CN113533582A

CN113533582A - UPLC fingerprint quality detection method of natural plant antibacterial growth-promoting feed additive and application thereof

Info

Publication number: CN113533582A
Application number: CN202110932277.8A
Authority: CN
Inventors: 姚浪群; 董素军; 高瑞琳; 王微; 刘萍
Original assignee: Hebei Ailiu Bioengineering Co ltd
Current assignee: Hebei Ailiu Bioengineering Co ltd
Priority date: 2021-08-13
Filing date: 2021-08-13
Publication date: 2021-10-22
Anticipated expiration: 2041-08-13
Also published as: CN113533582B

Abstract

The invention relates to the technical field of quality detection methods of feed additives, in particular to a UPLC fingerprint quality detection method of a natural plant antibacterial growth-promoting feed additive and application thereof. The UPLC fingerprint quality detection method of the natural plant antibacterial growth-promoting feed additive provided by the invention comprises the following steps: and (3) performing UPLC detection by using a gradient elution method by using acetonitrile as a mobile phase A and 0.15-0.25% phosphoric acid aqueous solution as a mobile phase B. The similarity between the UPLC fingerprint spectrum and the contrast spectrum of the natural plant antibacterial growth-promoting feed additive established by the method is more than 92 percent, and the characteristics of the natural plant antibacterial growth-promoting feed additive can be effectively represented. The invention provides an effective detection method for quality control and authenticity identification of the natural plant antibacterial growth-promoting feed additive product, has simple and easy operation, and is beneficial to monitoring the quality of the natural plant antibacterial growth-promoting feed additive product.

Description

UPLC fingerprint quality detection method of natural plant antibacterial growth-promoting feed additive and application thereof

Technical Field

The invention relates to the technical field of quality detection methods of feed additives, in particular to a UPLC fingerprint quality detection method of a natural plant antibacterial growth-promoting feed additive and application thereof.

Background

Compared with the traditional antibiotic additive, the natural feed additive derived from plants can well solve a series of problems caused by antibiotic addition. Chinese patent ZL201410834689.8 discloses a natural plant antibacterial growth-promoting feed additive and application thereof. The natural plant antibacterial growth-promoting feed additive has a relatively simple formula, and is stable and comprehensive in effect, safe and efficient; the feed additive has good killing effect on gram-positive bacteria and gram-negative bacteria, has the effects of inhibiting bacteria, killing bacteria, resisting viruses and improving immunity and disease resistance, can completely replace feed antibiotics, is green and environment-friendly, has moderate cost, and is simple and convenient in production method and easy to operate.

The establishment of the fingerprint of the feed additive has important significance for the quality control and the authenticity identification of the feed additive, and is beneficial to ensuring the quality, the efficacy and the safety of the feed additive. The natural plant extracts contain more known and unknown components and more complex components, and for feed additives containing various plant extracts, the establishment of fingerprint spectrums is particularly important for the quality control of the feed additives. At present, no targeted fingerprint quality detection method is available in the prior art for the natural plant antibacterial growth-promoting feed additive disclosed by ZL 201410834689.8.

Disclosure of Invention

The invention aims to provide a UPLC fingerprint quality detection method of a natural plant antibacterial growth-promoting feed additive and application thereof.

The invention discloses a UPLC fingerprint spectrum quality detection method aiming at a natural plant antibacterial growth-promoting feed additive disclosed by ZL201410834689.8, and the preparation raw materials of the natural plant antibacterial growth-promoting feed additive comprise astragalus extract, eucommia bark extract, honeysuckle extract, coptis extract, cinnamon extract, turmeric extract, quassia extract, dandelion extract, aloe extract, nutmeg extract and liquorice extract. Wherein the effective components include astragaloside A, chlorogenic acid, curcumin, caffeic acid, aloin, dehydrodiisobutyronitrile, liquiritin, chicoric acid, etc. In the research and development process, the invention discovers that the common liquid chromatography detection method aiming at one or more effective components such as chlorogenic acid, caffeic acid and the like is adopted to detect the natural plant antibacterial growth-promoting feed additive, or the problems that the detected components are few in variety and difficult to separate a plurality of components well exist, or the problems that the linearity, precision, repeatability or accuracy of detection are poor exist. Through continuous research and development, the invention discovers that the specific gradient elution is carried out by adopting acetonitrile (mobile phase A) -0.2% phosphoric acid aqueous solution (mobile phase B) as a mobile phase, so that the quantity of detected components can be obviously increased, the separation effect of each component is improved, and the accuracy, precision and repeatability of detection are improved. On the basis of the mobile phase selection, in cooperation with the optimization of other detection conditions, the invention provides a set of UPLC fingerprint quality detection method for the natural plant antibacterial growth-promoting feed additive, and the fingerprint of the natural plant antibacterial growth-promoting feed additive obtained by the method can be used for the authenticity identification and quality control of the natural plant antibacterial growth-promoting feed additive.

Specifically, the invention provides the following technical scheme:

the invention provides a UPLC fingerprint spectrum quality detection method of a natural plant antibacterial growth-promoting feed additive, which comprises the following steps: acetonitrile is used as a mobile phase A, 0.15-0.25% phosphoric acid aqueous solution is used as a mobile phase B, UPLC detection is carried out by adopting a gradient elution method,

the natural plant antibacterial growth-promoting feed additive is prepared from the following raw materials in parts by weight: 50-150 parts of astragalus extract, 50-150 parts of eucommia extract, 50-150 parts of honeysuckle extract, 50-150 parts of coptis extract, 50-100 parts of cinnamon extract, 50-100 parts of turmeric extract, 50-100 parts of quassia extract, 50-100 parts of dandelion extract, 50-100 parts of aloe extract, 50-100 parts of nutmeg extract and 50-100 parts of liquorice extract.

Aiming at the mobile phase, the invention screens and optimizes the elution program, and the program for finally obtaining gradient elution comprises the following steps: 0-9min, 10-16% of mobile phase A and 90-84% of mobile phase B; 9-18min, mobile phase A16-26%, mobile phase B84-74%; 18-24min, mobile phase A26-33%, mobile phase B74-67%; 24-28min, 33-10% of mobile phase A and 67-90% of mobile phase B; 28-30min, mobile phase A10% and mobile phase B90%.

The gradient elution is carried out by adopting the flowing and matching elution procedure, so that the effective components in the natural plant antibacterial growth-promoting feed additive can be detected more comprehensively, the effective components are well separated, and the detection accuracy and precision are ensured.

Preferably, the flow rate of the mobile phase is 0.4-0.8 mL-min^-1。

More preferably, the flow rate of the mobile phase is 0.4-0.5 mL-min^-1。

In the method, the wavelength of the UPLC detection is 270-290 nm; preferably 280 nm. Under the condition of the wavelength, the effective ingredients in the natural plant antibacterial growth-promoting feed additive can be detected more comprehensively, and meanwhile, the accuracy and precision of detection are ensured.

In the above method, the chromatographic column used for UPLC detection is a phenyl column. Compared with common chromatographic columns such as C18 and the like, the phenyl column is used for detecting the active ingredients in the natural plant antibacterial growth-promoting feed additive, can detect the active ingredients in the natural plant antibacterial growth-promoting feed additive more comprehensively, and has better separation effect.

Preferably, the chromatography column is a phenyl column having the following specification parameters: SB-Phenyl, 3.0 mm. times.150 mm, 3.5. mu.m.

The column temperature is set to 28-35 ℃ according to the detection conditions of the mobile phase, the elution program, the chromatographic column and the like. Preferably set at 30-32 deg.c.

In the above method, the sample size to be detected is 2 to 5. mu.L.

In the above method, before UPLC detection, a step of preparing a test solution is further included, where the preparing of the test solution includes: dissolving the sample in 75-85% methanol water solution, ultrasonic treating, mixing, and filtering.

Preferably, the test solution is prepared by the following method: dissolving the sample with 75-85% methanol water solution, treating with ultrasound (55-65W, 40-50Hz) for 15-25min, shaking, filtering, and collecting filtrate and filtering membrane as sample solution.

The method also comprises the step of preparing a reference substance solution before UPLC detection, wherein the reference substance comprises chlorogenic acid, caffeic acid, liquiritin and chicoric acid.

Preferably, the chlorogenic acid, caffeic acid, liquiritin and chicoric acid control are dissolved in 75-85% methanol water solution to prepare a control solution with concentration of 0.1-2 mg/mL.

By using the detection method, 17 common peaks are calibrated, wherein 4 known peaks are identified and are respectively chlorogenic acid, caffeic acid, liquiritin and chicoric acid.

As a preferable aspect of the present invention, the detection method includes the steps of:

(1) preparation of chlorogenic acid, caffeic acid, liquiritin and chicoric acid reference solution

Dissolving chlorogenic acid, caffeic acid, liquiritin and chicoric acid reference substances by using 75-85% methanol water solution to prepare a reference substance solution with the concentration of 0.1-2 mg/mL;

(2) preparation of test solution

Dissolving the sample with 75-85% methanol water solution, treating with ultrasound (55-65W, 40-50Hz) for 15-25min, shaking, filtering, and collecting filtrate and filtering membrane as sample solution;

(3) UPLC detection

A chromatographic column: phenyl column (SB-Phenyl, 3.0 mm. times.150 mm, 3.5 μm); mobile phase: acetonitrile-0.2% aqueous phosphoric acid, and gradient elution was performed according to the elution procedure shown in table 1: the flow rate is 0.4-0.5 mL/min^-1(ii) a The detection wavelength is 280 nm; column temperature: the sample size is 2-5 μ L at 30 ℃.

Table 1 mobile phase gradient elution procedure

Time program (min)	Mobile phase A (acetonitrile)%	Mobile phase B (0.2% aqueous phosphoric acid solution)%
			0	10	90
9	16	84
			18	26	74
24	33	67
			28	10	90
30	10	90

The invention also provides application of the detection method in quality control or authenticity identification of the natural plant antibacterial growth-promoting feed additive.

In the application, the natural plant antibacterial growth-promoting feed additive is prepared from the following raw materials in parts by weight: 50-150 parts of astragalus extract, 50-150 parts of eucommia extract, 50-150 parts of honeysuckle extract, 50-150 parts of coptis extract, 50-100 parts of cinnamon extract, 50-100 parts of turmeric extract, 50-100 parts of quassia extract, 50-100 parts of dandelion extract, 50-100 parts of aloe extract, 50-100 parts of nutmeg extract and 50-100 parts of liquorice extract.

The invention has the beneficial effects that: the invention adopts the method of establishing the UPLC fingerprint spectrum for the first time to identify the natural plant antibacterial growth-promoting feed additive product, the similarity between the UPLC fingerprint spectrum of the natural plant antibacterial growth-promoting feed additive established by the method and the comparison spectrum is more than 92 percent, and the characteristics of the feed additive can be effectively represented. The invention provides an effective detection method for quality control and authenticity identification of the natural plant antibacterial growth-promoting feed additive product, has simple and easy operation, and is beneficial to monitoring the quality of the natural plant antibacterial growth-promoting feed additive product.

Drawings

FIG. 1 is an ultra high performance liquid chromatogram of a chlorogenic acid control in example 1 of the present invention.

FIG. 2 is an ultra high performance liquid chromatogram of a caffeic acid control in example 1 of the invention.

FIG. 3 is an ultra-high performance liquid chromatogram of a liquiritin reference substance in example 1 of the present invention.

FIG. 4 is an ultra high performance liquid chromatogram of a chicoric acid control in example 1 of the present invention.

FIG. 5 is a fingerprint of 12 Elephantopi test samples in example 3 of the present invention.

FIG. 6 is an indication of peaks shared by the Epstein samples in example 3 of the present invention, wherein the 7 peak is chlorogenic acid, the 8 peak is caffeic acid, the 9 peak is liquiritin, the 14 peak is chicoric acid, and the others are unknown peaks.

Fig. 7 is an ultra-high performance liquid chromatogram of an eremosu sample and a compound product of a certain plant extract detected at a wavelength of 280nm in example 4 of the present invention, where a is a spectrum of the compound product of the certain plant extract (i.e., an unqualified product), b is a spectrum of the eremosu product, and R is a comparison fingerprint of the eremosu product.

FIG. 8 is an ultra-high performance liquid chromatogram of an Episrawhide sample detected at 237nm and 327nm in comparative example 1.

FIG. 9 is an ultra-high performance liquid chromatogram of an Eleusines sample detected at a wavelength of 280nm in comparative example 2.

FIG. 10 is an ultra-high performance liquid chromatogram of an Eleusines sample detected at a wavelength of 280nm in comparative example 3.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Example 1 establishment of UPLC fingerprint quality testing method for natural plant antibacterial growth promoting feed additive (1)

1. An experimental instrument: agilent 1260Prime hplc (quaternary pump, VWD detector, autosampler, online degassing); a double-frequency numerical control ultrasonic cleaner (KQ-500VD type); an electric heating blowing dry box (WGL-45B) and the like.

2. Experimental reagent: acetonitrile was chromatographically pure (Fisher corporation, USA); phosphoric acid is chromatographically pure (alatin reagent); methanol was chromatographically pure (Fisher corporation, USA); ultrapure water (ultrapure water machine: american oscapu).

3. Detecting an object: ZL201410834689.8 feed additive prepared in example 1 (alvingshu product, processed by the bioengineering ltd, alvinysu, bei alvinysu).

The formula of the feed additive is as follows: 120 parts of astragalus extract, 100 parts of eucommia bark extract, 120 parts of honeysuckle extract, 110 parts of coptis extract, 100 parts of cinnamon extract, 70 parts of turmeric extract, 80 parts of quassia extract, 90 parts of dandelion extract, 60 parts of aloe extract, 60 parts of nutmeg extract and 90 parts of liquorice extract.

The effective component contents of the above extracts are as follows. Astragalus extract: astragalus polysaccharide (UV) with content not less than 60-65%, and astragaloside (HPLC) with content not less than 0.1-0.2%; eucommia ulmoides extract: beverage grade, chlorogenic acid (HPLC) with content not less than 3%; and (3) honeysuckle extract: beverage grade, chlorogenic acid (HPLC) with content not less than 3-5%; the coptis chinensis extract: drinking grade (5: 1); cinnamon extract: drinking grade (10: 1); turmeric extract: curcumin (UV) with the content of more than or equal to 0.9 percent; and (3) bitter wood extract: drinking grade (15: 1); dandelion extract: a blending grade (10:1), caffeic acid (HPLC) with the content being more than or equal to 0.1%; aloe extract: drinking water grade, with the content of barbaloin (UV) more than or equal to 20%, and barbaloin (HPLC) more than or equal to 10%; nutmeg extract: dehydrodiisobutyronitrile (UV) with the content of more than or equal to 0.08 percent; and (3) liquorice extract: and (6:1) a material mixing level, wherein the content of glycyrrhizic acid (UV) is more than or equal to 10%, and the content of glycyrrhizic acid (HPLC) is more than or equal to 2%.

4. Experimental reference substances: chlorogenic acid (batch number: Y24J7K 16726); caffeic acid (batch number: Y17D6C 7672); glycyrrhizin (batch No. P25J7F 18328); chicoric acid (batch: Y24J11Y 119342). The above control samples were purchased from Shanghai leaf Biotech, Inc.

5. The experimental steps are as follows:

(1) preparation of control solutions

Chlorogenic acid: precisely weighing 5mg of chlorogenic acid reference substance, dissolving with 80% methanol, and diluting to 5mL volumetric flask to obtain chlorogenic acid reference substance solution;

caffeic acid: taking 5mg of caffeic acid reference substance, precisely weighing, dissolving with 80% methanol, and diluting to a volume of 5mL to obtain caffeic acid reference substance solution;

liquiritin: taking 5mg of liquiritin reference substance, precisely weighing, dissolving with 80% methanol, and diluting to a volume of 5mL volumetric flask to obtain liquiritin reference substance solution;

chicoric acid: precisely weighing 5mg of chicoric acid reference substance, dissolving with 80% methanol, and diluting to 5mL volumetric flask to obtain chicoric acid reference substance solution.

(2) Preparation of test solution

Taking about 2g of the Elephantopi sample to be detected, precisely weighing, dissolving with 20mL of 80% methanol, treating with ultrasound (60W, 45Hz) for 20min, shaking, filtering, and filtering the filtrate with 0.22 μm filter membrane as sample solution for use.

(3) Chromatographic conditions

A chromatographic column: phenyl column (SB-Phenyl, 3.0 mm. times.150 mm, 3.5 μm); mobile phase: acetonitrile (mobile phase a) -0.2% aqueous phosphoric acid (mobile phase B), gradient elution was performed according to the elution procedure shown in table 1: flow rate 0.43mL·min^-1(ii) a The detection wavelength is 280 nm; column temperature: at 30 ℃.

(4) Sample assay

And (3) respectively sucking 3 mu L of the reference substance and the test solution, injecting into an ultra high performance liquid chromatograph, and measuring according to the chromatographic conditions in the step (3), wherein the liquid chromatogram of the 4 reference substances are respectively shown in figure 1, figure 2, figure 3 and figure 4.

Recording chromatogram within 30min, and processing with fingerprint software to obtain fingerprint of the Ailvshu product.

Example 2 methodological investigation of the UPLC fingerprint quality detection method

The performance analysis of the UPLC fingerprint quality detection method of each example and comparative example is as follows:

and (3) calculating the relative retention time and the relative peak area of each common peak in the HPLC fingerprint of the Ericerus chinensis by taking chlorogenic acid (peak 7) as a reference peak, calculating the relative standard deviation RSD value of the Epicerus chinensis, and performing methodology investigation.

1. Precision survey

And taking the same Elephantopi test solution, carrying out continuous sample injection for 6 times according to the detection method of the embodiment 1, recording a chromatogram, and carrying out precision investigation. The RSD of the retention time of the peak area of each common peak is less than 0.10 percent and the RSD of the relative peak area is less than 2.70 percent, and the experimental result shows that the precision of the instrument is good and meets the requirement of establishing a fingerprint spectrum.

2. Stability survey

And taking the same Elephantopi test solution, injecting samples for analysis at 0h, 2h, 4h, 8h, 12h and 24h respectively according to the detection method of the embodiment 1, recording a chromatogram, and carrying out stability investigation. The relative retention time RSD of the peak area of each common peak is less than 1.11 percent, and the RSD of the relative peak area is less than 4.44 percent. The result shows that the test article has good measurement stability within 24 hours.

3. Repeatability survey

Taking 6 parts of Elephantopi sample in the same batch, respectively preparing 6 parts of the test solution in parallel according to the preparation method of the test solution in the example 1, carrying out sample injection analysis according to the detection method in the example 1, determining a chromatogram, and carrying out repeatability inspection. The RSD of the retention time of all the common peaks is less than 0.14 percent, and the RSD value of the relative peak area is less than 4.97 percent, and the result shows that the method has good repeatability.

Example 3 creation of Elephantopi sample UPLC fingerprint

Experimental samples: ZL201410834689.8 the feed additive prepared in example 1 (as described in example 1) was processed from Erelo bioengineering, Inc. to give 12 batches of Elelo samples (see Table 2 for batch number).

TABLE 2 sample batch number

According to the established UPLC detection method of the

embodiment

1, 12 batches of Elephantopi sample test solutions and 3 μ L of 4 reference solutions are respectively absorbed and injected into an ultra-high performance liquid chromatograph for UPLC detection, and the UPLC fingerprint spectrum is established as follows:

1. measurement and similarity evaluation of 12 batches of Elephantopi UPLC fingerprint

Referring to technical requirements (temporary) of traditional Chinese medicine injection fingerprint spectrum research, combining different batches of Elephantopi chromatographic peak conditions, selecting a common peak capable of reflecting chemical components in Elephantopi, and performing common peak identification on different batches of Elephantopi.

The peak area is screened by a median method, the UPLC contrast map (R) of the Elephantopi is established, the UPLC fingerprint map of the Elephantopi is shown in figure 5, and the matching result is shown in tables 3 and 4.

2. Identification of common peaks

According to the UPLC fingerprint analysis result of 12 batches of the Elephantopi, 17 common peaks are calibrated by the Elephantopi, the peak area of the common peak accounts for 90% of the total peak area, the selected common peaks can comprehensively reflect chemical component information in a sample, and 4 known peaks, namely 7, 8, 9 and 14 peaks, are respectively chlorogenic acid, caffeic acid, liquiritin and chicoric acid, and the others are unknown peaks, and are shown in a figure 6 in detail.

3. Similarity calculation

By taking the common mode as reference and calculating the similarity of 12 batches of EllLushu UPLC fingerprints through the traditional Chinese medicine fingerprint similarity calculation software, the similarity of 12 batches of EllLushu UPLC fingerprints is more than 90 percent, and compared with a reference map (R), the similarity is more than 92 percent, and the result is shown in Table 5.

TABLE 31-6 batches of Ellison's respective common peak retention time and peak area

TABLE 47-12 batches of Ellison's respective common peak retention time and peak area

TABLE 51-12 batch Ellison similarity

Batches of	S1	S2	S3	S4	S5	S6	S7	S8	S9	S10	S11	S12	Control
														S1	1.000	0.998	0.976	0.982	0.977	0.976	0.978	0.974	0.976	0.975	0.973	0.975	0.977
S2	0.998	1.000	0.975	0.999	0.982	0.976	0.975	0.972	0.975	0.973	0.974	0.974	0.975
														S3	0.976	0.975	1.000	0.974	0.935	0.987	0.956	0.964	0.987	0.952	0.986	0.946	0.982
S4	0.982	0.999	0.974	1.000	0.975	0.974	0.976	0.972	0.974	0.973	0.973	0.974	0.975
														S5	0.977	0.982	0.935	0.975	1.000	0.962	0.934	0.974	0.934	0.942	0.950	0.912	0.935
S6	0.976	0.976	0.987	0.974	0.962	1.000	0.995	0.941	0.929	0.933	0.924	0.930	0.943
														S7	0.978	0.975	0.956	0.976	0.934	0.995	1.000	0.943	0.931	0.945	0.935	0.941	0.928
S8	0.974	0.972	0.964	0.972	0.974	0.941	0.943	1.000	0.943	0.948	0.938	0.944	0.925
														S9	0.976	0.975	0.987	0.974	0.934	0.929	0.931	0.943	1.000	0.923	0.929	0.931	0.976
S10	0.975	0.973	0.952	0.973	0.942	0.933	0.945	0.948	0.923	1.000	0.924	0.935	0.976
														S11	0.973	0.974	0.986	0.973	0.950	0.924	0.935	0.938	0.929	0.924	1.000	0.920	0.971
S12	0.975	0.974	0.946	0.974	0.912	0.930	0.941	0.944	0.931	0.935	0.920	1.000	0.974
														Control	0.977	0.975	0.982	0.975	0.935	0.943	0.928	0.925	0.976	0.976	0.971	0.974	1.000

According to analysis results, the characteristic components in the Ulva's UPLC fingerprint spectrum are obvious and completely retained, 17 common peaks are provided, the separation effect of each characteristic peak is good, and the quality information of the product can be comprehensively reflected. The comparison chart generated by 12 batches of Elephantopi fingerprint spectrums can be used for authenticity identification and quality control of Elephantopi products, if the similarity between the fingerprint spectrum of the sample to be detected and the comparison fingerprint spectrum is more than 90%, the product is true, and if the similarity is more than 92%, the product is qualified.

Example 4 application of UPLC fingerprint detection

The detection method of example 1 is used for detecting commercial alunite products and other similar products, and the specific steps are as follows:

3. Detecting an object: ZL201410834689.8 the feed additive prepared in example 1 was processed by Eriseuseoul, Hebei Eisei bioengineering, Inc.; a compound product of a certain plant extract is an additive product of Feiheilvshu, which is processed by the biological engineering limited company of Hebei ai Lu, and the formula is as follows: 120 parts of astragalus extract, 100 parts of eucommia extract, 120 parts of honeysuckle extract, 110 parts of coptis extract, 100 parts of cinnamon extract, 70 parts of turmeric extract, 80 parts of quassia extract, 90 parts of dandelion extract, 60 parts of nutmeg extract and 150 parts of eucommia leaf powder. Wherein, the astragalus extract: astragalus polysaccharide (UV) with content not less than 60-65%, and astragaloside (HPLC) with content not less than 0.1-0.2%; eucommia ulmoides extract: beverage grade, chlorogenic acid (HPLC) with content not less than 3%; and (3) honeysuckle extract: beverage grade, chlorogenic acid (HPLC) with content not less than 3-5%; the coptis chinensis extract: drinking grade (5: 1); cinnamon extract: drinking grade (10: 1); turmeric extract: curcumin (UV) with the content of more than or equal to 0.9 percent; and (3) bitter wood extract: drinking grade (15: 1); dandelion extract: a blending grade (10:1), caffeic acid (HPLC) with the content being more than or equal to 0.1%; nutmeg extract: dehydrodiisobutyronitrile (UV) with the content of more than or equal to 0.08 percent; folium cortex eucommiae powder: the content is more than or equal to 70 percent of total polysaccharide (UV).

4. The experimental steps are as follows:

(1) preparation of test solution

Respectively taking 2g of the Elephantopi sample and a compound product of a certain plant extract, precisely weighing, respectively dissolving with 20mL of 80% methanol, treating with ultrasound (60W, 45Hz) for 20min, shaking up, filtering, and filtering the filtrate with 0.22 μm filter membrane as a test solution for later use.

(2) Chromatographic conditions

A chromatographic column: phenyl column (SB-Phenyl, 3.0 mm. times.150 mm, 3.5 μm); mobile phase: acetonitrile-0.2% phosphoric acid water, gradient elution according to table 1: flow rate 0.43 mL/min^-1(ii) a The detection wavelength is 280 nm; column temperature: at 30 ℃.

(3) Determination of Ailvshu sample and certain plant extract compound product

Sucking 2 sample solutions 3 μ L each, injecting into ultra high performance liquid chromatograph, and measuring according to the chromatographic conditions of step (2), wherein the liquid chromatogram is shown in FIG. 7.

Compared with the Elephantopi UPLC fingerprint, the Elephantopi product (qualified product) has better matching between the retention time of each common peak and the peak area, and the plant extract compound product (unqualified product) has poorer matching between the number of common peaks and the retention time. The similarity between the two products and the Elephantos UPLC fingerprint is analyzed by the traditional Chinese medicine fingerprint similarity calculation software by taking the common mode as a reference, and compared with a reference spectrum (R), the similarity of the Elephantos product (namely a qualified product) spectrum is 96 percent, so that the Elephantos product is a qualified product. The pattern similarity of a certain plant extract compound product (namely an unqualified product) is 84 percent, and the product is an unqualified product. The result shows that the established UPLC fingerprint spectrum can successfully identify the counterfeit product and is applied to the quality control of the Ailvshu product.

Comparative example 1

2. Experimental reagent: phosphoric acid is chromatographically pure (alatin reagent); methanol was chromatographically pure (Fisher corporation, USA); ultrapure water (ultrapure water machine: american oscapu).

3. Detecting an object: ZL201410834689.8 feed additive prepared in example 1 (same as in example 1 above).

4. The experimental steps are as follows:

(1) preparation of test solution

2g of Elephantopi sample is precisely weighed, dissolved in 20mL of 80% methanol, treated with ultrasound (60W, 45Hz) for 20min, shaken up, filtered, and the subsequent filtrate is taken to pass through a 0.22 mu m filter membrane to serve as a test solution for standby.

(2) Chromatographic conditions

A chromatographic column: SB-C18 column (3.0 mm. times.150 mm, 3.5 m); mobile phase: methanol-0.2% phosphoric acid water, gradient elution was performed according to the elution procedure of table 6: flow rate 0.43 mL/min^-1(ii) a Detection wavelengths are 237nm and 327 nm; column temperature: at 30 ℃.

TABLE 6 procedure for mobile phase gradient elution

Time program (min)	Mobile phase A (methanol)%	Mobile phase B (0.2% phosphoric acid water)%
			0	10	90
6	25	75
			12	30	70
18	60	40
			27	10	90
28	90	10
			30	10	90

(3) Determination of Ellison samples

Sucking 3 μ L of the sample solution, injecting into an ultra high performance liquid chromatograph, and measuring under the chromatographic conditions of step (2), wherein the liquid chromatogram is shown in FIG. 8.

The results showed that the separation effect was not achieved for the plurality of complex components in comparative example 1 and the unevenness of the base line occurred, as compared with example 1.

Comparative example 2

4. The experimental steps are as follows:

(1) preparation of test solution

(2) Chromatographic conditions

A chromatographic column: phenyl column (SB-Phenyl, 3.0 mm. times.150 mm, 3.5 μm); mobile phase: acetonitrile-0.2% phosphoric acid water, gradient elution was performed according to the elution procedure shown in table 7: flow rate 0.43 mL/min^-1(ii) a The detection wavelength is 280 nm; column temperature: at 30 ℃.

TABLE 7 procedure for mobile phase gradient elution

Time program (min)	Mobile phase A (acetonitrile)%	Mobile phase B (0.2% phosphoric acid water)%
			0	10	90
9	16	84
			21	28	72
25	45	55
			28	10	90
30	10	90

(3) Determination of Ellison samples

Sucking 3 μ L of the sample solution, injecting into an ultra high performance liquid chromatograph, and measuring under the chromatographic conditions of step (2), wherein the liquid chromatogram is shown in FIG. 9.

The result shows that compared with example 1, the chromatogram shows that a plurality of complex components achieve better separation effect, the base line is flatter, but the chlorogenic acid peak and the unknown component at 7.2min and the chicoric acid peak and the unknown component at 19.5min do not achieve better separation effect.

Comparative example 3

4. The experimental steps are as follows:

(1) preparation of test solution

(2) Chromatographic conditions

A chromatographic column: phenyl column (SB-Phenyl, 3.0 mm. times.150 mm, 3.5 μm); mobile phase: acetonitrile-0.2% phosphoric acid water, gradient elution was performed according to the elution procedure shown in table 8: flow rate 0.43 mL/min^-1(ii) a The detection wavelength is 280 nm; column temperature: at 30 ℃.

TABLE 8 procedure for mobile phase gradient elution

Time program (min)	Mobile phase A (acetonitrile)%	Mobile phase B (0.2% phosphoric acid water)%
			0	10	90
9	16	84
			22	32	68
26	45	55
			28	10	90
30	10	90

(3) Determination of Ellison samples

Sucking 3 μ L of the sample solution, injecting into an ultra high performance liquid chromatograph, and measuring under the chromatographic conditions of step (2), wherein the liquid chromatogram is shown in FIG. 10.

The results show that the chlorogenic acid peak at 7.2min does not achieve a better separation effect from the unknown components than in example 1.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. A UPLC fingerprint quality detection method of a natural plant antibacterial growth-promoting feed additive is characterized by comprising the following steps: acetonitrile is used as a mobile phase A, 0.15-0.25% phosphoric acid aqueous solution is used as a mobile phase B, UPLC detection is carried out by adopting a gradient elution method,

2. The detection method according to claim 1, wherein the procedure of gradient elution comprises: 0-9min, 10-16% of mobile phase A and 90-84% of mobile phase B; 9-18min, mobile phase A16-26%, mobile phase B84-74%; 18-24min, mobile phase A26-33%, mobile phase B74-67%; 24-28min, 33-10% of mobile phase A and 67-90% of mobile phase B; 28-30min, mobile phase A10% and mobile phase B90%.

3. The detection method according to claim 1 or 2, wherein the flow rate of the mobile phase is 0.4-0.8ml.min^-1。

4. The detection method according to any one of claims 1 to 3, wherein the wavelength of the UPLC detection is 270-290 nm; preferably 280 nm.

5. The detection method according to any one of claims 1 to 4, wherein the chromatographic column used in the UPLC detection is a phenyl column;

6. The detection method according to any one of claims 1 to 5, wherein the detected column temperature is 28 to 35 ℃; preferably 30-32 deg.c.

7. The method according to any one of claims 1 to 6, wherein the amount of the sample to be detected is 2 to 5. mu.L.

8. The detection method according to any one of claims 1 to 7, further comprising a step of preparing a test solution before the UPLC detection, wherein the preparation of the test solution comprises: dissolving the sample in 75-85% methanol water solution, ultrasonic treating, mixing, and filtering.

9. The assay of any one of claims 1 to 8, further comprising a step of preparing a solution of a control comprising chlorogenic acid, caffeic acid, liquiritin and chicoric acid prior to UPLC assay.

10. The use of the detection method of any one of claims 1 to 9 in quality control or authenticity identification of a natural plant antibacterial growth-promoting feed additive.