CN104698022B - A kind of method measuring arylnaphthalene lactone type lignanoid purity based on quantitative hydrogen nuclear magnetic resonance spectroscopy - Google Patents
A kind of method measuring arylnaphthalene lactone type lignanoid purity based on quantitative hydrogen nuclear magnetic resonance spectroscopy Download PDFInfo
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Abstract
The method that the present invention relates to measure arylnaphthalene lactone type lignanoid purity based on quantitative hydrogen nuclear magnetic resonance spectroscopy, can effectively solve that existing arylnaphthalene lactone type lignanoid purity detecting technical costs is high, reference substance is difficult to obtain, operational approach is complicated, the problem of time-consuming length, method is, weigh the internal standard substance of 10 14mg, it is dissolved in the DMSO d6 of 0.8 1.2 mL, is prepared as internal standard substance solution；Weigh the arylnaphthalene lactone type lignanoid of 10 50 mg, be dissolved in the DMSO d6 of 0.8 1.2 mL, be prepared as target analytes solution；Measure the internal standard substance solution of 80 120 μ L, the target analytes solution of 180 220 μ L, the DMSO d6 of 180 220 μ L, make sample solution, by sample solution suspendible 25 35s, carry out quantitative nuclear-magnetism test, measure aromatic and arylnaphthalene lactone type lignanoid H 2 on internal standard substance, the peak area of 6, draw the target analytes molal weight ratio relative to internal standard substance, then the mass ratio by target analytes with internal standard substance, low cost of the present invention, the purity of arylnaphthalene lactone type lignanoid can be evaluated fast and accurately, have good economic and social benefit.
Description
Technical field
The present invention relates to field of medicine and chemical technology, particularly a kind of based on quantitative hydrogen nuclear magnetic resonance spectroscopy mensuration arylnaphthalene lactone
The method of type lignanoid purity.
Background technology
Due to biosynthetic complexity, the structure of natural product is the most complicated various.Based on conventional chromatographic processes
Isolated and purified means be often difficult to come Separation of Natural Products closely similar for structure, and natural product is mixed with trace
Highly active impurity can cause bioactive mistake conclusion.According to relevant report, natural product ursolic acid has 120 kinds of biologies and lives
Property, some biological activity is antipodal in different documents, such as antibacterial activity, cytotoxic activity.Such conclusion occurs
Reason be that test ursolic acid sample used by activity is not single ursolic acid chemical entities, and the impurity coexisted with it is serious
Have impact on Activity Results.Hence set up necessity that perfect natural product Purity assessment method is its evaluated biological activity accurately
Condition.The most highly purified natural product is also necessary to relevant evaluation of medical materials' quality.Therefore the Purity assessment of natural product
It is very important.
Arylnaphthalene lactone type lignanoid is the lignans that the number found from plant is more, the most plants more than 40
Plant has been reported that.This compounds has the effects such as antitumor, antiviral, antibacterial, parasite killing.Arylnaphthalene lactone type lignanoids
Compound with podophyllotoxin as representative, its derivant etoposide (etoposide, VP-16) and teniposide
(teniposide, VM-26) is applied to clinic in eighties of last century the eighties, to breast carcinoma, carcinoma of testis, small cell lung cancer, lymph
The kinds cancer such as cancer, leukemia of children all has good therapeutic effect, the arylnaphthalene lactone type wood fat with podophyllotoxin as representative
Element is one of class natural product that research is the most deep at present, enjoys the extensive concern of Chinese scholars always.This compounds
Content in relevant medical material is mainly by thin layer chromatography, and high performance liquid chromatography, capillary electrophoresis chromatograph, wherein with ultraviolet
The efficient liquid-phase chromatography method of detector is the most universal, and this method cannot be examined for those compounds not having uv absorption
Surveying, it is necessary to have high-purity standard substance to compare, these standard substance are expensive, be difficult to acquisition, and this operational approach complicated and time consumption is long.
Quantitatively hydrogen nuclear magnetic resonance spectroscopy, due to it, operation is fast and convenient, sample consumption is few, provide structural information and calmly qualitatively simultaneously
Component analysis result, particularly quantitatively hydrogen nuclear magnetic resonance spectroscopy does not relies on the high-purity standard substance of measured object, therefore quantitative analysis
Cost be substantially reduced.Utilize quantitative hydrogen nuclear magnetic resonance spectroscopy, select internal standard substance to substitute and use the standard substance of target detection thing to do
It is a very important research work that outer target high performance liquid chromatography carries out the purity testing of arylnaphthalene lactone type lignanoid.
But it is not yet seen the open report of the purity using quantitative hydrogen nuclear magnetic resonance spectroscopy to measure arylnaphthalene lactone type lignanoid.
Summary of the invention
For above-mentioned situation, in order to overcome the defect of prior art, the present invention provides a kind of based on quantitative hydrogen nuclear magnetic resonance
Spectrometry measures the method for arylnaphthalene lactone type lignanoid purity, can effectively solve existing arylnaphthalene lactone type lignanoid purity detecting
Technical costs is high, reference substance is difficult to obtain, and operational approach is complicated, the problem of time-consuming length.
The technical scheme that the present invention solves is that the method comprises the following steps: weighs the internal standard substance of 10-14mg, is dissolved in
In the DMSO-d6 of 0.8-1.2mL, it is prepared as internal standard substance solution；Weigh the arylnaphthalene lactone type lignanoid of 10-50mg, be dissolved in
In the DMSO-d6 of 0.8-1.2mL, it is prepared as target analytes solution；Measure the internal standard substance solution of 80-120 μ L, 180-220 μ L
Target analytes solution, the DMSO-d6 of 180-220 μ L, transfer to, in centrifuge tube, make sample solution, by this sample solution
Suspendible 25-35s, is transferred in nuclear magnetic tube, carries out quantitative nuclear-magnetism test, the condition that quantitative nuclear-magnetism is tested: scanning times (NS), and 8
Or 16；Spectral width (SW), 15-25ppm；Relaxation time (D1), 16-35s；Test temperature, 20-30 DEG C；Pulse angle, 30 °,
Or 45 °, or 90 °；Acquisition time (AQ), 2.5-3.5s；Measure aromatic and arylnaphthalene lactone type lignanoid H-on internal standard substance
The peak area of 2,6, draws the target analytes molal weight ratio relative to internal standard substance, then by target analytes and internal standard substance
Mass ratio is according to formula:
Calculate purity P of target analytesX, wherein:
Ix is the peak area of target analytes；IStdFor interior target peak area；Nx is the nuclear number of target analytes
Mesh；NStdFor the nuclear number of interior target；Mx is the molal weight of target analytes；MStdFor interior target molal weight；Mx is
The quality of target analytes；mStdFor interior target quality；Px is the purity of target analytes；PStdPurity for internal standard substance；
Described arylnaphthalene lactone type lignanoid is deoxypodophyllotoxin, podophyllotoxin, 4-demethylpodophyllotoxin, podophyllotoxin
Element-7 '-O-β-D-pyranglucoside, any one in 4-demethylpodophyllotoxin-7 '-O-β-D-pyranglucoside；
Described internal standard substance is Isosorbide-5-Nitrae-dinitro benzene, 3,4,5-trichloropyridines, arbitrary in 1,3,5-tri-chloro-2-Nitrobenzol
Kind.
Low cost of the present invention, can evaluate the purity of arylnaphthalene lactone type lignanoid fast and accurately, have good economy and
Social benefit.
Accompanying drawing explanation
Fig. 1 is the quantitative nuclear magnetic spectrogram of the present invention.
Fig. 2 is 4-demethylpodophyllotoxin and 1,4-dinitro benzene mol ratio and the weight of the quantitative nmr for the determination of the present invention
The mol ratio linear regression result figure that component analysis method measures.
Detailed description of the invention
Below in conjunction with embodiment, the detailed description of the invention of the present invention is described in further detail.
The present invention can be given by following example in being embodied as.
The inventive method comprises the following steps: weigh the internal standard substance of 10-14mg, is dissolved in the DMSO-d of 0.8-1.2mL6
In, it is prepared as internal standard substance solution；Weigh the arylnaphthalene lactone type lignanoid of 10-50mg, be dissolved in the DMSO-d of 0.8-1.2mL6
In, it is prepared as target analytes solution；Measure the internal standard substance solution of 80-120 μ L, the target analytes solution of 180-220 μ L,
The DMSO-d of 180-220 μ L6, transfer to, in centrifuge tube, obtain sample solution, by this sample solution suspendible 25-35s, be transferred to
In nuclear magnetic tube, carry out quantitative nuclear-magnetism test, the condition of described quantitative nuclear-magnetism test: scanning times (NS), 8 or 16；Spectral width
Degree (SW), 15-25ppm；Relaxation time (D1), 16-35s；Test temperature, 20-30 DEG C；Pulse angle, 30 °, or 45 °, or
90°；Acquisition time (AQ), 2.5-3.5s；Measure aromatic and the face, peak of arylnaphthalene lactone type lignanoid H-2,6 on internal standard substance
Long-pending, draw the target analytes molal weight ratio relative to internal standard substance, then depended on by the mass ratio of target analytes with internal standard substance
The purity of target analytes is calculated according to formula；
Described arylnaphthalene lactone type lignanoid is deoxypodophyllotoxin, podophyllotoxin, 4-demethylpodophyllotoxin, podophyllotoxin
Element-7 '-O-β-D-pyranglucoside, 4-demethylpodophyllotoxin-7 '-O-β-D-pyranglucoside, in sinolignan A
Any one；
R1=CH3Or H；R2=H, OH, glc or 6-acetyl-glc
Described internal standard substance is Isosorbide-5-Nitrae-dinitro benzene, 3,4,5-trichloropyridines, arbitrary in 1,3,5-tri-chloro-2-Nitrobenzol
Kind；
Described arylnaphthalene lactone type lignanoid purity obtains according to equation below
Ix is the peak area of target analytes；IStdFor interior target peak area；Nx is the nuclear number of target analytes
Mesh；NStdFor the nuclear number of interior target；Mx is the molal weight of target analytes；MStdFor interior target molal weight；Mx is
The quality of target analytes；mStdFor interior target quality；Px is the purity of target analytes；PStdPurity for internal standard substance.
Embodiment 1
The inventive method comprises the following steps: weighs the Isosorbide-5-Nitrae-dinitro benzene of 12.47mg, is dissolved in the DMSO-d of 1.0mL6
In, it is prepared as Isosorbide-5-Nitrae-dinitro benzene internal standard substance solution；Weigh the deoxypodophyllotoxin of 28.61mg, be dissolved in the DMSO-of 1.0mL
d6In, it is prepared as deoxypodophyllotoxin target analytes solution；Measure the Isosorbide-5-Nitrae-dinitro benzene internal standard substance solution of 100 μ L, 200 μ L
Deoxypodophyllotoxin target analytes solution, the DMSO-d of 200 μ L6, transfer to, in centrifuge tube, obtain sample solution, should
Sample solution suspendible 30s, is transferred in nuclear magnetic tube, carries out quantitative nuclear-magnetism test, the condition of described quantitative nuclear-magnetism test: scanning
Number of times (NS), 16；Spectral width (SW), 20ppm；Relaxation time (D1), 16s；Test temperature, 25 DEG C；Pulse angle, 30 °；Adopt
The collection time (AQ), 3.2s；Measure aromatic and deoxypodophyllotoxin H-2 on Isosorbide-5-Nitrae-dinitro benzene, the peak area of 6, must go out
Oxygen podophyllotoxin is relative to the molal weight ratio of Isosorbide-5-Nitrae-dinitro benzene, then the matter by deoxypodophyllotoxin with Isosorbide-5-Nitrae-dinitro benzene
Amount calculates the purity of deoxypodophyllotoxin than according to formula.
Embodiment 2
The inventive method comprises the following steps: weighs the Isosorbide-5-Nitrae-dinitro benzene of 12.85mg, is dissolved in the DMSO-d of 1.0mL6
In, it is prepared as Isosorbide-5-Nitrae-dinitro benzene internal standard substance solution；Weigh the podophyllotoxin of 29.95mg, be dissolved in the DMSO-d of 1.0mL6In,
It is prepared as podophyllotoxin target analytes solution；Measure the Isosorbide-5-Nitrae-dinitro benzene internal standard substance solution of 100 μ L, the podophyllotoxin of 200 μ L
Element target analytes solution, the DMSO-d of 200 μ L6, transfer to, in centrifuge tube, obtain sample solution；By this sample solution suspendible
30s, is transferred in nuclear magnetic tube, carries out quantitative nuclear-magnetism test, the condition of described quantitative nuclear-magnetism test: scanning times (NS), and 16；
Spectral width (SW), 20ppm；Relaxation time (D1), 16s；Test temperature, 25 DEG C；Pulse angle, 30 °；Acquisition time (AQ),
3.2s；Measure aromatic and podophyllotoxin H-2 on Isosorbide-5-Nitrae-dinitro benzene, the peak area of 6, draw podophyllotoxin relative to Isosorbide-5-Nitrae-
The molal weight ratio of dinitro benzene, then calculate podophyllotoxin by the mass ratio of podophyllotoxin with Isosorbide-5-Nitrae-dinitro benzene according to formula
The purity of element.
Embodiment 3
The inventive method comprises the following steps: weighs the Isosorbide-5-Nitrae-dinitro benzene of 12.90mg, is dissolved in the DMSO-d of 1.0mL6
In, it is prepared as Isosorbide-5-Nitrae-dinitro benzene internal standard substance solution；Weigh the 4-demethylpodophyllotoxin of 29.35mg, be dissolved in 1.0mL's
DMSO-d6In, it is prepared as 4-demethylpodophyllotoxin target analytes solution；The 1,4-dinitro benzene internal standard substance measuring 100 μ L is molten
Liquid, the 4-demethylpodophyllotoxin target analytes solution of 200 μ L, the DMSO-d of 200 μ L6, transfer to, in centrifuge tube, obtain sample
Solution, by this sample solution suspendible 30s, is transferred in nuclear magnetic tube, carries out quantitative nuclear-magnetism test, described quantitative nuclear-magnetism test
Condition: scanning times (NS), 8；Spectral width (SW), 25ppm；Relaxation time (D1), 35s；Test temperature, 30 DEG C；Pulse angle
Degree, 90 °；Acquisition time (AQ), 3.5s；Measure aromatic and the peak of 4-demethylpodophyllotoxin H-2,6 on 1,4-dinitro benzene
Area, draws the 4-demethylpodophyllotoxin molal weight ratio relative to Isosorbide-5-Nitrae-dinitro benzene, then by 4-demethylpodophyllotoxin with
The mass ratio of 1,4-dinitro benzene calculates the purity of 4-demethylpodophyllotoxin according to formula.
Embodiment 4
The inventive method comprises the following steps: weighs the Isosorbide-5-Nitrae-dinitro benzene of 11.87mg, is dissolved in the DMSO-d of 1.0mL6
In, it is prepared as Isosorbide-5-Nitrae-dinitro benzene internal standard substance solution；Weigh 4-demethylpodophyllotoxin-7 '-O-β-D-glucopyra of 39.84mg
Glucosides, is dissolved in the DMSO-d of 1.0mL6In, it is prepared as 4-demethylpodophyllotoxin-7 '-O-β-D-pyranglucoside target and divides
Analysis thing solution；Measure the Isosorbide-5-Nitrae-dinitro benzene internal standard substance solution of 100 μ L, 4-demethylpodophyllotoxin-7 '-O-β-D-pyrrole of 200 μ L
Glucopyranoside glycosides target analytes solution, the DMSO-d of 200 μ L6, transfer to, in centrifuge tube, obtain sample solution, by this sample
Solution suspendible 30s, is transferred in nuclear magnetic tube, carries out quantitative nuclear-magnetism test, the condition of described quantitative nuclear-magnetism test: scanning times
(NS), 8；Spectral width (SW), 15ppm；Relaxation time (D1), 16s；Test temperature, 20 DEG C；Pulse angle, 30 °；During collection
Between (AQ), 3.2s；Measure aromatic and 4-demethylpodophyllotoxin-7 '-O-β-D-pyranglucoside on 1,4-dinitro benzene
H-2, the peak area of 6, draw 4-demethylpodophyllotoxin-7 '-O-β-D-pyranglucoside rubbing relative to Isosorbide-5-Nitrae-dinitro benzene
That mass ratio, then the mass ratio foundation by 4-demethylpodophyllotoxin-7 '-O-β-D-pyranglucoside with Isosorbide-5-Nitrae-dinitro benzene
Formula calculates the purity of 4-demethylpodophyllotoxin-7 '-O-β-D-pyranglucoside.
Embodiment 5
The inventive method comprises the following steps: weighs the Isosorbide-5-Nitrae-dinitro benzene of 11.80mg, is dissolved in the DMSO-d of 1.0mL6
In, it is prepared as Isosorbide-5-Nitrae-dinitro benzene internal standard substance solution；Weigh podophyllotoxin-7 '-O-β-D-pyranglucoside of 40.24mg,
It is dissolved in the DMSO-d of 1.0mL6In, it is prepared as podophyllotoxin-7 '-O-β-D-pyranglucoside target analytes solution；Amount
Take the Isosorbide-5-Nitrae-dinitro benzene internal standard substance solution of 100 μ L, podophyllotoxin-7 '-O-β-D-pyranglucoside goal analysis of 200 μ L
Thing solution, the DMSO-d of 200 μ L6, transfer to, in centrifuge tube, obtain sample solution, by this sample solution suspendible 30s, be transferred to
In nuclear magnetic tube, carry out quantitative nuclear-magnetism test, the condition of described quantitative nuclear-magnetism test: scanning times (NS), 16；Spectral width
(SW), 20ppm；Relaxation time (D1), 16s；Test temperature, 25 DEG C；Pulse angle, 30 °；Acquisition time (AQ), 3.2s；Measure
On Isosorbide-5-Nitrae-dinitro benzene, aromatic and podophyllotoxin-7 '-O-β-D-pyranglucoside H-2, the peak area of 6, draw Rhizoma Dysosmae Versipellis
Toxin-7 '-O-β-D-pyranglucoside is relative to the molal weight ratio of Isosorbide-5-Nitrae-dinitro benzene, then passes through podophyllotoxin-7 '-O-
β-D-pyranglucoside calculates podophyllotoxin-7 '-O-β-D-pyrans Portugal with the mass ratio of 1,4-dinitro benzene according to formula
The purity of polyglycoside.
Embodiment 6
The inventive method comprises the following steps: the method comprises the following steps: weigh the Isosorbide-5-Nitrae-dinitro benzene of 12.83mg,
It is dissolved in the DMSO-d of 1.0mL6In, it is prepared as Isosorbide-5-Nitrae-dinitro benzene internal standard substance solution；Weigh the sinolignan of 47.08mg
A, is dissolved in the DMSO-d of 1.0mL6In, it is prepared as sinolignan A target analytes solution；Measure the 1,4-dinitro of 100 μ L
Base benzene internal standard substance solution, the sinolignan A target analytes solution of 200 μ L, the DMSO-d of 200 μ L6, transfer to centrifuge tube
In, obtain sample solution, by this sample solution suspendible 30s, be transferred in nuclear magnetic tube, carry out quantitative nuclear-magnetism test, described determines
The condition of amount nuclear-magnetism test: scanning times (NS), 16；Spectral width (SW), 20ppm；Relaxation time (D1), 35s；Test temperature
Degree, 25 DEG C；Pulse angle, 90 °；Acquisition time (AQ), 3.5s；Measure aromatic and sinolignan on 1,4-dinitro benzene
The H-2 of A, the peak area of 6, draw the sinolignan A molal weight ratio relative to Isosorbide-5-Nitrae-dinitro benzene, then pass through
The mass ratio of sinolignan A and 1,4-dinitro benzene calculates the purity of sinolignan A according to formula.
Embodiment 7
The inventive method comprises the following steps: weigh the 3 of 10.91mg, and 4,5-trichloropyridines are dissolved in the DMSO-of 0.8mL
d6In, it is prepared as 3,4,5-trichloropyridine internal standard substance solution；Weigh the podophyllotoxin of 12.11mg, be dissolved in the DMSO-d of 0.8mL6
In, it is prepared as podophyllotoxin target analytes solution；Measure 3,4, the 5-trichloropyridine internal standard substance solution of 80 μ L, the ghost of 220 μ L
Mortar toxin target analytes solution, the DMSO-d of 220 μ L6, transfer to, in centrifuge tube, obtain sample solution, by this sample solution
Suspendible 25s, is transferred in nuclear magnetic tube, carries out quantitative nuclear-magnetism test, the condition of described quantitative nuclear-magnetism test: scanning times
(NS), 16；Spectral width (SW), 20ppm；Relaxation time (D1), 16s；Test temperature, 25 DEG C；Pulse angle, 30 °；During collection
Between (AQ), 3.2s；Measure aromatic and podophyllotoxin H-2 on Isosorbide-5-Nitrae-dinitro benzene, the peak area of 6, draw podophyllotoxin phase
For 3, the molal weight ratio of 4,5-trichloropyridines, then by podophyllotoxin and 3, the mass ratio of 4,5-trichloropyridines is according to formula
Calculate the purity of podophyllotoxin.
Embodiment 8
The inventive method comprises the following steps: weighs the 1 of 11.52mg, 3,5-tri-chloro-2-Nitrobenzol, is dissolved in 1.2mL's
DMSO-d6In, it is prepared as 1,3,5-tri-chloro-2-Nitrobenzol internal standard substance solution；Weigh the sinolignan A of 15.86mg, dissolve
DMSO-d in 1.2mL6In, it is prepared as sinolignan A target analytes solution；Measure the chloro-2-of 1,3,5-tri-of 120 μ L
Nitrobenzol internal standard substance solution, the sinolignan A target analytes solution of 180 μ L, the DMSO-d of 180 μ L6, transfer to be centrifuged
Guan Zhong, obtains sample solution, by this sample solution suspendible 35s, is transferred in nuclear magnetic tube, carries out quantitative nuclear-magnetism test, described
The quantitatively condition of nuclear-magnetism test: scanning times (NS), 16；Spectral width (SW), 20ppm；Relaxation time (D1), 16s；Test temperature
Degree, 25 DEG C；Pulse angle, 30 °；Acquisition time (AQ), 3.2s；Measure on 1,3,5-tri-chloro-2-Nitrobenzol aromatic and
The H-2 of sinolignan A, the peak area of 6, draw sinolignan A relative to 1, mole matter of 3,5-tri-chloro-2-Nitrobenzol
Amount ratio, then by sinolignan A and 1, the mass ratio of 3,5-tri-chloro-2-Nitrobenzol calculates sinolignan according to formula
The purity of A.
The parameter of above-described embodiment 1-8 and the result such as table 1 of arylnaphthalene lactone type lignanoid purity
The inventive method is reliable and stable, can measure the purity of arylnaphthalene lactone type lignanoid, relevant experiment money exactly
Expect as follows:
1. experiment material
4-demethylpodophyllotoxin is bought in Nanjing Qing Ze Pharmaceutical Technology Co., Ltd (purity is 98.0%).1,4-dinitro
Benzene is bought and is melted into Industrial Co., Ltd in Tokyo.Chromatograph methanol is bought in α Cygni friend fine chemicals company limited,
DMSO-d6 buys in Cambridge Isotope Laboratories.Quantitatively nuclear magnetic spectrogram uses Bruker Avance 500MHz nuclear magnetic resonance analyser
Measure.High performance liquid chromatography uses Waters Alliance 2489 piece-rate system and Waters 2695 ultraviolet detection system to enter
Row measures.
2. methodology confirmation
The most linearly investigate
Accurately weigh a certain amount of 4-demethylpodophyllotoxin and Isosorbide-5-Nitrae-dinitro benzene in the sample cell of 5ml, add 0.5ml's
DMSO-d6 dissolves, and obtains the 4-demethylpodophyllotoxin of different mol ratio example (0.946,0.6291,0.3078,1.516,2.947)
Hybrid standard sample solution with 1,4-dinitro benzene.By the 4-demethylpodophyllotoxin and 1 to quantitative nmr for the determination,
The mol ratio that 4-dinitro benzene mol ratio and gravimetry measure carries out linear regression, obtains linear equation, Y=1.002X
0.001, R2=1.Show that the method has good linear.The preparation of linear sample and test result are shown in Table 1 and Fig. 1.
The preparation of table 1 linear sample and test result
2.2. specificity
The special of the method is investigated by the nuclear-magnetism hydrogen signal of interference that may be present in bioassay standard sample solution
Attribute.Test result shows, chemical shift is from δ 6.2 to 9.0 in the range of this, and baseline is flat, the H-2 in target detection thing,
6 and Isosorbide-5-Nitrae-dinitro benzene on fragrant Hydrogen Proton signal do not receive the interference of any hydrogen signal, peak shape is symmetrical, and the two
The signal to noise ratio of signal is more than 1000.Therefore the specificity of the method is stronger.
2.3. accuracy
The accuracy of the method is investigated by the average recovery rate and relative standard deviation (RSD) measuring three groups of samples.
The results are shown in Table 2, three groups of samples be averagely recovered as 100.2%, relative standard deviation (RSD) is 0.51%.The method energy is described
The purity of mensuration arylnaphthalene lactone type lignanoid the most exactly.
The accuracy measurement result of table 2.4-demethylpodophyllotoxin,
| Group number | 4-demethylpodophyllotoxin (mg) | 1.4-dinitro benzene (mg) | Purity (%) | The response rate (%) |
| Set-1 | 5.94 | 1.29 | 98.9 | 100.9 |
| Set-1 | 5.77 | 1.25 | 97.3 | 99.3 |
| Set-1 | 6.67 | 1.38 | 98.7 | 100.7 |
| Set-2 | 9.85 | 1.07 | 97.8 | 99.8 |
| Set-2 | 10.00 | 1.09 | 98.3 | 100.3 |
| Set-2 | 11.13 | 1.16 | 98.6 | 100.6 |
| Set-3 | 15.03 | 1.06 | 97.9 | 99.9 |
| Set-3 | 17.10 | 1.18 | 98.1 | 100.1 |
| Set-3 | 15.64 | 1.09 | 98.3 | 100.3 |
| Mean | 98.2 | 100.2 | ||
| RSD (%) | 0.51 |
2.4. precision
The result of withinday precision and day to day precision is shown in Table 3 and table 4 respectively.Withinday precision and the phase of day to day precision
Standard deviation (RSD) is respectively 0.32% and 0.36%, shows that the method precision is good.
The withinday precision measurement result of table 3 4-demethylpodophyllotoxin
| Numbering | 4-demethylpodophyllotoxin (mg) | 1.4-dinitro benzene (mg) | Purity (%) |
| 1 | 5.94 | 1.29 | 98.3 |
| 2 | 6.02 | 1.34 | 98.2 |
| 3 | 5.87 | 1.30 | 97.6 |
| 4 | 5.96 | 1.33 | 98.0 |
| 5 | 6.07 | 1.34 | 98.0 |
| 6 | 6.01 | 1.30 | 97.5 |
| Mean | 97.9 | ||
| RSD (%) | 0.32 |
The day to day precision measurement result of table 4 4-demethylpodophyllotoxin
| Numbering | 4-demethylpodophyllotoxin (mg) | 1.4-dinitro benzene (mg) | Purity (%) |
| 1 | 6.04 | 1.32 | 98.2 |
| 2 | 6.01 | 1.30 | 98.3 |
| 3 | 5.85 | 1.29 | 97.6 |
| 4 | 5.86 | 1.31 | 98.1 |
| 5 | 6.07 | 1.36 | 98.0 |
| 6 | 6.15 | 1.34 | 97.4 |
| Mean | 97.9 | ||
| RSD (%) | 0.36 |
2.5. stability
The standard sample solution prepared measured purity respectively after 0,8,16 and 24 hours, and RSD is 0.34%, shows sample
Product solution was stable existence in 24 hours.The results are shown in Table 5.
The Stability Determination result of table 5 4-demethylpodophyllotoxin
2.6. quantitatively nuclear-magnetism algoscopy and the high performance liquid chromatography comparison to arylnaphthalene lactone type lignanoid purity testing
Research
For confirming further the accuracy of the method, use pure to arylnaphthalene lactone type lignanoid of high performance liquid chromatography
Degree is determined.Liquid phase chromatogram condition is, methanol-water gradient elution (35-65%, 0-35min；65-100%, 35-
50min)；Detection wavelength 210nm；Flow velocity 1.0ml/ml；Column temperature 25 DEG C.Statistical result shows, quantitative nuclear-magnetism algoscopy is with efficient
Liquid chromatography is basically identical to the measurement result of arylnaphthalene lactone type lignanoid purity.Specifically it is shown in Table 6.
The quantitative nuclear-magnetism algoscopy of table 6 and the high performance liquid chromatography measurement result to arylnaphthalene lactone type lignanoid purity
The analysis result tested according to this, compared with traditional efficient liquid-phase chromatography method, quantitative hydrogen nuclear magnetic resonance spectroscopy
Have the advantage that
The quickest.Pre-treatment and analysis for each quantitative nuclear magnetic resonance sample need 15min.And efficient liquid phase
The pre-treatment of each sample of chromatography and analysis amount to 65min (sample pre-treatments 5min；Chromatographic column balance 10min；Chromatography
50min).Each sample amounts hydrogen nuclear magnetic resonance spectroscopy can save 50min.
The most accurate.For lacking the compound of conjugated system, the high performance liquid chromatography with UV-detector is not ring
Answer.Therefore, when being mixed with the impurity of this kind of shortage conjugated system in target analytes, analysis result is inaccurate.Quantitatively core
Magnetic resonance hydrogen spectrometry detection be proton, for organic compound, proton generally exists, target analytes and and its
Whether the organic impurities coexisted contains conjugated system, and it can be detected by quantitative hydrogen nuclear magnetic resonance spectroscopy accurately.
The most cost-effective.Quantitatively hydrogen nuclear magnetic resonance spectroscopy moderate organic little point of simple in construction molecular weight cheap and easy to get
Son does internal standard, and this test uses Isosorbide-5-Nitrae-dinitro benzene to do internal standard, and requirement about 1mg, cost is 15 yuan；The DMSO-of 0.5ml
D6, cost is 20 yuan.For quantitative hydrogen nuclear magnetic resonance spectroscopy, each sample needs about 35 yuan.High performance liquid chromatography, chromatograph first
Alcohol 60ml, cost about 2 yuan；Needing the standard substance about 10mg of object, the cost of different arylnaphthalene lactone type lignanoid is the most not
The most identical, deoxypodophyllotoxin (335 yuan), podophyllotoxin (190 yuan), 4-demethylpodophyllotoxin (225 yuan), for efficient liquid phase
Chromatography, each sample needs 192-337 unit, cost-effective 157-302 unit.And arylnaphthalene lactone type lignanoid glucosides such as Rhizoma Dysosmae Versipellis
Toxin-7 '-O-β-D-Glucose glycosides, 4-demethylpodophyllotoxin-7 '-O-β-D-Glucose glycosides, sinolignan A is commercially
Being difficult to buy, therefore the absolute purity for these three arylnaphthalene lactone type lignanoid glucosides measures and cannot pass through high-efficient liquid phase color
Spectrometry is measured.
Above-mentioned experiment can clearly show, the invention discloses arylnaphthalene lactone type lignanoid method for detecting purity, fixed
Amount hydrogen nuclear magnetic resonance spectroscopy can measure the purity of arylnaphthalene lactone type lignanoid rapidly and accurately, and low cost, in arylnaphthalene
The quality control of ester type lignanoid evaluated biological activity the most accurately and relevant medical material provides early stage basis, for natural product
The Purity assessment of thing makes creative contribution, has good economic and social benefit.
Claims (9)
1. the method measuring arylnaphthalene lactone type lignanoid purity based on quantitative hydrogen nuclear magnetic resonance spectroscopy, it is characterised in that
The method comprises the following steps: weigh the internal standard substance of 10-14mg, is dissolved in the DMSO-d of 0.8-1.2mL6In, it is prepared as internal standard
Solution；Weigh the arylnaphthalene lactone type lignanoid of 10-50mg, be dissolved in the DMSO-d of 0.8-1.2mL6In, it is prepared as target and divides
Analysis thing solution；Measure the inner mark solution of 80-120 μ L, the target analytes solution of 180-220 μ L, the DMSO-d of 180-220 μ L6,
Transfer to, in centrifuge tube, obtain sample solution, by this sample solution suspendible 25-35s, be transferred in nuclear magnetic tube, carry out quantitative core
Magnetic tester, the condition of described quantitative nuclear-magnetism test: scanning times, 8 or 16；Spectral width, 15-25ppm；Relaxation time, 16-
35s；Test temperature, 20-30 DEG C；Pulse angle, 30 °, or 45 °, or 35 °；Acquisition time, 2.5-3.5s；Measure on internal standard substance
Aromatic and arylnaphthalene lactone type lignanoid H-2, the peak area of 6, draw the target analytes mole matter relative to internal standard substance
Amount ratio, then the mass ratio foundation formula by target analytes with internal standard substance:
Calculate purity P of target analytesX, wherein:
Ix is the peak area of target analytes；IStdFor interior target peak area；Nx is the nuclear number of target analytes；NStd
For the nuclear number of interior target；Mx is the molal weight of target analytes；MStdFor interior target molal weight；mStdFor interior target
Quality；Px is the purity of target analytes；PStdPurity for internal standard substance；
Described arylnaphthalene lactone type lignanoid is deoxypodophyllotoxin, podophyllotoxin, 4-demethylpodophyllotoxin, podophyllotoxin-
7 '-O-β-D-pyranglucoside, 4-demethylpodophyllotoxin-7 '-O-β-D-pyranglucoside, appointing in sinolignanA
A kind of；
Described internal standard substance is Isosorbide-5-Nitrae-dinitro benzene, 3,4,5-trichloropyridines, any one in 1,3,5-tri-chloro-2-Nitrobenzol.
One the most according to claim 1 measures arylnaphthalene lactone type lignanoid purity based on quantitative hydrogen nuclear magnetic resonance spectroscopy
Method, it is characterised in that the method comprises the following steps: weighs the Isosorbide-5-Nitrae-dinitro benzene of 12.47mg, is dissolved in 1.0mL's
DMSO-d6In, it is prepared as Isosorbide-5-Nitrae-dinitro benzene inner mark solution；Weigh the deoxypodophyllotoxin of 28.61mg, be dissolved in 1.0mL's
DMSO-d6In, it is prepared as deoxypodophyllotoxin target analytes solution；Measure the Isosorbide-5-Nitrae-dinitro benzene inner mark solution of 100 μ L,
The deoxypodophyllotoxin target analytes solution of 200 μ L, the DMSO-d of 200 μ L6, transfer to, in centrifuge tube, obtain sample solution,
By this sample solution suspendible 30s, it is transferred in nuclear magnetic tube, carries out quantitative nuclear-magnetism test, the condition of described quantitative nuclear-magnetism test:
Scanning times 16；Spectral width, 20ppm；Relaxation time, 16s；Test temperature, 25 DEG C；Pulse angle, 30 °；Acquisition time,
3.2s；Measure aromatic and deoxypodophyllotoxin H-2 on Isosorbide-5-Nitrae-dinitro benzene, the peak area of 6, draw deoxypodophyllotoxin phase
For the molal weight ratio of Isosorbide-5-Nitrae-dinitro benzene, then the mass ratio foundation formula by deoxypodophyllotoxin with Isosorbide-5-Nitrae-dinitro benzene
Calculate the purity of deoxypodophyllotoxin.
One the most according to claim 1 measures arylnaphthalene lactone type lignanoid purity based on quantitative hydrogen nuclear magnetic resonance spectroscopy
Method, it is characterised in that the method comprises the following steps: weighs the Isosorbide-5-Nitrae-dinitro benzene of 12.85mg, is dissolved in 1.0mL's
DMSO-d6In, it is prepared as Isosorbide-5-Nitrae-dinitro benzene inner mark solution；Weigh the podophyllotoxin of 29.95mg, be dissolved in the DMSO-of 1.0mL
d6In, it is prepared as podophyllotoxin target analytes solution；Measure the Isosorbide-5-Nitrae-dinitro benzene inner mark solution of 100 μ L, the Rhizoma Dysosmae Versipellis of 200 μ L
Toxin target analytes solution, the DMSO-d of 200 μ L6, transfer to, in centrifuge tube, obtain sample solution；This sample solution is mixed
Outstanding 30s, is transferred in nuclear magnetic tube, carries out quantitative nuclear-magnetism test, the condition of described quantitative nuclear-magnetism test: scanning times, and 16；Light
Spectral width, 20ppm；Relaxation time, 16s；Test temperature, 25 DEG C；Pulse angle, 30 °；Acquisition time, 3.2s；Measure 1,4-bis-
Aromatic and podophyllotoxin H-2, the peak area of 6 on Nitrobenzol, draw podophyllotoxin relative to Isosorbide-5-Nitrae-dinitro benzene mole
Mass ratio, then the purity of podophyllotoxin is calculated by the mass ratio foundation formula of podophyllotoxin with Isosorbide-5-Nitrae-dinitro benzene.
One the most according to claim 1 measures arylnaphthalene lactone type lignanoid purity based on quantitative hydrogen nuclear magnetic resonance spectroscopy
Method, it is characterised in that the method comprises the following steps: weighs the Isosorbide-5-Nitrae-dinitro benzene of 12.90mg, is dissolved in 1.0mL's
DMSO-d6In, it is prepared as Isosorbide-5-Nitrae-dinitro benzene inner mark solution；Weigh the 4-demethylpodophyllotoxin of 29.35mg, be dissolved in 1.0mL
DMSO-d6In, it is prepared as 4-demethylpodophyllotoxin target analytes solution；The 1,4-dinitro benzene internal standard measuring 100 μ L is molten
Liquid, the 4-demethylpodophyllotoxin target analytes solution of 200 μ L, the DMSO-d of 200 μ L6, transfer to, in centrifuge tube, obtain sample
Solution, by this sample solution suspendible 30s, is transferred in nuclear magnetic tube, carries out quantitative nuclear-magnetism test, described quantitative nuclear-magnetism test
Condition: scanning times, 8；Spectral width, 25ppm；Relaxation time, 35s；Test temperature, 30 DEG C；Pulse angle, 90 °；During collection
Between, 3.5s；Measure aromatic and 4-demethylpodophyllotoxin H-2, the peak area of 6 on Isosorbide-5-Nitrae-dinitro benzene, draw the nor-ghost of 4-
Mortar toxin is relative to the molal weight ratio of Isosorbide-5-Nitrae-dinitro benzene, then the quality by 4-demethylpodophyllotoxin with Isosorbide-5-Nitrae-dinitro benzene
Than the purity calculating 4-demethylpodophyllotoxin according to formula.
One the most according to claim 1 measures arylnaphthalene lactone type lignanoid purity based on quantitative hydrogen nuclear magnetic resonance spectroscopy
Method, it is characterised in that the method comprises the following steps: weighs the Isosorbide-5-Nitrae-dinitro benzene of 11.87mg, is dissolved in 1.0mL's
DMSO-d6In, it is prepared as Isosorbide-5-Nitrae-dinitro benzene inner mark solution；Weigh 4-demethylpodophyllotoxin-7 '-O-β-D-pyrrole of 39.84mg
Glucopyranoside glycosides, is dissolved in the DMSO-d of 1.0mL6In, it is prepared as 4-demethylpodophyllotoxin-7 '-O-β-D-pyranglucoside
Target analytes solution；Measure the Isosorbide-5-Nitrae-dinitro benzene inner mark solution of 100 μ L, 4-demethylpodophyllotoxin-7 '-O-β of 200 μ L-
D-pyranglucoside target analytes solution, the DMSO-d of 200 μ L6, transfer to, in centrifuge tube, obtain sample solution, should
Sample solution suspendible 30s, is transferred in nuclear magnetic tube, carries out quantitative nuclear-magnetism test, the condition of described quantitative nuclear-magnetism test: scanning
Number of times, 8；Spectral width, 15ppm；Relaxation time, 16s；Test temperature, 20 DEG C；Pulse angle, 30 °；Acquisition time, 3.2s；
Measure aromatic and the face, peak of 4-demethylpodophyllotoxin-7 '-O-β-D-pyranglucoside H-2,6 on 1,4-dinitro benzene
Long-pending, draw 4-demethylpodophyllotoxin-7 '-O-β-D-pyranglucoside molal weight ratio relative to Isosorbide-5-Nitrae-dinitro benzene, then
Calculated according to formula by the mass ratio of 4-demethylpodophyllotoxin-7 '-O-β-D-pyranglucoside with 1,4-dinitro benzene
The purity of 4-demethylpodophyllotoxin-7 '-O-β-D-pyranglucoside.
One the most according to claim 1 measures arylnaphthalene lactone type lignanoid purity based on quantitative hydrogen nuclear magnetic resonance spectroscopy
Method, it is characterised in that the method comprises the following steps: weighs the Isosorbide-5-Nitrae-dinitro benzene of 11.80mg, is dissolved in 1.0mL's
DMSO-d6In, it is prepared as Isosorbide-5-Nitrae-dinitro benzene inner mark solution；Weigh podophyllotoxin-7 '-O-β-D-glucopyra of 40.24mg
Glucosides, is dissolved in the DMSO-d of 1.0mL6In, it is prepared as podophyllotoxin-7 '-O-β-D-pyranglucoside target analytes molten
Liquid；Measure the Isosorbide-5-Nitrae-dinitro benzene inner mark solution of 100 μ L, podophyllotoxin-7 '-O-β-D-pyranglucoside target of 200 μ L
Analyte solution, the DMSO-d of 200 μ L6, transfer to, in centrifuge tube, obtain sample solution, by this sample solution suspendible 30s, turn
Move in nuclear magnetic tube, carry out quantitative nuclear-magnetism test, the condition of described quantitative nuclear-magnetism test: scanning times, 16；Spectral width,
20ppm；Relaxation time, 16s；Test temperature, 25 DEG C；Pulse angle, 30 °；Acquisition time, 3.2s；Measure 1,4-dinitro benzene
Upper aromatic and podophyllotoxin-7 '-O-β-D-pyranglucoside H-2, the peak area of 6, draw podophyllotoxin-7 '-O-β-
D-pyranglucoside is relative to the molal weight ratio of Isosorbide-5-Nitrae-dinitro benzene, then passes through podophyllotoxin-7 '-O-β-D-glucopyra
The mass ratio of glucosides and 1,4-dinitro benzene calculates the purity of podophyllotoxin-7 '-O-β-D-pyranglucoside according to formula.
One the most according to claim 1 measures arylnaphthalene lactone type lignanoid purity based on quantitative hydrogen nuclear magnetic resonance spectroscopy
Method, it is characterised in that the method comprises the following steps: weighs the Isosorbide-5-Nitrae-dinitro benzene of 12.83mg, is dissolved in 1.0mL's
DMSO-d6In, it is prepared as Isosorbide-5-Nitrae-dinitro benzene inner mark solution；Weigh the sinolignan A of 47.08mg, be dissolved in 1.0mL's
DMSO-d6In, it is prepared as sinolignan A target analytes solution；Measure the Isosorbide-5-Nitrae-dinitro benzene inner mark solution of 100 μ L,
The sinolignan A target analytes solution of 200 μ L, the DMSO-d of 200 μ L6, transfer to, in centrifuge tube, obtain sample solution,
By this sample solution suspendible 30s, it is transferred in nuclear magnetic tube, carries out quantitative nuclear-magnetism test, the condition of described quantitative nuclear-magnetism test:
Scanning times, 8；Spectral width, 20ppm；Relaxation time, 35s；Test temperature, 25 DEG C；Pulse angle, 90 °；Acquisition time,
3.5s；Measure aromatic and the H-2 of sinolignan A on Isosorbide-5-Nitrae-dinitro benzene, the peak area of 6, draw sinolignanA
Relative to the molal weight ratio of Isosorbide-5-Nitrae-dinitro benzene more public with the mass ratio foundation of Isosorbide-5-Nitrae-dinitro benzene by sinolignan A
Formula calculates the purity of sinolignan A.
One the most according to claim 1 measures arylnaphthalene lactone type lignanoid purity based on quantitative hydrogen nuclear magnetic resonance spectroscopy
Method, it is characterised in that the method comprises the following steps: weigh the 3 of 10.91mg, and 4,5-trichloropyridines are dissolved in 0.8mL
DMSO-d6In, it is prepared as 3,4,5-trichloropyridine inner mark solutions；Weigh the podophyllotoxin of 12.11mg, be dissolved in 0.8mL's
DMSO-d6In, it is prepared as podophyllotoxin target analytes solution；Measure 3,4, the 5-trichloropyridine inner mark solutions of 80 μ L, 220 μ L
Podophyllotoxin target analytes solution, the DMSO-d of 220 μ L6, transfer to, in centrifuge tube, obtain sample solution, by this sample
Solution suspendible 25s, is transferred in nuclear magnetic tube, carries out quantitative nuclear-magnetism test, the condition of described quantitative nuclear-magnetism test: scanning time
Number, 16；Spectral width, 20ppm；Relaxation time, 16s；Test temperature, 25 DEG C；Pulse angle, 30 °；Acquisition time, 3.2s；Survey
Determine aromatic and podophyllotoxin H-2, the peak area of 6 on Isosorbide-5-Nitrae-dinitro benzene, draw podophyllotoxin relative to 3,4,5-trichlorines
The molal weight ratio of pyridine, then by podophyllotoxin and 3, the mass ratio of 4,5-trichloropyridines calculates podophyllotoxin according to formula
Purity.
One the most according to claim 1 measures arylnaphthalene lactone type lignanoid purity based on quantitative hydrogen nuclear magnetic resonance spectroscopy
Method, it is characterised in that the method comprises the following steps: weighs the 1 of 11.52mg, 3,5-tri-chloro-2-Nitrobenzol, is dissolved in
The DMSO-d of 1.2mL6In, it is prepared as 1,3,5-tri-chloro-2-Nitrobenzol inner mark solutions；Weigh the sinolignan A of 15.86mg,
It is dissolved in the DMSO-d of 1.2mL6In, it is prepared as sinolignan A target analytes solution；Measure the 1,3,5-tri-of 120 μ L
Chloro-2-Nitrobenzol inner mark solution, the sinolignan A target analytes solution of 180 μ L, the DMSO-d of 180 μ L6, transfer to from
In heart pipe, obtain sample solution, by this sample solution suspendible 35s, be transferred in nuclear magnetic tube, carry out quantitative nuclear-magnetism test, described
Quantitative nuclear-magnetism test condition: scanning times, 16；Spectral width, 20ppm；Relaxation time, 16s；Test temperature, 25 DEG C；Arteries and veins
Angle of attack degree, 30 °；Acquisition time, 3.2s；Measure aromatic and the H-of sinolignan A on 1,3,5-tri-chloro-2-Nitrobenzol
The peak area of 2,6, draw sinolignan A relative to 1, the molal weight ratio of 3,5-tri-chloro-2-Nitrobenzol, then pass through
The mass ratio of sinolignan A Yu 1,3,5-tri-chloro-2-Nitrobenzol calculates the purity of sinolignan A according to formula.
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