A method for simultaneously determining the contents of tropisetron hydrochloride, methyl hesperidin and vitamin B6 in Naolvtong capsules

Abstract

The application belongs to the field of pharmaceutical analysis, and discloses a method for simultaneously determining the contents of tropisetron hydrochloride, methyl hesperidin and vitamin B6 in Naolvtong capsules, which comprises three steps of mixed control solution preparation, sample solution preparation and content determination, and the chromatographic conditions for the content determination are as follows: a C18 chromatographic column; a mobile phase of methanol (A)-0.1% glacial acetic acid solution (B), gradient elution: 96% B at 0-5 min, 96%-60% B at 5-6 min, 60%-50% B at 6-20 min, a volume flow rate of 1.0 ml / min, a column temperature of 30 DEG C, a detection wavelength of 291 nm, and a sample injection amount of 1 muL. The method can realize the synchronous detection of three components (tropisetron hydrochloride, methyl hesperidin and vitamin B6) with significant differences in physicochemical properties, and the chromatographic conditions are simple and the detection speed is fast.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis, and more specifically, relates to a method for simultaneously determining the contents of tolperidone hydrochloride, methyl hesperidin and vitamin B6 in Naoluotong capsules. Background Technology

[0002] With the increasing aging of the population, cerebrovascular diseases have become the second leading cause of death worldwide. Naoluotong capsules, a commonly used clinical compound preparation of traditional Chinese and Western medicine, are composed of Danshen extract, tolperidone hydrochloride, Chuanxiong extract, methyl hesperidin, Astragalus extract, and vitamin B6. They have the effect of dilating blood vessels and increasing cerebral blood flow, and are used for various cerebrovascular diseases such as cerebral thrombosis, cerebral arteriosclerosis, and sequelae of stroke, as well as headaches, dizziness, hemiplegia, limb numbness, and neurasthenia caused by qi deficiency and blood stasis. They play an important role in the treatment of cerebral infarction and cerebral arteriosclerosis. However, there are many current standards for Naoluotong capsules, and the quality control indicators of each standard are inconsistent, leading to significant differences in products from different companies. Currently, the existing standards for tolperidone hydrochloride all use chloroform extraction and non-aqueous titration to determine the content. Chloroform is a highly toxic and volatile solvent, and the sample pretreatment filtration speed is slow. The colors of products from different companies are inconsistent, making it difficult to effectively determine the titration endpoint, further affecting the accuracy of experimental results and the physical and mental health of experimental personnel. Previous reports on improving the quality standards of Naoluotong capsules are few, and those that exist only measure the content of a single indicator, making it difficult to comprehensively reflect the quality of the preparation. For example, the HPLC determination of topochlorite hydrochloride content in Naoluotong capsules published by Ma Zhenghong et al. used an ODS column as the stationary phase, methanol-water-triethylamine (160:39:1, pH adjusted to 7.5 with glacial acetic acid) as the mobile phase, and a detection wavelength of 261 nm. Guo Dongyan et al.'s HPLC determination of topochlorite hydrochloride content in Naoluotong capsules used an ODS C18 column (5 μm, 4.6 mm × 250 mm), acetonitrile-0.05 mol / L potassium dihydrogen phosphate (30:70) (pH adjusted to 3.0 with phosphoric acid) as the mobile phase, a detection wavelength of 256 nm, a flow rate of 1.0 ml / min, and a column temperature of room temperature. Zhang Chunyu et al. published "Determination of Methylhesperidin in Naoluotong Capsules by High Performance Liquid Chromatography," using a Zorbax C18 column (4.6×250mm, 5μm); mobile phase: methanol-0.3% glacial acetic acid (38:62); detection wavelength: 283nm; column temperature: 40℃. Su Mingwu et al. published "Determination of Vitamin B6 in Naoluotong Capsules by High Performance Liquid Chromatography," using a Dikma C18 column (250mm×4.6mm, 5μm); mobile phase: methanol-0.06% sodium heptanesulfonate aqueous solution (containing 1.2% glacial acetic acid and 0.12% triethylamine) (13:87); detection wavelength: 290nm; column temperature: 30℃.As the quality control of traditional Chinese medicine (TCM) gradually shifts from single-component to multi-component overall control, quantitative analysis of multiple indicators has become another approach to TCM quality evaluation. Li Jianming et al. published "Simultaneous determination of the contents of tolperidone hydrochloride and methyl hesperidin in Naoluotong capsules by HPLC". The chromatographic column was Shim-pack VP-ODS acetonitrile-0.025mol·L⁻¹ potassium dihydrogen phosphate (with 5mL of triethylamine added per 1L and pH adjusted to 3.0 with phosphoric acid) (25:75) as the mobile phase, the flow rate was 1.0mL·min⁻¹, the column temperature was 30℃, and the detection wavelengths were 261 and 283nm. Due to the significant differences in the physicochemical properties of components such as tolperone hydrochloride, methyl hesperidin, and vitamin B6 in Naoluotong capsules, for example, tolperone hydrochloride is prone to peak tailing in conventional mobile phases due to residual silanol groups, usually requiring the addition of additional chemical reagents to neutralize the activity of silanol groups; methyl hesperidin is a weakly acidic flavonoid glycoside, which is prone to peak broadening or bifurcation due to dissociation in non-ion-pair systems; vitamin B6 is a water-soluble vitamin with a high degree of protonation under acidic conditions, requiring pH optimization to balance its retention time and separation selectivity. This results in complex chromatographic conditions for determining multiple components in Naoluotong capsules, and makes it impossible to simultaneously determine the content of tolperone hydrochloride, methyl hesperidin, and vitamin B6. Summary of the Invention

[0003] In view of the shortcomings of the prior art, the purpose of this invention is to provide a method for simultaneously determining the content of tolperone hydrochloride, methyl hesperidin and vitamin B6 in Naoluotong capsules. This method can achieve the simultaneous detection of three components with significantly different physicochemical properties (tolperone hydrochloride, methyl hesperidin and vitamin B6), and the chromatographic conditions are simple and the detection speed is fast.

[0004] To achieve the above objectives, the present invention adopts the following technical solution:

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

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

[0007] Step 2. Preparation of test solution: Accurately weigh the Naoluotong powder, place it in a volumetric flask, add methanol, sonicate for 10-60 minutes, cool, add methanol to the mark, shake well, filter, and take the filtrate to obtain the test solution;

[0008] Step 3. Content determination:

[0009] Chromatographic conditions: C18 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] As a preferred embodiment of the present invention, the mixed reference solution is first dissolved in methanol to prepare a solution containing 0.0582 mg of vitamin B6, 0.2558 mg of tolperidone hydrochloride, and 0.0764 mg of methyl hesperidin per 1 mL, respectively. The solution is then shaken, filtered, and the solution is obtained.

[0011] As a preferred embodiment of the present invention, the specific method for preparing the test solution is as follows: take 0.1g of Naoluotong powder, accurately weigh it, place it in a 20ml volumetric flask, add methanol, and sonicate it for 30 minutes at a power of 140W and a frequency of 42kHZ.

[0012] As a preferred embodiment of the present invention, a high-performance liquid chromatography (HPLC) system or an ultra-high-performance liquid chromatography (UHPLC) system is used for the determination.

[0013] As a preferred embodiment of the present invention, the C18 chromatographic column includes a Kromasil 100-5 C18 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; or an Ecosil C18 chromatographic column with a specification of 5 μm and 4.6 × 250 mm.

[0014] This invention also provides a method for simultaneously determining the uniformity of the contents of tolperidone hydrochloride, methyl hesperidin, and vitamin B6 in Naoluotong capsules, the method comprising the following steps:

[0015] Step 1. Preparation of mixed reference solution: Prepare a mixed reference solution containing vitamin B6, tolperidone hydrochloride, and methyl hesperidin;

[0016] Step 2. Preparation of 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 a volumetric flask, add methanol, sonicate for 10-60 minutes, cool, make up to the mark, shake well, filter, and take the filtrate as the test solution.

[0017] Step 3. Content uniformity determination:

[0018] Chromatographic conditions: C18 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] As a preferred embodiment of the present invention, the specific method for preparing the test solution is as follows: randomly take 10 Naoluotong capsules, pour out the contents, and place them in a 100ml volumetric flask; sonicate at a power of 140W and a frequency of 42kHz for 30 minutes.

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

[0021] (1) This invention achieves, for the first time, the simultaneous detection of three components with significantly different physicochemical properties (vitamin B6, methyl hesperidin, and tolperidone hydrochloride) in Naoluotong capsules, and simultaneously establishes a method for determining the content uniformity of Naoluotong capsules. This addresses the industry pain points of current methods requiring multiple detections and balancing the detection of both traditional Chinese and Western medicines, improving detection efficiency by 200%. While ensuring an appropriate peak time for vitamin B6, baseline separation of methyl hesperidin and tolperidone hydrochloride is achieved (R>1.5). This method exhibits good specificity, repeatability, and practicality, providing a methodological reference for quality control research of compound preparations of traditional Chinese and Western medicines.

[0022] (2) This invention discovers that the acidity regulation of the mobile phase is a key influencing factor for improving the separation effect of Naoluotong capsules. It abandons the cumbersome operation of traditional phosphate buffer system + phosphoric acid to adjust pH, and develops a new simplified mobile phase system (methanol-0.1% glacial acetic acid) by reconstructing and innovating the mobile phase system. It innovatively uses a single acid regulator (glacial acetic acid) to achieve precise pH control for Naoluotong capsules, optimizes the methanol ratio to achieve dynamic balance between elution capacity and separation degree, and establishes a "simplified acidic mobile phase" design paradigm.

[0023] (3) The method provided by the present invention is not picky about instruments or columns, and has good versatility. It can flexibly adopt existing instruments and columns in different laboratories or analytical scenarios, which is conducive to popularization and promotion. Moreover, it can save 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 in this invention significantly improves separation efficiency and operational economy. This simplified acidic mobile phase system improves ease of operation and significantly enhances the robustness of the method. For large-scale sample detection, time and economy are optimized. The hydrolysis rate of the silica matrix under acidic conditions is controllable, and no ion-pairing reagents are added, which extends the service life of the chromatographic column. In addition, it can reduce the emission of toxic reagents, which is in line with the trend of green analytical chemistry. The simplified mobile phase system is easier to couple with mass spectrometry detectors, providing a technical basis for subsequent trace analysis or metabolic research.

[0025] (5) This invention overcomes the technical bottleneck of simultaneous separation of multiple components with different properties through the innovation of acidic mobile phase system, providing an efficient and low-cost solution for the quality control of compound preparations of Chinese and Western medicines, and has significant industrial application value. Attached Figure Description

[0026] Figure 1 The chromatogram is for the mixed reference standards;

[0027] Figure 2 The chromatogram of the test sample (S6);

[0028] Figure 3 The chromatogram is for a negative sample. Detailed Implementation

[0029] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto. It should be particularly noted that all similar substitutions and modifications will be obvious to those skilled in the art, and they are all considered to be included within the scope of protection 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 system (Millipore Corporation, USA); Vacucell 22 vacuum drying oven (Memmert GmbH, Germany); Kromasil 100-5C18 column (5μm, 4.6×250mm) (Kromasil GmbH, Sweden).

[0033] 1.2. Reagents and Tests: Vitamin B6 (batch number: 100116-201906, purity 99.9%) and methyl hesperidin (batch number: 111580-200503, purity 100%) reference standards were purchased from the China National Institutes for Food and Drug Control. Tolperidone hydrochloride (batch number: 02M-OTY-81-4, purity 99.0%) reference standard was purchased from PANPHY Corporation, USA. 21 batches of Naoluotong capsules (numbers S1-S21) were produced, with S1-S3 from manufacturer 1, S4-S6 from manufacturer 2, S7-S8 from manufacturer 3, and S9-S10 from manufacturer 4; S11-S12 from manufacturer 5, S13-S14 from manufacturer 6, S15-S17 from manufacturer 7, S18-S19 from manufacturer 8, and S20-S21 from manufacturer 9. Methanol was chromatographically pure (Fisher Laboratories, USA); glacial acetic acid and other reagents were analytically pure (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 solution: Accurately weigh appropriate amounts of vitamin B6, tolperidone hydrochloride, and methyl hesperidin reference standards, and prepare a solution containing 0.0582, 0.2558, and 0.0764 mg of each of the three in methanol per mL. Shake well, filter, and the solution is ready.

[0037] 2.1.2. Preparation of test solution: Weigh about 0.1g of this product (Naoluotong) powder accurately, place it in a 20ml volumetric flask, add an appropriate amount of methanol, sonicate (power 140W, frequency 42kHZ) for 30 minutes, cool, add methanol to the mark, shake well, filter, and take the filtrate to obtain the test solution.

[0038] 2.1.3. Preparation of negative sample solution: Prepare a negative sample lacking vitamin B6, tolperidone hydrochloride, and methyl hesperidin by simulating the preparation process according to the prescription ratio (excluding vitamin B6, tolperidone hydrochloride, and methyl hesperidin), and prepare it according to the method under "2.1.2".

[0039] 2.1.4. Shimadzu LC-20ADXR High Performance Liquid Chromatography (HPLC) Determination:

[0040] Chromatographic conditions: Kromasil 100-5C18 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, this embodiment also prepared reference stock solutions for subsequent recovery tests. The preparation method of the reference stock solutions is as follows: 29.15 mg of vitamin B6 reference standard and 19.11 mg of methyl hesperidin reference standard were placed in 50 ml volumetric flasks, and 51.68 mg of tolperidone hydrochloride reference standard was placed in 100 ml volumetric flasks. Methanol was added to dilute to the mark to prepare the vitamin B6 stock solution, methyl hesperidin stock solution, and tolperidone hydrochloride stock solution.

[0042] 2.2. Specificity test:

[0043] The reference standard, test sample (S6), and negative sample solution were prepared according to the method in section "2.1", and determined under the chromatographic conditions in section "2.1.4". The results are shown in [Figure 1]. Figures 1 to 3 Therefore, the absence of interference in negative samples indicates that this method has good specificity.

[0044] 2.3. Linear Relationship Examination:

[0045] Accurately weigh appropriate amounts of vitamin B6, tolperidone hydrochloride, and methyl hesperidin reference standards, and prepare reference standard solutions in methanol to achieve mass concentrations of 58.24, 535.59, and 76.44 μg / mL, respectively. These solutions were then diluted to six different mass concentrations and determined under the chromatographic conditions described in section "2.1.4". Regression was performed with the injection amount of each component as the abscissa (X) and the peak area as the ordinate (Y). The results are shown in Table 1, indicating that each component exhibits good linearity within its respective range.

[0046] Table 1 Linear Relationships of Each Component

[0047]

[0048] 2.4. Precision test:

[0049] The mixed reference solution was precisely pipetted and injected six times. The peak areas were recorded, and the RSDs of the peak areas of vitamin B6, tolperidone hydrochloride, and methyl hesperidin were 0.49%, 1.05%, and 0.63%, respectively, indicating that the instrument has good precision.

[0050] 2.5. Repeatability Test:

[0051] Approximately 0.1 g of Naoluotong capsules (product code: S6) was used to prepare test solutions in six portions. These solutions were injected into a high-performance liquid chromatograph (HPLC) and the peak areas were determined under the chromatographic conditions described above. The contents of vitamin B6, tolperidone hydrochloride, and methyl hesperidin in the Naoluotong capsules were 3.754 mg / g, 94.006 mg / g, and 11.805 mg / g, respectively, with RSDs all less than 3%, indicating good reproducibility of the method.

[0052] 2.6. Recovery test:

[0053] Take 0.05g of Naoluotong capsules (product code: S6), divide into 6 portions, and place them in 20ml volumetric flasks. Add 0.5ml of vitamin B6 stock solution, 10.0ml of tolperidone hydrochloride stock solution, and 1.5ml of methyl hesperidin stock solution to each flask. Add an appropriate amount of methanol, prepare the test solution according to "2.1.2", and analyze it according to the chromatographic conditions in "2.1.4". Calculate the recovery rate and RSD. The average recoveries of vitamin B6, tolperidone hydrochloride, and methyl hesperidin in Naoluotong capsules were 102.1%, 101.8%, and 99.3%, respectively, with RSDs of 1.6%, 2.6%, and 2.2%, respectively. The results indicate that the recovery rate meets the requirements.

[0054] 2.7. Stability test: Take one part of the test solution under section “2.1.2” and inject it at room temperature for 0, 2, 5, 8, 10, 14, 17, 20 and 24 h under the chromatographic conditions of section “2.1.4”. The peak areas RSD of vitamin B6, tolperidone hydrochloride and methyl hesperidin were found to be 2.12%, 2.26% and 2.32%, respectively, indicating that the solution has good stability within 24 h.

[0055] 2.8. Sample content determination:

[0056] Take appropriate amounts of samples from 21 batches of products from 9 manufacturers, prepare test solutions according to the method in section "2.1.2", and determine the content under the chromatographic conditions in section "2.1.4". The results are shown in Table 2.

[0057] Table 2 Sample Measurement Results (mg / capsule)

[0058]

[0059] Currently, domestically produced methyl hesperidin raw materials are made from naturally derived hesperidin, which is methylated to obtain a mixture of flavonoids, primarily methyl hesperidin. Besides methyl hesperidin, it also includes other flavonoid components. The average content of methyl hesperidin is 61% (based on area normalization). Adjusting for a 20% fluctuation based on the average content of the raw material and applying it to the formulation, the limit for methyl hesperidin (C29H36O15) is 4.8–7.3 mg. Referring to the limits for vitamin B6 and tolperidone hydrochloride under the National Drug Standard WS3-B-3667-98-3 for Naoluotong Capsules issued by the State Food and Drug Administration, each capsule of this product should contain 1.7–2.3 mg of vitamin B6 (C8H11NO3·HCl) and 45–55 mg of tolperidone hydrochloride (C16H23NO·HCl). The 21 batches of samples in this example showed significant differences between different manufacturers. Of the 21 batches of samples, 3 batches of vitamin B6 were substandard, all batches of toperazine hydrochloride were qualified, and 5 batches of methyl hesperidin were substandard.

[0060] 2.9. Content uniformity check:

[0061] 2.9.1. Chromatographic conditions are the same as in "2.1.4".

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

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

[0064] Sample measurement results:

[0065] Take appropriate amounts of samples from 21 batches of products from 9 manufacturers, prepare test solutions according to the method in section "2.9.3", and determine the content uniformity under the chromatographic conditions in section "2.1.4". The results are shown in Table 3.

[0066] Table 3 Results of Content Uniformity Measurement

[0067]

[0068]

[0069] In this invention, 21 batches of samples were tested. Referring to the content uniformity test method in Chapter 0941 of the 2020 edition of the Chinese Pharmacopoeia, the relative content of each single dose was calculated with the labeled amount as 100. The content uniformity was judged using the results of A+2.2S and A+S (A is the absolute value of the difference between the labeled amount and the mean; S is the standard deviation). It is recommended that the L value for tolperone hydrochloride and methyl hesperidin be 15. Because the content of vitamin B6 in capsules is low, an L value of 20 is recommended. Referring to the limits of the National Drug Standard WS3-B-3667-98-3 issued by the State Food and Drug Administration, the content of vitamin B6 should be 85.0%–115.0% of the labeled amount, the content of tolperone hydrochloride should be 90.0%–110.0% of the labeled amount, and the content of methyl hesperidin should be 85.0%–115.0% of the labeled amount (the content uniformity of methyl hesperidin is based on the content determination result as the labeled amount). Of the 21 batches of samples, 15 batches met the uniformity requirements for vitamin B6 content, 15 batches met the uniformity requirements for tolperidone hydrochloride content, 20 batches met the uniformity requirements for methyl hesperidin content, and 12 batches met all three requirements.

[0070] Therefore, in order to effectively control the quality of Naoluotong capsules, the content of vitamin B6, tolperidone hydrochloride, and methyl hesperidin should be determined and the content uniformity should be checked.

[0071] Example 2:

[0072] The Shimadzu LC-20ADXR HPLC system (Shimadzu Corporation, Japan) was replaced with a Thermo Fisher U3000 ultra-high performance liquid chromatography (UHPLC) system (Thermo Fisher Scientific (China) Co., Ltd.) for determination, under the same chromatographic conditions as in Example 1. The results showed that the determination of vitamin B6, tolperone hydrochloride, and methyl hesperidin under the same chromatographic conditions was also achievable. Furthermore, the determination of vitamin B6, tolperone hydrochloride, and methyl hesperidin was also achievable using a Dionex U3000 HPLC system and an Agilent 1260 HPLC system.

[0073] Example 3:

[0074] The Kromasil 100-5C18 column was replaced with a Venusil MP C18 column (5μm, 4.6×250mm) (Agela, USA), and other chromatographic conditions were the same as in Example 1. The results showed that the content of vitamin B6, tolperidone hydrochloride, and methyl hesperidin could also be determined under these chromatographic conditions.

[0075] Example 4:

[0076] The Kromasil 100-5C18 column was replaced with an Ecosil C18 column (5μm, 4.6×250mm) (Guangzhou Lvbaicao Scientific Instruments Co., Ltd.), and other chromatographic conditions were the same as in Example 1. The results showed that the content of vitamin B6, tolperidone hydrochloride, and methyl hesperidin could also be determined under these chromatographic conditions.

[0077] As can be seen from Examples 1 to 5, the method provided by the present invention has good versatility and can flexibly adopt existing instruments, equipment and columns in different laboratories or analytical 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 as described in Example 1.

[0080] Comparative Example 2:

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

[0082] Comparative Example 3:

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

[0084] Under the mobile phases of the above comparative examples 1 to 3, there are multiple problems, such as the separation defect of vitamin B6 and solvent peak overlap, the difficulty in coordinating the peak elution time of multiple components in complex component systems, and the contradiction between the complexity and stability of mobile phase preparation.

[0085] This invention also conducted other experiments on mobile phase selection, which will not be listed here. Through summarizing and analyzing failed experiments, it was unexpectedly discovered that the acidity control of the mobile phase is a key influencing factor for improving the separation effect of Naoluotong capsules. Therefore, the traditional phosphate buffer system and the cumbersome operation of adjusting pH with phosphoric acid 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 a dynamic balance between elution capacity and separation degree, establishing a "simplified acidic mobile phase" design paradigm. In addition, the peak time control technology for vitamin B6 can be extended to the quality control of other Chinese and Western medicine compound prescriptions containing components with different polarities.

[0086] Based on the above research, this invention investigated the volumetric flow rate (0.8, 1.0, 1.2 mL / min), column temperature (25, 30, 35℃), gradient elution conditions, etc., and determined the optimal parameters (volumetric flow rate 1.0 mL / min; column temperature 30℃; 0–5 min, 96% B; 5–6 min, 96%–60% B; 6–20 min, 60%–50% B) according to the resolution and analysis time.

[0087] In addition, the present invention has optimized the extraction solvent (methanol, 60% methanol, water) and extraction method (sonication for 10 minutes, sonication for 30 minutes, sonication for 60 minutes, and heating under reflux for 30 minutes) in the preparation of the test solution. The optimal scheme (methanol, sonication for 30 minutes) was determined based on the detection amount of each component and the ease of operation.

[0088] This invention marks the first time that three components with significantly different physicochemical properties (vitamin B6, methyl hesperidin, and tolperidone hydrochloride) in Naoluotong capsules have been simultaneously detected. A method for determining the content uniformity of Naoluotong capsules is also established simultaneously, addressing the industry pain points of current methods requiring multiple detections and balancing the detection of both traditional Chinese and Western medicines. The detection efficiency is improved by 200%, and baseline separation of methyl hesperidin and tolperidone hydrochloride (R>1.5) is achieved while ensuring an appropriate peak time for vitamin B6. This method exhibits good specificity and practicality, providing a methodological reference for quality control research of traditional Chinese and Western medicine compound preparations.

[0089] The above description is a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for simultaneously determining the contents of tolperidone hydrochloride, methyl hesperidin, and vitamin B6 in Naoluotong capsules, characterized in that, The method includes the following steps: Step 1. Preparation of mixed reference solution: Prepare a mixed reference solution containing vitamin B6, tolperidone hydrochloride, and methyl hesperidin; Step 2. Preparation of test solution: Accurately weigh the Naoluotong powder, place it in a volumetric flask, add methanol, sonicate for 10-60 minutes, cool, add methanol to the mark, shake well, filter, and take the filtrate to obtain the test solution; Step 3. Content determination: Chromatographic conditions: C18 column; mobile phase A: methanol; mobile phase B: 0.1% glacial acetic acid solution; 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℃; detection wavelength: 291 nm; injection volume: 1 μL. When preparing the mixed reference solution, first dissolve it in methanol to prepare a solution containing 0.0582 mg of vitamin B6, 0.2558 mg of tolperidone hydrochloride, and 0.0764 mg of methyl hesperidin per mL, respectively. Shake well and filter to obtain the solution.

2. The method for simultaneously determining the contents of tolperidone 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 0.1g of Naoluotong powder, accurately weigh it, place it in a 20ml volumetric flask, add methanol, and sonicate it for 30 minutes at a power of 140W and a frequency of 42kHz.

3. The method for simultaneously determining the contents of tolperidone hydrochloride, methyl hesperidin, and vitamin B6 in Naoluotong capsules according to claim 1, characterized in that, High performance liquid chromatography (HPLC) or ultra-high performance liquid chromatography (UHPLC) system is used for content determination.

4. The method for simultaneously determining the contents of tolperidone hydrochloride, methyl hesperidin and vitamin B6 in Naoluotong capsules according to claim 1, characterized in that, The C18 columns include Kromasil 100-5 C18 columns with dimensions of 5µm and 4.6×250mm; Venusil MP C18 columns with dimensions of 5µm and 4.6×250mm; and Ecosil C18 columns with dimensions of 5µm and 4.6×250mm.

5. A method for simultaneously determining the uniformity of the contents of tolperidone hydrochloride, methyl hesperidin, and vitamin B6 in Naoluotong capsules, characterized in that, The method includes the following steps: Step 1. Preparation of mixed reference solution: Prepare a mixed reference solution containing vitamin B6, tolperidone hydrochloride, and methyl hesperidin; Step 2. Preparation of 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 a volumetric flask, add methanol, sonicate for 10-60 minutes, cool, make up to the mark, shake well, filter, and take the filtrate as the test solution. Step 3. Content uniformity determination: Chromatographic conditions: C18 column; mobile phase A: methanol; mobile phase B: 0.1% glacial acetic acid solution; 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℃; detection wavelength: 291 nm; injection volume: 1 μL.

6. The method for simultaneously determining the uniformity of the contents of tolperidone hydrochloride, methyl hesperidin, and vitamin B6 in Naoluotong capsules according to claim 5, characterized in that, The specific method for preparing the test solution is as follows: randomly take 10 Naoluotong capsules, pour out the contents, and place them in a 100ml volumetric flask; sonicate at a power of 140W and a frequency of 42kHz for 30 minutes.

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