CN111562340B - Method for rapidly carrying out species analysis and content determination on anthocyanin in tomato fruits - Google Patents
Method for rapidly carrying out species analysis and content determination on anthocyanin in tomato fruits Download PDFInfo
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Abstract
The invention discloses a method for rapidly carrying out species analysis and content determination on anthocyanin in tomato fruits. The method comprises the steps of using methanol/formic acid (9, v/v) as a solvent, adding an internal standard, carrying out liquid-liquid extraction on anthocyanin in the tomato fruits, extracting at 4 ℃ for 12 hours, centrifuging to obtain a supernatant, passing through a membrane, simultaneously collecting mass spectrum information and spectrum information of the anthocyanin, quantifying the anthocyanin in the tomato fruits, and carrying out structure analysis. The detection method has the advantages of simple pretreatment process, accurate detection result and no need of complicated pretreatment and purification steps. The method can be used for carrying out qualitative and quantitative analysis on anthocyanin in tomato fruits.
Description
Technical Field
The invention relates to a method for rapidly carrying out species analysis and content determination on anthocyanin in tomato fruits.
Background
Because of the oxygen radical absorption capacity, anthocyanins are a class of substances beneficial to human health and also have medicinal value. In plants, anthocyanins can improve the stress tolerance of plants. Therefore, the identification, purification and structural analysis of anthocyanins have been the focus of research. The structural mother nucleus of anthocyanidin is 2-phenylbenzopyran cation, and mainly comprises cyanidin (cyanidin), pelargonidin (pelargonidin), delphinidin (delphinidin), peonidin (peonidin), petuniadin (petuniadin) and malvidin (malvidin). The structural analysis of anthocyanins is important, but the analysis is difficult because of their high structural similarity.
Anthocyanins are synthesized via flavonoid metabolic pathways, as shown in figure 1. Phenylalanine under the action of a series of enzymes synthesizes anthocyanin, including PAL, phenylalanine ammonia lyase; CA4H, cinnamate 4-hydroxylase; 4CL, 4-Coa-coumarate ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone-3-hydroxylase; FLS, flavonol synthase; f3'H, flavonoid 3' -hydroxylase; f3'5' H, flavonoid-3 ',5' -hydroxylase; OMT1, flavone 3' -O-methyltransferase 1; DFR, flavanonol reductase, and the like. Finally, the anthocyanin is formed through glycosylation modification and acylation modification and then is transported to a vacuole for storage.
Anthocyanins in tomato are biosynthesized under light induction. At present, there are 9 anthocyanins reported in tomato. Wherein petunidin-3- (cis-p-coumaric acid acyloxy) -rutinoside-5-glucoside is the main component of the tomato anthocyanin. Tomato is a very popular fruit in the market, but its fruit anthocyanin content is not optimized. Therefore, it is necessary to optimize the content of anthocyanins in tomato by studying the mechanism of anthocyanins synthesis. Although researchers have attempted to increase the anthocyanin content in tomato fruits, precise regulation is difficult. Among them, one important reason is that the detailed structure of anthocyanins synthesized in tomatoes has not been completely resolved. Structural modifications of anthocyanins include hydroxylation, methylation, glycosylation and acylation. Hydroxylation and methylation are direct modifications of the anthocyanin structure by hydroxyl and methyl groups. Glycosylation is the modification of the hydroxyl group of the 2-phenylbenzopyran cation by a monosaccharide, disaccharide, or trisaccharide. The acylation modification is to further add coumaric acid, caffeic acid, ferulic acid, etc. to the hydroxyl group of the glycoside after glycosylation modification. These structural modifications will increase the stability of the anthocyanins.
The pretreatment and detection processes have important influence on the rapid quantification of anthocyanin and the accurate analysis of anthocyanin structure. In previous studies, samples were mostly processed by lyophilization. Due to the longer lyophilization time, some of the anthocyanin is lost during this process. The anthocyanin is purified by column chromatography or solid phase extraction. Column chromatography requires a special column, and the experimental technical requirements are high. The solid phase extraction method requires special equipment and purification columns, is expensive, and comprises the steps of activation, balancing, sample loading, cleaning, elution and the like. Due to the long process flow, less anthocyanins, such as malvidin-like anthocyanins, may be lost during purification. To ensure that there is sufficient anthocyanin for detection after pretreatment, a large sample, such as 1kg of tomato peel, needs to be taken. Meanwhile, a large amount of solvent is also used for achieving the purpose of complete extraction. In addition, the addition of acid to the extraction solvent increases the stability of anthocyanins, but mineral acids may lead to degradation of the sample, for example hydrochloric acid may lead to hydrolysis of glycosidic bonds.
Disclosure of Invention
The invention aims to provide a method for rapidly carrying out species analysis and content measurement on anthocyanin in tomato fruits.
The invention provides a method for carrying out species analysis and content determination on anthocyanin in tomato fruits, which comprises the following steps:
1) Freezing and grinding the raw materials, and extracting with an extractant a to obtain an extracting solution a; centrifuging, collecting supernatant, blowing with nitrogen, adding extractant b for redissolving, filtering, and collecting filtrate to obtain extract b;
the raw materials are separated tomato peel and tomato pulp;
2) Analyzing the anthocyanin species of the extracting solution b by using liquid chromatography and mass spectrometry;
3) And (4) carrying out quantitative analysis on the content of different kinds of anthocyanin obtained by the extracting solution b, and completing the kind analysis and the content determination of the anthocyanin in the tomato fruits.
The invention also provides a method for carrying out species analysis on anthocyanin in tomato fruits, which comprises the following steps: step 1) and step 2) of the aforementioned method.
The invention also provides a method for measuring the content of anthocyanin in tomato fruits, which comprises the following steps: step 1) -step 3) of the aforementioned method.
In the freezing step in the step 1) of the method, the freezing mode is liquid nitrogen freezing;
in the extraction step, the extractant a is a solution containing an internal standard; the internal standard is paeoniflorin 3-glucoside (namely peonidin-3-glucoside chloride or chloridized peonidin-3-O-glucoside);
in the extractant a, a solvent is a mixed solution consisting of methanol and formic acid; the volume ratio of the methanol to the formic acid is 7-9:1; specifically, the method comprises the following steps of (1);
in the extractant a, the concentration of an internal standard is 1-5 mug/ml; specifically 3.75 mug/ml;
the dosage ratio of the extractant a to the raw materials is specifically 0.2-0.8g:1-4ml; specifically, 0.4g:4ml;
in the extraction step, the temperature is-20 ℃ to 5 ℃; in particular to 4 ℃; the time is 10-14h; in particular 12h;
the extractant b is a mixed solution consisting of methanol and formic acid; the volume ratio of the methanol to the formic acid is 7-9:1; specifically, the method comprises the following steps of (1); the volume ratio of the extractant b to the supernatant is as follows: 0.5 to 2; specifically, 1; the specific dosage of the supernatant is 2ml;
in the centrifugation step, the rotating speed is 2500-4500rpm; specifically 3900rpm; the time is 18-22min; specifically 20min;
in the filtering step, the aperture of the filter membrane is 0.22 μm.
In the step 2), the conditions of the liquid chromatography detection are as follows:
type of column: ACQUITY UPLC CSH C18;
chromatographic column parameters: 2.1mm × 100mm i.d.,1.7 μm;
mobile phase A: a mixed solution consisting of acetonitrile and formic acid with a volume ratio of 95;
mobile phase B: a mixed solution composed of water and formic acid in a volume ratio of 95;
the gradient elution procedure was: the initial volume proportion of the mobile phase A is 2.5%, the mobile phase A is increased from 2.5% to 10% within 5min, then is increased from 10% to 25% within 15min, is kept for 5min, and then is changed from 25% to 2.5% within 5min;
flow rate: 0.15ml/min;
column temperature: 25 ℃;
autosampler temperature: 20 ℃;
sample introduction volume: 1 μ l.
In the step 2), the mass spectrometry detection conditions are as follows:
the mass spectrum is in a positive ion mode;
an ion source: an ESI source;
capillary voltage: 2.0kV;
ion source temperature: 100 ℃;
desolvation gas temperature: 300 ℃;
desolventizing air flow rate: 800L/h;
taper hole airflow: 50L/h:
mass spectrum acquisition mode: fast DDA mode;
analysis mode: a resolution mode;
the collection range of the parent ions is as follows: 900m/z to 1000m/z;
the collection range of the daughter ions is as follows: 50m/z to 1000m/z;
selecting at most 5 daughter ions for qualitative analysis;
the collision energy range is 6V-80V;
spectral conditions: the wavelength range of the diode array detector is 190nm-800nm.
In the step 2) of the method, the step, the obtained anthocyanidin can be selected from delphinidin-3- (cis-p-coumaroyloxy) -rutinoside-5-glucoside, petunidin-3- (caffeic acid acyloxy) -rutinoside-5-glucoside, delphinidin-3- (trans-p-coumaroyloxy) -rutinoside-5-glucoside, delphinidin-3- (ferulic acid acyloxy) -rutinoside-5-glucoside, petunidin-3- (cis-p-coumaroyloxy) -rutinoside-5-glucoside, petunidin-3- (trans-p-coumaroyloxy) -rutinoside-5-glucoside, petunidin-3- (ferulic acid acyloxy) -rutinoside-5-glucoside, malvidin-3- (cis-p-coumaroyloxy) -rutinoside-5-glucoside, malvidin-3- (trans-p-coumaric acid acyloxy) -5-glucoside, malvidin-5-glucoside, and trans-malvidin-3- (trans-p-coumarinyloxy) -rutinoside-5-glucoside 5-glucoside, a vanilloid;
wherein the UV retention time of delphinidin-3- (cis-p-coumaric acid acyloxy) -rutinoside-5-glucoside is 11.41min; the MS retention time is 11.55min;
the UV retention time of the petunidin-3- (caffeic acid acyloxy) -rutinoside-5-glucoside is 11.71min; the MS retention time is 11.83min;
the UV retention time of delphinidin-3- (trans-p-coumaric acyloxy) -rutinoside-5-glucoside is 11.86min; the MS retention time is 11.97min;
the UV retention time of delphinidin-3- (ferulic acid acyloxy) -rutinoside-5-glucoside is 12.45min; the MS retention time is 12.58min;
the UV retention time of the petunidin-3- (cis-p-coumaric acyloxy) -rutinoside-5-glucoside is 13.01min; the MS retention time is 13.16min;
the UV retention time of the petunidin-3- (trans-p-coumaric acid acyloxy) -rutinoside-5-glucoside is 13.35min; the MS retention time is 13.52min;
the UV retention time of the petunidin-3- (ferulic acid acyloxy) -rutinoside-5-glucoside is 13.95min; the MS retention time is 14.11min;
the UV retention time of the malvidin-3- (cis-p-coumaric acyloxy) -rutinoside-5-glucoside is 14.61min; the MS retention time is 14.77min;
the UV retention time of the malvidin-3- (trans-p-coumaric acyloxy) -rutinoside-5-glucoside is 14.99min; the MS retention time is 15.08min;
the UV retention time of the malvidin-3- (ferulic acid acyloxy) -rutinoside-5-glucoside is 15.33min; the MS retention time is 15.68min;
the UV retention time of the petunidin-3- (trans-p-coumaric acid acyloxy-rhamnoside) -glucoside-5-glucoside is 15.73min; the MS retention time is 15.90min;
the UV retention time of the malvidin-3- (p-methoxy-trans-p-coumaric acid acyloxy) -rutinoside-5-glucoside is 16.07min; the MS retention time was 16.18min.
In the quantitative analysis in the step 3), the quantitative method is to perform semi-quantitative analysis on the internal standard equivalent; specifically, the peak area of the ultraviolet absorption chromatogram of the anthocyanin at 535nm is used for quantification.
In the step 3), the standard curve is y =221.941x-346.284; wherein y is the peak area of the internal standard substance; x is the concentration value of the internal standard substance and the unit is mu g/mL;
the linear range is 1-1000 mug/mL;
limit of quantitation < 1. Mu.g/mL.
Specifically, the anthocyanidin is selected from the group consisting of delphinidin-3- (cis-p-coumaric acyloxy) -rutinoside-5-glucoside, petuniain-3- (caffeic acyloxy) -rutinoside-5-glucoside, delphinidin-3- (trans-p-coumaric acyloxy) -rutinoside-5-glucoside, delphinidin-3- (ferulic acyloxy) -rutinoside-5-glucoside, petuniain-3- (cis-p-coumaric acyloxy) -rutinoside-5-glucoside, petuniain-3- (trans-p-coumaric acyloxy) -rutinoside-5-glucoside, petuniain-3- (ferulic acyloxy) -rutinoside-5-glucoside, malvidin-3- (cis-p-coumaric acyloxy) -rutinoside-5-glucoside, malvidin-3- (trans-p-coumaric acyloxy) -rutinoside, malvidin-5-glucoside, and trans-malvidin-3- (trans-p-coumaric acyloxy) -rutinoside-5-glucoside At least one of glucoside;
more specifically, the compound is at least one selected from the group consisting of petunidin-3- (cis-p-coumaric acid acyloxy) -rutinoside-5-glucoside, malvidin-3- (cis-p-coumaric acid acyloxy) -rutinoside-5-glucoside, petunidin-3- (trans-p-coumaric acid acyloxy-rhamnoside) -glucoside-5-glucoside and malvidin-3- (p-methoxy-trans-p-coumaric acid acyloxy) -rutinoside-5-glucoside.
The tomatoes described in the present invention are various known species of tomatoes; specifically, the extract can be indigo tomato.
The invention establishes a method for quickly quantifying anthocyanin in tomato fruits, and simultaneously accurately analyzes the substance structure of the anthocyanin. According to the method, methanol/formic acid (9, v/v) is used as a solvent, internal standard is added, liquid-liquid extraction is carried out on anthocyanin in tomato fruits, after 12h of extraction at 4 ℃, supernatant is obtained by centrifugation, after membrane filtration, mass spectrum information and spectrum information of anthocyanin are collected simultaneously, the anthocyanin in the tomato fruits is quantified, and structure analysis is carried out. The method has the advantages of small sample amount, less solvent, environmental friendliness and high extraction rate; the pretreatment process is simple, the detection result is accurate, and complex pretreatment and purification steps are not needed. The study was carried out using indigo tomato as experimental material. The research results show that 12 anthocyanins are detected in the indigo tomato, and 4 anthocyanins are not reported. The research provides a good experimental basis for further improvement of anthocyanin in tomatoes. The method can be used for carrying out qualitative and quantitative analysis on the anthocyanin in the tomato fruits.
Drawings
FIG. 1 is the biosynthetic pathway of anthocyanin in fruits. PAL, phenylalanine ammonia lyase; CA4H, cinnamate 4-hydroxylase; 4CL, 4-Coumaric acid coenzyme A ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone-3-hydroxylase; FLS, flavonol synthase; f3'H, flavonoid 3' -hydroxylase; f3'5' H, flavonoid-3 ',5' -hydroxylase; OMT1, flavone 3' -O-methyltransferase 1; DFR, flavanonol reductase.
FIG. 2 is a photograph of fruit red ripeness stage of indigo-blue tomato. This figure shows the fruit on the plant (a), top view of the fruit (b), bottom view of the fruit (c), after peel removal (d) and cross-sectional view (e). The scale bar of the picture is 2cm.
Fig. 3 is a chromatogram of the pericarp (a) and the pulp (b) of the indigo tomato fruit collected by liquid chromatography-time-of-flight mass spectrometry. In addition, the ultraviolet spectrogram of the anthocyanin is also collected in the experiment. The ultraviolet spectra of anthocyanins at retention times of 13.35min (c) and 14.99min (d) are shown. The experiment also collects chromatograms of the pericarp (e) and the pulp (f) of the indigo tomato fruit at 535nm of the ultraviolet absorption peak.
FIG. 4 shows the secondary mass spectra of 12 anthocyanosides (from FIG. a to FIG. i).
Detailed Description
The present invention will be further illustrated with reference to the following examples, but the present invention is not limited to the following examples. The method is a conventional method unless otherwise specified. The starting materials are commercially available from the open literature unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
The instrument comprises the following steps: UPLC-QTOF/PDA (Waters ACQUITY UPLC I-Class-Xevo G2-XS QTOF/PDA eLambda Detector)
Indigo tomato: purchased from Johnny's Selected Seeds (http:// www. Johnnyses. Com /);
balance: purchased from Sartorius.
Example 1 analysis of the type and content of anthocyanins in tomato fruit by liquid-liquid extraction-ultra high performance liquid chromatography-quadrupole time of flight mass spectrometry (LLE-UPLC-QTOF)
1) The indigo tomato fruit is washed and the peel and pulp are separated, and the photo of the indigo tomato fruit during the red ripeness stage is shown in fig. 2, which shows the fruit on the plant (a), the top view (b) of the fruit, the bottom view (c) of the fruit, the peel after being torn off (d) and the transverse view (e).
Freezing pericarp and pulp in liquid nitrogen, and grinding.
Separately, 0.4g each of the pericarp and pulp was weighed into a 15ml brown glass centrifuge tube.
4ml of a methanol/formic acid (9, 1, v/v) solution containing 15. Mu.g of peonidin 3-glucoside (i.e. peonidin-3-glucoside or chlorinated peonidin-3-O-glucoside) was added, which increased the stability of anthocyanins without hydrolysis. The equivalent of the internal standard is used for the quantification of the anthocyanin, and the loss rate of the internal standard can be converted into the loss rate of the sample so as to reduce the using amount of the solvent.
Shaking the extractive solution.
Extracting at 4 deg.C for 12h.
Centrifuge 15ml glass centrifuge tubes 3900rpm for 20min.
2ml of the supernatant was transferred to a new 15ml brown glass centrifuge tube.
2ml of the supernatant was blow-dried with nitrogen.
The blow dried sample was redissolved by adding 1ml of methanol/formic acid (9.
The dissolved solution is filtered through a 0.22 mu m organic filter membrane to a 2ml brown glass sample bottle for purification and then used for detection, thereby greatly simplifying the purification steps.
2) Chromatographic conditions are as follows: the column was run with an ACQUITY UPLC CSH C18 (2.1 mm x 100mm i.d.,1.7 μm), mobile phase A was acetonitrile/formic acid (95, 5,v/v), mobile phase B was water/formic acid (95, 5,v/v), flow rate 0.15ml/min, initial volume fraction of mobile phase A was 2.5%, mobile phase A rose from 2.5% to 10% within 5min, then from 10% to 25% within 15min, held for 5min, then changed from 25% to 2.5% within 5 min. The column oven temperature was maintained at 25 ℃ and the autosampler temperature was maintained at 20 ℃. The injection volume was 1. Mu.l.
Mass spectrum conditions: the mass spectrum is in positive ion mode, and the parameters of the ESI ion source are as follows: capillary voltage, 2.0kV; ion source temperature: 100 ℃; desolventizing gas temperature: 300 ℃; desolventizing air flow rate: 800L/h; taper hole airflow: 50L/h. The mass spectral acquisition used the Fast DDA mode. The analysis mode is a resolution mode. The collection range of the parent ions is 900m/z to 1000m/z. The sub-ion collection range is 50m/z to 1000m/z. A maximum of 5 daughter ions were selected for qualitative analysis. The collision energy ranges from 6V to 80V.
Spectral conditions: the diode array detector wavelength range is 190nm to 800nm.
The chromatogram of the peel and pulp of the fruit of the indigo tomato collected by liquid chromatography-time-of-flight mass spectrometry is shown in fig. 3a and 3 b. In addition, the ultraviolet spectrogram of the anthocyanin is also collected in the experiment. The ultraviolet spectra of anthocyanins at retention times of 13.35min (c) and 14.99min (d) are shown in FIGS. 3c and 3d, and the maximum ultraviolet absorption peaks are both 535nm. Chromatograms of the pericarp and pulp of the indigo tomato fruit at 535nm of the absorption peak of the maximum absorption peak of ultraviolet light are shown in fig. 3e and 3 f.
The spectrograms and mass spectrum information of the anthocyanidins are analyzed, a total of 12 anthocyanidins are detected, and the information of the daughter ions of the 12 anthocyanidins is shown in fig. 4a to 4 i.
The daughter ions and the putative anthocyanin structures of FIGS. 4 a-4 i are shown in Table 1.
Table 1, anthocyanins contained in indigo tomatoes found in chromatograms and their secondary structure information.
* Dpd, delphinidin; ptd, petunidin; mv, malvidin; glc, glucoside; rha, rhamnoside; pCouR, (p-coumaric acid acyloxy) -rutinoside; cafR, (caffeic acid acyloxy) -rutinoside; ferR, (Ferulic acid acyloxy) -rutinose
The anthocyanin retention time in MS is somewhat delayed from that in UV because it passes through the diode array detector before entering the mass spectrometer for detection during sample collection.
The detected anthocyanins are, sequentially from front to back, delphinidin-3- (cis-p-coumaroyloxy) -rutinoside-5-glucoside, petunidin-3- (caffeic acid acyloxy) -rutinoside-5-glucoside, delphinidin-3- (trans-p-coumaroyloxy) -rutinoside-5-glucoside, delphinidin-3- (ferulic acid acyloxy) -rutinoside-5-glucoside, petunidin-3- (cis-p-coumaroyloxy) -rutinoside-5-glucoside, petunidin-3- (trans-p-coumaroyloxy) -rutinoside-5-glucoside, petunidin-3- (ferulic acid acyloxy) -rutinoside-5-glucoside, malvidin-3- (cis-p-coumaroyloxy) -rutinoside-5-glucoside, malvidin-3- (trans-p-coumaroyloxy) -rutinoside-5-glucoside, and trans-malvidin-3- (trans-p-coumaroyloxy) -rutinoside-5-glucoside -a glucoside. Among them, the four anthocyanidins in bold in table 1 are the ones newly found in the present invention.
After the qualitative analysis of 12 anthocyanidins, the equivalent of the internal standard is adopted to carry out the quantitative analysis. The results of the anthocyanin quantification are shown in table 2.
TABLE 2 anthocyanin content (mg/kg FW) in indigo tomato peel and pulp.
* Results are mean ± standard deviation of biological replicates (n = 3).
The anthocyanin content in the peel of the indigo tomato is 3977.93mg/kg, and the anthocyanin content in the pulp is 88.44mg/kg. Wherein the petunidin-3- (trans-p-coumaric acyloxy) -rutinoside-5-glucoside and the malvidin-3- (trans-p-coumaric acyloxy) -rutinoside-5-glucoside are main components of anthocyanin in pericarp, and respectively account for 68.7 percent and 14.2 percent of the total amount of the anthocyanin in the pericarp.
Claims (9)
1. A method for carrying out species analysis and content determination on anthocyanin in tomato fruits comprises the following steps:
1) Freezing and grinding the raw materials, and extracting with an extractant a to obtain an extracting solution a; centrifuging, collecting supernatant, blowing with nitrogen, adding extractant b for redissolving, filtering, and collecting filtrate to obtain extract b;
in the freezing step in the step 1), the freezing mode is liquid nitrogen freezing;
in the extraction step, the extractant a is a solution containing an internal standard; the internal standard is paeoniflorin 3-glucoside;
in the extractant a, a solvent is a mixed solution consisting of methanol and formic acid; the volume ratio of the methanol to the formic acid is 7-9:1; in the extractant a, the concentration of an internal standard is 1-5 mug/mL;
the extractant b is a mixed solution composed of methanol and formic acid; the volume ratio of the methanol to the formic acid is 7-9:1;
in the filtering step, the aperture of the filter membrane is 0.22 mu m;
the raw materials are separated tomato peel and tomato pulp;
2) Analyzing the anthocyanin species of the extracting solution b by using liquid chromatography and mass spectrometry;
in the step 2), the conditions of the liquid chromatography detection are as follows:
type of chromatography column: ACQUITY UPLC CSH C18;
chromatographic column parameters: 2.1mm × 100mm i.d.,1.7 μm;
mobile phase A: a mixed solution consisting of acetonitrile and formic acid with a volume ratio of 95;
and (3) mobile phase B: a mixed solution composed of water and formic acid in a volume ratio of 95;
the gradient elution procedure was: the initial volume proportion of the mobile phase A is 2.5%, the mobile phase A is increased from 2.5% to 10% within 5min, then is increased from 10% to 25% within 15min, is kept for 5min, and then is changed from 25% to 2.5% within 5min;
flow rate: 0.15mL/min;
column temperature: at 25 ℃;
autosampler temperature: at 20 ℃;
sample introduction volume: 1. mu L;
in the step 2), the mass spectrum detection conditions are as follows:
the mass spectrum is in a positive ion mode;
an ion source: an ESI source;
capillary voltage: 2.0kV;
ion source temperature: 100 ℃;
desolventizing gas temperature: 300 ℃ is carried out;
desolventizing air flow rate: 800L/h;
taper hole airflow: 50L/h:
mass spectrum acquisition mode: fast DDA mode;
analysis mode: a resolution mode;
the collection range of the parent ions is as follows: 900m/z to 1000m/z;
the collection range of the daughter ions is as follows: 50m/z to 1000m/z;
the collision energy range is 6V-80V;
spectral conditions: the wavelength range of the diode array detector is 190nm-800 nm;
in the step 2), the obtained anthocyanidin is resolved into various types including delphinidin-3- (cis-p-coumaroyloxy) -rutinoside-5-glucoside, petunidin-3- (caffeic acid acyloxy) -rutinoside-5-glucoside, delphinidin-3- (trans-p-coumaroyloxy) -rutinoside-5-glucoside, delphinidin-3- (ferulic acid acyloxy) -rutinoside-5-glucoside, petunidin-3- (cis-p-coumaroyloxy) -rutinoside-5-glucoside, petunidin-3- (trans-p-coumaroyloxy) -rutinoside-5-glucoside, petunidin-3- (ferulic acid acyloxy) -rutinoside-5-glucoside, malvidin-3- (cis-p-coumaroyloxy) -rutinoside-5-glucoside, malvidin-3- (trans-p-coumaroyloxy) -rutinoside-5-glucoside, and trans-malvidin-p-coumarine-5-glucoside 5-glucoside, a vanilloid;
3) And (4) carrying out quantitative analysis on the content of different kinds of anthocyanin obtained from the extracting solution b, and completing the kind analysis and the content determination of the anthocyanin in the tomato fruits.
2. The method of claim 1, wherein: the volume ratio of the methanol to the formic acid is 9;
in the extraction agent a, the concentration of an internal standard is 3.75 mug/mL;
the dosage ratio of the extracting agent a to the raw materials is 0.2-0.8g:1-4mL;
in the extraction step, the temperature is-20 ℃ to 5 ℃; the time is 10-14h; in the extractant b, the volume ratio of the methanol to the formic acid is 9; the volume ratio of the extractant b to the supernatant is as follows: 0.5 to 2;
in the centrifugation step, the rotating speed is 2500-4500rpm; the time is 18-22min.
3. The method of claim 2, wherein: in the extraction step, the temperature is 4 ℃; the time is 12h;
in the centrifugation step, the rotating speed is 3900rpm; the time is 20min.
4. A method according to any one of claims 1-3, characterized in that: the UV retention time of delphinidin-3- (cis-p-coumaric acid acyloxy) -rutinoside-5-glucoside is 11.41min; the MS retention time is 11.55min;
the UV retention time of the petunidin-3- (caffeic acid acyloxy) -rutinoside-5-glucoside is 11.71min; the MS retention time is 11.83min;
the UV retention time of delphinidin-3- (trans-p-coumaric acid acyloxy) -rutinoside-5-glucoside is 11.86min; the MS retention time is 11.97min;
the UV retention time of the delphinidin-3- (ferulic acid acyloxy) -rutinoside-5-glucoside is 12.45min; the MS retention time is 12.58min;
the UV retention time of the petunidin-3- (cis-p-coumaric acid acyloxy) -rutinoside-5-glucoside is 13.01min; the MS retention time is 13.16min;
the UV retention time of the petunidin-3- (trans-p-coumaric acyloxy) -rutinoside-5-glucoside is 13.35min; the MS retention time is 13.52min;
the UV retention time of the petunidin-3- (ferulic acid acyloxy) -rutinoside-5-glucoside is 13.95min; the MS retention time is 14.11min;
the UV retention time of the malvidin-3- (cis-p-coumaric acyloxy) -rutinoside-5-glucoside is 14.61min; the MS retention time is 14.77min;
the UV retention time of the malvidin-3- (trans-p-coumaric acid acyloxy) -rutinoside-5-glucoside is 14.99min; the MS retention time is 15.08min;
the UV retention time of the malvidin-3- (ferulic acid acyloxy) -rutinoside-5-glucoside is 15.33min; the MS retention time is 15.68min;
the UV retention time of the petunidin-3- (trans-p-coumaric acid acyloxy-rhamnoside) -glucoside-5-glucoside is 15.73min; the MS retention time is 15.90min;
the UV retention time of malvidin-3- (p-methoxy-trans-p-coumaric acyloxy) -rutinoside-5-glucoside is 16.07min; the MS retention time was 16.18min.
5. A method according to any one of claims 1-3, characterized in that: in the quantitative analysis in the step 3), the quantitative method is to perform semi-quantitative analysis on the internal standard equivalent; and (4) quantifying by using the peak area of the ultraviolet absorption chromatogram of the anthocyanin at 535nm.
6. A method according to any one of claims 1-3, characterized in that: in the step 3), the linear range is 1-1000 mug/mL;
the standard curve is y =221.941x-346.284; wherein y is the peak area of the internal standard substance; x is the concentration value of the internal standard substance, and the unit is mu g/mL;
limit of quantitation < 1. Mu.g/mL.
7. A method according to any one of claims 1-3, characterized in that: the anthocyanidin is at least one selected from petunidin-3- (cis-p-coumaric acid acyloxy) -rutinoside-5-glucoside, malvidin-3- (cis-p-coumaric acid acyloxy) -rutinoside-5-glucoside, petunidin-3- (trans-p-coumaric acid acyloxy-rhamnoside) -glucoside-5-glucoside and malvidin-3- (p-methoxy-trans-p-coumaric acid acyloxy) -rutinoside-5-glucoside.
8. A method for carrying out species analysis on anthocyanin in tomato fruits comprises the following steps: step 1) and step 2) as set forth in any one of claims 1 to 7.
9. A method for measuring the content of anthocyanin in tomato fruits comprises the following steps: the steps 1) -3) of any one of claims 1-7.
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| CN110108830A (en) * | 2019-05-06 | 2019-08-09 | 中国科学院东北地理与农业生态研究所 | It is a kind of while qualitative and quantitative detection method is carried out to 9 kinds of anthocyanidin in indigo fruit |
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