Method for detecting 3-chloroaniline in trazodone hydrochloride tablet

Abstract

The invention discloses a method for detecting 3-chloroaniline in a trazodone hydrochloride tablet, the detection method is a high performance liquid chromatography, an octadecylsilane bonded silica gel packed column is used, a phosphoric acid aqueous solution with a volume fraction of 0.1 + / -0.02% is used as a mobile phase A, acetonitrile is used as a mobile phase B, gradient elution is carried out, and the elution procedure is as follows: 0-7th minutes, 0-7th minutes, 0-7th minutes, 0-7th minutes, 0-7th minutes, 0-7th minutes, 0-7th minutes, 0-7th minutes, 0-7th minutes, 0-7th minutes and 0-7th the mobile phase A and the mobile phase B with the constant volume fraction are used for isocratic elution, and the volume fraction of the mobile phase B is 2-10%; the volume fraction of the mobile phase B is increased for gradient elution for 12 minutes, and the volume fraction of the mobile phase B is 68%; performing isocratic elution for 17 minutes by keeping the volume fraction of the mobile phase B to be 68%; reducing the volume fraction of the mobile phase B to 5% for gradient elution for 18 minutes; and finally, keeping the volume fraction of the mobile phase B at 5%, performing isocratic elution for 25 minutes, and ending. In the elution process, the total volume fraction of the mobile phase A and the mobile phase B is 100%. Qualitative and quantitative analysis of 3-chloroaniline can be completed only by using high performance liquid chromatography.

Description

Technical Field

[0001] This invention relates to a column chromatography analysis method, and more particularly to a method for detecting 3-chloroaniline in trazodone hydrochloride tablets. Background Technology

[0002] Trazodone hydrochloride (below) Figure 1 Triazolylpyridine derivatives, with the structure 2-[3-[4-(3-chlorophenyl)-1-piperazinyl]propyl]-1,2,4-triazol[4,3-a]pyridine-3(2H)-hydrochloric acid, have five main synthetic routes (M. Maziti et al. Purified forms of trazodone and trazodone hydrochloride [P], CN101772490A.2010.), one of which uses m-chloroaniline / 3-chloroaniline as raw material (Xue Xuming, He Xin, Liu Changchun, et al. Synthesis of trazodone hydrochloride [J]. China Pharmaceutical Industry Journal, 2008, (11):808-810.), but 3-chloroaniline has genotoxicity.

[0003]

[0004] In the prior art, invention patent CN 117470987 A discloses a method for detecting related substances of trazodone hydrochloride, but the substances being detected do not include 3-chloroaniline. Invention patent application CN 115201377 A discloses a method for detecting the migration of 17 genotoxic aromatic amines in a corn starch lunchbox food simulation liquid, and the substances being detected include 3-chloroaniline. However, this method requires coupling with mass spectrometry and has a high detection cost. Invention patent application CN 114594192A discloses a method for detecting chloroaniline and chloronitrobenzylaniline isomers in posaconazole starting materials. Similarly, this method uses liquid chromatography-mass spectrometry (LC-MS). The prior art lacks a method for directly detecting intermediate chloroaniline in trazodone hydrochloride tablets using liquid chromatography. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide an efficient and low-cost method for detecting 3-chloroaniline in trazodone hydrochloride tablets.

[0006] Technical Solution: The present invention provides a method for detecting 3-chloroaniline in trazodone hydrochloride tablets. The method is high-performance liquid chromatography (HPLC) using an octadecylsilane-bonded silica gel column, a diode array detector, and a 0.1±0.02% (v / v) phosphoric acid aqueous solution as mobile phase A and acetonitrile as mobile phase B, with gradient elution. The elution program is as follows:

[0007] From minute 0 to minute 7, isocratic elution was performed using mobile phases A and B with constant volume fractions, where mobile phase B had a volume fraction of 2–10%. Then, a gradient elution with increasing volume fraction of mobile phase B was performed until minute 12, at which point the volume fraction of mobile phase B was 68%. This was followed by isocratic elution with mobile phase B at 68% volume fraction until minute 17. Next, a gradient elution with decreasing volume fraction of mobile phase B was performed until minute 18, at which point the volume fraction of mobile phase B was 5%. Finally, isocratic elution with mobile phase B at 5% volume fraction was performed until minute 25, at which point the elution was complete. During the elution process, the total volume fraction of mobile phases A and B was 100%.

[0008] In this invention, by rationally controlling the concentration gradient and duration of gradient elution, trazodone hydrochloride, tablet excipients, and the impurity 3-chloroaniline are efficiently separated. Qualitative and quantitative analysis can be completed using only high-performance liquid chromatography, reducing the cost of detection instruments.

[0009] Preferably, in the elution process, the volume fraction of mobile phase B is 3-7% from minute 0 to minute 7. Within this range, the target peak elution is not affected by blank or extraneous peaks.

[0010] Preferably, the detection wavelength of the diode array detector is 203–207 nm.

[0011] Preferably, in the elution process, the packed column temperature is 20-30℃, the parameters are 4.6mm×150mm, and the packing particle size is 3.5μm.

[0012] Preferably, in the elution process, the flow rate of the mobile phase is 1.1 to 1.3 mL / min, and the injection volume is 10 to 20 μL.

[0013] Preferably, the detection method further includes a sample preparation step: preparing a trazodone hydrochloride tablet test solution and a 3-chloroaniline reference solution respectively, and obtaining the spectra of the test solution and the reference solution respectively using high performance liquid chromatography. The absorption peak with the same retention time in the spectra of the test solution and the reference solution is 3-chloroaniline.

[0014] Preferably, the trazodone hydrochloride tablets contain pharmaceutically acceptable excipients, wherein the excipients are at least one selected from sucrose, carnauba wax, povidone, and magnesium stearate.

[0015] Preferably, the concentration of the test solution is 2-3 mg / mL, and the concentration of the reference solution is 0.2-0.3 μg / mL.

[0016] Preferably, methanol is used as the solvent for preparing the test solution and the reference solution.

[0017] Preferably, the content of 3-chloroaniline in the test solution is calculated based on the ratio of the peak area of ​​3-chloroaniline in the spectrum of the test solution to the peak area of ​​3-chloroaniline in the spectrum of the reference solution.

[0018] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. Good separation of 3-chloroaniline peak and good sensitivity: effectively separates trazodone hydrochloride, solvent and excipients that affect the identification of 3-chloroaniline peak; 2. Effective extraction of analytes from trazodone hydrochloride: using methanol as the extraction solvent, the impurities in trazodone hydrochloride are dissolved and extracted well; 3. High accuracy, meeting the detection requirements. Attached Figure Description

[0019] Figure 1 This is a chromatogram of the first elution gradient test solution of the present invention;

[0020] Figure 2 This is the chromatogram of the first elution gradient reference solution of the present invention;

[0021] Figure 3 This is the chromatogram of the second elution gradient reference solution of the present invention;

[0022] Figure 4 This is the chromatogram of the third elution gradient reference solution of the present invention;

[0023] Figure 5 The chromatograms of the fourth elution gradient reference, test sample, and sensitivity solution at a detection wavelength of 203 nm are shown.

[0024] Figure 6 The chromatograms of the fourth elution gradient reference, test sample, and sensitivity solution at a detection wavelength of 205 nm are shown.

[0025] Figure 7 The chromatograms of the fourth elution gradient reference, test sample, and sensitivity solution at a detection wavelength of 207 nm are shown.

[0026] Figure 8 The chromatograms are of the fourth elution gradient reference, test sample, and sensitivity solution at a flow rate of 1.1 mL / min.

[0027] Figure 9 The chromatograms are of the fourth elution gradient reference, test sample, and sensitivity solution at a flow rate of 1.3 mL / min.

[0028] Figure 10 The chromatograms are of the fourth elution gradient reference standard, test sample, and sensitivity solution at a column temperature of 20℃.

[0029] Figure 11 The chromatograms are of the fourth elution gradient reference standard, test sample, and sensitivity solution at a column temperature of 30℃.

[0030] Figure 12 The chromatograms are of the fourth elution gradient reference, test sample, and sensitivity solution at an initial concentration of 3% in phase B.

[0031] Figure 13 The chromatograms are of the fourth elution gradient reference, test sample, and sensitivity solution at an initial concentration of 7% in phase B. Detailed Implementation

[0032] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0033] Example 1:

[0034] 1. Sample preparation

[0035] 3-Chloroaniline stock solution: Weigh approximately 20 mg of 3-chloroaniline accurately, place it in a 100 mL volumetric flask, dissolve and dilute to the mark with methanol, and shake well to obtain the 3-chloroaniline stock solution (200 μg / mL).

[0036] 3-Chloroaniline stock solution: Measure 1.0 mL of 3-chloroaniline stock solution, place it in a 100 mL volumetric flask, dilute to the mark with methanol, and shake well to obtain the 3-chloroaniline stock solution (2 μg / mL).

[0037] Reference solution: Measure 5.0 mL of 3-chloroaniline stock solution, place it in a 50 mL volumetric flask, dilute to the mark with methanol, and shake well to obtain the reference solution with a concentration of 0.2 μg / mL.

[0038] Test solution: Take 10 trazodone hydrochloride sustained-release tablets (approximately equivalent to 40 mg of trazodone hydrochloride), grind them into powder, place them in a 20 mL volumetric flask, add methanol, sonicate to dissolve, dilute with methanol to the mark, shake well, filter, and take the filtrate to obtain (2 mg / mL).

[0039] Spiked test solution: Take 10 trazodone hydrochloride sustained-release tablets (approximately equivalent to 40 mg of trazodone hydrochloride), grind them into powder, place them in a 20 mL volumetric flask, add an appropriate amount of reference solution, sonicate to dissolve, dilute to the mark with reference solution, shake well, filter, and take the filtrate to obtain (2 mg / mL + 0.2 μg / mL).

[0040] Sensitivity solution (limit of quantitation solution): Measure 2.5 mL of the reference solution, place it in a 10 mL volumetric flask, dilute to the mark with methanol, and shake well to obtain the 3-chloroaniline concentration of 0.05 μg / mL.

[0041] 2. Filtering by criteria

[0042] 2.1 Gradient Filtering

[0043] Instrument conditions: Agilent 1260 high performance liquid chromatograph; diode array detector; C18 column, 4.6*150mm, 3.5μm; flow rate 1.2mL / min; column temperature 25℃; injection volume 10μL; detection wavelength 205nm; mobile phase A: 0.1% phosphoric acid aqueous solution; mobile phase B: acetonitrile.

[0044] Take the reference solution and the test solution respectively and perform gradient elution according to the gradient program in Table 1. The test results of the test solution are as follows: Figure 1 As shown.

[0045] Table 1:

[0046] T(min) Mobile phase A (%) Mobile phase B (%) 0 90 10 12 32 68 12.1 90 10 20 90 10

[0047] Depend on Figure 1 It can be seen that under these conditions, the 3-chloroaniline peak is interfered with by the excipient peak or other substances in the sample, and cannot be accurately integrated.

[0048] Take the reference solution and the test solution respectively and perform gradient elution according to the gradient program in Table 1 above, changing the injection volume to 50 μL. The test results for the reference solution are as follows: Figure 2 As shown. Under these conditions, the 3-chloroaniline peak in the reference solution splits, making accurate quantification impossible.

[0049] Take the reference solution and the test solution respectively, and perform gradient elution according to the gradient program in Table 2. The test results for the reference solution are as follows: Figure 3 As shown.

[0050] Table 2:

[0051] T(min) Mobile phase A (%) Mobile phase B (%) 0 80 20 5 80 20 12 32 68 17 32 68 18 80 20 25 80 20

[0052] Depend on Figure 3 It can be seen that under these conditions, the 3-chloroaniline peak is interfered with by the blank solvent peak and cannot be accurately integrated.

[0053] Take the reference solution and the test solution respectively and perform gradient elution according to the gradient program in Table 3. The test results for the reference solution are as follows: Figure 4 As shown.

[0054] Table 3:

[0055]

[0056]

[0057] like Figure 4 As shown, under these conditions, the 3-chloroaniline peak is interfered with by the excipient peak / peaks of other substances in the sample, and cannot be accurately integrated.

[0058] Take the reference solution and the test solution respectively and perform gradient elution according to the gradient program in Table 4.

[0059] Table 4:

[0060] T(min) Mobile phase A (%) Mobile phase B (%) 0 95 5 7 95 5 12 32 68 17 32 68 18 95 5 25 95 5

[0061] Under these conditions, the retention time of the 3-chloroaniline peak is approximately 7.5 min, unaffected by excipients or other impurities, and the specificity meets the requirements; the limit of quantitation of 3-chloroaniline is approximately 0.05 μg / mL, which is about 1 / 4 of the limit concentration, and the sensitivity meets the requirements; the recovery rate is ≥98%, and the accuracy meets the requirements.

[0062] 3. Durability test

[0063] (1) Selection of detection wavelength

[0064] Take appropriate amounts of the reference solution, test solution, and sensitivity solution, and record the absorption spectra at detection wavelengths of 203 nm, 205 nm, and 207 nm. The results are as follows: Figure 5 , Figure 6 , Figure 7 As shown, both the 3-chloroaniline sample and the test solution exhibit absorption peaks in the wavelength range of 203–207 nm.

[0065] (2) Flow velocity investigation

[0066] The chromatographic separation effects of the reference solution, test solution, and sensitivity solution were investigated at flow rates of 1.1 mL / min and 1.3 mL / min, respectively. The results are as follows: Figure 8 , Figure 9 As shown in the figure, the samples exhibited good separation performance under different flow rate conditions, meeting the detection requirements.

[0067] (3) Column temperature investigation

[0068] The chromatographic separation effects of the reference solution, test solution, and sensitivity solution were investigated at column temperatures of 20℃ and 30℃, respectively. The results are as follows: Figure 10 , Figure 11 As shown in the figure. The results indicate that the separation effect is better within the range of 20–30℃, and the temperature fluctuation can meet the separation requirements.

[0069] (4) Investigation of the initial proportion of phase B in gradient elution

[0070] The chromatographic separation effects of the reference solution, test solution, and sensitivity solution were investigated under initial phase B ratios of 3% and 7%, respectively. The results are as follows: Figure 12 , Figure 13 As shown in the figure. The results indicate that the separation effect is better when the initial proportion of phase B is within 3% to 7%, and the target peak is not affected by blank or impurity peaks, which can meet the requirements for qualitative and quantitative detection.

[0071] (5) Accuracy assessment

[0072] The sensitivity and recovery rate of 3-chloroaniline after fine-tuning of wavelength, column temperature, and flow rate are shown in the table below:

[0073] Table 5:

[0074] condition Sensitivity to solution response (S / N) Recovery rate (%) Standard conditions 23.5 100.1 wavelength 203nm 17.1 100.9 wavelength 207nm 25.4 99.5 Flow rate 1.1 mL / min 18.6 99.4 Flow rate 1.3 mL / min 22.1 100.7 Column temperature 20℃ 20.0 101.2 Column temperature 30℃ 18.9 100.7 Gradient initial ratio B phase 3% 28.9 99.0 Gradient initial ratio B phase 7% 30.7 100.8

[0075] As shown in the table, the recovery rates are all within acceptable ranges, and the accuracy meets the requirements.

Claims

1. A method for detecting 3-chloroaniline in trazodone hydrochloride tablets, characterized in that, The detection method is high-performance liquid chromatography (HPLC), using an octadecylsilane-bonded silica gel packed column, a diode array detector, and a mobile phase of 0.1 ± 0.02% (v / v) phosphoric acid aqueous solution as mobile phase A and acetonitrile as mobile phase B. The elution program is as follows: From minute 0 to minute 7, elute with mobile phase A and mobile phase B at constant volume fractions, where the volume fraction of mobile phase B is 2-10%. Increase the volume fraction of mobile phase B in a gradient elution until minute 12, when the volume fraction of mobile phase B is 68%. Maintain isocratic elution with mobile phase B at 68% for minute 17. Decrease the volume fraction of mobile phase B in a gradient elution until minute 18, when the volume fraction of mobile phase B is 5%. Maintain isocratic elution with mobile phase B at 5% for minute 25, then end the elution. During the elution process, the total volume fraction of mobile phase A and mobile phase B is 100%.

2. The detection method according to claim 1, characterized in that, In the elution process, from minute 0 to minute 7, mobile phase B has a volume fraction of 3-7%.

3. The detection method according to claim 1, characterized in that, The detection wavelength of the diode array detector is 203–207 nm.

4. The detection method according to claim 1, characterized in that, The packed column temperature is 20–30°C, the packed column parameters are 4.6 mm × 150 mm, and the packed particle size is 3.5 μm.

5. The detection method according to claim 1, characterized in that, In the elution procedure, the flow rate of the mobile phase is 1.1–1.3 mL / min, and the injection volume is 10–20 μL.

6. The detection method according to any one of claims 1 to 5, characterized in that, Prepare separate test solutions of trazodone hydrochloride tablets and reference solutions of 3-chloroaniline. Use high performance liquid chromatography to obtain the test solution and reference solution respectively. The absorption peak with the same retention time in the obtained test solution and reference solution chromatograms is 3-chloroaniline.

7. The detection method according to claim 6, characterized in that, The trazodone hydrochloride tablets contain pharmaceutically acceptable excipients, which are at least one of sucrose, carnauba wax, povidone, and magnesium stearate.

8. The detection method according to claim 6, characterized in that, The concentration of the test solution is 2–3 mg / mL, and the concentration of the reference solution is 0.2–0.3 μg / mL.

9. The detection method according to claim 8, characterized in that, Methanol was used as the solvent to prepare the test solution and the reference solution.

10. The detection method according to claim 7, characterized in that, In step (3), the concentration of 3-chloroaniline in the test solution is calculated based on the ratio of the peak area of ​​3-chloroaniline in the spectrum of the test solution to the peak area of ​​3-chloroaniline in the spectrum of the reference solution.

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