Method for determining chiral isomer impurities in tadalafil

Abstract

The invention discloses a method for determining chiral isomer impurities in tadalafil. The method comprises the following steps: carrying out liquid chromatographic analysis on tadalafil to realize detection of main components and chiral isomer impurities; the detection conditions of the liquid chromatographic analysis are as follows: a chromatographic column takes an amylose-tri (3, 5-dimethylphenyl carbamate) derivative as a filling agent; the mobile phase is composed of normal hexane and isopropanol in a volume ratio of 1: (0.95-1.05); the elution mode is isocratic elution; and the detection wavelength is 222nm. The method provided by the invention can effectively separate tadalafil and three chiral isomers and oxide impurities thereof, is high in separation degree, can realize quantitative detection, and is high in detection sensitivity, wide in linear range and good in repeatability.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and specifically relates to a method for detecting related substances of tadalafil, and is particularly suitable for detecting chiral isomer impurities of tadalafil. Background Art

[0002] Tadalafil, chemical name: (6R-12aR)-6-(1,3-benzodioxol-5-yl)-2-methyl-2,3,6,7,12,12a-hexahydropyrazino[1',2'-1,6]-pyrido[3,4-b]indole-1,4-dione, molecular formula: C 22 H 19 N3O4, relative molecular mass: 389.41. Tadalafil tablets are selective reversible inhibitors of cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase 5 (PDE-5), which can specifically inhibit PDE-5 and are now mainly used to treat erectile dysfunction (ED). Compared with the PDE-5 inhibitors currently used in clinical practice, tadalafil has a longer half-life, good cardiovascular safety, does not increase the risk and severity of cardiovascular events, and has good safety and tolerability. In addition, PDE-5 inhibitors can also effectively treat pulmonary hypertension, improve patients' exercise tolerance, quality of life and survival rate; in addition, tadalafil can also inhibit acute hypoxic pulmonary vasoconstriction and inhibit hypoxia-induced pulmonary artery expression of tumor necrosis factor. In the treatment of hypoxic pH, it has certain advantages over the other two PDE-5 inhibitors, sildenafil and vardenafil; in the study of animal models of metabolic syndrome, it was found that tadalafil can reduce visceral fat accumulation and improve insulin sensitivity. There are two chiral centers in the molecular structure of tadalafil. The optical isomers include tadalafil, isomer A, isomer B and isomer C (structures as follows), among which tadalafil isomer B and isomers A and C are enantiomers and diastereomers of tadalafil, respectively.

[0003]

[0004] There are significant differences in the absorption, distribution, metabolism and excretion of chiral drug enantiomers in the body, which can cause them to produce different or even completely opposite pharmacological and toxicological effects. The use of single enantiomers as drugs has always attracted the attention of pharmaceutical companies and research institutions. In the development of chiral drugs, enantiomeric impurities are crucial to the quality and safety of chiral drugs. The similarity of the physical and chemical properties between a pair of enantiomers makes the separation and detection of enantiomeric impurities a research hotspot and difficulty in drug analysis.

[0005] The chemical structures of diastereomers are exactly the same, only the configurations of the chiral carbon atoms are different, so their reversed-phase chromatographic behaviors are also very similar, and the separation degree is often difficult to meet the detection requirements. The impurity spectrum of drugs often involves many organic impurities, so the detection method must ensure that all impurities can be effectively detected on the one hand, and on the other hand, the separation degree between all impurities must be high enough so that each specific impurity can be quantitatively detected separately. Therefore, it is of great practical value to develop a quantitative detection method that can detect a large number of impurities, has high separation degree and high sensitivity. Summary of the invention

[0006] The purpose of the present invention is to provide a method for determining chiral isomer impurities in tadalafil. The chromatographic column of the method uses an amylose-tris (3,5-xylylcarbamate) derivative as a filler, and the mobile phase consists of n-hexane and isopropanol in a volume ratio of 1:1. Tadalafil and its three chiral isomers can be effectively separated with high separation degree, quantitative detection can be achieved at the same time, and the detection sensitivity is high, the linear range is wide, and the repeatability is good.

[0007] To achieve the above object, the present invention adopts the following technical solution:

[0008] A method for determining impurities in tadalafil comprises the following steps:

[0009] Liquid chromatography analysis of tadalafil samples was performed to achieve detection of major components and impurities;

[0010] The impurities include chiral isomer impurities of tadalafil;

[0011] The detection conditions of the liquid chromatography analysis include:

[0012] Chromatographic column: Amylose-tris (3,5-dimethylphenylcarbamate) derivative is used as filler;

[0013] Mobile phase: composed of n-hexane and isopropanol in a volume ratio of 1: (0.95-1.05);

[0014] Elution mode: isocratic elution;

[0015] Detection wavelength: 222nm.

[0016] Furthermore, the detection conditions of the liquid chromatography analysis also include:

[0017] Flow rate: 0.6~0.9mL / min;

[0018] Column temperature: 25-35°C;

[0019] Injection volume: 20 μL.

[0020] In the above method, the chromatographic column can be specifically CHIRALCEL AD-H 4.6mm×250mm, 5μm or a chromatographic column with equivalent performance.

[0021] In the above method, the chiral isomer impurities include three chiral isomers of tadalafil, which are enantiomers and diastereomers of tadalafil.

[0022] In the above method, the detection is a qualitative detection.

[0023] Furthermore, the impurities also include oxidized impurities of tadalafil, the structural formula of which is as follows:

[0024]

[0025] In the above method, the detection is quantitative detection.

[0026] In the above method, preferably, in the qualitative detection and the quantitative detection, a system consisting of n-hexane, isopropanol and acetonitrile in a volume ratio of 2:2:1 is used as the solvent for the reference substance and the test substance.

[0027] The present invention has the following beneficial effects:

[0028] The method of the invention can perform qualitative and quantitative detection on chiral isomer impurities in tadalafil, can effectively separate tadalafil and its three chiral isomers, can detect a large number of impurities with high separation, and can also simultaneously realize qualitative detection of tadalafil oxidation impurities. The detection limit of WTD-IM-A is 0.003 μg / ml, and the quantitative limit is 0.005 μg / ml. The detection limit of WTD is 0.003 μg / ml, and the quantitative limit is 0.005 μg / ml. The detection limit of WTD-IM-B is 0.005 μg / ml, and the quantitative limit is 0.010 μg / ml. The detection limit of WTD-IM-C is 0.010 μg / ml, and the quantitative limit is 0.020 μg / ml. The detection sensitivity of each impurity is 0. The linear range of WTD-IM-A is 0.005μg / ml~0.815μg / ml (r=1.0000), the linear range of WTD is 0.005μg / ml~0.757μg / ml (r=0.9999), the linear range of WTD-IM-B is 0.010μg / ml~0.778μg / ml (r=0.9999), the linear range of WTD-IM-C is 0.020μg / ml~0.767μg / ml (r=0.9999), each impurity and main component are within the range of quantitative limit~200%, and the linear relationship between the detection of each impurity and its concentration is good; the recovery rate of each impurity is between 90% and 110%, and the repeatability is good. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the system suitability chromatogram of Experimental Group 1 in Example 1 of the present invention, 1-Impurity A, 2-Tadalafil, 3-Oxidized impurity, 4-Impurity B, 5-Impurity C.

[0030] Figure 2 These are the chromatograms of the WTD-IM-A reference solution (a), the WTD-IM-P reference solution (oxidized impurities) (b), the WTD-IM-B reference solution (c), and the WTD-IM-C reference solution (d) in Example 1 of the present invention. DETAILED DESCRIPTION

[0031] As described in the background technology, a quantitative detection method with a large number of detected impurities, high separation and high sensitivity needs to be further developed. The method for determining impurities in tadalafil provided by the present invention comprises the following steps: performing liquid chromatography analysis on a tadalafil sample to detect the main component and impurities; the impurities include chiral isomer impurities of tadalafil; the detection conditions of the liquid chromatography analysis include: chromatographic column: using amylose-tris (3,5-xylylcarbamate) derivative as a filler; mobile phase: composed of n-hexane and isopropanol in a volume ratio of 1: (0.95-1.05), such as n-hexane and isopropanol in a volume ratio of 1: 1; elution mode: isocratic elution; detection wavelength: 222nm. Further, the detection conditions of the liquid chromatography analysis also include: flow rate: 0.6-0.9mL / min, such as 0.75mL / min; column temperature: 25-35°C, such as 30°C; injection volume: 20μL. In a specific embodiment of the present invention, the chromatographic column is CHIRALCEL AD-H 4.6mm×250mm, 5μm or a chromatographic column with equivalent performance. That is, the length of the chromatographic column (such as CHIRALCEL AD-H chromatographic column) used is 250mm, the inner diameter is 4.6mm, and the particle size of the stationary phase is 5μm.

[0032] According to the present invention, the chiral isomer impurities include three chiral isomers of tadalafil, which are enantiomers and diastereomers of tadalafil. Among them, the structures of tadalafil (WTD), isomer A (WTD-IM-A), isomer B (WTD-IM-B) and isomer C (WTD-IM-C) are as follows:

[0033]

[0034] In at least one embodiment of the present invention, the detection is a qualitative detection. The qualitative detection comprises the following steps: using the standard substances corresponding to the main component and the impurity as reference substances respectively; using the tadalafil sample as a test substance; comparing the retention time of the chromatographic peak in the chromatogram of the test substance with the retention time of the chromatographic peak of the reference substance to achieve the qualitative detection. Specifically as follows:

[0035] Compare the retention time of the chromatographic peak in the chromatogram of the test sample with the retention time of the chromatographic peak of the chiral isomer impurity reference substance represented by formula WTD-IM-A. If a chromatographic peak with the same retention time as the chromatographic peak of the chiral isomer impurity reference substance represented by formula WTD-IM-A appears in the chromatogram of the test sample, then the test sample contains the chiral isomer impurity represented by formula WTD-IM-A.

[0036] Compare the retention time of the chromatographic peak in the chromatogram of the test sample with the retention time of the chromatographic peak of the chiral isomer impurity reference substance represented by formula WTD-IM-B. If a chromatographic peak with the same retention time as the chromatographic peak of the chiral isomer impurity reference substance represented by formula WTD-IM-B appears in the chromatogram of the test sample, then the test sample contains the chiral isomer impurity represented by formula WTD-IM-B;

[0037] Compare the retention time of the chromatographic peak in the chromatogram of the test sample with the retention time of the chromatographic peak of the chiral isomer impurity reference substance represented by formula WTD-IM-C. If a chromatographic peak with the same retention time as the chromatographic peak of the chiral isomer impurity reference substance represented by formula WTD-IM-C appears in the chromatogram of the test sample, then the test sample contains the chiral isomer impurity represented by formula WTD-IM-C.

[0038] In the present invention, WTD-IM-A, tadalafil, WTD-IM-B, and WTD-IM-C are peaked in sequence. In a specific embodiment of the present invention, the mobile phase is composed of n-hexane and isopropanol in a volume ratio of 1:1, the flow rate is 0.75 mL / min, and the column temperature is 30°C; the retention times of WTD-IM-A, tadalafil, WTD-IM-B, and WTD-IM-C are 9.198 min, 11.494 min, 17.369 min, and 24.888 min, respectively.

[0039] In at least one embodiment of the present invention, the impurities further include oxidized impurities of tadalafil, the structural formula of which is as follows:

[0040]

[0041] The method of the present invention can simultaneously realize the qualitative detection of the oxidation impurities of tadalafil.

[0042] The details are as follows:

[0043] Compare the retention time of the chromatographic peak in the chromatogram of the test sample with the retention time of the chromatographic peak of the oxidized impurity reference substance of tadalafil. If a chromatographic peak with the same retention time as the chromatographic peak of the oxidized impurity reference substance of tadalafil appears in the chromatogram of the test sample, the oxidized impurity of tadalafil is contained in the test sample.

[0044] In the present invention, WTD-IM-A, tadalafil, tadalafil oxidized impurities, WTD-IM-B, and WTD-IM-C are peaked in sequence. In a specific embodiment of the present invention, the mobile phase is composed of n-hexane and isopropanol in a volume ratio of 1:1, the flow rate is 0.75 mL / min, and the column temperature is 30°C; the retention times of WTD-IM-A, tadalafil, tadalafil oxidized impurities, WTD-IM-B, and WTD-IM-C are 9.198 min, 11.494 min, 14.713 min, 17.369 min, and 24.888 min, respectively.

[0045] The method of the present invention can simultaneously detect tadalafil chiral isomer impurities and oxidized impurities, and can achieve quality control of tadalafil chiral isomer impurities and oxidized impurities.

[0046] In at least one embodiment of the present invention, the detection is a quantitative detection. The quantitative detection can be performed using a principal component self-control method, comprising the following steps:

[0047] Measure the peak area of ​​each impurity in the chromatogram of the test solution and compare it with the peak area of ​​the main component of the control solution to calculate the content of each impurity;

[0048] Preparation of control solution: Accurately measure 0.5 ml of the test solution, place it in a 100 ml volumetric flask, dilute to the scale with diluent, shake well, and obtain.

[0049] In at least one embodiment of the present invention, in the qualitative detection and the quantitative detection, a system consisting of n-hexane, isopropanol and acetonitrile in a volume ratio of 2:2:1 is used as the solvent for the reference substance and the test substance.

[0050] In at least one embodiment of the present invention, the linear range of the chiral isomer impurity represented by formula WTD-IM-A is 0.005 μg / ml to 0.815 μg / ml;

[0051] The linear range of the chiral isomer impurity represented by formula WTD-IM-B is 0.010 μg / ml to 0.778 μg / ml;

[0052] The linear range of the chiral isomer impurity represented by formula WTD-IM-C is 0.020 μg / ml to 0.767 μg / ml.

[0053] In at least one embodiment of the present invention, the concentration of the test solution is 0.25 mg / mL.

[0054] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0055] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0056] Example 1. System Applicability

[0057] 1. Solution preparation and determination method

[0058] Solvent: n-hexane-isopropanol-acetonitrile (v:v:v=2:2:1)

[0059] Take an appropriate amount of the product for test solution, weigh it accurately, add solvent to dissolve it by ultrasonic, and quantitatively dilute it to make a solution containing about 0.25 mg per 1 ml.

[0060] For the control solution, accurately measure an appropriate amount of the test sample solution and quantitatively dilute it with solvent to make a solution containing approximately 1.25 μg per 1 ml.

[0061] System suitability solution: Take appropriate amounts of tadalafil reference substance and impurity WTD-IM-A, WTD-IM-B, and WTD-IM-C reference substances, add solvent to dissolve by ultrasonication, and dilute to produce a solution containing approximately 0.25 mg of tadalafil and approximately 0.375 μg of each of the other impurities per 1 ml.

[0062] Determination method: Accurately measure the test solution and control solution and inject them into the liquid chromatograph respectively.

[0063] 2. Determination of chromatographic conditions

[0064] Experimental group 1: The column was filled with amylose-tris (3,5-diphenylylcarbamate) derivative (CHIRALCELAD-H 4.6 mm × 250 mm, 5 μm) and n-hexane-isopropanol (1:1) was used as the mobile phase;

[0065] Comparative group 1: Octylsilane bonded silica gel was used as filler (Agilent ZORBAX SB-C8 4.6 mm × 150 mm, 3.5 μm) as the chromatographic column, and acetonitrile: trifluoroacetic acid TFA: water (350:1:650) was used as the mobile phase.

[0066] The above experimental group 1 and comparative group 1 were subjected to liquid chromatography analysis under the conditions of a flow rate of 0.75 ml per minute, a column temperature of 30°C, a detection wavelength of 222 nm, and an injection volume of 20 μl. The chromatogram of the experimental group 1 is shown in FIG. Figure 1 As shown, impurities WTD-IM-A, tadalafil, WTD-IM-B, and WTD-IM-C eluted in sequence with retention times of 9.198 min, 11.494 min, 17.369 min, and 24.888 min, respectively. The retention time of the oxidized impurity was 14.713 min. Each impurity could be completely separated from the main peak.

[0067] Figure 2 The chromatograms are WTD-IM-A reference solution (a), WTD-IM-P reference solution (oxidized impurities) (b), WTD-IM-B reference solution (c), and WTD-IM-C reference solution (d). The blank solvent does not interfere with the detection of impurities.

[0068] The system suitability results of each experimental group are shown in Tables 1 and 2.

[0069] Table 1. System suitability of experimental group 1

[0070] name Theoretical plate number Separation Tailing Factor WTD-IM-A 6883 -- 1.152 WTD 7006 4.511 1.150 WTD-IM-B 6542 3.384 1.072 WTD-IM-C 5634 6.605 1.062

[0071] Table 2. System suitability of comparison group 1

[0072] name Theoretical plate number Separation Tailing Factor WTD / WTD-IM-B 13849 -- 1.0 WTD-IM-A / C 14955 4.4 1.0

[0073] It can be seen from Tables 1 and 2 above that when the stationary phase and mobile phase in Experimental Group 1 are used, the theoretical plate number of the main peak is the highest, and the separation degree between the impurity and the main peak is the highest, indicating that the system has good applicability and the best separation effect. However, when the stationary phase and mobile phase in Comparative Group 1 are used, the chiral impurities cannot be effectively separated.

[0074] Example 2: Injection Precision

[0075] Take an appropriate amount of WTD and dilute it with solvent to make a standard solution containing about 1.25μg concentration in each 1ml. Inject it continuously for 5 times. The retention times are 11.521min, 11.513min, 11.514min, 11.498min, and 11.478min, respectively. The RSD of retention time is 0.1%. The peak areas are 226187, 220866, 221807, 222937, and 224374, respectively, and the RSD of peak area is 0.9%. From the above results, it can be seen that the RSD of the peak area of ​​the main peak is less than 2.0%, and the RSD of the retention time is less than 1.0%, indicating that the injection precision is good.

[0076] Example 3, linear range

[0077] Take WTD-IM-A standard solution and dilute it with solvent to a standard solution with a concentration of 0.005μg / ml to 0.836μg / ml. The concentration gradient is 0.0056, 0.0418, 0.0836, 0.2091, 0.4182, 0.6273, 0.8364μg / ml, a total of 7 concentrations of standard solution, record the peak area of ​​the chromatographic peak at each concentration. Use concentration as the horizontal axis and peak area as the vertical axis to establish a standard curve.

[0078] Take the WTD standard solution and dilute it with solvent to a standard solution with a concentration of 0.005μg / ml to 0.757μg / ml. The concentration gradient is 0.0050, 0.0378, 0.0757, 0.1892, 0.3784, 0.5676, 0.7568μg / ml, a total of 7 concentrations of standard solution, record the peak area of ​​the chromatographic peak at each concentration. Use concentration as the horizontal axis and peak area as the vertical axis to establish a standard curve.

[0079] Take WTD-IM-B standard solution and dilute it with solvent to a standard solution with a concentration of 0.010μg / ml to 0.778μg / ml. The concentration gradient is 0.0104, 0.0391, 0.0781, 0.1953, 0.3907, 0.5860, 0.7813μg / ml, a total of 7 concentrations of standard solution, record the peak area of ​​the chromatographic peak at each concentration. Use concentration as the horizontal axis and peak area as the vertical axis to establish a standard curve.

[0080] Take the WTD-IM-C standard solution and dilute it with solvent to a standard solution with a concentration of 0.020μg / ml to 0.768μg / ml. The concentration gradient is 0.0204, 0.0384, 0.0768, 0.1920, 0.3840, 0.5760, 0.7681μg / ml, a total of 7 concentrations of standard solution, record the peak area of ​​the chromatographic peak at each concentration. Use concentration as the horizontal axis and peak area as the vertical axis to establish a standard curve.

[0081] The experimental results are shown in the following table:

[0082] Table 3. Linear range results of main components and chiral impurities

[0083]

[0084] Conclusion: The impurities and main components were within the range of quantitative limit ~200%, and the detection of each impurity had a good linear relationship with its concentration.

[0085] Example 3, detection limit

[0086] Take appropriate amounts of tadalafil reference substance, WTD-IM-A reference substance, WTD-IM-B reference substance and WTD-IM-C reference substance, accurately weigh them, dissolve them in solvent, and dilute them step by step with solvent to appropriate concentrations. Under the chromatographic conditions of experimental group 1 in the above embodiment 1, the detection limit is determined by the injection volume of the chromatograph at the concentration corresponding to the signal-to-noise ratio of >3:1. The detection limit results of each impurity are shown in the following table.

[0087] Table 4. Chromatographic peak results of main components and detection limits of various chiral impurities

[0088]

[0089] Conclusion: The detection limits of each impurity meet the requirements and the sensitivity is high.

[0090] Example 4, Quantitation Limit

[0091] Take appropriate amounts of tadalafil reference substance, WTD-IM-A reference substance, WTD-IM-B reference substance and WTD-IM-C reference substance, accurately weigh them, dissolve them in solvent, and dilute them step by step with solvent to appropriate concentrations. Under the chromatographic conditions of Experimental Group 1 in Example 1 above, the detection limit is determined by the injection volume of the chromatograph at a concentration corresponding to a signal-to-noise ratio of >10:1. The detection limit results of each impurity are shown in the following table.

[0092] Table 5. Chromatographic peak results of main components and quantitative limits of each chiral impurity

[0093]

[0094] Conclusion: The quantitative limits of each impurity meet the requirements and the sensitivity is high.

[0095] Example 5: Accuracy

[0096] Preparation of impurity stock solution: Accurately weigh appropriate amount of impurity WTD-IM-A, WTD-IM-B, WTD-IM-C reference substances, dissolve in solvent and quantitatively dilute to make a solution containing about 18.75 μg per 1 ml, shake well, and use as impurity stock solution.

[0097] Preparation of test solution: Accurately weigh an appropriate amount of tadalafil, dissolve it in solvent and quantitatively dilute it to make a solution containing about 0.25 mg per 1 ml, shake well, and use it as the test solution.

[0098] Preparation of reference solution: Accurately measure 0.5 ml of impurity stock solution, place in a 25 ml volumetric flask, dilute to scale with solvent, shake well, and use as reference solution. Prepare 2 portions in parallel.

[0099] Preparation of accuracy sample solution (limit 50%): accurately weigh an appropriate amount of tadalafil, add solvent and shake to dissolve, then add an appropriate amount of impurity stock solution, quantitatively dilute with solvent to make a solution containing about 0.25 mg of tadalafil and 0.1875 μg of each impurity per 1 ml, shake well, and prepare 3 copies in parallel.

[0100] Preparation of accuracy sample solution (limit 100%): accurately weigh an appropriate amount of tadalafil, add solvent and shake to dissolve, then add an appropriate amount of impurity stock solution, quantitatively dilute with solvent to make a solution containing about 0.25 mg of tadalafil and 0.375 μg of each impurity per 1 ml, shake well, and prepare 3 copies in parallel.

[0101] Preparation of accuracy sample solution (limit 150%): accurately weigh an appropriate amount of tadalafil, add solvent and shake to dissolve, then add an appropriate amount of impurity stock solution, quantitatively dilute with solvent to make a solution containing about 0.25 mg of tadalafil and 0.5625 μg of each impurity per 1 ml, shake well, and prepare 3 copies in parallel.

[0102] The experimental results are shown in the following table.

[0103] Table 6. Results of impurity recovery

[0104] name Average recovery rate% RSD% WTD-IM-A 102.4 2.1 WTD-IM-B 99.31 4.3 WTD-IM-C 102.1 2.9

[0105] Conclusion: The recovery rates of all impurities were between 90% and 110%, indicating that the method of the present invention has good accuracy.

[0106] Example 6, Repeatability

[0107] Prepare 6 spiked test solutions in parallel

[0108] Preparation of impurity stock solution: Accurately weigh appropriate amount of impurity WTD-IM-A, WTD-IM-B, WTD-IM-C reference substances, dissolve in solvent and quantitatively dilute to make a solution containing about 18.75 μg per 1 ml, shake well, and use as impurity stock solution.

[0109] Spiked test solution: accurately weigh an appropriate amount of tadalafil, add solvent and shake to dissolve, then add an appropriate amount of impurity stock solution, quantitatively dilute with solvent to make a solution containing about 0.25 mg of tadalafil and 0.375 μg of each impurity per 1 ml, shake well, and the solution is ready. Prepare 6 portions in parallel.

[0110] Control solution: Accurately measure 0.5 ml of the test solution, place it in a 100 ml volumetric flask, dilute to the scale with solvent, shake well, and obtain.

[0111] The experimental results are shown in the following table:

[0112] Table 7. Repeatability results of each impurity

[0113] name Repeatability RSD% WTD-IM-A 3.4 WTD-IM-B 3.0 WTD-IM-C 3.3

[0114] Conclusion,It can be seen from the above table that the RSD of each impurity detected in the spiked test solution is less than 10.0%, indicating that the method has good repeatability.

[0115] Example 7, intermediate precision

[0116] Two experimenters prepared 6 portions of spiked test solution in parallel at different times and using different instruments, totaling 12 portions.

[0117] Preparation of impurity stock solution: Accurately weigh appropriate amount of impurity WTD-IM-A, WTD-IM-B, WTD-IM-C reference substances, dissolve in solvent and quantitatively dilute to make a solution containing about 18.75 μg per 1 ml, shake well, and use as impurity stock solution.

[0118] Spiked test solution: accurately weigh an appropriate amount of tadalafil, add solvent and shake to dissolve, then add an appropriate amount of impurity stock solution, quantitatively dilute with solvent to make a solution containing about 0.25 mg of tadalafil and 0.375 μg of each impurity per 1 ml, shake well, and the solution is ready. Prepare 6 portions in parallel.

[0119] Control solution: Accurately measure 0.5 ml of the test solution, place it in a 100 ml volumetric flask, dilute to the scale with solvent, shake well, and obtain.

[0120] The experimental results are shown in the following table:

[0121] Table 8. Intermediate precision test results of various impurities

[0122]

[0123]

[0124] Conclusion,It can be seen from the above table that the RSD of each impurity detected in the spiked test solution is less than 10.0%, indicating that the intermediate precision of this method is good.

[0125] Example 8, Durability

[0126] Durability examines the extent to which the results are not affected by slight changes in chromatographic conditions, with the aim of providing a basis for considerations when the method is used for routine testing. The durability of this method mainly examines factors such as column temperature, mobile phase ratio and flow rate in chromatographic conditions.

[0127] The measurement conditions vary as follows:

[0128] Table 9. Durability range of inspection and measurement conditions

[0129] Chromatographic parameters Specified value Scope of change Column temperature 30℃ ±5℃ Mobile phase ratio Hexane and isopropanol (1:1) ±5％ Flow rate 0.75mL / min ±0.15ml / min

[0130] Prepare the test solution and examine the changes in the results of the relevant substances of the batch of samples under the above different chromatographic conditions to examine the influence of slight changes in chromatographic conditions on the determination results. The experimental process is as follows:

[0131] Preparation of impurity stock solution: Accurately weigh appropriate amount of impurity WTD-IM-A, WTD-IM-B, WTD-IM-C reference substances, dissolve in solvent and quantitatively dilute to make a solution containing about 18.75 μg per 1 ml, shake well, and use as impurity stock solution.

[0132] System suitability solution: Accurately weigh an appropriate amount of tadalafil reference substance, add solvent and shake to dissolve, then add an appropriate amount of impurity stock solution, quantitatively dilute with solvent to make a solution containing approximately 0.25 mg of tadalafil and 0.375 μg of each impurity per 1 ml, shake well, and obtain.

[0133] Spiked test solution: Accurately weigh an appropriate amount of tadalafil, add solvent and shake to dissolve, then add an appropriate amount of impurity stock solution, quantitatively dilute with solvent to make a solution containing approximately 0.25 mg of tadalafil and 0.375 μg of each impurity per 1 ml, shake well, and obtain.

[0134] Control solution: Accurately measure 0.5 ml of the test solution, place it in a 100 ml volumetric flask, dilute to the scale with solvent, shake well, and obtain.

[0135] The experimental results are as follows:

[0136] Table 10. Column temperature durability - separation

[0137]

[0138]

[0139] Table 11. Column temperature durability - test results

[0140]

[0141] Conclusion: Within the temperature range of 30±5℃, the separation between peaks meets the requirements and there is no significant change in the detection results of impurities.

[0142] Table 12. Flow rate durability - separation

[0143]

[0144] Table 13. Flow rate durability - test results

[0145]

[0146]

[0147] Conclusion: Within the flow rate range of 0.75±0.15ml / min, the separation between peaks meets the requirements and there is no significant change in the detection results of impurities.

[0148] Table 14. Mobile phase ratio durability-separation

[0149]

[0150] Table 15. Mobile phase ratio durability - test results

[0151]

[0152] Conclusion: When the volume ratio of n-hexane to isopropanol of the mobile phase is within the range of 1:(0.95-1.05), the separation between the peaks meets the requirements and there is no significant change in the detection results of the impurities.

[0153] Example 9, determination of samples

[0154] The chromatographic conditions in Experimental Group 1 of Example 1 were used to detect the content of each impurity in the representative batch samples of tadalafil. The test solution, the reference solution and the control solution were respectively injected into the liquid chromatograph, and the chromatogram was recorded. If there was a chromatographic peak in the chromatogram of the test solution with the same retention time as the reference solution, its content was calculated by the self-control method. The test results are as follows:

[0155] Table 16. Test results of test products

[0156] Batch No. / Content% WTD-IM-A WTD-IM-B WTD-IM-C 20210421-1 Not detected Not detected Not detected 210918 Not detected Not detected Not detected A08-03-220301 Not detected Not detected Not detected

[0157] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principle of the present invention, the application is intended to include any changes, uses or improvements of the present invention, including departing from the disclosed scope in the application and the changes made with conventional techniques known in the art.

Claims

1. A method for determining impurities in tadalafil, comprising the following steps: Tadalafil samples were subjected to liquid chromatography analysis to achieve the detection of major components and impurities; The impurities include chiral isomer impurities of tadalafil; The detection conditions of the liquid chromatography analysis include: Chromatographic column: Amylose-tris (3,5-dimethylphenylcarbamate) derivative as filler; Mobile phase: composed of n-hexane and isopropanol in a volume ratio of 1: (0.95-1.05); Elution mode: isocratic elution; Detection wavelength: 222nm.

2. The method according to claim 1, characterized in that: The detection conditions of the liquid chromatography analysis also include at least one of the following: Flow rate: 0.6~0.9mL / min; Column temperature: 25-35°C; Injection volume: 20 μL.

3. The method according to claim 1 or 2, characterized in that: The chromatographic column is CHIRALCEL AD-H 4.6 mm×250 mm, 5 μm.

4. The method according to any one of claims 1 to 3, characterized in that: The chiral isomer impurities include three chiral isomers of tadalafil, which are enantiomers and diastereomers of tadalafil.

5. The method according to any one of claims 1 to 4, characterized in that: The detection is a qualitative detection.

6. The method according to any one of claims 1 to 5, characterized in that: The impurities also include oxidized impurities of tadalafil, the structural formula of which is as follows:

7. The method according to any one of claims 1 to 4, characterized in that: The detection is a quantitative detection.

8. The method according to any one of claims 5 to 7, characterized in that: In the qualitative detection and the quantitative detection, a system consisting of n-hexane, isopropanol and acetonitrile in a volume ratio of 2:2:1 is used as the solvent for the reference substance and the test substance.

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