A method for detecting a traditional Chinese medicine composition for activating meridians and reducing blood sugar by GC-MS

Abstract

The application provides a kind of traditional Chinese medicine composition GC-MS quality detection method for Tongmai Jiangtang, which has the advantages of simple operation, accurate detection and high stability. The volatile components in Tongmai Jiangtang are determined, and through the determination of 15 batches of content, it is known that the content of moxa atractylodes alcohol in Tongmai Jiangtang granules is 0.0205-0.1893 mg / g, the content of beta-eudesmol is 0.0643-0.5737 mg / g, the content of palmitic acid is 0.7319-5.1163 mg / g, the content of myrobalan is 0.0743-0.2356 mg / g, and the content of danshensu is 0.0777-0.8459 mg / g. The above detection method can be used as the internal quality detection standard of the product to further ensure the product quality. The detection method can also be used for the chemical composition and identification of Tongmai Jiangtang traditional Chinese medicine composition, and has good market prospect.

Description

Technical Field

[0001] This invention belongs to the field of quality testing of chemical components of traditional Chinese medicine, and in particular relates to a GC-MS quality testing method for a traditional Chinese medicine composition for promoting blood circulation and lowering blood sugar. Background Technology

[0002] Tongmai Jiangtang Capsules are an oral solid dosage form prepared from ingredients such as Codonopsis pilosula, Salvia miltiorrhiza, Coptis chinensis, Astragalus membranaceus, Gynostemma pentaphyllum, Dioscorea opposita, Atractylodes lancea, Scrophularia ningpoensis, Hirudo medicinalis, Malva verticillata, and Pueraria lobata. It has the effects of nourishing Yin and clearing heat, and promoting blood circulation. It is used for diabetes mellitus caused by deficiency of both Qi and Yin and obstruction of the meridians. Among the herbs involved in Tongmai Jiangtang, Codonopsis pilosula contains cyclic peptides, sugars, glycosides, fatty acids, oils, and volatile oils, and its polysaccharides have hypoglycemic activity. Salvia miltiorrhiza contains tanshinone and salvianolic acid components, which have hypoglycemic effects. Coptis chinensis contains alkaloids, flavonoids, lignins, organic acids, and volatile oils, and its alkaloid and polysaccharide components have hypoglycemic effects. Astragalus membranaceus contains polysaccharides, saponins, and flavonoids, and its polysaccharides and flavonoid components have hypoglycemic effects. Gynostemma pentaphyllum contains gypenosides, polysaccharides, flavonoids, and other components. Gynostemma pentaphyllum saponins, polysaccharides, and crude extracts have hypoglycemic effects. Dioscorea opposita contains polysaccharides, proteins, amino acids, esters, and other components. Its polysaccharides have a hypoglycemic effect. Atractylodes lancea contains sesquiterpenes, alkenes, organic acids, and other components. Atractylodes lancea water extract, alcohol extract, and ethyl acetate extract have hypoglycemic effects. Scrophularia ningpoensis contains iridoid glycosides, phenylpropanoids, flavonoids, polysaccharides, and other components. Scrophularia ningpoensis polysaccharides and alcohol extracts of different concentrations all have hypoglycemic effects. Malva verticillata fruit contains acidic compounds, flavonoids, sterols, and other compounds. Its flavonoid components have a hypoglycemic effect. Pueraria lobata contains flavonoids, organic acids, triterpenoids, and other chemical components. Its flavonoid components have a hypoglycemic effect.

[0003] A review of existing technical literature on Tongmai Jiangtang Capsules reveals that content detection methods primarily focus on the study of water-soluble and alcohol-soluble chemical components. For example, He Yaling, Che Xiaoyan, et al., published in *West China Pharmaceutical Journal*, March 30, 1998, on the determination of astragaloside A content in Tongmai Jiangtang Granules using thin-layer chromatography. This paper employed thin-layer chromatography with a chloroform-methanol-water (13:6:2) mixed developing solvent. After development and drying, the plate was uniformly moistened with sulfuric acid ethanol solution. The detection wavelengths were 500 nm and 700 nm to determine the astragaloside A content. The standard for Tongmai Jiangtang Capsules, WS-10116(ZD-0116)-2002, discloses the use of methanol-water (15:5) as the mobile phase and a detection wavelength of 270 nm. Accurately pipette the reference solution and test solution, inject them into the liquid chromatograph, and determine the content of tanshinone IIA (C19H18O3) to be not less than 0.15 mg. Xue Li, Determination of berberine hydrochloride content in Tongmai Jiangtang granules by high performance liquid chromatography, Chinese Journal of Hospital Pharmacy, using acetonitrile-water (55:35) as the mobile phase and a detection wavelength of 345 nm to determine the content of berberine hydrochloride.

[0004] However, there are currently no reports on the volatile oil components of Tongmai Jiangtang Capsules. This study uses GC-MS to quantitatively analyze the content of volatile oils, providing a reference for the determination of volatile substances in Tongmai Jiangtang Capsules. Summary of the Invention

[0005] This invention provides a GC-MS quality detection method for a traditional Chinese medicine composition for promoting blood circulation and lowering blood sugar. This method has the advantages of simple operation, accurate detection, and high stability. The present invention determines the volatile components in the blood circulation and lowering blood sugar composition. After content determination of 15 batches, the contents of the granules are as follows: atractylodes acetyl alcohol 0.0205–0.1893 mg / g, β-cineole 0.0643–0.5737 mg / g, palmitic acid 0.7319–5.116 mg / g, mirosol 0.0743–0.2356 mg / g, and tanshinone IIA 0.0777–0.8459 mg / g. The above detection method can be used as an internal quality control standard for this product, further ensuring product quality. Furthermore, this detection method can also be used for the chemical composition and identification of the traditional Chinese medicine composition for promoting blood circulation and lowering blood sugar, showing good market application prospects.

[0006] The technical solution of this invention patent application is as follows:

[0007] A GC-MS quality detection method for a traditional Chinese medicine composition for promoting blood circulation and lowering blood sugar, characterized in that the detection method includes the following steps:

[0008] (1) Preparation of test solution: Weigh the Chinese herbal medicine composition, accurately add ethyl acetate, weigh the sample, sonicate for 20-40 min, sonic power 150-250 W, sonic frequency 30-50 Hz, let it stand at room temperature, make up the lost weight, filter, and take the filtrate to obtain the test solution.

[0009] (2) Preparation of mixed reference solution: Weigh out the reference standards of atractylodes lancea alcohol, β-eucalyptol, palmitic acid, mirosol, and tanshinone IIA, respectively, and dilute to the mark with ethyl acetate. Shake well to prepare a reference solution with a concentration of 0.5-5.0 μg / mL for atractylodes lancea alcohol, 1.0-5.0 μg / mL for β-eucalyptol, 1.0-20.0 μg / mL for palmitic acid, 0.5-5.0 μg / mL for mirosol, and 2.0-20.0 μg / mL for tanshinone IIA.

[0010] (3) Gas chromatography conditions:

[0011] A quartz capillary column was used, with a temperature range of 320–350℃. The temperature program was as follows: initial temperature 100℃; increasing to 142℃ at 10.0℃ / min; increasing to 150℃ at 1.5℃ / min and holding for 2 min; increasing to 170℃ at 10.0℃ / min and holding for 3 min; increasing to 177℃ at 0.8℃ / min; increasing to 200℃ at 5.0℃ / min and holding for 2 min; increasing to 207℃ at 1.0℃ / min and holding for 2 min; increasing to 235℃ at 5.0℃ / min. Hold for 3 min; increase temperature to 240℃ at 0.8℃ / min; increase temperature to 270℃ at 5.0℃ / min); injection port temperature is 270~290℃; carrier gas is high-purity helium; carrier gas flow rate is 1.0~2.0mL / min; split ratio is 8~12:1; solvent delay is 1~3 min; pressure is 120~130KPa; total flow rate is 22~27mL / min; column flow rate is 1.0~2.5mL / min; linear velocity is 40~60cm / s; purge flow rate is 2.0~4.0mL / min.

[0012] (4) Mass spectrometry conditions: EI ion source was used; ion source temperature was 270–290℃, interface temperature was 240–260℃; scan range was m / z 50–750.

[0013] (5) Content determination: Take the mixed reference solution from step (2) and the test solution from step (1), inject the sample, determine the peak area, and calculate the content.

[0014] Preferably, the ultrasonic time in step (1) of the detection method is 30 min, the ultrasonic power is 200 W, and the ultrasonic frequency is 40 Hz.

[0015] Preferably, in step (2) of the detection method, the concentrations of atractylodes acetonide, β-eucalyptol, palmitic acid, milnosine, and tanshinone IIA in the mixed reference solution are 2.0758 μg / mL, 2.1834 μg / mL, 9.9510 μg / mL, 2.0360 μg / mL, and 10.6067 μg / mL.

[0016] Preferably, in step (3) of the detection method, the quartz capillary column is an Agilent DB-5MS with column specifications of 0.25mm × 30m and 0.25μm.

[0017] Preferably, in step (3) of the detection method, the injection port temperature is 280℃; the carrier gas is high-purity helium; the carrier gas flow rate is 1.5 mL / min; the split ratio is 10:1; the solvent delay is 2 min; the pressure is 125.4 kPa; the total flow rate is 25.4 mL / min; the column flow rate is 2.04 mL / min; the linear velocity is 52.1 cm / s; and the purge flow rate is 3.0 mL / min.

[0018] Preferably, in step (4), the ion source temperature is 280°C and the interface temperature is 250°C.

[0019] Preferably, the mass content determination of the traditional Chinese medicine composition of the present invention is carried out according to the gas chromatography and mass spectrometry conditions in step (3) and (4), wherein the content of atractylodes acetonitrile is 0.0205-0.1893 mg / g, the content of β-eudesminol is 0.0643-0.5737 mg / g, the content of palmitic acid is 0.7319-5.1163 mg / g, the content of mirosol is 0.0743-0.2356 mg / g, and the content of tanshinone IIA is 0.0777-0.8459 mg / g.

[0020] Preferably, the mass content determination of the traditional Chinese medicine composition of the present invention is carried out according to the gas chromatography and mass spectrometry conditions in step (3) and (4), wherein the content of atractylodes acetonitrile is 0.0838 mg / g, the content of β-eudesminol is 0.2493 mg / g, the content of palmitic acid is 1.5572 mg / g, the content of mirosol is 0.1211 mg / g, and the content of tanshinone IIA is 0.1949 mg / g.

[0021] Preferably, the detection method is used in the quality detection, intermediate determination, content determination, and identification of effective components of traditional Chinese medicine for blood sugar lowering and blood sugar control.

[0022] To further illustrate the inventiveness of the detection method for Tongmai Jiangtang granules of the present invention, the experimental contents of the screening of the technical solution of the present invention are summarized as follows.

[0023] 1.1 Selection of extraction solvent

[0024] 1.1.1 Extraction of volatile oils

[0025] 1.1.1.1 Solvent extraction of volatile oils

[0026] Take 1g of Tongmai Jiangtang Capsules, make 3 parallel portions, accurately weigh them, add 100mL of n-hexane, ethyl acetate and anhydrous ethanol respectively, sonicate (30min, 400W, 40Hz), cool, add anhydrous sodium sulfate and filter, wash the filter residue and filter paper repeatedly with the extraction solvent, collect the filtrate and washings, and dry.

[0027] 1.1.1.2 Sample Methylation Treatment

[0028] Take a small amount of the extracted volatile oil from the Tongmai Jiangtang capsules, add 5 mL of 0.5 M KOH methanol solution, and place in a 70 °C water bath for saponification for 10 min. Shake occasionally during the saponification process to ensure complete reaction of the oil. Cool for 3 min, add 3 mL of BF3 methanol solution, and place in a 70 °C water bath for saponification for 5 min to ensure complete methylation. Then add 10 mL of saturated sodium chloride solution and a small amount of chromatographic grade n-hexane. Allow to stand and separate into layers. Take the supernatant and filter it through a 0.22 μm organic microporous filter to obtain the test solution.

[0029] 1.1.2 GC-MS Analysis Conditions for Volatile Oils

[0030] 1.1.2.1 Gas Chromatography Conditions

[0031] The following temperature program was used: SH-RXi-5SilMS quartz capillary column (0.25 mm × 30 m, 0.25 μm, temperature range 320 / 350 °C); temperature program (initial temperature 60 °C, equilibration 3 min; increase to 100 °C at 5.0 °C / min, hold for 5 min; increase to 150 °C at 3.0 °C / min, hold for 5 min; increase to 200 °C at 3.0 °C / min, hold for 5 min; increase to 250 °C at 3.0 °C / min, hold for 5 min; increase to 270 °C at 5.0 °C / min, hold for 2 min); injection port temperature 280 °C; carrier gas high-purity helium (99.999%); carrier gas flow rate 1.5 mL / min; split ratio 10:1; solvent delay 2 min. Pressure 125.4 kPa, total flow rate 25.4 mL / min, column flow rate 2.04 mL / min, linear velocity 52.1 cm / s, purge flow rate 3.0 mL / min. Injection 1 μL.

[0032] 1.1.2.2 Mass Spectrometry Conditions

[0033] An EI ion source was used; a triple quadrupole mass analyzer was used; the interface temperature was 250℃; the ion source temperature was 280℃; and the scanning range was m / z 50~750.

[0034] 1.1.3 Results

[0035] Similarity searches revealed 40, 38, and 38 compounds in hexane, ethyl acetate, and anhydrous ethanol, respectively. Anhydrous ethanol showed a higher concentration of fatty acid compounds. Hexane and ethyl acetate yielded 7 identical components, with the ethyl acetate extract showing the highest relative abundance of these components. The total ion chromatogram is shown below. Figure 1-A (B, C).

[0036] 1.2 Determination of Extraction Solvent

[0037] 1.2.1.1 Gas Chromatography Conditions

[0038] An Agilent DB-5MS quartz capillary column (0.25 mm × 30 m, 0.25 μm, temperature range 320 / 350 °C) was used; the temperature program was as follows: initial temperature 60 °C, equilibration for 30 min; increase to 100 °C at 5.0 °C / min; increase to 150 °C at 3.0 °C / min, hold for 5 min; increase to 200 °C at 3.0 °C / min, hold for 5 min; increase to 250 °C at 3.0 °C / min, hold for 5 min; increase to 270 °C at 5.0 °C / min, hold for 2 min); the injection port temperature was 280 °C; the carrier gas was high-purity helium (99.999%); the carrier gas flow rate was 1.5 mL / min; the split ratio was 10:1; and the solvent delay was 2 min. Pressure 125.4 kPa, total flow rate 25.4 mL / min, column flow rate 2.04 mL / min, linear velocity 52.1 cm / s, purge flow rate 3.0 mL / min.

[0039] 1.2.1.2 Mass Spectrometry Conditions

[0040] An EI ion source, a triple quadrupole detector, an interface temperature of 250℃, an ion source temperature of 280℃, and a scan range of m / z 50–750 were used. 1 μL of sample was injected.

[0041] 1.2.1.3 Preparation of the test solution

[0042] (1) Hexane extraction

[0043] Accurately weigh 0.5g of the sample into two portions, add 50mL of n-hexane to each portion, weigh them, sonicate for 30min (temperature 20℃, power 200W, frequency 40Hz), let them cool to room temperature, make up the lost weight, filter with anhydrous sodium sulfate, take the filtrate, filter it through a 0.22μm microporous membrane, and take the filtrate to obtain the final product.

[0044] (2) Ethyl acetate extraction

[0045] Accurately weigh 1g of sample, accurately add 10mL of ethyl acetate, weigh the sample, sonicate for 30min (temperature 20℃, power 200W, frequency 40Hz), let it cool to room temperature, make up the lost weight, filter with anhydrous sodium sulfate, take the filtrate, filter it through a 0.22μm microporous membrane, and take the filtrate.

[0046] 1.2.1.4 Results

[0047] Hexane extraction yields fewer volatile components, requires a high solid-liquid ratio, and has a significant impact from baseline noise. Ethyl acetate, with a 1:10 solid-liquid ratio, produces a wider variety of components and has less noise impact. See chromatographic results for details. Figure 2-A , Figure 2-B Therefore, ethyl acetate was chosen as the extraction solvent. Atractylodes lancea alcohol, β-cineole, palmitic acid, mirosinol, and tanshinone IIA were selected as the components to be analyzed.

[0048] 1.3 Selection of Solvent Amount

[0049] 1.3.1 Chromatographic and Mass Spectrometry Conditions

[0050] 1.3.1.1 Gas Chromatography Conditions

[0051] An Agilent DB-5MS quartz capillary column (0.25 mm × 30 m, 0.25 μm, temperature range 320 / 350 °C) was used; the temperature program was as follows: initial temperature 60 °C, equilibration for 30 min; increase to 100 °C at 5.0 °C / min; increase to 150 °C at 3.0 °C / min, hold for 5 min; increase to 200 °C at 3.0 °C / min, hold for 5 min; increase to 250 °C at 3.0 °C / min, hold for 5 min; increase to 270 °C at 5.0 °C / min, hold for 2 min); the injection port temperature was 280 °C; the carrier gas was high-purity helium (99.999%); the carrier gas flow rate was 1.5 mL / min; the split ratio was 10:1; and the solvent delay was 2 min. Pressure 125.4 kPa, total flow rate 25.4 mL / min, column flow rate 2.04 mL / min, linear velocity 52.1 cm / s, purge flow rate 3.0 mL / min. Injection 1 μL.

[0052] 1.3.1.2 Mass Spectrometry Conditions

[0053] An EI ion source was used; triple quadrupole mass spectrometry was employed; the interface temperature was 250℃; the ion source temperature was 280℃; and the scanning range was m / z 50~750.

[0054] 1.3.2 Preparation of the test solution

[0055] Accurately weigh 1g of sample into 3 portions, and accurately add 10, 12, and 14mL of ethyl acetate respectively. Weigh the portions, sonicate for 30min (temperature 20℃, power 200W, frequency 40Hz), let cool to room temperature, replenish the lost weight, filter with anhydrous sodium sulfate, collect the filtrate, filter through a 0.22μm microporous membrane, and collect the filtrate to obtain the final product.

[0056] 1.3.3 Results

[0057] The sample contained high levels of atractylodes lancea alcohol, β-cineole, palmitic acid, mirosol, and tanshinone IIA, hence the selection of a 1:10 solid-liquid ratio. The test results show... Figure 3-A (B, C).

[0058] 1.4 Optimization of chromatographic conditions

[0059] 1.4.1 Optimization of Chromatographic Methods 2

[0060] (1) Gas chromatography conditions

[0061] An Agilent DB-5MS quartz capillary column (0.25 mm × 30 m, 0.25 μm, temperature range 320 / 350 °C) was used; the temperature program was as follows: initial temperature 100 °C; increase to 142 °C at 10.0 °C / min; increase to 150 °C at 2.0 °C / min and hold for 5 min; increase to 170 °C at 10.0 °C / min and hold for 5 min; increase to 176 °C at 1.0 °C / min and hold for 3 min; increase to 10 °C / min and hold for 3 min. The temperature was increased from 0℃ / min to 195℃; increased to 200℃ at 1.0℃ / min and held for 2 min; increased to 230℃ at 10.0℃ / min; increased to 245℃ at 1.0℃ / min and held for 5 min; increased to 270℃ at 10.0℃ / min; the injection port temperature was 280℃; the carrier gas was high-purity helium (99.999%); the carrier gas flow rate was 1.5 mL / min; the split ratio was 10:1; and the solvent delay was 2 min. The pressure was 125.4 kPa, the total flow rate was 25.4 mL / min, the column flow rate was 2.04 mL / min, the linear velocity was 52.1 cm / s, the purge flow rate was 3.0 mL / min, and the injection volume was 1 μL.

[0062] (2) Mass spectrometry conditions

[0063] An EI ion source was used; a triple quadrupole mass analyzer was used; the interface temperature was 250℃; the ion source temperature was 280℃; and the scanning range was m / z 50~750.

[0064] (3) Preparation of the test solution

[0065] Accurately weigh 1g of sample, accurately add 10mL of ethyl acetate, weigh the sample, sonicate for 30min (temperature 20℃, power 200W, frequency 40Hz), allow to cool to room temperature, replenish the lost weight, filter with anhydrous sodium sulfate, collect the filtrate, filter through a 0.22μm microporous membrane, and collect the filtrate. This is the test solution for optimizing chromatographic conditions.

[0066] (4) Results

[0067] Palmitic acid, mirosol, and tanshinone IIA peaks were poorly separated; see chromatogram. Figure 4 .

[0068] 1.4.2 Optimization of Chromatographic Methods 3

[0069] (1) Gas chromatography conditions

[0070] An Agilent DB-5MS quartz capillary column (0.25 mm × 30 m, 0.25 μm, temperature range 320 / 350 °C) was used; the temperature program was as follows: initial temperature 100 °C; increase to 142 °C at 10.0 °C / min; increase to 150 °C at 1.5 °C / min and hold for 5 min; increase to 170 °C at 10.0 °C / min and hold for 5 min; increase to 177 °C at 0.8 °C / min and hold for 3 min; increase to 5 °C / min. The temperature was increased from 0℃ / min to 200℃; increased to 210℃ at 2.0℃ / min and held for 3 min; increased to 235℃ at 5.0℃ / min; increased to 240℃ at 1.0℃ / min and held for 3 min; increased to 270℃ at 5.0℃ / min; the injection port temperature was 280℃; the carrier gas was high-purity helium (99.999%); the carrier gas flow rate was 1.5 mL / min; the split ratio was 10:1; and the solvent delay was 2 min. The pressure was 125.4 kPa, the total flow rate was 25.4 mL / min, the column flow rate was 2.04 mL / min, the linear velocity was 52.1 cm / s, the purge flow rate was 3.0 mL / min, and the injection volume was 1 μL.

[0071] (2) Mass spectrometry conditions

[0072] An EI ion source was used; triple quadrupole mass spectrometry was employed; the interface temperature was 250℃; the ion source temperature was 280℃; and the scanning range was m / z 50~750.

[0073] (3) Preparation of the test solution

[0074] The test solution prepared under section 2 was optimized using chromatographic conditions.

[0075] (4) Results

[0076] Palmitic acid, mirosol, and tanshinone IIA peaks were poorly separated; see chromatogram. Figure 5 .

[0077] 1.4.3 Optimization of Chromatographic Methods 4

[0078] (1) Gas chromatography conditions

[0079] An Agilent DB-5MS quartz capillary column (0.25 mm × 30 m, 0.25 μm, temperature range 320 / 350 °C) was used; the temperature program was as follows: initial temperature 100 °C; increase to 142 °C at 10.0 °C / min; increase to 150 °C at 1.5 °C / min and hold for 5 min; increase to 170 °C at 10.0 °C / min and hold for 5 min; increase to 177 °C at 0.5 °C / min and hold for 3 min; increase to 5 °C / min and hold for 3 min. The temperature was increased from 0℃ / min to 200℃; increased to 210℃ at 1.5℃ / min and held for 3 min; increased to 235℃ at 5.0℃ / min; increased to 240℃ at 0.8℃ / min and held for 3 min; increased to 270℃ at 10.0℃ / min; the injection port temperature was 280℃; the carrier gas was high-purity helium (99.999%); the carrier gas flow rate was 1.5 mL / min; the split ratio was 10:1; and the solvent delay was 2 min. The pressure was 125.4 kPa, the total flow rate was 25.4 mL / min, the column flow rate was 2.04 mL / min, the linear velocity was 52.1 cm / s, the purge flow rate was 3.0 mL / min, and the injection volume was 1 μL.

[0080] (2) Mass spectrometry conditions

[0081] An EI ion source was used; the interface temperature was 250℃; the ion source temperature was 280℃; and the scanning range was 50~750 m / z.

[0082] (3) Preparation of the test solution

[0083] The test solution prepared under section 2 was optimized using chromatographic conditions.

[0084] (4) Results

[0085] The chromatographic peaks of palmitic acid and tanshinone IIA were poorly separated.

[0086] 1.4.4 Optimization of Chromatographic Methods 5

[0087] (1) Gas chromatography conditions

[0088] An Agilent DB-5MS quartz capillary column (0.25 mm × 30 m, 0.25 μm, temperature range 320 / 350 °C) was used; the temperature program was as follows: initial temperature 100 °C; increase to 142 °C at 10.0 °C / min; increase to 150 °C at 1.5 °C / min and hold for 2 min; increase to 170 °C at 10.0 °C / min and hold for 5 min; increase to 177 °C at 0.8 °C / min and hold for 3 min; increase to 5.0 °C / min and hold for 3 min. The temperature was increased to 200℃; then increased to 210℃ at 1.0℃ / min and held for 3 min; then increased to 235℃ at 5.0℃ / min; then increased to 240℃ at 1.0℃ / min and held for 3 min; finally increased to 270℃ at 10.0℃ / min and held for 5 min; the injection port temperature was 280℃; the carrier gas was high-purity helium (99.999%); the carrier gas flow rate was 1.5 mL / min; the split ratio was 10:1; and the solvent delay was 2 min. The pressure was 125.4 kPa, the total flow rate was 25.4 mL / min, the column flow rate was 2.04 mL / min, the linear velocity was 52.1 cm / s, and the purge flow rate was 3.0 mL / min. 1 μL of sample was injected.

[0089] (2) Mass spectrometry conditions

[0090] An EI ion source was used; triple quadrupole mass spectrometry was employed; the interface temperature was 250℃; the ion source temperature was 280℃; and the scanning range was m / z 50~750.

[0091] (3) Preparation of the test solution

[0092] The test solution prepared under section 2 was optimized using chromatographic conditions.

[0093] (4) Results

[0094] Palmitic acid and tanshinone IIA peaks were poorly separated; see chromatogram. Figure 7 .

[0095] 1.4.5 Optimization of Chromatographic Methods 6

[0096] (1) Gas chromatography conditions

[0097] An Agilent DB-5MS quartz capillary column (0.25 mm × 30 m, 0.25 μm, temperature range 320 / 350 °C) was used; the temperature program was as follows: initial temperature 100 °C; increase to 142 °C at 10.0 °C / min; increase to 150 °C at 1.5 °C / min and hold for 2 min; increase to 170 °C at 10.0 °C / min and hold for 3 min; increase to 177 °C at 0.8 °C / min; increase to 5.0 °C / min. The temperature was increased to 200℃ and held for 2 min; then increased to 207℃ at 1.0℃ / min and held for 2 min; then increased to 235℃ at 5.0℃ / min and held for 3 min; then increased to 240℃ at 0.8℃ / min; and finally increased to 270℃ at 5.0℃ / min. The injection port temperature was 280℃; the carrier gas was high-purity helium (99.999%); the carrier gas flow rate was 1.5 mL / min; the split ratio was 10:1; and the solvent delay was 2 min. The pressure was 125.4 kPa, the total flow rate was 25.4 mL / min, the column flow rate was 2.04 mL / min, the linear velocity was 52.1 cm / s, and the purge flow rate was 3.0 mL / min. 1 μL of sample was injected.

[0098] (2) Mass spectrometry conditions

[0099] An EI ion source was used; triple quadrupole mass spectrometry was employed; the interface temperature was 250℃; the ion source temperature was 280℃; and the scanning range was m / z 50~750.

[0100] (3) Preparation of the test solution

[0101] The test solution prepared under section 2 was optimized using chromatographic conditions.

[0102] (4) Results

[0103] The five components, atractylodes acetyl alcohol, β-cineole, palmitic acid, mirosinol, and tanshinone IIA, were separated at baseline; see chromatogram. Figure 8 .

[0104] The beneficial effects of the patented technical solution of this invention are as follows:

[0105] (1) Through extensive experimentation and trial and error, the optimal gas chromatography conditions were finally found: Agilent DB-5MS quartz capillary column (0.25mm × 30m, 0.25μm, temperature range 320 / 350℃); programmed temperature rise (initial temperature 100℃; increase to 142℃ at 10.0℃ / min; increase to 150℃ at 1.5℃ / min, hold for 2 min; increase to 170℃ at 10.0℃ / min, hold for 3 min; increase to 142℃ at 0.8℃ / min). The temperature was increased to 77℃; then increased to 200℃ at 5.0℃ / min and held for 2 min; then increased to 207℃ at 1.0℃ / min and held for 2 min; then increased to 235℃ at 5.0℃ / min and held for 3 min; then increased to 240℃ at 0.8℃ / min; then increased to 270℃ at 5.0℃ / min. The injection port temperature was 280℃; the carrier gas was high-purity helium (99.999%); the carrier gas flow rate was 1.5 mL / min; the split ratio was 10:1; and the solvent delay was 2 min. The pressure was 125.4 kPa, the total flow rate was 25.4 mL / min, the column flow rate was 2.04 mL / min, the linear velocity was 52.1 cm / s, and the purge flow rate was 3.0 mL / min.

[0106] (2) Methodological investigation revealed that the RSD of the peak areas for atractylodes lancea, β-cineole, palmitic acid, mirosol, and tanshinone IIA ranged from 0.52% to 3.06%. This indicates that the RSD is less than 5%, demonstrating good precision of the instrument. Repeatability tests showed that the RSD of the peak areas for atractylodes lancea, β-cineole, palmitic acid, mirosol, and tanshinone IIA ranged from 1.28% to 2.58%. Stability tests, conducted at 0, 12, 20, 24, 34, 43, and 48 hours, showed peak area RSDs (%) ranging from 2.54% to 5.08%. These data indicate that the stability meets the requirements. Spike recovery tests showed RSDs ranging from 2.16% to 4.14%.

[0107] (3) Content determination in 15 batches revealed that the content of atractylodes acetylcholine in the Tongmai Jiangtang granules of this invention is 0.0205–0.1893 mg / g, β-cineole 0.0643–0.5737 mg / g, palmitic acid 0.7319–5.1163 mg / g, mirosol 0.0743–0.2356 mg / g, and tanshinone IIA 0.0777–0.8459 mg / g. The above detection methods can serve as internal quality control standards for this product, further ensuring its intrinsic quality. Attached Figure Description

[0108] Figure 1. Total ion chromatogram of volatile oil from Tongmai Jiangtang Capsules: A: Total ion chromatogram of n-hexane extract, B: Total ion chromatogram of ethyl acetate extract, C: Total ion chromatogram of anhydrous ethanol extract.

[0109] Figure 2. Total ion chromatograms of the n-hexane and ethyl acetate extracts. A: Total ion chromatogram of the n-hexane extract; B: Total ion chromatogram of the ethyl acetate extract.

[0110] Figure 3. Total ion chromatograms of the ethyl acetate extract at different solid-liquid ratios, where A: 10 times solid-liquid ratio; B: 12 ​​times solid-liquid ratio; C: 14 times solid-liquid ratio;

[0111] Figure 4 Total ion chromatogram of chromatographic optimization method 2;

[0112] Figure 5 Total ion chromatogram of chromatographic optimization method 3;

[0113] Figure 6 Total ion chromatogram of chromatographic optimization method 4;

[0114] Figure 7 Total ion chromatogram of chromatographic optimization method 5;

[0115] Figure 8 6. Total ion chromatogram of optimized chromatographic method;

[0116] Figure 9 MC diagram of ethyl acetate;

[0117] Figure 10 Mixed standard MC diagram;

[0118] Figure 11 Atractylodes lancea negative MC diagram;

[0119] Figure 12 , Danshen negative MC image. Detailed Implementation

[0120] Unless otherwise defined, the technical or scientific terms used in the specification and claims of this patent application shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0121] Example 1: Construction of the GC-MS quality detection method for the blood vessel clearing and blood sugar lowering traditional Chinese medicine composition of the present invention

[0122] 1. Experimental Instruments and Materials

[0123] 1.1 Instruments and Equipment

[0124] Gas chromatograph-mass spectrometer (GCMS-TQ8040NX, Shimadzu, Japan); high-power ultrasonic cleaner (KQ5200DE, Kunshan Ultrasonic Instrument Co., Ltd.); electronic balance (TE124S, Sartorius); electronic balance (SQP-SECURA225D-1CN, Sartorius).

[0125] 1.2 Experimental Materials Atractylodes lancea alcohol (Chengdu Efa Biotechnology, batch number: AFBF3002, 97%), β-eucalyptol (Chengdu Efa Biotechnology, AFDK0701, 98%), palmitic acid (China National Institute for Food and Drug Control, batch number: 190029-201001), mirocinol (Shanghai Yuanye, batch number: A23IB222781, purity 96%), tanshinone IIA (China National Institute for Food and Drug Control, batch number 110766-202323, 99.5%). Tongmai Jiangtang Capsules were provided by Baoding Tianhao Pharmaceutical Co., Ltd.; n-hexane was chromatographically pure, and ethyl acetate, n-hexane, anhydrous ethanol, anhydrous sodium sulfate, etc., were all analytically pure.

[0126] 2. Experimental methods and results

[0127] 2.1 Establishment of SIM determination method

[0128] 2.1.1 Preparation of the reference solution

[0129] Accurately weigh atractylodes lancea alcohol, β-eucalyptol, palmitic acid, mirosol, and tanshinone IIA reference standards, and prepare reference solutions with concentrations of 0.81408, 0.83032, 1.09172, 1.6318, and 1.3268 mg / mL, respectively. The preparation tables are shown in Table 1.

[0130] Table 1 Preparation of reference solution

[0131]

[0132] 2.1.2 Preparation of mixed reference solution

[0133] Accurately measure 5, 4, 15, 5, and 13 μL of stock solutions of atractylodes lancea alcohol, β-eucalyptol, palmitic acid, mirosol, and tanshinone IIA reference standards, respectively, and place them in a uniform 2 mL volumetric flask. Dilute to the mark with ethyl acetate and shake well to obtain mixed reference standard solutions with concentrations of 2.0758, 2.1834, 9.9510, 2.0360, and 10.6067 μg / mL, respectively.

[0134] 2.1.3 Determination Method

[0135] Chromatographic method 6 was used, followed by filtration, and the injection volume was 1 μL.

[0136] 2.1.4 Results

[0137] Based on the results of the mixed reference standard determination, a compound component table was created, peaks to be processed were selected, background was subtracted, a similarity search was performed, target components were identified, and a SIM quantitative method was established. Target ions and reference ions are shown in Table 2. The default ion tolerance was set to 50%.

[0138] Table 2 Mass-charge ratio of target ion and reference ion

[0139]

[0140]

[0141] 2.2 Methodological Validation

[0142] 2.2.1 Chromatographic and Mass Spectrometry Conditions

[0143] 2.2.1.1 Gas Chromatography Conditions

[0144] An Agilent DB-5MS quartz capillary column (0.25 mm × 30 m, 0.25 μm, temperature range 320 / 350 °C) was used; the temperature program was as follows: initial temperature 100 °C; increase to 142 °C at 10.0 °C / min; increase to 150 °C at 1.5 °C / min and hold for 2 min; increase to 170 °C at 10.0 °C / min and hold for 3 min; increase to 177 °C at 0.8 °C / min; increase to 5.0 °C / min. The temperature was increased to 200℃ and held for 2 min; then increased to 207℃ at 1.0℃ / min and held for 2 min; then increased to 235℃ at 5.0℃ / min and held for 3 min; then increased to 240℃ at 0.8℃ / min; and finally increased to 270℃ at 5.0℃ / min. The injection port temperature was 280℃; the carrier gas was high-purity helium (99.999%); the carrier gas flow rate was 1.5 mL / min; the split ratio was 10:1; and the solvent delay was 2 min. The pressure was 125.4 kPa, the total flow rate was 25.4 mL / min, the column flow rate was 2.04 mL / min, the linear velocity was 52.1 cm / s, and the purge flow rate was 3.0 mL / min. The injection volume was 1 μL.

[0145] 2.2.1.2 Mass Spectrometry Conditions

[0146] An EI ion source was used; the ion source temperature was 280℃, and the interface temperature was 250℃; the scanning range was 50~750 m / z.

[0147] 2.2.1.3 Preparation of reference stock solution

[0148] Accurately weigh 4.28 mg, 5.27 mg, 33.17 mg, 4.24 mg, and 8.20 mg of atractylodes acetyl alcohol, β-cineole, palmitic acid, mirosinol, and tanshinone IIA, respectively, and prepare reference solutions with concentrations of 0.8303 mg / mL, 1.0917 mg / mL, 1.3268 mg / mL, 0.8144 mg / mL, and 1.6318 mg / mL, respectively, using ethyl acetate as the solvent. These solutions are used as reference stock solutions.

[0149] 2.2.1.4 Preparation of the test solution

[0150] Accurately weigh 1.0 g of the negative test sample, accurately add 10 mL of ethyl acetate, weigh the sample, sonicate for 30 min (temperature 20℃, power 200W, frequency 40Hz), let it cool to room temperature, make up the lost weight, filter with anhydrous sodium sulfate, take the filtrate, filter it through a 0.22 μm microporous membrane, and inject the filtrate into the sample.

[0151] 2.2.1.5 Measurement

[0152] Take 1 μL of the reference solution and the test solution, inject them, determine the peak area, and calculate the content.

[0153] 2.2.2 System Adaptability Experiment

[0154] Take the test sample and negative samples lacking Atractylodes lancea and Salvia miltiorrhiza, and prepare the test solution according to the test solution preparation method. Take the above-mentioned reference solution, test solution, and negative test solution lacking Atractylodes lancea and Salvia miltiorrhiza respectively, and measure the results. It was found that the negative samples did not interfere with the determination of atractylodes lancea alcohol, mirosol, and tanshinone IIA.

[0155] 2.2.3 Precision Test

[0156] Prepare mixed reference solutions with concentrations of 99.6384, 98.2548, 99.5100, 97.7290, and 97.9080 μg / mL using the reference stock solution, atractylodes lancea, β-cineole, palmitic acid, mirosol, and tanshinone IIA, respectively. Inject 5 samples and calculate the RSD (%) of the peak area. The required RSD is less than 5%. The results of this experiment show that the RSD is less than 5%, indicating that the instrument's precision meets the requirements. The results are shown in Table 3.

[0157] Table 3 Precision Measurement Results

[0158]

[0159] 2.2.4 Examination of Linear Relationships

[0160] Accurately pipette 2.5, 5.0, 12, 25, 50, 120, and 240 μL of the above reference stock solutions; 9, 18, 35, 90, 130, 150, and 180 μL of the β-cineole reference solution; 30, 75, 150, 225, 300, 377, and 450 μL of the palmitic acid reference solution; 12, 25, 50, 120, 170, 197, and 245 μL of the mirosinol reference solution; and tanshinone II. AReference solutions of 2.5, 6.0, 12, 25, 60, 85, and 120 μL were sequentially placed in 2 mL volumetric flasks, and ethyl acetate was added to the mark. Linear regression was performed with the peak area of ​​the quantitative ion as the ordinate (Y) and the mass of the injected sample (μg) as the abscissa (X). The results are shown in Table 4. The limits of detection and quantitation are also listed.

[0161] Table 4. Linear equations, limits of detection, and limits of quantitation for the five components.

[0162]

[0163] 2.2.5 Repeatability Test

[0164] Accurately weigh 1.0 g of the same batch of Tongmai Jiangtang Capsules, make five parallel samples, accurately add 10 mL of ethyl acetate, weigh, sonicate for 30 min (temperature 20℃, power 200W, frequency 40Hz), let stand at room temperature, replenish the lost weight, filter with anhydrous sodium sulfate, collect the filtrate, filter through a 0.22 μm microporous membrane, collect the filtrate, inject the sample, determine the peak area, and calculate the content using the linear equation. For contents between 0.1% and 0.01%, the RSD (%) of repeatability should be less than 4%; for contents between 0.01% and 0.001%, the RSD (%) of repeatability should be less than 6%. The test results show that the repeatability meets the requirements. The results are shown in Table 5.

[0165] Table 5 Results of Repeatability Experiments

[0166]

[0167] 2.2.6 Stability Test

[0168] Prepare one test solution according to the method for preparing the test solution. Inject the sample at 0, 12, 20, 24, 34, 43, and 48 hours, and measure the peak area. Calculate the RSD (%) of the peak area. For concentrations between 0.1% and 0.01%, the repeatability RSD (%) should be less than 8%; for concentrations between 0.01% and 0.001%, the repeatability RSD (%) should be less than 11%. The test results show that the stability meets the requirements. The results are shown in Table 6.

[0169] Table 6. Stability test results

[0170]

[0171]

[0172] 2.2.7 Spike Recovery Experiment

[0173] Accurately measure 0.25 g of the known content of the hypoglycemic and blood vessel-clearing capsule powder, prepare six parallel aliquots, add an appropriate amount of mixed reference solution, and prepare the test solution according to the preparation method of the test solution. Calculate the recovery rate and RSD. For content between 0.1% and 0.01%, the recovery rate should be 85%–110%, and the RSD (%) should be less than 4%; for content between 0.01% and 0.001%, the recovery rate should be 80%–115%, and the repeatability RSD (%) should be less than 6%. The test results show that the requirements are met. The results are shown in Table 7.

[0174] Table 7 Results of Spike Recovery Test

[0175]

[0176] 2.2.8 Content Determination

[0177] Fifteen batches of Tongmai Jiangtang Capsules were collected, and test solutions were prepared according to the test solution preparation method. The solutions were injected, peak areas were measured, and the contents were calculated by substituting the results into the linear equation. The average contents of atractylodes lancea alcohol, β-cineole, palmitic acid, mirosol, and tanshinone IIA were 0.0838 mg / g, 0.2493 mg / g, 1.5572 mg / g, 0.1211 mg / g, and 0.1949 mg / g, respectively. The results are shown in Table 8.

[0178] Table 8. Results of content determination in Tongmai Jiangtang Capsules samples

[0179]

[0180] 3. Summary and Discussion

[0181] 3.1 The preparation method of the test sample for Tongmai Jiangtang Capsules was optimized. The extraction solvent, the material-liquid ratio, the ultrasonic time, and the ultrasonic power were selected to obtain the preparation method of the test sample.

[0182] 3.2 Methodological optimization of Tongmai Jiangtang Capsules was carried out, including optimization of chromatographic column, temperature gradient, and retention time, to obtain chromatographic and mass spectrometric conditions suitable for the determination of Tongmai Jiangtang Capsules samples.

[0183] 3.3 Methodological validation was performed, and the accuracy, precision, and linearity met the requirements. This method can be used to determine the content of atractylodes lancea alcohol, β-eudesminol, palmitic acid, mirosol, and tanshinone IIA in Tongmai Jiangtang capsules.

Claims

1. A GC-MS quality detection method for a traditional Chinese medicine composition for promoting blood circulation and lowering blood sugar, characterized in that, The detection method includes the following steps: (1) Preparation of test solution: Weigh the blood sugar-lowering capsules, accurately add ethyl acetate, weigh the contents, sonicate for 20-40 min, sonic power 150-250 W, sonic frequency 30-50 Hz, let it stand at room temperature, replenish the lost weight, filter, and take the filtrate to obtain the test solution. (2) Preparation of mixed reference solution: Weigh out the reference standards of atractylodes lancea alcohol, β-eucalyptol, palmitic acid, mirosol, and tanshinone IIA, respectively, and dilute to the mark with ethyl acetate. Shake well to prepare a reference solution with a concentration of 0.5-5.0 μg / mL for atractylodes lancea alcohol, 1.0-5.0 μg / mL for β-eucalyptol, 1.0-20.0 μg / mL for palmitic acid, 0.5-5.0 μg / mL for mirosol, and 2.0-20.0 μg / mL for tanshinone IIA. (3) Gas chromatography conditions: A quartz capillary column, model Agilent DB-5MS, with column specifications of 0.25 mm × 30 m and 0.25 μm, was used. The temperature range was 320–350 °C, and the temperature program was as follows: initial temperature 100 °C; increased to 142 °C at 10.0 °C / min; increased to 150 °C at 1.5 °C / min and held for 2 min; increased to 170 °C at 10.0 °C / min and held for 3 min; increased to 177 °C at 0.8 °C / min; increased to 200 °C at 5.0 °C / min and held for 2 min; increased to 207 °C at 1.0 °C / min and held for 2 min. The temperature was increased to 235℃ at 5.0℃ / min and held for 3 min, then increased to 240℃ at 0.8℃ / min, and finally increased to 270℃ at 5.0℃ / min. The injection port temperature was 270–290℃, the carrier gas was high-purity helium, the carrier gas flow rate was 1.0–2.0 mL / min, the split ratio was 8–12:1, the solvent delay was 1–3 min, the pressure was 120–130 kPa, the total flow rate was 22–27 mL / min, the column flow rate was 1.0–2.5 mL / min, the linear velocity was 40–60 cm / s, and the purge flow rate was 2.0–4.0 mL / min. (4) Mass spectrometry conditions: EI ion source was used; ion source temperature was 270–290℃, interface temperature was 240–260℃; scan range was m / z 50–750. (5) Content determination: Take the mixed reference solution from step (2) and the test solution from step (1), inject the sample, determine the peak area, and calculate the content.

2. The quality inspection method as described in claim 1, characterized in that, The detection method step (1) specifies an ultrasonic time of 30 min, an ultrasonic power of 200 W, and an ultrasonic frequency of 40 Hz.

3. The quality inspection method as described in claim 1, characterized in that, In step (2) of the detection method, the concentrations of atractylodes acetonide, β-eucalyptol, palmitic acid, mitochondriol, and tanshinone IIA in the mixed reference solution are 2.0758 μg / mL, 2.1834 μg / mL, 9.9510 μg / mL, 2.0360 μg / mL, and 10.6067 μg / mL.

4. The quality inspection method as described in claim 1, characterized in that, In step (3) of the detection method, the injection port temperature is 280℃, the carrier gas is high-purity helium, the carrier gas flow rate is 1.5 mL / min, the split ratio is 10:1, the solvent delay is 2 min, the pressure is 125.4 kPa, the total flow rate is 25.4 mL / min, the column flow rate is 2.04 mL / min, the linear velocity is 52.1 cm / s, and the purge flow rate is 3.0 mL / min.

5. The quality inspection method as described in claim 1, characterized in that, According to step 4, the ion source temperature is 280℃ and the interface temperature is 250℃.

6. The quality inspection method as described in claim 1, characterized in that, The mass content of the Tongmai Jiangtang Capsules of the present invention was determined according to the gas chromatography and mass spectrometry conditions in steps (3) and (4). The contents of the capsules were: atractylodes acetonitrile 0.0205-0.189 mg / g, β-eucalyptol 0.0643-0.5737 mg / g, palmitic acid 0.7319-5.1163 mg / g, mirosol 0.0743-0.2356 mg / g, and tanshinone IIA 0.0777-0.8459 mg / g.

7. The quality inspection method as described in claim 6, characterized in that, The mass content of the traditional Chinese medicine composition of the present invention was determined according to the gas chromatography and mass spectrometry conditions in step (3) and (4), wherein the content of atractylodes lancea alcohol was 0.0838 mg / g, the content of β-eudesminol was 0.2493 mg / g, the content of palmitic acid was 1.5572 mg / g, the content of mirocin was 0.1211 mg / g, and the content of tanshinone IIA was 0.1949 mg / g.

8. The quality inspection method as described in claim 1, characterized in that, The detection method is used in the quality detection, intermediate determination, content determination, and identification of effective components of traditional Chinese medicine compositions for promoting blood circulation and lowering blood sugar.

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