CN112858491A - Rhodiola rosea medicinal material fingerprint spectrum determination method - Google Patents
Rhodiola rosea medicinal material fingerprint spectrum determination method Download PDFInfo
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- CN112858491A CN112858491A CN201911100467.2A CN201911100467A CN112858491A CN 112858491 A CN112858491 A CN 112858491A CN 201911100467 A CN201911100467 A CN 201911100467A CN 112858491 A CN112858491 A CN 112858491A
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The invention provides a rhodiola rosea HPLC fingerprint, which provides a feasible method for the comprehensive evaluation of rhodiola rosea quality and provides a basis for further drug effect substance basic research.
Description
Technical Field
The invention belongs to the field of traditional Chinese medicine detection, and particularly relates to an RP-HPLC fingerprint spectrum determination method for rhodiola rosea medicinal materials.
Background
The radix Rhodiolae is dry root and rhizome of Rhodiola crenulata Rhodiola crenulata (hook.f. et Thoms.) H.Ohba. It is sweet and bitter in flavor, neutral in nature, and enters lung and heart meridians. It is used for controlling plateau, treating nervous system diseases and relieving fatigue. The rhodiola rosea also has the pharmacological actions of removing free radicals, improving the activity of antioxidant enzyme, activating the signal transduction of phosphoinositide kinase/serine kinase and the like; the chemical components of rhodiola are complex, mainly including polysaccharides, sterols, tannins, flavones and the like, which together form the basis of the medicinal substances of rhodiola.
At present, salidroside is generally used as the only indicative component for quality control, but due to the complexity of the components of traditional Chinese medicinal materials, the single component is not enough to comprehensively reflect the inherent quality of rhodiola; the fingerprint spectrum technology is an analysis technical means for systematically grasping the quality of medicinal materials from the perspective of the overall material basis of the medicinal materials; provides a reliable quality control method for the effective control of the quality of medicinal materials.
The invention patent with Chinese patent application number 200310112581X provides a method for constructing HPLC fingerprint of rhodiola Tibet, which adopts different chromatographic conditions and sample processing method from the invention, calibrates 8 chromatographic peaks, and does not identify specific components.
Patent application No. 201110064022.0: the quality detection method of rhodiola crenulata medicinal material provides a HPLC fingerprint detection method of rhodiola crenulata, and is completely different from the sample treatment method and chromatographic condition adopted by said invention, said method can calibrate 26 characteristic peaks, and can not make identification of specific component due to base line and peak type.
The HPLC fingerprint determination method of rhodiola rosea is disclosed in the study of HPLC fingerprint research of rhodiola rosea medicinal materials produced in Alba, Sichuan, the southwest university, and is different from the sample treatment method and chromatographic conditions adopted by the invention, particularly mobile phase, and 19 peaks are marked but are not identified.
The invention adopts HPLC method to research the quality of rhodiola root medicinal material, establishes fingerprint of rhodiola root, lays foundation for improving quality standard of rhodiola root medicinal material, and provides basis for further medicinal effect material basic research.
Disclosure of Invention
The invention provides a method for measuring an HPLC fingerprint spectrum of a rhodiola rosea medicinal material. The methodology investigation of the HPLC fingerprint accords with the relevant requirements, 17 common peaks of the HPLC fingerprint of the rhodiola rosea medicinal material are calibrated together, 12 characteristic peaks of salidroside, gallic acid, caffeic acid, tyrosol, p-coumaric acid, protocatechuic acid, catechin, vanillin, isoquercitrin, quercitrin, chrysophanol-8-O-glucoside and kaempferol are identified, and the similarity of the fingerprint of 13 batches of the rhodiola rosea medicinal material is 0.863-0.984. The established rhodiola rosea HPLC fingerprint spectrum can provide a basis for establishing the quality standard of the rhodiola rosea.
The rhodiola rosea fingerprint detection method specifically comprises the following steps:
(1) preparation of a test solution: weighing 1-3g of rhodiola rosea powder, precisely adding 20 mL of 75% methanol into a 50mL conical flask with a plug, weighing, ultrasonically extracting for 15-30 min, weighing again, supplementing the weight loss with 75% methanol, shaking up, centrifuging, taking supernatant, filtering with a 0.45 mu m microporous membrane, and taking subsequent filtrate;
(2) preparation of control solutions: precisely weighing a proper amount of each reference substance, placing in a volumetric flask with a plug, adding 75% methanol for dissolving, performing ultrasonic treatment, and preparing into solution containing salidroside, tyrosol, catechin, chrysophanol-8-O-glucoside reference substance about 0.50 mg, vanillin, p-coumaric acid, isoquercitrin, quercitrin, kaempferol reference substance about 1.00mg, gallic acid, protocatechuic acid, and caffeic acid about 1.50mg in 75% methanol per 1mL as reference substance solution;
(3) chromatographic parameters: RP18 chromatographic column, the column temperature is 25-40 ℃, the flow rate is 0.5-1.5mL min < -1 >, the detection wavelength is 270-290 nm, and the sample injection amount is 8-15 mu L; the mobile phase A is acetonitrile, the mobile phase B is 0.1 percent glacial acetic acid water solution, the gradient elution procedure is 0-18 min, and the concentration of the mobile phase A is 10-24 percent; 24-25% of A for 18-30 min; 30-45 min, 25% -35% A; 45-55 min, 35-45% A; 55-60 min, 45-55% A; 60-65 min, 55% -95% A; 65-70 min, 95% -100% A;
(4) quality control of the fingerprint spectrum: according to the chromatographic conditions, respectively injecting the test solution and the reference solution into a high performance liquid chromatograph for detection, comparing the obtained chromatogram with the reference fingerprint of the rhodiola root medicinal material, calculating the similarity, identifying the number of the common absorption peaks, determining the similarity, and realizing the identification and quality control of the rhodiola root medicinal material.
Preferably, the rhodiola rosea medicinal material fingerprint spectrum detection method specifically comprises the following steps:
(1) preparation of a test solution: weighing rhodiola rosea powder 1g, precisely adding 75% methanol 20 mL into a 50mL conical flask with a plug, weighing, ultrasonically extracting for 15min, weighing again, supplementing the lost weight with 75% methanol, shaking up, centrifuging for 5min, taking supernatant, filtering with 0.45 μm microporous membrane, and taking the subsequent filtrate;
(2) preparation of control solutions: precisely weighing a proper amount of each reference substance, placing in a volumetric flask with a plug, adding 75% methanol for dissolving, performing ultrasonic treatment, and preparing into solution containing salidroside, tyrosol, catechin, chrysophanol-8-O-glucoside reference substance about 0.50 mg, vanillin, p-coumaric acid, isoquercitrin, quercitrin, kaempferol reference substance about 1.00mg, gallic acid, protocatechuic acid, and caffeic acid about 1.50mg in 75% methanol per 1mL as reference substance solution;
(3) chromatographic parameters: an RP18 chromatographic column with the column temperature of 30 ℃, the flow rate of 1mL min < -1 >, the detection wavelength of 276 nm and the sample injection amount of 10 mu L; the mobile phase A is acetonitrile, the mobile phase B is 0.1 percent glacial acetic acid water solution, the gradient elution procedure is 0-18 min, and the concentration of the mobile phase A is 10-24 percent; 24-25% of A for 18-30 min; 30-45 min, 25% -35% A; 45-55 min, 35-45% A; 55-60 min, 45-55% A; 60-65 min, 55% -95% A; 65-70 min, 95% -100% A;
(4) quality control of the fingerprint spectrum: according to the chromatographic conditions, respectively injecting the test solution and the reference solution into a high performance liquid chromatograph for detection, comparing the obtained chromatogram with the reference fingerprint of the rhodiola root medicinal material, calculating the similarity, identifying the number of the common absorption peaks, determining the similarity, and realizing the identification and quality control of the rhodiola root medicinal material.
In the method for detecting the fingerprint spectrum of the rhodiola rosea, in the step of preparing the test solution, the rhodiola rosea powder needs to be screened by a third sieve; the ultrasonic power is 500W, and the frequency is 40 kHz; centrifuging at 3500 r/min.
According to the fingerprint detection method of the rhodiola rosea medicinal material, the RP18 chromatographic column parameter is 5 mu m and 4.6 multiplied by 250 mm.
The rhodiola rosea medicinal material fingerprint detection method provided by the invention is used for detecting rhodiola rosea, namely rhodiola crenulata.
The HPLC fingerprint spectrum detection method of the rhodiola rosea medicinal material has the advantages of simple sample treatment, different mobile phase from the traditional acetonitrile phosphoric acid solution system, and safer and more nontoxic performance due to the adoption of the acetonitrile glacial acetic acid solution system. And the applicant creatively selects a 0.1% glacial acetic acid solution as a mobile phase, and obtains a spectrum with a smooth baseline, a large capacity and a best separation effect. And 17 characteristic peaks were confirmed.
The following experimental studies were carried out in order to confirm the stability, accuracy and feasibility of the method of the invention:
1 Instrument and reagent
Waters e2695 model high performance liquid chromatograph (PDA detector, column oven, autosampler; Empower Pro chromatographic workstation) (Waters, USA); model AL204 1/ten thousand and model AB 135-S1/10 ten thousand electronic balances (mettlelto instruments ltd); KQ-500DB type digital control ultrasonic cleaner (Kunshan ultrasonic instruments Co., Ltd.); chromatographic column Waters Xbridge Shield RP18 (5 μm, 4.6X 250 mm) (Waters, USA); DL-5200B-II type low-speed large-capacity multi-tube centrifuge (Shanghai' an pavilion scientific instruments and plants) with a centrifugation radius of 19.6 cm; Milli-Q ultrapure water treatment systems (Millipore, USA); 0.45 μm microporous membrane (Tianjin Shi laboratory instruments Co., Ltd.).
Salidroside (batch number: 1001210553, sigma, usa); gallic acid (batch No. 1110831-201605, China institute for food and drug testing); tyrosol (batch No. 111676) -200602, China pharmaceutical biologicals institute); caffeic acid (batch number: 1001098874, sigma, usa); p-coumaric acid (batch number: 112037-201801, China institute for testing and testing of food and drug); protocatechuic acid (batch number: 809-; catechin (batch No. 13051511, Chengdu Biochemical Co., Ltd.); vanillin (batch number: 20180712, shinylen research institute); isoquercitrin (batch No. 111809 201403, China institute for testing food and drug); quercetin (batch No. 111538-200504, China pharmaceutical biologicals institute); chrysophanol-8-O-glucoside (batch number: B20239-20mg, Shanghai-derived leaf Biotech Co., Ltd.); kaempferol (batch number: 1354900, USP 12601 Twinbrook Pkwy, Rockville, Md.); methanol (chromatographically pure, Fisher, usa); acetonitrile (chromatographically pure, Fisher, usa), the remaining reagents were analytically pure, and water was ultrapure water.
The Rhodiola rosea medicinal material is identified as Rhodiola rosea Rhodioa crenulata (hook.f. et Thoms.) H.Ohba by the Chinese medicine resource of Ling pharmaceutical industries Limited, Chaojiazhuang, Zusanheng high-level engineer. The source of the medicinal materials is shown in table 1.
2 methods and results
2.1 chromatographic conditions
Waters Xbridge Shield RP18 (5 μm, 4.6X 250 mm); the mobile phase is acetonitrile (A) -0.1% glacial acetic acid (B); gradient elution (see table 2); column temperature: c, 30 ℃; the detection wavelength is as follows: 276 nm; sample introduction amount: 10 muL.
2.2 preparation of test solutions
Weighing rhodiola rosea powder (passing through a No. three sieve) about 1g, precisely adding 20 mL of 75% methanol into a 50mL conical flask with a plug, weighing, ultrasonically extracting (with the power of 500W and the frequency of 40 kHz) for 15min, weighing again, supplementing the lost weight with 75% methanol, shaking up, centrifuging at 3500 r/min for 5min, taking the supernatant, filtering with a 0.45-micron microporous membrane, and taking the subsequent filtrate to obtain the rhodiola rosea powder.
2.3 preparation of control solutions
Precisely weighing appropriate amount of each control, placing in a volumetric flask with a plug, adding 75% methanol to dissolve, performing ultrasonic treatment for 5min, and making into solution containing salidroside, tyrosol, catechin, chrysophanol-8-O-glucoside about 0.50 mg, vanillin, p-coumaric acid, isoquercitrin, quercitrin, kaempferol about 1.00mg, gallic acid, protocatechuic acid, and caffeic acid about 1.50mg in 75% methanol per 1mL as control solution.
2.4 methodological considerations
2.4.1 precision test sample solution R8 was sampled for 6 times, the RSD of each chromatogram peak relative to the retention time (salidroside No. 2) was less than 0.26%, the RSD of the relative peak area was less than 4.35%, the similarity of the obtained chromatograms was 0.998, 0.999, 0.999, 0.999, 0.998, RSD was 0.06%, indicating that the precision of the instrument is good.
2.4.2 stability test A test solution R8 is taken and injected for measurement at 0, 2, 4, 6, 8, 10, 12 and 24 h respectively, RSD of relative (salidroside No. 2) retention time of each common chromatographic peak is less than 0.34 percent, RSD of relative peak area is less than 5.55 percent, similarity of 16 common peaks of obtained chromatogram is 0.997, 0.999, 0.999, 0.995, 0.996, 1.000, 0.997 and RSD is 0.18 percent respectively, which indicates that the test solution is stable within 24 h.
2.4.3 repeatability test samples R8 were taken, 6 test solutions were prepared in parallel, and the RSD of the relative (salidroside No. 2) retention time of each common chromatographic peak was determined to be less than 0.34%, the RSD of the relative area was determined to be less than 2.17%, the similarities of the chromatograms obtained were 0.999, 0.999, 0.999, 1.000, 1.000, 0.998, and the RSD was 0.08%, indicating that the method is good in repeatability.
2.5 establishment of fingerprint
HPLC detection results of 13 rhodiola rosea in different batches show that nearly 30 common peaks (shown in figure 1) are obtained, and 17 chromatographic peaks in the common peaks with good separation degree and high response value are selected as common peaks for evaluating the quality of rhodiola rosea medicinal materials.
After 17 common peaks are determined, the retention time of the chromatographic peak of the reference solution is compared with the retention time of the chromatographic peak of the test solution and the ultraviolet absorption spectrogram (figure 1). The ultraviolet spectra (figure 2) of 12 chromatographic peaks in the identified test solution are characterized in that salidroside of a chromatographic peak No. 2 with a large peak area and stable peak appearance is taken as a reference peak, similarity evaluation is carried out by adopting a Chinese medicine chromatography fingerprint similarity evaluation system (2012.130723 edition) of the State pharmacopoeia Committee to establish a common mode, and superposition spectra and comparison spectra of different batches of rhodiola rosea medicinal material samples are generated (figure 3, the similarity value of the generated comparison spectra (R) is determined to be 1, the similarity of the fingerprint spectra of each batch of rhodiola rosea medicinal material samples is calculated, and the result shows that the similarity is 0.863-0.984 (see table 3).
2.6 fingerprint analysis
According to the matching results, the similarity of 13 batches of rhodiola rosea medicinal materials is better, the fingerprint contains 17 common peaks, salidroside No. 2 is taken as a reference peak (S), the relative retention time of each common peak and the S peak is calculated, and the average value of the relative retention time of the 17 common peaks is shown in the table 4. The average value of the relative retention time is taken as a specified value, and the relative retention time of 17 common peaks of 13 batches of rhodiola rosea fingerprints is within +/-5% of the specified value.
Discussion of 3
The PDA detector is adopted to carry out sample analysis in the full wavelength range, wherein the chromatogram detected at 276 nm has the most chromatographic peaks, larger peak values and smoother base line. The detection wavelength was therefore determined to be 276 nm.
Five different chromatographic columns of three brands are considered, and the Waters Xbridge Shield RP18 chromatographic column is selected by the method in consideration of the peak shape, the separation degree and the application universality of the chromatographic columns. Meanwhile, the influence of different mobile phases and chromatographic column temperature on the obtained fingerprint chromatogram is compared, and the result shows that when the column temperature is 30 ℃, acetonitrile-0.1% glacial acetic acid is taken as the mobile phase for gradient elution, the chromatographic peak information capacity in the chromatogram is large, and the separation effect is best. The time of the separation method is prolonged to 2 times (140 min), and the result shows that no other chromatographic peak appears after 70 min, so that the gradient elution time is determined to be 70 min.
In the optimization process of the extraction process, methanol and ethanol with different proportions are used as solvents, and the extraction mode and time are respectively considered. As a result, chromatographic peak information obtained by ultrasonic treatment of 75% methanol for 15min is optimal. Therefore, the method is used for extracting the sample.
The rhodiola rosea fingerprint is established by adopting HPLC-PDA method gradient elution, and meanwhile, chromatographic conditions and a preparation method of a test solution are optimized and methodology investigation is carried out; the RSD of the relative retention time-precision, repeatability and stability is less than 0.4 percent. And a common mode of the rhodiola rosea medicinal material fingerprint is established, more reliable quality support of the raw medicinal material is provided for a company to smoothly pass through the traditional Chinese medicine in FDA second-stage clinic, and a foundation is laid for improving the quality standard of the rhodiola rosea medicinal material.
Drawings
FIG. 1: rhodiola rosea HPLC fingerprint
FIG. 2: 12 ultraviolet spectra assigned to common peaks
FIG. 3: 13 batches of rhodiola rosea chromatographic fingerprint with different numbers
Example 1:
the HPLC detection method of the rhodiola root medicinal material comprises the following steps:
(1) preparation of a test solution: weighing radix Rhodiolae powder about 1g, adding 75% methanol 20 mL into 50mL conical flask with plug, weighing, ultrasonic extracting for 20 min, weighing again, supplementing with 75% methanol, shaking, centrifuging at 3500 r/min for 5min, collecting supernatant, filtering with 0.45 μm microporous membrane, and collecting filtrate.
(2) Preparation of control solutions: precisely weighing appropriate amount of each control, placing in a volumetric flask with a plug, adding 75% methanol to dissolve, performing ultrasonic treatment for 5min, and making into solution containing salidroside, tyrosol, catechin, chrysophanol-8-O-glucoside about 0.50 mg, vanillin, p-coumaric acid, isoquercitrin, quercitrin, kaempferol about 1.00mg, gallic acid, protocatechuic acid, and caffeic acid about 1.50mg in 75% methanol per 1mL as control solution.
(3) The chromatographic parameters are as follows: waters Xbridge Shield RP18 (5 μm, 4.6X 250 mm); the mobile phase is acetonitrile (A) -0.1% glacial acetic acid (B); gradient elution; column temperature: c, 30 ℃; the detection wavelength is as follows: 270 nm; sample introduction amount: 10 muL. The mobile phase gradient elution parameters were:
(4) as a result: the rhodiola fingerprint analysis meets the relevant requirements.
Example 2
The HPLC detection method of the rhodiola root medicinal material comprises the following steps:
(1) preparation of a test solution: weighing about 1g of rhodiola rosea powder passing through a third sieve, precisely adding 20 mL of 75% methanol into a 50mL conical flask with a plug, weighing, ultrasonically extracting (power 500W and frequency 40 kHz) for 15min, weighing again, supplementing the lost weight with 75% methanol, shaking up, centrifuging at 3500 r/min for 5min, taking supernatant, filtering with a 0.45-micron microporous membrane, and taking the subsequent filtrate.
(2) Preparation of control solutions: precisely weighing appropriate amount of each control, placing in a volumetric flask with a plug, adding 75% methanol to dissolve, performing ultrasonic treatment for 5min, and making into solution containing salidroside, tyrosol, catechin, chrysophanol-8-O-glucoside about 0.50 mg, vanillin, p-coumaric acid, isoquercitrin, quercitrin, kaempferol about 1.00mg, gallic acid, protocatechuic acid, and caffeic acid about 1.50mg in 75% methanol per 1mL as control solution.
(3) The chromatographic parameters are as follows: waters Xbridge Shield RP18 (5 μm, 4.6X 250 mm); the mobile phase is acetonitrile (A) -0.1% glacial acetic acid (B); gradient elution; column temperature: c, 30 ℃; the detection wavelength is as follows: 276 nm; sample introduction amount: 10 muL. The mobile phase gradient elution parameters were:
(4) as a result: the rhodiola fingerprint analysis meets the relevant requirements.
Example 3
The HPLC detection method of the rhodiola root medicinal material comprises the following steps:
(1) preparation of a test solution: weighing rhodiola rosea powder (passing through a No. three sieve) about 3g, precisely adding 20 mL of 75% methanol into a 50mL conical flask with a plug, weighing, ultrasonically extracting (with the power of 500W and the frequency of 40 kHz) for 30min, weighing again, supplementing the lost weight with 75% methanol, shaking up, centrifuging at 3500 r/min for 5min, taking the supernatant, filtering with a 0.45-micron microporous membrane, and taking the subsequent filtrate to obtain the rhodiola rosea powder.
(2) Preparation of control solutions: precisely weighing appropriate amount of each control, placing in a volumetric flask with a plug, adding 75% methanol to dissolve, performing ultrasonic treatment for 5min, and making into solution containing salidroside, tyrosol, catechin, chrysophanol-8-O-glucoside about 0.50 mg, vanillin, p-coumaric acid, isoquercitrin, quercitrin, kaempferol about 1.00mg, gallic acid, protocatechuic acid, and caffeic acid about 1.50mg in 75% methanol per 1mL as control solution.
(3) The chromatographic parameters are as follows: waters Xbridge Shield RP18 (5 μm, 4.6X 250 mm); the mobile phase is acetonitrile (A) -0.1% glacial acetic acid (B); gradient elution; column temperature: c, 40 ℃; the detection wavelength is as follows: 290 nm; sample introduction amount: 10 muL. The mobile phase gradient elution parameters were:
(4) as a result: the rhodiola fingerprint analysis meets the relevant requirements.
Claims (6)
1. A rhodiola fingerprint detection method is characterized by comprising the following steps:
(1) preparation of a test solution: weighing 1-3g of rhodiola rosea powder, adding 20 mL of 75% methanol into a 50mL conical flask with a plug, weighing, ultrasonically extracting for 15-30 min, weighing again, supplementing the lost weight with 75% methanol, shaking up, centrifuging, taking supernatant, filtering with a 0.45 mu m microporous membrane, and taking subsequent filtrate;
(2) preparation of control solutions: precisely weighing a proper amount of each reference substance, placing the reference substances in a volumetric flask with a plug, adding 75% methanol for dissolving, and ultrasonically processing to obtain a solution containing about 0.50 mg of control substances of salidroside, tyrosol, catechin and chrysophanol-8-O-glucoside, about 1.00mg of control substances of vanillin, p-coumaric acid, isoquercitrin, quercitrin and kaempferol and about 1.50mg of gallic acid, protocatechuic acid and caffeic acid in each 1mL of 75% methanol as a reference substance solution;
(3) chromatographic parameters: RP18 chromatographic column, the column temperature is 25-40 ℃, the flow rate is 0.5-1.5mL min < -1 >, the detection wavelength is 270-290 nm, and the sample injection amount is 8-15 mu L; the mobile phase A is acetonitrile, the mobile phase B is 0.1 percent glacial acetic acid water solution, the gradient elution procedure is 0-18 min, and the concentration of the mobile phase A is 10-24 percent; 24-25% of A for 18-30 min; 30-45 min, 25% -35% A; 45-55 min, 35-45% A; 55-60 min, 45-55% A; 60-65 min, 55% -95% A; 65-70 min, 95% -100% A;
(4) quality control of the fingerprint spectrum: according to the chromatographic conditions, respectively injecting the test solution and the reference solution into a high performance liquid chromatograph for detection, comparing the obtained chromatogram with the reference fingerprint of the rhodiola root medicinal material, calculating the similarity, identifying the number of the common absorption peaks, determining the similarity, and realizing the identification and quality control of the rhodiola root medicinal material.
2. The rhodiola rosea fingerprint detection method according to claim 1, which is characterized by comprising the following steps:
(1) preparation of a test solution: weighing rhodiola rosea powder 1g, adding 75% methanol 20 mL into a 50mL conical flask with a plug, weighing, ultrasonically extracting for 15min, weighing again, supplementing the lost weight with 75% methanol, shaking up, centrifuging for 5min, taking supernatant, filtering with 0.45 μm microporous membrane, and taking the subsequent filtrate;
(2) preparation of control solutions: precisely weighing a proper amount of each reference substance, placing in a volumetric flask with a plug, adding 75% methanol for dissolving, performing ultrasonic treatment, and preparing into solution containing salidroside, tyrosol, catechin, chrysophanol-8-O-glucoside reference substance about 0.50 mg, vanillin, p-coumaric acid, isoquercitrin, quercitrin, kaempferol reference substance about 1.00mg, gallic acid, protocatechuic acid, and caffeic acid about 1.50mg in 75% methanol per 1mL as reference substance solution;
(3) chromatographic parameters: an RP18 chromatographic column with the column temperature of 30 ℃, the flow rate of 1mL min < -1 >, the detection wavelength of 276 nm and the sample injection amount of 10 mu L; the mobile phase A is acetonitrile, the mobile phase B is 0.1 percent glacial acetic acid water solution, the gradient elution procedure is 0-18 min, and the concentration of the mobile phase A is 10-24 percent; 24-25% of A for 18-30 min; 30-45 min, 25% -35% A; 45-55 min, 35-45% A; 55-60 min, 45-55% A; 60-65 min, 55% -95% A; 65-70 min, 95% -100% A;
(4) quality control of the fingerprint spectrum: according to the chromatographic conditions, respectively injecting the test solution and the reference solution into a high performance liquid chromatograph for detection, comparing the obtained chromatogram with the reference fingerprint of the rhodiola root medicinal material, calculating the similarity, identifying the number of the common absorption peaks, determining the similarity, and realizing the identification and quality control of the rhodiola root medicinal material.
3. The method for detecting the fingerprint of the rhodiola rosea medicinal material according to claim 1 or 2, wherein in the step of preparing the test solution, the rhodiola rosea powder needs to be screened by a third sieve.
4. The method for detecting the fingerprint of the rhodiola rosea medicinal material according to claim 1 or 2, wherein in the step (1), the ultrasonic power is 500W, and the frequency is 40 kHz; the centrifugation parameter is 3500 r/min.
5. The method for detecting the fingerprint of the rhodiola rosea medicinal material according to claim 1 or 2, wherein in the step (3), chromatographic parameters are as follows: the RP18 chromatographic column parameter is 5 mu m, 4.6 multiplied by 250 mm.
6. The method for detecting the fingerprint of the rhodiola rosea medicinal material according to any one of claims 1 to 5, wherein the rhodiola rosea is rhodiola crenulata.
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