JP5858515B2 - Method for measuring partition coefficient using quantitative analysis by NMR - Google Patents
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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.
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