JPS58223062A - Separation and analysis of physiological active substance and/or medicine - Google Patents
Separation and analysis of physiological active substance and/or medicineInfo
- Publication number
- JPS58223062A JPS58223062A JP57106309A JP10630982A JPS58223062A JP S58223062 A JPS58223062 A JP S58223062A JP 57106309 A JP57106309 A JP 57106309A JP 10630982 A JP10630982 A JP 10630982A JP S58223062 A JPS58223062 A JP S58223062A
- Authority
- JP
- Japan
- Prior art keywords
- protein
- eluent
- column
- analysis
- molecular weight
- 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.)
- Pending
Links
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
- B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
- B01J20/287—Non-polar phases; Reversed phases
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
- B01J20/28002—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their physical properties
- B01J20/28004—Sorbent size or size distribution, e.g. particle size
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
- B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
- B01J20/3204—Inorganic carriers, supports or substrates
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
- B01J20/3214—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the method for obtaining this coating or impregnating
- B01J20/3217—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
- B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
- B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
- B01J20/3244—Non-macromolecular compounds
- B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
- B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
- B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
- B01J20/3268—Macromolecular compounds
- B01J20/3272—Polymers obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
- B01J20/3274—Proteins, nucleic acids, polysaccharides, antibodies or antigens
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2220/00—Aspects relating to sorbent materials
- B01J2220/50—Aspects relating to the use of sorbent or filter aid materials
- B01J2220/54—Sorbents specially adapted for analytical or investigative chromatography
Abstract
Description
【発明の詳細な説明】
本発明は生体液等の蛋白質を含有する溶液中に存在する
低分子量の生理活性物質及び／又は薬物あるいはこれら
両者の伏射物を高精度で分離分析する方法に関する。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for highly accurate separation and analysis of low-molecular-weight physiologically active substances and/or drugs, or hidden substances of both, present in solutions containing proteins such as biological fluids.
尿、血漿、血清、髄液、唾液、羊水、各種腺分泌液、組
織ホ七ジナイプド液、菌細胞倍養液等々の蛋白質を含む
生体液中に含まれる低分子量の生理活性物質、薬剤を迅
速、容易に分離し、更にこれらを定最することは医学上
あるいは生物工業技術上大きな意義があることは周知の
とおりである。Rapidly removes low-molecular weight physiologically active substances and drugs contained in protein-containing biological fluids such as urine, plasma, serum, cerebrospinal fluid, saliva, amniotic fluid, various gland secretions, tissue culture fluid, bacterial cell culture fluid, etc. It is well known that it is of great medical or biotechnological significance to easily separate and standardize these substances.
従来、上記の如き生体液中の低分子量成分を分析するに
は、これらの被検溶液中に含まれる蛋白質を除去せずに
これを行なう場合は、酵素法または免疫学的方法等の生
物学的方法が、又前記蛋白質を抽出等の前処理を行なっ
て除蛋白質操作を行なう場合はガスクロマドづラフイー
、電気泳動法、液体クロマトづラフイー等の分離分析法
が用いられる。しかし、いずれにしてもとれらの方法は
以下のような欠点を有している。Conventionally, in order to analyze low molecular weight components in biological fluids such as those mentioned above, biological methods such as enzymatic methods or immunological methods have been used to analyze the low molecular weight components in biological fluids without removing the proteins contained in these test solutions. Alternatively, when the protein is subjected to pretreatment such as extraction to remove proteins, separation analysis methods such as gas chromatography, electrophoresis, liquid chromatography, etc. are used. However, in any case, these methods have the following drawbacks.
生物学的方法では反応物質が生物学的物質であるので検
体質料の長期保存、安定性、結果の再現性、分析の操作
性等において不利となる場合が多い。In biological methods, since the reactant is a biological substance, there are many disadvantages in terms of long-term storage of specimen materials, stability, reproducibility of results, operability of analysis, etc.
また分離分析法では上記の検体をこれらの装置に注入す
る前に、除蛋白質、即ちオーづシカラムを用いる検体の
粗精製・濃縮・抽出、有機溶媒・塩析を用いる抽出等の
煩雑なる操作を通常は手操作で行なう必要がある。この
ため測定結果の再現性、測定に関する時間、要求検体量
および経済性の点からも不利となる。In addition, in the separation analysis method, before injecting the above-mentioned specimen into these devices, complicated operations such as protein removal, that is, rough purification, concentration, and extraction of the specimen using an OD column, and extraction using an organic solvent and salting out, are performed. Normally, this must be done manually. This is disadvantageous in terms of reproducibility of measurement results, time required for measurement, amount of sample required, and economic efficiency.
本発明者らは、これらの事情に鑑み、生体液等の蛋白質
を含有する溶液中の低分子量の生理活性物質、薬剤、こ
れら両者の代謝物等々の測定が、Ｋ 畦の検体で高精度
でなしうる方法を鋭意研究した結果、従来使用されてい
なかった特殊処理をした液体クロマトづラフイー用充填
剤カラムを用いることにより可能となるとの知見を見い
出し、本発明に到った。In view of these circumstances, the present inventors have determined that the measurement of low molecular weight physiologically active substances, drugs, metabolites of both, etc. in solutions containing proteins such as biological fluids can be performed with high precision using K. As a result of intensive research on possible methods, it was discovered that this could be achieved by using a specially treated liquid chromatography packing column, which had not been previously used, leading to the present invention.
すなわち本発明の要旨は、蛋白質含有溶液中に一存在す
る低分子量の生理活性物質、薬物等を分離し定数する際
、予め蛋白質で処理しだ逆相り０マドづラフイー用充填
剤を用いてこれらを分離分析することを特徴とする方法
にある。That is, the gist of the present invention is that when separating and quantifying low-molecular-weight physiologically active substances, drugs, etc. present in a protein-containing solution, it is necessary to pre-treat with protein and use a reverse-phase 0-mud rough-fi packing material. The method is characterized by separating and analyzing these.
以下、本発明を更に詳しく説明する。The present invention will be explained in more detail below.
充填剤を処理するために使用される蛋白質としては、人
又は動物由来の血漿、血清等が使用でき、例えば人血清
、人血漿、牛血清Ｐ ＩＬ、づ三シ、免血漿、Ｐルづ三
シ、／７０づリシ等である。As the protein used to treat the filler, human or animal-derived plasma, serum, etc. can be used, such as human serum, human plasma, bovine serum PIL, zusanshi, immune plasma, PL shi, /70zu rishi, etc.
この蛋白質の分子量としては１００００〜１ｏｏｏｏｏ
ｏ。The molecular weight of this protein is 10,000 to 1oooooo
o.
好ましくは１００００〜６０００００、更に好ましくは
１００００〜５０００００のものである。Preferably it is 10,000 to 600,000, more preferably 10,000 to 500,000.
液体り０マドクラフイー用逆相充填剤としては、オクタ
デシル基、大り千１し基、又はフェニル基を共有結合し
たシリカゲル又は有機ビニル系高分子骨格をもつゲルの
微粒子である。この粒径としては３〜１０〇五りＤシ、
好ましくは１０〜５〇三クロシ、ポアサイズは２０〜２
００Ａ。The reversed phase filler for liquid flow is fine particles of silica gel or gel having an organic vinyl polymer skeleton to which an octadecyl group, an octadecyl group, or a phenyl group is covalently bonded. The particle size is 3 to 1005 D,
Preferably 10 to 503 kuroshi, pore size 20 to 2
00A.
好ましくは５０〜２００Ａのものが使用できる。Preferably, one with a capacity of 50 to 200 A can be used.
特に、オクタデシル基又はフェニル基を結合した パ
１′・シリガゲ１しが好ましい。Particularly preferred is Pa1'-Siligage-1, which has an octadecyl group or a phenyl group bonded to it.
このゲルに蛋白質を吸着させるだめの処理法としては、
特に制限はないが、例えばゲルを充填したカラムＫ、同
容駿の血清の如き蛋白質を通過させることにより、ゲ）
し表面に蛋白質を飽ゝ和させることができる。又、ゲル
を蛋白質溶液に十分浸漬した後カラムに充填する方法で
も゛よい゛。The treatment method for adsorbing proteins to this gel is as follows:
Although there are no particular limitations, for example, gel-filled column K can be used to pass proteins such as serum of the same volume.
The surface can be saturated with protein. Alternatively, it is also possible to immerse the gel sufficiently in a protein solution and then fill it into a column.
このように本発明は、蛋白質で処理した充填剤をもちい
ることにより、生体液等の検体を除蛋白質処理すること
なく、直接測定できる方法である。As described above, the present invention is a method that allows direct measurement of samples such as biological fluids without protein removal treatment by using a packing material treated with proteins.
分離分析する低分子量の生理活性物質及び／又は薬物と
しては、通常、分子量が３０００以下、好ま、シ＜は１
０００以下のもので、例えば神経伝達物質、ホル七シ、
生理・薬理作用のある化合物、制ｆｊシ剤、抗腫瘍剤、
麻薬、鎮痛剤、精神剤等の血中薬物濃度の測定が必要と
されるものに適用できる。The low molecular weight physiologically active substances and/or drugs to be separated and analyzed usually have a molecular weight of 3000 or less, preferably a molecular weight of less than 1.
000 or less, such as neurotransmitters, hormones,
Compounds with physiological and pharmacological effects, antifj drugs, antitumor agents,
It can be applied to things that require measurement of blood drug concentrations such as narcotics, analgesics, and psychotropic drugs.
検体中に多種の対象低分子物質が存在する場合には、更
に第二カラムをもちいて低分子物質群同志を分離しても
よい。この場合用いられる充填剤としては、いかなる種
類のものも利用でき、たとえばポリスチしシタ１シ、シ
リカ系ゲル、ポリヒニ）し系′ｆ）し等々である０
参考までに本発明で使用する充填剤に充分に蛋白質を吸
着させると、もはや蛋白質を吸着１７なくなるが、この
状態においても低分子量の有機化合物は充分に吸着され
る。このとき飽和吸着した蛋白質は充填剤に強固に吸着
されている。If a variety of target low-molecular substances are present in the sample, a second column may be further used to separate the low-molecular substance groups. The filler used in this case can be of any kind, such as polyethylene, silica gel, polyhinyl, etc.For reference, the filler used in the present invention When the protein is sufficiently adsorbed on the adsorbent, the protein is no longer adsorbed, but even in this state, low molecular weight organic compounds are sufficiently adsorbed. At this time, the saturated adsorbed protein is firmly adsorbed on the filler.
このことは表−１に示したように充分に蛋白質を吸着さ
せた後、充分に水洗いし、乾燥させた充填剤の元素分析
値からも明らかである。またこの蛋白質を飽和吸着した
充填剤をカラムより取り出してＰ三ドづラック東側で処
理すると、染色し得ることからも明らかである。This is also clear from the elemental analysis values of the filler which was sufficiently adsorbed with protein, washed thoroughly with water, and dried as shown in Table 1. It is also clear that if the packing material that has saturated the protein adsorption is taken out of the column and treated on the east side of the P3D rack, it can be stained.
表−１オクタプシル化シリカゲルの元素分析値次に本発
明を更に実施により説明するが、これらの実施例のみに
限定されるものでない。Table 1: Elemental analysis values of octapsilated silica gel Next, the present invention will be further explained based on practical examples, but is not limited to these examples.
内、実施例に用いた液体クロマトづラフイー（商品名ｒ
ＨＬＯ８０３ＤＪ東洋曹達工業株式会社製）のフローを
図２−１又は図２−２に示し、図中の第一カラム（２）
には、サイズ内径４謔、長さ５０間で、前もって人血漿
で処理したＬＳ ４１００ＤＳ （商品名、東洋曹達工
業株式会社製）、２０〜３０μを充填して用いた。流速
は１．５ ｍｔ／ｍｉｎである。図中の第二カラム（６
）には、低分子量物質を分離するいかなるカラムも用い
るが、記載の実施例１．ではＬＳ４１００ＤＳを充填し
た、サイズ内径７．５咽、長さ７５鰭のカラムを用いた
。Among them, liquid chromatography (product name: r) used in the examples.
The flow of HLO803DJ (manufactured by Toyo Soda Kogyo Co., Ltd.) is shown in Figure 2-1 or Figure 2-2, and the first column (2) in the figure
LS 4100DS (trade name, manufactured by Toyo Soda Kogyo Co., Ltd.), which had an inner diameter of 4 mm and a length of 50 mm, was filled with 20 to 30 µm of LS 4100DS (trade name, manufactured by Toyo Soda Kogyo Co., Ltd.), which had been previously treated with human plasma. The flow rate is 1.5 mt/min. The second column (6
), any column that separates low molecular weight substances can be used, but the method described in Example 1. In this case, a column filled with LS4100DS and having an inner diameter of 7.5 mm and a length of 75 fins was used.
実施例１
０．３μ２の６−メルカづトつリンを免血清１ｍｌに溶
解したものを検体とし、その２０μｔを図２−１に示し
たフローの装置に注入し、分離した。検出は３２６ ｎ
ｍの波長の吸光光度法によった。使用した溶離液はｐＨ
３，０，０，１５Ｎリン酸緩衝液である。その結果得ら
れたり０マドづうムを、図１□に蛋白質を含む人血漿に
より処理したオクタプシル化五ノリ力ゲル充填カラムを
用いて得た６−メルカづトづリンのり０マトジラムとこ
の処理前に得たり１コマトダラムを比較して示しだ。人
血漿で処理したカラムを用いると６−メルカづトづリン
の保持体積は若干減少するが、充分に保持されているこ
とがわかる。Example 1 A sample was prepared by dissolving 0.3 µ2 of 6-merca-dutrin in 1 ml of immune serum, and 20 µt of the sample was injected into an apparatus having the flow shown in Figure 2-1 and separated. Detection is 326n
By spectrophotometry at a wavelength of m. The eluent used was pH
3,0,0,15N phosphate buffer. The results are shown in Figure 1 □, and the 6-merca-durin paste obtained using an octapsid gel-packed column treated with protein-containing human plasma and the pre-treatment This is a comparison of 1 komato dalam. It can be seen that when a column treated with human plasma is used, the retention volume of 6-mercadurine is slightly reduced, but it is retained sufficiently.
まだ人血漿中の蛋白質飽和吸着が、液体りＤマドづラフ
イー用逆相系充填剤の低分子量有機化合物の吸着をほと
んど妨害Ｉ〜ないことを示している。従って蛋白質を含
む溶液中に存在する低分子量有機化合物の定量分析の際
、蛋白質を飽和吸着させた逆相系充填剤力うムを組み込
んだ液体り０マドクラフイーを用いると、蛋白質はカラ
ムに保持されず、はぼ死容積付近に容出さ隨るが、一方
低分子喰有機化合物は蛋白質よりもこのカラムに保持さ
れ、充分分離されるの ・で定量測定は可能で
あることを示している。This shows that the saturated adsorption of proteins in human plasma hardly interferes with the adsorption of low molecular weight organic compounds by the reverse phase packing material for liquid media. Therefore, when performing quantitative analysis of low-molecular-weight organic compounds present in solutions containing proteins, proteins are retained in the column by using a liquid column containing a reversed-phase packing material that has saturated protein adsorption. However, low-molecular-weight organic compounds are retained in this column more than proteins and are sufficiently separated, indicating that quantitative measurements are possible.
従って、人血清蛋白質、牛血清Ｐルづ三シ等を飽和吸着
した逆相系カラムを用いる液体クロマトシラフィーを低
分子量有機化合物の定量分析に供する際は、検体を除蛋
白質せずに用いうろことはあきらかである。Therefore, when using liquid chromatography using a reversed-phase column with saturated adsorption of human serum protein, bovine serum protein, etc. for quantitative analysis of low molecular weight organic compounds, it is necessary to use scales without deproteinizing the sample. It is obvious.
実施例２
人血漿１ｍｔ当り、５．４μｍ０づロ力イシアミドを溶
解したものを検体とし、この混合検体２０μｔを図２−
１のフＤ−の装置に注入した検出は２８０ ｎｍの波長
の吸光光度法によった。Example 2 The sample was prepared by dissolving 5.4 μm of isyamide per 1 mt of human plasma, and 20 μt of this mixed sample was used as shown in Figure 2-
Detection injected into the apparatus of No. 1 was by spectrophotometry at a wavelength of 280 nm.
もちいだ溶離液は、溶離液ＡにはｐＨ７，４，０，１５
Ｎのリン酸５緩衝液に１０％（ｖｏｔ／ｖｏｚ）のサリ
シを混合したものを溶離液ＢにはｐＨ３，０，０，１５
Ｎのリシ酸緩、衝液に３％のＰｔトニトリルを混合した
ものである。検体注入後、溶離液Ａを４分間カラム（２
）に通過させた後、三方バルー５ （４’）を切り替え
て溶離液Ｂを通し、得られたり０マドづラムを図３−１
に示した。Eluent A has a pH of 7,4,0,15.
Eluent B was a mixture of 10% (vot/voz) Salicy in N phosphate 5 buffer at pH 3,0,0,15.
This is a mixture of 3% Pt tonitrile in a N ricinic acid buffer solution. After injecting the sample, eluent A was applied to the column (2
), then switch the three-way valve 5 (4') to pass the eluent B, and the obtained or 0 column is shown in Figure 3-1.
It was shown to.
実施例３
人血漿１ ｍｌ当り、０．３μｍの６−メ１し力づトづ
リンを溶解したものを検体とし、この混合検体２０μｔ
を図２−１の７０−の装置に注入した。検出は３２６
ｎｍの波長の吸光光度法によった。もちいた溶離液はｐ
Ｈ３，０，０，１５Ｎのリン酸緩衝液である。得られた
クロマトづラムを図３−２にした。Example 3 A sample was prepared by dissolving 0.3 μm of 6-methane per ml of human plasma, and 20 μt of this mixed sample was prepared.
was injected into the device 70- in Figure 2-1. Detection is 326
By spectrophotometry at a wavelength of nm. The eluent used was p
H3, 0,0,15N phosphate buffer. The obtained chromatogram is shown in Figure 3-2.
実施例４
人血漿１ｍｌ当り、２．０μＶのメソトし＋セードを溶
解したものを検体とし、この混合検体５０μｔを図２−
１のフローの装置に注入した。Example 4 The sample was prepared by dissolving 2.0 μV of mesoton + Sade per 1 ml of human plasma, and 50 μt of this mixed sample was used as shown in Figure 2-
It was injected into a flow device of 1.
検出は３１０ ｎｍの波長の吸光光度法によった。Detection was by spectrophotometry at a wavelength of 310 nm.
溶離液ＡにはｐＨ７，４，０，１５Ｎのリン酸緩衝液９
体積に１体積のサリシを混合したものを、溶離液Ｂには
溶１１ｆ液Ａ９２体積に８体積のア七ト二トリルを混合
したものを用いた。検体注入後、溶離液Ａを４分間カラ
ム（２）に通過させた後、三方パルづ（４）を切り替え
て溶離液Ｂを通し、得られたクロマトジラムを図３−３
に示した。Eluent A contains phosphate buffer 9 with pH 7, 4, 0, 15N.
For the eluent B, a mixture of 92 volumes of solution 11f solution A and 8 volumes of acetonitrile was used. After injecting the sample, eluent A was allowed to pass through the column (2) for 4 minutes, and then the three-way pulse (4) was switched to allow eluent B to pass through, and the resulting chromatodiram was shown in Figure 3-3.
It was shown to.
実施例５
人血ｉ１ｍｔ当り、０．６μ２のタウ０ルじシシを溶解
したものを検体とし、この混合検体１００μｔを図２−
１のフ０−の装置に注入した。検出は、入射光４７５ｎ
ｍ、螢光５８０ ｎｍの波長の螢光光度法によった。溶
離液ＡにはｐＨ３，０，０、１５Ｎのリシ酸緩衝液を、
溶離液Ｂには溶離液Ａ３体積に１休檀のｒｔトニトリル
を混合したものを用いた。検体注入後、溶離液Ａを４分
間第Ｎカラム（２）に通過させた後、三方爪）しづ（４
）を切り替えて溶離７ｉＢを通し、得られたりＯマドグ
ラムを図３−４に示した。Example 5 The sample was prepared by dissolving 0.6 μ2 of tau per 1 mt of human blood, and 100 μt of this mixed sample was used in Figure 2-
It was injected into the apparatus of No.1. Detection is performed using 475n of incident light.
m, by fluorescence photometry at a wavelength of 580 nm. Eluent A contains lysic acid buffer of pH 3, 0, 0, 15N,
As eluent B, a mixture of 3 volumes of eluent A and 1 part of rt tonitrile was used. After injecting the sample, let the eluent A pass through the N column (2) for 4 minutes, and then
), the elution 7iB was passed, and the O madogram obtained is shown in Figure 3-4.
実施例６
人血漿１ ｒｎＬ当り、９．０μ２のテオフイリシを溶
解したものを検体と１７、この混合検体１０μｔを図２
−５１のフローの装置に注入した。検出は２８０ ｎｍ
の波長の吸光光度法によった。溶離液ＡにはｐＨ７，４
，０，１５Ｎのリシ酸緩衝液９体積に１休檀の１リシを
混合したものを、溶離液Ｂには溶離液Ａ９４体積に６休
檀のアセトニトリルを混合したものを用いた。検体注入
後、溶離液Ａを４分間カラム（２）に通過させた後、三
方爪１しづ（４）を切り替えて溶離液Ｂを通し、得られ
たり０マドシラ八を図３−５に示しだ。Example 6 Sample 17 was prepared by dissolving 9.0μ2 of theophyllis per rnL of human plasma, and 10μt of this mixed sample was used in Figure 2.
-51 flow into the device. Detection is at 280 nm
by spectrophotometry at a wavelength of . Eluent A has a pH of 7.4.
For the eluent B, a mixture of 9 volumes of a 0.15N lysic acid buffer and 1 h of acetonitrile was used. After injecting the sample, let the eluent A pass through the column (2) for 4 minutes, then switch the three-way hook (4) and let the eluent B pass through. .
実施例７
人血漿１ ｍｌ当り、０．３μｆのづＯづラフ０−ルを
溶解したものを検体とし、この混合検体１００μＬを図
２−１のフローの装置に注入した。検出は入射光２８７
ｎｍ、螢光３４０ ｎｍの波長の螢光光度法により分析
した。溶離液ＡにはｐＨ７，４，０，１５Ｎのリシ酸緩
衝液９体積にサリン１体積を混合したものを、解離液Ｂ
には溶離液Ａ６５体積にＰｔ）ニトリ１し３５５休檀混
合したものを、溶離液Ｂ′にはｐＨ３，０、０，１５Ｎ
のリシ酸緩衝液４体積に１休檀のア七ト二トリルを混合
したものを用いた。検体注入後、溶離液Ａを４分間カラ
ム（２）に通過させた後、三方バ１しづ（４）を切り替
えて、溶離液Ｂを通し、更に４分後再び三方パルづ（４
′）を切り替えて、Ｐ４離液Ｂ′を通しだ。得られたり
Ｏマドクラムを図３−６に示しだ。Example 7 A sample was prepared by dissolving 0.3 μf of 0.3 μf of human plasma per 1 ml of human plasma, and 100 μL of this mixed sample was injected into the apparatus having the flow shown in FIG. 2-1. Detection is incident light 287
analyzed by fluorophotometry at a wavelength of 340 nm. Eluent A is a mixture of 9 volumes of lysic acid buffer at pH 7, 4, 0, 15N and 1 volume of sarin, and dissociation solution B is
For eluent A, 65 volumes were mixed with Pt) Nitri 1 and 355, and for eluent B', pH was 3.0 and 0.15N.
A mixture of 4 volumes of lysic acid buffer and 1 part of acetatonitrile was used. After injecting the sample, let the eluent A pass through the column (2) for 4 minutes, then switch the three-way bar 1 (4) to pass the eluent B, and after another 4 minutes, pass the eluent A through the column (4) again.
') and pass P4 synergic liquid B'. The resulting O-mad crumb is shown in Figure 3-6.
実施例８
イシドール化合物群の測定を図２−２のフローの装置罠
より分析した。カラム（２）には内径４間、１番さ４０
鰭にオクタイシ）しシラン処理シリカゲルを充填ｊ〜、
イｑ白１グ処理したものを、カラム（６）には内径７．
５ｍ、長さ７５間にオクタプシル処理シリカゲルを充填
したものを用いた。溶離液Ａ′にはｐＨ３，０，０，Ｉ
Ｎのり、７酸緩衝液に０．５チトリクＤ１し酢酸をｆ？
８１解したものを、溶離液ＣにはｐＨ３，０，０，１５
Ｎ、のリン酸緩衝液９体積に１休檀のＰｔｌニトリｌし
を混合したものを、溶離液りにはｐＨ３，０，０，１５
Ｎのリシ酸緩衝液７体積に３休檀のＰ七トニトリルを混
合したものを用い、検体パルづ（１）を通過させて装置
に注入後７外間、溶離液Ａをカラム（２）に通過さす。Example 8 The measurement of isidol compound group was analyzed using the apparatus trap of the flow shown in FIG. 2-2. Column (2) has an inner diameter of 4 mm and a diameter of 40 mm.
The fins are filled with silanized silica gel.
Column (6) had an inner diameter of 7.
A 5 m long tube with a length of 75 mm filled with octapsil-treated silica gel was used. Eluent A' has pH 3,0,0,I
N glue, 0.5 Titric D1 in 7-acid buffer and acetic acid f?
Eluent C has pH 3,0,0,15.
A mixture of 9 volumes of N. phosphate buffer and 1 portion of Ptl nitrile was added to the eluent solution at pH 3,0,0,15.
Using a mixture of 7 volumes of N lysic acid buffer and 3 volumes of P7 tonitrile, pass through the sample pulse (1) and inject it into the device, then pass the eluent A through the column (2) for 7 hours. As expected.
その後パルづ（７）を切り替えて溶離液Ｃをカラム（２
）に２９分間通した。その後ノ’＋ルづ（９）を切り替
えて、溶ｌｉ！（ｔ ｉ夜りをカラム（６）に通した。After that, switch the pulse (7) and transfer the eluent C to the column (2).
) for 29 minutes. After that, switch to No' + Luzu (9) and let it go! (t i night was passed through column (6).
検出は入射光２８７ｎｍ、螢光３４０ ｎｍの波長の螢
光光度法により分析し語られたりＯマドづラムを図３−
７に示した。Detection is performed by analyzing the incident light at a wavelength of 287 nm and fluorescent light at a wavelength of 340 nm.
7.
比較例
本発明法の実施例２〜７までの薬物の回収率と、従来法
として１０チドリクロル酸酸溶液を各検体と同体積添加
して除蛋白した後、図５２−１のフローの装置によシ同
様に分離分析した後の回収率との比較を表−２に示した
。Comparative Example The recovery rate of the drug in Examples 2 to 7 of the method of the present invention and the conventional method in which the same volume of 10 tidlychloric acid solution was added to each sample to remove protein, and then the results were shown in the apparatus of the flow shown in Figure 52-1. Table 2 shows a comparison with the recovery rate after separation and analysis in the same manner as above.
表−２ は本発明方法に用いる装置の構成フロー図である。Table-2 1 is a configuration flow diagram of an apparatus used in the method of the present invention.
（１）：検体注入装置 （２）：第一カラム（３）二ポ
ジづ （４）、（４’） ：三方パルづ（５）
：検出器 （６）：第二カラム（７）二六方パ
ルづ （８）；ポジづ（９）、三方バ１しづ
００：記録計（Ｔ］）、α１−゛ア士１ムレター
（１オＡ、（１２Ｂ、０→Ｃ１（１のＤ：各々溶離液Ａ
、Ｂ、Ｏ，Ｄ第１
人匍ｊＪＩ処１里４走
わ−メルｈブトプリン
Ｕ ］ Ｚ ブ丁人前１邊ヌ
１王里Ｊ’１
第３＝１図
↑ Ｐ、ブロカインア汁０
４ ６ ノ２介
第３−３図 第８−４図
第３−５図 第３−６図(1): Sample injection device (2): First column (3) two-position (4), (4'): three-way pulse (5)
: Detector (6): Second column (7) 26-way pulse (8); Positive (9), 3-way bar 1 pulse
00: Recorder (T)), α1-A 1 meter (1 OA, (12B, 0→C1 (1 D: Eluent A)
, B, O, D 1st person 4 j JI place 1 ri 4 running - meru h butpurin U] Z buto 1 person 1 bye nu 1 ori J'1 3rd = 1 figure ↑ P, brocain a soup 0
4 6 No. 2 Figure 3-3 Figure 8-4 Figure 3-5 Figure 3-6
Claims (1)
又は薬物を分離分析するＫあたり、蛋白質で処理した液
体クロマイづラフイー用逆相充填剤を用いることを特徴
とする生理活性物質及び／又は薬物の分離分析方法。 ２ 充填剤がオクタデシル基、オクチル基又はフエニ４
し基の少なくとも一種を共有結合した粒径３〜１０〇三
り０シのシリカゲルであることを特徴とする特許請求の
範囲第１項に記載した生理活性物質及び／又は薬物の分
離分析方法。[Claims] 1. Low molecular weight physiologically active substances and/or in protein-containing solutions
Or, a method for separating and analyzing physiologically active substances and/or drugs, which is characterized in that a protein-treated liquid chromafluoride reverse-phase packing material is used for separating and analyzing drugs. 2 The filler is an octadecyl group, an octyl group, or a phenylene group.4
2. The method for separating and analyzing physiologically active substances and/or drugs as set forth in claim 1, wherein the silica gel is made of silica gel having a particle size of 3 to 100,000 to which at least one group is covalently bonded.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP57106309A JPS58223062A (en) | 1982-06-21 | 1982-06-21 | Separation and analysis of physiological active substance and/or medicine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP57106309A JPS58223062A (en) | 1982-06-21 | 1982-06-21 | Separation and analysis of physiological active substance and/or medicine |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPS58223062A true JPS58223062A (en) | 1983-12-24 |
Family
ID=14430385
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP57106309A Pending JPS58223062A (en) | 1982-06-21 | 1982-06-21 | Separation and analysis of physiological active substance and/or medicine |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS58223062A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6165159A (en) * | 1984-08-31 | 1986-04-03 | パーデュー・リサーチ・ファウンデーション | Inner-surface reversed-phase filling material for liquid chromatograph-column and manufacture thereof |
| JPS63126735A (en) * | 1986-11-04 | 1988-05-30 | スタミカーボン・ベスローテン・ベンノートシヤツプ | Manufacture of polyamide product from melt |
| JPS63163275A (en) * | 1986-12-26 | 1988-07-06 | Sekisui Chem Co Ltd | Method for fractionating and measuring bilirubin in blood |
| JPH01315348A (en) * | 1987-10-27 | 1989-12-20 | Yokogawa Electric Corp | Production of cation exchange resin |
| WO1992010508A1 (en) * | 1990-12-06 | 1992-06-25 | Sumitomo Electric Industries, Ltd. | Method of separating protein and apparatus therefor |
| WO1999011650A1 (en) * | 1997-08-28 | 1999-03-11 | Mercian Corporation | Method of purifying daunomycin |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS53140097A (en) * | 1977-04-26 | 1978-12-06 | Elf Aquitaine | Separation and purification of protein by chromatography |
| JPS5760261A (en) * | 1980-09-29 | 1982-04-12 | Sekisui Chem Co Ltd | Manufacture of filler for liquid chromatograph |
-
1982
- 1982-06-21 JP JP57106309A patent/JPS58223062A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS53140097A (en) * | 1977-04-26 | 1978-12-06 | Elf Aquitaine | Separation and purification of protein by chromatography |
| JPS5760261A (en) * | 1980-09-29 | 1982-04-12 | Sekisui Chem Co Ltd | Manufacture of filler for liquid chromatograph |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6165159A (en) * | 1984-08-31 | 1986-04-03 | パーデュー・リサーチ・ファウンデーション | Inner-surface reversed-phase filling material for liquid chromatograph-column and manufacture thereof |
| JPS63126735A (en) * | 1986-11-04 | 1988-05-30 | スタミカーボン・ベスローテン・ベンノートシヤツプ | Manufacture of polyamide product from melt |
| JPS63163275A (en) * | 1986-12-26 | 1988-07-06 | Sekisui Chem Co Ltd | Method for fractionating and measuring bilirubin in blood |
| JPH01315348A (en) * | 1987-10-27 | 1989-12-20 | Yokogawa Electric Corp | Production of cation exchange resin |
| WO1992010508A1 (en) * | 1990-12-06 | 1992-06-25 | Sumitomo Electric Industries, Ltd. | Method of separating protein and apparatus therefor |
| WO1999011650A1 (en) * | 1997-08-28 | 1999-03-11 | Mercian Corporation | Method of purifying daunomycin |
| US6388058B1 (en) | 1997-08-28 | 2002-05-14 | Mercian Corporation | Method of purifying daunomycin |
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