Quantitative quantitative analysis (HPLC) method for isocratic elution of brain-benefiting capsules by quantitative analysis of double components by single marker

Abstract

The invention relates to an isocratic elution HPLC (high performance liquid chromatography) quantitative determination method for QAMS (quantitative evaluation of double components by single marker) of a brain-benefiting capsule. The method is characterized in that a test solution is obtained by a simple, convenient and rapid pretreatment method, the contents of ganoderic acid A and schizandrin in a sample are simultaneously determined at 255 + / -1nm by adopting a common chromatographic instrument and an isocratic elution mode and taking acetonitrile-methanol-0. 1% phosphoric acid with the volume ratio of 28: 20: 52 as a mobile phase, the wave crest separation is good, the sample injection is performed once, and the peak appearance is completed within 40 minutes. Gradient elution for measuring one component of ganoderic acid A is replaced by common isocratic elution, the content of schizandrin is measured at the same time, and the practical purposes of single-measurement double-evaluation, high efficiency, low consumption and easy popularization are achieved. The method for simultaneously determining ganoderic acid A and schizandrin is reported for the first time, and manifests the novelty and creativity of the technology and the practical value of low cost and double-index detection with half the effort.

Description

Technical Field

[0001] The present invention relates to a one-measurement, two-evaluation, isocratic elution HPLC quantitative determination method for Yinao capsules, that is, a common high performance liquid chromatograph is used to simultaneously determine the contents of ganoderic acid A and schisandrin A in Yinao capsules in an isocratic elution manner. Background Art

[0002] Yinao Capsule is an OTC drug with National Medical Approval Number Z10983042. It has the effects of replenishing Qi and Yin, nourishing the kidneys and strengthening the brain, improving intelligence and calming the mind. It is used for neurasthenia, fatigue, dizziness, insomnia and dreaminess. The formula composition and preparation process are as follows:

[0003] Prescription: Tortoise Shell Glue 38.6g, Polygala 193.3g, Dragon Bone 387.3g, Ganoderma Lucidum 387.3g Schisandra chinensis 49.3g, Ophiopogon japonicus 193.3g, Acorus calamus 193.3g, Codonopsis pilosula 111.0g Ginseng 66.6g, Poria 387.3g

[0004] Preparation process: The above ten ingredients, except tortoise shell glue, ginseng and Poria cocos (25%) are crushed into fine powder respectively. The remaining Poria cocos and the other seven ingredients including Polygala tenuifolia are decocted in water twice, the first time for 3 hours and the second time for 2 hours. Filter, combine the filtrate, and concentrate it into a thick paste. Add ginseng, Poria cocos powder and dissolved tortoise shell glue, mix thoroughly, dry, grind into fine powder, sieve, and put into capsules to make 1000 capsules.

[0005] Despite being a ten-herb formula, the quality standard for Yinao Capsules only includes thin-layer chromatography (TLC) identification, with no quantitative determination. Even Yinao Tablets, which uses the same formula and process and has the most testing items in the 2020 edition of the Chinese Pharmacopoeia, only has TLC identification for three herbs and gradient HPLC quantitative determination of schisandrin A, a test that takes an hour to run. Current quality standards make it difficult to control the intrinsic quality of a preparation.

[0006] In order to ensure the high quality of the therapeutic effect of Yinao Capsules, the water extraction process was strictly monitored, and a simultaneous quantitative study was conducted on the active ingredients of Schisandra chinensis and Ganoderma lucidum, Schisandrin A and Ganoderic A in the capsules.

[0007] According to the literature, ganoderic acid A and schisandrin A are two active ingredients with completely different chemical structures and properties. Their structural formulas are as follows: From the above structural formula, we know that ganoderic acid A has high water solubility and contains many interfering components. Therefore, even for the determination of single ganoderic acid A, a gradient elution of 60 minutes is required (see Figure 1); Schisandrin A is much more lipid-soluble, and its quantitative determination by mass standard also uses gradient elution, with a run time of 55 minutes. Regardless of the method, the resolution is less than ideal. Due to the difference in polarity, it is difficult to visualize the two in the same chromatogram within a short retention time. Therefore, to date, no reports have been found on the simultaneous determination of ganoderic acid A and schisandrin A in Yinao Capsules. Summary of the Invention

[0008] Against this backdrop, the present invention, designed to improve detection efficiency, reduce detection costs, and minimize environmental pollution, utilizes a simple and rapid pretreatment method to obtain a test sample solution. Using a conventional chromatograph and isocratic elution, with a mobile phase of acetonitrile-methanol-0.1% phosphoric acid in a volume ratio of 28:20:52, the ganoderic acid A and schisandrin A content in the sample were simultaneously determined at 255±1 nm. Peak separation was excellent, and a single injection yielded peaks in 40 minutes. This conventional isocratic elution method not only replaced the gradient elution method used for the determination of ganoderic acid A alone, but also simultaneously determined schisandrin A content, achieving a single measurement with dual evaluation, high efficiency, low cost, and ease of popularization. This simultaneous determination of ganoderic acid A and schisandrin A is the first reported method, highlighting the novelty, creativity, and practicality of the technology for low-cost, dual-index detection with twice the result with half the effort.

[0009] Through methodological investigation, the injection amount of ganoderic acid A was 56.02-560.20 ng, and the injection amount of schisandrin A was 39.64-396.40 ng, which showed a good linear relationship with the peak area (see Table 1, Table 2, Figure 2 、 Figure 3 ), the regression equations were: Y = 861.22X-3629.4, r = 0.99999 for ganoderic acid A; Y = 1729.5X-6806.8, r = 0.99999 for schisandrin A. The results of the sample recovery experiment showed that the average recovery rate of ganoderic acid A for 6 determinations was 96.66%, with an RSD of 1.31% (see Table 3); the average recovery rate of schisandrin A for 6 determinations was 99.68%, with an RSD of 1.55% (see Table 4). Precision (see Table 5), stability (see Table 6), repeatability (see Table 7), and UV spectra of the determined components (see Figure 4 、 5 ), specificity (see Figure 6 、 7 The experimental data for the determination of three sample batches (Table 8), extraction solvent selection (Table 9), and ultrasonic time investigation (Table 10) all met the methodological requirements. This method is simple, accurate, and efficient and can be used for the simultaneous quantitative determination of ganoderic acid A and schisandrin A in Yinao Capsules.

[0010] The technical solution adopted by the present invention to solve its technical problem is:

[0011] (1) A. Chromatographic Conditions and System Suitability Test: Octadecylsilane bonded silica gel was used as the filler; acetonitrile-methanol-0.1% phosphoric acid (volume ratio: 28:20:52) was used as the mobile phase; the detection wavelength was 255±1 nm; the column temperature was 30°C; the theoretical plate number calculated based on the ganoderic acid A peak was not less than 5000;

[0012] B. Preparation of Reference Solution: Accurately weigh appropriate amounts of ganoderic acid A and schisandrin A reference substances, add methanol to each to make a mixed solution containing 25 μg of each per 1 ml, and shake well to obtain the solution.

[0013] C. Preparation of Test Solution: Take 20 Yinao Capsules, pour out the contents, grind finely, and weigh approximately 2.0 g. Place in a stoppered conical flask, accurately add 25 ml of 90% methanol, stopper tightly, and weigh the solution. Ultrasonicate at 300 W and 40 kHz for 30 minutes. Allow to cool, weigh again, and make up the loss with 90% methanol. Shake well, filter, and collect the filtrate.

[0014] D. Determination method: Accurately aspirate 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, determine, and calculate the content of the two components respectively.

[0015] The principles of the present invention are as follows:

[0016] According to the extraction principle of like dissolves like, combined with the water decoction extraction process, the extraction yield of water-soluble ganoderic acid A is high, while the extraction yield of fat-soluble schisandrin A is low. Figure 3 、 4 ) as the detection wavelength, not only does it optimize the size and area of ​​the two component peaks, but it also significantly reduces interfering peaks at this detection wavelength, resulting in a stable chromatographic baseline and excellent peak separation, paving the way for simultaneous determination of the two components. Furthermore, quantitative determination is possible based on the good linear relationship between the injection volume of the two components and their respective peak areas within a certain range.

[0017] The innovative features and beneficial effects of the present invention are as follows:

[0018] (1) Using conventional isocratic elution, acetonitrile-methanol-0.1% phosphoric acid in a volume ratio of 28:20:52 was used as the mobile phase; the contents of ganoderic acid A and schisandrin A in the sample were simultaneously determined at 255±1nm, enabling the two active ingredients of traditional Chinese medicine with completely different properties and structures to be quantitatively determined in one run using the same mobile phase. The quantitative chromatograms of the two components showed a stable baseline and good peak separation. The peak retention time of ganoderic acid A was approximately 22 minutes, and the peak retention time of schisandrin A was approximately 33 minutes. This method involves no extraction, no concentration, and no evaporation. It is simple, rapid, accurate, and reproducible, making it easy to popularize and master, effectively improving detection efficiency, reducing detection costs, and minimizing environmental pollution.

[0019] (2) According to the literature, the determination of ganoderic acid A and schisandrin A is currently done by separate determination of single components, and most of them are gradient elution. It takes 60 minutes to run one injection of ganoderic acid A ( Figure 1 ), schisandrin A takes 55 minutes; not only is the separation unsatisfactory, but the reproducibility is also poor. This is the first report of a method using high-performance liquid chromatography with isocratic elution to simultaneously determine the contents of ganoderic acid A and schisandrin A. Detection efficiency is greatly improved, and instrument costs are significantly reduced. A common liquid chromatograph replaces a high-end instrument that requires gradient elution. Not only that, but more importantly, the one-test-one-evaluation method has been upgraded to one-test-two-evaluations, achieving twice the result with half the effort. Its novelty, innovation, resource conservation, and practical value in improving detection phase rate and detection indicators are obvious to all.

[0020] (3) The advent of the method of the present invention not only provides a method for the simultaneous detection of ganoderic acid A and schisandrin A in Yinao Capsules, but also provides a reference basis and research ideas for the simultaneous determination of the two components in other compound preparations, which has a representative and exemplary role.

[0021] (4) The key technology of the present invention is to utilize the fact that methanol in the three mobile phases has the property of delaying the peak retention time of various chemical components, while acetonitrile has the property of delaying the peak retention time of various chemical components. The addition of phosphoric acid solution can make the acidic components exist as molecules, eliminating the tailing of ions. Through the study of the optimal ratio between methanol, acetonitrile and phosphoric acid at multiple concentrations, it was found that isocratic elution can make two chemical components with large polarity differences appear in the same mobile phase, and the peak retention time is appropriate. The retention time of ganoderic acid A is 22 minutes, and the retention time of schisandrin A is 33 minutes. The baseline is stable and the reproducibility is good. The running time is also shortened by 20 minutes compared with the gradient elution of a single component. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Gradient elution HPLC chromatogram of ganoderic acid A in Yinao capsule reported in the literature

[0023] Figure 2Linear relationship diagram of Ganoderic acid A

[0024] Figure 3 Linear relationship diagram of Schisandrin A

[0025] Figure 4 UV spectrum of ganoderic acid A

[0026] Figure 5 UV spectrum of schisandrin A

[0027] Figure 6 HPLC chromatograms of ganoderic acid A and schisandrin A reference substances

[0028] Figure 7 HPLC chromatogram of Yinao Capsule sample

[0029] Figure 8 HPLC chromatogram of a blank sample without Ganoderma lucidum

[0030] Figure 9 HPLC chromatogram of blank sample without Schisandra chinensis

[0031] Figure 2 、 3 In the figure, the vertical axis is the peak area; the horizontal axis is the injection amount (ng)

[0032] Figure 4 、 5 In the figure, the vertical axis is the absorption intensity (mAU); the horizontal axis is the absorption wavelength (nm).

[0033] Figure 1 、 6 In Figures 9 and 10, the horizontal axis is the retention time (minutes), the vertical axis is the response value (uV), 1. is the peak of ganoderic acid A, 2. is the peak of schisandra alcohol A Specific embodiments of the present invention Example

[0035] (1) A. Chromatographic Conditions and System Suitability Test: Octadecylsilane bonded silica gel was used as the filler; acetonitrile-methanol-0.1% phosphoric acid (volume ratio: 28:20:52) was used as the mobile phase; the detection wavelength was 255±1 nm; the column temperature was 30°C; the theoretical plate number calculated based on the ganoderic acid A peak was not less than 5000;

[0036] B. Preparation of Reference Solution: Accurately weigh appropriate amounts of ganoderic acid A and schisandrin A reference substances, add methanol to each to make a mixed solution containing 25 μg of each per 1 ml, and shake well to obtain the solution.

[0037] C. Preparation of Test Solution: Take 20 Yinao Capsules, pour out the contents, grind finely, and weigh approximately 2.0 g. Place in a stoppered conical flask, accurately add 25 ml of 90% methanol, stopper tightly, and weigh the solution. Ultrasonicate at 300 W and 40 kHz for 30 minutes. Allow to cool, weigh again, and make up the loss with 90% methanol. Shake well, filter, and collect the filtrate.

[0038] D. Determination method: Accurately aspirate 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, determine, and calculate the content of the two components respectively.

[0039] Table 1 Injection amount and peak area of ​​ganoderic acid A

[0040]

[0041] Table 2 Schisandrin A injection amount and peak area

[0042]

[0043] Table 3 Recovery test results of ganoderic acid A in samples

[0044]

[0045] Table 4 Recovery test results of Schisandrin A in samples

[0046]

[0047] Table 5 Precision test results

[0048]

[0049] Table 6 Stability test results

[0050]

[0051] Table 7 Repeatability test results (mg / g)

[0052]

[0053] Table 8 Determination results of the contents of two ingredients in three batches of Yinao Capsules (mg / g, n=2)

[0054]

[0055] Table 9 Extraction solvent investigation (mg / g)

[0056]

[0057] Table 10 Ultrasonic time investigation (mg / g)

[0058]

Claims

1. A one-test, two-evaluation, equal-degree elution HPLC quantitative determination method for Yinao Capsules, characterized in that: A. Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler; acetonitrile-methanol-0.1% phosphoric acid with a volume ratio of 28:20:52 was used as mobile phase; detection wavelength: 255±1nm; column temperature: 30℃; theoretical plate number calculated based on the ganoderic acid A peak was not less than 5000; B. Preparation of reference solution: Accurately weigh appropriate amount of ganoderic acid A and schisandra alcohol A reference substances, accurately weigh them, add methanol to make a mixed solution containing 25 μg of each per 1 ml, shake well, and obtain; C. Preparation of test solution Take 20 capsules of Yinao capsules, pour out the contents, grind them into powder, weigh about 2.0g, accurately weigh, put them into a stoppered conical bottle, accurately add 25ml of 90% methanol, stopper it, weigh it, treat it with ultrasound at a power of 300w and a frequency of 40kHz for 30 minutes, let it cool, weigh it again, make up the lost weight with 90% methanol, shake it well, filter it, and take the filtrate to obtain the solution; D. Determination method: Accurately pipette 10μl of reference solution and test solution respectively, inject into liquid chromatograph, determine, and calculate the contents of the two components respectively.

2. The one-test-two-evaluation equal-degree elution HPLC quantitative determination method of Yinao Capsule according to claim 1, characterized in that: The one-test-two-evaluation method refers to one-test to simultaneously evaluate the contents of two components, ganoderic acid A and schisandrae alcohol A.

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