Detection method of bear gall powder or preparation thereof and construction method of fingerprint spectrum

Abstract

The invention relates to the field of traditional Chinese medicine detection, in particular to a detection method of bear gall powder or a preparation thereof and a construction method of a fingerprint spectrum. The detection method provided by the invention can accurately analyze the bear gall powder or the preparation thereof and construct the fingerprint spectrum, not only can accurately identify the quality and authenticity of different batches of bear gall powder, but also can ensure the uniform and stable quality of the bear gall powder preparation. Experiments show that the fingerprint spectrums of the bear gall powder and the bear gall eye drops are successfully constructed, the relative retention time RSD of all common peaks in the obtained fingerprint spectrums of the bear gall powder is 0.02%-0.15%, and the relative peak area RSD of all the common peaks is 0.48%-2.62%; the relative retention time RSD of each common peak in the obtained fingerprint of the bear gall eye drops is 0.01%-0.21%, and the relative peak area RSD of each common peak is 0.11%-9.72%; the fingerprint detection requirements of the bear gall powder and the preparation thereof can be met.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine testing, specifically a method for detecting bear bile powder or its preparations and a method for constructing fingerprint spectra. Background Technology

[0002] Bear bile eye drops have the effects of clearing heat and detoxifying, removing pterygium and improving vision. They are an ophthalmic preparation made from bear bile powder through extraction and formulation. Bear bile powder is a dried product obtained from the bile drained by gallbladder surgery of the black bear (Selenaretos thibetanus Cuvier). It has the effects of clearing heat and detoxifying, calming the liver and improving vision, killing parasites and stopping bleeding. Clinically, it is often used to treat liver, gallbladder, and ophthalmic diseases. Its main components are bile acids, primarily taurine-bound bile acids, mainly tauroursodeoxycholic acid (TUDCA) and taurourchenodeoxycholic acid (TCDCA). It also contains free ursodeoxycholic acid and chenodeoxycholic acid, as well as small amounts of cholic acid and deoxycholic acid. Its characteristic component, TUDCA, can cross the blood-vitreous barrier to reach the aqueous humor and vitreous humor, reaching the corresponding target tissues. It has good therapeutic effects on lens opacity, vitreous opacity, fundus hemorrhage, and retrobulbar optic neuritis. Bear bile powder is included in the standards issued by the Ministry of Health of the People's Republic of China, standard number: WS3-09(B-09)-96(Z).

[0003] The composition of bear bile powder varies due to numerous factors, including climate, season, bear habits, diet, physical condition, drainage methods, and cycles. Furthermore, the formulation of bear bile powder preparations is subject to frequent variations, making quality comparisons before and after these changes extremely important. The quality standard for bear bile powder (standard number: WS3-09(B-09)-96(Z)) includes quantitative studies on tauroursodeoxycholic acid and qualitative studies on ursodeoxycholic acid and chenodeoxycholic acid; other components are not included. To better ensure the stability of the preparation's quality and reduce batch-to-batch variations in bear bile eye drops, it is urgent to strengthen the quality control of bear bile powder, increase research on its fingerprint spectrum, and establish internal quality control standards. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is a detection method for bear bile powder or its preparations and the construction of a fingerprint spectrum. The detection method provided by the present invention can accurately analyze bear bile powder or its preparations and construct a fingerprint spectrum, ensuring the uniformity and stability of the quality of bear bile powder preparations.

[0005] This invention provides a method for detecting bear bile powder or its preparations, comprising:

[0006] The test solution of bear bile powder or its preparation was detected by HPLC. The chromatographic conditions for HPLC were as follows:

[0007] Octadecylsilane-bonded silica gel was used as the stationary phase;

[0008] Acetonitrile was used as the mobile phase A, and a mixed solution of sodium dihydrogen phosphate and methanol with a volume ratio of (89-91):(9-11) was used as the mobile phase B.

[0009] In the HPLC chromatographic conditions described in this invention, the gradient elution program is as follows:

[0010]

[0011]

[0012] In one embodiment of the present invention, the gradient elution procedure is as follows:

[0013] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 0 100 60 60 40 70 60 40 75 0 100 90 0 100

[0014] In the HPLC chromatographic conditions described in this invention, octadecylsilane-bonded silica gel is used as the stationary phase. In some embodiments of this invention, the stationary phase is Welch Ultisil. TM C 18 Column or Welch Ultimate XB-C 18 Chromatographic column. In some embodiments of the present invention, the column temperature of the stationary phase is 30°C to 40°C.

[0015] In the HPLC chromatographic conditions described in this invention, acetonitrile is used as mobile phase A, and a mixed solution of sodium dihydrogen phosphate and methanol with a volume ratio of (89-91):(9-11) is used as mobile phase B, preferably a mixed solution of sodium dihydrogen phosphate and methanol with a volume ratio of 90:10. In mobile phase B, the concentration of sodium dihydrogen phosphate in the sodium dihydrogen phosphate solution is 0.025 mol / L to 0.035 mol / L, preferably 0.030 mol / L; the pH of the sodium dihydrogen phosphate solution is 4.2-4.6, preferably 4.4. In some embodiments of this invention, the sodium dihydrogen phosphate solution is a phosphoric acid-containing sodium dihydrogen phosphate solution, and the phosphoric acid is used to adjust the pH of the sodium dihydrogen phosphate solution.

[0016] In the HPLC chromatographic conditions described in this invention, the flow rate of the mobile phase is 0.5 mL / min to 1 mL / min, preferably 0.8 mL / min. The detection wavelength of the HPLC described in this invention is 210 nm to 220 nm. When using HPLC for detection in this invention, the injection volume of the test solution is 5 μL to 15 μL.

[0017] This invention does not impose any particular limitation on the instrument used for the HPLC. In some embodiments of this invention, the HPLC instrument is an Agilent 1100 high-performance liquid chromatograph or a Shimadzu LC-2030CPLUS high-performance liquid chromatograph.

[0018] This invention relates to the detection of test solutions of bear bile powder or its preparations, wherein the bear bile powder is selected from the bile powder of the black bear (Selenaretos thibetanus Cuvier), and the preparations include eye drops, decoction pieces, soup, or granules. In some embodiments of this invention, the bear bile powder preparation is a multi-dose bear bile eye drop, comprising bear bile powder, boric acid, borax, sodium chloride, and ethylparaben. In other embodiments of this invention, the bear bile powder preparation is a single-dose bear bile eye drop, comprising bear bile powder, boric acid, borax, and sodium chloride.

[0019] The test solution of bear bile powder or its preparations according to the present invention is prepared by the following method: dissolving the bear bile powder or its preparations in water as an extraction solvent, filtering the resulting solution using an aqueous filter membrane to obtain the test solution of the bear bile powder or its preparations. The aqueous filter membrane of the present invention is selected from polyethersulfone filter membranes. In some embodiments of the present invention, the extraction time is 5 min to 30 min.

[0020] The detection method for bear bile powder or its preparations provided in this application uses the above-mentioned mobile phase A and mobile phase B as binary mobile phases to perform gradient elution HPLC detection on the test solution of bear bile powder or its preparations. This method can accurately analyze bear bile powder or its preparations and can construct a fingerprint spectrum that meets the detection requirements.

[0021] This invention also provides a method for constructing a fingerprint spectrum of bear bile powder or its preparations, comprising the following steps:

[0022] S1) Detect different batches of bear bile powder or its preparations according to the detection method described in any of the above technical solutions;

[0023] S2) Identify the common peak;

[0024] S3) The fingerprint spectrum of bear bile powder or its preparations was established using the median method.

[0025] Specifically, this invention uses the detection method described in any of the above technical solutions to detect test solutions of different batches of bear bile powder or its preparations, identifies the common peaks in each HPLC chromatogram, selects a reference peak, and establishes a fingerprint chromatogram of bear bile powder or its preparations using the median method in the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version). The bear bile powder or its preparations described in this invention are the same as described above and will not be repeated. The preparation method of the test solution described in this invention is the same as described above and will not be repeated.

[0026] This invention establishes a fingerprint spectrum of bear bile powder or its preparations using sodium tauroursodeoxycholate as a reference peak. The relative retention time of sodium tauroursodeoxycholate is 1.000. The fingerprint spectrum includes at least six common peaks, and the relative retention times of the common peaks are 0.629–0.650, 0.814–0.830, 0.845–0.860, 0.971–0.990, 1.170–1.180, and 1.747–1.800, respectively.

[0027] In one embodiment of the present invention, test solutions of different batches of bear bile powder medicinal materials are detected according to the detection method described in any of the above technical solutions. A fingerprint spectrum of bear bile eye drops is established with sodium tauroursodeoxycholate as a reference peak. The relative retention time of sodium tauroursodeoxycholate is 1.000. The fingerprint spectrum includes at least 6 common peaks, and the relative retention times of the common peaks are 0.630-0.649, 0.815-0.829, 0.846-0.857, 0.972-0.982, 1.171-1.180, and 1.747-1.782, respectively.

[0028] In one embodiment of the present invention, test solutions of different batches of multi-dose bear bile eye drops are detected according to the detection method described in any of the above technical solutions. A fingerprint spectrum of bear bile eye drops is established using sodium tauroursodeoxycholate as a reference peak. The relative retention time of sodium tauroursodeoxycholate is 1.000. The fingerprint spectrum includes at least 8 common peaks, and the relative retention times of the common peaks are 0.453–0.482, 0.495–0.521, 0.629–0.649, 0.814–0.828, 0.845–0.856, 0.971–0.981, 1.170–1.178, and 1.748–1.781, respectively.

[0029] In one embodiment of the present invention, test solutions of single-dose bear bile eye drops from different batches are detected according to the detection method described in any of the above technical solutions. A fingerprint spectrum of bear bile eye drops is established using sodium tauroursodeoxycholate as a reference peak. The relative retention time of sodium tauroursodeoxycholate is 1.000. The fingerprint spectrum includes at least 6 common peaks, and the relative retention times of the common peaks are 0.630–0.650, 0.820–0.830, 0.850–0.860, 0.970–0.990, 1.170–1.180, and 1.750–1.800, respectively.

[0030] This invention provides a method for detecting bear bile powder or its preparations and a method for constructing fingerprint chromatograms. The detection method provided by this invention can accurately analyze bear bile powder or its preparations and construct fingerprint chromatograms, not only accurately identifying the quality and authenticity of different batches of bear bile powder, but also ensuring the uniformity and stability of the quality of bear bile powder preparations. Experiments show that this invention successfully explored the optimal chromatographic conditions for the HPLC detection method of bear bile powder or its preparations, and based on these chromatographic conditions, detected bear bile powder and bear bile eye drops, successfully constructing fingerprint chromatograms for both. The RSD of the relative retention time of each common peak in the obtained fingerprint chromatogram of bear bile powder is 0.02%–0.15%, and the RSD of the relative peak area of ​​each common peak is 0.48%–2.62%; the RSD of the relative retention time of each common peak in the obtained fingerprint chromatogram of bear bile eye drops is 0.01%–0.21%, and the RSD of the relative peak area of ​​each common peak is 0.11%–9.72%, meeting the requirements for fingerprint chromatogram detection of bear bile powder and its preparations. Attached Figure Description

[0031] Figure 1 HPLC chromatogram of bear bile powder;

[0032] Figure 2 The 203nm chromatogram of bear bile powder;

[0033] Figure 3 The 210nm chromatogram of bear bile powder;

[0034] Figure 4 The 230nm spectra of bear bile powder;

[0035] Figure 5 The 254nm spectra of bear bile powder;

[0036] Figure 6 Superimposed chromatograms for comparing the precision similarity of bear bile powder sample injection;

[0037] Figure 7 A superimposed spectrum comparing the similarity of repeatability tests of bear bile powder medicinal materials;

[0038] Figure 8 Superimposed spectrum for comparison of similarity in intermediate precision tests of bear bile powder medicinal materials;

[0039] Figure 9 This is a superimposed spectrum comparing the similarity of bear bile powder in stability tests.

[0040] Figure 10 Superimposed graph of similarity comparison of bear bile powder medicinal materials across a range of tests;

[0041] Figure 11 A superimposed spectrum comparing the similarity of fine-tuned pH values ​​in the durability test of bear bile powder medicinal materials;

[0042] Figure 12 A superimposed spectrum comparing the similarity of the mobile phase ratio for a durability test of bear bile powder medicinal materials.

[0043] Figure 13 A superimposed spectrum comparing the similarity of fine-tuned salt concentrations in a durability test of bear bile powder medicinal materials.

[0044] Figure 14 Superimposed chromatograms comparing the similarity of chromatographic columns from different batches of bear bile powder in a durability test of the medicinal material.

[0045] Figure 15 A superimposed spectrum comparing the similarity of different batches of bear bile powder.

[0046] Figure 16 Fingerprint spectrum of bear bile powder as a reference material;

[0047] Figure 17 A superimposed graph comparing the similarity of bear bile powder from different origins;

[0048] Figure 18 Overlay of similarity graphs for bear bile powder medicinal materials from different harvesting seasons;

[0049] Figure 19 The HPLC-ELSD chromatogram of blank excipients for the detection of multi-dose bear bile eye drops;

[0050] Figure 20 HPLC-ELSD method for detecting sodium tauroursodeoxycholate in multi-dose bear bile eye drops;

[0051] Figure 21 The HPLC-ELSD method is used to detect the chromatogram of bear bile eye drops in multi-dose bear bile eye drops.

[0052] Figure 22 The chromatogram of the test sample for multi-dose bear bile eye drops by HPLC-UV method;

[0053] Figure 23 Fingerprint spectroscopy of multi-dose bear bile eye drops;

[0054] Figure 24 The chromatogram of the test solution of multi-dose bear bile eye drops at a flow rate of 1.2 mL / min is shown.

[0055] Figure 25 The chromatogram of the test solution of multi-dose bear bile eye drops at a flow rate of 1.0 mL / min is shown.

[0056] Figure 26 The chromatogram of the test solution of multi-dose bear bile eye drops at a flow rate of 0.8 mL / min is shown.

[0057] Figure 27The chromatogram of the test solution of multi-dose bear bile eye drops at 203 nm;

[0058] Figure 28 The chromatogram of the test solution of multi-dose bear bile eye drops at 210 nm;

[0059] Figure 29 The chromatogram of the test solution of multi-dose bear bile eye drops at 230 nm;

[0060] Figure 30 The chromatogram of the test solution of multi-dose bear bile eye drops at 254 nm;

[0061] Figure 31 To adopt Agilent ZORBAX SB-C 18 Chromatogram of the multi-dose bear bile eye drops test solution on a chromatographic column;

[0062] Figure 32 To use Kromasil C 18 Chromatogram of the multi-dose bear bile eye drops test solution on a chromatographic column;

[0063] Figure 33 To use Diamonsil C 18 Chromatogram of the multi-dose bear bile eye drops test solution on a chromatographic column;

[0064] Figure 34 To use Welch Ultimate LP-C 18 Chromatogram of the multi-dose bear bile eye drops test solution on a chromatographic column;

[0065] Figure 35 To adopt Welch Ultisil TM C 18 Chromatogram of the multi-dose bear bile eye drops test solution on a chromatographic column;

[0066] Figure 36 To adopt Welch Ultimate XB-C 18 Chromatogram of the multi-dose bear bile eye drops test solution on a chromatographic column;

[0067] Figure 37 Superimposed graphs for comparing the similarity of multiple dose bear bile eye drops range tests;

[0068] Figure 38 Superimposed spectral data for comparing the precision similarity of multiple doses of bear bile eye drops injection;

[0069] Figure 39 A superimposed graph comparing the similarity of repeatability tests of multi-dose bear bile eye drops;

[0070] Figure 40A superimposed graph comparing the similarity of intermediate precision tests of multi-dose bear bile eye drops;

[0071] Figure 41 Superimposed graphs showing the similarity of multiple-dose bear bile eye drops test solutions in stability testing.

[0072] Figure 42 Superimposed graphs comparing the similarity of pH values ​​in a durability test of multi-dose bear bile eye drops.

[0073] Figure 43 Superimposed graphs showing the similarity of fine-tuned mobile phase ratios in a durability test of multi-dose bear bile eye drops.

[0074] Figure 44 Superimposed graphs showing the similarity of fine-tuned salt concentrations in a durability test of multi-dose bear bile eye drops.

[0075] Figure 45 Superimposed chromatograms comparing the similarity of chromatographic columns from different batches in a durability test of multi-dose bear bile eye drops;

[0076] Figure 46 A superimposed spectrum for similarity comparison of multiple doses of bear bile eye drops;

[0077] Figure 47 Superimposed spectral data for comparing the precision similarity of single-dose bear bile eye drops injection;

[0078] Figure 48 A superimposed graph comparing the similarity of repeatability tests of single-dose bear bile eye drops;

[0079] Figure 49 Superimposed graphs showing the similarity of intermediate precision tests for single-dose bear bile eye drops;

[0080] Figure 50 Superimposed graphs showing the similarity of single-dose bear bile eye drops test solution stability test samples.

[0081] Figure 51 A superimposed graph comparing the similarity of single-dose bear bile eye drops range tests;

[0082] Figure 52 Superimposed graphs comparing the similarity of pH values ​​fine-tuned in the durability test of single-dose bear bile eye drops;

[0083] Figure 53 Superimposed graphs showing the similarity of fine-tuned salt concentrations in a single-dose bear bile eye drop durability test.

[0084] Figure 54 Superimposed graphs showing the similarity of fine-tuned mobile phase ratios in a durability test of single-dose bear bile eye drops.

[0085] Figure 55Superimposed chromatograms comparing the similarity of chromatographic columns from different batches in the durability test of single-dose bear bile eye drops.

[0086] Figure 56 A superimposed graph showing the similarity of different batches of single-dose bear bile eye drops.

[0087] Figure 57 The fingerprint spectrum of reference solution for batch number 20240620 was constructed using the ternary mobile phase method;

[0088] Figure 58 The fingerprint spectrum of the test sample solution with batch number 02211102 was constructed using the ternary mobile phase method;

[0089] Figure 59 The fingerprint spectrum of the test sample solution of batch number 02211104 was constructed using the ternary mobile phase method;

[0090] Figure 60 Superimposed comparative spectra of test sample solutions with batch numbers 02230801, 02211102 and 02211104 constructed using the ternary mobile phase method;

[0091] Figure 61 The fingerprint spectrum of the reference solution for batch number 20211203 was constructed using the binary mobile phase method;

[0092] Figure 62 The fingerprint spectrum of the test sample solution with batch number 02211102 was constructed using the binary mobile phase method;

[0093] Figure 63 The fingerprint spectrum of the test sample solution of batch number 02211104 was constructed using the binary mobile phase method;

[0094] Figure 64 To compare the fingerprint spectrum using different system-reference solutions;

[0095] Figure 65 Comparison of fingerprint spectra of different systems and test sample solutions (batch number 02211102);

[0096] Figure 66 This is a comparison of fingerprint spectra of different systems and test solutions (batch number 02211104). Detailed Implementation

[0097] This invention discloses a method for detecting bear bile powder or its preparations and a method for constructing a fingerprint spectrum. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0098] The present invention will be further described below with reference to the embodiments:

[0099] Example 1

[0100] Establishment and validation of fingerprint analysis method for bear bile powder: The research object of this embodiment is bear bile powder.

[0101] (1) Establishment of analytical methods

[0102] 1. Experimental Instruments and Materials

[0103] 1.1 Sample and Reference Standard

[0104] As shown in Table 1:

[0105] Table 1

[0106]

[0107] 1.2 Reagents

[0108] As shown in Table 2:

[0109] Table 2

[0110] name level source methanol HPLC BCL Sodium dihydrogen phosphate AR Chengdu Jinshan Chemical Reagent Co., Ltd. Phosphoric acid AR Chengdu Kelong Chemical Reagent Factory / Chengdu Jinshan Chemical Reagent Co., Ltd. Acetonitrile HPLC BCL

[0111] 1.3 Instruments

[0112] As shown in Table 3:

[0113] Table 3

[0114] Instrument Name model source High Performance Liquid Chromatography Agilent 1100 Agilent Electronic balance CP225D Sartorius (Shanghai) Trading Co., Ltd. Electronic balance HZT-A+300 Fuzhou Huazhi Scientific Instruments Co., Ltd. pH meter PB-10 Sartorius (Shanghai) Trading Co., Ltd.

[0115] 1.4 Chromatographic conditions

[0116] 1.4.1 Chromatographic conditions and system suitability test

[0117] Welch Ultimate XB-C uses octadecylsilane-bonded silica gel as a filler. 18The chromatographic column (15 cm long, 4.6 mm inner diameter, 5 μm particle size) was used. Acetonitrile was used as mobile phase A, and 0.03 mol / L sodium dihydrogen phosphate solution (adjusted to pH 4.4 with phosphoric acid)-methanol (90:10 v / v) was used as mobile phase B. Gradient elution was performed according to the specifications in Table 4. The detection wavelength was 210 nm; the column temperature was 35 °C; and the flow rate was 0.8 mL / min. The theoretical plate number, calculated based on the sodium tauroursodeoxycholate peak, should be no less than 2500.

[0118] Table 4

[0119] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 0 100 60 60 40 70 60 40 75 0 100 90 0 100

[0120] 1.4.2 Adaptability of the Method

[0121] Preparation of reference solution: Take an appropriate amount of sodium tauroursodeoxycholate (batch number: 110816-201509), accurately weigh it, and add methanol to prepare a solution containing about 1 mg of sodium tauroursodeoxycholate per 1 mL.

[0122] Preparation of the test solution: Take about 25 mg of bear bile powder (batch number: 201610-1), place it in a 5 mL volumetric flask, add water to dissolve by sonication and dilute to the mark, shake well, filter, and take the filtrate.

[0123] Chromatographic peak integration parameters: No integration is performed in the first 5 minutes, and the minimum peak area is no less than 1.0% of the total peak area.

[0124] Take 10 μL each of the reference solution and the test solution, inject them into the liquid chromatograph, determine the chromatogram, and record the result. Figure 1 As shown, Figure 1 This is the HPLC chromatogram of bear bile powder. The chromatogram shows that, after deducting the solvent blank peak, the retention time of sodium tauroursodeoxycholate in the bear bile powder test solution is 36.208 minutes, with a theoretical plate number of 77948. A total of 9 absorption peaks were detected in the system. The solvent peak and gradient peaks did not interfere with the detection, indicating that this method can be used for fingerprint chromatographic studies of bear bile powder.

[0125] 1.4.3 Selection of detection wavelength

[0126] Take approximately 25 mg of bear bile powder (batch number: 201610-1), place it in a 5 mL volumetric flask, add water, sonicate to dissolve and dilute to the mark, shake well, filter, and use the filtrate as the test solution. Accurately measure 10 μL and inject it into the liquid chromatograph. Detect using a diode array detector (DAD), record the chromatogram, and compare the peak area and number of peaks of sodium tauroursodeoxycholate in the test sample as evaluation indicators. Statistically analyze the data at wavelengths of 203 nm, 210 nm, 230 nm, and 254 nm. The results are shown in Table 5 and... Figures 2-5 As shown. Figure 2The 203nm chromatogram of bear bile powder; Figure 3 The 210nm chromatogram of bear bile powder; Figure 4 The 230nm spectra of bear bile powder; Figure 5 The 254nm spectra of bear bile powder;

[0127] Table 5

[0128] Wavelength (nm) 203 210 230 254 Number of detection peaks 6 9 21 15 Sodium tauroursodeoxycholate 7153731 2213780 122317 -- Total peak area 14101489 4985502 1020676 1064264

[0129] From Table 5 and Figures 2-5 The results showed that the absorption of each chromatographic peak was stronger closer to the lower wavelength, and vice versa. Based on a comprehensive analysis of the number of chromatographic peaks, absorption intensity, and full-wavelength scan chromatograms, 210 nm was selected as the detection wavelength for bear bile powder fingerprint chromatograms, using the same method as the finished product fingerprint chromatogram.

[0130] 1.5 Screening of test sample solution preparation methods

[0131] 1.5.1 Selection of Extraction Solvent

[0132] Since bear bile eye drops are obtained by dissolving bear bile powder in water and then purifying and preparing them, water is preferred as the extraction solvent for fingerprint analysis of bear bile powder to facilitate comparison with the formulation.

[0133] 1.5.2 Examination of extraction time

[0134] Approximately 50 mg of finely powdered bear bile (batch number: 201610-1) was divided into four portions and placed in separate 10 mL volumetric flasks. Appropriate amounts of water were added, and the flasks were ultrasonically extracted for 5 min, 10 min, 20 min, and 30 min, respectively. The extracts were cooled, diluted to the mark with water, shaken well, filtered, and the filtrates were used as the test solutions. 10 μL of each solution was accurately measured and injected into the liquid chromatograph for analysis. The chromatograms were recorded and compared. The sodium tauroursodeoxycholate peak in the test solution was used as the reference peak. The ratio of the peak area of ​​the reference peak to the sample amount (mg) (A / M) and the number of detected peaks were used as evaluation indicators. The results are shown in Table 6.

[0135] Table 6

[0136] Time (min) 5 10 20 30 RSD (%) Number of detection peaks 9 9 9 9 / A / M 41258.0 40860.5 41271.4 41175.4 0.47

[0137] As shown in Table 6, there was no significant difference in the number of absorption peaks and A / M ratio in the test solution for different ultrasonic times, indicating that ultrasonic time has no significant effect on extraction. It is tentatively determined that ultrasonic extraction with water for 5 minutes is sufficient for this product.

[0138] 1.5.3 Selection of Filter Membrane

[0139] Take the test solution from test item "1.5.2" and sonicate it for 5 min. Filter it using a water-based membrane (PES) and an organic membrane (Nylon), respectively. Collect the filtrate as the test solution. Accurately measure 10 μL of each test solution and inject it into the liquid chromatograph. Record the chromatogram. The total absorption peak area and the number of detection peaks of each test solution are used as evaluation indicators. The results are shown in Table 7.

[0140] Table 7

[0141] Filter membrane PES Nylon Number of detection peaks 9 6 Total peak area 4579072 3961930

[0142] As shown in Table 7, the number of peaks detected and the total peak area were both higher when using the PES filter membrane than when using the Nylon filter membrane; therefore, the test solution of this product was filtered using an aqueous filter membrane (PES).

[0143] 1.5.4 Selection of Injection Volume

[0144] Take approximately 25 mg of bear bile powder (batch number: 201610-1), place it in a 5 mL volumetric flask, add water, sonicate to dissolve and dilute to the mark, shake well, filter using an aqueous membrane filter (PES), and use the filtrate as the test solution. Accurately measure 5 μL, 10 μL, and 15 μL of the test solution, inject them into the liquid chromatograph, record the chromatograms, and compare them. The number of detected peaks and the total peak area were used as evaluation indicators. The results are shown in Table 8.

[0145] Table 8

[0146] Injection volume (μL) 5 10 15 Total peak area 2401812 4790844 7187853 Number of detection peaks 9 9 9

[0147] As shown in Table 8, the number of peaks detected is the same for different injection volumes, and the total peak area increases exponentially with the injection volume. This indicates that the detection capability is comparable within the injection volume range of 5 μL to 15 μL. Therefore, 10 μL is temporarily selected as the injection volume for fingerprint chromatograms.

[0148] 1.6 Determination of the fingerprinting method for bear bile powder

[0149] Based on the above experimental results, the tentative method for determining the fingerprint spectrum of bear bile powder is as follows:

[0150] Chromatographic conditions and system suitability test: using octadecylsilane-bonded silica gel as the stationary phase, Welch Ultimate XB-C 18 The chromatographic column (15 cm long, 4.6 mm inner diameter, 5 μm particle size) was used. Acetonitrile was used as mobile phase A, and 0.03 mol / L sodium dihydrogen phosphate solution (adjusted to pH 4.4 with phosphoric acid)-methanol (90:10 v / v) was used as mobile phase B. Gradient elution was performed according to the specifications in the table below. The detection wavelength was 210 nm; the column temperature was 35 °C; and the flow rate was 0.8 mL / min. The theoretical plate number, calculated based on the sodium tauroursodeoxycholate peak, should be no less than 2500.

[0151] Table 9

[0152] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 0 100 60 60 40 70 60 40 75 0 100 90 0 100

[0153] Reference solution: Accurately weigh sodium tauroursodeoxycholate reference standard, add methanol to prepare a solution containing approximately 1 mg of sodium tauroursodeoxycholate per 1 mL.

[0154] Test solution: Weigh approximately 50 mg of bear bile powder accurately, place it in a 10 mL volumetric flask, add water and sonicate for 5 min to dissolve and dilute to the mark, shake well, filter (using 0.45 μm aqueous polyethersulfone PES), discard the initial filtrate, and collect the subsequent filtrate to obtain the test solution.

[0155] Determination method: Accurately pipette 10 μL each of the reference solution and the test solution into the liquid chromatograph, measure and record the chromatogram to obtain the result.

[0156] According to the similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine, the chromatographic peaks after 5 minutes of retention time should be calculated. The similarity between the fingerprint chromatogram of the test sample and the fingerprint chromatogram of the control sample should not be less than 0.90.

[0157] Chromatographic peak integration parameters: No integration is performed in the first 5 minutes, and the minimum peak area is no less than 1.0% of the total peak area.

[0158] (2) Validation of analytical methods

[0159] Table 10 summarizes the validation of fingerprint analysis methods for bear bile powder.

[0160] Table 10

[0161]

[0162]

[0163]

[0164] 2. Method Validation

[0165] 2.1 Specificity

[0166] Specificity will be examined to determine the interference of solvents on fingerprint detection.

[0167] Take an appropriate amount of bear bile powder (batch number: 201610-1) and prepare the reference solution and test solution according to the method determined in “1.6” of (1) above.

[0168] Take 10 μL of each solution and solvent, inject them into the liquid chromatograph, and record the chromatograms. The results show that the solvent has no obvious absorption at the main peak position of the reference solution and does not affect the determination of the bear bile powder fingerprint chromatogram.

[0169] 2.2 Precision Test

[0170] 2.2.1 Instrument precision test

[0171] Take an appropriate amount of bear bile powder (batch number: 201610-1), and prepare the reference solution and test solution according to the method determined in "1.6" of (1) above. Take 10 μL of each solution and inject it repeatedly 6 times. Record the chromatograms. Use the solvent as a blank, subtract the blank, and perform data processing. Use the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" to generate the reference fingerprint chromatogram using the median method. Calculate the similarity between the fingerprint chromatogram of each test sample and the generated reference fingerprint chromatogram. Count the common peaks of each test sample solution and number them. The reference peak is represented by S, and the remaining common peaks are represented by numbers in sequence. Take sodium tauroursodeoxycholate as the reference peak S in each test sample solution, and calculate the relative retention time and relative peak area of ​​each common peak according to formula 1 and formula 2.

[0172]

[0173]

[0174] The results of the reference solution injection precision test are shown in Table 11:

[0175] Table 11

[0176]

[0177] The results of the sample injection precision similarity comparison are as follows: Figure 6 As shown, Figure 6 The superimposed chromatograms show the similarity of the injection precision of bear bile powder medicinal materials; the similarity results of the instrument precision test of the test solution are shown in Table 12.

[0178] Table 12

[0179] serial number 1 2 3 4 5 6 Similarity 1.000 1.000 1.000 1.000 1.000 1.000

[0180] The relative retention times of the test solutions are shown in Table 13:

[0181] Table 13

[0182]

[0183]

[0184] The relative peak areas of the test solution are shown in Table 14:

[0185] Table 14

[0186]

[0187] Conclusions: As shown in Table 11, after six consecutive injections of the reference solution, the RSDs for retention time and main peak area were 0.06% and 0.17%, respectively. As shown in Tables 13 and 14, after six consecutive injections of the test sample solution, the RSDs for retention time and peak area of ​​sodium tauroursodeoxycholate (S peak) were 0.03% and 0.19%, respectively, and the RSD for the total peak area was 0.27%. The relative retention time RSDs of the common peaks in the test sample ranged from 0.02% to 0.15%, and the relative peak area RSDs ranged from 0.48% to 2.62%. As shown in Table 12, the similarity calculated using the chromatographic fingerprint similarity evaluation system for traditional Chinese medicine was 1.000, indicating good instrument injection precision.

[0188] 2.2.2 Repeatability Test

[0189] Take an appropriate amount of bear bile powder (batch number: 201610-1), and prepare a reference solution and 6 test solutions according to the method determined in "1.6" of (1) above. Take 10 μL of each solution and inject it into the liquid chromatograph. Record the chromatogram. Use the solvent as a blank and subtract the blank for data processing. Use the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" to generate a reference fingerprint chromatogram using the median method. Calculate the similarity between the fingerprint chromatogram of each test sample and the generated reference fingerprint chromatogram. Count the common peaks of each test sample solution and number them. The reference peak is represented by S, and the remaining common peaks are represented by numbers in sequence. Take sodium tauroursodeoxycholate as the reference peak S in each test sample solution and calculate the relative retention time and relative peak area of ​​each common peak.

[0190] The similarity results of the repeatability test of each test sample solution are shown in Table 15. Figure 7 Superimposed graph of similarity comparison in repeatability tests of bear bile powder medicinal materials:

[0191] Table 15

[0192] serial number 1 2 3 4 5 6 Similarity (median) 1.000 1.000 1.000 1.000 1.000 1.000

[0193] The relative retention times of the repeatability tests for each test solution are shown in Table 16.

[0194] Table 16

[0195]

[0196] The results of the relative peak areas of the repeatability tests for each test solution are shown in Table 17.

[0197] Table 17

[0198]

[0199]

[0200] Conclusion: As shown in Tables 16 and 17, the relative retention times (RSDs) of the common peaks in the six test solutions ranged from 0.02% to 0.07%, and the relative peak areas (RSDs) ranged from 0.46% to 5.88%. As shown in Table 15, the similarity calculated using the chromatographic fingerprint similarity evaluation system for traditional Chinese medicine was 1.000 for all samples, indicating that the method has good repeatability.

[0201] 2.2.3 Intermediate Precision Test

[0202] To investigate the effect of random variation factors on precision, six test solutions and reference solutions were prepared by different analysts on different dates, and the reference solutions and test solutions were prepared according to the method determined in “1.6” of (1) above.

[0203] Take 10 μL of each solution and inject it into the liquid chromatograph. Record the chromatogram. Using the solvent as a blank, subtract the blank and perform data processing. Take 12 chromatograms of the test samples for repeatability and intermediate precision tests. Use the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" to generate a reference fingerprint chromatogram using the median method. Calculate the similarity between the fingerprint chromatograms of each test sample and the generated reference fingerprint chromatogram. Count the common peaks of each test sample solution and number them. The reference peak is represented by S, and the remaining common peaks are represented by numbers in sequence. Using sodium tauroursodeoxycholate as the reference peak S in each test sample solution, calculate the relative retention time and relative peak area of ​​each common peak.

[0204] The similarity results of the intermediate precision test for each test solution are shown in Table 18 and Figure 8 As shown; Figure 8 Superimposed spectrum for comparison of similarity in intermediate precision tests of bear bile powder medicinal materials;

[0205] Table 18

[0206]

[0207] The results of the relative peak areas of the intermediate precision test for each test sample solution are shown in Table 19.

[0208] Table 19

[0209]

[0210]

[0211] The relative retention times of the intermediate precision tests for each test solution are shown in Table 20.

[0212] Table 20

[0213]

[0214] Conclusions: As shown in Tables 19 and 20, the repeatability and intermediate precision tests revealed that, under different time periods, personnel, and instruments, the relative retention times (RSDs) of the common peaks in the 12 test sample solutions ranged from 0.09% to 3.57%, and the relative peak areas (RSDs) ranged from 0.87% to 7.94%. Table 18 showed that, using the similarity evaluation system for traditional Chinese medicine chromatographic fingerprints, the similarity was within the range of 0.999 to 1.000. This indicates that the intermediate precision of this method for fingerprint determination is good and meets the requirements for fingerprint detection.

[0215] 2.2.4 Solution stability

[0216] Take an appropriate amount of bear bile powder (batch number: 201610-1), prepare the reference solution and the test solution according to the method determined in "1.6" of (1) above, place them at room temperature, accurately measure 10 μL at different times, inject them into the liquid chromatograph, record the chromatogram, use the solvent as a blank, subtract the blank, and perform data processing; use the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System", take the fingerprint chromatogram of the test sample at 0 hours as the reference chromatogram, compare the fingerprint chromatogram of the test sample at each time point with the reference chromatogram, and calculate the similarity. Count the common peaks of the test sample at each time point, and number the common peaks. The reference peak is represented by S, and the remaining common peaks are represented by numbers in sequence. Take sodium tauroursodeoxycholate as the reference peak S in each test sample, and calculate the relative retention time and relative peak area of ​​each common peak.

[0217] The similarity results of the stability test of the test sample solution are shown in Table 21 and Figure 9 As shown; Figure 9 This is a superimposed spectrum comparing the similarity of bear bile powder in stability tests.

[0218] Table 21

[0219]

[0220] The relative peak area results of the stability test of the test sample solution are shown in Table 22;

[0221] Table 22

[0222]

[0223] The relative retention times of the test sample solutions in the stability test are shown in Table 23.

[0224] Table 23

[0225]

[0226]

[0227] The results of the stability test of the reference solution are shown in Table 24:

[0228] Table 24

[0229]

[0230] Conclusions: As shown in Table 24, the RSDs of the main peak retention time and peak area of ​​the reference solution after different injection times at room temperature were 0.18% and 1.95%, respectively, indicating that the reference solution was stable for 27 hours at room temperature. As shown in Tables 22 and 23, the RSDs of the retention time and peak area of ​​sodium tauroursodeoxycholate (S peak) of the test solution after different injection times at room temperature were 0.14% and 1.54%, respectively. The RSDs of the relative retention times of common peaks in each chromatogram ranged from 0.02% to 0.36%, and the RSDs of the relative peak areas of each common peak ranged from 0.42% to 4.89%. As shown in Table 21, the RSD of the total peak area was 1.36%. Using the similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine, with the 0-hour fingerprint as the reference chromatogram, the similarity was calculated to be 1.000 for all values, indicating that the test solution was stable for 97 hours at room temperature.

[0231] 2.2.5 Range Test

[0232] The range test mainly examines the applicability of the method to the analyte in the sample within a certain concentration range (higher concentration, lower concentration).

[0233] Take an appropriate amount of bear bile powder (batch number: 201610-1), and prepare the reference solution and test solution according to the method determined in "1.6" of (1) above. Take 8 μL, 10 μL and 15 μL respectively, inject them into the liquid chromatograph, and record the chromatograms. Use the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" and take the 10 μL fingerprint chromatogram as the reference chromatogram. Compare the fingerprint chromatograms of each test sample with the reference chromatogram and calculate the similarity. Count the common peaks and number them. The reference peak is represented by S, and the remaining common peaks are represented by numbers in sequence. Take sodium tauroursodeoxycholate as the reference peak S in each test sample and calculate the relative retention time and relative peak area of ​​each common peak.

[0234] The similarity results of the range test are shown in Table 25 and Figure 10 As shown; Figure 10 Superimposed graph of similarity comparison of bear bile powder medicinal materials across a range of tests;

[0235] Table 25

[0236] Injection volume (μL) 8 8 8 10 15 15 15 Similarity 1.000 1.000 1.000 1.000 1.000 1.000 1.000

[0237] The relative retention time results of the range test are shown in Table 26:

[0238] Table 26

[0239]

[0240] The relative peak area results of the range test are shown in Table 27:

[0241] Table 27

[0242]

[0243] Conclusions: As shown in Tables 26 and 27, within the injection volume range of 8 μL (80% of the current sample concentration) to 15 μL (150% of the current sample concentration), the relative retention time RSD of the common peaks in each chromatogram was 0.05% to 0.34%, and the relative peak area RSD of each common peak was 0.36% to 8.77%. As shown in Table 25, the similarity calculated using the similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine was 1.000 for all values. This indicates that the test results within the range of this method meet the requirements.

[0244] 2.2.6 Durability

[0245] Take an appropriate amount of bear bile powder (batch number: 201610-1), and prepare the reference solution and test solution according to the method determined in "1.6" of (1) above. Fine-tune the salt concentration and pH value, and replace the chromatographic column with a different batch number from the same manufacturer, and perform detection. Use the "Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine" to compare the fingerprint chromatogram of the test sample under each condition with the reference chromatogram and calculate the similarity.

[0246] The robustness test results of the bear bile powder fingerprint analysis method are shown in Table 28 and Figures 11-14 As shown: Figure 11 A superimposed spectrum comparing the similarity of fine-tuned pH values ​​in the durability test of bear bile powder medicinal materials; Figure 12 A superimposed spectrum comparing the similarity of the mobile phase ratio for a durability test of bear bile powder medicinal materials. Figure 13 A superimposed spectrum comparing the similarity of fine-tuned salt concentrations in a durability test of bear bile powder medicinal materials. Figure 14 Superimposed chromatograms comparing the similarity of chromatographic columns from different batches in the durability test of bear bile powder medicinal materials.

[0247] Table 28

[0248]

[0249] Conclusion: The results above show that the fingerprint similarity of the chromatographic columns from different batches of the same manufacturer is above 0.90 when the salt concentration is 0.025-0.035 mol / L (±0.005M), the pH is 4.4±0.2, the organic phase ratio of the mobile phase is 9%-11% (±1%), and the column concentration is 4.4±0.2. This indicates that the method has good robustness.

[0250] (3) Validation of the standard fingerprint spectrum of bear bile powder

[0251] 3.1 Investigation of different batches of bear bile powder

[0252] Take 15 batches of bear bile powder and prepare reference solution and test solution according to the method determined in "1.6" of (1) above. Inject 10 μL of each prepared solution into the chromatograph and record the chromatogram. Use the solvent as a blank, subtract the blank, and perform data processing. Import the chromatogram data of 15 batches of bear bile powder into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" in sequence. Use the median method to generate the reference fingerprint spectrum. Compare the similarity between the fingerprint spectrum of each test sample and the reference fingerprint spectrum and calculate the similarity.

[0253] Similarity data of different batches of bear bile powder are shown in Table 29 and Figure 15 As shown; Figure 15 A superimposed spectrum comparing the similarity of different batches of bear bile powder.

[0254] Table 29

[0255] batch number Similarity batch number Similarity 201608-1 0.999 201612-1 0.997 201611-1 0.997 201701-1 0.996 201702-1 0.997 201610-1 0.994 201703-1 0.999 201709-1 0.980 201704-1 0.998 201801-1 0.987 201705-1 0.996 201802-1 0.992 201706-1 0.979 201803-1 0.993 201707-1 0.994 / /

[0256] The results above show that the similarity between the fingerprint chromatograms of each batch of medicinal materials and the control fingerprint chromatograms ranged from 0.979 to 0.999, indicating that the differences between the 15 batches of medicinal materials were not significant, and the control fingerprint chromatograms generated from these 15 batches were representative. Therefore, the control fingerprint chromatograms generated from these 15 batches of medicinal materials were used as the control fingerprint chromatograms for bear bile powder and incorporated into the internal quality control standards for investigation of the origin and harvesting season of the medicinal materials and for subsequent quality control of the medicinal materials. Figure 16 As shown, Figure 16 Fingerprint spectrum of bear bile powder as a reference material.

[0257] 3.2 Investigation of Bear Bile Powder from Different Origins

[0258] Fingerprint studies were conducted on bear bile powder from different origins to investigate the influence of origin. Appropriate amounts of bear bile powder from different origins were taken, and reference solutions and test solutions were prepared according to the method determined in “1.6” of (1) above. 10 μL of each prepared solution was injected into the chromatograph, and the chromatograms were recorded. The solvent was used as a blank, and the blank was subtracted for data processing. The fingerprint chromatograms of each test sample were compared with the reference fingerprint chromatogram of bear bile powder using the “Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine”.

[0259] The similarity results of bear bile powder from different origins are shown in Table 30 and Figure 17 As shown; Figure 17 A superimposed graph comparing the similarity of bear bile powder from different origins;

[0260] Table 30

[0261]

[0262]

[0263] Conclusion: The data above show that the similarity of the fingerprint spectra of bear bile powder from different manufacturers and production areas to the control bear bile powder is greater than 0.90, indicating that there is no significant difference in the quality of bear bile powder from different production areas.

[0264] 3.3 Comparison of different harvesting seasons

[0265] Take bear bile powder from Zhangfeng Pharmaceutical Factory in Longchuan County, Yunnan Province, harvested in different seasons, and take appropriate amounts of each. Prepare reference solution and test solution according to the method determined in "1.6" of (1) above. Take 10 μL of each solution and inject it into the liquid chromatograph. Record the chromatogram. Use the solvent as a blank and subtract the blank for data processing. Use the "Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine" to compare the similarity between the fingerprint chromatogram of the test sample and the reference fingerprint chromatogram of bear bile powder.

[0266] The similarity results of bear bile powder from different harvesting seasons are shown in Table 31 and Figure 18 As shown; Figure 18 Overlay of similarity graphs for bear bile powder medicinal materials from different harvesting seasons;

[0267] Table 31

[0268] Harvest season batch number Similarity November 201611-1 0.997 February 201702-1 0.998 May 201705-1 0.998 July 201707-1 0.992

[0269] Conclusion: The data above show that the similarity of the fingerprint spectra of bear bile powder from different harvesting seasons with that of bear bile powder control is greater than 0.90, indicating that the harvesting season has no significant impact on the quality of bear bile powder.

[0270] Example 2

[0271] Establishment and validation of fingerprint analysis method for multi-dose bear bile eye drops: The research object of this embodiment is multi-dose bear bile eye drops, the prescription of which is: 1000mL of preparation contains 5.0g bear bile powder, 12.0g boric acid, 0.6g borax, 2.2g sodium chloride, 0.8g ethylparaben, and appropriate amount of water for injection.

[0272] (1) Establishment of analytical methods

[0273] 1.1 Detector Selection

[0274] This study compares and selects between two different detectors: HPLC-ELSD and HPLC-UV.

[0275] 1.1.1 HPLC-ELSD

[0276] This part of the experiment was commissioned to the Institute of Biology, China Academy of Testing Technology.

[0277] Chromatographic conditions:

[0278] Column: Alltima TM 250×4.6mm, 5μm

[0279] Detector: ELSD with digital-to-analog conversion, Alltech 2000ES, ELSD 2000, evaporative light detector, drift tube temperature: 110℃; compressed air as evaporative gas, airflow velocity 3.0L / min.

[0280] Flow rate: 1.0 mL / min

[0281] Column temperature: 35℃

[0282] Mobile phase A: 0.03 mol / L sodium dihydrogen phosphate (pH adjusted to 4.4 with phosphoric acid)

[0283] Mobile phase B: Acetonitrile

[0284] Perform gradient elution according to the gradient elution procedure specified in Table 32:

[0285] Table 32

[0286] Time (min) Mobile phase A Mobile phase B 0 95 5 10 95 5 60 30 70 70 30 70 72 90 5 80 90 5

[0287] Reference solution: Take an appropriate amount of sodium tauroursodeoxycholate (batch number: 110816-201509) reference standard and dissolve it in methanol.

[0288] Test solution: Use bear bile eye drops (batch number: 20180712) as the test solution.

[0289] Blank excipient solution: Take blank excipient (batch number: 20180911) to prepare blank excipient solution.

[0290] Accurately measure 10 μL of each solution and inject it into the chromatograph for analysis. The results are as follows: Figures 19-21 As shown, Figure 19 This is the HPLC-ELSD chromatogram of blank excipients for the detection of multi-dose bear bile eye drops. Figure 20 This is a chromatogram of sodium tauroursodeoxycholate detected in multi-dose bear bile eye drops using HPLC-ELSD method. Figure 21 This is a chromatogram of bear bile eye drops detected by HPLC-ELSD in a multi-dose bear bile eye drop solution. Figures 19-21 It can be seen that, excluding the blank excipient absorption peak, bear bile eye drops have 3 obvious absorption peaks in the ELSD detector.

[0291] 1.1.2 HPLC-UV

[0292] Our company uses HPLC-UV method for testing, as follows:

[0293] Octadecylsilane-bonded silica gel was used as the packing material; mobile phase A was methanol-0.03 mol / L sodium dihydrogen phosphate (pH adjusted to 4.4 with phosphoric acid) at a volume ratio of 10:90; acetonitrile was used as mobile phase B, and gradient elution was performed; the detection wavelength was 210 nm.

[0294] Perform gradient elution according to the gradient elution procedure specified in Table 33.

[0295] Table 33

[0296]

[0297]

[0298] Reference solution: Take an appropriate amount of sodium tauroursodeoxycholate reference standard (batch number: 110816-201509) and dissolve it in methanol.

[0299] Test solution: Use bear bile eye drops (batch number: 20180712) as the test solution.

[0300] Accurately measure 10 μL of each solution and inject it into the chromatograph for analysis. The results are as follows: Figure 22 As shown, Figure 22 This is the chromatogram of the test sample for multi-dose bear bile eye drops using HPLC-UV method. Figure 22 It can be seen that HPLC-UV method detected 30 absorption peaks in the test solution.

[0301] 1.1.3 Comparison of Results

[0302] As can be seen from the above spectra, the absorption of this product on the UV detector is better than that on the ELSD detector. Therefore, the UV detector is selected for the fingerprint spectrum detection of this product.

[0303] 1.2 Comparison and Selection of Different Detection Systems

[0304] 1.2.1 Selection of reference solution

[0305] Sodium tauroursodeoxycholate is one of the main active ingredients in this product. It is present in high concentrations and is stable. Therefore, sodium tauroursodeoxycholate was selected as a reference.

[0306] Accurately weigh sodium tauroursodeoxycholate (batch number: 110816-201509) reference standard, and prepare a solution containing approximately 1 mg of sodium tauroursodeoxycholate per 1 mL with methanol as the reference solution.

[0307] 1.2.2 Preparation of the test solution

[0308] Test solution: Take this product (batch number: 20180712), filter, and collect the filtrate.

[0309] Blank excipient solution: Take blank excipient (batch number: 20180911), filter, and collect the filtrate.

[0310] 1.2.3 Comparison of different detection systems

[0311] Referring to the elution systems of methods 1 to 3 shown in Table 34, the samples were tested to compare the detection capabilities of different systems. The detection wavelength was 210 nm and the flow rate was 1.0 mL / min.

[0312] Table 34

[0313]

[0314]

[0315] Note 1: The elution system of Method 2 was adjusted to a similar binary system for gradient elution. The specific method is as follows: Mobile phase A: 0.03 mol / L sodium dihydrogen phosphate (pH adjusted to 4.4 with phosphoric acid) - methanol (volume ratio 90:10); Mobile phase B: acetonitrile. Gradient elution was performed with the following gradient program: 0–60 min mobile phase A 90% → 40%, 60–70 min mobile phase A 40%, 70–75 min mobile phase A 40% → 90%, 75–80 min mobile phase A 90%.

[0316] The reference solution and the test solution were tested under the above testing conditions, and the results are shown in Table 35.

[0317] Table 35

[0318] method Retention time of sodium tauroursodeoxycholate (min) Number of detection peaks Method 1 8.161 11 Method 2 30.928 30 Method 3 21.562 26

[0319] Conclusion: Based on the above results, Method 2 has the highest number of peaks detected, and further research will be conducted based on this method.

[0320] 1.2.4 Screening of chromatographic elution methods

[0321] The mobile phase A was 0.03 mol / L sodium dihydrogen phosphate (pH adjusted to 4.4 with phosphoric acid)-methanol (90:10 v / v); the mobile phase B was acetonitrile; the detection wavelength was 210 nm; the minimum peak area was 0.5% of the total peak area; different gradient elution conditions were used in the experiment. The specific parameters of gradient elution conditions 1 to 5 are shown in Table 36. The chromatographic column for gradient elution conditions 1 to 4 was C1. 18 (250mm × 4.6mm, 5μm), gradient elution condition 5, column: C10 ... 18 (150mm×4.6mm, 5μm).

[0322] Table 36

[0323]

[0324]

[0325] Accurately inject 10 μL each of the reference solution, test solution, and blank excipient solution into the liquid chromatograph, and conduct experiments using the gradient elution conditions described above. The results are shown in Table 37.

[0326] Table 37

[0327]

[0328] Note: To facilitate the viewing of the separation between sodium tauroursodeoxycholate and excipient peaks in each test sample, the excipients adjacent to sodium tauroursodeoxycholate were integrated in the chromatograms of the test samples under gradient elution conditions 1 to 4. When counting the number of peaks, the excipient peaks were not included.

[0329] Results: The results above show that the overall detection capabilities of gradient elution conditions 4 and 5 are comparable, with the same number of detection peaks and the most detection absorption peaks. In gradient elution condition 5, the reference peak (sodium tauroursodeoxycholate) in the test sample has better separation from the adjacent absorption peak.

[0330] Based on comprehensive evaluation, gradient elution condition 5 was selected for determining the fingerprint spectrum of bear bile eye drops. This condition yielded the highest number of peaks, showed good separation between the reference peak and adjacent absorption peaks, and did not interfere with the determination. The method was then optimized. The finalized method is as follows:

[0331] Column: C 18 Chromatographic column (Yuxu, Ultisil) TM C 18 (4.6×150mm, 5μm)

[0332] Detection wavelength: 210nm

[0333] Flow rate: 1.0 mL / min

[0334] Column temperature: 35℃

[0335] Mobile phase A: Acetonitrile

[0336] Mobile phase B: 0.03 mol / L sodium dihydrogen phosphate (pH adjusted to 4.4 with phosphoric acid) - methanol (volume ratio 90:10)

[0337] The fingerprint spectrum of bear bile eye drops (batch number: 20180712) was analyzed according to the above chromatographic conditions and the gradient elution procedure specified in Table 38. The results are as follows: Figure 23 As shown, Figure 23 Fingerprint spectroscopy of multi-dose bear bile eye drops;

[0338] Table 38

[0339] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 0 100 60 60 40 70 60 40 75 0 100 90 0 100

[0340] from Figure 23 Chromatographic analysis revealed 14 peaks exceeding 1% of the total peak area after excluding the blank excipient peak. Among these, two peaks exceeded 20% of the total peak area, while the remaining absorption peaks had areas less than 1% of the total peak area. Because this method employs low-wavelength detection and gradient elution, minor fluctuations in the HPLC instrument system (such as baseline noise and gradient elution) significantly affect small absorption peaks. However, small absorption peaks have a relatively small impact on fingerprint comparison; therefore, peaks smaller than 1.0% of the total peak area were not integrated during peak processing. Chromatographic comparison showed that peaks prior to 5 minutes consisted of densely packed small peaks that were difficult to separate further. These peaks constituted a small proportion of the total peak area and did not affect fingerprint comparison; therefore, peaks prior to 5 minutes were not integrated during peak processing.

[0341] 1.3 Method Optimization

[0342] Through the above experiments, the basic conditions for fingerprint analysis were determined. Based on these basic conditions, the column temperature, flow rate, mobile phase salt concentration, mobile phase pH, injection volume, detection wavelength, and chromatographic column were optimized to determine a detection method suitable for the fingerprint spectrum of this product.

[0343] 1.3.1 Column Temperature Optimization

[0344] Bear bile eye drops (batch number: 20180712) and blank excipient (batch number: 20180911) were tested at column temperatures of 30℃, 35℃, and 40℃, according to the method determined under "1.2.4 Screening of Chromatographic Elution Methods". Chromatograms were recorded. The test solution was analyzed using the blank excipient as a blank and after subtracting the blank. The results are shown in Table 39.

[0345] Table 39

[0346]

[0347]

[0348] Conclusion: The results above show that different column temperatures have no significant effect on the retention time of sodium tauroursodeoxycholate. The number of peaks detected decreases at 40℃, and the separation of sodium tauroursodeoxycholate from the preceding and following absorption peaks is smaller at 30℃ than at 35℃. Therefore, the column temperature for fingerprint spectroscopy detection is tentatively set at 35℃.

[0349] 1.3.2 Flow rate optimization

[0350] Bear bile eye drops (batch number: 20180712) and blank excipient (batch number: 20180911) were tested at different flow rates under a column temperature of 35℃, following the method determined in section "1.2.4 Screening of Chromatographic Elution Methods". Chromatograms were recorded. The test solution was analyzed using the blank excipient as a blank and after subtracting the blank. The results are shown in Table 40. Among them, as... Figure 24 , Figure 25 and Figure 26 As shown, Figure 24 The chromatogram of the test solution of multi-dose bear bile eye drops at a flow rate of 1.2 mL / min is shown. Figure 25 The chromatogram of the test solution of multi-dose bear bile eye drops at a flow rate of 1.0 mL / min is shown. Figure 26 The chromatogram of the test solution of multi-dose bear bile eye drops at a flow rate of 0.8 mL / min is shown.

[0351] Table 40

[0352] Flow rate (mL / min) Retention time of sodium tauroursodeoxycholate (min) Number of detection peaks 0.8 36.482 11 1.0 34.547 11 1.2 33.537 9

[0353] Conclusion: The results show that the number of absorption peaks decreases when the flow rate is 1.2 mL / min; the number of absorption peaks detected is the same when the flow rate is 0.8 mL / min and 1.0 mL / min. The chromatogram shows that the blank excipient peak in the test solution is better separated from the absorption peaks before and after when the flow rate is 0.8 mL / min. Therefore, the flow rate for fingerprint chromatogram detection is tentatively set at 0.8 mL / min.

[0354] 1.3.3 Optimization of mobile phase salt concentration

[0355] Bear bile eye drops (batch number: 20180712) and blank excipient (batch number: 20180911) were tested using mobile phases with different salt concentrations at a column temperature of 35℃ and a flow rate of 0.8 mL / min, following the method determined in section 1.2.4 "Screening of Chromatographic Elution Methods". Chromatographic chromatograms were recorded. The test solution was analyzed using the blank excipient as a blank and after subtracting the blank. The results are shown in Table 41.

[0356] Table 41

[0357] Salt concentration Retention time of sodium tauroursodeoxycholate (min) Number of detection peaks 0.015M 36.049 8 0.03M 36.482 11 0.045M 36.305 9

[0358] Conclusion: The results above show that different salt concentrations have no significant effect on the retention time of sodium tauroursodeoxycholate; the absorption peaks decreased at salt concentrations of 0.015M and 0.045M, so the salt concentration in the mobile phase is tentatively set at 0.03M for fingerprint analysis.

[0359] 1.3.4 pH Optimization

[0360] Bear bile eye drops (batch number: 20180712) and blank excipient (batch number: 20180911) were tested using the method determined under "1.2.4 Screening of Chromatographic Elution Methods". The mobile phase was prepared with different pH values ​​of salt solutions at a column temperature of 35℃, a flow rate of 0.8 mL / min, and a salt concentration of 0.03 M. Chromatograms were recorded. The test solution was analyzed using the blank excipient as a blank and after subtracting the blank. The results are shown in Table 42.

[0361] Table 42

[0362] pH of salt solution Retention time of sodium tauroursodeoxycholate (min) Number of detection peaks Without adjusting the pH value (4.83) 36.572 9 pH 3.9 36.532 8 pH 4.4 36.482 11

[0363] Conclusion: The results above show that different pH values ​​have no significant effect on the retention time of sodium tauroursodeoxycholate; the number of detection peaks at pH 3.9 and without pH adjustment are 8 and 9, respectively; the number of detection peaks is 11 at pH 4.4, which is the highest number of absorption peaks detected. Therefore, the pH of the salt solution in the mobile phase is tentatively set at 4.4 for fingerprint spectroscopy detection.

[0364] 1.3.5 Optimization of Injection Volume

[0365] Bear bile eye drops (batch number: 20180712) and blank excipient (batch number: 20180911) were taken and precisely injected into the liquid chromatograph at 5 μL, 10 μL and 20 μL respectively, according to the experimental conditions determined above. The chromatograms were recorded and the detection of each absorption peak under different injection volumes was investigated. The results are shown in Table 43.

[0366] Table 43

[0367] Injection volume (μL) 5 10 20 Number of detection peaks 11 11 11

[0368] Conclusion: The results above show that different injection volumes have no significant effect on the number of peaks detected. Therefore, the conventional injection volume of 10 μL was selected as the injection volume for this method.

[0369] 1.3.6 Optimization of detection wavelength

[0370] Bear bile eye drops (batch number: 20180712) and blank excipients (batch number: 20180911) were tested under the conditions determined above. 10 μL of each sample was accurately injected into the liquid chromatograph, and detection was performed using a diode array detector (DAD). The chromatogram was recorded, and the peak area, total peak area (Area Total), and number of detected peaks of sodium tauroursodeoxycholate in the sample were compared as evaluation indicators. Data at wavelengths of 203 nm, 210 nm, 230 nm, and 254 nm were statistically analyzed. The results are shown in Table 44 and... Figures 27-30 As shown, Figure 27 The chromatogram of the test solution of multi-dose bear bile eye drops at 203 nm; Figure 28The chromatogram of the test solution of multi-dose bear bile eye drops at 210 nm; Figure 29 The chromatogram of the test solution of multi-dose bear bile eye drops at 230 nm; Figure 30 The 254 nm chromatogram of the test solution for multi-dose bear bile eye drops.

[0371] Table 44

[0372] Wavelength (nm) 203 210 230 254 Number of detection peaks (number) 5 11 18 19 Sodium tauroursodeoxycholate Area 6100522 1877655 97312 16780 Area Total 13137510 4411903 609302 619941

[0373] Conclusion: The results above show that the absorption of each chromatographic peak is stronger closer to the lower wavelength, and vice versa. Based on the comprehensive analysis of the number of chromatographic peaks detected, the absorption intensity, and the full wavelength scan, 210 nm was selected as the detection wavelength for the fingerprint spectrum of this product.

[0374] 1.3.7 Column Optimization

[0375] Reference solution, test solution, and blank excipient solution were tested using chromatographic columns of different brands, and chromatograms were recorded. The number of peaks detected in the test sample was used as the evaluation index for comparison. The results are shown in Table 45 and... Figures 31-36 As shown, Figure 31 To adopt Agilent ZORBAX SB-C 18 Chromatogram of the multi-dose bear bile eye drops test solution on a chromatographic column. Figure 32 To use Kromasil C 18 Chromatogram of the multi-dose bear bile eye drops test solution on a chromatographic column. Figure 33 To use Diamonsil C 18 Chromatogram of the multi-dose bear bile eye drops test solution on a chromatographic column. Figure 34 To use Welch Ultimate LP-C 18 Chromatogram of the multi-dose bear bile eye drops test solution on a chromatographic column. Figure 35 To adopt Welch Ultisil TM C 18 Chromatogram of the multi-dose bear bile eye drops test solution on a chromatographic column. Figure 36 To adopt Welch Ultimate XB-C 18 Chromatogram of the test solution of multi-dose bear bile eye drops on a chromatographic column.

[0376] Table 45

[0377]

[0378] Conclusion: The results above show that using Welch Ultisil... TM C 18 Welch Ultimate XB-C18 Chromatographic column analysis showed that all absorption peaks of the test sample were well separated from the gradient peaks and blank excipient peaks, and the number of detected peaks was the highest. Research on Welch Ultisil... TM C 18 Since the manufacturer has currently ceased production of the chromatographic column, the Welch Ultimate XB-C, which has comparable analytical capabilities, was selected. 18 A chromatographic column (150×4.6mm, 5μm) was used as the column for detecting the fingerprint spectrum of this product.

[0379] 1.3.8 Study on the preparation of blank excipients for multi-dose bear bile eye drops

[0380] Take all excipients and simulate production. Weigh out 2.41132g of boric acid, 0.46637g of borax, 0.44808g of sodium chloride, and 0.16042g of ethylparaben according to the prescription ratio. Place them in the same beaker, add about 160mL of water, boil to dissolve, cool to room temperature, and then make up to 200mL. Heat under reflux for different times, take samples, and test. The results are shown in Table 46.

[0381] Table 46

[0382]

[0383] Results: The data above show that the blank excipient solution prepared for simulated production has three obvious absorption peaks. Peak 1 shows an increasing trend in peak area with the extension of heating time, while peak 2 shows a decreasing trend in peak area with the extension of heating time. The total peak area does not change significantly. In subsequent experiments, the blank excipient for the comparative formulation was prepared by heating, dissolving and refluxing for 30 minutes, similar to the production process.

[0384] 1.4 Determination of fingerprinting method for multi-dose bear bile eye drops

[0385] Based on the above experimental results, the fingerprint spectroscopy determination method is determined as follows:

[0386] Chromatographic conditions and system suitability test: using octadecylsilane-bonded silica gel as the stationary phase, Welch Ultimate XB-C 18 The chromatographic column (15 cm long, 4.6 mm inner diameter, 5 μm particle size) was used. Acetonitrile was used as mobile phase A, and 0.03 mol / L sodium dihydrogen phosphate solution (adjusted to pH 4.4 with phosphoric acid): methanol (90:10 v / v) was used as mobile phase B. Gradient elution was performed according to the gradient elution program specified in Table 47. The detection wavelength was 210 nm; the column temperature was 35 °C; and the flow rate was 0.8 mL / min. The theoretical plate number, calculated based on the sodium tauroursodeoxycholate peak, should be no less than 2500.

[0387] Table 47

[0388] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 0 100 60 60 40 70 60 40 75 0 100 90 0 100

[0389] Preparation of reference solution: Accurately weigh sodium tauroursodeoxycholate reference standard and add methanol to prepare a solution containing approximately 1 mg of sodium tauroursodeoxycholate per 1 mL.

[0390] Preparation of test solution: Take this product, filter it, and use the filtrate as the test solution.

[0391] Preparation of blank excipient solution: Take blank excipient solution, filter, and use the filtrate as test solution; or take an appropriate amount of blank excipient and prepare it according to the production process and prescription ratio.

[0392] Assay: Accurately pipette 10 μL each of blank excipient solution, reference solution and test solution into the liquid chromatograph, measure and record the chromatogram to obtain the result.

[0393] Chromatographic peak integration parameters: No integration is performed in the first 5 minutes of the chromatogram of the test sample. After deducting the blank excipient peak, the minimum peak area shall not be less than 1.0% of the total peak area.

[0394] (2) Validation of analytical methods

[0395] 2.1 Validation of fingerprint analysis method for multi-dose bear bile eye drops

[0396] 2.1.1 Sample and Reference Standard

[0397] As shown in Table 48:

[0398] Table 48

[0399] name batch number source Sodium tauroursodeoxycholate 110816-201509 China National Institutes for Food and Drug Control Tauroursodeoxycholic acid 110816-202110 China National Institutes for Food and Drug Control Bear bile eye drops 20180712 Yunnan Dai Medicine Co., Ltd. Bear bile eye drops 01211010 Yunnan Dai Medicine Co., Ltd. Blank supplementary materials 20180911 Yunnan Dai Medicine Co., Ltd. boric acid 200901 Zigong Honghe Pharmaceutical Co., Ltd. Borax 200908 Hebei Huachen Pharmaceutical Group Co., Ltd. Sodium chloride 20200208 Taishan Xinning Pharmaceutical Co., Ltd. Ethylparaben 103620210701 Hunan Ercon Pharmaceutical Co., Ltd.

[0400] 2.1.2 Reagents

[0401] As shown in Table 49:

[0402] Table 49

[0403] name level source methanol HPLC BCL / Merck Sodium dihydrogen phosphate AR Chengdu Jinshan Chemical Reagent Co., Ltd. Phosphoric acid AR Chengdu Jinshan Chemical Reagent Co., Ltd. Acetonitrile HPLC BCL

[0404] 2.1.3 Instruments

[0405] As shown in Table 50:

[0406] Table 50

[0407] Instrument Name model source High Performance Liquid Chromatography LC-2030C PLUS Shimadzu Malaysia Factory High Performance Liquid Chromatography Agilent 1100 Agilent Electronic balance CP225D Sartorius (Shanghai) Trading Co., Ltd. Electronic balance HZT-A+300 Fuzhou Huazhi Scientific Instruments Co., Ltd. pH meter PB-10 Sartorius (Shanghai) Trading Co., Ltd.

[0408] 2.1.4 Chromatographic column

[0409] As shown in Table 51:

[0410] Table 51

[0411] Serial Number Column name 1 <![CDATA[Welch Ultimate XB-C 18 ;SN:60190100660]]> 2 <![CDATA[Welch Ultimate XB-C 18 ;SN:60190801792]]> 3 <![CDATA[Welch Ultimate XB-C 18 ;SN:60190401100]]>

[0412] 2.1.5 The summary table of analytical method validation is shown in Table 52:

[0413] Table 52

[0414]

[0415]

[0416]

[0417] 2.1.6 Fingerprint Spectrum Determination Method

[0418] Chromatographic conditions and system suitability test: using octadecylsilane-bonded silica gel as the stationary phase, Welch Ultimate XB-C 18 The chromatographic column (15 cm long, 4.6 mm inner diameter, 5 μm particle size) was used. Acetonitrile was used as mobile phase A, and 0.03 mol / L sodium dihydrogen phosphate solution (adjusted to pH 4.4 with phosphoric acid): methanol (90:10 v / v) was used as mobile phase B. Gradient elution was performed according to the specifications in Table 53. The detection wavelength was 210 nm. The column temperature was 35 °C, and the flow rate was 0.8 mL / min. The theoretical plate number, calculated based on the sodium tauroursodeoxycholate peak, should be no less than 2500.

[0419] Table 53

[0420] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 0 100 60 60 40 70 60 40 75 0 100 90 0 100

[0421] Preparation of reference solution: Accurately weigh sodium tauroursodeoxycholate reference standard and add methanol to prepare a solution containing approximately 1 mg of sodium tauroursodeoxycholate per 1 mL.

[0422] Preparation of test solution: Take this product, filter it, and use the filtrate as the test solution.

[0423] Preparation of blank excipient solution: Take blank excipient solution, filter, and use the filtrate as test solution; or take an appropriate amount of blank excipient and prepare according to the prescription ratio.

[0424] Determination method: Accurately pipette 10 μL each of the reference solution and the test solution into the liquid chromatograph, measure and record the chromatogram to obtain the result.

[0425] Chromatographic peak integration parameters: No integration is performed in the first 5 minutes of the chromatogram of the test sample. After deducting the blank excipient peak, the minimum peak area shall not be less than 1.0% of the total peak area.

[0426] According to the similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine, the similarity between the fingerprint chromatogram of the test sample and the fingerprint chromatogram of the reference sample shall not be less than 0.90.

[0427] Note: Tauroursodeoxycholic acid and sodium tauroursodeoxycholate have the same retention time under chromatographic conditions. Since October 2021, the China National Institutes for Food and Drug Control has only been able to purchase tauroursodeoxycholic acid reference standard. Therefore, the fingerprint reference standard for subsequent tests in October 2021 was changed from sodium tauroursodeoxycholate to tauroursodeoxycholic acid.

[0428] 2.1.7 Method Validation

[0429] 2.1.7.1 Specificity Verification

[0430] Specificity will be examined to determine the interference of blank excipients on fingerprint spectrum detection.

[0431] Reference solution: Accurately weigh sodium tauroursodeoxycholate (batch number: 110816-201509) reference standard, and add methanol to prepare a solution containing about 1 mg of sodium tauroursodeoxycholate per 1 mL.

[0432] Test solution: Take this product (batch number: 20180712), filter it, and use the filtrate as the test solution.

[0433] Blank excipient solution: Take blank excipient solution (batch number: 20180911), filter it, and use the filtrate as the test solution.

[0434] Take 10 μL each of the reference solution, the test solution, and the blank excipient solution, inject them into the liquid chromatograph, and record the chromatogram.

[0435] Results: The blank excipient showed no significant absorption at the corresponding position of the main peak in the reference solution, and did not interfere with the detection of the test solution when compared with the chromatogram of the test solution.

[0436] 2.1.7.2 Scope

[0437] The range test mainly examines the applicability of the method to the analyte in the sample within a certain concentration range (higher concentration, lower concentration).

[0438] Take bear bile eye drops (batch number: 20180712), prepare the test solution according to section "2.1.6", and inject 8 μL, 10 μL, and 15 μL into the liquid chromatograph respectively. Record the chromatograms, using the blank excipient as a blank, subtract the blank, and perform data processing. Using the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System", with the chromatogram of the 10 μL test sample as the reference chromatogram, compare the similarity between the fingerprint chromatograms of each test sample and the reference chromatogram. The results are shown in Table 54 and... Figure 37 As shown, Figure 37This is a superimposed spectrum for comparing the similarity of multiple doses of bear bile eye drops. Common peaks were counted and numbered, with the reference peak denoted by S, and the remaining common peaks denoted by numbers in sequence. Sodium tauroursodeoxycholate was used as the reference peak for each test sample, and the relative retention time and relative peak area of ​​each common peak were calculated. The results are shown in Tables 55-56.

[0439] Table 54

[0440] Injection volume (μL) 8 8 8 10 15 15 15 Similarity 1.000 1.000 1.000 1.000 1.000 1.000 1.000

[0441] Table 55

[0442]

[0443] Table 56

[0444]

[0445] Conclusions: Tables 55 and 56 show that within the injection volume range of 8 μL (80% of the current sample concentration) to 15 μL (150% of the current sample concentration), the relative retention time RSD of the common peaks in each chromatogram is 0.02% to 0.24%, and the relative peak area RSD of each common peak is 0.32% to 8.61%. Table 54 shows that the similarity calculated using the similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine is 1.000 for all values. This indicates that the test results within the range of this method meet the requirements.

[0446] 2.1.7.3 Precision Test

[0447] ① Instrument precision test

[0448] Reference solution: Weigh an appropriate amount of sodium tauroursodeoxycholate (batch number: 110816-201509) reference standard, dissolve it in methanol, and prepare a solution containing approximately 1 mg of sodium tauroursodeoxycholate per 1 mL.

[0449] Test solution: Take bear bile eye drops (batch number: 20180712), filter, and use the filtrate as the test solution.

[0450] Blank excipient solution: Take the blank excipient solution (batch number: 20180911), filter it, and take the filtrate as the blank excipient solution.

[0451] Accurately measure 10 μL each of the test solution and reference solution, and inject them according to the detection method under section "2.1.6". Inject each solution six times consecutively, record the chromatograms, and subtract the blank excipient solution. Perform data processing, and calculate the retention time and peak area of ​​the main peak of the reference solution according to formulas 3 and 4, and calculate the RSD. The results are shown in Table 57. Using the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System", a reference fingerprint chromatogram was generated using the median method. The similarity between the fingerprint chromatograms of each test sample and the generated reference fingerprint chromatogram was calculated. The results are shown in Table 58. Figure 38 As shown, Figure 38 A superimposed chromatogram was used to compare the similarity of the injection precision of multiple doses of bear bile eye drops. Common peaks were counted and numbered. The reference peak was denoted by S, and the remaining common peaks were denoted by numbers in sequence. Sodium tauroursodeoxycholate was used as the reference peak S in each test solution. The relative retention time and relative peak area of ​​each common peak were calculated. The results are shown in Tables 59 and 60.

[0452]

[0453]

[0454] Table 57

[0455]

[0456] Table 58

[0457] serial number 1 2 3 4 5 6 Similarity 1.000 1.000 1.000 1.000 1.000 1.000

[0458] Table 59

[0459]

[0460] Table 60

[0461]

[0462]

[0463] Conclusions: As shown in Table 57, with six consecutive injections of the reference solution, the RSDs for retention time and main peak area were 0.06% and 0.17%, respectively. As shown in Tables 59 and 60, with six consecutive injections of the test sample solution, the RSDs for retention time and peak area of ​​sodium tauroursodeoxycholate (S peak) were 0.09% and 0.25%, respectively. The RSDs for relative retention time of each common peak in the test sample ranged from 0.01% to 0.21%, and the RSDs for relative peak area of ​​each common peak ranged from 0.11% to 9.72%. As shown in Table 58, the similarity was calculated using the similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine, and the similarity was 1.000 for all values, indicating good instrument injection precision.

[0464] ② Repeatability test

[0465] Reference solution: Weigh an appropriate amount of sodium tauroursodeoxycholate (batch number: 110816-201509) reference standard, dissolve it in methanol, and prepare a solution containing approximately 1 mg of sodium tauroursodeoxycholate per 1 mL.

[0466] Test solution: Take bear bile eye drops (batch number: 20180712), filter, and use the filtrate as the test solution. (Prepare 6 samples using the same method)

[0467] Blank excipient solution: Take the blank excipient solution (batch number: 20180911), filter it, and take the filtrate as the blank excipient solution.

[0468] Take 10 μL of each of the above solutions and inject them into the liquid chromatograph. Record the chromatograms. Using the blank excipient solution as a blank, subtract the blank and perform data processing. The "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" is used to generate a reference fingerprint chromatogram using the median method. The similarity between the fingerprint chromatograms of each test sample and the generated reference fingerprint chromatogram is calculated, and the results are shown in Table 61. Figure 39 As shown, Figure 39 This is a superimposed chromatogram for comparing the similarity of repeatability tests of multiple doses of bear bile eye drops. Common peaks in each test solution were counted and numbered, with the reference peak denoted by S, and other common peaks indicated by numbers in sequence. Using sodium tauroursodeoxycholate as the reference peak S in each test solution, the relative retention time and relative peak area of ​​each common peak were calculated. The statistical results are shown in Tables 62 and 63.

[0469] Table 61

[0470] serial number 1 2 3 4 5 6 Similarity 1.000 1.000 1.000 1.000 1.000 1.000

[0471] Table 62

[0472]

[0473] Table 63

[0474]

[0475] Conclusions: As shown in Tables 62 and 63, the relative retention times (RSDs) of the common peaks in the six test solutions ranged from 0.02% to 0.04%, and the relative peak areas (RSDs) ranged from 0.10% to 5.18%. As shown in Table 61, the similarity was calculated using the similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine, and the similarity was 1.000 for all results, indicating that the method has good repeatability.

[0476] ③ Intermediate precision test

[0477] To investigate the effect of random variation on precision, six test sample solutions and reference solutions were prepared by different analysts on different days. The experiment was conducted according to section "2.1.6". 10 μL of each solution was injected into the liquid chromatograph, and the chromatograms were recorded. A blank excipient solution was used as a blank, and the blank was subtracted before data processing. Twelve test sample chromatograms were used for repeatability and intermediate precision tests. Using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine", a reference fingerprint chromatogram was generated using the median method. The similarity between each test sample fingerprint chromatogram and the generated reference fingerprint chromatogram was calculated. The results are shown in Table 64. Figure 40 As shown, Figure 40 This is a superimposed chromatogram for comparing the similarity of intermediate precision tests of multiple doses of bear bile eye drops. Common peaks in each test solution were counted and numbered. The reference peak was denoted by S, and the remaining common peaks were numbered sequentially. Sodium tauroursodeoxycholate was used as the reference peak S in each test solution. The relative retention time and relative peak area of ​​each common peak were calculated, and the results are shown in Tables 65 and 66.

[0478] Table 64

[0479]

[0480] Table 65

[0481]

[0482]

[0483] Table 66

[0484]

[0485] Conclusions: As shown in Tables 65 and 66, the repeatability and intermediate precision tests revealed that, for fingerprint chromatograms determined at different times, by different personnel, and using different instruments, the relative retention time RSD of each common peak in the 12 test sample solutions ranged from 0.07% to 3.94%, and the relative peak area RSD ranged from 0.46% to 8.20%. As shown in Table 64, the similarity calculated using the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System was 0.999 for all samples. This indicates that the intermediate precision of this method for fingerprint chromatogram determination is good and meets the requirements for fingerprint chromatogram detection.

[0486] 2.1.7.4 Solution stability

[0487] Reference solution: Weigh an appropriate amount of sodium tauroursodeoxycholate (batch number: 110816-201509) reference standard, dissolve it in methanol, and prepare a solution containing approximately 1 mg of sodium tauroursodeoxycholate per 1 mL.

[0488] Test solution: Take bear bile eye drops (batch number: 20180712), filter, and use the filtrate as the test solution.

[0489] The reference solution and the test solution were placed at room temperature, and 10 μL was precisely measured at different time points and injected into the liquid chromatograph. The chromatograms were recorded. The blank excipient solution was used as a blank, and the blank was subtracted. Data processing was performed, and the results are shown in Table 67. The "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" was used, with the 0-hour fingerprint chromatogram of the test sample as the reference chromatogram. The fingerprint chromatograms of the test sample at each time point were compared with the reference chromatogram, and the similarity was calculated. The results are shown in Table 68 and 69. Figure 41 As shown, Figure 41 This is a superimposed chromatogram comparing the similarity of stability test solutions of multiple dose bear bile eye drops. Common peaks at each time point were counted and numbered. The reference peak was denoted by S, and the remaining common peaks were numbered sequentially. Using sodium tauroursodeoxycholate as the reference peak S in each test sample, the relative retention time and relative peak area of ​​each common peak were calculated. The results are shown in Tables 69 and 70.

[0490] Table 67

[0491]

[0492] Table 68

[0493] Placement time (h) 0 3 12 32 60.5 94 Similarity 1.000 1.000 1.000 1.000 1.000 1.000

[0494] Table 69

[0495]

[0496] Table 70

[0497]

[0498]

[0499] Conclusions: As shown in Table 67, the RSDs of the main peak retention time and main peak area of ​​the reference solution after different injection times at room temperature were 0.18% and 1.95%, respectively, indicating that the reference solution was stable for 27 hours at room temperature. As shown in Tables 69 and 70, the RSDs of the retention time and peak area of ​​sodium tauroursodeoxycholate (S peak) after different injection times at room temperature were 0.14% and 1.17%, respectively, with a total peak area RSD of 1.69%. The RSDs of the relative retention times of common peaks in each chromatogram ranged from 0.04% to 0.39%, and the RSDs of the relative peak areas of each common peak ranged from 0.34% to 7.26%. As shown in Table 68, the similarity was calculated using the chromatographic fingerprint similarity evaluation system for traditional Chinese medicine, with the 0-hour chromatogram as the reference chromatogram. The similarity was 1.000 for all values, indicating that the test solution was stable for 94 hours at room temperature.

[0500] 2.1.7.5 Durability

[0501] ① Fine-tune the salt concentration, pH value, and mobile phase ratio.

[0502] Reference solution: Weigh an appropriate amount of sodium tauroursodeoxycholate reference standard (batch number: 110816-201509), dissolve it in methanol, and prepare a solution containing approximately 1 mg of sodium tauroursodeoxycholate per 1 mL.

[0503] Test solution: Take bear bile eye drops (batch number: 20180712), filter, and use the filtrate as the test solution.

[0504] Blank excipient solution: Take the blank excipient solution (batch number: 20180911), filter it, and take the filtrate as the blank excipient solution.

[0505] To examine the robustness of this method, the chromatographic conditions were fine-tuned, and 10 μL of each solution was injected into the liquid chromatograph for detection. Using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines," the fingerprint chromatograms of the test samples under each condition were compared with the reference chromatogram, and the similarity was calculated. The results are shown in Table 71 and... Figures 42-44 As shown, Figure 42 A superimposed graph comparing the similarity of pH values ​​adjusted for durability testing of multi-dose bear bile eye drops. Figure 43 A superimposed graph comparing the similarity of the mobile phase ratio for a durability test of multi-dose bear bile eye drops. Figure 44 This is a superimposed graph comparing the similarity of fine-tuned salt concentrations in a durability test of multi-dose bear bile eye drops.

[0506] Table 71

[0507]

[0508]

[0509] ②Chromatographic columns from different batches

[0510] Reference solution: Weigh an appropriate amount of tauroursodeoxycholic acid (batch number: 110816-202110) reference standard, dissolve it in methanol, and prepare a solution containing approximately 1 mg of tauroursodeoxycholic acid per 1 mL.

[0511] Test solution: Use bear bile eye drops (batch number: 01211010) as the test solution.

[0512] Blank excipient solution: Take an appropriate amount of blank excipient and prepare a blank excipient solution according to the production process and prescription ratio.

[0513] Welch Ultimate XB-C using different batch numbers 18A chromatographic column (15 cm long, 4.6 mm inner diameter, 5 μm particle size) was used. 10 μL of each solution was injected into the liquid chromatograph for detection. The "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" was used, with the original conditions as the reference chromatogram. The fingerprint chromatograms of the test samples under each condition were compared with the reference chromatogram, and the similarity was calculated. The results are shown in Table 72. Figure 45 As shown, Figure 45 Superimposed chromatograms comparing the similarity of chromatographic columns from different batches in a durability test of multi-dose bear bile eye drops.

[0514] Table 72

[0515]

[0516] ③ Conclusion

[0517] The results above show that even with fine adjustments to salt concentration, pH value, mobile phase ratio, and replacement with different batches of the same chromatographic column, the fingerprint similarity remains at 1.000, indicating that this method has good robustness.

[0518] 2.1.7.6 Sample Testing

[0519] Take appropriate amounts of samples from three batches of bear bile eye drops (batch numbers: 01211010, 01211011, and 01211012), and prepare the test solution and reference solution according to section "2.1.6". Inject 10 μL of each solution into the liquid chromatograph and record the chromatograms. Using the blank excipient solution as a blank, subtract the blank and perform data processing. Use the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" to generate a reference fingerprint using the median method. Calculate the similarity between the fingerprint chromatograms of each batch of test samples and the generated reference fingerprint chromatogram. The results are shown in Table 73. Figure 46 As shown, Figure 46 This is a superimposed graph comparing the similarity of multiple doses of bear bile eye drops samples.

[0520] Table 73

[0521] batch number 01211010 01211011 01211012 Similarity 1.000 1.000 1.000

[0522] Conclusion: The similarity of the three batches of samples was greater than 0.90, which meets the requirements.

[0523] Example 3

[0524] Establishment and validation of fingerprint analysis method for single-dose bear bile eye drops: The research object of this embodiment is single-dose bear bile eye drops, the prescription of which is: 1000mL of preparation contains 5.0g bear bile powder, 12.0g boric acid, 0.6g borax, 2.2g sodium chloride, and appropriate amount of water for injection.

[0525] (1) Establishment of analytical methods

[0526] From a production process perspective, the extraction and purification processes for single-dose bear bile eye drops and multi-dose bear bile eye drops are consistent. From a formulation perspective, the formulation of single-dose bear bile eye drops is identical except for the absence of the antibacterial agent (ethylparaben). To investigate the consistency of the material basis between single-dose and multi-dose bear bile eye drops, it is proposed that both single-dose and multi-dose bear bile eye drops undergo fingerprint spectroscopy studies using the same method, i.e., the detection method under "2.1.6" in Example 2 will be used. The methodological validation of the fingerprint spectroscopy determination methods for multi-dose and single-dose bear bile eye drops will be conducted separately.

[0527] (2) Validation of analytical methods

[0528] 1.1 Samples and Reference Standards

[0529] As shown in Table 74:

[0530] Table 74

[0531] name batch number batch source Tauroursodeoxycholic acid 110816-202110 / China National Institutes for Food and Drug Control Bear bile eye drops 02211102 250L Yunnan Dai Medicine Co., Ltd. boric acid 200901 / Zigong Honghe Pharmaceutical Co., Ltd. Borax 200908 / Hebei Huachen Pharmaceutical Group Co., Ltd. Sodium chloride 20200208 / Taishan Xinning Pharmaceutical Co., Ltd.

[0532] 1.2 Reagents

[0533] As shown in Table 75:

[0534] Table 75

[0535]

[0536]

[0537] 1.3 Instruments

[0538] As shown in Table 76:

[0539] Table 76

[0540] Instrument Name model source High Performance Liquid Chromatography LC-2030C PLUS Shimadzu Malaysia Factory High Performance Liquid Chromatography Agilent 1100 Agilent Electronic balance CP225D Sartorius (Shanghai) Trading Co., Ltd. Electronic balance HZT-A+300 Fuzhou Huazhi Scientific Instruments Co., Ltd. pH meter PB-10 Sartorius (Shanghai) Trading Co., Ltd.

[0541] 1.4 Chromatographic Column

[0542] As shown in Table 77:

[0543] Table 77

[0544] Serial Number Column name 1 <![CDATA[Welch Ultimate XB-C 18 ;SN:60190100660]]> 2 <![CDATA[Welch Ultimate XB-C 18 ;SN:60190801792]]> 3 <![CDATA[Welch Ultimate XB-C 18 ;SN:60190401100]]>

[0545] 1.5 The summary table of analytical method validation is shown in Table 78.

[0546] Table 78

[0547]

[0548]

[0549]

[0550] 1.6 Fingerprint Spectrum Detection Method

[0551] Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the stationary phase (Welch Ultimate XB-C). 18 The chromatographic column (15 cm long, 4.6 mm inner diameter, 5 μm particle size) was used. Acetonitrile was used as mobile phase A, and 0.03 mol / L sodium dihydrogen phosphate solution (adjusted to pH 4.4 with phosphoric acid)-methanol (90:10 v / v) was used as mobile phase B, with gradient elution according to Table 79. The detection wavelength was 210 nm; the column temperature was 35 °C; and the flow rate was 0.8 mL / min. The theoretical plate number, calculated based on the tauroursodeoxycholic acid peak, should be no less than 2500.

[0552] Table 79

[0553]

[0554]

[0555] Preparation of reference solution: Accurately weigh tauroursodeoxycholic acid reference standard and add methanol to prepare a solution containing approximately 1 mg of tauroursodeoxycholic acid per 1 mL.

[0556] Preparation of test solution: Take this product, filter it, and use the filtrate as the test solution.

[0557] Determination method: Accurately pipette 10 μL each of the reference solution and the test solution into the liquid chromatograph, measure and record the chromatogram to obtain the result.

[0558] Chromatographic peak integration parameters: No integration is performed in the first 5 minutes, and the minimum peak area is no less than 1.0% of the total peak area.

[0559] According to the similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine, the similarity between the fingerprint chromatogram of the test sample and the fingerprint chromatogram of the reference sample shall not be less than 0.90.

[0560] 1.7 Method Validation

[0561] 1.7.1 Exclusivity

[0562] Specificity will be examined to determine the interference of blank excipients on fingerprint spectrum detection.

[0563] Preparation of blank excipient solution: Weigh 1.20501g of boric acid, 0.06242g of borax, and 0.22638g of sodium chloride, place them in the same 100mL volumetric flask, add water to dissolve and dilute to the mark, shake well, filter, and take the filtrate.

[0564] Preparation of reference solution: Weigh an appropriate amount of tauroursodeoxycholic acid (batch number: 110816-202110) reference standard, dissolve it in methanol to prepare a solution containing about 1 mg of tauroursodeoxycholic acid per 1 mL.

[0565] Test solution: Take bear bile eye drops (batch number: 02211102), filter, and use the filtrate as the test solution.

[0566] Take each solution and perform the test according to the detection method under section "2.1.6", and record the chromatogram.

[0567] Conclusion: The blank excipient solution showed no significant absorption at the corresponding position of the main peak in the reference solution; it did not interfere with the detection when compared with the test solution. This indicates that the blank excipient does not interfere with the fingerprint determination of this product.

[0568] 1.7.2 Precision Test

[0569] ① Sample injection precision

[0570] Preparation of reference solution: Weigh an appropriate amount of tauroursodeoxycholic acid (batch number: 110816-202110) reference standard, dissolve it in methanol, and prepare a solution containing approximately 1 mg of tauroursodeoxycholic acid per 1 mL.

[0571] Test solution: Take bear bile eye drops (batch number: 02211102), filter, and use the filtrate as the test solution.

[0572] Accurately measure 10 μL each of the test solution and the reference solution, and inject them according to the detection method under "2.1.6" in Example 2. Inject each solution six times consecutively, and record the chromatograms. The results are shown in Table 80. The retention time and peak area of ​​the main peak in the reference solution were calculated according to Formulas 3 and 4, and the RSD was calculated. The fingerprint chromatograms of each test sample were compared with the reference fingerprint chromatogram using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine," and the median method was used to generate the control fingerprint chromatogram. The similarity between the fingerprint chromatograms of each test sample and the control fingerprint chromatogram was calculated, and the results are shown in Tables 81 and 82. Figure 47 As shown, Figure 47 The superimposed chromatograms are used to compare the similarity of the injection precision of single-dose bear bile eye drops. Common peaks were counted and numbered, with the reference peak denoted by S, and the remaining common peaks denoted by sequential numbers. Tauroursodeoxycholic acid was used as the reference peak S in each test solution, and the relative retention time and relative peak area of ​​each common peak were calculated. The results are shown in Tables 82 and 83.

[0573] Table 80

[0574]

[0575] Table 81

[0576] Number of measurements 1 2 3 4 5 6 Similarity (median) 1.000 1.000 1.000 1.000 1.000 1.000

[0577] Table 82

[0578]

[0579] Table 83

[0580]

[0581]

[0582] Conclusions: Table 80 shows that after six consecutive injections of the reference solution, the RSDs for retention time and main peak area were 0.04% and 0.36%, respectively. Tables 82 and 83 show that after six consecutive injections of the test solution, the RSDs for retention time and peak area of ​​tauroursodeoxycholic acid in the S peak were 0.04% and 0.18%, respectively. The RSDs for relative retention time of each common peak in the test sample ranged from 0.01% to 0.04%, the RSDs for relative peak area of ​​each common peak ranged from 0.17% to 4.89%, and the RSD for total peak area was 0.20%. Table 81 shows that the similarity calculated using the similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine was 1.000, indicating that the instrument's injection precision was good.

[0583] ②Repetitiveness

[0584] Preparation of reference solution: Weigh an appropriate amount of tauroursodeoxycholic acid (batch number: 110816-202110) reference standard, dissolve and dilute with methanol to prepare a solution containing approximately 1 mg of tauroursodeoxycholic acid per mL. Test solution: Take bear bile eye drops (batch number: 02211102), filter, and use the filtrate as the test solution. (Prepare 6 portions using the same method.)

[0585] Take 10 μL of each of the above solutions and perform the determination according to the method under "2.1.6" in Example 2, and record the chromatograms. Use the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" to generate a reference fingerprint chromatogram using the median method. The similarity results of the fingerprint chromatograms of each test sample and the generated reference fingerprint chromatogram are shown in Table 84. Figure 48 As shown, Figure 48 This is a superimposed chromatogram comparing the similarity of repeatability tests of single-dose bear bile eye drops. Common peaks for each test sample were counted and numbered, with the reference peak denoted by S, and the remaining common peaks denoted by numbers in sequence. Using tauroursodeoxycholic acid as the reference peak S in each test sample solution, the relative retention time and relative peak area of ​​each common peak were calculated. The results are shown in Tables 85 and 86.

[0586] Table 84

[0587]

[0588] Table 85

[0589]

[0590] Table 86

[0591]

[0592] Conclusions: As shown in Tables 85 and 86, the relative retention times (RSDs) of the common peaks in the six test solutions ranged from 0.00% to 0.03%, and the relative peak areas (RSDs) ranged from 0.01% to 0.99%. As shown in Table 84, the similarity was calculated using the similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine, and the similarity was 1.000 for all results, indicating that the method has good repeatability.

[0593] ③ Intermediate precision test

[0594] To investigate the effect of random variation on precision, different analysts collected bear bile eye drops (batch number: 02211102) on different dates and performed the same procedures as described in section "② Repeatability" above, following the method in section "2.1.6" of Example 2. A total of 12 chromatograms of the test samples were collected for repeatability and intermediate precision tests. Using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine," a control fingerprint chromatogram was generated using the median method. The similarity between the fingerprint chromatograms of each test sample and the generated control fingerprint chromatogram was calculated, and the results are shown in Table 87. Figure 49 As shown, Figure 49 This is a superimposed chromatogram comparing the similarity of intermediate precision tests for single-dose bear bile eye drops. Common peaks in each test solution were counted and numbered, with the reference peak denoted by S, and the remaining common peaks represented by numbers in sequence. Using tauroursodeoxycholic acid as the reference peak S in each test solution, the relative retention time and relative peak area of ​​each common peak were calculated. The results are shown in Tables 88 and 89.

[0595] Table 87

[0596]

[0597] Table 88

[0598]

[0599] Table 89

[0600]

[0601] Conclusions: Tables 88 and 89 show that, for fingerprint chromatograms determined at different times, by different personnel, and using different instruments, the relative retention time RSD of each common peak in the 12 test sample solutions ranged from 0.44% to 1.15%, and the relative peak area RSD ranged from 0.70% to 8.36%. Table 87 shows that, using the similarity evaluation system for traditional Chinese medicine chromatographic fingerprint chromatograms, the similarity was 1.000 for all samples. This indicates that the intermediate precision of this method for fingerprint chromatogram determination is good and can meet the requirements for fingerprint chromatogram detection.

[0602] 1.7.3 Solution Stability

[0603] Reference solution: Weigh an appropriate amount of tauroursodeoxycholic acid (batch number: 110816-202110) reference standard, dissolve it in methanol, and prepare a solution containing approximately 1 mg of tauroursodeoxycholic acid per 1 mL.

[0604] Test solution: Take bear bile eye drops (batch number: 02211102), filter, and use the filtrate as the test solution.

[0605] The test solution and reference solution were placed at room temperature. The determination was performed according to the method described in section "2.1.6" of Example 2, and the chromatograms were recorded. The retention time and peak area of ​​the main peak in the reference solution were statistically analyzed, and the results are shown in Table 90. The fingerprint chromatograms of each test sample were compared with the reference chromatogram using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine." The fingerprint chromatogram of the test sample at 0 hours was used as the reference chromatogram. The similarity was calculated, and the results are shown in Tables 91 and 92. Figure 50 As shown, Figure 50 This is a superimposed chromatogram comparing the similarity of single-dose bear bile eye drops solution stability test samples. Common peaks at each time point were counted and numbered, with the reference peak denoted by S, and the remaining common peaks numbered sequentially. Using tauroursodeoxycholic acid as the reference peak S in each sample, the relative retention time and relative peak area of ​​each common peak were calculated, and the results are shown in Tables 92 and 93.

[0606] Table 90

[0607] Time (h) 0 10.5 13.5 16.5 18 Retention time (min) 34.796 34.797 34.816 34.804 34.832 Peak area 1492141 1482778 1479960 1488153 1483188 Time (h) 27 46.5 49.5 54 RSD(%, n=9) Retention time (min) 34.819 34.825 34.850 34.819 0.05 Peak area 1480394 1488740 1493532 1471568 0.46

[0608] Table 91

[0609]

[0610] Table 92

[0611]

[0612]

[0613] Table 93

[0614]

[0615] Conclusions: Table 90 shows that the RSDs of the main peak retention time and peak area of ​​the reference solution after different injection times at room temperature were 0.05% and 0.46%, respectively, indicating that the reference solution was stable for 54 hours at room temperature. Tables 92 and 93 show that the RSDs of the retention time and peak area of ​​tauroursodeoxycholic acid (S peak) of the test solution after different injection times at room temperature were 0.07% and 0.15%, respectively, the RSD of the total peak area was 0.16%, the RSDs of the relative retention times of common peaks in each chromatogram ranged from 0.01% to 0.06%, and the RSDs of the relative peak areas of each common peak ranged from 0.11% to 3.95%. Table 91 shows that the similarity calculated using the chromatographic fingerprint similarity evaluation system for traditional Chinese medicine with the 0-hour chromatogram as the reference chromatogram was 1.000, indicating that the test solution was stable for 49.5 hours at room temperature.

[0616] 1.7.4 Range Test

[0617] The range test mainly examines the applicability of the method to the analyte in the sample within a certain concentration range (higher concentration, lower concentration).

[0618] Reference solution: Weigh an appropriate amount of tauroursodeoxycholic acid (batch number: 110816-202110) reference standard, dissolve and dilute it with methanol to prepare a solution containing approximately 1 mg of tauroursodeoxycholic acid per 1 mL.

[0619] Test solution: Take bear bile eye drops (batch number: 02211102), filter, and use the filtrate as the test solution.

[0620] Take 6 μL, 8 μL, 10 μL, and 12 μL of the test solution respectively, and inject them according to the detection method under "2.1.6" in Example 2, and record the chromatograms. Using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine," with the 10 μL fingerprint chromatogram as the reference chromatogram, the results are shown in Table 94. Figure 51 As shown, Figure 51 This is a superimposed chromatogram for comparing the similarity of single-dose bear bile eye drops. The fingerprint chromatograms of each test sample were compared with the reference chromatogram to calculate the similarity. Common peaks were identified and numbered; the reference peak was denoted by S, and the remaining common peaks were numbered sequentially. Using tauroursodeoxycholic acid as the reference peak S in each test sample, the relative retention time and relative peak area of ​​each common peak were calculated. The results are shown in Tables 95 and 96.

[0621] Table 94

[0622] Injection volume (μL) 6 8 10 12 Similarity 1.000 1.000 1.000 1.000

[0623] Table 95

[0624]

[0625] Table 96

[0626]

[0627] Conclusions: Tables 95 and 96 show that within the injection volume range of 6 μL (60% of the current sample concentration) to 12 μL (120% of the current sample concentration), the relative retention time RSD of the common peaks in each chromatogram is 0.03% to 0.22%, and the relative peak area RSD of each common peak is 0.64% to 5.96%. Table 94 shows that the similarity calculated using the similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine is 1.000, indicating that the test results within the range of this method meet the requirements.

[0628] 1.7.5 Durability

[0629] Reference solution: Accurately weigh tauroursodeoxycholic acid (batch number: 110816-202110) reference standard, and add methanol to prepare a solution containing about 1 mg of tauroursodeoxycholic acid per 1 mL.

[0630] Test solution: Take bear bile eye drops (batch number: 02211102), filter, and use the filtrate as the test solution.

[0631] To examine the robustness of the detection method under section "2.1.6" of Example 2, the chromatographic conditions will be slightly adjusted below using different batches of Welch Ultimate XB-C. 18 A chromatographic column (15 cm long, 4.6 mm inner diameter, 5 μm particle size) was used for determination. The "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" was employed. Using the original conditions as the reference chromatogram, the fingerprint chromatograms of the test samples under each condition were compared with the reference chromatogram, and the similarity was calculated. The results are shown in Table 97. Figures 52-55 As shown, Figure 52 This is a superimposed graph comparing the similarity of pH values ​​adjusted in a durability test of single-dose bear bile eye drops. Figure 53 This is a superimposed graph comparing the similarity of fine-tuned salt concentrations in a durability test of single-dose bear bile eye drops. Figure 54 This is a superimposed graph comparing the similarity of the fine-tuned mobile phase ratio in a durability test of single-dose bear bile eye drops. Figure 55 This is a superimposed chromatogram comparing the similarity of chromatographic columns from different batches in a durability test of single-dose bear bile eye drops.

[0632] Table 97

[0633]

[0634] Conclusion: As shown in Table 97, by finely adjusting the pH (±0.2), salt concentration (±0.005M), organic phase ratio of mobile phase B (±1%), and changing different chromatographic columns, the similarity of the test solution was 1.000 under each chromatographic condition, indicating that this method has good robustness.

[0635] 1.7.6 Test Results

[0636] Three batches of bear bile eye drops (batch numbers: 02211102, 02211103, 02211104) were tested according to the method described in section "2.1.6" of Example 2. Chromatograms were recorded, and a reference fingerprint was generated using the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" with the median method. The similarity between the fingerprints of each batch of test samples and the reference fingerprint was compared. The results are shown in Table 98. Figure 56 As shown, Figure 56 The above is a superimposed graph comparing the similarity of different batches of single-dose bear bile eye drops. As shown in Table 98, the similarity scores are all greater than 0.90.

[0637] Table 98

[0638] batch number 02211102 02211103 02211104 Similarity 1.000 1.000 1.000

[0639] Comparative Example 1

[0640] The fingerprint spectrum of the single-dose bear bile eye drops described in Example 3 was constructed using a ternary mobile phase method, as detailed below:

[0641] (1) Sample and reference standard

[0642] As shown in Table 99:

[0643] Table 99

[0644] name batch number source Tauroursodeoxycholic acid 110816-202110 China National Institutes for Food and Drug Control Bear bile eye drops 02211102、02211104、02230801 Yunnan Dai Medicine Co., Ltd.

[0645] (2) Reagents

[0646] As shown in Table 100:

[0647] Table 100

[0648] name level source methanol HPLC Merck Group Limited Partnership Acetonitrile HPLC Merck Group Limited Partnership Phosphoric acid AR Chengdu Kelong Chemical Co., Ltd. Sodium dihydrogen phosphate AR Tianjin Fengchuan Chemical Reagent Technology Co., Ltd.

[0649] (3) Instruments

[0650] As shown in Table 101:

[0651] Table 101

[0652] Instrument Name model source High Performance Liquid Chromatography LC-2030 Shimadzu Corporation, Japan Electronic balance BP-221S Sertoris, Germany Electronic balance XS-205 Mettler Toledo Instruments (Shanghai) Co., Ltd. pH meter pHS-3C Shanghai Precision Scientific Instruments Co., Ltd.

[0653] (4) Chromatographic column

[0654] As shown in Table 102:

[0655] Table 102

[0656] Serial Number Column name 1 <![CDATA[Welch Ultimate XB-C 18 ;SN:60190401100]]>

[0657] (5) Chromatographic conditions

[0658] Mobile phase A: Acetonitrile;

[0659] Mobile phase B: Methanol;

[0660] Mobile phase C: 0.03 mol / L sodium dihydrogen phosphate (pH adjusted to 4.4 with phosphoric acid)

[0661] Gradient elution was performed according to the specifications in Table 103; the detection wavelength was 210 nm; the column temperature was 35 °C; and the flow rate was 0.8 mL per minute.

[0662] Table 103

[0663] Time (minutes) Mobile phase A (%) Mobile phase B (%) Mobile phase C (%) 0 0 10 90 60 60 4 36 70 60 4 36 75 0 10 90 90 0 10 90

[0664] Preparation of reference solution: Accurately weigh tauroursodeoxycholic acid reference standard and add methanol to prepare a solution containing approximately 1 mg of tauroursodeoxycholic acid per 1 mL.

[0665] Preparation of test solution: Take this product, filter it, and use the filtrate as the test solution.

[0666] Determination method: Accurately pipette 10 μL each of the reference solution and the test solution into the liquid chromatograph, measure and record the chromatogram to obtain the result.

[0667] Chromatographic peak integration parameters: No integration is performed in the first 5 minutes, and the minimum peak area is no less than 1.0% of the total peak area.

[0668] (6) Results

[0669] Following the method in (5), a ternary elution system was used for gradient elution and detection conditions. The reference solution and the test solution were detected separately, and the detection chromatograms are shown below. Figures 57-60 As shown, Figure 57 This is the fingerprint spectrum of the reference solution for batch number 20240620, constructed using the ternary mobile phase method. Figure 58 The fingerprint spectrum of the test sample solution with batch number 02211102 was constructed using the ternary mobile phase method. Figure 59 The fingerprint spectrum of the test sample solution with batch number 02211104 was constructed using the ternary mobile phase method. Figure 60 The superimposed comparison spectra of test sample solutions with batch numbers 02230801, 02211102 and 02211104 are constructed using the ternary mobile phase method.

[0670] Example 4

[0671] Fingerprint mapping was constructed for the single-dose bear bile eye drops described in Example 3:

[0672] (1) Sample and control: Same as Comparative Example 1

[0673] (2) Reagents: Same as Comparative Example 1

[0674] (3) Instruments: Same as Comparative Example 1

[0675] (4) Chromatographic column:

[0676] Welch Ultimate XB-C uses octadecylsilane-bonded silica gel as a filler. 18 Chromatographic column (15cm length, 4.6mm inner diameter, 5μm particle size);

[0677] (5) Chromatographic conditions:

[0678] Mobile phase A: Acetonitrile;

[0679] Mobile phase B: 0.03 mol / L sodium dihydrogen phosphate solution (pH adjusted to 4.4 with phosphoric acid): methanol (volume ratio 90:10).

[0680] Gradient elution was performed according to the specifications in Table 104; the detection wavelength was 210 nm; the column temperature was 35 °C; and the flow rate was 0.8 mL per minute.

[0681] Table 104

[0682] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 0 100 60 60 40 70 60 40 75 0 100 90 0 100

[0683] Preparation of reference solution: Accurately weigh tauroursodeoxycholic acid reference standard and add methanol to prepare a solution containing approximately 1 mg of tauroursodeoxycholic acid per 1 mL.

[0684] Preparation of test solution: Take this product, filter it, and use the filtrate as the test solution.

[0685] Determination method: Accurately pipette 10 μL each of the reference solution and the test solution into the liquid chromatograph, measure and record the chromatogram to obtain the result.

[0686] Chromatographic peak integration parameters: No integration is performed in the first 5 minutes, and the minimum peak area is no less than 1.0% of the total peak area.

[0687] (6) Results

[0688] Gradient elution was performed according to the method and detection conditions for a binary elution system as described in (5). The reference solution and the test solution were detected separately, and the detection chromatograms are shown below. Figures 61-63 As shown, Figure 61This is the fingerprint spectrum of the reference solution for batch number 20211203, constructed using the binary mobile phase method. Figure 62 The fingerprint spectrum of the test sample solution with batch number 02211102 was constructed using the binary mobile phase method. Figure 63 This is the fingerprint spectrum of the test sample solution of batch number 02211104 constructed using the binary mobile phase method.

[0689] The comparison of the detection spectra of Comparative Example 1 and Example 3 is as follows: Figures 64-66 As shown, Figure 64 To compare the fingerprint spectrum using different system-reference solutions. Figure 65 To compare the fingerprint spectra of different systems and test solutions (batch number 02211102), Figure 66 This is a comparison of fingerprint spectra of different systems and test solutions (batch number 02211104).

[0690] Results: The comparison of the above spectra shows that the number of absorption peaks is almost identical when using binary and ternary mobile phases. However, the baseline is more stable when using the binary mobile phase, while the baseline drift is larger when using the ternary mobile phase. The baseline is relatively stable from 0 to 10 min, but gradually drifts downwards from 10 to 60 min, failing to meet the baseline stability requirements for liquid chromatography detection. In summary, the binary mobile phase is more suitable for detecting bear bile eye drops fingerprints due to its stable baseline compared to the ternary mobile phase.

[0691] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for detecting bear bile powder or its preparations, characterized in that, include: The test solution of bear bile powder or its preparation was detected by HPLC. The chromatographic conditions for HPLC were as follows: Octadecylsilane-bonded silica gel was used as the stationary phase; Acetonitrile was used as the mobile phase A, and a mixed solution of sodium dihydrogen phosphate and methanol with a volume ratio of (89-91):(9-11) was used as the mobile phase B. The gradient elution procedure is as follows: 。 2. The detection method according to claim 1, characterized in that, The concentration of sodium dihydrogen phosphate in the sodium dihydrogen phosphate solution is 0.025 mol / L to 0.035 mol / L.

3. The detection method according to claim 1, characterized in that, In mobile phase B, the sodium dihydrogen phosphate solution is a sodium dihydrogen phosphate solution containing phosphoric acid; the pH of the sodium dihydrogen phosphate solution is 4.2 to 4.

6.

4. The detection method according to claim 1, characterized in that, The flow rate of the mobile phase is 0.5 mL / min to 1 mL / min.

5. The detection method according to claim 1, characterized in that, The stationary phase is Welch Ultisil. TM C 18 Column or Welch Ultimate XB-C 18 Chromatographic column.

6. The detection method according to claim 1, characterized in that, The column temperature of the stationary phase is 30℃~40℃.

7. The detection method according to claim 1, characterized in that, The detection wavelength of the HPLC is 210 nm to 220 nm.

8. The detection method according to claim 1, characterized in that, The test solution of the bear bile powder or its preparation is prepared by the following method: The bear bile powder or its preparation was dissolved in water as an extraction solvent, and the resulting solution was filtered through an aqueous filter membrane to obtain a test solution of the bear bile powder or its preparation.

9. A method for constructing a fingerprint spectrum of bear bile powder or its preparations, characterized in that, Includes the following steps: S1) Detect different batches of bear bile powder or its preparations using the detection method according to any one of claims 1 to 8; S2) Identify the common peak; S3) The fingerprint spectrum of bear bile powder or its preparations was established using the median method.

10. The construction method according to claim 9, characterized in that, A fingerprint spectrum of bear bile powder or its preparations was established using sodium tauroursodeoxycholate as a reference peak. The relative retention time of sodium tauroursodeoxycholate was 1.

000. The fingerprint spectrum included at least six common peaks, and the relative retention times of the common peaks were 0.629–0.650, 0.814–0.830, 0.845–0.860, 0.971–0.990, 1.170–1.180, and 1.747–1.800, respectively.

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