METHOD FOR IDENTIFYING GRAPE SEED EXTRACT AUTHENTICITY USING HPLC FINGERPRINT SPECTRUM

Abstract

Disclosed is a method for identifying grape seed extract authenticity using the HPLC fingerprint spectrum, and in particular, a method for identifying the adulteration of a pine bark extract or a peanut skin extract in a grape seed extract. The method comprises the following steps: 1) establishing the HPLC fingerprint spectrums of the grape seed extract, the pine bark extract and the peanut skin extract, respectively; 2) determining a characteristic peak of pine bark extract and a characteristic peak of peanut skin extract; 3) subjecting the grape seed extract sample to be tested to liquid chromatography detection, identifying the adulteration of the pine bark extract or the peanut skin extract in the sample to be tested according to whether the chromatogram contains the characteristic peaks of the pine bark extract and/or the peanut skin extract, wherein the addition of more than 3% of adulterants can be accurately identified. The method has good stability and reproducibility, high efficiency, obvious identification characteristics, provides a theoretical basis for the identification of the plant sources of grape seed extracts, and is conducive to promoting the healthy development of the plant extract industry.

Claims

1. A method for identifying grape seed extract authenticity using HPLC fingerprint spectrum, characterized in that, the method comprises the following steps: S1: establishing HPLC fingerprint spectrums of a grape seed extract, an adulterant pine bark extract and an adulterant peanut skin extract, respectively; S2: comparing the HPLC fingerprint spectrum of the pine bark extract with the HPLC fingerprint spectrum of the grape seed extract to determine the characteristic peak of the pine bark extract; comparing the HPLC fingerprint spectrum of the peanut skin extract with the HPLC fingerprint spectrum of the grape seed extract to determine the characteristic peak of the peanut skin extract; S3: determining a grape seed extract sample to be tested using high performance liquid chromatography, and identifying the adulteration of the pine bark extract or the peanut skin extract in the sample to be tested according to the presence or absence of characteristic peaks.

2. The method according to claim 1, characterized in that, the specific process of S1 is as follows: S11: preparation of a test solution: accurately weighing appropriate amounts of standard substances of grape seed extract, pine bark extract, and peanut skin extract, respectively, adding aqueous ethanol solution, performing ultrasonic dissolving, cooling and diluting to a fixed volume, so as to prepare a test solution with a concentration of 5 to 20 mg/mL; and S12: HPLC determination: respectively, accurately drawing the test solution of grape seed extract, the test solution of pine bark extract, and the test solution of peanut skin extract, injecting the solutions into a liquid chromatograph, performing detection using liquid chromatography, and respectively establishing a HPLC fingerprint spectrum of the grape seed extract, a HPLC fingerprint spectrum of the pine bark extract and a HPLC fingerprint spectrum of the peanut skin extract with a time period of 0 to 30 min, wherein, HPLC chromatographic conditions are as follows: octadecylsilane bonded silica gel column is used as the stationary phase, and acetonitrile and formic acid aqueous solution are used as the mobile phase for gradient elution; the flow rate is 1.2 mL/min; the detection wavelength is 270 to 310 nm; and the temperature of the chromatographic column is 25 to 40 C.

3. The method according to claim 1, characterized in that, the HPLC fingerprint spectrum of the pine bark extract is compared with the HPLC fingerprint spectrum of the grape seed extract in S2, a characteristic peak PB1 of the pine bark extract is determined at 10 to 15 min, and the retention time of the characteristic peak PB1 is 11.71 min.

4. The method according to claim 1, characterized in that, the HPLC fingerprint spectrum of the peanut skin extract is compared with the HPLC fingerprint spectrum of the grape seed extract in S2, characteristic peaks PS1 and PS2 of the peanut skin extract are determined at 10 to 15 min, and the retention times of characteristic peaks PS1 and PS2 are 11.51 min and 11.71 min, respectively.

5. The method according to claim 1, characterized in that, the specific process of S3 is as follows: if a peak occurs at 11.71 min, indicating that the sample to be tested is adulterated with the pine bark extract; and if peaks occur both at 11.51 min and 11.71 min, indicating that the sample to be tested is adulterated with the peanut skin extract or with both the peanut skin extract and the pine bark extract.

6. The method according to claim 2, characterized in that, the concentration of the aqueous ethanol solution in S11 is 20 to 80 v/v %.

7. The method according to claim 2, characterized in that, the ultrasonic conditions in S11 are as follows: the ultrasonic power is 600 W with a frequency of 40 KHz and an ultrasonic time of 2 to 5 minutes, wherein the ultrasonic is started for 2 to 3 seconds, and stopped for 3 to 5 seconds.

8. The method according to claim 2, characterized in that, the conditions for gradient elution in S12 are as follows: 0 to 15 min: 10% to 18% acetonitrile; 15 to 23 min: 18% to 60% acetonitrile; 23 to 24 min: 60% to 10% acetonitrile; and 24 to 30 min: 10% acetonitrile.

9. The method according to claim 2, characterized in that, the specifications of the silica gel column in S12 are as follow: a length of 150 mm, an inner diameter of 4.6 mm, and a particle size of 5 m for an octadecylsilane bonded silica gel layer.

10. The method according to claim 2, characterized in that, the volume ratio of formic acid to water in the formic acid aqueous solution in S12 is 0.1:100.

11. The method according to claim 2, characterized in that, the HPLC fingerprint spectrum of the pine bark extract is compared with the HPLC fingerprint spectrum of the grape seed extract in S2, a characteristic peak PB1 of the pine bark extract is determined at 10 to 15 min, and the retention time of the characteristic peak PB1 is 11.71 min.

12. The method according to claim 3, characterized in that, the HPLC fingerprint spectrum of the peanut skin extract is compared with the HPLC fingerprint spectrum of the grape seed extract in S2, characteristic peaks PS1 and PS2 of the peanut skin extract are determined at 10 to 15 min, and the retention times of characteristic peaks PS1 and PS2 are 11.51 min and 11.71 min, respectively.

13. The method according to claim 4, characterized in that, the specific process of S3 is as follows: if a peak occurs at 11.71 min, indicating that the sample to be tested is adulterated with the pine bark extract; and if peaks occur both at 11.51 min and 11.71 min, indicating that the sample to be tested is adulterated with the peanut skin extract or with both the peanut skin extract and the pine bark extract.

14. The method according to claim 5, characterized in that, the concentration of the aqueous ethanol solution in S11 is 20 to 80 v/v%.

15. The method according to claim 6, characterized in that, the ultrasonic conditions in S11 are as follows: the ultrasonic power is 600 W with a frequency of 40 KHz and an ultrasonic time of 2 to 5 minutes, wherein the ultrasonic is started for 2 to 3 seconds, and stopped for 3 to 5 seconds.

16. The method according to claim 7, characterized in that, the conditions for gradient elution in S12 are as follows: 0 to 15 min: 10% to 18% acetonitrile; 15 to 23 min: 18% to 60% acetonitrile; 23 to 24 min: 60% to 10% acetonitrile; and 24 to 30 min: 10% acetonitrile.

17. The method according to claim 8, characterized in that, the specifications of the silica gel column in S12 are as follow: a length of 150 mm, an inner diameter of 4.6 mm, and a particle size of 5 m for an octadecylsilane bonded silica gel layer.

18. The method according to claim 9, characterized in that, the volume ratio of formic acid to water in the formic acid aqueous solution in S12 is 0.1:100.

19. The method according to claim 2, characterized in that, the HPLC fingerprint spectrum of the peanut skin extract is compared with the HPLC fingerprint spectrum of the grape seed extract in S2, characteristic peaks PS1 and PS2 of the peanut skin extract are determined at 10 to 15 min, and the retention times of characteristic peaks PS1 and PS2 are 11.51 min and 11.71 min, respectively.

20. The method according to claim 3, characterized in that, the specific process of S3 is as follows: if a peak occurs at 11.71 min, indicating that the sample to be tested is adulterated with the pine bark extract; and if peaks occur both at 11.51 min and 11.71 min, indicating that the sample to be tested is adulterated with the peanut skin extract or with both the peanut skin extract and the pine bark extract.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIG. 1 shows the HPLC fingerprint spectrum established in Example 1 of the present invention; wherein, A represents grape seed extract, B represents pine bark extract, and C represents peanut skin extract;

[0030] FIGS. 2-5 shows the HPLC chromatograms of grape seed extracts adulterated with 3%, 10%, 15% and 20% pine bark extract in Example 2 of the present invention, respectively.

[0031] FIG. 6 shows the HPLC chromatogram of unadulterated grape seed extract in Example 2 of the present invention;

[0032] FIGS. 7-10 shows the HPLC chromatograms of grape seed extracts adulterated with 3%, 10%, 15% and 20% peanut skin extracts in Example 3 of the present invention, respectively;

[0033] FIG. 11 shows the HPLC chromatogram of unadulterated grape seed extract in Example 3 of the present invention.

SPECIFIC MODES FOR CARRYING OUT THE EMBODIMENTS

[0034] The following Examples are intended to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the Examples are conventional means well known to a person skilled in the art, and the raw materials are all commercially available products.

Example 1: Establishment of HPLC Fingerprint Spectrums of Grape Seed Extract, Pine Bark Extract and Peanut Skin Extract

[0035] 1, Instruments and Reagents

[0036] 1.1 Instruments

[0037] Agilent 1260 high performance liquid chromatograph, and Zorbax SB-C18 chromatographic column (5 m, 150 mm4.6mm).

[0038] 1.2 Reagents

[0039] Ethanol used for extraction was an analytically pure reagent; and acetonitrile used as a reagent for the liquid phase analysis was a chromatographically pure reagent. Grape seed extract, pine bark extract, and peanut skin extract were provided by Chenguang Biotech Group Co., Ltd.

[0040] 2. Methods and Results

[0041] For the separation conditions of liquid chromatography, see USP 38, Grape Seeds Oligomeric Proanthocyanidins, and the chromatographic conditions were as follows:

[0042] (1) The stationary phase was an octadecylsilane bonded silica gel column (150 mm4.6 mm, 5 m);

[0043] (2) The mobile phase was (A) acetonitrile, (B) water/formic acid (100/0.1, v/v); and the gradient of the mobile phase was as follows: 0-15 min: 10 to 18% A; 15-23 min: 18 to 60% A; 23-24 min: 60 to 10% A; and 24-30min: 10% A;

[0044] (3) Flow rate: 1.2 mL/min;

[0045] (4) Detection wavelength: 278 nm; and

[0046] (5) Injection volume: 5 L.

[0047] 3. Determination of HPLC Fingerprint Spectrum

[0048] 3.1 Establishment of HPLC Fingerprint Spectrum of Grape Seed Extract

[0049] (a) Preparation of a test solution: an appropriate amount of the grape seed extract standard was accurately weighed, and added into a aqueous ethanol solution with a certain volume fraction of ethanol, subjected to ultrasonic dissolving and cooling, and then the obtained solution was diluted to a fixed volume so as to prepare the test solution with a concentration of 10 mg/mL; and the ultrasonic conditions were as follows: the ultrasonic power was 600 W; the frequency was 40 KHz; the ultrasonic time was 2 to 5 minutes, wherein the ultrasonic is started for 2 to 3 seconds, and stopped for 3 to 5 seconds.

[0050] (b) The chromatographic conditions were as follows: octadecylsilane bonded silica gel was used as the stationary phase; gradient elution was performed by using acetonitrile and formic acid aqueous solution; the flow rate was 1.2 mL/min; the detection wavelength was 278 nm; and the temperature of the chromatographic column was 40 C.

[0051] (c) Measurement: the test solution of the grape seed extract was accurately drawn, and injected into a liquid chromatograph, and determined by liquid chromatography, so as to obtain a HPLC fingerprint spectrum of the grape seed extract (FIG. 1A).

[0052] 3.2 Establishment of HPLC Fingerprint spectrum of Pine Bark Extract

[0053] The HPLC fingerprint spectrum of pine bark extract was establish by the same method in 3.1 (a) to (c).

[0054] 3.3 Establishment of HPLC Fingerprint Spectrum of Peanut Skin Extract

[0055] The HPLC fingerprint spectrum of peanut skin extract was established by the same method in 3.1 (a) to (c).

[0056] 4. Determination of the Characteristic Peaks

[0057] 4.1 Determination of a Characteristic Peak PB1 of Pine Bark Extract

[0058] It was determined that a peak appearing at 11.7 min1 min was peculiar to pine bark extract by comparing the fingerprint spectrum of the grape seed extract with the fingerprint spectrum of the pine bark extract (FIG. 1B), whereas the grape seed extract showed no chromatographic peak at this retention time.

[0059] 4.2 Determination of characteristic peaks PS1 and PS2 of peanut skin extract

[0060] It was determined that the peaks at 11.5 minlmin and 11.7 minlmin were peculiar to peanut skin extract by comparing the fingerprint spectrum of the grape seed extract with the fingerprint spectrum of the peanut skin extract (FIG. 1C), whereas the grape seed extract showed no chromatographic peak at these retention times.

Example 2: Identification of Grape Seed Extract Samples Adulterated with Pine Bark Extract

[0061] The identification includes the following steps:

[0062] Step (1): the fingerprint spectrum of the grape seed extract and a fingerprint spectrum of the pine bark extract were established by the same method as in 3.1 and 3.2 of Example 1.

[0063] Step (2): the grape seed extract was adulterated with pine bark extract, and subjected to relevant detection. Homemade grape seed extract was weighed as 1.02 g, 1.10 g, 1.05 g, and 1.09 g, respectively; and 0.03 g, 0.1 g, 0.15 g, and 0.2 g of pine bark extracts were added respectively, i.e. the adulteration amount were 3%, 10%, 15%, and 20%, respectively. The adulterated grape seed extracts were subjected to liquid chromatography detection by the same method as in 3.1 of Example 1.

[0064] The results were shown in FIGS. 2 to 6. It can be seen from the HPLC chromatograms that, the chromatograms of the extracts adulterated with pine bark extract had a significant chromatographic peak at about 11.7 min1 min, while the grape seed extract without pine bark extract had no obvious chromatographic peak at this time point, and the characteristic peak increased as the proportion of the added pine bark extract increased.

Example 3: Identification of Grape Seed Extract Samples Adulterated with Peanut Skin Extract

[0065] The identification included the following steps:

[0066] Step (1): a fingerprint spectrum of the grape seed extract and a fingerprint spectrum of the peanut skin extract were established by the same methods as in 3.1 and 3.3 of Example 1.

[0067] Step (2): the grape seed extract was adulterated with peanut skin extract, and subjected to relevant detection. Homemade grape seed extract was weighed as 1.01 g, 1.0.5 g, 1.08 g, and 1.09 g, respectively; and 0.03 g, 0.1 g, 0.15 g, and 0.2 g of peanut skin extract were added respectively, i.e. the adulteration amount were 3%, 10%, 15%, and 20%, respectively. The adulterated grape seed extracts were subjected to liquid chromatography detection by the same method as in 3.1 of Example 1.

[0068] The results were shown in FIGS. 7 to 11. It can be seen from the HPLC chromatograms that, the chromatograms of the extracts adulterated with pine bark extract had significant chromatographic peaks at about 11.5 min1 min and 11.7 min1 min, while the grape seed extract without pine bark extract had no obvious chromatographic peak at 11.5 min1 min or 11.7 min1 min, and the characteristic peaks increased as the proportion of the added peanut skin extract increased.

[0069] Although the present invention has been described above in detail with general description and specific embodiments, it is obvious to a person skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the protection scope of the present invention.

INDUSTRIAL APPLICABILITY

[0070] The present invention provides a method for identifying grape seed extract authenticity using an HPLC fingerprint spectrum, and in particular, provides a method for identifying whether the grape seed extract was adulterated with pine bark extract or peanut skin extract. The method has good stability and reproducibility, high efficiency, obvious identification characteristics, provides a theoretical basis for the identification of the plant sources of grape seed extracts, and is conducive to promoting the healthy development of the plant extract industry.