CN109632994B - Method for establishing UPLC (ultra Performance liquid chromatography) spectrum of cowherb seed and fried cowherb seed and application of UPLC spectrum - Google Patents
Method for establishing UPLC (ultra Performance liquid chromatography) spectrum of cowherb seed and fried cowherb seed and application of UPLC spectrum Download PDFInfo
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- 238000004704 ultra performance liquid chromatography Methods 0.000 title claims abstract description 40
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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Abstract
The invention provides a method for establishing a UPLC (ultra performance liquid chromatography) spectrum of cowherb seed and fried cowherb seed and application thereof. The invention relates to a method for establishing a UPLC (ultra performance liquid chromatography) spectrum of cowherb seeds or fried cowherb seeds, which comprises the following steps: preparation of a test solution: taking the cowherb seed or the test sample powder of the fried cowherb seed, adding 75% ethanol, carrying out ultrasonic treatment, filtering, and taking the filtrate to obtain a test sample solution; detecting by a high performance liquid chromatograph: and detecting the test solution by using a Waters high performance liquid chromatograph to establish a UPLC spectrum. During identification, the cowherb seed flavonoid glycoside of the UPLC spectrum is taken as a reference peak, the relative peak area of a characteristic peak with the relative retention time within the range of 1.25 +/-10% is calculated, the cowherb seed is taken as the relative peak area within the range of 0.068-0.149, and the cowherb seed is fried within the range of 0.183-0.411.
Description
Technical Field
The invention relates to a method for establishing a UPLC (ultra performance liquid chromatography) spectrum of cowherb seed and fried cowherb seed and an application method for identifying cowherb seed and fried cowherb seed.
Background
Dried mature seed of Vaccaria segetalis (Neck.) Garcke of Caryophyllaceae is named as semen Vaccariae, Vaccinium vitis-idaea, flos Daturae Metelis, herba Scissiae Japonicae, flos Impatientis, and fructus Vaccinii Vitis-idaeae. The cowherb seed contains various chemical components, such as triterpenoid saponin, cyclic peptide, flavone, alkaloid, phenolic acid, steroid, volatile oil, etc., wherein the content of the triterpenoid saponin is the highest, and the flavone and the cyclic peptide are the second.
At present, the processing method of the cowherb seed in the pharmacopoeia is a stir-frying method. The ancient statement of "stir-frying when eating twice" is provided, but at present, the bioavailability of the sub-class of traditional Chinese medicines is considered to be possibly increased after being stir-fried. Few reports about the processing mechanism research of the stir-fried cowherb seed are reported. Researches show that after the seed is fried, the extract content of the seed cowherb is greatly increased. However, how the main active ingredients change in the processing and dissolution processes and whether the water decoction dissolution rate can be increased or not are not discussed at present. And the research literature on the characteristic spectrum of the (fried) cowherb seed is less at present. Fingerprint spectrums of roasted cowherb seeds in different producing areas are subjected to correlation research by high performance liquid chromatography, and the result shows that the similarity of the fingerprint spectrums of the roasted cowherb seeds in 10 batches of samples in different producing areas and the comparison fingerprint spectrum is more than 0.99 (Huangyi, Ouyangbo, and the like, HPLC fingerprint spectrums and content determination research of the roasted cowherb seeds in different producing areas [ J ]. Hunan: "Zhongnan pharmacy", 2017,15(7): 879-882). Chenlin et al established fingerprint of parched semen Vaccariae formula granule by high performance liquid chromatography, and provided basis for quality standard of parched semen Vaccariae formula granule (Chenlin, Huchangjiang, Von Jian, etc., high performance liquid fingerprint research of parched semen Vaccariae formula granule [ J ]. Asia-Pacific traditional medicine, 2013, (4):16-18.DOI:10.3969/J. issn.1673-2197.2013.04.009). A characteristic spectrum method of semen Vaccariae is established by HPLC in hong Kong Chinese medicinal material Standard. Sunwerin et al measured the change of flavonoid glycoside content in processed semen Vaccariae by UPLC method, and after processing, the content of Vaccarin (Vaccarin) in semen Vaccariae was greatly reduced (Sunwerin, tomahawk, Xurea, UPLC method measured the content of Vaccarin in semen Vaccariae before and after processing [ J ]. Yatai traditional medicine (2015 (20): 20-21).
Disclosure of Invention
The invention aims to provide a method for establishing a UPLC (ultra performance liquid chromatography) spectrum of the cowherb seeds or the fried cowherb seeds, which has good repeatability and accuracy.
Another object of the present invention is to provide a method for identifying whether a sample is cowherb seed or parched cowherb seed.
In one aspect, the invention provides a method for establishing a UPLC (ultra performance liquid chromatography) spectrum of cowherb seeds or fried cowherb seeds, which comprises the following steps:
preparation of a test solution: taking the cowherb seed or the test sample powder of the fried cowherb seed, adding 75% ethanol, carrying out ultrasonic treatment, filtering, and taking the filtrate to obtain a test sample solution;
detecting by a high performance liquid chromatograph: and detecting the test solution by using a Waters high performance liquid chromatograph to establish a UPLC spectrum.
According to the specific embodiment of the present invention, in the method for establishing the UPLC spectrum of the cowherb seeds or the parched cowherb seeds, in the preparation process of the test solution, the ratio of the test powder to the ethanol is as follows: 1g of test powder: 25ml of 75% ethanol. Preferably, the test article powder passes through a third sieve.
According to the specific embodiment of the invention, in the establishing method of the UPLC spectrum of the cowherb seeds or the fried cowherb seeds, the ultrasonic treatment conditions in the preparation process of the test solution are as follows: the power is 250W, the frequency is 33KHz, and the ultrasonic treatment is carried out for 30 minutes.
According to the specific embodiment of the invention, in the establishing method of the UPLC spectrum of the cowherb seeds or the fried cowherb seeds, YMC Triart C18 is taken as a chromatographic column in the detection process of a high performance liquid chromatograph. Preferably, the YMC Triart C18 chromatography column is of the following specification: 100 mm. times.2.1 mm, 1.9 μm.
According to the specific embodiment of the invention, in the establishing method of the UPLC spectrum of the cowherb seed or the fried cowherb seed, the sample injection amount is 1 mul when the detection is carried out by a high performance liquid chromatograph.
According to the specific embodiment of the invention, in the establishing method of the UPLC spectrum of the cowherb seed or the fried cowherb seed, acetonitrile-water is taken as a mobile phase for gradient elution in the detection process of a high performance liquid chromatograph, and the flow rate is 0.35ml per minute; the column temperature was 35 ℃; the detection wavelength was 219 nm. More specifically, in the detection process of the high performance liquid chromatograph, acetonitrile is used as a mobile phase A, an aqueous solution is used as a mobile phase B, and gradient elution is carried out according to the following conditions: 0-5.5 min, 10% → 15% A; 5.5-11 min, 15% → 30% A; 11-16.5 min, 30% → 70% A; 16.5-16.51 min, 70% → 10% A; 16.51-23 min, 10% A.
The method for establishing the UPLC spectrum of the cowherb seeds or the fried cowherb seeds has the advantages that the prepared test solution has good stability, the UPLC detection separation degree is good, the detection time is greatly shortened, and the repeatability and the accuracy are good.
In another aspect, the present invention provides a method for identifying whether a sample is cowherb seed or parched cowherb seed, the method comprising:
preparing a sample solution of a sample according to the establishing method of the UPLC spectrum of the cowherb seed or the fried cowherb seed and establishing the UPLC spectrum;
and identifying the sample as the cowherb seed or the fried cowherb seed according to the characteristic peak of the UPLC spectrum of the sample.
In the research of the inventor, the UPLC spectrums of a large number of cowherb seed medicinal materials and the fried cowherb seed decoction pieces are established according to the method, and the analysis and research show that the relative peak area of a characteristic peak (referred to as peak 3 in the invention) with the relative retention time of 1.25 +/-10% by taking the cowherb seed flavonoid glycoside as a reference peak is obviously different from that of cowherb seed and fried cowherb seed. The relative peak area of the peak 3 of the cowherb seed medicinal material is 0.068-0.149, the relative peak area of the peak 3 of the fried cowherb seed decoction piece is 0.183-0.411, and the relative peak area of the peak 3 of the fried cowherb seed decoction piece is larger than that of the cowherb seed, which shows that the peak area of the peak 3 of the cowherb seed is correspondingly increased after the processing. The peak 3 can distinguish the cowherb seed from the fried cowherb seed. Thus, according to an embodiment of the present invention, the method for identifying whether the sample is cowherb seed or cowherb seed parched further comprises: and (3) taking the flavonoid glycoside of the cowherb seeds as a reference peak, calculating the relative peak area of the characteristic peak with the relative retention time within the range of 1.25 +/-10%, and identifying the sample as the cowherb seeds or fried cowherb seeds according to the relative peak area. More specifically, the relative peak area is within the range of 0.068-0.149, and the sample is identified as the cowherb seed; and identifying the sample as the fried cowherb seed when the relative peak area is within the range of 0.183-0.411.
In conclusion, the invention provides the method for establishing the UPLC spectrum of the cowherb seed or the fried cowherb seed, the prepared test solution has good stability and better UPLC detection separation degree, the detection time is greatly shortened, and the method has good repeatability and accuracy. The invention also provides a method for identifying whether the sample is the cowherb seed or the fried cowherb seed, and the identification method can simply, accurately and reliably distinguish the cowherb seed from the fried cowherb seed.
Drawings
Fig. 1 is an overlay chromatogram of 20 different sources/batches of vaccaria segetalis medicinal material. Wherein, peak 1: erythrina base, peak 2 (S): semen Vaccariae flavonoid glycoside.
FIG. 2 is a comparison characteristic spectrum of a cowherb seed medicinal material.
FIG. 3 is an overlay chromatogram of 20 batches of parched semen Vaccariae decoction pieces from different sources/batches. Wherein, peak 1: erythrina base, peak 2 (S): semen Vaccariae flavonoid glycoside.
FIG. 4 is a reference characteristic spectrum of parched semen Vaccariae decoction pieces.
Detailed Description
The method of the present invention is described below with reference to specific examples to make it easier to understand and understand the technical solution of the present invention, but the present invention is not limited thereto.
Example 1
1 Material
1.1 instruments and reagents
The instrument comprises the following steps: waters high performance liquid chromatograph (Waters corporation), YMCTriartC18(2.1 × 100mm, 1.9 μm), millionth balance (mettler-toledo, ME204E), millionth of a day (mettler-toledo, XP 26).
Reagent: ethanol (Yongda chemical reagents, Inc. of Tianjin) is analytical grade, methanol (Guangdong Guanghua science and technology, Inc.) is analytical grade, acetonitrile (Merck, Inc.) is chromatographic grade, and water is ultrapure water (Mili-Qdirect, Inc.).
Reagent testing: erythrine (Shanghai Yuan leaf Biotechnology Co., Ltd., content: 98.0%, batch number: P26N6F6550), and cowherb seed flavonoid glycoside (China food and drug testing institute, content: 99.7%, batch number: 111853-201704).
2. Method and results
2.1 UPLC characteristic map of cowherb seed
2.1.1 chromatographic conditions
Gradient elution is carried out by taking YMCC18(100mm multiplied by 2.1mm, 1.9 μm) as a chromatographic column, acetonitrile as a mobile phase A and an aqueous solution as a mobile phase B (0-5.5 min, 10% → 15% A; 5.5-11 min, 15% → 30% A; 11-16.5 min, 30% → 70% A; 16.5-16.51 min, 70% → 10% A; 16.51-23 min, 10% A); the flow rate was 0.35ml per minute; the column temperature was 35 ℃; the detection wavelength is 219 nm; the amount of sample was 1. mu.l.
2.1.2 preparation of control solutions
Respectively taking appropriate amount of erythrine reference substance and cowherb seed flavonoid glycoside reference substance, adding methanol to obtain solutions containing erythrine 32 μ g per 1ml, and adding 70% methanol to obtain solutions containing cowherb seed flavone 150 μ g per 1 ml.
2.1.3 preparation of test solutions
Precisely weighing about 1g of semen Vaccariae powder (sieved by a third sieve), placing into a conical flask with a plug, precisely adding 25ml of 75% ethanol, weighing, ultrasonically treating (power 250W, frequency 33KHz) for 30 min, cooling, shaking, filtering, and collecting the filtrate.
2.2 verification of characteristic spectrum methodology of seed of cowherb
2.2.1 precision test
Repeatedly injecting the cowherb seed sample solution for 6 times under the condition of '2.1' item of chromatography. The cowherb seed flavonoid glycoside is taken as a reference peak, the relative retention time and the relative peak area are calculated, and the result shows that the relative retention time and the relative peak area RSD of the 4 common peaks are both less than 3 percent, which indicates that the precision of the instrument is good.
2.5.2 stability test
Sampling and analyzing the cowherb seed sample solution for 0, 2, 4, 6, 12 and 24 hours respectively according to the chromatographic conditions of the item 2.1. The cowherb seed flavonoid glycoside is taken as a reference peak, the relative retention time and the relative peak area are calculated, and the result shows that the relative retention time and the relative peak area RSD of the 4 common peaks are less than 3 percent, which shows that the sample is stable within 24 h.
2.5.3 repeatability test
Taking 6 parts of the cowherb seed sample solution, treating according to the preparation method of the sample solution of item 2.1.3, and measuring according to the chromatographic condition of item 2.1. The relative retention time and the relative peak area are calculated by taking the cowherb seed flavonoid glycoside as a reference peak, and the result shows that the relative retention time and the relative peak area RSD of the 4 common peaks are both less than 3 percent. The method is shown to have good repeatability.
2.3 sample determination
Taking 20 batches of cowherb crude drug samples and fried cowherb decoction piece samples, preparing a sample solution according to a sample solution preparation method determined under the item 2.1.3, carrying out sample injection determination according to chromatographic conditions determined under the item 2.1, carrying out common peak identification on the characteristic spectrums of the cowherb crude drugs and the fried cowherb decoction pieces of 20 batches of cowherb crude drugs by using traditional Chinese medicine chromatographic fingerprint similarity evaluation software, and producing a reference spectrum.
The overlapped chromatogram of 20 batches of the cowherb seed medicinal material is shown in figure 1, the comparison characteristic spectrum of the cowherb seed medicinal material is shown in figure 2, the overlapped chromatogram of 20 batches of the fried cowherb seed decoction pieces is shown in figure 3, and the comparison characteristic spectrum of the fried cowherb seed decoction pieces is shown in figure 4. The results of the relative peak areas of the common peaks of the 20 batches of the cowherb seeds and the stir-fried cowherb seeds are shown in the table 1 and the table 2.
Table 1: 20 batches of cowherb seed medicinal material characteristic map (relative peak area)
Table 2: feature spectrum (relative peak area) of 20 batches of fried cowherb seed decoction pieces
The relative peak area ranges of the characteristic spectrums of 20 batches of the cowherb seeds and the fried cowherb seeds are shown in a table 3.
Table 3: the relative peak area range of the characteristic peak of the cowherb seed medicinal material and the fried cowherb seed decoction piece
| Peak number | Cowherb seed medicinal material | Decoction pieces of parched |
| Peak | ||
| 1 | 0.829～1.333 | 0.901～1.539 |
| Peak 2(S) | 1.000 | 1.000 |
| |
0.068～0.149 | 0.183～0.411 |
| Peak 4 | 0.129～0.247 | 0.128～0.283 |
| Peak 5 | 0.048～0.082 | 0.062～0.122 |
As can be seen from the comparison results of the data in the table 3, in the cowherb seed medicinal material and the fried cowherb seed decoction pieces, the relative peak area of the peak 3 of the cowherb seed medicinal material is 0.068-0.149, the relative peak area of the peak 3 of the fried cowherb seed decoction pieces is 0.183-0.411, and the relative peak area of the peak 3 of the fried cowherb seed decoction pieces is larger than that of the cowherb seed, which indicates that the peak area of the peak 3 is correspondingly increased after the cowherb seed is processed. The peak 3 can distinguish the cowherb seed from the fried cowherb seed. And the relative peak area of the peak 3 is within the range of 0.068-0.149, namely the cowherb seeds are fried, and the relative peak area of the peak is within the range of 0.183-0.411, namely the cowherb seeds are fried.
Claims (4)
1. A method for establishing a UPLC (ultra Performance liquid chromatography) spectrum of cowherb seeds or fried cowherb seeds comprises the following steps:
preparation of a test solution: taking the cowherb seed or the test sample powder of the fried cowherb seed, adding 75% ethanol, carrying out ultrasonic treatment, filtering, and taking the filtrate to obtain a test sample solution;
detecting by a high performance liquid chromatograph: detecting the test solution by using a Waters high performance liquid chromatograph to establish a UPLC spectrum;
wherein, in the preparation process of the test solution, the ratio of the test powder to the ethanol is as follows: 1g of test powder: 25ml of 75% ethanol;
in the detection process of the high performance liquid chromatograph, the YMC Triart C18 is used as a chromatographic column, and the specification of the YMC Triart C18 chromatographic column is as follows: 100mm × 2.1mm, 1.9 mm; performing gradient elution with acetonitrile-water as mobile phase at flow rate of 0.35 ml/min; the column temperature was 35 ℃; the detection wavelength is 219 nm;
in the detection process of the high performance liquid chromatograph, acetonitrile is used as a mobile phase A, aqueous solution is used as a mobile phase B, and gradient elution is carried out according to the following conditions: 0-5.5 min, 10% → 15% A; 5.5-11 min, 15% → 30% A; 11-16.5 min, 30% → 70% A; 16.5-16.51 min, 70% → 10% A; 16.51-23 min, 10% A.
2. The method of claim 1, wherein the sonication conditions during the preparation of the test solution are: the power is 250W, the frequency is 33KHz, and the ultrasonic treatment is carried out for 30 minutes.
3. The method of claim 1, wherein the amount of sample is 1 μ l in HPLC detection.
4. A method of identifying whether a sample is cowherb seed or parched cowherb seed, the method comprising:
preparing a sample test solution of a sample and establishing a UPLC spectrum according to the method of any one of claims 1 to 3;
identifying whether the sample is the cowherb seed or the fried cowherb seed according to the characteristic peak of the UPLC spectrum of the sample;
wherein, taking the cowherb seed flavonoid glycoside as a reference peak, calculating the relative peak area of a characteristic peak within the range of relative retention time of 1.25 +/-10 percent, and identifying whether the sample is cowherb seed or fried cowherb seed according to the relative peak area;
identifying the sample as the cowherb seed when the relative peak area is within the range of 0.068-0.149; and identifying the sample as the fried cowherb seed when the relative peak area is within the range of 0.183-0.411.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009117828A1 (en) * | 2008-03-26 | 2009-10-01 | National Research Counsil Of Canada | Saponin extract from saponaria spp. and uses thereof |
| CN102399252A (en) * | 2011-12-15 | 2012-04-04 | 成都普思生物科技有限公司 | Preparation method of cowherb seed flavonoid glycoside monomer |
| CN103983704A (en) * | 2013-07-30 | 2014-08-13 | 雷允上药业有限公司 | Fingerprint detection method of cyclomastopathy eliminating pill preparation |
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2018
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| WO2009117828A1 (en) * | 2008-03-26 | 2009-10-01 | National Research Counsil Of Canada | Saponin extract from saponaria spp. and uses thereof |
| CN102399252A (en) * | 2011-12-15 | 2012-04-04 | 成都普思生物科技有限公司 | Preparation method of cowherb seed flavonoid glycoside monomer |
| CN103983704A (en) * | 2013-07-30 | 2014-08-13 | 雷允上药业有限公司 | Fingerprint detection method of cyclomastopathy eliminating pill preparation |
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