KR100926223B1 - Simultaneous determination of two or more Amadori compounds by high-performance anion-exchange chromatography with pulsed amperometric detection - Google Patents

Abstract

The present invention is an analysis method of two or more Amadori compounds produced by the Maillard reaction during the preparation of red ginseng, which is an arginine-fructo which is a red ginseng extract, red ginseng extract-containing composition, or amarium compound in blood samples. A method for simultaneously and accurately analyzing os-glucose (Arg-Fru-Glc) or arginine-fructose (Arg-Fru) by simultaneously selecting the stationary and mobile phases of ion chromatography is disclosed. The simultaneous analysis method can be applied to the quality control of red ginseng because it has advantages in reproducibility, accuracy, analysis time, cost and simplicity, arginine-fructose (Arg-Fru), arginine-fructose-glucose (Arg-Fru- It is also widely applicable to pharmacokinetic studies using Glc). In addition, it can be applied to analysis of other natural extracts other than red ginseng or biological samples other than blood and effectively applied to pseudo-Amadori compounds.

Amadori Compound, Ion Chromatography (IC), Arginine-Fructose, Arginine-Fructose-Glucose, Red Ginseng, Blood

Description

Simultaneous determination of two or more Amadori compounds by high-performance anion-exchange chromatography with pulsed amperometric detection}

The present invention relates to the analysis of two or more Amadori compounds produced by the Maillard reaction during the preparation of red ginseng, specifically, red ginseng extract, red ginseng extract-containing composition or blood sample in blood samples A method for simultaneously and accurately analyzing phosphorus arginine-fructose-glucose (Arg-Fru-Glc) or arginine-fructose (Arg-Fru) by appropriately selecting the stationary and mobile phases of ion chromatography.

Ginseng is a traditional medicinal plant that has been used in East Asia for the purpose of Jangsaeng, Shinshin and Tonic. Among them, red ginseng is processed by steaming raw ginseng with heated steam and drying with sunlight or hot air, which is known to have a greater pharmacological effect than ginseng. Known pharmacological effects of red ginseng include blood pressure lowering, increased blood flow, antithrombotic effects, anticancer and immune function. In addition, it is known to have anti-oxidation and antioxidative effects, anti-oxidation effect and anti-radiation effect related to aging inhibition.

Among the various components of red ginseng, Amadori compound, a non-saponin-based component, is dried by drying ginseng and processed into red ginseng, and a Maillard reaction, which is a condensation reaction between amino acids and sugars, occurs by heat treatment. Is a 1-deoxy-1-amino-fructopyranose compound. In other words, the amadori compound is an intermediate produced during the process of producing the final reaction product as a result of non-enzymatic glycation. Typically, the aldehyde group of a sugar combines with the amino group of an amino acid to form an imine or Schiff base, and the Schiff base is rearranged so that the hydroxyl group next to the carbon-nitrogen double bond changes to a ketone group. Li compound is produced. Oxidation of the amadori compounds yields the final product of non-enzymatic glycosylation. Specifically, about 2% of free amino acid is contained in fresh ginseng, and arginine content is present in a considerably high ratio of 60-70%. Therefore, arginine in red ginseng is used for heat treatment when preparing red ginseng. As a result, a new amino acid derivative, Amadori, is produced by binding to sugar. Arginine and maltose, Arginine and Arginine and Glucose in the ginseng combine Arginine-Fructose and Arginine-Fructose-Glucose. Is generated.

This Maillard reaction is called nonenzymatic browning reaction, which affects the color, flavor, and aroma of food. The products of Armadori are antioxidants and antibiotics. It is reported that there is a physiological activity such as (antibiotic) action. In addition, Arginine-Fructose-Glucose and Arginine-Fructose inhibit maltase enzyme activity to inhibit or delay glucose uptake, thereby suppressing rapid blood sugar rise. It has been studied to have pharmacological activity of anti-obesity and anti-diabetes.

The present invention evaluates the quality of red ginseng or red ginseng processed products, two or more amarium compounds, especially arginine-fructose, in red ginseng extract and compositions or blood samples containing the same for various research purposes on physiological activity. And an arginine-fructose-glucose (Arginine-Fructose-Glucose) components to provide an optimal ion chromatography (IC) method for simultaneous and accurate analysis.

The present invention provides a method for simultaneously analyzing two or more Amadori compounds, comprising the steps of: a) separating an Amadori compound from a sample containing two or more Amadori compounds; b) preparing a strong basic anion exchanger column as a stationary phase of ion chromatography, and injecting the separated Amadori compound into the column; And c) preparing a mixed solvent of water and sodium hydroxide as a mobile phase of ion chromatography, and analyzing two or more Amadori compounds by ion chromatography using the mobile phase. ), Wherein the Amadori compound is arginine-fructose-glucose (Arg-Fru-Glc) or arginine-fructose (Arg-Fru) yl in red ginseng extract, red ginseng extract-containing composition or blood sample. Can be.

In another aspect, the present invention provides a red ginseng quality evaluation method comprising the steps of: detecting the content of the amadori compound in red ginseng using the analysis method of the amadori compound; And red ginseng quality evaluation method comprising the step of evaluating that the red ginseng degree of the red ginseng is high if the amount of the Amadori compound is large.

Simultaneous analysis of two or more Amadori compounds according to one embodiment of the present invention is advantageous in reproducibility, accuracy, analysis time, cost and simplicity, and thus can be applied to quality control of red ginseng, and arginine-fructose ( Arg-Fru) and arginine-fructose-glucose (Arg-Fru-Glc) can be widely applied to pharmacokinetic studies. In addition, it can be applied to analysis of other natural extracts other than red ginseng or biological samples other than blood and effectively applied to pseudo-Amadori compounds.

Hereinafter, the present invention will be described in detail.

Arginine-Fructose, Arginine-Fructose-Glucose (Arginine-Fructose-Glucose) simultaneous analysis according to an embodiment of the present invention consists of the following steps.

a) Arginine-Fructose, Arginine-Fructose, Arginine-Fructose-Glucose from a sample containing Arginine-Fructose, Arginine-Fructose-Glucose Separating (Arginine-Fructose-Glucose);

b) preparing a strong basic anion exchanger column as a stationary phase of ion chromatography, and arginine-fructose and arginine-fructose-glucose separated from the separated arginine-fructose-glucose. Injecting; And

c) preparing a mixed solvent of water and sodium hydroxide as a mobile phase of ion chromatography, and qualitatively and quantitatively analyzing the two Amadori compounds by ion chromatography using the mobile phase.

In one embodiment of the present invention, Arginine-Fructose and Arginine-Fructose-Glucose in the sample vary the column passage time by the difference in affinity for the column filler. do. The difference in retention time due to migration is characteristic of each compound (ion state), which is represented by an increase in conductivity in the electrical conductivity detector, which is called a peak. The analyte is subjected to component identification (e.g. qualitative analysis) by comparison of elution time with standard material, and the amount contained by the height or area of the peak is calculated (quantitative analysis).

Arginine-Fructose (Argine-Fru) and Arginine-Fructose-Glucose (Argine-Fru-Glc), which are Amadori compounds, are represented by the formula Has a structure.

In one embodiment of the present invention, the sample in step a may be red ginseng extract, red ginseng extract containing composition or blood. When the sample is a red ginseng extract or a red ginseng extract-containing composition, the amadori compound is extracted from the sample using water as the extraction solvent in step a, and when the sample is blood, the water and trichloroacetic acid in step a. A 9: 1 (volume ratio) mixture can be used to remove blood proteins from the sample and to separate the Amadori compounds.

In one embodiment of the present invention, the column used in step b may be filled with a polymer resin having the characteristics of a strong basic anion exchanger suitable for sugar analysis, specifically, the filler of the column is PS / DVB ( Polystyrene / Divinyl benzene). In order to effectively separate the monosaccharide structure and the disaccharide structure among the molecular structures of Arginine-Fructose (Arg-Fru) and Arginine-Fructose-Glucose (Arg-Fru-Glc), Using a column of a strong base anion exchanger rather than an amine (column with a NH ₂ functional group) column increases separation and thus allows accurate analysis.

In an embodiment of the present invention, the mobile phase in step c may simultaneously move arginine-fructose (Arg-Fru) and arginine-fructose-glucose (Arg-Fru-Glc) of monosaccharide structure and disaccharide structure. For this purpose, a mixed solvent of water and sodium hydroxide in a 400 mM concentration of sodium hydroxide: water in a 9: 1 (volume ratio) may be used at a flow rate of 0.7-1 ml / min. When the flow rate of the mobile phase is less than 0.7 ml / min, arginine-fructose-glucose (Arg-Fru-Glc) overlaps the interference component in the sample and exceeds 1 ml / min in the red ginseng extract and the red ginseng extract-containing composition sample. In the case of arginine-fructose (Arg-Fru) and arginine-fructose-glucose (Arg-Fru-Glc) in the red ginseng extract, red ginseng extract-containing composition or blood sample, it is difficult to accurately analyze.

On the other hand, in the case of using a mixed solvent of sodium hydroxide + sodium acetate + water used in analyzing the sugar in ion chromatography, it is difficult to accurately analyze the overlapping components of the blood sample.

Arginine-Fructose (Arg-Fru), Arginine-Fructose in Red Ginseng Extract, Compositions Comprising the Same, and Blood Samples Using Simultaneous Analysis of Two or More Amadori Compounds According to One Embodiment of the Present Invention As a result of quantitative determination of -glucose (Arg-Fru-Glc), the two components in the calibration curve showed good linearity of R ² = 0.999 or more in the concentration range of 0.05 to 20 μg / ml.

In one embodiment of the present invention, the degree of browning of red ginseng can be assessed using the Amadori compound analysis method. The degree of browning of red ginseng is an important evaluation criterion for the quality of red ginseng products. As the intensity of the color increases, the red ginseng can be classified into high quality red ginseng. That is, the higher the content of Arginine-Fructose-Glucose and Arginine-Fructose, which are the reaction products by Maillard reaction, are classified as products with higher red ginseng degree. Can be. Therefore, the quality of the red ginseng can be evaluated by analyzing the contents of the two components.

Hereinafter, preferred examples and test examples of the present invention will be described in more detail. However, the following embodiments are merely preferred embodiments of the present invention, and the present invention is not limited to such embodiments.

Example 1 Selection of Optimum Conditions for Analysis

In order to accurately analyze arginine-fructose (Arg-Fru) and arginine-fructose-glucose (Arg-Fru-Glc) with high degree of separation, the following experiments were performed to determine the optimum conditions.

1. Selection of analytical methods

In order to separate Arginine-Fructose (Arg-Fru) and Arginine-Fructose-Glucose (Arg-Fru-Glc) with high separation, various analytical methods were used. First, Arginine-Fructose (Arg-Fru) and Arginine-Fructose-Glucose (Arg-Fru-Glc) are compounds of the form of the amino acid arginine and sugar in the molecule are very polar, As there is no special UV absorption function in the molecule, it was difficult to apply an analysis method using a reverse phase nonpolar column (C18) and an ultraviolet absorption spectrophotometer (UV), which are used in conventional liquid chromatography. Also in the case of using amino acid derivatization, Arginine-Fructose (Arg-Fru) and Arginine-Fructose-Glucose (Arg-Fru-Glc) are fluorescent reagents for amino acid derivatization due to the sugar structure in the molecule. The reactivity of was greatly reduced and the sensitivity was lower than that of the general amino acid derivatization reaction, and the separation degree between the two compounds was lowered, and the analytical process was not simple and complicated by the pretreatment process called derivatization. In order to separate and analyze sugar using sugar structure in the molecule, liquid chromatography using amine column (column with NH ₂ functional group) and differential index detector is applied. The separation of arginine-fructose with monosaccharide structure and arginine-fructose glucose with disaccharide structure was not suitable for simultaneous analysis of two components. Due to its characteristics, the detection sensitivity was not good, making it difficult to apply to trace analysis such as in blood samples. In the method of derivatizing the sugar part in the molecule by trimethylsilylation by gas chromatography (GC) method, in order to convert the Amadori compound into a volatile compound, a pretreatment called derivatization Separation is complicated because the process is essential and tautomers are produced as reaction byproducts.

 Therefore, in one embodiment of the present invention, an electrochemical detector for ion chromatography capable of analyzing ions using the structural properties of an Amadori compound having a cationic or anionic charge depending on pH conditions. The high performance anion exchange chromatography / pulsed amperometric detection (HPAEC-PAD) method is applied, and the pulsed amperometric detection method is one of the electrochemical detectors. That's the way. In one embodiment of the present invention, using a strong basic anion exchanger as a stationary phase of a high performance ion exchange chromatography / electrochemical detector to analyze anions in various inorganic and organic compounds with high selectivity and sensitivity, and mobile phase of basic pH The sugar was ionized to have a weak anionic property, and each component was separated using affinity with the column fixed phase.

2. Optimal Column Selection

The stationary phase used in ion chromatography introduce | transduces the functional group which has ion exchange ability into polystyrene or a silica gel, and consists of a base material which forms a backbone, and an exchanger which has ion exchange ability. There are four types of ion exchangers: strong acid, weak acid, weak base, and strong base, but to analyze arginine-fructose (Arg-Fru) and arginine-fructose-glucose (Arg-Fru-Glc) under strong base conditions A separation mechanism was applied to ionize the sugar to have a weak anionicity and to use a binding force with a column composed of a strong basic anion exchanger.

On the other hand, in strong base condition (high pH), it cannot be applied because the silica-based column cycle agent used in the conventional liquid chromatography (HPLC) is melted, and the pH range is 0-14, which is stable even at high pH. Column periodic agents consisting of polymer bases can be used. Specifically, the column periodic agent may be a copolymer resin of polystyrene / divinyl benzene (PS / DVB). In one embodiment of the present invention was selected Carbopac PA1 column.

3. Setting mobile phase condition to improve separation

In ion chromatography, an aqueous solution is mainly used, and the retention time of ions can be controlled by adjusting the ionic strength and pH of the mobile phase. Anionic solvents are used for the cation exchanger and cationic solvents are used for the anion exchanger. When the ionic strength of the mobile phase increases, the competition between the sample ions for the ion exchanger of the ion exchanger and the opposing ions of the mobile phase increases, and the retention time of the sample ions decreases. In the case of adjusting the dissociation degree of the sample by changing the pH of the mobile phase, the ratio of ionic species in the sample is changed, and at high pH, the retention time of the sample ions increases in anion exchange chromatography.

The present inventors have found that high pH sodium hydroxide as a mobile phase to increase the retention time of sample ions to simultaneously move arginine-fructose (Arg-Fru) and arginine-fructose-glucose (Arg-Fru-Glc) A mixed solvent of + sodium acetate + water and a mixed solvent of water + sodium hydroxide were applied. First, when a mixed solvent of sodium hydroxide + sodium acetate + water was used, the endogenous substance and arginine-fructose-glucose (Arg-Fru-Glc) were measured in plasma samples when the mobile phase ionic strength was adjusted in the concentration range of 100 mM to 500 mM. ) Did not separate, and the internal standard did not separate well from the disturbing components of the sample, so that the exact analysis conditions could not be established.

Second, when a mixed solvent of water and sodium hydroxide was used, the endogenous substance and arginine-fructose-glucose (Arg-Fru-Glc) in plasma samples were gradually increased in the concentration of sodium hydroxide in the range of 100 mM to 500 mM. ) And the concentration at which the internal standard is separated. As a result, it was confirmed that the most effective analysis at a concentration of 400 mM. In addition, impurities eluted in the sample were effectively eluted and did not affect the analysis at the next injection of the sample. In addition, arginine-fructose and arginine-fructo at a rate of 0.7-1 ml per minute, preferably 0.7 ml per minute, when the flow rate of the mobile phase is adjusted to improve the retention time and separation of ions Os-glucose (Arg-Fru-Glc) and internal standards were separated within 15 minutes with satisfactory separation and retention time.

4. Optimum Conditions for Analysis

Therefore, two kinds of Amadori components, arginine-fructose (Arg-Fru) and arginine-fructose-glucose (Arg-Fru-Glc), have high performance ion exchange chromatography / pulse current detection (high performance). Analysis was performed by anion exchange chromatography / pulsed amperometric detection (HPAEC-PAD). Specific conditions are described in Table 1 below, in which the components separated from the sample were injected into ion chromatography, and the fixed phase was a Carbopac PA1 (length: 25 cm, inner diameter: 4.0 mm) column, and the mobile phase was 400 mM sodium hydroxide. Water was used. The flow rate of the mobile phase was 0.7 mL / min, the sample injection amount was 25 μl, and an electrochemical detector was used using an electrode composed of a working electrode (gold) and a reference electrode (silver / silver chloride) used for pulse current detection. The column temperature was 30 ° C during analysis, and the pulsed amperometric detection method used for electrochemical detection was a quadruple potential waveform method, and the conditions are shown in Table 2. That is, after a 0.2 minute delay time at a potential of 0.1 V, the current generated by oxidizing sugar on the surface of the working electrode (gold) at a potential of 0.1 V for 0.2 to 0.4 minutes is measured by integrating. For 0.41-0.5 minutes, the surface of the electrode is cleaned by changing the potential difference of Table 2 to reduce the gold oxide on the surface of the electrode produced by the oxidation of sugar back to the original gold.

column Carbopac PA1 Column (4.0 mm x 25 cm) Carbopac PA1 Guard Column (4.0 mm x 5 cm) Mobile phase Solvent A (water): Solvent B (400 mM sodium hydroxide) = 10:90 Flow rate 0.7 mL / min Column temperature 30 ℃ Injection volume 25 μl Detector ECD Working electrode Gold Reference electrode Ag / AgCl

Minutes Potential (V) Integration 0.0 +0.1 0.2 +0.1 start 0.4 +0.1 End 0.41 -2.0 0.42 -2.0 0.43 +0.6 0.44 -0.1 0.50 -0.1

Example 2 Simultaneous Analysis of Arginine-Fructose and Arginine-Fructose-Glucose

Red ginseng extract or a composition comprising the same, and arginine-fructose (Arg-Fru) and arginine-fructose-glucose (Arg-Fru-Glc) which are actually present in the blood using the optimal conditions selected in Example 1 above. In order to analyze the compounds simultaneously, the following experiment was performed.

1. Analyzer (HPAEC-PAD)

The ion chromatography used in this example was a ICS2500 manufactured by Dionex, which used a system connected with an electrochemical detector (ECD) of ED50. The column was used by connecting a CarboPac PA1 guard column (4.0 mm x 5 cm) from Dionex and a Carbopac PA1 column (4.0 mm x 25 cm), and data processing was performed using Dionex's Chromeleon 6.5 program. .

Ion chromatograms obtained from standards of arginine-fructose (Arg-Fru) and arginine-fructose-glucose (Arg-Fru-Glc) are shown in FIG. The elution order was Arginine-Fructose (Arg-Fru), Arginine-Fructose-Glucose (Arg-Fru-Glc), and N-acetylneuraminic acid (N-acetylneuraminic acid) used as an internal standard.

2. Standard

Standards of Arginine-Fructose (Arg-Fru) and Arginine-Fructose-Glucose (Arg-Fru-Glc) were synthesized separately and separated by column chromatography, nuclear magnetic resonance (NMR) and liquid chromatography / mass spectrometers. What was identified using was used. Specifically, it was performed as follows.

10 g of arginine (arginnin), 20 g of maltose (maltose), 10 g of arginine (arginnine) and 20 g of glucose (glucose) were put into an empty flask, and 50 ml of acetic acid was added thereto and heated at 80 ° C. for 20 minutes. After cooling to room temperature, the reaction was centrifuged at 1000 g for 10 minutes to remove the precipitate, and only the supernatant was taken up and concentrated under reduced pressure. 50 ml of distilled water was added to the concentrated solution, and the eluate which was loaded on the ion exchanger column was lyophilized. The silica gel column was separated and purified again, and the column eluate was confirmed by TLC, and each fraction was concentrated under reduced pressure. Arginine-fructose (Arg-Fru) and arginine-fructose-glucose (Arg-Fru-Glc) obtained were subjected to 300 MHz nuclear magnetic resonance (Gemini 2000, Varian) and liquid chromatography / mass spectrometer (LCQ-Deca). XP, Thermo Fisher Scientific) was identified and the molecular weight was confirmed that the purity is more than 95%. Mass spectra of arginine-fructose (Arg-Fru) and arginine-fructose-glucose (Arg-Fru-Glc) are shown in FIG. 2.

3. Simultaneous Analysis of Arginine-Fructose (Arg-Fru) and Arginine-Fructose-Glucose (Arg-Fru-Glc) in Red Ginseng Extract and Red Ginseng Extract Composition

Take 20 mg of each sample from commercially available red ginseng products and self-produced red ginseng extract, add N-acetylneuraminic acid, an internal standard, and the final concentration of internal standard Dilute with distilled water to 5 μg / ml. The samples were ultrasonically extracted for 15 minutes, dispersed well, and filtered through a 0.2 μm PVDF membrane filter without any other pretreatment. 25 μl of the sample solution was injected into a high performance ion exchange chromatography / electrochemical detector and analyzed. The chromatogram obtained as a result of the analysis is shown in FIG. 4, and the content measurement results are shown in Table 3 below.

sample Content of Arg-Fru (%) Arg-Fru-Glc (%) Red Ginseng Extract 1 0.95 2.64 Red Ginseng Extract 2 1.51 2.33 Product 1 (JL) 2.51 1.32 Product 2 (JG) 1.80 0.96 Product 3 (LTJS) 1.62 1.05 Product 4 (DH) 0.35 0.29 Product 5 (NH) 1.57 0.25 Product 6 (RGHK) 0.85 0.77

Calibration curves for the determination of arginine-fructose (Arg-Fru) and arginine-fructose-glucose (Arg-Fru-Glc) compounds ranged from 0.05 to 20 μg / ml, with R = 0.99 The above good linearity was shown and shown in FIG.

As a result, the detection limits of Arginine-Fructose (Arg-Fru) and Arginine-Fructose-Glucose (Arg-Fru-Glc) compounds in red ginseng extract and products were 0.1 ㎍ / g, respectively.

On the other hand, in order to confirm the results of the content analysis in the sample, a certain amount of standard was added to the red ginseng extract, which knows the content of arginine-fructose (Arg-Fru) and arginine-fructose-glucose (Arg-Fru-Glc) compounds. The recovery rate was determined by analyzing. Table 4 shows the initial concentrations of the arginine-fructose (Arg-Fru) and arginine-fructose-glucose (Arg-Fru-Glc) compounds in the red ginseng extract, the concentrations of the standard products added and the recovery rate.

compound Initial concentration (㎍ / ㎖) Added amount (㎍ / ㎖) Measured amount (µg / mL) % Recovery Arg-Fru 4.8 16.0 21.14 102.1 10.0 14.78 99.8 1.0 5.82 101.5 0.2 4.99 94.7 Arg-Fru-Glc 4.5 16.0 20.40 99.4 10.0 14.56 100.6 1.0 5.52 101.5 0.2 4.69 94.5

4. Simultaneous Analysis of Arginine-Fructose (Arg-Fru) and Arginine-Fructose-Glucose (Arg-Fru-Glc) in Blood

In order to simultaneously analyze the two compounds in the blood of rats, 6 to 8-week old male rats (270 ± 30 g) were collected from the blood and centrifuged at 13,000 rpm for 5 minutes to separate plasma and stored at -20 ℃. It was.

Spike 10 μl of a mixed solution of arginine-fructose-glucose (Arg-Fru-Glc) and arginine-fructose (Arg-Fru) into 490 μl of the plasma to obtain a final concentration of 0.5, 1 , 5, 10 ㎍ / ㎖ concentration, 100 μl of the plasma spiked (standard) was taken, and 10 ㎕ of N-acetylneuraminic acid (N-acetylneuraminic acid) of 100 ㎍ / ㎖ concentration. In order to remove the protein in plasma, 90 μl of 10% trichloroacetic acid was added and mixed, and the supernatant was separated by centrifugation at 4 ° C. and 12,000 rpm for 10 minutes.

The separated supernatant was filtered through a 0.2 μm PVDF membrane filter and 25 μl was injected into a high performance ion exchange chromatography / electrochemical detector and analyzed. In order to verify whether accurate measurement of the analyte is possible in the presence of interfering or endogenous substances that may exist in the matrix of blood samples, a comparison between the blank plasma sample and the plasma sample spiked with the standard solution is performed. It was observed whether there was no phenomenon. 5 shows a chromatogram (top) for a plasma sample and a chromatogram (bottom) for a plasma sample spiked with a standard solution. The peak retention position of the components was confirmed from the standard solution (chromatogram of FIG. 1), and the observation of the influence of the matrix in the blood showed arginine-fructose (Arg-Fru) and arginine-fructose-glucose (Arg- Fru-Glc) it was confirmed that there is no other peculiar peak in the position of the components. The detection limits of arginine-fructose-glucose (Arg-Fru-Glc) and arginine-fructose (Arg-Fru) compounds present in blood were 0.1 µg / ml, respectively.

In addition, to confirm the recovery rate of the arginine-fructose-glucose (Arg-Fru-Glc) and arginine-fructose (Arg-Fru) spiked in the blood to confirm the analysis results, for each concentration Five sampling samples were used for intraday and interday measurements and averaged. Table 5 below shows the results of the recovery of arginine-fructose (Arg-Fru) in the blood, and table 6 in the arginine-fructose-glucose (Arg-Fru-Glc) compound.

Measure Spiked concentration (µg / ml) Measured concentration (µg / mL) (mean ± standard deviation) Recovery (%, n = 5) Intraday 10 10.21 ± 0.14 102.1 5 4.97 ± 0.12 99.4 One 1.03 ± 0.03 102.6 0.5 0.48 ± 0.02 96.4 Interday 10 10.24 ± 0.19 102.4 5 5.02 ± 0.13 100.3 One 1.02 ± 0.02 101.5 0.5 0.48 ± 0.02 95.5

Measure Spiked concentration (µg / ml) Measured concentration (µg / mL) (mean ± standard deviation) Recovery (%, n = 5) Intraday 10 9.88 ± 0.15 98.8 5 4.80 ± 0.10 96.1 One 1.02 ± 0.03 101.7 0.5 0.49 ± 0.02 97.6 Interday 10 9.95 ± 0.15 99.5 5 4.94 ± 0.13 98.7 One 1.01 ± 0.03 100.8 0.5 0.48 ± 0.02 95.4

1 shows two kinds of Amadori compounds, Arginine-Fructose (Arg-Fru) and Arginine-Fructose-Glucose (Arg-Fru-Glc), and Enacetylnu, an internal standard (IS). The standard solution ion chromatogram of N-acetylneuraminic acid is shown.

Figure 2 shows the mass spectrum of the standards of arginine-fructose (Arg-Fru) and arginine-fructose-glucose (Arg-Fru-Glc).

Figure 3 shows the calibration curve for the concentration and signal strength (peak area) of arginine-fructose-glucose (Arg-Fru-Glc) and arginine-fructose (Arg-Fru).

Figure 4 shows a chromatogram of simultaneous analysis of arginine-fructose-glucose (Arg-Fru-Glc) and arginine-fructose (Arg-Fru) in red ginseng extract samples.

FIG. 5 is a plasma sample and a plasma sample spiked with a standard to verify the accuracy of analysis of arginine-fructose-glucose (Arg-Fru-Glc) and arginine-fructose (Arg-Fru) in blood samples. In the plasma), a chromatogram showing the simultaneous analysis of arginine-fructose-glucose (Arg-Fru-Glc) and arginine-fructose (Arg-Fru) is shown.

Claims (9)

As a method of simultaneously analyzing two or more Amadori compounds, a) separating an Amadori compound from a sample containing at least two Amadori compounds; b) preparing a strong basic anion exchanger column as a stationary phase of ion chromatography, and injecting the separated Amadori compound into the column; And c) preparing a mixed solvent of water and sodium hydroxide as a mobile phase of ion chromatography, and analyzing two or more Amadori compounds by ion chromatography using the mobile phase. Assay of Compounds. The method of claim 1, In the step a, the sample is selected from the group consisting of red ginseng extract, red ginseng extract containing composition and blood, analysis of Amadori compound. The method of claim 2, If the sample is a red ginseng extract or red ginseng extract-containing composition in step a to extract the amadori compound from the sample using water as an extraction solvent, the analysis method of the Amadori compound. The method of claim 2, If the sample is blood in step a, using a mixture of water and trichloroacetic acid to remove proteins in the blood from the sample and to separate the Amadori compound, Amadori compound assay. The method of claim 1, The amadori compound in step a is arginine-fructose-glucose (Arg-Fru-Glc) or arginine-fructose (Arg-Fru), the analysis method of the Amadori compound. The method of claim 1, The filler of the column in step b is a copolymer resin of polystyrene / Divinyl benzene (PS / DVB), analysis method of the Amadori compound. The method of claim 1, The c and the mixed solvent of water and sodium hydroxide in step c is a solvent mixed so that the volume ratio of sodium hydroxide: water of 400 mM concentration of 9: 1, Amadori compound analysis method. The method of claim 1, The flow rate of the mobile phase in step c is 0.7-1 ml / min, Amadori compound assay. As a red ginseng quality evaluation method, Detecting the content of amadori compound in red ginseng using the assay of the amadori compound according to any one of claims 1 to 3 and 5 to 8; And Red ginseng quality evaluation method comprising the step of evaluating the degree of red ginseng according to the amount of detection of the Amadori compound.

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