Detection method of deoxycholic acid intermediate

Abstract

The invention provides a deoxycholic acid intermediate detection method, which comprises: dissolving a deoxycholic acid intermediate in a diluent to obtain a sample solution, and detecting the sample solution through an HPLC method. The detection method provided by the invention can effectively separate deoxycholic acid intermediates and impurities so as to effectively control the quality of deoxycholic acid products, and the method completely meets acceptance standards in the aspects of specificity, linearity, range, accuracy, precision, detection limit, quantitation limit, durability, solution stability, system precision, system applicability and the like and has good performance.

Description

Technical Field

[0001] The present invention belongs to the technical field of chemical analysis, and particularly relates to a detection method for deoxycholic acid intermediates. Background Art

[0002] Deoxycholic acid is a bile acid lacking a hydroxyl group at C-7. It is a free bile acid derived from cholic acid by losing one oxygen atom. In bile, it mainly exists in the form of conjugation with taurine and glycine. Deoxycholic acid is the world's first local lipolytic drug, which has strong surface activity, can destroy and dissolve cell membranes, and reduce the dissolution of local subcutaneous fat in a small area. In clinical trials, deoxycholic acid can effectively eliminate submental fat and improve the overall appearance.

[0003] At present, the preparation methods of deoxycholic acid face problems such as poor stability of intermediates, difficulty in product purification, low yield of products or intermediates during the synthesis process, and unstable yield. Therefore, how to reduce impurities in the reaction process, obtain more stable intermediates, and deoxycholic acid that is easier to purify has become an urgent problem to be solved.

[0004] The inventors of the present invention have developed a preparation method of deoxycholic acid. The purification methods of the intermediates and products prepared by this preparation method are simple, and stable intermediates and high product yields can be obtained. At the same time, the prepared deoxycholic acid can meet the quality requirements of bulk drugs. On this basis, the inventors further studied the detection method of the intermediate. Summary of the Invention

[0005] In order to overcome the deficiencies of the prior art, the present invention provides a detection method for deoxycholic acid intermediates. The related substances of deoxycholic acid intermediates are detected by HPLC method, and method validation is carried out. It has been proved by experiments that the separation degree between deoxycholic acid intermediates and adjacent impurities in the detection method of the present invention is high, the specificity is strong, and the separation degree and system precision are good, thereby effectively controlling the quality of deoxycholic acid products.

[0006] In the first aspect of the present invention, a detection method for deoxycholic acid intermediates is provided. The detection method includes the following steps: dissolving the deoxycholic acid intermediate in a diluent to obtain a sample solution, and detecting the sample solution by HPLC method;

[0007] The deoxycholic acid intermediate is selected from the compounds shown by the following formula:

[0008]

[0009] wherein, P is a hydroxyl protecting group, preferably any one of acetyl, C1-C3 alkyl or benzoyl;

[0010] R is a C1-C6 alkyl group, preferably a C1-C3 alkyl group, more preferably methyl or ethyl.

[0011] Preferably, the deoxycholic acid intermediate is selected from the compounds represented by the following formula:

[0012]

[0013] Furthermore, the diluent is selected from one or more of acetonitrile (ACN), methanol (MeOH), ethanol (EtOH), tetrahydrofuran (THF), triphenylphosphine (PPh 3 ), formic acid (FA), and water.

[0014] Preferably, the diluent is a triphenylphosphine acetonitrile solution at 0.01 - 2 g / L, specifically such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.22, 0.24, 0.26, 0.28, 0.3, 0.32, 0.34, 0.36, 0.38, 0.4, 0.42, 0.44, 0.46, 0.48, 0.5, 0.55, 1, 1.5, 2 g / L, preferably 0.05 - 0.5 g / L.

[0015] Preferably, the diluent is acetonitrile.

[0016] Preferably, the diluent is a mixed solution of methanol and tetrahydrofuran with a volume ratio of 0.1 - 10:1, specifically such as 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.2:1, 2.4:1, 2.6:1, 2.8:1, 3:1, 3.2:1, 3.4:1, 3.6:1, 3.8:1, 4:1, 4.2:1, 4.4:1, 4.6:1, 4.8:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, 8.5:1, 9:1, 9.5:1, 10:1, preferably 0.5 - 5:1.

[0017] In some embodiments of the present invention, the deoxycholic acid intermediate is The diluent is a 0.1 g / L triphenylphosphine acetonitrile solution.

[0018] In some embodiments of the present invention, the deoxycholic acid intermediate is The diluent described above is acetonitrile.

[0019] In some embodiments of the present invention, the deoxycholic acid intermediate is The diluent described above is a mixed solution of methanol and tetrahydrofuran with a volume ratio of 1:1.

[0020] In some embodiments of the present invention, the deoxycholic acid intermediate is The diluent described above is a mixed solution of methanol and tetrahydrofuran with a volume ratio of 1:1.

[0021] Furthermore, in the HPLC method described above, the chromatographic conditions are as follows:

[0022] The stationary phase of the chromatographic column is octadecylsilyl-bonded silica gel;

[0023] The mobile phase includes mobile phase A and mobile phase B;

[0024] The mobile phase A is an aqueous formic acid solution with a volume percentage concentration of 0.01 - 5%, specifically such as 0.01, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5%, preferably 0.05 - 2%;

[0025] The mobile phase B is an acetonitrile solution of formic acid with a volume percentage concentration of 0.01 - 5%, specifically such as 0.01, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5%, preferably 0.05 - 2%;

[0026] A gradient elution program is adopted, and the gradient elution program is as follows:

[0027] At t = 0 min, the volume percentage of mobile phase B is 20 - 30%;

[0028] When 0 < t ≤ 2 min, the volume percentage of mobile phase B is 40 - 50%;

[0029] When 2 < t ≤ 14 min, the volume percentage of mobile phase B is 53 - 63%;

[0030] When 14 < t ≤ 24 min, the volume percentage of mobile phase B is 90 - 99%;

[0031] When 24 < t ≤ 35 min, the volume percentage of mobile phase B is 90 - 99%.

[0032] The described gradient elution program may further include the following steps:

[0033] 35 < t ≤ 35.1 min, the volume percentage of mobile phase B is 20 - 30%;

[0034] 35.1 < t ≤ 45 min, the volume percentage of mobile phase B is 20 - 30%.

[0035] Preferably, the gradient elution program is as follows:

[0036] t = 0 min, the volume percentage of mobile phase B is 25%;

[0037] 0 < t ≤ 2 min, the volume percentage of mobile phase B is 45%;

[0038] 2 < t ≤ 14 min, the volume percentage of mobile phase B is 58%;

[0039] 14 < t ≤ 24 min, the volume percentage of mobile phase B is 95%;

[0040] 24 < t ≤ 35 min, the volume percentage of mobile phase B is 95%.

[0041] The described gradient elution program may further include the following steps:

[0042] 35 < t ≤ 35.1 min, the volume percentage of mobile phase B is 25%;

[0043] 35.1 < t ≤ 45 min, the volume percentage of mobile phase B is 25%.

[0044] In some embodiments of the present invention, the specifications of the chromatographic column are 150 mm * 4.6 mm, 3 μm.

[0045] In some embodiments of the present invention, the mobile phase A is an aqueous solution of 0.1% formic acid, and the mobile phase B is an acetonitrile solution of 0.1% formic acid.

[0046] Furthermore, the flow rate of the mobile phase is 0.5 - 2 mL / min, specifically 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0 mL / min.

[0047] Furthermore, the column temperature of the chromatographic column is 20 - 50 °C, specifically 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 °C.

[0048] Further, the injection volume of the sample solution is 1 - 200 μL, specifically 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 μL.

[0049] Further, the temperature of the injection disk is 1 - 30 °C, specifically 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 °C.

[0050] In some embodiments of the present invention, the flow rate is 1.0 mL / min.

[0051] In some embodiments of the present invention, the column temperature is 40 °C.

[0052] In some embodiments of the present invention, the deoxycholic acid intermediate is The injection volume is 5 μL.

[0053] In some embodiments of the present invention, the deoxycholic acid intermediate is The injection volume is 3 μL.

[0054] In some embodiments of the present invention, the deoxycholic acid intermediate is The temperature of the injection disk is 25 °C.

[0055] In some embodiments of the present invention, the deoxycholic acid intermediate is The temperature of the injection disk is 5 °C.

[0056] Further, in the HPLC method, the detector is a CAD detector.

[0057] In some embodiments of the present invention, the parameters of the CAD detector: Power Function: 1.00; Data Collection rate: 10 Hz; Filter: 1.0; Control Evaporator temperature: 50 °C.

[0058] Further, the deoxycholic acid intermediate is In the HPLC method described above, secondary separation and detection are also included, which can effectively separate the key substances C-12, C-13, and C-14 during the reaction process.

[0059] Preferably, in the secondary separation and detection, the deoxycholic acid intermediate is

[0060] Furthermore, in the secondary separation and detection, the chromatographic conditions are as follows:

[0061] The stationary phase of the chromatographic column is octadecylsilyl-bonded silica gel;

[0062] The mobile phase includes mobile phase A and mobile phase B;

[0063] The mobile phase A is an aqueous formic acid solution with a volume percentage concentration of 0.01-5%, specifically such as 0.01, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5%, preferably 0.05-2%;

[0064] The mobile phase B is a mixed solution of methanol and tetrahydrofuran with a volume ratio of 1-10:1, specifically such as 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, 8.5:1, 9:1, 9.5:1, 10:1, 10.5:1, 11:1, 11.5:1, 12:1, 12.5:1, 13:1, 13.5:1, 14:1, 14.5:1, 15:1, 15.5:1, 16:1, 16.5:1, 17:1, 17.5:1, 18:1, 18.5:1, 19:1, 19.5:1, 20:1, preferably 3-5:1;

[0065] A gradient elution program is adopted, and the gradient elution program is as follows:

[0066] At t = 0 min, the volume percentage of mobile phase B is 50-70%;

[0067] When 0 < t ≤ 2 min, the volume percentage of mobile phase B is 50-70%;

[0068] When 2 < t ≤ 25 min, the volume percentage of mobile phase B is 90-99%;

[0069] When 25 < t ≤ 35 min, the volume percentage of mobile phase B is 90-99%.

[0070] The described gradient elution procedure may further include the following steps:

[0071] 35 < t ≤ 35.1 min, the volume percentage of mobile phase B is 50 - 70%;

[0072] 35.1 < t ≤ 45 min, the volume percentage of mobile phase B is 50 - 70%.

[0073] Preferably, the described gradient elution procedure is as follows:

[0074] t = 0 min, the volume percentage of mobile phase B is 60%;

[0075] 0 < t ≤ 2 min, the volume percentage of mobile phase B is 60%;

[0076] 2 < t ≤ 25 min, the volume percentage of mobile phase B is 95%;

[0077] 25 < t ≤ 35 min, the volume percentage of mobile phase B is 95%.

[0078] The described gradient elution procedure may further include the following steps:

[0079] 35 < t ≤ 35.1 min, the volume percentage of mobile phase B is 60%;

[0080] 35.1 < t ≤ 45 min, the volume percentage of mobile phase B is 60%.

[0081] In some embodiments of the present invention, in the secondary separation detection, the specification of the chromatographic column is 150 mm * 4.6 mm, 3 μm.

[0082] In some embodiments of the present invention, in the secondary separation detection, the mobile phase A is 0.1% formic acid aqueous solution, and the mobile phase B is a mixed solution of methanol and tetrahydrofuran with a volume ratio of 4:1.

[0083] Further, in the secondary separation detection, the flow rate of the mobile phase is 0.5 - 2 mL / min, specifically such as 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0 mL / min.

[0084] Further, in the secondary separation detection, the column temperature of the chromatographic column is 50 - 70 °C, specifically such as 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 °C

[0085] Further, in the secondary separation detection, the injection volume of the sample solution is 1 - 200 μL, specifically 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 μL.

[0086] Further, in the secondary separation detection, the temperature of the injection tray is 1 - 30 °C, specifically 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 °C.

[0087] In some embodiments of the present invention, in the secondary separation detection, the flow rate is 1.0 mL / min.

[0088] In some embodiments of the present invention, in the secondary separation detection, the column temperature is 60 °C.

[0089] In some embodiments of the present invention, in the secondary separation detection, the injection volume of the sample solution is 5 μL.

[0090] In some embodiments of the present invention, in the secondary separation detection, the temperature of the injection tray is 25 °C.

[0091] Further, in the secondary separation detection, the detector is a CAD detector.

[0092] In some embodiments of the present invention, in the secondary separation detection, the parameters of the CAD detector are: Power Function: 1.00; Data Collection rate: 10 Hz; Filter: 1.0; Control Evaporator temperature: 50 °C.

[0093] In the second aspect of the present invention, there is provided a method for detecting a deoxycholic acid intermediate, and the detection method includes the following steps: dissolving the deoxycholic acid intermediate in a diluent to obtain a sample solution, and detecting the sample solution by HPLC method;

[0094] The deoxycholic acid intermediate is selected from the compounds shown by the following formula:

[0095]

[0096] Among them, P is a hydroxyl protecting group, preferably any one selected from acetyl, C1-C3 alkyl or benzoyl;

[0097] R is C1-C6 alkyl, preferably C1-C3 alkyl, more preferably methyl or ethyl.

[0098] Preferably, the deoxycholic acid intermediate is selected from the compounds shown by the following formula:

[0099]

[0100] Furthermore, the diluent is as described in the first aspect of the present invention.

[0101] Furthermore, in the HPLC method, the chromatographic conditions are as described in the chromatographic conditions in the secondary separation and detection in the first aspect of the present invention (for example, stationary phase, mobile phase, gradient elution program, specifications of the chromatographic column, flow rate, column temperature, sample injection volume, sample tray temperature, detector and its parameters).

[0102] In the third aspect of the present invention, a method for detecting a deoxycholic acid intermediate is provided. The detection method includes the following steps: dissolving the deoxycholic acid intermediate in a diluent to obtain a sample solution, and detecting the sample solution by HPLC method;

[0103] The deoxycholic acid intermediate is selected from the compounds shown by the following formula:

[0104]

[0105] Among them, P is a hydroxyl protecting group, preferably any one selected from acetyl, C1-C3 alkyl or benzoyl;

[0106] R is C1-C6 alkyl, preferably C1-C3 alkyl, more preferably methyl or ethyl.

[0107] Preferably, the deoxycholic acid intermediate is selected from the compounds shown by the following formula:

[0108]

[0109] Furthermore, the diluent is as described in the first aspect of the present invention.

[0110] Furthermore, in the HPLC method, the chromatographic conditions are as described in the chromatographic conditions in the secondary separation and detection in the first aspect of the present invention (for example, stationary phase, mobile phase, gradient elution program, specifications of the chromatographic column, flow rate, column temperature, sample injection volume, sample tray temperature, detector and its parameters).

[0111] In a fourth aspect of the present invention, a method for detecting a deoxycholic acid intermediate is provided. The detection method includes the following steps: dissolving the deoxycholic acid intermediate in a diluent to obtain a sample solution, and detecting the sample solution by HPLC method;

[0112] The deoxycholic acid intermediate is a compound represented by the following formula:

[0113]

[0114] Wherein, P is a hydroxyl protecting group, preferably any one selected from acetyl group, C1-C3 alkyl group or benzoyl group.

[0115] Preferably, the deoxycholic acid intermediate is a compound represented by the following formula:

[0116]

[0117] Further, the diluent is selected from one or more of acetonitrile (ACN), methanol (MeOH), ethanol (EtOH), tetrahydrofuran (THF), triphenylphosphine (PPh 3 3), formic acid (FA), and water.

[0118] In some embodiments of the present invention, the diluent is acetonitrile.

[0119] Further, in the HPLC method, the chromatographic conditions are as follows:

[0120] The stationary phase of the chromatographic column is phenylhexyl-bonded silica gel;

[0121] The mobile phase includes mobile phase A and mobile phase B;

[0122] The mobile phase A is an aqueous solution of formic acid with a volume percentage concentration of 0.01-5%, specifically such as 0.01, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5%, preferably 0.05-2%;

[0123] The mobile phase B is a mixed solution of acetonitrile and methanol with a volume ratio of 1 - 20:1, specifically 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, 8.5:1, 9:1, 9.5:1, 10:1, 10.5:1, 11:1, 11.5:1, 12:1, 12.5:1, 13:1, 13.5:1, 14:1, 14.5:1, 15:1, 15.5:1, 16:1, 16.5:1, 17:1, 17.5:1, 18:1, 18.5:1, 19:1, 19.5:1, 20:1, preferably 5 - 15:1;

[0124] A gradient elution program is adopted, and the gradient elution program is as follows:

[0125] At t = 0 min, the volume percentage of mobile phase B is 35 - 45%;

[0126] When 0 < t ≤ 2 min, the volume percentage of mobile phase B is 35 - 45%;

[0127] When 2 < t ≤ 25 min, the volume percentage of mobile phase B is 65 - 75%;

[0128] When 25 < t ≤ 35 min, the volume percentage of mobile phase B is 85 - 95%;

[0129] When 35 < t ≤ 40 min, the volume percentage of mobile phase B is 85 - 95%.

[0130] The gradient elution program may further include the following steps:

[0131] When 40 < t ≤ 40.1 min, the volume percentage of mobile phase B is 35 - 45%;

[0132] When 40.1 < t ≤ 50 min, the volume percentage of mobile phase B is 35 - 45%.

[0133] Preferably, the gradient elution program is as follows:

[0134] At t = 0 min, the volume percentage of mobile phase B is 40%;

[0135] When 0 < t ≤ 2 min, the volume percentage of mobile phase B is 40%;

[0136] When 2 < t ≤ 25 min, the volume percentage of mobile phase B is 70%;

[0137] When 25 < t ≤ 35 min, the volume percentage of mobile phase B is 90%;

[0138] When 35 < t ≤ 40 min, the volume percentage of mobile phase B is 90%.

[0139] The gradient elution program described above may further include the following steps:

[0140] When 40 < t ≤ 40.1 min, the volume percentage of mobile phase B is 40%;

[0141] When 40.1 < t ≤ 50 min, the volume percentage of mobile phase B is 40%.

[0142] In some embodiments of the present invention, the specifications of the chromatographic column are 150 mm * 4.6 mm, 3.5 μm.

[0143] In some embodiments of the present invention, mobile phase A is an aqueous solution of 0.1% formic acid, and mobile phase B is a mixed solution of acetonitrile and methanol with a volume ratio of 9:1.

[0144] Further, the flow rate of the mobile phase is 0.5 - 2 mL / min, specifically 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0 mL / min.

[0145] Further, the column temperature of the chromatographic column is 20 - 50 °C, specifically 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 °C.

[0146] Further, the injection volume of the sample solution is 1 - 200 μL, specifically 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 μL.

[0147] Further, the temperature of the injection tray is 1 - 30 °C, specifically 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 °C.

[0148] In some embodiments of the present invention, the flow rate is 1.0 mL / min.

[0149] In some embodiments of the present invention, the column temperature is 40 °C.

[0150] In some embodiments of the present invention, the sample injection volume is 5 μL.

[0151] In some embodiments of the present invention, the sample injection tray temperature is 25 °C.

[0152] Further, in the HPLC method, the detector is a CAD detector.

[0153] In some embodiments of the present invention, the parameters of the CAD detector are as follows: Power Function: 1.00; Data Collection rate: 10 Hz; Filter: 1.0; Control Evaporator temperature: 50 °C.

[0154] In a fifth aspect of the present invention, there is provided the use of the detection method described in the first, second, third or fourth aspect of the present invention in the quality control or quality evaluation of deoxycholic acid intermediates, and in the quality control or quality evaluation of the preparation process of deoxycholic acid.

[0155] The present invention has the following beneficial effects:

[0156] The detection method of the present invention can effectively separate deoxycholic acid intermediates and impurities, thereby effectively controlling the quality of deoxycholic acid products, and this method fully meets the acceptance criteria and performs well in terms of specificity, linearity, range, accuracy, precision, detection limit, quantitation limit, durability, solution stability, system precision, system suitability, etc. Description of the Drawings

[0157] Figure 1 Shown is the NMR spectrum of commercially available deoxycholic acid.

[0158] Figure 2 Shown is the NMR spectrum of deoxycholic acid prepared by the preparation method of the present invention.

[0159] Figures 3 - 15 Shown is the NMR spectrum of the intermediate product of deoxycholic acid prepared by the method of the embodiment of the present invention.

[0160] Figure 16 Shown is the chromatogram of the resolution solution C-10-RS and the blank solution.

[0161] Figure 17 Shown is the chromatogram of the resolution solution C-11-RS and the blank solution.

[0162] Figure 18The chromatograms of the resolution solution C-12-RS and the blank solution are shown as follows.

[0163] Figure 19 The chromatograms of the resolution solution C-13-RS-1 and the blank solution are shown as follows.

[0164] Figure 20 The chromatograms of the resolution solution C-13-RS-2 and the blank solution are shown as follows.

[0165] Figure 21 The chromatograms of the resolution solution C-14-RS-1 and the blank solution are shown as follows.

[0166] Figure 22 The superimposed chromatograms of the resolution solutions C-12-RS and C-14-RS-1 are shown as follows.

[0167] Figure 23 The chromatograms of the resolution solution C-14-RS-2 and the blank solution are shown as follows. Detailed implementation mode

[0168] Unless otherwise defined, all scientific and technical terms used in the present invention have the same meaning as commonly understood by those skilled in the technical field to which the present invention pertains.

[0169] In the present invention, the term "room temperature" refers to the temperature of an article being close to or the same as the temperature of the space (e.g., the location of the fume hood in which the article is located). In pharmaceutical inspection, "room temperature" generally refers to about 10°C to about 30°C, or about 22°C to 27°C, or about 25°C.

[0170] The impurity reference substances used in the examples of the present invention are shown in Table 1 and are provided by Aimeike and commissioned by Wuhan WuXi AppTec Co., Ltd.

[0171] Table 1 Summary of impurity reference substances

[0172]

[0173]

[0174] The embodiments of the present invention will be described in detail below in conjunction with the examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. For those not specified in the examples, they are carried out under conventional conditions or conditions recommended by the manufacturer. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0175] Example 1

[0176]

[0177]

[0178] (1) Synthesis of Compound C-2

[0179] Dissolve Compound C-1 (150 g, 1.0 eq) in pyridine (2.25 L, 5V). Under nitrogen protection, add 10% Pd / C (30 g, 0.2 w / w). Replace nitrogen three times and replace hydrogen three times. Stir and react at 15 °C under 15 psi for 12 hours. After the reaction is completed, filter the reaction solution, concentrate it to dryness, slurry with n-heptane to obtain a solid, filter, and wash to obtain 119.4 g of solid C-2, with a yield of 79.0%.

[0180] NMR test conditions 1 HNMR (400 MHz, CDCl 3 ) The test results are as Figure 3 shown; Mass (m / z): [M + H] + : 289.3.

[0181] (2) Synthesis of Compound C-3

[0182] Dissolve Compound C-2 (1.9 kg) in THF (9.5 L, 5V). Slowly add a THF solution of LiAlH(t - OBu) 3 (2.0 kg dissolved in 7.9 L of THF, 1.2 eq) dropwise to the reaction flask and react at -5 °C for 3 h. After the reaction is completed, add (16 L) of 15% aqueous sodium potassium tartrate solution to the reaction solution and stir for 0.5 h. Separate the layers, extract the aqueous phase with EA (5 L) once, combine the organic phases, wash with 10% anhydrous sodium sulfate to obtain the organic phase, concentrate it to 5 L, add n-heptane (20 L) and stir for 1.5 h, filter to obtain a solid. The weight of Compound C-3 is 1.56 kg, with a yield of 81.4%.

[0183] NMR test conditions 1 HNMR (400 MHz, CDCl 3 ) The test results are as Figure 4 shown; Mass (m / z): [M - H 2 O + H] + : 291.1.

[0184] (3) Synthesis of Compound C-4

[0185] Add 1.55 kg of Compound C-3 (1.0 eq) to methyl tert-butyl ether MTBE (7.5 L, 5V), then add 2-aminomethylpyridine (1.73 kg, 3 eq) and triethyl orthoformate CH(OEt) 3(1.58 kg, 2.0 eq), the temperature of the system was raised to 60 °C, and the reaction was stirred for 16 hours. After the reaction was complete, the temperature of the system was lowered to 20 °C, saturated ammonium chloride solution (6 L) was added, and the mixture was stirred for 0.5 h, filtered to obtain a filtrate. It was washed with saturated brine, separated by liquid-liquid extraction, the organic phase was concentrated, and it was stirred with MTBE and n-heptane and filtered to obtain a solid. 1.09 kg of compound C-4 was obtained, and the yield was 53.7%.

[0186] NMR test conditions 1 HNMR (400 MHz, CDCl 3 ) The test results are as Figure 5 shown; Mass (m / z): [M + H] + : 381.3.

[0187] (4) Synthesis of compound C-5

[0188] 1.5 L of acetone and 1.5 L of ethanol were added to the reaction flask, and the temperature was lowered to 0 - 10 °C. Cu(oTf) 2 (427.70 g, 1.50 eq) was added to the reaction solution in batches, and the temperature was controlled at 5 °C (added in about 40 minutes). Compound C-4 (300 g, 1 eq) was added to the reaction solution, and then 312.35 g of sodium ascorbate (2 eq) was added to the reaction solution. It was purged with nitrogen 3 times and then with oxygen 3 times. The temperature was controlled at 45 °C, and the reaction was carried out under an oxygen pressure of 15 psi for 17 hours. The reaction solution was cooled to 20 °C. 627.8 g of EDTA-4Na (2 eq) was dissolved in 1884 mL of water and added dropwise to the reaction solution. The reaction solution was heated to 45 °C and stirred for reaction. After the reaction was completed, water and ethyl acetate were added to the reaction solution, stirred for 10 minutes, allowed to stand for liquid-liquid extraction, the organic phase was collected, concentrated under reduced pressure to obtain an oil, dissolved in ethyl acetate, washed with aqueous citric acid solution, dried over anhydrous sodium sulfate, filtered, the mother liquor was evaporated to dryness to obtain a crude product, and the solid was obtained by slurrying with MTBE. The filter cake was dried to obtain 128.76 g of compound C-5, and the yield was 53.3%.

[0189] NMR test conditions 1 HNMR (400 MHz, CDCl 3 ) The test results are as Figure 6 shown; Mass (m / z): [M - H 2 O + H] + : 289.1.

[0190] (5) Synthesis of compound C-6

[0191] Add 1.8 L of THF and 726.9 g of ethyltriphenylphosphonium bromide (5 eq) to the reaction flask. Replace the air with nitrogen three times. Cool the reaction solution to 15 °C. Add 219.7 g of potassium tert-butoxide (5 eq) to the reaction flask in portions. Heat the mixture to 40 °C and stir for 1 hour. Then cool it to 20 °C. Add 120 g of compound C-5 (1 eq) to the reaction solution, and then add 600 mL of THF. Heat the system to 65 °C and stir for 17 hours. Cool the reaction solution to 20 °C. Slowly add the reaction solution to saturated ammonium chloride solution to quench the reaction. Stir the mixture, let it stand, and separate the layers. Wash the organic layer with saturated brine, dry the organic layer with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure at 45 °C to dryness to obtain 543.35 g of an oily substance. Purify it by column chromatography to obtain 70.23 g of compound C-6, with a yield of 56.3%.

[0192] NMR test conditions 1 HNMR(400MHz, CDCl 3 ) The test results are as Figure 7 shown; Mass(m / z): [M-H 2 O+H] + : 301.2.

[0193] (6) Synthesis of compound C-7

[0194] Add 68 g of compound C-6 (1 eq) to 1020 mL of DCM. Replace the air with nitrogen three times. Cool the mixture to 5 °C. Add 33.78 g of pyridine (2 eq), then add 2.6 g of DMAP (0.1 eq), and then add 26.18 g of acetic anhydride (1.2 eq). Control the temperature at 5 °C and react for 9 hours. After the reaction is completed, add 2N HCl to the reaction solution, stir for 10 minutes, let it stand, and separate the layers. Collect the organic layer. Dry the organic layer with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure at 40 °C to dryness to obtain 79.32 g of a crude oily product of compound C-7, with a yield of 103.0%.

[0195] NMR test conditions 1 HNMR(400MHz, CDCl 3 ) The test results are as Figure 8 shown; Mass(m / z): [M-H 2 O+H] + : 343.2.

[0196] (7) Synthesis of compound C-8

[0197] Method 1: Add 1125 mL of DCM and 39.61 g (1.50 eq) of oxalyl chloride to a reaction flask and cool the temperature to -55 °C. Add 39 g of DMSO (2.4 eq) to 225 mL of DCM, mix well and slowly add it dropwise to the reaction solution, and stir at -55 °C for 20 minutes. Dissolve 75 g (1 eq) of compound C-7 in 450 mL of DCM, add it dropwise to the reaction solution at -55 °C, react for 1 hour, then add 338 mL of TEA dropwise to the reaction solution, stir at -55 °C for 30 minutes, and the reaction is completed. Add 95 mL of water to the reaction solution, let it stand for liquid separation, wash the organic phase with saturated brine (95 mL) once, dry the organic phase with anhydrous sodium sulfate, filter, concentrate the filtrate to dryness, and purify it by column chromatography to obtain 43.9 g of compound C-8 with a yield of 58.8%.

[0198] NMR test conditions 1 HNMR(400MHz, CDCl 3 ) The test results are as Figure 9 shown; Mass(m / z): [M+H] + : 359.3.

[0199] Method 2: Add 142.5 mL of DCM and 5.05 g (1.50 eq) of oxalyl chloride to a reaction flask and cool the temperature to -55 °C. Add 4.95 g of DMSO (2.4 eq) to 5 mL of DCM, mix well and slowly add it dropwise to the reaction solution, and stir at -55 °C for 15 minutes. Dissolve 9.5 g (1 eq) of compound C-7 in 57 mL of DCM, add it dropwise to the reaction solution at -55 °C, react for 1 hour, then add 43 mL of TEA dropwise to the reaction solution, stir at -55 °C for 2 hours, and the reaction is completed. Add 95 mL of water to the reaction solution, let it stand for liquid separation, wash the organic phase with saturated brine (95 mL) once, dry the organic phase with anhydrous sodium sulfate, filter, concentrate the filtrate to dryness, and purify it by column chromatography to obtain 5.60 g of compound C-8 with a yield of 59.2%.

[0200] (8) Synthesis of compound C-9

[0201] Method 1: Dissolve 20 g of compound C-8 (1 eq) in 400 mL of ethyl acetate, cool the temperature to 5 °C, add 20 g of paraformaldehyde (12 eq) to the reaction solution, and slowly add 30.98 g of BF 3 ·Et 2 O (1.8 eq) dropwise to the reaction solution, stir at 5 °C for 2 hours, and the reaction is completed. Add 100 mL of saturated NaHCO 3The solution was stirred for 10 min, filtered, and the filtrate was allowed to stand for liquid separation. The aqueous phase was extracted twice with 80 mL of ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the mother liquor was rotary evaporated to obtain 18.00 g of crude product of compound C-9 with a yield of 83.3%.

[0202] NMR test conditions 1 HNMR (400 MHz, CDCl 3 ) The test results are as Figure 10 shown; Mass (m / z): [M - H 2 O + H] + : 371.4.

[0203] Method 2: 200 mg of compound C-8 (1 eq) was dissolved in 0.5 mL of DCM, the temperature was lowered to -5 °C, 200 mg of paraformaldehyde (12 eq) was added to the reaction solution, and 310 mg of BF 3 ·Et 2 O (1.8 eq) was slowly added dropwise to the reaction solution, and the mixture was stirred at -5 °C for 2 hours to complete the reaction. 2 mL of saturated NaHCO 3 solution was added to the reaction solution, stirred for 10 min, filtered, the filtrate was allowed to stand for liquid separation, the aqueous phase was extracted with 2 mL of DCM to obtain the organic phase, dried over anhydrous sodium sulfate, filtered, and the mother liquor was rotary evaporated to obtain 156 mg of crude product of compound C-9 with a yield of 72.2% and a purity of 90.5%.

[0204] (9) Synthesis of compound C-10

[0205] Method 1: 3.5 g (1 eq) of compound C-9 was added to 25 mL of EtOH and 25 mL of EtOAc, stirred until dissolved and clear, purged with nitrogen 3 times, 0.7 g of Pt / C (0.2 w / w) was added to the reaction flask, purged with hydrogen 3 times, and stirred at 25 °C and 15 psi for 2 hours to complete the reaction. The reaction solution was filtered, the mother liquor was collected, and the mother liquor was concentrated to dryness at 40 °C to obtain a colorless oil. 4.5 mL of THF was added, the temperature was raised to 50 °C and stirred until dissolved and clear, 45 mL of n-heptane was added, the temperature was lowered to 25 °C and stirred for 1 hour, filtered, and the filter cake was dried to obtain 3.23 g of white solid compound C-10 with a yield of 91.79%.

[0206] NMR test conditions 1 HNMR (400 MHz, CDCl 3 ) The test results are as Figure 11 shown; Mass (m / z): [M + H] + : 391.4.

[0207] Method 2: Add 450.0 g (1 eq) of Compound C-9 to 2250 mL of EtOH and 2250 mL of EtOAc, stir until dissolved and clear, change the gas three times with nitrogen. Add 90.0 g of Pt / C (0.2 w / w) to the reaction flask, change the gas three times with hydrogen, control the temperature at 5 °C, stir and react at 15 psi for 2 hours until the reaction is complete. Filter the reaction solution, collect the mother liquor, concentrate the mother liquor to dryness, add 900 mL of THF, heat up to 55 °C and stir until dissolved and clear, add 3.6 L of n-heptane, cool down to 25 °C, stir for 2 h. After drying the filter cake, the mass of the white solid Compound C-10 is 321 g, the yield is 70.85%, and the purity is 98.73%.

[0208] (10) Synthesis of Compound C-11

[0209] Method 1: Add 1 g of Compound C-10 (1 eq) to 5 mL of DCM and 5 mL of water, cool down to 5 °C, and then successively add 120 mL of TEMPO (0.3 eq), 215 mg of NaHCO 3 (1 eq), 131 mg of NaBr (0.5 eq), slowly dropwise add the NaClO solution (1.83 g, 1.2 eq), control the temperature at 5 °C and finish dropping, continue to react for 1.5 h. After the reaction is complete, dissolve Na 2 S 2 O 3 (387 mg, 1.2 eq) in 10 mL of water, add it to the reaction solution, stir for 10 minutes, let it stand for liquid separation, extract the aqueous phase twice with 20 mL of DCM, combine the organic phases, wash once with 5 mL of saturated NaCl solution, dry with anhydrous sodium sulfate, filter, and rotary evaporate the mother liquor to obtain 0.97 g of the crude product of Compound C-11, yield: 97.1%.

[0210] NMR test conditions 1 HNMR (400 MHz, CDCl 3 ) The test results are as Figure 12 shown; Mass (m / z): [M + H] + : 389.4.

[0211] Method 2: Add 1.00 g of Compound C-10 (1 eq) to 15 mL of DCM, cool down to 5 °C, and then successively add 2.00 g of DMSO (10 eq), 1.99 g of DIEA (6 eq), 1.43 g of SO 3.Py (3.5 eq), react at 5 °C for 1.5 h. After the reaction is complete, add 15 mL of 20% citric acid solution to the reaction solution, stir for 10 minutes, let it stand for liquid separation. The aqueous phase is extracted twice with 10 mL of DCM, the organic phases are combined, washed once with 10 mL of saturated NaCl solution, dried over anhydrous sodium sulfate, filtered, and the mother liquor is rotary evaporated to obtain 0.94 g of crude product of compound C-11, yield: 94.5%, purity 83.3%.

[0212] (11) Synthesis of compound C-12

[0213] Method 1: Add 325 mg of diethyl methylphosphonoacetate (1.2 eq) to 5 mL of THF and stir until clear. Cool the temperature to 10 °C, slowly drop 1.54 mL of LiHMDS (1 M, 1.2 eq) into the reaction flask, and react at 10 °C for 1 h. Dissolve 0.50 g (1 eq) of compound C-11 in 2.5 mL of THF, slowly drop it into the reaction solution, and stir and react at 10 °C for 2 h. After the reaction is completed, add 10 mL of water to the reaction solution, stir for 0.5 h, then add 0.20 g of ammonium chloride to the reaction solution, stir at 25 °C for 0.5 h, let it stand for liquid separation. The aqueous phase is extracted twice with 20 mL of ethyl acetate, the organic phases are combined, dried over anhydrous sodium sulfate, filtered, and the mother liquor is rotary evaporated to obtain 0.5 g of crude product of compound C-12, yield: 87.2%.

[0214] NMR test conditions 1 HNMR (400 MHz, CDCl 3 ) The test results are as Figure 13 shown; Mass (m / z): [M + H 2 O] + : 462.4.

[0215] Method 2: Add 50.0 g of compound C-11 to 500 mL of DCM and stir until clear. Add 86.05 g of methoxycarbonylmethylenetriphenylphosphine (2.0 eq) to the reaction flask, heat up to 35 °C, and react at 35 °C for 36 h. After the reaction is completed, add 250 mL of 30% citric acid solution to the reaction solution, stir for 20 minutes, let it stand for liquid separation. The organic phase is dried over anhydrous sodium sulfate, filtered, and the mother liquor is rotary evaporated to obtain 49.38 g of crude product of compound C-12. After purification with ethanol, a solid is obtained, yield: 86.3%, purity 95.1%.

[0216] (12) Synthesis of compound C-13

[0217] Method 1: Add 2.5 g of C-12 (1 eq) to 25 mL of THF, cool down to 5 °C, and slowly add the lithium tri-tert-butoxyaluminum hydride solution in THF (1 M in THF, 6.75 mL, 1.2 eq) dropwise to the reaction solution. React at 5 °C for 2 hours until the reaction is complete. Add 25 mL of 20% potassium sodium tartrate solution to the reaction solution, stir for 30 minutes, let it stand and separate the layers. Extract the aqueous phase with 50 mL of ethyl acetate twice, combine the organic phases, wash once with saturated brine, dry over anhydrous sodium sulfate, filter, concentrate the mother liquor to dryness, and obtain 2.47 g of the crude product as a colorless oily compound C-13 with a yield of 98.41%.

[0218] NMR test conditions 1 HNMR(400MHz, CDCl 3 ) The test results are as Figure 14 shown; Mass(m / z): [M+H 2 O] + : 464.5.

[0219] Method 2: Add 260 g of C-12 (1 eq) to 2.6 L of THF, cool down to 5 °C, and slowly add the lithium tri-tert-butoxyaluminum hydride solution in THF (1 M in THF, 1.17 L, 2.0 eq) dropwise to the reaction solution. React at 5 °C for 2 hours until the reaction is complete. Add 2.3 L of 20% potassium sodium tartrate solution to the reaction solution, stir for 20 minutes, let it stand and separate the layers. Extract the aqueous phase with 1.3 L of ethyl acetate twice, combine the organic phases, dry over anhydrous sodium sulfate, filter, concentrate the mother liquor to dryness, and refine with methanol to obtain 228 g of C-13 with a yield of 87.3% and a purity of 78.8%.

[0220] (13) Synthesis of compound C-14

[0221] Method 1: Dissolve 0.5 g (1 eq) of compound C-13 in 3.5 mL of MeOH and 3.5 mL of EtOAc, purge with nitrogen three times, add 0.10 g of Pt / C (0.2 w / w), purge with hydrogen 3 times, control the temperature at 25 °C, and react under a hydrogen pressure of 50 psi for 19 h. Filter, collect the filtrate, and concentrate the filtrate under reduced pressure to dryness to obtain 0.49 g of the crude product as a colorless oily compound C-14 with a yield of 97.6%.

[0222] NMR test conditions 1 HNMR(400MHz, CDCl 3 ) The test results are as Figure 15 shown; Mass(m / z): [M+H 2 O] + : 466.5.

[0223] Method 2: Dissolve 170 g (1 eq) of compound C-13 in 1.7 L of THF, purge with nitrogen three times, add 34.0 g of Pt / C (0.2 w / w), purge with hydrogen 3 times, control the temperature at 25 °C, react under a hydrogen pressure of 15 psi for 19 h, filter, collect the filtrate, concentrate the filtrate under reduced pressure to dryness to obtain a colorless oil, and purify it with ethanol to obtain 125.3 g of compound C-14, with a yield of 73.4% and a purity of 88.6%.

[0224] (14) Synthesis of compound C-15

[0225] Method 1: Add compound C-14 (0.2 g) to 2 mL of THF and 2 mL of MeOH, stir until dissolved clearly, then add 0.4 mL of water and 75 mg of LiOH·H 2 O (4 eq), control the temperature at 25 °C and stir for 12 hours to complete the reaction. Concentrate the reaction solution to dryness under reduced pressure to obtain a white solid. Add 1 mL of water, stir to dissolve, then adjust the pH to 3.5 with 4N HCl, continue to stir for 1 hour, filter, and after drying the filter cake, obtain 0.15 g of white solid, which is the crude product of compound C-15, with a yield of 85.7%. The HNMR of the product is as Figure 2 shown, and by comparison, it is consistent with the HNMR of Figure 1 purchased deoxycholic acid.

[0226] NMR test conditions 1 The HNMR (400 MHz, MeOD) test results are as Figure 2 shown; Mass (m / z): [2M + H] + : 785.7.

[0227] Method 2: Add compound C-14 (12.0 g) to 60 mL of THF and 60 mL of MeOH, stir until dissolved clearly, then add 60 mL of water and 4.50 g of LiOH·H 2 O (4 eq), control the temperature at 25 °C and stir for 3 hours to complete the reaction. Concentrate the reaction solution to dryness under reduced pressure to obtain a white solid. Adjust the pH to 3.5 with 6N HCl, continue to stir for 1 hour, filter, and purify the filter cake with methanol / DCM and ethanol / water respectively to obtain 8.31 g of white solid, which is compound C-15, with a yield of 79.1% and a purity greater than 99%.

[0228] The following describes the detection methods for the important intermediates C-10, C-11, C-12, C-13, and C-14 in the synthesis process of Example 1, which can effectively separate deoxycholic acid intermediates and impurities, and have been investigated in terms of specificity, limit of quantitation, limit of detection, etc.

[0229] Detection method for deoxycholic acid intermediate C-10 in Example 2

[0230] Related impurities during the reaction of Table 2C - 9 to C - 10

[0231]

[0232] 2.1 Solution preparation

[0233] (1) Blank solution (diluent):

[0234] Measure 1000 mL of acetonitrile into a mobile phase bottle, mix well, and degas by sonication.

[0235] (2) Mobile phase:

[0236] Mobile phase A (0.1% FA aqueous solution, v / v): Measure 1000 mL of purified water and 1 mL of formic acid into a mobile phase bottle, mix well, and degas by sonication.

[0237] Mobile phase B (ACN:MeOH = 9:1, v / v): Measure 900 mL of acetonitrile and 100 mL of methanol into a mobile phase bottle, mix well, and degas by sonication.

[0238] (3) Resolution solution:

[0239] Take about 5 mg of C - 9 impurity reference substance, accurately weigh it, place it in a 10 - mL volumetric flask, dissolve it with diluent and dilute to the mark, shake well, and label it as C - 9 impurity reference substance. Prepare C - 16 impurity reference substance, C - 17 impurity reference substance, C - 18 impurity reference substance, and C - 19 impurity reference substance in the same way.

[0240] Weigh about 25 mg of C - 10 reference substance accurately, place it in a 5 - mL volumetric flask, respectively measure 0.5 mL of each of the solutions of C - 9 impurity reference substance, C - 16 impurity reference substance, C - 17 impurity reference substance, C - 18 impurity reference substance, and C - 19 impurity reference substance and add them into it, dissolve with diluent and dilute to the mark, shake well, and label it as C - 10 - RS.

[0241] (4) Sample solution:

[0242] Take about 25 mg of C - 10 sample, accurately weigh it, place it in a 5 - mL volumetric flask, dissolve it with diluent and dilute to the mark, shake well, and label it as C - 10 - SPL.

[0243] (5) Sample control solution (1% self - control solution):

[0244] Accurately measure 1 mL of C - 10 - SPL solution into a 100 - mL volumetric flask, dilute it to the mark with diluent, shake well, and label it as C - 10 - SPL - 1%.

[0245] 2.2 HPLC conditions

[0246] HPLC: Thermo Ultimate 3000 series high performance liquid chromatograph, CAD detector;

[0247] Chromatographic column: Waters Xselect CSH phenyl Hexyl 150mm * 4.6mm, 3.5μm;

[0248] CAD: Power Function: 1.00;

[0249] Data Collection rate: 10Hz;

[0250] Filter: 1.0;

[0251] Control Evaporator temperature: High / 50°C;

[0252] Mobile phase: Mobile phase A: 0.1% FA aqueous solution (v / v)

[0253] Mobile phase B: ACN:MeOH = 9:1 (v / v);

[0254] Sample tray temperature: Room temperature;

[0255] Column temperature: 40°C;

[0256] Flow rate: 1.0 mL / min;

[0257] Injection volume: 5 μL;

[0258] Gradient elution program is shown in Table 3 (after 40 min is the column flushing and system equilibration stage):

[0259] Table 3 Gradient elution program

[0260] Time (min) B(％) 0.0 40 2.0 40 25.0 70 35.0 90 40.0 90 40.1 40 50.0 40

[0261] 2.3 Experimental results

[0262] The chromatogram of the resolution solution C-10-RS is as Figure 16 shown, where the peak valley ratio of the main peak is 22.2, and the resolution between the known impurity and the adjacent peak ≥ 1.0. The retention times of the main peak and the known impurity, and the LOQ and LOD data of C-10 are shown in Table 4.

[0263] The RSD of the C-10 peak area in the C-10-SPL-1% chromatograms of 3 consecutive injections is 0.3%, and the RSD of the C-10 retention time is 0.8%.

[0264] Table 4 Experimental data of C-10

[0265]

[0266] According to the above detection results, it can be known that this detection method can effectively separate deoxycholic acid intermediate C-10 and impurities C-9, C-16, C-17, C-18, and C-19.

[0267] Detection method for deoxycholic acid intermediate C-11 in Example 3

[0268] 3.1 Solution preparation

[0269] (1) Blank solution (diluent):

[0270] 0.1 g / L triphenylphosphine acetonitrile solution: Weigh about 0.1 g of triphenylphosphine, accurately weigh it, place it in a mobile phase bottle, measure 1000 mL of acetonitrile and add it thereto, and ultrasonically dissolve it and mix well.

[0271] (2) Mobile phase:

[0272] Mobile phase A (0.1% FA aqueous solution, v / v): Measure 1000 mL of purified water and 1 mL of formic acid into a mobile phase bottle, and ultrasonically degas.

[0273] Mobile phase B (0.1% FA acetonitrile solution, v / v): Measure 1000 mL of acetonitrile and 1 mL of formic acid into a mobile phase bottle, and ultrasonically degas.

[0274] (3) Resolution solution:

[0275] Take about 5 mg of C-10 impurity reference substance, accurately weigh it and place it in a 10 mL volumetric flask, dissolve it with the diluent and dilute it to the scale, shake well, and label it as C-10 impurity reference substance.

[0276] Weigh about 25 mg of C-11 reference substance, accurately weigh it, place it in a 5 mL volumetric flask, measure 0.5 mL of the C-10 impurity reference substance solution and add it thereto, dissolve it with the diluent and dilute it to the scale, shake well, and label it as C-11-RS.

[0277] (4) Sample solution:

[0278] Take about 25 mg of C-11 sample, accurately weigh it, place it in a 5 mL volumetric flask, dissolve it with the diluent and dilute it to the scale, shake well, and label it as C-11-SPL.

[0279] (5) Sample control solution (1% self-control solution):

[0280] Accurately measure 1 mL of the C-11-SPL solution into a 100 mL volumetric flask, dilute it to the scale with the diluent, shake well, and label it as C-11-SPL-1%.

[0281] 3.2 Liquid Phase Conditions

[0282] HPLC: Thermo Ultimate 3000 series high performance liquid chromatograph, CAD detector;

[0283] Chromatographic column: YMC Triart C18 - ExRS 150mm * 4.6mm, 3μm;

[0284] CAD: Power Function: 1.00;

[0285] Data Collection rate: 10Hz;

[0286] Filter: 1.0;

[0287] Control Evaporator temperature: High / 50°C;

[0288] Mobile phase: Mobile phase A: 0.1% FA aqueous solution (v / v)

[0289] Mobile phase B: 0.1% FA acetonitrile solution (v / v);

[0290] Injector tray temperature: Room temperature;

[0291] Column temperature: 40°C;

[0292] Flow rate: 1.0 mL / min;

[0293] Injection volume: 5 μL;

[0294] Gradient elution program is shown in Table 5 (after 35 min is the column flushing and system equilibration stage):

[0295] Table 5 Gradient Elution Program

[0296] Time (min) B(％) 0.0 25 2.0 45 14.0 58 24.0 95 35.0 95 35.1 25 45.0 25

[0297] 3.3 Experimental Results

[0298] The chromatogram of the resolution solution C - 11 - RS is as shown in Figure 17 which, the resolution between the main peak and the adjacent peak ≥ 3.0, and the resolution between the known impurity C - 10 and the adjacent peak ≥ 2.4. The retention times of the main peak and the known impurities, and the LOQ and LOD data of C - 11 are shown in Table 6.

[0299] In the chromatograms of C - 11 - SPL - 1% for 3 consecutive injections, the RSD of the C - 11 peak area = 2.4%, and the RSD of the C - 11 retention time = 0.0%.

[0300] Table 6 Experimental Data of C - 11

[0301]

[0302] According to the above test results, it can be known that this test method can effectively separate deoxycholic acid intermediate C-11 and impurity C-10.

[0303] Test Method for Deoxycholic Acid Intermediate C-12 in Example 4

[0304] Table 7 Related Impurities during the Reaction from C-11 to C-12

[0305]

[0306] 4.1 Solution Preparation

[0307] (1) Blank Solution (Diluent):

[0308] ACN: Measure 1000 mL of acetonitrile into a diluent bottle and degas it by ultrasonic treatment.

[0309] (2) Mobile Phase:

[0310] Same as in Example 3.

[0311] (3) Resolution Solution:

[0312] Take about 5 mg of C-10 impurity reference substance, accurately weigh it and place it in a 5 mL volumetric flask. Add diluent to dissolve and dilute to the scale, shake well, and label it as C-10 impurity reference substance. Prepare C-11 impurity reference substance, POPh 3 impurity reference substance, and C-20 impurity reference substance in the same way.

[0313] Weigh about 50 mg of C-12 reference substance accurately, place it in a 5 mL volumetric flask, measure 0.5 mL of each of the C-10 impurity reference substance solution, C-11 impurity reference substance solution, POPh 3 impurity reference substance solution, and C-20 impurity reference substance solution and add them thereto. Add diluent to dissolve and dilute to the scale, shake well, and label it as C-12-RS.

[0314] (4) Sample Solution:

[0315] Take about 50 mg of C-12 sample, accurately weigh it, place it in a 5 mL volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and label it as C-12-SPL.

[0316] (5) Sample Control Solution (1% Self-Control Solution):

[0317] Accurately measure 1 mL of C-12-SPL solution into a 100 mL volumetric flask, add diluent to dilute to the scale, shake well, and label it as C-12-SPL-1%.

[0318] 4.2 Liquid Phase Conditions

[0319] HPLC, chromatographic column, CAD, mobile phase, gradient elution program, flow rate, and column temperature are the same as in Example 3.

[0320] Injector tray temperature: 5 °C;

[0321] Sample injection volume: 3 μL.

[0322] 4.3 Experimental Results

[0323] The chromatogram of Resolution Solution C-12-RS is as Figure 18 shown. Among them, the resolution between the main peak and the adjacent peak is ≥ 12.5, and the resolution between the known impurity and the adjacent peak is ≥ 12.5. The retention times of the main peak and the known impurity, and the LOQ and LOD data of C-12 are shown in Table 8.

[0324] In the chromatogram of C-12-SPL-1% for 3 consecutive injections, the RSD of the C-12 peak area is 0.9%, and the RSD of the C-12 retention time is 0.0% (N / A).

[0325] Table 8 Experimental Data of C-12

[0326]

[0327]

[0328] According to the above detection results, it can be known that this detection method can effectively separate deoxycholic acid intermediate C-12 and impurities C-10, C-11, C-20, POPh 3 .

[0329] Detection Method for Deoxycholic Acid Intermediate C-13 in Example 5

[0330] 5.1 Primary Separation Detection

[0331] Table 9 Related Impurities in the Reaction Process of C-12 to C-13

[0332]

[0333] 5.1.1 Solution Preparation

[0334] (1-1) Blank Solution (Diluent):

[0335] MeOH:THF = 1:1: Measure 500 mL of methanol and 500 mL of tetrahydrofuran and mix them well in a mobile phase bottle.

[0336] (1-2) Mobile Phase:

[0337] Same as in Example 3.

[0338] (1 - 3) Resolution solution:

[0339] Take about 5 mg of C - 12 impurity reference substance, accurately weigh it and place it in a 10 - mL volumetric flask. Add diluent to dissolve and dilute to the mark, shake well, and label it as C - 12 impurity reference substance. Prepare C - 14 impurity reference substance, C - 21 impurity reference substance, C - 22 impurity reference substance, C - 23 impurity reference substance, C - 24 impurity reference substance, and C - 25 impurity reference substance in the same way.

[0340] Weigh about 25 mg of C - 13 reference substance, accurately weigh it, place it in a 5 - mL volumetric flask. Respectively measure 0.5 mL of each of the solutions of C - 12 impurity reference substance, C - 14 impurity reference substance, C - 21 impurity reference substance, C - 22 impurity reference substance, C - 23 impurity reference substance, C - 24 impurity reference substance, and C - 25 impurity reference substance and add them thereto. Add diluent to dissolve and dilute to the mark, shake well, and label it as C - 13 - RS - 1.

[0341] (1 - 4) Sample solution:

[0342] Take about 25 mg of C - 13 sample, accurately weigh it, place it in a 5 - mL volumetric flask. Add diluent to dissolve and dilute to the mark, shake well, and label it as C - 13 - SPL - 1.

[0343] (1 - 5) Sample control solution (1% self - control solution):

[0344] Accurately measure 1 mL of the C - 13 - SPL - 1 solution and transfer it to a 100 - mL volumetric flask. Add diluent to dilute to the mark, shake well, and label it as C - 13 - SPL - 1 - 1%.

[0345] 5.1.2 Liquid phase conditions

[0346] HPLC, chromatographic column, CAD, mobile phase, gradient elution program, flow rate, and column temperature are the same as in Example 3.

[0347] Injector tray temperature: 5°C;

[0348] Injection volume: 5 μL.

[0349] 5.1.3 Experimental results

[0350] The chromatogram of the resolution solution C - 13 - RS - 1 is as Figure 19 shown. Among them, the resolution between the main peak and the adjacent peak is ≥2.1, and the resolution between the known impurity and the adjacent peak is ≥2.1. The retention times of the main peak and the known impurity, and the LOQ and LOD data of C - 13 are shown in Table 10.

[0351] The RSD of the peak area of C-13 in the chromatogram of three consecutive injections of C-13-SPL-1-1% was 0.8%, and the RSD of the retention time of C-13 was 0.0%.

[0352] Table 10 Experimental data of C-13

[0353]

[0354]

[0355] According to the above test results, it can be seen that this test method can effectively separate deoxycholic acid intermediate C-13 and impurities C-14, C-21, C-22, C-23, C-24, C-25. In order to further separate the key impurity C-12, secondary HPLC separation and detection were carried out.

[0356] 5.2 Secondary separation and detection

[0357] 5.2.1 Solution preparation

[0358] (2-1) Blank solution (diluent):

[0359] Same as (1-1) in the solution preparation of 5.1.1 in Example 5.

[0360] (2-2) Mobile phase:

[0361] Mobile phase A (0.1% FA aqueous solution, v / v): Measure 1000 mL of purified water and 1 mL of formic acid into a mobile phase bottle, and perform ultrasonic degassing.

[0362] Mobile phase B (MeOH:THF = 8:2, v / v): Measure 800 mL of methanol and 200 mL of tetrahydrofuran into a mobile phase bottle, and perform ultrasonic degassing.

[0363] (2-3) Resolution solution:

[0364] Take about 5 mg of C-12 impurity reference substance, accurately weigh it and place it in a 10 mL volumetric flask, dissolve it with diluent and dilute it to the mark, shake well, and label it as C-12 impurity reference substance;

[0365] Weigh about 25 mg of C-13 reference substance, accurately weigh it, place it in a 5 mL volumetric flask, add 0.5 mL of C-12 impurity reference substance solution to it, dissolve it with diluent and dilute it to the mark, shake well, and label it as C-13-RS-2.

[0366] (2-4) Sample solution:

[0367] Same as (1-4) in the solution preparation of 5.1.1 in Example 5, and label it as C-13-SPL-2.

[0368] (2-5) Sample control solution (1% self-control solution):

[0369] Same as (1-5) in the solution preparation of 5.1.1 in Example 5, labeled as C-13-SPL-2-1%.

[0370] 5.2.2 Liquid phase conditions

[0371] HPLC, chromatographic column, CAD, and flow rate are the same as in Example 3.

[0372] Mobile phase: Mobile phase A: 0.1% FA in H 2 O (v / v)

[0373] Mobile phase B: MeOH:THF = 8:2 (v / v);

[0374] Sampling tray temperature: Room temperature;

[0375] Column temperature: 60 °C;

[0376] Injection volume: 5 μL;

[0377] Gradient elution program is shown in Table 11 (after 35 min is the column flushing and system equilibration stage):

[0378] Table 11 Gradient elution program

[0379] Time (min) B(％) 0.0 60 2.0 60 25.0 95 35.0 95 35.1 60 45.0 60

[0380] 5.2.3 Experimental results

[0381] The chromatogram of the resolution solution C-13-RS-2 is as Figure 20 shown. Among them, the peak valley ratio of the main peak is 8.3, and the resolution between the known impurity and the adjacent peak ≥ 1.6. The retention times of the main peak and the known impurity, and the LOQ and LOD data of C-13 are shown in Table 12.

[0382] The RSD of the C-13 peak area in the chromatograms of C-13-SPL-2-1% for 3 consecutive injections is 0.2%, and the RSD of the C-13 retention time is 0.0%.

[0383] Table 12 Experimental data of C-13

[0384]

[0385] According to the above detection results, it can be known that this detection method can effectively separate deoxycholic acid intermediate C-13 and impurity C-12. In order to directly separate C-12 and C-13, the secondary HPLC separation and detection method in 5.2 can be directly adopted.

[0386] Example 6 Detection method for deoxycholic acid intermediate C-14

[0387] 6.1 Primary separation detection

[0388] Table 13C-13 to C-14 Related impurities that may be generated during the reaction

[0389]

[0390] 6.1.1 Solution preparation

[0391] (1) Blank solution (diluent):

[0392] MeOH:THF = 1:1: Measure 500 mL of methanol and 500 mL of tetrahydrofuran into a mobile phase bottle and mix well.

[0393] (2) Mobile phase:

[0394] Same as Example 3.

[0395] (3) Resolution solution:

[0396] Take about 5 mg of C-13 impurity reference substance, accurately weigh it and place it in a 10 mL volumetric flask. Dissolve it with diluent and dilute to the mark, shake well, and label it as C13 impurity reference substance. Prepare C-24 impurity reference substance, C-25 impurity reference substance, C-26 impurity reference substance, C-27 impurity reference substance, and C-28 impurity reference substance in the same way.

[0397] Weigh about 25 mg of C-14 reference substance, accurately weigh it, and in a 5 mL volumetric flask, respectively measure 0.5 mL of each of the solutions of C13 impurity reference substance, C-22 impurity reference substance, C-24 impurity reference substance, C-25 impurity reference substance, C-26 impurity reference substance, C-27 impurity reference substance, and C-28 impurity reference substance and add them thereto. Dissolve with diluent and dilute to the mark, shake well, and label it as C-14-RS-1.

[0398] (4) Sample solution:

[0399] Take about 25 mg of C-14 sample, accurately weigh it, place it in a 5 mL volumetric flask, dissolve it with diluent and dilute to the mark, shake well, and label it as C-14-SPL-1.

[0400] (5) Sample control solution (1% self-control solution):

[0401] Accurately measure 1 mL of C-14-SPL-1 solution into a 100 mL volumetric flask, dilute to the mark with diluent, shake well, and label it as C-14-SPL-1-1%.

[0402] 6.1.2 Liquid phase conditions

[0403] The HPLC, chromatographic column, CAD, mobile phase, gradient elution program, flow rate, and column temperature were the same as in Example 3.

[0404] Sample tray temperature: 5 °C;

[0405] Sample volume: 5 μL.

[0406] 6.1.3 Experimental results

[0407] The chromatogram of Resolution Solution C-14-RS-1 is as Figure 21 shown, where the resolution between the main peak and the adjacent peak is ≥2.6, and the resolution between the known impurity and the adjacent peak is ≥1.7. The retention times of the main peak and the known impurity, and the LOQ and LOD data of C-14 are shown in Table 14.

[0408] In the chromatograms of C-14-SPL-1-1% for 3 consecutive injections, the RSD of the C-14 peak area was 0.3%, and the RSD of the C-14 retention time was 0.0%.

[0409] Table 14 Experimental data of C-14

[0410]

[0411] The chromatographic system and gradient elution program for deoxycholic acid intermediate C-12 were the same as those for the first separation and detection of C-14. The chromatogram of C-12-RS was superimposed on the chromatogram of C-14-RS-1, as Figure 22 shown. It can be seen that when C-14 was separated and detected once, deoxycholic acid intermediates C-14 and impurities C-10, C-11, C-12, and C-13 could be effectively separated. Among them, C-12 and C-13 were co-eluted, which further indicated that the detection method of the present application could achieve the separation and localization of key intermediates and had continuity.

[0412] 6.2 Second separation and detection

[0413] 6.2.1 Solution preparation

[0414] (2-1) Blank solution (diluent):

[0415] Same as (1-1) in Solution Preparation in 5.1.1 of Example 5.

[0416] (2-2) Mobile phase:

[0417] Same as (2-2) in Solution Preparation in 5.2.1 of Example 5.

[0418] (2-3) Resolution solution:

[0419] Take about 5 mg of the C-13 impurity reference substance, accurately weigh it, place it in a 10 mL volumetric flask, dissolve it with the diluent and dilute it to the mark, shake well, and label it as the C-13 impurity reference substance; prepare the C-27 impurity reference substance in the same way.

[0420] Weigh about 50 mg of the C-14 reference substance, accurately weigh it, place it in a 10 mL volumetric flask, add 1 mL each of the C-13 impurity reference substance solution and the C-27 impurity reference substance solution thereto, dissolve it with the diluent and dilute it to the mark, shake well, and label it as C-14-RS-2.

[0421] (2-4) Sample solution:

[0422] Same as (1-4) in the solution preparation of 6.1.1 in Example 6, and label it as C-14-SPL-2.

[0423] (2-5) Sample control solution (1% self-control solution):

[0424] Same as (1-5) in the solution preparation of 6.1.1 in Example 6, and label it as C-14-SPL-2-1%.

[0425] 6.2.2 Liquid phase conditions

[0426] The HPLC, chromatographic column, CAD, injection tray temperature, column temperature, flow rate, injection volume, mobile phase, and gradient elution program are the same as those in the liquid phase conditions of 5.2.2 in Example 5.

[0427] 6.2.3 Experimental results

[0428] The chromatogram of the resolution solution C-14-RS-2 is as Figure 23 shown, in which the minimum resolution between the main peak and the adjacent peak is 1.9, and the minimum resolution between the known impurity and the adjacent peak is 1.9. The retention times of the main peak and the known impurity, and the LOQ and LOD data of C-14 are shown in Table 15.

[0429] The RSD of the C-14 peak area in the chromatograms of C-14-SPL-2-1% for 3 consecutive injections is 0.9%, and the RSD of the C-14 retention time is 0.0%.

[0430] Table 15 Experimental data of C-14

[0431]

[0432] According to the above detection results, it can be known that this detection method can effectively separate the deoxycholic acid intermediate C-14 and the impurities C-13 and C-27. In order to directly separate the key intermediates C-13 and C-14, the secondary HPLC separation and detection method in 6.2 can be directly adopted.

[0433] In summary, the detection method of the deoxycholic acid intermediate of the present application can effectively detect the target substance and separate related impurities, thereby effectively controlling the quality of deoxycholic acid products. Moreover, this method fully meets the acceptance criteria and performs well in terms of specificity, linearity, range, accuracy, precision, detection limit, quantitation limit, durability, solution stability, system precision, system suitability, etc.

[0434] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some or all of the technical features. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting a deoxycholic acid intermediate, the detection method comprising the following steps: dissolving the deoxycholic acid intermediate in a diluent to obtain a sample solution, and detecting the sample solution by high performance liquid chromatography (HPLC). The deoxycholic acid intermediate is selected from the compounds represented by the following formula: Wherein, P is a hydroxyl protecting group, preferably any one of acetyl, C1-C3 alkyl or benzoyl; R is C1-C6 alkyl, preferably C1-C3 alkyl, more preferably methyl or ethyl; Preferably, the deoxycholic acid intermediate is selected from the compounds represented by the following formula:

2. According to the detection method described in claim 1, It is characterized in that, In the HPLC method, the stationary phase of the chromatographic column is octadecylsilyl-bonded silica gel.

3. According to the detection method described in claim 1, It is characterized in that, In the HPLC method, the mobile phase comprises mobile phase A and mobile phase B; The mobile phase A is an aqueous formic acid solution with a volume percentage concentration of 0.01-5%, preferably 0.05-2%, more preferably 0.1%; The mobile phase B is a formic acid acetonitrile solution with a volume percentage concentration of 0.01-5%, preferably 0.05-2%, more preferably 0.1%; Preferably, in the HPLC method, a gradient elution program is adopted, and the gradient elution program is as follows: t = 0 min, the volume percentage of mobile phase B is 20-30%; 0 < t ≤ 2 min, the volume percentage of mobile phase B is 40-50%; 2 < t ≤ 14 min, the volume percentage of mobile phase B is 53-63%; 14 < t ≤ 24 min, the volume percentage of mobile phase B is 90-99%; 24 < t ≤ 35 min, the volume percentage of mobile phase B is 90-99%; More preferably, the gradient elution program is as follows: t = 0 min, the volume percentage of mobile phase B is 25%; 0 < t ≤ 2 min, the volume percentage of mobile phase B is 45%; 2 < t ≤ 14 min, the volume percentage of mobile phase B is 58%; 14 < t ≤ 24 min, the volume percentage of mobile phase B is 95%; 24 < t ≤ 35 min, the volume percentage of mobile phase B is 95%.

4. According to the detection method described in claim 1, It is characterized in that, The diluent is selected from one or more of acetonitrile, methanol, ethanol, tetrahydrofuran, triphenylphosphine, formic acid, water; Preferably, the deoxycholic acid intermediate is preferably The diluent is a 0.01 - 2 g / L triphenylphosphine acetonitrile solution, preferably a 0.05 - 0.5 g / L triphenylphosphine acetonitrile solution, and more preferably a 0.1 g / L triphenylphosphine acetonitrile solution; Preferably, the deoxycholic acid intermediate is preferably The diluent is acetonitrile; Preferably, the deoxycholic acid intermediate is preferably The diluent is a mixed solution of methanol and tetrahydrofuran with a volume ratio of 0.1 - 10:1, preferably a mixed solution of methanol and tetrahydrofuran with a volume ratio of 0.5 - 5:1, and more preferably a mixed solution of methanol and tetrahydrofuran with a volume ratio of 1:1; Preferably, the deoxycholic acid intermediate is preferably The diluent is a mixed solution of methanol and tetrahydrofuran with a volume ratio of 0.1 - 10:1, preferably a mixed solution of methanol and tetrahydrofuran with a volume ratio of 0.5 - 5:1, and more preferably a mixed solution of methanol and tetrahydrofuran with a volume ratio of 1:

1.

5. According to the detection method described in any one of claims 1-4, It is characterized in that, The deoxycholic acid intermediate is In the HPLC method described above, secondary separation and detection are also included; Preferably, the deoxycholic acid intermediate is Preferably, in the secondary separation detection, the stationary phase of the chromatographic column is octadecylsilyl-bonded silica gel; The mobile phase comprises mobile phase A and mobile phase B; The mobile phase A is an aqueous formic acid solution with a volume percentage concentration of 0.01-5%, preferably 0.05-2%, more preferably 0.1%; The mobile phase B is a mixed solution of methanol and tetrahydrofuran with a volume ratio of 1-10:1, preferably 3-5:1, more preferably 4:1; A gradient elution program is adopted, and the gradient elution program is as follows: t = 0 min, the volume percentage of mobile phase B is 50-70%; 0 <t≤2min,流动相B的体积百分比为50-70%; 2 <t≤25min,流动相B的体积百分比为90-99%; 25 <t≤35min,流动相B的体积百分比为90-99%; Preferably, the gradient elution procedure is as follows: t = 0 min, the volume percentage of mobile phase B is 60%; 0 <t≤2min,流动相B的体积百分比为60%; 2 <t≤25min,流动相B的体积百分比为95%; 25 <t≤35min,流动相B的体积百分比为95%。 6. A method for detecting a deoxycholic acid intermediate, the method comprising the following steps: dissolving the deoxycholic acid intermediate in a diluent to obtain a sample solution, and detecting the sample solution by HPLC; The deoxycholic acid intermediate is a compound shown in the following formula: in, P is a hydroxyl protecting group, preferably any one selected from acetyl, C1-C3 alkyl or benzoyl; Preferably, the deoxycholic acid intermediate is a compound represented by the following formula:

7. The detection method according to claim 6, It is characterized in that The diluent is selected from one or more of acetonitrile, methanol, ethanol, tetrahydrofuran, triphenylphosphine, formic acid and water; Preferably, the diluent is acetonitrile.

8. The detection method according to claim 6, It is characterized in that In the HPLC method, the stationary phase of the chromatographic column is phenylhexyl bonded silica gel.

9. The detection method according to claim 6, It is characterized in that In the HPLC method, the mobile phase includes mobile phase A and mobile phase B; The mobile phase A is a formic acid aqueous solution with a volume percentage concentration of 0.01-5%, preferably 0.05-2%, more preferably 0.1%; The mobile phase B is a mixed solution of acetonitrile and methanol in a volume ratio of 1-20:1, preferably 5-15:1, more preferably 9:1; Preferably, in the HPLC method, a gradient elution program is used, and the gradient elution program is as follows: t = 0 min, the volume percentage of mobile phase B is 35-45%; 0 <t≤2min,流动相B的体积百分比为35-45%; 2 <t≤25min,流动相B的体积百分比为65-75%; 25 <t≤35min,流动相B的体积百分比为85-95%; 35 <t≤40min,流动相B的体积百分比为85-95%; More preferably, the gradient elution procedure is as follows: t = 0 min, the volume percentage of mobile phase B is 40%; 0 <t≤2min,流动相B的体积百分比为40%; 2 <t≤25min,流动相B的体积百分比为70%; 25 <t≤35min,流动相B的体积百分比为90%; 35 <t≤40min,流动相B的体积百分比为90%。 10. Use of the detection method according to any one of claims 1 to 9 in the quality control or quality evaluation of deoxycholic acid intermediates, or the quality control or quality evaluation of the preparation process of deoxycholic acid.

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