JP2526600B2 - Method for analyzing reducing sugar and / or sialic acid - Google Patents
Method for analyzing reducing sugar and / or sialic acidInfo
- Publication number
- JP2526600B2 JP2526600B2 JP62209271A JP20927187A JP2526600B2 JP 2526600 B2 JP2526600 B2 JP 2526600B2 JP 62209271 A JP62209271 A JP 62209271A JP 20927187 A JP20927187 A JP 20927187A JP 2526600 B2 JP2526600 B2 JP 2526600B2
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- Japan
- Prior art keywords
- sialic acid
- reducing sugar
- reaction
- present
- fluorescence
- Prior art date
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- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Description
【発明の詳細な説明】 （産業上の利用分野） 本発明は、1,2−ジアリルエチレンジアミン誘導体を
蛍光誘導体化試薬として用いる還元糖及び／又はシアル
酸の分析方法に関する。TECHNICAL FIELD The present invention relates to a method for analyzing reducing sugar and / or sialic acid using a 1,2-diallylethylenediamine derivative as a fluorescent derivatization reagent.
（従来の技術） 生体中で糖やシアル酸は微量であるが重要な役割を果
しているものが多いため、高感度な分析方法が求められ
ている。(Prior Art) Since a small amount of sugar and sialic acid play an important role in a living body, a highly sensitive analysis method is required.
糖の分析方法として、ガスクロマトグラフィーが古く
から用いられているが、糖の誘導体化反応が困難であ
り、また多糖類の誘導体は揮発性でないなどの難点を有
していた。近年高速液体クロマトグラフィー（HPLCと略
称する）の進歩に伴い示差屈折計と組合せた糖の分析方
法が数多く報告されている。しかし、示差屈折計は感度
が低く、選択性に乏しいことや他の検出記に比べて温度
や流速の影響を受けやすいなどの欠点を有している。そ
のためカラムで分離した後、紫外部に吸収を持つものや
蛍光性を有するものに誘導体化する（ポストカラム誘導
体化と略称する）方法を利用する分析方法の開発が試み
られている。Gas chromatography has been used as a method for analyzing sugars for a long time, but it has drawbacks such that the derivatization reaction of sugars is difficult and the derivatives of polysaccharides are not volatile. In recent years, with the progress of high performance liquid chromatography (abbreviated as HPLC), many methods for analyzing sugar combined with a differential refractometer have been reported. However, the differential refractometer has drawbacks such as low sensitivity, poor selectivity, and being more susceptible to temperature and flow velocity than other detection methods. Therefore, attempts have been made to develop an analytical method that uses a method of derivatizing into a substance having absorption in the ultraviolet region or a substance having fluorescence (abbreviated as post-column derivatization) after separation on a column.
糖の誘導体化試薬は次の２種類に大別することができ
る。ひとつは５−ヒドロキシテトラロン（T.Momose et
al,Chem.Pham.Bull.,7,31（1959））、レゾルシノール
（C.G.Rodgers etal,Anal.Chem.,38,1851（1966））、
オルシノール（R.B.Kesler,Anal.Chem.,39,1416（196
7））などで、反応に強酸を使用するものである。いま
一つはエチレンジアミン（S.Honda et al,Anal.Chim.Ac
ta,70,133（1974））、シアノアセトアミド（S.Honda e
t al,Anal.Chem.,52,1079（1980））、アルギニン（H.M
ikami et al,Bunseki Kagaku,37,E207（1983））などで
アルカリ性で加熱するものである。Sugar derivatization reagents can be broadly classified into the following two types. One is 5-hydroxytetralone (T. Momose et
al, Chem.Pham.Bull., 7,31 (1959)), resorcinol (CGRodgers et al, Anal.Chem., 38,1851 (1966)),
Orcinol (RB Kesler, Anal. Chem., 39, 1416 (196
In 7)), etc., a strong acid is used for the reaction. Another is ethylenediamine (S.Honda et al, Anal.Chim.Ac.
ta, 70,133 (1974), cyanoacetamide (S. Honda e
t al, Anal.Chem., 52,1079 (1980)), arginine (HM
ikami et al, Bunseki Kagaku, 37, E207 (1983)) and the like are alkaline heating.
（発明が解決しようとする問題点） 従来の方法として前者を使用した場合には、強酸を使
用するために取扱いが困難であったり、強酸に耐える特
別な装置が必要となるといった問題を有しており、又後
者の方法では、反応に長時間を要するためポストカラム
誘導体化方法を適用した場合、感度が低下するといった
問題点を有している。従って簡便でかつ高感度な還元糖
及び／又はシアル酸の分析方法が望まれている。(Problems to be Solved by the Invention) When the former method is used as a conventional method, there are problems that it is difficult to handle because a strong acid is used and a special device that withstands a strong acid is required. However, the latter method has a problem that the sensitivity is lowered when the post-column derivatization method is applied because the reaction takes a long time. Therefore, a simple and highly sensitive method for analyzing reducing sugar and / or sialic acid is desired.
本発明の目的は、簡便な方法で還元糖またはシアル酸
の誘導体化反応を行ない、かつ高感度での分析を可能と
する還元糖及び／又はシアル酸の分析方法を提供するこ
とにある。An object of the present invention is to provide a method for analyzing a reducing sugar and / or sialic acid, which enables derivatization reaction of a reducing sugar or sialic acid by a simple method and enables analysis with high sensitivity.
（問題点を解決するための手段） 本発明者らは、上記の問題点を解決すべく鋭意検討し
た結果、ｐ−（又はｍ−）置換フェニル基を有するエチ
レンジアミン誘導体が還元糖及び／又はシアル酸と反応
して青緑色蛍光を有する化合物となり、この際に溶媒中
で塩基存在下反応させることにより誘導体化反応が容易
に進行することを見出し本発明を完成した。(Means for Solving Problems) The inventors of the present invention have conducted extensive studies to solve the above problems, and as a result, found that an ethylenediamine derivative having a p- (or m-)-substituted phenyl group was a reducing sugar and / or a sialic acid. The present inventors have completed the present invention by finding that a compound having blue-green fluorescence is reacted with an acid and a derivatization reaction proceeds easily by reacting in the presence of a base in a solvent.
すなわち本発明は、一般式 RC（NH2）Ｃ（NH2）Ｒ （Ｒはｐ−メトキシフェニル、ｐ−エトキシフェニル、
ｐ−クロロフェニル、ｐ−フルオロフェニル、ｐ−シア
ノフェニル、ｐ−メチルフェニル、ｍ−メチルフェニル
を表わす）で示される1,2−ジアリルエチレンジアミン
誘導体と還元糖及び／又はシアル酸を、溶媒中で塩基の
存在下反応させ、反応生成物の蛍光を測定することを特
徴とする還元糖及び／又はシアル酸の分析方法に関する
ものである。That is, the present invention provides a compound represented by the general formula RC (NH 2 ) C (NH 2 ) R (R is p-methoxyphenyl, p-ethoxyphenyl,
1,2-diallylethylenediamine derivative represented by p-chlorophenyl, p-fluorophenyl, p-cyanophenyl, p-methylphenyl, m-methylphenyl) and a reducing sugar and / or sialic acid are used as bases in a solvent. The present invention relates to a method for analyzing reducing sugars and / or sialic acid, which comprises reacting in the presence of the above and measuring the fluorescence of the reaction product.
本発明方法では、食品、血液、尿などの試料中に含ま
れる還元糖及び／又はシアル酸の分析に適用することが
できる。The method of the present invention can be applied to the analysis of reducing sugars and / or sialic acid contained in samples such as food, blood and urine.
本発明で使用される誘導体化試薬としての1,2−ジア
リルエチレンジアミン誘導体（DAEsと略称する）は、RC
（NH2）Ｃ（NH2）Ｒの一般式で示されるもので、その置
換基Ｒとしてはｐ−メトキシフェニル、ｐ−エトキシフ
ェニル、ｐ−クロロフェニル、ｐ−フルオロフェニル、
ｐ−シアノフェニル、ｐ−メチルフェニル又はｍ−メチ
ルフェニルのｐ−（又はｍ−）置換フェニル基である。
これらの中でも1,2−ビス（４−メトキシフエニル）エ
チレンジアミン（ｐ−MOEDと略称する）がもっとも強い
蛍光を与え、好ましい誘導体化試薬てある。The 1,2-diallylethylenediamine derivative (abbreviated as DAEs) as a derivatization reagent used in the present invention is RC
(NH 2 ) C (NH 2 ) R is represented by the general formula, and the substituent R is p-methoxyphenyl, p-ethoxyphenyl, p-chlorophenyl, p-fluorophenyl,
It is a p- (or m-)-substituted phenyl group of p-cyanophenyl, p-methylphenyl or m-methylphenyl.
Among these, 1,2-bis (4-methoxyphenyl) ethylenediamine (abbreviated as p-MOED) gives the strongest fluorescence and is a preferable derivatization reagent.
本発明のDAEsと還元糖及び／又はシアル酸との反応
は、溶媒中塩基存在下で行われなければならない。The reaction of the DAEs of the present invention with a reducing sugar and / or sialic acid must be performed in a solvent in the presence of a base.
本発明で使用する溶媒とは、DAEsが水に不溶であるた
めに添加するもので、水溶性の有機溶媒から選択され
る。例えば、メタノール、エタノール、イソプロパノー
ル、アセトン、メチルセロソルブなどであるが、特に強
い蛍光を与えるエタノールが好ましい。The solvent used in the present invention is added because DAEs are insoluble in water, and is selected from water-soluble organic solvents. For example, methanol, ethanol, isopropanol, acetone, methyl cellosolve and the like are used, but ethanol which gives particularly strong fluorescence is preferable.
又本発明では、反応を短時間に進行させるために、反
応液を塩基性にする必要がある。そのために添加する塩
基としては、水酸化ナトリウムや水酸化カリウムなど通
常使用されるものから選択されるが、水酸化ナトリウム
が特に強い蛍光を与えるので好ましい。Further, in the present invention, it is necessary to make the reaction solution basic in order to proceed the reaction in a short time. The base to be added for that purpose is selected from those usually used such as sodium hydroxide and potassium hydroxide, but sodium hydroxide is preferable because it gives particularly strong fluorescence.
本発明の反応は、90〜150℃の温度、15〜80分の時間
範囲で適宜選択される。The reaction of the present invention is appropriately selected at a temperature of 90 to 150 ° C. and a time range of 15 to 80 minutes.
以上の本発明方法によれば、還元糖及び／又はシアル
酸をnmol/ml以下のオーダーで分析することができる。According to the method of the present invention described above, reducing sugar and / or sialic acid can be analyzed in the order of nmol / ml or less.
なお本発明方法をポストカラム誘導体化HPLCに適用す
ることにより、還元糖類の高感度な自動分析を行なうこ
とも可能である。第２図にその場合の装置の概略を示す
が、溶離液１を溶離液送液ポンプ２によりカラム流路に
導入し、試料導入バルブ３にて試料を溶離液流に導入す
る。分離カラム４で各試料中の還元糖成分を分離後、反
応液として溶媒に溶解した誘導体化試薬溶液５及び塩基
溶液６を送液ポンプ7,8により溶離液流路に添加混合
し、反応コイル９で反応させる。反応生成物の蛍光を蛍
光検出器10により測定し、記録計11で記録する。例え
ば、ホウ酸緩衝液を溶離液とし、陰イオンイオン交換カ
ラムで還元糖類を分離する。分離カラムで分離された各
還元糖成分を含む溶出液にｐ−MOED溶液と水酸化ナトリ
ウム溶液をオンラインで添加混合し反応コイルで誘導体
化反応させ、反応生成物の蛍光を蛍光検出器により検出
すれば良い。By applying the method of the present invention to post-column derivatization HPLC, it is possible to perform highly sensitive automatic analysis of reducing sugars. FIG. 2 shows the outline of the apparatus in that case. The eluent 1 is introduced into the column flow path by the eluent liquid feed pump 2, and the sample is introduced into the eluent flow by the sample introduction valve 3. After the reducing sugar component in each sample is separated in the separation column 4, the derivatization reagent solution 5 and the base solution 6 which are dissolved in the solvent as the reaction solution are added and mixed in the eluent flow path by the solution feeding pumps 7 and 8, and the reaction coil React at 9. The fluorescence of the reaction product is measured by the fluorescence detector 10 and recorded by the recorder 11. For example, using a borate buffer as an eluent, reducing saccharides are separated by an anion ion exchange column. To the eluate containing each reducing sugar component separated by the separation column, p-MOED solution and sodium hydroxide solution are added online and mixed, and a derivatization reaction is carried out in the reaction coil, and the fluorescence of the reaction product is detected by a fluorescence detector. Good.
（発明の効果） 以上説明したように、本発明の誘導体化方法を用いれ
ば、還元糖及び／又はシアル酸を簡便な反応条件 で蛍光を発する反応性生成物にでき、高感度に各還元糖
成分を分析することができる。(Effects of the Invention) As described above, when the derivatization method of the present invention is used, reducing sugar and / or sialic acid can be made into a reactive product that fluoresces under simple reaction conditions, and each reducing sugar can be highly sensitive. The components can be analyzed.
又ポストカラム誘導体化方法にも適用でき、還元糖類
の自動分析も可能である。It can also be applied to a post-column derivatization method, and automatic analysis of reducing sugars is also possible.
（実施例） 以下本発明を実施例によりさらに説明するが、本発明
はこれらの実施例にのみ限定されるものではない。(Examples) The present invention will be further described below with reference to examples, but the present invention is not limited to these examples.
実施例１ DAEsのｐ−置換フェニル基として表１の置換基を使用
した。Ｄ−グルコース溶液（100nmol/ml）1mlにDAEs溶
液（10mM;50％エタノール溶液）0.5ml及び水酸化ナトリ
ウム溶液（0.01M）1mlを添加、撹拌後、100℃で30分間
反応させた。反応生成物の蛍光を表１の励起波長
（λex）および発光極大波長（λem）で測定した。結果
として得られた相対蛍光強度を表１に示す。Example 1 The substituents shown in Table 1 were used as the p-substituted phenyl group of DAEs. 0.5 ml of DAEs solution (10 mM; 50% ethanol solution) and 1 ml of sodium hydroxide solution (0.01 M) were added to 1 ml of D-glucose solution (100 nmol / ml), stirred and reacted at 100 ° C. for 30 minutes. The fluorescence of the reaction product was measured at the excitation wavelength (λ ex ) and emission maximum wavelength (λ em ) in Table 1. The relative fluorescence intensities obtained as a result are shown in Table 1.
実施例２ 表２に記載した還元糖の17種類の水溶液（100nmol/m
l）1mlにｐ−MOED（20mM;50％エタノール溶液）0.5ml及
び水酸化ナトリウム溶液（0.02M）1mlを添加、撹拌後、
140℃で30分間加熱反応させた。反応生成物の蛍光は励
起波長330nm及び発光極大波長460nmとして測定した。得
られた相対蛍光強度及び検出限界（nmol/ml）を表２に
示す。 Example 2 17 types of aqueous solutions of reducing sugars listed in Table 2 (100 nmol / m
l) 0.5 ml of p-MOED (20 mM; 50% ethanol solution) and 1 ml of sodium hydroxide solution (0.02M) were added to 1 ml, and after stirring,
The mixture was heated and reacted at 140 ° C for 30 minutes. The fluorescence of the reaction product was measured at an excitation wavelength of 330 nm and an emission maximum wavelength of 460 nm. Table 2 shows the obtained relative fluorescence intensity and detection limit (nmol / ml).
実施例３ 表３に記載した11種類の還元糖及び２種類のシアル酸
を用いた以外は、実施例２同様にして各糖の分析を行っ
た。得られた相対蛍光強度及び検出限界（nmol/ml）を
表３に示す。Example 3 Each sugar was analyzed in the same manner as in Example 2 except that 11 kinds of reducing sugars and 2 kinds of sialic acid shown in Table 3 were used. Table 3 shows the obtained relative fluorescence intensity and detection limit (nmol / ml).
実施例４ 試料の還元糖として、セロビオース、マルトース、Ｄ
−リボース、Ｄ−マンノース、Ｄ−アラビノース、Ｄ−
ガラクトース、Ｄ−キシロース及びＤ−グルコースの８
種類を用いた。還元糖濃度20nmol/ml（Ｄ−ガラクトー
スのみ10nmol/ml）に調整した溶液100μを第２図のHP
LCに注入した。分離カラムで分離された各成分を含む溶
出液に反応液としてｐ−MOED溶液（15mM;50％エタノー
ル溶液）と水酸化ナトリウム溶液（0.75M）をオンライ
ンで添加混合し、反応カラムで誘導体化反応させ、反応
生成物の蛍光を励起波長330nm、発光極大波長460nmで測
定した。得られたクロマトグラムを第１図に示す。 Example 4 Cellobiose, maltose, D were used as the reducing sugars of the sample.
-Ribose, D-mannose, D-arabinose, D-
8 of galactose, D-xylose and D-glucose
The type used. A solution of 100 μm adjusted to a reducing sugar concentration of 20 nmol / ml (D-galactose only 10 nmol / ml) was used as the HP of Fig. 2.
Injected into LC. To the eluate containing each component separated in the separation column, p-MOED solution (15 mM; 50% ethanol solution) and sodium hydroxide solution (0.75M) were added online as a reaction solution and mixed, and the derivatization reaction was carried out in the reaction column. Then, the fluorescence of the reaction product was measured at an excitation wavelength of 330 nm and an emission maximum wavelength of 460 nm. The obtained chromatogram is shown in FIG.
HPLCの測定条件は以下のとおりである。 The HPLC measurement conditions are as follows.
分離カラム:TSKgel SugerAXG（4.6mmI.D.×15cm）東洋
曹達工業（株）製 分離温度:60℃ 溶離液:0.5Mホウ酸塩緩衝液（pH8.7） 流速（溶離液）:0.4ml/min 流速（反応液）:0.15ml/min 反応コイル:0.25mmI.D.×20m 反応温度:140℃ 実施例５ 尿250mlに活性炭1gと0.1Mトリス塩酸緩衝液（pH8.5）
1mlを添加した。遠心分離後、得られた上清100mlを実施
例４と同様にして分析した。Separation column: TSKgel SugerAXG (4.6mmI.D. × 15cm) manufactured by Toyo Soda Kogyo Co., Ltd. Separation temperature: 60 ℃ Eluent: 0.5M borate buffer (pH8.7) Flow rate (eluent): 0.4ml / min Flow rate (reaction liquid): 0.15 ml / min Reaction coil: 0.25 mm I.D. × 20 m Reaction temperature: 140 ° C. Example 5 250 g of urine, 1 g of activated carbon and 0.1 M Tris-HCl buffer (pH 8.5)
1 ml was added. After centrifugation, 100 ml of the resulting supernatant was analyzed as in Example 4.
得られたクロマトグラムを第３図に示す。 The obtained chromatogram is shown in FIG.
【図面の簡単な説明】 第１図は本発明実施例により得られたクロマトグラム、
第２図は本発明方法をHPLCに適用した場合の装置の概略
図、第３図は尿試料を分析した本発明実施例５で得られ
たクロマトグラムである。 １……溶離液、２……送液ポンプ ３……試料導入、４……分離カラム ５……誘導体化試薬、６……塩基溶液 ７……送液ポンプ、８……送液ポンプ ９……反応コイル、10……蛍光検出器 11……記録計BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a chromatogram obtained by an example of the present invention,
FIG. 2 is a schematic diagram of an apparatus when the method of the present invention is applied to HPLC, and FIG. 3 is a chromatogram obtained in Example 5 of the present invention in which a urine sample was analyzed. 1 ... Eluent, 2 ... Delivery pump 3 ... Sample introduction, 4 ... Separation column 5 ... Derivatization reagent, 6 ... Basic solution 7 ... Delivery pump, 8 ... Delivery pump 9 ... … Reaction coil, 10 …… Fluorescence detector 11 …… Recorder
Claims (1)
ｐ−クロロフェニル、ｐ−フルオロフェニル、ｐ−シア
ノフェニル、ｐ−メチルフェニル、ｍ−メチルフェニル
を表わす）で示される1,2−ジアリルエチレンジアミン
誘導体と還元糖及び／又はシアル酸とを、溶媒中で塩基
の存在下反応させ、反応生成物の蛍光を測定することを
特徴とする還元糖及び／又はシアル酸の分析方法。1. A general formula RC (NH 2 ) C (NH 2 ) R (R is p-methoxyphenyl, p-ethoxyphenyl,
a 1,2-diallylethylenediamine derivative represented by p-chlorophenyl, p-fluorophenyl, p-cyanophenyl, p-methylphenyl and m-methylphenyl) and a reducing sugar and / or sialic acid in a solvent. A method for analyzing reducing sugar and / or sialic acid, which comprises reacting in the presence of a base and measuring the fluorescence of the reaction product.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62209271A JP2526600B2 (en) | 1987-08-25 | 1987-08-25 | Method for analyzing reducing sugar and / or sialic acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62209271A JP2526600B2 (en) | 1987-08-25 | 1987-08-25 | Method for analyzing reducing sugar and / or sialic acid |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS6454233A JPS6454233A (en) | 1989-03-01 |
| JP2526600B2 true JP2526600B2 (en) | 1996-08-21 |
Family
ID=16570181
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP62209271A Expired - Fee Related JP2526600B2 (en) | 1987-08-25 | 1987-08-25 | Method for analyzing reducing sugar and / or sialic acid |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2526600B2 (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0737971B2 (en) * | 1989-12-27 | 1995-04-26 | 株式会社島津製作所 | Analysis method for amino compounds |
| JP2005024336A (en) * | 2003-06-30 | 2005-01-27 | Japan Science & Technology Corp | Method and kit for analyzing acidic polysaccharides |
| JP4997487B2 (en) * | 2005-08-03 | 2012-08-08 | 独立行政法人農業・食品産業技術総合研究機構 | Apparatus for liquid-liquid extraction or solid-liquid extraction and extraction method using the same |
| JP5279286B2 (en) * | 2008-02-13 | 2013-09-04 | 学校法人北里研究所 | Fluorescent labeling method of sialic acid, sialic acid-containing carbohydrate, or sialic acid-containing complex carbohydrate, and fluorescently labeled sialic acid, sialic acid-containing carbohydrate, or sialic acid-containing complex sugar obtained by the above method quality |
-
1987
- 1987-08-25 JP JP62209271A patent/JP2526600B2/en not_active Expired - Fee Related
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| Publication number | Publication date |
|---|---|
| JPS6454233A (en) | 1989-03-01 |
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