JPS62230761A - Method for separating hippuric acid and methylhippuric acid in urine - Google Patents
Method for separating hippuric acid and methylhippuric acid in urineInfo
- Publication number
- JPS62230761A JPS62230761A JP7313786A JP7313786A JPS62230761A JP S62230761 A JPS62230761 A JP S62230761A JP 7313786 A JP7313786 A JP 7313786A JP 7313786 A JP7313786 A JP 7313786A JP S62230761 A JPS62230761 A JP S62230761A
- Authority
- JP
- Japan
- Prior art keywords
- acid
- urine
- methylhippuric
- flow path
- hippuric acid
- 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
- QIAFMBKCNZACKA-UHFFFAOYSA-N N-benzoylglycine Chemical compound OC(=O)CNC(=O)C1=CC=CC=C1 QIAFMBKCNZACKA-UHFFFAOYSA-N 0.000 title claims abstract description 88
- 210000002700 urine Anatomy 0.000 title claims abstract description 31
- UAQVHNZEONHPQG-ZETCQYMHSA-N N-benzoyl-L-alanine Chemical compound OC(=O)[C@H](C)NC(=O)C1=CC=CC=C1 UAQVHNZEONHPQG-ZETCQYMHSA-N 0.000 title claims abstract description 30
- 238000000034 method Methods 0.000 title claims description 17
- 239000011347 resin Substances 0.000 claims abstract description 10
- 229920005989 resin Polymers 0.000 claims abstract description 10
- HVVWZTWDBSEWIH-UHFFFAOYSA-N [2-(hydroxymethyl)-3-prop-2-enoyloxy-2-(prop-2-enoyloxymethyl)propyl] prop-2-enoate Chemical compound C=CC(=O)OCC(CO)(COC(=O)C=C)COC(=O)C=C HVVWZTWDBSEWIH-UHFFFAOYSA-N 0.000 claims abstract description 9
- 239000007853 buffer solution Substances 0.000 claims abstract description 6
- 230000000717 retained effect Effects 0.000 claims description 5
- 238000000926 separation method Methods 0.000 abstract description 13
- 238000004128 high performance liquid chromatography Methods 0.000 abstract description 8
- 239000002253 acid Substances 0.000 abstract 1
- 239000000126 substance Substances 0.000 abstract 1
- 238000004458 analytical method Methods 0.000 description 18
- 238000004891 communication Methods 0.000 description 13
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 6
- 238000012856 packing Methods 0.000 description 5
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 4
- 238000007796 conventional method Methods 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 238000004140 cleaning Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 239000005711 Benzoic acid Substances 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 235000010233 benzoic acid Nutrition 0.000 description 2
- 239000000356 contaminant Substances 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000000945 filler Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- YKAKNMHEIJUKEX-UHFFFAOYSA-N m-methylhippuric acid Chemical compound CC1=CC=CC(C(=O)NCC(O)=O)=C1 YKAKNMHEIJUKEX-UHFFFAOYSA-N 0.000 description 2
- NRSCPTLHWVWLLH-UHFFFAOYSA-N p-methylhippuric acid Chemical compound CC1=CC=C(C(=O)NCC(O)=O)C=C1 NRSCPTLHWVWLLH-UHFFFAOYSA-N 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 229910000162 sodium phosphate Inorganic materials 0.000 description 2
- 239000001488 sodium phosphate Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 238000000638 solvent extraction Methods 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 2
- 230000002485 urinary effect Effects 0.000 description 2
- -1 1-methylhippuric acid Chemical compound 0.000 description 1
- 229920000858 Cyclodextrin Polymers 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- LEHOTFFKMJEONL-UHFFFAOYSA-N Uric Acid Chemical compound N1C(=O)NC(=O)C2=C1NC(=O)N2 LEHOTFFKMJEONL-UHFFFAOYSA-N 0.000 description 1
- TVWHNULVHGKJHS-UHFFFAOYSA-N Uric acid Natural products N1C(=O)NC(=O)C2NC(=O)NC21 TVWHNULVHGKJHS-UHFFFAOYSA-N 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 101150041626 ipi gene Proteins 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 230000003908 liver function Effects 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- YOEBAVRJHRCKRE-UHFFFAOYSA-N o-methylhippuric acid Chemical compound CC1=CC=CC=C1C(=O)NCC(O)=O YOEBAVRJHRCKRE-UHFFFAOYSA-N 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 235000004252 protein component Nutrition 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 1
- HFHDHCJBZVLPGP-UHFFFAOYSA-N schardinger α-dextrin Chemical compound O1C(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(O)C2O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC2C(O)C(O)C1OC2CO HFHDHCJBZVLPGP-UHFFFAOYSA-N 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229940116269 uric acid Drugs 0.000 description 1
Landscapes
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
Description
【発明の詳細な説明】
（イ）産業上の利用分野
本発明は、尿中の馬尿酸及Ｖ／又はメチル馬尿酸の分離
方法に関し、特に、尿中の馬尿酸又はメチル馬尿酸の分
析方法における馬尿酸又はメチル馬尿酸の分離方法に関
する。Detailed Description of the Invention (a) Industrial Application Field The present invention relates to a method for separating hippuric acid and V/or methylhippuric acid in urine, and in particular, a method for analyzing hippuric acid or methylhippuric acid in urine. The present invention relates to a method for separating hippuric acid or methylhippuric acid.
（ロ）従来の技術
肝臓は、安息香酸とグリシンから馬尿酸を合成する機能
を有しており、肝機能が障害されると、馬尿酸及びメチ
ル馬尿酸の生成量が減少するので、肝機能検査の馬尿酸
試験では、一定量の安息香酸を与えて、尿中に排泄され
る馬尿酸を測定している。この尿中の馬尿酸分析は、ま
ず、尿を酸性にして、尿中の馬尿酸を酢酸エチル等の溶
媒で抽出し、この抽出液から溶媒を蒸発し去って、これ
を移動相に溶解して、高速液体クロマトグラフィによっ
て分析することによって行われている。(b) Conventional technology The liver has the function of synthesizing hippuric acid from benzoic acid and glycine, and when liver function is impaired, the amount of hippuric acid and methylhippuric acid produced decreases. In the hippuric acid test, a certain amount of benzoic acid is given and the amount of hippuric acid excreted in the urine is measured. In this analysis of hippuric acid in urine, first, the urine is made acidic, the hippuric acid in the urine is extracted with a solvent such as ethyl acetate, the solvent is evaporated from this extract, and it is dissolved in a mobile phase. This is done by analyzing the results using high-performance liquid chromatography.
（ハ）発明が解決しようとする問題点
しかし、このような従来の尿中の馬尿酸分析においては
、高速液体クロマトグラフィ分析に先立って、尿中の馬
尿酸成分を、酢酸エチル等による溶媒抽出によって、蛋
白質成分と分離する工程を要しており、しかも、この工
程は操作が繁雑であって用手操作によらねばならず、ま
た、抽出効率も不安定であって内部標準による補正が不
可欠であり、熟練を要して問題であった。(c) Problems to be solved by the invention However, in such conventional analysis of hippuric acid in urine, the hippuric acid component in urine is extracted by solvent extraction with ethyl acetate etc. prior to high performance liquid chromatography analysis. However, this process requires a process to separate protein components, and this process is complicated and must be performed manually. Furthermore, the extraction efficiency is unstable and correction using an internal standard is essential. This was a problem because it required skill.
本発明は、このような従来の尿中の馬尿酸の分析法にお
ける尿中の馬尿酸の分離についての問題点を解決するこ
とを目的としている。The present invention aims to solve the problems associated with the separation of hippuric acid in urine in such conventional methods for analyzing hippuric acid in urine.
（ニ）問題点を解決するための手段
本発明は、尿中の馬尿酸及び／又はメチル馬尿酸の分析
に適し、推作が簡単で分離精度が高く、しかも、自動化
が容易な尿中の馬尿酸の分離法及び分析法を提供するも
のである。(d) Means for Solving the Problems The present invention is suitable for analyzing hippuric acid and/or methylhippuric acid in urine, is easy to estimate, has high separation accuracy, and is easy to automate. The present invention provides a method for separating and analyzing hippuric acid.
すなわち、本発明は、尿中の馬尿酸又はメチル馬尿酸を
分離する方法において、尿をＰＨ２〜４の緩衝溶液の存
在下に、テトラメチロールメタントリアクリレート架橋
樹脂と接触させ、馬尿酸又はメチル馬尿酸を該樹脂に保
持させることを特徴とする尿中の馬尿酸又はメチル馬尿
酸の分離方法にある。That is, the present invention provides a method for separating hippuric acid or methyl hippuric acid in urine by contacting urine with a tetramethylolmethane triacrylate crosslinked resin in the presence of a buffer solution of pH 2 to 4, and separating hippuric acid or methyl hippuric acid from urine. A method for separating hippuric acid or methylhippuric acid in urine, which comprises retaining uric acid in the resin.
本発明において、尿の用語は希釈尿又は尿を意味し、こ
こで使用されるテトラメチロールメタントリアクリレー
ト架橋樹脂は、高速液体クロマトグラフィ充填剤として
市販のものが使用できる。In the present invention, the term "urine" means diluted urine or urine, and the tetramethylolmethane triacrylate crosslinked resin used here can be one commercially available as a high performance liquid chromatography packing material.
このような充填剤を充填した高速液体クロマトグラフィ
分離用カラムとしては、例えば、Ｓｈｉｍ−ｐａｃｋ
５ＰＣ−ＲＰ １をあげることができる。この場合、
移動相のｐＨは約２乃至４の範囲とされる。ＰＨ４以上
のｐＨ領域では、馬尿酸及びメチル馬尿酸の保持が充分
でなく、ｐＨ２以下の酸性領域では、テトラメチロール
メタントリアクリレート架橋樹脂充填剤が損傷される。Examples of high-performance liquid chromatography separation columns filled with such a packing material include Shim-pack.
5PC-RP 1 can be given. in this case,
The pH of the mobile phase ranges from about 2 to 4. In a pH range of 4 or higher, retention of hippuric acid and methylhippuric acid is insufficient, and in an acidic range of pH 2 or lower, the tetramethylolmethane triacrylate crosslinked resin filler is damaged.
したがって、本発明では、緩衝溶液の存在下に尿中の馬
尿酸及び／又はメチル馬尿酸の分離が行われる。Therefore, in the present invention, hippuric acid and/or methylhippuric acid in urine is separated in the presence of a buffer solution.
緩衝溶液としては、級！ＩｐＨ値が２乃至４の範囲内に
あれば、如何なる緩衝溶液も使用することができる。As a buffer solution, it's class! Any buffer solution with an IpH value in the range 2-4 can be used.
（ホ）作用
本発明は、尿中の馬尿酸及び／又はメチル馬尿酸を直接
例えばカラムに充填されたテトラメチロールメタントリ
アクリレート架橋樹脂に保持させて、尿中馬尿酸及びメ
チル馬尿酸を馬尿酸及びメチル馬尿酸より極性の高い夾
雑物蛋白質等と分離できるので、この保持された馬尿酸
及び／又はメチル馬尿酸を、移動相に溶出させて、高速
液体クロマドグ９フ４分離用カラムに導入して分離分析
を行うことができる。(e) Effect The present invention directly retains urinary hippuric acid and/or methylhippuric acid, for example, in a tetramethylolmethane triacrylate crosslinked resin packed in a column, thereby converting urinary hippuric acid and methylhippuric acid into hippuric acid. The retained hippuric acid and/or methylhippuric acid can be separated from proteins and other contaminants that are more polar than methylhippuric acid, so the retained hippuric acid and/or methylhippuric acid is eluted into the mobile phase and introduced into a high performance liquid chromadog 9F 4 separation column. Separation and analysis can be performed using
したがって、本発明においては、カラムへの液の移動繰
作により簡単に処理操作を行うことができる。Therefore, in the present invention, processing operations can be easily performed by moving the liquid to the column.
（へ）実施例
以下、添付図面を参照して本発明の馬尿酸及１メチル馬
尿酸の分離方法の一実施例の態様について説明するが、
本発明は、この説明及び例示により何ら制限されるもの
ではない。(F) Example Hereinafter, an embodiment of the method for separating hippuric acid and 1-methylhippuric acid of the present invention will be described with reference to the accompanying drawings.
The present invention is not limited in any way by this description and exemplification.
第１図は、本発明の一実施例を示す流路図であり、高速
液体クロマトグフフイ分析装置に適用した一例を示す。FIG. 1 is a flow path diagram showing an embodiment of the present invention, and shows an example of application to a high performance liquid chromatography analyzer.
本例の装置は、試料導入部１、六方流路切換パルプ２、
前処理カラム３、分析カラム４、検出器５を備えている
。試料導入部１は、導入された尿検体を前処理カラム３
に導入するために、前処理用移動相容器６及ゾボンプ７
に流路８を介して接続している。本例の流路８には、洗
浄用移動相容器９に流路８を連通させるための流路切換
バルブ１１と分析カラム４の充填物と同一の充填物が充
填されたプレカラム１０が設けられている。流路８他端
は、六方流路切換バルブ２の流路１２に接続している。The device of this example includes a sample introduction section 1, a hexagonal flow path switching pulp 2,
It is equipped with a pretreatment column 3, an analysis column 4, and a detector 5. The sample introduction section 1 transfers the introduced urine sample to the pretreatment column 3.
In order to introduce the pretreatment mobile phase container 6 and Zobonp 7
It is connected to via a flow path 8. The flow path 8 in this example is provided with a flow path switching valve 11 for communicating the flow path 8 with the cleaning mobile phase container 9, and a precolumn 10 filled with the same packing as the analytical column 4. ing. The other end of the flow path 8 is connected to a flow path 12 of the hexagonal flow path switching valve 2 .
六方流路切換パルプ２には、連絡路１３を有するロータ
が備えられている。ロータの回動により、連絡路１３は
実線の位置と鎖線の位置をとることができる。連絡路１
３が実線の位置にあるときは分析段階であり、鎖線の位
置にあるときは、前処理段階又は前処理カラムの洗浄段
階である。六方流路切換バルブの流路１２は連絡路１３
を介して流路１４から排出路１５に連通している。The hexagonal channel switching pulp 2 is equipped with a rotor having a communication channel 13. By rotating the rotor, the communication path 13 can take the position shown by the solid line and the position shown by the chain line. Communication route 1
When 3 is at the solid line position, it is the analysis stage, and when it is at the chain line position, it is the pretreatment stage or the pretreatment column washing stage. The flow path 12 of the hexagonal flow path switching valve is the communication path 13
The flow path 14 communicates with the discharge path 15 via.
分析用移動相容器１６は流路１７を介してポンプ１８に
連通しており、また流路１７には、前処理カラム３の充
填物と同一の充填物が充填されたプレカラム１９が設け
られている。流路１７は、六方流路切換バルブ２の流路
２０に接続しており、この流路２０は、ロータの連絡路
１３を介して、流路２１に連通している。前処理カラム
３の入口は六方流路切換パルプ２の流路２１に流路２２
を介して接続しており、前処理カラム３の出口は、六方
流路切換パルプ２の流路２３に流路２４を介して接続し
ている。六方流路切換パルプの残る流路２５は、流路２
６を介して、分析カラム４に接続している。分析カラム
４の出口側は流路２７に接続しており、この流路２７に
は検出器５が設けられている。この流路２７は検出器５
を過ぎて排出路２８に接続している。The analytical mobile phase container 16 communicates with a pump 18 via a channel 17, and the channel 17 is provided with a precolumn 19 filled with the same packing as the pretreatment column 3. There is. The flow path 17 is connected to a flow path 20 of the hexagonal flow path switching valve 2, and this flow path 20 communicates with a flow path 21 via the communication path 13 of the rotor. The inlet of the pretreatment column 3 is connected to the flow path 21 of the hexagonal flow path switching pulp 2 through the flow path 22.
The outlet of the pretreatment column 3 is connected to the channel 23 of the hexagonal channel switching pulp 2 via a channel 24. The channel 25 where the hexagonal channel switching pulp remains is the channel 2
6 to the analytical column 4. The outlet side of the analytical column 4 is connected to a flow path 27, and this flow path 27 is provided with a detector 5. This flow path 27 is connected to the detector 5
It passes through and connects to the discharge path 28.
本例は以上のように構成されており、前処理カラム３に
は、テトラメチロールメタントリアクリレート架１１ｉ
１！脂を充填剤として充填したものであり、例えばＳｈ
ｉｍ−ｐａｃｋ ５ＰＣ−ＲＰ ｉカラム、内径４ｍ
、長さ３０ｍが使用されている。This example is constructed as described above, and the pretreatment column 3 includes a tetramethylolmethane triacrylate rack 11i.
1! Filled with fat as a filler, for example Sh
im-pack 5PC-RP i column, inner diameter 4m
, a length of 30 m is used.
分析カラム４は逆相液体クロマトグラフィ分離用カラム
であり、例えば、ＯＤＳシリカを充填剤とするものであ
り、例えば、Ｓｈｉｍ−ｐａｃｋＣＬＯ−ＯＤＳカラム
、内径６ｍ、氏さ１５０覇が使用されている。検出器５
は紫外吸収計であり、測定波長は２４０覇ｍである。The analytical column 4 is a column for reversed-phase liquid chromatography separation, and uses, for example, ODS silica as a packing material, such as a Shim-pack CLO-ODS column with an inner diameter of 6 m and a height of 150 cm. Detector 5
is an ultraviolet absorption meter, and the measurement wavelength is 240 ham.
前処理に先立って、六方流路切換パルプ２は、ロータを
回して、連絡路１３をＭＩＩＡの位置にしである。Prior to pretreatment, the hexagonal flow path switching pulp 2 is prepared by rotating the rotor to set the communication path 13 to the MIIA position.
５０４の尿検体は、試料導入部１から、ＰＨ２，１の１
００ｍＭのリン酸ナトリウム溶液の前処理移動相中に導
入される。該移動相の流量は、１−ｎ７分である。尿検
体は、前処理移動相と共に、流路１２、連絡路１３、流
路２１及び流路２２を経由して、前処理カラム３に導入
される。前処理カラム３に馬尿酸及びメチル馬尿酸が保
持されたところで、六方流路切換バルブ２のロータを切
換えて、連絡路１３を実線の位置にする。１０ｍＭのβ
−シクロデキストリンをｐＨ２，１の１００ｍＭのリン
酸ナトリウム溶液に混合した溶液２部に対しメタノール
１部を混合した混合溶液の分析用移動相は、容器１６か
らポンプ１８によって、プレカラム１９及び流路１７か
ら、六方流路切換バルブの流路２０に入り、流路１２、
流路２１及び流路２２から前処理カラム３に入り、保持
されている馬尿酸及びメチル馬尿酸を溶出する。分析用
移動相の流量は１ｄ／分である。溶出液は、流路２５、
六方流路切換パルプ２の流路２３、連絡路１３及び流路
２５を経て、分析カラム４に入り、互に分離さ°れて流
路２７から流出し、検出器の紫外吸収 、計５により測
定されろ。分析結果は、第３図に示される。The urine sample No. 504 is transferred from the sample introduction section 1 to a pH of 2.1.
00 mM sodium phosphate solution is introduced into the pretreatment mobile phase. The flow rate of the mobile phase is 1-n7 minutes. The urine specimen is introduced into the pretreatment column 3 along with the pretreatment mobile phase via the flow path 12, the communication path 13, the flow path 21, and the flow path 22. When hippuric acid and methylhippuric acid are retained in the pretreatment column 3, the rotor of the hexagonal flow path switching valve 2 is switched to set the communication path 13 to the position shown by the solid line. 10mM β
- An analytical mobile phase of a mixed solution of 1 part of methanol mixed with 2 parts of a solution of cyclodextrin in a 100 mM sodium phosphate solution at pH 2.1 is transferred from the container 16 to the precolumn 19 and the flow path 17 by the pump 18. , enters the flow path 20 of the hexagonal flow path switching valve, flows into the flow path 12,
It enters the pretreatment column 3 through the channels 21 and 22, and the retained hippuric acid and methylhippuric acid are eluted. The flow rate of the analytical mobile phase is 1 d/min. The eluate flows through the channel 25,
The pulp enters the analysis column 4 through the flow path 23, communication path 13, and flow path 25 of the hexagonal flow path switching pulp 2, is separated from each other, flows out from the flow path 27, and is absorbed by the ultraviolet absorption of the detector. Be measured. The analysis results are shown in FIG.
第２図は、馬尿酸、Ｏ−メチル馬尿酸、ｍ−メチル馬尿
酸、ｐ−メチル馬尿酸の標品各ＩＰｇについての分離デ
ータである。FIG. 2 shows separation data for each IPg of hippuric acid, O-methylhippuric acid, m-methylhippuric acid, and p-methylhippuric acid.
馬尿酸又はメチル馬尿酸が、前処理カラム３から溶出し
終えた段階で、六方流出切換パルプのロータを回して、
連絡路を点線の位置し、分析用移動相を、流路１７、流
路２０、連絡路１３、流路２５及び流路２６と流して、
分析カラム５に流入させて、馬尿酸及びメチル馬尿酸の
分析を続ける。When hippuric acid or methyl hippuric acid has finished eluting from the pretreatment column 3, the rotor of the hexagonal flow switching pulp is rotated.
The communication channel is positioned as indicated by the dotted line, and the mobile phase for analysis is caused to flow through channel 17, channel 20, communication channel 13, channel 25, and channel 26,
Flow into analytical column 5 to continue analysis of hippuric acid and methylhippuric acid.
一方、この開に、流路切換パルプ１０のロータを時計廻
りに９０°回して、洗浄用移動相容器９内の洗浄用移動
相を流路８、流路１２、連絡路１３、流路２１及び流路
２２から前処理カラム３に流して、馬尿酸及びメチル馬
尿酸より極性の低い夾雑物を該カラムで洗浄する。洗浄
し終えたところで、流路切換パルプ１０のロータを反時
計廻りに回して、前処理用移動相容器６から前処理用移
動相を前処理カラム３に送り、該カラムを再生し、次の
尿検体の分離に備える。On the other hand, in this opening, the rotor of the flow path switching pulp 10 is rotated 90 degrees clockwise to transfer the cleaning mobile phase in the cleaning mobile phase container 9 to the flow path 8, the flow path 12, the communication path 13, and the flow path 21. It is then flowed through the flow path 22 to the pretreatment column 3, and impurities having a polarity lower than that of hippuric acid and methylhippuric acid are washed in the column. When washing is finished, the rotor of the flow path switching pulp 10 is rotated counterclockwise to send the pretreatment mobile phase from the pretreatment mobile phase container 6 to the pretreatment column 3, regenerate the column, and perform the next process. Prepare for separation of urine specimen.
（ト）発明の効果
本発明は、馬尿酸及び／又はメチル馬尿酸を直接、例え
ばカラムに充填されたテトラメチロールメタントリアク
リレート架橋ｍｍに保持させて、尿中の馬尿酸及びメチ
ル馬尿酸をこれらよｌ）極性の高い夾雑物や蛋白質等と
分離させるので、従来法における溶媒抽出のような複雑
な前処理工程を必要としない。したがって、分離時間が
著しく短縮される。(G) Effects of the Invention The present invention directly retains hippuric acid and/or methylhippuric acid in, for example, tetramethylolmethane triacrylate crosslinked mm packed in a column, thereby removing hippuric acid and methylhippuric acid from urine. y) Since highly polar contaminants and proteins are separated, there is no need for complicated pretreatment steps such as solvent extraction in conventional methods. The separation time is therefore significantly reduced.
しかも、本発明においては、前処理工程を簡単な操作で
カラム内で行う上に、更に前処理された試料のカラム間
の移動をパルプ操作で簡単に行えるので、高速液体クロ
マトグラフィ分析に前処理手段として適用すると、分析
工程全体の操作が、簡単なパルプ操作等で行えることに
なり、分析時間が短縮されると共に本分析工程の自動化
が容易となる。Furthermore, in the present invention, the pretreatment step can be carried out within the column with a simple operation, and the pretreated sample can be easily transferred between columns using a pulp operation. When applied as a method, the entire analysis process can be performed by simple pulp manipulation, etc., which shortens analysis time and facilitates automation of the analysis process.
また、本発明においては、分離工程がカラムクロマトグ
ラフィ分＃Ｉ′Ｐｊ、作で行われるために、従来法に比
して、分離効率が高く、高速液体クロマトグラフィ分析
に、前処理手段として適用した場合、分析精度の向上を
はかることができ、内部標準を必要とするには至らない
。In addition, in the present invention, since the separation step is performed by column chromatography fraction #I'Pj, the separation efficiency is higher than that of conventional methods, and when applied as a pretreatment means to high performance liquid chromatography analysis. , it is possible to improve analytical accuracy, and it does not require an internal standard.
以上のように、本発明は、従来法に比してすぐれており
、分離手段、精製手段又は分析手段等として、その及ぼ
す影響は大きい。As described above, the present invention is superior to conventional methods, and has a great influence as a separation means, purification means, analysis means, etc.
第１図は、本発明の一実施例を示す流路図であり、高速
液体クロマトグラフィ分析装置に適用した一例を示す。
第２図は、馬尿酸、０−メチル馬尿酸、ｍ−メチル馬尿
酸、ｐ−メチル馬尿酸の楳品各ＩＰＩ！についての分離
データである。、第３図は、分析結果である。
１は試料導入部、２は六方流路切換パルプ、３は前処理
カラム、４は分析カラム、５は検出器又は紫外吸収計、
６は前処理用移動相容器、７及び１８はポンプ、８．１
２．１４．１７，２０゜２１．２２，２３．２４，２５
．２６及び２７は流路、９は洗浄用移動相容器、１０は
流路切換パルプ、１１及び１９はプレカラム、１３は連
絡路、１５及び２８は排出路、１６は分析用移動相容器
である。FIG. 1 is a flow path diagram showing an embodiment of the present invention, and shows an example of application to a high performance liquid chromatography analyzer. Figure 2 shows the IPIs of hippuric acid, 0-methylhippuric acid, m-methylhippuric acid, and p-methylhippuric acid. This is separated data about . , FIG. 3 shows the analysis results. 1 is a sample introduction part, 2 is a hexagonal flow path switching pulp, 3 is a pretreatment column, 4 is an analysis column, 5 is a detector or an ultraviolet absorption meter,
6 is a mobile phase container for pretreatment, 7 and 18 are pumps, 8.1
2.14.17, 20°21.22, 23.24, 25
．． 26 and 27 are channels, 9 is a mobile phase container for washing, 10 is a channel switching pulp, 11 and 19 are pre-columns, 13 is a communication channel, 15 and 28 are discharge channels, and 16 is a mobile phase container for analysis.
Claims (1)
、尿をｐＨ２〜４の緩衝溶液の存在下に、テトラメチロ
ールメタントリアクリレート架橋樹脂と接触させ、馬尿
酸又はメチル馬尿酸を該樹脂に保持させることを特徴と
する尿中の馬尿酸又はメチル馬尿酸の分離方法。In a method for separating hippuric acid or methylhippuric acid in urine, urine is brought into contact with a tetramethylolmethane triacrylate crosslinked resin in the presence of a buffer solution of pH 2 to 4, and hippuric acid or methylhippuric acid is retained in the resin. A method for separating hippuric acid or methylhippuric acid in urine, which comprises:
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP7313786A JPS62230761A (en) | 1986-03-31 | 1986-03-31 | Method for separating hippuric acid and methylhippuric acid in urine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP7313786A JPS62230761A (en) | 1986-03-31 | 1986-03-31 | Method for separating hippuric acid and methylhippuric acid in urine |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPS62230761A true JPS62230761A (en) | 1987-10-09 |
Family
ID=13509514
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP7313786A Pending JPS62230761A (en) | 1986-03-31 | 1986-03-31 | Method for separating hippuric acid and methylhippuric acid in urine |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS62230761A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2013066390A (en) * | 2011-09-20 | 2013-04-18 | Nipro Corp | Method for measuring each concentration of hippuric acid and methylhippuric acid in biological sample |
-
1986
- 1986-03-31 JP JP7313786A patent/JPS62230761A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2013066390A (en) * | 2011-09-20 | 2013-04-18 | Nipro Corp | Method for measuring each concentration of hippuric acid and methylhippuric acid in biological sample |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| McDowall | Sample preparation for biomedical analysis | |
| CN110208401B (en) | Online analysis system and method of solid phase dehydration extraction-supercritical fluid chromatography-mass spectrometry | |
| CN102262163B (en) | Rapid and automatic determination method and device for tripolycyanamide content in dairy products | |
| US5135718A (en) | Apparatus for simultaneously analyzing vanillylmandelic acid, homovanillic acid and creatinine | |
| JPH05281222A (en) | Method and apparatus for analysis of hemoglobin and column deterioration-suppressing liquid used for them | |
| JPS6263858A (en) | Method and instrument for analyzing bitterness component derived from hop | |
| AU1420997A (en) | Multi-cycle loop injection for trace analysis by ion chromatography apparatus and method | |
| Lingeman et al. | Sample pretreatment procedures | |
| JPH01126544A (en) | Biochemical analysis method and apparatus | |
| JPS62230761A (en) | Method for separating hippuric acid and methylhippuric acid in urine | |
| Karger et al. | Automated sample cleanup in HPLC using column-switching techniques | |
| EP0359322A2 (en) | Liquid chromatography | |
| Mao et al. | Online extraction and column switching techniques in LC‐MS bioanalysis | |
| CN107014935A (en) | A kind of IgG sugar-type detection batch pre-treating method of blood plasma or serum | |
| JP3092627B2 (en) | High-performance liquid chromatograph | |
| CN101358946A (en) | Anionic polymer grafting coatings capillary pipe and analytical method for on-line enrichment for protein | |
| Henzel et al. | Reversed‐phase isolation of peptides | |
| JP3628495B2 (en) | Catecholamine analysis method and analyzer | |
| JPH0797111B2 (en) | Method for analyzing vanillyl mandelic acid and homovanillic acid | |
| Stegehuis et al. | Bioanalysis of the peptide de-enkephalin-γ-endorphin: On-line sample pretreatment using membrane dialysis and solid-phase isolation | |
| Turnell et al. | Automated pre-column derivatization and its application to amino-acid analysis using high-performance liquid chromatography | |
| JP2001056326A (en) | Method for refining and measuring serotonin and 5- hydroxyindole acetic acid | |
| JPH02141658A (en) | Analysis of biochemical component and apparatus therefor | |
| Farouk et al. | Sensitivity Enhancement for Separation-Based Analytical Techniques Utilizing Large Volume Injection and Analyte Derivatization for Trace Analysis in Bio-Fluids | |
| Wells | Sample preparation for mass spectrometry |