CN110376311B

CN110376311B - Method for identifying angelica and mixed counterfeit angelica

Info

Publication number: CN110376311B
Application number: CN201910768796.8A
Authority: CN
Inventors: 唐志书; 刘妍如; 宋忠兴; 杨宁娟; 李晓红; 江大海
Original assignee: Shaanxi University of Chinese Medicine
Current assignee: Shaanxi University of Chinese Medicine
Priority date: 2019-08-20
Filing date: 2019-08-20
Publication date: 2021-07-27
Anticipated expiration: 2039-08-20
Also published as: CN110376311A

Abstract

The invention discloses a method for identifying angelica and mixed counterfeit angelica, which is specifically established by adopting a technology of combining chromatography and mass spectrum, and finally determines the specificity identification and detection indexes of the angelica and the mixed counterfeit angelica, namely: ligustilide in angelica sinensis: the signal/noise of osthole, i.e. the ratio of S/N is more than 1, ligustilide in the mixed counterfeit angelica sinensis: the S/N ratio of osthole is less than 1. The method can accurately and rapidly identify the angelica and the mixed counterfeit angelica, and provides a scientific and effective technical identification means for the medication safety of the Chinese medicinal angelica.

Description

Method for identifying angelica and mixed counterfeit angelica

Technical Field

The invention relates to a method for identifying angelica and mixed counterfeit angelica, in particular to an angelica identification method based on chromatography-bark making combination, belonging to the technical field of quality detection of traditional Chinese medicines.

Background

Angelica sinensis is the dried root of Angelica sinensis (Oliv.) Diels, a biennial herb of Umbelliferae, and its medicinal record is originally reported in Shennong Ben Cao Jing, which has been known for over 2000 years. The angelica has strong fragrance, sweet, pungent and slightly bitter taste, and has the effects of enriching blood and activating blood, regulating menstruation and relieving pain, and relaxing bowel. It is mainly used for treating blood deficiency and sallow complexion, vertigo and palpitation, irregular menstruation, amenorrhea and dysmenorrhea, deficiency cold and abdominal pain, rheumatic arthralgia, traumatic injury, superficial infection, pyocutaneous disease, intestinal dryness and constipation. After the resource general survey in 1983, the national medical administration has clearly regulated that the use of levisticum officinale, levisticum officinale and the like instead of levisticum officinale is prohibited, and the source of levisticum officinale in the edition of the Chinese pharmacopoeia 2005 is only the dry root of levisticum officinale belonging to the family Umbelliferae. Therefore, the plant-based source of the Angelica is very clear and is a single plant-based source and is a special species in China, namely, the plant of the Angelica-based source is Angelica (Angelica sinensis (Oliv.) Diels) which is an Umbelliferae plant.

Dong gui (a. acutiloba Kitag) is the main counterfeit drug of chinese angelica. Donggui is root of Angelica gigas nakai of Angelica gigas of Leguminosae (Sieb. et Zucc). In the prior art, methods such as character, microscopic and physical and chemical identification are mostly adopted to identify angelica and Donggui, for example, the prior published scientific and technical documents: wanghanhua, Hushufeng, the identification of Dang Gui and European Dang Gui, common counterfeits thereof [ J ]. Chinese medical guidelines, 2012.10(5): 4-5. In addition, the ITS2 barcode can be used to effectively identify the chinese traditional medicine angelica and ITS mixed counterfeit, and PCR technology is mostly used to utilize the sequence of the transcribed spacer region (ITS2) in the ribosomal r RNA gene from the molecular biology level, for example, the prior published scientific and technical literature: zhang Chun, Wang Xiao Li, Ju He Ye, etc. rDNA ITS sequence analysis and identification of Chinese angelica and ITS mixed counterfeit product [ J ] academic newspaper of Sichuan agricultural university, 2011.29(2): 218-. However, the above existing angelica identification techniques all have certain practical drawbacks, wherein the traditional identification techniques are affected by the growth environment, growth period and processing technique of the original plants; the DNA bar code technology is affected by the quality of the DNA template affected by processing and processing. Therefore, the traditional identification technology and molecular biology technology have the defects that the authenticity identification of the medicinal material is difficult to realize stably, reliably and quickly.

The chromatography-mass spectrometry combined identification technology is a technology which combines the identification and the quantification performance efficiently. Therefore, the technical scheme of the invention is based on the high-efficiency separation performance of the chromatogram and the abundant component structure information obtained by the mass spectrum technology, and the organic combination of the high-efficiency separation performance and the mass spectrum technology can promote the formation of the traditional Chinese medicine identification technology of the system, is favorable for promoting the obvious improvement of the selectivity and the sensitivity of the traditional detector, and can also qualitatively determine the unknown chemical components in the traditional Chinese medicine sample. And by combining with multivariate statistical analysis technology, the discrimination capability of medicinal materials from different sources and production places can be improved, and the method is more suitable for traditional Chinese medicine samples which are easy to change and damage after processing and concocting. Therefore, the invention creatively establishes a method for rapidly and accurately identifying the angelica and the counterfeit angelica thereof through repeated exploration and verification.

Disclosure of Invention

The invention aims to provide a chromatography-mass spectrometry combined identification method of angelica and a confounding angelica thereof based on principal component analysis, which finally adopts ligustilide: S/N ratio (S/N) of osthole_Ligustilide:S/N_Osthole) As the identification index for identifying the adulterant and the adulterant.

In order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:

a method for identifying radix Angelicae sinensis and radix Angelicae sinensis of mixed pseudo-product comprises selecting radix Angelicae sinensis as medicinal material or decoction pieces; the identification method is characterized in that ligustilide in angelica is used: the S/N ratio of osthole is used as a detection index for identifying osthole and angelica sinensis. The S/N ratio in the scheme of the present invention is the signal/noise ratio of the chromatographic peak.

Further, the identification method of the angelica and the mixed counterfeit angelica is characterized in that: the determination indexes comprise that ligustilide in angelica sinensis: the S/N ratio of osthole is more than 1, and the content of ligustilide in the angelica sinensis is as follows: the S/N ratio of osthole is less than 1.

Still further, the method for identifying angelica and the mixed counterfeit angelica is established by adopting a technology of combining chromatography and mass spectrometry.

Still further, the identification method of angelica and mixed counterfeit angelica sinensis of the invention comprises the following chromatographic conditions: by C₁₈A chromatographic column; the mobile phase is the volume ratio of 0.1 percent formic acid-water solution to acetonitrile; the flow rate is 300 muL/min, the column temperature is 30 ℃, the sample injection amount is 5 muL, the detection wavelength is 254nm, and the gradient elution parameters are as follows: the elution time is 0-1min, and the volume ratio of 0.1 percent formic acid-water solution is 98 percent; the elution time is 1-18min, and the volume ratio of 0.1 percent formic acid-water solution is from 98 percent to 0 percent; the elution time is 18-20min, and the volume ratio of 0.1 percent formic acid-water solution is 0 percent; the elution time is 20-23min, and the volume ratio of 0.1 percent formic acid-water solution is from 0 percent to 98 percent; the elution time is 23-25min, and the volume ratio of 0.1 percent formic acid-water solution is 98 percent.

Furthermore, the identification method of the angelica and the mixed counterfeit angelica provided by the invention has the following mass spectrum conditions: the ion source is an electrospray ion source, data are acquired in a positive and negative ion mode, the injection voltage of the positive and negative ion source is 5500V, -4500V respectively, the atomization temperature is 550 ℃, the atomization gas and the auxiliary gas are nitrogen gas, both are 50psi, and the gas curtain gas is 35 psi; the scanning range of parent ion is 50-1600m/z, and MS is carried out on 8 strongest peaks exceeding 100cps²Data are collected, and the scanning range of the sub-ions is 50-1500 m/z.

Further, the identification method of the angelica and the mixed counterfeit angelica sinensis provided by the invention comprises the following steps of: weighing 0.2g of angelica or Donggui powder, precisely weighing, placing in a conical flask, precisely adding 20mL of 70% methanol, weighing, refluxing in water bath at 85 ℃ for 30 minutes, standing, cooling, weighing, adding a solvent to supplement the reduced weight, shaking uniformly, standing, taking supernatant, filtering, and taking subsequent filtrate.

In addition, in the technical scheme of the invention, the liquid chromatography conditions are optimized as follows:

the mobile phase is compared with two solvent systems of acetonitrile-water and methanol-water respectively, and the result shows that the base line of the binary gradient elution system of acetonitrile-water is even compared with the base line of the binary gradient elution system of methanol-water, and the time consumption is short. The analysis time is only 30min, and the 1h spectrogram of the sample shows that no characteristic peak appears after 30 min. In terms of Σ Rs, r (× 10)^-10) Φ, HCRF evaluation of the separation efficiency of the samples:

evaluation of chromatographic separation quality

Evaluation of amount of chromatography information

HCRF＝1,000,000n+100,00R_min+(t_m-t₁) (formula 3)^*: layered chromatography response values

^*R: degree of separation between two peaks, n: number of theoretical plates, R_min: minimum degree of separation, t_m-t₁: difference between maximum and minimum retention time, a: chromatographic peak area.

Σ Rs refers to the sum of resolutions of two adjacent peaks based on USP resolution, and is used mainly to evaluate chromatographic separation quality. Since Σ Rs is usually determined by the spectral peak with the maximum resolution, which tends to be less correlated to the total efficiency value of fingerprint separation. Therefore, r (× 10) was used^-5) The (formula 1) value may maintain the resolution of all spectral peaks in the fingerprint at a normalized state. The other parameter for evaluating the separation quality of the fingerprint is a phi value (formula 2), which is also called a chromatographic information value and is a chromatographic interaction information value for establishing the fingerprint based on the chromatographic peak area, retention time and theoretical plate number. The phi value is beneficial to the fingerprint analysis with short analysis time and more active pharmaceutical ingredients. The standard also adopts a layered chromatography response value (HCRF, formula 3) to evaluate the number of separation peaks, the separation degree and the analysis time performance in the method. The HCRF value can reasonably evaluate the fingerprint with poor separation degree and separation time. Therefore, in order to evaluate the quality of the fingerprint from multiple aspects, the scheme of the invention adopts the four response factors to evaluate the quality of the fingerprint.

In order to obtain the best parameter settings, the column temperature (25-35 ℃), the flow rate (0.2-0.5mL/min), the buffer concentration (0.1-0.2% formic acid, 0.05-0.1% acetic acid), the initial proportion of the aqueous phase (95-99%), the column type (column 1: 50 mm. times.2.1 mm, 1.7 μm; column 2: 100 mm. times.2.1 mm, 1.7 μm; column 3: 100 mm. times.2.1 mm, 2.5 μm) were chosen as the optimization parameters, and the results are given in Table 1.

TABLE 1 chromatographic Condition optimization results

The optimization result shows that the buffer system is 0.1% formic acid-water solution, the initial proportion of the water phase is 98% when the column temperature is 30 ℃, the optimization result of parameters of each peak separation degree and the number of the tower plates is better when the flow rate is 0.3ml/min, and therefore the optimization result is used as the determined chromatographic condition for sample determination.

The invention has the advantages of

1. The technical scheme of the invention establishes a detection index for identifying the angelica and the mixed counterfeit angelica by using a combined chromatography-mass spectrometry technology, namely ligustilide: the S/N ratio of the osthole has accurate and rapid technical effects, and obviously overcomes the technical defects of the traditional identification method and molecular biology in reality.

2. According to the technical scheme, the comprehensive evaluation concept of the ingredients to the effect of the Chinese medicament is fully followed, and a one-sided mode determined for a single ingredient in the prior art is abandoned, so that the medication safety and the curative effect quality of the Chinese medicament angelica are effectively ensured.

Drawings

FIG. 1 is a total ion flow diagram of the Angelica sinensis and Angelica acutiloba positive ion mode of one embodiment of the present invention;

FIG. 2 is a scatter plot of Angelica sinensis and Angelica acutiloba PCA fit according to one embodiment of the present invention;

FIG. 3 is a graph of the differential loading of the components of Angelica sinensis and Angelica acutiloba according to one embodiment of the present invention;

FIG. 4 is a graph of the maximum difference ion ratio of Angelica sinensis and Angelica acutiloba according to one embodiment of the present invention;

FIG. 5 is a osthole chromatogram of an embodiment of the invention;

FIG. 6 is a first order mass spectrum of osthole according to one embodiment of the present invention;

FIG. 7 is a secondary mass spectrum of osthole according to one embodiment of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Example a method for identifying angelica and mixed counterfeit angelica

(1) Preparation of sample solution

1) Preparing a Chinese angelica medicinal material solution: weighing about 0.2g of angelica powder (passing through a third sieve), precisely weighing, placing in a conical flask, precisely adding 20mL of 70% methanol, weighing, refluxing in water bath at 85 ℃ for 30 minutes, standing, cooling, weighing, adding a solvent to supplement the reduced weight, uniformly shaking, standing, taking supernatant, filtering, and taking a subsequent filtrate for later use.

2) Preparing a Donggui solution: weighing about 0.2g of Donggui powder (sieved by a third sieve), precisely weighing, placing in a conical flask, precisely adding 20mL of 70% methanol, weighing, refluxing in water bath at 85 ℃ for 30 minutes, standing, cooling, weighing, adding solvent to supplement the reduced weight, shaking uniformly, standing, taking supernatant, filtering, and taking a subsequent filtrate for later use.

(2) UPLC-Q-TOF/MS qualitative analysis

1) Mass spectrum conditions: the ion source is an electrospray ion source, data are acquired in a positive and negative ion mode, the injection voltage of the positive and negative ion source is 5500V, -4500V respectively, the atomization temperature is 550 ℃, the atomization gas and the auxiliary gas are nitrogen gas, both are 50psi, and the gas curtain gas is 35 psi; data are acquired by adopting an Information Dependent Acquisition (IDA), a Dynamic Background Subtraction (DBS) and a high-sensitivity mode, a scanning range of a parent ion (TOF-MS) is 50-1600m/z, and MS is carried out on 8 strongest peaks exceeding 100cps²And collecting data, wherein the scanning range of the sub-ions is 50-1500 m/z.

2) The liquid phase method comprises the following steps: using Acquity UPLC BEH C₁₈Column (50 mm. times.2.1 mm, 1.7 μm) chromatography column; the mobile phase is 0.1% formic acid-water solution A and acetonitrile B; the flow rate is 300 muL/min, the column temperature is 30 ℃, the sample injection amount is 5 muL, and the detection wavelength is 254 nm; gradient elution conditions, as shown in table 2.

TABLE 2 gradient elution conditions

3) Determination of characteristic detected ions

Triple TOFTM 5600⁺The accurate mass number and isotope kurtosis ratio of the compound can be provided, the collected data is checked by Peakview 2.2, non-targeted mode component search is carried out in a database by Masterview, and the parameters are set as follows: set Mass Error (Mass deviation)<5.0, weight 30%; isotope (Isotope distribution difference)<10.0 percent and 30 percent of weight; fomular Finder Score (molecular formula search)>70, weight 40%. The compound information is determined by accurately extracting the quasi-molecular ions, mass deviation, retention time and secondary mass spectrogram of the compound. The total ion flow chart of the positive ion mode of Angelica sinensis and Angelica acutiloba is shown in FIG. 1 (solid line-Angelica acutiloba; dotted line-Angelica acutiloba).

And (3) carrying out compound matching on the data acquired by the IDA and a traditional Chinese medicine Component database, and importing the data into Markerview1.3 for Principal Component Analysis (PCA) to find out characteristic difference components. After the data is preprocessed through scaling (autoscaling), a PCA model of positive and negative ion mode data is established, and as can be seen from a main component scatter diagram of medicinal material classification, in the positive ion mode, the Donggui sampling sample point shows a larger difference from other batches of Angelica sinensis genuine medicinal materials, which is specifically shown in FIG. 2. According to the generated classification result, compound information with larger difference among groups is extracted, compound ions with larger contribution to clustering difference are screened, the screening rule is that Fold difference (FC) > 1.5, t test result p is less than 0.01, and the result is shown in figure 3. According to the mass-to-charge ratio and retention time of the differential compounds, matching with an AB SCIEX (applied biosystems, USA) traditional Chinese medicine compound mass spectrum database to retrieve accurate mass spectrum information, finding out important differential components of osthole (mass-to-charge ratio 245.1m/z, retention time 11.4min), and the component difference of osthole in angelica and angelica, wherein the specific result is shown in figure 4.

According to the above experiment results, a reference solution of 0.01mg/mL of osthole is prepared, the sample injection is carried out according to the above sample injection conditions, the retention time of the osthole is 11.440min, and the result is shown in FIG. 5. According to the first-order mass spectrum result, an ion pair which is good in response and relatively stable is selected as a detection index, and the collision energy of each ion is optimized by adopting an ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry method, and the result is shown in fig. 6. Wherein the characteristic positive ions are m/z245.1 → 189.0, m/z245.1 → 131.0, CE:22.8, DP: 79.6; CXP: 6.15, the results are shown in FIG. 7.

(3) Determination of identification method of angelica and Donggui

1) And (3) special investigation: the solution without angelica is used as a negative sample, the angelica sinensis reference medicinal material and the sample to be detected are used as positive samples, characteristic ion peaks m/z245.1 → 189.0 and m/z245.1 → 131.0 are selected as detection ion pairs for determination, and in the chromatogram in the negative sample, no corresponding chromatographic peak exists at the position corresponding to the reference medicinal material and the sample, thereby indicating that the specificity of the method is good.

2) Examination of detection limit: an angelica and angelicae sinensis identification solution was prepared according to the sample solution described in (1) above, and the signal/noise (S/N) ratio of chromatographic peaks of osthole and ligustilide in angelica and angelicae sinensis was examined using ligustilide (quantitative parameter: m/z191.0 → 115.0) as a main component as a reference peak, and the results are shown in table 3.

Table 3 angelica ligustilide: S/N ratio (S/N) of osthole_Ligustilide:S/N_Osthole) Results

The results show that: angelicin ligustilide: S/N ratio (S/N) of osthole_Ligustilide:S/N_Osthole) Less than 1, and 15 batches of angelica ligustilide: the S/N ratio of osthole is greater than 1. Therefore, in the chromatography of angelica sinensis, the ligustilide (ligustilide) is preparedC₁₂H₁₄O₂) With osthole (C)₁₅H₁₆O₃) The signal peak ratio of (A) is not less than 1, and can be used as the test standard for identification of radix Angelicae sinensis and radix Angelicae sinensis.

Claims

1. A method for identifying angelica and mixed counterfeit angelica sinensis is characterized in that: the angelica is angelica medicinal materials or decoction pieces; the identification method is characterized in that ligustilide in angelica is used: the S/N ratio of osthole is used as a detection index for identifying osthole and angelica;

the detection indexes of the identification method comprise that ligustilide in angelica sinensis: the S/N ratio of osthole is more than 1, and ligustilide in pseudo-product Donggui: the S/N ratio of osthole is less than 1;

the identification method is established by adopting a technology of combining chromatography and mass spectrometry.

2. The method for discriminating angelica and pseudo-mix angelicae gigantis according to claim 1, wherein the method comprises the following steps: the chromatographic conditions of the identification method are as follows: by C₁₈A chromatographic column; the mobile phase is the volume ratio of 0.1 percent formic acid-water solution to acetonitrile; the flow rate is 300 muL/min, the column temperature is 30 ℃, the sample injection amount is 5 muL, the detection wavelength is 254nm, and the gradient elution parameters are as follows: the elution time is 0-1min, and the volume ratio of 0.1 percent formic acid-water solution is 98 percent; the elution time is 1-18min, and the volume ratio of 0.1 percent formic acid-water solution is from 98 percent to 0 percent; the elution time is 18-20min, and the volume ratio of 0.1 percent formic acid-water solution is 0 percent; the elution time is 20-23min, and the volume ratio of 0.1 percent formic acid-water solution is from 0 percent to 98 percent; the elution time is 23-25min, and the volume ratio of 0.1 percent formic acid-water solution is 98 percent.

3. The method for discriminating angelica and pseudo-mix angelicae gigantis according to claim 2, wherein: the mass spectrum condition of the identification method is as follows:

the ion source is an electrospray ion source, data are acquired in a positive and negative ion mode, the injection voltage of the positive and negative ion source is 5500V, -4500V respectively, the atomization temperature is 550 ℃, the atomization gas and the auxiliary gas are nitrogen gas, both are 50psi, and the gas curtain gas is 35 psi; parent ionScanning range 50-1600m/z, MS for 8 strongest peaks exceeding 100cps²Data are collected, and the scanning range of the sub-ions is 50-1500 m/z.

4. The method for discriminating angelica and pseudo-mix angelicae gigantis according to claim 3, wherein: the identification method comprises the following steps of preparing an angelica or angelica dong sample solution:

weighing 0.2g of angelica or Donggui powder, precisely weighing, placing in a conical flask, precisely adding 20mL of 70% methanol, weighing, refluxing in water bath at 85 ℃ for 30 minutes, standing, cooling, weighing, adding a solvent to supplement the reduced weight, shaking uniformly, standing, taking supernatant, filtering, and taking subsequent filtrate.