JP4473999B2 - Column cleaning solution - Google Patents

Description

【００３３】
【表２】

【００３４】
ｐＨ７．０〜１２．０で緩衝能を有する物質とは、少なくとも一つのｐＫａ（酸解離定数）が５．３〜１１．５であるものが好ましく、より好ましくは５．５〜１１．０のものが好ましい。
【００３５】
上記緩衝剤の洗浄液中の濃度、及びこれら緩衝剤を複数種用いる場合には、これら複数種の合計の濃度は、洗浄液のｐＨを２．０〜４．５、または、７．０〜１２．０にする緩衝作用があれば良く、その濃度としては、１〜１０００ｍＭが好ましく、更には１０〜５００ｍＭが好ましい。
【００３６】
また、上記洗浄液には、以下の物質を添加しても良い。
【００３７】
（１）無機塩類（塩化ナトリウム、塩化カリウム、硫酸ナトリウム、硫酸カリウム、リン酸ナトリウム等）を添加しても良い。これらの塩類の濃度は、特に限定されないが、好ましくは１〜１５００ｍＭである。
【００３８】
（２）ｐＨ調節剤として、公知の酸、塩基を加えても良い。酸としては、例えば、塩酸、リン酸、硝酸、硫酸等が、塩基としては、水酸化ナトリウム、水酸化カリウム、水酸化リチウム、水酸化マグネシウム、水酸化バリウム、水酸化カルシウム等が挙げられる。これらの酸、塩基の濃度は、特に限定されないが、好ましくは、０．００１〜５００ｍＭである。
【００３９】
（３）メタノール、エタノール、アセトニトリル、アセトン等の水溶性有機溶媒を混合しても良い。これらの有機溶媒の濃度は、特に限定されないが、好ましくは０〜８０％（ｖ／ｖ）であり、カオトロピックイオン、無機酸、有機酸、これらの塩等が析出しない程度であって、かつ、分離パターンに悪影響を与えない範囲で用いるのが好ましい。
【００４０】
（４）アジ化ナトリウム、チモール等の防腐剤を添加しても良い。
上記のｐＨ２．０〜４．５で緩衝能を持つ緩衝剤とｐＨ７．０〜１２．０で緩衝能を持つ緩衝剤を同一洗浄液中に添加して用いると同一洗浄液のｐＨを酸性側、または、アルカリ側に変えるだけで２種類の洗浄液を調製出来るメリットがある。
また、本発明の洗浄液を１検体測定後のカラム洗浄液としても用いても良い。その場合、勾配溶出法、あるいは段階溶出法によって送液しても良い。
【００４１】
また、本発明におけるカラム洗浄液は、従来公知のカラム再生方法に用いるカラム洗浄液として用いて良い。
【００４２】
発明におけるカラム洗浄液は、液体クロマトグラフ装置の特定の局面で、少なくとも１種の溶離液としても用いて良い。
【００４３】
［充填剤］
本発明において充填剤は、公知のものでよく特に制限はない。 このような、充填剤としては、例えば、シリカ、ジルコニアなどの無機系粒子；セルロース、ポリアミノ酸、キトサンなどの天然高分子粒子；ポリスチレン、ポリアクリル酸エステルなどの合成高分子粒子などが挙げられる。
但し、上記シリカ等は、アルカリで溶解するので、上記カラム洗浄液のｐＨは、これらが溶解しない範囲で設定することが好ましい。
【００４４】
また、ＬＣの分離は、充填剤の表面官能基と測定対象試料との相互作用により決まるため、充填剤表面の官能基種は、適用するＬＣの分離モードによって、以下のように選択され、これらの官能基を持つ充填剤が使用される。
【００４５】
（イオン交換クロマトグラフィー用充填剤）
上記充填剤には、イオン交換基として、カチオン交換基又は、アニオン交換基を含有するものを用いるのが好ましい。
【００４６】
上記カチオン交換基は、公知のものでよく特に制限はない。例えば、カルボキシル基、スルホン酸基、リン酸基などのカチオン交換基等が挙げられる。また、このカチオン交換基は、複数種導入しても良い。
【００４７】
上記アニオン交換基は、公知のものでよく特に制限はない。例えば、３級アミノ基や４級アミノ基などのアニオン交換基等が挙げられる。また、このアニオン交換基は、複数種導入しても良い。
【００４８】
（ゲル浸透クロマトグラフィー用充填剤）
上記充填剤には、水酸基、エポキシ基等の非イオン性親水基を含有するものを用いるのが好ましい。
【００４９】
（逆相クロマトグラフィー用充填剤）
上記充填剤には、オクチル基やオクタデシル基などのアルキル基、あるいはフェニル基等の疎水性基を含有するものを用いるのが好ましい。
【００５０】
上記充填剤は、単独に、あるいは複数種の充填剤を組み合わせたものに、本カラム洗浄液は用いることができる。
【００５１】
上記粒子の直径は、好ましくは０．５〜２０μｍ、より好ましくは１〜１０μｍである。
また、粒度分布は、変動係数値（ＣＶ値）（粒径の標準偏差÷平均直径×１００（％））として、好ましくは４０％以下、より好ましくは３０％以下である。
【００５２】
［カラム］
上記充填剤はカラムに充填されて液体クロマトグラフィー測定に用いられる。カラムのサイズは、内径０．１〜５０ｍｍ、長さ１〜３００ｍｍのものが好ましく、より好ましくは、内径０．２〜３０ｍｍ、長さ５〜２００ｍｍである。
【００５３】
上記カラムの素材としては、特に限定されず、公知のステンレス製等の金属、ガラス製、ＰＥＥＫ等の樹脂製などが用いられる。
また、カラムの充填剤とカラム本体が接する部位を、不活性な素材で被覆してもよく、その素材としては、例えばＰＥＥＫ、ポリエチレン、テフロン、チタン化合物、珪素化合物、シリコン膜等が挙げられる。
【００５４】
［装置］
本発明に使用されるＬＣ装置は、公知のもので良く、例えば、送液ポンプ、試料注入装置（サンプラ）、カラム、検出器等から構成される。また、他の付属装置（カラム恒温槽や溶離液の脱気装置等）が適宜付加されても良い。
【００５５】
また、本発明の測定方法における溶出方法は、公知の方法でよく、段階溶出法、勾配溶出、ステップアップグラディエント溶出を行ってもよい。
【００５６】
上記測定方法における、他の測定条件としては、公知の条件でよく、溶離液の流速は、好ましくは０．０５〜５ｍｌ／分、より好ましくは０．２〜３ｍｌ／分である。ヘモグロビン類の検出は、４１５ｎｍの可視光が好ましいが、特にこれのみに限定されるわけではない。測定試料は、通常、界面活性剤など溶血活性を有する物質を含む溶液により溶血された溶血液を希釈したものを用いる。液体クロマトグラフへの試料注入量は、希釈倍率により異なるが、好ましくは０．１〜１００μｌ程度である。
【００５７】
［溶離液］
本発明の溶離液は、液体クロマトグラフ装置において測定対象成分に応じて用いられている適宜の溶離液により構成されていることが好ましい。
【００５８】
（ヘモグロビン類の分析の場合）
特にヘモグロビン類の分析の場合、ＨｂＡ０の溶出に際し、すなわち、ＨｂＡ１ｃより強く充填剤に保持されたＨｂＡ等から成る「ＨｂＡ０成分」を溶出するためには、カラムに流入する際のｐＨをヘモグロビンの等電点よりアルカリ側になるように設定した溶離液を用いるのが好ましい。この条件を実現するには、ｐＨがヘモグロビンの等電点よりアルカリ側であるひとつの溶離液を送液する方法や、ｐＨの異なる２種以上の溶離液を用いる方法がある。
【００５９】
ヘモグロビンはｐＨが等電点より酸性側からアルカリ側になると、総荷電がプラスからマイナスに変わるため、充填剤の陽イオン交換基との「電気的反発力によってＨｂＡ０成分を溶出」させることができる。
【００６０】
なお、理化学辞典（第４版、１９８７年９月、岩波書店、久保亮五ら編集）、１１７８頁に記載されているように、ヘモグロビンの等電点はｐＨ６．８〜７．０である。そのため、ＨｂＡ０成分を溶出するために、カラムに流入する際の溶離液のｐＨを６．８以上にすることがより好ましい。
【００６１】
この条件を満たすため、測定に用いる溶離液の内、少なくともひとつの溶離液のｐＨが６．８以上であることが必要である。本溶離液のｐＨは望ましくは７．０〜１２．０であり、７．５〜１１．０がより好ましく、更には８．０〜９．５が好ましい。溶離液のｐＨが６．８未満になるとＨｂＡ０成分の溶出が不十分となる。溶離液のｐＨは、用いる充填剤の分解が起こらない範囲に設定すれば良い。
【００６２】
ＨｂＡ０成分の溶出に好適に用いられる、ｐＨが６．８以上で緩衝能をもつ溶離液としては、例えば、リン酸、ホウ酸、炭酸等の無機酸または、その塩；クエン酸等のヒドロキシカルボン酸、β、β−ジメチルグルタル酸等のカルボン酸誘導体、マレイン酸等のジカルボン酸、カコジル酸、等の有機酸または、その塩からなる緩衝液が挙げられる。その他、２−（Ｎ−モリホリノ）エタンスルホン酸（ＭＥＳ）、Ｎ−２−ヒドロキシエチルピペラジン−Ｎ’−エタンスルホン酸（ＨＥＰＥＳ）、ビス（２−ヒドロキシエチル）イミノトリス−（ヒドロキシメチル）メタン（Ｂｉｓｔｒｉｓ）、Ｔｒｉｓ、ＡＤＡ、ＰＩＰＥＳ、Ｂｉｓｔｒｉｓｐｒｏｐａｎｅ、ＡＣＥＳ、ＭＯＰＳ、ＢＥＳ、ＴＥＳ、ＨＥＰＥＳ、ＨＥＰＰＳ、Ｔｒｉｃｉｎｅ、Ｂｉｃｉｎｅ、グリシルグリシン、ＴＡＰＳ、ＣＡＰＳ等の一般にグッド（Ｇｏｏｄ）の緩衝液といわれるものも使用できる。また、ＢｒｉｔｔｏｎとＲｏｂｉｎｓｏｎの緩衝液；ＧＴＡ緩衝液も使用できる。また、イミダゾール等のイミダゾール類；エチレンジアミン、メチルアミン、エチルアミン、トリエチルアミン、ジエタノールアミン、トリエタノールアミン等のアミン類；グリシン、β−アラニン、アスパラギン酸、アスパラギン等のアミノ酸類；等の有機物も使用できる。
【００６３】
また、無機酸；有機酸；無機酸または有機酸の塩；有機物は、複数混合して用いても良く、また、有機酸、無機酸及び有機物を混合しても良い。
【００６４】
（作用）
本発明１のカラム洗浄液は、カオトロピックイオンを含み、かつ、ｐＨが２．０〜４．５及び／又は、ｐＨが７．０〜１２．０で緩衝能を持つ緩衝剤からなるため、カラム中の充填剤に非特異的に吸着した蛋白質・脂質を十分に洗浄する効果があるので、カラム再生及び洗浄において容易に用いることができるばかりでなく、幅広いカラムに使用できる汎用性の高いものである。
【００６５】
【実施例】
次に、実施例、比較例を挙げて本発明を詳しく説明するが、本発明はこれらの実施例にのみ限定されるものではない。
【００６６】
（実施例１）
カチオン交換クロマトグラフィーにより、ヒト血液中のＨｂ類の測定を行った。
（充填剤の調製）
テトラエチレングリコールジメタクリレート（新中村化学製）４００ｇ、メタクリル酸（和光純薬）１５０ｇの混合物に過酸化ベンゾイル（和光純薬製）１．５ｇを溶解した。これを４重量％のポリビニルアルコール（日本合成化学製）水溶液２５００ｍＬに分散させ、撹拌しながら窒素雰囲気下で７５℃に昇温し、８時間重合した。重合後、洗浄し乾燥した後、分級して平均粒径６μm の粒子を得た（充填剤１）。
（カラムの充填）
得られた充填剤１を以下の方法でカラムに充填した。粒子０．７ｇを、５０ｍＭリン酸緩衝液（ｐＨ５．８）３０ｍＬに分散し、５分間超音波処理した後、よく撹拌した。全量をステンレス製空カラム（内径４．６×３５mm）を接続したパッカー（梅谷精機製）に注入した。パッカーに送液ポンプ（サヌキ工業製）を接続し、圧力３００ｋｇ／ｃｍ² で定圧充填した。
【００６７】

【００６８】
（測定試料の調整）
健常人血をフッ化ナトリウム採血した全血検体を溶血希釈液Ｘ（０．１重量％トリトンＸ−１００のリン酸緩衝液溶液（ｐＨ７．０））で溶血し、１５０倍に希釈して測定試料とした。
（測定結果）
上記測定条件により、試料を測定して得られたクロマトグラムを図１に示した。ピーク１はＨｂＡ１ａおよびＨｂＡ１ｂ、ピーク２はＨｂＦ、ピーク３は不安定型ＨｂＡ１ｃ、ピーク４は安定型ＨｂＡ１ｃ、ピーク５はＨｂＡ０を示す。図から解るように、いずれのピークも良好に分離された。
（カラム耐久性の評価）
測定試料を２００検体測定する毎に、本発明のカラム洗浄液を１分間流し、溶離液Ａ（専用溶離液５０Ａ）を２分間流し、測定試料を４０００検体測定した。
上記測定試料を１０００検体、２０００検体、３０００検体、４０００検体測定したときのヘモグロビンＡ１ｃ値の変動を図２に示した。これより実施例１は、測定試料を４０００検体測定してもＨｂＡ１ｃ値の変動は、初期に比べて測定値の３％以内と測定値の変化がなく良好な耐久性を有する。
【００６９】
（比較例１）
カラム洗浄液として５０ｍＭリン酸緩衝液（ｐＨ１２．０）を用いた以外は、実施例１と同様にしてカラム耐久性を評価した。
（カラム耐久性評価結果）
結果を図２に示した。比較例１では、ＨｂＡ１ｃの保持時間が早くなり、ＨｂＡ１ｃ値の変動が次第に大きくなりカラム耐久性が悪い。
【００７０】
（実施例２）
ゲル浸透クロマトグラフィーによりリポ蛋白の測定を行った。
（カラム）
リポ蛋白専用カラム（ＬＩＰＯＰＲＯＰＫ, 内径７．５ｍｍ長さ３０ｃｍ：東ソー社製）

上記システムにより、血清５μＬを注入し、カラムからの溶出液に酵素液を送り込み４５℃で反応（テフロンチューブ内径０．４ｍｍ、長さ７．５ｍｍ）させた後、吸光度５５０ｎｍで検出した。
【００７１】
（測定試料）
健常人血の血清を用いた。
（カラム耐久性の評価）
測定試料を５０検体測定する毎に、本発明のカラム洗浄液を１分間流し、専用溶離液を２分間流し、測定試料を１０００検体測定した。結果を図３に示した。
上記測定試料を３００検体、６００検体、１０００検体測定したときのリポタンパク値（ＨＤＬ値）の変動の度合で評価した。
これより実施例２は、測定試料を１０００検体測定してもＨＤＬ値の変動は、初期に比べて測定値の３％以内と測定値の変化がなく良好な耐久性を有する。
【００７２】
（比較例２）
カラム洗浄液として５０ｍＭリン酸緩衝液（ｐＨ１２．０）を用いた以外は、実施例２と同様にしてカラム耐久性を評価した。
（カラム耐久性評価結果）
結果を図３に示した。比較例２では、ＨＤＬ値の変動が次第に大きくなりカラム耐久性が悪い。
【００７３】
（実施例３）
陰イオン交換クロマトグラフィーにより蛋白アルブミンの測定を行った。
（カラム）
Ｓｈｏｄｅｘ ＩＥＣ ＤＥＡＤ−４２０Ｎ, 内径４．６ｍｍ長さ３５ｃｍ：昭和電工社製）

【００７４】
（測定試料）
卵白アルブンミン水溶液（１０ｍｇ／ｍＬ）
（カラム耐久性の評価）
測定試料を５０検体測定する毎に、本発明のカラム洗浄液を１分間流し、溶離液Ａを２分間流し、測定試料を１０００検体測定した。結果を図４に示した。 上記測定試料を３００検体、６００検体、１０００検体測定したときのアルブミン値の変動で評価した。
これより実施例３は、測定試料を１０００検体測定してもアルブミン値の変動は、初期に比べて測定値の３％以内と測定値の変化がなく良好な耐久性を有する。
【００７５】
（比較例３）
カラム洗浄液として５０ｍＭリン酸緩衝液（ｐＨ２．０）を用いた以外は、実施例３と同様にしてカラム耐久性を評価した。
（カラム耐久性評価結果）
結果を図４に示した。比較例３では、アルブミン値の変動は、次第に大きくなりカラム耐久性が悪かった。
【００７６】
（実施例４）
逆相クロマトグラフィーにより、リボヌクレアーゼの測定を行った。
（カラム）
ＴＳＫｇｅｌ ＯＤＳ−８０Ｔｓ，内径６．０ｍｍ長さ１５０ｍｍ：昭和電工社製）

【００７７】
（測定試料）
リボヌクレアーゼ水溶液（１０ｍｇ／ｍＬ）
（カラム耐久性の評価）
測定試料を５０検体測定する毎に、本発明のカラム洗浄液を１分間流し、溶離液Ａを２分間流し、測定試料を１０００検体測定した。結果を図５に示した。 上記測定試料を３００検体、６００検体、１０００検体測定したときのリボヌクレアーゼの値の変動で評価した。
これより実施例４は、測定試料を１０００検体測定してもリボヌクレアーゼ値の変動は、測定値の３％以内と測定値の変化がなく良好な耐久性を有する。
【００７８】
（比較例４）
カラム洗浄液として５０ｍＭリン酸緩衝液（ｐＨ２．０）を用いた以外は、実施例４と同様にしてカラム耐久性を評価した。
（カラム耐久性評価結果）
結果を図５に示した。比較例４では、リボヌクレアーゼ値の変動は、次第に大きくなりカラム耐久性が悪かった。
【００７９】
（実施例５）
液体クロマトグラフ装置の自動洗浄を行い測定を行った。
（装置類）
高速液体クロマトグラフ装置（京都第一科学社製、自動グリコヘモグロビン測定装置ＨＡ−８１３１）を改良し、図６に示した液体クロマトグラフ装置を構成した。分離カラム１３としては、上記液体クロマトグラフ装置専用のカラム（積水化学工業社製、カラムユニット−ＨＳIII ）を用いた。ここでは、検体の測定回数が、予め決められた基準回数になった場合に、カラムの洗浄操作を行うように改良したクロマトグラフ装置を用いた。
【００８０】
図６に示した液体クロマトグラフ装置の動作説明に従って複数の検体を測定した。この場合、段階溶出法を用い、溶離液１を１０８秒、溶離液２を１２秒、溶離液１を６０秒送液することにより１回の測定に際しての送液を行った。
【００８１】
（溶離液）
溶離液１，２としては、上記液体クロマトグラフ装置専用溶離液である積水化学工業社製溶離液１（５０ｍＭの過塩素酸を含有するリン酸緩衝液、ｐＨ＝５．３）及び溶離液２（３００ｍＭの過塩素酸を含有するリン酸緩衝液、ｐＨ＝８．５）を用いた。なお、洗浄液としては、上記溶離液１，溶離液２を下記の方法で用いた。
（洗浄液）
すなわち、洗浄液の代わりに溶離液１，２を以下のように送液した。すなわち、洗浄液として溶離液１，２を用い、溶離液２を１０８秒送液し、次に溶離液１を７２秒送液することにより洗浄操作を行った。
【００８２】
血液試料を調製するのに、赤血球を溶血し希釈するため溶血洗浄液（上記液体クロマトグラフ装置専用溶血洗浄液）を用いた。
測定及び洗浄に際しての測定流路を流れる溶離液１，２の流速はいずれも２ｍｌ／分とした。
【００８３】
（測定試料）
検体としては、抗凝固剤としてＥＤＴＡ−２Ｋが収容された採血管（積水化学工業社製、商品名：インセパック−Ｅ）で採血した全血を検体とした。本液体クロマトグラフ装置では、採血管から全血が採取され、この全血が上記溶血洗浄液により溶血されて自動的に溶血希釈試料が調製される。
【００８４】
（カラム耐久性評価結果）
洗浄操作を行う基準回数を３００とし、１５００回の測定を行った。この場合、分離カラム１３の初期状態のＨｂＡ１ｃの保持時間を１００とし、各測定回数ごとに測定されたＨｂＡ１ｃの保持時間を記録した。結果を図７に実線Ａで示す。実線Ａから明らかなように、１５００回測定した場合であっても、ＨｂＡ１ｃの保持時間はほとんど変動しなかった。なお、洗浄に際しての分離カラム流入部の溶離液ｐＨを測定したところ、８．２であった。
【００８５】
（比較例５）
洗浄操作を行わなかったことを除いては、実施例５と同様にして測定を行った。結果を図７に破線で示す。
図７から明らかなように、洗浄操作を行わなかった場合には、ＨｂＡ１ｃの保持時間は、測定回数が増加するにつれて早くなることがわかる。
【００８６】
（実施例６）
測定カウンタを使用しないこと及びＨｂＡ１ｃの保持時間が、定められた範囲からはずれている場合に、カラムの洗浄操作を行うように改良したことを除いては、実施例５と同じ装置を用い、実施例５と同様にして溶離液１，２を送液した。すなわち、分離カラム１３の初期状態のＨｂＡ１ｃの保持時間を１００とし、該保持時間が９５以上を設定範囲とした。従って、保持時間が９５未満になった場合に、洗浄操作を開始することとした。
【００８７】
また、洗浄に際しては、段階溶出法に従って、溶離液２を１０８秒送液し、次に溶離液１を７２秒送液した。すなわち洗浄液は用いなかった。
実施例５と同様にして実施例５で用いたのと同じ試料を２５００回測定した。結果を図８に示す。図８から明らかなように、約４５０回測定した後、保持時間が速くなり、約８００回測定したところで基準値を下回ったので自動洗浄が開始された。自動洗浄後には保持時間はもとにレベルに復帰した。従って、最終的に２５００回測定したところ、自動洗浄が３回行われ、２５００回の測定まで、高精度にＨｂＡ１ｃを測定し得ることが確かめられた。
【００８８】
【発明の効果】
本発明のカラム洗浄液は、カオトロピックイオンを含み、かつ、ｐＨが２．０〜４．５、及び／又は、ｐＨが７．０〜１２．０で緩衝能を持つ緩衝剤からなるので、カラム中の充填剤に非特異的に吸着した蛋白質・脂質を十分に洗浄する効果があり、カラム再生及び洗浄において容易に用いることができるばかりでなく、幅広いカラムに使用できる汎用性の高いものである。
【図面の簡単な説明】
【図１】実施例１の測定条件により、ヘモグロビン類の測定を行った際に得られたクロマトグラムを示す図。
【図２】実施例１及び比較例１における測定回数の増加にともなうＨｂＡ１ｃ値の変動を示す図。
【図３】実施例２及び比較例２における測定回数の増加にともなうＨＤＬ値の変動を示す図。
【図４】実施例３及び比較例３における測定回数の増加にともなうアルブミンの変動を示す図。
【図５】実施例４及び比較例４における測定回数の増加にともなうリボヌクレアーゼの変動を示す図。
【図６】実施例５に係る液体クロマトグラフ装置の概略ブロック図。
【図７】実施例５及び比較例５における測定回数の増加にともなうＨｂＡ１ｃの保持時間の変化を示す図。
【図８】実施例６において、測定回数の増加によるＨｂＡ１ｃの保持時間の変化を示す図。
【符号の説明】
１ ＨｂＡ１ａ及びｂのピーク
２ ＨｂＦのピーク
３ 不安定型ＨｂＡ１ｃのピーク
４ 安定型ＨｂＡ１ｃのピーク
５ ＨｂＡ０のピーク
１１ 送液手段
１２ 制御手段
１３ 分離カラム
１４ 検出手段
１５ 試料注入手段
１６ 検体検知手段
１７ 測定カウンタ[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a cleaning liquid for a liquid chromatograph column whose analysis target is, for example, a biological component.
[0002]
[Prior art]
Recently, liquid chromatograph (LC) has been widely used in the clinical laboratory field, and in particular, a component to be measured is separated from contaminant components contained in a blood sample, or a plurality of components having similar properties. Are used to separate and analyze the components from each other and to separate specific components.
[0003]
In the liquid chromatograph apparatus, the sample-containing eluent is supplied to the separation column for separating various components as described above, and due to the difference in the interaction between the filler in the separation column and the various components in the sample, The components are separated.
[0004]
The components contained in the sample are eluted from the separation column in order from the component with the weakest interaction. Therefore, chromatographic information can be obtained by measuring the eluate with a detection means such as an absorptiometer. By analyzing this chromatographic information, qualitative analysis and quantitative analysis of the measurement target component can be performed.
[0005]
On the other hand, among blood samples, glycated hemoglobin, particularly hemoglobin A1c (hereinafter referred to as HbA1c) is widely used as an index for diabetes diagnosis. HbA1c is produced by irreversibly binding to hemoglobin after sugar in the blood enters the erythrocytes, and reflects the average sugar concentration in the blood over the past 1-2 months.
And as a measuring method of this HbA1c, the above-mentioned LC method and immunization method are generally used.
[0006]
The measurement of HbA1c by the liquid chromatography method is mainly performed by the cation exchange liquid chromatography method (Japanese Patent Publication No. 8-7198 etc.). When a hemolyzed sample is separated by cation exchange liquid chromatography, usually, hemoglobin A1a (hereinafter referred to as HbA1a) and hemoglobin A1b (hereinafter referred to as HbA1b), hemoglobin F (hereinafter referred to as HbF), unstable HbA1c, stable HbA1c, and Peaks such as hemoglobin A0 (hereinafter referred to as HbA0) appear. In addition, HbA1c used as an index for diagnosis of diabetes is recently stable HbA1c, and the ratio is expressed as the ratio (%) of the area of stable HbA1c peak to the total hemoglobin peak area. It has been demanded.
[0007]
However, when many HbA1c values in the blood sample are measured, the LC separation column gradually deteriorates. For this reason, the peak retention time (Rt) is increased, the position of the peak is moved, each peak is broadened, and the shape of the chromatogram is changed. As a result, the HbA1c value also changes and there is a problem that the variation of the HbA1c value becomes large.
[0008]
Furthermore, blood samples are rich in proteins such as albumin and lipids such as cholesterol. When analyzing an analyte in a blood sample by LC, proteins and lipids other than the analyte are non-specifically adsorbed on the filler in the separation column, and the analysis in the filler and blood sample in the separation column is performed. The interaction with the object changes (generally, the interaction becomes smaller), the separation pattern is deteriorated, and accurate measurement values cannot be obtained (deterioration of the column).
In addition, dedicated separation columns that can analyze and quantify glycated albumin, glycated hemoglobin, and cholesterol in a short time have recently been sold, but they are very expensive. And lipids adsorb nonspecifically, and the durability of the separation column is poor.
If the durability of the separation column is improved, there is a merit that the cost (running cost) related to the measurement becomes very low.
[0009]
For example, when the separation column deteriorates in the measurement of HbA1c, the component that is strongly retained in the filler in the blood sample is gradually accumulated in the filler, and the effective ion exchange groups in the filler are reduced. It is. As a result, the retention behavior of each hemoglobin component in the separation column changes.
[0010]
In order to solve the above problems, as a conventional column regeneration method, high-performance liquid chromatographic analysis (revised edition) (Edited by the Japan Society for Analytical Chemistry, Kanto Branch, Editor: Japan Society for Analytical Chemistry, Kanto Branch, Issuer: Katsuhisa Morita, Revised May 30, 1985, Second Edition, p104, 7.5 Column Life and Regeneration) describes the following methods 1 and 2.
In the first method, in the case where irreversible adsorption occurs, the property of the surface of the filler changes, and the elution pattern of the standard sample changes, a solvent that dissolves the substance coating the surface well. It recovers only by washing with.
In the second method, the ion exchange resin is washed with 1N hydrochloric acid and 1N sodium hydroxide, equilibrated with 1 to 2N saline, and then washed with an ultrasonic cleaner.
Usually, this method may be used. However, when protein or the like is strongly adsorbed, it is treated with 1-2N sodium hydroxide or separated and eluted using an enzyme such as protease. If recovery does not occur after the above treatment, treatment with about 10% sodium hypochlorite recovers in most cases, but the exchange capacity may change slightly.
[0011]
In addition to the above, there is a method for improving the column durability by the following method.
[0012]
For example, as a third method, in order to wash the separation column when HbA1c is quantified, a method of passing a washing solution composed of 0.5% SDS + 0.5 M NaOH in the reverse direction to the measurement method at the time of measurement. (Pharmacia, FPLC: Application file No. 13 “Quantification of HbA1c”, published in March 1985).
[0013]
In JP-A-5-281222, as a fourth method, a phosphate buffer solution containing S-carboxyalkyl-L-cysteine at a concentration of 100 mM or less when analyzing hemoglobins in a blood sample by chromatography. A method of supplying a separation column in the middle of an analysis step is disclosed. In this method, it is indicated that the liquid may be used as an eluent in the measurement, and the separation column may be used as a washing liquid.
[0014]
As a fifth method, Japanese Patent Application Laid-Open No. 6-9469 discloses a packing material in a separation column in order to prevent the separation column for liquid chromatography from being deteriorated due to a change in functional group during storage or use. A method is disclosed in which a solution containing a component having the same structure as the chemical modification group is used as a separation column cleaning solution.
[0015]
[Problems to be solved by the invention]
However, in the first method, the separation pattern changes unless the washed solvent is completely removed from the filler. In order to remove the solvent, it is necessary to dry the packing material and refill the column, which is very complicated.
The column regeneration is insufficient only by treatment with hydrochloric acid or sodium hydroxide in the second method. Moreover, after using an enzyme such as a protease, the method of removing the enzyme is very complicated, and removal of lipids and the like is insufficient only by the protease treatment. When treated with about 10% sodium hypochlorite, the exchange capacity may change slightly.
In the third method, since the cleaning liquid contains a surfactant, bubbles enter the column and cause column deterioration.
[0016]
In the fourth and fifth methods, the eluent is designed so that hemoglobins can be separated in a short time and with high resolution when the organic compound is added for the purpose of washing. May be difficult.
[0017]
An object of the present invention is to provide a column cleaning solution that eliminates the above-mentioned drawbacks of the prior art, has a strong column cleaning capability, can sufficiently regenerate the column, and can improve the durability of the column.
[0018]
[Means for Solving the Problems]
In the first aspect of the present invention (hereinafter referred to as the present invention 1), the washing liquid for the column for liquid chromatography contains chaotropic ions and has a pH of 2.0 to 4.5 and / or a pH of 7 Provided is a column washing liquid (hereinafter sometimes abbreviated as a washing liquid) containing a buffer having a buffer capacity of 0 to 12.0.
In the present invention according to claim 2 (hereinafter referred to as present invention 2), the washing liquid for the column for liquid chromatography contains chaotropic ions and has a buffering capacity at pH 7.0 to 12.0. There is provided a column cleaning liquid characterized by being used for cleaning a column comprising a packing material containing an agent and having at least a cation exchange group.
Further, in the present invention according to claim 3 (hereinafter referred to as present invention 3), the washing liquid for the column for liquid chromatography contains a chaotropic ion and has a buffering capacity at a pH of 2.0 to 4.5. There is provided a column washing liquid characterized by being used for washing a column comprising a packing material containing an agent and having at least an anion exchange group.
Hereinafter, the present invention will be described in detail.
[0019]
[Column washing solution]
(Chaotropic ion)
The chaotropic ion in the present invention is an ion generated by dissociation when a compound is dissolved in an aqueous solution, which destroys the structure of water and suppresses the decrease in entropy of water that occurs when a hydrophobic substance and water come into contact with each other. It is.
[0021]
Above Chaotropic Io As an Chlorate ion Is Used.
[0022]
If the concentration of chaotropic ions in the washing solution is lower than 1 mM, the column washing effect is small, and even if it is higher than 3000 mM, the column washing effect cannot be expected. The concentration of chaotropic ions in the washing solution is preferably 1 mM to 3000 mM, more preferably 10 mM to 1000 mM, and further preferably 50 mM to 500 mM.
Moreover, you may use the said chaotropic ion in mixture of multiple types.
The pH of the column cleaning solution of the present invention is preferably 2.0 to 4.5 or 7.0 to 12.0.
[0023]
(Buffering agent)
In the present invention, the buffer having a buffering ability at pH of 2.0 to 4.5 or pH of 7.0 to 12.0 includes the following.
[0024]
Examples of the substance having a buffer capacity at pH 2.0 to 4.5 include inorganic acids, organic acids or salts thereof.
As said inorganic acid, phosphoric acid etc. are mentioned, for example. Examples of the organic acid include carboxylic acid, dicarboxylic acid, carboxylic acid derivative, hydroxycarboxylic acid, amino acid, pyrophosphoric acid and the like.
[0025]
Examples of the carboxylic acid include acetic acid and propionic acid.
Examples of the dicarboxylic acid include oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, maleic acid, fumaric acid, and phthalic acid.
Examples of the carboxylic acid derivative include β, β-dimethylglutaric acid, barbituric acid, aminobutyric acid, and the like.
Examples of the hydroxycarboxylic acid include citric acid, tartaric acid, and lactic acid.
Examples of the amino acid include aspartic acid and asparagine.
[0026]
The substance having a buffer capacity at pH 2.0 to 4.5 preferably has at least one pKa (acid dissociation constant) of 1.5 to 5.2, more preferably 1.8 to 5.0. Are preferred.
[0027]
The inorganic acid or organic acid salt may be a known salt, and examples thereof include a sodium salt and a potassium salt.
[0028]
The inorganic acid, organic acid or salt thereof may be used as a mixture of two or more kinds, or a mixture of inorganic acid and organic acid may be used.
[0029]
Examples of substances having a buffer capacity at pH 7.0 to 12.0 include phosphoric acid, boric acid, carbonic acid and the like as inorganic substances, and examples of organic substances include dicarboxylic acid, carboxylic acid derivatives, hydroxycarboxylic acids, amino acids, and amines. And organic substances such as imidazoles and alcohols. In addition, ethylenediaminetetraacetic acid, pyrophosphoric acid, cacodylic acid, glycerol phosphoric acid, 2,4,6-collidine, N-ethylmorpholine, morpholine, 4-aminopyridine, ammonia, ephedrine, hydroxyproline, tris (hydroxymethyl) aminomethane And glycylglycine.
[0030]
Examples of the dicarboxylic acid include malonic acid, succinic acid, maleic acid, and the like.
Examples of the carboxylic acid derivative include β, β′-dimethylglutaric acid, 5,5-diethylbarbituric acid, γ-aminobutyric acid, ε-aminocaproic acid, and the like.
As said hydroxycarboxylic acid, a citric acid etc. are mentioned, for example.
Examples of the amino acid include aspartic acid, asparagine, glycine, α-alanine, β-alanine, histidine, serine, leucine and the like.
Examples of the amines include ethylenediamine, ethanolamine, trimethylamine, and diethanolamine. Examples of the imidazoles include imidazole, 5 (4) -hydroxyimidazole, 5 (4) -methylimidazole, and 2,5 (4) -dimethylimidazole.
Examples of the alcohols include 2-amino-2-methyl-1,3-propanediol, 2-amino-2-ethyl-1,3-propanediol, and 2-amino-2-methyl-1-propanol. Is mentioned.
[0031]
Examples of the buffer include 2- (N-morpholino) ethanesulfonic acid (MES), bis (2-hydroxyethyl) iminotris- (hydroxymethyl) methane (Bistris), N- (2-acetamido) imidodiacetic acid ( ADA), piperazine-N, N′-bis (2-ethanesulfonic acid) (PIPES), 1,3-bis (tris (hydroxymethyl) -methylamino) propane (Bistrispropane), N- (acetamido) -2- Aminoethanesulfonic acid (ACES), 3- (N-morpholine) propanesulfonic acid (MOPS), N, N′-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid (BES), N-tris ( Hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES), N-2-hydroxyethane Lupiperazine-N′-ethanesulfonic acid (HEPES), N-2-hydroxyethylpiperazine-N′-propanesulfonic acid (HEPPS), N-tris (hydroxymethyl) methylglycine (Tricine), tris (hydroxymethyl) aminoethane ( Tris), N, N′-bis (2-hydroxyethyl) glycine (Bicine), glycylglycine, N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPS), glycine, cyclohexylaminopropanesulfonic acid Substances that make up what is commonly referred to as Good's buffer, such as (CAPS), can also be used. The pKa values of these substances are shown in Tables 1 and 2 (cited literature: Takeichi Horio and Nihei Yamashita Protein and enzyme basic experiment method, Nanedo 1985).
[0032]
[Table 1]

[0033]
[Table 2]

[0034]
The substance having a buffer capacity at pH 7.0 to 12.0 is preferably one having at least one pKa (acid dissociation constant) of 5.3 to 11.5, more preferably 5.5 to 11.0. Those are preferred.
[0035]
The concentration of the buffer in the cleaning solution, and when a plurality of these buffering agents are used, the total concentration of the plurality of buffering agents is such that the pH of the cleaning solution is 2.0 to 4.5, or 7.0 to 12. Any buffering effect may be used as long as it is 0, and the concentration is preferably 1 to 1000 mM, more preferably 10 to 500 mM.
[0036]
In addition, the following substances may be added to the cleaning liquid.
[0037]
(1) Inorganic salts (such as sodium chloride, potassium chloride, sodium sulfate, potassium sulfate, and sodium phosphate) may be added. The concentration of these salts is not particularly limited, but is preferably 1 to 1500 mM.
[0038]
(2) A known acid or base may be added as a pH regulator. Examples of the acid include hydrochloric acid, phosphoric acid, nitric acid, and sulfuric acid, and examples of the base include sodium hydroxide, potassium hydroxide, lithium hydroxide, magnesium hydroxide, barium hydroxide, and calcium hydroxide. The concentration of these acids and bases is not particularly limited, but is preferably 0.001 to 500 mM.
[0039]
(3) A water-soluble organic solvent such as methanol, ethanol, acetonitrile, or acetone may be mixed. The concentration of these organic solvents is not particularly limited, but is preferably 0 to 80% (v / v), and does not precipitate chaotropic ions, inorganic acids, organic acids, salts thereof, and the like, and It is preferable to use in a range that does not adversely affect the separation pattern.
[0040]
(4) Preservatives such as sodium azide and thymol may be added.
When the buffer having a buffer capacity at pH 2.0 to 4.5 and the buffer having a buffer capacity at pH 7.0 to 12.0 are added to the same cleaning solution and used, the pH of the same cleaning solution is set to the acidic side, or There is an advantage that two types of cleaning liquids can be prepared simply by changing to the alkali side.
Further, the cleaning liquid of the present invention may be used as a column cleaning liquid after measuring one sample. In that case, the solution may be sent by a gradient elution method or a step elution method.
[0041]
Further, the column cleaning liquid in the present invention may be used as a column cleaning liquid used in a conventionally known column regeneration method.
[0042]
The column cleaning liquid in the invention may be used as at least one eluent in a specific aspect of the liquid chromatograph apparatus.
[0043]
[filler]
In the present invention, the filler may be a known one and is not particularly limited. Examples of such fillers include inorganic particles such as silica and zirconia; natural polymer particles such as cellulose, polyamino acid and chitosan; and synthetic polymer particles such as polystyrene and polyacrylate.
However, since the silica and the like are dissolved in an alkali, it is preferable to set the pH of the column cleaning solution within a range in which they are not dissolved.
[0044]
In addition, since LC separation is determined by the interaction between the surface functional group of the filler and the sample to be measured, the functional group type on the surface of the filler is selected as follows depending on the LC separation mode to be applied. A filler having the following functional group is used.
[0045]
(Filler for ion exchange chromatography)
The filler preferably contains a cation exchange group or an anion exchange group as an ion exchange group.
[0046]
The cation exchange group may be a known one and is not particularly limited. Examples thereof include cation exchange groups such as carboxyl group, sulfonic acid group, and phosphoric acid group. A plurality of these cation exchange groups may be introduced.
[0047]
The anion exchange group may be a known one and is not particularly limited. For example, anion exchange groups such as a tertiary amino group and a quaternary amino group can be mentioned. A plurality of these anion exchange groups may be introduced.
[0048]
(Filler for gel permeation chromatography)
It is preferable to use a filler containing a nonionic hydrophilic group such as a hydroxyl group or an epoxy group as the filler.
[0049]
(Filler for reverse phase chromatography)
It is preferable to use a filler containing an alkyl group such as an octyl group or an octadecyl group, or a hydrophobic group such as a phenyl group.
[0050]
The column cleaning solution can be used alone or in combination of a plurality of types of packing materials.
[0051]
The diameter of the particles is preferably 0.5 to 20 μm, more preferably 1 to 10 μm.
The particle size distribution is preferably 40% or less, more preferably 30% or less, as a coefficient of variation (CV value) (standard deviation of particle size ÷ average diameter × 100 (%)).
[0052]
[column]
The packing material is packed in a column and used for liquid chromatography measurement. The column is preferably 0.1 to 50 mm in inner diameter and 1 to 300 mm in length, more preferably 0.2 to 30 mm in inner diameter and 5 to 200 mm in length.
[0053]
The column material is not particularly limited, and a known metal such as stainless steel, glass, resin such as PEEK, or the like is used.
Further, the portion where the column filler and the column main body are in contact may be coated with an inert material, and examples of the material include PEEK, polyethylene, Teflon, titanium compound, silicon compound, silicon film and the like.
[0054]
[apparatus]
The LC device used in the present invention may be a well-known device and includes, for example, a liquid feed pump, a sample injection device (sampler), a column, a detector, and the like. Further, other attached devices (such as a column thermostat or an eluent degassing device) may be added as appropriate.
[0055]
The elution method in the measurement method of the present invention may be a known method, and step elution, gradient elution, or step-up gradient elution may be performed.
[0056]
Other measurement conditions in the above measurement method may be known conditions, and the flow rate of the eluent is preferably 0.05 to 5 ml / min, more preferably 0.2 to 3 ml / min. The detection of hemoglobin is preferably visible light of 415 nm, but is not limited to this. As the measurement sample, a solution obtained by diluting hemolyzed blood with a solution containing a substance having hemolytic activity such as a surfactant is usually used. The amount of sample injection into the liquid chromatograph varies depending on the dilution factor, but is preferably about 0.1 to 100 μl.
[0057]
[Eluent]
The eluent of the present invention is preferably composed of an appropriate eluent that is used in accordance with the component to be measured in the liquid chromatograph apparatus.
[0058]
(For analysis of hemoglobin)
Particularly in the case of hemoglobin analysis, in order to elute HbA0, that is, in order to elute the “HbA0 component” composed of HbA and the like held in the packing material stronger than HbA1c, the pH at the time of flowing into the column is equal to that of hemoglobin. It is preferable to use an eluent set so as to be on the alkali side from the electric point. In order to realize this condition, there are a method of feeding one eluent whose pH is alkaline from the isoelectric point of hemoglobin, and a method of using two or more eluents having different pH.
[0059]
When the pH of the hemoglobin changes from the acidic side to the alkaline side from the isoelectric point, the total charge changes from positive to negative, so that the HbA0 component can be eluted with the cation exchange group of the filler. .
[0060]
In addition, as described in Rikagaku Dictionary (4th edition, September 1987, edited by Iwanami Shoten, Ryogo Kubo et al.), Page 1178, the isoelectric point of hemoglobin is pH 6.8 to 7.0. Therefore, in order to elute the HbA0 component, the pH of the eluent when flowing into the column is more preferably 6.8 or more.
[0061]
In order to satisfy this condition, it is necessary that at least one of the eluents used for the measurement has a pH of 6.8 or higher. The pH of the eluent is desirably 7.0 to 12.0, more preferably 7.5 to 11.0, and even more preferably 8.0 to 9.5. When the pH of the eluent is less than 6.8, elution of the HbA0 component becomes insufficient. The pH of the eluent may be set within a range in which the filler used does not decompose.
[0062]
Examples of an eluent suitably used for elution of the HbA0 component and having a buffer capacity at a pH of 6.8 or more include inorganic acids such as phosphoric acid, boric acid and carbonic acid, or salts thereof; hydroxycarboxylic acids such as citric acid; Examples include acids, carboxylic acid derivatives such as β, β-dimethylglutaric acid, dicarboxylic acids such as maleic acid, organic acids such as cacodylic acid, and buffer solutions thereof. In addition, 2- (N-morpholino) ethanesulfonic acid (MES), N-2-hydroxyethylpiperazine-N′-ethanesulfonic acid (HEPES), bis (2-hydroxyethyl) iminotris- (hydroxymethyl) methane (Bistris) ), Tris, ADA, PIPES, Bistrispropane, ACES, MOPS, BES, TES, HEPES, HEPPS, Tricine, Bicine, glycylglycine, TAPS, CAPS, and the like, which are generally referred to as good buffer solutions, can also be used. Britton and Robinson buffer; GTA buffer can also be used. Organic substances such as imidazoles such as imidazole; amines such as ethylenediamine, methylamine, ethylamine, triethylamine, diethanolamine, and triethanolamine; amino acids such as glycine, β-alanine, aspartic acid, and asparagine;
[0063]
In addition, an inorganic acid; an organic acid; a salt of an inorganic acid or an organic acid; and a plurality of organic substances may be used as a mixture.
[0064]
(Function)
Since the column washing solution of the present invention 1 comprises chaotropic ions and is composed of a buffer having a pH of 2.0 to 4.5 and / or a pH of 7.0 to 12.0 and a buffering capacity, Because it has the effect of washing proteins and lipids adsorbed nonspecifically to the packing material, it can be easily used for column regeneration and washing, and it can be used for a wide range of columns. .
[0065]
【Example】
EXAMPLES Next, although an Example and a comparative example are given and this invention is demonstrated in detail, this invention is not limited only to these Examples.
[0066]
Example 1
Hb in human blood was measured by cation exchange chromatography.
(Preparation of filler)
1.5 g of benzoyl peroxide (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in a mixture of 400 g of tetraethylene glycol dimethacrylate (manufactured by Shin-Nakamura Chemical Co., Ltd.) and 150 g of methacrylic acid (Wako Pure Chemical Industries, Ltd.). This was dispersed in 2500 mL of a 4% by weight aqueous solution of polyvinyl alcohol (manufactured by Nippon Synthetic Chemical), heated to 75 ° C. in a nitrogen atmosphere with stirring, and polymerized for 8 hours. After polymerization, washing and drying, classification was performed to obtain particles having an average particle size of 6 μm (filler 1).
(Packing the column)
The obtained packing material 1 was packed in a column by the following method. 0.7 g of the particles were dispersed in 30 mL of 50 mM phosphate buffer (pH 5.8), sonicated for 5 minutes, and then stirred well. The whole amount was injected into a packer (made by Umeda Seiki) connected with an empty stainless steel column (inner diameter 4.6 × 35 mm). A liquid feed pump (manufactured by Sanuki Industry) is connected to the packer, and the pressure is 300 kg / cm. ² At a constant pressure.
[0067]

[0068]
(Measurement sample adjustment)
A whole blood sample obtained by collecting sodium fluoride from healthy human blood was dissolved in hemolysis diluent X (0.1 wt% Triton X-100 phosphate buffer solution (pH 7.0)), diluted 150 times, and measured. A sample was used.
(Measurement result)
A chromatogram obtained by measuring the sample under the above measurement conditions is shown in FIG. Peak 1 is HbA1a and HbA1b, Peak 2 is HbF, Peak 3 is unstable HbA1c, Peak 4 is stable HbA1c, and Peak 5 is HbA0. As can be seen from the figure, both peaks were well separated.
(Evaluation of column durability)
Each time 200 samples were measured, the column cleaning liquid of the present invention was allowed to flow for 1 minute, the eluent A (dedicated eluent 50A) was allowed to flow for 2 minutes, and 4000 samples were measured.
FIG. 2 shows the change in hemoglobin A1c value when 1000, 2000, 3000, and 4000 samples of the measurement sample were measured. Thus, in Example 1, even when 4000 specimens were measured, the variation in the HbA1c value was within 3% of the measured value compared to the initial value, and the measured value did not change and had good durability.
[0069]
(Comparative Example 1)
Column durability was evaluated in the same manner as in Example 1 except that 50 mM phosphate buffer (pH 12.0) was used as the column washing solution.
(Column durability evaluation results)
The results are shown in FIG. In Comparative Example 1, the retention time of HbA1c is shortened, the fluctuation of the HbA1c value is gradually increased, and the column durability is poor.
[0070]
(Example 2)
Lipoprotein was measured by gel permeation chromatography.
(column)
Lipoprotein dedicated column (LIPOPROPK, inner diameter 7.5 mm, length 30 cm: manufactured by Tosoh Corporation)

Using the above system, 5 μL of serum was injected, the enzyme solution was fed into the eluate from the column, reacted at 45 ° C. (Teflon tube inner diameter 0.4 mm, length 7.5 mm), and then detected at an absorbance of 550 nm.
[0071]
(Measurement sample)
Serum of healthy human blood was used.
(Evaluation of column durability)
Every time 50 samples were measured, the column cleaning solution of the present invention was flowed for 1 minute, the dedicated eluent was flowed for 2 minutes, and 1000 samples were measured. The results are shown in FIG.
The measurement sample was evaluated based on the degree of change in lipoprotein value (HDL value) when 300, 600, and 1000 samples were measured.
Thus, in Example 2, even when 1000 samples are measured, the HDL value fluctuation is within 3% of the measured value compared to the initial value, and the measured value does not change and has good durability.
[0072]
(Comparative Example 2)
Column durability was evaluated in the same manner as in Example 2 except that 50 mM phosphate buffer (pH 12.0) was used as the column washing solution.
(Column durability evaluation results)
The results are shown in FIG. In Comparative Example 2, the HDL value fluctuates gradually and the column durability is poor.
[0073]
(Example 3)
Protein albumin was measured by anion exchange chromatography.
(column)
Shodex IEC DEAD-420N, inner diameter 4.6 mm, length 35 cm: Showa Denko)

[0074]
(Measurement sample)
Egg white albumin aqueous solution (10mg / mL)
(Evaluation of column durability)
Every time 50 samples were measured, the column cleaning solution of the present invention was flowed for 1 minute, the eluent A was flowed for 2 minutes, and 1000 samples were measured. The results are shown in FIG. The measurement sample was evaluated by fluctuations in albumin values when 300, 600, and 1000 samples were measured.
Thus, in Example 3, even when 1000 specimens are measured, the variation in albumin value is within 3% of the measured value compared to the initial value, and the measured value does not change and has good durability.
[0075]
(Comparative Example 3)
Column durability was evaluated in the same manner as in Example 3 except that 50 mM phosphate buffer (pH 2.0) was used as the column washing solution.
(Column durability evaluation results)
The results are shown in FIG. In Comparative Example 3, the albumin value fluctuated gradually and the column durability was poor.
[0076]
Example 4
Ribonuclease was measured by reverse phase chromatography.
(column)
TSKgel ODS-80Ts, inner diameter 6.0 mm, length 150 mm: Showa Denko)

[0077]
(Measurement sample)
Ribonuclease aqueous solution (10 mg / mL)
(Evaluation of column durability)
Every time 50 samples were measured, the column cleaning solution of the present invention was flowed for 1 minute, the eluent A was flowed for 2 minutes, and 1000 samples were measured. The results are shown in FIG. The measurement sample was evaluated by the change in ribonuclease value when 300, 600, and 1000 samples were measured.
Thus, in Example 4, even if 1000 samples are measured, the ribonuclease value is within 3% of the measured value, and the measured value does not change and has good durability.
[0078]
(Comparative Example 4)
Column durability was evaluated in the same manner as in Example 4 except that 50 mM phosphate buffer (pH 2.0) was used as the column washing solution.
(Column durability evaluation results)
The results are shown in FIG. In Comparative Example 4, the ribonuclease value fluctuated gradually and the column durability was poor.
[0079]
(Example 5)
The liquid chromatograph was automatically cleaned and measured.
(Equipment)
A high-performance liquid chromatograph apparatus (manufactured by Kyoto Daiichi Kagaku Co., Ltd., automatic glycohemoglobin measuring apparatus HA-8131) was improved to constitute the liquid chromatograph apparatus shown in FIG. As the separation column 13, a column dedicated to the liquid chromatograph apparatus (Sekisui Chemical Co., Ltd., column unit-HSIII) was used. Here, a chromatographic apparatus improved so as to perform a column washing operation when the number of times of measurement of the sample reaches a predetermined reference number was used.
[0080]
A plurality of samples were measured according to the operation description of the liquid chromatograph apparatus shown in FIG. In this case, using the step elution method, the eluent 1 was sent for 108 seconds, the eluent 2 was sent for 12 seconds, and the eluent 1 was sent for 60 seconds to carry out liquid feeding for one measurement.
[0081]
(Eluent)
As eluents 1 and 2, eluent 1 (phosphate buffer containing 50 mM perchloric acid, pH = 5.3) manufactured by Sekisui Chemical Co., Ltd., which is an eluent dedicated to the liquid chromatograph apparatus, and eluent 2 (Phosphate buffer containing 300 mM perchloric acid, pH = 8.5) was used. As the cleaning liquid, the above eluent 1 and eluent 2 were used in the following manner.
(Cleaning solution)
That is, the eluents 1 and 2 were sent as follows instead of the cleaning liquid. That is, using the eluents 1 and 2 as the cleaning liquid, the eluent 2 was sent for 108 seconds, and then the eluent 1 was sent for 72 seconds to perform the cleaning operation.
[0082]
In preparing the blood sample, a hemolysis washing liquid (the above-mentioned hemolysis washing liquid dedicated to the liquid chromatograph apparatus) was used to hemolyze and dilute red blood cells.
The flow rates of the eluents 1 and 2 flowing through the measurement channel during measurement and washing were both 2 ml / min.
[0083]
(Measurement sample)
As a specimen, whole blood collected with a blood collection tube (trade name: Insepack-E, manufactured by Sekisui Chemical Co., Ltd.) containing EDTA-2K as an anticoagulant was used as a specimen. In the present liquid chromatograph, whole blood is collected from a blood collection tube, and this whole blood is hemolyzed by the hemolysis washing solution to automatically prepare a hemolyzed diluted sample.
[0084]
(Column durability evaluation results)
The reference number of times for the washing operation was set to 300, and the measurement was performed 1500 times. In this case, the retention time of HbA1c in the initial state of the separation column 13 was set to 100, and the retention time of HbA1c measured for each measurement number was recorded. The result is shown by a solid line A in FIG. As is clear from the solid line A, the retention time of HbA1c hardly changed even when measured 1500 times. The eluent pH at the separation column inflow portion at the time of washing was measured and found to be 8.2.
[0085]
(Comparative Example 5)
Measurement was performed in the same manner as in Example 5 except that the washing operation was not performed. The results are shown by broken lines in FIG.
As is clear from FIG. 7, it can be seen that when the cleaning operation is not performed, the retention time of HbA1c becomes faster as the number of measurements increases.
[0086]
(Example 6)
Using the same apparatus as in Example 5 except that the measurement counter was not used and the column was washed so that the HbA1c retention time deviated from the predetermined range. In the same manner as in Example 5, Eluents 1 and 2 were fed. That is, the retention time of HbA1c in the initial state of the separation column 13 was set to 100, and the retention time was set to 95 or more. Accordingly, the cleaning operation is started when the holding time becomes less than 95.
[0087]
In washing, the eluent 2 was fed for 108 seconds according to the step elution method, and then the eluent 1 was fed for 72 seconds. That is, no cleaning solution was used.
In the same manner as in Example 5, the same sample as used in Example 5 was measured 2500 times. The results are shown in FIG. As is apparent from FIG. 8, after measuring about 450 times, the holding time became faster, and when it was measured about 800 times, it fell below the reference value, so automatic cleaning was started. After the automatic cleaning, the level returned to the original level based on the holding time. Therefore, when the final measurement was performed 2500 times, automatic cleaning was performed 3 times, and it was confirmed that HbA1c could be measured with high accuracy up to 2500 measurements.
[0088]
【The invention's effect】
The column washing solution of the present invention contains chaotropic ions and has a pH of 2.0 to 4.5 and / or a buffer having a buffer capacity at a pH of 7.0 to 12.0. It has the effect of sufficiently washing proteins and lipids adsorbed non-specifically to the packing material, and can be easily used in column regeneration and washing, and can be used in a wide range of columns.
[Brief description of the drawings]
1 is a diagram showing a chromatogram obtained when hemoglobins were measured under the measurement conditions of Example 1. FIG.
FIG. 2 is a diagram showing a change in HbA1c value with an increase in the number of measurements in Example 1 and Comparative Example 1.
FIG. 3 is a diagram showing a change in HDL value with an increase in the number of measurements in Example 2 and Comparative Example 2.
FIG. 4 is a graph showing the change in albumin with an increase in the number of measurements in Example 3 and Comparative Example 3.
FIG. 5 is a graph showing fluctuations in ribonuclease with increase in the number of measurements in Example 4 and Comparative Example 4.
6 is a schematic block diagram of a liquid chromatograph apparatus according to Embodiment 5. FIG.
7 is a graph showing a change in the retention time of HbA1c with an increase in the number of measurements in Example 5 and Comparative Example 5. FIG.
FIG. 8 is a diagram showing a change in retention time of HbA1c due to an increase in the number of measurements in Example 6.
[Explanation of symbols]
1 HbA1a and b peaks
2 HbF peak
3 Peak of unstable HbA1c
4 Stable HbA1c peak
5 HbA0 peak
11 Liquid feeding means
12 Control means
13 Separation column
14 Detection means
15 Sample injection means
16 Sample detection means
17 Measurement counter

Claims (3)

After measuring the measurement sample by liquid chromatography, it is passed through the column for liquid chromatography, and the washing liquid of the column for liquid chromatography for washing the protein or lipid adsorbed nonspecifically to the packing material in the column,
A column washing solution containing a perchlorate ion and a buffer having a buffer capacity at a pH of 2.0 to 4.5 and / or a pH of 7.0 to 12.0. Liquid chromatography that measures a sample to be measured by liquid chromatography and then flows it through a column for liquid chromatography consisting of at least a cation exchange group-containing packing material to wash nonspecifically adsorbed proteins or lipids in the packing material in the column In the column column cleaning solution,
Include perchlorate ion, and including Column Wash buffers the pH with buffering capacity at 7.0 to 12.0. Liquid chromatography that measures a sample to be measured by liquid chromatography and then flows it to a column for liquid chromatography consisting of at least a filler having an anion exchange group to wash the non-specifically adsorbed protein or lipid in the column. In the column column cleaning solution,
Include perchlorate ion, and including Column Wash buffers the pH with buffering capacity at 2.0 to 4.5.

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