KR100942988B1 - Analysis method for glycosides using reversed-phase high-performance liquid chromatography and pulsed amperometric detection - Google Patents

Abstract

The present invention relates to a method for analyzing glycosides using reversed phase high performance liquid chromatography and pulsed electrochemical detectors. Unlike conventional methods, the glycoside analysis method of the present invention is simple because no pretreatment step is required, and shows high selectivity, good sensitivity, and reproducibility with respect to glycosides. The glycoside analysis method of the present invention enables the analysis of preparations containing complex components, in particular the quality evaluation of herbal preparations.

Glycosides, Ginsenosides, Reversed-Phase High Performance Liquid Chromatography, Pulsed Electrochemical Detectors, Glycosides

Description

Analysis method for glycosides using reversed-phase high-performance liquid chromatography and pulsed amperometric detection}

The present invention relates to methods for analyzing glycosides using reversed phase high performance liquid chromatography and pulsed electrochemical detectors.

Most plant components are found in combination with other components, and glycosides refer to forms in which sugars and organic active compounds, such as aglycone and genin, are combined.

Among the plants, ginseng is the most widely used herb in the East as root and root of Panax gensing or its related plant. Traditionally, ginseng has been used as a tonic tonic and actually has immune enhancing, anti-inflammatory and vasodilating effects. Recently, it is widely used as food and medicine not only in the Orient but also in many countries around the world. Saponins are a glycoside, and plant saponins are divided into three groups, depending on their chemical structure, Protopanaxadiol (PD), Protopanaxatriol (PT), and oleanolic acid. Most saponins in the plant family are oleanane, and ginseng saponins are triterpenoid saponins of the dammarane family, which are rarely found in other plants, and are distinctive from other plant saponins. Because it has a structure, it is called ginsenoside in the sense of Ginseng Glycoside to distinguish it. Among these ginsenosides, the non-glycosides of the ginsenosides Rb ₁ , Rb ₂ , Rc, Rd and the protopanaxatriol type ginsenoside Rg ₁ , It can be divided into Rg ₂ , Re, Rf and its structure is shown in FIG. Such ginsenosides exhibit different effects depending on the type of non-saccharide and the type of sugar. Therefore, isolating and quantifying ginsenosides in ginseng is important in estimating its efficacy.

High performance liquid chromatography-ultraviolet detector (hereinafter referred to as HPLC-UV) method, high performance liquid chromatography-vaporized light scattering detector (hereinafter referred to as HPLC-ELSD) method, and high performance liquid chromatography-Zanlan for analysis of glycosides including ginsenosides Many papers have been published, including analytical spectrum (HPLC-MS) methods.

The most commonly used glycoside analysis method is HPLC-UV method. Flavonoid glycosides have chromophores, so they show good sensitivity when analyzed by absorbance method, but terpenoids occupy the largest part of glycosides. Since terpenoid glycosides do not have chromophores, they can be detected at low wavelengths in the 200-210 nm range. However, at this wavelength, many interferences are difficult to apply to herbal medicines and preparations, and their sensitivity is limited. HPLC-ELSD method is suitable for analyzing glycosides that are difficult to analyze by absorbance method, but its sensitivity is poor. The HPLC-MS method is suitable for analyzing trace materials due to its high sensitivity, but the equipment is expensive and difficult to apply to quality evaluation of Daerang. Therefore, development of a new glycoside analysis method is required.

Pulsed electrochemical detectors were originally developed to analyze sugars in high-performance anion-exchange chromatography (HPAEC). Its detection principle is an electrochemical detector based on the oxidation of carbohydrates at the gold electrode, taking advantage of the fact that sugar hydroxyls are ionized under strong base conditions. However, this method is not suitable for the separation and analysis of components other than sugars and amino acids because an anion exchange column and sodium hydroxide are used as mobile phases.

In the glycoside analysis, the present inventors have used a reversed-phased high performance liquid chromatography-pulse amperometric detection (RP-HPLC-PAD) for the selective and high sensitivity separation assay of glycosides. The present invention has been completed by developing possible assays.

It is an object of the present invention to provide a method for analyzing glycosides using a reversed phase high performance liquid chromatography-pulsed electrochemical detector.

In order to achieve the above object, the present invention provides a combination of reverse phase high performance liquid chromatography and pulsed electrochemical detector.

The reversed phase high performance liquid chromatography method provides a method for detecting glycosides by a pulsed electrochemical detector by separating glycosides using a C-18 column and then supplying a strong base solution after passing through the column.

In one embodiment, the present invention relates to (a) passing a sample through a reversed phase column using an organic solution, which is a first eluent, and (b) passing the eluent passed through the column, using a T-shaped mixer, to form a second eluent, sodium hydroxide. A method for glycoside analysis comprising mixing with a solution and (c) detecting the mixed eluent with a pulsed electrochemical detector.

In the present invention, the first eluent uses a sequential gradient elution method in which the volume ratio of the 10% acetonitrile solution and the 60% acetonitrile solution is 80:20 to 60:40 and again 80:20.

In the present invention, the flow rate of the first eluent is 0.2 mL / min, it is preferable that the flow rate of the second eluent is 0.8 mL / min. In addition, it is preferable to use a six-potential waveform as the pulsed electrochemical detector.

According to the present invention, a method for analyzing glycosides using reversed phase high performance liquid chromatography and a pulsed electrochemical detector can be completely separated according to mobile phase conditions while the non-saccharide portion of the glycosides stays in a column, and easily with amino acids or sugar alcohols. Separation results in high selectivity for glycosides. In addition, the conventional glycoside analysis method uses a detection wavelength of about 210 nm mainly through absorption detection in a manner that focuses on the detection of non-saccharides rather than the detection of sugars. However, the detection wavelength of this region is not selective because there are many components detected, there is a disadvantage that the sensitivity is low. However, the glycoside analysis method of the present invention has the advantage of separating the glycosides selectively and with high sensitivity.

Hereinafter, the present invention will be described in detail. Those skilled in the art will appreciate that various modifications and variations can be made in the present invention without departing from the spirit and scope of the invention as set forth in the claims below.

The glycoside analysis method of the present invention is a method of analyzing glycosides using reverse phase high performance liquid chromatography and a pulsed electrochemical detector, and a schematic diagram of the method is shown in FIG. 2. Passing the sample through a reverse phase column using an organic solution as the first eluent to separate glycosides, and mixing the eluate passed through the column with a sodium hydroxide solution as a second eluent using a T-shaped mixer. And a third step of detecting the glycoside using the pulsed electrochemical detector. A more detailed description of the glycoside analysis method of the present invention is as follows.

1. The first step

HPLC equipment including Model Nanospace SI-2 / 3001 pump and 3004 column oven was used from Shiseido (Tokyo, Japan). The column used Hypersil Gold 3 μm C18 (150 mm × 2.1 mm I.D .; 3 μm, Thermo, Waltham). The mobile phase was analyzed by gradient elution of 10% acetonitrile (solvent A) and 60% acetonitrile (solvent B). Elution isocratic with A: B (80:20) for 18 minutes, gradient elution from A: B (80:20) to A: B (60:40) for 18 to 20 minutes, A for 20 to 60 minutes : Elution of isocratic with B (60:40), gradient elution from A: B (60:40) to (80:20) for 60 to 62 minutes and equilibration to 70 minutes. Under these solvent conditions, all seven kinds of ginseng saponins were separated in a reversed-phase C18 column. The flow rate was set to 0.2 mL / min and the separation temperature was set to 30 ° C.

2. Second Step

The eluent passed through the column was mixed with a second eluent, 200 mM sodium hydroxide solution, using a T-shaped mixer. When strong alkali condition is achieved by sodium hydroxide, when anionized, the sugar portion of the isolated glycoside may be anionized and detected by an electrochemical detector. The flow rate of the second eluent is 0.8 mL / min.

3. The third step

Glycosides were detected using the mixed eluent using a pulsed electrochemical detector. Dionex (CA, USA) ICS-3000 series pulsed electrochemical detector with gold electrode and Ag / AgCl auxiliary electrode was used, E1 = -0.2V; E2 = 0V; E3 = +0.22 V; E4 = 0V; E5 = -2V; And a potential of E6 = + 0.6V. Each potential E1, E2 is classified into an absorption and delay phase, potentials E3 and E4 are detected, and potentials E5 and E6 are washed and activated. Data was analyzed using the Chromeleon client program provided by Dionex.

Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are merely to illustrate the content of the present invention is not limited to the scope of the invention.

Example 1 Chromatogram Comparison of Ultraviolet and Pulsed Electrochemical Detectors for Ginseng and Sagunja-tang

Herbal medicines such as ginseng, bokyeong, licorice, and baekchul were purchased in Gyeongdong market in accordance with Korean Pharmacopoeia (VIII) regulations. For preparing the standard solution, the sample solution, and the mobile phase, 18 MΩ tertiary distilled water prepared by automatic aquarius AW-1001 (Top Trading Co., Seoul, Korea) was used. Millipore membrane filter (type HA, pore size 0.45 μm) was used to filter the solvent. All sample fluids were filtered through a MFS 13 disposable syringe filter before injection. The weight of each sample was measured using a METTLER TOLEDO (AX 105 DeltaRange) microbalance. Sagunja-tang, a traditional oriental prescription, consists of ginseng, 10 Fuling, 9 licorice, 6 Baekchul, and 9, which represents the mass ratio in the prescription.

1 g of ginseng powder and 100 mL of water were added to a 250 ml spherical flask, and the resultant boiled for 1 hour was filtered with a nonwoven fabric, and the filtrate was lyophilized to obtain 0.56 g of dried product. The lyophilisate dissolved in 20% acetonitrile was centrifuged at 12000 rpm for 20 minutes, filtered using a Millipore membrane filter (type HA, pore size 0.45 μm) and subjected to high performance liquid chromatography. 1 g of ginseng, white ginseng, Bogyeong, 0.9 g, licorice 0.6 g and 100 mL of water were added to a 250 ml spherical flask, and the mixture was boiled for 1 hour and filtered with a nonwoven fabric. The lyophilisate was dissolved in 20% acetonitrile and subjected to high performance liquid chromatography through the same pretreatment as ginseng. The amount of sample injected was 28 μg for ginseng extract and 61 μg for Sagunja-tang.

In order to compare with the HPLC-UV method, the ginseng extract and Sagunja-tang were concentrated by taking a saturated butanol layer through partition extraction with saturated butanol, and then dissolving the residue in 20% acetonitrile solution and then performing the same preliminary treatment to perform high performance liquid chromatography. It was applied to graphy. Figure 3 is a chromatogram obtained by using an ultraviolet detector and a pulsed electrochemical detector for ginseng and Sagunja-tang. It is difficult to quantify the UV detector in the ginseng chromatogram (A) because the peaks of ginsenosides are low due to their low sensitivity, and it is difficult to quantify them. Side peaks were too small or buried due to their interference, making it difficult to identify. Therefore, trace amounts of ginsenosides in Sagunjatang could not be determined. On the other hand, in the case of using a pulsed electrochemical detector, only the peaks of ginsenosides appeared in the chromatogram of ginseng (C), and the sensitivity was very high compared to the ultraviolet detection method. In addition, only peaks of ginsenosides appeared in the chromatogram (D) of Sagunja-tang so that there was no difference with the chromatogram of ginseng, and there was no disturbing peak. Therefore, it can be seen that the glycoside analysis method using the reverse phase high performance liquid chromatography and the pulsed electrochemical detector of the present invention enables the analysis of the trace components in the herbal preparations due to the high selectivity and sensitivity to ginsenosides.

Example 2: Chromatogram Comparison of Ultraviolet and Pulsed Electrochemical Detectors for Simultaneous Analysis of Glycosides and Non-saccharides

To determine whether the glycoside analysis method using reversed-phase high performance liquid chromatography and pulsed electrochemical detectors were selective for glycosides, glycosides and non-saccharides were simultaneously analyzed using an ultraviolet detector and a pulsed electrochemical detector. 4A and 4B show chromatograms of glycoside D-amigdalin and its non-saccharide mandelonitrile detected by an ultraviolet detector and a pulsed electrochemical detector. For reference, for the analysis of (A) and (B), 40% acetonitrile was used for the mobile phase, and 35% acetonitrile for the analysis of (C), (D) and (E), respectively. The injection amount of the sample was 10 µl. In the chromatogram obtained by the ultraviolet detector, all peaks appeared without selectivity, but the chromatogram obtained by the pulsed electrochemical detector clearly showed the glycoside D-amigdaline, but the mandelonitrile was very small, and the glycosylated non-glycoside (schizandrin) did not appear at all. (C), (D) and (E) of Figure 4 is a comparison of the glycosides naringin (naringin) and its non-glycosides naringenin (naringenin), while in the chromatogram by the ultraviolet detector both peaks appear without selectivity In the case of applying six-potential waveform in the chromatogram (D) through the pulsed electrochemical detector, the glycoside, naringin was clearly seen, but the nonglycoside, naringenin, was very small. On the other hand, in the chromatogram (D) of the pulsed electrochemical detector detected by applying a quadruple-potential waveform, the naringenin peak disappeared completely. Therefore, the glycoside analysis method using the reversed-phase high performance liquid chromatography and the pulsed electrochemical detector of the present invention is considerably selective for glycosides when analyzing a sample containing complex glycosides, non-saccharides, or other non-glycosides. Can be.

Example 3: Glycoside Selectivity Experiments on Sugars, Amino Acids, Sugar Alcohols, and Ginsenoside Mixtures

Current materials that can be detected by analyzing the gold electrode of the pulse type electrochemical detector include carbohydrates such as monosaccharides and polysaccharides, amino acids and sugar alcohols. In order to confirm the selectivity of glycosides, 10 sugar mixtures, 40 amino acid mixtures, 3 sugar alcohol mixtures, and ginsenosides were mixed and applied to high performance liquid chromatography. The chromatogram is shown in FIG. 5 and ginsenosides were clearly separated from sugars, amino acids, sugar alcohols, etc. which do not stay in the C-18 column. When quantifying ginseng and ginseng preparations by absorption spectrophotometry, pretreatment such as saturated butanol extraction or solide-phase extraction was required to remove the interference components, but when quantifying them by pulsed electrochemical detector method Complex pretreatment was omitted because they were not affected.

As described above, the glycoside analysis method of the present invention is simple because no pretreatment step is required, and shows high selectivity, good sensitivity, and reproducibility with respect to the glycoside. The glycoside analysis method of the present invention will be particularly useful for the analysis of preparations containing complex components and can contribute to the quality evaluation of herbal preparations and thus have high industrial applicability.

1 is a structural formula of glycosides and non-glycosides ((A) ginsenosides, (B) aspartaloside IV, (C) D-amygdalin, mandellonitrile ( mandelonitrile), (D) schizandrin and (E) naringin, naringenin).

2 is a schematic diagram of a reverse phase high performance liquid chromatography and pulsed electrochemical detector (RP-HPLC-PAD) method.

Figure 3 is a diagram comparing the chromatogram of the ultraviolet detector for ginseng and Sagunja-tang and the pulsed electrochemical detector of the present invention ((A) ginseng extract obtained by the ultraviolet detector at 203nm wavelength, (B) ultraviolet at 203nm wavelength Sagunja-tang obtained by detector, (G) Ginseng extract obtained by pulsed electrochemical detector and Sagunja-tang obtained by (D) pulse electrochemical detector Peak 1: G-Rg ₁ , 2: G-Re, 3: G-Rf, 4: G-Rb ₁ , 5: G-Rc, 6: G-Rb _2. 7: G-Rd, IS: AST IV).

4 is a chromatogram of the ultraviolet detector and the pulsed electrochemical detector of the present invention for the simultaneous analysis of glycosides and non-saccharides ((A): amygdalin (amygdalin), mandel obtained through the ultraviolet detector at 214 nm) Chromogram (mandelonitrile) and schizandrarin (schizandrin), (B): chromatogram obtained by pulsed electrochemical detector (six-potential), (C): naringin obtained by ultraviolet detector at 280 nm chromatograms of naringin) and naringenin and (D): chromatograms obtained via pulsed electrochemical detector (six-potential) (E): chromatograms obtained by pulsed electrochemical detector (quadruple-potential) Peak 1: amygdalin, 2: mandelonitrile, 3: schizandrin, 4: naringin, 5: naringenin.

5 is a chromatogram of sugar, amino acid, sugar alcohol and ginsenoside mixtures analyzed by glycoside analysis method of the present invention (Peak 1: G-Rg ₁ 2: G-Re, 3: G-Rf, 4: G -Rb ₁ , 5: G-Rc, 6: G-Rb ₂ , 7: G-Rd, 8: carbohydrates, free amino acids mixture and sugar alcohols, IS: AST IV).

Claims (9)

As a method of analysis using reverse phase high performance liquid chromatography and pulsed electrochemical detector, (a) passing the sample through a reversed phase column using an organic solution that is a first eluent; (b) mixing the eluent passed through the column with the strong base solution, which is the second eluent, using a T-shaped mixer; And (C) a glycoside analysis method comprising the step of detecting the glycosides using the pulse type electrochemical detector in the eluent mixed in step (b). The method of claim 1, wherein the glycoside is ginsenoside. The method of claim 1 wherein the reversed phase column is a C 18 column. The glycoside analysis of claim 1, wherein the first eluent has a sequential gradient elution in which the volume ratio of the 10% acetonitrile solution and the 60% acetonitrile solution is 80:20 to 60:40 and again 80:20. Way. The glycoside analysis method according to claim 1, wherein the second eluent uses sodium hydroxide solution. The method of claim 1, wherein the flow rate of the first eluent is 0.2 mL / min. The glycoside analysis method according to claim 1, wherein the flow rate of the second eluent is 0.8 mL / min. The method of claim 1, wherein a six-potential waveform is used as a pulsed electrochemical detector. 7. The six-potential waveform of claim 6 wherein each potential has a potential of E1 = -0.2V, E2 = 0V, E3 = + 0.22V, E4 = 0V, E5 = -2V and E6 = +0.6 Glycoside analysis method characterized in that V.

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