Method for detecting related substances of chloral hydrate

Abstract

The invention relates to the field of drug detection and analysis, in particular to a method for detecting degraded impurities such as carboxylic acids and aldehydes of chloral hydrate. According to the detection method, high performance liquid chromatography is adopted, octadecyl silane bonded silica gel is adopted as a filling agent, an ultraviolet detector is adopted, and the detection wavelength is 195 nm + / -2 nm. According to the detection method, effective separation of auxiliary material peaks and related substances can be achieved, peak patterns of all peaks are good, the separation degree between impurity peaks and main peaks and the separation degree between the impurity peaks meet the requirements of pharmacopeia, and specificity is good. The method is suitable for drug registration quality research of chloral hydrate and preparations thereof.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis technology, and specifically to a method for detecting related substances in chloral hydrate or its preparations. Background Technology

[0002] Chloral hydrate syrup is a commonly used hypnotic and sedative drug in pediatric examinations. It has a rapid onset of action, high safety, and induces sleep similar to physiological sleep. Therapeutic doses do not suppress respiration and have no obvious side effects. Its chemical name is 2,2,2-trichloro-1,1-ethylenediol, CAS number: 92478-27-8; its chemical structural formula is as follows:

[0003]

[0004] The main excipients used in the production of chloral hydrate syrup include sucrose, anhydrous citric acid, and flavorings. Chloral hydrate is easily degraded under conditions of heat, oxidation, and light, and is unstable, especially in aqueous solutions. Chloral hydrate decomposes slowly in aqueous solution, and decomposition is accelerated by heating, oxidation, and light, producing degradation products such as chloroform, dichloroacetaldehyde, trichloroacetic acid, and acetic acid. Sucrose slowly degrades in solution to produce 5-hydroxymethylfurfural. Currently, chloral hydrate is primarily used clinically both domestically and internationally as a syrup or oral solution, and because it is frequently used in pediatrics, the quality of this product requires careful consideration.

[0005] Neither chloral hydrate raw material nor chloral hydrate oral solution includes related substance testing items in domestic or international pharmacopoeia standards, and chloral hydrate syrup is not included in either domestic or international pharmacopoeia standards. Existing technology CN115453025A discloses a method for detecting related substances in chloral hydrate, but it can only detect some impurities. Therefore, developing a more scientific, accurate, and widely applicable detection method is of great significance. Summary of the Invention

[0006] The purpose of this invention is to provide a method for sample preparation that is simple, accurate, specific, and highly sensitive, and can simultaneously and effectively separate and determine chloral hydrate and its impurities, thereby achieving effective quality control of chloral hydrate preparations.

[0007] Extensive research has revealed that chloral hydrate or its syrup formulations are prone to generating various impurities during production or storage, including dichloroacetaldehyde hydrate, 5-hydroxymethylfurfural, formic acid, acetic acid, trichloroacetic acid, and dichloroacetic acid. According to the International Council for Pharmaceutical Registration Technical Requirements for Human Use (ICH Guidelines), all of the aforementioned impurities require detection and control, including genotoxic impurities that require strict limit control. The chemical structures of these impurities and the basis for their control are summarized in Table 1.

[0008] Table 1 Summary of relevant impurities, structural formulas, and control criteria for chloral hydrate preparations

[0009]

[0010] However, the applicant discovered that some of the aforementioned impurities have not been reported or studied in existing chloral hydrate or its syrup formulations, and existing detection methods for chloral hydrate or syrup formulations cannot simultaneously detect and analyze all of the aforementioned impurities. Furthermore, certain excipients in syrup formulations often interfere with the detection. Therefore, the applicant, after extensive research, has developed an analytical method capable of simultaneously detecting and analyzing the aforementioned impurities. This method boasts high sensitivity, strong specificity, is unaffected by excipient interference, and is simple and accurate.

[0011] This invention provides a method for detecting related substances in chloral hydrate, the method employing liquid chromatography and comprising the following steps:

[0012] Accurately weigh an appropriate amount of chloral hydrate or its preparation, and dilute it with a solvent to prepare a test solution.

[0013] Inject the test solution into the high-performance liquid chromatograph and detect it according to the chromatographic conditions;

[0014] The chromatographic conditions include: using octadecylsilane-bonded silica gel as the packing material, employing a UV detector with a detection wavelength of 195nm±2nm, using an acidic aqueous solution as mobile phase A and acetonitrile as mobile phase B, and eluting according to a gradient.

[0015] Furthermore, the concentration of the test solution is 16–24 mg / ml, and even more specifically, 20 mg / ml.

[0016] Further, the preparation of the test solution includes: taking an appropriate amount of chloral hydrate syrup, diluting it with a solvent to the required concentration, shaking well, and obtaining a test solution with a chloral hydrate concentration of 16-24 mg / ml.

[0017] Furthermore, the solvent is a disodium hydrogen phosphate solution, and even more specifically, a 0.005 mol / L disodium hydrogen phosphate solution.

[0018] Furthermore, the injection volume of the detection method is 5–100 μl, and even more specifically, 20 μl.

[0019] Furthermore, the flow rate of the detection method is 0.9–1.1 ml / min, and even more specifically 1.0 ml / min.

[0020] Furthermore, the mobile phase A comprises an aqueous solution of phosphoric acid.

[0021] Further, the concentration of the mobile phase A is selected from 0.05% to 0.5%; further, from 0.10% to 0.30%; and even further, from 0.1%, 0.2%, or 0.3%.

[0022] Furthermore, the gradient elution includes the following procedure: at 0 minutes, the proportion of mobile phase A is 95-100, the proportion of mobile phase B is 0-5, and this is maintained for 10-15 minutes.

[0023] Furthermore, the gradient elution includes the following procedure:

[0024]

[0025] Furthermore, the gradient elution includes the following procedure:

[0026]

[0027] Furthermore, the gradient elution includes the following procedure:

[0028]

[0029] Furthermore, the gradient elution includes the following procedure:

[0030]

[0031] Furthermore, the gradient elution includes the following procedure:

[0032]

[0033] Furthermore, the related substances include formic acid, acetic acid, dichloroacetaldehyde hydrate, dichloroacetic acid, trichloroacetic acid, and 5-hydroxymethylfurfural.

[0034] Furthermore, the filler is selected from Waters XBridge Shield RP18 or JADE-PAK KP-C18.

[0035] Further, the column temperature of the detection method is selected from 25 to 70°C; further, the column temperature is 25°C to 27°C or 40 to 50°C; even further, the column temperature is 25°C to 27°C or 42°C to 45°C.

[0036] Furthermore, in the chromatogram of the test solution, if any relevant substances are present, the formic acid content shall not exceed 0.5%; or / and the acetic acid content shall not exceed 0.5%; or / and the dichloroacetic acid content shall not exceed 0.012%; or / and the trichloroacetic acid content shall not exceed 0.05%; or / and the dichloroacetaldehyde hydrate shall not exceed 0.10%; or / and the 5-hydroxymethylfurfural content shall not exceed 0.02%; or / and other individual impurities shall not exceed 0.15%; or / and the total amount of impurities shall not exceed 1.0%.

[0037] Furthermore, the chromatographic column has an inner diameter of 3.0–5.0 mm, a length of 100–250 mm, and a packing particle size of 2–5 μm; even further, the chromatographic column has an inner diameter of 4.6 mm, a length of 250 mm, and a packing particle size of 5 μm.

[0038] Further, the chloral hydrate formulation includes syrup, oral solution, enema solution, suppository, or solution to be administered, preferably a syrup. In some embodiments of this application, the chloral hydrate syrup comprises chloral hydrate, sucrose, citric acid, glycerin, flavoring, and water; more specifically, the weight ratio of chloral hydrate, sucrose, and glycerin is 1:5:3.

[0039] In this invention, the negative blank solution refers to the test solution prepared according to the prescription amount of the excipients and diluted with a diluent to a concentration consistent with the sample solution (e.g., reference solution, test solution) as needed for detection. It is mainly used as a blank control in analytical testing to eliminate interference from excipients and solvents, and to accurately detect and quantify the components in the test sample.

[0040] In this invention, the gradient elution can effectively separate all known impurities after 40 minutes. The purpose of extending the elution time is to elute the residual excipients and trace amounts of unknown impurities and to protect the column chromatography column.

[0041] Beneficial effects:

[0042] 1. The detection method of the present invention can effectively separate excipient peaks from related substances. Each peak has a good shape, and the separation degree between impurity peaks and main peaks, as well as between impurity peaks, meets the pharmacopoeia requirements, exhibiting excellent specificity.

[0043] 2. The detection wavelength is determined based on the ultraviolet absorption characteristics of the target analyte, which is the terminal absorption wavelength. This results in low impurity response and a low limit, enabling effective impurity detection while maintaining method specificity, with excellent sensitivity. The range of quality control impurities is wider than existing technologies, including known haloalkanes, aldehydes, carboxylic acids, and unknown impurities, with the unknown impurities separated from the main peak baseline.

[0044] 3. The impurity calculation method adopts the external standard method of impurity reference standard, which has high accuracy and can save analysis time when multiple batches of samples are tested at the same time (the number of injection needles is reduced compared with the main component self-reference method), which is of great significance for controlling time costs.

[0045] The detection method of this invention can comprehensively detect organic impurities with ultraviolet absorption in chloral hydrate preparations. This method has excellent specificity, detection sensitivity, accuracy and robustness. It can effectively detect the impurity status before and after product degradation and is a detection method with stability indication capability, which provides a guarantee for the quality of chloral hydrate syrup during production, transportation and shelf life.

[0046] Instruction manual illustrations

[0047] Figure 1-1 Example 1 “2. Screening of chromatographic conditions - (3) Selection of column temperature” Chromatogram of spiked test sample solution

[0048] Figure 1 Chromatogram of negative blank solution in Experiment Example 2, "2 Specificity - (1) System Suitability"

[0049] Figure 2 Chromatogram of the reference solution in Experiment Example 2, “2 Specificity - (1) System Suitability”

[0050] Figure 3 Chromatogram of the specific solution in Experimental Example 2, “2 Specificity - (1) System Suitability”

[0051] Figure 4 Experimental Example 2 "2 Specificity - (1) System Suitability" Colorimetric spectrum of the test sample

[0052] Figure 5 Chromatogram of acid degradation of chloral hydrate syrup in Experiment Example 2, "2 specificity - (2) degradation specificity".

[0053] Figure 6 Chromatogram of alkaline degradation of chloral hydrate syrup in Experiment Example 2, "2 specificity - (2) degradation specificity".

[0054] Figure 7 Chromatogram of oxidative degradation of chloral hydrate syrup in Experiment Example 2, "2 specificity - (2) degradation specificity".

[0055] Figure 8 Chromatogram of high-temperature degradation of chloral hydrate syrup in Experiment Example 2, "2 specificity - (2) degradation specificity".

[0056] Figure 9 Chromatogram of light-induced degradation of chloral hydrate syrup in Experiment Example 2, "2 specificity - (2) degradation specificity".

[0057] Figure 10 Experimental Example 2: Detection Limits and Quantification Limits – Detection Limits for Chloral Hydrate and Various Impurities (Graph)

[0058] Figure 11 Experimental Example 2, "3. Limits of Detection and Limits of Quantification": Limits of Quantification for Chloral Hydrate and Various Impurities (Graph)

[0059] Figure 12 Chromatograms of comparative sample detection Specific Implementation

[0060] The materials and instruments used in the following experimental examples and embodiments are as follows:

[0061] Thermo U3000 HPLC system, Agilent 1260 HPLC system, Mettler XS205DU, ME204 electronic balance, disodium hydrogen phosphate (analytical grade), phosphoric acid (chromatographic grade), acetonitrile (chromatographic grade), and purified water.

[0062] Formic acid and acetic acid were purchased from TCI; dichloroacetaldehyde hydrate and dichloroacetic acid were purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; trichloroacetic acid was purchased from Beijing Bailingwei Technology Co., Ltd.; 5-hydroxymethylfurfural was purchased from the National Institutes for Food and Drug Control; and chloral hydrate was purchased from Qingdao Yulong Algae Co., Ltd.

[0063] The preparation method of chloral hydrate syrup described in the following test examples and embodiments is as follows: Take the prescribed amount of sucrose, add an appropriate amount of water, heat and boil to dissolve, add glycerin and flavoring and mix evenly, then add chloral hydrate to dissolve, and adjust the pH to 2.2 with hydrochloric acid and anhydrous citric acid to obtain the syrup.

[0064] Methodological Investigation of Related Substances Detection Method for Chloral Hydrate Syrup, Example 1

[0065] 1. Solution preparation and chromatographic detection conditions

[0066] (1) Negative blank solution: Take 5g of excipient sucrose, 3g of glycerol, 20mg of citric acid and 50mg of flavoring respectively, add 10ml of water and stir to dissolve to obtain excipient solution. Weigh 2.6g of excipient solution, put it in a 10ml volumetric flask, dilute to the mark with solvent and shake well.

[0067] (2) Test solution: Take about 2.6g of chloral hydrate syrup (about equivalent to 200mg of chloral hydrate), weigh accurately, put it in a 10ml volumetric flask, dilute it to the mark with solvent, shake well, and you will get a solution containing about 20mg of chloral hydrate per 1ml.

[0068] (3) Reference solution: Take appropriate amounts of formic acid, acetic acid, dichloroacetaldehyde hydrate, dichloroacetic acid, trichloroacetic acid, 5-hydroxymethylfurfural and chloral hydrate reference standards, accurately weigh them, dissolve them in solvent and quantitatively dilute them to prepare a mixed solution containing approximately 100 μg formic acid, 100 μg acetic acid, 20 μg dichloroacetaldehyde hydrate, 2.4 μg dichloroacetic acid, 10 μg trichloroacetic acid, 10 μg 5-hydroxymethylfurfural and 30 μg chloral hydrate per 1 ml.

[0069] (4) Spiked test solution: Weigh 2.6g of chloral hydrate syrup and place it in a 10ml volumetric flask. Add appropriate amounts of the above-mentioned impurity reference standards and prepare a mixed solution containing approximately 20mg of chloral hydrate, 100μg of formic acid, 100μg of acetic acid, 20μg of dichloroacetaldehyde hydrate, 2.4μg of dichloroacetic acid, 10μg of trichloroacetic acid, and 10μg of 5-hydroxymethylfurfural per 1ml.

[0070] (5) High temperature destruction of the test solution: Take about 2.6g of chloral hydrate syrup (about equivalent to 200mg of chloral hydrate), accurately weigh it, put it in a 10ml volumetric flask, dilute it to the mark with solvent, shake it well, place it in a water bath for high temperature destruction for a certain period of time, take it out, cool it to room temperature, and the test solution is obtained.

[0071] The solvent described above is a phosphate solution: Weigh 0.7g of disodium hydrogen phosphate, add 1000ml of water to dissolve, mix well, and a solution with a concentration of 0.005mol / L is obtained. All solvents within the scope of this invention are applicable.

[0072] Chromatographic conditions

[0073] Octadecylsilane-bonded silica gel was used as the packing material (JADE-PAK KP-C18 4.6mm×250mm, 5μm column or equivalent column); phosphoric acid aqueous solution was used as mobile phase A and acetonitrile was used as mobile phase B. Gradient elution was performed according to the table below, with a flow rate of 1.0 ml per minute. Screening was conducted at detection wavelengths of 192~284nm, mobile phase A concentrations of 0.1%~0.2%, and column temperatures of 25℃~45℃.

[0074]

[0075] 2. Screening of chromatographic conditions

[0076] (1) Wavelength selection

[0077] The UV absorption spectra of each impurity were determined using a DAD detector. The maximum UV absorption wavelengths for each substance are shown in Table 1. Formic acid showed maximum absorption at 208 nm, acetic acid at 202 nm, while dichloroacetic acid, trichloroacetic acid, dichloroacetaldehyde hydrate, and chloral hydrate (the main component) all showed terminal absorption. Additionally, 5-hydroxymethylfurfural exhibited characteristic absorptions at 192 nm, 228 nm, and 284 nm. At a wavelength of 195 nm, dichloroacetaldehyde hydrate, with the lowest response, was used as the sensitivity indicator. The signal-to-noise ratio for the limit concentration of dichloroacetaldehyde hydrate was 50–60, and its detection limit was 5% of the limit.

[0078] The results show that, due to the low limits of each impurity and the slight differences in response, a wavelength of 195 nm is sufficient to ensure the detection sensitivity of each impurity.

[0079] (2) Selection of mobile phase A

[0080] All known impurities are highly polar, and the impurities formic acid, acetic acid, dichloroacetic acid, and trichloroacetic acid are all strongly acidic. In order to suppress the ionization of acidic compounds, improve peak shape, and increase retention, different concentrations of phosphoric acid were added to the aqueous phase, and the above solutions were injected and analyzed, and chromatograms were collected.

[0081] The results showed that under chromatographic conditions with different concentrations of phosphoric acid aqueous solution as mobile phase A, when the phosphoric acid concentration was 0.1%, the blank excipient solution interfered with the elution position of formic acid, and high temperature destroyed other impurities in the test sample solution, interfering with the elution of dichloroacetic acid and hydrated dichloroacetaldehyde, resulting in poor specificity. When the phosphoric acid concentration was 0.2%, the main peak and impurity peaks, as well as the impurity peaks themselves, could be effectively separated, with good peak shapes and good specificity.

[0082] (3) Selection of column temperature

[0083] The peaks of the formulation excipients were complex, posing a significant challenge to the specificity of known impurities. Column temperature had little effect on the separation between 5-hydroxymethylfurfural and the main component, with baseline separation achieved in both cases. Furthermore, increasing column temperature affected the peaks of hydrated dichloroacetaldehyde and dichloroacetic acid. Experiments were conducted at different column temperatures, ranging from 25℃ to 45℃, under the same elution gradient conditions. The spiked sample solution was injected and analyzed, and chromatograms were collected. Detailed results are shown in the appendix. Figure 1-1 .

[0084] The results show that under column temperature conditions between 25℃ and 27℃ and between 42℃ and 45℃, the main peak and impurity peaks, as well as the impurity peaks themselves, can be effectively separated, and each peak has a good shape.

[0085] (4) Selection of elution program

[0086] The formulation excipients are complex in composition, and the known impurities are highly polar. Gradients have a significant impact on their retention. To ensure the separation between excipient peaks and between excipient peaks and impurities, gradients are screened.

[0087] Gradient elution procedure 1:

[0088]

[0089] Gradient elution procedure 2:

[0090]

[0091] Gradient elution procedure 3:

[0092]

[0093] Gradient elution procedure 4:

[0094]

[0095] The results showed that in elution programs 1 and 2, with an initial organic phase (mobile phase B) proportion ≥10%, all excipient peaks, impurity peaks, and the main peak were completely eluted within 20 minutes, but there was no separation between peaks, indicating poor specificity and failing to meet the detection requirements. By gradually reducing the initial organic phase proportion from 5% to 0%, the separation between excipient peaks and between excipient peaks and impurities gradually improved. When mobile phase A was maintained at 95% / 100% for 13-15 minutes, baseline separation was achieved between impurities such as formic acid, acetic acid, hydrated dichloroacetaldehyde, dichloroacetic acid, and trichloroacetic acid and the excipients. Gradually decreasing the organic phase proportion from 50% to 30% and slowly increasing it facilitated the separation of impurity peaks and excipient peaks after the main peak (5-hydroxymethylfurfural with the main peak, and unknown impurity peaks with the excipient peaks). Elution programs 3 and 4 met the detection requirements for the formulation. Under the conditions of elution programs 3 and 4, excipient peaks and impurity peaks, the main peak and impurity peaks, and impurity peaks were effectively separated, exhibiting good specificity.

[0096] Experimental Example 2: Methodological Validation

[0097] 1. Analytical method to be verified

[0098] Chromatographic conditions

[0099] Octadecylsilane-bonded silica gel was used as the packing material (JADE-PAK KP-C18 4.6mm×250mm, 5μm column or equivalent column); 0.2% phosphoric acid aqueous solution was used as mobile phase A, acetonitrile was used as mobile phase B, gradient elution was performed according to Table 2, the flow rate was 1.0 ml per minute, the column temperature was 43℃, the detection wavelength was 195 nm, and the injection volume was 20 μl.

[0100] Table 2 Gradient elution program

[0101]

[0102] Negative blank solution: Take 5g of excipient sucrose, 3g of glycerol, 20mg of citric acid and 50mg of flavoring respectively, add 10ml of water and stir to dissolve to obtain excipient solution. Weigh 2.6g of excipient solution, put it in a 10ml volumetric flask, dilute to the mark with solvent and shake well.

[0103] Test solution: Weigh approximately 2.6g of chloral hydrate syrup (approximately equivalent to 200mg of chloral hydrate) accurately, place it in a 10ml volumetric flask, dilute to the mark with solvent, and shake well to obtain a solution containing approximately 20mg of chloral hydrate per 1ml.

[0104] Reference solution: Take appropriate amounts of formic acid, acetic acid, dichloroacetaldehyde hydrate, dichloroacetic acid, trichloroacetic acid, 5-hydroxymethylfurfural and chloral hydrate reference standards, accurately weigh them, dissolve them in solvent and quantitatively dilute them to prepare a mixed solution containing approximately 100 μg formic acid, 100 μg acetic acid, 20 μg dichloroacetaldehyde hydrate, 2.4 μg dichloroacetic acid, 10 μg trichloroacetic acid, 10 μg 5-hydroxymethylfurfural and 30 μg chloral hydrate per 1 ml.

[0105] Specific solution: Weigh 2.6g of chloral hydrate syrup and place it in a 10ml volumetric flask. Add appropriate amounts of the above-mentioned impurity reference standards and prepare a mixed solution containing approximately 20mg of chloral hydrate, 100μg of formic acid, 100μg of acetic acid, 20μg of dichloroacetaldehyde hydrate, 2.4μg of dichloroacetic acid, 10μg of trichloroacetic acid, and 10μg of 5-hydroxymethylfurfural per 1ml. This solution is used as the specific solution.

[0106] The solvent described above is a phosphate solution: Weigh 0.7g of disodium hydrogen phosphate, add 1000ml of water to dissolve, mix well, and a solution with a concentration of 0.005mol / L is obtained. All solvents within the scope of this invention are applicable.

[0107] In the chromatogram of the reference solution, dichloroacetaldehyde hydrate, dichloroacetic acid, trichloroacetic acid, 5-hydroxymethylfurfural, and chloral hydrate should elute sequentially, and the resolution between each peak should meet the requirements. In the specific solution, the excipient peaks should not interfere with the target impurity peaks for the detection of the main components.

[0108] For the assay, take the test solution and the reference solution, inject them into the liquid chromatograph respectively, and record the chromatograms.

[0109] Acceptable limits: For known impurity limits, please refer to Table 1. Other individual unknown impurities shall not exceed 0.15%, and total impurities shall not exceed 1.0%.

[0110] 2. Exclusivity

[0111] (1) System applicability

[0112] Solvent: 0.005 mol / L disodium hydrogen phosphate buffer.

[0113] Reference stock solution: Take appropriate amounts of formic acid, acetic acid, dichloroacetaldehyde hydrate, dichloroacetic acid, trichloroacetic acid, 5-hydroxymethylfurfural hydrate, and chloral hydrate (to replace other individual impurities), accurately weigh them, place them in different volumetric flasks, add appropriate amounts of water to dissolve them, and dilute them separately to prepare solutions containing approximately 10 mg of formic acid, 10 mg of acetic acid, 2 mg of dichloroacetaldehyde hydrate, 1.2 mg of dichloroacetic acid, 1 mg of trichloroacetic acid, 1 mg of 5-hydroxymethylfurfural, and 3 mg of chloral hydrate per 1 ml.

[0114] Reference standard positioning solution: Take an appropriate amount of the reference standard stock solution and place it in different volumetric flasks, then dilute it 10 times with solvent.

[0115] Reference solution: Take appropriate amounts of the reference stock solution and place them in the same volumetric flask, then dilute 100 times with solvent.

[0116] Sensitivity solution: Take an appropriate amount of dichloroacetaldehyde hydrate positioning solution and dilute it with solvent to prepare a solution containing approximately 6 μg per 1 ml.

[0117] Test solution: Weigh approximately 2.6g of chloral hydrate syrup (approximately equivalent to 200mg of chloral hydrate) accurately, place it in a 10ml volumetric flask, dilute to the mark with solvent, and shake well to obtain a solution containing approximately 20mg of chloral hydrate per ml.

[0118] Specific solution: Weigh approximately 2.6g of chloral hydrate syrup (approximately equivalent to 200mg of chloral hydrate) accurately, place it in a 10ml volumetric flask, add 0.1ml of each impurity stock solution, dilute to the mark with solvent, and shake well.

[0119] Negative blank solution: Following the preparation method of negative blank solution in Example 1, weigh approximately 2.6g of excipient solution, place it in a 10ml volumetric flask, dissolve and dilute to the mark with solvent, and shake well to obtain the solution.

[0120] Take 20 μl each of the above negative blank solution, reference solution, test solution, and specific solution, and inject them into the liquid chromatograph. The chromatographic conditions are the same as the analytical method to be verified in Example 2. Record the chromatogram of the sample detection. See the appendix for results. Figure 1 ~Appendix Figure 4 The excipient peaks do not interfere with the detection of known impurities and main components, and the separation effect between components in the chromatogram of the reference solution is good.

[0121] (2) Degradation specificity

[0122] ① Undamaged test solution: Same as test solution in (1).

[0123] ② Acid destruction of the test solution: Weigh about 2.6g of chloral hydrate syrup (equivalent to 200mg of chloral hydrate), place it in a 10ml volumetric flask, add 1ml of hydrochloric acid solution of appropriate concentration, mix well, let stand at room temperature, dilute to the mark with solvent, shake well, and you will get a solution containing about 20mg of chloral hydrate per ml.

[0124] ③ Alkali destruction of the test solution: Weigh about 2.6g of chloral hydrate syrup (equivalent to 200mg of chloral hydrate), place it in a 10ml volumetric flask, add 1ml of appropriate concentration of sodium hydroxide solution, mix well, let stand at room temperature, dilute to the mark with solvent, shake well, and you will get a solution containing about 20mg of chloral hydrate per ml.

[0125] ④ Oxidative destruction of the test solution: Weigh about 2.6g of chloral hydrate syrup (equivalent to 200mg of chloral hydrate), place it in a 10ml volumetric flask, add 1ml of hydrogen peroxide solution of appropriate concentration, let it stand at room temperature, dilute to the mark with solvent, and shake well to obtain a solution containing about 20mg of chloral hydrate per ml.

[0126] ⑤ High-temperature destruction of the test solution: Remove the outer packaging from one bottle of this product and place it in a 60℃ oven for 30 days. Take it out and weigh 2.6g of the destroyed sample (equivalent to 200mg of chloral hydrate), place it in a 10ml volumetric flask, dilute it to the mark with solvent, and shake well to obtain a solution containing approximately 20mg of chloral hydrate per ml.

[0127] ⑥ Light damage to the test solution: One bottle of this product, with the outer packaging removed, is placed in a light chamber (incandescent lamp energy 5000±500LUX, near-ultraviolet energy not less than 83μ / m²). 2 Place the sample in a container for 15 days, remove it, weigh 2.6g of the destroyed sample (equivalent to 200mg of chloral hydrate), place it in a 10ml volumetric flask, dilute to the mark with solvent, and shake well to obtain a solution containing approximately 20mg of chloral hydrate per ml.

[0128] Take 20 μl of each of the above solutions (②~⑥) and inject them into the liquid chromatograph.

[0129] Table 3 Degradation specificity results (%)

[0130]

[0131] Note: LOD indicates below the detection limit.

[0132] Experimental results show that, under all degradation conditions, baseline separation was achieved between the main peak and adjacent impurity peaks, and between each known degradation impurity peak and adjacent impurity peaks in the chromatogram of the test sample solution. Material balance was achieved under all degradation conditions. Figures 5-9 In summary, this method exhibits good specificity and provides stability indicators.

[0133] 3. Limit of Detection and Limit of Quantification

[0134] Take appropriate amounts of each of the above reference standard positioning solutions, place them in volumetric flasks, and dilute with solvent until the signal-to-noise ratio (S / N) of each peak is greater than 10 to obtain the limit of quantitation solution.

[0135] Take an appropriate amount of the limit of quantitation solution and place it in a volumetric flask. Dilute it with solvent until the signal-to-noise ratio (S / N) of each peak is between 3 and 10 to obtain the limit of detection solution.

[0136] Table 4 Detection Limit Results

[0137]

[0138] Table 5 Results of Limit of Quantification

[0139]

[0140] The results above show that the detection limit concentrations of all components are below 10% of the limit, and the quantitation limit concentrations of all components are below 30% of the limit. Figures 10-11 This indicates that the method has good detection sensitivity and can meet the requirements for detecting various impurities in chloral hydrate syrup.

[0141] 4. Linear range

[0142] Take appropriate amounts of formic acid, acetic acid, dichloroacetaldehyde hydrate, dichloroacetic acid, trichloroacetic acid, 5-hydroxymethylfurfural, and chloral hydrate (replacing other individual impurities) reference standards, dissolve them in 0.005 mol / L phosphate solution, and prepare a series of standard solutions at the limit of quantitation, 50%, 100%, 150%, 200%, and 300% based on the impurity limits. Inject and determine according to the planned chromatographic conditions, and perform linear regression on the peak area (Y) against the corresponding solvent mass concentration (X, μg / mL).

[0143] Table 6 Linear Results

[0144]

[0145] The results above show that the linear relationship of each component is good within the range of limit of quantitation to 300% concentration, and the correlation coefficients are all greater than 0.999, indicating that the method has good linearity.

[0146] 5. Repeatability

[0147] Mixed impurity reference standard stock solution: Take appropriate amounts of formic acid, acetic acid, dichloroacetaldehyde hydrate, dichloroacetic acid, trichloroacetic acid, and 5-hydroxymethylfurfural reference standard stock solutions under the specific item "2", place them in the same volumetric flask, and dilute 10 times with solvent.

[0148] Impurity reference solution: Measure an appropriate amount of the mixed impurity reference stock solution and place it in the same volumetric flask. Dilute 10 times with solvent. Spiked test solution: Accurately weigh approximately 2.6 g of the test sample and place it in a 10 ml volumetric flask. Add approximately 3 ml of solvent, then accurately add 1.0 ml of the mixed impurity reference stock solution. Dilute to the mark with solvent and mix well. Prepare 6 parallel solutions.

[0149] Accurately measure 20 μl each of the impurity reference solution and the spiked test solution, inject them into the liquid chromatograph, record the chromatogram, and calculate the content of each known degradation impurity using the external standard method of impurity reference. For other individual impurities, calculate the impurity content using the principal component reference method.

[0150] Table 7 Repeatability Results

[0151]

[0152] The results above show that the RSD of the results obtained from the detection of the six spiked test solutions is less than 10.0%, and the range of the results obtained from other single impurities and each known impurity (≤0.1%) is less than 30% of the corresponding impurity limit, indicating that the method has good repeatability.

[0153] 6. Accuracy

[0154] Impurity reference solution: Same as under item "5" repeatability.

[0155] Accuracy solution: Accurately weigh about 2.6 g of the test sample and place it in a 10 ml volumetric flask. Add about 3 ml of solvent and mix well. Add 0.5 ml, 1.0 ml and 1.5 ml of the mixed impurity reference stock solution under item "5" repeatability, respectively. Dilute to the mark with solvent and shake well. Prepare 3 portions for each concentration.

[0156] Accurately measure 20 μl of each impurity reference solution and each accuracy solution, inject them into the liquid chromatograph, record the chromatogram, and calculate the recovery rate of each known degradation impurity.

[0157] Table 8 Accuracy Results

[0158]

[0159] The results in the table above show that, at different limit concentrations (50%, 100%, and 150% limit concentrations), the recovery rates of each impurity are all between 90.0% and 110.0%, and the RSD (n=9) values ​​are all less than 10.0%, indicating that the method has good accuracy.

[0160] 7. Durability of chromatographic conditions

[0161] Based on the standard chromatographic conditions (the analytical method to be verified in this embodiment), the effects of changes in flow rate (0.9 ml / min and 1.1 ml / min), column temperature (44℃ and 42℃), and mobile phase A (0.19% phosphoric acid solution and 0.21% phosphoric acid solution) on the analytical method were investigated.

[0162] Negative blank solution, control solution, and sensitivity solution: Prepare the same solutions as those under the specificity section of Example 2 in this test.

[0163] Spiked test solution: Prepared in the same manner as the corresponding solution under the repeatability section of this embodiment.

[0164] Under each chromatographic condition, 20 μl of each of the above solutions was accurately measured and injected into the liquid chromatograph. Chromatograms were recorded to examine the specificity and the detection of various impurities in the test solution. The results are detailed in Table 9-10.

[0165] Table 9 Results of Chromatographic Condition Robustness Test—Resolution of Reference Solution

[0166]

[0167] Table 10 Results of Chromatographic Condition Robustness Test—Impurity Content in Test Solution

[0168]

[0169] The experimental results showed that under various chromatographic conditions, the separation of components in the reference solution was good, and the detection results of the content of various impurities in the test solution and the spiked test solution were small (the maximum range was 0.01%), indicating that the method has good robustness when the chromatographic conditions are slightly changed.

[0170] Comparative example:

[0171] Solution preparation:

[0172] (1) Blank solution / solvent: 0.1% phosphoric acid aqueous solution - acetonitrile (95:5).

[0173] (2) Reference stock solution: Take appropriate amounts of formic acid reference standard, acetic acid reference standard, dichloroacetaldehyde hydrate reference standard, dichloroacetic acid reference standard, trichloroacetic acid reference standard and 5-hydroxymethylfurfural reference standard, weigh them accurately, place them in different volumetric flasks, add appropriate amount of water to dissolve them and dilute them separately to prepare solutions containing approximately 10 mg of formic acid, 10 mg of acetic acid, 2 mg of dichloroacetaldehyde hydrate, 1.2 mg of dichloroacetic acid, 1 mg of trichloroacetic acid and 1 mg of 5-hydroxymethylfurfural per ml.

[0174] (3) Reference standard positioning solution: Take an appropriate amount of the reference standard stock solution and place it in different volumetric flasks, then dilute it 10 times with solvent.

[0175] (4) Test solution: Take about 2.6g of chloral hydrate syrup (about equivalent to 200mg of chloral hydrate), weigh accurately, put it in a 10ml volumetric flask, dilute it to the mark with solvent, shake well, and you will get a solution containing about 20mg of chloral hydrate per 1ml.

[0176] (5) Blank excipient solution: Refer to the preparation method of negative blank solution in Experiment Example 1. Take 2.6g of excipient solution, put it in a 10ml volumetric flask, dilute it to the mark with solvent, and shake well.

[0177] Take 20 μl each of the above blank solution / solvent, blank excipient solution, and reference standard positioning solution, and inject them into the liquid chromatograph. Perform the chromatographic conditions as per condition 4 of the detection method in patent (CN115453025A), record the chromatogram of the sample, and see the attached results. Figure 12 .

[0178] The spectra show that the peak positions of dichloroacetic acid and hydrated dichloroacetaldehyde overlap. Furthermore, magnified overlay images reveal other impurities at similar peak positions, interfering with the detection of dichloroacetic acid and hydrated dichloroacetaldehyde. Additionally, formic acid and acetic acid peaks are both present in the blank excipient peaks and cannot be separated. Hydrated chloral hydrate shows poor separation from its preceding and following impurities, indicating poor overall specificity.

[0179] Example 1: Detection of related substances in chloral hydrate syrup

[0180] According to the method for detecting related substances in chloral hydrate syrup of the present invention, the content of each impurity in the related substances of three batches of preparation samples was determined.

[0181] (1) Chromatographic conditions refer to Experimental Example 2

[0182] (2) Solution preparation

[0183] Negative blank solution: Prepared according to Experimental Example 2.

[0184] Test solution: Take an appropriate amount of each batch of chloral hydrate syrup and prepare it according to Test Example 2.

[0185] Reference solution: Prepared according to Experimental Example 2.

[0186] (3) Measurement method

[0187] Accurately measure the test solution and the reference solution, inject them separately into the liquid chromatograph, and record the chromatograms.

[0188] (4) Calculation methods and limits

[0189] In the chromatogram of the test solution, excluding peaks older than 5 minutes and excipient peaks (refer to the attached diagram of the negative blank solution; if a corresponding excipient peak or excipient degradation peak is present at the corresponding position in the chromatogram of the test solution, it should be deducted; local identification may be necessary), if there are chromatographic peaks with retention times consistent with formic acid, acetic acid, dichloroacetaldehyde hydrate, dichloroacetic acid, and trichloroacetic acid in the chromatogram of the reference solution, they should be calculated based on peak area using the external standard method for impurity reference standards and converted to density (1.3 g / ml). These peaks should not exceed 0.5%, 0.5%, 0.10%, 0.012%, and 0.05% of the labeled amount of chloral hydrate, respectively. Other individual impurities should be calculated using the external standard method for the main component reference standards and should not exceed 0.15% of the labeled amount of chloral hydrate. The total impurity amount should not exceed 1.0%. 5-hydroxymethylfurfural and other individual impurities with a detection amount less than 0.05% in the chromatogram of the test solution are not included in the total impurity count.

[0190] If a chromatographic peak with the same retention time as 5-hydroxymethylfurfural is found, its peak area should be calculated using the external standard method and converted to the density of this product (1.3 g / ml). The peak area should not exceed 0.02% of the sucrose content (this product contains 5 g of sucrose per 10 ml).

[0191] (5) Calculation results

[0192] The specific solution chromatogram showed good separation between components (in the order of elution of formic acid, acetic acid, dichloroacetaldehyde hydrate, dichloroacetic acid, trichloroacetic acid, and 5-hydroxymethylfurfural, the excipient peaks did not interfere with the detection of known impurities and the main peak), therefore the system suitability test met the requirements.

[0193] The results of related substance tests for three batches of chloral hydrate syrup are shown in Table 11.

[0194] Table 11 Results of related substances detection in different batches

[0195]

Claims

1. A method for detecting related substances in chloral hydrate or its preparations, employing liquid chromatography, characterized in that, The method includes the following steps: Accurately weigh an appropriate amount of chloral hydrate or its preparation, and dilute it with a solvent to prepare a test solution; Inject the test solution into the high-performance liquid chromatograph and detect it according to the chromatographic conditions; The chromatographic conditions include: using octadecylsilane-bonded silica gel as the packing material, employing a UV detector with a detection wavelength of 195nm±2nm, using an acidic aqueous solution as mobile phase A and acetonitrile as mobile phase B, and eluting according to a gradient.

2. The detection method according to claim 1, characterized in that, The mobile phase A comprises an aqueous solution of phosphoric acid.

3. The detection method according to claim 1, characterized in that, The concentration of the mobile phase A is selected from 0.05% to 0.5%; more specifically, it is selected from 0.10% to 0.30%.

4. The detection method according to claim 1, characterized in that, The gradient elution includes the following procedure:

5. The detection method according to claim 1, characterized in that, The related substances include formic acid, acetic acid, dichloroacetaldehyde hydrate, dichloroacetic acid, trichloroacetic acid, and 5-hydroxymethylfurfural.

6. The detection method according to claim 1, characterized in that, The filler is selected from Waters XBridgeShield RP18 or JADE-PAK KP-C18.

7. The detection method according to claim 1, characterized in that, The column temperature is selected from 25 to 70°C, further, the column temperature is 25°C or 40 to 50°C; even further, the column temperature is 25°C to 27°C or 42°C to 45°C.

8. The detection method according to claim 1, characterized in that, In the chromatogram of the test solution, if any relevant substances are present, the formic acid content shall not exceed 0.5%; or / and the acetic acid content shall not exceed 0.5%; or / and the dichloroacetic acid content shall not exceed 0.012%; or / and the trichloroacetic acid content shall not exceed 0.05%; or / and the dichloroacetaldehyde hydrate shall not exceed 0.10%; or / and the 5-hydroxymethylfurfural content shall not exceed 0.02%; or / and other individual impurities shall not exceed 0.15%; or / and the total amount of impurities shall not exceed 1.0%.

9. The detection method according to claim 1, characterized in that, The chromatographic column has an inner diameter of 3.0–5.0 mm, a length of 100–250 mm, and a packing particle size of 2–5 μm; further, the chromatographic column has an inner diameter of 4.6 mm, a length of 250 mm, and a packing particle size of 5 μm.

10. The detection method according to claim 1, characterized in that, The chloral hydrate preparations include syrups, oral solutions, enema solutions, suppositories, or solutions to be administered, with syrups being preferred.

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