CN112557486B - Anthocyanin type analysis and identification method and quantitative detection method - Google Patents
Anthocyanin type analysis and identification method and quantitative detection method Download PDFInfo
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Abstract
The invention relates to an anthocyanin type analysis and identification method, which comprises the following steps: (1) Performing secondary mass spectrometry on cyanidin, malvidin, delphinidin, petuniin, paeoniflorin and pelargonidin respectively to determine typical fragment ions; the typical fragment ions are determined by resolving the fragment structure and combining the ion abundance; (2) Crushing a sample to be detected, performing ultrasonic extraction on anthocyanin by using an ethanol-water strong acid solution, and performing hydrolysis by using a boiling water bath to obtain anthocyanin-containing liquid to be detected; (3) Filtering the anthocyanin-containing liquid to be detected, carrying out secondary mass spectrometry on the filtrate, and respectively comparing the anthocyanin in the liquid to be detected with typical fragment ions of cyanidin, malvidin, delphinidin, petuniin, paeoniflorin and pelargonidin to identify the variety of the anthocyanin. The anthocyanin type analysis and identification method can accurately distinguish the types of the anthocyanin, and is simple and convenient to identify.
Description
Technical Field
The invention belongs to the technical field of anthocyanin analysis, and particularly relates to an anthocyanin type analysis and identification method and a quantitative detection method.
Background
Anthocyanin is a plant-derived pigment widely existing in nature, belongs to a flavonoid compound taking 2-phenylbenzopyran (C6-C3-C6) as a parent nucleus, and has the following basic structure:
due to R 1 And R is 2 The groups and the types of anthocyanin were different, and are shown in Table 1.
TABLE 1 kinds of anthocyanidins
| Sequence number | Anthocyanidin | R 1 | R 2 |
| 1 | Pelargonium pigment | H | H |
| 2 | Delphinium pigment | OH | OH |
| 3 | Cornflower pigment | OH | H |
| 4 | Morning glory pigment | OCH 3 | OH |
| 5 | Paeonia lactiflora pigment | OCH 3 | H |
| 6 | Malva pigment | OCH 3 | OCH 3 |
Anthocyanins are formed when the hydroxyl groups in the 3,5,7 positions of the anthocyanidin a ring are bound to the glycosyl glycoside groups. Anthocyanin exists in the form of anthocyanin in plants, and the types, the amounts and the positions of bound sugar are different, so that the types of formed anthocyanin are different. The phenolic hydroxyl group on the mother nucleus structure is acidic and reductive, and the conjugated double bond on the pyran ring of the phenolic hydroxyl group is easy to undergo B-ring electron displacement. Most non-acylated or monoacylated anthocyanins exist in solution at a balance of 4 anthocyanins: the quinoid base, yellow-melted cationic pseudobase and chalcone, and the environmental factors such as acid, alkali, oxidant, light radiation and the like can all react with anthocyanin molecules to change the chemical structures such as quinoid base, pseudobase, chalcone, addition, ring opening, hydration and the like.
At present, the anthocyanin quantification method mainly adopts a pH differential method and a liquid chromatography method.
The pH differential method is calculated according to the molar absorptivity of anthocyanin monomers and the molar absorptivity of anthocyanin monomers under different pH values and specific wavelengths; for example: patent document publication No. CN107996908A discloses a pH differential method for detecting anthocyanin content; however, the pH differential method is only a rough quantitative method for anthocyanin detection, and is limited by the molar absorptivity of which anthocyanin monomer is selected in the calculation formula, so that the calculation result is greatly affected.
The liquid chromatography is to draw a standard curve according to the separation characteristics of different anthocyanin standard substances on a chromatographic column so as to quantitatively calculate the anthocyanin content; for example: the patent document with publication number CN104764846B discloses HPLC detection to identify anthocyanin, but chromatographic peaks formed by anthocyanin monomer substances in the chromatographic separation process are easily affected by molecules or groups with the same structure, and the selected standard substances are only representative ones and cannot cover all anthocyanin types, so that the accuracy of the quantitative method is greatly compromised.
Therefore, how to effectively identify anthocyanin types and accurately quantitatively detect the anthocyanin types is a problem to be solved in the field.
Disclosure of Invention
Based on the defects and shortcomings in the prior art, the invention provides an anthocyanin type analysis and identification method and a quantitative detection method.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
an anthocyanin type analysis and identification method comprises the following steps:
(1) Performing secondary mass spectrometry on cyanidin, malvidin, delphinidin, petuniin, paeoniflorin and pelargonidin respectively to determine typical fragment ions; the typical fragment ions are determined by resolving the fragment structure and combining the ion abundance;
(2) Crushing a sample to be detected, performing ultrasonic extraction on anthocyanin by using an ethanol-water strong acid solution, and performing hydrolysis by using a boiling water bath to obtain anthocyanin-containing liquid to be detected;
(3) Filtering the anthocyanin-containing liquid to be detected, carrying out secondary mass spectrometry on the filtrate, and respectively comparing the anthocyanin in the liquid to be detected with typical fragment ions of cyanidin, malvidin, delphinidin, petuniin, paeoniflorin and pelargonidin to identify the variety of the anthocyanin.
Preferably, the conditions for the secondary mass spectrometry of the procyanidin are as follows:
selecting a standard product of the procyanidin, wherein the mass-to-core ratio of procyanidin monomer ions is 287, and the procyanidin monomer ions form fragment ions under the 28-30eV collision energy in a mass spectrum positive ion mode; analyzing the fragment structure and combining the ion abundance, and selecting a plurality of fragment ions with strong ion abundance as typical fragment ions of the procyanidine pigment.
Preferably, the condition of the secondary mass spectrometry of the malvidin is as follows:
selecting a standard product of the malvidin, wherein the mass-to-core ratio of the malvidin monomer ions is 331, and the malvidin monomer ions form fragment ions under the collision energy of 25-30eV in a mass spectrum positive ion mode; analyzing the fragment structure and combining the ion abundance, and selecting a plurality of fragment ions with strong ion abundance as typical fragment ions of the malvidin.
Preferably, the condition of the secondary mass spectrometry analysis of the delphinium pigment is as follows:
selecting a standard product of the delphinium pigment, wherein the mass-to-core ratio of the delphinium pigment monomer ion is 303, and the delphinium pigment monomer ion forms fragment ions under the collision energy of 26-30eV in a mass spectrum positive ion mode; analyzing the fragment structure and combining the ion abundance, and selecting a plurality of fragment ions with strong ion abundance as typical fragment ions of the delphinium pigment.
Preferably, the conditions for the secondary mass spectrometry of petunia pigment are as follows:
selecting a standard product of petuniin, wherein the mass-nuclear ratio of the petuniin pigment monomer ions is 317, and the petuniin pigment monomer ions form fragment ions under the collision energy of 25-30eV in a mass spectrum positive ion mode; analyzing the fragment structure and combining the ion abundance, and selecting a plurality of fragment ions with strong ion abundance as typical fragment ions of petunia pigment.
Preferably, the conditions of the secondary mass spectrometry of the paeoniflorin are as follows:
selecting standard products of paeoniflorin, wherein the mass-nuclear ratio of paeoniflorin monomer ions is 301, and the paeoniflorin monomer ions form fragment ions under the collision energy of 22-35eV in a mass spectrum positive ion mode; analyzing the fragment structure and combining the ion abundance, and selecting a plurality of fragment ions with strong ion abundance as typical fragment ions of the paeoniflorin.
Preferably, the secondary mass spectrometry analysis of the pelargonidin comprises the following conditions:
selecting a standard substance of the pelargonium pigment, wherein the mass-to-core ratio of pelargonium pigment monomer ions is 271, and the pelargonium pigment monomer ions form fragment ions under the collision energy of 30-40eV in a mass spectrum positive ion mode; analyzing the fragment structure and combining the ion abundance, and selecting a plurality of fragment ions with strong ion abundance as typical fragment ions of the pelargonidin.
The invention also provides a quantitative detection method of anthocyanin, which comprises the following steps:
(1) Performing secondary mass spectrometry on cyanidin, malvidin, delphinidin, petuniin, paeoniflorin and pelargonidin respectively to determine typical fragment ions; the typical fragment ions are determined by resolving the fragment structure and combining the ion abundance;
(2) Taking ions with the strongest ion abundance in typical fragment ions of cornflower pigment, mallow pigment, delphinium pigment, petunia pigment, paeonia lactiflora pigment and pelargonium pigment as respective quantitative ions, and establishing standard curves between the ion abundance of different concentrations of various pigments and the corresponding quantitative ions;
(3) Crushing a sample to be detected, performing ultrasonic extraction on anthocyanin by using an ethanol-water strong acid solution, and performing hydrolysis by using a boiling water bath to obtain anthocyanin-containing liquid to be detected;
(4) Carrying out liquid chromatographic separation on the anthocyanin-containing liquid to be detected, and then carrying out multi-channel mass spectrometry analysis to obtain typical fragment ions and ion abundance of quantitative ions corresponding to the sample to be detected;
(5) Comparing typical fragment ions of a sample to be detected with typical fragment ions of cyanidin, malvidin, delphinidin, petuniin, paeoniflorin and pelargonidin respectively to identify the type of anthocyanin;
(6) And matching the ion abundance of the quantitative ions with a standard curve of the corresponding anthocyanin types to obtain the corresponding anthocyanin concentration.
Preferably, the conditions for the liquid chromatography separation include:
mobile phase a is 0.1% formic acid, mobile phase B is acetonitrile;
the gradient elution procedure was: 0-1.1min,5-10% mobile phase B;1.1-5.5min,10-50% mobile phase B;5.5-6.9min,5-50% mobile phase B; 6.9-7.5 min,5% mobile phase B, balance 1-3min.
Preferably, the conditions for mass spectrometry of the multiple paths include:
ion source: electrospray esi+; the detection mode is as follows: multi-reaction detection MRM; capillary voltage: 1.0kV; ion source temperature: 150 ℃; taper hole reverse blowing flow rate: 50L/h; desolventizing gas temperature: 500 ℃; desolventizing gas flow rate: 1000L/h; collision gas: 0.15mL/min; ion residence time: 0.02s.
Compared with the prior art, the invention has the beneficial effects that:
according to the anthocyanin type analysis and identification method, the anthocyanin type is identified by analyzing the secondary mass spectrum ions of the anthocyanin monomer standard substance and the sample to be detected and analyzing the consistency of the anthocyanin type mainly contained in the sample to be detected and the secondary mass spectrum ion distribution condition of procyanidine pigment, malvidin pigment, delphinidin pigment, petuniin pigment, paeoniflorin pigment and pelargonidin pigment. The anthocyanin type analysis and identification method can accurately distinguish the types of the anthocyanin, and is simple and convenient to identify.
According to the quantitative detection method of anthocyanin, the separation degree caused by overlapping of chromatographic peaks or similar chromatographic peaks can be generated on the basis of separation detection by utilizing liquid chromatography, and the separation degree can not meet the detection and quantification requirements, and the multi-path mass spectrometry means is used for accurately determining and quantifying the parent ions of substances and the child ions under specific collision energy, so that the disadvantages of liquid chromatography detection can be well avoided.
Drawings
FIG. 1 is an MRM spectrum of six anthocyanin classes according to an embodiment of the present invention.
Detailed Description
The technical scheme of the invention is further explained by the following specific examples.
The anthocyanin type analysis and identification method provided by the embodiment of the invention comprises the following steps:
(1) Performing secondary mass spectrometry on cyanidin, malvidin, delphinidin, petuniin, paeoniflorin and pelargonidin respectively to determine typical fragment ions; wherein, the typical fragment ions are determined by analyzing the fragment structure and combining the ion abundance, and the number of the typical fragment ions is a plurality;
(2) Crushing a sample to be detected, performing ultrasonic extraction on anthocyanin by using an ethanol-water strong acid solution, and performing hydrolysis by using a boiling water bath to obtain anthocyanin-containing liquid to be detected; specifically, 5g of a crushed sample to be detected is weighed, placed in a 25mL colorimetric tube, mixed extract of absolute ethyl alcohol, water and hydrochloric acid (the ratio of 2:1:1) is added, the volume is fixed, ultrasonic extraction is carried out for 30min, hydrolysis is carried out in a boiling water bath for 1h, cooling is carried out, the volume is fixed by using an extraction solution, standing is carried out, and supernatant fluid is taken, thus obtaining the anthocyanin-containing liquid to be detected.
(3) Filtering the anthocyanin-containing liquid to be detected with a 0.22 mu m filter membrane, performing secondary mass spectrometry on the filtrate, and comparing the anthocyanin in the liquid to be detected with typical fragment ions of cyanidin, malvidin, delphinidin, petuniin, paeoniflorin and pelargonidin respectively to identify the anthocyanin type.
The conditions for performing the secondary mass spectrometry in the on-machine test comprise:
sample injection mode: combine; taper hole voltage 35V, ionization voltage 1V, desolventizing temperature 450 ℃; desolventizing gas flow rate: 1000L/h mobile phase A (20% methanol solution) 90%, mobile phase B (90% methanol solution) 10%, flow rate 0.2mL/min, column temperature 35 ℃, sample injection amount 250 μL, sample injection speed 5 μL/min.
Specifically, the conditions for secondary mass spectrometry analysis of procyanidins are:
selecting a standard chlorinated procyanidin of procyanidin, obtaining procyanidin monomer ions under a solution environment, wherein the mass-to-core ratio of the procyanidin monomer ions is 287, and fragment ions are formed under the collision energy of 30eV (optionally 28-30 eV) in a mass spectrum positive ion mode of the procyanidin monomer ions; by resolving the fragment structure, combining the ion abundance, typical fragment ions for several procyanidins are determined. For example: typical fragment ions of six procyanidins were selected with mass-to-core ratios of 137, 213, 149, 241, 157, 185, respectively.
When the sample to be detected is analyzed, parent ions with the same nuclear ratio 287 as the procyanidine pigment are selected, and enter a secondary mass spectrum for analysis, and if the distribution of the fragment ions of the secondary mass spectrum is consistent with the typical fragment ion distribution of the standard product of the procyanidine pigment, the procyanidine pigment in the sample to be detected is identified; otherwise, no procyanidin is present.
Specifically, the conditions for the secondary mass spectrometry of malvidin are:
selecting a standard substance of malvidin to chloridize the malvidin, obtaining malvidin monomer ions in a solution environment, wherein the mass-to-core ratio of the malvidin monomer ions is 331, and the malvidin monomer ions form fragment ions under the collision energy of 30eV (optionally 25-30 eV) in a mass spectrum positive ion mode; by resolving the fragment structure, combining the ion abundance, typical fragment ions for several malvidins are determined. For example: six typical fragment ions of malvidin were selected with mass-to-core ratios of 315, 287, 242, 270, 299, 150, respectively.
When the analysis of the sample to be detected is carried out, parent ions with the same nuclear ratio 331 as the malvidin are selected, and enter a secondary mass spectrum for analysis, and if the distribution of the fragment ions of the secondary mass spectrum is consistent with the typical fragment ion distribution of the standard substance of the malvidin, the malvidin is identified to exist in the sample to be detected; otherwise, no malvidin is present.
Specifically, the conditions for the secondary mass spectrometry of delphinium pigment are:
the method comprises the steps of selecting a standard substance of delphinium pigment, namely, chloridizing the delphinium pigment, obtaining delphinium pigment monomer ions in a solution environment, wherein the mass-to-core ratio of the delphinium pigment monomer ions is 303, and forming fragment ions under the collision energy of 30eV (can be selected arbitrarily between 26 and 30 eV) in a mass spectrum positive ion mode of the delphinium pigment monomer ions; by resolving the fragment structure, combining with the ion abundance, typical fragment ions of several delphinium pigments are determined. For example: typical fragment ions of six delphinium pigments were selected with mass-to-core ratios 229, 257, 153, 173, 109, 81, respectively.
When a sample to be detected is analyzed, parent ions with the same nuclear ratio 331 as that of the delphinium pigment are selected, and enter a secondary mass spectrum for analysis, and if the distribution of the fragment ions of the secondary mass spectrum is consistent with the typical fragment ion distribution of a standard product of the delphinium pigment, the existence of the delphinium pigment in the sample to be detected is identified; otherwise, delphinium pigment is not present.
Specifically, the conditions for the secondary mass spectrometry of petuniin pigment are:
chloridizing petuniin serving as a standard product of petuniin is selected, petuniin monomer ions are obtained in a solution environment, the mass-to-nuclear ratio of the petuniin monomer ions is 317, and fragment ions are formed under the collision energy of 30eV (optionally 25-30 eV) in a mass spectrum positive ion mode of the petuniin monomer ions; typical fragment ions of several petunia pigments were determined by resolving the fragment structure, combined with ion abundance. For example: the typical ion of fragments of six petunia pigments was selected with mass-to-nuclear ratios of 302, 274, 245, 217, 203, 228, respectively.
When the sample to be detected is analyzed, parent ions with the same nuclear ratio 331 as petuniin are selected, and enter a secondary mass spectrum for analysis, and if the distribution of the fragment ions of the secondary mass spectrum is consistent with the typical fragment ion distribution of the standard product of the petuniin, the petuniin in the sample to be detected is identified; otherwise, petunia pigment is not present.
Specifically, the conditions for the secondary mass spectrometry of paeoniflorin are:
selecting standard product paeoniflorin of paeoniflorin, wherein the paeoniflorin is used for obtaining paeoniflorin monomer ion in a solution environment, the mass-nuclear ratio of the paeoniflorin monomer ion is 301, and fragment ions are formed under the collision energy of 30eV (optionally 22-35 eV) in a mass spectrum positive ion mode of the paeoniflorin monomer ion; by analyzing the fragment structure and combining the ion abundance, typical fragment ions of a plurality of paeoniflorin are determined. For example: typical fragment ions of six paeoniflorin are selected, and the mass-to-nuclear ratio is 201, 202, 286, 258, 229 and 230 respectively.
When the analysis of the sample to be detected is carried out, parent ions with the same nuclear ratio 331 as the paeoniflorin are selected, and enter a secondary mass spectrum for analysis, and if the distribution of fragment ions of the secondary mass spectrum is consistent with the typical fragment ion distribution of the standard substance of the paeoniflorin, the paeoniflorin is identified to exist in the sample to be detected; otherwise, no paeoniflorin is present.
Specifically, the conditions for the secondary mass spectrometry of the pelargonidin are:
selecting standard products of the pelargonium pigment, namely chlorinated pelargonium pigment, wherein the chlorinated pelargonium pigment obtains pelargonium pigment monomer ions under a solution environment, the mass-to-core ratio of the pelargonium pigment monomer ions is 271, and the pelargonium pigment monomer ions form fragment ions under the collision energy of 30eV (optionally 30-40 eV) in a mass spectrum positive ion mode; by resolving the fragment structure, combining the ion abundance, typical fragment ions for several pelargonidins are determined. For example: typical fragment ions of six pelargonidins were selected with mass to core ratios of 121, 141, 65, 93, 197, 169, respectively.
When the sample to be detected is analyzed, parent ions with the same nuclear ratio 331 as the pelargonium pigment are selected, and enter a secondary mass spectrum for analysis, and if the distribution of the fragment ions of the secondary mass spectrum is consistent with the typical fragment ion distribution of the standard sample of the pelargonium pigment, the pelargonium pigment is identified to exist in the sample to be detected; otherwise, no pelargonidin is present.
The embodiment of the invention also provides a quantitative detection method of anthocyanin, which comprises the following steps:
(1) Performing secondary mass spectrometry on cyanidin, malvidin, delphinidin, petuniin, paeoniflorin and pelargonidin respectively to determine typical fragment ions; the typical fragment ions are determined by resolving the fragment structure and combining the ion abundance;
(2) Taking ions with the strongest ion abundance in typical fragment ions of cornflower pigment, mallow pigment, delphinium pigment, petunia pigment, paeonia lactiflora pigment and pelargonium pigment as respective quantitative ions, and establishing standard curves between the ion abundance of different concentrations of various pigments and the corresponding quantitative ions;
(3) Crushing a sample to be detected, performing ultrasonic extraction on anthocyanin by using an ethanol-water strong acid solution, and performing hydrolysis by using a boiling water bath to obtain anthocyanin-containing liquid to be detected;
(4) Carrying out liquid chromatographic separation on the anthocyanin-containing liquid to be detected, and then carrying out multi-channel mass spectrometry analysis to obtain typical fragment ions and ion abundance of quantitative ions corresponding to the sample to be detected;
(5) Comparing typical fragment ions of a sample to be detected with typical fragment ions of cyanidin, malvidin, delphinidin, petuniin, paeoniflorin and pelargonidin respectively to identify the type of anthocyanin;
(6) And matching the ion abundance of the quantitative ions with a standard curve of the corresponding anthocyanin types to obtain the corresponding anthocyanin concentration.
Specifically, the conditions for liquid chromatography separation include:
chromatographic column: waters ACQUITY BEH C18 column (1.7 um,2.1 x 100 mm);
column temperature: 40 ℃;
sample injection volume: 2. Mu.L;
flow rate: 0.5ml/min;
mobile phase a is 0.1% formic acid, mobile phase B is acetonitrile;
the gradient elution procedure was: 0-1.1min,5-10% mobile phase B;1.1-5.5min,10-50% mobile phase B;5.5-6.9min,5-50% mobile phase B;6.9 to 7.5min,5% mobile phase B, 1min (optionally 1-3 min).
Specifically, the conditions for mass spectrometry of the multiple passes include:
ion source: electrospray esi+; the detection mode is as follows: multi-reaction detection MRM; capillary voltage: 1.0kV; ion source temperature: 150 ℃; taper hole reverse blowing flow rate: 50L/h; desolventizing gas temperature: 500 ℃; desolventizing gas flow rate: 1000L/h; collision gas: 0.15mL/min; ion residence time: 0.02s. The method comprises the steps of carrying out a first treatment on the surface of the The corresponding monitored ion pairs (m/z), cone voltage and other parameters are shown in Table 2 below.
Table 2 table of parameters of mass spectrometry analysis of various anthocyanins
Note that: in the sub-ions, the band is a quantitative ion, the others are qualitative ions, and the quantitative ions are also qualitative ions.
The six kinds of azurin monomers in the sample to be detected can have similar or overlapped chromatographic column separation retention time, and the separation detection of liquid phase chromatography can generate overlapped chromatographic peaks or similar chromatographic peaks, so that the separation degree can not meet the detection and quantification requirements; the disadvantage of liquid chromatography can be well avoided by adopting a multi-channel mass spectrometry method to accurately determine and quantify the parent ions and the child ions of the substances under the specific collision energy, and six anthocyanin substances can be well separated in different channels, as shown in figure 1.
Standard curves of six anthocyanin monomers 5, 10, 20, 50, 100 and 200 mug/L are established, the linear correlation coefficient of the standard curves can reach more than 0.99, the RSD is less than 15%, and the standard adding recovery rate is 85-110%, as shown in table 3.
TABLE 3 Standard curves for six anthocyanin substances
| Compounds of formula (I) | Linear regression equation | Correlation coefficient | Linear range |
| Compound | Regression equationg | r | μg/L |
| Cornflower pigment | y＝4082.61*x-8800.58 | 0.9973 | 5.0～200 |
| Delphinium pigment | y＝1156.13*x-2101.56 | 0.9978 | 5.0～200 |
| Paeonia lactiflora pigment | y＝12768.5*x-18376.3 | 0.9960 | 5.0～200 |
| Petunia pigment | y＝12411.9*x-38053.7 | 0.9916 | 5.0～200 |
| Malva pigment | y＝4237.98*x-8818.26 | 0.9978 | 5.0～200 |
| Pelargonium pigment | y＝14081.2*x+11078.3 | 0.9989 | 5.0～200 |
Note that: x is the concentration and y is the ion abundance.
The foregoing is only illustrative of the preferred embodiments and principles of the present invention, and changes in specific embodiments will occur to those skilled in the art upon consideration of the teachings provided herein, and such changes are intended to be included within the scope of the invention as defined by the claims.
Claims (3)
1. The quantitative detection method of anthocyanin is characterized by comprising the following steps of:
(1) Performing secondary mass spectrometry on cyanidin, malvidin, delphinidin, petuniin, paeoniflorin and pelargonidin respectively to determine typical fragment ions; the typical fragment ions are determined by resolving the fragment structure and combining the ion abundance;
(2) Taking ions with the strongest ion abundance in typical fragment ions of cornflower pigment, mallow pigment, delphinium pigment, petunia pigment, paeonia lactiflora pigment and pelargonium pigment as respective quantitative ions, and establishing standard curves between the ion abundance of different concentrations of various pigments and the corresponding quantitative ions;
(3) Crushing a sample to be detected, performing ultrasonic extraction on anthocyanin by using an ethanol-water strong acid solution, and performing hydrolysis by using a boiling water bath to obtain anthocyanin-containing liquid to be detected;
(4) Carrying out liquid chromatographic separation on the anthocyanin-containing liquid to be detected, and then carrying out multi-channel mass spectrometry analysis to obtain typical fragment ions and ion abundance of quantitative ions corresponding to the sample to be detected;
(5) Comparing typical fragment ions of a sample to be detected with typical fragment ions of cyanidin, malvidin, delphinidin, petuniin, paeoniflorin and pelargonidin respectively to identify the type of anthocyanin;
(6) And matching the ion abundance of the quantitative ions with a standard curve of the corresponding anthocyanin types to obtain the corresponding anthocyanin concentration.
2. The method for quantitative detection of anthocyanin according to claim 1, wherein the conditions of liquid chromatography separation include:
mobile phase a is 0.1% formic acid, mobile phase B is acetonitrile;
the gradient elution procedure was: 0-1.1min,5-10% mobile phase B;1.1-5.5min,10-50% mobile phase B;5.5-6.9min,5-50% mobile phase B; 6.9-7.5 min,5% mobile phase B, equilibrated for 1-3min.
3. The method for quantitative detection of anthocyanin according to claim 1, wherein the conditions of mass spectrometry of the multipass include:
ion source: electrospray esi+; the detection mode is as follows: multi-reaction detection MRM; capillary voltage: 1.0kV; ion source temperature: 150 ℃; taper hole reverse blowing flow rate: 50L/h; desolventizing gas temperature: 500 ℃; desolventizing gas flow rate: 1000L/h; collision gas: 0.15mL/min; ion residence time: 0.02s.
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