Method for detecting O-desmethyltramadol and its related substances by HPLC

Abstract

This invention belongs to the field of drug detection technology, specifically relating to a method for detecting O-nordrod and its related substances using HPLC. The test solution, blank solution, reference solution, and impurity reference solution are injected into the HPLC instrument, and the chromatograms are recorded. The content of O-nordrod and its related substances in the test solution is calculated using the peak area normalization method. The related substances include one or more of impurities A, B, C, D, or E. This invention exhibits good specificity, robustness, and stability, as well as excellent precision and accuracy.

Description

Technical Field

[0001] This invention belongs to the field of drug detection technology, specifically relating to a method for detecting O-nordtramadol and related substances using HPLC. Background Technology

[0002] Postoperative pain is acute pain that occurs immediately after surgery and is the most common and urgent type of acute pain in clinical practice. If it is not adequately controlled in its initial acute stage, it can easily develop into chronic pain. Opioids are commonly used to treat postoperative pain, but they have adverse effects such as respiratory depression and addiction. Local anesthetics are also the most important analgesics in clinical practice, but existing local anesthetics have a relatively short duration of action and require repeated administration. Therefore, there is an urgent clinical need to develop analgesics and anesthetics with fewer side effects that can still exert their therapeutic effects.

[0003] Tramadol is a weak opioid analgesic. Due to its good tolerability, including low short-term addictive potential and low risk of respiratory depression, it is generally considered a low-risk drug for moderate to severe pain. Tramadol is effective for a variety of pain types, including neuropathic pain, postoperative pain, fibromyalgia, and pain associated with osteoarthritis and cancer.

[0004] Tramadol undergoes two main metabolic pathways in the human body: O-demethylation catalyzed by CYP2D6 to O-nortramadol, and N-demethylation catalyzed by CYP3A4 to N-nortramadol. However, different patients exhibit varying pharmacokinetic characteristics after taking tramadol, attributed to polymorphisms in the CYP2D6 gene, which affect the enzymatic activity of CYP2D6. Patients with high CYP2D6 enzyme activity produce high concentrations of the active metabolite O-nortramadol, posing a risk of tramadol toxicity. Patients with low CYP2D6 enzyme activity have low concentrations of the active metabolite, leading to insufficient analgesic effect. Tramadol may cause individual differences in efficacy and even the risk of toxicity.

[0005] O-nortramadol, as the active metabolite of tramadol, can produce analgesic effects without undergoing biological metabolism. It can overcome the risk of overdose poisoning in strong metabolizers and the problem of insufficient analgesia in weak metabolizers during tramadol administration, making it easier to control the accuracy of drug dosage in clinical practice and having extremely high clinical application value.

[0006] O-nortramadol, chemically named (±)-3-[(1RS,2RS)-2-[(N,N-dimethylamino)methylene]-1-hydroxycyclohexyl]phenol, molecular formula: C 15 H 23 NO2, molecular weight: 249.35, its structural formula is: .

[0007] O-nordtramadol sample was prepared by the following process: a Grignard reagent was prepared by m-methoxybromobenzene, which was then reacted with 2-dimethylaminomethylcyclohexanone to obtain tramadol, which was then purified by a reduction process to obtain O-nordtramadol.

[0008]

[0009] The synthesis of O-nortramadol involves various process impurities that play a crucial role in the quality of the product. Therefore, necessary quality control should be implemented to ensure the safety of clinical use. Summary of the Invention

[0010] The purpose of this invention is to provide a method for detecting O-nordrotrodine and related substances using HPLC, which has good specificity, robustness and stability, and excellent precision and accuracy.

[0011] The method for detecting O-nordromadol and its related substances using HPLC according to the present invention involves injecting the test solution, blank solution, reference solution, and impurity reference solution into a liquid chromatograph, recording the chromatograms, and calculating the content of O-nordromadol and its related substances in the test solution using the peak area normalization method; wherein, the related substances include one or more of impurities A, B, C, D, or E. HPLC chromatographic conditions include the mobile phase and gradient elution program; The mobile phase consists of acetate buffer as mobile phase A and methanol as mobile phase B; The gradient elution program is as follows: 0-10 minutes, mobile phase A accounts for 65-75% of the total mobile phase volume, and mobile phase B accounts for 25-35% of the total mobile phase volume; 10-15 minutes, mobile phase A accounts for 45-55% of the total mobile phase volume, and mobile phase B accounts for 45-55% of the total mobile phase volume; 15-35 minutes, mobile phase A accounts for 45-55% of the total mobile phase volume, and mobile phase B accounts for 45-55% of the total mobile phase volume; 35-35.1 minutes, mobile phase A accounts for 65-75% of the total mobile phase volume, and mobile phase B accounts for 25-35% of the total mobile phase volume; 35.1-45 minutes, mobile phase A accounts for 65-75% of the total mobile phase volume, and mobile phase B accounts for 25-35% of the total mobile phase volume.

[0012] The total flow rate of mobile phase A and mobile phase B is 0.95-1.05 mL / min.

[0013] HPLC chromatographic conditions also include the chromatographic column, detection wavelength, and detector; the chromatographic column is a C18 column with octadecylsilane-bonded silica gel as the packing material, and the column temperature is 28-32℃; the detection wavelength is 269-273nm, and the detector is an ultraviolet detector or a PDA detector.

[0014] The blank solution is a diluent.

[0015] The preparation method of the reference solution is to weigh O-nordtramadol reference standard, dissolve it in methanol, and then dilute it with diluent to prepare the reference solution; wherein, the ratio of O-nordtramadol reference standard to methanol is 10:2-5, O-nordtramadol reference standard is expressed in mg, and methanol is expressed in mL; the concentration of O-nordtramadol in the reference solution is 0.3-0.5 mg / mL.

[0016] The impurity reference solution is prepared by mixing impurity A stock solution, impurity B stock solution, impurity C stock solution, impurity D stock solution and impurity E stock solution and then diluting with a diluent to prepare the impurity reference solution; the concentration of impurity A in the impurity reference solution is 0.32-20 μg / mL, and the concentrations of impurities B, C, D and E are all 0.16-10 μg / mL.

[0017] The preparation method of impurity A stock solution is as follows: weigh impurity A reference standard, dissolve it in methanol, and dilute it with diluent to prepare impurity A stock solution; wherein, the ratio of impurity A reference standard to methanol is 10:2-5, impurity A reference standard is expressed in mg, and methanol is expressed in mL; the concentration of impurity A in impurity A stock solution is 3.2-500 μg / mL; The impurity B stock solution is prepared by weighing the impurity B reference standard, dissolving it in methanol, and then diluting it with a diluent to prepare the impurity B stock solution; wherein, the ratio of impurity B reference standard to methanol is 10:2-5, the impurity B reference standard is expressed in mg, and the methanol is expressed in mL; the concentration of impurity B in the impurity B stock solution is 1.6-250 μg / mL. The impurity C stock solution is prepared by weighing the impurity C reference standard, dissolving it in methanol, and then diluting it with a diluent to prepare the impurity C stock solution; wherein, the ratio of impurity C reference standard to methanol is 10:2-5, the impurity C reference standard is expressed in mg, and the methanol is expressed in mL; the concentration of impurity C in the impurity C stock solution is 1.6-250 μg / mL; The preparation method of impurity D stock solution is to weigh impurity D reference standard, dissolve it in methanol, and then dilute it with diluent to prepare impurity D stock solution; wherein, the ratio of impurity D reference standard to methanol is 10:2-5, impurity D reference standard is expressed in mg, and methanol is expressed in mL; the concentration of impurity D in impurity D stock solution is 1.6-250 μg / mL. The method for preparing the impurity E stock solution is to weigh the impurity E reference standard, dissolve it in methanol, and then dilute it with a diluent to prepare the impurity E stock solution; wherein, the ratio of impurity E reference standard to methanol is 10:2-5, the impurity E reference standard is expressed in mg, and the methanol is expressed in mL; the concentration of impurity E in the impurity E stock solution is 1.6-250 μg / mL.

[0018] The test solution is prepared by weighing O-nordtramadol sample, dissolving it in methanol, and then diluting it with a diluent. The ratio of O-nordtramadol sample to methanol is 10:2-5. The O-nordtramadol sample is expressed in mg and the methanol in mL. The concentration of O-nordtramadol in the test solution is 0.3-0.5 mg / mL.

[0019] The diluent is a mixture of acetate buffer and methanol. The acetate buffer is prepared by sodium acetate, glacial acetic acid and water, and the volume ratio of acetate buffer to methanol is 65-75:25-35.

[0020] The structural formula of O-nordrol is as follows:

[0021]

[0022] The structural formula of impurity A is as follows:

[0023]

[0024] The structural formula of impurity B is as follows:

[0025]

[0026] The structural formula of impurity C is as follows:

[0027]

[0028] The structural formula of impurity D is as follows:

[0029]

[0030] The structural formula of impurity E is as follows:

[0031] .

[0032] The beneficial effects of this invention are as follows: (1) This invention has good specificity, durability and stability, and excellent precision and accuracy, providing technical means for the study of key quality properties of O-nordrod.

[0033] (2) The present invention can simultaneously determine the content of O-nortramadol and the content of related substances, and the separation between the main peak and each impurity peak meets the requirements. Attached Figure Description

[0034] Figure 1 This is the 1H NMR spectrum of impurity A.

[0035] Figure 2 This is the carbon NMR spectrum of impurity A.

[0036] Figure 3 This is the 1H NMR spectrum of impurity B.

[0037] Figure 4 This is the carbon NMR spectrum of impurity B.

[0038] Figure 5 This is the 1H NMR spectrum of impurity C.

[0039] Figure 6 This is the carbon NMR spectrum of impurity C.

[0040] Figure 7 This is the 1H NMR spectrum of impurity D.

[0041] Figure 8 This is the carbon NMR spectrum of impurity D.

[0042] Figure 9 This is the 1H NMR spectrum of impurity E.

[0043] Figure 10 This is the carbon NMR spectrum of impurity E.

[0044] Figure 11 The NMR spectrum of O-nordrotrod is shown in the form of hydrogen NMR.

[0045] Figure 12 This is the carbon NMR spectrum of O-nortramadol.

[0046] Figure 13 This is the HPLC chromatogram of the impurity reference solution.

[0047] Figure 14 This is the HPLC chromatogram of the blank solution.

[0048] Figure 15 This is the HPLC chromatogram of the test solution.

[0049] Figure 16 This is the HPLC chromatogram of the spiked solution of the test sample.

[0050] Figure 17 The HPLC chromatogram is for the solution containing impurity A.

[0051] Figure 18The HPLC chromatogram is for the solution containing impurity B.

[0052] Figure 19 The HPLC chromatogram is for the solution containing impurity C.

[0053] Figure 20 The HPLC chromatogram of the solution containing impurity D is shown.

[0054] Figure 21 The HPLC chromatogram is for the solution containing impurity E.

[0055] Figure 22 This is the HPLC chromatogram of the reference solution. Detailed Implementation

[0056] The present invention will be further described below with reference to embodiments.

[0057] Example 1 O-nortramadol and its related substances are shown in Table 1. The NMR spectra of impurities A, B, C, D, and E, and O-nortramadol are shown in [Table 1]. Figure 1-12 .

[0058] Table 1 O-nordrodol and related substances

[0059] (1) Solution preparation: Diluent: A mixture of acetate buffer (18g sodium acetate, 9.8ml glacial acetic acid, diluted with water to 1000ml) and methanol, with a volume ratio of acetate buffer to methanol of 70:30; Blank solution: diluent; Reference solution: Weigh 20 mg of O-nordtramadol reference standard, place it in a 50 mL volumetric flask, add 4 mL of methanol to dissolve it, then dilute to the mark with diluent and shake well; Impurity A stock solution: Weigh 8 mg of impurity A reference standard, dissolve it in 2 mL of methanol, and dilute with diluent to prepare a solution containing 400 μg of impurity A per 1 mL; Impurity B stock solution: Weigh 4 mg of impurity B reference standard, dissolve it in 1 mL of methanol, and dilute with diluent to prepare a solution containing 200 μg of impurity B per mL; Impurity C stock solution: Weigh 4 mg of impurity C reference standard, dissolve it in 1 mL of methanol, and dilute with diluent to prepare a solution containing 200 μg of impurity C per 1 mL; Impurity D stock solution: Weigh 4 mg of impurity D reference standard, dissolve it in 1 mL of methanol, and dilute with diluent to prepare a solution containing 200 μg of impurity D per 1 mL; Impurity E stock solution: Weigh 4 mg of impurity E reference standard, dissolve it in 1 mL of methanol, and dilute with diluent to prepare a solution containing 200 μg of impurity E per 1 mL; Impurity A positioning solution: Accurately measure 1 mL of impurity A stock solution, place it in a 50 mL volumetric flask, dilute to the mark with diluent, and shake well; Impurity B positioning solution: Accurately measure 1 mL of impurity B stock solution, place it in a 50 mL volumetric flask, dilute to the mark with diluent, and shake well; Impurity C positioning solution: Accurately measure 1 mL of impurity C stock solution, place it in a 50 mL volumetric flask, dilute to the mark with diluent, and shake well; Impurity D positioning solution: Accurately measure 1 mL of impurity D stock solution, place it in a 50 mL volumetric flask, dilute to the mark with diluent, and shake well; Impurity E positioning solution: Accurately measure 1 mL of impurity E stock solution, place it in a 50 mL volumetric flask, dilute to the mark with diluent, and shake well; Impurity reference solution: Accurately measure 1 mL of impurity A stock solution, 1 mL of impurity B stock solution, 1 mL of impurity C stock solution, 1 mL of impurity D stock solution and 1 mL of impurity E stock solution, place them in the same 50 mL volumetric flask, dilute to the mark with diluent, and shake well; Test solution: Weigh 20 mg of O-nordtramadol sample, dissolve in 4 mL of methanol, and dilute with diluent to prepare a solution containing 0.4 mg of O-nordtramadol per mL; Spiked solution for test sample: Weigh 20 mg of O-nordrotrodine sample and place it in a 50 mL volumetric flask. Add 4 mL of methanol to dissolve the sample. Then, add 1 mL each of the stock solutions of impurity A, impurity B, impurity C, impurity D, and impurity E. Dilute to the mark with diluent and shake well.

[0060] (2) Chromatographic conditions: Instrument: Shimadzu LC-20AT liquid chromatograph with PDA detector; Column: Agilent SB-C18 4.6×150mm 5μm; Mobile phase A: Acetate buffer (take 18g of sodium acetate, add 9.8ml of glacial acetic acid, and dilute with water to 1000ml). Mobile phase B: Methanol; Column temperature: 30℃; Detection wavelength: 271nm; Flow rate: 1.0 mL / min; Injection volume: 20 μL; The gradient elution procedure is shown in Table 2; Table 2 Gradient elution procedure for Example 1

[0061] (3) Determination method: Accurately measure 20 μL each of the test solution, spiked test solution, blank solution, localization solutions of each impurity, reference solution and impurity reference solution and inject them into the liquid chromatograph. Record the chromatograms. The results are shown in the figure. Figure 13-22 The resolution between O-nortramadol and adjacent impurities is greater than 1.5, and the resolution among all impurities is greater than 1.5, indicating good specificity of this invention. The blank solution showed no interference at the O-nortramadol main peak and the chromatographic peaks of all impurities. Figure 15 It can be seen that the test solution contains impurities A, B, and C. The chromatographic data are shown in Table 3, and the resolution between each chromatographic peak is greater than 1.5. Figure 16 As can be seen, impurities C, O-nordtramadol, impurities A, D, B and E eluted in sequence, and the chromatographic data are shown in Table 4.

[0062] Table 3 Chromatographic results of the test solution

[0063] Table 4 Chromatographic results of the spiked solution of the test sample

[0064] The methodology was validated as follows: 1. Limit of detection and limit of quantitation tests (1) Solution preparation Diluent: A mixture of acetate buffer (18g sodium acetate, 9.8ml glacial acetic acid, diluted with water to 1000ml) and methanol, with a volume ratio of acetate buffer to methanol of 70:30; Blank solution: diluent; O-nordtramadol stock solution: Accurately weigh 4 mg of O-nordtramadol reference standard, dissolve it in 1 mL of methanol, dilute it to 20 mL with diluent, and shake well.

[0065] Mixed solution: Accurately measure 1.0 ml each of the stock solutions of each impurity (same as in Example 1) and the O-nordtramadol stock solution and place them in the same 25 ml volumetric flask. Dilute to the mark with diluent and shake well. Limit of Quantitation (LOQ) test solution: Accurately measure 1.0 ml of the mixed solution into a 50 ml volumetric flask, dilute to the mark with diluent, and shake well; Detection limit test solution: Take 3.0 ml of the quantitation limit test solution and place it in a 10 ml volumetric flask. Add diluent to dilute to the mark and shake well.

[0066] (2) Chromatographic conditions: Instrumentation: Shimadzu LC-20AT liquid chromatograph with UV detector; other chromatographic conditions were the same as in Example 1. (3) Determination: Take blank solution, limit of quantitation test solution and limit of detection test solution, inject them according to chromatographic conditions, record the chromatogram, and the detection results are shown in Table 5.

[0067] Table 5 Results of Limit of Quantitation and Limit of Detection

[0068] 2. Linearity test (1) Linearity of O-nordtramadol content determination: Accurately weigh 16 mg, 18 mg, 20 mg, 22 mg, and 24 mg of O-nordtramadol reference standards, place them in 50 mL volumetric flasks respectively, dissolve them in about 4 mL of methanol, dilute to the mark with diluent, and shake well. These are used as linearity test solutions ①-⑤ for the determination of O-nordtramadol content. Accurately measure 20 μL of each of the above solutions, inject them into the liquid chromatograph under the same chromatographic conditions as the limit of detection and limit of quantitation tests, and plot the peak area against the concentration. Calculate the regression equation and correlation coefficient. The results are shown in Table 6.

[0069] Table 6 Results of the content linearity test

[0070] (2) Linearity of related substances: Take the stock solutions of impurity A, impurity B, impurity C, impurity D, impurity E, and O-nordtramadol and place them in the same volumetric flask. Dilute to the limit of quantitation concentration with diluent to prepare linearity test solution ①; Take 0.5 ml each of the stock solutions of impurity A, impurity B, impurity C, impurity D, impurity E, and O-nordtramadol and place them in a 50 ml volumetric flask. Dilute to the mark with diluent and shake well to prepare linearity test solution ②; Take the stock solutions of impurity A, impurity B, impurity C, impurity D, impurity E, and O-nordtramadol and place them in a 50 ml volumetric flask. Dilute to the mark with diluent and shake well to prepare linearity test solution ②. Take 0.8 ml of each of the impurity stock solutions and place them together in a 50 ml volumetric flask. Dilute to the mark with diluent and mix well to prepare linearity test solution ③. Take 1.0 ml each of the impurity A, impurity B, impurity C, impurity D, impurity E, and O-nordtramadol stock solutions and place them together in a 50 ml volumetric flask. Dilute to the mark with diluent and mix well to prepare linearity test solution ④. Take 2.0 ml each of the impurity A, impurity B, impurity C, impurity D, impurity E, and O-nordtramadol stock solutions and place them together in a 50 ml volumetric flask. Dilute to the mark with diluent and mix well to prepare linearity test solution ⑤. Accurately measure 20 μL of each of the above solutions and test them under the same chromatographic conditions as the limit of detection and limit of quantitation tests. Plot a curve of peak area against concentration, calculate the regression equation and correlation coefficient, and the results are shown in Table 7.

[0071] Table 7 Results of linear experiments on related substances

[0072] 3. Repeatability test Weigh 8 mg of impurity A, 4 mg of impurity B, 4 mg of impurity C, 4 mg of impurity D, and 4 mg of impurity E into a 20 mL volumetric flask, dissolve in 5 mL of methanol, dilute to the mark with diluent, and mix well to prepare the impurity stock solution. Weigh 20 mg of O-nordtrandol into a 50 mL volumetric flask, dissolve in 4 mL of methanol, accurately add 1.0 mL of the impurity stock solution, dilute to the mark with diluent, and mix well to prepare the repeatability test solution. Prepare 6 aliquots using the same method. Accurately measure 20 μL of each repeatability test solution and test under the same chromatographic conditions for the limit of detection and limit of quantitation tests. The experimental results are shown in Table 8.

[0073] Table 8 Results of Repeatability Experiments

[0074] 4. Intermediate precision test With a different testing personnel, solutions were prepared for dates different from the repeatability tests. 8 mg of impurity A, 4 mg of impurity B, 4 mg of impurity C, 4 mg of impurity D, and 4 mg of impurity E were weighed and placed in a 20 mL volumetric flask. 5 mL of methanol was added to dissolve the impurities, and the solution was diluted to the mark with diluent and shaken well. This was used as the impurity stock solution. 20 mg of O-nortramadol was weighed and placed in a 50 mL volumetric flask. 4 mL of methanol was added to dissolve the impurities, and 1.0 mL of the impurity stock solution was accurately added. The solution was diluted to the mark with diluent and shaken well. This was used as the intermediate precision test solution. Six aliquots were prepared using the same method. 20 μL of each intermediate precision test solution was accurately measured and tested under the same chromatographic conditions as the limit of detection and limit of quantitation tests. The experimental results are shown in Table 9.

[0075] Table 9 Results of intermediate precision determination

[0076] 5. Accuracy Test (1) Accuracy of O-nordtramadol content: Weigh approximately 20 mg of O-nordtramadol reference standard into a 50 mL volumetric flask, dissolve it in approximately 4 mL of methanol, dilute to the mark with diluent, and shake well to obtain the reference solution. Weigh 16 mg, 20 mg, and 24 mg of O-nordtramadol reference standard into 50 mL volumetric flasks respectively, dissolve them in approximately 4 mL of methanol, dilute to the mark with diluent, and shake well. Prepare three parallel solutions for each concentration, which are used as O-nordtramadol content accuracy test solutions 1-3. Accurately measure 20 μL of each solution and test it under the same chromatographic conditions for the limit of detection and limit of quantitation tests. The experimental results are shown in Table 10.

[0077] Table 10 Content accuracy test results

[0078] (2) Accuracy of related substances (known impurities): Take 1.0 ml each of impurity A stock solution, impurity B stock solution, impurity C stock solution, impurity D stock solution and impurity E stock solution and place them in a 50 ml volumetric flask. Dilute to the mark with diluent and shake well to prepare the impurity reference solution. Weigh about 20 mg of O-nordtramadol sample and place it in a 50 ml volumetric flask. Dissolve it in about 4 mL of methanol and dilute to the mark with diluent. Shake well to prepare the test solution. Take 1.0 ml each of impurity A stock solution, impurity B stock solution, impurity C stock solution, impurity D stock solution and impurity E stock solution and place them in a 25 ml volumetric flask. Dilute to the mark with diluent and shake well. Accurately measure 1.0 ml of each solution and place it in a 50 ml volumetric flask. Dilute to the mark with diluent and shake well. Prepare 3 parallel solutions as accuracy test solution 1. Weigh approximately 20 mg of O-nortramadol sample and place it in a 50 mL volumetric flask. Dissolve it in approximately 4 mL of methanol, then add 0.5 mL each of impurity A, impurity B, impurity C, impurity D, and impurity E stock solutions. Dilute to the mark with diluent, shake well, and prepare three parallel aliquots as accuracy test solution 2. Weigh approximately 20 mg of O-nortramadol sample and place it in a 50 mL volumetric flask. Dissolve it in approximately 4 mL of methanol, then add 1.0 mL each of impurity A, impurity B, impurity C, impurity D, and impurity E stock solutions. Dilute to the mark with diluent, shake well, and prepare three parallel aliquots as accuracy test solution 3. Weigh approximately 20 mg of O-nortramadol sample and place it in a 50 mL volumetric flask. Dissolve the sample in approximately 4 mL of methanol, then add 1.2 mL each of impurity A, impurity B, impurity C, impurity D, and impurity E stock solutions. Dilute to the mark with diluent, shake well, and prepare three parallel aliquots as accuracy test solutions 4. Inject the above test solutions, impurity reference solutions, and each accuracy test solution under the same chromatographic conditions for detection and quantitation tests, and record the chromatograms. The recoveries of each accuracy test solution were between 80% and 120%, and the RSD of the peak area of ​​the impurity reference solution after six injections was ≤5.0%. The results are shown in Tables 11-15.

[0079] Table 11 Accuracy Test Results for Impurity A

[0080] Table 12 Accuracy Test Results of Impurity B

[0081] Table 13 Accuracy Test Results of Impurity C

[0082] Table 14 Accuracy Test Results of Impurity D

[0083] Table 15 Accuracy Test Results for Impurity E

[0084] (3) Accuracy of related substances (unknown impurities): Weigh approximately 4 mg of O-nordrodrodo reference standard into a 20 ml volumetric flask, dissolve in approximately 1 mL of methanol, dilute to the mark with diluent, and mix well to obtain the stock solution. Accurately measure 1.0 ml of the stock solution into a 50 ml volumetric flask, dilute to the mark with diluent, and mix well to obtain the reference solution. Accurately measure 1.0 ml of the stock solution into a 25 ml volumetric flask, dilute to the mark with diluent, and mix well. Then accurately measure 1.0 ml into a 50 ml volumetric flask, dilute to the mark with diluent, and mix well. Prepare 3 parallel solutions as accuracy test solution 1. Accurately measure 0.5 ml of the stock solution into a 50 ml volumetric flask, dilute to the mark with diluent, and mix well. Prepare 3 parallel solutions as accuracy test solution 2. Accurately measure 1.0 ml of the stock solution into a 50 ml volumetric flask, dilute to the mark with diluent, and mix well. Prepare three parallel aliquots as accuracy test solution 3. Accurately measure 1.2 ml of the stock solution into a 50 ml volumetric flask, dilute to the mark with diluent, and mix well. Prepare three parallel aliquots as accuracy test solution 4. Accurately measure 20 μL of each solution and test under the same chromatographic conditions as the limit of detection and limit of quantitation tests. The experimental results are shown in Table 16.

[0085] Table 16 Accuracy Test Results for Related Substances (Unknown Impurities)

[0086] 6. Solution stability test Weigh 8 mg of impurity A, 4 mg of impurity B, 4 mg of impurity C, 4 mg of impurity D, and 4 mg of impurity E into a 20 mL volumetric flask, dissolve in 5 mL of methanol, dilute to the mark with diluent, and mix well to prepare the impurity stock solution. Weigh 20 mg of O-nordtrandol into a 50 mL volumetric flask, dissolve in 4 mL of methanol, accurately add 1.0 mL of the impurity stock solution, dilute to the mark with diluent, and mix well to prepare the stability test solution. The stability test solution was measured at 0, 8, 24, 48, 72, 96, and 120 hours, and determined under the same chromatographic conditions as the limit of detection and limit of quantitation tests. The experimental results are shown in Table 17. The results indicate that the stability test solution is stable for at least 120 hours.

[0087] Table 17 Stability Test Results

[0088] 7. Durability test (1) Solution preparation: Diluent: A mixture of acetate buffer (18g sodium acetate, 9.8ml glacial acetic acid, diluted with water to 1000ml) and methanol, with a volume ratio of acetate buffer to methanol of 70:30; Blank solution: diluent; Impurity stock solution: Weigh 8 mg of impurity A, 4 mg of impurity B, 4 mg of impurity C, 4 mg of impurity D and 4 mg of impurity E and place them in a 20 ml volumetric flask. Dissolve them in 5 mL of methanol, then dilute to the mark with diluent and shake well. Durability test solution: Weigh 20 mg of O-nortramadol into a 50 ml volumetric flask, dissolve in 4 mL of methanol, accurately add 1.0 ml of impurity stock solution, dilute to the mark with diluent, and shake well.

[0089] (2) Chromatographic conditions: The tests were conducted under the original chromatographic conditions for the limit of detection and limit of quantitation, wavelength ±2nm (269nm, 273nm), column temperature ±2℃ (28℃, 32℃), flow rate ±5% (0.95ml / min, 1.05ml / min), and initial mobile phase methanol ±5%.

[0090] (3) Determination method: Accurately measure 20 μL of blank solution and durability test solution and inject them into the liquid chromatograph. Record the chromatogram and the determination results are shown in Table 18. The range of each impurity result is not greater than 0.3%, the range of the total impurity result is not greater than 0.5%, and the RSD of the O-nortramadol content result is 0.10%. The experimental results show that the present invention has good durability.

[0091] Table 18 Durability Test Results

[0092] 8. Sample testing (1) Solution preparation: Diluent: A mixture of acetate buffer (18g sodium acetate, 9.8ml glacial acetic acid, diluted with water to 1000ml) and methanol, with a volume ratio of acetate buffer to methanol of 70:30; Blank solution: diluent; Reference solution: Weigh 20 mg of O-nordrodrodo reference standard, place it in a 50 mL volumetric flask, add 4 mL of methanol to dissolve it, then dilute to the mark with diluent and shake well.

[0093] Impurity reference solution: Weigh 8 mg of impurity A, 4 mg of impurity B, 4 mg of impurity C, 4 mg of impurity D and 4 mg of impurity E and place them in a 20 mL volumetric flask. Dissolve them in 5 mL of methanol, then dilute to the mark with diluent and shake well to prepare the impurity stock solution. Accurately measure 1 mL of the impurity stock solution and place it in a 50 mL volumetric flask. Dilute to the mark with diluent and shake well.

[0094] Test solution: Accurately weigh 20 mg of O-nordtrandoline, place it in a 50 ml volumetric flask, add 4 mL of methanol to dissolve it, then dilute to the mark with diluent and shake well.

[0095] (2) Chromatographic conditions: Same as the limit of detection and limit of quantitation tests; (3) Determination method: Accurately measure the test solution, blank solution, reference solution and impurity reference solution and inject them into the liquid chromatograph. Record the chromatograms and calculate the contents of impurity A (relative retention time 2.6), impurity B (relative retention time 3.7), impurity C (relative retention time 0.8), total impurities and O-nordrodo in the test solution by peak area normalization method. The results are shown in Table 19.

[0096] Table 19 Determination results of the test solution

Claims

1. A method for detecting O-nordtramadol and its related substances by HPLC, characterized in that... The test solution, blank solution, reference solution, and impurity reference solution were injected into the liquid chromatograph, and the chromatograms were recorded. The content of O-nordromadol and its related substances in the test solution was calculated by the peak area normalization method. Among them, the related substances include one or more of impurities A, B, C, D, or E. HPLC chromatographic conditions include the mobile phase and gradient elution program; The mobile phase consists of acetate buffer as mobile phase A and methanol as mobile phase B; The gradient elution program is as follows: 0-10 minutes, mobile phase A accounts for 65-75% of the total mobile phase volume, and mobile phase B accounts for 25-35% of the total mobile phase volume; 10-15 minutes, mobile phase A accounts for 45-55% of the total mobile phase volume, and mobile phase B accounts for 45-55% of the total mobile phase volume; 15-35 minutes, mobile phase A accounts for 45-55% of the total mobile phase volume, and mobile phase B accounts for 45-55% of the total mobile phase volume; 35-35.1 minutes, mobile phase A accounts for 65-75% of the total mobile phase volume, and mobile phase B accounts for 25-35% of the total mobile phase volume; 35.1-45 minutes, mobile phase A accounts for 65-75% of the total mobile phase volume, and mobile phase B accounts for 25-35% of the total mobile phase volume.

2. The method for detecting O-nordtramadol and related substances by HPLC according to claim 1, characterized in that... The total flow rate of mobile phase A and mobile phase B is 0.95-1.05 mL / min.

3. The method for detecting O-nordtramadol and related substances by HPLC according to claim 1, characterized in that... HPLC chromatographic conditions also include the chromatographic column, detection wavelength, and detector; the chromatographic column is a C18 column with octadecylsilane-bonded silica gel as the packing material, and the column temperature is 28-32℃; the detection wavelength is 269-273nm, and the detector is an ultraviolet detector or a PDA detector.

4. The method for detecting O-nordtramadol and related substances by HPLC according to claim 1, characterized in that... The blank solution is a diluent.

5. The method for detecting O-nordtramadol and related substances by HPLC according to claim 1, characterized in that... The preparation method of the reference solution is to weigh O-nordtramadol reference standard, dissolve it in methanol, and then dilute it with diluent to prepare the reference solution; wherein, the ratio of O-nordtramadol reference standard to methanol is 10:2-5, O-nordtramadol reference standard is expressed in mg, and methanol is expressed in mL; the concentration of O-nordtramadol in the reference solution is 0.3-0.5 mg / mL.

6. The method for detecting O-nordtramadol and related substances by HPLC according to claim 1, characterized in that... The impurity reference solution is prepared by mixing impurity A stock solution, impurity B stock solution, impurity C stock solution, impurity D stock solution and impurity E stock solution and then diluting with a diluent to prepare the impurity reference solution. The concentration of impurity A in the impurity reference solution is 0.32-20 μg / mL, and the concentrations of impurities B, C, D and E are all 0.16-10 μg / mL.

7. The method for detecting O-nordtramadol and related substances by HPLC according to claim 6, characterized in that... The preparation method of impurity A stock solution is as follows: weigh impurity A reference standard, dissolve it in methanol, and dilute it with diluent to prepare impurity A stock solution; wherein, the ratio of impurity A reference standard to methanol is 10:2-5, impurity A reference standard is expressed in mg, and methanol is expressed in mL; the concentration of impurity A in impurity A stock solution is 3.2-500 μg / mL; The impurity B stock solution is prepared by weighing the impurity B reference standard, dissolving it in methanol, and then diluting it with a diluent to prepare the impurity B stock solution; wherein, the ratio of impurity B reference standard to methanol is 10:2-5, the impurity B reference standard is expressed in mg, and the methanol is expressed in mL; the concentration of impurity B in the impurity B stock solution is 1.6-250 μg / mL. The impurity C stock solution is prepared by weighing the impurity C reference standard, dissolving it in methanol, and then diluting it with a diluent to prepare the impurity C stock solution; wherein, the ratio of impurity C reference standard to methanol is 10:2-5, the impurity C reference standard is expressed in mg, and the methanol is expressed in mL; the concentration of impurity C in the impurity C stock solution is 1.6-250 μg / mL; The preparation method of impurity D stock solution is to weigh impurity D reference standard, dissolve it in methanol, and then dilute it with diluent to prepare impurity D stock solution; wherein, the ratio of impurity D reference standard to methanol is 10:2-5, impurity D reference standard is expressed in mg, and methanol is expressed in mL; the concentration of impurity D in impurity D stock solution is 1.6-250 μg / mL. The method for preparing the impurity E stock solution is to weigh the impurity E reference standard, dissolve it in methanol, and then dilute it with a diluent to prepare the impurity E stock solution; wherein, the ratio of impurity E reference standard to methanol is 10:2-5, the impurity E reference standard is expressed in mg, and the methanol is expressed in mL; the concentration of impurity E in the impurity E stock solution is 1.6-250 μg / mL.

8. The method for detecting O-nordtramadol and related substances by HPLC according to claim 1, characterized in that... The test solution is prepared by weighing O-nordtramadol sample, dissolving it in methanol, and then diluting it with a diluent. The ratio of O-nordtramadol sample to methanol is 10:2-5. The O-nordtramadol sample is expressed in mg and the methanol in mL. The concentration of O-nordtramadol in the test solution is 0.3-0.5 mg / mL.

9. The method for detecting O-nordtramadol and related substances by HPLC according to any one of claims 4-8, characterized in that... The diluent is a mixture of acetate buffer and methanol. The acetate buffer is prepared by sodium acetate, glacial acetic acid and water, and the volume ratio of acetate buffer to methanol is 65-75:25-35.

10. The method for detecting O-nordtramadol and related substances by HPLC according to claim 1, characterized in that... The structural formula of O-nordrol is as follows: The structural formula of impurity A is as follows: The structural formula of impurity B is as follows: The structural formula of impurity C is as follows: The structural formula of impurity D is as follows: The structural formula of impurity E is as follows: 。

Images