JP6002567B2 - Sample pretreatment method - Google Patents
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Description
本発明は、最終糖化産物の分析のための試料の前処理方法に関する。 The present invention relates to a sample pretreatment method for analysis of final glycation products.
従来、分子量１０００以下の生体由来の低分子を測定および分析する場合、高速液体クロマトグラフィー（ＨＰＬＣ）と質量分析（ＭＳ）とを組み合わせたＬＣ−ＭＳやＬＣ−ＭＳ／ＭＳシステムが用いられている。従来、生体試料をＬＣ−ＭＳ分析する場合は、一般に、生体試料をＨＰＬＣにアプライする前に、酸で加水分解した後、夾雑成分の除去および分離のためのＣ１８担体等を用いた逆層カラム処理にかけることにより、分析対象物質の初発純度を向上させていた。しかし、対象物質によっては、上記逆層カラム処理では夾雑成分を十分に除去できないことがあり、そのため分析結果に多くのノイズが検出されて、十分な検出感度や精度が得られないことがあった。 Conventionally, when measuring and analyzing a biologically-derived small molecule having a molecular weight of 1000 or less, an LC-MS or LC-MS / MS system combining high performance liquid chromatography (HPLC) and mass spectrometry (MS) has been used. . Conventionally, when LC-MS analysis of a biological sample is performed, generally, a biological sample is hydrolyzed with an acid before being applied to HPLC, and then a reverse layer column using a C18 carrier or the like for removing and separating contaminant components. By applying the treatment, the initial purity of the analyte was improved. However, depending on the target substance, the above-described reverse layer column treatment may not sufficiently remove the contaminating component, and as a result, a lot of noise is detected in the analysis result, and sufficient detection sensitivity and accuracy may not be obtained. .
より精度の高い分析を行うため、試料の前処理方法の改良が求められている。例えば、特許文献１には、生体試料中のデスモシンとイソデスモシンのＬＣ−ＭＳ／ＭＳ分析の測定精度を向上させるために、生体試料を、陽イオン交換樹脂にかけ、非酸性条件下で自然落下にて溶出させ、得られたサンプルをＬＣ−ＭＳ／ＭＳ分析にかける方法が記載されている。しかしながら、有効な前処理方法は物質によって異なることがあるため、測定する目的物質にとって適切な前処理方法を見出すことは容易ではない。 In order to perform analysis with higher accuracy, improvement of a sample pretreatment method is demanded. For example, in Patent Document 1, in order to improve the measurement accuracy of LC-MS / MS analysis of desmosine and isodesmosine in a biological sample, the biological sample is subjected to a cation exchange resin and is allowed to fall naturally under non-acidic conditions. A method for eluting and subjecting the resulting sample to LC-MS / MS analysis is described. However, since effective pretreatment methods may vary depending on the substance, it is not easy to find a suitable pretreatment method for the target substance to be measured.
最終糖化産物（Advanced Glycation Endproduct：ＡＧＥ）は、体内で蛋白質と糖との反応により生成される物質の総称であり、糖尿病合併症などの指標として知られている。ＡＧＥを精度よく検出および定量できれば、これらの疾患の診断や研究のために有利であるが、従来、正確な測定は容易ではなかった。 Advanced glycation end product (AGE) is a general term for substances produced by the reaction of protein and sugar in the body, and is known as an indicator of diabetic complications. Accurate detection and quantification of AGE is advantageous for diagnosis and research of these diseases, but accurate measurement has not been easy.
非特許文献１には、最終糖化産物であるメチルグリオキサール由来ハイドロイミダゾロン（ＭＧ−Ｈ）を定量するために、生体試料（血漿）を酸加水分解処理を行わずに等量の４質量％のリン酸溶液を添加した後、希釈した試料を固相抽出カラムにかけ、水酸化アンモニウム溶液で溶出して得られたサンプルをＬＣ−ＭＳ／ＭＳにかけたことが記載されている。しかし一般に、ＡＧＥを測定する場合、特定のＡＧＥを認識・結合するモノクローナル抗体を利用したＥＬＩＳＡ測定法か、モノクローナル抗体が確立できていなければ、糖化された蛋白質を完全加水分解し、ＨＰＬＣ分析、ＬＣ−ＭＳ分析、またはＬＣ−ＭＳ／ＭＳ分析により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。また一般に、蛋白質の完全加水分解は、試料を水を加えた有機または無機の強酸（塩酸、硫酸、トリフルオロ酢酸、パラ−ベンゼンスルホン酸、メタンスルホン酸など）と混合し、気相で加水分解することで実施されている。例えば、リボヌクレアーゼのアミノ酸組成分析のための強酸による完全加水分解法として、減圧封管した試験管内で定沸点塩酸（５．７Ｎ ＨＣl）処理する方法、１１０℃に２０〜７２時間加熱する加水分解法（非特許文献２）、および塩酸からの汚染物質の混入を防ぐために６Ｎ塩酸を封管試験管内で直接蒸留する変法（気相加水分解法）が広く用いられている。さらに、強アルカリ（水酸化ナトリウム、水酸化カリウムなど）による蛋白質の加水分解法も知られている。また、非特許文献３には、血清蛋白中のメチル化アルギニンを完全酸加水分解の後にＨＰＬＣで測定するために、生体試料（マウス心臓および腎臓組織）のホモジネートをＴＣＡ処理し、変性し不溶化した蛋白質を６Ｎ塩酸で１１０℃、１６時間、気相塩酸加水分解法で処理したことが記載されている。しかし、上記のような強酸または強アルカリによる分解は、目的物であるアミノ酸がさらに分解されてしまうことがあるため、理想的な加水分解法とはいえないのが現状である。例えば、塩酸加水分解ではセリン、スレオニン、メチオニン、システイン、トリプトファンが変性または分解してしまうことが知られている。診断や研究に応用するためには、様々なＡＧＥが分解を受けることなく、より高感度且つ高精度に検出、測定できることが望ましい。 Non-Patent Document 1 discloses that in order to quantify methylglyoxal-derived hydroimidazolone (MG-H), which is the final glycation product, an equal amount of 4% by mass without subjecting a biological sample (plasma) to acid hydrolysis treatment. It is described that after adding a phosphoric acid solution, the diluted sample was applied to a solid phase extraction column, and the sample obtained by elution with an ammonium hydroxide solution was subjected to LC-MS / MS. However, in general, when measuring AGE, an ELISA measurement method using a monoclonal antibody that recognizes and binds to a specific AGE, or if a monoclonal antibody has not been established, glycated protein is completely hydrolyzed, HPLC analysis, LC A method of identifying and quantifying by detecting AGE by -MS analysis or LC-MS / MS analysis and comparing it with a standard is performed. In general, protein is completely hydrolyzed by mixing the sample with a strong organic or inorganic acid (hydrochloric acid, sulfuric acid, trifluoroacetic acid, para-benzenesulfonic acid, methanesulfonic acid, etc.) with water and hydrolyzing it in the gas phase. It is carried out by doing. For example, as a complete hydrolysis method with strong acid for analysis of amino acid composition of ribonuclease, a method of treating with constant boiling hydrochloric acid (5.7N HCl) in a test tube sealed under reduced pressure, a hydrolysis method of heating to 110 ° C. for 20 to 72 hours (Non-Patent Document 2) and a modified method (gas phase hydrolysis method) in which 6N hydrochloric acid is directly distilled in a sealed tube test tube are widely used in order to prevent contamination from hydrochloric acid. Furthermore, a method for hydrolyzing proteins with a strong alkali (sodium hydroxide, potassium hydroxide, etc.) is also known. In Non-Patent Document 3, in order to measure methylated arginine in serum protein by HPLC after complete acid hydrolysis, a biological sample (mouse heart and kidney tissue) homogenate was treated with TCA, denatured and insolubilized. It is described that the protein was treated with 6N hydrochloric acid at 110 ° C. for 16 hours by a gas phase hydrochloric acid hydrolysis method. However, the degradation with strong acids or strong alkalis as described above is not an ideal hydrolysis method because the target amino acid may be further degraded. For example, it is known that hydrolysis with hydrochloric acid results in denaturation or degradation of serine, threonine, methionine, cysteine, and tryptophan. For application to diagnosis and research, it is desirable that various AGEs can be detected and measured with higher sensitivity and higher accuracy without being decomposed.
より高感度且つ高精度な生体由来最終糖化産物（ＡＧＥ）の分析、およびそれを可能にするより高純度な試料を調製するための方法が求められている。 There is a need for more sensitive and accurate analysis of biologically derived final glycation products (AGE) and methods for preparing higher purity samples that allow it.
本発明者らは、ＡＧＥ分析のためのより高純度な試料を調製する方法について検討した結果、生体試料を液相で酸処理した後、強酸性陽イオン交換樹脂を用いて精製し、得られた試料をＬＣ−ＭＳ／ＭＳ分析にかけることにより、測定データのノイズが大幅に低減され、高感度および高精度でＡＧＥを分析することができることを見出した。 As a result of studying a method for preparing a higher-purity sample for AGE analysis, the present inventors obtained a biological sample after acid treatment in a liquid phase and then purification using a strongly acidic cation exchange resin. It was found that by subjecting the obtained sample to LC-MS / MS analysis, noise in the measurement data was greatly reduced, and AGE could be analyzed with high sensitivity and high accuracy.
すなわち本発明は、最終糖化産物分析のための試料の調製方法であって、
生体試料を液相で塩酸を用いて処理すること、および
酸処理した試料を、強酸性陽イオン交換樹脂に添加し、非酸性条件下で溶出すること
を含む方法を提供する。
また本発明は、上記方法により調製された試料を液体クロマトグラフィー−質量分析することを含む、最終糖化産物の分析方法を提供する。
That is, the present invention is a method for preparing a sample for final glycation product analysis,
There is provided a method comprising treating a biological sample with hydrochloric acid in a liquid phase, and adding the acid-treated sample to a strongly acidic cation exchange resin and eluting under non-acidic conditions.
The present invention also provides a method for analyzing a final glycation product, which comprises subjecting a sample prepared by the above method to liquid chromatography-mass spectrometry.
本発明の試料の調製方法により調製された試料をＬＣ−ＭＳ／ＭＳ分析すれば、夾雑物質によるピークを除去してノイズを低減することができるだけでなく、ＡＧＥの検出レベルを向上することができるので、分析対象とするＡＧＥを誤判読する危険性が低下し、高感度且つ高精度なＡＧＥ分析が可能になる。 If the sample prepared by the sample preparation method of the present invention is analyzed by LC-MS / MS, not only can peaks due to contaminants be removed to reduce noise, but also the detection level of AGE can be improved. Therefore, the risk of misreading the AGE to be analyzed is reduced, and highly sensitive and highly accurate AGE analysis becomes possible.
本発明の試料の調製方法で調製される試料は、ＡＧＥ分析のための試料として有用である。本発明の試料の調製方法で調製される試料から分析されるＡＧＥとしては、Ｎ−ε−（カルボキシメチル）リジン、Ｓ−（２−スクシニル）システインおよびメチルグリオキサール−イミダゾロンが挙げられる。 The sample prepared by the sample preparation method of the present invention is useful as a sample for AGE analysis. Examples of AGEs analyzed from the sample prepared by the sample preparation method of the present invention include N-ε- (carboxymethyl) lysine, S- (2-succinyl) cysteine, and methylglyoxal-imidazolone.
本発明の試料の調製方法においては、まず、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、および体液、例えば、皮膚、筋肉、骨、脂肪組織、脳神経系、心臓および血管等の循環器系、肺、肝臓、脾臓、膵臓、腎臓、消化器系、胸腺、リンパ、血液、全血、血清、血漿、リンパ液、唾液、尿、腹水、喀痰等、ならびにそれらの培養物が挙げられる。このうち、全血、血清、血漿が好ましく、血清がより好ましい。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。 In the sample preparation method of the present invention, first, a biological sample is treated with an acid. The biological sample may be collected from a healthy person or collected from a diseased patient. The biological sample includes all cells, tissues, and body fluids collected from the living body, for example, skin, muscle, bone, adipose tissue, cerebral nervous system, circulatory system such as heart and blood vessels, lung, liver, spleen, pancreas, Examples include kidney, digestive system, thymus, lymph, blood, whole blood, serum, plasma, lymph, saliva, urine, ascites, sputum, etc., and cultures thereof. Among these, whole blood, serum, and plasma are preferable, and serum is more preferable. The biological sample may be homogenized as necessary before being subjected to acid treatment, and then subjected to centrifugation or filtration to remove in advance contaminants such as cell debris and insoluble substances.
酸処理に使用される酸は、有機酸でも無機酸でもよいが、塩酸が好ましい。使用する酸のｐＨは、好ましくはｐＨ０．５〜１．５、より好ましくはｐＨ０．８〜１．２である。酸処理においては、例えば、上記生体試料に酸の溶液を添加し、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。さらに、加水分解中に反応液を加温すると好ましい。また必要に応じて、途中で試料を攪拌して、反応容器の壁に付着した試料を再度酸溶液に混合してもよい。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。酸処理に使用される酸の量、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種類に応じて決定すればよい。例えば血液試料を用いる場合、血清試料１ｍＬに対して酸が５〜８Ｎであればよい。酸処理の一例においては、血清試料１００μＬに対して、２０〜２９質量％塩酸溶液を１ｍＬ〜４ｍＬを添加し、攪拌した後、６５〜１００℃で６〜２４時間、試料を加水分解させればよい。 The acid used for the acid treatment may be an organic acid or an inorganic acid, but hydrochloric acid is preferred. The pH of the acid used is preferably pH 0.5 to 1.5, more preferably pH 0.8 to 1.2. In the acid treatment, for example, an acid solution may be added to the biological sample, and after shaking or stirring as necessary, the sample may be allowed to stand to hydrolyze the sample. Furthermore, it is preferable to warm the reaction solution during hydrolysis. If necessary, the sample may be stirred in the middle, and the sample adhering to the wall of the reaction vessel may be mixed with the acid solution again. 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. The amount of acid used for the acid treatment, the reaction time, and the temperature may be any conditions that allow the biological sample to be sufficiently dissolved and the reaction solution is in a liquid phase, depending on the type of biological sample and acid used. To decide. For example, when a blood sample is used, the acid may be 5 to 8 N with respect to 1 mL of the serum sample. In an example of acid treatment, 1 to 4 mL of 20 to 29% by mass hydrochloric acid solution is added to 100 μL of a serum sample and stirred, and then the sample is hydrolyzed at 65 to 100 ° C. for 6 to 24 hours. Good.
酸処理された試料は、必要に応じて遠心または濾過されて沈殿物を除去した後、エバポレーター等により乾固処理され、酸を除去される。乾固処理した試料は、次の強酸性陽イオン交換樹脂による精製処理まで室温保存しておくことができる。 The acid-treated sample is centrifuged or filtered as necessary to remove the precipitate, and then dried and solidified by an evaporator or the like to remove the acid. The dry-treated sample can be stored at room temperature until the next purification treatment with a strongly acidic cation exchange resin.
次いで、上記で得られた酸処理された試料を、強酸性陽イオン交換樹脂により精製する。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えばよい。すなわち、該樹脂に酸処理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。 The acid-treated sample obtained above is then purified with a strongly acidic cation exchange resin. The purification treatment with a strongly acidic cation exchange resin may basically be performed according to a usual method. That is, after the acid-treated sample is added to the resin, the resin is washed, and then the substance adsorbed on the resin is eluted with an eluent, and the eluate is recovered.
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、例えば血液試料を用いる場合、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 strong acid cation exchange resin, a sulfonic acid type strong acid cation exchange resin is preferable. As the strong acid cation exchange resin, a commercially available strong acid cation exchange resin can be used. For example, Diaion (registered trademark) UBK-550, Diaion (registered trademark) SK1B (Mitsubishi Chemical), Oasis (trademark) MCX (Nippon Waters), STRATA (trademark) X-C (Phenomenex), Amberlite (registered) (Trademark) IR120B, Amberlite (registered trademark) 200C, Dowex (registered trademark) MSC-1 (The Dow Chemical Company), Duolite C26 (Rohm and Haas), LEWATIT (registered trademark) SP-112 (LANXESS Distribution GmbH) Etc. can be suitably used. For example, when a blood sample is used, the amount of the resin used for purification is preferably 50 mg to 300 mg, more preferably 70 mg to 150 mg with respect to 1 mL of the serum sample.
強酸性陽イオン交換樹脂は、酸処理した試料を添加する前に、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、必要に応じて樹脂量の５０倍容量以上の溶離液に用いる酸溶液で、次いで樹脂量の５０倍容量以上の純水で、試料を添加する前のカラムに通液させ、樹脂を洗浄する。 The strongly acidic cation exchange resin is preferably washed in advance before adding the acid-treated sample. For example, add the sample with 100% methanol of 50 times the volume of the resin, as needed, with an acid solution used for the eluent of 50 times the volume of the resin, and then with pure water of 50 times the volume of the resin Pass through the column before washing to wash the resin.
酸処理した試料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、試料を含む液体を通液させればよい。一例としては、上述した手順で酸処理され乾固処理された試料には、液体を添加し、乾固物を溶解させておく。強イオン交換樹脂の場合、試料を溶解させる液体は、ｐＨ５〜９の弱酸性〜弱塩基性の塩濃度の低い液体であればよいが、ｐＨ６〜８の中性付近のｐＨを有する塩濃度の低い液体がより好ましく、特に純水が好ましい。必要に応じて、試料を溶解させた液体をさらに遠心し、上清を回収して使用してもよい。得られた試料を含む液体を、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、自然滴下程度の速度が好ましい。さらに好ましくは１ｍＬ／ｍｉｎ以下が好ましい。カラムに添加した試料中のＡＧＥを含む目的物質は、強酸性陽イオン交換樹脂に吸着する。 The method for adding the acid-treated sample to the strongly acidic cation exchange resin is not particularly limited. For example, the liquid containing the sample may be passed through a column filled with the strongly acidic cation exchange resin. As an example, a liquid is added to the sample that has been acid-treated and dried to solidification in the above-described procedure, and the dried solid is dissolved. In the case of a strong ion exchange resin, the liquid in which the sample is dissolved may be a weakly acidic to weakly basic liquid having a pH of 5 to 9, but a salt concentration having a pH near neutral of pH 6 to 8. A low liquid is more preferable, and pure water is particularly preferable. If necessary, the liquid in which the sample is dissolved may be further centrifuged, and the supernatant may be recovered and used. The liquid containing the obtained sample is dropped onto a column packed with a strongly acidic cation exchange resin and allowed to pass therethrough. The speed of liquid passage is not particularly limited, but a speed of natural dropping is preferable. More preferably, it is 1 mL / min or less. The target substance containing AGE in the sample added to the column is 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、または上記希酸終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。溶出処理は、非酸性条件下で行うことが望ましい。例えば、洗浄処理後の樹脂に、揮発性で中性〜塩基性、好ましくはｐＨ７以上１３以下の溶離液を添加し、樹脂に吸着した目的物質を溶出させる。好ましい溶離液としては、純水、アンモニア溶液、およびこれらとメタノールの混合溶液などを挙げることができ、より好ましくは５〜１０質量％アンモニア含有溶液が挙げられる。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ればよいが、樹脂に吸着した目的物質が回収される量および濃度であればよい。一般的には、樹脂体積の２０〜５００倍量使用すればよい。溶離液は、カラムに自然滴下し、通液させればよい。 Next, the resin on which the target substance is adsorbed is washed. For washing, dilute acid, for example, 0.05 to 0.2N hydrochloric acid solution, or 1.5 to 2.5% by mass formic acid solution, or a mixed solution of equal amounts of dilute acid and methanol with the above dilute acid final concentration is added. And passing through the column. Since the contaminants in the column are removed by the washing, the target substance can be selectively recovered by the subsequent elution treatment. The elution treatment is desirably performed under non-acidic conditions. For example, a volatile, neutral to basic, preferably pH 7 or more and 13 or less eluent is added to the washed resin to elute the target substance adsorbed on the resin. Preferred eluents include pure water, ammonia solution, and a mixed solution of these with methanol, and more preferably 5 to 10% by mass ammonia-containing solution. The amount and concentration of the eluent may be optimized depending on the type of the sample and the resin, but may be an amount and concentration at which the target substance adsorbed on the resin is recovered. Generally, it may be used in an amount 20 to 500 times the resin volume. The eluent may be spontaneously dropped onto the column and allowed to pass through.
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。 The eluate may use the entire fraction as a sample for AGE analysis, but it is preferable to selectively collect a fraction having a high content of the target substance and use it as a sample for AGE analysis. The fraction with a high content of the target substance should be determined in advance by performing column purification using a standard solution and examining the content of the target substance for each fraction of the eluate collected over time. Can do.
イオン交換樹脂に液体を通過させる場合、該樹脂を充填したカラムの上から液体を滴下して自然に落下させることで樹脂に液体を通過させてもよいが、カラムをバキュームマニホールドなどにセットし、減圧することで、効率よく液体をカラム内に導入することができる。 When passing the liquid through the ion exchange resin, the liquid may be allowed to pass through the resin by dropping it from the top of the column filled with the resin, but the column is set in a vacuum manifold or the like, By reducing the pressure, the liquid can be efficiently introduced into the column.
溶離液により強酸性陽イオン交換樹脂から溶出された溶出液は、好ましくはさらなる精製処理に供される。例えば、上記手順にて得られた溶出液を、乾固処理し、次いで適切な溶媒に溶解させた後、濾過処理する。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、または限外濾過を行うことができる。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）などのフィルターを、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）など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、乾固試料を溶解した溶媒に対して溶媒耐性があれば特に限定されない。 The eluate eluted from the strongly acidic cation exchange resin by the eluent is preferably subjected to further purification treatment. For example, the eluate obtained by the above procedure is dried and then dissolved in a suitable solvent, followed by filtration. As the filtration treatment, for example, microfiltration by ultracentrifugation or reduced pressure treatment, or ultrafiltration can be performed. For microfiltration, filters such as Excrodisc 13CR (pore size 0.2 μm, Nippon Pall), Minisalto RC4 (pore size 0.2 μm, Sartorius), Milex LG (pore size 0.2 μm, Merck Millipore) are limited. For external filtration, a filter such as Nanosep UF (fractionated molecular weight 3K to 300K, Nihon Pall) or Vivaspin 500 (fractionated molecular weight 3K to 1000K, Sartorius) can be used. The filter to be used is not particularly limited as long as it has solvent resistance to the solvent in which the dried sample is dissolved.
上記の手順により精製された試料は、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され得るため、その形態は特に限定されない。 The sample purified by the above procedure is prepared into a form suitable for AGE analysis and subjected to AGE analysis. Since the sample for AGE analysis can be appropriately prepared according to the AGE analysis method and the equipment to be used, its form is not particularly limited.
ＡＧＥ分析の方法としては、ＡＧＥが測定可能な方法であれば特に限定されないが、液体クロマトグラフィーと質量分析とを組み合わせた分析方法が好ましく、例えば、液体クロマトグラフィー−質量分析（例えば、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 AGE analysis method is not particularly limited as long as AGE can be measured, but an analysis method combining liquid chromatography and mass spectrometry is preferable. For example, liquid chromatography-mass spectrometry (for example, LC-MS Method, LC-MS / MS, LC-MS / MS / MS, etc.) method. In order to further improve the detection sensitivity, liquid chromatography-tandem mass spectrometry such as LC-MS / MS method or LC-MS / MS / MS method is more preferable.
液体クロマトグラフィー−質量分析のための乾固試料溶解用の適切な溶媒としては、液体クロマトグラフィーの移動相の最終条件と同じ溶媒を用いることが好ましい。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が、特に限定されない。より具体的には、本発明の試料の調製方法により調製された試料を乾固処理し、８０体積％アセトニトリル＋０．１質量％ギ酸水溶液に溶解させて、ＡＧＥ分析用試料とする。あるいは、本発明の試料の調製方法により調製された試料を乾固処理し、８０体積％アセトニトリル＋０．１質量％ギ酸溶液に溶解させた後、前述の孔径０．２μｍのフィルターを用いた精密濾過処理にかけ、回収した濾液に等量の８０体積％アセトニトリル＋０．１質量％ギ酸溶液を添加して２倍希釈させ、ＡＧＥ分析用試料とする。血清試料１００μＬから精製された試料に対して、５００〜２０００μＬ程度の８０体積％アセトニトリル＋０．１質量％ギ酸溶液を添加するとよい。 As a suitable solvent for dissolving a dry sample for liquid chromatography-mass spectrometry, it is preferable to use the same solvent as the final condition of the mobile phase of liquid chromatography. For example, an aqueous solution of methanol, an aqueous solution of acetonitrile, a mixed aqueous solution of acetonitrile and trifluoroacetic acid, a mixed aqueous solution of acetonitrile and formic acid, and the like can be mentioned. More specifically, the sample prepared by the sample preparation method of the present invention is dried and dissolved in 80% by volume acetonitrile + 0.1% by weight formic acid aqueous solution to obtain a sample for AGE analysis. Alternatively, after the sample prepared by the sample preparation method of the present invention is dried and dissolved in 80% by volume acetonitrile + 0.1% by mass formic acid solution, microfiltration using the aforementioned filter having a pore diameter of 0.2 μm is performed. In the treatment, an equal volume of 80% by volume acetonitrile + 0.1% by mass formic acid solution is added to the collected filtrate to dilute it twice to obtain a sample for AGE analysis. A sample purified from 100 μL of serum sample may be added with about 500 to 2000 μL of 80 volume% acetonitrile + 0.1 mass% formic acid solution.
液体クロマトグラフィー−質量分析計により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、上述の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 a liquid chromatography-mass spectrometer are appropriately set by those skilled in the art based on ordinary knowledge according to the type of AGE, the type of equipment, the state of the sample, etc. That's fine. The conditions of liquid chromatography vary depending on the sample to be provided. For example, for the above-mentioned 80% by volume acetonitrile + 0.1% by weight formic acid solution, it is preferable to form a gradient with an aqueous formic acid solution and an aqueous formic acid solution in the mobile phase. . Examples of the mass spectrometer include, but are not limited to, a double focusing magnetic field mass spectrometer, an ion trap mass spectrometer, and a quadrupole mass spectrometer.
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。 AGE derived from a biological sample can be quantified by comparing the measured value for AGE in the sample measured by the above procedure with the measured value from the standard solution measured by the same procedure. Specifically, a calibration curve is created based on the measurement result from a standard solution containing a predetermined concentration of AGE. When creating a calibration curve, it is preferable to calibrate each measurement value using an internal standard because a calibration curve with higher accuracy can be obtained.
以下、実施例を挙げて本発明を詳細に説明するが、本発明は何らこれらの実施例に限定されるものではない。 EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated in detail, this invention is not limited to these Examples at all.
実施例１
（試料の調製）
−８０℃のディープフリーザで凍結保存されていた血清を室温で溶解した。２ｍＬチューブに、解凍した血清を５０μＬずつ分注した。全てのサンプルに２０質量％塩酸溶液を１ｍＬずつ添加し、よく懸濁して塊をつぶした後、チューブをボルテックスにかけ、血清の塊がなくなるまでしっかりと溶かした。次いで、サンプルを入れたチューブをドライバスにセットし、１００℃で一晩（１８時間）、加水分解を行った。反応中にチューブの上部に塊が形成した場合、チューブを指で弾いて塊を液中に落とした。加水分解反応後、試料を遠心エバポレーターにて乾固させ、室温保存した。
Example 1
(Sample preparation)
Serum that had been cryopreserved in a deep freezer at −80 ° C. was dissolved at room temperature. 50 μL of thawed serum was dispensed into a 2 mL tube. To all samples, 1 mL of 20% by mass hydrochloric acid solution was added and suspended well to crush the clot, and then the tube was vortexed to dissolve completely until the serum clot disappeared. Next, the tube containing the sample was set in a dry bath and hydrolyzed at 100 ° C. overnight (18 hours). If a lump formed at the top of the tube during the reaction, the tube was flipped with a finger to drop the lump into the liquid. After the hydrolysis reaction, the sample was dried with a centrifugal evaporator and stored at room temperature.
（強酸性陽イオン交換樹脂による精製）
強酸性陽イオン交換樹脂充填カラム（Ｏａｓｉｓ（商標）ＭＣＸ １ｃｃ、型番１８６００１８８１、日本ウォーターズ社）をサンプル数分用意し、バキュームマニホールドにセットした。カラムの洗浄のため、カラムにメタノールを１ｍＬずつ滴下し、バキュームマニホールドの減圧を開始した状態でチューブとの接続部のコックを開いてメタノールを通過させた。続いて、カラムに超純水を１ｍＬずつ滴下し、通過させた。
乾固サンプルの入った２ｍＬチューブに１ｍＬの純水を添加し、ソニケーターをかけて乾固サンプルを溶解させた。乾固サンプルが完全に溶けたら、遠心機で１０，０００ｒｐｍで５分間遠心した。上清を回収し、洗浄済みカラムへ滴下して通過させた。続いて、洗浄液（０．１Ｎ塩酸水溶液）を３ｍＬを通過させて洗浄を行った。
バキュームマニホールドのそれぞれのカラムの下に、溶出液回収用チューブをセットした。カラムに溶離液（７質量％アンモニア水溶液）３ｍＬを滴下し、溶出した液を回収した。回収した溶出液は２ｍＬチューブに１ｍＬ分注し、吹付式試験管濃縮装置にセットして乾固させた。
(Purification with strongly acidic cation exchange resin)
A strongly acidic cation exchange resin packed column (Oasis (trademark) MCX 1 cc, model number 186001881, Nippon Waters) was prepared for each sample and set in a vacuum manifold. In order to wash the column, 1 mL of methanol was added dropwise to the column, and while the vacuum manifold was started to be depressurized, the cock connected to the tube was opened to allow methanol to pass through. Subsequently, 1 mL of ultrapure water was added dropwise to the column and allowed to pass through.
1 mL of pure water was added to a 2 mL tube containing the dried sample, and the dried sample was dissolved using a sonicator. When the dried sample was completely dissolved, it was centrifuged for 5 minutes at 10,000 rpm in a centrifuge. The supernatant was collected and dropped to a washed column. Subsequently, 3 mL of a cleaning liquid (0.1N hydrochloric acid aqueous solution) was passed through to perform cleaning.
An eluate collection tube was set under each column of the vacuum manifold. 3 mL of eluent (7% by mass ammonia aqueous solution) was dropped into the column, and the eluted liquid was collected. 1 mL of the recovered eluate was dispensed into a 2 mL tube, set in a spray type test tube concentrator, and dried.
（精密濾過処理）
乾固したサンプルを、ボルテックスまたはソニケーターを使って８０体積％アセトニトリル＋０．１質量％ギ酸水溶液５００μＬに溶解した。孔径０．２２μｍのフィルター付き遠心チューブに溶解液を全量入れ、遠心機で１０，０００ｒｐｍで５分間遠心した。フィルターを通過した溶液を回収し、回収液に８０体積％アセトニトリル＋０．１質量％ギ酸水溶液５００μＬを添加した。この溶液をＡＧＥ分析用試料とした。
(Microfiltration processing)
The dried sample was dissolved in 500 μL of 80 vol% acetonitrile + 0.1 wt% formic acid aqueous solution using a vortex or sonicator. The entire amount of the lysate was placed in a centrifuge tube with a pore size of 0.22 μm and centrifuged at 10,000 rpm for 5 minutes in a centrifuge. The solution that passed through the filter was recovered, and 500 μL of 80 volume% acetonitrile + 0.1 mass% formic acid aqueous solution was added to the recovered liquid. This solution was used as a sample for AGE analysis.
（標準試料の調製）
〔Ｎ−ε−（カルボキシメチル）リジン（ＣＭＬ）〕
市販品（PolyPeptide Laboratories社、ＵＳ、品番：ＳＣ１５０５）を標準試料とした。
〔Ｓ−（２−スクシニル）システイン（２ＳＣ）〕
チューブにＮ‐ａｃｅｔｙｌ-Ｃｙｓ（４８．９ｍｇ）、Ｎ-ｅｔｈｙｌｍａｌｅｉｍｉｄｅ（７５ｍｇ）を入れ、純水で１０ｍＬまでメスアップした。室温で２時間インキュベートした後、等量ずつ４本のネジ栓付チューブに分け、遠心式濃縮装置で乾燥させた。各乾燥サンプルに６Ｎ塩酸水溶液を１ｍＬ加え１１０℃で２４時間加水分解させた後、加水分解産物を遠心濃縮した。各乾燥サンプルを５ｍＬの１質量％ＴＦＡに溶解させた。予め１０質量％塩酸水溶液で洗浄し、約５倍量の１質量％ＴＦＡで平衡化させたＤＯＷＥＸ５０カラム（The Dow Chemical Company）にサンプルのＴＦＡ溶液をアプライし、通過画分を回収した。１質量％ＴＦＡを流し、回収液量が５０ｍＬ程度になるまで通過画分を回収した。次に、５Ｍアンモニア水溶液（約２５ｍＬ）を添加後、溶出液を吸着画分として回収した。薄層クロマトグラフィーで２ＳＣの存在を確認した。吸着画分は凍結乾燥させ、これを標準試料とした。
〔メチルグリオキサール−イミダゾロン（ＭＧ−Ｈ）〕
１Ｎ水酸化ナトリウム溶液（４９ｍＬ）と４０質量％Ｍｅｔｈｙｌ Ｇｌｙｏｘａｌ溶液（８６２μｌ）を混合し、これにＬ-Ａｒｇｉｎｅ（８７１ｍｇ）を添加して攪拌し、３７℃で１時間インキュベートした。塩酸でサンプル溶液を中性にした。予め１０質量％ピリジン水溶液と１０質量％塩酸水溶液で洗浄し、純水で平衡化させたＤＯＷＥＸ５０カラム（The Dow Chemical Company）にサンプル溶液２５ｍＬをアプライし、カラム２倍量の純水を流した後、１０質量％ピリジン水溶液で溶出させ、約３ｍＬずつ分取した。各分画を濾紙にスポットし、ここに０．５質量％ニンヒドリン−水飽和ブタノール溶液を噴霧し、加熱、発色させた。発色した画分とＭＧ−Ｈのアイソフォーム１の市販品（PolyPeptide Laboratories社、ＵＳ、品番：ＳＣ１５２８）とをシリカゲルプレートにスポットし、クロロホルム：メタノール：水＝４：６：２の混合溶媒で展開させ、ここに０．５質量％ニンヒドリン−水飽和ブタノール溶液を噴霧し、加熱、発色させた。市販品と同じ位置に発色した部分を削り取って回収し、エバポレーターでピリジンを蒸発させた後に−８０℃で凍結させ、次に凍結乾燥をさせた。この凍結乾燥サンプル５００ｍｇを約１ｍＬの純水に溶解させ、シリカゲルカラム（内径１７ｍｍ、ベッド高４４０ｍｍ）にアプライし、９０体積％アセトニトリル（５０ｍＬ）で、次に８０体積％アセトニトリル（１５０ｍＬ）で、次に７０体積％アセトニトリル（１５０ｍＬ）で溶出を行い、溶出液を約８ｍＬずつ分取した。分取した各画分を濾紙にスポットし、ここに０．５質量％ニンヒドリン−水飽和ブタノール溶液を噴霧した。発色した画分を選択し、前述の条件で薄層クロマトグラフィーを行い、ＭＧ−Ｈの存在を確認した。選択した画分は、凍結乾燥させ、再度シリカゲルカラムで精製を行い、分取した各画分を濾紙にスポットし、ここに０．５質量％ニンヒドリン−水飽和ブタノール溶液を添加した。発色した画分を選択し、前述の条件で薄層クロマトグラフィーを行い、ＭＧ−Ｈの存在を確認した。確認できた画分を集め、これを標準試料とした。
ＣＭＬ、２ＳＣ、ＭＧ−Ｈの各標準試料は、質量分析計にて分子量を調べ、目的とする物質であることを確認した。
(Preparation of standard sample)
[N-ε- (carboxymethyl) lysine (CML)]
A commercially available product (PolyPeptide Laboratories, US, product number: SC1505) was used as a standard sample.
[S- (2-succinyl) cysteine (2SC)]
N-acetyl-Cys (48.9 mg) and N-ethylmaleimide (75 mg) were added to the tube, and the volume was made up to 10 mL with pure water. After incubating at room temperature for 2 hours, it was divided into four equal amounts of tubes with screw caps and dried with a centrifugal concentrator. 1 mL of 6N hydrochloric acid aqueous solution was added to each dried sample and hydrolyzed at 110 ° C. for 24 hours, and then the hydrolyzate was concentrated by centrifugation. Each dry sample was dissolved in 5 mL of 1 wt% TFA. A sample TFA solution was applied to a DOWEX50 column (The Dow Chemical Company) previously washed with a 10% by mass hydrochloric acid aqueous solution and equilibrated with about 5 times the amount of 1% by mass TFA, and the passing fraction was collected. 1% by mass TFA was flowed, and the flow-through fraction was collected until the amount of the collected liquid reached about 50 mL. Next, 5M ammonia aqueous solution (about 25 mL) was added, and the eluate was recovered as an adsorbed fraction. The presence of 2SC was confirmed by thin layer chromatography. The adsorbed fraction was freeze-dried and used as a standard sample.
[Methylglyoxal-imidazolone (MG-H)]
1N sodium hydroxide solution (49 mL) and 40% by mass Methyl Glyoxal solution (862 μl) were mixed, and L-Argine (871 mg) was added thereto, stirred, and incubated at 37 ° C. for 1 hour. The sample solution was neutralized with hydrochloric acid. After applying 25 mL of the sample solution to a DOWEX 50 column (The Dow Chemical Company) previously washed with a 10% by mass pyridine aqueous solution and a 10% by mass hydrochloric acid aqueous solution and equilibrated with pure water, and then flowing 2 column volumes of pure water. Elution was performed with a 10% by mass pyridine aqueous solution, and about 3 mL was collected. Each fraction was spotted on a filter paper, and 0.5% by mass of a ninhydrin-water saturated butanol solution was sprayed thereon to heat and develop color. The colored fraction and a commercial product of MG-H isoform 1 (PolyPeptide Laboratories, US, product number: SC1528) were spotted on a silica gel plate and developed with a mixed solvent of chloroform: methanol: water = 4: 6: 2. The solution was sprayed with 0.5% by mass ninhydrin-water saturated butanol solution, and heated to develop color. The colored portion at the same position as the commercial product was scraped off and collected. After evaporation of pyridine with an evaporator, it was frozen at −80 ° C. and then freeze-dried. 500 mg of this lyophilized sample was dissolved in about 1 mL of pure water, applied to a silica gel column (inner diameter: 17 mm, bed height: 440 mm), then added with 90% by volume acetonitrile (50 mL), then with 80% by volume acetonitrile (150 mL), Was eluted with 70% by volume acetonitrile (150 mL), and about 8 mL of the eluate was collected. Each fraction collected was spotted on a filter paper, and a 0.5% by mass ninhydrin-water saturated butanol solution was sprayed thereon. The colored fraction was selected and thin layer chromatography was performed under the conditions described above to confirm the presence of MG-H. The selected fractions were lyophilized, purified again with a silica gel column, and each fraction collected was spotted on a filter paper, and a 0.5 mass% ninhydrin-water saturated butanol solution was added thereto. The colored fraction was selected and thin layer chromatography was performed under the conditions described above to confirm the presence of MG-H. The confirmed fractions were collected and used as a standard sample.
Each standard sample of CML, 2SC, and MG-H was examined for molecular weight by a mass spectrometer, and confirmed to be a target substance.
実施例２
実施例１で得られたＡＧＥ分析用試料の全量をＬＣ−ＭＳ／ＭＳにかけ、ＣＭＬ、２ＳＣおよびＭＧ−Ｈを測定した。対照として、酸加水分解を行わなかった以外は実施例１と同じ手順で調製した試料、および強酸性陽イオン交換樹脂の代わりにＣ１８化シリカ担体充填カラムＳｅｐ−Ｐａｋ（登録商標）Ｃ１８（日本ウォーターズ社）を用いた以外は実施例１と同じ手順で調製した試料について、同様に測定した。
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）、２ＳＣ（約１０分）、ＣＭＬ（約１２分）
（質量分析条件）
イオン化方法：Ｈ−ＥＳＩ
インジェクション量：１０μＬ
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））
ＭＧ−Ｈ：７０（２３）、１６６（１４）、１１４（１４）
２ＳＣ：１２１（１９）、１０３（２５）
ＣＭＬ：８４（１６）、１３０（１１）
Example 2
The whole amount of the AGE analysis sample obtained in Example 1 was subjected to LC-MS / MS, and CML, 2SC and MG-H were measured. As a control, a sample prepared by the same procedure as in Example 1 except that acid hydrolysis was not performed, and a Sep 18 P18 (registered trademark) C18 (Nippon Waters) column packed with a C18-silica support instead of a strongly acidic cation exchange resin. A sample prepared in the same procedure as in Example 1 except that was used was measured in the same manner.
The conditions for LC-MS / MS measurement are as follows.
(HPLC conditions)
Chromatography column: SeQuant, ZIC-HILIC, 150 × 2.1 mm, 5 μm, 200 A Peak Hplc Column
Column temperature: 40 ° C
Mobile phase: A: 0.1% by mass formic acid aqueous solution, B: 0.1% by mass formic acid-containing acetonitrile solution Gradient condition: A: 10% + B: 90%
Flow rate: 200 μL / min
Injection volume: 10μL
Analysis time: 20 minutes Elution time: MG-H (about 12 minutes), 2SC (about 10 minutes), CML (about 12 minutes)
(Mass spectrometric conditions)
Ionization method: H-ESI
Injection volume: 10μL
Capillary temperature: 300 ° C
Ionization energy: About 3500V (at the time of positive ionization)
(Detection peak (m / z))
MG-H: 70 (23), 166 (14), 114 (14)
2SC: 121 (19), 103 (25)
CML: 84 (16), 130 (11)
測定結果を図１〜３に示す。塩酸加水分解と強酸性陽イオン交換樹脂による精製を行った試料は、目的物質のピーク（図中、矢印）が明瞭に検出された。一方、酸加水分解をしなかった試料およびシリカ充填カラムで精製した試料は、目的物質のピーク以外のピークやノイズが発生していた。特に２ＳＣは、塩酸加水分解をしない場合、目的のピークが全く検出されなかった。 The measurement results are shown in FIGS. In the sample that had been subjected to hydrochloric acid hydrolysis and purified with a strongly acidic cation exchange resin, the peak of the target substance (in the figure, the arrow) was clearly detected. On the other hand, the sample that was not acid-hydrolyzed and the sample purified by the silica packed column had peaks and noises other than the peak of the target substance. In particular, in 2SC, the target peak was not detected at all when hydrochloric acid hydrolysis was not performed.
Claims (7)
生体試料を液相において塩酸を用いて処理すること、および
液相において処理した試料を強酸性陽イオン交換樹脂に添加し、非酸性条件下で溶出すること
を含む試料の調製方法。 A method for preparing a sample for final glycation product analysis comprising:
Treating with hydrochloric acid a biological sample in liquid phase, and
A method for preparing a sample, comprising adding a sample treated in a liquid phase to a strongly acidic cation exchange resin and eluting under a non-acidic condition.
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