CN106370763A - UPLC method for detecting components in radix puerariae, radix puerariae extract and radix puerariae-containing preparation - Google Patents
UPLC method for detecting components in radix puerariae, radix puerariae extract and radix puerariae-containing preparation Download PDFInfo
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Abstract
The present invention provides a UPLC method for qualitatively and quantitatively detecting the contents of six components such as 3'-hydroxy puerarin, puerarin, puerarin-6"-O-xyloside, 3'-methoxy puerarin, mirificin and daidzin in radix puerariae, radix puerariae extracts and radix puerariae-containing preparations by adopting puerarin as a reference substance. The UPLC method comprises: preparing a reference substance solution and a testing sample solution, and carrying out gradient elution by adopting 0.1% acetic acid as a mobile phase A and adopting acetonitrile as a mobile phase B, wherein the detection wavelength is 250 nm, the chromatographic column is SB-C18, RRHD 1.8 [mu]m, 2.1*100 mm, the column temperature is 31 DEG C, the flow rate is 0.4 mL/min, and the injection volume is 1 [mu]L; comparing the obtained chromatogram with a radix puerariae herb finger print, wherein the main peak is puerarin, and other components are corresponding to various peaks in a one-to-one manner so as to identify the herb; and calculating the contents of the six components in the radix puerariae by combining relative calibration factors (the relative calibration factor of 3'-hydroxy puerarin is 1.253, the relative calibration factor of puerarin is 1.000, the relative calibration factor of puerarin-6"-O-xyloside is 1.422, the relative calibration factor of 3'-methoxy puerarin is 1.297, the relative calibration factor of mirificin is 1.332, and the relative calibration factor of daidzin is 1.030) and the peak area of puerarin in the contrast solution.
Description
Technical field
The invention belongs to field of medicaments, it is related to Chinese medicinal material extract and extractive technique, and the uplc rapid analysis method of preparation.
Background technology
Radix Puerariae is the dry root of legume pueraria lobata, is all distributed in national most laboratories, and Hubei is an important producing region.Radix Puerariae deep processing is become extract, preferable social effect and economic benefit can be produced.Meanwhile, the main component in Radix Puerariae extract is osajin, and pharmacological action ratio is wide, more prominent to the effect ratio of cardiovascular and cerebrovascular disease aspect, home and abroad wide market.All using Radix Puerariae as principal agent in multiple Chinese medicine prescriptions, and select puerarin therein as quality control index.Chinese medicine ingredients are complicated, and during treating disease, these compositions produce synergism when most.Therefore, determine Chinese crude drug and its corresponding product quality should be a combination of wherein various composition.But, past is due to the restriction of condition, the quality controlling Chinese crude drug often chooses the composition (index composition) that one of which is easy to detect, so may result in some problems, on the one hand, the effect of medical material is not necessarily produced by a kind of this composition, therefore can not fully reflect the quality of medical material；On the other hand, illegal Producer is in order to seek exorbitant profit, need the composition of detection using adding touchstone in adulterant or poor products, therefore, select the Multiple components (index composition or active component) in medical material material setting up corresponding quality control standard, thus can be very good to prevent artificial doping and adulterated, also can instruct commercial production it is ensured that the quality of product.Uplc has sensitivity height, and separation efficiency is high and speed is fast, the few advantage of solvent consumption, can complete within a short period of time to analyze simultaneously, can be used not only for the mensure of Multiple components in single medical material, can also realize the mensure of various ingredients in compound recipe.
Content of the invention
The task of the present invention is to provide a kind of detection Radix Puerariae or Radix Puerariae extract or the uplc method containing kudzu vine root preparation composition.
The present invention provides and detects that Radix Puerariae or Radix Puerariae extract or the uplc method containing composition in kudzu vine root preparation are: with puerarin for comparison, qualitative and detection by quantitative Radix Puerariae, Radix Puerariae extract and containing 3'-hydroxypuerarin, puerarin, puerarin -6 in kudzu vine root preparation "-o- xyloside, the uplc method of 3 '-methoxy puerarin, celery glycosyl puerarin glycosides and 6 kinds of component contents of daiazi; reference substance and need testing solution are prepared in inclusion; with 0.1% acetic acid for mobile phase a; carry out gradient elution with acetonitrile for mobile phase b, and Detection wavelength is 250nm；Chromatographic column is sb-c18, rrhd, 1.8um, 2.1*100mm；Column temperature is 31oc；Flow velocity is 0.4ml/min；Sample size is 1ul；Gained chromatogram is compared with Radix Puerariae medicinal materials fingerprint, main peak is puerarin, remaining composition corresponds, medical material can be differentiated, the peak area in combination with puerarin in relative correction factor (3'-hydroxypuerarin is 1.253, puerarin is 1.000, puerarin -6 "-o- xyloside be 1.422,3 '-methoxy puerarin be that 1.297, celery glycosyl puerarin glycosides is 1.332 and daiazi is 1.030) and contrast solution can calculate the content of 6 kinds of compositions in Radix Puerariae.
Detection Radix Puerariae or Radix Puerariae extract or the uplc method containing kudzu vine root preparation composition that the present invention provides, comprise the following steps:
Step one, preparation reference substance solution: weigh puerarin reference substance in right amount in volumetric flask, plus methanol dissolves and is diluted to about 125ug/ml；
Step 2, prepare need testing solution: weigh Radix Puerariae powder (crossing No. three sieves) 0.15g, add 40% ethanol 50ml, weigh, supersound extraction 30min, let cool to room temperature, complement to original weight with 40% ethanol, shake up, 0.22um membrane filtration obtains final product；
Step 3, by the following method sample detection: chromatograph: uplc (agilent 1290, dad detector)；
Chromatographic condition: gradient elution, mobile phase a:0.1% acetic acid mobile phase b: acetonitrile
| Time (min) | Mobile phase a (%) | Mobile phase b (%) |
| 0 | 92 | 8 |
| 0.5 | 88.5 | 11.5 |
| 3.5 | 88.5 | 11.5 |
| 7 | 77 | 23 |
| 10 | 50 | 50 |
| 12 | 10 | 90 |
| 14 | 10 | 90 |
Detection wavelength: 250nm；Chromatographic column: sb-c18, rrhd, 1.8um, 2.1*100mm；Column temperature: 31 DEG C；Flow velocity: 0.4ml/min；Sample size: 1ul；
Step 4, gained chromatogram is compared with Radix Puerariae medicinal materials fingerprint, main peak is puerarin, remaining composition corresponds, and medical material can be differentiated.
In comparing gained chromatogram with Radix Puerariae medicinal materials fingerprint described in above-mentioned steps four, the peak area in conjunction with puerarin in relative correction factor and contrast solution can calculate the content of 6 kinds of compositions in Radix Puerariae；In Radix Puerariae, the relative correction factor of 6 kinds of compositions is: 3'-hydroxypuerarin is 1.253；Puerarin is 1.000；Puerarin -6 "-o- xyloside is 1.422；3 '-methoxy puerarin is 1.297；Celery glycosyl puerarin glycosides is 1.332；Daiazi is 1.030.
The method that the present invention provides quickly and accurately can carry out quality control to the Radix Puerariae origin components in Radix Puerariae medical material, extract and the Chinese patent medicine preparation containing Radix Puerariae, and required time is shorter, can complete to analyze within 14min.The sensitivity of method is higher, has higher actual application value.
Experimental data
1. the foundation of analysis method
Chromatograph: uplc (agilent 1290, dad detector)
Chromatographic condition: gradient elution, mobile phase a:0.1% acetic acid mobile phase b: acetonitrile
| Time (min) | Mobile phase a (%) | Mobile phase b (%) |
| 0 | 92 | 8 |
| 0.5 | 88.5 | 11.5 |
| 3.5 | 88.5 | 11.5 |
| 7 | 77 | 23 |
| 10 | 50 | 50 |
| 12 | 10 | 90 |
| 14 | 10 | 90 |
Detection wavelength: 250nm；Chromatographic column: sb-c18, rrhd, 1.8um, 2.1*100mm；Column temperature: 31oc；Flow velocity: 0.4ml/min；Sample size: 1ul
In several Radix Puerariaes such as selection 3'-hydroxypuerarin, puerarin, puerarin -6 "-o- xyloside, 3 '-methoxy puerarin, celery glycosyl puerarin glycosides and daiazi, contained isoflavone is as standard substance.Each composition ultraviolet absorpting spectrum (dad scanning) sees that Fig. 2 is shown in by Fig. 1, mixing reference substance and sample collection of illustrative plates,
2. analysis method checking
The pre-treatment of 2.1 samples
2.1.1 the selection of Extraction solvent: precision weighs 0.15g Radix Puerariae medicinal powder (crossing No. three sieves), is separately added into different concentration ethanol solution (30%, 40%, 50%, 60% and 70%) 50ml, weighs, supersound extraction 30min, taking-up lets cool to room temperature, complement to original weight with corresponding concentration ethanol solution, shake up, 0.22um membrane filtration, take 1ul sample introduction, be compared with peak area/sample weighting amount.
The selection of table 1 solvent strength
The ratio of wherein 40% ethanol extraction is all larger, therefore selects 40% ethanol as Extraction solvent.
2.1.2 the determination of ultrasonic time: precision weighs 0.15g Radix Puerariae medicinal powder (crossing No. three sieves), is separately added into 40% ethanol 50ml, weighs, investigates ultrasonic different time (20min, 30min, 40min and 50min) to the impact extracted.It is shown in Table 2.
The determination of table 2 ultrasonic time
During ultrasonic 30min, ratio is maximum, determines that ultrasonic time is 30min.
The preparation of 2.2 solution: need testing solution preparation: weigh Radix Puerariae powder 0.15g, add 40% ethanol 50ml, weigh, supersound extraction 30min, let cool to room temperature, complement to original weight with 40% ethanol, shake up, 0.22um membrane filtration obtains final product.(preparation process of Radix Puerariae extract and Chinese patent medicine preparation is shown in subsequent instance).Prepared by reference substance stock solution: weigh 3 '-hydroxyl puerarin 4.13mg respectively, puerarin 6.54mg, puerarin -6 "-o- xyloside 4.78mg, 3 '-methoxy puerarin 4.34mg, celery glycosyl puerarin glycosides 4.79mg, daiazi 6.10mg is in 25ml volumetric flask, with methanol ultrasonic dissolution and be diluted to scale, (each reference substance content is respectively 3 '-hydroxyl puerarin 98.5% to obtain final product reference substance stock solution, puerarin 95.5%, puerarin -6 "-o- xyloside 98.0%, 3 '-methoxy puerarin 98.5%, celery glycosyl puerarin glycosides 98.5%, daiazi 91.3%).
2.3 stabilities of solution: take Radix Puerariae powder (lot number: a40805) 0.15g, plus 40% ethanol 50ml, weigh, supersound extraction 30min, let cool to room temperature, complement to original weight with 40% ethanol, shake up, 0.22um membrane filtration obtains final product.Take 1ul sample introduction, obtain final product 0h peak area, room temperature is placed, and in 2h, 4h, 8h, 12h and 24h take 1ul sample introduction respectively, the results are shown in Table 3, have been respectively compared the rate of change that each composition of each time point is with respect to 0h.
Table 3 solution room temperature stability is investigated
Have upper table to understand: 3 '-hydroxyl puerarin is unstable, room temperature (25oc) is placed and easily degraded, should in 12h sample introduction, such as need to preserve for a long time, should be saved in (it is experimentally confirmed that sample preservation has good stability in refrigerator) in refrigerator.
2.4 specificity tests: take blank solvent (40% ethanol solution), contrast solution and each 1ul of need testing solution sample introduction respectively, by aforementioned analytical methods sample introduction, record chromatogram.Result shows, sample solvent does not interfere to measuring composition.
2.5 linear, quantitative limit and test limit: take reference substance stock solution, dilute 2 times successively, 5 times, 10 times, 25 times, 50 times, 100 times, 250 times, 625 times, take 1ul sample introduction respectively.Record chromatogram, the results are shown in Table 4 and table 5.
The each reference substance concentration of table 4 and corresponding peak area
The each component linear relation of table 5, quantitative limit and test limit
The linear equation intercept of above-mentioned each composition is set to zero, the slope of each composition is respectively as follows: 3 '-hydroxyl puerarin 8.3993, puerarin 10.435, puerarin -6 "-o- xyloside 7.3135,3 '-methoxy puerarin 8.0432; celery glycosyl puerarin glycosides 7.0811; daiazi 10.221, because puerarin is main component in Radix Puerariae it is easy to preparation; therefore select it as object of reference, determine the relative correction factor that other compositions are with respect to puerarin.Relative correction factor as daiazi is 10.435/10.221=1.021, relative correction factor is determinand and the response ratio of object of reference unit concentration solution, the concentration of determinand similar to internal standard method, when lacking determinand reference substance, can be determined with reference to relative correction factor by referring to thing.Prepare two standard curves, measure relative correction factor result as follows:
Table 6 relative correction factor
2.6 precision tests: continuous three days, weigh three parts of Radix Puerariae powder, every part of 0.15g daily, prepare need testing solution with 2.2, take 1ul sample introduction, record chromatogram, calculate peak area rsd experimental result and be shown in Table 7.
Table 7: peak area is in a few days and day to day precision
2.7 accuracy tests (average recovery): precision weighs reference substance puerarin -6 "-o- xyloside 3.98mg is in 20ml volumetric flask, plus methanol ultrasonic dissolution be diluted to scale.In addition weigh reference substance 3 '-hydroxyl puerarin 5.41mg, puerarin 18.82mg, 3 '-methoxy puerarin 4.06mg, celery glycosyl puerarin glycosides 4.33mg, daiazi 3.12mg is in 25ml volumetric flask, plus proper amount of methanol ultrasonic dissolution, pipette the aforesaid puerarin -6 of 5ml "-o- xyloside contrast solution 5ml, plus methanol dilution to scale obtains final product accuracy mark-on mother solution.
The need testing solution 5ml measuring preparation under 2.2 is in 10ml volumetric flask, totally 9 parts, above 3 parts add the above-mentioned mother solution of 0.4ml (80% concentration), middle three parts add 0.5ml (100% concentration), last three parts add 0.6ml (120% concentration), are settled to scale with 40% ethanol solution.Shake up, after 0.22um membrane filtration, take 1ul sample introduction respectively, record chromatogram, experiment with computing the results are shown in Table 8 (notes: the response rate=(amount in the test liquid of actual measured amount-addition)/addition reference substance amount * 100%)
Table 8: average recovery
2.8 serviceability tests take need testing solution 1ul sample introduction, and the concentration investigating acetic acid in (1) adjustment mobile phase a is 0.09%, 0.1% and 0.11%；(2) adjustment flow velocity is 0.38ml/min, 0.40ml/min and 0.42ml/min；(3) adjustment column temperature is 29oc, the impact to each composition separating degree for 31oc and 33oc, only changes a condition every time, and other conditions keep constant.Experimental result is shown in Table 9
Table 9 serviceability test result
The application of 3 analysis methods
3.1 Radix Puerariae medicinal materials fingerprints
Collect the Radix Puerariae medical material of different provinces (ground such as Hubei, Henan, Guangxi, Shaanxi, Zhejiang and Anhui), weigh powder 0.15g, plus 40% ethanol 50ml, weigh, ultrasonic 30min, let cool to room temperature, complement to original weight with 40% ethanol, shake up, 0.22um membrane filtration obtains final product, take 1ul sample introduction respectively, record chromatograph.Chromatogram is imported Chinese Pharmacopoeia Commission's similarity evaluation, sets up the reference fingerprint of Radix Puerariae medical material by this system, have 12 common characteristic peaks (wherein 7 characteristic peaks are pointed out), result is shown in Fig. 3.
The detection of 3.2 Radix Puerariae extracts: buy 2 parts of commercially available Radix Puerariae extracts (all indicating puerarin content ＞ 10%, total flavones ＞ 40%).Weigh each extract 30mg respectively in 50ml volumetric flask, add 40% EtOH Sonicate to dissolve and be settled to scale, after 0.22um membrane filtration, take 1ul sample introduction.Separately take Radix Puerariae control medicinal material powder (National Institute for Food and Drugs Control's lot number: 121551-201103) 0.15g, plus 40% ethanol 50ml, weigh, ultrasonic 30min, let cool to room temperature, complement to original weight with 40% ethanol, shake up, 1ul sample introduction is taken after 0.22um membrane filtration, result is shown in Fig. 4, and (testing result of the product that two companies produce is consistent, only list a result), as illustrated, contain in Radix Puerariae extract 6 kinds of osajin composition (3'-hydroxypuerarins (1)；Puerarin (2)；Puerarin -6 "-o- xyloside (3)；3 '-methoxy puerarin (4)；Celery glycosyl puerarin glycosides (5)；Daiazi (6)) consistent with Radix Puerariae control medicinal material, show that this analysis method is applied to detection and the real and fake discrimination (if adulterant, then inspection does not measure above-mentioned 6 kinds of compositions) of Radix Puerariae extract.
It is shown in Table 10 with the content that the testing result of one of producer calculates the wherein content of puerarin and above-mentioned several known flavone.
10 two kinds of calculations of table compare
Note: (1) calculates each constituent concentration by comparison liquid and is calculated by bringing each Component peak area into corresponding linear equation.
(2) made with reference to computing formula by puerarin: c one-tenths=r becomes * f to become/(r Pueraria lobota right/c Pueraria lobota to): c one-tenth: the concentration of certain composition to be measured；R becomes: the peak area of this composition；F becomes: the relative correction factor of this composition；C Pueraria lobota pair: the concentration of puerarin in comparison liquid；R Pueraria lobota pair: the peak area of puerarin in comparison liquid collection of illustrative plates.Making reference calculating concentration as 3 '-hydroxyl puerarin by puerarin is: c=121.18544*1.253/ (1234.49182/124.914)=15.36ug/ml.
This batch extract sample weighting amount is 30.03mg, and discounting for moisture, then the content of puerarin is 77.79*50/1000/30.03=12.95% ＞ 10%, is consistent with labelled amount.Because total flavones adopt ultraviolet spectrophotometry to detect, not verify herein.
3.3 detections containing Radix Puerariae Chinese patent medicine preparation
3.3.1 Yuquan granule (prescription forms: Radix Trichosanthis, Radix Puerariae, Radix Ophiopogonis, Radix Ginseng, Poria, Fructus Mume, the Radix Astragali, Radix Glycyrrhizae, Radix Rehmanniae and Fructus Schisandrae Chinensis etc.)
Prepared by need testing solution: by Yuquan particulate abrasive to powder, weigh in 0.45g and 50ml volumetric flask, add 40% appropriate amount of ethanol, let cool, be settled to scale with 40% ethanol, take 1ul sample detection after 0.22um membrane filtration after ultrasonic 30min.Result is shown in Fig. 5.As shown in the figure, can detect that 6 kinds of osajin compositions in Radix Puerariae control medicinal material in the granule of Yuquan, show that this analysis method is applied to Chinese patent medicine preparation detection and the quality control (controlling by the content of puerarin and total isoflavone (above-mentioned 6 kinds of isoflavones components summations)) that Radix Puerariae makees principal agent.
3.3.2 Kang Xin piece (mainly containing soybean isoflavone, soybean phospholipid, Fructus Lycii, Radix Puerariae, Aloe etc.) need testing solution preparation: by Kang Xin piece grinds, weigh 0.1g in 50ml volumetric flask, let cool to room temperature after adding 40% EtOH Sonicate, it is settled to scale with 40% ethanol, shake up.1ul sample introduction is taken after 0.22um membrane filtration.Record chromatogram, result is shown in Fig. 6.As shown in the figure, 6 kinds of isoflavones components in Kang Xin piece are corresponded with Radix Puerariae control medicinal material, the soybean isoflavone constituents that contain between 6 kinds of compositions and in preparation separate good, show that the method is suitable for inclusion in the detection of preparation and the quality control of Radix Puerariae and soybean isoflavone constituents.
3.3.3 TANGMAIKANG KELI (prescription includes the Radix Astragali, Radix Rehmanniae, Radix Paeoniae Rubra, Radix Salviae Miltiorrhizae, Radix Puerariae, Folium Mori, Herba Epimedii etc.).Prepared by need testing solution: TANGMAIKANG KELI is pulverized, weighs 0.4g in 50ml volumetric flask, let cool to room temperature, 40% ethanol dilution, to scale, shakes up, 0.22um membrane filtration, takes 1ul sample introduction after adding 40% EtOH Sonicate 30min.Record chromatogram, result is shown in Fig. 7.As illustrated, can detect that 7 kinds of Radix Puerariae isoflavone constituents in TANGMAIKANG KELI, Radix Puerariae does not make principal agent in said preparation, shows that the sensitivity of the method is higher.
To sum up, the method that the present invention provides quickly and accurately can carry out quality control to the Radix Puerariae origin components in Radix Puerariae medical material, extract and the Chinese patent medicine preparation containing Radix Puerariae, and required time is shorter, can complete to analyze within 14min.The sensitivity of method is higher, has higher actual application value.
Brief description
In several Radix Puerariaes such as Fig. 1, Fig. 2: choose 3'-hydroxypuerarin, puerarin, puerarin -6 "-o- xyloside, 3 '-methoxy puerarin, celery glycosyl puerarin glycosides and daiazi, contained isoflavone is as standard substance, Fig. 1 position each composition ultraviolet absorpting spectrum (dad scanning).Fig. 2 is mixing reference substance and sample collection of illustrative plates: a is mixed reference substance solution, and b is sample solution.
Fig. 3 Radix Puerariae medicinal materials fingerprint peak 3:3 '-hydroxyl puerarin；Peak 5: puerarin；Peak 6: puerarin -6 "-o- xyloside；Peak 7:3 '-methoxy puerarin；Peak 8: celery glycosyl puerarin glycosides；Peak 9: daiazi；Peak 12: daidzein (because reference substance is only for differentiating, not setting up its standard curve) ".
Fig. 4 Radix Puerariae extract and Radix Puerariae medical material comparison collection of illustrative plates.
Fig. 5 Yuquan granule compares collection of illustrative plates with Radix Puerariae medical material.
Fig. 6 Kang Xin piece and Radix Puerariae medical material comparison collection of illustrative plates.
Fig. 7: TANGMAIKANG KELI and Radix Puerariae medical material comparison collection of illustrative plates.
Specific embodiment
Embodiment 1: the detection of Radix Puerariae extract
Buy 2 parts of commercially available Radix Puerariae extracts (all indicating puerarin content ＞ 10%, total flavones ＞ 40%).Weigh each extract 30mg respectively in 50ml volumetric flask, add 40% EtOH Sonicate to dissolve and be settled to scale, after 0.22um membrane filtration, take 1ul sample introduction.Separately take Radix Puerariae control medicinal material powder (National Institute for Food and Drugs Control's lot number: 121551-201103) 0.15g, plus 40% ethanol 50ml, weigh, ultrasonic 30min, let cool to room temperature, complement to original weight with 40% ethanol, shake up, 1ul sample introduction is taken after 0.22um membrane filtration, result is shown in Fig. 3, and (testing result of the product that two companies produce is consistent, only list a result), as illustrated, contain in Radix Puerariae extract 6 kinds of osajin composition (3'-hydroxypuerarins (1)；Puerarin (2)；Puerarin -6 "-o- xyloside (3)；3 '-methoxy puerarin (4)；Celery glycosyl puerarin glycosides (5)；Daiazi (6)) consistent with Radix Puerariae control medicinal material, show that this analysis method is applied to detection and the real and fake discrimination (if adulterant, then inspection does not measure above-mentioned 6 kinds of compositions) of Radix Puerariae extract
It is shown in Table 10 with the content that the testing result of one of producer calculates the wherein content of puerarin and above-mentioned several known flavone.
10 two kinds of calculations of table compare
Note: (1) calculates each constituent concentration by comparison liquid and is calculated by bringing each Component peak area into corresponding linear equation.
(2) made with reference to computing formula by puerarin: c one-tenths=r becomes * f to become/(r Pueraria lobota right/c Pueraria lobota to): c one-tenth: the concentration of certain composition to be measured；R becomes: the peak area of this composition；F becomes: the relative correction factor of this composition；C Pueraria lobota pair: the concentration of puerarin in comparison liquid；R Pueraria lobota pair: the peak area of puerarin in comparison liquid collection of illustrative plates.Making reference calculating concentration as 3 '-hydroxyl puerarin by puerarin is: c=121.18544*1.253/ (1234.49182/124.914)=15.36ug/ml.
This batch extract sample weighting amount is 30.03mg, and discounting for moisture, then the content of puerarin is 77.79*50/1000/30.03=12.95% ＞ 10%, is consistent with labelled amount.Because total flavones adopt ultraviolet spectrophotometry to detect, not verify herein.
Embodiment 2: the detection of the Yuquan of Chinese patent medicine preparation containing Radix Puerariae granule
Yuquan granule (prescription forms: Radix Trichosanthis, Radix Puerariae, Radix Ophiopogonis, Radix Ginseng, Poria, Fructus Mume, the Radix Astragali, Radix Glycyrrhizae, Radix Rehmanniae and Fructus Schisandrae Chinensis etc.).Prepared by need testing solution: by Yuquan particulate abrasive to powder, weigh in 0.45g and 50ml volumetric flask, add 40% appropriate amount of ethanol, let cool, be settled to scale with 40% ethanol, take 1ul sample detection after 0.22um membrane filtration after ultrasonic 30min.Result is shown in Fig. 5.As shown in the figure, can detect that 6 kinds of osajin compositions in Radix Puerariae control medicinal material in the granule of Yuquan, show that this analysis method is applied to Chinese patent medicine preparation detection and the quality control (controlling by the content of puerarin and total isoflavone (above-mentioned 6 kinds of isoflavones components summations)) that Radix Puerariae makees principal agent.
Embodiment 3: the detection of the Kang Xin piece of Chinese patent medicine preparation containing Radix Puerariae
Kang Xin piece (mainly contains soybean isoflavone, soybean phospholipid, Fructus Lycii, Radix Puerariae, Aloe etc..Prepared by need testing solution: Kang Xin piece grinds weigh 0.1g in 50ml volumetric flask, lets cool to room temperature, be settled to scale with 40% ethanol, shake up after adding 40% EtOH Sonicate.1ul sample introduction is taken after 0.22um membrane filtration.Record chromatogram, result is shown in Fig. 6.As shown in the figure, 6 kinds of isoflavones components in Kang Xin piece are corresponded with Radix Puerariae control medicinal material, the soybean isoflavone constituents that contain between 6 kinds of compositions and in preparation separate good, show that the method is suitable for inclusion in the detection of preparation and the quality control of Radix Puerariae and soybean isoflavone constituents.
Embodiment 4: the detection of the TANGMAIKANG KELI of Chinese patent medicine preparation containing Radix Puerariae
TANGMAIKANG KELI (prescription includes the Radix Astragali, Radix Rehmanniae, Radix Paeoniae Rubra, Radix Salviae Miltiorrhizae, Radix Puerariae, Folium Mori, Herba Epimedii etc.).Prepared by need testing solution: TANGMAIKANG KELI is pulverized, weighs 0.4g in 50ml volumetric flask, let cool to room temperature, 40% ethanol dilution, to scale, shakes up, 0.22um membrane filtration, takes 1ul sample introduction after adding 40% EtOH Sonicate 30min.Record chromatogram, result is shown in Fig. 7.As illustrated, can detect that 7 kinds of Radix Puerariae isoflavone constituents in TANGMAIKANG KELI, Radix Puerariae does not make principal agent in said preparation, shows that the sensitivity of the method is higher.
Claims (2)
1. one kind with puerarin be control test Radix Puerariae, Radix Puerariae extract or containing 3'-hydroxypuerarin in kudzu vine root preparation, puerarin,
Puerarin -6 "-o- xyloside, the uplc method of 3 '-methoxy puerarin, celery glycosyl puerarin glycosides and 6 kinds of compositions of daiazi,
Comprise the following steps:
Step one, reference substance solution preparation: weigh puerarin reference substance in right amount in volumetric flask, plus methanol dissolves and is diluted to about
125ug/ml；
Step 2, need testing solution preparation: weigh Radix Puerariae powder (crossing No. three sieves) 0.15g, add 40% ethanol 50ml,
Weigh, supersound extraction 30min, let cool to room temperature, complement to original weight with 40% ethanol, shake up, 0.22um membrane filtration is
?；
Step 3, by the following method sample detection: chromatograph: uplc (agilent 1290, dad detector)；
Chromatographic condition: gradient elution, mobile phase a:0.1% acetic acid mobile phase b: acetonitrile
Detection wavelength: 250nm；Chromatographic column: sb-c18, rrhd, 1.8um, 2.1*100mm；Column temperature: 31 DEG C；Flow velocity:
0.4ml/min；Sample size: 1ul；
Step 4, gained chromatogram is compared with Radix Puerariae medicinal materials fingerprint, main peak is puerarin, remaining composition corresponds,
Medical material can be differentiated.
2. method according to claim 1 it is characterised in that described in step 4 by gained chromatogram and Radix Puerariae medicine
Material finger printing relatively in, the peak area in conjunction with puerarin in relative correction factor and contrast solution can calculate 6 kinds in Radix Puerariae
The content of composition；In Radix Puerariae, the relative correction factor of 6 kinds of compositions is: 3'-hydroxypuerarin is 1.253；Puerarin is 1.000；
Puerarin -6 "-o- xyloside is 1.422；3 '-methoxy puerarin is 1.297；Celery glycosyl puerarin glycosides is 1.332；Semen sojae atricolor
Glycosides is 1.030.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510433533.3A CN106370763B (en) | 2015-07-22 | 2015-07-22 | UPLC method for detecting kudzu root, kudzu root extract and kudzu root preparation component |
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| CN113406241A (en) * | 2021-07-14 | 2021-09-17 | 鲁南制药集团股份有限公司 | Quality detection method of Xintong granules |
| CN113406241B (en) * | 2021-07-14 | 2022-09-13 | 鲁南制药集团股份有限公司 | Detection method of Xintong granules |
| CN116196347A (en) * | 2022-11-24 | 2023-06-02 | 齐鲁工业大学 | Green extraction method of five active ingredients in kudzuvine root |
| CN116196347B (en) * | 2022-11-24 | 2023-12-19 | 齐鲁工业大学 | Green extraction method of five active ingredients in kudzuvine root |
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