CN110297061B - Method for determining contents of chlorogenic acid, caffeic acid and luteolin in Ixeris denticulata by one-test-multiple-evaluation method - Google Patents
Method for determining contents of chlorogenic acid, caffeic acid and luteolin in Ixeris denticulata by one-test-multiple-evaluation method Download PDFInfo
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Abstract
The invention relates to the technical field of traditional Chinese medicine component detection, in particular to a method for determining the content of chlorogenic acid, caffeic acid and luteolin in Ixeris denticulata by adopting a one-test-multiple-evaluation method, which comprises the following steps: (1) preparation of control solution (2) preparation of test solution (3) chromatographic conditions and system suitability test (4). The invention adopts chlorogenic acid as an internal reference substance to perform one test and multiple evaluation, establishes a relative correction factor among the chlorogenic acid, caffeic acid and luteolin, and calculates the contents of the chlorogenic acid, the caffeic acid and the luteolin in the Ixeris denticulata through the relative correction factor. The method is simple, convenient to operate, high in practicability and accurate in result, and can realize synchronous determination of the contents of chlorogenic acid, caffeic acid and luteolin in the Ixeris denticulata, so that the quality of Ixeris denticulata medicinal materials is effectively evaluated and controlled, and scientific basis is provided for ensuring the safety and effectiveness of clinical medication and the development of medicinal material resources.
Description
Technical Field
The invention relates to the technical field of detection of traditional Chinese medicine components, in particular to a method for determining the content of chlorogenic acid, caffeic acid and luteolin in Ixeris denticulata by adopting a one-test-multiple-evaluation method.
Background
Ixeris denticulata (A) and (B)Ixeridiumchinense(Thunb.) Tzvel) is a perennial herb of Ixeris in Compositae, the whole herb is used as a medicine, is called Chinese Ixeris chinensis, and Ixeris chinensis, is distributed in the north, south and east provinces, is pharmaceutically acceptable and edible, has the effects of clearing heat and detoxicating, diminishing inflammation and cooling blood, relieving pain and swelling, resisting tumors and the like, and is used for treating unknown swelling and pain, abdominal abscess, dysentery, appendicitis, pneumonia, arthritis and other symptoms.
The chemical components of the mountain sow thistle such as Liuchang are subjected to in-depth systematic research to find that the mountain sow thistle contains flavonoid components, wherein the flavonoid components have strong pharmacological activity; mainly comprises the following components: luteolin, luteolin-7-O-beta-D-glucoside, apigenin-7-O-beta-D-glucoside, secalin, luteolin-7-O-beta-D-glucoside acetate, apigenin, caffeic acid and the like (Liuchang, Naming, Shaoshaishuai, etc. research progress of chemical components and pharmacological actions of sowthistle herb, Chinese journal of experimental prescriptions, 2015). In order to better utilize the Ixeris denticulata resources, the Zhongtao and the like establish an HPLC method for measuring the content of the luteolin in the Ixeris denticulata, and measure the content of the luteolin in the Ixeris denticulata in 6 different production places to obtain that the content of the luteolin in the Ixeris denticulata in the 6 different production places is 1.2812.288 mg g < -1 > and the average content is 1.780 mg g < -1 > (Zhongtao, Suilu, that mulberry, and the like). The study of chemical components of the alternative name of Chinese endive (mountain endive) is carried out by Zhonghonglei, etc., 5 compounds are separated from alcohol extract (the study of chemical components of Zhonghonglei, Yuanjirong, Chinese endive [ J ]. Chinese herbal medicine, 1996).
In the aspect of pharmacological research, the Ixeris denticulata has various pharmacological activities including anti-inflammatory and liver-protecting effects, antiviral and anti-leukemia effects, and effects of treating diabetes; the research on chemical components shows that the Ixeris denticulata contains various flavonoid compounds, has the effects of resisting tumors, resisting oxidation and the like, under the optimal extraction process conditions, the Ixeris denticulata total flavone is obtained by extraction, and the content of the Ixeris denticulata total flavone is measured by adopting a visible spectrophotometry, so that the result shows that the rutin concentration range is in good linearity (Taiwenjie, Chenopdiaceae, Ganxui sea. the research on the extraction and the antioxidant activity of the total flavone in the Ixeris denticulata in Guizhou State academic institute [ J ]. 2016). Zhouli and the like report that Chinensiolide A, which is an alias of Chinese ixeris (ixeris sonchifolia hance), can effectively inhibit the growth of lung adenocarcinoma A549 cells, liver cancer Ble-7402 cells and LoVo cells in vitro and has stronger antitumor activity (Zhouli, Zhao Ying, Wang Yu, and the like. The research of the Wangli and the like discovers that the alias of the Chinese ixeris (Ixeris denticulata) has good effects of resisting virus, reducing blood fat, reducing cholesterol and the like (the Wangli, the Yangli, the Liyue birch and the like. Chengqiang and the like find that the Chinese Ixeris denticulata extract has obvious blood sugar reducing effect in researching the alias of the Chinese Ixeris denticulata (Ixeris denticulata) in the Chinese Ixeris denticulata; the chlorogenic acid component is found in the process of the experiment, and the component is researched and explored; the research on the preparation of the general flavone extract of the Ixeris chinensis and the drug effect and mechanism for treating type 2 diabetes [ D ]. Heilongjiang Chinese medicine university, 2016).
As can be seen from the above, the Ixeris denticulata is an important and commonly used Chinese medicament in China, the medicinal value is high, and the quality of the Ixeris denticulata is closely related to the health benefits of people. The quality of the traditional Chinese medicine materials is influenced by a plurality of factors, so that the quality control of the traditional Chinese medicine materials in the market is difficult, and the quality of the traditional Chinese medicine materials is evaluated only by a single component, so that the traditional Chinese medicine materials cannot well reflect the excellent quality of the traditional Chinese medicine materials. Due to the complexity of phytochemicals, multi-index content determination has become a consensus for quality control of plant-derived drugs. The traditional determination method for simultaneously determining a plurality of components in the traditional Chinese medicinal materials is difficult to obtain as a reference substance, long in inspection period, energy-consuming and time-consuming. The first-measurement multi-evaluation method is characterized in that one component (a reference substance is easy to obtain, cheap and effective) is measured through an internal functional relation and a proportional relation method existing among effective components of the traditional Chinese medicine, so that synchronous measurement of a plurality of components (the reference substance is difficult to obtain or difficult to supply) is realized; the method not only solves the problem of lack of reference substances in multi-component quantitative and multi-index quality control of the traditional Chinese medicine, but also realizes multi-index synchronous quality control reaction on the quality of the traditional Chinese medicine. At present, the related research on the quality analysis of the Ixeris denticulata is less, and no report exists at home and abroad for measuring the content of chlorogenic acid, caffeic acid and luteolin in the Ixeris denticulata by a multi-evaluation method according to the inspection of documents.
Disclosure of Invention
The invention aims to provide a method for measuring Ixeris denticulata (A) by adopting a one-time multi-evaluation method aiming at the defects of the prior artIxeridiumchinense(Thunb.) Tzvel) method for determining the content of chlorogenic acid, caffeic acid and luteolin, which is simple and accurate, and convenient to operateThe method has high practicability and accurate result, and can realize synchronous determination of the contents of chlorogenic acid, caffeic acid and galuteolin in Ixeris denticulata, thereby effectively evaluating and controlling Ixeris denticulata (L.) RoxbIxeridiumchinense(Thunb.) Tzvel.) the quality of the medicinal materials provides scientific basis for ensuring the safety and the effectiveness of clinical medication and the development of medicinal material resources.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for determining contents of chlorogenic acid, caffeic acid and luteolin in Ixeris denticulata by one-time evaluation comprises the following steps:
(1) preparation of control solutions: precisely weighing 11.59mg of chlorogenic acid reference substance, 6.72mg of caffeic acid reference substance and 13.60mg of luteolin reference substance, respectively adding into a 25mL volumetric flask, adding a proper amount of methanol to dilute to a scale, dissolving by ultrasonic, shaking up, precisely weighing 1.5mL of the chlorogenic acid solution, 1.5mL of the caffeic acid solution and 6.0mL of the luteolin solution, placing into the same 10mL volumetric flask, fixing the volume to the scale by methanol, and shaking up to obtain a mixed reference substance solution, wherein each 1mL of the mixed reference substance solution contains 69.54 μ g of chlorogenic acid, 40.32 μ g of caffeic acid and 326.4 μ g of luteolin;
(2) preparation of a test solution: taking 2g of medicinal herb powder of ixeris sonchifolia, putting the medicinal herb powder into a 100mL conical flask, adding 30mL of methanol with the volume concentration of 70%, weighing, ultrasonically extracting for 60min at the temperature of 30 ℃ and the frequency of 70kHz, cooling, complementing the loss weight with the methanol with the volume concentration of 70%, filtering, and filtering a subsequent filtrate by using a microporous filter membrane with the particle size of 0.45 mu m to obtain the medicinal herb powder;
(3) chromatographic conditions and system applicability test: a chromatographic column: CAPCELLPAK TYPE MG II SIZE-C18(4.6X 250mm,5 μm); the volume flow rate is 1.0 ml.min-1(ii) a The detection wavelength is 330 nm; the column temperature was 25 ℃; the sample size is 5 muL; gradient elution is carried out on the mobile phase by adopting methanol-0.2 percent phosphoric acid aqueous solution, the elution procedure is shown in table 1, and the theoretical plate number is not lower than 5000 according to the peak calculation of chlorogenic acid;
(4) and (3) determination: precisely absorbing 5 mu L of each of a reference solution and a test solution respectively, injecting the reference solution and the test solution into a liquid chromatograph, measuring under the chromatographic condition of the system adaptability, calculating by adopting an internal standard method, keeping the retention time of chromatographic peaks of chlorogenic acid, caffeic acid and luteoloside in the test solution consistent with that of the reference solution, and enabling the separation degree of the chromatographic peaks and other coexisting components to be more than 1.5, and enabling a main peak and an impurity peak to achieve baseline separation; the characteristic spectrum of the test solution shows the peak values of chlorogenic acid, caffeic acid and luteolin, the chlorogenic acid is taken as an internal reference, the relative correction factors of the chlorogenic acid, the caffeic acid and the luteolin are calculated, and the contents of the chlorogenic acid, the caffeic acid and the luteolin in the Ixeris denticulata are calculated through the relative correction factors.
The method for measuring the contents of chlorogenic acid, caffeic acid and luteolin in the Ixeris denticulata by adopting a one-measurement-multiple-evaluation method comprises the following steps of: respectively sucking 0.4 mL, 0.6 mL, 0.8 mL, 1.0mL, 1.2 mL and 1.6 mL of the mixed reference substance solution prepared in the step (1), respectively placing the mixed reference substance solution in a 2.0 mL measuring flask, adding 70% methanol to fix the volume to the scale, shaking up to prepare a series of mixed reference substance solutions with different concentrations, respectively injecting 5 muL of the mixed reference substance solution according to chromatographic conditions, respectively measuring the peak areas of caffeic acid and luteolin in the Ixeris chinensis by taking chlorogenic acid as an internal standard, calculating a formula fac = f a/fc = (Aa/Ca)/(Ac/Cc) according to a relative correction factor, and calculating a relative correction factor fac of the caffeic acid and the luteolin, wherein the reference substance is used for calculation, Aa is the peak area of the chlorogenic acid of the internal reference substance, Ca is the concentration of the chlorogenic acid of the internal reference substance, and Ac is a certain component to be measured; the relative correction factor fac is the average of the data obtained from each experiment.
The invention has the beneficial effects that:
1. the method for determining the content of chlorogenic acid, caffeic acid and luteolin in the Ixeris denticulata by adopting a one-test-multiple-evaluation method meets the requirement of methodology verification, can be applied to quality evaluation of the Ixeris denticulata under the condition of shortage of reference substances, can be used for determining the content of chlorogenic acid, caffeic acid and luteolin in the Ixeris denticulata, is simple, convenient and quick, has good repeatability, can save resource loss, reduces solvent consumption and analysis time, is more beneficial to environmental protection, and provides a basis for establishing quality evaluation of the golden camellia medicinal material.
2. The method for determining the content of chlorogenic acid, caffeic acid and luteolin in the Ixeris denticulata by adopting a one-test-multiple-evaluation method is simple, accurate in determination, convenient to operate, high in practicability, accurate in result, repeatable in method and good in durability, and can be used for synchronously determining the content of chlorogenic acid, caffeic acid and luteolin in the Ixeris denticulata, so that the quality of the Ixeris denticulata medicinal material can be evaluated and controlled better and more comprehensively and effectively, the quality control of the Ixeris denticulata medicinal material can be used for providing scientific basis for ensuring the safety and effectiveness of clinical medication and the development of medicinal material resources; and simultaneously avoids the cost and operation problems caused by measuring the content of multiple components.
Drawings
FIG. 1 is a graph of the standard curve of chlorogenic acid;
FIG. 2 is a standard graph of caffeic acid;
FIG. 3 is a standard curve of luteolin;
FIG. 4 is a chromatogram of a mixed control, in which 1, chlorogenic acid, 2, caffeic acid, 3, and luteolin are present;
FIG. 5 is a chromatogram of Ixeris denticulata sample, including 1 chlorogenic acid, 2 caffeic acid, 3 and luteolin.
Detailed Description
Example 1
The method for measuring the content of chlorogenic acid, caffeic acid and luteolin in the Ixeris denticulata is adopted to detect 10 batches of Ixeris denticulata medicinal materials, and the specific measurement conditions are as follows:
1 Experimental materials, instruments and reagents
1.1 Experimental materials
The area and date of picking the traditional Chinese medicine of Ixeris denticulata is shown in Table 2, and is identified as Ixeris denticulata (R) A. denticulata of Ixeris of Compositae by West Sonchus professor of Guangxi university of traditional Chinese medicineIxeridiumchinense(Thunb.) Tzvel.) of whole grass.
1.2 instruments
U3000 high performance liquid chromatograph (Thermo Fisher, USA); 1260 high performance liquid chromatography (Agilent, usa); chromatographic column CAPCELLPAK TYPE II SIZE C18(4.6mm I.D.. times.250 mm,5 μm), Agilent Eclipse plus C18 (4.6X 250mm,5 μm), Waters Atlantis T3 (4.6X 250mm,5 μm); XS-205Du electronic analytical balance (Mettler TOLEDO, Switzerland); KQ-500GDV ultrasonic cleaner (ultrasonic instruments, Inc. of Kunshan); 501 super constant temperature water bath (jin Tan City medical instrument factory); a Synergy ultra-pure water machine (Millipore, USA).
1.3 reagent
Chlorogenic acid (110753-201415, the content is 96.2%), caffeic acid (110885-200102) and trifoliflorin (111720-201307, the content is 94.0%) were purchased from China food and drug testing institute. Acetonitrile, methanol (chromatographic purity, Fisher company, USA), phosphoric acid (batch No. 20150327, super grade purity, shin-Shen-Shih-Mi-Kong-Fine chemical research institute), ultrapure water as the water for the liquid chromatography, and analytically pure reagents used in the liquid chromatography.
2 experimental methods and results
2.1 the method for measuring the content of chlorogenic acid, caffeic acid and luteolin in the Ixeris denticulata by adopting a one-measurement-multiple-evaluation method comprises the following steps:
(1) preparation of control solutions: precisely weighing 11.59mg of chlorogenic acid reference substance, 6.72mg of caffeic acid reference substance and 13.60mg of luteolin reference substance, respectively adding into a 25mL volumetric flask, adding a proper amount of methanol to dilute to a scale, dissolving by ultrasonic, shaking up, precisely weighing 1.5mL of the chlorogenic acid solution, 1.5mL of the caffeic acid solution and 6.0mL of the luteolin solution, placing into the same 10mL volumetric flask, fixing the volume to the scale by methanol, and shaking up to obtain a mixed reference substance solution, wherein each 1mL of the mixed reference substance solution contains 69.54 μ g of chlorogenic acid, 40.32 μ g of caffeic acid and 326.4 μ g of luteolin;
(2) preparation of a test solution: taking 2g of medicinal herb powder of ixeris sonchifolia, putting the medicinal herb powder into a 100mL conical flask, adding 30mL of methanol with the volume concentration of 70%, weighing, ultrasonically extracting for 60min at the temperature of 30 ℃ and the frequency of 70kHz, cooling, complementing the loss weight with the methanol with the volume concentration of 70%, filtering, and filtering a subsequent filtrate by using a microporous filter membrane with the particle size of 0.45 mu m to obtain the medicinal herb powder;
(3) chromatographic conditions and system applicability test: a chromatographic column: CAPCELLPAK TYPE MG II SIZE-C18(4.6X 250mm,5 μm); the volume flow rate is 1.0 ml.min-1(ii) a The detection wavelength is 330 nm; the column temperature was 25 ℃; the sample size is 5 muL; gradient elution is carried out on the mobile phase by adopting methanol-0.2 percent phosphoric acid aqueous solution, the elution procedure is shown in table 1, and the theoretical plate number is not lower than 5000 according to the peak calculation of chlorogenic acid;
(4) and (3) determination: precisely absorbing 5 mu L of each of a reference solution and a test solution respectively, injecting the reference solution and the test solution into a liquid chromatograph, measuring under the chromatographic condition of the system adaptability, calculating by adopting an internal standard method, keeping the retention time of chromatographic peaks of chlorogenic acid, caffeic acid and luteoloside in the test solution consistent with that of the reference solution, and enabling the separation degree of the chromatographic peaks and other coexisting components to be more than 1.5, and enabling a main peak and an impurity peak to achieve baseline separation; the characteristic spectrum of the test solution shows the peak values of chlorogenic acid, caffeic acid and luteolin, the chlorogenic acid is taken as an internal reference, the relative correction factors of the chlorogenic acid, the caffeic acid and the luteolin are calculated, and the contents of the chlorogenic acid, the caffeic acid and the luteolin in the Ixeris denticulata are calculated through the relative correction factors.
The method for measuring the contents of chlorogenic acid, caffeic acid and luteolin in the Ixeris denticulata by adopting a one-measurement-multiple-evaluation method comprises the following steps of: respectively sucking 0.4 mL, 0.6 mL, 0.8 mL, 1.0mL, 1.2 mL and 1.6 mL of the mixed reference substance solution prepared in the step (1), respectively placing the mixed reference substance solution in a 2.0 mL measuring flask, adding 70% methanol to fix the volume to the scale, shaking up to prepare a series of mixed reference substance solutions with different concentrations, respectively injecting 5 muL of the mixed reference substance solution according to chromatographic conditions, respectively measuring the peak areas of caffeic acid and luteolin in the Ixeris chinensis by taking chlorogenic acid as an internal standard, calculating a formula fac = f a/fc = (Aa/Ca)/(Ac/Cc) according to a relative correction factor, and calculating a relative correction factor fac of the caffeic acid and the luteolin, wherein the reference substance is used for calculation, Aa is the peak area of the chlorogenic acid of the internal reference substance, Ca is the concentration of the chlorogenic acid of the internal reference substance, and Ac is a certain component to be measured; the relative correction factor fac is the average of the data obtained from each experiment.
2.2 methodology of the method for determining contents of chlorogenic acid, caffeic acid and luteolin in Ixeris denticulata with one-test-multiple-evaluation method of the invention is examined as follows:
2.2.1 examination of the Linear Range
Respectively sucking 0.4 mL, 0.6 mL, 0.8 mL, 1.0mL, 1.2 mL and 1.6 mL of the mixed reference substance solution prepared in the step (1) of the invention, respectively placing the mixed reference substance solution in a 2.0 mL measuring flask, adding 70% methanol to fix the volume to the scale, shaking up to prepare a series of mixed reference substance solutions with different concentrations, respectively injecting 5 muL of the mixed reference substance solution according to the chromatographic conditions defined by the invention, respectively injecting the sample, taking the peak area as the ordinate and the concentration as the abscissa, and observing the linear regression range in a table 3, wherein the results are shown in the figures 1, 2 and 3. From the results, it can be obtained: the linear range investigation was good in this experiment.
2.2.2 precision test
Taking 2g of Ixeris denticulata powder of No. S4, preparing the test solution according to the preparation method of the test solution, continuously feeding samples for six times according to chromatographic conditions, recording sample maps and calculating the area of the peak of the Ixeris denticulata. The results are shown in Table 4: the mean peak area values of chlorogenic acid, caffeic acid and luteolin were 15.617, 5.467 and 31.040, respectively, and the RSD was 2.0%, 2.1% and 2.1%, respectively, resulting in good precision of the instrument.
2.2.3 repeatability test
Taking 6 parts of the Ixeris denticulata powder of No. S4, each 2g, preparing a test sample according to the preparation method of the test sample solution, measuring according to chromatographic conditions, observing a spectrum peak, recording the sample data of the Ixeris denticulata, and calculating the content of chlorogenic acid, caffeic acid and luteolin in the Ixeris denticulata. The results are shown in Table 5: the average content of chlorogenic acid is 1.167mg/g, the RSD is 1.7%, the average content of caffeic acid is 0.222mg/g, the RSD is 2.3%, the average content of luteolin is 2.510mg/g, and the RSD is 2.3%. Indicating that the method has good repeatability.
2.2.4 stability test
Taking a test solution in a repeatability test, determining at 1, 2, 4, 6, 8, 12 and 24h according to chromatographic conditions, recording the spectrum and peak area of the Ixeris denticulata sample, calculating the content data of the Ixeris denticulata, and inspecting the stability of the test solution. The results are shown in Table 6: the average content of chlorogenic acid is 1.146mg/g, the RSD is 5.1%, the average content of caffeic acid is 0.213mg/g, the RSD is 4.8%, the average content of luteolin is 2.416mg/g, and the RSD is 5.1%. The stability of the test solution within 24h is good.
2.2.5 sample recovery test
Precisely weighing 6 parts of the determined content (1 g of 1.167mg of chlorogenic acid, 0.222mg of caffeic acid and 2.510mg of luteolin) of the Ixeris denticulata sample, and weighing 1g of the Ixeris denticulata sample; precisely weighing 6.86mg of chlorogenic acid control, 1.34mg of caffeic acid control and 16.24mg of luteolin control, placing in a 200ml measuring flask, adding appropriate amount of 70% methanol, ultrasonic dissolving, diluting to scale, and shaking to obtain mixed control stock solution (each 1ml contains 33.0 μ g of chlorogenic acid, 6.7 μ g of caffeic acid and 76.3 μ g of luteolin). Respectively and precisely adding 30ml of reference substance stock solution, preparing a Ixeris denticulata sample adding and recovering solution under the item of 2.5, measuring according to chromatographic conditions, recording peak areas of chlorogenic acid, caffeic acid and luteolin, and calculating the recovery rate to obtain the result that the RSD is less than 3. The results are shown in tables 7, 8 and 9.
2.3 calculation of relative correction factor of index to be measured in Ixeris denticulata
Under the chromatographic conditions defined by the invention, the mixed reference solution under the item of '2.2.1 linear range investigation result' is precisely absorbed, chlorogenic acid is taken as an internal standard, the peak areas of caffeic acid and luteoloside in the ixeris sonchifolia hance are measured, the relative correction factors of the caffeic acid and the luteoloside are calculated according to a relative correction factor calculation formula, the result is shown in table 10, and the relative correction factors are averaged. Fac = fa/fc = (Aa/Ca)/(Ac/Cc), wherein the calculation is carried out by using a reference substance, the sum of Aa is the peak area of an internal reference substance (chlorogenic acid), Ca is the concentration of the internal reference substance (chlorogenic acid), and Ac is the peak area of a component to be measured; cc is the concentration of a certain component to be measured.
2.4 investigation of System durability
The method is characterized in that the mixed reference substance solution under the item of '2.2.1 linear range investigation result' is precisely absorbed for determination under the chromatographic condition defined by the invention, the relative correction factor and the relative retention value of the content of the component to be detected in the reference substance are calculated for investigating durability, the positioning of chromatography is investigated, the obtained result can be better evaluated, and the application of the one-test-multiple-evaluation method in the future is verified.
2.4.1 different sample size investigation
Under the item of '2.4', the influence of different sample sizes of 4, 5 and 6 mu L is examined, and the result is shown in a table 11. The different volume flows have no significant influence on the device, and the durability is good.
2.4.2 investigation of different instruments, columns
ThermoUlltiMate 3000, Agilnt 1260, YPE MG II SIZE-C18, Agilent Eclipse plus-C18, Atlantis T3-C18 chromatographic columns (4.6X 250mm 5 μm) were selected under the condition of item "2.4" and the results were examined and shown in Table 12. It can be seen that different instruments and chromatographic columns have no obvious influence on the device, and the device has good durability.
2.4.3 different volume flow investigation
The effect of the volume flow rates of 0.9, 1.0, 1.1ml/min on the relative correction factors and relative retention values was examined under the term "2.4" and the results are shown in Table 13. The influence degree of different volume flow rates on the device is not obvious, and the durability is good.
2.4.4 investigation of different column temperatures
The effect of column temperatures of 20, 25, 30, ° c on the relative correction factors and relative retention values was examined under the term "2.4" and the results are shown in table 14. The influence degree of different column temperatures is not obvious, and the durability is good.
2.4.5 different wavelength investigation
Under the term "2.4", the influence of the detection wavelengths of 220, 254 and 330nm on the relative correction factors and relative retention values was examined, and the results are shown in Table 15. The detection wavelength has no obvious influence on the detection wavelength, and the durability is good.
2.5 positioning of chromatographic Peak to be measured by one-measurement-multiple-evaluation method
The influence of the relative retention values in different instruments and columns under the term "2.4" and the term "2.4.2" was examined under the term "2.4", and the results are shown in Table 16. As can be seen from the table, the relative retention value fluctuation is small, and the RSD of caffeic acid and luteolin is 1.4% and 6.8% respectively, so that different instruments and chromatographic columns are selected as the chromatographic peak positioning indexes.
2.6 comparison of content measurement results between one-test-multiple-evaluation method and external standard method
After methodological optimization and investigation, the content of caffeic acid and luteolin in 10 batches of Ixeris denticulata extract is determined by the established one-test-multiple-evaluation method, and the content of caffeic acid and the content of luteolin are respectively determined by injecting sample according to the determination method of the invention and the reference substance solution under the item of 2.2.1 (figure 4 and figure 5). The results of the comparison using the external standard method and the relative correction factor are shown in Table 17.
In order to verify the accuracy of the QAMS method, the QAMS method is compared with the external standard method. The feasibility of the established one-test-multiple-evaluation method was evaluated with an Accuracy (Accuracy) calculated by the following formula: accuracy (%) = CS/CE × 100%, where CS represents the content calculated by the one-test-multiple-scoring method and CE represents the content calculated by the external standard method.
The experimental results are as follows: the content of chlorogenic acid, caffeic acid and luteolin in the ixeris sonchifolia hance in 10 batches of production areas is measured by a one-test-multiple evaluation method in the experiment, and the results show that the production areas with higher content of chlorogenic acid are S10, S6 and S4, the production areas with higher content of caffeic acid are S10, S5, S6, S4 and S2, the production areas with higher content of luteolin are S10, S5, S4 and S6, and the content of the ixeris sonchifolia hance chlorogenic acid, caffeic acid and luteolin in the production areas of S10, S6 and S4 is higher.
Discussion of 3
3.1 selection of the Mobile phase
In the experiment, the content of the ixeris sonchifolia hance extract is examined to determine the contained components, and the content of the components is determined by examining documents, wherein 0.1 percent of phosphoric acid: acetonitrile; 0.1% phosphoric acid: methanol; 0.2% phosphoric acid: acetonitrile; 0.2% phosphoric acid: methanol; water: investigating various fluidity of methanol and the like; in the experimental process, the addition of acid is considered because the polarity of water is higher, the peak-generating components are less and the peaks are piled up together; the separation effect cannot be achieved when 0.1% phosphoric acid is used, so 0.2% phosphoric acid is selected; when selecting acetonitrile and methyl alcohol for use, the acetonitrile is narrow and the separation effect is relatively poor when separating, selects 0.2% phosphoric acid: the separation effect is better when the methanol adopts gradient elution.
3.2 preparation of test articles
The preparation of a test sample is determined by observing extraction conditions, and in different extraction methods, different solvent concentrations of methanol, different material-liquid ratios and different extraction times, the extracted solution directly passes through a microporous filter membrane, and is directly injected after being filtered, and experiments show that the peak base line obtained by using 30ml of 70% methanol for ultrasonic treatment for 60min is stable, and the chromatogram has more detection components and better peak shape. When the reflux method and the ultrasonic method are examined, the difference of the extraction contents of the reflux method and the ultrasonic method is small, and the ultrasonic extraction method is favored when the literature is consulted.
3.3 selection of detection wavelength
In the experiment, the mobility and chromatographic conditions are determined when a sample is inspected, the chlorogenic acid, caffeic acid and luteolin are contained in the Ixeris denticulata is determined by adopting a reference substance, and when the determined reference substance and medicinal material powder are scanned at full wavelength under the determined chromatographic conditions, a common peak appears at 330nm, the retention time is the same, and the chromatographic peak shape is better, so that 330nm is used as the detection wavelength.
3.4 internal standard screening
In the experiment, the ixeris sonchifolia hance contains more flavonoid components, 3 contents of the ixeris sonchifolia hance are calculated by an external standard method, the content of chlorogenic acid and the content of the 3 components are stable, a chlorogenic acid reference substance in the three contents measured under the condition of limiting the reference substance is easy to purchase, the availability is high, and the chlorogenic acid is used as an internal reference substance to establish a relative correction factor of caffeic acid and luteolin. Experiments show that the chlorogenic acid peak-producing time is moderate and the retention time difference fluctuation is small on different instruments and chromatographic columns, and the RSD is within 5 percent; because the caffeic acid has low content and is unstable, systematic analysis is not suitable; the retention time of luteolin is more than 5%, so it is not suitable to position the components. Therefore, chlorogenic acid is selected as an internal standard, and the relative correction factors in the QAMS method are used for calculating the content of caffeic acid and luteolin.
In conclusion, the content determination method established by the experiment is convenient to operate and accurate in result, and can be used for synchronously determining the content of chlorogenic acid, caffeic acid and luteoloside in the ixeris hance
3.5 examination of the durability of the "QAMS" method
In the experiment, a QAMS method is adopted to select and investigate Ultimate3000 and Agilent1260, different chromatographic conditions are adopted to investigate the durability of the method, the measured influence of each component on a relative correction factor and a relative retention value is used to verify the durability of the method on the quality control of the Ixeris denticulata. The results show that: under the condition of the shortage of the reference substances, the repeatability in the 'one-test-multiple-evaluation method' is ideal, and the loss of resources can be saved.
3.6 chromatographic Peak localization
In the experiment, when only a single reference substance is used, the problems of chromatographic peaks of 2 components of caffeic acid and trifolioside measured in the ixeris sonchifolia hance can be correctly positioned by adopting a one-test-and-multiple-evaluation method, the retention time difference, the relative retention value and the separation degree of the components are selected as positioning standards, and the two are investigated by using different instruments and chromatographic columns. The results show that the relative retention values can more accurately locate the chromatographic peaks.
3.7 evaluation of external Standard method and one-test-multiple-evaluation method
The content of the medicinal material of the Ixeris denticulata in 10 batches of producing areas is analyzed, the results of the one-test multi-evaluation method are compared by an external standard method, the obtained results are approximately the same, and the results show that the one-test multi-evaluation method is simple and convenient, high in accuracy and high in feasibility.
4 conclusion
Comparing 10 batches of medicinal materials of the ixeris sonchifolia hance by an external standard method and a one-test-multiple evaluation method, and obtaining the results that the content of the medicinal materials of the ixeris sonchifolia hance before each production place is obviously different, and different places have higher content and lower content, thereby explaining the importance of genuine medicinal materials; the method is simple and accurate, and convenient to operate, so that the method can be used for content determination under the condition of reference substance shortage, can reduce solvent consumption and analysis time, and is more beneficial to environmental protection.
Claims (1)
1. The method for determining the content of chlorogenic acid, caffeic acid and luteolin in the Ixeris denticulata by adopting a one-test-multiple-evaluation method is characterized by comprising the following steps of:
(1) preparation of control solutions: precisely weighing 11.59mg of chlorogenic acid reference substance, 6.72mg of caffeic acid reference substance and 13.60mg of luteolin reference substance, respectively adding into a 25mL volumetric flask, adding a proper amount of methanol to dilute to a scale, dissolving by ultrasonic, shaking up, precisely weighing 1.5mL of the chlorogenic acid solution, 1.5mL of the caffeic acid solution and 6.0mL of the luteolin solution, placing into the same 10mL volumetric flask, fixing the volume to the scale by methanol, and shaking up to obtain a mixed reference substance solution, wherein each 1mL of the mixed reference substance solution contains 69.54 μ g of chlorogenic acid, 40.32 μ g of caffeic acid and 326.4 μ g of luteolin;
(2) preparation of a test solution: taking 2g of medicinal herb powder of ixeris sonchifolia, putting the medicinal herb powder into a 100mL conical flask, adding 30mL of methanol with the volume concentration of 70%, weighing, ultrasonically extracting for 60min at the temperature of 30 ℃ and the frequency of 70kHz, cooling, complementing the loss weight with the methanol with the volume concentration of 70%, filtering, and filtering a subsequent filtrate by using a microporous filter membrane with the particle size of 0.45 mu m to obtain the medicinal herb powder;
(3) chromatographic conditionsAnd (3) carrying out system suitability test: a chromatographic column: CAPCELLPAK TYPE MG II SIZE-C18(ii) a The volume flow rate is 1.0 ml.min-1(ii) a The detection wavelength is 330 nm; the column temperature was 25 ℃; the sample size is 5 muL; gradient elution is carried out on the mobile phase by adopting methanol-0.2 percent phosphoric acid aqueous solution, the elution procedure is shown in table 1, and the theoretical plate number is not less than 5000 according to the peak calculation of chlorogenic acid;
(4) and (3) determination: precisely absorbing 5 muL of each of a reference solution and a test solution, respectively, injecting into a liquid chromatograph, measuring under the adaptive chromatographic condition of the system, calculating by adopting an internal standard method, wherein the characteristic map of the test solution shows peak values of chlorogenic acid, caffeic acid and luteolin, calculating relative correction factors of the chlorogenic acid, the caffeic acid and the luteolin by taking the chlorogenic acid as an internal reference, and calculating the contents of the chlorogenic acid, the caffeic acid and the luteolin in the Ixeris denticulata through the relative correction factors;
the Latin chemical name of the Ixeris denticulata is as follows: ixeridium chinensis (Thunb.) Tzvel;
the calculation method of the relative correction factor comprises the following steps: respectively sucking 0.4 mL, 0.6 mL, 0.8 mL, 1.0mL, 1.2 mL and 1.6 mL of the mixed reference substance solution prepared in the step (1), respectively placing the mixed reference substance solution in a 2.0 mL measuring flask, adding 70% methanol to fix the volume to the scale, shaking up to prepare a series of mixed reference substance solutions with different concentrations, respectively injecting 5 muL of the mixed reference substance solution according to chromatographic conditions, respectively measuring the peak areas of caffeic acid and luteolin in the Ixeris chinensis by taking chlorogenic acid as an internal standard, calculating a formula fac = f a/fc = (Aa/Ca)/(Ac/Cc) according to a relative correction factor, and calculating a relative correction factor fac of the caffeic acid and the luteolin, wherein the reference substance is used for calculation, Aa is the peak area of the chlorogenic acid of the internal reference substance, Ca is the concentration of the chlorogenic acid of the internal reference substance, and Ac is the peak area of a certain component to be measured; cc is the concentration of a certain component to be measured; the relative correction factor fac is the average of the data obtained from each experiment.
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