JP2022135201A - Analysis method of advanced glycation end product - Google Patents

Analysis method of advanced glycation end product Download PDF

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JP2022135201A
JP2022135201A JP2021034846A JP2021034846A JP2022135201A JP 2022135201 A JP2022135201 A JP 2022135201A JP 2021034846 A JP2021034846 A JP 2021034846A JP 2021034846 A JP2021034846 A JP 2021034846A JP 2022135201 A JP2022135201 A JP 2022135201A
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JP7460566B2 (en
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竜児 永井
Ryoji Nagai
洋介 菊池
Yosuke Kikuchi
通代 杉本
Michiyo Sugimoto
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Tokai University
Nisshin Seifun Group Inc
Oriental Yeast Co Ltd
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Nisshin Seifun Group Inc
Oriental Yeast Co Ltd
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Abstract

To provide an analysis method of an advanced glycation end product capable of highly sensitively analyzing AGE contained in a biological sample and significantly shortening a time required for the analysis.SOLUTION: An analysis method of an advanced glycation end product includes: an acid treatment step of treating a biological sample with hydrochloric acid solution; a refining step of bringing a sample-containing liquid after the acid treatment into contact with a strongly acidic cationic resin and then eluting and refining the sample with eluent; and an analysis step of subjecting eluate by the eluent to LC-MS analysis. In the acid treatment step, the sample is treated by the hydrochloric acid solution with a final concentration of 1-12 N. In the refining process, diluted solution obtained by diluting, with water, etc., the sample-containing liquid after the acid treatment, is brought into contact with the strongly acidic cationic resin without performing drying/solidification treatment, and eluted by using, as the eluent, an aqueous solution containing an organic solvent for use in the LC-MS analysis of 20 mas% or more and 80 mass% or less, and a basic compound of 1 mass% or more and 7 mass% or less. In the analysis step, the eluate obtained in the refining step is subjected to the LC-MS analysis without performing drying/solidification treatment.SELECTED DRAWING: None

Description

本発明は、終末糖化産物の分析方法に関する。 The present invention relates to a method for analyzing advanced saccharification products.

タンパク質中に存在するアミノ基とグルコース等の還元糖のアルデヒド基とが、非酵素的に反応すると糖化産物が形成される。この反応は、非酵素的糖化反応又はメイラード反応と称される。非酵素的糖化反応は、生体内においても起きている。非酵素的糖化反応は、シッフ塩基を経てアマドリ転位生成物が形成される前期反応と、複雑な開裂や縮合等が起こる後期反応との2段階で起こると考えられ、後期反応では、終末糖化産物(Advanced Glycation End-product:AGE)と呼ばれる、単一ではない様々な物質が生成する。 Glycation products are formed when amino groups present in proteins and aldehyde groups of reducing sugars such as glucose react non-enzymatically. This reaction is called the non-enzymatic saccharification reaction or Maillard reaction. Non-enzymatic saccharification reactions also occur in vivo. The non-enzymatic saccharification reaction is thought to occur in two stages: the early reaction, in which Amadori rearrangement products are formed via Schiff bases, and the late reaction, in which complex cleavage and condensation occur. (Advanced Glycation End-product: AGE), various non-single substances are produced.

前期反応の生成物であるHbA1cは、糖尿病患者の血糖コントロールの指標として世界的に測定されて利用されているが、HbA1cの値の高さと合併症の発症率とが必ずしも一致しないことから、糖尿病の合併症の発症や進展について他の因子の関与も指摘されている。 HbA1c, which is a product of the prophase reaction, is measured and used worldwide as an index of blood sugar control in diabetic patients. The involvement of other factors in the onset and progression of complications in pneumonia has also been pointed out.

またAGEは、糖尿病や老化に伴う各種の症状や疾患等との関連性が報告されている。また、様々な生活習慣病との関連も報告されている。しかしながら、AGEは、HbA1cとは異なり、構造が多様でかつ不安定な構造のものも多く、生体における様々なAGEを正確に定量することは困難である。
AGEの分析方法としては、分析対象のAGEを認識・結合するモノクローナル抗体が利用できる場合は、それを利用したELISA測定法を用いることが可能であるが、そのようなモノクローナル抗体が確立できていない場合は、糖化された蛋白質を、酸又はアルカリによって加水分解し、酸又はアルカリの除去後に、HPLC分析、LC-MS分析、LC-MS/MS分析などによりAGEを検出し、標品と比較することで特定、定量する方法が行われている。
AGEs have also been reported to be associated with various symptoms and diseases associated with diabetes and aging. In addition, association with various lifestyle-related diseases has also been reported. However, unlike HbA1c, many AGEs have diverse and unstable structures, making it difficult to accurately quantify various AGEs in vivo.
As an AGE analysis method, if a monoclonal antibody that recognizes and binds to the AGE to be analyzed is available, it is possible to use an ELISA measurement method using it, but such a monoclonal antibody has not been established. In the case, the saccharified protein is hydrolyzed with acid or alkali, and after removing the acid or alkali, AGE is detected by HPLC analysis, LC-MS analysis, LC-MS/MS analysis, etc., and compared with the standard product. A method to identify and quantify is performed.

本出願人は先に、質量分析による高感度且つ高精度なAGEの分析を可能にする試料の前処理方法として、生体試料を液相で酸処理するステップ、及び、酸処理した試料を強酸性陽イオン交換樹脂に添加して非酸性条件下で溶出するステップを含む調製方法を提案している(特許文献1参照)。この調製方法によって調整した試料は、AGEの純度が高く、該試料をLC-MS/MS等の質量分析にかけると、夾雑物に由来するノイズが大幅に低減された測定データが得られる。 The present applicant has previously proposed a sample pretreatment method that enables highly sensitive and highly accurate analysis of AGEs by mass spectrometry. They have proposed a preparation method including the step of adding to a cation exchange resin and eluting under non-acidic conditions (see Patent Document 1). A sample prepared by this preparation method has high AGE purity, and when the sample is subjected to mass spectrometry such as LC-MS/MS, measurement data with greatly reduced noise derived from contaminants can be obtained.

また本出願人は、終末糖化産物分析のための試料の調製方法として、蛋白質の完全加水分解を要しない方法も提案している(特許文献2参照)。特許文献2の方法は、生体試料に対し、限外濾過膜で濾過処理する工程及び還元処理を施す工程を含み、生体試料に対して酸処理を施す工程を含まない。 The present applicant has also proposed a method for preparing samples for analysis of advanced glycation end products that does not require complete hydrolysis of proteins (see Patent Document 2). The method of Patent Document 2 includes a step of filtering the biological sample with an ultrafiltration membrane and a step of subjecting the biological sample to a reduction treatment, but does not include a step of subjecting the biological sample to an acid treatment.

特開2014-119370号公報JP 2014-119370 A 特開2017-049024号公報JP 2017-049024 A

特許文献1に記載の方法では、質量分析によるAGE分析の前処理手法として、タンパク質を分解するための塩酸による加水分解処理や、夾雑物の除去のための陽イオン交換カラム処理を行っている。しかしながら、塩酸による加水分解処理後に、塩酸を除去するために乾固処理を行っており、例えば、遠心濃縮装置を用いて1mLの塩酸を除去する場合、10時間以上の時間を要する等、乾固処理には時間が掛かる。また使用する塩酸が高濃度であると、遠心濃縮装置にダメージを与えることになり、処理時間の長期化のみならず、装置の維持のための労力的又は経済的な負担という課題もある。 In the method described in Patent Document 1, hydrolysis treatment with hydrochloric acid for degrading proteins and cation exchange column treatment for removing contaminants are performed as pretreatment methods for AGE analysis by mass spectrometry. However, after hydrolysis treatment with hydrochloric acid, drying treatment is performed to remove hydrochloric acid. For example, when removing 1 mL of hydrochloric acid using a centrifugal concentration device, it takes 10 hours or more. Processing takes time. Moreover, if the hydrochloric acid used has a high concentration, it will damage the centrifugal concentrator, which not only prolongs the treatment time, but also poses the problem of labor and economic burdens for maintaining the apparatus.

また特許文献1には、陽イオン交換カラム処理の工程においては、樹脂に吸着した物質を溶出させるために用いる溶出液として、アンモニア溶液やアンモニア溶液とメタノールの混合溶液が記載されており、例えば、実施例では7質量%アンモニア水溶液という比較的アンモニア濃度の高い溶離液を用いているため、回収した溶出液を吹付式試験管濃縮装置にセットして乾固させてアンモニアを除去している。しかしながら、例えば、吹付式試験管濃縮装置を用いて3mLのアンモニア水溶液を乾固する場合、10時間以上の時間を要する等、この段階の乾固処理にも時間が掛かる。
特許文献1の方法のように、質量分析に供する試料の前処理に時間を要することは、AGEの分析値を、臨床的に使用することの障害にもなる。
また蛋白質の加水分解処理に高濃度の塩酸を用いることは、AGEの分析のための、試料の前処理又は前処理を含む一連の処理を自動化装置で行うことを考えたときに、機器の腐食や有毒ガスの放出等の不都合を生じ得るため、分析の自動化の点でも好ましくない。
Further, Patent Document 1 describes an ammonia solution or a mixed solution of ammonia solution and methanol as an eluent used for eluting substances adsorbed on a resin in the step of cation exchange column treatment. In the examples, an eluent having a relatively high concentration of ammonia, ie, a 7% by mass ammonia aqueous solution, is used, so the collected eluate is set in a spray-type test-tube concentrator and dried to remove ammonia. However, for example, when 3 mL of aqueous ammonia solution is dried using a spray-type test tube concentrator, it takes 10 hours or more.
The time required for pretreatment of samples subjected to mass spectrometry, as in the method of Patent Document 1, is also an obstacle to the clinical use of analytical values of AGEs.
In addition, the use of high-concentration hydrochloric acid for protein hydrolysis may cause corrosion of equipment when considering pretreatment of samples or a series of treatments including pretreatment for AGE analysis using automated equipment. This is not preferable from the point of view of automating the analysis because it may cause inconveniences such as release of toxic gas and the like.

他方、特許文献2に記載の方法によれば、生体試料に対して酸処理を施す工程を不要とすることができる。しかしながら、酸処理を行わないことによって、酵素加水分解に用いる酵素の試薬代によって測定費用が高額となり、更に酵素に含まれるAGEsによって測定値が過剰に見積もられるという点において改善の余地がある。また、多数の生体試料の分析を行うためには、限外濾過膜を備えた部品が多量に必要となったり、その交換を高頻度に行う必要が生じる等の不都合がある。斯かる不都合は、例えば短時間での多量の生体試料の分析やロボットによる分析を図る等の観点から好ましくない。 On the other hand, according to the method described in Patent Document 2, the step of acid-treating the biological sample can be eliminated. However, there is room for improvement in that measurement costs are high due to the cost of reagents for the enzymes used in the enzymatic hydrolysis and the measured values are overestimated due to the AGEs contained in the enzymes, due to the lack of acid treatment. In addition, in order to analyze a large number of biological samples, there are disadvantages such as the need for a large number of parts with ultrafiltration membranes and the need to replace them frequently. Such an inconvenience is undesirable from the viewpoint of, for example, analyzing a large amount of biological samples in a short period of time or analyzing by a robot.

本発明の解決課題は、生体試料に含まれるAGEの高感度な分析が可能であり、且つ該分析に要する時間を大幅に短縮可能な終末糖化産物の分析方法を提供することにある。 The problem to be solved by the present invention is to provide a method for analyzing advanced saccharification products that enables highly sensitive analysis of AGEs contained in a biological sample and greatly shortens the time required for the analysis.

本発明は、生体試料を塩酸溶液で処理する酸処理工程、酸処理後の試料含有液を強酸性陽イオン樹脂と接触させ、次いで溶離液で試料を溶出させて精製する精製工程、及び前記溶離液による溶出液をLC-MS分析に供する分析工程を備える終末糖化産物の分析方法であって、前記酸処理工程においては、終濃度が1~12Nの塩酸溶液で処理し、前記精製工程においては、酸処理後の試料含有液を水、又は揮発性で中性から塩基性の化合物を含む溶液で希釈し、該試料を含む酸処理後の希釈液を、乾固処理することなく前記強酸性陽イオン樹脂と接触させ、且つ、前記溶離液として、前記LC-MS分析に用いる有機溶媒を20質量%以上80質量%以下含み、且つ塩基性化合物を1質量%以上7質量%以下含む水溶液を用いて溶出させ、前記分析工程においては、前記精製工程で得られた溶出液を、乾固処理することなく前記LC-MS分析に供する、終末糖化産物の分析方法を提供することにより、上記の目的を達成したものである。 The present invention includes an acid treatment step of treating a biological sample with a hydrochloric acid solution, a purification step of contacting a sample-containing liquid after acid treatment with a strongly acidic cation resin and then eluting the sample with an eluent for purification, and the elution. A method for analyzing advanced saccharification products comprising an analysis step of subjecting an eluate by liquid to LC-MS analysis, wherein in the acid treatment step, a hydrochloric acid solution having a final concentration of 1 to 12N is used, and in the purification step , the sample-containing liquid after acid treatment is diluted with water or a solution containing a volatile neutral to basic compound, and the diluted solution containing the sample after acid treatment is subjected to the strong acidity without drying treatment An aqueous solution that is brought into contact with a cationic resin and contains, as the eluent, 20% by mass or more and 80% by mass or less of the organic solvent used in the LC-MS analysis and 1% by mass or more and 7% by mass or less of a basic compound. By providing a method for analyzing advanced saccharification products, in which the eluate obtained in the purification step is subjected to the LC-MS analysis without drying treatment in the analysis step, the above It has achieved its purpose.

本発明の終末糖化産物の分析方法によれば、生体試料に含まれるAGEの高感度な分析が可能であり、且つ該分析に要する時間を大幅に短縮可能である。 According to the method for analyzing advanced saccharification products of the present invention, AGEs contained in biological samples can be analyzed with high sensitivity, and the time required for the analysis can be greatly shortened.

図1(a)は、実施例1の分析方法における処理工程図であり、図1(b)は、実施例1の分析方法により得られたLC-MS/MS法による質量分析結果を示すグラフである。FIG. 1(a) is a processing step diagram in the analysis method of Example 1, and FIG. 1(b) is a graph showing the mass spectrometry results by the LC-MS/MS method obtained by the analysis method of Example 1. is. 図2(a)は、比較例1の分析方法における処理工程図であり、図2(b)は、比較例1の分析方法により得られたLC-MS/MS法による質量分析結果を示すグラフである。FIG. 2(a) is a processing step diagram in the analysis method of Comparative Example 1, and FIG. 2(b) is a graph showing the mass spectrometry results by the LC-MS/MS method obtained by the analysis method of Comparative Example 1. is.

以下、本発明をその好ましい実施態様に基づいて説明する。
本発明の終末糖化産物の分析方法は、生体試料に含まれる終末糖化産物を分析する方法である。終末糖化産物の分析には、生体試料に含まれる終末糖化産物についての何らかの情報が得られる分析が広く包含され、生体試料に含まれる終末糖化産物の特定や定量、複数の終末糖化産物の比率の測定等であってもよく、また終末糖化産物の有無を問わずチャートを得るのみであってもよい。
The present invention will be described below based on its preferred embodiments.
The method for analyzing advanced glycation end products of the present invention is a method for analyzing advanced glycation end products contained in a biological sample. Analysis of advanced glycation end products broadly includes analyzes that provide some information about advanced glycation end products contained in biological samples, such as identification and quantification of advanced glycation end products contained in biological samples, and determination of the ratio of multiple advanced glycation end products. It may be measurement or the like, or simply obtaining a chart regardless of the presence or absence of advanced saccharification products.

本発明で分析する対象の終末糖化産物(以下AGEという)としては、例えば、N-ε-(カルボキシメチル)リジン、N-ε-(カルボキシエチル)リジン、S-(2-スクシニル)システイン、メチルグリオキサール-イミダゾロン、カルボキシエチルアルギニン、S-(2-スクシニル)システイン(以下、2SCともいう)が挙げられる。AGEは、生体中では、タンパク質若しくはペプチドと結合した非遊離体、又はタンパク質及びペプチドの何れとも結合していない遊離体として存在する。
本発明において分析するAGEは、通常、酸処理工程を経て、遊離体のAGEとなっている。
Advanced glycation end products (hereinafter referred to as AGEs) to be analyzed in the present invention include, for example, N-ε-(carboxymethyl)lysine, N-ε-(carboxyethyl)lysine, S-(2-succinyl)cysteine, methyl Glyoxal-imidazolone, carboxyethylarginine, S-(2-succinyl)cysteine (hereinafter also referred to as 2SC). AGEs exist in vivo as non-free forms bound to proteins or peptides, or as free forms not bound to either proteins or peptides.
The AGEs analyzed in the present invention are usually converted to free AGEs through an acid treatment step.

本発明の終末糖化産物の分析方法は、酸処理工程及び精製工程を含む前処理工程と、前記精製工程により得られる前記溶離液による溶出液をLC-MS分析に供する分析工程とを備えている。以下、本発明における各工程について説明する。 The method for analyzing advanced saccharification products of the present invention comprises a pretreatment step including an acid treatment step and a purification step, and an analysis step of subjecting the eluate from the eluent obtained by the purification step to LC-MS analysis. . Each step in the present invention will be described below.

[酸処理工程]
前記酸処理工程においては、生体試料を酸で処理する。生体試料は、健常人から採取されたものであっても、疾病罹患患者から採取されたものでも良い。また生体試料としては、生体から採取されたあらゆる細胞、組織、及び体液、例えば、皮膚、筋肉、骨、毛髪、爪、脂肪組織、脳神経系、心臓及び血管等の循環器系、肺、肝臓、脾臓、膵臓、腎臓、消化器系、胸腺、血管、リンパ管、血液試料(全血、血清、血漿)、リンパ液、唾液、尿、精液、腹水、喀痰等、並びにそれらの培養物が挙げられる。このうち、低侵襲性の観点から血液試料(全血、血清、血漿)、リンパ液、尿、皮膚、末梢血管、筋肉、脂肪組織等が好ましく、血清、血漿がより好ましい。上記生体試料は、酸処理にかける前に、必要に応じてホモジナイズした後、遠心や濾過にかけ、細胞片や不溶性物質などの夾雑物を予め除去しておいてもよい。
[Acid treatment process]
In the acid treatment step, the biological sample is treated with acid. A biological sample may be one obtained from a healthy individual or one obtained from a diseased patient. In addition, biological samples include all cells, tissues, and body fluids collected from living organisms, such as skin, muscles, bones, hair, nails, adipose tissue, cranial nervous system, circulatory system such as heart and blood vessels, lungs, liver, Spleen, pancreas, kidney, digestive system, thymus, blood vessels, lymph vessels, blood samples (whole blood, serum, plasma), lymph, saliva, urine, semen, ascites, sputum, etc., and cultures thereof. Among these, blood samples (whole blood, serum, plasma), lymph, urine, skin, peripheral blood vessels, muscle, adipose tissue, etc. are preferred from the viewpoint of minimal invasiveness, and serum and plasma are more preferred. Prior to the acid treatment, the biological sample may be homogenized, if necessary, and then centrifuged or filtered to remove contaminants such as cell debris and insoluble substances.

酸処理には、塩酸を用いる。酸処理においては、例えば、上記生体試料に塩酸の溶液を添加し、必要に応じて振盪又は攪拌した後、静置し、試料を加水分解させればよい。上記酸処理は、液相において行われる。すなわち、液体の状態の反応液中で試料の加水分解反応を進行させる。蛋白質が充分に加水分解するようにする観点、及び乾固処理することなく希釈のみで酸の影響を軽減できるようにする観点から、塩酸は、塩酸溶液の終濃度が1~12N、より好ましくは3~12Nとなるように加える。酸処理に使用される塩酸の量、反応時間および温度の条件は、生体試料を十分に溶解できる条件且つ反応液が液相となる条件であればよく、使用する生体試料や酸の濃度等に応じて決定すればよい。酸処理は、例えば、血清試料が5μLの場合は、200μLの6N塩酸を添加し、塩酸の添加を行った試料について、80~120℃で10~48時間、好ましくは100℃以上で18時間以上の加水分解処理を行う。 Hydrochloric acid is used for the acid treatment. In the acid treatment, for example, a hydrochloric acid solution may be added to the biological sample, shaken or stirred as necessary, and allowed to stand still to hydrolyze the sample. The acid treatment is performed in the liquid phase. That is, the hydrolysis reaction of the sample is allowed to proceed in the liquid reaction solution. From the viewpoint of sufficiently hydrolyzing the protein and from the viewpoint of reducing the effect of the acid only by dilution without drying treatment, hydrochloric acid has a final concentration of 1 to 12N, more preferably Add so that it becomes 3-12N. The amount of hydrochloric acid used for the acid treatment, the reaction time, and the temperature conditions may be any conditions as long as the biological sample can be sufficiently dissolved and the reaction solution is in a liquid phase. You can decide accordingly. For acid treatment, for example, when the serum sample is 5 μL, 200 μL of 6N hydrochloric acid is added, and the sample to which hydrochloric acid has been added is treated at 80 to 120° C. for 10 to 48 hours, preferably at 100° C. or higher for 18 hours or longer. is hydrolyzed.

[還元処理]
前記酸処理工程においては、酸処理工程の前に、生体試料に還元処理を施す還元処理を行うことも好ましい。還元処理を行う理由は次の通りである。従来、AGEを含む試料の質量分析には、液体クロマトグラフから溶離する液体試料をエレクトロスプレープローブ(ESIプローブ)によりイオン化して質量分析計に導入する、エレクトロスプレーイオン化質量分析法を利用することが好ましい。例えば、このESI法を利用してAGE分析を行うと、その分析中に、液体試料に含まれていたアマドリ転位生成物がESIプローブにてAGEへと変化してしまい、分析結果の信頼性が低下するという問題がある。生体試料に還元処理を施すことにより、ESI法による質量分析中に生体試料中のカルボニル基のアマドリ転位を防止し、結果として生体試料中で非AGE成分がAGEに変化することを防止し、高感度且つ高精度のAGE分析の実現を図ることができる。
[Reduction treatment]
In the acid treatment step, it is also preferable to carry out a reduction treatment, in which the biological sample is subjected to a reduction treatment, before the acid treatment step. The reason for carrying out the reduction treatment is as follows. Conventionally, for mass spectrometry of samples containing AGEs, electrospray ionization mass spectrometry can be used, in which a liquid sample eluted from a liquid chromatograph is ionized by an electrospray probe (ESI probe) and introduced into a mass spectrometer. preferable. For example, when AGE analysis is performed using this ESI method, the Amadori rearrangement product contained in the liquid sample is changed to AGE by the ESI probe during the analysis, and the reliability of the analysis result is reduced. There is a problem of lowering By subjecting the biological sample to reduction treatment, Amadori rearrangement of carbonyl groups in the biological sample is prevented during mass spectrometry by the ESI method, and as a result, non-AGE components in the biological sample are prevented from changing to AGE, and high Realization of sensitive and highly accurate AGE analysis can be achieved.

還元処理としては、還元処理剤としてヒドリド還元剤を用いたヒドリド還元(hydride reduction)処理が好ましい。ヒドリド還元とは、化合物の還元を求核剤としての水素供与体により行う還元反応である。ヒドリド還元剤としては公知の物を適宜使用可能であり、例えば、水素化ホウ素ナトリウム〔NaBH4〕、シアノ水素化ホウ素ナトリウム〔NaBH3CN〕、水素化トリエチルホウ素リチウム〔LiBH(C2H5)3〕、水素化トリ(sec-ブチル)ホウ素リチウム〔LiBH(sec-C4H9)3〕、水素化トリ(sec-ブチル)ホウ素カリウム〔KBH(sec-C4H9)3〕、水素化ホウ素リチウム、水素化ホウ素亜鉛、アセトキシ水素化ホウ素ナトリウム、水素化アルミニウムリチウム〔(LAH) LiAlH4〕、水素化ビス(2-メトキシエトキシ)アルミニウムナトリウム〔NaAlH2(OC2H4OCH3)2〕等が挙げられる。これらのヒドリド還元剤の中でも特に、水素化ホウ素ナトリウムは、還元力が高く、且つカルボニル基を還元するが、エステルやアミド基は還元しないため、本発明で好ましく用いられる。 As the reduction treatment, a hydride reduction treatment using a hydride reducing agent as a reducing treatment agent is preferred. Hydride reduction is a reduction reaction in which a compound is reduced with a hydrogen donor as a nucleophile. As the hydride reducing agent , known substances can be used as appropriate. 3 ], lithium tri(sec-butyl)borohydride [LiBH(sec- C4H9 ) 3 ], potassium tri(sec-butyl)borohydride [KBH(sec -C4H9)3 ] , borohydride Lithium, zinc borohydride, sodium acetoxyborohydride, lithium aluminum hydride [(LAH) LiAlH 4 ], sodium bis(2-methoxyethoxy)aluminum hydride [NaAlH 2 (OC 2 H 4 OCH 3 ) 2 ], etc. is mentioned. Among these hydride reducing agents, sodium borohydride is particularly preferably used in the present invention because it has a high reducing power and reduces a carbonyl group but does not reduce an ester or an amide group.

還元処理は、例えば、生体試料に還元剤処理剤を含む溶液を添加し、必要に応じて振盪又は攪拌した後、所定時間静置することで実施できる。還元処理に使用される還元処理剤の量、反応時間、温度等の条件は、使用する生体試料や還元処理剤の種類等に応じて決定すれば良い。例えば、生体試料として全血、血清、血漿等の血液試料を用い、その膜透過画分に対してヒドリド還元処理を施す際に、ヒドリド還元剤として2mMのNaBH4溶液を用いる場合、血清試料の1/10容量程度で良い。 The reduction treatment can be carried out by, for example, adding a solution containing a reducing agent treatment to the biological sample, shaking or stirring it as necessary, and allowing it to stand still for a predetermined period of time. Conditions such as the amount of the reducing agent used for the reduction treatment, the reaction time, and the temperature may be determined according to the type of the biological sample and the reducing agent used. For example, when a blood sample such as whole blood, serum, or plasma is used as a biological sample, and a membrane-permeable fraction thereof is subjected to hydride reduction treatment, a 2 mM NaBH 4 solution is used as a hydride reducing agent. About 1/10 capacity is sufficient.

〔精製工程〕
酸処理後の試料含有液は、水、又はアンモニア等の揮発性で中性から塩基性の化合物を含む溶液を加えて希釈する。この希釈により酸の影響を軽減することで、乾固処理することなく、強酸性陽イオン交換樹脂と接触させることが可能となる。希釈するための水としては、純水を用いることが好ましい。純水としては、蒸留水、イオン交換水、RO水等が挙げられる。比抵抗値が18MΩ・cm以上の超純水を用いてもよい。希釈に、アンモニア等の揮発性で中性から塩基性の化合物を含む溶液を用いることもでき、当該希釈用液のpHは、2~7であることが好ましい。
酸処理後の試料含有液は、その容量の20倍から100倍の希釈液で希釈することが好ましく、その容量の25倍から60倍の希釈液で希釈することが更に好ましい。例えば、酸処理後の試料含有液が200μLである場合、5mL以上の純水、又はアンモニア等の揮発性で中性から塩基性の化合物を含む溶液を加えて希釈する。
[Purification process]
The acid-treated sample-containing liquid is diluted by adding water or a solution containing a volatile, neutral to basic compound such as ammonia. By reducing the influence of the acid by this dilution, it becomes possible to contact with the strongly acidic cation exchange resin without dryness treatment. Pure water is preferably used as water for dilution. Pure water includes distilled water, ion-exchanged water, RO water, and the like. Ultrapure water with a specific resistance of 18 MΩ·cm or more may be used. A solution containing a volatile, neutral to basic compound such as ammonia can also be used for dilution, and the pH of the diluent is preferably 2-7.
The acid-treated sample-containing solution is preferably diluted with a diluent that is 20 to 100 times its volume, more preferably 25 to 60 times its volume. For example, when the sample-containing liquid after acid treatment is 200 μL, it is diluted by adding 5 mL or more of pure water or a solution containing a volatile, neutral to basic compound such as ammonia.

酸処理後の試料含有液は、上述のように希釈により酸の影響を軽減した後、エバポレーター等により乾固処理することなく、強酸性陽イオン樹脂と接触させる精製工程に供される。以下、酸処理後の試料含有液を希釈したものを酸処理後の希釈液ともいう。
強酸性陽イオン交換樹脂による精製処理は、基本的には、通常の方法に従って行うことができる。強酸性陽イオン交換樹脂による精製処理は、通常、強酸性陽イオン交換樹脂に、酸処理後に希釈した試料を添加した後、該樹脂を洗浄し、その後、溶離液により該樹脂に吸着した物質を溶出させ、溶出液を回収することで実施される。
The acid-treated sample-containing liquid is subjected to a purification step of contacting with a strongly acidic cation resin without being subjected to dryness treatment by an evaporator or the like after reducing the influence of the acid by dilution as described above. Hereinafter, a solution obtained by diluting the sample-containing liquid after acid treatment is also referred to as a diluted solution after acid treatment.
Purification treatment with a strongly acidic cation exchange resin can basically be carried out according to a normal method. In the purification treatment using a strongly acidic cation exchange resin, a sample diluted after acid treatment is usually added to a strongly acidic cation exchange resin, the resin is washed, and then the substances adsorbed on the resin are removed by an eluent. It is carried out by eluting and collecting the eluate.

強酸性陽イオン交換樹脂としては、スルホン酸型強酸性陽イオン交換樹脂が好ましい。強酸性陽イオン交換樹脂は、市販品の強酸性陽イオン交換樹脂を使用することができる。例えば、ダイヤイオン(登録商標)UBK-550、ダイヤイオン(登録商標)SK1B(三菱化学)、Oasis(商標)MCX(日本ウォーターズ社)、STRATA(商標)X-C(Phenomenex)、アンバーライト(登録商標)IR120B、アンバーライト(登録商標)200C、ダウエックス(登録商標)MSC-1(The Dow Chemical Company)、デュオライトC26(Rohm and Haas)、LEWATIT(登録商標)SP-112(LANXESS Distribution GmbH)等が好適に使用され得る。精製に使用する樹脂の量としては、例えば血清試料を用いる場合、血清試料1mLに対して50mg~300mgが好ましく、70mg~150mgがより好ましい。 As the strongly acidic cation exchange resin, a sulfonic acid type strongly acidic cation exchange resin is preferred. As the strongly acidic cation exchange resin, a commercially available strongly acidic cation exchange resin can be used. For example, Diaion (registered trademark) UBK-550, Diaion (registered trademark) SK1B (Mitsubishi Chemical), Oasis (registered trademark) MCX (Nippon Waters Co., Ltd.), STRATA (registered trademark) XC (Phenomenex), Amberlite (registered trademark) Trademarks) IR120B, Amberlite® 200C, Dowex® MSC-1 (The Dow Chemical Company), Duolite C26 (Rohm and Haas), LEWATIT® SP-112 (LANXESS Distribution GmbH) etc. can be preferably used. The amount of resin used for purification is preferably 50 mg to 300 mg, more preferably 70 mg to 150 mg, per 1 mL of serum sample, for example, when a serum sample is used.

強酸性陽イオン交換樹脂は、酸処理後の希釈液を添加する前に、予め洗浄しておくことが好ましい。例えば、樹脂量の50倍容量以上の100%メタノールを、試料を添加する前のカラムに通液させて洗浄することが好ましく、100%メタノールの通液後、樹脂量の50倍容量以上の純水を通液させて、該樹脂を平衡化させることも好ましい。 The strongly acidic cation exchange resin is preferably washed in advance before adding the acid-treated diluent. For example, it is preferable to pass 100% methanol of 50 times the volume or more of the resin amount through the column before adding the sample to wash it, and after passing 100% methanol, the pure column of 50 times the volume or more of the resin amount It is also preferred to equilibrate the resin by passing water through it.

酸処理後の希釈液を強酸性陽イオン交換樹脂に添加する方法は特に限定されないが、例えば、強酸性陽イオン交換樹脂を充填したカラムに、酸処理後に希釈した液体を通液させればよい。酸処理後の希釈液は、例えば強酸性陽イオン交換樹脂を充填したカラムに滴下し、通液させる。通液の速度は、特に限定されないが、例えば減圧ポンプを用いた真空マニホールドを用いて1mL/30秒以下が好ましい。カラムに添加した試料中のAGE等の目的物質は、強酸性陽イオン交換樹脂に吸着する。 The method of adding the diluted solution after acid treatment to the strongly acidic cation exchange resin is not particularly limited, but for example, the diluted solution after acid treatment may be passed through a column packed with a strongly acidic cation exchange resin. . The diluted solution after the acid treatment is added dropwise to, for example, a column filled with a strongly acidic cation exchange resin, and passed through the column. The flow rate is not particularly limited, but is preferably 1 mL/30 seconds or less using a vacuum manifold using a decompression pump, for example. Target substances such as AGEs in the sample applied to the column are adsorbed on the strongly acidic cation exchange resin.

次いで、目的物質が吸着した樹脂を洗浄する。洗浄は、希酸、例えば0.05~0.2N塩酸溶液、または1.5~2.5質量%ギ酸溶液、または終濃度が上記濃度となる希酸とメタノールの等量混合溶液を添加し、カラムを通過させればよい。洗浄によりカラム中の夾雑物が除去されるので、その後の溶出処理により、目的物質を選択的に回収することが可能となる。 Next, the resin on which the target substance has been adsorbed is washed. Washing is carried out by adding a dilute acid such as a 0.05 to 0.2N hydrochloric acid solution, a 1.5 to 2.5% by mass formic acid solution, or an equal mixed solution of a dilute acid and methanol whose final concentration is the above concentration. , should be passed through the column. Since contaminants in the column are removed by washing, the target substance can be selectively recovered by the subsequent elution treatment.

次いで、強酸性陽イオン交換樹脂に吸着させた試料を溶出させる。溶出処理に用いる溶離液としては、後の分析工程で行う質量分析装置に使用可能な有機溶媒を含む水溶液を基本とし、非酸性条件とするべく塩基性化合物をわずかに加えた溶離液を用いることが好ましい。より具体的には、LC-MS分析に用いる有機溶媒を20質量%以上80質量%以下含み、且つ塩基性化合物を1質量%以上7質量%以下含む水溶液を用いることが好ましい。LC-MS分析に用いる有機溶媒とは、アセトニトリル、メタノール等の、LC-MS分析において、液体クロマトグラフィーの移動相(以下、LCの移動相ともいう)に用いられる有機溶媒のことであり、LCの移動相として、アセトニトリルを主成分とするものを用いる場合は、溶離液に含まれる有機溶媒もアセトニトリルであることが好ましく、LCの移動相として、メタノールを主成分とするものを用いる場合は、溶離液に含まれる有機溶媒もメタノールであることが好ましい。 Then, the sample adsorbed on the strongly acidic cation exchange resin is eluted. The eluent used in the elution process should be basically an aqueous solution containing an organic solvent that can be used in the mass spectrometer used in the subsequent analysis process, and an eluent to which a slight amount of a basic compound is added to create non-acidic conditions. is preferred. More specifically, it is preferable to use an aqueous solution containing 20% by mass or more and 80% by mass or less of an organic solvent used for LC-MS analysis and containing 1% by mass or more and 7% by mass or less of a basic compound. The organic solvent used for LC-MS analysis is an organic solvent used in the mobile phase of liquid chromatography (hereinafter also referred to as LC mobile phase) in LC-MS analysis, such as acetonitrile and methanol, and LC When using a mobile phase containing acetonitrile as a main component, the organic solvent contained in the eluent is preferably acetonitrile, and when using a mobile phase containing methanol as a main component, The organic solvent contained in the eluent is also preferably methanol.

前記塩基性化合物としては、例えば、アンモニア等が挙げられる。溶出処理用の溶離液に塩基性化合物を含ませることにより、樹脂に吸着したAGEsを溶出させることができる。塩基性化合物は、測定物質のイオンを妨害しない点から揮発性であることが好ましい。ここでいう、揮発性とは、室温でのイオン化の段階でも容易に気化する性質を有することをいう。
溶出溶液は、中性から塩基性であることが好ましく、pH7以上13以下であることが好ましい。
溶離液の量や濃度は、試料や樹脂の種類によって最適化することができ、樹脂に吸着した目的物質が回収され、質量分析装置にて目的とするAGE構造が検出可能な量及び濃度とすることが好ましい。
Examples of the basic compound include ammonia and the like. By including a basic compound in the eluent for elution treatment, AGEs adsorbed on the resin can be eluted. The basic compound is preferably volatile so as not to interfere with the ions of the substance to be measured. Volatile as used herein means having a property of being easily vaporized even in the stage of ionization at room temperature.
The elution solution is preferably neutral to basic, and preferably has a pH of 7 or higher and 13 or lower.
The amount and concentration of the eluent can be optimized depending on the type of sample and resin, and the target substance adsorbed on the resin is recovered, and the amount and concentration are such that the target AGE structure can be detected by the mass spectrometer. is preferred.

[濾過工程]
前記手順で得られた溶出液は、好ましくは更なる精製処理に供される。
前記手順で得られた溶出液は、そのまま液体クロマトグラフィー-質量分析法による分析(LC-MS分析)に用いることができるが、夾雑物質等の所望しない混入物を除去する目的で、前記手順で得られた溶出液に対して、濾過処理を施してもよい。濾過処理としては、例えば、遠心や減圧処理による精密濾過、又は限外濾過を行うことができる。
精密濾過で用いる濾過膜の孔径は、分析装置のカラム流路及び質量分析装置のイオン化部等に干渉する物質を除去する観点から、好ましくは0.45μm以下、より好ましくは0.3μm以下、更に好ましくは0.2μm以下である。
[Filtration step]
The eluate obtained by the above procedure is preferably subjected to further purification treatment.
The eluate obtained by the above procedure can be used as it is for analysis by liquid chromatography-mass spectrometry (LC-MS analysis). The obtained eluate may be filtered. As the filtration treatment, for example, microfiltration by centrifugation or decompression treatment, or ultrafiltration can be performed.
The pore size of the filtration membrane used in microfiltration is preferably 0.45 μm or less, more preferably 0.3 μm or less, and more preferably 0.3 μm or less, from the viewpoint of removing substances that interfere with the column flow path of the analyzer and the ionization part of the mass spectrometer. It is preferably 0.2 μm or less.

濾過工程で用いる限外濾過膜の分画分子量は、遊離体のAGE、特に修飾アミノ酸を通過させ、非遊離体のAGE、特に修飾アミノ酸以外の高分子化合物を除去する観点から、好ましくは10000以下、より好ましくは5000以下、更に好ましくは3000以下である。精密濾過膜及び限外濾過膜としては市販のものも好ましく用いられ、例えば、精密濾過には、エキクロディスク13CR(孔径0.2μm、日本ポール社)、ミニザルトRC4(孔径0.2μm、ザルトリウス社)、マイレクスLG(孔径0.2μm、メルクミリポア社)等のフィルターを用いることができ、限外濾過には、ナノセップUF(分画分子量3K~300K、日本ポール社)、ビバスピン500(分画分子量3K~1000K、ザルトリウス社)等のフィルターを用いることができる。使用するフィルターは、試料を溶解した溶媒に対して溶媒耐性を有するものを特に制限なく用いることができる。 The cutoff molecular weight of the ultrafiltration membrane used in the filtration step is preferably 10,000 or less from the viewpoint of allowing free AGEs, particularly modified amino acids, to pass through and removing non-free AGEs, particularly polymer compounds other than modified amino acids. , more preferably 5,000 or less, and still more preferably 3,000 or less. Commercially available microfiltration membranes and ultrafiltration membranes are also preferably used. ), Millex LG (pore size 0.2 μm, Merck Millipore) and the like can be used. Filters such as 3K to 1000K (Sartorius) can be used. Any filter having solvent resistance to the solvent in which the sample is dissolved can be used without particular limitation.

[分析工程]
前記精製工程で得られた溶出液は、全画分をAGE分析用試料として使用してもよいが、目的物質の含有量の高い画分を選択的に回収してAGE分析用試料として使用することが好ましい。目的物質の含有量の高い画分は、標準溶液を用いてカラム精製を行い、経時的に分取した溶出液の各画分について目的物質の含有量を調べることによって、予め決定しておくことができる。
前記精製工程で得られた溶出液は、AGE分析に適切な形態へと調製され、AGE分析に供される。AGE分析用試料は、AGE分析の方法や使用する機器に応じて適宜調製し得る。
[Analysis process]
Although all fractions of the eluate obtained in the purification step may be used as samples for AGE analysis, fractions with a high content of the target substance are selectively collected and used as samples for AGE analysis. is preferred. Fractions with a high target substance content should be determined in advance by performing column purification using a standard solution and examining the content of the target substance in each fraction of the eluate collected over time. can be done.
The eluate obtained in the purification step is prepared into a form suitable for AGE analysis and subjected to AGE analysis. A sample for AGE analysis can be appropriately prepared according to the method of AGE analysis and the equipment to be used.

本発明の方法においては、前記の精製工程で得られた溶出液を、乾固処理することなくLC-MS分析に供する。すなわち、本発明においては、前記の溶出処理に用いる溶離液として、LC-MS分析において液体クロマトグラフィーの移動相として用いられる有機溶媒を含むものを用いることによって、強酸性陽イオン交換樹脂から遊離した成分を含む溶出液を、乾固処理することなく、LC-MS分析に供することが可能である。 In the method of the present invention, the eluate obtained in the purification step is subjected to LC-MS analysis without drying. That is, in the present invention, the eluent used for the elution treatment contains an organic solvent that is used as a mobile phase for liquid chromatography in LC-MS analysis. The eluate containing the components can be subjected to LC-MS analysis without dryness treatment.

本発明におけるAGE分析の手法は、LC-MS分析である。すなわち、液体クロマトグラフィーと質量分析とを組み合わせたLC-MS法によりAGEの分析を行う。LC-MS法としては、LC-MS法、LC-MS/MS、LC-MS/MS/MS等法が挙げられる。検出感度をより向上させるためには、LC-MS/MS法や、LC-MS/MS/MS法などの液体クロマトグラフィー-タンデム型質量分析法がより好ましい。 The method of AGE analysis in the present invention is LC-MS analysis. That is, the AGEs are analyzed by the LC-MS method combining liquid chromatography and mass spectrometry. The LC-MS method includes LC-MS method, LC-MS/MS, LC-MS/MS/MS and the like. Liquid chromatography-tandem mass spectrometry such as the LC-MS/MS method and the LC-MS/MS/MS method is more preferable in order to further improve the detection sensitivity.

LC-MS分析の液体クロマトグラフィーの移動相としては、前述した溶離液に含まれる有機溶媒を用いることが好ましい。溶離液に、アセトニトリルを含む水溶液を用いた場合の移動相、好ましくは最終条件の移動相としては、例えば、メタノールの水溶液やアセトニトリルの水溶液、アセトニトリルとトリフルオロ酢酸の混合水溶液、アセトニトリルとギ酸の混合水溶液などが挙げられる。 As the mobile phase for liquid chromatography for LC-MS analysis, it is preferable to use the organic solvent contained in the eluent described above. The mobile phase when an aqueous solution containing acetonitrile is used as the eluent, preferably the mobile phase of the final conditions, includes, for example, an aqueous solution of methanol, an aqueous solution of acetonitrile, a mixed aqueous solution of acetonitrile and trifluoroacetic acid, and a mixture of acetonitrile and formic acid. Aqueous solutions and the like can be mentioned.

LC-MS分析計によりAGEを測定する際の測定条件は、目的とするAGEの種類や、機器の型、試料の状態等に応じて、当業者が通常の知識に基づいて適宜設定すればよい。液体クロマトグラフィーの条件は供される試料によって異なるが、例えば、80体積%アセトニトリル+0.1質量%ギ酸溶液に対しては、移動相にギ酸水溶液とギ酸アセトニトリル溶液でグラジエントを形成させると好ましい。質量分析計としては、二重収束磁場型質量分析計、イオントラップ型質量分析計、四重極型質量分析計などが挙げられるが、これらに限定されない。 The measurement conditions for measuring AGE with an LC-MS analyzer can be appropriately set by those skilled in the art based on their ordinary knowledge, depending on the type of target AGE, the type of instrument, the state of the sample, etc. . The conditions of liquid chromatography vary depending on the sample to be provided. For example, for 80% by volume acetonitrile + 0.1% by mass formic acid solution, it is preferable to form a gradient of formic acid aqueous solution and formic acid acetonitrile solution as the mobile phase. Mass spectrometers include, but are not limited to, double focusing magnetic field mass spectrometers, ion trap mass spectrometers, quadrupole mass spectrometers, and the like.

上記手順で測定された試料中のAGEに関する測定値を、同様の手順で測定された標準溶液からの測定値と比較することによって、生体試料由来のAGEを定量することができる。具体的には、所定濃度のAGEを含有する標準溶液からの測定結果に基づいて、検量線を作成する。検量線作成の際には、内部標準を用いて各測定値を校正しておくと、より精度の高い検量線が得られるため好ましい。 By comparing the measured value of AGE in the sample measured by the above procedure with the measured value from the standard solution measured by the same procedure, the AGE derived from the biological sample can be quantified. Specifically, a calibration curve is created based on the measurement results from a standard solution containing a predetermined concentration of AGE. When creating a calibration curve, it is preferable to calibrate each measured value using an internal standard, because a more accurate calibration curve can be obtained.

上述した本発明の好ましい実施形態によれば、高濃度の塩酸溶液で処理することによって、生体試料中の蛋白質が加水分解されるため、LC-MS分析によるAGEの分析を高精度に行うことができる上に、酸処理後の酸除去のための乾固処理や強酸性陽イオン樹脂からの溶出液の乾固処理を不要とでき、LC-MS分析に供する試料の調整に要する工程や時間を大幅に軽減できる。斯かる効果が奏されることは、AGEの測定の臨床的な使用を容易とする。また酸処理後の乾固処理が不要であること等は、塩酸による装置の劣化を防止することも可能となり、ロボットにより前処理及び一連の分析処理を行うことも容易となる。 According to the preferred embodiment of the present invention described above, the treatment with a high-concentration hydrochloric acid solution hydrolyzes the proteins in the biological sample, so that AGE analysis by LC-MS analysis can be performed with high accuracy. In addition, drying treatment for acid removal after acid treatment and drying treatment of the eluate from the strongly acidic cation resin can be eliminated, and the steps and time required for sample preparation to be subjected to LC-MS analysis can be reduced. can be greatly reduced. Having such an effect facilitates the clinical use of AGE measurement. In addition, the elimination of drying treatment after acid treatment makes it possible to prevent deterioration of the apparatus due to hydrochloric acid, and facilitates pretreatment and a series of analysis treatments by robot.

以下、実施例を基に本発明を更に詳述するが、本発明は以下の実施例に限定されるものではない。 The present invention will be described in more detail below based on examples, but the present invention is not limited to the following examples.

〔実施例1〕
ヒト血清5μLに、蒸留水5μL及びそれらと等量(10μL)のホウ酸ナトリウム緩衝液(0.2Mホウ酸、2mM DTPA、pH9.0)を添加し、更に水素化ホウ素ナトリウム溶液(2mM NaBH4、0.1N NaOH)を、該緩衝液の1/10量(1μL)添加し、軽く撹拌し遠心した後、室温で4時間放置して還元処理を行った。
還元処理後、各AGE及びLysineの内部標準を添加し、6Nの無鉄塩酸を200μL加え、100℃で18時間加熱し、加水分解を行った。
加水分解後、蒸留水によって10mLまで倍希釈したサンプルを、陽イオン交換カラムであるStrata-X-Cカラム(Phenomenex, Torrance, CA, USA)にアプライして分画を行なった。カラムは1mLのMeOHにて洗浄後、1mLの蒸留水で平衡化した後、サンプルを全量通過させた後、2%ギ酸3mLで洗浄し、1%のアンモニアを含む20%アセトニトリル1mLで溶出した。溶出した画分を0.2μmのポアフィルターによって濾過し、それをAGE分析用試料として、LC-MS/MS(TSQ Quantiva triple stage quadrupole mass spectrometer (Thermo Fisher Scientific, Waltham, MA))にて測定を行った。LC-MS/MSのLCの移動相は、0.1%のギ酸を含む蒸留水と0.1%のギ酸を含むアセトニトリルを用いた。
[Example 1]
To 5 μL of human serum, 5 μL of distilled water and an equivalent amount (10 μL) of sodium borate buffer (0.2 M boric acid, 2 mM DTPA, pH 9.0) were added, and further sodium borohydride solution (2 mM NaBH4, 0.1N NaOH) was added to 1/10 volume (1 μL) of the buffer solution, gently stirred and centrifuged, and then allowed to stand at room temperature for 4 hours for reduction treatment.
After reduction treatment, an internal standard of each AGE and Lysine was added, 200 μL of 6N iron-free hydrochloric acid was added, and the mixture was heated at 100° C. for 18 hours for hydrolysis.
After hydrolysis, the sample diluted to 10 mL with distilled water was applied to a cation exchange column, Strata-XC column (Phenomenex, Torrance, Calif., USA) for fractionation. The column was washed with 1 mL of MeOH, equilibrated with 1 mL of distilled water, allowed to pass through the entire sample, washed with 3 mL of 2% formic acid, and eluted with 1 mL of 20% acetonitrile containing 1% ammonia. The eluted fraction was filtered through a 0.2 μm pore filter, and measured with LC-MS/MS (TSQ Quantiva triple stage quadrupole mass spectrometer (Thermo Fisher Scientific, Waltham, Mass.)) as a sample for AGE analysis. gone. Distilled water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid were used as mobile phases for LC-MS/MS LC.

LC-MS/MS測定の条件は以下のとおりである。
(HPLC条件)
クロマトグラフィーカラム:SeQuant、ZIC-HILIC,150×2.1mm、5μm、200A Peek Hplc Column
カラム温度:40℃
移動相:A:0.1質量%ギ酸水溶液、B:0.1質量%ギ酸含有アセトニトリル溶液
グラジュエント条件:A:10%+B:90%
流速:200μL/min
インジェクション量:10μL
分析時間:20分
溶出時間:MG-H(約12分)、CMA(約13分)、CML(約12分)
(質量分析条件)
イオン化方法:H-ESI
キャピラリー温度:300℃
イオン化エネルギー:約3500V(陽性イオン化時)
(検出ピーク(m/z))
Lysine:147m/z→84m/z
Lysine d6:153m/z→89m/z
CML :205m/z→130m/z
CML d2:207m/z→130m/z
MG-H1 :229m/z→114m/z
MG-H1 d3:232m/z→117m/z
CMA :233m/z→116m/z又は118m/z
CMA d6:239m/z→119m/z又は121m/z
The conditions for LC-MS/MS measurement are as follows.
(HPLC conditions)
Chromatography column: SeQuant, ZIC-HILIC, 150×2.1mm, 5μm, 200A Peek Hplc Column
Column temperature: 40°C
Mobile phase: A: 0.1 wt% formic acid aqueous solution, B: acetonitrile solution containing 0.1 wt% formic acid Gradient conditions: A: 10% + B: 90%
Flow rate: 200 μL/min
Injection volume: 10 μL
Analysis time: 20 minutes Elution time: MG-H (about 12 minutes), CMA (about 13 minutes), CML (about 12 minutes)
(mass spectrometry conditions)
Ionization method: H-ESI
Capillary temperature: 300°C
Ionization energy: about 3500 V (at positive ionization)
(Detected peak (m/z))
Lysine: 147m/z→84m/z
Lysine d6: 153m/z→89m/z
CML: 205m/z→130m/z
CML d2: 207m/z→130m/z
MG-H1: 229m/z → 114m/z
MG-H1 d3: 232 m/z → 117 m/z
CMA: 233m/z→116m/z or 118m/z
CMA d6: 239 m/z → 119 m/z or 121 m/z

〔比較例1〕
ヒト血清5μLに蒸留水15μL、等量(20μL)のホウ酸ナトリウム緩衝液(0.2Mホウ酸、2mM DTPA、pH9.0)を添加し、水素化ホウ素ナトリウム溶液(2mM NaBH、0.1N NaOH)を、該緩衝液の1/10量(2μL)添加し、軽く撹拌し遠心した後、室温で4時間放置して還元処理を行った。還元処理後、各AGE及びLysineの内部標準を添加し、6Nの無鉄塩酸を1mL 加え、100℃で18時間加熱し、加水分解を行った。加水分解後、遠心濃縮により乾固させたサンプルを1mLの蒸留水に溶解し、陽イオン交換カラムであるStrata-X-Cカラム(Phenomenex, Torrance, CA, USA)を用いて分画を行った。カラムは1mLのMeOHにて洗浄後、1mLの蒸留水で平衡化した後、サンプルを全量通過させ、2%ギ酸3mLで洗浄、7%アンモニア3mLで溶出した。溶出した画分を乾固し、0.1%ギ酸を含む20%アセトニトリル1mLにて溶解後、0.2μmのポアフィルターによって濾過し、LC-MS/MS(TSQ Quantiva triple stage quadrupole mass spectrometer (Thermo Fisher Scientific, Waltham, MA))にて測定した。
[Comparative Example 1]
15 μL of distilled water and an equal volume (20 μL) of sodium borate buffer (0.2 M boric acid, 2 mM DTPA, pH 9.0) were added to 5 μL of human serum, and sodium borohydride solution (2 mM NaBH 4 , 0.1 N) was added. NaOH) was added to 1/10 volume (2 μL) of the buffer solution, gently stirred and centrifuged, and then allowed to stand at room temperature for 4 hours for reduction treatment. After the reduction treatment, an internal standard of each AGE and Lysine was added, 1 mL of 6N iron-free hydrochloric acid was added, and the mixture was heated at 100° C. for 18 hours for hydrolysis. After hydrolysis, the sample dried by centrifugal concentration was dissolved in 1 mL of distilled water and fractionated using a cation exchange column, Strata-XC column (Phenomenex, Torrance, CA, USA). . After washing the column with 1 mL of MeOH and equilibrating with 1 mL of distilled water, the entire sample was passed through, washed with 3 mL of 2% formic acid, and eluted with 3 mL of 7% ammonia. The eluted fraction was dried, dissolved in 1 mL of 20% acetonitrile containing 0.1% formic acid, filtered through a 0.2 μm pore filter, and subjected to LC-MS/MS (TSQ Quantiva triple stage quadrupole mass spectrometer (Thermo Fisher Scientific, Waltham, Mass.)).

実施例1におけるLC-MS/MSによる測定(分析)開始までの各工程の進行実績及びLC-MS/MSによるAGE及びLysineの測定(分析)結果を図1に示し、比較例1におけるLC-MS/MSによる測定(分析)開始までの各工程の進行実績及びLC-MS/MSによるAGE及びLysineの測定(分析)結果を図2に示した。
図1(b)に示すように、実施例1の方法によれば、各種のAGE及びLysineの各ピークが明瞭に検出され、目的物質のピーク以外のピークやノイズの発生も僅かであった。また、図2(a)に示すように、比較例1の方法によれば、LC-MS/MSによる測定(分析)開始までに4日間要したのに対して、図1(a)に示すように、実施例1の方法によれば、LC-MS/MSによる測定(分析)開始までに2日のみ要しており、実施例の方法によれば、生体試料に含まれるAGEの高感度な分析を可能であること、及び該分析に要する時間を大幅に短縮可能であることが判る。
The results of the progress of each step until the start of measurement (analysis) by LC-MS/MS in Example 1 and the measurement (analysis) results of AGE and Lysine by LC-MS/MS are shown in FIG. FIG. 2 shows the progress of each step up to the start of measurement (analysis) by MS/MS and the measurement (analysis) results of AGE and Lysine by LC-MS/MS.
As shown in FIG. 1(b), according to the method of Example 1, each peak of various AGEs and Lysine was clearly detected, and peaks other than the peak of the target substance and noise were slightly generated. Further, as shown in FIG. 2(a), according to the method of Comparative Example 1, it took 4 days to start measurement (analysis) by LC-MS/MS, whereas FIG. 1(a) shows Thus, according to the method of Example 1, it takes only two days to start measurement (analysis) by LC-MS/MS, and according to the method of Example, high sensitivity of AGEs contained in biological samples can be obtained. It can be seen that it is possible to perform an accurate analysis, and that the time required for the analysis can be greatly shortened.

Claims (4)

生体試料を塩酸溶液で処理する酸処理工程、酸処理後の試料含有液を強酸性陽イオン樹脂と接触させ、次いで溶離液で試料を溶出させて精製する精製工程、及び前記溶離液による溶出液をLC-MS分析に供する分析工程を備える終末糖化産物の分析方法であって、
前記酸処理工程においては、終濃度が1~12Nの塩酸溶液で処理し、
前記精製工程においては、酸処理後の試料含有液を水、又は揮発性で中性から塩基性の化合物を含む溶液で希釈し、該試料を含む酸処理後の希釈液を、乾固処理することなく前記強酸性陽イオン樹脂と接触させ、且つ、
前記溶離液として、前記LC-MS分析に用いる有機溶媒を20質量%以上80質量%以下含み、且つ塩基性化合物を1質量%以上7質量%以下含む水溶液を用いて溶出させ、
前記分析工程においては、前記精製工程で得られた溶出液を、乾固処理することなく前記LC-MS分析に供する、終末糖化産物の分析方法。
An acid treatment step of treating a biological sample with a hydrochloric acid solution, a purification step of contacting a sample-containing liquid after acid treatment with a strongly acidic cation resin, then eluting the sample with an eluent for purification, and an elution solution with the eluent. A method for analyzing advanced saccharification products comprising an analysis step of subjecting to LC-MS analysis,
In the acid treatment step, treatment with a hydrochloric acid solution having a final concentration of 1 to 12N,
In the purification step, the acid-treated sample-containing liquid is diluted with water or a solution containing a volatile, neutral to basic compound, and the acid-treated diluted liquid containing the sample is dried to dryness. without contacting with the strongly acidic cation resin, and
As the eluent, an aqueous solution containing 20% by mass or more and 80% by mass or less of the organic solvent used in the LC-MS analysis and containing 1% by mass or more and 7% by mass or less of a basic compound is used for elution,
In the analysis step, the eluate obtained in the purification step is subjected to the LC-MS analysis without drying treatment.
前記酸処理工程の前に、生体試料に対し、還元処理を施す工程を具備する、請求項1に記載の終末糖化産物の分析方法。 2. The method for analyzing advanced saccharification products according to claim 1, comprising a step of subjecting the biological sample to reduction treatment before said acid treatment step. 前記分析する終末糖化産物が、糖修飾アミノ酸である、請求項1又は2に記載の終末糖化産物の分析方法。 3. The method for analyzing advanced glycation products according to claim 1 or 2, wherein the advanced glycation products to be analyzed are sugar-modified amino acids. 前記LC-MS分析が液体クロマトグラフィー-タンデム型質量分析法である、請求項1~3の何れか1項に記載の終末糖化産物の分析方法。
The method for analyzing advanced glycation end products according to any one of claims 1 to 3, wherein the LC-MS analysis is liquid chromatography-tandem mass spectrometry.
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