CN112903887B - Method for establishing HPLC-VWD-ELSD characteristic spectrum of ginkgo leaf drop pills and characteristic spectrum thereof - Google Patents
Method for establishing HPLC-VWD-ELSD characteristic spectrum of ginkgo leaf drop pills and characteristic spectrum thereof Download PDFInfo
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Abstract
The invention relates to a traditional Chinese medicine characteristic fingerprint analysis method, in particular to a method for establishing a ginkgo leaf dripping pill HPLC-VWD-ELSD characteristic spectrum and the characteristic spectrum obtained by the method. The invention discloses a method for establishing a characteristic spectrum of ginkgo leaf dripping pills by HPLC-VWD-ELSD, which comprises the steps of preparing a test sample solution, establishing and measuring characteristic spectrum measuring conditions, wherein 11 characteristic common peaks are identified by the characteristic spectrum through a reference substance. The invention uses ultrafiltration tube to pre-treat sample, which can basically remove polyethylene glycol 4000 (PEG 4000), and reduce the influence of the polyethylene glycol 4000 on the response of lactone components in ELSD chromatogram.
Description
Technical Field
The invention relates to a traditional Chinese medicine characteristic fingerprint analysis method, in particular to a method for establishing a ginkgo leaf dripping pill HPLC-VWD-ELSD characteristic spectrum and the characteristic spectrum obtained by the method.
Background
The ginkgo leaf drop pill is loaded in the 2020 edition (first part) of Chinese pharmacopoeia, is prepared by heating and dissolving ginkgo leaf extract and polyethylene glycol 4000, mixing, dripping into methyl silicone oil coolant, removing surface oil trace, or coating film, and is mainly used for treating chest stuffiness and pain, apoplexy, hemiplegia and glossolalia caused by blood stasis and obstruction of collaterals; coronary heart disease, stable angina pectoris and cerebral infarction with the symptoms.
The traditional Chinese medicine characteristic fingerprint refers to a chromatogram or spectrogram which can reflect the chemical characteristics of the traditional Chinese medicine or the preparation thereof after being properly processed by a certain analysis method. The chromatographic fingerprint can well reflect the comprehensiveness and complexity of the traditional Chinese medicine, and is an effective method for evaluating the authenticity and consistency of the traditional Chinese medicine.
The flavonoid and lactone components are main chemical components in the ginkgo leaf drop pills, and are also index components for content measurement in Chinese pharmacopoeia, and the quality control method of the ginkgo leaf drop pills at present mainly comprises the steps of measuring the flavonoid and lactone components singly or establishing a fingerprint of a single component. Because the flavonoid component has good ultraviolet absorption, but the lactone does not have ultraviolet absorption, the flavonoid component and the lactone component can be detected simultaneously by using an ultraviolet detector and an evaporative light scattering detector, and the HPLC-VWD-ELSD characteristic spectrum of the ginkgo leaf dripping pill is established, so that the chemical components of the ginkgo She Di pill can be comprehensively characterized from the whole angle.
Disclosure of Invention
In order to achieve the above purpose, the invention provides a method for establishing a characteristic spectrum of ginkgo leaf dripping pills HPLC-VWD-ELSD, which comprises the following steps:
(1) Preparation of test solution:
pretreatment of an ultrafiltration membrane: 400 μL of 50% methanol is placed in a Mitigo ultrafiltration tube (outer tube 1.5mL, 3K), centrifuged for 20min at 15000r, the filtrate is discarded, the inner tube is placed in the outer tube reversely, and centrifuged for 1min at 1000r, and the liquid in the inner tube is removed.
Taking appropriate amount of ginkgo leaf drop pills, grinding, taking 0.25g of powder, precisely weighing, placing into a conical bottle with a plug, precisely adding 20mL of 50% methanol, performing ultrasonic treatment for 60min (40 KHz, 500W), taking out, cooling to room temperature, shaking uniformly, 15000r, centrifuging for 20min, taking 400 mu L of supernatant, placing into a pretreated ultrafiltration tube, 15000r, centrifuging for 20min, and taking out the solution in a filtrate collecting tube.
(2) Preparation of a control solution: taking reference substances of bilobalide, bilobalide J, bilobalide C, bilobalide A, bilobalide B, rutin, quercetin-3-O-glucosyl (1- & gt 2) rhamnoside, kaempferol-3-O-rutin, narcissus, quercetin-3-O-2 '- (6' -p-coumaroyl) -glucosyl-rhamnoside and kaempferol-3-O-2 '- (6' -p-coumaroyl) -glucosyl-rhamnoside, adding 50% methanol to prepare mother solution with proper concentration, and properly diluting to prepare a mixed reference substance solution with the final concentration of 10-100 mug/mL.
(3) Ultraviolet-evaporative light scattering detector assay: chromatographic column: agilent InfinityLab Poroshell 120EC-C18 (3.0X105 mm,2.7 μm); mobile phase: gradient elution was performed using 0.05% formic acid in water as mobile phase A and acetonitrile-methanol (8:2) as mobile phase B, flow rates as follows: 0.4mL/min, column temperature: 35 ℃, detection wavelength: 360nm; evaporative light scattering detector: n (N) 2 Flow rate: 1.2SLM, evaporator tube temperature: 80 ℃; atomizer temperature: 80 ℃.
(4) Establishing a characteristic map: preparing 20 batches of ginkgo leaf drop pills into a sample solution according to the step (1), injecting the sample solution into a high performance liquid chromatograph, detecting according to the chromatographic conditions of the step (3), recording chromatograms respectively, analyzing by adopting software of a traditional Chinese medicine chromatographic fingerprint similarity evaluation system 2012 edition to obtain a VWD-ELSD standard fingerprint (R) of the ginkgo leaf drop pills, determining 11 common peaks, wherein the lactone uses peak 1 (bilobalide) as a reference peak, and the flavonoid glycoside uses peak 4 (rutin) as a reference peak.
In addition, the invention also provides an HPLC-VWD-ELSD characteristic map of the ginkgo leaf drop pill, as shown in figure 8.
The profile shares peaks with 11 features, where peak 1: bilobalide; peak 2: ginkgolide J; peak 3: ginkgolide C; peak 4: rutin; peak 5: quercetin-3-O-glucosyl (1- > 2) rhamnoside; peak 6: kaempferol-3-O-rutinoside; peak 7: narcissus glucoside; peak 8: ginkgolide A; peak 9: ginkgolide B; peak 10: quercetin-3-O-2 "- (6" -p-coumaroyl) -glucosyl-rhamnoside; peak 11: kaempferol-3-O-2 "- (6" -p-coumaroyl) -glucosyl-rhamnoside.
The invention discloses a method for establishing a characteristic spectrum of ginkgo leaf dripping pills by HPLC-VWD-ELSD, which comprises the steps of preparing a test sample solution, establishing and measuring characteristic spectrum measuring conditions, wherein 11 characteristic common peaks are identified by the characteristic spectrum through a reference substance.
Compared with the prior art, the invention has the following improvement points:
1. the ultrafiltration tube is used for sample pretreatment, so that the polyethylene glycol 4000 (PEG 4000) serving as an auxiliary material can be basically removed, and the influence of the polyethylene glycol 4000 on the response of lactone components in an ELSD chromatogram is reduced.
2. The chemical components in the ginkgo She Di pill can be comprehensively analyzed by using the combination of an ultraviolet detector and an evaporative light scattering detector to simultaneously detect compounds with ultraviolet absorption (flavonoid components) and compounds without ultraviolet absorption (lactone components).
3. The method has the advantages of good precision, stability and repeatability, and the obtained ginkgo leaf drop pill has high similarity of characteristic patterns, is accurate and reliable, can more comprehensively reflect the integral characteristics of the chemical composition of the ginkgo She Di pill, and can more comprehensively evaluate the quality of the ginkgo She Di pill.
Drawings
FIG. 1 is a comparison chart of different parameter selections of a sample solution preparation method;
wherein, the conditions of extraction are different in FIG. 1A, the solvents are different in FIG. 1B, the time of extraction is different in FIG. 1C, and the feed liquid ratio is different in FIG. 1D.
FIG. 2 chromatograms before and after ultrafiltration membrane filtration (before and after removal of PEG 4000)
Figure 3 is a comparison of three different chromatographic columns used in the example of the invention.
FIG. 4 is a graph comparing various mobile phase systems used in the example of the present invention.
FIG. 5 is a graph showing a comparison of different aqueous phases in a mobile phase system for an embodiment of the present invention.
FIG. 6 is a graph comparing various flow rate systems used in the embodiment of the present invention.
FIG. 7 is a graph comparing various column temperature systems used in the examples of the present invention.
FIG. 8A is a standard characteristic map of ginkgo leaf drop pills (11 characteristic common peaks 1-11)
Fig. 9 ginkgo leaf drop pill reference substance map
Fig. 10 is a graph showing 20 batches of characteristic maps of ginkgo leaf dripping pills
Wherein, peak 1: bilobalide; peak 2: ginkgolide J; peak 3: ginkgolide C; peak 4: rutin; peak 5: quercetin-3-O-glucosyl (1- > 2) rhamnoside; peak 6: kaempferol-3-O-rutinoside; peak 7: narcissus glucoside; peak 8: ginkgolide A; peak 9: ginkgolide B; peak 10: quercetin-3-O-2 "- (6" -p-coumaroyl) -glucosyl-rhamnoside; peak 11: kaempferol-3-O-2 "- (6" -p-coumaroyl) -glucosyl-rhamnoside
Detailed Description
The present invention will be further described with reference to examples below to provide a more detailed understanding of the present invention.
Example 1
1 instrument and reagent
1.1 instruments
One ten thousandth analytical balance (sartorius), agilent 1260InfinityVWD-ELSD combination, milli-Q deionized water, high-speed refrigerated centrifuge.
1.2 reagents
Ginkgo She Di pill is provided by Wanbond pharmaceutical Co., ltd, and the sample lot number is shown in Table 1. Methanol, acetonitrile and formic acid are chromatographic purity, water is ultrapure water, and the rest reagents are analytical purity.
TABLE 1 information table of ginkgo leaf drop pills
2 methods and results
2.1 examination of the preparation method of the sample solution
The preparation method of the sample solution is examined, and comprises the steps of extracting conditions (ultrasonic wave and reflux), extracting solvent (water, 25% methanol and 50% methanol), extracting time (20 min, 40min and 60 min) and extracting feed liquid ratio (1-40, 1-80 and 1-120), and determining the final extracting method as shown in the figure 1: 0.25g of the sample is weighed and extracted with 20mL of 50% methanol by ultrasound for 60min (the extraction solvent is considered to be 50% methanol solution because the ultrafiltration membrane material can only pass through < 60% methanol solution).
Preparation of test solution:
pretreatment of an ultrafiltration membrane: 400 μL of 50% methanol is taken in a Mitigo ultrafiltration tube (outer tube 1.5mL, 3K), centrifuged for 20min at 15000r, the filtrate is discarded, the inner tube is placed in the outer tube reversely, and centrifuged for 1min at 1000r, and the residual liquid in the inner tube is removed.
Taking appropriate amount of ginkgo leaf drop pills, grinding, taking 0.25g of powder, precisely weighing, placing into a conical bottle with a plug, precisely adding 20mL of 50% methanol, performing ultrasonic treatment for 60min (40 KHz, 500W), taking out, cooling to room temperature, shaking uniformly, 15000r, centrifuging for 20min, taking 400 mu L of supernatant, placing into a pretreated ultrafiltration tube, 15000r, centrifuging for 20min, and taking out the solution in a filtrate collecting tube.
Chromatograms before and after ultrafiltration membrane filtration (before and after removal of PEG 4000) are shown in fig. 2.
2.2 optimization and selection of chromatographic methods
The mobile phase examined water-acetonitrile, acid water-acetonitrile-methanol systems, the columns examined Agilent ZORBAX SB-C18 (2.1X100 mm,1.8 μm), agilent InfinityLab Poroshell EC-C18 (2.1X100 mm,2.7 μm) and Agilent InfinityLab Poroshell EC-C18 (3.0X105 mm,2.7 μm) three columns, agilent InfinityLab Poroshell 120EC-C18 (3.0X105 mm,2.7 μm) were selected as analytical columns, and the parameter parts of ELSD were examined, including evaporation temperature, atomization temperature and N 2 Flow rate, etc., determines optimal ELSD parameters.
Chromatographic column: agilent InfinityLab Poroshell 120EC-C18 (3.0X105 mm,2.7 μm), mobile phase: taking 0.05% formic acid aqueous solution as a mobile phase A, acetonitrile-methanol (8:2) as a mobile phase B,gradient elution was performed as follows, flow rate: 0.4mL/min, column temperature: 35 ℃, detection wavelength: 360nm; evaporative light scattering detector: n (N) 2 Flow rate: 1.2SLM, evaporator tube temperature: 80 ℃, atomizer temperature: 80 ℃.
TABLE 2 elution gradient table
2.2.1 investigation of different chromatographic columns
Three different chromatographic columns were examined for their separation effect (columns 1:Agilent ZORBAX SB-C18 (2.1X100 mm,1.8 μm), 2:Agilent InfinityLab Poroshell 120EC-C18 (2.1X100 mm,2.7 μm) and 3:Agilent InfinityLab Poroshell 120EC-C18 (3.0X105 mm,2.7 μm)), and eluted with a gradient according to Table 2. As can be seen from FIG. 3, the overall peak shape and the degree of separation of the chromatogram obtained in column 3 were good, and therefore Agilent InfinityLab Poroshell 120-EC-C18 (3.0X105 mm,2.7 μm) was selected as the analytical column.
2.2.2 investigation of different Mobile phase systems
2.2.2.1 examination of mobile phase System-organic phase-0.05% formic acid Water
The influence of the change of the organic phase in the mobile phase system on the chromatographic peak was examined, and the elution was carried out in a gradient manner according to Table 2. Considering both VWD and ELSD chromatograms (fig. 4), the separation effect of flavonoid glycosides was poor for the first 10min when pure acetonitrile was used as the organic phase; after mixing methanol, the retention is enhanced, and the separation degree is increased; the ratio of acetonitrile to methanol was further examined when acetonitrile: methanol=8: 2, the separation degree and peak shape of each of the identified peaks are good, and the elution time is appropriate, so acetonitrile is selected: methanol=8: 2 as organic phase.
2.2.2.2 investigation of mobile phase System-acetonitrile: methanol=8: 2-aqueous phase
The effect of the change of the water phase in the mobile phase system on the chromatographic peak was examined and eluted with a gradient according to table 2. The peak shape was improved by adding formic acid to the aqueous phase. As can be seen from FIG. 5, the degree of separation of peak 7 (Narcissus) was improved by adding 0.05% formic acid to the aqueous phase; formic acid concentration is increased to 0.1%, and the difference is not too great with 0.05% formic acid, so acetonitrile is selected to be used under the condition of ensuring the acidity bearing capacity of a chromatograph and a chromatographic column: methanol=8: 2-0.05% formic acid aqueous solution as mobile phase.
2.2.3 investigation of different flow Rate systems
The effect of different flow rates (0.3 mL/min, 0.4mL/min and 0.5 mL/min) on the separation degree of each chromatographic peak was examined by using an Agilent 1260 high performance liquid chromatograph, and the elution was carried out in a gradient manner according to Table 2. Experimental results show (as in FIG. 6), in the VWD chromatogram, both peak 4 and peak 7 are small peaks, and in the ELSD chromatogram, a better separation degree (peak 2 and peak 3) is obtained at a flow rate of 0.4mL/min, and a flow rate of 0.4mL/min is selected under comprehensive consideration.
2.2.4 investigation of different column temperature systems
The effect of different column temperatures (30 ℃,35 ℃ and 40 ℃) on the separation degree of each chromatographic peak was examined by using an Agilent 1260 high performance liquid chromatograph, and the elution was carried out in a gradient manner according to Table 2. As is clear from FIG. 7, when the column temperature was 30℃and 40 ℃, the separation effect of the peak 4 and the peak 11 was poor, and when the separation of each peak was good at 35 ℃, the detection temperature was selected to be 35℃in consideration of the temperature resistance of the column.
2.2.5 investigation of ELSD parameters
The evaporation temperature (60 ℃, 70 ℃ and 80 ℃) of ELSD, the atomization temperature (60 ℃, 70 ℃ and 80 ℃) and N were examined 2 Flow rates (1.2 SLM, 1.4SLM and 1.6 SLM), as will be seen from the following table, when the vaporization temperature is 80℃and the vaporization temperature is 80℃N 2 At a flow rate of 1.2SLM, the peak area of each lactone component is high.
3 methodology investigation
3.1 precision experiments
0.25g of ginkgo leaf drop pills (S12) is taken, precisely weighed, a sample solution is prepared according to the sample solution preparation method, 2 mu L of subsequent filtrate is precisely sucked, the filtrate is injected into a liquid chromatograph, continuous sample injection is carried out for 6 times, retention time and peak area of 11 common peaks are recorded, relative retention time, relative peak area and RSD value are calculated, and the results are shown in tables 3 and 4. Experimental results show that the method has good precision.
TABLE 3 precision investigation results (relative retention time)
Table 4 results of precision investigation (relative peak area)
3.2 repeatability experiments
Taking 6 parts of ginkgo leaf dropping pills (S12) in the same batch, precisely weighing 0.25g of each part, preparing a test solution according to the test solution preparation method, precisely sucking 2 mu L of the subsequent filtrate, injecting the filtrate into a liquid chromatograph, recording retention time and peak area of 11 common peaks, and calculating relative retention time, relative peak area and RSD value of the peak area, wherein the results are shown in tables 5 and 6. Experimental results show that the method is good in repeatability.
TABLE 5 repeatability test results (relative retention time)
TABLE 6 repeatability test results (relative peak area)
3.3 stability test
Taking the next sample solution of the repeatability item, precisely sucking 2 mu L of the sample solution at 0h,4h,6h,10h,12h and 24h, injecting the sample solution into a liquid chromatograph, recording retention time and peak area of 11 common peaks, and calculating relative retention time, relative peak area and RSD value of the peak area, wherein the results are shown in tables 7 and 8. Experimental results show that the method is good in stability.
TABLE 7 stability investigation results (relative retention time)
TABLE 8 stability investigation results (relative peak area)
4. Sample measurement
Preparing 20 batches of ginkgo leaf drop pills into a sample solution according to the sample solution preparation method, carrying out sample injection analysis according to the chromatographic method, respectively recording chromatograms of the sample solution, carrying out analysis by adopting software of a traditional Chinese medicine chromatographic fingerprint similarity evaluation system 2012 edition, generating a control map by a median method (figure 10), obtaining an HPLC-VWD-ELSD standard characteristic map (R) of the ginkgo leaf drop pills (figure 8), determining 11 common peaks, wherein the lactone uses peak 1 as a reference peak, the flavonoid glycosides use peak 4 as a reference peak, the relative retention time of each peak is shown in table 9, analyzing the similarity of the chromatograms of 20 batches of ginkgo leaf drop pills and the control characteristic map, and the similarity data is shown in tables 10 and 11, wherein the results show that the similarity of She Di pills of different batches and the control map is higher (more than 0.9).
TABLE 9 relative retention time of 11 common fingerprint peaks in 20 ginkgolic leaf drops
Table 10 similarity results (VWD) for 20 batches of ginkgo leaf drops
TABLE 11 20 results of similarity of ginkgo leaf drops (ELSD)
Claims (2)
1. A method for establishing HPLC-VWD-ELSD characteristic spectrum of ginkgo leaf drop pill comprises the following steps:
(1) Preparation of test solution:
pretreatment of an ultrafiltration membrane: taking 400 mu L of 50% methanol in a Mitigo ultrafiltration tube, centrifuging for 20min at 15000/r, discarding filtrate, reversely placing the inner tube in the outer tube at 1000/r/min, centrifuging for 1min, and removing liquid in the inner tube;
grinding folium Ginkgo dripping pill, collecting powder 0.25g, precisely weighing, placing into conical flask with plug, precisely adding 50% methanol 20mL, ultrasonic treating for 60min, taking out, cooling to room temperature, shaking uniformly, 15000 r/min, centrifuging for 20min, collecting 400 μl supernatant, placing into pretreated ultrafiltration tube, 15000 r/min, centrifuging for 20min, and collecting filtrate collecting tube solution;
(2) Preparation of a control solution:
collecting control bilobalide, bilobalide J, bilobalide C, bilobalide A, bilobalide B, rutin, and quercetin-3-OGlucosyl (1.fwdarw.2) rhamnoside, kaempferol-3-ORutin, narcissus and quercetin-3-O-2"-(6"-pCoumaroyl) -glucosyl-rhamnoside and kaempferol-3-O-2"-(6"-p-coumaroyl) -glucosyl-rhamnoside with proper amount, adding 50% methanol to prepare mother solution with proper concentration, and properly diluting to prepare mixed reference substance solution with final concentration of 10-100 mug/mL;
(3) High performance liquid chromatography-evaporative light scattering detector assay:
chromatographic column: agilent InfinityLab Poroshell 120 EC-C18; mobile phase: the gradient elution was carried out with 0.05% formic acid aqueous solution as mobile phase A, acetonitrile-methanol as mobile phase B, acetonitrile-methanol at a volume ratio of 8:2, and flow rates as follows: 0.4mL/min, column temperature: 35. c, detecting wavelength: 360nm; evaporative light scattering detector: n (N) 2 Flow rate: 1.2SLM, evaporating tube temperature: 80. the temperature is lower than the temperature; atomizer temperature: 80. the temperature is lower than the temperature;
(4) Establishing a characteristic map:
preparing 20 batches of ginkgo leaf drop pills into a sample solution according to the step (1), injecting the sample solution into a high performance liquid chromatograph, detecting according to the chromatographic conditions of the step (3), recording chromatograms respectively, analyzing by adopting software of a traditional Chinese medicine chromatographic fingerprint similarity evaluation system 2012 edition to obtain a VWD-ELSD standard fingerprint of the ginkgo leaf drop pills, determining 11 common peaks, wherein the lactone uses peak 1 bilobalide as a reference peak and the flavonoid glycoside uses peak 4 rutin as a reference peak.
2. The ginkgo leaf drop pill HPLC-VWD-ELSD characteristic map obtained by the method of claim 1.
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