CN111679020A - HPLC method for determination of main flavonoids in buckwheat - Google Patents

Abstract

The invention discloses an HPLC detection method for the content of main flavonoids in buckwheat, comprising: (1) preparing standard products of rutin, kaempferol-3-O-rutinoside and quercetin, respectively drawing rutin and kaempferol ‑3‑O‑ The standard curve of rutinoside and quercetin and determine its linear regression equation; 2) The buckwheat sample solution to be detected is analyzed by high performance liquid chromatography, the sample is subjected to gradient elution, and the elution is determined respectively (3) The contents of rutin, kaempferol-3-O-rutinoside and quercetin in the buckwheat samples to be detected were calculated according to the standard curve and linear regression equation. The HPLC detection method for the content of main flavonoids in buckwheat established by the invention has 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

HPLC method for determination of main flavonoids in buckwheat

technical field

The invention relates to a method for detecting the content of flavonoids in buckwheat, in particular to an HPLC detection method for the content of main flavonoids in buckwheat, and belongs to the field of detecting the content of main flavonoids in buckwheat.

Background technique

Buckwheat, also known as triangular wheat, black wheat, and gray barley, is an annual or perennial dicotyledonous herb of the family Polygonaceae (Fagopyrum). Buckwheat contains a variety of biologically active substances, the most important of which include flavonoids, polyphenols, and active ingredients such as buckwheat sugar alcohol, D-chiro-inositol, and active peptides. In particular, flavonoids, as one of the most important active substances in buckwheat, are natural free radical scavengers that have attracted widespread attention, and have the effects of anti-oxidation, anti-aging, and anti-three highs.

With the improvement of people's living standards, buckwheat has been paid more and more attention as an important functional crop. The content and fields of its trait evaluation and quality identification have been broadened, but there are still problems such as ambiguous evaluation standards, incomplete systems, and difficult to unify analysis methods, resulting in the lack of a standard identification system in the entire industry, and the pace of development is slow. .

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide an HPLC (high performance liquid chromatography) detection method for the content of main flavonoids in buckwheat.

The object of the present invention is achieved through the following technical solutions,

An HPLC method for detecting the content of main flavonoids in buckwheat, comprising:

(1) Prepare standard products of rutin, kaempferol-3-O-rutinoside and quercetin, draw mixed standard products of different concentrations and analyze by high performance liquid chromatograph. The area is the ordinate and the standard curve is drawn respectively to determine the linear regression equation of rutin, kaempferol-3-O-rutinoside and quercetin;

(2) the buckwheat sample solution to be detected is analyzed by high performance liquid chromatography, the sample is subjected to gradient elution, and the peak area of the dominant peak of the eluate is determined;

(3) Calculate the contents of rutin, kaempferol-3-O-rutinoside and quercetin in the buckwheat sample to be detected by the linear regression equation of step (1).

Among them, the standard curves of rutin, quercetin and kaempferol-3-O-rutinoside standard products are shown in Figure 11-Figure 13 respectively, and the determined linear regression equation is as follows:

Rutin: Y ₂ =14878X ₂ +15.349, R ² =0.9999;

Quercetin: Y ₃ =19731X ₃ -13.076, R ² =0.9999;

Kaempferol-3-O-rutinoside: Y ₄ =13124X ₄ -11.909, R ² =0.9995;

The preparation of the buckwheat sample solution described in step (2) includes: drying the buckwheat seeds, crushing and sieving them, adding methanol aqueous solution for ultrasonic extraction to obtain the sample solution to be detected; wherein, the concentration of the methanol aqueous solution is preferably 55% -85%; the conditions for the ultrasonic extraction are preferably: the temperature is 30-60° C., the ultrasonic time is 15-35 min, and the ultrasonic frequency is 30-60 kHz.

In step (1) or (3), the chromatographic conditions for analysis by high performance liquid chromatograph are preferably: the stationary phase is a chromatographic column with octadecyl-bonded silica gel as filler; the mobile phase A is an aqueous solution containing 0.1% formic acid, Mobile phase B is methanol solution containing 0.1% formic acid; flow rate: 0.3-1.5 mL/min; detection wavelength: 320-380 nm; injection volume: 5-20 μL; column temperature 33-40 °C; gradient elution, gradient washing The removal procedure is as follows:

Time: 0～13.0min, mobile phase A: 80, mobile phase B: 20; time min: 13.0～13.5, mobile phase A: 50, mobile phase B: 50; time: 13.5～17min, mobile phase A: 80, Mobile phase B: 20; time: 17-18 min, mobile phase A: 80, mobile phase B: 20; time: 18.01 min, stop.

The invention has carried out methodological verification on the established HPLC detection method for the content of main flavonoids in buckwheat, including specificity test, precision test, stability test, repeatability test, sample addition and recovery test, and the verification test results show that this The detection method established by the invention is good and the instrument is precise, and it is feasible to detect the content of the main flavonoids in the sample under the established detection conditions.

The HPLC detection method for the content of main flavonoids in buckwheat established by the invention has 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 of drawings

Figure 1. Test mass spectrum total ion current diagram.

Figure 2 The first order mass spectrum (H ⁺ ) of the positive ion of compound 1 m/z 611.1624.

Figure 3 Secondary mass spectrum (H ⁺ ) of the positive ion of compound 1 m/z 611.1624.

Figure 4 Positive ion (H ⁺ ) cleavage pathway of rutin.

Figure 5 First order mass spectrum (H ⁺ ) of compound 2m/z 595.1666 positive ion.

Figure 6 Secondary mass spectrum (H ⁺ ) of the positive ion of compound 2m/z 595.1666.

Figure 7 Positive ion (H ⁺ ) cleavage pathway of kaempferol-3-O-rutinoside.

Figure 8 First order mass spectrum (H ⁺ ) of the positive ion of compound 3m/z 303.0508.

Figure 9 Secondary mass spectrum (H ⁺ ) of the positive ion of compound 3m/z 303.0508.

Figure 10 Positive ion (H ⁺ ) cleavage pathway of quercetin.

Figure 11 Standard curve of rutin.

Figure 12 Quercetin standard curve.

Figure 13 kaempferol-3-O-rutinoside standard curve.

Figure 14. The chromatogram of the test solution.

Figure 15. Chromatogram of reference solution.

Figure 16 Blank solvent chromatogram.

Detailed ways

The present invention will be further described below with reference to specific embodiments, and the advantages and characteristics of the present invention will become more apparent with the description. However, these examples are only exemplary and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

Example 1 Establishment of a method for determining the content of main flavonoids in buckwheat

(1) Preparation of sample solution: Take an appropriate amount of buckwheat seeds, bake to constant weight at 65°C, pulverize, and pass through a 40-80 mesh sieve. The fine powder was accurately weighed, methanol solution was added, and ultrasonic extraction was performed. The methanol solution concentration is 55-85%, the temperature is 30-60 DEG C, the ultrasonic time is 15-35min, and the ultrasonic frequency is 30-60kHz.

(2) The determination method of main flavonoids content was established: the stationary phase was a chromatographic column filled with octadecyl-bonded silica gel; the mobile phase A was an aqueous solution (containing 0.1% formic acid), and the mobile phase B was a methanol solution (containing 0.1% formic acid). % formic acid); flow rate: 0.3-1.5 mL/min; detection wavelength: 320-380 nm; injection volume: 5-20 μL;

Table 1 Gradient elution procedure

(3) Mass spectrometry detection: Electrospray ionization (ESI) was used for detection in positive and negative ion mode. Carrier gas: N ₂ , carrier gas temperature: 300°C; drying gas flow rate: 8.0L/min-1; nebulizer pressure: 35psi; capillary voltage: 3.5kV; capillary outlet voltage: 175V; cone voltage: 65V; collision Energy: 10V-80V; mass spectrum scanning range: m/z 50～1000. Figure 1 is a total ion current diagram of the mass spectrum for the test.

The content and composition of the main flavonoids in the samples were determined by HPLC with gradient elution of methanol and aqueous solution (both containing 0.1% formic acid), and three dominant peaks were detected, named tR1 (compound 1), tR2 ( Compound 2), tR3 (compound 3), using HPLC-ESI-Q-TOF/MS technology, the mass spectrometry fragmentation rule was studied, and its structure was deduced and confirmed.

Verification of mass spectrometry structure: RT1, RT2, RT3 were used to verify the results of mass spectrometry inference, and the three compounds were determined to be rutin, kaempferol-3-O-rutinoside, and quercetin.

Compound 1 mass spectrometry structure verification:

The quasi-molecular ion peak is m/z 611.1621, which is the [M+H] ⁺ peak. The secondary mass spectrometry shows that the [M+H] ⁺ peak m/z611.1621 has undergone collision-induced dissociation, and the mouse at the end of the oligosaccharide chain is mainly lost. Linosyl (Rha, 146Da) yields the m/z 465.1051 ion, ie [M-Rha+H] ⁺ . In addition, [M+H] ⁺ generates m/z 303.0508 by losing the entire oligosaccharide chain neutral fragment (308Da), so the loss of terminal rhamnosyl and disoside is the main reaction (Fig. 2-Fig. 4), verifying Compound 1 is rutin.

Structure confirmation of compound 2 by mass spectrometry

The quasi-molecular ion peak is m/z 595.1666, which is the [M+H] ⁺ peak, and the second mass spectrum shows that the glycosidic bond of the [M+H] ⁺ peak ion is broken to generate fragments m/z 287.0562 and m/z 449.1096, among which fragments m/z 287.0562 indicates that the aglycone of compound 2 is kaempferol, that is, a disoside is attached to the flavonoid aglycone. In addition, the terminal rhamnosyl group was lost when the glycosidic bond between the disglycosides was broken, that is, the fragment m/z 449.1096 was generated (Fig. 5-Fig. 7). Combined with the cracking rule of the reference mass spectrum, it was confirmed that compound 2 was kaempferol-3-O-rutinoside.

Compound 3 mass spectrometry structure verification

The quasi-molecular ion peak is m/z 303.0508, which is the [M+H] ⁺ peak, and the secondary mass spectrum shows that the [M+H] ⁺ peak is that quercetin lost a molecule of H ₂ O to form a fragment m/z 285.0356, and then lost C ring carbonyl forms fragment m/z 257.0470. After the structural rearrangement of fragment m/z 257.0470, C ring carbonyl continues to lose to form fragment m/z 229.0487 (Figure 8-Figure 10), thus verifying that compound 3 is quercetin .

Test Example 1 Methodological Verification

(1) Establishment of standard curve

Precisely weigh 9.8 mg of rutin standard, 3.0 mg of kaempferol-3-O-rutinoside standard, and 3.2 mg of quercetin standard, and use 80% methanol solution to dilute to 10 mL to obtain rutin, kaempferol- 3-O-rutinoside and quercetin mixed reference stock solution. Take 5 mL of the stock solution and dilute it to 10 mL with 80% methanol, dilute it in equal times successively, filter it with a 0.22 μm microporous membrane, take the subsequent filtrate, inject 5 μL each time for detection, record the peak area, and take the peak area Y as the ordinate , the standard concentration X (mg/mL) is the abscissa to draw a standard curve.

The regression equation for obtaining the standard products of rutin, quercetin and kaempferol-3-O-rutinoside is as follows:

Rutin: Y ₂ =14878X ₂ +15.349, R ² =0.9999

Quercetin: Y ₃ =19731X ₃ -13.076, R ² =0.9999

Kaempferol-3-O-rutinoside: Y ₄ =13124X ₄ -11.909, R ² =0.9995

It shows that the linear relationship between rutin, quercetin and kaempferol-3-O-rutinoside is good, and the standard curves are shown in Figure 11-Figure 13 respectively.

(2) Specificity test

Precisely pipet the mixed reference solution M ₁ of rutin, 0.16 mg/mL quercetin and 0.15 mg/mL kaempferol-3-O-rutinoside with concentrations of 0.49 mg/mL, respectively, and filter them at 0.22 μm. The chromatographic conditions of Example 1 were determined.

The specificity test results are shown in Figure 14 to Figure 16, indicating that the blank solvent does not interfere with the determination of the main substances.

(3) Precision test

The mixed reference solution M ₁ was taken, and the sample was repeatedly injected 6 times with a sample injection volume of 5 μL, measured according to the chromatographic conditions of Example 1, the peak area was recorded, and the RSD was calculated.

The test results showed that the peak area RSD (%) of rutin, quercetin and kaempferol-3-O-rutinoside were all less than 1.0%, indicating that the instrument had good precision.

Table 2 Precision test results

(4) Stability test

Take the mixed reference solution M ₁ , inject samples at 0, 2, 4, 6, 8, 10, 12, and 24 h respectively, with a sample injection volume of 5 μL, measure according to the chromatographic conditions of Example 1, and record its peak area, Calculate RSD.

The test results show that the RSD (%) of the peak areas of rutin, quercetin and kaempferol-3-O-rutinoside are all less than 1.0%, indicating that the solution has good stability within 24 hours and can meet the measurement requirements.

Table 3 Stability test results

(4) Repeatability test

A 200 mg buckwheat seed powder sample was extracted, which was precise and stable, and the volume was adjusted to 20 mL with 80% methanol, and the sample solution was filtered at 0.22 μm. Six sample solutions were prepared in parallel, and the injection volume was 5 μL. According to the chromatographic conditions of Example 1, the peak area was recorded, and the RSD was calculated.

Table 4 Repeatability test results

(5) Sample recovery test

Accurately weigh a certain amount of rutin, quercetin, and kaempferol-3-O-rutinoside standard (dissolve and repack to constant volume) and place them in 6 conical flasks, respectively, and accurately add a known content of buckwheat. Seed powder 0.2g, the contents of rutin, quercetin and kaempferol-3-O-rutinoside were 4.064mg, 0.068mg, 0.247mg respectively, and the volume was 20ml. The injection volume was 5 μL. Measure according to the chromatographic conditions of Example 1, record the peak area, and calculate the recovery and RSD according to formula (1).

The test results are shown in Table 5. The results showed that the recovery rates of rutin, quercetin and kaempferol-3-O-rutinoside were all between 90 and 100%, and the average recoveries were 98.88%, 97.32% and 98.02%, respectively. RSD All are less than 1.0%, indicating that the method established by the present invention is reliable and meets the requirements of content determination.

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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