CN111505162A - Method for measuring content of 6 polyphenol compounds in compound red skin blood replenishing oral liquid - Google Patents

Method for measuring content of 6 polyphenol compounds in compound red skin blood replenishing oral liquid Download PDF

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CN111505162A
CN111505162A CN202010399615.1A CN202010399615A CN111505162A CN 111505162 A CN111505162 A CN 111505162A CN 202010399615 A CN202010399615 A CN 202010399615A CN 111505162 A CN111505162 A CN 111505162A
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白青山
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Abstract

The invention belongs to the technical field of chemical detection, and particularly relates to a method for determining the content of 6 polyphenols in compound red-skin blood-enriching oral liquid, wherein the polyphenols comprise caffeic acid, gallic acid, p-hydroxycinnamic acid, ferulic acid, catechin and epicatechin, and the method for determining the content is UP L C method, methodological experiment results show that the linear ranges of caffeic acid, gallic acid, p-hydroxycinnamic acid, ferulic acid, catechin and epicatechin are respectively 2.35-235.00 mu g/m L, 0.20-20.00 mu g/m L, 0.27-27.00 mu g/m L, 0.63-63.00 mu g/m L, 1.80-180.00 mu g/m L, 0.26-260.00 mu g/m L, the quantification limits are respectively 105, 100, 135, 315, 90 and 130ng/m L, the detection limits are respectively 36, 33, 45, 105, 30, 43ng/m L, the average recovery rate of RSD and the average recovery rate of RSD is less than 98.07 percent.

Description

Method for measuring content of 6 polyphenol compounds in compound red skin blood replenishing oral liquid
Technical Field
The invention belongs to the technical field of chemical detection, and particularly relates to a method for determining the content of 6 polyphenol compounds in a compound red skin blood replenishing oral liquid.
Background
The compound red skin blood replenishing oral liquid is refined by using peanut red skin, edible fungus, Chinese date and Chinese wolfberry as raw materials, has the effects of replenishing blood, benefiting qi and strengthening spleen, and is used for the auxiliary treatment of iron-deficiency anemia, wherein the peanut red skin is the main component of the compound red skin, namely peanut skin, peanut skin and peanut skin, is the seed coat of Arachis hypogaea (Arachis hypogaea L inn.) of Leguminosae, is sweet in taste, slightly bitter and neutral in nature, has the effects of stopping bleeding, dissipating blood stasis and relieving swelling, and polyphenol substances are general terms of polyphenol hydroxyl compounds and have various physiological and pharmacological activities such as bacteriostasis, anti-inflammation, antioxidation, radiation protection and blood pressure reduction.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for measuring the content of 6 polyphenol compounds in compound oral liquid for enriching blood, which adopts a UP L C method to simultaneously measure 6 polyphenol components of Caffeic acid (Caffeic acid), Gallic acid (Gallic acid), p-Hydroxy-cinnamic acid (p-Hydroxy-cinamic), Ferulic acid (Ferulic acid), Catechin (Catechin) and Epicatechin (Epicatechin) in the compound oral liquid for enriching blood, thereby providing a reliable method for the quality control of the compound oral liquid for enriching blood.
The invention solves the technical problems through the following technical scheme:
a method for measuring the content of 6 polyphenols in a compound oral liquid for enriching blood with red skin comprises the steps of measuring the content of caffeine and caffeic acid, gallic acid, p-hydroxycinnamic acid, ferulic acid, catechin and epicatechin by a UP L C method.
The content determination method of the 6 polyphenol compounds in the compound red skin blood replenishing oral liquid comprises the following steps:
(1) chromatographic conditions and system applicability tests;
(2) preparing a mixed reference substance solution;
(3) preparing a test solution;
(4) and (4) measuring.
In the method for measuring the content of the 6 polyphenol compounds in the compound red skin blood replenishing oral liquid, the chromatographic conditions and the system applicability test are as follows:
a chromatographic column: waters Symmetry C18(4.6mm × 150mm, 3.5 microns), a mobile phase of methanol (A) -0.1% formic acid solution (B), gradient elution and an elution program are shown in the table, the flow rate is 0.5m L/min, the detection wavelengths are 250nm (caffeic acid, gallic acid, p-hydroxycinnamic acid and ferulic acid) and 280nm (catechin and epicatechin), the column temperature is 30 ℃, the sample injection amount is 5 microns L, under the chromatographic condition, the 6 components have normal peak shapes, the separation degrees of the 6 components and adjacent chromatographic peaks are more than 1.5, the baseline separation is achieved, and the number of theoretical plates is more than 10000;
Figure BDA0002488936060000021
in the method for measuring the content of 6 polyphenol compounds in the compound red skin blood-enriching oral liquid, caffeic acid, gallic acid, p-hydroxycinnamic acid and ferulic acid are detected under the wavelength of 250 nm; detecting catechin and epicatechin at the wavelength of 280 nm. The Waters Symmetry C18The size of the column was 4.6mm × 150mm, 3.5 μm.
In the method for measuring the content of the 6 polyphenol compounds in the compound red skin blood replenishing oral liquid, the preparation method of the reference substance solution comprises the following steps:
precisely weighing 117mg of caffeic acid reference substance, 11mg of gallic acid reference substance, 13mg of p-hydroxycinnamic acid reference substance, 31mg of ferulic acid reference substance, 90mg of catechin reference substance and 13mg of epicatechin reference substance, respectively placing in 50m L measuring bottles, adding methanol for dissolving, fixing the volume, shaking up to obtain reference substance stock solutions, precisely weighing 1m L of the reference substance stock solutions, placing in 100m L measuring bottles, diluting with methanol to scale, and shaking up to obtain a mixed reference substance solution.
In the method for measuring the content of the 6 polyphenol compounds in the compound Hongyi blood-enriching oral liquid, the preparation method of the test solution comprises the steps of precisely measuring 1ml of a sample, passing the sample through an AB-8 type macroporous adsorption resin column (the inner diameter is 1.5cm, the length is 12cm), eluting with water 70m L, discarding water solution, eluting with 70% ethanol 50m L, collecting eluent, evaporating to dryness, dissolving with methanol, transferring to a 100m L measuring flask, fixing the volume to scale, and shaking up to obtain the test solution;
the content determination method of the 6 polyphenol compounds in the compound red skin blood replenishing oral liquid comprises the following detailed steps:
(1) chromatographic conditions and system applicability
A chromatographic column: waters Symmetry C18(4.6mm × 150mm, 3.5 microns), a mobile phase of methanol (A) -0.1% formic acid solution (B), gradient elution and an elution program are shown in the table, the flow rate is 0.5m L/min, the detection wavelengths are 250nm (caffeic acid, gallic acid, p-hydroxycinnamic acid and ferulic acid) and 280nm (catechin and epicatechin), the column temperature is 30 ℃, the sample injection amount is 5 microns L, under the chromatographic condition, the 6 components have normal peak shapes, the separation degrees of the 6 components and adjacent chromatographic peaks are more than 1.5, the baseline separation is achieved, and the number of theoretical plates is more than 10000;
Figure BDA0002488936060000031
(2) preparation of Mixed control solutions
Precisely weighing 117.85mg of caffeic acid reference substance, 11.00mg of gallic acid reference substance, 13.80mg of p-hydroxycinnamic acid reference substance, 31.80mg of ferulic acid reference substance, 90.75mg of catechin reference substance and 13.05mg of epicatechin reference substance, respectively placing in a L measuring flask of 50m, adding methanol for dissolving, fixing the volume and shaking up to obtain reference substance storage solutions, precisely weighing 1m L of each reference substance storage solution, placing in a L measuring flask of 100m, diluting with methanol to scale, and shaking up to obtain mixed reference substance solutions with the concentrations of 23.50, 2.00, 2.70, 6.30, 18.00 and 2.60 μ g/m L respectively;
(3) preparing a test solution, precisely measuring 1ml of a sample, passing through an AB-8 type macroporous adsorption resin column (with the inner diameter of 1.5cm and the length of 12cm), eluting with 70m L water, discarding water solution, eluting with 70% ethanol 50m L, collecting eluent, evaporating to dryness, dissolving with methanol, transferring to a 100m L measuring flask, fixing the volume to the scale, and shaking up to obtain the test solution;
(4) the determination method comprises precisely sucking 5 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and determining.
The invention has the beneficial effects that:
the method for measuring the contents of 6 polyphenol compounds in the compound red skin blood-enriching oral liquid disclosed by the invention has the linear ranges of 2.35-235.00 mu g/m L (r is 1.0000), 0.20-20.00 mu g/m L (r is 0.9998), 0.27-27.00 mu g/m L (r is 0.9999), 0.63-63.00 mu g/m L (r is 0.9999), 1.80-180.00 mu g/m L (r is 1.0000), 0.26-260.00 mu g/m L (r is 0.9999), the quantitative limits are 105, 100, 135, 315, 90, 130, L, the detection limits are 36, 33, 45, 105, 43, 30, 25, 9 percent and the simple recovery rate of the compound red skin blood-enriching oral liquid (RSD is equal to 1.97%, the content of the compound red skin blood-enriching oral liquid is equal to 2.35-235, 9.00% and the content of the compound red skin blood-enriching oral liquid is equal to 9.97% (RSD), the simple recovery rate of the compound red skin blood-enriching oral liquid is equal to 3, the content of the compound red skin blood-enriching oral liquid is equal to 9.97% (RSD 13.97, 9, equal to 3, equal to 10.97, equal to 10.7, equal to 10, equal to 10.9.9.7, equal to 10.7, equal to 10, equal to.
Drawings
FIG. 1 shows the high performance liquid chromatogram of the control solution for determining the content of 6 polyphenols in the compound oral liquid for enriching blood with red skin of the invention (note: 1. caffeic acid; 2. gallic acid; 3. p-hydroxycinnamic acid; 4. ferulic acid; 5. catechin; 6. epicatechin).
FIG. 2 shows the high performance liquid chromatogram of the test solution for determining the content of 6 polyphenols in the compound oral liquid for enriching blood with red skin of the invention (note: 1. caffeic acid; 2. gallic acid; 3. p-hydroxycinnamic acid; 4. ferulic acid; 5. catechin; 6. epicatechin).
Detailed Description
The invention will be further described with reference to the accompanying drawings and specific embodiments so that those skilled in the art may better understand the invention, but the invention is not limited thereto.
EXAMPLE 1 method for determining the content of 6 polyphenols in Compound HONGYIBUXUE oral liquid
1 Material
1.1 instruments
1290 model ultra-high performance liquid chromatograph, comprising an on-line vacuum degasser, a quaternary gradient pump, an autosampler, a column oven and a DAD detector (Agilent, usa); Mettler-Toledo XP 205 electronic analytical balance (Mettler-Toledo company).
1.2 drugs and reagents
Caffeic acid reference (batch No. 110885-201703, purity: 99.7%), gallic acid reference (batch No. 110831-201605, purity: 90.8%), ferulic acid reference (batch No. 110773-201614, purity: 99.0%), puerarin reference (batch No. 110877-201604, purity: 99.2%), epicatechin reference (batch No. 110878-201703, purity: 99.7%) were purchased from China institute of food and drug assay; a p-hydroxycinnamic acid reference (batch No. DD0057, purity: 98.0%) was purchased from Doctorite Biotechnology Ltd; methanol as chromatographically pure (Merck, Germany); ethanol is analytically pure (chemical reagents of national drug group, Inc.); the water is ultrapure water; the other reagents were analytical grade (national pharmaceutical group chemical reagents, Ltd.). The compound oral liquid for enriching blood is provided by Xiangyu pharmaceutical industry GmbH (batch No. 1801011, 1802011, 1803011).
2 methods and results
2.1 chromatographic conditions and System applicability
A chromatographic column: waters Symmetry C18(4.6mm × 150mm, 3.5 microns), a mobile phase of methanol (A) -0.1% formic acid solution (B), gradient elution and an elution program are shown in table 1, the flow rate is 0.5m L/min, the detection wavelength is 250nm (caffeic acid, gallic acid, p-hydroxycinnamic acid and ferulic acid) and 280nm (catechin and epicatechin), the column temperature is 30 ℃, the sample injection amount is 5 microns L, under the chromatographic condition, the 6 components have normal peak shapes, the separation degrees from adjacent chromatographic peaks are more than 1.5, the baseline separation is achieved, the number of theoretical plates is more than 10000, and the chromatogram is shown in fig. 1 and fig. 2.
TABLE 1 gradient elution procedure
Tab 1 Gradient elution procedure
Figure BDA0002488936060000051
2.2 preparation of the solution
2.2.1 Mixed reference solution 117.85mg of caffeic acid reference, 11.00mg of gallic acid reference, 13.80mg of p-hydroxycinnamic acid reference, 31.80mg of ferulic acid reference, 90.75mg of catechin reference and 13.05mg of epicatechin reference are precisely weighed and respectively placed in a 50m L measuring flask, methanol is added for dissolving and constant volume, and the mixture is shaken up to obtain reference stock solutions, 1m L of each reference stock solution is precisely weighed and placed in a 100m L measuring flask, the reference stock solutions are diluted to scale with methanol and shaken up to obtain mixed reference stock solutions with the concentrations of 23.50, 2.00, 2.70, 6.30, 18.00 and 2.60 mu g/m L respectively.
2.2.2 sample solution 1ml sample is measured precisely, and is passed through AB-8 type macroporous adsorbent resin column (inner diameter 1.5cm, length 12cm), eluted with water 70m L, water solution is discarded, then eluted with 70% ethanol 50m L, eluent is collected, evaporated to dryness, dissolved with methanol and transferred to 100m L measuring flask, and constant volume is determined to scale, shaken well, and filtered with 0.45 μm microporous membrane before sample injection.
2.3 Linear relationship inspection
Respectively and precisely measuring 0.1, 0.2, 0.5, 1, 2, 5 and 10m L of the mixed reference substance storage solution under the item of '2.2.1', respectively placing the mixed reference substance storage solution into a 100m L measuring flask, diluting the mixed reference substance storage solution to a scale with methanol, shaking the mixed reference substance storage solution uniformly to obtain a series of concentration mixed reference substance solutions, respectively carrying out sample injection measurement according to chromatographic conditions under the item of '2.1', and carrying out linear regression by taking the concentration (x, mu g/m L) of the solution as a horizontal coordinate and taking a peak area (y) as a vertical coordinate to obtain a regression equation and a correlation coefficient (r) result which are shown in a table 2.
TABLE 2 regression equation and Linear Range
Tab.2 Regression equations and linear range
Figure BDA0002488936060000052
Figure BDA0002488936060000061
2.4 quantitative Limit and detection Limit investigation
Taking a proper amount of the mixed reference substance solution under the item of '2.2.1', gradually diluting, carrying out sample injection measurement according to the chromatographic condition under the item of '2.1', recording peak areas, obtaining the limit of quantitation when the signal-to-noise ratio is 10: 1, obtaining the limit of detection when the signal-to-noise ratio is 3: 1, and obtaining the result, wherein the limit of quantitation of caffeic acid, gallic acid, p-hydroxycinnamic acid, ferulic acid, catechin and epicatechin is respectively 105, 100, 135, 315, 90 and 130ng/m L, and the limit of detection is respectively 36, 33, 45, 105, 30 and 43ng/m L.
2.5 precision test
Taking the mixed reference substance solution, continuously feeding sample for 6 times according to the chromatographic condition under the item of 2.1, and recording the peak area. As a result, the RSD of the peak areas of caffeic acid, gallic acid, p-hydroxycinnamic acid, ferulic acid, catechin and epicatechin is below 1.5%, which indicates that the precision of the instrument is good.
2.6 stability test
The same sample solution (lot number: 1801011) was sampled and measured under the chromatographic conditions of "2.1" at room temperature for 0, 2, 4, 8, 12, and 24 hours, and the peak area was recorded. As a result, the RSD of the peak areas of caffeic acid, gallic acid, p-hydroxycinnamic acid, ferulic acid, catechin and epicatechin were all 1.5% or less, indicating that the sample solutions were stable when left at room temperature for 24 hours.
2.7 repeatability test
Taking the product, preparing a sample solution according to the method under the item 2.2.2, adding 6 parts in total, carrying out sample injection determination according to the chromatographic condition under the item 2.1, recording peak area and calculating the content of the sample. As a result, the RSD of the contents of caffeic acid, gallic acid, p-hydroxycinnamic acid, ferulic acid, catechin and epicatechin is below 2.0%, which shows that the repeatability of the method is good.
2.8 sample recovery test
Taking the same lot of sample (lot No. 1801011), precisely measuring about 0.5ml, and 9 parts in total, adding reference substance storage solution 0.4, 0.5, and 0.6ml respectively, preparing recovery rate test solutions with low, medium, and high concentrations according to the preparation method of test solution under item "2.2.2", respectively sampling and measuring, and calculating average recovery rates of caffeic acid, gallic acid, p-hydroxycinnamic acid, ferulic acid, catechin, and epicatechin, the results are shown in Table 3.
Table 3 sample recovery test results (n ═ 9)
Tab 3 Results of recovery tests(n＝9)
Figure BDA0002488936060000062
Figure BDA0002488936060000071
Figure BDA0002488936060000081
2.9 spectroscopic purity investigation
And (3) detecting the spectral purity of chromatographic peaks of caffeic acid, gallic acid, p-hydroxycinnamic acid, ferulic acid, catechin and epicatechin by using a DAD detector spectral purity detection function of a high performance liquid chromatograph. The results show that: the spectroscopic purity values for the 6 components were all within the threshold range given by the instrument. The chromatographic peaks of caffeic acid, gallic acid, p-hydroxycinnamic acid, ferulic acid, catechin and epicatechin are single chromatographic peaks, and no chromatographic peak overlapping interference exists.
2.10 sample assay
Taking samples, preparing test solution according to the method under item 2.2.2, respectively, sampling for determination, recording peak area, and calculating the content of caffeic acid, gallic acid, p-hydroxycinnamic acid, ferulic acid, catechin and epicatechin by external standard method, the result is shown in Table 4.
Table 4 assay results/mg/g (n ═ 2, mg/ml)
Tab 4 Determination results of samples(n＝2，mg/ml)
Figure BDA0002488936060000082

Claims (8)

1. A method for measuring the content of 6 polyphenol compounds in compound red skin blood-enriching oral liquid is characterized in that the content measurement method is a UP L C method.
2. The method for measuring the content of 6 polyphenols in the compound red skin blood-enriching oral liquid according to claim 1, wherein the method for measuring the content comprises the following steps:
(1) chromatographic conditions and system applicability tests;
(2) preparing a mixed reference substance solution;
(3) preparing a test solution;
(4) and (4) measuring.
3. The method for measuring the content of 6 polyphenols in the compound oral liquid for enriching blood according to claim 2, wherein the chromatographic conditions and the system applicability test are as follows:
a chromatographic column: waters Symmetry C18(4.6mm × 150mm, 3.5 microns), a mobile phase of methanol (A) -0.1% formic acid solution (B), gradient elution and an elution program are shown in the table, the flow rate is 0.5m L/min, the detection wavelengths are 250nm (caffeic acid, gallic acid, p-hydroxycinnamic acid and ferulic acid) and 280nm (catechin and epicatechin), the column temperature is 30 ℃, the sample injection amount is 5 microns L, under the chromatographic condition, the 6 components have normal peak shapes, the separation degrees of the 6 components from adjacent chromatographic peaks are more than 1.5, the baseline separation is achieved, and the number of theoretical plates is more than 10000;
Figure FDA0002488936050000011
4. the method for measuring the content of 6 polyphenols in the compound oral liquid for enriching blood of red skin according to claim 3, wherein caffeic acid, gallic acid, p-hydroxycinnamic acid and ferulic acid are detected at the wavelength of 250 nm; detecting catechin and epicatechin at the wavelength of 280 nm.
5. The method for determining the content of 6 polyphenols in the compound red skin blood-enriching oral liquid according to claim 3, wherein the Waters Symmetry C is18The size of the column was 4.6mm × 150mm, 3.5 μm.
6. The method for measuring the content of 6 polyphenols in the compound red skin blood-enriching oral liquid according to claim 3, wherein the preparation method of the reference solution comprises the following steps:
precisely weighing 117mg of caffeic acid reference substance, 11mg of gallic acid reference substance, 13mg of p-hydroxycinnamic acid reference substance, 31mg of ferulic acid reference substance, 90mg of catechin reference substance and 13mg of epicatechin reference substance, respectively placing in 50m L measuring bottles, adding methanol for dissolving, fixing volume, shaking up to obtain reference substance stock solutions, precisely weighing 1m L of the reference substance stock solutions, placing in 100m L measuring bottles, diluting with methanol to scale, and shaking up to obtain mixed reference substance solutions.
7. The method for measuring the content of 6 polyphenols in the compound HONGYIBUXUE oral liquid as claimed in claim 3, wherein the test solution is prepared by precisely measuring 1ml of sample, passing through AB-8 type macroporous adsorbent resin column (diameter 1.5cm, length 12cm), eluting with 70m L of water, discarding water solution, eluting with 70% ethanol 50m L, collecting eluate, evaporating to dryness, dissolving with methanol, transferring to 100m L measuring flask, metering to desired volume, and shaking to obtain test solution.
8. The method for measuring the content of 6 polyphenols in the compound oral liquid for enriching blood of red skin according to claim 3, wherein the detailed steps of the method for measuring the content are as follows:
(1) chromatographic conditions and system applicability
A chromatographic column: waters Symmetry C18(4.6mm × 150mm, 3.5 μm), a mobile phase of methanol (A) -0.1% formic acid solution (B), gradient elution and an elution program are shown in the following table, the flow rate is 0.5m L/min, the detection wavelength is 250nm (caffeic acid, gallic acid, p-hydroxycinnamic acid, ferulic acid) and 280nm (catechin and epicatechin), the column temperature is 30 ℃, the sample injection amount is 5 μ L, under the chromatographic condition, the peaks of 6 components are normal, the separation degree of the peaks and adjacent chromatographic peaks is more than 1.5, the baseline separation is achieved, and the number of theoretical plates is more than 10000;
Figure FDA0002488936050000021
(2) preparation of Mixed control solutions
Precisely weighing 117.85mg of caffeic acid reference substance, 11.00mg of gallic acid reference substance, 13.80mg of p-hydroxycinnamic acid reference substance, 31.80mg of ferulic acid reference substance, 90.75mg of catechin reference substance and 13.05mg of epicatechin reference substance, respectively placing in a L measuring flask of 50m, adding methanol for dissolving, fixing the volume and shaking up to obtain reference substance storage solutions, precisely weighing 1m L of each reference substance storage solution, placing in a L measuring flask of 100m, diluting with methanol to scale, and shaking up to obtain mixed reference substance solutions with the concentrations of 23.50, 2.00, 2.70, 6.30, 18.00 and 2.60 μ g/m L respectively;
(3) preparing a test solution, precisely measuring 1ml of a sample, passing through an AB-8 type macroporous adsorption resin column (with the inner diameter of 1.5cm and the length of 12cm), eluting with 70m L water, discarding water solution, eluting with 70% ethanol 50m L, collecting eluent, evaporating to dryness, dissolving with methanol, transferring to a 100m L measuring flask, fixing the volume to the scale, and shaking up to obtain the test solution;
(4) the determination method comprises precisely sucking 5 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and determining.
CN202010399615.1A 2020-05-12 2020-05-12 Method for measuring content of 6 polyphenol compounds in compound red skin blood replenishing oral liquid Withdrawn CN111505162A (en)

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Cited By (2)

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Publication number Priority date Publication date Assignee Title
CN115192597A (en) * 2022-08-08 2022-10-18 青海大学 Pharmaceutical composition for preventing and treating pulmonary hypertension, preparation method and application thereof
CN115524410A (en) * 2022-07-14 2022-12-27 贵州中医药大学 Method for measuring content of blood ginseng ethanol extract

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115524410A (en) * 2022-07-14 2022-12-27 贵州中医药大学 Method for measuring content of blood ginseng ethanol extract
CN115192597A (en) * 2022-08-08 2022-10-18 青海大学 Pharmaceutical composition for preventing and treating pulmonary hypertension, preparation method and application thereof

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Application publication date: 20200807