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WO2018082647A1 - 一种中药组合物制剂中薄荷脑的含量测定方法 - Google Patents

一种中药组合物制剂中薄荷脑的含量测定方法 Download PDF

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WO2018082647A1
WO2018082647A1 PCT/CN2017/109262 CN2017109262W WO2018082647A1 WO 2018082647 A1 WO2018082647 A1 WO 2018082647A1 CN 2017109262 W CN2017109262 W CN 2017109262W WO 2018082647 A1 WO2018082647 A1 WO 2018082647A1
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Prior art keywords
menthol
preparation
solution
test solution
temperature
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PCT/CN2017/109262
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English (en)
French (fr)
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张水英
毕丹
陈育鹏
赵倩
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石家庄以岭药业股份有限公司
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Priority to RS20230668A priority Critical patent/RS64450B1/sr
Priority to CA3042612A priority patent/CA3042612C/en
Priority to EP17868270.4A priority patent/EP3537147B1/en
Priority to KR1020197015749A priority patent/KR102227057B1/ko
Priority to US16/347,465 priority patent/US11333637B2/en
Priority to RU2019112906A priority patent/RU2712775C1/ru
Publication of WO2018082647A1 publication Critical patent/WO2018082647A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
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    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
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    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
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    • G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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    • G01N30/02—Column chromatography
    • G01N30/60—Construction of the column
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    • G01N30/6073—Construction of the column body in open tubular form
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    • G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
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    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N2030/062—Preparation extracting sample from raw material
    • G—PHYSICS
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    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/30—Control of physical parameters of the fluid carrier of temperature
    • G01N2030/3076—Control of physical parameters of the fluid carrier of temperature using specially adapted T(t) profile
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/50—Conditioning of the sorbent material or stationary liquid
    • G01N30/52—Physical parameters
    • G01N30/54—Temperature

Definitions

  • the invention relates to a method for determining the content of menthol in a preparation of a traditional Chinese medicine composition.
  • Gas chromatography is to vaporize the analytical sample in the inlet, and then carry the carrier gas into the column. By separating the column with different retention properties of the components to be tested, the components are separated and sequentially introduced into the detector. To obtain a detection signal of each component. According to the order of introduction of the detectors, after comparison, it is possible to distinguish what components are, and the content of each component can be calculated according to the peak height or the peak area.
  • the menthol is extracted from the leaves and stems of mint, white crystals, which are the main constituents of peppermint and peppermint essential oils.
  • India is the main producer of natural mint.
  • Both menthol and racemic menthol can be used as toothpastes; perfumes; flavoring agents for beverages and candies. It is used as a stimulant in medicine, and it acts on the skin or mucous membranes. It has a cooling and antipruritic effect.
  • Oral administration can be used as a wind-driven medicine for headache and nose; throat; laryngitis. Its esters are used in perfumes and pharmaceuticals.
  • gas chromatography is widely used in the quality control of traditional Chinese medicine, especially in the detection of volatile components.
  • the quality control and evaluation of compound Chinese medicine is one of the key issues in the modernization of traditional Chinese medicine. It is also a difficult point and hot spot in the research of traditional Chinese medicine, especially the control of volatile chemical components.
  • the purpose of quality control of traditional Chinese medicine is to ensure the effectiveness and safety of traditional Chinese medicine.
  • the quality control of traditional Chinese medicine is to monitor the pharmacodynamic substances, toxic substances and their changing laws of traditional Chinese medicine. It is based on a series of quality standards to conduct quality testing and control on each link in the production process of traditional Chinese medicine, while the techniques, methods and strategies for quality evaluation of traditional Chinese medicines Research is the basis for the development of scientific, rational and advanced quality standards.
  • the invention claims a method for determining the content of menthol in a traditional Chinese medicine composition, which comprises the following medicines: forsythia, honeysuckle, radix isatidis, bitter almond, menthol, houttuynia, rhubarb, patchouli, cotton Ma Guanzhong, Rhodiola, Ephedra, Licorice, Gypsum, the content determination method, using gas chromatography to determine the content of menthol in the square, can effectively control the content of menthol in the square, this method can save energy and reduce analysis Cost, and the technical content is not disclosed in the prior art.
  • the invention provides a method for determining the content of menthol in a preparation of a traditional Chinese medicine composition, which is prepared from the following raw materials by weight: forsythia 200-300, ephedra 60-100, rhubarb 40-60, surimi Grass 200-300, Honeysuckle 200-300, Ban GmbH 200-300, Patchouli 60-100, Mianma Guanzhong 200-300, Rhodiola 60-100, Menthol 5-9, Bitter Almond 60-100, Licorice 60-100, gypsum 200-300, wherein the content of the menthol is determined as follows:
  • the menthol reference substance is prepared as a menthol having a menthol concentration of 4.80 ⁇ g/mL or more, preferably 17.65 ⁇ g/mL or more, more preferably 0.2-0.3 mg/mL.
  • the column is a weakly polar capillary column; the column temperature is programmed to temperature: initial temperature 80-100 ° C, hold for 10-15 min, rise to 120-160 ° C at a rate of 6-10 ° C per minute, keep 1.5-3.5 min, Then increase to 240-300 ° C at a rate of 100-160 ° C for 5-20 min.
  • the mass-to-volume ratio of the formulation of the traditional Chinese medicine composition to the non-polar solvent used when preparing the test solution is (1 g: 400 mL) to (1 g: 50 mL).
  • the weakly polar capillary chromatography column has a phenyl-methyl polysiloxane as a stationary phase, and preferably the phenyl-methyl polysiloxane has a phenyl content of 1 to 10%. More preferably, it is 5%, that is, a capillary column having a 5% phenyl-95% methylpolysiloxane as a stationary phase. Particularly preferably, the invention uses an HP-5 or DB-5 capillary column.
  • the injection amount of the reference solution and the test solution is 0.5-2 ⁇ L, respectively, and may be 0.5 ⁇ L, 1 ⁇ L, 1.5 ⁇ L or 2 ⁇ L, or the like.
  • the reference solution and the test solution are subjected to split injection, and the split ratio is in the range of 50:1 to 10:1, preferably 30:1 to 20:1, for example. 25:1.
  • the gas ratio also includes a gas ratio. It is generally known to those skilled in the art that the amount and ratio of gas used in gas chromatography is related to the detector used in the chromatograph. In some embodiments of the invention, the gas ratio is from 8:1 to 12:1, preferably from 9:1 to 11:1, most preferably 10:1.
  • the chromatographic conditions also include a detector temperature and an inlet temperature of 300-400 ° C, respectively. In some embodiments of the invention, the chromatographic conditions further include a carrier gas and a flow rate thereof, the carrier gas being nitrogen at a flow rate of 0.8-1.2 mL/min.
  • the non-polar solvent used to dissolve the control and the test article is selected from the group consisting of a non-polar saturated alkane or a halogenated saturated alkane, and a non-polar ester solvent, particularly n-hexane, two. Methyl chloride, petroleum ether or ethyl acetate, preferably n-hexane.
  • the preparation method of the test solution is specifically: grinding and mixing a certain amount of the traditional Chinese medicine composition preparation, placing it in a narrow-mouthed container, adding a non-polar organic solvent, shaking 20 More than two seconds, filtered, the test solution was obtained.
  • the filtration step is carried out using a 0.2-0.4 [mu]m microporous filter, preferably using a 0.22 [mu]m microporous filter.
  • the method of preparing the test solution further includes the step of soaking or ultrasonically extracting at room temperature before the shaking step.
  • the reference solution of the present invention can be prepared using methods conventionally used in the art.
  • the menthol reference substance is appropriately and accurately weighed, and the menthol concentration is 4.80 ⁇ g/mL or more, preferably 17.65 ⁇ g/mL or more, more preferably 0.2-0.3 mg/mL, especially when a non-polar organic solvent is added. It is a 0.23 mg/mL menthol reference solution.
  • the preparation of the traditional Chinese medicine composition to be tested can be directly used as the content of the marketed broccoli preparation (for example, a tablet or a capsule), or can be prepared by the following method:
  • the pharmaceutically acceptable excipient is starch.
  • a method of determining the content of menthol in a traditional Chinese medicine composition is provided.
  • the traditional Chinese medicine composition is prepared from the following raw materials by weight: Forsythia 200-300, Ephedra 60-100, Rhubarb 40-60, Houttuynia 200-300, Honeysuckle 200-300, Radix Isatidis 200-300, Patchouli 60-100, Mianma Guanzhong 200-300, Rhodiola 60-100, menthol 5-9, bitter almond 60-100, licorice 60-100, gypsum 200-300, the content of the menthol is determined as follows :
  • test solution The contents of the product under the difference of the amount of the preparation, finely ground, mixed, take 0.2-0.5g, accurately weighed, placed in a conical flask, precision added 20-30mL of n-hexane, Shake it counterclockwise for about 20-50 seconds, take an appropriate amount of solution, filter through a 0.22 ⁇ m microporous membrane to obtain a test solution;
  • Preparation of reference solution Take appropriate amount of menthol reference substance, accurately weighed, add n-hexane to make 0.23mg menthol solution per 1mL, that is;
  • Chromatographic conditions column: Agilent J&W Scientific HP-5 capillary column (30m ⁇ 0.25mm, 0.25 ⁇ m); column temperature is programmed temperature: initial temperature 98 ° C, maintained for 12 min, at a rate of 8 ° C per minute to 140 ° C, Hold for 2.5 min, then increase to 280 ° C at 140 ° C for 5-20 min; detector temperature is 300-400 ° C; inlet temperature is 300-400 ° C; carrier gas is nitrogen, flow rate: 0.8-1.2 mL /min; split injection, split ratio: 25:1; injection volume: 0.5-2 ⁇ L; gas ratio: air-hydrogen (450:45);
  • Determination method respectively, accurately draw the reference solution and the test solution 0.5-2 ⁇ L each, inject into the gas chromatograph, determine, that is.
  • the formulation of the traditional Chinese medicine composition of the invention is made from the following bulk parts:
  • Forsythia 200 Honeysuckle 300, Ban GmbH 200, Rhubarb 40, Patchouli 60, Mianma Guanzhong 300, Rhodiola 100, Menthol 9, Ephedra 60, Bitter Almond 100, Houttuynia 200, Licorice 100, Gypsum 200 .
  • preferred parts of the formulation of the traditional Chinese medicine composition of the invention are as follows:
  • Forsythia 300 Honeysuckle 200, Ban GmbH 300, Rhubarb 60, Patchouli 100, Mianma Guanzhong 200, Rhodiola 60, Menthol 5, Ephedra 100, Bitter Almond 60, Houttuynia 300, Licorice 60, Gypsum 300 .
  • preferred parts of the formulation of the traditional Chinese medicine composition of the invention are as follows:
  • preferred parts of the formulation of the traditional Chinese medicine composition of the invention are as follows:
  • the preparation method of the traditional Chinese medicine composition preparation of the present invention is:
  • the method for determining the content of menthol in the preparation of the traditional Chinese medicine composition of the present invention is:
  • test solution The contents of the product under the difference of the amount of the preparation, finely researched, mixed, taken 0.3g, accurately weighed, placed in a conical flask, precision added with 25mL of n-hexane, counterclockwise delivery Shake for about 30 seconds, take an appropriate amount of solution, filter through a 0.22 ⁇ m microporous membrane to obtain a test solution;
  • Preparation of reference solution Take appropriate amount of menthol reference substance, accurately weighed, add n-hexane to make 0.23mg menthol solution per 1mL, that is;
  • Chromatographic conditions column: Agilent J&W Scientific HP-5 capillary column (30m ⁇ 0.25mm, 0.25 ⁇ m); column temperature is programmed temperature: initial temperature 98 ° C, maintained for 12 min, at a rate of 8 ° C per minute to 140 ° C, Hold for 2.5 min, then increase to 280 ° C at 140 ° C for 10 min; detector temperature is 300 ° C; inlet temperature is 300 ° C; carrier gas is nitrogen, flow rate: 1 mL / min; split injection, split ratio :25:1; injection volume: 1 ⁇ L; gas ratio: air-hydrogen (450:45);
  • Determination method respectively, accurately draw 1 ⁇ L of the reference solution and the test solution, inject into a gas chromatograph, and measure, that is, obtain.
  • test solution the contents of the product under the difference of the amount of the preparation, finely researched, mixed, taken 0.2g, accurately weighed, In a stoppered conical flask, precisely add 20 mL of n-hexane, shake it counterclockwise for about 20 seconds, take an appropriate amount of solution, and filter through a 0.22 ⁇ m microporous membrane to obtain a test solution;
  • Preparation of reference solution Take appropriate amount of menthol reference substance, accurately weighed, add n-hexane to make 0.23mg menthol solution per 1mL, that is;
  • Chromatographic conditions column: Agilent J&W Scientific HP-5 capillary column (30m ⁇ 0.25mm, 0.25 ⁇ m); column temperature is programmed temperature: initial temperature 98 ° C, maintained for 12 min, at a rate of 8 ° C per minute to 140 ° C, Hold for 2.5 min, then increase to 280 ° C at 140 ° C for 5-20 min; detector temperature is 350 ° C; inlet temperature is 350 ° C; carrier gas is nitrogen, flow rate: 0.8 mL / min; split injection , split ratio: 25:1; injection volume: 0.5 ⁇ L; gas ratio: air-hydrogen (450:45);
  • Determination method respectively, accurately draw the reference solution and 0.5 ⁇ L of the test solution, inject into the gas chromatograph, and measure.
  • test solution The contents of the product under the difference of the amount of the preparation, finely ground, mixed, take 0.5g, accurately weighed, placed in a conical flask, precision added 30mL of n-hexane, handed in counterclockwise Shake for about 50 seconds, take an appropriate amount of solution, filter through a 0.22 ⁇ m microporous membrane to obtain a test solution;
  • Preparation of reference solution Take appropriate amount of menthol reference substance, accurately weighed, add n-hexane to make 0.23mg menthol solution per 1mL, that is;
  • Chromatographic conditions column: Agilent J&W Scientific HP-5 capillary column (30m ⁇ 0.25mm, 0.25 ⁇ m); column temperature is programmed temperature: initial temperature 98 ° C, maintained for 12 min, at a rate of 8 ° C per minute to 140 ° C, Hold for 2.5 min, then increase to 280 ° C at 140 ° C for 5-20 min; detector temperature is 400 ° C; inlet temperature is 400 ° C; carrier gas is nitrogen, flow rate: 1.2 mL / min; split injection , split ratio: 25:1; injection volume: 2 ⁇ L; gas ratio: air-hydrogen (450:45);
  • Assay method accurately draw 2 ⁇ L of each of the reference solution and the test solution, and inject into a gas chromatograph to measure.
  • the method for determining the content of the traditional Chinese medicine composition of the present invention is evaluated from various aspects, and the evaluation method is as follows:
  • Reagents n-hexane (chromatographically pure, Fisher, USA); petroleum ether, dichloromethane, ethyl acetate (analytical grade, Beijing Chemical Plant).
  • Drug menthol reference substance (purchased SIGMA-ALORICH, lot number: M2772-100G-A, purity 99%).
  • the content of the traditional Chinese medicine composition preparation under the difference of the loading amount is finely ground and mixed, and 0.3g is taken, accurately weighed, placed in a 100mL stoppered conical flask, and two different extraction solvents are parallel in parallel, respectively, and the extracting solvent is precisely extracted.
  • Petroleum ether 60 ⁇ 90 ° C
  • dichloromethane n-hexane
  • shake gently in reverse counterclockwise for about 30 seconds take an appropriate amount of solution, 0.22 ⁇ m microporous filter filtered, that is to test Product solution.
  • the content of the preparation of the traditional Chinese medicine composition under the difference of the loading amount is finely ground and mixed, and 0.3g is taken, accurately weighed, placed in a 100mL stoppered conical flask, cold soaked (ie, immersed at room temperature) and ultrasonically extracted. 2 copies in parallel.
  • Cold soaking extraction precision soaking 50ml of n-hexane, after cold soaking for 20min, shake it counterclockwise for about 30 seconds
  • ultrasonic extraction precision soaking 50ml of n-hexane, weighing before sonication, sonicating for 20min, adding n-hexane Make up the weight.
  • a suitable amount of the solution under each extraction method was filtered through a 0.22 ⁇ m microporous membrane to obtain a test solution.
  • the content of the traditional Chinese medicine composition preparation under the difference of the loading amount is finely ground and mixed, and 0.3g is taken, accurately weighed, and placed in a 100mL stoppered conical flask.
  • the cold soaking extraction time is 0min, 20min, 40min, respectively.
  • the extraction time was 2 times in parallel, and the shaking was repeated in the counterclockwise direction for about 30 seconds.
  • An appropriate amount of the solution was taken and filtered through a 0.22 ⁇ m microporous membrane to obtain a test solution.
  • the content of the preparation of the traditional Chinese medicine composition under the difference of the loading amount is finely ground and mixed, and 0.3g is taken, accurately weighed, and placed in a conical flask.
  • the amount of different solvents is 2 in parallel, and the amount of precision solvent is 25 mL respectively. 50mL, 75mL, shaking in the counterclockwise direction for about 30 seconds, taking an appropriate amount of solution, 0.22 ⁇ m microporous membrane filtration, that is, the test solution is obtained.
  • the preparation method of the test solution is finally determined as follows: the content of the preparation of the traditional Chinese medicine composition under the difference of the amount of the preparation is finely ground, mixed, and taken, 0.3 g, accurately weighed, and placed with a cone-shaped flask In the middle, precise addition of 25 mL of n-hexane, shaking in a counterclockwise direction for about 30 seconds, taking an appropriate amount of the solution, and filtering through a 0.22 ⁇ m microporous membrane to obtain a test solution.
  • Aspirate solvent ie blank reagent
  • negative control solution refers to a solution prepared without the menthol preparation according to the preparation method of the test solution.
  • the results showed that the negative control had no interference with the measured components and the specificity was good.
  • solvent, negative control, reference, chromatogram of the test sample are shown in Figures 2 to 5).
  • menthol reference substance put it in a 50mL volumetric flask, dissolve it with n-hexane and dilute to the mark, shake it up, and use it as a stock solution, then accurately measure 1mL, 1.5mL, 2mL, 2.5mL, 3mL respectively.
  • 3.5mL was placed in a 25mL volumetric flask, diluted with n-hexane to the mark, and the concentrations were 0.0804mg/mL, 0.1206mg/mL, 0.1608mg/mL, 0.2010mg/mL, 0.2412mg/mL, 0.2814mg.
  • the reference solution of menthol 0.0804 mg/ml was further diluted to 28.944 ⁇ g/mL, 24.120 ⁇ g/mL, 19.296 ⁇ g/mL, 17.688 ⁇ g/mL, 16.080 ⁇ g/mL, 12.060 ⁇ g/mL, 8.040 ⁇ g/mL, 6.432.
  • the detection limits and quantitation limits of menthol were 4.824 ⁇ g/mL and 17.688 ⁇ g/mL, respectively.
  • the test solution was taken, and other chromatographic conditions were fixed, and the FID detector temperature was measured at 295 ° C, 300 ° C and 305 ° C, respectively, and the menthol reference substance was used as a control to calculate the content of menthol in the traditional Chinese medicine composition of the present invention. Compare the influence of the temperature change of the FID detector on the measurement results. The results are shown in Table 11.
  • test solution was taken, other chromatographic conditions were fixed, and different capillary columns such as Agilent DB-5 and Agilent HP-5 were used for measurement, and menthol reference substance was used as a control to calculate the content of menthol in the traditional Chinese medicine composition of the present invention. Compare the effects of different columns on the measurement results. The results are shown in Table 13.
  • the invention establishes a method for determining the content of menthol in the traditional Chinese medicine composition of the invention, and the above experimental results show that the method has good precision and stability, and repeatability, and provides a kind of quality control for improving the traditional Chinese medicine composition. new method.
  • Figure 2 Blank reagent chromatogram
  • Reagents n-hexane (chromatographically pure, Fisher, USA); petroleum ether, dichloromethane, ethyl acetate (analytical grade, Beijing Chemical Plant).
  • Drug menthol reference substance (purchased SIGMA-ALORICH, lot number: M2772-100G-A, purity 99%).
  • the auxiliary material may be 35 g of starch
  • test solution The contents of the product under the difference of the amount of the preparation, finely researched, mixed, taken 0.3g, accurately weighed, placed in a conical flask, precision added with 25mL of n-hexane, counterclockwise delivery Shake for about 30 seconds, take an appropriate amount of solution, filter through a 0.22 ⁇ m microporous membrane to obtain a test solution;
  • Preparation of reference solution Take appropriate amount of menthol reference substance, accurately weighed, add n-hexane to make 0.23mg menthol solution per 1mL, that is;
  • Chromatographic conditions column: Agilent J&W Scientific HP-5 capillary column (30m ⁇ 0.25mm, 0.25 ⁇ m); column temperature is programmed temperature: initial temperature 98 ° C, maintained for 12 min, at a rate of 8 ° C per minute to 140 ° C, Hold for 2.5 min, then increase to 280 ° C at 140 ° C for 10 min; detector temperature is 300 ° C; inlet temperature is 300 ° C; carrier gas is nitrogen, flow rate: 1 mL / min; split injection, split ratio :25:1; injection volume: 1 ⁇ L; gas ratio: air-hydrogen (450:45);
  • Determination method respectively, accurately draw 1 ⁇ L of the reference solution and the test solution, inject into a gas chromatograph, and measure, that is, obtain.
  • the auxiliary material may be 35 g of starch
  • test solution The contents of the product under the difference of the amount of the preparation, finely researched, mixed, taken 0.2g, accurately weighed, placed in a conical flask, precision added 20mL of n-hexane, counterclockwise delivery Shake for about 20 seconds, take an appropriate amount of solution, filter through a 0.22 ⁇ m microporous membrane to obtain a test solution;
  • Preparation of reference solution Take appropriate amount of menthol reference substance, accurately weighed, add n-hexane to make 0.23mg menthol solution per 1mL, that is;
  • Chromatographic conditions column: Agilent J&W Scientific HP-5 capillary column (30m ⁇ 0.25mm, 0.25 ⁇ m); column temperature is programmed temperature: initial temperature 98 ° C, maintained for 12 min, at a rate of 8 ° C per minute to 140 ° C, Hold for 2.5 min, then increase to 280 ° C at 140 ° C for 5-20 min; detector temperature is 350 ° C; inlet temperature is 350 ° C; carrier gas is nitrogen, flow rate: 0.8 mL / min; split injection , split ratio: 25:1; injection volume: 0.5 ⁇ L; gas ratio: air-hydrogen (450:45);
  • Determination method respectively, accurately draw the reference solution and 0.5 ⁇ L of the test solution, inject into the gas chromatograph, and measure.
  • the raw material formula is: Forsythia 278g, Honeysuckle 294g, Radix Isatidis 285g, Rhubarb 55g, Patchouli 95g, Mianma Guanzhong 290g, Rhodiola 87g, Menthol 8.5g, Ephedra 88g, Bitter Almond 80g, Houttuynia 284g , licorice 95g, gypsum 277g, according to the following process:
  • the auxiliary material may be 35 g of starch
  • test solution The contents of the product under the difference of the amount of the preparation, finely ground, mixed, take 0.5g, accurately weighed, placed in a conical flask, precision added 30mL of n-hexane, handed in counterclockwise Shake for about 50 seconds, take an appropriate amount of solution, filter through a 0.22 ⁇ m microporous membrane to obtain a test solution;
  • Preparation of reference solution Take appropriate amount of menthol reference substance, accurately weighed, add n-hexane to make 0.23mg menthol solution per 1mL, that is;
  • Chromatographic conditions column: Agilent J&W Scientific HP-5 capillary column (30m ⁇ 0.25mm, 0.25 ⁇ m); column temperature is programmed temperature: initial temperature 98 ° C, maintained for 12 min, at a rate of 8 ° C per minute to 140 ° C, Hold for 2.5 min, then increase to 280 ° C at 140 ° C for 5-20 min; detector temperature is 400 ° C; inlet temperature Degree is 400 ° C; carrier gas is nitrogen, flow rate: 1.2 mL / min; split injection, split ratio: 25:1; injection volume: 2 ⁇ L; gas ratio: air - hydrogen (450: 45);
  • Assay method accurately draw 2 ⁇ L of each of the reference solution and the test solution, and inject into a gas chromatograph to measure.
  • the raw material formula is: weighed according to the ratio: Forsythia 255g, Honeysuckle 255g, Radix Isatidis 255g, Rhubarb 51g, Patchouli 85g, Mianma Guanzhong 255g, Rhodiola 85g, Menthol 7.5g, Ephedra 85g, Bitter Almond 85g Houttuynia 255g, licorice 85g, gypsum 255g, extracted according to the following process:
  • the auxiliary material may be 35 g of starch
  • test solution The contents of the product under the difference of the amount of the preparation, finely ground, mixed, take 0.4g, accurately weighed, placed in a conical flask, precision added 20mL of hexane, handed counterclockwise Shake for about 40 seconds, take an appropriate amount of solution, filter through a 0.22 ⁇ m microporous membrane to obtain a test solution;
  • Preparation of reference solution Take appropriate amount of menthol reference substance, accurately weighed, add n-hexane to make 0.23 per 1mL Mg menthol solution, that is;
  • Chromatographic conditions column: Agilent J&W Scientific HP-5 capillary column (30m ⁇ 0.25mm, 0.25 ⁇ m); column temperature is programmed temperature: initial temperature 98 ° C, maintained for 12 min, at a rate of 8 ° C per minute to 140 ° C, Hold for 2.5 min, then increase to 280 ° C at 140 ° C for 5-20 min; detector temperature is 350 ° C; inlet temperature is 350 ° C; carrier gas is nitrogen, flow rate: 1.1 mL / min; split injection , split ratio: 25:1; injection volume: 1 ⁇ L; gas ratio: air-hydrogen (450:45);
  • Determination method respectively, accurately draw 1 ⁇ L of the reference solution and the test solution, inject into a gas chromatograph, and measure, that is, obtain.

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Abstract

一种中药组合物中薄荷脑的含量测定方法，该中药组合物由以下药材组成：连翘、金银花、板蓝根、苦杏仁、薄荷脑、鱼腥草、大黄、广藿香、绵马贯众、红景天、麻黄、甘草、石膏，该含量测定方法，采用气相色谱法测定方中薄荷脑的含量，可以有效的控制方中薄荷脑的含量，该方法可节约能源，降低分析成本。

Description

一种中药组合物制剂中薄荷脑的含量测定方法 技术领域
本发明涉及到一种中药组合物制剂中薄荷脑的含量测定方法。
背景技术
气相色谱法是将分析样品在进样口中气化后，由载气带入色谱柱，通过对欲检测混合物中组分有不同保留性能的色谱柱，使各组分分离，依次导入检测器，以得到各组分的检测信号。按照导入检测器的先后次序，经过对比，可以区别出是什么组分，根据峰高度或峰面积可以计算出各组分含量。
薄荷脑系由薄荷的叶和茎中所提取，白色晶体，为薄荷和欧薄荷精油中的主要成分。在世界上，印度是主要的天然薄荷生产国。薄荷脑和消旋薄荷脑均可用作牙膏；香水；饮料和糖果等的赋香剂。在医药上用作刺激药，作用于皮肤或粘膜，有清凉止痒作用；内服可作为驱风药，用于头痛及鼻；咽；喉炎症等。其酯用于香料和药物。目前气相色谱在中药的质量控制方面应用较多，特别是在挥发性成分检测方面的应用。复方中药的质量控制和评价是中药现代化的关键问题之一，也是中药研究的难点和热点，特别是易挥发性化学成分的控制，中药质量控制的目的是保证中药的有效性和安全性。中药质量控制就是监测中药的药效物质、有毒物质及其变化规律，是依据一系列质量标准对中药生产过程中的每一个环节进行质量检测与控制，而中药质量评价的技术、方法和策略的研究是制订科学、合理、先进的质量标准的基础。
本发明要求保护一种中药组合物中薄荷脑的含量测定方法，该中药组合物由以下药材组成：连翘、金银花、板蓝根、苦杏仁、薄荷脑、鱼腥草、大黄、广藿香、绵马贯众、红景天、麻黄、甘草、石膏，该含量测定方法，采用气相色谱法测定方中薄荷脑的含量，可以有效的控制方中薄荷脑的含量，该方法可节约能源，降低分析成本，而现有技术中并没有公开该技术内容。
发明内容
本发明提供一种中药组合物制剂中薄荷脑的含量测定方法，该中药组合物制剂由如下重量份的原料药制成：连翘200-300、麻黄60-100、大黄40-60、鱼腥草200-300、金银花200-300、板蓝根200-300、广藿香60-100、绵马贯众200-300、红景天60-100、薄荷脑5-9、苦杏仁60-100、甘草60-100、石膏200-300，其中所述薄荷脑的含量测定方法如下：
1)使用非极性溶剂对所述中药组合物制剂进行提取，得到供试品溶液；
2)使用与步骤1)中相同的非极性溶剂，将薄荷脑对照品制备成薄荷脑浓度在4.80μg/mL以上，优选17.65μg/mL以上，更优选0.2-0.3mg/mL的薄荷脑对照品溶液；
3)分别取等量对照品溶液与供试品溶液，注入气相色谱仪，测定薄荷脑含量，其中色谱条件为：
色谱柱为弱极性毛细管色谱柱；柱温为程序升温：初始温度80-100℃，保持10-15min，以每分钟6-10℃的速率升至120-160℃，保持1.5-3.5min，再以100-160℃的速率升至240-300℃，保持5-20min。
在本发明的一些实施方案中，制备供试品溶液时，使用的中药组合物制剂与非极性溶剂的质量对体积比为(1g:400mL)～(1g:50mL)。
在本发明的一些实施方案中，弱极性毛细管色谱柱以苯基-甲基聚硅氧烷为固定相，优选所述苯基-甲基聚硅氧烷中苯基含量为1～10％，更优选5％，即以5％苯基-95％甲基聚硅氧烷为固定相的毛细管柱。特别优选地，本发明使用HP-5或DB-5毛细管色谱柱。
在本发明的一些实施方案中，对照品溶液与供试品溶液的进样量分别为0.5-2μL，可以是0.5μL、1μL、1.5μL或2μL等。
在本发明的一些实施方案中，在色谱测试时，对照品溶液与供试品溶液采用分流进样，分流比处于50:1～10:1范围内，优选30:1～20:1，例如25:1。
在本发明的一些实施方案中，色谱条件中还包括燃气比例。本领域普通技术人员所普遍知晓的是，气相色谱中所述燃气用量与比例与色谱仪所使用的检测器相关。在本发明一些实施方案中，燃气比例为空气对氢气比为8:1～12:1，优选9:1～11:1，最优选10:1。
在本发明的一些实施方案中，色谱条件还包括分别为300-400℃的检测器温度和进样口温度。在本发明的一些实施方案中，色谱条件还包括载气及其流速，所述载气为氮气，流速为0.8-1.2mL/min。
在本发明的一些实施方案中，用于溶解对照品和供试品的非极性溶剂选自非极性饱和烷烃或卤代饱和烷烃，以及非极性酯类溶剂，特别是正己烷、二氯甲烷、石油醚或乙酸乙酯，优选正己烷。
在本发明的一些实施方案中，供试品溶液的制备方法具体为：将一定量的中药组合物制剂研磨混匀后置于具塞窄口容器中，加入非极性有机溶剂，振摇20秒以上，过滤，得供试品溶液。在一些优选的实施方案中，过滤步骤使用0.2-0.4μm微孔滤膜进行，优选使用0.22μm微孔滤膜过滤。在一些优选的实施方案中，供试品溶液的制备方法在振摇步骤前，还包括室温下浸泡或超声提取的步骤。
本发明的对照品溶液可使用本领域常规使用的方法进行制备。优选的方式为取薄荷脑对照品适量、精密称定，加非极性有机溶剂制成薄荷脑浓度在4.80μg/mL以上，优选17.65μg/mL以上，更优选0.2-0.3mg/mL，特别是0.23mg/mL的薄荷脑对照品溶液。
在本发明的实施方案中，待检测的中药组合物制剂可直接使用已上市的连花清瘟制剂(例如片剂或胶囊剂)的内容物，也可使用以下方法制备：
(1)按照原料药重量比例称取中药材，净选、碎断；
(2)广藿香，加8-12倍量水(水体积对药材质量比，v/w)提取挥发油，提油时间6-10小时，收集挥发油，备用；提取液过滤后，留滤液备用；
(3)连翘、麻黄、鱼腥草、大黄，用10-14倍量(水体积对药材质量比，v/w)的60-80％(v/v)的乙醇提取2-4次，每次1.5-3小时，提取液合并过滤，滤液备用；
(4)金银花、石膏、板蓝根、绵马贯众、甘草、红景天，加10-14倍量水(水体积对药材质量比，v/w)煎煮至沸，加入苦杏仁，煎煮2-4次，每次0.5-2小时，提取液合并过滤，所得滤液与步骤(2)广藿香提油后的滤液合并，浓缩成在50-70℃时测定相对密度为1.10-1.15的清膏，加入乙醇，调节至醇浓度为65-80％，冷藏放置，过滤，至滤渣无醇味，得清膏备用；
(5)将步骤(4)所得清膏与步骤(3)所得醇提液合并，浓缩至在50-70℃时测定相对密度为1.15-1.20的清膏，干燥，得干膏粉，备用；
(6)将步骤(5)所得干膏粉加入适当药学上可接受的辅料制粒；
(7)将薄荷脑、步骤(2)所得挥发油加入乙醇溶解，喷入步骤(6)所得颗粒，密闭，混匀，压片或者装胶囊、或者装袋。
在本发明的一些实施方案中，所述药学上可接受的辅料为淀粉。
在本发明的一些实施方案中，提供了一种中药组合物中薄荷脑的含量测定方法。该中药组合物由如下重量份的原料药制成：连翘200-300、麻黄60-100、大黄40-60、鱼腥草200-300、金银花200-300、板蓝根200-300、广藿香60-100、绵马贯众200-300、红景天60-100、薄荷脑5-9、苦杏仁60-100、甘草60-100、石膏200-300，所述薄荷脑的含量测定方法如下：
供试品溶液制备：取装量差异项下的本品内容物，研细，混匀，取0.2-0.5g，精密称定，置具塞锥形瓶中，精密加入正己烷20-30mL，顺逆时针递次振摇约20-50秒钟，取适量溶液，0.22μm微孔滤膜滤过，即得供试品溶液；
对照品溶液的制备：取薄荷脑对照品适量，精密称定，加正己烷制成每1mL含0.23mg薄荷脑溶液，即得；
色谱条件：色谱柱：Agilent J&W Scientific HP-5毛细管柱(30m×0.25mm,0.25μm)；柱温为程序升温：初始温度98℃，保持12min，以每分钟8℃的速率升至140℃，保持2.5min，再以140℃的速率升至280℃，保持5-20min；检测器温度为300-400℃；进样口温度为300-400℃；载气为氮气，流速：0.8-1.2mL/min；分流进样，分流比：25:1；进样量：0.5-2μL；燃气比例：空气-氢气(450:45)；
测定法：分别精密吸取对照品溶液与供试品溶液各0.5-2μL，注入气相色谱仪，测定，即得。
在本发明的一些实施方案中，本发明所述中药组合物制剂是由如下重量份的原料药制成：
连翘200、金银花300、板蓝根200、大黄40、广藿香60、绵马贯众300、红景天100、薄荷脑9、麻黄60、苦杏仁100、鱼腥草200、甘草100、石膏200。
在本发明的一些实施方案中，本发明所述中药组合物制剂优选的重量份如下：
连翘300、金银花200、板蓝根300、大黄60、广藿香100、绵马贯众200、红景天60、薄荷脑5、麻黄100、苦杏仁60、鱼腥草300、甘草60、石膏300。
在本发明的一些实施方案中，本发明所述中药组合物制剂优选的重量份如下：
连翘278、金银花294、板蓝根285、大黄55、广藿香95、绵马贯众290、红景天87、薄荷脑8.5、麻黄88、苦杏仁80、鱼腥草284、甘草95、石膏277。
在本发明的一些实施方案中，本发明所述中药组合物制剂优选的重量份如下：
连翘255、金银花255、板蓝根255、大黄51、广藿香85、绵马贯众255、红景天85、薄荷脑7.5、麻黄85、苦杏仁85、鱼腥草255、甘草85、石膏255。
在本发明的一些实施方案中，本发明所述中药组合物制剂的制备方法为：
(1)按照原料药重量比例称取中药材，净选，酌情碎断；
(2)广藿香碎断，加10倍量水提取挥发油，提油时间8小时，收集挥发油，备用；提取液过滤后，残渣弃去，滤液备用；
(3)连翘、麻黄、鱼腥草、大黄，用12倍量70％的乙醇提取3次，每次2.5小时，提取液合并过滤，回收乙醇，滤液备用；
(4)金银花、石膏、板蓝根、绵马贯众、甘草、红景天，加12倍量水煎煮至沸，加入苦杏仁，煎煮2次，每次1小时，提取液合并过滤，所得滤液与步骤(2)广藿香提油后的滤液合并，浓缩成在60℃时测定相对密度为1.10-1.15的清膏，加入乙醇，调节至醇浓度为70％，冷藏放置，过滤，回收乙醇至无醇味，得清膏备用；
(5)将步骤(4)所得清膏与步骤(3)所得醇提液合并，浓缩至在60℃时测定相对密度为1.15-1.20的清膏，干燥，得干膏粉，备用；
(6)将步骤(5)所得干膏粉加入适当药学上可接受的辅料制粒；
(7)将薄荷脑、步骤(2)所得挥发油加入乙醇溶解，喷入步骤(6)所得颗粒，密闭，混匀，压片或者装胶囊、或者装袋。
在本发明的一些实施方案中，本发明所述中药组合物制剂中薄荷脑的含量测定方法为：
供试品溶液制备：取装量差异项下的本品内容物，研细，混匀，取0.3g，精密称定，置具塞锥形瓶中，精密加入正己烷25mL，顺逆时针递次振摇约30秒钟，取适量溶液，0.22μm微孔滤膜滤过，即得供试品溶液；
对照品溶液的制备：取薄荷脑对照品适量，精密称定，加正己烷制成每1mL含0.23mg薄荷脑溶液，即得；
色谱条件：色谱柱：Agilent J&W Scientific HP-5毛细管柱(30m×0.25mm,0.25μm)；柱温为程序升温：初始温度98℃，保持12min，以每分钟8℃的速率升至140℃，保持2.5min，再以140℃的速率升至280℃，保持10min；检测器温度为300℃；进样口温度为300℃；载气为氮气，流速：1mL/min；分流进样，分流比：25:1；进样量：1μL；燃气比例：空气-氢气(450:45)；
测定法：分别精密吸取对照品溶液与供试品溶液各1μL，注入气相色谱仪，测定，即得。
或者本发明所述含量测定方法如下：
供试品溶液制备：取装量差异项下的本品内容物，研细，混匀，取0.2g，精密称定， 置具塞锥形瓶中，精密加入正己烷20mL，顺逆时针递次振摇约20秒钟，取适量溶液，0.22μm微孔滤膜滤过，即得供试品溶液；
对照品溶液的制备：取薄荷脑对照品适量，精密称定，加正己烷制成每1mL含0.23mg薄荷脑溶液，即得；
色谱条件：色谱柱：Agilent J&W Scientific HP-5毛细管柱(30m×0.25mm,0.25μm)；柱温为程序升温：初始温度98℃，保持12min，以每分钟8℃的速率升至140℃，保持2.5min，再以140℃的速率升至280℃，保持5-20min；检测器温度为350℃；进样口温度为350℃；载气为氮气，流速：0.8mL/min；分流进样，分流比：25:1；进样量：0.5μL；燃气比例：空气-氢气(450:45)；
测定法：分别精密吸取对照品溶液与供试品溶液各0.5μL，注入气相色谱仪，测定，即得。
或者本发明所述含量测定方法如下：
供试品溶液制备：取装量差异项下的本品内容物，研细，混匀，取0.5g，精密称定，置具塞锥形瓶中，精密加入正己烷30mL，顺逆时针递次振摇约50秒钟，取适量溶液，0.22μm微孔滤膜滤过，即得供试品溶液；
对照品溶液的制备：取薄荷脑对照品适量，精密称定，加正己烷制成每1mL含0.23mg薄荷脑溶液，即得；
色谱条件：色谱柱：Agilent J&W Scientific HP-5毛细管柱(30m×0.25mm,0.25μm)；柱温为程序升温：初始温度98℃，保持12min，以每分钟8℃的速率升至140℃，保持2.5min，再以140℃的速率升至280℃，保持5-20min；检测器温度为400℃；进样口温度为400℃；载气为氮气，流速：1.2mL/min；分流进样，分流比：25:1；进样量：2μL；燃气比例：空气-氢气(450:45)；
测定法：分别精密吸取对照品溶液与供试品溶液各2μL，注入气相色谱仪，测定，即得。
测试方法可行性评价
用本发明实施例1制备的中药组合物制剂，对本发明中药组合物的含量测定方法从多个方面进行可行性评价，评价方法如下：
1仪器与试药
仪器：PerKinElmer Clarus 680气相色谱仪、AL204型和AB135-S电子天平、Agilent J&W Scientific HP-5毛细管柱(30m×0.25mm,0.25μm)、数控超声波清洗器(型号：KQ-500DB，500W，40KHZ)、0.22μm微孔滤膜(天津市津腾实验设备有限公司)。
试剂：正己烷(色谱纯，美国Fisher)；石油醚、二氯甲烷、乙酸乙酯(分析纯，北京化工厂)。
药品：薄荷脑对照品(购SIGMA-ALORICH，批号：M2772-100G-A，纯度为99％)。
2色谱条件
色谱柱：Agilent J&W Scientific HP-5毛细管柱(30m×0.25mm,0.25μm)；柱温为程序升温：初始温度98℃，保持12min，以每分钟8℃的速率升至140℃，保持2.5min，再以140℃的速率升至280℃，保持10min；检测器温度为300℃；进样口温度为300℃；载气为氮气，流速：1mL/min；分流进样，分流比：25:1；进样量：1μL；燃气比例：空气-氢气(450:45)。
3供试品溶液和对照品溶液的制备
3.1供试品溶液制备方法考察
3.1.1提取溶媒的考察
取装量差异项下的中药组合物制剂内容物，研细，混匀，取0.3g，精密称定，置100mL具塞锥形瓶中，不同提取溶媒各平行2份，分别精密吸取提取溶媒石油醚(60～90℃)、二氯甲烷、正己烷、乙酸乙酯50mL，顺逆时针递次振摇约30秒钟，取适量溶液，0.22μm微孔滤膜滤过，即得供试品溶液。
分别吸取供试品溶液1μL，注入气相色谱仪，并以薄荷脑对照品做对照，比较供试品溶液中薄荷脑的百分含量，结果显示以正己烷为提取溶媒时，待测成分的百分含量较高，故选择正己烷作为提取溶媒。见表1。
表1提取溶媒的考察结果(n＝2)
溶媒 百分含量(％)
石油醚 1.90
二氯甲烷 1.91
正己烷 1.93
乙酸乙酯 1.83
3.1.2提取方法的考察
取装量差异项下的中药组合物制剂内容物，研细，混匀，取0.3g，精密称定，置100mL具塞锥形瓶中，冷浸(即，室温浸泡提取)与超声提取各平行2份。冷浸提取：精密吸取正己烷50ml，冷浸20min后，顺逆时针递次振摇约30秒钟；超声提取：精密吸取正己烷50ml，超声处理前称重，超声处理20min后，加正己烷补足重量。取适量各提取方法下的溶液，0.22μm微孔滤膜滤过，即得供试品溶液。
分别吸取供试品溶液1μL，注入气相色谱仪，并以薄荷脑对照品做对照，比较供试品溶液中薄荷脑的百分含量，结果显示两种提取方法效果相当，考虑到冷浸提取方便，故选择冷浸提取。见表2。
表2提取方法的考察结果(n＝2)
提前方法 百分含量(％)
冷浸 1.93
超声 1.96
3.1.3提取时间的考察
取装量差异项下的中药组合物制剂内容物，研细，混匀，取0.3g，精密称定，置100mL具塞锥形瓶中，冷浸提取时间分别为0min、20min、40min，不同提取时间各平行2份，顺逆时针递次振摇约30秒钟，取适量溶液，0.22μm微孔滤膜滤过，即得供试品溶液。
分别吸取供试品溶液1μL，注入气相色谱仪，并以薄荷脑对照品做对照，比较供试品溶液中薄荷脑的百分含量，结果显示三种提取时间下薄荷脑的百分含量相当，故选择冷浸0min。见表3。
表3提取时间的考察结果(n＝2)
时间(min) 百分含量(％)
0 1.94
20 1.96
40 1.93
3.1.4溶媒用量的考察
取装量差异项下的中药组合物制剂内容物，研细，混匀，取0.3g，精密称定，置塞锥形瓶中，不同溶媒量各平行2份，精密吸取溶媒量分别为25mL、50mL、75mL，顺逆时针递次振摇约30秒钟，取适量溶液，0.22μm微孔滤膜滤过，即得供试品溶液。
分别吸取供试品溶液1μL，注入气相色谱仪，并以薄荷脑对照品做对照，比较供试品溶液中薄荷脑的百分含量，结果显示25mL、50mL、75mL提取效果相当，故选择溶媒量为25mL。见表4。
表4溶媒量的考察结果(n＝2)
溶媒量(mL) 百分含量(％)
25 1.87
50 1.90
75 1.87
3.1.5供试品溶液制备方法的确定
根据上述试验结果，最终确定供试品溶液的制备方法为：取装量差异项下的中药组合物制剂内容物，研细，混匀，取0.3g，精密称定，置具塞锥形瓶中，精密加入正己烷25mL，顺逆时针递次振摇约30秒钟，取适量溶液，0.22μm微孔滤膜滤过，即得供试品溶液。
3.2对照品溶液制备方法考察
对照品溶液的制备
取薄荷脑对照品适量，精密称定，加正己烷制成每1mL含0.23mg薄荷脑溶液，即得。
3.2.1专属性考察
吸取溶剂(即空白试剂)、阴性对照溶液、对照品溶液、供试品溶液，按照确定的色 谱条件进行检测，其中阴性对照溶液指不加薄荷脑的制剂按照供试品溶液的制备方法制备的溶液。结果显示阴性对照对所测成分无干扰，专属性良好。(溶剂、阴性对照、对照品、供试品色谱图见附图2～5)。
3.2.2线性关系考察
精密称取薄荷脑对照品100.51mg，置50mL容量瓶中，用正己烷溶解并定容至刻度，摇匀，作为储备液，再从中分别精密量取1mL、1.5mL、2mL、2.5mL、3mL、3.5mL置于25mL的容量瓶中，加正己烷稀释至刻度，分别制成浓度为0.0804mg/mL、0.1206mg/mL、0.1608mg/mL、0.2010mg/mL、0.2412mg/mL、0.2814mg/mL的系列对照品溶液，精密吸取上述对照品溶液各1μL，注入气相色谱仪，检测峰面积，以进样浓度(mg/mL)为横坐标，以峰面积为纵坐标进行线性回归，结果表明薄荷脑在0.0804mg/mL～0.2814mg/mL范围内线性良好，回归方程为y＝48583.7941x+142.5821(R2＝0.9996)，结果见表5和图1。
表5薄荷脑对照品浓度与峰面积关系
Figure PCTCN2017109262-appb-000001
3.2.3检测限和定量限的确定
将薄荷脑0.0804mg/ml的对照品溶液进一步稀释成28.944μg/mL、24.120μg/mL、19.296μg/mL、17.688μg/mL、16.080μg/mL、12.060μg/mL、8.040μg/mL、6.432μg/mL、4.824μg/mL、3.216μg/mL的系列对照品溶液，分别精密吸取上述对照品溶液各1μL，注入气相色谱仪，记录信号强度与噪音强度的比值，将信号强度和噪音强度之比等于3时的浓度作为检测限，将信号强度与噪音强度之比等于10时的浓度作为定量限。薄荷脑的检测限和定量限分别为4.824μg/mL和17.688μg/mL。
4.方法学考察
4.1精密度考察
取本发明中药组合物适量，研细，混匀，取0.3g，精密称定，按照供试品溶液制备方法制备供试品溶液，取薄荷脑对照品溶液(0.2292mg/mL)，按确定的色谱条件连续进样6次，记录薄荷脑的峰面积，计算其RSD，结果见表6。
表6精密度的考察结果
Figure PCTCN2017109262-appb-000002
表6结果显示供试品溶液和对照品溶液中薄荷脑的峰面积RSD小于2％，表明仪器 精密度良好。
4.2稳定性试验
取本发明中药组合物适量，研细，取0.3g，精密称定，按照供试品溶液制备方法制备供试品溶液，按照确定的色谱条件，分别在0h、2h、4h、6h、8h、10h、12h、24h进样，记录薄荷脑的峰面积，计算其RSD，考察溶液的稳定性，结果见表7。
表7稳定性的试验结果
进样时间(h) 峰面积(μv*sec)
0 10259.94
2 10236.74
4 10492.14
6 10494.76
8 10486.51
10 10565.91
12 10506.19
24 11204.67
RSD(％) 2.83
由表7结果显示，供试品溶液中薄荷脑峰面积在24h内RSD小于3％，表明供试品溶液在24h内稳定。
4.3重复性试验
取本发明中药组合物适量，研细，分别按供试品溶液制备方法中取样量的80％、100％和120％取样，即分别取约0.24、0.3、0.36g，各平行3份，共9份样品，按照供试品溶液的制备方法制备供试品溶液，按照确定的色谱条件测定，计算供试品溶液中薄荷脑的含量(％)，计算其RSD，结果见表8。
表8重复性的试验结果
Figure PCTCN2017109262-appb-000003
由表8结果显示，9份供试品溶液中薄荷脑的平均含量为1.86％，其含量的RSD小于3％，表明该方法具有良好的重复性。
4.4加样回收率试验
按取样量0.3g的二分之一，即0.15g称取样品，精密称定，平行9份，取已知含量的薄荷脑对照品溶液，按照样品含量的80％、100％、120％的比例分别加入样品中，各平行三份，按照供试品溶液的制备方法制备供试品溶液，按照确定的色谱条件测定，计算本发明中药组合物中薄荷脑的加样回收率，计算其RSD，结果见表9。
表9薄荷脑加样回收率的试验结果
Figure PCTCN2017109262-appb-000004
由表9结果显示，9份供试品溶液中薄荷脑的回收率平均值为97.68％，RSD为2.49％，表明该法回收率良好。
4.5系统耐用性试验
4.5.1载气流速对薄荷脑含量测定的影响
取本发明中药组合物适量，研细，混匀，取0.3g，精密称定，平行取样2份，按照供试品溶液的制备方法制备供试品溶液，固定其他色谱条件，分别在载气流速为0.80、1.00和1.20mL/min进行测定(每份样品平行进样两针)，并以薄荷脑对照品做对照，计算本发明中药组合物中薄荷脑的的含量，比较载气流速变化对测定结果的影响，结果见表10。
表10载气流速变化对薄荷脑含量的影响
Figure PCTCN2017109262-appb-000005
由上述结果可知，载气流速变化后，薄荷脑含量测定结果的RSD值小于3％，说明载气流速的变化，对样品含量测定结果影响较小。
4.5.2检测器温度变化对薄荷脑含量测定的影响
取供试品溶液，固定其他色谱条件，分别在FID检测器温度为295℃、300℃和305℃进行测定，并以薄荷脑对照品做对照，计算本发明中药组合物中薄荷脑的的含量，比较FID检测器温度变化对测定结果的影响，结果见表11。
表11 FID检测器温度变化对薄荷脑含量的影响
Figure PCTCN2017109262-appb-000006
由上述结果可知，检测器温度变化后，薄荷脑含量测定结果的RSD值小于3％，说明检测器温度的变化，对样品含量测定结果影响较小。
4.5.3进样口温度变化对薄荷脑含量测定的影响
取供试品溶液，固定其他色谱条件，分别在进样口温度为295℃、300℃和305℃进行测定，并以薄荷脑对照品做对照，计算本发明中药组合物中薄荷脑的的含量，比较进样口温度变化对测定结果的影响，结果见表12。
表12进样口温度变化对薄荷脑含量的影响
Figure PCTCN2017109262-appb-000007
由上述结果可知，进样口温度变化后，薄荷脑含量测定结果的RSD值小于3％，说明进样口温度的变化，对样品含量测定结果影响小。
4.5.3不同毛细管柱对薄荷脑含量测定的影响
取供试品溶液，固定其他色谱条件，分别用Agilent DB-5、Agilent HP-5等不同的毛细管柱进行测定，并以薄荷脑对照品做对照，计算本发明中药组合物中薄荷脑的含量，比较不同的色谱柱对测定结果的影响，结果见表13。
表13不同毛细管柱对薄荷脑含量的影响
Figure PCTCN2017109262-appb-000008
由上述结果可知，选择同一品牌不同型号的毛细管柱，薄荷脑含量测定结果的RD值小于3％，说明此含量测定方法的耐用性好。
4.5.4不同批次本发明中药组合物制剂中薄荷脑的含量测定结果
用所制定的含量测定方法，对石家庄以岭药业股份有限公司提供的10个批次(具体 批号见下表)的本发明中药组合物中所含薄荷脑进行了测定，结果见表14。
表14 10批本发明中药组合物制剂薄荷脑的含量测定结果
批号 薄荷脑含量(％)
A1601015 1.88
A1602014 1.87
A1601030 1.95
A1601028 1.85
A1602012 1.81
A1601018 1.84
A1602011 1.94
A1602009 1.91
A1601022 1.83
A1601020 1.93
本发明建立了本发明中药组合物的薄荷脑的含量测定方法，由以上实验结果可知，本方法具有良好的精密度和稳定性，以及重复性，为提高该中药组合物的质量控制提供一种新方法。
附图说明
图1：薄荷脑的标准曲线
图2：空白试剂色谱图
图3：阴性对照溶液色谱图
图4：对照品溶液色谱图
图5：供试品溶液色谱图
具体实施方式
仪器与试药
仪器：PerKinElmer Clarus 680气相色谱仪、AL204型和AB135-S电子天平、Agilent J&W Scientific HP-5毛细管柱(30m×0.25mm,0.25μm)、数控超声波清洗器(型号：KQ-500DB，500W，40KHZ)、0.22μm微孔滤膜(天津市津腾实验设备有限公司)。
试剂：正己烷(色谱纯，美国Fisher)；石油醚、二氯甲烷、乙酸乙酯(分析纯，北京化工厂)。
药品：薄荷脑对照品(购SIGMA-ALORICH，批号：M2772-100G-A，纯度为99％)。
以下各实施例各按所列条件分别平行进行三次，编号分别记为1、2和3。
实施例1
按比例称取：连翘200g、金银花300g、板蓝根200g、大黄40g、广藿香60g、绵马 贯众300g、红景天100g、薄荷脑9g、麻黄60g、苦杏仁100g、鱼腥草200g、甘草100g、石膏200g，按照以下工艺提取：
(1)按照原料药重量比例称取中药材，净选，酌情碎断；
(2)广藿香碎断，加10倍量水提取挥发油，提油时间8小时，收集挥发油，备用；提取液过滤后，残渣弃去，滤液备用；
(3)连翘、麻黄、鱼腥草、大黄，用12倍量70％的乙醇提取3次，每次2.5小时，提取液合并过滤，回收乙醇，滤液备用；
(4)金银花、石膏、板蓝根、绵马贯众、甘草、红景天，加12倍量水煎煮至沸，加入苦杏仁，煎煮2次，每次1小时，提取液合并过滤，所得滤液与步骤(2)广藿香提油后的滤液合并，浓缩成在60℃时测定相对密度为1.15的清膏，加入乙醇，调节至醇浓度为70％，冷藏放置，过滤，回收乙醇至无醇味，得清膏备用；
(5)将步骤(4)所得清膏与步骤(3)所得醇提液合并，浓缩至在60℃时测定相对密度为1.20的清膏，干燥，得干膏粉，备用；
(6)将步骤(5)所得干膏粉加入适当辅料制粒；该辅料可以是35g淀粉；
(7)将薄荷脑、步骤(2)所得挥发油加入乙醇溶解，喷入步骤(6)所得颗粒，装胶囊，即得。
薄荷脑含量测定方法：
供试品溶液制备：取装量差异项下的本品内容物，研细，混匀，取0.3g，精密称定，置具塞锥形瓶中，精密加入正己烷25mL，顺逆时针递次振摇约30秒钟，取适量溶液，0.22μm微孔滤膜滤过，即得供试品溶液；
对照品溶液的制备：取薄荷脑对照品适量，精密称定，加正己烷制成每1mL含0.23mg薄荷脑溶液，即得；
色谱条件：色谱柱：Agilent J&W Scientific HP-5毛细管柱(30m×0.25mm,0.25μm)；柱温为程序升温：初始温度98℃，保持12min，以每分钟8℃的速率升至140℃，保持2.5min，再以140℃的速率升至280℃，保持10min；检测器温度为300℃；进样口温度为300℃；载气为氮气，流速：1mL/min；分流进样，分流比：25:1；进样量：1μL；燃气比例：空气-氢气(450:45)；
测定法：分别精密吸取对照品溶液与供试品溶液各1μL，注入气相色谱仪，测定，即得。
组合物中薄荷脑的的含量测定结果
Figure PCTCN2017109262-appb-000009
结论：结果薄荷脑分离度较好，可以用于控制该中药组合物的质量。
实施例2
按比例称取：连翘300g、金银花200g、板蓝根300g、大黄60g、广藿香100g、绵马贯众200g、红景天60g、薄荷脑5g、麻黄100g、苦杏仁60g、鱼腥草300g、甘草60g、石膏300g，按照以下工艺提取：
(1)按照原料药重量比例称取中药材，净选，酌情碎断；
(2)广藿香碎断，加10倍量水提取挥发油，提油时间8小时，收集挥发油，备用；提取液过滤后，残渣弃去，滤液备用；
(3)连翘、麻黄、鱼腥草、大黄，用12倍量70％的乙醇提取3次，每次2.5小时，提取液合并过滤，回收乙醇，滤液备用；
(4)金银花、石膏、板蓝根、绵马贯众、甘草、红景天，加12倍量水煎煮至沸，加入苦杏仁，煎煮2次，每次1小时，提取液合并过滤，所得滤液与步骤(2)广藿香提油后的滤液合并，浓缩成在60℃时测定相对密度为1.10的清膏，加入乙醇，调节至醇浓度为70％，冷藏放置，过滤，回收乙醇至无醇味，得清膏备用；
(5)将步骤(4)所得清膏与步骤(3)所得醇提液合并，浓缩至在60℃时测定相对密度为1.15的清膏，干燥，得干膏粉，备用；
(6)将步骤(5)所得干膏粉加入适当药学上可接受的辅料制粒；该辅料可以是35g淀粉；
(7)将薄荷脑、步骤(2)所得挥发油加入乙醇溶解，喷入步骤(6)所得颗粒，压片，即得。
薄荷脑含量测定方法：
供试品溶液制备：取装量差异项下的本品内容物，研细，混匀，取0.2g，精密称定，置具塞锥形瓶中，精密加入正己烷20mL，顺逆时针递次振摇约20秒钟，取适量溶液，0.22μm微孔滤膜滤过，即得供试品溶液；
对照品溶液的制备：取薄荷脑对照品适量，精密称定，加正己烷制成每1mL含0.23mg薄荷脑溶液，即得；
色谱条件：色谱柱：Agilent J&W Scientific HP-5毛细管柱(30m×0.25mm,0.25μm)；柱温为程序升温：初始温度98℃，保持12min，以每分钟8℃的速率升至140℃，保持2.5min，再以140℃的速率升至280℃，保持5-20min；检测器温度为350℃；进样口温度为350℃；载气为氮气，流速：0.8mL/min；分流进样，分流比：25:1；进样量：0.5μL；燃气比例：空气-氢气(450:45)；
测定法：分别精密吸取对照品溶液与供试品溶液各0.5μL，注入气相色谱仪，测定，即得。
组合物中薄荷脑的的含量测定结果
Figure PCTCN2017109262-appb-000010
结论：结果薄荷脑分离度较好，可以用于控制该中药组合物的质量。
实施例3
原料药配方为：连翘278g、金银花294g、板蓝根285g、大黄55g、广藿香95g、绵马贯众290g、红景天87g、薄荷脑8.5g、麻黄88g、苦杏仁80g、鱼腥草284g、甘草95g、石膏277g，按照以下工艺提取：
(1)按照原料药重量比例称取中药材，净选，酌情碎断；
(2)广藿香碎断，加10倍量水提取挥发油，提油时间8小时，收集挥发油，备用；提取液过滤后，残渣弃去，滤液备用；
(3)连翘、麻黄、鱼腥草、大黄，用12倍量70％的乙醇提取3次，每次2.5小时，提取液合并过滤，回收乙醇，滤液备用；
(4)金银花、石膏、板蓝根、绵马贯众、甘草、红景天，加12倍量水煎煮至沸，加入苦杏仁，煎煮2次，每次1小时，提取液合并过滤，所得滤液与步骤(2)广藿香提油后的滤液合并，浓缩成在60℃时测定相对密度为1.13的清膏，加入乙醇，调节至醇浓度为70％，冷藏放置，过滤，回收乙醇至无醇味，得清膏备用；
(5)将步骤(4)所得清膏与步骤(3)所得醇提液合并，浓缩至在60℃时测定相对密度为1.18的清膏，干燥，得干膏粉，备用；
(6)将步骤(5)所得干膏粉加入适当药学上可接受的辅料制粒；该辅料可以是35g淀粉；
(7)将薄荷脑、步骤(2)所得挥发油加入乙醇溶解，喷入步骤(6)所得颗粒，装袋，即得。
含量测定方法：
供试品溶液制备：取装量差异项下的本品内容物，研细，混匀，取0.5g，精密称定，置具塞锥形瓶中，精密加入正己烷30mL，顺逆时针递次振摇约50秒钟，取适量溶液，0.22μm微孔滤膜滤过，即得供试品溶液；
对照品溶液的制备：取薄荷脑对照品适量，精密称定，加正己烷制成每1mL含0.23mg薄荷脑溶液，即得；
色谱条件：色谱柱：Agilent J&W Scientific HP-5毛细管柱(30m×0.25mm,0.25μm)；柱温为程序升温：初始温度98℃，保持12min，以每分钟8℃的速率升至140℃，保持2.5min，再以140℃的速率升至280℃，保持5-20min；检测器温度为400℃；进样口温 度为400℃；载气为氮气，流速：1.2mL/min；分流进样，分流比：25:1；进样量：2μL；燃气比例：空气-氢气(450:45)；
测定法：分别精密吸取对照品溶液与供试品溶液各2μL，注入气相色谱仪，测定，即得。
组合物中薄荷脑的的含量测定结果
Figure PCTCN2017109262-appb-000011
结果：结果薄荷脑分离度较好，可以用于控制该中药组合物的质量。
实施例4
原料药配方为：按比例称取：连翘255g、金银花255g、板蓝根255g、大黄51g、广藿香85g、绵马贯众255g、红景天85g、薄荷脑7.5g、麻黄85g、苦杏仁85g、鱼腥草255g、甘草85g、石膏255g，按照以下工艺提取：
(1)按照原料药重量比例称取中药材，净选，酌情碎断；
(2)广藿香碎断，加10倍量水提取挥发油，提油时间8小时，收集挥发油，备用；提取液过滤后，残渣弃去，滤液备用；
(3)连翘、麻黄、鱼腥草、大黄，用12倍量70％的乙醇提取3次，每次2.5小时，提取液合并过滤，回收乙醇，滤液备用；
(4)金银花、石膏、板蓝根、绵马贯众、甘草、红景天，加12倍量水煎煮至沸，加入苦杏仁，煎煮2次，每次1小时，提取液合并过滤，所得滤液与步骤(2)广藿香提油后的滤液合并，浓缩成在60℃时测定相对密度为1.14的清膏，加入乙醇，调节至醇浓度为70％，冷藏放置，过滤，回收乙醇至无醇味，得清膏备用；
(5)将步骤(4)所得清膏与步骤(3)所得醇提液合并，浓缩至在60℃时测定相对密度为1.19的清膏，干燥，得干膏粉，备用；
(6)将步骤(5)所得干膏粉加入适当药学上可接受的辅料制粒；该辅料可以是35g淀粉；
(7)将薄荷脑、步骤(2)所得挥发油加入乙醇溶解，喷入步骤(6)所得颗粒，装胶囊，即得。
含量测定方法：
供试品溶液制备：取装量差异项下的本品内容物，研细，混匀，取0.4g，精密称定，置具塞锥形瓶中，精密加入正己烷20mL，顺逆时针递次振摇约40秒钟，取适量溶液，0.22μm微孔滤膜滤过，即得供试品溶液；
对照品溶液的制备：取薄荷脑对照品适量，精密称定，加正己烷制成每1mL含0.23 mg薄荷脑溶液，即得；
色谱条件：色谱柱：Agilent J&W Scientific HP-5毛细管柱(30m×0.25mm,0.25μm)；柱温为程序升温：初始温度98℃，保持12min，以每分钟8℃的速率升至140℃，保持2.5min，再以140℃的速率升至280℃，保持5-20min；检测器温度为350℃；进样口温度为350℃；载气为氮气，流速：1.1mL/min；分流进样，分流比：25:1；进样量：1μL；燃气比例：空气-氢气(450:45)；
测定法：分别精密吸取对照品溶液与供试品溶液各1μL，注入气相色谱仪，测定，即得。
组合物中薄荷脑的的含量测定结果
Figure PCTCN2017109262-appb-000012
结果：结果薄荷脑分离度较好，可以用于控制该中药组合物的质量。

Claims (18)

  1. 一种中药组合物制剂中薄荷脑的含量测定方法，所述中药组合物制剂由如下重量份的原料药制成：连翘200-300、麻黄60-100、大黄40-60、鱼腥草200-300、金银花200-300、板蓝根200-300、广藿香60-100、绵马贯众200-300、红景天60-100、薄荷脑5-9、苦杏仁60-100、甘草60-100、石膏200-300，其特征在于，所述薄荷脑的含量测定方法如下：
    1)使用非极性溶剂对所述中药组合物制剂进行提取，得到供试品溶液；
    2)使用与步骤1)中相同的非极性溶剂，将薄荷脑对照品制备成薄荷脑浓度在4.80μg/mL以上，优选17.65μg/mL以上，更优选0.2-0.3mg/mL的薄荷脑对照品溶液；
    3)分别取等量对照品溶液与供试品溶液，注入气相色谱仪，测定薄荷脑含量，其中色谱条件为：
    色谱柱为弱极性毛细管色谱柱；柱温为程序升温：初始温度80-100℃，保持10-15min，以每分钟6-10℃的速率升至120-160℃，保持1.5-3.5min，再以100-160℃的速率升至240-300℃，保持5-20min。
  2. 根据权利要求1所述的方法，其特征在于，所述供试品溶液制备时，使用的所述中药组合物制剂与非极性溶剂的质量对体积比为(1g:400mL)～(1g:50mL)。
  3. 根据权利要求1或2所述的方法，其特征在于，所述弱极性毛细管色谱柱以苯基-甲基聚硅氧烷为固定相，优选所述苯基-甲基聚硅氧烷中苯基含量为1～10％，更优选5％；进一步优选所述弱极性色谱柱毛细管色谱柱为HP-5型或DB-5型色谱柱。
  4. 根据前述任一项权利要求所述的方法，其特征在于，所述注入气相色谱仪中的对照品溶液与供试品溶液分别为0.5-2μL。
  5. 根据前述任一项权利要求所述的方法，其特征在于，在所述薄荷脑含量测定中，采取分流进样，分流比为50:1～10:1，优选30:1～20:1，更优选25:1。
  6. 根据前述任一项权利要求所述的方法，其特征在于，所述色谱条件还包括分别为300-400℃的检测器温度和进样口温度。
  7. 根据前述任一项权利要求所述的方法，其特征在于，所述色谱条件还包括载气及其流速，所述载气为氮气，流速为0.8-1.2mL/min。
  8. 根据前述任一项权利要求所述的方法，其特征在于，所述色谱条件还包括燃气比例，所述燃气比例为空气对氢气比为8:1～12:1，优选9:1～11:1，更优选10:1。
  9. 根据前述任一项权利要求所述的方法，其特征在于，所述非极性溶剂选自非极性饱和烷烃或卤代饱和烷烃，以及非极性酯类溶剂；优选所述非极性溶剂选自正己烷、二氯甲烷、石油醚和乙酸乙酯，更优选正己烷。
  10. 根据前述任一项权利要求中所述的方法，其特征在于，所述供试品溶液的制备方法具体为：将所述中药组合物制剂研磨混匀后置于具塞窄口容器中，加入所述非极性有机溶剂，振摇20秒以上，过滤，得所述供试品溶液；优选所述过滤步骤使用0.2-0.4μm微孔滤膜进行，更优选使用0.22μm微孔滤膜过滤；优选在振摇步骤前，还包括室温下浸 泡或超声提取的步骤。
  11. 根据前述任一项权利要求所述的方法，其特征在于，所述中药组合物的制备方法为：
    (1)按照原料药重量比例称取中药材，净选、碎断；
    (2)广藿香，加8-12倍量水提取挥发油，提油时间6-10小时，收集挥发油，备用；提取液过滤后，残渣弃去，滤液备用；
    (3)连翘、麻黄、鱼腥草、大黄，用10-14倍量60-80％的乙醇提取2-4次，每次1.5-3小时，提取液合并过滤，回收乙醇，滤液备用；
    (4)金银花、石膏、板蓝根、绵马贯众、甘草、红景天，加10-14倍量水煎煮至沸，加入苦杏仁，煎煮2-4次，每次0.5-2小时，提取液合并过滤，所得滤液与步骤(2)广藿香提油后的滤液合并，浓缩成在50-70℃时测定相对密度为1.10-1.15的清膏，加入乙醇，调节至醇浓度为65-80％，冷藏放置，过滤，回收乙醇至无醇味，得清膏备用；
    (5)将步骤(4)所得清膏与步骤(3)所得醇提液合并，浓缩至在50-70℃时测定相对密度为1.15-1.20的清膏，干燥，得干膏粉，备用；
    (6)将步骤(5)所得干膏粉加入适当药学上可接受的辅料制粒；
    (7)将薄荷脑、步骤(2)所得挥发油加入乙醇溶解，喷入步骤(6)所得颗粒，密闭，混匀，压片或者装胶囊、或者装袋。
  12. 根据权利要求11所述的方法，其特征在于，所述药学上可接受的辅料是淀粉。
  13. 根据前述任一项权利要求所述的方法，所述中药组合物制剂由如下重量份的原料药制成：连翘200-300、麻黄60-100、大黄40-60、鱼腥草200-300、金银花200-300、板蓝根200-300、广藿香60-100、绵马贯众200-300、红景天60-100、薄荷脑5-9、苦杏仁60-100、甘草60-100、石膏200-300，其特征在于所述薄荷脑的含量测定方法如下：
    供试品溶液制备：取本品内容物，研细，混匀，取0.2-0.5g，精密称定，置具塞锥形瓶中，精密加入正己烷20-30mL，顺逆时针递次振摇约20-50秒钟，取适量溶液，0.22μm微孔滤膜滤过，即得供试品溶液；
    对照品溶液的制备：取薄荷脑对照品适量，精密称定，加正己烷制成每1mL含0.23mg薄荷脑溶液，即得；
    色谱条件：色谱柱：Agilent J&W Scientific HP-5毛细管柱(30m×0.25mm,0.25μm)；柱温为程序升温：初始温度98℃，保持12min，以每分钟8℃的速率升至140℃，保持2.5min，再以140℃的速率升至280℃，保持5-20min；检测器温度为300-400℃；进样口温度为300-400℃；载气为氮气，流速：0.8-1.2mL/min；分流进样，分流比：25:1；进样量：0.5-2μL；燃气比例：空气-氢气(450:45)；
    测定法：分别精密吸取对照品溶液与供试品溶液各0.5-2μL，注入气相色谱仪，测定，即得。
  14. 根据前述任一项权利要求所述的方法，其特征在于，所述中药组合物制剂是由如下重量份的原料药制成：
    连翘200、金银花300、板蓝根200、大黄40、广藿香60、绵马贯众300、红景天100、薄荷脑9、麻黄60、苦杏仁100、鱼腥草200、甘草100、石膏200；或者所述中药组合物制剂由下列重量份的原料药制成：
    连翘300、金银花200、板蓝根300、大黄60、广藿香100、绵马贯众200、红景天60、薄荷脑5、麻黄100、苦杏仁60、鱼腥草300、甘草60、石膏300；或者所述中药组合物制剂由下列重量份的原料药制成：
    连翘278、金银花294、板蓝根285、大黄55、广藿香95、绵马贯众290、红景天87、薄荷脑8.5、麻黄88、苦杏仁80、鱼腥草284、甘草95、石膏277；或者所述中药组合物制剂由下列重量份的原料药制成：
    连翘255、金银花255、板蓝根255、大黄51、广藿香85、绵马贯众255、红景天85、薄荷脑7.5、麻黄85、苦杏仁85、鱼腥草255、甘草85、石膏255。
  15. 根据前述任一项权利要求所述的方法，其特征在于，所述中药组合物制剂的制备方法为：
    (1)按照原料药重量比例称取中药材，净选，酌情碎断；
    (2)广藿香碎断，加10倍量水提取挥发油，提油时间8小时，收集挥发油，备用；提取液过滤后，残渣弃去，滤液备用；
    (3)连翘、麻黄、鱼腥草、大黄，用12倍量70％的乙醇提取3次，每次2.5小时，提取液合并过滤，回收乙醇，滤液备用；
    (4)金银花、石膏、板蓝根、绵马贯众、甘草、红景天，加12倍量水煎煮至沸，加入苦杏仁，煎煮2次，每次1小时，提取液合并过滤，所得滤液与步骤(2)广藿香提油后的滤液合并，浓缩成在60℃时测定相对密度为1.10-1.15的清膏，加入乙醇，调节至醇浓度为70％，冷藏放置，过滤，回收乙醇至无醇味，得清膏备用；
    (5)将步骤(4)所得清膏与步骤(3)所得醇提液合并，浓缩至在60℃时测定相对密度为1.15-1.20的清膏，干燥，得干膏粉，备用；
    (6)将步骤(5)所得干膏粉加入适当药学上可接受的辅料制粒；
    (7)将薄荷脑、步骤(2)所得挥发油加入乙醇溶解，喷入步骤(6)所得颗粒，密闭，混匀，压片或者装胶囊、或者装袋。
  16. 根据前述任一项权利要求所述的方法，其特征在于，所述含量测定方法如下：
    供试品溶液制备：取装量差异项下的本品内容物，研细，混匀，取0.3g，精密称定，置具塞锥形瓶中，精密加入正己烷25mL，顺逆时针递次振摇约30秒钟，取适量溶液，0.22μm微孔滤膜滤过，即得供试品溶液；
    对照品溶液的制备：取薄荷脑对照品适量，精密称定，加正己烷制成每1mL含0.23 mg薄荷脑溶液，即得；
    色谱条件：色谱柱：Agilent J&W Scientific HP-5毛细管柱(30m×0.25mm,0.25μm)；柱温为程序升温：初始温度98℃，保持12min，以每分钟8℃的速率升至140℃，保持2.5min，再以140℃的速率升至280℃，保持10min；检测器温度为300℃；进样口温度为300℃；载气为氮气，流速：1mL/min；分流进样，分流比：25:1；进样量：1μL；燃气比例：空气-氢气(450:45)；
    测定法：分别精密吸取对照品溶液与供试品溶液各1μL，注入气相色谱仪，测定，即得。
  17. 根据前述任一项权利要求所述的方法，其特征在于，所述含量测定方法如下：
    供试品溶液制备：取装量差异项下的本品内容物，研细，混匀，取0.2g，精密称定，置具塞锥形瓶中，精密加入正己烷20mL，顺逆时针递次振摇约20秒钟，取适量溶液，0.22μm微孔滤膜滤过，即得供试品溶液；
    对照品溶液的制备：取薄荷脑对照品适量，精密称定，加正己烷制成每1mL含0.23mg薄荷脑溶液，即得；
    色谱条件：色谱柱：Agilent J&W Scientific HP-5毛细管柱(30m×0.25mm,0.25μm)；柱温为程序升温：初始温度98℃，保持12min，以每分钟8℃的速率升至140℃，保持2.5min，再以140℃的速率升至280℃，保持5min；检测器温度为350℃；进样口温度为350℃；载气为氮气，流速：0.8mL/min；分流进样，分流比：25:1；进样量：0.5μL；燃气比例：空气-氢气(450:45)；
    测定法：分别精密吸取对照品溶液与供试品溶液各0.5μL，注入气相色谱仪，测定，即得。
  18. 根据前述任一项权利要求所述的方法，其特征在于，所述含量测定方法如下：
    供试品溶液制备：取装量差异项下的本品内容物，研细，混匀，取0.5g，精密称定，置具塞锥形瓶中，精密加入正己烷30mL，顺逆时针递次振摇约50秒钟，取适量溶液，0.22μm微孔滤膜滤过，即得供试品溶液；
    对照品溶液的制备：取薄荷脑对照品适量，精密称定，加正己烷制成每1mL含0.23mg薄荷脑溶液，即得；
    色谱条件：色谱柱：Agilent J&W Scientific HP-5毛细管柱(30m×0.25mm,0.25μm)；柱温为程序升温：初始温度98℃，保持12min，以每分钟8℃的速率升至140℃，保持2.5min，再以140℃的速率升至280℃，保持20min；检测器温度为400℃；进样口温度为400℃；载气为氮气，流速：1.2mL/min；分流进样，分流比：25:1；进样量：2μL；燃气比例：空气-氢气(450:45)；
    测定法：分别精密吸取对照品溶液与供试品溶液各2μL，注入气相色谱仪，测定，即得。
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