Detection method of compound sodium picosulfate oral solution

Abstract

The invention provides a method for detecting a compound sodium picosulfate oral solution. The method is used for detecting at least one of methylparaben, propylparaben and 4-hydroxybenzoic acid in the compound sodium picosulfate oral solution. A high performance liquid chromatography method is adopted for determination, and the chromatographic conditions of the high performance liquid chromatography method are as follows: a chromatographic column comprises a reverse C18 chromatographic column or an efficacy equivalent column; the mobile phase comprises a mobile phase A and a mobile phase B, the mobile phase A is a mixed solution of an ammonium acetate buffer solution and methanol, and the mobile phase B is acetonitrile; the elution mode is gradient elution. According to the detection method, methylparaben, propylparaben and 4-hydroxybenzoic acid can be effectively separated and accurately quantified, interference generated by a blank solvent and an auxiliary material solution can be eliminated, and the quality controllability of a product is ensured.

Description

Technical Field

[0001] The present application relates to the technical field of drug analysis, and in particular to a method for detecting compound sodium picosulfate oral solution. Background Art

[0002] Sodium picosulfate is a stimulant laxative that is metabolized by colonic bacteria into bis(p-hydroxyphenyl)-pyridyl-2-methane (BHPM). BHPM effectively promotes intestinal motility and is primarily used to treat constipation. Compound sodium picosulfate oral solution contains various preservatives, such as methylparaben and propylparaben. 4-hydroxybenzoic acid is produced by the degradation of preservatives under conditions such as acidity and alkalinity. Excessive 4-hydroxybenzoic acid can irritate the skin, eyes, and gastrointestinal tract, and can disrupt the endocrine system. Therefore, monitoring the levels of preservatives and 4-hydroxybenzoic acid is necessary.

[0003] Although the Chinese Pharmacopoeia records the detection method for methylparaben, it cannot be applied to compound sodium picosulfate oral solution because 4-hydroxybenzoic acid has a high polarity, weak retention and is interfered by excipients. In addition, the polarity difference between propylparaben and 4-hydroxybenzoic acid is large, and detection in the same method will extend the running time. Summary of the Invention

[0004] Based on this, it is necessary to provide a detection method for compound sodium picosulfate oral solution to simultaneously detect methylparaben, propylparaben and 4-hydroxybenzoic acid in compound sodium picosulfate oral solution.

[0005] The technical solution of this application includes the following:

[0006] A method for detecting a compound sodium picosulfate oral solution, for detecting at least one of methylparaben, propylparaben, and 4-hydroxybenzoic acid in the compound sodium picosulfate oral solution; the method employs high performance liquid chromatography (HPLC), wherein the chromatographic conditions of the HPLC method include:

[0007] The chromatographic column includes a reversed-phase C18 column or a column of equivalent performance;

[0008] The mobile phase includes mobile phase A and mobile phase B, wherein the mobile phase A is a mixture of ammonium acetate buffer and methanol, and the mobile phase B is acetonitrile;

[0009] Gradient elution was used, and the gradient elution procedure included: from 0 to 16 minutes, the volume percentage of the mobile phase A was reduced from 85%-95% to 70%-80%, and the volume percentage of the mobile phase B was increased from 5%-15% to 20%-30%; from 16 minutes to 16.1 minutes, the volume percentage of the mobile phase A was reduced from 70%-80% to 55%-65%, and the volume percentage of the mobile phase B was increased from 20%-30% to 35%-45%; from 16.1 minutes to 25 minutes n, maintain the volume percentage of the mobile phase A at 55%-65%, and the volume percentage of the mobile phase B at 35%-45%; 25min-25.1min, increase the volume percentage of the mobile phase A from 55%-65% to 85%-95%, and reduce the volume percentage of the mobile phase B from 35%-45% to 5%-15%; 25.1min-35min, maintain the volume percentage of the mobile phase A at 85%-95%, and the volume percentage of the mobile phase B at 5%-15%.

[0010] In some embodiments, the column temperature of the HPLC method is 25°C-35°C.

[0011] In some embodiments, the detection wavelength of the HPLC method is 240 nm-263 nm.

[0012] In some embodiments, in the mobile phase A, the volume proportion of the ammonium acetate buffer is 90%-95%, and the volume proportion of the methanol is 5%-10%.

[0013] In some embodiments, the ammonium acetate buffer comprises ammonium acetate at a molar concentration of 25 mmol / L-100 mmol / L.

[0014] In some embodiments, the pH of the ammonium acetate buffer is 3.85-4.15.

[0015] In some embodiments, the flow rate of the HPLC method is 0.9 mL / min-1.1 mL / min.

[0016] In some embodiments, the injection volume of the HPLC method is 10 μL-60 μL.

[0017] In some embodiments, the reversed-phase C18 chromatography column has an inner diameter and a length of 4.6 mm and 150 mm, respectively, and a filler particle size of 3 μm.

[0018] In some embodiments, the chromatography column further comprises a ghost peak trapping column.

[0019] In some embodiments, the gradient elution procedure includes: 0-16 min, reducing the volume percentage of the mobile phase A from 90% to 75%, and increasing the volume percentage of the mobile phase B from 10% to 25%; 16 min-16.1 min, reducing the volume percentage of the mobile phase A from 75% to 60%, and increasing the volume percentage of the mobile phase B from 25% to 40%; 16.1 min-25 min, maintaining the volume percentage of the mobile phase A at 60%, and the volume percentage of the mobile phase B at 40%; 25 min-25.1 min, increasing the volume percentage of the mobile phase A from 60% to 90%, and reducing the volume percentage of the mobile phase B from 40% to 10%; 25.1 min-35 min, maintaining the volume percentage of the mobile phase A at 90%, and the volume percentage of the mobile phase B at 10%.

[0020] In some embodiments, the column temperature of the HPLC method is 28°C-32°C.

[0021] In some embodiments, the sample measured by high performance liquid chromatography is diluted with a diluent comprising methanol and water, wherein the volume proportion of the methanol is 0-10%. In some embodiments, the sample measured by high performance liquid chromatography is diluted with a diluent comprising methanol and water, wherein the volume proportion of the methanol is 5%-10%.

[0022] The above-mentioned detection method for compound sodium picosulfate oral solution adopts mobile phase A of ammonium acetate buffer and methanol mixture and mobile phase B of acetonitrile, in conjunction with a specific gradient elution program, and can simultaneously detect methylparaben, propylparaben and 4-hydroxybenzoic acid in compound sodium picosulfate oral solution, solves the problem that 4-hydroxybenzoic acid is interfered with by excipients, can effectively separate and accurately quantify methylparaben, propylparaben and 4-hydroxybenzoic acid, and shortens the detection time by one half, is conducive to batch detection of compound sodium picosulfate oral solution, and improves detection efficiency. In addition, this detection method can eliminate the interference generated by blank solvent and excipient solution, can be used for the preparation process of compound sodium picosulfate oral solution and the quality control of final product, ensures the quality controllability of product, and reduces safety risk. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more fully understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present application. Those skilled in the art can also derive other drawings based on these drawings without inventive effort.

[0024] Figure 1is the chromatogram of the blank solution of Example 1;

[0025] Figure 2 It is the chromatogram of the blank excipient solution of Example 1;

[0026] Figure 3 is the chromatogram of the mixed reference solution of Example 1;

[0027] Figure 4 is the chromatogram of the 4-hydroxybenzoic acid reference substance solution of Example 1;

[0028] Figure 5 is the chromatogram of the test solution of Example 1;

[0029] Figure 6 This is the chromatogram of the test solution of Comparative Example 1;

[0030] Figure 7 This is the chromatogram of the test solution of method 1;

[0031] Figure 8 Chromatogram of the spiked solution of the test sample for Method 2;

[0032] Figure 9 Chromatogram of the test sample spiked solution for Method 3;

[0033] Figure 10 Detect the chromatogram of the blank excipient spiked solution;

[0034] Figure 11 The following are the detection chromatograms of the test solution prepared with diluents having different methanol volume ratios. DETAILED DESCRIPTION

[0035] To facilitate understanding of the present application, the present application will be described more fully below with reference to the relevant embodiments. The following provides preferred embodiments of the present application. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing specific embodiments only and are not intended to limit this application.

[0037] The terms "and / or", "or / and", and "and / or" used herein include any one of two or more related listed items, and also include any and all combinations of the related listed items, wherein the any and all combinations include any combination of two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND" and technical solutions connected by "logical OR".

[0038] In this application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0039] In this application, when referring to numerical ranges, unless otherwise specified, the numerical ranges are considered continuous and include the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe a feature or characteristic, the ranges can be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges subsumed therein.

[0040] Only certain numerical ranges are specifically disclosed herein. However, any lower limit may be combined with any upper limit to form an unspecified range; and any lower limit may be combined with other lower limits to form an unspecified range, and similarly, any upper limit may be combined with any other upper limit to form an unspecified range. In addition, each individually disclosed point or single value may itself serve as a lower limit or upper limit and be combined with any other point or single value, or with other lower limits or upper limits, to form an unspecified range.

[0041] The temperature parameters in this application, unless otherwise specified, allow for both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows for temperature fluctuations within the precision range of instrument control. Fluctuations within ranges such as ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are permitted.

[0042] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc. shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0043] In this application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be understood as limiting the scope of protection of this application.

[0044] In this application, the terms "optionally," "optional," and "optional" mean optional or dispensable, i.e., they refer to either option being selected from two parallel options: "with" or "without." If a technical solution contains multiple "optional" clauses, each "optional" clause is independent unless otherwise specified and there are no contradictions or constraints.

[0045] In the description of the application, "multiple" means at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

[0046] Unless otherwise specified, all embodiments and optional embodiments of the present application can be combined with each other to form a new technical solution. Unless otherwise specified, all technical features and optional technical features of the present application can be combined with each other to form a new technical solution.

[0047] Unless otherwise specified, all steps of the present application can be performed sequentially or randomly, preferably sequentially.

[0048] The prior art lacks a method for efficiently determining the content of 4-hydroxybenzoic acid and preservatives in compound sodium picosulfate oral solution. The Chinese Pharmacopoeia "Methylparaben" method can detect 4-hydroxybenzoic acid, methylparaben, and ethylparaben, but this method is not suitable for compound sodium picosulfate oral solution. The main reason is that 4-hydroxybenzoic acid has a high polarity and is subject to interference from excipients, while propylparaben has a significant polarity difference from 4-hydroxybenzoic acid, and detecting them in the same method would extend the run time.

[0049] Based on the above problems, one or more embodiments of the present application provide a method for detecting a compound sodium picosulfate oral solution, which is used to detect at least one of methylparaben, propylparaben, and 4-hydroxybenzoic acid in the compound sodium picosulfate oral solution; the determination is performed using high performance liquid chromatography, and the chromatographic conditions of the high performance liquid chromatography include: a chromatographic column including a reversed-phase C18 chromatographic column or a column with equivalent performance; a mobile phase including a mobile phase A and a mobile phase B, wherein mobile phase A is a mixture of ammonium acetate buffer and methanol, and mobile phase B is acetonitrile; the elution mode is gradient elution, and the gradient elution procedure includes: from 0 to 16 minutes, the volume percentage of mobile phase A is reduced from 85%-95% to 70%-80%, and the volume percentage of mobile phase B is increased from 5%-15% to 20% -30%; 16min-16.1min, the volume percentage of mobile phase A is reduced from 70%-80% to 55%-65%, and the volume percentage of mobile phase B is increased from 20%-30% to 35%-45%; 16.1min-25min, the volume percentage of mobile phase A is maintained at 55%-65%, and the volume percentage of mobile phase B is 35%-45%; 25min-25.1min, the volume percentage of mobile phase A is increased from 55%-65% to 85%-95%, and the volume percentage of mobile phase B is reduced from 35%-45% to 5%-15%; 25.1min-35min, the volume percentage of mobile phase A is maintained at 85%-95%, and the volume percentage of mobile phase B is maintained at 5%-15%.

[0050] It should be noted that the "C18" mentioned above refers to a chromatographic column filled with octadecylsilane bonded silica gel.

[0051] Understandably, the above-mentioned detection method for compound sodium picosulfate oral solution adopts mobile phase A of ammonium acetate buffer and methanol mixture and mobile phase B of acetonitrile, in combination with a specific gradient elution program, which can detect methylparaben, propylparaben and 4-hydroxybenzoic acid in compound sodium picosulfate oral solution in a short time and simultaneously, solving the problem of 4-hydroxybenzoic acid being interfered with by excipients, and can effectively separate and accurately quantify methylparaben, propylparaben and 4-hydroxybenzoic acid, thereby shortening the detection time by half, which is beneficial to batch detection of compound sodium picosulfate oral solution and improving detection efficiency. In addition, the detection method can eliminate the interference caused by blank solvent and excipient solution, and can be used for quality control of the preparation process of compound sodium picosulfate oral solution and the final product, ensuring the quality controllability of the product and reducing safety risks.

[0052] This assay boasts excellent specificity, resolution, sensitivity, and reproducibility, making it crucial for evaluating formulation processes, controlling final product quality, and monitoring stability samples. Furthermore, this assay is suitable for determining the content of 4-hydroxybenzoic acid, methylparaben, and propylparaben in intermediate and finished products of compound sodium picosulfate oral solution, ensuring product quality and demonstrating its practicality in quality control.

[0053] In some embodiments, the HPLC flow rate is 0.9 mL / min to 1.1 mL / min, for example, but not limited to, 0.9 mL / min, 0.95 mL / min, 1 mL / min, 1.05 mL / min, 1.1 mL / min, or a range between any two of the foregoing flow rates. When the HPLC flow rate is within the foregoing range, the 4-hydroxybenzoic acid, methylparaben, and propylparaben peaks are well retained.

[0054] As a possible embodiment, the column temperature of the HPLC method is 25°C-35°C; for example, it can be, but is not limited to, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, or a range between any two of the above column temperatures. This ensures effective separation of the 4-hydroxybenzoic acid, methylparaben, and propylparaben peaks, and provides a reliable guarantee for accurate quantitative analysis.

[0055] In some optional embodiments, the column temperature of the HPLC method is 28°C-32°C.

[0056] In some embodiments, the detection wavelength of the high performance liquid chromatography is 240 nm-263 nm; for example, it can be but is not limited to 240 nm, 242 nm, 244 nm, 246 nm, 248 nm, 250 nm, 252 nm, 254 nm, 256 nm, 258 nm, 260 nm, 263 nm or a range between any two of the above detection wavelengths.

[0057] In some embodiments, in mobile phase A, the volume proportion of ammonium acetate buffer is 90%-95%, and the volume proportion of methanol is 5%-10%; for example, the volume proportion of ammonium acetate buffer in mobile phase A can be, but is not limited to, 90%, 91%, 92%, 93%, 94%, 95%, or a range between any two of the above values. The volume proportion of methanol in mobile phase A can be, but is not limited to, 5%, 6%, 7%, 8%, 9%, 10%, or a range between any two of the above values. Reasonable regulation of the volume proportions of ammonium acetate buffer and methanol in mobile phase A can ensure that there is no solvent peak interference near the retention time range of the 4-hydroxybenzoic acid peak and achieve effective separation of the 4-hydroxybenzoic acid peak from the adjacent excipient peak.

[0058] As a possible embodiment, the ammonium acetate buffer comprises ammonium acetate at a molar concentration of 25 mmol / L-100 mmol / L. As a non-limiting example, the molar concentration of ammonium acetate in the ammonium acetate buffer may be, but is not limited to, 25 mmol / L, 30 mmol / L, 35 mmol / L, 40 mmol / L, 45 mmol / L, 50 mmol / L, 55 mmol / L, 60 mmol / L, 65 mmol / L, 70 mmol / L, 75 mmol / L, 80 mmol / L, 85 mmol / L, 90 mmol / L, 95 mmol / L, 100 mmol / L, or a range between any two of the above molar concentrations. This helps ensure that the retention time of 4-hydroxybenzoic acid is within a reasonable range and improves the resolution of each main peak.

[0059] In some embodiments, the pH value of the ammonium acetate buffer is 3.85-4.15. Without limitation, the pH value of the ammonium acetate buffer can be, but is not limited to, 3.85, 3.9, 3.95, 4.0, 4.05, 4.1, 4.15, or a range between any two of the above pH values. When the pH value of the ammonium acetate buffer is within the above range, it is beneficial to achieve good retention and separation of the 4-hydroxybenzoic acid, methylparaben, and propylparaben peaks; at the same time, it can effectively inhibit the ion exchange effect of the stationary phase, reduce peak tailing, improve peak symmetry, and ensure the accuracy and reproducibility of the test results.

[0060] As a possible embodiment, the injection volume of high performance liquid chromatography is 10 μL-60 μL; for example, it can be but not limited to 10 μL, 15 μL, 20 μL, 25 μL, 30 μL, 35 μL, 40 μL, 45 μL, 50 μL, 55 μL, 60 μL or a range between any two of the above injection volumes.

[0061] In some embodiments, the reversed-phase C18 chromatography column has an inner diameter and a length of 4.6 mm and 150 mm, respectively, and a filler particle size of 3 μm.

[0062] As a possible implementation, the model of the chromatographic column is Ultimate XB-C18.

[0063] In some embodiments, the chromatographic column further comprises a ghost peak capture cartridge. It should be noted that the ghost peak capture cartridge mentioned above is an online cleanup column for high performance liquid chromatography systems, specifically designed to capture impurities ("ghost peaks") in the mobile phase or sample matrix to prevent them from interfering with the analytical column and test results.

[0064] As a possible embodiment, the ghost peak capture column of the high performance liquid chromatography is a reverse C18 chromatographic column. In some optional embodiments, the inner diameter and length of the ghost peak capture column are 4.6 mm and 50 mm respectively.

[0065] In some embodiments, the ghost peak capture column is a Welch Ghost-Buster Column.

[0066] In some embodiments, the gradient elution procedure includes: 0-16 min, reducing the volume percentage of mobile phase A from 90% to 75%, and increasing the volume percentage of mobile phase B from 10% to 25%; 16 min-16.1 min, reducing the volume percentage of mobile phase A from 75% to 60%, and increasing the volume percentage of mobile phase B from 25% to 40%; 16.1 min-25 min, maintaining the volume percentage of mobile phase A at 60%, and the volume percentage of mobile phase B at 40%; 25 min-25.1 min, increasing the volume percentage of mobile phase A from 60% to 90%, and reducing the volume percentage of mobile phase B from 40% to 10%; 25.1 min-35 min, maintaining the volume percentage of mobile phase A at 90%, and the volume percentage of mobile phase B at 10%.

[0067] As a possible embodiment, the sample measured by high performance liquid chromatography is diluted with a diluent, the diluent includes methanol and water, and the volume proportion of methanol is 0-10%. In some optional embodiments, the sample measured by high performance liquid chromatography is diluted with a diluent, the diluent includes methanol and water, and the volume proportion of methanol is 5%-10%.

[0068] In some embodiments, the method for preparing an ammonium acetate buffer solution comprises: mixing ammonium acetate with water, and then adding glacial acetic acid to adjust the pH value of the mixture to 3.85-4.15 to prepare the ammonium acetate buffer solution.

[0069] The following is a detailed description of the technical solutions of the present application in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. For experimental methods in the following examples where specific conditions are not specified, reference should be made to the instructions given in this application, or to experimental manuals or conventional conditions in the field, or to conditions recommended by the manufacturer, or to experimental methods known in the art.

[0070] In the following specific examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operational accuracy are allowed.

[0071] 1. Instruments and Reagents

[0072] 1. Main instruments

[0073] Instrument: Waters Arc-2998 series high performance liquid chromatograph;

[0074] Chromatographic columns: Ultimate XB-C18 4.6*150mm, 3μm; Ghost-Buster Column, 50mm×4.6mm;

[0075] Electronic balance: GL224I-1SCN (Sartorius);

[0076] pH meter: FE28 (Mettler).

[0077] 2. Main reagents

[0078] Ammonium acetate (analytical grade), glacial acetic acid (analytical grade), methanol (chromatographic grade), acetonitrile (chromatographic grade).

[0079] 3. Reference substances

[0080] 4-Hydroxybenzoic acid reference substance: sourced from China National Institute for Food and Drug Control, content 100%, batch number 101149-202204.

[0081] Propylparaben reference substance: sourced from China National Institute for Food and Drug Control, content 99.9%, batch number 100444-202306.

[0082] Methylparaben reference substance: sourced from China National Institute for Food and Drug Control, content 100%, batch number 100278-202307.

[0083] 4. Sample information

[0084] Test sample: Compound sodium picosulfate oral solution, source: Huayi Pharmaceutical Technology (Anhui) Co., Ltd., content specification: 175mL: sodium picosulfate 10mg; batch number: 2411097

[0085] Blank excipient: provided by Shenzhen Beimei Pharmaceutical Co., Ltd., batch number: 241203; the blank excipient is an oral solution that does not contain 4-hydroxybenzoic acid, methylparaben and propylparaben, and contains the following substances: citric acid, magnesium oxide, sucralose, glycine, alanine, acesulfame potassium and sodium hydroxide.

[0086] 2. Solution Preparation

[0087] 1. Blank solution: 10% methanol in water (V / V).

[0088] 2. Diluent: Take appropriate amount of methanol and water, mix them in the ratio of 10:90 (V / V).

[0089] 3. Stock solution of propylparaben reference substance: Weigh approximately 17.5 mg of propylparaben reference substance accurately, place in a 100 mL volumetric flask, add appropriate amount of methanol, ultrasonically dissolve and dilute to the scale, shake well, and obtain.

[0090] 4. Methylparaben reference substance stock solution: Weigh approximately 60 mg of methylparaben reference substance accurately, place in a 50 mL volumetric flask, add appropriate amount of methanol, ultrasonically dissolve and dilute to the scale, shake well, and obtain.

[0091] 5. 4-Hydroxybenzoic acid reference substance stock solution ①: Weigh approximately 19 mg (calculated as C7H6O3) of 4-hydroxybenzoic acid reference substance, accurately weigh it, place it in a 100 mL volumetric flask, ultrasonically dissolve it and dilute it to the scale with a diluent, shake well, and the solution is ready.

[0092] 6. 4-Hydroxybenzoic acid reference stock solution ②: Accurately measure 3 mL of 4-hydroxybenzoic acid reference stock solution ① and place it in a 50 mL volumetric flask. Dilute to the mark with diluent and shake well.

[0093] 7. Mixed reference solution: Accurately measure 4 mL of methylparaben reference stock solution and 5 mL of propylparaben reference stock solution, place them in the same 50 mL volumetric flask, dilute to the scale with diluent, shake well, and obtain.

[0094] 8. 4-Hydroxybenzoic acid reference solution: Accurately measure ②5mL of 4-hydroxybenzoic acid reference stock solution and place it in a 50mL volumetric flask. Dilute to the mark with diluent and shake well.

[0095] 9. Test solution: Take the product and shake well. Filter through a 0.45μm PES filter. Discard at least 1mL of the initial filtrate and use the subsequent filtrate as the sample solution. Accurately measure 5mL of the sample solution and place it in a 50mL volumetric flask. Dilute to the mark with diluent and shake well to obtain the test solution.

[0096] 10. Blank excipient solution: Take the blank excipient and shake it evenly. Filter it with a 0.45 μm PES filter membrane. Discard 1 mL of the primary filtrate. Accurately measure 5 mL of the subsequent filtrate and place it in a 50 mL volumetric flask. Dilute it to the mark with diluent and shake it evenly.

[0097] 3. Test methods and conditions

[0098] A method for detecting compound sodium picosulfate oral solution is used to detect at least one of methylparaben, propylparaben, and 4-hydroxybenzoic acid. The detection method uses high-performance liquid chromatography, and the conditions of the high-performance liquid chromatography include:

[0099] (1) The chromatographic column includes a reversed-phase C18 column or a column of equivalent performance;

[0100] (2) Mobile phase A: The volume ratio of ammonium acetate buffer to methanol is 90-95:5-10. The pH value of the ammonium acetate buffer is adjusted to 3.85-4.15 with glacial acetic acid. The ammonium acetate concentration of the ammonium acetate buffer is 25 mmol / L-100 mmol / L.

[0101] (3) Mobile phase B: acetonitrile;

[0102] (4) Flow rate: 0.9 mL / min-1.1 mL / min;

[0103] (5) The detection wavelength is 240nm-263nm;

[0104] (6) Injection volume is 10 μL-60 μL;

[0105] (7) Column temperature is 25℃-35℃;

[0106] (8) Gradient elution. The elution procedure is shown in Table 1.

[0107] Table 1 Liquid phase elution program

[0108] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 85％-95％ 5％-15％ 16 70％-80％ 20％-30％ 16.1 55％-65％ 35％-45％ 25 55％-65％ 35％-45％ 25.1 85％-95％ 5％-15％ 35 85％-95％ 5％-15％

[0109] 4. Calculation formula

[0110] (1) External standard method

[0111]

[0112] Concentration (μg / ml) = As × F 均 ×n S ×1000

[0113]

[0114] in,

[0115] M st The sample weight of the reference substance (mg);

[0116] P st is the content of reference substance (%);

[0117] V st is the dilution volume of the reference substance (mL);

[0118] A st is the main peak area in the reference solution;

[0119] A s is the main peak area in the test solution;

[0120] n s is the dilution multiple of the test sample;

[0121] F 均 is the average F value of the reference solution (n=5);

[0122] V is the volume of the finished product package (175 mL);

[0123] L is the nominal amount.

[0124] (2) Recovery rate

[0125]

[0126] 3)Relative standard deviation (RSD)

[0127]

[0128] Where S is the standard deviation, is the arithmetic mean of the measurement results.

[0129] V. Specific test content

[0130] Example 1

[0131] A method for detecting compound sodium picosulfate oral solution is used to detect at least one of methylparaben, propylparaben, and 4-hydroxybenzoic acid. The method uses high performance liquid chromatography for determination, and the chromatographic conditions of the high performance liquid chromatography include:

[0132] Column: Ultimate XB-C18, 150 × 4.6 mm, 3 μm;

[0133] Ghost-Buster Column: Ghost-Buster Column, 50mm×4.6mm;

[0134] Detection wavelength: 255nm;

[0135] Flow rate: 1.0 mL / min;

[0136] Column temperature: 30°C;

[0137] Injection volume: 30 μL;

[0138] Mobile phase A: ammonium acetate buffer-methanol (volume ratio 95:5); wherein the ammonium acetate buffer was prepared by weighing 7.7 g of ammonium acetate, adding 1000 mL of water to dissolve it, and adjusting the pH to 4.0 with glacial acetic acid. The concentration of the ammonium acetate buffer was 0.1 mol / L.

[0139] Mobile phase B: acetonitrile;

[0140] Elution method: gradient elution. The elution program is shown in Table 2.

[0141] The blank solution, blank excipient solution, mixed reference solution, 4-hydroxybenzoic acid reference solution and test solution were respectively injected into the high performance liquid chromatograph, and the contents of methylparaben, propylparaben and 4-hydroxybenzoic acid were calculated respectively by the external standard method.

[0142] Table 2 Liquid phase elution program

[0143] Time (min) Mobile phase A (%) Mobile phase B (%) 0 90 10 16 75 25 16.1 60 40 25 60 40 25.1 90 10 35 90 10

[0144] Example 2

[0145] Compared with Example 1, the column temperature was adjusted to 28° C. and the flow rate was adjusted to 1.1 mL / min. Other chromatographic conditions remained unchanged.

[0146] Example 3

[0147] Compared with Example 1, the column temperature was adjusted to 32° C. and the flow rate was adjusted to 0.9 mL / min. Other chromatographic conditions remained unchanged.

[0148] Example 4

[0149] Compared with Example 1, the column temperature was adjusted to 25° C. Other chromatographic conditions remained unchanged.

[0150] Example 5

[0151] Compared with Example 1, the column temperature was adjusted to 35° C. Other chromatographic conditions remained unchanged.

[0152] Example 6

[0153] Compared with Example 1, the wavelength was adjusted to 240 nm, while other chromatographic conditions remained unchanged.

[0154] Example 7

[0155] Compared with Example 1, the wavelength was adjusted to 263 nm, while other chromatographic conditions remained unchanged.

[0156] Example 8

[0157] Compared with Example 1, the injection volume was adjusted to 10 μl, while other chromatographic conditions remained unchanged.

[0158] Example 9

[0159] Compared with Example 1, the injection volume was adjusted to 60 μl, while other chromatographic conditions remained unchanged.

[0160] Example 10

[0161] Compared with Example 1, the pH of the mobile phase buffer salt was adjusted to 3.85. Other chromatographic conditions remained unchanged.

[0162] Example 11

[0163] Compared with Example 1, the pH of the mobile phase buffer salt was adjusted to 4.15. Other chromatographic conditions remained unchanged.

[0164] Example 12

[0165] Compared with Example 1, the elution procedure was adjusted, as shown in Table 3. Other chromatographic conditions remained unchanged.

[0166] Table 3 Liquid phase elution program

[0167] Time (min) Mobile phase A (%) Mobile phase B (%) 0 95 5 16 80 20 16.1 55 45 25 55 45 25.1 95 5 35 95 5

[0168] Example 13

[0169] Compared with Example 1, the elution procedure was adjusted, as shown in Table 4. Other chromatographic conditions remained unchanged.

[0170] Table 4 Liquid phase elution program

[0171] Time (min) Mobile phase A (%) Mobile phase B (%) 0 85 15 16 70 30 16.1 65 35 25 65 35 25.1 85 15 35 85 15

[0172] Comparative Example 1

[0173] Comparative Example 1 provides another method for detecting compound sodium picosulfate oral solution, which is determined by high performance liquid chromatography. The chromatographic conditions of the high performance liquid chromatography include:

[0174] Column: Ultimate XB-C18, 150 × 4.6 mm, 3 μm;

[0175] Detection wavelength: 263nm;

[0176] Flow rate: 1.0 mL / min;

[0177] Column temperature: 30°C;

[0178] Injection volume: 10 μL;

[0179] Mobile phase A: 0.1% glacial acetic acid;

[0180] Mobile phase B: acetonitrile;

[0181] Elution method: gradient elution. The elution program is shown in Table 5.

[0182] The test solution was injected into the high performance liquid chromatography instrument for detection.

[0183] Table 5 Liquid phase elution program

[0184] Time (min) Mobile phase A (%) Mobile phase B (%) 0 85 15 19 80 20 19.1 60 40 30 60 40 30.1 90 10 35 90 10

[0185] Comparative Example 2

[0186] Compared with Example 1, the column temperature was adjusted to 20° C. Other chromatographic conditions remained unchanged.

[0187] Comparative Example 3

[0188] Compared with Example 1, the column temperature was adjusted to 40° C. Other chromatographic conditions remained unchanged.

[0189] The test results of the test solution of this embodiment and the comparative example are shown in Table 6, and the chromatograms of the blank solution, blank excipient solution, mixed reference solution, 4-hydroxybenzoic acid reference solution and test solution of Example 1 are shown in Table 6. Figures 1 to 5 The chromatogram of the test solution of Comparative Example 1 is shown in Figure 6 .

[0190] Table 6 Test solution data of each embodiment and comparative example

[0191]

[0192] Note: The “minimum separation from adjacent peaks” in Table 6 refers to the minimum value of the separation between the methylparaben peak, propylparaben peak, 4-hydroxybenzoic acid peak and their respective adjacent peaks.

[0193] As shown in Table 6, Examples 1 to 11 adopt the high performance liquid chromatography detection conditions of the present application. By analyzing the test solution, the minimum separation between each main peak and the adjacent peak is 31.8-37.8, which is greater than 1.5, indicating that the methylparaben, propylparaben, and 4-hydroxybenzoic acid peaks can be effectively separated. Moreover, the peaks can be completely separated in 25 minutes, thereby greatly shortening the detection time of the methylparaben, propylparaben, and 4-hydroxybenzoic acid peaks in the compound sodium picosulfate oral solution, and improving the detection efficiency.

[0194] See Figure 6 , the mobile phase A of Comparative Example 1 adopts a glacial acetic acid system. Although it can separate methylparaben and propylparaben, 4-hydroxybenzoic acid is easily affected by adjacent peaks and has poor durability. Comparative Example 2, based on Example 1, adjusts the column temperature to 20°C, and obtains a minimum separation degree of 1.4 between each main peak and adjacent peak, which is less than 1.5, and the 4-hydroxybenzoic acid peak and the excipient peak cannot be effectively separated. Comparative Example 3, based on Example 1, adjusts the column temperature to 40°C, and obtains a minimum separation degree of 23.1 between each main peak and adjacent peak, which is greater than 1.5, and the 4-hydroxybenzoic acid peak and the solvent peak cannot be effectively separated, and the peak purity is unqualified. It can be seen that the present application provides a method for effectively detecting methylparaben, propylparaben and 4-hydroxybenzoic acid in compound sodium picosulfate oral solution at the same time.

[0195] 6. Detection Method Development Process

[0196] Method 1: A method for detecting compound sodium picosulfate oral solution, using high performance liquid chromatography for determination. The chromatographic conditions of the high performance liquid chromatography include:

[0197] Column: Ultimate XB-C18, 150 × 4.6 mm, 3 μm;

[0198] Detection wavelength: 263nm;

[0199] Flow rate: 1.0 mL / min;

[0200] Column temperature: 45°C;

[0201] Injection volume: 10 μL;

[0202] Mobile phase: Mobile phase A: ammonium acetate buffer-methanol (volume ratio 95:5); the ammonium acetate buffer was prepared by weighing 7.7 g of ammonium acetate, dissolving it in 1000 mL of water, and adjusting the pH to 4.0 with glacial acetic acid to obtain an ammonium acetate buffer concentration of 0.1 mol / L; Mobile phase B: acetonitrile;

[0203] Elution procedure: isocratic elution, mobile phase A:mobile phase B ratio of 88:12 (V / V).

[0204] Preparation method of test solution: Take an appropriate amount of test sample, filter it with a 0.45μm PES filter membrane, discard 1ml of the initial filtrate, and take the subsequent filtrate. Figure 7 .

[0205] from Figure 7 It can be seen that methylparaben, propylparaben and 4-hydroxybenzoic acid were not separated.

[0206] Method 2: Based on the above method 1, adjust to a gradient elution program, see the elution program in Table 7.

[0207] Table 7 Liquid phase elution program

[0208] Time (minutes) Mobile phase A (%) Mobile phase B (%) 15 88 12 16 70 30 46 70 30 47 88 12 55 88 12

[0209] Preparation method of test sample spiked solution: Take an appropriate amount of test sample, filter it with a 0.45μm PES filter membrane, discard 1ml of the initial filtrate, take 0.2ml of the subsequent filtrate and 0.2ml of methylparaben reference solution and 0.2ml of propylparaben reference solution, mix them and obtain the result. The chromatogram of test sample spiked solution is shown in Figure 8 .

[0210] from Figure 8 It can be seen that although the peak shapes of methylparaben and propylparaben are good and there is no interference from excipients, 4-hydroxybenzoic acid cannot be separated, the detection time is long, and the baseline fluctuation is large, which affects the quantification of methylparaben.

[0211] Method 3: Based on the above method 1, adjust the column temperature to 30°C, isocratic elution, and mobile phase A:mobile phase B ratio to 70:30 (V / V).

[0212] Preparation of the test sample spiked solution: Accurately weigh 5 ml of methylparaben stock solution and 5 ml of propylparaben stock solution into a 25 ml volumetric flask, dilute to the mark with the test sample. Then transfer 5 ml of the above solution into a 50 ml volumetric flask, dilute to the mark with water, and shake well. See the chromatogram of the test sample spiked solution for details. Figure 9 .

[0213] from Figure 9 As can be seen, the baseline is stable and the tailing factor of 1.1 for methylparaben meets the requirements. However, this method cannot separate 4-hydroxybenzoic acid.

[0214] Method 4: Based on the above method 3, adjust to a gradient elution program, see the elution program in Table 8, and add a ghost peak capture column: Ghost-Buster Column, 50 mm × 4.6 mm.

[0215] Table 8 Liquid phase elution program

[0216] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 90 10 16 75 25 16.10 60 40 25 60 40 25.1 90 10 30 90 10

[0217] Reference substance stock solution: Take appropriate amount of methylparaben reference substance and propylparaben reference substance, and dilute them with methanol to a solution containing 0.98 mg of methylparaben and 0.2 mg of propylparaben per 1 ml.

[0218] Preparation method of blank excipient spiked solution: Take an appropriate amount of blank excipient, filter through a 0.45μm PES filter membrane, discard 1ml of the initial filtrate, take 1ml of the subsequent filtrate and 1ml of the reference substance stock solution in a 10ml volumetric flask, and dilute with water to the scale (containing about 98μg / ml of methylparaben and about 20μg / ml of propylparaben). The chromatogram of the blank excipient spiked solution is shown in Figure 10 .

[0219] from Figure 10 As can be seen, 4-hydroxybenzoic acid elutes at approximately 5 minutes without interference from the excipient peaks; both methylparaben and propylparaben peaks can be separated, and the run time is significantly shortened, with a good baseline. Therefore, this detection method was selected.

[0220] 7. Verification of diluent

[0221] Using the detection method of Example 1, diluents with a methanol volume ratio of 5%, 10%, 15%, 20%, and 50% were prepared, and the test solution was tested to verify the effect of different volumes of methanol diluents on the detection. The chromatographic results are shown in Figure 2. Figure 11 .

[0222] from Figure 11As can be seen, as the proportion of organic phase increases, the baseline effect becomes greater and the 4-hydroxybenzoic acid tailing becomes more severe. Using a diluent of 5-10% methanol (v / v) not only increases the solubility of the test sample but also improves the 4-hydroxybenzoic acid tailing, avoiding abnormal chromatographic peak shape caused by large differences in solvent strength.

[0223] 8. Methodological Validation

[0224] The determination method of Example 1 was used for methodological verification to verify its feasibility and stability.

[0225] 1. System adaptability

[0226] Prepare 4-hydroxybenzoic acid sensitivity solution: Accurately measure 5 ml of 4-hydroxybenzoic acid reference solution, place it in a 200 ml volumetric flask, add diluent to dilute to the scale, shake well, and obtain.

[0227] Two mixed reference solutions were prepared in parallel, designated as mixed reference solution 1 and reference solution 2, and a 4-hydroxybenzoic acid reference solution was prepared. The chromatographic conditions of Example 1 were used and the test was performed in the following order: 5 injections of mixed reference solution 1, 2 injections of mixed reference solution 2, 1 injection of 4-hydroxybenzoic acid sensitivity solution, and 5 injections of 4-hydroxybenzoic acid reference solution were performed. A PDA detector was selected and a chromatogram was recorded. The system suitability results are shown in Table 9.

[0228] Table 9 System suitability results

[0229]

[0230]

[0231] Note: N / A means not applicable.

[0232] From the above results, it can be seen that: the mixed reference solution 1 was injected continuously for 5 times, and the peak area RSDs of methylparaben and propylparaben were 0.2% and 0.2%, respectively, both of which were no more than 1.5%; in the chromatogram of the first injection of the mixed reference solution, the tailing factors of methylparaben and propylparaben were 0.9 and 0.9, respectively, ranging from 0.8 to 1.8; the recoveries of methylparaben and propylparaben in the mixed reference solution 2 relative to the mixed reference solution 1 were 99.0% and 100.4%, respectively, ranging from 98.0% to 102.0%.

[0233] The signal-to-noise ratio of the 4-hydroxybenzoic acid sensitivity solution was 53, which was greater than 10; in five consecutive injections, the 4-hydroxybenzoic acid tailing factor of the first injection of the 4-hydroxybenzoic acid reference solution was 1.0, which was between 0.8 and 1.8; the 4-hydroxybenzoic acid reference solution was injected five times continuously, and the RSD of the peak area of ​​4-hydroxybenzoic acid was 0.2%, which was no greater than 2.0%.

[0234] In the 6 injections of mixed reference solution 1 (including the return needle solution), the RSDs (n=6) of the peak areas of methylparaben and propylparaben were 0.2% and 0.3%, respectively, both less than 1.5%; in the 6 injections of 4-hydroxybenzoic acid reference solution (including the return needle solution), the RSD (n=6) of the peak area of ​​4-hydroxybenzoic acid was 0.2%, less than 2.0%.

[0235] Therefore, the system applicability of this method meets the requirements.

[0236] 2. Exclusivity

[0237] (1) Solution preparation

[0238] Blank excipient solution: Take the blank excipient solution and shake it evenly, filter it with a 0.45μm PES filter membrane, discard 2mL of the initial filtrate, accurately measure 5mL of the subsequent filtrate, place it in a 50mL volumetric flask, dilute it to the scale with methanol-water (the volume ratio of methanol and water is 10:90), shake it evenly, and obtain it.

[0239] 4-Hydroxybenzoic acid reference substance stock solution ③: Weigh approximately 28.5 mg (calculated as C7H6O3) of 4-hydroxybenzoic acid reference substance, accurately weigh it, place it in a 50 mL volumetric flask, ultrasonically dissolve it and dilute it to the scale with methanol-water (the volume ratio of methanol to water is 10:90), shake well, and the solution is ready.

[0240] Mixed spiked solution: Accurately measure 2 mL of 4-hydroxybenzoic acid reference stock solution ③ into a 100 mL volumetric flask. Dilute to the mark with this product, shake well, and filter through a 0.45 μm PES filter. Discard 2 mL of the initial filtrate and prepare the subsequent filtrate. Accurately measure 5 mL of the subsequent filtrate into a 50 mL volumetric flask, dilute to the mark with methanol-water (10:90), and shake well.

[0241] (2) 30 μL of each solution was injected into a liquid chromatograph, the PDA detector was selected, and the chromatogram was recorded. The acceptance criteria are shown in Table 10. The results are shown in Table 11.

[0242] Table 10 Acceptance criteria

[0243]

[0244] Table 11 Specificity verification results

[0245]

[0246]

[0247] Note: N / A means not applicable.

[0248] The above results indicate that neither the blank solution nor the blank excipient solution interfered with the assay. The minimum resolutions of the 4-hydroxybenzoic acid, methylparaben, and propylparaben peaks in the test solution and their adjacent peaks were 13.1, 30.5, and 30.5, respectively, all no less than 1.5. The target peak purity angles were all below the purity threshold, indicating peak purity met the requirements. The retention times of the target peaks were consistent with those of the reference solution.

[0249] In the spiked solution of the test sample, the minimum separation between the 4-hydroxybenzoic acid peak, methylparaben and propylparaben and the adjacent peaks were 4.0, 32.5 and 32.5, respectively, all not less than 1.5; the peak purity angles of the target peaks were also less than the purity threshold, and the peak purity met the requirements; the retention time of the target peak was consistent with that of the reference solution.

[0250] Therefore, this method has good specificity.

[0251] 3. Limit of Quantitation and Limit of Detection

[0252] Preparation of quantitation limit solution: Accurately measure an appropriate amount of 4-hydroxybenzoic acid reference substance stock solution ② and dilute it step by step to an appropriate concentration. The quantitation limit solution is obtained when the signal-to-noise ratio (S / N) of the 4-hydroxybenzoic acid peak height is greater than 10.

[0253] Preparation of detection limit solution: Accurately measure an appropriate amount of 4-hydroxybenzoic acid reference stock solution ② and dilute it step by step to an appropriate concentration. The detection limit solution is when the signal-to-noise ratio (S / N) of the 4-hydroxybenzoic acid peak height is greater than 3.

[0254] Accurately measure 30 μl of each of the above solutions and inject them into a liquid chromatograph. Record the chromatogram. The acceptance criteria are shown in Table 12. The results are shown in Table 13.

[0255] Table 12 Acceptance criteria

[0256]

[0257] Table 13 Results of detection limit and quantification limit tests for 4-hydroxybenzoic acid

[0258]

[0259] Note: N / A means not applicable.

[0260] The above results indicate that the quantification limit for 4-hydroxybenzoic acid is 2.6% of the limit and no greater than 30% of the limit. Six consecutive injections of the 4-hydroxybenzoic acid limit of quantification solution yielded a minimum signal-to-noise ratio of 49, no less than 10. The RSD for the 4-hydroxybenzoic acid peak area was 2.5%, no greater than 30.0%. In the detection limit solution, the signal-to-noise ratio for 4-hydroxybenzoic acid was 30, no less than 3.

[0261] Therefore, the detection limit and quantification limit of 4-hydroxybenzoic acid were in compliance with the standards.

[0262] 4. Accuracy

[0263] Blank excipient filtrate: Take the blank excipient solution and shake it evenly, filter it with a 0.45μm PES filter membrane, discard 2mL of the initial filtrate, and take the subsequent filtrate to obtain.

[0264] 4-Hydroxybenzoic Acid - 2.5% Accurate Solution (Prepare 3 times in parallel): Accurately measure 5 mL of the 4-hydroxybenzoic acid reference solution into a 20 mL volumetric flask. Dilute to the mark with the blank excipient filtrate and shake well. Accurately measure 5 mL of the above solution into a 50 mL volumetric flask. Dilute to the mark with methanol-water (methanol:water ratio of 10:90 by volume) and shake well.

[0265] 4-Hydroxybenzoic Acid - 100% Accuracy Solution (Prepare 3 replicates): Accurately measure 3 mL of the 4-hydroxybenzoic acid reference stock solution (①) into a 50 mL volumetric flask. Dilute to the mark with the blank excipient filtrate and shake well. Accurately measure 5 mL of the above solution into a 50 mL volumetric flask. Dilute to the mark with methanol-water (methanol:water ratio of 10:90 by volume) and shake well.

[0266] 4-Hydroxybenzoic Acid - 200% Accuracy Solution (Prepare three times in parallel): Accurately measure 3 mL of the 4-hydroxybenzoic acid reference stock solution ① into a 25 mL volumetric flask. Dilute to the mark with the blank excipient filtrate and shake well. Accurately measure 5 mL of the above solution into a 50 mL volumetric flask. Dilute to the mark with methanol-water (methanol:water ratio of 10:90 by volume) and shake well.

[0267] Propylparaben linear stock solution: weigh about 28 mg of propylparaben reference substance (according to C 10 H 12 O3 meter), accurately weighed, placed in a 25mL volumetric flask, ultrasonically dissolved with methanol and diluted to the scale, shaken to obtain.

[0268] Propylparaben accurate stock solution: In a 50mL volumetric flask, accurately add 10mL of propylparaben linear stock solution, dilute to the scale with methanol-water (the volume ratio of methanol and water is 10:90), shake well, and obtain.

[0269] Propylparaben-25% accuracy solution (prepare 3 portions in parallel): Accurately measure 10 mL of blank excipient filtrate and 2 mL of propylparaben accuracy stock solution, place them in a 100 mL volumetric flask, dilute to the scale with methanol-water (the volume ratio of methanol and water is 10:90), shake well, and obtain the solution.

[0270] Propylparaben-100% accuracy solution (prepare 3 portions in parallel): Accurately measure 2.5 mL of blank excipient filtrate and 2 mL of propylparaben stock solution, place them in a 25 mL volumetric flask, dilute to the scale with methanol-water (methanol and water volume ratio is 10:90), shake well, and obtain.

[0271] Propylparaben-150% accuracy solution (prepare 3 portions in parallel): Accurately measure 2.5 mL of blank excipient filtrate and 3 mL of propylparaben accuracy stock solution, place them in a 25 mL volumetric flask, dilute to the scale with methanol-water (methanol and water volume ratio is 10:90), shake well, and obtain.

[0272] Methylparaben-75% accuracy solution (prepare 3 portions in parallel): Accurately measure 5 mL of blank excipient filtrate and 3 mL of methylparaben stock solution, place them in a 50 mL volumetric flask, dilute to the scale with methanol-water (methanol and water volume ratio is 10:90), shake well, and obtain.

[0273] Methylparaben-100% accuracy solution (prepare 3 portions in parallel): Accurately measure 5 mL of blank excipient filtrate and 4 mL of methylparaben stock solution, place them in a 50 mL volumetric flask, dilute to the scale with methanol-water (methanol and water volume ratio is 10:90), shake well, and obtain.

[0274] Methylparaben-150% accuracy solution (prepare 3 portions in parallel): Accurately measure 2.5 mL of blank excipient filtrate and 3 mL of methylparaben stock solution, place them in a 25 mL volumetric flask, dilute to the scale with methanol-water (methanol and water volume ratio is 10:90), shake well, and obtain.

[0275] 30 μl of each of the above solutions was injected into a liquid chromatograph, and the chromatogram was recorded. The recovery and RSD of each solution were calculated using the external standard method. The acceptance criteria are shown in Table 14. The results are shown in Table 15.

[0276] Table 14 Acceptance criteria

[0277]

[0278]

[0279] Table 15 Accuracy test results

[0280]

[0281] Note: N / A means not applicable.

[0282] From the above results, it can be seen that the single recovery rate of 4-hydroxybenzoic acid-2.5% accuracy solution is between 96.7% and 100.1%, and the RSD (n=3) is 1.8%; the single recovery rate of 4-hydroxybenzoic acid-100% accuracy solution is between 99.4% and 99.8%, and the RSD (n=3) is 0.3%; the single recovery rate of 4-hydroxybenzoic acid-200% accuracy solution is between 99.8% and 100.3%, and the RSD (n=3) is 0.3%; the single recovery rate and RSD values ​​of its 2.5% accuracy solution, 100% accuracy solution and 200% accuracy solution all meet the requirements, indicating that 4-hydroxybenzoic acid has good accuracy in the range of 0.02916 μg / mL to 2.333 μg / mL (equivalent to 2.6% to 204.6% of the limit concentration).

[0283] In 9 portions of propylparaben-accuracy solutions, the individual recoveries of propylparaben were between 100.9% and 101.8%, the average recovery was 101.3%, and the RSD was 0.4%. The individual recovery, average recovery, and RSD values ​​all met the requirements, indicating that propylparaben had good accuracy in the range of 4.557 μg / mL to 27.34 μg / mL (equivalent to 25.7% to 154.4% of the limit concentration).

[0284] In 9 methylparaben accuracy solutions, the individual recoveries of methylparaben were between 99.3% and 101.5%, the average recovery was 100.3%, and the RSD was 0.9%. The individual recovery, average recovery, and RSD values ​​all met the requirements, indicating that the accuracy of methylparaben was good in the range of 74.7 μg / mL to 149.4 μg / mL (equivalent to 76.2% to 152.5% of the limit concentration).

[0285] According to the above test results, 4-hydroxybenzoic acid, propylparaben and methylparaben all meet the acceptable standards, and the accuracy of the detection method of compound sodium picosulfate oral solution of the present application is good.

[0286] 5. Repeatability

[0287] 4-Hydroxybenzoic acid reference substance stock solution ③: Weigh approximately 28.5 mg (calculated as C7H6O3) of 4-hydroxybenzoic acid reference substance, accurately weigh it, place it in a 50 mL volumetric flask, ultrasonically dissolve it and dilute it to the scale with methanol-water (the volume ratio of methanol to water is 10:90), shake well, and the solution is ready.

[0288] Test sample spiked stock solution: Accurately measure 32 mL of 4-hydroxybenzoic acid reference stock solution and place it in a 100 mL volumetric flask. Dilute to the mark with this product and shake well.

[0289] Test sample spike solution: Take an appropriate amount of the test sample spiked stock solution and filter through a 0.45 μm PES filter. Discard 2 mL of the initial filtrate. Accurately measure 5 mL of the subsequent filtrate into a 50 mL volumetric flask. Dilute to the mark with methanol-water (methanol:water ratio of 10:90 by volume) and shake well.

[0290] Prepare 6 test sample spiked solutions, inject 30 μl of each solution into the liquid chromatograph, record the chromatogram, and calculate the content and RSD of the 6 test samples by the external standard method.

[0291] (1) Acceptable standards are as follows:

[0292] a) The RSD of the detected amount X of 4-hydroxybenzoic acid shall meet the requirements of Table 16:

[0293] Table 16

[0294] Test results Relative standard deviation (RSD) X<quantification limit No comparison Limit of quantification ≤X<0.1μg / mL RSD≤12% 0.1μg / mL≤X<0.5μg / mL RSD≤10% 0.5 μg / mL≤X<1.0 μg / mL RSD≤8% 1.0μg / mL≤X<10μg / mL RSD≤6% 10 μg / mL≤X RSD≤4%

[0295] b) The RSD of methylparaben and propylparaben content in 6 replicate solutions is ≤ 2.0%.

[0296] (2) The results of the repeatability test are shown in Table 17.

[0297] Table 17 Repeatability test results

[0298]

[0299]

[0300] The above results show that in the six sample spiked stock solutions, the mean concentration of methylparaben was 1245.0 μg / mL with an RSD of 0.1%; the mean concentration of propylparaben was 169.8 μg / mL with an RSD of 0.4%. The RSD values ​​of methylparaben and propylparaben were both less than 2.0%. The mean concentration of 4-hydroxybenzoic acid was 11.5 μg / mL with an RSD of 0.5% (<4%).

[0301] All of the above meet the requirements, indicating that the repeatability of the detection method for compound sodium picosulfate oral solution of this application meets the requirements.

[0302] 6. Linearity and range

[0303] 4-Hydroxybenzoic acid-10% linear solution: Accurately measure 2 mL of the 4-hydroxybenzoic acid reference stock solution (2) and place it in a 200 mL volumetric flask. Dilute to the mark with methanol-water (10:90) and shake well. (4-Hydroxybenzoic acid: 0.1140 μg / mL)

[0304] 4-Hydroxybenzoic acid-50% linear solution: Accurately measure 25 mL of the 4-hydroxybenzoic acid reference stock solution into a 100 mL volumetric flask. Dilute to the mark with methanol-water (10:90), shake well, and the solution is ready. (4-Hydroxybenzoic acid: 0.5700 μg / mL)

[0305] 4-Hydroxybenzoic acid - 100% linear solution: Accurately measure 25 mL of 4-hydroxybenzoic acid reference stock solution into a 50 mL volumetric flask. Dilute to the mark with methanol-water (10:90), shake well, and the solution is ready. (4-Hydroxybenzoic acid: 1.140 μg / mL)

[0306] 4-Hydroxybenzoic acid-200% linear solution: Accurately measure 25 mL of the 4-hydroxybenzoic acid reference stock solution into a 25 mL volumetric flask. Dilute to the mark with methanol-water (10:90), shake well, and the solution is ready. (4-Hydroxybenzoic acid: 2.280 μg / mL)

[0307] Methylparaben linear stock solution: Weigh approximately 62 mg of methylparaben reference substance accurately into a 50 mL volumetric flask. Ultrasonicate and dissolve in methanol and dilute to the mark. Shake well to obtain the solution. (Methylparaben: 1240 μg / mL)

[0308] Methylparaben-20% linear solution: Accurately measure 5 mL of methylparaben-100% linear solution into a 25 mL volumetric flask. Dilute to the mark with methanol-water (10:90) and shake well. (Propylparaben: 19.9 μg / mL)

[0309] Methylparaben-50% linear solution: Accurately measure 2 mL of methylparaben linear stock solution and place it in a 50 mL volumetric flask. Dilute to the mark with methanol-water (10:90) and shake well. (Propylparaben: 49.8 μg / mL)

[0310] Methylparaben-75% linear solution: Accurately measure 3 mL of methylparaben linear stock solution and place it in a 50 mL volumetric flask. Dilute to the mark with methanol-water (10:90) and shake well. (Propylparaben: 74.7 μg / mL)

[0311] Methylparaben-100% linear solution: Accurately measure 2 mL of methylparaben linear stock solution and place it in a 25 mL volumetric flask. Dilute to the mark with methanol-water (10:90) and shake well. (Propylparaben: 99.6 μg / mL)

[0312] Methylparaben-150% linear solution: Accurately measure 3 mL of methylparaben linear stock solution and place it in a 25 mL volumetric flask. Dilute to the mark with methanol-water (10:90) and shake well. (Propylparaben: 149.4 μg / mL)

[0313] Propylparaben linear stock solution ①: weigh about 28 mg of propylparaben reference substance (according to C 10 H 12 O3 meter), accurately weigh, place in a 25mL volumetric flask, ultrasonically dissolve and dilute to the mark with methanol, shake well, and obtain. (Propylparaben: 1120μg / mL)

[0314] Propylparaben linear stock solution ②: In a 50mL volumetric flask, accurately add 10mL of propylparaben linear stock solution ①, dilute to the mark with methanol-water (10:90), and shake well to obtain. (Propylparaben: 224.0μg / mL)

[0315] Propylparaben-25% linear solution: Accurately measure 2 mL of the propylparaben linear stock solution into a 100 mL volumetric flask. Dilute to the mark with methanol-water (10:90), shake well, and the solution is ready. (Propylparaben: 4.480 μg / mL)

[0316] Propylparaben-50% linear solution: Accurately measure 2 mL of the propylparaben linear stock solution into a 50 mL volumetric flask. Dilute to the mark with methanol-water (10:90) and shake well. (Propylparaben: 8.960 μg / mL)

[0317] Propylparaben-75% linear solution: Accurately measure 3 mL of the propylparaben linear stock solution ② and place it in a 50 mL volumetric flask. Dilute to the mark with methanol-water (10:90) and shake well. (Propylparaben: 13.44 μg / mL)

[0318] Propylparaben-100% linear solution: Accurately measure 2 mL of the propylparaben linear stock solution into a 25 mL volumetric flask. Dilute to the mark with methanol-water (10:90) and shake well. (Propylparaben: 17.92 μg / mL)

[0319] Propylparaben-150% linear solution: Accurately measure 3 mL of the propylparaben linear stock solution ② and place it in a 25 mL volumetric flask. Dilute to the mark with methanol-water (10:90) and shake well. (Propylparaben: 26.88 μg / mL)

[0320] Accurately measure 30 μl of each of the above solutions and inject them into a liquid chromatograph. Record the chromatogram. Using concentration as the abscissa and peak area as the ordinate, calculate the regression equation, correlation coefficient, intercept (percentage of response at the limiting concentration), and residual sum of squares using the least squares method. Acceptance criteria are shown in Table 18. The results are shown in Tables 19-21.

[0321] Table 18 Acceptance criteria

[0322]

[0323] Table 19 4-Hydroxybenzoic acid linear test results

[0324]

[0325] Table 20 Methylparaben linearity test results

[0326]

[0327] Table 21 Propylparaben linearity test results

[0328]

[0329]

[0330] From the above data, the linear regression equation of 4-hydroxybenzoic acid is y=162439.8560x-134.2398, r=1.0000, the ratio of the y-axis intercept to the 100% concentration response value is 0.1%, and the residual sum of squares is 185300, indicating that 4-hydroxybenzoic acid has a good linear relationship in the range of 0.02916 μg / mL-2.333 μg / mL (equivalent to the limit concentration of 2.6%-204.6%).

[0331] The linear regression equation for methylparaben was y=48772.0714x-36468.1354, r=1.0000, the ratio of the y-axis intercept to the 100% concentration response value was 0.1%, and the residual sum of squares was 18256342, indicating that methylparaben had a good linear relationship in the range of 19.92 μg / mL-149.4 μg / mL (equivalent to 20.33%-152.45% of the test sample concentration).

[0332] The linear regression equation of propylparaben was y=148460.0856x-8112.5946, r=1.0000, the ratio of the y-axis intercept to the 100% concentration response value was 0.3%, and the residual sum of squares was 181343814, indicating that propylparaben had a good linear relationship in the range of 4.557 μg / mL-27.34 μg / mL (equivalent to 25.7% to 154.4% of the test sample concentration).

[0333] In summary, the system adaptability, specificity, limit of quantification and detection limit, accuracy, repeatability, linearity, and range of the detection method of this application all meet the requirements. It can effectively separate and accurately quantify methylparaben, propylparaben, and 4-hydroxybenzoic acid in compound sodium picosulfate oral solution. The method has good specificity, good resolution, and good accuracy. Therefore, the detection method of this application can be used for routine testing of methylparaben, propylparaben, and 4-hydroxybenzoic acid content in compound sodium picosulfate oral solution and for testing during sample preparation.

[0334] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0335] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A method for detecting compound sodium picosulfate oral solution, characterized in that: Used to detect at least one of methylparaben, propylparaben and 4-hydroxybenzoic acid in the compound sodium picosulfate oral solution; the determination is performed using high performance liquid chromatography, and the chromatographic conditions of the high performance liquid chromatography include: The chromatographic column includes a reversed-phase C18 column or a column of equivalent performance; The mobile phase includes mobile phase A and mobile phase B, wherein the mobile phase A is a mixture of ammonium acetate buffer and methanol, and the mobile phase B is acetonitrile; Gradient elution was used, and the procedure of the gradient elution included: From 0 to 16 minutes, the volume percentage of the mobile phase A is reduced from 85%-95% to 70%-80%, and the volume percentage of the mobile phase B is increased from 5%-15% to 20%-30%; 16 min-16.1 min, the volume percentage of the mobile phase A is reduced from 70%-80% to 55%-65%, and the volume percentage of the mobile phase B is increased from 20%-30% to 35%-45%; 16.1 min-25 min, maintaining the volume percentage of the mobile phase A at 55%-65%, and the volume percentage of the mobile phase B at 35%-45%; 25 min-25.1 min, the volume percentage of the mobile phase A is increased from 55%-65% to 85%-95%, and the volume percentage of the mobile phase B is reduced from 35%-45% to 5%-15%; 25.1 min-35 min, the volume percentage of the mobile phase A is maintained at 85%-95%, and the volume percentage of the mobile phase B is maintained at 5%-15%.

2. The detection method according to claim 1, wherein The column temperature of the high performance liquid chromatography is 25°C-35°C.

3. The detection method according to claim 2, wherein The detection wavelength of the high performance liquid chromatography is 240nm-263nm.

4. The detection method according to claim 3, wherein In the mobile phase A, the volume proportion of the ammonium acetate buffer is 90%-95%, and the volume proportion of the methanol is 5%-10%.

5. The detection method according to claim 4, wherein The ammonium acetate buffer comprises ammonium acetate with a molar concentration of 25 mmol / L-100 mmol / L.

6. The detection method according to claim 5, wherein The pH value of the ammonium acetate buffer is 3.85-4.

15.

7. The detection method according to claim 6, wherein The chromatographic conditions of the high performance liquid chromatography method also include one or more of the following conditions: (1) Flow rate: 0.9 mL / min-1.1 mL / min; (2) Injection volume is 10 μL-60 μL; (3) The inner diameter and length of the reverse-phase C18 column were 4.6 mm and 150 mm, respectively, and the particle size of the filler was 3 μm; (4) The chromatographic column also includes a ghost peak capture column.

8. The detection method according to claim 7, wherein The gradient elution procedure includes: From 0 to 16 minutes, the volume percentage of the mobile phase A is reduced from 90% to 75%, and the volume percentage of the mobile phase B is increased from 10% to 25%; 16 min-16.1 min, the volume percentage of the mobile phase A is reduced from 75% to 60%, and the volume percentage of the mobile phase B is increased from 25% to 40%; 16.1 min-25 min, maintaining the volume percentage of the mobile phase A at 60% and the volume percentage of the mobile phase B at 40%; 25 min-25.1 min, the volume percentage of the mobile phase A is increased from 60% to 90%, and the volume percentage of the mobile phase B is reduced from 40% to 10%; From 25.1 min to 35 min, the volume percentage of the mobile phase A was maintained at 90%, and the volume percentage of the mobile phase B was maintained at 10%.

9. The detection method according to claim 7, wherein The column temperature of the high performance liquid chromatography is 28°C-32°C.

10. The detection method according to any one of claims 1 to 9, wherein: The sample measured by the high performance liquid chromatography method is diluted with a diluent, and the diluent includes methanol and water, and the volume proportion of the methanol is 5%-10%.

Images