KR101476144B1 - Highly sensitive method of determining the quantity of herbal medicine-derived components - Google Patents

Abstract

A method for quantifying a herbal medicine-derived component with high sensitivity is a method in which a mixed substance of a biological sample mixed with an alkali or an alcohol is injected into a solid phase having a reverse phase distribution function and an anion exchange function and then the solid phase is reacted with water, , And then eluting the solid phase from the solid phase with an acidic alcohol to prepare an extract containing the herbal medicine-derived component; At least one selected from the group consisting of glycyrrhizin, glycyrrhizinic acid, glycyrrhizin and metabolites of glycyrrhizic acid, glycyrrhizin and glycyrrhizic acid, a saponin component in licorice, and a pharmaceutically acceptable salt thereof, And a step of detecting and quantifying one species by mass spectrometry.

Description

HIGHLY SENSITIVE METHOD OF DETERMINING THE QUANTITY OF HERBAL MEDICINE-DERIVED COMPONENTS [0002]

The present invention relates to a method for quantitatively measuring glycyrrhizin or its metabolites in a biological sample with high sensitivity.

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

BACKGROUND ART [0002] Glycyrrhizin (hereinafter abbreviated as " GL ") and its salts are used in a wide variety of applications, including anti-allergic and anti-inflammatory actions as well as immunomodulating action, hepatocyte injury inhibiting action, hepatocyte proliferation promoting action, It is a liquorice root extract ingredient with physiological activity. In Japan, it has been used as a clinical drug for herbal medicine and the like and is widely used for the treatment of chronic liver disease, eczema / dermatitis, pediatric stroplus, alopecia areata, various allergies, inflammation and the like. In addition to the use of pharmaceuticals, GL has an effect of reducing the salty taste and has a sweetening effect, and therefore is widely used as a sweetener in a wide range of foods such as pickles and seasonings.

From the results of nonclinical studies, it is believed that GL is easily accumulated in the liver and is rapidly excreted in the bile, and is discharged through metabolism and long circulation. Particularly, in oral administration, GL, a glycoside and a water-soluble polar substance, exhibits low water absorption, and its concentration in peripheral blood is very low due to metabolism or first pass effect by intestinal bacteria. For this reason, in order to measure the blood circulation of GL, various methods have been tried so far. However, in a herbal preparation containing GL and licorice containing a GL, it is difficult to detect blood GL when taken, It has long been regarded as unclear.

For example, there is a method in which plasma is taken from a normal human being orally administered GL and the amount of GL in the plasma is measured by enzyme immunoassay (EIA) after removing the protein by methanol treatment, (For example, non-patent documents 1 to 4) in which a column having a reversed-phase distribution function such as a conjugated-type (ODS) column is used for measurement by high performance liquid chromatography (HPLC). However, in these measurement methods, although the quantitative limit value was at most 0.1 μg / ml and glycyrrhizic acid (hereinafter abbreviated as "GA"), which is a major metabolite, could be detected, GL could not be detected. For this reason, it has been proved indirectly that GL is transferred to blood as an example of detection from urine (see Non-Patent Document 4). Regarding the drug efficacy of the GL-containing preparation, the quantifiable GA is an evaluation index instead of the GL.

 Ishiwata et al., 7, Biological and Pharmaceutical Bulletin, 2000, Vol. 23, No. 8, pp. 904-905.  De Groot et al., Journal of Chromatography, 1988, 456, pp. 71-81.  Nakata et al., Journal of Korean Oriental Medicine, 1986, Vol. 3, No. 3, pp. 278-279.  Yamamura et al., 7, Journal of Pharmaceutical Sciences, 1992, Vol. 81, No. 10, pp. 1042-1046.

The pharmacokinetics of the unchanged GL, which is the active body, is useful in determining appropriate use in terms of efficacy and safety. Therefore, it is preferable that the amount of GL in a biological specimen such as blood plasma can be quantified without using GA as an evaluation target.

The present invention has been made in view of the above problems, and an object thereof is to provide a method for detecting and quantifying GL in a biological sample such as plasma.

As a result of intensive studies to solve the above problems, the present inventors have found that, after extracting GL-containing components from biological samples by a solid phase extraction method using a solid phase having a reverse phase distribution function and an anion exchange function, Mass spectrometry, whereby GL in the biological sample can be detected and quantified, thereby completing the present invention.

That is,

(1) A method in which a mixture of a biological sample and an alkali or alcohol is injected into a solid phase having a reversed phase distribution function and an anion exchange function, the solid phase is washed at least once with a washing liquid, An extracting step of preparing an extract containing the herbal medicine-derived component by eluting from the solid phase,

A group consisting of glycyrrhizin, glycyrrhizinic acid, glycyrrhizin and glycyrrhizin acid metabolites, glycyrrhizin and glycyrrhizin acid, a saponin component contained in licorice, and a pharmaceutically acceptable salt thereof in the extract extracted by the above extraction step And a quantitative step of detecting and quantifying at least one species selected from the group consisting of

Wherein the cleaning liquid is a mixed solution of one or more kinds selected from the group consisting of water, alkali, alcohol, and acetonitrile,

(2) In the above-mentioned step (1), the washing of the solid phase is carried out by washing with a mixture of alkali, alcohol and water, followed by washing with a mixture of alcohol, alcohol and water or acetonitrile. A method for quantifying a high sensitivity of the herbal medicine-

(3) The method for quantitatively determining the herbal medicine-derived component according to (2) above, wherein the mixed solution of the alkali, alcohol and water is prepared by mixing 0.5 to 28% by volume of ammonia water and methanol at a ratio of 99: 1 to 1: ,

(4) A method for quantitatively determining the high sensitivity of a herbal medicine-derived component according to any one of (1) to (3), wherein the biological sample is blood, plasma, or tissue extract.

The GL of the biological sample having a very small content can be detected and quantified by the method for quantitatively determining the herbal medicine-derived component of the present invention. Therefore, by using the high sensitivity assay method of the herbal medicine-derived component of the present invention, it is possible to quantitatively measure the GL in a biological specimen collected from a human who ingests a GL-containing food or a food or drink, .

1A is a graph showing the chromatogram of GL in a blank plasma sample in Example 6. Fig.
1B is a graph showing the chromatogram of GA in the blank plasma sample in Example 6. Fig.
1C is a chromatogram of the internal standard (IS) in the blank plasma sample in Example 6. FIG.
2A is a graph showing the chromatogram of GL in a plasma sample (amount of GL added in plasma: 0.5 ng / ml) to which standard solution (G) is added in Example 6. Fig.
2B is a graph showing the chromatogram of GA in a plasma sample (GA addition amount in plasma: 2 ng / ml) to which standard solution (G) is added in Example 6. Fig.
2C is a chromatogram showing the IS in the plasma sample to which the standard solution (G) is added in Example 6. Fig.
Fig. 3 is a graph showing a change in average plasma concentration of GL in Example 7. Fig.
4 is a graph showing the average plasma concentration change of GA in Example 7. Fig.

The high sensitivity determination method of the herbal medicine-derived component of the present invention comprises extracting GL in a biological sample by a solid phase extraction method using a solid phase having a reverse phase distribution function and an anion exchange function and then quantifying the GL content in the obtained extract by mass spectrometry . The detection sensitivity of the GL can be increased by quantitatively using mass spectrometry instead of the conventional HPLC method or the like. Further, by preparing a solid phase having a reversed 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 as in the conventional ODS column, more contaminants are obtained from the extract provided for mass analysis The sensitivity of the mass spectrometry can be dramatically improved.

Specifically, the high-sensitivity determination method of the herbal medicine-derived component of the present invention comprises injecting a mixture of a biological sample mixed with alkali or alcohol into a solid phase having a reversed-phase distribution function and an anion exchange function, And extracting the extract containing the herbal medicine-derived component by eluting the solid from the solid phase with an acidic alcohol, and a step of preparing an extract containing the herbal medicine-derived component, wherein GL, GA, GL And at least one member selected from the group consisting of a metabolite of GA, a substance of GL and a substance of GA, a saponin component contained in licorice, and a pharmaceutically acceptable salt thereof by mass spectrometry And the cleaning liquid is a mixture of one or more kinds selected from the group consisting of water, alkali, alcohol, and acetonitrile .

It is preferable that an extract containing at least two herbal medicine-derived components is prepared in the extraction step, and at least two kinds of components are detected and quantified by one measurement (separately from the detection mode) in the quantification step.

The herbal medicine-derived component quantified by the method for high-sensitivity determination of the herbal medicine-derived component of the present invention includes GL, GA, GL and GA metabolites, GL and GA soft substances, saponin components contained in licorice, (Hereinafter sometimes referred to as " GL, etc. ").

Examples of the metabolites of GL and GA include 3-monoglucuronyl-glychrretinic acid (20β-Carboxy-11-oxo-30-norleucine-12-en-3β-yl-β-D-glucopyranosiduronic acid) 3-oxo-GA (3,11-Dioxoolean-12-ene-30-oic acid), monoglucuronyl-glychrretinic acid (3? -Hydroxy-11-oxoolean-12- 3α-GA (3α-Hydroxy-11-oxoolean-12-en-30-oic acid), 3β-Hydroxysulfonyloxy-11-oxoolean-12-en-30-oic acid, -Dihydroxy-11-oxoolean-12-en-30-oic acid, and 3β, 24-Dihydroxy-11-oxoolean-12-en-30-oic acid.

Further, as a flexible substance of GL and GA, for example, 20? -Carboxy-11-oxo-29-norore-12-en-3? -Yl (? - D-glucopyranosyluronic acid) -D-glucopyranosiduronic acid, 20β-Carboxy-24-hydroxy-11-oxo-30-nor-12-ene-3β-yl (β-D-glucopyranosyluronic acid) D-glucopyranosiduronic acid) - (1 → 2) -β-D-glucopyranosiduronic acid), 18α-GL (20β-Carboxy-11-oxo- , 18α-GA (3β-Hydroxy-11-oxo- (18αH) -delete-12-en-30-oic acid).

Examples of the saponin component contained in licorice include licorice-saponin C1 (20-carboxy-11-oxo-30-nor-12-ene- 1? 2) -? - D-glucopyranosiduronic acid).

The pharmaceutically acceptable salt of GL or the like used in the present invention is not particularly limited as long as it is a salt having the same pharmacological effect as GL or the like in a living body. Specific examples thereof include ammonium salts, sodium salts, potassium salts and the like.

In the present invention, one type of GL or the like may be quantified, or two or more types may be quantified by one operation. In the present invention, it is particularly preferable to simultaneously quantify GL and GA.

The biological sample to be provided in the high sensitivity determination method of the herbal medicine-derived component of the present invention may be any sample collected from a living body, preferably collected from an animal such as a human, a mouse, or a rat. (Tissue extract) such as blood, plasma, serum, urine, ascites, pleural fluid, joint fluid, bone marrow fluid, bile, etc., and tissue pieces collected from tissues such as liver, pancreas and kidney. Extracts such as tissue pieces can be prepared by homogenizing the tissue pieces or the like by a conventional method. In the present invention, blood, plasma, urine, or tissue extract is preferable, and plasma is more preferable.

Hereinafter, a high sensitivity determination method of the herbal medicine-derived component of the present invention will be described step by step.

As an extraction step, first, a hydrolyzate in which a biological sample is mixed with an alkali or an alcohol is prepared. The alkali to be added to the biological sample is not particularly limited as long as the pH of the resultant mixture is alkaline, and examples thereof include ammonia, aqueous ammonia, sodium hydroxide solution and sodium hydrogen carbonate solution. The alcohol is preferably a lower alcohol having 1 to 6 carbon atoms, and examples thereof include methanol, ethanol and isopropanol. It may also be an alcohol-diluted solution. In the present invention, the alkali or alcohol to be added to the biological sample is preferably ammonia, aqueous ammonia, methanol, or aqueous methanol solution, more preferably ammonia or ammonia water.

The concentration of the ammonia water to be added to the biological sample is not particularly limited and can be appropriately adjusted in consideration of the kind of the biological sample, the kind of the solid used thereafter, the ammonia concentration of the resulting mixture, and the like. In the present invention, the concentration of ammonia or ammonia water to be added to the biological sample is preferably such that the ammonia concentration of the obtained mixture is from 0.01 to 30% by volume, more preferably from 0.05 to 25% by volume , And more preferably 0.05 to 20% by volume.

Subsequently, the obtained mixture is injected into a solid phase having a reverse phase distribution function and an anion exchange function to adsorb the GL and the like in the solid phase to the solid phase. Examples of the solid phase having a reverse phase distribution function and an anion exchange function include a reverse phase distribution-anion exchange mixed mode solid phase cartridge, Oasis MAX (manufactured by Waters Corporation), and the like.

GL or the like is washed once or more than twice with a cleaning liquid to remove non-specifically bound components. The cleaning liquid is a mixture of one or more kinds selected from the group consisting of water, alkali, alcohol, and acetonitrile. Examples of the alkali or alcohol used as the cleaning liquid include the same alkali and alcohol added to the biological sample. The cleaning of the solid phase may be carried out twice or more times with a same kind of cleaning liquid, or may be sequentially cleaned with different kinds of cleaning liquid.

In the present invention, the solid phase washing is preferably carried out by washing with a mixture of alkali, alcohol and water, a mixed solution of alkali and water, or water, followed by washing with a mixed solution of alcohol, alcohol and water or acetonitrile. And a mixture of alcohol and water, followed by washing with a mixture of alcohol, alcohol and water, or acetonitrile. The mixture of alkali, alcohol and water is preferably a mixture of ammonia, alcohol and water, more preferably a mixture of ammonia, methanol and water.

The composition ratio of the respective components in the mixture of ammonia, methanol and water is not particularly limited as far as it can maintain GL adsorbed on the solid phase. For example, the methanol concentration in the mixed liquid is preferably 1 to 75% by volume and the ammonia concentration is preferably 0.1 to 21% by volume, more preferably 25 to 75% by volume methanol concentration and 0.1 to 21% by volume ammonia concentration . The mixed solution having such a composition ratio can be prepared, for example, by mixing 0.5 to 28% by volume of ammonia water and methanol in a ratio of 99: 1 to 1: 3.

It is preferable to use alcohol as the cleaning liquid to be used for the second cleaning after the first cleaning by the mixture of the alkali and the alcohol and water, more preferably to use methanol or ethanol, and to use methanol More preferable.

Thereafter, the extract is eluted from the solid phase with an acidic alcohol to prepare an extract containing a herbal medicine-derived component. Examples of the acidifying agent include formic acid alcohol, hydrochloric acid, trifluoroacetic acid, and the like. In the present invention, it is preferable to use formic acid alcohol. The formic acid 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 more preferably formic acid methanol. Further, the obtained eluate is evaporated to dryness for subsequent mass analysis.

Next, as a quantification step, GL in the extract extracted by the extraction step is detected and quantified by mass spectrometry. The mass analysis is preferably performed by an LC-MS (liquid chromatography / mass spectrometry) method or an LC-MS / MS (liquid chromatography / tandem mass spectrometry) method. Specifically, the eluate which has been evaporated to dryness is dissolved in the mobile phase of LC, LC is performed, and MS is carried out. LC-MS and LC-MS / MS can be carried out by using a device in which a combination of HPLC and mass spectrometry are used.

As described in Patent Documents 1 to 4, the LC is preferably carried out using a column having a reverse phase distribution function such as an ODS column. Further, the MS can be carried out by a conventional method. For example, a sample is ionized by an ESI (Electrospray Ionization) method or an APCI (atmospheric pressure chemical ionization) method, and then each ion is separated and detected by a device such as a magnetic field deflection type, quadrupole type or flight time type .

By performing the mass analysis by the LC-MS method, the quantitative limit of GL, etc. in blood, plasma, or serum can be improved to about 20 ng / ml, for example. Similarly, by the LC-MS / MS method, the quantitative limit of GL and the like in blood and the like can be improved to about 10 ng / ml or less, for example, about 0.5 ng / ml.

It has been reported that the GL concentration in plasma in the oral administration of a commercial reagent (1600 mg as GL) is about 500 ng / ml (Environmental Health Perspectives, 102 (9), 65-68, 1994). As a result of the proportional calculation based on the knowledge, the estimated blood concentration of the clinical dose of 75 mg, which is a clinical dose, is calculated to be about 23 ng / ml for the purpose of treating liver disease. That is, by the high sensitivity determination method of the herbal medicine-derived component of the present invention, GL in the blood which can not be detected by the conventional method can be quantified. In particular, when the GL concentration in the blood after oral ingestion of a GL formulation is large and the quantitative limit is insufficient, there is a case in which GL is not detected depending on the blood specimen, or a case where the long-term pharmacokinetics can not be measured However, in the high sensitivity determination method of the herbal medicine-derived component of the present invention, particularly, the mass analysis is performed by the LC-MS / MS method to obtain a more reliable measurement result .

Example

EXAMPLES The present invention will be described in further detail with reference to the following examples, but the invention is not limited to the following examples.

[Example 1]

The method for determining the high sensitivity of the herbal medicine-derived component of the present invention was such that the quantitative limit value of GL and GA in plasma was measured by mass spectrometry by the LC-MS method, and a calibration curve was also prepared.

<Preparation of Standard Solution>

First, 10.0 mg of GL (manufactured by Tokiwa Phytochemical Laboratories) was precisely measured and dissolved in methanol to make exactly 100 ml, and a 100 μg / ml GL standard stock solution was prepared. In the same manner, 10.0 mg of GA (manufactured by Alps Pharmaceutical Co., Ltd.) was precisely measured and dissolved in methanol to make exactly 100 ml, and a 100 μg / ml GA standard stock solution was prepared.

Subsequently, 2 ml of the GL standard stock solution and 8 ml of the GA standard stock solution were accurately separated and taken to be exactly 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 diluted with methanol successively to prepare standard solutions (B) to (F) shown in Table 1. Standard stock solutions and standard solutions were prepared and refrigerated (5 ± 4 ° C), respectively. The dispenser was made of glass.

<Preparation of plasma sample>

50 μl of the standard solution (A) was added to 0.5 ml of human plasma, and sufficiently mixed was used as a plasma sample. Plasma samples were also prepared in the same manner for the standard solutions (B) to (F). To these plasma samples, 50 내부 of internal standard solution (IS) was further added. In this example, α-Hederin was used as an internal standard substance. Further, 100 μl of methanol was added to 0.5 ml of human plasma as a blank plasma sample.

<Extraction Process>

To the respective plasma samples, 1 ml of 0.56 vol% ammonia water was added and sufficiently mixed (ammonia concentration in the mixed solution: 0.37 vol%), and the resulting mixture was subjected to Oasis MAX (Waters Co., ). &Lt; / RTI &gt; The MAX was conditioned by injecting 2 vol% formic acid / methanol solution before injecting the plasma sample and then injecting water.

After the plasma sample was injected, the MAX was washed with 0.56 vol% ammonia water / methanol solution (mixed solution of volume ratio 1: 1), and then washed again with methanol.

Thereafter, a 2 volume% formic acid / methanol solution was injected to elute the GL adsorbed to the MAX. The eluate (extract) was evaporated to dryness with nitrogen gas at 40 ° C.

<Quantitative Process>

The evaporated and dried extract was dissolved in 250 쨉 l of the following mobile phase A solution / B solution (mixture of 1: 1 by volume). 20 μl of the solution was injected into the analysis column, and LC was performed under the following conditions.

Apparatus: Alliance HT 2695 (Waters)

Analysis column: CAPCELL PAK C18 (UG120, 5 mu m, 1.5 mm x 150 mm, manufactured by Shiseido)

Mobile phase: liquid A 0.1 vol% formic acid, liquid B 0.1 vol% formic acid acetonitrile, linear gradient (Table 2)

Flow rate: 0.30 ml / min.

Column temperature: 40 DEG C

Sample injection amount: 20 μl

MS was then performed on the fractions containing GL and the like obtained by LC under the following conditions.

Apparatus: ZQ2000 (manufactured by Waters Corporation)

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

Desolvation Temperature: 350 ° C

Desolvation Gas Flow: 350 l / hr

Cone Gas Flow: 50 l / hr

As a result, the measurement result of the LC-MS was increased depending on the GL or GA concentration in the plasma sample, and the calibration curve obtained by measuring the GL or GA concentration in the plasma sample and the LC-MS showed a linearity. That is, from these results, it can be seen that the GL concentration in the biological sample such as plasma is 20 to 400 ng / ml and the GA concentration is 80 to 1600 ng / ml, and by the method for quantitatively determining the herbal medicine- It is clear that GL in the sample can be quantified with high accuracy.

<Review of specificity, linearity, and intra-day reproducibility>

In addition, specificity, linearity, and intra-day reproducibility were examined. As a result, the high sensitivity assay method of the herbal medicine-derived component of the present invention using LC-MS showed reproducibility with good linearity (correlation coefficient of the calibration curve was 0.9979) in the range of 20 to 400 ng / ml and 80 to 1600 ng / . As a result of the intra-day reproducibility, as shown in Table 4, the progress of the negative mode (GL-) of GL is -1.2 to + 0.6%, the precision is 5.2 to 6.0%, and the progress of the positive mode (GL + 5.9 to + 5.9%, and precision to 3.3 to 7.7%. The progress of the GA was -7.0 to + 7.2% and the accuracy was 3.0 to 7.7%. 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.

[Example 2]

The influence of the kind of alkali or alcohol blended in the biological sample on the quantitative sensitivity of the method for quantifying high-sensitivity of the herbal medicine-derived component of the present invention was examined.

To 0.5 ml of human plasma, 50 占 퐇 of the standard solution (E) of Example 1 and 50 占 퐇 of IS were added and sufficiently mixed to obtain a plasma sample.

Specifically, GL and GA were quantified in the same manner as in Example 1, except that 1 ml of the solution described in Table 5 was added to a plasma sample and a mixture to be applied to MAX was prepared.

The recovery rate (relative value) of GL, GA, and IS of each plasma sample was calculated with the quantification result when the solution mixed with the plasma sample was 0.56 vol% ammonia water as the recovery rate of 100%, respectively. The results are shown in Table 5. The values in parentheses after ammonia water in Table 5 indicate the ammonia concentration (volume%) in the mixed product obtained by adding the ammonia water.

As a result, in the case of using an alkali or an alcohol, the recovery was better than in the case of using water. Conversely, when an acidic solution such as phosphoric acid or perchloric acid was added, GL was not detected and GA could hardly be recovered. Among the alcohols, the recoveries of all of GL and GA were also better when methanol was used than ethanol. Among the alkalis, the case of using ammonia water was better than the case of using an aqueous solution of sodium hydroxide or sodium hydrogencarbonate. However, the difference by the concentration of ammonia water was not observed much.

[Example 3]

The influence of the kind of washing liquid on the solid phase on the quantitative sensitivity of the method for quantifying high sensitivity of the herbal medicine component of the present invention was examined.

To 0.5 ml of human plasma, 50 占 퐇 of the standard solution (E) of Example 1 and 50 占 퐇 of IS were added and sufficiently mixed to obtain a plasma sample.

Specifically, GL and GA were quantified in the same manner as in Example 1, except that MAX after the injection of the plasma sample was washed with the solution described in Table 6, and then washed again with methanol.

GA, and IS recovery rates of each plasma sample with a recovery rate of 100% for the quantification results when the cleaning solution of MAX after injection of the plasma sample was a 0.56 vol% ammonia water / methanol solution (mixture of 1: 1 volume ratio) (Relative value) was calculated. The calculation results are shown in Table 6. In Table 6, the ratio in parentheses after the solution name indicates the mixing ratio of each solution.

As a result, the recovery rate of GA was slightly lower only when the sample was washed with a 0.0025M sodium hydroxide aqueous solution / methanol solution (mixed solution of 1: 1 by volume). However, even if any of the mixed solution of water, ammonia water and ammonia water and methanol was used as the cleaning liquid , GL, GA, and IS were all good.

[Example 4]

The influence of the kind of washing liquid on the solid phase on the quantitative sensitivity of the method of high sensitivity determination of the herbal medicine-derived component of the present invention was examined.

To 0.5 ml of human plasma, 50 占 퐇 of the standard solution (E) of Example 1 and 50 占 퐇 of IS were added and sufficiently mixed to obtain a plasma sample.

Specifically, MAX after the injection of the plasma sample was washed with 0.56 vol% ammonia water / methanol solution (mixture of 1: 1 volume ratio) and then washed again with ethanol, methanol, 50 vol% methanol aqueous solution or acetonitrile GL and GA were quantified in the same manner as in Example 1 except for that.

The recovery rate (relative value) of GL, GA, and IS of each plasma sample was calculated assuming that the second washing liquid was methanol. Table 7 shows the calculation results. As a result, even when the solution was washed with any solution, the recovery rate of GL was good at 80% or more. Also, the recovery rate of GA was better than 80% except for 50 vol% methanol aqueous solution. In particular, methanol was the best.

[Example 5]

The influence of the kind of eluate from the solid phase on the quantitative sensitivity of the method for quantifying high-sensitivity of the herbal medicine-derived component of the present invention was examined.

To 0.5 ml of human plasma, 50 占 퐇 of the standard solution (E) of Example 1 and 50 占 퐇 of IS were added and sufficiently mixed to obtain a plasma sample.

Specifically, GL and GA were quantified in the same manner as in Example 1, except that the elution from MAX was carried out using the solution described in Table 8.

The recovery rate (relative value) of GL, GA, and IS of each plasma sample was calculated assuming that the eluent was methanol with formic acid as 100%. The results are shown in Table 8. As a result, methanol was the most recovered formic acid. In addition, even when formic acid ethanol was used, GL and GA could be recovered well, though not methanol formate. On the other hand, when the eluate was acetonitrile formate or methanol, GL could not be detected.

[Comparative Example 1]

GL and GA in plasma were quantified using a solid phase having only a reversed phase distribution function instead of MAX.

To 0.5 ml of human plasma, 50 占 퐇 of the standard solution (E) of Example 1 and 50 占 퐇 of IS were added and sufficiently mixed to obtain a plasma sample.

Specifically, Sep-Pak (3 cc, 60 mg, 30 탆, manufactured by Waters Corporation), which is an ODS column, was used instead of MAX. Conditioning of the column was carried out with methanol and then with water, (Volume ratio = 1: 3, containing 1 M tetrabutylammonium bromide) as a solid cleaning liquid, or an eluate from a solid phase (water / acetonitrile / water) Was prepared in the same manner as in Example 1 except that 2 volume% formic acid methanol, 2 volume% acetonitrile formate / water (volume ratio = 1: 1) or 2 volume% methanol / water (volume ratio = 1: Respectively.

The recovery rate (relative value) of GL, GA, and IS of each plasma sample was calculated assuming that the amount of the solution mixed in the plasma sample in Example 2 was 0.56 vol% ammonia water and the recovery rate was 100%. Table 9 shows the calculation results. In Table 9, "1-1" represents 0.56% by volume ammonia water, "1-2" represents 0.1N hydrochloric acid, "2-1" represents water, "2-2" represents acetonitrile / water (Volume ratio = 1: 1), &quot; 3-3 &quot; is 2% by volume of acetone, Vol.% Formic acid methanol / water (volume ratio = 1: 1).

As a result, it was found that the recovery rate of GL was at most 50% at most even when the recovery rate was the best among the combinations of diluent and the like, and the detection sensitivity of GL and the like was much lower than that when MAX was used.

[Comparative Example 2]

GL and GA in the plasma were quantified using a solid phase having a reversed phase distribution function and a polarity interaction function in place of the MAX.

To 0.5 ml of human plasma, 50 占 퐇 of the standard solution (E) of Example 1 and 50 占 퐇 of IS were added and sufficiently mixed to obtain a plasma sample.

Specifically, Oasis HLB (3 cc, 60 mg, 30 탆, manufactured by Waters), which is a porous polymer, was used instead of MAX. The conditioning of the column was carried out with methanol and then with water, The same procedure as in Example 1 was carried out except that 0.56 vol% ammonia water or 0.1 N hydrochloric acid was used as a solution (diluting solution) for preparing cargo, a 5 vol% methanol aqueous solution was used as a solid phase washing solution and methanol was used as an eluent from the solid phase Respectively.

The recovery rate (relative value) of GL, GA, and IS of each plasma sample was calculated assuming that the amount of the solution mixed in the plasma sample in Example 2 was 0.56 vol% ammonia water and the recovery rate was 100%. The calculation results are shown in Table 10.

As a result, when 0.1 N hydrochloric acid was used as a diluent, no GL was detected, and the recovery rate of GA was also very low. On the other hand, even when 0.56 vol% ammonia water was used, the recovery rate of GL was at most 55%, and the detection sensitivity of GL and the like was much lower than that of using MAX.

[Comparative Example 3]

GL and GA in the plasma were quantified using a solid phase having a reversed phase distribution function and a cation exchange function in place of the MAX.

To 0.5 ml of human plasma, 50 占 퐇 of the standard solution (E) of Example 1 and 50 占 퐇 of IS were added and sufficiently mixed to obtain a plasma sample.

Specifically, Oasis MCX (3 cc, 60 mg, 30 탆, manufactured by Waters) was used in place of MAX. Conditioning of the column was carried out with methanol, followed by water, and preparation of a mixture to be applied to the column Except that 0.1N hydrochloric acid was used as a solution (diluting solution) for the solid phase, 0.1N hydrochloric acid was used as a solid phase washing solution, and 2 vol% ammonia water / methanol solution was used as an eluent from the solid phase.

The recovery rate (relative value) of GL, GA, and IS of each plasma sample was calculated assuming that the amount of the solution mixed in the plasma sample in Example 2 was 0.56 vol% ammonia water and the recovery rate was 100%. Table 11 shows the calculation result and the average value of the 5 independent runs.

As a result, it was found that the detection sensitivity of GL and GA was far lower than that of the case where the recovery rate did not reach 50% and MAX was used.

[Example 6]

The high-sensitivity determination method of the herbal medicine-derived component of the present invention was carried out by measuring the quantitative limit value of GL and GA in plasma when the mass analysis was carried out by the LC-MS / MS method, and also 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 solutions used in Example 1 were diluted with methanol in the same manner as in Example 1 to prepare the standard solutions (B) to (G) shown in Table 12.

<Preparation and Extraction of Plasma Samples>

A human plasma sample and a blank plasma sample to which standard solutions (B) to (G) were added were prepared in the same manner as in Example 1, and these plasma samples were applied to MAX, respectively, to adsorb GL and the like, Washed with ammonia water / methanol solution and methanol, eluted with 2 vol% formic acid / methanol solution, further evaporated to dryness.

<Quantitative Process>

The evaporated and dried extract was dissolved in 250 쨉 l of the following mobile phase A solution / B solution (mixture of 1: 1 by volume). One μl of the solution was injected into the analysis column, and subjected to LC-MS / MS under the following conditions.

LC-MS / MS system: API4000 system (manufactured by Applied Biosystems)

(LC)

Apparatus: LC-20A (manufactured by Shimadzu Corporation)

Analytical column: Inertsil ODS-3 (2.1 mm x 150 mm, particle diameter 5 m, manufactured by Gell Science)

Flow rate: 0.50 ml / min.

Column temperature: 40 DEG C

Auto sampler temperature: 5 ℃

Sample injection amount: 1 μl

Mobile phase: liquid A 0.1 vol% formic acid, liquid B 0.1 vol% formic acid acetonitrile, linear gradient (Table 13)

(MS / MS)

Device: API4000 (Applied Biosystems)

Ionization method: ESI (Turbo Spray)

Detection method: Static ion detection MRM (Multiple reaction monitoring)

Ion Spray Voltage: 5500V (positive)

Ion source temperature (Temperature): 400 ° C

Curtain gas: 10 (nitrogen)

Ion Source Gas 1: 70 (nitrogen)

Ion Source Gas 2: 50 (nitrogen)

Collision Gas: 8 (Nitrogen)

Figures 1A-1C show chromatograms of blank plasma samples. 1B shows the position of the peak of GA (GA retention time; m / z 471 to 149), FIG. 1C shows the position of peak of GA (IS / (The retention time of IS: m / z 751? 455), respectively. 2A to 2C show chromatograms of plasma samples (GL concentration in plasma: 0.5 ng / ml, GA concentration: 2 ng / ml) to which standard solution (G) was added. Fig. 2A shows a peak of GL (indicated by an arrow in the figure), Fig. 2B shows a peak of GA (indicated by an arrow in the figure) and Fig. 2C shows a peak of IS (indicated by an arrow in the figure). The retention times of GL, GA, and IS were about 1.2, 3.1, and 1.4 minutes, respectively. No peaks were observed on the chromatogram of the blank plasma samples, and the peak shapes of GL, GA and IS were good.

The quantitative results are shown in Table 15. Table 15 also shows the relative error between the actually added concentration and the measured value (quantitative value in the table). As a result, the relative error between both GL and GA was within 15%. That is, from these results, it can be seen that when the concentration of GL in the biological sample such as plasma is 0.5 to 200 ng / ml and the concentration of GA is 2 to 800 ng / ml, the high sensitivity of the herbal medicine- It is clear that GL in the biological sample can be quantified with high accuracy by the quantification method.

<Review of specificity, linearity, daily and daytime reproducibility>

In addition, according to the US Food and Drug Administration's Guidance for Industry ("Bioanalytical Method Validation", US Department of Health and Human Services, Food and Drug Administration, May 2001), specificity, linearity, Respectively. In addition, the stability evaluation test (storage stability, freeze-thaw stability, stability during measurement, standard solution stability in a biological sample in cryopreservation for 3 hours at -20 ° C and -80 ° C for 4 hours at room temperature) was also performed. Table 16 shows the results of the review. As a result, the method for quantitatively determining the herbal medicine-derived component of the present invention using LC-MS / MS showed good linearity and reproducibility in the range of 0.5 to 200 ng / ml and 2 to 800 ng / ml, respectively. As a result of intra-day reproducibility, the progress of GL was -12.8 ~ + 4.8%, precision was 4.0 ~ 9.5%, GA was -13.4 ~ + 4.4% and accuracy was 2.2 ~ 9.0%. On the other hand, in the daily reproducibility, the progress of GL was -9.4 to + 2.0%, precision was 2.1 to 5.5%, progress of GA was -10.9 to + 8.1%, and precision was 1.3 to 9.1%. From the results of reproducibility, it was confirmed that the quantitative limit concentrations of GL and GA were 0.5 ng / ml and 2 ng / ml, respectively.

In addition, it was stable within ± 15% in all stability evaluation tests (storage stability in living body samples, freeze-thaw stability, stability during measurement, standard solution stability) compared with the initial value. From the above results, it has been confirmed that the method of high sensitivity determination of the herbal medicine-derived component of the present invention is a reliable assay method in accordance with the assay validation guidance of the US Food and Drug Administration.

[Example 7]

Detection of GL in plasma after oral administration of GL-containing preparation and pharmacokinetics were measured.

In addition, the present embodiment is based on the ethical principle based on the Helsinki Declaration, Article 14 (3), Article 80-2 of the Pharmaceutical Affairs Law, and "Holy Spirit Concerning the Practice Standards for Clinical Trials of Drugs (1997) . Specifically, the oral administration of the GL-containing preparation and the collection of the plasma sample are carried out by the APS Co., Ltd. and the medical corporation Nursing YAMAGUCHI Hospitals by the request of the applicant, after approval by the third party committee, . Measurement of GL and GA in the obtained plasma sample was carried out by the Eco-Techno business division of the Japanese medical clinical research laboratory Co., Ltd. (currently, the present invention was analyzed by the applicant).

In addition, the subjects were instructed to take the test in the following manner. Six healthy Japanese men (age: 20 to 35 years old, pre-test BMI: 18.5 to 25.0 Or less).

A glycyrrhizin acid ammonium salt, a glycine, and a DL-methionine-containing sugar solution "Glycyrone (registered trademark) compounded form" were used as the GL-containing preparation. Glycyrrone formulations were administered in triplicate oral doses according to the clinical dose with sufficient water. Table 17 shows the test schedule such as administration and blood collection time. The obtained blood was collected by centrifugation (4 ° C, 3000 rpm, 10 minutes) within 30 minutes and 1 ml or more was recovered as plasma. The blood was stored frozen at -20 ° C or lower until the concentration was measured.

1 ml of 0.56 vol% ammonia water was added to 0.5 ml of the collected plasma sample and sufficiently mixed. Thereafter, the obtained mixture was applied to the MAX in the same manner as in Example 1 to adsorb the GL and the like, and a 0.56% by volume aqueous ammonia / Methanol solution and methanol, and then eluted with a 2 vol% formic acid / methanol solution, and further evaporated to dryness.

Then, in the same manner as in Example 6, LC-MS / MS was performed on the evaporated and dried extract, and GL concentration and GA concentration in each plasma sample were measured. FIG. 3 is a graph showing the average plasma concentration change of GL, and FIG. 4 is a graph showing the average plasma concentration change of GA. 3 and 4 show an average value and a standard deviation of six specimens. As a result, it was confirmed that the plasma concentration of the unchanged GL itself, as well as the GA, which is conventionally regarded as a major index, can be directly measured by the high sensitivity determination method of the herbal medicine-derived component of the present invention. That is, according to the present invention, for the first time in the detection of a trace amount of GL in plasma, the blood's dynamics of GL, which has been unclear for a long time, became clear.

<Analysis of Drug Dynamics>

From the measurement data of GL and GA, the maximum plasma concentration (Cmax) and the maximum plasma concentration reaching time (Tmax) were calculated. The withdrawal rate constant (kel) and the disappearance half-life (t1 / 2) of the plasma-concentration-time curve backward (AUC0 → 48h) up to 48 hours after administration were calculated by trapezoidal rule, Respectively. In addition, plasma concentration-time curves (AUC0-∞) and in-vivo retention times (MRT) up to 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 showed a correlation between the dose of GL and the plasma concentration in that it was almost equivalent to the predicted value (about 23 ng / ml) based on the large-scale oral administration example of the above-mentioned commercial reagent. Also, Tmax was 4.5 hours on average, but subjects showing multiple concentration peaks such as before (1 to 2 hours) or after (12 hours) were also observed. The posterior concentration peak appeared after the meal, suggesting that dietary intake may also affect GL absorption or intestinal circulation.

On the other hand, 4 hours of lag was confirmed until it was confirmed in the plasma, and the plasma concentration (about 20 times as high on the molar basis) of GL was detected about 10 times of GL after 6 hours. In vitro, GL was rapidly incubated with 3-monoglucuronyl-glychrretinic acid as a result of in vitro incubation under optimal conditions using human liver microsomes, but the production of GA was minute (Biochemical Pharmacology, 42 (6 / 7) 1025-1029, 1991). In addition, as a result of oral administration of GL in germ-free rats, it is considered that intestinal bacteria are predominantly responsible for the generation of GA in vivo, since GA is not confirmed among plasma and feces. Pharm. Pharmacol., 46, 135-137, 1994). Therefore, the absorption lag is presumed to be the time until contact with intestinal flora, and the dietary condition (or fasting condition) affects the movement of the drug in the digestive tract, the metabolic ability of the intestinal flora and digestive tract absorption, . Observation of the GA after absorption showed that the metabolites appeared to be weak in the subjects with high unchanged body concentration and that the metabolites appeared to be strong in the subjects whose unchanged body concentration was low. At this point, the absorption of digestive tracts of unchanged GL, as well as GA, was implicated in the metabolic ability of intestinal flora, suggesting that it could be expressed as individual differences (data not shown).

The high sensitivity determination method of the herbal medicine-derived component of the present invention can quantify GL in the blood after ingesting the GL-containing agent or the food or the like of the clinical dose by the high sensitivity assay method of the present invention. , Appropriate use of medicines, oriental medicine, safety evaluation, analysis of drug behavior, development of new GL formulation, and the like.

Claims (4)

A mixed substance in which a biological sample is mixed with an alkali or an alcohol is injected into a solid phase having a reversed phase distribution function and an anion exchange function and then the solid phase is washed at least once with a washing liquid and then the solid phase is removed from the solid phase An extraction step of preparing an extract containing the herbal medicine-derived component by elution,
A group consisting of glycyrrhizin, glycyrrhizic acid, glycyrrhizin and glycyrrhizin acid metabolites, glycyrrhizin and glycyrrhizin acid, a saponin component contained in licorice, and a pharmaceutically acceptable salt thereof, in the extract extracted by the above extraction step And a quantitative step of detecting and quantifying at least one species selected from the group consisting of
Wherein the cleaning liquid is one or more kinds of liquids selected from the group consisting of water, alkali, alcohol, and acetonitrile,
In the extraction step, washing of the solid phase is carried out by washing with a mixture of alkali, alcohol and water, a mixed solution of alkali and water, or water, followed by washing with a mixed solution of alcohol, alcohol and water or acetonitrile,
Wherein the biological sample is blood, plasma, or tissue extract. The method according to claim 1,
Wherein the solid phase washing is carried out by washing with a mixed solution of alkali and alcohol and water in the extraction step and then washing with a mixed solution of alcohol, alcohol and water or acetonitrile. 3. The method of claim 2,
Wherein the mixed solution of the alkali, alcohol and water is prepared by mixing 0.5 to 28% by volume of ammonia water and methanol in a ratio of 99: 1 to 1: 3. delete

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