CN110579544A - Ultra-high performance liquid phase detection method for gentiana straminea - Google Patents
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Abstract
The invention discloses an ultra-high performance liquid detection method of gentiana straminea, which comprises the following steps: (1) preparing a test solution; (2) ultra performance liquid chromatography analysis. The method can be effectively applied and used for distinguishing the gentiana lineolata flowers, similar varieties and mixed counterfeit products thereof; the method can be used for overall evaluation and control of the quality of the gentiana straminea flowers, and the one-sidedness caused by comparing only individual chemical components in the total components when judging the authenticity and the quality of the gentiana straminea flowers is avoided; the detection method has good reproducibility and high separation degree, and is very suitable for the determination of the index components of the gentiana straminea and the establishment of a fingerprint spectrum; the ultra-high performance liquid phase can accept higher throughput and smaller sample size, providing better selectivity and better detection and quantification limits.
Description
Technical Field
the invention belongs to a quality control method of traditional Chinese medicinal materials, and particularly relates to an ultra-high performance liquid detection method of gentiana lineata.
Background
Gentiana belongs to Gentianales Gentianaceae, more than 400 Gentianaceae are available worldwide, and are widely distributed in the global world and mainly produced in the northern temperate zone and tropical mountain areas. I have 22 genera and about more than 500, and the total number of the genera is 12 and 70 for medical use. Gentian is an annual or perennial herb. The plants of the genus gentian of the family gentianaceae are the most diverse, and gentian plants can be used for various purposes such as ornamental and medical purposes, and are mostly traditional Tibetan medicines. The Tibetan medicine 'Banjian' is a general name of various medicinal plants of gentian, is a representative common large Tibetan medicinal material, has the effects of treating virus diseases, various pyretic symptoms and laryngitis fever, and has definite curative effect and wide application.
Gentiana straminea (academic name:Gentiana farreribalf, f.) also called blue flower gentian, "beng jian wu yan bao", gentian flower, which is distributed in the four Sichuan, Tibet, Gansu, Qinghai, etc. areas of continental China, growing at an altitude of 2, 410 m to 4, 600 m, with small leaves, light blue flowers, growing mostly in bushes, mountain meadows and swamp beaches. The Bingzhong is derived from various medicinal plants of gentiana of Gentianaceae, is a representative common large Tibetan medicinal material, and has the effects of treating virus diseases, various pyretic symptoms and laryngitis. At present, more than 10 Tibetan medicine enterprises have 14 Tibetan medicine compound preparations with national medicine standard word sizes, namely ten-ingredient gentiana granule (capsule), three-ingredient gentiana tablet pill and fifteen-ingredient gentiana pill (powder); the gentian flower is sold in medicinal material markets of Qinghai, Sichuan, Tibet, Gansu and the like and local specialty door markets, and is one of the most common Tibetan medicinal materials in China. If the efficacy is different according to the classification of 'between charts', the 'between charts' nature is rather cool, and the situation that the basic varieties of 'between charts' are complex and are mutually substituted and used is seen in the current situation of use.
application of Dingwenya and the like to gentianella trifida by HPLC methodG. trifloraRadix Gentianae MacrophyllaeG. crassicaulisRadix Gentianae, DAWULIG. dahuricagentian root of blue jade hairpinG. veitchiorumGentiana macrophyllaG. macrophyllaThe content of gentiopicroside is measured. Paeonia ostii Hayata et al by HPLCG.ferreriThe oleanolic acid, mangiferin and swertisin were separated and analyzed. Zhang Xingwang establishes the cloud gentianG. nubigertaHPLC content determination method of swertiamarin and isoorientin is provided. And determining contents of swertiamarin and gentiopicroside in Gentiana yunnanensis flowers in different regions. Zhang Xiaolong, etc. applied HPLC method to determine the content of swertia amara, gentiopicrin, swertiamara and isoorientin in 10 wild medicinal materials of Gentiana glauca, Gentiana rigescens, Gentiana spicata, Gentiana yunnanensis, Gentiana striolata, Gentiana lineolata, Gentiana crassipes, Gentiana macrophylla, etc. in Qinghai-Tibet plateau. Huang Jie et al, Dan Min county GentianaG.purdomiiand herba Hedyotis Auriculariae, and thin layer chromatography identification is performed with oleanolic acid and gentiopicrin as reference substances. Liu Yuan and the like establish the effect of compound gentiana baihua and gentiana regale in Baihua GentianaG. purdomiithe TCL method of (1), and gentiopicroside as a control. As can be seen, most of various medicinal plants in Gentiana take gentiopicroside as an index component for thin-layer identification and content determination.
At present, only old and old farmers, Huntingnong, xu-pass plum and the like exist in China: the method has coarse and shallow researches on the aspects of histology, embryology, chemistry and clinical application of gentiana filiformis flowers, and has only a few researches on the aspects of resources, distribution, ecological current situation, multi-index effective components and pharmacological action. However, the Tibetan medicine is still lack of systematic research, the variety is complex, the medicinal parts are unclear, the classification is disordered, the use condition of the gentiana filiformis flowers needs to be comprehensively mastered urgently, the disordered variety is clarified, and the mainstream is cleared.
Disclosure of Invention
the invention aims to provide an ultra-high performance liquid detection method of gentiana straminea to solve the problems.
In order to achieve the purpose, the invention adopts the technical scheme that:
the ultra-high performance liquid detection method of gentiana straminea is characterized by comprising the following steps:
(1) Preparing a test solution: picking up weeds, crushing gentiana lineolata flowers to be detected, sieving the crushed gentiana lineolata flowers with a 60-mesh sieve, weighing 1g of gentiana lineolata flower powder to be detected, ultrasonically extracting the powder with methanol, filtering an extracting solution, and filtering the extracting solution with a microporous filter membrane to obtain a test solution;
(2) Ultra-high performance liquid chromatography analysis: injecting the sample solution into an ultra-high performance liquid chromatograph, and performing gradient elution by using methanol as a mobile phase A and water as a mobile phase B; the conditions of the ultra-high performance liquid chromatography are as follows: the chromatographic column is an ultra-high performance liquid C18 chromatographic column; the flow rate is 0.3 mL/min; the sample injection amount is 1 mu L; the detection wavelength is 242 nm; the column temperature is 30 ℃; gradient elution procedure is 0-10min, A is 5% -17%; 10-13min, wherein A is 17% -19%; 13-24min, wherein A is 19-35%; 24-32min, wherein A is 35% -90%; analyzing the test solution under the conditions to obtain an ultra-high performance liquid chromatogram of the gentiana straminea, and comparing the chromatogram with a chromatogram of a standard substance.
Wherein the standard substance in the step (2) comprises swertiamarin, loganin, gentiamarin, isoorientin, 6' -O-beta-D-glucosyl gentiamarin and isovitexin.
In order to better implement the present invention, the methanol in the step (1) is 60% methanol.
in order to better implement the invention, the ultrasonic time in the step (1) is 40 min.
in order to better implement the invention, the filter paper used in the filtration in the step (1) is a medium-speed filter paper with a length of 12.5 cm.
in order to better implement the invention, the microporous filter membrane in the step (1) adopts a 0.22 μm microporous filter membrane.
Compared with the prior art, the invention has the beneficial effects that:
1. The ultra-high performance liquid chromatogram obtained by the detection method can be effectively applied to and used for distinguishing Gentiana lineolata and similar varieties and mixed counterfeit products thereof.
2. The ultra-high performance liquid chromatogram obtained by the invention can be used for overall evaluation and control of the quality of the gentiana lineata flower, and the one-sidedness caused by comparing only individual chemical components in the total components when judging the authenticity and the quality of the gentiana lineata flower is avoided.
3. The detection method has good reproducibility and high separation degree, and is very suitable for the determination of the index components of the gentiana straminea and the establishment of a fingerprint.
4. The ultra-high performance liquid phase is able to accept higher throughput and smaller sample sizes, thus providing better selectivity and better detection and quantitation limits for use. The experimental time and the experimental result show that the experimental time can be greatly shortened by the ultra-high-efficiency liquid phase under the same condition, the detection rate is improved, the detection result is more accurate, the consumption of the organic solvent is obviously reduced, the analysis cost can be effectively reduced, and the experimental efficiency is improved.
5. In analysis, the ultra-high performance liquid phase can greatly shorten the analysis period of a sample, and can obviously enhance the separation degree and sensitivity in chromatographic peak detection. And has good reproducibility, high accuracy, good specificity, novel method, and lower detection limit and quantification limit.
Drawings
FIG. 1 is an ultra-high performance liquid chromatogram of a Gentiana lineolata flower-like product obtained by the detection method of the invention, wherein 1-7 in the chromatogram are loganin, swertiamarin, 6' -O-beta-D-glucosyl gentiamarin, swertiamarin, isoorientin and isovitexin in sequence;
FIG. 2 is a chromatogram of loganin acid standard;
FIG. 3 is a chromatogram of a swertiamarin standard sample;
FIG. 4 is a chromatogram of a 6' -O- β -D-glucosyl gentiopicroside standard;
FIG. 5 is a chromatogram of a gentiopicroside control sample;
FIG. 6 is a chromatogram of a reference sample of sweroside;
FIG. 7 is a chromatogram of a isoorientin reference substance;
FIG. 8 is a chromatogram of a control of isovitexin;
FIG. 9 is a chromatogram of a mixed control, wherein the numbers 1-7 are as follows: 1. loganin acid; 2. swertiamarin; 3. 6' -O-beta-D-glucosyl gentiopicroside; 4. gentiopicroside; 5. swertiamarin of swertia; 6. isoorientin; 7. isovitexin.
Detailed Description
The invention is further described with reference to the following figures and detailed description.
The ultra-high performance liquid detection method of gentiana straminea is characterized by comprising the following steps:
(1) Preparing a test solution: picking up weeds, crushing gentiana lineolata flowers to be detected, sieving the crushed gentiana lineolata flowers with a 60-mesh sieve, weighing 1g of gentiana lineolata flower powder to be detected, ultrasonically extracting the powder with methanol, filtering an extracting solution, and filtering the extracting solution with a microporous filter membrane to obtain a test solution;
(2) ultra-high performance liquid chromatography analysis: injecting the sample solution into an ultra-high performance liquid chromatograph, and performing gradient elution by using methanol as a mobile phase A and water as a mobile phase B; the conditions of the ultra-high performance liquid chromatography are as follows: the chromatographic column is an ultra-high performance liquid C18 chromatographic column Acquity UPLC BEH C18(50mm multiplied by 2.1mm,1.7 μm); the flow rate is 0.3 mL/min; the sample injection amount is 1 mu L; the detection wavelength is 242 nm; the column temperature is 30 ℃; gradient elution procedure is 0-10min, A is 5% -17%; 10-13min, wherein A is 17% -19%; 13-24min, wherein A is 19-35%; 24-32min, wherein A is 35% -90%; analyzing the test solution under the conditions to obtain an ultra-high performance liquid chromatogram of the gentiana straminea, and comparing the chromatogram with a chromatogram of a standard substance.
Wherein the standard substance in the step (2) comprises swertiamarin, loganin, gentiamarin, isoorientin, 6' -O-beta-D-glucosyl gentiamarin and isovitexin.
In order to better implement the present invention, the methanol in the step (1) is 60% methanol.
In order to better implement the invention, the ultrasonic time in the step (1) is 40 min.
In order to better implement the invention, the filter paper used in the filtration in the step (1) is a medium-speed filter paper with a length of 12.5 cm.
In order to better implement the invention, the microporous filter membrane in the step (1) adopts a 0.22 μm microporous filter membrane.
experimental example:
1. Test instruments and reagents:
(1) Test apparatus (see table 1):
TABLE 1 test instrument information sheet
(2) Reagent:
Methanol (chromatographically pure, Saimer Feishell science and technology Co., Ltd.), methanol (analytically pure, Dacron chemical reagent factory, Tianjin), and purified water (distilled water of Drech.).
2. sample and control:
(1) The sample is selected from flower of Gentiana veitchiana (Gentiana veitchiana) of Gentiana of Gentianaceae, collected from Manri Maxiang of Maqu county of Gansu provinceGentiana farreri Balf. f.
(2) the control samples were purchased from Shanghai leaf Biotech, Inc. and the information is shown in Table 2.
TABLE 2 reference information sheet
3. Preparing a test solution: picking up weeds, crushing gentiana straminea maxim to be detected, sieving with a 60-mesh sieve, weighing 1g of gentiana straminea maxim powder to be detected, carrying out ultrasonic extraction for 40min by using 60% methanol, filtering an extracting solution, and filtering with a microporous filter membrane to obtain a sample solution;
4. Ultra-high performance liquid chromatography analysis: injecting the sample solution into an ultra-high performance liquid chromatograph, and performing gradient elution by using methanol as a mobile phase A and water as a mobile phase B; the conditions of the ultra-high performance liquid chromatography are as follows: the column was Acquity UPLC BEH C18(50 mm. times.2.1 mm,1.7 μm); the flow rate is 0.3 mL/min; the sample injection amount is 1 mu L; the detection wavelength is 242 nm; the column temperature is 30 ℃; gradient elution procedure is 0-10min, A is 5% -17%; 10-13min, wherein A is 17% -19%; 13-24min, wherein A is 19-35%; 24-32min, wherein A is 35% -90%; analyzing the sample solution under the above conditions to obtain ultra high performance liquid chromatogram of Gentiana lineolata (FIG. 1), and comparing with chromatogram of standard product (FIGS. 2-8) and chromatogram of mixed control product (FIG. 9).
The standard substance comprises swertiamarin, loganin, gentiamarin, isoorientin, 6' -O-beta-D-glucosyl gentiamarin and isovitexin.
wherein the filter paper used for filtration is medium-speed filter paper with the diameter of 12.5 cm.
Wherein the microporous filter membrane is a 0.22 μm microporous filter membrane.
5. methodology investigation:
(1) Preparation of a reference solution:
Accurately weighing a proper amount of a swertiamarin reference substance, a strychnide reference substance, a gentiopicrin reference substance, an isoorientin reference substance, a 6' -O-beta-D-glucosyl gentiopicrin reference substance and an isovitexin reference substance respectively in a 5mL volumetric flask, adding a proper amount of methanol for dissolving, and after dissolving, fixing the volume to scale by using the methanol to obtain the stock solutions of the reference substances. The stock solutions were each 1mL each in a 10mL volumetric flask, diluted to the scale with methanol, and mixed control solutions at mass concentrations of 0.022, 0.030, 0.040, 0.020, 0.018, and 0.024 mg/mL were prepared, and filtered through a 0.22 μm microfiltration membrane, for use.
(2) Determination of gradient elution procedure:
During the process of adjusting the elution program, the separation degrees of the 1-peak loganin, the 2-peak swertiamarin, the 5-peak swertiamarin, the 6-peak isoorientin and the 7-peak isovitexin are high. The peak emergence time of the No. 3 peak 6 '-O-beta-D-glucosyl gentiopicroside is very similar to that of the No. 4 peak gentiopicroside, because the two compounds belong to iridoid glycoside compounds, hydrogen in the gentiopicroside structure is replaced by glucosyl to be changed into 6' -O-beta-D-glucosyl gentiopicroside, the structures are very similar, the separation difficulty of the two compounds is large, the elution is selected at the isocratic of 10-13min at first, the elution effect is not ideal, and the separation effect is good after the methanol proportion is increased from 17% to 19% through trying. Through multiple experiments, the best effect was found under the following elution conditions, and finally the gradient elution procedure was determined as shown in table 3.
TABLE 3 procedure for mobile phase gradient elution
(3) Determination of column temperature
respectively setting column temperature gradients of 20 ℃, 25 ℃, 30 ℃ and 35 ℃ in sequence for tests, and finding that the peak shape is widened and the peak-out time is prolonged at a lower temperature; when the temperature is too high, many components are difficult to separate, and the phenomenon of adhesion of chromatographic peaks occurs. The separation effect is best because the peak shape is sharp and the separation degree is high at 30 ℃, so that the column temperature is finally determined to be 30 DEG C
(4) Determination of detection wavelength
According to the data, the detection wavelengths of gentiopicrin, swertiamarin and the like are mostly concentrated between 240-250nm, and the detection wavelengths are sequentially set to be 240nm, 242nm, 244nm, 246nm, 248nm and 250 nm. The baseline is stable when the wavelength is 242nm and the resolution is high through respective sample injection, and the detection wavelength is finally determined to be 242 nm.
(5) Determination of flow rate
The flow rates were set to 0.15mL/min, 0.2mL/min, 0.25mL/min, and 0.3mL/min in this order. When the flow rate was 0.3mL/min, the peak appearance was fast and the separation effect was good, so that it was determined that the flow rate was 0.3 mL/min.
Chromatograms for separating the test sample and the standard sample under the conditions of mobile phase, elution procedure, column temperature, detection wavelength and flow rate chromatogram determined in the above experiment are shown in fig. 1-8.
(6) Finger print conditional exploration
further exploring fingerprint spectrum conditions on the basis of the prior UPLC chromatographic conditions, and finding out through experiments that gradient elution is carried out by taking methanol-water as a mobile phase, wherein the mobile phase A: methanol, mobile phase B: and (3) water. The gradient elution procedure was: 0-10min, A is 5% -17%; 10-13min, wherein A is 17% -19%; 13-24min, wherein A is 19-35%; 24-32min, wherein A is 35-90%. The flow rate is 0.3mL/min, the detection wavelength is 242nm, and the column temperature: 30 ℃; sample introduction amount: 1 μ L. The separation effect is better. The chromatograms of the test sample and the mixed reference are shown in FIGS. 1 and 9.
Claims (6)
1. The ultra-high performance liquid detection method of gentiana straminea is characterized by comprising the following steps:
(1) Preparing a test solution: picking up weeds, crushing gentiana lineolata flowers to be detected, sieving the crushed gentiana lineolata flowers with a 60-mesh sieve, weighing 1g of gentiana lineolata flower powder to be detected, ultrasonically extracting the powder with methanol, filtering an extracting solution, and filtering the extracting solution with a microporous filter membrane to obtain a test solution;
(2) ultra-high performance liquid chromatography analysis: injecting the sample solution into an ultra-high performance liquid chromatograph, and performing gradient elution by using methanol as a mobile phase A and water as a mobile phase B; the conditions of the ultra-high performance liquid chromatography are as follows: the chromatographic column is an ultra-high performance liquid C18 chromatographic column; the flow rate is 0.3 mL/min; the sample injection amount is 1 mu L; the detection wavelength is 242 nm; the column temperature is 30 ℃; gradient elution procedure is 0-10min, A is 5% -17%; 10-13min, wherein A is 17% -19%; 13-24min, wherein A is 19-35%; 24-32min, wherein A is 35% -90%; analyzing the test solution under the conditions to obtain an ultra-high performance liquid chromatogram of the gentiana straminea, and comparing the chromatogram with a chromatogram of a standard substance.
2. the ultra-high performance liquid detection method of gentiana lineata according to claim 1, wherein: the standard substance in the step (2) comprises swertiamarin, loganin, gentiamarin, isoorientin, 6' -O-beta-D-glucosyl gentiamarin and isovitexin.
3. The ultra-high performance liquid detection method of gentiana lineata according to claim 1 or 2, characterized in that: the concentration of the methanol in the step (1) is 60%.
4. The ultra-high performance liquid detection method of gentiana lineata according to claim 3, wherein: the ultrasonic time in the step (1) is 40 min.
5. The ultra-high performance liquid detection method of gentiana lineata according to claim 4, wherein: the filter paper used for the filtration in the step (1) is medium-speed filter paper with the diameter of 12.5 cm.
6. The ultra-high performance liquid detection method of gentiana lineata according to claim 5, wherein: in the step (1), the microfiltration membrane adopts a 0.22 mu m microfiltration membrane.
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| CN117074595A (en) * | 2023-07-25 | 2023-11-17 | 广东一方制药有限公司 | Method for constructing characteristic spectrum of swertia Japonica Makino and application thereof |
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Application publication date: 20191217 |