, this invention selects 220 nm as the main detection wavelength (to ensure impurity detection sensitivity) and 278 nm as the reference wavelength (to assist in qualitative analysis and reduce baseline interference), and adopts a dual-wavelength detection mode.

[0109] Performance testing: Experiment 1: Repeatability Test of Test Solution Accurately weigh 50 mg of the test sample (consistent with the test sample used in the example), place it in a 20 mL volumetric flask, dilute to the mark with acetonitrile, shake well to obtain the test sample solution, prepare 6 parallel aliquots, and perform repeatability experiments. Other operations are the same as in Example 1. The repeatability results of each detection target in the test sample are shown in Table 8 below. Figure 10 .

[0110] Table 8: Repeatability results of each detection target in the test sample

[0111] N / A means not detected. The results are shown in Table 8: In this experimental method, the minimum concentration of the test sample was selected, and the range of the content of each known single impurity was less than 0.05%, and the range of the total impurities was less than 0.2%, indicating that the detection error of the present invention is small and has good repeatability.

[0112] Experiment 2: Repeatability test of mixed solution containing all target analytes Accurately weigh 52.7 mg of p-chlorophenol, 50.4 mg of toluene, 51.5 mg of 2,5-dichloronitrobenzene, 54.0 mg of 4,4'-dichloro-2-hydroxydiphenyl ether, 50.8 mg of 4,4'-dichloro-2-aminodiphenyl ether, 49.8 mg of 4,4'-dichloro-2-nitrodiphenyl ether, and 49.5 mg of 2,8-dichlorodibenzofuran into 20 mL volumetric flasks. Dissolve each flask in acetonitrile and dilute to the mark with acetonitrile to obtain stock solutions of each individual target analyte. Take 1 mL of each stock solution and place it into a 50 mL volumetric flask. Dilute to the mark with acetonitrile and mix well to obtain a mixed solution containing all the target analytes. Prepare six parallel aliquots for repeatability experiments, following the same procedures as in Example 1. The repeatability results are shown in Table 9 below. Figure 11 .

[0113] Table 9: Repeatability results of each target analyte in the mixed solution

[0114] In this experimental method, the repeatability RSD of each detection target is less than 1%, indicating that the method of the present invention has small error and good repeatability.

[0115] Experiment 3: Examination of Linear Relationship and Plotting of Standard Curve Following the method in Example 1, after the instrument baseline stabilized, quantification was performed using the concentration-peak area external standard method. A series of linear solutions of each single target analyte were prepared and injected for analysis. The detection wavelength was 220 nm, and other operations were the same as in Example 1. In the linear equation, x represents the concentration (μg / mL), and y represents the peak area (mAU·s). The results are shown in Tables 10-16 below. Figures 12-18 As shown. Among them, Figure 12 This is a linear graph of p-chlorophenol at 220 nm in Experiment 3 of this invention. Figure 13 This is a linear graph of toluene at 220 nm in Experiment 3 of this invention. Figure 14 This is a linear graph of 2,5-dichloronitrobenzene at 220 nm in Experiment 3 of this invention. Figure 15 This is a linear graph of 4,4'-dichloro-2-hydroxydiphenyl ether at 220 nm in Experiment 3 of this invention. Figure 16 This is a linear graph of 4,4'-dichloro-2-aminodiphenyl ether at 220 nm in Experiment 3 of this invention. Figure 17 This is a linear graph of 4,4'-dichloro-2-nitrodiphenyl ether at 220 nm in Experiment 3 of this invention. Figure 18 This is a linear graph of 2,8-dichlorodibenzofuran at 220 nm in Experiment 3 of this invention.

[0116] Table 10: Linearity results for p-chlorophenol

[0117] Table 11: Linearity Results for Toluene

[0118] Table 12: Linearity Results of 2,5-Dichloronitrobenzene

[0119] Table 13: Linearity results for 4,4'-dichloro-2-hydroxydiphenyl ether

[0120] Table 14: Linearity results for 4,4'-dichloro-2-aminodiphenyl ether

[0121] Table 15: Linearity results for 4,4'-dichloro-2-nitrodiphenyl ether

[0122] Table 16: Linearity Results for 2,8-Dichlorodibenzofuran

[0123] From Tables 10-16 and Figures 12-18 This indicates that in this experimental method, the correlation coefficient R of each detected target analyte is [value missing]. 2 All values ​​are greater than 0.999, indicating that the method of this invention exhibits good linearity within the target analyte concentration range of 10 μg / mL to 81 μg / mL, meeting the requirements for quantitative analysis. Furthermore, Tables 10-16 and... Figures 12-18 The linear results can be directly used as the content-peak area standard curve of the target analyte.

[0124] The correction factor for each detected target was calculated based on the linear data, and the results of the correction factor are shown in Table 17 below.

[0125] Table 17: Correction Factor Results for Each Detected Target

[0126] As shown in Table 17, the correction factor for toluene is 17.8, which is greater than 5.0. Therefore, it needs to be calculated by peak area using the external standard method. The correction factors for other detection targets are all between 0.2 and 5.0, and are calculated by the area normalization method with correction factors.

[0127] Experiment 4: Detection Limit Experiment Take the mixed solution containing all the target analytes prepared in Experiment 2, dilute it to a concentration where the signal-to-noise ratio (S / N) of each target analyte is greater than 3, and then perform the limit of detection experiment. Other procedures are the same as in the example. The detection results and limit of detection concentrations are shown in Table 18 below. Figure 19 .

[0128] Table 18: Detection Limits for Each Target Specimen

[0129] As shown in Table 18, in this experimental method, at 220 nm, the detection limit for all targets except toluene (5.0 μg / mL) is 0.1 μg / mL; while at 278 nm, there is no detection signal for toluene. This indicates that the method of the present invention has high detection sensitivity for most related substances.

[0130] Experiment 5: Method Robustness Test The chromatographic detection results of Examples 2-7 are shown in Table 19 and Figure 20-25 As shown, Examples 2-7, compared to Example 1, fine-tuned the flow rate, column temperature, and initial volume fraction of mobile phase B. The robustness of the method was tested by examining the resolution between each target analyte. Other operations were the same as in Example 1. The chromatographic detection results of Examples 2-7 are shown in Table 19 below. Among them, Figure 20 The chromatogram for detecting related substances in the test sample solution is shown in Example 2. Figure 21 The chromatogram for detecting related substances in the test sample solution is shown in Example 3. Figure 22 The chromatogram for detecting related substances in the test sample solution is shown in Example 4. Figure 23 The chromatogram for detecting related substances in the test sample solution is shown in Example 5. Figure 24 The chromatogram for detecting related substances in the test sample solution is shown in Example 6. Figure 25 The chromatogram for detecting related substances in the test sample solution is shown in Example 7.

[0131] Table 19: Chromatographic detection results of Examples 2-7

[0132] As shown in Table 19, after fine-tuning the flow rate, column temperature, and initial volume fraction of mobile phase B, the separation degree of each detected target is greater than 1.5, which indicates that the method of the present invention has good durability.

[0133] Experiment 6: Suitability Test of Test Samples from Different Manufacturers To further verify the universality and applicability of the method of the present invention to diclosan products from different sources, diclosan test samples were purchased from three different manufacturers (commercially available test sample 1 from Dixin Chemical, commercially available test sample 2 from Didu Pharmaceutical Chemical, and commercially available test sample 3 from Xinjiecheng Chemical). The same chromatographic conditions and operating methods as in Example 1 were used for detection. The separation of 4,4'-dichloro-2-hydroxydiphenyl ether from its intermediates and impurities was examined in each test sample. The resolution between each target analyte was used as the evaluation index to test the applicability of the method of the present invention to test samples from different manufacturers. The chromatographic detection results of the test samples from the three manufacturers are as follows: Figure 26-28 And Table 20, in which, Figure 26 The chromatogram of commercially available test sample 1 produced by Dixin Chemical. Figure 27 The chromatogram of commercially available test sample 2 produced by Didu Pharmaceutical Chemical Co., Ltd. Figure 28 The chromatogram is of commercially available test sample 3 produced by Xinjiecheng Chemical. The results show that no interference from unknown impurity peaks to known target peaks was observed in the chromatograms of the test samples from the three manufacturers. Each known impurity peak and its adjacent unknown impurity peaks were able to achieve baseline separation and did not affect the accurate quantification of each target analyte.

[0134] Table 20 Detection results of various target substances in test samples from different manufacturers

[0135] Table 20 and Figure 26-28 This indicates that the method of the present invention is universal for dichloroanisole products from different sources.

[0136] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0137] The above embodiments merely illustrate several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of the present invention should be determined by the appended claims.

[0138] The above are merely preferred embodiments of the present invention and are not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for simultaneously determining 4,4'-dichloro-2-hydroxydiphenyl ether and its various intermediates and impurities, characterized in that: Includes the following steps: S1 prepares a series of mixed standard solutions containing the standard of the detection target, wherein the detection target is 4,4'-dichloro-2-hydroxydiphenyl ether, 4,4'-dichloro-2-nitrodiphenyl ether, 4,4'-dichloro-2-aminodiphenyl ether, p-chlorophenol, 2,5-dichloronitrobenzene, toluene and 2,8-dichlorodibenzofuran; S2 is used to prepare the test solution; S3 was detected using high-performance liquid chromatography (HPLC) under the following chromatographic conditions: Column: Packed with octadecylsilane-bonded silica gel; Mobile phase A: Ammonium acetate-phosphoric acid aqueous solution; Mobile phase B: A mixed solution of methanol and acetonitrile; Gradient elution is employed, and the gradient elution procedure is as follows: within a time range of 0 to 40 minutes, the volume fraction of mobile phase A is switched from a first ratio to a second ratio and then restored to the first ratio, wherein the first ratio is 38% to 42% and the second ratio is 13% to 17%. Detection wavelengths: dual wavelengths of 220nm and 278nm.

2. The method for simultaneous determination of 4,4'-dichloro-2-hydroxydiphenyl ether and its various intermediates and impurities according to claim 1, characterized in that: In the ammonium acetate-phosphoric acid aqueous solution of the mobile phase A, the concentration of ammonium acetate is 18~22 mmol / L and the volume fraction of phosphoric acid aqueous solution is 0.09%~0.11%.

3. The method for simultaneous determination of 4,4'-dichloro-2-hydroxydiphenyl ether and its various intermediates and impurities according to claim 1, characterized in that: In the mobile phase B, the volume ratio of methanol to acetonitrile is 49~51:51~49.

4. The method for simultaneously determining 4,4'-dichloro-2-hydroxydiphenyl ether and its various intermediates and impurities according to claim 2 or 3, characterized in that: The mobile phase A is a 20 mmol / L ammonium acetate-0.10% vol phosphoric acid solution; the mobile phase B is a 50% vol methanol-50% vol acetonitrile solution.

5. The method for simultaneous determination of 4,4'-dichloro-2-hydroxydiphenyl ether and its various intermediates and impurities according to claim 1, characterized in that: The gradient elution procedure is shown in Table 1 below: Table 1 Gradient elution program 。 6. The method for simultaneous determination of 4,4'-dichloro-2-hydroxydiphenyl ether and its various intermediates and impurities according to claim 1, characterized in that: The series of mixed standard solutions are prepared by the following method: dissolving the standards in acetonitrile and making up to volume to prepare stock solutions of a single target analyte, and then mixing and diluting the stock solutions to obtain a series of mixed standard solutions.

7. The method for simultaneous determination of 4,4'-dichloro-2-hydroxydiphenyl ether and its various intermediates and impurities according to claim 1, characterized in that: The test solution is prepared as follows: 4,4'-dichloro-2-hydroxydiphenyl ether is taken, dissolved in acetonitrile and diluted to volume to prepare a test solution with a concentration of 2.5~5 mg / mL.

8. The method for simultaneous determination of 4,4'-dichloro-2-hydroxydiphenyl ether and its various intermediates and impurities according to claim 1, characterized in that: The chromatographic conditions in S3 also include: a flow rate of 0.9~1.1 mL / min, a column temperature of 25~35 ℃, and an injection volume of 5~15 μL.

9. The method for simultaneous determination of 4,4'-dichloro-2-hydroxydiphenyl ether and its various intermediates and impurities according to claim 1, characterized in that: Qualitative analysis was performed based on the retention time of each target analyte in the dual-wavelength detection spectrum at 220 nm and 278 nm, and quantitative analysis was performed based on the peak area of ​​each target analyte at 220 nm. During the quantitative analysis, the content of toluene was calculated using the external standard method, and the content of the other target analytes was calculated using the area normalization method with a correction factor.

10. The method for simultaneously determining 4,4'-dichloro-2-hydroxydiphenyl ether and its various intermediates and impurities as described in any one of claims 1 to 9, in the safety and quality control of 4,4'-dichloro-2-hydroxydiphenyl ether raw material.

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