A method for determining methylamine in tadalafil by high performance liquid chromatography

Abstract

The present invention provides a method for determining methylamine in tadalafil by high performance liquid chromatography, and the chromatographic conditions are: the chromatographic column is C18; the mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A is 0.1% phosphoric acid, and mobile phase B is acetonitrile; the detection wavelength is 226nm, the column temperature is 30℃±5℃, and the flow rate is 1.0mL / min±0.1mL / min. The present invention provides a detection method for determining methylamine in tadalafil by high performance liquid chromatography, and the separation between methylamine and adjacent peaks is greater than 1.5, the detection limit of methylamine is 0.00301µg / mL, which is approximately equivalent to 0.0006% of the sample concentration, and the quantitative limit is 0.01002µg / mL, which is approximately equivalent to 0.002% of the sample concentration. The method has high sensitivity, good repeatability, accurate results, and can be used for quality control of tadalafil raw material production.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis and detection, and specifically relates to a method for determining methylamine in tadalafil by adopting high performance liquid chromatography. Background Art

[0002] Tadalafil is a selective, reversible inhibitor of cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase 5 (PDE5). It has a rapid onset of action and is not affected by high-fat diet and alcohol intake. It is the only long-acting PDE5 inhibitor approved by the FDA and CFDA. In addition to its indications for erectile dysfunction (ED), it is also approved for the treatment of pulmonary hypertension and benign prostatic hyperplasia. This year, drug quality and safety issues have received much attention, so there are higher requirements for the quality control of tadalafil API.

[0003] In the synthesis process of tadalafil, it is necessary to add an excess of methylamine to increase the yield. Methylamine is a low-toxic alkaline substance that is irritating and corrosive to the skin and mucous membranes, can affect the excitability of the central nervous system, and cause allergic reactions such as asthma. Therefore, it is necessary to strictly control the residual amount of methylamine in tadalafil. Chinese patent CN108169395B discloses an analytical detection method for tadalafil oil tubing substances, which uses high-performance liquid chromatography combined with an ultraviolet detector for impurity detection, and can separate tadalafil from impurities with high sensitivity. Xue Fengfeng et al. used gas chromatography to determine the residual amount of methylamine and triethylamine in tadalafil raw materials. The injection port temperature was 200°C, the detector temperature was 260°C, and the flow rate was 3.46mL / min. However, this condition was close to the tolerance condition of the gas chromatography column, resulting in a significant decrease in the column efficiency of the chromatographic column during use, making the method less reproducible. At the same time, since methylamine is a volatile gas and has no ultraviolet absorption, it is considered to derivatize methylamine and then use HPLC to detect the target peak after derivatization. Summary of the invention

[0004] The purpose of the present invention is to provide a method for determining the content of methylamine in tadalafil. The method has high sensitivity, good repeatability and accurate results, and can be used for quality control of methylamine in tadalafil production.

[0005] The technical solution of the present invention is as follows:

[0006] A method for determining the methylamine content in tadalafil: firstly derivatizing the methylamine and then performing high performance liquid chromatography detection.

[0007] Furthermore, the derivatization method is to add p-toluenesulfonyl chloride and methylamine to tadalafil, shake it, then add KOH solution, shake it well and dilute it to a fixed volume.

[0008] Furthermore, in the derivatized solution, the concentration of tadalafil is 1-2 mg / mL, the concentration of p-toluenesulfonyl chloride is 0.02-0.04 mg / mL, the concentration of methylamine is 20-40 mg / mL, the concentration of KOH is 0.005-0.01 mg / mL, and the mass fraction of methylamine is 25%-30%.

[0009] Furthermore, when preparing the solution after derivatization, various reagents are first prepared into stock solutions, and then diluted with a solvent to a fixed volume; the solvent is acetonitrile-water (50:50).

[0010] Furthermore, the chromatographic conditions of the HPLC are as follows: the chromatographic column is C18, the mobile phase A is 0.1% phosphoric acid, the mobile phase B is acetonitrile, the detection wavelength is 210-254nm, the column temperature is 30℃±5℃, and the flow rate is 1.0mL / min±0.1mL / min.

[0011] Furthermore, the detection wavelength was 226 nm, the column temperature was 30° C., the flow rate was 1.0 mL / min, and the injection volume was 10 μL.

[0012] Furthermore, the chromatographic column is a Waters XbridgeC18 chromatographic column, with octadecylsilane bonded silica gel as the filler stationary phase.

[0013] Furthermore, during the detection process, the mobile phase was used for gradient elution, and the specific elution procedure is shown in Table 1:

[0014] Table 1

[0015]

[0016] The present invention also provides an application of the method in the production quality control of tadalafil raw material medicine.

[0017] The present invention has the following beneficial effects:

[0018] The method for determining methylamine in tadalafil by high-performance liquid chromatography provided by the present invention derivatizes methylamine impurities in tadalafil raw materials into N-methyl-p-toluenesulfonamide, and achieves a separation degree of 2.34 from adjacent impurities, which is determined to be greater than 1.5, successfully separates methylamine from other impurities, and accurately detects the content of methylamine. The detection limit LOD is 0.00301 μg / mL, which is approximately equivalent to 0.0006% of the sample concentration, and the quantitative limit is 0.01002 μg / mL, which is approximately equivalent to 0.002% of the sample concentration. At the same time, a linear experiment proves that the method has good linearity within the range of the quantitative limit to 150% of the limit concentration (0.0100 μg / mL-0.7514 μg / mL).

[0019] The detection method has high accuracy. The average recovery rate of the target peak in multiple experiments is 100.1%, and the RSD is 2.1%, which is less than 3.0%. It also has good repeatability and can meet the needs of actual sample detection. It is suitable for the determination of methylamine impurities in tadalafil raw materials. The determination results are reliable, which improves the product quality standards, controls the risk of exceeding the methylamine impurity standard, and is conducive to ensuring the safety and effectiveness of synthetic drug products. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is the test spectrum of Example 1, in which A is a chromatogram with o-tolualdehyde as the derivatization reagent and 0.1% trifluoroacetic acid as the mobile phase A, B is a chromatogram with o-tolualdehyde as the derivatization reagent and pure water as the mobile phase A, and C is a chromatogram with p-toluenesulfonyl chloride as the derivatization reagent and 0.1% trifluoroacetic acid as the mobile phase A.

[0021] Figure 2 This is the test spectrum for determining the detection wavelength in Example 3.

[0022] Figure 3 This is the detection spectrum of different mobile phase types in Example 3, where A is trifluoroacetic acid and B is phosphoric acid.

[0023] Figure 4 This is the detection spectrum of Example 3 using gradient elution from 3-1 to 3-6.

[0024] Figure 5 This is the linear relationship diagram of N-methyl-p-toluenesulfonamide in Example 4. DETAILED DESCRIPTION

[0025] The embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention.

[0026] The liquid chromatograph used in the following examples is Waters e2695, UV and PDA detectors; the electronic analytical balance is Mettler-Toledo XSE 205; tadalafil samples, reference substances (SM01, SM02, TL01, TL02, IP-A, IP-C, IP-D, IP-I, isomer A, impurity E, impurity H, impurity G); methylamine aqueous solution (AR grade); o-methylbenzaldehyde (AR grade), p-toluenesulfonyl chloride (AR grade), acetonitrile (HPLC grade), potassium hydroxide (AR grade), phosphoric acid (AR grade).

[0027] The formula of each solution commonly used in the following embodiments is as follows:

[0028] Methylamine stock solution (concentration is 0.05 mg / mL, where the concentration of methylamine aqueous solution is calculated as 25%): Accurately weigh 2.0292 g of methylamine aqueous solution, place it in a 100 mL volumetric flask, add purified water to make up to volume and shake well; accurately measure 1.0 mL of the solution and place it in a 100 mL volumetric flask, add purified water to make up to volume and shake well.

[0029] Preparation of p-toluenesulfonyl chloride (TsCl) stock solution (concentration 0.5 mg / mL): Accurately weigh 50 mg of TsCl, place it in a 100 mL volumetric flask, add acetonitrile to dilute to the scale, and shake well to obtain p-toluenesulfonyl chloride stock solution.

[0030] Preparation of KOH stock solution (concentration is 0.1 mg / mL): Accurately weigh 25 mg of KOH, place it in a 100 mL volumetric flask, add purified water to dilute to the scale, and shake well to obtain the KOH stock solution.

[0031] Preparation of test sample solution: Take 25 mg of tadalafil sample and place it in a 50 mL volumetric flask, accurately measure 1.0 mL of p-toluenesulfonyl chloride stock solution, cover the bottle cap, shake for 1-2 minutes, add 1.0 mL of potassium hydroxide stock solution, shake well, let it stand for 10 minutes, then dilute to the scale with acetonitrile-water (50:50), and shake well to obtain the test sample solution.

[0032] Preparation of control solution: Accurately measure 1.0 mL of p-toluenesulfonyl chloride stock solution and place it in a 50 mL volumetric flask. Quickly and accurately measure 1.0 mL of methylamine stock solution, cover the bottle cap, shake for about 1 min, add 1.0 mL of potassium hydroxide stock solution, shake well, let it stand for 10 min, then dilute to the scale with acetonitrile-water (50:50), and shake well to obtain a control solution.

[0033] Accuracy 80% solution: Accurately weigh 25 mg of tadalafil sample and place it in a 50 mL volumetric flask. Accurately measure 1.0 mL of p-toluenesulfonyl chloride stock solution, then quickly and accurately measure 0.8 mL of methylamine stock solution. Cover the bottle cap and shake for 1 minute. Add 1.0 mL of KOH stock solution and shake well. After leaving it for 10 minutes, dilute it to the scale with acetonitrile-water (50:50) and shake well.

[0034] Accuracy 100% solution: Accurately weigh 25 mg of tadalafil sample and place it in a 50 mL volumetric flask. Accurately measure 1.0 mL of p-toluenesulfonyl chloride stock solution, then quickly and accurately measure 1.0 mL of methylamine stock solution. Cover the bottle cap and shake for 1 min. Add 1.0 mL of KOH stock solution and shake well. After leaving it for 10 min, dilute it to the scale with acetonitrile-water (50:50) and shake well.

[0035] Accuracy 120% solution: Accurately weigh 25 mg of tadalafil sample and place it in a 50 mL volumetric flask. Accurately measure 1.0 mL of p-toluenesulfonyl chloride stock solution, then quickly and accurately measure 1.2 mL of methylamine stock solution. Cover the bottle cap and shake for 1 min. Add 1.0 mL of KOH stock solution and shake well. After leaving it for 10 min, dilute it to the scale with acetonitrile-water (50:50) and shake well.

[0036] In the following examples, octadecylsilane bonded silica gel (Waters Xbridge C18, 4.6*150 mm, 5 μm) was used as the stationary phase in the chromatographic column.

[0037] Example 1 Selection of derivatization reagents

[0038] O-tolualdehyde and p-toluenesulfonyl chloride were used as derivatization reagents, respectively, and the peak area was detected to quantitatively detect methylamine and determine the most suitable derivatization reagent. The method is as follows:

[0039] (1) Preparation of o-methylbenzaldehyde derivatization reagent: Add 2 mL o-methylbenzaldehyde (concentration: 0.5 mg / ml), 1 mL methylamine aqueous solution (0.05 mg / ml), and 1 mL KOH stock solution into a headspace bottle, cap it, react at room temperature for 1 h, and then dilute it with 50% acetonitrile aqueous solution to obtain a derivatization reagent;

[0040] (2) Preparation of p-toluenesulfonyl chloride derivatization reagent: accurately weigh 50 mg of tadalafil (API) and place it in a 50 mL volumetric flask, measure 2 mL of p-toluenesulfonyl chloride stock solution, accurately measure 1 mL of methylamine stock solution, cover the bottle cap and shake for 30 seconds, then add 1 mL of KOH stock solution, shake well, dilute to the mark with acetonitrile-water (50:50), shake well and use as the derivatization reagent;

[0041] (3) Take 10 μL of each of the two derivatization reagents and inject them into the chromatograph, use C18 as the filler, 0.1% trifluoroacetic acid or pure water as the mobile phase A, acetonitrile as the mobile phase B, perform gradient elution according to Table 2, and the detection wavelength is 278 nm.

[0042] Table 2 Gradient elution

[0043] Time (min) A B Flow rate (mL / min) 0 75 25 1.0 2 75 25 1.0 4 75 25 1.0 6 75 25 1.0 8 25 75 1.0 10 25 75 1.0 12 25 75 1.0 14 25 75 1.0 16 25 75 1.0 18 75 25 1.0 20 75 25 1.0 22 75 25 1.0 24 75 25 1.0

[0044] When trichloroacetic acid is used as the mobile phase ( Figure 1 A), the target peak generated after adding methylamine and the peak of o-methylbenzaldehyde shifted forward, so the method needed to be optimized.

[0045] Pure water was used instead of 0.1% trichloroacetic acid ( Figure 1B), it was found that the target peak at 4.0 min was smaller than that at 4.0 min. This may be because the reaction is a reversible reaction and the generated target product is unstable and easily decomposed. Therefore, o-methylbenzaldehyde is not suitable as a derivatization reagent.

[0046] p-Toluenesulfonyl chloride was used as a derivatization reagent ( Figure 1 C), the reaction proceeds normally in the trifluoroacetic acid system, so it can be used as a derivatization reagent, but the response of the target peak is low, the retention time is 9.379min, and the peak area is 170465. Therefore, when p-toluenesulfonyl chloride is used as a derivatization reagent, its derivatization method and chromatographic conditions need to be optimized.

[0047] Example 2 Optimization of derivatization reaction conditions

[0048] p-Toluenesulfonyl chloride was used as a derivatization reagent, C18 was used as a filler, 0.1% trifluoroacetic acid was used as a mobile phase A, acetonitrile was used as a mobile phase B, elution was performed according to the gradient in Table 1, and the detection wavelength was 226 nm.

[0049] 1. Determination of KOH dosage

[0050] Since the reaction of p-toluenesulfonyl chloride and methylamine will generate HCl, which inhibits the forward progress of the reaction, KOH needs to be added as an acid-binding agent. Therefore, under the premise of adding excessive p-toluenesulfonyl chloride, the amount of KOH needs to be investigated and determined. The amount of KOH and the chromatographic results are shown in Table 3.

[0051] Methylamine + TsCl + API: Weigh 50 mg of API and place it in a 50 mL volumetric flask. Measure 2 mL of p-methanesulfonyl chloride stock solution and accurately measure 1 mL of methylamine stock solution. Cover the bottle cap, shake well, dilute to the mark with acetonitrile-water (50:50), and shake well to obtain.

[0052] Methylamine + TsCl + KOH + API: Weigh 50 mg of API and place it in a 50 mL volumetric flask. Measure 2 mL of p-methanesulfonyl chloride stock solution and accurately measure 1 mL of methylamine stock solution. Cover the bottle cap and shake for 3 seconds. Add 1 mL of KOH stock solution and shake well. Dilute to the mark with acetonitrile-water (50:50) and shake well to obtain.

[0053] Methylamine + TsCl + KOH: Measure 2 mL of p-methanesulfonyl chloride stock solution, then accurately measure 1 mL of methylamine stock solution, cover the bottle cap, shake for 3 seconds, then add different amounts of KOH stock solution, shake well, dilute to the scale with acetonitrile-water (50:50), and shake well to obtain.

[0054] Table 3 KOH dosage and chromatographic peak area

[0055]

[0056]

[0057] The results show that as the molar ratio of methylamine:TsCl:KOH gradually increases, the area of ​​the target peak first increases and then decreases. When the molar ratio is about 1:3:5, the area of ​​the target peak is the largest. Therefore, a molar ratio of methylamine:TsCl:KOH of about 1:3:5 is selected for derivatization.

[0058] 2. Determination of Derivatization Reaction Time

[0059] Since the reaction time of toluenesulfonyl chloride and methylamine may affect the degree of reaction, after adding KOH solution to the reaction solution and leaving it for different time periods, chromatography was performed to explore the effect of derivatization time on the recovery rate. The results are shown in Table 3. After adding KOH solution to the toluenesulfonyl chloride derivatization reagent, it was immediately diluted to a fixed volume, and then 6 injections were continuously injected for chromatographic detection to determine the peak area RSD. The results are shown in Table 4, where the molar ratio of methylamine:TsCl:KOH was 1:3:5.

[0060] Table 4

[0061]

[0062]

[0063] Table 5

[0064] Serial number 1 2 3 4 5 6 RSD Peak area 186789 187748 187763 188339 188544 188511 0.36％

[0065] The experimental results show (Table 4) that when the KOH solution is added and diluted immediately, the peak area in the control solution is smaller due to the difference in operation time, and the recovery rate is between 107.6% and 114.4%, which is larger, indicating that the reaction is still going on during the operation time. However, when the immediately diluted solution is poured into the injection vial and injected 6 times continuously, the peak area RSD is 0.36% (Table 5), indicating that the reaction is basically stable after dilution and no longer goes on. Therefore, the placement time before dilution should be focused on.

[0066] When diluted after 10 minutes, the recovery rate was between 101.8% and 104.2%, indicating that after 10 minutes, the reaction basically tended to equilibrium (Table 4). Therefore, 10 minutes was selected as the reaction time.

[0067] In summary, the reaction conditions for derivatization are to mix methylamine and TsCl in a molar ratio of 1:3, then add a 5-fold molar ratio of KOH solution, mix well to prepare a derivatization reagent, then leave it for 10 minutes and then dilute it, and finally subject the diluted solution to high performance liquid chromatography.

[0068] Example 3 Determination of liquid chromatography conditions

[0069] p-Toluenesulfonyl chloride was used as a derivatization reagent, C18 was used as a filler, and acetonitrile was used as a mobile phase B for elution. The detection wavelength, type of mobile phase, elution gradient, sample concentration, and injection volume were determined.

[0070] 1. Determination of wavelength

[0071] 0.1% trifluoroacetic acid was used as mobile phase A, and elution was performed according to the gradient in Table 1 to obtain the LCMS results, and 9.35 min was determined to be the target peak of N-methyl-p-toluenesulfonamide, and the MS+1 (molecular weight) was 186.16. According to the scanning diagram of the target peak in the PDA detector, the detection wavelength of the target peak was determined to be 226 nm ( Figure 2 ).

[0072] 2. Determination of mobile phase

[0073] 0.1% trifluoroacetic acid and 0.1% phosphoric acid were used as mobile phase A, respectively, and elution was performed according to the gradient in Table 2 to detect the baseline shift.

[0074] The results are as follows Figure 3 As shown, trifluoroacetic acid has terminal absorption at low wavelengths. When the wavelength of 226 nm is selected in this method, the baseline shift is large, while when 0.1% phosphoric acid is used as mobile phase A, the baseline shift is small. Therefore, phosphoric acid is used instead of trifluoroacetic acid as mobile phase A in subsequent experiments to improve the sensitivity of the method.

[0075] 3. Determination of elution gradient

[0076] In the HPLC method, tadalafil API and its impurities will emit peaks at a wavelength of 226 nm, which may interfere with the detection of methylamine derivatives. Therefore, when examining the specificity of the method, the appropriate elution gradient is selected based on the separation of the impurity peak and the target peak in the impurity mixed derivatization solution.

[0077] SM01 stock solution: Take about 2 mg of SM01 reference substance, place it in a 10 mL EP tube, add 4 mL of diluent to dissolve it, and shake well. Prepare SM02, TL01, TL02, IP-A, IP-C, IP-D, IP-I, impurity E, impurity H, impurity G, and isomer A stock solutions in the same way.

[0078] SM01 localization solution: Take 1 mL of SM01 stock solution, place it in a 10 mL volumetric flask, add diluent to make up the volume, and shake well. Prepare SM02, TL01, TL02, IP-A, IP-C, IP-D, IP-I, impurity E, impurity H, impurity G, and isomer A localization solutions in the same way.

[0079] Preparation of impurity mixed derivatization solution: accurately weigh 50 mg of tadalafil, place in a 50 mL volumetric flask, measure 1 mL of p-toluenesulfonyl chloride stock solution, and then measure 0.5 mL of each of the impurity stock solutions (SM01, SM02, TL01, TL02, IP-A, IP-C, IP-D, IP-I, impurity E, impurity H, impurity G), cover the bottle cap, shake for about 1 min, add 1 mL of KOH stock solution, shake well, let stand for 10 min, then dilute to the scale with acetonitrile-water (50:50), shake well.

[0080] All other testing conditions remained unchanged. The same batch of samples was used and divided into 6 groups, namely 3-1, 3-2, 3-3, 3-4, 3-5 and 3-6. Under different gradient conditions, the impurity mixed derivatization solution was subjected to chromatographic testing. The specific elution gradients are shown in Table 6-11:

[0081] Table 6 3-1 Corresponding gradient conditions

[0082] Time (min) Mobile phase A (%) Mobile phase B (%) 0 70 30 10.0 10 90 10.5 70 30 15.0 10 90

[0083] Table 7 3-2 Corresponding gradient conditions

[0084] Time (min) Mobile phase A (%) Mobile phase B (%) 0 70 30 4.0 46 54 7.0 46 54 10.0 10 90 10.5 70 30 15.0 70 30

[0085] Table 8 3-3 Corresponding gradient conditions

[0086] Time (min) Mobile phase A (%) Mobile phase B (%) 0 70 30 4.0 55 45 7.0 55 45 10.0 10 90 10.5 70 30 15.0 70 30

[0087] Table 9 3-4 corresponding gradient conditions

[0088] Time (min) Mobile phase A (%) Mobile phase B (%) 0 60 40 5.0 60 40 10.0 10 10 10.5 60 40 15.0 60 40

[0089] Table 10 3-5 corresponding gradient conditions

[0090] Time (min) Mobile phase A (%) Mobile phase B (%) 0 65 35 7.0 65 35 10.0 10 90 10.5 65 35 15.0 65 35

[0091] Table 11 3-6 corresponding gradient conditions

[0092] Time (min) Mobile phase A (%) Mobile phase B (%) 0 70 30 7.0 70 30 8.0 10 90 10.0 10 90 10.5 70 30 15.0 70 30

[0093] The results show that ( Figure 4 ), SM02, IP-D and the target peak are adjacent to each other, and the positions of the three are slightly changed by the influence of the elution gradient; under the gradient elution of 3-1, SM02 and IP-D overlap, with a retention time of 4.590min, which will interfere with the detection of the target peak ( Figure 4 A); Under the gradient elution of 3-2, SM02 and IP-D overlapped, with a retention time of 4.581 min, which still interfered with the detection of the target peak and did not improve ( Figure 4B); Under the gradient elution of 3-3, SM02 and IP-D were separated, and the retention times were 4.868min and 5.166min, respectively. At this time, the target peak between the two would still interfere with the detection of the target peak ( Figure 4 C); Under the gradient elution of 3-4, IP-D and SM02 are separated, and the IP-D peak is before SM02, with retention times of 3.620min and 3.955min respectively. At this time, the target peak is still between the two and will be interfered ( Figure 4 D); Under the gradient elution of 3-5, SM02 and IP-D were separated, and the retention times were 4.515min and 4.800min, respectively. At this time, the target peak between the two would still interfere with the detection of the target peak ( Figure 4 E); Under the gradient elution of 3-6, the peak order is SM02, target peak, IP-D. It can be found that the three substances have been completely separated, indicating that the elution gradient can meet the experimental requirements ( Figure 4 F).

[0094] 4. Determination of sample concentration and injection volume

[0095] When acetonitrile-water (30:70) was used to prepare a 1 mg / mL solution of the tadalafil sample, the sample was completely dissolved, but after being placed for 1 hour, solids precipitated. Therefore, after entering the chromatograph, the sample solution may precipitate. Therefore, the sample concentration was determined to be adjusted to 0.5 mg / mL, the methylamine limit was 0.10%, and the concentration was 0.5 μg / mL. A control solution was prepared according to this concentration, and 10 μL and 20 μL were injected respectively to test the sensitivity of the high performance liquid chromatography. The results are shown in Table 12:

[0096] Table 12 Peak area of ​​target peaks at different injection volumes and S / N of target peaks in reference solutions

[0097] Injection volume Peak area S / N 10μL 271474 9037 20μL 133073 5283

[0098] The results show (Table 12) that when the sample concentration is reduced and the injection volume is 10 μL, the target peak S / N is 5293, and the concentration is equivalent to 0.05% of the sample concentration. The sensitivity meets the requirements, so the sample concentration is determined to be 0.5 mg / mL and the injection volume is 10 μL.

[0099] Example 5

[0100] (1) Chromatographic conditions: The stationary phase of the chromatographic column was octadecylsilane bonded silica gel (Waters Xbridge C18, 4.6 mm × 250 mm, 5 μm or a chromatographic column with equivalent performance); mobile phase A: 0.1% phosphoric acid, mobile phase B: acetonitrile; flow rate: 1.0 mL / min; column temperature: 30°C; detection wavelength: 226 nm; injection volume: 10 μL; elution gradient is shown in Table 10.

[0101] (2) Solution preparation:

[0102] Blank solution / diluent: acetonitrile-water (50:50)

[0103] Derivatization blank solution: Accurately measure 1.0 mL of p-toluenesulfonyl chloride stock solution, place it in a 50 mL volumetric flask, cover the flask, shake for 1 min, add 1.0 mL of KOH stock solution, shake well, let stand for 10 min, then dilute to the scale with acetonitrile-water (50:50), and shake well.

[0104] Test solution: Weigh about 25 mg of tadalafil sample and place it in a 50 mL volumetric flask. Accurately measure 1.0 mL of p-toluenesulfonyl chloride stock solution, cover the bottle cap, shake for 1 min, add 1.0 mL of KOH stock solution, shake well, let stand for 10 min, then dilute to the scale with acetonitrile-water (50:50), and shake well.

[0105] (3) Specificity experiment

[0106] The blank solution, the derivatized blank solution, the SM01 positioning solution in Example 3 (III) and the impurity mixed derivatized solution were subjected to chromatography, and the peak retention time and the separation between the peaks were calculated. The results are shown in Table 13:

[0107] Table 13 Specificity test results

[0108]

[0109] The results showed that the blank solution and the derivatized blank solution had no interference with the detection of the target peak; in the impurity mixed derivatized solution, the separation between the target peak and the adjacent peaks was 2.34, which was greater than 1.5, and had specificity.

[0110] (4) System adaptability

[0111] Take 6 consecutive samples of the reference solution and put them into the chromatograph to detect the target peak area and RSD. The results are shown in Table 14:

[0112] Table 14 System suitability results

[0113] Serial number Retention time (min) Peak area 1 6.249 126064 2 6.246 125752 3 6.241 125516 4 6.240 125315 5 6.250 125176 6 6.249 125071 average 6.246 125482.3 RSD(%) / 0.30

[0114] The results showed that the peak area RSD of the target peak in 6 consecutive reference solutions was 0.30%, which was less than 1.0%, indicating that the method had good system applicability.

[0115] (5) Limit of Quantification and Limit of Detection Experiments

[0116] Quantitation limit solution: Accurately measure 1.0 mL of p-toluenesulfonyl chloride stock solution and place it in a 50 mL volumetric flask. Quickly and accurately measure an appropriate amount of methylamine stock solution, cover the bottle cap and shake for 1 min. Add 1.0 mL of KOH stock solution, shake well and let stand for 10 min, then dilute to the scale with acetonitrile-water (50:50), shake well, so that the signal-to-noise ratio of the target peak is greater than 10, and the quantitation limit solution is obtained; prepare 6 solutions in parallel.

[0117] Detection limit solution: Accurately measure 1.0 mL of p-toluenesulfonyl chloride stock solution and place it in a 50 mL volumetric flask. Quickly and accurately measure an appropriate amount of methylamine stock solution, cover the bottle cap and shake for 1 min. Add 1.0 mL of KOH stock solution, shake well and let stand for 10 min, then dilute to the scale with acetonitrile-water (50:50), shake well so that the signal-to-noise ratio of the target peak is greater than 3, and the detection limit solution is obtained.

[0118] The quantitative limit and detection limit solutions were chromatographed separately, the relevant parameters of the target peaks were detected and the concentrations of the quantitative limit and detection limit were calculated.

[0119] Table 15 Quantitative limit and detection limit results

[0120]

[0121]

[0122] The results showed that the average S / N value of the target peak in the six detection limit solutions was 6.03, and the detection limit was 3.01 ng / mL, equivalent to 0.0006% of the sample concentration.

[0123] The average S / N value of the target peak in the quantitation limit solution was 37.60, the RSD of the peak area was 0.61%, and the quantitation limit was 10.02 ng / mL, equivalent to 0.002% of the sample concentration.

[0124] (6) Linear experiment

[0125] The linear range was designed to be from the quantitative limit concentration to 150% of the limit concentration. Six concentration points were designed, namely the quantitative limit, 10%, 50%, 80%, 100%, and 150% of the reference solution concentration.

[0126] Linear concentration 10%: Accurately measure 1.0 mL of p-toluenesulfonyl chloride stock solution and place it in a 50 mL volumetric flask. Quickly and accurately measure 0.1 mL of methylamine stock solution, cover the bottle cap and shake for 1 min. Add 1.0 mL of KOH stock solution and shake well. After leaving it for 10 min, dilute it to the scale with acetonitrile-water (50:50) and shake well.

[0127] Linear concentration 50%: Accurately measure 1.0 mL of p-toluenesulfonyl chloride stock solution and place it in a 50 mL volumetric flask. Quickly and accurately measure 0.5 mL of methylamine stock solution, cover the bottle cap and shake for 1 min. Add 1.0 mL of KOH stock solution and shake well. After leaving it for 10 min, dilute it to the scale with acetonitrile-water (50:50) and shake well.

[0128] Linear concentration 80%: Accurately measure 1.0 mL of p-toluenesulfonyl chloride stock solution and place it in a 50 mL volumetric flask. Quickly and accurately measure 0.8 mL of methylamine stock solution, cover the bottle cap and shake for 1 minute. Add 1.0 mL of KOH stock solution and shake well. After leaving it for 10 minutes, dilute it to the scale with acetonitrile-water (50:50) and shake well.

[0129] Linear concentration 100%: Accurately measure 1.0 mL of p-toluenesulfonyl chloride stock solution and place it in a 50 mL volumetric flask. Quickly and accurately measure 1.0 mL of methylamine stock solution, cover the bottle cap, shake for 1 min, add 1.0 mL of KOH stock solution and shake well. After leaving it for 10 min, dilute it to the scale with acetonitrile-water (50:50) and shake well.

[0130] Linear concentration 150%: Accurately measure 1.0 mL of p-toluenesulfonyl chloride stock solution and place it in a 50 mL volumetric flask. Quickly and accurately measure 1.5 mL of methylamine stock solution, cover the bottle cap and shake for 1 min. Add 1.0 mL of KOH stock solution and shake well. After leaving it for 10 min, dilute it to the scale with acetonitrile-water (50:50) and shake well.

[0131] The reference substance solutions at the above 6 concentration points were chromatographed respectively, and two parallel experiments were performed, and the peak areas were counted. The results are shown in Table 16:

[0132] Table 16 Linearity test results

[0133]

[0134] The results showed (Table 16 and Figure 5 ), the linear equation was y=231669x+2923.7, the correlation coefficient R was 0.9962, and the Y-axis intercept accounted for 100% of the limit response concentration ratio was less than 10%, indicating that the linearity was good in the range of quantitation limit to 150% of the limiting concentration (0.0100μg / mL-0.7514μg / mL).

[0135] (7) Accuracy experiment

[0136] Blank sample: accurately weigh 25 mg of tadalafil sample and place it in a 50 mL volumetric flask. accurately measure 1.0 mL of p-toluenesulfonyl chloride stock solution, cover the bottle cap and shake for 1 min, add 1.0 mL of KOH stock solution and shake well. After leaving it for 10 min, dilute it to the scale with acetonitrile-water (50:50) and shake well.

[0137] Solutions with accuracy of 80%, 100% and 120% were taken for chromatography respectively. The experiment was repeated 3 times for each accuracy solution. The peak area was recorded and the recovery rate and RSD were calculated. The results are shown in Table 17:

[0138] Table 17 Accuracy experimental results

[0139]

[0140] The results showed that the recoveries of the target peaks of 9 samples in 3 accuracy solutions were all between 95.92% and 102.17%, with an average recovery of 100.1% and an RSD of 2.1%, which was less than 3.0%, indicating high accuracy.

[0141] (8) Repeatability experiment

[0142] The control solution and the 100% accuracy solution in (7) were taken and divided into 6 equal parts, and then respectively put into the liquid chromatograph for chromatography, the peak area in the chromatogram was recorded, and the repeatability of the content detection experiment was calculated. The results are shown in Table 18:

[0143] Table 18 Repeatability test results

[0144]

[0145] The average content of the target peak in 6 replicate samples was 0.10%, and the RSD was 1.8%, which was less than 3.0%, indicating that the method had good repeatability.

[0146] (9) Solution stability test

[0147] The control solution and the 100% accuracy solution in (7) were placed at room temperature for a period of time and then placed in a liquid chromatograph to detect the content of the target peak in the solution and investigate the stability of the solution. The results are shown in Table 19:

[0148] Table 19 Solution stability test results

[0149]

[0150] The control solution and the 100% accuracy solution were placed at room temperature for 26 hours. In the 100% accuracy solution, the RSD of the target peak content was 0.45%, which was less than 3.0%, indicating that the solution had good stability after being placed at room temperature for 26 hours.

[0151] (10) Durability test

[0152] Derivatization blank solution, impurity mixed derivatization solution, control solution and accuracy 100% solution were taken to investigate the durability of different chromatographic conditions. The chromatographic conditions were: column temperature: temperature ± 2C; flow rate: 1mL / min ± 0.1mL / min; mobile phase: ratio ± 2%.

[0153] Table 20 Durability test results

[0154]

[0155] The results showed that the derivatized blank solution had no interference with the detection of the target peak. Under each chromatographic condition, the separation between the target peak and the adjacent impurities in the impurity mixed derivatized solution was greater than 1.5. Under each chromatographic condition, the RSD of the target peak content in the 100% accuracy solution was 0.09%.

[0156] (10) Derivatization method durability test

[0157] The control solution and the 100% accuracy solution were taken to investigate the robustness of the derivatization method. The derivatization conditions were changed as follows:

[0158] 1) Rapidly add methylamine and shake for 0.5 min, 1 min, 1.5 min;

[0159] 2) Standing time after adding KOH: 5min, 10min, 15min.

[0160] Table 21 Derivatization method durability test results

[0161]

[0162] In the solution with 100% accuracy, the recovery rate of the target peak after rapid addition of methylamine and shaking for 1±0.5min was between 96.46% and 99.39%, indicating that the shaking time had little effect on the recovery rate and had little effect on the accuracy of the results. The recovery rate of the target peak after adding KOH and standing for 10±5min was between 95.85% and 99.39%, indicating that the standing time had little effect on the recovery rate and had little effect on the accuracy of the results. This indicates that the derivatization method has good durability.

Claims

1. A method for determining methylamine in tadalafil by high performance liquid chromatography, characterized in that: First, methylamine is derivatized and then detected by HPLC; The derivatization method comprises adding p-toluenesulfonyl chloride and methylamine to tadalafil, shaking, then adding KOH solution, shaking well, and then diluting to a fixed volume; The chromatographic conditions of the HPLC are as follows: the chromatographic column is C18, the mobile phase A is 0.1% phosphoric acid, the mobile phase B is acetonitrile, the detection wavelength is 210-254 nm, the column temperature is 30°C ± 5°C, and the flow rate is 1.0 mL / min ± 0.1 mL / min; During the detection process, the mobile phase was used for gradient elution, and the elution procedure was: 。 2. The method for determining methylamine in tadalafil by high performance liquid chromatography according to claim 1, characterized in that: In the derivatized solution, the concentration of tadalafil is 1-2 mg / mL, the concentration of p-toluenesulfonyl chloride is 0.02-0.04 mg / mL, the concentration of methylamine is 20-40 mg / mL, the concentration of KOH is 0.005-0.01 mg / mL, and the mass fraction of methylamine is 25%-30%.

3. The method for determining methylamine in tadalafil by high performance liquid chromatography according to claim 2, characterized in that: When preparing the solution after derivatization, various reagents are first prepared into stock solutions, and then diluted with a solvent to a fixed volume; the solvent is acetonitrile-water in a ratio of 50:

50.

4. The method for determining methylamine in tadalafil by high performance liquid chromatography according to claim 1, characterized in that: The detection wavelength was 226 nm, the column temperature was 30 °C, the flow rate was 1.0 mL / min, and the injection volume was 10 μL.

5. The method for determining methylamine in tadalafil by high performance liquid chromatography according to claim 1, characterized in that: The chromatographic column is a Waters Xbridge C18 chromatographic column, with octadecylsilane bonded silica gel as the filler stationary phase.

6. Application of the method according to any one of claims 1 to 5 in the production quality control of tadalafil raw material.

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