Method for detecting impurities in tadalafil bulk drug and / or tadalafil intermediate

Abstract

The invention belongs to the technical field of analysis and detection, and discloses a method for detecting impurities in a tadalafil bulk drug and / or a tadalafil intermediate. The method for detecting the impurities in the tadalafil bulk drug and / or the tadalafil intermediate comprises the following steps: taking the tadalafil bulk drug and / or the tadalafil intermediate to be detected, and preparing a sample solution; taking an impurity standard substance, and preparing a standard sample solution; and detecting the impurities in the sample solution by adopting an ultra-high performance liquid chromatography-mass spectrometry method. According to the detection method, two N-nitroso genotoxic impurities and ten other process impurities in tadalafil raw materials and / or tadalafil intermediates can be determined at the same time, the specificity is high, the sensitivity is high, results of multiple different types of impurities can be obtained at the same time in one-time determination, the analysis efficiency is effectively improved, and the method is suitable for popularization and application. Powerful guarantee is provided for analyzing generation factors influencing impurities, effectively controlling medicine quality and guaranteeing medicine use safety.

Description

Technical Field

[0001] This invention relates to the field of analytical testing technology, specifically to a method for detecting impurities in tadalafil active pharmaceutical ingredient and / or tadalafil intermediates. Background Technology

[0002] Tadalafil (see details for formula) Figure 1 As a phosphodiesterase V (PDE5) inhibitor, it plays a crucial role in the treatment of erectile dysfunction and pulmonary hypertension in men. However, in the manufacturing process of its active pharmaceutical ingredient, the final product or intermediates are prone to contain two types of N-nitroso genotoxic impurities with serious hazards: one is N-nitrosodimethylamine (NDMA) introduced by the degradation of the recrystallization solvent N,N-dimethylformamide (DMF), and the other is N-nitrosation impurity (NO-TDLF02) generated during the N-nitrosation reaction of intermediate #2 (TDLF02) in the conversion to intermediate #3 (TDLF03). According to the U.S. Food and Drug Administration (FDA), the acceptable limit for NDMA is 96 ng / day. Based on a maximum daily dose of tadalafil of 40 mg, this equates to a limit of 2.4 ppm in the active pharmaceutical ingredient (API). While there is no clearly defined limit for NO-TDLF02, the acceptable limit, calculated using the computer-aided toxicology (quantitative) structure-activity relationship ((Q)SAR) method recommended in ICH M7 guidelines, is 1500 ng / day. Based on a maximum daily dose of tadalafil of 40 mg, this equates to 37.5 ppm. Furthermore, the production and storage of tadalafil involve the generation of various process impurities and degradation impurities, further increasing the complexity of quality control.

[0003] Currently, existing methods for detecting tadalafil-related substances have significant drawbacks: First, existing methods commonly add peak shape improvers such as trifluoroacetic acid to the mobile phase, and even tetrabutylammonium hydroxide and trifluoroacetic acid are added to the USP system suitability solution. These non-volatile or ion-suppressing additives not only cause significant damage to the chromatographic column, but also, due to their non-volatility or ion source suppression effect, are completely incompatible with mass spectrometry detectors, making it impossible to detect trace genotoxic impurities or to effectively identify unknown impurities, seriously hindering the tracing of impurity sources and corresponding process improvement research. Second, existing methods lack a universal scheme for simultaneously detecting N-nitroso genotoxic impurities and conventional process / degradation impurities, and existing impurity detection methods also generally have the following problems: (1) some methods require the use of derivatization reagents, which are cumbersome and complicated to operate; (2) a single detection can only determine a few impurities, resulting in low efficiency; (3) the mobile phase system contains peak shape improvers or non-volatile salt components, which are not suitable for mass spectrometry detectors, seriously limiting the applicability of the methods. For example, while the detection method disclosed in Chinese patent CN 115184531B ​​achieves the separation and determination of three specific impurities, it requires pre-derivatization treatment and has a limited range of detectable impurities, making it difficult to meet the needs of complex impurity spectrum analysis. Furthermore, because N-nitroso impurities have significantly different detection sensitivity requirements compared to other impurities, existing methods typically employ entirely different liquid chromatography systems. This separation and analysis mode is not only inefficient and costly, but also hinders in-depth research into the influencing factors of N-nitroso genotoxic impurities and makes it difficult to achieve comprehensive quality control of related products.

[0004] Therefore, developing a method that does not require derivatization, does not use non-volatile additives, can be coupled with mass spectrometry, and can simultaneously and accurately determine N-nitroso genotoxic impurities and other conventional process impurities and degradation impurities in tadalafil, enabling rapid and accurate detection of impurities in tadalafil raw materials or intermediates under the same system, is of vital importance for improving the quality control level of tadalafil products, ensuring drug safety, and guiding the improvement of production processes. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for detecting impurities in tadalafil raw materials and / or tadalafil intermediates.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] This invention provides a method for detecting impurities in tadalafil active pharmaceutical ingredient and / or tadalafil intermediate. The method involves taking the tadalafil active pharmaceutical ingredient and / or tadalafil intermediate to be tested and preparing a sample solution; taking impurity standards and preparing a standard solution; and using ultra-high performance liquid chromatography-mass spectrometry to detect the impurities in the sample solution.

[0008] In the ultra-high performance liquid chromatography-mass spectrometry method, the chromatographic conditions include: the column packing material is core-shell silica gel with pentafluorophenylpropyl groups bonded to its surface; the mass spectrometry conditions include: the ionization method is electrospray ionization or heated electrospray ionization; and the scanning mode includes at least one of parallel reaction monitoring, target ion monitoring, full scan monitoring, and multiple reaction monitoring.

[0009] The method for detecting impurities in tadalafil raw material and / or tadalafil intermediates provided by this invention can simultaneously determine two N-nitroso genotoxic impurities and ten other process impurities in tadalafil raw material and / or tadalafil intermediates.

[0010] The tadalafil intermediate is methyl(1R,3R)-1-(benzod1,3-dioxolane-5-yl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1H-pyrido-3,4-b-indole-3-carboxylic acid ester, the structural formula of which is shown in formula (I):

[0011]

[0012] The two N-nitroso genotoxic impurities include: impurity NDMA as shown in formula (II) and impurity NO-TDLF02 as shown in formula (III).

[0013]

[0014] The 10 other process impurities include: impurity A as shown in formula (IV), impurity C as shown in formula (V), impurity D as shown in formula (VI), impurity E as shown in formula (VII), impurity F as shown in formula (VIII), impurity G as shown in formula (IX), impurity H as shown in formula (X), impurity I as shown in formula (XI), impurity 422-Z407 as shown in formula (XII), and impurity methylated SM2 as shown in formula (XIII).

[0015]

[0016]

[0017] The detection method of this invention is highly specific and sensitive. The detection limit for impurity NDMA is 0.21 ppm, equivalent to 9% of the acceptable limit (2.4 ppm); the detection limit for impurity NO-TDLF02 is 0.22 ppm, equivalent to 0.6% of the acceptable limit (37.5 ppm); and the detection limits for other process impurities are between 0.05 ppm and 0.62 ppm, equivalent to 0.005% to 0.06% of the acceptable limit (0.10%). This method meets the relevant requirements of ICH and can obtain the results of multiple impurities in a single determination, effectively improving analytical efficiency and reducing analytical costs. It provides strong support for analyzing factors affecting the generation of impurities, effectively controlling drug quality, and ensuring the safety of drug use.

[0018] The detection method of the present invention includes qualitative detection and / or quantitative detection; the qualitative detection method is to determine the presence of target impurities in the sample solution based on the retention time and mass-to-charge ratio of impurities in the standard solution; the quantitative detection method is to calculate the content of target impurities in the sample solution based on the peak area of ​​impurities in the standard solution according to the external standard method or the standard curve method.

[0019] In a preferred embodiment of the method for detecting impurities in tadalafil raw material and / or tadalafil intermediates according to the present invention, the solvent of the sample solution and / or the standard solution includes at least one of methanol, acetonitrile, ethanol, and isopropanol.

[0020] In a preferred embodiment of the method for detecting impurities in tadalafil raw material or tadalafil intermediates according to the present invention, the concentration of tadalafil raw material and / or tadalafil intermediate in the sample solution is 0.9 mg / mL to 1.1 mg / mL.

[0021] As a preferred embodiment of the method for detecting impurities in tadalafil raw material and / or tadalafil intermediates described in this invention, the ultra-high performance liquid chromatography-mass spectrometry method uses a quadrupole-high resolution electrostatic field orbital trap mass spectrometer or a triple quadrupole tandem linear ion trap mass spectrometer.

[0022] As a preferred embodiment of the method for detecting impurities in tadalafil raw material and / or tadalafil intermediates according to the present invention, the chromatographic conditions include: a Kinetex F5 column; mobile phase A is methanol; mobile phase B is an aqueous solution of formic acid containing ammonium acetate; flow rate is 0.3 mL / min-0.6 mL / min; and column temperature is 40℃-60℃.

[0023] Preferably, the chromatographic conditions include: a Kinetex F5 column, 100 mm × 4.6 mm, 2.6 μm; a flow rate of 0.5 mL / min; and a column temperature of 50 °C.

[0024] Preferably, the concentration of ammonium acetate in the mobile phase B is 0.009 mol / L to 0.015 mol / L.

[0025] More preferably, the concentration of ammonium acetate in the mobile phase B is 0.01 mol / L.

[0026] Preferably, the volume percentage of methanol in the mobile phase B is 0.05%-0.15%.

[0027] More preferably, the volume percentage of methanol in the mobile phase B is 0.1%.

[0028] In a preferred embodiment of the method for detecting impurities in tadalafil raw material and / or tadalafil intermediates according to the present invention, the chromatographic conditions include: gradient elution; the gradient elution program is as follows: with elution time T, 0 ≤ T < 6 min, the volume percentage of mobile phase A is 2%; 6 ≤ T < 9 min, the volume percentage of mobile phase A is 2%-55%; 9 ≤ T < 31 min, the volume percentage of mobile phase A is 55%; 31.1 ≤ T < 36 min, the volume percentage of mobile phase A is 2%-55%; 36 min, the volume percentage of mobile phase A is 2%.

[0029] Preferably, the gradient elution procedure is as follows: with elution time T, 0 ≤ T < 6 min, the volume percentage of mobile phase A is 2%, and the volume percentage of mobile phase B is 98%; 6 ≤ T < 9 min, the volume percentage of mobile phase A increases from 2% to 55%, and the volume percentage of mobile phase B decreases from 98% to 45%; 9 ≤ T < 31 min, the volume percentage of mobile phase A is 55%, and the volume percentage of mobile phase B is 45%; 31.1 ≤ T < 36 min, the volume percentage of mobile phase A decreases from 55% to 2%, and the volume percentage of mobile phase B increases from 45% to 98%; after 36 min, the volume percentage of mobile phase A is 2%, and the volume percentage of mobile phase B is 98%.

[0030] As a preferred embodiment of the method for detecting impurities in the tadalafil active pharmaceutical ingredient and / or tadalafil intermediates of the present invention, the chromatographic conditions include: isocratic elution; and the volume percentage of mobile phase A in the isocratic elution is 50%-60%.

[0031] Preferably, the volume percentage of the isocratic elution mobile phase A is 55%.

[0032] As a preferred embodiment of the method for detecting impurities in tadalafil raw material and / or tadalafil intermediates according to the present invention, the mass spectrometry conditions include: ionization mode is heated electrospray ionization; sheath gas flow rate is 50 arb-55 arb; auxiliary gas flow rate is 12 arb-15 arb; purge gas flow rate is 2 units-5 units; spray voltage is 2 kV-3 kV; ion transfer tube temperature is 250℃-350℃; dryer temperature is 400℃-450℃; number of repeated scans is 1-5; and maximum injection time is 150 ms-250 ms.

[0033] As a preferred embodiment of the method for detecting impurities in tadalafil raw material and / or tadalafil intermediates according to the present invention, the mass spectrometry conditions include: the scanning mode being target ion monitoring and full scan monitoring.

[0034] Preferably, the mass spectrometry conditions include: ionization method is heated electrospray ionization; sheath gas flow rate is 53 arb; auxiliary gas flow rate is 14 arb; purge gas flow rate is 3 units; spray voltage is 2.5 kV; ion transmission tube temperature is 300 °C; dryer temperature is 438 °C; number of repeated scans is 3; and maximum injection time is 200 ms.

[0035] As a preferred embodiment of the method for detecting impurities in tadalafil raw material and / or tadalafil intermediates according to the present invention, the mass spectrometry conditions include: the scanning modes are parallel reaction monitoring, target ion monitoring and full scan monitoring; the collision energy is 8-15; preferably, the collision energy is 10.

[0036] As a preferred embodiment of the method for detecting impurities in tadalafil raw material and / or tadalafil intermediates according to the present invention, the mass spectrometry conditions include: ionization mode of electrospray ionization; curtain gas pressure of 30psi-40psi; nebulization voltage of 5kV-6kV; source temperature of 450℃-550℃; spray gas pressure of 55psi-65psi; scanning mode of multiple reaction monitoring; collision energy of 25-35; and injection time of 80ms-120ms.

[0037] Preferably, the mass spectrometry conditions include: ionization mode is electrospray ionization; curtain gas pressure is 35 psi; nebulization voltage is 5.5 kV; source temperature is 500 °C; spray gas pressure is 60 psi; scanning mode is multiple reaction monitoring; collision energy is 30; and injection time is 100 ms.

[0038] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0039] First, the detection method of this invention can simultaneously determine two N-nitroso genotoxic impurities and ten non-genotoxic impurities in tadalafil raw materials and / or tadalafil intermediates. It exhibits high specificity and sensitivity, with a detection limit of 0.21 ppm for impurity NDMA, equivalent to 9% of the acceptable limit (2.4 ppm), and a detection limit of 0.22 ppm for impurity NO-TDLF02, equivalent to 0.6% of the acceptable limit (37.5 ppm). The detection limits for other impurities are between 0.05 ppm and 0.62 ppm, equivalent to 0.005% to 0.06% of the acceptable limit (0.10%). This meets the relevant ICH requirements and allows for the simultaneous acquisition of results for multiple impurities in a single assay, effectively improving analytical efficiency and reducing analytical costs. It provides strong support for analyzing factors influencing impurity formation, effectively controlling drug quality, and ensuring drug safety.

[0040] Secondly, the detection method of this invention verified the accuracy of impurity NO-TDLF02 at the limit concentration (37.5 ppm). Using quadrupole-high-resolution electrostatic field orbital trap mass spectrometry, the recovery rate of impurity NO-TDLF02 in tadalafil raw material was between 93.4% and 100.0%, and the recovery rate in intermediates was 92.5% to 100.3%. Using triple quadrupole linear tandem ion trap mass spectrometry, the recovery rate of impurity NO-TDLF02 in tadalafil raw material was between 98.4% and 102.9%, and the recovery rate in intermediates was 98.5% to 105.7%. Simultaneously, two N-nitroso genotoxic impurities and ten non-genotoxic impurities showed good linearity over a very wide concentration range (r...). 2 ≥0.9996), and among which NDMA, NO-TDLF02, impurity F, impurity I, and SM2 have r 2 A value as high as 0.9999 or 1 means that the detection method of the present invention can be used for accurate quantitative analysis of impurities in tadalafil raw material and tadalafil intermediates, providing reliable technical support for drug quality control.

[0041] Therefore, the detection method of the present invention is an efficient, accurate and reliable method for detecting impurities in tadalafil raw materials and / or tadalafil intermediates, and has broad application prospects in environmental monitoring, drug safety and other fields. Attached Figure Description

[0042] Figure 1 This is a schematic diagram of the structure of tadalafil of the present invention;

[0043] Figure 2 The extracted ion chromatograms obtained by high-resolution mass spectrometry in PRM mode under gradient elution conditions in Example 1 of this invention are shown below. Figure 2 a is the spectrum of the blank solvent. Figure 2b is the chromatogram of the NDMA reference standard. Figure 2 c is the chromatogram of the active pharmaceutical ingredient (API) used in the test sample. Figure 2 d is the chromatogram of the intermediate test sample;

[0044] Figure 3 The spectrum obtained by high-resolution mass spectrometry in Targeted-SIM mode under gradient elution conditions in Embodiment 1 of the present invention is shown below. Figure 3 a is the spectrum of the blank solvent. Figure 3 b is the chromatogram of NO-TDLF02 reference standard. Figure 3 c represents the chromatogram of the active pharmaceutical ingredient test sample. Figure 3 d is the chromatogram of the intermediate test sample;

[0045] Figure 4 This is the blank solvent solution extraction ion chromatogram acquired by high-resolution mass spectrometry in FullMS-SIM mode under gradient elution conditions in Example 1 of the present invention;

[0046] Figure 5 This is the extraction ion chromatogram of tadalafil and other impurity reference solution acquired by high-resolution mass spectrometry in Full MS-SIM mode under gradient elution conditions in Example 1 of the present invention. Figure 5 a is the chromatogram of impurity F reference standard. Figure 5 b is the chromatogram of impurity E reference standard. Figure 5 c is the chromatogram of impurity H reference standard. Figure 5 d is the chromatogram of impurity 422-407 reference standard. Figure 5 e is the chromatogram of impurity D reference standard. Figure 5 f is the chromatogram of impurity G reference standard. Figure 5 g is the chromatogram of impurity I reference standard. Figure 5 h represents the chromatogram of tadalafil reference standard;

[0047] Figure 6 This is the extraction ion chromatogram of the tadalafil raw material test solution acquired by high-resolution mass spectrometry in Full MS-SIM mode under gradient elution conditions in Example 1 of the present invention.

[0048] Figure 7 This is the extraction ion chromatogram of the tadalafil intermediate test solution acquired by high-resolution mass spectrometry in Full MS-SIM mode under gradient elution conditions in Example 1 of the present invention.

[0049] Figure 8 This is a blank solvent selected ion pair spectrum acquired by triple quadrupole mass spectrometry in MRM mode under isocratic elution conditions in Example 2 of the present invention.

[0050] Figure 9The selected ion pair spectrum of the reference solution was acquired by triple quadrupole mass spectrometry in MRM mode under isocratic elution conditions in Example 2 of the present invention.

[0051] Figure 10 The selected ion pair spectrum of the tadalafil raw material test solution was acquired by triple quadrupole mass spectrometry in MRM mode under isocratic elution conditions in Example 2 of the present invention.

[0052] Figure 11 The selected ion pair spectrum of the tadalafil intermediate test solution was acquired by triple quadrupole mass spectrometry in MRM mode under isocratic elution conditions in Example 2 of the present invention.

[0053] Figure 12 The spectrum obtained by high-resolution mass spectrometry in Targeted-SIM mode under isocratic elution conditions in Example 3 of the present invention is shown below. Figure 12 a is the spectrum of the blank solvent. Figure 12 b is the chromatogram of the NO-TDLF02 reference solution. Figure 12 c is the chromatogram of the tadalafil raw material test solution. Figure 12 d is the chromatogram of the tadalafil intermediate test solution;

[0054] Figure 13 The blank solvent selected ion spectrum acquired by high-resolution mass spectrometry in Full MS-SIM mode under isocratic elution conditions in Example 3 of this invention;

[0055] Figure 14 The selected ion spectra of tadalafil and impurity reference solutions were acquired by high-resolution mass spectrometry in Full MS-SIM mode under isocratic elution conditions in Example 3 of this invention. Figure 14 a is the chromatogram of impurity F reference standard. Figure 14 b is the chromatogram of impurity E reference standard. Figure 14 c is the chromatogram of impurity H reference standard. Figure 14 d is the chromatogram of impurity 422-407 reference standard. Figure 14 e is the chromatogram of impurity D reference standard. Figure 14 f represents the chromatogram of impurity G reference standard solution. Figure 14 g is the chromatogram of impurity I reference standard. Figure 14 h represents the chromatogram of tadalafil reference standard;

[0056] Figure 15 The selected ion spectrum of the tadalafil raw material test solution was acquired by high-resolution mass spectrometry in Full MS-SIM mode under isocratic elution conditions in Example 3 of the present invention.

[0057] Figure 16The selected ion spectrum of the tadalafil intermediate test solution was acquired by high-resolution mass spectrometry in Full MS-SIM mode under isocratic elution conditions in Example 3 of the present invention.

[0058] Figure 17 This is a comparison chart showing the separation of impurities E, F, and H in Example 1, Comparative Example 1, and Comparative Example 2 of the present invention. Detailed Implementation

[0059] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described below with reference to specific embodiments. Those skilled in the art should understand that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0060] The following description, in conjunction with specific embodiments, illustrates the practical effects of the present invention.

[0061] Unless otherwise specified, the experimental methods used in the examples are conventional methods; the materials, reagents, equipment, etc. used are all commercially available unless otherwise specified.

[0062] The instruments and reagents used in the embodiments and comparative examples of this invention are as follows:

[0063] Instruments: Q Exactive quadrupole-high resolution electrostatic track trap LC-MS (Thermo Fisher Scientific, USA), QTRAP6500+ triple quadrupole tandem linear ion trap mass spectrometer (SCIEX, USA), UltiMate 3000 ultra-high performance liquid chromatograph (Thermo Fisher Scientific, USA), Compound Discover 3.3 and Mass Frontier 8.0 software (Thermo Fisher Scientific, USA).

[0064] Reagents: Methanol (LC / MS grade), ammonium acetate (LC / MS grade), formic acid (LC / MS grade).

[0065] Standards: Tadalafil and impurity NDMA were purchased from the China National Institutes for Food and Drug Control; impurities

[0066] NO-TDLF02 was provided by DYG Pharmaceuticals; impurity D was purchased from QCSRM; impurities E, G, H and I were purchased from Wuxi Hailun; impurity F was purchased from Sinco Pharmachem; impurity 422-Z407 was provided by ZHLB Pharmaceuticals; and methylated SM2 was purchased from Aladdin.

[0067] Example 1: A method for detecting impurities in tadalafil active pharmaceutical ingredient and / or tadalafil intermediates

[0068] Instruments: Q Exactive quadrupole-high resolution electrostatic track trap liquid chromatography-mass spectrometry system, UltiMate 3000 ultra-high performance liquid chromatograph;

[0069] Software: Compound Discover 3.3 and Mass Frontier 8.0 software (Thermo Fisher Scientific, USA).

[0070] 1. Preparation of test solution and reference solution

[0071] Tadalafil raw material test solution: Grind tadalafil into a fine powder, weigh 10 mg of the fine powder, add 10 mL of methanol, and vortex for 10 min.

[0072] Tadalafil intermediate test solution: Grind TDP03 into a fine powder, weigh 10 mg of the powder, add 10 mL of methanol, and shake for 90 min.

[0073] NDMA reference solution: Take 20 mg of NDMA reference standard, place it in a 100 mL volumetric flask, dissolve and dilute to the mark with methanol, mix well, and the solution has a concentration of 200 μg / mL. -1 Prepare a stock solution of NDMA reference standard. Take a certain amount of the NDMA reference standard stock solution and dilute it stepwise with methanol to a concentration of 100 ng / mL. -1 50 ng·mL -1 40 ng·mL -1 20 ng / mL -1 10 ng·mL -1 5 ng / mL -1 2ng·mL -1 1 ng·mL -1 0.5 ng / mL -1 and 0.2 ng·mL -1 A series of reference solutions.

[0074] NO-TDLF02 reference solution: Take 10 mg of NO-TDLF02 reference standard, place it in a 100 mL volumetric flask, dissolve and dilute to the mark with methanol, mix well, and the solution has a concentration of 100 μg / mL. -1 NO-TDLF02 reference standard stock solution. Take a certain amount of NO-TDLF02 reference standard stock solution and dilute it stepwise with methanol to a concentration of approximately 100 ng / mL. -1 50 ng·mL -1 20 ng / mL -1 10 ng·mL-1 5 ng / mL -1 2ng·mL -1 1 ng·mL -1 0.5 ng / mL -1 0.2 ng / mL -1 and 0.1 ng·mL -1 A series of reference solutions.

[0075] Tadalafil reference solution: Take 10 mg of tadalafil reference standard, place it in a 100 mL volumetric flask, dissolve and dilute to the mark with methanol, mix well, and the concentration is 100 μg / mL. -1 Tadalafil reference stock solution. Take a certain amount of tadalafil reference stock solution and dilute it stepwise with methanol to a concentration of approximately 1000 ng / mL. -1 50 ng·mL -1 20 ng / mL -1 10 ng·mL -1 5 ng / mL -1 2ng·mL -1 and 1 ng·mL -1 A series of reference solutions.

[0076] Reference solutions for impurities D, E, F, G, H, I, 422-Z407, and methylated SM2: Take 1-5 mg of the above reference standards, place them in a 20 mL volumetric flask, dissolve and dilute to the mark with methanol, mix well, and obtain concentrations of 70-250 μg·mL. -1 Prepare a reference standard stock solution. Take a certain amount of the reference standard stock solution and dilute it stepwise with methanol to a concentration of 500 ng / mL. -1 250 ng / mL -1 100 ng·mL -1 50 ng·mL -1 40 ng·mL -1 20 ng / mL -1 10 ng·mL -1 5 ng / mL -1 2ng·mL -1 1 ng·mL -1 and 0.5 ng·mL -1 A series of reference solutions.

[0077] 2. HPLC-HRMS analysis

[0078] (1) Chromatographic conditions:

[0079] The column packing material was pentafluorophenylpropyl silica gel with a core-shell matrix; the column was a Kinetex F5, 100 mm × 4.6 mm, 2.6 μm.

[0080] Mobile phase A is methanol; mobile phase B is an aqueous solution of formic acid containing ammonium acetate, with the concentration of ammonium acetate in mobile phase B being 0.01 mol·L⁻¹. -1 The volume percentage of formic acid is 0.1%.

[0081] The column temperature was 50℃, and the flow rate was 0.5 mL / min. -1 The injection volume was 5 μL, the injector temperature was 10 °C, the solvent was methanol, and the mobile phase was either isocratic or gradient elution (gradient elution was used when it was necessary to monitor NDMA, NO-TDLF02 and other impurities simultaneously, and isocratic elution was used when it was not necessary to detect NDMA and only needed to monitor NO-TDLF02 and other impurities. In isocratic elution, the volume fraction of mobile phase A was 55% and the volume fraction of mobile phase B was 45%).

[0082] The gradient elution procedure is as follows:

[0083] From 0 to 6 minutes, the volume percentage of mobile phase A was 2%, and the volume percentage of mobile phase B was 98%.

[0084] Over 6-9 minutes, the volume percentage of mobile phase A increased from 2% to 55%, while the volume percentage of mobile phase B decreased from 98% to 45%.

[0085] 9-31 min, the volume percentage of mobile phase A is 55%, and the volume percentage of mobile phase B is 45%;

[0086] From 31.1 to 36 min, the volume percentage of mobile phase A decreased from 55% to 2%, while the volume percentage of mobile phase B increased from 45% to 98%.

[0087] (2) Mass spectrometry conditions:

[0088] Ionization method: Heated electrospray ionization (HESI+); Sheath gas flow rate: 53 arb; Auxiliary gas flow rate: 14 arb; Purge gas flow rate: 3 units; Spray voltage: 2.50 kV; Ion transfer tube temperature: 300℃; Dryer temperature: 438℃;

[0089] Scanning modes: 0.0-6.0 min for parallel reaction monitoring (PRM), 27.0-30.0 min for targeted ion monitoring (Targeted-SIM), and 0.0-36.0 min for full scan monitoring (Full MS-SIM);

[0090] Quantitative ion m / z: 75.0553±10ppm for 1-6 min, 380.1241±10ppm for 27-30 min, 422.1347±10ppm for 27-30 min, 408.1555±10ppm for 27-30 min, 404.1242±10ppm for 27-30 min, 406.1397±10ppm for 27-30 min, and 165.0552±10ppm for 27-30 min.

[0091] Resolution: 17500 (PRM) / 70000 (Targeted-SIM and Full MS-SIM);

[0092] Automatic gain control target ion number: 1e5 (PRM) / 5e4 (Targeted-SIM) / 3e6 (Full MS-SIM);

[0093] Number of repeated scans (Mcroscans): 3;

[0094] Isolation window: 1.0m / z (PRM) / 0.5m / z (SIM);

[0095] Collision energy (NCE): 10 (PRM);

[0096] Maximum injection time: 200ms;

[0097] Switch to mass spectrometry for 1-16 min and 21-31 min, and switch to waste liquid for the rest of the time (except when determining tadalafil reference solution).

[0098] (3) Use the test conditions of step (2) to perform qualitative and quantitative detection on the test solution and reference solution obtained in step (1);

[0099] Quantitative detection includes the external standard method and the standard curve method:

[0100] Standard curve method:

[0101] A series of reference solutions of impurities were tested sequentially using HPLC-HRMS. The peak area corresponding to each concentration point was recorded. Linear regression analysis was performed with concentration (X) as the abscissa and peak area (Y) as the ordinate to obtain the standard curve and linear equation.

[0102] The test solution was tested using HPLC-HRMS, and the content of each impurity in the test solution was calculated based on the peak area of ​​each impurity in the test solution.

[0103] External standard method for reference standards:

[0104] The calculation formula for the external standard method is as follows:

[0105]

[0106] The impurities include NDMA, NO-TDLF02, impurity A, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, 422-Z407, and methylated SM2.

[0107] 3. Evaluation parameters and results of the method

[0108] (1) Exclusivity

[0109] Take the test solution and the reference solution, inject them into the Q Exactive quadrupole-high-resolution electrostatic track trap liquid chromatography-mass spectrometry instrument, record the mass spectra, and the localization results of each component are shown in Table 1.

[0110] The blank solvent chromatogram, NDMA reference solution chromatogram, raw material test solution chromatogram, and intermediate test solution chromatogram under PRM mode are shown below. Figure 2 This mode is used to determine NDMA, with a scan time range of 0-10 min, an extracted ion mass-to-charge ratio range of m / z 75.0545-75.0561, an NDMA retention time of 4.7 min, and a base peak mass-to-charge ratio of m / z 75.0552 in the reference solution.

[0111] The chromatograms of the blank solvent, NO-TDLF02 reference solution, raw material test solution, and intermediate test solution under Targeted-SIM mode are shown below. Figure 3 This mode was used to determine NO-TDLF02, with a scan time range of 20-35 min, a selected monitoring ion range of m / z 380.122-380.1260, a NO-TDLF02 retention time of 28.4 min, and a base peak mass-to-charge ratio of m / z 380.1231 in the reference solution.

[0112] The blank solvent spectrum in Full MS-SIM mode is shown below. Figure 4 The tadalafil raw material test solution is shown below. Figure 6 The tadalafil intermediate test solution is shown below. Figure 7 This mode is used to determine other impurities in tadalafil, with a scan time range of 0-35 min.

[0113] in, Figure 4 The range of ions extracted is m / z 422.1343-422.1351. Figure 4 The range of ions extracted by b is m / z 408.1551-408.1559. Figure 4 c. Extractable ion range m / z 404.1238-404.1246. Figure 4 The range of d-extractable ions is m / z 406.1397-406.1405. Figure 4 The range of e-extractable ions is m / z 390.1445-390.1453;

[0114] Figure 5 Solution a is the reference solution of impurity F, with a retention time of 12.5 min and a base peak mass-to-charge ratio of m / z 422.1347. Figure 5 b is the reference solution for impurity E, with a retention time of 12.7 min and a base peak mass-to-charge ratio of m / z 422.1345. Figure 5 c is the reference solution of impurity H, with a retention time of 13.1 min and a base peak mass-to-charge ratio of m / z 422.1343. Figure 5 d is the reference solution for impurity 422-407, with a retention time of 13.4 min and a base peak mass-to-charge ratio of m / z 408.1552. Figure 5 e is the reference solution of impurity D, with a retention time of 14.1 min and a base peak mass-to-charge ratio of m / z 422.1349. Figure 5 f is the reference solution of impurity G, with a retention time of 14.2 min and a base peak mass-to-charge ratio of m / z 404.1242. Figure 5 g is the impurity I reference solution, with a retention time of 15.9 min and a base peak mass-to-charge ratio of m / z 406.1395. Figure 5 h is the tadalafil reference solution, with a retention time of 17.7 min and a base peak mass-to-charge ratio of m / z 390.1446;

[0115] Figure 6 The range of ions extracted is m / z 422.1305-422.1389. Figure 6 The range of ions extracted by b is m / z 408.1514-408.1596. Figure 6 The range of ions extracted by c is m / z 404.1202-404.1282. Figure 6 The range of d-extractable ions is m / z 406.1360-406.1442. Figure 6 The range of e-extractable ions is m / z 390.1410-390.1448.

[0116] Table 1. Localization and quantitative analysis of each analyte component, m / z deviation of ions, and conditions of the test sample solution.

[0117]

[0118] The results showed that the blank solvent, tadalafil API and intermediates did not interfere with the analyte. The deviation of the quantitative ion m / z from the theoretical value was between 0.0 and 5.1 ppm. For the analyte detected, the retention time of the analyte in the reference solution and the test solution was consistent, indicating good specificity.

[0119] (2) Linear relationship

[0120] A series of concentrations of NDMA, NO-TDLF02, NO-TDLF, impurities D, E, F, G, H, I, and 422-Z407 reference solutions were injected into a Q Exactive quadrupole-high-resolution electrostatic track trap LC-MS / MS instrument. Mass spectra were recorded, and the linear range, linear equation, detection limit, and quantitation limit were investigated. The results are shown in Table 2.

[0121] Table 2. Test results of linearity, linear range, limit of detection, and limit of quantitation for each analyte component.

[0122]

[0123] Since there is no corresponding reference standard for impurities A / C, and impurities A / C are non-corresponding isomers of tadalafil with similar responses, their content can be calculated using tadalafil reference standards.

[0124] As shown in Table 2, the impurities of this invention exhibit good linearity over a very wide concentration range (r). 2 ≥0.9996), and among which NDMA, NO-TDLF02, impurity F, impurity I, and SM2 have r 2 A value as high as 0.9999 or 1 means that the detection method of the present invention can be used for accurate quantitative analysis of tadalafil raw material and its impurities, providing reliable technical support for drug quality control.

[0125] (3) Precision

[0126] Take the reference solutions of each impurity and inject them repeatedly 6 times. Calculate the peak area RSD. The results are shown in Table 3.

[0127] Table 2. Results of injection precision tests for each analyte component.

[0128]

[0129]

[0130] As shown in Table 3, the results of this invention, which examines the stability of the instrument for detecting the same component by calculating the relative standard deviation (RSD) of the peak area through six consecutive injections of the same impurity reference solution, demonstrate that the detection method of this invention has good precision for most impurity components and can be used for the quantitative analysis of impurities in tadalafil raw material and its intermediates, ensuring that the results meet quality control requirements.

[0131] (4) Repeatability

[0132] Take the tadalafil raw material test solution, perform 6 parallel operations, and determine each impurity. The results are shown in Table 4.

[0133] Table 4. Repeatability test results (ppm) for each impurity component to be tested.

[0134]

[0135]

[0136] As shown in Table 4, this invention verifies the detection results of impurity content in the same batch of samples under the same operator, instrument, and laboratory conditions by preparing multiple test solutions in parallel and measuring them independently. It can be seen that the detection method of this invention has good repeatability for most quantifiable impurities, and the results are highly reliable, which can ensure the consistency of detection results for different batches or different operators.

[0137] (5) Accuracy

[0138] Take 10 mg of tadalafil raw material and place it in a 10 mL volumetric flask. Add a specific amount of each impurity reference solution, then add methanol to 10 mL and vortex for 10 min. Perform the operation in parallel for 6 times and calculate the recovery rate. The results are shown in Table 5.

[0139] Table 5. Accuracy test results (ppm) for each impurity component to be tested.

[0140]

[0141] As shown in Table 5, the results of this invention, which verifies whether the detection method can accurately reflect the true content of impurities in a sample by adding a specific amount of impurity reference standard to a sample with known content and measuring the recovery rate (i.e., the proportion of added impurities that are detected), show that the detection method of this invention has good accuracy for most impurities and can provide an accuracy reference for the formulation of impurity limits in pharmaceuticals, ensuring that the detection results meet regulatory requirements.

[0142] (6) Sample determination

[0143] Different batches of tadalafil raw material and intermediate samples were tested, and the results are shown in Table 6.

[0144] Table 6 Sample Test Results (ppm)

[0145] batch number 202410104 202410102 4222304002 23010307 22033004 NDMA ＜0.21 ＜0.21 0.4 0.3 ＜0.21 NO-TDLF02 ＜0.22 ＜0.22 ＜0.22 ＜0.22 ＜0.22 Impurities A / C ＜0.28 ＜0.28 20.2 125.5 1.4 Impurity D ＜0.26 ＜0.26 ＜0.26 ＜0.26 ＜0.26 Impurity E 33.2 9.1 2.3 3.7 4.9 impurity F 4.3 4.4 1.0 1.1 ＜0.26 Impurity G 3.3 1.7 0.3 0.6 ＜0.13 impurity H 28.2 174.4 48.1 38.4 70.0 Impurity I ＜0.56 44.8 6.3 25.7 1.7 422-Z407 ＜0.26 ＜0.26 ＜0.26 ＜0.26 ＜0.26 SM2 ＜0.62 ＜0.62 ＜0.62 ＜0.62 ＜0.62

[0146] As shown in Table 6, the detection method of the present invention can accurately detect the content of various impurities in different batches (active pharmaceutical ingredient and intermediate), confirm the stability of the production process and the consistency of quality between batches, and provide data support for process stability and product quality.

[0147] Example 2: A method for detecting NO-TDLF02, an impurity, in tadalafil raw material and tadalafil intermediates.

[0148] The differences between this embodiment and Embodiment 1 include:

[0149] Replace the Q Exactive quadrupole-high resolution electrostatic track trap LC-MS with the QTRAP6500+ triple quadrupole tandem linear ion trap mass spectrometer.

[0150] Chromatographic conditions:

[0151] Isocratic elution was performed with mobile phase A comprising 55±5% and mobile phase B comprising 45±5%.

[0152] Mass spectrometry conditions:

[0153] Ionization method: Electrospray ionization (ESI+); Air curtain pressure: 35.0 psi; Atomization voltage: 5500; Source temperature: 500℃; Spray pressure: 60.0 psi;

[0154] Injection time: 100ms; Collision energy: 30;

[0155] Scanning mode: Multiple Response Monitoring (MRM);

[0156] Quantitative ion pairs: m / z 380.2-m / z 275.2;

[0157] Qualitative ion pairs: m / z 380.2-m / z 334.2;

[0158] Switch to mass spectrometry from 10.0 to 18.0 min, and switch to waste liquid for the rest of the time.

[0159] Evaluation parameters and results of the method:

[0160] (1) Exclusivity

[0161] Take the test solution and the reference solution, inject them into the QTRAP6500+ triple quadrupole tandem linear ion trap mass spectrometer, and record the mass spectra.

[0162] See blank solvent spectrum. Figure 8 The selected ion pair is m / z 380.2-m / z 275.2;

[0163] The chromatogram of the NO-TDLF02 reference solution is shown below. Figure 9 The selected ion pair was m / z 380.2-m / z 275.2, and the retention time of NO-TDLF02 was 12.63 min.

[0164] The chromatogram of the active pharmaceutical ingredient test solution is shown below. Figure 10 The selected ion pair is m / z 380.2-m / z 275.2;

[0165] The chromatogram of the intermediate test solution is shown in [reference needed]. Figure 11 The selected ion pair is m / z 380.2-m / z 275.2.

[0166] The results showed that the blank solvent, tadalafil API and intermediates did not interfere with the analyte, indicating good specificity.

[0167] (2) Linear relationship

[0168] A series of NO-TDLF02 reference solutions of different concentrations were injected into a QTRAP6500+ triple quadrupole tandem linear ion trap mass spectrometer, and the mass spectra were recorded to investigate the linear range, linear equation, detection limit, and lower limit of quantitation.

[0169] The results showed that NO-TDLF02 was effective in the range of 0.54-53.98 ng·mL⁻¹. -1 The linearity is good within the range, and the linear regression equation is Y = 34333X + 5051.3, r 2 =0.9998, the limit of detection (LOD) is 0.04 ppm, and the limit of quantitation (LOQ) is 0.06 ppm.

[0170] (3) Accuracy

[0171] Take 10 mg of tadalafil raw material and place it in a 10 mL volumetric flask. Add 20 ng / mL of the solution. -1 Add 1 mL of NO-TDLF02 reference solution and 9 mL of methanol, then vortex for 10 min;

[0172] Take 10 mg of tadalafil intermediate and place it in a 10 mL volumetric flask. Add 20 ng / mL of the solution. -1 Add 1 mL of NO-TDLF02 reference solution and then 9 mL of methanol, and vortex for 10 min.

[0173] Perform the experiment in parallel for 6 samples, and take another sample with a concentration of 20 ng / mL. -1 1 mL of NO-TDLF02 reference solution was placed in a 10 mL volumetric flask and the same procedure was followed to prepare the reference solution. The recovery rate was calculated by peak area ratio and standard curve method, respectively. The results are shown in Table 7.

[0174] Table 7 Accuracy Test Results

[0175]

[0176] As shown in Table 7, the detection method of this embodiment has high accuracy and good precision in detecting NO-TDLF02 impurities in tadalafil raw material and intermediates, and is suitable for quality control.

[0177] (4) Sample determination

[0178] Different batches of tadalafil raw material and intermediate samples were tested, and the results are shown in Table 8.

[0179] Table 8 Sample Test Results (ppm)

[0180] batch number 202410104 202410102 4222304002 23010307 22033004 NO-TDLF02 ＜0.22 ＜0.22 ＜0.22 ＜0.22 ＜0.22

[0181] As shown in Table 8, the detection method of this embodiment can accurately detect the content of various impurities in different batches (active pharmaceutical ingredient and intermediate), confirm the stability of the production process and the consistency of quality between batches, and provide data support for process stability and product quality.

[0182] Example 3: A method for detecting impurities in tadalafil raw material or tadalafil intermediates

[0183] The differences between this embodiment and Embodiment 1 include:

[0184] Chromatographic conditions:

[0185] Isocratic elution was performed with mobile phase A comprising 55±5% and mobile phase B comprising 45±5%.

[0186] Mass spectrometry conditions:

[0187] Ionization method: Heated electrospray ionization (HESI+); Sheath gas flow rate: 53 arb; Auxiliary gas flow rate: 14 arb; Purge gas flow rate: 3 units; Spray voltage: 2.50 kV; Ion transfer tube temperature: 300℃; Dryer temperature: 438℃;

[0188] Scanning modes: 0.0-20.0 min for Targeted Ion Monitoring (SIM) and Full MS-SIM;

[0189] Quantitative ion m / z: 380.1241±10ppm, 422.1347±10ppm, 408.1559±10ppm, 404.1241±10ppm and 406.1397±10ppm; resolution: 70000;

[0190] Automatic gain control target ion number: 5e4 (Targeted-SIM) / 3e6 (Full MS-SIM);

[0191] Number of repeated scans (Mcroscans): 3;

[0192] Isolation window: 0.5m / z;

[0193] Maximum injection time: 200ms;

[0194] Switch to mass spectrometry for 0.5-17 minutes, and switch to waste liquid for the rest of the time.

[0195] Evaluation parameters and results of the method:

[0196] (1) Exclusivity

[0197] Take the test solution and the reference solution, inject them into the Q Exactive quadrupole-high-resolution electrostatic track trap liquid chromatography-mass spectrometry instrument, record the mass spectra, and the localization results of each component are shown in Table 5.

[0198] The chromatograms of the blank solvent, NO-TDLF02 reference solution, raw material test solution, and intermediate test sample under Targeted-SIM mode are shown below. Figure 12 This mode is used to determine NO-TDLF02, with a scan time range of 0-20 min and a selected monitoring ion range of m / z 380.122-380.1260;

[0199] The blank solvent spectrum in Full MS-SIM mode is shown below. Figure 13 This mode is used to determine other impurities in tadalafil, with a scan time range of 0-20 min. Figure 13 The range of ions extracted is m / z 422.1305-422.1389. Figure 13 The range of ions extracted by b is m / z 408.15141-408.1596. Figure 13 c Extraction ion range m / z 404.1202-404.1282, Figure 13 The range of d-extractable ions is m / z 406.1360-406.1442. Figure 13 The range of ions extracted by e is m / z 390.1410-390.1488. Figure 13 The range of ions extracted is m / z 380.1203-380.1279;

[0200] chromatogram of tadalafil other impurity reference solution Figure 14 The scanning time range is 0-20 minutes, among which, Figure 14 Solution a is the reference solution of impurity F, with a retention time of 2.8 min and a base peak mass-to-charge ratio of m / z 422.1349. Figure 14 b is the reference solution for impurity E, with a retention time of 3.0 min and a base peak mass-to-charge ratio of m / z 422.1342. Figure 14 c is the reference solution of impurity H, with a retention time of 3.0 min and a base peak mass-to-charge ratio of m / z 422.1349. Figure 14 d is the reference solution for impurity 422-407, with a retention time of 4.1 min and a base peak mass-to-charge ratio of m / z 408.1555. Figure 14 e is the reference solution of impurity D, with a retention time of 5.1 min and a base peak mass-to-charge ratio of m / z 422.1348. Figure 14 f is the reference solution of impurity G, with a retention time of 5.3 min and a base peak mass-to-charge ratio of m / z 404.1243. Figure 14 g is the reference solution for impurity I, with a retention time of 8.9 min and a base peak mass-to-charge ratio of m / z 406.1400. Figure 14 h is the tadalafil reference solution, with a retention time of 13.3 min and a base peak mass-to-charge ratio of m / z 390.1447;

[0201] The chromatogram of the tadalafil API test solution is shown below. Figure 15 This mode is used to determine other impurities in tadalafil, with a scan time range of 0-20 min. Figure 15 The range of ions extracted is m / z 422.1307-422.1391. Figure 15 The range of ions extracted by b is m / z 408.1514-408.1596. Figure 15 The range of ions extracted by c is m / z 404.1203-404.1283. Figure 15 The range of d-extractable ions is m / z 406.1359-406.1441. Figure 15 The range of e-extractable ions is m / z 390.1403-390.1488;

[0202] The chromatogram of the tadalafil intermediate test solution is shown below. Figure 16 This mode is used to determine other impurities, with a scan time range of 0-20 min. Figure 16 The range of ions extracted is m / z 422.1307-422.1391. Figure 16 The range of ions extracted by b is m / z 408.1514-408.1596. Figure 16 The range of ions extracted by c is m / z 404.1203-404.1283. Figure 16 The range of d-extractable ions is m / z 406.1359-406.1441, among which... Figure 16 The range of e-extractable ions is m / z 390.1403-390.1488.

[0203] Table 9. Location and quantitative deviation of each impurity component ion m / z, and condition of the test sample solution.

[0204]

[0205] The results showed that the blank solvent, tadalafil API and intermediates did not interfere with the analyte. The retention times of the analyte in the reference solution and the test solution were consistent, and the m / z deviation of the quantitative ion was between 0.0 and 3.2, indicating good specificity.

[0206] Comparative Example 1: A method for detecting impurities in tadalafil raw material and tadalafil intermediates

[0207] The only difference between this comparative example and Example 1 is that the chromatographic column was replaced with an ACE Excel 3C18 column. Comparative Example 2: A method for detecting impurities in tadalafil active pharmaceutical ingredient and tadalafil intermediates.

[0208] The only difference between this comparative example and Example 1 is that the chromatographic column was replaced with a Titank C18 column.

[0209] like Figure 17 As shown, using the separation of three isomers E, F and H, which are difficult to separate, as the evaluation criterion, the detection methods of Comparative Examples 1 and 2, which used ACE Excel 3C18 columns or Titank C18 columns, could not completely separate the above three impurities.

[0210] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for detecting impurities in tadalafil active pharmaceutical ingredient and / or tadalafil intermediates, characterized in that, Take the tadalafil active pharmaceutical ingredient and / or tadalafil intermediate to be tested and prepare a sample solution; take the impurity standard and prepare a standard solution. Impurities in the sample solution were detected using ultra-high performance liquid chromatography-mass spectrometry. In the ultra-high performance liquid chromatography-mass spectrometry method, the chromatographic conditions include: the column packing material is core-shell silica gel with pentafluorophenylpropyl groups bonded to its surface; the mass spectrometry conditions include: the ionization method is electrospray ionization or heated electrospray ionization; and the scanning mode includes at least one of parallel reaction monitoring, target ion monitoring, full scan monitoring, and multiple reaction monitoring.

2. The method for detecting impurities in tadalafil active pharmaceutical ingredient and / or tadalafil intermediate as described in claim 1, characterized in that, The structural formula of the tadalafil intermediate is shown in formula (I):

3. The method for detecting impurities in tadalafil active pharmaceutical ingredient and / or tadalafil intermediate as described in claim 1, characterized in that, The chromatographic conditions include: a Kinetex F5 column; mobile phase A is methanol; mobile phase B is an aqueous solution of formic acid containing ammonium acetate; flow rate is 0.3 mL / min-0.6 mL / min; and column temperature is 40℃-60℃.

4. The method for detecting impurities in tadalafil active pharmaceutical ingredient and / or tadalafil intermediate as described in claim 3, characterized in that, The concentration of ammonium acetate in the mobile phase B is 0.009 mol / L to 0.015 mol / L.

5. The method for detecting impurities in tadalafil active pharmaceutical ingredient and / or tadalafil intermediate as described in claim 1, characterized in that, The chromatographic conditions include: gradient elution; the gradient elution program is as follows: with elution time T, 0 ≤ T < 6 min, mobile phase A volume percentage is 2%; 6 ≤ T < 9 min, mobile phase A volume percentage is 2%-55%; 9 ≤ T < 31 min, mobile phase A volume percentage is 55%; 31.1 ≤ T < 36 min, mobile phase A volume percentage is 2%-55%; 36 min, mobile phase A volume percentage is 2%.

6. The method for detecting impurities in tadalafil active pharmaceutical ingredient and / or tadalafil intermediate as described in claim 1, characterized in that, The chromatographic conditions include: isocratic elution; and the volume percentage of mobile phase A in the isocratic elution is 50%-60%.

7. The method for detecting impurities in tadalafil active pharmaceutical ingredient and / or tadalafil intermediate as described in claim 1, characterized in that, The mass spectrometry conditions include: ionization method is heated electrospray ionization; sheath gas flow rate is 50-55 alb; auxiliary gas flow rate is 12-15 alb; purge gas flow rate is 2-5 units; spray voltage is 2-3 kV; ion transmission tube temperature is 250-350℃; desiccator temperature is 400-450℃; number of repeated scans is 1-5; and maximum injection time is 150-250 ms.

8. The method for detecting impurities in tadalafil active pharmaceutical ingredient and / or tadalafil intermediate as described in claim 7, characterized in that, The mass spectrometry conditions include: scanning modes of parallel reaction monitoring, target ion monitoring, and full scan monitoring; and collision energy of 8-15.

9. The method for detecting impurities in tadalafil active pharmaceutical ingredient and / or tadalafil intermediate as described in claim 7, characterized in that, In the mass spectrometry conditions, the scanning modes are target ion monitoring and full scan monitoring.

10. The method for detecting impurities in tadalafil active pharmaceutical ingredient and / or tadalafil intermediate as described in claim 1, characterized in that, The mass spectrometry conditions include: ionization mode is electrospray ionization; curtain gas pressure is 30psi-40psi; nebulization voltage is 5kV-6kV; source temperature is 450℃-550℃; spray gas pressure is 55psi-65psi; scanning mode is multiple reaction monitoring; collision energy is 25-35; injection time is 80ms-120ms.

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