WO2012067090A1 - 生薬由来成分の高感度定量方法 - Google Patents
生薬由来成分の高感度定量方法 Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2560/00—Chemical aspects of mass spectrometric analysis of biological material
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- the present invention relates to a method for quantifying glycyrrhizin or a metabolite thereof in a biological sample with high sensitivity.
- Glycyrrhizin (hereinafter abbreviated as GL) and its salts have various anti-allergic and anti-inflammatory effects, as well as immunoregulatory effects, hepatocellular injury inhibiting effects, hepatocyte proliferation promoting effects, virus proliferation inhibiting / inactivating effects, etc. It is a licorice root extract component having various physiological activities. In Japan, it has long been used as a clinical medicine such as Chinese herbal medicine, and is now widely used for the treatment of chronic liver disease, eczema / dermatitis, childhood stroflus, alopecia areata, various allergies, inflammation and the like. Further, as a use other than pharmaceuticals, GL has a salting effect, and since it has a sweetening effect, it is frequently used as a sweetener in a wide range of foods such as pickles and seasonings.
- GL is easy to accumulate in the liver and is rapidly excreted in bile, and is excreted while undergoing metabolism and enterohepatic circulation.
- GL which is a glycoside and a water-soluble polar substance, exhibits low absorbability, and its concentration in peripheral blood is extremely low due to metabolism by the enteric bacteria and the first-pass effect.
- various methods have been tried so far to measure the kinetics of GL in the blood.
- Non-Patent Documents 1 to 4 a method of measuring the amount of GL in the plasma by enzyme immunization (EIA) after treating the plasma collected from a healthy person orally administered with GL and removing the protein by methanol treatment.
- EIA enzyme immunization
- HPLC high performance liquid chromatography
- ODS octadecyl group chemical bond type
- the pharmacokinetics of unchanged GL which is the active substance, is useful in determining proper use from the efficacy and safety. For this reason, it is desirable that the amount of GL in a biological sample such as plasma can be quantified instead of using GA as an evaluation target.
- the present invention has been made in view of the above problems, and an object of the present invention is to provide a method for detecting and quantifying GL in a biological sample such as plasma.
- the present inventors firstly, a component containing GL from a biological sample by a solid phase extraction method using a solid phase having a reverse phase distribution function and an anion exchange function. After extraction, the GL in the extract was subjected to mass spectrometry, and it was found that GL in a biological sample could be detected and quantified, thereby completing the present invention.
- the present invention (1) After injecting a mixture obtained by mixing a biological sample with alkali or alcohol into a solid phase having a reverse phase distribution function and an anion exchange function, the solid phase is washed at least once with a washing solution, and then an acidic alcohol An extraction step of preparing an extract containing herbal medicine-derived components by eluting from the solid phase with Glycyrrhizin, glycyrrhetinic acid, metabolites of glycyrrhizin and glycyrrhetinic acid, related substances of glycyrrhizin and glycyrrhetinic acid, saponin components contained in licorice, and pharmaceutically acceptable products thereof in the extract extracted by the extraction process
- a quantitative step of detecting and quantifying at least one selected from the group consisting of salts by mass spectrometry; Have A highly sensitive quantitative determination method for crude drug-derived components, wherein the washing solution is one or a mixture of two or more selected from the
- (1) a highly sensitive quantitative method for herbal medicine-derived components
- (3) The mixed solution of (2), wherein the mixed solution of 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.
- a highly sensitive method for quantitative determination of herbal medicine-derived components as described in (4) The highly sensitive method for quantifying a crude drug-derived component according to any one of (1) to (3), wherein the biological sample is blood, plasma, or tissue extract, Is to provide.
- GL can be detected and quantified in a biological sample having a very low content by the highly sensitive quantification method of herbal medicine-derived components of the present invention. For this reason, by using the high-sensitivity quantification method for herbal medicine-derived components of the present invention, GL in a biological sample collected from a human who has ingested a preparation containing GL or a food or drink is quantified, and the pharmacokinetics is analyzed accurately. can do.
- Example 6 it is the figure which showed the chromatogram of GL in a blank plasma sample. In Example 6, it is the figure which showed the chromatogram of GA in a blank plasma sample. In Example 6, it is the figure which showed the chromatogram of the internal standard (IS) in a blank plasma sample. In Example 6, it is the figure which showed the chromatogram of GL in the plasma sample (GL addition amount in plasma: 0.5 ng / mL) which added the standard solution (G). In Example 6, it is the figure which showed the chromatogram of GA in the plasma sample (The amount of GA addition in plasma: 2 ng / mL) which added the standard solution (G). In Example 6, it is the figure which showed the chromatogram of IS in the plasma sample which added the standard solution (G). In Example 7, it is the figure which showed the average plasma concentration transition of GL. In Example 7, it is the figure which showed the average plasma concentration transition of GA.
- GL in a biological sample is extracted by a solid phase extraction method using a solid phase having a reverse phase distribution function and an anion exchange function, and then in the obtained extract.
- the GL content of is quantitatively determined by mass spectrometry.
- the detection sensitivity of GL can be increased by quantifying using mass spectrometry instead of the conventional HPLC method.
- a solid phase having a reverse phase distribution function and an anion exchange function instead of a solid phase extraction method using a solid phase having only a reverse phase distribution function like a conventional ODS column, More impurities can be removed from the extract used for analysis, and the sensitivity of mass spectrometry can be dramatically improved.
- the high-sensitivity quantification method for herbal medicine-derived components of the present invention after injecting a mixture obtained by mixing a biological sample with alkali or alcohol into a solid phase having a reverse phase distribution function and an anion exchange function, The solid phase is washed once or twice with a washing solution and then eluted from the solid phase with acidic alcohol to prepare an extract containing herbal medicine-derived components, and the extraction extracted by the extraction step At least one selected from the group consisting of GL, GA, GL and GA metabolites, GL and GA analogs, saponin components contained in licorice, and pharmaceutically acceptable salts thereof , Having a quantitative step of detecting and quantifying by mass spectrometry, wherein the cleaning liquid is one or a mixture of two or more selected from the group consisting of water, alkali, alcohol, and acetonitrile And wherein the door. It is preferable that an extract containing at least two kinds of herbal medicine-derived components is prepared in the extraction step, and at least two components are detected and
- the herbal medicine-derived components quantified by the high-sensitivity quantification method for herbal medicine-derived components of the present invention include GL, GA, GL and GA metabolites, GL and GA related substances, saponin components contained in licorice, and these It is at least one selected from the group consisting of pharmaceutically acceptable salts (hereinafter sometimes referred to as “GL etc.”).
- metabolites of GL and GA include 3-monoglycuronic-glycryretinic acid (20 ⁇ -Carboxy-11-oxo-30-norolean-12-en-3 ⁇ -yl- ⁇ -D-glucopyranosiduronic acid), 30-monoglycuric acid.
- glychreretinic acid (3 ⁇ -Hydroxy-11-oxoolan-12-en-30-oyl- ⁇ -D-glucopyranosiduronic acid), 3-oxo-GA (3,11-Dioxolene-12-en-30-oic acid), 3 ⁇ -GA (3 ⁇ -Hydroxy-11-oxolean-12-en-30-ic acid), 3-position sulfate conjugate (3 ⁇ -Hydr oxysulfonyloxy-11-oxo-12-en-30-ic acid), 3 ⁇ , 22 ⁇ -Dihydroxy-11-oxo-12-en-30-ic acid, and 3 ⁇ , 24-Dihydroxy-11-oxolean-12-en- 30-oic acid and the like.
- GL and GA for example, 20 ⁇ -Carboxy-11-oxo-29-norlean-12-en-3 ⁇ -yl ( ⁇ -D-glucopyranosyluronic acid)-(1 ⁇ 2) - ⁇ -D -Glucopyranosiduronic acid, 20 ⁇ -Carboxy-24-hydroxy-11-oxo-30-norolean-12-en-3 ⁇ -yl ( ⁇ -D-glucopynosyluronic acid)-(1 ⁇ 2) - ⁇ -D-glucopyranicidic18, -GL (20 ⁇ -Carboxy-11-oxo- (18 ⁇ H) -30-norolean-12-en-3 ⁇ -yl ( ⁇ -D-glucopyranosyluronic cid) - (1 ⁇ 2) - ⁇ -D-glucopyranosiduronic acid), 18 ⁇ -GA (3 ⁇ -Hydroxy-11-oxo- (18 ⁇ H) -olean-12-en-30-oic acid) and the
- saponin component contained in licorice examples include, for example, liquorice-saponin20C1 (20 ⁇ -Carboxy-11-oxo-30-norolean-12-en-3 ⁇ -yl ( ⁇ -D-glucopyranyluronic acid)-(1 ⁇ 2) - ⁇ -D-glucopyranosiduronic acid) and the like.
- the pharmaceutically acceptable salt such as GL used in the present invention is not particularly limited as long as it has a pharmacological effect similar to that of GL in vivo. Specifically, ammonium salt, sodium salt, potassium salt etc. are mentioned, for example.
- one type of GL or the like may be quantified, and two or more types may be quantified by one operation.
- the biological sample used in the high-sensitivity quantification method for herbal medicine-derived components of the present invention may be a sample collected from a living body, and is preferably collected from an animal such as a human, mouse, or rat. It may also be blood, plasma, serum, urine, ascites, pleural effusion, joint fluid, bone marrow fluid, bile, etc., and extracts such as tissue fragments collected from tissues such as liver, pancreas, kidney (tissue extract) ). An extract such as a tissue piece can be prepared by homogenizing a tissue piece or the like by a conventional method. In the present invention, blood, plasma, urine, or tissue extract is preferable, and plasma is more preferable.
- an admixture obtained by mixing a biological sample with alkali or alcohol is prepared.
- the alkali added to the biological sample is not particularly limited as long as the pH of the resulting mixture can be made alkaline.
- ammonia, aqueous ammonia, sodium hydroxide solution, sodium bicarbonate Examples include solutions.
- the alcohol is preferably a lower alcohol having 1 to 6 carbon atoms, and examples thereof include methanol, ethanol, and isopropanol.
- the alcohol solution diluted with water may be sufficient.
- the alkali or alcohol added to the biological sample is preferably ammonia, aqueous ammonia, methanol, or an aqueous methanol solution, and more preferably ammonia or aqueous ammonia.
- the concentration of ammonia water added to the biological sample is not particularly limited, and can be appropriately adjusted in consideration of the type of biological sample, the type of solid phase used thereafter, the ammonia concentration of the resulting mixture, and the like. .
- the concentration of ammonia or aqueous ammonia added to the biological sample is preferably such that the resulting mixture has an ammonia concentration of 0.01 to 30% by volume, and 0.05 to 25% by volume. % Is more preferable, and a concentration of 0.05 to 20% by volume is more preferable.
- the obtained mixture is injected into a solid phase having a reverse phase distribution function and an anion exchange function, and GL and the like in the mixture are adsorbed on the solid phase.
- the solid phase having the reverse phase distribution function and the anion exchange function include a reverse phase distribution-anion exchange mixed mode solid phase cartridge, Oasis MAX (manufactured by Waters) and the like.
- the cleaning liquid is one or a mixture of two or more selected from the group consisting of water, alkali, alcohol, and acetonitrile.
- alkali and alcohol used as the cleaning liquid include the same alkali and alcohol added to the biological sample.
- the solid phase may be washed twice or more with the same kind of washing solution, or sequentially with different kinds of washing solutions.
- the solid phase is washed with a mixture of alkali, alcohol and water, a mixture of alkali and water, or water, and then washed with alcohol, a mixture of alcohol and water, or acetonitrile. It is preferable to carry out by washing, and it is more preferred to wash by washing with alcohol, a mixture of alcohol and water, or acetonitrile after washing with a mixture of alkali, alcohol and water.
- the mixed solution of alkali, alcohol and water is preferably a mixed solution of ammonia, alcohol and water, and more preferably a mixed solution of ammonia, methanol and water.
- the composition ratio of each component in the mixed solution of ammonia, methanol, and water is not particularly limited as long as it can maintain the state in which GL or the like is adsorbed to the solid phase.
- the methanol concentration in the mixed solution is preferably 1 to 75% by volume and the ammonia concentration is preferably 0.1 to 21% by volume, the methanol concentration is 25 to 75% by volume, and the ammonia concentration is 0.1 to 21% by volume. It is more preferable that A mixed liquid having such a composition ratio can be prepared, for example, by mixing 0.5 to 28% by volume of ammonia water and methanol at 99: 1 to 1: 3.
- the washing liquid used for the second washing is preferably alcohol, more preferably methanol or ethanol, and methanol. Further preferred.
- an extract containing herbal medicine-derived components is prepared by elution from the solid phase with acidic alcohol.
- the acidifying solvent include formic alcohol, hydrochloric acid, trifluoroacetic acid and the like.
- formic alcohol is preferred.
- the formic alcohol used as the eluent is preferably an ester of formic acid and a lower alcohol having 1 to 6 carbon atoms, more preferably formic acid methanol or formic acid ethanol, and further preferably formic acid methanol.
- the obtained eluate is evaporated to dryness for subsequent mass analysis.
- GL or the like in the extract extracted by the extraction process is detected and quantified by mass spectrometry.
- the mass spectrometry is preferably carried out by LC-MS (liquid chromatography / mass spectrometry) method or LC-MS / MS (liquid chromatography / tandem mass spectrometry) method. Specifically, the eluate evaporated to dryness is dissolved in the LC mobile phase, followed by LC and MS. LC-MS and LC-MS / MS can be performed by using an apparatus that combines HPLC and a mass spectrometer.
- LC is preferably performed using a column having a reverse phase distribution function, such as an ODS column, as described in Patent Documents 1 to 4.
- MS can be performed by a conventional method. For example, after ionizing a sample by an ESI (electrospray ionization) method or an APCI (atmospheric pressure chemical ionization) method, each ion is separated and detected by a magnetic field deflection type, quadrupole type, time-of-flight type device or the like. .
- ESI electrospray ionization
- APCI atmospheric pressure chemical ionization
- the limit of quantification of GL or the like in blood, plasma, or serum can be improved to about 20 ng / mL.
- the limit of quantification of GL or the like in blood or the like can be improved to 10 ng / mL or less, for example, about 0.5 ng / mL.
- the plasma GL concentration in a large-scale oral administration (1600 mg as GL) of a commercially available reagent was about 500 ng / mL (Environmental Health Perspectives, 102 (9), 65-68, 1994).
- the estimated blood concentration equivalent to a clinical dose of 75 mg is calculated to be about 23 ng / mL. That is, GL in blood that could not be detected by the conventional method can be quantified by the high-sensitivity quantification method for herbal medicine-derived components of the present invention.
- the GL concentration in the blood after oral intake of GL preparations, etc. has a large individual difference and the measurement limit is insufficient.
- the high-sensitivity quantification method of herbal medicine-derived components of the present invention particularly by performing mass spectrometry by LC-MS / MS method, the measurement accuracy and the reliability of measurement results are insufficient. A more reliable measurement result can be obtained.
- Example 1 When the high-sensitivity quantification method for herbal medicine-derived components of the present invention was performed by mass spectrometry using the LC-MS method, the quantification limit values of GL and GA in plasma were measured, and a calibration curve was prepared.
- ⁇ Preparation of standard solution> First, 10.0 mg of GL (manufactured by Tokiwa Phytochemical Laboratories) was accurately weighed and dissolved in methanol, and exactly 100 mL was prepared to prepare a 100 ⁇ g / mL GL standard stock solution.
- a plasma sample was prepared by adding 50 ⁇ L of the standard solution (A) to 0.5 mL of human plasma and mixing well.
- standard solutions (B) to (F) plasma samples were prepared in the same manner.
- 50 ⁇ L of internal standard solution (IS) was further added.
- ⁇ -Hederin was used as an internal standard substance.
- a blank plasma sample was prepared by adding 100 ⁇ L of methanol to 0.5 mL of human plasma.
- the measurement result of LC-MS increases depending on the GL or GA concentration in the plasma sample, and the calibration curve obtained from the GL or GA concentration in the plasma sample and the measurement result of LC-MS is linear. showed that. That is, from these results, the concentration of GL in a biological sample such as plasma is 20 to 400 ng / mL, the concentration of GA is 80 to 1600 ng / mL, and the high concentration of the crude drug-derived component of the present invention using LC-MS. It is clear that GL and the like in a biological sample can be quantified with high accuracy by the sensitivity quantification method.
- the accuracy of the positive mode (GL +) is -5.9 to + 5.9%, the accuracy is 3.3 to 7.7%, the accuracy of GA is -7.0 to + 7.2%, and the accuracy is 3. 0.0 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 effect of the type of alkali or alcohol mixed with the biological sample on the quantitative sensitivity of the high-sensitivity quantitative method for herbal medicine-derived components of the present invention was examined.
- a plasma sample was prepared by adding 50 ⁇ L of the standard solution (E) of Example 1 and 50 ⁇ L of IS to 0.5 mL of human plasma and mixing them well.
- GL and GA were quantified in the same manner as in Example 1 except that 1 mL of the solution shown in Table 5 was added to the plasma sample to prepare a mixture to be applied to MAX.
- Example 3 The effect of the type of the solid phase washing solution on the quantitative sensitivity of the high-sensitivity quantitative method for herbal medicine-derived components of the present invention was examined.
- a plasma sample was prepared by adding 50 ⁇ L of the standard solution (E) of Example 1 and 50 ⁇ L of IS to 0.5 mL of human plasma and mixing them well.
- GL and GA were quantified in the same manner as in Example 1 except that MAX after injection of the plasma sample was washed with the solution shown in Table 6 and further washed with methanol.
- the quantification result when the MAX washing solution after injecting the plasma sample was 0.56% by volume ammonia water / methanol solution (mixed solution with a volume ratio of 1: 1) was defined as a recovery rate of 100%.
- GA, and IS recovery rates were calculated. Table 6 shows the calculation results. In Table 6, the ratio in parentheses after the solution name indicates the mixing ratio of each solution.
- the GA recovery rate was slightly low only when washed with 0.0025M sodium hydroxide aqueous solution / methanol solution (volume ratio 1: 1 mixture), but water, ammonia water, ammonia water and methanol When any of the mixed solutions was used as a cleaning solution, the recovery rates of GL, GA, and IS were all good.
- Example 4 The effect of the type of the solid phase washing solution on the quantitative sensitivity of the high-sensitivity quantitative method for herbal medicine-derived components of the present invention was examined.
- a plasma sample was prepared by adding 50 ⁇ L of the standard solution (E) of Example 1 and 50 ⁇ L of IS to 0.5 mL of human plasma and mixing them well. Specifically, after the plasma sample is injected, MAX is washed with 0.56% by volume ammonia water / methanol solution (mixed solution with a volume ratio of 1: 1), and then ethanol, methanol, 50% by volume methanol aqueous solution, or acetonitrile. GL and GA were quantified in the same manner as in Example 1 except that the GL and GA were further washed.
- the recovery rate (relative value) of GL, GA, and IS of each plasma sample was calculated assuming that the second washing solution was methanol as 100%. Table 7 shows the calculation results. As a result, the GL recovery rate was 80% or more, which was good, regardless of which solution was used for washing. Also, the GA recovery rate was 80% or higher except in the case of 50% by volume methanol aqueous solution, which was good. In particular, methanol was the best.
- Example 5 The effect of the type of eluate from the solid phase on the quantitative sensitivity of the high-sensitivity quantitative method for crude drug-derived components of the present invention was examined.
- a plasma sample was prepared by adding 50 ⁇ L of the standard solution (E) of Example 1 and 50 ⁇ L of IS to 0.5 mL of human plasma and mixing them well.
- GL and GA were quantified in the same manner as in Example 1 except that elution from MAX was performed using the solutions shown in Table 8.
- GL and GA in plasma were quantified using a solid phase having only a reverse phase distribution function instead of MAX.
- a plasma sample was prepared by adding 50 ⁇ L of the standard solution (E) of Example 1 and 50 ⁇ L of IS to 0.5 mL of human plasma and mixing them well.
- Example 2 when the solution mixed with the plasma sample was 0.56% by volume of ammonia water, the recovery rate was 100%, and the recovery rates of GL, GA, and IS of each plasma sample (relative values) was calculated. Table 9 shows the calculation results.
- the recovery rate of GL is at most about 50%, and the detection sensitivity of GL and the like is far higher than when MAX is used. I found it inferior.
- GL and GA in plasma were quantified using a solid phase having a reverse phase distribution function and a polar interaction function instead of MAX.
- a plasma sample was prepared by adding 50 ⁇ L of the standard solution (E) of Example 1 and 50 ⁇ L of IS to 0.5 mL of human plasma and mixing them well.
- Example 2 when the solution mixed with the plasma sample was 0.56% by volume of ammonia water, the recovery rate was 100%, and the recovery rates of GL, GA, and IS of each plasma sample (relative values) was calculated. Table 10 shows the calculation results.
- Oasis MCX (3 cc, 60 mg, 30 ⁇ m, manufactured by Waters) was used instead of MAX, the conditioning of the column was performed with methanol and then water, and the mixture applied to the column Except for using 0.1N hydrochloric acid as a solution (diluent) for preparation, 0.1N hydrochloric acid as a solid phase washing solution, and 2% by volume ammonia water / methanol solution as an eluent from the solid phase, respectively. The same operation as in Example 1 was performed.
- Example 2 when the solution mixed with the plasma sample was 0.56% by volume of ammonia water, the recovery rate was 100%, and the recovery rates of GL, GA, and IS of each plasma sample (relative values) was calculated. Table 11 shows the calculation results and average values of five independent trials. As a result, it was found that the recovery rate of GL and GA did not reach 50%, and the detection sensitivity of GL or the like was far inferior to the case of using MAX.
- Example 6 When the high-sensitivity quantification method for crude drug-derived components of the present invention was performed by mass spectrometry using the LC-MS / MS method, the quantification limit values of GL and GA in plasma were measured, and a calibration curve was prepared. At the same time, the accuracy and reliability of the method were verified.
- ⁇ Plasma sample preparation and extraction process> In the same manner as in Example 1, human plasma samples and blank plasma samples to which standard solutions (B) to (G) were added were prepared, and each of these plasma samples was applied to MAX to adsorb GL and the like. After washing with 56 volume% aqueous ammonia / methanol solution and methanol, elution was carried out with 2 volume% formic acid / methanol solution, followed by evaporation to dryness.
- LC-MS / MS system API4000 system (Applied Biosystems) (LC) Equipment: LC-20A (manufactured by Shimadzu Corporation) Analytical column: Inertsil ODS-3 (2.1 mm ⁇ 150 mm, particle size 5 ⁇ m, manufactured by GL Sciences Inc.) Flow rate: 0.50 mL / min. Column temperature: 40 ° C Autosampler temperature: 5 ° C Sample injection volume: 1 ⁇ L Mobile phase: Liquid A 0.1 vol% formic acid, liquid B 0.1 vol% formic acid acetonitrile, linear gradient (Table 13)
- Ionization method ESI (Turbo Spray) Detection method: Positive ion detection MRM (Multiple reaction monitoring) Ion spray voltage (IonSpray Voltage): 5500V (positive) Ion source temperature (Temperature): 400 ° C.
- 1A to 1C show chromatograms of blank plasma samples.
- 1A shows the position of the GL peak (GL retention time; m / z 823 ⁇ 453)
- FIG. 1B shows the GA peak position (GA retention time; m / z 471 ⁇ 149)
- FIG. 1C shows the IS.
- the peak positions (IS retention time; m / z 751 ⁇ 455) are shown.
- 2A to 2C show chromatograms of plasma samples to which the standard solution (G) was added (GL concentration in plasma: 0.5 ng / mL, GA concentration: 2 ng / mL).
- 2A shows a GL peak (indicated by an arrowhead in the figure)
- FIG. 2B shows a GA peak (indicated by an arrowhead in the figure), and FIG. 2C shows an IS peak (indicated by an arrowhead in the figure).
- Retention times of GL, GA, and IS are about 1.2 minutes, 3.1 minutes, and 1.4 minutes, respectively, and no peak interfering with quantification is observed on the chromatogram of the blank plasma sample.
- the peak shapes of GA and IS were good.
- the high-sensitivity quantification method for crude drug-derived components of the present invention using LC-MS / MS shows good linearity and reproducibility in the range of 0.5 to 200 ng / mL and 2 to 800 ng / mL, respectively. It was.
- the accuracy of GL is -12.8 to + 4.8%, the accuracy is 4.0 to 9.5%, and the accuracy of GA is -13.4 to + 4.4%, the accuracy Was 2.2 to 9.0%.
- the accuracy of GL is -9.4 to + 2.0%, the accuracy is 2.1 to 5.5%, and the accuracy of GA is -10.9 to + 8.1%.
- Example 7 The detection and pharmacokinetics of GL in plasma after oral administration of a GL-containing preparation were measured. This example is based on the ethical principles based on the Declaration of Helsinki, Article 14, Paragraph 3 of the Pharmaceutical Affairs Law, Article 80-2, and “Ministerial Ordinance on Standards for Conducting Clinical Trials of Drugs (GCP)” (1997 Ministry of Health and Welfare) This was carried out in accordance with Ordinance No. 28). Specifically, oral administration of GL-containing preparations and collection of plasma samples will be conducted at the request of the applicant, by EPS Co., Ltd.
- a sugar-coated tablet “Glycylon (registered trademark) combination tablet” containing monoammonium glycyrrhizinate, glycine and DL-methionine was used as a GL-containing preparation.
- the glycyrone combination tablets were given as a single oral administration of 3 tablets according to the clinical normal dose together with sufficient water. Test schedules such as administration and blood collection time are shown in Table 17. The obtained blood was centrifuged within 30 minutes (4 ° C., 3000 rpm, 10 minutes) to collect 1 mL or more of plasma and stored frozen at ⁇ 20 ° C. or less until the concentration measurement.
- FIG. 3 is a graph showing changes in the average plasma concentration of GL, and FIG.
- the high-sensitivity quantification method for herbal medicine-derived components of the present invention directly measures the plasma concentration of GL itself, which is not changed, as well as the main metabolite GA, which has conventionally been an alternative index. It was confirmed that That is, the present invention succeeded for the first time in the detection of a very small amount of plasma GL, and revealed the blood kinetics of GL, which has long been unknown.
- GA which is the main metabolite of GL
- a plasma concentration about 10 times that of GL (about 20 times in terms of mole) is detected. It was.
- In vitro as a result of incubating GL under optimal conditions using human liver microsomes, it was immediately converted to 3-monoglucouronyl-glycrretic acid, an intermediate metabolite, but there was little GA production ( Biochemical® Pharmacology, 42 (6/7) 1025-1029, 1991.).
- the absorption lag is estimated to be the time to contact the intestinal flora, and the dietary conditions (or fasting conditions) affect the movement of drugs in the gastrointestinal tract, the metabolic capacity of the intestinal flora, gastrointestinal absorption, It was also considered to be a factor affecting the GA exposure.
- the high-sensitivity quantification of the crude drug-derived component of the present invention can be quantified by the high-sensitivity quantification method of the crude drug-derived component of the present invention.
- the method can be used mainly for proper use of pharmaceuticals and Kampo, safety evaluation, pharmacokinetic analysis, development of new GL preparations, and the like.
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Abstract
Description
本願は、日本国において、2010年11月16日に出願された特願2010-256187号に基づき優先権を主張し、その内容をここに援用する。
(1) 生体試料をアルカリ又はアルコールに混和した混和物を、逆相分配機能及び陰イオン交換機能を備える固相に注入した後、前記固相を洗浄液で少なくとも1回洗浄し、その後、酸性アルコールにより前記固相から溶出することにより、生薬由来成分を含む抽出物を調製する抽出工程と、
前記抽出工程により抽出された抽出物中のグリチルリチン、グリチルレチン酸、グリチルリチン及びグリチルレチン酸の代謝物、グリチルリチン及びグリチルレチン酸の類縁物質、甘草に含有されているサポニン成分、並びにこれらの薬学的に許容される塩からなる群より選択される少なくも1種を、質量分析法により検出し定量する定量工程と、
を有し、
前記洗浄液が、水、アルカリ、アルコール、及びアセトニトリルからなる群より選択される1種又は2種以上の混合液であることを特徴とする生薬由来成分の高感度定量方法、
(2) 前記抽出工程において、固相の洗浄を、アルカリとアルコールと水の混合液により洗浄した後、アルコール、アルコールと水の混合液、又はアセトニトリルにより洗浄することによって行うことを特徴とする前記(1)に記載の生薬由来成分の高感度定量方法、
(3) 前記アルカリとアルコールと水の混合液が、0.5~28体積%アンモニア水とメタノールを99:1~1:3で混合してなるものであることを特徴とする前記(2)に記載の生薬由来成分の高感度定量方法、
(4)前記生体試料が、血液、血漿、又は組織抽出物であることを特徴とする前記(1)~(3)のいずれか一つに記載の生薬由来成分の高感度定量方法、
を提供するものである。
前記抽出工程にて少なくとも2種の生薬由来成分を含む抽出物を調製し、前記定量工程にて少なくとも2種の成分を一回の測定(検出モードは別)で検出し定量することが好ましい。
GL及びGAの代謝物としては、例えば、3-monoglucuronyl-glychrretinic acid(20β-Carboxy-11-oxo-30-norolean-12-en-3β-yl-β-D-glucopyranosiduronic acid)、30-monoglucuronyl-glychrretinic acid(3β-Hydroxy-11-oxoolean-12-en-30-oyl-β-D-glucopyranosiduronic acid)、3-oxo-GA(3,11-Dioxoolean-12-en-30-oic acid)、3α-GA(3α-Hydroxy-11-oxoolean-12-en-30-oic acid)、3位硫酸抱合体(3β-Hydroxysulfonyloxy-11-oxoolean-12-en-30-oic acid)、3β,22α-Dihydroxy-11-oxoolean-12-en-30-oic acid、及び3β,24-Dihydroxy-11-oxoolean-12-en-30-oic acid等が挙げられる。
抽出工程として、まず、生体試料をアルカリ又はアルコールに混和した混和物を調製する。生体試料に添加するアルカリとしては、得られる混和物のpHをアルカリ性にすることができるものであれば、特に限定されるものではなく、例えば、アンモニア、アンモニア水、水酸化ナトリウム溶液、炭酸水素ナトリウム溶液等が挙げられる。また、アルコールとしては、炭素数1~6の低級アルコールであることが好ましく、メタノール、エタノール、イソプロパノール等が挙げられる。また、水で希釈されたアルコール溶液であってもよい。本発明においては、生体試料に添加するアルカリ又はアルコールとしては、アンモニア、アンモニア水、メタノール、又はメタノール水溶液であることが好ましく、アンモニア又はアンモニア水であることがより好ましい。
本発明の生薬由来成分の高感度定量方法を、質量分析をLC-MS法によりを行った場合の、血漿中のGL及びGAの定量限界値を測定し、かつ検量線を作成した。
<標準溶液の調製>
まず、GL(常磐植物化学研究所製)10.0mgを正確に量り、メタノールに溶解した後、正確に100mLとし、100μg/mLのGL標準原液を調製した。同様に、GA(アルプス薬品工業株式会社製)10.0mgを正確に量り、メタノールに溶解した後、正確に100mLとし、100μg/mLのGA標準原液を調製した。
次いで、GL標準原液2mL及びGA標準原液8mLを正確に分取し、メタノールで正確に50mLとして、GL濃度が4000ng/mL、GA濃度が16000ng/mLである標準溶液(A)を調製した。この標準溶液Aをメタノールで順次希釈し、表1に記載の標準溶液(B)~(F)を調製した。標準原液及び標準溶液はそれぞれ調製後冷蔵保存(5±4℃)した。調製器具はガラス製のものを用いた。
ヒト血漿0.5mLに、標準溶液(A)50μLを添加し、十分に混和したものを血漿サンプルとした。標準溶液(B)~(F)についても、それぞれ同様にして血漿サンプルを調製した。これらの血漿サンプルには、内部標準液(IS)50μLをさらに添加した。なお、本実施例では、内部標準物質としてα-Hederinを用いた。また、ブランク血漿サンプルとして、ヒト血漿0.5mLにメタノールを100μL添加したものを調製した。
各血漿サンプルに、0.56体積%アンモニア水1mLを添加して十分に混和した後(混和物中のアンモニア濃度:0.37体積%)、得られた混和物を全量、Oasis MAX(Waters社製)(以下、単に「MAX」)に注入(アプライ)した。当該MAXは、血漿サンプルを注入する前に予め、2体積%ギ酸/メタノール溶液を注入した後、水を注入することにより、コンディショニングしておいた。
血漿サンプルを注入後、当該MAXに0.56体積%アンモニア水/メタノール溶液(体積比1:1の混合液)で洗浄した後、メタノールでさらに洗浄した。
その後、2体積%ギ酸/メタノール溶液を注入し、当該MAXに吸着していたGL等を溶出させた。溶出液(抽出物)は、40℃加温下、窒素ガスにて蒸発乾固させた。
蒸発乾固させた抽出物を、下記移動相A液/B液(体積比1:1の混合液)250μLに溶解させた。このうち20μLを分析カラムに注入し、下記に示す条件でLCを行った。
装置: Alliance HT 2695(Waters社製)
分析カラム: CAPCELL PAK C18(UG120、5μm、1.5mm×150mm、資生堂社製)
移動相:A液 0.1体積%ギ酸、B液 0.1体積%ギ酸アセトニトリル、リニアグラジエント(表2)
流速: 0.30mL/min.
カラム温度: 40℃
試料注入量: 20μL
装置: ZQ2000(Waters社製)
イオン化法: ESI(Turbo Spray)
検出法: GL(負、正イオン検出)、GA・IS(正イオン検出)、MRM(Multiple reaction monitoring)
Capillary電圧: 2.5kV
Extractor: 2V
RF lens: 0.2V
Source Temperature: 110℃
Desolvation Temperature: 350℃
Desolvation Gas Flow: 350L/hr
Cone Gas Flow: 50L/hr
さらに、特異性、直線性、及び日内再現性を検討した。この結果、LC-MSを用いた本発明の生薬由来成分の高感度定量方法は、それぞれ20~400ng/mL及び80~1600ng/mLの範囲で良好な直線性(検量線の相関係数は0.9979)と再現性を示した。また、日内再現性の結果、表4に示すように、GLのネガティブモード(GL-)の真度は-1.2~+0.6%、精度は5.2~6.0%であり、ポジティブモード(GL+)の真度は-5.9~+5.9%、精度は3.3~7.7%であり、GAの真度は-7.0~+7.2%、精度は3.0~7.7%であった。以上の結果より、GL及びGAの定量限界濃度はそれぞれ、20ng/mL、80ng/mLであることが確認された。
生体試料に混和するアルカリ又はアルコールの種類が、本発明の生薬由来成分の高感度定量方法の定量感度に及ぼす影響を調べた。
ヒト血漿0.5mLに、実施例1の標準溶液(E)50μL及びIS50μLを添加し、十分に混和したものを血漿サンプルとした。
具体的には、表5に記載の溶液1mLを血漿サンプルに添加してMAXにアプライする混和物を調製した以外は実施例1と同様にして、GL及びGAを定量した。
血漿サンプルに混和した溶液が0.56体積%アンモニア水であった場合の定量結果をそれぞれ回収率100%とし、各血漿サンプルのGL、GA、及びISの回収率(相対値)を算出した。算出結果を表5に示す。なお、表5中のアンモニア水の後ろの括弧書内の数値は、当該アンモニア水を添加して得られた混和物中のアンモニア濃度(体積%)を示す。
固相の洗浄液の種類が、本発明の生薬由来成分の高感度定量方法の定量感度に及ぼす影響を調べた。
ヒト血漿0.5mLに、実施例1の標準溶液(E)50μL及びIS50μLを添加し、十分に混和したものを血漿サンプルとした。
具体的には、血漿サンプルを注入後のMAXを表6に記載の溶液で洗浄した後、メタノールでさらに洗浄した以外は実施例1と同様にして、GL及びGAを定量した。
血漿サンプルを注入後のMAXの洗浄液が0.56体積%アンモニア水/メタノール溶液(体積比1:1の混合液)であった場合の定量結果をそれぞれ回収率100%とし、各血漿サンプルのGL、GA、及びISの回収率(相対値)を算出した。算出結果を表6に示す。なお、表6中、溶液名の後ろの括弧書内の比率は、各溶液の混合比を示す。
固相の洗浄液の種類が、本発明の生薬由来成分の高感度定量方法の定量感度に及ぼす影響を調べた。
ヒト血漿0.5mLに、実施例1の標準溶液(E)50μL及びIS50μLを添加し、十分に混和したものを血漿サンプルとした。
具体的には、血漿サンプルを注入後のMAXを0.56体積%アンモニア水/メタノール溶液(体積比1:1の混合液)で洗浄した後、エタノール、メタノール、50体積%メタノール水溶液、又はアセトニトリルでさらに洗浄した以外は実施例1と同様にして、GL及びGAを定量した。
2度目の洗浄液がメタノールであった場合を100%とし、各血漿サンプルのGL、GA、及びISの回収率(相対値)を算出した。算出結果を表7に示す。この結果、いずれの溶液で洗浄した場合であっても、GLの回収率は80%以上であり、良好であった。また、GAの回収率も、50体積%メタノール水溶液の場合を除き80%以上であり、良好であった。特に、メタノールが最も良好であった。
固相からの溶出液の種類が、本発明の生薬由来成分の高感度定量方法の定量感度に及ぼす影響を調べた。
ヒト血漿0.5mLに、実施例1の標準溶液(E)50μL及びIS50μLを添加し、十分に混和したものを血漿サンプルとした。
具体的には、MAXからの溶出を表8に記載の溶液を用いて行った以外は実施例1と同様にして、GL及びGAを定量した。
MAXに代えて、逆相分配機能のみを備える固相を用いて、血漿中のGL及びGAの定量を行った。
ヒト血漿0.5mLに、実施例1の標準溶液(E)50μL及びIS50μLを添加し、十分に混和したものを血漿サンプルとした。
具体的には、MAXに代えてODSカラムであるSep-Pak(3cc、60mg、30μm、Waters社製)を用いたこと、当該カラムのコンディショニングをメタノール、次いで水で行ったこと、及び、カラムにアプライする混和物を調製するための溶液(希釈液)として0.56体積%アンモニア水若しくは0.1N塩酸、固相の洗浄液として水若しくはアセトニトリル/水(体積比=1:3、1Mテトラブチルアンモニウムブロミド含有)、及び固相からの溶出液として2体積%ギ酸メタノール、2体積%ギ酸アセトニトリル/水(体積比=1:1)若しくは2体積%ギ酸メタノール/水(体積比=1:1)をそれぞれ用いた以外は、実施例1と同様にして行った。
実施例2において血漿サンプルに混和した溶液が0.56体積%アンモニア水であった場合の定量結果をそれぞれ回収率100%とし、各血漿サンプルのGL、GA、及びISの回収率(相対値)を算出した。算出結果を表9に示す。なお、表9中、「1-1」は0.56体積%アンモニア水、「1-2」は0.1N塩酸、「2-1」は水、「2-2」はアセトニトリル/水(体積比=1:3、1Mテトラブチルアンモニウムブロミド含有)、「3-1」は2体積%ギ酸メタノール、「3-2」は2体積%ギ酸アセトニトリル/水(体積比=1:1)、「3-3」は2体積%ギ酸メタノール/水(体積比=1:1)である。
MAXに代えて、逆相分配機能と極性相互作用機能とを備える固相を用いて、血漿中のGL及びGAの定量を行った。
ヒト血漿0.5mLに、実施例1の標準溶液(E)50μL及びIS50μLを添加し、十分に混和したものを血漿サンプルとした。
具体的には、MAXに代えて多孔性ポリマーであるOasis HLB(3cc、60mg、30μm、Waters社製)を用いたこと、当該カラムのコンディショニングをメタノール、次いで水で行ったこと、及び、カラムにアプライする混和物を調製するための溶液(希釈液)として0.56体積%アンモニア水若しくは0.1N塩酸を、固相の洗浄液として5体積%メタノール水溶液を、及び固相からの溶出液としてメタノールをそれぞれ用いた以外は、実施例1と同様にして行った。
実施例2において血漿サンプルに混和した溶液が0.56体積%アンモニア水であった場合の定量結果をそれぞれ回収率100%とし、各血漿サンプルのGL、GA、及びISの回収率(相対値)を算出した。算出結果を表10に示す。
MAXに代えて、逆相分配機能と陽イオン交換機能とを備える固相を用いて、血漿中のGL及びGAの定量を行った。
ヒト血漿0.5mLに、実施例1の標準溶液(E)50μL及びIS50μLを添加し、十分に混和したものを血漿サンプルとした。
具体的には、MAXに代えてOasis MCX(3cc、60mg、30μm、Waters社製)を用いたこと、当該カラムのコンディショニングをメタノール、次いで水で行ったこと、及び、カラムにアプライする混和物を調製するための溶液(希釈液)として0.1N塩酸を、固相の洗浄液として0.1N塩酸を、及び固相からの溶出液として2体積%アンモニア水/メタノール溶液をそれぞれ用いた以外は、実施例1と同様にして行った。
この結果、GLとGAのいずれも回収率が50%に届かず、MAXを用いた場合よりも遥かにGL等の検出感度が劣ることが分かった。
本発明の生薬由来成分の高感度定量方法を、質量分析をLC-MS/MS法によりを行った場合の、血漿中のGL及びGAの定量限界値を測定し、かつ検量線を作成した。また、同時に、当該方法の精度や信頼性を検証した。
<標準溶液の調製>
実施例1で用いた標準原液を実施例1と同様にしてメタノールにより希釈し、表12に記載の標準溶液(B)~(G)を調製した。
実施例1と同様にして、標準溶液(B)~(G)を添加したヒト血漿サンプルとブランク血漿サンプルを調製し、これらの血漿サンプルをそれぞれMAXにアプライして、GL等を吸着させ、0.56体積%アンモニア水/メタノール溶液及びメタノールで洗浄した後に、2体積%ギ酸/メタノール溶液で溶出させ、さらに蒸発乾固した。
蒸発乾固させた抽出物を、下記移動相A液/B液(体積比1:1の混合液)250μLに溶解させた。このうち1μLを分析カラムに注入し、下記に示す条件でLC-MS/MSを行った。
LC-MS/MSシステム: API4000システム(アプライドバイオシステムズ社製)
(LC)
装置: LC-20A(島津製作所製)
分析カラム: Inertsil ODS-3(2.1mm×150mm、粒径5μm、ジーエルサイエンス社製)
流速: 0.50mL/min.
カラム温度: 40℃
オートサンプラー温度: 5℃
試料注入量: 1μL
移動相:A液 0.1体積%ギ酸、B液 0.1体積%ギ酸アセトニトリル、リニアグラジエント(表13)
装置: API4000(アプライドバイオシステムズ)
イオン化法: ESI (Turbo Spray)
検出法: 正イオン検出 MRM(Multiple reaction monitoring)
イオンスプレー電圧(IonSpray Voltage): 5500V(正)
イオン源温度(Temperature): 400℃
カーテンガス (Curtain Gas): 10 (窒素)
イオンソースガス1(Ion Source Gas 1): 70 (窒素)
イオンソースガス2(Ion Source Gas 2): 50(窒素)
コリジョンガス(Collision Gas): 8(窒素)
さらに、米国食品医薬品局の分析法バリデーションのガイダンス(”Guidance for Industry, Bioanalytical Method Validation”, U.S. Department of Health and Human Services, Food and Drug Administration, May 2001)に従い、特異性、直線性、日内、日間再現性を検討した。また、安定性評価試験(室温下4時間及び-20℃及び-80℃下3カ月凍結保存における生体試料中保存安定性、凍結融解安定性、測定実試料中安定性、標準溶液安定性)についても実施した。検討結果を表16に示す。この結果、LC-MS/MSを用いた本発明の生薬由来成分の高感度定量方法は、それぞれ0.5~200ng/mL及び2~800ng/mLの範囲で良好な直線性と再現性を示した。日内再現性の結果、GLの真度は-12.8~+4.8%、精度は4.0~9.5%であり、GAの真度は-13.4~+4.4%、精度は2.2~9.0%であった。一方、日間再現性においても、GLの真度は-9.4~+2.0%、精度は2.1~5.5%、GAの真度は-10.9~+8.1%、精度は1.3~9.1%であった。再現性の結果より、GL及びGAの定量限界濃度はそれぞれ、0.5ng/mL、2ng/mLである事を確認した。
また、全ての安定性評価試験(生体試料中保存安定性、凍結融解安定性、測定実試料中安定性、標準溶液安定性)において、初期値と比較し±15%以内であり安定であった。以上の結果から、米国食品医薬品局の分析法バリデーションのガイダンスに則り、本発明の生薬由来成分の高感度定量方法が信頼性の確保された定量法であることが確認された。
GL含有製剤を経口投与後の血漿中のGLの検出及び薬物動態を測定した。
なお、本実施例は、ヘルシンキ宣言に基づく倫理的原則、薬事法第14条第3項、第80条の2及び「医薬品の臨床試験の実施の基準(GCP)に関する省令」(平成9年厚生省令第28号)に従い実施されたものである。具体的には、GL含有製剤の経口投与及び血漿サンプルの採取は、出願人の依頼により、イーピーエス株式会社及び医療法人社団育生會山口病院によって、第三者委員会における承認の後、試験責任医師の管理の下、実施された。また、得られた血漿サンプルにおけるGL及びGAの測定は、同じく出願人の依頼により、株式会社日本医学臨床検査研究所エコテクノ事業部(現医薬香粧品分析事業部)によって行われた。
また、被験者は、試験の目的、試験内容、プライバシーの保護等について十分説明された後、書面にて試験への参加を同意した健康な日本人男性6名(年齢:20歳以上35歳以下、事前検査時BMI:18.5以上25.0以下)を対象とした。
GL含有製剤として、グリチルリチン酸一アンモニウム、グリシン、DL-メチオニンを配合した糖衣錠「グリチロン(登録商標)配合錠」を用いた。グリチロン配合錠は、十分な水分と共に臨床常用量に従い、3錠の単回経口投与とした。投与及び採血時間などの試験スケジュールを表17に示す。得られた血液は、30分以内に遠心分離処理(4℃、3000rpm、10分間)により血漿として1mL以上を回収し、濃度測定時まで-20℃以下で凍結保存した。
次いで、実施例6と同様にして、蒸発乾固させた抽出物に対してLC-MS/MSを行い、各血漿サンプル中のGL濃度及びGA濃度を測定した。図3は、GLの平均血漿中濃度推移を示した図であり、図4は、GAの平均血漿中濃度推移を示した図である。なお、図3及び図4は、6検体の平均値及び標準偏差を示している。この結果、本発明の生薬由来成分の高感度定量方法により、従来から代替的な指標とされてきた主代謝物であるGAのみならず、未変化体であるGL自体の血漿中濃度を直接測定し得ることが確認された。すなわち、本発明により、血漿中GLの微量検出に初めて成功し、長らく不明であったGLの血中動態が明らかにされた。
GL及びGAの測定データから、最高血漿中濃度(Cmax)、最高血漿中濃度到達時間(Tmax)を算出した。また、投与後48時間までの血漿中濃度-時間曲線下面積(AUC0→48h)は台形法により、消失速度定数(kel)及び消失半減期(t1/2)は消失相から最小二乗法により算出した。また、投与後無限大時間までの血漿中濃度-時間曲線下面積(AUC0―∞)、体内滞留時間(MRT)についても算出した。
この結果、本実施例では、GLのCmaxは約10~40ng/mLの範囲にあり、平均値として24.8ng/mLであった。これは、前述の市販試薬の大量経口投与例に基づいた予測値(約23ng/mL)とほぼ同等であったことから、GLの投与量と血漿中濃度の相関性が示された。また、Tmaxは平均4.5時間であったが、それ以前(1~2時間)あるいは以後(12時間)など複数の濃度ピークを示す被験者も観察された。後方の濃度ピークは食後に出現したことから、食事摂取がGLの吸収あるいは腸肝循環に影響を与える可能性も示唆された。
Claims (4)
- 生体試料をアルカリ又はアルコールに混和した混和物を、逆相分配機能及び陰イオン交換機能を備える固相に注入した後、前記固相を洗浄液で少なくとも1回洗浄し、その後、酸性アルコールにより前記固相から溶出することにより、生薬由来成分を含む抽出物を調製する抽出工程と、
前記抽出工程により抽出された抽出物中のグリチルリチン、グリチルレチン酸、グリチルリチン及びグリチルレチン酸の代謝物、グリチルリチン及びグリチルレチン酸の類縁物質、甘草に含有されているサポニン成分、並びにこれらの薬学的に許容される塩からなる群より選択される少なくも1種を、質量分析法により検出し定量する定量工程と、
を有し、
前記洗浄液が、水、アルカリ、アルコール、及びアセトニトリルからなる群より選択される1種又は2種以上の混合液であることを特徴とする生薬由来成分の高感度定量方法。 - 前記抽出工程において、固相の洗浄を、アルカリとアルコールと水の混合液により洗浄した後、アルコール、アルコールと水の混合液、又はアセトニトリルにより洗浄することによって行うことを特徴とする請求項1に記載の生薬由来成分の高感度定量方法。
- 前記アルカリとアルコールと水の混合液が、0.5~28体積%アンモニア水とメタノールを99:1~1:3で混合してなるものであることを特徴とする請求項2に記載の生薬由来成分の高感度定量方法。
- 前記生体試料が、血液、血漿、又は組織抽出物であることを特徴とする請求項1~3のいずれか一項に記載の生薬由来成分の高感度定量方法。
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