Method for detecting 2, 6-butylated hydroxytoluene in ropivacaine hydrochloride sustained release preparation

Abstract

The invention provides a method for detecting 2, 6-butylated hydroxytoluene in a ropivacaine hydrochloride sustained release preparation, and belongs to the field of medicine detection. The method for detecting the 2, 6-butylated hydroxytoluene in the ropivacaine hydrochloride sustained release preparation comprises the following steps: (1) carrying out pretreatment; and 2) detecting. According to the method, the ropivacaine hydrochloride sustained release preparation containing the 2, 6-butylated hydroxytoluene is pretreated, other fat-soluble auxiliary materials in the ropivacaine hydrochloride sustained release preparation are removed, the content of the 2, 6-butylated hydroxytoluene in the ropivacaine hydrochloride sustained release preparation is measured through the high performance liquid chromatography, the method is high in specificity and sensitivity and good in accuracy and linearity, and the ropivacaine hydrochloride sustained release preparation containing the 2, 6-butylated hydroxytoluene is obtained. The method is completely suitable for measuring the content of 2, 6-butylated hydroxytoluene in complex components of the ropivacaine sustained release preparation.

Description

Technical Field

[0001] The invention belongs to the field of detection chemistry and relates to a detection method for a ropivacaine hydrochloride sustained-release preparation, and specifically relates to a detection method for 2,6-di-tert-butyl-p-cresol in a ropivacaine hydrochloride sustained-release preparation. Background Art

[0002] Surgical pain is acute pain caused by damage to subcutaneous tissue, nerves, blood vessels, and internal organs due to the trauma of the surgery itself. It is a comprehensive response to soft tissue trauma during the hypersensitive surgery that stimulates the central nervous system. Patients often experience various emotional abnormalities due to pain stimulation, such as depression, anxiety, irritability, sleep disorders, etc. Sometimes it will have a significant impact on the functions of the patient's organs, causing a series of physiological and pathological changes, and leading to various complications, seriously affecting the patient's postoperative recovery.

[0003] Postoperative analgesia can not only relieve the pain of patients after surgery, but also stabilize the mood of patients, reduce the fluctuation or abnormality of various physiological indicators, reduce postoperative complications, shorten the hospital stay, etc. Commonly used postoperative analgesics in clinical practice include non-steroidal analgesics, such as ibuprofen, aspirin, etc., opioid narcotic analgesics, such as morphine, fentanyl, etc., and amide local anesthetics, such as ropivacaine, bupivacaine, etc. Among them, non-steroidal analgesics can have side effects such as liver damage, kidney damage, and gastrointestinal adverse reactions; opioid analgesics have adverse reactions such as respiratory depression and addiction; and amide local anesthetics have the advantages of low cardiac toxicity, obvious separation of sensory block and motor block, and dual effects of anesthesia and analgesia when used for postoperative analgesia, so they are often preferred in clinical practice.

[0004] Ropivacaine is the first pure L-amide local anesthetic. It was first developed by Astra in Sweden in 1995 and first launched in the Netherlands in 1996. Ropivacaine can reduce the permeability of sodium ions on nerve cell membranes, thereby reversibly blocking the conduction of nerve impulses, achieving the dual effects of anesthesia and analgesia. Compared with bupivacaine, ropivacaine has significantly lower toxicity to the cardiovascular and central nervous systems, so it is safer for clinical use.

[0005] Since ropivacaine has good water solubility, the clinical dosage forms developed in the early stage are mainly ordinary water injections. For example, in Patent Document 1 (Yue Feng, Liang Chengbiao, etc., A High-Safety Ropivacaine Hydrochloride Injection and Its Preparation Method [P], Patent No. CN201210019757.6), the prescription composition of ropivacaine hydrochloride injection is ropivacaine hydrochloride, sodium chloride, sodium hydroxide or hydrochloric acid, and water for injection. In Patent Document 2 (Shi Jianguo, Zhang Yu, etc., Ropivacaine Hydrochloride Injection and Its Preparation Process [P], Patent No. CN201210111806.9), the prescription composition of ropivacaine hydrochloride injection is ropivacaine hydrochloride, sodium chloride and water.

[0006] Although the aqueous injection preparation of ropivacaine hydrochloride has a simple preparation process, good product stability and a rapid onset of action, the analgesic effect lasts for a short time, generally 3-6 hours. If the patient needs long-term analgesia after surgery, repeated administration of the drug is required, resulting in poor patient compliance and sometimes causing peak and trough effects in blood drug concentrations, resulting in toxic and side effects. Therefore, the use of a sustained-release preparation of ropivacaine hydrochloride can better meet the actual needs of clinicians and patients.

[0007] Since ropivacaine has low lipid solubility, when developing a sustained-release preparation, natural phospholipids, non-aqueous carriers, and viscosity regulators are usually used in the prescription. For example: Patent Document 3 (Tong Xinyong, Zou Aifeng, et al., A sustained-release lipid composition and its preparation method [P], Patent No. CN202080045830.X) provides a method for preparing a proliposomal sustained-release preparation of ropivacaine hydrochloride, wherein the natural phospholipids include egg yolk lecithin, soybean lecithin, and dipalmitoyl phosphatidylcholine, and the non-aqueous carriers include medium-chain triglycerides and capric triglycerides.

[0008] Since the fatty acid chains in these phospholipids or non-aqueous carriers contain unsaturated double bonds, oxidation and deterioration are prone to occur after contact with air during the production, transportation or storage of the preparation product, resulting in product denaturation or the generation of toxic impurities, such as anisidine impurities, N-oxide impurities, etc. Therefore, when developing the above-mentioned ropivacaine sustained-release preparation, a certain amount of antioxidant components are often added to the prescription, such as the antioxidant components added in Patent Document 3 (Tong Xinyong, Zou Aifeng, etc., a sustained-release lipid composition and its preparation method [P], patent number CN202080045830.X) include α-tocopherol, butylated hydroxyanisole, tert-butylhydroquinone, dibutylhydroxytoluene (BHT), etc.; these antioxidant components will gradually consume and reduce as the ropivacaine sustained-release preparation sample is stored. Therefore, its content must be accurately measured during the production release and stability study of the sample to determine whether it has an antioxidant effect.

[0009] In the prior art, when determining the content of antioxidant components in ropivacaine sustained-release preparation samples, the determination method is easily affected by other fat-soluble excipients, such as phospholipids, triglycerides or other vegetable oils, and there are certain difficulties. Currently, the prior art has not reported a method for determining the content of antioxidants in ropivacaine sustained-release preparation samples. During the early research and development process, our company developed a ropivacaine hydrochloride sustained-release preparation, whose antioxidant component is butylated hydroxytoluene (BHT). In order to accurately control the quality of sample production and release products, and determine the accurate content of butylated hydroxytoluene (BHT) in samples during stability inspections, so as to ensure the safety and effectiveness of the products leaving the factory, it is necessary to develop a method for detecting the accurate content of BHT in ropivacaine hydrochloride sustained-release preparation samples. Summary of the invention

[0010] The present invention aims at the problems existing in the prior art and provides a method for detecting 2,6-di-tert-butyl-p-cresol in a ropivacaine hydrochloride sustained-release preparation. The ropivacaine hydrochloride sustained-release preparation containing 2,6-di-tert-butyl-p-cresol is pre-treated to remove other fat-soluble excipients therein, and then the content of 2,6-di-tert-butyl-p-cresol therein is determined by high performance liquid chromatography. The method has high specificity, high sensitivity, good accuracy and linearity, and is fully applicable to the determination of the content of 2,6-di-tert-butyl-p-cresol in the complex components of the ropivacaine sustained-release preparation.

[0011] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0012] In a first aspect, the present invention provides a method for detecting 2,6-di-tert-butyl-p-cresol in a ropivacaine hydrochloride sustained-release preparation, comprising the following steps:

[0013] 1) Pretreatment: dissolving a ropivacaine hydrochloride sustained-release preparation in an alcohol organic solvent to obtain a sample solution, mixing a mixed solution of acetonitrile and water with the sample solution, removing the precipitate, and obtaining a sample to be tested, wherein the ropivacaine hydrochloride sustained-release preparation contains 2,6-di-tert-butyl-p-cresol and other fat-soluble excipients;

[0014] 2) Detection: The content of 2,6-di-tert-butyl-p-cresol in the sample to be tested is detected by high performance liquid chromatography, and the conditions of the high performance liquid chromatography are:

[0015] A C18 chromatographic column is used, one or two of acetonitrile and methanol are used as mobile phase A, and one or more of water, phosphate buffer and acetate buffer are used as mobile phase B for isocratic elution.

[0016] The step 1) is a pre-treatment step, specifically:

[0017] First, a ropivacaine hydrochloride sustained-release preparation is dissolved in an alcohol organic solvent, wherein the ropivacaine hydrochloride sustained-release preparation contains 2,6-di-tert-butyl-p-cresol and other fat-soluble excipients, wherein the other fat-soluble excipients include but are not limited to vegetable oils, and the vegetable oils may be phospholipids, triglycerides or other vegetable oils. The alcohol organic solvent is any type well known to those skilled in the art, including but not limited to ethanol, isopropanol, hexafluoroisopropanol, etc. The alcohol organic solvent may be one or more, and may be any one component, a system of any two components mixed, or a system of any two or more components mixed. The mixing ratio may be any ratio, so that the sample is completely dissolved, and the method for complete dissolution includes but is not limited to strong shaking, ultrasound or vortexing, etc., to obtain a sample solution.

[0018] Then, the sample solution is mixed with a mixed solution of acetonitrile and water, and the volume ratio of the acetonitrile and water is 5:5-9:1, preferably 8:2-9:1. Since BHT and main components in the ropivacaine hydrochloride sustained-release preparation sample are soluble in acetonitrile, and other fat-soluble adjuvants such as phospholipids are difficult to dissolve in acetonitrile and water, the sample solution of the ropivacaine hydrochloride sustained-release preparation completely dissolved is taken, mixed with a mixed solution of acetonitrile and water, so that other fat-soluble adjuvants in the ropivacaine hydrochloride sustained-release preparation are precipitated. Further, in order to more thoroughly remove other fat-soluble adjuvants, it is preferred that the sample solution be mixed with a mixed solution of acetonitrile and water, and then stand at room temperature for at least 30 minutes, or be centrifuged, with a centrifugal rate of 5000-12000rpm, preferably 10000rpm, and a centrifugal time of at least 5 minutes, preferably 10 minutes. The precipitate is removed to obtain a sample to be tested, wherein the concentration of the ropivacaine hydrochloride sustained-release preparation in the sample to be tested is 5.0-25.0 mg / mL, preferably 10-25 mg / mL, and more preferably 20-25 mg / mL.

[0019] Preferably, it also includes: after removing the precipitate, filtering with an organic filter membrane with a pore size of 0.45 μm to obtain a sample to be tested. In one embodiment, after the sample solution and the mixed solution of acetonitrile and water are allowed to stand or centrifuged, an appropriate amount of the supernatant is taken. Since the supernatant contains more organic solvents and also suspends some other unprecipitated fat-soluble excipients, it is necessary to filter with an organic filter membrane with a pore size of 0.45 μm, and the organic filter membrane includes but is not limited to a nylon filter membrane, etc., and the sample dilution is preferably filtered with a nylon filter membrane with a pore size of 0.45 μm, and 2 mL of the initial filtrate is discarded, and the filtrate is taken to obtain the sample to be tested.

[0020] In one embodiment, the sample liquid is mixed with a mixed solution of acetonitrile and water, the volume ratio of the acetonitrile and water is 8:2, and the volume ratio of the sample liquid to the mixed solution of acetonitrile and water is 8:2 to obtain a test sample, wherein the concentration of the ropivacaine hydrochloride sustained-release preparation in the test sample is 5.0-25.0 mg / mL, preferably 20-25 mg / mL, and more preferably 20 mg / mL or 25 mg / mL.

[0021] The step 2) is a detection step, specifically:

[0022] The content of 2,6-di-tert-butyl-p-cresol in the sample to be tested was detected by high performance liquid chromatography, and the conditions of the high performance liquid chromatography were:

[0023] A C18 chromatographic column is used for high performance liquid chromatography detection, and the C18 chromatographic column includes but is not limited to analytical columns of brands and specifications such as XB-C18 (250mm×4.6mm, 5μm) or Welch Ultimate XB-C18 (250mm×4.6mm, 5μm), and XB-C18 (250mm×4.6mm, 5μm) is preferably used. The column temperature will affect the peak shape of 2,6-di-tert-butyl-p-cresol and the column efficiency of the chromatographic column. The column temperature of the C18 chromatographic column is 25-40°C, preferably 30°C.

[0024] The mobile phase A is acetonitrile or methanol, or a mixed solution of acetonitrile and methanol. The mixed solution of acetonitrile and methanol can be a mixture in any proportion. Pure acetonitrile or a mixed solution of acetonitrile and methanol is preferably used. The volume ratio of acetonitrile to methanol in the mixed solution of acetonitrile and methanol is 5-9:1-5, preferably 9:1.

[0025] The mobile phase B is one or more of water, phosphate buffer and acetate buffer, the water is purified water, the phosphate buffer includes but is not limited to potassium dihydrogen phosphate buffer, dipotassium hydrogen phosphate buffer, sodium dihydrogen phosphate buffer, disodium hydrogen phosphate buffer or ammonium dihydrogen phosphate buffer or diammonium hydrogen phosphate buffer, the acetate buffer includes but is not limited to ammonium acetate buffer, sodium acetate buffer or potassium acetate buffer, the concentration of the phosphate buffer and the acetate buffer is preferably 10-30mmol / L, and the pH value of the mobile phase B is preferably 4.5-6.5. Preferably, the mobile phase B is one or more of water and phosphate buffer, the phosphate buffer is potassium dihydrogen phosphate buffer, dipotassium hydrogen phosphate buffer, sodium dihydrogen phosphate buffer, disodium hydrogen phosphate buffer or ammonium dihydrogen phosphate buffer or diammonium hydrogen phosphate buffer. In one embodiment, mobile phase B uses a 20mmol / L potassium dihydrogen phosphate solution with a pH value of 5.0, and the symmetry of the BHT peak is good when the sample to be tested is detected.

[0026] Mobile phase A and mobile phase B are used for isocratic elution, wherein the volume ratio of mobile phase A and mobile phase B is 70:30-95:5, preferably 90:10-95:5, and the elution rate of the isocratic elution is 0.7-1.5mL / min, preferably 0.7-1.2mL / min, more preferably 1.0mL / min or 0.9mL / min. In one embodiment, the volume ratio of mobile phase A and mobile phase B is 90:10. In one embodiment, the volume ratio of mobile phase A and mobile phase B is 95:5. When the content of 2,6-di-tert-butyl-p-cresol is determined, if the flow rate of the mobile phase is too low, the peak type of 2,6-di-tert-butyl-p-cresol will be broadened or tailed, and the symmetry of the peak will be poor; if the flow rate of the mobile phase is too high, the column pressure will be too large, the chromatographic column cannot tolerate it, and the column efficiency and service life of the chromatographic column will be reduced.

[0027] The detector for the HPLC detection is a VWD detector (variable wavelength detector), a DAD detector (diode array detector), or a PDA detector (diode array detector). The detection wavelength is 270-290nm, preferably 280nm, which can ensure that the detection method has a high sensitivity.

[0028] In one embodiment, the injection volume range of the HPLC detection is 5-50 μL, preferably 20 μL. If the injection volume is too small, the detection sensitivity, accuracy and method repeatability of 2,6-di-tert-butyl-p-cresol will be affected. If the injection volume is too large, the solvent effect or chromatographic column overload will be caused, resulting in tailing of the 2,6-di-tert-butyl-p-cresol peak, affecting the peak integration and data calculation.

[0029] In one embodiment, in high performance liquid chromatography detection, mobile phase A and mobile phase B with a volume ratio of 70:30-95:5 are used for elution, and the elution time is not less than 20 minutes. Preferably, mobile phase A and mobile phase B with a volume ratio of 90:10 are used for isocratic elution, and the elution time is 20 minutes. This can ensure that the 2,6-di-tert-butyl-p-cresol peak can be effectively separated from other potential impurities or auxiliary material peaks, and ensure that the retention time of the 2,6-di-tert-butyl-p-cresol peak is appropriate, thereby improving the analysis efficiency.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] (1) The prescription composition of the ropivacaine hydrochloride sustained-release preparation contains other fat-soluble excipients, such as phospholipids, triglycerides or other vegetable oils, etc. These components have a strong adsorption effect on the C18 column and are difficult to elute, which affects the column efficiency. The present invention removes other fat-soluble excipients from the ropivacaine hydrochloride sustained-release preparation including 2,6-di-tert-butyl-p-cresol by pre-treating it, thereby reducing the impact on the detection;

[0032] (2) The content of 2,6-di-tert-butyl-p-cresol (BHT) in the sample was determined by high performance liquid chromatography. This method has high specificity, high sensitivity, good accuracy and linearity, and can accurately determine the content of 2,6-di-tert-butyl-p-cresol in the complex components of ropivacaine sustained-release preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is the chromatogram of the BHT reference substance solution in Example 1;

[0034] Figure 2 This is a typical chromatogram of the accuracy test solution in Example 1;

[0035] Figure 3 is the BHT linear correlation diagram of Example 1;

[0036] Figure 4 is the chromatogram of the BHT reference solution in Example 2;

[0037] Figure 5 This is a typical chromatogram of the accuracy test solution in Example 2;

[0038] Figure 6 is the BHT linear correlation diagram of Example 2;

[0039] Figure 7 This is the first typical chromatogram of the accuracy test solution in Comparative Example 1;

[0040] Figure 8 This is the second typical chromatogram of the accuracy test solution in Comparative Example 1;

[0041] Fig. 9 This is a state diagram of the preparation sample solution in Comparative Example 2 being diluted with a mixture of acetonitrile and water. DETAILED DESCRIPTION

[0042] It is worth noting that, unless otherwise specified, the raw materials used in the present invention are all common commercially available products, and their sources are not specifically limited.

[0043] The following sources of raw materials are provided for illustrative purposes:

[0044] The sample of ropivacaine hydrochloride sustained-release preparation was prepared by Jiali (Shenzhen) Biotechnology Co., Ltd., and the prescription composition was: ropivacaine hydrochloride, sesame oil, phosphatidylcholine, ethanol, and BHT in a mass ratio of 2%:25%:52.5%:7.5%:0.01%.

[0045] Example 1

[0046] 1. Preparation of BHT reference solution: Take about 15 mg of 2,6-di-tert-butyl-p-cresol (BHT) reference substance, accurately weigh it, place it in a 50 mL volumetric flask, dissolve it with acetonitrile and dilute it to the mark as the reference substance stock solution. Accurately transfer 1.0 mL of the reference substance stock solution to a 50 mL volumetric flask, dilute it to the mark with acetonitrile, shake it well, and use it as the reference substance solution.

[0047] 2. Preparation of quantitative limit solution: Accurately measure 1.0 mL of BHT reference solution, place it in a 50 mL volumetric flask, dilute to the scale with acetonitrile, shake well, and obtain.

[0048] 3. Preparation of accuracy test solution: Take about 2.0g of ropivacaine hydrochloride sustained-release preparation sample, weigh accurately, place in a 50mL volumetric flask, dissolve and dilute to the mark with ethanol, shake vigorously to mix, accurately measure 5.0mL, place in a 10mL volumetric flask, add 0.2mL of BHT reference substance stock solution, then dilute to the mark with a mixture of acetonitrile and water (volume ratio 8:2), shake vigorously to mix, and finally transfer to a centrifuge tube, centrifuge at 10000rpm for 10 minutes, take an appropriate amount of supernatant, filter with a nylon filter membrane with a pore size of 0.45μm, discard 2.0mL of the initial filtrate, and take the subsequent filtrate. (Prepare 6 portions in parallel)

[0049] 4. Preparation of linear test solution:

[0050] Linear solution 1: is the quantitative limit solution (the preparation method is the same as the above “Preparation of quantitative limit solution”).

[0051] Linear solution 2: Accurately pipette 0.5 mL of BHT reference stock solution into a 50 mL volumetric flask, dilute to the mark with acetonitrile, and shake well.

[0052] Linear solution 3: Accurately pipette 1.0 mL of BHT reference stock solution into a 50 mL volumetric flask, dilute to the mark with acetonitrile, and shake well.

[0053] Linear solution 4: Accurately pipette 1.5 mL of BHT reference stock solution into a 50 mL volumetric flask, dilute to the mark with acetonitrile, and shake well.

[0054] Linear solution 5: Accurately pipette 2.0 mL of BHT reference stock solution into a 50 mL volumetric flask, dilute to the mark with acetonitrile, and shake well.

[0055] 5.HPLC determination method:

[0056] Chromatographic column: XB-C18 (250mm×4.6mm, 5μm) chromatographic column;

[0057] Mobile phase A: acetonitrile;

[0058] Mobile phase B: 20 ​​mmol / L potassium dihydrogen phosphate buffer, adjusted to pH 5.0 with phosphoric acid or 2 M potassium hydroxide solution;

[0059] Detection wavelength: 280nm;

[0060] Flow rate: 1.0 mL / min;

[0061] Column temperature: 30°C;

[0062] Injection volume: 20 μl;

[0063] Elution mode: isocratic elution, mobile phase A-mobile phase B (volume ratio of 90:10), elution for 20 minutes.

[0064] 6. Typical spectra of BHT reference solution and accuracy test solution are shown in Figure 1 and Figure 2 As shown, Figure 1 is the chromatogram of the BHT reference substance solution in Example 1, Figure 2 is a typical chromatogram of the accuracy investigation solution in Example 1, wherein Figure 1 The chromatographic peak with a retention time of 7.725 min is the BHT peak. The experimental results show that the tailing factor of the BHT peak is 1.11, the peak has good symmetry, the theoretical plate number is 19853, and the separation column efficiency is good. Figure 2 The chromatographic peak with a retention time of 7.739 min is the BHT peak. From the experimental spectrum, it can be seen that there are no solvent peaks, other impurity peaks, and other auxiliary material peaks before and after the BHT peak to interfere with the detection of BHT, so the specificity of this method is good.

[0065] 7. Repeatability test results: Take an appropriate amount of BHT reference solution and perform continuous sampling and detection 6 times according to the above HPLC determination method. The results of BHT peak area determination are shown in Table 1.

[0066] The experimental results show that the RSD of the BHT peak area after 6 consecutive injections is 1.28%, indicating that the detection method has good injection repeatability.

[0067] Table 1 Results of the BHT injection repeatability experiment in Example 1

[0068] Injection times 1 2 3 4 5 6 RSD(%) BHT peak area 56162 58159 57291 57991 57484 57955 1.28

[0069] 8. Sensitivity test results: Take an appropriate amount of the limit of quantitation solution (LOQ solution) and perform the test according to the above HPLC test method. The sensitivity test results of BHT are shown in Table 2.

[0070] The experimental results show that when the BHT concentration is 0.1251 μg / mL, which is equivalent to 0.0006% of the sample solution concentration, the peak height signal-to-noise ratio of BHT is 15.0, which is greater than 10.0 and meets the requirements of quantitative determination. Therefore, the detection method of BHT has good sensitivity.

[0071] Table 2 Results of the sensitivity test of BHT in Example 1

[0072]

[0073] 9. Results of the accuracy test: Take an appropriate amount of the 6 parallel prepared accuracy test solutions and perform the test according to the above HPLC method. The results of the BHT recovery rate test are shown in Table 3.

[0074] The experimental results show that in the six parallel spiked test solutions, the recovery of BHT is in the range of 99.8% to 100.9%, and the RSD of the recovery is 0.40%, indicating that the detection method has good accuracy and precision.

[0075] Table 3 Accuracy experimental results of BHT in Example 1

[0076]

[0077] 10. Results of linearity test solution: Take appropriate amount of prepared linearity test solution of each concentration and test it according to the above HPLC test method. The results of linearity test of BHT are shown in Table 4 and Figure 3 , Figure 3 : is the BHT linear correlation diagram of Example 1, wherein the straight line represents the connection between data points, and the dotted line represents the fitted straight line.

[0078] The experimental results show that the concentration of BHT is linearly correlated with the response peak area within the range of the quantitative limit concentration to 200% standard concentration, that is, within the concentration range of 0.1251-12.5075 μg / mL. The linear equation is Y=9567.3105X+96.5534, the correlation coefficient is 1.0000, and the linear correlation is good.

[0079] Table 4 Linearity test results of BHT in Example 1

[0080] Linearity test solution BHT concentration (μg / mL) Peak area of ​​BHT (mAU*S) LOQ solution 0.1251 1209 50% standard concentration solution 3.1269 30071 100% standard concentration solution 6.2537 59781 150% standard concentration solution 9.3806 90296 2000% standard concentration solution 12.5075 119480

[0081] Example 2

[0082] 1. Preparation of BHT reference solution: Take about 15 mg of 2,6-di-tert-butyl-p-cresol (BHT) reference substance, accurately weigh it, place it in a 50 mL volumetric flask, dissolve it with a mixed solution of acetonitrile-methanol (9:1) and dilute it to the mark as the reference substance stock solution. Accurately pipette 1.0 mL of the reference substance stock solution, place it in a 50 mL volumetric flask, dilute it to the mark with a mixed solution of acetonitrile-methanol (volume ratio is 9:1), shake it well, and use it as the reference substance solution.

[0083] 2. Preparation of quantitative limit solution: Accurately measure 1.0 mL of BHT reference solution, place it in a 50 mL volumetric flask, dilute to the scale with acetonitrile-methanol (volume ratio of 9:1) mixed solution, shake well, and obtain.

[0084] 3. Preparation of the accuracy test solution: Take about 1.25g of the sample of ropivacaine hydrochloride sustained-release preparation, weigh it accurately, put it in a 50mL volumetric flask, dissolve it with ethanol and dilute it to the mark, shake it vigorously to mix it, accurately measure 8.0mL, put it in a 10mL volumetric flask, add 0.2mL of BHT reference substance stock solution, and then dilute it to the mark with a mixture of acetonitrile-water (volume ratio of 5:5), shake it vigorously to mix it, let it stand at room temperature for 2 hours, take an appropriate amount of supernatant, filter it with a nylon filter membrane with a pore size of 0.45μm, discard 2.0mL of the initial filtrate, and take the subsequent filtrate. (Prepare 6 portions in parallel)

[0085] 4. Preparation of linear test solution:

[0086] Linear solution 1: is the quantitative limit solution (the preparation method is the same as the above “Preparation of quantitative limit solution”).

[0087] Linear solution 2: Accurately pipette 0.5 mL of BHT reference stock solution into a 50 mL volumetric flask, dilute to scale with a mixed solution of acetonitrile-methanol (volume ratio of 9:1), and shake well.

[0088] Linear solution 3: Accurately pipette 1.0 mL of BHT reference stock solution into a 50 mL volumetric flask, dilute to scale with a mixed solution of acetonitrile-methanol (volume ratio of 9:1), and shake well.

[0089] Linear solution 4: Accurately pipette 1.5 mL of BHT reference stock solution into a 50 mL volumetric flask, dilute to scale with a mixed solution of acetonitrile-methanol (volume ratio of 9:1), and shake well.

[0090] Linear solution 5: Accurately pipette 2.0 mL of BHT reference stock solution into a 50 mL volumetric flask, dilute to scale with a mixed solution of acetonitrile-methanol (volume ratio of 9:1), and shake well.

[0091] 5.HPLC determination method:

[0092] Chromatographic column: Welch Ultimate XB-C18 (250 mm × 4.6 mm, 5 μm) column;

[0093] Mobile phase A: acetonitrile-methanol (volume ratio 9:1) mixed solution;

[0094] Mobile phase B: purified water;

[0095] Detection wavelength: 280nm;

[0096] Flow rate: 0.9 mL / min;

[0097] Column temperature: 25°C;

[0098] Injection volume: 20 μl;

[0099] Elution mode: isocratic elution, mobile phase A-mobile phase B (volume ratio of 95:5), elution for 20 minutes.

[0100] 6. Typical spectra of BHT reference solution and accuracy test solution are shown in Figure 4 and Figure 5 As shown, Figure 4 is the chromatogram of the BHT reference substance solution in Example 2, Figure 5 is a typical chromatogram of the accuracy investigation solution in Example 2, wherein Figure 4 The chromatographic peak with a retention time of 8.392 min is the BHT peak. The experimental results show that the tailing factor of the BHT peak is 0.98, the peak has good symmetry, the theoretical plate number is 21917, and the separation column efficiency is good. Figure 5 The chromatographic peak with a retention time of 8.375 min is the BHT peak. From the experimental spectrum, it can be seen that there are no solvent peaks, other impurity peaks, and other auxiliary material peaks before and after the BHT peak to interfere with the detection of BHT, so the specificity of this method is good.

[0101] 7. Repeatability test results: Take an appropriate amount of BHT reference solution and inject it continuously for 6 times according to the above HPLC determination method. The results of BHT peak area determination are shown in Table 5. From the experimental results, it can be seen that the RSD of BHT peak area after 6 consecutive injections is 0.08%, indicating that the injection repeatability of the detection method is good.

[0102] Table 5 Results of the injection repeatability test of BHT in Example 2

[0103] Injection times 1 2 3 4 5 6 RSD(%) BHT peak area 61291 61358 61332 61279 61223 61349 0.08

[0104] 8. Sensitivity test results: Take an appropriate amount of the quantitative limit solution and inject it according to the above HPLC determination method. The sensitivity determination results of BHT are shown in Table 6. The experimental results show that when the BHT concentration is 0.1239 μg / mL, which is equivalent to 0.0006% of the sample solution concentration, the peak height signal-to-noise ratio of BHT is 13.2, which is greater than 10.0, meeting the requirements of quantitative determination, so the BHT detection method has good sensitivity.

[0105] Table 6 Sensitivity test results of BHT in Example 2

[0106]

[0107] 9. Results of the accuracy test experiment: Take an appropriate amount of the 6 parallel prepared accuracy test solutions, and perform the injection test according to the above HPLC test method. The results of the BHT recovery rate test are shown in Table 7. The experimental results show that in the 6 parallel spiked test solutions, the recovery rate of BHT is in the range of 98.9% to 100.5%, and the RSD of the recovery rate is 0.58%, indicating that the accuracy of the detection method is good, and the precision of the method is also good.

[0108] Table 7 Accuracy experimental results of BHT in Example 2

[0109]

[0110] 10. Results of linearity test solution: Take appropriate amount of prepared linearity test solution of each concentration and test it according to the above HPLC test method. The results of linearity test of BHT are shown in Table 8 and Figure 6 , Figure 6 The figure is a linear correlation diagram of BHT in Example 2, wherein the straight line represents the connection between data points and the dotted line represents the fitted straight line. The experimental results show that within the range of the quantitative limit concentration to 200% standard concentration, i.e., within the concentration range of 0.1239-12.3902 μg / mL, the concentration of BHT is linearly correlated with the response peak area, the linear equation is Y=9393.3694X+618.3573, the correlation coefficient is 0.9996, and the linear correlation is good.

[0111] Table 8 Linearity test results of BHT in Example 2

[0112] Linearity test solution BHT concentration (μg / mL) Peak area of ​​BHT LOQ solution 0.1239 1174 50% standard concentration solution 3.0975 29978 100% standard concentration solution 6.1951 58661 150% standard concentration solution 9.2926 89825 2000% standard concentration solution 12.3902 115581

[0113] Comparative Example 1

[0114] The difference from Example 1 is that the "method for preparing the accuracy investigation solution" in Example 1 is replaced by "preparation of the accuracy investigation solution: take about 2.0 g of the sample of ropivacaine hydrochloride sustained-release preparation, accurately weigh it, put it in a 50 mL volumetric flask, dissolve it with ethanol and dilute it to the scale, shake it vigorously to mix, accurately measure 5.0 mL, put it in a 10 mL volumetric flask, add 0.2 mL of BHT reference substance stock solution, then dilute it with ethanol to the scale, and shake it vigorously to mix. (Prepare 6 portions in parallel)", and the other steps and operations are the same as Example 1.

[0115] Typical spectra of two consecutive accuracy test solutions are shown in Figure 7 and Figure 8 As shown, Figure 7 The chromatographic peak with a retention time of 7.705 min is the BHT peak. Figure 8 The chromatographic peak with a retention time of 8.899 min is the BHT peak. The experimental results show that if the sample solution of the ropivacaine hydrochloride sustained-release preparation does not use a mixed solution of acetonitrile and water to precipitate the auxiliary material components, the auxiliary material will appear a large miscellaneous peak after the BHT main peak. Although the miscellaneous peak does not interfere with the integration of the BHT peak, the spiked recovery rates of the six parallel samples are also in the range of 98% to 102% (the spiked recovery data are shown in Table 9). However, due to the small polarity of the auxiliary material components, they are easily retained in the chromatographic column and are difficult to be eluted cleanly, affecting the BHT peak retention time of the next injection (two consecutive injections, BHT Therefore, in order to avoid serious drift of the retention time of the BHT main peak and prevent the chromatographic column from being contaminated and blocked by the excipient peak when used for a long time, the detection method stipulates that the prepared ropivacaine hydrochloride sustained-release preparation sample solution is pre-treated, that is, the preparation sample solution is diluted with a mixed solution of acetonitrile and water (the mixing ratio is in the range of 5:5 to 9:1), and after strong shaking, it is allowed to stand at room temperature for at least 30 minutes, or centrifuged at a centrifugal speed of 5000-12000rpm for at least 5 minutes to remove the precipitate and obtain the sample solution to be tested.

[0116] Table 9 Accuracy experimental results of BHT in Comparative Example 1

[0117]

[0118] Comparative Example 2

[0119] The difference from Example 1 is that the "method for preparing the solution for accuracy investigation" in Example 1 is replaced by "taking about 2.0 g of ropivacaine hydrochloride sustained-release preparation sample, accurately weighing, placing in a 50 mL measuring bottle, dissolving and diluting to the scale with ethanol, vigorously shaking to mix, accurately measuring 5.0 mL, placing in a centrifuge tube, adding 0.2 mL of BHT reference substance stock solution, then diluting to 10 ml with a mixed solution of acetonitrile and water (volume ratio 4:6), vigorously shaking, and finally the sample solution forms a gel-like substance, see Fig. 9 As shown, the sample solution cannot be separated by precipitation. Therefore, when the ratio of acetonitrile to water in the diluent is lower than 5:5, it is not suitable for the pretreatment of the sample solution of ropivacaine hydrochloride sustained-release preparation, because there is too much water and the auxiliary material components cross-link with water to form a gel.

[0120] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A method for detecting 2,6-di-tert-butyl-p-cresol in a ropivacaine hydrochloride sustained-release preparation, characterized in that: The following steps are involved: 1) Pretreatment: dissolving a ropivacaine hydrochloride sustained-release preparation in an alcohol organic solvent to obtain a sample solution, mixing the sample solution with a mixed solution of acetonitrile and water, removing the precipitate, and obtaining a sample to be tested, wherein the ropivacaine hydrochloride sustained-release preparation contains 2,6-di-tert-butyl-p-cresol and other fat-soluble excipients; 2) Detection: The content of 2,6-di-tert-butyl-p-cresol in the sample to be tested is detected by high performance liquid chromatography, and the conditions of the high performance liquid chromatography are: A C18 chromatographic column is used, one or two of acetonitrile and methanol are used as mobile phase A, and one or more of water, phosphate buffer and acetate buffer are used as mobile phase B for isocratic elution.

2. The detection method according to claim 1, characterized in that: In the step 1), the volume ratio of acetonitrile to water is 5:5-9:

1.

3. The detection method according to claim 2, characterized in that: In the step 1), the volume ratio of the sample solution to the mixed solution of acetonitrile and water is 5:5-9:

1.

4. The detection method according to claim 3, characterized in that: In the step 1), the concentration of the ropivacaine hydrochloride sustained-release preparation in the sample to be tested is 5.0-25.0 mg / mL.

5. The detection method according to claim 4, characterized in that: The step 1) further comprises: after removing the precipitate, filtering with an organic filter membrane having a pore size of 0.45 μm to obtain a sample to be tested.

6. The detection method according to claim 1, characterized in that: In the step 2), the volume ratio of mobile phase A to mobile phase B is 70:30-95:

5.

7. The detection method according to claim 1, characterized in that: In the step 2), the mobile phase A is acetonitrile or a mixed solution of acetonitrile and methanol, and the volume ratio of acetonitrile to methanol in the mixed solution of acetonitrile and methanol is 5-9:1-5.

8. The detection method according to claim 1, characterized in that: In the step 2), the mobile phase B is one or more of water and phosphate buffer; the phosphate buffer is potassium dihydrogen phosphate buffer, dipotassium hydrogen phosphate buffer, sodium dihydrogen phosphate buffer, disodium hydrogen phosphate buffer or ammonium dihydrogen phosphate buffer or diammonium hydrogen phosphate buffer; the pH value of the mobile phase B is 4.5-6.

5.

9. The detection method according to claim 1, characterized in that: In the step 2), the detection wavelength is 270-290 nm.

10. The detection method according to claim 1, characterized in that: In the step 2), the column temperature of the C18 chromatographic column is 25-40° C.; the elution rate of the isocratic elution is 0.7-1.5 mL / min.

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