JP3848459B2 - Method for measuring hemoglobins - Google Patents

Description

【００６４】
測定試料
健常人血をフッ化ナトリウム採血し、以下の試料を調製した。
試料ａ（糖負荷血）：健常人血に、５００ｍｇ／ｄｌとなるようグルコース水溶液を添加し、３７℃で３時間反応させたもの。
試料ｂ（カルバミル化ヘモグロビン（ＣＨｂ）含有試料）：健常人血１０ｍｌに、０．３重量％のシアン酸ナトリウムの生理食塩水溶液１ｍｌを添加し、３７℃で１時間反応させたもの。
試料ｃ（アセチル化ヘモグロビン（ＡＨｂ）含有試料）：健常人血１０ｍｌに、０．３重量％のアセトアルデヒドの生理食塩水溶液１ｍｌを添加し、室温で３時間反応させたもの。
試料ｄ：上記試料ｂと試料ｃを等量混合したもの。
【００６５】
次いで、上記試料ａと試料ｄをそれぞれ以下のように処理して試料Ａと試料Ｄを作製し測定試料とした。
試料Ａ：上記試料ａ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倍に希釈して試料Ａとした。
試料Ｄ：上記試料ｄを、溶血希釈液Ｙ（溶血希釈液Ｘにポリリン酸ナトリウム（太平化学社製）を０．０５重量％となるよう添加したもの）で１５０倍に溶血希釈し、３５℃で２分間加温して試料Ｄとした。
【００６６】
測定結果
上記測定条件により、試料Ａを測定して得られたクロマトグラムを図１に、試料Ｄを測定して得られたクロマトグラムを図２に示す。ピーク１はＨｂＡ１ａ及び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、ピーク６はＡＨｂ、ピーク７はＣＨｂを示す。
図１では、ピーク３及び４が良好に分離されている。また、図２ではＡＨｂやＣＨｂのような修飾ヘモグロビンも良好に分離されていることがわかる。
【００６７】
（実施例２）リン酸基を有する充填剤を用いるもの
テトラエチレングリコールジメタクリレート（新中村化学社製）４００ｇ及び（２−メタクリロイルオキシエチル）アシッドホスフェート（共栄社化学社製）１００ｇの混合物に、過酸化ベンゾイル（和光純薬社製）１．５ｇを溶解した。これを４重量％ポリビニルアルコール水溶液２５００ｍｌに分散した。撹拌しながら窒素雰囲気下で８０℃に昇温し、８０℃で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（以下、充填剤２という）を得た。
参考例１と同様にして、充填剤２をカラムに充填し、ヘモグロビン類の測定を行った。
溶離液Ａとして１００ｍＭリン酸緩衝液（ｐＨ５．８）、溶離液Ｂとして３００ｍＭリン酸緩衝液（ｐＨ７．０）を用い参考例１に準じて測定を行ったところ、得られたクロマトグラムは図１及び図２と同様、良好であった。
【００６８】
（実施例３）スルホン酸基を有する充填剤を用いるもの
特公平８−７１９７号公報に記載の方法により、充填剤を調製した。
テトラエチレングリコールジメタクリレート３００ｇ、グリセロールジメタクリレート（新中村化学社製）１００ｇに過酸化ベンゾイル１．５ｇを溶解し、４重量％ポリビニルアルコール水溶液２５００ｍｌに分散した。撹拌しながら窒素雰囲気下で昇温し、８０℃で１時間重合した。１時間後、２−アクリルアミド−２−メチルプロパンスルホン酸１００ｇを添加し、さらに６５℃で３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（以下、充填剤３という）を得た。イオン交換容量を測定したところ、乾燥粒子１ｇ当たり１１．３μｅｑであった。参考例１と同様にして充填剤３をカラムに充填し、ヘモグロビン類の測定を行った。溶離液ＡとしてＭＥＳを１００ｍＭ濃度で、食塩を１００ｍＭ濃度で含む水溶液（ｐＨ５．７）、溶離液Ｂとして３００ｍＭリン酸緩衝液（ｐＨ７．０）を用い参考例１に準じて測定を行ったところ、得られたクロマトグラムは図１及び図２と同様、良好であった。
【００６９】
（比較例１）
参考例１と同様にして充填剤１をカラムに充填し、溶離液Ａとして１００ｍＭリン酸緩衝液（ｐＨ６．０）及び溶離液Ｂとして３００ｍＭリン酸緩衝液（ｐＨ７．０）を用いて、参考例１に準じて（溶離液Ａ及びＢの通流時間は、実用的な測定時間の範囲内で、できるだけ良好な分離ができるように変えた）、ヘモグロビン類の測定を行った。得られたクロマトグラムを図３（試料Ａ）及び図４（試料Ｄ）に示す。
図１及び図２に比較して、測定時間が長いにもかかわらず、分離能が悪いことが分かる。
【００７０】
（比較例２）
参考例１と同様にして充填剤２をカラムに充填し、溶離液ＡとしてＭＥＳを１００ｍＭ濃度で、食塩を１００ｍＭ濃度で含む水溶液（ｐＨ５．９）、溶離液ＢとしてＭＥＳを１００ｍＭ濃度で、食塩を５００ｍＭ濃度で含む水溶液（ｐＨ５．９）を用いて、参考例１に準じて（溶離液Ａ及びＢの通流時間は、実用的な測定時間の範囲内で、できるだけ良好な分離ができるように変えた）、ヘモグロビン類の測定を行ったところ、得られたクロマトグラムは図３及び図４と同様であった。
【００７１】
（比較例３）
参考例１と同様にして充填剤３をカラムに充填し、溶離液Ａとして２０ｍＭクエン酸ナトリウム（和光純薬社製）水溶液（ｐＨ６．０）及び溶離液Ｂとして３００ｍＭリン酸緩衝液（ｐＨ７．０）を用いて、参考例１に準じて（溶離液Ａ及びＢの通流時間は、実用的な測定時間の範囲内で、できるだけ良好な分離ができるように変えた）、ヘモグロビン類の測定を行った。得られたクロマトグラムは、図３及び図４と同様であった。
【００７２】
（実施例４）
テトラエチレングリコールジメタクリレート（新中村化学社製）４００ｇ及び２−アクリルアミド−２−メチルプロパンスルホン酸（東京化成社製）１００ｇの混合物に過酸化ベンゾイル（和光純薬社製）１．５ｇを溶解した。これを４重量％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８０℃に昇温し、８０℃で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（以下、充填剤４という）の粒子を得た。
【００７３】
参考例１と同様にして充填剤４をカラムに充填し、溶離液ＡとしてＭＥＳを１０ｍＭ濃度で、食塩を１００ｍＭ濃度で含む水溶液（ｐＨ５．７）、溶離液Ｂとして３００ｍＭリン酸緩衝液（ｐＨ７．２）を用いて、参考例１に準じて（但し、流速は１．５ｍｌ／分とした）ヘモグロビン類の測定を行った。試料Ａを用いて得られたクロマトグラムを図５に、試料Ｄを用いて得られたクロマトグラムを図６に示した。図５では、ピーク３及び４が良好に分離されている。また、図６ではＡＨｂやＣＨｂのような修飾ヘモグロビンも良好に分離されていることがわかる。
【００７４】
（実施例５）
特公平８−７１９７号公報に記載の方法により、充填剤を調製した。
トリエチレングリコールジメタクリレート（新中村化学社製）４００ｇに過酸化ベンゾイル１．５ｇを溶解し、４重量％ポリビニルアルコール水溶液２５００ｍｌに分散した。撹拌しながら窒素雰囲気下で昇温し、８０℃で１時間重合した。１時間後メタクリル酸１５０ｇを添加し、さらに１時間８０℃で重合した。重合後、洗浄し乾燥した後、分級して平均粒径６μｍの粒子（以下、充填剤５という）を得た。
参考例１と同様にして充填剤５をカラムに充填し、溶離液Ａとして２０ｍＭ濃度でＢｉｓ−Ｔｒｉｓ（和光純薬社製）を含む１１０ｍＭ食塩水溶液（ｐＨ６．０）及び溶離液Ｂとして３００ｍＭリン酸緩衝液（ｐＨ７．０）を用いて、実施例４に準じてヘモグロビン類の測定を行った。得られたクロマトグラムは、図５及び図６と同様であった。
【００７５】
（比較例４）
実施例４と同様にして充填剤４をカラムに充填し、溶離液Ａとして１００ｍＭリン酸緩衝液（ｐＨ５．５）及び溶離液Ｂとして３００ｍＭリン酸緩衝液（ｐＨ７．０）を用いて、実施例４に準じて（溶離液Ａ及びＢの通流時間は、実用的な測定時間の範囲内で、できるだけ良好な分離ができるように変えた）、ヘモグロビン類の測定を行った。得られたクロマトグラムを図７（試料Ａ）及び図８（試料Ｄ）に示す。
図５及び図６に比較して、測定時間が長いにもかかわらず、分離能が悪いことが分かる。
【００７６】
（比較例５）
実施例５と同様にして充填剤５をカラムに充填し、溶離液Ａとして１００ｍＭリン酸緩衝液（ｐＨ５．９）及び溶離液Ｂとして３００ｍＭリン酸緩衝液（ｐＨ７．０）を用いて、実施例４に準じて（溶離液Ａ及びＢの通流時間は、実用的な測定時間の範囲内で、できるだけ良好な分離ができるように変えた）、ヘモグロビン類の測定を行った。得られたクロマトグラムは図７及び図８と同様であった。
【００７７】
（参考例６）
実施例４と同様にして充填剤４をカラムに充填し、溶離液Ａとして１０ｍＭの濃度でコハク酸を含む１００ｍＭ食塩水溶液（ｐＨ５．３）、溶離液Ｂとして１０ｍＭの濃度でコハク酸を含む２００ｍＭ食塩水溶液（ｐＨ５．３）、溶離液Ｃとして１０ｍＭの濃度でコハク酸を含む４００ｍＭ食塩水溶液（ｐＨ５．８）を用いて、測定開始より０〜０．３分の間は溶離液Ａを流し、０．３〜１．０分の間は溶離液Ｂを流し、１．０〜１．２分の間は溶離液Ｃを流し、１．２〜２．０分の間は溶離液Ａを再び流したことの他は、実施例４に準じて（但し、試料注入量は１０μｌ）ヘモグロビン類の測定を行った。試料Ａを用いて得られたクロマトグラムを図９に、試料Ｄを用いて得られたクロマトグラムを図１０に示した。図９では、ピーク３及び４が良好に分離されている。また、図１０ではＡＨｂやＣＨｂのような修飾ヘモグロビンも良好に分離されていることがわかる。
【００７８】
（比較例６）
参考例６と同様にして充填剤４をカラムに充填し、溶離液Ａとして１００ｍＭリン酸緩衝液（ｐＨ５．３）、溶離液Ｂとして１２０ｍＭリン酸緩衝液（ｐＨ５．３）及び溶離液Ｃとして４００ｍＭリン酸緩衝液（ｐＨ５．９）を用いて、参考例６に準じて（溶離液Ａ、Ｂ及びＣの通流時間は、実用的な測定時間の範囲内で、できるだけ良好な分離ができるように変えた）、ヘモグロビン類の測定を行った。試料Ａを用いて得られたクロマトグラムを図１１に、試料Ｄを用いて得られたクロマトグラムを図１２に示した。図９及び図１０に比較して、測定時間が長いにもかかわらず、分離能が悪いことが分かる。
【００７９】
【発明の効果】
本発明１の測定方法によれば、従来のヘモグロビン類の測定法における問題点であった測定時間の延長や、分離性能が解決され、短時間内に、かつ高精度にヘモグロビン類を分離できる。
【００８０】
本発明２の測定方法によれば、従来短時間では分離が困難であった不安定型ＨｂＡ１ｃを安定型ＨｂＡ１ｃから分離できるため、従来より短時間で、精度良く、ヘモグロビン類を測定できる。また、ＡＨｂ、ＣＨｂのような修飾ヘモグロビンも安定型ＨｂＡ１ｃより分離でき、またＡＨｂとＣＨｂも分離できるため、それぞれを定量することが可能となった。
【図面の簡単な説明】
【図１】 参考例１の測定条件により、試料Ａの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】 参考例１の測定条件により、試料Ｄの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３】比較例１の測定条件により、試料Ａの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４】比較例１の測定条件により、試料Ｄの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５】実施例４の測定条件により、試料Ａの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６】実施例４の測定条件により、試料Ｄの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７】比較例４の測定条件により、試料Ａの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８】比較例４の測定条件により、試料Ｄの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９】 参考例６の測定条件により、試料Ａの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１０】 参考例６の測定条件により、試料Ｄの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１１】比較例６の測定条件により、試料Ａの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１２】比較例６の測定条件により、試料Ｄの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。
【符号の説明】
１ ＨｂＡ１ａ及びＨｂＡ１ｂ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
６ ＡＨｂのピーク
７ ＣＨｂのピーク[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for measuring hemoglobin by liquid chromatography.
[0002]
[Prior art]
Glycated hemoglobin, particularly glycated hemoglobin A1c (hereinafter referred to as HbA1c) is widely measured as an index for diagnosis of diabetes. Glycated hemoglobin is produced by combining sugar in blood with erythrocytes after entering the erythrocytes in proportion to its concentration. The glycated hemoglobin in the hemolyzed blood sample reflects the average sugar concentration in the blood over the past 1 to 2 months, so the glycated hemoglobin in the hemolyzed blood is regarded as an index for the diagnosis of diabetes, The HbA1c value (ratio of HbA1c to the total of glycated hemoglobin and non-glycated hemoglobin (%)) using glycated hemoglobin A1c (hereinafter referred to as HbA1c) is widely used as an optimal index for diagnosis of diabetes.
[0003]
As a method for measuring HbA1c, a liquid chromatography method or an immunization method is generally used. The liquid chromatography method is most widely used because of its high accuracy.
[0004]
The measurement of HbA1c by a liquid chromatography method is mainly performed by a cation exchange liquid chromatography method (Japanese Patent Publication No. 8-7198). When the hemolyzed blood 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; HbA0 is non-glycated hemoglobin and the like) appear. HbA1c used as an index for diagnosis of diabetes is stable HbA1c, and is determined as a ratio (%) of the area of stable HbA1c peak to the total hemoglobin peak area.
[0005]
However, since it is difficult to separate the stable HbA1c peak from the unstable HbA1c peak, it is usually difficult to accurately measure only the stable HbA1c peak. Therefore, various methods for eliminating the influence of the unstable HbA1c peak are considered and implemented as follows, but each method still has drawbacks. That is, for example, there is a method of removing unstable HbA1c by adding a reagent described in JP-A-63-298063, but this method has a problem that the pretreatment operation for removal becomes complicated. . In addition, for example, by using a filler as described in JP-B-8-7197, a method of separating the peaks on the chromatogram taking a relatively long time can be considered, but the measurement time becomes long. Is a problem.
[0006]
In this liquid chromatography method, as described above, a cation exchange resin is generally used as the filler, and a carboxyl group and a sulfonic acid group are used as the ion exchange group. As the eluent, a phosphate buffer is mostly used.
[0007]
In this liquid chromatography method, the measurement time has been shortened, and measurement can be performed within a few minutes per sample, but the separation of each peak is still insufficient.
[0008]
Further, it is said that the measurement by the liquid chromatography method is affected by modified hemoglobin such as acetylated hemoglobin (hereinafter referred to as AHb) and carbamylated hemoglobin (hereinafter referred to as CHb). Furthermore, since the peaks of AHb and CHb elute near the stable HbA1c peak, it is difficult to separate in a short time.
[0009]
[Problems to be solved by the invention]
An object of the present invention is to provide a method for measuring hemoglobin with a short time and with high accuracy, without the above-mentioned disadvantages of the conventional method.
[0010]
Invention of Claim 1 and Invention of Claim 2(Hereinafter referred to as “the present invention 1”) is a method for measuring hemoglobin by liquid chromatography, wherein a buffer solution containing the same ion species as the ion exchange group of the filler is used as an eluent. This is a measurement method.
[0011]
Claim 3In the invention (hereinafter referred to as the present invention 2), the filler isA filler having a carboxyl groupThe eluent isHas a carboxyl groupIt is a good (Good) buffer solution.RuheThis is a method for measuring moglobins.
[0013]
Hereinafter, the details of the present invention 1 will be described.
The filler used in the present invention 1 is a filler composed of particles having at least one ion exchange group. The ion exchange group-containing filler is produced by introducing ion exchange groups into polymer particles, as will be described in detail later. The ion exchange group is, for example, a cationic functional group such as a carboxyl group, a sulfonic acid group, or a phosphoric acid group, and an anionic functional group such as a tertiary amine or a quaternary ammonium group. A plurality of functional groups may be mixed. The term “mixing” includes not only the case where one particle has a plurality of functional groups, but also the case where a plurality of particles having one functional group are used in combination. The amount of these ion exchange groups introduced is not particularly limited, but is preferably 0.1 μeq to 100 meq per 1 g dry weight of the filler. The diameter of the filler particles is preferably 0.5 to 20 μm, more preferably 1 to 10 μm. The particle size distribution of the filler is preferably 20% or less, more preferably 15% or less, as a coefficient of variation (CV value) (standard deviation of particle size ÷ average particle size × 100).
[0014]
The method for producing the filler used in the present invention 1 is not particularly limited. For example, (a) a method of introducing an ion exchange group into polymer particles, and (b) a monomer having an ion exchange group is polymerized. (C) Polymerizable ion exchange group-containing ester (methyl (meth) acrylate, ethyl (meth) acrylate, etc.) is mixed with a crosslinkable monomer, and a polymerization initiator is present. Examples thereof include a method in which the obtained particles are subjected to hydrolysis treatment to convert esters to ion exchange groups after polymerization under the following conditions.
[0015]
The above (a) method for introducing ion exchange groups into the polymer particles will be described below.
Examples of the polymer particles 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.
[0016]
As the polymer particles, the constituent components other than the ion exchange group to be introduced are preferably more hydrophilic, and those having a high degree of crosslinking are preferred from the viewpoint of pressure resistance and swelling resistance.
[0017]
When the polymer particles are silica particles, the silica particles can be obtained, for example, by adding an alkoxylane derivative to an alcohol / water mixed solvent and polymerizing in the presence of a catalyst. Furthermore, an appropriate functional group can also be introduce | transduced by making a functional group containing silane coupling agent react as needed.
[0018]
When the polymer particles are cellulose particles, the cellulose particles can be obtained, for example, by dissolving cellulose triacetate in an organic solvent, adding an aqueous gelatin solution to remove the organic solvent, and then hydrolyzing under alkaline conditions. it can.
[0019]
When the polymer particles are polyamino acid particles, the polyamino acid particles can be obtained, for example, by polymerizing N-carboxylic acid anhydride.
[0020]
When the polymer particles are synthetic polymer particles, the synthetic polymer particles include, for example, styrene, dibilbenzene, divinyltoluene; alkyl (meth) acrylates such as methyl (meth) acrylate and ethyl (meth) acrylate; polyethylene glycol It can be obtained by polymerizing monomers such as di (meth) acrylate and polypropylene glycol di (meth) acrylate in the presence of a polymerization initiator.
[0021]
The method of introducing an ion exchange group into the polymer particles may be a case where the polymer particles have a reactive functional group (for example, a hydroxyl group, an amino group, a carboxyl group, an epoxy group, etc., hereinafter referred to as a reactive group). For example, a compound having both a functional group having reactivity with the reactive group and an ion exchange group may be chemically bonded to the polymer particle. In order to prepare the polymer particles having the reactive group, for example, a hydroxyl group-containing monomer (2-hydroxyethyl (meth) acrylate, 2-hydroxymethyl (meth) acrylate, hydroxypropyl (meth) acrylate, (poly ) Ethylene glycol mono (meth) acrylate, glycerol mono (meth) acrylate, etc.]; amino group-containing monomers ((meth) acrylamide, 2-aminoethyl (meth) acrylate, 2-aminomethyl (meth) acrylate, aminopropyl) (Meth) acrylate, dimethylaminoethyl (meth) acrylate, diethylaminoethyl (meth) acrylate, allylamine, etc.); carboxyl group-containing monomer ((meth) acrylic acid, itaconic acid, etc.); or epoxy group-containing monomer ( Glycidyl (meth) acryl The chromatography etc. g) was mixed with such a crosslinkable monomer may be polymerized in the presence of a polymerization initiator.
[0022]
In the case of polymer particles that do not have the reactive group, a reactive group may be introduced into the polymer particles using various known chemical reactions.
[0023]
The ion exchange group introduced into the polymer particles may be a known one and is not particularly limited. Examples include cation exchange groups such as carboxyl groups, sulfonic acid groups, and phosphoric acid groups; and anion exchange groups such as tertiary amino groups and quaternary ammonium groups.
[0024]
The method (b) for polymerizing the monomer having an ion exchange group to obtain polymer particles will be described below.
Examples of the monomer having an ion exchange group include (meth) acrylic acid, itaconic acid, fumaric acid, maleic acid, and the like if the ion exchange group is a carboxyl group. For example, styrene sulfonic acid, 3-sulfopropyl (meth) acrylic acid, 3-sulfopropyl (meth) acrylate, 3-sulfopropyl itacolate, N- (3-sulfopropyl) -N- (meth) Examples include acryloyloxyethyl-N, N-dimethylammonium betaine, 2-acrylamido-2-methylpropanesulfonic acid, and allylsulfonic acid. In the case of a phosphoric acid group, for example, ((meth) acryloyloxymethyl) Acid phosphate, (2- (meth) acryloyloxyethyl) acid phosphate, ( -(Meth) acryloyloxypropyl) acid phosphate, (3- (meth) acryloyloxypropyl) acid phosphate, and the like. For example, in the case of a tertiary amino group or a quaternary ammonium group, dimethylaminoethyl (Meth) acrylate, diethylaminoethyl (meth) acrylate, (meth) acrylate hydroxypropyltrimethylammonium chloride, (meth) acrylate dimethylaminoethyltrimethylammonium chloride, etc. are mentioned.
[0025]
Examples of the polymerization method include a method in which the monomer and a crosslinkable monomer are mixed and polymerized in the presence of a polymerization initiator.
[0026]
In carrying out the measurement method of the present invention 1, the packing material is packed in a column and used for liquid chromatography measurement. The column is not particularly limited, such as a known stainless steel, glass or resin. The column size is preferably 0.5 to 10 mm in inner diameter and 5 to 300 mm in length. Any known method can be used for packing the packing material into the column, but a slurry packing method is more preferable. Specifically, for example, it is performed by press-fitting a slurry in which filler particles are dispersed in a buffer solution such as an eluent into a column using a liquid feed pump or the like.
[0027]
The liquid chromatograph used for the measurement may be a known one, and includes, for example, a liquid feed pump, a sample injection device (sampler), a column, a detector, and the like. In addition, other attachment devices (such as a column thermostat or an eluent degassing device) may be attached as appropriate.
[0028]
The eluent used in the measurement method of the present invention 1 is composed of a buffer solution containing the same ion species (hereinafter referred to as the same ion) as the ion exchange group of the filler used. For example, when a filler having a carboxyl group is used as the filler, a compound having a carboxyl group [specifically, for example, amino acids such as glycine; acids such as acetic acid, citric acid and phthalic acid; N- (acetamide] A buffer solution containing Good's buffer solution such as 2-iminodiacetic acid (hereinafter referred to as ADA), N-tris (hydroxymethyl) methylglycine (hereinafter referred to as TRICINE), and the like; When a filler having a sulfonic acid group is used, a compound having a sulfonic acid group [specifically, for example, N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (hereinafter referred to as HEPES), 2- ( N-morpholino) ethanesulfonic acid (hereinafter referred to as MES), 3- (N-morpholino) propanesulfonic acid (hereinafter referred to as MOPS) N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (hereinafter referred to as TES), N, N′-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid (hereinafter referred to as BES) In the case where a filler having a phosphate group is used as the filler, a compound having a phosphate group [specifically, for example, sodium phosphate, potassium phosphate, lithium phosphate, phosphorus A buffer solution containing magnesium acid or the like] is used.
[0029]
The concentration of the eluent varies depending on the filler, the sample to be measured, and other measurement conditions, but is preferably 1 to 1000 mM, more preferably 5 to 800 mM. Further, a plurality of the same kind of ion buffer solutions may be mixed and used.
[0030]
In the measurement method of the present invention 1, the above-mentioned homogeneous ion buffer is always contained in the eluent when glycated hemoglobin is eluted during the analysis of hemoglobins. However, it is not necessary to be composed of only the same type of ions, and different types of ions (ion exchange groups of the filler and different types of ions) may be mixed. In this case as well, it is necessary that the same type of ions be contained in an amount of preferably 1 to 1000 mM, more preferably 5 to 800 mM.
[0031]
Further, when a filler having two or more different ion exchange groups is used as the filler, the same kind of ions with either one of the ion exchange group species may be used as the eluent. For example, when a filler having both a carboxyl group and a sulfonic acid group is used, a buffer solution having a carboxyl group, a buffer solution having a sulfonic acid group, or a mixed buffer of both may be used.
[0032]
In addition, the following compounds may be added to the same kind of ion buffer.
(1) Inorganic salts (sodium chloride, potassium chloride, sodium sulfate, etc.) may be added. The concentration of these salts is not particularly limited, but is preferably 1 to 1500 mM.
(2) Inorganic acids such as hydrochloric acid and organic acids such as acetic acid may be added. Although the density | concentration of these inorganic acids and organic acids is not specifically limited, Preferably it is 0.001-100 mM.
(3) A base such as sodium hydroxide or potassium hydroxide may be added. The concentration of these bases is not particularly limited, but is preferably 0.001 to 100 mM.
(4) You may mix with water-soluble organic solvents, such as methanol, ethanol, and acetonitrile. The concentration of these organic solvents is not particularly limited, but is preferably 0 to 80% (V / V).
[0033]
Further, the eluent may be switched during the measurement, or gradient elution may be performed. In this case, as described above, at the time of elution of glycated hemoglobins, it is necessary to perform separation with an eluent containing the above-mentioned homogeneous ion buffer. It is not necessary to include a buffer component. The above-mentioned "at the time of elution of glycated hemoglobin" refers to the time of elution of so-called first fraction including HbA1a, HbA1b, HbF, unstable HbA1c, and stable HbA1c in this order. However, in this case, the case where other substances are mixed as a peak is included.
[0034]
In the measurement method of the present invention 1, other measurement conditions may be known conditions, and the flow rate of the eluent is preferably 0.05 to 5 ml / min, more preferably 0.1 to 3 ml / min. The detection of hemoglobins is preferably visible light at 415 nm. 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 used. The amount of sample injection into the liquid chromatograph varies depending on the dilution factor, but is preferably about 1 to 50 μl.
[0035]
Hereinafter, the details of the present invention 2 will be described.
The filler used in the present invention 2 is the same as the filler used in the present invention 1 except that it corresponds to a cation exchange resin among the fillers used in the present invention 1, and its production method This is also the same as the description of the first invention.
[0036]
When the measurement method of the present invention 2 is carried out, the packing material is packed in a column and used for liquid chromatography measurement, and the liquid chromatograph used for the measurement is the same as in the present invention 1.
[0037]
The eluent used in the measurement method of the present invention 2 is selected from those generally referred to as Good's buffer. Specific examples include bis (2-hydroxyethyl) iminotris- (hydroxymethyl) methane (hereinafter referred to as Bis-Tris), HEPES, MES, MOPS, TES, and the like.
[0038]
The concentration of the eluent varies depending on other measurement conditions, but is preferably 1 to 1000 mM, more preferably 5 to 800 mM. A plurality of the above buffer solutions may be mixed and used. Other buffers (inorganic buffers including phosphates, borates, etc., organic acid buffers including citric acid, succinic acid, etc.); salts (sodium chloride, potassium chloride, sodium sulfate, etc.); An inorganic acid such as hydrochloric acid; an organic acid such as acetic acid; a base such as sodium hydroxide or potassium hydroxide; and a water-soluble organic solvent such as methanol, ethanol, or acetonitrile may be used as a mixture.
[0039]
Further, the eluent may be switched during the measurement, or gradient elution may be performed. At this time, elution of glycated hemoglobins needs to be performed with an eluent containing the above-mentioned Good's buffer, but it is not always necessary to contain Good's buffer components when eluting other fractions (for example, HbA0 fraction). Absent.
[0040]
In the measurement method of the present invention 2, other measurement conditions may be known conditions, and the flow rate of the eluent is preferably 0.05 to 5 ml / min, more preferably 0.1 to 3 ml / min. The detection of hemoglobins is preferably visible light at 415 nm. 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 used. The amount of sample injection into the liquid chromatograph varies depending on the dilution factor, but is preferably about 1 to 50 μl.
[0057]
(Function)
In the measurement method of the present invention 1, since a buffer solution containing the same ion species as the ion exchange group of the filler is used as an eluent, the measurement time can be extended or separated, which has been a problem in the conventional methods for measuring hemoglobins. The performance is solved, and hemoglobins can be separated with high accuracy within a short time.
[0058]
The measurement method of the present invention 2 is a method for measuring hemoglobins of the present invention 1 characterized in that the filler is a cation exchange resin and the eluent is a Good's buffer solution. In addition, high-accuracy measurement of stable HbA1c in a short time and separation and quantification of AHb and CHb became possible.
[0060]
【Example】
Hereinafter, the present invention will be further clarified by giving non-limiting examples of the present invention.
[0061]
(Reference example1) Using a filler having a carboxyl group
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 (manufactured by Wako Pure Chemical Industries, Ltd.). This was dispersed in 2500 ml of a 4% by weight 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 diameter of 6 μm (hereinafter referred to as “filler 1”).
[0062]
Column packing
The obtained packing material 1 was packed in the column as follows. 0.7 g of the particles were dispersed in 30 ml of 50 mM phosphate buffer (pH 6.0), sonicated for 5 minutes, and then stirred well. The whole amount was injected into a packer (Umeya Seiki Co., Ltd.) connected with a stainless steel empty column (4.6φ × 35 mm). A liquid feed pump (manufactured by Sanuki Industry Co., Ltd.) is connected to the packer, and the pressure is 300 kg / cm.² At a constant pressure.
[0063]
Measurement of hemoglobins
Using the obtained column, hemoglobins were measured under the following measurement conditions. Measurement condition

[0064]
Measurement sample
Healthy human blood was collected from sodium fluoride and the following samples were prepared.
Sample a (sugar-loaded blood): A blood obtained by adding an aqueous glucose solution to a healthy human blood to 500 mg / dl and reacting at 37 ° C. for 3 hours.
Sample b (sample containing carbamylated hemoglobin (CHb)): 1 ml of a physiological saline solution of 0.3% by weight of sodium cyanate added to 10 ml of healthy human blood and reacted at 37 ° C. for 1 hour.
Sample c (acetylated hemoglobin (AHb) -containing sample): A sample obtained by adding 1 ml of a 0.3% by weight physiological saline solution of acetaldehyde to 10 ml of healthy human blood and reacting at room temperature for 3 hours.
Sample d: A mixture of the above sample b and sample c in equal amounts.
[0065]
Next, the sample a and the sample d were respectively processed as follows to prepare a sample A and a sample D, and used as measurement samples.
Sample A: The sample a was hemolyzed with a hemolysis diluent X (0.1 wt% Triton X-100 phosphate buffer solution (pH 7.0)) and diluted 150 times to obtain a sample A.
Sample D: Sample d was hemolyzed and diluted 150 times with hemolysis diluent Y (hemolysis dilution X with sodium polyphosphate (Tahei Kagaku Co., Ltd.) added to 0.05 wt%) at 35 ° C. The sample D was heated for 2 minutes.
[0066]
Measurement result
FIG. 1 shows a chromatogram obtained by measuring the sample A under the above measurement conditions, and FIG. 2 shows a chromatogram obtained by measuring the sample D. Peak 1 is HbA1a and HbA1b, Peak 2 is HbF, Peak 3 is unstable HbA1c, Peak 4 is stable HbA1c, Peak 5 is HbA0, Peak 6 is AHb, and Peak 7 is CHb.
In FIG. 1, peaks 3 and 4 are well separated. FIG. 2 also shows that modified hemoglobins such as AHb and CHb are well separated.
[0067]
(Example 2) Using a filler having a phosphate group
  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 100 g of (2-methacryloyloxyethyl) acid phosphate (manufactured by Kyoeisha Chemical Co., Ltd.). . This was dispersed in 2500 ml of a 4% by weight aqueous polyvinyl alcohol solution. While stirring, the temperature was raised to 80 ° C. in a nitrogen atmosphere, and polymerization was carried out at 80 ° C. for 8 hours. After polymerization, washing and drying, classification was performed to obtain particles having an average particle diameter of 6 μm (hereinafter referred to as “filler 2”).
  Reference exampleIn the same manner as in Example 1, packing material 2 was packed in a column, and hemoglobins were measured.
  100 mM phosphate buffer (pH 5.8) is used as eluent A, and 300 mM phosphate buffer (pH 7.0) is used as eluent B.Reference exampleWhen the measurement was performed according to No. 1, the obtained chromatogram was good as in FIG. 1 and FIG.
[0068]
(Example 3) Using a filler having a sulfonic acid group
  A filler was prepared by the method described in JP-B-8-7197.
  1.5 g of benzoyl peroxide was dissolved in 300 g of tetraethylene glycol dimethacrylate and 100 g of glycerol dimethacrylate (manufactured by Shin-Nakamura Chemical Co., Ltd.) and dispersed in 2500 ml of a 4% by weight polyvinyl alcohol aqueous solution. While stirring, the temperature was raised in a nitrogen atmosphere, and polymerization was performed at 80 ° C. for 1 hour. After 1 hour, 100 g of 2-acrylamido-2-methylpropanesulfonic acid was added, and the mixture was further polymerized at 65 ° C. for 3 hours. After polymerization, washing and drying, classification was performed to obtain particles having an average particle size of 6 μm (hereinafter referred to as filler 3). The ion exchange capacity was measured and found to be 11.3 μeq / g dry particle.Reference exampleIn the same manner as in 1, the packing material 3 was packed in a column, and hemoglobins were measured. As an eluent A, an aqueous solution (pH 5.7) containing MES at a concentration of 100 mM and sodium chloride at a concentration of 100 mM is used, and as an eluent B, a 300 mM phosphate buffer (pH 7.0) is used.Reference exampleWhen the measurement was performed according to No. 1, the obtained chromatogram was good as in FIG. 1 and FIG.
[0069]
(Comparative Example 1)
  Reference exampleIn the same manner as in Example 1, packing 1 is packed in a column, and 100 mM phosphate buffer (pH 6.0) is used as eluent A and 300 mM phosphate buffer (pH 7.0) is used as eluent B.Reference exampleThe hemoglobins were measured according to No. 1 (the flow times of the eluents A and B were changed so that separation was as good as possible within the practical measurement time range). The obtained chromatogram is shown in FIG. 3 (Sample A) and FIG. 4 (Sample D).
  Compared to FIG. 1 and FIG. 2, it can be seen that the resolution is poor despite the long measurement time.
[0070]
(Comparative Example 2)
  Reference exampleIn the same manner as in Example 1, packing 2 is packed into a column, an aqueous solution (pH 5.9) containing MES at a concentration of 100 mM and sodium chloride at a concentration of 100 mM as eluent A, and MES at a concentration of 100 mM as eluent B and sodium chloride at 500 mM. Using an aqueous solution containing a concentration (pH 5.9),Reference exampleAccording to 1 (the eluents A and B flow times were changed so that separation was as good as possible within the practical measurement time range), hemoglobins were obtained. The chromatogram was the same as in FIGS.
[0071]
(Comparative Example 3)
  Reference exampleIn the same manner as in No. 1, the packing material 3 is packed in a column, and 20 mM sodium citrate (Wako Pure Chemical Industries, Ltd.) aqueous solution (pH 6.0) as eluent A and 300 mM phosphate buffer (pH 7.0) as eluent B Using,Reference exampleThe hemoglobins were measured according to No. 1 (the flow times of the eluents A and B were changed so that separation was as good as possible within the practical measurement time range). The obtained chromatogram was the same as in FIGS.
[0072]
(Example 4)
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 100 g of 2-acrylamido-2-methylpropanesulfonic acid (manufactured by Tokyo Chemical Industry Co., Ltd.). . This was dispersed in 2500 ml of a 4% by weight polyvinyl alcohol (manufactured by Nippon Synthetic Chemical), heated to 80 ° C. in a nitrogen atmosphere with stirring, and polymerized at 80 ° C. for 8 hours. After polymerization, washing and drying, classification was performed to obtain particles having an average particle diameter of 6 μm (hereinafter referred to as filler 4).
[0073]
  Reference exampleIn the same manner as in Example 1, packing 4 is packed in a column, an aqueous solution (pH 5.7) containing MES at a concentration of 10 mM and sodium chloride at a concentration of 100 mM as eluent A, and 300 mM phosphate buffer (pH 7.2) as eluent B. )Using,Reference exampleThe hemoglobins were measured according to 1 (however, the flow rate was 1.5 ml / min). A chromatogram obtained using the sample A is shown in FIG. 5, and a chromatogram obtained using the sample D is shown in FIG. In FIG. 5, peaks 3 and 4 are well separated. FIG. 6 also shows that modified hemoglobins such as AHb and CHb are well separated.
[0074]
(Example 5)
  A filler was prepared by the method described in JP-B-8-7197.
  1.5 g of benzoyl peroxide was dissolved in 400 g of triethylene glycol dimethacrylate (manufactured by Shin-Nakamura Chemical Co., Ltd.) and dispersed in 2500 ml of a 4% by weight aqueous polyvinyl alcohol solution. While stirring, the temperature was raised in a nitrogen atmosphere, and polymerization was performed at 80 ° C. for 1 hour. After 1 hour, 150 g of methacrylic acid was added, and the mixture was further polymerized at 80 ° C. for 1 hour. After polymerization, washing and drying, classification was performed to obtain particles having an average particle size of 6 μm (hereinafter referred to as filler 5).
  Reference exampleIn the same manner as in Example 1, packing 5 is packed in a column, 110 mM saline solution (pH 6.0) containing Bis-Tris (manufactured by Wako Pure Chemical Industries, Ltd.) at a concentration of 20 mM as eluent A, and 300 mM phosphate buffer as eluent B Hemoglobins were measured according to Example 4 using the solution (pH 7.0). The obtained chromatogram was the same as in FIGS.
[0075]
(Comparative Example 4)
In the same manner as in Example 4, the column was packed with the packing material 4 and 100 mM phosphate buffer (pH 5.5) was used as eluent A and 300 mM phosphate buffer (pH 7.0) was used as eluent B. The hemoglobins were measured according to Example 4 (the flow times of the eluents A and B were changed so that separation was as good as possible within the practical measurement time range). The obtained chromatogram is shown in FIGS. 7 (sample A) and 8 (sample D).
Compared to FIG. 5 and FIG. 6, it can be seen that the separation performance is poor despite the long measurement time.
[0076]
(Comparative Example 5)
In the same manner as in Example 5, the column was packed with the packing material 5, and using 100 mM phosphate buffer (pH 5.9) as eluent A and 300 mM phosphate buffer (pH 7.0) as eluent B In accordance with Example 4 (the flow times of eluents A and B were changed so that separation was as good as possible within the practical measurement time range), hemoglobins were measured. The obtained chromatogram was the same as in FIGS.
[0077]
(Reference example6)
  In the same manner as in Example 4, the column was packed with packing material 4, and 100 mM saline solution (pH 5.3) containing succinic acid at a concentration of 10 mM as eluent A and 200 mM containing succinic acid at a concentration of 10 mM as eluent B Saline solution (pH 5.3), 400 mM saline solution (pH 5.8) containing succinic acid at a concentration of 10 mM as eluent C, and eluent A was allowed to flow for 0 to 0.3 minutes from the start of measurement. Eluent B is allowed to flow for 0.3 to 1.0 minutes, eluent C is allowed to flow for 1.0 to 1.2 minutes, and eluent A is again allowed to flow for 1.2 to 2.0 minutes. Except for flowing, hemoglobins were measured according to Example 4 (however, the sample injection volume was 10 μl). A chromatogram obtained using the sample A is shown in FIG. 9, and a chromatogram obtained using the sample D is shown in FIG. In FIG. 9, peaks 3 and 4 are well separated. FIG. 10 also shows that modified hemoglobins such as AHb and CHb are well separated.
[0078]
(Comparative Example 6)
  Reference exampleIn the same manner as in No. 6, the column is packed with the packing material 4, and 100 mM phosphate buffer (pH 5.3) is used as eluent A, 120 mM phosphate buffer (pH 5.3) is used as eluent B, and 400 mM phosphorus is used as eluent C. Using acid buffer (pH 5.9),Reference exampleThe hemoglobins were measured according to No. 6 (the flow times of the eluents A, B, and C were changed so that separation was as good as possible within the practical measurement time range). A chromatogram obtained using Sample A is shown in FIG. 11, and a chromatogram obtained using Sample D is shown in FIG. Compared to FIG. 9 and FIG. 10, it can be seen that the separation performance is poor despite the long measurement time.
[0079]
【The invention's effect】
According to the measurement method of the present invention 1, the extension of the measurement time and the separation performance, which have been problems in the conventional hemoglobin measurement method, are solved, and hemoglobins can be separated with high accuracy within a short time.
[0080]
According to the measurement method of the present invention 2, unstable HbA1c, which has conventionally been difficult to separate in a short time, can be separated from stable HbA1c, so that hemoglobins can be measured in a shorter time and with higher accuracy. In addition, modified hemoglobins such as AHb and CHb can be separated from stable HbA1c, and AHb and CHb can also be separated, so that each can be quantified.
[Brief description of the drawings]
[Figure 1]Reference exampleThe figure which shows the chromatogram obtained when the hemoglobin of sample A was measured on the measurement conditions of 1. FIG.
[Figure 2]Reference exampleThe figure which shows the chromatogram obtained when the hemoglobin of the sample D was measured on the measurement conditions of 1. FIG.
3 is a diagram showing a chromatogram obtained when the hemoglobins of sample A were measured under the measurement conditions of Comparative Example 1. FIG.
4 is a diagram showing a chromatogram obtained when the hemoglobins of sample D were measured under the measurement conditions of Comparative Example 1. FIG.
5 is a diagram showing a chromatogram obtained when measurement of hemoglobin of sample A under the measurement conditions of Example 4. FIG.
6 is a diagram showing a chromatogram obtained when hemoglobins of sample D are measured under the measurement conditions of Example 4. FIG.
7 is a diagram showing a chromatogram obtained when measuring hemoglobin of sample A under the measurement conditions of Comparative Example 4. FIG.
8 is a diagram showing a chromatogram obtained when measuring hemoglobin of sample D under the measurement conditions of Comparative Example 4. FIG.
FIG. 9Reference exampleFIG. 6 is a diagram showing a chromatogram obtained when the hemoglobins of sample A are measured under the measurement conditions of 6.
FIG. 10Reference exampleThe figure which shows the chromatogram obtained when the hemoglobin of the sample D was measured on the measurement conditions of 6. FIG.
11 is a diagram showing a chromatogram obtained when measurement of hemoglobin of sample A under the measurement conditions of Comparative Example 6. FIG.
12 is a diagram showing a chromatogram obtained when measuring hemoglobin of Sample D under the measurement conditions of Comparative Example 6. FIG.
[Explanation of symbols]
1 HbA1a and HbA1b peaks
2 HbF peak
3 Peak of unstable HbA1c
4 Stable HbA1c peak
5 HbA0 peak
6 AHb peak
7 CHb peak

Claims (3)

  A method for measuring hemoglobin by liquid chromatography, comprising using a filler having a sulfonic acid group and using a buffer containing a compound having a sulfonic acid group as an eluent .   A method for measuring hemoglobin by liquid chromatography, comprising using a filler having a phosphate group and using a buffer containing a compound having a phosphate group as an eluent. .   A method for measuring hemoglobin by liquid chromatography, comprising using a filler having a carboxyl group and using Good's buffer having a carboxyl group as an eluent .

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