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 PDF

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WO2012067090A1
WO2012067090A1 PCT/JP2011/076248 JP2011076248W WO2012067090A1 WO 2012067090 A1 WO2012067090 A1 WO 2012067090A1 JP 2011076248 W JP2011076248 W JP 2011076248W WO 2012067090 A1 WO2012067090 A1 WO 2012067090A1
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alcohol
herbal medicine
water
solid phase
plasma
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French (fr)
Japanese (ja)
Inventor
加代子 鈴木
恒陽 鈴木
路子 塚原
裕子 春田
明 八田
雄二 浜田
井上　秀雄
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Minophagen Pharmaceutical Co Ltd
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Minophagen Pharmaceutical Co Ltd
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Priority to KR1020137012465A priority Critical patent/KR101476144B1/ko
Priority to RU2013125221/15A priority patent/RU2558042C2/ru
Priority to CN201180054812.9A priority patent/CN103210308B/zh
Publication of WO2012067090A1 publication Critical patent/WO2012067090A1/ja
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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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2560/00—Chemical aspects of mass spectrometric analysis of biological material

Definitions

  • 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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JP2019074494A (ja) * 2017-10-19 2019-05-16 株式会社あすか製薬メディカル ビタミンｄ代謝物の選択的測定法
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CN113984916A (zh) * 2021-09-28 2022-01-28 天津中医药大学第一附属医院 一种正髓丸及正髓丸药材含量测定方法
CN113984916B (zh) * 2021-09-28 2024-04-19 天津中医药大学第一附属医院 一种正髓丸及正髓丸药材含量测定方法

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