RU2558042C2 - High-sensitivity method for measuring number of components recovered from medicinal herbs - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to a high-sensitivity method for measuring the amount of individual's blood plasma glycyrrhizin, glycyrrhetinic acid and their pharmaceutically acceptable salts. The high-sensitivity method for measuring the amount of glycyrrhizin, glycyrrhetinic acid and their pharmaceutically acceptable salts is characterised by the fact that a mixture of individual's blood plasma with methanol or ammonia water in the specific concentration is introduced into a solid phase having the reverse-phase distribution function and the anion exchange function; the solid phase is then washed with a cleaning fluid that is a single-component fluid or a mixed fluid of at least two components specified in a group containing water, alkali, alcohol and acetonitrile. That is followed by elution from the solid phase in acid alcohol specified in formic acid - methanol or formic acid - ethanol; that is followed by the stage of measuring glycyrrhizin, glycyrrhetinic acid and their pharmaceutically acceptable salts by liquid chromatography - mass spectrometry or liquid chromatography - mass spectrometry/mass spectrometry.

EFFECT: high-sensitivity method enables detecting and measuring the amounts of individual's blood plasma glycyrrhizin, glycyrrhetinic acid and their pharmaceutically acceptable salts.

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Description

FIELD OF THE INVENTION

The present invention relates to a highly sensitive method for the quantitative determination of glycyrrhizin, its metabolites or the like, in biological samples.

Priority is claimed for the present invention in Japanese Patent Application No. 2010-256187, filed November 16, 2010, the contents of which are incorporated herein by reference.

State of the art

Glycyrrhizin (hereinafter abbreviated as “GL”) and its salts are components of licorice root extract having various physiological properties, such as anti-allergenic and anti-inflammatory properties, as well as immunoregulatory properties, hepatoprotective properties, the property of stimulating the growth of hepatocytes and the property of inhibiting / inactivating the growth of viruses. In Japan, GL and its salts have long been used as clinical medicinal products, for example, in Chinese medicine of medicinal herbs, and are now widely used in the treatment of chronic liver diseases, weeping eczema, childhood strofulus, focal alopecia, with various allergies, inflammatory diseases and etc. For applications other than pharmaceuticals, GL is often used as a sweetener in a wide range of foods, such as marinades and seasonings, due to its salt-ripening and sweetening properties.

Based on non-clinical studies, it can be concluded that GL has a tendency to accumulate in the liver, is rapidly secreted with bile and is thrown out, undergoing metabolic and hepatic-intestinal recirculation. In particular, upon oral administration, GL, which is a glycoside and a water-soluble polar substance, is poorly absorbed, and its concentration in the peripheral blood is extremely low due to the effect of the first passage and metabolism of intestinal bacteria. As a result, various methods for measuring the kinetics of GL in blood have been tried before, but detecting GL in the blood during administration of formulations containing GL or Chinese licorice based herbal formulations is very difficult, and its kinetics have long been unclear.

For example, there are methods in which methanol is processed to remove proteins from blood plasma collected from a healthy person who was orally administered GL, followed by measuring the amount of GL in blood plasma by: enzyme-linked immunosorbent assay (ELISA); or measurements using high performance liquid chromatography (HPLC) using a column operating in reverse phase, such as a column with a carrier such as a chemically bonded group of octadecyl (ODS) (see, for example, non-patent documents 1-4). However, with such measurement methods, the quantitative detection limit is at most about 0.1 μg / ml, and GL is not detected, even though glycyrrhetinic acid (hereinafter abbreviated "GA"), which is the main metabolite, can be detected. For this reason, the migration of GL in the blood was only indirectly confirmed by detection in the urine (see Non-Patent Document 4). With regard to the pharmacological efficacy of formulations containing GL, instead of GL, the quantitatively measured GA was an evaluation indicator.

Background Documents

Non-Patent Document 1: Ishiwata and 7 co-authors. Biological and Pharmaceutical Bulletin, 2000, Vol.23, No.8, pp. 904-905.

Non-Patent Document 2: De Groot and one co-author. Journal of Chromatography, 1988, Vol. 456, pp. 71-81.

Non-Patent Document 3: Nakata, and 3 co-authors. Medical and Pharmaceutical Society for Wakan-Yaku, 1986, Vol. 3, No. 3, pp. 278-279.

Non-Patent Document 4: Yamamura and 7 Collaborators. Journal of Pharmaceutical Sciences, 1992, Vol. 81, No.10, pp. 1042-1046.

Disclosure of invention

Tasks Solved by the Present Invention

The pharmacokinetics of unchanged GL, which is an activator, is useful for proper use in terms of efficacy and safety. Thus, it is desirable to have a method for quantifying the amount of GL in biological samples, such as blood plasma, not linked to a GA study.

The present invention has been made in light of the above problem, and an object of the invention is to provide a method for detecting and quantifying GL in biological samples such as blood plasma.

Means of solving these problems

As a result of painstaking studies aimed at solving the above problem, the authors of the present invention found that GL in biological samples can be detected and quantified by extraction of components including GL from a biological sample by solid phase extraction using a solid phase with a reversed-phase distribution function and anion exchange function, followed by directing GL as an extract to mass spectrometry. Thus was developed the present invention.

Thus, the present invention describes:

(1) a highly sensitive method for determining the amount of components derived from medicinal herbs, including:

an extraction stage in which an extract containing components obtained from medicinal herbs is prepared as follows: a mixture containing a biological sample with alkali or alcohol is introduced into the solid phase having a reversed-phase distribution function and anion exchange function; the solid phase is washed at least once with a cleaning liquid; and then carry out the elution from the solid phase with acidic alcohol, and

a quantitative determination step in which at least one component selected from the group consisting of glycyrrhizin, glycyrrhetinic acid, metabolites of glycyrrhizin and glycyrrhetinic acid, compounds related to glycyrrhizin and glycyrrhetinic acid, components of saponin contained in licorice (licorice), and their pharmacologically acceptable salts, and contained in the extract extracted at the stage of extraction, is detected and quantified by mass spectrometry;

where the cleaning liquid is a single component liquid or a liquid mixture of at least two components selected from the group comprising water, alkali, alcohol and acetonitrile;

(2) a highly sensitive method for determining the amount of components derived from medicinal herbs according to (1) above, wherein, in the extraction step, the solid phase is washed with a liquid mixture of alkali, alcohol and water, followed by washing with alcohol, a liquid mixture of alcohol and water, or acetonitrile;

(3) a highly sensitive method for determining the amount of components obtained from medicinal herbs according to (2) above, wherein in a liquid mixture of alkali, alcohol and water, 0.5-28 vol.% Aqueous ammonia and methanol are mixed in a ratio of 99: 1 to 1: 3, and also

(4) a highly sensitive method for determining the amount of components derived from medicinal herbs according to any one of (1) to (3) above, wherein the biological sample is blood, blood plasma or tissue extract.

Advantages of the Invention

In accordance with a highly sensitive method for determining the amount of herbal components of the present invention, very small amounts of GL in a biological sample can be detected and quantified. As a result, when using the highly sensitive method for determining the amount of the components obtained from medicinal herbs of the present invention, it is possible to quantify GL in biological samples taken from people who absorbed GL-containing compounds or foods or drinks, and its pharmacokinetics can be accurately analyzed.

Brief Description of the Drawings

Figa is a graphical image corresponding to the chromatogram GL in the control sample of blood plasma according to example 6.

Figv is a graphical image corresponding to the chromatogram GA in the control sample of blood plasma according to example 6.

Fig. 1C is a graphical representation corresponding to an internal standard (IS) chromatogram in a control plasma sample of Example 6.

Fig. 2A is a graphical representation corresponding to a GL chromatogram in a blood plasma sample to which a standard solution (G) is added (blood plasma GL content: 0.5 ng / ml) according to Example 6.

FIG. 2B is a graphical representation corresponding to a GA chromatogram in a blood plasma sample to which a standard solution (G) (plasma GA content: 2 ng / ml) was added, as in Example 6.

FIG. 2C is a graphical representation corresponding to an internal standard (IS) chromatogram in a blood plasma sample to which a standard solution (G) is added, as in Example 6.

Figure 3 is a graphical representation that illustrates changes in the average concentration of GL in blood plasma in example 7.

Figure 4 is a graphical image that illustrates the changes in the average concentration of GA in blood plasma in example 7.

The implementation of the invention

A highly sensitive method for determining the amount of components obtained from medicinal herbs of the present invention involves the extraction of GL from a biological sample by solid phase extraction using a solid phase having a reversed-phase distribution function and anion exchange function, followed by quantitative determination of the GL content in the obtained extract by mass spectrometry . GL detection sensitivity is increased by quantification using mass spectrometry instead of the usual HPLC method and the like. In addition, in contrast to solid phase extraction using a solid phase having only a reversed-phase distribution function, as a normal ODS column, the method according to the invention is carried out using a solid phase having both a reversed-phase distribution function and an anion-exchange function, which allows you to remove from the extract sent to mass spectrometry, a lot of foreign substances, and significantly increase the sensitivity of mass spectrometry.

In particular, a highly sensitive method for determining the amount of components obtained from medicinal herbs of the present invention comprises an extraction step in which an extract is prepared containing components obtained from medicinal herbs by feeding a mixture of a biological sample with alkali or alcohol to a solid phase having a reversed-phase distribution function and anion exchange function, and then the solid phase is washed one, two or more times with a cleaning liquid, and then elution is carried out from the solid acid alcohol phase; and a quantification step for detecting and quantifying by mass spectrometry a substance of at least one type selected from the group consisting of GL, GA, GL and GA metabolites, related substances GL and GA, components of saponin contained in licorice and their pharmacologically acceptable salts, in the extract obtained in the extraction step; wherein the cleaning liquid is a one-component liquid or a liquid mixture of at least two components selected from the group consisting of water, alkali, alcohol and acetonitrile.

It is preferable that an extract containing at least two components derived from medicinal herbs be prepared at the extraction stage, with subsequent detection and quantification of at least two components obtained from a lower grade medicinal product, at the stage of quantification in one measurement (c using various detection methods).

Herbal components that can be quantified by a highly sensitive method for determining the amount of herbal components of the present invention are at least one component selected from the group consisting of GL, GA, GL and GA metabolites, related compounds GL and GA, components of saponin contained in licorice, as well as their pharmacologically acceptable salts (hereinafter abbreviated as "GL, etc.").

Examples of GL and GA metabolites include 3-monoglucuronyl-glycyrrhetinic acid (20β-carboxy-11-oxo-30-norolean-12-en-3β-yl-β-D-glucopyranosiduronic acid), 30-monoglucuronyl-glycyrrhetinic acid (3β- hydroxy-11-oxo-olean-12-en-30-oyl-β-D-glucopyranosiduronic acid), 3-oxo-GA (3,11-dioxoleolean-12-en-30-acid), 3α-GA (3α- hydroxy-11-oxo-olean-12-en-30-oic acid), triple sulfoconjugate (3β-hydroxysulfonyloxy-11-oxo-olean-12-en-30-oic acid), 3β, 22α-dihydroxy-11-oxoolean-12-en -30-acid, 3β, 24-dihydroxy-11-oxoolean-12-en-30-acid, and the like Noe.

Examples of related compounds GL and GA 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-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) and the like.

Examples of licorice saponin components include licorice-saponin C1 (20β-carboxy-11-oxo-30-norolean-12-en-3β-yl (β-D-glucopyranosyluronic acid) - (1 → 2) -β- D-glucopyranosiduronic acid) and the like.

There are no particular restrictions on the pharmacologically acceptable salts of GL, etc. used in the present invention, provided that they are salts having the same in vivo pharmacological properties as GL, etc. Specific examples of salts include ammonium salts, sodium salts, potassium salts and the like.

In the present invention, one type of GL, etc. may be quantified. or at least two types of GL, etc. can be quantified in one operation. In a particularly preferred embodiment of the present invention, GL and GA are simultaneously quantified.

Although the biological samples used in the highly sensitive method for determining the amount of the components of the present invention derived from medicinal herbs can be any samples taken from a living organism, in a preferred embodiment, the biological samples are samples taken from humans or animals, such as mice or rats. Biological samples may be blood, blood plasma, blood serum, urine, peritoneal fluid, pleural fluid, synovial fluid, bone marrow, bile, extracts of tissue fragments or the like (tissue extracts) collected from tissues such as the liver, pancreas, or kidneys, or the like. Tissue extracts can be prepared by homogenizing tissue fragments or the like. using conventional methods. In the present invention, the biological samples are preferably blood, blood plasma, urine or tissue extracts, and more preferably blood plasma.

The following is a description of each of the stages of a highly sensitive method for determining the amount of components of the present invention obtained from medicinal herbs.

At the extraction stage, a mixture of the biological sample with alkali or alcohol is first prepared. There is no particular limitation on the alkali that is mixed with the biological sample, provided that the pH of the resulting mixture can be adjusted in the alkaline region. Examples of alkalis include ammonia, ammonia water (aqueous ammonia solution), sodium hydroxide solution, sodium hydrogen carbonate solution and the like. As the alcohol, lower alcohols with a carbon number of 1-6 are preferred. Examples of alcohols include methanol, ethanol, isopropanol and the like. Alcohol solutions diluted with water can also be used. The present invention preferably uses ammonia, ammonia water, methanol or an aqueous methanol solution, and more preferably, ammonia or ammonia water is used as the alkali or alcohol mixed with the biological sample.

There are no specific restrictions on the concentration of ammonia water added to the biological sample, and this concentration can be adjusted accordingly, taking into account factors such as the type of biological sample and solid phase used and the ammonia concentration of the resulting mixture. In the present invention, the concentration of ammonia or ammonia water added to the biological sample is preferably such that the concentration of ammonia in the resulting mixture is 0.01-30 vol.%, More preferably 0.05-25 vol.%, And even more preferably 0.05-20 vol.%.

Then, the resulting mixture is introduced into the solid phase, which has a reversed-phase distribution function and anion-exchange function, for the adsorption of GL, etc. from the mixture to the solid phase. Examples of solid phases having a reversed-phase distribution function and anion-exchange function include an Oasis MAX mixed phase solid-state extraction / reverse-phase distribution / anion-exchange cartridge (from Waters Corp.,) and the like.

The solid phase in which GL is adsorbed, etc., is washed one, two or more times with a cleaning liquid to remove non-specifically bound components. A cleaning liquid is a one-component liquid or a liquid mixture of at least two components selected from the group consisting of water, alkali, alcohol and acetonitrile. As the alkali or alcohol used as the cleaning fluid, the exact same alkali or alcohol that can be added to the biological sample can be used. The washing of the solid phase can be carried out twice or more times using one type of cleaning liquid or with the sequential use of different types of cleaning liquids.

In the present invention, the solid phase is preferably washed with a liquid mixture of alkali, alcohol and water, a liquid mixture of alkali and water, or water, followed by washing with alcohol, a liquid mixture of alcohol and water, or acetonitrile, and more preferably the solid phase is washed with a liquid mixture of alkali, alcohol and water, followed by washing with alcohol, a liquid mixture of alcohol and water, or acetonitrile. Preferably, the liquid mixture of alkali, alcohol and water is a liquid mixture of ammonia, alcohol and water, and even more preferably a liquid mixture of ammonia, methanol and water.

There are no specific restrictions on the ratio of the amounts of the respective components of a liquid mixture of ammonia, methanol and water, provided that a state is maintained in which GL, etc. adsorbed into the solid phase. For example, it is preferable that the concentration of methanol in the liquid mixture is 1-75 vol.%, And the concentration of ammonia in the liquid phase is 0.1-21 vol.%, And even more preferably, the concentration of methanol in the liquid phase is 25-75 vol. %, and the concentration of ammonia in the liquid phase was 0.1-21 vol.%. A liquid mixture with such a ratio of the amounts of the components can, for example, be prepared by mixing 0.5-28 vol.% Ammonia water with methanol in a ratio of from 99: 1 to 1: 3.

Preferably, an alcohol, more preferably methanol or ethanol, and even more preferably methanol, is used as a cleaning liquid for the second washing after performing the first washing with a liquid mixture of alkali, alcohol and water.

Then, by elution from the solid phase with acidic alcohol, an extract is obtained containing components obtained from medicinal herbs. Examples of solvents for acidification include formic acid-alcohol, hydrochloric acid, trifluoroacetic acid and the like. In the present invention, formic acid-alcohol is a preferred embodiment. As the formic acid-alcohol used for elution, an ester of formic acid and a lower alcohol with a carbon number of 1-6 is preferably used, more preferably formic acid-methanol or formic acid-ethanol, and even more preferably formic acid-methanol . The resulting eluted product was evaporated to dryness for subsequent mass spectrometry.

Then GL, etc. in the elution product extracted at the extraction stage, detect and quantify by mass spectrometry at the quantification stage. As the mass spectrometry method, the LC-MS (liquid chromatography / mass spectrometry) method or the LC-MS / MS (liquid chromatography / tandem mass spectrometry) method is preferably used. In particular, one stripped off to dryness the product of elution is dissolved in the mobile phase of LC, followed by LC and MS. LC-MS and LC-MS / MS can be carried out using a device combining HPLC with a mass spectrometer.

As noted in Non-Patent Documents 1-4, LC is preferably carried out using a column having a reversed-phase distribution function, such as ODS columns. MS can be carried out using conventional methods. For example, samples are ionized by ESI (electrospray ionization) or APCI (chemical atmospheric pressure ionization), followed by separation and detection of the corresponding ions using a magnetic deflection device, quadrupole type, time-of-flight type, or the like.

When performing mass spectrometry by LC-MS, a quantitatively determined level of GL, etc. in blood, plasma or serum can be improved up to 20 ng / ml. Similarly, when performing mass spectrometry by LC-MS / MS, a quantitatively determined level of GL, etc. in blood, etc. can be improved to not more than 10 ng / ml, for example to about 0.5 ng / ml.

It has been previously reported that plasma concentrations of GL are approximately 500 ng / ml when a commercially available reagent is administered orally at a high dose (1600 mg, calculated as GL) (Environmental Health Perspectives, 102 (9), 65-68, 1994 ) As a result of the proportional calculation based on the aforementioned observation, it can be concluded that the blood concentration when administered at a dose of 75 mg (normal clinical dose) is estimated to be approximately 23 ng / ml when the goal is to treat liver disease. Thus, a highly sensitive method for determining the amount of components obtained from medicinal herbs of the present invention allows to quantify the level of GL in the blood, which cannot be detected by conventional methods. Although blood GL concentrations after oral administration of formulations including GL or the like are characterized by significant variability, there are times when measurement methods having insufficient quantitative detection limits may make it impossible or impossible to detect GL in some blood samples will measure the long-term pharmacokinetics of GL, which will lead to insufficient measurement accuracy or insufficient reliability of the measurement results, and a highly sensitive method for determining the amount of Twa components obtained from herbs of the present invention make it possible to obtain a higher reliability of measurement results, in particular by mass spectrometry by LC-MS / MS.

Examples

The following examples are presented in order to describe the present invention in more detail, but the present invention is not limited to these examples.

Example 1

The limiting detectable amounts of GL and GA in the blood plasma were measured, and calibration curves were constructed for the case when a highly sensitive method for determining the amount of components obtained from medicinal herbs of the present invention was carried out using LC-MS mass spectrometry.

Getting a standard solution

First, 10.0 mg of GL (manufactured by Tokiwa Phytochemical., Ltd.) was accurately weighed and then dissolved in methanol. After that, the volume of the solution was precisely set to 100 ml, obtaining 100 μg / ml of a standard GL stock solution. Similarly, 10.0 mg GA (manufactured by Alps Pharmaceutical Industries Co., Ltd.) was weighed accurately and then dissolved in methanol. After that, the volume of the solution was precisely set to 100 ml, obtaining 100 μg / ml of a standard GA stock solution.

Then, 2 ml of standard GL stock solution and 8 ml of GA standard stock solution were precisely mixed. The total volume was adjusted with methanol to exactly 50 ml to obtain a standard solution (A) in which the GL concentration was 4000 ng / ml and the GA concentration was 16000 ng / ml. The resulting standard solution A was successively diluted with methanol to obtain standard solutions (B) - (F), are shown in table 1. The original standard solutions and the resulting standard solutions were stored at low temperatures (5 ± 4 ° C) after preparation. In the preparation used glass tools.

Table 1 Standard solutions, ng / ml BUT AT FROM D E F GL 4000 2000 1500 1000 500 200 GA 16000 8000 6000 4000 2000 800

Obtaining blood plasma samples

50 μl of the standard solution (A) was added to 0.5 ml of human blood plasma, and thoroughly mixed to obtain a sample of blood plasma. The same procedure was carried out respectively with standard solutions (B) - (F) to obtain the corresponding samples of blood plasma. Then, 50 μl of an internal standard (IS) solution was added to the appropriate blood plasma samples. In the present example, α-hederin (α-Hederin) was used as the internal standard substance. Control plasma samples were obtained by adding 100 μl of methanol to 0.5 ml of human blood plasma.

Stage of extraction

1 ml of ammonia water (0.56 vol.%) Was added to each blood plasma sample and mixed thoroughly (ammonia concentration in the mixture: 0.37 vol.%), Followed by the introduction of the entire mixture into Oasis MAX (manufactured by Waters Corp.) ( hereinafter abbreviated as ″ MAX ″). Before the introduction of the blood plasma sample, the MAX was pretreated by introducing a solution of formic acid-methanol (2 vol.%) Into it, followed by the introduction of water.

After the introduction of a blood plasma sample, the MAX was washed with a solution of ammonia water / methanol with a concentration of 0.56 vol% (liquid mixture with a volume ratio of 1: 1), followed by additional washing with methanol.

Then, to elute adsorbed GL, etc. a solution of formic acid / methanol with a concentration of 2% vol. The eluate (extract) was evaporated to dryness using nitrogen gas while heating at 40 ° C.

Quantification step

Evaporated to dryness, the extract was dissolved in 250 μl of the following mobile phase: liquid A / liquid B (liquid mixture with a volume ratio of 1: 1). 20 μl of the liquid mixture was introduced into the analytical column and subjected to LC under the conditions given below.

Device: Alliance HT 2695 (manufactured by Waters Corp.)

Analytical column: CAPCELL CANCER C 18 (UG120, 5 μl, 1.5 mm × 150 mm, manufactured by Shiseido Co., Ltd.)

Mobile phase: liquid A represents 0.1% vol. Formic acid, liquid B represents 0.1% vol. Formic acid / acetonitrile, gradient mode (table 2)

Flow rate: 0.30 ml / min

Column temperature: 40 ° C

The volume of the injected sample: 20 μl

table 2 Time (minutes) Mobile phase A (%) Mobile phase B (%) 0.00 60 40 2.00 10 90 4.00 10 90 4.01 60 40 14.00 60 40

Then in relation to the fraction containing GL, etc. and obtained by LC, MS was carried out under the following conditions.

Device: ZQ2000 (manufactured by Waters Corp.)

Ionization Method: ESI (Turbo Spray)

Detection method: GL (positive and negative ion detection), GA and IS (positive ion detection), MRM (multiple reaction monitoring)

Capillary voltage: 2.5 kV

Extractor: 2 B

Radio Frequency Lens: 0.2 V

Initial temperature: 110 C

Desolvation Temperature: 350 ° C

Desolvating gas flow: 350 l / h

Cone gas flow: 50 l / h

Table 3 Name of substance Ion detection Weight (m / z) Taper Voltage (B) GL Negative 821 60 Positive 823 thirty GA Positive 471 60 IS Positive 751 thirty

The results of LC-MS measurements increased depending on the concentrations of GL or GA in the blood plasma samples, and the calibration curves obtained from the measurements of LC-MS and the concentrations of GL or GA in the blood plasma samples were linear. Thus, based on the results, the concentration of GL in biological samples of blood plasma was 20-400 ng / ml, and the concentration of GA was 80-1600 ng / ml, from which it is clearly seen that a highly sensitive quantitative determination of GL, etc., was carried out. in biological samples using a highly sensitive method for determining the amount obtained from medicinal herbs of the components of the present invention using LC-MS.

The study of specificity, linearity and reproducibility throughout the day

Next, a study was conducted of specificity, linearity and reproducibility throughout the day. As a result, a highly sensitive method for detecting the amount of components obtained from medicinal herbs of the present invention using LC-MS showed satisfactory linearity (the correlation coefficient of the calibration curve was 0.9979) and reproducibility within, respectively, the concentration ranges of 20-400 ng / ml and 80- 1600 ng / ml. As for the results of daily reproducibility, then, as shown in Table 4, the correctness of the negative mode for determining GL (GL-) was from -1.2 to 0.6%, and the accuracy from 5.2 to 6.0%; the correctness of the positive mode for determining GL (GL +) was from -5.9 to + 5.9%, and the accuracy was from 3.3 to 7.7%; the accuracy of the GA determination was from -7.0 to + 7.2%, and the accuracy was from 3.0 to 7.7%. From the above results, it was concluded that the limits of the determined concentrations of GL and GA were, respectively, 20 ng / ml and 80 ng / ml.

Table 4 GL GA GL- GL + Additive Concentration (ng / ml) Accuracy (% CV) Correctness (% RE) Accuracy (% CV) Correctness (% RE) Additive Concentration (ng / ml) Accuracy (% CV) Correctness (% RE) twenty 5.7 0.6 3.3 5.9 80 7.7 -1.8 one hundred 6.0 -1.2 77 0.5 400 3.0 7.2 400 5.2 -0.9 6.1 -5.9 1600 4.7 -7.0

Example 2

A study was conducted of the effect of the type of alkali or alcohol, which is mixed with a biological sample, on the quantitative sensitivity of a highly sensitive method for determining the amount of components obtained from medicinal herbs of the present invention.

50 μl of the standard solution (E) from Example 1 and 50 μl IS were added to 0.5 ml of human blood plasma, and then thoroughly mixed to obtain blood plasma samples.

GL and GA were quantified by the same procedure as in Example 1, except that 1 ml of the solutions shown in Table 5 were added to each blood plasma sample to prepare the mixture for introduction into MAX.

Considering each of the results of the quantitative determination, taking into account the fact that the case when the solution mixed with the blood plasma sample was 0.56 vol.% Ammonia water was taken as 100% of the degree of extraction, the relative degrees of extraction of GL, GA and IS were calculated for each blood plasma sample. The calculation results are shown in table 5. The numbers shown in brackets after “ammonia water” in table 5 show the concentration of ammonia (vol.%) In the mixture obtained by adding ammonia water.

Table 5 The degree of extraction (relative value) GL- (%) GL + (%) GA (%) IS (%) Water 67.6 62.9 73,4 72,4 0.14% ammonia water (0.09%) 94.7 102.5 76.3 82.5 0.56% ammonia water (0.37%) 100.0 100.0 100.0 100.0 2.8% ammonia water (1.86%) 102,4 110.6 103.9 110.6 5.6% ammonia water (3.72%) 94.7 103.6 101.1 113.6 28% ammonia water (18.6%) 87.2 102.8 99.8 103.3 0.0025M sodium hydroxide aqueous solution 73.7 86.1 74.0 69.1 0.1% aqueous solution of sodium bicarbonate 80.5 75.0 93.2 81.9 The degree of extraction (relative value) GL- (%) GL + (%) GA (%) IS (%) Methanol 93.1 88.5 96.5 94.7 Ethanol 77.9 76.6 77.1 53.7 2% phosphoric acid 0,0 0,0 5.9 0,0 2% perchloric acid 0,0 0,0 3,7 0,0

As a result, when alkali or alcohol was used, the degree of extraction was better than when water was used. Conversely, when an acidic solution such as phosphoric acid or perchloric acid was added, GL could not be detected, and GA was practically not recovered. Among the alcohols, the recovery rates for both GL and GA were better when methanol was used than when ethanol was used. Among the alkalis, the results were better when using ammonia water (aqueous ammonia) than when using an aqueous sodium hydroxide solution or an aqueous sodium hydrogen carbonate solution. However, differences at different concentrations of ammonia water were barely observed.

Example 3

A study was conducted of the influence of the type of cleaning liquid for the solid phase on the quantitative sensitivity of a highly sensitive method for determining the amount of components obtained from medicinal herbs of the present invention.

50 μl of the standard solution (E) and 50 μl of IS from Example 1 were added to 0.5 ml of human blood plasma, and then thoroughly mixed to obtain blood plasma samples.

GL and GA were quantified in the same manner as in Example 1, except that after washing with MAX solutions in Table 6, an additional washing with methanol was carried out after administration of a blood plasma sample.

Considering each of the results of the quantitative determination, taking into account the fact that the case when the cleaning fluid MAX after the introduction of a blood plasma sample consisted of 0.56 vol.% Ammonia water (aqueous ammonia solution) and methanol solution (liquid mixture with a volume ratio of 1: 1) , taken as 100% degree of extraction, calculated the relative degree of extraction of GL, GA and IS for each sample of blood plasma. The calculation results are shown in table 6. The ratios shown in parentheses after the name of the solution correspond to the mixture ratio of the corresponding solution.

Table 6 Relative degree of extraction GL- (%) GL + (%) GA (%) IS (%) Water 94.8 101.3 92.0 98.3 0.56% ammonia water 92.3 89.8 93.5 99.8 1.4% ammonia water 96.3 90.6 106.9 103.5 0.56% ammonia water / methanol (1: 1) 100.0 100.0 100.0 100.0 2.8% ammonia water / methanol (1: 1) 83.0 81.2 121,2 101.8 5.6% ammonia water / methanol (1: 1) 104.1 116.0 119.9 112.9 28% ammonia water / methanol (1: 1) 103.0 99.3 111.8 94.9 0.56% ammonia water / methanol (3: 1) 108.1 117.7 93.7 98.6 0.56% ammonia water / methanol (1: 3) 95.9 110.5 107.4 102.7 0.0025M aqueous solution of sodium hydroxide / methanol (1: 1) 101,4 94.2 78.8 94.8

As a result, although the degree of extraction of GA was somewhat low only when washing was carried out with a 0.0025 M aqueous solution of sodium hydroxide / methanol solution (liquid mixture with a volume ratio of 1: 1), the recovery rates of GL, GA and IS were satisfactory in all cases when water, ammonia water or a mixed solution of ammonia water and methanol were used as a cleaning liquid.

Example 4

A study was conducted of the influence of the type of cleaning liquid for the solid phase on the quantitative sensitivity of a highly sensitive method for determining the amount of components obtained from medicinal herbs of the present invention.

50 μl of the standard solution (E) and 50 μl of IS from Example 1 were added to 0.5 ml of human blood plasma, and then thoroughly mixed to obtain a blood plasma sample.

The amounts of GL and GA were determined in the same manner as in Example 1, except that after washing with a MAX of 0.56 vol.% Ammonia water / methanol solution (liquid mixture with a volume ratio of 1: 1) following the injection of a blood plasma sample , carried out additional washing with ethanol, methanol, 50 vol.% aqueous methanol or acetonitrile.

Considering the case when the second cleaning fluid was methanol, for 100%, the relative degrees of extraction of GL, GA, and IS were calculated for each blood plasma sample. The calculation results are shown in table 7. As a result, when washing was carried out with any of the solvents used, the degree of GL recovery was satisfactory - 80% or more. The degree of GA recovery was also satisfactory — 80% or more, with the exception of one case where a 50 vol% aqueous methanol solution was used. In particular, the use of methanol gave the most favorable results.

Table 7 The degree of extraction (relative value) GL- (%) GL + (%) GA (%) IS (%) Methanol 100.0 100.0 100.0 100.0 Ethanol 94.7 93.5 83.0 92.7 50% methanol 92.7 87.8 70.3 82.3 Acetonitrile 85,2 83.3 81.4 85.6

Example 5

A study was conducted of the influence of the type of eluate from the solid phase on the quantitative sensitivity of a highly sensitive method for determining the amount obtained from medicinal herbs of the components of the present invention.

50 μl of the standard solution (E) and 50 μl of IS from Example 1 were added to 0.5 ml of human blood plasma, and then thoroughly mixed to obtain a blood plasma sample.

The amount of GL and GA was determined in the same manner as in example 1, except that the elution from MAX was carried out using the solutions shown in table 8.

Table 8 The degree of extraction (relative value) GL- (%) GL + (%) GA (%) IS (%) Formic Acid-Methanol 100.0 100.0 100.0 100.0 Formic Acid-Ethanol 82,2 87.7 77.9 88.5 Formic Acid-Acetonitrile 0,0 0,0 94.8 102,4 Phosphoric Acid-Methanol 0,0 0,0 0,0 0,0

Considering the case when the eluate was formic acid-methanol, for 100%, the relative degrees of extraction of GL, GA, and IS were calculated for each blood plasma sample. The calculation results are presented in table 8. As a result, the recovery rates were the highest when using formic acid-methanol. GL and GA were also recovered with satisfactory results when using formic acid-ethanol, although not to the same extent as in the case of formic acid-methanol. In contrast, when the eluate was formic acid-acetonitrile or phosphoric acid-methanol, GL could not be detected.

Comparative Example 1

Quantitative determination of GL and GA in blood plasma was carried out using, instead of MAX, a solid phase with only a reversed-phase distribution function.

50 μl of the standard solution (E) and 50 μl of IS from Example 1 were added to 0.5 ml of human blood plasma and mixed thoroughly to obtain blood plasma samples.

In general, the same procedure was performed as in Example 1, with the exception of the following: Sep-Pak (3 cm ³ , 60 mg, 30 μm, manufactured by Waters Corp.), which is an ODS column, was used instead of MAX; the required conditions in the column were established first with methanol and then with water; 0.56 vol.% Ammonia water or 0.1 N hydrochloric acid was used as a solution (diluent) to obtain a mixture for supply to the column; water or a mixture of acetonitrile / water (volume ratio = 1: 3, contains 1 M tetrabutylammonium bromide) was used as a cleaning liquid for the solid phase; and as a eluate for the solid phase, 2 vol.% formic acid-methanol, 2 vol.% formic acid-acetonitrile / water (1: 1 volume ratio) or 2 vol. formic acid-methanol / water (volume ratio 1: 2) were used. one).

Considering the case when the solution mixed with the blood plasma sample in Example 2 was 0.56 vol% ammonia water, for 100% recovery, the relative recovery rates of GL, GA, and IS were calculated for each blood plasma sample. The calculation results are shown in table 9. In table 9, "1-1" means 0.56 vol.% Ammonia water (aqueous ammonia solution), "1-2" means 0.1 N hydrochloric acid, "2-1" means water , "2-2" means acetonitrile / water (volume ratio = 1: 3, contains 1 M tetrabutylammonium bromide), "3-1" means 2 vol.% Formic acid-methanol, "3-2" means 2 vol.% formic acid-acetonitrile / water (volume ratio = 1: 1), and "3-3" means 2% vol. formic acid-methanol / water (volume ratio 1: 1).

Table 9 Diluent Cleansing fluid Eluent Relative degree of extraction GL- (%) GL + (%) GA (%) IS (%) 1-1 2-1 3-1 36,4 0,0 36,4 60.8 1-2 2-1 3-1 50,2 0,0 31.5 91.5 1-1 2-2 3-1 0,0 0,0 0,0 0,0 1-1 2-1 3-2 0,0 0,0 13.8 41.6 1-1 2-1 3-3 17.8 0,0 7.8 73.1

As a result, the GL recovery rate was at most 50%, even in the case of the highest recovery rate among combinations of diluent, etc., from which it is clearly seen that the detection sensitivity with respect to GL, etc. It was quite low compared with the cases when MAX was used.

Reference Example 2

Quantitative determination of the levels of GL and GA in blood plasma was carried out using, instead of the MAX, a solid phase with a reversed-phase distribution function and a polar interaction function.

50 μl of the standard solution (E) and 50 μl of IS from Example 1 were added to 0.5 ml of human blood plasma and mixed thoroughly to obtain blood plasma samples.

In general, the same procedure was performed as in Example 1, with the exception of the following: instead of MAX, Oasis HLB (3 cm ³ , 60 mg, 30 μm manufactured by Waters Corp.) was used, which was a porous polymer; the required conditions in the column were established first with methanol and then with water; 0.56 vol.% ammonia water or 0.1 N hydrochloric acid was used as a solution (diluent) to obtain a mixture for feeding into the column; A 5 vol.% Aqueous methanol solution was used as a cleaning liquid for the solid phase.

Considering the case when the solution mixed with the blood plasma sample in Example 2 was 0.56 vol% ammonia water (aqueous ammonia solution) as a 100% recovery, the relative recovery levels of GL, GA, and IS were calculated for each plasma sample blood. The calculation results are shown in table 10.

Table 10 Diluent The degree of extraction (relative value) GL- (%) GL + (%) GA (%) IS (%) 0.56% aqueous ammonia 51,2 58.0 37.9 103,2 0.1 N hydrochloric acid 0,0 0,0 39.1 94.3

As a result, in the case where 0.1 N hydrochloric acid was used as a diluent, GL could not be detected, and the degree of GA recovery was very low. On the other hand, even when a 0.56 vol.% Aqueous solution of ammonia was used, the degree of GL recovery did not exceed 55%, which clearly implies that the sensitivity to GL, etc. was much lower than when MAX was used.

Reference Example 3

Quantitative determination of GL and GA in blood plasma was carried out using instead of MAX a solid phase with reverse phase distribution function and positive ion exchange function.

50 μl of the standard solution (E) and 50 μl of IS from Example 1 were added to 0.5 ml of human blood plasma and mixed thoroughly to obtain blood plasma samples.

In general, the same procedure was performed as in Example 1, with the exception of the following: instead of MAX, Oasis MOX was used (3 cm ³ , 60 mg, 30 μm, manufactured by Waters Corp.,); the required conditions in the column were created first using methanol and then water; 0.1 N hydrochloric acid was used as a solution (diluent) to obtain a mixture for feeding into the column; 0.1 N hydrochloric acid was used as a cleaning liquid for the solid phase; and 2 vol% ammonia water / methanol solution was used as the eluate for the solid phase.

Table 11 The degree of extraction (relative value) 1 GL- (%) GL + (%) GA (%) IS (%) one 40,0 40.5 40.7 56.4 2 33.9 37.7 38.9 56.7 3 42.3 39.8 42.1 60.0 four 38.6 46.5 41.6 61.1 5 32.9 39.2 41.1 55.8 average 37.6 40.7 40.9 58.0

Considering the case when the solution mixed with the blood plasma sample in Example 2 was a 0.56 vol.% Aqueous ammonia solution as a 100% recovery, the relative recovery rates of GL, GA, and IS were calculated for each blood plasma sample. The calculation results for 5 independent tests and their average values are presented in table 11.

As a result, the recovery rates of GL and GA did not reach 50%, which clearly indicates that the detection sensitivity with respect to GL, etc. was much worse than when MAX was used.

Example 6

The limiting detectable amounts of GL and GA in the blood plasma were measured and calibration curves were constructed for the case when the highly sensitive method for determining the amount of components obtained from medicinal herbs of the present invention was carried out using the LC-MS / MS method as a mass spectrometry method. In addition, the accuracy and reliability of the method of the invention was confirmed.

Getting a standard solution

The standard stock solutions used in Example 1 were diluted with methanol in the same procedure as Example 1 to prepare the standard solutions (B) - (G) shown in Table 12.

Table 12 Standard solutions, ng / ml AT FROM D E F G GL 2000 500 200 fifty twenty 5 GA 8000 2000 800 200 80 twenty

Preparation of blood plasma samples and stage extraction

Human blood plasma samples, to which, respectively, standard solutions (B) - (G) were added, as well as control (without additives) human plasma samples, were obtained according to the same procedure as in Example 1. Blood plasma samples were, respectively introduced into the MAX to achieve adsorption on it GL, etc., and then washed with 0.56 vol.% solution of ammonia water / methanol, and then with pure methanol, and then eluted with 2 vol.% solution of formic acid / methanol and the resulting mixture was evaporated to dryness.

Quantification step

Evaporated to dryness, the extract was dissolved in 250 μl of the following mobile phase: solutions A / solutions B (liquid mixtures with a volume ratio of 1: 1). 1 μl of each product was introduced into the analytical column and carried out LC-MS / MS under the conditions given below.

LC-MS / MS system: API 4000 system (manufactured by Applied Biosystems Corp.)

(LC)

Device: LC-20A (manufactured by Shimadzu, Ltd.)

Analytical column: Inertsil ODS-3 (2.1 mm × 150 mm, 5µ grain size, manufactured by GL Sciences, Inc.)

Flow rate: 0.50 ml / min.

Column temperature: 40 ° C

Automatic dispenser temperature: 5 ° C

Amount of sample injected: 1 μl

Mobile phase: solution A - 0.1% vol. Formic acid; Solution B 0.1 vol.% formic acid-acetonitrile; linear gradient (table 13)

Table 13 Time (minutes) Mobile phase A (%) Mobile phase B (%) 0.00 40 60 2.00 10 90 4.00 10 90 4.01 40 60 6.00 40 60

(MS / MS)

Device: API 4000 (Applied Biosystems) Ionization method: ESI (turbo-spray)

Detection method: detection of positive MRM ions (monitoring of multiple reactions)

Ion Spray Voltage: 5500 V (positive)

Ion Source Temperature (Temperature): 400 ° C

Protecting gas flow: 10 (nitrogen)

Gaseous ion source 1: 70 (nitrogen)

Gaseous ion source 2: 50 (nitrogen)

Collision gas: 8 (nitrogen)

Table 14 The studied ions and other parameters Substance Q1 Weight (m / z) Q3 Weight (m / z) DP (B) EP (B) CE (eB) CXP (B) GL 823 453 126 10 35 fourteen GA 471 149 116 10 57 10 IS 751 455 71 10 25 12

1A-1C are chromatograms of control plasma samples. FIG. 1A shows the position of the peak GL (retention time GL; M / z 823 → 453), FIG. 1B shows the position of the peak GA (retention time GA; m / z 471 → 149), and FIG. 1C shows the position of the peak IS (time retention IS; m / z 751 → 455). 2A-FIG. 2C are chromatograms of blood plasma samples to which a standard solution (G) was added (plasma concentration of GL: 0.5 ng / ml, GA concentration: 2 ng / ml). FIG. 2A shows the peak GL (shown by the arrow in the figure), FIG. 2B shows the peak GA (shown by the arrow in the figure), and FIG. 2C shows the peak IS (shown by the arrow in the figure). The retention times of GL, GA, and IS were approximately 1.2 minutes, 3.1 minutes, and 1.4 minutes, respectively; no peaks hindering quantification were observed in the chromatograms of the initial plasma samples, and the peak shapes of GL, GA, and IS were satisfactory.

Table 15 shows the results of quantification. The relative errors in the actual added concentrations and measured values (quantitative values in the table) are also shown in Table 15. As a result, the relative errors of both GL and GA were within 15%. Thus, the results show that a highly sensitive method for determining the amount obtained from medicinal herbs of the components of the present invention using LC-MS / MS allows you to quantify GL, etc. in biological samples with a high degree of accuracy in the case where the GL concentration is 0.5-200 ng / ml and the GA concentration is 2-800 ng / ml for such biological samples as blood plasma.

Table 15 GL GA Additive Concentration (ng / ml) Quantitative value (ng / ml) Relative error (%) Additive Concentration (ng / ml) Quantitative value (ng / ml) Relative error (%) 0.5 0.545 +9.0 2 1,831 -8.5 2 1,992 -0.4 8 8.235 +2.9 5 4,915 -1.7 twenty 20,796 +4.0 twenty 18,315 -8.4 80 77,434 -3.2 fifty 50,504 +1.0 200 212,042 + 6.0 200 201,230 +0.6 800 789,661 -1.3

The study of specificity, linearity and reproducibility throughout the day / on different days

Then, a specificity, linearity and reproducibility study was carried out during the day / on different days according to the Guidance for Industry, Bioanalytical Method Validation, US Department of Health and Human Services, Food and Drug Administration, May 2001. In addition, a stability study was carried out ( maintaining stability in biological samples at room temperature for 4 hours and at low temperature storage for three months at a temperature of -20 ° C and -80 ° C, stability during freezing and thawing, stability in the measurement of real samples and standard stability solution). The results are shown in table 16. As a result, a highly sensitive method for determining the amount of components obtained from medicinal herbs of the present invention using LC-MS / MS showed satisfactory linearity and reproducibility in the ranges of 0.5-200 ng / ml and 2-800 ng / ml . As for the reproducibility results during the day, the correctness of the results for GL was from -12.8 to + 4.8%, and the accuracy was from 4.0 to 9.5%; correctness for GA ranged from -13.4 to + 4.4%, and accuracy ranged from 2.2 to 9.0%. On the other hand, with regard to reproducibility on different days, the correctness of GL was from -9.4 to + 2.0%, and the accuracy was 2.1 to 5.5%, the correctness for GA was from -10.9 up to + 8.1%, and the accuracy was from 1.3 to 9.1%. The reproducibility results confirmed that the limits of the determined concentrations of GL and GA are, respectively, 0.5 ng / ml and 2 ng / ml.

In all stability tests (maintaining stability in biological samples, stability during freezing and thawing, stability in measuring real samples and stability of a standard solution), the results were within ± 15% of the initial value. From the above results, it was concluded that in accordance with the Guidance for Industry, Bioanalytical Method Validation of the US Food and Drug Administration, a highly sensitive method for determining the amount of components obtained from medicinal herbs of the present invention is a quantitative determination method with proven reliability.

Table 16 GL GA Additive Concentration (ng / ml) Quantitative value (ng / ml) Accuracy (% CV) Correctness (% RE) Additive Concentration (ng / ml) Quantitative value (ng / ml) Accuracy (% CV) Correctness (% RE) Reproducibility throughout the day Reproducibility throughout the day 0.5 0.436 8.0 -12.8 2 1,733 9.0 -13.4 one 0.938 9.5 -6.2 four 3,700 6.6 -7.5 10 10,295 4.0 +3.0 40 41,779 4.4 +4.4 160 167,721 4.4 +4.8 640 614,862 2.2 -3.9 Repeatability on different days Repeatability on different days 0.5 0.453 5.1 -9.4 2 1,783 9.1 -10.9 one 0.954 2.1 -4.6 four 3,853 6.6 -3.7 10 10,197 3.9 +2.0 40 43,249 3.0 +8.1 160 160,408 5.5 +0.3 640 622,379 1.3 -2.8

Example 7

Plasma GL was detected after oral administration of GL-containing formulations and its pharmacokinetics were measured.

This experiment was carried out in accordance with ethical principles based on the Helsinki Declaration, in accordance with Section 14, Section 3 and Section 80 (2) of the Pharmaceutical Law, as well as in accordance with the Ministerial Ordinance on Good Clinical Practice (GCP) (Ministry of Health and Welfare Ordinance No. 28, 1997). In particular, the oral administration of a GL-containing composition and the collection of blood plasma samples was carried out at the request of the authors at EPS Corporation Clinic and Yamaguchi under the supervision of a lead researcher after obtaining the approval of an independent committee. Measurement of GL and GA in the obtained plasma samples was performed using Ecotechno Division (currently Pharmaceutical, Perfumery and Cosmetics Analysis Division) of Japan Clinical Laboratories, Inc.

As for the test subjects, after providing a full explanation of the goals and contents of the research, as well as the policy for protecting personal information and the like, six healthy Japanese men were selected (age: 20 to 35, BMI (body mass index) during preliminary inspection: from 18.5 or more to 25.0 or less), which gave written consent to participate in the study as a participant.

Sugar-coated tablets with the name "GLYCYRON tablets (trademark)" containing monoammonium glycyrrhizinate, glycine and DL-methionine were used as GL-containing formulations. GLYCYRON tablets were administered orally once in a normal dose of three tablets with a sufficient amount of water. Table 17 shows the schedule of the study, including the time of administration and time of blood sampling. 1 ml or more of blood plasma was recovered by subjecting the collected blood to centrifugal separation (4 ° C, 3000 rpm, 10 min) for 30 minutes from the time of blood collection, and stored in a frozen state at -20 ° C or lower until the moment of concentration measurement.

Table 17 timetable Time Past tense Condition Blood collection Validation / Safety Study Previous day 18:00 -15 h Hospitalization 19:00 -14 h Eating First day 7 a.m. - 9 a.m. from 2 hours to 0 hours • • 9 a.m. 0 h Introduction 9:15 0.25 h • 9:30 a.m. 0.5 h • quarter to ten 0.75 h • 10 a.m. 1 hour • 11:00 2 h • 13:00 4 h Meal * • 3 p.m. 6 h • 5 p.m. 8 h • 19:00 10 h Meal * • 9 p.m. 12 h • Second day 3 o'clock 18 h • 9 a.m. 24 h Meal * • 13:00 28 h Eating 19:00 34 h Eating 9 p.m. 36 h • The third day 9 a.m. 48 h Hospital discharge • • * Eating after blood collection

After adding 1 ml of a 0.56 vol.% Aqueous ammonia solution to 0.5 ml of the collected blood plasma sample and subsequent thorough mixing, the same procedure was carried out as in example 1, during which the resulting mixture was deposited on MAX to adsorb GL on it etc. and washed with 0.56 vol.% aqueous ammonia / methanol and then methanol, followed by elution with 2 vol.% formic acid / methanol, followed by evaporation to dryness.

Then, an LC-MS / MS analysis was performed as described in Example 6 with respect to the evaporated to dryness of the extract, and the GL concentration and GA concentration in each blood plasma sample were measured. Figure 3 illustrates changes in the average concentration of GL in plasma, and Figure 4 illustrates changes in the average concentration of GA in plasma. Figure 3 and figure 4 show the average values and standard deviations for 6 samples. As a result, it was confirmed that, using a highly sensitive method for determining the amount of the components obtained from medicinal herbs of the present invention, it is possible to directly measure the plasma concentration of not only GA, which is the main metabolite previously used as an alternative indicator, but also directly GL, which is unmodified active substance. That is, the present invention for the first time allows the detection of small amounts of GL in blood plasma and clarifies the kinetics of blood GL, which has long been unclear.

Pharmacokinetics Analysis

The maximum plasma concentration (Cmax) and the time to reach it (Tmax) were calculated on the basis of GL and GA measurements. The area under the dependence (AUC) of blood plasma concentration on time up to 48 hours after administration (AUC 0 → 48 hours) was calculated by the trapezoidal method, and the excretion rate constant (Cal) and half-life (t1 / 2) were calculated by the least squares method according to the elimination phase . In addition, the area was calculated under the dependence of blood plasma concentration on time up to an infinite time after administration (AUC 0-∞) and average retention time (MRT).

As a result, in the present example, Cmax GL was in the range of about 10-40 ng / ml, and its average value was 24.8 ng / ml. Since the value was almost identical to the value predicted on the basis of the above experiment on the oral administration of large doses of a commercially available reagent (approximately 23 ng / ml), a correlation between the dosage of GL and the concentration in blood plasma was illustrated. Tmax averaged 4.5 hours, but subjects who observed many concentration peaks before (1-2 hours) or after (12 hours) of this point were also observed. Since the last peak in concentration occurred after ingestion, it was suggested that food intake may affect GL uptake or that enterohepatic circulation occurs.

On the other hand, with respect to GA, which is the main metabolite of GL, it was confirmed that there was a 4-hour delay until its presence in plasma was confirmed, and also that its plasma concentration after 6 hours was about ten times (about 20 times in terms of moles) more than the concentration of GL. As a result of in vitro incubation of GL under optimal conditions using human liver microsomes, despite conversion to 3-monoglucuronyl-glycyrrhetinic acid, which is an intermediate metabolite, conversion to a small amount of GA was also confirmed (Biochemical Pharmacology, 42 (6/7), 1025-1029, 1991). Oral administration of GL to sterile rats failed to confirm the presence of GA in blood plasma or feces, and therefore it was concluded that GA production in vivo is regulated by intestinal bacteria (Ref .: J. Pharm. Pharmacol. 46, 135-137, 1994). Therefore, it is assumed that the delay in absorption corresponds to the time required for contact with the intestinal flora, and it is believed that eating (or starvation conditions) negatively affects the “visibility” of GA by affecting the promotion of the drug inside the gastrointestinal tract, the metabolic ability of the intestinal tract flora, and absorption in the digestive tract. During observation of GA kinetics by the subject after absorption, it was found that the appearance of metabolites was less pronounced in subjects with a high concentration of unchanged substance and vice versa, the appearance of metabolites was more pronounced in subjects with a low concentration of unchanged substance. Thus, it was suggested that, along with GA, the absorption of GL, which is an invariable substance, in the gastrointestinal tract is also associated with the metabolic ability of the intestinal flora, which leads to corresponding individual differences (data not published).

Industrial applicability

A highly sensitive method for determining the amount of components obtained from medicinal herbs of the present invention allows to determine the level of GL in the blood after oral administration of a clinical dose of a GL-containing composition, food product or the like, and therefore, this method can be used to control the use of pharmaceuticals or Chinese herbal medicine, safety tests, pharmacokinetic analyzes, the development of new GL formulations and the like.

Claims (1)

A highly sensitive method for determining the amount of glycyrrhizin, glycyrrhetinic acid and their pharmacologically acceptable salts present in human blood plasma, characterized in that this method includes:
the introduction of a mixture of human blood plasma with methanol or a solution of ammonia water with such a concentration that the concentration of ammonia in the resulting mixture was 0.05-20 vol. %, in the solid phase having a reversed-phase distribution function and anion exchange function;
washing the solid phase with a cleaning liquid, which is a single component liquid or a liquid mixture of at least two components selected from the group consisting of water, alkali, alcohol and acetonitrile; and
subsequent elution from the solid phase with an acid alcohol selected from formic acid-methanol or formic acid-ethanol; and
a step for the quantitative determination of glycyrrhizin, glycyrrhetinic acid and their pharmacologically acceptable salts by LC-MS or LC-MS / MS.

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