JP5193266B2 - Sensitive quantification method for herbal medicine - Google Patents
Sensitive quantification method for herbal medicine Download PDFInfo
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- JP5193266B2 JP5193266B2 JP2010256187A JP2010256187A JP5193266B2 JP 5193266 B2 JP5193266 B2 JP 5193266B2 JP 2010256187 A JP2010256187 A JP 2010256187A JP 2010256187 A JP2010256187 A JP 2010256187A JP 5193266 B2 JP5193266 B2 JP 5193266B2
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Images
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2560/00—Chemical aspects of mass spectrometric analysis of biological material
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
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- Biotechnology (AREA)
- Food Science & Technology (AREA)
- General Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
Description
本発明は、生体試料中のグリチルリチンやその代謝物等を高感度に定量するための方法に関する。 The present invention relates to a method for quantifying glycyrrhizin or a metabolite thereof in a biological sample with high sensitivity.
グリチルリチン（以下、ＧＬと略記する）及びその塩は、抗アレルギー作用、抗炎症作用と共に、免疫調節作用、肝細胞障害抑制作用、肝細胞増殖促進作用、ウィルス増殖抑制・不活性化作用等の多様な生理活性を有する甘草根抽出成分である。日本では、古くから漢方薬等の臨床薬として用いられており、現在では、慢性肝疾患、湿疹・皮膚炎、小児ストロフルス、円形脱毛症、各種アレルギー、炎症等の治療に幅広く使用されている。また、医薬品以外の使用として、ＧＬは塩なれ効果をもち、甘味効果があることから甘味料として漬物や調味料など広範囲の食品に多用されている。 Glycyrrhizin (hereinafter abbreviated as GL) and its salts have various anti-allergic and anti-inflammatory effects, as well as immunoregulatory effects, hepatocellular injury inhibiting effects, hepatocyte proliferation promoting effects, virus proliferation inhibiting / inactivating effects, etc. It is a licorice root extract component having various physiological activities. In Japan, it has long been used as a clinical medicine such as Chinese herbal medicine, and is now widely used for the treatment of chronic liver disease, eczema / dermatitis, childhood stroflus, alopecia areata, various allergies, inflammation and the like. Further, as a use other than pharmaceuticals, GL has a salting effect, and since it has a sweetening effect, it is frequently used as a sweetener in a wide range of foods such as pickles and seasonings.
非臨床試験の結果から、ＧＬは肝臓に集積しやすく、且つ速やかに胆汁中へ排泄される特性があり、代謝及び腸肝循環を受けながら排出されると考えられている。特に経口投与において、配糖体で水溶性極性物質であるＧＬは低吸収性を示すと共に、腸内細菌による代謝や初回通過効果により、末梢血における濃度が極めて低い。このため、ＧＬの血中動態を測定するために、現在までに様々な方法が試みられているが、ＧＬを含有する製剤、甘草を配合する漢方製剤において、服用時の血中ＧＬは検出困難であり、その動態も長い間不明とされてきた。 From the results of non-clinical studies, it is considered that GL is easy to accumulate in the liver and is rapidly excreted in bile, and is excreted while undergoing metabolism and enterohepatic circulation. In particular, in oral administration, GL, which is a glycoside and a water-soluble polar substance, exhibits low absorbability, and has extremely low concentration in peripheral blood due to metabolism by the enteric bacteria and the first-pass effect. For this reason, various methods have been tried so far to measure the kinetics of GL in the blood. However, it is difficult to detect blood GL at the time of taking in GL-containing preparations and Chinese medicine preparations containing licorice. And the dynamics have been unknown for a long time.
例えば、ＧＬを経口投与された健常者から採取された血漿に対して、メタノール処理を行ってタンパク質を除去した後、当該血漿中のＧＬの量を、酵素免疫法（ＥＩＡ）により測定する方法やオクタデシル基化学結合型（ＯＤＳ）カラム等の逆相分配機能を備えるカラムを用いた高速液体クロマトグラフィー（ＨＰＬＣ）を用いて測定する方法（例えば、非特許文献１〜４）がある。しかしながら、これらの測定方法では、定量限界値がせいぜい０．１μｇ／ｍＬ程度であり、主代謝物であるグリチルレチン酸（以下、ＧＡと略記する。）は検出することができても、ＧＬは検出することはできなかった。このため、尿中からの検出例をもって間接的にＧＬの血中移行が証明された（引用文献４参照。）。そして、ＧＬ含有製剤薬効については、ＧＬの代わりに、定量可能なＧＡが評価指標とされている。 For example, a method of measuring the amount of GL in the plasma by enzyme immunization (EIA) after treating the plasma collected from a healthy person orally administered with GL and removing the protein by methanol treatment, There are methods (for example, Non-Patent Documents 1 to 4) for measurement using high performance liquid chromatography (HPLC) using a column having a reverse phase distribution function such as an octadecyl group chemical bond type (ODS) column. However, in these measurement methods, the quantification limit value is at most about 0.1 μg / mL, and GL is detected even though glycyrrhetinic acid (hereinafter abbreviated as GA), which is the main metabolite, can be detected. I couldn't. For this reason, GL blood transfer was indirectly proved with a detection example from urine (see cited document 4). For GL-containing drug efficacy, quantifiable GA is used as an evaluation index instead of GL.
活性本体である未変化体ＧＬの薬物動態は、有効性、安全性から適正使用を判断する上で有用である。このため、ＧＡを評価対象にするのではなく、血漿等の生体試料中のＧＬ量を定量できることが望ましい。 The pharmacokinetics of unchanged GL, which is the active substance, is useful in determining proper use from the efficacy and safety. For this reason, it is desirable that the amount of GL in a biological sample such as plasma can be quantified instead of using GA as an evaluation target.
本発明は、上記課題を鑑みてなされたものであって、血漿等の生体試料中のＧＬを検出し定量するための方法を提供することを目的とする。 This invention is made | formed in view of the said subject, Comprising: It aims at providing the method for detecting and quantifying GL in biological samples, such as plasma.
本発明者らは、上記課題を解決するために鋭意研究した結果、まず、逆相分配機能及び陰イオン交換機能を備える固相を用いた固相抽出法により、生体試料中からＧＬを含む成分を抽出した後、抽出物中のＧＬを質量分析法に供することにより、生体試料中のＧＬを検出し定量できることを見出し、本発明を完成させた。 As a result of diligent research to solve the above problems, the present inventors firstly, a component containing GL from a biological sample by a solid phase extraction method using a solid phase having a reverse phase distribution function and an anion exchange function. After extraction, the GL in the extract was subjected to mass spectrometry, and it was found that GL in a biological sample could be detected and quantified, thereby completing the present invention.
すなわち、本発明は、
（１） 生体試料をアルカリ又はアルコールに混和した混和物を、逆相分配機能及び陰イオン交換機能を備える固相に注入した後、前記固相を洗浄液で２回以上洗浄し、その後、酸性アルコールにより前記固相から溶出することにより、生薬由来成分を含む抽出物を調製する抽出工程と、
前記抽出工程により抽出された抽出物中のグリチルリチン、グリチルレチン酸、グリチルリチン及びグリチルレチン酸の代謝物、グリチルリチン及びグリチルレチン酸の類縁物質、甘草に含有されているサポニン成分、並びにこれらの薬学的に許容される塩からなる群より選択される少なくも１種以上を、質量分析法により検出し定量する工程、
を有し、
前記洗浄液が、水、アルカリ、アルコール、及びアセトニトリルからなる群より選択される１種又は２種以上の混合液であり、
前記抽出工程において、固相の洗浄を、アルカリとアルコールと水との混合液、又はアルカリと水との混合液により洗浄した後、アルコール、アルコールと水の混合液、又はアセトニトリルにより洗浄することによって行うことを特徴とする生薬由来成分の高感度定量方法、
（２） 前記抽出工程において、固相の洗浄を、アルカリとアルコールと水の混合液により洗浄した後、アルコール、アルコールと水の混合液、又はアセトニトリルにより洗浄することによって行うことを特徴とする前記（１）に記載の生薬由来成分の高感度定量方法、
（３） 前記アルカリとアルコールと水の混合液が、０．５〜２８％アンモニア水とメタノールを９９：１〜１：３であることを特徴とする前記（２）に記載の生薬由来成分の高感度定量方法、
（４）前記生体試料が、血液、血漿、又は組織抽出物であることを特徴とする前記（１）〜（３）のいずれか一つに記載の生薬由来成分の高感度定量方法、
を提供するものである。
That is, the present invention
(1) a blend of the biological sample were mixed in alkali or alcohol, was poured into the solid phase with a reversed-phase function and an anion exchange function, the solid phase is washed twice more with washing solution, then, the acidic alcohol An extraction step of preparing an extract containing herbal medicine-derived components by eluting from the solid phase with
Glycyrrhizin, glycyrrhetinic acid, metabolites of glycyrrhizin and glycyrrhetinic acid, related substances of glycyrrhizin and glycyrrhetinic acid, saponin components contained in licorice, and pharmaceutically acceptable substances thereof in the extract extracted by the extraction process Detecting and quantifying at least one selected from the group consisting of salts by mass spectrometry,
Have
The cleaning solution is one or a mixture of two or more selected from the group consisting of water, alkali, alcohol, and acetonitrile;
In the extraction step, the solid phase is washed with a mixture of alkali, alcohol and water, or a mixture of alkali and water, and then washed with alcohol, a mixture of alcohol and water, or acetonitrile. A highly sensitive quantitative method for herbal medicine-derived components,
(2) In the extraction step, the solid phase is washed with a mixture of alkali, alcohol and water, and then washed with alcohol, a mixture of alcohol and water, or acetonitrile. (1) a highly sensitive quantitative method for herbal medicine-derived components,
(3) The mixed solution of the herbal medicine according to (2), wherein the mixture of alkali, alcohol and water is 99: 1 to 1: 3 of 0.5 to 28% ammonia water and methanol. High-sensitivity quantification method,
(4) The high-sensitivity quantification method for crude drug-derived components according to any one of (1) to (3), wherein the biological sample is blood, plasma, or tissue extract,
Is to provide.
本発明の生薬由来成分の高感度定量方法により、含有量が非常に少ない生体試料中のＧＬを検出し、定量することができる。このため、本発明の生薬由来成分の高感度定量方法を用いることにより、ＧＬを含有する製剤や飲食品を摂取したヒトから採取された生体試料中のＧＬを定量し、薬物動態を精度よく解析することができる。 The highly sensitive quantification method for herbal medicine-derived components of the present invention can detect and quantify GL in a biological sample with a very low content. For this reason, by using the high-sensitivity quantification method for herbal medicine-derived components of the present invention, GL in a biological sample collected from a human who has ingested a preparation containing GL or a food or drink is quantified, and the pharmacokinetics is analyzed accurately. can do.
本発明の生薬由来成分の高感度定量方法は、生体試料中のＧＬを逆相分配機能及び陰イオン交換機能を備える固相を用いた固相抽出法により抽出した後、得られた抽出物中のＧＬ含有量を質量分析法により定量することを特徴とする。従来のＨＰＬＣ法等に代えて質量分析法を利用して定量することにより、ＧＬの検出感度を高められる。また、従来のＯＤＳカラムのような逆相分配機能のみを備える固相を用いた固相抽出法ではなく、逆相分配機能及び陰イオン交換機能を備える固相を用いて調製することにより、質量分析に供される抽出物からより多くの夾雑物を除去することができ、質量分析の感度を飛躍的に向上させることができる。 In the method for sensitive quantification of crude drug-derived components of the present invention, GL in a biological sample is extracted by a solid phase extraction method using a solid phase having a reverse phase distribution function and an anion exchange function, and then in the obtained extract. The GL content of is quantitatively determined by mass spectrometry. The detection sensitivity of GL can be increased by quantifying using mass spectrometry instead of the conventional HPLC method. In addition, by using a solid phase having a reverse phase distribution function and an anion exchange function instead of a solid phase extraction method using a solid phase having only a reverse phase distribution function like a conventional ODS column, More impurities can be removed from the extract used for analysis, and the sensitivity of mass spectrometry can be dramatically improved.
具体的には、本発明の生薬由来成分の高感度定量方法は、生体試料をアルカリ又はアルコールに混和した混和物を、逆相分配機能及び陰イオン交換機能を備える固相に注入した後、前記固相を洗浄液で１回又は２回以上洗浄し、その後、酸性アルコールにより前記固相から溶出することにより、生薬由来成分を含む抽出物を調製する抽出工程と、前記抽出工程により抽出された抽出物中のＧＬ、ＧＡ、ＧＬ及びＧＡの代謝物、ＧＬ及びＧＡの類縁物質、甘草に含有するサポニン成分、並びにこれらの薬学的に許容される塩からなる群より選択される少なくも１種以上を、質量分析法により検出し定量する工程を有し、前記洗浄液が、水、アルカリ、アルコール、及びアセトニトリルからなる群より選択される１種又は２種以上の混合液であることを特徴とする。 Specifically, in the high-sensitivity quantification method for herbal medicine-derived components of the present invention, after injecting a mixture obtained by mixing a biological sample with alkali or alcohol into a solid phase having a reverse phase distribution function and an anion exchange function, The solid phase is washed once or twice with a washing solution and then eluted from the solid phase with acidic alcohol to prepare an extract containing herbal medicine-derived components, and the extraction extracted by the extraction step At least one selected from the group consisting of GL, GA, GL and GA metabolites in the product, GL and GA related substances, saponin components contained in licorice, and pharmaceutically acceptable salts thereof The washing liquid is one or a mixture of two or more selected from the group consisting of water, alkali, alcohol, and acetonitrile. And wherein the door.
本発明の生薬由来成分の高感度定量方法により定量される生薬由来成分は、ＧＬ、ＧＡ、ＧＬ及びＧＡの代謝物、ＧＬ及びＧＡの類縁物質、甘草に含有されているサポニン成分、並びにこれらの薬学的に許容される塩からなる群（以下、「ＧＬ等」ということがある。）より選択される少なくも１種以上である。
ＧＬ及びＧＡの代謝物としては、例えば、３−ｍｏｎｏｇｌｕｃｕｒｏｎｙｌ−ｇｌｙｃｈｒｒｅｔｉｎｉｃ ａｃｉｄ（２０β−Ｃａｒｂｏｘｙ−１１−ｏｘｏ−３０−ｎｏｒｏｌｅａｎ−１２−ｅｎ−３β−ｙｌ−β−Ｄ−ｇｌｕｃｏｐｙｒａｎｏｓｉｄｕｒｏｎｉｃ ａｃｉｄ）、３０−ｍｏｎｏｇｌｕｃｕｒｏｎｙｌ−ｇｌｙｃｈｒｒｅｔｉｎｉｃ ａｃｉｄ（３β−Ｈｙｄｒｏｘｙ−１１−ｏｘｏｏｌｅａｎ−１２−ｅｎ−３０−ｏｙｌ−β−Ｄ−ｇｌｕｃｏｐｙｒａｎｏｓｉｄｕｒｏｎｉｃ ａｃｉｄ）、３−ｏｘｏ−ＧＡ（３，１１−Ｄｉｏｘｏｏｌｅａｎ−１２−ｅｎ−３０−ｏｉｃ ａｃｉｄ）、３α−ＧＡ（３α−Ｈｙｄｒｏｘｙ−１１−ｏｘｏｏｌｅａｎ−１２−ｅｎ−３０−ｏｉｃ ａｃｉｄ）、３位硫酸抱合体（３β−Ｈｙｄｒｏｘｙｓｕｌｆｏｎｙｌｏｘｙ−１１−ｏｘｏｏｌｅａｎ−１２−ｅｎ−３０−ｏｉｃ ａｃｉｄ）、３β，２２α−Ｄｉｈｙｄｒｏｘｙ−１１−ｏｘｏｏｌｅａｎ−１２−ｅｎ−３０−ｏｉｃ ａｃｉｄ、及び３β，２４−Ｄｉｈｙｄｒｏｘｙ−１１−ｏｘｏｏｌｅａｎ−１２−ｅｎ−３０−ｏｉｃ ａｃｉｄ等が挙げられる。
The herbal medicine-derived components quantified by the high-sensitivity quantification method for herbal medicine-derived components of the present invention include GL, GA, GL and GA metabolites, GL and GA related substances, saponin components contained in licorice, and these It is at least one selected from the group consisting of pharmaceutically acceptable salts (hereinafter sometimes referred to as “GL etc.”).
Examples of metabolites of GL and GA include 3-monoglycuronic-glycryretinic acid (20β-Carboxy-11-oxo-30-norolean-12-en-3β-yl-β-D-glucopyranosiduronic acid), 30-monoglucuronic acid. glychlororetinic acid (3β-Hydroxy-11-oxoolan-12-en-30-oyl-β-D-glucopyranosiduronic acid), 3-oxo-GA (3,11-Dioxolene-12-en-30-oic acid), 3α -GA (3α-Hydroxy-11-oxolean-12-en-30-ic acid), 3-position sulfate conjugate (3β-Hydr oxysulfonyloxy-11-oxo-12-en-30-ic acid), 3β, 22α-Dihydroxy-11-oxo-l-12-en-30-ic acid, and 3β, 24-Dihydroxy-11-ox-oline-12-en- 30-oic acid and the like.
また、ＧＬ及びＧＡの類縁物質としては、例えば、２０α−Ｃａｒｂｏｘｙ−１１−ｏｘｏ−２９−ｎｏｒｏｌｅａｎ−１２−ｅｎ−３β−ｙｌ（β−Ｄ−ｇｌｕｃｏｐｙｒａｎｏｓｙｌｕｒｏｎｉｃ ａｃｉｄ）−（１→２）−β−Ｄ−ｇｌｕｃｏｐｙｒａｎｏｓｉｄｕｒｏｎｉｃ ａｃｉｄ、２０β−Ｃａｒｂｏｘｙ−２４−ｈｙｄｒｏｘｙ−１１−ｏｘｏ−３０−ｎｏｒｏｌｅａｎ−１２−ｅｎ−３β−ｙｌ（β−Ｄ−ｇｌｕｃｏｐｙｒａｎｏｓｙｌｕｒｏｎｉｃ ａｃｉｄ）−（１→２）−β−Ｄ−ｇｌｕｃｏｐｙｒａｎｏｓｉｄｕｒｏｎｉｃ ａｃｉｄ、１８α−ＧＬ（２０β−Ｃａｒｂｏｘｙ−１１−ｏｘｏ−（１８αＨ）−３０−ｎｏｒｏｌｅａｎ−１２−ｅｎ−３β−ｙｌ（β−Ｄ−ｇｌｕｃｏｐｙｒａｎｏｓｙｌｕｒｏｎｉｃ ａｃｉｄ）−（１→２）−β−Ｄ−ｇｌｕｃｏｐｙｒａｎｏｓｉｄｕｒｏｎｉｃ ａｃｉｄ）、１８α−ＧＡ（３β−Ｈｙｄｒｏｘｙ−１１−ｏｘｏ−（１８αＨ）−ｏｌｅａｎ−１２−ｅｎ−３０−ｏｉｃ ａｃｉｄ）等が挙げられる。 Examples of GL and GA related substances include 20α-Carboxy-11-oxo-29-norolean-12-en-3β-yl (β-D-glucopyranosyluronic acid)-(1 → 2) -β-D. -Glucopyranosiduronic acid, 20β-Carboxy-24-hydroxy-11-oxo-30-norolean-12-en-3β-yl (β-D-glucopyranosyluronic acid)-(1 → 2) -β-D-glucopyranicidic acid, 18- -GL (20β-Carboxy-11-oxo- (18αH) -30-norlean-12-en-3β-yl (β-D-glucopyranosyluronic aci ) - (1 → 2) -β-D-glucopyranosiduronic acid), 18α-GA (3β-Hydroxy-11-oxo- (18αH) -olean-12-en-30-oic acid) and the like.
また、甘草に含有されているサポニン成分としては、例えば、ｌｉｃｏｒｉｃｅ−ｓａｐｏｎｉｎ Ｃ１（２０β−Ｃａｒｂｏｘｙ−１１−ｏｘｏ−３０−ｎｏｒｏｌｅａｎ−１２−ｅｎ−３β−ｙｌ（β−Ｄ−ｇｌｕｃｏｐｙｒａｎｏｓｙｌｕｒｏｎｉｃ ａｃｉｄ）−（１→２）−β−Ｄ−ｇｌｕｃｏｐｙｒａｎｏｓｉｄｕｒｏｎｉｃ ａｃｉｄ）等が挙げられる。 Moreover, as a saponin component contained in licorice, for example, licorice-saponin C 1 (20β-Carboxy-11-oxo-30-norolean-12-en-3β-yl (β-D-glucopyranyluronic acid)-( 1 → 2) -β-D-glucopyranosiduronic acid) and the like.
本発明において用いられるＧＬ等の薬学的に許容される塩は、生体内において、ＧＬ等と同様の薬理効果を有する塩であれば、特に限定されるものではない。具体的には、例えば、アンモニウム塩、ナトリウム塩、カリウム塩等が挙げられる。 The pharmaceutically acceptable salt such as GL used in the present invention is not particularly limited as long as it has a pharmacological effect similar to that of GL in vivo. Specifically, ammonium salt, sodium salt, potassium salt etc. are mentioned, for example.
本発明においては、ＧＬ等のうち、１種類を定量してもよく、２種類以上を一の操作で定量してもよい。本発明においては、特に、ＧＬとＧＡを同時に定量することが好ましい。 In the present invention, one type of GL or the like may be quantified, and two or more types may be quantified by one operation. In the present invention, it is particularly preferable to quantify GL and GA at the same time.
本発明の生薬由来成分の高感度定量方法に供される生体試料は、生体から採取された試料であればよく、ヒトやマウス、ラット等の動物から採取されたものであることが好ましい。また、血液、血漿、血清、尿、腹水、胸水、関節液、骨髄液、胆汁等であってもよく、肝臓や膵臓、腎臓等の組織から採取された組織片等の抽出物（組織抽出物）であってもよい。組織片等の抽出物は、組織片等を常法によりホモジナイズすることにより調製することができる。本発明においては、血液、血漿、尿、又は組織抽出物であることが好ましく、血漿であることがより好ましい。 The biological sample used in the high-sensitivity quantification method for herbal medicine-derived components of the present invention may be a sample collected from a living body, and is preferably collected from an animal such as a human, mouse, or rat. It may also be blood, plasma, serum, urine, ascites, pleural effusion, joint fluid, bone marrow fluid, bile, etc., and extracts such as tissue fragments collected from tissues such as liver, pancreas and kidney (tissue extract) ). An extract such as a tissue piece can be prepared by homogenizing a tissue piece or the like by a conventional method. In the present invention, blood, plasma, urine, or tissue extract is preferable, and plasma is more preferable.
以下、本発明の生薬由来成分の高感度定量方法を工程ごとに説明する。
抽出工程として、まず、生体試料をアルカリ又はアルコールに混和した混和物を調製する。生体試料に添加するアルカリとしては、得られる混和物のｐＨをアルカリ性にすることができるものであれば、特に限定されるものではなく、例えば、アンモニア、アンモニア水、水酸化ナトリウム溶液、炭酸水素ナトリウム溶液等が挙げられる。また、アルコールとしては、炭素数１〜６の低級アルコールであることが好ましく、メタノール、エタノール、イソプロパノール等が挙げられる。また、水で希釈されたアルコール溶液であってもよい。本発明においては、生体試料に添加するアルカリ又はアルコールとしては、アンモニア、アンモニア水、メタノール、又はメタノール水溶液であることが好ましく、アンモニア又はアンモニア水であることがより好ましい。
Hereinafter, the highly sensitive determination method of the crude drug origin component of this invention is demonstrated for every process.
As an extraction step, first, an admixture obtained by mixing a biological sample with alkali or alcohol is prepared. The alkali added to the biological sample is not particularly limited as long as the pH of the resulting mixture can be made alkaline. For example, ammonia, aqueous ammonia, sodium hydroxide solution, sodium bicarbonate Examples include solutions. Moreover, as alcohol, it is preferable that it is a C1-C6 lower alcohol, and methanol, ethanol, isopropanol, etc. are mentioned. Moreover, the alcohol solution diluted with water may be sufficient. In the present invention, the alkali or alcohol added to the biological sample is preferably ammonia, aqueous ammonia, methanol, or an aqueous methanol solution, and more preferably ammonia or aqueous ammonia.
生体試料に添加するアンモニア水の濃度は特に限定されるものではなく、生体試料の種類、その後に使用する固相の種類、得られる混和物のアンモニア濃度等を考慮して適宜調整することができる。本発明においては、生体試料に添加するアンモニア又はアンモニア水の濃度は、得られる混和物のアンモニア濃度が０．０１〜３０％となるような濃度であることが好ましく、０．０５〜２５％となるような濃度であることがより好ましく、０．０５〜２０％となるような濃度であることがさらに好ましい。 The concentration of ammonia water added to the biological sample is not particularly limited, and can be appropriately adjusted in consideration of the type of biological sample, the type of solid phase used thereafter, the ammonia concentration of the resulting mixture, and the like. . In the present invention, the concentration of ammonia or aqueous ammonia added to the biological sample is preferably such that the resulting mixture has an ammonia concentration of 0.01 to 30%, and 0.05 to 25%. It is more preferable that the concentration is such that the concentration is 0.05 to 20%.
次いで、得られた混和物を、逆相分配機能及び陰イオン交換機能を備える固相に注入し、当該混和物中のＧＬ等を固相に吸着させる。逆相分配機能及び陰イオン交換機能を備える固相としては、例えば、Ｏａｓｉｓ ＭＡＸ（Ｗａｔｅｒｓ社製）等が挙げられる。 Next, the obtained mixture is injected into a solid phase having a reverse phase distribution function and an anion exchange function, and GL and the like in the mixture are adsorbed on the solid phase. Examples of the solid phase having a reverse phase distribution function and an anion exchange function include Oasis MAX (manufactured by Waters).
ＧＬ等を吸着させた固相を洗浄液で１回又は２回以上洗浄し、非特異的に結合している成分を除去する。洗浄液は、水、アルカリ、アルコール、及びアセトニトリルからなる群より選択される１種又は２種以上の混合液である。洗浄液として用いられるアルカリやアルコールとしては、生体試料に添加されるアルカリやアルコールと同様のものが挙げられる。また、固相の洗浄は、同種の洗浄液で２回以上行ってもよく、異なる種類の洗浄液で順次洗浄してもよい。 The solid phase on which GL or the like is adsorbed is washed once or twice with a washing solution to remove non-specifically bound components. The cleaning liquid is one or a mixture of two or more selected from the group consisting of water, alkali, alcohol, and acetonitrile. Examples of the alkali and alcohol used as the cleaning liquid include the same alkali and alcohol added to the biological sample. In addition, the solid phase may be washed twice or more with the same kind of washing solution, or sequentially with different kinds of washing solutions.
本発明においては、固相の洗浄を、アルカリとアルコールと水との混合液、アルカリと水との混合液、又は水により洗浄した後、アルコール、アルコールと水の混合液、又はアセトニトリルにより洗浄することによって行うことが好ましく、アルカリとアルコールと水との混合液により洗浄した後、アルコール、アルコールと水の混合液、又はアセトニトリルにより洗浄することによって行うことがより好ましい。アルカリとアルコールと水の混合液としては、アンモニアとアルコールと水の混合液であることが好ましく、アンモニアとメタノールと水の混合液であることがより好ましい。 In the present invention, the solid phase is washed with a mixture of alkali, alcohol and water, a mixture of alkali and water, or water, and then washed with alcohol, a mixture of alcohol and water, or acetonitrile. It is preferable to carry out by washing, and it is more preferred to wash by washing with alcohol, a mixture of alcohol and water, or acetonitrile after washing with a mixture of alkali, alcohol and water. The mixed solution of alkali, alcohol and water is preferably a mixed solution of ammonia, alcohol and water, and more preferably a mixed solution of ammonia, methanol and water.
アンモニアとメタノールと水の混合液中の各成分の組成比は、ＧＬ等を固相に吸着させた状態を保持することができるものであれば特に限定されるものではない。例えば、混合液中のメタノール濃度が１〜７５％、アンモニア濃度が０．１〜２１％であることが好ましく、メタノール濃度が２５〜７５％、アンモニア濃度が０．１〜２１％であることがより好ましい。このような組成比の混合液は、例えば、０．５〜２８％アンモニア水とメタノールを９９：１〜１：３で混合することにより、調製することができる。 The composition ratio of each component in the mixed solution of ammonia, methanol, and water is not particularly limited as long as it can maintain the state in which GL or the like is adsorbed to the solid phase. For example, the methanol concentration in the mixed solution is preferably 1 to 75% and the ammonia concentration is preferably 0.1 to 21%, the methanol concentration is 25 to 75%, and the ammonia concentration is 0.1 to 21%. More preferred. A liquid mixture having such a composition ratio can be prepared, for example, by mixing 0.5 to 28% ammonia water and methanol at 99: 1 to 1: 3.
一回目の洗浄をアルカリとアルコールと水の混合液により行った後、２回目の洗浄に用いる洗浄液としては、アルコールを用いることが好ましく、メタノール又はエタノールを用いることがより好ましく、メタノールを用いることがさらに好ましい。 After the first washing is performed with a mixed solution of alkali, alcohol and water, the washing liquid used for the second washing is preferably alcohol, more preferably methanol or ethanol, and methanol. Further preferred.
その後、酸性アルコールにより前記固相から溶出することにより、生薬由来成分を含む抽出物を調製する。酸性化する溶媒としては、ギ酸アルコール、塩酸、トリフルオロ酢酸等が挙げられる。本発明においては、ギ酸アルコールであることが好ましい。溶出液として用いるギ酸アルコールとしては、ギ酸と炭素数１〜６の低級アルコールとのエステルであることが好ましく、ギ酸メタノール又はギ酸エタノールであることがより好ましく、ギ酸メタノールであることがさらに好ましい。また、得られた溶出物は、以降の質量分析のために、蒸発乾固させる。 Then, the extract containing a crude drug origin component is prepared by eluting from the solid phase with acidic alcohol. Examples of the acidifying solvent include formic alcohol, hydrochloric acid, trifluoroacetic acid and the like. In the present invention, formic alcohol is preferred. The formic alcohol used as the eluent is preferably an ester of formic acid and a lower alcohol having 1 to 6 carbon atoms, more preferably formic acid methanol or formic acid ethanol, and further preferably formic acid methanol. The obtained eluate is evaporated to dryness for subsequent mass analysis.
次いで、定量工程として、抽出工程により抽出された抽出物中のＧＬ等を、質量分析法により検出し定量する。質量分析法としては、ＬＣ−ＭＳ（液体クロマトグラフィー／質量分析）法又はＬＣ−ＭＳ／ＭＳ（液体クロマトグラフィー／タンデム質量分析）法により行うことが好ましい。具体的には、蒸発乾固させた溶出物をＬＣの移動相に溶解させた後、ＬＣを行い、ＭＳを行う。ＬＣ−ＭＳ及びＬＣ−ＭＳ／ＭＳは、ＨＰＬＣと質量分析計を組み合わせた装置を用いることにより行うことができる。 Next, as a quantification step, GL or the like in the extract extracted by the extraction step is detected and quantified by mass spectrometry. The mass spectrometry is preferably carried out by LC-MS (liquid chromatography / mass spectrometry) or LC-MS / MS (liquid chromatography / tandem mass spectrometry). Specifically, the eluate evaporated to dryness is dissolved in the LC mobile phase, followed by LC and MS. LC-MS and LC-MS / MS can be performed by using an apparatus that combines HPLC and a mass spectrometer.
ＬＣは、特許文献１〜４に記載されているように、ＯＤＳカラム等の逆相分配機能を有するカラムを用いて行うことが好ましい。また、ＭＳは常法により行うことができる。例えば、ＥＳＩ（エレクトロスプレーイオン化）法やＡＰＣＩ（大気圧化学イオン化）法により試料をイオン化した後、磁場偏向型、四重極型、飛行時間型等の装置により、各イオンを分離して検出する。 As described in Patent Documents 1 to 4, LC is preferably performed using a column having a reverse phase distribution function such as an ODS column. Moreover, MS can be performed by a conventional method. For example, after ionizing a sample by an ESI (electrospray ionization) method or an APCI (atmospheric pressure chemical ionization) method, each ion is separated and detected by a magnetic field deflection type, quadrupole type, time-of-flight type device or the like. .
質量分析をＬＣ−ＭＳ法により行うことにより、例えば血液、血漿、又は血清中のＧＬ等の定量限界を２０ｎｇ／ｍＬ程度にまで改善することができる。同様に、ＬＣ−ＭＳ／ＭＳ法により行うことによって、血液等中のＧＬ等の定量限界を１０ｎｇ／ｍＬ以下、例えば０．５ｎｇ／ｍＬ程度にまで向上させることができる。 By performing mass spectrometry by the LC-MS method, for example, the limit of quantification of GL or the like in blood, plasma, or serum can be improved to about 20 ng / mL. Similarly, by performing the LC-MS / MS method, the limit of quantification of GL or the like in blood or the like can be improved to 10 ng / mL or less, for example, about 0.5 ng / mL.
市販試薬の大量経口投与（ＧＬとして１６００ｍｇ）における血漿中ＧＬ濃度は約５００ｎｇ／ｍＬ程度であった、という報告がなされている（Environmental Health Perspectives, 102(9), 65-68, 1994）。当該知見に基づいた比例計算の結果、肝疾患治療を目的とした場合、臨床常用量である投与量７５ｍｇ相当の推定血中濃度は約２３ｎｇ／ｍＬと算出される。つまり、本発明の生薬由来成分の高感度定量方法により、従来法では検出不可能であった血液中のＧＬが定量可能となる。特に、ＧＬ製剤等を経口摂取後の血液中のＧＬ濃度は、個体差が大きく、定量限界が不十分な測定法では、血液検体によってはＧＬが検出されない場合や、長時間の薬物動態を測定することができず、測定の精度や測定結果の信頼性が不十分となるが、本発明の生薬由来成分の高感度定量方法では、特に質量分析をＬＣ−ＭＳ／ＭＳ法により行うことによって、より信頼性の高い測定結果を得ることができる。 It has been reported that the plasma GL concentration in a large-scale oral administration (1600 mg as GL) of a commercially available reagent was about 500 ng / mL (Environmental Health Perspectives, 102 (9), 65-68, 1994). As a result of proportional calculation based on this finding, when the aim is to treat liver disease, the estimated blood concentration equivalent to a clinical dose of 75 mg is calculated to be about 23 ng / mL. That is, GL in blood that could not be detected by the conventional method can be quantified by the high-sensitivity quantification method for herbal medicine-derived components of the present invention. In particular, the GL concentration in the blood after oral intake of GL preparations, etc. has a large individual difference and the measurement limit is insufficient. However, in the high-sensitivity quantification method of herbal medicine-derived components of the present invention, particularly by performing mass spectrometry by LC-MS / MS method, the measurement accuracy and the reliability of measurement results are insufficient. A more reliable measurement result can be obtained.
次に実施例を示して本発明をさらに詳細に説明するが、本発明は以下の実施例に限定されるものではない。 EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example.
［実施例１］
本発明の生薬由来成分の高感度定量方法を、質量分析をＬＣ−ＭＳ法によりを行った場合の、血漿中のＧＬ及びＧＡの定量限界値を測定し、かつ検量線を作成した。
＜標準溶液の調製＞
まず、ＧＬ（常磐植物化学研究所製）１０．０ｍｇを正確に量り、メタノールに溶解した後、正確に１００ｍＬとし、１００μｇ／ｍＬのＧＬ標準原液を調製した。同様に、ＧＡ（アルプス薬品工業株式会社製）１０．０ｍｇを正確に量り、メタノールに溶解した後、正確に１００ｍＬとし、１００μｇ／ｍＬのＧＡ標準原液を調製した。
次いで、ＧＬ標準原液２ｍＬ及びＧＡ標準原液８ｍＬを正確に分取し、メタノールで正確に５０ｍＬとして、ＧＬ濃度が４０００ｎｇ／ｍＬ、ＧＡ濃度が１６０００ｎｇ／ｍＬである標準溶液（Ａ）を調製した。この標準溶液Ａをメタノールで順次希釈し、表１に記載の標準溶液（Ｂ）〜（Ｆ）を調製した。標準原液及び標準溶液はそれぞれ調製後冷蔵保存（５±４℃）した。調製器具はガラス製のものを用いた。
[Example 1]
When the high-sensitivity quantification method for herbal medicine-derived components of the present invention was performed by mass spectrometry using the LC-MS method, the quantification limit values of GL and GA in plasma were measured, and a calibration curve was prepared.
<Preparation of standard solution>
First, 10.0 mg of GL (manufactured by Tokiwa Phytochemical Laboratories) was accurately weighed and dissolved in methanol, and exactly 100 mL was prepared to prepare a 100 μg / mL GL standard stock solution. Similarly, 10.0 mg of GA (manufactured by Alps Pharmaceutical Co., Ltd.) was accurately weighed and dissolved in methanol, and exactly 100 mL was prepared to prepare a 100 μg / mL GA standard stock solution.
Subsequently, 2 mL of the GL standard stock solution and 8 mL of the GA standard stock solution were accurately collected and adjusted to 50 mL with methanol to prepare a standard solution (A) having a GL concentration of 4000 ng / mL and a GA concentration of 16000 ng / mL. This standard solution A was sequentially diluted with methanol to prepare standard solutions (B) to (F) shown in Table 1. The standard stock solution and the standard solution were each refrigerated after preparation (5 ± 4 ° C.). The preparation instrument was made of glass.
＜血漿サンプルの調製＞
ヒト血漿０．５ｍＬに、標準溶液（Ａ）５０μＬを添加し、十分に混和したものを血漿サンプルとした。標準溶液（Ｂ）〜（Ｆ）についても、それぞれ同様にして血漿サンプルを調製した。これらの血漿サンプルには、内部標準液（ＩＳ）５０μＬをさらに添加した。なお、本実施例では、内部標準物質としてα−Ｈｅｄｅｒｉｎを用いた。また、ブランク血漿サンプルとして、ヒト血漿０．５ｍＬにメタノールを１００μＬ添加したものを調製した。
<Preparation of plasma sample>
A plasma sample was prepared by adding 50 μL of the standard solution (A) to 0.5 mL of human plasma and mixing well. For standard solutions (B) to (F), plasma samples were prepared in the same manner. To these plasma samples, 50 μL of internal standard solution (IS) was further added. In this example, α-Hederin was used as an internal standard substance. In addition, a blank plasma sample was prepared by adding 100 μL of methanol to 0.5 mL of human plasma.
＜抽出工程＞
各血漿サンプルに、０．５６％アンモニア水１ｍＬを添加して十分に混和した後（混和物中のアンモニア濃度：０．３７％）、得られた混和物を全量、Ｏａｓｉｓ ＭＡＸ（Ｗａｔｅｒｓ社製）（以下、単に「ＭＡＸ」）に注入（アプライ）した。当該ＭＡＸは、血漿サンプルを注入する前に予め、２％ギ酸／メタノール溶液を注入した後、水を注入することにより、コンディショニングしておいた。
血漿サンプルを注入後、当該ＭＡＸに０．５６％アンモニア水／メタノール溶液（体積比１：１の混合液）で洗浄した後、メタノールでさらに洗浄した。
その後、２％ギ酸／メタノール溶液を注入し、当該ＭＡＸに吸着していたＧＬ等を溶出させた。溶出液（抽出物）は、４０℃加温下、窒素ガスにて蒸発乾固させた。
<Extraction process>
After adding 1 mL of 0.56% aqueous ammonia to each plasma sample and mixing well (ammonia concentration in the mixture: 0.37%), the total amount of the resulting mixture was Oasis MAX (manufactured by Waters). (Hereinafter simply referred to as “MAX”). The MAX was previously conditioned by injecting water after injecting a 2% formic acid / methanol solution before injecting the plasma sample.
After injecting the plasma sample, the MAX was washed with 0.56% aqueous ammonia / methanol solution (mixed solution with a volume ratio of 1: 1), and further washed with methanol.
Thereafter, a 2% formic acid / methanol solution was injected to elute GL adsorbed on the MAX. The eluate (extract) was evaporated to dryness with nitrogen gas while heating at 40 ° C.
＜定量工程＞
蒸発乾固させた抽出物を、下記移動相Ａ液／Ｂ液（体積比１：１の混合液）２５０μＬに溶解させた。このうち２０μＬを分析カラムに注入し、下記に示す条件でＬＣを行った。
装置： Ａｌｌｉａｎｃｅ ＨＴ ２６９５（Ｗａｔｅｒｓ社製）
分析カラム： ＣＡＰＣＥＬＬ ＰＡＫ Ｃ１８（ＵＧ１２０、５μｍ、１．５ｍｍ×１５０ｍｍ、資生堂社製）
移動相：Ａ液 ０．１％ギ酸、Ｂ液 ０．１％ギ酸アセトニトリル、リニアグラジエント（表２）
流速： ０．３０ｍＬ／ｍｉｎ．
カラム温度： ４０℃
試料注入量： ２０μＬ
<Quantitative process>
The extract obtained by evaporation to dryness was dissolved in 250 μL of the following mobile phase liquid A / liquid B (mixed liquid having a volume ratio of 1: 1). Of this, 20 μL was injected into the analytical column, and LC was performed under the conditions shown below.
Apparatus: Alliance HT 2695 (Waters)
Analysis column: CAPCELL PAK C18 (UG120, 5 μm, 1.5 mm × 150 mm, manufactured by Shiseido Co., Ltd.)
Mobile phase: Liquid A 0.1% formic acid, Liquid B 0.1% formic acid acetonitrile, linear gradient (Table 2)
Flow rate: 0.30 mL / min.
Column temperature: 40 ° C
Sample injection volume: 20 μL
次いで、ＬＣにより得られたＧＬ等を含む画分に対して、下記に示す条件でＭＳを行った。
装置： ＺＱ２０００（Ｗａｔｅｒｓ社製）
イオン化法： ＥＳＩ（Ｔｕｒｂｏ Ｓｐｒａｙ）
検出法： ＧＬ（負、正イオン検出)、ＧＡ・ＩＳ（正イオン検出)、ＭＲＭ（Ｍｕｌｔｉｐｌｅ ｒｅａｃｔｉｏｎ ｍｏｎｉｔｏｒｉｎｇ）
Ｃａｐｉｌｌａｒｙ電圧： ２．５ｋＶ
Ｅｘｔｒａｃｔｏｒ： ２Ｖ
ＲＦ ｌｅｎｓ： ０．２Ｖ
Ｓｏｕｒｃｅ Ｔｅｍｐｅｒａｔｕｒｅ： １１０℃
Ｄｅｓｏｌｖａｔｉｏｎ Ｔｅｍｐｅｒａｔｕｒｅ： ３５０℃
Ｄｅｓｏｌｖａｔｉｏｎ Ｇａｓ Ｆｌｏｗ： ３５０Ｌ／ｈｒ
Ｃｏｎｅ Ｇａｓ Ｆｌｏｗ： ５０Ｌ／ｈｒ
Subsequently, MS was performed on the fraction containing GL and the like obtained by LC under the conditions shown below.
Apparatus: ZQ2000 (manufactured by Waters)
Ionization method: ESI (Turbo Spray)
Detection method: GL (negative and positive ion detection), GA / IS (positive ion detection), MRM (Multiple reaction monitoring)
Capillary voltage: 2.5 kV
Extractor: 2V
RF lens: 0.2V
Source Temperature: 110 ° C
Desolation Temperature: 350 ° C
Desolvation Gas Flow: 350L / hr
Cone Gas Flow: 50L / hr
この結果、ＬＣ−ＭＳの測定結果は血漿サンプル中のＧＬ又はＧＡ濃度依存的に増大しており、血漿サンプル中のＧＬ又はＧＡ濃度とＬＣ−ＭＳの測定結果により得られた検量線は直線性を示した。すなわち、これらの結果から、血漿等の生体試料中のＧＬの濃度が２０〜４００ｎｇ／ｍＬ、ＧＡの濃度が８０〜１６００ｎｇ／ｍＬであり、ＬＣ−ＭＳを用いた本発明の生薬由来成分の高感度定量方法により、生体試料中のＧＬ等を高精度に定量可能であることが明らかである。 As a result, the measurement result of LC-MS increases depending on the GL or GA concentration in the plasma sample, and the calibration curve obtained from the GL or GA concentration in the plasma sample and the measurement result of LC-MS is linear. showed that. That is, from these results, the concentration of GL in a biological sample such as plasma is 20 to 400 ng / mL, the concentration of GA is 80 to 1600 ng / mL, and the high concentration of the crude drug-derived component of the present invention using LC-MS. It is clear that GL and the like in a biological sample can be quantified with high accuracy by the sensitivity quantification method.
＜特異性、直線性、日内再現性の検討＞
さらに、特異性、直線性、及び日内再現性を検討した。この結果、ＬＣ−ＭＳを用いた本発明の生薬由来成分の高感度定量方法は、それぞれ２０〜４００ｎｇ／ｍＬ及び８０〜１６００ｎｇ／ｍＬの範囲で良好な直線性（検量線の相関係数は０．９９７９）と再現性を示した。また、日内再現性の結果、表４に示すように、ＧＬのネガティブモードの真度は−１.２〜＋０．６％、精度は５．２〜６．０％であり、ポジティブモードの真度は−５．９〜＋５．９％、精度は３．３〜７．７％であり、ＧＡの真度は−７．０〜＋７．２％、精度は３．０〜７．７％であった。以上の結果より、ＧＬ及びＧＡの定量限界濃度はそれぞれ、２０ｎｇ／ｍＬ、８０ｎｇ／ｍＬであることが確認された。
<Examination of specificity, linearity, and intraday reproducibility>
Furthermore, specificity, linearity, and intraday reproducibility were examined. As a result, the high-sensitivity quantification method for crude drug-derived components of the present invention using LC-MS has good linearity in the range of 20 to 400 ng / mL and 80 to 1600 ng / mL, respectively (the correlation coefficient of the calibration curve is 0). .. 9799). Moreover, as shown in Table 4, the accuracy of the negative mode of GL is -1.2 to + 0.6% and the accuracy is 5.2 to 6.0% as shown in Table 4. Degree is -5.9 to + 5.9%, accuracy is 3.3 to 7.7%, GA accuracy is -7.0 to + 7.2%, accuracy is 3.0 to 7.7% Met. From the above results, it was confirmed that the quantitative limit concentrations of GL and GA were 20 ng / mL and 80 ng / mL, respectively.
［実施例２］
生体試料に混和するアルカリ又はアルコールの種類が、本発明の生薬由来成分の高感度定量方法の定量感度に及ぼす影響を調べた。
ヒト血漿０．５ｍＬに、実施例１の標準溶液（Ｅ）５０μＬ及びＩＳ５０μＬを添加し、十分に混和したものを血漿サンプルとした。
具体的には、表５に記載の溶液１ｍＬを血漿サンプルに添加してＭＡＸにアプライする混和物を調製した以外は実施例１と同様にして、ＧＬ及びＧＡを定量した。
血漿サンプルに混和した溶液が０．５６％アンモニア水であった場合の定量結果をそれぞれ回収率１００％とし、各血漿サンプルのＧＬ、ＧＡ、及びＩＳの回収率（相対値）を算出した。算出結果を表５に示す。なお、表５中のアンモニア水の後ろの括弧書内の数値は、当該アンモニア水を添加して得られた混和物中のアンモニア濃度を示す。
[Example 2]
The effect of the type of alkali or alcohol mixed with the biological sample on the quantitative sensitivity of the high-sensitivity quantitative method for herbal medicine-derived components of the present invention was examined.
A plasma sample was prepared by adding 50 μL of the standard solution (E) of Example 1 and 50 μL of IS to 0.5 mL of human plasma and mixing them well.
Specifically, GL and GA were quantified in the same manner as in Example 1 except that 1 mL of the solution shown in Table 5 was added to the plasma sample to prepare a mixture to be applied to MAX.
The quantification result when the solution mixed with the plasma sample was 0.56% aqueous ammonia was set as a recovery rate of 100%, and the recovery rates (relative values) of GL, GA, and IS of each plasma sample were calculated. Table 5 shows the calculation results. In addition, the numerical value in the parenthesis after the ammonia water in Table 5 indicates the ammonia concentration in the mixture obtained by adding the ammonia water.
この結果、アルカリやアルコールを用いた場合は、水を用いた場合よりも回収率が良好であった。逆に、リン酸や過塩素酸等の酸性溶液を添加した場合には、ＧＬは検出されず、ＧＡもほとんど回収できなかった。また、アルコールの中では、エタノールよりもメタノールを用いた場合のほうが、ＧＬとＧＡのどちらの回収率も良好であった。アルカリの中では、水酸化ナトリウム水溶液や炭酸水素ナトリウム溶液を用いた場合よりも、アンモニア水を用いた場合のほうが、良好であった。ただし、アンモニア水の濃度による違いはあまり観察されなかった。 As a result, the recovery rate was better when alkali or alcohol was used than when water was used. Conversely, when an acidic solution such as phosphoric acid or perchloric acid was added, GL was not detected and GA was hardly recovered. Further, among alcohols, both GL and GA recoveries were better when methanol was used than ethanol. Among alkalis, the use of aqueous ammonia was better than the use of an aqueous sodium hydroxide solution or a sodium bicarbonate solution. However, the difference due to the concentration of ammonia water was not observed so much.
［実施例３］
固相の洗浄液の種類が、本発明の生薬由来成分の高感度定量方法の定量感度に及ぼす影響を調べた。
ヒト血漿０．５ｍＬに、実施例１の標準溶液（Ｅ）５０μＬ及びＩＳ５０μＬを添加し、十分に混和したものを血漿サンプルとした。
具体的には、血漿サンプルを注入後のＭＡＸを表６に記載の溶液で洗浄した後、メタノールでさらに洗浄した以外は実施例１と同様にして、ＧＬ及びＧＡを定量した。
血漿サンプルを注入後のＭＡＸの洗浄液が０．５６％アンモニア水／メタノール溶液（体積比１：１の混合液）であった場合の定量結果をそれぞれ回収率１００％とし、各血漿サンプルのＧＬ、ＧＡ、及びＩＳの回収率（相対値）を算出した。算出結果を表６に示す。なお、表６中、溶液名の後ろの括弧書内の比率は、各溶液の混合比を示す。
[Example 3]
The effect of the type of the solid phase washing solution on the quantitative sensitivity of the high-sensitivity quantitative method for herbal medicine-derived components of the present invention was examined.
A plasma sample was prepared by adding 50 μL of the standard solution (E) of Example 1 and 50 μL of IS to 0.5 mL of human plasma and mixing them well.
Specifically, GL and GA were quantified in the same manner as in Example 1 except that MAX after injection of the plasma sample was washed with the solution shown in Table 6 and further washed with methanol.
When the MAX washing solution after injecting the plasma sample was a 0.56% aqueous ammonia / methanol solution (mixed solution with a volume ratio of 1: 1), the recovery rate was 100%, and the GL, The recovery rates (relative values) of GA and IS were calculated. Table 6 shows the calculation results. In Table 6, the ratio in parentheses after the solution name indicates the mixing ratio of each solution.
この結果、０．００２５Ｍ 水酸化ナトリウム水溶液／メタノール溶液（体積比１：１の混合液）で洗浄した場合にのみ、ＧＡの回収率がやや低かったものの、水、アンモニア水、アンモニア水とメタノールの混合溶液のいずれを洗浄液とした場合も、ＧＬ、ＧＡ、及びＩＳの回収率は全て良好であった。 As a result, only when washed with 0.0025M sodium hydroxide aqueous solution / methanol solution (mixed solution with a volume ratio of 1: 1), although the GA recovery rate was slightly low, water, ammonia water, ammonia water and methanol When any of the mixed solutions was used as a cleaning solution, the recovery rates of GL, GA, and IS were all good.
［実施例４］
固相の洗浄液の種類が、本発明の生薬由来成分の高感度定量方法の定量感度に及ぼす影響を調べた。
ヒト血漿０．５ｍＬに、実施例１の標準溶液（Ｅ）５０μＬ及びＩＳ５０μＬを添加し、十分に混和したものを血漿サンプルとした。
具体的には、血漿サンプルを注入後のＭＡＸを０．５６％アンモニア水／メタノール溶液（体積比１：１の混合液）で洗浄した後、エタノール、メタノール、５０％メタノール水溶液、又はアセトニトリルでさらに洗浄した以外は実施例１と同様にして、ＧＬ及びＧＡを定量した。
２度目の洗浄液がメタノールであった場合を１００％とし、各血漿サンプルのＧＬ、ＧＡ、及びＩＳの回収率（相対値）を算出した。算出結果を表７に示す。この結果、いずれの溶液で洗浄した場合であっても、ＧＬの回収率は８０％以上であり、良好であった。また、ＧＡの回収率も、５０％メタノールの場合を除き８０％以上であり、良好であった。特に、メタノールが最も良好であった。
[Example 4]
The effect of the type of the solid phase washing solution on the quantitative sensitivity of the high-sensitivity quantitative method for herbal medicine-derived components of the present invention was examined.
A plasma sample was prepared by adding 50 μL of the standard solution (E) of Example 1 and 50 μL of IS to 0.5 mL of human plasma and mixing them well.
Specifically, after the plasma sample was injected, MAX was washed with a 0.56% aqueous ammonia / methanol solution (mixed solution with a volume ratio of 1: 1), and then further added with ethanol, methanol, a 50% aqueous methanol solution, or acetonitrile. GL and GA were quantified in the same manner as in Example 1 except for washing.
The recovery rate (relative value) of GL, GA, and IS of each plasma sample was calculated assuming that the second washing solution was methanol as 100%. Table 7 shows the calculation results. As a result, the GL recovery rate was 80% or more, which was good, regardless of which solution was used for washing. Also, the GA recovery rate was 80% or higher except for 50% methanol, which was good. In particular, methanol was the best.
［実施例５］
固相からの溶出液の種類が、本発明の生薬由来成分の高感度定量方法の定量感度に及ぼす影響を調べた。
ヒト血漿０．５ｍＬに、実施例１の標準溶液（Ｅ）５０μＬ及びＩＳ５０μＬを添加し、十分に混和したものを血漿サンプルとした。
具体的には、ＭＡＸからの溶出を表８に記載の溶液を用いて行った以外は実施例１と同様にして、ＧＬ及びＧＡを定量した。
[Example 5]
The effect of the type of eluate from the solid phase on the quantitative sensitivity of the high-sensitivity quantitative method for crude drug-derived components of the present invention was examined.
A plasma sample was prepared by adding 50 μL of the standard solution (E) of Example 1 and 50 μL of IS to 0.5 mL of human plasma and mixing them well.
Specifically, GL and GA were quantified in the same manner as in Example 1 except that elution from MAX was performed using the solutions shown in Table 8.
溶出液がギ酸メタノールであった場合を１００％とし、各血漿サンプルのＧＬ、ＧＡ、及びＩＳの回収率（相対値）を算出した。算出結果を表８に示す。この結果、ギ酸メタノールが最も回収率が高かった。また、ギ酸エタノールを用いた場合も、ギ酸メタノールには及ばないものの、ＧＬとＧＡを良好に回収することができた。これに対して、溶出液がギ酸アセトニトリルやリン酸メタノールであった場合には、ＧＬは検出できなかった。 When the eluate was methanol formate, the recovery rate (relative value) of GL, GA, and IS of each plasma sample was calculated as 100%. Table 8 shows the calculation results. As a result, methanol formate had the highest recovery rate. Also, when formic acid ethanol was used, GL and GA could be recovered satisfactorily, although not as good as formic acid methanol. In contrast, when the eluent was acetonitrile formate or methanol phosphate, GL could not be detected.
［比較例１］
ＭＡＸに代えて、逆相分配機能のみを備える固相を用いて、血漿中のＧＬ及びＧＡの定量を行った。
ヒト血漿０．５ｍＬに、実施例１の標準溶液（Ｅ）５０μＬ及びＩＳ５０μＬを添加し、十分に混和したものを血漿サンプルとした。
具体的には、ＭＡＸに代えてＯＤＳカラムであるＳｅｐ−Ｐａｋ（３ｃｃ、６０ｍｇ、３０μｍ、Ｗａｔｅｒｓ社製）を用いたこと、当該カラムのコンディショニングをメタノール、次いで水で行ったこと、及び、カラムにアプライする混和物を調製するための溶液（希釈液）として０．５６％アンモニア水若しくは０．１Ｎ塩酸、固相の洗浄液として水若しくはアセトニトリル／水（体積比＝１：３、１Ｍテトラブチルアンモニウムブロミド含有）、及び固相からの溶出液として２％ギ酸メタノール、２％ギ酸アセトニトリル／水（体積比＝１：１）若しくは２％ギ酸メタノール／水（体積比＝１：１）をそれぞれ用いた以外は、実施例１と同様にして行った。
実施例２において血漿サンプルに混和した溶液が０．５６％アンモニア水であった場合の定量結果をそれぞれ回収率１００％とし、各血漿サンプルのＧＬ、ＧＡ、及びＩＳの回収率（相対値）を算出した。算出結果を表９に示す。なお、表９中、「１−１」は０．５６％アンモニア水、「１−２」は０．１Ｎ塩酸、「２−１」は水、「２−２」はアセトニトリル／水（体積比＝１：３、１Ｍテトラブチルアンモニウムブロミド含有）、「３−１」は２％ギ酸メタノール、「３−２」は２％ギ酸アセトニトリル／水（体積比＝１：１）、「３−３」は２％ギ酸メタノール／水（体積比＝１：１）である。
[Comparative Example 1]
GL and GA in plasma were quantified using a solid phase having only a reverse phase distribution function instead of MAX.
A plasma sample was prepared by adding 50 μL of the standard solution (E) of Example 1 and 50 μL of IS to 0.5 mL of human plasma and mixing them well.
Specifically, Sep-Pak (3 cc, 60 mg, 30 μm, manufactured by Waters), which is an ODS column, was used instead of MAX, conditioning of the column was performed with methanol and then water, and 0.56% aqueous ammonia or 0.1N hydrochloric acid as a solution (diluent) for preparing a mixture to be applied, water or acetonitrile / water as a solid phase washing solution (volume ratio = 1: 3, 1M tetrabutylammonium bromide) And 2% methanol formate, 2% acetonitrile formate / water (volume ratio = 1: 1) or 2% methanol formate / water (volume ratio = 1: 1) as eluents from the solid phase. Was carried out in the same manner as in Example 1.
In Example 2, when the solution mixed with the plasma sample was 0.56% ammonia water, the recovery rate was 100%, and the recovery rates (relative values) of GL, GA, and IS for each plasma sample were as follows. Calculated. Table 9 shows the calculation results. In Table 9, “1-1” is 0.56% ammonia water, “1-2” is 0.1N hydrochloric acid, “2-1” is water, “2-2” is acetonitrile / water (volume ratio). = 1: 3, containing 1M tetrabutylammonium bromide), “3-1” is 2% methanol formate, “3-2” is 2% acetonitrile / water formate (volume ratio = 1: 1), “3-3” Is 2% methanol formate / water (volume ratio = 1: 1).
この結果、希釈液等の組み合わせの中で最も回収率が良好な場合であっても、ＧＬの回収率はせいぜい５０％程度であり、ＭＡＸを用いた場合よりも遥かにＧＬ等の検出感度が劣ることが分かった。 As a result, even when the recovery rate is the best among the combinations of diluents and the like, the recovery rate of GL is at most about 50%, and the detection sensitivity of GL and the like is far higher than when MAX is used. I found it inferior.
［比較例２］
ＭＡＸに代えて、逆相分配機能と極性相互作用機能とを備える固相を用いて、血漿中のＧＬ及びＧＡの定量を行った。
ヒト血漿０．５ｍＬに、実施例１の標準溶液（Ｅ）５０μＬ及びＩＳ５０μＬを添加し、十分に混和したものを血漿サンプルとした。
具体的には、ＭＡＸに代えて多孔性ポリマーであるＯａｓｉｓ ＨＬＢ（３ｃｃ、６０ｍｇ、３０μｍ、Ｗａｔｅｒｓ社製）を用いたこと、当該カラムのコンディショニングをメタノール、次いで水で行ったこと、及び、カラムにアプライする混和物を調製するための溶液（希釈液）として０．５６％アンモニア水若しくは０．１Ｎ塩酸を、固相の洗浄液として５％メタノール水溶液を、及び固相からの溶出液としてメタノールをそれぞれ用いた以外は、実施例１と同様にして行った。
実施例２において血漿サンプルに混和した溶液が０．５６％アンモニア水であった場合の定量結果をそれぞれ回収率１００％とし、各血漿サンプルのＧＬ、ＧＡ、及びＩＳの回収率（相対値）を算出した。算出結果を表１０に示す。
[Comparative Example 2]
GL and GA in plasma were quantified using a solid phase having a reverse phase distribution function and a polar interaction function instead of MAX.
A plasma sample was prepared by adding 50 μL of the standard solution (E) of Example 1 and 50 μL of IS to 0.5 mL of human plasma and mixing them well.
Specifically, instead of MAX, a porous polymer Oasis HLB (3 cc, 60 mg, 30 μm, manufactured by Waters) was used, the conditioning of the column was performed with methanol and then water, and the column 0.56% aqueous ammonia or 0.1N hydrochloric acid is used as a solution (diluent) for preparing the mixture to be applied, 5% aqueous methanol solution is used as the solid phase washing solution, and methanol is used as the eluent from the solid phase. The same procedure as in Example 1 was performed except that it was used.
In Example 2, when the solution mixed with the plasma sample was 0.56% ammonia water, the recovery rate was 100%, and the recovery rates (relative values) of GL, GA, and IS for each plasma sample were as follows. Calculated. Table 10 shows the calculation results.
この結果、希釈液として０．１Ｎ塩酸を用いた場合には、ＧＬは検出されず、ＧＡの回収率も非常に低かった。一方、０．５６％アンモニア水を用いた場合であってさえ、ＧＬの回収率はせいぜい５５％程度であり、ＭＡＸを用いた場合よりも遥かにＧＬ等の検出感度が劣ることが分かった。 As a result, when 0.1N hydrochloric acid was used as the diluent, GL was not detected, and the GA recovery rate was very low. On the other hand, even when 0.56% ammonia water was used, the recovery rate of GL was about 55% at most, and it was found that the detection sensitivity of GL and the like was far inferior to that when MAX was used.
［比較例３］
ＭＡＸに代えて、逆相分配機能と陽イオン交換機能とを備える固相を用いて、血漿中のＧＬ及びＧＡの定量を行った。
ヒト血漿０．５ｍＬに、実施例１の標準溶液（Ｅ）５０μＬ及びＩＳ５０μＬを添加し、十分に混和したものを血漿サンプルとした。
具体的には、ＭＡＸに代えてＯａｓｉｓ ＭＣＸ（３ｃｃ、６０ｍｇ、３０μｍ、Ｗａｔｅｒｓ社製）を用いたこと、当該カラムのコンディショニングをメタノール、次いで水で行ったこと、及び、カラムにアプライする混和物を調製するための溶液（希釈液）として０．１Ｎ塩酸を、固相の洗浄液として０．１Ｎ塩酸を、及び固相からの溶出液として２％アンモニア水／メタノール溶液をそれぞれ用いた以外は、実施例１と同様にして行った。
[Comparative Example 3]
Instead of MAX, GL and GA in plasma were quantified using a solid phase having a reverse phase distribution function and a cation exchange function.
A plasma sample was prepared by adding 50 μL of the standard solution (E) of Example 1 and 50 μL of IS to 0.5 mL of human plasma and mixing them well.
Specifically, Oasis MCX (3 cc, 60 mg, 30 μm, manufactured by Waters) was used instead of MAX, the conditioning of the column was performed with methanol and then water, and the mixture applied to the column The procedure was carried out except that 0.1N hydrochloric acid was used as a solution (diluent) for preparation, 0.1N hydrochloric acid was used as the solid phase washing solution, and 2% aqueous ammonia / methanol solution was used as the eluent from the solid phase. Performed as in Example 1.
実施例２において血漿サンプルに混和した溶液が０．５６％アンモニア水であった場合の定量結果をそれぞれ回収率１００％とし、各血漿サンプルのＧＬ、ＧＡ、及びＩＳの回収率（相対値）を算出した。独立した５回の試行の算出結果及び平均値を表１１に示す。この結果、ＧＬとＧＡのいずれも回収率が５０％に届かず、ＭＡＸを用いた場合よりも遥かにＧＬ等の検出感度が劣ることが分かった。 In Example 2, when the solution mixed with the plasma sample was 0.56% ammonia water, the recovery rate was 100%, and the recovery rates (relative values) of GL, GA, and IS for each plasma sample were as follows. Calculated. Table 11 shows the calculation results and average values of five independent trials. As a result, it was found that the recovery rate of GL and GA did not reach 50%, and the detection sensitivity of GL or the like was far inferior to the case of using MAX.
［実施例６］
本発明の生薬由来成分の高感度定量方法を、質量分析をＬＣ−ＭＳ／ＭＳ法によりを行った場合の、血漿中のＧＬ及びＧＡの定量限界値を測定し、かつ検量線を作成した。また、同時に、当該方法の精度や信頼性を検証した。
＜標準溶液の調製＞
実施例１で用いた標準原液を実施例１と同様にしてメタノールにより希釈し、表１２に記載の標準溶液（Ｂ）〜（Ｇ）を調製した。
[Example 6]
When the high-sensitivity quantification method for crude drug-derived components of the present invention was performed by mass spectrometry using the LC-MS / MS method, the quantification limit values of GL and GA in plasma were measured, and a calibration curve was prepared. At the same time, the accuracy and reliability of the method were verified.
<Preparation of standard solution>
The standard stock solution used in Example 1 was diluted with methanol in the same manner as in Example 1 to prepare standard solutions (B) to (G) shown in Table 12.
＜血漿サンプルの調製及び抽出工程＞
実施例１と同様にして、標準溶液（Ｂ）〜（Ｇ）を添加したヒト血漿サンプルとブランク血漿サンプルを調製し、これらの血漿サンプルをそれぞれＭＡＸにアプライして、ＧＬ等を吸着させ、０．５６％アンモニア水／メタノール溶液及びメタノールで洗浄した後に、２％ギ酸／メタノール溶液で溶出させ、さらに蒸発乾固した。
<Plasma sample preparation and extraction process>
In the same manner as in Example 1, human plasma samples and blank plasma samples to which standard solutions (B) to (G) were added were prepared, and these plasma samples were each applied to MAX to adsorb GL and the like. After washing with .56% aqueous ammonia / methanol solution and methanol, elution was carried out with 2% formic acid / methanol solution and further evaporated to dryness.
＜定量工程＞
蒸発乾固させた抽出物を、下記移動相Ａ液／Ｂ液（体積比１：１の混合液）２５０μＬに溶解させた。このうち１μＬを分析カラムに注入し、下記に示す条件でＬＣ−ＭＳ／ＭＳを行った。
ＬＣ−ＭＳ／ＭＳシステム： ＡＰＩ４０００システム（アプライドバイオシステムズ社製）
（ＬＣ）
装置： ＬＣ−２０Ａ（島津製作所製）
分析カラム： Ｉｎｅｒｔｓｉｌ ＯＤＳ−３（２．１ｍｍ×１５０ｍｍ、粒径５μｍ、ジーエルサイエンス社製）
流速： ０．５０ｍＬ／ｍｉｎ．
カラム温度： ４０℃
オートサンプラー温度： ５℃
試料注入量： １μＬ
移動相：Ａ液 ０．１％ギ酸、Ｂ液 ０．１％ギ酸アセトニトリル、リニアグラジエント（表１３）
<Quantitative process>
The extract obtained by evaporation to dryness was dissolved in 250 μL of the following mobile phase liquid A / liquid B (mixed liquid having a volume ratio of 1: 1). 1 μL of this was injected into the analytical column, and LC-MS / MS was performed under the conditions shown below.
LC-MS / MS system: API4000 system (Applied Biosystems)
(LC)
Device: LC-20A (manufactured by Shimadzu Corporation)
Analysis column: Inertsil ODS-3 (2.1 mm × 150 mm, particle size 5 μm, manufactured by GL Sciences Inc.)
Flow rate: 0.50 mL / min.
Column temperature: 40 ° C
Autosampler temperature: 5 ° C
Sample injection volume: 1 μL
Mobile phase: Solution A 0.1% formic acid, Solution B 0.1% formic acid acetonitrile, linear gradient (Table 13)
（ＭＳ／ＭＳ）
装置： ＡＰＩ４０００（アプライドバイオシステムズ）
イオン化法： ＥＳＩ （Ｔｕｒｂｏ Ｓｐｒａｙ）
検出法： 正イオン検出 ＭＲＭ（Ｍｕｌｔｉｐｌｅ ｒｅａｃｔｉｏｎ ｍｏｎｉｔｏｒｉｎｇ）
イオンスプレー電圧（ＩｏｎＳｐｒａｙ Ｖｏｌｔａｇｅ）： ５５００Ｖ（正)
イオン源温度（Ｔｅｍｐｅｒａｔｕｒｅ）： ４００℃
カーテンガス （Ｃｕｒｔａｉｎ Ｇａｓ）： １０ (窒素)
イオンソースガス１（Ｉｏｎ Ｓｏｕｒｃｅ Ｇａｓ １）： ７０ (窒素)
イオンソースガス２（Ｉｏｎ Ｓｏｕｒｃｅ Ｇａｓ ２）： ５０（窒素)
コリジョンガス（Ｃｏｌｌｉｓｉｏｎ Ｇａｓ）： ８（窒素)
(MS / MS)
Device: API4000 (Applied Biosystems)
Ionization method: ESI (Turbo Spray)
Detection method: Positive ion detection MRM (Multiple reaction monitoring)
Ion spray voltage (IonSpray Voltage): 5500V (positive)
Ion source temperature (Temperature): 400 ° C.
Curtain Gas: 10 (nitrogen)
Ion source gas 1 (Ion Source Gas 1): 70 (nitrogen)
Ion source gas 2 (Ion Source Gas 2): 50 (nitrogen)
Collision Gas: 8 (nitrogen)
図１に、ブランク血漿サンプルのクロマトグラムを示す。図１（Ａ）はＧＬのピークの位置（ＧＬの保持時間；ｍ／ｚ ８２３→４５３）を、図１（Ｂ）はＧＡのピークの位置（ＧＡの保持時間；ｍ／ｚ ４７１→１４９）を、図１（Ｃ）はＩＳのピークの位置（ＩＳの保持時間；ｍ／ｚ ７５１→４５５）をそれぞれ示す。また、図２に、標準溶液（Ｇ）を添加した血漿サンプル（血漿中のＧＬ濃度：０．５ｎｇ／ｍＬ、ＧＡ濃度：２ｎｇ／ｍＬ）のクロマトグラムを示す。図２（Ａ）はＧＬのピーク（図中、矢頭で示す）を、図２（Ｂ）はＧＡのピーク（図中、矢頭で示す）を、図２（Ｃ）はＩＳのピーク（図中、矢頭で示す）を、それぞれ示す。ＧＬ、ＧＡ、ＩＳの保持時間は、それぞれ約１．２分、３．１分、１．４分であり、ブランク血漿サンプルのクロマトグラム上には定量を妨害するピークは認められず、ＧＬ、ＧＡ、ＩＳのピーク形状は良好であった。 FIG. 1 shows a chromatogram of a blank plasma sample. 1A shows the position of the GL peak (GL retention time; m / z 823 → 453), and FIG. 1B shows the position of the GA peak (GA retention time; m / z 471 → 149). FIG. 1C shows the IS peak position (IS retention time; m / z 751 → 455). FIG. 2 shows a chromatogram of a plasma sample (GL concentration in plasma: 0.5 ng / mL, GA concentration: 2 ng / mL) to which the standard solution (G) was added. 2A shows a GL peak (indicated by an arrow in the figure), FIG. 2B shows a GA peak (indicated by an arrow in the figure), and FIG. 2C shows an IS peak (in the figure). , Indicated by an arrowhead). Retention times of GL, GA, and IS are about 1.2 minutes, 3.1 minutes, and 1.4 minutes, respectively, and no peak interfering with quantification is observed on the chromatogram of the blank plasma sample. The peak shapes of GA and IS were good.
定量結果を表１５に示す。また、実際の添加濃度と測定値（表中、定量値）との相対誤差も同じく表１５に示した。この結果、ＧＬとＧＡの両方とも、相対誤差が１５％以内であった。すなわち、これらの結果から、血漿等の生体試料中のＧＬの濃度が０．５〜２００ｎｇ／ｍＬ、ＧＡの濃度が２〜８００ｎｇ／ｍＬである場合に、ＬＣ−ＭＳ／ＭＳを用いた本発明の生薬由来成分の高感度定量方法により、生体試料中のＧＬ等を高精度に定量可能であることが明らかである。 Table 15 shows the quantitative results. The relative error between the actual added concentration and the measured value (quantitative value in the table) is also shown in Table 15. As a result, both GL and GA had a relative error within 15%. That is, based on these results, the present invention using LC-MS / MS when the concentration of GL in a biological sample such as plasma is 0.5 to 200 ng / mL and the concentration of GA is 2 to 800 ng / mL. It is clear that GL and the like in a biological sample can be quantified with high accuracy by a high-sensitivity quantification method for herbal medicine-derived components.
＜特異性、直線性、日内、日間再現性の検討＞
さらに、米国食品医薬品局の分析法バリデーションのガイダンス（”Guidance for Industry, Bioanalytical Method Validation”, U.S. Department of Health and Human Services, Food and Drug Administration, May 2001）に従い、特異性、直線性、日内、日間再現性を検討した。また、安定性評価試験（室温下４時間及び−２０℃及び−８０℃下３カ月凍結保存における生体試料中保存安定性、凍結融解安定性、測定実試料中安定性、標準溶液安定性）についても実施した。検討結果を表１６に示す。この結果、ＬＣ−ＭＳ／ＭＳを用いた本発明の生薬由来成分の高感度定量方法は、それぞれ０．５〜２００ｎｇ／ｍＬ及び２〜８００ｎｇ／ｍＬの範囲で良好な直線性と再現性を示した。日内再現性の結果、ＧＬの真度は−１２．８〜＋４．８％、精度は４．０〜９．５％であり、ＧＡの真度は−１３．４〜＋４．４％、精度は２．２〜９．０％であった。一方、日間再現性においても、ＧＬの真度は−９．４〜＋２．０％、精度は２．１〜５．５％、ＧＡの真度は−１０．９〜＋８．１％、精度は１．３〜９．１％であった。再現性の結果より、ＧＬ及びＧＡの定量限界濃度はそれぞれ、０．５ｎｇ／ｍＬ、２ｎｇ／ｍＬである事を確認した。また、全ての安定性評価試験（生体試料中保存安定性、凍結融解安定性、測定実試料中安定性、標準溶液安定性）において、初期値と比較し±１５％以内であり安定であった。以上の結果から、米国食品医薬品局の分析法バリデーションのガイダンスに則り、本発明の生薬由来成分の高感度定量方法が信頼性の確保された定量法であることが確認された。
<Examination of specificity, linearity, intraday, daily reproducibility>
In addition, according to the US Food and Drug Administration analytical method validation guidance (“Guidance for Industry, Bioanalytical Method Validation”, US Department of Health and Human Services, Food and Drug Administration, May 2001) The reproducibility was examined. In addition, regarding stability evaluation tests (storage stability in biological samples, freeze-thaw stability, stability in actual measurement samples, stability in standard solutions) at 4 hours at room temperature and at -20 ° C and -80 ° C for 3 months. Was also implemented. The examination results are shown in Table 16. As a result, the high-sensitivity quantification method for crude drug-derived components of the present invention using LC-MS / MS shows good linearity and reproducibility in the range of 0.5 to 200 ng / mL and 2 to 800 ng / mL, respectively. It was. As a result of intraday reproducibility, the accuracy of GL is −12.8 to + 4.8%, the accuracy is 4.0 to 9.5%, and the accuracy of GA is −13.4 to + 4.4%, the accuracy. Was 2.2 to 9.0%. On the other hand, in terms of daily reproducibility, the accuracy of GL is -9.4 to + 2.0%, the accuracy is 2.1 to 5.5%, and the accuracy of GA is -10.9 to + 8.1%. Was 1.3 to 9.1%. From the reproducibility results, it was confirmed that the quantitative limit concentrations of GL and GA were 0.5 ng / mL and 2 ng / mL, respectively. In all stability evaluation tests (storage stability in biological samples, freeze-thaw stability, stability in actual measurement samples, standard solution stability), the stability was within ± 15% compared to the initial value. . From the above results, it was confirmed that the high-sensitivity quantification method for herbal medicine-derived components of the present invention was a reliable quantification method in accordance with the guidance of the US Food and Drug Administration analytical method validation.
［実施例７］
ＧＬ含有製剤を経口投与後の血漿中のＧＬの検出及び薬物動態を測定した。
なお、本実施例は、ヘルシンキ宣言に基づく倫理的原則、薬事法第１４条第３項、第８０条の２及び「医薬品の臨床試験の実施の基準（ＧＣＰ）に関する省令」（平成９年厚生省令第２８号）に従い実施されたものである。具体的には、ＧＬ含有製剤の経口投与及び血漿サンプルの採取は、出願人の依頼により、イーピーエス株式会社及び医療法人社団育生會山口病院によって、第三者委員会における承認の後、試験責任医師の管理の下、実施された。また、得られた血漿サンプルにおけるＧＬ及びＧＡの測定は、同じく出願人の依頼により、株式会社日本医学臨床検査研究所エコテクノ事業部（現医薬香粧品分析事業部）によって行われた。
また、被験者は、試験の目的、試験内容、プライバシーの保護等について十分説明された後、書面にて試験への参加を同意した健康な日本人男性６名（年齢：２０歳以上３５歳以下、事前検査時ＢＭＩ：１８．５以上２５．０以下）を対象とした。
ＧＬ含有製剤として、グリチルリチン酸一アンモニウム、グリシン、ＤＬ−メチオニンを配合した糖衣錠「グリチロン（登録商標）配合錠」を用いた。グリチロン配合錠は、十分な水分と共に臨床常用量に従い、３錠の単回経口投与とした。投与及び採血時間などの試験スケジュールを表１７に示す。得られた血液は、３０分以内に遠心分離処理（４℃、３０００ｒｐｍ、１０分間）により血漿として１ｍＬ以上を回収し、濃度測定時まで−２０℃以下で凍結保存した。
[Example 7]
The detection and pharmacokinetics of GL in plasma after oral administration of a GL-containing preparation were measured.
This example is based on the ethical principles based on the Declaration of Helsinki, Article 14, Paragraph 3 of the Pharmaceutical Affairs Law, Article 80-2, and “Ministerial Ordinance on Standards for Conducting Clinical Trials of Drugs (GCP)” (1997 Ministry of Health and Welfare) This was carried out in accordance with Ordinance No. 28). Specifically, oral administration of GL-containing preparations and collection of plasma samples will be conducted at the request of the applicant, by EPS Co., Ltd. and the medical corporation Ikusei Yamaguchi Hospital, after approval by a third party committee, Conducted under the control of In addition, the measurement of GL and GA in the obtained plasma sample was performed by the Eco-Techno Division (currently Pharmaceutical Cosmetics Analysis Division) of the Japan Medical Clinical Laboratory Laboratories at the request of the applicant.
The subjects were 6 healthy Japanese men (age: 20 to 35 years old, who agreed to participate in the study in writing after having fully explained the purpose of the study, study content, privacy protection, etc. Preliminary inspection BMI: 18.5 to 25.0).
As a GL-containing preparation, a sugar-coated tablet “Glycylon (registered trademark) combination tablet” containing monoammonium glycyrrhizinate, glycine, and DL-methionine was used. The glycyrone combination tablets were given as a single oral administration of 3 tablets according to the clinical normal dose together with sufficient water. Test schedules such as administration and blood collection time are shown in Table 17. The obtained blood was collected by centrifugation (4 ° C., 3000 rpm, 10 minutes) within 30 minutes, and 1 mL or more of plasma was collected and stored frozen at −20 ° C. or less until the concentration measurement.
採取された血漿サンプル０．５ｍＬに、０．５６％アンモニア水１ｍＬを添加して十分に混和した後、実施例１と同様にして、得られた混和物をＭＡＸにアプライしてＧＬ等を吸着させ、０．５６％アンモニア水／メタノール溶液及びメタノールで洗浄した後に、２％ギ酸／メタノール溶液で溶出させ、さらに蒸発乾固した。
次いで、実施例６と同様にして、蒸発乾固させた抽出物に対してＬＣ−ＭＳ／ＭＳを行い、各血漿サンプル中のＧＬ濃度及びＧＡ濃度を測定した。図３は、ＧＬの平均血漿中濃度推移を示した図であり、図４は、ＧＡの平均血漿中濃度推移を示した図である。なお、図３及び図４は、６検体の平均値及び標準偏差を示している。この結果、本発明の生薬由来成分の高感度定量方法により、従来から代替的な指標とされてきた主代謝物であるＧＡのみならず、未変化体であるＧＬ自体の血漿中濃度を直接測定し得ることが確認された。すなわち、本発明により、血漿中ＧＬの微量検出に初めて成功し、長らく不明であったＧＬの血中動態が明らかにされた。
Add 0.5 mL of 0.56% aqueous ammonia to 0.5 mL of the collected plasma sample and mix well, then apply the resulting mixture to MAX in the same manner as in Example 1 to adsorb GL, etc. The mixture was washed with 0.56% aqueous ammonia / methanol solution and methanol, eluted with 2% formic acid / methanol solution, and evaporated to dryness.
Subsequently, in the same manner as in Example 6, LC-MS / MS was performed on the evaporated and dried extract, and the GL concentration and the GA concentration in each plasma sample were measured. FIG. 3 is a graph showing changes in the average plasma concentration of GL, and FIG. 4 is a graph showing changes in the average plasma concentration of GA. 3 and 4 show the average value and standard deviation of six samples. As a result, the high-sensitivity quantification method for herbal medicine-derived components of the present invention directly measures the plasma concentration of GL itself, which is not changed, as well as the main metabolite GA, which has conventionally been an alternative index. It was confirmed that That is, the present invention succeeded for the first time in the detection of a very small amount of plasma GL, and revealed the blood dynamics of GL, which has been unknown for a long time.
＜薬物動態の解析＞
ＧＬ及びＧＡの測定データから、最高血漿中濃度（Ｃｍａｘ）、最高血漿中濃度到達時間（Ｔｍａｘ）を算出した。また、投与後４８時間までの血漿中濃度-時間曲線下面積（ＡＵＣ０→４８ｈ）は台形法により、消失速度定数（ｋｅｌ）及び消失半減期（ｔ１／２）は消失相から最小二乗法により算出した。また、投与後無限大時間までの血漿中濃度−時間曲線下面積（ＡＵＣ０―∞）、体内滞留時間（ＭＲＴ）についても算出した。
この結果、本実施例では、ＧＬのＣｍａｘは約１０〜４０ｎｇ／ｍＬの範囲にあり、平均値として２４．８ｎｇ／ｍＬであった。これは、前述の市販試薬の大量経口投与例に基づいた予測値（約２３ｎｇ／ｍＬ）とほぼ同等であったことから、ＧＬの投与量と血漿中濃度の相関性が示された。また、Ｔｍａｘは平均４．５時間であったが、それ以前（１〜２時間）あるいは以後（１２時間）など複数の濃度ピークを示す被験者も観察された。後方の濃度ピークは食後に出現したことから、食事摂取がＧＬの吸収あるいは腸肝循環に影響を与える可能性も示唆された。
<Pharmacokinetic analysis>
From the measurement data of GL and GA, the maximum plasma concentration (Cmax) and the maximum plasma concentration arrival time (Tmax) were calculated. In addition, the area under the plasma concentration-time curve (AUC 0 → 48h ) up to 48 hours after administration is determined by the trapezoidal method, and the elimination rate constant (kel) and elimination half-life (t 1/2 ) are calculated from the elimination phase using the least squares method. Calculated by In addition, the area under the plasma concentration-time curve (AUC 0-∞ ) and the residence time in the body (MRT) until infinite time after administration were also calculated.
As a result, in this example, the Cmax of GL was in the range of about 10 to 40 ng / mL, and the average value was 24.8 ng / mL. This was almost the same as the predicted value (about 23 ng / mL) based on the large-scale oral administration example of the above-mentioned commercially available reagent, and thus a correlation between the dose of GL and the plasma concentration was shown. In addition, Tmax was an average of 4.5 hours, but subjects who showed multiple concentration peaks before (1-2 hours) or after (12 hours) were also observed. The backward concentration peak appeared after meals, suggesting that dietary intake may affect GL absorption or enterohepatic circulation.
一方、ＧＬの主代謝物であるＧＡは、血漿中に確認されるまで４時間のラグが確認され、６時間後にＧＬの約１０倍の血漿中濃度(モル換算で約２０倍)が検出された。ｉｎ ｖｉｔｒｏにおいては、ヒト肝ミクロソームを用いてＧＬを至適条件下でインキュベートした結果、速やかに中間代謝物である３−ｍｏｎｏｇｌｕｃｕｒｏｎｙｌ−ｇｌｙｃｈｒｒｅｔｉｎｉｃ ａｃｉｄへ変換されたが、ＧＡの生成は僅かであった（Biochemical Pharmacology,42(6/7)1025-1029,1991。）。また、ｇｅｒｍ−ｆｒｅｅラットにおいてＧＬを経口投与した結果、血漿及び糞便中にＧＡが確認されないことから、生体内でのＧＡ生成は腸内細菌が支配的に担っていると考えられている（引用：J. Pharm. Pharmacol. 46, 135-137, 1994）。したがって、吸収のラグは腸内細菌叢へ接触するまでの時間と推測され、食事条件（あるいは絶食条件）が薬物の消化管内の移動、腸内細菌叢の代謝能、消化管吸収に影響し、ＧＡ曝露量を左右する要因とも考えられた。吸収後のＧＡ動態を被験者別に観察すると、未変化体濃度が高い被験者において代謝物の出現が弱く、反対に未変化体濃度が低い被験者において代謝物の出現が強い傾向が認められた。このことから、ＧＡと同時に未変化体であるＧＬの消化管吸収も腸内細菌叢の代謝能と関連し、それが個人差として発現する可能性が示唆された（データ不掲載）。 On the other hand, GA, which is the main metabolite of GL, is confirmed to have a lag of 4 hours until it is confirmed in plasma, and after 6 hours, a plasma concentration about 10 times that of GL (about 20 times in terms of mole) is detected. It was. In vitro, as a result of incubating GL under optimal conditions using human liver microsomes, it was rapidly converted to 3-monoglucouronyl-glycrhetic acid, an intermediate metabolite, but there was little GA production ( Biochemical Pharmacology, 42 (6/7) 1025-1029, 1991.). Moreover, as a result of oral administration of GL in germ-free rats, GA is not confirmed in plasma and feces, and therefore it is considered that intestinal bacteria are predominantly responsible for the production of GA in vivo. : J. Pharm. Pharmacol. 46, 135-137, 1994). Therefore, the absorption lag is estimated to be the time to contact the intestinal flora, and the dietary conditions (or fasting conditions) affect the movement of drugs in the gastrointestinal tract, the metabolic capacity of the intestinal flora, gastrointestinal absorption, It was also considered to be a factor affecting the GA exposure. When the GA dynamics after absorption was observed for each subject, the appearance of metabolites was weak in subjects with a high unchanged substance concentration, and conversely, the appearance of metabolites was strong in subjects with a low unchanged substance concentration. This suggests that the gastrointestinal absorption of GL, which is unchanged as well as GA, is also related to the metabolic ability of the intestinal flora, which may be expressed as individual differences (data not shown).
本発明の生薬由来成分の高感度定量方法により、臨床用量のＧＬ含有製剤や飲食品等を摂取した後の血液中のＧＬを定量することができるため、本発明の生薬由来成分の高感度定量方法は、主に、医薬品・漢方の適正使用、安全性評価、薬物動態解析、新規のＧＬ製剤開発等に利用することが可能である。 Since the GL in the blood after ingesting a clinical-dose GL-containing preparation or food / drink can be quantified by the high-sensitivity quantification method of the crude drug-derived component of the present invention, the sensitive quantification of the crude drug-derived component of the present invention The method can be used mainly for proper use of pharmaceuticals and Kampo, safety evaluation, pharmacokinetic analysis, development of new GL preparations, and the like.
Claims (4)
前記抽出工程により抽出された抽出物中のグリチルリチン、グリチルレチン酸、グリチルリチン及びグリチルレチン酸の代謝物、グリチルリチン及びグリチルレチン酸の類縁物質、甘草に含有されているサポニン成分、並びにこれらの薬学的に許容される塩からなる群より選択される少なくも１種以上を、質量分析法により検出し定量する定量工程と、
を有し、
前記洗浄液が、水、アルカリ、アルコール、及びアセトニトリルからなる群より選択される１種又は２種以上の混合液であり、
前記抽出工程において、固相の洗浄を、アルカリとアルコールと水との混合液、又はアルカリと水との混合液により洗浄した後、アルコール、アルコールと水の混合液、又はアセトニトリルにより洗浄することによって行うことを特徴とする生薬由来成分の高感度定量方法。 A mixture obtained by mixing a biological sample with an alkali or alcohol is injected into a solid phase having a reverse phase distribution function and an anion exchange function, and then the solid phase is washed with a washing solution at least twice, and then the solid is washed with acidic alcohol. An extraction step of preparing an extract containing herbal medicine-derived components by eluting from the phase;
Glycyrrhizin, glycyrrhetinic acid, metabolites of glycyrrhizin and glycyrrhetinic acid, related substances of glycyrrhizin and glycyrrhetinic acid, saponin components contained in licorice, and pharmaceutically acceptable substances thereof in the extract extracted by the extraction process A quantitative step of detecting and quantifying at least one selected from the group consisting of salts by mass spectrometry;
Have
The cleaning solution is one or a mixture of two or more selected from the group consisting of water, alkali, alcohol, and acetonitrile;
In the extraction step, the solid phase is washed with a mixture of alkali, alcohol and water, or a mixture of alkali and water, and then washed with alcohol, a mixture of alcohol and water, or acetonitrile. A highly sensitive method for quantitative determination of crude drug-derived components, which is performed.
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| KR1020137012465A KR101476144B1 (en) | 2010-11-16 | 2011-11-15 | Highly sensitive method of determining the quantity of herbal medicine-derived components |
| RU2013125221/15A RU2558042C2 (en) | 2010-11-16 | 2011-11-15 | High-sensitivity method for measuring number of components recovered from medicinal herbs |
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