JP2008170428A - Hplc analysis for sugar and sugar-alcohol - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a simultaneous analytical method for sugar and/or sugar-alcohol capable of benzoylating the sugar and/or sugar-alcohol in a sample, and capable of removing a contaminant by solid phase extraction to be separation-analyzed by a reversed-phase high-performance liquid chromatography. <P>SOLUTION: The sample containing the sugar and/or sugar-alcohol is benzoylated in this analytical method for analyzing the sugar and/or sugar-alcohol in the sample, the contaminant other than the sugar and/or sugar-alcohol is removed by the solid phase extraction without using liquid-liquid extraction (vacuum drying-up), the sugar and/or sugar-alcohol in the sample is separation by the reversed-phase high-performance liquid chromatography, and ultraviolet absorption is measured to be analyzed. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for analyzing sugars and sugar alcohols in the chemical field and the medical field, and more particularly to a method for analyzing sugars and sugar alcohols present in trace amounts in biological samples and foods.

  In recent years, with the generalization of the concept of metabolic syndrome, interest in diabetes has increased, and research on the metabolism of sugar present in the living body has attracted attention. The amount of sugar present in the living body is extremely small (on the order of ppm), and techniques for qualitative and quantitative determination at that level have not been generalized.

  Sugars and sugar alcohols do not have UV absorption and cannot be detected with an HPLC system equipped with a normal UV detector. Therefore, when a trace amount sugar and sugar alcohol are quantified by HPLC, the further device is needed. Microanalysis of sugars and sugar alcohols by current HPLC is a method in which sugars and sugar alcohols are ionized under strong alkali, separated by HPLC using an ion exchange column, and detected by an electrochemical detector (Non-patent Document 1). And a method of reacting a fluorescent substance after separating sugar and sugar alcohol with a reverse phase or normal phase column and detecting with a fluorescence detector (see Non-Patent Document 2).

  However, in order to perform these methods, it is necessary to prepare special equipment. Moreover, since analysis is performed under strong alkali, it is dangerous.

  On the other hand, when sugars and sugar alcohols are benzoylated, they have UV absorption and can be detected with a normal UV detector. This principle can be applied to HPLC to analyze sugars and sugar alcohols. (See Non-Patent Documents 3-8). The detection of sugar and sugar alcohol by this method is excellent in convenience because it can be carried out using ordinary HPLC.

Watanabe N et al. J Chromatogr 330; 333-338, 1985 Yuh YS et al. J Pharm Biomed Anal 16; 1059-1066, 1998 Oehlke J et al. J Chromatogr B Biomed Sci Appl 655; 105-111,1994 Jentofft N et al. Anal Biochem 148; 424-433, 1985 Dethy JM et al. Anal Biochem 143; 119-124, 1984 Daniel PF et al. Carbohydr Res 97; 161-180, 1981 Nojiri S et al. J AOAC Int 82; 134-140, 1999 Kwang-Hyok S et al. Clinica Chimica Acta 354; 41-47, 2005

  The present inventors tried to analyze sugars and sugar alcohols according to Non-Patent Document 8, but it was extremely difficult to accurately analyze trace amounts of sugars and sugar alcohols present in the order of ppm in the sample. Furthermore, in the method based on benzoylation as described in Non-Patent Documents 3 to 8, it is necessary to use an organic solvent such as ethyl acetate when recovering the benzoylated sugar. In this case, it was found that organic solvents and organic substances in biological samples were mixed into the HPLC sample, which caused baseline instability, column contamination, and the like. In particular, baseline instability was fatal for the determination of trace substances, which was considered to be the reason why this method was not common.

  The problem to be solved by the present invention is to improve the problem of saccharide microanalysis based on benzoylation, which has the feature of excellent convenience, and to analyze sugars and sugar alcohols existing in the order of ppm in vivo. It is possible to provide an analysis method that is possible and convenient.

  The present inventors, after benzoylating a sample containing a sugar component, by using a solid-phase extraction cartridge (by performing solid-phase extraction), an HPLC sample with less contamination without using an organic solvent Enabled the preparation of Furthermore, by reducing the contamination, it is possible to use high resolution columns with a high particle number of 3μm and high theoretical plate number (50000 plates / tube) for HPLC analysis. As a result, the inventors have obtained a new finding that extremely high sensitivity, that is, a small amount of a sugar component present in a sample can be accurately analyzed. These two improvements made it possible to perform stable sugar microanalysis that can be performed with ordinary HPLC equipment.

That is, the method of the present invention is as follows.
[1] A method for analyzing sugar and / or sugar alcohol in a sample, wherein a sample containing sugar and / or sugar alcohol is benzoylated, and other than sugar and sugar alcohol by a solid phase extraction method without using an organic solvent. A method for analyzing sugar and / or sugar alcohol in a sample, comprising removing impurities, separating sugar and / or sugar alcohol in a sample by reversed-phase high performance liquid chromatography, and measuring and analyzing ultraviolet absorption.
[2] The method for analyzing sugar and / or sugar alcohol in a sample according to [1], wherein protein removal treatment is performed before benzoylating the sample.

[3] The sugar and / or sugar alcohol in the sample is one or more sugars or sugar alcohols selected from the group consisting of ribose, mannose, glucose, fructose, myo-inositol, mannitol and sorbitol [1] or [2] Of analyzing sugar and / or sugar alcohol in a sample.
[4] A method for analyzing sugar and / or sugar alcohol in a sample according to any one of [1] to [3], wherein the sample is a biological sample.

[5] The solid phase extraction is performed using a stationary phase for reverse phase chromatography or a stationary phase for ion exchange chromatography. The sugar and / or sugar alcohol in the sample of any one of [1] to [4] is analyzed. how to.
[6] The solid phase extraction is performed using a stationary phase having an octadecylsilyl group or an octyl group, and the elution of sugar and / or sugar alcohol is performed using acetonitrile. [5] The sugar and / or sugar in the sample of [5] How to analyze alcohol.

[7] Reversed phase high performance liquid chromatography is performed using a column containing a packing material having an octadecylsilyl group or an octyl group, and elution of sugar and / or sugar alcohol is performed using acetonitrile. [6] The method for analyzing a sugar and / or sugar alcohol according to any one of [6].
[8] The method for analyzing sugar and / or sugar alcohol according to [7], wherein reverse phase high performance liquid chromatography is performed using a column containing a packing material having an octadecylsilyl group having a particle size of 3 μm.
[9] A method for analyzing the sugar and / or sugar alcohol according to any one of [1] to [8], wherein reverse phase high performance liquid chromatography is performed by isocratic elution.

[10] In the method for analyzing a sugar and / or sugar alcohol according to any one of [1] to [9], a benzoylated sugar and / or sugar alcohol organic solvent separated by reverse phase high performance liquid chromatography is used as a solid phase. A method of concentrating benzoylated sugar and / or sugar alcohol in a sample at a high recovery rate by exchanging the organic solvent with a concentration of 100% using an extraction method and evaporating the organic solvent.

[11] A method for analyzing sugar and / or sugar alcohol in a sample, comprising analyzing the sugar and / or sugar alcohol concentrated by the method of [10] by NMR.
[12] In the method of any one of [1] to [9] and [11], a compound having an OH group in a sample is benzoylated instead of sugar and / or a sugar alcohol, and the sample has an OH group in the sample A method for analyzing compounds.
[13] A method for concentrating a compound having an OH group in a sample, wherein the compound having an OH group in a sample is benzoylated instead of sugar and / or sugar alcohol in the method of [11].

[14] A method for concentrating sugar and / or sugar alcohol in a sample, wherein the sample containing sugar and / or sugar alcohol is benzoylated and subjected to a solid-phase extraction method using an organic solvent without subjecting to vacuum drying. A step of removing impurities other than sugar and sugar alcohol, a step of separating sugar and / or sugar alcohol in a sample by reversed-phase high performance liquid chromatography, and using a solid phase extraction method for the separated sugar and / or sugar alcohol solvent. In this method, the benzoylated sugar and / or sugar alcohol in the sample is concentrated at a high recovery rate by exchanging with 100% organic solvent and evaporating the organic solvent.
[15] A method for analyzing sugar and / or sugar alcohol in a sample, comprising analyzing the sugar and / or sugar alcohol concentrated by the method of [14] by NMR.

  According to the method of the present invention, a stable HPLC baseline can be obtained, and sugars and sugar alcohols present in trace amounts in a sample can be analyzed with high sensitivity.

  According to the method of the present invention, sugars such as ribose, mannose, glucose, fructose, and myo-inositol, and sugar alcohols such as mannitol and sorbitol in various samples such as biological samples are not subjected to the conventional gradient method, but the isocratic method. Can be analyzed quickly.

  By the method of the present invention, it is possible to simultaneously analyze the above sugars and sugar alcohols in one analysis. In particular, glucose, mannose, fructose, myo-inositol, sorbitol and the like in a biological sample can be analyzed at a time. For example, sugars and sugar alcohols in erythrocytes can be analyzed at once.

By the method of the present invention, the sample to be measured can be prepared more easily than the conventional method.
Moreover, since analysis is possible if there is a HPLC apparatus equipped with a commonly used UV detector, analysis can be performed with good convenience.

Furthermore, the method of the present invention has a low running cost.
By this method, a trace amount of sugar contained in a living body can be efficiently collected and concentrated, and structural analysis such as NMR can be performed.

Hereinafter, the present invention will be described in detail.
The analysis method of the present invention is characterized in that sugar and / or sugar alcohol contained in a trace amount in a sample can be analyzed with high sensitivity.

  In the present invention, the analysis sample is not limited, but includes biological samples, food additives such as foods, food materials, beverages and supplements, pharmaceuticals, quasi drugs and the like. Examples of biological samples include whole blood, serum, plasma, red blood cells in blood, and various tissue samples. Examples of foods include food extracts, brewed foods, and fermented foods.

  The sugar and sugar alcohol that can be analyzed by the method of the present invention are not particularly limited. Specifically, the sugar includes monosaccharides such as glucose, galactose, fructose, ribose, ribulose, maltose, lactose, sucrose, and the like. And polysaccharides such as oligosaccharides such as disaccharides, fructooligosaccharides, galactooligosaccharides, and dairy oligosaccharides. However, since disaccharides and higher are insoluble when subjected to benzoylation in an aqueous system, it is necessary to set analysis conditions different from those for monosaccharides. A person skilled in the art can appropriately determine the analysis conditions for a saccharide more than a disaccharide. Also included are amino sugars such as deoxyribose and fucose. Examples of the sugar alcohol include sorbitol, mannitol, galactitol, maltitol, lactitol, inositol, myo-inositol, and the like. In particular, it can be preferably used for analysis of samples containing ribose, mannose, glucose, and fructose as sugars, and myo-inositol, mannitol, and sorbitol as sugar alcohols, and these seven types can be analyzed simultaneously. .

In the method of the present invention, the sugar and sugar alcohol to be analyzed are benzoylated before analysis. Benzoylation refers to introduction of a benzoyl group (C ₆ H ₅ CO-) mainly into the hydroxyl group of sugar and sugar alcohol. Benzoylation can be carried out by a known method, for example, using benzochloride or nitrobenzochloride.

  In the method of HPLC UV detection of sugar by benzoylation, when a sample such as a biological sample containing a large amount of protein is used as the sample, the protein is also benzoylated. For this reason, since contamination with benzoylated protein affects the analysis of sugar and sugar alcohol, it is necessary to remove the protein from the sample in advance.

  Deproteinization can be performed by various known methods, for example, deproteinization due to protein denaturation or the like. In this method, acids such as perchloric acid, trichloroacetic acid, and metaphosphoric acid are added to the sample, or organic solvents such as acetone, acetonitrile, methanol, and ethanol sugar are added to the sample to denature the protein, and the denatured protein is filtered. It can be achieved by removal by treatment or centrifugation. In addition, the protein may be physically deproteinized by ultrafiltration, gel filtration, dialysis, ultracentrifugation, or the like.

  Another feature of the present invention is to perform protein removal treatment as necessary as described above, and to remove impurities other than sugar and / or sugar alcohol contained in the benzoylated sample by solid phase extraction. is there. Here, solid phase extraction refers to selective use of a specific target component from a sample having a complex composition using a stationary phase (solid phase, adsorbent) such as chemically bonded silica gel, porous polymer, alumina, activated carbon and the like. This refers to a method of extracting, separating and purifying, thereby removing impurities in the sample. In the conventional method of analyzing benzoylated sugars, a pretreatment such as reduced-pressure drying is required. However, in the method of the present invention, the reduced-pressure drying treatment is unnecessary, and solid-phase extraction is sufficient. By solid phase extraction, sugar and sugar alcohol are selectively extracted using the charge and hydrophobicity of benzoylated sugar and sugar alcohol. For solid phase extraction, a stationary phase for reverse phase partition chromatography, a stationary phase for ion exchange chromatography, or the like can be widely used. As stationary phase for reversed phase chromatography, octadecylsilyl group (C18), octyl group (C8), butyl group (C4), trimethyl group (C3), phenyl group (Ph), butyl group, triacontyl group (C30), etc. A filler having an octadecylsilyl group (C18) or an octyl group (C8) is preferable among them. The solvent used for eluting the sugar and sugar alcohol is not particularly limited as long as it does not absorb low wavelength UV. Examples of such include acetonitrile, methanol, and tetrahydrofuran. Acetonitrile is preferred because it does not absorb UV light. Moreover, you may mix and use these extract liquids. When acetonitrile is selected as the eluting solvent, the amount of acetanilyl for eluting benzoylated sugar from the stationary phase is reduced when the octyl group is used compared to the case where the octadecylsilyl group is used as the stationary phase. Therefore, the concentration of the sample is increased and the analysis sensitivity is improved. Therefore, it is more preferable to use a filler having an octyl group as the stationary phase. As the cartridge used for the solid phase extraction, a commercially available syringe type solid phase cartridge or a disk type solid phase cartridge can be used. Examples of commercially available cartridge types include Sep-Pak (registered trademark, Waters), Autoprep (registered trademark, Showa Denko), Discovery DSC-18 (SUPELCO), Discovery DSC-8 (SUPELCO), and the like. .

  When using a stationary phase for ion exchange chromatography, as the anion exchange group, a diethylaminoethyl (DEAE) group, a quaternary aminoethyl (QAE) group having a stronger basicity, etc. are used as the cation exchange group. A carboxymethyl (CM) group, a sulfopropyl (SP) group having a higher acidity, a phosphoric acid group, etc. may be selected.

  In a generally available solid phase extraction cartridge or the like, since the stationary phase for solid phase extraction is dry-packed, it is necessary to add a benzoylated sample after conditioning (washing). Conditioning (washing) of the stationary phase for solid phase extraction can be performed by first washing several times with 100% acetonitrile and then substituting with water. The benzoylated sample is then added to the stationary phase. The amount of the sample to be added can be appropriately selected depending on the size of the stationary phase to be used and the type of the sample to be used. For example, in the case of a blood sample, the benzoylated sample may be added to a 100 to 1000 μL commercially available stationary extraction cartridge. . After adding the sample, the sample is washed with water and the above extraction solution. For example, after washing with water, wash with 40-80% acetonitrile, preferably 60% diluted acetonitrile, then wash with 80-100%, preferably 100% acetonitrile, then extract with further 100% acetonitrile. The collected eluate is taken as a sample. Solid phase extraction can be performed at room temperature, but is preferably performed at 25 ° C. ± 2 ° C.

  In the present invention, the sample eluted by solid phase extraction is then analyzed by HPLC. HPLC is performed by reverse phase or normal phase chromatography. As the column, a column having a polymer gel such as silica gel, alumina, styrene-divinylbenzene copolymer, polymethacrylate or the like and having an octadecylsilyl group (C18) is preferable, but an octyl group (C8), a butyl group (C4), A column having a group such as a trimethyl group (C3) aminopropyl group (NH2) can also be used. Among these, an ODS column (C18 column) having silica gel as a carrier and having an octadecyl group is preferable in that analysis with higher accuracy can be performed. When a C18 column is used, the separation of each sugar peak is improved, and the confidence interval in the analysis is expanded both at the upper and lower limits. In addition, fructose and myo-inositol, mannitol and sorbitol, which cannot be separated by other columns, can be separated by an isocratic method. A commercially available apparatus may be used for HPLC, and a commercially available column may be used. The carrier packed in the column can be spherical or crushed, and its particle size is 3 to 10 μm, and it is required to have a high number of theoretical plates from the viewpoint of high-accuracy analysis. Further, 3 μm is preferable. In the conventional method for carrying out the vacuum drying treatment, when a column having a particle diameter of 3 μm is used, clogging occurs due to impurities in the sample, resulting in an increase in pressure that affects the measurement. On the other hand, in the method of the present invention, since there is a solid phase extraction step in sample preparation, the inflow of contaminants that cause clogging into the column can be minimized. Therefore, it is possible to use a column packed with 3 μ diameter particles for a long period of time without using a guard column. The size of the column to be used depends on the volume of the sample to be applied as a sample. For example, a column having an inner diameter of 4 to 6 mm and a length of 2.5 to 30 cm may be used. Preferably, those having an inner diameter of 4.6 mm and a length of 25 cm are used.

  As the mobile phase in the HPLC analysis, one having no or little UV absorption is used. As such, acetonitrile, methanol, tetrahydrofuran and the like can be mentioned. Acetonitrile is preferable in that there is no UV absorption. Moreover, you may mix and use these extract liquids. The concentration of the mobile phase such as acetonitrile is 60 to 80%, preferably 70 to 80%, more preferably 77.5 to 78%. Although elution may be performed by gradient elution in which the composition and concentration of the mobile phase are gradually changed, an isocratic method that does not change the composition and concentration of the mobile phase is preferable in that it can be rapidly analyzed. The flow rate of the mobile phase at the time of analysis is 0.1 mL / min to 5 mL / min, preferably 0.5 mL / min to 2 mL / min.

  According to the method of the present invention, a sample from which benzoylation and impurities are removed by solid phase extraction can be analyzed in 10 to 60 minutes.

  The injection amount of the sample in the HPLC analysis can be appropriately determined depending on the type of sample and the column to be used, and is several tens of μL to several hundred μL, for example, 10 μL to 200 μL, preferably 10 μL to 100 μL.

  The separated benzoylated sugar and sugar alcohol may be measured for UV absorption using a UV detector. The measurement wavelength is preferably a wavelength near the absorption maximum of benzoylated sugar and sugar alcohol, and is 200 to 250 nm, preferably 220 to 240 nm, and more preferably 228 nm.

  The separated sugar and sugar alcohol are fractionated, and the separated sugar and sugar alcohol can be used as materials for synthesizing drugs and various compounds. Separated sugar and sugar alcohol can be collected, concentrated and analyzed by the following method.

  The benzoylated sugar and / or sugar alcohol fractionated using HPLC is contained in an organic solvent used as a mobile phase such as acetonitrile of 60 to 80%, preferably 70 to 80%, more preferably 77.5 to 78%. Exists. Thus, when it is intended to concentrate benzoylated sugar and / or sugar alcohol in 60-80% organic solvent, concentration from 60-80% organic solvent increases benzoylated sugar and / or concentration as the concentration proceeds. Alternatively, sugar alcohol is adsorbed on the wall of the container, and the recovery rate is deteriorated. This is expected to occur because the organic solvent evaporates first in the concentration step and the organic solvent concentration becomes thin. However, it is necessary to prepare a high-concentration sugar fraction when conducting structural analysis of sugars and / or sugar alcohols using NMR or the like. In addition, when analyzing sugar and / or sugar alcohol contained in a trace amount in a biological sample, it is necessary to prepare a high concentration fraction by concentrating sugar and / or sugar alcohol.

  Therefore, 95% or more, preferably 98% or more, more preferably 99% or more, particularly preferably 100% of the organic solvent in 60 to 80% containing the benzoylated sugar and / or sugar alcohol separated by HPLC. Replace with the organic solvent. The exchange of the organic solvent can be performed by subjecting the sample to solid phase extraction again. In this case, 60-80% organic solvent containing the collected benzoylated sugar and / or sugar alcohol is diluted to prepare a final concentration of 55-65%, preferably 60%, and the preparation solution is solid-phase extracted. Contact with the carrier for carrier, bind to the carrier for solid phase extraction, and then add 100% organic solvent to separate and recover the benzoylated sugar and / or sugar alcohol bound to the carrier for solid phase extraction from the carrier . Thereafter, the benzoylated sugar and / or sugar alcohol is concentrated by evaporating the organic solvent. In this case, the solid phase extraction carrier to be used is as described above. A column having silica gel as a carrier and having an octyl group (C8 column) or an ODS column having an octadecyl group (C18 column) is preferable. preferable. As a carrier for solid phase extraction, a commercially available cartridge type carrier can be used, and examples thereof include Discovery DSC-18 (SUPELCO), Discovery DSC-8 (SUPELCO) and the like.

  Thus, once the concentration of the organic solvent containing the benzoylated sugar and / or sugar alcohol fractionated by HPLC is lowered, the benzoylated sugar and / or sugar alcohol is significantly adsorbed on the wall of the container. It does not occur and binds to a solid phase extraction carrier. Replacing the benzoylated sugar and / or sugar alcohol organic solvent with 100% organic solvent by eluting the benzoylated sugar and / or sugar alcohol bound to the solid phase extraction carrier with 100% organic solvent. Is possible. Concentration of benzoylated sugar and / or sugar alcohol after exchange with 100% organic solvent hardly caused adsorption to the container wall, so the structure of benzoylated sugar and / or sugar alcohol was maintained. As it is, it is possible to concentrate at a high recovery rate while minimizing the adsorption to the wall of the container. For example, when 100% organic solvent is used, it can be recovered with a recovery rate of almost 100%. As a result, according to the series of steps of the present invention, sugar and / or sugar alcohol can be separated from a biological sample and structural analysis using NMR or the like can be performed. In addition, it is possible to purify and concentrate sugar and / or sugar alcohol present in a minute amount in a living body from a large amount of specimen. The present invention also includes a method for isolating and recovering sugar and / or sugar alcohol from a sample.

  Here, 100% organic solvent means a high-purity organic solvent supplied from a reagent manufacturer, and actually includes a solvent having a purity of about 99%.

  In this way, the concentrated benzoylated sugar and / or sugar alcohol can be analyzed by nuclear magnetic resonance (NMR) or mass spectrometry. Moreover, the sugar and sugar alcohol obtained by concentration can be fractionated, and the fractionated sugar and / or sugar alcohol can be used as a material for synthesizing drugs and various compounds.

  The sugar and / or sugar alcohol of the present invention is benzoylated, and impurities other than sugar and sugar alcohol are removed by solid phase extraction without subjecting to vacuum drying, and the sugar and / or sugar alcohol in the sample is separated by HPLC. The method and the method of concentrating after separation by HPLC can also be used for other compounds having an OH group. Examples of other compounds having an OH group include various alcohols, amino acids, and fatty acids. In this case, what is necessary is just to change conditions suitably according to the characteristic of the compound used as object.

  The present invention will be specifically described by the following examples, but the present invention is not limited to these examples.

Example 1 Analysis of sugars and sugar alcohols in normal human erythrocytes Sugars and sugar alcohols in normal human erythrocytes were analyzed.

Sample preparation 2 mL of blood was collected in a blood collection tube containing heparin or EDTA-2Na, and immediately mixed with ice after cooling. At this time, the hematocrit value and the red blood cell count were measured. 1 mL of well-mixed whole blood was accurately placed in a centrifuge tube (15 mL), centrifuged at 4 ° C. and 3000 rpm for 10 minutes, and the plasma was discarded. Washed 3 times with 4 ° C. physiological saline. The first and second washes were performed by centrifuging at 1500 rpm for 5 minutes at 4 ° C. The third washing was performed at 2000 rpm, and the physiological saline in the supernatant was discarded.

Deproteinization of the sample Deproteinization was performed using perchloric acid as follows.
1 mL of 1N perchloric acid was added to the washed erythrocytes, immediately mixed well using a vortex mixer, and allowed to stand for 15 minutes. Subsequently, centrifugation was performed at 4 ° C. and 3000 rpm for 10 minutes, and 0.8 mL of the supernatant was collected in another centrifuge tube. The collected supernatant was neutralized with 380 μL of 1.25 mol / L K ₂ CO ₃ . The mixture was further centrifuged at 4 ° C. and 3000 rpm for 5 minutes, and the supernatant was transferred to another 2.0 mL Eppendorf tube and stored at −80 ° C.
The standard protein for the calibration curve and the water for Blank were also subjected to the above deproteinization operation in the same manner as for erythrocytes.

Benzoylation of the sample The deproteinized sample was benzoylated by the following method.
200 μL each of the standard solution for the calibration curve after deproteinization and the sample were taken in a polypropylene tube with a lid. 1 mol / L KH ₂ PO ₄ 60 μL was added thereto, and lightly mixed with a vortex mixer. Furthermore, 10 μL of benzoyl chloride was added, and the mixture was capped and mixed well. Subsequently, 45 μL of 8 mol / L sodium hydroxide was added and the mixture was capped, and mixed for 50 seconds using a vortex mixer. After mixing, the mixture was allowed to stand for 10 minutes after the addition. After exactly 10 minutes, 45 μL of 16 mL / 100 mL phosphoric acid aqueous solution was added and mixed well using a vortex mixer.

Conditioning cartridges for solid phase extraction
A Teflon disposable valve liner was attached to Discovery DSC-8 (SUPELCO). Extraction was done by natural fall, and after confirming that the fall was gone, we proceeded to the next operation. The cartridge was washed 3 times with 1 mL of 100% acetonitrile. In the first washing, in order to prevent channeling of the filler, the pressure was reduced with a vacuum pump to generate bubbles in the filler. Thereafter, it was washed twice or more with water.

Solid Phase Extraction A benzoylated sample was added to a solid phase extraction cartridge after conditioning. After adding the sample, it was washed twice with 1 mL of water. Subsequently, it was washed once with 0.8 mL of 60% acetonitrile and further washed once with 0.3 mL of 100% acetonitrile. Next, 0.5 mL of 100% acetonitrile was added, and the material eluted at this time was collected and measured by HPLC.

Analysis by HPLC The configuration of the HPLC apparatus used was as follows.
HPLC System Controller by Shimadzu: SCL-10A _VP
UV Detector: SPD-10A _VP
Pump: LC-10AD _VP
Auto Sampler: SIL-10AD _VP
Integrator: C-R8A
GL Science column oven; CO 631C

  The HPLC column is Cadenza (5) CD-C18 (C8 column, particle diameter 3 μm 4.6Φ × 250 mm, manufactured by Imtakt) or Inertsil ODS3 (C8 column, particle diameter 5 μm, 4.6Φ × 250 mm manufactured by GL Sciences). Using.

  50 μl of the solid phase extracted sample was injected, and 77% acetonitrile was used as the mobile phase. The flow rate during HPLC analysis was 1 mL / min. The column oven was set at 25 ° C. Detection was performed by measuring UV absorption at 228 nm. The time taken to analyze one sample was 40 minutes.

  The standard solutions for determining the limit of quantification and linearity of quantification by HPLC analysis are ribose, mannose, glucose, myo-inositol, mannitol, and sorbitol standard products dissolved in water, 0.01, 0.025, 0.05, 0.10. , 0.25, 0.5, 1.0, 2.5, 5.0 and 10.0 μg / mL. Fructose was prepared in double volume. As the standard solution for the calibration curve, 0.5, 1.0, 2.5, 5.0 and 10.0 μg / mL standard solutions (fructose was doubled) were subjected to protein removal treatment in the same manner as the measurement sample. The final concentrations were 0.097, 0.194, 0.485, 0.970 and 1.940 μg / mL (fructose was doubled).

  As a comparative example, a sample prepared by a conventional method was also measured. That is, it measured using the sample benzoylated and dried under reduced pressure. In the conventional method, elution was performed by the gradient method. Hereinafter, a method in which a sample containing sugar and / or sugar alcohol is benzoylated, subjected to vacuum drying and eluted by a gradient method using HPLC is called a conventional benzoylation method.

  Benzoylation is the same as the method used in solid-phase extraction, and the conditions under which measurement sensitivity increases with some investigations, with reference to Kwang-Hyok S et al. Clinica Chimica Acta 354; 41-47, 2005 Was found and then applied to dryness under reduced pressure.

  Vacuum drying was conducted with reference to Kwang-Hyok S et al. Clinica Chimica Acta 354; 41-47, 2005, and several conditions were examined to find conditions for increasing measurement sensitivity. That is, 150 μL of ethyl acetate was added to the benzoylated sample and mixed well by vortexing, and then 100 μL of the ethyl acetate layer was collected in another polypropylene tube and sucked with a vacuum pump for 1 hour or more to dry. Next, a sample redissolved with 100 μL of 100% acetonitrile was used as a sample.

  The gradient conditions were determined through examination and improvement with reference to Kwang-Hyok S et al. Clinica Chimica Acta 354; 41-47, 2005. The analytical column described in Kwang-Hyok S et al. Clinica Chimica Acta 354; 41-47, 2005 and the analytical column used by the inventors are both standard ODS columns and have the same resolution. Though possible, it was not the same, so the optimum condition had to be determined. The conditions were as follows.

  From 0 to 3 minutes, 82% acetonitrile, the next 3 to 36 minutes was 78.5% acetonitrile, and from 36 minutes to 82% acetonitrile step gradient, and the flow rate was 1 mL / min. The analysis time for one sample was 70 minutes.

  FIG. 1 shows a chromatogram when a standard solution containing 0.5 μg / mL of each saccharide is benzoylated, followed by solid-phase extraction and using a C18 column (Cadenza CD-C18) as an analytical column. Ribose, mannose, glucose, fructose, myo-inositol, mannitol and sorbitol added as standard substances in the method of the present invention can be separated and analyzed all at once.

  FIG. 2 shows a chromatogram of normal human erythrocytes. FIG. 2A shows the result of the conventional benzoylation method, ie, benzoylation of a deproteinized sample, and then redissolving the evaporated and dried sample in 100% acetanilyl and analyzing it with Inertsil ODS3, a standard ODS column. . FIG. 2B shows the results of the method 1 of the present invention, that is, a sample obtained by subjecting a deproteinized sample to benzoylation and then solid phase extraction using a C8 cartridge was analyzed with an Inertsil ODS3 column. FIG. 2C shows the result of analysis using Cadenza (5) CD-C18 having a high number of theoretical plates by changing the analytical column to Inertsil ODS3 column in the method 2 of the present invention. In the conventional benzoylation method, the analysis took 1 hour or more, whereas in the method of the present invention, the analysis was completed in 40 to 50 minutes.

  As can be seen by comparing FIG. 2A and FIG. 2B, the method of the present invention using a solid-phase extraction column has a stable base line and more accurate analysis than the conventional benzoylation method. Furthermore, FIG. 2C shows that the separation of fructose and myo-inositol and mannitol and sorbitol is improved by changing the analytical column to Cadenza (5) CD-C18.

  The linearity of quantification in the method of FIG. 2B is up to 2.5 μg / mL for sugars and sugar alcohols other than fructose, and up to 5 μg / mL for fructose, whereas in the method of FIG. The sugar was up to 5 μg / mL and the fructose was up to 10 μg / mL.

  On the other hand, the limit of quantification in the method of FIG. 2B was 100 ng / mL for fructose, 25 ng / mL for mannitol and sorbitol, and 50 ng / mL for other sugars, whereas in the method of FIG. 2C, fructose and myo-inositol were used. The measurement sensitivity was improved by 50 ng / mL, 10 ng / mL for mannitol and sorbitol, and 25 ng / mL for other sugars.

  By using the method of the present invention, the sample preparation time was shortened. In the conventional benzoylation method, since a vacuum drying method is used, it takes about 60 minutes to prepare a sample for HPLC analysis, but in the method of the present invention, solid phase extraction is performed without vacuum drying. Therefore, the sample could be prepared in about 30 minutes. In addition, contaminants such as organic solvents were not mixed into the sample analyzed by HPLC, the HPLC baseline was stabilized, and the accuracy of sugar quantification was improved. Furthermore, in the HPLC analysis, a gradient for washing out contaminants was not necessary, and it was possible to carry out isocratic. Therefore, the analysis time per sample was shortened. The conventional benzoylation method took about 70 minutes, but the method of the present invention took about 40 minutes. When the sample preparation time and the analysis time by HPLC were combined, the analysis by the conventional benzoylation method took about 130 minutes, but the method of the present invention took about 70 minutes.

  In addition, the method of the present invention does not perform the drying and drying process under reduced pressure, and the contamination of solids and fine particles is reduced by solid phase extraction, so that the advantage of extending the life of the analytical column is recognized. Further, the organic Cadenza CD-C18 The particle size of the packing material such as a column is 3 μm, and it is possible to use a column that is high performance but easily clogged.

  Furthermore, in the method of the present invention, the amount of acetanilyl used in the HPLC analysis is reduced, which is economical and meaningful from the viewpoint of environmental conservation.

Example 2 Reduction in Recovery of Benzoylated Sugar and Sugar Alcohol with Acetonitrile Glucose, fructose, mannitol and sorbitol were benzoylated and dissolved in 5, 10, 20, 50, 60, 75 and 100% acetonitrile, 30 It was collected after minutes and the sugar concentration was measured by HPLC.

  FIG. 3A shows the sugar and sugar alcohol concentration after 30 minutes at each acetonitrile concentration, and FIG. 3B shows the relative concentration at each acetonitrile concentration when the concentration of each sugar and sugar alcohol after 30 minutes in 100% acetonitrile is 100. The concentration of each typical sugar and sugar alcohol is shown. Furthermore, FIG. 4 shows the stability such as benzoylation in 100% acetonitrile. As shown in FIGS. 3A and 3B, in all the sugars examined, the lower the acetonitrile concentration, the lower the recovery rate of sugar and sugar alcohol, and the reduction became remarkable when the acetonitrile concentration was less than 50%. As shown in FIG. 4, when the acetonitrile concentration was 100%, no reduction in the recovery rate of benzoylated sugar and sugar alcohol was observed.

Example 3 Analysis of Sugar and Sugar Alcohol in Normal Human Red Blood Cells (Part 2)
Preparation of sample Heparinized blood is dispensed into a 50 mL centrifuge tube by 20 mL, centrifuged at 4 ° C. and 3000 rpm for 20 minutes, and then the plasma is discarded. The precipitate was washed three times with cooled physiological saline, an appropriate amount of saline was added, mixed by inversion, centrifuged at 4 ° C. and 3000 rpm for 20 minutes, and the supernatant was discarded. To the remaining red blood cells, 1N perchloric acid was added to red blood cells 4 at a ratio of 10 perchloric acid while mixing with a Voltex mixer to deproteinize. After standing for 10 minutes, the mixture was centrifuged at 4 ° C. and 3000 rpm for 20 minutes. 3 mL of the deproteinized supernatant was dispensed into a 15 mL centrifuge tube, and 850 μL of 1.25 M K ₂ CO ₃ was added. At this time, if the pH test paper showed acidity, the amount of K ₂ CO ₃ was increased. Centrifugation was performed at 4 ° C. and 3000 rpm for 10 minutes, and the supernatant was transferred to another 50 mL centrifuge tube by decantation. The prepared sample could be stored at -20 ° C or -80 ° C until examination.

Benzoylation Samples and reagents were mixed as shown in Table 1 to benzoylate sugars and sugar alcohols in the samples.

  In Table 1, “normal scale” is a protocol when the amount of the sample is several mL or less, and scale-up is a protocol when the amount of the sample is 10 mL or more. Table 1 shows a case where the scale-up is 200 times.

  In the case of a normal scale, the reagents 1) to 3) in Table 1 were mixed and stirred with voltex for 1 minute, then sodium hydroxide was added, neutralized with 16% phosphoric acid, and stirred for several seconds. Benzoylation is preferably performed in a glass tube.

A separate protocol was used when handling a large number of samples by scaling up during structural analysis.
When scaling up to 200 times, after adding reagents 1) to 3) in Table 1, a 4 cm stir bar was placed in a 200 mL conical flask with a stopper, and the stirrer was rotated for 10 minutes. The rotation speed was set to the maximum speed at which the stirrer could rotate without jumping. Thereafter, sodium hydroxide was added, neutralized with 16% phosphoric acid, and stirred for several seconds.

Solid phase extraction
Discovery CD-C18 500 mg / 6 mL (5 bottles) was washed 3 times with 5 mL of 100% acetonitrile and twice with water. A 73 mL sample was all applied to five cartridges in several batches. The cartridge was then washed twice with 6 mL of water. Then, after washing with 2.5 mL of 75% acetonitrile and 0.85 mL of 100% acetonitrile, the sample in the cartridge was collected with 1.7 mL of 100% acetonitrile.
In the case of normal-scale sugar measurement, the collected sample was applied to HPLC.

Concentration of specimen In the case of scale-up for separating a large amount of sugar, the specimen was concentrated in advance.
Since the sample collected after the solid phase extraction is in 100% acetonitrile, it can be concentrated as it is.

  The collected samples were collected into one centrifuge tube and subdivided into 1 mL aliquots in 2 mL Eppendorf tubes for concentration again. The aliquoted tube was set in a small vacuum concentrator (Micro Vac MV-100) and concentrated for about 50 minutes. The obtained concentrated liquid was put together in one tube. A small amount of 100% acetonitrile was added to the emptied tube, vortexed and washed, and the washing solution was transferred to the next tube and washed successively. The liquid after the last tube was washed was transferred to the first 3 tube to increase the sample recovery rate. The finally obtained sample was applied to HPLC.

Sample preparation by HPLC
The HPLC conditions were as follows:
Mobile phase A: 100% acetonitrile mobile phase B: Distilled water gradient
0 to 35 minutes B Conc. 22.5% (separated)
35 to 45 minutes B Conc. 0% (washing)
45 to 58 minutes B Conc. 22.5% (return to initial state)
Column: Cadenza CD-C18 3μm 4.6φ × 250mm
Flow rate: 1mL / min
Injection; 25μL
Detection 228nm
Temperature: 25 ℃

  ADVANTEC CHF122SC was used as a sorting device. The number of Eppendorf tubes for 1.5 mL required for sorting was set in a sorting device. Next, piping from the HPLC detector was set in a preparative device, and necessary conditions were input to perform preparative operation.

Re-solid phase extraction and concentration
It was found that when the sample collected in the mobile phase of 77.5% acetonitrile was concentrated as it was with a small vacuum concentrator, the recovery rate of the benzoylated sugar deteriorated. This was presumed to be due to the fact that acetonitrile was first evaporated and water was increased, that is, the concentration of acetonitrile was lowered, and as a result, the benzoylated sugar was adsorbed on the wall of the container. Actually, the lower the concentration of acetonitrile, the lower the recovery rate of benzoylated sugar and benzoylated sugar alcohol.

Therefore, 77.5% of the water in the mobile phase was removed and replaced with 100% acetonitrile again using a Discovery CD-C18 cartridge. At this time, when the sample in the mobile phase of 77.5% was applied to the cartridge as it was, most of the sample was not adsorbed by the cartridge and was not effectively recovered. Therefore, the sample solvent does not significantly adsorb to the wall of the container, but it is diluted to 60% that can be sufficiently adsorbed by the cartridge, adsorbed to the cartridge, and then recovered with 100% acetonitrile. Replaced with 100% acetonitrile.
That is, after fractionation, the solvent was replaced with 100% acetonitrile as follows.

  The collected benzoylated saccharide fraction was collected and diluted from 77.5% (HPLC mobile phase concentration) to 60% acetonitrile (for example, 1.4 mL of 20% acetonitrile for 3 mL of sugar or sugar alcohol fraction). added). Discovery CD-C18 was conditioned by washing three times with 5 mL of 100% acetonitrile, twice with water, and once with 2 mL of 60% acetonitrile.

  The sample replaced with 100% acetonitrile was applied to a conditioned cartridge. The sample was washed successively with 2 mL of 60% acetonitrile and 0.85 mL of 100% acetonitrile. The benzoylated sugar fraction in the cartridge was collected with 2.0 mL of 100% acetonitrile. Divide the collected sample into 1 mL aliquots of 2 mL Eppendorf tubes, set in a small vacuum concentrator (Micro Vac MV-100), and concentrate for about 60 minutes. The specimen was applied to NMR analysis and analyzed.

  The results are shown in FIG. 5A shows the results of NMR analysis of two types of sugar alcohols (X and Y, respectively) separated by the method of this example. B shows the results of NMR analysis of two types of benzoylated sugar alcohol X preparations, and C shows the results of NMR analysis of benzoylated sugar alcohol Y preparations. According to the method of the present invention, benzoylated sugar and sugar alcohol in erythrocytes were suppressed to adsorption to the wall of the container to the minimum, recovered efficiently, and concentrated to a high concentration. Therefore, analysis by NMR became possible.

  By the method of the present invention, sugars and sugar alcohols in various samples such as biological samples can be analyzed, and can be used in the fields of food, medicine and medicine.

It is a figure which shows the chromatogram of the sugar analysis at the time of performing a solid-phase extraction using Discovery C8 cartridge, using a Cadenza CD-C18 column as an analytical column, after benzoylating the 0.5 microgram / mL standard solution. It is a figure which shows the chromatogram of a normal human erythrocyte. FIG. 2A shows the analysis results when Inertsil ODS3 was used as the analytical column after deproteinization of the sample, followed by benzoylation and drying under reduced pressure. FIG. 2B shows the results when Inertsil ODS3 was used as the analytical column after deproteinization of the sample, benzoylation, and solid phase extraction by the method of the present invention. FIG. 2C shows the results when Cadenza (5) CD-C18 was used as an analytical column in the method of the present invention. It is a figure which shows the collection | recovery rate of the benzoylated sugar and sugar alcohol by acetonitrile. It is a figure which shows the time-dependent recovery rate of the benzoylated sugar and sugar alcohol in 100% acetonitrile. It is a figure which shows the result of the analysis by NMR of the sugar and sugar alcohol which were benzoylated, refine | purified by HPLC, and was further concentrated.

Claims (13)

  A method for analyzing sugar and / or sugar alcohol in a sample, wherein a sample containing sugar and / or sugar alcohol is benzoylated, and impurities other than sugar and sugar alcohol are obtained by solid-phase extraction without subjecting to vacuum drying. A method for analyzing sugar and / or sugar alcohol in a sample, comprising separating sugar and / or sugar alcohol in a sample by reversed-phase high-performance liquid chromatography, and measuring and analyzing ultraviolet absorption.   The method for analyzing sugar and / or sugar alcohol in a sample according to claim 1, wherein a deproteinization treatment is performed before the sample is benzoylated.   The sample according to claim 1 or 2, wherein the sugar and / or sugar alcohol in the sample is one or more sugars or sugar alcohols selected from the group consisting of ribose, mannose, glucose, fructose, myo-inositol, mannitol and sorbitol. Of analyzing sugars and / or sugar alcohols.   The method for analyzing sugar and / or sugar alcohol in a sample according to any one of claims 1 to 3, wherein the sample is a biological sample.   The solid phase extraction is performed using a stationary phase for reverse phase chromatography or a stationary phase for ion exchange chromatography, and the sugar and / or sugar alcohol in the sample according to any one of claims 1 to 4 is analyzed. Method.   6. The sugar and / or sugar alcohol in the sample according to claim 5, wherein the solid phase extraction is performed using a stationary phase having an octadecylsilyl group or an octyl group, and the elution of sugar and / or sugar alcohol is performed using acetonitrile. How to analyze.   The reverse phase high performance liquid chromatography is performed using a column containing a packing material having an octadecylsilyl group or an octyl group, and elution of sugar and / or sugar alcohol is performed using acetonitrile. A method for analyzing the sugar and / or sugar alcohol according to claim 1.   The method for analyzing sugar and / or sugar alcohol according to claim 7, wherein the reversed-phase high performance liquid chromatography is performed using a column containing a packing material having an octadecylsilyl group having a particle size of 3 µm.   The method for analyzing sugar and / or sugar alcohol according to any one of claims 1 to 8, wherein reverse phase high performance liquid chromatography is carried out by isocratic elution.   The method for analyzing sugar and / or sugar alcohol according to any one of claims 1 to 9, wherein the organic solvent of the benzoylated sugar and / or sugar alcohol separated by reverse phase high performance liquid chromatography is subjected to solid phase extraction. A method of concentrating a benzoylated sugar and / or sugar alcohol in a sample with a high recovery rate by exchanging the organic solvent with a concentration of 100% using a method and evaporating the organic solvent.   A method for analyzing sugar and / or sugar alcohol in a sample, comprising analyzing the sugar and / or sugar alcohol concentrated by the method according to claim 10 by NMR.   12. The method according to any one of claims 1 to 9 and 11, wherein a compound having an OH group in a sample is analyzed by benzoylating the compound having an OH group in the sample instead of sugar and / or sugar alcohol. how to.   The method according to claim 11, wherein the compound having an OH group in the sample is concentrated by benzoylating the compound having an OH group in the sample instead of sugar and / or sugar alcohol.

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