CN111351891A - Method for detecting nucleoside and amino acid components in safflower - Google Patents

Method for detecting nucleoside and amino acid components in safflower Download PDF

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CN111351891A
CN111351891A CN202010372446.2A CN202010372446A CN111351891A CN 111351891 A CN111351891 A CN 111351891A CN 202010372446 A CN202010372446 A CN 202010372446A CN 111351891 A CN111351891 A CN 111351891A
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safflower
amino acid
nucleoside
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solution
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唐于平
濮宗进
周桂生
严辉
乐世俊
郭盛
陈艳琰
宿树兰
段金廒
彭国平
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Nanjing University of Chinese Medicine
Shaanxi University of Chinese Medicine
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Shaanxi University of Chinese Medicine
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • 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
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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Abstract

The invention discloses a method for detecting nucleoside and amino acid components in safflower, which is screened by a large number of experiments and adopts
Figure DDA0002478644810000011
The detection method can simultaneously detect 28 nucleoside, alkali and amino acid components in safflower. The method has high detection sensitivity and good stability, can objectively, comprehensively and accurately evaluate the quality of the safflower medicinal material and the extract and preparation thereof, and has important significance for controlling the quality and ensuring the curative effect.

Description

Method for detecting nucleoside and amino acid components in safflower
Technical Field
The invention relates to a quality control method of a traditional Chinese medicinal material, in particular to a detection method of nucleoside and amino acid components in safflower.
Background
Safflower (Carthami flowers) is a dry tubular flower of Carthamus tinctorius L.of Compositae, and is originated from Mediterranean region and distributed in America, India, China, Iran, Turkey, etc., and Xinjiang, Henan, Gansu, Sichuan, Yunnan, etc. of China become the main producing areas of safflower medicinal materials. Safflower is used as a traditional Chinese medicine, is recorded in Kai Bao Ben Cao, has pungent taste and warm nature, enters heart and liver meridians, is a traditional good medicine for promoting blood circulation to remove blood stasis, removing blood stasis and relieving pain, and is clinically used for treating dysmenorrheal, amenorrhea, blood vessel occlusion, traumatic injury, coronary heart disease, hypertension, angina pectoris and the like. At present, more than 200 compounds have been isolated and identified from safflower, including components of quinoid chalcones, flavonoids, alkaloids, polyacetylenes, etc., wherein the quinoid chalcones and flavonoids are generally considered as pharmaceutically relevant active ingredients. Therefore, many studies have been made to quantitatively detect the active ingredient of safflower. In addition, over the past 50 years, seeds were also harvested from commercially planted safflower and seed oil was extracted. Safflower seed oil is colorless and odorless, and is considered as a source of healthy edible oil. Therefore, the application of safflower is mainly focused on the dyeing of food and clothes, the extraction of pharmaceutical preparations and safflower seed oil. However, there are few reports on the edible value of safflower, and amino acids, nucleosides, polysaccharides, lipids, vitamins, and the like contained in safflower are very important for the development of food products of safflower.
The nucleoside components have good physiological activity, and are essential components for cell to maintain life activity. Amino acid components are also important living matters and are basic units of enzymes and proteins. Has effects in enhancing immunity, relieving fatigue, and regulating metabolism. At present, the research on detecting nucleoside and amino acid in ginkgo and Chinese date clarifies the value of nutrition and health and provides a basis for further developing food. Like ginkgo and jujube, safflower is also a plant used as both medicine and food, and has the potential to become a functional food. Therefore, the composition of the nucleoside and amino acid components in the safflower is clarified, so that the theoretical basis is provided for further development and utilization of the safflower, and the method has very important significance. However, there is no research focused on the nucleoside, base, and amino acid components in safflower. The determination of free amino acids is limited, most of which require derivatization and the process is cumbersome.
The method established by the invention can simultaneously detect the nucleoside, alkali and amino acid components in the safflower, and has important significance for controlling the quality of the safflower.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to overcome the defects of the prior art, and adopts UPLC-
Figure BDA0002478644790000011
/MS2The method can simultaneously detect 28 kinds of nucleoside, basic and amino acid components in the safflower. The method has high detection sensitivity and good stability, can objectively, comprehensively and accurately evaluate the quality of the safflower medicinal material and the extract and preparation thereof, and has important significance for controlling the quality and ensuring the curative effect.
The technical scheme is as follows: in order to achieve the above purpose, the invention adopts the technical scheme that:
a method for detecting nucleoside and amino acid components in safflower comprises the following steps:
step (1) preparation of control solution
Accurately weighing uracil, guanine, adenine, cytidine, guanosine, inosine, thymidine, thymine, uridine, hypoxanthine, xanthine, L-glutamic acid, trans-4-hydroxy-L-proline, L-lysine, L-glutamine, L-serine, L (+) -arginine, L-histidine, L-asparagine, L-leucine, L-methionine, L-phenylalanine, L-proline, L-threonine, L-tryptophan, L-tyrosine, L-valine and L-citrulline which are dried to constant weight, preparing a mixed standard storage solution, and storing at 4 ℃;
step (2) preparation of test solution
Precisely weighing safflower sample powder, placing in a conical flask with a plug, precisely adding deionized water, weighing, ultrasonically extracting, supplementing weight loss with deionized water, centrifuging, collecting supernatant, storing at 4 deg.C, and filtering with 0.22 μm microporous membrane before sample injection;
step (3) establishment of linear regression equation
Taking the mixed standard stock solution in the step (1), sequentially diluting by 5 times, filtering through a 0.22 mu m cellulose membrane to obtain mixed reference substance solutions with series concentrations, and sequentially injecting UPLC-
Figure BDA0002478644790000021
/MS2Taking the concentration of the series of reference substances as a horizontal coordinate X and the corresponding peak area as a vertical coordinate Y, performing linear regression analysis on each chemical component and calculating a linear regression equation;
step (4) content measurement
Taking the safflower test solution in the step (2), and injecting UPLC-
Figure BDA0002478644790000022
/MS2And (4) analyzing, substituting the peak areas into the linear regression equation in the step (3), and calculating the content of the nucleoside, the base and the amino acid in the sample solution.
Preferably, in the method for detecting nucleoside and amino acid components in safflower described above, the method for preparing the reference solution in step (1) is:
uracil (Ura, 1), guanine (Gua, 2), adenine (Ade, 3), cytidine (Cyt, 4), guanosine (Guad, 5), inosine (Ino, 6), thymidine (Thyd, 7), thymine (Thy, 8), uridine (Uri, 9), hypoxanthine (Hyp, 10), xanthine (Xan, 11), L-glutamic acid (Glu, 12), trans-4-hydroxy-L-proline (Hpro, 13), L-lysine (Lys, 14), L-glutamine (Gln, 15), L-serine (Ser, 16), L (+) -arginine (Arg, 17), L-histidine (His, 18), L-asparagine (Asp, 19), L-leucine (Leu, 20), L-methionine (Met, 21), L-phenylalanine (Phe), 22) l-proline (Pro, 23), L-threonine (Thr, 24), L-tryptophan (Try, 25), L-tyrosine (Tyr, 26), L-valine (Val, 27) and L-citrulline (Cit, 28) in a 9: 1 volume ratio acetonitrile/water at a concentration of 35.000. mu.g/mL, 3.900. mu.g/mL, 36.667. mu.g/mL, 34.667. mu.g/mL, 37.333. mu.g/mL, 40.333. mu.g/mL, 37.000. mu.g/mL, 33.333. mu.g/mL, 34.000. mu.g/mL, 4.000. mu.g/mL, 4.033. mu.g/mL, 46.333. mu.g/mL, 35.667. mu.g/mL, 45.333. mu.g/mL, 55.667. mu.g/mL, 50.667. mu.g/mL, 38.333. mu.g/mL, 43.667. mu.g/mL, 47.667. mu.g/mL, 33.333. mu.g/mL, and/mL, 47.000 μ g/mL, 43.000 μ g/mL, 70.000 μ g/mL, 34.667 μ g/mL, 33.333 μ g/mL, 36.333 μ g/mL, 32.000 μ g/mL and 41.667 μ g/mL of mixed standard stock solutions, stored at 4 ℃;
preferably, in the method for detecting nucleoside and amino acid components in safflower described above, the method for preparing the test solution in step (2) is:
precisely weighing 1.0g of safflower sample powder, placing the powder in a 50mL conical flask with a plug, precisely adding 20mL of deionized water, weighing, carrying out ultrasonic treatment at 30 ℃ for 40 minutes and at 40kHz, complementing the weight loss by using the deionized water, centrifuging at 13000rpm for 10min, taking supernatant, storing at 4 ℃, and filtering through a 0.22-micron microporous filter membrane before sample injection.
Preferably, the method for detecting nucleoside and amino acid components in safflower described above, UPLC-
Figure BDA0002478644790000033
/MS2The chromatographic conditions of (A) are as follows:
the chromatographic column is Acquity UPLC BEHAmide with the specification of 2.1 × 100mM and the thickness of 1.7 mu m, the mobile phase comprises an A phase (5mM ammonium formate and ammonium acetate and 0.2% formic acid) and a B phase (acetonitrile and 1mM ammonium formate, ammonium acetate and 0.2% formic acid), the gradient elution procedure is that 10% A is used for 0-3 min, 10% A is used for 3-9 min, 10% A is used for 9-15 min, 18% A is used for 18% to 20% A, 15-16 min is used for 20% A is used for 46% A for 16-18 min, 46% A is used for 18-20 min, the flow rate is 0.40mL/min, the sample feeding amount is 1 mu L, the column temperature is 30 ℃, and the sample temperature is 4 ℃;
the positive ions and the negative ions adopt a Multiple Reaction Monitoring (MRM) mode, and the capillary voltage: 5kV, temperature: 550 ℃, desolventizing air flow rate: 1000L/h, cone airflow rate: 50L/h, ion source temperature: at 150 ℃. The mass spectrum conditions of the 28 reference substances are shown in the following table 1:
TABLE 1 comparison of quality Spectrum conditions
Figure BDA0002478644790000031
In the method for detecting nucleoside and amino acid components in safflower described above, the linear regression equation of the 28 reference substances in step (3) is as follows:
TABLE 2 Linear regression equation
Figure BDA0002478644790000032
Figure BDA0002478644790000041
Methodology investigation
(1) Mixed standard solutions were prepared at a range of concentrations to obtain detection Limits (LOD) and quantitation (LOQ) for 28 compounds at signal to noise ratios (S/N) of 3 and 10, respectively, as in table 2 above. The peak height is divided by the background noise value as S/N.
(2) Precision of the method
Precision within and between one day was examined for one day and three consecutive days, respectively, and RSD was calculated according to the chromatographic condition test described above. The results are shown in Table 3.
(3) Repeatability of
Six batches of S1 sample solutions were prepared according to the test sample solution method described above, and the RSD was calculated by performing condition detection according to the chromatography described above. The results are shown in Table 3.
(4) Stability of
The test solution is prepared according to the method, and analyzed for 0, 2, 4, 6, 8 and 10 hours respectively to calculate RSD. The results are shown in Table 3.
(5) Sample recovery rate
To the sample S1 solution, 28 standard solutions performed by adding standard solutions of low concentration (80% of known amount), medium concentration (same of known amount) and high concentration (120% of known amount) to the representative sample S1 solution, adding standard samples according to the above method, and calculating RSD. The results are shown in Table 3.
TABLE 3 precision, repeatability, stability, sample recovery
Figure BDA0002478644790000042
Figure BDA0002478644790000051
Has the advantages that: compared with the prior art, the detection method for the nucleoside and amino acid components in the safflower provided by the invention has the following advantages:
1. according to the invention, based on the structures and property characteristics of 28 nucleic acid nucleotides, alkali groups and amino acid components with different structures and properties in safflower, through a large number of experimental screening, chromatographic and mass spectrum conditions are optimized, and the separation effects of an Acquisetic UPLC BEH C18(2.1 × mm,1.7 μm), a Thermo Scientific Hypersil GOLD (3 × 100mm, 1.9 μm) chromatographic column and an UPLC BEH HILIC (2.1 × mm,1.7 μm) chromatographic column are examined.
2. In addition, the invention screens mass spectrum conditions through a large number of experiments and adopts the positive ion mode [ M + H ]]+In (3), the parent ion (Q1) is cleaved by energy to give the daughter ion (Q3). Cyt, Guad, Ino, Thyd and Uri lose ribose (132Da) to give the corresponding product ions. Ura, Gua and Ade through [ M + H-42]+The product ion Phe, Leu, Met, Val, Pro, Tyr, Thr, Ser, His, and Asp (involving the carboxyl group and the α -amino group) is obtained via [ M + H-46]+Abundant product ions are obtained because formic acid is rearranged. Gln and Lys at [ M + H-46-17]+Has abundant product ions. Q3 of Glu is [ M + H-46-18]+。
3. The methodological investigation shows that the method has high detection sensitivity, good stability, good reproducibility and high detection accuracy, can simultaneously detect 28 nucleosides, bases and amino acid components, can objectively, comprehensively and accurately evaluate the quality of the safflower medicinal material, the extract thereof and the preparation, and has important significance for controlling the quality and ensuring the clinical curative effect.
Drawings
FIG. 1 shows MRM chromatograms of 28 control components.
Detailed Description
The present invention is further illustrated by the following examples, which are intended to be purely exemplary and are not intended to limit the scope of the invention, as various equivalent modifications of the invention will occur to those skilled in the art upon reading the present disclosure and fall within the scope of the appended claims.
Example 1
The instruments and materials used in this example
Apparatus and device
The WatersAcquity UPLC system was equipped with a four-pump solvent system, an in-line degasser, and an autosampler (Waters, Milford, USA); AB SCIEX Triple Quad 6500plus (AB SCIEX Corp., Massachusetts, USA). An electronic balance (BP211D, d 0.01mg, sartorius scientific instruments ltd); centrifuge (Anke TGL-16B, Shanghai' an Tint scientific Instrument factory); an ultrasonic cleaner (model KH-500DB, kunshan grass ultrasonic instrument ltd); high-speed Chinese medicine grinder (XFB-200 type, good pharmaceutical machinery factory in Ji' e city).
Reagents and materials used in the examples
Reagent and reagent
The 28 controls were: uracil (Ura, 1), guanine (Gua, 2), adenine (Ade, 3), cytidine (Cyt, 4), guanosine (Guad, 5), inosine (Ino, 6), thymidine (Thyd, 7), thymine (Thy, 8), uridine (Uri, 9), hypoxanthine (Hyp, 10), xanthine (Xan, 11), L-glutamic acid (Glu, 12), trans-4-hydroxy-L-proline (Hpro, 13), L-lysine (Lys, 14), L-glutamine (Gln, 15), L-serine (Ser, 16), L (+) -arginine (Arg, 17), L-histidine (His, 18), L-asparagine (Asp, 19), L-leucine (Leu, 20), L-methionine (Met, 21), L-phenylalanine (Phe, 22), l-proline (Pro, 23), L-threonine (Thr, 24), L-tryptophan (Try, 25), L-tyrosine (Tyr, 26), L-valine (Val, 27) and L-citrulline (Cit, 28) were all purchased from Sigma Chemical Co. Acetonitrile and formic acid (HPLC grade) were purchased from Merck (Darmstadt, Germany). Ammonium formate and ammonium acetate (HPLC grade) were purchased from national pharmaceutical chemicals of stock limited (shanghai, china).
30 batches of safflower samples from different regions were identified as tubular flowers of safflower, and the specimen was stored in the plant specimen chamber of the university of traditional Chinese medicine of Nanjing, China. The samples were crushed, sieved (50 mesh) and stored under dry conditions at room temperature. The sample information is shown in table 4.
Table 430 batch safflower medicinal material information table
Figure BDA0002478644790000061
Figure BDA0002478644790000071
A method for detecting nucleoside and amino acid components in safflower comprises the following steps:
step (1) preparation of control solution
Uracil (Ura, 1), guanine (Gua, 2), adenine (Ade, 3), cytidine (Cyt, 4), guanosine (Guad, 5), inosine (Ino, 6), thymidine (Thyd, 7), thymine (Thy, 8), uridine (Uri, 9), hypoxanthine (Hyp, 10), xanthine (Xan, 11), L-glutamic acid (Glu, 12), trans-4-hydroxy-L-proline (Hpro, 13), L-lysine (Lys, 14), L-glutamine (Gln, 15), L-serine (Ser, 16), L (+) -arginine (Arg, 17), L-histidine (His, 18), L-asparagine (Asp, 19), L-leucine (Leu, 20), L-methionine (Met, 21), L-phenylalanine (Phe), 22) l-proline (Pro, 23), L-threonine (Thr, 24), L-tryptophan (Try, 25), L-tyrosine (Tyr, 26), L-valine (Val, 27) and L-citrulline (Cit, 28) in a 9: 1 volume ratio acetonitrile/water at a concentration of 35.000. mu.g/mL, 3.900. mu.g/mL, 36.667. mu.g/mL, 34.667. mu.g/mL, 37.333. mu.g/mL, 40.333. mu.g/mL, 37.000. mu.g/mL, 33.333. mu.g/mL, 34.000. mu.g/mL, 4.000. mu.g/mL, 4.033. mu.g/mL, 46.333. mu.g/mL, 35.667. mu.g/mL, 45.333. mu.g/mL, 55.667. mu.g/mL, 50.667. mu.g/mL, 38.333. mu.g/mL, 43.667. mu.g/mL, 47.667. mu.g/mL, 33.333. mu.g/mL, and/mL, 47.000 μ g/mL, 43.000 μ g/mL, 70.000 μ g/mL, 34.667 μ g/mL, 33.333 μ g/mL, 36.333 μ g/mL, 32.000 μ g/mL and 41.667 μ g/mL of mixed standard stock solutions, stored at 4 ℃;
step (2) preparation of test solution
Precisely weighing 1.0g of safflower sample powder of 30 batches in the table 4, respectively placing the powder into 50mL conical bottles with stoppers, respectively and precisely adding 20mL of deionized water, weighing, sequentially carrying out ultrasonic treatment at 30 ℃ for 40 minutes and at 40kHz, using the deionized water to complement and reduce the weight loss, centrifuging at 13000rpm for 10min, taking supernatant, and storing at 4 ℃ to obtain 30 safflower sample solutions. The sample was passed through a 0.22 μm microporous membrane.
Step (3) establishment of linear regression equation
Taking the mixed standard stock solution in the step (1), sequentially diluting by 5 times, filtering through a 0.22 mu m cellulose membrane to obtain mixed reference substance solutions with series concentrations, and sequentially injecting UPLC-
Figure BDA0002478644790000073
/MS2MRM of 28 reference ComponentsThe chromatogram is shown in FIG. 1. Taking the concentration of the series of reference substances as a horizontal coordinate X and the corresponding peak area as a vertical coordinate Y, carrying out linear regression analysis on each chemical component and calculating a linear regression equation as follows;
Figure BDA0002478644790000072
Figure BDA0002478644790000081
step (4) content measurement
Taking 30 batches of safflower sample solution in the step (2), and respectively injecting UPLC-
Figure BDA0002478644790000084
/MS2And (4) carrying out analysis, substituting the peak areas into the linear regression equation in the step (3), and calculating the content of the nucleoside, the base and the amino acid in the test solution, as shown in the following tables 5 and 6.
TABLE 530 safflower batches with 14 ingredient contents (mg/g)
Figure BDA0002478644790000082
Table 630 additional safflower carthamus ingredients (mg/g)
Figure BDA0002478644790000083
Figure BDA0002478644790000091
UPLC-
Figure BDA0002478644790000093
/MS2The chromatographic conditions of (A) are as follows:
the chromatographic column is Acquity UPLC BEHAmide with the specification of 2.1 × 100mM and the thickness of 1.7 mu m, the mobile phase comprises an A phase (5mM ammonium formate and ammonium acetate and 0.2% formic acid) and a B phase (acetonitrile and 1mM ammonium formate, ammonium acetate and 0.2% formic acid), the gradient elution procedure is that 10% A is used for 0-3 min, 10% A is used for 3-9 min, 10% A is used for 9-15 min, 18% A is used for 18% to 20% A, 15-16 min is used for 20% A is used for 46% A for 16-18 min, 46% A is used for 18-20 min, the flow rate is 0.40mL/min, the sample feeding amount is 1 mu L, the column temperature is 30 ℃, and the sample temperature is 4 ℃;
the positive ions and the negative ions adopt a Multiple Reaction Monitoring (MRM) mode, and the capillary voltage: 5kV, temperature: 550 ℃, desolventizing air flow rate: 1000L/h, cone airflow rate: 50L/h, ion source temperature: at 150 ℃. The 28 control quality spectrum conditions are as follows:
Figure BDA0002478644790000092
Figure BDA0002478644790000101
the results in tables 5 and 6 show that the method established by the invention can simultaneously detect the content of 28 components. And compared with the content of nucleoside and base components in the safflower, the content of Ura, Cyt and Guad in the safflower is very high, so the safflower not only has medicinal value, but also has certain edible value. The total nucleoside and nucleobase content of Gansu safflower is highest, with the content of Ura, Ade, Thyd, Uri, Xan higher than that of samples elsewhere. The total nucleoside and nucleobase content was the lowest in the samples from Henan province. The total content of nucleoside and nucleobase of the newly-built safflower in Yunnan, Sichuan and China is not greatly different.
The content of total essential amino acids of Gansu safflower is highest, and the content of Henan samples is lowest. The content of His, Thr and Val in Gansu safflower is higher than that of samples in other areas.
The detection method provided by the invention can be used for simultaneously and accurately detecting 28 nucleoside, basic group and amino acid components in the safflower, and the quality control method of the safflower and the extract thereof provided by the invention has the advantages of high precision, high sensitivity, high stability and high accuracy, can be used for objectively, comprehensively and accurately evaluating the quality of the safflower medicinal material, the extract and the preparation thereof, and has important significance for accurately controlling the quality of the safflower medicinal material and the preparation thereof.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (6)

1. A method for detecting nucleoside and amino acid components in safflower is characterized by comprising the following steps:
step (1) preparation of control solution
Accurately weighing uracil, guanine, adenine, cytidine, guanosine, inosine, thymidine, thymine, uridine, hypoxanthine, xanthine, L-glutamic acid, trans-4-hydroxy-L-proline, L-lysine, L-glutamine, L-serine, L (+) -arginine, L-histidine, L-asparagine, L-leucine, L-methionine, L-phenylalanine, L-proline, L-threonine, L-tryptophan, L-tyrosine, L-valine and L-citrulline which are dried to constant weight, preparing a mixed standard storage solution, and storing at 4 ℃;
step (2) preparation of test solution
Precisely weighing safflower sample powder, placing in a conical flask with a plug, precisely adding deionized water, weighing, ultrasonically extracting, supplementing weight loss with deionized water, centrifuging, collecting supernatant, storing at 4 deg.C, and filtering with 0.22 μm microporous membrane before sample injection;
step (3) establishment of linear regression equation
Sequentially diluting the mixed standard stock solution obtained in the step (1) by 5 times, filtering through a 0.22 mu m cellulose membrane to obtain mixed reference substance solutions with serial concentrations, and sequentially injecting the mixed reference substance solutions into the mixed standard stock solution
Figure FDA0002478644780000011
/MS2Taking the concentration of the series of reference substances as a horizontal coordinate X and the corresponding peak area as a vertical coordinate Y, performing linear regression analysis on each chemical component and calculating a linear regression equation;
step (4) content measurement
Taking the safflower sample solution in the step (2), and injecting the safflower sample solution
Figure FDA0002478644780000012
/MS2And (4) analyzing, substituting the peak areas into the linear regression equation in the step (3), and calculating the content of the nucleoside, the base and the amino acid in the sample solution.
2. The method according to claim 1, wherein the method for detecting glycosides and amino acids in safflower,
the preparation method of the reference substance solution in the step (1) comprises the following steps:
uracil (Ura, 1), guanine (Gua, 2), adenine (Ade, 3), cytidine (Cyt, 4), guanosine (Guad, 5), inosine (Ino, 6), thymidine (Thyd, 7), thymine (Thy, 8), uridine (Uri, 9), hypoxanthine (Hyp, 10), xanthine (Xan, 11), L-glutamic acid (Glu, 12), trans-4-hydroxy-L-proline (Hpro, 13), L-lysine (Lys, 14), L-glutamine (Gln, 15), L-serine (Ser, 16), L (+) -arginine (Arg, 17), L-histidine (His, 18), L-asparagine (Asp, 19), L-leucine (Leu, 20), L-methionine (Met, 21), L-phenylalanine (Phe), 22) l-proline (Pro, 23), L-threonine (Thr, 24), L-tryptophan (Try, 25), L-tyrosine (Tyr, 26), L-valine (Val, 27) and L-citrulline (Cit, 28) in a 9: 1 volume ratio acetonitrile/water at a concentration of 35.000. mu.g/mL, 3.900. mu.g/mL, 36.667. mu.g/mL, 34.667. mu.g/mL, 37.333. mu.g/mL, 40.333. mu.g/mL, 37.000. mu.g/mL, 33.333. mu.g/mL, 34.000. mu.g/mL, 4.000. mu.g/mL, 4.033. mu.g/mL, 46.333. mu.g/mL, 35.667. mu.g/mL, 45.333. mu.g/mL, 55.667. mu.g/mL, 50.667. mu.g/mL, 38.333. mu.g/mL, 43.667. mu.g/mL, 47.667. mu.g/mL, 33.333. mu.g/mL, and/mL, 47.000. mu.g/mL, 43.000. mu.g/mL, 70.000. mu.g/mL, 34.667. mu.g/mL, 33.333. mu.g/mL, 36.333. mu.g/mL, 32.000. mu.g/mL and 41.667. mu.g/mL of mixed standard stock solutions, stored at 4 ℃.
3. The method according to claim 1, wherein the method for detecting glycosides and amino acids in safflower,
the preparation method of the test solution in the step (2) comprises the following steps:
precisely weighing 1.0g of safflower sample powder, placing the powder in a 50mL conical flask with a plug, precisely adding 20mL of deionized water, weighing, carrying out ultrasonic treatment at 30 ℃ for 40 minutes and at 40kHz, complementing the weight loss by using the deionized water, centrifuging at 13000rpm for 10min, taking supernatant, storing at 4 ℃, and filtering through a 0.22-micron microporous filter membrane before sample injection.
4. The method according to claim 1, wherein the method for detecting glycosides and amino acids in safflower,
in the step (3) and the step (4)
Figure FDA0002478644780000023
/MS2The chromatographic conditions of (A) are as follows:
the chromatographic column is an Acquity UPLC BEH Amide with the specification of 2.1 × 100mM and the thickness of 1.7 mu m, the mobile phase comprises an A phase (5mM ammonium formate and ammonium acetate, 0.2% formic acid) and a B phase (acetonitrile and 1mM ammonium formate, ammonium acetate and 0.2% formic acid), the gradient elution procedure comprises the following steps of 0-3 min: 10% A, 3-9 min: 10% A-18% A, 9-15 min: 18% A-20% A, 15-16 min: 20% A-46% A, 16-18 min: 46% A, 18-20 min: 46% A-10% A, the flow rate is 0.40mL/min, the sample introduction amount is 1 mu L, the column temperature is 30 ℃, and the sample temperature is 4 ℃;
the positive ions and the negative ions adopt a multi-reaction monitoring mode, and the capillary voltage is as follows: 5kV, temperature: 550 ℃, desolventizing air flow rate: 1000L/h, cone airflow rate: 50L/h, ion source temperature: at 150 ℃.
5. The method for detecting nucleoside and amino acid components in safflower as claimed in claim 4, wherein the mass spectrometric conditions of 28 reference substances are as follows:
Figure FDA0002478644780000021
6. the method for detecting nucleoside and amino acid components in safflower according to claim 1, wherein the linear regression equation of 28 kinds of reference substances in step (3) is as follows:
Figure FDA0002478644780000022
Figure FDA0002478644780000031
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