A method for determining clove content and its application

Abstract

The present invention belongs to the technical field of traditional Chinese medicine detection, and specifically relates to a method for determining the content of cloves and its application. The method for determining the content of cloves of the present invention comprises the following steps: preparation of a reference solution; preparation of a sample solution to be tested; gas chromatography separation; determination of the retention time of the chromatographic peaks of four components in cloves; and quantitative detection. The method for determining the content of cloves provided by the present invention can solve the problem of a large number of reference substances required for determination by a conventional multi-index content determination method; it can also effectively solve the problem that the relative retention time of sample chromatographic peaks in different capillary columns is difficult to reproduce when determining by a one-measurement-multiple-evaluation method or a substitute reference substance method; at the same time, it can be well used for the quality control of cloves, ensuring the uniformity of the quality and efficacy of cloves.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a method for determining clove content and application thereof. Background Art

[0002] Clove is the dried flower bud of Eugenia caryophyllata Thunb., a plant of the Myrtaceae family. It is commonly known as male clove and is a commonly used Chinese medicine. It is pungent and warm in nature and flavor; it enters the spleen, stomach, lung, and kidney meridians; it has the effects of warming the middle and lowering adverse qi, tonifying the kidney and assisting yang; it is mainly used for spleen and stomach deficiency and cold, hiccups and vomiting, poor appetite, vomiting and diarrhea, cold pain in the heart and abdomen, and impotence due to kidney deficiency. Modern research shows that clove volatile oil has strong antibacterial and anti-inflammatory, free radical scavenging and anti-tumor effects. It is mainly used to treat cardiovascular and cerebrovascular system, respiratory system, and digestive system diseases. In addition, it can also be used externally and for oral care, or developed into deodorants, flavoring agents, etc., and is widely used in the pharmaceutical and spice industries. Clove is native to Indonesia, Madagascar, Tanzania and other places, and is cultivated in Yunnan and Hainan, China. The quality of clove medicinal materials on the market is uneven, so how to quickly and effectively determine the volatile components of cloves, objectively evaluate and control their quality is an urgent problem to be solved.

[0003] The volatile oils contained in cloves mainly include eugenol, β-caryophyllene, α-caryophyllene, eugenol acetate and other components, and their peak order and relative content are characteristic and easy to identify. In the clove standards of the prior art, only eugenol is controlled; but since clove stems also contain eugenol, cloves on the market are mixed with clove stems, while clove stems do not contain eugenol acetate. This doping phenomenon makes the quality of cloves unguaranteed. In view of this, it is necessary to provide a detection method for determining the content of the above four components of cloves to make up for the shortcomings of the prior art. Summary of the invention

[0004] The purpose of the present invention is to solve the deficiencies of the prior art and provide a method for determining the content of cloves and its application. The present invention establishes a method for determining the content of volatile components in cloves by a "double-label multiple-measurement" method, that is, by determining one or two components (eugenol or β-caryophyllene), establishing a relative correction factor between the component and other effective components (α-caryophyllene, eugenol acetate), calculating the content of each component, realizing the simultaneous determination of multiple index components, and locating each chromatographic peak using relative retention time. The determination results of this method have no significant difference with those of the external standard method in terms of qualitative and quantitative determination of multiple index components, and can be used as a simple and accurate quality evaluation model for the determination of the content of volatile components in cloves.

[0005] The technical solution of the present invention to solve the above technical problem is as follows: A method for determining the clove content comprises the following steps:

[0006] Step 1: Preparation of reference solution:

[0007] Accurately weigh reference substances β-caryophyllene and eugenol, respectively, add n-hexane to dissolve and dilute to obtain β-caryophyllene reference substance solution and eugenol reference substance solution respectively;

[0008] Step 2: Preparation of sample solution to be tested:

[0009] Accurately weigh the clove sample to be tested, accurately add n-hexane, and pre-treat to obtain the sample solution to be tested;

[0010] Step 3, gas chromatography separation:

[0011] The chromatographic conditions were as follows: the chromatographic column was a capillary column with polyethylene glycol as the stationary phase; the column temperature was 170°C; the injection port temperature was 280°C; the detector temperature was 300°C;

[0012] Step 4: Determination of the chromatographic peak retention times of the four components in cloves:

[0013] Accurately pipetting the β-caryophyllene reference substance solution obtained in step 1, the eugenol reference substance, and the sample solution to be tested obtained in step 2, respectively, injecting them into a gas chromatograph for determination, plotting a retention time standard curve with the standard retention time of β-caryophyllene and eugenol as the abscissa and the measured retention time of β-caryophyllene and eugenol as the ordinate, and obtaining the reference retention times of the four components of β-caryophyllene, α-caryophyllene, eugenol acetate, and eugenol, respectively;

[0014] In the gas chromatogram of the clove sample to be tested, chromatographic peaks corresponding to the control retention times of the four components of β-caryophyllene, α-caryophyllene, eugenyl acetate and eugenol appeared;

[0015] Step 5: Quantitative detection:

[0016] Accurately pipette the eugenol reference solution obtained in step 1 and the sample solution obtained in step 2, respectively, inject them into the gas chromatograph for determination, and use the peak area and correction factor, and then calculate according to the formula The contents of the four components of β-caryophyllene, α-caryophyllene, eugenol acetate and eugenol were calculated; where C is the concentration of the component to be measured; f is the correction factor; A is the peak area of ​​the component to be measured; A i is the peak area of ​​eugenol reference substance; C i is the concentration of eugenol reference solution.

[0017] The technical principle of the present invention is:

[0018] (1) Selection of reference substances: Among the active ingredients of cloves, β-caryophyllene, α-caryophyllene, eugenol, and eugenol acetate, reference substances of β-caryophyllene and eugenol are relatively common, while the other two reference substances are difficult to prepare and unstable. Therefore, in step 1 of the present invention, β-caryophyllene and eugenol, two readily available substances, are used as reference substances, which solves the problems of expensive and unstable reference substances.

[0019] (2) Investigation of the linear equation of retention time and selection of standard retention time: On different capillary columns, the retention time of components has a linear relationship: Under the same analytical conditions, even if different gas chromatography systems and different capillary columns are used, the retention time of components has a simple linear relationship. In practice, this linear relationship can be applied to improve the reproducibility of retention time.

[0020] (3) Differences in chemical composition between cloves and clove pedicels: Cloves contain eugenol acetate, while clove pedicels do not. The quality of cloves can be effectively controlled by controlling the eugenol acetate content.

[0021] In the experiment, the inventor first investigated whether the separation of β-caryophyllene, α-caryophyllene, eugenol and eugenol acetate in the same capillary column conforms to a linear relationship. Using different capillary columns, the retention time of the above four components was measured, and different retention times were obtained, and the average value was taken as its standard retention time; the standard retention time of the four components was used as the horizontal coordinate, and the retention time in each capillary column was used as the vertical coordinate to draw the standard curve of the retention time of each component in each capillary column. The results show that the retention time of the four components in each capillary column is linear, and the retention time linear equation can be used to judge the retention time of the chromatographic peak of the component to be measured. In addition, the standard retention time of the four components is obtained, and the standard retention time of the four components is respectively determined as: the standard retention time of β-caryophyllene is 3.225min, the standard retention time of α-caryophyllene is 3.621min, the standard retention time of eugenol is 12.031min, and the standard retention time of eugenol acetate is 15.721.

[0022] (4) Determination of the retention time of the chromatographic peak of the sample to be tested: Since the retention time of the above four components to be tested is in the same capillary column and has a certain linear relationship with the standard retention time, two components (β-caryophyllene and eugenol) can be selected as controls when measuring the sample to be tested, and a retention time standard curve can be drawn using the standard retention time and the measured retention time. The standard retention times of α-caryophyllene and eugenol acetate in the two components to be tested are substituted into the standard curve to obtain the control retention time of each component. In the gas chromatogram of the sample to be tested, chromatographic peaks corresponding to the above four retention times appear, thereby determining the retention times of the chromatographic peaks of the four components to be tested in the sample to be tested.

[0023] (5) Calculation method of content: The “relative correction factor” of the surrogate reference substance relative to the reference substance is used. That is, the “relative correction factor” is obtained by testing the real reference substance and the surrogate reference substance. The content of the components of the sample being tested is then tested using the surrogate reference substance combined with the correction factor, thereby controlling the quality of the drug.

[0024] Furthermore, in step 1 of the above technical solution, the concentration of β-caryophyllene in the β-caryophyllene reference solution is 0.4 mg / mL; the concentration of eugenol in the eugenol reference solution is 2 mg / mL.

[0025] Furthermore, in step 2 of the above technical solution, the clove sample is pretreated before use; the weight of the clove is 0.3-0.5 g, and the volume of the n-hexane is 20-50 mL.

[0026] Furthermore, in the above technical solution, the pretreatment is ultrasonic treatment or heating reflux, the power of the ultrasonic treatment is 500W, the frequency is 40kHz, and the time is 15min; the time of the heating reflux is 30min.

[0027] Furthermore, in step 3 of the above technical solution, the capillary column is any one of a DB-WAX capillary column, an Rtx-WAX capillary column, and an HP-INNOWAX capillary column. The present invention adopts the above-mentioned multiple capillary columns to sequentially measure the four components of β-caryophyllene, α-caryophyllene, eugenol, and eugenol acetate, and obtains the average retention time of the above four components in different capillary columns as the standard retention time of each component.

[0028] Furthermore, in the above technical solution, the DB-WAX capillary column has a column length of 30 m, an inner diameter of 0.32 mm, and a membrane thickness of 0.25 μm or 0.50 μm; the Rtx-WAX capillary column has a column length of 30 m, an inner diameter of 0.32 mm, and a membrane thickness of 0.50 μm; the HP-INNOWAX capillary column has a column length of 30 m, an inner diameter of 0.32 mm, and a membrane thickness of 0.50 μm.

[0029] Furthermore, in step 5 of the above technical solution, the correction factor f is calculated according to the following formula: In the formula,

[0030] f is the correction factor;

[0031] A i is the peak area of ​​eugenol reference substance;

[0032] A r is the peak area of ​​the reference substance of the component to be tested;

[0033] Ci is the concentration of eugenol reference;

[0034] C r The present invention obtains a "relative correction factor" by testing the real reference substance and the substitute reference substance, and then uses the substitute reference substance combined with the correction factor to test the content of the sample component to control the quality of the drug.

[0035] The present invention also provides an application of the above-mentioned clove content determination method in the quality control of clove medicines. The determination method is used to detect the content of each active ingredient in clove medicines, which can make up for the technical defect that only eugenol is detected in the existing clove standards and adulteration occurs, and provides a reliable method for the quality control of cloves, which can be used for the quality assessment of cloves.

[0036] Advantages compared to the prior art:

[0037] 1. The method for determining the clove content of the present invention comprises the following steps: first, using β-caryophyllene and eugenol as reference substances, and after drawing a retention time standard curve, the retention times of the chromatographic peaks of the four components in the sample to be tested are judged, wherein the retention times of the chromatographic peaks of β-caryophyllene and eugenol can be judged by using reference substances, and the retention times of the chromatographic peaks of the other two components are obtained by calculating the retention time standard curve under the chromatographic conditions; second, using the eugenol reference substance as a reference substance, and using the peak area and the correction factors of the above four components relative to eugenol for quantitative determination.

[0038] 2. The method for determining the clove content provided by the present invention can not only solve the problem of a large number of reference substances required for conventional multi-index content determination methods, but also solve the problem of difficulty in reproducing the relative retention times of sample chromatographic peaks in different capillary columns when using a one-measurement-multiple-evaluation method or a reference substance replacement method; at the same time, it can be well used for the quality control of cloves, ensuring the uniformity of the quality and efficacy of cloves.

[0039] 3. The determination method of the present invention is simple, accurate and rapid, and can be effectively used to determine the content of volatile components in cloves, and can provide a basis for the quality control of cloves. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 It is a linear graph of the retention times of the four components of the clove sample to be tested in Example 1 of the present invention in a DB-WAX (film thickness of 0.50 μm) capillary column;

[0041] Figure 2 This is a linear graph of the retention times of the four components of the clove sample to be tested in Example 1 of the present invention in a DB-WAX (film thickness of 0.25 μm) capillary column;

[0042] Figure 3It is a linear graph of the retention times of the four components of the clove sample to be tested in Example 1 of the present invention in a Rtx-WAX (film thickness of 0.50 μm) capillary column;

[0043] Figure 4 It is a linear graph of the retention times of the four components of the clove sample to be tested in Example 1 of the present invention in a HP-INNOWAX (film thickness of 0.50 μm) capillary column;

[0044] Figure 5 The gas chromatogram (PEG-20M gas chromatographic column) of the β-caryophyllene reference substance solution in Example 1 of the present invention;

[0045] Figure 6 The gas chromatogram (PEG-20M gas chromatographic column) of the eugenol reference substance solution in Example 1 of the present invention;

[0046] Figure 7 The gas chromatogram (PEG-20M gas chromatographic column) of the clove sample to be tested of the present invention;

[0047] Figure 8 The gas chromatogram (PEG-20M gas chromatographic column) of the mixed reference substance of the comparative example of the present invention. DETAILED DESCRIPTION

[0048] The above-mentioned technical features of the present invention and the technical features specifically described below (such as implementation cases) can be combined with each other to form new or preferred technical solutions, but the present invention is not limited to these embodiments, and these embodiments do not limit the present invention in any way.

[0049] The experimental methods in the following examples are conventional methods unless otherwise specified. The preparations involved in the following examples are common commercial products unless otherwise specified and can be purchased from the market.

[0050] The present invention is further described in detail below in conjunction with the drawings and embodiments:

[0051] Experimental reagents

[0052] Instruments and reagents

[0053] Instruments: Agilent 6890N gas chromatograph (Agilent Technologies, Inc., USA); Agilent DB-WAX (30 m × 0.32 mm, 0.25 μm; Agilent Technologies, Inc., USA) capillary column; Agilent DB-WAX (30 m × 0.32 mm, 0.50 μm; Agilent Technologies, Inc., USA) capillary column; Rtx-WAX (30 m × 0.32 mm, 0.50 μm; Shimadzu Corporation, Japan) capillary column; HP-INNOWAX (30 m × 0.32 mm, 0.50 μm; Agilent Technologies, Inc., USA) capillary column; PEG-20M (30 m × 0.32 mm, 0.25 μm; China Science and Technology Antai Co., Ltd.) capillary column; BT25S electronic balance, BSA124S-CW electronic balance (Sartorius (Beijing) Co., Ltd.).

[0054] Reagents: All reagents were of analytical grade; eugenol reference substance (110725-201615, content 99.3%) was purchased from China Food and Drug Inspection Institute; β-caryophyllene reference substance (0001321406, content 98.5%) was purchased from SIGMA, USA; α-caryophyllene reference substance (Z25M8H36799, content 93.0%) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; eugenol acetate reference substance (Z25M8H36798, content 98.0%) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0055] Example 1

[0056] A method for determining clove content in this embodiment comprises the following steps:

[0057] Step 1: Preparation of reference solution

[0058] Accurately weigh 10 mg of reference substance β-caryophyllene and 50 mg of eugenol, respectively, and place them in 25 mL volumetric flasks, add n-hexane to dissolve them to the scale, shake well, and obtain β-caryophyllene reference solution and eugenol reference solution, respectively.

[0059] Step 2: Preparation of sample solution

[0060] Take about 0.3 g of clove sample powder (passed through No. 2 sieve), accurately weigh, accurately add 20 mL of n-hexane, weigh, ultrasonically treat for 15 minutes, let it cool to room temperature, weigh again, make up the lost weight with n-hexane, shake well, filter, and take the filtrate to obtain.

[0061] Step 3: Gas chromatography separation

[0062] The capillary column with polyethylene glycol as the stationary phase is used as the chromatographic column, and the models of the capillary columns are: the DB-WAX capillary column has a column length of 30m, an inner diameter of 0.32mm, and a film thickness of 0.50μm; the DB-WAX capillary column has a column length of 30m, an inner diameter of 0.32mm, and a film thickness of 0.25μm; the Rtx-WAX capillary column has a column length of 30m, an inner diameter of 0.32mm, and a film thickness of 0.50μm; the HP-INNOWAX capillary column has a column length of 30m, an inner diameter of 0.32mm, and a film thickness of 0.50μm; and the PEG-20M capillary column has a column length of 30m, an inner diameter of 0.32mm, and a film thickness of 0.25μm.

[0063] The column temperature was 170°C; the injection port temperature was 280°C; and the detector temperature was 300°C.

[0064] The injection volume is 1 μL, the split ratio is 10:1, and the theoretical plate number calculated based on the eugenol peak should be no less than 9000.

[0065] Step 4: Determination of the retention times of the chromatographic peaks of the four components in cloves

[0066] Accurately pipette the β-caryophyllene reference solution and eugenol reference solution obtained in step 1 and the clove sample solution obtained in step 2, respectively, and inject them into a gas chromatograph for determination.

[0067] The above four capillary columns can well separate the four components of β-caryophyllene, α-caryophyllene, eugenol and eugenol acetate. The average retention time of the same component on the four columns is taken as the standard retention time, which is 3.225 min for β-caryophyllene, 3.621 min for α-caryophyllene, 12.031 min for eugenol and 15.721 min for eugenol acetate, as shown in Table 1.

[0068] Table 1 Peak elution time of four components in different capillary columns (min)

[0069]

[0070] The standard retention time of the four components of β-caryophyllene, α-caryophyllene, eugenol, and eugenol acetate was used as the horizontal axis, and the measured retention time on each capillary column was used as the vertical axis to draw a standard curve. The linear equation and correlation coefficient of each capillary column are shown in Table 2. The linear graph is shown in Figure 1 , Figure 2 , Figure 3 and Figure 4 shown.

[0071] Table 2 Linear equations and correlation coefficients of retention time on different capillary columns

[0072]

[0073]

[0074] From Table 2 and Figure 1-4 It can be seen that the standard retention time and the measured retention time of the four components of β-caryophyllene, α-caryophyllene, eugenol and eugenol acetate show a good linear relationship in the four capillary columns, and the separation of the four components of β-caryophyllene, α-caryophyllene, eugenol and eugenol acetate in each capillary column shows a linear relationship.

[0075] After establishing the standard retention times of all the components to be analyzed, the retention time linear equation is used to predict the components in the sample to be analyzed.

[0076] First, β-caryophyllene, eugenol reference solution and the sample solution to be tested were measured on a PEG-20M gas chromatography column, wherein the chromatograms of the β-caryophyllene reference solution, eugenol reference solution and the sample solution to be tested are as follows: Figure 5-7 As shown, the chromatographic peak positioning was performed by the above two reference substances to obtain the actual retention times of β-caryophyllene and eugenol in the sample to be tested, which were 2.918 min and 12.039 min, respectively.

[0077] Then, using the standard retention time of β-caryophyllene and eugenol reference substances (3.225 min, 12.031 min) as the abscissa and the measured retention time (2.918 min, 12.039 min) as the ordinate, two points were obtained, and then the two-point linear equation was obtained: y=1.0358x-0.4224.

[0078] Finally, the standard retention times of α-caryophyllene and eugenol acetate peaks (3.621min and 15.721min) were substituted into the equation, and the predicted retention times of α-caryophyllene and eugenol acetate in the sample were 3.328min and 15.861min, respectively. Compared with the measured retention times (3.386min and 15.958min) obtained above, they were both within the specified range, indicating that this method is feasible.

[0079] Among them, 4 corresponding chromatographic peaks are extracted from the high performance gas chromatography of the sample to be tested as the components to be tested for determination, and the obtained chromatogram is as follows: Figure 7 shown.

[0080] Step 5: Quantitative testing

[0081] 1 μL of the eugenol reference solution obtained in step 1 and 1 μL of the clove sample solution obtained in step 2 were accurately pipetted and injected into the gas chromatograph for determination. The contents of the four components of β-caryophyllene, α-caryophyllene, eugenol, and eugenol acetate were calculated using the peak area and correction factor according to the following formulas.

[0082] Correction Factor Where f is the correction factor; A i is the peak area of ​​eugenol reference substance; A r is the peak area of ​​the reference substance of the component to be tested; C i is the concentration of eugenol reference substance; C r is the concentration of the reference substance of the component to be tested. β-石竹烯 is 0.8769,f α-石竹烯 is 0.07585, f 丁香酚 is 1.000, f 乙酸丁香酚酯 It is 1.0806.

[0083] Where, C is the concentration of the component to be measured; f is the correction factor; A is the peak area of ​​the component to be measured; A i is the peak area of ​​eugenol reference substance; C i is the concentration of the eugenol reference solution. The content of β-caryophyllene was calculated to be 2.35 mg / g, the content of α-caryophyllene was 0.247 mg / g, the content of eugenol was 12.90 mg / g, and the content of eugenol acetate was 2.99 mg / g.

[0084] Comparative Example

[0085] The method for determining the clove content in this comparative example is different from that in Example 1 in that step 1 is to mix the reference solution, which contains four components: β-caryophyllene, α-caryophyllene, eugenol, and eugenol acetate.

[0086] Step 1: Preparation of mixed reference solution

[0087] Accurately weigh 13.30 mg of α-caryophyllene reference substance, place it in a 10 mL volumetric flask, add n-hexane to dissolve and dilute to the scale, shake well, and obtain the α-caryophyllene reference substance solution; accurately weigh 10.22 mg of β-caryophyllene reference substance, 53.05 mg of eugenol, and 11.09 mg of eugenol acetate, and accurately measure 1 mL of the above α-caryophyllene reference substance solution, place it in the same 25 mL volumetric flask, add n-hexane to dissolve and dilute to the scale, shake well, and obtain the mixed reference substance solution.

[0088] Step 2: Same as Example 1.

[0089] Step 3: Same as Example 1.

[0090] Step 4: Determination of the retention times of the chromatographic peaks of the four components in cloves

[0091] 1 μL of the mixed reference solution obtained in step 1 and 1 μL of the clove sample solution obtained in step 2 were accurately pipetted and injected into the gas chromatograph for determination. In the gas chromatogram, 4 chromatographic peaks corresponding to the reference chromatogram appeared, which were used as the components to be determined.

[0092] Step 5: Quantitative testing

[0093] Accurately pipette 1 μL of the mixed reference solution obtained in step 1 and 1 μL of the clove sample solution obtained in step 2, inject them into the gas chromatograph for determination. The chromatograms are as follows: Figure 8 and Figure 7 As shown, the contents of the four components to be tested, namely β-caryophyllene, α-caryophyllene, eugenol and eugenol acetate, were calculated by peak area and external standard method.

[0094] The calculated contents of β-caryophyllene, α-caryophyllene, eugenol, and eugenol acetate were 2.30 mg / g, 0.250 mg / g, 12.90 mg / g, and 3.04 mg / g, respectively.

[0095] Comparing the contents of β-caryophyllene, α-caryophyllene, eugenol and eugenol acetate measured in Example 1 and the comparative example, the absolute errors are 0.05 mg / g, 0.003 mg / g, 0 mg / g and 0.05 mg / g respectively; the relative errors are 0.8%, 0.6%, 0% and 0.8% respectively. It can be seen that the results measured in Example 1 are consistent with the results measured in the comparative example.

[0096] While in the determination process of the comparative example, four reference substances, namely β-caryophyllene, α-caryophyllene, eugenol and eugenol acetate, are required, the embodiment only requires two reference substances, namely β-caryophyllene and eugenol. Considering the complex and unstable preparation process of the reference substances, the inspection cost can be greatly saved.

[0097] It can be seen that the method for determining the clove content provided by the present invention can not only solve the problem of a large number of reference substances required for determination in the conventional multi-index content determination method, but also solve the problem that the relative retention time of sample chromatographic peaks in different capillary columns is difficult to reproduce when using the one-measurement-multiple-evaluation method or the reference substance replacement method; at the same time, it can be well used for the quality control of cloves, ensuring the uniformity of the quality and efficacy of cloves, and at low cost.

[0098] Finally, it should be emphasized that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various changes and modifications. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for determining clove content, characterized in that: The steps include: Step 1: Preparation of reference solution: Accurately weigh reference substances β-caryophyllene and eugenol, respectively, add n-hexane to dissolve and dilute to obtain β-caryophyllene reference substance solution and eugenol reference substance solution respectively; Step 2: Preparation of sample solution to be tested: Accurately weigh the clove sample to be tested, accurately add n-hexane, and pre-treat to obtain the sample solution to be tested; Step 3, gas chromatography separation: The chromatographic conditions were as follows: the chromatographic column was a capillary column with polyethylene glycol as the stationary phase; the column temperature was 170°C; the injection port temperature was 280°C; the detector temperature was 300°C; Step 4: Determination of the chromatographic peak retention times of the four components in cloves: Accurately pipetting the β-caryophyllene reference substance solution obtained in step 1, the eugenol reference substance, and the sample solution to be tested obtained in step 2, respectively, injecting them into a gas chromatograph for determination, plotting a retention time standard curve with the standard retention time of β-caryophyllene and eugenol as the abscissa and the measured retention time of β-caryophyllene and eugenol as the ordinate, and obtaining the reference retention times of the four components of β-caryophyllene, α-caryophyllene, eugenol acetate, and eugenol, respectively; In the gas chromatogram of the clove sample to be tested, chromatographic peaks corresponding to the control retention times of the four components of β-caryophyllene, α-caryophyllene, eugenyl acetate and eugenol appeared; Step 5: Quantitative detection: Accurately pipette the eugenol reference solution obtained in step 1 and the sample solution obtained in step 2, respectively, inject them into the gas chromatograph for determination, and use the peak area and correction factor, and then calculate according to the formula The contents of the four components of β-caryophyllene, α-caryophyllene, eugenol acetate and eugenol were calculated; where C is the concentration of the component to be measured; f is the correction factor; A is the peak area of ​​the component to be measured; A i is the peak area of ​​eugenol reference substance; C i is the concentration of eugenol reference solution.

2. The method for determining clove content according to claim 1, characterized in that: In step 1, the concentration of β-caryophyllene in the β-caryophyllene reference solution is 0.4 mg / mL; the concentration of eugenol in the eugenol reference solution is 2 mg / mL.

3. The method for determining clove content according to claim 1, characterized in that: In step 2, the clove sample is pretreated before use; the weight of the clove is 0.3-0.5 g, and the volume of the n-hexane is 20-50 mL.

4. The method for determining clove content according to claim 3, characterized in that: The pretreatment is ultrasonic treatment or heating reflux, the power of the ultrasonic treatment is 500W, the frequency is 40kHz, and the time is 15min; the time of the heating reflux is 30min.

5. The method for determining clove content according to claim 1, characterized in that: In step 3, the capillary column is any one of a DB-WAX capillary column, an Rtx-WAX capillary column, and a HP-INNOWAX capillary column.

6. The method for determining clove content according to claim 5, characterized in that: The DB-WAX capillary column has a length of 30 m, an inner diameter of 0.32 mm, and a membrane thickness of 0.25 μm or 0.50 μm; the Rtx-WAX capillary column has a length of 30 m, an inner diameter of 0.32 mm, and a membrane thickness of 0.50 μm; the HP-INNOWAX capillary column has a length of 30 m, an inner diameter of 0.32 mm, and a membrane thickness of 0.50 μm.

7. The method for determining clove content according to claim 1, characterized in that: In step 5, the correction factor f is calculated according to the following formula: In the formula, f is the correction factor; A i is the peak area of ​​eugenol reference substance; A r is the peak area of ​​the reference substance of the component to be tested; C i is the concentration of eugenol reference; C r is the concentration of the reference substance of the component to be tested.

8. Application of the method for determining clove content according to any one of claims 1 to 7 in the quality control of clove medicines.

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