JPH0238858A - Monosaccharide analysis for dabsylhydrazine - Google Patents

Abstract

PURPOSE: To enhance accuracy and reproducibility by taking advantage of reaction between dansyl hydrazine and monosaccharide (possibly existing in a biological specimen of fruit, serum or other humor) in an acidic solution. CONSTITUTION: A monosaccharide reacts easily on dansylhydrazine to produce dansylhydrazine of various kinds of monosaccharides and the mixture thereof is proportional to the initial concentration. Production rate of hydrazine is reduced significantly in a solution of low acidity and when the system is heated up to 60 deg.C, hydrazine is produced completely in 60min. Since the dansylhydrazine of saccharide is significantly stable in an acidic solution, it can be employed in the analysis of high efficiency liquid phase chromatography or thin film chromatography.

Description

DETAILED DESCRIPTION OF THE INVENTION [III Field of the Industry] The present invention relates to the chromatographic analysis of monosaccharides using dabnorhydrazino.

: Conventional technology] Dabsylhydrazine
) easily reacts with various moieties including glucose, milk sugar, mannan, kinloose, arahia sugar, cough tong, deoxidized nucleic sugar, and propylene aldehyde, producing various double hydrazones. This compound has a very strong absorption at 425ni.

Such coloring agents can be used in monosaccharide analysis methods using thin layer chromatography and high efficiency liquid phase chromatography.

Dabnorhydrazino has the following structural formula.

4-((,1,-(dimethyl)aminohenzeno]-abhenzene)zulponylhydranone.

Currently, there is no such method.
If (I h' - and iil in the otsu kind fruit juice)
fI'J Analyze the sugar concentration, do something like that. The milk sugar and mannan content extracted after serum glycoprotein hydrolysis can be assayed in one minute using this newly developed dabsylhydrazo method.

Since 1B sugars are generally present in the biosphere, the search for stable and quantitative analysis of carbohydrate compounds in samples has become a challenging topic for many biochemistry researchers. It has become. Recently, several new methods have been developed for the analysis of these 11 quantities. Is gas-liquid phase chromatography even simpler after some improvements? It is now possible to analyze up to 100% of nanometers, and the sensitivity is even higher. (Vawhinney, T.P.
, ; Feathers, M, S, ; Barher
o, G, J,; Marthez, J, R,
Anal, BIochea+, 19go, 1
01 112-117. ). Mr. using an anion exchange resin in high performance liquid phase chromatography (II P 1., C).

chek, J. E.; Dinsmore, S.
R; Vaalkes, T., PChin, Chc.
m, (Winskon-Sales, N, C,)
1975.21゜1314-1322, (Kcsl
er, R, B, Anal, Chem, 19673
9, 1416-1422. ) or to connect the phase Noricon rubber tube column 1.1nden, J, C,: i, a
vhead, C, L, J, Chr.

IIlatogr, 1975.105.125-13
3. XRabel, F, M,: Caputo, ^,
G: Butts, E, T, J, Chrom
aLogr, 1976+26731740, Bind
er, H, J, Chromatogr,! 9801
89414-420), the sample preparation steps can be reduced, greatly increasing the area of use. However, the refractive index and sensitivity of ultraviolet absorption spectrometers are quite low for simple sugars. Researchers have previously used dansylhydrazine to attach at the reducing end of sugars to create fluorescent labels (A
vigad, G, J, ChromaLogr, 1
977, 139, 243-247), that is, the monosaccharide dabcylhydranone, was produced and used for the analysis of )l PLC. (Alp
enfeis, W, F, Anal, Biochem
The fluorescence emitted by such dansyl derivatives is somewhat unstable, thereby reducing its accuracy and reproducibility.

Clearly, saccharide analysis is in great need of stable chromatographic reagents.

[Three problems to be solved and their means] When dabcylhydranone is reacted with a monosaccharide, the monosaccharide reacts with dabcylhydranone very easily, producing various monosaccharides of dabcylhydranone, and the mixture is It is directly proportional to its initial concentration (as shown in the figure). In less acidic solutions, the rate of hydrone formation is greatly reduced, reducing the system up to 60° (゛1JII M-4
Then, hydrazone is completely generated within 60 minutes. The sugar dabcylhydranone is very stable in acid solution and can be used in high efficiency liquid phase chromatography or thin layer chromatography.

Many: Reducing sugars (e.g. glucose, milk sugar, mannan,
Arahia sugar, quinolose, nuclear sugar, deacidified nuclear sugar, I-t
, Yamato )'s house [The hydracin that has been removed has a degree of mobility that can be applied to the silicone rubber fibers. ＃Rixocarbon sugar I [
The production rate and yield of the hydrazone is similar to that of glucose, and 11 ura sugar and pentose sugar (quinolose arabiale 11, nuclear kq, acetic acid (containing sugar) are converted to dab, /ruhydranone under the above conditions. The reaction with hydrazone is extremely fast, and it also generates Okawa's hydrazone.If the heating time is extended and the hydrazone is kept at room temperature, the yield of hydrazone will still increase, but the fact that it will require 5 reactions is that hydrazine The action of fructose and dabcylhydranone is extremely slow when the conditions for generating -3' are set at a pH value of 4 or higher; It can promote the reaction of fructose.

<Example 1> Glucose in grape juice is analyzed by high-efficiency liquid-liquid chromatography. The original version of Califusnia Green Grapes (a sweet seedless variety) is obtained by gentle pressing. Completely freeze-dry 0.4 ml of the clarified juice in a glass bottle. The remaining residue was dissolved in 2++ liters of warm absolute alcohol (50
℃) and collect 4 millipores (iillipore
) Filter through a filter film (045ia). React the filtrate of 111 and IH dabcylhydranone in the alcohol of 11 at 60°C for 1 hour. The reacted mixture was further cooled to room temperature (2, filtered once through Millipore filter film, 1-
10 ml of filtrate is injected into a high 2 IJ rate liquid phase chromatograph and analyzed. Besides this, 0. An alcoholic solution of the Img/it glucose standard is run through this procedure and serves as a control.

The fruit juice of the pond, pineapple, pear, watermelon, nana and lily are all homogenized and analyzed using the same procedure.

Example 2 The procedure for analyzing human serum using high-efficiency liquid phase chromatography is to first homogenize human serum (0,2+al) using 5II absolute alcohol to remove proteins. The mixture is allowed to stand at room temperature for 30 minutes and then passed through a millibore filter (0.45++ m). I m1if! Take the liquid 51
Mix with absolute alcohol and pass through the millibore filter again.

Take the filtrate and add 1 + ag of dabcylhydrazine.
React for 1 hour in lIl alcohol and write at 60°C. Adding 0.1% acetic acid to the mixture clearly accelerates the reaction. After the reaction, the mixture was kept at room temperature and passed through a millibore filter membrane. Finally, l of 1-5ii! The liquid is taken and injected into high-efficiency liquid phase chromatography for analysis. An identification class glucose (0°1~1mg/g+I) alcohol solution and double hydrazine are operated in the same manner, and this is used as a standard product for analysis by high-efficiency liquid phase chromatography.

<Example 3> Serum glycoproteins such as milk sugar and mannan were analyzed using a high-efficiency liquid phase analysis method.
) etc. Purify using method (2). However, some minor corrections will be made.

Human body surface i'ff (0.05m1) and 9 of 0.55a+l
Add 5% alcohol to Ebendorf (F, ppendorr)
Mix thoroughly in >H, centrifuge at 5000 rpm for 15 minutes, wash the precipitate at the bottom of the tube with 0.6+Al alcohol, and add 0.1ml of 0. Dissolve in I N sodium chloride solution, mix the mixture with 0121 double distilled water and 30 ml concentrated malic acid (approximately 12 N), and add 5 ml of nitrogen gas.
Degas for a minute, then cover the septum and seal tightly. Thereafter, hydrolysis was carried out in an electric dryer at 100° C. for 4 hours, and 0.01 ml of the hydrolysis solution was taken out and freeze-dried. The residue was reacted with an alcoholic solution containing 1ii of IB gubnorhydranone, and 60
Hold at ℃ for 1 hour. The reaction mixture is passed through a millibore filter membrane (045NMIIAW P ) at room temperature. Finally 1i
The filtrate of i is taken and analyzed by high-efficiency chromatography.

The equipment used in high-efficiency liquid phase chromatography is a Waters company, a model 6000 double pump solvent transfer system, a model 06K manual TT gun,
Model 450 absorption measuring instrument (tL length is 425 n1Bj
:Includes Model 660 /1g cutting transportation pattern and system. The recorder is a model B5000 (Texas Austin Hyuston Equipment) Omnisc.
ribe) It is a long paper marking device. The analysis 71 note used is Nova-P~K Cl8 (3,91
1+l'yKc111), iff element 4nm or μBo
nd -apak Cl8(4+u+X 30 cta
), particles - reverse phase tube columns of about 10 nm. Concave line colored line pattern (curve, line 7 or 8) = A solvent, water methyl noanide (78/22 body ratio), [3 dissolved? I, methylnoanide (10-85%, 45 min or 15-85%, 4
5 minutes). The flow rate under general conditions is 1.2II+l
/min, AUFS (1α during absorption of all gauges) is 0.02~
0.04゜〈Actual Example 1〉 Separate the monosaccharide dabnorhydrazone by thin layer chromatography. As mentioned above, the monosaccharide dabcylhydrazine is produced by reacting various monosaccharides with dabcylhydrazine. Reaction mixture (2~lOn+o.

(containing 05 to 100 μmol of sugar) was applied to the starting point of the rubber plate and placed in the mouth according to the procedure described in the experimental part.
The nrla of dabnorhydrazone of these sugars, which advance the foliage, is listed in Table 1. -1 hydrazone glue (J
Chromatographic properties can be provided on a thin plate to quickly measure the signature of these monosaccharides; the chromatographic points on the plate are within the visible light range, and the lowest 1811 constant limit is 0. 1 to 10 μmol of glucose dabcylhydrazone.

Table 1 At layer chromatographic data of monosaccharide-derived dabnolhydrazone Saccharide dabnolhydrazone@sugar Milk sugar Manonano sugar fructose Nucleic sugar Removed acid Nucleic sugar Quinolose sugar Arabic sugar 1 to oil sugar a, solvent system: A , I butyl alcohol (saturated water)
-diethylamine (30.1 volume ratio): B, noazmethane-1-butylcol-ethane (20:2:1 volume ratio):
0, Noatsumethane-benzene-ethyl acetate (l 5:I
5:1 volume ratio) 2I], anomethane butyl alcohol-ethyl acetate (20:2:1 volume ratio), E, benzene-chloroform-ethyl alcohol (15:l 5.
10 volume ratio). The Rr value is obtained by averaging three measurements.

The linear relationship and sensitivity of the quantitative analysis is one simple, reproducible, and highly sensitive method for measuring the monosaccharide dabcylhydrazone using visible light absorbance. A mixture produced by reacting eight types of standard sugars with different concentrations and dabcyl hydranone, and analyzed by high-efficiency L& chromatography, the results are shown as the absorbance of six types of monosaccharide derivatives and This shows that there is a good linear relationship between the a changes.

The hydrazone produced after reacting dabcylhydranone with a mixture containing heterogeneous alf was analyzed by high-efficiency liquid phase chromatography in the experimental part. In the figure, the dissimilarity rate of the valley return line probably indicates that the dissimilar M-induced reaction has dissimilar equilibrium constants. However, this good linear relationship indicates that this method can be used for quantitative analysis.

We have discovered that the PLL value during reaction of a mixture influences the production rate of hydrazone. The best pu range for the reaction is p
H is around 2, as shown in FIG. It is worth noting that the production rate of glucose dabcyl hydrazone when pll is 2 is 11 times faster than when pH is 7. However, the reaction between liquid sugar and Arabic sugar is p+-t or 3-6
When it's in between, it doesn't have much of an effect.

After derivatizing 100 mmol of monosaccharides in the method described above, the absolute sensitivity of the method is determined. This method uses −
The actual minimum measurement limit for facet sugar or pond sugar determined in this way is approximately At 10 mmol (2 μg), such sensitivity can be said to be as good as, or even better than, that of a gas-liquid phase chromatograph f.

, the accuracy and yield of the invented analytical method and the accuracy of this method are the same on the same day! I@sugar dabnorhydrazone (glu
cascdat>5ylhydrazone) The solution is repeatedly operated to t! ＞ j This is the case. A 100 μm sample was injected to determine the coefficient of variation (Coeffi).
client or variation) is l, 7
'% (repeated 6 times), Ura Shusuke (0°2) was added to the grape juice.
20 μg added to 11 fruit tV) and measured reaction 2
2) The yield is 97 to 102% (repeated 7 times) 6 The analysis method of the present invention is used to measure blood-borne insects in human serum, as described in Mentsu. be. Measured Ai+R
, pineapple, banana, pear, watermelon/')
mj sucrose a If ha+'! i 5, 7, l015
．． 109.55.4.3 and 2.9 mg/g.

[Brief explanation of the drawing]

Figure '55I is a diagram showing the relationship between the concentration and absorbance of three types of monosaccharide derivatives according to the analytical method of the present invention, and Figure 2 is a diagram showing the relationship between pH 1a and the monosaccharide dabcylhydrazone produced in the reaction of the present invention,
I) It is a figure showing the relationship with the absorbance of 425nn. Figure 2

Claims (1)

[Scope of Claims] 1) A method for analyzing monosaccharides by thin layer chromatography or high-efficiency liquid phase chromatography, which utilizes a reaction between dabcylhydrazine and monosaccharides in an acidic solution. 2) The monosaccharide analysis method according to claim 1, wherein the monosaccharide can be present in biological specimens such as fruit, serum or other body fluids.