CN107764908B - Method for measuring content of alkaloid components in blood-nourishing and brain-refreshing water extract - Google Patents
Method for measuring content of alkaloid components in blood-nourishing and brain-refreshing water extract Download PDFInfo
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Abstract
The invention relates to a method for measuring the content of alkaloid components in a blood-nourishing and brain-refreshing water extract, which comprises the following steps: the quality control method has the advantages that the preparation of the test solution, the preparation of the reference solution and the content measurement can effectively remove interfering components, meanwhile, the alkaloid components can be effectively enriched, the method is efficient and rapid, good in stability and high in sensitivity, the effective separation of the four alkaloid components can be realized within 5 minutes, the quality control method is proved to be good in precision, reproducibility and stability, and the alkaloid components in the blood-nourishing brain-refreshing water extract can be effectively measured.
Description
Technical Field
The invention relates to a method for measuring the content of active ingredients in a traditional Chinese medicine extract, in particular to a method for measuring the content of alkaloid ingredients in a blood-nourishing and brain-refreshing water extract.
Background
The blood-nourishing and brain-refreshing granules and the blood-nourishing and brain-refreshing pills are compound traditional Chinese medicines prepared from eleven traditional Chinese medicines of angelica, ligusticum wallichii, radix paeoniae alba, prepared rehmannia root, uncaria, caulis spatholobi, selfheal, semen cassiae, nacre, rhizoma corydalis and asarum and auxiliary materials. The water extract for nourishing blood and refreshing brain is the intermediate of the preparation of blood-nourishing and refreshing granule and pill. The preparation process comprises the following steps: decocting radix rehmanniae Preparata, ramulus Uncariae cum uncis, caulis Spatholobi, Prunellae Spica, Concha Margaritifera, and herba asari in water, filtering, concentrating, adding ethanol, standing, filtering, recovering ethanol, and concentrating to appropriate amount to obtain water extract for nourishing blood and refreshing brain.
The Chinese pharmacopoeia 2015 edition discloses content detection of two preparations for nourishing blood and refreshing brain, which are used for detecting paeoniflorin, and the preparation method of the test sample comprises the following steps: taking the content of the product, grinding, taking about 0.08g, precisely weighing, placing in a 20ml beaker, adding 0.2% sodium bicarbonate, carrying out ultrasonic treatment for 5 minutes, passing through a D101 macroporous adsorption resin column, eluting with water, discarding the eluent, eluting with methanol, collecting the eluent, adding water to the scale, shaking up, centrifuging for 5 minutes, taking the supernatant, filtering, and taking the subsequent filtrate to obtain the product; the chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as filler, isopropanol-methanol citric acid solution (2: 18: 80) is used as mobile phase, the detection wavelength is 240nm, and the number of theoretical plates is not less than 2000 calculated according to paeoniflorin peak.
In the blood-nourishing and brain-refreshing preparation, uncaria plays an important role in the formula, has the efficacies of clearing heat and calming the liver, calming endogenous wind and arresting convulsion, and is matched with the efficacies of the blood-nourishing and brain-refreshing preparation, namely blood-nourishing and liver-calming, blood circulation promoting and collateral dredging. The alkaloid is the main active ingredient of the uncaria rhynchophylla medicinal material, and the detection of the uncaria rhynchophylla ingredient in the blood-nourishing and brain-refreshing preparation is not reported in pharmacopoeia.
The inventor carries out deep research on alkaloid components in the blood-nourishing and brain-refreshing water extract in the early period, establishes a separation and purification method of the alkaloid components by utilizing an SPE-UPLC method, and realizes effective separation of the alkaloid components. The alkaloid components in the blood-nourishing and brain-refreshing water extract are structurally identified by adopting a series quadrupole time-of-flight mass spectrometer (Q-TOF/MS) LC MS, and the alkaloid components mainly containing four alkaloid components, namely rhynchophylline, isocoubine, corynoxeine and isocoubine, are determined by analyzing primary fragments and secondary fragments and comparing reference substances.
A whole set of comprehensive process quality control system is established for the blood-nourishing and brain-refreshing water extract, but no quality analysis method for alkaloid components exists.
In order to further improve the quality control level of the product and comprehensively master the product quality, the inventor establishes a content determination method for alkaloid components in the blood-nourishing brain-refreshing water extract and performs methodology verification.
Disclosure of Invention
The invention relates to a method for measuring the content of alkaloid components in a blood-nourishing and brain-refreshing water extract, which adopts ultra-high performance liquid chromatography.
The method comprises the following steps: preparing a test solution, preparing a reference solution and measuring the content of the reference solution.
Wherein, the preparation of the test solution comprises the following steps: taking about 0.2g of blood-nourishing and brain-refreshing water extract, precisely weighing, ultrasonically dissolving with 10mL of 35-45% methanol, and adding into treated ProElut C18-U (500mg/6mL) column, washing with 5mL of 35% -45% methanol, discarding the washing solution, eluting with 4.5mL of methanol, collecting the eluent, diluting to 5mL with methanol, and shaking up to obtain the final product;
wherein the preparation of the reference solution comprises the following steps:
preparing a solution containing 0.008-0.012mg, 0.01-0.02mg, 0.001-0.002mg and 0.004-0.006mg of rhynchophylline, isorhynchophylline, corynoxeine and isocorynoxeine per milliliter by using rhynchophylline, isocorynoxeine, corynoxeine and isocorynoxeine as reference substances and using methanol to obtain the solution;
wherein, the content determination comprises the following steps: respectively taking 1-3ul of each of the reference solution and the test solution, injecting into a liquid chromatograph, recording peak areas, and calculating the content of rhynchophylline, isocoumarine, corynoxeine and isocoumarine in the test solution by an external standard method;
wherein, the chromatographic conditions of the liquid chromatograph in the content detection are as follows: chromatographic column ACQUITY UPLC CSH C18(2.1X 100mm), gradient elution, with mobile phase: the mobile phase A is 0.02-0.2% ammonium acetate water solution, and the mobile phase B is acetonitrile; flow rate: 0.35-0.45 ml/min; detection wavelength: 240 and 250 nm.
The ammonium acetate of the mobile phase is preferably 0.02-01%.
Preferably, the method of the present invention comprises the steps of:
step 1: preparation of control solutions
Accurately weighing appropriate amount of rhynchophylline, isocoumarin, corynoxeine and isocoumarin, respectively placing in volumetric flasks, adding methanol for dissolving, and respectively preparing solutions containing rhynchophylline, isocoumarin, corynoxeine and isocoumarin of 0.01mg, 0.015mg, 0.0016mg and 0.005mg per ml to obtain the final product;
step 2: preparation of test solution
Taking about 0.2g of blood-nourishing and brain-refreshing water extract, precisely weighing, ultrasonically dissolving with 10mL of 35-45% methanol, and adding into treated ProElut C18-U (500mg/6mL) column, washing with 5mL of 35% -45% methanol, discarding the washing solution, eluting with 4.5mL of methanol, collecting the eluent, diluting to 5mL with methanol, and shaking up to obtain the final product;
and step 3: assay method
Respectively taking 2ul of reference solution and sample solution, injecting into a liquid chromatograph, recording peak area, and calculating content by external standard method;
the chromatographic conditions of the liquid chromatograph are as follows:
chromatographic column ACQUITY UPLC CSH C18(2.1X 100mm), mobile phase: the mobile phase A is 0.02-0.2% ammonium acetate aqueous solution, the mobile phase B is acetonitrile, and the gradient elution procedure is shown in table 1; flow rate: 0.35-0.45ml/min, detection wavelength: 240-250 nm;
table 1 mobile phase gradient procedure
Most preferably, the method of the present invention comprises the steps of:
step 1: preparation of control solutions
Accurately weighing appropriate amount of rhynchophylline, isocoumarin, corynoxeine and isocoumarin, respectively placing in volumetric flasks, adding methanol for dissolving, and respectively preparing solutions containing rhynchophylline, isocoumarin, corynoxeine and isocoumarin of 0.01mg, 0.015mg, 0.0016mg and 0.005mg per ml to obtain the final product;
step 2: preparation of test solution
Taking about 0.2g of blood-nourishing and brain-refreshing water extract, precisely weighing, ultrasonically dissolving with 10mL of 40% methanol, and adding into treated ProElut C18-U (500mg/6mL) column, washing with 5mL of 40% methanol, discarding the washing solution, eluting with 4.5mL of methanol, collecting the eluent, diluting to 5mL with methanol, and shaking up to obtain the final product;
and step 3: assay method
Respectively taking 2ul of reference solution and sample solution, injecting into a liquid chromatograph, recording peak area, and calculating content by external standard method;
the chromatographic conditions of the liquid chromatograph are as follows:
chromatographic column ACQUITY UPLC CSH C18(2.1X 100mm), mobile phase: mobile phase a was 0.05% ammonium acetate aqueous solution, mobile phase B was acetonitrile, and the gradient elution procedure is shown in table 1; flow rate: 0.35-0.45ml/min, detection wavelength: 246 nm.
The method provided by the invention comprises the following steps:
the methanol concentration is not limited and is defined as 100% methanol, 40% methanol, specified in pharmacopoeia, wherein the percentage means volume percentage, for example, 40ml methanol in 100ml solution.
The preferred method and chromatographic conditions of the invention are obtained by screening, and the screening process is as follows:
1. establishment of pretreatment method
1.1 selection of test sample solvent
Taking 0.2g of blood-nourishing and brain-refreshing water extract, performing solid phase extraction with 20% methanol, 30% methanol, 40% methanol and 50% methanol respectively, and performing sample injection, wherein the chromatogram is shown in figure 3 (figure 3 is UPLC spectra obtained by dissolving samples with different solvents, wherein A is the UPLC spectrum obtained by dissolving a sample with 20% methanol, B is the UPLC spectrum obtained by dissolving the sample with 30% methanol, C is the UPLC spectrum obtained by dissolving the sample with 40% methanol, and D is the UPLC spectrum obtained by dissolving the sample with 50% methanol).
From the chromatogram, it is clear that the sample dissolved in 50% methanol has a leak, and the peak area is smaller than that of 40% methanol. The 20% and 30% methanol-solubilized samples did not dissolve alkaloids well and the recovery rate was low in addition to the turbidity of the sample solution. For comprehensive consideration, it is appropriate to select a sample dissolved with 40% methanol.
1.2 selection of SPE cartridges
Respectively adopting selection ProElut C18-U、ProElut C18The ProElut PXW solid phase extraction column purifies the sample solution, and the result shows that ProElut C18The selectivity and the recovery rate of the-U solid phase extraction column are optimal. ProElut C18The column U is a silica gel bond C18Reversed phase C without subsequent capping18And (4) an extraction column. The residual silicon hydroxyl on the surface of the silica gel substrate has polar interaction with polar compounds, so that the retention of the polar compounds, particularly amine compounds, is enhanced, and the method is suitable for extracting polar and non-polar compounds. So the inventor chooses ProElut C18-a U SPE column.
1.2.1 SPE sample application speed selection
Weighing 0.2g of blood nourishing and brain refreshing water extract, ultrasonic dissolving with 10mL of 40% methanol, and respectively activating medical methanol and water activated ProElut C at flow rates of 0.5mL/min, 1.0mL/min and 1.5mL/min18the-U SPE column was loaded and methanol eluted to collect five ml. The recovery rates are respectively measured, and the recovery rates of the rhynchophylline are 100.5%, 100.0% and 94.5%; the recovery rate of isorhynchophylline is 100.2%, 100.8% and 96.7%. The sample loading speed is preferably 1.0 mL/min.
1.2.2 selection of the wash volume on SPE
Weighing 0.2g of extract of blood nourishing and brain refreshing water, ultrasonic dissolving in 10mL of 40% methanol, and adding into ProElut C activated with methanol and water at flow rate of 1.0mL/min18the-U SPE cartridge was loaded and eluted with methanol to collect 3mL, 5mL, 6mL, 10mL, respectively. The recovery rates of the rhynchophylline are respectively measured, and the recovery rates of the rhynchophylline are 96.5%, 99.3%, 99.5% and 99.4%; the recovery rate of isorhynchophylline is 95.2%, 100.5%, 100.9% and 101.2%. To illustrate that 5mL is sufficient to elute the sample and save elution time, an elution volume of 5mL was chosen.
2.2 chromatographic conditions
2.2.1 column selection
Respectively adopted is ACQUITY UPLC CSH C18And an ACQUITY UPLC HSS T3 column.
FIG. 4 (FIG. 4 is a UPLC profile using an ACQUITY UPLC HST 3 column) and FIG. 5 (FIG. 5 is a UPLC CSH C using ACQUITY UPLC18UPLC spectrum of chromatographic column) shows that, ACQUITY UPLC CSH C18The column has good resolution and good peak shape, so the ACQUITY UPLC CSH C is selected18A chromatographic column.
2.2.2 selection of wavelength
The mixed standard was prepared according to the method in "2.1.1", and uv-scanned to obtain a spectrum, and the results are shown in fig. 5.
As shown in fig. 6 (fig. 6 is an ultraviolet scan of a mixed reference product of rhynchophylline and isocorynine), since the maximum ultraviolet absorption is observed at 246nm, 246nm was selected as the detection wavelength in the experiment.
2.2.3 selection of the proportions of the mobile phases
Taking 0.2g of blood-nourishing and brain-clearing water extract, preparing the extract according to the method under the item of '2.1.6', and respectively using a mobile phase acetonitrile solution (A) and a 0.05% ammonium acetate aqueous solution (B) according to the initial proportion of the mobile phase of 25: 75. 35: 65. 35: 55, 2 mu L of sample is injected by a chromatographic method, and a chromatogram is shown in figure 7 (figure 7 is UPLC spectra of blood-nourishing and brain-refreshing water extract under three initial proportions of mobile phases, wherein A is the UPLC spectrum of the blood-nourishing and brain-refreshing water extract under the initial proportion of the mobile phase of 45: 55, B is the UPLC spectrum of the blood-nourishing and brain-refreshing water extract under the initial proportion of the mobile phase of 35: 65, and C is the UPLC spectrum of the blood-nourishing and brain-refreshing water extract under the initial proportion of the mobile phase of 25: 75).
The results in fig. 7 show that when the mobile phase ratio is 35: at 65, the best separation was achieved with a smooth baseline.
2.3 structural confirmation of the detection component
The method comprises the following steps of (1) measuring a sample by using a UPLC-Q-TOF/MS combined instrument, wherein the mass spectrum condition is a flight tube detection mode V type, a positive ion (ESI +) scanning mode is adopted, the capillary voltage is 3.5/kV (+), the cone hole voltage is 4/30V (+), the extraction cone hole voltage is 5.0V, the ion source temperature is 100 ℃, the desolvation gas temperature is 500 ℃, the cone hole air flow is 1L/h, the desolvation gas is 594L/h, the Resolution ratio is a Resolution mode, the collision cell inlet voltage is 25V, the collision cell outlet voltage is 60V, the scanning time is 11min, the scanning time interval is 0.3s, and the mass-to-charge ratio range: m/z is 50-1200; a data acquisition form Centroid; sensitivity Normal; the locked mass number POS 556.2771 the sample was prepared under the heading "2.1.6" and the conditions of the liquid phase were as defined in the heading "2.2". Obtaining mass spectrum information (figure 8 is a total ion flow diagram of the extract of blood-nourishing and brain-refreshing water extracts).
Through comparison and analysis with the reference quality spectrum data, the extract for nourishing blood and refreshing brain is determined to mainly contain four alkaloid components, namely rhynchophylline, isocoumarin, corynoxeine and isocoumarin.
2.4 methodological validation
2.4.1 Linearity and Range
Accurately weighing appropriate amount of rhynchophylline, isocourine, corynoxeine and isocourine reference substances, measuring peak areas, and determining reference substance maps shown in figure 2 (because there is interference between the reference substances, there are two reference substance maps, namely figures 2-1 and 2-2, figure 2-1 is corynoxeine and isocourine, and figure 2-2 is corynoxeine and isocourine). The method for measuring the peak area is as follows: adding methanol to prepare a mixed control solution containing 0.1mg, 0.04g, 0.08g and 0.1mg of rhynchophylline, isocorynoxeine and isocorynoxeine in each milliliter of reference substance, respectively measuring 0.5, 1, 2, 5 and 10ml of the mixed control solution, placing the mixed control solution in a 50ml measuring flask, adding methanol to the scale, uniformly mixing to prepare a series of solutions, injecting the solutions into a liquid chromatograph, measuring, and performing linear regression on the measured peak area according to the concentration to obtain a regression equation. The results are shown in table 2, fig. 9 (fig. 8 shows a rhynchophylline linear relationship), table 3, fig. 10 (fig. 9 shows an isocoumarin linear relationship), table 4, fig. 11 (fig. 11 shows a rhynchophylline linear relationship), table 5, and fig. 12 (fig. 12 shows an isocoumarin linear relationship).
TABLE 2 Linear relationship of rhynchophylline
| Concentration (mg/ml) | 0.001028 | 0.002056 | 0.004112 | 0.008224 | 0.02056 |
| Peak area | 9185 | 21377 | 43923 | 50960 | 100892 |
TABLE 3 Linear relationship of isocoumarines
| Concentration (mg/ml) | 0.00405 | 0.0081 | 0.01619 | 0.04048 | 0.08096 |
| Peak area | 4643 | 10937 | 21940 | 26222 | 51814 |
TABLE 4 Linear relationship of rhynchophylline
| Concentration (mg/ml) | 0.04106 | 0.08212 | 0.16424 | 0.32848 | 0.8212 |
| Peak area | 10040 | 23143 | 46188 | 54043 | 107382 |
TABLE 5 Linear Isodehydrorhynchophylline relationship
| Concentration (mg/ml) | 0.00716 | 0.01431 | 0.03578 | 0.07155 | 0.1431 |
| Peak area | 13614 | 31705 | 62675 | 73378 | 145242 |
2.4.2 sample injection precision test:
taking samples of the batch D20140204, preparing a test solution according to the method, repeatedly injecting samples for 6 times, recording peak areas of rhynchophylline, isocorynoxeine, corynoxeine and isocorynoxeine, and calculating relative standard deviation. The results are shown in Table 6.
TABLE 6 precision of injection of rhynchophylline, isocoumarine, rhynchophylline and isocoumarine
2.4.3 reproducibility test:
taking samples of batch D20140204, preparing 6 parts of test solution according to a method, respectively measuring the contents of rhynchophylline, isocorynoxeine, corynoxeine and isocorynoxeine, and calculating relative standard deviation. The results are shown in Table 7.
TABLE 7 reproducibility of rhynchophylline, isocoumarin, rhynchophylline and isocoumarin
2.4.4 recovery test:
precisely weighing 6 parts of the D20140204 batch of extract, each part is about 0.1g, placing the 6 parts into a 50ml measuring flask, respectively and precisely adding 2ml of mixed reference substance solution (each ml contains 0.01384mg, 0.0936mg, 0.0631mg and 0.1618mg of rhynchophylline, isocorynine, corynoxeine and isocorynoxeine), treating the mixture according to the method under the content determination item from the point of adding a proper amount of 70% methanol solution and ultrasonically dissolving the mixture to obtain a test solution, injecting 2ul of the test solution into a chromatograph, and respectively calculating the recovery rates of the rhynchophylline, the isocorynine, the corynoxeine and the isocorynoxeine. The results are shown in tables 8, 9, 10 and 11.
Table 8 recovery of rhynchophylline (mg, n ═ 6)
Table 9 isorhynchophylline recovery (mg, n ═ 6)
TABLE 10 recovery of rhynchophylline (mg, n ═ 6)
TABLE 11 recovery of isocorynoxeine (mg, n ═ 6)
2.4.5 stability
Preparing a test solution from batch D20140204, injecting samples for 0, 2, 4, 8, 12 and 24 hours respectively, recording peak areas of rhynchophylline, isorhynchophylline, corynoxeine and isorhynchophylline, and calculating relative standard deviation. The results are shown in Table 12.
TABLE 12 reproducibility of rhynchophylline, isocoumarin, rhynchophylline and isocoumarin
2.4.6 sample determination
Samples were taken and the test sample profile was determined according to the method provided by the present invention (see FIG. 1).
And (5) verifying and concluding:
the method for simultaneously measuring four components of rhynchophylline, isocorynoxeine, corynoxeine, isocorynoxeine and the like in the blood-nourishing and brain-refreshing water extract is verified by methodology, and the result shows that the measuring method has good linear relation and recovery rate, the precision, the reproducibility and the sample stability meet the verification requirements, and can be used for measuring alkaloid components in the blood-nourishing and brain-refreshing water extract.
2.5 detection Condition Range
Test sample solvent: 35-45% methanol solution; mobile phase solvent a: 0.02-0.2% ammonium acetate aqueous solution; solid phase extraction column: available Waters Oasis MAX, Cleanert S C18And substituting the equal type numbers.
The invention has the advantages of
1. The method adopts the ultra-high performance liquid chromatography for determination, has high efficiency, high speed, good stability and high sensitivity, and can realize effective separation of four alkaloid components within 5 minutes.
2. The invention adopts a solid phase extraction technology, can effectively remove interfering components and can effectively enrich alkaloid components.
3. The prior art is as follows:
CN103630614A (application No. 201210301559.9, hereinafter abbreviated as 2012 patent) discloses a high performance liquid chromatography detection method for serum-nourishing brain particles, which comprises the following steps: the sample is prepared by ultrasonic extraction with 25% methanol as solvent; the mobile phase A is 0.01-0.05% phosphoric acid solution and B is acetonitrile in the chromatographic condition. Compared with the 2012 patent: the main difference in this document is the difference in the components measured, which are: gallic acid, protocatechuic acid, chlorogenic acid, caffeic acid, ferulic acid, paeoniflorin, albiflorin, and the invention is directed to rhynchophylline, isocoumarine, corynoxeine and isocoumarine.
In the method for determining the content of rhynchophylline in samples of different extraction processes by using ultra-high performance liquid chromatography (Anemone chinensis, Oldham (Spirodela Lischneid), Wang Ying, university of capital university, No. 2 of 32), ACQUITY UPLC BEH C is adopted18The column was eluted isocratically with methanol-5 mmol/L ammonium acetate aqueous solution adjusted to pH 9.8 (60: 40) with ammonia as the mobile phase at a detection wavelength of 240nm, determined as rhynchophylline, and the peak time was 4.87 minutes. Compared with the method, the method has the advantages of higher analysis speed, better separation degree, more analysis components and simpler mobile phase composition.
The invention has the advantages that:
1) aims at the problems of complex components, more interfering substances and increased determination difficulty of compound preparations for nourishing blood and refreshing brain.
2) The method has the advantages that the four alkaloid components are detected simultaneously, the composition and content proportion of the alkaloid components are comprehensively mastered, the quick detection is realized, the separation difficulty is high, and the efficiency is high. Improves the quality control of the product and provides data support for the research of the pharmacodynamic ingredients of the product.
3) In the preparation of the test sample: ultrasonically dissolving with methanol, and dissolving with ProElut C18And the-U solid phase extraction column is used for purification, so that interference impurities are effectively removed, enrichment of a measured target substance is realized, the chromatographic peak separation degree is improved, and the service life of the chromatographic column is prolonged.
4) The selection of the mobile phase in the chromatographic condition, the screening through a chromatographic column and the mobile phase and the composition through a simple mobile phase achieve perfect peak shape and high resolution, and overcome the defect that the alkaloid is easy to tailing in the reverse phase chromatographic separation.
5. The quality control method provided by the invention has good precision, reproducibility and stability, and can effectively measure the alkaloid components in the blood-nourishing and brain-refreshing water extract.
Drawings
FIG. 1 is a sample map;
FIGS. 2-1 and 2-2 are maps of reference substances;
fig. 3 is a UPLC spectrum obtained by dissolving a sample in different solvents, wherein a: dissolving a sample in 20% methanol to obtain a UPLC spectrum; b: dissolving a sample in 30% methanol to obtain a UPLC spectrum; c: dissolving a sample with 40% methanol to obtain a UPLC spectrum; d: dissolving a sample in 50% methanol to obtain a UPLC spectrum;
FIG. 4 is a UPLC spectrum using an ACQUITY UPLC HST 3 column;
FIG. 5 shows a CSH C using ACQUITY UPLC18A UPLC spectrum of a chromatographic column;
FIG. 6 is a UV scanning chart of a reference substance of rhynchophylline and isocorynine;
fig. 7 is UPLC spectra of blood-nourishing and brain-refreshing water extracts at three initial proportions of mobile phases, wherein a: initial mobile phase ratio of 45: UPLC spectrum of the blood-nourishing and brain-refreshing water extract under 55 deg.C; b: initial mobile phase ratio was 35: UPLC spectrum of the blood-nourishing and brain-refreshing water extract under 65 deg.C; c: initial mobile phase ratio 25: UPLC spectrum of water extract for nourishing blood and refreshing brain under 75 deg.C;
FIG. 8 is a total ion flow diagram of a blood-nourishing brain-refreshing water extract;
FIG. 9 is a rhynchophylline linear relationship;
FIG. 10 is a linear relationship of isorhynchophylline;
FIG. 11 is a linear relationship for corynoxeine;
FIG. 12 is a linear isocorynoxeine relationship.
Detailed Description
The invention is further illustrated by the following examples.
Example 1: method for measuring content of alkaloid components in blood-nourishing and brain-refreshing water extract
1. Chromatographic conditions and system suitability test: the apparatus is Waters ACQUITY UPLC, and ACQUITY UPLC CSH C is adopted18(2.1X 100mm) column, mobile phase A with 0.05% aqueous ammonium acetate and mobile phase B with acetonitrile, gradient setup as shown in Table 1; the detection wavelength was 246 nm.
2. Preparation of control solutions: accurately weighing appropriate amount of rhynchophylline, isocoumarin, corynoxeine and isocoumarin, respectively placing in volumetric flasks, adding methanol for dissolving, and respectively preparing solutions containing rhynchophylline, isocoumarin, corynoxeine and isocoumarin of 0.01mg, 0.015mg, 0.0016mg and 0.005mg per ml to obtain the final product.
3. Preparation of a test solution: taking about 0.2g of blood-nourishing and brain-refreshing water extract, precisely weighing, ultrasonically dissolving with 10mL of 40% methanol, and adding into treated ProElut C18Washing a U (500mg/6mL) column with 5mL of 40% methanol, discarding a washing solution, eluting with 4.5mL of methanol, collecting an eluent, metering the volume to 5mL with methanol, and shaking up to obtain the compound.
4. The determination method comprises the following steps: respectively taking 2ul of reference solution and sample solution, injecting into liquid chromatograph, recording peak area, and calculating content by external standard method.
5. And (3) detection results: three batches of on-line blood-nourishing and brain-refreshing water extracts are taken for determination, and the results are shown in Table 13.
TABLE 13 content of alkaloid component in blood-nourishing and brain-refreshing water extracts measured by the method of example 1
| Number (C) | Rhynchophylline | Isorhynchophylline | | Isodehydrorhynchophylline | |
| 1 | 0.264 | 0.095 | 0.040 | 0.152 | |
| 2 | 0.246 | 0.083 | 0.032 | 0.130 | |
| 3 | 0.239 | 0.086 | 0.041 | 0.149 |
Example 2: method for measuring content of alkaloid components in blood-nourishing and brain-refreshing water extract
1. Chromatographic conditions and system suitability test: the apparatus is Waters ACQUITY UPLC, and ACQUITY UPLC CSH C is adopted18(2.1X 100mm) column, mobile phase A with 0.1% aqueous ammonium acetate and mobile phase B with acetonitrile, gradient setup as shown in Table 1; the detection wavelength was 240nm.
2. Preparation of control solutions: accurately weighing appropriate amount of rhynchophylline, isocoumarin, corynoxeine and isocoumarin, respectively placing in volumetric flasks, adding methanol for dissolving, and respectively preparing solutions containing rhynchophylline, isocoumarin, corynoxeine and isocoumarin of 0.01mg, 0.015mg, 0.0016mg and 0.005mg per ml to obtain the final product.
3. Preparation of a test solution: taking about 0.2g of blood-nourishing and brain-refreshing water extract, precisely weighing, ultrasonically dissolving with 10mL of 40% methanol, and adding into treated ProElut C18-U (500mg/6mL) column. Washing with 5mL of 40% methanol, discarding washing liquid, eluting with 4.5mL of methanol, collecting eluent, diluting to 5mL with methanol, and shaking up to obtain the final product.
4. The determination method comprises the following steps: respectively taking 2ul of each of the reference solution and the sample solution, injecting into a liquid chromatograph, and recording peak area. And calculating the content by an external standard method.
5. And (3) detection results: three batches of on-line blood-nourishing and brain-refreshing water extracts are taken for determination, and the results are shown in table 14:
TABLE 14 content of alkaloid component in the blood-nourishing and brain-refreshing aqueous extract of example 2
| Serial number | Rhynchophylline | Isorhynchophylline | | Isodehydrorhynchophylline | |
| 1 | 0.258 | 0.097 | 0.041 | 0.155 | |
| 2 | 0.241 | 0.085 | 0.033 | 0.125 | |
| 3 | 0.245 | 0.084 | 0.041 | 0.146 |
Example 3: method for measuring content of alkaloid components in blood-nourishing and brain-refreshing water extract
1. Chromatographic conditions and system suitability test:
the apparatus is Waters ACQUITY UPLC, and ACQUITY UPLC CSH C is adopted18(2.1X 100mm) column, mobile phase A with 0.02% aqueous ammonium acetate and mobile phase B with acetonitrile, gradient setup as shown in Table 1; the detection wavelength was 250 nm.
2. Preparation of control solutions: accurately weighing appropriate amount of rhynchophylline, isocoumarin, corynoxeine and isocoumarin, respectively placing in volumetric flasks, adding methanol for dissolving, and respectively preparing solutions containing rhynchophylline, isocoumarin, corynoxeine and isocoumarin of 0.01mg, 0.015mg, 0.0016mg and 0.005mg per ml to obtain the final product.
3. Preparation of a test solution: taking about 0.2g of blood-nourishing and brain-refreshing water extract, precisely weighing, ultrasonically dissolving with 10mL of 40% methanol, and adding into treated ProElut C18-U (500mg/6mL) column. Washing with 5mL of 40% methanol, discarding washing liquid, eluting with 4.5mL of methanol, collecting eluent, diluting to 5mL with methanol, and shaking up to obtain the final product.
4. The determination method comprises the following steps: respectively taking 2ul of each of the reference solution and the sample solution, injecting into a liquid chromatograph, and recording peak area. And calculating the content by an external standard method.
5. And (3) detection results: three batches of on-line blood-nourishing and brain-refreshing water extracts are taken for determination, and the results are shown in table 15:
TABLE 15 content of alkaloid component in the blood-nourishing and brain-refreshing aqueous extract of example 3
| Serial number | Rhynchophylline | Isorhynchophylline | | Isodehydrorhynchophylline | |
| 1 | 0.260 | 0.096 | 0.040 | 0.156 | |
| 2 | 0.247 | 0.084 | 0.033 | 0.127 | |
| 3 | 0.240 | 0.081 | 0.040 | 0.143 |
Example 4: method for measuring content of alkaloid components in blood-nourishing and brain-refreshing water extract
1. Chromatographic conditions and system suitability test:
the apparatus is Waters ACQUITY UPLC, and ACQUITY UPLC CSH C is adopted18(2.1X 100mm) column, mobile phase A with 0.05% aqueous ammonium acetate and mobile phase B with acetonitrile, gradient setup as shown in Table 1; the detection wavelength was 246 nm.
2. Preparation of control solutions: accurately weighing appropriate amount of rhynchophylline, isocoumarin, corynoxeine and isocoumarin, respectively placing in volumetric flasks, adding methanol for dissolving, and respectively preparing solutions containing rhynchophylline, isocoumarin, corynoxeine and isocoumarin of 0.01mg, 0.015mg, 0.0016mg and 0.005mg per ml to obtain the final product.
3. Preparation of a test solution: taking about 0.2g of blood-nourishing and brain-clearing water extract, precisely weighing, ultrasonically dissolving with 10mL of 45% methanol, and adding into the processed Cleanert S C18(500mg/6mL) column. Washing with 5mL of 45% methanol, discarding washing liquid, eluting with 4.5mL of methanol, collecting eluent, diluting to 5mL with methanol, and shaking up to obtain the final product.
4. The determination method comprises the following steps: respectively taking 2ul of reference solution and sample solution, injecting into liquid chromatograph, recording peak area, and calculating content by external standard method.
5. And (3) detection results: three batches of on-line blood-nourishing and brain-refreshing water extracts are taken for determination, and the results are shown in table 16:
TABLE 16 content of alkaloid component in the blood-nourishing and brain-refreshing aqueous extract of example 4
| Serial number | Rhynchophylline | Isorhynchophylline | | Isodehydrorhynchophylline | |
| 1 | 0.261 | 0.093 | 0.041 | 0.153 | |
| 2 | 0.244 | 0.084 | 0.033 | 0.129 | |
| 3 | 0.237 | 0.088 | 0.042 | 0.147 |
Example 5: method for measuring content of alkaloid components in blood-nourishing and brain-refreshing water extract
1. Chromatographic conditions and system suitability test:
the apparatus is Waters ACQUITY UPLC, and ACQUITY UPLC CSH C is adopted18(2.1X 100mm) column, mobile phase A with 0.05% aqueous ammonium acetate and mobile phase B with acetonitrile, gradient setup as shown in Table 1; the detection wavelength was 246 nm.
2. Preparation of control solutions: accurately weighing appropriate amount of rhynchophylline, isocoumarin, corynoxeine and isocoumarin, respectively placing in volumetric flasks, adding methanol for dissolving, and respectively preparing solutions containing rhynchophylline, isocoumarin, corynoxeine and isocoumarin of 0.01mg, 0.015mg, 0.0016mg and 0.005mg per ml to obtain the final product.
3. Preparation of a test solution: taking about 0.25g of blood-nourishing and brain-clearing water extract, precisely weighing, ultrasonically dissolving with 10mL of 35% methanol, and adding into treated Waters Sep-pak C18(500mg/6mL) column. Washing with 5mL of 35% methanol, discarding washing liquid, eluting with 4.5mL of methanol, collecting eluent, diluting to 5mL with methanol, and shaking up to obtain the final product.
4. The determination method comprises the following steps: respectively taking 2ul of reference solution and sample solution, injecting into liquid chromatograph, recording peak area, and calculating content by external standard method.
5. And (3) detection results: three batches of on-line blood-nourishing and brain-refreshing water extracts are taken for determination, and the results are shown in table 17:
TABLE 17 content of alkaloid component in the blood-nourishing and brain-refreshing aqueous extract of example 5
| Serial number | Rhynchophylline | Isorhynchophylline | | Isodehydrorhynchophylline | |
| 1 | 0.265 | 0.093 | 0.041 | 0.151 | |
| 2 | 0.251 | 0.082 | 0.032 | 0.132 | |
| 3 | 0.237 | 0.081 | 0.041 | 0.146 |
Example 6
1. Chromatographic conditions and system suitability test:
the apparatus is Waters ACQUITY UPLC, and ACQUITY UPLC CSH C is adopted18(2.1X 100mm) column, mobile phase A with 0.05% aqueous ammonium acetate and mobile phase B with acetonitrile, gradient setup as shown in Table 1; the detection wavelength was 246 nm.
2. Preparation of control solutions: accurately weighing appropriate amount of rhynchophylline, isocoumarin, corynoxeine and isocoumarin, respectively placing in volumetric flasks, adding methanol for dissolving, and respectively preparing solutions containing rhynchophylline, isocoumarin, corynoxeine and isocoumarin of 0.01mg, 0.015mg, 0.0016mg and 0.005mg per ml to obtain the final product.
3. Preparation of a test solution: taking about 0.15g of blood-nourishing and brain-clearing water extract, precisely weighing, ultrasonically dissolving with 10mL of 40% methanol, and adding into treated Waters Sep-pak C18(500mg/6mL) column. Washing with 5mL of 40% methanol, discarding washing liquid, eluting with 4.5mL of methanol, collecting eluent, diluting to 5mL with methanol, and shaking up to obtain the final product.
4. The determination method comprises the following steps: respectively taking 2ul of each of the reference solution and the sample solution, injecting into a liquid chromatograph, and recording peak area. And calculating the content by an external standard method.
5. And (3) detection results: three batches of on-line blood-nourishing and brain-refreshing water extracts are taken for determination, and the results are shown in table 18:
TABLE 18 content of alkaloid component in the blood-nourishing and brain-refreshing aqueous extracts of EXAMPLE 6
| Serial number | Rhynchophylline | Isorhynchophylline | | Isodehydrorhynchophylline | |
| 1 | 0.255 | 0.094 | 0.038 | 0.154 | |
| 2 | 0.242 | 0.085 | 0.032 | 0.131 | |
| 3 | 0.232 | 0.088 | 0.040 | 0.146 |
A summary of all the above example data is provided in tables 19-1, 19-2, 19-3:
table 19-1: 01 batch of test data
| Serial number | Rhynchophylline | Isorhynchophylline | Corynoxeine hydrochloride | Isodehydrorhynchophylline |
| Example 1 | 0.264 | 0.095 | 0.040 | 0.152 |
| Example 2 | 0.258 | 0.097 | 0.041 | 0.155 |
| Example 3 | 0.260 | 0.096 | 0.040 | 0.156 |
| Example 4 | 0.261 | 0.093 | 0.041 | 0.153 |
| Example 5 | 0.265 | 0.093 | 0.041 | 0.151 |
| Example 6 | 0.255 | 0.094 | 0.038 | 0.154 |
| Mean value of | 0.261 | 0.095 | 0.040 | 0.154 |
| RSD％ | 1.4 | 1.7 | 1.9 | 1.2 |
Table 19-2: 02 lot test data
| Serial number | Rhynchophylline | Isorhynchophylline | Corynoxeine hydrochloride | Isodehydrorhynchophylline |
| Example 1 | 0.246 | 0.083 | 0.032 | 0.130 |
| Example 2 | 0.241 | 0.085 | 0.033 | 0.125 |
| Example 3 | 0.247 | 0.084 | 0.033 | 0.127 |
| Example 4 | 0.244 | 0.084 | 0.033 | 0.129 |
| Example 5 | 0.251 | 0.082 | 0.032 | 0.132 |
| Example 6 | 0.242 | 0.085 | 0.032 | 0.131 |
| Mean value of | 0.245 | 0.084 | 0.033 | 0.129 |
| RSD％ | 1.5 | 1.4 | 1.7 | 2.0 |
Tables 19 to 3: 03 batch of test data
| Serial number | Rhynchophylline | Isorhynchophylline | Corynoxeine hydrochloride | Isodehydrorhynchophylline |
| Example 1 | 0.239 | 0.086 | 0.041 | 0.149 |
| Example 2 | 0.245 | 0.084 | 0.041 | 0.146 |
| Example 3 | 0.240 | 0.086 | 0.040 | 0.143 |
| Example 4 | 0.237 | 0.088 | 0.042 | 0.147 |
| Example 5 | 0.237 | 0.085 | 0.041 | 0.146 |
| Example 6 | 0.232 | 0.088 | 0.040 | 0.146 |
| Mean value of | 0.238 | 0.086 | 0.041 | 0.146 |
| RSD％ | 1.8 | 1.9 | 1.8 | 1.3 |
The results in tables 19-1, 19-2 and 19-3 show that the results of the above three batches have no significant difference and all can obtain correct results, as determined by the embodiments in the examples.
Claims (4)
1. A method for measuring the content of alkaloid components in a blood-nourishing brain-refreshing water extract comprises the following steps: preparing a test solution, preparing a reference solution and measuring the content of the reference solution,
wherein, the preparation of the test solution comprises the following steps: taking about 0.2g of blood-nourishing and brain-refreshing water extract, precisely weighing, ultrasonically dissolving with 10mL of 35-45% methanol, and adding into processed ProElut C with specification of 500mg/6mL18A U column is washed by 5mL of 35-45% methanol, washing liquid is discarded, 4.5mL of methanol is used for elution, eluent is collected, the volume is determined to be 5mL by methanol, and the mixture is shaken up to obtain the compound preparation,
wherein the preparation of the reference solution comprises the following steps: taking rhynchophylline, isocoumarin, corynoxeine and isocoumarin as reference substances, preparing a solution containing 0.008-0.012mg, 0.01-0.02mg, 0.001-0.002mg and 0.004-0.006mg of rhynchophylline, isocoumarin, corynoxeine and isocoumarin per milliliter with methanol to obtain the final product,
wherein, the content determination comprises the following steps: respectively taking 1-3ul of each of the reference solution and the test solution, injecting into a liquid chromatograph, recording peak area, calculating the content of rhynchophylline, isocoumarin, corynoxeine and isocoumarin in the test solution by an external standard method,
the chromatographic conditions of the liquid chromatograph during the content detection are as follows: chromatographic column ACQUITY UPLC CSH C18Specification 2.1 × 100mm, gradient elution, with mobile phase: the mobile phase A is 0.02-0.2% ammonium acetate water solution, and the mobile phase B is acetonitrile; flow rate: 0.35-0.45 ml/min; detection wavelength: 240-250nm of the molecular weight of the silicon,
the gradient elution procedure was as follows:
2. the method according to claim 1, characterized in that the ammonium acetate of the mobile phase is preferably 0.02-0.1%.
3. Method according to claim 1, characterized in that it comprises the following steps:
step 1: preparation of control solutions
Accurately weighing appropriate amount of rhynchophylline, isocoumarin, corynoxeine and isocoumarin, respectively placing in volumetric flasks, adding methanol for dissolving, and respectively preparing solutions containing rhynchophylline, isocoumarin, corynoxeine and isocoumarin of 0.01mg, 0.015mg, 0.0016mg and 0.005mg per ml to obtain the final product;
step 2: preparation of test solution
Taking about 0.2g of blood-nourishing and brain-refreshing water extract, precisely weighing, ultrasonically dissolving with 10mL of 35-45% methanol, and adding into processed ProElut C with specification of 500mg/6mL18-U column, washing with 5mL of 35% -45% methanol, discarding washing solution, eluting with 4.5mL of methanol, collecting eluent, diluting to 5mL with methanol, and shaking up to obtain the final product;
and step 3: assay method
Respectively taking 2ul of reference solution and sample solution, injecting into a liquid chromatograph, recording peak area, and calculating content by external standard method;
the chromatographic conditions of the liquid chromatograph are as follows:
chromatographic column ACQUITY UPLC CSH C18Specification 2.1 × 100mm, mobile phase: the mobile phase A is 0.02-0.2% ammonium acetate aqueous solution, the mobile phase B is acetonitrile, and the gradient elution procedure is shown in the following table; flow rate: 0.35-0.45ml/min, detection wavelength: 240-250 nm;
4. method according to claim 1, characterized in that it comprises the following steps:
step 1: preparation of control solutions
Accurately weighing appropriate amount of rhynchophylline, isocoumarin, corynoxeine and isocoumarin, respectively placing in volumetric flasks, adding methanol for dissolving, and respectively preparing solutions containing rhynchophylline, isocoumarin, corynoxeine and isocoumarin of 0.01mg, 0.015mg, 0.0016mg and 0.005mg per ml to obtain the final product;
step 2: preparation of test solution
Taking about 0.2g of blood-nourishing and brain-refreshing water extract, precisely weighing, ultrasonically dissolving with 10mL of 40% methanol, and adding into processed ProElut C with specification of 500mg/6mL18-U column, washing with 5mL of 40% methanol, discarding washing solution, eluting with 4.5mL of methanol, collecting eluent, diluting to 5mL with methanol, and shaking up to obtain the final product;
and step 3: assay method
Respectively taking 2ul of reference solution and sample solution, injecting into a liquid chromatograph, recording peak area, and calculating content by external standard method;
the chromatographic conditions of the liquid chromatograph are as follows:
chromatographic column ACQUITY UPLC CSH C18Specification 2.1 × 100mm, mobile phase: the mobile phase A is 0.05% ammonium acetate water solution, the mobile phase B is acetonitrile, and the gradient elution procedure is shown in the following table; flow rate: 0.35-0.45ml/min, detection wavelength: 246 nm;
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