JPH0481665A - Method for analyzing bioamino acid - Google Patents
Method for analyzing bioamino acidInfo
- Publication number
- JPH0481665A JPH0481665A JP2082277A JP8227790A JPH0481665A JP H0481665 A JPH0481665 A JP H0481665A JP 2082277 A JP2082277 A JP 2082277A JP 8227790 A JP8227790 A JP 8227790A JP H0481665 A JPH0481665 A JP H0481665A
- Authority
- JP
- Japan
- Prior art keywords
- amino acid
- amino acids
- secondary amino
- mobile phase
- primary amino
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000000034 method Methods 0.000 title description 15
- 239000002253 acid Substances 0.000 title 1
- 125000000467 secondary amino group Chemical group [H]N([*:1])[*:2] 0.000 claims abstract description 36
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims abstract description 22
- 238000004458 analytical method Methods 0.000 claims abstract description 12
- ZWLUXSQADUDCSB-UHFFFAOYSA-N phthalaldehyde Chemical compound O=CC1=CC=CC=C1C=O ZWLUXSQADUDCSB-UHFFFAOYSA-N 0.000 claims abstract description 12
- DKIDEFUBRARXTE-UHFFFAOYSA-N 3-mercaptopropanoic acid Chemical compound OC(=O)CCS DKIDEFUBRARXTE-UHFFFAOYSA-N 0.000 claims abstract description 9
- 150000001413 amino acids Chemical class 0.000 claims description 24
- 238000004366 reverse phase liquid chromatography Methods 0.000 claims description 9
- WTKZEGDFNFYCGP-UHFFFAOYSA-N Pyrazole Chemical compound C=1C=NNC=1 WTKZEGDFNFYCGP-UHFFFAOYSA-N 0.000 claims 1
- 238000004128 high performance liquid chromatography Methods 0.000 abstract description 14
- 238000001514 detection method Methods 0.000 abstract description 12
- 238000007796 conventional method Methods 0.000 abstract description 5
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Substances C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 abstract description 5
- 230000014759 maintenance of location Effects 0.000 abstract description 4
- 229940054441 o-phthalaldehyde Drugs 0.000 abstract 1
- 238000010828 elution Methods 0.000 description 11
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 9
- 230000005284 excitation Effects 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 5
- 238000001212 derivatisation Methods 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 238000001917 fluorescence detection Methods 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 238000004140 cleaning Methods 0.000 description 2
- 239000000356 contaminant Substances 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- IRXSLJNXXZKURP-UHFFFAOYSA-N fluorenylmethyloxycarbonyl chloride Chemical compound C1=CC=C2C(COC(=O)Cl)C3=CC=CC=C3C2=C1 IRXSLJNXXZKURP-UHFFFAOYSA-N 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- LQNUZADURLCDLV-UHFFFAOYSA-N nitrobenzene Chemical compound [O-][N+](=O)C1=CC=CC=C1 LQNUZADURLCDLV-UHFFFAOYSA-N 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 2
- 239000001509 sodium citrate Substances 0.000 description 2
- XNWFRZJHXBZDAG-UHFFFAOYSA-N 2-METHOXYETHANOL Chemical compound COCCO XNWFRZJHXBZDAG-UHFFFAOYSA-N 0.000 description 1
- IGHBXJSNZCFXNK-UHFFFAOYSA-N 4-chloro-7-nitrobenzofurazan Chemical compound [O-][N+](=O)C1=CC=C(Cl)C2=NON=C12 IGHBXJSNZCFXNK-UHFFFAOYSA-N 0.000 description 1
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- -1 biochemistry Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000007599 discharging Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 238000002795 fluorescence method Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 125000004079 stearyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
Description
【発明の詳細な説明】
（イ）産業上の利用分野
本発明は、高速液体クロマトグラフィによる生体アミノ
酸の分析方法に関し、特に蛋白質を構成しているアミノ
酸の高速液体クロマトグラフィによる分析方法に関する
。DETAILED DESCRIPTION OF THE INVENTION (a) Field of Industrial Application The present invention relates to a method for analyzing biological amino acids by high-performance liquid chromatography, and particularly to a method for analyzing amino acids constituting proteins by high-performance liquid chromatography.
また、本発明は、食品、医薬品、生化学並びに医学の分
野における生体アミノ酸の分析方法に関する。The present invention also relates to a method for analyzing biological amino acids in the fields of food, medicine, biochemistry, and medicine.
（ロ）従来の技術
例えば、蛋白質を構成するアミノ酸についての高速液体
クロマトグラフィによる分析は、アミノ酸が、β−メル
カプトプロピオン酸のような還元剤の存在下に、蛍光誘
導体化剤のオルト−フタルアルデヒドと反応して発蛍光
の誘導体を形成することを利用して、アミノ酸を前もっ
てラベル化しておき、このラベル化されたアミノ酸を高
速液体クロマトグラフィにより分離して、蛍光法により
夫々のアミノ酸を定量している。(b) Conventional technology For example, in the analysis of amino acids constituting proteins by high performance liquid chromatography, amino acids are treated with ortho-phthalaldehyde, a fluorescent derivatizing agent, in the presence of a reducing agent such as β-mercaptopropionic acid. Utilizing the reaction to form fluorescent derivatives, amino acids are pre-labeled, the labeled amino acids are separated using high performance liquid chromatography, and each amino acid is quantified using a fluorescence method. .
くハ）発明が解決しようとする課題
しかし、蛍光誘導体化剤のオルト−フタルアルデヒドは
、−級アミノ酸について発蛍光の誘導体を形成するが、
二級アミノ酸については、発蛍光しないので、二級アミ
ノ酸については同時に分析することができず問題とされ
ている。C) Problems to be Solved by the Invention However, the fluorescent derivatizing agent ortho-phthalaldehyde forms fluorescent derivatives of -class amino acids;
Since secondary amino acids do not emit fluorescence, they cannot be analyzed at the same time, which poses a problem.
そこで、試料中の二級アミノ酸をＦＭＯＣ（９フルオレ
ニルメチルクロロ示ルマート）と反応させて発蛍光誘導
体を形成させて、高速液体クロマトグラフィ分析法によ
り、二級アミノ酸について分析する方法が提案されてい
る。Therefore, a method has been proposed in which secondary amino acids in a sample are reacted with FMOC (9-fluorenyl methyl chloroformate) to form a fluorescent derivative, and secondary amino acids are analyzed using high-performance liquid chromatography analysis. There is.
この方法は、−級アミノ酸をオルト−フタルアルデヒド
により発蛍光誘導体化させ、次いでＦＭ○Ｃにより、二
級アミノ酸を発蛍光の誘導体化させて、高速液体クロマ
トグラフィにより分離するものである。In this method, -class amino acids are fluorescently derivatized with ortho-phthalaldehyde, and then secondary amino acids are fluorescently derivatized with FM○C and separated by high performance liquid chromatography.
この方法では、まず−級アミノ酸の誘導体の夫々が順次
溶出し、次ぎに二級アミノ酸が溶出するので、まず−級
アミノ酸の誘導体について、励起波長３４０ｎｍ、検出
蛍光波長４５０ｎｍ ″Ｃ測定し、その後、二級アミノ
酸について、検出波長を、励起波長が２６６ ｎ、ｍ、
蛍光波長が３０５ｎｍ に切り替えて測定が行われる。In this method, each derivative of the -grade amino acid is eluted sequentially, and then the secondary amino acid is eluted, so first, the derivative of the -grade amino acid is measured at an excitation wavelength of 340 nm and a detection fluorescence wavelength of 450 nm. For secondary amino acids, the detection wavelength is 266 n, m, and the excitation wavelength is 266 n, m,
Measurement is performed with the fluorescence wavelength switched to 305 nm.
しかし、この方法は、二級アミノ酸の溶出が一級アミノ
酸の溶出の後であるために、分析時間が長くなり、また
、二級アミノ酸の発蛍光誘導体の検出波長が一級アミノ
酸誘導体の場合より著しく短波長側であり、夾雑物によ
り影響を受は易く問題が残っている。However, in this method, the elution of secondary amino acids occurs after the elution of primary amino acids, which increases the analysis time, and the detection wavelength of fluorescent derivatives of secondary amino acids is significantly shorter than that of primary amino acid derivatives. Since it is on the wavelength side, it is easily affected by contaminants, and problems remain.
本発明は、高速液体クロマトグラフィによるアミノ酸分
析における二級アミノ酸の分析に基づく課題を解決する
ことを目的としている。The present invention aims to solve problems based on secondary amino acid analysis in amino acid analysis using high performance liquid chromatography.
（ニ）課題を解決するための手段
本発明は、試料中の一級アミノ酸及び二級アミノ酸につ
いて、プレカラム法で異なる検出波長で同時に測定する
ことができるアミノ酸分析法を提案するものである。(d) Means for Solving the Problems The present invention proposes an amino acid analysis method that can simultaneously measure primary and secondary amino acids in a sample using a pre-column method at different detection wavelengths.
即ち、本発明は、試料中の一級アミノ酸を βメルカプ
トプロピオン酸の存在下にオルト−フタルアルデヒドと
反応させて発蛍光誘導体化させ、次いで、試料中の二級
アミノ酸を４−クロロ−７二トロベンゾー２−オキサ−
１，３−ジアゾールと反応させて発蛍光誘導体化させ、
この発蛍光誘導体化された試料を逆相クロマトグラフィ
相に通し、該逆相クロマトグラフィ相から溶出する発蛍
光誘導体について、異なる二つの波長帯域において同時
に蛍光検出することを特徴とする生体アミノ酸分析方法
にある。That is, in the present invention, primary amino acids in a sample are reacted with ortho-phthalaldehyde in the presence of β-mercaptopropionic acid to fluorescein derivatize, and then secondary amino acids in the sample are derivatized with 4-chloro-7 nitrobenzene. 2-oxa-
React with 1,3-diazole to make a fluorescent derivative,
The method for analyzing biological amino acids is characterized in that the fluorescent derivatized sample is passed through a reversed phase chromatography phase, and the fluorescent derivatives eluted from the reversed phase chromatography phase are simultaneously detected for fluorescence in two different wavelength bands. .
本発明は、−級アミノ酸を β−メルカプトプロピオン
酸の存在下にオルトフタルアルデヒドにより発蛍光誘導
体化させ、次いで二級アミノ酸を２−オルト−フタルア
ルデヒドにより発蛍光誘導体化させることによって、二
級アミノ酸を一級アミノ酸と共に、高速液体クロマトグ
ラフィ法、特に逆相クロマトグラフィ法により測定する
ものである。The present invention provides a secondary amino acid by fluorescently derivatizing a -class amino acid with ortho-phthalaldehyde in the presence of β-mercaptopropionic acid, and then fluorescently derivatizing a secondary amino acid with 2-ortho-phthalaldehyde. together with primary amino acids by high-performance liquid chromatography, particularly reversed-phase chromatography.
本発明において、試料中の一級アミノ酸の発誘導体化は
、従来法と同様にプレカラム法により、オルトフタルア
ルデヒドを、β−メルカプトプロピオン酸の存在下に試
料中の一級アミノ酸に反応させて行われる。この場合、
反応液のｐＨ値を８乃至１０とすると反応が円滑に進行
するので好ましい。In the present invention, the derivatization of the primary amino acid in the sample is carried out by reacting orthophthalaldehyde with the primary amino acid in the sample in the presence of β-mercaptopropionic acid by a precolumn method similar to the conventional method. in this case,
It is preferable to set the pH value of the reaction solution to 8 to 10 because the reaction proceeds smoothly.
また、本発明において、試料中の二級アミノ酸の発誘導
体化は、誘導体化剤として４−クロロ７−ニトロベンゾ
−２−オキサ−１，３−ジアゾールを使用して、プレカ
ラム法により行われる。Further, in the present invention, derivatization of secondary amino acids in a sample is performed by a precolumn method using 4-chloro7-nitrobenzo-2-oxa-1,3-diazole as a derivatizing agent.
この二級アミノ酸の発蛍光誘導体化反応はｐＨ８乃至９
で行われるのが、反応の進行が円滑になって好ましい。This fluorescent derivatization reaction of secondary amino acids occurs at pH 8 to 9.
It is preferred that the reaction proceed smoothly.
逆相クロマトグラフィにより第１級アミノ酸及び第２級
アミノ酸の分離を行うのに、使用される固定相がオクタ
デシル基を有しており、移動相としてｐＨ６乃至７の緩
衝液と有機溶媒の混合液を使用するのが、第２級アミノ
酸を第１級アミノ酸と同時に検出することができるので
好ましい。The stationary phase used to separate primary and secondary amino acids by reversed phase chromatography has an octadecyl group, and the mobile phase is a mixture of a buffer solution with a pH of 6 to 7 and an organic solvent. It is preferable to use this method because secondary amino acids can be detected simultaneously with primary amino acids.
（ホ）作用
本発明は、−級アミノ酸の発誘導体化を、従来法と同様
に、β−メルカプトプロピオン酸の存在下においてオル
トフタルアルデヒドにより行い二級アミノ酸の発誘導体
化を、４−クロロ−７ニトロベンゾー２−オキサル１．
３−ジアゾールにより行うので、二級アミノ酸の保持時
間を一級アミノ酸の保持時間の範囲内に設定することが
できることとなり、二級アミノ酸の検出蛍光波長と一級
アミノ酸の検出蛍光波長帯域を異ならせることにより、
二級アミノ酸を一級アミノ酸と同時に蛍光検出すること
ができる。(E) Effect The present invention performs the derivatization of a -class amino acid with orthophthalaldehyde in the presence of β-mercaptopropionic acid in the same manner as the conventional method, and derivatizes a secondary amino acid with 4-chloro- 7 Nitrobenzo 2-oxal 1.
Since it is carried out using 3-diazole, the retention time of secondary amino acids can be set within the range of the retention time of primary amino acids, and by making the detection fluorescence wavelength of secondary amino acids and the detection fluorescence wavelength band of primary amino acids different. ,
Secondary amino acids can be fluorescently detected simultaneously with primary amino acids.
したがって、本発明によると、高速液体クロマトグラフ
ィ法により、−級アミノ酸の蛍光検出する時間内に二級
アミノ酸の蛍光検出を行うことができる。Therefore, according to the present invention, by high performance liquid chromatography, it is possible to detect the fluorescence of a secondary amino acid within the time it takes to detect the fluorescence of a -class amino acid.
くホ）実施例
以下、添付図面を参照して、本発明の実施の態様の−に
ついて説明するが、本発明は、以下の説明及び例示によ
って何等限定されるものではない。(iv) Examples Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings, but the present invention is not limited in any way by the following explanations and examples.
第１図は本発明の一実施例の高速液体クロマトグラフィ
装置の一例を示す概略の説明図であり。FIG. 1 is a schematic explanatory diagram showing an example of a high performance liquid chromatography apparatus according to an embodiment of the present invention.
第２図は第１図に示される実施例のクロマトクラムを示
す図である。FIG. 2 is a diagram showing the chromatogram of the embodiment shown in FIG. 1.
第１図の高速液体クロマトグラフィ分析装置１において
、メインカラム２は、内径６．００ ｍｗで長さ１５０
ｍｍの逆相クロマトグラフィ用カラムＳｈｉｍ−ｐａ
ｃｋ ＣＬＣ−ＯＤＳ（商品名：株式会社島津製作所製
）が、高温槽ＣＴＯ−６＾（商品名８株式会社島津製作
所製）３内に収容されて使用された。このメインカラム
２の溶出口４の下流側には、二基の蛍光分光光度計ＲＦ
−５３５（商品名１株式会社島津製作所製）５及び６が
設けられている。本例において、蛍光分光光度計５は一
級アミノ酸の検出用であって、励起波長か３４０ ｎｍ
で、検出蛍光波長は４６０ ｒ＋ｎに設定されており、
蛍光分光光度計６は二級アミノ酸の検出用であって、励
起波長が４９０ ｎｍで、検出蛍光波長５３０ ｎｍに
設定されている。In the high performance liquid chromatography analyzer 1 shown in FIG. 1, the main column 2 has an inner diameter of 6.00 mw and a length of 150 mw.
mm column for reversed phase chromatography Shim-pa
ck CLC-ODS (trade name: manufactured by Shimadzu Corporation) was housed in a high temperature tank CTO-6^ (trade name 8 manufactured by Shimadzu Corporation) 3 and used. On the downstream side of the elution port 4 of this main column 2, there are two fluorescence spectrophotometers RF.
-535 (product name 1 manufactured by Shimadzu Corporation) 5 and 6 are provided. In this example, the fluorescence spectrophotometer 5 is for detecting primary amino acids and has an excitation wavelength of 340 nm.
The detection fluorescence wavelength was set to 460 r+n,
The fluorescence spectrophotometer 6 is used for detecting secondary amino acids, and is set to have an excitation wavelength of 490 nm and a detection fluorescence wavelength of 530 nm.
一方、前記メインカラム２の流入ロアには、内径４．Ｏ
ｌ、長さ １０ ｍｍのガードカラムＳｈｉｍｐａｃｋ
Ｃ；−０ＤＳ （４） （商品名°株式会社島津製作
所製）８が接続されており、そのガードカラム８の流入
側９は、試料の注入及び前処理用のオートサンプラー５
ＩＬ−６Ｂ （商品名二株式会社島津製作所製）１０に
接続されている。オートサンプラー１０は、試料の導入
部と誘導体化を行うところであり、混合機能を備えてい
る。オートサンプラー１０の導入側１１には、メインカ
ラム２の保護用の、内径４．０ ｍｍ、長さ ２５０
ｍｍプレカラムＳｈ ｉｍｐａｃｋ ＧＲＤ−ＯＤＳ
（商品名６株式会社島津製作所製）１２が接続されてい
る。プレカラム１２の流入側１３は、ミキシングユニッ
ト１４の流出口１５に接続している。ミキシングユニッ
ト １４には、二つの流入口１６及び１７が形成されて
おり、夫々移動相送出ポンプＩ８及び１９の移動相流出
側２０及び２１に接続している。On the other hand, the inflow lower of the main column 2 has an inner diameter of 4. O
l, length 10 mm guard column Shimpack
C;-0DS (4) (Product name: Shimadzu Corporation) 8 is connected, and the inflow side 9 of the guard column 8 is connected to an autosampler 5 for sample injection and pretreatment.
It is connected to IL-6B (product name 2, manufactured by Shimadzu Corporation) 10. The autosampler 10 is a part for introducing and derivatizing a sample, and has a mixing function. The introduction side 11 of the autosampler 10 has an inner diameter of 4.0 mm and a length of 250 mm for protecting the main column 2.
mm precolumn Sh impack GRD-ODS
(Product name 6 manufactured by Shimadzu Corporation) 12 is connected. The inlet side 13 of the precolumn 12 is connected to the outlet 15 of the mixing unit 14 . Two inlets 16 and 17 are formed in the mixing unit 14 and are connected to mobile phase outlet sides 20 and 21 of mobile phase delivery pumps I8 and 19, respectively.
移動相送出ポンプ１８の移動相吸引側２２は、メチルセ
ロソルブ５ ｍｌを、くえん酸ナトリウム１０ ｍＭ浴
溶液ｐＨ６，８） ２容量とアセトニトリル１容量の溶
液に溶解して、全容を１１にした移動相２３に先端が浸
漬されている管路２４に接続している。The mobile phase suction side 22 of the mobile phase delivery pump 18 is used to transfer 5 ml of methyl cellosolve to a solution of 2 volumes of sodium citrate 10 mM bath solution (pH 6,8) and 1 volume of acetonitrile to bring the total volume to 11. It is connected to a conduit 24 whose tip is immersed in phase 23 .
他方の移動相送出ポンプ１９の移動相吸引側２５は、流
路切換弁ＦＣＶ−３＾Ｌ（商品名・株式会社島津製作所
製）２６の流出路２７に接続している。The mobile phase suction side 25 of the other mobile phase delivery pump 19 is connected to the outflow path 27 of a flow path switching valve FCV-3^L (trade name, manufactured by Shimadzu Corporation) 26.
流路切換弁２６の二つの流入側の流路２８及び２９の一
方の流路２８は、くえん酸ナトリウム１゜１溶液（ｐＨ
６，８）の移動相３０に先端が浸漬されている管路３１
に接続しており、他方の流入側流路２９は、洗浄用の８
０％アセトニトリル溶液３２に先端が浸漬されている管
路３３に接続している。この洗浄用のアセトニトリル溶
液は、分析終了時に分析装置内を清浄にするために流さ
れるものである。One of the two flow paths 28 and 29 on the inflow side of the flow path switching valve 26 is filled with a 1.1 sodium citrate solution (pH
A pipe line 31 whose tip is immersed in the mobile phase 30 of 6, 8)
, and the other inflow side flow path 29 is connected to 8 for cleaning.
It is connected to a conduit 33 whose tip is immersed in a 0% acetonitrile solution 32. This cleaning acetonitrile solution is flushed to clean the inside of the analyzer at the end of analysis.
本例においては、各移動相の送出量を制御すると共に、
恒温槽３の温度等を制御するためにンステムコントロー
ラ５ＣＬ−６８（商品名３株式会社島津製作所製）３４
が設けられている。また、二基の蛍光分光光度計５及び
６の蛍光検出データを処理して分析値を算出するように
、蛍光分光光度計５及び６に接続してデータ処理装置Ｃ
−Ｒ４八（商品名株式会社島津製作所製）３５が設けら
れている。In this example, while controlling the delivery amount of each mobile phase,
In order to control the temperature etc. of the constant temperature bath 3, a system controller 5CL-68 (product name 3 manufactured by Shimadzu Corporation) 34 is used.
is provided. Further, the data processing device C is connected to the fluorescence spectrophotometers 5 and 6 so as to process the fluorescence detection data of the two fluorescence spectrophotometers 5 and 6 to calculate an analysis value.
-R48 (trade name, manufactured by Shimadzu Corporation) 35 is provided.
本例は、上記第１図に示される逆相クロマトクラフイ装
置を用いて、血清中のアミノ酸について分析が行われた
。In this example, amino acids in serum were analyzed using the reversed phase chromatography apparatus shown in FIG. 1 above.
オートサンプラー１０に、血清試料１０μ！を吸引採取
し、これにβ−メルカプト試薬（β−メルカプトプロピ
オン酸１０μｌと １００ ｍＭはう酸塩緩衝液（ｐＨ
８，９＞ １０ ｍｌの混合液）２５０μｐを吸引混合
し、続いてオルト−フタルアルデヒド試薬〈オルト−フ
タルアルデヒド５ＩＩＩＩ？、エタノール３社及び１０
０ ＩＩＭのほう酸塩Ｍ樹液（ｐＨ８，９）１０ｍｌの
混合液）〈ＯＰΔ試薬＞２５０μＺを吸引混合して、第
一アミノ酸についての誘導体化が室温において行われた
。続いて、この試薬混合試料に、ＮＢＤ−ＣＩ試薬（４
−クロロ−７−ニトロベンゾ２−オキサ−１，３−ジア
ゾール１００ ｍｇとエタノール１０ ｍＲの混合液）
２５０μｌを加え混合する。混合操作は、混合液を吸引
し次いで吐出して行われた。４回混合後、混合液は、３
分間放置され注入された
一方、システムコントローラ３４により移動相送出用の
ポンプ１９を作動させ、移動相３０をメインカラム２に
送出する。Autosampler 10, serum sample 10μ! was collected by suction, and mixed with β-mercapto reagent (β-mercaptopropionic acid 10 μl and 100 mM phosphate buffer (pH
8,9 > 10 ml of mixture) 250 μp was mixed by suction, followed by ortho-phthalaldehyde reagent (ortho-phthalaldehyde 5III?). , 3 ethanol companies and 10
Derivatization for the first amino acid was carried out at room temperature by suction mixing a mixture of 10 ml of 0 IIM borate M sap (pH 8,9) (OPΔ reagent) 250 μZ. Next, NBD-CI reagent (4
-Mixture of 100 mg of -chloro-7-nitrobenzo2-oxa-1,3-diazole and 10 mR of ethanol)
Add 250 μl and mix. The mixing operation was performed by suctioning and then discharging the mixed liquid. After mixing 4 times, the mixture is 3
While the system controller 34 operates the mobile phase delivery pump 19, the mobile phase 30 is delivered to the main column 2.
前記の混合試料の１０μ！は、オートサンプラー １０
から分析装置１内に導入され、ガードカラム８を通して
移動相３０と共にメインカラム２内に流入し、分析が開
始される。10μ of the above mixed sample! is autosampler 10
It is introduced into the analyzer 1 through the guard column 8 and flows into the main column 2 along with the mobile phase 30, and analysis is started.
恒温槽３の温度は予め４５℃に、調整されている。移動
相の流量は１．０ ｍｅ ７分であり、システムコント
ローラ３４により、最初、移動相３０が分析装置内に流
され、次いで、グラジェント溶出が行われる。グラジェ
ント溶出のために、移動相送出ポンプ１８の作動が開始
され、移動相３０へ移動相２３の混合が開始される。移
動相３０と移動相２３の混合はミキシングユニット１６
において行われる。移動相３０への移動相２３の混合比
は、順次増加され、グラジェント溶出か行われる。The temperature of the constant temperature bath 3 is adjusted in advance to 45°C. The mobile phase flow rate is 1.0 me 7 minutes, and the system controller 34 first flows the mobile phase 30 into the analyzer and then performs gradient elution. For gradient elution, operation of the mobile phase delivery pump 18 is started, and mixing of the mobile phase 23 into the mobile phase 30 is started. The mobile phase 30 and the mobile phase 23 are mixed in the mixing unit 16.
It will be held in The mixing ratio of mobile phase 23 to mobile phase 30 is increased sequentially, and gradient elution is performed.
グラジェント溶出の移動相３０と移動相２３の混合比の
調整は、システムコントローラ３４により移動相送出ポ
ンプ１８及び１９の送出液量を制御して行われた。最終
的には、移動相送出ポンプ２６の作動が停止されて、移
動相２３のみがメインカラム２に送液された。The mixing ratio of the mobile phase 30 and the mobile phase 23 for gradient elution was adjusted by controlling the amount of liquid delivered by the mobile phase delivery pumps 18 and 19 by the system controller 34. Eventually, the operation of the mobile phase delivery pump 26 was stopped, and only the mobile phase 23 was delivered to the main column 2.
メインカラム２の溶出口４からの溶出液についての蛍光
検出は、蛍光分光光度計５及び６により行われた。−級
アミノ酸については、励起波長が３４０ ｎｍで、検出
蛍光波長が４６０ ｒｕｎで、蛍光分光光度計５により
測定された。二級アミン酸については、励起波長が４９
０ ｎｍで、検出蛍光波長が５３０ ｎｍで蛍光分光光
度計６により測定された。これらの測定結果は、データ
処理装置３３によりデータ処理されて、第２図に示され
るクロマトグラムが得られた。Fluorescence detection of the eluate from the elution port 4 of the main column 2 was performed using fluorescence spectrophotometers 5 and 6. - grade amino acids were measured using a fluorescence spectrophotometer 5 with an excitation wavelength of 340 nm and a detection fluorescence wavelength of 460 runs. For secondary amino acids, the excitation wavelength is 49
0 nm, and the detection fluorescence wavelength was measured by fluorescence spectrophotometer 6 at 530 nm. These measurement results were data processed by the data processing device 33 to obtain the chromatogram shown in FIG. 2.
第２図において、上段のクロマトグラムは一級アミノ酸
についてのクロマトグラムであり、下段は二級アミノ酸
についてのクロマトグラムである。In FIG. 2, the upper chromatogram is a chromatogram for primary amino acids, and the lower chromatogram is a chromatogram for secondary amino acids.
本例に示されるように、二級アミノ酸の溶出は、−級ア
ミノ酸の溶出と平行して、同時に測定されている。As shown in this example, the elution of secondary amino acids is measured simultaneously in parallel with the elution of -class amino acids.
本例において、二級アミノ酸の蛍光検出波長は、５３０
ｎｍであり、従来法における蛍光検出波長の３０５
ｎｍに比較して長波長であり夾雑物の影響を受は難い。In this example, the fluorescence detection wavelength of secondary amino acids is 530
nm, which is the fluorescence detection wavelength in the conventional method of 305 nm.
It has a longer wavelength than nm and is less susceptible to the effects of contaminants.
（ト）発明の効果
本発明は、−級アミノ酸の発話導体化を、従来法と同様
に、β−メルカプトプロピオン酸の存在下においてオル
トフタルアルデヒドにより行うが、二級アミノ酸の発話
導体化を、４−クロロ−７二トロベンゾー２−オキサ−
１３−ジアゾールにより行うので、従来の逆相クロマト
グラフィ法と比較して、アミノ酸−級及び二級アミノ酸
の分析時間を著しく短縮することができる。(G) Effects of the Invention In the present invention, the speech conductorization of -class amino acids is carried out using orthophthalaldehyde in the presence of β-mercaptopropionic acid, as in the conventional method, but the speech conductorization of secondary amino acids is 4-chloro-7nitrobenzo-2-oxa-
Since it is carried out using 13-diazole, the analysis time for amino acid-class and secondary amino acids can be significantly shortened compared to conventional reversed-phase chromatography methods.
第１図は本発明の一実施例の高速液体クロマトグラフィ
装置の一例を示す概略の説明図であり。
第２図は第１図に示される実施例のクロマトグラフィ
符号については、１は高速液体クロマトグラフィ分析装
置、２はメインカラム、３は恒温槽、４はメインカラム
の溶出口、５及び６は蛍光分光光度計、７はメインカラ
ムの流入口、８はガードカラム、９はガードカラムの流
入側、１０はオートサンプラー、１１はオートサンプラ
ーの導入側、１２はプレカラム、１３はプレカラムの流
入側、１４ミキシングユニツト、１５はミキシングユニ
ットの流出側、１６及び１７ミキシングユニツトの流出
口、１８及び１９は移動相送出ポンプ、２０及び２１は
移動相送出ポンプの流出側、２２及び２５は移動相流出
ポンプの移動相吸引側、２３は移動相、２４．３１及び
３３は管路、２６は流路切換弁、２７は流出路、２８及
び２９は流路切換弁の流路、３０は移動相、３２は洗浄
用のアセトニトリル溶液、３４はシステムコントローラ
、３５はデータ処理装置、である。
代
埋入FIG. 1 is a schematic explanatory diagram showing an example of a high performance liquid chromatography apparatus according to an embodiment of the present invention. FIG. 2 shows the chromatography codes of the example shown in FIG. 1. 1 is a high-performance liquid chromatography analyzer, 2 is a main column, 3 is a constant temperature bath, 4 is an elution port of the main column, and 5 and 6 are fluorescence spectrometers. Photometer, 7 is the inlet of the main column, 8 is the guard column, 9 is the inlet side of the guard column, 10 is the autosampler, 11 is the inlet side of the autosampler, 12 is the precolumn, 13 is the inlet side of the precolumn, 14 mixing 15 is the outflow side of the mixing unit, 16 and 17 are the outflow ports of the mixing unit, 18 and 19 are the mobile phase delivery pumps, 20 and 21 are the outflow sides of the mobile phase delivery pump, and 22 and 25 are the mobile phase outflow pumps. mobile phase suction side, 23 is a mobile phase, 24. 31 and 33 are pipes, 26 is a flow path switching valve, 27 is an outflow path, 28 and 29 are flow paths of the flow path switching valve, 30 is a mobile phase, 32 34 is a system controller, and 35 is a data processing device. substitution embedding
Claims (1)
ン酸の存在下にオルト−フタルアルデヒドと反応させて
発蛍光誘導体化させ、次いで、試料中の二級アミノ酸を
４−クロロ−７−ニトロベンゾ−２−オキサ−１，３−
ジアゾールと反応させて発蛍光誘導体化させ、この発蛍
光誘導体化された試料を逆相クロマトグラフィ相に通し
、該逆相クロマトグラフィ相から溶出する発蛍光誘導体
について、異なる二つの波長帯域において同時に蛍光検
出することを特徴とする生体アミノ酸分析方法。(1) The primary amino acids in the sample are reacted with ortho-phthalaldehyde in the presence of β-mercaptopropionic acid to produce a fluorescent derivative, and then the secondary amino acids in the sample are converted into 4-chloro-7-nitrobenzo-2 -Oxa-1,3-
The sample is reacted with a diazole to generate a fluorescent derivative, and the fluorescently derivatized sample is passed through a reversed phase chromatography phase, and the fluorescent derivative eluted from the reversed phase chromatography phase is simultaneously detected for fluorescence in two different wavelength bands. A biological amino acid analysis method characterized by the following.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2082277A JPH0481665A (en) | 1990-03-29 | 1990-03-29 | Method for analyzing bioamino acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2082277A JPH0481665A (en) | 1990-03-29 | 1990-03-29 | Method for analyzing bioamino acid |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH0481665A true JPH0481665A (en) | 1992-03-16 |
Family
ID=13770006
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2082277A Pending JPH0481665A (en) | 1990-03-29 | 1990-03-29 | Method for analyzing bioamino acid |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0481665A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006038674A (en) * | 2004-07-28 | 2006-02-09 | Kazuaki Kakehi | Analysis method of glycoprotein sugar chain, and manufacturing method of unlabeled sugar chain |
| JP2010071986A (en) * | 2008-08-20 | 2010-04-02 | Kazuhiro Imai | Fluorescence reagent |
| WO2010046624A1 (en) * | 2008-10-22 | 2010-04-29 | Oxtox Limited | Assay method for detecting primary amines |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5776455A (en) * | 1980-10-30 | 1982-05-13 | Kazuhiro Imai | Novel method for detection and determination of amino acid and/or amine |
| JPS6180051A (en) * | 1984-09-28 | 1986-04-23 | Toyo Soda Mfg Co Ltd | Analysis of amino acid |
| JPS62209055A (en) * | 1986-02-26 | 1987-09-14 | Yokogawa Hewlett Packard Ltd | Amino acid derivative mixture and production thereof |
-
1990
- 1990-03-29 JP JP2082277A patent/JPH0481665A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5776455A (en) * | 1980-10-30 | 1982-05-13 | Kazuhiro Imai | Novel method for detection and determination of amino acid and/or amine |
| JPS6180051A (en) * | 1984-09-28 | 1986-04-23 | Toyo Soda Mfg Co Ltd | Analysis of amino acid |
| JPS62209055A (en) * | 1986-02-26 | 1987-09-14 | Yokogawa Hewlett Packard Ltd | Amino acid derivative mixture and production thereof |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006038674A (en) * | 2004-07-28 | 2006-02-09 | Kazuaki Kakehi | Analysis method of glycoprotein sugar chain, and manufacturing method of unlabeled sugar chain |
| JP4568551B2 (en) * | 2004-07-28 | 2010-10-27 | 一晃 掛樋 | Method for analyzing glycoprotein sugar chain and method for producing unlabeled sugar chain |
| JP2010071986A (en) * | 2008-08-20 | 2010-04-02 | Kazuhiro Imai | Fluorescence reagent |
| WO2010046624A1 (en) * | 2008-10-22 | 2010-04-29 | Oxtox Limited | Assay method for detecting primary amines |
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