JPH0225147B2 - - Google Patents
Info
- Publication number
- JPH0225147B2 JPH0225147B2 JP57105644A JP10564482A JPH0225147B2 JP H0225147 B2 JPH0225147 B2 JP H0225147B2 JP 57105644 A JP57105644 A JP 57105644A JP 10564482 A JP10564482 A JP 10564482A JP H0225147 B2 JPH0225147 B2 JP H0225147B2
- Authority
- JP
- Japan
- Prior art keywords
- protein
- column
- analysis
- substance
- adsorbed
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
- 102000004169 proteins and genes Human genes 0.000 claims description 75
- 108090000623 proteins and genes Proteins 0.000 claims description 75
- 239000000126 substance Substances 0.000 claims description 34
- 238000004458 analytical method Methods 0.000 claims description 22
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 21
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 18
- 239000003480 eluent Substances 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 15
- 239000000463 material Substances 0.000 claims description 12
- 238000012856 packing Methods 0.000 claims description 12
- 239000003960 organic solvent Substances 0.000 claims description 11
- 239000013076 target substance Substances 0.000 claims description 7
- 239000010419 fine particle Substances 0.000 claims description 5
- 239000012488 sample solution Substances 0.000 claims description 3
- 229920001477 hydrophilic polymer Polymers 0.000 claims description 2
- 235000018102 proteins Nutrition 0.000 description 64
- 239000000523 sample Substances 0.000 description 23
- 210000002966 serum Anatomy 0.000 description 18
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 9
- 239000007788 liquid Substances 0.000 description 6
- 239000000945 filler Substances 0.000 description 5
- 239000002245 particle Substances 0.000 description 5
- 229920000642 polymer Polymers 0.000 description 5
- HVVWZTWDBSEWIH-UHFFFAOYSA-N [2-(hydroxymethyl)-3-prop-2-enoyloxy-2-(prop-2-enoyloxymethyl)propyl] prop-2-enoate Chemical compound C=CC(=O)OCC(CO)(COC(=O)C=C)COC(=O)C=C HVVWZTWDBSEWIH-UHFFFAOYSA-N 0.000 description 4
- 239000000178 monomer Substances 0.000 description 4
- 230000001919 adrenal effect Effects 0.000 description 3
- 229940088597 hormone Drugs 0.000 description 3
- 239000005556 hormone Substances 0.000 description 3
- LTHJXDSHSVNJKG-UHFFFAOYSA-N 2-[2-[2-[2-(2-methylprop-2-enoyloxy)ethoxy]ethoxy]ethoxy]ethyl 2-methylprop-2-enoate Chemical compound CC(=C)C(=O)OCCOCCOCCOCCOC(=O)C(C)=C LTHJXDSHSVNJKG-UHFFFAOYSA-N 0.000 description 2
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- PQSUYGKTWSAVDQ-ZVIOFETBSA-N Aldosterone Chemical compound C([C@@]1([C@@H](C(=O)CO)CC[C@H]1[C@@H]1CC2)C=O)[C@H](O)[C@@H]1[C@]1(C)C2=CC(=O)CC1 PQSUYGKTWSAVDQ-ZVIOFETBSA-N 0.000 description 2
- PQSUYGKTWSAVDQ-UHFFFAOYSA-N Aldosterone Natural products C1CC2C3CCC(C(=O)CO)C3(C=O)CC(O)C2C2(C)C1=CC(=O)CC2 PQSUYGKTWSAVDQ-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- CERQOIWHTDAKMF-UHFFFAOYSA-N Methacrylic acid Chemical compound CC(=C)C(O)=O CERQOIWHTDAKMF-UHFFFAOYSA-N 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 229960002478 aldosterone Drugs 0.000 description 2
- 239000012491 analyte Substances 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 239000012472 biological sample Substances 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 235000004252 protein component Nutrition 0.000 description 2
- 229910001220 stainless steel Inorganic materials 0.000 description 2
- 239000010935 stainless steel Substances 0.000 description 2
- 238000010557 suspension polymerization reaction Methods 0.000 description 2
- -1 ultisol Chemical compound 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 1
- 239000004342 Benzoyl peroxide Substances 0.000 description 1
- OMPJBNCRMGITSC-UHFFFAOYSA-N Benzoylperoxide Chemical compound C=1C=CC=CC=1C(=O)OOC(=O)C1=CC=CC=C1 OMPJBNCRMGITSC-UHFFFAOYSA-N 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 239000004372 Polyvinyl alcohol Substances 0.000 description 1
- YWYQTGBBEZQBGO-BERLURQNSA-N Pregnanediol Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](O)C)[C@@]2(C)CC1 YWYQTGBBEZQBGO-BERLURQNSA-N 0.000 description 1
- YWYQTGBBEZQBGO-UHFFFAOYSA-N UC1011 Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(O)C)C1(C)CC2 YWYQTGBBEZQBGO-UHFFFAOYSA-N 0.000 description 1
- 239000000935 antidepressant agent Substances 0.000 description 1
- 239000007900 aqueous suspension Substances 0.000 description 1
- 235000019400 benzoyl peroxide Nutrition 0.000 description 1
- FFGPTBGBLSHEPO-UHFFFAOYSA-N carbamazepine Chemical compound C1=CC2=CC=CC=C2N(C(=O)N)C2=CC=CC=C21 FFGPTBGBLSHEPO-UHFFFAOYSA-N 0.000 description 1
- 229960000623 carbamazepine Drugs 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000005345 coagulation Methods 0.000 description 1
- 230000015271 coagulation Effects 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 238000007334 copolymerization reaction Methods 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 229940011871 estrogen Drugs 0.000 description 1
- 239000000262 estrogen Substances 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- STVZJERGLQHEKB-UHFFFAOYSA-N ethylene glycol dimethacrylate Substances CC(=C)C(=O)OCCOC(=O)C(C)=C STVZJERGLQHEKB-UHFFFAOYSA-N 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- DDBREPKUVSBGFI-UHFFFAOYSA-N phenobarbital Chemical compound C=1C=CC=CC=1C1(CC)C(=O)NC(=O)NC1=O DDBREPKUVSBGFI-UHFFFAOYSA-N 0.000 description 1
- 229960002695 phenobarbital Drugs 0.000 description 1
- 239000002861 polymer material Substances 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 229920002451 polyvinyl alcohol Polymers 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- OIGNJSKKLXVSLS-VWUMJDOOSA-N prednisolone Chemical compound O=C1C=C[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 OIGNJSKKLXVSLS-VWUMJDOOSA-N 0.000 description 1
- 229960005205 prednisolone Drugs 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000003270 steroid hormone Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/16—Injection
- G01N30/20—Injection using a sampling valve
Description
【発明の詳細な説明】
本発明は蛋白質を含んでいる血清等の試料中の
非蛋白物質を液体クロマトグラフ等分析カラムを
用いる手法により分析する分析方法に関する。血
清等に含まれるアルドステロン、アルドステロ
ン、ユルチゾール、プレグナンジオール、エスト
ロゲン等のステロイド系ホルモンなどや、各種薬
剤等の非蛋白質成分を、病理学的な研究や診断に
利用するために液体クロマトグラフ等により分析
することが広く行われているが、血清等の生体試
料中には一般に蛋白質が含まれている。この蛋白
質の存在により、分析精度や分析カラム機能が低
下したり、該分析カラムの寿命が短かくなつたり
するので、非蛋白質成分の分析においては、予め
例えばエチルアルコールのような有機溶媒を使用
して蛋白質を沈殿除去する除蛋白操作により蛋白
質を除いた試料を用いるのが普通である。しかし
ながら、この方法では除蛋白操作に多大の時間、
労力を要する、該操作の段階で分析成分が多量に
損失したり、若しくは変性したりする等の欠点が
あつた。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an analytical method for analyzing non-protein substances in a sample such as serum containing proteins by a method using an analytical column such as a liquid chromatograph. Analyze steroid hormones such as aldosterone, aldosterone, ultisol, pregnanediol, and estrogen contained in serum, etc., and non-protein components of various drugs using liquid chromatography for use in pathological research and diagnosis. However, biological samples such as serum generally contain proteins. The presence of this protein reduces analytical accuracy and analytical column functionality, and shortens the life of the analytical column. Therefore, when analyzing non-protein components, it is recommended to use an organic solvent such as ethyl alcohol in advance. It is common to use a sample from which proteins have been removed by a protein removal procedure in which proteins are precipitated and removed. However, this method requires a lot of time and
This method has disadvantages such as being labor-intensive and causing a large amount of analytical components to be lost or denatured during the operation.
又、試料のより簡便な処理方法として、例えば
特開昭53−15197号公報に記載されているように、
前処理カラムを使用する方法も知られている。こ
れは、環境試料や生体試料中の微量成分の分析に
際し、分析目的成分以外の多量成分を吸着せず、
分析目的の微量成分を吸着するような充填剤を収
容した前処理カラムに試料を流し、分析目的成分
のみを前処理カラムに吸着させた後、次に溶離液
を流して脱着せしめ、それを分離カラムに送り分
析目的成分を分離定量する方法である。このよう
な方法においては、前処理カラムの充填剤として
一般にポーラスポリマー型のものが使用されてい
る。本発明者らは、ポーラスポリマー型の充填剤
として良く知られているスチレン−ジビニルベン
ゼン共重合ポリマーを使用して、血清試料の除蛋
白をすべく種々条件を検討したが、この場合は非
蛋白物質のみならず蛋白質も吸着してしまい、除
蛋白目的を達成できないことが分つた。 In addition, as a simpler method for processing samples, for example, as described in JP-A-53-15197,
Methods using pretreatment columns are also known. When analyzing trace components in environmental samples or biological samples, this technology does not adsorb large amounts of components other than those targeted for analysis.
The sample is passed through a pretreatment column containing a packing material that adsorbs trace components of interest for analysis, and only the components of interest for analysis are adsorbed onto the pretreatment column.Then, an eluent is passed through to desorb and separate them. This method separates and quantifies the target component by sending it to a column. In such a method, a porous polymer type is generally used as the packing material for the pretreatment column. The present inventors investigated various conditions for deproteinizing serum samples using a styrene-divinylbenzene copolymer, which is well known as a porous polymer type filler. It was found that not only substances but also proteins were adsorbed, making it impossible to achieve the purpose of protein removal.
又、シリカODSタイプの充填剤についても、
原理的に使用可能と考えられ検討してみたが、こ
の場合も非蛋白物質のみならず蛋白質も吸着し、
カラムが短時間のうちに詰まり目的を達成できな
いことが分つた。又、蛋白質を吸着しないものと
して、アガロースやデキストランのような多糖類
を主成分とする架橋された充填剤についても検討
してみたが、この場合は、蛋白質のみならず、分
析目的の非蛋白物質も吸着せず、目的を達成でき
ないことが分つた。 Also, regarding silica ODS type fillers,
We considered it possible to use it in principle, but in this case as well, it adsorbs not only non-protein substances but also proteins.
It was found that the column became clogged within a short period of time and could not achieve its purpose. We also investigated cross-linked packing materials mainly composed of polysaccharides such as agarose and dextran, which do not adsorb proteins. However, it was found that the target could not be achieved as the adsorption was not achieved.
このように、従来の充填剤では、血清等蛋白質
を含む試料中の蛋白質を吸着せず、非蛋白物質の
みを吸着するようなものがなく、除蛋白には、効
率の悪い有機溶媒沈殿法などを使用せざるを得な
かつた。 In this way, conventional packing materials do not adsorb proteins in samples containing proteins such as serum, but only non-protein substances, and methods such as organic solvent precipitation, which is inefficient, are required for protein removal. I had no choice but to use .
本発明は上記の如き従来法の欠点にかんがみ、
簡単な操作でかつ有効に除蛋白が行える分析方法
を提供することを目的としてなされたものであ
り、その要旨は、非蛋白物質を分析カラムを通過
させることにより分析を実質的に有機溶剤を含ま
ない水の存在下では非蛋白分析目的物質は吸着す
るが蛋白質は吸着せず、そして吸着した非蛋白分
析目的物質を、有機溶剤としてメタノール又はア
セトニトリルを含有する水の存在下では溶離する
性質を有する、親水性重合体物質の微粒子からな
る充填剤が充填された除蛋白カラムに、分析目的
物質及び蛋白質を含む試料液を通して、該充填剤
に分析目的物質を含む非蛋白物質を吸着させると
共に、吸着しなかつた蛋白質を系外に除去したの
ち、該除蛋白カラムに有機溶剤としてメタノール
又はアセトニトリルを含有する水からなる溶離液
を流して、吸着されていた上記非蛋白物質を脱着
させ、これを分析カラムに直接チヤージすること
を特徴とする非蛋白物質の分析方法に存する。 The present invention takes into consideration the drawbacks of the conventional method as described above, and
It was developed with the aim of providing an analytical method that can effectively remove proteins with simple operations, and its gist is that by passing non-protein substances through an analytical column, the analysis can be carried out using substances that do not substantially contain organic solvents. In the presence of free water, non-protein analyte substances are adsorbed, but proteins are not adsorbed, and in the presence of water containing methanol or acetonitrile as an organic solvent, the adsorbed non-protein analyte substances are eluted. A sample solution containing the substance of interest and protein is passed through a protein removal column filled with a packing material made of fine particles of a hydrophilic polymer substance, and the non-protein substance containing the substance of interest is adsorbed onto the packing material. After removing the unresolved proteins from the system, an eluent consisting of water containing methanol or acetonitrile as an organic solvent is passed through the protein removal column to desorb the adsorbed non-protein substances, and this is analyzed. A method for analyzing non-protein substances characterized by direct charging to a column.
以下、図面を参照しながら本発明につき説明す
る。 Hereinafter, the present invention will be explained with reference to the drawings.
第１図は本発明の一実施態様を示す接続図であ
り、接続端Ａ〜Ｆを有する６方バルブ４に、除蛋
白カラム５が接続端Ｂ，Ｅを介して接続され、水
槽１中のイオン交換水若しくは緩衝液がポンプ２
によつて試料インジエクター３，接続端Ａ，Ｂを
経由して除蛋白カラム５に送られ、そこから接続
端Ｅ，Ｆを通り系外に排出するようになされてい
る。 FIG. 1 is a connection diagram showing one embodiment of the present invention, in which a protein removal column 5 is connected to a six-way valve 4 having connection ends A to F via connection ends B and E, and a Ion exchange water or buffer solution is pumped 2
The sample is sent to the protein removal column 5 via the sample injector 3 and connection ends A and B, and from there, it is discharged from the system through connection ends E and F.
一方、溶離液槽６中の溶難液はポンプ７によ
り、接続端Ｄ，Ｃを経由して液体クロマトグラフ
の分析カラム８へ送られる様になされている。上
記除蛋白カラム５には特定の条件、すなわち実質
的に有機溶剤を含まない水の存在下では非蛋白質
である分析目的物質は吸着するが、蛋白質は吸着
せず、そして吸着した非蛋白分析目的物質を、有
機溶剤としてメタノール又はアセトニトリルを含
有する水の存在下では溶離する性質を有する充填
剤が充填されている。 On the other hand, the refractory solution in the eluent tank 6 is sent by a pump 7 to an analysis column 8 of a liquid chromatograph via connection ends D and C. Under certain conditions, that is, in the presence of water substantially free of organic solvents, the protein removal column 5 adsorbs non-protein analysis target substances, but does not adsorb proteins; It is filled with a filler which has the property of eluting substances in the presence of water containing methanol or acetonitrile as organic solvents.
本発明においては、分析目的物質に応じて上記
充填剤が選定されるのであるが、一般に充填剤と
して上記性質を有する高分子物質の多孔質微粒子
状のものを採用するのが好ましい。又、該充填剤
の分析目的物質に対する吸着性に関しては、該物
質を分析するための分析カラムに充填される充填
剤よりも吸着性が低いことが好ましく、この様な
組合せの除蛋白カラム及び分析カラムを用いるこ
とによつて、除蛋白カラムに吸着されていた目的
物質を溶離液によつて完全に溶離させ、これをそ
のまゝ分析カラムに流して該分析カラムで精度よ
く分離、分析することが出来る。しかして、除蛋
白カラム５に充填される充填剤の好適な例として
は、例えば血清試料中の非蛋白物質分析用として
テトラメチロールメタントリアクリレートやｎ−
エチレングリコールジメタクリレート（ｎは２〜
４）の単独重合あるいはこれらのモノマーを含む
モノマー混合物の共重合によつて得られる水との
親和性を有する重合体物質の粒状物が挙げられ、
なかんずく、懸濁重合によつて得られる粒径５〜
20μ程度の多孔質微細粒子が好ましい。なお、多
孔質の重合体粒子は、水懸濁重合において、重合
性モノマーに希釈剤例えばトルエン等を適量混合
して重合に供し、得られた重合体粒子から微粒子
中に残存する上記トルエン等を洗浄等により除去
することにより得ることが出来る。 In the present invention, the above-mentioned filler is selected depending on the substance to be analyzed, and it is generally preferable to employ porous fine particles of a polymeric substance having the above-mentioned properties as the filler. In addition, it is preferable that the adsorptivity of the packing material for the substance to be analyzed is lower than that of the packing material packed in the analytical column for analyzing the substance, and such a combination of protein removal column and analysis By using a column, the target substance adsorbed on the protein removal column is completely eluted with an eluent, which is then passed directly to the analytical column for accurate separation and analysis. I can do it. Therefore, suitable examples of the packing material packed in the protein removal column 5 include tetramethylolmethane triacrylate and n-
Ethylene glycol dimethacrylate (n is 2-
Examples include granules of polymeric substances having an affinity for water obtained by homopolymerization of 4) or copolymerization of monomer mixtures containing these monomers,
Above all, the particle size obtained by suspension polymerization is 5~
Porous fine particles of about 20μ are preferred. In addition, porous polymer particles are produced by mixing an appropriate amount of a diluent such as toluene with a polymerizable monomer in water suspension polymerization, and subjecting the resulting polymer particles to the toluene remaining in the fine particles. It can be obtained by removing it by washing or the like.
しかして、本発明方法では第１図に示される上
述の接続状態において、試料インジエクター３よ
り、試料例えば除蛋白処理を行つていない血清試
料を注入すると、該血清試料はポンプ２によりイ
オン交換水若しくは緩衝液と共に移送されて除蛋
白カラム５に達し、ここで血清試料中の非蛋白分
析目的物質は該カラムの充填剤に吸着され、一
方、該試料中の蛋白質は吸着されないで該カラム
５を通過し、６方バルブ４の接続端Ｅ，Ｆを経由
して系外に排出される。 According to the method of the present invention, when a sample, for example, a serum sample that has not been subjected to protein removal treatment, is injected from the sample injector 3 in the above-mentioned connection state shown in FIG. Alternatively, it is transferred together with the buffer solution and reaches the protein removal column 5, where the non-protein analysis target substance in the serum sample is adsorbed to the packing material of the column, while the protein in the sample is not adsorbed and passes through the column 5. It passes through and is discharged to the outside of the system via the connection ends E and F of the six-way valve 4.
ここで６方バルブ４を切換えて第２図の如くに
接続すると、ポンプ７により溶離液槽６中の有機
溶剤としてメタノール又はアセトニトリルを含有
する水からなる溶離液が接続端Ｄ，Ｅを経由して
除蛋白カラム５を通過し、かくして上記において
吸着された非蛋白物質が溶離されて溶離液と共に
移送され、接続端Ｂ，Ｃを経由して分析カラム８
に達し、この分析カラム８の作用によつて分析目
的物質が分離され、分析カラム８以降に設けられ
た適宜な検出器により検出され、分析が行われる
のである。 When the six-way valve 4 is switched and connected as shown in Fig. 2, the eluent consisting of water containing methanol or acetonitrile as an organic solvent in the eluent tank 6 is pumped by the pump 7 via the connecting ends D and E. The non-protein substances adsorbed above are eluted and transferred together with the eluent, and then passed through the analytical column 8 via connecting ends B and C.
The substance to be analyzed is separated by the action of the analytical column 8, detected by an appropriate detector provided after the analytical column 8, and analyzed.
なお、本実施例では除蛋白カラム５におけるポ
ンプ２によるイオン交換水の進行方向と、ポンプ
７による溶離液の進行方向とはそれぞれ反対方向
になる様になされたが、これに限られる必要はな
く、これらの進行方向が同じ方向となる様な接続
方式が採用されてもよい。又、第２図の接続に切
換えたのち、分析目的物質が６方バルブ４の接続
端Ｃを通過して分析カラム８に達した頃を見計ら
つて、６方バルブ４を第１図に示される接続にさ
らに切換えることも可能であり、この様に切換え
ることにより、分析カラム８で分析を行いなが
ら、次の試料を除蛋白カラム５に通し次の分析の
ための除蛋白処理を平行して行うことが出来るの
で能率的である。 In this example, the direction of movement of the ion-exchanged water by the pump 2 in the protein removal column 5 and the direction of movement of the eluent by the pump 7 were set to be opposite to each other, but there is no need to be limited to this. , a connection method may be adopted in which these traveling directions are the same. After switching to the connection shown in FIG. 2, the six-way valve 4 is connected as shown in FIG. It is also possible to further switch to a connection that is similar to that shown in Figure 1. By switching in this way, while performing analysis in analytical column 8, the next sample can be passed through protein removal column 5 and protein removal processing for the next analysis can be performed in parallel. It is efficient because it can be done easily.
本発明の非蛋白物質の分析方法は上述の通りの
方法であり、分析目的物質を損失したり、変質さ
せたりすることなく簡単な操作で試料中の蛋白質
を除去することが出来るので、血清等蛋白質を含
む試料の非蛋白物質を分析するために採用されて
有効なるものである。 The method for analyzing non-protein substances of the present invention is as described above, and since proteins in a sample can be removed with a simple operation without loss or deterioration of the substance to be analyzed, serum etc. It is effective when employed to analyze non-protein substances in samples containing proteins.
以下本発明につき実施例により説明する。 The present invention will be explained below with reference to Examples.
実施例 １
高圧６方バルブ４に、除蛋白カラム５，液体ク
ロマトグラフ（ウオーターズ、モデル6000A）に
組み込まれた分析カラム８等を第１図に示す通り
に接続した。Example 1 A protein removal column 5, an analytical column 8 incorporated in a liquid chromatograph (Waters, model 6000A), etc. were connected to a high-pressure six-way valve 4 as shown in FIG.
なお、除蛋白カラム５としては、内径４mm，長
さ1.5cmのステンレス製カラムに、４重量％ポリ
ビニルアルコール水溶液400ml中にテトラエチレ
ングリコールジメタクリレート40ｇ，テトラメチ
ロールメタントリアクリレート10ｇ、メタクリル
酸50ｇ、トルエン40ｇ及びベンゾイルパーオキサ
イド1.5ｇを供給し、400rPmで撹拌しながら80℃
で10時間反応させ、反応後熱水及びアセトンで洗
浄して得た粒子径５〜20μの高分子球状多孔体を
充填したものを用いた。 The protein removal column 5 was a stainless steel column with an inner diameter of 4 mm and a length of 1.5 cm, containing 40 g of tetraethylene glycol dimethacrylate, 10 g of tetramethylolmethane triacrylate, 50 g of methacrylic acid, and toluene in 400 ml of a 4% by weight polyvinyl alcohol aqueous solution. 40g and 1.5g of benzoyl peroxide were supplied and heated to 80℃ while stirring at 400rPm.
A spherical porous polymer material having a particle diameter of 5 to 20 μm obtained by reacting for 10 hours and washing with hot water and acetone after the reaction was used.
ポンプ２により水槽１中のイオン交換水を1.0
c.c.／分の流速で６方バルブ４の接続端Ａ，Ｂを経
由して除蛋白カラム５に流しながら、試料インジ
エクター３から除蛋白処理をしていない試料血清
10μを注入した。 Pump 2 pumps ion-exchanged water in tank 1 to 1.0
Sample serum that has not been subjected to protein removal treatment is supplied from the sample injector 3 while flowing through the connection ends A and B of the six-way valve 4 to the protein removal column 5 at a flow rate of cc/min.
10μ was injected.
該試料血清は正常男子血清に副腎質ホルモン、
プレドニゾロンが10PPm添加されたものである。 The sample serum contains normal male serum, adrenal hormone,
Contains 10PPm of prednisolone.
上記注入の５分後に、６方バルブ４を切換えて
第２図の如く接続し、ポンプ７により、溶離液槽
６中のメタノール対水が70：30の溶離液を0.5
c.c.／分の流速で、６方バルブ接続端Ｄ，Ｅ，除蛋
白カラム５，６方バルブ接続端Ｂ，Ｃを経由して
液体クロマトグラフの分析カラム８へと通し、除
蛋白カラム５において吸着されていたが上記溶離
液の流通により溶離した非蛋白物質の分析を行つ
た。 Five minutes after the above injection, the six-way valve 4 is switched and connected as shown in Figure 2, and the pump 7 pumps 0.5 of the eluent containing 70:30 methanol to water in the eluent tank 6.
At a flow rate of cc/min, it passes through the six-way valve connections D and E, the protein removal column 5, and the six-way valve connections B and C to the analytical column 8 of the liquid chromatograph, and is adsorbed in the protein removal column 5. However, the non-protein substances eluted by the flow of the above-mentioned eluent were analyzed.
なお、分析カラムとしてはSHANDON社製
HYPERSIL−ODS（商品名、カラムサイズ内径
６mm；長さ10cm）を用い、検出は240nmの吸収強
度によつて行つた。得られたクロマトグラムは第
３図Ａに示される通りであり、副腎質ホルモンを
添加しない正常男子血清を試料として用いる以外
は上記と同様にして分析操作を行つて得られた第
１図Ｂに示されるクロマトグラムとの対比により
明らかなる様に、第１図Ａのクロマトグラムにお
いては血清中の副腎質ホルモンがピークとして
明確に検出されている。 The analytical column is manufactured by SHANDON.
Detection was performed using HYPERSIL-ODS (trade name, column size, inner diameter 6 mm; length 10 cm) based on absorption intensity at 240 nm. The obtained chromatogram is as shown in Figure 3A, and the chromatogram in Figure 1B was obtained by performing the analysis procedure in the same manner as above, except that normal male serum to which no adrenal hormone was added was used as the sample. As is clear from the comparison with the chromatogram shown, the adrenal hormone in the serum is clearly detected as a peak in the chromatogram shown in FIG. 1A.
実施例 ２
実施例１で用いたのと同じステンレス製カラム
に、実施例１で使用した混合モノマー（テトラエ
チレングリコールジメタクリレート、テトラメチ
ロールメタントリアクリレート及びメタクリル
酸）に代えてテトラメチロールメタントリアクリ
レート100ｇを用いる以外は実施例１と同様に重
合を行つて用意した粒子径５〜20μの高分子球状
多孔体を充填して除蛋白カラムを用意した。この
除蛋白カラムを用いる以外は実施例１と同様な分
析装置を用いた。ポンプ２により水槽１中の
0.01N Na2CO3水溶液を1.0c.c.／分の流速で、第
１図の如く６方バルブ４の接続端Ａ，Ｂを経由し
て除蛋白カラム５に流しながら、試料インジエク
ター３から除蛋白処理をしていない試料血清10μ
を注入した。Example 2 Into the same stainless steel column as used in Example 1, 100 g of tetramethylolmethane triacrylate was added instead of the mixed monomers (tetraethylene glycol dimethacrylate, tetramethylolmethane triacrylate, and methacrylic acid) used in Example 1. A protein removal column was prepared by filling a polymer spherical porous body with a particle size of 5 to 20 μm prepared by polymerization in the same manner as in Example 1, except that the column was used. The same analyzer as in Example 1 was used except for using this protein removal column. The water in tank 1 is pumped by pump 2.
Protein removal treatment is carried out from the sample injector 3 while flowing a 0.01N Na 2 CO 3 aqueous solution at a flow rate of 1.0 cc/min to the protein removal column 5 via the connecting ends A and B of the 6-way valve 4 as shown in Figure 1. Sample serum 10 μ without
was injected.
該試料血清は抗テンカン剤服用患者の血清であ
る。 The sample serum is from a patient taking an anti-depressant drug.
試料注入の５分後に、６方バルブ４を切換えて
第２図の如く接続し、ポンプ７により溶離液槽６
中のアセトニトリル対水が35：65の溶離液を1.0
c.c.／分の流速で、６方バルブ接続端Ｄ，Ｅ，除蛋
白カラム５，６方バルブ接続端Ｂ，Ｃを経由して
液体クロマトグラフの分析カラム８へと通し、除
蛋白カラム５に吸着されていた非蛋白物質を溶離
させて分析カラム８を通過させて該物質の検出を
行つた。なお、分析カラムとしては実施例１で用
いたのと同じカラムを用い、検出は220nmの吸収
強度によつて行つた。得られたクロマトグラムは
第４図Ａに示される通りである。一方、第４図Ｂ
は上記患者血清を用いる代りに正常人の血清を試
料として用いる以外は上記と同様にして分析操作
を行つて得られたクロマトグラムであり、第４図
Ａのクロマトグラムには、同図Ｂでは検出されて
いないピーク１２，１３，１４が検出されている
ことが認められる。 Five minutes after sample injection, the six-way valve 4 is switched and connected as shown in Figure 2, and the eluent tank 6 is pumped by the pump 7.
Eluent of 35:65 acetonitrile to water in 1.0
At a flow rate of cc/min, it passes through the six-way valve connection ends D and E, the protein removal column 5, and the six-way valve connection ends B and C to the analytical column 8 of the liquid chromatograph, and is adsorbed on the protein removal column 5. The non-protein substances that had been present were eluted and passed through an analytical column 8 to detect the substances. The analytical column used was the same as that used in Example 1, and detection was performed based on absorption intensity at 220 nm. The obtained chromatogram is as shown in FIG. 4A. On the other hand, Fig. 4B
is a chromatogram obtained by performing the same analytical procedure as above except that normal human serum was used as the sample instead of the patient serum described above. It is recognized that undetected peaks 12, 13, and 14 are detected.
そして、該ピーク１２，１３，及び１４はそれ
ぞれ抗テンカン剤中に含まれるフエノバルビター
ル、カルバマゼピン及びフエニイトンであること
が同定の結果判明した。 As a result of identification, it was found that peaks 12, 13, and 14 were phenobarbital, carbamazepine, and pheniitone, respectively, which are contained in anti-temperature drugs.
比較例 １
実施例１において用いた除蛋白カラムを用いず
して、実施例１と同じ血清試料を同じ液体クロマ
トグラムの分析カラム８に通したが、該カラム内
で血清蛋白の凝固による沈積が生じ、カラム内圧
の上昇等が起つて分析困難となつた。Comparative Example 1 The same serum sample as in Example 1 was passed through analytical column 8 of the same liquid chromatogram without using the protein removal column used in Example 1, but no sedimentation due to coagulation of serum proteins occurred in the column. This caused an increase in column internal pressure, making analysis difficult.
第１図及び第２図は本発明の一実施態様を示す
接続図であり、第３図Ａ，Ｂは本発明実施例１で
得られたクロマトグラム、第４図Ａ，Ｂは本発明
実施例２で得られたクロマトグラムである。
１……水槽、２，７……ポンプ、３……試料イ
ンジエクター、４……６方バルブ、５……除蛋白
カラム、６……溶離液槽、８……分析カラム。
Figures 1 and 2 are connection diagrams showing one embodiment of the present invention, Figures 3A and B are chromatograms obtained in Example 1 of the present invention, and Figures 4A and B are chromatograms obtained in Example 1 of the present invention. This is a chromatogram obtained in Example 2. 1... Water tank, 2, 7... Pump, 3... Sample injector, 4... 6-way valve, 5... Protein removal column, 6... Eluent tank, 8... Analysis column.
Claims (1)
より分析を行うに際し、実質的に有機溶剤を含ま
ない水の存在下では非蛋白分析目的物質は吸着す
るが蛋白質は吸着せず、そして吸着した非蛋白分
析目的物質を、有機溶剤としてメタノール又はア
セトニトリルを含有する水の存在下では溶離する
性質を有する、親水性重合体物質の微粒子からな
る充填剤が充填された除蛋白カラムに、分析目的
物質及び蛋白質を含む試料液を通して、該充填剤
に分析目的物質を含む非蛋白物質を吸着させると
共に、吸着しなかつた蛋白質を系外に除去したの
ち、該除蛋白カラムに有機溶剤としてメタノール
又はアセトニトリルを含有する水からなる溶離液
を流して、吸着されていた上記非蛋白物質を脱着
させ、これを分析カラムに直接チヤージすること
を特徴とする非蛋白物質の分析方法。1 When performing analysis by passing a non-protein substance through an analytical column, in the presence of water that does not substantially contain an organic solvent, the target substance for non-protein analysis is adsorbed, but proteins are not adsorbed, and the adsorbed non-protein substance is The target substance of analysis and protein are loaded into a protein removal column packed with a packing material made of fine particles of a hydrophilic polymer substance that has the property of eluting the target substance of analysis in the presence of water containing methanol or acetonitrile as an organic solvent. After passing the sample solution containing the sample solution to the packing material to adsorb non-protein substances including the target substance of analysis and removing unadsorbed proteins from the system, methanol or acetonitrile is added as an organic solvent to the protein removal column. A method for analyzing non-protein substances, characterized in that the adsorbed non-protein substance is desorbed by flowing an eluent consisting of water, and then directly charged to an analytical column.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP10564482A JPS58223061A (en) | 1982-06-18 | 1982-06-18 | Assay of non-protein material |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP10564482A JPS58223061A (en) | 1982-06-18 | 1982-06-18 | Assay of non-protein material |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS58223061A JPS58223061A (en) | 1983-12-24 |
| JPH0225147B2 true JPH0225147B2 (en) | 1990-05-31 |
Family
ID=14413159
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP10564482A Granted JPS58223061A (en) | 1982-06-18 | 1982-06-18 | Assay of non-protein material |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS58223061A (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS60165551A (en) * | 1984-02-08 | 1985-08-28 | Yokogawa Hokushin Electric Corp | Method and device for measuring urea nitrogen using immobilized enzyme |
| JPS62229065A (en) * | 1986-03-31 | 1987-10-07 | Shimadzu Corp | Analysis of low-molecular component in high-molecular substance and device therefor |
| JPH0718856B2 (en) * | 1986-12-26 | 1995-03-06 | 積水化学工業株式会社 | Fractional measurement of blood bilirubin |
| JPH02216453A (en) * | 1989-02-17 | 1990-08-29 | Sekisui Chem Co Ltd | Method for measuring cyclosporin |
| JP2011505560A (en) * | 2007-11-30 | 2011-02-24 | ウオーターズ・テクノロジーズ・コーポレイシヨン | Determining the presence or absence of mineralcorticoids in a sample |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5551354A (en) * | 1978-10-11 | 1980-04-15 | Toyo Soda Mfg Co Ltd | Method and device for catechol amine analysis |
-
1982
- 1982-06-18 JP JP10564482A patent/JPS58223061A/en active Granted
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5551354A (en) * | 1978-10-11 | 1980-04-15 | Toyo Soda Mfg Co Ltd | Method and device for catechol amine analysis |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS58223061A (en) | 1983-12-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| McDowall | Sample preparation for biomedical analysis | |
| Rashid et al. | Preliminary evaluation of a molecular imprinted polymer for solid-phase extraction of tamoxifen | |
| US6794148B2 (en) | High speed, automated, continuous flow, multi-dimensional molecular selection and analysis | |
| Moors et al. | Analyte isolation by solid phase extraction (SPE) on silica-bonded phases: classification and recommended practices (Technical Report) | |
| JP3444900B2 (en) | Liquid chromatography and column packing | |
| JP2782470B2 (en) | Glycohemoglobin separation method and separation apparatus and separation column | |
| JPH0225147B2 (en) | ||
| Lingeman et al. | Sample pretreatment procedures | |
| JPH0338891B2 (en) | ||
| JP2510302B2 (en) | Method for measuring amines in biological fluids | |
| JPH0774799B2 (en) | Method for measuring blood components | |
| Bourque et al. | Automated HPLC analyses of drugs of abuse via direct injection of biological fluids followed by simultaneous solid‐phase extraction and derivatization with fluorescence detection | |
| Mehta | Sample pretreatment in the trace determination of drugs in biological fluids | |
| JP3050951B2 (en) | Packing material for affinity chromatography | |
| Tamai et al. | High-performance liquid chromatographic drug analysis by direct injection of whole blood samples: I. Determination of moderately hydrophobic drugs incorporated into blood corpuscles | |
| Law et al. | Fundamental studies in reversed-phase liquid—solid extraction of basic drugs; III: Sample matrix effects | |
| JP2830107B2 (en) | Anion exchange resin | |
| Tamai et al. | High-performance liquid chromatographic drug analysis by direct injection of whole blood samples: III. Determination of hydrophobic drugs adsorbed on blood cell membranes | |
| JPS6073461A (en) | Analyzing method of non-protein material | |
| JP3435265B2 (en) | Method for separating proteins having an isoelectric point of 3.3 or less | |
| Boardman | Ion-exchange chromatography | |
| JPH0610675B2 (en) | Packing agent for separating non-protein substances | |
| Shintani | Comparison of solid-phase extraction and dialysis on pretreatment efficiency of blood urea analysis | |
| Tian et al. | Synthesis of molecularly imprinted co-polymers for recognition of ephedrine | |
| JP3628495B2 (en) | Catecholamine analysis method and analyzer |