JP2021128005A - Blood pretreatment method and examination method - Google Patents
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- 238000002203 pretreatment Methods 0.000 title claims description 20
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Abstract
Description
本発明は、血液中の薬物濃度をモニタリングする方法に関する。また本発明は血液中の薬物濃度を精度よくモニタリングするための、前処理方法および該前処理に用いるキットに関する。 The present invention relates to a method for monitoring a drug concentration in blood. The present invention also relates to a pretreatment method and a kit used for the pretreatment for accurately monitoring the drug concentration in blood.
臨床で用いられる薬剤には定められた固有の用法・用量がある。しかし、たとえ同じ用量の薬剤を投与したとしても、患者により効果が違ってくることがある。その理由は、薬物の体内動態には個人差があるためである。そのため個人ごとに適した薬用量や薬物の投与設計のために、患者の血中濃度のモニタリングを行い、その結果や臨床所見からより適切な血中濃度となるように薬物動態学的な解析をふまえて用法・用量を調節していく手法がとられる場合がある。 Drugs used clinically have specific dosages and administrations. However, even if the same dose of drug is given, the effect may vary from patient to patient. The reason is that there are individual differences in the pharmacokinetics of drugs. Therefore, in order to design the dosage and administration of the drug suitable for each individual, the blood concentration of the patient is monitored, and the pharmacokinetic analysis is performed so that the blood concentration becomes more appropriate based on the results and clinical findings. In some cases, a method of adjusting the dosage and administration may be taken.
このような薬物血中濃度をモニタリングする方法（以下、ＴＤＭと略する場合がある）として、高速液体クロマトグラフィーやガスクロマトグラフィーによる分離分析法、蛍光偏光免疫測定法、酵素免疫測定法または放射性免疫測定法による免疫学的手法、および原子吸光光度法が知られている。
上記方法のなかでも分離分析法は一回の分析で複数種の薬物を定性・定量できることから、非常に有用な方法である。
As a method for monitoring the blood concentration of such a drug (hereinafter, may be abbreviated as TDM), a separation analysis method by high performance liquid chromatography or gas chromatography, a fluorescently polarized immunoassay, an enzyme immunoassay or a radioimmunoassay Immunological methods based on measurement methods and atomic absorptiometry are known.
Among the above methods, the separation analysis method is a very useful method because it can qualify and quantify a plurality of kinds of drugs in one analysis.
上記分離分析法では薬物の同定、定量は通常、質量分析計を用いて質量分析により行われる。質量分析計を用いて血液中の薬物濃度を測定する場合、通常、分析精度を高めるために血液中のたんぱく質を除くための前処理として除タンパクが必要である。除タンパクに用いられる除タンパク液には通常、塩が含まれており、この塩を含んだままの血液検体で分離分析法を行うと、質量分析時の感度低下を引き起こす。 In the above separation analysis method, drug identification and quantification are usually performed by mass spectrometry using a mass spectrometer. When measuring the drug concentration in blood using a mass spectrometer, deproteinization is usually required as a pretreatment for removing proteins in blood in order to improve the analysis accuracy. The deproteinizing solution used for deproteinization usually contains a salt, and if a separation analysis method is performed on a blood sample containing this salt, the sensitivity at the time of mass spectrometry is lowered.
したがって血液を除タンパクした後、血液から塩を除去する必要がある。このような塩や血液中に含まれる夾雑物を除去するための前処理として、質量分析計の前にトラップカラムと呼ばれるカラムを用いて、塩や夾雑物を除去する方法が知られている。 Therefore, after deproteinizing the blood, it is necessary to remove the salt from the blood. As a pretreatment for removing such impurities contained in salt and blood, a method of removing salts and impurities by using a column called a trap column in front of a mass spectrometer is known.
分離分析法には、移動相の組成比を一定の割合で送液するイソクラティック溶離と、移動相の組成比を時間によって変化させながら送液するグラジエント溶離が知られている。イソクラティック溶離で分離分析を行う場合、トラップカラムと液体クロマトグラフ質量分析計（以下、ＬＣ−ＭＳ／ＭＳと称する場合がある）を組み合わせた分析装置、分析キットが知られている（例えば、島津製作所製のＬＣ−ＭＳ／ＭＳ用免疫抑制剤分析キットDOSIMMUNE（登録商標））。ＬＣ−ＭＳ／ＭＳは、混合物を空間的に分離する高速液体クロマトグラフ（ＨＰＬＣ）と、対象化合物の選択的な検出が可能な質量分析計（ＭＳ／ＭＳ）を連結したハイブリッド型分析装置である。 As the separation analysis method, isocratic elution in which the composition ratio of the mobile phase is sent at a constant ratio and gradient elution in which the composition ratio of the mobile phase is changed with time are known. Analytical devices and analysis kits that combine a trap column and a liquid chromatograph mass spectrometer (hereinafter, may be referred to as LC-MS / MS) are known for performing separation analysis by isocratic elution (for example,). Shimadzu LC-MS / MS immunosuppressant analysis kit DOSIMMUNE (registered trademark). LC-MS / MS is a hybrid analyzer that connects a high performance liquid chromatograph (HPLC) that spatially separates a mixture and a mass spectrometer (MS / MS) that can selectively detect a target compound. ..
このイソクラティック溶離で用いる装置等をグラジエント溶離に適用しようとしても、分析用カラムの前にバルブが必要であったり、移動相の流路構成の切り替えが煩雑であったりして、装置の改良が大掛かりなものとなる。 Even if the device used for this isocratic elution is applied to gradient elution, a valve is required in front of the analysis column, and switching of the mobile phase flow path configuration is complicated, so the device is improved. Becomes a large scale.
したがって、グラジエント溶離に適用できるような、除タンパクした後の血液から塩や夾雑物を除去するための前処理と分離分析手法が一体となった、簡便な薬物血中濃度をモニタリングする方法およびその装置の開発が求められていた。 Therefore, a simple method for monitoring drug blood concentration, which is applicable to gradient elution, is a combination of pretreatment and separation analysis method for removing salts and impurities from the deproteinized blood, and a method thereof. The development of the device was required.
本発明は血液の前処理と分離分析方法が一体となった簡便な薬物血中濃度のモニタリング方法を提供することを目的とする。
また本発明は、上記モニタリング方法を達成するための簡便な血液検体の前処理方法および該前処理方法に用いられるキットを提供することを目的とする。
An object of the present invention is to provide a simple method for monitoring a drug blood concentration in which a blood pretreatment and a separation analysis method are integrated.
Another object of the present invention is to provide a simple blood sample pretreatment method for achieving the above monitoring method and a kit used for the pretreatment method.
すなわち、本発明は、
除タンパク処理された血液検体をコンディショニングされた固相抽出カラムへ添加し、該血液検体が固相抽出カラムを通過後、固相抽出カラムを洗浄し、その後固相抽出カラムから吸着物を溶出用液にて溶出させ、得られた溶出液をＬＣ−ＭＳ／ＭＳ分析することを含む、薬物血中濃度のモニタリング方法に関する。
That is, the present invention
The deproteinized blood sample is added to the conditioned solid-phase extraction column, the blood sample passes through the solid-phase extraction column, the solid-phase extraction column is washed, and then the adsorbent is eluted from the solid-phase extraction column. The present invention relates to a method for monitoring a drug blood concentration, which comprises eluting with a liquid and subjecting the obtained eluate to LC-MS / MS analysis.
また本発明は、
除タンパク処理された血液検体をコンディショニングされた固相抽出カラムへ添加し、該血液検体が固相抽出カラムを通過後、固相抽出カラムを洗浄し、その後固相抽出カラムから吸着物を溶出させることを含む血液検体の前処理方法、および
固相抽出カラム、遠沈管、コンディショニング液、洗浄液、および溶出液を含む前処理キット、
に関する。
Further, the present invention
The deproteinized blood sample is added to the conditioned solid-phase extraction column, the blood sample passes through the solid-phase extraction column, the solid-phase extraction column is washed, and then the adsorbent is eluted from the solid-phase extraction column. Pretreatment methods for blood samples, including solid-phase extraction columns, centrifuge tubes, conditioning fluids, wash fluids, and pretreatment kits containing eluates.
Regarding.
本発明によれば、血液の前処理と分離分析方法が一体となった簡便な薬物血中濃度のモニタリング方法を提供することができる。
また本発明によれば、上記モニタリング方法を達成するための簡便な血液検体の前処理方法および該前処理方法に用いられるキットを提供することができる。
According to the present invention, it is possible to provide a simple method for monitoring a drug blood concentration in which a blood pretreatment and a separation analysis method are integrated.
Further, according to the present invention, it is possible to provide a simple pretreatment method for blood samples and a kit used for the pretreatment method for achieving the above monitoring method.
本発明に用いられる血液検体は予め除タンパクされた血液である。除タンパクの方法は、例えば、除たんぱく剤を使う方法、水と混和可能な有機溶媒による変性法、加熱または冷却による熱変性法、などである。効果的にタンパク質が除かれるという観点から、変性法が好ましい。 The blood sample used in the present invention is pre-deproteinized blood. The method for removing protein includes, for example, a method using a protein removing agent, a denaturation method using an organic solvent that can be mixed with water, and a heat denaturation method by heating or cooling. The denaturation method is preferred from the viewpoint of effectively removing proteins.
変性法に用いる有機溶媒としては、アセトニトリルやメタノール、エタノール等が挙げられる。また除タンパクを効率的に行う観点から、硫酸亜鉛を添加してもよい。 Examples of the organic solvent used in the modification method include acetonitrile, methanol, ethanol and the like. Further, zinc sulfate may be added from the viewpoint of efficiently removing protein.
除タンパク前の血液に上述のような除タンパク剤や変性法に用いる有機溶媒を添加し、撹拌後遠心分離により、上澄み液を回収して除タンパクされた血液検体を得ることができる。得られた上澄み液をさらにカラム処理し、以後の操作に用いてもよい。 The above-mentioned deproteinizing agent and the organic solvent used in the denaturation method are added to the blood before deproteinization, and the supernatant is recovered by centrifugation after stirring to obtain a deproteinized blood sample. The obtained supernatant liquid may be further column-treated and used for subsequent operations.
上記方法により得られた除タンパクされた血液検体はコンディショニングされた固相抽出カラムに添加される。コンディショニングは固相抽出カラムの充填剤を濡らし、充填剤の官能基を活性化させる目的で行われる。
コンディショニングは、まず、メタノールやアセトニトリル等の極性有機溶媒を充填剤体積の約３倍から１０倍程度、充填剤に流す。その後、分離分析法で用いる移動相と同じ組成の溶媒を用い、充填剤を洗浄する。
The deproteinized blood sample obtained by the above method is added to a conditioned solid-phase extraction column. Conditioning is performed for the purpose of wetting the packing material of the solid-phase extraction column and activating the functional groups of the packing material.
For conditioning, first, a polar organic solvent such as methanol or acetonitrile is flowed through the filler in an amount of about 3 to 10 times the volume of the filler. Then, the filler is washed with a solvent having the same composition as the mobile phase used in the separation analysis method.
極性有機溶媒を充填剤に流し、溶媒で洗浄するコンディショニング操作は、除タンパクされた血液検体を固相抽出カラムに添加する前に、少なくとも１回は行う。コンディショニング操作は２回以上行ってもよいが、コンディショニングを行った後、充填剤が再び乾燥する前に、除タンパクされた血液検体を固相抽出カラムに添加する必要がある。 The conditioning operation of flushing the polar organic solvent through the filler and washing with the solvent is performed at least once before adding the deproteinized blood sample to the solid phase extraction column. The conditioning operation may be performed more than once, but after the conditioning, the deproteinized blood sample must be added to the solid-phase extraction column before the filler dries again.
用いる固相抽出カラムは、固相抽出に用いられるカラムである。固相抽出とは、溶液や懸濁液に含まれる分析対象物とそれ以外の不純物とを分離するために、充填剤を充填したカートリッジに溶液や懸濁液を通し目的物と不純物を分離する方法である。固相抽出カラムは、このような充填剤を充填したカートリッジである。 The solid-phase extraction column used is a column used for solid-phase extraction. Solid-phase extraction is to separate the target substance and impurities by passing the solution or suspension through a cartridge filled with a filler in order to separate the analysis target contained in the solution or suspension from other impurities. The method. A solid-phase extraction column is a cartridge filled with such a filler.
固相抽出カラムの充填剤としては、不純物を保持し、対象物質を流出させる充填剤と、対象物質を保持しその後、別の種類の移動相を流し、対象物質を溶出させる充填剤とがあるが、対象物質を保持する充填剤を有する固相抽出カラムが、結果的に対象物質を濃縮することができ、血中に含まれる薬物のように低濃度の対象物質を定量分析するのに適していることから、本発明のモニタリング方法に適している。 The filler of the solid phase extraction column includes a filler that retains impurities and causes the target substance to flow out, and a filler that retains the target substance and then flows another type of mobile phase to elute the target substance. However, a solid phase extraction column with a filler that retains the target substance can result in the concentration of the target substance, which is suitable for quantitative analysis of low-concentration target substances such as drugs contained in blood. Therefore, it is suitable for the monitoring method of the present invention.
充填剤としては主成分にシリカモノリスを含む充填剤が用いられ、シリカモノリスにオクタデシル基が結合した充填剤、フェニル基が結合した充填剤、トリメチルアミノプロピル基が結合した充填剤、プロピルベンゼンスルホン酸が結合した充填剤、カルボキシル基が結合した充填剤等がある。なお、シリカモノシルとはマイクロメートルオーダーの網目状の骨格が繋がった特徴的な構造をもつ一体型のシリカ多孔質体である。
例えば、充填剤としてＧＬサイエンス社製のＭｏｎｏｓｐｉｎ Ｃ８、Ｍｏｎｏｓｐｉｎ Ｃ１８、Ｍｏｎｏｐｉｎ Ｃ１８ＦＦ、Ｍｏｎｏｓｐｉｎ Ｐｈ、（Ｍｏｎｏｓｐｉｎは登録商標）などが知られている。
As the filler, a filler containing silica monolith as a main component is used, and a filler in which an octadecyl group is bonded to silica monolith, a filler in which a phenyl group is bonded, a filler in which a trimethylaminopropyl group is bonded, and a propylbenzenesulfonic acid. There are fillers to which is bonded, fillers to which a carboxyl group is bonded, and the like. Silica monosyl is an integrated silica porous body having a characteristic structure in which micrometer-order mesh-like skeletons are connected.
For example, Monospin C8, Monospin C18, Monopin C18FF, Monospin Ph, (Monospin is a registered trademark) manufactured by GL Science Co., Ltd. are known as fillers.
上記方法によりコンディショニングされた固相抽出カラムに上述の除タンパクされた血液検体を添加し、当該血液検体を充填剤に通過させる。上述のとおり、本発明のモニタリング方法で固相抽出カラムが対象物質を保持させる充填剤を有している場合、充填剤を血液検体が通過する過程で、目的とする血中薬物が充填剤に吸着する。 The above-mentioned deproteinized blood sample is added to the solid-phase extraction column conditioned by the above method, and the blood sample is passed through a filler. As described above, when the solid-phase extraction column has a filler that retains the target substance in the monitoring method of the present invention, the target blood drug becomes the filler in the process of passing the blood sample through the filler. Adsorb.
目的とする血中薬物を充填剤に効率よく吸着させるために、モニタリングを目的とする血中薬物の種類により充填剤の種類を予め選定すればよい。血中薬物が疎水性の高い薬物の場合、疎水性相互作用を利用して分離する逆相カラムが選定される。また血中薬物の疎水性がそれほど高くない場合、順相カラムを選定すればよい。
例えばモニタリングする薬物が、後述する免疫抑制剤またはリンパ脈管筋腫症治療剤の一種であるシロリムス、エベロリムス、タクロリムス、シクロスポリンの少なくとも１種である場合、シリカモノリスにオクタデシル基が結合した充填剤を用いた固相抽出カラムが好適に使用できる。
In order to efficiently adsorb the target blood drug to the filler, the type of filler may be selected in advance according to the type of blood drug for monitoring. When the blood drug is a highly hydrophobic drug, a reversed-phase column that separates using hydrophobic interaction is selected. If the hydrophobicity of the drug in the blood is not so high, a normal phase column may be selected.
For example, if the drug to be monitored is at least one of immunosuppressive drug or lymphangioleiomyomatosis therapeutic agent, which will be described later, sirolimus, everolimus, tacrolimus, and cyclosporine, a filler in which an octadecyl group is bound to silica monolith is used. The solid-phase extraction column that has been used can be preferably used.
血液検体を充填剤に通過させる方法としては、自然に血液検体が重力により落下して充填剤を通過する自然落下法、血液検体をシリンジ等で加圧して通過させる加圧法、固相抽出カラムの出口側を減圧または吸引して血液検体を充填剤に通過させる吸引法、固相抽出カラムと溶液を回収する容器を組み合わせて、遠心機にかける遠心分離法があるが、簡便性の観点から遠心分離法が好ましい。 As a method of passing the blood sample through the filler, a natural drop method in which the blood sample naturally falls due to gravity and passes through the filler, a pressurization method in which the blood sample is pressurized by a syringe or the like and passed through, a solid-phase extraction column There are a suction method in which the outlet side is decompressed or aspirated to pass the blood sample through the filler, and a centrifugation method in which a solid-phase extraction column and a container for collecting the solution are combined and centrifuged, but from the viewpoint of convenience, centrifugation is performed. The separation method is preferred.
除タンパクされた血液検体が固相抽出カラムを通過することで、充填剤にモニタリングを目的とする血中薬物が吸着され、血液検体に含まれていた夾雑物や塩はそのまま流出する。流出した液は廃液として処理される。 When the deproteinized blood sample passes through the solid-phase extraction column, the blood drug for monitoring is adsorbed on the filler, and the impurities and salts contained in the blood sample flow out as they are. The spilled liquid is treated as waste liquid.
除タンパクされた血液検体が固相抽出カラムを通過後、当該固相抽出カラムを洗浄する。洗浄することで、余分な吸着物を除去し、モニタリングを目的する血中薬物のみが吸着される。
洗浄の回数は１回以上であれば特に制限はないが、洗浄の回数が多すぎるとモニタリングを目的する血中薬物まで洗い出されてしまうので、通常、洗浄の回数は１回である。
After the deproteinized blood sample passes through the solid-phase extraction column, the solid-phase extraction column is washed. Washing removes excess adsorbates and adsorbs only blood drugs for monitoring purposes.
The number of washings is not particularly limited as long as the number of washings is one or more, but if the number of washings is too large, even the blood drug for the purpose of monitoring is washed out, so the number of washings is usually one.
洗浄液は使用する固相カラムの性質により適宜選定され、上述したように逆相カラムを用いる場合、洗浄液は水と有機溶媒との混合液が好ましい。洗浄液中の水の含有量は８０質量％から１００質量％が好ましい。 The cleaning liquid is appropriately selected depending on the properties of the solid phase column to be used, and when a reverse phase column is used as described above, the cleaning liquid is preferably a mixed liquid of water and an organic solvent. The content of water in the cleaning liquid is preferably 80% by mass to 100% by mass.
また本発明のモニタリング方法は、後述するＬＣ−ＭＳ／ＭＳの分析がグラジエント溶離で行われるモニタリング方法に好適に適用できるが、グラジエント溶離の場合は、用いる分析カラムの性質にもよるが、逆相カラムの場合は一般的に水とメタノールまたはアセトニトリルとの混合液が用いられる。メタノールまたはアセトニトリルの含有量は８０質量％から１００質量％が好ましい。 Further, the monitoring method of the present invention can be suitably applied to a monitoring method in which LC-MS / MS analysis described later is performed by gradient elution, but in the case of gradient elution, the phase is reversed, depending on the nature of the analysis column used. In the case of a column, a mixed solution of water and methanol or acetonitrile is generally used. The content of methanol or acetonitrile is preferably 80% by mass to 100% by mass.
固相抽出カラムを洗浄後、固相抽出カラムに吸着した吸着物を溶出させる。溶出に用いる溶出用液は、吸着物を溶解して溶出させる溶媒であり、モニタリングを目的とする血中薬物を溶出させる有機溶媒である。例えばアセトニトリル、メタノールである。 After washing the solid-phase extraction column, the adsorbent adsorbed on the solid-phase extraction column is eluted. The elution liquid used for elution is a solvent that dissolves and elutes the adsorbent, and is an organic solvent that elutes the blood drug for the purpose of monitoring. For example, acetonitrile and methanol.
上記により溶出した溶出液はＬＣ−ＭＳ／ＭＳ分析に供され、分離分析され血中に含まれている薬物の同定および定量が行われる。ＬＣ−ＭＳ／ＭＳ分析では、移動相の組成比を時間によって変化させながら送液するグラジエント溶離が分析時間を短縮できる観点から好ましい。 The eluate eluted as described above is subjected to LC-MS / MS analysis, separated and analyzed, and the drug contained in the blood is identified and quantified. In LC-MS / MS analysis, gradient elution, in which the liquid is fed while changing the composition ratio of the mobile phase with time, is preferable from the viewpoint of shortening the analysis time.
本発明の血中薬物のモニタリング方法において、モニタリングの対象となる薬物は、ＴＤＭの対象となる薬物である。ＴＤＭは、例えば、薬物体内動態の把握、医薬品の適正量の投与、多剤併用の可否、中毒・副作用の早期発見、を目的として行われるものであり、これらが必要な薬物がモニタリングの対象となる薬物である。 In the blood drug monitoring method of the present invention, the drug to be monitored is the drug to be monitored by TDM. TDM is performed for the purpose of grasping the pharmacokinetics of drugs, administering an appropriate amount of drugs, whether or not multiple drugs can be used in combination, and early detection of poisoning / side effects. Is a drug.
ＴＤＭの対象となる薬物の例としては、特定薬剤治療管理料の対象薬物が挙げられ、例えば、ジギタリス製剤、テオフィリン製剤、不整脈用剤、抗てんかん剤、アミノ配糖体抗生物質、グリコペプチド系抗生物質、トリアゾール系抗真菌剤、免疫抑制剤、サリチル酸系製剤、抗悪性腫瘍剤、ハロペリドール製剤、ブロムペリドール製剤、リチウム製剤、リンパ脈管筋腫症治療剤からなる群から選ばれる少なくとも１種が挙げられる。 Examples of drugs subject to TDM include drugs subject to specific drug treatment management fees, such as digitalis preparations, theophylline preparations, arrhythmic agents, antiepileptic agents, aminoglycosyl antibiotics, and glycopeptide antibiotics. At least one selected from the group consisting of substances, triazole antifungal agents, immunosuppressive agents, salicylic acid-based preparations, antineoplastic agents, haloperidol preparations, bromperidol preparations, lithium preparations, and lymphovascular myomatosis therapeutic agents. Be done.
上記薬剤の中でも、免疫抑制剤、リンパ脈管筋腫症治療剤からなる群から選ばれる少なくとも１種に対して、本発明のモニタリング方法が好適に適用できる。また、シロリムス、エベロリムス、タクロリムス、シクロスポリンの少なくとも１種を含む血液検体に対しても、本発明のモニタリング方法は好適に適用できる。 Among the above agents, the monitoring method of the present invention can be suitably applied to at least one selected from the group consisting of immunosuppressive agents and therapeutic agents for lymphangioleiomyomatosis. The monitoring method of the present invention can also be suitably applied to a blood sample containing at least one of sirolimus, everolimus, tacrolimus, and cyclosporine.
また除タンパク処理された血液検体をコンディショニングされた固相抽出カラムへ添加し、該血液検体が固相抽出カラムを通過後、固相抽出カラムを洗浄し、その後固相抽出カラムから吸着物を溶出させることを含む血液検体の前処理方法により、除タンパクされた血液検体に含まれる塩や夾雑物を効率的に除去することができる。
上記前処理方法において、それぞれの操作は上述のとおりである。
Further, the deproteinized blood sample is added to the conditioned solid-phase extraction column, the blood sample passes through the solid-phase extraction column, the solid-phase extraction column is washed, and then the adsorbate is eluted from the solid-phase extraction column. By the blood sample pretreatment method including elution, salts and impurities contained in the deproteinized blood sample can be efficiently removed.
In the above pretreatment method, each operation is as described above.
上記前処理方法は、固相抽出カラム、遠沈管、コンディショニング液、洗浄液、および溶出液を含む前処理キットを用いることで、効率的に行うことができる。 The pretreatment method can be efficiently performed by using a pretreatment kit containing a solid-phase extraction column, a centrifuge tube, a conditioning liquid, a cleaning liquid, and an eluate.
固相抽出カラムは上述の充填剤を含むカラムであり、シリンジバレル型、ルアーバレル型、ウェルプレート型等の形状がある。キットに含まれる固相抽出カラムの数は１以上であればよい。 The solid-phase extraction column is a column containing the above-mentioned filler, and has shapes such as a syringe barrel type, a luer barrel type, and a well plate type. The number of solid-phase extraction columns included in the kit may be one or more.
遠沈管は、上記固相抽出カラムと組み合わせて使用され、除タンパク処理された血液検体がコンディショニングされた固相抽出カラムを通過し流出した液、固相抽出カラムを洗浄した際の流出液、固相抽出カラムから吸着物を溶出させた時の溶出液を受容する役割を果たす。血液検体の充填剤の通過を上述の遠心分離法により行った場合、遠沈管は遠心分離管の役割も果たすことが好ましい。 The centrifuge tube is used in combination with the above-mentioned solid-phase extraction column, and the liquid that the deproteinized blood sample passes through the conditioned solid-phase extraction column and flows out, the eluate when the solid-phase extraction column is washed, and the solid. It plays a role of receiving the eluate when the adsorbate is eluted from the phase extraction column. When the filler of the blood sample is passed by the above-mentioned centrifugation method, the centrifuge tube preferably also serves as a centrifuge tube.
遠沈管は上記前処理方法の各操作を通じて、同じものを用いてもよいが、各操作からのコンタミネーションを避ける観点から、操作毎に遠沈管を交換することが好ましい。したがって、遠沈管の数は固相抽出カラム１本に対して、各操作の数だけあることが好ましく、固相抽出カラム１本に対して、２本以上あればよい。コストの観点から固相抽出カラム１本に対して遠沈管は２本がより好ましい。 The same centrifuge tube may be used throughout each operation of the pretreatment method, but it is preferable to replace the centrifuge tube for each operation from the viewpoint of avoiding contamination from each operation. Therefore, the number of centrifuge tubes is preferably as many as the number of each operation for one solid-phase extraction column, and may be two or more for one solid-phase extraction column. From the viewpoint of cost, two centrifuge tubes are more preferable for one solid-phase extraction column.
遠沈管の容量は洗浄液、溶出液の量にもよるが、汎用の卓上型遠心機を用いて遠心できる大きさが好ましく、通常、約１．５ｍＬから約２ｍＬである。遠沈管が複数ある場合は、各遠沈管の容量は同じでも互いに異なっていてもよいが、取扱の観点から同じであることが好ましい。 The capacity of the centrifuge tube depends on the amount of the washing liquid and the eluate, but it is preferably a size that can be centrifuged using a general-purpose desktop centrifuge, and is usually about 1.5 mL to about 2 mL. When there are a plurality of centrifuge tubes, the capacities of the centrifuge tubes may be the same or different from each other, but they are preferably the same from the viewpoint of handling.
上記固相抽出カラムおよび遠沈管の容器は材質、形状、容量等が互いに異なっていてもよいし、同じでもよい。材質については取り扱いが簡便で耐薬品性および加工性に優れる材質が好ましい。また視認性に優れる材質が好ましい。これら材質の例としては、ポリプロピレンが挙げられる。また特殊コーティングが施され、ペプチド等の吸着が抑制された、島津ジエルシー社製ＴＯＲＡＳＴ−Ｈ Ｂｉｏ Ｖｉａｌ（ＴＯＲＡＳＴ−Ｈは登録商標）なども好適に使用される。
また蓋をすることで、そのままＨＰＬＣに導入できる容器形状、例えば島津ジエルシー社製ＴＯＲＡＳＴ Ｖｉａｌ（ＴＯＲＡＳＴは登録商標）も好適に使用できる。
The material, shape, capacity, etc. of the solid-phase extraction column and the container of the centrifuge tube may be different from each other, or may be the same. As for the material, a material that is easy to handle and has excellent chemical resistance and workability is preferable. Further, a material having excellent visibility is preferable. Examples of these materials include polypropylene. Further, TORAST-H Bio Vial (TORAST-H is a registered trademark) manufactured by Shimadzu GLC Co., Ltd., which is coated with a special coating and suppresses adsorption of peptides and the like, is also preferably used.
Further, a container shape that can be directly introduced into HPLC by covering it with a lid, for example, a TORAST Vial manufactured by Shimadzu GLC Co., Ltd. (TORAST is a registered trademark) can also be preferably used.
形状、容量等は例えば、取り扱いの観点からすべて同じであることが好ましいが、色彩、符号、番号、バーコード等により各容器を識別できるようしておくことが検体や各混合物の添加漏れ等のヒューマンエラーを抑制する観点から好ましい。 For example, it is preferable that the shapes, volumes, etc. are all the same from the viewpoint of handling, but it is necessary to be able to identify each container by color, code, number, bar code, etc. It is preferable from the viewpoint of suppressing human error.
コンディショニング液は固相抽出カラムの充填剤を濡らし、充填剤の官能基を活性化させる目的で使用される液であり、例えば、メタノールやアセトニトリル等の極性有機溶媒が挙げられる。 The conditioning liquid is a liquid used for the purpose of wetting the packing material of the solid-phase extraction column and activating the functional groups of the packing material, and examples thereof include polar organic solvents such as methanol and acetonitrile.
洗浄液は除タンパクされた血液検体が固相抽出カラムを通過後、余分な吸着物を除去する目的で使用される液であり、ＬＣ−ＭＳ／ＭＳの分析で移動相として使用される液体と同じ成分であることが好ましい。洗浄液は使用する固相カラムの性質により適宜選定される。上述したように逆相カラムを用いる場合、洗浄液は水の含有量が８０質量％から１００質量％である。 The washing solution is a solution used for the purpose of removing excess adsorbent after the deproteinized blood sample has passed through the solid-phase extraction column, and is the same as the liquid used as the mobile phase in LC-MS / MS analysis. It is preferably an ingredient. The cleaning solution is appropriately selected according to the properties of the solid phase column used. When a reverse phase column is used as described above, the cleaning liquid has a water content of 80% by mass to 100% by mass.
溶出用液は固相抽出カラムに吸着した吸着物を溶出させる目的で使用される液であり、モニタリングを目的とする血中薬物を溶出させる有機溶媒である。例えばアセトニトリル、メタノールである。 The elution liquid is a liquid used for the purpose of eluting the adsorbent adsorbed on the solid-phase extraction column, and is an organic solvent for eluting the blood drug for the purpose of monitoring. For example, acetonitrile and methanol.
上述の前処理キットを用いていることで、本発明の前処理方法を効率的に行うことができる。その結果、本発明のモニタリング方法を効率的に行うことができる。 By using the above-mentioned pretreatment kit, the pretreatment method of the present invention can be efficiently performed. As a result, the monitoring method of the present invention can be efficiently performed.
次に実施例を挙げて本発明を詳細に説明するが、本発明の範囲はこれらによって限定されない。 Next, the present invention will be described in detail with reference to examples, but the scope of the present invention is not limited thereto.
［除タンパク操作］
薬物としてシロリムス、エベロリムス、タクロリムス、およびシクロスポリンを含む全血５０μＬに、シロリムス、エベロリムス、タクロリムス、シクロスポリンを安定同位体標識した内部標準試薬２５μＬを添加し、硫酸亜鉛、アセトニトリル、メタノール等の有機溶媒を含む抽出液（製品名：ＤＯＳＩＭＭＵＮＥ 抽出液、ＤＯＳＩＭＭＵＮＥは登録商標））３５０μＬを加え、６０秒間、撹拌した。撹拌後、７分間、遠心分離し得られた上澄み液をＣ１８ ＦＦカラムを通過させ、通過液を回収し、除タンパクされた血液検体を得た。
[Protein removal operation]
To 50 μL of whole blood containing sirolimus, everolimus, tacrolimus, and cyclosporine as drugs, 25 μL of an internal standard reagent labeled with stable isotopes of sirolimus, everolimus, tacrolimus, and cyclosporine is added, and an organic solvent such as zinc sulfate, acetonitrile, and methanol is contained. 350 μL of an extract (product name: DOSIMMUNE extract, DOSIMMUNE is a registered trademark) was added, and the mixture was stirred for 60 seconds. After stirring, the supernatant obtained by centrifugation for 7 minutes was passed through a C18 FF column, and the passing solution was collected to obtain a deproteinized blood sample.
［固相抽出カラムのコンディショニング］
固相抽出カラムＣ１８にコンディショニング液としてメタノールを１００μＬ添加し、遠心分離法により充填剤に１００μＬのＤＯＳＩＭＭＵＮＥ 移動相A（商品名、ＤＯＳＩＭＭＵＮＥは登録商標）を通過させてコンディショニングを行った。
[Conditioning of solid-phase extraction column]
100 μL of methanol was added to the solid-phase extraction column C18 as a conditioning liquid, and 100 μL of DOSIMMUNE mobile phase A (trade name, DOSIMMUNE is a registered trademark) was passed through the filler by a centrifugation method for conditioning.
［除タンパクされた血液検体の前処理］
上記除タンパクされた血液検体を上記コンディショニングされた固相抽出カラムに添加し、血液検体中の薬物を充填剤に吸着させた。充填剤を通過した液は遠沈管で受容し、廃液として廃棄した。ＤＯＳＩＭＭＵＮＥ 移動相A（商品名、ＤＯＳＩＭＭＵＮＥは登録商標）を洗浄液として１００μＬを固相抽出カラムに添加し、遠心分離法で通過させた。通過した液は遠沈管で受容し、廃液とした。次に溶出用液としてアセトニトリル２００μＬを固相抽出カラムに添加し、再び遠心分離法で溶出用液を通過させ、通過した液を新しい遠沈管で受容した。受容した溶出液をＬＳ−ＭＳ／ＭＳでグラジエント溶離により分析した。分析条件は以下のとおりである。
[Pretreatment of deproteinized blood samples]
The deproteinized blood sample was added to the conditioned solid-phase extraction column, and the drug in the blood sample was adsorbed on the filler. The liquid that passed through the filler was received by the centrifuge tube and discarded as waste liquid. 100 μL of DOSIMMUNE mobile phase A (trade name, DOSIMMUNE is a registered trademark) was added to the solid-phase extraction column as a washing solution, and the mixture was passed through by centrifugation. The passed liquid was received by the centrifuge tube and used as waste liquid. Next, 200 μL of acetonitrile was added to the solid-phase extraction column as an elution solution, and the elution solution was passed again by a centrifugation method, and the passed solution was received by a new centrifuge tube. The received eluate was analyzed by gradient elution with LS-MS / MS. The analysis conditions are as follows.
使用機器：以下の機器を使用した。
ＨＰＬＣ:株式会社島津製作所製 ＬＣ−３０ＡＤ×２、ＳＩＬ−３０ＡＣ、ＣＴＯ−２０ＡＣ、ＣＢＭ−２０
ＬＣ−ＭＳ／ＭＳ:株式会社島津製作所 ＬＣＭＳ−８０５０
Equipment used: The following equipment was used.
HPLC: LC-30AD x 2, SIL-30AC, CTO-20AC, CBM-20 manufactured by Shimadzu Corporation
LC-MS / MS: Shimadzu Corporation LCMS-8050
分析条件：以下の分析条件で行った。
ＨＰＬＣ条件
移動相 ＤＯＳＩＭＭＵＮＥ（登録商標）キット
分析カラム ＤＯＳＩＭＭＵＮＥ（登録商標）キット
カラムオーブン ６５℃
分析モード グラジエントモード
注入量 ５μＬ
ポンプ流量 ０．４５ ｍｌ／ｍｉｎ
タイムプログラム：以下のとおり移動相Ｂの濃度を変化させた
スタート時を７５%とし、１０％／分の割合で移動相Ｂの濃度を増加させ、２．５分で１００％とした。３．５分まで１００％を維持した。その後１分間で７５％まで移動相Ｂの濃度を下げ、４．５分まで７５％を維持した。
Analytical conditions: The following analytical conditions were used.
HPLC Condition Mobile Phase DOSIMMUNE® Kit Analytical Column DOSIMMUNE® Kit Column Oven 65 ° C.
Analysis mode Gradient mode Injection volume 5 μL
Pump flow rate 0.45 ml / min
Time program: The concentration of mobile phase B was changed as follows. The starting time was 75%, the concentration of mobile phase B was increased at a rate of 10% / min, and the concentration was set to 100% in 2.5 minutes. It was maintained at 100% for up to 3.5 minutes. After that, the concentration of mobile phase B was lowered to 75% in 1 minute, and maintained at 75% until 4.5 minutes.
ＭＳ条件：以下の条件で行った。
イオン化法：エレクトロスプレー（ＥＳＩ） ポジティブ
ＭＳ／ＭＳモード：
ＭＲＭ（Ｍｕｌｔｉｐｌｅ Ｒｅａｃｔｉｏｎ Ｍｏｎｉｔｏｒｉｎｇ）
インターフェースパラメーターおよびＭＲＭパラメーターは表１のとおり。
MS condition: The following conditions were used.
Ionization method: Electrospray (ESI) Positive MS / MS mode:
MRM (Multiple Reaction Monitoring)
Table 1 shows the interface parameters and MRM parameters.
上記分析の結果、除タンパクされた血液検体、血液検体中の薬物を充填剤に吸着し洗浄後、および固相抽出カラムから溶出させた後、それぞれのシロリムス、エベロリムス、タクロリムス、およびシクロスポリンの量を定量した。
表２は各成分のＮ＝５での回収率の平均値である。エベロリムス、シロリムス、タクロリムス、シクロスポリンＡを添加した全血を上記手順に従い処理し、溶出時の回収率を計算した。回収率はいずれの成分も９２%以上であり、標準偏差を平均値で除した値、ＣＶは２．０%以下と良好な再現性を示した。なお回収率は２回連続で溶出し、１回目の溶出でどれだけ溶出できているか各成分の面積値から計算した。
As a result of the above analysis, the deproteinized blood sample, the drug in the blood sample was adsorbed on the filler and washed, and after elution from the solid-phase extraction column, the amounts of sirolimus, everolimus, tacrolimus, and cyclosporine were determined. Quantified.
Table 2 shows the average recovery rate of each component at N = 5. Whole blood supplemented with everolimus, sirolimus, tacrolimus, and cyclosporin A was treated according to the above procedure, and the recovery rate at the time of elution was calculated. The recovery rate was 92% or more for all the components, the standard deviation was divided by the average value, and the CV was 2.0% or less, showing good reproducibility. The recovery rate was calculated from the area value of each component to determine how much elution was achieved by elution twice in a row and the first elution.
表３はエベロリムス、シロリムス、タクロリムス、シクロスポリンAの安定同位体を添加した全血を上記手順に従い処理し、上記と同様に溶出時の回収率を計算した。回収率は各安定同位体のＮ＝５での回収率の平均値である。いずれの成分も回収率９６％以上、ＣＶ=０．６％以下の良好な再現性を示した。 In Table 3, whole blood supplemented with stable isotopes of everolimus, sirolimus, tacrolimus, and cyclosporin A was treated according to the above procedure, and the recovery rate at elution was calculated in the same manner as above. The recovery rate is the average value of the recovery rates of each stable isotope at N = 5. All of the components showed good reproducibility with a recovery rate of 96% or more and CV = 0.6% or less.
上記の結果から本発明の前処理による血液検体中の薬物の損失はほとんどないことがわかる。また上記前処理は本発明の前処理キットを用いることで、簡便に行うことができる。したがって、本発明の薬物血中濃度のモニタリング方法は、薬物体内動態の把握、医薬品の適正量の投与、多剤併用の可否、中毒・副作用の早期発見等、を目的として行われるＴＤＭに好適に適用することができる。 From the above results, it can be seen that there is almost no loss of the drug in the blood sample due to the pretreatment of the present invention. Further, the above pretreatment can be easily performed by using the pretreatment kit of the present invention. Therefore, the method for monitoring the blood concentration of a drug of the present invention is suitable for TDM performed for the purpose of grasping the pharmacokinetics of a drug, administering an appropriate amount of a drug, whether or not a multidrug can be used in combination, and early detection of poisoning / side effects. Can be applied.
［態様］
上述した例示的な実施形態は、以下の態様の具体例であることが当業者により理解される。
[Aspect]
It will be understood by those skilled in the art that the above-described exemplary embodiments are specific examples of the following embodiments.
［１］除タンパク処理された血液検体をコンディショニングされた固相抽出カラム へ添加し、該血液検体が固相抽出カラムを通過後、固相抽出カラムを洗浄し、その後固相抽出カラムから吸着物を溶出用液にて溶出させ、得られた溶出液をＬＣ−ＭＳ／ＭＳ分析することを含む、薬物血中濃度のモニタリング方法。 [1] The deproteinized blood sample is added to the conditioned solid-phase extraction column, the blood sample passes through the solid-phase extraction column, the solid-phase extraction column is washed, and then the adsorbent is removed from the solid-phase extraction column. A method for monitoring the blood concentration of a drug, which comprises eluting with an elution solution and LC-MS / MS analysis of the obtained eluate.
上記発明［１］によれば、血液の前処理と分離分析方法が一体となった簡便な薬物血中濃度のモニタリング方法を提供することができる。 According to the above invention [1], it is possible to provide a simple method for monitoring a drug blood concentration in which a blood pretreatment and a separation analysis method are integrated.
［２］前記洗浄に用いる洗浄液が水または、水、メタノールおよびギ酸アンモニウムからなる群から選ばれる少なくとも１種を含む混合液である、前記［１］に記載のモニタリング方法。
［３］前記洗浄液が水である、前記［２］に記載のモニタリング方法。
［４］前記溶出に用いる溶出用液がメタノールまたはアセトニトリルの少なくとも１種である、前記［１］から［３］のいずれか１に記載のモニタリング方法。
［５］前記溶出用液がアセトニトリルである、前記［４］に記載のモニタリング方法。
[2] The monitoring method according to the above [1], wherein the cleaning liquid used for the cleaning is water or a mixed liquid containing at least one selected from the group consisting of water, methanol and ammonium formate.
[3] The monitoring method according to the above [2], wherein the cleaning liquid is water.
[4] The monitoring method according to any one of [1] to [3] above, wherein the elution liquid used for the elution is at least one of methanol and acetonitrile.
[5] The monitoring method according to the above [4], wherein the elution liquid is acetonitrile.
上記発明［２］から［５］によれば、より精度の高い薬物血中濃度のモニタリング方法が提供される。 According to the above inventions [2] to [5], a more accurate method for monitoring a drug blood concentration is provided.
［６］前記血液検体が特定薬剤治療管理料の対象薬物を含んでいる前記［１］に記載のモニタリング方法。
［７］前記血液検体が、ジギタリス製剤、テオフィリン製剤、不整脈用剤、抗てんかん剤、アミノ配糖体抗生物質、グリコペプチド系抗生物質、トリアゾール系抗真菌剤、免疫抑制剤、サリチル酸系製剤、抗悪性腫瘍剤、ハロペリドール製剤、ブロムペリドール製剤、リチウム製剤、リンパ脈管筋腫症治療剤からなる群から選ばれる少なくとも１種を含んでいる、前記［１］に記載のモニタリング方法。
［８］前記特定薬剤治療管理料の対象薬物が、免疫抑制剤、リンパ脈管筋腫症治療剤からなる群から選ばれる少なくとも１種である、前記［６］に記載のモニタリング方法。
［９］前記特定薬剤治療管理料の対象薬物が、シロリムス、エベロリムス、タクロリムス、シクロスポリンの少なくとも１種である、前記［６］に記載のモニタリング方法。
[6] The monitoring method according to the above [1], wherein the blood sample contains a target drug for a specific drug treatment management fee.
[7] The blood sample is a digitalis preparation, a theophylline preparation, an arrhythmia preparation, an antiepileptic agent, an aminoglycosyl antibiotic, a glycopeptide antibiotic, a triazole antifungal agent, an immunosuppressant, a salicylic acid preparation, and an anti. The monitoring method according to the above [1], which comprises at least one selected from the group consisting of a malignant tumor agent, a haloperidol preparation, a bromperidol preparation, a lithium preparation, and a therapeutic agent for lymphovascular myomatosis.
[8] The monitoring method according to the above [6], wherein the target drug of the specific drug treatment management fee is at least one selected from the group consisting of an immunosuppressant and a lymphangioleiomyomatosis therapeutic agent.
[9] The monitoring method according to the above [6], wherein the target drug of the specific drug treatment management fee is at least one of sirolimus, everolimus, tacrolimus, and cyclosporine.
上記発明［６］から［９］によれば、薬物体内動態の把握、医薬品の適正量の投与、多剤併用の可否、中毒・副作用の早期発見等、を目的として行われるＴＤＭに好適に適用できる。 According to the above inventions [6] to [9], it is suitably applied to TDM performed for the purpose of grasping the pharmacokinetics of a drug, administering an appropriate amount of a drug, whether or not a multidrug can be used in combination, and early detection of poisoning / side effects. can.
［１０］前記ＬＣ−ＭＳ／ＭＳ分析がグラジエント溶離で行われる前記［１］から［９］のいずれか１に記載のモニタリング方法。 [10] The monitoring method according to any one of [1] to [9], wherein the LC-MS / MS analysis is performed by gradient elution.
上記発明［１０］によれば、グラジエント溶離で行われるＬＣ−ＭＳ／ＭＳ分析が迅速に行われる。 According to the above invention [10], LC-MS / MS analysis performed by gradient elution is rapidly performed.
［１１］除タンパク処理された血液検体をコンディショニングされた固相抽出カラムへ添加し、該血液検体が固相抽出カラムを通過後、固相抽出カラムを洗浄し、その後固相抽出カラムから吸着物を溶出させることを含む血液検体の前処理方法。 [11] The deproteinized blood sample is added to the conditioned solid-phase extraction column, the blood sample passes through the solid-phase extraction column, the solid-phase extraction column is washed, and then the adsorbent is removed from the solid-phase extraction column. A method for pretreating a blood sample, which comprises eluting.
上記発明［１１］によれば、除タンパクされた血液検体に含まれる塩や夾雑物を効率的に除去することができ、上記［１］から［１０］の薬物血中濃度のモニタリングを精度よく行うことができる。 According to the above invention [11], salts and impurities contained in the deproteinized blood sample can be efficiently removed, and the drug blood concentration of the above [1] to [10] can be accurately monitored. It can be carried out.
［１２］前記洗浄に用いる洗浄液が水または、水、メタノールおよびギ酸アンモニウムからなる群から選ばれる少なくとも１種を含む混合液である、前記［１１］に記載の前処理方法。
［１３］前記洗浄液が水である、前記［１２］に記載の前処理方法。
［１４］前記溶出に用いる溶出液がメタノールまたはアセトニトリルの少なくとも１種である、前記［１１］から［１３］のいずれか１に記載の前処理方法。
［１５］前記溶出液がアセトニトリルである、前記［１４］に記載の前処理方法。
[12] The pretreatment method according to the above [11], wherein the cleaning liquid used for the cleaning is water or a mixed liquid containing at least one selected from the group consisting of water, methanol and ammonium formate.
[13] The pretreatment method according to the above [12], wherein the cleaning liquid is water.
[14] The pretreatment method according to any one of [11] to [13] above, wherein the eluate used for the elution is at least one of methanol and acetonitrile.
[15] The pretreatment method according to the above [14], wherein the eluate is acetonitrile.
上記発明［１２］から［１５］によれば、除タンパクされた血液検体に含まれる塩や夾雑物を効率的に除去することができる。 According to the above inventions [12] to [15], salts and impurities contained in the deproteinized blood sample can be efficiently removed.
［１６］固相抽出カラム、遠沈管 、コンディショニング液、洗浄液、および溶出液を含む前処理キット。 [16] A pretreatment kit containing a solid-phase extraction column, a centrifuge tube, a conditioning solution, a cleaning solution, and an eluate.
上記発明の［１６］の前処理キットは上記発明［１１］から［１５］の前処理工程を効率的に行うことができる。 The pretreatment kit of the above invention [16] can efficiently perform the pretreatment steps of the above inventions [11] to [15].
［１７］前記遠沈管の容量が１．５ｍｌから２ｍｌである前記[１６]に記載の前処理キット。
［１８］前記前記遠沈管の数が２である、前記［１７］に記載の前処理キット。
[17] The pretreatment kit according to the above [16], wherein the volume of the centrifuge tube is 1.5 ml to 2 ml.
[18] The pretreatment kit according to [17], wherein the number of the centrifuge tubes is 2.
上記発明［１７］から［１８］によれば、上記発明［１１］から［１５］の前処理工程を効率的に行うことができる。
According to the inventions [17] to [18], the pretreatment steps of the inventions [11] to [15] can be efficiently performed.
Claims (18)
The pretreatment kit according to claim 17, wherein the number of the centrifuge tubes is 2.
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| CN115219611A (en) * | 2022-03-10 | 2022-10-21 | 上海义准生物有限公司 | Kit for determining 7 antifungal drugs in serum by ultra-high performance liquid chromatography-tandem mass spectrometry and application thereof |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2008170428A (en) * | 2006-12-12 | 2008-07-24 | Keio Gijuku | Hplc analysis for sugar and sugar-alcohol |
| JP2010002416A (en) * | 2008-05-22 | 2010-01-07 | Ono Pharmaceut Co Ltd | Biomarker for prostaglandin d2-related disease treatment and measurement method of the same |
| WO2010018810A1 (en) * | 2008-08-12 | 2010-02-18 | 和光純薬工業株式会社 | Polymer for filler for preprocessing column |
| JP2011095069A (en) * | 2009-10-29 | 2011-05-12 | Hitachi High-Technologies Corp | Clinical examination system, clinical examination method, and equipment for pretreatment |
| JP2012002593A (en) * | 2010-06-15 | 2012-01-05 | Hitachi High-Technologies Corp | Method and device for pretreatment of biological sample |
| JP2013541009A (en) * | 2010-10-13 | 2013-11-07 | スフェア メディカル リミテッド | Sample detection method |
| JP2019522658A (en) * | 2016-06-22 | 2019-08-15 | ノバルティス アーゲー | Wnt inhibitor for use in the treatment of fibrosis |
-
2020
- 2020-02-12 JP JP2020021117A patent/JP2021128005A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2008170428A (en) * | 2006-12-12 | 2008-07-24 | Keio Gijuku | Hplc analysis for sugar and sugar-alcohol |
| JP2010002416A (en) * | 2008-05-22 | 2010-01-07 | Ono Pharmaceut Co Ltd | Biomarker for prostaglandin d2-related disease treatment and measurement method of the same |
| WO2010018810A1 (en) * | 2008-08-12 | 2010-02-18 | 和光純薬工業株式会社 | Polymer for filler for preprocessing column |
| JP2011095069A (en) * | 2009-10-29 | 2011-05-12 | Hitachi High-Technologies Corp | Clinical examination system, clinical examination method, and equipment for pretreatment |
| JP2012002593A (en) * | 2010-06-15 | 2012-01-05 | Hitachi High-Technologies Corp | Method and device for pretreatment of biological sample |
| JP2013541009A (en) * | 2010-10-13 | 2013-11-07 | スフェア メディカル リミテッド | Sample detection method |
| JP2019522658A (en) * | 2016-06-22 | 2019-08-15 | ノバルティス アーゲー | Wnt inhibitor for use in the treatment of fibrosis |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115219611A (en) * | 2022-03-10 | 2022-10-21 | 上海义准生物有限公司 | Kit for determining 7 antifungal drugs in serum by ultra-high performance liquid chromatography-tandem mass spectrometry and application thereof |
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