A method for determining the content of tert-butyl hydroperoxide in deoxycholic acid or deoxycholic acid intermediates

Abstract

The application discloses a method for determining the content of potential genotoxic impurity tert-butyl hydroperoxide in deoxycholic acid bulk drug or an intermediate thereof, which adopts pre-column derivatization high performance liquid chromatography for detection, and adopts triphenylphosphine for derivatization treatment of the sample in pre-column derivatization; the detection conditions of the high performance liquid chromatography are as follows: a chromatographic column is an Agilent Eclipse Plus C18 column or a Waters Symmetry C18 column; a mobile phase A is 0.01% to 0.10% phosphoric acid solution; a mobile phase B is acetonitrile; a flow rate is 0.9 to 1.1 ml / min; a detection wavelength is 210 to 250 nm; a sample injection volume is 5 to 10 ul; a column temperature is 30 to 40 DEG C; a diluent is methanol or acetonitrile; and an elution mode is gradient elution. The method has the characteristics of low cost, simple operation, high accuracy and good sensitivity, and is suitable for quality control of medicines.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of pharmaceutical analysis, and particularly relates to a method for determining the content of potential genotoxic impurity tert-butyl hydroperoxide (TBHP) in deoxycholic acid or deoxycholic acid intermediates. BACKGROUND

[0002] Deoxycholic acid, with the chemical name of 3α, 12α-dihydroxy-5β-cholanic acid, has the structural formula shown in formula (I), and is an injectable cell lysing drug. Deoxycholic acid injection was first developed by the American Celura Biopharmaceutical Company, and was approved for marketing by the U.S. Food and Drug Administration (FDA) in April 2015, with the trade name of Belkyra, and is the first fat-dissolving needle approved by the FDA for treating lower jaw fat accumulation.

[0003]

[0004] Deoxycholic acid widely exists in animals, and deoxycholic acid from animal sources has a lower cost. However, deoxycholic acid from animal sources may contain pathogens, and therefore there is an urgent need for deoxycholic acid obtained by a plant source or a chemical synthesis method.

[0005] CN106146593B discloses a method for preparing deoxycholic acid, and the reaction route is as follows:

[0006]

[0007]

[0008] However, it was found in the production process that the oxidant TBHP used in the step of synthesizing intermediate 4 might remain in intermediate 4 or the bulk drug deoxycholic acid. TBHP contains an -O-O- group, which is a potential genotoxic alert structure. In order to better control product quality and verify the toxicity of impurities, the applicant commissioned a third party to analyze and evaluate 31 process and degradation impurities including TBHP. Specifically, according to the International Conference on Harmonization Guidelines for Good Clinical Practice (ICH M7), the toxicity of impurities was evaluated by two complementary (Q)SAR prediction methods (one based on expert knowledge rules and the other based on statistics), and the evaluation was performed using the Derek Nexus & Sarah Nexus evaluation software of Lhasa limited company. This method is a toxicity evaluation method that uses computational methods to analyze, simulate, visualize or predict the toxicity of chemicals, which has obvious advantages in time, cost and animal welfare, can be used to predict the results of bacterial mutagenicity tests, and has been generally accepted by FDA, EMA and CDE, etc. If the bacterial mutagenicity (Ames) test is positive, control measures need to be taken for the impurity, or if the level of the impurity cannot be controlled within a proper acceptable limit, an in vivo gene mutation test is recommended to conduct further hazard assessment. The evaluation results show that both the two complementary prediction methods and the Ames test show that the mutagenicity of the impurity TBHP is positive, which is a mutagenic impurity Figure 1 A mutagenic impurity refers to a genotoxic impurity that can directly cause DNA damage and lead to DNA mutation at a lower level, which may trigger cancer. Therefore, in the production process of deoxycholic acid, the content of TBHP in deoxycholic acid intermediates and bulk drug is detected, and the residual amount of TBHP is strictly controlled to be within a proper acceptable range, which is crucial for the quality control and safety of the drug.

[0009] In clinical practice, deoxycholic acid injection (10 mg / ml) is intermittently administered once a month, with a maximum administration volume of 10 ml (100 mg) each time, and the total administration time span is longest about 6 months (i.e. administration for 6 days, less than 1 month), so the acceptable intake of a single impurity in the drug is 120 μg / day. It is calculated that the limit of TBHP impurity in deoxycholic acid is 1200 ppm.

[0010] There are many literatures reported the methods for detecting the content of TBHP. For example, document 1 (Research on the determination of organic peroxide by graphene modified electrode, Journal of Wuhan Textile University, 2013, Vol. 26, No. 6, pp. 23-26) discloses an electrochemical method for determining the content of organic peroxide TBHP; document 2 (Journal of Analytical Chemistry, 2016, 71(9), 932-943) discloses a method for determining the content of organic peroxide TBHP in micelles and aqueous solution using a spectrophotometric biosensor under the catalysis of peroxidase; document 3 (Org. Process Res. Dev. 2019, 23, 2538-2542) studies the applicability of using an impregnated test paper semi-quantitative test paper to determine the content of TBHP in organic solvents; document 4 (Org. Process Res. Dev. 2020, 24, 1321-1327) discloses a method for detecting organic hydroperoxide based on liquid chromatography-ultraviolet. The above methods or steps are complicated, the experimental conditions are harsh, or the sensitivity and repeatability of TBHP detection are low, which are difficult to implement in a conventional drug analysis laboratory. Therefore, it is of great significance to develop a high-sensitivity TBHP content analysis method suitable for drug analysis experiments for the quality control of drugs. SUMMARY

[0011] The present application aims to overcome the above problems and provide a method for determining the content of tert-butyl hydroperoxide (TBHP) based on pre-column derivatization high performance liquid chromatography. The method generates triphenyl phosphine oxide (TPPO) by derivatization reaction of TBHP and triphenyl phosphine (TPP), and detects TBHP by determining the amount of TPPO, which can be used to determine the low-level content of TBHP (e.g. ppm level), has the advantages of low cost, simple operation, high accuracy and good sensitivity, and is very suitable for determining the content of mutagenic impurity TBHP in deoxycholic acid or deoxycholic acid intermediates.

[0012] To achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0013] A method for determining the content of potential genotoxic impurity tert-butyl hydroperoxide (TBHP) in deoxycholic acid or deoxycholic acid intermediates, characterized in that the method uses pre-column derivatization high performance liquid chromatography for detection, and triphenyl phosphine is used for derivatization treatment of the sample; the detection conditions of high performance liquid chromatography are as follows:

[0014] Chromatographic column: octadecylsilane-bonded silica gel as the filler; preferably Agilent Eclipse Plus C18 column or Waters Symmetry C18 column; more preferably, the specification of the chromatographic column is 4.6x100mm, 3.5μm;

[0015] Mobile phase A: 0.01-0.10% phosphoric acid aqueous solution; preferably 0.04-0.06% phosphoric acid aqueous solution; more preferably 0.05% phosphoric acid aqueous solution;

[0016] Mobile phase B: acetonitrile;

[0017] Flow rate: 0.9-1.1ml / min; preferably 1.0ml / min;

[0018] Detection wavelength: 210-250nm; preferably 223-227nm; more preferably 225nm;

[0019] Injection volume: 5-10μl;

[0020] Column temperature: 30-40℃; preferably 35℃;

[0021] The gradient elution program is as follows:

[0022]

[0023]

[0024] Preferably, the gradient elution program is as follows:

[0025]

[0026] More preferably, the gradient elution program is as follows:

[0027]

[0028] The method of the present application pre-treats the sample to be measured (e.g. deoxycholic acid, deoxycholic acid intermediate) by pre-column derivatization, and then determines the content of TBHP by high performance liquid chromatography. Specifically, the sample is derivatized by using the derivatization reagent triphenylphosphine (TPP), so that TPP reacts with TBHP in the sample to be measured to generate triphenylphosphine oxide (TPPO); the content of TPPO is determined by high performance liquid chromatography, so as to calculate the content of TBHP in the sample to be measured. The derivatization reaction mechanism is as follows:

[0029]

[0030] In one embodiment, the diluent / blank solvent is methanol or acetonitrile, and further, the diluent / blank solvent can be selected according to the solubility of the sample to be detected. For example, when the sample is deoxycholic acid, the diluent is methanol; when the sample is deoxycholic acid intermediate, the diluent is acetonitrile.

[0031] In one embodiment, the temperature of the derivatization treatment is 25-40°C.

[0032] In one embodiment, the sample is deoxycholic acid, the diluent is methanol, and the derivatization treatment includes derivatizing the sample with triphenylphosphine in methanol, and the reaction time is 1.5-2.5 hours, preferably 2 hours; or, the sample is deoxycholic acid intermediate, the diluent is acetonitrile, and the derivatization treatment includes derivatizing the sample with triphenylphosphine in acetonitrile, and the reaction time is 13-17 hours, preferably 15 hours.

[0033] In one embodiment, the method includes using a triphenylphosphine oxide control solution. Specifically, triphenylphosphine oxide control is dissolved in the diluent to prepare a triphenylphosphine oxide control solution with a concentration of 1-5 μg / ml; preferably, the concentration of the triphenylphosphine oxide control solution is 3 μg / ml.

[0034] In one embodiment, the method includes using a test sample solution. Specifically, the test sample is dissolved in the diluent with triphenylphosphine to obtain a solution with a test sample concentration of 5 mg / ml and a triphenylphosphine concentration of 0.1 mg / ml; the TBHP and TPP present in the test sample of the solution undergo derivatization reaction at 25-40°C to obtain a derivatized test sample solution; preferably, the derivatization reaction temperature is 40°C.

[0035] In one embodiment, the time of the derivatization reaction is related to the solvent. When the diluent is methanol, the time of the derivatization reaction is 1.5-2.5 hours, preferably 2 hours; when the diluent is acetonitrile, the time of the derivatization reaction is 13-17 hours, preferably 15 hours.

[0036] In one embodiment, the method includes using a blank derivatization reagent. Specifically, triphenylphosphine is dissolved in the diluent to obtain a 0.1 mg / ml triphenylphosphine solution, which is then placed at 25-40°C for the same time as the derivatization reaction to obtain a blank derivatization reagent. The use of the blank derivatization reagent can prove that the background of TPPO in the blank derivatization reagent is small and does not interfere with the detection of the sample; it can also prove that after water bath treatment, the background of TPPO is basically unchanged compared to 0, i.e., the blank derivatization reagent remains stable at this temperature. The blank derivatization reagent can eliminate the influence of the background of TPPO in the blank derivatization reagent on the detection results, correct the experimental results, and improve the accuracy of the detection results.

[0037] In one embodiment, the content of TBHP in deoxycholic acid or deoxycholic acid intermediate is calculated by the following formula:

[0038]

[0039] Wherein:

[0040] A spl is the peak area of TPPO in the test sample solution (after deducting the blank correction) ;

[0041] A std is the peak area of TPPO in the TPPO control solution;

[0042] P std is the purity of TPPO control solution, %;

[0043] M std is the sample weight of TPPO control solution, mg;

[0044] M spl is the sample weight, mg;

[0045] S std is the dilution factor of TPPO control solution;

[0046] S spl is the dilution factor of the sample;

[0047] M TBHP is the relative molecular mass of TBHP, about 90.12;

[0048] M TPPO is the relative molecular mass of TPPO, about 278.28;

[0049] M TBHP The ratio of M TPPO is the conversion factor.

[0050] In one exemplary embodiment, the present application provides a method for determining the content of TBHP in deoxycholic acid or deoxycholic acid intermediate, characterized in that the method uses pre-column derivatization high performance liquid chromatography for detection, and the detection conditions are as follows:

[0051] Chromatographic column: Agilent Eclipse Plus C18 column or Waters Symmetry C18 column, specifications 4.6x100mm, 3.5μm;

[0052] Mobile phase A: 0.04-0.06% phosphoric acid aqueous solution;

[0053] Mobile phase B: acetonitrile;

[0054] Flow rate: 0.9-1.1ml / min;

[0055] Detection wavelength: 223-227 nm;

[0056] Injection volume: 5 μl;

[0057] Column temperature: 30-40 °C;

[0058] The gradient elution program is as follows:

[0059]

[0060] In another exemplary embodiment, the present application provides a method for determining the content of TBHP in deoxycholic acid or deoxycholic acid intermediate, characterized in that the method uses pre-column derivatization high performance liquid chromatography for detection, and the detection conditions are as follows:

[0061] Chromatographic column: Agilent Eclipse Plus C18 column or Waters Symmetry C18 column, with a size of 4.6 x 100 mm, 3.5 μm;

[0062] Mobile phase A: 0.05% phosphoric acid aqueous solution;

[0063] Mobile phase B: acetonitrile;

[0064] Flow rate: 1.0 ml / min;

[0065] Detection wavelength: 225 nm;

[0066] Injection volume: 5 μl;

[0067] Column temperature: 35 °C;

[0068] Diluent: methanol or acetonitrile;

[0069] The gradient elution program is as follows:

[0070]

[0071] In another exemplary embodiment, the method of the present application includes the following solution preparation:

[0072] The diluent / blank solvent is methanol or acetonitrile; when the test product is deoxycholic acid, the diluent is methanol; when the test product is deoxycholic acid intermediate, the diluent is acetonitrile;

[0073] Preparation of triphenylphosphine reference solution: dissolve the triphenylphosphine reference in the diluent to prepare a triphenylphosphine reference solution with a concentration of 3 μg / ml;

[0074] Preparation of the test sample solution: the test sample and triphenylphosphine are dissolved in the diluent, wherein the concentration of the test sample is 5 mg / ml, and the concentration of triphenylphosphine is 0.1 mg / ml; the test sample and triphenylphosphine are subjected to a derivatization reaction at 40°C, and then cooled to room temperature to obtain the test sample solution; wherein when the diluent is methanol, the time of the derivatization reaction is 2 hours; when the diluent is acetonitrile, the time of the derivatization reaction is 15 hours;

[0075] Preparation of the blank derivatization reagent: triphenylphosphine is dissolved in the diluent to prepare a solution with a concentration of 0.1 mg / ml, which is placed at 40°C for the same time as the derivatization reaction, and then cooled to room temperature to obtain the blank derivatization reagent.

[0076] In another exemplary embodiment, the method of the present application comprises sample detection and content calculation. Specifically, the blank derivatization reagent, the triphenylphosphine reference solution, and the test sample solution are injected into the liquid chromatograph, respectively, and the chromatogram is recorded; and the content of TBHP is calculated according to the following formula:

[0077]

[0078] Wherein:

[0079] A spl is the peak area of TPPO in the test sample solution (after blank correction);

[0080] A std is the peak area of TPPO in the TPPO reference solution;

[0081] P std is the purity of the TPPO reference solution, %;

[0082] M std is the sample weight of the TPPO reference solution, mg;

[0083] M spl is the sample weight, mg;

[0084] S std is the dilution multiple of the TPPO reference solution;

[0085] S spl is the dilution multiple of the sample;

[0086] M TBHP is the relative molecular mass of TBHP, about 90.12;

[0087] M TPPO is the relative molecular mass of TPPO, about 278.28.

[0088] The present application adopts pre-column derivatization high performance liquid chromatography to detect the content of the mutagenic impurity TBHP with potential genotoxicity, and the method has the advantages of simple operation, high accuracy, high sensitivity, good durability, etc. The method of the present application can be used to determine any sample that may contain TBHP, and is especially suitable for detecting drugs containing trace amounts of TBHP, and can be specifically used to determine deoxycholic acid or intermediates (such as intermediate 4) in the preparation process of deoxycholic acid. By detecting and controlling the amount of TBHP in the raw material drug deoxycholic acid or its intermediates (such as intermediate 4), the quality control of deoxycholic acid can be realized, and the safety of drug use can be ensured. BRIEF DESCRIPTION OF DRAWINGS

[0089] Figure 1 is a graph of the toxicity evaluation result report of TBHP impurities; wherein Figure 1 A shows that the Sarah model predicts the mutagenicity of TBHP to be 100% positive, Figure 1 B shows that the Sarah and Derek models predict the mutagenicity of TBHP to be positive, and the combined results of the bacterial mutagenesis (Ames) test determine that TBHP is mutagenic positive;

[0090] Figure 2 is an HPLC chromatogram of the blank solvent in Example 1;

[0091] Figure 3 is an HPLC chromatogram of the blank derivatization reagent in Example 1;

[0092] Figure 4 is an HPLC chromatogram of the TPPO reference solution in Example 1;

[0093] Figure 5 is an HPLC chromatogram of the deoxycholic acid test sample solution in Example 1;

[0094] Figure 6 is an HPLC chromatogram of the blank solvent in Example 2;

[0095] Figure 7 is an HPLC chromatogram of the blank derivatization reagent in Example 2;

[0096] Figure 8 is an HPLC chromatogram of the TPPO reference solution in Example 2;

[0097] Figure 9 is an HPLC chromatogram of the deoxycholic acid intermediate test sample solution in Example 2;

[0098] Figure 10 is an HPLC chromatogram of the blank sensitivity solution in Example 3;

[0099] Figure 11HPLC chromatogram of the sensitivity solution in Example 3;

[0100] Figure 12 HPLC chromatogram of the TBHP control solution in Example 3;

[0101] Figure 13 HPLC chromatogram of the LOQ solution in Example 4;

[0102] Figure 14 HPLC chromatogram of the LOD solution in Example 4;

[0103] Figure 15 Time course plot of derivatization reaction in Example 7 (in methanol);

[0104] Figure 16 Time course plot of derivatization reaction in Example 8 (in acetonitrile);

[0105] Figure 17 Gas chromatogram of the TBHP control solution in Comparative Example 2;

[0106] Figure 18 HPLC chromatogram of the TBHP control solution without derivatization in Comparative Example 3. DETAILED DESCRIPTION

[0107] The features and advantages of the present technical solution will be better illustrated by the following examples. It should be understood that the examples of the present application are only used for explanation and illustration, and are not used to limit the protection scope of the present application. Unless otherwise specified, the methods used in the examples are conventional methods, which can be carried out according to conventional conditions or according to the conditions recommended by the manufacturers; the materials, reagents, etc. used can be obtained from commercial channels. Unless otherwise defined, all professional and scientific terms used herein have the same meaning as that familiar to those skilled in the art, for example, the retention time is abbreviated as RT, and the peak area unit is μV*sec.

[0108] In order to better control the product quality of deoxycholic acid and its intermediates, the applicant commissioned Beijing Xinglingjian Pharmaceutical Science and Technology Development Co., Ltd. to analyze and evaluate 31 process impurities and degradation impurities including TBHP. Specifically, the toxicity of the impurities was evaluated by two complementary (Q)SAR prediction methods (one based on expert knowledge rules and the other based on statistics). The evaluation was carried out using the Derek Nexus & Sarah Nexus evaluation software of Lhasa limited company. The results showed that the Derek evaluation result was PLAUSIBLE, and the compound contained a warning structure of hydrogen peroxide; the Sarah evaluation result was POSITIVE (100%), and the Ames test was positive, i.e. the evaluation result showed that the mutagenicity of TBHP was positive, and this mutagenic impurity was a potential genotoxic impurity ( Figure 1

[0109] The deoxycholic acid or deoxycholic acid intermediate 4 (see Background Art) sample used in the examples was prepared according to the method of document CN106146593B. The total yield of deoxycholic acid synthesized from deoxycholic acid intermediate 4 in this method was about 63.1%. The applicant took the residual amount of TBHP in intermediate 4 as no removal and all transferred to the final product deoxycholic acid to calculate the TBHP content limit in intermediate 4, so as to strictly monitor the quality of intermediate 4. The TBHP impurity limit in deoxycholic acid is 1200 ppm; after conversion, the strict TBHP content limit in intermediate 4 is 758 ppm.

[0110] Experimental materials:

[0111] TBHP reference substance: purchased from Sigma-Aldrich, batch number: SHBP3312. It is a solution of TBHP in n-decane, and its COA shows that the concentration of TBHP is 5.4 mol / L, i.e. 486 mg / ml. In order to improve the stability of TBHP, n-heptane is used as the solvent, and the TBHP reference substance stock solution with a concentration of about 486 μg / ml is prepared and stored at 2-8℃.

[0112] TPPO reference substance: purchased from aladdin. Batch number: F2214480, purity: 98%.

[0113] Example 1. Detection of TBHP content in deoxycholic acid

[0114] 1. Instruments and analysis conditions

[0115] High performance liquid chromatograph: Waters Acquity Arc;

[0116] ​​Column: Agilent Eclipse Plus C18, 3.5 μm, 4.6 x 100 mm;

[0117] Detector: Ultraviolet detector;

[0118] Detection wavelength: 225 nm;

[0119] Mobile phase A: 0.05% phosphoric acid solution;

[0120] Mobile phase B: Acetonitrile;

[0121] Column temperature: 35 °C;

[0122] Flow rate: 1.0 ml / min;

[0123] Injection volume: 5 μl;

[0124] Elution procedure:

[0125]

[0126]

[0127] 2. Preparation of solutions

[0128] Mobile phase A (0.05% phosphoric acid solution): measure 0.5 ml of phosphoric acid and 1000 ml of water, mix well, and ultrasonicate to obtain the solution.

[0129] Diluent / blank solvent: methanol.

[0130] Derivatization reagent (1 mg / ml): weigh about 100 mg of triphenylphosphine, place it in a 100-ml brown volumetric flask, add methanol to dissolve and dilute to the mark, and shake well to obtain the solution.

[0131] Blank derivatization reagent (0.1 mg / ml): accurately measure 1 ml of the derivatization reagent, place it in a 10-ml brown volumetric flask, dilute to the mark with methanol, shake well, and place it in a 40 °C water bath for 2 hours, then take it out and cool to room temperature to obtain the solution.

[0132] TPPO control solution (3 μg / ml): accurately weigh about 30 mg of TPPO standard, place it in a 100-ml brown volumetric flask, add methanol to dissolve and dilute to the mark, shake well, accurately measure 1 ml of the solution, place it in a 100-ml brown volumetric flask, dilute to the mark with methanol, and shake well to obtain the solution.

[0133] Deoxycholic acid test solution (5 mg / ml): accurately weigh about 50 mg of deoxycholic acid test sample, place it in a 10-ml brown volumetric flask, add 1 ml of derivatization reagent, continue to add methanol to dissolve and dilute to the mark, shake well, place it in a 40 °C water bath for 2 hours, then take it out and cool to room temperature to obtain the solution.

[0134] 3. Experimental procedure and results

[0135] 1) Take 5 μL of blank solvent, inject into the HPLC, record the chromatogram, basically as shown in Figure 1. The blank solvent should have no interference at the TPPO peak, if so, the peak area is not more than 50% of the TPPO peak area in the blank derivatization reagent. Figure 2

[0136] 2) Take 5 μL of blank derivatization reagent, inject into the HPLC, record the chromatogram, basically as shown in Figure 2. Figure 3

[0137] 3) Take 5 μL of TPPO control solution, inject into the HPLC, record the chromatogram, basically as shown in Figure 3. Figure 4

[0138] 4) Take 5 μL of test sample solution, inject into the HPLC, record the chromatogram, basically as shown in Figure 4. Figure 5

[0139] The content of TBHP in deoxycholic acid is calculated according to the following formula:

[0140]

[0141] Wherein:

[0142] A spl is the TPPO peak area in the test sample solution (after blank correction) ;

[0143] A std is the TPPO peak area in the TPPO control solution;

[0144] P std is the purity of TPPO control solution, %;

[0145] M std is the sample weight of TPPO control solution, mg;

[0146] M spl is the sample weight, mg;

[0147] S std is the dilution multiple of TPPO control solution;

[0148] S spl is the dilution multiple of the sample;

[0149] M TBHP is the relative molecular mass of TBHP, about 90.12;

[0150] M TPPO is the relative molecular mass of TPPO, about 278.28. ​​​​

[0151] In this embodiment, the deoxycholic acid test sample has better solubility in methanol, so methanol is selected as the diluent / blank solvent.

[0152] The experimental results show that the blank solvent has no interference at the TPPO peak (RT about 3.6-3.7 min). According to the external standard method, the peak area of TPPO in the TPPO control solution (about 3 μg / ml) is 78678, and the peak area of TPPO in the deoxycholic acid test sample solution is 18020 (after blank correction), which is about 5 mg / ml. It is calculated that the content of TBHP in the deoxycholic acid sample is about 46 ppm, which is lower than the limit requirement, showing that the quality of this batch of product is controllable.

[0153] Example 2. Detection of the content of TBHP in deoxycholic acid intermediate 4

[0154] 1. Instruments and analysis conditions:

[0155] The same as example 1.

[0156] 2. Solution preparation

[0157] Mobile phase A, derivatization reagent: measure 0.5 ml of phosphoric acid and 1000 ml of water, mix well, and ultrasonic, to obtain.

[0158] Diluent / blank solvent: acetonitrile.

[0159] Derivatization reagent (1 mg / ml): weigh about 100 mg of triphenylphosphine, put it into a 100 ml brown volumetric flask, add acetonitrile to dissolve and dilute to the mark, shake well, and obtain.

[0160] Blank derivatization reagent (0.1 mg / ml): accurately measure 2 ml of derivatization reagent, put it into a 20 ml brown volumetric flask, dilute to the mark with acetonitrile, shake well, and after 15 hours of reaction in a 40°C water bath, take it out and cool to room temperature, to obtain.

[0161] TPPO control solution (3 μg / ml): take about 30 mg of TPPO control, accurately weigh, put it into a 100 ml brown volumetric flask, add acetonitrile to dissolve and dilute to the mark, shake well; accurately measure 1 ml of the above solution, put it into a 100 ml brown volumetric flask, dilute to the mark with acetonitrile, shake well, and obtain.

[0162] Deoxycholic acid intermediate 4 test sample solution (5 mg / ml): accurately weigh about 50 mg of deoxycholic acid intermediate 4, put it into a 10 ml brown volumetric flask, add 1 ml of derivatization reagent to it, continue to add acetonitrile to dissolve and dilute to the mark, shake well, and after derivatization reaction in a 40°C water bath for 15 hours, take it out and cool to room temperature, to obtain.

[0163] 3. Experimental process and results

[0164] 1) Take 5 μL of the blank solvent and inject into the HPLC. Record the chromatogram, which is substantially as shown in Figure 1. Figure 6 The blank solvent should have no interference at the TPPO peak. If there is interference, the peak area should be no more than 50% of the peak area of the TPPO peak in the blank derivatization reagent.

[0165] 2) Take 5 μL of the blank derivatization reagent and inject into the HPLC. Record the chromatogram, which is substantially as shown in Figure 2. Figure 7

[0166] 3) Take 5 μL of the TPPO control solution and inject into the HPLC. Record the chromatogram, which is substantially as shown in Figure 3. Figure 8

[0167] 4) Take 5 μL of the sample solution and inject into the HPLC. Record the chromatogram, which is substantially as shown in Figure 4. Figure 9

[0168] The content of TBHP in deoxycholic acid intermediate 4 is calculated according to the calculation formula shown in Example 1.

[0169] In this example, the solubility of the deoxycholic acid intermediate 4 sample in acetonitrile is better, so acetonitrile is selected as the diluent / blank solvent. The experimental results show that, according to the external standard method, the peak area of TPPO in the TPPO control solution (about 3 μg / ml) is 79767, and the peak area of TPPO in the deoxycholic acid intermediate 4 sample solution is 44039 (after blank correction), and the concentration is about 5 mg / ml. It is calculated that the content of TBHP in the deoxycholic acid intermediate 4 sample is about 110 ppm, which is lower than the limit requirement, indicating that the TBHP content of the intermediate 4 batch is controllable.

[0170] Example 3. System suitability and specificity of the analysis method of the present application

[0171] 1. Instruments and analysis conditions: same as in Example 1.

[0172] 2. Solution preparation

[0173] The preparation of the diluent / blank solvent, derivatization reagent, blank derivatization reagent, and TPPO control solution is the same as in Example 2.

[0174] TBHP control stock solution (486 μg / ml): 200 μl of TBHP control was taken using a pipette and placed in a 20 ml brown volumetric flask containing an appropriate amount of n-heptane (about 10 ml). Dilute to the mark with n-heptane and shake well. Take 1 ml of the above solution accurately and place it in a 10 ml brown volumetric flask. Dilute to the mark with n-heptane and shake well. Store at 2-8°C.

[0175] ​​​TBHP control solution (1 μg / ml): precisely pipette 2 ml of the derivatization reagent into a 20 ml brown volumetric flask, pipette 40 μl of the TBHP control stock solution into the same flask using a pipette gun, dilute to the mark with acetonitrile, shake well; after derivatization at 40°C water bath for 15 hours, take out and cool to room temperature, and it is ready.

[0176] Sensitivity solution: precisely pipette 2.0 ml of the TBHP control solution into a 10 ml brown volumetric flask, dilute to the mark with acetonitrile, shake well, and it is ready as the sensitivity solution.

[0177] Sensitivity blank solution: precisely pipette 2.0 ml of the blank derivatization reagent into a 10 ml brown volumetric flask, dilute to the mark with acetonitrile, shake well, and it is ready as the sensitivity blank solution.

[0178] 3. Experimental process and results

[0179] Inject at least 1 needle of the blank solvent, 1 needle of the blank derivatization reagent, 1 needle of the sensitivity blank solution, 1 needle of the sensitivity solution, 1 needle of the TBHP control solution and 1 needle of the TPPO control solution, respectively. Record the chromatogram and peak area data of each sample.

[0180] 1) Take 5 μL of the blank solvent, inject into the high performance liquid chromatograph, and record the chromatogram (not shown, basically consistent with Figure 6 ).

[0181] 2) Take 5 μL of the blank derivatization reagent, inject into the high performance liquid chromatograph, and record the chromatogram (not shown, basically consistent with Figure 7 ).

[0182] 3) Take 5 μL of the sensitivity blank solution, inject into the high performance liquid chromatograph, and record the chromatogram, basically as Figure 10 shown.

[0183] 4) Take 5 μL of the sensitivity solution, inject into the high performance liquid chromatograph, and record the chromatogram, basically as Figure 11 shown.

[0184] 5) Take 5 μL of the TBHP control solution, inject into the high performance liquid chromatograph, and record the chromatogram, basically as Figure 12 shown.

[0185] 6) Take 5 μL of the TPPO control solution, inject into the high performance liquid chromatograph, and record the chromatogram (not shown, basically consistent with Figure 8 ).

[0186] Wherein, TPPO is the product of the reaction of TBHP with excess TPP, the amount of TPPO generated by the derivatization reaction is calculated by the external standard method, and compared with the theoretical value to determine the derivatization efficiency of TBHP in the TBHP control solution. ). The following equation was used for the calculation:

[0187]

[0188]

[0189] In the above equation:

[0190] C' TPPO : Concentration of TPPO (derivatized) in the TBHP standard solution, μg / ml;

[0191] A' TPPO : Peak area of TPPO (derivatized) in the chromatogram of the TBHP standard solution;

[0192] A TPPO : Peak area of TPPO in the chromatogram of the TPPO standard solution;

[0193] C TPPO : Concentration of TPPO in the TPPO standard solution, μg / ml;

[0194] C TBHP : Concentration of TBHP in the TBHP standard solution, μg / ml;

[0195] M TBHP : Relative molecular mass of TBHP, about 90.12;

[0196] M TPPO : Relative molecular mass of TPPO, about 278.28;

[0197] Derivatization efficiency, %.

[0198] The system suitability acceptance criteria were as follows: 1) the blank solvent had no interference at the TPPO peak, and if there was interference, the peak area was not more than 50% of the peak area of TPPO in the blank derivatization reagent; 2) the peak area of TPPO in the blank sensitivity solution was not more than 50% of the peak area of TPPO in the sensitivity solution; there were no other interfering peaks in the blank sensitivity solution, and if there were, the separation degree from TPPO was not less than 1.5; 3) the signal-to-noise ratio of the TPPO peak in the sensitivity solution was ≥10; 4) the tailing factor of the TPPO peak in the TBHP standard solution was ≤1.5; and 5) the derivatization efficiency of TBHP was between 80% and 120%.

[0199] The experimental results show that: the blank solvent TPPO has no interference at the peak position; the interference of the blank sensitivity solution: the peak area of TPPO in the blank sensitivity solution is 3380, which is less than 50% of the peak area of TPPO in the sensitivity solution (20525); the signal-to-noise ratio of the TPPO peak in the sensitivity solution is 39, which is greater than 10; the tailing factor of the TPPO peak in the TBHP control solution is 1.2, which is less than 1.5; the derivatization efficiency of TBHP is 105.0%, which is between 80% and 120%. In summary, the system applicability and specificity of the method of the application meet the requirements.

[0200] Example 4. Quantification limit and detection limit of the analysis method of the application

[0201] 1. Instrument and analysis conditions: same as in Example 1.

[0202] 2. Solution preparation

[0203] The TBHP control solution is the same as in Example 3.

[0204] Quantification limit blank solution: same as the sensitivity blank solution in Example 3.

[0205] Detection limit blank solution: accurately measure 3.0 ml of the quantification limit blank solution, and dilute to the mark in a 10-ml volumetric flask with acetonitrile, shake well, and use as the detection limit blank solution.

[0206] Quantification limit solution (LOQ): accurately measure 2.0 ml of the TBHP control solution, and dilute to the mark in a 10-ml volumetric flask with acetonitrile, shake well. Prepare 6 replicates.

[0207] Detection limit solution (LOD): accurately measure 3.0 ml of the quantification limit solution, and dilute to the mark in a 10-ml volumetric flask with acetonitrile, shake well. Prepare 3 replicates.

[0208] 3. Experimental process and results

[0209] After the system applicability is verified to be qualified according to the method of this Example 3, 1 needle of the quantification limit blank solution and 1 needle of the detection limit blank solution are injected, respectively, and the chromatogram is recorded to investigate the interference of the TPPO background on the quantification limit and the detection limit. 6 needles of the quantification limit solution are injected, respectively, and the chromatogram, retention time, peak area, and signal-to-noise ratio are recorded; 3 needles of the detection limit solution are injected, and the chromatogram and signal-to-noise ratio are recorded. The detection limit concentration, quantification limit concentration, and level are calculated.

[0210] The concentration level calculation formula is as follows:

[0211]

[0212] Acceptance criteria: 1) The average signal-to-noise ratio of the TPPO peak in 3 replicate limit of detection solutions should not be less than 3; 2) The average signal-to-noise ratio of the TPPO peak in 6 replicate limit of quantitation solutions should not be less than 10; 3) The RSD of the retention time of the TPPO peak in 6 replicate limit of quantitation solutions should not be greater than 2.0%; 4) The RSD of the peak area of the TPPO peak in 6 replicate limit of quantitation solutions should not be greater than 5.0%.

[0213] The chromatograms of the limit of detection and the limit of quantitation are shown in Figure 13 , Figure 14 The results are shown in Table 1 and Table 2.

[0214] Table 1. Results of limit of detection (LOD)

[0215]

[0216] Table 2. Results of limit of quantitation (LOQ)

[0217]

[0218]

[0219] The experimental results show that the average signal-to-noise ratio of the TPPO peak in the limit of detection solution is greater than 3; the average signal-to-noise ratio of the TPPO peak in the limit of quantitation solution is greater than 10; the RSD of the retention time of the TPPO peak in 6 replicate limit of quantitation solutions is 0.2%, which is less than 2.0%; the RSD of the peak area of the TPPO peak in 6 replicate limit of quantitation solutions is 1.9%, which is less than 5.0%. The limit of detection and the limit of quantitation of TBHP are 0.05832 μg / ml and 0.1944 μg / ml, respectively, which are equivalent to 11 ppm and 38 ppm of the concentration of the test solution (5 mg / ml), respectively. It is shown that the limit of detection and the limit of quantitation of the analysis method meet the requirements for the control limit of genotoxic impurities in deoxycholic acid or intermediates thereof, and the detection method has high sensitivity.

[0220] Example 5. Robustness of the analysis method of the present application

[0221] In this example, the robustness of the analysis method of the present application is studied by investigating the influence of changes in the chromatographic conditions on the detection results of TBHP.

[0222] 1. Instruments and analysis method

[0223] Under other chromatographic conditions as shown in Example 2, the chromatographic conditions are adjusted and investigated singly. The specific investigation conditions (robustness conditions) are shown as follows:

[0224] 1) The concentration of phosphoric acid in mobile phase A (the concentration of phosphoric acid is 0.04%, 0.06%);

[0225] 2) The initial proportion of mobile phase B (the volume proportion of mobile phase B is 35%, 45%);

[0226] 3) flow rate (0.9 ml / min, 1.1 ml / min);

[0227] 4) detection wavelength (223 nm, 227 nm);

[0228] 5) column temperature (30℃, 40℃);

[0229] 6) column (Waters Symmetry C18, 4.6*100mm, 3.5μm was used for column 2)

[0230] 2. Solution preparation was the same as Example 3.

[0231] 3. Experimental procedure and results

[0232] The system suitability of each durability condition was investigated.

[0233] After adjusting the chromatographic conditions, at least one injection of blank solvent, one injection of blank derivatization reagent, one injection of sensitivity blank solution, one injection of sensitivity solution, one injection of TBHP control solution, one injection of TPPO control solution and one injection of sample solution were injected, respectively. The chromatogram and chromatographic data were recorded. The detection results under each durability condition were shown in Table 3 and Table 4.

[0234] Acceptable standard: under each durability condition, the system suitability requirements described in Example 3 were met, and the detection results of TBHP in the sample under different conditions should be consistent.

[0235] Table 3 Durability condition results-1

[0236]

[0237] Table 4 Durability condition results-2

[0238]

[0239]

[0240] The experimental results showed that under different conditions, there was no interference at the peak of blank solvent TPPO; the interference of blank sensitivity solution: the peak area of TPPO in the blank sensitivity solution was less than 50% of the peak area of TPPO in the sensitivity solution; the signal-to-noise ratio of TPPO peak in the sensitivity solution was greater than 10; the tailing factor of TPPO peak in the TBHP control solution was less than 1.5; the derivatization efficiency of TBHP was between 80%-120%. In summary, the durability conditions of the method of the present application met the system suitability requirements, and the detection results of TBHP in the sample under different conditions were basically consistent.

[0241] Example 6. Derivation temperature screening of the analysis method of the present application

[0242] In the present application, TBHP in the sample to be tested is allowed to react with TPP by derivatization to form TPPO, and the content of TBHP is indirectly determined by detecting the content of TPPO. In order to determine the interference of the background of TPPO in the derivatization process and thus determine the appropriate derivatization reaction temperature, the temperature of the derivatization reaction is investigated in this example.

[0243] 1. Instrument and analysis conditions are the same as in Example 1.

[0244] 2. Preparation of solutions

[0245] Blank solvent / diluent: acetonitrile.

[0246] Derivatization reagent (1 mg / ml): same as in Example 2.

[0247] Blank derivatization reagent (0.1 mg / ml): take an appropriate amount of derivatization reagent, dissolve and dilute with acetonitrile to prepare a 0.1 mg / ml triphenylphosphine acetonitrile solution, and divide it into three parts; after 5 hours in a 25°C, 40°C and 50°C water bath respectively, take an appropriate amount and investigate the effect of temperature.

[0248] 3. Experimental process and results

[0249] Take the diluent, the blank derivatization reagent at 0h and the blank derivatization reagent after derivatization at different temperatures for 5h, respectively, and record the chromatogram and peak area. The results are shown in Table 5.

[0250] Table 5 Results of different derivatization reaction temperatures

[0251]

[0252] The above experimental results show that when the derivatization reaction temperature is 25°C and 40°C, the peak area of TPPO in the blank derivatization reagent after derivatization for 5h is comparable to that of the blank derivatization reagent at 0h (chromatogram not shown, consistent with Figure 7 the results of Example 2), indicating that the 0.1 mg / ml blank derivatization reagent remains stable at this derivatization temperature; but when the derivatization temperature is 50°C, the peak area of TPPO in the 0.1 mg / ml blank derivatization reagent is significantly larger, being 126.0% of the peak area at 0h, indicating that when the derivatization temperature is too high, the blank derivatization reagent will degrade, thereby affecting the accuracy and sensitivity of the method.

[0253] Example 7. Derivation time selection of the analysis method of the present application (methanol as diluent)

[0254] In this example, the derivatization reaction time is investigated when the diluent is methanol.

[0255] 1. Instruments and analytical conditions: same as in Example 1.

[0256] 2. Preparation of solutions

[0257] Blank solvent / diluent: methanol.

[0258] Derivatization reagent: same as in Example 1.

[0259] TBHP control stock solution: same as in Example 3.

[0260] Blank derivatization reagent (0.1 mg / ml): take an appropriate amount of derivatization reagent, dissolve and dilute with methanol to prepare a 0.1 mg / ml solution of triphenylphosphine in methanol, place in a 40°C water bath, and at different time intervals, take an appropriate amount to investigate the effect of time.

[0261] TBHP control solution (1 μg / ml): accurately take 2 ml of derivatization reagent into a 20 ml brown volumetric flask, use a pipette to take 40 μl of TBHP control stock solution into the same volumetric flask, dilute to the mark with methanol, shake well, place in a 40°C water bath, and at different time intervals, take an appropriate amount to investigate the effect of time.

[0262] 3. Experimental procedure and results

[0263] Take the diluent, blank derivatization reagent at different time points, and TBHP control solution into the sample, respectively, and record the chromatogram. The change of derivatization reaction with time is shown in Figure 15 , wherein the concentration of TBHP is calculated according to the peak area of TPPO by external standard method, and then calculated by the conversion relationship of the reaction.

[0264] The experimental results show that when the temperature is 40°C and the diluent is methanol, the derivatization reaction of TBHP and TPP is rapid and can be basically completed in 1.5 h. It can be seen from Figure 15 that the peak area of TPPO in the TBHP control solution increases with the increase of derivatization time, and after 2 hours, the peak area reaches a platform, and there is no obvious change in the peak area of TPPO when the derivatization time is increased to 2.5 hours. In order to ensure complete derivatization, when methanol is used as the solvent, the derivatization time is preferably 1.5-2.5 hours, and more preferably 2 hours.

[0265] Example 8. Selection of derivatization time of the analysis method of the application (acetonitrile as diluent)

[0266] In this example, the derivatization reaction time when the diluent is acetonitrile is investigated.

[0267] 1. Instruments and analytical conditions: same as in Example 1.

[0268] 2. Preparation of solutions

[0269] Blank solvent / diluent: acetonitrile.

[0270] Derivatization reagent: same as Example 2.

[0271] TBHP control stock solution: same as Example 3.

[0272] Blank derivatization reagent (0.1 mg / ml): take an appropriate amount of derivatization reagent, dissolve and dilute with acetonitrile to prepare a 0.1 mg / ml triphenylphosphine acetonitrile solution, place in a 40°C water bath, and take samples at different time points for detection to investigate the effect of time.

[0273] TBHP control solution (1 μg / ml): accurately take 2 ml of derivatization reagent into a 20 ml brown volumetric flask, use a pipette to transfer 40 μl of TBHP control stock solution into the same volumetric flask, dilute to the mark with acetonitrile, shake well, place in a 40°C water bath, and take samples at different time points for detection to investigate the effect of time.

[0274] 3. Experimental process and results

[0275] Take the diluent, blank derivatization reagent at different time points, and TBHP control solution into the sample, respectively, and record the chromatogram. The change of derivatization reaction with time is shown in Figure 16 , wherein the concentration of TBHP is calculated according to the peak area of TPPO by external standard method, and then calculated by the conversion relationship of the reaction.

[0276] The experimental results show that the concentration of TBHP (calculated value) in the control solution gradually increases to about 1 μg / ml (after blank correction) with the extension of derivatization time, which is consistent with the theoretical value. As can be seen from the figure, after 13 hours of derivatization reaction, TBHP is basically completely derivatized, and the concentration does not change significantly with the extension of derivatization reaction time. Therefore, too short derivatization reaction time will lead to incomplete derivatization of TBHP, and too long derivatization reaction time will lead to a decrease in analysis efficiency. When measuring the TBHP content of intermediate 4, acetonitrile is used as the blank solvent, in order to ensure that TBHP is completely derivatized at 40°C, the reaction time is preferably 13-17 hours, and more preferably 15 hours.

[0277] Comparative Example 1.

[0278] This comparative example adjusts the concentration of blank derivatization reagent to study the effect of different concentrations of blank derivatization reagent on the sensitivity of the detection method.

[0279] 1. Instruments and analysis conditions: same as Example 1.

[0280] 2. Preparation of solutions

[0281] Blank solvent / diluent, derivatization reagent, TBHP control solution were the same as Example 2.

[0282] Blank derivatization reagent: an appropriate amount of derivatization reagent was measured, dissolved and diluted in acetonitrile to prepare a 0.1 mg / ml and a 0.3 mg / ml triphenylphosphine acetonitrile solution, respectively. After being derivatized in a 40°C water bath for 15 hours, the solution was taken out and cooled to room temperature.

[0283] TBHP control solution (1 μg / ml): 2 ml of derivatization reagent was accurately measured into a 20 ml brown volumetric flask, and 40 μl of TBHP control stock solution (the same as in Example 3) was pipetted into the same volumetric flask using a pipette gun, diluted to the mark with acetonitrile, and shaken well. After being derivatized in a 40°C water bath for 15 hours, the solution was taken out and cooled to room temperature.

[0284] 3. Experimental process and results

[0285] The diluent, blank derivatization reagent and TBHP control solution were injected separately, and the chromatogram and chromatographic data were recorded. The chromatographic data are shown in Table 6.

[0286] Table 6

[0287]

[0288] Note: The TBHP control solution was derived from 0.1 mg / ml TPP, and the background was not deducted.

[0289] The experimental results show that when the concentration of derivatization reagent is 0.3 mg / ml, the TPPO peak in the background is too high, accounting for about 46% of the area of the TPPO peak generated by the derivatization of the TBHP control solution. Generally, the smaller the background interference, the higher the accuracy of the measurement. Although increasing the concentration of derivatization reagent can improve the derivatization efficiency, excessive background interference will significantly reduce the sensitivity of the analysis. Therefore, the application preferably uses a 0.1 mg / ml triphenylphosphine solution as the derivatization reagent, which can significantly improve the accuracy and sensitivity of the analysis while ensuring the derivatization efficiency.

[0290] Comparative Example 2.

[0291] In this comparative example, the content of TBHP was directly detected by gas chromatography.

[0292] 1. Instruments and analysis conditions

[0293] Gas chromatograph: Agilent 7820A;

[0294] Chromatographic column: Agilent DB-624, 30 m x 0.32 mm x 1.8 μm;

[0295] Split ratio: 20:1;

[0296] Inlet pressure: 5.2213 psi;

[0297] Inlet temperature: 250 °C;

[0298] FID detector, 250 °C;

[0299] Carrier gas: nitrogen;

[0300] Makeup gas flow rate: 30 ml / min;

[0301] Air flow rate: 400 ml / min;

[0302] Hydrogen flow rate: 40 ml / min;

[0303] Control mode: constant pressure mode;

[0304] Temperature program: initial temperature of the column was 40 °C, hold for 1 min, ramp to 220 °C at 15 °C / min, hold for 5 min;

[0305] Injection mode: direct injection;

[0306] Injection volume: 1 μl;

[0307] Run time: 18 min

[0308] 2. Preparation of solutions

[0309] Blank solvent / diluent: acetonitrile.

[0310] TBHP reference solution (20 mg / ml): take an appropriate amount of TBHP reference, dissolve and dilute with acetonitrile to make a solution containing 20 mg per 1 ml.

[0311] 3. Experimental procedure and results

[0312] Take the diluent and reference solution respectively, record the chromatogram, which is substantially as shown in Figure 17 wherein t-butanol (t-BuOH) is at about 5.3 min, TBHP is at about 8.6 min, and n-decane (solvent contained in the TBHP reference) is at about 12.3 min.

[0313] The experimental results show that when TBHP is directly determined by gas chromatography under non-derivatization conditions, TBHP will be degraded or partially degraded to form t-butanol at the inlet of the instrument due to the high inlet temperature of 250 °C; when TBHP is at a lower concentration level, the effect of degradation on the determination of content will be more significant. Therefore, the determination of TBHP content by gas chromatography cannot be accurately quantified, and is even less suitable for the determination of ppm-level TBHP content.

[0314] Comparative Example 3.

[0315] This comparative example uses HPLC to determine the content of TBHP without derivatization.

[0316] 1. Instruments and analytical conditions

[0317] Liquid chromatograph: Waters Arc.

[0318] Chromatographic column: Agilent Eclipse Plus C18, 4.6 x 100 mm, 3.5 μm;

[0319] Mobile phase A: 0.1% phosphoric acid solution;

[0320] Mobile phase B: acetonitrile;

[0321] Isocratic elution: mobile phase A: mobile phase B = 40:60;

[0322] Detection wavelength: 215 nm;

[0323] Column temperature: 35°C;

[0324] Injection volume: 5 μl;

[0325] Diluent: acetonitrile;

[0326] Run time: 5 min.

[0327] 2. Preparation of solutions

[0328] Blank solvent / diluent: acetonitrile.

[0329] TBHP reference solution (25 μg / ml): take TBHP reference stock solution (as in Example 3) and dilute it with acetonitrile to obtain a solution containing about 25 μg per 1 ml.

[0330] 3. Experimental procedure and results

[0331] Inject the diluent and the reference solution separately and record the chromatograms, which are substantially as shown in Figure 18 where the TBHP peak is at about 1.6 min RT.

[0332] The experimental results show that, when TBHP is determined directly using liquid chromatography without derivatization, the limit of quantification is about 25 μg / ml (S / N = 22) even at 215 nm, and the sensitivity is significantly low, which is not suitable for the determination of ppm-level TBHP content.

[0333] It can be seen from the above examples that the method has the advantages of good resolution, high accuracy, low detection limit and quantitative limit, and high sensitivity, and can be used for determining the content of the mutagenic impurity TBHP in deoxycholic acid or deoxycholic acid intermediate, and is very suitable for quality research of deoxycholic acid bulk drug or deoxycholic acid intermediate. However, the present application is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for determining the content of tert-butyl hydrogen peroxide in deoxycholic acid or a deoxycholic acid intermediate, characterized in that, This method employs pre-column derivatization high-performance liquid chromatography (HPLC) for detection. Pre-column derivatization involves derivatizing the sample with triphenylphosphine. The HPLC detection conditions are as follows: Column: Packed with octadecylsilane-bonded silica gel; Mobile phase A: 0.01%~0.10% phosphoric acid solution; Mobile phase B: Acetonitrile; Flow rate: 0.9~1.1 ml / min; Detection wavelength: 210~250nm; Injection volume: 5~10 μl; Column temperature: 30~40℃; Diluent: Methanol or acetonitrile; The gradient elution procedure is as follows: The temperature for the derivatization process is 25~40℃; When the sample is deoxycholic acid, the diluent is methanol, and the derivatization treatment includes reacting the sample with triphenylphosphine in methanol for 1.5 to 2.5 hours. Alternatively, when the sample is a deoxycholic acid intermediate, the diluent is acetonitrile, and the derivatization process includes derivatizing the sample with triphenylphosphine in acetonitrile for 13 to 17 hours. The deoxycholic acid or deoxycholic acid intermediate sample was prepared according to the method in document CN106146593B. The structural formula of the deoxycholic acid intermediate is as follows: .

2. The method of claim 1, wherein the chromatographic column is an Agilent Eclipse Plus C18 column or a Waters Symmetry C18 column; with dimensions of 4.6 × 100 mm and 3.5 µm.

3. The method according to claim 1, wherein the mobile phase A is a 0.04~0.06% phosphoric acid solution; the detection wavelength is 223~227nm; and the column temperature is 35℃.

4. The method of claim 1, wherein the gradient elution procedure is as follows: 。 5. The method according to any one of claims 1 to 4, wherein the method comprises: Preparation of triphenylphosphine oxide reference solution: Dissolve triphenylphosphine oxide in diluent to obtain a reference solution with a triphenylphosphine oxide concentration of 1~5 μg / ml; Preparation of the test solution: Dissolve the test sample and triphenylphosphine in a diluent to obtain a solution with a test sample concentration of 5 mg / ml and a triphenylphosphine concentration of 0.1 mg / ml; the solution undergoes a derivatization reaction at 25~40℃ to obtain the test solution. Preparation of blank derivatization reagent: Dissolve triphenylphosphine in diluent to obtain a 0.1 mg / ml triphenylphosphine solution, and then place it at 25~40℃ for the same time as the derivatization reaction to obtain a blank derivatization reagent.

6. The method of claim 1, wherein when the sample is deoxycholic acid, the diluent is methanol and the derivatization reaction time is 2 hours; when the sample is a deoxycholic acid intermediate, the diluent is acetonitrile and the derivatization reaction time is 15 hours.

7. A method for determining the content of tert-butyl hydrogen peroxide in deoxycholic acid or a deoxycholic acid intermediate, characterized in that, 1) This method uses pre-column derivatization high-performance liquid chromatography for detection, and the detection conditions are as follows: Chromatographic column: Agilent Eclipse Plus C18 column or Waters Symmetry C18 column, 4.6×100mm, 3.5µm; Mobile phase A: 0.05% aqueous phosphoric acid solution; Mobile phase B: Acetonitrile; Flow rate: 1.0 ml / min; Detection wavelength: 225nm; Injection volume: 5 μl; Column temperature: 35℃; Diluent: Methanol or acetonitrile; Gradient elution procedure: ； 2) Solution preparation Preparation of triphenylphosphine oxide reference solution: Dissolve triphenylphosphine oxide reference standard in diluent to prepare a triphenylphosphine oxide reference solution with a concentration of 3 μg / ml; Preparation of the test solution: Dissolve the test sample and triphenylphosphine in a diluent, wherein the concentration of the test sample is 5 mg / ml and the concentration of triphenylphosphine is 0.1 mg / ml; the test sample and triphenylphosphine undergo a derivatization reaction at 40℃, and are cooled to room temperature to obtain the test solution; wherein when the diluent is methanol, the derivatization reaction time is 2 hours; when the diluent is acetonitrile, the derivatization reaction time is 15 hours; Preparation of blank derivatization reagent: Dissolve triphenylphosphine in diluent to prepare a solution with a concentration of 0.1 mg / ml. Place the solution at 40°C for the same time as the derivatization reaction, and then cool to room temperature to obtain the blank derivatization reagent. 3) Detection and calculation Inject blank derivatization reagent, triphenylphosphine oxide reference solution, and test solution into the liquid chromatograph and record the chromatograms; The TBHP content is calculated using the following formula: ; in: A spl The peak area of ​​triphenylphosphine oxide in the test solution after deducting blank correction; A std The peak area of ​​triphenylphosphine oxide in the triphenylphosphine oxide reference solution; P std The purity of triphenylphosphine oxide reference standard is % . M std Weigh the sample amount of triphenylphosphine oxide reference standard, in mg; M spl The sample weight is in mg. S std This refers to the dilution factor of triphenylphosphine oxide reference standard; S spl This refers to the sample dilution factor. M TBHP is the relative molecular mass of tert-butyl hydroperoxide; M TPPO is the relative molecular mass of triphenylphosphine oxide; The deoxycholic acid or deoxycholic acid intermediate sample was prepared according to the method in document CN106146593B. The structural formula of the deoxycholic acid intermediate is as follows: .

Images