Separation and quality control detection method for related substances in deoxycholic acid

Abstract

The invention relates to a separation and quality control detection method for related substances in deoxycholic acid, which adopts a high performance liquid chromatography method, a chromatographic column takes octadecyl bonded silica gel as a filler, 0.010 mol / L dipotassium phosphate solution as a mobile phase A and acetonitrile as a mobile phase B, linear gradient elution is carried out, and the related substances are subjected to separation and quality control detection. Wherein the related substance is pyridine. The quality control detection method for related substances in deoxycholic acid provided by the invention comprises the following steps: preparing a pyridine reference substance solution with a certain concentration, determining the peak area of pyridine in a reference substance and a test solution by using a high performance liquid chromatography, and calculating the content of pyridine by using an external standard method. Therefore, the content of pyridine in deoxycholic acid can be accurately and quantitatively determined.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of analytical chemistry, and particularly relates to a separation and quality control detection method of related substances in deoxycholic acid. BACKGROUND

[0002] Pyridine, CAS No. 110-86-1, English name Pyridine, is a six-membered heterocyclic compound containing one nitrogen atom. As one of the important nitrogen-containing heterocyclic compounds, pyridine has been widely used in the fields of photocatalysis and optoelectronic devices. In medicinal chemistry, pyridine is also commonly used as the parent structure of drugs. Pyridine has the functions and effects of solvent, basic catalyst, intermediate, protecting group remover, and stabilizing agent in organic synthesis.

[0003] Based on the wide application of pyridine, the residual amount of pyridine needs to be controlled in the purity detection of raw pharmaceuticals obtained by reaction involving pyridine. For example, pyridine is used in the process of deoxycholic acid raw material, so its residual amount must be studied to ensure the safety of the drug. However, there are few literature reports on the detection of the residual amount of pyridine in raw materials.

[0004] Currently, GC is commonly used to detect volatile organic solvent impurities, but we found that when using GC method, the acid radical in deoxycholic acid is easy to form salt with pyridine, which makes the GC injection port outlet of pyridine unable to vaporize, resulting in that the recovery rate of pyridine in GC method is not up to standard, making it difficult to use GC method. The use of ion chromatography or fluorescence chromatography or GC-MS or LC-MS and other analysis methods is not suitable for industrialized mass production due to the high price of the instruments used, resulting in low popularization rate.

[0005] As a class II residual solvent recorded in the Chinese Pharmacopoeia 2020 Edition Volume IV, the limit of pyridine is 0.02%, which is low and requires high detection accuracy. Since pyridine has certain ultraviolet absorption under the ultraviolet detector, the HPLC-UV quality control detection method is studied for the residual analysis of pyridine. SUMMARY

[0006] The technical problem to be solved by the present application is to overcome the deficiencies in the prior art and provide a separation and quality control detection method of related substances in deoxycholic acid. The method can detect the content of pyridine in deoxycholic acid, and the method is sensitive, specific, fast and accurate.

[0007] To solve the above technical problems, the technical scheme adopted by the present application is:

[0008] The first aspect of this invention provides a method for separating related substances from deoxycholic acid, which employs high performance liquid chromatography (HPLC). The chromatographic column is packed with octadecyl bonded silica gel, and the mobile phase is 0.010 mol / L dipotassium hydrogen phosphate solution. Acetonitrile is used as the mobile phase B, and linear gradient elution is performed to separate deoxycholic acid and related substances.

[0009] The relevant substance is pyridine.

[0010] In some implementations, the linear gradient elution procedure is as follows:

[0011]

[0012] Preferably, the chromatographic column is a Waters XBridge C18 or a column with equivalent performance.

[0013] Preferably, the chromatographic column has an inner diameter of 4.6 mm, a length of 250 mm, and a packing particle size of 5 μm.

[0014] Preferably, the high-performance liquid chromatography method employs an ultraviolet detector.

[0015] Preferably, the column temperature of the chromatographic column is 20℃~40℃, and more preferably 30℃.

[0016] Preferably, the injection volume for gradient elution is 10 μL to 30 μL, and more preferably 20 μL.

[0017] Preferably, the gradient elution flow rate is 0.5–1.5 mL / min, and more preferably 1.0 mL / min.

[0018] Preferably, the detection wavelength of the high-performance liquid chromatography is 210-260 nm, more preferably 240-260 nm, and even more preferably 256 nm.

[0019] A second aspect of the present invention provides a quality control detection method for deoxycholic acid-related substances, comprising the following steps:

[0020] (1) Preparation of reference solution: Dissolve the relevant substance reference in a solvent to prepare a reference solution;

[0021] (2) Preparation of test solution: Dissolve deoxycholic acid in solvent to prepare a test solution of deoxycholic acid;

[0022] (3) Determination method: The reference solution and the test solution prepared according to steps (1) and (2) are injected into a high performance liquid chromatograph for separation, detection and recording of chromatograms, and the separation method is as described above;

[0023] Record the peak area of ​​the main peak in the reference solution and the peak area of ​​related substances in the test solution;

[0024] (4) Calculation: Calculate the concentration and content of related substances in deoxycholic acid;

[0025] The relevant substance is pyridine.

[0026] Preferably, in steps 1) and 3), the solvent is one or more of methanol and acetonitrile, preferably methanol.

[0027] Preferably, in step 1), the concentration range of the related substance reference solution is 0.2–5 μg / mL, more preferably 1–3 μg / mL, and even more preferably 2 μg / mL.

[0028] Preferably, in step 3), the concentration range of the deoxycholic acid test solution is 5–25 mg / mL, more preferably 5–15 mg / mL, and even more preferably 10 mg / mL.

[0029] The third aspect of this invention provides an application of the quality control and detection method described above in the quality control of deoxycholic acid.

[0030] Due to the adoption of the above technical solutions, the present invention has the following advantages compared with the prior art:

[0031] The method for separation and quality control detection of related substances in deoxycholic acid provided by the present invention involves preparing a pyridine reference solution of a certain concentration, determining the peak area of ​​pyridine in the reference and test solutions by high performance liquid chromatography, and calculating the content of pyridine by external standard method. This method has high sensitivity and strong specificity, thus enabling accurate quantitative determination of the content of the related substance pyridine.

[0032] The detection limit of this quality control method is 0.1 μg / mL, which is equivalent to 0.001% of the theoretical concentration of the test sample. Pyridine at a concentration of 5% of the limit can be detected. Attached Figure Description

[0033] Figure 1 The chromatogram is of the pyridine reference solution in Example 1.

[0034] Figure 2 The chromatogram is of the deoxycholic acid test solution in Example 1.

[0035] Figure 3 The result is the linear fitting result of pyridine in Example 4. Detailed Implementation

[0036] To make the technical solution and beneficial effects of the present invention more apparent and understandable, a detailed description is provided below in conjunction with the accompanying drawings and specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any product identical or similar to the present invention, derived by any person under the guidance of the present invention or by combining the present invention with features of other prior art, falls within the protection scope of the present invention. The drawings are not necessarily drawn to scale; local features may be enlarged or reduced to more clearly show the details of the local features. Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application pertains.

[0037] The first aspect of this invention provides a method for separating related substances from deoxycholic acid, which employs high performance liquid chromatography (HPLC). The chromatographic column is packed with octadecyl bonded silica gel, and the mobile phase is 0.010 mol / L dipotassium hydrogen phosphate solution. Acetonitrile is used as the mobile phase B, and linear gradient elution is performed to separate deoxycholic acid and related substances.

[0038] The relevant substance is pyridine.

[0039] In some implementations, the linear gradient elution procedure is as follows:

[0040]

[0041] In some embodiments, the chromatographic column is a Waters XBridge C18 or a column with equivalent performance.

[0042] In some embodiments, the chromatographic column is a Waters XBridge C18.

[0043] In some embodiments, the chromatographic column has an inner diameter of 4.6 mm, a length of 250 mm, and a packing particle size of 5 μm.

[0044] In some embodiments, the high-performance liquid chromatography method employs an ultraviolet detector.

[0045] In some embodiments, the column temperature of the chromatographic column is 20°C to 40°C.

[0046] In some embodiments, the column temperature of the chromatographic column is 30°C.

[0047] In some embodiments, the injection volume for gradient elution is 10 μL to 30 μL.

[0048] In some embodiments, the injection volume for gradient elution is 20 μL.

[0049] In some embodiments, the gradient elution flow rate is 0.5 to 1.5 mL / min.

[0050] In some embodiments, the gradient elution flow rate is 1.0 mL / min.

[0051] In some embodiments, the detection wavelength of the high-performance liquid chromatography is 210–260 nm.

[0052] In some embodiments, the detection wavelength of the high-performance liquid chromatography is 240–260 nm.

[0053] In some embodiments, the detection wavelength of the high-performance liquid chromatography is 256 nm.

[0054] A second aspect of the present invention provides a quality control detection method for deoxycholic acid-related substances, comprising the following steps:

[0055] (1) Preparation of reference solution: Dissolve the relevant substance reference in a solvent to prepare a reference solution;

[0056] (2) Preparation of test solution: Dissolve deoxycholic acid in solvent to prepare a test solution of deoxycholic acid;

[0057] (3) Determination method: The reference solution and the test solution prepared according to steps (1) and (2) are injected into a high performance liquid chromatograph for separation, detection and recording of chromatograms, and the separation method is as described above;

[0058] Record the peak area of ​​the main peak in the reference solution and the peak area of ​​related substances in the test solution;

[0059] (4) Calculation: Calculate the concentration and content of related substances in deoxycholic acid;

[0060] The relevant substance is pyridine.

[0061] In some embodiments, in steps 1) and 3), the solvent is one or more of methanol and acetonitrile.

[0062] In some embodiments, the solvent in steps 1) and 3) is methanol.

[0063] In some embodiments, in step 1), the concentration range of the relevant substance reference solution is 0.2–5 μg / mL.

[0064] In some embodiments, in step 1), the concentration range of the relevant substance reference solution is 1–3 μg / mL.

[0065] In some embodiments, in step 1), the concentration range of the relevant substance reference solution is 2 μg / mL.

[0066] In some embodiments, in step 3), the concentration of the deoxycholic acid test solution ranges from 5 to 25 mg / mL.

[0067] In some embodiments, in step 3), the concentration of the deoxycholic acid test solution ranges from 5 to 15 mg / mL.

[0068] In some embodiments, in step 3), the concentration of the deoxycholic acid test solution is in the range of 10 mg / mL.

[0069] The third aspect of this invention provides an application of the quality control and detection method described above in the quality control of deoxycholic acid.

[0070] Definitions and Explanations

[0071] Unless otherwise stated, the following terms and phrases as used herein are intended to have the following meanings. A particular phrase or term should not be considered uncertain or unclear unless specifically defined, but should be understood in its ordinary sense. When trade names appear herein, they are intended to refer to the corresponding product or its active ingredient.

[0072] In this invention, the term "system suitability test," according to the definition in the Chinese Pharmacopoeia, typically includes five parameters for the suitability test of a chromatographic system: theoretical plate number, resolution, sensitivity, tailing factor, and repeatability. The main system suitability evaluation indicators in this invention are sensitivity and repeatability. The acceptable system suitability controls in this invention are: the signal-to-noise ratio of the main peak in the sensitivity solution should be no less than 10, and the relative standard deviation of the peak areas of all reference solutions should not exceed 5.0%.

[0073] In this invention, the term "accuracy" refers to the degree to which the result measured by the established method is close to the true value or reference value, and is generally expressed as recovery rate (%).

[0074] In this invention, the term "content" refers to the ratio of the mass of pyridine to the mass of deoxycholic acid, expressed as a percentage (%).

[0075] In this invention, the term "solvent" refers to methanol.

[0076] In this invention, unless otherwise stated, the water or aqueous solution referred to refers to analytical water commonly used in the analytical field, including but not limited to purified water, ultrapure water, deionized water, etc.

[0077] In this invention, regardless of whether the specific numerical value is preceded by "about," it refers to the fact that the specific numerical value can fluctuate within a range recognized in the art. Specifically, it can fluctuate within, for example, the absolute value of the specific numerical value within ±10%, ±9%, ±8%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1%. The invention will be specifically described below through embodiments, which are not intended to limit the invention in any way.

[0078] The solvent used in this invention is commercially available and can be used without further purification.

[0079] The following abbreviations are used in this invention: μg / mL represents micrograms per milliliter; r represents the linear correlation coefficient, the larger the linear correlation coefficient, the better the quantification ability; RSD represents the relative standard deviation.

[0080] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions. Percentages and parts are by weight unless otherwise stated.

[0081] Test methods in the following examples that do not specify specific conditions can be performed under standard conditions or as recommended by the manufacturer. Unless otherwise defined, all technical and scientific terms used herein are familiar to those skilled in the art.

[0082] Example 1: Quality Control Detection Method for Pyridine, a Related Substance in Deoxycholic Acid

[0083] 1. Instruments and analytical conditions

[0084] The high-performance liquid chromatograph is equipped with a UV detector, and the chromatographic column is a Waters column. A C18 250mm × 4.6mm, 5μm column or a column with equivalent performance was used. Mobile phase A was 0.01mol / L dipotassium hydrogen phosphate solution, and mobile phase B was acetonitrile. Linear gradient elution was performed according to Table 1: column temperature 30℃, detection wavelength 256nm, flow rate 1.0mL / min, and injection volume 20μl. Methanol was used as the solvent.

[0085] Table 1 Linear Gradient Elution Table

[0086]

[0087]

[0088] 2. Preparation of reference solution and test solution

[0089] Preparation of reference solution: Weigh approximately 100 mg of pyridine into a 50 mL volumetric flask containing an appropriate amount of solvent, dilute to the mark with solvent, and shake well; then accurately measure 1 mL into a 100 mL volumetric flask, dilute to the mark with solvent, and shake well; this is recorded as the reference stock solution; accurately transfer 1.0 mL of the reference stock solution into a 10 mL volumetric flask, dilute to the mark with solvent, and shake well.

[0090] Preparation of sensitivity solution: Transfer 1 mL of the reference solution to a 10 mL volumetric flask, dilute to the mark with solvent, and shake well.

[0091] Preparation of the test solution: Accurately weigh 100 mg of deoxycholic acid sample, place it in a 10 mL volumetric flask, add an appropriate amount of solvent, sonicate to dissolve and dilute to the mark, and shake well to obtain the solution.

[0092] 3. Experimental Procedure

[0093] Take 20 μL of solvent, inject it into the high-performance liquid chromatograph, and record the chromatogram. The solvent should be free from interference.

[0094] Take 20 μL of the sensitivity solution, inject it into the high performance liquid chromatograph, and record the chromatogram. The signal-to-noise ratio of the main peak in the sensitivity solution should not be less than 10.

[0095] Inject 20 μL of the test solution into the high-performance liquid chromatograph and record the chromatogram. Calculate the concentration and content of pyridine in deoxycholic acid using the following formulas ① and ②.

[0096] ①The formula for calculating the concentration of pyridine in deoxycholic acid is:

[0097]

[0098] ②The formula for calculating the pyridine content in deoxycholic acid is:

[0099]

[0100] In equations ① and ② above,

[0101] Cs: Concentration of the reference solution (μg / mL), Ct: Concentration of the sample solution (μg / mL);

[0102] At: Pyridine peak area in the sample solution; As: Pyridine peak area in the reference solution;

[0103] Vt: Dilution volume of the sample (mL), Mt: Sample weight (mg);

[0104] 1000: Conversion factor from mg to μg for sample weighing units;

[0105] P: Content of reference standard (e.g., if the content is 99.9%, subtract 0.999 for calculation).

[0106] Example 2: Specificity and System Suitability Test

[0107] The purpose of this experiment is to confirm whether the quality control detection method for pyridine in Example 1 meets the requirements of system suitability. The acceptable criteria for the system suitability test are: the RSD of the pyridine peak area in the reference solution should be ≤5.0%, and the signal-to-noise ratio of the main peak in the sensitivity solution should be not less than 10.

[0108] 1. Instruments and analytical conditions: Same as in Example 1.

[0109] 2. Solution preparation: Prepare the reference solution, sensitivity solution and test solution according to the method in Example 1.

[0110] 3. Experimental procedure:

[0111] Take 20 μL of solvent and inject it into the high performance liquid chromatograph. Record the chromatogram. The chromatogram shows that the solvent has no interference and the method has strong specificity.

[0112] Take 20 μL of the sensitivity solution and inject it into the high-performance liquid chromatograph to perform a sensitivity test, then record the chromatogram. The signal-to-noise ratio of the main peak in the sensitivity solution is 21.

[0113] Inject 20 μL of the reference solution into the high performance liquid chromatograph. The injection precision was assessed by injecting the reference solution five times consecutively. The specific results are shown in Table 2 below.

[0114] Table 2. Injection precision results

[0115] Injection times 1 2 3 4 5 RSD Peak area 76.059 78.797 77.022 76.353 76.502 1.5％

[0116] Based on the above experiments and results, it can be seen that: in the sensitivity test, the signal-to-noise ratio of the main peak in the sensitivity solution is 21, which is not less than 10, and the injection precision is good. In the precision test, the RSD of the main peak area of ​​the 6-pair reference solution is 1.5%, which is much lower than 5.0%.

[0117] Example 3: Limit of Detection and Limit of Quantification Test

[0118] The purpose of this experiment is to determine the minimum limit (limit of quantitation) and the minimum limit (limit of detection) at which the analyte can be accurately quantified under the conditions of this method.

[0119] 1. The instruments and analytical conditions are the same as in Example 1.

[0120] 2. Solution preparation

[0121] Limit of Quantitation Solution: Accurately weigh approximately 100 mg of pyridine into a 50 mL volumetric flask pre-filled with an appropriate amount of solvent, dilute to the mark with solvent, and mix well. Then accurately measure 1 mL into a 100 mL volumetric flask, dilute to the mark with solvent, and mix well. Finally, accurately transfer 1.0 mL into a 100 mL volumetric flask, dilute to the mark with solvent, and mix well.

[0122] Detection limit solution: Transfer 5 mL of the quantitation limit solution to a 10 mL volumetric flask, dilute to the mark with solvent, and shake well.

[0123] 3. Experimental Procedure and Results

[0124] After the system suitability was verified as qualified according to the method in Example 2, 20 μL of each of the 6 limit of quantitation (LOQ) and 1 limit of detection (LOD) solutions were injected into the high performance liquid chromatograph, the chromatograms were recorded, the peak areas were recorded, and the signal-to-noise ratio of the LOQ and LOD and the relative standard deviation of the peak area of ​​the LOQ solution were examined. The specific results are shown in Tables 3 and 4 below.

[0125] The signal-to-noise ratio of the pyridine peak in the first-injection detection limit solution was 10.3, which is greater than 3, thus passing the test.

[0126] The average signal-to-noise ratio of the pyridine peak in the 6-needle limit-of-quantitation solution was 22.7, which is greater than 10, thus passing the test.

[0127] The RSD of the peak area of ​​the pyridine peak in the 6-needle limit solution was 8.2%, which is less than 15.0%, and thus passed.

[0128] Table 3: Limit of Quantitation Results for Pyridine

[0129]

[0130] Table 4: Detection Limits for Pyridine

[0131] Name Peak area mAU*S Signal to noise Concentration Limit level Detection limit solution 3.999 10.3 0.1056 μg / mL 5％

[0132] The above experiments and results show that the detection limit concentration of the pyridine quality control detection method of the present invention is 0.1056 μg / mL, which is 5% of the limit level (0.02%), and the quantitation limit concentration is 0.2111 μg / mL, which is 10% of the limit level (0.02%). In summary, the pyridine quality control detection method of the present invention can accurately and sensitively detect pyridine.

[0133] Example 4 Linearity Test

[0134] The purpose of this experiment is to determine the linear relationship between the measurement results and concentration of the test solution within a pre-defined range.

[0135] 1. The instruments and analytical conditions are the same as in Example 1.

[0136] 2. Solution preparation

[0137] Linear stock solution: Take about 100 mg of pyridine and place it in a 50 mL volumetric flask with an appropriate amount of solvent added beforehand. Accurately weigh the solution, dilute it to the mark with the solvent, and shake well. Then accurately measure 1 mL of the solution and place it in a 100 mL volumetric flask. Dilute it to the mark with the solvent and shake well.

[0138] 200% linear solution: Accurately transfer 2.0 mL of the linear stock solution into a 10 mL volumetric flask, dilute to the mark with solvent, and mix well. Record as L-200%.

[0139] 150% linear solution: Accurately transfer 3.0 mL of the linear stock solution into a 20 mL volumetric flask, dilute to the mark with solvent, and mix well. Record as L⁻¹⁵⁰.

[0140] 100% linear solution: Accurately transfer 1 mL of the linear stock solution into a 10 mL volumetric flask, dilute to the mark with solvent, and mix well. Record as L⁻¹⁰⁰%.

[0141] 80% linear solution: Accurately transfer 2 mL of the linear stock solution into a 25 mL volumetric flask, dilute to the mark with solvent, and mix well. Record as L-80%.

[0142] 50% linear solution: Accurately transfer 1 mL of the linear stock solution into a 20 mL volumetric flask, dilute to the mark with solvent, and mix well. Record as L-50%.

[0143] 10% linear solution: Accurately transfer 1 mL of the linear stock solution into a 100 mL volumetric flask, dilute to the mark with solvent, and mix well. Record as L⁻¹⁰%.

[0144] 3. Experimental Procedure and Results

[0145] After the system suitability was verified as qualified according to the method in Example 2, 20 μL of linear solutions with concentrations of 10%, 50%, 80%, 100%, 150%, and 200% were injected into the high-performance liquid chromatograph, respectively. Chromatograms and peak areas were recorded. Linear regression was performed with peak area as the ordinate and concentration (μg / mL) as the abscissa. The correlation coefficient r should be ≥0.990. The linearity results are shown in Table 5 below.

[0146] Table 5: Linearity Results

[0147]

[0148] The above experiments and results show that pyridine exhibits a good linear relationship with peak area in the concentration range of 0.2111–4.2216 μg / mL, with a linear correlation coefficient r of 0.9995.

[0149] Example 5 Accuracy Test

[0150] The purpose of this experiment is to determine the difference between the measurement results and the true values ​​of the chromatographic method within the expected linear range, thereby confirming whether the method can obtain accurate test results.

[0151] 1. The instruments and analytical conditions are the same as in Example 1.

[0152] 2. Solution preparation

[0153] Reference solution: Weigh approximately 100 mg of pyridine into a 50 mL volumetric flask containing an appropriate amount of solvent, dilute to the mark with solvent, and shake well; then accurately measure 1 mL into a 100 mL volumetric flask, dilute to the mark with solvent, and shake well; this is recorded as the reference stock solution; accurately transfer 1.0 mL into a 10 mL volumetric flask, dilute to the mark with solvent, and shake well.

[0154] Accuracy stock solution (1000% limit level): that is, the reference stock solution used in the preparation of the above reference solution.

[0155] Preparation of accuracy solution:

[0156] Unspecified solution: Accurately weigh 100 mg of deoxycholic acid sample, place it in a 10 mL volumetric flask, add an appropriate amount of solvent, sonicate to dissolve and dilute to the mark, and shake well to obtain the solution.

[0157] 20% Level Accuracy Solution: Accurately weigh 500 mg of deoxycholic acid sample into a 50 mL volumetric flask, add an appropriate amount of solvent and sonicate to dissolve, then precisely add 1 mL of accuracy stock solution, and dilute to the mark with solvent. Shake well and prepare three parallel portions.

[0158] 50% accuracy solution: Accurately weigh 200 mg of deoxycholic acid sample into a 20 mL volumetric flask, add an appropriate amount of solvent and sonicate to dissolve, then precisely add 1 mL of accuracy stock solution, and dilute to the mark with solvent. Shake well and prepare six parallel aliquots.

[0159] 100% accuracy solution: Accurately weigh 100 mg of deoxycholic acid sample into a 10 mL volumetric flask, add an appropriate amount of solvent and sonicate to dissolve, then precisely add 1 mL of accuracy stock solution, and dilute to the mark with solvent. Shake well and prepare three parallel portions.

[0160] 150% spiking solution: Accurately weigh 200 mg of deoxycholic acid sample into a 20 mL volumetric flask, add an appropriate amount of solvent and sonicate to dissolve, then accurately add 3 mL of accuracy stock solution, and dilute to the mark with solvent. Shake well and prepare three parallel portions.

[0161] After the system suitability was verified as qualified according to the method in Example 2, 20 μL of each of the following solutions were injected: 20% limit level accuracy solution, 50% limit level accuracy spiked solution, 100% limit level accuracy spiked solution, and 150% limit level accuracy spiked solution. The content and recovery rate were calculated. The individual and mean recovery rates of pyridine in the 20% limit level accuracy solution should be between 70.0% and 130.0%, and the RSD of the recovery rate should not exceed 15.0%. The individual and mean recovery rates of pyridine in the 50%–150% limit level accuracy solutions should be between 85.0% and 110.0%. The RSD of the pyridine recovery rate in the nine accuracy solutions at the 50%–150% limit level should not exceed 10.0%.

[0162] 3. Experimental Procedure and Results

[0163] The accuracy results are shown in Table 6 below.

[0164]

[0165] In the formula:

[0166] Cm: Mass of pyridine measured in the spiked test solution, in μg;

[0167] Cc: Mass of pyridine in the spiked test solution before spiking, in μg;

[0168] Cg: Mass of pyridine added to the spiked test solution, in μg.

[0169] Table 6 Accuracy Results

[0170]

[0171] The experiment shows that:

[0172] (1) At the 20% limit level, the single-valued recoveries of the solutions were between 91.4% and 93.8% (between 70.0% and 130.0%); the mean recoveries were 92.2% (between 70.0% and 130.0%); and the RSD was 1.6% (≤15.0%).

[0173] (2) The individual recoveries of the nine accuracy solutions at the 50%–150% limit level were between 91.6% and 101.9% (between 85.0% and 110.0%); the mean recovery was 96.7% (between 85.0% and 110.0%).

[0174] (3) The RSD of the recovery rate of the nine accuracy solutions at the 50% to 150% limit level was 3.4% (≤10.0%).

[0175] The method demonstrates good accuracy, proving its accuracy and feasibility in determining pyridine, a related substance in deoxycholic acid.

[0176] Example 6 Repeatability Test

[0177] The purpose of this experiment is to examine the random error of the experimental method and evaluate its precision.

[0178] 1. The instruments and analytical conditions are the same as in Example 1.

[0179] 2. Solution preparation

[0180] Reference solution: Weigh approximately 100 mg of pyridine into a 50 mL volumetric flask containing an appropriate amount of solvent, dilute to the mark with solvent, and shake well; then accurately measure 1 mL into a 100 mL volumetric flask, dilute to the mark with solvent, and shake well; this is recorded as the reference stock solution; accurately transfer 1.0 mL into a 10 mL volumetric flask, dilute to the mark with solvent, and shake well.

[0181] Accuracy stock solution (1000% limit level): that is, the reference stock solution used in the preparation of the above reference solution.

[0182] Preparation of repeatability solution: Same as the 100% limit level accuracy solution in Example 5. Prepare 6 parallel solutions.

[0183] After the system suitability was verified to be qualified according to the method in Example 2, 20 μL of repeatability solution was injected into the high performance liquid chromatograph, the chromatogram was recorded, the pyridine peak area was recorded, and the pyridine content and its 6 RSD values ​​were calculated.

[0184] 3. Experimental Procedure and Results

[0185] The repeatability results are shown in Table 7 below.

[0186] Table 7 Repeatability Results

[0187]

[0188] The experiment showed that the RSD of pyridine content in the 6 replicate solutions was 0.0% (≤10.0%).

[0189] It should be understood that the above embodiments are exemplary and are not intended to encompass all possible implementations included in the claims. Various modifications and changes can be made to the above embodiments without departing from the scope of this disclosure. Similarly, the various technical features of the above embodiments can be arbitrarily combined to form other embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only illustrate several implementations of the present invention and do not limit the scope of protection of this patent.

Claims

1. A method for separating related substances from deoxycholic acid, characterized in that, High performance liquid chromatography (HPLC) was used with an octadecyl bonded silica column as the packing material, 0.010 mol / L dipotassium hydrogen phosphate solution as mobile phase A, and acetonitrile as mobile phase B, to separate deoxycholic acid and related substances by linear gradient elution. The relevant substance is pyridine.

2. The method for separating deoxycholic acid-related substances according to claim 1, characterized in that, The linear gradient elution procedure is as follows:

3. The method for separating deoxycholic acid-related substances according to claim 1, characterized in that, The chromatographic column is a Waters XBridge C18 or a column with equivalent performance. Preferably, the chromatographic column has an inner diameter of 4.6 mm, a length of 250 mm, and a packing particle size of 5 μm; Preferably, the high-performance liquid chromatography method employs an ultraviolet detector.

4. The method for separating deoxycholic acid-related substances according to claim 1, characterized in that, The column temperature of the chromatographic column is 20℃~40℃, preferably 30℃.

5. The method for separating deoxycholic acid-related substances according to claim 1, characterized in that, The injection volume for gradient elution is 10 μL to 30 μL, preferably 20 μL; Preferably, the gradient elution flow rate is 0.5–1.5 mL / min, and more preferably 1.0 mL / min.

6. The method for separating deoxycholic acid-related substances according to claim 1, characterized in that, The detection wavelength of the high-performance liquid chromatography is 210-260 nm, preferably 240-260 nm, and more preferably 256 nm.

7. A quality control detection method for deoxycholic acid-related substances, characterized in that, Includes the following steps: (1) Preparation of reference solution: Dissolve the relevant substance reference in a solvent to prepare a reference solution; (2) Preparation of test solution: Dissolve deoxycholic acid in solvent to prepare deoxycholic acid test solution; (3) Determination method: The reference solution and the test solution prepared according to steps (1) and (2) are injected into a high performance liquid chromatograph for separation, detection and recording of chromatograms, wherein the separation method is as described in any one of claims 1-6; Record the peak area of ​​the main peak in the reference solution and the peak area of ​​related substances in the test solution; (4) Calculation: Calculate the concentration and content of related substances in deoxycholic acid; The relevant substance is pyridine.

8. The quality control and detection method for related substances in deoxycholic acid according to claim 7, characterized in that, In steps 1) and 3), the solvent is one or more of methanol and acetonitrile, preferably methanol.

9. The quality control detection method for related substances in deoxycholic acid according to any one of claims 7-8, characterized in that, In step 1), the concentration range of the related substance reference solution is 0.2–5 μg / mL, preferably 1–3 μg / mL, and more preferably 2 μg / mL; Preferably, in step 3), the concentration range of the deoxycholic acid test solution is 5–25 mg / mL, more preferably 5–15 mg / mL, and even more preferably 10 mg / mL.

10. The application of a quality control and testing method according to any one of claims 7-9 in the quality control of deoxycholic acid.

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