Method for detecting acetone and triethylamine in ursodesoxycholic acid

Abstract

The invention discloses a method for detecting acetone and triethylamine in ursodesoxycholic acid. According to the method, headspace sampling gas chromatography is adopted for detection. According to the method, a 0.4 mol / L sodium hydroxide solution is used as a solvent of ursodesoxycholic acid, and acetone and triethylamine in a to-be-detected sample are completely separated from ursodesoxycholic acid and impurities under the chromatographic conditions of a specific heating program, a sample inlet temperature, a detector temperature, a flow rate and headspace parameters, so that accurate determination of acetone and triethylamine in ursodesoxycholic acid is realized. The method disclosed by the invention is high in accuracy and sensitivity and good in repeatability, and has practical popularization and application values.

Description

Technical Field

[0001] The invention belongs to the field of drug detection, and particularly relates to a method for detecting acetone and triethylamine in ursodeoxycholic acid. Background Art

[0002] Ursodeoxycholic acid, also known as 3a,7β-dihydroxy-5β-cholestane-24-acid, is an organic compound that is odorless, bitter, easily soluble in ethanol, insoluble in chloroform, easily soluble in glacial acetic acid, and soluble in sodium hydroxide solution. It is referred to as UDCA. Its chemical formula is C 24 H 40 O4, molecular weight is 392.58, planar structure is:

[0003]

[0004] The three-dimensional structure is:

[0005]

[0006] Ursodeoxycholic acid is used medically to increase bile acid secretion, change bile composition, reduce cholesterol and cholesterol esters in bile, and facilitate the gradual dissolution of cholesterol in gallstones. It is used to treat gallstones, cholestatic liver disease, fatty liver, various types of hepatitis, toxic liver disorders, cholecystitis, choledochitis, bile dyspepsia, bile reflux gastritis, eye diseases, etc.

[0007] The refining process of ursodeoxycholic acid requires the use of solvents acetone and triethylamine, of which acetone is a colorless transparent liquid with a special pungent smell; triethylamine is a colorless oily liquid with a strong ammonia smell. According to the Chinese Pharmacopoeia "0861 Determination of Residual Solvents", acetone and triethylamine are both Class III solvents, and the drug GMP quality requirements limit the use of them to less than 0.5ppm. However, there is no standard method for detecting acetone and triethylamine residues in ursodeoxycholic acid at home and abroad, so the residual amount of acetone and triethylamine in ursodeoxycholic acid cannot be accurately detected. Therefore, it is urgent to develop a reliable and accurate method for detecting acetone and triethylamine residues in ursodeoxycholic acid, clarify the release standards, and supervise the detection of acetone and triethylamine residues in ursodeoxycholic acid. It is also very necessary and has extremely important practical significance. Summary of the invention

[0008] In order to solve the above problems, the present invention provides a method for detecting acetone and triethylamine in ursodeoxycholic acid, which comprises the following steps:

[0009] (1) Preparation of reference solution: Take acetone and / or triethylamine reference solution and dissolve it in sodium hydroxide solution to obtain a reference solution;

[0010] (2) Prepare the test solution: Take ursodeoxycholic acid and dissolve it in sodium hydroxide solution;

[0011] (3) Detection: Detection was performed by headspace gas chromatography; gas chromatography conditions: chromatographic column DB-624, heating program: starting temperature was 60°C, maintained for 3 min, then heated to 130°C at a rate of 15°C / min and maintained for 5 min.

[0012] Furthermore, in step (1), the mass volume ratio of acetone to sodium hydroxide solution is 60-100 mg:250 ml, preferably 75 mg:250 ml; the mass volume ratio of triethylamine to sodium hydroxide solution is 10-50 mg:250 ml, preferably 25 mg:250 ml.

[0013] Furthermore, in step (2), the mass volume ratio of ursodeoxycholic acid to sodium hydroxide solution is 100-200 mg:20 ml.

[0014] Furthermore, the concentration of the sodium hydroxide solution is 0.1% to 0.4%.

[0015] Furthermore, the concentration of the sodium hydroxide solution is 0.4%.

[0016] Furthermore, in step (3), the specifications of the chromatographic column DB-624 are 30m×0.32mm, 1.8μm.

[0017] Furthermore, in step (3), the gas chromatography conditions include an injection port temperature of 160°C to 220°C, an injection volume of 0.5 to 1.5 ml, a split ratio of 20 to 30:1, a carrier gas flow rate of 1.5 to 2.5 ml / min, a detector temperature of 150°C to 280°C, a headspace equilibrium temperature of 80 to 100°C, a quantitative loop temperature of 80 to 120°C; and a transmission line temperature of 100 to 120°C.

[0018] Furthermore, the gas chromatography conditions include an injection port temperature of 130°C to 182°C, an injection volume of 1.0 to 1.5 ml, a split ratio of 25 to 30:1, a carrier gas flow rate of 1.9 to 2.1 ml / min, a detector temperature of 245°C to 255°C, a headspace equilibrium temperature of 88 to 92°C, a quantitative loop temperature of 100 to 120°C, and a transmission line temperature of 110 to 120°C.

[0019] Furthermore, the gas chromatography conditions include an injection port temperature of 180°C, an injection volume of 1.0 ml, a split ratio of 30:1, a carrier gas flow rate of 2.0 ml / min, a detector temperature of 250°C, a headspace equilibrium temperature of 90°C, a quantitative loop temperature of 100°C, and a transmission line temperature of 110°C.

[0020] Furthermore, the chromatogram of the test solution presents a chromatographic peak with a retention time consistent with that of the chromatographic peak in the chromatogram of the reference solution, thereby confirming that ursodeoxycholic acid contains acetone and / or triethylamine, which are components corresponding to the reference solution;

[0021] The content of acetone and / or triethylamine in the ursodeoxycholic acid is calculated by peak area using an external standard method.

[0022] The invention discloses a method for detecting acetone and triethylamine in ursodeoxycholic acid. By using 0.4 mol / L sodium hydroxide solution as a solvent for ursodeoxycholic acid, under the chromatographic conditions of a specific heating program, injection port temperature, detector temperature, flow rate and headspace parameters, acetone and triethylamine in a sample to be detected are completely separated from ursodeoxycholic acid and impurities, thereby realizing accurate determination of acetone and triethylamine in ursodeoxycholic acid. The method has high accuracy and sensitivity, good repeatability and practical promotion and application value.

[0023] The above contents of the present invention are further described in detail below through specific implementation methods in the form of examples. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Spectra of acetone and triethylamine reference solution;

[0025] Figure 2 Residual spectra of acetone and triethylamine in ursodeoxycholic acid samples;

[0026] Figure 3 Acetone quantification limit spectrum;

[0027] Figure 4 Triethylamine quantitation limit spectrum;

[0028] Figure 5 Acetone 100% linear solution spectrum;

[0029] Figure 6 Triethylamine 100% linear solution spectrum;

[0030] Figure 7 Changing durability changes the detector temperature map;

[0031] Figure 8 Spectrum when N,N-dimethylformamide is selected as solvent;

[0032] Fig. 9 The spectrum when 0.1mol / L sodium hydroxide solution is selected as the solvent;

[0033] Fig.10 The spectrum when 0.2mol / L sodium hydroxide solution is selected as the solvent;

[0034] Fig.11 The spectrum when 0.3 mol / L sodium hydroxide solution is selected as the solvent;

[0035] Fig.12 The spectrum when 0.4 mol / L sodium hydroxide solution is selected as the solvent;

[0036] Fig.13 Spectrum when HP-1 (30m×0.32mm×0.25μm) column is selected;

[0037] Fig.14 Select the spectrum when the injection port temperature is 230°C;

[0038] Fig.15 The programmed temperature condition was selected as 60°C for 3 min, then increased to 150°C at a heating rate of 30°C / min and maintained for 3 min. DETAILED DESCRIPTION

[0039] Example 1 Qualitative Detection of Acetone and Triethylamine in Ursodeoxycholic Acid of the Present Invention

[0040] 1) Prepare the test solution

[0041] Take 150 mg of ursodeoxycholic acid test sample, weigh accurately, place in a 20 ml headspace bottle, add 1 ml of 0.4 mol / L sodium hydroxide solution to dissolve, seal the bottle mouth, and obtain;

[0042] 2) Preparation of reference solution

[0043] Take 75 mg of acetone reference substance and 25 mg of triethylamine reference substance, weigh accurately, place in a 50 ml volumetric flask containing 10 ml of 0.4 mol / L sodium hydroxide solution, then transfer completely to a 250 ml volumetric flask, dilute with 0.4 mol / L sodium hydroxide solution and make up to the mark, mix well, accurately measure 1 ml, place in a 20 ml headspace bottle, seal the bottle mouth, and you have it;

[0044] 3) Pipette the reference solution and the test solution into the headspace gas chromatograph respectively. The chromatographic conditions are as follows:

[0045] Chromatographic column: DB-624 (30m×0.32mm, 1.8μm); column temperature: 60℃ for 3min, then heated to 130℃ for 5min at a heating rate of 15℃ / min; injection port: 180℃; injection volume: 1.0ml; split ratio: 30:1; carrier gas flow rate: 2.0ml / min; detector: hydrogen flame ionization detector 250℃; heating box: 90℃; quantitative loop: 100℃; transmission line: 110℃;

[0046] 3) The chromatogram of the test solution shows a chromatographic peak with the same retention time as the chromatographic peak in the chromatogram of the reference solution, and it is determined that ursodeoxycholic acid contains acetone and / or triethylamine. The content of acetone and / or triethylamine is calculated by the external standard method based on the peak area. The specific formula is as follows:

[0047]

[0048] In the formula: X—residual acetone and triethylamine, %; A2—peak area of ​​residual acetone / triethylamine in the test solution, pA*s; A1—peak area of ​​acetone / triethylamine in the reference solution, pA*s; C2—theoretical concentration of the test sample, mg / ml; C1—actual concentration of acetone / triethylamine in the reference solution, mg / ml.

[0049] The following test examples are used to demonstrate the beneficial effects of the present invention.

[0050] Experimental Example 1 Study on the Detection Method of Acetone and Triethylamine in Ursodeoxycholic Acid

[0051] 1. Instruments and reagents

[0052] 1) Reagents and Materials

[0053] Acetone reference substance (chromatographically pure manufacturer: Sichuan Xilong Science)

[0054] Triethylamine reference substance (analytical pure manufacturer: Sinopharm Chemical Reagent Co., Ltd.)

[0055] Sodium hydroxide: (analytical grade manufacturer: Sinopharm Chemical Reagent Co., Ltd.)

[0056] Chromatographic column (model: DB-624, specification: 30m×0.32mm, 1.8μm, manufacturer: Agilent Technologies, Inc.);

[0057] Volumetric flask (Specifications: 25ml, 50ml, 100ml, 250100ml Manufacturer: Tianjin Tianke Glass Instrument Manufacturing Co., Ltd.).

[0058] 2) Main instruments

[0059] Hydrogen generator: (SPH-300 Beijing Zhonghuipu);

[0060] Air generator: (GCK3302 Beijing Zhonghuipu);

[0061] Electronic balance (XSR105 Mettler Toledo);

[0062] Workstation: (Agilent Chemstation);

[0063] Gas chromatograph (Agilent 7890B Agilent Technologies Co., Ltd.);

[0064] 2. Method screening and results

[0065] 2.1 Screening of sample solvents

[0066] Take 150 mg of ursodeoxycholic acid test sample, accurately weigh it, put it in a 20 ml headspace bottle, dissolve it with 1 ml of N,N-dimethylformamide, 0.1 mol / L sodium hydroxide solution, 0.2 mol / L sodium hydroxide solution, 0.3 mol / L sodium hydroxide solution and 0.4 mol / L sodium hydroxide solution respectively, seal the bottle mouth, and inject it into the headspace injection gas chromatograph as the test solution. The chromatographic conditions are as follows:

[0067] Chromatographic column: DB-624 (30m×0.32mm, 1.8μm); column temperature: 60℃ for 3min, then increase to 130℃ at a heating rate of 15℃ / min and maintain for 5min; injection port: 180℃; injection volume: 1.0ml; split ratio: 30:1; detector: hydrogen flame ionization detector 250℃; heating box: 90℃; quantitative loop: 100℃; transfer line: 110℃.

[0068] The results of the test according to the above method are as follows: when N,N-dimethylformamide is dissolved, triethylamine does not appear in the chromatogram of the ursodeoxycholic acid sample. When 0.1-0.4 mol / L sodium hydroxide solution is selected for dissolution, acetone and triethylamine appear normally in the chromatogram of the ursodeoxycholic acid sample. The tailing factor of triethylamine is different. As the concentration of sodium hydroxide solution increases, the tailing factor decreases, which are 13.1, 9.3, 6.8, and 2.1, respectively. When 0.4 mol / L sodium hydroxide solution is used as the solvent for ursodeoxycholic acid, the sample dissolution effect is the best, and the peak shapes of acetone and triethylamine are good. The tailing factor of triethylamine is less than 5, and the two target peaks can achieve baseline separation, which can meet the daily detection needs ( Figures 8 to 12 ), so 0.4 mol / L sodium hydroxide solution was selected as the solvent of ursodeoxycholic acid.

[0069] 2.2 Screening of chromatographic conditions

[0070] Take 150 mg of ursodeoxycholic acid test sample, accurately weigh it, put it in a 20 ml headspace bottle, add 1 ml of 0.4 mol / L sodium hydroxide solution to dissolve it, seal the bottle mouth, and inject it into the headspace injection gas chromatograph as the test solution. The chromatographic conditions are as follows:

[0071] Chromatographic conditions ①, the only difference from the chromatographic conditions in "2.1 Screening of sample solvents" is that the chromatographic column is: HP-1 (30m×0.32mm×0.25μm);

[0072] Chromatographic condition ② is different from the chromatographic condition in “2.1 Screening of sample solvents” only in that the injection port temperature is 230°C:

[0073] Chromatographic condition ③ is different from the chromatographic condition in "2.1 Screening of sample solvents" only in that the programmed temperature condition is 60°C for 3 min, and then the temperature is increased to 150°C at a rate of 30°C / min and maintained for 3 min:

[0074] Note: The USP Pharmacopoeia requires that the tailing factor should be between 0.8 and 1.8. In the Chinese Pharmacopoeia, there is no specific requirement for the tailing factor in the peak area quantitative method, but it is stipulated that when the peak area is used as a quantitative parameter, if severe tailing affects the judgment of the baseline and the start and end of the chromatographic peak and the accuracy of the peak area integration, the tailing factor should be specified according to the variety. Since the tailing factor of triethylamine cannot meet the requirements of the USP Pharmacopoeia in this system, general detection requirements are stipulated, and the tailing factor should be less than 5.

[0075] According to the above method, when the chromatographic column is HP-1 (30m×0.32mm×0.25μm), the tailing factor of triethylamine is 88.1 ( Fig.13 ), which does not meet the general detection requirements; when the injection port temperature is 230℃, the elution time of acetone and triethylamine is earlier, and the tailing factor of triethylamine is 22.0( Fig.14 ), which does not meet the general detection requirements; when the program temperature is maintained at 60℃ for 3min, the temperature is increased to 150℃ at a rate of 30℃ / min and maintained for 3min, the tailing factor of triethylamine is 4.0 ( Fig.15 ), the detection effect is not as good as maintaining the temperature at 60℃ for 3min. Heating the temperature to 130℃ at a rate of 15℃ / min and maintaining it for 5min will not affect the baseline separation, but will affect the judgment of the start and end of the chromatographic peak and the accuracy of the peak area integration.

[0076] From the screening results, it can be seen that good detection of acetone and triethylamine in ursodeoxycholic acid can be achieved only under the following detection conditions:

[0077] Take ursodeoxycholic acid, place it in a headspace bottle, dissolve it with 1 ml of 0.4 mol / L sodium hydroxide solution, seal the bottle mouth, and inject it into the headspace injection gas chromatograph as the test solution. The chromatographic conditions are as follows:

[0078] Chromatographic column: DB-624 (30m×0.32mm, 1.8μm); column temperature: 60℃ for 3min, then increase to 130℃ at a heating rate of 15℃ / min and maintain for 5min; injection port: 180℃; injection volume: 1.0ml; split ratio: 30:1; carrier gas flow rate 2.0ml / min; detector: hydrogen flame ionization detector 250℃; heating box: 90℃; quantitative loop: 100℃; transmission line: 110℃.

[0079] 3. Methodological investigation

[0080] 3.1 Precision experiment

[0081] 1) Preparation of test solution

[0082] Weigh about 150 mg of ursodeoxycholic acid test sample, a total of 6 portions, put them into 20 ml headspace bottles, add 1 ml of 0.4 mol / L sodium hydroxide solution to dissolve, seal the bottle mouth, and use it as the test solution;

[0083] 2) Chromatographic conditions

[0084] Injection method: headspace injection;

[0085] Chromatographic column: DB-624 (30m×0.32mm, 1.8μm)

[0086] Column temperature: 60°C for 3 min, then increase to 130°C at a rate of 15°C / min and hold for 5 min;

[0087] Inlet: 180℃

[0088] Injection volume: 1.0ml

[0089] Split ratio: 30:1

[0090] Carrier gas flow rate 2.0ml / min

[0091] Detector: Hydrogen flame ionization detector 250℃

[0092] Heating box: 90℃

[0093] Quantitative ring: 100°C

[0094] Transmission line: 110℃

[0095] 4) Calculation method:

[0096]

[0097] Where:

[0098] X—Acetone and triethylamine residues, %;

[0099] A2—peak area of ​​acetone / triethylamine residue in the test solution, pA*s;

[0100] A1—peak area of ​​acetone / triethylamine in reference solution, pA*s;

[0101] C2—theoretical concentration of the test sample, mg / ml;

[0102] C1—Actual concentration of acetone / triethylamine in the reference solution, mg / ml.

[0103] 4) According to the chromatographic conditions of 2) above, 6 portions of the test solution were injected into the gas chromatograph respectively, the peak areas of acetone and triethylamine were recorded, and the residual acetone and triethylamine were calculated by the calculation formula of 3).

[0104] 5) Test results

[0105] Table 1 Precision test results

[0106]

[0107] Conclusion: After 6 consecutive injections, the RSD of the acetone residue result was 1.84%, and the RSD of the triethylamine residue result was 3.62%, indicating good repeatability ( Figures 1-2 ).

[0108] 3.2 Limit of quantitation experiment

[0109] 1) 100% acetone linear solution: Accurately weigh 150 mg of acetone into a 100 ml volumetric flask, dilute to the mark with 0.4 mol / L sodium hydroxide solution and shake well to obtain.

[0110] 2) 100% triethylamine linear solution: Accurately weigh 75 mg of triethylamine in a 500 ml volumetric flask, dilute to the mark with 0.4 mol / L sodium hydroxide solution and shake well to obtain.

[0111] 3) Quantitation limit solution: Accurately measure 0.1 ml of 100% acetone and triethylamine linear solution into a 100 ml volumetric flask, dilute to the mark with 0.4 mol / L sodium hydroxide solution and shake well to obtain acetone and triethylamine quantitation limit solutions (acetone and triethylamine quantitation limit solutions are prepared separately).

[0112] 4) Chromatographic conditions and calculation methods: Same as the chromatographic conditions and calculation methods under “3.1 Precision experiment”.

[0113] 5) According to the chromatographic conditions, take acetone and triethylamine quantitative limit solution and inject them 6 times respectively, and record the chromatogram ( Figures 3-4 ), calculate the RSD of the peak areas of acetone and triethylamine in the 6 injections of the quantitative limit solution.

[0114] Table 2 Results of quantitation limit experiment

[0115]

[0116] Conclusion: After 6 consecutive injections, the quantification limit of acetone residue was 0.0003 mg / ml, and the RSD of 6 consecutive injections was 1.60%; the quantification limit of triethylamine residue was 0.0001 mg / ml, and the RSD of 6 consecutive injections was 4.97%, both of which met the determination requirements.

[0117] 3.3 Linearity experiment

[0118] 1) Preparation of acetone linear stock solution: Accurately weigh 150 mg of acetone, place in a 100 ml volumetric flask, quantitatively dilute with 0.4 mol / L sodium hydroxide solution and dilute to the scale, shake well, and obtain the linear stock solution.

[0119] 2) 150%, 100%, 80%, 50%, 10%, 1% acetone linear solutions: accurately pipette 7.5ml, 5.0ml, 4.0ml of acetone linear stock solution into 10ml volumetric flasks respectively, add 0.4mol / L sodium hydroxide solution to dilute to the scale to obtain 150%, 100%, 80% acetone linear solutions; take 5.0ml of 100% acetone linear solution, 2.0ml of 50% acetone linear solution, 1.0ml of 10% acetone linear solution into 10ml volumetric flasks, add 0.4mol / L sodium hydroxide solution to dilute to the scale to obtain 50%, 10%, 1% acetone linear solutions.

[0120] 3) Preparation of triethylamine linear stock solution: Accurately weigh 75 mg of triethylamine, place in a 500 ml volumetric flask, dilute to volume with 0.4 mol / L sodium hydroxide solution and shake well to obtain.

[0121] 4) 150%, 100%, 80%, 50%, 10%, 1% triethylamine linear solutions: accurately pipette 4.5ml, 3.0ml, 2.4ml of triethylamine linear stock solution into 10ml volumetric flasks respectively, add 0.4mol / L sodium hydroxide solution to dilute to the scale to obtain 150%, 100%, 80% triethylamine linear solutions; take 5.0ml of 100% triethylamine linear solution, 2.0ml of 50% triethylamine linear solution, 1.0ml of 10% triethylamine linear solution into 10ml volumetric flasks, add 0.4mol / L sodium hydroxide solution to dilute to the scale to obtain 50%, 10%, 1% triethylamine linear solutions.

[0122] 5) Chromatographic conditions: Same as those in “3.1 Precision experiment”.

[0123] 6) Injecting acetone and triethylamine linear solutions into the gas chromatograph from low concentration to high concentration in sequence according to the chromatographic conditions, recording the chromatogram, calculating the peak area and concentration of ursodeoxycholic acid acetone and triethylamine residual solvents within the range of the quantitative limit concentration to 150% limit concentration, and obtaining the regression equation and correlation coefficient.

[0124] 7) Test results

[0125] Table 3 Linearity test results

[0126]

[0127] Conclusion: Based on the above table and Figures 5-6It can be seen that the correlation coefficients of the linear regression equations of acetone and triethylamine are both 1.00, indicating that the method has good linearity.

[0128] 3.4 Accuracy Experiment

[0129] 1) Preparation of test solution

[0130] Weigh about 150 mg of ursodeoxycholic acid test sample, a total of 3 portions (1, 2, 3), put them into 20 ml headspace bottles, add 1 ml of 0.4 mol / L sodium hydroxide solution to dissolve, seal the bottle mouth, and use them as the test solution;

[0131] 2) Preparation of stock solution

[0132] Accuracy stock solution of the test sample: Accurately weigh about 7500 mg of ursodeoxycholic acid, place it in a 100 ml volumetric flask, add 50 ml of 0.4 mol / L sodium hydroxide solution to dissolve it, shake well, and obtain the solution.

[0133] Acetone reference accuracy stock solution: Accurately weigh 184 mg of acetone into a 25 ml volumetric flask containing 10 ml of 0.4 mol / L sodium hydroxide solution, make up to volume with 0.4 mol / L sodium hydroxide solution, and shake well.

[0134] Triethylamine reference accuracy stock solution: Accurately weigh 50 mg of triethylamine into a 50 ml volumetric flask containing 10 ml of 0.4 mol / L sodium hydroxide solution, then transfer to a 250 ml volumetric flask, make up to volume with 0.4 mol / L sodium hydroxide solution, and shake well.

[0135] 3) Preparation of accuracy solution

[0136] Accurately pipette 5.0ml of the sample stock solution, add 0.6ml, 1.7ml, and 2.8ml of acetone and triethylamine stock solutions into a 10ml volumetric flask, dilute with 0.4mol / L sodium hydroxide solution, make up to volume, and shake well. Prepare 3 portions of each serial number, a total of 9 mixed samples (serial numbers: 1-9). Prepare acetone and triethylamine accuracy solutions separately.

[0137] 4) Chromatographic conditions: Same as those in “3.1 Precision experiment”.

[0138] 5) Carry out the test according to the chromatographic conditions, inject 3 test solutions once each, and inject 3 different concentrations of sample solutions 3 times each. Record the chromatogram, calculate the contents of acetone and triethylamine in the sample solutions respectively, and calculate the recovery rate, the average recovery rate of each sample solution, and the RSD according to the following formula.

[0139]

[0140] Where:

[0141] C loading - the concentration of acetone or triethylamine in the loading solution, mg / ml;

[0142] C supply - the concentration of acetone or triethylamine in the test sample in the sample solution, mg / ml;

[0143] C control - the concentration of acetone or triethylamine added to the sample solution, mg / ml.

[0144] 6) Test results

[0145] Table 4 Acetone accuracy test results

[0146]

[0147]

[0148] Conclusion: The recovery rates of 3 acetone accuracy solutions with different concentrations were 93.56%, 93.18%, and 91.36%, and the recovery rate results were between 80.0% and 120.0%, which met the requirements; the RSD values ​​of the recovery rates of 3 acetone accuracy solutions with different concentrations were 0.082%, 1.13%, and 1.01%, respectively, and the results were ≤10.0%, which met the requirements. This method was confirmed to have good accuracy in detecting acetone residues.

[0149] Table 5 Triethylamine accuracy test results

[0150]

[0151] Conclusion: The recovery rates of three triethylamine accuracy solutions with different concentrations were 81.79%, 87.18%, and 88.03%, and the recovery rate results were between 80.0% and 120.0%, which met the requirements; the RSD values ​​of the recovery rates of three triethylamine accuracy solutions with different concentrations were 1.57%, 2.08%, and 1.17%, respectively, and the results were ≤10.0%, which met the requirements. This method was confirmed to have good accuracy in detecting triethylamine residues.

[0152] 3.5 Durability test

[0153] 1) Preparation of test solution

[0154] Weigh about 150 mg of ursodeoxycholic acid test sample, place it in a 20 ml headspace bottle, add 1 ml of 0.4 mol / L sodium hydroxide solution to dissolve it, and seal the bottle mouth to use as the test solution;

[0155] 2) Chromatographic conditions and calculation methods: Same as the chromatographic conditions and technical methods under “3.1 Precision experiment”.

[0156] 4) Determination method: The chromatographic behavior of the instrument (flow rate ±0.1 ml / min, injection port temperature ±2°C, starting temperature ±2°C, detector temperature ±5°C, heating box temperature ±2°C) and the changes in chromatographic columns of different batches were investigated respectively. Under each condition, the residual acetone and triethylamine in one batch of test solution were detected.

[0157] 5) Measurement results

[0158] Table 6 Acetone and triethylamine durability test results

[0159]

[0160]

[0161] Conclusion: By changing the flow rate, injection port temperature, column temperature, detector temperature, heating box temperature and other conditions in the original test, the residual acetone and triethylamine in the product were detected and compared with the results of acetone and triethylamine detected under the original test conditions. The relative deviation of the results of acetone and triethylamine detected by the two methods was calculated, and both met the requirements ( Figure 7 ).

[0162] In summary, the present invention uses gas chromatography to determine the residual acetone and triethylamine in ursodeoxycholic acid. The method verification results show that the detection method of the present invention has the advantages of high sensitivity, high separation efficiency, good selectivity, good renaturation and high accuracy, and can be used for the continuous and stable detection of residual acetone and triethylamine in ursodeoxycholic acid.

Claims

1. A method for detecting acetone and triethylamine in ursodeoxycholic acid, characterized in that: It includes the following steps: (1) Preparation of reference solution: Take acetone and / or triethylamine reference solution and dissolve it in sodium hydroxide solution to obtain a reference solution; (2) Prepare the test solution: Take ursodeoxycholic acid and dissolve it in sodium hydroxide solution; (3) Detection: Detection was performed by headspace gas chromatography; gas chromatography conditions: chromatographic column DB-624, heating program: starting temperature was 60°C, maintained for 3 min, then heated to 130°C at a rate of 15°C / min and maintained for 5 min.

2. The detection method according to claim 1, characterized in that: In step (1), the mass volume ratio of the acetone to the sodium hydroxide solution is 60-100 mg:250 ml, preferably 75 mg:250 ml; the mass volume ratio of the triethylamine to the sodium hydroxide solution is 10-50 mg:250 ml, preferably 25 mg:250 ml.

3. The detection method according to claim 1, characterized in that: In step (2), the mass volume ratio of ursodeoxycholic acid to sodium hydroxide solution is 100-200 mg:20 ml.

4. The detection method according to claim 1, characterized in that: The concentration of the sodium hydroxide solution is 0.1% to 0.4%.

5. The detection method according to claim 3, characterized in that: The concentration of the sodium hydroxide solution is 0.4%.

6. The detection method according to claim 1, characterized in that: In step (3), the specifications of the chromatographic column DB-624 are 30m×0.32mm, 1.8μm.

7. The detection method according to claim 1, characterized in that: In step (3), the gas chromatography conditions include an injection port temperature of 160°C to 220°C, an injection volume of 0.5 to 1.5 ml, a split ratio of 20 to 30:1, a carrier gas flow rate of 1.5 to 2.5 ml / min, a detector temperature of 150°C to 280°C, a headspace equilibrium temperature of 80 to 100°C, a quantitative loop temperature of 80 to 120°C; and a transmission line temperature of 100 to 120°C.

8. The detection method according to claim 7, characterized in that: The gas chromatography conditions include an injection port temperature of 130°C to 182°C, an injection volume of 1.0 to 1.5 ml, a split ratio of 25 to 30:1, a carrier gas flow rate of 1.9 to 2.1 ml / min, a detector temperature of 245°C to 255°C, and a headspace equilibrium temperature of 88 to 92°C. Quantitative loop temperature: 100~120℃; transfer line temperature 110~120℃.

9. The detection method according to claim 8, characterized in that: The gas chromatography conditions include an injection port temperature of 180°C, an injection volume of 1.0 ml, a split ratio of 30:1, a carrier gas flow rate of 2.0 ml / min, a detector temperature of 250°C, and a headspace equilibrium temperature of 90°C. Quantitative loop temperature: 100°C; transfer line temperature: 110°C.

10. The detection method according to any one of claims 1 to 8, characterized in that: The chromatogram of the test solution shows a chromatographic peak with the same retention time as the chromatographic peak in the chromatogram of the reference solution, confirming that ursodeoxycholic acid contains acetone and / or triethylamine, which are components corresponding to the reference solution; The content of acetone and / or triethylamine in the ursodeoxycholic acid is calculated by peak area using an external standard method.

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