Content detection method for finger citruses

Abstract

The invention discloses a method for detecting the content of finger citruses, which comprises the following steps: preparing a reference solution, preparing a test solution, respectively and precisely sucking the reference solution and the test solution, injecting into a liquid chromatograph, and taking octadecylsilane chemically bonded silica as a filler; acetonitrile is used as a mobile phase A, a phosphoric acid aqueous solution is used as a mobile phase B, and gradient elution is performed.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and in particular relates to a method for detecting the content of traditional Chinese medicine. Background Art

[0002] Five-finger orange is the dried whole plant of Vitex negundo L., a plant of the Verbenaceae family. It is mainly distributed in areas south of the Yangtze River in my country and reaches the Qinling Mountains and Huaihe River in the north. It grows on hillsides, roadsides, streams and small shrubs in sparse forests. It prefers areas with plenty of sunshine and is often cultivated. It can be harvested in all seasons.

[0003] The outer skin of the root of the five-fingered mandarin orange is yellowish-white to gray-brown, often flaking. The stems and branches are yellowish-brown to tan, distinctly quadrangular in shape above and sub-cylindrical in shape below, densely covered with short hairs. The palmately compound leaves are opposite, with five or three leaflets, often wrinkled. When flattened, the complete leaflet becomes elliptical-ovate. The three central leaflets are larger, while the lateral leaflets are smaller and sessile. The apex is acuminate, the base is cuneate, and the margin is entire or with 2-5 coarse serrations on both sides. The upper surface is light green, the lower surface is off-white, and both surfaces are covered with short hairs along the veins. The inflorescence is terminal. The calyx is bell-shaped, densely covered with short white hairs, with five teeth. The corolla is pale purple, hairy, and bilabiate. The fruit is spherical or obovate, with the lower half enclosed in the persistent calyx. It has a slightly odor and a bitter, slightly astringent taste.

[0004] The functions and indications of Wuzhigan are to dispel exterior pathogens and clear away heat, dissipate dampness and eliminate phlegm, relieve cough and asthma, regulate qi and relieve pain, and stop malaria and kill parasites. It is used for colds, coughs and asthma, liver disease, abdominal distension and pain, diarrhea, dysentery, malaria, pinworms, and externally for carbuncles, swellings, sores and ringworms.

[0005] The "Guangdong Provincial Traditional Chinese Medicine Standards Volume 3" includes Wuzhigan. Its quality standard identification items only include microscopic identification, thin-layer chromatography identification methods, extracts, inspections, and medicinal pieces. There is no content determination item, which needs further improvement.

[0006] 4-Hydroxybenzoic acid in tangerine peel is a compound with a phenolic acid structure. It has multiple pharmacological effects such as anti-inflammatory and antibacterial, which are consistent with its functions and main indications. It is a key quality attribute component. In addition, 4-Hydroxybenzoic acid is highly water-soluble and is a specific component of tangerine peel. Therefore, it is urgent to develop a detection method for the content of 4-Hydroxybenzoic acid in tangerine peel. Summary of the Invention

[0007] In order to solve the problems existing in the prior art, the present invention provides a method for detecting the content of five-finger orange, comprising the following steps:

[0008] Step A: Preparation of Reference Solution: Take 4-hydroxybenzoic acid reference substance and add methanol solution to prepare a solution to obtain a reference solution;

[0009] Step B Preparation of the Test Solution Take the five-fingered orange powder passed through a No. 3 sieve, accurately weigh it, accurately add the extraction solvent, weigh it, extract it ultrasonically or under reflux, let it cool, weigh it again, make up the lost weight with the extraction solvent, shake it well, filter it, and take the filtrate to obtain the test solution;

[0010] Step C Determination Method: Accurately pipette the reference solution obtained in step A and the test solution obtained in step B, inject them into a liquid chromatograph, and determine the chromatographic column according to the following chromatographic conditions: octadecylsilane bonded silica gel as the filler; acetonitrile as mobile phase A, phosphoric acid aqueous solution as mobile phase B, and perform gradient elution as specified in the table below;

[0011]

[0012] Preferably, in step A, the reference solution is prepared by adding a methanol solution to a 4-hydroxybenzoic acid reference substance to prepare a solution containing 10 μg per ml to obtain the reference solution.

[0013] Preferably, the extraction solvent in step B is water or methanol, wherein the concentration of methanol can be 10%-100%, such as 10% methanol, 20% methanol, 50% methanol, methanol.

[0014] Preferably, in step A, preparation of the reference solution is performed by adding 50% methanol to a 4-hydroxybenzoic acid reference solution containing 10 μg per ml to obtain the reference solution.

[0015] Preferably, in step B, preparation of the test solution is to take about 0.2 g of five-fingered orange powder passed through a No. 3 sieve, accurately weigh it, place it in a stoppered conical flask, accurately add 50 ml of water, seal it tightly, weigh it, and extract it ultrasonically or under reflux for 30-180 minutes (for example, 30, 60, 90, 120, 180 minutes), let it cool, weigh it again, make up the lost weight with water, shake it well, filter it, and take the filtrate to obtain it.

[0016] Preferably, the column temperature in the chromatographic conditions of step C is 20-40°C, such as 30°C, 28°C, or 32°C.

[0017] Preferably, the chromatographic condition flow rate in step C is: 0.5-1.5 mL / min, such as 1 mL / min, 0.9 mL / min, 1.1 mL / min.

[0018] Preferably, the chromatographic detection wavelength in step C is 240-260 nm, such as 254 nm, 250 nm, or 258 nm.

[0019] Preferably, the chromatographic conditions in step C are 0.05-0.3% phosphoric acid aqueous solution as mobile phase B, such as 0.1% phosphoric acid aqueous solution, 0.05% phosphoric acid aqueous solution, or 0.2% phosphoric acid aqueous solution.

[0020] Preferably, in step C, 5-20 μl (eg, 10 μl) of the reference solution and the test solution are accurately aspirated and injected into the liquid chromatograph.

[0021] The present invention is a method for screening the content of five-fingered orange medicinal materials obtained through a large number of screening tests.

[0022] 1. Instruments and test drugs

[0023] Instruments: Agilent 1260-VWD, Agilent 1260-DAD high performance liquid chromatographs; Sartorius BCE224I-1CCN, Sartorius BCE223I-1CCN, Sartorius MSA6.6S-OCE-DM electronic balances.

[0024] Reagents: Gradient methanol, phosphoric acid, acetonitrile of chromatographic grade, methanol of analytical grade, distilled water, Watsons purified water. Sample: Wuzhigan batch number: Wuzhigan B230403, provided by Guangzhou Wanglaoji Pharmaceutical Co., Ltd.

[0025] Reference substance: 4-hydroxybenzoic acid, China Food and Drug Inspection Institute, batch number: 101149-202204, content 100%.

[0026] 2. Selection of chromatographic conditions

[0027] 2.1 Detection method

[0028] According to the general rule 0512 of Part IV of the 2020 edition of the Chinese Pharmacopoeia, the HPLC method was used to determine the content of 4-hydroxybenzoic acid, the main component of five-fingered orange.

[0029] 2.2 Exploration of analytical methods

[0030] A. Selection of chromatographic conditions

[0031] Through continuous experimentation, the mobile phase was selected as an acetonitrile-0.1% phosphoric acid solution system, and octadecylsilane bonded silica gel was finally selected as the filler; the chromatographic column used acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, and gradient elution was performed as specified in the table below; the detection wavelength was 254 nm, and the column temperature was 30°C.

[0032]

[0033] BDetermination of detection wavelength

[0034] Take the 4-hydroxybenzoic acid reference solution and perform ultraviolet absorption spectrum scanning between 190 and 400 nm. The results show that the maximum absorption wavelength of 4-hydroxybenzoic acid is at 254 nm, so 254 nm is selected as the wavelength for determining the content of 4-hydroxybenzoic acid in five-fingered orange.

[0035] 3. Preparation and inspection of test samples

[0036] 3.1 Investigation of extraction methods

[0037] Take 4 portions of 5-fingered orange powder (passed through No. 3 sieve) from batch B230403 of 5-fingered orange, each about 0.2 g, accurately weighed, placed in a round-bottom flask, accurately added 50 ml of methanol, weighed, ultrasonically extracted (power 500 W, frequency 40 kHz) and heated under reflux for 120 minutes, cooled, weighed again, supplemented with methanol to make up the lost weight, shaken well, filtered, and the filtrate was obtained. The results are shown in Table 1.

[0038] Table 1 Extraction method investigation test results

[0039]

[0040] Test results: The extraction method has a greater impact on the content of 4-hydroxybenzoic acid. The content of 4-hydroxybenzoic acid extracted by heating reflux is higher than that by ultrasonic extraction. Therefore, heating reflux extraction is selected as the extraction method.

[0041] 3.2 Investigation of extraction solvent

[0042] Take 10 portions of 5-fingered tangerine powder (passed through No. 3 sieve) from batch B230403 of 5-fingered tangerine, about 0.2 g per portion, accurately weigh, and place in a round-bottom flask. Accurately add 50 ml each of 10% methanol, 20% methanol, 50% methanol, methanol, and water, weigh the weight, heat and reflux for 120 minutes, cool, and weigh again. Make up the lost weight with each solvent, shake well, filter, and take the filtrate to obtain the result. See Table 2.

[0043] Table 2 Extraction solvent investigation test results

[0044]

[0045] Test results: When water is used as solvent, the content of 4-hydroxybenzoic acid is the highest, so water is selected as the extraction solvent.

[0046] 3.3 Investigation of extraction time

[0047] Take 10 portions of 5-fingered orange powder (passed through No. 3 sieve) from batch B230403 of 5-fingered orange, about 0.2 g per portion, accurately weigh, place in a round-bottom flask, accurately add 50 ml of water each, weigh the weight, heat and reflux for 30, 60, 90, 120, and 180 minutes respectively, cool, weigh again, make up the lost weight with water, shake well, filter, and take the filtrate to obtain the result. See Table 3.

[0048] Table 3 Extraction time investigation test results

[0049]

[0050] Experimental results: The extraction was complete after heating under reflux for 120 min, so the extraction time was selected as 120 min.

[0051] Conclusion: The preparation method of the test sample is to take about 0.2g of citrus aurantium powder (passed through No. 3 sieve), accurately weigh it, put it in a round-bottom flask, accurately add 50ml of water respectively, weigh the weight, heat and reflux for 120 minutes, cool, weigh the weight again, make up the lost weight with water, shake well, filter, and take the filtrate to obtain the product.

[0052] Glossary

[0053] When the dosage is "about", it means that the dosage should not exceed ±10% of the specified amount. For example, if about 0.2g of wuzhigan powder passes through a No. 3 sieve, it means that 0.18g-0.22g of wuzhigan powder passes through a No. 3 sieve.

[0054] Beneficial effects of the present invention

[0055] First, it has strong specificity. The HPLC method for the determination of 4-hydroxybenzoic acid, through specific chromatographic conditions and gradient elution procedures, can accurately separate and determine the target component, avoiding interference from other components. Existing standard methods such as thin-layer chromatography identification have relatively weak specificity.

[0056] Second, it is quantitatively accurate. This method can accurately determine the content of 4-hydroxybenzoic acid, providing accurate data for quality control. Existing standards such as extractables can only roughly reflect the content of the ingredient. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 This is the HPLC chromatogram of the reference substance in Example 1

[0058] Figure 2 This is the HPLC chromatogram of the test sample in Example 1

[0059] Figure 3 This is the standard curve of 4-hydroxybenzoic acid content DETAILED DESCRIPTION

[0060] Example 1

[0061] Preparation of reference solution: Take an appropriate amount of 4-hydroxybenzoic acid reference substance and add 50% methanol to prepare a solution containing 10 μg per ml.

[0062] Preparation of the test solution: Take about 0.2 g of citrus aurantium powder (passed through No. 3 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 50 ml of water, stopper it, weigh it, reflux and extract it for 120 minutes, cool it, weigh it again, make up the lost weight with water, shake it well, filter it, and take the filtrate to obtain the solution.

[0063] Determination method: Accurately aspirate 10 μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine according to the following chromatographic conditions. The HPLC chromatogram is shown in the figure. Figure 1 、 Figure 2 ;

[0064] Chromatographic conditions: Chromatographic column: octadecylsilane bonded silica gel as filler (4.6*250 mm, 5 μm); acetonitrile as mobile phase A, 0.1% phosphoric acid aqueous solution as mobile phase B, gradient elution as specified in the table below; column temperature: 30°C; flow rate: 1.0 mL / min; detection wavelength: 254 nm.

[0065]

[0066] Calculate the content, the results are shown in Table 4

[0067] Table 4 Test results of five-finger orange

[0068]

[0069] Example 2 Methodological Investigation

[0070] Investigation of linear relationships

[0071] Take 5 mg of 4-hydroxybenzoic acid reference substance, accurately weigh it, place it in a 50 ml volumetric flask, add 50% methanol to make a solution containing 0.1 mg per 1 ml, and obtain the reference substance stock solution (0.10048 mg / ml). Accurately draw 0.25, 0.5, 1, 2.5, 5, 10, and 25 ml of the reference substance stock solution into a 25 ml volumetric flask, add 50% methanol to dilute to the scale, shake well, draw 10 μl of each of the above solutions, inject into the liquid chromatograph, and measure the peak area according to the chromatographic conditions of Example 1. The results are shown in Table 5. Figure 3 .

[0072] Table 5 Linear relationship investigation test results

[0073]

[0074] Test results: Linear equation: Y=61.817X+11.035, r=1; the injection concentration of 4-hydroxybenzoic acid is in the range of 1.0048~100.48μg / ml, showing a good linear relationship with the peak area.

[0075] Precision test

[0076] Take the same test solution of Wuzhigan (Wuzhigan B230403 batch) and inject it into the liquid chromatograph for 6 consecutive times. The peak area is determined according to the chromatographic conditions of Example 1. The results are shown in Table 6.

[0077] Table 6 Precision test results

[0078]

[0079] Test results: The RSD of the precision test is 0.55%, n=6, and the instrument precision is good.

[0080] Solution stability investigation

[0081] The same sample solution of Ophiopogon japonicus (Ophiopogon japonicus B230403 batch) was taken and injected into the liquid chromatograph at different times. The chromatographic conditions of Example 1 were used for determination, the peak areas were recorded, and the RSD results were calculated. See Table 7.

[0082] Table 7 Solution stability test results

[0083]

[0084]

[0085] Test results: After the test solution was placed for 24 hours, the average value of the 4-hydroxybenzoic acid peak area was 469.484, and the RSD was 0.15%. The test solution was stable at room temperature for 24 hours.

[0086] Repeatability test

[0087] Take 6 portions of 5-fingered citrus (5-fingered citrus B230403 batch) powder (passed through a No. 3 sieve), approximately 0.2 g each, accurately weigh, place in a round-bottom flask, accurately add 50 ml of water to each portion, weigh the weight, heat and reflux for 120 minutes, cool, weigh again, make up the lost weight with water, shake well, filter, and collect the filtrate. Take 10 μl each of the reference solution and the test solution, inject into a liquid chromatograph, and measure according to the chromatographic conditions of Example 1. Record the chromatogram and calculate the content. The results are shown in Table 8.

[0088] Table 8 Repeatability test results

[0089]

[0090] Test results: The average value of 4-hydroxybenzoic acid content was 0.193%, and the RSD was 2.48%, which was in line with the requirements of the pharmacopoeia, indicating that the repeatability test results were good.

[0091] Sample recovery test

[0092] Take 6 portions of five-fingered orange (five-fingered orange B230403 batch) powder (passed through No. 3 sieve), accurately weighed, each portion is about 0.1 g, and the other half is accurately added with a reference solution equivalent to 100% of the added amount. The remaining steps are carried out according to the method of Example 1. Take 10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, record the chromatogram, and calculate the recovery rate. The results are shown in Table 9.

[0093] Table 9 Sample recovery test

[0094]

[0095] Test results: The average recovery rate of 4-hydroxybenzoic acid content determination was 98.09%, ranging from 95.80% to 101.26%, with an RSD of 2.01%, which was in line with the requirements of the pharmacopoeia and showed that the experimental method was feasible.

[0096] Durability test

[0097] When the determination conditions (wavelength, column temperature, flow rate, ratio of mobile phase, amount of phosphoric acid and chromatographic column) change slightly, the change in content is examined.

[0098] a Wavelength change

[0099] Take about 0.2 g of citrus aurantium (Citrus aurantium B230403 batch) powder (passed through a No. 3 sieve) and operate according to the method of Example 1 except for the wavelength. When the wavelength changes slightly, sample is injected respectively, the chromatogram is recorded, and the content of the sample is calculated. The results are shown in Table 10.

[0100] Table 10 Wavelength change test results

[0101]

[0102]

[0103] Test results: When the wavelength changed slightly, the average value of 4-hydroxybenzoic acid content was 0.180% and the RSD was 0.32%, which had little effect on the content determination results.

[0104] b Column temperature change

[0105] Take about 0.2 g of citrus aurantium (Citrus aurantium B230403 batch) powder (passed through a No. 3 sieve) and operate according to the method of Example 1 except for the column temperature. When the column temperature changes slightly, sample is injected respectively, the chromatogram is recorded, and the content of the sample is calculated. The results are shown in Table 11.

[0106] Table 11 Column temperature change test results

[0107]

[0108] Test results: When the column temperature changed slightly, the average value of 4-hydroxybenzoic acid content was 0.179% and the RSD was 0.65%, which had little effect on the content determination results.

[0109] cFlow rate change

[0110] Take about 0.2 g of citrus aurantium (Citrus aurantium B230403 batch) powder (passed through a No. 3 sieve) and operate according to the method of Example 1 except the flow rate. When the initial flow rate changes slightly, sample is injected respectively, the chromatogram is recorded, and the content of the sample is calculated. The results are shown in Table 12.

[0111] Table 12 Flow rate change test results

[0112]

[0113] Test results: When the flow rate changes slightly, the average value of 4-hydroxybenzoic acid content is 0.180%, and the RSD is 0.32, which has little effect on the content determination results.

[0114] d Change in the initial mobile phase ratio

[0115] Take about 0.2 g of citrus aurantium (Citrus aurantium B230403 batch) powder (passed through a No. 3 sieve) and operate according to the method of Example 1 except for the ratio of the initial mobile phase. When the ratio of the initial mobile phase changes slightly, the sample is injected separately, the chromatogram is recorded, and the content of the sample is calculated. The results are shown in Table 13.

[0116] Table 13 Results of the initial mobile phase ratio change test

[0117]

[0118] Test results: When the proportion of the mobile phase changed slightly, the average value of the 4-hydroxybenzoic acid content was 0.185% and the RSD was 4.22%, which had little effect on the content determination results.

[0119] eChanges in the amount of phosphoric acid used in the mobile phase

[0120] About 0.2 g of citrus aurantium (Citrus aurantium B230403 batch) powder (passed through a No. 3 sieve) was taken. Except for the amount of phosphoric acid used in the mobile phase, the remaining steps were carried out according to the method of Example 1. When the amount of phosphoric acid used in the mobile phase changed slightly, samples were injected separately, the chromatograms were recorded, and the content of the samples was calculated. The results are shown in Table 14.

[0121] Table 14 Results of the experiment on the change of the amount of phosphoric acid used in the mobile phase

[0122]

[0123] Test results: When the amount of phosphoric acid in the mobile phase changes slightly, the average value of 4-hydroxybenzoic acid content is 0.185% and the RSD is 4.22%, which has little effect on the content determination results.

[0124] f Column changes

[0125] Take about 0.2 g of citrus aurantium (Citrus aurantium B230403 batch) powder (passed through a No. 3 sieve), select different chromatographic columns, and operate according to the method of Example 1. When the chromatographic column changes, inject the sample separately, record the chromatogram, and calculate the content of the sample. The results are shown in Table 15.

[0126] Table 15 Results of investigation on chromatographic column changes

[0127]

[0128] Test results: When the chromatographic column was changed, the average 4-hydroxybenzoic acid content was 0.180% and the RSD was 0, which had little effect on the content determination results. Conclusion: When the determination conditions (wavelength, column temperature, flow rate, initial mobile phase ratio, mobile phase phosphoric acid amount, and chromatographic column) were slightly changed, the results of the 4-hydroxybenzoic acid determination were basically unaffected. The robustness test results of this method are good.

[0129] Intermediate precision

[0130] Six portions of 5-fingered citrus (5-fingered citrus B230403 batch) powder (passed through a No. 3 sieve) were accurately weighed and placed in a round-bottom flask. 50 ml of water was accurately added to each portion and the weight was determined. The mixture was heated under reflux for 120 minutes, cooled, and weighed again. The remaining weight was made up with water, shaken well, filtered, and the filtrate was collected. The filtrate was injected into a liquid chromatograph and analyzed according to the chromatographic conditions of Example 1. The chromatogram was recorded and the content was calculated. The results are shown in Table 16.

[0131] Table 16 Intermediate precision test results of tangerine

[0132]

[0133] Test results: The average value of the intermediate precision of the 4-hydroxybenzoic acid content determination was 0.188%, and the RSD was 4.38%, which met the requirements of the pharmacopoeia, indicating that the intermediate precision test results were good. The above test results show that the high performance liquid chromatography method for determining the content of 4-hydroxybenzoic acid in tangerine peel has a good linear relationship, and the precision, repeatability, stability, sample recovery rate and intermediate precision test results meet the requirements. The durability test results are good. Therefore, the high performance liquid chromatography method for detecting the content of 4-hydroxybenzoic acid in tangerine peel is accurate, reliable and durable.

Claims

1. A method for detecting the content of tangerine peel, comprising the following steps: Step A: Preparation of Reference Solution: Take 4-hydroxybenzoic acid reference substance and add methanol solution to prepare a solution to obtain a reference solution; Step B Preparation of the Test Solution Take the five-fingered orange powder passed through a No. 3 sieve, accurately weigh it, accurately add the extraction solvent, weigh it, extract it ultrasonically or under reflux, let it cool, weigh it again, make up the lost weight with the extraction solvent, shake it well, filter it, and take the filtrate to obtain the test solution; Step C determination method: Accurately aspirate the reference solution obtained in step A and the test solution obtained in step B, inject them into the liquid chromatograph, and determine them according to the following chromatographic conditions: Chromatographic column: Octadecylsilane bonded silica gel as filler; acetonitrile as mobile phase A, phosphoric acid aqueous solution as mobile phase B, gradient elution as specified in the table below; 2. The content detection method according to claim 1, characterized in that Step A: Preparation of Reference Solution: Take 4-hydroxybenzoic acid reference substance and add methanol solution to prepare a solution containing 10 μg per ml to obtain the reference solution.

3. The method for detecting the content of a compound according to any one of claims 1 to 2, wherein: The extraction solvent in step B is water or methanol, wherein the concentration of methanol can be 10%-100%.

4. The content detection method according to claim 1, characterized in that Step A: Preparation of Reference Solution: Take 4-hydroxybenzoic acid reference substance and add 50% methanol to prepare a solution containing 10 μg per ml to obtain the reference solution.

5. The content detection method according to claim 1, characterized in that Step B Preparation of the test solution: Take about 0.2 g of five-fingered orange powder passed through a No. 3 sieve, accurately weigh it, place it in a stoppered conical flask, accurately add 50 ml of water, stopper it, weigh it, and extract it ultrasonically or under reflux for 30-180 minutes. Let it cool, weigh it again, make up the lost weight with water, shake it well, filter it, and take the filtrate to obtain the product.

6. The content detection method according to claim 1, characterized in that: The column temperature in the chromatographic conditions of step C is 20-40°C.

7. The content detection method according to claim 1, characterized in that: The chromatographic condition flow rate in step C is 0.5-1.5 mL / min.

8. The content detection method according to claim 1, characterized in that: The chromatographic conditions in step C include a detection wavelength of 240-260 nm.

9. The content detection method according to claim 1, characterized in that: The chromatographic conditions of step C are as follows: 0.05-0.3% phosphoric acid aqueous solution as mobile phase B.

10. The content detection method according to claim 1, characterized in that: In step C, 5-20 μl of the reference solution and the test solution are accurately aspirated and injected into the liquid chromatograph.

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