CN105987978B - Method that is a kind of while measuring 7 kinds of compounds contents in cortex periplocae - Google Patents
Method that is a kind of while measuring 7 kinds of compounds contents in cortex periplocae Download PDFInfo
- Publication number
- CN105987978B CN105987978B CN201510065617.6A CN201510065617A CN105987978B CN 105987978 B CN105987978 B CN 105987978B CN 201510065617 A CN201510065617 A CN 201510065617A CN 105987978 B CN105987978 B CN 105987978B
- Authority
- CN
- China
- Prior art keywords
- compound
- reference substance
- solution
- acetonitrile
- sample
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention discloses a kind of methods for measuring 7 kinds of compounds contents in cortex periplocae simultaneously, belong to Pharmaceutical Analysis field, this method is made of following steps: the preparation of reference substance solution, the formulation of chromatographic condition and standard curve, the preparation of sample solution, calculated result.This method measures the contents of Multiple components in the Chinese medicine composition simultaneously, reproducible, precision is high, analysis speed is fast, and the content of each ingredient can be detected within the shorter period, can more easily control the quality of drug.
Description
Technical field
The invention belongs to Pharmaceutical Analysis fields, and in particular to the measuring method of active ingredient of Chinese herbs.
Background technique
Cortex periplocae, Chinese medicine name.For the dry root skin of Asclepiadaceae plant periploca spium.Spring, the excavation of two season of autumn, root skin is stripped, is dried,
Acrid flavour, hardship, it is warm-natured, there is the effect of inducing diuresis for removing edema, wind-damp dispelling, strengthening the bones and muscles.It include many chemical components, C21 steroid in cortex periplocae
Body, the main active that cardiac glycosides are cortex periplocae, this has important role for treatment related disease.Wherein cortex periplocae
In several compounds such as: compound 1: Δ 5- pregnene -3 β, 16 β, 20(R)-triol -20-O- β-D- glucopyranosyl -
(1 → 6)-β-D- glucopyranosyl-(1 → 2)-β-D- pyrans digitalose glycoside compound 2: Glucoperiplocymarin, compound 3: Δ
5- pregnene -3 β, 16 α, 20(S)-triol -20-O- β-D- glucopyranosyl-(1 → 6)-β-D- glucopyranosyl-(1 →
2)-β-D- pyrans digitalis glycoside, compound 4: periplocoside H2, compound 5: periplocoside H1, compound 6: periploca sepium glycosides M,
Compound 7: periplocoside P.Wherein compound 1,3,4,5,6,7 is C21 steroid compound, and compound 2 is cardiac glycosides, this
Seven kinds of compound structures are as follows:
Can be by the content of these compounds in measurement cortex periplocae, this has very big side for the quality control of cortex periplocae
It helps.
Summary of the invention
The object of the present invention is to provide a kind of methods for measuring 7 kinds of compounds contents in cortex periplocae simultaneously, and this method is simultaneously
Measure 7 kinds of ingredients, high sensitivity.
The technical scheme adopted by the invention is that:
Method that is a kind of while measuring 7 kinds of compounds contents in cortex periplocae, the method are made of following steps:
A, the preparation of reference substance solution: -3 β of Δ 5- pregnene, 16 β, 20(R are weighed respectively)-triol -20-O- β-D- pyrans
Glucosyl group-(1 → 6)-β-D- glucopyranosyl-(1 → 2)-β-D- pyrans digitalis glycoside, Glucoperiplocymarin, the pregnant steroid of Δ 5-
Alkene -3 β, 16 α, 20(S)-triol -20-O- β-D- glucopyranosyl-(1 → 6)-β-D- glucopyranosyl-(1 → 2)-β -
D- pyrans digitalis glycoside, periplocoside H2, periplocoside H1, periploca sepium glycosides M, periplocoside P reference substance, are dissolved with methanol, are obtained
Each reference substance stock solution draws above-mentioned 7 kinds single reference substance stock solutions respectively, sets in same volumetric flask, add methanol dilution, obtain
Mixed reference substance solution is diluted to a series of concentration step by step to get reference substance solution by mixed reference substance solution；
B, the formulation of chromatographic condition and standard curve: chromatographic column is C18 column, and mobile phase is acetonitrile, 0.1% formic acid water, stream
Speed: 0.4ml/min；Sample volume is 2 μ L；Gradient program: the acetonitrile of 0-4min, 10-47%, 4-6min, 47% acetonitrile, 6-
The acetonitrile of 7min, 47-100%；40 DEG C of column temperature；
Mass Spectrometry Conditions: mass spectrum uses MRM mode, and the source ESI negative ions mode monitors simultaneously, is provided that capillary
Voltage positive ion mode is 3.0 kV, and negative ion mode is -2.5 kV, and 450 °C of source temperature, residence time is arranged automatically, from
Son to being respectively as follows: 863.4/817.4,741.4/695.2,1209.6/1163.6,1171.3/525.0,627.4/469.3,
833.3/311.2 orifice potential 40-80v, cracking voltage is 20-90ev；
By ultra high efficiency chromatograph and mass spectrograph in a series of reference substance solution injection step B of various concentrations in step A
Middle analysis, using chromatographic peak area as ordinate, concentration is abscissa, obtains the standard curve of each reference substance；
C, the preparation of sample solution: weighing cortex periplocae sample powder, adds methanol, and ultrasonic extraction is placed room temperature, mended with methanol
Sufficient less loss weight, filtering, obtains sample solution；
D, calculated result: sample solution being injected in ultra high efficiency chromatograph and is analyzed, and brings each Component peak area into standard song
In line, the content of compound 1-7 is obtained.
It is preferred that the method is made of following steps:
A, the preparation of reference substance solution: -3 β of Δ 5- pregnene, 16 β, 20(R are weighed respectively)-triol -20-O- β-D- pyrans
Glucosyl group-(1 → 6)-β-D- glucopyranosyl-(1 → 2)-β-D- pyrans digitalis glycoside, Glucoperiplocymarin, the pregnant steroid of Δ 5-
Alkene -3 β, 16 α, 20(S)-triol -20-O- β-D- glucopyranosyl-(1 → 6)-β-D- glucopyranosyl-(1 → 2)-β -
D- pyrans digitalis glycoside, periplocoside H2, periplocoside H1, periploca sepium glycosides M, periplocoside P reference substance, are dissolved with methanol, are obtained
Each reference substance stock solution draws each reference substance stock solution as in same 10mL volumetric flask respectively, adds methanol dilution, mixed
Reference substance solution, concentration are respectively as follows: compound 1:1.33 μ g/mL, compound 2:3.10 μ g/mL, compound 3:30.42 μ g/
ML, compound 4:12.18 μ g/mL, compound 5:14.66 μ g/mL, compound 6:40.87 μ g/mL, compound 7:44.40
Mixed reference substance solution is diluted to a series of concentration step by step to get the reference substance solution of various concentration by μ g/mL；
B, the formulation of chromatographic condition and standard curve: chromatographic column is C18 column, and mobile phase is acetonitrile, 0.1% formic acid water, stream
Speed: 0.4ml/min；Sample volume is 2 μ L；Gradient program: the acetonitrile of 0-4min, 10-47%, 4-6min, 47% acetonitrile, 6-
The acetonitrile of 7min, 47-100%；40 DEG C of column temperature；
Mass Spectrometry Conditions: mass spectrum uses MRM mode, and the source ESI negative ions mode monitors simultaneously, is provided that capillary
Voltage positive ion mode is 3.0 kV, and negative ion mode is -2.5 kV, and 450 °C of source temperature, residence time is arranged automatically, from
Son to being respectively as follows: 863.4/817.4,741.4/695.2,1209.6/1163.6,1171.3/525.0,627.4/469.3,
833.3/311.2 orifice potential 40-80v, cracking voltage is 20-90ev；
By ultra high efficiency chromatograph and mass spectrograph in a series of reference substance solution injection step B of various concentrations in step A
Middle analysis, using chromatographic peak area as ordinate, concentration is abscissa, obtains the standard curve of each reference substance；
C, the preparation of sample solution: weighing cortex periplocae sample powder 0.1-0.3g, adds methanol 50ml, ultrasonic extraction 30min,
Room temperature is placed, less loss weight is supplied with methanol, 0.22um filter membrane is crossed, obtains sample solution；
D, calculated result: sample solution being injected in ultra high efficiency chromatograph and is analyzed, and brings each Component peak area into standard song
In line, the content of compound 1-7 is obtained.
In the step B chromatographic column be Waters AQUITY UPLC BEH C18 chromatographic column, specification be 2.1 mm ×
50 mm, 1.7 µm。
In order to verify the feasibility and accuracy of method of the invention, make following test:
1, material
Glucoperiplocymarin is purchased from National Institute for Food and Drugs Control, remaining six kinds of compound separates from cortex periplocae to be mentioned
It obtains, content is greater than 95%.
For the chromatographic grade formic acid that UPLC-ESI-MS/MS system uses, acetonitrile is purchased from U.S. Fisher company, and flowing makes
It is made with the super purification system of Millipore 10A, sample treatment is public purchased from Tianjin Ke Miou reagent using pure methanol is analyzed
Department.
26 batch cortex periplocae samples are collected in China Shanxi, Henan, 26 different regions in Hebei, through Hebei province's drug inspection
The dry root skin of be accredited as Periploca Plants periploca spium.Detailed sample acquisition information is shown in Table 1.
2, Mass Spectrometry Conditions
Instrument: U.S.'s Waters ACQUITY Ultra Performance Liquid Chromatography instrument, the triple level four bars of Waters XEVO TQD
Mass spectrum, 4.1 software of MassLynx.
Mass spectrum uses MRM mode, and the source ESI negative ions mode monitors simultaneously, is mainly provided that capillary voltage just
Ion mode is 3.0 kV, and negative ion mode is -2.5 kV, and 450 °C of source temperature, residence time is arranged automatically.Parent ion,
Daughter ion, orifice potential, collision voltage are shown in Fig. 1.
3, the preparation of standard curve: the reference substance of accurate Weigh Compound 1-7 respectively is dissolved with methanol, obtains seven kinds of standards
The stock solution of product.Precision draws 7 kinds of reference substance stock solutions as in same 10mL volumetric flask, adds methanol constant volume, must mix control
Product solution (each compound concentration is respectively as follows: compound 1:1.33 μ g/mL, compound 2:3.10 μ g/mL, compound 3:
30.42 μ g/mL, compound 4:12.18 μ g/mL, compound 5:14.66 μ g/mL, compound 6:40.87 μ g/mL, chemical combination
Object 7:44.40 μ g/mL.This mixed reference substance solution is diluted to a series of concentration step by step, standard curve is produced, presses
It is measured according to chromatographic mass spectrometry condition, using concentration as abscissa, peak area is that ordinate carries out regression analysis.Regression equation and line
Property range is shown in Table 2.
3, detection limit and quantitative limit
Above-mentioned reference substance stock solution is gradually diluted with methanol, concentration when signal-to-noise ratio is 3 is detection limit, signal-to-noise ratio 10
When as quantitative limit.Specifying information is shown in Table 2.
4, specificity
Blank solvent (methanol), reference substance solution and cortex periplocae sample solution, respectively into UPLC-ESI-MS/MS system into
Row analysis.Compare chromatographic peak, observes at identical retention time with the presence or absence of impurity peaks.If without if the specificity of illustration method it is good
It is good.Specificity result is shown in Fig. 2.
5, precision repeatability and stability
Precision includes withinday precision and day to day precision.Withinday precision: a reference substance solution continuous sample introduction 5
It is secondary, the relative standard deviation of 7 kinds of compound concentrations is obtained, day to day precision: a reference substance solution sample introduction 6 in continuous three days
It is secondary, obtain the relative standard deviation of 7 kinds of compound concentrations.Repeatability: 6 parts of cortex periplocae sample solutions, sample introduction, obtains each compound respectively
The relative standard deviation of content.Stability: a cortex periplocae sample solution is placed at room temperature, respectively in 0 h, 4h, 8h,
12h obtains the relative standard deviation of sample concentration for 24 hours into UPLC-ESI-MS/MS network analysis.Result above is shown in Table 3, explanation
Instrument precision is good, and the repeatability of method is good, and sample has good stability.
3 precision of table, repeatability, stability
6, sample recovery rate
Accurately weighed 9 parts of 0.1g cortex periplocae powder, be separately added into 0.5mL reference substance solution (it is low, in, high concentration each three
Part, it is shown in Table 4).Sample solution is prepared according to the method for the present invention.2 μ L of sample introduction records peak area, and the sample-adding for calculating each reference substance returns
Yield the results are shown in Table 5.
The sample recovery rate and matrix effect of 57 kinds of compounds of table
7, matrix effect
Sample solution (doing three parts in parallel) is handled according to the method for the present invention, takes 1mL sample solution, 1mL mixing control is added
Product solution (it is low, in, 4) high concentration is shown in Table, obtain solution A, separately takes 1mL sample solution, and 1mL methanol is added, obtains solution B, then take
1mL methanol is added in the above-mentioned mixed reference substance solution of 1mL, obtains solution C.Solution A, B, C distinguish sample introduction 2uL, calculate matrix effect.Knot
Fruit is shown in Table 5.
8, principal component analysis and clustering
Principal component analysis and cluster point are carried out to 7 kinds of compounds in 26 place of production cortex periplocaes using 11.5 software of SPSS
Analysis.The result of principal component analysis is consistent with cluster analysis result, and 26 batches of samples one are divided into 6 major class, wherein sample 10,
12 ~ 15,25,26 batches totally 7 samples be first major class, totally 11 samples are the second major class to 5,8,9,16 ~ 22,24 batches, sample,
Totally 5 samples are third major class to 1 ~ 4,6 batch, sample, and remaining 7,23,11 batches of samples are respectively fourth, fifth, six major class.Cluster point
, principal component analysis visual result significant related to the place of production of cortex periplocae sample of classifying as the result is shown is analysed, it is satisfactory.Conclusion, it is fragrant
Adding the compounds content in skin is very effective index of tracing to the source for its place of production, utilizes compound quantitative analysis analytical technology pair
The differentiation of cortex periplocae provenance is feasible.Principal component analysis and cluster analysis result figure such as Fig. 3, Fig. 4.
By adopting the above-described technical solution, the technological progress achieved by the present invention is:
The present invention measures the contents of Multiple components in the Chinese medicine composition simultaneously, reproducible, precision is high, analysis speed
Fastly, the content of each ingredient can be detected within the shorter period, can more easily control the quality of drug.
Detailed description of the invention
Fig. 1 is the second order ms figure of compound 1-7, and parent ion, daughter ion unit are m/z, orifice potential v, cracking electricity
Pressure is ev；
Fig. 2 is specificity figure, (A: blank solution, B: reference substance, C: sample)；
Fig. 3 is principal component analysis figure；
Fig. 4 is clustering figure.
Specific embodiment
The present invention is described in further details With reference to embodiment:
Embodiment 1
Method that is a kind of while measuring 7 kinds of compounds contents in cortex periplocae, this method are made of following steps:
A, the preparation of reference substance solution: -3 β of Δ 5- pregnene, 16 β, 20(R are weighed respectively)-triol -20-O- β-D- pyrans
Glucosyl group-(1 → 6)-β-D- glucopyranosyl-(1 → 2)-β-D- pyrans digitalis glycoside (compound 1), Glucoperiplocymarin
(compound 2), Δ 5- pregnene -3 β, 16 α, 20(S)-triol -20-O- β-D- glucopyranosyl-(1 → 6)-β-D- pyrans
Glucosyl group-(1 → 2)-β-D- pyrans digitalis glycoside (compound 3), periplocoside H2(compound 4), periplocoside H1(compound
5), periploca sepium glycosides M(compound 6), periplocoside P(compound 7) reference substance, dissolved with methanol, obtain each reference substance deposit
Liquid draws each reference substance stock solution as in same 10mL volumetric flask respectively, adds methanol dilution, obtain mixed reference substance solution,
Concentration is respectively as follows: compound 1:1.33 μ g/mL, compound 2:3.10 μ g/mL, compound 3:30.42 μ g/mL, compound
4:12.18 μ g/mL, compound 5:14.66 μ g/mL, compound 6:40.87 μ g/mL, compound 7:44.40 μ g/mL will
Mixed reference substance solution is diluted to a series of concentration step by step to get the reference substance solution of various concentration；
B, the formulation of chromatographic condition and standard curve: chromatographic column is Waters AQUITY UPLC BEH C18 column, rule
Lattice are the mm of 2.1 mm × 50, and 1.7 μm, mobile phase is acetonitrile, 0.1% formic acid water, flow velocity: 0.4ml/min；Sample volume is 2 μ
L；Gradient program: the acetonitrile of 0-4min, 10-47%, 4-6min, 47% acetonitrile, the acetonitrile of 6-7min, 47-100%；Column temperature 40
℃；
Mass Spectrometry Conditions: mass spectrum uses MRM mode, and the source ESI negative ions mode monitors simultaneously, is provided that capillary
Voltage positive ion mode is 3.0 kV, and negative ion mode is -2.5 kV, and 450 °C of source temperature, residence time is arranged automatically, respectively
The ion pair of compound, orifice potential, cracking voltage are shown in Fig. 1,
By ultra high efficiency chromatograph and mass spectrograph in a series of reference substance solution injection step B of various concentrations in step A
Middle analysis, using chromatographic peak area as ordinate, concentration is abscissa, obtains the standard curve of each reference substance；
C, the preparation of sample solution: weighing In Luoning, Henan cortex periplocae sample powder 0.2g, and methanol 50ml is added to dissolve, and ultrasound mentions
30min is taken, supersonic frequency is 40 kHz, places room temperature, supplies less loss weight with methanol, crosses 0.22um filter membrane, it is molten to obtain sample
Liquid,
D, calculated result: sample solution is injected in ultra high efficiency chromatograph and mass spectrograph and is analyzed, by each Component peak area band
Enter in standard curve, obtains the content of compound 1-7, the content of compound 1 is 0.012mg/g, and the content of compound 2 is
1.07 mg/g, the content of compound 3 are 0.025 mg/g, and the content of compound 4 is 3.254 mg/g, the content of compound 5
For 5.10 mg/g, the content of compound 6 is 0.058 mg/g, and the content of compound 7 is 0.025 mg/g.
Embodiment 2
Method that is a kind of while measuring 7 kinds of compounds contents in cortex periplocae, this method are made of following steps:
A, the preparation of reference substance solution: -3 β of Δ 5- pregnene, 16 β, 20(R are weighed respectively)-triol -20-O- β-D- pyrans
Glucosyl group-(1 → 6)-β-D- glucopyranosyl-(1 → 2)-β-D- pyrans digitalis glycoside (compound 1), Glucoperiplocymarin
(compound 2), Δ 5- pregnene -3 β, 16 α, 20(S)-triol -20-O- β-D- glucopyranosyl-(1 → 6)-β-D- pyrans
Glucosyl group-(1 → 2)-β-D- pyrans digitalis glycoside (compound 3), periplocoside H2(compound 4), periplocoside H1(compound
5), periploca sepium glycosides M(compound 6), periplocoside P(compound 7) reference substance, dissolved with methanol, obtain each reference substance deposit
Liquid draws each reference substance stock solution as in same 10mL volumetric flask respectively, adds methanol dilution, obtain mixed reference substance solution,
Concentration is respectively as follows: compound 1:1.33 μ g/mL, compound 2:3.10 μ g/mL, compound 3:30.42 μ g/mL, compound
4:12.18 μ g/mL, compound 5:14.66 μ g/mL, compound 6:40.87 μ g/mL, compound 7:44.40 μ g/mL will
Mixed reference substance solution is diluted to a series of concentration step by step to get the reference substance solution of various concentration；
B, the formulation of chromatographic condition and standard curve: chromatographic column is Waters AQUITY UPLC BEH C18 column, rule
Lattice are the mm of 2.1 mm × 50, and 1.7 μm, mobile phase is acetonitrile, 0.1% formic acid water, flow velocity: 0.4ml/min；Sample volume is 2 μ
L；Gradient program: the acetonitrile of 0-4min, 10-47%, 4-6min, 47% acetonitrile, the acetonitrile of 6-7min, 47-100%；Column temperature 40
℃；
Mass Spectrometry Conditions: mass spectrum uses MRM mode, and the source ESI negative ions mode monitors simultaneously, is provided that capillary
Voltage positive ion mode is 3.0 kV, and negative ion mode is -2.5 kV, and 450 °C of source temperature, residence time is arranged automatically, respectively
The ion pair of compound, orifice potential, cracking voltage are shown in Fig. 1,
By ultra high efficiency chromatograph and mass spectrograph in a series of reference substance solution injection step B of various concentrations in step A
Middle analysis, using chromatographic peak area as ordinate, concentration is abscissa, obtains the standard curve of each reference substance；
C, the preparation of sample solution: weighing Hebei Handan cortex periplocae sample powder 0.3g, and methanol 50ml is added to dissolve, and ultrasound mentions
30min is taken, supersonic frequency is 40 kHz, places room temperature, supplies less loss weight with methanol, crosses 0.22um filter membrane, it is molten to obtain sample
Liquid,
D, calculated result: sample solution is injected in ultra high efficiency chromatograph and mass spectrograph and is analyzed, by each Component peak area band
Enter in standard curve, obtains the content of compound 1-7, the content of compound 1 is 0.013mg/g, and the content of compound 2 is
1.05 mg/g, the content of compound 3 are 0.021 mg/g, and the content of compound 4 is 3.234 mg/g, the content of compound 5
For 5.13 mg/g, the content of compound 6 is 0.056 mg/g, and the content of compound 7 is 0.028 mg/g.
Embodiment 3
Method that is a kind of while measuring 7 kinds of compounds contents in cortex periplocae, this method are made of following steps:
A, the preparation of reference substance solution: -3 β of Δ 5- pregnene, 16 β, 20(R are weighed respectively)-triol -20-O- β-D- pyrans
Glucosyl group-(1 → 6)-β-D- glucopyranosyl-(1 → 2)-β-D- pyrans digitalis glycoside (compound 1), Glucoperiplocymarin
(compound 2), Δ 5- pregnene -3 β, 16 α, 20(S)-triol -20-O- β-D- glucopyranosyl-(1 → 6)-β-D- pyrans
Glucosyl group-(1 → 2)-β-D- pyrans digitalis glycoside (compound 3), periplocoside H2(compound 4), periplocoside H1(compound
5), periploca sepium glycosides M(compound 6), periplocoside P(compound 7) reference substance, dissolved with methanol, obtain each reference substance deposit
Liquid draws each reference substance stock solution as in same 10mL volumetric flask respectively, adds methanol dilution, obtain mixed reference substance solution,
Concentration is respectively as follows: compound 1:1.33 μ g/mL, compound 2:3.10 μ g/mL, compound 3:30.42 μ g/mL, compound 4:
12.18 μ g/mL, compound 5:14.66 μ g/mL, compound 6:40.87 μ g/mL, compound 7:44.40 μ g/mL will be mixed
It closes reference substance solution and is diluted to a series of concentration step by step to get the reference substance solution of various concentration；
B, the formulation of chromatographic condition and standard curve: chromatographic column is Waters AQUITY UPLC BEH C18 column, rule
Lattice are the mm of 2.1 mm × 50, and 1.7 μm, mobile phase is acetonitrile, 0.1% formic acid water, flow velocity: 0.4ml/min；Sample volume is 2 μ
L；Gradient program: the acetonitrile of 0-4min, 10-47%, 4-6min, 47% acetonitrile, the acetonitrile of 6-7min, 47-100%；Column temperature 40
℃；
Mass Spectrometry Conditions: mass spectrum uses MRM mode, and the source ESI negative ions mode monitors simultaneously, is provided that capillary
Voltage positive ion mode is 3.0 kV, and negative ion mode is -2.5 kV, and 450 °C of source temperature, residence time is arranged automatically, respectively
The ion pair of compound, orifice potential, cracking voltage are shown in Fig. 1,
By ultra high efficiency chromatograph and mass spectrograph in a series of reference substance solution injection step B of various concentrations in step A
Middle analysis, using chromatographic peak area as ordinate, concentration is abscissa, obtains the standard curve of each reference substance；
C, the preparation of sample solution: the cortex periplocae sample powder 0.1g in village, Yuncheng, Shanxi Wenxi County is weighed, adds methanol 50ml molten
Solution, ultrasonic extraction 30min, supersonic frequency are 40 kHz, place room temperature, supply less loss weight with methanol, cross 0.22um filter membrane, obtain
To sample solution,
D, calculated result: sample solution is injected in ultra high efficiency chromatograph and mass spectrograph and is analyzed, by each Component peak area band
Enter in standard curve, obtains the content of compound 1-7, the content of compound 1 is 0.011mg/g, and the content of compound 2 is
1.08 mg/g, the content of compound 3 are 0.024 mg/g, and the content of compound 4 is 3.253 mg/g, the content of compound 5
For 5.09 mg/g, the content of compound 6 is 0.057 mg/g, and the content of compound 7 is 0.022 mg/g.
Claims (3)
1. a kind of method for measuring 7 kinds of compounds contents in cortex periplocae simultaneously, it is characterised in that the method is by following steps structure
At:
A, the preparation of reference substance solution: difference Weigh Compound 1: Δ 5- pregnene -3 β, 16 β, 20(R)-triol -20-O- β-D-
Glucopyranosyl-(1 → 6)-β-D- glucopyranosyl-(1 → 2)-β-D- pyrans digitalis glycoside, compound 2: periploca spium poison
Glycosides, compound 3: Δ 5- pregnene -3 β, 16 α, 20(S)-triol -20-O- β-D- glucopyranosyl-(1 → 6)-β-D- pyrans
Glucosyl group-(1 → 2)-β-D- pyrans digitalis glycoside, compound 4: periplocoside H2, compound 5: periplocoside H1, compound 6:
Periploca sepium glycosides M, compound 7: periplocoside P reference substance is dissolved with methanol, is obtained each reference substance stock solution, is drawn above-mentioned 7 respectively
The single reference substance stock solution of kind, sets in same volumetric flask, adds methanol dilution, obtain mixed reference substance solution, will mix reference substance
Solution is diluted to a series of concentration step by step to get reference substance solution；
B, the formulation of chromatographic condition and standard curve: chromatographic column C18Column, mobile phase be acetonitrile, 0.1% formic acid water, flow velocity:
0.4ml/min；Sample volume is 2 μ L；Gradient program: the acetonitrile of 0-4min, 10-47%, 4-6min, 47% acetonitrile, 6-7min,
The acetonitrile of 47-100%；40 DEG C of column temperature；
Mass Spectrometry Conditions: mass spectrum uses MRM mode, and the source ESI negative ions mode monitors simultaneously, is provided that capillary voltage
Positive ion mode is 3.0 kV, and negative ion mode is -2.5 kV, and 450 °C of source temperature, residence time is arranged automatically, ion pair
It is respectively as follows: 863.4/817.4,741.4/695.2,1209.6/1163.6,1171.3/525.0,627.4/469.3,833.3/
311.2, orifice potential 40-80v, cracking voltage are 20-90ev；
By in ultra high efficiency chromatograph in a series of reference substance solution injection step B of various concentrations in step A and mass spectrograph points
Analysis, using chromatographic peak area as ordinate, concentration is abscissa, obtains the standard curve of each reference substance；
C, the preparation of sample solution: weighing cortex periplocae sample powder, adds methanol, and ultrasonic extraction is placed room temperature, supplied and subtracted with methanol
Weight loss, filtering, obtains sample solution；
D, calculated result: sample solution being injected in ultra high efficiency chromatograph and is analyzed, each Component peak area is brought into standard curve,
Obtain the content of compound 1-7.
2. according to the method described in claim 1, it is characterized in that the method is made of following steps:
A, the preparation of reference substance solution: difference Weigh Compound 1: Δ 5- pregnene -3 β, 16 β, 20(R)-triol -20-O- β-D-
Glucopyranosyl-(1 → 6)-β-D- glucopyranosyl-(1 → 2)-β-D- pyrans digitalis glycoside, compound 2: periploca spium poison
Glycosides, compound 3: Δ 5- pregnene -3 β, 16 α, 20(S)-triol -20-O- β-D- glucopyranosyl-(1 → 6)-β-D- pyrrole
Glucopyranoside base-(1 → 2)-β-D- pyrans digitalis glycoside, compound 4: periplocoside H2, compound 5: periplocoside H1, compound
6: periploca sepium glycosides M, compound 7: periplocoside P reference substance is dissolved with methanol, obtains each reference substance stock solution, is drawn respectively each
Reference substance stock solution adds methanol dilution as in same 10mL volumetric flask, obtains mixed reference substance solution, being respectively as follows: of concentration
Object 1:1.33 μ g/mL, compound 2:3.10 μ g/mL, compound 3:30.42 μ g/mL, compound 4:12.18 μ g/mL are closed,
Compound 5:14.66 μ g/mL, compound 6:40.87 μ g/mL, compound 7:44.40 μ g/mL, by mixed reference substance solution
It is diluted to a series of concentration step by step to get the reference substance solution of various concentration；
B, the formulation of chromatographic condition and standard curve: chromatographic column C18Column, mobile phase be acetonitrile, 0.1% formic acid water, flow velocity:
0.4ml/min；Sample volume is 2 μ L；Gradient program: the acetonitrile of 0-4min, 10-47%, 4-6min, 47% acetonitrile, 6-7min,
The acetonitrile of 47-100%；40 DEG C of column temperature；
Mass Spectrometry Conditions: mass spectrum uses MRM mode, and the source ESI negative ions mode monitors simultaneously, is provided that capillary voltage
Positive ion mode is 3.0 kV, and negative ion mode is -2.5 kV, and 450 °C of source temperature, residence time is arranged automatically, ion pair
It is respectively as follows: 863.4/817.4,741.4/695.2,1209.6/1163.6,1171.3/525.0,627.4/469.3,833.3/
311.2, orifice potential 40-80v, cracking voltage are 20-90ev；
By in ultra high efficiency chromatograph in a series of reference substance solution injection step B of various concentrations in step A and mass spectrograph points
Analysis, using chromatographic peak area as ordinate, concentration is abscissa, obtains the standard curve of each reference substance；
C, the preparation of sample solution: weighing cortex periplocae sample powder 0.1-0.3g, adds methanol 50ml, ultrasonic extraction 30min, places
Room temperature supplies less loss weight with methanol, crosses 0.22um filter membrane, obtain sample solution；
D, calculated result: sample solution being injected in ultra high efficiency chromatograph and is analyzed, each Component peak area is brought into standard curve,
Obtain the content of compound 1-7.
3. method according to claim 1 or 2, it is characterised in that: chromatographic column is Waters AQUITY in the step B
UPLC BEH C18Chromatographic column, specification be the mm of 2.1 mm × 50,1.7 μm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510065617.6A CN105987978B (en) | 2015-02-09 | 2015-02-09 | Method that is a kind of while measuring 7 kinds of compounds contents in cortex periplocae |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510065617.6A CN105987978B (en) | 2015-02-09 | 2015-02-09 | Method that is a kind of while measuring 7 kinds of compounds contents in cortex periplocae |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105987978A CN105987978A (en) | 2016-10-05 |
| CN105987978B true CN105987978B (en) | 2019-03-05 |
Family
ID=57036370
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510065617.6A Active CN105987978B (en) | 2015-02-09 | 2015-02-09 | Method that is a kind of while measuring 7 kinds of compounds contents in cortex periplocae |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105987978B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113588818B (en) * | 2021-07-22 | 2023-08-29 | 安领生物医药(苏州)有限公司 | Method for measuring content of acanthopanax bark glycoside M in SD rat plasma |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0393724A (en) * | 1989-09-02 | 1991-04-18 | Taiyo Kagaku Co Ltd | External preparation |
| JPH08283143A (en) * | 1995-04-14 | 1996-10-29 | Kose Corp | Skin preparation for external use |
| CN1593503A (en) * | 2004-07-13 | 2005-03-16 | 天津中医学院 | Effective extract of Chinese silkuine bark with cardotonic function and its preparing process and uasge |
| CN101498702A (en) * | 2008-02-02 | 2009-08-05 | 天津天士力制药股份有限公司 | Method for analyzing fatty acid chemical composition in cortex periplocae radicis |
| CN101745091A (en) * | 2010-01-05 | 2010-06-23 | 安徽安科余良卿药业有限公司 | Method for detecting quality of blood-activating analgesic plaster |
-
2015
- 2015-02-09 CN CN201510065617.6A patent/CN105987978B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0393724A (en) * | 1989-09-02 | 1991-04-18 | Taiyo Kagaku Co Ltd | External preparation |
| JPH08283143A (en) * | 1995-04-14 | 1996-10-29 | Kose Corp | Skin preparation for external use |
| CN1593503A (en) * | 2004-07-13 | 2005-03-16 | 天津中医学院 | Effective extract of Chinese silkuine bark with cardotonic function and its preparing process and uasge |
| CN101498702A (en) * | 2008-02-02 | 2009-08-05 | 天津天士力制药股份有限公司 | Method for analyzing fatty acid chemical composition in cortex periplocae radicis |
| CN101745091A (en) * | 2010-01-05 | 2010-06-23 | 安徽安科余良卿药业有限公司 | Method for detecting quality of blood-activating analgesic plaster |
Non-Patent Citations (4)
| Title |
|---|
| A new pregnane glycoside from the root bark of Periploca sepium;Bao-jun Shi et al;《Chemistry of Natural Compounds》;20140131;第49卷(第6期);1043-4047 |
| HPLC法测定香加皮提取物中杠柳毒苷和杠柳苷元;刘虹 等;《中草药》;20060930;第37卷(第9期);1355-1356 |
| 香加皮中甾体类化学成分研究;殷志琦 等;《中国药学杂志》;20090731;第44卷(第13期);968-971 |
| 高效液相色谱法测定中药香加皮中7种孕甾烷糖苷类成分的含量;赵祎武 等;《世界科学技术》;20150131;第17卷(第1期);138-141 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105987978A (en) | 2016-10-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Xu et al. | Pharmacokinetics and bioavailability of ginsenoside Rb1 and Rg1 from Panax notoginseng in rats | |
| CN104597160B (en) | HPLC (High Performance Liquid Chromatography) method for simultaneously determining content of six organic acids in pinellia ternata | |
| CN107607649B (en) | Method for detecting periploca forrestii schltr | |
| CN104914199B (en) | The content assaying method of 12 kinds of compositions in a kind of Chinese medicinal composition preparation | |
| CN109324126B (en) | Method for simultaneously determining 9 chemical components in spina date seeds by using UPLC-MS/MS | |
| CN108181396A (en) | The detection method of 17 kinds of triterpenoid contents in a kind of ganoderma lucidum | |
| CN104777243B (en) | It is a kind of at the same determine the tuber of pinellia in organic acid, nucleosides and ephedrine HPLC methods | |
| CN102520079A (en) | Method for rapidly measuring content of solanesol in tobaccos by using UPLC (Ultra Performance Liquid Chromatography) | |
| Xie et al. | Simultaneous determination of six main components in Bushen Huoxue prescription by HPLC-CAD | |
| CN109239224A (en) | 9 kinds of method for quantitatively determining while enter blood component in semen ziziphi spinosae water extract | |
| Huang et al. | Systematic investigation on the distribution of four hidden toxic Aconitum alkaloids in commonly used Aconitum herbs and their acute toxicity | |
| CN105628835B (en) | The multicomponent content assaying method of Sheepear Inula Herb medicinal material | |
| CN110455961A (en) | Multicomponent high-flux detection method in a kind of health liquor | |
| CN105987978B (en) | Method that is a kind of while measuring 7 kinds of compounds contents in cortex periplocae | |
| CN105891403A (en) | Anemone flaccida medicinal material HPLC-UV characteristic spectrum construction method | |
| CN110261512B (en) | Metabonomics-based quality evaluation method for Uighur coreopsis tinctoria | |
| CN105510452B (en) | Multi-target ingredient assay, fingerprint map construction and the preparation method of liver-benefiting eye-brightening oral liquid | |
| CN107941955B (en) | The method of 9 kinds of functional active components in a kind of while quick detection YEDAO LUGUI JIU | |
| CN110988198A (en) | Content determination method of bi-tong ning capsules | |
| CN110243969A (en) | HPLC method that is a kind of while measuring 7 kinds of organic acids in RHIZOMA ARISAEMATIS | |
| CN105334273B (en) | Detection method of anisetree bark | |
| CN109212111A (en) | Obtain the method and its application of Paris polyphylla extracting solution | |
| CN107607665A (en) | One seedling medicine subprostrate sophora and its Chinese medicine preparation method of quality control | |
| CN102353731A (en) | Method for detecting astragalus-chickpea particles | |
| Xu et al. | Quantitative Determination of 15 Active Components in Lepidium meyenii with UHPLC-PDA and GC-MS |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |