A method for constructing gas phase characteristic spectrum of volatile components in Banxia Baizhu Tianma decoction

Abstract

The invention belongs to the technical field of quality detection of traditional Chinese medicines, and specifically relates to a method for constructing a gas phase characteristic spectrum of volatile components in a Banxia Baizhu Tianma decoction. A test solution is determined by gas chromatography in a programmed temperature manner specified in an instruction manual, and a characteristic spectrum of volatile components in the Banxia Baizhu Tianma decoction is established, and effective separation of multiple characteristic peaks is achieved. Moreover, the obtained characteristic spectrum has a stable baseline and a good characteristic peak shape. The method is stable, reliable, durable, and highly specific, and has guiding significance for quality detection and evaluation of the variety.

Description

Technical Field

[0001] The invention belongs to the technical field of quality detection of traditional Chinese medicines, and particularly relates to a method for constructing a gas phase characteristic spectrum of volatile components in a Banxia Baizhu Tianma decoction. Background Art

[0002] Banxia Baizhu Tianma Decoction comes from "Medical Enlightenment". It is composed of 8 herbs: Banxia, ​​Tianma, Baizhu, Tangerine Peel, Poria, Licorice, Ginger, and Jujube. It has the effects of dispelling wind and resolving phlegm, strengthening the spleen and stopping dizziness. It is a classic prescription for treating dizziness, mainly suitable for dizziness caused by wind-phlegm disturbance and phlegm turbidity blocking the middle. In the prescription, Banxia dries dampness and resolves phlegm, descends adverse and stops vomiting; Tianma calms the liver and extinguishes wind and stops dizziness. The two are used together as the main medicine, which is the key medicine for treating dizziness and headache caused by wind-phlegm. Baizhu strengthens the spleen and dries dampness; Poria strengthens the spleen and penetrates dampness, and assists in treating the root of phlegm. It is combined with Banxia and Tianma to strengthen the power of resolving phlegm and calming wind, and they are all ministers. Tangerine Peel regulates qi and resolves phlegm, makes qi smooth and phlegm disappear, and is an adjuvant. Licorice is used to harmonize the various herbs, and ginger and jujube are added to harmonize the spleen and stomach. The combination of various herbs has the effects of resolving phlegm and extinguishing wind, strengthening the spleen and removing dampness.

[0003] According to literature research, the volatile components in Banxia Baizhu Tianma Decoction are directly related to the effects of the prescription in regulating qi, resolving phlegm and strengthening the spleen. The characteristic spectrum of traditional Chinese medicine refers to the spectrum of common peaks that can mark the characteristics of various component groups in the traditional Chinese medicine after proper treatment and certain analytical methods. It is a new quality control mode for traditional Chinese medicine. The authenticity of traditional Chinese medicine can be effectively identified by the characteristic characteristics of the characteristic spectrum; the quality of traditional Chinese medicine can be effectively controlled by regulating the area and proportion of the main characteristic peaks of the characteristic spectrum. With the gradual update of the version of the "Chinese Pharmacopoeia", the characteristic spectrum detection methods of multiple varieties have been added. After the characteristic spectrum is used as a control indicator, the relative stability of the overall quality of traditional Chinese medicine and Chinese patent medicine can be guaranteed.

[0004] According to literature search, there are few reports on the control of chemical components of each herb in Banxia Baizhu Tianma Decoction. Among them, the HPLC method is mainly used to control the content of each chemical component or the overall quality (fingerprint) in Banxia Baizhu Tianma Decoction, and there is a lack of control methods for volatile components in Banxia Baizhu Tianma Decoction. Banxia Baizhu Tianma Decoction is composed of 8 Chinese medicines: Pinellia, Atractylodes, Tangerine Peel, Licorice, Gastrodia, Poria, Ginger, and Jujube. This prescription contains many volatile components, and the composition of volatile components is complex, with large polarity, weak ultraviolet absorption, and low boiling point. Among them, 3 herbs contain volatile components, Atractylodes contains atractylodesone and other components, and Tangerine Peel and Ginger both contain limonene and other components. Therefore, it is urgent to establish a method for detecting or controlling volatile components in Banxia Baizhu Tianma Decoction. Summary of the invention

[0005] Therefore, the purpose of the present invention is to provide a characteristic spectrum of volatile components in Banxia Baizhu Tianma Decoction and a method for establishing the same. The method establishes a characteristic spectrum of the variety according to the characteristics of Banxia Baizhu Tianma Decoction, achieves effective separation of the characteristic peaks corresponding to the volatile components, and provides a scientific basis for comprehensively establishing the quality control standards of Banxia Baizhu Tianma Decoction.

[0006] The present invention provides a method for constructing a gas phase characteristic spectrum of volatile components in Banxia Baizhu Tianma decoction, comprising the following steps:

[0007] Preparation of test solution;

[0008] Gas chromatography detection: The test solution is measured by gas chromatography using a programmed temperature method to obtain a characteristic spectrum; the programmed temperature method includes:

[0009] 1) The initial temperature is 35-80°C and maintained for 6-10 minutes;

[0010] 2) Raise the temperature to 110-130°C at 2.8-3.2°C / min and maintain for 9-11 minutes;

[0011] 3) Raise the temperature to 140-180°C at 1-2°C / min and keep for 0-20min; or, raise the temperature to 135-140°C at 1-3°C / min and keep for 10-20min; then raise the temperature to 150-200°C at 0.5-4°C / min and keep for 0-10min;

[0012] 4) Raise the temperature to 210-230°C at 2-5°C / min and maintain for 5-10 min.

[0013] Furthermore, the method for preparing the test solution is to extract the test sample with an extractant, take the extractant layer, and obtain the test solution; preferably, the extractant is selected from ethyl acetate or n-hexane, and preferably, the volume ratio of the extractant to the test sample is 5:30-80.

[0014] Furthermore, the preparation method of the test solution comprises: mixing the test sample and ethyl acetate, extracting volatile oil by steam distillation, and collecting the ethyl acetate layer to obtain;

[0015] In order to increase the number of characteristic peaks and enrich the characteristic spectrum information, preferably, the volume ratio of the test sample to ethyl acetate is 200-2600:0.5-1.5; more preferably 400-600:0.5-1.5;

[0016] Preferably, a reflux condenser is connected during steam distillation, and the solution is heated to a slight boil and maintained for 4-6 hours;

[0017] Preferably, after collecting the ethyl acetate layer, the process further comprises the steps of diluting with ethyl acetate and / or filtering.

[0018] Furthermore, the programmed temperature rising method includes: the programmed temperature rising method includes:

[0019] The initial temperature is 70°C and maintained for 8 minutes; the temperature is increased to 120°C at 3°C / min and maintained for 10 minutes; the temperature is increased to 135-140°C at 2°C / min and maintained for 15-20 minutes; the temperature is increased to 155-200°C at 0.5-4°C / min and maintained for 0-10 minutes, and then increased to 220°C at 2-5°C / min and maintained for 5-10 minutes; or, the programmed temperature increase method includes: the initial temperature is 70°C and maintained for 8 minutes; the temperature is increased to 120°C at 3°C / min and maintained for 10 minutes; the temperature is increased to 140-180°C at 1-2°C / min and maintained for 0-20 minutes; the temperature is increased to 220°C at 3.2-5°C / min and maintained for 10 minutes.

[0020] Furthermore, the programmed temperature rising method includes:

[0021] The initial temperature was maintained at 70°C for 8 min; then increased to 120°C at 3°C / min and maintained for 10 min; increased to 140°C at 2°C / min and maintained for 15 min; increased to 200°C at 4°C / min and maintained for 10 min, then increased to 220°C at 2°C / min and maintained for 5 min.

[0022] Furthermore, during the gas chromatography detection process, the injection port temperature was 235-245° C., the detector temperature was 295-305° C., and the carrier flow rate was 1.4-1.6 ml / min.

[0023] Furthermore, the construction method also includes the step of using a solvent to dissolve at least one of limonene and atractylodesone to prepare a reference solution, and the step of detecting the reference solution by gas chromatography according to any of the above-mentioned construction methods to obtain a reference spectrum of the reference substance. Preferably, the solvent is selected from at least one of methanol, ethanol and water; and each milliliter of the reference solution contains 50-70 μg of limonene and / or 80-120 μg of atractylodesone.

[0024] Furthermore, the gas phase characteristic spectrum has 8 characteristic peaks, the peak corresponding to the limonene reference peak is the S1 peak, the peak corresponding to the atractylodes ketone reference peak is the S2 peak, the relative retention times of peaks 1 to 4 and the S1 peak, and the relative retention times of peaks 5 to 6 and the S2 peak are all within the range of ±10% of the specified values, and the specified values ​​are: 0.43 (peak 1), 0.64 (peak 2), 1.00 (peak 3), 3.14 (peak 4), 1.00 (peak 5), 1.23 (peak 6), 1.26 (peak 7), and 1.58 (peak 8).

[0025] Furthermore, the Banxia Baizhu Tianma Decoction contains Pinellia, Gastrodia, Atractylodes, Tangerine Peel, Poria, Licorice, ginger and jujube. Preferably, in parts by weight, the Banxia Baizhu Tianma Decoction contains 4.5-6.7 parts of Pinellia, 3.0-4.5 parts of Gastrodia, 9.0-13.4 parts of Atractylodes, 3.0-4.5 parts of Tangerine Peel, 3.0-4.5 parts of Poria, 1.5-2.2 parts of Licorice, 0.8-1.2 parts of ginger and 4.8-7.2 parts of jujube.

[0026] The present invention also provides a quality detection method for Banxia Baizhu Tianma Decoction, comprising the step of comparing a gas phase characteristic spectrum of a Banxia Baizhu Tianma Decoction product to be tested with a control characteristic spectrum of the Banxia Baizhu Tianma Decoction, wherein the characteristic spectrum of the Banxia Baizhu Tianma Decoction product to be tested is constructed using the Banxia Baizhu Tianma Decoction product to be tested according to any of the above-mentioned construction methods, and the control characteristic spectrum of the Banxia Baizhu Tianma Decoction is selected from any one of the following (1)-(3):

[0027] (1) It has 8 characteristic peaks, the peak corresponding to the limonene reference peak is the S1 peak, the peak corresponding to the atractylodesone reference peak is the S2 peak, the relative retention times of peaks 1 to 4 and S1 peak, and the relative retention times of peaks 5 to 6 and S2 peak are within ±10% of the specified values, and the specified values ​​are: 0.43 (peak 1), 0.64 (peak 2), 1.00 (peak 3), 3.14 (peak 4), 1.00 (peak 5), 1.23 (peak 6), 1.26 (peak 7), and 1.58 (peak 8);

[0028] (2) a characteristic spectrum of Banxia Baizhu Tianma decoction obtained by using a single batch or multiple batches of Banxia Baizhu Tianma decoction according to any of the construction methods described above;

[0029] (3) The characteristic spectra obtained by using multiple batches of Banxia Baizhu Tianma decoction according to any of the construction methods described above are used to prepare a control characteristic spectrum by using the average value or median method.

[0030] The gas chromatography method used a chromatographic column with (14%-cyanopropyl-phenyl)methylpolysiloxane as the stationary phase.

[0031] The technical solution of the present invention has the following advantages:

[0032] 1. The method for constructing the gas phase characteristic spectrum of the volatile components in the Banxia Baizhu Tianma decoction provided by the present invention is to determine the test solution by gas chromatography using the following programmed temperature increase method: 1) the initial temperature is 35-80°C and maintained for 6-10 min; 2) the temperature is increased to 110-130°C at 2.8-3.2°C / min and maintained for 9-11 min; 3) the temperature is increased to 140-180°C at 1-2°C / min and maintained for 0-20 min; or, the temperature is increased to 135-140°C at 1-3°C / min. ℃, maintain for 10-20min; then increase to 150-200℃ at 0.5-4℃ / min, maintain for 0-10min; 4) increase to 210-230℃ at 2-5℃ / min, maintain for 5-10min, and establish the characteristic spectrum of volatile components in Banxia Baizhu Tianma Decoction, achieving effective separation of multiple characteristic peaks, and the baseline of the obtained characteristic spectrum is stable and the characteristic peak shape is good. This method is stable, reliable, durable and highly specific, and has guiding significance for quality detection and evaluation of this variety.

[0033] 2. The method for constructing the gas phase characteristic spectrum of the volatile components in the Banxia Baizhu Tianma decoction provided by the present invention, wherein the programmed temperature rise method comprises: maintaining the initial temperature at 70°C for 8 min; increasing the temperature to 120°C at 3°C / min and maintaining it for 10 min; increasing the temperature to 135-140°C at 2°C / min and maintaining it for 15-20 min; increasing the temperature to 155-200°C at 0.5-4°C / min and maintaining it for 0-10 min, and then increasing the temperature to 220°C at 2-5°C / min and maintaining it for 5-10 min; or, the programmed temperature rise method comprises: maintaining the initial temperature at 70°C for 8 min; increasing the temperature to 120°C at 3°C / min and maintaining it for 10 min; increasing the temperature to 140°C at 1-2°C / min and maintaining it for 15-20 min; ~180℃, keep for 0~20min; increase to 220℃ at 3.2-5℃ / min, keep for 10min, especially adopting the programmed temperature rising method including: initial temperature 70℃ keep for 8min; increase to 120℃ at 3℃ / min, keep for 10min; increase to 140℃ at 2℃ / min, keep for 15min; increase to 200℃ at 4℃ / min, keep for 10min, and then increase to 220℃ at 2℃ / min, keep for 5min, further improve the uniformity and separation of characteristic peak distribution, realize the effective separation of citric acid and atractylodesone, accurately locate the peak positions of citric acid and atractylodesone, and fully reflect the integrity and characteristic of Banxia Baizhu Tianma Decoction.

[0034] 3. The method for constructing the gas phase characteristic spectrum of the volatile components in the Banxia Baizhu Tianma Decoction provided by the present invention can prepare the test solution by extraction and steam distillation, especially extracting the volatile components from the Banxia Baizhu Tianma Decoction by steam distillation, and then collecting them with ethyl acetate to prepare the test solution containing more volatile components; taking limonene and atractylodesone reference substances and dissolving them in methanol to prepare a reference solution; detecting by gas chromatograph to obtain a characteristic spectrum, and each chromatographic peak in the characteristic spectrum can be attributed to the prescription The three medicinal flavors of Atractylodes macrocephala, tangerine peel and ginger can effectively control the volatile components in Banxia Baizhu Tianma Decoction as a whole. The chromatographic peaks in the characteristic spectrum of volatile components of Banxia Baizhu Tianma Decoction were attributed and analyzed, and it was determined that the chromatographic peaks presented in the characteristic spectrum of volatile components of Banxia Baizhu Tianma Decoction mainly came from the three medicinal flavors of Atractylodes macrocephala, tangerine peel and ginger. Among them, characteristic peaks 1 to 4 are tangerine peel-specific components, characteristic peaks 5 to 8 are Atractylodes macrocephala-specific components, and the peak area of ​​ginger-specific components is small and was not selected as the characteristic peak. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0036] Figure 1 The spectrum obtained by the chromatographic condition optimization experiment of the single-dose decoction in Example 1 of the present invention, from bottom to top, are: blank (ethyl acetate), test solution method 1, test solution method 2, and test solution method 3;

[0037] Figure 2 The spectrum obtained by method 3 in the chromatographic condition optimization experiment of decoction of multiple prescription amounts in Example 1 of the present invention;

[0038] Figure 3 The spectrum obtained by methods 4-6 in the chromatographic condition optimization experiment of the decoction of multiple prescription amounts in Example 1 of the present invention, from bottom to top: method 6, method 5, method 4;

[0039] Figure 4 The spectra obtained by methods 7-11 in the chromatographic condition optimization experiment of decoction of multiple prescription amounts in Example 1 of the present invention are, from bottom to top: method 11, method 10, method 9, method 8, method 7.

[0040] Figure 5 The results of the solvent extraction method in Example 1 of the present invention are as follows, from bottom to top: n-hexane, ethyl acetate, methanol, atractylodesone reference solution, limonene reference solution, test sample 1, and test sample 2;

[0041] Figure 6 The results of the distillation method in Example 1 of the present invention are as follows, from bottom to top: ethyl acetate, test sample 3, atractylodesone reference solution, and limonene reference solution;

[0042] Figure 7 This is a comparison chart of the number of extractions, from bottom to top: ethyl acetate, first extraction, second extraction;

[0043] Figure 8 The chromatograms for confirmation of the chemical components of the characteristic spectrum are, from bottom to top: blank (ethyl acetate), atractylodesone reference substance, limonene reference substance, freeze-dried powder of Banxia Baizhu Tianma decoction, and decoction of Banxia Baizhu Tianma decoction;

[0044] Fig. 9 This is the spectrum of the peak attribution of the medicinal flavor of Atractylodes macrocephala, from bottom to top: blank (ethyl acetate), blank (methanol), limonene and atractylodesone mixed reference solution, Atractylodes macrocephala single flavor 21120101, Banxia Baizhu Tianma decoction (without Atractylodes macrocephala) 21120104, Banxia Baizhu Tianma decoction;

[0045] Fig.10 This is the spectrum of the peak attribution of the medicinal flavor of tangerine peel, from bottom to top: blank (ethyl acetate), blank (methanol), mixed reference solution of limonene and atractylodesone, tangerine peel alone 21113003, Banxia Baizhu Tianma decoction (lacking tangerine peel) 21113006, Banxia Baizhu Tianma decoction;

[0046] Fig.11 The attribution spectrum of the peaks of ginger flavor, from bottom to top, are: blank (ethyl acetate), blank (methanol), mixed reference solution of limonene and atractylodesone, ginger alone 21120102, Banxia Baizhu Tianma decoction (lacking ginger) 21120105, Banxia Baizhu Tianma decoction;

[0047] Fig.12 This is a specificity comparison chart, from bottom to top: blank (ethyl acetate), blank (methanol), limonene and atractylodesone mixed reference solution, Banxia Baizhu Tianma decoction 21121001;

[0048] Fig.13 This is a stability test comparison chart, from bottom to top: blank, 0h, 7h, 14h, 21h, 29h;

[0049] Fig.14 This is a comparison chart of the carrier gas flow rate, from bottom to top: the carrier gas flow rate is 1.4ml / min, 1.6ml / min, 1.5ml / min;

[0050] Fig.15This is a comparison chart of the detector temperature investigation, from bottom to top: detector temperature 300℃, 295℃, 305℃;

[0051] Fig.16 This is a comparison chart of injection port temperature investigation, from bottom to top: injection port temperature 240℃, 235℃, 245℃;

[0052] Fig.17 This is a comparison chart of the durability of different instruments and chromatographic columns. S1 to S3 are: Thermo Fisher instrument-chromatographic column FX-GC-007, Thermo Fisher instrument-chromatographic column FX-186, Agilent instrument-chromatographic column FX-GC-007;

[0053] Fig.18 The results of the characteristic spectrum test of volatile components of 15 batches of Banxia Baizhu Tianma decoction were compared;

[0054] Fig.19 Fitting reference characteristic spectra of volatile components of 15 batches of Banxia Baizhu Tianma decoction. DETAILED DESCRIPTION

[0055] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0056] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0057] Example 1 Construction of characteristic spectrum

[0058] 1. Investigation of chromatographic conditions

[0059] (1) Drug testing

[0060] The decoction of Banxia Baizhu Tianma decoction is prepared by the existing conventional method. For example, the decoction of Banxia Baizhu Tianma decoction in the present invention can be prepared by the following prescription and method, prescription: 5.60g of Qing Banxia, ​​3.73g of Tianma, 11.19g of Baizhu, 3.73g of Poria, 3.73g of Tangerine peel, 1.87g of Licorice, 1.0g of Ginger, 6.0g of Jujube. Preparation method: add water to the above medicinal flavors and boil twice, add 12 times the amount of water for the first time and boil for 30 minutes, add 10 times the amount of water for the second time and boil for 25 minutes, filter the decoction, combine the filtrate, and obtain. The multiple refers to the number of milliliters of water added to each gram of medicinal material.

[0061] (2) Optimization of chromatographic conditions for single-dose decoction

[0062] Preparation of test solution: Take 260ml of Pinellia, Atractylodes and Gastrodia decoction, put it in a round-bottom flask, connect it to a volatile oil analyzer, add water from the upper end of the analyzer to the scale and overflow into the flask, then add 1ml of ethyl acetate, connect a reflux condenser, heat to a slight boil, and keep it for 5 hours, cool, take 0.5ml of the ethyl acetate layer, add 1ml of ethyl acetate to dilute, mix well and pass it through a 0.45μm microporous filter membrane, take the filtrate, and obtain the test solution.

[0063] The test solution and the blank (ethyl acetate) were subjected to gas chromatography detection using an Agilent chromatographic column (DB-1701 capillary column [14% -cyanopropyl-phenylmethylpolysiloxane as stationary phase], column length 30m, inner diameter 0.32mm, film thickness 0.25μm), the test solution was tested according to the chromatographic conditions of Methods 1-3 in Table 1, and the blank (ethyl acetate) was tested according to the chromatographic conditions of Method 3 in Table 1, and the injection volume was 1 μl.

[0064] Table 1 Optimization of chromatographic conditions for decoction with single-dose prescription

[0065]

[0066] Table 2 Chromatographic peak information of method 1

[0067]

[0068]

[0069] Table 3 Chromatographic peak information of method 2

[0070]

[0071] Table 4 Chromatographic peak information of method 3

[0072]

[0073]

[0074] After the above three methods were used to test the test products, the comparison chart is shown in Figure 1 As shown. Methods 1-3 have fewer characteristic peaks and insufficient peak information, and are unable to identify the active ingredients limonene and atractylodesone. Compared with methods 1 and 2, the distribution of the larger chromatographic peaks in the chromatogram of the test sample in method 3 is more uniform, the separation is better, and the total detection time is shorter, saving detection time, so it will continue to be optimized in the future.

[0075] (3) Optimization of chromatographic conditions for decoctions with multiple prescription amounts

[0076] Preparation of multiple prescription decoction: Prescription: 5.60g of Pinellia ternata, 3.73g of Gastrodia elata, 11.19g of Atractylodes macrocephala, 3.73g of Poria cocos, 3.73g of Citrus aurantium, 1.87g of Licorice root, 1.0g of Ginger, 6.0g of Jujube. Preparation method: Add water to the above medicinal ingredients and boil them twice. Boil them for 30 minutes with 12 times the amount of water for the first time and for 25 minutes with 10 times the amount of water for the second time. Filter the decoction and combine the filtrate to obtain 1 portion of decoction. Prepare 5 portions in the same way and mix them well.

[0077] Preparation of test solution: Take 2550ml of Pinellia, Atractylodes and Gastrodia decoction, place it in a round-bottom flask, connect it to a volatile oil analyzer, add water from the top of the analyzer to the scale and overflow into the flask, then add 1ml of ethyl acetate, connect a reflux condenser, heat to a slight boil, and keep it for 5 hours, cool, and separate the ethyl acetate layer to obtain the test solution.

[0078] Gas chromatography was performed using an Agilent column (DB-1701 capillary column [14% -cyanopropyl-phenylmethylpolysiloxane as stationary phase], column length 30 m, inner diameter 0.32 mm, film thickness 0.25 μm), and the tests were performed according to the chromatographic conditions of Method 3 in Table 1 and the chromatographic conditions of Table 2, respectively, with an injection volume of 1 μL.

[0079] Table 5 Optimization of chromatographic conditions for decoctions with multiple prescription amounts

[0080]

[0081]

[0082] Table 6 Chromatographic peak information of method 7

[0083]

[0084]

[0085]

[0086] Table 7 Chromatographic peak information of method 8

[0087]

[0088]

[0089]

[0090] Table 8 Chromatographic peak information of method 9

[0091]

[0092]

[0093]

[0094] Table 9 Chromatographic peak information of method 10

[0095]

[0096]

[0097]

[0098] Table 10 Method 11 Chromatographic peak information

[0099]

[0100]

[0101]

[0102] The chromatogram obtained by method 3 is shown in Figure 2 As shown, the chromatogram obtained by method 4-6 is as follows Figure 3 As shown, the chromatogram obtained by method 7-11 is shown in Figure 2 As shown. The results show that the chromatographic peaks obtained by method 3 are concentrated between 30-35min, with poor separation and poor distribution uniformity of each chromatographic peak in the characteristic spectrum. After the improvement of methods 4-6, the retention time of the chromatographic peaks originally concentrated at 30-35min was extended to 40-60min, and the separation was improved to a certain extent, but the heating rate still needs to be further reduced to separate these chromatographic peaks in order to improve the separation of the chromatographic peaks and the uniformity of the distribution of the chromatographic peaks. It has been found that when the initial temperature is increased to 70°C, the solvent peak can be eluted at 0-5min, and the limonene peak position is around 10min. Therefore, the initial temperature is preferably 70°C. When the final temperature is raised to 220°C, there is basically no obvious chromatographic peak, so the final temperature is preferably 220°C.

[0103] Comparing method 7-11 with method 3-6, it can be seen that method 7-11 further reduces the heating rate of 120℃~180℃, which can improve the separation of more chromatographic peaks in the middle of the spectrum. The uniformity of chromatographic peak distribution in method 7-11 is significantly improved. From the perspective of the overall chromatographic peak distribution, the above method 11 is the optimal method for the detection of volatile components in Banxia Baizhu Tianma Decoction.

[0104] 2. Investigation of extraction methods

[0105] (1) Investigation of the extraction method of the test sample

[0106] The volatile components in Banxia Baizhu Tianma decoction were extracted by solvent extraction and distillation respectively.

[0107] ①Solvent extraction method: Take 2 portions of Banxia Baizhu Tianma decoction, 50 ml each, extract one portion with 5 ml of n-hexane once, collect the n-hexane layer to obtain sample 1; extract one portion with 5 ml of ethyl acetate once, collect the ethyl acetate layer to obtain sample 2.

[0108] ②Distillation method: Take 510ml of the decoction of Pinellia, Atractylodes and Gastrodia elata (equivalent to the single prescription amount), place it in a round-bottom flask, connect it to a volatile oil measuring instrument, add water from the upper end of the measuring instrument until it reaches the scale and overflows into the flask, then add 1ml of ethyl acetate, connect a reflux condenser, heat to a slight boil, and keep it for 5 hours, cool, take about 0.5ml of the ethyl acetate layer, add 1ml of ethyl acetate to dilute, mix well and pass it through a 0.45μm microporous filter membrane, take the filtrate to obtain the test sample 3.

[0109] Limonene reference solution: Take an appropriate amount of limonene reference, weigh accurately, and add methanol to make a solution containing 0.06 mg of limonene per 1 ml.

[0110] Atractylodes ketone reference solution: Take an appropriate amount of atractylodes ketone reference, weigh it accurately, and add methanol to make a solution containing 0.1 mg of atractylodes ketone per 1 ml.

[0111] The above three test samples, solvents n-hexane, ethyl acetate, methanol, and atractylodes ketone reference solution and limonene reference solution were tested under the chromatographic conditions of method 11 in this embodiment, and the results are as follows: Figure 5-6 The results showed that after removing the chromatographic peaks in the blank solvent, there were fewer chromatographic peaks in the n-hexane extract (test sample 1), indicating that the ingredients in Banxia Baizhu Tianma decoction had low solubility in n-hexane; in contrast, there were more chromatographic peaks in the ethyl acetate extract (test sample 2), among which the reference sample was used to locate and determine that limonene was present; Figure 6 It can be seen that compared with the extraction method, the sample 3 obtained after distillation contains more volatile substances. After positioning with the reference substance, it can be determined that it contains limonene and atractylodesone.

[0112] (2) Investigation of the number of extractions of the test sample

[0113] The above results confirmed that the distillation method can better achieve the purpose of extracting and enriching the volatile components in Banxia Baizhu Tianma Decoction. In order to investigate whether the volatile components in the decoction were completely extracted, the number of extractions was investigated.

[0114] Preparation of test solution: Take 510ml of Banxia Baizhu Tianma decoction (equivalent to the single prescription amount), put it in a round-bottom flask, connect it to a volatile oil analyzer, add water from the upper end of the analyzer to the scale and overflow into the flask, then add 1ml of ethyl acetate, connect a reflux condenser, heat to a slight boil, and keep it for 5 hours, let it cool, and separate the ethyl acetate layer to obtain the first extraction test sample; use the remaining decoction after the above extraction as the raw material, extract it for the second time in the same way, and obtain the second extraction test sample. The test sample obtained by the second extraction was tested under the chromatographic conditions of method 11 in this embodiment, and the results are shown in Tables 11 and 11 below. Figure 7 shown.

[0115] Table 11 Results of the investigation on the number of extractions

[0116]

[0117] Calculation formula: Peak area decrease ratio % = (peak area of ​​the first extraction - peak area of ​​the second extraction) / peak area of ​​the first extraction * 100%

[0118] From the above results, it can be seen that the peak area of ​​the known components in the volatile oil obtained by distilling the decoction after volatile oil extraction decreased significantly. Limonene could no longer be detected in the second extraction, and the peak area of ​​atractylodesone decreased by 91.89% compared with the first extraction, indicating that these two components were basically extracted completely during the 5-hour extraction process; however, some chromatographic peaks were still detected in the characteristic spectrum of the second extraction result (such as Figure 7 The possible reasons are: ① the content of this component is relatively high and it cannot be completely extracted in one extraction; ② this component may be converted from other components in the decoction, and continuous heating during the extraction may lead to continued conversion reaction.

[0119] (3) Determined method for preparing test solution

[0120] Take about 500ml of the decoction of Pinellia, Atractylodes and Gastrodia elata (equivalent to the single prescription amount), put it in a round-bottom flask, connect it to a volatile oil measuring instrument, add water from the upper end of the measuring instrument until it reaches the scale and overflows into the flask, then add 1ml of ethyl acetate, connect a reflux condenser, heat to a slight boil, and keep it for 5 hours, cool it, separate the ethyl acetate layer, filter it through a 0.45μm microporous filter membrane, and take the filtrate to obtain the product.

[0121] 3. Chemical composition confirmation and peak attribution

[0122] (1) Chemical composition confirmation

[0123] Banxia Baizhu Tianma Decoction: Prescription: Banxia 5.60g, Tianma 3.73g, Baizhu 11.19g, Fuling 3.73g, Tangerine 3.73g, Licorice 1.87g, Ginger 1.0g, Jujube 6.0g. Preparation: Add water to the above medicinal ingredients and boil twice. The first time, add 12 times the amount of water and boil for 30 minutes. The second time, add 10 times the amount of water and boil for 25 minutes. Filter the decoction and combine the filtrate to obtain 1 portion of decoction. Prepare 5 portions in the same way and mix well.

[0124] Freeze-dried powder of Banxia Baizhu Tianma decoction: Prescription: 5.60g of Banxia, ​​3.73g of Tianma, 11.19g of Baizhu, 3.73g of Fuling, 3.73g of Tangerine peel, 1.87g of Licorice, 1.0g of Ginger, 6.0g of Jujube. Preparation method: Add water to the above medicinal flavors and decoct twice. The first time, add 12 times the amount of water and decoct for 30 minutes, and the second time, add 10 times the amount of water and decoct for 25 minutes. Filter the decoction, combine the filtrate, and obtain the decoction. The obtained decoction is freeze-dried in a vacuum freeze dryer.

[0125] The test solution of the decoction of Banxia, ​​Baizhu and Tianma: take the decoction of Banxia, ​​Baizhu and Tianma prepared above and place it in a round-bottom flask, connect it to a volatile oil measuring instrument, add water from the upper end of the measuring instrument until it reaches the scale and overflows into the flask, then add 1 ml of ethyl acetate, connect a reflux condenser, heat to a slight boil, and keep it for 5 hours, cool, take the ethyl acetate solution, filter it through a 0.45 μm microporous filter membrane, and take the filtrate to obtain the test solution.

[0126] The test solution of the lyophilized powder of the decoction of Pinellia, Atractylodes and Gastrodia: Place the lyophilized powder of the decoction of Pinellia, Atractylodes and Gastrodia prepared above in a round-bottom flask, add 500 ml of water, shake to mix, connect the volatile oil analyzer, add water from the upper end of the analyzer to the scale and overflow into the flask, then add 1 ml of ethyl acetate, connect a reflux condenser, heat to a slight boil, and keep it for 5 hours, cool, separate the ethyl acetate solution, pass it through a 0.45 μm microporous filter membrane, and take the filtrate to obtain the solution.

[0127] Limonene reference solution: Take an appropriate amount of limonene reference, weigh accurately, and add methanol to make a solution containing 0.06 mg of limonene per 1 ml.

[0128] Atractylodes ketone reference solution: Take an appropriate amount of atractylodes ketone reference, weigh it accurately, and add methanol to make a solution containing 0.1 mg of atractylodes ketone per 1 ml.

[0129] The decoction of Banxia Baizhu Tianma decoction and the freeze-dried powder were tested with the limonene and atractylodesone reference solutions according to the detection method 11 under item 1 (3) of Example 1. The results are as follows: Figure 8 The test sample contained chromatographic peaks with the same retention time as the limonene and atractylodesone reference substances, indicating that the volatile components of Banxia Baizhu Tianma Decoction contained limonene and atractylodesone.

[0130] (2) Chromatographic peak attribution

[0131] Atractylodes macrocephala single ingredient: Prescription: Atractylodes macrocephala 11.19g. Preparation method: Same as the preparation method of Banxia Baizhu Tianma decoction.

[0132] Banxia Baizhu Tianma Decoction (without Baizhu): Prescription: 5.60g of Banxia, ​​3.73g of Tianma, 3.73g of Fuling, 3.73g of Tangerine Peel, 1.87g of Licorice, 1.0g of Ginger, 6.0g of Jujube. Preparation method: the same as that of Banxia Baizhu Tianma Decoction.

[0133] Tangerine peel single ingredient: Prescription: Tangerine peel 3.73g. Preparation method: Same as the preparation method of Banxia Baizhu Tianma decoction.

[0134] Banxia Baizhu Tianma Decoction (without orange peel): Prescription: 5.60g of Banxia, ​​3.73g of Tianma, 11.19g of Baizhu, 3.73g of Fuling, 1.87g of Licorice, 1.0g of Ginger, 6.0g of Jujube. Preparation method: the same as that of Banxia Baizhu Tianma Decoction.

[0135] Ginger single ingredient: Prescription: 1.0g ginger. Preparation method: Same as the preparation method of Banxia Baizhu Tianma decoction.

[0136] Banxia Baizhu Tianma Decoction (without ginger): Prescription: Banxia 5.60g, Tianma 3.73g, Baizhu 11.19g, Fuling 3.73g, Tangerine peel 3.73g, Licorice 1.87g, Jujube 6.0g. Preparation method: the same as that of Banxia Baizhu Tianma Decoction.

[0137] Banxia Baizhu Tianma Decoction: Prescription: Banxia 5.60g, Tianma 3.73g, Baizhu 11.19g, Fuling 3.73g, Tangerine peel 3.73g, Licorice 1.87g, Ginger 1.0g, Jujube 6.0g. Preparation method: Add water to the above medicinal ingredients and boil twice. The first time, add 12 times the amount of water and boil for 30 minutes. The second time, add 10 times the amount of water and boil for 25 minutes. Filter the decoction and combine the filtrate to obtain 1 portion of decoction. Prepare 5 portions in the same way and mix well.

[0138] Mixed reference solution of limonene and atractylodesone: Take appropriate amount of limonene and atractylodesone reference substances, weigh accurately, add methanol to prepare a mixed solution containing 0.06 mg of limonene and 0.1 mg of atractylodesone per 1 ml.

[0139] Take Banxia Baizhu Tianma Decoction, Baizhu single ingredient, Tangerine peel single ingredient, ginger single ingredient, Banxia Baizhu Tianma Decoction (lacking Baizhu), Banxia Baizhu Tianma Decoction (lacking Tangerine peel) and Banxia Baizhu Tianma Decoction (lacking ginger) and prepare Banxia Baizhu Tianma Decoction volatile oil (whole recipe volatile oil), Baizhu single ingredient volatile oil, Tangerine peel single ingredient volatile oil, ginger single ingredient volatile oil and Banxia Baizhu Tianma Decoction (lacking Baizhu) test sample solution, Banxia Baizhu Tianma Decoction (lacking Tangerine peel) test sample solution and Banxia Baizhu Tianma Decoction (lacking ginger) test sample solution according to item 2 (3) of Example 1, and then test these solutions with ethyl acetate, methanol, limonene and atractylodesone mixed reference solution according to the detection method 11 under item 1 (3) of Example 1, and the results are as follows: Figures 9 to 11 .

[0140] contrast Fig. 9 and 11 It can be seen that the chromatographic peaks presented in the characteristic spectrum of the volatile components of Banxia Baizhu Tianma Decoction mainly come from the three medicinal flavors of Baizhu, Tangerine Peel and Ginger.

[0141] There are many chromatographic peaks in the volatile oil of Atractylodes macrocephala alone, mainly concentrated in 35-100 min; comparing the results of the missing and whole formulas, there are obvious differences in the number of chromatographic peaks and responses between the two, among which peak group 1, peak group 2, peak group 3, peak group 4, etc. are only present in the whole formula and single ingredient, indicating that these chromatographic peaks are all specific components of Atractylodes macrocephala, and peak group 2 contains atractylodesone.

[0142] The chromatographic peaks presented in the single-flavor volatile oil of tangerine peel are mainly concentrated in 0-35min, including limonene. Comparing the results of the missing flavor and the whole formula, there are certain differences in the number of chromatographic peaks and responses between the two. There is no obvious chromatographic peak at the position of limonene in the missing tangerine peel, indicating that limonene in the characteristic spectrum of the volatile oil of Banxia Baizhu Tianma Decoction is mainly provided by tangerine peel; in addition, peaks 1, 2, and 3 are present in the whole formula and the single missing flavor test sample, indicating that the component is provided by tangerine peel, but it is not a specific component of tangerine peel; and peaks 4 and 5 are only present in the single flavor and the whole formula, indicating that these two components are specific components of tangerine peel.

[0143] The chromatographic peaks of ginger volatile oil are mainly concentrated in 0-30min. Comparing the results of the whole formula with the lack of flavor, peak 1 and peak 2 only appear in the whole formula and the single flavor, indicating that this component is a specific component of ginger.

[0144] 4. Construction of characteristic spectra of volatile components of 15 batches of Banxia Baizhu Tianma decoction

[0145] Take 15 batches of Banxia Baizhu Tianma decoction, and test the characteristic spectrum of volatile components according to the determined detection method. The results are compared as follows Fig.18. Visual analysis of the number of chromatographic peaks presented in the characteristic spectra of each batch showed no significant difference, but the chromatographic peak response was significantly different. The difference between batches was obvious when evaluated by fingerprint similarity or relative peak area of ​​characteristic peaks. The reason is that it is difficult to perform quantitative control during the preparation of the test solution of this detection method, so it has a greater impact on the peak area of ​​the test result. The volatile oil of Banxia Baizhu Tianma Decoction is a complex mixed substance. Qualitative control of the types of components it contains can more comprehensively evaluate its quality. Therefore, it was preliminarily determined that the relative retention time of the characteristic peaks was used as the evaluation index.

[0146] The AIA data of the characteristic spectrum detection of volatile components of 15 batches of Banxia Baizhu Tianma Decoction were imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012.130723 version, National Pharmacopoeia Committee)" computing software, and the parameters were set as follows: data clipping 0-3min solvent peak, S1 as reference spectrum, generation method as median, time window as 0.1; multi-point correction (3), full peak matching of peaks with peak area accounting for more than 0% of the total peak area, and 15 batches of Banxia Baizhu Tianma Decoction volatile component fitting control characteristic spectrum were generated, and the common peaks were matched. The common peaks with a total peak area of ​​more than 2.5% in the control characteristic spectrum were selected as characteristic peaks, marked as peaks 1 to 8 in the figure below, and the durability of the volatile component characteristic spectrum was evaluated. According to the test results, the relative retention time of each characteristic peak in the characteristic spectrum obtained by different instruments and chromatographic columns did not exceed ±10% of the mean. Therefore, the specified value in the proposed quality standard is the mean under the durability item, that is, 8 characteristic peaks should be presented in the characteristic spectrum of the test sample. The peak corresponding to the limonene reference peak is the S1 peak, and the peak corresponding to the atractylodes ketone reference peak is the S2 peak. The relative retention time of peaks 1 to 4 and S1 peak, and the relative retention time of peaks 6 to 8 and S2 peak are calculated. The relative retention time should be within the range of ±10% of the specified value. The specified values ​​are: 0.43 (peak 1), 0.64 (peak 2), 3.14 (peak 4), 1.23 (peak 6), 1.26 (peak 7), and 1.58 (peak 8).

[0147] The relative retention time of each characteristic peak in the characteristic spectra of volatile components of 15 batches of Banxia Baizhu Tianma Decoction benchmark samples was statistically analyzed. The results are shown in the following table. The RSD value of the relative retention time of each characteristic peak in the characteristic spectra of 15 batches of benchmark samples did not exceed 0.1%. The differences between the characteristic spectra of each batch of benchmark samples evaluated by relative retention time were small.

[0148] Table 12 Relative retention time of characteristic spectra of 15 batches of reference samples

[0149]

[0150] Example 2 Methodological Investigation

[0151] Methodological validation was carried out in accordance with the guidelines for validation of analytical methods for pharmaceutical quality standards in General Chapter 9101 of the 2020 edition of the "Chinese Pharmacopoeia" and the technical requirements for research and formulation of quality standards for traditional Chinese medicines in the "Chinese Pharmacopoeia". The results showed that the method had good specificity and durability, and can be used for characteristic spectrum detection of benchmark samples of Banxia Baizhu Tianma Decoction.

[0152] A. Exclusiveness Investigation

[0153] Take the decoction of Banxia Baizhu Tianma decoction, prepare the test solution according to the determined method, and inject it with blank solvent (ethyl acetate, methanol) and mixed reference solution of limonene and atractylodesone respectively for detection. The results are as follows Fig.12 The blank solvent did not produce a peak at the corresponding position of the test sample chromatogram, but peaks with the same retention time as limonene and atractylodesone were found in the test sample chromatogram, indicating that this method has good specificity.

[0154] B. Durability test

[0155] a. Solution stability

[0156] Take the Banxia Baizhu Tianma decoction, prepare the test solution according to the method determined under item 2 (3) of Example 1, and inject and detect according to method 11 under item 1 (3) of Example 1 at 0h, 7h, 14h, 21h, and 29h, and record the chromatogram. Statistically calculate the retention time of characteristic peaks 1 to 8, take peak 3 (limonene) as S1, and calculate the relative retention time of peaks 1 to 4; take peak 5 (atractylodes ketone) as S2, and calculate the relative retention time of peaks 6 to 8, as shown in Figure 1. Fig.13 shown.

[0157] Results: The relative retention time RSD values ​​of the eight characteristic peaks determined in Experimental Example 1 in the characteristic spectra measured at different time points were all less than 0.2%, and the relative peak area RSD values ​​were all less than 3.9%, indicating that the test solution was stable within 29 hours.

[0158] b. Carrier gas flow rate

[0159] The effect of small changes in the carrier gas flow rate on the characteristic spectrum measurement results was investigated.

[0160] Take the Banxia Baizhu Tianma decoction, prepare the test solution according to the method determined in item 2 (3) of Example 1, set the carrier gas flow rate in the chromatographic conditions to 1.5 ± 1 ml per minute, and the other chromatographic conditions are all sampled and tested according to method 11 under item 1 (3) of Example 1, and record the chromatogram. Statistically calculate the retention time of characteristic peaks 1 to 8, take peak 3 (limonene) as S1, calculate the relative retention time of peaks 1 to 4; take peak 5 (atractylodes ketone) as S2, calculate the relative retention time of peaks 6 to 8, as shown in Figure 1. Fig.14 shown.

[0161] Results: In the characteristic spectra obtained by detection at different carrier gas flow rates, the relative retention time RSD values ​​of the 8 characteristic peaks determined in Experimental Example 1 were all less than 2.4%, and the relative peak area RSD values ​​were all less than 2.0%, indicating that the method has good durability when the carrier gas flow rate varies in the range of 1.4 ml to 1.6 ml per minute.

[0162] c. Detector temperature

[0163] The influence of small changes in detector temperature on characteristic spectrum measurement results in the investigation method was investigated.

[0164] Take the Banxia Baizhu Tianma decoction, prepare the test solution according to the method determined in item 2 (3) of Example 1, set the detector temperature in the chromatographic conditions to 300 ± 5 ° C, and the other chromatographic conditions are all sampled and tested according to method 11 in item 1 (3) of Example 1, and record the chromatogram. Statistically calculate the retention time of characteristic peaks 1 to 8, take peak 3 (limonene) as S1, and calculate the relative retention time of peaks 1 to 4; take peak 5 (atractylodes ketone) as S2, and calculate the relative retention time of peaks 6 to 8, such as Fig.15 shown.

[0165] Results: In the characteristic spectra obtained by detection at different detector temperatures, the relative retention time RSD values ​​of the 8 characteristic peaks determined in Experimental Example 1 were all less than 0.1%, and the relative peak area RSD values ​​were all less than 3.1%, indicating that the method has good durability when the detector temperature varies in the range of 295℃~305℃.

[0166] d. Inlet temperature

[0167] The influence of small changes in the injection port temperature on the characteristic spectrum measurement results was investigated.

[0168] Take the Banxia Baizhu Tianma decoction, prepare the test solution according to the method determined in item 2 (3) of Example 1, set the injection port temperature in the chromatographic conditions to 240 ± 5 ° C, and the other chromatographic conditions are all sampled and tested according to method 11 in item 1 (3) of Example 1, and record the chromatogram. Statistical retention time of characteristic peaks 1 to 8, taking peak 3 (limonene) as S1, calculate the relative retention time of peaks 1 to 4; taking peak 5 (atractylodes ketone) as S2, calculate the relative retention time of peaks 6 to 8, such as Fig.16 shown.

[0169] Results: In the characteristic spectra obtained by detecting different injection port temperatures, the relative retention time RSD values ​​of the 8 characteristic peaks determined in Experimental Example 1 were all less than 0.1%, and the relative peak area RSD values ​​were all less than 2.4%, indicating that the method has good durability when the injection port temperature varies within the range of 235℃~245℃.

[0170] e. Durability of different numbered columns and different brands of instruments

[0171] The effects of using instruments of different brands and chromatographic columns of the same brand with different numbers on the characteristic spectra were investigated.

[0172] The decoction of Banxia Baizhu Tianma decoction was used to prepare a test solution according to the method determined under item 2(3) of Example 1. According to the chromatographic conditions of method 11 under item 1(3) of Example 1, the same instrument was used to examine the effects of different brands of instruments (Thermo Fisher Trace 1300; Agilent GC-7890B) on the characteristic spectrum with different chromatographic columns of the same brand (Agilent DB-1701 capillary column, 30m*0.32mm, 0.25μm, SN: US0363121H; Agilent DB-1701 capillary column, 30m*0.32mm, 0.25μm, SN: UST283827H) and the same chromatographic column was used to examine the effects of different brands of instruments (Thermo Fisher Trace 1300; Agilent GC-7890B) on the characteristic spectrum, and the chromatograms were recorded. The characteristic spectrum AIA data detected by different instruments and chromatographic columns were imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012.130723 version, National Pharmacopoeia Committee)" computing software, and the parameters were set as follows: data clipping 0-4min solvent peak, S1 as reference spectrum, generation method as average, time window as 0.1; multi-point correction (15), full peak matching of peaks with peak area accounting for more than 0% of the total peak area, and generating a comparison chart of durability inspection results. The retention time of characteristic peaks 1 to 8 was statistically calculated, with peak 3 (limonene) as S1, and the relative retention time of peaks 1 to 4 was calculated; with peak 5 (atractylodes ketone) as S2, the relative retention time of peaks 6 to 8 was calculated, such as Fig.17 shown.

[0173] Results: The RSD values ​​of the relative retention times of the eight characteristic peaks determined in Experimental Example 1 in the characteristic spectra of instruments of different brands and chromatographic columns of different numbers were all less than 1.2%. Affected by different instruments and chromatographic columns, the relative peak areas of individual characteristic peaks differed greatly, but did not affect the identification of the overall characteristic peaks, indicating that instruments of different brands and chromatographic columns of different numbers had little effect on the characteristic spectrum results.

[0174] C. Determination of the number of theoretical plates

[0175] The theoretical plate numbers of the limonene peak in each characteristic spectrum under the durability of different instruments and columns were calculated respectively. The theoretical plate number of the Thermo Fisher instrument-column 2 was 90685, the theoretical plate number of the Thermo Fisher instrument-column 1 was 88834, and the theoretical plate number of the Agilent instrument-column 2 was 80182. It can be seen that the theoretical plate numbers of the limonene peak under the determined detection method are all greater than 80000. It is tentatively decided that a decrease of 50% in the theoretical plate number is the minimum standard to determine the limit of the theoretical plate number, that is, calculated according to the limonene peak, the theoretical plate number should not be less than 40000.

[0176] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for constructing a gas phase characteristic spectrum of volatile components in Banxia Baizhu Tianma decoction, characterized in that: The steps include: Preparation of test solution; Gas chromatography detection: gas chromatography is used to measure the test solution by programmed temperature to obtain a characteristic spectrum; the programmed temperature method includes: 1) The initial temperature is 35~80℃ and maintained for 6~10 minutes; 2) Raise the temperature to 110-130°C at 2.8-3.2°C / min and maintain for 9-11 minutes; 3) Raise the temperature to 140~180℃ at 1~2℃ / min and keep it for 0~20min; or, raise the temperature to 135~140℃ at 1~3℃ / min and keep it for 10~20min; then raise the temperature to 150~200℃ at 0.5~4℃ / min and keep it for 0~10min; 4) Raise the temperature to 210-230℃ at 2-5℃ / min and maintain for 5-10min; According to the weight percentage, the Banxia Baizhu Tianma decoction contains 4.5-6.7 parts of Banxia, ​​3.0-4.5 parts of Tianma, 9.0-13.4 parts of Baizhu, 3.0-4.5 parts of Tangerine peel, 3.0-4.5 parts of Poria, 1.5-2.2 parts of Licorice, 0.8-1.2 parts of ginger and 4.8-7.2 parts of jujube; the gas phase characteristic spectrum has 8 characteristic peaks corresponding to the peaks of the limonene reference substance The peak corresponding to the atractylodes ketone reference peak is the S1 peak, and the peak corresponding to the atractylodes ketone reference peak is the S2 peak. The relative retention times of peaks 1 to 4 and S1 peak, as well as the relative retention times of peaks 5 to 6 and S2 peak are all within ±10% of the specified values. The specified values ​​are: peak 1 is 0.43, peak 2 is 0.64, peak 3 is 1.00, peak 4 is 3.14, peak 5 is 1.00, peak 6 is 1.23, peak 7 is 1.26, and peak 8 is 1.

58.

2. The method for constructing the gas phase characteristic spectrum of the volatile components in the Banxia Baizhu Tianma decoction according to claim 1, characterized in that: The preparation method of the test sample solution is to extract the test sample with an extractant, and take the extractant layer to obtain the test sample solution.

3. The method for constructing the gas phase characteristic spectrum of the volatile components in Banxia Baizhu Tianma decoction according to claim 2, characterized in that: The extractant is selected from ethyl acetate or n-hexane.

4. The method for constructing the gas phase characteristic spectrum of the volatile components in Banxia Baizhu Tianma decoction according to claim 2, characterized in that: The volume ratio of the extractant to the test sample is 5:30-80.

5. The method for constructing the gas phase characteristic spectrum of the volatile components in Banxia Baizhu Tianma decoction according to claim 1, characterized in that: The preparation method of the test solution comprises: mixing the test sample and ethyl acetate, extracting volatile oil by steam distillation, and collecting the ethyl acetate layer to obtain; The volume ratio of the test sample to ethyl acetate is 200-2600: 0.5-1.5; During steam distillation, connect a reflux condenser and heat the solution to a slight boil, maintaining this temperature for 4-6 hours. After collecting the ethyl acetate layer, the method further comprises the steps of adding ethyl acetate to dilute and / or filtering.

6. The method for constructing the gas phase characteristic spectrum of the volatile components in Banxia Baizhu Tianma decoction according to claim 1, characterized in that: The volume ratio of the test sample to ethyl acetate is 400-600: 0.5-1.

5.

7. The method for constructing the gas phase characteristic spectrum of the volatile components in the Banxia Baizhu Tianma decoction according to any one of claims 1 to 6, characterized in that: The programmed temperature rising method includes: Initial temperature 70℃, hold for 8min; increase to 120℃ at 3℃ / min, hold for 10min; increase to 135~140℃ at 2℃ / min, hold for 15~20min; increase to 155-200℃ at 0.5-4℃ / min, hold for 0-10min, then increase to 220℃ at 2-5℃ / min, hold for 5-10min; or, The programmed temperature rising method includes: maintaining the initial temperature at 70°C for 8 minutes; increasing the temperature to 120°C at 3°C / min and maintaining it for 10 minutes; increasing the temperature to 140-180°C at 1-2°C / min and maintaining it for 0-20 minutes; increasing the temperature to 220°C at 3.2-5°C / min and maintaining it for 10 minutes.

8. The method for constructing the gas phase characteristic spectrum of the volatile components in Banxia Baizhu Tianma decoction according to claim 7, characterized in that: The programmed temperature rising method includes: The initial temperature was maintained at 70°C for 8 min; then increased to 120°C at 3°C / min and maintained for 10 min; increased to 140°C at 2°C / min and maintained for 15 min; increased to 200°C at 4°C / min and maintained for 10 min, then increased to 220°C at 2°C / min and maintained for 5 min.

9. The method for constructing the gas phase characteristic spectrum of the volatile components in the Banxia Baizhu Tianma decoction according to any one of claims 1 to 6, characterized in that: During the gas chromatography detection process, the injection port temperature was 235~245°C, the detector temperature was 295~305°C, and the carrier flow rate was 1.4~1.6ml / min.

10. The method for constructing the gas phase characteristic spectrum of the volatile components in the Banxia Baizhu Tianma decoction according to any one of claims 1 to 6, characterized in that: The construction method also includes the step of preparing a reference solution by dissolving at least one of limonene and atractylodesone in a solvent, and the step of obtaining a reference spectrum of the reference solution by detecting the reference solution by gas chromatography according to the construction method described in any one of claims 1 to 6.

11. The method for constructing the gas phase characteristic spectrum of the volatile components in Banxia Baizhu Tianma decoction according to claim 10, characterized in that: The solvent is selected from at least one of methanol, ethanol and water; each milliliter of the reference solution contains 50-70 µg of limonene and / or 80-120 µg of atractylodes ketone.

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