Method for determining components in gushen dingchuan pills and application thereof

Abstract

The application provides a method for determining components in Gushen Dingchuan Pills and application thereof. In the preparation process of the sample to be determined, the mixed solvent composed of water and organic solvent is used to ultrasonically extract the Gushen Dingchuan Pill powder, and then cold soaking and layering are performed, so that the influence of other components on the separation and stability of volatile components in the Gushen Dingchuan Pill can be significantly reduced, the separation stability of the volatile components in the Gushen Dingchuan Pill is significantly improved, and the separation time is shortened. The method provided by the application can quantitatively analyze seven chemical components (borneol acetate, camphor, borneol, cinnamic aldehyde, paeonol, alpha-pinene and linalool) in the Gushen Dingchuan Pill, and realizes qualitative and quantitative determination at the same time.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical analysis, and particularly to a method for determining the components in Gushen Dingchuan Pills and its application. Background Art

[0002] Gushen Dingchuan Pills are processed from 13 Chinese herbal pieces such as cinnamon, prepared rehmannia root, plantain seed, psoralea corylifolia salted, aconite, semen alpiniae oxyphyllae salted, etc. It warms the kidney and receives qi, strengthens the spleen and resolves phlegm. Clinically, it is used for cough, asthma, especially aggravated by movement caused by deficiency of the lung and spleen qi and failure of the kidney to receive qi; chronic bronchitis, pulmonary emphysema, bronchial asthma with the above syndromes. The prescription is derived from the ancient formula in "Songya Zunshengshu", originally named "Spleen and Kidney Pills" and included in "Dictionary of Chinese Medicine". The original formula was used to treat senile consumptive disease with asthma in the elderly. This formula is also evolved from Jisheng Shenqi Pills with added ingredients. The original formula warms the yin and tonifies the kidney, resolves phlegm, and promotes diuresis. It is combined with semen alpiniae oxyphyllae to warm the spleen and kidney and astringe essence; psoralea corylifolia tonifies the kidney and nourishes yin, and amomum villosum promotes qi movement and resolves dampness. The combination of these herbs enhances the effects of warming the kidney and receiving qi, and strengthening the spleen and resolving phlegm.

[0003] Clinical trial studies have shown that Gushen Dingchuan Pills can significantly improve the symptoms of fatigue, shortness of breath / wheezing and cough during the recovery period of viral infection. In addition, it also shows good clinical effects in the treatment of chronic obstructive pneumonia, heart failure, and cough variant asthma. Due to the large number of prescription herbs in Gushen Dingchuan Pills, rich volatile components, and complex chemical components, relying solely on its quality standards and existing literature reports for liquid-phase quantitative analysis of one or several components cannot comprehensively reflect the material basis and the integrity and complexity of the chemical component group of Gushen Dingchuan Pills, and it is difficult to comprehensively control the internal quality of Gushen Dingchuan Pills. The Chinese medicine fingerprint has the characteristics of overall, macroscopic, and fuzzy analysis. According to the characteristics of the overall comprehensive action of multiple components and multiple targets of Chinese medicine, starting from the perspective of "all components", by describing the overall characteristics of Chinese medicine, quality problems that are difficult to detect by conventional inspections can be revealed. It is an important modern quality control method and is more comprehensive for the quality control of drugs with non-single components. The quality standard of Gushen Dingchuan Pills is included in the "Chinese Pharmacopoeia" (Part I) of 2020 edition. At present, its quality standard lacks systematic qualitative research. For traditional Chinese medicine compound preparations rich in volatile oil components, the detection indicators cannot reflect the overall quality of traditional Chinese medicine preparations, and there are certain limitations in ensuring the consistency of product quality.

[0004] Therefore, it is necessary to establish the GC fingerprint of Gushen Dingchuan Pills to complete the qualitative analysis of the volatile components in Gushen Dingchuan Pills, so as to comprehensively and effectively monitor the quality of Gushen Dingchuan Pills. Summary of the Invention

[0005] The purpose of this invention is to overcome the deficiency in the existing technology of lacking systematic qualitative and quantitative analysis standards for the volatile components in Gushen Dingchuan Pills, and to provide a method for determining the components in Gushen Dingchuan Pills. This method allows for the simultaneous quantitative analysis of seven volatile oil chemical components in Gushen Dingchuan Pills, and the attribution and identification of the chromatographic peaks of each single volatile medicinal material in Gushen Dingchuan Pills. It enables effective monitoring from the source of raw materials, strictly controls the quality of raw medicinal materials, and can be used for quality control in the production process, thereby ensuring the safety and effectiveness of clinical medication.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A method for determining the components of Gushen Dingchuan Pills includes the following steps:

[0008] Preparation of the test sample:

[0009] The powder of Gushen Dingchuan Pills is dissolved in a mixed solvent obtained by mixing water and an organic solvent. After ultrasonic treatment, it is placed in an environment of 2-10°C for cold soaking to obtain an organic layer solution and an aqueous layer solution. The organic solvent includes at least one of diethyl ether and chloroform.

[0010] After removing the solvent from the organic layer solution, residue A is obtained; the aqueous layer solution is extracted with ethyl acetate, and the ethyl acetate layer is collected, concentrated, and recorded as solution B; residue A is dissolved in ethyl acetate and combined with solution B to obtain the sample to be tested;

[0011] Preparation of standard samples:

[0012] Each target analyte is dissolved in ethyl acetate to obtain a standard sample of the corresponding target analyte.

[0013] S2. The test sample and standard sample obtained in step S1 are tested by gas chromatography at a temperature of 40-150℃ to separate and identify the target components.

[0014] In the sample preparation process of this invention, a mixed solvent consisting of water and organic solvent is first used to ultrasonically extract the powder of Gushen Dingchuan Pills, followed by cold soaking and layering. This method can fully extract the less polar components such as bornyl acetate, cinnamaldehyde, and α-pinene from Gushen Dingchuan Pills, reducing their impact on the extraction, separation, and stability of the more polar volatile components in Gushen Dingchuan Pills. This is beneficial to improving the extraction and separation efficiency of the more polar volatile components in subsequent extraction steps of Gushen Dingchuan Pills.

[0015] Preferably, the amount of water added in step S1 is: water:Gushen Dingchuan Pill powder = (0.6~15) mL / g, more preferably (1.5~6) mL / g, and even more preferably 4 mL / g.

[0016] Preferably, the amount of organic solvent added in step S1 is: organic solvent: Gushen Dingchuan pill powder = (1.2~30) mL / g, more preferably (3~12) mL / g, and even more preferably 8 mL / g.

[0017] Adding appropriate amounts of water and organic solvents can further improve the separation stability of volatile components in Gushen Dingchuan Pills.

[0018] Preferably, the frequency of the ultrasound in step S1 is 35–45 kHz; the duration of the ultrasound is 60–90 min; and the power of the ultrasound is 300–600 W. During the high-frequency ultrasound process, the Gushen Dingchuan Pill powder can fully contact the solvent under high-frequency mechanical force, which is beneficial for the extraction of components.

[0019] Preferably, the cold soaking time in step S1 is 8–14 hours. Cold soaking is a simple operation that not only separates oil and water but also utilizes the different solubilities of different active ingredients at different temperatures to extract volatile oil components with fewer impurities and higher purity. Compared to high-temperature extraction methods such as direct reflux extraction and Soxhlet extraction, cold soaking is more suitable for extracting the less polar volatile components from Gushen Dingchuan Pills.

[0020] Preferably, the solvent removal method in step S1 can be rotary evaporation. The temperature of the rotary evaporation is 35–55°C.

[0021] It should be noted that, in the preparation of the standard sample in step S1 of this invention, the target analyte includes at least: bornyl acetate, camphor, borneol, cinnamaldehyde, paeonol, α-pinene, and linalool.

[0022] Preferably, in step S2, the temperature program for gas chromatography is as follows: initial temperature 42-48℃, hold for 5-7 min, increase to 76-80℃ at a rate of 2.5-4℃ / min → increase to 101-103℃ at a rate of 1.8-2.2℃ / min → increase to 110-112℃ at a rate of 0.8-1.2℃ / min, hold for 3-5 min → increase to 119-121℃ at a rate of 0.8-1.2℃ / min, hold for 4-6 min → increase to 139-141℃ at a rate of 2.8-3.2℃ / min, hold for 2-4 min.

[0023] The preferred temperature program for the gas chromatograph is as follows: initial temperature 45℃, hold for 5 min, increase to 78℃ at a rate of 3℃ / min; increase to 102℃ at a rate of 2℃ / min; increase to 111℃ at a rate of 1℃ / min, hold for 4 min; increase to 120℃ at a rate of 1℃ / min, hold for 5 min; increase to 140℃ at a rate of 3℃ / min, hold for 3 min.

[0024] A suitable heating program can significantly improve the separability of volatile components and greatly shorten the separation time.

[0025] The selection of other parameters in gas chromatography can also affect the separation of volatile components.

[0026] Preferably, the gas chromatograph uses a split injection method with a split ratio of (5-15):1, more preferably 10:1; the temperature of the injection port is 220-250℃, more preferably 230℃; and the injection volume is 0.5-1μL, more preferably 1μL.

[0027] Preferably, in step S2, the gas chromatographic column is an Agilent HP-5 column with dimensions of 30m × 0.32mm and 0.25μm.

[0028] Preferably, in step S2, the carrier gas of the gas chromatograph is at least one of nitrogen, argon, and helium, and more preferably nitrogen with a purity ≥ 99.999%; the flow rate of the carrier gas is 1.6 to 2.0 mL / min, and more preferably 1.8 mL / min.

[0029] The above-described determination method of the present invention can also perform quantitative analysis of each component. The quantitative analysis steps are as follows: Prepare a series of standard sample solutions of different concentrations for each target component, and analyze them using gas chromatography to obtain the linear relationship between the content and peak area of ​​the seven components in the standard reference solution. Plot a corresponding standard working curve with the peak area of ​​each component corresponding to its corresponding concentration, and calculate the regression equation for each standard working curve. Then, detect the sample to be tested using gas chromatography, obtain the peak areas of the seven components in the sample to be tested, and substitute them into the regression equations of the respective standard working curves to calculate the corresponding content of the seven components in the test solution.

[0030] This invention also protects the application of the above-described method for determining the components of Gushen Dingchuan Pills, wherein the method is applied to the quality testing of the components of Gushen Dingchuan Pills.

[0031] Specifically, the gas chromatographic fingerprint of Gu Shen Ding Chuan Wan was obtained using the aforementioned method for determining the GC fingerprint of Gu Shen Ding Chuan Wan. The similarity of this gas chromatographic fingerprint was then compared with that of a standard gas chromatographic fingerprint of Gu Shen Ding Chuan Wan obtained under the same detection conditions. When comparing the obtained gas chromatographic fingerprint of Gu Shen Ding Chuan Wan with its standard gas chromatographic fingerprint, the software "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" published by the National Pharmacopoeia Commission was used. The similarity was ≥0.98, indicating that the method of this invention for determining the components of Gu Shen Ding Chuan Wan can be used for the quality detection of the components of Gu Shen Ding Chuan Wan.

[0032] In this invention, when matching the common peaks of the gas phase fingerprint spectrum of the Gushen Dingchuan Pill with the standard gas phase fingerprint spectrum, multi-point correction is adopted, and automatic matching is performed at a time window width of 0.05 to 0.15 min, preferably 0.1 min. The gas phase fingerprint spectrum and the standard gas phase fingerprint spectrum are generated using the averaging method.

[0033] Compared with the prior art, the beneficial effects of the present invention are:

[0034] The method for determining the components of Gushen Dingchuan Pill provided by this invention is the first method to establish a GC fingerprint spectrum for Gushen Dingchuan Pill. It has good precision and repeatability. The fingerprint spectrum identifies 16 common peaks. The RSD of the relative retention time of each common peak is less than 0.10%. The similarity evaluation results of multiple batches of samples are all greater than 0.98. It can maintain good stability of the test solution within 24 hours.

[0035] Furthermore, the method provided by this invention can quantitatively analyze seven chemical components (borneol acetate, camphor, borneol, cinnamaldehyde, paeonol, α-pinene, and linalool) in Gushen Dingchuan Pills, achieving simultaneous qualitative and quantitative determination in a single sample analysis.

[0036] The method provided by this invention can also identify and assign chromatographic peaks of multiple single medicinal materials in Gushen Dingchuan Pills. The gas chromatographic fingerprint spectrum reveals characteristic peaks of cinnamon, amomum villosum, peony bark, salt-processed Alpinia oxyphylla, salt-processed Psoralea corylifolia, Rehmannia glutinosa, Alisma plantago-aquatica, and Rosa laevigata. This method enables effective monitoring from the source of raw materials, strictly controls the quality of raw medicinal materials, and can be applied to quality control in the production process, thereby ensuring the safety and effectiveness of clinical medication. Attached Figure Description

[0037] Figure 1 The above are the superimposed GC fingerprint chromatograms (i.e., gas chromatograms) of 16 batches of Gushen Dingchuan Pills in this invention, where S1 to S16 are 16 batches of Gushen Dingchuan Pills samples, and R is the standard fingerprint chromatogram of Gushen Dingchuan Pills.

[0038] Figure 2 This is the GC fingerprint of the standard reference material of Gushen Dingchuan Pill in this invention, wherein peak 3 is α-pinene, peak 4 is linalool, peak 5 is camphor, peak 6 is borneol, peak 8 is cinnamaldehyde, peak 9 is borneol acetate, and peak 12 is paeonol.

[0039] Figure 3 This is the standard GC fingerprint spectrum of the kidney-tonifying and asthma-relieving pill in this invention, wherein peak 3 is α-pinene, peak 4 is linalool, peak 5 is camphor, peak 6 is borneol, peak 8 is cinnamaldehyde, peak 9 is borneol acetate, and peak 12 is paeonol.

[0040] Figure 4The GC fingerprint of Gushen Dingchuan Pill (batch number R03001) was obtained by testing the chromatographic conditions in Example 4 (1).

[0041] Figure 5 The GC fingerprint of Gushen Dingchuan Pill (batch number R03001) was obtained by testing the chromatographic conditions in Example 4 (2).

[0042] Figure 6 The GC fingerprint of Gushen Dingchuan Pill (batch number R03001) was obtained by testing under the chromatographic conditions of Example 1.

[0043] Figure 7 This is a superimposed chromatogram comparing the standard GC fingerprint of the Gushen Dingchuan Pill and the GC fingerprint of each individual herb in this invention; S1: Rehmannia glutinosa (processed); S2: Aconitum carmichaelii (processed); S3: Paeonia suffruticosa; S4: Achyranthes bidentata; S5: Psoralea corylifolia (processed with salt); S6: Amomum villosum; S7: Plantago asiatica; S8: Poria cocos; S9: Alpinia oxyphylla (processed with salt); S10: Cinnamomum cassia; S11: Dioscorea opposita; S12: Alisma plantago-aquatica; S13: Rosa laevigata (processed with salt). Detailed Implementation

[0044] To better illustrate the purpose, technical solution, and advantages of this invention, the invention will be further described below with reference to specific embodiments and accompanying drawings. However, the embodiments do not limit the invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, all reagents and materials used in this invention are commercially available.

[0045] The reagents and instruments used in the following examples are as follows:

[0046] 1. Reagents

[0047] The samples to be tested: 16 batches of Gushen Dingchuan Pills (batch number S1: R03001, batch number S2: R03002, batch number S3: R03003, batch number S4: R03004, batch number S5: R03005, batch number S6: R03006, batch number S7: K10024, batch number S8: K10025, batch number S9: K10026, batch number S10: K10027, batch number S11: K10028, batch number S12: K10029, batch number S13: K10030, batch number S14: K12031, batch number S15: K12032, batch number S16: K12033) were all provided by Guangzhou Baiyunshan Jingxiutang Pharmaceutical Co., Ltd.

[0048] Standard samples: α-pinene (batch number: 110897-202204, content: 98.40%), linalool (batch number: 111503-202004, content: 98.80%), camphor (batch number: 110747-201810, content: 99.00%), borneol (batch number: 110881-201709, content: 99.60%), cinnamaldehyde (batch number: 110710-202022, content: 99.50%), bornyl acetate (batch number: 110759-202007, content: 99.30%), and paeonol (batch number: 110708-201908, content: 99.80%) were all purchased from the China National Institutes for Food and Drug Control.

[0049] Reagents: Ethyl acetate (Guangzhou Chemical Reagent Factory); Ultrapure water (prepared using the Micra ultrapure water treatment system, Guangzhou Wengdi Instrument Co., Ltd.)

[0050] 2. Instruments

[0051] 6890N gas chromatograph (Agilent Technologies, USA); ME204T analytical electronic balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.); Micra pure water system (Guangzhou Wengdi Instrument Co., Ltd.).

[0052] Example 1

[0053] This embodiment provides a method for determining the components in Gushen Dingchuan Pills, including the following steps:

[0054] S1. Preparation of the test sample and standard sample:

[0055] Preparation of the test sample:

[0056] Weigh 25g of Gushen Dingchuan Pill powder, place it in a container, add 100mL of water and 200mL of chloroform, and extract by ultrasonication (power 600W, frequency 40kHz) for 90min. Then place it in an explosion-proof refrigerator (4℃) for cold soaking for 12h to separate the organic layer solution and the aqueous layer solution. After recovering chloroform by rotary evaporation of the organic layer solution at 55℃, the residue A obtained is dissolved in ethyl acetate to obtain solution A; the aqueous layer solution is extracted twice with ethyl acetate at a ratio of 1:1, and the extracts are combined. The extracts are concentrated by rotary evaporation at 55℃ to obtain solution B; solutions A and B are combined into a 20mL volumetric flask and diluted to the mark with ethyl acetate to obtain the sample to be tested.

[0057] Preparation of standard samples:

[0058] Take the reference standards of borneol acetate, camphor, borneol, cinnamaldehyde, paeonol, α-pinene, and linalool, dissolve them in ethyl acetate, and prepare a mixed standard (reference) sample solution containing 500 μg of each of the following per 1 mL: borneol acetate, camphor, borneol, cinnamaldehyde, paeonol, α-pinene, and linalool.

[0059] S2. The test sample and standard sample obtained in step S1 are tested by gas chromatography. The specific test conditions are as follows:

[0060] Agilent HP-5 column (30m × 0.32mm, 0.25μm); injection port temperature: 230℃; injection volume: 1μL; carrier gas: high-purity nitrogen, purity ≥99.999%; carrier gas flow rate: 1.80mL / min; injection method: split injection, split ratio: 10:1;

[0061] The heating program is as follows: initial temperature 45℃, hold for 5 min, increase to 78℃ at a rate of 3℃ / min; increase to 102℃ at a rate of 2℃ / min; increase to 111℃ at a rate of 1℃ / min, hold for 4 min; increase to 120℃ at a rate of 1℃ / min, hold for 5 min; increase to 140℃ at a rate of 3℃ / min, hold for 3 min.

[0062] The gas chromatographic fingerprints of the test sample and the mixed reference solution were determined separately using the above chromatographic conditions to obtain the gas chromatographic fingerprints of the test sample and the standard reference solution. The gas chromatographic fingerprints of the test sample and the standard reference solution were then compared. Based on the known characteristic peaks of the gas chromatographic fingerprint of the standard reference solution (see...),... Figure 2 By identifying the corresponding characteristic peaks in the gas phase fingerprint spectrum of the test sample through relative retention time, the gas phase fingerprint spectrum of the test sample is obtained (see...). Figure 1 The index components were assigned and located to obtain the GC gas phase fingerprint spectrum of Gushen Dingchuan Pill.

[0063] The established GC fingerprinting method for Gushen Dingchuan Pills was used to determine the GC fingerprinting of 16 batches of Gushen Dingchuan Pills (S1-S16). Chromatograms were recorded, and the GC fingerprint data of the samples were imported into the "Similarity Evaluation System for Chromatogram Fingerprints of Traditional Chinese Medicine" software published by the National Pharmacopoeia Commission. After multi-point correction, automatic matching was performed (time window 0.1 min). The GC fingerprints of the samples and the standard GC fingerprint of Gushen Dingchuan Pills were generated using the averaging method. Specific chromatograms are shown in [link to chromatogram]. Figure 1 , 3 .from Figure 3As can be seen from the data, the standard gas phase fingerprint spectrum of this Gushen Dingchuan Pill includes 16 common fingerprint peaks. Taking peak 9 as the reference peak (S peak, relative retention time of 1.000, relative peak area of ​​1.000), the relative retention times and relative peak areas of the other 15 common fingerprint peaks are as follows:

[0064] Peak 1 (relative retention time 0.098±0.002, relative peak area 0.246±0.122);

[0065] Peak 2 (relative retention time 0.177±0.004, relative peak area 0.109±0.026);

[0066] Peak 3 (relative retention time 0.342±0.002, relative peak area 0.084±0.042);

[0067] Peak 4 (relative retention time 0.613±0.005, relative peak area 0.068±0.018);

[0068] Peak 5 (relative retention time 0.696±0.003, relative peak area 0.757±0.105);

[0069] Peak 6 (relative retention time 0.741±0.004, relative peak area 0.166±0.029);

[0070] Peak 7 (relative retention time 0.934±0.002, relative peak area 0.100±0.211);

[0071] Peak 8 (relative retention time 0.969±0.003, relative peak area 2.604±0.104);

[0072] Peak 10 (relative retention time 1.167±0.005, relative peak area 0.092±0.012);

[0073] Peak 11 (relative retention time 1.251±0.006, relative peak area 0.108±0.063);

[0074] Peak 12 (relative retention time 1.326±0.004, relative peak area 3.702±0.092);

[0075] Peak 13 (relative retention time 1.442±0.007, relative peak area 0.141±0.007);

[0076] Peak 14 (relative retention time 1.471±0.005, relative peak area 0.135±0.050);

[0077] Peak 15 (relative retention time 1.547±0.005, relative peak area 0.236±0.050);

[0078] Peak 16 (relative retention time 1.764±0.005, relative peak area 0.390±0.009).

[0079] The gas chromatographic fingerprint of the standard Gushen Dingchuan pill and the gas chromatographic fingerprint of the standard reference solution ( Figure 2 By comparing the peaks, peak 3 was identified as the fingerprint peak of α-pinene, peak 4 as the fingerprint peak of linalool, peak 5 as the fingerprint peak of camphor, peak 6 as the fingerprint peak of borneol, peak 8 as the fingerprint peak of cinnamaldehyde, peak 9 as the fingerprint peak of borneol acetate, and peak 12 as the fingerprint peak of paeonol.

[0080] Example 2

[0081] This embodiment provides a method for determining the components in Gushen Dingchuan Pills. After the qualitative analysis of the sample to be tested in Example 1, the following analysis is further performed:

[0082] 1. Similarity evaluation:

[0083] The similarity between the gas phase fingerprint spectrum of the sample to be tested and the standard gas phase fingerprint spectrum of Gushen Dingchuan Pill obtained under the same fingerprint spectrum detection conditions was compared and the similarity was calculated. The specific calculation results are shown in Table 1.

[0084] Table 1 Similarity Calculation Results

[0085]

[0086] Data shows that the gas phase fingerprint similarity of all 16 batches of Gushen Dingchuan Pills was above 0.98, which meets the requirements.

[0087] 2. Batch stability test of Gushen Dingchuan Pills:

[0088] The relative retention times (based on peak 9) of each peak in different test samples were compared, and the RSD was calculated. Simultaneously, the relative peak areas (based on peak 9) of each peak in different test samples were compared, and the RSD was calculated. The results are shown in Table 2.

[0089] Table 2. Batch stability results of Gushen Dingchuan Pills

[0090]

[0091] Table 2 shows that the RSD of the relative retention time for different samples was less than 0.15%; the RSD of the relative peak area ranged from 10.86% to 39.32%. The results indicate that the elution times of common substances in each batch of samples were basically the same, and the composition was relatively stable, but their contents varied considerably.

[0092] Example 3

[0093] This embodiment evaluates the repeatability, precision, and stability of the method for determining the components of Gushen Dingchuan Pill provided in Example 1:

[0094] 1. Repeatability

[0095] Six samples of Gushen Dingchuan Pills (batch number R03001) were taken, and test solutions were prepared according to step 1 of Example 1 above. The tests were performed according to step 3 of Example 1 above, using peak 9 as the reference peak. The relative retention time and relative peak area RSD of each common peak were calculated. The results showed that the RSD of the relative retention time of all 16 common peaks was less than 0.13%, and the RSD of the relative peak area was less than 1.2%, indicating that the method in this invention has good repeatability and high accuracy.

[0096] 2. Precision

[0097] One sample of Gushen Dingchuan Pills (batch number R03001) was taken, and the test solution was prepared according to step 1 in Example 1 above. The sample was then analyzed according to step 3 in Example 1 above, with six consecutive injections. The chromatogram was recorded, and peak 9 was used as the reference peak to examine the relative retention times of the common peaks specified in the fingerprint chromatogram. The results showed that the RSD of the relative retention times of the 16 common peaks was less than 0.09%, and the RSD of the relative retention areas was less than 1.0%, indicating that the instrument had good precision.

[0098] 3. Stability

[0099] A sample of Gushen Dingchuan Pills (batch number R03001) was prepared according to step 1 in Example 1 above. The sample was then left to stand for 0h, 3h, 6h, 9h, 12h, and 24h, and then measured according to step 3 in Example 1 above. The results showed that, using peak 9 as the reference peak, the relative retention time RSD of all 16 common peaks was less than 0.07%, and the relative peak area RSD was less than 1.0%, indicating that the sample exhibited good stability within 24h.

[0100] Example 4

[0101] This embodiment uses the determination steps of Example 1 as a baseline, and changes the split ratio and temperature program of gas chromatography to study their effects on the gas chromatographic fingerprint (also known as CG fingerprint) of Gushen Dingchuan Pills. The prepared Gushen Dingchuan Pills (batch number R03001) sample was used for determination. Chromatograms were recorded after determination under the following chromatographic conditions, and the results are shown in [Figure 1]. Figures 4-6 .

[0102] (1) Chromatographic conditions: Agilent HP-5 column (30m×0.32mm, 0.25μm); sample port temperature 220℃; injection volume 1μL; carrier gas high-purity nitrogen with a purity ≥99.999%; carrier gas flow rate 1.80mL / min; injection method split injection with a split ratio of 1:20.

[0103] The heating process is as follows:

[0104] The initial temperature was 50℃. The temperature was increased to 102℃ at a rate of 3℃ / min and held for 1 min. The temperature was increased to 120℃ at a rate of 2℃ / min and held for 20 min. The temperature was increased to 140℃ at a rate of 3℃ / min and held for 30 min.

[0105] Test results are available Figure 4 As can be seen, most chromatographic peaks eluted before 10 min, and the relative retention time RSD of the 16 common peaks was less than 0.18%, and the relative peak area RSD was less than 1.5%, indicating that the method in this invention has good repeatability and high accuracy.

[0106] (2) Chromatographic conditions: Agilent HP-5 column (30m×0.32mm, 0.25μm). Sample port temperature was 220℃; injection volume was 1μL; carrier gas was high-purity nitrogen with a purity ≥99.999%; carrier gas flow rate was 1.80mL / min; injection method was split injection with a split ratio of 1:5.

[0107] The heating process is as follows:

[0108] The initial temperature was 45℃; the temperature was increased to 78℃ at a rate of 3℃ / min and held for 3 min; then increased to 102℃ at a rate of 2℃ / min; finally increased to 120℃ at a rate of 2℃ / min and held for 25 min; and then increased to 140℃ at a rate of 3℃ / min and held for 20 min. The results are shown below. Figure 5 .

[0109] Depend on Figure 5 It can be seen that under the above chromatographic conditions, most chromatographic peaks can be effectively separated; the relative retention time RSD of the 16 common peaks is less than 0.17%, and the relative peak area RSD is less than 1.6%, indicating that the method in this invention has good repeatability and high accuracy.

[0110] Example 5

[0111] This embodiment provides a method for screening the gas phase fingerprint spectra of multiple medicinal materials in Gushen Dingchuan Pill, specifically including the following steps:

[0112] (1) Preparation of single herbal samples: Any one or more of the 13 herbs in Gushen Dingchuan Pill, namely Rehmannia glutinosa, Paeonia suffruticosa, Psoralea corylifolia (salted), Plantago asiatica, Alpinia oxyphylla (salted), Dioscorea opposita, Rosa laevigata, Aconitum carmichaelii (black aconite), Achyranthes bidentata, Amomum villosum, Poria cocos, Cinnamomum cassia, and Alisma plantago-aquatica, were prepared according to step 1 of the method for determining the GC fingerprint spectrum of Gushen Dingchuan Pill, and at least one single herbal sample was obtained.

[0113] (2) Determination: The gas chromatographic fingerprint of the single herbal sample was determined using the same chromatographic conditions as in step 3 of the method for determining the GC fingerprint of Gushen Dingchuan Pill (i.e., the gas chromatographic conditions of Example 1) to obtain the gas fingerprint of the single herbal sample.

[0114] (3) Quality Inspection: The gas phase fingerprint of the single herbal sample was compared with the standard gas phase fingerprint of Gushen Dingchuan Pill. By using the relative retention time, the corresponding characteristic peaks of the single herbal sample in the standard gas phase fingerprint of Gushen Dingchuan Pill were identified, thereby assigning and locating the characteristic peaks in the gas phase fingerprint of the single herbal sample. The test results are shown in […]. Figure 7 And Table 3.

[0115] Table 3. Fingerprint chromatograms of each single herb in Gu Shen Ding Chuan Wan (a traditional Chinese medicine formula) show the total number of fingerprint peaks.

[0116]

[0117]

[0118] Among the aforementioned fingerprint peaks, peak 3 is identified as the fingerprint peak of α-pinene, peak 4 as the fingerprint peak of linalool, peak 5 as the fingerprint peak of camphor, peak 6 as the fingerprint peak of borneol, peak 8 as the fingerprint peak of cinnamaldehyde, peak 9 as the fingerprint peak of borneol acetate, and peak 12 as the fingerprint peak of paeonol.

[0119] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for determining the components in Gushen Dingchuan Pills, characterized in that, Includes the following steps: S1. Preparation of test samples and standard samples Preparation of the test sample: The powder of Gushen Dingchuan Pills is dissolved in a mixed solvent obtained by mixing water and an organic solvent. After ultrasonic treatment, it is placed in an environment of 2-10°C for cold soaking to obtain an organic layer solution and an aqueous layer solution. The organic solvent includes at least one of diethyl ether and chloroform. After removing the solvent from the organic layer solution, residue A is obtained; the aqueous layer solution is extracted with ethyl acetate, and the ethyl acetate layer is collected, concentrated, and recorded as solution B; residue A is dissolved in ethyl acetate and combined with solution B to obtain the sample to be tested. The aforementioned kidney-tonifying and asthma-relieving pills contain borneol acetate, camphor, borneol, cinnamaldehyde, paeonol, α-pinene, and linalool. Preparation of standard samples: Each target analyte was dissolved in ethyl acetate to obtain a standard sample of the corresponding target analyte; the target analytes included at least the following: bornyl acetate, camphor, borneol, cinnamaldehyde, paeonol, α-pinene, and linalool; S2. The test sample and standard sample obtained in step S1 are tested by gas chromatography at a temperature of 40~150℃ to separate and identify the target components. The temperature program for gas chromatography was as follows: initial temperature 45℃, hold for 5 min, increase to 78℃ at a rate of 3℃ / min; increase to 102℃ at a rate of 2℃ / min; increase to 111℃ at a rate of 1℃ / min, hold for 4 min; increase to 120℃ at a rate of 1℃ / min, hold for 5 min; increase to 140℃ at a rate of 3℃ / min, hold for 3 min. The gas chromatography column was an Agilent HP-5 column with dimensions of 30m × 0.32mm and 0.25μm.

2. The method for determining the components of Gushen Dingchuan Pill according to claim 1, characterized in that, The amount of water added in step S1 is: water: Gushen Dingchuan Pill powder = 0.6~15mL / g; the amount of organic solvent added in step S1 is: organic solvent: Gushen Dingchuan Pill powder = 1.2~30mL / g.

3. The method for determining the components of Gushen Dingchuan Pill according to claim 1, characterized in that, The frequency of the ultrasound in step S1 is 35-45 kHz; the duration of the ultrasound is 60-90 min.

4. The method for determining the components of Gushen Dingchuan Pill according to claim 1, characterized in that, The cold soaking time in step S1 is 8~14 hours.

5. The method for determining the components of Gushen Dingchuan Pill according to claim 1, characterized in that, In step S2, the carrier gas for gas chromatography is at least one of nitrogen, argon, and helium; the flow rate of the carrier gas is 1.6 to 2.0 mL / min.

6. The method for determining the components of Gushen Dingchuan Pill according to claim 1, characterized in that, In step S2, the gas chromatography satisfies at least one of the following conditions: 1) The injection method is split injection; 2) The temperature of the injection port is 220~250℃; 3) The injection volume is 0.5~1μL; 4) The split ratio is 5 to 15:

1.

7. The application of the method for determining the components of Gushen Dingchuan Pill according to any one of claims 1 to 6, characterized in that, The method described above is used in the quality testing of the components of Gushen Dingchuan Pill.

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