CN111679020A - HPLC (high Performance liquid chromatography) detection method for content of main flavonoid compounds in buckwheat - Google Patents
HPLC (high Performance liquid chromatography) detection method for content of main flavonoid compounds in buckwheat Download PDFInfo
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- buckwheat
- kaempferol
- quercetin
- rutinoside
- rutin
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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Abstract
The invention discloses an HPLC (high performance liquid chromatography) detection method for the content of main flavonoid compounds in buckwheat, which comprises the following steps: (1) preparing rutin, kaempferol-3-O-rutinoside and quercetin standard substances, respectively drawing standard curves of the rutin, the kaempferol-3-O-rutinoside and the quercetin, and determining linear regression equations of the standard curves; 2) analyzing a buckwheat sample solution to be detected by adopting a high performance liquid chromatograph, carrying out gradient elution on the sample, and respectively determining peak areas of three dominant peaks of an eluate; (3) and calculating the contents of rutin, kaempferol-3-O-rutinoside and quercetin in the buckwheat sample to be detected by contrasting a standard curve and a linear regression equation. The HPLC detection method for the content of the main flavonoid compounds in the buckwheat, which is established by the invention, has the advantages of good selectivity, high sensitivity, accuracy and reliability, and provides a new analysis method and basis for quality analysis and quality control of buckwheat products.
Description
Technical Field
The invention relates to a method for detecting the content of flavonoids in buckwheat, in particular to an HPLC (high performance liquid chromatography) detection method for the content of main flavonoids in buckwheat, belonging to the field of detection of the content of main flavonoids in buckwheat.
Background
Buckwheat is also called Triticum Aestivum, rye, and Triticum aestivum, and belongs to Polygonaceae (Polygonaceae) Fagopyrum genus (Fagopyrum) annual or perennial dicotyledonous herbaceous plant. Buckwheat contains various bioactive substances, wherein the most important substances comprise flavonoid compounds, polyphenol compounds, and active ingredients such as buckwheat sugar alcohol, D-chiro-inositol, active peptide and the like. In particular to flavonoid compounds, which are one of the most important active substances of buckwheat, are natural free radical scavengers widely paid attention to by people and have the effects of resisting oxidation, preventing aging and resisting three highs.
With the improvement of the national living standard, buckwheat is increasingly regarded as an important functional crop. The content and the field of character evaluation and quality identification are widened, but the problems of fuzzy evaluation standard, incomplete system, difficult unification of analysis methods and the like still exist at present, so that the whole industry is totally lack of a standard identification system, and the development pace is slow.
Disclosure of Invention
The invention aims to provide an HPLC (high performance liquid chromatography) detection method for the content of main flavonoid compounds in buckwheat.
The object of the invention is achieved by the following solution,
an HPLC detection method for the content of main flavonoid compounds in buckwheat comprises the following steps:
(1) preparing standard substances of rutin, kaempferol-3-O-rutinoside and quercetin, absorbing mixed standard substances with different concentrations, analyzing by using a high performance liquid chromatograph, respectively drawing standard curves by taking the sample concentration as a horizontal coordinate and taking the peak area as a vertical coordinate, and determining a linear regression equation of the rutin, the kaempferol-3-O-rutinoside and the quercetin;
(2) analyzing the buckwheat sample solution to be detected by adopting a high performance liquid chromatograph, carrying out gradient elution on the sample, and determining the peak area of the dominant peak of the eluate;
(3) and (2) calculating the contents of rutin, kaempferol-3-O-rutinoside and quercetin in the buckwheat sample to be detected through the linear regression equation in the step (1).
Wherein the standard curves of rutin, quercetin and kaempferol-3-O-rutinoside standard substance are respectively shown in FIGS. 11-13, and the determined linear regression equation is as follows:
rutin: y is2＝14878X2+15.349，R2＝0.9999；
And (3) quercetin: y is3＝19731X3-13.076，R2＝0.9999；
kaempferol-3-O-rutinoside: y is4＝13124X4-11.909，R2＝0.9995；
The preparation of the buckwheat sample solution in the step (2) comprises the following steps: drying buckwheat seeds, crushing and sieving the dried buckwheat seeds, adding a methanol aqueous solution into the crushed buckwheat seeds, and performing ultrasonic extraction to obtain a sample solution to be detected; wherein, the concentration of the methanol aqueous solution is preferably 55-85%; the ultrasonic extraction conditions are preferably that the temperature is 30-60 ℃, the ultrasonic time is 15-35min, and the ultrasonic frequency is 30-60 kHz.
The chromatographic conditions for the analysis by the high performance liquid chromatograph in the step (1) or (3) are preferably: the stationary phase is a chromatographic column taking octadecyl bonded silica gel as a filler; the mobile phase A is a 0.1% formic acid aqueous solution, and the mobile phase B is a 0.1% formic acid methanol solution; flow rate: 0.3-1.5 mL/min; detection wavelength: 320-380 nm; sample introduction amount: 5-20 μ L; the column temperature is 33-40 ℃; gradient elution was used, the procedure for gradient elution being as follows:
time: 0-13.0 min, mobile phase A: 80, mobile phase B: 20; time min: 13.0-13.5, mobile phase A: 50, mobile phase B: 50; time: 13.5-17 min, mobile phase A: 80, mobile phase B: 20; time: 17-18 min, mobile phase A: 80, mobile phase B: 20; time: and (4) stopping for 18.01 min.
The established HPLC detection method for the content of the main flavonoid compounds in the buckwheat is subjected to methodology verification including a specificity test, a precision test, a stability test, a repeatability test and a sample adding recovery test, and the verification test result shows that the established detection method is good and the instrument is precise, and the detection of the content of the main flavonoid compounds in the sample under the established detection condition has feasibility.
The HPLC detection method for the content of the main flavonoid compounds in the buckwheat, which is established by the invention, has the advantages of good selectivity, high sensitivity, accuracy and reliability, and provides a new analysis method and basis for quality analysis and quality control of buckwheat products.
Drawings
FIG. 1 is a total ion flow diagram of a mass spectrum of a test sample.
FIG. 2 first order mass spectrum (H) of 1m/z 611.1624 positive ion of compound+)。
FIG. 3 Secondary Mass Spectrum (H) of 1m/z 611.1624 positive ion of Compound No. 3+)。
Figure 4 positive ion (H) of rutin+) A cleavage pathway.
FIG. 5 Primary mass spectrum (H) of 2m/z 595.1666 positive ion of compound+)。
FIG. 6 Secondary Mass Spectrum (H) of 2m/z 595.1666 positive ion of Compound No. 6+)。
FIG. 7 Positive ion (H) of kaempferol-3-O-rutinoside+) A cleavage pathway.
FIG. 8 first order mass spectrum (H) of compound 3m/z 303.0508 positive ion+)。
FIG. 9 Secondary Mass Spectrum (H) of 3m/z 303.0508 positive ion of Compound+)。
Fig. 10 positive ion of quercetin: (H+) A cleavage pathway.
Figure 11 rutin standard curve.
Fig. 12 quercetin standard curve.
FIG. 13 Standard Curve of Kaempferol-3-O-rutinoside.
FIG. 14 is a chromatogram of a sample solution.
FIG. 15 chromatogram of control solution.
Figure 16 blank solvent chromatogram.
Detailed Description
The invention is further described below in conjunction with specific embodiments, the advantages and features of which will become apparent from the description. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.
Example 1 method for determining content of main flavonoids in buckwheat
(1) Preparation of sample solution: taking an appropriate amount of buckwheat seeds, drying at 65 ℃ to balance weight, crushing, and sieving with a 40-80 mesh sieve. Precisely weighing the fine powder, adding methanol solution, and performing ultrasonic extraction. The concentration of the methanol solution is 55-85%, the temperature is 30-60 ℃, the ultrasonic time is 15-35min, and the ultrasonic frequency is 30-60 kHz.
(2) The method for measuring the content of the main flavonoid compounds comprises the following steps: the stationary phase is a chromatographic column taking octadecyl bonded silica gel as a filler; the mobile phase A is aqueous solution (containing 0.1% formic acid), and the mobile phase B is methanol solution (containing 0.1% formic acid); flow rate: 0.3-1.5 mL/min; detection wavelength: 320-380 nm; sample introduction amount: 5-20 μ L; the column temperature was 33-40 ℃ and the gradient elution procedure is shown in Table 1.
TABLE 1 gradient elution procedure
(3) Mass spectrum detection: adopting electrospray ionization (ESI) mode to perform positive and negative ionizationAnd detecting a sub-mode. Carrier gas: n is a radical of2The carrier gas temperature: 300 ℃; flow rate of drying gas: 8.0L/min-1; atomizer pressure: 35 psi; capillary voltage: 3.5 kV; capillary exit voltage: 175V; taper hole voltage: 65V; collision energy: 10V-80V; mass spectrum scanning range: m/z is 50 to 1000. FIG. 1 is a total ion flow diagram of a mass spectrum of a test sample.
By utilizing an HPLC (high performance liquid chromatography) technology, methanol and an aqueous solution (both containing 0.1% formic acid) are used for gradient elution, the content and the composition of main flavonoid compounds in a sample are determined, 3 dominant peaks are detected in total and named as tR1 (compound 1), tR2 (compound 2) and tR3 (compound 3), the mass spectrum cracking rule is researched by adopting an HPLC-ESI-Q-TOF/MS technology, and the structure is deduced and confirmed.
And (3) verifying the mass spectrum structure: and (3) verifying mass spectrum inference results of RT1, RT2 and RT3 by using standard substances, and determining that the three compounds are rutin, kaempferol-3-O-rutinoside and quercetin respectively.
Mass spectrum structure verification of compound 1:
the peak of the excimer ion is M/z611.1621, which is the [ M + H ] thereof]+Peak, second order Mass Spectrometry shows [ M + H]+Peak M/z611.1621 Collision-induced dissociation of the rhamnosyl group (Rha, 146Da) mainly missing at the end of the oligosaccharide chain, leading to M/z465.1051 ions, i.e. [ M-Rha + H ]]+. In addition, [ M + H ]]+M/z303.0508 was generated by loss of the entire oligosaccharide chain neutral fragment (308Da), thus loss of terminal rhamnosyl and the diglycoside was the main reaction (fig. 2-4), verifying that compound 1 is rutin.
Mass spectrum structure confirmation of compound 2
The peak of the excimer ion is M/z 595.1666, which is the [ M + H ] thereof]+Peak, second order Mass Spectrometry shows [ M + H]+The glycosidic bond of the peak ion is broken to generate fragments m/z 287.0562 and m/z 449.1096, wherein the fragment m/z 287.0562 indicates that the aglycone of the compound 2 is kaempferol, namely, the diglucoside is connected on the flavonoid aglycone. In addition, the terminal rhamnosyl group is lost when the glycosidic bond between the disaccharides is broken, i.e. the fragment m/z 449.1096 is generated (FIGS. 5-7). And (3) confirming that the compound 2 is kaempferol-3-O-rutinoside by combining with the quality spectrum cracking rule of a control.
The peak of the excimer ion is M/z303.0508, which is the [ M + H ] thereof]+Peak, second order Mass Spectrometry shows [ M + H]+The peak is the loss of one molecule of H from quercetin2O formed fragment m/z 285.0356, then lost C-cyclic carbonyl formed fragment m/z257.0470, fragment m/z257.0470 underwent structural rearrangement, and continued to lose C-cyclic carbonyl formed fragment m/z 229.0487 (fig. 8-10), thereby verifying that compound 3 was quercetin.
Test example 1 methodological verification
(1) Preparation of standard curve
Precisely weighing 9.8mg of rutin standard, 3.0mg of kaempferol-3-O-rutinoside standard and 3.2mg of quercetin standard, and fixing the volume to 10mL by using 80% methanol solution to obtain a mixed reference substance stock solution of rutin, kaempferol-3-O-rutinoside and quercetin. Diluting 5mL of stock solution with 80% methanol to constant volume of 10mL, sequentially diluting at equal times, filtering with 0.22 μm microporous membrane, collecting filtrate, detecting 5 μ L of sample, recording peak area, and drawing standard curve with peak area Y as ordinate and standard substance concentration X (mg/mL) as abscissa.
The regression equation of the obtained rutin, quercetin and kaempferol-3-O-rutinoside standard substance is as follows:
rutin: y is2＝14878X2+15.349，R2＝0.9999
And (3) quercetin: y is3＝19731X3-13.076，R2＝0.9999
kaempferol-3-O-rutinoside: y is4＝13124X4-11.909，R2＝0.9995
Shows that the linear relation among rutin, quercetin and kaempferol-3-O-rutinoside is good, and the standard curves are respectively shown in figures 11-13.
(2) Specificity test
Precisely transferring mixed reference solution M of rutin with concentration of 0.49mg/mL, quercetin with concentration of 0.16mg/mL, and kaempferol-3-O-rutinoside with concentration of 0.15mg/mL10.22 μm filtration and determination according to the chromatographic conditions of example 1.
The results of the specificity test are shown in FIGS. 14 to 16, which indicate that the blank solvent does not interfere with the measurement of the main substance.
(3) Precision test
Taking mixed reference substance solution M1The sample introduction was repeated 6 times in an amount of 5. mu.L, and the RSD was calculated by measuring the peak area under the chromatographic conditions of example 1 and recording the peak area.
The test result shows that: the peak areas RSD (%) of rutin, quercetin and kaempferol-3-O-rutinoside are all less than 1.0%, which indicates that the precision of the instrument is good.
TABLE 2 results of precision test
(4) Stability test
Taking mixed reference substance solution M1Samples were taken at 0, 2, 4, 6, 8, 10, 12, and 24 hours, respectively, at 5. mu.L, and the peak area was recorded and the RSD was calculated by measuring the sample under the chromatographic conditions of example 1.
Test results show that the RSD (%) of the peak areas of rutin, quercetin and kaempferol-3-O-rutinoside is less than 1.0 percent, which indicates that the solution has good stability within 24 hours and can meet the measurement requirements.
TABLE 3 stability test results
(4) Repeatability test
A buckwheat seed powder sample of 200mg is extracted, precision and stability are realized, the volume is adjusted to 20mL by 80% methanol, a sample solution is obtained by filtering with the diameter of 0.22 μm, and 6 sample solutions are prepared in parallel, and the sample injection amount is 5 μ L. The RSD was calculated by measuring the chromatographic conditions of example 1 and recording the peak area.
TABLE 4 results of the repeatability tests
(5) Sample application recovery test
Precisely weighing a certain amount of rutin, quercetin and kaempferol-3-O-rutinoside standard substance (subpackaging and fixing the volume after dissolving) respectively, placing into 6 conical bottles, respectively and precisely adding 0.2g of buckwheat seed powder with known content, wherein the content of the rutin, the quercetin and the kaempferol-3-O-rutinoside is respectively 4.064mg, 0.068mg and 0.247mg, and fixing the volume to 20 ml. The amount of sample was 5. mu.L. The peak area was recorded by measurement under the chromatographic conditions of example 1, and the recovery rate and RSD were calculated according to the formula (1).
The test results are shown in table 5. The result shows that the sample adding recovery rates of rutin, quercetin and kaempferol-3-O-rutinoside are all 90-100%, the average recovery rates are 98.88%, 97.32% and 98.02%, and the RSD is less than 1.0%, which shows that the method established by the invention is reliable and meets the requirement of content measurement.
TABLE 5 sample recovery test results
Claims (10)
1. An HPLC detection method for the content of main flavonoid compounds in buckwheat is characterized by comprising the following steps:
(1) preparing standard substances of rutin, kaempferol-3-O-rutinoside and quercetin, absorbing mixed standard substances with different concentrations, analyzing by using a high performance liquid chromatograph, respectively drawing a standard curve of the rutin, the kaempferol-3-O-rutinoside and the quercetin by taking the sample concentration as a horizontal coordinate and taking the peak area as a vertical coordinate, and determining a linear regression equation of the rutin, the kaempferol-3-O-rutinoside and the quercetin;
(2) analyzing a buckwheat sample solution to be detected by adopting a high performance liquid chromatograph, carrying out gradient elution on the sample, and respectively determining peak areas of three dominant peaks of an eluate;
(3) and (3) calculating the contents of rutin, kaempferol-3-O-rutinoside and quercetin in the buckwheat sample to be detected by contrasting the standard curve in the step (1) and a linear regression equation.
2. The HPLC detecting method according to claim 1, wherein the standard curves of rutin, quercetin, kaempferol-3-O-rutinoside as standard substances are shown in FIG. 11, FIG. 12 and FIG. 13, respectively.
3. The HPLC detection method of claim 1, wherein the linear regression equations for rutin, quercetin, kaempferol-3-O-rutinoside as the standard are as follows:
rutin: y is2＝14878X2+15.349，R2＝0.9999；
And (3) quercetin: y is3＝19731X3-13.076，R2＝0.9999；
kaempferol-3-O-rutinoside: y is4＝13124X4-11.909，R2＝0.9995。
4. The HPLC detecting method according to claim 1, wherein the preparation of the buckwheat sample solution in the step (2) comprises: drying buckwheat seeds, crushing and sieving the dried buckwheat seeds, adding a methanol water solution into the crushed buckwheat seeds, and performing ultrasonic extraction to obtain a sample solution to be detected.
5. An HPLC detection method as claimed in claim 4, wherein the concentration of said aqueous methanol solution is 55-85%.
6. An HPLC detection method as described in claim 4, wherein the ultrasonic extraction conditions are 30-60 ℃ for 15-35min and 30-60 kHz.
7. The HPLC detecting method according to claim 1, wherein the chromatographic conditions for the analysis by the high performance liquid chromatograph in step (1) or (3) are: the mobile phase A is a 0.1% formic acid aqueous solution, and the mobile phase B is a 0.1% formic acid methanol solution; gradient elution.
8. An HPLC detection method according to claim 7, characterized in that the gradient elution procedure is as follows:
time: 0-13.0 min, mobile phase A: 80, mobile phase B: 20; time: 13.0-13.5 min, mobile phase A: 50, mobile phase B: 50; time: 13.5-17 min, mobile phase A: 80, mobile phase B: 20; time min: 17-18, mobile phase A: 80, mobile phase B: 20; time min: 18.01, terminate.
9. An HPLC detection method according to claim 7, wherein the stationary phase in the chromatographic condition is a chromatographic column using octadecyl bonded silica gel as a filler.
10. An HPLC detection method as recited in claim 7, wherein the chromatographic conditions further include: flow rate: 0.3-1.5 mL/min; detection wavelength: 320-380 nm; sample introduction amount: 5-20 μ L; the column temperature is 33-40 ℃.
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