A method for constructing a compound health-care wine fingerprint spectrum

Abstract

The application discloses a method for constructing a compound health-care wine fingerprint spectrum, which comprises the following steps: preparing a test sample solution, preparing a control sample solution, performing multi-index quantitative determination, determining the fingerprint spectrum, and performing fingerprint spectrum analysis and processing. The method can simultaneously detect 21 characteristic fingerprint peaks in the compound health-care wine, and 4 fingerprint peaks are identified by comparison with the control sample, which are chlorogenic acid, bitter crocins, p-coumaric acid, ferulic acid and the like; and more fingerprint spectrum characteristic peaks can be obtained in the shortest possible separation time.

Description

Technical Field

[0001] This invention relates to the field of analytical testing technology, specifically to a method for constructing a fingerprint spectrum of a compound health wine. Background Technology

[0002] Health wine refers to a beverage with health benefits made by blending, mixing, and refining edible or medicinal ingredients or food additives with distilled spirits, fermented wines, or edible alcohol as the base. It belongs to the category of liqueurs.

[0003] Traditional Chinese medicine (TCM) herbs constitute a complex system, and health-enhancing wines contain both the chemical substances found in baijiu (Chinese liquor) and those found in TCM herbs, making the system even more complex. Therefore, a comprehensive method for characterizing the quality of health-enhancing wines, providing rich information on compounds, is essential. Currently, quality monitoring of health-enhancing wines mainly focuses on indicators such as total flavonoids, total saponins, and crude polysaccharides. These indicators primarily represent the total amount of a class of substances and are insufficient to interpret the intrinsic quality of health-enhancing wines. Furthermore, the detection of these indicators mainly relies on ultraviolet spectrophotometry, which, while simple to operate, is susceptible to interference. TCM fingerprinting is a comprehensive and quantifiable identification method. Applying TCM fingerprinting methods and content detection to health-enhancing wines can comprehensively interpret the compound information of health-enhancing wines and is an effective method for evaluating the authenticity, stability, and quality of health-enhancing wines. Summary of the Invention

[0004] The purpose of this invention is to provide a method for constructing a fingerprint spectrum of a compound health wine. The constructed fingerprint spectrum can comprehensively reflect the characteristics of the health wine, with good separation of each characteristic peak, providing a scientific method for the quality control of the health wine. The health wine involved in this invention is mainly prepared by extracting and using high-quality white wine as the base, with ginseng, polygonatum, eucommia male flowers, saffron, poria cocos, lily, black wolfberry, cordyceps militaris, etc., as the main medicinal materials.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A method for constructing a fingerprint spectrum of a compound health wine includes the following steps:

[0007] 1) Preparation of reference solution: Take chlorogenic acid, crocin, p-coumaric acid and ferulic acid reference standards, dissolve them in 50% methanol to obtain the reference solution;

[0008] 2) Preparation of the test solution: Measure 5-15 mL of the sample to be tested, evaporate it to near dryness in a water bath at 40-60℃, load the sample onto a Copure HLB solid phase extraction column, wash with 30-55 mL of water, elute with 5-10 mL of ethyl acetate, collect the eluent, take the ethyl acetate layer, evaporate it to dryness at 40-60℃, dissolve the residue in 1 mL of 50% methanol, shake well, filter through a 0.22 μm syringe filter to obtain the test solution;

[0009] 3) Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the stationary phase; detection wavelength: 270 nm; mobile phase: acetonitrile as mobile phase A and 0.1% formic acid aqueous solution as mobile phase B; gradient elution program: 0–12 min, 5%–11% A; 12–16 min, 11%–18% A; 16–19 min, 18%–18% A; 19–28 min, 18%–38% A; 28–33 min, 38%–100% A; 33–37 min, 100%–100% A;

[0010] 4) Inject the reference solution and the test solution into the ultra-high performance liquid chromatograph, measure and record the chromatograms.

[0011] Preferably, the reference solution in step 1) contains 5-15 μg of chlorogenic acid, 60-150 μg of crocin, 40-100 μg of p-coumaric acid, and 3-10 μg of ferulic acid per 1 ml.

[0012] Preferably, the sample volume to be tested in step 2) is 10 ml.

[0013] Preferably, the water bath temperature in step 2) is 50°C.

[0014] Preferably, the amount of water used for washing in step 2) is 20 ml.

[0015] Preferably, the ethyl acetate elution volume in step 2) is 7 ml.

[0016] Preferably, in step 3), the chromatographic conditions are as follows: the chromatographic column is an Agilent ZORBAX Eclipse XDB-C18, 2.1×150mm, 1.8um, the column temperature is 25~35℃, the injection volume is 1~5μL, and the flow rate is 0.1~0.4mL / min.

[0017] Preferably, in step 3), the chromatographic conditions are: column temperature 30°C, injection volume 2 μL, and flow rate 0.3 mL / min.

[0018] Compared with the prior art, the present invention has the following effects:

[0019] This invention provides a method for detecting the fingerprint spectrum of compound health wine using ultra-high performance liquid chromatography (UHPLC). The method employs a solid-phase extraction column to remove impurities (such as sugars) from the sample, ensuring a stable fingerprint baseline. Ethyl acetate is used to extract the chemical components of the compound health wine, yielding more compound information. The purified sample is then concentrated and diluted to a suitable volume to improve the sensitivity and response value of the fingerprint peaks, increasing the number of identified characteristic peaks and providing more comprehensive compound information. The method exhibits good system applicability, repeatability, and stability, providing abundant chromatographic peak information, meeting the requirements for fingerprint spectrum construction, and offers advantages such as ease of operation and reliable results. Attached Figure Description

[0020] Figure 1 This is the chromatogram of the test solution under condition 1 in Example 1;

[0021] Figure 2 This is the chromatogram of the test solution under condition 2 in Example 1;

[0022] Figure 3 This is the chromatogram of the test solution under condition 3 in Example 1;

[0023] Figure 4 This is the chromatogram of the test solution under condition 4 in Example 1;

[0024] Figure 5 This is the chromatogram of the test solution under condition 5 in Example 1;

[0025] Figure 6 This is the chromatogram of the test solution under condition 6 in Example 2;

[0026] Figure 7 This is the chromatogram of the test solution under condition 7 in Example 2;

[0027] Figure 8 This is the chromatogram of the test solution under condition 8 in Example 2;

[0028] Figure 9 UPLC fingerprint overlay images of 10 batches of compound health wine;

[0029] Figure 10 The common pattern of UPLC fingerprint spectrum of 10 batches of compound health wine;

[0030] Figure 11 UPLC chromatograms of four reference standards and compound health wine. Detailed Implementation

[0031] The embodiments of this application will be described in further detail below with reference to the accompanying drawings and examples. The terminology used in the embodiments section of this application is only used to explain the specific embodiments of this application and is not intended to limit this application.

[0032] Example 1

[0033] This embodiment provides a method for detecting the fingerprint spectrum of compound health wine, as detailed below:

[0034] 1. Preparation of the test solution—Investigation of the eluent

[0035] 1.1 Preparation: Accurately measure 10 mL of the sample to be tested, evaporate it to near dryness in a 50℃ water bath, and load the sample onto a Copure HLB solid-phase extraction column (specification: 200 mg / 6 mL, activated with 6 mL methanol and 12 mL water before loading). Wash with 40 mL of water, then elute with 7 mL of organic solvent. Collect the eluent, take the organic solvent layer, evaporate it to dryness at 50℃, dissolve the residue in 1 mL of 50% methanol, shake well, and filter through a 0.22 μm syringe filter to obtain the test solution.

[0036] Condition 1: 1.1 The organic solvent used in the preparation is ethyl acetate, see [link to preparation details]. Figure 1 ;

[0037] Condition 2: 1.1 The organic solvent used in the preparation is benzene, see Figure 2 ;

[0038] Condition 3: 1.1 The organic solvent used in the preparation is dichloromethane, see [link to preparation details]. Figure 3 ;

[0039] Condition 4: 1.1 The organic solvent used in the preparation is chloroform, see [link to preparation details]. Figure 4 ;

[0040] Condition 5: 1.1 The organic solvent used in the preparation is diethyl ether, see [link to preparation details]. Figure 5 ;

[0041] according to Figures 1-5 The experimental results show that ethyl acetate is the best overall extraction agent because it provides more chromatographic peak information, fewer impurity peaks, and a higher signal intensity. Therefore, ethyl acetate is the preferred extraction agent.

[0042] Example 2

[0043] 1. Preparation of the test solution—Investigation of the solid-phase extraction column

[0044] 1.1 Preparation: Accurately measure 10 mL of the sample to be tested, evaporate it to near dryness in a 50℃ water bath, load the sample onto a solid-phase extraction column, wash with 40 mL of water, then elute with 7 mL of organic solvent, collect the eluent, take the organic solvent layer, evaporate it to dryness at 50℃, dissolve the residue in 1 mL of 50% methanol, shake well, filter through a 0.22 μm syringe filter to obtain the test solution.

[0045] Condition 6: 1.1 The solid-phase extraction column used in the preparation was Copure HLB, 200 mg / 6 mL, see [link to preparation]. Figure 6 ;

[0046] Condition 7: 1.1 The solid-phase extraction column used in the preparation was RayCure HLB, 200 mg / 6 mL, see [link to preparation]. Figure 7 ;

[0047] Condition 8: 1.1 The solid-phase extraction column used in the preparation was Waters HLB, 200 mg / 6 mL, see [link to preparation]. Figure 8 ;

[0048] according to Figures 6-8 The experimental results show that when using the Copure HLB, 200mg / 6mL solid-phase extraction column, there are more chromatographic peaks, fewer impurity peaks, and a higher signal intensity. Therefore, the Copure HLB, 200mg / 6mL solid-phase extraction column is the preferred choice.

[0049] Example 3

[0050] Methodological investigation

[0051] 1. Materials and Methods

[0052] 1.1 Instruments: Agilent 1290 Infinity II ultra-high performance liquid chromatograph (Agilent Technologies, USA); XPR2 1 / 1,000,000 electronic balance (Mettler Tridonic, Switzerland); Agilent ZORBAX Eclipse XDB-C18 (1.8µm, 2.1×150mm) column; RE-2002 rotary evaporator (Zhengzhou Keda Machinery Instrument Equipment Co., Ltd.); PURELAB ultrapure water system (ELGA, UK);

[0053] 1.2 The reagents for 10 batches of compound health wine were provided by Jinpai Co., Ltd.; the reference standards for chlorogenic acid (batch number 110753-201716, mass fraction 99.3%), crocin (batch number 112056-202102, mass fraction 97.3%), p-coumaric acid (batch number 112037-202102, mass fraction 99.3%), and ferulic acid (batch number 110773-201313, mass fraction 99.6%) were all from the China National Institutes for Food and Drug Control.

[0054] Formic acid and acetonitrile were chromatographic grade, water was ultrapure water, and other reagents were analytical grade.

[0055] 2. Methods and Results

[0056] 2.1 Chromatographic conditions: An Agilent ZORBAX Eclipse XDB-C18 column (2.1 × 150 mm, 1.8 μm) was used; acetonitrile was used as mobile phase A, and 0.1% formic acid aqueous solution was used as mobile phase B, with gradient elution: 0–12 min, 5%–11% A; 12–16 min, 11%–18% A; 16–19 min, 18%–18% A; 19–28 min, 18%–38% A; 28–33 min, 38%–100% A; 33–37 min, 100%–100% A; column temperature 30℃; injection volume 2 μL; flow rate 0.3 mL / min; detection wavelength 270 nm.

[0057] 2.2 Preparation of reference solutions: Take appropriate amounts of chlorogenic acid, crocin, p-coumaric acid, and ferulic acid, add 50% methanol, and prepare chlorogenic acid reference solutions with a concentration of 10 μg / ml, crocin with a concentration of 120 μg / ml, p-coumaric acid with a concentration of 70 μg / ml, and ferulic acid with a concentration of 5 μg / ml.

[0058] 2.3 Preparation of the test solution: Accurately measure 10 mL of the sample to be tested, evaporate it to near dryness in a water bath at 50 °C, and load the sample onto a Copure HLB solid-phase extraction column (specification: 200 mg / 6 mL, activated with 6 mL methanol and 12 mL water before loading). Wash with 40 mL of water, then elute with 7 mL of ethyl acetate. Collect the eluent, take the ethyl acetate layer, evaporate it to dryness at 50 °C, dissolve the residue in 1 mL of 50% methanol, shake well, and filter through a 0.22 μm syringe filter to obtain the test solution.

[0059] Under optimized conditions, chlorogenic acid, crocin, p-coumaric acid, and ferulic acid reference solutions were injected sequentially. The results showed that crocin had a large peak area and a moderate retention time; therefore, the crocin peak was selected as the reference peak.

[0060] 2.4 System suitability test: Under the above chromatographic conditions, the reference solution was injected repeatedly 6 times, and the relative retention time (RRT) and relative peak area (RPA) were recorded. The RSD (%) was calculated. The results are shown in Table 1.

[0061] Table 1 System adaptability test results

[0062]

[0063] The results showed that the repeatability RSD of the reference solution was less than 2%, indicating good system adaptability.

[0064] 2.5 Precision Test: The same test solution (S1) was injected six times under the chromatographic conditions described in section "2.1". Using crocin as the reference peak, the relative retention time and relative peak area (RSD) of the 21 common peaks were calculated to be less than 2%, indicating good instrument precision.

[0065] 2.6 Stability Test: The same test solution (S1) was injected and analyzed at 0h, 3h, 6h, 9h, 12h, and 15h under the chromatographic conditions described in section "2.1". Using crocin as a reference peak, the relative retention times and relative peak areas (RSDs) of the 21 common peaks were all less than 2%. This indicates that the test solution exhibits good stability within 15h.

[0066] 2.7 Repeatability Test: Six portions of the compound health wine (S1) were prepared as test solutions according to the method described in section "2.3", and the chromatographic conditions described in section "2.1" were used for determination. Using crocin as a reference peak, the relative retention times and relative peak areas (RSDs) of the 21 common peaks were all less than 2%, indicating good repeatability of the method.

[0067] 2.8 Establishment and Similarity Evaluation of UPLC Fingerprints Ten batches of compound health wine samples (S1–S10) were prepared into test solutions according to the method described in section “2.3”, and injected sequentially for determination under the chromatographic conditions described in section “2.1”. Fingerprint analysis was performed using the “Similarity Evaluation Software for Chromatographic Fingerprints of Traditional Chinese Medicines” (2012 version). Using S1 as the reference fingerprint, Mark peak matching was performed after multi-point correction, and the fingerprint was generated using the mean method. The results are shown in [Figure number missing]. Figure 9 And generate a comparative fingerprint spectrum, see Figure 10 The chromatograms of 10 batches of compound health wine were compared with the control fingerprint chromatograms for similarity evaluation. The similarity of S1 to S10 was greater than 0.98. The results are shown in Table 2.

[0068] Table 2. Similarity evaluation of fingerprint spectra of compound health wine samples.

[0069]

[0070] 2.9 Identification and Assignment of Common Peaks in UPLC Fingerprint Spectra The compound health wine (S1) was used. The test solution was prepared according to the method described in section "2.3", and the reference solutions were prepared according to the method described in section "2.2". The samples were injected and analyzed under the chromatographic conditions described in section "2.1". By comparing retention time and UV spectra, 21 common peaks were identified, and 4 chromatographic peaks were assigned. Peak 6 was chlorogenic acid, peak 9 was crocin, peak 10 was p-coumaric acid, and peak 12 was ferulic acid. The UPLC chromatograms of the reference standards and the compound health wine sample are shown below. Figure 11 .

[0071] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application and are not intended to limit it. Although this application has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of this application without departing from the spirit and scope of the technical solutions of this application, and all such modifications or substitutions should be covered within the scope of the claims of this application.

Claims

1. A method for constructing a compound health care wine fingerprint, characterized in that, The method comprises the following steps: 1) Preparation of the control solution: take chlorogenic acid, picric acid, p-coumaric acid, ferulic acid control, and dissolve in 50% methanol to obtain the control solution; 2) Preparation of the test solution: take 5-15 mL of the sample to be tested, and rotate evaporate in a 40-60°C water bath to near dryness, and then load on a Copure HLB solid phase extraction column, wash with 30-55 mL of water, elute with 5-10 mL of ethyl acetate, collect the eluate, take the ethyl acetate layer, and rotate evaporate at 40-60°C to dryness, dissolve the residue in 1 mL of 50% methanol, shake well, and filter with a 0.22 μm needle filter to obtain the test solution; 3) Chromatographic conditions: octadecylsilane-bonded silica gel as the filler; detection wavelength: 270 nm; mobile phase: acetonitrile as the mobile phase A and 0.1% formic acid aqueous solution as the mobile phase B; gradient elution program 0-12 min, 5%-11% A; 12-16 min, 11%-18% A; 16-19 min, 18%-18% A; 19-28 min, 18%-38% A; 28-33 min, 38%-100% A; 33-37 min, 100%-100% A; 4) Take the control solution and the test solution respectively and inject into the ultra-high performance liquid chromatograph for determination, and record the chromatogram; The compound health-care wine is a health-care wine prepared by extracting and preparing medicinal materials of ginseng, rhizoma polygonati, eucommia ulmoides male flower, saffron, poria cocos, lily, lycium ruthenicum murr, cordyceps militaris and high-quality baijiu as the wine base.

2. The construction method of claim 1, wherein: The control solution in step 1) contains 5-15 μg of chlorogenic acid, 60-150 μg of picric acid, 40-100 μg of p-coumaric acid and 3-10 μg of ferulic acid per 1 ml.

3. The method of construction of claim 1, wherein: The sample to be tested in step 2) is 10 ml.

4. The method of construction of claim 1, wherein: The water bath temperature in step 2) is 50°C.

5. The method of construction of claim 1, wherein: The water washing amount in step 2) is 20 ml.

6. The method of construction of claim 1, wherein: The ethyl acetate elution amount in step 2) is 7 ml.

7. The method of construction of claim 1, wherein: In step 3), the chromatographic column is Agilent ZORBAX Eclipse XDB-C18, 2.1*150mm, 1.8um, the column temperature is 25-35°C, the injection amount is 1-5 μL, and the flow rate is 0.1-0.4 mL / min.

8. The method of construction of claim 1, wherein: In step 3), the column temperature is 30°C, the injection amount is 2 μL, and the flow rate is 0.3 mL / min.

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