WO2014046163A1 - Procédé de mesure de l'acide 2-amino-3-[2-(α-mannopyranosyl)indol-3-yl]propionique dans un échantillon - Google Patents

Procédé de mesure de l'acide 2-amino-3-[2-(α-mannopyranosyl)indol-3-yl]propionique dans un échantillon Download PDF

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Publication number
WO2014046163A1
WO2014046163A1 PCT/JP2013/075260 JP2013075260W WO2014046163A1 WO 2014046163 A1 WO2014046163 A1 WO 2014046163A1 JP 2013075260 W JP2013075260 W JP 2013075260W WO 2014046163 A1 WO2014046163 A1 WO 2014046163A1
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WIPO (PCT)
Prior art keywords
man
trp
sample
measuring
phase hplc
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PCT/JP2013/075260
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English (en)
Japanese (ja)
Inventor
晃 堀口
修 草田
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協和メデックス株式会社
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Priority to JP2014536893A priority Critical patent/JP6193241B2/ja
Publication of WO2014046163A1 publication Critical patent/WO2014046163A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/32—Bonded phase chromatography
    • B01D15/325—Reversed phase
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
    • B01J20/287—Non-polar phases; Reversed phases
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
    • G01N2030/8818—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/89—Inverse chromatography

Definitions

  • the present invention relates to a method for measuring amino-3- [2- ( ⁇ -mannopyranosyl) indol-3-yl] propionic acid in a sample by high performance liquid chromatography.
  • Trp-Man 2-Amino-3- [2- ( ⁇ -mannopyranosyl) indol-3-yl] propionic acid
  • Trp-Man 2-Amino-3- [2- ( ⁇ -mannopyranosyl) indol-3-yl] propionic acid
  • Trp-Man is known to serve as an index of biological functions, particularly kidney function, central nervous function, fetal growth function, and glomerular filtration rate (see Patent Document 2).
  • HPLC high performance liquid chromatography
  • impurities in the specimen are often present.
  • accurate measurement was not possible.
  • various fluorescent contaminants contained in urine often hinder accurate measurement of Trp-Man.
  • An object of the present invention is to provide a method for accurately measuring Trp-Man in a sample by reverse phase HPLC.
  • the present inventors used a mobile phase containing a sulfonic acid-based ion-pairing reagent as a mobile phase, whereby impurities in the sample and Trp-
  • the present invention was completed by finding the knowledge that Man can be efficiently separated. That is, the present invention relates to the following [1] to [4].
  • [1] A method for measuring Trp-Man in a sample by reversed-phase HPLC, wherein a mobile phase containing a sulfonic acid ion-pairing reagent is used as the mobile phase.
  • the present invention provides an accurate method for measuring Trp-Man in a sample by reversed-phase HPLC.
  • FIG. 3 shows a chromatogram of measurement of Trp-Man in a urine sample by reverse phase HPLC using a mobile phase containing sodium 1-hexanesulfonate as an ion pair reagent.
  • 2 shows a chromatogram of Trp-Man measurement in a serum sample by reversed-phase HPLC using a mobile phase containing sodium 1-hexanesulfonate as an ion-pairing reagent.
  • the sample in the present invention is not particularly limited as long as it can measure Trp-Man by HPLC of the present invention, and examples thereof include urine, whole blood, serum, plasma, spinal fluid, amniotic fluid, cell extract and the like. Urine and serum are preferred.
  • a biological sample itself or a pretreated biological sample may be used as the specimen.
  • pretreatment of biological specimens include centrifugation, filter treatment, reducing agent treatment, and acid treatment.
  • reducing agent used by a reducing agent process ascorbic acid etc. are mentioned, for example.
  • the acid used in the acid treatment include hydrochloric acid and perchloric acid.
  • the reverse phase HPLC in the present invention is not particularly limited as long as it is a reverse phase HPLC capable of measuring Trp-Man.
  • the stationary phase in reversed phase HPLC include silica gel bonded with an octadecylsilyl (ODS) group.
  • the mobile phase in reverse phase HPLC is a mobile phase containing a sulfonic acid ion pair reagent, and examples thereof include a mixed solvent of an organic solvent and a buffer solution containing the ion pair reagent.
  • the organic solvent include acetonitrile.
  • the buffer solution examples include a phosphate buffer solution, a methanesulfonate buffer solution, a formate buffer solution, an acetate buffer solution, and a citrate buffer solution.
  • the ratio of the organic solvent to the buffer in the mobile phase can be selected as appropriate, and the ratio can be changed to obtain a mobile phase having a concentration gradient.
  • a known detection method such as fluorescence, light absorption, differential refraction, electrochemical detection or the like is used.
  • the measurement of Trp-Man in a sample by reverse phase HPLC may be a method using an internal standard material or a method not using an internal standard material.
  • an internal standard substance in the method using the internal standard substance for example, a fluorescent substance such as N-methyl serotonin is used.
  • the measurement of Trp-Man in a specimen using an internal standard substance can be performed, for example, by the following steps.
  • a step of preparing a specimen by adding an internal standard substance of a known concentration to a biological sample or a biological sample subjected to the above pretreatment (2) A step of subjecting the specimen prepared in step (1) to reverse phase HPLC; (3) a step of calculating the area of the peak corresponding to Trp-Man and the area of the peak corresponding to the internal standard substance in the obtained chromatogram; (4) A step of determining the concentration of Trp-Man in the sample from the ratio of the peak area corresponding to Trp-Man calculated in step (3) and the peak area corresponding to the internal standard substance.
  • Measurement of Trp-Man in a specimen without using an internal standard substance can be performed, for example, by the following steps. (1) preparing a standard solution of Trp-Man at a known concentration; (2) subjecting the standard solution prepared in step (1) to reverse phase HPLC; (3) a step of calculating an area of a peak corresponding to Trp-Man in the obtained chromatogram; (4) A step of creating a calibration curve representing the relationship between the Trp-Man concentration and the peak area corresponding to Trp-Man from the area of the peak corresponding to Trp-Man calculated in step (3); (5) A step of subjecting the specimen to HPLC; (6) a step of calculating an area of a peak corresponding to Trp-Man in the obtained chromatogram; (7) A step of determining the Trp-Man concentration in the specimen by comparing the calibration curve created in the step (4) with the area calculated in the above (6).
  • Examples of the sulfonic acid ion-pairing reagent in the present invention include alkyl sulfonates, aryl sulfonates, alkyl sulfates, and the like, and alkyl sulfonates are preferable.
  • Examples of the alkyl sulfonate include propane sulfonate, butane sulfonate, pentane sulfonate, hexane sulfonate, heptane sulfonate, octane sulfonate, nonane sulfone having an alkyl having 3 to 18 carbon atoms.
  • Acid salt decane sulfonate, undecane sulfonate, dodecane sulfonate, tridecane sulfonate, tetradecane sulfonate, pentadecane sulfonate, hexadecane sulfonate, heptadecane sulfonate, octadecane sulfonate, etc. Is mentioned.
  • the aryl sulfonate include 2-naphthalene sulfonate.
  • alkyl sulfate ester salt include dodecyl sulfate.
  • the salt in the alkyl sulfonate, aryl sulfonate, and alkyl sulfate include sodium salt and potassium salt.
  • the concentration of the ion-pairing reagent in the mobile phase is not particularly limited as long as it allows the measurement of Trp-Man. For example, it is 0.005 to 1.0 mol / L, and preferably 0.01 to 0.5 mol / L.
  • Trp-Man was dissolved in distilled water to prepare an aqueous solution of 1 mg / mL Trp-Man.
  • Trp-Man solutions, an aqueous solution of 1 mg / mL Trp-Man and the mobile phase (Trp-Man 0 mg / mL) in (2) above were used as standard solutions.
  • Trp-Man peak was well separated from the peaks of other fluorescent substances. Therefore, it was found that Trp-Man in a urine sample can be accurately measured by reversed-phase HPLC using a mobile phase containing an ion pair reagent.
  • Trp-Man in a serum sample by reversed-phase HPLC As a sample, instead of the urine sample prepared in (4) of Example 1, a serum sample prepared by the following method was used. By the method, Trp-Man in the serum sample was measured by reverse phase HPLC. The chromatogram obtained by reverse phase HPLC is shown in FIG.
  • Trp-Man peak was well separated from the peaks of other fluorescent substances in reversed-phase HPLC using a mobile phase containing an ion-pairing reagent. Therefore, it was found that Trp-Man in a serum sample can be accurately measured by reverse phase HPLC using a mobile phase containing an ion pair reagent.
  • the present invention provides a method for measuring Trp-Man in a specimen by reverse phase HPLC.
  • the measurement method of the present invention is effective for diagnosis of kidney function and the like.

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  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)

Abstract

L'invention concerne un procédé de mesure de l'acide 2-amino-3-[2-(α-mannopyranosyl)indol-3-yl]propionique (référé en tant que « Trp-Man », ci-après) dans un échantillon de façon précise par chromatographie liquide haute performance en phase inverse (référée en tant que « HPLC en phase inverse », ci-après). L'invention concerne un procédé de mesure de Trp-Man dans un échantillon par HPLC en phase inverse, ledit procédé étant caractérisé en ce qu'une phase mobile à utiliser est une contenant un réactif de paire d'ions de type acide sulfonique. Le procédé de mesure de Trp-Man par HPLC en phase inverse selon la présente invention est efficace pour le diagnostic d'une fonction rénale et similaire.
PCT/JP2013/075260 2012-09-20 2013-09-19 Procédé de mesure de l'acide 2-amino-3-[2-(α-mannopyranosyl)indol-3-yl]propionique dans un échantillon WO2014046163A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2014536893A JP6193241B2 (ja) 2012-09-20 2013-09-19 検体中の２−アミノ−３−［２−（α−マンノピラノシル）インドール−３−イル］プロピオン酸の測定方法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2012206492 2012-09-20
JP2012-206492 2012-09-20

Publications (1)

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WO2014046163A1 true WO2014046163A1 (fr) 2014-03-27

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4180790B2 (ja) * 1997-08-20 2008-11-12 協和メデックス株式会社 新規物質２−アミノ−３−［２−（α−マンノピラノシル）インドール−３−イル］プロピオン酸およびその製造方法並びにその新規物質を用いた生体機能の検査方法

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4180790B2 (ja) * 1997-08-20 2008-11-12 協和メデックス株式会社 新規物質２−アミノ−３−［２−（α−マンノピラノシル）インドール−３−イル］プロピオン酸およびその製造方法並びにその新規物質を用いた生体機能の検査方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
HIROAKI KUBO: "Determination of furosemide in serum and urine by high performance liquid chromatography", JOURNAL OF JAPAN SOCIETY FOR ANALYTICAL CHEMISTRY, vol. 35, no. 3, 5 March 1986 (1986-03-05), pages 259 - 262 *

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JPWO2014046163A1 (ja) 2016-08-18

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