Method for detecting related substances of trazodone hydrochloride sustained release tablets

Abstract

The invention discloses a method for detecting related substances of trazodone hydrochloride sustained release tablets, and belongs to the technical field of pharmaceutical analysis. According to the detection method, high performance liquid chromatography is adopted, and the chromatographic condition is that an octadecyl bonded silica gel column is used as a chromatographic column; the column temperature is 25-35 DEG C, and the flow velocity is 0.8-1.2 ml / min; the sample size is 5-15 [mu] l, and the detection wavelength is 252-256 nm; the mobile phase A is a potassium hexafluorophosphate buffer solution; the mobile phase B is acetonitrile; and carrying out gradient elution. Compared with trazodone hydrochloride sustained-release tablet imported drug registration standards and United States Pharmacopoeia related substance detection methods of trazodone hydrochloride bulk drugs, the method abandons a tailing-sweeping agent triethylamine suitable for an alkaline sample, adjusts the pH value of a mobile phase to be acidic, improves the peak shape and increases retention of trazodone hydrochloride and impurities through a chaotropic agent potassium hexafluorophosphate buffer solution, and improves the quality of trazodone hydrochloride sustained-release tablets. The method has the advantages of moderate impurity retention, good separation effect, excellent peak shape, long service life of the chromatographic column and reduction of the analysis cost. The method has the advantages of strong specificity, high sensitivity, high accuracy and good durability.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis technology, and relates to a method for detecting related substances in trazodone hydrochloride sustained-release tablets. Background Technology

[0002] Trazodone Hydrochloride Sustained-Release Tablets are an antidepressant manufactured by AZ.CHIM.RIUN.ANGELINI.FRANCESCO.ACRAF.SPA in Italy, and were approved for marketing in China on December 2, 2020. Its mechanism of action is to inhibit the reuptake of the neurotransmitter serotonin, thereby exerting its antidepressant effect. Its structural formula is shown in Formula I below:

[0003]

[0004] Related substances in pharmaceutical preparations primarily study degradation impurities resulting from interactions between the active pharmaceutical ingredient (API) and excipients and packaging materials, as well as degradation impurities inherent in the API itself. Process impurities already controlled in the API are generally not controlled in the finished product. Research on related substances is crucial for ensuring the safety of the preparation and provides data support for excipient screening, formulation process research, stability studies, and the selection of packaging and storage methods. Therefore, related substance detection is particularly important throughout the entire lifecycle of the preparation, and accurate and reliable detection methods are a prerequisite and essential guarantee for preparation quality research.

[0005] Trazodone hydrochloride extended-release tablets have been approved for marketing in China and have an import drug registration standard (standard number: JX20201028). The impurities controlled in the standard are impurity A hydrochloride and impurity L hydrochloride. The structural formulas of impurity A and impurity L are shown in Formula II and Formula III, respectively:

[0006]

[0007] The controlled process impurities of the active pharmaceutical ingredient trazodone hydrochloride are impurity B, impurity C, impurity D, impurity E, impurity G, impurity H, and impurity J, with their structural formulas as follows: (Ⅳ), (Ⅴ), (Ⅵ), (Ⅶ), (Ⅷ), (Ⅸ), (Ⅹ) respectively.

[0008]

[0009] Trazodone is strongly alkaline, leading to poor peak shape and severe tailing in liquid chromatography analysis. In the related substances detection method of the imported drug registration standard for trazodone hydrochloride sustained-release tablets, two impurities show weak retention, with impurity L exhibiting severe tailing. The use of triethylamine as a tailing agent in the mobile phase results in short column life and a lack of buffering capacity in the mobile phase. The active pharmaceutical ingredient (API) of trazodone hydrochloride sustained-release tablets is trazodone hydrochloride, which is listed in the United States Pharmacopeia (USP) NF 2021. The USP chromatographic method also uses triethylamine as a tailing agent in the mobile phase to improve peak shape. While trazodone and other impurities show good peak shapes in the alkaline system of triethylamine, the problems of short column life, high column wear, and high analytical costs due to triethylamine persist. Summary of the Invention

[0010] The purpose of this invention is to establish a method for detecting related substances in trazodone hydrochloride sustained-release tablets. This method solves the problem of peak tailing for trazodone and other impurities, and under acidic mobile phase conditions, it can significantly extend the life of the chromatographic column, reducing the cost of routine testing. This method can accurately and reliably detect related substances in trazodone hydrochloride sustained-release tablets, and can better control the quality of trazodone hydrochloride sustained-release tablets.

[0011] To achieve the above objectives, the present invention provides the following technical solution:

[0012] A method for detecting related substances in trazodone hydrochloride sustained-release tablets employs high-performance liquid chromatography (HPLC). The chromatographic conditions are as follows: octadecyl-bonded silica column; column temperature 25-35℃; flow rate 0.8-1.2 ml / min; detection wavelength 252-256 nm; injection volume 5-15 μl; mobile phase A: potassium hexafluorophosphate buffer; mobile phase B: acetonitrile; gradient elution is performed. The elution program for the gradient elution is set as follows:

[0013]

[0014] Furthermore, the pH range of the potassium hexafluorophosphate buffer solution is 1.8-2.2.

[0015] Further, the concentration of the potassium hexafluorophosphate buffer solution is 90 mmol / L to 110 mmol / L; more preferably, the concentration of the potassium hexafluorophosphate buffer solution is 100 mmol / L.

[0016] Furthermore, the chromatographic column is selected as a Waters SunFire C18 column. More preferably, the specifications of the Waters SunFire C18 column are: particle size × inner diameter × column length: 3.5μm × 4.6mm × 150mm.

[0017] In some embodiments, the column temperature is preferably 30°C;

[0018] In some embodiments, the flow rate is preferably 1.0 ml / min;

[0019] In some embodiments, the detection wavelength is preferably 254 nm;

[0020] In some embodiments, the injection volume is preferably 10 μl;

[0021] In some embodiments, the pH of potassium hexafluorophosphate buffer is adjusted using phosphoric acid.

[0022] In some embodiments, the pH of the mobile phase A buffer is preferably 2.0.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] This invention enables the detection of related substances in trazodone hydrochloride sustained-release tablets via high-performance liquid chromatography (HPLC). Compared with the imported drug registration standard for trazodone hydrochloride sustained-release tablets and the related substance detection method in the United States Pharmacopeia (USP) for trazodone hydrochloride raw material, this method eliminates the need for triethylamine, a tailing agent suitable for alkaline samples. Instead, it adjusts the mobile phase pH to acidic and uses potassium hexafluorophosphate buffer as a dissociation agent to improve peak shape and increase the retention of trazodone and other impurities. The retention of each impurity is moderate, resulting in good separation, excellent peak shape, long column life, and reduced analytical costs. This method is highly specific, sensitive, accurate, and robust. Attached Figure Description

[0025] Figure 1 This is a system suitability chromatogram of related substances for the trazodone hydrochloride sustained-release tablets of the present invention in Example 1.

[0026] Figure 2 This is a chromatogram of the mixed impurity localization solution of trazodone hydrochloride raw material in Example 2 of the present invention;

[0027] Figure 3 The graph shows the linear relationship of impurity A.

[0028] Figure 4 The graph shows the linear relationship between impurity L;

[0029] Figure 5 The graph shows the linear relationship of trazodone hydrochloride.

[0030] Figure 6 The system suitability chromatogram of related substances for the standard method for registration of imported drugs in Comparative Example 1;

[0031] Figure 7 for Figure 6 A partial view with a retention time of 0-4 minutes;

[0032] Figure 8The system suitability chromatogram of related substances for the USP method in trazodone hydrochloride raw material in Comparative Example 2;

[0033] Figure 9 This is a system applicability graph of related substances in the ammonium acetate system of Comparative Example 3.

[0034] Figure 10 This is a map showing the applicability of the method to the relevant material system in Comparative Example 4; Detailed Implementation

[0035] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be described in detail below through specific embodiments, but the content and scope of protection claimed by the present invention are not limited to the following embodiments.

[0036] All reagents used in this invention are commercially available or can be prepared by the methods described herein. Impurity A reference standard was purchased from the China National Institutes for Food and Drug Control; impurity L (hydrochloride), impurity B, impurity C, impurity D, impurity E, impurity G, impurity H, and impurity J reference standards were all purchased from Guangzhou Jiatu Technology Co., Ltd.; trazodone hydrochloride reference standard was purchased from the China National Institutes for Food and Drug Control; and trazodone hydrochloride sustained-release tablets (75 mg) were purchased from AZ. CHIM. RIUN. ANGELINI FRANCESCO ACRAFS. PA, Italy.

[0037] Example 1 System Suitability Test

[0038] (1) Chromatographic conditions

[0039] Chromatographic column: Waters SunFire C18, 4.6 mm × 150 mm, 3.5 μm

[0040] Mobile phase A: 100 mmol / L potassium hexafluorophosphate buffer, pH 2.0

[0041] Mobile phase B: Acetonitrile

[0042] Column temperature: 30℃

[0043] Flow rate: 1.0 ml / min

[0044] Detection wavelength: 254nm

[0045] Injection volume: 10 μl

[0046] Gradient procedure:

[0047]

[0048] (2) Solution preparation

[0049] Solvent: Take 400ml acetonitrile and 600ml water, mix well, and degas by sonication.

[0050] Mobile phase A: Dissolve 18.41 g of potassium hexafluorophosphate in 1000 ml of water, adjust the pH to 2.0 with phosphoric acid, and filter.

[0051] Mobile phase B: Take 1000 ml of acetonitrile, shake well and sonicate.

[0052] Impurity A reference standard stock solution: Weigh approximately 22.5 mg of impurity A reference standard accurately, place it in a 100 ml volumetric flask, add solvent and sonicate to dissolve and dilute to the mark, then shake well; accurately measure 5 ml of this solution, place it in a 50 ml volumetric flask, add solvent and dilute to the mark, then shake well.

[0053] Impurity L reference standard stock solution: Weigh approximately 17.6 mg of impurity L hydrochloride reference standard (approximately equivalent to 15 mg of impurity L), accurately weigh it, place it in a 100 ml volumetric flask, add solvent, sonicate to dissolve and dilute to the mark, and shake well; accurately measure 5 ml of this solution, place it in a 50 ml volumetric flask, add solvent to dilute to the mark, and shake well.

[0054] System suitability solution: Weigh approximately 37.5 mg of trazodone hydrochloride reference standard accurately, place it in a 50 ml volumetric flask, add an appropriate amount of solvent, sonicate to dissolve, accurately add 5 ml each of impurity A and impurity L reference standard stock solutions, dilute to the mark with solvent, and shake well.

[0055] (3) Measurement methods and test results

[0056] Set the chromatographic conditions and, after the system stabilizes, inject the system suitability solution into the chromatograph and record the chromatogram. The system suitability chromatogram is shown below. Figure 1 The elution order corresponds to impurity L, trazodone hydrochloride, and impurity A, respectively. Table 1 shows the results of the system suitability solution determination.

[0057] Table 1 Results of System Suitability Solution Tests

[0058] name Retention time (min) Resolution Tail Factor impurity L 11.482 — 1.09 Trazodone Hydrochloride 16.651 17.8 1.19 Impurity A 18.528 5.9 1.03

[0059] Experimental results show that under these conditions, impurities A and L controlled by this product are well separated from the main component trazodone hydrochloride, with symmetrical peak shapes.

[0060] Example 2 Specificity Test

[0061] Under the above experimental conditions, process impurities in trazodone hydrochloride raw material were located, and a forced destruction test was conducted on trazodone hydrochloride sustained-release tablets to examine the specificity of the analytical method.

[0062] (1) Chromatographic conditions were the same as in Example 1.

[0063] (2) Solution preparation

[0064] The solvent, mobile phase A, and mobile phase B were prepared in the same manner as in Example 1.

[0065] Impurity A stock solution: Weigh about 15 mg of impurity A reference standard, place it in a 100 ml volumetric flask, add an appropriate amount of solvent and sonicate to dissolve, cool to room temperature, and dilute to the mark with solvent.

[0066] Impurity B stock solution: Weigh about 15 mg of impurity B reference standard, place it in a 100 ml volumetric flask, add an appropriate amount of solvent and sonicate to dissolve, cool to room temperature, and dilute to the mark with solvent.

[0067] Impurity C stock solution: Weigh about 15 mg of impurity C reference standard, place it in a 100 ml volumetric flask, add an appropriate amount of solvent and sonicate to dissolve, cool to room temperature, and dilute to the mark with solvent.

[0068] Impurity D stock solution: Weigh about 15 mg of impurity D reference standard, place it in a 100 ml volumetric flask, add an appropriate amount of solvent and sonicate to dissolve, cool to room temperature, and dilute to the mark with solvent.

[0069] Impurity E stock solution: Weigh about 15 mg of impurity E reference standard, place it in a 100 ml volumetric flask, add an appropriate amount of solvent and sonicate to dissolve, cool to room temperature, and dilute to the mark with solvent.

[0070] Impurity G stock solution: Weigh about 15 mg of impurity G reference standard, place it in a 100 ml volumetric flask, add an appropriate amount of solvent and sonicate to dissolve, cool to room temperature, and dilute to the mark with solvent.

[0071] Impurity H stock solution: Weigh about 15 mg of impurity H reference standard, place it in a 100 ml volumetric flask, add an appropriate amount of solvent and sonicate to dissolve, cool to room temperature, and dilute to the mark with solvent.

[0072] Impurity J stock solution: Weigh about 15 mg of impurity J reference standard, place it in a 100 ml volumetric flask, add an appropriate amount of solvent and sonicate to dissolve, cool to room temperature, and dilute to the mark with solvent.

[0073] Impurity L stock solution: Weigh about 17.6 mg of impurity L hydrochloride reference standard (approximately equivalent to 15 mg of impurity L), place it in a 100 ml volumetric flask, add an appropriate amount of solvent and sonicate to dissolve, cool to room temperature, and dilute to the mark with solvent.

[0074] Mixed impurity positioning solution: Weigh approximately 75 mg of trazodone hydrochloride reference standard accurately, place it in a 100 ml volumetric flask, add an appropriate amount of solvent and sonicate to dissolve, then accurately add 1 ml of each impurity stock solution, and dilute to the mark with solvent.

[0075] Acid-degrading solution: Take 5 trazodone hydrochloride sustained-release tablets, place them in a 100ml volumetric flask, add an appropriate amount of solvent (about 70% of the volumetric flask volume), sonicate for 5 minutes, shake for 30 seconds, sonicate again for 15 minutes, cool to room temperature, add 2ml of 0.4mol / L hydrochloric acid solution, let stand at room temperature for 5 hours, neutralize with 2ml of 0.4mol / L sodium hydroxide solution, dilute to the mark with solvent, shake well, filter through a 0.22μm PTFE filter membrane, discard at least 3ml of the initial filtrate, and take the subsequent filtrate; accurately measure 5ml of the subsequent filtrate, place it in a 25ml volumetric flask, dilute to the mark with solvent, and shake well.

[0076] Alkali-degrading solution: Take 5 trazodone hydrochloride sustained-release tablets, place them in a 100ml volumetric flask, add an appropriate amount of solvent (about 70% of the volumetric flask volume), sonicate for 5 minutes, shake for 30 seconds, sonicate again for 15 minutes, cool to room temperature, add 5ml of 0.4mol / L sodium hydroxide solution, let stand at room temperature for 5 hours, neutralize with 5ml of 0.4mol / L hydrochloric acid solution, dilute to the mark with solvent, shake well, filter through a 0.22μm PTFE filter membrane, discard at least 3ml of the initial filtrate, and take the subsequent filtrate; accurately measure 5ml of the subsequent filtrate, place it in a 25ml volumetric flask, dilute to the mark with solvent, and shake well.

[0077] Oxidative Degradation Solution: Take 5 trazodone hydrochloride sustained-release tablets, place them in a 100ml volumetric flask, add an appropriate amount of solvent (about 70% of the volumetric flask volume), sonicate for 5 minutes, shake for 30 seconds, sonicate again for 15 minutes, cool to room temperature, add 1ml of 30% hydrogen peroxide solution, let stand at room temperature for 5 hours, dilute to the mark with solvent, shake well, filter through a 0.22μm PTFE filter membrane, discard at least 3ml of the initial filtrate, and take the subsequent filtrate; accurately measure 5ml of the subsequent filtrate, place it in a 25ml volumetric flask, dilute to the mark with solvent, and shake well.

[0078] Solid high-temperature destruction: Take 10 trazodone hydrochloride sustained-release tablets, grind them into a fine powder, take an appropriate amount of the powder in a weighing bottle, spread it into a thin layer ≤5mm thick, place it in an oven at 85℃ for 5 hours, take it out, cool it to room temperature, take about 720mg of the powder, accurately weigh it, place it in a 100ml volumetric flask, add an appropriate amount of solvent (about 70% of the volumetric flask volume), sonicate for 5 minutes, shake for 30 seconds, sonicate for another 15 minutes, cool to room temperature, dilute to the mark with solvent, shake well, filter through a 0.22μm PTFE filter membrane, discard 3ml of the initial filtrate, take the subsequent filtrate; accurately measure 5ml of the subsequent filtrate, place it in a 25ml volumetric flask, dilute to the mark with solvent, and shake well.

[0079] High-temperature destruction of solution: Take 5 trazodone hydrochloride sustained-release tablets, place them in a 100ml volumetric flask, add an appropriate amount of solvent (about 70% of the volumetric flask volume), sonicate for 5 minutes, shake for 30 seconds, sonicate again for 15 minutes, place in a water bath at 85℃ for 5 hours, cool to room temperature, dilute to the mark with solvent, shake well, filter through a 0.22μm PTFE filter membrane, discard at least 3ml of the initial filtrate, and take the subsequent filtrate; accurately measure 5ml of the subsequent filtrate, place it in a 25ml volumetric flask, dilute to the mark with solvent, and shake well.

[0080] Solid-state photodegradation: Take 10 trazodone hydrochloride sustained-release tablets, grind them into a fine powder, take an appropriate amount of the powder in a weighing bottle, spread it into a thin layer ≤5mm thick, place it under an illuminance of 4500±500lx for 7 days, take about 720mg of the powder, accurately weigh it, place it in a 100ml volumetric flask, add an appropriate amount of solvent (about 70% of the volumetric flask volume), sonicate for 5 minutes, shake for 30 seconds, sonicate again for 15 minutes, cool to room temperature, dilute to the mark with solvent, shake well, filter through a 0.22μm PTFE filter membrane, discard at least 3ml of the initial filtrate, take the subsequent filtrate; accurately measure 5ml of the subsequent filtrate, place it in a 25ml volumetric flask, dilute to the mark with solvent, and shake well.

[0081] Photodegradation of solution: Take 5 trazodone hydrochloride sustained-release tablets, place them in a 100ml volumetric flask, add an appropriate amount of solvent (about 70% of the volumetric flask volume), sonicate for 5 minutes, shake for 30 seconds, sonicate again for 15 minutes, cool to room temperature, dilute to the mark with solvent, shake well, place under 4500±500lx illuminance for 1 hour, remove, filter through a 0.22μm PTFE filter membrane, discard at least 3ml of the initial filtrate, and take the subsequent filtrate; accurately measure 5ml of the subsequent filtrate, place it in a 25ml volumetric flask, dilute to the mark with solvent, and shake well.

[0082] High humidity destruction: Take 10 trazodone hydrochloride sustained-release tablets, grind them into a fine powder, take an appropriate amount of the powder in a weighing bottle, spread it into a thin layer ≤5mm thick, place it under 75%RH±5%RH conditions for 5 days, take about 720mg of the powder, accurately weigh it, put it in a 100ml volumetric flask, add an appropriate amount of solvent (about 70% of the volumetric flask volume), sonicate for 5 minutes, shake for 30 seconds, sonicate again for 15 minutes, cool to room temperature, dilute with solvent to the mark, shake well, filter through a 0.22μm PTFE filter membrane, discard at least 3ml of the initial filtrate, take the subsequent filtrate; accurately measure 5ml of the subsequent filtrate, put it in a 25ml volumetric flask, dilute with solvent to the mark, and shake well.

[0083] (3) Measurement methods and test results

[0084] Set the chromatographic conditions, and after the system stabilizes, inject the mixed impurity localization solution and the destructive solutions under each condition into the chromatograph, and record the chromatograms. The chromatogram of the mixed impurity localization solution is shown below. Figure 2 Table 2 shows the results of the mixed impurity localization solution determination, and Table 3 shows the results of the forced destruction experiment.

[0085] The controlled process impurities in the active pharmaceutical ingredient do not interfere with the detection of impurities A and L, and the separation of each impurity is good. Impurity A is degraded under oxidative degradation conditions, and a larger impurity is degraded under solution light degradation conditions, with a detection amount of 0.86%. This degraded impurity can be well separated from trazodone hydrochloride, as well as impurities A and L, indicating that the analytical method has good specificity.

[0086] Table 2 Results of the determination of mixed impurities in the localization solution

[0087] name Retention time / min Resolution Impurity J 1.683 N / A Impurity B 10.238 61.36 impurity L 11.436 4.40 Trazodone hydrochloride / Impurity C 16.636 15.88 Impurity D 18.161 4.91 Impurity A 18.509 2.1 Impurity E 26.692 30.67 Impurity G 31.795 22.14 impurity H 32.225 2.21

[0088] Table 3 Results of the forced destruction experiment

[0089]

[0090]

[0091] Example 3: Limit of Detection and Limit of Quantification Test

[0092] (1) Chromatographic conditions were the same as in Example 1.

[0093] (2) Solution preparation

[0094] The solvent, mobile phase A, and mobile phase B were prepared in the same manner as in Example 1.

[0095] Linear stock solution: Accurately weigh approximately 22.5 mg of impurity A reference standard, approximately 17.6 mg of impurity L hydrochloride reference standard (approximately equivalent to 15 mg of impurity L), and approximately 15 mg of trazodone hydrochloride reference standard. Place them in the same 100 ml volumetric flask, add an appropriate amount of solvent (approximately 70% of the volumetric flask volume), sonicate to dissolve, cool to room temperature, and dilute to the mark with solvent. Accurately transfer 5 ml of the above solution to a 50 ml volumetric flask and dilute to the mark with solvent.

[0096] Limit of Quantitation Solution (equivalent to 20% of the limit concentration): Accurately transfer 1 ml of the linear stock solution into a 50 ml volumetric flask and dilute to the mark with solvent.

[0097] Detection limit solution (equivalent to 5% limit concentration): Accurately transfer 2.5 ml of the quantitation limit solution into a 10 ml volumetric flask and dilute to the mark with solvent.

[0098] (3) Measurement methods and test results

[0099] After setting the chromatographic conditions and waiting for the system to stabilize, inject the limit of detection (LOD) solution and the limit of quantitation (LOQ) solution into the chromatograph and record the chromatograms. Table 4 shows the experimental results for the LOD and LQ.

[0100] Table 4. Experimental results for limit of quantitation and limit of detection.

[0101] Limit of Quantification Solution S / N Detection limit solution S / N Impurity A 52 14 impurity L 58 16 Trazodone Hydrochloride 31 8

[0102] The signal-to-noise ratios (S / N) of the quantitation limit solutions for impurities A and L are both greater than 10, and the signal-to-noise ratios (S / N) of the detection limit solutions are both greater than 3, far exceeding the requirements of the quantitation limit and detection limit, indicating that the method of the present invention has high sensitivity.

[0103] Example 4: Linear Correlation Test

[0104] (1) Chromatographic conditions were the same as in Example 1.

[0105] (2) Solution preparation

[0106] The solvent, mobile phase A, and mobile phase B were prepared in the same manner as in Example 1.

[0107] Linear stock solution: Accurately weigh approximately 22.5 mg of impurity A reference standard, approximately 17.6 mg of impurity L hydrochloride reference standard (approximately equivalent to 15 mg of impurity L), and approximately 15 mg of trazodone hydrochloride. Place them in the same 100 ml volumetric flask, add an appropriate amount of solvent, sonicate to dissolve, cool to room temperature, and dilute to the mark with solvent. Accurately transfer 5 ml of the above solution to a 50 ml volumetric flask and dilute to the mark with solvent.

[0108] Linear solution with limit of quantitation: Same as the solution with limit of quantitation in Example 3.

[0109] 50% linear solution: Accurately measure 2.5 ml of the linear stock solution and place it in a 50 ml volumetric flask. Dilute to the mark with solvent.

[0110] 80% linear solution: Accurately measure 4 ml of the linear stock solution and place it in a 50 ml volumetric flask. Dilute to the mark with solvent.

[0111] 100% Linear Solution: Accurately measure 5 ml of the linear stock solution and place it in a 50 ml volumetric flask. Dilute to the mark with solvent.

[0112] 150% Linear Solution: Accurately measure 7.5 ml of the linear stock solution and place it in a 50 ml volumetric flask. Dilute to the mark with solvent.

[0113] 200% linear solution: Accurately measure 10 ml of the linear stock solution and place it in a 50 ml volumetric flask. Dilute to the mark with solvent.

[0114] (3) Measurement methods and test results

[0115] Set the chromatographic conditions, and after the system stabilizes, inject each linear solution into the chromatograph and record the chromatograms. Figure 3 , Figure 4 and Figure 5The figures show the linear relationships between impurity A, impurity L, and trazodone hydrochloride. The results indicate good linearity between impurity A, impurity L, and trazodone hydrochloride, with a correction factor of 1.02 for impurity A and 0.65 for impurity L.

[0116] Example 5 Accuracy Test

[0117] Spiking recovery tests were conducted on impurities A and L to examine the accuracy of the analytical method.

[0118] (1) Chromatographic conditions were the same as in Example 1.

[0119] (2) Solution preparation

[0120] The solvent, mobile phase A, and mobile phase B were prepared in the same manner as in Example 1.

[0121] Accuracy impurity stock solution: Weigh approximately 22.5 mg of impurity A reference standard and approximately 17.6 mg of impurity L hydrochloride reference standard (approximately equivalent to 15 mg of impurity L), accurately weigh them, place them in the same 100 ml volumetric flask, add an appropriate amount of solvent, sonicate to dissolve, cool to room temperature, and dilute to the mark with solvent.

[0122] Quantitative limit accuracy solution: Take 5 trazodone hydrochloride sustained-release tablets, place them in a 100ml volumetric flask, add an appropriate amount of solvent (approximately 70% of the volumetric flask volume), sonicate for 5 minutes, shake for 30 seconds, sonicate again for 15 minutes, cool to room temperature, accurately add 1ml of accuracy impurity stock solution, dilute to the mark with solvent, shake well, filter through a 0.22μm PTFE filter membrane, discard at least 3ml of the initial filtrate, and collect the subsequent filtrate; accurately measure 5ml of the subsequent filtrate, place it in a 25ml volumetric flask, dilute to the mark with solvent, and shake well. Prepare 3 parallel portions.

[0123] 50% Accuracy Solution: Take 5 trazodone hydrochloride sustained-release tablets, place them in a 100ml volumetric flask, add an appropriate amount of solvent (approximately 70% of the volumetric flask volume), sonicate for 5 minutes, shake for 30 seconds, sonicate again for 15 minutes, cool to room temperature, accurately add 2.5ml of accuracy impurity stock solution, dilute to the mark with solvent, shake well, filter through a 0.22μm PTFE membrane, discard at least 3ml of the initial filtrate, and collect the subsequent filtrate; accurately measure 5ml of the subsequent filtrate, place it in a 25ml volumetric flask, dilute to the mark with solvent, and shake well. Prepare 3 parallel solutions.

[0124] 100% Accuracy Solution: Take 5 trazodone hydrochloride sustained-release tablets, place them in a 100ml volumetric flask, add an appropriate amount of solvent (approximately 70% of the volumetric flask volume), sonicate for 5 minutes, shake for 30 seconds, sonicate again for 15 minutes, cool to room temperature, accurately add 5ml of accuracy impurity stock solution, dilute to the mark with solvent, shake well, filter through a 0.22μm PTFE membrane, discard at least 3ml of the initial filtrate, and collect the subsequent filtrate; accurately measure 5ml of the subsequent filtrate, place it in a 25ml volumetric flask, dilute to the mark with solvent, and shake well. Prepare 3 parallel solutions.

[0125] 150% Accuracy Solution: Take 5 trazodone hydrochloride sustained-release tablets, place them in a 100ml volumetric flask, add an appropriate amount of solvent (approximately 70% of the volumetric flask volume), sonicate for 5 minutes, shake for 30 seconds, sonicate again for 15 minutes, cool to room temperature, accurately add 7.5ml of accuracy impurity stock solution, dilute to the mark with solvent, shake well, filter through a 0.22μm PTFE filter membrane, discard at least 3ml of the initial filtrate, and collect the subsequent filtrate; accurately measure 5ml of the subsequent filtrate, place it in a 25ml volumetric flask, dilute to the mark with solvent, and shake well. Prepare 3 parallel solutions.

[0126] (3) Measurement methods and test results

[0127] After setting the chromatographic conditions and stabilizing the system, the solutions of each accuracy level were injected into the chromatograph, and the chromatograms were recorded. Tables 5 and 6 show the accuracy test results for impurity A and impurity L, respectively. The accuracy of both impurities was between 95% and 105%, indicating that the method of the present invention has high accuracy.

[0128] Table 5. Accuracy test results for impurity A

[0129]

[0130] Table 6. Results of Accuracy Test for Impurity L

[0131]

[0132] Example 6

[0133] Minor variations in chromatographic conditions were made to examine the robustness of the analytical method.

[0134] (1) Solution preparation

[0135] Solvent: Take 400ml acetonitrile and 600ml water, mix well, and degas by sonication.

[0136] Mobile phase A: Dissolve an appropriate amount of potassium hexafluorophosphate in 1000 ml of water, adjust the pH with phosphoric acid, and filter.

[0137] Mobile phase B: Take 1000 ml of acetonitrile, shake well and sonicate.

[0138] Durability impurity stock solution: Weigh approximately 22.5 mg of impurity A reference standard and approximately 17.6 mg of impurity L hydrochloride reference standard (approximately equivalent to 15 mg of impurity L), accurately weigh them, place them in the same 100 ml volumetric flask, add an appropriate amount of solvent, sonicate to dissolve, cool to room temperature, and dilute to the mark with solvent.

[0139] Durability solution: Take 5 trazodone hydrochloride sustained-release tablets, place them in a 100ml volumetric flask, add an appropriate amount of solvent (about 70% of the volumetric flask volume), sonicate for 5 minutes, shake for 30 seconds, sonicate again for 15 minutes, cool to room temperature, accurately add 5ml of durability impurity stock solution, dilute to the mark with solvent, shake well, filter through a 0.22μm PTFE filter membrane, discard at least 3ml of the initial filtrate, and take the subsequent filtrate; accurately measure 5ml of the subsequent filtrate, place it in a 25ml volumetric flask, dilute to the mark with solvent, and shake well.

[0140] (3) Measurement methods and test results

[0141] Set the chromatographic conditions (the standard conditions are the same as those in Example 1), and after the system stabilizes, inject the robust solution into the chromatograph and record the chromatogram. Table 7 shows the robustness test results. The results indicate that minor changes in chromatographic conditions have no effect on the detection results of impurities A and L, and the method has good robustness.

[0142] Table 7 Durability Test Results

[0143]

[0144] Comparative Example 1

[0145] (1) Chromatographic conditions

[0146] Column: Suplex pKb-100, 4.6 mm × 150 mm, 5 μm

[0147] Mobile phase A: Acetic acid solution, pH 7.0

[0148] Mobile phase B: Acetonitrile

[0149] Column temperature: 35℃

[0150] Flow rate: 1.0 ml / min

[0151] Detection wavelength: 254nm

[0152] Injection volume: 10 μl

[0153] Mobile phase: Mobile phase A: Mobile phase B = 60:40

[0154] (2) Solution preparation

[0155] The preparation of solvent, mobile phase B, impurity A reference standard stock solution, impurity L reference standard stock solution, and system suitability solution were all consistent with those in Example 1.

[0156] Mobile phase A: Take 6.3 ml of glacial acetic acid, 14.0 ml of triethylamine, add 850 ml of water, adjust the pH to 7.0 with triethylamine, and dilute with water to 1000 ml.

[0157] (3) Measurement methods and test results

[0158] Set the chromatographic conditions and, after the system stabilizes, inject the system suitability solution into the chromatograph and record the chromatogram. The system suitability chromatogram is shown below. Figure 6 The elution order corresponds to impurity A, impurity L, and trazodone hydrochloride, respectively. Figure 7 for Figure 6 Partial images of retention times from 0 to 4 minutes. Table 8 shows the results of the system suitability solution determination.

[0159] Table 8 Results of System Suitability Solution Tests

[0160] name Retention time (min) Resolution Tail Factor Impurity A 2.103 — 1.17 impurity L 2.397 3.3 2.38 Trazodone Hydrochloride 10.436 40.2 0.96

[0161] This method is the detection method for imported drug registration standards of trazodone hydrochloride sustained-release tablets. Under these chromatographic conditions, the retention of impurities A and L is weak, posing a risk of solvent peak interference. Furthermore, the peak shape of impurity L is severely tailed, with a tailing factor of 2.38.

[0162] Comparative Example 2

[0163] (1) Chromatographic conditions

[0164] Chromatographic column: Waters Xbridge C18, 4.6 mm × 75 mm, 3.5 μm

[0165] Mobile phase A: Triethylamine-water (0.1:1000, v / v)

[0166] Mobile phase B: Triethylamine-acetonitrile (0.1:1000, v / v)

[0167] Column temperature: 35℃

[0168] Flow rate: 1.0 ml / min

[0169] Detection wavelength: 254nm

[0170] Injection volume: 10 μl

[0171] Gradient procedure:

[0172]

[0173] (2) Solution preparation

[0174] The preparation of the solvent, impurity A reference standard stock solution, impurity L reference standard stock solution, and system suitability solution were all consistent with those in Example 1.

[0175] Mobile phase A: Take 1000ml of water, add 0.1ml of triethylamine, mix well, and degas by sonication.

[0176] Mobile phase B: Take 1000 ml of acetonitrile, add 0.1 ml of triethylamine, mix well, and degas by sonication.

[0177] (3) Measurement methods and test results

[0178] Set the chromatographic conditions and, after the system stabilizes, inject the system suitability solution into the chromatograph and record the chromatogram. The system suitability chromatogram is shown below. Figure 8 The elution order corresponds to impurity A, impurity L, and trazodone hydrochloride, respectively. Table 9 shows the results of the system suitability solution determination.

[0179] Table 9 Results of System Suitability Solution Tests

[0180] name Retention time (min) Resolution Tail Factor Impurity A 4.018 — 1.23 impurity L 6.526 15.60 1.42 Trazodone Hydrochloride 9.817 18.52 1.24

[0181] This method is for the detection of trazodone hydrochloride raw material USP. Under these conditions, the separation effect of impurities A and L from the main component trazodone hydrochloride is comparable to that of the method of this invention, but impurity L shows slight tailing. Under these chromatographic conditions, triethylamine is used in the mobile phase, and the pH is not adjusted. The high pH of the mobile phase affects the column life for a prolonged period, resulting in a shorter column life in this method compared to the analytical method of this invention.

[0182] Comparative Example 3

[0183] Ammonium acetate is also commonly used in the analysis of alkaline samples to suppress tailing. An attempt was made to replace triethylamine with ammonium acetate to improve the peak shape of trazodone hydrochloride.

[0184] (1) Chromatographic conditions

[0185] Chromatographic column: Waters SunFire C18, 4.6 mm × 150 mm, 3.5 μm

[0186] Mobile phase A: 100 mmol / L ammonium acetate buffer (pH adjusted to 5.0 with acetic acid)

[0187] Mobile phase B: Acetonitrile

[0188] Column temperature: 35℃

[0189] Flow rate: 1.0 ml / min

[0190] Detection wavelength: 254nm

[0191] Injection volume: 10 μl

[0192] Gradient procedure:

[0193]

[0194] (2) Solution preparation

[0195] The preparation of the solvent, impurity A reference standard stock solution, impurity L reference standard stock solution, and system suitability solution were all consistent with those in Example 1.

[0196] Mobile phase A: Weigh approximately 7.7g of ammonium acetate accurately, add 1000ml of water, sonicate to dissolve, adjust the pH to 5.0 with acetic acid, and filter.

[0197] Mobile phase B: Take 1000 ml of acetonitrile and degas it by sonication.

[0198] (3) Measurement methods and test results

[0199] Set the chromatographic conditions and, after the system stabilizes, inject the system suitability solution into the chromatograph and record the chromatogram. The system suitability chromatogram is shown below. Figure 9 The elution order corresponds to impurity L, trazodone hydrochloride, and impurity A, respectively. Table 10 shows the results of the system suitability solution determination.

[0200] Table 10 Results of System Suitability Solution Tests

[0201] name Retention time (min) Resolution Tail factor impurity L 5.457 — 0.97 Trazodone Hydrochloride 8.022 15.60 2.02 Impurity A 9.105 6.63 1.13

[0202] Under these chromatographic conditions, the main peak of trazodone hydrochloride exhibited severe tailing, with a tailing factor of 2.02 (generally, 0.9–1.3 is considered optimal).

[0203] Comparative Example 4

[0204] (1) Chromatographic conditions

[0205] Chromatographic column: Waters SunFire C18, 4.6 mm × 150 mm, 3.5 μm

[0206] Mobile phase A: 20 mmol / L potassium hexafluorophosphate buffer

[0207] Column temperature: 30℃

[0208] Flow rate: 1.0 ml / min

[0209] Detection wavelength: 254nm

[0210] Injection volume: 10 μl

[0211] Gradient procedure:

[0212]

[0213] (2) Solution preparation

[0214] Mobile phase A: Dissolve 3.68 g of potassium hexafluorophosphate in 1000 ml of water, adjust the pH to 2.0 with phosphoric acid, and filter.

[0215] Mobile phase B: Take 1000 ml of acetonitrile, shake well and sonicate.

[0216] The preparation of the solvent, impurity A reference standard stock solution, impurity L reference standard stock solution, and system suitability solution were all consistent with those in Example 1.

[0217] (3) Measurement methods and test results

[0218] Set the chromatographic conditions and, after the system stabilizes, inject the system suitability solution into the chromatograph and record the chromatogram. The system suitability chromatogram is shown below. Figure 10 The elution order corresponds to impurity L, trazodone hydrochloride, and impurity A, respectively. Table 11 shows the results of the system suitability solution determination.

[0219] Table 11 Results of System Suitability Solution Tests

[0220] name Retention time (min) Resolution Tail Factor impurity L 7.560 — 1.06 Trazodone Hydrochloride 10.108 14.0 1.76 Impurity A 11.120 5.4 1.10

[0221] Under these chromatographic conditions, the concentration of potassium hexafluorophosphate buffer was 20 mmol / L. Compared with the method of the present invention, the main peak trazodone showed a tailing effect with a tailing factor of 1.76.

Claims

1. A method for detecting related substances in trazodone hydrochloride sustained-release tablets, characterized in that, High-performance liquid chromatography (HPLC) was used. The chromatographic conditions were as follows: octadecyl-bonded silica column; column temperature 25-35℃; flow rate 0.8-1.2 ml / min; detection wavelength 252-256 nm; injection volume 5-15 μl; mobile phase A: potassium hexafluorophosphate buffer; mobile phase B: acetonitrile; gradient elution was performed. The elution program for the gradient elution was set as follows:

2. The detection method according to claim 1, characterized in that, The pH range of the potassium hexafluorophosphate buffer solution is 1.8-2.

2.

3. The detection method according to claim 1 or 2, characterized in that, The concentration of the potassium hexafluorophosphate buffer solution is 90 mmol / L to 110 mmol / L.

4. The detection method according to claim 1, characterized in that, The chromatographic column selected was a Waters SunFire C18 column.

5. The detection method according to claim 4, characterized in that, The specifications of the Waters SunFire C18 column are: particle size × inner diameter × column length: 3.5μm × 4.6mm × 150mm.

6. The detection method according to claim 2, characterized in that, The pH of the potassium hexafluorophosphate buffer solution was adjusted using phosphoric acid.

7. The detection method according to claim 1 or 2, characterized in that, The pH value of the potassium hexafluorophosphate buffer solution is 2.

0.

8. The detection method according to claim 1, characterized in that, The chromatographic conditions were as follows: column temperature 30°C, flow rate 1.0 ml / min, and detection wavelength 254 nm.

9. The detection method according to claim 1, characterized in that, The relevant substances include impurity A and impurity L, and the structural formulas of impurity A and impurity L are respectively shown in Formula II and Formula III:

Images