JPS58187857A - Analyzing method for sweetening component of stevia leaf - Google Patents

Abstract

PURPOSE:To enable the simultaneous quantification of six sweetening components of leaf with high sensitivity in one column, by using a high-speed liquid chromatography having a moving phase prepared by adding a small quantity of salt to a mixture solvent of acetonitrile/water and a fixed phase of amino-base- combined type silica. CONSTITUTION:The moving phase of a high-speed liquid chromatography is prepared by a method wherein a phosphate such as potassium dihydrogen phosphate, sulphate or the like is added to the mixture liquid of acetonitrile/water in the volume ratio of 75/25 to 90/10 so that the added salt accounts for 1- 5millimol/l. For the fixed phase thereof, amino-base chemical combination type silica (PNH2-10/S2504 prepared by Shimazu Seisakusho) is used. Components extracted from stevia leaf are quantified by the high-speed liquid chromatography thus prepared. By this method, it becomes possible to separate and quantify precisely the rebaudeoside B and steviol bioside from other stevioside, rebaudeoside A and C, and zurcoside A which can not be separated and quantified by conventional methods, together with the other four components.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for steady-state analysis of the main components of 6 maple extracted from stevia leaves.

Stevia leaves, which are the raw material for this invention, are Stevia rebaudiana, a plant of the Asteraceae family native to Paraguay in South America.
-(3tevia Rebaudiana Berto
ni) are the leaves of a shrub. Stevia leaves contain several types of sweetening components, including stevioside, which is famous as a natural sweetener and is said to be 800 times sweeter than sugar. Therefore, extracts from the leaves have been used as a harmless sweetener since ancient times, and in addition to being used in foods, with the regulation of synthetic sweeteners in Japan, it has attracted attention as a highly stable, low-calorie natural sweetener. It's starting to happen.

Against this background, it is necessary to know the quality of the sweet component of Stevia leaves, that is, the content of the sweet component. Conventionally, high-performance liquid chromatography using acetonitrile/water-based solvent mixture was performed using amino group chemically bonded silica as a packing material. I was worried. However, the conventional method has the drawback that only four of the six major components can be analyzed simultaneously.

The six components analyzed in this invention are stevioside (symbol: (I)) and rebaudioside A.

B and C (&3 (II), (lit) and (IV)
), dulcoside A (symbol: (V)), and steviolbioside (symbol: (Vl)). When performing the conventional method, that is, high performance liquid chromatography using an acetonitrile/water mixed solvent, (I)
, (11), (lV), (Y) (7) Minamata G-1 (
iiD, (■) Separation Hachikiru separates 6 components at the same time,
cannot be analyzed. (The reason why the surface and (■) are strongly retained by the filler is thought to be because both have carboxyl groups in their molecules, which cause an ion exchange effect with the amine groups of the filler stationary phase.

Under these circumstances, the inventors of the present invention discovered a method for simultaneously analyzing and analyzing six sweet components of stevia leaves, and thus accomplished the present invention. In this invention, the sweet component of stevia leaves is subjected to high performance liquid chromatography using a mixed solvent of acetonitrile/water and salts added in a saucer as the mobile phase and silica chemically bonded with amino groups as the stationary phase to obtain stevioside and red. This invention provides a method for analyzing the sweetness components of Stevia leaves by simultaneously placing six components: Baudioside A, B, and C1, Dulcoside, A1, and Steviol Bioside.

Examples of the low-volume salts to be added include phosphates, sulfates, etc., and dihydrogen phosphate is preferred. Further, the concentration is adjusted so that the mobile phase contains 1 to 5 mmol/l. The mobile phase acetonitrile/water mixing ratio is influenced by the type of column stationary phase, but is generally about 75/2.
5 to 90/10 is used. The separation power tends to decrease as the acetonitrile becomes lower than 75 parts by volume, while the separation time tends to increase as the volume exceeds 90 parts by volume.

According to the present invention, it is possible to simultaneously analyze six components of Stevia leaf sweetness by EJ, and it is possible to eliminate operational steps such as having to use different mobile phases to analyze the six components.

Next, the present invention will be explained in more detail using comparative examples and examples.

At a column temperature of 40C, acetonitrile/water = 60/40.70/80 and 80/20 as mobile phase.
Using a solvent with a mixing ratio of 1. The eluate was separated by high-performance liquid chromatography at a flow rate of 1 min.
UV detection was performed at m (U■ detector 5PD-2A). The results are shown in FIG. The vertical axis of the graph is the volume ratio (k'), and the horizontal axis is the mixing ratio of the solvents. Acetonitrile/water = 6
At 0/40 or 70/:110, (III) and (■) are eluted and separated, but the other four components (I), (■), (IV),
(V) does not separate, and at 80/20, 4 components (I),
(b), ff), and (V) can be separated by elution, but the two components (l
It is understood that [I) and (■) are not eluted.

Example - Inject the sample into the column of PNH2-10152504,
Using a mixed solvent of 82 parts by volume of acetonitrile and 18 parts by volume of water to which potassium dihydrogen phosphate was further added at a concentration of 8 mmol/l as the mobile phase, the column temperature was 40.
°C, 2. Separation was performed by high performance liquid chromatography at a flow rate of Od1 min, and the eluate was subjected to UV detection with 200 nIn (U■ detector 5PD-2A). The results are shown in FIG. The vertical axis represents the elution amount, and the horizontal axis represents the elution time.

Six components of stevia leaf sweetness were simultaneously analyzed with excellent sensitivity.

[Brief explanation of the drawing]

Figure 1 is a graph showing the state of separation by the conventional method;
The figure is a high performance liquid chromatogram according to the present invention. In Figure 1, the vertical axis is capacity ratio (k'), and the horizontal axis is acetonitrile/
The mixing ratio of water and the horizontal axis in FIG. 2 indicate the elution time. Figure 1 shows '1 □ Volume mixing of acetonitrile 77 ↓ Figure 2

Claims (1)

[Claims] l. The sweet component of Stevia extract is subjected to high-performance liquid chromatography using a mixed solvent of acetonitrile/water and a small amount of salt as the mobile phase and silica chemically bonded with amino groups as the stationary phase. A method for analyzing the taste components of stevia leaves, characterized in that six components of stevioside, rebaudioside A, B, and C1 dulcoside A1 steviol bioside are simultaneously established. 2. The method according to claim 1, characterized in that the salt phosphorus [j is added to the mobile phase so as to contain 1 to 5 mmol/l.