Method for simultaneously determining contents of tolperidone hydrochloride, methyl hesperidin and vitamin B6 in Naoluotong capsule

Abstract

The invention belongs to the field of pharmaceutical analysis, and discloses a method for simultaneously determining the contents of tolperidone hydrochloride, methyl hesperidin and vitamin B6 in Naoluotong capsules, the method comprises three steps of preparation of a mixed reference solution, preparation of a test solution and content determination, and the chromatographic conditions of the content determination are as follows: a C18 chromatographic column, a C18 chromatographic column, a C18 chromatographic column, a C18 chromatographic column, a C18 chromatographic column, a C18 chromatographic column, a C18 chromatographic column, a C18 chromatographic column and a C18 chromatographic column; a mobile phase is methanol (A)-0.1% glacial acetic acid solution (B), gradient elution is carried out for 0-5 min, and 96% of B is obtained; 5-6 min, 96%-60% of B is selected; 6-20 min, the content of B is 60%-50%; the volume flow rate is 1.0 ml / min; the column temperature is 30 DEG C; the detection wavelength is 291 nm; the sample size is 1 microliter. According to the method, synchronous detection of three components (tolperidone hydrochloride, methyl hesperidin and vitamin B6) with remarkable physicochemical property difference can be realized, the chromatographic condition is simple, and the detection speed is high.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical analysis, and more specifically, relates to a method for simultaneously determining the contents of tolperisone hydrochloride, hesperidin methylchalcone and vitamin B6 in Naoluotong capsules. Background Art

[0002] With the aggravation of population aging, cerebrovascular diseases have become the second leading cause of death globally. As a commonly used Chinese and Western medicine compound preparation in clinical practice, Naoluotong Capsule is composed of Salvia miltiorrhiza extract, Tolperisone Hydrochloride, Ligusticum wallichii extract, Hesperidin methylchalcone, Astragalus membranaceus extract, and Vitamin B6. It has the effect of dilating blood vessels and increasing cerebral blood flow, and is used for various cerebrovascular diseases such as cerebral thrombosis, cerebral arteriosclerosis, and sequelae of stroke, as well as symptoms such as headache, dizziness, hemiplegia, limb numbness, and nerve fatigue caused by qi deficiency and blood stasis syndrome. It plays an important role in the treatment of diseases such as cerebral infarction and cerebral arteriosclerosis. However, there are many current standards for Naoluotong Capsule, and the quality control indicators of each standard are inconsistent, resulting in significant differences in the products of each enterprise. Currently, in the current standards, Tolperisone Hydrochloride is extracted with chloroform and its content is determined by non-aqueous titration. The solvent chloroform is highly toxic and volatile, the filtration speed of sample pretreatment is slow, the colors of the products of each enterprise are different, and it is difficult to effectively determine the titration end point, further affecting the accuracy of experimental results and the physical and mental health of experimental personnel. In the past, there were few reports on the improvement of the quality standard of Naoluotong Capsule, and only single indicators were determined for their contents, making it difficult to comprehensively reflect the quality of the preparation. For example, Ma Zhenghong et al. published "Determination of the Content of Tolperisone Hydrochloride in Naoluotong Capsule by HPLC", using an ODS column as the stationary phase, methanol-water-triethylamine (160:39:1, adjusted to pH 7.5 with glacial acetic acid) as the mobile phase, and a detection wavelength of 261 nm. Guo Dongyan et al. published "Determination of the Content of Tolperisone Hydrochloride in Naoluotong Capsule by HPLC", using an ODS C18 column (5μm, 4.6mm×250mm), acetonitrile-0.05mol / L potassium dihydrogen phosphate (30∶70) (adjusted to pH 3.0 with phosphoric acid) as the mobile phase, a detection wavelength of 256 nm, a flow rate of 1.0ml / min, and a column temperature of room temperature. Zhang Chunyu et al. published "Determination of the Content of Hesperidin Methylchalcone in Naoluotong Capsule by High Performance Liquid Chromatography", with a chromatographic column of ZorbaxC18 column (4.6×250mm, 5μ); the mobile phase was methanol-0.3% glacial acetic acid (38:62); the detection wavelength was 283 nm, and the column temperature was 40℃. Su Mingwu et al. published "Determination of the Content of Vitamin B6 in Naoluotong Capsule by High Performance Liquid Chromatography", using high performance liquid chromatography, a Dikma C18 column (250mm×4.6mm, 5μm); the mobile phase: methanol-0.06% sodium heptanesulfonate aqueous solution (containing 1.2% glacial acetic acid and 0.12% triethylamine) (13:87); the detection wavelength: 290 nm; the column temperature: 30℃.With the gradual transformation of traditional Chinese medicine quality control from single-component to multi-component overall control, methods such as quantitative analysis of multi-index components have become another approach for the quality evaluation of traditional Chinese medicine. Li Jianming et al. published "Simultaneous Determination of Tropicamide Hydrochloride and Hesperidin Methyl Chalcone in Naoluotong Capsules by HPLC". The chromatographic column was Shim-pack VP-ODS, with acetonitrile - 0.025 mol·L-1 potassium dihydrogen phosphate (5 mL of triethylamine was added to every 1 L, and the pH was adjusted to 3.0 with phosphoric acid) (25:75) as the mobile phase, the flow rate was 1.0 mL·min-1, the column temperature was 30 °C, and the detection wavelengths were 261 and 283 nm. Since the physicochemical properties of components such as tropicamide hydrochloride, hesperidin methyl chalcone, and vitamin B6 in Naoluotong capsules vary greatly. For example, tropicamide hydrochloride is prone to peak tailing due to the residual effect of silanols in the conventional mobile phase, and usually additional chemical reagents need to be added to neutralize the activity of silanols; hesperidin methyl chalcone is a weakly acidic flavonoid glycoside, and in a non-ion pair system, it is prone to peak broadening or splitting due to dissociation; vitamin B6 is a water-soluble vitamin, with a high degree of protonation under acidic conditions, and the pH needs to be optimized to balance its retention time and separation selectivity. As a result, the current chromatographic conditions for determining multiple components in Naoluotong capsules are complex, and it is impossible to simultaneously determine the contents of tropicamide hydrochloride, hesperidin methyl chalcone, and vitamin B6. Summary of the Invention

[0003] In view of the deficiencies of the prior art, the purpose of the present invention is to provide a method for simultaneously determining the contents of tropicamide hydrochloride, hesperidin methyl chalcone, and vitamin B6 in Naoluotong capsules. This method can achieve the synchronous detection of three components (tropicamide hydrochloride, hesperidin methyl chalcone, and vitamin B6) with significantly different physicochemical properties, and has simple chromatographic conditions and a fast detection speed.

[0004] To achieve the above purpose, the present invention adopts the following technical solutions:

[0005] A method for simultaneously determining the contents of tropicamide hydrochloride, hesperidin methyl chalcone, and vitamin B6 in Naoluotong capsules, the method comprising the following steps:

[0006] Step 1. Preparation of a mixed reference solution: Prepare a mixed reference solution containing vitamin B6, tropicamide hydrochloride, and hesperidin methyl chalcone;

[0007] Step 2. Preparation of a test solution: Take Naoluotong powder, accurately weigh it, place it in a volumetric flask, add methanol, ultrasonically treat for 10 - 60 minutes, let it cool, add methanol to the scale, shake well, filter, and take the continuous filtrate to obtain the test solution;

[0008] Step 3. Content determination:

[0009] Chromatographic conditions: C18 chromatographic column; mobile phase: methanol (A) - 0.1% glacial acetic acid solution (B), gradient elution: 0 - 5 min, 96% B; 5 - 6 min, 96% - 60% B; 6 - 20 min, 60% - 50% B; flow rate 1.0 ml / min; column temperature 30 °C; detection wavelength 291 nm; injection volume 1 μL.

[0010] Preferably in the present invention, when preparing the mixed reference substance solution, first dissolve it with methanol to prepare a solution containing 0.0582, 0.2558, and 0.0764 mg of vitamin B6, tolperisone hydrochloride, and hesperidin methyl chalcone per 1 mL respectively, shake well, filter, and obtain it.

[0011] Preferably in the present invention, the specific method for preparing the test solution is: take 0.1 g of Naoluotong powder, accurately weigh it, place it in a 20 ml volumetric flask, add methanol, and ultrasonically treat it for 30 minutes at a power of 140 W and a frequency of 42 kHz.

[0012] Preferably in the present invention, a high performance liquid chromatography system or an ultra high performance liquid chromatography system is used for determination.

[0013] Preferably in the present invention, the C18 chromatographic column includes Kromasil 100 - 5 C18 chromatographic column, with a specification of 5 μm, 4.6 × 250 mm; Venusil MP C18 chromatographic column, with a specification of 5 μm, 4.6 × 250 mm; Ecosil C18 chromatographic column, with a specification of 5 μm, 4.6 × 250 mm.

[0014] The present invention also provides a method for simultaneously determining the content uniformity of tolperisone hydrochloride, hesperidin methyl chalcone, and vitamin B6 in Naoluotong capsules, and this method includes the following steps:

[0015] Step 1. Preparation of the mixed reference substance solution: Prepare a mixed reference substance solution containing vitamin B6, tolperisone hydrochloride, and hesperidin methyl chalcone.

[0016] Step 2. Preparation of the test solution: Randomly take multiple Naoluotong capsules, pour out the contents, place them in a volumetric flask, wash the capsule shells with methanol, transfer the washing solution to the volumetric flask, add methanol, ultrasonically treat it for 10 - 60 minutes, let it cool, make up the volume to the scale, shake well, filter, and take the subsequent filtrate as the test solution.

[0017] Step 3. Determination of content uniformity:

[0018] Chromatographic conditions: C18 chromatographic column; mobile phase: methanol (A) - 0.1% glacial acetic acid solution (B), gradient elution: 0 - 5 min, 96% B; 5 - 6 min, 96% - 60% B; 6 - 20 min, 60% - 50% B; flow rate 1.0 ml / min; column temperature 30 °C; detection wavelength 291 nm; injection volume 1 μL.

[0019] Preferably, the specific method for preparing the test sample solution is as follows: Randomly take 10 Naoluotong capsules, pour out the contents, and place them in a 100 ml volumetric flask; ultrasonically treat for 30 minutes at a power of 140 W and a frequency of 42 kHz.

[0020] Advantages and beneficial effects of the present invention:

[0021] (1) The present invention first realizes the synchronous detection of three components with significantly different physicochemical properties (vitamin B6, hesperidin methylchalcone, tolperisone hydrochloride) in Naoluotong capsules, and simultaneously establishes a method for determining the content uniformity of Naoluotong capsules, solving the industry pain points of the current method for Naoluotong capsules that requires separate detections and taking into account the detections of traditional Chinese and Western medicines. The detection efficiency is increased by 200%. On the premise of ensuring an appropriate peak time for vitamin B6, the baseline separation of hesperidin methylchalcone and tolperisone hydrochloride (R>1.5) is achieved. This method has good specificity, repeatability and practicability, and provides a methodological reference for the quality control research of traditional Chinese and Western medicine compound preparations.

[0022] (2) The present invention discovers that the regulation of the acidity of the mobile phase is the key influencing factor for improving the separation effect of Naoluotong capsules. It abandons the cumbersome operations such as the traditional phosphate buffer system + phosphoric acid for adjusting the pH, and through the innovation of reconstructing the mobile phase system, develops a new simplified mobile phase system (methanol - 0.1% glacial acetic acid). For Naoluotong capsules, a single acidic regulator (glacial acetic acid) is innovatively used to achieve precise pH control, and the methanol ratio is optimized to achieve the dynamic balance of the elution ability and separation degree, establishing a "simplified acidic mobile phase" design paradigm.

[0023] (3) The method provided by the present invention is not picky about instruments and columns, has good versatility, and in different laboratories or analysis scenarios, existing instruments and columns can be flexibly used, which is conducive to popularization and promotion. Moreover, it can save the time for adapting to specific instruments and columns and reduce the cost invested in specific instruments and columns.

[0024] (4) The "simplified acidic mobile phase" system proposed by the present invention significantly improves the separation efficiency and operation economy; this simplified acidic mobile phase system improves the operation simplicity, significantly improves the method robustness, optimizes the time and economy for large-scale sample detection, the hydrolysis rate of the silica matrix is controllable under acidic conditions, and no ion pair reagent is added, which prolongs the service life of the chromatographic column; in addition, it can reduce the emission of toxic reagents, conforming to the trend of green analytical chemistry; the simplified mobile phase system is more easily coupled with a mass spectrometry detector, providing a technical basis for subsequent trace analysis or metabolic research.

[0025] (5) Through the innovation of the acidic mobile phase system, the present invention overcomes the technical bottleneck of synchronous separation of components with multiple property differences, provides an efficient and low-cost solution for the quality control of traditional Chinese and Western medicine compound preparations, and has significant industrial application value. Description of the Drawings

[0026] Figure 1 It is the chromatogram of the mixed reference substance;

[0027] Figure 2 It is the chromatogram of the test sample (No. S6);

[0028] Figure 3 It is the chromatogram of the negative sample. Detailed Embodiments

[0029] The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the protection scope of the present invention.

[0030] Example 1:

[0031] 1. Materials

[0032] 1.1 Instruments: Shimadzu LC-20ADXR high-performance liquid chromatography system (Shimadzu Corporation, Japan); XPE-2015 electronic balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.); Milli-Advantuge A10 ultrapure water instrument (Millipore Corporation, USA); Vacucell22 vacuum drying oven (Memmert GmbH + Co. KG, Germany); Kromasil 100-5C18 chromatographic column (5μm, 4.6×250mm) (Kromasil AB, Sweden).

[0033] 1.2 Reagents and test drugs: Reference substances of vitamin B6 (batch number: 100116-201906, purity 99.9%) and hesperidin methylchalcone (batch number: 111580-200503, purity 100%) were purchased from the National Institutes for Food and Drug Control. The reference substance of tolperisone hydrochloride (batch number: 02M-OTY-81-4, purity 99.0%) was purchased from PANPHY Corporation, USA. 21 batches of Naoluotong capsules (No. S1-S21), among which S1-S3 were from manufacturer 1, S4-S6 were from manufacturer 2, S7-S8 were from manufacturer 3, S9-S10 were from manufacturer 4, among which S11-S12 were from manufacturer 5, S13-S14 were from manufacturer 6, S15-S17 were from manufacturer 7, S18-S19 were from manufacturer 8, S20-S21 were from manufacturer 9. Methanol was chromatographically pure (Fisher Scientific, USA); reagents such as glacial acetic acid were all of analytical grade (Sinopharm Chemical Reagent Co., Ltd.), and water was purified water.

[0034] 2. Methods and Results

[0035] 2.1 Determination of sample content

[0036] 2.1.1. Preparation of mixed reference substance solution: Weigh accurately appropriate amounts of reference substances of vitamin B6, tolperisone hydrochloride, and hesperidin methyl chalcone. Prepare a solution with methanol such that each 1 mL contains 0.0582 mg, 0.2558 mg, and 0.0764 mg of the three substances respectively. Shake well and filter to obtain the solution.

[0037] 2.1.2. Preparation of test solution: Take about 0.1 g of the powder of this product (Naoluotong), weigh accurately, place it in a 20 mL volumetric flask, add an appropriate amount of methanol, ultrasonically treat it (power 140 W, frequency 42 kHz) for 30 minutes, let it cool, add methanol to the scale, shake well, filter, and take the subsequent filtrate to obtain the solution.

[0038] 2.1.3. Preparation of negative sample solution: According to the prescription ratio (excluding vitamin B6, tolperisone hydrochloride, and hesperidin methyl chalcone), simulate the preparation process to make a negative sample lacking vitamin B6, tolperisone hydrochloride, and hesperidin methyl chalcone, and prepare it according to the method under item "2.1.2" to obtain the solution.

[0039] 2.1.4. Determination by Shimadzu LC-20ADXR high performance liquid chromatography (HPLC):

[0040] Chromatographic conditions: Kromasil 100-5 C18 chromatographic column (5 μm, 4.6×250 mm); mobile phase: methanol (A) - 0.1% glacial acetic acid solution (B), gradient elution (0 - 5 min, 96% B; 5 - 6 min, 96% - 60% B; 6 - 20 min, 60% - 50% B); flow rate 1.0 mL / min; column temperature 30 °C; detection wavelength 291 nm; injection volume 1 μL.

[0041] In addition, in this example, a reference substance stock solution was also prepared for the subsequent recovery test. The preparation method of the reference substance stock solution is as follows: Take 29.15 mg of vitamin B6 reference substance and 19.11 mg of hesperidin methyl chalcone reference substance and place them in 50 mL volumetric flasks respectively, and take 51.68 mg of tolperisone hydrochloride reference substance and place it in a 100 mL volumetric flask. Dilute them to the scale with methanol respectively to obtain vitamin B6 stock solution, hesperidin methyl chalcone stock solution, and tolperisone hydrochloride stock solution.

[0042] 2.2. Specificity test:

[0043] Prepare reference substance, test sample (No. S6), and negative sample solution according to the method under item "2.1", and conduct the determination under the chromatographic conditions in item "2.1.4". The results are shown in Figures 1 to 3 . It can be seen from this that the negative sample has no interference, indicating that this method has good specificity.

[0044] 2.3. Investigation of linear relationship:

[0045] Accurately weigh appropriate amounts of vitamin B6, tolperisone hydrochloride, and hesperidin methylchalcone reference substances, and prepare a reference substance solution with methanol to make the mass concentrations of the three substances 58.24, 535.59, and 76.44 μg / mL respectively. Then dilute it into 6 mass concentrations and determine under the chromatographic conditions in "2.1.4". Using the injection volume of each component as the abscissa (X) and the peak area of the chromatographic peak as the ordinate (Y) for regression, the results are shown in Table 1, indicating that the linear relationships of each component are good within their respective ranges.

[0046] Table 1 Linear relationships of each component

[0047]

[0048] 2.4. Precision test:

[0049] Precisely pipette the "mixed reference substance solution" and inject it repeatedly 6 times, record the peak area. The RSDs of the peak areas of vitamin B6, tolperisone hydrochloride, and hesperidin methylchalcone are 0.49%, 1.05%, and 0.63% respectively, indicating good precision of the instrument.

[0050] 2.5. Repeatability test:

[0051] Take about 0.1 g of Naoluotong Capsules (No.: S6), six portions each, prepare the test solution, inject it into the high-performance liquid chromatograph, and determine the peak area according to the above chromatographic conditions. The contents of vitamin B6, tolperisone hydrochloride, and hesperidin methylchalcone in Naoluotong Capsules are 3.754 mg / g, 94.006 mg / g, and 11.805 mg / g respectively, and the RSDs are all less than 3%, indicating good repeatability of the method.

[0052] 2.6. Recovery test:

[0053] Take 0.05 g of Naoluotong Capsules (No.: S6), six portions each, place them in 20-ml volumetric flasks, add 0.5 ml of vitamin B6 stock solution, 10.0 ml of tolperisone hydrochloride stock solution, and 1.5 ml of hesperidin methylchalcone stock solution respectively, add an appropriate amount of methanol, prepare the test solution according to "2.1.2" and analyze it under the chromatographic conditions of "2.1.4", and calculate the recovery rate and RSD. The average recovery rates of vitamin B6, tolperisone hydrochloride, and hesperidin methylchalcone in Naoluotong Capsules are 102.1%, 101.8%, and 99.3% respectively, and the RSDs are 1.6%, 2.6%, and 2.2% respectively, indicating that the recovery rates meet the requirements.

[0054] 2.7. Stability test: Take 1 portion of the test solution under item "2.1.2", inject and determine it under the chromatographic conditions of item "2.1.4" at 0, 2, 5, 8, 10, 14, 17, 20, and 24 h at room temperature. The RSDs of the peak areas of vitamin B6, tolperisone hydrochloride, and hesperidin methyl chalcone are 2.12%, 2.26%, and 2.32% respectively, indicating that the solution has good stability within 24 h.

[0055] 2.8. Determination of sample content:

[0056] Take appropriate amounts of 21 batches of samples from 9 production enterprises, prepare the test solution according to the method under item "2.1.2", inject and determine it under the chromatographic conditions of item "2.1.4", and calculate the content. The results are shown in Table 2.

[0057] Table 2 Results of sample determination / (mg / capsule)

[0058]

[0059] At present, domestic hesperidin methyl chalcone raw materials are prepared from natural hesperidin as raw materials and methylated to obtain a flavonoid mixture mainly composed of hesperidin methyl chalcone. In addition to hesperidin methyl chalcone, it also includes other flavonoid components. The average content of hesperidin methyl chalcone is 61% (by area normalization method). Calculated based on a 20% fluctuation on the basis of the average content in the raw material drug and converted into the preparation, the limit of hesperidin methyl chalcone (C29H36O15) is 4.8 - 7.3 mg. Referring to the limits of vitamin B6 and tolperisone hydrochloride under the national drug standard WS3-B-3667-98-3 Naoluotong Capsules of the State Food and Drug Administration of China, that is, each capsule of this product should contain 1.7 - 2.3 mg of vitamin B6 (C8H11NO3·HCl) and 45 - 55 mg of tolperisone hydrochloride (C16H23NO·HCl). There are significant differences among different manufacturers of the 21 batches of samples in this example. Among the 21 batches of samples, 3 batches of vitamin B6 are unqualified, all batches of tolperisone hydrochloride are qualified, and 5 batches of hesperidin methyl chalcone are unqualified.

[0060] 2.9. Content uniformity inspection:

[0061] 2.9.1. The chromatographic conditions are the same as those in "2.1.4"

[0062] 2.9.2. Preparation of the reference solution is the same as that in "2.1.1"

[0063] 2.9.3. Preparation of the test solution: Randomly take 10 capsules, carefully pour out the contents respectively, place them in a 100 ml volumetric flask, quickly wash the capsule shells with methanol, transfer the washing solution to the volumetric flask, add an appropriate amount of methanol, ultrasonically treat (power 140 W, frequency 42 kHZ) for 30 minutes, let it cool, make up to the mark, shake well, filter, and take the subsequent filtrate as the test solution.

[0064] Results of sample determination:

[0065] An appropriate amount of 21 batches of samples from 9 manufacturing enterprises was taken, and the test solution was prepared according to the method under item "2.9.3". The sample was injected and determined under the chromatographic conditions of item "2.1.4", and the content uniformity was calculated. The results are shown in Table 3.

[0066] Table 3 Results of content uniformity determination

[0067]

[0068]

[0069] In the present invention, 21 batches of samples were determined. Referring to the content uniformity inspection method of General Chapter 0941 in the Fourth Part of Chinese Pharmacopoeia 2020 Edition, the relative content of each single dose with the labeled amount as 100 was calculated, and the content uniformity was judged by the results of A + 2.2S and A + S (A is the absolute value of the difference between the labeled amount and the mean value; S is the standard deviation). It is recommended that the L value of tolperisone hydrochloride and hesperidin methylchalcone be 15. Since the content of vitamin B6 in the capsule is relatively low, it is recommended that the L value be 20. Referring to the limit of the national drug standard WS3-B-3667-98-3 of the State Food and Drug Administration, the content of vitamin B6 should be 85.0% - 115.0% of the labeled amount, the content of tolperisone hydrochloride should be 90.0% - 110.0% of the labeled amount, and the content of hesperidin methylchalcone should be 85.0% - 115.0% of the labeled amount (the content uniformity of hesperidin methylchalcone is based on the result of content determination as the labeled amount). Among the 21 batches of samples, the qualified batches of content uniformity of vitamin B6 were 15 batches, the qualified batches of content uniformity of tolperisone hydrochloride were 15 batches, the qualified batches of content uniformity of hesperidin methylchalcone were 20 batches, and the batches qualified for all the above three indicators were 12 batches.

[0070] Thus, in order to effectively control the quality of Naoluotong Capsules, the content determination and content uniformity inspection of vitamin B6, tolperisone hydrochloride, and hesperidin methylchalcone should be carried out.

[0071] Example 2:

[0072] The Thermo Fisher U3000 ultra-high performance liquid chromatography system (Thermo Fisher Scientific (China) Co., Ltd.) was used to replace the Shimadzu LC-20ADXR high performance liquid chromatography system (Shimadzu Corporation, Japan) for determination. The chromatographic conditions were the same as those in Example 1. The results showed that the content determination of vitamin B6, tolperisone hydrochloride, and hesperidin methylchalcone could also be achieved under the above chromatographic conditions. In addition, the Dionex U3000 high performance liquid chromatography system and the Agilent 1260 high performance liquid chromatography system could also achieve the content determination of vitamin B6, tolperisone hydrochloride, and hesperidin methylchalcone.

[0073] Embodiment 3:

[0074] A Venusil MP C18 chromatographic column (5 μm, 4.6×250 mm) (Agela, USA) was used to replace the Kromasil 100-5C18 chromatographic column. Other chromatographic conditions were the same as those in Example 1. The results showed that the contents of vitamin B6, tolperisone hydrochloride, and methyl hesperidin could also be determined under the chromatographic conditions.

[0075] Embodiment 4:

[0076] An Ecosil C18 column (5 μm, 4.6×250 mm) (Guangzhou Green Baicao Scientific Instrument Co., Ltd.) was used to replace the Kromasil100-5C18 column. Other chromatographic conditions were the same as those in Example 1. The results showed that the contents of vitamin B6, tolperisone hydrochloride, and methyl hesperidin could also be determined under these chromatographic conditions.

[0077] It can be seen from Examples 1 to 5 that the method provided by the present invention has good versatility and can flexibly adopt existing instruments and columns in different laboratories or analysis scenarios, which is conducive to popularization and promotion.

[0078] Comparative Example 1:

[0079] The mobile phase was adjusted to: acetonitrile-0.2% triethylamine (25:75) (pH adjusted to 3.5±0.1 with phosphoric acid), and other chromatographic conditions were the same as in Example 1.

[0080] Comparative Example 2:

[0081] The mobile phase was adjusted to: acetonitrile-0.05 mol / L potassium dihydrogen phosphate (5 ml of triethylamine was added to every 1 L, and the pH was adjusted to 3.0 with phosphoric acid) (25:75). The other chromatographic conditions were the same as those in Example 1.

[0082] Comparative Example 3:

[0083] The mobile phase was adjusted to: methanol-0.4% sodium dodecyl sulfate (containing 0.2% acetic acid) (5:95), and other chromatographic conditions were the same as in Example 1.

[0084] Under the mobile phases of Comparative Examples 1 to 3 above, there are multiple problems such as separation defects due to overlapping of vitamin B6 and solvent peaks, difficulty in coordinating the elution times of multiple components in a complex ingredient system, and contradiction between the complexity and stability of mobile phase preparation.

[0085] Other experiments were also conducted on the selection of the mobile phase in the present invention, and no further examples will be given in this application. Through summarizing and analyzing the failed experiments, it was unexpectedly found that the regulation of the acidity of the mobile phase is the key influencing factor for improving the separation effect of Naoluotong Capsules; therefore, the cumbersome operations such as the traditional phosphate buffer system + phosphoric acid for pH adjustment were boldly abandoned, the mobile phase system was reconstructed and innovated, and a new simplified mobile phase system (methanol - 0.1% glacial acetic acid) was developed. For Naoluotong Capsules, a single acidic regulator (glacial acetic acid) was innovatively used to achieve precise pH control, and the methanol ratio was optimized to achieve the dynamic balance of elution ability and resolution, establishing a "simplified acidic mobile phase" design paradigm. In addition, the technical solution for controlling the peak emergence time of vitamin B6 can be extended to the quality control of other traditional Chinese and Western medicine compound preparations containing components with polar differences.

[0086] On the basis of the above research, the present invention investigated the volume flow rate (0.8, 1.0, 1.2 mL / min), column temperature (25, 30, 35 °C), gradient elution conditions, etc., and determined the optimal parameters according to the resolution and analysis time (volume flow rate 1.0 ml / min; column temperature 30 °C; 0 - 5 min, 96% B; 5 - 6 min, 96% - 60% B; 6 - 20 min, 60% - 50% B).

[0087] In addition, when preparing the test solution, the present invention also optimized the extraction solvent (methanol, 60% methanol, water) and extraction method (ultrasonic for 10 minutes, ultrasonic for 30 minutes, ultrasonic for 60 minutes, heating under reflux for 30 minutes), and determined the optimal scheme (methanol, ultrasonic for 30 minutes) based on the detected amount of each component and the simplicity of operation.

[0088] The present invention first realized the synchronous detection of three components with significantly different physicochemical properties (vitamin B6, hesperidin methyl chalcone, tropicamide hydrochloride) in Naoluotong Capsules, and simultaneously established a method for determining the content uniformity of Naoluotong Capsules, solving the industry pain points of the current method for Naoluotong Capsules, which requires separate detections and takes into account the detections of traditional Chinese and Western medicines. The detection efficiency was increased by 200%. On the premise of ensuring an appropriate peak emergence time for vitamin B6, the baseline separation of hesperidin methyl chalcone and tropicamide hydrochloride was achieved (R > 1.5). This method has good specificity and practicability, and provides a methodological reference for the quality control research of traditional Chinese and Western medicine compound preparations.

[0089] The above are the preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for simultaneously determining the contents of tolperisone hydrochloride, methyl hesperidin and vitamin B6 in Naoluotong capsules, characterized in that: The method comprises the following steps: Step 1. Preparation of mixed reference solution: preparing a mixed reference solution containing vitamin B6, tolperisone hydrochloride, and methyl hesperidin; Step 2. Preparation of test solution: Take Naoluotong powder, accurately weigh it, put it in a volumetric bottle, add methanol, ultrasonically treat it for 10-60 minutes, cool it, add methanol to the scale, shake it well, filter it, and take the filtrate to obtain the solution; Step 3. Assay: Chromatographic conditions: C18 chromatographic column; mobile phase methanol (A)-0.1% glacial acetic acid solution (B), gradient elution: 0-5 min, 96% B; 5-6 min, 96%-60% B; 6-20 min, 60%-50% B; volume flow rate 1.0 ml / min; column temperature 30°C; detection wavelength 291 nm; injection volume 1 μL.

2. A method for simultaneously determining the contents of tolperisone hydrochloride, methyl hesperidin and vitamin B6 in Naoluotong capsules according to claim 1, characterized in that: When preparing the mixed reference solution, first dissolve it with methanol to make a solution containing 0.0582 mg, 0.2558 mg, and 0.0764 mg of vitamin B6, tolperisone hydrochloride, and methyl hesperidin per 1 mL, respectively. Shake well, filter, and obtain the solution.

3. A method for simultaneously determining the contents of tolperisone hydrochloride, methyl hesperidin and vitamin B6 in Naoluotong capsules according to claim 1, characterized in that: The specific method for preparing the test solution is as follows: take 0.1 g of Naoluotong powder, accurately weigh it, put it in a 20 ml volumetric flask, add methanol, and ultrasonically treat it at a power of 140 W and a frequency of 42 kHz for 30 minutes.

4. A method for simultaneously determining the contents of tolperisone hydrochloride, methyl hesperidin and vitamin B6 in Naoluotong capsules according to claim 1, characterized in that: A high performance liquid chromatography system or an ultra high performance liquid chromatography system is used for content determination.

5. A method for simultaneously determining the contents of Tolperisone Hydrochloride, Methyl Hesperidin and Vitamin B6 in Naoluotong Capsules according to claim 1, characterized in that: The C18 chromatographic column includes a Kromasil 100-5C18 chromatographic column with a specification of 5 μm and 4.6×250 mm; a Venusil MP C18 chromatographic column with a specification of 5 μm and 4.6×250 mm; and an Ecosil C18 chromatographic column with a specification of 5 μm and 4.6×250 mm.

6. A method for simultaneously determining the uniformity of the contents of tolperisone hydrochloride, methyl hesperidin and vitamin B6 in Naoluotong capsules, characterized in that: The method comprises the following steps: Step 1. Preparation of mixed reference solution: preparing a mixed reference solution containing vitamin B6, tolperisone hydrochloride, and methyl hesperidin; Step 2. Preparation of test solution: randomly take multiple Naoluotong capsules, pour out the contents, place in a volumetric flask, wash the capsule shell with methanol, transfer the washing solution to a volumetric flask, add methanol, ultrasonically treat for 10-60 minutes, cool, dilute to the scale, shake well, filter, and take the filtrate as the test solution; Step 3. Content uniformity determination: Chromatographic conditions: C18 chromatographic column; mobile phase methanol (A)-0.1% glacial acetic acid solution (B), gradient elution: 0-5 min, 96% B; 5-6 min, 96%-60% B; 6-20 min, 60%-50% B; volume flow rate 1.0 ml / min; column temperature 30°C; detection wavelength 291 nm; injection volume 1 μL.

7. A method for simultaneously determining the uniformity of the contents of Tolperisone Hydrochloride, Methyl Hesperidin and Vitamin B6 in Naoluotong Capsules according to claim 6, characterized in that: The specific method for preparing the test solution is: randomly take 10 Naoluotong capsules, pour out the contents, and place them in a 100 ml volumetric flask; ultrasonically treat them at a power of 140 W and a frequency of 42 kHz for 30 minutes.

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