JP5858515B2 - Method for measuring partition coefficient using quantitative analysis by NMR - Google Patents
Method for measuring partition coefficient using quantitative analysis by NMR Download PDFInfo
- Publication number
- JP5858515B2 JP5858515B2 JP2011041393A JP2011041393A JP5858515B2 JP 5858515 B2 JP5858515 B2 JP 5858515B2 JP 2011041393 A JP2011041393 A JP 2011041393A JP 2011041393 A JP2011041393 A JP 2011041393A JP 5858515 B2 JP5858515 B2 JP 5858515B2
- Authority
- JP
- Japan
- Prior art keywords
- compound
- sample solution
- aqueous phase
- organic phase
- nmr
- 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 - Fee Related
Links
Images
Landscapes
- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
Description
本発明は、ＮＭＲによる定量分析工程を含む、分配係数の測定方法に関する。 The present invention relates to a method for measuring a partition coefficient including a quantitative analysis step by NMR.
分配係数は分配比、分配率などともよばれ、化合物の疎水性を表す数値である。分配係数は化合物が水と有機溶媒の二相系に溶解したときの平衡溶解度比であり、下記式（１） The partition coefficient is also called a partition ratio, a partition rate, etc., and is a numerical value representing the hydrophobicity of a compound. The partition coefficient is an equilibrium solubility ratio when the compound is dissolved in a two-phase system of water and an organic solvent.
[式中、Ｐは分配係数、Ｃｏは有機相中の化合物の濃度、Ｃｗは水相中の化合物の濃度を表す]
で表される。一般的には、Ｐの常用対数である下記式（２）
[Wherein P is the partition coefficient, Co is the concentration of the compound in the organic phase, and Cw is the concentration of the compound in the aqueous phase]
It is represented by Generally, the following formula (2) which is a common logarithm of P
[式中、Ｐ、Ｃｏ、Ｃｗは前述と同義]
が分配係数として広く用いられている。分配係数は化合物の脂溶性を表すため、特に医薬品、農薬及び環境物質の体内吸収や残留性の指標となっている。例えば、化審法では、化合物の物性の指標として１−オクタノールと水の二相系での分配係数が採用されている。また、分配係数の測定法も日本工業規格（非特許文献１）やＯＥＣＤ Ｔｅｓｔ Ｇｕｉｄｅｌｉｎｅ（非特許文献２）によって標準化されている。
[Wherein P, Co, and Cw are as defined above]
Is widely used as a distribution coefficient. Since the partition coefficient represents the fat solubility of the compound, it is an index of in vivo absorption and persistence of pharmaceuticals, agricultural chemicals and environmental substances. For example, in the Chemical Substances Control Law, a distribution coefficient in a two-phase system of 1-octanol and water is adopted as an index of physical properties of a compound. The distribution coefficient measurement method is also standardized by Japanese Industrial Standard (Non-Patent Document 1) and OECD Test Guideline (Non-Patent Document 2).
ところで、今日用いられている分配係数の測定法（以下、従来法）では、有機相や水相中の化合物濃度を高速液体クロマトグラフィー（ＨＰＬＣ）や紫外可視吸光光度計で定量するため、検量線の作成が不可欠となる。検量線の作成には多量の化合物が必要であるが、化合物が希少である場合や環境への負荷を考慮した場合、化合物の使用量は少ないことが望ましい。また、検量線の作成は作業負荷が大きいため、種々の化合物の分配係数を測定する場合は特に問題となる。さらに従来法は、UVで化合物を定量する測定機器を用いる場合、ＨＰＬＣや紫外可視吸光光度計で化合物を定量するため、紫外吸収が弱いか、もしくは全くない化合物には使用できないという欠点がある。 By the way, in the distribution coefficient measurement method used today (hereinafter referred to as the conventional method), a calibration curve is used to quantify the concentration of a compound in an organic phase or an aqueous phase using high performance liquid chromatography (HPLC) or an ultraviolet-visible spectrophotometer. The creation of is essential. Although a large amount of compound is required to create a calibration curve, it is desirable that the amount of compound used be small when the compound is scarce or when considering the burden on the environment. Moreover, since the creation of a calibration curve has a large work load, it is particularly problematic when measuring the partition coefficients of various compounds. Further, the conventional method has a drawback that when a measuring instrument for quantifying a compound by UV is used, the compound is quantified by HPLC or an ultraviolet-visible spectrophotometer, so that it cannot be used for a compound having weak or no ultraviolet absorption.
また、化合物の分配係数を実測するのではなく、コンピューターを用いて分配係数を計算する方法も報告されているが、計算値は実測値と乖離することがあるため、信頼性の面から実測することが好ましい。 In addition, a method for calculating the distribution coefficient using a computer instead of actually measuring the distribution coefficient of the compound has been reported, but the calculated value may deviate from the actual measurement value. It is preferable.
ところで、近年、ＮＭＲを用いた化合物の定量分析法が報告されている（非特許文献３）。ＮＭＲを用いた定量分析法のひとつである内部標準法では、化合物と内標準物質の両方を含有する溶液をＮＭＲで測定し、ＮＭＲスペクトル上の化合物と内標準物質のそれぞれのシグナル面積を比較することで、化合物の定量を可能としている。 By the way, in recent years, a quantitative analysis method of a compound using NMR has been reported (Non-patent Document 3). In the internal standard method, which is one of the quantitative analysis methods using NMR, a solution containing both a compound and an internal standard is measured by NMR, and the signal areas of the compound and the internal standard on the NMR spectrum are compared. This makes it possible to quantify compounds.
従来の分配係数の測定法は、水相中及び有機相中の化合物の定量のために検量線の作成が必要である。しかしながら、検量線の作成には多量の化合物が必要であるため、環境への負荷が大きい。また、作業負荷が大きいことから簡便性、迅速性に欠ける点も課題である。また、ＵＶ検出器を備えたＨＰＬＣ又は紫外可視吸光光度計で測定する場合、紫外吸収が弱い若しくは全くない化合物には使用できない点も課題である。 Conventional methods for measuring partition coefficients require the creation of calibration curves for the quantification of compounds in the aqueous and organic phases. However, since a large amount of compound is required to create a calibration curve, the load on the environment is large. Another problem is the lack of simplicity and speed due to the large workload. Another problem is that when measuring with an HPLC or UV-visible absorptiometer equipped with a UV detector, it cannot be used for compounds with weak or no UV absorption.
本発明は、水相中及び有機相中の化合物の定量にＮＭＲを用いることで上記課題を解決した（以下、ＮＭＲ法）。すなわち、本発明は以下の通りである。 This invention solved the said subject by using NMR for fixed_quantity | quantitative_assay of the compound in an aqueous phase and an organic phase (henceforth, NMR method). That is, the present invention is as follows.
第１発明は、ＮＭＲを用いた定量分析工程を含む、分配係数の測定方法に関する。
第２発明は、以下の工程を含む分配係数の測定方法であって、
ｉ）化合物、重水及び重水素化有機溶媒を混合する工程、
ｉｉ）工程ｉの混合物を水相及び有機相に分離する工程、及び
ｉｉｉ）工程ｉｉの水相及び／又は有機相中の化合物の特定基のシグナル面積をNMRを用いて測定する工程、
を含む、第１発明記載の分配係数の測定方法に関する。
第３発明は、工程ｉｉｉが、水相及び有機相中の化合物の特定基のシグナル面積をNMRを用いて測定する工程である、第２発明記載の分配係数の測定方法に関する。
The first invention relates to a method for measuring a partition coefficient including a quantitative analysis step using NMR.
The second invention is a method for measuring a distribution coefficient including the following steps,
i) mixing the compound, deuterated water and deuterated organic solvent;
ii) a step of separating the mixture of step i into an aqueous phase and an organic phase, and iii) a step of measuring the signal area of a specific group of the compound in the aqueous phase and / or organic phase of step ii using NMR,
The distribution coefficient measuring method according to the first invention.
The third invention relates to the method for measuring a partition coefficient according to the second invention, wherein step iii is a step of measuring the signal area of a specific group of a compound in an aqueous phase and an organic phase using NMR.
第４発明は、以下の工程を含む分配係数の測定方法であって、
ｉ）化合物、飽和重水及び飽和重水素化有機溶媒を混合する工程、
ｉｉ）工程ｉの混合物を水相及び有機相に分離する工程、
ｉｉｉ）工程ｉｉで得た有機相に内標準物質を溶解させ有機相試料溶液とした後、NMRを用いて有機相試料溶液中の化合物及び内標準物質の特定基のシグナル面積を測定し、当該シグナル面積を用いて有機相中の化合物含量を算出する工程、
ｉｖ）工程ｉｉで得た水相に工程ｉｉｉと同一又は異なる内標準物質を溶解させ水相試料溶液とした後、NMRを用いて水相試料溶液中の化合物及び内標準物質の特定基のシグナル面積を測定し、当該シグナル面積を用いて水相中の化合物含量を算出する工程、及び
ｖ）工程ｉｉｉで算出した有機相中の化合物含量と、工程ｉｖで算出した水相中の化合物含量を用いて化合物の分配係数を算出する工程、
を含む、第３発明記載の分配係数の測定方法に関する。
A fourth invention is a method of measuring a distribution coefficient including the following steps,
i) mixing the compound, saturated deuterated water and saturated deuterated organic solvent;
ii) separating the mixture of step i into an aqueous phase and an organic phase;
iii) After dissolving the internal standard substance in the organic phase obtained in step ii to obtain an organic phase sample solution, the signal area of the specific group of the compound in the organic phase sample solution and the internal standard substance is measured using NMR. Calculating the compound content in the organic phase using the signal area,
iv) After dissolving the same or different internal standard substance as in step iii in the aqueous phase obtained in step ii to obtain an aqueous phase sample solution, the signals of the compounds in the aqueous phase sample solution and specific groups of the internal standard substance are measured using NMR. Measuring the area and calculating the compound content in the aqueous phase using the signal area; and v) the compound content in the organic phase calculated in step iii and the compound content in the aqueous phase calculated in step iv. Using to calculate the partition coefficient of the compound,
The distribution coefficient measuring method according to the third aspect of the invention.
第５発明は、以下の工程を含む分配係数の測定方法であって、
ｉ）化合物、飽和重水及び飽和重水素化有機溶媒を混合する工程、
ｉｉ）工程ｉの混合物を水相及び有機相に分離する工程、
ｉｉｉ）工程ｉｉで得た水相に重水素化酸、重水素化塩基若しくはシフト試薬を溶解させ水相試料溶液とし、工程ｉｉで得た有機相を有機相試料溶液とするか、或いは工程ｉｉで得た水相を水相試料溶液とし、工程ｉｉで得た有機相にシフト試薬を溶解させ有機相試料溶液とし、当該有機相試料溶液と水相試料溶液のうち一方を二重ＮＭＲ管の外管に採取し、他方を二重ＮＭＲ管の内管に採取した後、NMRを用いて有機相試料溶液及び水相試料溶液中の化合物の特定基のシグナル面積を測定する工程、及び
ｉｖ）工程ｉｉｉで測定した有機相試料溶液及び水相試料溶液中の化合物の特定基のシグナル面積を用いて化合物の分配係数を算出する工程、
を含む、第３発明記載の分配係数の測定方法に関する。
The fifth invention is a method of measuring a distribution coefficient including the following steps,
i) mixing the compound, saturated deuterated water and saturated deuterated organic solvent;
ii) separating the mixture of step i into an aqueous phase and an organic phase;
iii) Dissolving deuterated acid, deuterated base or shift reagent in the aqueous phase obtained in step ii to make an aqueous phase sample solution, and making the organic phase obtained in step ii an organic phase sample solution, or step ii The aqueous phase obtained in
The distribution coefficient measuring method according to the third aspect of the invention.
本発明によれば、検量線の作成が不要であること等から、従来法と比較して少量の化合物で分配係数を測定することが可能である。また、簡便、迅速に分配係数を測定することが可能である。さらに、本発明によれば、紫外吸収を示さない化合物の分配係数の測定も可能である。 According to the present invention, since it is not necessary to create a calibration curve, the partition coefficient can be measured with a small amount of compound as compared with the conventional method. Moreover, it is possible to measure the distribution coefficient simply and quickly. Furthermore, according to the present invention, it is possible to measure the partition coefficient of a compound that does not exhibit ultraviolet absorption.
本明細書中において使用される語句の定義は以下の通りである。 Definitions of terms used in the present specification are as follows.
化合物とは、酸性化合物、中性化合物、塩基性化合物、両性化合物等の任意の化合物を表す。なお、本発明においては、コンピューター（Ｐａｌｌａｓ３．０、インフォコム株式会社）を用いて計算し、酸塩基解離定数が６以下の化合物を酸性化合物、７．８以上の化合物を塩基性化合物、両方の性質を持ち合わせるものを両性化合物とし、それ以外を中性化合物と分類した。 A compound represents arbitrary compounds, such as an acidic compound, a neutral compound, a basic compound, and an amphoteric compound. In the present invention, a compound having a acid-base dissociation constant of 6 or less is an acidic compound, a compound having a compound of 7.8 or more is a basic compound, and both properties are calculated using a computer (Pallas 3.0, Infocom Corporation). Those with a saponification were classified as amphoteric compounds and the others were classified as neutral compounds.
重水とは、水分子の二つの水素原子を重水素原子に置き換えた水を表し、Ｄ２Ｏと表すこともある。 The heavy water represents water in which two hydrogen atoms of a water molecule are replaced with deuterium atoms, and may be represented as D 2 O.
重水素化有機溶媒とは、有機溶媒分子に含まれる水素原子の一部または全部を重水素原子に置き換えた有機溶媒を表す。重水素化有機溶媒としては、例えば、シクロヘキサン−ｄ１２、トルエン−ｄ８、ベンゼン−ｄ６、クロロホルム−ｄ、ジクロロメタン−ｄ２又は１−オクタノール−ｄ１８が挙げられる。 The deuterated organic solvent represents an organic solvent in which part or all of the hydrogen atoms contained in the organic solvent molecule are replaced with deuterium atoms. Examples of the deuterated organic solvent include cyclohexane-d 12 , toluene-d 8 , benzene-d 6 , chloroform-d, dichloromethane-d 2 or 1-octanol-d 18 .
内標準物質とは、化合物を定量するために、水相及び／又は有機相に添加する物質である。内標準物質としては、例えば、２，６−ジクロロベンズアルデヒド、２−ヒドロキシ−３，５−ジニトロ−安息香酸、テレフタル酸ジメチルエステル、フタル酸水素カリウム、安息香酸、１，３，５−トリオキサン、テトラメチルピラジン、ジメチルスルホン、酢酸ナトリウム、トリメチルシリルプロパンスルホン酸ナトリウム−ｄ６、トリメチルシリルプロピオン酸ナトリウム−ｄ４又は１，４−ビス（トリメチルシリル）ベンゼン−ｄ４が挙げられる。 The internal standard substance is a substance added to the aqueous phase and / or the organic phase in order to quantify the compound. Examples of internal standard substances include 2,6-dichlorobenzaldehyde, 2-hydroxy-3,5-dinitro-benzoic acid, dimethyl terephthalate, potassium hydrogen phthalate, benzoic acid, 1,3,5-trioxane, tetra Examples include methylpyrazine, dimethylsulfone, sodium acetate, sodium trimethylsilylpropanesulfonate-d 6 , sodium trimethylsilylpropionate-d 4 or 1,4-bis (trimethylsilyl) benzene-d 4 .
シグナル面積は、ピーク面積又は積分値と同義である。 The signal area is synonymous with the peak area or the integrated value.
特定基とは、化合物および内標準物質のそれぞれの任意の置換基を表す。 The specific group represents an arbitrary substituent of each of the compound and the internal standard substance.
飽和重水素化有機溶媒とは、重水が飽和した重水素化有機溶媒を表す。 The saturated deuterated organic solvent represents a deuterated organic solvent saturated with heavy water.
飽和重水とは、重水素化有機溶媒が飽和した重水を表す。 Saturated heavy water represents heavy water saturated with a deuterated organic solvent.
重水素化酸とは、酸分子に含まれる水素原子の一部または全部を重水素原子に置き換えた酸を表す。重水素化酸としては、例えば、蟻酸−ｄ２、酢酸−ｄ、酢酸−ｄ４、トリフルオロ酢酸−ｄ、塩酸−ｄ（重塩酸）、硫酸−ｄ２が挙げられる。 The deuterated acid represents an acid in which part or all of the hydrogen atoms contained in the acid molecule are replaced with deuterium atoms. Examples of the deuterated acid include formic acid-d 2 , acetic acid-d, acetic acid-d 4 , trifluoroacetic acid-d, hydrochloric acid-d (deuterated hydrochloric acid), and sulfuric acid-d 2 .
重水素化塩基とは、塩基分子に含まれる水素原子の一部または全部を重水素原子に置き換えた塩基を表す。重水素化塩基としては、例えば、水酸化アンモニウム−ｄ５、ピリジン−ｄ５、水酸化ナトリウム−ｄ、水酸化カリウム−ｄが挙げられる。 A deuterated base represents a base in which part or all of the hydrogen atoms contained in the base molecule are replaced with deuterium atoms. Examples of the deuterated base include ammonium hydroxide-d 5 , pyridine-d 5 , sodium hydroxide-d, and potassium hydroxide-d.
シフト試薬とは、有機配位子に配位結合した希土（ランタニド）類系列のイオンである。例えば、トリス（２、２、６、６−テトラメチル−３、５−ヘプタンジオナト）ユウロピウム （Ｅｕ（ｄｐｍ）３）、トリス（６、６、７、７、８、８、８−ヘプタフルオロ−２、２−ジメチル−３、５−オクタンジオナト）ユウロピウム （Ｅｕ（ｆｏｄ）３）、トリス［Ｎ、Ｎ−ビス（トリメチルシリル）アミド］ユウロピウム、トリス（４、４、４−トリフルオロ−１−（２−チエニル）−１、３−ブタンジオノ）ユウロピウム、トリス（６、６、７、７、８、８、８−ヘプタフルオロ−２、２−ジメチル−３、５−オクタンジオナト）イッテルビウム （Ｙｂ（ｆｏｄ）３）、トリス（２、２、６、６−テトラメチル−３、５−ヘプタンジオナト）ジスプロシウム（ＩＩＩ） （Ｄｙ（ｄｐｍ）３）、トリス（２、２、６、６‐テトラメチル‐３、５‐ヘプタンジオナト）ガドリニウム （Ｇｄ（ｄｐｍ）３）が挙げられる。
A shift reagent is a rare earth (lanthanide) series ion coordinated to an organic ligand. For example, tris (2,2,6,6-tetramethyl-3,5-heptanedionato) europium (Eu (dpm) 3 ), tris (6,6,7,7,8,8,8-heptafluoro-2 , 2-dimethyl-3,5-octandionato) europium (Eu (fod) 3 ), tris [N, N-bis (trimethylsilyl) amido] europium, tris (4,4,4-trifluoro-1- ( 2-thienyl) -1,3-butanediono) europium, tris (6,6,7,7,8,8,8-heptafluoro-2,2-dimethyl-3,5-octandionato) ytterbium (Yb ( fod) 3), tris (2,2,6,6-tetramethyl-3,5-heptanedionato) dysprosium (III) (Dy (dpm) 3), tris (2,2,6,6-
二重ＮＭＲ試料管とは、外管（アウターチューブ）と内管（インナーチューブ）を備えたＮＭＲ管を表す。外管と内管の各々に試料溶液を採取することができる。 A double NMR sample tube represents an NMR tube having an outer tube (outer tube) and an inner tube (inner tube). A sample solution can be collected in each of the outer tube and the inner tube.
本発明の好ましい形態は以下の通りである。 Preferred embodiments of the present invention are as follows.
化合物は、中性化合物及び両性化合物が、濃度による分配係数への影響が少ないため好ましい。また、より正確な分配係数が得られるため、ＬｏｇＰが−３〜３の範囲の化合物が好ましく、−２．５〜２．５の範囲の化合物が好ましく、−２〜２の範囲の化合物が特に好ましい。 As the compounds, neutral compounds and amphoteric compounds are preferable because the concentration coefficient has little influence on the partition coefficient. In addition, since a more accurate distribution coefficient can be obtained, a compound with LogP in the range of −3 to 3 is preferable, a compound in the range of −2.5 to 2.5 is preferable, and a compound in the range of −2 to 2 is particularly preferable. preferable.
化合物の濃度は、重水素化有機溶媒１ｍLに対し、好ましくは０．００８〜３０ ｍｇであり、更に好ましくは０．０２〜１０ ｍｇであり、特に好ましくは１．８〜１０ ｍｇである。また、重水１ｍLに対し、好ましくは０．００８〜３０ｍｇであり、更に好ましくは０．０２〜１０ｍｇであり、特に好ましくは１．１〜１０ｍｇである。 The concentration of the compound is preferably 0.008 to 30 mg, more preferably 0.02 to 10 mg, and particularly preferably 1.8 to 10 mg with respect to 1 mL of deuterated organic solvent. Moreover, with respect to 1 mL of heavy water, Preferably it is 0.008-30 mg, More preferably, it is 0.02-10 mg, Most preferably, it is 1.1-10 mg.
ＮＭＲは、測定感度の点からプロトンＮＭＲ（１Ｈ−ＮＭＲ）が好ましい。 NMR is preferably proton NMR ( 1 H-NMR) from the viewpoint of measurement sensitivity.
重水素化有機溶媒は、有機溶媒分子に含まれる水素原子の全部を重水素原子に置き換えた有機溶媒が好ましい。好ましくはシクロヘキサン−ｄ１２、トルエン−ｄ８、ベンゼン−ｄ６、クロロホルム−ｄ、ジクロロメタン−ｄ２又は１−オクタノール−ｄ１８であり、更に好ましくはクロロホルム−ｄ又は１−オクタノール−ｄ１８であり、特に好ましくはクロロホルム−ｄである。 The deuterated organic solvent is preferably an organic solvent in which all of the hydrogen atoms contained in the organic solvent molecule are replaced with deuterium atoms. Preferred are cyclohexane-d 12 , toluene-d 8 , benzene-d 6 , chloroform-d, dichloromethane-d 2 or 1-octanol-d 18 , more preferably chloroform-d or 1-octanol-d 18 . Particularly preferred is chloroform-d.
内標準物質は、そのＮＭＲシグナルのうち少なくとも１つのシグナルが、化合物のＮＭＲシグナルと重ならないものであれば、任意に選ぶことができる。有機相に添加する場合、好ましくは２，６−ジクロロベンズアルデヒド又は１，４−ビス（トリメチルシリル）ベンゼン−ｄ４である。水相に添加する場合、好ましくはトリメチルシリルプロピオン酸ナトリウム-ｄ４又はトリメチルシリルプロパンスルホン酸ナトリウム−ｄ６である。 The internal standard substance can be arbitrarily selected as long as at least one of the NMR signals does not overlap with the NMR signal of the compound. When added to the organic phase, 2,6-dichlorobenzaldehyde or 1,4-bis (trimethylsilyl) benzene-d 4 is preferred. When added to the aqueous phase, preferably sodium trimethylsilyl propionate sodium -d 4 or trimethylsilyl propanesulfonic acid -d 6.
重水素化酸は、好ましくは蟻酸−ｄ２、酢酸−ｄ、酢酸−ｄ４、トリフルオロ酢酸−ｄ、塩酸−ｄ（重塩酸）、硫酸−ｄ２であり、更に好ましくは酢酸−ｄ４、トリフルオロ酢酸−ｄ、塩酸−ｄ（重塩酸）であり、特に好ましくは塩酸−ｄ（重塩酸）である。 The deuterated acid is preferably formic acid-d 2 , acetic acid-d, acetic acid-d 4 , trifluoroacetic acid-d, hydrochloric acid-d (deuterated hydrochloric acid), sulfuric acid-d 2 , more preferably acetic acid-d 4. , Trifluoroacetic acid-d and hydrochloric acid-d (deuterated hydrochloric acid), particularly preferably hydrochloric acid-d (deuterated hydrochloric acid).
重水素化塩基は、好ましくは水酸化アンモニウム−ｄ５、ピリジン−ｄ５、水酸化ナトリウム−ｄ、水酸化カリウム−ｄである。 The deuterated base is preferably ammonium hydroxide-d 5 , pyridine-d 5 , sodium hydroxide-d, potassium hydroxide-d.
シフト試薬は、好ましくはトリス（２、２、６、６−テトラメチル−３、５−ヘプタンジオナト）ユウロピウム、トリス（６、６、７、７、８、８、８−ヘプタフルオロ−２、２−ジメチル−３、５−オクタンジオナト）ユウロピウムである。 The shift reagent is preferably tris (2,2,6,6-tetramethyl-3,5-heptanedionato) europium, tris (6,6,7,7,8,8,8-heptafluoro-2,2- Dimethyl-3,5-octandionato) europium.
特定基は任意に選択できるが、精確なシグナル面積を得るため、そのＮＭＲシグナルが他のＮＭＲシグナルと完全に分離している置換基から選ぶことが好ましい。 The specific group can be arbitrarily selected, but in order to obtain an accurate signal area, it is preferable to select from substituents whose NMR signals are completely separated from other NMR signals.
以下に本発明の一般的な実施形態を説明するが、本実施形態によって本発明の範囲が限定されるものではない。 In the following, general embodiments of the present invention will be described, but the scope of the present invention is not limited by these embodiments.
（工程１）飽和溶媒の作成
適当量の重水素化有機溶媒と重水を激しく振り混ぜた後、室温下、２４時間以上静置する。その後、二つの層を分離し、飽和重水素化有機溶媒と飽和重水を得る。
(Step 1) Preparation of saturated solvent A suitable amount of deuterated organic solvent and heavy water are vigorously shaken and then allowed to stand at room temperature for at least 24 hours. Thereafter, the two layers are separated to obtain a saturated deuterated organic solvent and saturated heavy water.
（工程２）化合物の分配操作
化合物を精秤し、これに工程１で得られた飽和重水素化有機溶媒を一定量正確に加えて化合物を完全に溶解させる。これに工程１で得られた飽和重水を、飽和重水素化有機溶媒と同容量加え、激しく混合後、２５℃で２４時間振とうする。これを遠心分離により二つの層に分離し、有機相と水相を得る。
(Step 2) Compound distribution operation The compound is precisely weighed, and a certain amount of the saturated deuterated organic solvent obtained in
（工程３）ＮＭＲによる、有機相及び／又は水相中の化合物の定量分析と分配係数の算出
有機相及び水相中の化合物の定量分析は、以下に示す個別定量法又は同時定量法により行うことができる。
(Step 3) Quantitative analysis of compounds in organic phase and / or aqueous phase and calculation of partition coefficient by NMR Quantitative analysis of compounds in organic phase and aqueous phase is performed by the following individual quantitative method or simultaneous quantitative method. be able to.
１．個別定量法
本方法では、工程２で得られた有機相及び／又は水相に内標準物質を添加し、得られた有機相試料溶液及び水相試料溶液についてＮＭＲを用いて個別に定量分析を行った後、分配係数を算出する。
1. Individual quantification method In this method, an internal standard substance is added to the organic phase and / or aqueous phase obtained in Step 2, and the obtained organic phase sample solution and aqueous phase sample solution are individually quantitatively analyzed using NMR. After that, the distribution coefficient is calculated.
１）有機相中の化合物の定量分析
内標準物質を精秤し、これに工程２で得られた有機相を一定量正確に加えて内標準物質を完全に溶解させ、これを有機相試料溶液とする。その後、有機相試料溶液をＮＭＲで測定する。この際、公知技術（平成２１年度成果報告書 基準認証研究開発事業「１対多型校正技術の研究開発」 平成２２年３月経済産業省産業技術環境局知的基盤課 委託先（独）産業技術総合研究所）等を参考にして測定条件を選択し、化合物及び内標準物質の特定基のシグナル面積を得る。下記式（３）
1) Quantitative analysis of compounds in the organic phase Weigh accurately the internal standard substance, add a certain amount of the organic phase obtained in step 2 to this accurately to dissolve the internal standard substance completely, and add this to the organic phase sample solution And Thereafter, the organic phase sample solution is measured by NMR. In this case, publicly known technology (2009 results report standard certification research and development project "Research and development of one-to-many proofreading technology" March 2010 Ministry of Economy, Trade and Industry Industrial Technology Environment Bureau Intellectual Infrastructure Division Contracted (Germany) Industry The measurement conditions are selected with reference to the Technical Research Institute, etc., and the signal areas of the specific groups of the compound and internal standard substance are obtained. Following formula (3)
に化合物及び内標準物質の特定基のシグナル面積、特定基のプロトン数、分子量及びＮＭＲ測定に用いた質量並びに内標準物質の純度を代入することにより、有機相中の化合物の含量を算出する。 The content of the compound in the organic phase is calculated by substituting for the signal area of the specific group of the compound and internal standard substance, the number of protons of the specific group, the molecular weight, the mass used for NMR measurement, and the purity of the internal standard substance.
２）水相中の化合物の定量分析
工程２で得られた水相中の化合物の定量分析は、以下に示す実測法又は差し引き法により行うことができる。適用可能な分配係数の範囲が広いこと及び正確な分配係数を得られることから実測法が好ましい。
＜実測法＞
工程２で得られた水相と、内標準物質を用いて、１）と同様の方法により水相試料溶液を作成後、ＮＭＲで測定し、式（３）に従って水相中の化合物含量を算出する。
＜差し引き法＞
下記式（４）
2) Quantitative analysis of the compound in the aqueous phase The quantitative analysis of the compound in the aqueous phase obtained in the step 2 can be performed by the following actual measurement method or subtraction method. The actual measurement method is preferable because the range of applicable distribution coefficients is wide and an accurate distribution coefficient can be obtained.
<Measurement method>
Using the aqueous phase obtained in step 2 and the internal standard substance, prepare an aqueous phase sample solution by the same method as in 1), then measure by NMR, and calculate the compound content in the aqueous phase according to formula (3) To do.
<Subtraction method>
Following formula (4)
により水相中の化合物含量を算出することができる。ここで、分配前の化合物含量とは、工程２の分配前の化合物純度を表す。例えば、工程２と同量の化合物と、一定量の内標準物質を精量し、これに工程２と同量の飽和重水素化有機溶媒を加えて完全に溶解させ試料溶液とした後、１）と同様の方法によりＮＭＲを測定し、式（３）に従って算出する。このようにして得た分配前の化合物含量と、１）で得た有機相中の化合物含量を式（４）に代入し、水相中の化合物含量を算出する。 Can be used to calculate the compound content in the aqueous phase. Here, the compound content before distribution represents the compound purity before distribution in Step 2. For example, the same amount of the compound as in step 2 and a certain amount of internal standard substance are weighed, and the same amount of saturated deuterated organic solvent as in step 2 is added and completely dissolved to obtain a sample solution. NMR is measured by the same method as in (1) and calculated according to formula (3). The compound content before distribution thus obtained and the compound content in the organic phase obtained in 1) are substituted into the formula (4) to calculate the compound content in the aqueous phase.
３）分配係数の算出
化合物含量の比は化合物濃度の比と等しいため、式（２）のＣｏに１）で算出した有機相中の化合物含量を代入し、Ｃｗに２）で算出した水相中の化合物含量を代入することで、化合物の分配係数を算出する。
3) Calculation of partition coefficient Since the ratio of compound content is equal to the ratio of compound concentration, the compound content in the organic phase calculated in 1) is substituted for Co in formula (2), and the water phase calculated in 2) is substituted for Cw. The partition coefficient of the compound is calculated by substituting the compound content in the compound.
２．同時定量法
本方法では、工程２で得られた有機相又は水相に、重水素化酸、重水素化塩基若しくはシフト試薬を加え、得られた有機相試料溶液及び水相試料溶液の一方を二重ＮＭＲ試料管の外管に、他方を二重ＮＭＲ試料管の内管に採取し、ＮＭＲを用いて定量分析を行った後、分配係数を算出する。本測定法は内標準物質を必要とせず、また、有機相試料溶液と水相試料溶液を同時に測定できるため、簡便である。
2. Simultaneous quantification method In this method, deuterated acid, deuterated base or shift reagent is added to the organic phase or aqueous phase obtained in step 2, and one of the obtained organic phase sample solution and aqueous phase sample solution is added. A sample is collected in the outer tube of the double NMR sample tube and the other is taken in the inner tube of the double NMR sample tube. After quantitative analysis using NMR, the distribution coefficient is calculated. This measurement method is simple because it does not require an internal standard substance and can simultaneously measure an organic phase sample solution and an aqueous phase sample solution.
１）補正値の算出
同一の化合物濃度を有する有機相試料溶液と水相試料溶液であっても、一方を二重ＮＭＲ管の外管に、他方を二重ＮＭＲ管の内管に採取し、ＮＭＲ測定した場合、種々の要因により化合物の特定基のシグナル面積に差異が生じる。したがって、当該差異を補正するための補正値を算出する。
化合物を重水素化有機溶媒に溶解し有機相試料溶液とする。また、有機相試料溶液中の化合物濃度と等しくなるように化合物及び重水素化酸、重水素化塩基若しくはシフト試薬を重水に溶解し水相試料溶液とする。或いは化合物及びシフト試薬を重水素化有機溶媒に溶解し有機相試料溶液とする。また、有機相試料溶液中の化合物濃度と等しくなるように化合物を重水に溶解し水相試料溶液とする。
有機相試料溶液を二重ＮＭＲ試料管の外管に、水相試料溶液を二重ＮＭＲ試料管の内管に採取し、ＮＭＲを測定し、水相試料溶液及び有機相試料溶液中の化合物の特定基のシグナル面積を得る。溶媒量、化合物の質量及びシグナル面積を下記式（５）
1) Calculation of correction value Even if it is an organic phase sample solution and an aqueous phase sample solution having the same compound concentration, one is collected in the outer tube of the double NMR tube, and the other is collected in the inner tube of the double NMR tube. When NMR measurement is performed, a difference occurs in the signal area of a specific group of the compound due to various factors. Therefore, a correction value for correcting the difference is calculated.
The compound is dissolved in a deuterated organic solvent to obtain an organic phase sample solution. In addition, a compound and a deuterated acid, a deuterated base, or a shift reagent are dissolved in heavy water so as to be equal to the compound concentration in the organic phase sample solution to obtain an aqueous phase sample solution. Alternatively, the compound and the shift reagent are dissolved in a deuterated organic solvent to obtain an organic phase sample solution. Further, a compound is dissolved in heavy water so as to be equal to the compound concentration in the organic phase sample solution to obtain an aqueous phase sample solution.
Collect the organic phase sample solution in the outer tube of the double NMR sample tube and the aqueous phase sample solution in the inner tube of the double NMR sample tube, measure the NMR, and analyze the compounds in the aqueous phase sample solution and the organic phase sample solution. Obtain the signal area of a specific group. The amount of the solvent, the mass of the compound and the signal area are represented by the following formula (5).
に代入し、補正値を算出する。 And the correction value is calculated.
２）化合物の定量分析
工程２で得られた水相に重水素化酸、重水素化塩基若しくはシフト試薬を加えて水相試料溶液とし、工程２で得られた有機相はそのまま有機相試料溶液とする。或いは工程２で得られた水相はそのまま水相試料溶液とし、工程２で得られた有機相にシフト試薬を加えて有機相試料溶液とする。有機相試料溶液を二重ＮＭＲ試料管の外管に、水相試料溶液を二重ＮＭＲ試料管の内管に入れ、ＮＭＲを測定し、水相試料溶液及び有機相試料溶液中の化合物の特定基のシグナル面積を得る。
2) Quantitative analysis of the compound Add the deuterated acid, deuterated base or shift reagent to the aqueous phase obtained in the step 2 to obtain an aqueous phase sample solution, and the organic phase obtained in the step 2 is used as it is. And Alternatively, the aqueous phase obtained in step 2 is directly used as an aqueous phase sample solution, and a shift reagent is added to the organic phase obtained in step 2 to obtain an organic phase sample solution. Put the organic phase sample solution in the outer tube of the double NMR sample tube and the aqueous phase sample solution in the inner tube of the double NMR sample tube, measure the NMR, and identify the compound in the aqueous phase sample solution and the organic phase sample solution Get the signal area of the group.
３）分配係数の算出
化合物の特定基のシグナル面積は化合物濃度に比例する。したがって、シグナル面積の比は化合物濃度の比と等しい。すなわち、分配係数は下記式（６）
3) Calculation of partition coefficient The signal area of a specific group of a compound is proportional to the compound concentration. Therefore, the signal area ratio is equal to the compound concentration ratio. That is, the distribution coefficient is the following formula (6).
により算出できる。
ここで、２）において水相に加える重水素化酸、重水素化塩基若しくはシフト試薬が水溶液の場合、水相は当該水溶液により希釈されていることから、水相試料溶液中の化合物の特定基のシグナル面積は、水相中の化合物の特定基のシグナル面積よりも希釈率分だけ小さい値となる。また、１）で述べたように、有機相試料溶液中の化合物の特定基のシグナル面積は補正する必要がある。これらを考慮し、分配係数は下記式（７）
Can be calculated.
Here, when the deuterated acid, deuterated base or shift reagent added to the aqueous phase in 2) is an aqueous solution, since the aqueous phase is diluted with the aqueous solution, the specific group of the compound in the aqueous phase sample solution The signal area of is a value smaller by the dilution rate than the signal area of the specific group of the compound in the aqueous phase. Further, as described in 1), it is necessary to correct the signal area of the specific group of the compound in the organic phase sample solution. Considering these, the distribution coefficient is the following formula (7)
[式中、Ｐ、Ｉｏ’、Ｉｗ’は前述と同義]
により算出できる。式（７）に１）で得られた補正値、２）で得られた化合物の特定基のシグナル面積、及び希釈率を代入することで、化合物の分配係数を算出する。なお、水相に加える重水素化酸、重水素化塩基若しくはシフト試薬が水溶液ではない場合は、希釈率は考慮しなくてよい。
[Wherein P, Io ′ and Iw ′ are as defined above]
Can be calculated. The distribution coefficient of the compound is calculated by substituting the correction value obtained in 1) into the equation (7), the signal area of the specific group of the compound obtained in 2), and the dilution rate. When the deuterated acid, deuterated base or shift reagent added to the aqueous phase is not an aqueous solution, the dilution rate need not be considered.
以下に本発明を実施例及び比較例にて更に詳細に説明するが、これらによって本発明の範囲が限定されるものではない。 The present invention will be described in more detail below with reference to examples and comparative examples, but the scope of the present invention is not limited by these examples.
＜比較例１＞
従来法による、アンチピリンの分配係数測定
<Comparative Example 1>
Measurement of antipyrine partition coefficient by conventional method
１．検量線の作成
以下、クロロホルム溶液及び水溶液中のアンチピリン濃度に関する検量線を作成した。
１）クロロホルム溶液中のアンチピリン濃度に関する検量線
アンチピリン（シグマ・アルドリッチ製、１０．６７ｍｇ）にクロロホルム（関東化学製、特級）を加えて２５ｍＬとし、原液（４２６．８μｇ／ｍＬ）とした。原液２ｍＬにクロロホルムを加えて２０ｍＬとし、溶液Ａとした（４２．６８μｇ／ｍＬ）。溶液Ａ４ｍＬにクロロホルムを加えて２０ｍＬとし、溶液Ｂとした（８．５３６μｇ／ｍＬ）。溶液Ｂ４ｍＬにクロロホルムを加えて２０ｍＬとし、溶液Ｃとした（１．７０７μｇ／ｍＬ）。溶液Ａ７ｍＬにクロロホルムを加えて１０ｍＬとし、溶液Ｄとした（２９．８８μｇ／ｍＬ）。溶液Ｂ４ｍＬにクロロホルムを加えて１０ｍＬとし、溶液Ｅとした（３．４１４μｇ／ｍＬ）。溶液Ｃ４ｍＬにクロロホルムを加えて１０ｍＬとし、溶液Ｆとした（０．６８２９μｇ／ｍＬ）。
溶液ＡからＦを紫外可視吸光光度計で測定し、約２７８ｎｍにおける吸光度について、ｘ軸を濃度、ｙ軸を吸光度とした回帰式を作成し、相関係数１．０００、切片０．００１５６４、傾き０．０５１７６の下記式（８）
1. Preparation of calibration curve A calibration curve relating to the concentration of antipyrine in a chloroform solution and an aqueous solution was prepared below.
1) Calibration curve concerning antipyrine concentration in chloroform solution Chloroform (manufactured by Kanto Chemical Co., Ltd., special grade) was added to antipyrine (manufactured by Sigma-Aldrich, 10.67 mg) to make 25 mL to obtain a stock solution (426.8 μg / mL). Chloroform was added to 2 mL of the stock solution to make 20 mL, thereby preparing Solution A (42.68 μg / mL). Chloroform was added to 4 mL of solution A to make 20 mL, and solution B was obtained (8.536 μg / mL). Chloroform was added to 4 mL of Solution B to make 20 mL, and Solution C was obtained (1.707 μg / mL). Chloroform was added to 7 mL of solution A to make 10 mL, and solution D was obtained (29.88 μg / mL). Chloroform was added to 4 mL of solution B to make 10 mL, and solution E was obtained (3.414 μg / mL). Chloroform was added to 4 mL of Solution C to make 10 mL, and Solution F was obtained (0.6829 μg / mL).
Solutions A to F were measured with a UV-visible absorptiometer, and a regression equation was created with the x-axis being the concentration and the y-axis being the absorbance for the absorbance at about 278 nm, with a correlation coefficient of 1.000, an intercept of 0.001564, and a slope. The following formula (8) of 0.05176
を得た。 Got.
２）水溶液中のアンチピリン濃度に関する検量線
アンチピリン（シグマ・アルドリッチ製、２０．４４ｍｇ）に超純水（ミリＱ水）を加えて５０ｍＬとし、原液（４０８．８μｇ／ｍＬ）とした。原液２ｍＬに超純水を加えて２０ｍＬとし、溶液Ａとした（４０．８８μｇ／ｍＬ）。溶液Ａ４ｍＬに超純水を加えて２０ｍＬとし、溶液Ｂとした（８．１７６μｇ／ｍＬ）。溶液Ｂ４ｍＬに超純水を加えて２０ｍＬとし、溶液Ｃとした（１．６３５μｇ／ｍＬ）。溶液Ａ５ｍＬに超純水を加えて１０ｍＬとし、溶液Ｄとした（２０．４４μｇ／ｍＬ）。溶液Ｂ４ｍＬに超純水を加えて１０ｍＬとし、溶液Ｅとした（３．２７０μｇ／ｍＬ）。溶液Ｃ４ｍＬに超純水を加えて１０ｍＬとし、溶液Ｆとした（０．６５４１μｇ／ｍＬ）。
溶液ＡからＦを紫外可視吸光光度計で測定し、約２４２ｎｍにおける吸光度について、ｘ軸を濃度、ｙ軸を吸光度とした回帰式を作成し、相関係数１．０００、切片０．００１５２１、傾き０．０４８６０の下記式（９）
2) Calibration curve for antipyrine concentration in aqueous solution Ultrapure water (Milli-Q water) was added to antipyrine (Sigma-Aldrich, 20.44 mg) to make 50 mL, to obtain a stock solution (408.8 μg / mL). Ultrapure water was added to 2 mL of the stock solution to make 20 mL, thereby preparing Solution A (40.88 μg / mL). Ultrapure water was added to 4 mL of Solution A to make 20 mL, and Solution B was obtained (8.176 μg / mL). Ultrapure water was added to 4 mL of Solution B to make 20 mL, and Solution C was obtained (1.635 μg / mL). Ultrapure water was added to 5 mL of Solution A to make 10 mL, and Solution D (20.44 μg / mL) was obtained. Ultrapure water was added to 4 mL of Solution B to make 10 mL, and Solution E was obtained (3.270 μg / mL). Ultra pure water was added to 4 mL of Solution C to make 10 mL, and Solution F (0.6541 μg / mL) was obtained.
Solutions A to F were measured with a UV-visible absorptiometer, and a regression equation was prepared for the absorbance at about 242 nm with the x-axis being the concentration and the y-axis being the absorbance. The correlation coefficient was 1.000, the intercept was 0.001521, the slope The following formula (9) of 0.04860
を得た。 Got.
２．分配係数測定
以下、クロロホルム／水系での分配係数を従来法により測定した。
超純水（ミリＱ水、２００ｍＬ）とクロロホルム（関東化学製、特級、２００ｍＬ）を分液漏斗で振り混ぜた後、２４時間以上放置し、上層と下層を分離した。得られた上層を飽和水、下層を飽和クロロホルムとした。
アンチピリン（シグマ・アルドリッチ製、２０１．２７ｍｇ）に飽和クロロホルムを加えて２０ｍＬとし，アンチピリン溶液とした。アンチピリン溶液１２ｍＬに飽和水１２ｍＬを加えて室温で３０分間激しく振とう後、２５℃で２４時間振とうし、遠心分離（３０００ｒｐｍ、１０分）して上層（水相）と下層（有機相）を完全に分離した。得られた上層（水相）４ｍＬに超純水を加えて２０ｍＬとし、溶液Aとした。溶液A ４ｍLに超純水を加えて２０ｍLとし、水相試料溶液とした。また、得られた下層（有機相）２ｍＬにクロロホルムを加えて１０ｍＬとし、溶液Bとした。溶液B ２ｍLにクロロホルムを加えて２０ｍLとし、溶液Cとした。溶液C ２mＬにクロロホルムを加えて２０mＬとし、有機相試料溶液とした。
有機相試料溶液と水相試料溶液の吸光度を紫外可視吸光光度計で測定した（有機相試料溶液：２７８ｎｍ、水相試料溶液：２４２ｎｍ）。それぞれの試料溶液から得られた吸光度を式（８）及び式（９）に代入し、有機相試料溶液中のアンチピリン濃度（Ｃｏ）と水相試料溶液中のアンチピリン濃度（Ｃｗ）を定量した。得られたＣｏ及びＣｗを式（２）に代入し、ＬｏｇＰ＝１．３５を得た。
2. Partition coefficient measurement Hereinafter, the partition coefficient in a chloroform / water system was measured by a conventional method.
Ultrapure water (Milli-Q water, 200 mL) and chloroform (Kanto Chemical Co., Ltd., special grade, 200 mL) were shaken and mixed with a separatory funnel and allowed to stand for 24 hours or more to separate the upper and lower layers. The obtained upper layer was saturated water and the lower layer was saturated chloroform.
Saturated chloroform was added to antipyrine (Sigma Aldrich, 201.27 mg) to make 20 mL, and an antipyrine solution was obtained. Add 12 mL of saturated water to 12 mL of antipyrine solution, shake vigorously at room temperature for 30 minutes, shake at 25 ° C. for 24 hours, and centrifuge (3000 rpm, 10 minutes) to separate the upper layer (aqueous phase) and lower layer (organic phase). Completely separated. Ultrapure water was added to 4 mL of the obtained upper layer (aqueous phase) to make 20 mL, and Solution A was obtained. Ultrapure water was added to 4 mL of Solution A to make 20 mL, and an aqueous phase sample solution was obtained. Moreover, chloroform was added to 2 mL of obtained lower layers (organic phase), and it was set as 10 mL, and it was set as the solution B. Chloroform was added to 2 mL of Solution B to make 20 mL, and Solution C was obtained. Chloroform was added to 2 mL of solution C to make 20 mL, and an organic phase sample solution was obtained.
The absorbance of the organic phase sample solution and the aqueous phase sample solution was measured with an ultraviolet-visible spectrophotometer (organic phase sample solution: 278 nm, aqueous phase sample solution: 242 nm). The absorbance obtained from each sample solution was substituted into the equations (8) and (9), and the antipyrine concentration (Co) in the organic phase sample solution and the antipyrine concentration (Cw) in the aqueous phase sample solution were quantified. The obtained Co and Cw were substituted into the formula (2) to obtain LogP = 1.35.
＜比較例２〜１０＞
その他９化合物についても比較例１と同様にして分配係数を測定した。
<Comparative Examples 2 to 10>
For the other 9 compounds, the distribution coefficient was measured in the same manner as in Comparative Example 1.
＜実施例１＞
ＮＭＲ法（実測法）による、アンチピリンの分配係数測定
重水（アクロス製、１７ｍＬ）とクロロホルム−ｄ（アクロス製、０．０３ｖ／ｖ％ＴＭＳ含有、１７ｍＬ）を分液漏斗で振り混ぜた後、２４時間以上放置し、上層と下層を分離した。得られた上層を飽和重水、下層を飽和クロロホルム−ｄとした。
アンチピリン（シグマ・アルドリッチ製、２１．７９ｍｇ）に飽和クロロホルム−ｄ２ｍＬを加えて溶解し、さらに飽和重水２ｍＬを加えて３０秒間激しく混合後、２５℃で２４時間振とうし、遠心分離（３０００ｒｐｍ、１０分）して上層（水相）と下層（有機相）を完全に分離した。
得られた下層（有機相）１ｍＬに内標準物質として２，６−ジクロロベンズアルデヒド（アクロス製、１０．８４ｍｇ）を溶解し、有機相試料溶液とした。得られた有機相試料溶液について表１の条件にて１Ｈ−ＮＭＲを測定した結果、アンチピリンの特定基のシグナル面積は３．００、２，６−ジクロロベンズアルデヒドの特定基のシグナル面積は１．１０であった。なお、アンチピリンの特定基は１，２−ジヒドロ−３Ｈ−ピラゾール−３−オンの５位のメチル基、２，６−ジクロロベンズアルデヒドの特定基はホルミル基を選択した。
<Example 1>
Antipyrine partition coefficient measurement by NMR method (actual measurement method) Heavy water (Across, 17 mL) and chloroform-d (Across, containing 0.03 v / v% TMS, 17 mL) were shaken and mixed in a separatory funnel, then 24 The upper layer and the lower layer were separated by standing for more than an hour. The obtained upper layer was saturated heavy water, and the lower layer was saturated chloroform-d.
Saturated chloroform-d (2 mL) was added to antipyrine (Sigma Aldrich, 21.79 mg) and dissolved. Further, 2 mL of saturated heavy water was added and mixed vigorously for 30 seconds, followed by shaking at 25 ° C. for 24 hours, and centrifugation (3000 rpm, 10 rpm). The upper layer (aqueous phase) and the lower layer (organic phase) were completely separated.
2,6-dichlorobenzaldehyde (manufactured by Acros, 10.84 mg) as an internal standard substance was dissolved in 1 mL of the obtained lower layer (organic phase) to obtain an organic phase sample solution. As a result of measuring 1 H-NMR for the obtained organic phase sample solution under the conditions shown in Table 1, the signal area of the specific group of antipyrine was 3.00, and the signal area of the specific group of 2,6-dichlorobenzaldehyde was 1. 10. The specific group of antipyrine was a methyl group at the 5-position of 1,2-dihydro-3H-pyrazol-3-one, and the specific group of 2,6-dichlorobenzaldehyde was a formyl group.
表１ １Ｈ−ＮＭＲ測定条件及び解析条件 Table 1 1 H-NMR measurement conditions and analysis conditions
得られた結果に式（３）を適用して、有機相中のアンチピリン含量（Ｐsample）を算出した。式（３）にＩsample＝３．００、Ｈsample＝３、Ｍsample＝１８８．２３、Ｗsample＝１０．９０、Ｉstd＝１．１０、Ｈstd＝１、Ｍstd＝１７５．０１、Ｗstd＝１０．８４、Ｐstd＝９９を代入し、Ｐsample＝９６．２７（％）を得た。これは有機相中のアンチピリン含量と等しい。
また、得られた上層（水相）１ｍＬに内標準物質としてトリメチルシリルプロピオン酸ナトリウム−ｄ４（ＩＳＯＴＥＣ製、１０．０１ｍｇ）を溶解し、水相試料溶液とした。得られた水相試料溶液について表１の条件にて１Ｈ−ＮＭＲを測定した結果、アンチピリンの特定基のシグナル面積は２．９１、トリメチルシリルプロピオン酸ナトリウム−ｄ４の特定基のシグナル面積は２４０．５２であった。なお、アンチピリンの特定基は１，２−ジヒドロ−３Ｈ−ピラゾール−３−オンの５位のメチル基、トリメチルシリルプロピオン酸ナトリウム−ｄ４の特定基はメチル基を選択した。
得られた結果に式（３）を適用して水相中のアンチピリン含量（Ｐsample）を算出した。式（３）にＩsample＝２．９１、Ｈsample＝３、Ｍsample＝１８８．２３、Ｗsample＝１０．９０、Ｉstd＝２４０．５２、Ｈstd＝９、Ｍstd＝１７２．２４、Ｗstd＝１０．０１、Ｐstd＝９８を代入し、Ｐsample＝３．５７０（％）を得た。これは水相中のアンチピリン含量と等しい。
式（２）に有機相中のアンチピリン含量９６．２７（％）と水相中のアンチピリン含量３．５７０（％）を代入し、ＬｏｇＰ＝１．４３を得た。
Formula (3) was applied to the obtained results to calculate the antipyrine content (Psample) in the organic phase. In formula (3), Isample = 3.00, Hsample = 3, Msample = 188.23, Wsample = 10.90, Istd = 1.10, Hstd = 1, Mstd = 175.01, Wstd = 10.84, Pstd = 99 was substituted to obtain Psample = 96.27 (%). This is equal to the antipyrine content in the organic phase.
Further, sodium trimethylsilylpropionate-d 4 (manufactured by ISOTEC, 10.01 mg) as an internal standard substance was dissolved in 1 mL of the obtained upper layer (aqueous phase) to obtain an aqueous phase sample solution. As a result of measuring 1 H-NMR for the obtained aqueous phase sample solution under the conditions shown in Table 1, the signal area of the specific group of antipyrine was 2.91, and the signal area of the specific group of sodium trimethylsilylpropionate-d 4 was 240. .52. The specific group of antipyrine was a methyl group at the 5-position of 1,2-dihydro-3H-pyrazol-3-one, and the specific group of sodium trimethylsilylpropionate-d 4 was a methyl group.
The antipyrine content (Psample) in the aqueous phase was calculated by applying formula (3) to the obtained results. In formula (3), Isample = 2.91, Hsample = 3, Msample = 188.23, Wsample = 10.90, Istd = 240.52, Hstd = 9, Mstd = 172.24, Wstd = 10.01, Pstd = 98 was substituted to obtain Psample = 3.570 (%). This is equal to the antipyrine content in the aqueous phase.
Substituting the antipyrine content 96.27 (%) in the organic phase and the antipyrine content 3.570 (%) in the aqueous phase into the formula (2), Log P = 1.43 was obtained.
＜実施例２〜１０＞
その他の９化合物についても実施例１と同様にして分配係数を測定した。
<Examples 2 to 10>
For the other 9 compounds, the distribution coefficient was measured in the same manner as in Example 1.
＜実施例１１＞
ＮＭＲ法（差し引き法）による、レボフロキサシンの分配係数測定
重水（アクロス製、２３ｍＬ）とクロロホルム−ｄ（アクロス製、０．０３ｖ／ｖ％ＴＭＳ含有、３２ｍＬ）を分液漏斗で振り混ぜた後、２４時間以上放置し、上層と下層を分離した。得られた上層を飽和重水、下層を飽和クロロホルム−ｄとした。
レボフロキサシン（東京化成製、２１．４２ｍｇ）に飽和重水２ｍＬを加えて溶解し、さらに飽和クロロホルム−ｄ２ｍＬを加えて３０秒間激しく混合後、２５℃で２４時間振とうし、遠心分離（３０００ｒｐｍ、１０分）して上層（水相）と下層（有機相）を完全に分離した。
得られた下層（有機相）１ｍＬに内標準物質として２，６−ジクロロベンズアルデヒド（アクロス製、１１．６３ｍｇ）を溶解し、有機相試料溶液とした。得られた有機相試料溶液について表１の条件にて１Ｈ−ＮＭＲを測定した結果、レボフロキサシンの特定基のシグナル面積は１．００、２，６−ジクロロベンズアルデヒドの特定基のシグナル面積は２．６４であった。なお、レボフロキサシンの特定基はレボフロキサシン５位のプロトン、２，６−ジクロロベンズアルデヒドの特定基はホルミル基を選択した。
得られた結果に式（３）を適用して、有機相中のレボフロキサシン含量（Ｐsample）を算出した。式（３）にＩsample＝１．００、Ｈsample＝１、Ｍsample＝３６１．３７、Ｗsample＝１０．７１、Ｉstd＝２．６４、Ｈstd＝１、Ｍstd＝１７５．０１、Ｗstd＝１１．６３、Ｐstd＝９９を代入し、Ｐsample＝８４．０８（％）を得た。これは有機相中のレボフロキサシン含量と等しい。
また、水相中のレボフロキサシン含量は、式（４）により算出した。分配前の含量は、レボフロキサシン１０．６９ｍｇ及び２，６−ジクロロベンズアルデヒド１２．３９ｍｇを飽和クロロホルム−ｄ１ｍＬに溶解し、表１の条件で１Ｈ−ＮＭＲを測定及び解析し、式（３）により算出した。１Ｈ−ＮＭＲ測定の結果、レボフロキサシンの特定基のシグナル面積は１．０１、２，６−ジクロロベンズアルデヒドの特定基のシグナル面積は２．５０であった。式（３）にＩsample＝１．０１、Ｈsample＝１、Ｍsample＝３６１．３７、Ｗsample＝１０．６９、Ｉstd＝２．５０、Ｈstd＝１、Ｍstd＝１７５．０１、Ｗstd＝１２．３９、Ｐstd＝９９を代入し、分配前のレボフロキサシン含量を算出したところ、Ｐsample＝９５．７２（％）を得た。式（４）に分配前のレボフロキサシン含量＝９５．７２（％）、有機相中のレボフロキサシン含量＝８４．０８（％）を代入し、算出したところ、水相中のレボフロキサシン含量１１．６４（％）を得た。
式（２）に有機相中のレボフロキサシン含量８４．０８（％）と水相中のレボフロキサシン含量１１．６４（％）を代入し、ＬｏｇＰ＝０．８５９を得た。
<Example 11>
Determination of partition coefficient of levofloxacin by NMR method (subtraction method) Heavy water (Across, 23 mL) and chloroform-d (Across, containing 0.03 v / v% TMS, 32 mL) were shaken and mixed with a separatory funnel, then 24 The upper layer and the lower layer were separated by standing for more than an hour. The obtained upper layer was saturated heavy water, and the lower layer was saturated chloroform-d.
Saturated heavy water (2 mL) was added to levofloxacin (Tokyo Kasei 21.42 mg) and dissolved. Saturated chloroform-d (2 mL) was added, and the mixture was vigorously mixed for 30 seconds, shaken at 25 ° C. for 24 hours, and centrifuged (3000 rpm, 10 minutes). The upper layer (aqueous phase) and the lower layer (organic phase) were completely separated.
2,6-dichlorobenzaldehyde (manufactured by Acros, 11.63 mg) as an internal standard substance was dissolved in 1 mL of the obtained lower layer (organic phase) to obtain an organic phase sample solution. As a result of measuring 1 H-NMR for the obtained organic phase sample solution under the conditions shown in Table 1, the signal area of the specific group of levofloxacin was 1.00, and the signal area of the specific group of 2,6-dichlorobenzaldehyde was 2. 64. The specific group of levofloxacin was selected as the proton at the 5-position of levofloxacin, and the specific group of 2,6-dichlorobenzaldehyde was selected as the formyl group.
Formula (3) was applied to the obtained results to calculate the levofloxacin content (Psample) in the organic phase. In equation (3), Isample = 1.00, Hsample = 1, Msample = 361.37, Wsample = 10.71, Istd = 2.64, Hstd = 1, Mstd = 175.01, Wstd = 11.63, Pstd = 99 was substituted to obtain Psample = 84.08 (%). This is equal to the levofloxacin content in the organic phase.
Further, the levofloxacin content in the aqueous phase was calculated by the formula (4). The content before distribution is calculated by the equation (3) by dissolving 10.69 mg of levofloxacin and 12.39 mg of 2,6-dichlorobenzaldehyde in saturated chloroform-d1 mL, measuring and analyzing 1 H-NMR under the conditions shown in Table 1. did. As a result of 1 H-NMR measurement, the signal area of the specific group of levofloxacin was 1.02, and the signal area of the specific group of 1,2,6-dichlorobenzaldehyde was 2.50. In equation (3), Isample = 1.01, Hsample = 1, Msample = 361.37, Wsample = 10.69, Istd = 2.50, Hstd = 1, Mstd = 175.01, Wstd = 12.39, Pstd = 99 was substituted and the levofloxacin content before distribution was calculated to obtain Psample = 95.72 (%). Substituting levofloxacin content before distribution = 95.72 (%) and levofloxacin content in the organic phase = 84.08 (%) into the formula (4) and calculating, the levofloxacin content in the aqueous phase was 11.64 (% )
Substituting the levofloxacin content of 84.08 (%) in the organic phase and the levofloxacin content of 11.64 (%) in the aqueous phase into the formula (2), Log P = 0.659 was obtained.
＜実施例１２＞
内標準物質を使用しない二重ＮＭＲ試料管による分配係数測定法（同時測定法）
<Example 12>
Partition coefficient measurement method using double NMR sample tube without internal standard (simultaneous measurement method)
１）補正値の算出
カフェイン（東京化成製、２．２９ｍｇ）に重水６００μＬ及び重塩酸（アクロス製、２０ｗｔ．％重水溶液、１００μＬ）を加えて溶解し、カフェイン重水試料溶液とした。 また、カフェイン１．９２ｍｇをクロロホルム−ｄ７００μＬに溶解し、カフェインクロロホルム−ｄ試料溶液とした。
カフェインクロロホルム−ｄ試料溶液を二重ＮＭＲ試料管（シゲミ製、特殊NMR同軸CELL、５ｍｍ）の外管に、カフェイン重水試料溶液を内管に入れ、表１の条件で１Ｈ−ＮＭＲを測定した。１Ｈ−ＮＭＲ測定の結果、カフェインクロロホルム−ｄ試料溶液の特定基のケミカルシフトは７．５０８ｐｐｍ、シグナル面積は８．３４であり、カフェイン重水試料溶液の特定基のケミカルシフトは８．８０６ｐｐｍ、シグナル面積は９９．６３であった。なお、カフェインの特定基は１，３，７−トリメチル−３，７−ジヒドロ−プリン−２，６−ジオンの８位のプロトンを選択した。
式（５）にＩｗ’＝９９．６３、Ｖｗ＝０．７、Ｗｗ＝２．２９、Ｉｏ’＝８．３４、Ｖｏ＝０．７、Ｗｏ＝１．９２を代入し、補正値＝１０．０１を得た。
1) Calculation of Correction Value Caffeine (manufactured by Tokyo Chemical Industry Co., Ltd., 2.29 mg) was dissolved by adding 600 μL of heavy water and deuterated hydrochloric acid (manufactured by Acros, 20 wt.% Heavy aqueous solution, 100 μL) to obtain a caffeine heavy water sample solution. Further, 1.92 mg of caffeine was dissolved in 700 μL of chloroform-d to prepare a caffeine chloroform-d sample solution.
Double NMR sample tube caffeine chloroform -d sample solution (Shigemi Ltd., special NMR coaxial CELL, 5 mm) to the outer tube, placed in the inner tube caffeine heavy water sample solution, a 1 H-NMR under the conditions of Table 1 It was measured. As a result of 1 H-NMR measurement, the chemical shift of the specific group in the caffeine chloroform-d sample solution was 7.508 ppm, the signal area was 8.34, and the chemical shift of the specific group in the caffeine heavy water sample solution was 8.806 ppm. The signal area was 99.63. As the specific group of caffeine, the proton at the 8-position of 1,3,7-trimethyl-3,7-dihydro-purine-2,6-dione was selected.
Substituting Iw ′ = 99.63, Vw = 0.7, Ww = 2.29, Io ′ = 8.34, Vo = 0.7, Wo = 1.92 into equation (5), and correction value = 10 .01 was obtained.
２）分配係数の測定
重水１０ｍＬとクロロホルム−ｄ１０ｍＬを分液漏斗で振り混ぜた後、２４時間以上放置し、上層と下層を分離した。得られた上層を飽和重水、下層を飽和クロロホルム−ｄとした。
カフェイン２１．７１ｍｇに飽和クロロホルム−ｄ２ｍＬを加えて溶解し、さらに飽和重水２ｍＬを加えて３０秒間激しく混合後、２５℃で２４時間振とうし、遠心分離（３０００ｒｐｍ、１０分）して上層（水相）と下層（有機相）を完全に分離した。得られた上層（水相）６００μＬに重塩酸１００μＬを加えて水相試料溶液とし、下層（有機相）はそのまま有機相試料溶液とした。有機相試料溶液を二重ＮＭＲ試料管の外管に、水相試料溶液を内管に入れ、表１の条件で１Ｈ−ＮＭＲを測定した。１Ｈ−ＮＭＲ測定の結果、有機相試料溶液中の化合物の特定基のシグナル面積は２９．６８であり、水相試料溶液中の化合物の特定基のシグナル面積は９．４６であった。
式（７）にＩｏ’＝２９．６８、Iｗ’＝９．４６、補正値＝１０．０１、希釈率＝６００／７００を代入し、LogP＝１．４３を得た。
2) Measurement of partition coefficient After shaking and mixing 10 mL of heavy water and 10 mL of chloroform-d with a separatory funnel, the mixture was allowed to stand for 24 hours or more, and the upper layer and the lower layer were separated. The obtained upper layer was saturated heavy water, and the lower layer was saturated chloroform-d.
Add 2 mL of saturated chloroform-d to 21.71 mg of caffeine, dissolve, add 2 mL of saturated heavy water, mix vigorously for 30 seconds, shake at 25 ° C. for 24 hours, and centrifuge (3000 rpm, 10 minutes) to form the upper layer ( The aqueous phase) and the lower layer (organic phase) were completely separated. To 600 μL of the obtained upper layer (aqueous phase), 100 μL of deuterated hydrochloric acid was added to prepare an aqueous phase sample solution, and the lower layer (organic phase) was used as it was as an organic phase sample solution. The organic phase sample solution was put in the outer tube of the double NMR sample tube, and the aqueous phase sample solution was put in the inner tube, and 1 H-NMR was measured under the conditions shown in Table 1. As a result of 1 H-NMR measurement, the signal area of the specific group of the compound in the organic phase sample solution was 29.68, and the signal area of the specific group of the compound in the aqueous phase sample solution was 9.46.
Substituting Io ′ = 29.68, Iw ′ = 9.46, correction value = 10.01, and dilution ratio = 600/700 into the equation (7), LogP = 1.43 was obtained.
従来法（比較例１〜１０）及びＮＭＲ法（実施例１〜１２）の結果を表２及び図１に示す。また、従来法とＮＭＲ法のそれぞれで求めた分配係数の相関を図２に示す。 The results of the conventional method (Comparative Examples 1 to 10) and the NMR method (Examples 1 to 12) are shown in Table 2 and FIG. Moreover, the correlation of the distribution coefficient calculated | required by each of the conventional method and NMR method is shown in FIG.
表２ 分配係数測定結果 Table 2 Results of distribution coefficient measurement
ＮＭＲ法と従来法の分配係数の間には良好な相関が認められた。したがって、ＮＭＲ法は、分配係数の測定法として信頼に足りる方法といえる。また、NMR法は酸性化合物、中性化合物、塩基性化合物、両性化合物のいずれの化合物にも使用可能であり、さらに、測定可能な分配係数の範囲も広い。なお、ＮＭＲ測定においてＳ／Ｎ比が大きい化合物ほどＮＭＲ法と従来法の分配係数が近くなることが判明した。したがって、有機相又は水相中の化合物濃度が低いためＳ／Ｎ比が小さい化合物については、積算回数を増やすことにより正確な分配係数が得られる。 A good correlation was observed between the NMR method and the conventional method. Therefore, the NMR method can be said to be a reliable method for measuring the distribution coefficient. Further, the NMR method can be used for any compound of an acidic compound, a neutral compound, a basic compound, and an amphoteric compound, and further has a wide range of measurable partition coefficients. In NMR measurement, it was found that a compound having a larger S / N ratio has a closer distribution coefficient between the NMR method and the conventional method. Therefore, for a compound having a low S / N ratio due to a low concentration of the compound in the organic phase or aqueous phase, an accurate distribution coefficient can be obtained by increasing the number of integrations.
＜比較例１３＞
表３に、ＮＭＲ法と従来法における所要時間を示す。表中、アンチピリンの従来法は紫外可視吸光光度計、リドカインの従来法はＨＰＬＣを用いた測定である。また、分配係数測定に要した時間は、使用前に溶媒を飽和させる操作は含まない。
<Comparative Example 13>
Table 3 shows the time required for the NMR method and the conventional method. In the table, the conventional method of antipyrine is a measurement using an ultraviolet-visible spectrophotometer, and the conventional method of lidocaine is a measurement using HPLC. Further, the time required for measuring the partition coefficient does not include an operation for saturating the solvent before use.
表３ 従来法及びＮＭＲ法の所要時間 Table 3 Time required for conventional method and NMR method
ＮＭＲ法は従来法と比較して、短時間で分配係数を測定することが可能であり、また、溶媒および化合物の使用量も少ない。 Compared with the conventional method, the NMR method can measure the partition coefficient in a short time, and uses less solvent and compounds.
本発明により、簡便に分配係数を測定することが可能となった。本発明によれば、測定に使用する化合物は少量で良く、また、迅速に測定することが可能である。また、本発明は、紫外吸収を示さない化合物にも使用可能であるため、適用範囲が広い。 According to the present invention, the distribution coefficient can be easily measured. According to the present invention, the amount of the compound used for the measurement may be small, and the measurement can be performed quickly. Moreover, since the present invention can be used for a compound that does not exhibit ultraviolet absorption, the applicable range is wide.
Claims (2)
ｉ）化合物、飽和重水及び飽和重水素化有機溶媒を混合する工程、
ｉｉ）工程ｉの混合物を水相及び有機相に分離する工程、
ｉｉｉ）工程ｉｉで得た有機相に内標準物質を溶解させ有機相試料溶液とした後、ＮＭＲを用いて有機相試料溶液中の化合物及び内標準物質の特定基のシグナル面積を測定し、当該シグナル面積を用いて有機相中の化合物含量を算出する工程、
ｉｖ）工程ｉｉで得た水相に工程ｉｉｉと同一又は異なる内標準物質を溶解させ水相試料溶液とした後、ＮＭＲを用いて水相試料溶液中の化合物及び内標準物質の特定基のシグナル面積を測定し、当該シグナル面積を用いて水相中の化合物含量を算出する工程、
ｖ）工程ｉｉｉで算出した有機相中の化合物含量と、工程ｉｖで算出した水相中の化合物含量を用いて化合物の分配係数を算出する工程、
を含む、検量線の作成が不要である、分配係数の測定方法。 A method for measuring a partition coefficient of a compound comprising the following steps,
i) mixing the compound, saturated deuterated water and saturated deuterated organic solvent;
ii) separating the mixture of step i into an aqueous phase and an organic phase;
iii) After dissolving the internal standard substance in the organic phase obtained in step ii to obtain an organic phase sample solution, the signal area of the specific group of the compound in the organic phase sample solution and the internal standard substance is measured using NMR, and Calculating the compound content in the organic phase using the signal area,
iv) After dissolving the same or different internal standard substance in step iii in the aqueous phase obtained in step ii to obtain an aqueous phase sample solution, the signals of the compounds in the aqueous phase sample solution and specific groups of the internal standard substance are measured using NMR. Measuring the area and calculating the compound content in the aqueous phase using the signal area;
v) calculating a compound partition coefficient using the compound content in the organic phase calculated in step iii and the compound content in the aqueous phase calculated in step iv;
A distribution coefficient measurement method that does not require the creation of a calibration curve.
ｉ）化合物、飽和重水及び飽和重水素化有機溶媒を混合する工程、
ｉｉ）工程ｉの混合物を水相及び有機相に分離する工程、
ｉｉｉ）工程ｉｉで得た水相に重水素化酸、重水素化塩基若しくはシフト試薬を溶解させ水相試料溶液とし、工程ｉｉで得た有機相を有機相試料溶液とするか、或いは工程ｉｉで得た水相を水相試料溶液とし、工程ｉｉで得た有機相にシフト試薬を溶解させ有機相試料溶液とし、当該有機相試料溶液と水相試料溶液のうち一方を二重ＮＭＲ管の外管に採取して、他方を二重ＮＭＲ管の内管に採取した後、ＮＭＲを用いて有機相試料溶液及び水相試料溶液中の化合物の特定基のシグナル面積を測定する工程、及び
ｉｖ）工程ｉｉｉで測定した有機相試料溶液及び水相試料溶液中の化合物の特定基のシグナル面積を用いて化合物の分配係数を算出する工程、
を含む、検量線の作成が不要であり、且つ、内標準物質を使用しない分配係数の測定方法。 A method for measuring a partition coefficient of a compound comprising the following steps,
i) mixing the compound, saturated deuterated water and saturated deuterated organic solvent;
ii) separating the mixture of step i into an aqueous phase and an organic phase;
iii) Dissolving deuterated acid, deuterated base or shift reagent in the aqueous phase obtained in step ii to make an aqueous phase sample solution, and making the organic phase obtained in step ii an organic phase sample solution, or step ii The aqueous phase obtained in step 1 is used as an aqueous phase sample solution, the shift reagent is dissolved in the organic phase obtained in step ii to obtain an organic phase sample solution, and one of the organic phase sample solution and the aqueous phase sample solution is used in a double NMR tube. Taking the outer tube and taking the other into the inner tube of the double NMR tube, and then measuring the signal area of the specific group of the compound in the organic phase sample solution and the aqueous phase sample solution using NMR, and iv ) Calculating the partition coefficient of the compound using the signal area of the specific group of the compound in the organic phase sample solution and the aqueous phase sample solution measured in step iii;
A method for measuring a partition coefficient that does not require the creation of a calibration curve and does not use an internal standard substance.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2011041393A JP5858515B2 (en) | 2011-02-28 | 2011-02-28 | Method for measuring partition coefficient using quantitative analysis by NMR |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2011041393A JP5858515B2 (en) | 2011-02-28 | 2011-02-28 | Method for measuring partition coefficient using quantitative analysis by NMR |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2012177643A JP2012177643A (en) | 2012-09-13 |
| JP5858515B2 true JP5858515B2 (en) | 2016-02-10 |
Family
ID=46979563
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2011041393A Expired - Fee Related JP5858515B2 (en) | 2011-02-28 | 2011-02-28 | Method for measuring partition coefficient using quantitative analysis by NMR |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP5858515B2 (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE102018203434B4 (en) * | 2018-03-07 | 2019-12-05 | Bernd Willi Karl-Heinz Diehl | Method for heteronuclear quantitative determination by NMR spectroscopy, reference substances therefor and method for determining the degree of deuteration of a deuterated compound |
| JP7329471B2 (en) * | 2020-03-11 | 2023-08-18 | 大陽日酸株式会社 | Isotope enrichment measurement method |
| CN111855730A (en) * | 2020-08-12 | 2020-10-30 | 黄河三角洲京博化工研究院有限公司 | Method for testing water content in butyl rubber glue solution |
| CN112345575B (en) * | 2020-11-10 | 2024-05-31 | 淮阴师范学院 | Through1Method for quantitatively determining L-carnitine fumarate by H NMR |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| IT1265440B1 (en) * | 1993-12-24 | 1996-11-22 | Bracco Spa | PARAMAGNETIC DIAGNOSTIC FORMULATIONS AND METHOD OF USE OF THEM |
| US5856591A (en) * | 1996-07-17 | 1999-01-05 | Eastman Chem Co | Process for the separation of 2-alkene-1,4-diols and 3-alkene-1,2-diols from catalysts |
| JP3592917B2 (en) * | 1997-12-10 | 2004-11-24 | 花王株式会社 | Method for determination of mixtures containing surfactants |
| US5952530A (en) * | 1998-02-02 | 1999-09-14 | Union Carbide Chemicals & Plastics Technology Corporation | Separation processes |
| JP2008224284A (en) * | 2007-03-09 | 2008-09-25 | Sumitomo Metal Mining Co Ltd | Identification and quantitative determination method for fluorine ion species in effluent by internal standard method using fourier transformation nuclear magnetic resonance spectrometry |
-
2011
- 2011-02-28 JP JP2011041393A patent/JP5858515B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JP2012177643A (en) | 2012-09-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Butts et al. | High precision NOEs as a probe for low level conformers—a second conformation of strychnine | |
| Yazdanpanah et al. | Analytical measurement of serum 25-OH-vitamin D3, 25-OH-vitamin D2 and their C3-epimers by LC–MS/MS in infant and pediatric specimens | |
| Kaiser et al. | Comparison of extraction methods for per-and polyfluoroalkyl substances (PFAS) in human serum and placenta samples—insights into extractable organic fluorine (EOF) | |
| JP5858515B2 (en) | Method for measuring partition coefficient using quantitative analysis by NMR | |
| CN102967568B (en) | Method for testing dual-wavelength of light splitting luminosity | |
| Rodziewicz et al. | Rapid determination of chloramphenicol residues in milk powder by liquid chromatography–elektrospray ionization tandem mass spectrometry | |
| Muchakayala et al. | Development and validation of a RP-UPLC method for the determination of betamethasone dipropionate impurities in topical formulations using a multivariate central composite design | |
| Peng et al. | Derivatization method for sensitive determination of fluorotelomer alcohols in sediment by liquid chromatography–electrospray tandem mass spectrometry | |
| Wells et al. | Dimethylsulfone as a universal standard for analysis of organics by QNMR | |
| Yip et al. | Assessment of the chemical and enantiomeric purity of organic reference materials | |
| US20120083039A1 (en) | Method for quantitatively determining impurities | |
| Piva et al. | Determination of perfluoroalkyl substances (PFAS) in human hair by liquid chromatography-high accurate mass spectrometry (LC-QTOF) | |
| Jacob et al. | Evaluation, optimization, and application of three independent suspect screening workflows for the characterization of PFASs in water | |
| Abraham et al. | The lipophilicity and hydrogen bond strength of pyridine-N-oxides and protonated pyridine-N-oxides | |
| Bonthu et al. | Simultaneous determination of candesartan and hydrochlorothiazide in human plasma by LC-MS/MS | |
| Hall et al. | High accuracy determination of malachite green and leucomalachite green in salmon tissue by exact matching isotope dilution mass spectrometry | |
| Christodouleas et al. | Determination of total antioxidant activity of edible oils as well as their aqueous and organic extracts by chemiluminescence | |
| Çaǧlar et al. | Two simple and rapid spectrophotometric methods for the determination of a new antihypertensive drug olmesartan in tablets | |
| CN105424853A (en) | LC-MS/MS kit for detecting nicotine and its metabolites in saliva | |
| Lin et al. | Use of 1H NMR to facilitate solubility measurement for drug discovery compounds | |
| Li et al. | The effects of isotope-labeled analogs on the LC-IDMS measurement by comparison of ESI responses and matrix effect of melamine, 13 C 3-melamine, 13 C 3+ 15 N 3-melamine, and 15 N 3-melamine | |
| Guillou et al. | On-the-fly analysis of chloride and bromide in drug samples using NMR spectroscopy | |
| El-Shaheny | Utility of a green fluorone-based turn-off fluorescence probe for submicromolar determination and stability testing of two macrolides. Insights into reaction thermodynamics, quenching mechanism, and identification of the oxidative degradation products by ESI+-MS | |
| Elečková et al. | Application of cinnamoyl derivative as a new ligand for dispersive liquid-liquid microextraction and spectrophotometric determination of cobalt | |
| Su et al. | Accurate analysis of trace pentachlorophenol in textiles by isotope dilution liquid chromatography‐mass spectrometry |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20140213 |
|
| A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20141119 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20141202 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20150119 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20150623 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20150706 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20151208 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20151211 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 5858515 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
| LAPS | Cancellation because of no payment of annual fees |