CN108362811A - The detection method of active ingredient in sweet wormwood - Google Patents
The detection method of active ingredient in sweet wormwood Download PDFInfo
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- CN108362811A CN108362811A CN201810131792.4A CN201810131792A CN108362811A CN 108362811 A CN108362811 A CN 108362811A CN 201810131792 A CN201810131792 A CN 201810131792A CN 108362811 A CN108362811 A CN 108362811A
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Abstract
The invention belongs to ultra performance liquid chromatography and mass spectrum are combined test or analysis of material technical field, and in particular to the detection method of active ingredient in a kind of sweet wormwood.It is described that detection method includes the following steps：By sample broke, sieving, ultrasonic extraction is then carried out, then carries out gradient elution with ultra performance liquid chromatography, then detected with mass spectrography, detected with peak area quantified by external standard method.This method can detect the content of qinghaosu in Artemisia annua, Arteannuic acid, Qinghaosu II, dihydroartemisinine, Kaempferol, cyanidenon and Quercetin simultaneously, and detection time is short, high sensitivity, specificity is strong, and accuracy is high, and linear relationship is good, precision is high, and stability is strong, reproducible.
Description
Technical field
The invention belongs to which ultra performance liquid chromatography and mass spectrum are combined test or analysis of material technical field, specifically
It is related to a kind of detection method of active ingredient in sweet wormwood.
Background technology
Sweet wormwood is the dry aerial parts of composite family annual herb plant artemisia annua (Artemisia annua L.),
Property bitter, Xin Han, have clear abnormal heat, cool blood except steaming, relieving summer-heat, preventing malaria and other effects (Pharmacopoeia of People's Republic of China (one)), state
Pharmacopoeia commission of family compiles, Beijing：China Medical Science Press, 2015 page 198, publication date on December 31st, 2015).
Sweet wormwood is widely distributed, Chongqing, Sichuan, Guizhou, Guangxi, Guangdong, Jiangxi, Hubei, Hunan, Jiangsu, Hebei, Henan, Shandong and Shan
West etc. area be distributed (" Discussion On Analyzing And Evaluation Methods For Lands Suitable For Artemisia Annua L. growth ", Liu Junming etc., Chinese agriculture resource and zoning,
The 4th phase of volume 27 in 2006, the 14-17 pages, publication date on 08 31st, 2006).Wherein, Chongqing possesses good sweet wormwood kind
Matter resource (" the sweet wormwood pharmaceutical ingredient content researchs of different sources ", snow etc., China Dispensary, the 15th phase of volume 20 in 2009,
The 1188-1191 pages, publication date on December 31st, 2009), for the maximum sweet wormwood planting base in the whole nation.
Also contain Arteannuic acid, Qinghaosu II in addition to qinghaosu, in Artemisia annua and with Kaempferol, cyanidenon, Quercetin
For the flavone compound of representative, these ingredients cooperate with qinghaosu to malaria have therapeutic effect (" sweet wormwood chemical analysis and its
With pharmacodynamic study of the qinghaosu 5 to mouse malaria ", discipline twilight etc., parasite and medical insect journal, 2008 years volume 15 the
4 phases, the 198-201 pages, publication date on December 31st, 2008).Research is it is also shown that dihydroartemisinine is thin to human lung adenocarcinoma A549
Born of the same parents have growth inhibition effect (" experimental study of the anti-human Lung Adenocarcinoma A 549 Cell growth of dihydroartemisinine ", Chen Wei in vivo and in vitro
It is strong etc., Chinese tuberculosis magazine, the 2nd phase of volume 8 in 2005, the 85-88 pages, publication date on 04 30th, 2005).Currently, sweet wormwood
The quality research of medicinal material is mainly the index containing survey, dihydroartemisinine, Kaempferol, sweet-scented osmanthus with qinghaosu, Arteannuic acid, Qinghaosu II
The research of the compounds such as careless element, Quercetin is less, and analysis method includes column front derivation-high performance liquid chromatography (English abbreviation column
Preceding derivative-HPLC), the ultraviolet combination method (English abbreviation HPLC-UV) of high performance liquid chromatography-, high performance liquid chromatography-evaporative light dissipate
Penetrate detector combination method (English abbreviation HPLC-ELSD) and high performance liquid chromatography-it is ultraviolet-evaporative light scattering detector method is (English
Abbreviation HPLC-UV-ELSD) (" column front derivation-RP-HPLC methods measure the content of Artemisinin in Artemisia annuna ", Liu Lifang etc., China are wild
Plant resource, the 6th phase of volume 23 in 2004, the 60-62 pages, publication date on December 31st, 2004；" UV methods are surveyed with HPLC methods
The method for determining content of artemisinin in sweet wormwood compares ", small jasmine of fourth etc., China Dispensary, the 3rd phase of volume 22 in 2011,246-248
Page, publication date on December 31st, 2011；" HPLC-ELSD measures the content of different sources Artemisinin in Artemisia annuna ", Hu Xiangrong etc.,
Modern food and drug magazine, the 3rd phase of volume 16 in 2006, the 34-36 pages, publication date on December 31st, 2006；“HPLC-
UV-ELSD methods measure the content of Artemisinin in Artemisia annuna, Qinghaosu II and Arteannuic acid simultaneously ", east etc., Acta Pharmaceutica Sinica, 2007
The 9th phase of volume 42, the 978-981 pages, publication date on December 31st, 2007).Wherein, column front derivation-HPLC and HPLC-UV be not
The content of qinghaosu, Arteannuic acid, Qinghaosu II, dihydroartemisinine, Kaempferol, cyanidenon and Quercetin can be measured simultaneously；
The detection time of HPLC-ELSD and HPLC-UV-ELSD is long, needs 30-60min, and sensitivity is low, content ten a ten thousandths with
Under composition detection do not go out.
Invention content
In view of this, the purpose of the present invention is to provide a kind of detection method of active ingredient in sweet wormwood, this method can
Detect qinghaosu in Artemisia annua, Arteannuic acid, Qinghaosu II, dihydroartemisinine, Kaempferol, cyanidenon and Quercetin simultaneously
Content, detection time is short, can detect that content, high sensitivity are capable of detecting when content in ten a ten thousandths in 15min
Ingredient below.
To achieve the above object, the technical scheme is that：
The detection method of active ingredient, includes the following steps in sweet wormwood：By sample broke, sieving, ultrasonic extraction is then carried out
It takes, then carries out gradient elution with ultra performance liquid chromatography, then detected with mass spectrography, detected with peak area quantified by external standard method.
Further, described be sieved referred to《Pharmacopoeia of People's Republic of China》No. 5 sieves of version in 2015.
Further, the solvent of the ultrasonic extraction is the composition of petroleum ether and acetone.
The petroleum ether is the petroleum ether that boiling point is 60-90 DEG C.
Further, it is 0.8-1.1: 0.8-1.1 that the solvent of the ultrasonic extraction, which is petroleum ether with acetone according to volume ratio,
Composition.
Further, the dosage of the solvent of the ultrasonic extraction is 33-37 times of sample quality.
Further, the parameter of the ultrasonic extraction is set as：Extraction power is 280-330W, time 40-50min.
Further, the chromatographic condition of the ultra performance liquid chromatography is,
Chromatographic column：Ultra performance liquid chromatography；Mobile phase：Mobile phase A is 90: 10-10: 90 according to volume ratio with Mobile phase B
Mixed solution, the mobile phase A be acetonitrile, the Mobile phase B is the acetic acid solution that volume fraction is 0.1%；
Gradient elution；
Flow velocity：0.1-0.4mL/min；Column temperature：20-35℃；
Sample size：2μL.
Further, the detailed process of the gradient elution is that elution time and mobile phase A volume ratio sequence are 0-1min
10% operation, 1-6min from 10% → 85%, 6-13min again with 85% operation, 13-14min, then from 85% → 10%, 14-
15min, with 10% operation.
Further, the Mass Spectrometry Conditions are：Electric spray ion source is detected using cation and negative ion mode；It is multiple anti-
Answer monitoring pattern, ion source temperature：550℃.
This method can detect qinghaosu in Artemisia annua, Arteannuic acid, Qinghaosu II, dihydroartemisinine, kaempferia galamga simultaneously
The content of phenol, cyanidenon and Quercetin, detection time is short, can detect that content, high sensitivity can detect in 15min
Go out content in ten a ten thousandths ingredient below.
Figure of description
Fig. 1 is qinghaosu contrast solution chromatogram and qinghaosu sample solution chromatogram in embodiment 2, wherein A is blueness
Artemisin contrast solution chromatogram, B are qinghaosu sample solution chromatogram；
Fig. 2 is Arteannuic acid contrast solution chromatogram and Arteannuic acid sample solution chromatogram in embodiment 2, wherein A is blueness
Artemisic acid contrast solution chromatogram, B are Arteannuic acid sample solution chromatogram；
Fig. 3 is Qinghaosu II contrast solution chromatogram and Qinghaosu II sample solution chromatogram in embodiment 2, wherein A
For Qinghaosu II contrast solution chromatogram, B is Qinghaosu II sample solution chromatogram；
Fig. 4 is dihydroartemisinine contrast solution chromatogram and dihydroartemisinine sample solution chromatogram in embodiment 2,
In, A is dihydroartemisinine contrast solution chromatogram, and B is dihydroartemisinine sample solution chromatogram；
Fig. 5 is Kaempferol contrast solution chromatogram and Kaempferol sample solution chromatogram in embodiment 2, wherein A is mountain
How phenol contrast solution chromatogram, B be Kaempferol sample solution chromatogram；
Fig. 6 is cyanidenon contrast solution chromatogram and cyanidenon sample solution chromatogram in embodiment 2, wherein A
For cyanidenon contrast solution chromatogram, B is cyanidenon sample solution chromatogram；
Fig. 7 is Quercetin contrast solution chromatogram and Quercetin sample solution chromatogram in embodiment 2, wherein A is Mongolian oak
Skin element contrast solution chromatogram, B are Quercetin sample solution chromatogram.
The beneficial effects of the present invention are：
This method can detect qinghaosu in Artemisia annua, Arteannuic acid, Qinghaosu II, dihydroartemisinine, kaempferia galamga simultaneously
The content of phenol, cyanidenon and Quercetin.
The detection time of this method is short, can detect that content in 15min.
The high sensitivity of this method is capable of detecting when content in ten a ten thousandths ingredient below.
The specificity of this method is strong.
The accuracy of this method is high, and it is 1.84%-3.03% to accelerate the relative standard deviation of recovery test.
The linear relationship of this method is good, and linear coefficient is up to 99.78%-99.99%.
The precision of this method is high, relative standard deviation 0.99%-2.37%.
The stability of this method is strong, and the relative standard deviation of stability test is 1.39%-2.47%.
This method it is reproducible, the relative standard deviation of reperformance test is 1.47%-2.50%.
Specific implementation mode
Illustrated embodiment be in order to preferably be illustrated to present disclosure, but be not present disclosure only
It is limited to illustrated embodiment.So those skilled in the art carry out non-intrinsically safe according to foregoing invention content to embodiment
Modifications and adaptations, still fall within protection scope of the present invention.
Following sweet wormwood sample collection is in Chongqing City sweet wormwood standardized planting base；Qinghaosu, Arteannuic acid control
Product, dihydroartemisinine reference substance, Kaempferol reference substance, cyanidenon reference substance and Quercetin reference substance are purchased from Chengdu Man Site
Bio tech ltd, product batch number be respectively MUST-13060203,13011614,13101302,13121112,
13121011 and 13072505), Qinghaosu II reference substance is purchased from the bio tech ltd Wei Keqi of Sichuan Province, product batch number
It is 120915；Water is deionized water, and acetone and petroleum ether are that analysis is pure, and methanol and acetonitrile are chromatographically pure；
" weighing " instrument as described below is the AW-220 type electronic balances of Japanese Shimadzu Corporation, " precision weighs " institute
With the AEG-45SM type electronic balances that instrument is Japanese Shimadzu Corporation；Ultrasonic extraction instrument, which is city of Kunshan's ultrasonic instrument, to be had
The KQ-250B type supersonic wave cleaning machines of limit company；Ultra Performance Liquid Chromatography instrument is that the LC-30AT types of Japanese Shimadzu Corporation are efficient
Liquid phase instrument；Mass spectrometer used in mass spectrography is the 4600 high-resolution matter of Triple T of of Applied biosystems
Spectra system.
The detection method of active ingredient in 1 sweet wormwood of embodiment, the specific steps are：
A. after taking the Artemisia annua sample of each district different sources in Chongqing is (as shown in table 1) to crush, mistake《The Chinese people are total
With state's pharmacopeia》(version in 2015) No. 5 sieves, then accurately weigh 3 parts of 0.50g sweet wormwoods sample, and filter paper package is respectively placed in 25mL
In measuring bottle, the Extraction solvent of 50% petroleum ether (boiling point of petroleum ether is 60-90 DEG C) -50% acetone (being volume ratio) is added
20mL, the ultrasonic extraction 45min under the conditions of power is 300W, continuous ultrasound extract 2 times, merge extracting solution, then with 50% stone
The Extraction solvent of oily ether (boiling point of petroleum ether is 60-90 DEG C) -50% acetone (being volume ratio) is settled to 100mL, shakes up,
0.1mL is taken, is placed in 100mL measuring bottles, evaporates into dry, is dissolved, and be settled to scale, is shaken up with methanol, with 0.22 μm of filter
Head, filtering, takes filtrate, as sample solution；
1 sample list of table
B. sample solution obtained by step A is surpassed and carries out gradient elution with high performance liquid chromatography, chromatographic condition is：
Chromatographic column：Nucleodur C18 chromatographic columns (100mm × 4.6mm, 3 μm of ID)；
Mobile phase：The mixed solution of mobile phase A and Mobile phase B, the mobile phase A are acetonitrile, and the Mobile phase B is body
The acetic acid solution that fraction is 0.1%；
Gradient elution, elution time and mobile phase A volume ratio sequence are that 0-1min 10% is run, 1-6min from 10% →
85%, 6-13min are again with 85% operation, 13-14min, then from 85% → 10%, 14-15min, are run with 10%；
Flow velocity：0.4mL/min；
Column temperature：30℃；
Sample size：2μL；
C. the sample solution for carrying out gradient elution is detected with mass spectrography again, is detected with peak area quantified by external standard method；EFI
Mist ion source is detected using cation and negative ion mode；Multiple reaction monitoring pattern, ion source temperature：550 DEG C, 7 kinds of mesh
Other mass spectral analysis parameters for marking ingredient are as shown in table 2.
2 Mass Spectrometry Conditions of table
D. standard curve making：Precision weighs Qinghaosu, Arteannuic acid reference substance, Qinghaosu II reference substance, double hydrogen
Qinghaosu, Kaempferol reference substance, cyanidenon reference substance and each 5mg of Quercetin reference substance, are respectively placed in 10mL measuring bottles
In, add methanol be configured to respectively a concentration of 0.964mg/mL, 1.023mg/mL, 0.990 mg/mL, 1.017mg/mL,
The solution of 0.990mg/mL, 1.000mg/mL and 1.040mg/mL are compareed respectively as Qinghaosu mother liquor, Arteannuic acid
Product mother liquor, Qinghaosu II reference substance mother liquor, dihydroartemisinine reference substance mother liquor, Kaempferol reference substance mother liquor, cyanidenon control
Product mother liquor and Quercetin reference substance mother liquor, with methanol dilution mother liquor obtain series concentration be 144.60 μ g/mL, 96.40 μ g/mL,
The qinghaosu solution of 48.20 μ g/mL, 24.10 μ g/mL, 19.28 μ g/mL, 14.46 μ g/mL and 9.64 μ g/mL；Series concentration
For 8.18 μ g/mL, 6.14 μ g/mL, 5.12 μ g/mL, 4.09 μ g/mL, 2.05 μ g/mL, 1.02 μ g/mL and 0.10 μ g/mL
Sweet wormwood acid solution；Series concentration be 0.99 μ g/mL, 0.74 μ g/mL, 0.50 μ g/mL, 0.25 μ g/mL, 0.050 μ g/mL,
The Qinghaosu II solution of 0.025 μ g/mL and 0.012 μ g/mL；Series concentration is 1.02 μ g/mL, 0.51 μ g/mL, 0.10 μ g/
The dihydroartemisinine solution of mL, 0.051 μ g/mL, 0.010 μ g/mL, 0.0051 μ g/mL and 0.0020 μ g/mL；Series concentration is
0.99 μ g/mL, 0.74 μ g/mL, 0.50 μ g/mL, 0.25 μ g/mL, 0.050 μ g/mL, 0.025 μ g/mL and 0.012 μ g/mL
Kaempferia galamga phenol solution；Series concentration be 1.0 μ g/mL, 0.50 μ g/mL, 0.10 μ g/mL, 0.050 μ g/mL, 0.010 μ g/mL,
The luteolin solution of 0.0050 μ g/mL and 0.0010 μ g/mL；1.04μg/mL、0.52μg/mL、0.10μg/mL、 0.052μ
The Quercetin solution of g/mL, 0.010 μ g/mL, 0.0052 μ g/mL and 0.0010 μ g/mL；The above series concentration solution is inhaled respectively
2 μ L are taken, injection Ultra Performance Liquid Chromatography instrument carries out gradient elution, then is detected with mass spectrography, and chromatographic condition is identical as step B, matter
Spectral condition is identical as step C；Using the concentration x of sample size as abscissa, mass spectroscopy signal strength peak area Y is ordinate, is painted
Standard curve processed, and regression equation is calculated, the results are shown in Table 3.
3 regression equation of table
As shown in Table 3, qinghaosu, Arteannuic acid, Qinghaosu II, dihydroartemisinine, Kaempferol, cyanidenon and Quercetin
Linear relationship is good in its corresponding sample introduction mass range respectively.
E. qinghaosu sample solution, Arteannuic acid sample solution, Qinghaosu II sample solution, dihydroartemisinine sample is molten
The chromatographic peak area substitution standard obtained of liquid, Kaempferol sample solution, cyanidenon sample solution and Quercetin sample solution
To get qinghaosu, Arteannuic acid, Qinghaosu II, dihydroartemisinine, Kaempferol, sweet-scented osmanthus in sweet wormwood sample in the regression equation of curve
The content of careless element and Quercetin, using the average value of three parts of samples as final result, the results are shown in Table 4.
4 content detection result of table
As shown in Table 4, method of the invention can detect simultaneously qinghaosu in Artemisia annua, Arteannuic acid, Qinghaosu II,
The content of dihydroartemisinine, Kaempferol, cyanidenon and Quercetin；And the high sensitivity of this method, it is capable of detecting when that content exists
Ten a ten thousandths ingredient below.
2 specificity of embodiment is analyzed
A. after taking the Artemisia annua sample comminution that Zhong County is newly stood, mistake《Pharmacopoeia of People's Republic of China》(version in 2015) No. 5 numbers
Sieve, then accurately weighs 3 parts of 0.50g sweet wormwoods sample, and filter paper package is respectively placed in 25mL measuring bottles, 50% petroleum ether is added
The Extraction solvent 20mL of (boiling point of petroleum ether be 60-90 DEG C) -50% acetone (being volume ratio), in power be 300W conditions
Lower ultrasonic extraction 45min, continuous ultrasound extract 2 times, merge extracting solution, and then with 50% petroleum ether, (boiling point of petroleum ether is
60-90 DEG C) Extraction solvent of -50% acetone (being volume ratio) is settled to 100mL, and it shakes up, takes 0.1mL, be placed in 100mL amounts
It in bottle, evaporates into dry, is dissolved, and be settled to scale, shaken up with methanol, with 0.22 μm of filter, filtering takes filtrate, as
Sample solution；
B. sample solution obtained by step A is surpassed and carries out gradient elution, chromatographic condition and embodiment 1 with high performance liquid chromatography
It is identical；
C. the sample solution for carrying out gradient elution is detected with mass spectrography again, Mass Spectrometry Conditions parameter setting and 1 phase of embodiment
Together；
D. Qinghaosu, Arteannuic acid reference substance, Qinghaosu II reference substance, dihydroartemisinine reference substance, kaempferia galamga are weighed
Phenol reference substance, cyanidenon reference substance and Quercetin reference substance 0.5g, methanol dissolving, are configured to the solution of a concentration of 1mg/mL,
Using 0.22 μm of filter, filtering takes filtrate, as a contrast product solution, draws 2 μ L reference substance solutions, injects ultra high efficiency liquid phase
Chromatograph carries out gradient elution, then is detected with mass spectrography, and chromatographic condition and Mass Spectrometry Conditions are same as Example 1；Fig. 1 is sweet wormwood
Plain contrast solution chromatogram and qinghaosu sample solution chromatogram, wherein A is qinghaosu contrast solution chromatogram, and B is sweet wormwood
Plain sample solution chromatogram；Fig. 2 is Arteannuic acid contrast solution chromatogram and Arteannuic acid sample solution chromatogram, wherein A is blueness
Artemisic acid contrast solution chromatogram, B are Arteannuic acid sample solution chromatogram；Fig. 3 is Qinghaosu II contrast solution chromatogram and blueness
Wormwood artemisia B prime sample solution chromatogram, wherein A is Qinghaosu II contrast solution chromatogram, and B is Qinghaosu II sample solution chromatography
Figure；Fig. 4 is dihydroartemisinine contrast solution chromatogram and dihydroartemisinine sample solution chromatogram, wherein A is double hydrogen sweet wormwoods
Plain contrast solution chromatogram, B are dihydroartemisinine sample solution chromatogram；Fig. 5 is Kaempferol contrast solution chromatogram and kaempferia galamga
Phenol sample solution chromatogram, wherein A is Kaempferol contrast solution chromatogram, and B is Kaempferol sample solution chromatogram；Fig. 6 is
Cyanidenon contrast solution chromatogram and cyanidenon sample solution chromatogram, wherein A is cyanidenon contrast solution chromatography
Figure, B are cyanidenon sample solution chromatogram；Fig. 7 is Quercetin contrast solution chromatogram and Quercetin sample solution chromatography
Figure, wherein A is Quercetin contrast solution chromatogram, and B is Quercetin sample solution chromatogram.
By Fig. 1-7 it is found that the retention time of sample solution and the chromatogram of reference substance solution is close, and retention time ＜
10min.Thus it proves, the specificity of method of the invention is strong, and detection time is short.
3 precision test of embodiment
2 gained Qinghaosu solution of extraction embodiment, Arteannuic acid reference substance solution, Qinghaosu II reference substance solution,
Dihydroartemisinine reference substance solution, Kaempferol reference substance solution, cyanidenon reference substance solution and Quercetin reference substance solution are each
2 μ L, injection Ultra Performance Liquid Chromatography instrument carry out gradient elution, then are detected with mass spectrography, chromatographic condition and Mass Spectrometry Conditions and implementation
Example 1 is identical, and continuous sample introduction 6 times measures the peak area of each reference substance, and calculates relative standard deviation, and the results are shown in Table 5.
4 stability test of embodiment
2 gained Qinghaosu solution of extraction embodiment, Arteannuic acid reference substance solution, Qinghaosu II reference substance solution,
Dihydroartemisinine reference substance solution, Kaempferol reference substance solution and each 2 μ L of Quercetin reference substance solution, respectively 0 hour, it is 4 small
When, 8 hours, 12 hours and 24 hours injection Ultra Performance Liquid Chromatography instruments carry out gradient elution, then detected with mass spectrography, chromatography
Condition and Mass Spectrometry Conditions are same as Example 1, measure the peak area of each reference substance, and relative standard deviation, as a result such as 5 institute of table
Show.
5 repetitive test of embodiment
After taking the Artemisia annua sample comminution that Zhong County is newly stood, mistake《Pharmacopoeia of People's Republic of China》(version in 2015) No. 5 numbers
Sieve, then accurately weighs 6 parts of 0.50g sweet wormwoods sample, and filter paper package is respectively placed in 25mL measuring bottles, 50% petroleum ether is added
The Extraction solvent 20mL of (boiling point of petroleum ether be 60-90 DEG C) -50% acetone (being volume ratio), in power be 300W conditions
Lower ultrasonic extraction 45min, continuous ultrasound extract 2 times, merge extracting solution, and then with 50% petroleum ether, (boiling point of petroleum ether is
60-90 DEG C) Extraction solvent of -50% acetone (being volume ratio) is settled to 100mL, and it shakes up, takes 0.1mL, be placed in 100mL amounts
It in bottle, evaporates into dry, is dissolved, and be settled to scale, shaken up with methanol, with 0.22 μm of filter, filtering takes filtrate, as
Sample solution measures containing for qinghaosu, Arteannuic acid, Qinghaosu II, dihydroartemisinine, Kaempferol, cyanidenon and Quercetin
Amount, method is same as Example 1, and calculates the relative standard deviation of acquired results, and the results are shown in Table 5.
5 precision of table, stability and repeated measurement result
| Qinghaosu | Arteannuic acid | Qinghaosu II | Dihydroartemisinine | Kaempferol | Reseda | Quercetin | |
| Precision (relative standard deviation)/% | 1.21 | 1.45 | 0.99 | 2.37 | 1.12 | 2.23 | 1.89 |
| Stability (relative standard deviation)/% | 1.81 | 1.98 | 1.53 | 2.47 | 1.39 | 2.02 | 1.72 |
| Repeatability (relative standard deviation)/% | 1.77 | 1.92 | 1.90 | 2.50 | 1.47 | 1.77 | 2.14 |
As shown in Table 5, the precision of method of the invention be 0.99%-2.37%, stability 1.39%-2.47%,
Repeatability is 1.47%-2.50%.Thus it proves, the precision of method of the invention is high, stability and reproducible.
6 sample recovery rate of embodiment is tested
Precision measure 9 parts measured qinghaosu, Arteannuic acid, Qinghaosu II, dihydroartemisinine, Kaempferol, cyanidenon and
Artemisia annua powder 0.25g is newly found in the Zhong County of quercetin content, and filter paper package divides 3 groups, every group 3 parts, each group is separately added into
High (120%), in (100%), low (80%) 3 mass concentration hybrid standard product, mistake《Pharmacopoeia of People's Republic of China》
(version in 2015) No. 5 sieves, are respectively placed in 25mL measuring bottles, and 50% petroleum ether (boiling point of petroleum ether is 60-90 DEG C)-is added
The Extraction solvent 20mL of 50% acetone (being volume ratio), the ultrasonic extraction 45min under the conditions of power is 300W, continuous ultrasound
Extraction 2 times merges extracting solution, (is then body with 50% petroleum ether (boiling point of petroleum ether is 60-90 DEG C) -50% acetone
Product ratio) Extraction solvent be settled to 100mL, shake up, take 0.1mL, be placed in 100mL measuring bottles, evaporate into dry, dissolved with methanol,
And it is settled to scale, it shakes up, with 0.22 μm of filter, filtering takes filtrate, as sample solution, injects ultra performance liquid chromatography
Instrument carries out gradient elution, then is detected with mass spectrography, and chromatographic condition and Mass Spectrometry Conditions are same as Example 1, obtain the peak of each reference substance
Area calculates containing for qinghaosu in sample, Arteannuic acid, Qinghaosu II, dihydroartemisinine, Kaempferol, cyanidenon and Quercetin
Amount, and the rate of recovery, average content and relative standard deviation are calculated, the results are shown in Table 6.
Table 6 is loaded recovery experiment result
As shown in Table 6, the relative standard deviation of the sample-adding recovery test of the method for the invention is 1.84%-3.03%.
Thus it proves, the accuracy of method of the invention is high.
In addition, it should be understood that although this specification is described in terms of embodiments, but not each embodiment is only
It contains an independent technical solution, this description of the specification is merely for the sake of clarity, and those skilled in the art answer
When considering the specification as a whole, the technical solutions in the various embodiments may also be suitably combined, forms people in the art
The other embodiment that member is appreciated that.
Claims (8)
1. the detection method of active ingredient in sweet wormwood, which is characterized in that include the following steps：By sample broke, sieving, then into
Row ultrasonic extraction then carries out gradient elution with ultra performance liquid chromatography, then is detected with mass spectrography, with peak area quantified by external standard method
Detection.
2. detection method according to claim 1, which is characterized in that the solvent of the ultrasonic extraction is petroleum ether and acetone
Composition.
3. detection method according to claim 2, which is characterized in that the solvent of the ultrasonic extraction is petroleum ether and acetone
The composition for being 0.8-1.1: 0.8-1.1 according to volume ratio.
4. detection method according to claim 2 or 3, which is characterized in that the dosage of the solvent of the ultrasonic extraction is sample
33-37 times of quality.
5. detection method according to claim 1,2,3 or 4, which is characterized in that the parameter of the ultrasonic extraction is set as：
Extraction power is 280-330W, time 40-50min.
6. according to the detection method described in claim 1,2,3,4 or 5, which is characterized in that the color of the ultra performance liquid chromatography
Spectral condition is,
Chromatographic column：Ultra performance liquid chromatography；
Mobile phase：The mixed solution that mobile phase A is 90: 10-10: 90 according to volume ratio with Mobile phase B, the mobile phase A are second
Nitrile, the Mobile phase B are the acetic acid solution that volume fraction is 0.1%；
Gradient elution；
Flow velocity：0.1-0.4mL/min；
Column temperature：20-35℃；
Sample size：2uL.
7. the detection method described in claim 6, which is characterized in that the detailed process of the gradient elution is, elution time and
Mobile phase A volume ratio sequence is that 0-1min 10% is run, and 1-6min is from 10% → 85%, 6-13min again with 85% operation, 13-
14min, then from 85% → 10%, 14-15min, with 10% operation.
8. according to the detection method described in claim 1,2,3,4,5,6 or 7, which is characterized in that the Mass Spectrometry Conditions are：EFI
Mist ion source is detected using cation and negative ion mode；Multiple reaction monitoring pattern, ion source temperature：550℃.
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