Method for detecting p-aminophenol in compound paracetamol and chlorpheniramine maleate granules and application

Abstract

The invention provides a method for detecting p-aminophenol in compound paracetamol and chlorpheniramine maleate granules and application, and belongs to the technical field of chemical drug analysis methods. The invention discloses a method for detecting p-aminophenol in compound paracetamol and chlorpheniramine maleate granules, which is high performance liquid chromatography, and the high performance liquid chromatography conditions are as follows: a chromatographic column is an octadecyl silane bonded silica gel chromatographic column; the detection wavelength is 255 to 259 nm; the flow velocity is 0.9 to 1.1 ml / min; the column temperature is 28-32 DEG C; a 0.025 mol / L disodium hydrogen phosphate solution (the pH is adjusted to 6.6 by phosphoric acid) is used as a mobile phase A, methanol is used as a mobile phase B, and gradient elution is performed for separation; the solvent is a 15% methanol water (containing 0.1 mg / ml ascorbic acid) solution. According to the analysis method, p-aminophenol in the compound paracetamol and chlorpheniramine maleate granules can be accurately and quantitatively detected, the separation degree of a p-aminophenol peak and an adjacent impurity peak meets the requirement, and the method is high in specificity, good in durability and high in sensitivity.

Description

Technical Field

[0001] This invention relates to a method for detecting para-aminophenol in compound acetaminophen and chlorpheniramine maleate granules, and relates to the field of chemical drug analysis methods. Background Technology

[0002] Drug purity reflects the quality of a drug. Related substances are the main factor affecting drug purity. These mainly include starting materials, reagents, intermediates, and byproducts introduced during drug production, as well as specific impurities such as degradation products, polymers, or crystal form transformations generated during production, transportation, and storage. Different synthetic routes and production processes produce different related substances. Therefore, it is necessary to establish scientific and comprehensive detection methods to accurately detect and monitor all related substances in drugs.

[0003] Compound acetaminophen and chlorpheniramine maleate granules are a cold medicine, a compound preparation that can effectively relieve various symptoms caused by the cold, such as fever, headache, sore throat, nasal congestion, runny nose, and sneezing. Its main components are acetaminophen, chlorpheniramine maleate, artificial bezoar, and caffeine. Acetaminophen primarily works by inhibiting prostaglandin synthesis, thus exerting its antipyretic and analgesic effects. It causes peripheral vasodilation, leading to sweating and thus reducing fever. In addition, it also has peripheral analgesic effects. However, acetaminophen accounts for 90.6% of the total of the four main components and is hepatotoxic; therefore, the rational use of acetaminophen should be carefully considered. As a starting material in the synthesis process of acetaminophen, incomplete acylation may carry over into the final product. Acetaminophen is highly toxic, causing skin and mucous membrane irritation, mutagenic and teratogenic effects, and can also cause acute poisoning. Therefore, it is necessary to control the content of para-aminophenol in compound acetaminophen and chlorpheniramine granules, but the current standard for compound acetaminophen and chlorpheniramine granules does not specify a method for detecting para-aminophenol impurities.

[0004] For the reasons mentioned above, this invention provides a method for detecting para-aminophenol in compound acetaminophen and chlorpheniramine maleate granules, which ensures efficient separation and accurate quantification of para-aminophenol from active ingredients or other unknown impurities, giving it good specificity, repeatability and accuracy, and better ensuring the safety and efficacy of the drug. Summary of the Invention

[0005] The purpose of this invention is to provide a method for detecting para-aminophenol in compound acetaminophen and chlorpheniramine granules, in order to solve the above-mentioned problems. By optimizing chromatographic conditions, para-aminophenol can be detected, thereby improving the quality control technology of compound acetaminophen and chlorpheniramine granules and thus improving the safety of medication for patients.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] On the one hand, this invention provides a method for detecting para-aminophenol in compound acetaminophen and chlorpheniramine maleate granules, which is a high-performance liquid chromatography method, and the chromatographic conditions are as follows:

[0008] Column: Non-polar column, packed with octadecylsilane-bonded silica gel;

[0009] Flow rate: 0.9-1.1 mL / min;

[0010] Column temperature: 28-32℃;

[0011] Detection wavelength: 255-259nm;

[0012] Mobile phase:

[0013] Mobile phase A: disodium hydrogen phosphate solution;

[0014] Mobile phase B: Methanol;

[0015] Solvent: 15% methanol solution;

[0016] The elution method is gradient elution, wherein the gradient elution is as follows:

[0017] 0-15 min, mobile phase A: 92%-88% changed to 75%, mobile phase B: 8%-12% changed to 25%;

[0018] 15-30 min, mobile phase A: 75% to 45%, mobile phase B: 25% to 55%;

[0019] 30-44 min, mobile phase A: 45%, mobile phase B: 55%;

[0020] 44.1-55 min, mobile phase A: 92%-88%, mobile phase B: 8%-12%.

[0021] Preferably, the chromatographic column is a Welch Xtimate. ® C18 4.6×250mm, 5μm or ThermoAcclaim TM 120 C18 4.6×250mm, 5μm; more preferably, the chromatographic column is a Welch Xtimate. ® C184.6×250mm, 5μm.

[0022] Preferably, the flow rate is 1.0 ml / min.

[0023] Preferably, the column temperature is 30°C.

[0024] Preferably, the detection wavelength is 257 nm.

[0025] Preferably, the injection volume is 20 μl.

[0026] Preferably, the concentration of the disodium hydrogen phosphate solution is 0.025 mol / L.

[0027] Preferably, the mobile phase A is a disodium hydrogen phosphate solution with a pH of 6.6.

[0028] Preferably, the pH of the disodium hydrogen phosphate solution is adjusted with phosphoric acid.

[0029] Preferably, ascorbic acid is added to the solvent as a stabilizer at a concentration of 0.1 mg / ml.

[0030] Preferably, the gradient elution is as follows:

[0031]

[0032] Preferably, the above detection method includes the following steps:

[0033] (1) Stock solution of p-aminophenol reference standard: Accurately weigh an appropriate amount of p-aminophenol reference standard and dilute it quantitatively with solvent to a concentration of 0.5 mg / ml.

[0034] (2) Reference solution: Take the para-aminophenol reference standard and dilute it quantitatively with solvent to a solution of 5 μg / ml.

[0035] (3) Test solution: Grind compound acetaminophen and chlorpheniramine granules into a fine powder, add solvent and dilute quantitatively to prepare a solution containing acetaminophen at a concentration of 5 mg / ml, and take the filtrate.

[0036] (4) Test sample + impurity solution: Grind compound acetaminophen and chlorpheniramine granules into a fine powder, accurately measure an appropriate amount of para-aminophenol reference stock solution, add solvent to quantitatively dilute to prepare a solution containing acetaminophen concentration of 5 mg / ml and para-aminophenol concentration of 50 μg / ml, and take the filtrate.

[0037] (5) Accurately measure 20 μl each of the reference solution, the test solution, and the test solution + impurity solution, and inject them into the liquid chromatograph for detection.

[0038] On the other hand, the present invention provides the application of the above-described detection method in the quality control of compound acetaminophen and chlorpheniramine granules.

[0039] This invention has the following advantages:

[0040] The technical solution of this invention uses gradient elution in a high-performance liquid chromatograph and adds disodium hydrogen phosphate to the mobile phase. This ensures effective separation of aminophenol and other impurities while also ensuring that the peak shapes of each chromatographic peak meet the requirements and accurately quantifies the related substances of the product. The method has high sensitivity, good specificity and robustness. Attached Figure Description

[0041] The accompanying drawings, which constitute a part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of this application. In the drawings:

[0042] Figure 1 This is the chromatogram of the blank solvent in Example 1.

[0043] Figure 2 The chromatogram is of the p-aminophenol reference standard in Example 1. The retention time of p-aminophenol is 5.151 min.

[0044] Figure 3 The chromatogram of the test sample in Example 1 shows that the retention time of p-aminophenol is 5.127 min.

[0045] Figure 4 The chromatogram for the test sample plus impurities in Example 1 shows that the retention time of p-aminophenol is 5.152 min.

[0046] Figure 5 This is the chromatogram of the blank solvent in Example 2.

[0047] Figure 6 This is the chromatogram of the blank excipient in Example 2.

[0048] Figure 7 The chromatogram of the para-aminophenol reference standard in Example 2 shows that the retention time of para-aminophenol is 5.217 min.

[0049] Figure 8 The chromatogram of the test sample in Example 2 shows that the retention time of p-aminophenol is 5.108 min.

[0050] Figure 9 The chromatogram for the test sample plus impurities in Example 2 shows that the retention time of p-aminophenol is 5.215 min.

[0051] Figure 10 This is the chromatogram of the blank solvent in Example 3.

[0052] Figure 11 This is the chromatogram of the blank excipient in Example 3.

[0053] Figure 12 The chromatogram of the p-aminophenol reference standard in Example 3 shows that the retention time of p-aminophenol is 5.082 min.

[0054] Figure 13 The chromatogram of the test sample in Example 3 shows that the retention time of p-aminophenol is 5.083 min.

[0055] Figure 14 The chromatogram for the test sample plus impurities in Example 3 shows that the retention time of p-aminophenol is 5.077 min.

[0056] Figure 15 This is the chromatogram of the blank solvent in Example 4.

[0057] Figure 16 This is the chromatogram of the blank excipient in Example 4.

[0058] Figure 17 The chromatogram of the para-aminophenol reference standard in Example 4 shows that the retention time of para-aminophenol is 5.697 min.

[0059] Figure 18 The chromatogram of the test sample in Example 4 shows that the retention time of p-aminophenol is 5.593 min.

[0060] Figure 19 The chromatogram for the test sample plus impurities in Example 4 shows that the retention time of p-aminophenol is 5.696 min.

[0061] Figure 20 This is the chromatogram of the blank solvent in Example 5.

[0062] Figure 21 This is the chromatogram of the blank excipient in Example 5.

[0063] Figure 22 The chromatogram of the para-aminophenol reference standard in Example 5 shows that the retention time of para-aminophenol is 4.692 min.

[0064] Figure 23 The chromatogram of the test sample in Example 5 shows that the retention time of p-aminophenol is 4.615 min.

[0065] Figure 24 The chromatogram for the sample and impurities in Example 5 shows that the retention time of p-aminophenol is 4.693 min.

[0066] Figure 25 This is the chromatogram of the blank solvent in Example 6.

[0067] Figure 26 This is the chromatogram of the blank excipient in Example 6.

[0068] Figure 27 The chromatogram of the para-aminophenol reference standard in Example 6 shows that the retention time of para-aminophenol is 5.153 min.

[0069] Figure 28 The chromatogram of the test sample in Example 6 shows that the retention time of p-aminophenol is 5.069 min.

[0070] Figure 29 The chromatogram for the test sample plus impurities in Example 6 shows that the retention time of p-aminophenol is 5.150 min.

[0071] Figure 30 This is the chromatogram of the blank solvent in Example 7.

[0072] Figure 31 This is the chromatogram of the blank excipient in Example 7.

[0073] Figure 32 The chromatogram is for the para-aminophenol reference standard in Example 7. The retention time of para-aminophenol is 5.151 min.

[0074] Figure 33 The chromatogram of the test sample in Example 7 shows that the retention time of p-aminophenol is 5.071 min.

[0075] Figure 34 The chromatogram for the test sample plus impurities in Example 7 shows that the retention time of p-aminophenol is 5.150 min.

[0076] Figure 35 This is the chromatogram of the blank solvent in Example 8.

[0077] Figure 36 This is the chromatogram of the blank excipient in Example 8.

[0078] Figure 37 The chromatogram is for the para-aminophenol reference standard in Example 8. The retention time of para-aminophenol is 5.558 min.

[0079] Figure 38 The chromatogram of the test sample in Example 8 shows that the retention time of p-aminophenol is 5.602 min.

[0080] Figure 39 The chromatogram for the sample and impurities in Example 8 shows that the retention time of p-aminophenol is 5.556 min.

[0081] Figure 40 This is the chromatogram of the blank solvent in Example 9.

[0082] Figure 41 The chromatogram of the para-aminophenol reference standard in Example 9 shows that the retention time of para-aminophenol is 4.768 min.

[0083] Figure 42 The chromatogram of the test sample in Example 9 shows that the retention time of p-aminophenol is 4.760 min.

[0084] Figure 43The chromatogram for the test sample plus impurities in Example 9 shows that the retention time of p-aminophenol is 4.754 min.

[0085] Figure 44 This is the chromatogram of the blank solvent in Example 10.

[0086] Figure 45 This is the chromatogram of the blank excipient in Example 10.

[0087] Figure 46 The chromatogram of the para-aminophenol reference standard in Example 10 shows that the retention time of para-aminophenol is 5.589 min.

[0088] Figure 47 The chromatogram of the test sample in Example 10 shows that the retention time of p-aminophenol is 5.565 min.

[0089] Figure 48 The chromatogram for the test sample + impurities in Example 10 shows that the retention time of p-aminophenol is 5.558 min.

[0090] Figure 49 This is a linear relationship graph for para-aminophenol. Detailed Implementation

[0091] To make the technical solution and advantages of this patent application clearer, various exemplary embodiments of the present invention are now described in detail with reference to the accompanying drawings. This detailed description should not be considered as a limitation of the present invention, but rather a more detailed description of certain aspects, features, and embodiments of the present invention.

[0092] Based on the embodiments described in the implementation details, other embodiments obtained by those skilled in the art without inventive effort are all within the scope of protection of this invention. It is worth noting that, unless specific conditions are specified in this invention, conventional conditions or the conditions recommended by the manufacturer of the equipment used can be followed. If the manufacturer of the reagents or instruments used is not specified, conventional products purchased from the market can be used. And if the technical means or methods involved are not specified with specific conditions, they should be carried out according to existing methods in the field. The technical and scientific terms used in the embodiments have meanings commonly understood by those skilled in the art to which this invention pertains.

[0093] The present invention will be further described in detail below with reference to specific embodiments. The preparation method of the blank excipient solution in the embodiments is as follows: take an appropriate amount of blank excipient, grind it into a fine powder, accurately weigh about 9.0g, place it in a 50ml volumetric flask, add an appropriate amount of solvent, sonicate to dissolve, and dilute to the mark, then shake well.

[0094] The sources of the p-aminophenol reference standard, test sample, and other reagents used in this invention are shown in the table below:

[0095]

[0096] Example 1

[0097] This embodiment provides a method for detecting para-aminophenol in compound acetaminophen and chlorpheniramine maleate granules, including the following steps:

[0098] (1) Reference solution, test solution, test solution + impurity solution

[0099] Solvent: Measure 850ml of water and 150ml of methanol, mix well, add 0.1g of ascorbic acid (stabilizer), sonicate to dissolve, and mix well to obtain the final product.

[0100] p-Aminophenol reference standard stock solution: Accurately weigh an appropriate amount of p-aminophenol reference standard, add solvent and quantitatively dilute to a concentration of 0.5 mg / ml.

[0101] Reference solution: Accurately weigh an appropriate amount of p-aminophenol reference standard, add solvent and quantitatively dilute to a solution containing approximately 5 μg per ml.

[0102] Test solution: Take an appropriate amount of compound acetaminophen and chlorpheniramine granules, grind them into a fine powder (approximately equivalent to 250 mg of acetaminophen), accurately weigh them, add solvent to quantitatively dilute to prepare a solution containing 5 mg / ml of acetaminophen, and collect the filtrate.

[0103] Test sample + impurity solution: Take compound acetaminophen and chlorpheniramine granules and grind them into a fine powder (approximately equivalent to 250 mg of acetaminophen). Accurately measure an appropriate amount of acetaminophen reference stock solution and add solvent to quantitatively dilute it to prepare a solution containing 5 mg / ml of acetaminophen and 50 μg / ml of acetaminophen. Take the filtrate.

[0104] (2) High performance liquid chromatography detection conditions

[0105] Instrument: Agilent 1260 Infinity high performance liquid chromatograph, DAD detector, detection wavelength: 257nm;

[0106] Column: Welch Xtimate ® C18 (4.6×250mm, 5μm) chromatographic column;

[0107] Mobile phases: Mobile phase A and mobile phase B;

[0108] The preparation of mobile phase A includes: preparing a 0.025 mol / L disodium hydrogen phosphate solution with pH 6.0 (take 8.95 g of disodium hydrogen phosphate dodecahydrate, add 1 L of water, sonicate to dissolve, and then adjust the pH to 6.6 with phosphoric acid); mobile phase B: methanol;

[0109] The flow rate was 1.0 mL / min; the column temperature was 30 °C; the detection wavelength was 257 nm; and the injection volume was 20 μL.

[0110] Gradient elution is performed according to the gradient table.

[0111] Table 1. Gradient elution program (volume ratio)

[0112]

[0113] (3) Detection steps

[0114] Accurately measure 20 μL each of blank solvent, p-aminophenol reference solution, test solution, and test solution + impurity solution, inject them into the liquid chromatograph, and perform detection according to the conditions in step (2). Record the chromatogram. Figure 1 , Figure 2 , Figure 3 and Figure 4 .exist Figure 2 In the study, the theoretical plate number of the para-aminophenol peak was 18132 > 5000, and the tailing factor was 1.19, which met the requirements. Figure 4 In the results, the theoretical plate number calculated based on the para-aminophenol peak is 17832 > 5000, and the resolution between the para-aminophenol and para-acetaminophen peaks is 30.15 > 1.5, which meets the requirements.

[0115] Example 2

[0116] This embodiment refers to the detection method of Embodiment 1, the only difference being that in the high-performance liquid chromatography (HPLC) detection conditions of step (2), the HPLC instrument is a Thermo U3000 and the column temperature is 28℃. Accurately measure 20 μL each of blank solvent, blank auxiliary solution, p-aminophenol reference solution, test solution, and test solution + impurity solution, inject them into the HPLC instrument, and record the chromatogram. See [link to HPLC method]. Figure 5 , Figure 6 , Figure 7 , Figure 8 and Figure 9 The results showed that changing the column temperature to this embodiment did not interfere with sample detection with the blank solvent and blank excipient. The theoretical plate number calculated based on p-aminophenol was greater than 5000. The recovery rate in the test sample + impurity solution was in the range of 90% to 108%, which met the acceptable standard, and the method had good robustness.

[0117] Table 2. Chromatogram data at a column temperature of 28℃

[0118]

[0119] Note: " / " indicates that the recovery rate of p-aminophenol is not calculated. The data for p-aminophenol reference standard and test sample + impurities are from the first injection of reference standard 1 solution and the first test sample + impurities solution, respectively.

[0120] Example 3

[0121] This embodiment refers to the detection method of Embodiment 1, with the only difference being: in the high-performance liquid chromatography (HPLC) detection conditions of step (2), the HPLC instrument is a Thermo U3000, the column temperature is 32℃, and 20 μL each of blank solvent, blank auxiliary solution, p-aminophenol reference solution, test solution, and test solution + impurity solution are accurately measured and injected into the HPLC instrument. The chromatogram is recorded. See Figure 10 , Figure 11 , Figure 12 , Figure 13 ,and Figure 14 The results showed that changing the column temperature to this embodiment did not interfere with sample detection with the blank solvent and blank excipient. The theoretical plate number calculated based on p-aminophenol was greater than 5000. The recovery rate in the test sample + impurity solution was in the range of 90% to 108%, which met the acceptable standard, and the method had good robustness.

[0122] Table 3. Chromatogram data at a column temperature of 32℃

[0123]

[0124] Note: " / " indicates that the recovery rate of p-aminophenol is not calculated. The data for p-aminophenol reference standard and test sample + impurities are from the first injection of reference standard 1 solution and the first test sample + impurities solution, respectively.

[0125] Example 4

[0126] This embodiment refers to the detection method of Example 1, with the only difference being: in the high-performance liquid chromatography (HPLC) detection conditions of step (2), the flow rate is 0.9 ml / min, and 20 μL each of blank solvent, blank auxiliary solution, p-aminophenol reference solution, test solution, and test solution + impurity solution are accurately measured and injected into the HPLC instrument. The chromatogram is recorded. See Figure 15 , Figure 16 , Figure 17 , Figure 18 and Figure 19 The results showed that changing the flow rate to this embodiment did not interfere with the sample detection with the blank solvent and blank excipient. The theoretical plate number calculated based on p-aminophenol was greater than 5000. The recovery rate in the test sample + impurity solution was in the range of 90% to 108%, which met the acceptable standard, and the method had good robustness.

[0127] Table 4. Chromatogram data at a flow rate of 0.9 ml / min

[0128]

[0129] Note: " / " indicates that the recovery rate of p-aminophenol is not calculated. The data for p-aminophenol reference standard and test sample + impurities are from the first injection of reference standard 1 solution and the first test sample + impurities solution, respectively.

[0130] Example 5

[0131] This embodiment refers to the detection method of Example 1, with the only difference being: in the high-performance liquid chromatography (HPLC) detection conditions of step (2), the flow rate is 1.1 ml / min, and 20 μL each of blank solvent, blank auxiliary solution, p-aminophenol reference solution, test solution, and test solution + impurity solution are accurately measured and injected into the HPLC instrument. The chromatogram is recorded. See Figure 20 , Figure 21 , Figure 22 , Figure 23 and Figure 24 The results showed that changing the flow rate to this embodiment did not interfere with the sample detection with the blank solvent and blank excipient. The theoretical plate number calculated based on p-aminophenol was greater than 5000. The recovery rate in the test sample + impurity solution was in the range of 90% to 108%, which met the acceptable standard, and the method had good robustness.

[0132] Table 5. Chromatogram data at a flow rate of 1.1 ml / min

[0133]

[0134] Note: " / " indicates that the recovery rate of p-aminophenol is not calculated. The data for p-aminophenol reference standard and test sample + impurities are from the first injection of reference standard 1 solution and the first test sample + impurities solution, respectively.

[0135] Example 6

[0136] This embodiment refers to the detection method of Embodiment 1, the only difference being: in the high performance liquid chromatography detection conditions of step (2), the detection wavelength is 255 nm, and 20 μL each of blank solvent, blank auxiliary solution, p-aminophenol reference solution, test solution, and test solution + impurity solution are accurately measured and injected into the liquid chromatograph, and the chromatogram is recorded. See Figure 25 , Figure 26 , Figure 27 , Figure 28 and Figure 29 The results showed that, when the wavelength was changed to that of this embodiment, the blank solvent and blank excipient did not interfere with the sample detection. The theoretical plate number calculated based on p-aminophenol was greater than 5000. The recovery rate in the test sample + impurity solution was in the range of 90% to 108%, which met the acceptable standard, and the method had good robustness.

[0137] Table 6. Chromatogram data under detection wavelength of 255 nm

[0138]

[0139] Note: " / " indicates that the recovery rate of p-aminophenol is not calculated. The data for p-aminophenol reference standard and test sample + impurities are from the first injection of reference standard 1 solution and the first test sample + impurities solution, respectively.

[0140] Example 7

[0141] This embodiment refers to the detection method of Embodiment 1, with the only difference being: in the high-performance liquid chromatography (HPLC) detection conditions of step (2), the detection wavelength is 259 nm. 20 μL each of the blank solvent, blank auxiliary solution, p-aminophenol reference solution, test solution, and test solution + impurity solution are accurately measured and injected into the HPLC instrument. The chromatogram is recorded. See [link to HPLC method]. Figure 30 , Figure 31 , Figure 32 , Figure 33 and 34 The results showed that, when the wavelength was changed to that of this embodiment, the blank solvent and blank excipient did not interfere with the sample detection. The theoretical plate number calculated based on p-aminophenol was greater than 5000. The recovery rate in the test sample + impurity solution was in the range of 90% to 108%, which met the acceptable standard, and the method had good robustness.

[0142] Table 7. Chromatogram data under detection wavelength of 259 nm

[0143]

[0144] Note: " / " indicates that the recovery rate of p-aminophenol is not calculated. The data for p-aminophenol reference standard and test sample + impurities are from the first injection of reference standard 1 solution and the first test sample + impurities solution, respectively.

[0145] Example 8

[0146] This embodiment refers to the detection method of Embodiment 1, the only difference being: in the high performance liquid chromatography detection conditions of step (2), the proportion of mobile phase B in the gradient elution program is reduced, and the gradient elution program table is as follows:

[0147] Table 8. Gradient elution program (volume ratio)

[0148]

[0149] Accurately measure 20 μL each of blank solvent, blank auxiliary solution, p-aminophenol reference solution, test solution, and test solution + impurity solution, inject them into the liquid chromatograph, and record the chromatogram. See [reference needed]. Figure 35 , Figure 36 , Figure 37 , Figure 38 and Figure 39 The results showed that changing the proportion of mobile phase B to that of this embodiment did not interfere with sample detection with the blank solvent and blank excipient. The theoretical plate number calculated based on p-aminophenol was greater than 5000. The recovery rate in the sample + impurity solution was in the range of 90% to 108%, which met the acceptable standard, and the method had good robustness.

[0150] Table 9. Chromatogram data under conditions of reduced proportion of mobile phase B in gradient elution program

[0151]

[0152] Note: " / " indicates that the recovery rate of p-aminophenol is not calculated. The data for p-aminophenol reference standard and test sample + impurities are from the first injection of reference standard 1 solution and the first test sample + impurities solution, respectively.

[0153] Example 9

[0154] This embodiment refers to the detection method of Embodiment 1, the only difference being: in the high performance liquid chromatography detection conditions of step (2), the proportion of mobile phase B in the gradient elution program is increased, and the gradient elution program table is as follows:

[0155] Table 10. Gradient elution program (volume ratio)

[0156]

[0157] Accurately measure 20 μL each of blank solvent, p-aminophenol reference solution, test solution, and test solution + impurity solution, inject them into the liquid chromatograph, and record the chromatogram. See [reference needed]. Figure 40 , Figure 41 , Figure 42 and Figure 43 The results showed that, by changing the proportion of mobile phase B to that of this embodiment, the blank solvent did not interfere with sample detection, the theoretical plate number calculated based on p-aminophenol was greater than 5000, and the recovery rate in the sample + impurity solution was in the range of 90% to 108%, which met the acceptable standard, and the method had good robustness.

[0158] Table 11. Chromatogram data under conditions of increased proportion of mobile phase B in gradient elution program

[0159]

[0160] Note: " / " indicates that the recovery rate of p-aminophenol is not calculated. The data for p-aminophenol reference standard and test sample + impurities are from the first injection of reference standard 1 solution and the first test sample + impurities solution, respectively.

[0161] Example 10

[0162] This embodiment refers to the detection method of Embodiment 1, the only difference being that: in the high performance liquid chromatography detection conditions of step (2), the chromatographic column is a Thermo Acclaim. TM For a 120 C18 HPLC system (4.6 × 250 mm, 5 μm), accurately measure 20 μL each of blank solvent, blank auxiliary solution, p-aminophenol reference solution, test solution, and test solution + impurity solution, inject them into the HPLC system, and record the chromatogram. See [reference needed]. Figure 44 , Figure 45 , Figure 46 , Figure 47 and Figure 48 The results showed that when the chromatographic column was changed to this embodiment, the blank solvent and blank excipient did not interfere with the sample detection. The theoretical plate number calculated based on p-aminophenol was greater than 5000. The recovery rate in the test sample + impurity solution was in the range of 90% to 108%, which met the acceptable standard, and the method had good robustness.

[0163] Table 12. Chromatogram data under the same specifications but different manufacturers' chromatographic column conditions

[0164]

[0165] Note: " / " indicates that the recovery rate of p-aminophenol is not calculated. The data for p-aminophenol reference standard and test sample + impurities are from the first injection of reference standard 1 solution and the first test sample + impurities solution, respectively.

[0166] Furthermore, in Examples 2-10, under each condition, the blank solvent and blank excipient do not interfere with the sample detection, and the theoretical plate number calculated based on para-aminophenol is greater than 5000; in the test sample + impurity solution, the solution recovery rate is in the range of 90% to 108%, and the RSD value is 1.84% which is less than 5.0%, meeting the acceptable standard, and the method has good robustness.

[0167] Test case

[0168] (1) Exclusivity

[0169] Forced degradation tests (including acid-base degradation, oxygen degradation, high temperature degradation at 60℃, and light degradation) were conducted on compound acetaminophen and chlorpheniramine granules to understand the degradation pathway and stability of the sample, and peak purity was calculated. Since the sample concentration was 5 mg / mL and the injection volume was 20 μL, the main peak was overloaded; therefore, each degradation solution needed to be diluted 1000 times to examine the purity of the main peak.

[0170] System suitability solution: Take the test solution and the para-aminophenol reference solution, and further dilute quantitatively with solvent to prepare a solution containing 0.25 mg / ml para-aminophenol and 2.5 μg / ml para-aminophenol.

[0171] Table 13. Results of acetaminophen peak purity

[0172]

[0173] Conclusion: Under the chromatographic conditions of Example 1, the system suitability solution injection results were as follows: the theoretical plate number calculated based on the p-aminophenol peak was 17832 > 5000, and the resolution between the p-aminophenol peak and the acetaminophen peak was 30.15 > 1.5, which met the requirements. Neither the blank solvent nor the blank auxiliary solvent interfered with sample detection. The degradation peaks of this product under acid, alkali, oxygen, high temperature (60℃), and light-induced degradation conditions did not interfere with the detection of the p-aminophenol peak, and the purity of the acetaminophen peak met the requirements. The method exhibits good specificity.

[0174] (2) Sample injection precision

[0175] Under the chromatographic conditions of Example 1, the system suitability of the method was tested by injecting the test sample and impurity solution (6 consecutive injections). The test results are shown in Table 14.

[0176] Table 14. Results of the injection precision test

[0177]

[0178] Conclusion: After six consecutive injections of the test sample and impurity solution, the RSD of the peak area of ​​para-aminophenol was 0.11% < 2.0%, which meets the acceptable standard, indicating that the injection precision of this method is good.

[0179] (3) Solution stability

[0180] Under the chromatographic conditions of Example 1, a p-aminophenol reference solution was prepared and measured at room temperature for different time periods (a total of 21 hours were investigated). The results are shown in Table 15:

[0181] Table 15. Stability results of p-aminophenol reference solution

[0182]

[0183] Note: " / " indicates that the change value (%) is not calculated.

[0184] Results: The change in peak area of ​​p-aminophenol reference solution within 21 h relative to 0 h was less than 2.0%, indicating that p-aminophenol reference solution was stable within 21 h at room temperature.

[0185] (4) Adsorption properties of filter membrane

[0186] Under the chromatographic conditions of Example 1, the test sample + impurity solution was prepared according to Table 16, filtered through a 0.45 μm organic filter membrane, and the adsorption capacity of the filter membrane for p-aminophenol was investigated. The results are shown in Table 17.

[0187] Table 16. Preparation of Filter Membrane Adsorption Solution

[0188]

[0189] Table 17. Results of the filter membrane adsorption test for the test sample + impurity solution

[0190]

[0191] Note: " / " indicates that the change value (%) is not calculated.

[0192] Conclusion: After filtration through a filter membrane, the changes in the peak area of ​​p-aminophenol compared to the peak area of ​​p-aminophenol after discarding 1 ml, 2 ml, and 5 ml of the initial filtrate were 0.49%, 0.62%, and 0.07%, respectively, all ≤ ±2.0%, meeting the acceptable standard. The test solution and the spiked test solution were prepared by filtering and discarding 2 ml of the initial filtrate.

[0193] (5) Limit of quantitation and limit of detection

[0194] Under the chromatographic conditions of Example 1, the p-aminophenol reference solution was gradually diluted to prepare limit of quantitation (LOQ) and limit of detection (LOD) solutions. The solution with a signal-to-noise ratio (SNR) of 10 ≤ S / N ≤ 15 was used as the LQ solution; the solution with a SNR of 3 ≤ S / N ≤ 5 was used as the LOD solution. The detection results of the LQ and LOD solutions are shown in Tables 18-19.

[0195] Table 18. Results of the limit of quantitation test for para-aminophenol

[0196]

[0197] Note: " / " indicates that the RSD (%) value is not calculated.

[0198] Table 19. Results of the detection limit test for para-aminophenol

[0199]

[0200] Conclusion: After six consecutive injections of the limit of quantitation solution, the signal-to-noise ratios (SNRs) of p-aminophenol were 11.16, 11.61, 10.08, 11.98, 12.46, and 10.37, respectively, all within the range of 10 to 15; the RSD of the peak area was 3.01% < 10.0%, which meets the acceptable standard, indicating that this method has good sensitivity.

[0201] (6) Linearity and Range

[0202] Under the chromatographic conditions of Example 1, the relationship between peak area and concentration in the range of limit of quantitation - 200% for para-aminophenol reference standard was investigated.

[0203] Linear stock solution: Take an appropriate amount of p-aminophenol reference standard and dilute it with solvent to a solution of 0.05 mg / ml.

[0204] Prepare linear solutions from linear stock solutions, as shown in Table 20:

[0205] Table 20. Preparation of linear solutions of p-aminophenol

[0206]

[0207] A linear regression was performed with concentration on the x-axis and peak area on the y-axis. The linear relationship is shown in [the diagram]. Figure 49 The results are shown in Table 21:

[0208] Table 21. Linearity and range results for p-aminophenol

[0209]

[0210] in conclusion:

[0211] The correlation coefficient r = 0.9999; the RSD of the response value is 4.53%, which is less than 5.0%.

[0212] The ratio of the Y-axis intercept to the 100% response value is 0.37%, which is less than 25.0%.

[0213] All of the above meet the acceptable criteria; that is, for para-aminophenol in the concentration range of 0.11~10.74μg / ml, the linear equation is: Y=7.4975x+0.1483, which shows a good linear relationship.

[0214] (7) Accuracy

[0215] Under the chromatographic conditions of Example 1, a certain amount of p-aminophenol reference standard stock solution was added to the test sample solution to prepare concentrations of 50%, 100%, and 150% for testing. The measured amount was calculated using the external standard method, and the ratio of the measured amount to the actual added amount was used as the recovery rate. The results are shown in Table 22.

[0216] Table 22. Results of Content Accuracy Test

[0217]

[0218] Conclusion: The maximum recovery rate of the nine solutions was 99.37%, the minimum was 95.75%, and the recovery rates at 50%, 100%, and 150% were 98.6%, all within the range of 90% to 108%. The RSD of the recovery rate of the nine accuracy solutions was 1.86%, which is less than 5.0%, meeting the acceptable standard and indicating that the method has good accuracy.

[0219] (8) Repeatability

[0220] Under the chromatographic conditions of Example 1, blank solvent, p-aminophenol stock solution, p-aminophenol reference solution, test solution, and test solution + impurity solution (prepared in parallel 6 times) were prepared. The precision of the detection results of the 6 test solution + impurity solution was investigated, and the results are shown in Table 23:

[0221] Table 23. Results of Repeatability Tests

[0222]

[0223] Conclusion: The average content of p-aminophenol in the six spiked test solutions was 100.38%, and the RSD value was 0.42%, which is less than 3.0%, meeting the acceptable standard, indicating that the method has good repeatability.

[0224] (9) Intermediate precision

[0225] Under the chromatographic conditions of Example 1, the repeatability test was conducted on different dates, by different analysts, and using different instruments. Six parallel test solutions (prepared as under the repeatability section) were prepared and compared with the repeatability results to examine the precision of the 12 sample determinations. The results are shown in Table 24.

[0226] Table 24. Results of intermediate precision tests

[0227]

[0228] Conclusion: The average content of p-aminophenol in the 12 spiked test solutions was 100.37%, and the RSD value was 0.56%, which is less than 6.0%, meeting the acceptable standard and indicating that the intermediate precision of this method is good.

[0229] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for detecting para-aminophenol in compound acetaminophen and chlorpheniramine maleate granules, characterized in that, High performance liquid chromatography (HPLC) was used, and the chromatographic conditions were as follows: Chromatographic column: Non-polar chromatographic column, packed with octadecylsilane-bonded silica gel; preferably, the chromatographic column is Welch Xtimate. ® C18 4.6×250mm, 5μm or Thermo Acclaim TM 120 C18 4.6×250mm, 5μm; Flow rate: 0.9-1.1 mL / min; preferably, flow rate: 1.0 mL / min; Column temperature: 28-32℃; preferably, column temperature: 30℃; Detection wavelength: 255-259nm; Mobile phase: Mobile phase A: disodium hydrogen phosphate solution; Mobile phase B: Methanol; Solvent: 15% methanol aqueous solution; The elution method is gradient elution, wherein the gradient elution is as follows: 0-15 min, mobile phase A: 92%-88% changed to 75%, mobile phase B: 8%-12% changed to 25%; 15-30 min, mobile phase A: 75% to 45%, mobile phase B: 25% to 55%; 30-44 min, mobile phase A: 45%, mobile phase B: 55%; 44.1-55 min, mobile phase A: 92%-88%, mobile phase B: 8%-12%.

2. The detection method according to claim 1, characterized in that, The mobile phase A is a disodium hydrogen phosphate solution with a pH of 6.

6.

3. The detection method according to claim 2, characterized in that, The concentration of the disodium hydrogen phosphate solution is 0.025 mol / L.

4. The detection method according to claim 1, characterized in that, The volume fraction of methanol and water in the solvent is 15%-85%.

5. The detection method according to claim 1, characterized in that, Ascorbic acid is added to the solvent as a stabilizer at a concentration of 0.1 mg / ml.

6. The detection method according to claim 1, characterized in that, The gradient elution is as follows:

7. The detection method according to any one of claims 1-8, characterized in that, Includes the following steps: (1) Stock solution of p-aminophenol reference standard: Accurately weigh an appropriate amount of p-aminophenol reference standard and dilute it quantitatively with solvent to a concentration of 0.5 mg / ml. (2) Reference solution: Take the para-aminophenol reference standard and dilute it quantitatively with solvent to a solution of 5 μg / ml. (3) Test solution: Grind compound acetaminophen and chlorpheniramine granules into a fine powder, add solvent and dilute quantitatively to prepare a solution containing acetaminophen at a concentration of 5 mg / ml, and take the filtrate. (4) Test sample + impurity solution: Grind compound acetaminophen and chlorpheniramine granules into a fine powder, accurately measure an appropriate amount of para-aminophenol reference stock solution, add solvent to quantitatively dilute to prepare a solution containing acetaminophen concentration of 5 mg / ml and para-aminophenol concentration of 50 μg / ml, and take the filtrate. (5) Accurately measure 20 μl each of the reference solution, the test solution, and the test solution + impurity solution, and inject them into the liquid chromatograph for detection.

8. The application of the detection method according to any one of claims 1-7 in the quality control of compound acetaminophen and chlorpheniramine granules.

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