Quality control and detection method for lumbricus soaked with liquorice

Abstract

The invention belongs to the technical field of liquid phase detection, and particularly relates to a quality control and detection method for lumbricus soaked with liquorice. According to the method, methanol water is used as a mobile phase, the content of hypoxanthine and inosine in the lumbricus glycyrrhiza is detected by high performance liquid chromatography, and the method has important significance on quality control of a lumbricus glycyrrhiza product. The detection method disclosed by the invention is good in system applicability, precision, reproducibility, recovery rate and stability, and meets the requirements of Chinese Pharmacopoeia, Chinese Pharmacopoeia, appendix of Chinese Pharmacopoeia, Chinese appendix of Chinese Pharmacopoeia, Chinese Pharmacopoeia, Chinese

Description

Technical Field

[0001] The present invention belongs to the technical field of liquid-phase detection. More specifically, it relates to a quality control detection method for earthworms soaked in licorice. Background Art

[0002] Earthworms are the dried bodies of the earthworm Pheretima aspergillum (E. Perrier), Pheretima vulgaris Chen, Pheretima guillelmi (Michaelsen), or Pheretima pectinifera Michaelsen of the family Megascolecidae. The first one is commonly known as "Guang earthworm", and the latter three are commonly known as "Hu earthworms". Earthworms are commonly used animal traditional Chinese medicines in clinical practice of traditional Chinese medicine. They were first recorded in "Shennong Ben Cao Jing" and were classified as inferior products. They are light in weight, slightly leathery, not easily broken, have a fishy smell, a salty taste, and are cold in nature. They belong to the liver, spleen, and bladder meridians. Guang earthworms are captured from spring to autumn, and Hu earthworms are captured in summer. The abdomen is promptly dissected, the internal organs and sediment are removed, washed, and dried in the sun or by low-temperature drying. In recent years, domestic and foreign studies have found that Guang earthworms are rich in nucleoside components such as hypoxanthine and inosine. These components are important players in the process of DNA metabolism, are the basic components for organisms to maintain life activities, have pharmacological activities such as regulating blood sugar, regulating immune function, and anti-ischemic injury, and are also the important active substance basis for the anti-asthmatic, bronchodilation, and antihistamine effects of earthworms, which have a certain correlation with the pharmacological effects of Guang earthworms.

[0003] Raw earthworms have a strong fishy smell and are not convenient for taking. Therefore, they are mostly used as medicine after processing. Earthworms soaked in licorice are a processed product of earthworms, which can reduce the fishy smell of raw earthworms and make it convenient for subsequent medication. In the present invention, earthworms soaked in licorice are prepared according to the "Guangdong Province Traditional Chinese Medicine Decoction Pieces Processing Specification". Specifically, take the purified earthworms, put them into warm licorice water, soak for 2 hours, and then take them out and dry; among them, for every 100 kg of earthworms, 20 kg of licorice is added to cook the licorice water. However, this processing method may cause a large loss of water-soluble substances in earthworms, such as nucleoside components, thereby reducing the medicinal efficacy of earthworms.

[0004] Therefore, in order to ensure that earthworms soaked in licorice with a relatively small fishy smell still have good pharmacological activities, there is an urgent need for a method to simultaneously detect the contents of xanthine and inosine in earthworms soaked in licorice as a quality monitoring method for earthworms soaked in licorice to ensure that the processed earthworm products still have good medicinal efficacy. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the defects and deficiencies of the prior art and provide a detection method for quality control of earthworms soaked in licorice.

[0006] The object of the present invention is to provide the application of the said detection method in the quality control of processed earthworm with licorice root.

[0007] The above object of the present invention is achieved by the following technical solutions:

[0008] The present invention provides a detection method for the quality control of processed earthworm with licorice root, characterized in that high performance liquid chromatography is used to detect the contents of hypoxanthine and inosine in the test solution, and the chromatographic conditions include:

[0009] Mobile phase A is pure water, and mobile phase B is methanol;

[0010] The elution gradient is shown in the following table:

[0011] Time (min) Mobile phase A (%) Mobile phase B (%) 0.01 90～95 5～10 15 90～95 5～10 15.01 0 100 25 0 100 25.01 90～95 5～10 35 90～95 5～10

[0012] The chromatographic column is a chromatographic column with octadecylsilyl bonded silica gel as the stationary phase;

[0013] The test sample is processed earthworm with licorice root.

[0014] To ensure the controllable quality, safety and effectiveness of the product, the present invention has studied and formulated a method for simultaneously determining the contents of hypoxanthine and inosine in processed earthworm with licorice root by high performance liquid chromatography. After the verification of the analytical method, its precision, reproducibility, recovery rate and stability meet the requirements of the "Guiding Principles for the Verification of Analytical Methods for the Quality Standards of Traditional Chinese Medicines" in Appendix I of the Chinese Pharmacopoeia 2020 Edition.

[0015] The present invention can accurately detect the contents of hypoxanthine and inosine in the test sample of processed earthworm with licorice root by using specific mobile phase, elution gradient and chromatographic column, which is of great significance for the quality control of processed earthworm with licorice root samples.

[0016] Preferably, the chromatographic conditions further include: the detection wavelength is 252 - 256 nm.

[0017] Preferably, the chromatographic conditions further include: the column temperature is 30 ± 1 °C.

[0018] Preferably, the chromatographic conditions further include: the flow rate is 0.8 - 1.2 mL / min.

[0019] Preferably, the chromatographic conditions further include: the injection volume is 10 - 15 μL.

[0020] Preferably, the chromatographic conditions further include: the detector is an ultraviolet detector.

[0021] Preferably, the test solution is prepared by the following method: take processed earthworm with licorice root, soak it in water and then extract it by ultrasonic wave to obtain the test solution.

[0022] Preferably, the mass - volume ratio of processed earthworm with licorice root to water is 1 g: 8 - 12 mL.

[0023] Preferably, the soaking time is 25 to 35 min.

[0024] Preferably, the ultrasonic time is 25 to 35 min.

[0025] Preferably, the ultrasonic power is 35 to 45 W.

[0026] Meanwhile, the present invention also protects the application of the detection method in the quality control of Glycyrrhiza uralensis Fisch. soaked earthworm.

[0027] The present invention has the following beneficial effects:

[0028] The present invention provides a quality control detection method for Glycyrrhiza uralensis Fisch. soaked earthworm. Using methanol-water as the mobile phase, the present invention uses high performance liquid chromatography to detect the contents of hypoxanthine and inosine in Glycyrrhiza uralensis Fisch. soaked earthworm, which is of great significance for the quality control of Glycyrrhiza uralensis Fisch. soaked earthworm products. The detection method of the present invention has good system suitability, precision, reproducibility, recovery rate and stability, and meets the requirements of the "Guiding Principles for the Validation of Analytical Methods for Chinese Medicine Quality Standards" in Appendix 1 of the Chinese Pharmacopoeia 2020 Edition. Description of the Drawings

[0029] Figure 1 They are chromatograms of the reference substance solution, the test solution and the blank solution. Detailed Embodiments

[0030] The following further illustrates the present invention with reference to the drawings in the specification and specific embodiments, but the embodiments do not impose any limitation on the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

[0031] Example 1

[0032] The chromatographic conditions are shown in Table 1:

[0033]

[0034]

[0035] Preparation of the test solution: Weigh 1 g of the powder of Glycyrrhiza uralensis Fisch. soaked earthworm, place it in a 10 mL stoppered graduated test tube, add 10 mL of ultrapure water, soak for 30 min, ultrasonicate for 30 min, the ultrasonic power is 40 W, after the ultrasonic treatment, centrifuge at 10000 r / min at room temperature for 15 min, take the supernatant, and filter it through a 0.45 μm filter membrane to obtain the test solution.

[0036] Detection steps:

[0037] Take 10 μL of the test solution for injection and perform high-performance liquid chromatography detection according to the above chromatographic conditions. Determine the inosine and hypoxanthine contents of 3 batches of the prepared Pheretima aspergillum soaked in Glycyrrhiza uralensis Fisch., with 4 parallel determinations for each batch. The results are shown in Table 2.

[0038] Table 2 Determination Results of Inosine and Hypoxanthine Contents in Pheretima aspergillum Soaked in Glycyrrhiza uralensis Fisch.

[0039]

[0040] Example 2

[0041] Chromatographic conditions: the same as those in Example 1;

[0042] Preparation of hypoxanthine reference solution: Weigh 1.2 mg of hypoxanthine reference substance, add 10 mL of ultrapure water to prepare a 0.12 mg / mL hypoxanthine reference solution.

[0043] Preparation of inosine reference solution: Weigh 2.1 mg of inosine reference substance, add 10 mL of ultrapure water to prepare a 0.21 mg / mL inosine reference solution.

[0044] Preparation of test solution: Weigh 1 g of the powder of Pheretima aspergillum soaked in Glycyrrhiza uralensis Fisch., place it in a 10 mL stoppered graduated test tube, add 10 mL of ultrapure water, soak for 30 min, sonicate for 30 min, centrifuge at 10000 r / min at room temperature for 15 min, take the supernatant, and filter through a 0.45 μm filter membrane to obtain the test solution.

[0045] Investigation of linear relationship:

[0046] Precisely pipette different volumes of the hypoxanthine reference solution and inosine reference solution respectively, and dilute them with the solvent (the solvent is 95% water + 5% methanol) to prepare solutions with different concentrations. Take 10 μL of each of the above solutions for injection, and perform analysis according to the above chromatographic conditions. The results are shown in Table 3.

[0047] Table 3

[0048]

[0049] Taking the mass concentration (X) as the abscissa and the peak area (Y) as the ordinate, draw the standard curve to obtain the regression equation. The regression equation of the inosine reference substance is y = 22815x + 4411, R 2 = 1; the regression equation of the hypoxanthine reference substance is y = 37177x + 24484, R 2 = 0.9999, indicating that the inosine reference substance has a good linear relationship in the range of 4.116 μg / mL - 205.8 μg / mL, and the hypoxanthine reference substance has a good linear relationship in the range of 2.352 μg / mL - 117.6 μg / mL. Investigation of precision:

[0050] Precisely pipette 10 μL of inosine reference solution and hypoxanthine reference solution respectively, and inject them continuously for 6 times under the above chromatographic conditions. The specific experimental results are shown in Table 4 as follows:

[0051] Table 4 Precision-related data of reference solution

[0052]

[0053] As shown in Table 4, the RSD value of the peak area of inosine reference solution for 6 consecutive injections is 0.07%, and the RSD value of the peak area of hypoxanthine reference solution is 0.02%. It is proved that the precision of this method is good and meets the detection requirements for the determination of sample content.

[0054] Specificity investigation:

[0055] Take 10 μL of inosine reference solution, hypoxanthine reference solution, test solution of Glycyrrhiza uralensis Fisch. soaked Pheretima aspergillum and blank solution (95% water + 5% methanol) and inject them into the high-performance liquid chromatograph, and analyze according to the above chromatographic conditions. The specificity chromatogram is as Figure 1 . As can be seen from Figure 1 , the elution times of inosine and hypoxanthine in the chromatogram of the test solution are consistent with those in the chromatograms of inosine reference solution and hypoxanthine reference solution. There is no interference from the blank, and the resolution and theoretical plate number of the main component chromatographic peaks in the chromatogram of the test solution both meet the requirements of the Chinese Pharmacopoeia, indicating that this method has good specificity.

[0056] Example 3

[0057] Chromatographic conditions: the same as those in Example 1;

[0058] Preparation of test solution: Weigh 1 g of the powder of Glycyrrhiza uralensis Fisch. soaked Pheretima aspergillum, place it in a 10 mL stoppered graduated test tube, add 10 mL of ultrapure water, soak for 30 min, sonicate for 30 min, centrifuge at 10000 r / min at room temperature for 15 min, take the supernatant, and filter it through a 0.45 μm filter membrane to obtain the test solution.

[0059] Investigation on solution stability:

[0060] Take the above test solution and inject it for analysis according to the above chromatographic conditions at 0, 2, 4, 8, 12, 20, and 24 h after preparation respectively. The results are shown in Table 5.

[0061] Table 5 Results of stability experiment

[0062]

[0063]

[0064] As shown in Table 5, in the range of 0 - 24 h, the RSD values of the peak areas of inosine and hypoxanthine in the test solution of earthworm soaked in licorice were 0.01% and 0.04% respectively, both less than 2%, indicating that the test solution of earthworm soaked in licorice had good solution stability within 24 hours.

[0065] Repeatability investigation:

[0066] Take the powder of the test sample of earthworm soaked in licorice, prepare 6 test solutions according to the method for preparing the test solution described above, analyze according to the above chromatographic conditions, and calculate the contents of hypoxanthine and inosine in the test sample. The results are shown in Table 6.

[0067] Table 6 Results of repeatability experiment of the test solution

[0068]

[0069]

[0070] Experimental results: The content of inosine measured in 6 test samples of earthworm soaked in licorice was 0.192%, and the RSD value was 0.655%; the content of hypoxanthine was 0.075%, and the RSD value was 1.575%, both less than 2%, indicating that this method had good repeatability.

[0071] Intermediate precision investigation:

[0072] Take the powder of the test sample of earthworm soaked in licorice, prepare the test solution according to the method for preparing the test solution described above under the conditions of different times and different experimenters, analyze according to the above chromatographic conditions, and calculate the contents of hypoxanthine and inosine in the test sample. The results are shown in Table 7.

[0073] Table 7 Results of intermediate precision experiment of the test solution

[0074]

[0075] Experimental results: Under the conditions of different times and different experimenters, the content of inosine measured in earthworm soaked in licorice was about 0.19% or 1.88 mg / g, and the RSD value was 0.04% (<3%); the content of hypoxanthine measured in earthworm soaked in licorice was about 0.08% or 0.79 mg / g, and the RSD value was 0.15% (<3%), indicating that this method had good intermediate precision under the conditions of different times and different experimenters.

[0076] Example 4

[0077] Spiked recovery test:

[0078] Accurately weigh about 0.5 g of the powder of earthworm soaked in licorice with known content, a total of 9 portions, accurately add hypoxanthine and inosine reference substance solutions respectively, prepare according to the preparation method of the test solution above, and analyze according to the chromatographic conditions described in Example 1. Calculate the recovery rate using the formula recovery rate % = (C - A) / B (where A is the content of the component to be measured in the test sample, B is the amount of the reference substance added, and C is the measured value), and calculate the content.

[0079] Table 8 Results of the spike recovery experiment

[0080]

[0081]

[0082]

[0083] The average recovery rate of the hypoxanthine reference substance was calculated to be 99.23%, and the RSD value was 3.18% (n = 9) < 5.0%; the average recovery rate of the inosine reference substance was 96.63%, and the RSD value was 2.73% (n = 9) < 5.0%, indicating that the recovery rate of this method is good.

[0084] Example 5

[0085] Preparation of hypoxanthine reference substance solution: Weigh 1.2 mg of hypoxanthine reference substance, add 10 mL of ultrapure water to prepare a 0.12 mg / mL hypoxanthine reference substance solution.

[0086] Preparation of inosine reference substance solution: Weigh 2.1 mg of inosine reference substance, add 10 mL of ultrapure water to prepare a 0.21 mg / mL inosine reference substance solution.

[0087] Determination of detection limit and quantitation limit: Continuously dilute the two reference substance solutions, and analyze according to the chromatographic conditions in Example 1 until the signal-to-noise ratio (S / N) = 3. At this time, the concentration is the detection limit; when the signal-to-noise ratio (S / N) = 10, the concentration at this time is the quantitation limit. The results are shown in Table 9 below.

[0088]

[0089] Analysis of experimental results:

[0090] For the inosine reference substance: When the concentration is 0.0108 μg / mL, the signal-to-noise ratio (S / N) is 3.16, indicating that the detection limit < 0.0108 μg / mL; when the concentration is 0.0361 μg / mL, the signal-to-noise ratio (S / N) is 10.66, indicating that the quantitation limit < 0.0361 μg / mL.

[0091] Hypoxanthine reference substance: When the concentration is 0.00286 μg / mL, the signal-to-noise ratio (S / N) is 3.68, indicating that the detection limit < 0.00286 μg / mL; when the concentration is 0.00953 μg / mL, the signal-to-noise ratio (S / N) is 11.79, indicating that the quantification limit < 0.00953 μg / mL.

[0092] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A detection method for the quality control of earthworm soaked in licorice, characterized in that, The contents of hypoxanthine and inosine in the test solution are detected by high performance liquid chromatography. The chromatographic conditions include: Mobile phase A is pure water, and mobile phase B is methanol; The elution gradient is shown in the following table: The chromatographic column is a chromatographic column with octadecylsilyl bonded silica gel as the stationary phase; The test sample is earthworm soaked in licorice.

2. The detection method according to claim 1, characterized in that The chromatographic conditions further include: the detection wavelength is 252 - 256 nm.

3. The detection method according to claim 1, wherein The chromatographic conditions further include: the column temperature is 30 ± 1 °C.

4. The detection method according to claim 1, wherein The chromatographic conditions further include: the flow rate is 0.8 - 1.2 mL / min.

5. The detection method according to claim 1, characterized in that The chromatographic conditions further include: the injection volume is 10 - 15 μL.

6. The detection method according to claim 1, wherein The chromatographic conditions further include: the detector is an ultraviolet detector.

7. The detection method according to claim 1, wherein The test solution is prepared by the following method: take earthworm soaked in licorice, soak it in water and then extract it by ultrasonic wave to obtain the test solution.

8. The detection method according to claim 7, characterized in that The mass - volume ratio of the earthworm soaked in licorice to water is 1 g: 8 - 12 mL.

9. The detection method according to claim 7, wherein The soaking time is 25 - 35 min.

10. Use of the detection method according to any one of claims 1 - 9 in the quality control of earthworm soaked in licorice.

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