A method for detecting p-chloro-m-cresol

Abstract

The present application relates to the technical field of chromatographic detection, and particularly to a detection method of p-chloro-m-xylenol. The detection method of p-chloro-m-xylenol comprises the following steps: (1) preparing a sample solution: dissolving a sample to be detected in chromatographic-grade acetonitrile, filtering with a microporous filter membrane to obtain a sample solution; (2) using high-performance liquid chromatography to analyze the sample solution obtained in step (1). Through improvement of specific solvents and chromatographic conditions, accurate and efficient determination of p-chloro-m-xylenol in reaction products is achieved.

Description

Technical Field

[0001] This invention relates to the field of chromatographic detection technology, and in particular to a method for detecting p-chloro-m-xylenol. Background Technology

[0002] 4-Chloro-3,5-dimethylphenol, also known as para-chloro-meta-xylenol (PCMX), is an important antimicrobial agent that can kill most Gram-positive and Gram-negative bacteria, fungi, and molds. PCMX has advantages such as mild properties, no oxidative bleaching effect, low toxicity, and stable performance. In addition to its use as a disinfectant, PCMX can also be used as a preservative and antifungal agent in the adhesive, coating, paint, textile, leather, and paper industries.

[0003] Given the wide application and important role of PCMX in disinfection, antiseptic, and mildew prevention, a method for determining p-chloro-m-xylenol in disinfectants has been established. The literature "Determination of p-chloro-m-xylenol in Disinfectants by High Performance Liquid Chromatography" (Article No.: 1005-3387(2007)03-0026-27) established a method for determining p-chloro-m-xylenol in disinfectants. A Kromasil C18 column was used, with a mobile phase of 0.2% acetonitrile-acetic acid aqueous solution at a flow rate of 1.0 mL / min and a detection wavelength of 220 nm. The recovery rate was between 98.52% and 100.66%, and the RSD (n=6) of the sample determination was 0.68%. Experimental results show that this method is rapid, simple, accurate, and reliable.

[0004] The literature "Determination of Salicylic Acid and Chloroxylenol in Hand Sanitizer by High Performance Liquid Chromatography" (Article No.: 10.3969 / j.issn.1008-6145.2021.01.009) established a method for the simultaneous determination of salicylic acid and chloroxylenol in hand sanitizer using high performance liquid chromatography. A Shim-pack Scepter C18 column (250 mm × 4.6 mm, 5 μm) was used. The mobile phase was methanol-0.1% phosphoric acid aqueous solution (v / v 70:30), the flow rate was 1.0 mL / min, the column temperature was room temperature, and the detector was a Shimadzu SPD-M20A diode array detector with a detection wavelength of 280 nm. Salicylic acid and chloroxylenol showed good linearity with the chromatographic peak area, with linear ranges of 40–400 μg / mL and 20–200 μg / mL, respectively, and correlation coefficients of R0 for both. 2=0.9999. The limit of detection of the method was 0.1 μg / mL. The relative standard deviations of the determination results of salicylic acid and p-chloro-m-xylenol were 0.79% and 1.39% (n=5), respectively, and the spiked recoveries were 96.9%–99.8%. This method is simple, rapid, highly sensitive, and reproducible, and can simultaneously and accurately detect the content of salicylic acid and p-chloro-m-xylenol in hand sanitizer.

[0005] Current analytical methods are limited to disinfectant personal care products, such as disinfectants and hand sanitizers, and lack rapid detection methods for the industrial production of PCMX. In conventional industrial processes, real-time monitoring of yield and content is essential for maintaining process standardization. For example, unpurified mixtures often contain structurally similar by-products. Incomplete or excessive chlorination may generate dichloro or polychlorinated meta-xylenol derivatives; the substituent positions may also change, such as generating isomers like o-chloro-meta-xylenol; or dechlorination may occur under high temperature or acidic conditions to generate meta-xylenol or other phenolic compounds.

[0006] Given the current lack of a standard detection method for chloro-m-xylenol (PCMX), and the urgent need for rapid and accurate quality monitoring in its industrial production process, there is a pressing need to develop a rapid detection method suitable for the PCMX production process to achieve efficient and accurate analysis of related products. Summary of the Invention

[0007] The purpose of this invention is to provide a rapid detection method for p-chloro-m-xylenol, which uses high performance liquid chromatography for analysis and detection. By improving specific solvents and chromatographic conditions, the method achieves accurate and efficient determination of p-chloro-m-xylenol in the reaction product.

[0008] In this invention, PCMX refers to 4-chloro-3,5-dimethylphenol, also known as p-chloro-m-xylenol.

[0009] To achieve the above-mentioned objectives, the technical solution of this invention is as follows:

[0010] A method for detecting p-chloro-meta-xylenol includes the following steps:

[0011] (1) Preparation of sample solution: Dissolve the sample to be tested in chromatographic grade acetonitrile and filter it with a microporous membrane to obtain the sample solution;

[0012] (2) The sample solution obtained in step (1) was analyzed using high performance liquid chromatography under the following chromatographic conditions:

[0013] Chromatographic column: C18 column;

[0014] Mobile phase: formic acid aqueous solution - acetonitrile;

[0015] Flow rate: 0.1-0.3 mL / min;

[0016] Wavelength: 200-220nm;

[0017] Column temperature: 22-28℃;

[0018] Injection volume: 5-20 μL;

[0019] Elution method: gradient elution.

[0020] Preferably, in step (1), dissolving the sample to be tested in chromatographic grade acetonitrile specifically involves dissolving the sample to be tested in chromatographic grade acetonitrile and diluting it.

[0021] More preferably, in step (1), dissolving the sample to be tested in chromatographic grade acetonitrile specifically means: dissolving the sample to be tested in chromatographic grade acetonitrile, wherein the mass-to-volume ratio of the sample to be tested and the chromatographic grade acetonitrile is 0.1-5:1-5, and the sample is diluted 5-10 times.

[0022] More preferably, in step (1), dissolving the sample to be tested in chromatographic grade acetonitrile specifically means: dissolving the sample to be tested in chromatographic grade acetonitrile, wherein the mass-to-volume ratio of the sample to be tested and the chromatographic grade acetonitrile is 1:1, and the sample is diluted 8 times.

[0023] Preferably, in step (1), the concentration of the sample solution is 0.5 mg / mL to 1.5 mg / mL; more preferably 1 mg / mL.

[0024] Preferably, in step (1), the microporous filter membrane has a size of 0.1-0.5 μm; more preferably, it has a size of 0.22 μm.

[0025] As a specific example of the present invention, step (1) specifically includes:

[0026] 10 mg of purified PCMX sample was dissolved in 10 mL of chromatographic grade acetonitrile. The resulting solution was diluted 8 times and then filtered through a 0.22 μm microporous membrane to obtain the PCMX sample solution to be tested.

[0027] Preferably, in step (2), the C18 column is selected from any one of the following: polar intercalation type C18 column, high density bonded C18 column, end-capped C18 column, and large pore size wide pH tolerant type C18 column.

[0028] More preferably, it is selected from any one of the following: RP-18 column, Acclaim PolarAdvantage IIC18 column, Thermo Scientific Accucore C18 column, Hypersil GOLD C18 column, Agilent ZORBAXEclipse Plus C18 column, Merck Purospher STAR RP-18 column, and Accucore aQ C18 column.

[0029] The preferred option is an RP-18 column.

[0030] Preferably, in step (2), the mobile phase is 0.05%-0.2% formic acid aqueous solution-acetonitrile, more preferably 0.08%-0.12% formic acid aqueous solution-acetonitrile, and most preferably 0.1% formic acid aqueous solution-acetonitrile.

[0031] Preferably, in step (2), the gradient elution is as follows:

[0032]

[0033] The best option is:

[0034]

[0035]

[0036] Preferably, in step (2), the chromatographic conditions are:

[0037] Column: RP-18;

[0038] Mobile phase: 0.08%-0.12% formic acid aqueous solution - acetonitrile;

[0039] Flow rate: 0.1-0.3 mL / min;

[0040] Wavelength: 200-220nm;

[0041] Column temperature: 22-28℃;

[0042] Injection volume: 5-20 μL;

[0043] Elution procedure: gradient elution,

[0044]

[0045] More preferably, in step (2), the chromatographic conditions are: column: RP-18;

[0046] Mobile phase: 0.1% formic acid aqueous solution - acetonitrile;

[0047] Flow rate: 0.1-0.3 mL / min;

[0048] Wavelength: 200-220nm;

[0049] Column temperature: 22-28℃;

[0050] Injection volume: 5-20 μL;

[0051] Elution procedure: gradient elution,

[0052]

[0053]

[0054] More preferably, in step (2), the chromatographic conditions are:

[0055] Column: RP-18 (50-2mm);

[0056] Mobile phase: 0.1% formic acid aqueous solution, acetonitrile;

[0057] Flow rate: 0.2 mL / min;

[0058] Wavelength: 210nm;

[0059] Column temperature: 25℃;

[0060] Injection volume: 10 μL;

[0061] Elution procedure: gradient elution,

[0062]

[0063] Preferably, the method for detecting p-chloro-m-xylenol further includes the step of preparing a reference solution, comprising:

[0064] Take PCMX reference standard, dissolve it in chromatographic grade acetonitrile, filter it through a microporous membrane to obtain PCMX stock solution with a concentration of 0.05-0.15 mg / mL, and perform isochronous dilution of the stock solution to obtain test reference standard solutions of different concentrations.

[0065] More preferably, it includes:

[0066] PCMX reference standard was dissolved in chromatographic grade acetonitrile and filtered through a microporous membrane to obtain a PCMX stock solution with a concentration of 0.1 mg / mL. The stock solution was then serially diluted to obtain test reference solutions with concentrations of 0.25 mg / mL, 0.125 mg / mL, 0.0625 mg / mL, 0.03125 mg / mL, and 0.015625 mg / mL, respectively.

[0067] The reference solution to be tested.

[0068] The beneficial effects of this invention are as follows:

[0069] (1) This invention provides a method for detecting p-chloro-m-xylenol, which uses high performance liquid chromatography for analysis and detection. By improving specific solvents and chromatographic conditions, the method achieves accurate and efficient determination of p-chloro-m-xylenol in the reaction product.

[0070] (2) The detection method of the present invention is applicable to chemical preparation or electrochemical preparation methods. It has been verified that it has the advantages of high precision, high detection sensitivity, good repeatability, solution stability, system applicability and specificity. Attached Figure Description

[0071] Figure 1 To prepare the PCMX obtained in Example 1 1 H NMR spectrum.

[0072] Figure 2 To prepare the PCMX obtained in Example 1 13 C10 NMR spectrum.

[0073] Figure 3 The HPLC chromatogram of PCMX standard in Example 1 is shown.

[0074] Figure 4 This is a standard curve of PCMX concentration versus peak area in Example 1.

[0075] Figure 5 The HPLC chromatogram of PCMX sample in Example 1 is shown.

[0076] Figure 6 The HPLC chromatogram of PCMX standard in Example 3 is shown.

[0077] Figure 7 This is a standard curve of PCMX concentration versus peak area in Example 3.

[0078] Figure 8 The HPLC chromatogram of PCMX sample in Example 3 is shown. Detailed Implementation

[0079] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the invention based on the disclosed content, which should also fall within the scope of protection claimed in this application.

[0080] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all chemical reagents used in the embodiments of the present invention were obtained through conventional commercial means. Unless otherwise specified, all contents mentioned below are mass contents. Unless otherwise specified, it is understood that the process was carried out at room temperature.

[0081] Preparation Example 1: Chemical Preparation of PCMX

[0082] Referring to the literature "Iron(III)-Catalyzed Chlorination of Activated Arenes" (Document No. 10.1021 / acs.joc.7b01225), PCMX was prepared by a chemical reaction method. The specific steps are as follows:

[0083] Ferric chloride (FeCl3, 0.4 g, 3 mmol) was added to a round-bottom flask containing a solution of 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide ([BMIM]NTf2, 4.0 g, 9 mmol). The mixture was stirred at room temperature for 0.5 h to obtain a potent Lewis acid tri(trifluoromethanesulfonyl)nitrogenous iron (Fe(NTf2)3) catalyst (i.e., trifluorosulfonamide iron(III) Lewis acid as described in the literature). Subsequently, the chlorine source N-chlorosuccinimide (NCS, 14 g, 105 mmol) and 150 mL of tetrahydrofuran (THF) solution were added to the system. After thorough mixing, 3,5-xylenol (1) (12 g, 100 mmol) was added, and the mixture was heated to 60 °C for 12 h. Under the action of the catalyst, 3,5-xylenol and the activated NCS underwent a chlorination reaction.

[0084] After the reaction was completed, water was added to quench the reaction, and the mixture was extracted with ethyl acetate. The upper organic phase was collected and washed three times with 1M sodium thiosulfate solution and saturated saline solution, respectively. The solvent was then removed by distillation under reduced pressure to obtain the crude product.

[0085] Purification was performed as follows: The crude product was added to 10 mL of methanol solution and heated under reflux at 70 °C until the product was completely dissolved. Then, 100 mL of n-hexane was added to the system, and the mixture was slowly cooled at room temperature to precipitate solid crystals. The mixed solution was filtered, and the filter cake was washed three times with pre-cooled n-hexane solution. The filter cake was collected, dried, and 12.5 g of PCMX product was obtained.

[0086] The chemical was then identified and analyzed by NMR. (PCMX) 1 H NMR, 13 The C NMR and mass spectrometry data are shown below. 1 H NMR and 13 The C NMR spectra are as follows: Figure 1 ,2 As shown.

[0087] PCMX: 1 H NMR (300MHz, DMSO-d6) δ9.40(s,1H),6.57(s,2H),2.22(s,6H). 13 C NMR(75MHz,DMSO-d6)δ155.45,136.28,123.18,115.53,20.39.MS(ESI,+ve):m / z 155.0[MH] - .

[0088] Example 1

[0089] 1.1 Preparation of PCMX reference solution

[0090] Accurately weigh 10 mg of PCMX reference standard into a 10 mL volumetric flask, dissolve it in chromatographic grade acetonitrile, and dilute to the mark. Then filter through a 0.22 μm microporous membrane to obtain a 1 mg / mL PCMX stock solution. Perform isostatic dilutions of the stock solution to obtain test standard solutions with concentrations of 0.25 mg / mL, 0.125 mg / mL, 0.0625 mg / mL, 0.03125 mg / mL, and 0.015625 mg / mL.

[0091] 1.2 Preparation of PCMX Sample Solution

[0092] Accurately weigh 10 mg of the PCMX product obtained in Preparation Example 1 and dissolve it in 10 mL of chromatographic grade acetonitrile to obtain a 1 mg / mL sample solution. Dilute the obtained solution 8 times and filter it through a 0.22 μm microporous membrane to obtain the test solution.

[0093] 1.3 Chromatographic Analysis

[0094] The test solution is injected into a high-performance liquid chromatograph for analysis and detection.

[0095] Chromatographic conditions:

[0096] Column: RP-18 (50-2mm)

[0097] Mobile phase: 0.1% formic acid aqueous solution, acetonitrile

[0098] Flow rate: 0.2 mL / min

[0099] Detector: PDA Detector

[0100] Wavelength: 210nm

[0101] Column temperature: 25℃

[0102] Injection volume: 10 μL

[0103] Running time: 20 minutes

[0104] Elution procedure: see Table 1.

[0105] Table 1. Gradient elution program table

[0106]

[0107] 1.4 Plotting Standard Curves

[0108] Under the chromatographic conditions described above, PCMX reference solutions with concentrations of 0.015625 mg / mL, 0.03125 mg / mL, 0.0625 mg / mL, 0.125 mg / mL, and 0.25 mg / mL were injected and analyzed. Each sample was analyzed three times, and the average peak area was recorded. The HPLC chromatogram of the PCMX standard is shown below. Figure 3 As shown in Table 2, the peak areas of each sample were obtained by integrating the peak times of the standard.

[0109] Table 2. Statistical Table of PCMX Concentration and Peak Area

[0110]

[0111] Standard curves were plotted with PCMX concentration (mg / mL) on the x-axis and peak area (A) on the y-axis. The results are shown in [Figure 1]. Figure 4 Therefore, the fitted linear regression equation can be obtained as y = 153953642.108x + 45151.021. R0 2 =0.996>0.99, indicating that the concentration of PCMX and the peak area have a good linear relationship in the range of 0.015625mg / mL to 0.25mg / mL.

[0112] 1.5 Sample Purity Determination

[0113] The PCMX sample solution was analyzed, and the integrated peak area was 19,088,782. Substituting this into the linear equation: y = 153,953,642.108x + 45,151.021, the calculated PCMX concentration was 0.1236 mg / mL, with a purity of 98.8%. The HPLC chromatogram of the sample is shown below. Figure 5 As shown.

[0114] 1.6 Precision

[0115] The PCMX content in the same sample solution was determined using this experimental method, with five consecutive injections. The RSD between the PCMX analysis results was 0.42%, indicating good instrument precision.

[0116] 1.7 Detection Limit

[0117] The PCMX content was detected by high performance liquid chromatography. The detection limit was calculated based on the amount of analyte equivalent to three times the noise level, and the detection limit was 20.8 ng / mL, which reflects extremely high sensitivity.

[0118] 1.8 Repeatability

[0119] The PCMX sample was tested in parallel five times according to the experimental method described herein. The RSD between the PCMX results was 0.83%, indicating that the method has good repeatability.

[0120] The PCMX content in the same sample solution was determined using this experimental method. The samples were injected at column temperatures of 22℃, 23℃, 24℃, 25℃, 26℃, 27℃, and 28℃, respectively. The RSD between the PCMX results was 0.35%, indicating that the method has good repeatability at different temperatures.

[0121] The PCMX content in the same sample solution was determined using this experimental method. Elution was performed at flow rates of 0.1, 0.2, and 0.3 mL / min, respectively. The RSD between the PCMX results was 0.66%, indicating that the method has good repeatability at different flow rates.

[0122] 1.9 Solution stability

[0123] The PCMX content in the same sample solution was determined using this experimental method. Samples were injected and detected at 0, 2, 4, 6, 8, 10, 12, and 24 hours. The RSD between the PCMX analysis results was 0.69%, indicating that the method has good repeatability.

[0124] 1.10 System suitability and specificity test

[0125] The reference solution and sample solution of PCMX were injected and analyzed separately. The results showed that the retention times of the PCMX sample and the reference solution were consistent, and there was no interference from the blank control. The resolution, tailing factor, and theoretical plate number of the reference chromatographic peak all met the requirements of the 2020 edition of the Chinese Pharmacopoeia.

[0126] Example 2

[0127] Following the same method, the PCMX content in the crude product was determined, and the purity was 75%. Example 2: Electrochemical preparation of PCMX.

[0128]

[0129] Under normal pressure, tetrabutylammonium perchlorate (n-Bu4N(ClO4), 17 g, 50 mmol) and 200 mL of dichloromethane (DCM) solution were added to a non-separated electrochemical electrolytic cell. After stirring until homogeneous, 3,5-xylenol (6 g, 50 mmol) was added to the mixture. Subsequently, the entire system was electrolyzed at a constant current of 2 A for 2.5 h, using copper and platinum as the cathode and anode, respectively.

[0130] Under the influence of electric current, the following reaction occurs at the anode: 2Cl - -2e - →Cl2↑, the generated chlorine gas acts as a chlorine source, directly reacting with 3,5-xylenol in a chlorination reaction to give product A.

[0131] After electrolysis, the solvent in the mixture was removed by vacuum distillation, followed by extraction with anhydrous diethyl ether. The extract was washed three times each with saturated sodium bicarbonate solution and saturated saline solution, and then dried with anhydrous sodium sulfate. The dried solution was concentrated under reduced pressure and then separated by silica gel column chromatography (using 200-300 mesh silica gel powder, 500 g per column, wet packing; the crude product was separated and purified by dry loading). The fractions were collected by elution with petroleum ether / ethyl acetate at a ratio of 20:1, and finally the solvent was removed by vacuum distillation to obtain product B.

[0132] The detection method was the same as in Example 1. The PCMX yield in the obtained product was 57.8%, and the purity was 99.7%.

[0133] The product was identified by PCMX NMR analysis, and the compound... 1 H NMR, 13 The C NMR and mass spectrometry data are shown below. 1 H NMR and 13 The C NMR spectra are as follows: Figure 1 , Figure 2 As shown.

[0134] PCMX: 1 H NMR (300MHz, DMSO-d6) δ9.40(s,1H),6.57(s,2H),2.22(s,6H). 13 C NMR(75MHz,DMSO-d6)δ155.47,136.29,123.20,115.54,20.40.MS(ESI,+ve):m / z 155.0[MH].

[0135] Example 3

[0136] The PCMX content in product B was detected using the same detection method as in Example 1.

[0137] 3.1 Plotting Standard Curves

[0138] PCMX reference solutions with concentrations of 0.03125 mg / mL, 0.0625 mg / mL, 0.125 mg / mL, 0.25 mg / mL, and 0.5 mg / mL were injected and analyzed. Each sample was analyzed three times, and the average peak area was recorded. The HPLC chromatogram of the PCMX standard is shown below. Figure 6 As shown in Table 3:

[0139] Table 3. Statistical Table of PCMX Concentration and Peak Area

[0140]

[0141] Standard curves were plotted with PCMX concentration (mg / mL) on the x-axis and peak area (A) on the y-axis. The results are shown in [Figure 1]. Figure 7 Therefore, the fitted linear regression equation is y = 105,332,515.333x + 4,108,256.854, R0. 2 =0.995>0.99, indicating that the concentration of PCMX and the peak area have a good linear relationship in the range of 0.03125mg / mL to 0.5mg / mL.

[0142] 3.2 Sample purity determination

[0143] Analysis of the PCMX sample solution yielded a peak area of ​​17,242,396. Substituting this into the linear equation: y = 105,332,515.333x + 4,108,256.854, the calculated PCMX concentration was 0.1246 mg / mL, with a purity of 99.7%. The HPLC chromatogram of the sample is shown below. Figure 8 As shown.

[0144] 3.3 Precision

[0145] The PCMX content in the same sample solution was determined using this experimental method, with five consecutive injections. The RSD between the measured PCMX results was 0.52%, indicating good instrument precision.

[0146] 3.4 Detection Limit

[0147] The PCMX content was detected by high performance liquid chromatography. The detection limit was calculated based on the amount of analyte equivalent to three times the noise level, and the detection limit was 20.8 ng / mL, which reflects extremely high sensitivity.

[0148] 3.5 Repeatability

[0149] Five PCMX samples were tested in parallel using this experimental method. The results showed that the RSD of PCMX was 0.69%, indicating that the method has good repeatability.

[0150] The PCMX content in the same sample solution was determined using this experimental method, and the analysis was performed at column temperatures of 22, 23, 24, 25, 26, 27, and 28 °C. The RSD between the measured PCMX results was 0.85%, indicating that the method has good repeatability.

[0151] The PCMX content in the same sample solution was determined using this experimental method at flow rates of 0.1, 0.2, and 0.3 mL / min. The RSD between the measured PCMX results was 0.67%, indicating that the method has good repeatability.

[0152] 3.6 Solution stability

[0153] The PCMX content in the same sample solution was determined using this experimental method. The samples were injected and analyzed at 0, 2, 4, 6, 8, 10, 12, and 24 hours. The RSD between the measured PCMX results was 0.39%, indicating that the method has good repeatability.

[0154] 3.7 System Suitability and Specificity Test

[0155] The reference solution and sample solution of PCMX were injected and analyzed separately. The results showed that the retention times of the PCMX sample and the reference solution were consistent, and there was no interference from the blank control. The resolution, tailing factor, and theoretical plate number of the reference chromatographic peak all met the requirements of the 2020 edition of the Chinese Pharmacopoeia.

[0156] Example 4

[0157] Using the same method, the PCMX content in product A was tested, and the purity was 62.9%.

[0158] Comparative Example 1

[0159] Unlike Example 3, the mobile phase was methanol-0.1% phosphoric acid aqueous solution, but everything else was the same.

[0160] 1.1 Linear

[0161] A standard curve was plotted with PCMX concentration (mg / mL) on the x-axis and peak area (A) on the y-axis. The fitted linear regression equation was then obtained as y = 109,181,977.70x + 4,462,423.52. R0 2 =0.959<0.99, indicating that the linear relationship between PCMX concentration and peak area is poor in the range of 0.03125mg / mL to 0.5mg / mL.

[0162] 1.2 Precision

[0163] The PCMX content in the same sample solution was determined using this experimental method, with five consecutive injections. The RSD between the measured PCMX results was 0.42%, indicating good instrument precision.

[0164] 1.3 Detection Limit

[0165] The PCMX content was detected by high performance liquid chromatography. The detection limit was calculated based on the amount of analyte equivalent to three times the noise level, and the detection limit was 25.8 ng / mL, which reflects extremely high sensitivity.

[0166] 1.4 Repeatability

[0167] The PCMX sample was tested in parallel five times according to the experimental method described herein. The results showed that the RSD of PCMX was 0.83%, indicating that the method has good repeatability.

[0168] The PCMX content in the same sample solution was determined using this experimental method, and the analysis was performed at column temperatures of 22, 23, 24, 25, 26, 27, and 28 °C. The RSD between the measured PCMX results was 0.35%, indicating that the method has good repeatability.

[0169] The PCMX content in the same sample solution was determined using this experimental method at flow rates of 0.1, 0.2, and 0.3 mL / min. The RSD between the measured PCMX results was 1.06%, indicating that the method has good repeatability.

[0170] 1.5 Solution stability

[0171] The PCMX content in the same sample solution was determined using this experimental method. The samples were injected and analyzed at 0, 2, 4, 6, 8, 10, 12, and 24 hours. The RSD between the measured PCMX results was 0.69%, indicating that the method has good repeatability.

[0172] 1.6 System Suitability and Specificity Test

[0173] The reference solution and sample solution of PCMX were injected and analyzed separately. The results showed that the retention times of the PCMX sample and the reference solution were consistent, and there was no interference from the blank control. The resolution, tailing factor, and theoretical plate number of the reference chromatographic peak all met the requirements of the 2020 edition of the Chinese Pharmacopoeia.

[0174] Comparative Example 2

[0175] Unlike Example 3, the mobile phase was acetonitrile-0.2% acetic acid aqueous solution, with isocratic elution, but otherwise the same.

[0176] 2.1 Linear

[0177] A standard curve was plotted with PCMX concentration (mg / mL) on the x-axis and peak area (A) on the y-axis. The fitted linear regression equation was then obtained as y = 154,292,566.83x + 504,317.688. R0 2 =0.988<0.99, indicating that the linear relationship between PCMX concentration and peak area is poor in the range of 0.03125mg / mL to 0.5mg / mL.

[0178] 2.2 Precision

[0179] The PCMX content in the same sample solution was determined using this experimental method, with five consecutive injections. The RSD between the measured PCMX results was 0.42%, indicating good instrument precision.

[0180] 2.3 Detection Limit

[0181] The PCMX content was detected by high performance liquid chromatography. The detection limit was calculated based on the amount of analyte equivalent to three times the noise level, and the detection limit was 20.8 ng / mL, which reflects extremely high sensitivity.

[0182] 2.4 Repeatability

[0183] The PCMX sample was tested in parallel five times according to the experimental method described herein. The results showed that the RSD of PCMX was 0.83%, indicating that the method has good repeatability.

[0184] The PCMX content in the same sample solution was determined using this experimental method, and the analysis was performed at column temperatures of 22, 23, 24, 25, 26, 27, and 28 °C. The RSD between the measured PCMX results was 0.35%, indicating that the method has good repeatability.

[0185] The PCMX content in the same sample solution was determined using this experimental method at flow rates of 0.1, 0.2, and 0.3 mL / min. The RSD between the measured PCMX results was 0.96%, indicating that the method has good repeatability.

[0186] 2.5 Solution stability

[0187] The PCMX content in the same sample solution was determined using this experimental method. The samples were injected and analyzed at 0, 2, 4, 6, 8, 10, 12, and 24 hours. The RSD between the measured PCMX results was 0.69%, indicating that the method has good repeatability.

[0188] 2.6 System Suitability and Specificity Test

[0189] The reference solution and sample solution of PCMX were injected and analyzed separately. The results showed that the retention times of the PCMX sample and the reference solution were consistent, and there was no interference from the blank control. The resolution, tailing factor, and theoretical plate number of the reference chromatographic peak all met the requirements of the 2020 edition of the Chinese Pharmacopoeia.

[0190] Comparative Example 3

[0191] Unlike Example 3, the mobile phase was acetonitrile-water (volume ratio 7:3), with isocratic elution, but otherwise the same.

[0192] 3.1 Linear

[0193] Standard curves were plotted with PCMX concentration (mg / mL) on the x-axis and peak area (A) on the y-axis. The results are shown in [Figure 1]. Figure 4 Therefore, the fitted linear regression equation is y = 152,405,255.011x - 204,848.979. R0 2 =0.986<0.99, indicating that the linear relationship between PCMX concentration and peak area is poor in the range of 0.03125mg / mL to 0.5mg / mL.

[0194] 3.2 Precision

[0195] The PCMX content in the same sample solution was determined using this experimental method, with five consecutive injections. The RSD between the measured PCMX results was 1.92%.

[0196] 3.3 Detection Limit

[0197] The PCMX content was detected by high performance liquid chromatography. The detection limit was calculated based on the amount of analyte equivalent to three times the noise level, and the detection limit was 22.8 ng / mL, which reflects extremely high sensitivity.

[0198] 3.4 Repeatability

[0199] The PCMX sample was tested in parallel five times according to the experimental method described herein. The results showed that the RSD of PCMX was 2.45%, indicating poor repeatability.

[0200] The PCMX content in the same sample solution was determined using this experimental method, and analysis was performed at column temperatures of 22, 23, 24, 25, 26, 27, and 28 °C. The RSD between the measured PCMX results was 1.75%.

[0201] The PCMX content in the same sample solution was determined using this experimental method at flow rates of 0.1, 0.2, and 0.3 mL / min. The RSD between the measured PCMX results was 2.63%, indicating poor repeatability of this method.

[0202] 3.5 Solution stability

[0203] The PCMX content in the same sample solution was determined using this experimental method. The samples were analyzed at 0, 2, 4, 6, 8, 10, 12, and 24 hours. The RSD between the measured PCMX results was 2.69%.

[0204] 3.6 System Suitability and Specificity Test

[0205] The reference solution and sample solution of PCMX were injected and analyzed separately. The results showed that the retention times of the PCMX sample and the reference solution were consistent, and there was no interference from the blank control. The resolution, tailing factor, and theoretical plate number of the reference chromatographic peak all met the requirements of the 2020 edition of the Chinese Pharmacopoeia.

[0206] Example 5

[0207] Unlike Example 3, the test solution preparation method uses a 0.45μm microporous membrane for filtration, but all other aspects are the same.

[0208] 4.1 Linear

[0209] Standard curves were plotted with PCMX concentration (mg / mL) on the x-axis and peak area (A) on the y-axis. The results are shown in [Figure 1]. Figure 4 Therefore, the fitted linear regression equation is y = 153663749.559x + 57235.345. R0 2 =0.995>0.99, indicating that the concentration of PCMX and the peak area have a good linear relationship in the range of 0.03125mg / mL to 0.5mg / mL.

[0210] 4.2 Precision

[0211] The PCMX content in the same sample solution was determined using this experimental method, with five consecutive injections. The RSD between the measured PCMX results was 0.45%, indicating good instrument precision.

[0212] 4.3 Detection Limit

[0213] The PCMX content was detected by high performance liquid chromatography. The detection limit was calculated based on the amount of analyte equivalent to three times the noise level, and the detection limit was 60.8 ng / mL, which reflects high sensitivity.

[0214] 4.4 Repeatability

[0215] According to the experimental method described herein, five parallel samples of PCMX were prepared. The results showed that the RSD of PCMX was 0.95%, indicating that the method has good repeatability.

[0216] The PCMX content in the same sample solution was determined using this experimental method, and the analysis was performed at column temperatures of 22, 23, 24, 25, 26, 27, and 28 °C. The RSD between the measured PCMX results was 0.45%, indicating that the method has good repeatability.

[0217] The PCMX content in the same sample solution was determined using this experimental method at flow rates of 0.1, 0.2, and 0.3 mL / min. The RSD between the PCMX results was 0.68%, indicating that the method has good repeatability.

[0218] 4.5 Solution stability

[0219] The PCMX content in the same sample solution was determined using this experimental method. The samples were injected and analyzed at 0, 2, 4, 6, 8, 10, 12, and 24 hours. The RSD between the measured PCMX results was 0.79%, indicating that the method has good repeatability.

[0220] 4.6 System Suitability and Specificity Test

[0221] The reference solution and sample solution of PCMX were injected and analyzed separately. The results showed that the retention times of the PCMX sample and the reference solution were consistent, and there was no interference from the blank control. The resolution, tailing factor, and theoretical plate number of the reference chromatographic peak all met the requirements of the 2020 edition of the Chinese Pharmacopoeia.

Claims

1. A method for detecting p-chloro-m-xylenol, characterized in that, Includes the following steps: (1) Preparation of sample solution: Dissolve the sample to be tested in chromatographic grade acetonitrile, then dilute the resulting solution and filter it with a microporous membrane to obtain the sample solution; (2) The sample solution obtained in step (1) was analyzed using high performance liquid chromatography under the following chromatographic conditions: Chromatographic column: C18 column; Mobile phase: 0.1% formic acid aqueous solution - acetonitrile; Flow rate: 0.1-0.3 mL / min; Wavelength: 200-220nm; Column temperature: 22-28℃; Injection volume: 5-20 μL; Elution method: gradient elution; The gradient elution is as follows: The sample to be tested is p-chloro-m-xylenol prepared by a chemical method. The specific preparation steps are as follows: 3 mmol of ferric chloride was added to a round-bottom flask containing 9 mmol of 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide solution, and the mixture was stirred at room temperature for 0.5 h. Then, 105 mmol of N-chlorosuccinimide and 150 mL of tetrahydrofuran solution were added to the system, and after stirring, 100 mmol of 3,5-xylenol was added. The mixture was then heated to 60 °C and reacted for 12 h. After the reaction was completed, the mixture was quenched with water, extracted with ethyl acetate, and the upper organic phase was collected. The organic phase was washed three times with 1 M sodium thiosulfate solution and saturated saline solution, respectively. The solvent was then removed by distillation under reduced pressure to obtain the crude product. The crude product was added to 10 mL of methanol solution and heated under reflux at 70 °C until the product was completely dissolved. Then, 100 mL of n-hexane was added to the system and the mixture was slowly cooled at room temperature to precipitate solid crystals. The mixed solution was filtered, and the filter cake was washed three times with pre-cooled n-hexane solution. The filter cake was collected, dried, and the PCMX product was obtained.

2. The detection method according to claim 1, characterized in that, In step (1), dissolving the sample to be tested in chromatographic grade acetonitrile specifically means: dissolving the sample to be tested in chromatographic grade acetonitrile, with a mass-to-volume ratio of the sample to acetonitrile of 0.1-5:1-5, and diluting it 5-10 times.

3. The detection method according to claim 2, characterized in that, In step (1), dissolving the sample to be tested in chromatographic grade acetonitrile specifically means: dissolving the sample to be tested in chromatographic grade acetonitrile, with a mass-to-volume ratio of the sample to chromatographic grade acetonitrile of 1:1, and diluting it 8 times.

4. The detection method according to claim 1, characterized in that, In step (1), the microporous filter membrane has a size of 0.1-0.5 μm.

5. The detection method according to claim 4, characterized in that, In step (1), the microporous filter membrane has a size of 0.22 μm.

6. The detection method according to claim 1, characterized in that, Step (1) is as follows: 10 mg of PCMX product is dissolved in 10 mL of chromatographic grade acetonitrile, the resulting solution is diluted 8 times and then filtered through a 0.22 μm microporous membrane to obtain the PCMX sample solution to be tested.

7. The detection method according to claim 1, characterized in that, In step (2), the C18 column is selected from any one of the following: RP-18 column, Acclaim PolarAdvantage II C18 column, Thermo Scientific Accucore C18 column, Hypersil GOLD C18 column, Agilent ZORBAX Eclipse Plus C18 column, Merck Purospher STAR RP-18 column, and Accucore aQ C18 column.

8. The detection method according to claim 7, characterized in that, In step (2), the C18 column is an RP-18 column.

9. The detection method according to claim 1, characterized in that, In step (2), the chromatographic conditions are as follows: Column: RP-18, 50mm x 2mm; Mobile phase: 0.1% formic acid aqueous solution, acetonitrile; Flow rate: 0.2 mL / min; Wavelength: 210 nm; Column temperature: 25℃; Injection volume: 10 μL.

10. The detection method according to any one of claims 1-9, characterized in that, The detection method for p-chloro-m-xylenol also includes the step of preparing a reference solution, which includes: taking PCMX reference standard, dissolving it in chromatographic grade acetonitrile, filtering it through a microporous membrane to obtain PCMX stock solution, and performing isochronous dilutions of the stock solution to obtain reference solutions of different concentrations.

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