Fingerprint detection method for fifteen-siwei saier pills

Abstract

The application discloses a kind of fifteen kinds of sereel Douwan fingerprint detection methods, it includes the following steps: a, preparation of reference solution: taking gallic acid, magnolia base, mangiferin, ellagic acid, berberine hydrochloride reference, add methanol to dissolve, namely obtained;B, preparation of test solution: take fifteen kinds of sereel Douwan, add methanol solution extraction, filter, take the filtrate, namely obtained;C, respectively, reference solution and test solution are pumped into liquid chromatograph and detected.The method first establishes the fingerprint of fifteen kinds of sereel Douwan, provides simple and accurate method for the detection and identification of fifteen kinds of sereel Douwan, guarantees the quality of fifteen kinds of sereel Douwan, also provides scientific basis for its clinical application, with good application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of drug detection and analysis, in particular to a fingerprint spectrum detection method of fifteen-flavor Sai'er pill. BACKGROUND

[0002] Fifteen-flavor Sai'er pill is a traditional preparation in Tibetan medicine, which is used for treating liver and gallbladder heat, cholecystitis, cholelithiasis and choledocholithiasis. It is widely used in clinical practice in minority areas. The only current quality standard is the National Food and Drug Administration of the State Drug Standard, standard number: WS-10738(ZD-0738)-2002. Many effective components are not controlled, the quality standard is relatively simple, and the uniformity, stability and controllability of the drug quality cannot be well reflected, which does not meet the requirements of modern drug quality.

[0003] Fifteen-flavor Sai'er pill is composed of fifteen Tibetan medicines such as Indian swertia herb, Terminalia chebula, pomegranate, and Berberis bark. Compound preparation is based on the comprehensive therapeutic effect of multiple chemical components, and has the characteristics of integrity and fuzziness. The traditional Chinese medicine quality evaluation method using single or several chemical components as indicators is difficult to achieve the purpose of Chinese medicine prescription quality control evaluation. The quality control of Chinese medicine prescription with more comprehensive chemical components can be more accurate and reliable. However, there is no research on the multiple components of fifteen-flavor Sai'er pill. SUMMARY

[0004] In order to solve the above problems, the present application provides a fingerprint spectrum detection method of fifteen-flavor Sai'er pill, characterized in that it comprises the following steps:

[0005] a. Preparation of reference solution: take gallic acid, magnoflorine, mangiferin, ellagic acid, and berberine hydrochloride reference substances, and dissolve them with methanol to obtain the reference solution;

[0006] b. Preparation of test solution: take fifteen-flavor Sai'er pill powder, add methanol solution for extraction, filter, and take the filtrate to obtain the test solution;

[0007] c. Respectively, the reference solution and the test solution are injected into the liquid chromatograph; the chromatographic conditions are as follows:

[0008] Chromatographic column: hydrophilic octadecylsilane bonded silica gel as filler; 0.1% phosphoric acid solution as mobile phase A, acetonitrile as mobile phase B, and gradient elution program as follows:

[0009]

[0010]

[0011] Further, each 1 mL of the reference solution described in step a contains 10–30 μg of gallic acid, 20–50 μg of magnoflorine, 1–10 μg of mangiferin, 10–30 μg of ellagic acid, and 5–25 μg of berberine hydrochloride.

[0012] Furthermore, the mass-to-volume ratio of the fifteen-flavor celery pills to the methanol solution in step b is 0.2–2 g: 50 mL.

[0013] Furthermore, the concentration of the methanol solution is 75%, mL / mL.

[0014] Further, the extraction described in step b is ultrasonic extraction, with a power ratio of 99%, a frequency of 45 kHz, and a time of 45–60 min.

[0015] Furthermore, in step c, the chromatographic conditions use a Swell column. C18, 5μm, 4.6×250mm, flow rate 0.6~1.0mL / min, injection volume 2~16μL, column temperature 25~35℃, wavelength 254±5nm.

[0016] Furthermore, the chromatographic conditions include a flow rate of 0.8 mL / min, a column temperature of 30 °C, and a wavelength of 254 nm.

[0017] Furthermore, the fingerprint spectrum of the fifteen-flavor Serdou pill should contain 13 common characteristic peaks, as detailed below. Figure 1 Among them, peak 2 is the gallic acid chromatographic peak, peak 8 is the ellagic acid chromatographic peak, peak 5 is the magnoflorine chromatographic peak, peak 10 is the berberine hydrochloride chromatographic peak, and peak 6 is the mangiferin chromatographic peak.

[0018] This invention provides a fingerprint chromatographic detection method for Shiwuwei Saierdou Pill. Through specific sample processing methods and chromatographic conditions, a fingerprint chromatogram of Shiwuwei Saierdou Pill has been established for the first time. This provides a simple and accurate method for the detection and identification of Shiwuwei Saierdou Pill, ensuring its quality and providing a scientific basis for its clinical application, thus showing good application prospects.

[0019] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0020] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0021] Figure 1 Superimposed fingerprint chromatograms (S1~S11) and control fingerprint chromatograms (R) of eleven batches of fifteen-flavor Serdou pills.

[0022] Figure 2 The fifteen-ingredient Serdou pill shared characteristic peaks (S1. Test sample; S2. lacking *Swertia spp.*; S3. lacking *Gnaphalium affine*; S4. lacking potassium nitrate; S5. lacking fennel; S6. lacking *Heliotropium indicum*; S7. lacking *Aconitum tanguticum*; S8. lacking pomegranate seeds; S9. lacking *Trichosanthes kirilowii* seeds; S10. lacking berberine bark; S11. lacking *Zanthoxylum bungeanum*; S12. lacking *Saussurea involucrata*; S13. lacking borneol; S14. lacking *Saussurea costus*; S15. lacking *Terminalia chebula*; S16. ...). Lacking gold-precipitated stone; S17. Indian swert; S18. golden sedge; S19. potassium nitrate; S20. fennel; S21. rabbit ear grass; S22. aconite; S23. pomegranate seed; S24. pine nut seed; S25. barley bark; S26. scutellaria barbata; S27. *Saussurea involucrata*; S28. borneol; S29. costus root; S30. chebula; S31. gold-precipitated stone; S32. mixed reference standard; S33. blank solution) Detailed Implementation

[0023] Example 1: Fingerprint Spectrum Detection of Fifteen-Flavor Serdou Pill

[0024] (1) Preparation of test solution

[0025] Take 0.5g of the powder of Fifteen Flavors of Seldou Pills, accurately weigh it, place it in a stoppered conical flask, accurately add 50ml of 70% methanol solution, sonicate (power ratio 99%, frequency 45kHz) for 45min, cool, filter, and take the filtrate and filter it through a 0.22μm microporous membrane to obtain the product.

[0026] (2) Preparation of reference solution

[0027] Accurately weigh 13.66 mg of gallic acid reference standard, 7.51 mg of magnoflorine reference standard, 6.42 mg of mangiferin reference standard, 5.07 mg of ellagic acid reference standard, and 8.67 mg of berberine hydrochloride reference standard, and dilute to the mark with methanol in 10 mL volumetric flasks to obtain reference standard stock solutions. Accurately pipette appropriate amounts of the above-prepared reference standard stock solutions into the same volumetric flask and dilute to the mark with methanol to obtain mixed reference standard solutions of gallic acid, magnoflorine, mangiferin, ellagic acid, and berberine hydrochloride, with mass concentrations of 27.32, 45.06, 6.42, 20.28, and 17.34 μg / mL, respectively.

[0028] (3) Inject 10 μL of the reference solution and the test solution into the liquid chromatograph, respectively; the chromatographic conditions are as follows:

[0029] Column: Swell C18 column (5 μm, 4.6 × 250 mm); column temperature: 30℃; flow rate: 0.8 mL / min; detection wavelength: 254 nm; mobile phase: acetonitrile (B) - 0.1% phosphoric acid solution (A), gradient elution program:

[0030]

[0031] (4) The fingerprint spectrum of the Fifteen-Flavor Serdou Pill should contain 13 common characteristic peaks. For details, see [link to relevant documentation]. Figure 1 Among them, peak 2 is the gallic acid chromatographic peak, peak 8 is the ellagic acid chromatographic peak, peak 5 is the magnoflorine chromatographic peak, peak 10 is the berberine hydrochloride chromatographic peak, and peak 6 is the mangiferin chromatographic peak.

[0032] The following experimental examples illustrate the beneficial effects of the present invention.

[0033] Experimental Example 1: Characteristic Mapping Study of Fifteen-Flavor Serdou Pills

[0034] 1. Experimental Materials

[0035] 1.1 Instruments

[0036] Table 1 Instruments

[0037]

[0038] 1.2 Fifteen-Flavor Seldouwan

[0039] The Fifteen-Ingredient Serdou Pill, with a specification of 0.5g / pill, is supplied by Qinghai Jiumei Tibetan Medicine Pharmaceutical Co., Ltd., and the batch number is shown in the table below. All fifteen medicinal ingredients in the Fifteen-Ingredient Serdou Pill are also supplied by Qinghai Jiumei Tibetan Medicine Pharmaceutical Co., Ltd.

[0040] Table 2. Eleventh batch of fifteen-flavor Saierdou pills.

[0041]

[0042]

[0043] 1.3 Reference Standard

[0044] Table 3 Reference Standards

[0045]

[0046] 1.4 Reagents and Materials

[0047] The water used in the experiment was ultrapure water.

[0048] Table 4 Reagents

[0049]

[0050] 1.5 Raw medicinal materials

[0051] To ensure the quality of the medicinal materials, the fifteen raw medicinal materials provided by Qinghai Jiumei Tibetan Medicine Pharmaceutical Co., Ltd. were tested according to the items specified in the current standards. The results are shown in Table 5.

[0052] Table 5. Results of Medicinal Material Inspection

[0053]

[0054]

[0055] 2. Characteristic Spectrum Study

[0056] 2.1 Chromatographic conditions

[0057] Column: Swell C18 column (5 μm, 4.6 × 250 mm); column temperature: 30℃; flow rate: 0.8 mL / min; detection wavelength: 254 nm; mobile phase: acetonitrile (B) - 0.1% phosphoric acid solution (A), gradient elution program:

[0058]

[0059] 2.2 Preparation method of test solution

[0060] Take about 0.5g of the powder of Fifteen Flavors of Seldou Pills, accurately weigh it, place it in a stoppered conical flask, accurately add 50ml of 70% methanol solution, sonicate (power ratio 99%, frequency 45kHz) for 45min, cool, filter, and take the filtrate to filter through a 0.22μm microporous membrane to obtain the test solution.

[0061] 2.3 Preparation of reference solution

[0062] Accurately weigh 13.66 mg of gallic acid reference standard, 7.51 mg of magnoflorine reference standard, 6.42 mg of mangiferin reference standard, 5.07 mg of ellagic acid reference standard, and 8.67 mg of berberine hydrochloride reference standard, and dilute to the mark with methanol in 10 mL volumetric flasks to obtain reference standard stock solutions. Accurately pipette appropriate amounts of the above-prepared reference standard stock solutions into the same volumetric flask and dilute to the mark with methanol to obtain a mixed reference standard solution of gallic acid, magnoflorine, mangiferin, ellagic acid, and berberine hydrochloride, with mass concentrations of 27.32, 45.06, 6.42, 20.28, and 17.34 μg / mL, respectively.

[0063] 2.4 Preparation of test solutions for single medicinal materials

[0064] Weigh each herb according to the prescription amount and prepare the test solution of each herb according to the preparation method in section "2.2".

[0065] 2.5 Preparation of negative control solution

[0066] According to the proportions of the 15-ingredient Saierdou Pill prescription, each medicinal material was weighed separately, and negative control solutions were prepared according to the method in section "2.2" that lacked Berberis bark, Gypsum fibrosum, Zha Xun, Chrysanthemum morifolium, Borneol, Fennel, Aconitum carmichaelii, Pomegranate seeds, Rabbit ear grass, Golden waist grass, Saltpeter, Swertia japonica, Terminalia chebula, Melon seeds, and Aucklandia lappa.

[0067] 2.6 Methodological Examination

[0068] 2.6.1 Precision Test

[0069] Take the test solution (batch 11) from the same batch as described in section "2.2", and inject it six times consecutively under the chromatographic conditions described in section "2.1". Record the retention time and peak area of ​​each common characteristic peak. Using ellagic acid (peak 8) as the reference peak, calculate the relative retention time and relative peak area of ​​the characteristic peaks (the retention time and peak area of ​​ellagic acid, i.e., the reference peak, are both calculated as 1). The results show that the RSD values ​​of the relative retention times of each common peak are all less than 0.26%. The RSD values ​​of the relative peak areas of common peaks whose single peak area accounts for more than 2% of the total peak area are all less than 3.44%. Chemometric evaluation was performed using the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2004A, and the similarity was 0.99, indicating good instrument precision.

[0070] 2.6.2 Stability Test

[0071] Take the test solution (batch 11) from the same batch as described in section "2.2" and inject it at 0, 2, 4, 8, 12, and 24 hours according to the chromatographic conditions described in section "2.1". Calculate the relative retention time and relative peak area of ​​the characteristic peaks. The results show that the RSD values ​​of the relative retention times of all common peaks are less than 0.41%. The RSD values ​​of the relative peak areas of common peaks whose single peak area accounts for more than 2% of the total peak area are all less than 3.21%, and the similarity evaluation is 0.99, indicating that the sample solution has good stability within 24 hours.

[0072] 2.6.3 Repeatability Test

[0073] Six aliquots of the same batch of test solution (batch eleven) were prepared according to the same method as described in section "2.2". They were injected and analyzed under the chromatographic conditions described in section "2.1", and the relative retention times and relative peak areas of the characteristic peaks were calculated. The RSD values ​​of the relative retention times of all common peaks were less than 0.11%. The RSD values ​​of the relative peak areas of common peaks whose single peak area accounted for more than 2% of the total peak area were all less than 4.86%, and the similarity evaluation was 0.99, indicating that the method has good repeatability.

[0074] 3. Establishment of the characteristic atlas of Fifteen Flavors of Seldou Pills

[0075] Following the chromatographic conditions described in section "2.1", eleven batches of the Fifteen-Flavor Serdou Pill test solution were sequentially injected, and chromatograms were recorded. Data matching was performed using the 2004A version of the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System. Peaks with good resolution and relatively high retention time matching among the eleven batches of test samples were selected as common characteristic peaks. In the chromatograms of the eleven batches of Fifteen-Flavor Serdou Pill samples, peak number 8 (ellagic acid), with a large peak area, moderate retention time, and relative stability, was selected as the reference peak (S peak). A reference chromatogram was generated using the median method, and matching data and similarity were calculated. Finally, a total of 13 common peaks were identified from the eleven batches of samples. The results are shown in […]. Figure 1 By comparing the chromatograms of the five mixed reference solutions and considering the retention times of these five components, some peaks could be identified: peak 2 was gallic acid, peak 5 was magnoflorine, peak 6 was mangiferin, peak 8 was ellagic acid, and peak 10 was berberine hydrochloride. The similarity scores of the chromatograms of the eleven batches of the fifteen-flavor Seldouwan sample and the reference chromatograms were 0.963, 0.957, 0.971, 0.976, 0.993, 0.994, 0.967, 0.980, 0.898, 0.948, and 0.905, respectively, all greater than 0.8. This indicates that the eleven batches of samples showed good similarity and good consistency and stability among different batches.

[0076] 4. Peak Attribution Analysis

[0077] The test solutions of each single medicinal material under section "2.4" and the negative control solutions under section "2.5" were injected and analyzed according to the chromatographic conditions under section "2.1". Chromatograms at 254 nm were recorded, and the chromatograms were imported into the Chinese herbal chromatographic fingerprint similarity evaluation system to analyze the attribution of 13 common characteristic peaks. Results are shown in […]. Figure 2 Peak 1 was found to be a solvent peak, present in all single-herb test solutions, negative control solutions, mixed control solutions, and blank solutions. Peaks 2 (gallic acid), 7, 8 (ellagic acid), and 9 originated from Terminalia chebula; peaks 3, 4, 5 (magnoliine), and 10 (berberine hydrochloride) originated from Berberis bark; peaks 6 (mangiferin) and 12 originated from Swertia xanthipes; peak 8 (ellagic acid) originated from pomegranate seeds; peak 11 originated from Lysimachia christinae; and peak 13 originated from Trichosanthes kirilowii seeds. Ellagic acid is a common component of Terminalia chebula and pomegranate seeds. Each characteristic chromatographic peak corresponds to the chromatographic peaks of the single-herb medicinal materials, providing a reference for the quality control study of Shiwuwei Saierdou Pill.

[0078] 5. Characteristic diagram of Fifteen Flavors of Seldouwan

[0079] Through preliminary experiments, it was found that the chromatographic peaks of Shiwuwei Saierdou Pills were abundant at 254 nm in liquid chromatography. Therefore, 254 nm was selected as the detection wavelength for the characteristic chromatogram of Shiwuwei Saierdou Pills. Given the complex composition of Shiwuwei Saierdou Pills, source analysis of its common characteristic peaks revealed that, except for peak 1 which was likely a solvent peak, the remaining 12 peaks could be attributed to their respective medicinal materials. Furthermore, the chemical components corresponding to 5 of these chromatographic peaks could be identified. Therefore, establishing the characteristic chromatogram of Shiwuwei Saierdou Pills using HPLC at a detection wavelength of 254 nm is accurate and feasible.

[0080] In summary, this invention, through specific sample processing methods and chromatographic conditions, has for the first time established the fingerprint spectrum of Shiwuwei Saierdou Pill, providing a simple and accurate method for the detection and identification of Shiwuwei Saierdou Pill, ensuring its quality, and providing a scientific basis for its clinical application, thus showing good application prospects.

Claims

1. A method for detecting the fingerprint spectrum of a 15-flavored salbutamol, characterized in that: It includes the following steps: a. Preparation of reference solutions: Take gallic acid, magnoflorine, mangiferin, ellagic acid, and berberine hydrochloride reference standards, dissolve them in methanol to obtain the solution; b. Preparation of the test solution: Take 15-flavor Seldou pills, extract with methanol aqueous solution, filter, and take the filtrate to obtain the test solution; c. Inject the reference solution and the test solution separately into the liquid chromatograph; the chromatographic conditions are as follows: Chromatographic column: Swell Chromplus® C18, 5 µm, 4.6 × 250 mm; mobile phase A: 0.1% phosphoric acid solution; mobile phase B: acetonitrile; flow rate: 0.8 mL / min; column temperature: 30 °C; wavelength: 254 nm; gradient elution program: The fingerprint chromatogram of the fifteen-flavor Serdou pill should have 13 common characteristic peaks, of which peak 2 is the gallic acid chromatographic peak, peak 8 is the ellagic acid chromatographic peak, peak 5 is the magnoflorine chromatographic peak, peak 10 is the berberine hydrochloride chromatographic peak, and peak 6 is the mangiferin chromatographic peak.

2. The fingerprint spectrum detection method for fifteen-flavored celestial pills according to claim 1, characterized in that: The reference solution described in step a contains 10-30 µg gallic acid, 20-50 µg magnoflorine, 1-10 µg mangiferin, 10-30 µg ellagic acid, and 5-25 µg berberine hydrochloride per 1 mL.

3. The fingerprint spectrum detection method for fifteen-flavor celestial pills according to claim 1, characterized in that: The mass-to-volume ratio of the fifteen-flavor Seldou pills to the methanol solution in step b is 0.2~2g:50mL.

4. The fingerprint spectrum detection method for fifteen-flavor celestial pills according to claim 3, characterized in that: The concentration of the methanol aqueous solution is 75%, mL / mL.

5. The fingerprint spectrum detection method for fifteen-flavor celestial pills according to claim 1, characterized in that: The extraction described in step b is ultrasonic extraction, with a power ratio of 99%, a frequency of 45kHz, and a time of 45~60min.

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