Detection method of Rhodiola rosea extract

Abstract

The invention belongs to the technical field of drug analysis, and specifically relates to a detection method for a rhodiola rosea extract, comprising the following steps: grinding a to-be-detected object, taking a fine powder of the to-be-detected object, adding an extracting solution, ultrasonically dissolving, cooling to room temperature, centrifuging, taking a supernatant, passing through a microporous filter membrane, adding an extracting solution to dilute, and shaking well to use a test solution; weighing a rhodiola rosea extract reference substance, adding the extracting solution, ultrasonically dissolving, cooling to room temperature, adding an extracting solution to dilute, and shaking well to use a reference substance solution; using a high performance liquid chromatograph, first needle detection of the reference substance solution, recording a chromatogram, injecting a test solution under the same chromatographic conditions, recording the chromatogram and data of the test solution, and using an external standard method to quantitatively analyze the rhodiola rosea extract in the test solution; the invention overcomes the problems of unstable separation between the rhodiola rosea extract and adjacent unknown impurities, inability to accurately locate and calculate the rhodiola rosea extract, long detection cycle and high cost, and has good reproducibility.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and particularly relates to a detection method for a rhodiola rosea extract. Background Art

[0002] Chinese name: Rhodiola rosea extract, alias: Salidroside, Salidroside, active ingredient: Salidroside, common specifications and contents: 1%, 3%, 10%, physical properties: brown powder. Rhodiola rosea extract is derived from the Crassulaceae plant Rhodiola rosea, and is a natural extract that has been refined, concentrated, and dried. It is widely used in medicine, health care products, and dosage forms such as Anrui tablets and Anpu capsules. Rhodiola rosea extract is an extract from the root of Rhodiola rosea, with a sweet aroma and a bitter taste. The main ingredients are salidroside and aglycones tyrosol and rosavidin.

[0003] Other reports suggest that the use of Rhodiola rosea extract can protect cardiovascular tissues in rats from stress-induced catecholamine release and reduce adrenaline-induced arrhythmias. The anti-arrhythmic effects of Rhodiola rosea extract are thought to be secondary to its ability to induce opioid biosynthesis and involve stimulation of peripheral kappa opioid receptors. Rhodiola rosea extract has also shown efficacy against heart problems caused or exacerbated by stress. The effects on these conditions are based on its ability to reduce the amount of catecholamines and steroids released by the adrenal glands in response to stress. Abnormal secretion of these stress hormones can lead to an increase in blood pressure, cholesterol, and potassium levels and increase the risk of heart disease. Rhodiola rosea has been found to reduce harmful blood lipids and therefore reduce the risk of heart disease. It also reduces the amount of cyclic AMP (c-AMP) released into heart cells. Cyclic AMP is related to ATP (adenosine triphosphate), the main energy molecule in the human body. C-AMP acts as a "second messenger" or liaison between the intracellular and extracellular environments. It helps intracellular calcium flow into the heart, greatly increasing the potential for heart muscle contraction. Rhodiola rosea thus regulates the heartbeat and prevents heart rhythm disturbances. Rhodiola rosea has potent antioxidant properties. By limiting the adverse effects of free radical damage, it can effectively protect against diseases caused by aging.

[0004] Rhodiola rosea extract not only has the effects of enhancing immune function, protecting cardiovascular and cerebrovascular, and anti-cancer and anti-depression, but also has certain effects on liver damage, can reduce serum. Reduce the degree of degeneration and necrosis of liver cells; has the effect of delaying aging, and has strong antioxidant function, which can reduce the activity of acidic enzymes. It can also promote cell metabolism, improve the body's immune ability and muscle exercise ability; can resist radiation, has a good effect of inhibiting induced mutations, and can also promote the generation of germ cells. Rhodiola rosea extract has been shown to alleviate stress-induced cardiovascular tissue damage and dysfunction. Rhodiola rosea extract can prevent the decrease in cardiac contractility secondary to environmental pressure in the state of rapid freezing and help stabilize contractility. Pretreatment with rhodiola rosea appears to induce a beneficial adaptive response to this type of stress. When mice taking rhodiola rosea were exposed to the state of rapid freezing, the decrease in cardiac contractility was prevented, and the stable contractility of cardiac tissue continued to appear during the perfusion period.

[0005] Salidroside is a compound extracted from the dried roots and rhizomes or dried whole grass of the Crassulaceae plant Rhodiola rosea. It has the functions of preventing tumors, enhancing immune function, delaying aging, resisting fatigue, resisting hypoxia, preventing radiation, bidirectionally regulating the central nervous system, repairing and protecting the body, etc. It is usually used to treat chronic patients and weak and susceptible patients. It is clinically used to treat neurasthenia and neurosis, improve attention and memory, high-altitude polycythemia, and hypertension; as a stimulant, it is used to improve intelligence, improve autonomic nerve-vascular tension disorders with muscle weakness, etc., and is used for diseases with increased free radicals, such as tumors, radiation damage, emphysema, senile cataracts, etc. It is also used as a tonic for impotence, etc. Salidroside preparations are also used in sports medicine and aerospace medicine, and for the health protection of workers under various special environmental conditions. Rhodiola rosea extract has the functions of adaptogen and bidirectional regulation. It has a wide range of clinical treatments, few side effects, a large market space, and good clinical effects.

[0006] The current editions of domestic and foreign pharmacopoeias, patents and references contain very few detection methods for Rhodiola rosea extracts, which restricts the application and development of Rhodiola rosea extracts in pharmaceutical, chemical, medical beauty, health care and other products. Summary of the invention

[0007] In view of the deficiencies in the prior art, the object of the present invention is to provide a method for detecting a Rhodiola rosea extract, which has stable separation from adjacent unknown impurities and short operating time.

[0008] The detection method of the Rhodiola rosea extract of the present invention comprises the following steps:

[0009] (1) Grind the substance to be tested into powder, add the extract, dissolve by ultrasonic, cool to room temperature, centrifuge, take the supernatant, filter through a microporous membrane, dilute with the extract, shake well, and use it as the test solution;

[0010] (2) Weigh the Rhodiola rosea extract reference substance and add it to the extract solution, dissolve it by ultrasonication, cool it to room temperature, dilute it with the extract solution, and shake it well to prepare the reference substance solution;

[0011] (3) Using a high performance liquid chromatograph, the reference solution was tested as the first injection, and the chromatogram was recorded as an evaluation index of the tailing factor of the chromatographic system. Under the same chromatographic conditions, the test solution was injected, and the chromatogram and data of the test solution were recorded. The Rhodiola rosea extract in the test solution was quantitatively analyzed using the external standard method;

[0012] During the detection process of the high performance liquid chromatograph, mobile phase A and mobile phase B are used for gradient elution, wherein the mobile phase A is methanol-0.02 mol / L sodium acetate solution in a volume ratio of 9:91; the mobile phase B is methanol-0.02 mol / L sodium acetate solution in a volume ratio of 50:50, the injection volume is 10 μL, the mobile phase flow rate is 1.0 mL / min, the detection wavelength is 215 nm, and the column temperature is 30°C.

[0013] Step (1) includes the following steps:

[0014] Grind the object to be tested, take 2.5g of the theoretical content of Rhodiola rosea extract, put it in a 50mL volumetric flask, add the extract, dissolve it by ultrasonic, cool to room temperature, dilute it to the scale with the extract, shake well, centrifuge, take the supernatant, and filter it through a microporous filter membrane. Accurately measure 5mL of the filtrate and put it in a 20mL volumetric flask, add the extract to dilute it to the scale, shake well, and use it as the test solution.

[0015] The centrifugal speed is 8000 rpm and the centrifugal time is 10 minutes.

[0016] The pore size of the microporous filter membrane is 0.45 μm.

[0017] Step (2) includes the following steps:

[0018] Weigh 20 mg of Rhodiola rosea extract reference substance, place it in a 100 mL volumetric flask, add the extract, dissolve it by ultrasonic, cool to room temperature, dilute to the scale with methanol, shake well, measure 5 mL and place it in a 20 mL volumetric flask, add the extract to dilute to the scale, shake well, as the reference substance solution.

[0019] The extract is methanol.

[0020] Step (3) includes the following steps:

[0021] Using a high performance liquid chromatograph, the reference solution was detected in the first injection, and the chromatogram was recorded as an evaluation index of the tailing factor of the chromatographic system. The content of Rhodiola rosea extract in the test solution was calculated by the external standard method. Under the same chromatographic conditions, the test solution was injected, and the chromatogram and data of the test solution were recorded.

[0022] The tailing factor T 拖尾 ≤1.5.

[0023] The high performance liquid chromatograph is preferably Shimadzu LC-20AT or LC-20AD; the detector is SPD-20A; and the chromatographic column is Agilent ZORBAX Eclipse XDB-C18 column (4.6×250 mm, 5 μm).

[0024] The gradient elution sequence of the mobile phase A and the mobile phase B is:

[0025] 0→45min, mobile phase A 100 parts, mobile phase B 0 parts;

[0026] 45.1→50min, mobile phase A 100 parts→0 parts, mobile phase B 0 parts→100 parts;

[0027] 50.1→120min, mobile phase A 0 parts, mobile phase B 100 parts;

[0028] 120.1→125min, mobile phase A 0 parts→100 parts, mobile phase B 100 parts→0 parts;

[0029] 125.1→160min, mobile phase A 100 parts, mobile phase B 0 parts.

[0030] Take the detection of Rhodiola rosea extract in Anrui Tablets as an example. The specific formula of Anrui Tablets is made of Rhodiola rosea extract, American ginseng extract, Ganoderma lucidum extract, Pueraria extract, microcrystalline cellulose, and magnesium stearate as raw materials and auxiliary materials. Non-clinical trials have shown that this product has auxiliary protective function against chemical liver damage.

[0031] The theoretical weight of Anrui tablets is 1g / tablet, of which 100g contains 0.18g of Rhodiola rosea. Take 20 tablets of Anrui tablets, grind them into fine powder, about 2.5g, put them in a 50mL volumetric bottle, add an appropriate amount of methanol, ultrasonically dissolve for 10 minutes, cool to room temperature, dilute to the mark with methanol, shake well, centrifuge at 8000 rpm for 10 minutes, take the supernatant, filter through a 0.45μm filter membrane, accurately measure 5mL of the filtrate and put it in a 20mL volumetric bottle, dilute to the mark with methanol, shake well, and use it as the test solution.

[0032] Weigh 20 mg of Rhodiola rosea extract reference substance, place it in a 100 mL volumetric flask, add an appropriate amount of methanol, dissolve by ultrasonication, cool to room temperature, dilute to the mark with methanol, shake well, measure 5 mL and place it in a 20 mL volumetric flask, dilute to the mark with methanol, shake well, as the reference substance solution;

[0033] Take the first needle of the reference solution for testing and record the chromatogram as the evaluation index of the tailing factor of the chromatographic system. The tailing factor T 拖尾 ≤1.5, calculate the content of Rhodiola rosea extract in the test solution by the external standard method, inject the test solution under the same chromatographic conditions, and record the chromatogram and data of the test solution.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] 1) The present invention provides a method for detecting a Rhodiola rosea extract, which can accurately and effectively detect and calculate the Rhodiola rosea extract, thereby ensuring stricter control of the Rhodiola rosea extract and improving the quality of the product.

[0036] 2) In the detection method provided by the present invention, the column temperature is selected to be 30°C under the chromatographic conditions to reduce the load of the instrument system and more effectively ensure the service life of the chromatographic column.

[0037] 3) In the detection method provided by the present invention, the running time is 160 minutes, which greatly reduces the sample running time compared with the prior art method, saves mobile phase, and reduces the discharge of wastewater.

[0038] 4) In the detection method provided by the present invention, the enrichment operation process of the Rhodiola rosea extract, that is, the test solution is centrifuged at a speed of 8000 rpm for 10 minutes and the supernatant is taken, which effectively improves the clarity of the test solution, increases the permeability of the filter membrane, and extends the service life of the filter membrane.

[0039] 5) In the detection method provided by the present invention, methanol-0.02 mol / L sodium acetate solution = 50:50 is used as the mobile phase B under the chromatographic conditions, which overcomes the problems of unstable separation degree with adjacent unknown impurities and inability to accurately locate and calculate the Rhodiola rosea extract, as well as the problem that it takes 260 minutes to complete a one-shot determination of the test solution, the running time is too long, and the detection cycle and cost are high.

[0040] 6) In the detection method provided by the present invention, gradient elution is used to solve the problem that the same component is sampled and measured twice, the results are very different, the reproducibility is poor, and the separation degree with adjacent unknown impurities is unstable, making it impossible to accurately locate and calculate the Rhodiola rosea extract. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The present invention provides a typical chromatogram of the Rhodiola rosea extract reference solution detection method.

[0042] Figure 2 This is a typical chromatogram for intermediate precision detection of the method provided by the present invention.

[0043] Figure 3 The method provided by the present invention measures a typical chromatogram of a commercially available Anrui tablet sample (batch number: 2007011).

[0044] Figure 4 This is a typical chromatogram of the isocratic detection method of Rhodiola rosea extract in Comparative Example 1.

[0045] Figure 5 This is a gradient elution chromatogram under the chromatographic conditions of Comparative Example 2 with methanol-0.02 mol / L sodium acetate solution = 30:70 as mobile phase B. DETAILED DESCRIPTION

[0046] The present invention will be further described below with reference to the embodiments.

[0047] Unless otherwise specified, all raw materials used in the examples are commercially available.

[0048] Preparation of typical chromatograms for detection of Rhodiola rosea extract reference solution:

[0049] (1) Rhodiola rosea extract reference solution: Weigh 20 mg of Rhodiola rosea extract reference solution into a 100 mL volumetric flask, add an appropriate amount of methanol, dissolve by ultrasonication, cool to room temperature, dilute to the mark with methanol, shake well, measure 5 mL into a 20 mL volumetric flask, dilute to the mark with methanol, shake well, and use as the reference solution.

[0050] (2) The HPLC instrument used was Shimadzu LC-20AT; the detector was SPD-20A; the chromatographic column was Agilent ZORBAX Eclipse XDB-C18 column (4.6×250mm, 5μm). The mobile phase A for HPLC detection was methanol-0.02mol / L sodium acetate solution = 9:91, and the mobile phase B was methanol-0.02mol / L sodium acetate solution = 50:50 under the chromatographic conditions; the injection volume was 10μL, the flow rate was 1.0mL / min, the detection wavelength was 215nm; and the column temperature was 30℃.

[0051] In the detection method provided in the present application, gradient elution is adopted under the chromatographic conditions, and the gradient elution order of mobile phase A and mobile phase B is:

[0052] 0→45min, mobile phase A 100 parts, mobile phase B 0 parts;

[0053] 45.1→50min, mobile phase A 100 parts→0 parts, mobile phase B 0 parts→100 parts;

[0054] 50.1→120min, mobile phase A 0 parts, mobile phase B 100 parts;

[0055] 120.1→125min, mobile phase A 0 parts→100 parts, mobile phase B 100 parts→0 parts;

[0056] 125.1→160min, mobile phase A 100 parts, mobile phase B 0 parts.

[0057] Take a needle of the reference solution for testing and record the chromatogram. Figure 1 :The method provided by the present invention detects a typical chromatogram of a reference solution of a Rhodiola rosea extract. Technical features: It overcomes the defects of large deviation and poor reproducibility of the results of two injections of the same sample. It takes 160 minutes to complete a single injection of the reference solution, which greatly shortens the injection time and reduces the inspection cost. The tailing factor of the main peak is ≤1.5, and the maximum tailing factor of the main peak is only 0.98.

[0058] Example 1

[0059] The detection method of the Rhodiola rosea extract comprises the following steps:

[0060] (1) Test solution: The theoretical weight of commercially available Anrui tablets (batch number: 2007011) is 1g / tablet, of which 100g contains 0.18g of Rhodiola rosea. Take 20 Anrui tablets, grind them into powder, take 2.5g of fine powder, put it in a 50mL volumetric flask, add an appropriate amount of methanol, sonicate for 10 minutes to dissolve, cool to room temperature, dilute to scale with methanol, shake well, centrifuge at 8000 rpm for 10 minutes, take the supernatant, filter through a 0.45μm filter membrane, accurately measure 5mL of the filtrate and put it in a 20mL volumetric flask, dilute to scale with methanol, shake well, and use it as the test solution. Prepare four replicates in parallel.

[0061] (2) The HPLC instrument used was Shimadzu LC-20AD (replaced with another HPLC instrument); the detector was SPD-20A; the chromatographic column was Agilent ZORBAX Eclipse XDB-C18 column (4.6×250mm, 5μm, replaced with another chromatographic column of the same brand, model and different batch number). The mobile phase A used for HPLC detection was: methanol-0.02mol / L sodium acetate solution = 9:91, and the mobile phase B was methanol-0.02mol / L sodium acetate solution = 50:50 under the chromatographic conditions; the injection volume was 10μL, the flow rate was 1.0mL / min, the detection wavelength was 215nm; the column temperature was 30℃.

[0062] Gradient elution was used under chromatographic conditions, and the gradient elution sequence of mobile phase A and mobile phase B was:

[0063] 0→45min, mobile phase A 100 parts, mobile phase B 0 parts;

[0064] 45.1→50min, mobile phase A 100 parts→0 parts, mobile phase B 0 parts→100 parts;

[0065] 50.1→120min, mobile phase A 0 parts, mobile phase B 100 parts;

[0066] 120.1→125min, mobile phase A 0 parts→100 parts, mobile phase B 100 parts→0 parts;

[0067] 125.1→160min, mobile phase A 100 parts, mobile phase B 0 parts.

[0068] Take a needle of the test solution and record the chromatogram. Figure 2 :The typical chromatogram of intermediate precision detection of the method provided by the present invention, Figure 2 The chromatogram data are shown in Table 1 below.

[0069] Table 1 Chromatographic data

[0070]

[0071] Technical features: The peak area and peak time of Rhodiola rosea extract measured 4 times in parallel on the same sample are relatively stable, and the slight fluctuation is within the error tolerance range; and the tailing factor of the main peak is ≤1.5, and the maximum tailing factor of the main peak is only 0.98, which meets the requirements. The tailing factor and theoretical plate number are stable when the chromatographic column is replaced, indicating that the intermediate precision result is good under the chromatographic conditions. The method of the present invention is applicable to instruments of different detection models, and the results are indistinguishable, the intermediate precision result is good, and the tolerance of the method of the present invention is good.

[0072] Example 2

[0073] The detection method of the Rhodiola rosea extract comprises the following steps:

[0074] (1) Test solution: The theoretical weight of commercially available Anrui tablets (batch number: 2007011) is 1g / tablet, of which 100g contains 0.18g of Rhodiola rosea. Take 20 commercially available Anrui tablets (batch number: 2007011), grind them into powder, take 2.5g of fine powder, put it into a 50mL volumetric flask, add an appropriate amount of methanol, sonicate for 10 minutes to dissolve, cool to room temperature, dilute to scale with methanol, shake well, centrifuge at 8000 rpm for 10 minutes, take the supernatant, filter through a 0.45μm filter membrane, accurately measure 5mL of the filtrate and put it into a 20mL volumetric flask, dilute to scale with methanol, shake well, and use it as the test solution.

[0075] (2) The HPLC instrument used was Shimadzu LC-20AD (replaced with another HPLC instrument); the detector was SPD-20A; the chromatographic column was Agilent ZORBAX Eclipse XDB-C18 column (4.6×250mm, 5μm, replaced with another chromatographic column of the same brand, model and different batch number). The mobile phase A used for HPLC detection was: methanol-0.02mol / L sodium acetate solution = 9:91, and the mobile phase B was methanol-0.02mol / L sodium acetate solution = 50:50 under the chromatographic conditions; the injection volume was 10μL, the flow rate was 1.0mL / min, the detection wavelength was 215nm; the column temperature was 30℃.

[0076] Gradient elution was used under chromatographic conditions, and the gradient elution sequence of mobile phase A and mobile phase B was:

[0077] 0→45min, mobile phase A 100 parts, mobile phase B 0 parts;

[0078] 45.1→50min, mobile phase A 100 parts→0 parts, mobile phase B 0 parts→100 parts;

[0079] 50.1→120min, mobile phase A 0 parts, mobile phase B 100 parts;

[0080] 120.1→125min, mobile phase A 0 parts→100 parts, mobile phase B 100 parts→0 parts;

[0081] 125.1→160min, mobile phase A 100 parts, mobile phase B 0 parts.

[0082] Take a needle of the test solution and record the chromatogram. Figure 3 :The method provided by the present invention detects a typical chromatogram of a commercially available Anrui tablet sample (batch number: 2007011). The main peak tailing factor is 0.90, which is less than 1.5 and meets the requirements. Technical features: The specificity test results are good, and the blank excipients, blank solvents, and adjacent impurity peaks do not interfere with the determination at the peak time of the Rhodiola rosea extract, and thus the detection method can perform quantitative analysis on the Rhodiola rosea extract.

[0083] Comparative Example 1

[0084] The detection method of Rhodiola rosea extract comprises the following steps:

[0085] Test solution: The theoretical weight of Anrui tablets is 1g / tablet, of which 100g contains 0.18g of Rhodiola rosea. Take 20 tablets of Anrui tablets, grind them into fine powder, put 2.5g into a 50mL volumetric bottle, add an appropriate amount of methanol, sonicate for 10 minutes to dissolve, cool to room temperature, dilute to scale with methanol, shake well, centrifuge at 3000 rpm for 10 minutes, take the supernatant, filter through a 0.45μm filter membrane, accurately measure 5mL of the filtrate and put it into a 20mL volumetric bottle, dilute to scale with methanol, shake well, and use it as the test solution. (2) The high performance liquid chromatograph used is Shimadzu LC-20AT; the detector is SPD-20A; the chromatographic column is Agilent ZORBAX Eclipse XDB-C18 column (4.6×250mm, 5μm). The HPLC detection conditions used were: mobile phase: methanol-0.02 mol / L sodium acetate solution = 9:91, isocratic elution. The HPLC detection conditions used were: detection wavelength 215 nm; column temperature 30°C, flow rate 1.0 mL / min, injection volume 10 μL.

[0086] Take one needle of the reference solution and one needle of the test solution for testing and measure twice in parallel, record the chromatogram, and record it as Figure 4 :Comparative Example 1 Typical chromatogram of isocratic detection method of Rhodiola rosea extract. Technical features: It takes 45 minutes to complete the one-shot determination of the test solution and the reference solution. The same component was sampled and determined twice, and the results were very different, with poor reproducibility. The peak area of ​​the same sample became smaller, and the Rhodiola rosea extract drifted to the unknown impurity peak, and the separation degree with the adjacent unknown impurities was unstable, making it impossible to accurately locate and calculate the Rhodiola rosea extract.

[0087] Figure 4 The chromatogram data are shown in Table 2 below. Figure 4 From bottom to top in the middle are the control solution, the first injection of comparative example 1, and the second injection of comparative example 1.

[0088] Table 2 Chromatographic data

[0089]

[0090] Comparative Example 2

[0091] The detection method of Rhodiola rosea extract comprises the following steps:

[0092] (1) Test solution: The theoretical weight of Anrui tablets is 1g / tablet, of which 100g contains 0.18g of Rhodiola rosea. Take 20 tablets of Anrui tablets, grind them into fine powder, put 2.5g of fine powder into a 50mL volumetric flask, add an appropriate amount of methanol, sonicate for 10 minutes to dissolve, cool to room temperature, dilute to scale with methanol, shake well, centrifuge at 3000 rpm for 10 minutes, take the supernatant, filter through a 0.45μm filter membrane, accurately measure 5mL of the filtrate into a 20mL volumetric flask, dilute to scale with methanol, shake well, and use it as the test solution.

[0093] (2) The HPLC instrument used was Shimadzu LC-20AT; the detector was SPD-20A; the chromatographic column was Agilent ZORBAX Eclipse XDB-C18 column (4.6×250mm, 5μm). The mobile phase A for HPLC detection was methanol-0.02mol / L sodium acetate solution = 9:91, and the mobile phase B was methanol-0.02mol / L sodium acetate solution = 30:70 under the chromatographic conditions; the injection volume was 10μL, the flow rate was 1.0mL / min, the detection wavelength was 215nm, and the column temperature was 30℃.

[0094] When the test solution is sampled and tested, the gradient elution order of mobile phase A and mobile phase B is:

[0095] 0→45min, mobile phase A 100 parts, mobile phase B 0 parts;

[0096] 45.1→50min, mobile phase A 100 parts→0 parts, mobile phase B 0 parts→100 parts;

[0097] 50.1→220min, mobile phase A 0 parts, mobile phase B 100 parts;

[0098] 220.1→230min, mobile phase A 0 parts→100 parts, mobile phase B 100 parts→0 parts;

[0099] 230.1→260min, mobile phase A 100 parts, mobile phase B 0 parts;

[0100] Take a needle of the test solution and record the chromatogram. Figure 5 : Comparative Example 2: Under the chromatographic conditions, a gradient elution chromatogram was performed with methanol-0.02mol / L sodium acetate solution = 30:70 as mobile phase B. Technical features: It overcomes the defects of large deviation and poor reproducibility of the results of twice injecting the same sample, but the separation degree with adjacent unknown impurities is unstable, and it is impossible to accurately locate and calculate the Rhodiola rosea extract. It takes 260 minutes to complete the one-shot determination of the test solution, the running time is too long, and the detection cycle and cost are high.

Claims

1. A method for detecting a Rhodiola rosea extract, characterized in that: The steps include: (1) Grind the substance to be tested into powder, add the extract, dissolve by ultrasonic, cool to room temperature, centrifuge, take the supernatant, filter through a microporous membrane, dilute with the extract, shake well, and use it as the test solution; (2) Weigh the Rhodiola rosea extract reference substance and add it to the extract solution, dissolve it by ultrasonication, cool it to room temperature, dilute it with the extract solution, and shake it well to prepare the reference substance solution; (3) Using a high performance liquid chromatograph, the reference solution was tested as the first injection, and the chromatogram was recorded as an evaluation index of the tailing factor of the chromatographic system. Under the same chromatographic conditions, the test solution was injected, and the chromatogram and data of the test solution were recorded. The Rhodiola rosea extract in the test solution was quantitatively analyzed using the external standard method; During the detection process of the high performance liquid chromatograph, mobile phase A and mobile phase B are used for gradient elution, wherein the mobile phase A is methanol-0.02 mol / L sodium acetate solution in a volume ratio of 9:91; the mobile phase B is methanol-0.02 mol / L sodium acetate solution in a volume ratio of 50:50, the injection volume is 10 μL, the mobile phase flow rate is 1.0 mL / min, the detection wavelength is 215 nm; the column temperature is 30° C.; The centrifugal speed is 8000 rpm and the centrifugal time is 10 minutes; The pore size of the microporous filter membrane is 0.45 μm; The extract is methanol; The tailing factor T 拖尾 ≤1.5; The chromatographic column used in step (3) is an Agilent ZORBAX Eclipse XDB-C18 column: 4.6×250 mm, 5 μm; The gradient elution sequence of the mobile phase A and the mobile phase B is: 0→45min, mobile phase A 100 parts, mobile phase B 0 parts; 45.1→50min, mobile phase A 100 parts→0 parts, mobile phase B 0 parts→100 parts; 50.1→120min, mobile phase A 0 parts, mobile phase B 100 parts; 120.1→125min, mobile phase A 0 parts→100 parts, mobile phase B 100 parts→0 parts; 125.1→160min, mobile phase A 100 parts, mobile phase B 0 parts.

2. The method for detecting the Rhodiola rosea extract according to claim 1, characterized in that: Step (1) includes the following steps: Grind the object to be tested, take 2.5g of the theoretical content of Rhodiola rosea extract, put it in a 50mL volumetric flask, add the extract, dissolve it by ultrasonic, cool to room temperature, dilute it to the scale with the extract, shake well, centrifuge, take the supernatant, and filter it through a microporous filter membrane. Accurately measure 5mL of the filtrate and put it in a 20mL volumetric flask, add the extract to dilute it to the scale, shake well, and use it as the test solution.

3. The detection method of Rhodiola rosea extract according to claim 1, characterized in that: Step (2) includes the following steps: Weigh 20 mg of Rhodiola rosea extract reference substance, place it in a 100 mL volumetric flask, add the extract, dissolve it by ultrasonic, cool to room temperature, dilute to the scale with methanol, shake well, measure 5 mL and place it in a 20 mL volumetric flask, add the extract to dilute to the scale, shake well, as the reference substance solution.

4. The detection method of Rhodiola rosea extract according to claim 1, characterized in that: Step (3) includes the following steps: Using a high performance liquid chromatograph, the reference solution was detected in the first injection, and the chromatogram was recorded as an evaluation index of the tailing factor of the chromatographic system. The content of Rhodiola rosea extract in the test solution was calculated by the external standard method. Under the same chromatographic conditions, the test solution was injected, and the chromatogram and data of the test solution were recorded.

Images