阅读说明：本技术 一种高效液相色谱-荧光检测法测定葡萄酒中聚天冬氨酸钾含量的方法 (Method for determining content of potassium polyaspartate in wine by high performance liquid chromatography-fluorescence detection method ) 是由 马义虔 李红洲 杨燕红 张建 梁桂娟 陈兴林 龙四红 谈晓君 彭小东 陈大鹏 陈 于 2020-09-23 设计创作，主要内容包括：本发明属于葡萄酒中聚天冬氨酸钾分析技术领域,公开了一种高效液相色谱-荧光检测法测定葡萄酒中聚天冬氨酸钾含量的方法,包括以下步骤：1.葡萄酒水解：依次加入偏重亚硫酸钠溶液、葡萄酒和盐酸溶液,盖紧瓶盖,在电热板上加热后转移至容量瓶中,加入氢氧化钠溶液,用超纯水定容至刻度。2.内标溶液的加入：分别移取标准工作液、未水解的葡萄酒和步骤1中水解后的葡萄酒于容量瓶中,加入内标溶液,用超纯水定容至刻度。3.衍生化反应：移取步骤2中3种溶液,加入衍生化溶液,混匀,过滤。4.高效液相色谱-荧光检测器分析检测。本发明可避免高浓度的衍生化试剂对目标物的干扰,为葡萄酒中聚天冬氨酸钾含量的检测提供了一个稳定、准确的方法。(The invention belongs to the technical field of analysis of potassium polyaspartate in wine, and discloses a method for determining the content of potassium polyaspartate in wine by a high performance liquid chromatography-fluorescence detection method, which comprises the following steps: 1. and (3) wine hydrolysis: sequentially adding sodium metabisulfite solution, wine and hydrochloric acid solution, covering the bottle cap tightly, heating on an electric hot plate, transferring to a volumetric flask, adding sodium hydroxide solution, and metering to the desired volume with ultrapure water. 2. Addition of internal standard solution: and (3) respectively transferring the standard working solution, the unhydrolyzed wine and the hydrolyzed wine in the step (1) into volumetric flasks, adding an internal standard solution, and metering the volume to the scale by using ultrapure water. 3. And (3) derivatization reaction: and (3) transferring the solution obtained in the step (2), adding a derivatization solution, uniformly mixing, and filtering. 4. And (4) analyzing and detecting by using a high performance liquid chromatography-fluorescence detector. The invention can avoid the interference of high-concentration derivatization reagent to the target object, and provides a stable and accurate method for detecting the content of the potassium polyaspartate in the wine.)

1. A method for determining the content of potassium polyaspartate in wine by high performance liquid chromatography-fluorescence detection method comprises the following steps;

step 1, hydrolyzing a wine sample: sequentially transferring a sodium metabisulfite solution, a wine sample and a hydrochloric acid solution into a 4mL brown small bottle, covering the bottle cap tightly, heating on an electric heating plate, transferring the solution into a 10mL A-grade volumetric flask, adding a sodium hydroxide solution, and metering the volume to the scale by using ultrapure water;

step 2, adding an internal standard solution: respectively transferring the aspartic acid standard working solution, the unhydrolyzed wine sample and the hydrolyzed wine solution in the step 1 into a 25mL A-grade volumetric flask, adding an internal standard solution, and then fixing the volume to the scale by using ultrapure water;

and 3, derivatization reaction: respectively transferring the 3 solutions in the step 2, adding a derivatization solution, uniformly mixing, filtering by using a 0.2 mu m acetate fiber filter membrane, and detecting;

and 4, analyzing and detecting by using a high performance liquid chromatography-fluorescence detector (HPLC-FLD).

2. An assay according to claim 1, wherein in step 1, said method of hydrolyzing a wine sample comprises:

(1) the sodium metabisulfite solution is prepared by the following method: weighing 5g of sodium metabisulfite in a 500mL A-level volumetric flask, and performing constant volume to scale with ultrapure water to obtain a sodium metabisulfite solution with the concentration of 10 g/L;

(2) the 6M hydrochloric acid solution was prepared by the following method: weighing 109.5g of hydrochloric acid in a 500mL A-level volumetric flask, and metering the volume to a scale with ultrapure water to obtain a hydrochloric acid solution with the concentration of 6M;

(3) the 5M sodium hydroxide solution was prepared by the following method: weighing 10g of sodium hydroxide solid in a 500mL A-level volumetric flask, and performing constant volume to a scale by using ultrapure water to obtain a sodium hydroxide solution with the concentration of 5M;

(4) the volumes of the sodium metabisulfite solution, the wine sample, the hydrochloric acid solution and the sodium hydroxide solution are respectively as follows: 0.2mL, 2mL, 2.5 mL;

(5) the temperature of the electric heating plate is 108 ℃, and the heating time is 72 h.

3. The assay method according to claim 1, wherein the addition of the internal standard solution in step 2 comprises:

(1) the aspartic acid standard working solution is prepared by the following method: preparing an aspartic acid stock solution 1: preparing 5000mg/L aspartic acid solution by using ultrapure water; preparing an aspartic acid stock solution 2: 200mg/L aspartic acid solution was prepared with ultrapure water. Preparing a standard working solution: sucking up 0.25mL, 1.25mL and 6.25mL of stock solutions 2 respectively, and diluting to 25mL with ultrapure water to obtain use solutions STD1, STD2 and STD3 with concentrations of 2, 10 and 50mg/L respectively; 0.5mL, 1.25mL and 2.5mL of stock solutions 1 were aspirated, and the volume was adjusted to 25mL with ultrapure water, to obtain use solutions STD4, STD5 and STD6 at concentrations of 100, 250 and 500mg/L, respectively.

(2) The internal standard solution (aminocaproic acid) was prepared by the following method: weighing 1g of aminocaproic acid in a 1000mL A-level volumetric flask, and using ultrapure water to perform constant volume to scale, thereby obtaining an aminocaproic acid internal standard solution with the concentration of 1000 mg/L.

(3) The volumes of the standard working solution of aspartic acid, the unhydrolyzed wine sample, and the 6.25mL hydrolyzed wine solution of step 1 of claim 1, and the internal standard solution of aminocaproic acid were 1.25mL, 6.25mL, and 0.25mL, respectively.

4. The assay method according to claim 1, wherein in step 3, the derivatization reaction comprises:

(1) the sodium tetraborate decahydrate buffer solution is prepared by the following method: weighing 19.1g of sodium tetraborate decahydrate in a 500mL A-level volumetric flask, and fixing the volume to a scale (regulating the pH value of the solution by hydrochloric acid or sodium hydroxide) by using ultrapure water to obtain a sodium tetraborate decahydrate buffer solution (the pH value is 10.5) with the concentration of 0.1M;

(2) the derivatization solution is prepared by the following method: 100mg of o-phthalaldehyde (OPA), 200. mu.L of mercaptoethanol and 1mL of methanol were added to a 10mL A-grade volumetric flask, and the volume was fixed to the scale (prepared before use) with 0.1M sodium tetraborate decahydrate buffer solution (pH 10.5);

(3) the volume of the 3 solutions removed in step 2 of claim 1 is 20 mL;

(4) the volumes of the derivatization solution, the mercaptoethanol and the methanol are respectively 10mL, 200 mu L and 1 mL;

(5) the mixing time was 60 s.

5. An assay according to claim 1, wherein in step 4, the HPLC-FLD assay comprises:

(1) the solution was prepared by the following method: weighing 2.05g of anhydrous sodium acetate solid in a 500mL A-level volumetric flask, and fixing the volume to a scale by using ultrapure water to obtain an anhydrous sodium acetate buffer solution with the concentration of 0.05M;

(2) the chromatographic conditions were as follows: the chromatographic column is C18 polar column (such as Syncronis aQ 4.6 × 250mm,5 μm); column temperature: 40 ℃; FLD wavelength (λ): λ ex 340 nm; λ em-450 nm; sample introduction amount: 10 mu L of the solution; the column flow rate was 1.1 mL/min; mobile phase A: ultrapure water; mobile phase B: 0.05M sodium acetate anhydrous buffer-tetrahydrofuran (96:4, v: v); mobile phase C: methanol; mobile phase D: acetonitrile; gradient elution procedure: 0-3 min, 100% B; 3-15 min, 50% of B-25% of C-25% of D; 15-17 min, 84% of B-8% of C-8% of D; 17-18 min, 100% B.

Technical Field

The invention belongs to the technical field of analysis of potassium polyaspartate (KPA) in wine, and particularly relates to a method for determining the content of potassium polyaspartate (KPA) in wine by a high performance liquid chromatography-fluorescence method.

Background

The grape wine contains various amino acids, anthocyanin, mineral substances, vitamins and other substances with high nutritional value, and the substances are all nutritional ingredients which are needed to be supplemented and absorbed by human bodies. However, the macromolecular substances such as tartaric acid, tannin, pigment, protein and the like are the reasons for the uncleanness and instability of the wine. The stability of tartrate is the most influential one to the stability of wineThe main factor is that the tartaric acid content in the wine is 2-6 g/L (Waterhouse AL, Sacks GL, Jeffery DW. unrestrained wine chemistry [ J ]].John Wiley&Sons,2016.)，K⁺The content is 125-2040 mg/L, Ca²⁺The content of the surfactant is 50-300 mg/L (Felie P, Yaneri M-G, Andrea V, et al, the use of the location exchange resins in the wires: Effects on pH, tartrate stability, and metal center [ J ])]Victualulture and technology, 2018,45(1):82-92.) if no better treatment is obtained before bottling, crystals such as potassium tartrate and calcium tartrate precipitate after bottling will be leached out in wine. Wine bottled and stored for a certain period of time has some precipitates which are normal but cause visual defects and affect the sensory evaluation of wine by consumers (Teas V, Correia A C, Jord O, et al. wine tartrate stabilization by secondary differential levels of wine for appearance of wine, and cosmetic film and cosmetic properties of red wires [ J. As shown in FIGS. ]]Food research International 2015,69: 364-. The addition of stabilizer can make the wine not easily separate out tartrate, can reduce the treatment to the maximum extent, and maintain the quality of the wine (Large C. cellulose gum, an effective solution for a Tartrate incorporation in wine J)].Australian and New Zealand Grape Grower and Winemaker,2016,632:58-61.)。

Potassium polyaspartate (KPA) is a polyamide produced by thermal polymerization of L-aspartic acid. The eu (eu committee regulation 2017/1399) approved 2016 for the additive of wine and received positive acceptance by the European Food Safety Agency (EFSA). Can be used for red and white wine, the common dosage range is 100-200 mg/L according to the unstable degree of the wine to be treated, and the highest safe dosage is recommended to be 300mg/L (Official journal of the european unit [ J ]]Commission Regulation (EU),2017,1399: 8-11.). Potassium polyaspartate is a negatively charged polymer that inhibits crystal nucleation and growth, and is of vital importance in modifying the morphology of the forming crystals to reduce the brewing function of wine instability. (EFSA Panel on Food Additives and Nutrient Sources added to Food safety of Food polymeric (A-5D K/SD) for useas a plasmid in wire [ J-5].EFSA joural2016,14(3):4435. and Colombo, F., Di Lorenzo, C., Casalegno, C., et al, Further experimental data supporting the security of a food prepared used in wire stabilization [ C. ]].41^stWorld Congress of Vine and wine, Bio of Coniferces, France EDP Sciences,2019:1-5.) thus developed a composition that is effective and stable. The technical method for accurately determining the potassium polyaspartate in the wine is beneficial to the stability and the visual quality of the wine, and has very important practical significance for promoting the healthy development of the wine industry.

Disclosure of Invention

(1) Problems to be solved

A method for quickly, stably and accurately measuring the content of the potassium polyaspartate in the wine is established.

In short, the method for measuring the content of the potassium polyaspartate in the wine is realized based on a high performance liquid chromatography-fluorescence detector (HPLC-FLD), and the blank of the potassium polyaspartate content measuring technology in the field of wine is filled. The invention promotes the technical progress of the content of the potassium polyaspartate in the wine and provides a technical method for establishing a detection standard in the future.

(2) Detailed description of the invention

The method combines the liquid chromatography separation theory and the fluorescence detection technology to realize the stable and accurate determination of the content of the potassium polyaspartate in the wine.

Specifically, the method for determining the content of the potassium polyaspartate in the wine comprises the following steps:

1) wine hydrolysis

0.2mL of 10g/L sodium metabisulfite solution, 2mL of wine sample and 2mL of 6M hydrochloric acid solution are sequentially transferred into a 4mL brown bottle, the bottle cap is tightly covered, the solution is completely transferred into a 10mL A-grade volumetric flask after being heated on an electric hot plate at 108 ℃ for 72 hours, 2.5mL of 5M sodium hydroxide solution is added, and the volume is fixed to the scale by ultrapure water.

2) Addition of the internal Standard solution

Respectively transferring 1.25mL of aspartic acid standard working solution, unhydrolyzed wine and 6.25mL of the wine solution hydrolyzed in the step 1 into a 25mL A-grade volumetric flask, adding 0.25mL of an internal standard (aminocaproic acid) solution, and then using ultrapure water to fix the volume to the scale.

3) Derivatization reaction

And (3) respectively transferring 20mL of the solution obtained in the step (2), adding 10mL of the derivatization solution, uniformly mixing for 60s, filtering by using a 0.2-micrometer acetate fiber filter membrane, and detecting.

4) HPLC-FLD analytical detection

In the above steps, the apparatus is prepared to adjust various parameters to a working state: the chromatographic column is a polar liquid phase chromatographic column; column temperature: 40 ℃; FLD wavelength (λ): λ ex 340 nm; λ em 450nm

The chromatographic condition is C18 polar column (such as Syncronis aQ 4.6X 250mm,5 μm); sample introduction amount: 10 mu L of the solution; the column flow rate was 1.1 mL/min; mobile phase A: ultrapure water; mobile phase B: 0.05M sodium acetate anhydrous-tetrahydrofuran (96:4, v: v); mobile phase C: methanol; mobile phase D: acetonitrile; gradient elution procedure: 0-3 min, 100% B; 3-15 min, 50% of B-25% of C-25% of D; 15-17 min, 84% of B-8% of C-8% of D; 17-18 min, 100% B.

(3) Advantageous effects

The invention realizes the high-precision analysis and determination of the content of the potassium polyaspartate in the wine according to the physical and chemical properties of the wine and the technical characteristics of the liquid chromatogram-fluorescence detector, and has the advantages of simplicity, rapidness, accuracy, small sample consumption and the like. The invention aims at the colleges and universities engaged in the wine analysis at home and abroad, and various food detection institutions promote the technical progress of the determination of the content of the potassium polyaspartate in the wine.

Drawings

FIG. 1 schematic diagram of aspartic acid derivatization

FIG. 2 aspartic acid standard curve

FIG. 3 HPLC-FLD chromatogram of aspartic acid in red wine

Detailed Description

In order to more clearly illustrate the invention, the invention is further described below in connection with preferred embodiments. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.

The first embodiment is as follows:

1. instruments and reagents

A high performance liquid chromatography system (comprising a quaternary pump, an automatic sampler, a column chamber with a thermostat and FLD), an electric hot plate, a 4mL brown small bottle, a 0.1-1.0 mL liquid transfer device, a 0.2 mu m acetate fiber filter membrane, an electronic balance, a volumetric flask and a Milli-Q ultrapure water system.

Aspartic acid (DL-aspartic acid C)₄H₇NO₄Purity is not less than 99%, CAS: 617-45-8), sodium metabisulfite (Na)₂S₂O₅CAS: 7681-57-4), hydrochloric acid solution (HCl, CAS: 7647-01-0); sodium hydroxide (NaOH, CAS: 1310-73-2), aminocaproic acid (C)₆H₁₃NO₂Purity is not less than 99%, CAS: 60-32-2), sodium tetraborate decahydrate (solid, purity > 99%, CAS: 1303-96-4), o-phthalaldehyde (OPA) (C)₈H₆O₂Purity is not less than 99%, CAS: 643-79-8), mercaptoethanol (C)₂H₆OS, purity not less than 99%, CAS: 60-24-2)

2. HPLC-FLD conditions

The chromatographic column is C18 polar column (such as Syncronis aQ 4.6 × 250mm,5 μm); column temperature: 40 ℃; FLD wavelength (λ): λ ex 340 nm; λ em-450 nm; sample introduction amount: 10 mu L of the solution; the column flow rate was 1.1 mL/min; mobile phase A: ultrapure water; mobile phase B: 0.05M sodium acetate anhydrous-tetrahydrofuran (96:4, v: v); mobile phase C: methanol; mobile phase D: acetonitrile; gradient elution procedure: 0-3 min, 100% B; 3-15 min, 50% of B-25% of C-25% of D; 15-17 min, 84% of B-8% of C-8% of D; 17-18 min, 100% B.

3. Solution preparation

1)10g/L sodium metabisulfite solution: weighing 5g of sodium metabisulfite into a 500mL A-level volumetric flask, and metering the volume to the scale with ultrapure water.

2)6M hydrochloric acid solution: 109.5g of hydrochloric acid is weighed into a 500mL A-level volumetric flask, and ultrapure water is added to the volume to the scale.

3)5M sodium hydroxide solution: weighing 10g of sodium hydroxide solid in a 500mL A volumetric flask, and metering to the scale with ultrapure water.

4) Preparation of standard working solution of aspartic acid

Preparing a stock solution 1: preparing 5000mg/L aspartic acid by using ultrapure water; preparing a stock solution 2: 200mg/L aspartic acid was prepared with ultrapure water.

Preparing a standard working solution: sucking up 0.25mL, 1.25mL and 6.25mL of stock solutions 2 respectively, and diluting to 25mL with ultrapure water to obtain use solutions STD1, STD2 and STD3 with concentrations of 2, 10 and 50mg/L respectively; 0.5mL, 1.25mL and 2.5mL of stock solutions 1 were aspirated, and the volume was adjusted to 25mL with ultrapure water, to obtain use solutions STD4, STD5 and STD6 at concentrations of 100, 250 and 500mg/L, respectively.

5) Aminocaproic acid stock solution: 1000mg/L aminocaproic acid internal standard solution was prepared with ultrapure water.

6)0.1M sodium tetraborate decahydrate buffer (pH 10.5): 19.1g of sodium tetraborate decahydrate is weighed into a grade A volumetric flask, and the volume is fixed to the scale by ultrapure water.

7) Derivatization solution: 100mg OPA, 200. mu.L mercaptoethanol and 1mL methanol were added to a 10mL class A volumetric flask and the volume was brought to the mark with 0.1M sodium tetraborate decahydrate buffer (ready before use).

8)0.05M anhydrous sodium acetate buffer: 2.05g of anhydrous sodium acetate is weighed into a grade A volumetric flask, and the volume is fixed to the scale by ultrapure water.

4. Sample pretreatment and HPLC-FLD analysis detection

1) Wine sample hydrolysis

0.2mL of 10g/L sodium metabisulfite solution, 2mL of wine sample and 2mL of 6M hydrochloric acid solution are sequentially transferred into a 4mL brown bottle, the bottle cap is tightly covered, the solution is completely transferred into a 10mL A-grade volumetric flask after being heated on an electric hot plate at 108 ℃ for 72 hours, 2.5mL of 5M sodium hydroxide solution is added, and the volume is fixed to the scale by ultrapure water.

2) Addition of the internal Standard solution

Respectively transferring 1.25mL of aspartic acid standard working solution, unhydrolyzed wine and 6.25mL of the wine solution hydrolyzed in the step 1 into a 25mL A-grade volumetric flask, adding 0.25mL of an internal standard (aminocaproic acid) solution, and then using ultrapure water to fix the volume to the scale.

3) Derivatization reaction

Transferring 20mL of the 3 solutions obtained in the step 2, adding 10mL of derivatization solution (adding 100mg of OPA, 200 μ L of mercaptoethanol and 1mL of methanol, using 0.1M sodium tetraborate decahydrate buffer solution to fix the volume to 10mL), mixing uniformly for 60s, filtering with 0.2 μ M acetate fiber filter membrane, and detecting.

4) HPLC-FLD analytical detection

5. Content calculation

The addition amount of potassium polyaspartate (KPA) was determined by the difference in aspartic acid content between the hydrolyzed and unhydrolyzed samples, and the formula (1) was calculated:

X(mg/L)＝(C_{hydrolysis-grape wine}-C_{Unhydrolyzed wine})×f_KPA (1)

Wherein: x is the addition amount (mg/L) of potassium polyaspartate (KPA); c_{Hydrolysis-grape wine}The content (mg/L) of aspartic acid after the wine is hydrolyzed; c_{Unhydrolyzed wine}The content (mg/L) of the aspartic acid in the unhydrolyzed wine; f. of_KPAThe factor for converting the potassium polyaspartate into the aspartic acid is calculated by the ratio of the molecular mass of the potassium polyaspartate monomer to the molecular mass of the aspartic acid, and the formula (2) is calculated:

6. detection limit and quantification limit

Deriving 2, 10, 50, 100, 250 and 500mg/L aspartic acid standard working solution, analyzing and detecting by HPLC-FLD (high performance liquid chromatography-liquid Crystal display), drawing a standard curve by using the concentration-peak area of the aspartic acid to obtain a regression equation y which is 5.954x-6.5241 and a correlation coefficient R²＝0.9998。

Since aspartic acid is naturally contained in wine, it was determined to measure it, and the detection limit LOD and the quantification limit LOQ (LOQ ═ 3LOD) were determined from the signal-to-noise ratio of the wine sample, and the results are shown in table 1.

TABLE 1 detection limit and quantitation limit results for determination of aspartic acid in wine by HPLC-FLD

7. Substrate effects on recovery results

1) Aqueous solution

The recovery rates of the acid hydrolysis and derivatization processes were verified by comparing the solutions before and after hydrolysis of aspartic acid. Aspartic acid solutions of known concentration (25, 100 and 200mg/L) were prepared, and 3 determinations of each concentration were averaged; the results shown in Table 2 were obtained:

TABLE 2 results of the effect of aqueous matrix on recovery

2) Grape wine

Standard addition methods were used to verify the effect of matrix interference on KPA measurements in white and red wines (50 mg/L and 200mg/L potassium polyaspartate (KPA), respectively). Repeat tests were performed 5 times per level. The results shown in Table 3 were obtained:

TABLE 3 results of the effect of wine samples on recovery

Remarking: the optimal recovery rate range is as follows: 80-110%.

Finally, it should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.