Method for quantitative determination of hexahydropyridine hydrochloride in tolperidone hydrochloride by high performance liquid chromatography

Abstract

The invention belongs to the field of quality standards and analysis technology detection of drugs, and particularly relates to a method for quantitative determination of hexahydropyridine hydrochloride in tolperidone hydrochloride by high performance liquid chromatography. According to the detection method, a derivatization reagent and an acid-binding agent are used for carrying out a derivatization reaction on hexahydropyridine hydrochloride in tolperidone hydrochloride, so that hexahydropyridine hydrochloride in a sample is converted into a derivative with an ultraviolet absorption functional group, acetonitrile is used as a mobile phase A, a sodium dihydrogen phosphate solution with the pH value adjusted to a specific value by sodium hydroxide is used as a mobile phase B, and the ultraviolet absorption functional group is detected by using a chromatographic column. Elution is carried out in a reversed-phase analysis column, and a high performance liquid chromatography system is matched with an ultraviolet detector to serve as a means for detecting target components. The detection method provided by the invention has the advantages of good separation effect on target components, symmetric chromatographic peak patterns, reasonable retention time and good reproducibility, and provides data support and reference for quality standard formulation of products and rapid screening of preparation processes.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a method for quantitatively determining piperidine hydrochloride in tolperisone hydrochloride by utilizing high performance liquid chromatography. Background Art

[0002] Tolperisone hydrochloride is a chemical drug with central muscle relaxant and vasodilator effects, primarily used to treat cerebrovascular disease and ischemic vascular disease. This product is chemically synthesized from p-methylpropiophenone and hexahydropyridine through a series of reactions, salt formation, and purification processes. Failure to fully optimize reaction conditions or inadequate post-processing steps may result in trace amounts of unreacted hexahydropyridine hydrochloride or degradation products remaining, ultimately present as hydrochloride impurities in the finished product. Unlike other APIs, the hexahydropyridine hydrochloride in tolperisone hydrochloride possesses unique biological activity that directly impacts the overall efficacy of tolperisone hydrochloride.

[0003] The production standards for Tolperisone Hydrochloride have strict requirements for the hexahydropyridine hydrochloride content, typically not exceeding 0.1%. This extremely low concentration requirement is rare in other drugs and is subject to interference from coexisting components, making its detection relatively difficult. Derivatization-based detection requires a more sensitive, stable, and effective quantitative method. Currently, control measures for hexahydropyridine hydrochloride in Tolperisone Hydrochloride are included as an inspection item in the national drug standard "Tolperisone Hydrochloride" promulgated by the State Food and Drug Administration, as well as in the Japanese Pharmacopoeia "Tolperisone Hydrochloride." This method is not a quantitative detection method. The specific operation is to react the tolperisone hydrochloride sample with the hexahydropyridine hydrochloride reference substance using copper sulfate solution, concentrated ammonia solution, etc. to generate a substance with ultraviolet absorption, and extract the color-developing substance using a non-aqueous solvent. The ultraviolet-visible spectrophotometry is used as a detection method to compare the absorbance of the test solution with that of the hexahydropyridine hydrochloride reference solution of a certain concentration. When the absorbance of the test solution is less than that of the control solution, the sample is judged to be qualified. However, after implementation, the reproducibility of this method is poor. When multiple sample solutions are prepared in parallel for the same sample, the absorbance values ​​measured are relatively large. In addition, this method is mainly used for inspection and comparison and cannot be used to quantify hexahydropyridine hydrochloride.

[0004] The structural formula of hexahydropyridine hydrochloride does not have a UV-absorbing functional group, making it impossible to directly measure it using the HPLC-UV method. According to literature reports, based on current technology, the quantitative determination methods for hexahydropyridine substances mainly utilize gas chromatography, ion chromatography, etc., but the equipment described has high requirements for laboratory hardware and is expensive. There is currently no relevant literature reporting on the quantitative determination method of hexahydropyridine hydrochloride in Tolperisone Hydrochloride. Therefore, developing a detection method that can effectively quantitatively determine hexahydropyridine hydrochloride in Tolperisone Hydrochloride is of great significance to improving the quality research and safety control of Tolperisone Hydrochloride. Summary of the Invention

[0005] The present invention aims to address the common industry problem that finished Tolperisone Hydrochloride products and intermediates in the process may contain trace amounts of unreacted hexahydropyridine hydrochloride or degradation product residues, while existing technologies lack relevant quantitative detection methods. After extensive experimental exploration, the present invention provides a method for the quantitative determination of hexahydropyridine hydrochloride in Tolperisone Hydrochloride using high-performance liquid chromatography. The method is simple to operate. After a derivatization reaction is performed on a Tolperisone Hydrochloride sample, quantitative analysis and detection are performed using a standard curve external standard method using reversed-phase liquid chromatography combined with an ultraviolet detector. This method is economical, environmentally friendly, and highly safe, and can be used as an auxiliary means for online quality control in the Tolperisone Hydrochloride production process.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions: A method for quantitatively determining piperidine hydrochloride in tolperisone hydrochloride using high performance liquid chromatography comprises: first performing a derivatization treatment on a sample to obtain a derivatized sample, and then eluting, separating, and detecting the derivatized sample; the elution and separation mobile phase system comprises a mobile phase A and a mobile phase B; the mobile phase A comprises acetonitrile; and the mobile phase B comprises an aqueous solution of sodium dihydrogen phosphate.

[0007] Preferably, in the mobile phase system, the volume ratio of the mobile phase A to the mobile phase B is 30-40:70-60; and the concentration of sodium dihydrogen phosphate in the mobile phase B is 0.05 mol / L.

[0008] Preferably, the pH of the mobile phase B includes 6.5, which is adjusted by sodium hydroxide; the elution separation detection includes high performance liquid chromatography elution separation detection; the conditions of the high performance liquid chromatography elution separation detection include: isocratic elution, the chromatographic column stationary phase adopts a reverse phase analytical column, and the detector adopts an ultraviolet detector or a diode array detector.

[0009] Preferably, the flow rate of the isocratic elution is 0.9-1.1 ml / min, the injection volume is 10 μl, and the column temperature is 25-35° C.; the reversed-phase analytical column comprises an octadecylsilane bonded silica gel packing column; and the detection wavelength of the detector is 370-390 nm.

[0010] The reversed-phase analytical column is a reversed-phase liquid chromatography column using octadecylsilane bonded silica gel filler and having a carbon loading parameter greater than 10. An Hpersil ODS C18 analytical column or other chromatographic columns with equivalent performance can be selected.

[0011] Preferably, the method for quantitative determination of piperidine hydrochloride in tolperisone hydrochloride by high performance liquid chromatography is characterized in that the derivatization treatment comprises: mixing the sample with a derivatization reagent and an acid binding agent, heating to carry out a derivatization reaction, then adding an acidic reagent to stop the reaction and diluting to obtain the derivatized sample.

[0012] Preferably, the derivatization reagent includes 2,4-dinitrofluorobenzene; the acid-binding agent includes diethylamine; the acidic reagent includes a hydrochloric acid-acetonitrile solution; the sample is dissolved in an organic solvent before being mixed with the derivatization reagent and the acid-binding agent; and the organic solvent includes acetonitrile.

[0013] Preferably, the derivatization treatment comprises: S1. Place the sample in a conical flask, add an organic solvent, shake and dissolve, add a 5 mg / ml 2,4-dinitrofluorophenylacetonitrile solution and diethylamine, heat in a water bath at 40°C for the derivatization reaction for 1 hour, remove and allow to stand at room temperature, then add a 5% by volume hydrochloric acid acetonitrile solution to stop the reaction to obtain a reaction solution; the volume-to-mass ratio of the sample, the organic solvent, the 2,4-dinitrofluorophenylacetonitrile solution, and the diethylamine is as follows: 200 mg: 35 ml: 0.8-1.2 ml: 0.08-0.12 ml; S2. Transfer the reaction solution to a volumetric flask, dilute to volume with a 5% by volume hydrochloric acid-acetonitrile solution, and filter through a 0.45 μm filter membrane to obtain the derivatized sample; the ratio of the volume of the volumetric flask to the mass of the sample is 50 ml:200 mg.

[0014] Preferably, after the elution, separation and detection, a chromatogram is recorded, the hexahydropyridine hydrochloride derivative in the sample is qualitatively analyzed according to the retention time of the hexahydropyridine hydrochloride derivative, and the content of the hexahydropyridine hydrochloride in the sample is determined from a standard curve.

[0015] Preferably, the standard curve is obtained by performing the elution separation detection under the same conditions using a standard curve solution, and the preparation of the standard curve solution comprises: A1. Place a hexahydropyridine hydrochloride reference substance in a conical flask, add an organic solvent, and shake to dissolve. Add a 5 mg / ml 2,4-dinitrofluorophenylacetonitrile solution and diethylamine. Heat in a 40°C water bath to carry out the derivatization reaction for 1 hour. After standing at room temperature, add a 5% by volume hydrochloric acid acetonitrile solution to stop the reaction to obtain a reaction solution. A2. Transfer the reaction solution to a volumetric flask, dilute to volume with 5% hydrochloric acid and acetonitrile solution, and filter through a 0.45 μm filter membrane to obtain a derivatization control sample; A3. The derivatized control sample was further diluted 25-fold with acetonitrile to serve as a stock solution of the hexahydropyridine hydrochloride derivative reference substance, and also as a reference solution with a concentration of 9.6 μg / ml, serving as standard solution V; B. Accurately pipette the reference stock solution of the hexahydropyridine hydrochloride derivative and dilute it 2-fold with acetonitrile to prepare a reference solution with a concentration of 4.8 μg / ml, which serves as standard curve IV. C. Accurately pipette the reference stock solution of the hexahydropyridine hydrochloride derivative and dilute it 5-fold with acetonitrile to prepare a reference solution with a concentration of 1.92 μg / ml, which serves as standard curve III. D. Accurately pipette the reference stock solution of the hexahydropyridine hydrochloride derivative and dilute it 10-fold with acetonitrile to prepare a reference solution with a concentration of 0.96 μg / ml, which serves as standard curve II. E. Accurately pipette the reference stock solution of the hexahydropyridine hydrochloride derivative and dilute it 20-fold with acetonitrile to prepare a reference solution with a concentration of 0.48 μg / ml, which serves as standard curve I.

[0016] Preferably, in step A1, the volume mass ratio of the hexahydropyridine hydrochloride reference substance, the organic solvent, the 2,4-dinitrofluorophenylacetonitrile solution, and the diethylamine includes: 12 mg: 35 ml: 1 ml: 0.1 ml; in step A2, the ratio of the volume of the volumetric flask to the mass of the hexahydropyridine hydrochloride reference substance includes 50 ml: 12 mg.

[0017] Compared with the prior art, the present invention has the following beneficial effects: 1. For the first time, a method for accurately quantifying the content of piperidine hydrochloride in tolperisone hydrochloride is disclosed, which solves the problem that the existing technology can only use ultraviolet-visible spectrophotometry to check and compare it, and the absorbance measurement method has poor reproducibility.

[0018] 2. Utilizing an acetonitrile-water-phosphate system at an appropriate ratio for elution, and using phosphate as a buffer system, the pH of the mobile phase is maintained stable, preventing changes in the compound's ionization state due to pH fluctuations, thereby improving peak shape. The phosphate system also possesses high ionic strength and chemical stability. When combined with the organic phase at an appropriate elution ratio, it provides excellent adsorption and desorption, improving the separation capability of reversed-phase chromatography, ensuring a reasonable retention time for the hexahydropyridine hydrochloride derivative and achieving good separation from other polar impurities. Selecting a specific detection wavelength range eliminates interference from the main component of tolperisone hydrochloride.

[0019] 3. The quality requirements for Tolperisone Hydrochloride place strict requirements on the content of piperidine hydrochloride, typically not exceeding 0.1%. This extremely low concentration requirement is rare in other drugs and is subject to interference from coexisting components, making its detection relatively difficult. The present invention provides a quantitative method with good stability, high sensitivity, and simple operation.

[0020] 4. The method used in the present invention is simple to operate, economical and environmentally friendly, highly safe, and has low requirements for the configuration of detection equipment. The method can be implemented using reversed-phase liquid chromatography and an ordinary C18 analytical column, has good economic benefits, and can be widely promoted and applied. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the chromatogram of No. 1 in the mobile phase condition investigation in Example 3; Figure 2 This is the chromatogram of No. 2 in the mobile phase condition investigation in Example 3; Figure 3 This is the chromatogram No. 3 in the mobile phase condition investigation in Example 3; Figure 4 This is the chromatogram No. 4 in the mobile phase condition investigation in Example 3; Figure 5 This is the chromatogram No. 5 in the mobile phase condition investigation in Example 3; Figure 6 This is the chromatogram No. 6 in the mobile phase condition investigation in Example 3; Figure 7 This is the chromatogram No. 7 in the mobile phase condition investigation in Example 3; Figure 8 This is the chromatogram No. 8 in the mobile phase condition investigation in Example 3; Figure 9 This is a chromatogram of the specificity and system suitability test in Example 4. DETAILED DESCRIPTION

[0022] To make the purpose, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the methods, steps or conditions of the present invention are within the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0023] Example 1 A method for quantitatively determining piperidine hydrochloride in tolperisone hydrochloride using high performance liquid chromatography comprises the following steps: (I) Preparation of test solution: Weigh approximately 200 mg of tolperisone hydrochloride sample and place it in a 100 ml conical flask. Add 35 ml of acetonitrile and gently shake to dissolve the solid. Accurately add 1 ml of 5 mg / ml 2,4-dinitrofluorobenzeneacetonitrile solution as a derivatization reagent and 0.1 ml of diethylamine as an acid-binding agent. Mix thoroughly and place in a 40°C water bath to react for 1 hour. After removing from the flask and allowing it to stand at room temperature, add an appropriate amount of 5% by volume hydrochloric acid and acetonitrile to terminate the reaction. Transfer the reaction solution to a 50 ml volumetric flask and dilute to the mark with 5% hydrochloric acid and acetonitrile. Shake well and filter the solution through a 0.45 μm filter membrane for later use.

[0024] (2) HPLC conditions Mobile phase: acetonitrile was used as mobile phase A, 0.05 mol / L sodium dihydrogen sulfate solution (pH adjusted to 6.5 with sodium hydroxide solution) was used as mobile phase B, and the isocratic elution was performed. The volume ratio of the mobile phase A to the mobile phase B was 35:65.

[0025] Chromatographic column: Hypersil ODS C18 column (Elite, 4.6×250mm, 5μm) Detection wavelength: 380nm; Column temperature: 30°C; Flow rate: 1.0 ml / min; Injection volume: 10 μl; (III) Preparation of standard solution: A. Accurately weigh 12 mg of hexahydropyridine hydrochloride reference substance and place it in a 100 ml conical flask. Add 35 ml of acetonitrile and gently shake to dissolve the solid. Accurately add 1 ml of 5 mg / ml 2,4-dinitrofluorobenzene acetonitrile solution as a derivatization reagent and 0.1 ml of diethylamine as an acid-binding agent. Mix thoroughly and place in a 40°C water bath for 1 hour. Remove and allow to stand at room temperature. Add 5% by volume hydrochloric acid and acetonitrile to terminate the reaction. Transfer the reaction solution to a 50 ml volumetric flask and dilute to 50 ml with 5% hydrochloric acid and acetonitrile. Shake thoroughly and filter through a 0.45 μm filter to obtain a derivatized reference sample. Dilute it 25-fold with acetonitrile to prepare a stock solution of the hexahydropyridine hydrochloride derivative reference substance with a concentration of 9.6 μg / ml, which is used as standard solution V. B. Accurately pipette the reference stock solution of the hexahydropyridine hydrochloride derivative and dilute it 2-fold with acetonitrile to prepare a reference solution with a concentration of 4.8 μg / ml, which serves as standard curve IV. C. Accurately pipette the reference stock solution of hexahydropyridine hydrochloride derivative and dilute it 5-fold with acetonitrile to prepare a reference solution with a concentration of 1.92 μg / ml, which serves as standard curve III. D. Accurately pipette the stock solution of the hexahydropyridine hydrochloride derivative reference substance and dilute it 10-fold with acetonitrile to prepare a reference solution with a concentration of 0.96 μg / ml, which serves as standard curve II. E. Accurately aspirate the reference stock solution of hexahydropyridine hydrochloride derivative and dilute it 20-fold with acetonitrile to prepare a reference solution with a concentration of 0.48 μg / ml, which serves as standard curve I.

[0026] (IV) Preparation of accompanying blank solution: Take blank solvent and prepare it according to the above scheme (I).

[0027] (V) Preparation of sample spike solution: Take the above test solution and standard curve IV and mix them in a volume ratio of 1:1.

[0028] (VI) Take 10 μl each of acetonitrile, accompanying blank sample solution, standard curve solution I to standard curve solution V, test solution, and sample spiked solution, and inject them into the liquid chromatograph according to the chromatographic conditions in (II) of the technical plan to record the chromatogram. Perform qualitative analysis based on the retention time of the main peak in the standard curve solution. Calculate the concentration of hexahydropyridine hydrochloride in the test solution by the external standard method of the standard curve, and calculate the corresponding content. The specificity spectrum is as follows: Figure 8 shown.

[0029] Example 2 Determination of sample pretreatment method (1) Determination of reaction temperature Take the same batch of uniform tolperisone hydrochloride sample, accurately weigh 200 mg of the sample, place it in a 100 ml conical flask, accurately add a certain amount of acetonitrile solution containing hexahydropyridine hydrochloride, add 35 ml of acetonitrile, gently shake to dissolve the solid, accurately add 1 ml of 5 mg / ml 2,4-dinitrofluorobenzene acetonitrile solution as a derivatization reaction reagent, add 0.1 ml of diethylamine as an acid binding agent, mix well, and prepare three parallel portions. Derivatization reactions were carried out under the following conditions: room temperature reaction for 1 hour; reaction in a water bath at 40°C for 1 hour; reaction in a water bath at 60°C for 1 hour; the remaining conditions were the same as the technical scheme in Example 1. The content of hexahydropyridine hydrochloride at different reaction temperatures was measured. The results are shown in Table 1.

[0030] Table 1 Investigation of derivatization reaction temperature in Tolperisone hydrochloride

[0031] Analysis of the data in the table shows that, for a 1-hour reaction time, the room temperature reaction yielded lower derivative conversions (measured by the peak area of ​​the hexahydropyridine hydrochloride derivative) than the heated reaction. The conversions were comparable for the reactions heated at 40°C and 60°C. Because the 60°C reaction temperature might increase the number of impurity peaks in the sample solution, 40°C was selected as the reaction temperature.

[0032] (2) Determination of reaction time Take the same batch of uniform tolperisone hydrochloride sample, accurately weigh 200 mg of the sample, place it in a 100 ml conical flask, accurately add a certain amount of acetonitrile solution containing hexahydropyridine hydrochloride, add 35 ml of acetonitrile, gently shake to dissolve the solid, accurately add 1 ml of 5 mg / ml 2,4-dinitrofluorobenzene acetonitrile solution as a derivatization reaction reagent, add 0.1 ml of diethylamine as an acid binding agent, mix well, prepare three parallel portions, and carry out the derivatization reaction under the following conditions: reaction in a water bath at 40°C for 45 min; reaction in a water bath at 40°C for 1 h; reaction in a water bath at 40°C for 1 h 15 min; the other conditions are the same as the technical scheme of Example 1, and the content of hexahydropyridine hydrochloride at different reaction times is measured. The results are shown in Table 2.

[0033] Table 2 Investigation of derivatization reaction time in Tolperisone hydrochloride

[0034] From the analysis of the data in the table, it can be seen that when the reaction temperature is constant and the reaction time is between 45min and 75min, the conversion rate of hexahydropyridine hydrochloride derivatives (with the peak area of ​​hexahydropyridine hydrochloride derivatives as the evaluation indicator) does not change much, and the middle value is selected as the final experimental condition.

[0035] (III) Determination of the amount of derivatization reagent and acid binding agent added Take the same batch of uniform tolperisone hydrochloride sample, accurately weigh 200 mg of the sample, place it in a 100 ml conical flask, accurately add a certain amount of acetonitrile solution containing hexahydropyridine hydrochloride, add 35 ml of acetonitrile, gently shake to dissolve the solid, prepare three parallel portions, and add the derivatization reaction reagent and acid binding agent according to the following conditions: accurately add 0.8 ml of 5 mg / ml 2,4-dinitrofluorobenzene acetonitrile solution and 0.08 ml of diethylamine; accurately add 1 ml of 5 mg / ml 2,4-dinitrofluorobenzene acetonitrile solution and 0.1 ml of diethylamine; accurately add 1.2 ml of 5 mg / ml 2,4-dinitrofluorobenzene acetonitrile solution and 0.12 ml of diethylamine; the other conditions are the same as the technical scheme of Example 1, and the corresponding hexahydropyridine hydrochloride content is measured under different amounts of derivatization reaction reagent and acid binding agent added. The results are shown in Table 3.

[0036] Table 3 Investigation of the amount of derivatization reagents and acid-binding agents added to Tolperisone hydrochloride

[0037] Analysis of the data in the table shows that when the reaction temperature and reaction time are constant, slight changes in the amount of derivatization reagent and acid-binding agent added have little effect on the conversion rate of hexahydropyridine hydrochloride derivatives (using the peak area of ​​hexahydropyridine hydrochloride derivatives as the evaluation indicator). Therefore, the intermediate value, which is easier to operate, was selected as the final experimental condition.

[0038] (IV) Determination of the concentration of hydrochloric acid solution for terminating the reaction The derivatization reaction of 2,4-dinitrofluorobenzene typically requires a weakly alkaline environment. When the derivatization reaction needs to be stopped, an acidic reagent can be added to neutralize the weakly alkaline reaction system to a neutral to acidic state, thereby stopping the reaction. If the acidic reagent is added at an insufficient concentration, the reaction cannot be completely terminated, potentially converting non-hydrochloric acid hexahydropyridine substances into derivatives, leading to inaccurate measurement results. If the acidic concentration is too high, the pH value of the solution decreases too rapidly. When the solution is injected into the liquid phase, the highly acidic solution can damage the life of the chromatographic column and deteriorate the main peak shape. Therefore, the amount of acidic reagent added to terminate the reaction needs to be carefully considered.

[0039] Take the same batch of uniform tolperisone hydrochloride samples, accurately weigh 200 mg of the sample, place it in a 100 ml conical flask, accurately add a certain amount of acetonitrile solution containing hexahydropyridine hydrochloride, add 35 ml of acetonitrile, gently shake to dissolve the solid, accurately add 1 ml of 5 mg / ml 2,4-dinitrofluorobenzene acetonitrile solution as a derivatization reaction reagent, add 0.1 ml of diethylamine as an acid binding agent, mix well, place in a 40 ° C water bath to react for 1 hour, take out and let it stand at room temperature, prepare 3 copies in parallel, and terminate the derivatization conditions according to the following conditions: add A 1% by volume hydrochloric acid acetonitrile solution was added to terminate the reaction, a 5% by volume hydrochloric acid acetonitrile solution was added to terminate the reaction, and a 10% by volume hydrochloric acid acetonitrile solution was added to terminate the reaction. The reaction solution was transferred to a 50 ml volumetric flask. The remaining conditions were the same as those in the technical scheme of Example 1 to prepare a test sample spiked solution, which was placed at room temperature for 15 h. The sample was repeatedly injected at 0 h, 5 h, 10 h, and 15 h, respectively, to determine the peak area percentage of hexahydropyridine hydrochloride at different reaction times, paying attention to peak parameters (such as peak width and asymmetry). The results are shown in Table 4.

[0040] Table 4 Investigation of hydrochloric acid solution concentration for termination reaction

[0041] Within 15 hours, the peak area percentage of hexahydropyridine hydrochloride derivatives is used as a measure of the formation of side reaction products. If the peak area percentage of hexahydropyridine hydrochloride derivatives gradually decreases, the amount of side reaction products gradually increases, indicating that the derivatization reaction is not completely terminated and the solution stability is poor. Otherwise, the derivatization reaction is completely terminated and the solution stability is good. According to data analysis, when the hydrochloric acid concentration is 5% and 10%, the peak area percentage of hexahydropyridine hydrochloride derivatives of the test solution within 15 hours is relatively stable, indicating that the amount of side reaction has not gradually increased, the derivatization reaction is completely terminated and the solution stability is good; considering that the closer the asymmetry of the main peak of the hexahydropyridine hydrochloride derivative is to 1, the better the peak shape, the concentration of 5% hydrochloric acid solution is selected as the termination reaction concentration.

[0042] Example 3 (1) Selection of mobile phase conditions Currently, no authoritative standards or publicly available literature supports the quantitative analysis of piperidine hydrochloride in tolperisone hydrochloride using high-performance liquid chromatography-ultraviolet (HPLC-UV) methods. For the determination of piperidine compounds in other products, gas chromatography and ion chromatography are often used. However, these methods require high-quality laboratory equipment and are expensive, making them less than optimal.

[0043] After extensive trial and error, the inventors developed a method for isocratic elution using acetonitrile as mobile phase A and 0.05 mol / L sodium dihydrogen phosphate solution (adjusted to pH 6.5 with 5% sodium hydroxide solution) as mobile phase B. This method achieved a main peak retention time of 14-18 minutes, a peak elution time that was reasonable and distinguishable from the solvent peak retention time, and eliminated interference from other polar components on the main peak, achieving accurate quantitative determination of the target component. The chromatographic conditions developed are shown in (2) of the technical solution in Example 1. The results of the chromatographic condition selection experiments are summarized in Table 5.

[0044] Table 5 Mobile phase ratio investigation test

[0045] The chromatogram of the mobile phase ratio investigation is shown in Figures 1 to 8 From the measured data and the spectrum in Table 4, it can be seen that if the experiment is carried out within the mobile phase ratio range of the chromatographic conditions (II) in the technical solution of Example 1, the measured substances can all be well separated and symmetrical peak shapes can be obtained; if the ratio of the two phases is further changed, such as No. 3, although detection can be achieved, the retention time is too long, the efficiency is reduced, and the cost is increased; according to literature review, using the potassium dihydrogen phosphate solution-acetonitrile system, the chromatograms obtained in the reference solution, sample solution, and sample spiked solution cannot distinguish the main peak of the hexahydropyridine hydrochloride derivative, and it is judged that the main peak is not retained under these conditions; if only the organic phase-water system is used without adding buffer salts for elution, the main peak is very close to the solvent peak and the polar component with a small retention time, and it is impossible to integrate and perform quantitative detection; adding volatile salts such as diethylamine to the organic phase-water system for elution also cannot change the situation of a small retention time and inability to integrate and quantify.

[0046] (2) Selection of chromatographic columns The inventors have conducted a large number of exploratory tests and examined commercially available chromatographic columns. Under the chromatographic conditions described in the technical solution (ii) of Example 1, experiments were conducted on a C18 column commonly used in the laboratory. Good peak shapes and relatively reasonable retention times and separations were obtained, with only slight differences in peak morphology. Specific parameters are shown in Table 6.

[0047] Table 6 Column Conditions

[0048] Example 4 Specificity and system suitability experiments Take 10 μl of acetonitrile, accompanying blank sample solution, unreacted test solution, hexahydropyridine hydrochloride reference substance derivatization solution (reference substance stock solution), derivatized test solution, and sample spike solution, respectively, and perform liquid chromatography determination according to the chromatographic conditions described in (II) of the technical scheme of Example 1, and record the chromatogram, as shown below. Figure 9 shown.

[0049] The results showed that acetonitrile, the accompanying blank sample solution, and the unreacted test solution did not interfere with the main peak determination. The derivatized solution of the hexahydropyridine hydrochloride reference substance, the derivatized test solution, and the sample spike solution showed no interference near the main peak, and the retention time of the main peak was consistent. The main peak purity was greater than 950. These results demonstrate that the determination method has good specificity and that the system adaptability meets the requirements.

[0050] Table 7 System suitability and specificity test data

[0051] Example 5 Limit of detection and limit of quantification A reference substance, hexahydropyridine hydrochloride, was derivatized as described in (I) of Example 1 to prepare a reference stock solution, which was then diluted with acetonitrile to prepare a solution with a signal-to-noise ratio of approximately 3:1, which served as a detection limit reference solution; and a solution with a signal-to-noise ratio of approximately 10:1, which served as a quantification limit reference solution. The chromatographic conditions of the technical scheme in Example 1 were used for injection and measurement, and the chromatograms were recorded. The quantification limit of this method was 0.0039 μg, and the detection limit was 0.0013 μg.

[0052] Example 6 Linear relationship experiment According to the chromatographic conditions in the technical solution of Example 1, 10 μL of each of the standard solutions I to V was accurately drawn and injected into the liquid chromatograph. The peak area of ​​each component was used as the ordinate and the concentration was used as the abscissa to calculate the regression equation. From the results, it can be seen that the standard linear equation of hexahydropyridine hydrochloride is y=944.7950x+0.002513, and the correlation coefficient r 2 =0.9998; this shows that the method presents a good linear relationship.

[0053] Example 7 Precision test Take the same batch of tolperisone hydrochloride samples and prepare the test solution according to the "Technical Scheme of Example 1", and measure according to the chromatographic conditions, record the chromatogram, and determine the content of hexahydropyridine hydrochloride in tolperisone hydrochloride by the external standard method based on the measured standard curve, and calculate the relative standard deviation. RSD should be ≤3% as a repeatability test.

[0054] The same batch of tolperisone hydrochloride samples were sampled using the same method and determined by different personnel on different days. The hexahydropyridine hydrochloride content and its relative standard deviation (RSD) in tolperisone hydrochloride were determined. The RSD should be ≤3%, which serves as an intermediate precision test. Comparing the results of this experiment with those of the repeatability test, the RSD of the 12 calculated data points should be ≤3%. The precision test results, shown in Table 8, demonstrate that the precision meets the requirements.

[0055] Table 8 Precision test results (unit: %)

[0056] Example 8 Accuracy test According to three levels (low, medium, and high), a batch of tolperisone hydrochloride samples was taken, 200 mg of the sample was accurately weighed, and placed in a 100 ml conical flask. Nine portions were prepared in parallel and numbered 1 to 9. To the low-level inspection samples (samples 1 to 3), 0.12 mg of hexahydropyridine hydrochloride reference substance was added; to the medium-level inspection samples (samples 4 to 6), 0.25 mg of hexahydropyridine hydrochloride reference substance was added; to the high-level inspection samples (samples 7 to 9), 0.49 mg of hexahydropyridine hydrochloride reference substance was added, and then 35 ml of acetonitrile was added to dissolve the solid, 1 ml of 5 mg / ml 2,4-dinitrofluorobenzene acetonitrile solution was accurately added as a derivatization reaction reagent, and 0 0.1 ml of diethylamine was used as an acid-binding agent, mixed thoroughly, and reacted in a 40°C water bath for 1 hour. After being taken out and allowed to stand at room temperature, an appropriate amount of 5% by volume hydrochloric acid and acetonitrile was added to terminate the reaction. The reaction solution was transferred to a 50 ml volumetric flask and the volume was adjusted to the mark with 5% hydrochloric acid and acetonitrile. The solution was filtered through a 0.45 μm filter membrane to obtain a sample solution. The samples were measured according to the above chromatographic conditions, and the chromatograms were recorded. The actual measured amount of hexahydropyridine hydrochloride was calculated respectively. The accuracy data was converted based on the spiked amount. See Table 9 for details.

[0057] Table 9 Accuracy test results

[0058] Example 9 Durability test to examine chromatographic conditions (1) Solution stability test Take the same batch of tolperisone hydrochloride samples and prepare one test solution according to the method of Example 1. According to the chromatographic conditions of the detection method of the present invention, the samples were injected once at 0, 2, 4, 8, 12, 18, and 24 hours respectively. The relative standard deviation of the hexahydropyridine hydrochloride content was compared. The RSD should be ≤3%. The results are shown in Table 10 below. The results show that the test solution is stable within 24 hours.

[0059] Table 10 Solution stability test results

[0060] (2) System durability The same batch of tolperisone hydrochloride samples were taken and the detection method of Example 1 was used. Other chromatographic conditions remained unchanged, but different column temperatures (25°C, 30°C, 35°C), different flow rates (0.9 ml / min, 1.0 ml / min, 1.1 ml / min), different detection wavelengths (370 nm, 380 nm, 390 nm), mobile phase ratios (mobile phase A-mobile phase B = 30:70, mobile phase A-mobile phase B = 35:65, mobile phase A-mobile phase B = 40:60), and different brands of chromatographic columns were set for determination. The chromatograms were recorded and the relative standard deviation of the hexahydropyridine hydrochloride content in the samples was calculated. The RSD should be ≤3%. The results are shown in Table 11 below.

[0061] Table 11 System durability test results

[0062] The test results show that the test solution has good stability within 24 hours; the determination of hexahydropyridine hydrochloride in tolperisone hydrochloride has no effect on the determination under slight changes in column temperature, flow rate, mobile phase ratio and chromatographic column.

[0063] Example 10 Several batches of pilot samples from the process study of Tolperisone Hydrochloride were taken, and the piperidine hydrochloride in Tolperisone Hydrochloride was quantitatively determined according to the method of Example 1. The determination results are shown in Table 12.

[0064] Table 12 The measurement results of multiple batches of Tolperisone Hydrochloride hourly samples

[0065] From the determination results, it can be seen that the method of the present invention can be used to quickly quantitatively determine the hexahydropyridine hydrochloride in tolperisone hydrochloride. Therefore, the present invention can be used as a means of product production process and product online quality control, providing data support for the industrialization of the product and a basis for the establishment of product quality standards.

[0066] The above disclosure is merely a preferred embodiment of the present invention and certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims

1. A method for quantitatively determining piperidine hydrochloride in tolperisone hydrochloride by high performance liquid chromatography, characterized in that: include: First, the sample is subjected to a derivatization treatment to obtain a derivatized sample, and then the derivatized sample is eluted, separated, and detected; The mobile phase system for elution and separation includes mobile phase A and mobile phase B; the mobile phase A includes acetonitrile; and the mobile phase B includes an aqueous solution of sodium dihydrogen phosphate.

2. The method for quantitative determination of piperidine hydrochloride in tolperisone hydrochloride by high performance liquid chromatography according to claim 1, wherein: In the mobile phase system, the volume ratio of the mobile phase A to the mobile phase B is 30-40:70-60; and the concentration of sodium dihydrogen phosphate in the mobile phase B is 0.05 mol / L.

3. The method for quantitative determination of piperidine hydrochloride in tolperisone hydrochloride by high performance liquid chromatography according to claim 1, wherein: The pH of the mobile phase B includes 6.5, which is adjusted by sodium hydroxide; the elution separation detection includes high performance liquid chromatography elution separation detection; The conditions for elution, separation and detection by high performance liquid chromatography include: isocratic elution, a reverse phase analytical column as the stationary phase of the chromatographic column, and an ultraviolet detector or a diode array detector as the detector.

4. The method for quantitative determination of piperidine hydrochloride in tolperisone hydrochloride by high performance liquid chromatography according to claim 3, wherein: The flow rate of the isocratic elution is 0.9-1.1 ml / min, the injection volume is 10 μl, and the column temperature is 25-35° C.; the reversed-phase analytical column includes an octadecylsilane bonded silica gel filler column; and the detection wavelength of the detector is 370-390 nm.

5. The method for quantitative determination of piperidine hydrochloride in tolperisone hydrochloride by high performance liquid chromatography according to claim 1, wherein: The derivatization treatment includes: mixing the sample with a derivatization reagent and an acid-binding agent, heating to carry out a derivatization reaction, then adding an acidic reagent to stop the reaction and diluting the sample to obtain the derivatized sample.

6. The method for quantitative determination of piperidine hydrochloride in tolperisone hydrochloride by high performance liquid chromatography according to claim 5, wherein: The derivatization reagent includes 2,4-dinitrofluorobenzene; the acid binding agent includes diethylamine; the acidic reagent includes hydrochloric acid acetonitrile solution; the sample is first dissolved in an organic solvent before being mixed with the derivatization reagent and the acid binding agent; and the organic solvent includes acetonitrile.

7. The method for quantitative determination of piperidine hydrochloride in tolperisone hydrochloride by high performance liquid chromatography according to claim 5, wherein: The derivatization treatment includes: S1. Place the sample in a conical flask, add an organic solvent, shake and dissolve, add a 5 mg / ml 2,4-dinitrofluorophenylacetonitrile solution and diethylamine, heat in a water bath at 40°C for the derivatization reaction for 1 hour, remove and allow to stand at room temperature, then add a 5% by volume hydrochloric acid acetonitrile solution to stop the reaction to obtain a reaction solution; the volume-to-mass ratio of the sample, the organic solvent, the 2,4-dinitrofluorophenylacetonitrile solution, and the diethylamine is as follows: 200 mg: 35 ml: 0.8-1.2 ml: 0.08-0.12 ml; S2. Transfer the reaction solution to a volumetric flask, dilute to volume with a 5% by volume hydrochloric acid-acetonitrile solution, and filter through a 0.45 μm filter membrane to obtain the derivatized sample; the ratio of the volume of the volumetric flask to the mass of the sample is 50 ml:200 mg.

8. The method for quantitative determination of piperidine hydrochloride in tolperisone hydrochloride by high performance liquid chromatography according to claim 1, wherein: After the elution, separation and detection, a chromatogram is recorded, and the hexahydropyridine hydrochloride derivative in the sample is qualitatively analyzed according to the retention time of the hexahydropyridine hydrochloride derivative. The content of the hexahydropyridine hydrochloride in the sample is determined by the standard curve external standard method.

9. The method for quantitative determination of piperidine hydrochloride in tolperisone hydrochloride by high performance liquid chromatography according to claim 8, wherein: The standard curve is obtained by performing the elution separation and detection under the same conditions on the standard curve solution, and the preparation of the standard curve solution includes: A1. Place a hexahydropyridine hydrochloride reference substance in a conical flask, add an organic solvent, and shake to dissolve. Add a 5 mg / ml 2,4-dinitrofluorophenylacetonitrile solution and diethylamine. Heat in a 40°C water bath to carry out the derivatization reaction for 1 hour. After standing at room temperature, add a 5% by volume hydrochloric acid acetonitrile solution to stop the reaction to obtain a reaction solution. A2. Transfer the reaction solution to a volumetric flask, dilute to volume with 5% hydrochloric acid and acetonitrile solution, and filter through a 0.45 μm filter membrane to obtain a derivatization control sample; A3. The derivatized control sample was further diluted 25-fold with acetonitrile to serve as a stock solution of the hexahydropyridine hydrochloride derivative reference substance, and also as a reference solution with a concentration of 9.6 μg / ml, serving as standard solution V; B. Accurately pipette the reference stock solution of the hexahydropyridine hydrochloride derivative and dilute it 2-fold with acetonitrile to prepare a reference solution with a concentration of 4.8 μg / ml, which serves as standard curve IV. C. Accurately pipette the reference stock solution of the hexahydropyridine hydrochloride derivative and dilute it 5-fold with acetonitrile to prepare a reference solution with a concentration of 1.92 μg / ml, which serves as standard curve III. D. Accurately pipette the reference stock solution of the hexahydropyridine hydrochloride derivative and dilute it 10-fold with acetonitrile to prepare a reference solution with a concentration of 0.96 μg / ml, which serves as standard curve II. E. Accurately pipette the reference stock solution of the hexahydropyridine hydrochloride derivative and dilute it 20-fold with acetonitrile to prepare a reference solution with a concentration of 0.48 μg / ml, which serves as standard curve I.

10. The method for quantitative determination of piperidine hydrochloride in tolperisone hydrochloride by high performance liquid chromatography according to claim 9, wherein: In step A1, the volume-to-mass ratio of the hexahydropyridine hydrochloride reference substance, the organic solvent, the 2,4-dinitrofluorophenylacetonitrile solution, and the diethylamine is 12 mg: 35 ml: 1 ml: 0.1 ml. In step A2, the ratio of the volume of the volumetric flask to the mass of the hexahydropyridine hydrochloride reference substance is 50 ml: 12 mg.

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