Method for improving detection stability of health-care wine

Abstract

The application discloses a method for improving the detection stability of health-care wine, which comprises the following steps: preparation of a sample solution, preparation of a control solution, multi-index quantitative determination, and different storage modes of the sample, etc. The method improves the quality stability of the health-care wine by innovating the storage mode of the health-care wine, thereby improving the detection stability. The method has high detection efficiency, can simultaneously detect eight effective components in the health-care wine, and provides technical support for quality guarantee of the health-care wine.

Description

Technical Field

[0001] This invention relates to the field of analytical testing technology, and specifically to a method for improving the testing stability of health wines. 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] Health tonics are a complex system, a product of the organic combination of baijiu (Chinese white liquor) and medicinal herbs that are both food and medicine. The active ingredients in the medicinal herbs dissolve into the baijiu, giving it certain specific effects. However, these active ingredients have their own chemical properties, such as sensitivity to light and heat and easy decomposition. Therefore, ensuring the quality and stability of health tonics is of great practical significance.

[0004] CN110455961A discloses a high-throughput detection method for multiple components in health wine, and CN111443141A discloses a method for detecting the content of effective components in health wine. The above invention patents only detect the effective components in health wine and do not involve the stability of the effective components, which is insufficient to fully characterize the quality and characteristics of health wine. Summary of the Invention

[0005] This invention provides a method to improve the detection stability of health wines, which are mainly made from ginseng, polygonatum, eucommia male flowers, saffron, poria cocos, lily, black goji berries, and / or cordyceps militaris as the main medicinal materials and extracted using high-quality baijiu as the base. This method can simultaneously detect eight active ingredients in the health wine, with good peak separation for each component, providing a scientific approach for the quality control of health wines.

[0006] Therefore, the technical solution of the present invention is as follows:

[0007] A method for improving the stability of health wine testing includes the following steps:

[0008] 1) Preparation of reference solution: 5-hydroxymethylfurfural, adenosine, cordycepin, N6-(2-hydroxyethyl)adenosine, crocin, rutin, crocin I, and crocin II reference standards are dissolved in 50% methanol to obtain the reference solution.

[0009] 2) Preparation of test solution: Measure 2-7 mL of the sample to be tested into a 10 mL volumetric flask, add water to make up to the mark, shake well, and filter with a 0.22 μm needle filter to obtain the test solution;

[0010] 3) Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the stationary phase; detection wavelength: 260 nm for 0-68 min, 440 nm for 68-80 min; mobile phase: acetonitrile as mobile phase A, 0.1% aqueous acetic acid as mobile phase B; gradient elution program: 0-10 min, 2%-2% A; 10-15 min, 2%-4% A; 15-25 min, 4%-7% A; 25-35 min, 7%-10% A; 35-40 min, 10%-10% A; 40-60 min, 10%-18% A; 60-80 min, 18%-35% A.

[0011] 4) Inject the reference solution and the test solution into the high-performance liquid chromatograph separately, determine and record the content values;

[0012] 5) The health wine was stored in different containers in different environments for 90 days, and the eight active ingredients in the health wine were tested.

[0013] Preferably, each 1 ml of the reference solution in step 1) contains 20-35 μg of 5-hydroxymethylfurfural, 20-50 μg of adenosine, 40-80 μg of cordycepin, 40-80 μg of N6-(2-hydroxyethyl)adenosine, 40-80 μg of crocin, 5-20 μg of rutin, 10-40 μg of crocin I, and 2-20 μg of crocin II.

[0014] Preferably, the sample volume to be tested in step 2) is 5 ml.

[0015] Preferably, in step 3), the chromatographic conditions are as follows: the chromatographic column is an Agilent SB-Aq-C18, 4.6×250mm, 5µm, the column temperature is 25–35℃, the injection volume is 5–15µL, and the flow rate is 0.5–1.5mL / min.

[0016] Preferably, in step 3), the chromatographic conditions are: column temperature 30°C, injection volume 10 μL, and flow rate 1.0 mL / min.

[0017] Preferably, the storage container is one or more of transparent glass bottles, ceramic bottles, and stainless steel bottles, and the storage environment is one of room temperature, high temperature, low temperature, and light exposure.

[0018] Preferably, the high temperature is 50°C and the low temperature is 2-4°C.

[0019] Compared with the prior art, the present invention has the following effects:

[0020] This invention provides a method for improving the stability of health wine testing. It is the first to study the effective components in such health wines and can quantitatively detect 5-hydroxymethylfurfural, adenosine, cordycepin, N6-(2-hydroxyethyl)adenosine, crocin, rutin, crocin I, and crocin II, achieving more comprehensive quality control of health wines through a more comprehensive analysis of their chemical composition. Simultaneously, this invention, through stability research, clarifies the optimal storage method for health wines, further improving the stability of the testing method. Therefore, this invention provides data support for the stability of health wine quality from two dimensions and has practical application value. Attached Figure Description

[0021] Figure 1 The chromatogram of the sample in Example 1;

[0022] Figure 2 This is a chromatogram of the mixed standard from Example 1. Detailed Implementation

[0023] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention.

[0024] Example 1

[0025] This embodiment provides a method for detecting the content of multiple components in health wine, and the methodological investigation method is as follows:

[0026] 1. Materials and Methods

[0027] Instruments: UltiMate 3000 series high performance liquid chromatograph (Thermo Fisher Scientific, USA); SQP electronic balance (Sartorius GmbH, Germany); Agilent SB-Aq C18-C18 (5µm, 4.6×250mm) column; PURELAB ultrapure water system (ELGA, UK);

[0028] The 10 batches of health wine were prepared using ginseng, polygonatum, eucommia male flowers, saffron, poria cocos, lily, black goji berries, cordyceps militaris, and other medicinal materials as the main ingredients. They were made through scientific ingredient ratios and extracted with high-quality white wine as the base. The ingredients were provided by Jingpai Co., Ltd.

[0029] 5-Hydroxymethylfurfural (batch number 111626-201711, mass fraction 97.3%), crocin (batch number 112056-202102, mass fraction 97.3%), adenosine (batch number 110879-201703, mass fraction 99.7%), cordycepin (batch number 110858-201704, mass fraction 99.4%), rutin (batch number 100080-201... The reference standards 610 (91.1% by mass), crocin I (batch number 111588-201704, 88.4% by mass), and crocin II (batch number 111589-201705, 92.2% by mass) were all from the National Institutes for Food and Drug Control; N6-(2-hydroxyethyl) adenosine (batch number C10092454, 98% by mass) was from Merck & Co., Ltd.

[0030] Acetic acid and acetonitrile were chromatographic grade, water was ultrapure water, and other reagents were analytical grade.

[0031] 2. Methods and Results

[0032] 2.1 Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the stationary phase; Agilent ZORBAX SB-Aq C18, 4.6*250mm, 5μm, column temperature 30℃, injection volume 10μL, flow rate 1.0mL / min, detection wavelength: 0-70min 260nm, 70-80min 440nm. Mobile phase: Acetonitrile as mobile phase A, 0.1% acetic acid aqueous solution as mobile phase B; gradient elution program: 0-10min, 2%-2% A; 10-15min, 2%-4% A; 15-25min, 4%-7% A; 25-35min, 7%-10% A; 35-40min, 10%-10% A; 40-60min, 10%-18% A; 60-80min, 18%-35% A.

[0033] 2.2 Preparation of mixed reference solution: Accurately weigh appropriate amounts of 5-hydroxymethylfurfural, adenosine, cordycepin, N6-(2-hydroxyethyl)adenosine, crocin, rutin, crocin I, and crocin II reference standards, dissolve them in 50% methanol to prepare a mixed reference solution containing 27ug of 5-hydroxymethylfurfural, 35ug of adenosine, 51ug of cordycepin, 51ug of N6-(2-hydroxyethyl)adenosine, 54ug of crocin, 15ug of rutin, 34ug of crocin I, and 17ug of crocin II per ml.

[0034] 2.3 Preparation of test solution: Take 5 ml of the sample to be tested, put it into a 10 ml volumetric flask, add water to make up to the mark, shake well, and the test solution is obtained.

[0035] 2.4 System Suitability Study: Take the health wine (S1), process it according to the test solution preparation method determined in section "2.3", prepare the mixed reference standard stock solution according to the method in section "2.2", and inject and determine it according to the chromatographic conditions in section "2.1". The chromatogram is shown in [reference needed]. Figures 1-2 ,in, Figure 1 This is the chromatogram of a health wine sample. Figure 2 This is a chromatogram of a mixed reference standard.

[0036] 2.5 Linearity and Detection Limit: The mixed reference standard stock solution from section "2.2" was gradually diluted to different concentrations and injected for determination according to the chromatographic conditions from section "2.1". The peak areas were recorded. A linear regression was performed with the concentration of each reference standard solution as the x-axis and the corresponding peak area as the y-axis to obtain the regression equation. See Table 1.

[0037] Table 1. Standard curve regression equations, linear ranges, limits of detection, and limits of quantitation for the eight active ingredients.

[0038]

[0039]

[0040] 2.6 Precision Test: The health wine (S1) was prepared according to the sample preparation method in section "2.3". Under the chromatographic conditions in section "2.1", the sample was continuously injected and analyzed six times. The peak areas of 5-hydroxymethylfurfural, adenosine, cordycepin, N6-(2-hydroxyethyl)adenosine, crocin, rutin, crocin I, and crocin II were obtained for each analysis. The RSD values ​​of the peak areas were calculated. The peak areas and RSD values ​​of each component in the precision test are shown in Table 2. The results show that the RSD values ​​of each component are all <3%, indicating good instrument precision.

[0041] Table 2 Precision Test Results

[0042]

[0043] 2.7 Stability Test: The same health wine sample (S1) was analyzed six times at 0h, 3h, 7.5h, 16.5h, 25.5h, and 40.5h under the above conditions. The peak areas of 5-hydroxymethylfurfural, adenosine, cordycepin, N6-(2-hydroxyethyl)adenosine, crocin, rutin, crocin I, and crocin II were obtained for each analysis. The RSD values ​​of the peak areas were calculated, and the results are shown in Table 3. The results show that the RSD values ​​of all components were <2%, indicating that the method has good stability.

[0044] Table 3 Stability test results

[0045]

[0046]

[0047] 2.8 Repeatability Test: Six samples of the same compound health wine (S1) were prepared according to the test sample preparation method in section "2.3". These samples were then injected and analyzed under the chromatographic conditions in section "2.1". The contents of 5-hydroxymethylfurfural, adenosine, cordycepin, N6-(2-hydroxyethyl)adenosine, crocin, rutin, crocin I, and crocin II were obtained for each analysis, and the RSD values ​​were calculated. The contents and RSD values ​​of each component in the repeatability test are shown in Table 4. The results show that the RSD values ​​of each component are all <2%, indicating good repeatability.

[0048] Table 4 Results of Repeatability Tests

[0049]

[0050] 2.9 Spike Recovery Test: Take 2.5 ml of the same batch of health wine (S1), divide it into 3 groups, with 3 replicates per group. Add the mixed reference standard to the 3 groups at mass ratios of 1:0.5, 1:1, and 1:1.5 respectively. Prepare 9 test solutions according to the method in section "2.3". Determine the chromatographic conditions according to section "2.1", record the peak area, and calculate the recovery rate of each component. The results showed that the recoveries of 5-hydroxymethylfurfural, adenosine, cordycepin, N6-(2-hydroxyethyl)adenosine, crocin, rutin, crocin I, and crocin II were 102.2%, 101.4%, 94.7%, 100%, 96.2%, 100.2%, 98.7%, and 97.9%, respectively, with RSD values ​​of 2.05%, 1.51%, 1.15%, 1.59%, 2.18%, 2.01%, 2.74%, and 3.04%, respectively. This indicates good recovery rates.

[0051] Example 2

[0052] The health wine samples were dispensed into brown glass bottles and stored at room temperature for 90 days. The contents of 5-hydroxymethylfurfural, adenosine, cordycepin, N6-(2-hydroxyethyl)adenosine, crocin, rutin, crocin I, and crocin II in the samples were then determined using the method described in Example 1. The results are as follows:

[0053] Table 5. Detection results (mg / L) of 8 active ingredients

[0054]

[0055] Example 3

[0056] The health wine sample from Example 2 was packaged in transparent glass bottles and stored at room temperature for 90 days. The contents of 5-hydroxymethylfurfural, adenosine, cordycepin, N6-(2-hydroxyethyl)adenosine, crocin, rutin, crocin I, and crocin II in the sample were then tested using the method described in Example 1.

[0057] Table 6. Detection results (mg / L) of 8 active ingredients

[0058]

[0059]

[0060] Example 4

[0061] The health wine sample from Example 2 was packaged in brown glass bottles and stored at low temperature for 90 days. The contents of 5-hydroxymethylfurfural, adenosine, cordycepin, N6-(2-hydroxyethyl)adenosine, crocin, rutin, crocin I, and crocin II in the sample were then tested using the method described in Example 1.

[0062] Table 7. Detection results (mg / L) of 8 active ingredients

[0063]

[0064] Example 5

[0065] The health wine sample from Example 2 was packaged in transparent glass bottles and stored under light. The total illuminance was set to 1.2 × 10⁶ Lux·hr. The contents of 5-hydroxymethylfurfural, adenosine, cordycepin, N6-(2-hydroxyethyl)adenosine, crocin, rutin, crocin I, and crocin II in the sample were then detected using the method described in Example 1.

[0066] Table 8. Detection results (mg / L) of eight active ingredients.

[0067]

[0068]

[0069] Example 6

[0070] The health wine sample from Example 2 was packaged in brown glass bottles and stored at high temperature for 90 days. The contents of 5-hydroxymethylfurfural, adenosine, cordycepin, N6-(2-hydroxyethyl)adenosine, crocin, rutin, crocin I, and crocin II in the sample were then tested using the method described in Example 1.

[0071] Table 9. Detection results (mg / L) of 8 active ingredients

[0072]

[0073] Examples 2-6 show that rutin, crocin I, and crocin II in the health tonic are sensitive to temperature and high light intensity, with their contents decreasing as temperature and light intensity increase, while the content of 5-hydroxymethylfurfural increases with increasing temperature. In low-temperature environments, the eight active ingredients show no significant changes, but precipitation occurs, which is detrimental to product quality. To ensure the stability of the health tonic and improve the stability of the detection method, the samples were stored in brown glass bottles at room temperature.

[0074] This invention is not limited to the above embodiments. Based on the technical solutions disclosed in this invention, those skilled in the art can make some substitutions and modifications to some of the technical features without creative effort, and all such substitutions and modifications are within the protection scope of this invention.

Claims

1. A method for detecting health-enhancing wine, characterized in that, Includes the following steps: 1) Preparation of reference solution: 5-hydroxymethylfurfural, adenosine, cordycepin, N6-(2-hydroxyethyl)adenosine, crocin, rutin, crocin I, and crocin II reference standards are dissolved in 50% methanol to obtain the reference solution. 2) Preparation of test solution: Measure 2-7 mL of the sample to be tested into a 10 mL volumetric flask, add water to make up to the mark, shake well, and filter with a 0.22 μm syringe filter to obtain the test solution; 3) Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the stationary phase; detection wavelength: 260 nm for 0-68 min, 440 nm for 68-80 min; mobile phase: acetonitrile as mobile phase A, 0.1% aqueous acetic acid as mobile phase B; gradient elution program: 0-10 min, 2%-2% A; 10-15 min, 2%-4% A; 15-25 min, 4%-7% A; 25-35 min, 7%-10% A; 35-40 min, 10%-10% A; 40-60 min, 10%-18% A; 60-80 min, 18%-35% A. 4) Inject the reference solution and the test solution into the high-performance liquid chromatograph separately, determine and record the content values; The reference solution described in step 1) contains 20-35 μg of 5-hydroxymethylfurfural, 20-50 μg of adenosine, 40-80 μg of cordycepin, 40-80 μg of N6-(2-hydroxyethyl)adenosine, 40-80 μg of crocin, 5-20 μg of rutin, 10-40 μg of crocin I, and 2-20 μg of crocin II per 1 ml. In step 2), the sample volume to be tested is 5 ml. In step 3), the chromatographic conditions are as follows: the column is an Agilent SB-Aq-C18, 4.6×250mm, 5µm, the column temperature is 25–35℃, the injection volume is 5–15µL, and the flow rate is 0.5–1.5mL / min. The health wine mentioned is made from ginseng, polygonatum, eucommia male flowers, saffron, poria cocos, lily, black goji berries, and cordyceps militaris as medicinal materials, and high-quality white wine as the base wine.

2. The method according to claim 1, characterized in that, In step 3), the chromatographic conditions are: column temperature 30℃, injection volume 10μL, and flow rate 1.0mL / min.

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