GC-MS (Gas Chromatography-Mass Spectrometer) quality detection method of traditional Chinese medicine composition capable of dredging

Abstract

The invention provides a GC-MS (Gas Chromatography-Mass Spectrometer) quality detection method for a pulse-invigorating and blood-glucose-reducing traditional Chinese medicine composition. The detection method has the advantages of simple and convenient operation steps, accurate detection and high stability. According to the preparation method, volatile components in the blood-glucose-promoting and blood-glucose-reducing granules are determined, and 15 batches of content determination shows that the content of atractylenol in the blood-glucose-promoting and blood-glucose-reducing granules is 0.0205 to 0.1893 mg / g, the content of beta-eudesmol in the blood-glucose-promoting and blood-glucose-reducing granules is 0.0643 to 0.5737 mg / g, the content of palmitic acid in the blood-glucose-promoting and blood-glucose-reducing granules is 0.7319 to 5.1163 mg / g, the content of ferruginol in the blood-glucose-promoting and blood-glucose-reducing granules is 0.0743 to 0.2356 mg / g, and the content of tanshinone IIA in the blood- The detection method can be used as an internal control quality detection standard of the product to further guarantee the product quality. And the detection method can also be used for identifying the chemical components of the traditional Chinese medicine composition for dredging collaterals and reducing blood glucose, and has a good market prospect.

Description

Technical Field

[0001] The present invention belongs to the field of quality detection of chemical components of traditional Chinese medicines, and in particular relates to a GC-MS quality detection method for a meridian-opening and blood sugar-lowering traditional Chinese medicine composition. Background Art

[0002] Tongmai Jiangtang Capsule is an oral solid preparation prepared from Radix Pseudostellariae, Radix Salviae Miltiorrhizae, Rhizoma Coptidis, Radix Astragali, Gynostemma Pentaphyllum, Rhizoma Dioscoreae, Rhizoma Atractylodis Macrocephalae, Radix Scrophulariae, Hirudo, Fructus Malvae, Radix Puerariae, etc., which has the effects of nourishing yin and clearing away heat, clearing away heat and activating blood circulation. It is used for diabetes mellitus caused by deficiency of both qi and yin and blood stasis. Regarding the medicinal materials involved in Tongmai Jiangtang, Radix Pseudostellariae contains cyclic peptides, sugars, glycosides, fatty acids, oils and fats, volatile oils and other ingredients, and its polysaccharides have hypoglycemic activity. The tanshinones and salvianolic acids in Radix Salviae Miltiorrhizae have hypoglycemic effects. Rhizoma Coptidis contains alkaloids, flavonoids, lignins, organic acids, volatile oils and other ingredients, and its alkaloids and polysaccharides have hypoglycemic effects. Radix Astragali contains polysaccharides, saponins, and flavonoids, and its polysaccharides and flavonoids have hypoglycemic effects. Gynostemma pentaphyllum contains gypenosides, polysaccharides, and flavonoids. Gynostemma pentaphyllum saponins, polysaccharides, and crude extracts have hypoglycemic effects. Chinese yam contains polysaccharides, proteins, amino acids, esters, and other components. Its polysaccharides have a blood sugar-lowering effect. Atractylodes lancea contains sesquiterpenes, enynes, and organic acids. Its aqueous, ethanolic, and ethyl acetate extracts have hypoglycemic effects. Scrophularia ningpoensis contains iridoid ether glycosides, phenylpropanoids, flavonoids, and polysaccharides. Its polysaccharides and various alcohol extracts have hypoglycemic effects. Winter mallow fruit contains acidic compounds, flavonoids, and sterols. Its flavonoids have hypoglycemic effects. Pueraria root contains flavonoids, organic acids, and triterpenes. Its flavonoids have a blood sugar-lowering effect.

[0003] A review of existing technical literature on Tongmai Jiangtang Capsules reveals that most content determination methods focus on water-soluble and alcohol-soluble chemical components. For example, He Yaling, Che Xiaoyan, et al., "Determination of Astragaloside I in Tongmai Jiangtang Granules by Thin-Layer Chromatography," published on March 30, 1998, employed thin-layer chromatography (TLC) with a developing solvent of chloroform-methanol-water (13:6:2). The plate was then developed, air-dried, and sprayed with ethanolic sulfuric acid to ensure uniform wetting. Detection wavelengths were set at 500 nm and 700 nm to determine the content of astragaloside I. The Jiangtang Tongmai Capsules standard, WS-10116 (ZD-0116)-2002, discloses the use of methanol-water (15:5) as the mobile phase and a detection wavelength of 270 nm. The reference solution and the test solution were accurately aspirated and injected into a liquid chromatograph to determine the content of danshen (calculated as tanshinone IIA (C₁₆H₁₈O₃), which must not be less than 0.15 mg. Xue Li, Determination of berberine hydrochloride in Tongmai Jiangtang Granules by HPLC, Chinese Journal of Hospital Pharmacy, acetonitrile and water (55:35) as mobile phase, detection wavelength 345nm, detection of berberine hydrochloride content.

[0004] However, there are no relevant reports on the study of volatile oil components in Tongmai Jiangtang Capsules. This study used 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] The present invention provides a GC-MS quality detection method for a Tongmai Jiangtang Chinese medicine composition, which has the advantages of simple operating steps, accurate detection, and high stability. The present invention determines the volatile components in Tongmai Jiangtang. After 15 batches of content determination, it can be seen that the content of atractylodesin in the Tongmai Jiangtang granules of the present invention is 0.0205-0.1893 mg / g, the content of β-eudesmol is 0.0643-0.5737 mg / g, the content of palmitic acid is 0.7319-5.116 mg / g, the content of silanol is 0.0743-0.2356 mg / g, and the content of tanshinone IIA is 0.0777-0.8459 mg / g. The above detection method can be used as an internal control quality detection standard for the product to further ensure product quality. In addition, the detection method can also be used for the chemical composition and identification of Tongmai Jiangtang Chinese medicine compositions, and has good market application prospects.

[0006] The technical solution of the present invention patent application is:

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

[0008] (1) Preparation of test solution: Weigh the Chinese medicine composition, accurately add ethyl acetate, weigh the weight, ultrasonic time 20-40 min, ultrasonic power 150-250 W, ultrasonic frequency 30-50 Hz, let it cool to room temperature, make up the lost weight, filter, and take the filtrate;

[0009] (2) Preparation of mixed reference solution: Weigh the reference substances of atractylodesinol, β-eudesmol, palmitic acid, mirocinol, and tanshinone IIA respectively, dilute to the mark with ethyl acetate, shake well, and prepare a reference solution with atractylodesinol concentration of 0.5-5.0 μg / mL, β-eudesmol concentration of 1.0-5.0 μg / mL, palmitic acid concentration of 1.0-20.0 μg / mL, mirocinol concentration of 0.5-5.0 μg / mL, and tanshinone IIA concentration of 2.0-20.0 μg / mL.

[0010] ⑶. Gas chromatography conditions:

[0011] A quartz capillary column was used, with a temperature range of 320-350°C and a temperature program of: initial temperature 100°C; heating to 142°C at 10.0°C / min; heating to 150°C at 1.5°C / min, holding for 2 min; heating to 170°C at 10.0°C / min, holding for 3 min; heating to 177°C at 0.8°C / min; heating to 200°C at 5.0°C / min, holding for 2 min; heating to 207°C at 1.0°C / min, holding for 2 min; heating to 235°C at 5.0°C / min , hold for 3 minutes; heat up to 240°C at 0.8°C / min; heat up to 270°C at 5.0°C / min); inlet temperature 270-290°C; carrier gas is high-purity helium; carrier gas flow rate 1.0-2.0 mL / min; split ratio 8-12:1; solvent delay 1-3 minutes, pressure 120-130 kPa, total flow rate 22-27 mL / min, column flow rate 1.0-2.5 mL / min, linear velocity 40-60 cm / s, purge flow rate 2.0-4.0 mL / min;

[0012] (4) Mass spectrometry conditions: EI ion source; ion source temperature 270-290°C, interface temperature 240-260°C; scan range m / z 50-750;

[0013] ⑸. Determination of content: Take the mixed reference solution in step ⑵ and the test solution in step ⑴, inject them, determine the peak area, and calculate the content.

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

[0015] Preferably, in step ⑵ of the detection method, the concentration of atractylodesin in the mixed reference solution is 2.0758 μg / mL, the concentration of β-eudesmol is 2.1834 μg / mL, the concentration of palmitic acid is 9.9510 μg / mL, the concentration of mirabelle is 2.0360 μg / mL, and the concentration of tanshinone IIA is 10.6067 μg / mL.

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

[0017] Preferably, in step (3) of the detection method, the injection port temperature is 280°C; 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 chromatographic 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, the ion source temperature in step (4) is 280°C and the interface temperature is 250°C.

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

[0020] Preferably, the mass content of the traditional Chinese medicine composition of the present invention is determined according to the gas chromatography and mass spectrometry conditions in step (3), wherein the atractylodesinol content is 0.0838 mg / g, the β-cineole content is 0.2493 mg / g, the palmitic acid content is 1.5572 mg / g, the silanol content is 0.1211 mg / g, and the tanshinone IIA content is 0.1949 mg / g.

[0021] Preferably, the detection method is used for quality detection, intermediate and content determination of Tongmai Jiangtang, and identification of effective substance components of traditional Chinese medicine.

[0022] In order to further illustrate the creativity of the detection method of Tongmai Jiangtang Granules of the present invention, some experimental contents screened by 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 oil

[0025] 1.1.1.1 Solvent extraction of volatile oil

[0026] Take Tongmai Jiangtang Capsules, 1 g each, and make 3 parallel portions. Accurately weigh and add 100 mL of n-hexane, ethyl acetate, and anhydrous ethanol respectively. Ultrasonic treatment (time 30 min, power 400 W, frequency 40 Hz) was performed. The mixture was cooled and filtered through anhydrous sodium sulfate. The filter residue and filter paper were repeatedly washed with the extraction solvent. The filtrate and washing liquid were collected and dried.

[0027] 1.1.1.2 Sample methylation treatment

[0028] Take a small amount of the extracted Tongmai Jiangtang Capsule volatile oil paste, add 5 mL of 0.5 M KOH methanol solution, place in a 70°C water bath for saponification reaction for 10 minutes, and shake occasionally during the saponification reaction to allow the oil to fully react. Cool for 3 minutes, add 3 mL of BF3 methanol solution, and place in a 70°C water bath for saponification reaction for 5 minutes to fully methylate. Then add 10 mL of saturated sodium chloride solution and a small amount of chromatographic grade n-hexane. Let it stand and separate the 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] A SH-RXi-5SilMS quartz capillary column (0.25 mm × 30 m, 0.25 μm, temperature range 320 / 350°C) was used; programmed temperature (initial temperature 60°C, equilibration 3 min; heating to 100°C at 5.0°C / min, hold 5 min; heating to 150°C at 3.0°C / min, hold 5 min; heating to 200°C at 3.0°C / min, hold 5 min; heating to 250°C at 3.0°C / min, hold 5 min; heating to 270°C at 5.0°C / min, hold 2 min); injection port temperature 280°C; carrier gas was high-purity helium (99.999%); carrier gas flow rate was 1.5 mL / min; split ratio was 10:1; solvent delay was 2 min. Pressure 125.4 kPa, total flow 25.4 mL / min, column flow 2.04 mL / min, linear velocity 52.1 cm / s, purge flow 3.0 mL / min. Inject 1 μL.

[0032] 1.1.2.2 Mass spectrometry conditions

[0033] An EI ion source and a triple quadrupole mass analyzer were used. The interface temperature was 250°C, the ion source temperature was 280°C, and the scanning range was m / z 50-750.

[0034] 1.1.3 Results

[0035] Similarity search showed that 40, 38, and 38 compounds were analyzed for n-hexane, ethyl acetate, and anhydrous ethanol, respectively. Anhydrous ethanol analyzed more fatty acid compounds. Seven identical components were analyzed for n-hexane and ethyl acetate, and the components with relatively high contents were those extracted from ethyl acetate. The total ion current is shown in ( 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; programmed temperature (initial temperature 60 °C, equilibration 30 min; heating to 100 °C at 5.0 °C / min; heating to 150 °C at 3.0 °C / min, hold for 5 min; heating to 200 °C at 3.0 °C / min, hold for 5 min; heating to 250 °C at 3.0 °C / min, hold for 5 min; heating to 270 °C at 5.0 °C / min, hold for 2 min); injection port temperature of 280 °C; carrier gas was high-purity helium (99.999%); carrier gas flow rate was 1.5 mL / min; split ratio was 10:1; solvent delay was 2 min. Pressure 125.4KPa, total flow 25.4mL / min, column flow 2.04mL / min, linear velocity 52.1cm / s, purge flow 3.0mL / min,

[0039] 1.2.1.2 Mass spectrometry conditions

[0040] An EI ion source and a triple quadrupole detector were used; the interface temperature was 250°C; the ion source temperature was 280°C; and the scan range was m / z 50–750. A 1 μL injection was used.

[0041] 1.2.1.3 Preparation of test solution

[0042] (1) Hexane extraction

[0043] Accurately weigh 0.5 g of the sample, 2 portions, accurately add 50 mL of n-hexane to each portion, weigh the weight, ultrasonicate for 30 min (temperature 20°C, power 200 W, frequency 40 Hz), let it cool to room temperature, make up for the lost weight, filter with anhydrous sodium sulfate, take the filtrate, filter with a 0.22 μm microporous filter membrane, and take the filtrate to obtain the product.

[0044] (2) Ethyl acetate extraction

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

[0046] 1.2.1.4 Results

[0047] The volatile oil extracted with n-hexane has fewer components, a large solid-liquid ratio, and a greater impact of baseline noise. Ethyl acetate uses a 1:10 solid-liquid ratio, has a wider variety of components, and has a smaller impact of noise. Chromatographic determination results can be found in Figure 2-A , Figure 2-B Therefore, ethyl acetate was selected as the extraction solvent. Atractylodesinol, β-eudesmol, palmitic acid, mirabelle, and tanshinone ⅡA were selected as the components to be tested.

[0048] 1.3 Selection of solvent dosage

[0049] 1.3.1 Chromatographic 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; programmed temperature (initial temperature 60 °C, equilibration 30 min; heating to 100 °C at 5.0 °C / min; heating to 150 °C at 3.0 °C / min, hold for 5 min; heating to 200 °C at 3.0 °C / min, hold for 5 min; heating to 250 °C at 3.0 °C / min, hold for 5 min; heating to 270 °C at 5.0 °C / min, hold for 2 min); injection port temperature of 280 °C; carrier gas was high-purity helium (99.999%); carrier gas flow rate was 1.5 mL / min; split ratio was 10:1; solvent delay was 2 min. Pressure 125.4 kPa, total flow 25.4 mL / min, column flow 2.04 mL / min, linear velocity 52.1 cm / s, purge flow 3.0 mL / min. Inject 1 μL.

[0052] 1.3.1.2 Mass spectrometry conditions

[0053] An EI ion source and a triple quadrupole mass spectrometer were used. The interface temperature was 250°C, the ion source temperature was 280°C, and the scanning range was m / z 50-750.

[0054] 1.3.2 Preparation of test solution

[0055] Accurately weigh 1 g of the sample, 3 portions, accurately add 10, 12, and 14 mL of ethyl acetate respectively, weigh the weight, ultrasonicate for 30 min (temperature 20°C, power 200 W, frequency 40 Hz), let it cool to room temperature, make up for the lost weight, filter with anhydrous sodium sulfate, take the filtrate, filter with a 0.22 μm microporous filter membrane, and take the filtrate to obtain the product.

[0056] 1.3.3 Results

[0057] The material-liquid ratio is 1:10. The content of atractylodesinol, β-eudesmol, palmitic acid, mirabeneol and tanshinone ⅡA in the test sample is high, so the material-liquid ratio of 1:10 is selected. The test results show that Figure 3-A , B, C).

[0058] 1.4 Optimization of chromatographic conditions

[0059] 1.4.1 Chromatographic method optimization 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 was programmed (initial temperature 100 °C; heating at 10.0 °C / min to 142 °C; heating at 2.0 °C / min to 150 °C, hold for 5 min; heating at 10.0 °C / min to 170 °C, hold for 5 min; heating at 1.0 °C / min to 176 °C, hold for 3 min; heating at 10. The temperature was increased from 0°C / min to 195°C; from 1.0°C / min to 200°C, held for 2 min; from 10.0°C / min to 230°C; from 1.0°C / min to 245°C, held for 5 min; and from 10.0°C / min to 270°C. The inlet 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. 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 and a triple quadrupole mass analyzer were used. The interface temperature was 250°C, the ion source temperature was 280°C, and the scanning range was m / z 50-750.

[0064] (3) Preparation of test solution

[0065] Accurately weigh 1 g of sample, accurately add 10 mL of ethyl acetate, weigh the sample, and sonicate for 30 min (temperature 20°C, power 200 W, frequency 40 Hz). Allow to cool to room temperature, make up for the loss, filter through anhydrous sodium sulfate, and filter the filtrate through a 0.22 μm microporous membrane. This filtrate is used as the test solution for chromatographic condition optimization.

[0066] (4) Results

[0067] The chromatographic peaks of palmitic acid, silanol, and tanshinone ⅡA are not well separated. See chromatographic Figure 4 .

[0068] 1.4.2 Chromatographic method optimization 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 was programmed (initial temperature 100 °C; heating at 10.0 °C / min to 142 °C; heating at 1.5 °C / min to 150 °C, holding for 5 min; heating at 10.0 °C / min to 170 °C, holding for 5 min; heating at 0.8 °C / min to 177 °C, holding for 3 min; heating at 5. The temperature was increased from 0°C / min to 200°C; from 2.0°C / min to 210°C, held for 3 min; from 5.0°C / min to 235°C; from 1.0°C / min to 240°C, held for 3 min; and from 5.0°C / min to 270°C. The inlet 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. 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 and a triple quadrupole mass spectrometer were used. The interface temperature was 250°C, the ion source temperature was 280°C, and the scanning range was m / z 50-750.

[0073] (3) Preparation of test solution

[0074] Use the test solution prepared under chromatographic condition optimization 2.

[0075] (4) Results

[0076] The chromatographic peaks of palmitic acid, silanol, and tanshinone ⅡA are not well separated. See chromatographic Figure 5 .

[0077] 1.4.3 Chromatographic method optimization 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 was programmed (initial temperature 100 °C; heating at 10.0 °C / min to 142 °C; heating at 1.5 °C / min to 150 °C, holding for 5 min; heating at 10.0 °C / min to 170 °C, holding for 5 min; heating at 0.5 °C / min to 177 °C, holding for 3 min; heating at 5. The temperature was increased from 0°C / min to 200°C; from 1.5°C / min to 210°C, held for 3 min; from 5.0°C / min to 235°C; from 0.8°C / min to 240°C, held for 3 min; and from 10.0°C / min to 270°C. The inlet 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. 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°C; the ion source temperature was 280°C, and the scanning range was m / z 50-750.

[0082] (3) Preparation of test solution

[0083] Use the test solution prepared under chromatographic condition optimization 2.

[0084] (4) Results

[0085] The chromatographic peaks of palmitic acid and tanshinone ⅡA were not well separated.

[0086] 1.4.4 Chromatographic method optimization 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 was programmed (initial temperature 100 °C; heating to 142 °C at 10.0 °C / min; heating to 150 °C at 1.5 °C / min, holding for 2 min; heating to 170 °C at 10.0 °C / min, holding for 5 min; heating to 177 °C at 0.8 °C / min, holding for 3 min; heating to 177 °C at 5.0 °C / min, holding for 3 min). The temperature was raised to 200°C at 1.0°C / min; then to 210°C, held for 3 minutes; then to 235°C at 5.0°C / min; then to 240°C at 1.0°C / min, held for 3 minutes; then to 270°C at 10.0°C / min, held for 5 minutes. The inlet temperature was 280°C. The carrier gas was high-purity helium (99.999%) at a flow rate of 1.5 mL / min. The split ratio was 10:1, and the solvent delay was 2 minutes. 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. A 1 μL injection was performed.

[0089] (2) Mass spectrometry conditions

[0090] An EI ion source and a triple quadrupole mass spectrometer were used. The interface temperature was 250°C, the ion source temperature was 280°C, and the scanning range was m / z 50-750.

[0091] (3) Preparation of test solution

[0092] Use the test solution prepared under chromatographic condition optimization 2.

[0093] (4) Results

[0094] The chromatographic peaks of palmitic acid and tanshinone ⅡA are not well separated. See chromatographic Figure 7 .

[0095] 1.4.5 Chromatographic method optimization 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 was programmed (initial temperature 100 °C; heating to 142 °C at 10.0 °C / min; heating to 150 °C at 1.5 °C / min, holding for 2 min; heating to 170 °C at 10.0 °C / min, holding for 3 min; heating to 177 °C at 0.8 °C / min; heating to 177 °C at 5.0 °C / min, holding for 3 min). Heat to 200°C, hold for 2 minutes; increase the temperature at 1.0°C / min to 207°C, hold for 2 minutes; increase the temperature at 5.0°C / min to 235°C, hold for 3 minutes; increase the temperature at 0.8°C / min to 240°C; and increase the temperature at 5.0°C / min to 270°C. Injection port temperature was 280°C. Carrier gas was high-purity helium (99.999%). Carrier gas flow rate was 1.5 mL / min. Split ratio was 10:1. Solvent delay was 2 minutes. Pressure was 125.4 kPa, total flow rate was 25.4 mL / min, column flow rate was 2.04 mL / min, linear velocity was 52.1 cm / s, and purge flow rate was 3.0 mL / min. Injection was 1 μL.

[0098] (2) Mass spectrometry conditions

[0099] An EI ion source and a triple quadrupole mass spectrometer were used. The interface temperature was 250°C, the ion source temperature was 280°C, and the scanning range was m / z 50-750.

[0100] (3) Preparation of test solution

[0101] Use the test solution prepared under chromatographic condition optimization 2.

[0102] (4) Results

[0103] Baseline separation of the five components: atractylodesinol, β-eudesmol, palmitic acid, mirabeneol, and tanshinone IIA. See chromatogram. Figure 8 .

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

[0105] ⑴. After a lot of experimental exploration and attempts, the present invention finally found the optimal gas chromatography conditions: Agilent DB-5MS quartz capillary column (0.25mm×30m, 0.25μm, temperature range 320 / 350℃); programmed temperature (initial temperature 100℃; heating to 142℃ at 10.0℃ / min; heating to 150℃ at 1.5℃ / min, holding for 2min; heating to 170℃ at 10.0℃ / min, holding for 3min; heating to 1 at 0.8℃ / min, holding for 1min). 77°C; increase the temperature at 5.0°C / min to 200°C, hold for 2 min; increase the temperature at 1.0°C / min to 207°C, hold for 2 min; increase the temperature at 5.0°C / min to 235°C, hold for 3 min; increase the temperature at 0.8°C / min to 240°C; increase the temperature at 5.0°C / min to 270°C); inlet 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.

[0106] (2) Methodological investigations revealed that the RSDs for the peak areas of atractylol, β-eudesmol, palmitic acid, mirocinol, and tanshinone IIA ranged from 0.52% to 3.06%. This indicates that the RSDs are less than 5%, indicating good instrument precision. Repeatability experiments revealed that the RSDs for the peak areas of atractylol, β-eudesmol, palmitic acid, mirocinol, and tanshinone IIA ranged from 1.28% to 2.58%. Stability experiments revealed that the peak area RSDs (%) for injections at 0, 12, 20, 24, 34, 43, and 48 hours were 2.54% to 5.08%. These data demonstrate that stability meets the requirements. Spike recovery experiments revealed RSDs of 2.16% to 4.14%.

[0107] (3) After 15 batches of content determination, it was found that the content of atractylodesin in the Tongmai Jiangtang Granules of the present invention was 0.0205-0.1893 mg / g, the content of β-eudesmol was 0.0643-0.5737 mg / g, the content of palmitic acid was 0.7319-5.1163 mg / g, the content of silanol was 0.0743-0.2356 mg / g, and the content of tanshinone IIA was 0.0777-0.8459 mg / g. The above detection method can be used as the internal control quality standard of the product to further ensure the intrinsic quality of the product. BRIEF DESCRIPTION OF THE DRAWINGS

[0108] Figure 1, total ion currents of the volatile oil of Tongmai Jiangtang Capsules, A: total ion current of the n-hexane extract, B: total ion current of the ethyl acetate extract, C: total ion current of the anhydrous ethanol extract;

[0109] Figure 2, total ion currents of n-hexane and ethyl acetate extracts, A: total ion current of n-hexane extract; B: total ion current of ethyl acetate extract;

[0110] Figure 3, total ion currents of 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 current profile 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 , total ion current profile of chromatographic optimization method 6;

[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 diagram. DETAILED DESCRIPTION

[0120] Unless otherwise defined, the technical or scientific terms used in the patent application specification and claims of the present invention shall have the common meanings understood by persons having ordinary skills in the field to which the invention belongs.

[0121] Example 1 Construction of GC-MS quality detection method for the Tongmai Jiangtangsugar 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 crusher (KQ5200DE, Kunshan Ultrasonic Instrument Co., Ltd.); electronic balance (TE124S, Sartorius), electronic balance (SQP-SECURA225D-1CN, Sartorius).

[0125] 1.2 Experimental Materials: Atractylodesinol (Chengdu Aifa Biotechnology, batch number: AFBF3002, 97%), β-eudesmol (Chengdu Aifa Biotechnology, AFDK0701, 98%), palmitic acid (National Institute for the Control of Traditional Chinese Medicine and Food and Drug Administration, batch number: 190029-201001), mirabeneol (Shanghai Yuanye, batch number: A23IB222781, purity 96%), and tanshinone IIA (National Institute for the Control of Traditional Chinese Medicine and Food and Drug Administration, batch number: 110766-202323, 99.5%). Tongmai Jiangtang Capsules were provided by Baoding Tianhao Pharmaceutical Co., Ltd.; n-hexane was chromatographically pure; ethyl acetate, n-hexane, anhydrous ethanol, and anhydrous sodium sulfate were all analytically pure.

[0126] 2 Experimental methods and results

[0127] 2.1 Establishment of SIM determination method

[0128] 2.1.1 Preparation of reference solution

[0129] Accurately weigh atractylodesinol, β-eudesmol, palmitic acid, mirocine, and tanshinone IIA reference substances and prepare reference substance solutions with concentrations of 0.81408, 0.83032, 1.09172, 1.6318, and 1.3268 mg / mL, respectively. The preparation table is 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 the reference substance stock solutions of atractylodesin, β-eudesmol, palmitic acid, miltiotropium, and tanshinone IIA, respectively, place them in a unified 2 mL volumetric flask, dilute to the mark with ethyl acetate, and shake well to obtain mixed reference substance 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, filtered, and the injection volume was 1 μL.

[0136] 2.1.4 Results

[0137] Based on the results of the mixed reference sample assay, create a compound component table, select peaks to be processed, subtract background, perform a similarity search, identify target components, and establish a SIM quantification method. The target and reference ions are listed in Table 2. The default ion tolerance is set to 50%.

[0138] Table 2 Charge-to-mass ratio of target ions and reference ions

[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 was programmed (initial temperature 100 °C; heating to 142 °C at 10.0 °C / min; heating to 150 °C at 1.5 °C / min, holding for 2 min; heating to 170 °C at 10.0 °C / min, holding for 3 min; heating to 177 °C at 0.8 °C / min; heating to 177 °C at 5.0 °C / min, holding for 3 min). Heat to 200°C, hold for 2 min; increase at 1.0°C / min to 207°C, hold for 2 min; increase at 5.0°C / min to 235°C, hold for 3 min; increase at 0.8°C / min to 240°C; and increase at 5.0°C / min to 270°C. Inlet 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 volume: 1 μL.

[0145] 2.2.1.2 Mass spectrometry conditions

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

[0147] 2.2.1.3 Preparation of reference substance stock solution

[0148] Accurately weigh 4.28 mg, 5.27 mg, 33.17 mg, 4.24 mg, and 8.20 mg of atractylodesinol, β-eudesmol, palmitic acid, tanshinone IIA, and use ethyl acetate as solvent to 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, as reference stock solutions.

[0149] 2.2.1.4 Preparation of test solution

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

[0151] 2.2.1.5 Determination

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

[0153] 2.2.2 System adaptability experiment

[0154] Prepare the test solution using the test sample and negative samples lacking Atractylodes lancea and Salvia miltiorrhiza according to the test solution preparation method. Separately, take the control solution, the test sample solution, and the negative test solution lacking Atractylodes lancea and Salvia miltiorrhiza. The results show that the negative samples do not interfere with the determination of atractylol, silanol, and tanshinone IIA.

[0155] 2.2.3 Precision test

[0156] Mixed reference solutions of atractylodesinol, β-eudesmol, palmitic acid, schizonephenol, and tanshinone IIA were prepared at concentrations of 99.6384, 98.2548, 99.5100, 97.7290, and 97.9080 μg / mL, respectively. Five injections were made, and the relative standard deviations (RSDs) of the peak areas were calculated. The RSD requirement was less than 5%, which was observed in this test, indicating that the instrument met the requirements. The results are shown in Table 3.

[0157] Table 3 Precision determination results

[0158]

[0159] 2.2.4 Investigation of linear relationship

[0160] Take the above-mentioned reference substance stock solution, accurately draw 2.5, 5.0, 12, 25, 50, 120, 240 μL of atractylodes alcohol reference solution, 9, 18, 35, 90, 130, 150, 180 μL of β-eudesmol reference solution, 30, 75, 150, 225, 300, 377, 450 μL of palmitic acid reference solution, 12, 25, 50, 120, 170, 197, 245 μL of tanshinone II A2.5, 6.0, 12, 25, 60, 85, and 120 μL of the reference solution were placed in 2 mL volumetric flasks, and ethyl acetate was added to the mark. Linear regression was performed using the peak area of ​​the quantification ion as the ordinate (Y) and the mass of the injected sample (μg) as the abscissa (X). The results are shown in Table 4. Detection limit and quantification limit.

[0161] Table 4 Linear equations and detection limits and quantification limits of five components

[0162]

[0163] 2.2.5 Repeatability test

[0164] Take Tongmai Jiangtang Capsules of the same batch number, accurately weigh 1.0g of the test sample, parallel 5 portions, accurately add 10mL of ethyl acetate, weigh the weight, ultrasonicate 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 with a 0.22μm microporous filter membrane, take the filtrate, inject, measure the peak area, and calculate the content by linear equation. The content is between 0.1% and 0.01%, and the RSD (%) of the repeatability is less than 4%. The content is between 0.01 and 0.001%, and the RSD (%) of the repeatability is less than 6%. The test results show that the repeatability meets the requirements. The results are shown in Table 5.

[0165] Table 5 Repeatability test results

[0166]

[0167] 2.2.6 Stability test

[0168] Prepare one aliquot of the test solution according to the test solution preparation method. Inject the sample at 0, 12, 20, 24, 34, 43, and 48 hours. Measure the peak area and calculate the RSD (%) of the peak area. For concentrations between 0.1% and 0.01%, the RSD (%) of repeatability should be less than 8%. For concentrations between 0.01% and 0.001%, the RSD (%) of repeatability should be less than 11%. These test results demonstrate that stability meets the requirements. See Table 6 for the results.

[0169] Table 6 Stability test results

[0170]

[0171]

[0172] 2.2.7 Spike recovery experiment

[0173] Precisely measure 0.25g of Jiangtang Tongmai Capsule powder of known content in six replicates, add an appropriate amount of the mixed reference solution, and prepare the test solution according to the test solution preparation method. Calculate the recovery and RSD. For content between 0.1% and 0.01%, the recovery should be 85-110% with an RSD of less than 4%. For content between 0.01% and 0.001%, the recovery should be 80-115% with a repeatability RSD of less than 6%. As can be seen from the test results, these requirements are met. See Table 7 for the results.

[0174] Table 7 Spiked recovery test results

[0175]

[0176] 2.2.8 Content determination

[0177] Fifteen batches of Tongmai Jiangtang Capsules were prepared using the test solution preparation method. Samples were injected, and the peak areas were measured. The peak areas were substituted into the linear equation to calculate the content. The average contents of atractylodesinol, β-eudesmol, palmitic acid, silanol, 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 Tongmai Jiangtang Capsule sample content determination results

[0179]

[0180] 3 Summary and Discussion

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

[0182] 3.2 The methodological optimization of Tongmai Jiangtang Capsules was carried out, and the chromatographic column, temperature gradient, and retention time were optimized to obtain the chromatographic mass spectrometry conditions that can be used 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 contents of atractylodesinol, β-eudesmol, palmitic acid, miltiotropium, 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 reducing blood sugar, characterized in that: The detection method comprises the following steps: (1) Preparation of test solution: Weigh the Chinese medicine composition, accurately add ethyl acetate, weigh the weight, ultrasonic time 20-40 min, ultrasonic power 150-250 W, ultrasonic frequency 30-50 Hz, let it cool to room temperature, make up the lost weight, filter, and take the filtrate; (2) Preparation of mixed reference solution: Weigh the reference substances of atractylodesinol, β-eudesmol, palmitic acid, mirocinol, and tanshinone IIA respectively, dilute to the mark with ethyl acetate, shake well, and prepare a reference solution with atractylodesinol concentration of 0.5-5.0 μg / mL, β-eudesmol concentration of 1.0-5.0 μg / mL, palmitic acid concentration of 1.0-20.0 μg / mL, mirocinol concentration of 0.5-5.0 μg / mL, and tanshinone IIA concentration of 2.0-20.0 μg / mL. ⑶. Gas chromatography conditions: A quartz capillary column was used, with a temperature range of 320-350°C and a temperature program of: initial temperature 100°C; heating to 142°C at 10.0°C / min; heating to 150°C at 1.5°C / min, holding for 2 min; heating to 170°C at 10.0°C / min, holding for 3 min; heating to 177°C at 0.8°C / min; heating to 200°C at 5.0°C / min, holding for 2 min; heating to 207°C at 1.0°C / min, holding for 2 min; heating to 235°C at 5.0°C / min , hold for 3 minutes, heat up to 240°C at 0.8°C / min, heat up to 270°C at 5.0°C / min), inlet temperature 270-290°C, carrier gas is high-purity helium, carrier gas flow rate is 1.0-2.0 mL / min, split ratio is 8-12:1, solvent delay is 1-3 minutes, pressure is 120-130 KPa, total flow rate is 22-27 mL / min, column flow rate is 1.0-2.5 mL / min, linear velocity is 40-60 cm / s, and purge flow rate is 2.0-4.0 mL / min; (4) Mass spectrometry conditions: EI ion source; ion source temperature 270-290°C, interface temperature 240-260°C; scan range m / z 50-750; ⑸. Determination of content: Take the mixed reference solution in step ⑵ and the test solution in step ⑴, inject them, determine the peak area, and calculate the content.

2. The quality inspection method according to claim 1, wherein: 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.

3. The quality inspection method according to claim 1, wherein: In step ⑵ of the detection method, the concentration of atractylodesin in the mixed reference solution is 2.0758 μg / mL, the concentration of β-eudesmol is 2.1834 μg / mL, the concentration of palmitic acid is 9.9510 μg / mL, the concentration of malathionol is 2.0360 μg / mL, and the concentration of tanshinone IIA is 10.6067 μg / mL.

4. The quality inspection method according to claim 1, wherein: The quartz capillary column model in step (3) of the detection method is: Agilent DB-5MS, and the chromatographic column specifications are: 0.25 mm×30 m, 0.25 μm.

5. The quality inspection method according to claim 1, wherein: In step (3) of the detection method, the injection port temperature is 280°C, the carrier gas is high-purity helium, and 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 chromatographic 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.

6. The quality inspection method according to claim 1, wherein: According to step (4), the ion source temperature is 280°C and the interface temperature is 250°C.

7. The quality inspection method according to claim 1, wherein: According to the gas chromatography and mass spectrometry conditions in step (3), the mass content of the traditional Chinese medicine composition of the present invention was determined, wherein the atractylodesinol content was 0.0205-0.189 mg / g, the β-cineole content was 0.0643-0.5737 mg / g, the palmitic acid content was 0.7319-5.1163 mg / g, the silanol content was 0.0743-0.2356 mg / g, and the tanshinone IIA content was 0.0777-0.8459 mg / g.

8. The quality inspection method according to claim 7, wherein: According to the gas chromatography and mass spectrometry conditions in step ⑶, the mass content of the traditional Chinese medicine composition of the present invention was determined, wherein the atractylodesinol content was 0.0838 mg / g, the β-cineole content was 0.2493 mg / g, the palmitic acid content was 1.5572 mg / g, the silanol content was 0.1211 mg / g, and the tanshinone IIA content was 0.1949 mg / g.

9. The quality inspection method according to claim 1, wherein: The detection method is used in quality detection, intermediate and content determination of traditional Chinese medicine compositions for promoting blood circulation and reducing blood sugar, and identification of effective substance components of traditional Chinese medicines.

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