A HPLC method for simultaneous determination of 10 components in Xianren mushroom oral liquid

Abstract

The application discloses a kind of HPLC methods for simultaneously determining the content of 10 components in Pholiota namei oral liquid, comprising the following steps: using Dikma Diamonsil Plus C 18 Column, eluted with acetonitrile-0.1% glacial acetic acid aqueous solution gradient; The flow rate is 1.0 mL / min, the column temperature is 30 DEG C, the detection wavelength is 270 nm, and the injection amount is 10 μL. The method is simple, accurate and reproducible. It is suitable for the determination of the content of 10 kinds of effective components in Pholiota namei oral liquid, such as adenosine, cordycepin, rutin, ononin, damianol B, damianol C, icariin, calycosin, ganoderic acid A and baohuoside I, and can be used as a quality control method for Pholiota namei oral liquid.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of chemical analysis, and particularly relates to a HPLC method for simultaneously determining the contents of 10 components in Xianren mushroom oral liquid. BACKGROUND

[0002] Xianren Gu oral liquid is a self-made preparation in the hospital of the Second Military Medical University. The prescription is composed of eight kinds of Chinese herbs, including Herba Epimedii, Radix Astragali, Radix Ginseng, Rhizoma Atractylodis Macrocephalae (Fried), Cordyceps sinensis, Boletus, Ganoderma lucidum, and Lycium chinense[[1]Shi J, Xu L, Qin ZF, et al. Clinical observation of Xianren Gu oral liquid in the treatment of middle and advanced gastric cancer[J]. Journal of Chengdu University of Traditional Chinese Medicine, 2002, 25(1): 15-16.]. It is a natural interferon inducer and immunomodulator developed by Professor Wei Pin Kang based on years of clinical experience, with the effects of tonifying qi and filling essence, and supporting healthy energy and consolidating the body[[2]He LJ, Jiang J, Chen G, et al. Research progress of pharmacological effects and clinical application of Herba Epimedii[J]. Clinical Research of Traditional Chinese Medicine, 2020, 12(02): 17-20.[3]Zhongyi Cong, Qing Zhao, Bo Yang, et al. Ginsenoside Rh2 Inhibits Proliferation and Induces Apoptosis of Colorectal Cancer Cells[J]. Pharmacology, 2020, 105(5-6).[4]Wang ZX, Ban JF, Ke R. Network Meta-analysis of TCM Injection Combined with Chemotherapy in the Treatment of Non-small Cell Lung Cancer[J]. Chinese Journal of Pharmacy and Clinic, 2020, 26(16): 167-175.]. Previous studies have shown that this product can promote human protein synthesis, improve liver cell activity[[5]Liu TT, Yu DH, Liu SM. Herbal textual research and modern research progress of Boletus sinensis[J]. Chinese Pharmacy, 2020, 31(24): 3055-3059.], reduce the content of circulating immune complexes in patients[[6]Chen TC, Zhang ZF, Qin ZF, et al. Observation of Xianren Gu oral liquid on the efficacy and toxicity reduction of 59 patients with malignant tumor during chemotherapy[J]. Journal of Chinese Medicine, 2007, 48(3): 247.], and improve the immune function of the body, thereby improving the quality of life of patients and playing a synergistic and toxicity-reducing role with other drugs[[7]Li YH, Liu JL, Wang TT, et al. Research progress of antitumor immunomodulatory mechanism of Ganoderma lucidum polysaccharides[J]. Chinese Journal of Immunology, 2021, 37(04): 511-514.].It is mainly used for treating and improving chronic consumptive disease, fatigue syndrome, etc. in clinic. It has auxiliary therapeutic effect on anemia and low immune function caused by radiotherapy and chemotherapy of malignant tumor. It has been clinically applied for more than 20 years and has definite curative effect. [9] Jing Zhao, Tao Pang, Jianpeng Jiao, et al. Xiaotan Jieyu Prescription Alleviates Breast Precancerous Lesions through PI3K_Akt Signaling Pathway [J]. Evidence-Based Complementary and Alternative Medicine 2020, Article ID 4129461.

[10] Tao Pang, Jun Pei, Aihua Chen, Wenkun Lu. Analysis of Clinical Application of Traditional Chinese Medicine Preparations in Our Hospital from 2014 to 2016 [J]. Frontier of Medicine, 2018, 10: 340-342.

[0003] At present, the quality standard of Xianren Gu oral liquid mainly adopts thin layer chromatography to qualitatively identify five kinds of medicinal materials such as Astragalus, Herba Epimedii, Fructus Lycii, Atractylodes and Ginseng.

[11] Le Jia-mei, Xiong Xiao-juan, Lu Wen-kun, et al. Quality Standard of Xianren Gu Oral Liquid [J]. China Pharmacy, 2015, 26(27): 3855-3859.

[12] Liang Li-juan, Zhao Kui-jun, Tu Peng-fei, et al. Simultaneous Determination of Four Flavonoids in Astragalus by HPLC [J]. China Pharmacy, 2010, 15: 1385-1387. High performance liquid chromatography (HPLC) is used to determine the content of icariin, the main component of Herba Epimedii.

[13] Yang Juan-juan, Li Ye, Wei Qiao-rong, et al. Determination of Ganoderic Acid A in Ganoderma lucidum by RP-HPLC [J]. Fujian Journal of Chinese Medicine, 2011, 33(3): 56-58.

[14] Xia Wen-juan, Zeng Xiao-ying, Yuan Hai-long, et al. Determination of Adenosine in Cordyceps sinensis from Different Producing Areas [J]. Chinese Journal of Chinese Materia Medica, 2001, 26(8): 540-542. Since the oral liquid is a compound preparation, it is difficult to reflect the overall quality of Xianren Gu oral liquid by determining the content of only one main component. Therefore, it is necessary to establish a rapid and accurate multi-component content determination method to perfect the quality standard. SUMMARY

[0004] The purpose of the present application is to provide an HPLC method for simultaneously determining the contents of 10 components in Xianren Gu oral liquid.

[0005] In order to achieve the above object, the technical scheme adopted by the present application is as follows:

[0006] The first aspect of the present application provides an HPLC method for simultaneously determining the contents of 10 components in a Hericium erinaceus oral liquid, comprising the following steps:

[0007] The Hericium erinaceus oral liquid is ultrasonically treated for 5-30 min, a solvent is added and centrifuged, the volume ratio of the solvent to the Hericium erinaceus oral liquid is 1:1, to obtain supernatant; then the solvent is added to the remaining liquid and centrifuged again, the above steps are repeated at least twice, the obtained supernatants are combined, the solvent is removed, and the contents of 10 components in the Hericium erinaceus oral liquid are simultaneously determined by HPLC method;

[0008] The solvent is selected from dichloromethane, trichloromethane, methyl tert-butyl ether, n-hexane and ethyl acetate;

[0009] The HPLC method for simultaneously determining the contents of 10 components in the Hericium erinaceus oral liquid is as follows:

[0010] a Dikma Diamonsil Plus C 18 column is used, gradient elution is performed, the flow rate is 0.5-2 mL / min, the column temperature is 25-35℃, the detection wavelength is 270-300 nm, and the injection amount is 5-15 μL;

[0011] The 10 components in the Hericium erinaceus oral liquid are selected from adenosine, cordycepin, icariin, rutin, calycosin, epimedium brevicornu B, epimedium brevicornu C, calycosin, ganoderic acid A and baohuoside I.

[0012] The mobile phase used in the gradient elution is selected from acetonitrile-phosphoric acid aqueous solution, acetonitrile-glacial acetic acid aqueous solution, acetonitrile-ammonium acetate solution, acetonitrile-formic acid aqueous solution and acetonitrile-water.

[0013] The centrifugation time is 1-10 min.

[0014] Preferably, the HPLC method for simultaneously determining the contents of 10 components in the Hericium erinaceus oral liquid is as follows: a Dikma Diamonsil Plus C 18 column is used, gradient elution is performed with acetonitrile-0.1% glacial acetic acid aqueous solution; the flow rate is 1.0 mL / min, the column temperature is 30℃, the detection wavelength is 270 nm, and the injection amount is 10 μL.

[0015] The contents of the 10 components in the Hericium erinaceus oral liquid are selected from:

[0016] Adenosine is 0.250-16.0 μg / ml, cordycepin and icariin is 6.25-400 μg / ml, rutin is 1.25-80.0 μg / ml, formononetin is 0.500-32.0 μg / ml, damianol B is 0.375-24.0 μg / ml, damianol C is 3.12-200 μg / ml, calycosin and ganoderic acid A is 0.625-40.0 μg / ml, baohuoside I is 0.125-8.00 μg / ml.

[0017] Due to the adoption of the above technical scheme, the present application has the following advantages and beneficial effects:

[0018] The method of the present application is simple in operation, high in accuracy and good in repeatability, and is suitable for the content determination of 10 kinds of effective components, i.e., adenosine, cordycepin, rutin, formononetin, damianol B, damianol C, icariin, calycosin, ganoderic acid A and baohuoside I in the Pholiota nameko oral liquid, and can be used as a quality control method for the Pholiota nameko oral liquid.

[0019] The present application establishes a rapid and accurate high performance liquid chromatography (HPLC) method for simultaneously determining the contents of 10 kinds of effective components, i.e., adenosine, cordycepin, rutin, formononetin, damianol B, damianol C, icariin, calycosin, ganoderic acid A and baohuoside I in the Pholiota nameko oral liquid. The method of the present application is simple in operation, high in accuracy and good in repeatability, and can achieve the purpose of rapidly and accurately controlling the quality of the Pholiota nameko oral liquid. The quality markers involved cover the main components in the prescription, and provide guarantee for the safety and effectiveness in clinic. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 is a schematic diagram of the HPLC chromatogram of the Pholiota nameko oral liquid. DETAILED DESCRIPTION

[0021] In order to more clearly illustrate the present application, the present application will be further described below in combination with preferred embodiments. It should be understood by those skilled in the art that the specific description below is illustrative rather than limiting, and should not limit the protection scope of the present application.

[0022] Example 1

[0023] The present application adopts the HPLC method to determine the contents of 10 kinds of components, i.e., adenosine, cordycepin, rutin, formononetin, damianol B, damianol C, icariin, calycosin, ganoderic acid A and baohuoside I in the Pholiota nameko oral liquid, and provides a scientific basis for the establishment of the quality standard of the Pholiota nameko oral liquid.

[0024] 1 Materials and methods

[0025] 1.1 Instruments

[0026] Agilent 1200 high performance liquid chromatography system, including G1311A (quaternary pump) infusion pump, G1313A autosampler, G1316A column oven, G1314A VWD detector, Chemstation chromatographic workstation. Ten millionth electronic balance, CPA225D type (Sartorius Scientific Instruments Co., Ltd.); ultrasonic instrument, SK7200H type (Shanghai Keduo Ultrasonic Instrument Co., Ltd.); desktop high-speed refrigerated centrifuge (Germany Eppendorf 5810R Co., Ltd.). Pipettor, 10 μL, 20 μL, 100 μL, 200 μL, 1000 μL (Germany Eppendorf Co., Ltd.); vortex shaker mixer, WL-901 type (Haimen Qishengbel Instrument Manufacturing Co., Ltd.).

[0027] 1.2 Reagents

[0028] Icariin (batch number T09N6B5664), epimedin B (batch number P25F8F30154), epimedin C (batch number C18J3G1), baohuoside I (batch number R11M8F31212), cordycepin (batch number N12M7W14489), calycosin (batch number P29M6R2), ganoderenic acid A (batch number Z15O6X4326), adenosine (batch number Z23S7J21814) reference substances were purchased from Shanghai Yuanye Biological Technology Co., Ltd. (HPLC≥98%). Ononin (batch number MB6579), rutin (batch number MB5118) reference substances were purchased from Dalian Melun Biotechnology Co., Ltd. (HPLC≥98%). Hericium erinaceus oral liquid was produced by the Preparation Room of Changzheng Hospital, the Second Military Medical University, batch numbers: 210328, 210416, 210520.

[0029] Methanol, acetonitrile were chromatographically pure, produced by Thermo Fisher Scientific (China) Co., Ltd., and water was Wahaha pure water.

[0030] 1.3 Chromatographic conditions

[0031] The chromatographic column was Dikma Diomomsil Plus C 18 column (4.6 mm x 250 mm, 5 μm); the mobile phase was acetonitrile-0.1% glacial acetic acid aqueous solution, gradient elution, the elution program was shown in Table 1; the flow rate was 1 mL / min; the column temperature was 30℃; the detection wavelength was 270 nm; the injection volume was 10 μL.

[0032] Table 1 Gradient elution program of mobile phase

[0033] Time (min) Acetonitrile (%) 0.1% glacial acetic acid (%) 0 2 98 4 5 95 7 5 95 18 20 80 21 30 70 38 30 70 48 90 10

[0034] 1.4 Preparation of solutions

[0035] 1.4.1 Preparation of reference solution

[0036] Accurately weigh 2.01 mg of icariin, 2.03 mg of Tsushimiside B, 2.02 mg of Tsushimiside C, 2.01 mg of Baohuoside I, 2.04 mg of Mutamycin, 2.02 mg of Calycosin, 2.03 mg of Ganoderic acid A, 2.04 mg of Rutin, 2.02 mg of Adenosine, respectively, and put them into 2 mL volumetric flasks, dissolve and dilute with methanol to prepare reference solutions of about 1.00 mg / mL; accurately weigh about 2.02 mg of cordycepin, put it into a 2 mL volumetric flask, add 20% methanol to prepare a cordycepin reference solution of 1.01 mg / mL.

[0037] 1.4.2 Preparation of test solution

[0038] Take a suitable amount of Pholiota nameko oral liquid, ultrasonic for 15 min, accurately take 200 μL, put it into a 1.5 mL centrifuge tube, add 200 μL of ethyl acetate, vortex for 30 s, and centrifuge at high speed at 13400 rpm for 5 min, take 200 μL of supernatant, repeat the above operation twice. After combining the supernatants of the three times, dry them in a dry instrument, redissolve with 1000 μL of initial mobile phase, centrifuge at high speed at 13400 rpm for 5 min, take 10 μL of supernatant for sample injection, and perform HPLC analysis, and calculate the sample content by external standard method.

[0039] 1.5 Methodology investigation

[0040] 1.5.1 Investigation of specificity

[0041] Take the blank solution with only the mobile phase, the mixed reference solution and the test solution according to the chromatographic conditions under item “1.3”, respectively, and determine whether there is an interfering peak in the corresponding position.

[0042] 1.5.2 Preparation of standard curve

[0043] Accurately take a suitable amount of the above-mentioned reference stock solution, put it into a 1 ml volumetric flask, and dilute to the mark with the initial mobile phase, shake well to obtain a series of concentration of reference mixed solution, and sequentially and continuously inject samples according to the chromatographic conditions under item “1.3”, inject samples in parallel for 3 times, take the liquid chromatographic peak area as the ordinate (Y) and the concentration as the abscissa (X) to perform linear regression.

[0044] 1.5.3 Precision test

[0045] Take the adenosine, cordycepin, rutin, formononetin, epimedium B, epimedium C, icariin, calycosin, ganoderic acid A, treasure I of mixed control solution (standard curve of the 4th mixed control) of the mixture of baohuo glycoside, according to the chromatographic conditions under "1.3" parallel sample 6 times. Calculate the RSD of adenosine, cordycepin, rutin, formononetin, epimedium B, epimedium C, icariin, calycosin, ganoderic acid A, treasure I.

[0046] 1.5.4 repeatability

[0047] Take the test sample (210416) of Xianren mushroom oral liquid, prepare 6 test sample solutions under the conditions of "1.4.2" parallel, sample under the chromatographic conditions of "1.3", measure the peak area, and calculate the RSD of adenosine, cordycepin, rutin, formononetin, calycosin, ganoderic acid A, treasure I of the content of baohuo glycoside B, C, icariin, respectively, to verify the repeatability of the method.

[0048] 1.5.5 stability

[0049] Take the test sample (210416) of Xianren mushroom oral liquid, prepare the test sample solution under the conditions of "1.4.2", sample at 0, 4, 6, 8, 12, 24 h, record the peak area under the chromatographic conditions of "1.3", calculate the RSD of adenosine, cordycepin, rutin, formononetin, epimedium B, epimedium C, icariin, calycosin, ganoderic acid A, treasure I of the content of baohuo glycoside, to verify the stability of the method.

[0050] 1.5.6 sample recovery rate experiment

[0051] Precisely take the test sample (210416) of Xianren mushroom oral liquid, add adenosine, cordycepin, rutin, formononetin, epimedium B, epimedium C, icariin, calycosin, ganoderic acid A, treasure I of the content of baohuo glycoside, that is, the sample amount is halved, and then add each component with approximately equal content of each component to be investigated, sample under the chromatographic conditions of "1.3", calculate the sample recovery rate and RSD value of each component.

[0052] 1.6 sample determination

[0053] Prepare the test sample solution under the conditions of "1.4.2", sample under the chromatographic conditions of "1.3" to measure the peak area, and calculate the content of adenosine, cordycepin, rutin, formononetin, epimedium B, epimedium C, icariin, calycosin, ganoderic acid A, treasure I of baohuo glycoside in three batches of Xianren mushroom oral liquid according to the standard curve.

[0054] 2 results

[0055] 2.1 specificity

[0056] The specificity investigation results show that the mixed control sample is completely separated, and the blank solution has no interfering peaks at the corresponding positions, as shown in Figure 1 Figure 1 is a HPLC chromatogram of Xianren Gu oral liquid, wherein A is a blank solution, B is a mixed control sample solution, and C is a Xianren Gu oral liquid test sample. 1 is adenosine, 2 is cordycepin, 3 is rutin, 4 is astragalin, 5 is damianol B, 6 is damianol C, 7 is icariin, 8 is calycosin, 9 is ganoderic acid A, and 10 is baohuoside I.

[0057] 2.2 Investigation of standard curve

[0058] The standard curve results show that the linear regression of each component in the range meets the methodological requirements (r = 0.9992-0.9998), wherein adenosine is 0.250-16.0 μg / mL; cordycepin and icariin are 6.25-400 μg / mL; rutin is 1.25-80.0 μg / mL; astragalin is 0.500-32.0 μg / mL; damianol B is 0.375-24.0 μg / mL; damianol C is 3.12-200 μg / mL; calycosin and ganoderic acid A are 0.625-40.0 μg / mL; baohuoside I is 0.125-8.00 μg / mL; sequentially and continuously sample according to the chromatographic conditions under item 2.1, sample in parallel for 3 times, take the liquid chromatography peak area as the ordinate (Y), and the concentration as the abscissa (X) to perform linear regression.

[0059] Table 2 Linear range, regression equation, and correlation coefficient of 10 components in Xianren Gu oral liquid

[0060]

[0061]

[0062] 2.3 Precision test

[0063] The precision test results show that the RSD of adenosine, cordycepin, rutin, astragalin, damianol B, damianol C, icariin, calycosin, ganoderic acid A, and baohuoside I is 0.30%, 0.23%, 1.77%, 0.22%, 0.42%, 1.91%, 0.42%, 0.34%, 0.32%, and 0.93% (n = 6) respectively, and the results show that the precision of the method is good.

[0064] 2.4 Reproducibility

[0065] ​The results of the repeatability test showed that the RSD values of the contents of adenosine, cordycepin, rutin, formononetin, calycosin, epimedium B, epimedium C, icariin, ganoderic acid A and baohuoside I were 3.54%, 0.86%, 2.59%, 1.06%, 1.14%, 0.11%, 0.11%, 1.72%, 3.73% and 1.44% respectively, indicating that the method was good in repeatability.

[0066] 2.5 Stability

[0067] The stability results showed that the RSD values of the contents of adenosine, cordycepin, rutin, formononetin, epimedium B, epimedium C, icariin, calycosin, ganoderic acid A and baohuoside I were 1.70%, 0.98%, 2.84%, 1.57%, 1.97%, 0.56%, 0.24%, 3.0%, 3.96% and 1.99% respectively, indicating that the test sample solution could be stably stored under the above conditions.

[0068] 2.6 Sample addition recovery rate test

[0069] The sample addition recovery rate results showed that the average recovery rate of adenosine was 97.64% with an RSD of 3.74%; the average recovery rate of cordycepin was 96.32% with an RSD of 0.86%; the average recovery rate of rutin was 97.46% with an RSD of 2.59%; the average recovery rate of formononetin was 98.48% with an RSD of 1.69%; the average recovery rate of epimedium B was 98.92% with an RSD of 2.41%; the average recovery rate of epimedium C was 99.07% with an RSD of 1.74%; the average recovery rate of icariin was 99.37% with an RSD of 0.79%; the average recovery rate of calycosin was 96.72% with an RSD of 3.57%; the average recovery rate of ganoderic acid A was 99.60% with an RSD of 0.73%; and the average recovery rate of baohuoside I was 97.50% with an RSD of 3.15%, meeting the requirements of the analysis method.

[0070] 2.7 Sample determination

[0071] The contents of adenosine, cordycepin, rutin, formononetin, epimedium B, epimedium C, icariin, calycosin, ganoderic acid A and baohuoside I in three batches of Xianrenmugou oral liquid were determined according to the law, and the results are shown in Table 3.

[0072] Table 3 Determination results of the contents of 10 components in Xianrenmugou oral liquid samples (n = 3, μg / mL)

[0073]

[0074]

[0075] 3 Conclusion

[0076] The present application establishes a method for simultaneously determining the contents of 10 components in Xianren mushroom oral liquid by HPLC. The contents of adenosine, cordycepin, rutin, formononetin, epimedin B, epimedin C, icariin, calycosin, ganoderic acid A and baohuoside I in Xianren mushroom oral liquid are determined by HPLC. The methodological investigation shows that the method is simple in operation, has good repeatability, stability and precision, and the recovery range meets the relevant standards, which can provide a reference for the quality standard of Xianren mushroom oral liquid.

[0077] 4 DISCUSSION

[0078] 4.1 Selection of detection wavelength

[0079] Under the condition that other chromatographic conditions remain unchanged, the mixed standard is gradient eluted at 270 nm, 280 nm, 290 nm and 300 nm respectively. The peak results of the mixed standard are compared, and the peak effect at 270 nm is better than that at other wavelengths, so 270 nm is selected as the detection wavelength.

[0080] 4.2 Investigation of mobile phase

[0081] The present application carries out chromatographic separation on 10 kinds of to-be-tested compounds. In the early stage of the experiment, under the condition of 270 nm detection wavelength, flow rate 1 mL / min, injection volume 10 μL and column temperature 30℃, the elution effects of the conventional mobile phases acetonitrile-phosphoric acid aqueous solution, acetonitrile-glacial acetic acid aqueous solution, acetonitrile-ammonium acetate solution, acetonitrile-formic acid aqueous solution and acetonitrile-water are compared. It is found that the acetonitrile-glacial acetic acid aqueous solution can realize good chromatographic separation of the to-be-tested substances on the basis of shortening the peak time, and the viscosity of acetonitrile is small, which can effectively reduce the system pressure. Further comparison of the glacial acetic acid concentration shows that the separation effect of 0.1% glacial acetic acid is the best. The present application uses acetonitrile and 0.1% glacial acetic acid aqueous solution as the mobile phase, which can completely separate the 10 kinds of to-be-tested compounds, and the preparation operation of the mobile phase is simple.

[0082] 4.3 Selection of extraction method

[0083] 1 mL of Xianren mushroom oral liquid is precisely taken in three portions and treated by the following three methods. ① Direct injection method: placed in a 5 mL volumetric flask, added with mobile phase to the mark, shaken, filtered, and the filtrate was taken. ② Ultrasonic injection method: placed in a 5 mL volumetric flask, added with mobile phase to the mark, ultrasonically treated for 30 min, cooled, added with mobile phase to the mark, shaken, filtered, and the filtrate was taken. ③ Extraction method: added with ethyl acetate for extraction for 3 times, 1 mL each time, combined the ethyl acetate liquid, evaporated, and the residue was dissolved in 5 mL of mobile phase. Under the same chromatographic conditions, the chromatographic baseline is stable when the extraction method is used, and the peak effect is better than that of the other two methods, so the extraction method is selected as the extraction method of the sample components of Xianren mushroom oral liquid.

[0084] 4.3.1 Selection of extraction solvent

[0085] 200 μL of the P. cubensis oral liquid was precisely pipetted 5 times, and was extracted with dichloromethane, chloroform, methyl tert-butyl ether, n-hexane and ethyl acetate, respectively, and was injected under the same chromatographic conditions. The results showed that the peaks were more and the response was higher when ethyl acetate was used for extraction, so ethyl acetate was selected as the extraction solvent.

[0086] 4.3.2 Selection of extraction volume

[0087] 200 μL of the P. cubensis oral liquid was precisely pipetted 3 times, and was extracted with 200 μL and 400 μL of ethyl acetate each time for 3 times, and was injected under the same chromatographic conditions. The results showed that the peaks were better when 200 μL of ethyl acetate was used for extraction each time, so 1:1 extraction was selected.

[0088] 4.3.3 Selection of extraction times

[0089] 200 μL of the P. cubensis oral liquid was precisely pipetted 3 times, and was extracted with 600 μL of ethyl acetate for 1 time and with 200 μL of ethyl acetate for 3 times, and was injected under the same chromatographic conditions. The results showed that the chromatogram had less impurity peaks and the separation effect was better when 200 μL of ethyl acetate was used for extraction for 3 times, so 200 μL of ethyl acetate was selected for extraction for 3 times as the pretreatment method of the P. cubensis oral liquid.

[0090] The above only describes the preferred embodiments of the present application and does not limit the present application in any form. Although the present application has been disclosed as the above preferred embodiments, it is not intended to limit the present application. Any person skilled in the art can make some changes or modifications to the above-mentioned technical content without departing from the technical solution of the present application, and any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present application are still within the scope of the present application.

Claims

1.A HPLC method for simultaneously determining the contents of 10 components in a Termitomyces oral liquid, characterized in that, The method comprises the following steps: Preparation of the sample solution: take a proper amount of the test sample, and ultrasonically treat it for 15 min. Then, 200 μL of the sample solution is precisely taken and placed in a 1.5 mL centrifuge tube. 200 μL of ethyl acetate is added, and the mixture is vortexed for 30 s. The mixture is centrifuged at 13,400 rpm for 5 min. 200 μL of the supernatant is taken, and the above operation is repeated twice. The supernatants obtained in the three times are combined, and placed in a rotary evaporator for evaporation. The residue is dissolved with 1000 μL of the initial mobile phase, and centrifuged at 13,400 rpm for 5 min. 10 μL of the supernatant is taken for HPLC analysis. The content of the sample is calculated by using an external standard method. The HPLC analysis conditions are as follows: Chromatographic column: Dikma Diomomsil Plus C 18 Column: Dikma Diomomsil Plus C18, 4.6 mm x 250 mm, 5 μm; mobile phase: acetonitrile-0.1% glacial acetic acid aqueous solution, gradient elution, flow rate: 1 mL / min; column temperature: 30 °C; detection wavelength: 270 nm; injection volume: 10 μL; The gradient elution program of the mobile phase is as follows: The 10 components in the test sample are selected from adenosine, cordycepin, icariin, rutin, ononin, epimedin B, epimedin C, calycosin, ganoderic acid A, and baohuoside I. 2.The HPLC method for simultaneously determining the contents of 10 components in Xianren mushroom oral liquid according to claim 1, characterized in that, The content of the 10 components in the test sample is selected from: adenosine is 0.250-16.0 μg / ml, cordycepin and icariin are 6.25-400 μg / ml, rutin is 1.25-80.0 μg / ml, ononin is 0.500-32.0 μg / ml, epimedin B is 0.375-24.0 μg / ml, epimedin C is 3.12-200 μg / ml, calycosin and ganoderic acid A are 0.625-40.0 μg / ml, and baohuoside I is 0.125-8.00 μg / ml.

Images