GCMSMS detection method for simultaneously detecting 16 volatile components in Da-collateral-activating pills

Abstract

The invention relates to the technical field of biology, in particular to a GCMSMS (Gas Chromatography Mass Spectrometry) detection method for simultaneously detecting 16 volatile components in Da collateral activating pills, which comprises the following steps: respectively investigating different extraction modes, different extraction solvents, different material-liquid ratios, different extraction time, different chromatographic conditions and different mass spectrum conditions, and finally selecting dichloromethane as an extraction solvent to detect the 16 volatile components in the Da collateral activating pills. A test solution is prepared through ultrasonic treatment for 30 min according to the material-liquid ratio of 10: 25, chromatographic peak separation is good when the chromatographic condition is 3, and sample elution is complete; the linear relation investigation shows that each component has a good linear relation in the respective range; the instrument precision is good; the test solution has good stability within 24 hours; the test repeatability is good; the method is good in accuracy, can effectively avoid the behavior that few or no materials are fed due to the fact that individual standard medicines do not have a quality control method in a production enterprise, has important practical significance in guaranteeing the medicine quality, and has wide application prospects.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a GCMS MS detection method for simultaneously detecting 16 volatile components in Dahuoluo pills. Background Art

[0002] Dahuoluo Pills are mainly composed of 48 medicinal ingredients, including Acuta acutus, Black-tailed Snake, Clematis chinensis, Gastrodia elata, Scorpion, Earthworm, processed Aconitum kusnezoffii, cinnamon, Asarum, Ephedra, Notopterygium wilfordii, Saposhnikovia divaricata, rosin, patchouli, cardamom, Bombyx batryticatus (fried), Arisaema consanguineum (processed), Bezoar, Linderae scutellariae, costusroot, Aquilaria sinensis, Clove, Citrus aurantium, Cyperus rotundus (processed with vinegar), benzoin, borneol, Coptis chinensis, Scutellaria baicalensis, Cyperus rotundus, Pueraria lobata, Rhubarb, Red ginseng, Rehmannia glutinosa, Polygonum multiflorum, Tortoise shell (quenched in vinegar), Scrophularia ningpoensis, Licorice, Atractylodes macrocephala, Angelica sinensis, Drynaria fortunei (scalded and dehaired), Rhizoma Anemarrhenae, Paeonia lactiflora, Myrrh (processed), frankincense, Sanguisorba officinalis, buffalo horn, leopard bone (crisped in oil), and musk. It has the effects of dispelling wind and relieving pain, removing dampness and phlegm, relaxing muscles and activating collaterals, and is suitable for paralysis, foot atrophy and pain, tendon and vein stiffness, waist and leg pain, bruises, difficulty walking, chest pain, etc. caused by stroke and phlegm. Its prescription comes from the "Experienced Prescriptions" of the Ming Dynasty.

[0003] There are three dosage forms: large honey pills, small honey pills and concentrated pills. The existing standard for large honey pills of Dahuoluowan is WS3-B-1082-92, which is included in the sixth volume of the drug standard for Chinese medicine formula preparations. It only controls the inspection items and does not control other items; the existing standard for concentrated pills is the standard of the Chinese patent medicine landmark upgraded to the national standard (meridians, limbs and brain system volume) numbered WS-10999 (ZD-0999)-2002, which includes microscopic identification and 4 thin layer identifications ((licorice, ginseng (ginsenoside Rb1, Re, Rg1), angelica, coptis (berberine hydrochloride), costus root (dehydrocostus lactone), dragon's blood)) and the determination of alcohol-soluble extracts. The existing standard for Xiaomiwan is the standard numbered YBZ01252009, which includes the inspection of ester alkaloids and aristolochic acid A, and the content determination of total alkaloids under the content determination item. None of these three standards effectively controls the volatile components. In addition, the resinous medicinal materials in Dahuoluowan include frankincense, agarwood, myrrh, benzoin, dragon's blood, rosin, and lindera, the medicinal materials containing volatile oils include musk, angelica root, cinnamon bark, asarum, myrrh, cardamom, and angelica, and the medicinal materials containing volatile components include patchouli, cloves, cardamom, and borneol. It can be seen that there are about 20 medicinal materials in the Dahuoluowan formula that contain volatile components, and there are a large number of literature reports that these volatile components are the key components for the medicinal materials to exert their therapeutic effects. Therefore, it is necessary to establish a control method for the volatile components of Dahuoluowan for the quality control of Dahuoluowan finished products.

[0004] Therefore, in order to improve the quality standard of Dahuoluo Pills, this study used gas chromatography-mass spectrometry to establish a gas chromatography quantitative method for 13 Chinese medicinal materials in Dahuoluo Pills (qianghuo, cardamom, borneol, benzoin, clove, cyperus rotundus, cardamom, patchouli, agarwood, cinnamon, musk, angelica, and frankincense) to quantitatively control the volatile components in Dahuoluo Pills. Summary of the invention

[0005] The primary purpose of the present invention is to provide a GCMSMS detection method for simultaneously detecting 16 volatile components in Dahuoluo Pills, comprising the following steps:

[0006] (1) Preparation of reference solution: accurately weigh eucalyptol, limonene, isoborneol, borneol, α-cyperone, patchouli alcohol, paeonol, eugenol, patchouli ketone, methyl eugenol, borneol, ligustilide, cinnamic acid, musk ketone, vanillin, and octyl acetate reference substances, and add dichloromethane to prepare a stock solution containing 1 μg of the reference substance per 1 ml;

[0007] (2) Preparation of the test solution: accurately weigh a sample of Dahuoluo Pills, place it in a stoppered conical flask, add a solvent, let it stand, shake it, and extract it by heating reflux extraction or ultrasonic extraction to obtain the test sample mother solution; take the test sample mother solution, add ethyl acetate to the volume, filter it with an organic filter membrane, and take the filtrate to obtain;

[0008] (3) Chromatographic conditions: initial temperature of 60 °C, maintained for 5 min, heated to 90 °C at a rate of 10 °C / min, maintained for 10 min, then heated to 160 °C at a rate of 10 °C / min, maintained for 20 min; injection port temperature: 250 °C; MS transmission line temperature: 250 °C; ion source temperature: 280 °C;

[0009] (4) Mass spectrometry conditions:

[0010]

[0011] Preferably, the solvent in step (2) is ethyl acetate, anhydrous ethanol, dichloromethane or n-hexane.

[0012] Preferably, the solvent in step (2) is dichloromethane.

[0013] Preferably, the amount of solvent added in step (2) is 25 ml to 35 ml.

[0014] Preferably, the material-liquid ratio in step (2) is 10:25.

[0015] Preferably, the extraction method in step (2) is ultrasonic extraction, specifically: ultrasonic treatment, filtration, and taking the filtrate.

[0016] Preferably, the ultrasonic treatment time in step (2) is 30 min to 90 min.

[0017] Preferably, the organic filter membrane in step (2) is 0.45 μm.

[0018] The beneficial effects of the present invention are as follows: (1) The present invention provides a GCMSMS detection method for simultaneously detecting 16 volatile components in Dahuoluo Pills, and investigates different extraction methods (ultrasonic extraction, hot reflux extraction), different extraction solvents (ethyl acetate, anhydrous ethanol, dichloromethane, n-hexane), different solid-liquid ratios (10:25, 10:30, 10:35), different extraction times (30 min, 60 min, 90 min) and different chromatographic conditions and different mass spectrometry conditions. Finally, dichloromethane is selected as the extraction solvent, and the test solution is prepared by ultrasonication for 30 min at a solid-liquid ratio of 10:25. When the chromatographic condition is 3, the chromatographic peak separation is better, and the sample elution is more complete.

[0019] (2) The linear relationship investigation showed that the correlation coefficients of limonene, eucalyptol, isoborneol, borneol, octyl acetate, benzoic acid, 5-hydroxymethylfurfural, eugenol, methyl eugenol, cinnamic acid, vanillin, patchouli alcohol, α-cyperone, patchouli ketone, musk ketone, and (E)-ligustilide were 0.9948, 0.9997, 0.9999, 0.9998, 0.9995, 0.9972, 0.9920, 0.9973, 0.9978, 0.9932, 0.9982, 0.9998, 0.9948, 0.9907, 0.9995, and 0.9970, respectively, indicating a good linear relationship.

[0020] (3) The RSDs of the peak areas of limonene, eucalyptol, isoborneol, borneol, octyl acetate, benzoic acid, 5-hydroxymethylfurfural, eugenol, methyl eugenol, cinnamic acid, vanillin, patchouli alcohol, α-cyperone, patchouli ketone, musk ketone and (E)-ligustilide after six injections were 0.66%, 1.52%, 1.62%, 1.38%, 1.49%, 1.52%, 0.52%, 0.53%, 1.83%, 1.71%, 1.70%, 1.55%, 1.94%, 1.23%, 1.73% and 1.69%, respectively, indicating that the instrument had good precision.

[0021] (4) The peak area RSDs of limonene, eucalyptol, isoborneol, borneol, octyl acetate, benzoic acid, 5-hydroxymethylfurfural, eugenol, methyl eugenol, cinnamic acid, vanillin, patchouli alcohol, α-cyperone, patchouli ketone, musk ketone and (E)-ligustilide at 0, 6, 12, 18 and 24 h were 1.02%, 0.81%, 1.19%, 1.44%, 1.15%, 1.46%, 0.96%, 1.74%, 1.37%, 0.86%, 1.66%, 2.02%, 1.79%, 0.80%, 1.93% and 1.75%, respectively, indicating that the test solution had good stability within 24 h.

[0022] (5) The RSDs of the mass fractions of six parallel samples of limonene, eucalyptol, isoborneol, borneol, octyl acetate, benzoic acid, 5-hydroxymethylfurfural, eugenol, methyl eugenol, cinnamic acid, vanillin, patchouli alcohol, α-cyperone, patchouli ketone, musk ketone and (E)-ligustilide were 0.17%, 1.20%, 1.64%, 1.01%, 1.92%, 1.46%, 0.40%, 1.45%, 1.49%, 1.12%, 1.92%, 1.51%, 2.07%, 0.80%, 1.51% and 1.09%, respectively, indicating that the test had good repeatability.

[0023] (6) The average recoveries of limonene, eucalyptol, isoborneol, borneol, octyl acetate, benzoic acid, 5-hydroxymethylfurfural, eugenol, methyl eugenol, cinnamic acid, vanillin, patchouli alcohol, α-cyperone, patchouli ketone, musk ketone, and (E)-ligustilide were 104.14%, 102.66%, 101.45%, 101.54%, 101.29%, 103.17%, 101.25%, 101.19%, 101.60%, and 102. .07%, 101.12%, 101.91%, 101.36%, 101.39%, 101.40%, and 100.82%, and the RSDs were 0.39%, 0.46%, 0.56%, 0.54%, 0.69%, 0.70%, 0.81%, 0.96%, 0.88%, 0.93%, 0.87%, 0.89%, 0.91%, 0.83%, 0.91%, and 0.98%, respectively, indicating that the method has good accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Chromatograms of different extraction methods

[0025] Note: A: Ultrasonic extraction B: Heating reflux extraction

[0026] Figure 2 Chromatograms of different extraction solvents

[0027] Note: A: Ethyl acetate B: Anhydrous methanol C: Dichloromethane D: Hexane

[0028] Figure 3 Chromatograms of different solid-liquid ratios

[0029] Note: A: material-liquid ratio 10:20 B: material-liquid ratio 10:25 C: material-liquid ratio 10:30

[0030] Figure 4 Chromatograms at different ultrasound times

[0031] Note: A: Ultrasound 30min B: Ultrasound 60min C: Ultrasound 90min

[0032] Figure 5 Different chromatographic conditions

[0033] Note: A: Chromatographic condition 1 B: Chromatographic condition 2 C: Chromatographic condition 3

[0034] Figure 6 Ion pair optimization chromatogram Note: A: Full Scan Discovery B: SRM Discovery 1 C: SRMDiscovery 2 D: Production Discovery 1 E: Production Discovery 2 F: ProductionDiscovery 3

[0035] Figure 7 Exclusive inspection

[0036] Note: A: Mixed reference solution B: Dahuoluo Pills test solution C: Blank solution DETAILED DESCRIPTION

[0037] In order to make the purpose, technical solution and effect of the present invention clearer and more specific, the present invention is further described in detail with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0038] In the following examples, unless otherwise specified, all reagents are commercially available.

[0039] In the following examples, unless otherwise specified, the methods used are all conventional methods.

[0040] GC-MS-MS is Gas Chromatography-Tandem Mass Spectrometry, which includes gas chromatography, mass spectrometry (MS) and tandem mass spectrometry (MS-MS). The gas chromatography (GC) part uses the difference in the distribution coefficients of different substances between the stationary phase and the mobile phase to separate the components in the mixture in the gas chromatography column in order. The sample is first injected into the injection port, and then enters the chromatographic column with the carrier gas (usually an inert gas such as helium) after gasification. After repeated adsorption, desorption and other distribution processes in the column, the components are separated, and then flow out of the chromatographic column into the mass spectrometer part in turn. The mass spectrometry (MS) part detects and analyzes the separated components flowing out of the gas chromatography column. The principle is to ionize the sample molecules in the ion source to form ions with different mass-to-charge ratios (m / z), and then these ions are separated according to the mass-to-charge ratio in the mass analyzer and detected and recorded to obtain a mass spectrum. The mass spectrum can provide information such as the molecular weight and structural fragments of the compound, which is helpful for qualitative analysis of the compound. The tandem mass spectrometry (MS-MS) part is based on the primary mass spectrometry, selecting specific ions (parent ions) in the primary mass spectrometry, and further fragmenting and analyzing the generated daughter ions. By changing parameters such as collision energy, the parent ion is fragmented to generate more fragment ion information about the structural details of the compound, which is very helpful for the structural analysis of compounds in complex mixtures, differentiation of isomers, and detection of trace substances.

[0041] Example 1: Optimization of GCMSMS detection method for Dahuoluo Pills

[0042] 1. Instruments and Materials

[0043] 1.1 Instrument

[0044] Thermo Fisher gas chromatograph-mass spectrometer (model: TRACE1310-TSQ9000), Mettler 1 / 100,000 analytical balance (model: SECURA125-1CN), Mettler 1 / 10,000 analytical balance (model: QUINTIX224-1CN), CNC ultrasonic cleaner (manufacturer: Kunshan Ultrasonic Instrument Co., Ltd.; model: KQ-300DB).

[0045] 1.2 Reagents and drugs

[0046] Limonene (Batch No.: 100470-2015030), eucalyptol (Batch No.: 110788-201707), isoborneol (Batch No.: 111512-202205), borneol (Batch No.: 110881-201709), octyl acetate (Batch No.: 111671-201403), benzoic acid (Batch No.: 100419-201703), 5-hydroxymethylfurfural (Batch No.: 111626-201912), eugenol (Batch No.: 110725-201917), cinnamic acid (Batch No.: 110786-201604), vanillin (Batch No.: 100491-201902), patchouli alcohol (Batch No.: : 110772-201608), α-cyperone (batch number: 110748-201815), patchouli ketone (batch number: 111822-201904), musk ketone (batch number: 110719-202117), (E)-ligustilide (batch number: 111737-201608), and methyl eugenol (batch number: 111642-200301) were all purchased from China Food and Drug Inspection Institutes; commercially available Dahuoluo Pills samples, chromatographic grade dichloromethane (Merck), analytical grade anhydrous ethanol (Sinopharm Group Co., Ltd.), analytical grade ethyl acetate (Sinopharm Group Co., Ltd.), and 0.45 μm organic filter membrane (Millipore Corporation, USA).

[0047] 2. Condition Optimization

[0048] 2.1 Selection of extraction method

[0049] Chromatographic conditions: Agilent DB-17MS capillary column with cross-linked 5% phenylmethylpolysiloxane as the stationary phase (column length: 30 m, inner diameter: 0.25 mm, film thickness: 0.25 μm); constant pressure mode, pressure: 146 kPa; programmed temperature: initial temperature 70°C, hold for 4 minutes, increase the temperature to 88°C at a rate of 5°C per minute, hold for 2 minutes, then increase the temperature to 100°C at a rate of 1°C, then increase the temperature to 120°C at a rate of 2°C, then increase the temperature to 145°C at a rate of 10°C, then increase the temperature to 160°C at a rate of 2°C, then increase the temperature to 220°C at a rate of 5°C, then increase the temperature to 250°C at a rate of 2°C, then increase the temperature to 300°C at a rate of 20°C, hold for 2 min; injection volume 1 μL.

[0050] Preparation of test solution:

[0051] (1) Hot reflux extraction: Weigh 10g of Dahuoluo pills sample accurately (weigh the sample of large honey pills first, then add appropriate amount of diatomaceous earth to grind evenly, crush the small honey pills first, then weigh the sample). Place it in a 150ml stoppered conical flask, accurately add 25ml of ethyl acetate solution, and weigh the weight. After heating and reflux extraction for 45min, cool and weigh again. Then make up the lost weight with ethyl acetate, shake well, and the mother liquor of the test sample is obtained. Take 50ul of the mother liquor of the test sample, add chromatographic grade dichloromethane to make up to 1ml, filter with a 0.45μm organic filter membrane, and take the filtrate to obtain.

[0052] (2) Ultrasonic extraction: Weigh 10 g of Dahuoluo pills (weigh the sample of large honey pills first, then add appropriate amount of diatomaceous earth to grind evenly, crush the small honey pills first, then weigh the sample), place in a conical flask, accurately add 25 ml of ethyl acetate solvent, let stand for 1 hour, shake from time to time during the standing process, ultrasonically treat for 30 minutes, filter, and take the filtrate to obtain the mother solution of the test sample. Take 50 ul of the mother solution of the test sample, add ethyl acetate to make the volume to 1 ml, filter with a 0.45 μm organic filter membrane, and take the filtrate to obtain.

[0053] like Figure 1 As shown in the figure, the chromatographic behaviors of the test solutions prepared by the two extraction methods are very similar. Combined with the indicators such as chromatographic peak shape and separation, and taking into account the convenience of operation, ultrasonic extraction was finally selected as the better choice.

[0054] 2.2 Selection of extraction solvent

[0055] Chromatographic conditions: Agilent DB-17MS capillary column with cross-linked 5% phenylmethylpolysiloxane as the stationary phase (column length: 30 m, inner diameter: 0.25 mm, film thickness: 0.25 μm); constant pressure mode, pressure: 146 kPa; programmed temperature: initial temperature 70°C, hold for 4 minutes, increase the temperature to 88°C at a rate of 5°C per minute, hold for 2 minutes, then increase the temperature to 100°C at a rate of 1°C, then increase the temperature to 120°C at a rate of 2°C, then increase the temperature to 145°C at a rate of 10°C, then increase the temperature to 160°C at a rate of 2°C, then increase the temperature to 220°C at a rate of 5°C, then increase the temperature to 250°C at a rate of 2°C, then increase the temperature to 300°C at a rate of 20°C, hold for 2 min; injection volume 1 μL.

[0056] Preparation of test solution: accurately weigh 10g of Dahuoluo pill sample (weigh the sample of large honey pill first, then add appropriate amount of diatomaceous earth to grind evenly, crush the small honey pill first, then weigh the sample), weigh 4 samples in parallel, place them in 150ml stoppered conical flasks, accurately add ethyl acetate, anhydrous ethanol, dichloromethane, and n-hexane solvent 25ml, let stand for 1 hour, shake from time to time during the standing process, ultrasonically treat for 30 minutes, filter, and take the filtrate to obtain the mother solution of the test sample. Take 50ul of the mother solution of the test sample, add ethyl acetate to 1ml, filter with a 0.45μm organic filter membrane, and take the filtrate.

[0057] like Figure 2 As shown in the figure, when anhydrous ethanol is used as the solvent, the chromatographic peak shape is poor, and when n-hexane is used as the solvent, the dissolved components are few and the content is low. After comparison, the difference between the two is small. Ethyl acetate and dichloromethane have the best peak effect. After comparison, dichloromethane has a good separation effect and can dissolve more volatile components. Therefore, dichloromethane is determined as the extraction solvent.

[0058] 2.3 Selection of extraction solid-liquid ratio

[0059] Chromatographic conditions: Agilent DB-17MS capillary column with cross-linked 5% phenylmethylpolysiloxane as the stationary phase (column length: 30 m, inner diameter: 0.25 mm, film thickness: 0.25 μm); constant pressure mode, pressure: 146 kPa; programmed temperature: initial temperature 70°C, hold for 4 minutes, increase the temperature to 88°C at a rate of 5°C per minute, hold for 2 minutes, then increase the temperature to 100°C at a rate of 1°C, then increase the temperature to 120°C at a rate of 2°C, then increase the temperature to 145°C at a rate of 10°C, then increase the temperature to 160°C at a rate of 2°C, then increase the temperature to 220°C at a rate of 5°C, then increase the temperature to 250°C at a rate of 2°C, then increase the temperature to 300°C at a rate of 20°C, hold for 2 min; injection volume 1 μL.

[0060] Preparation of test solution: accurately weigh 10g of Dahuoluo pill sample (weigh the sample of large honey pill first, then add appropriate amount of diatomaceous earth to grind evenly, crush the small honey pill first, then weigh the sample), weigh 3 samples in parallel, place them in 150ml stoppered conical flasks, accurately add 25ml, 30ml, and 35ml of dichloromethane solvent respectively, let stand for 1 hour, shake from time to time during the standing process, ultrasonically treat for 30 minutes, filter, and take the filtrate to obtain the mother solution of the test sample. Take 50ul of the mother solution of the test sample, add ethyl acetate to make the volume to 1ml, filter with a 0.45μm organic filter membrane, and take the filtrate.

[0061] like Figure 3 As shown, it was found that when the material-liquid ratio was 10:25, the components to be measured had better peak shape and separation, so the material-liquid ratio was selected to be 10:25.

[0062] 2.4 Selection of extraction time

[0063] Chromatographic conditions: Agilent DB-17MS capillary column with cross-linked 5% phenylmethylpolysiloxane as the stationary phase (column length: 30 m, inner diameter: 0.25 mm, film thickness: 0.25 μm); constant pressure mode, pressure: 146 kPa; programmed temperature: initial temperature 70°C, hold for 4 minutes, increase the temperature to 88°C at a rate of 5°C per minute, hold for 2 minutes, then increase the temperature to 100°C at a rate of 1°C, then increase the temperature to 120°C at a rate of 2°C, then increase the temperature to 145°C at a rate of 10°C, then increase the temperature to 160°C at a rate of 2°C, then increase the temperature to 220°C at a rate of 5°C, then increase the temperature to 250°C at a rate of 2°C, then increase the temperature to 300°C at a rate of 20°C, hold for 2 min; injection volume 1 μL.

[0064] Preparation of test solution: accurately weigh 10g of Dahuoluo pill sample (weigh the sample of large honey pill first, then add appropriate amount of diatomaceous earth to grind evenly, crush the small honey pill first, then weigh the sample), weigh 3 samples in parallel, place them in 150ml stoppered conical flasks, accurately add 25ml of dichloromethane solvent, let stand for 1 hour, shake from time to time during the standing process, ultrasonically treat for 30min, 60min, and 90min respectively, filter, take the filtrate, and get the mother solution of the test sample. Take 50ul of the mother solution of the test sample, add ethyl acetate to 1ml, filter with a 0.45μm organic filter membrane, and take the filtrate.

[0065] like Figure 4 As shown, it was found that there was no significant difference in the volatile components dissolved at 30 min and 60 min, and chloroform volatilized significantly at 90 min, so ultrasonication for 30 min was selected, and the lost mass was supplemented after ultrasonication.

[0066] 2.5 Selection of chromatographic conditions

[0067] Preparation of the test solution: accurately weigh 10g of Dahuoluo pill sample (weigh the sample first for large honey pills, then add appropriate amount of diatomaceous earth to grind evenly, crush the small honey pills first, then weigh the sample), place it in a 150ml stoppered conical flask, accurately add 25ml of dichloromethane solvent, let it stand for 1 hour, shake it from time to time during the standing process, ultrasonically treat it for 30min respectively, filter, take the filtrate, and get the mother solution of the test sample. Take 50ul of the mother solution of the test sample, add ethyl acetate to make it up to 1ml, filter it with a 0.45μm organic filter membrane, and take the filtrate.

[0068] Chromatographic conditions 1: Initial temperature is 60℃, maintained for 5 min, then heated to 90℃ at a rate of 10℃ / min, maintained for 10 min, then heated to 160℃ at a rate of 10℃ / min, maintained for 20 min, then heated to 280℃ at a rate of 5℃ / min, maintained for 10 min, then heated to 300℃ at a rate of 20℃ / min, maintained for 20 min. Inlet temperature: 250℃; MS transmission line temperature: 250℃; ion source temperature: 280℃.

[0069] Chromatographic condition 2: Initial temperature is 60℃, maintained for 5 min, then heated to 90℃ at a rate of 10℃ / min, maintained for 10 min, then heated to 160℃ at a rate of 10℃ / min, maintained for 20 min, then heated to 250℃ at a rate of 10℃ / min, maintained for 5 min. Inlet temperature: 250℃; MS transfer line temperature: 250℃; ion source temperature: 280℃.

[0070] Chromatographic condition 3: Initial temperature is 60℃, maintained for 5 min, then heated to 90℃ at a rate of 10℃ / min, maintained for 10 min, then heated to 160℃ at a rate of 10℃ / min, maintained for 20 min. Inlet temperature: 250℃; MS transfer line temperature: 250℃; ion source temperature: 280℃.

[0071] like Figure 5 As shown, under chromatographic condition 1, the components to be measured basically peak within 60 minutes, and after 60 minutes, the components to be measured are few and the content is low; under chromatographic condition 2, the components to be measured basically peak within 40 minutes, and after 40 minutes, the content of the components to be measured is low, and the components with low content can be ignored, thus reducing the measurement time; under chromatographic condition 3, the measurement time is short, the chromatographic peaks are better separated, and the sample is eluted more completely, so chromatographic condition 3 is selected as the optimal chromatographic condition.

[0072] 2.6 Optimization of mass spectrometry conditions

[0073] Preparation of reference solution: accurately weigh appropriate amount of eucalyptol, limonene, isoborneol, borneol, α-cyperone, patchouli alcohol, paeonol, eugenol, patchouli ketone, methyl eugenol, borneol, ligustilide, cinnamic acid, musk ketone, vanillin, and octyl acetate reference substances, add dichloromethane to prepare a mother solution of the test substance containing 1ug of reference substance per 1ml. Filter with a 0.45μm organic filter membrane (Millipore, USA), and take the filtrate to obtain the solution.

[0074] Chromatographic conditions: initial temperature 60°C, hold for 5 min, increase to 90°C at a rate of 10°C / min, hold for 10 min, then increase to 160°C at a rate of 10°C / min, hold for 20 min. Inlet temperature: 250°C; MS transfer line temperature: 250°C; ion source temperature: 280°C.

[0075] Using the AUTO SRM software provided by Thermo Fisher Scientific, the SCAN mode was used to establish the method, optimize the ion pairs and energy, and the results were as follows: Figure 6 The ion pairs and optimal collision energy of each compound to be tested were obtained, and then the MRM method for the determination of 16 volatile components in Dahuoluo Pills was established.

[0076] This example investigates the extraction method, and the results show that there is no significant difference in the peak content to be measured between heating reflux and ultrasonic 30min, so the more convenient ultrasonic extraction method is selected. At the same time, four different extraction solvents, ethyl acetate, methanol, anhydrous ethanol and dichloromethane, were investigated, and it was found that more volatile components can be dissolved in dichloromethane. At the same time, different ultrasonic times of 30, 60, and 90min were also investigated, and it was found that there was no significant difference in the volatile components dissolved at 30min and 60min, and chloroform volatilized significantly at 90min, so ultrasonic 30min was selected, and the lost mass was supplemented after ultrasonic. Finally, different material-liquid ratios of 10:20, 10:25, and 10:30 were investigated, and it was found that each component to be measured had a better peak shape and separation when the material-liquid ratio was 10:25, so the material-liquid ratio was selected as 10:25. Based on the above investigation results, dichloromethane was finally selected as the extraction solvent, and the test solution was prepared by ultrasonic 30min at a material-liquid ratio of 10:25.

[0077] The chromatographic conditions such as starting temperature, heating rate and running time were also investigated, and finally chromatographic condition 3 was selected (initial temperature was 60°C, maintained for 5 minutes, heated to 90°C at a rate of 10°C / min, maintained for 10 minutes, and then heated to 160°C at a rate of 10°C / min, maintained for 20 minutes. Inlet temperature: 250°C; MS transmission line temperature: 250°C; ion source temperature: 280°C). The chromatographic peaks were better separated and the sample eluted more completely.

[0078] Thermo Fisher's own AUTO SRM software was used to establish a method in full scan (SCAN) mode to optimize ion pairs and energy, obtain the ion pairs and optimal collision energy for each compound to be tested, and then establish an MRM method for the determination of 16 volatile components in Dahuoluo Pills.

[0079] Example 2: A method for rapidly determining the contents of 16 volatile components in Dahuoluo Pills by GCMSMS 1. Preparation of solution

[0080] 1.1 Preparation of reference solution and standard curve

[0081] Accurately weigh appropriate amounts of eucalyptol, limonene, isoborneol, borneol, α-cyperone, patchouli alcohol, paeonol, eugenol, patchouli ketone, methyl eugenol, borneol, ligustilide, cinnamic acid, musk ketone, vanillin, and octyl acetate reference substances, and add dichloromethane to prepare a mother solution of the test substance containing 1ug of the reference substance per 1ml. Take 5μl, 10μl, 20μl, 50μl, 100μl, 200μl, and 500μl of the mother solution respectively, dilute to 1ml with dichloromethane, filter with a 0.45μm organic filter membrane (Millipore, USA), and take the filtrate to obtain the product.

[0082] 1.2 Preparation of test solution

[0083] Accurately weigh 10g of Dahuoluo Pills sample (weigh the sample first for large honey pills, then add appropriate amount of diatomaceous earth to grind evenly, crush the small honey pills first, then weigh the sample), place in a conical flask, accurately add 25ml of dichloromethane solvent, let stand for 1 hour, shake from time to time during the standing process, ultrasonically treat for 30 minutes, filter, and take the filtrate to obtain the mother solution of the test sample. Take 50ul of the mother solution of the test sample, add chromatographic grade dichloromethane to make the volume to 1ml, filter with a 0.45μm organic filter membrane, and take the filtrate to obtain.

[0084] 2 Chromatographic conditions and system suitability test

[0085] Agilent DB-17MS capillary column with 5% phenylmethylpolysiloxane cross-linked as the stationary phase (column length: 30m, inner diameter: 0.25mm, film thickness: 0.25μm); constant pressure mode, pressure: 146kPa; program temperature: initial temperature 60℃, hold for 5 minutes, increase to 90℃ at a rate of 10℃ per minute, hold for 10 minutes, increase to 160℃ at a rate of 10℃ per minute, hold for 20 minutes; injection port temperature: 250℃, transfer line temperature: 250℃, ion source temperature: 280℃. Dwell time: 2.5min; solvent delay time: 5min; acquisition parameters: mass spectrometry multiple reaction monitoring (MRM, parameters see Table 1); TIC diagram of the mixed standard solution of 16 components to be tested and TIC diagram of the Dahuoluo pill sample are shown in Figure 7 .

[0086] Table 1 CAS numbers and MRM parameters of 16 components to be analyzed

[0087]

[0088]

[0089] 3 Methodological validation

[0090] 3.1 Specificity test

[0091] Take appropriate amount of blank solution, reference solution and test solution, and perform the test under the chromatographic conditions of item "2". The results are shown in Figure 7 It can be seen that there is no interference in the blank, indicating that the method has good specificity.

[0092] 3.2 Investigation of linear relationship

[0093] Accurately pipette an appropriate amount of the mixed reference solution under item "2.1.1", dilute it into 6 different mass concentrations, and inject it for determination under the chromatographic conditions under item "2.2". Perform linear regression with the mass concentration of the reference as the abscissa (X) and the peak area as the ordinate (Y), and obtain the regression equation, and use the signal-to-noise ratio (S / N) of 10:1 as the quantitative limit and the signal-to-noise ratio (S / N) of 3:1 as the detection limit. The results are shown in Table 2, and it can be seen that the linear relationship of each component is good within its respective range.

[0094] Table 2 Retention time, linear regression equation and correlation coefficient of 16 components to be analyzed

[0095]

[0096] 3.3 Precision test

[0097] Take an appropriate amount of Dahuoluo Pills sample X1, prepare the test solution according to the method under "1.2", and inject and measure 6 times under the chromatographic conditions under "2", with 1 μL each time. The peak area RSDs of limonene, eucalyptol, isoborneol, borneol, octyl acetate, benzoic acid, 5-hydroxymethylfurfural, eugenol, methyl eugenol, cinnamic acid, vanillin, patchouli alcohol, α-cyperone, patchouli ketone, musk ketone and (E)-ligustilide were 0.66%, 1.52%, 1.62%, 1.38%, 1.49%, 1.52%, 0.52%, 0.53%, 1.83%, 1.71%, 1.70%, 1.55%, 1.94%, 1.23%, 1.73% and 1.69%, respectively, indicating that the instrument has good precision.

[0098] 3.4 Stability test

[0099] Take an appropriate amount of Dahuoluo Pills sample X1, prepare the test solution according to the method under "1.2", place it at 25°C, and inject and measure under the chromatographic conditions under "2" at 0, 6, 12, 18, and 24 hours, respectively, to measure limonene, eucalyptol, isoborneol, borneol, octyl acetate, benzoic acid, 5-hydroxymethylfurfural, eugenol, methyl eugenol, cinnamic acid, vanillin, patchouli alcohol, α-cyperone, and patchouli ketone. The RSDs of the peak areas of musk ketone and (E)-ligustilide were 1.02%, 0.81%, 1.19%, 1.44%, 1.15%, 1.46%, 0.96%, 1.74%, 1.37%, 0.86%, 1.66%, 2.02%, 1.79%, 0.80%, 1.93% and 1.75%, respectively, indicating that the test solution had good stability within 24 h.

[0100] 3.5 Repeatability test

[0101] Take an appropriate amount of Dahuoluo Pills sample X1, weigh 6 samples in parallel, prepare the test solution according to the method under "1.2", and inject the sample under the chromatographic conditions under "2". Each sample is injected with 1 injection, 1 μL each time, and the RSDs of the mass fractions of limonene, eucalyptol, isoborneol, borneol, octyl acetate, benzoic acid, 5-hydroxymethylfurfural, eugenol, methyl eugenol, cinnamic acid, vanillin, patchouli alcohol, α-cyperone, patchouli ketone, musk ketone and (E)-ligustilide were 0.17%, 1.20%, 1.64%, 1.01%, 1.92%, 1.46%, 0.40%, 1.45%, 1.49%, 1.12%, 1.92%, 1.51%, 2.07%, 0.80%, 1.51% and 1.09%, respectively, indicating that the test has good repeatability.

[0102] 3.6 Sample recovery test

[0103] Accurately pipette appropriate amount of the reference substance stock solution under the above "1.1" to prepare a solution containing 0.425μg / mL of limonene, 1.791μg / mL of eucalyptol, 180.864μg / mL of isoborneol, 236.390μg / mL of borneol, 1.156μg / mL of benzoic acid, 29.814μg / mL of octyl acetate, 575.520μg / mL of 5-hydroxymethylfurfural, and 104. The mixed reference solution was composed of 24.998μg / mL, cinnamic acid 5.870μg / mL, vanillin 24.998μg / mL, methyl eugenol 5.898μg / mL, patchouli ketone 30.221μg / mL, patchouli alcohol 26.726μg / mL, α-cyperone 15.298μg / mL, (E)-ligustilide 127.625μg / mL, and musk ketone 6.968μg / mL.

[0104] Weigh 6 portions of Dahuoluo Pills X1 samples with determined contents of each component, weigh accurately, and place in a 25mL volumetric flask. Accurately pipette 2.5mL of the above mixed reference solution according to the ratio of 100% of the content of each component, prepare the test solution according to the method under "1.2", inject and determine under the chromatographic conditions under "2", and calculate the recovery rate. Results The average recovery rates of limonene, eucalyptol, isoborneol, borneol, octyl acetate, benzoic acid, 5-hydroxymethylfurfural, eugenol, methyl eugenol, cinnamic acid, vanillin, patchouli alcohol, α-cyperone, patchouli ketone, musk ketone, and (E)-ligustilide were 104.14%, 102.66%, 101.45%, 101.54%, 101.29%, 103.17%, 101.25%, 101.19%, 101.60%, 102 .07%, 101.12%, 101.91%, 101.36%, 101.39%, 101.40%, and 100.82%, and the RSDs were 0.39%, 0.46%, 0.56%, 0.54%, 0.69%, 0.70%, 0.81%, 0.96%, 0.88%, 0.93%, 0.87%, 0.89%, 0.91%, 0.83%, 0.91%, and 0.98%, respectively, indicating that the method has good accuracy.

[0105] 4 Results

[0106] The test solution was prepared according to the method under "1.2", the reference solution was prepared according to the method under "1.1", and the sample was injected and determined under the chromatographic conditions under "2". The results are shown in Table 3. Through the determination of 20 batches of Dahuoluo Pills samples, it was found that 16 volatile components could be quantitatively determined. This method can be used for the rapid determination and control of volatile components in Dahuoluo Pills samples.

[0107] Table 3 Detection results of 16 volatile components in Dahuoluo pills samples

[0108]

[0109]

[0110] In summary, the present invention provides a GCMSMS detection method for simultaneously detecting 16 volatile components in Dahuoluo Pills, and different extraction methods (ultrasonic extraction, hot reflux extraction), different extraction solvents (ethyl acetate, anhydrous ethanol, dichloromethane, n-hexane), different solid-liquid ratios (10:25, 10:30, 10:35), different extraction times (30min, 60min, 90min) and different chromatographic conditions and different mass spectrometry conditions are investigated. Finally, dichloromethane is selected as the extraction solvent, and the test solution is prepared by ultrasonication for 30min at a solid-liquid ratio of 10:25. When the chromatographic condition is 3, the chromatographic peak separation is better, and the sample elution is more complete; the linear relationship The investigation showed that the linear relationship of each component in its respective range was greater than 0.99, indicating a good linear relationship; the peak area RSDs of limonene, eucalyptol, isoborneol, borneol, octyl acetate, benzoic acid, 5-hydroxymethylfurfural, eugenol, methyl eugenol, cinnamic acid, vanillin, patchouli alcohol, α-cyperone, patchouli ketone, musk ketone, and (E)-ligustilide were 0.66%, 1.52%, 1.62%, 1.38%, 1.49%, 1.52%, 0.52%, 0.53%, 1.83%, 1.71%, 1.70%, 1.55%, 1.94%, 1.23%, 1.73%, and 1.69%, respectively, indicating good instrument precision. The RSDs of the peak areas of limonene, eucalyptol, isoborneol, borneol, octyl acetate, benzoic acid, 5-hydroxymethylfurfural, eugenol, methyl eugenol, cinnamic acid, vanillin, patchouli alcohol, α-cyperone, patchouli ketone, musk ketone and (E)-ligustilide were 1.02%, 0.81%, 1.19%, 1.44%, 1.15%, 1.46%, 0.96%, 1.74%, 1.37%, 0.86%, 1.66%, 2.02%, 1.79%, 0.80%, 1.93% and 1.75%, respectively, indicating that the test solution had good stability within 24 hours. The RSDs of the mass fractions of limonene, eucalyptol, isoborneol, borneol, octyl acetate, benzoic acid, 5-hydroxymethylfurfural, eugenol, methyl eugenol, cinnamic acid, vanillin, patchouli alcohol, α-cyperone, patchouli ketone, musk ketone and (E)-ligustilide were 0.17%, 1.20%, 1.64%, 1.01%, 1.92%, 1.46%, 0.40%, 1.45%, 1.49%, 1.12%, 1.92%, 1.51%, 2.07%, 0.80%, 1.51% and 1.09%, respectively, indicating that the test had good repeatability.The average recoveries of limonene, eucalyptol, isoborneol, borneol, octyl acetate, benzoic acid, 5-hydroxymethylfurfural, eugenol, methyl eugenol, cinnamic acid, vanillin, patchouli alcohol, α-cyperone, patchouli ketone, musk ketone and (E)-ligustilide were 104.14%, 102.66%, 101.45%, 101.54%, 101.29%, 103.17%, 101.25%, 101.19%, 101.60%, 102. The accuracy of the three methods was as follows: 0.07%, 101.12%, 101.91%, 101.36%, 101.39%, 101.40%, and 100.82%, and the RSDs were 0.39%, 0.46%, 0.56%, 0.54%, 0.69%, 0.70%, 0.81%, 0.96%, 0.88%, 0.93%, 0.87%, 0.89%, 0.91%, 0.83%, 0.91%, and 0.98%, respectively, indicating that the method has good accuracy.

[0111] The present invention uses a gas chromatography-mass spectrometer to establish a quantitative control method for 16 volatile components in Dahuo Luo Wan. The methodological investigation results show that the method has strong inspection specificity for Dahuo Luo Wan samples, good repeatability and accuracy, can achieve quality control of volatile medicinal materials in Dahuo Luo Wan, and the method has a low detection limit, can be used for rapid determination of volatile components of Dahuo Luo Wan, and provides a new method for the multi-component quality control mode of Dahuo Luo Wan. This method can effectively prevent manufacturers from under-feeding or not feeding because there is no quality control method for individual standard medicinal flavors, has important practical significance for ensuring the quality of drugs, and has broad application prospects.

Claims

1. A GCMSMS detection method for simultaneously detecting 16 volatile components in Dahuoluo Pills, characterized in that: The steps include: (1) Preparation of reference solution: accurately weigh eucalyptol, limonene, isoborneol, borneol, α-cyperone, patchouli alcohol, paeonol, eugenol, patchouli ketone, methyl eugenol, borneol, ligustilide, cinnamic acid, musk ketone, vanillin, and octyl acetate reference substances, and add dichloromethane to prepare a stock solution containing 1 μg of the reference substance per 1 ml; (2) Preparation of the test solution: accurately weigh a sample of Dahuoluo Pills, place it in a stoppered conical flask, add a solvent, let it stand, shake it, and extract it by heating reflux extraction or ultrasonic extraction to obtain the test sample mother solution; take the test sample mother solution, add ethyl acetate to the volume, filter it with an organic filter membrane, and take the filtrate to obtain; (3) Chromatographic conditions: initial temperature of 60 °C, maintained for 5 min, heated to 90 °C at a rate of 10 °C / min, maintained for 10 min, then heated to 160 °C at a rate of 10 °C / min, maintained for 20 min; injection port temperature: 250 °C; MS transmission line temperature: 250 °C; ion source temperature: 280 °C; (4) Mass spectrometry conditions:

2. The detection method according to claim 1, characterized in that The solvent in step (2) is ethyl acetate, anhydrous ethanol, dichloromethane or n-hexane.

3. The detection method according to claim 2, characterized in that The solvent in step (2) is dichloromethane.

4. The detection method according to claim 1, characterized in that The amount of solvent added in step (2) is 25ml to 35ml.

5. The detection method according to claim 1, characterized in that The material-liquid ratio in step (2) is 10:

25.

6. The detection method according to claim 1, characterized in that The extraction method described in step (2) is ultrasonic extraction, specifically: ultrasonic treatment, filtration, and taking the filtrate.

7. The detection method according to claim 6, characterized in that The ultrasonic treatment time in step (2) is 30 min to 90 min.

8. The detection method according to claim 1, characterized in that The organic filter membrane described in step (2) is 0.22 μm or 0.45 μm.

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