GC-MS technology-based method for simultaneously determining contents of various components in Yinhui cold powder

Abstract

The invention relates to the field of medicine analysis, in particular to a GC-MS (gas chromatography-mass spectrometry) technology-based method for simultaneously determining contents of various components in Yinhua cold powder. The method comprises a method for simultaneously determining the contents of various components in the Yinhua cold powder or volatile oil. The method comprises the following steps: (1) carrying out a GC analysis program on a medicinal powder extracting solution or volatile oil of the Yinhua cold powder; and (2) carrying out mass spectrum identification by utilizing a mass spectrum library under the mass spectrum conditions that an ion source is electron bombardment ionization (EI), the energy is 70eV, the temperature of the ion source is 200 DEG C, the interface temperature is 250 DEG C, a full scanning mode m / z is 45-500, the electron multiplication voltage is 1.5 kV, and collision gas is argon (99.99%). According to the method, the GC-MS technology is established for simultaneously determining the contents of various components in the Yinhui cold powder or volatile oil, more than 50 compounds are identified in the volatile oil of the Yinhui cold powder, and more than 25 compounds are identified in a normal hexane extracting solution of the powder.

Description

Technical Field

[0001] The invention relates to the field of drug analysis, and in particular to a method for simultaneously determining the contents of multiple components in Yinhu Ganmao powder based on GC-MS technology. Background Art

[0002] Yinhu Ganmao Powder (Yinhu Ganmao Powder,) is composed of 12 medicinal materials such as Pinus lanceolata, Eucalyptus globulus, Honeysuckle, Forsythia suspensa, Nepeta tenuifolia, Bupleurum, Artemisia annua, Mint, Patchouli, Tangerine peel, Platycodon grandiflorum, and Artemisia argyi. It is an external patch with the effects of dispelling wind and relieving exterior symptoms, clearing away heat and detoxifying. It is usually used for chills and fever, nasal congestion, sneezing, coughing, headache, and general discomfort caused by wind-heat colds. It plays a great role in the treatment of pneumonia caused by the new coronavirus. The volatile oil of Yinhu Ganmao Powder is composed of powder and oil. During the preparation process, the volatile oil of each medicinal material is first extracted by steam distillation, and the volatile oil is mixed to become oil. After the volatile oil is extracted from the medicinal materials such as Honeysuckle and Forsythia suspensa, the remaining dregs are crushed and dried to make powder. Before using the volatile oil of Yinhu Ganmao Powder, the medicated oil needs to be poured into the powder and applied to the navel for transdermal absorption to enter the human body for therapeutic effect. The pharmacological material basis of Yinhu Ganmao Powder volatile oil comes from multiple active ingredients in multiple medicinal flavors. However, the current quality standard of Yinhu Ganmao Powder volatile oil only determines the content of chlorogenic acid, and lacks an effective method to evaluate the overall quality of Yinhu Ganmao Powder volatile oil. Therefore, it is necessary to establish a comprehensive evaluation method for multi-component quantitative determination of Yinhu Ganmao Powder volatile oil.

[0003] So far, no report has been found on a method for simultaneously determining the contents of multiple components in Yinhu Ganmao San powder or volatile oil based on GC-MS technology. Summary of the invention

[0004] In order to overcome the shortcomings of the prior art, the present invention provides a method for simultaneously determining the contents of multiple components in Yinhu Ganmao Powder based on GC-MS technology.

[0005] The purpose of the present invention can be achieved by the following technical solutions:

[0006] A method for simultaneously determining the contents of multiple components in Yinhu Ganmao Powder based on GC-MS technology, the method comprising a method for simultaneously determining the contents of multiple components in Yinhu Ganmao Powder powder or volatile oil, the method comprising the following steps:

[0007] (1) subjecting the extract or volatile oil of Yinhu Ganmao San medicinal powder to a GC analysis procedure;

[0008] (2) Mass spectrometry identification was performed using a mass spectrometry library. The mass spectrometry conditions were as follows: the ion source was electron impact ionization (EI), the energy was 70 eV, the ion source temperature was 200°C, the interface temperature was 250°C, the full scan mode was m / z 45-500, the electron multiplier voltage was 1.5 kV, and the collision gas was argon (99.99%).

[0009] Preferably, the mass spectrum library includes one or a combination of two or more of NIST20-1, NIST20-2, and NIST20s.

[0010] Preferably, the GC analysis procedure of the volatile oil of Yinhu Ganmao Powder is: the column temperature is maintained at 80°C for 10 min, increased to 150°C at a rate of 10°C / min and maintained for 30 min, increased to 200°C at a rate of 25°C / min and maintained for 15 min, the carrier gas is nitrogen (99.99%), the split ratio is 10:1, the injection volume is 1 μL, the injection port temperature is 200°C, and the detector temperature is 250°C.

[0011] Preferably, the Yinhu Ganmao Powder extract is a n-hexane extract of Yinhu Ganmao Powder, and the GC analysis procedure of the n-hexane extract of Yinhu Ganmao Powder is as follows: the column temperature is maintained at 50°C for 1 min, increased to 180°C at a rate of 10°C / min and maintained for 30 min, increased to 240°C at a rate of 10°C / min and maintained for 30 min, the carrier gas is nitrogen (99.99%), the split ratio is 10:1, the injection volume is 1 μL, the injection port temperature is 200°C, and the detector temperature is 250°C.

[0012] Preferably, the preparation of the volatile oil of Yinhu Ganmao Powder: take Pinus kirilowii and Eucalyptus globulus leaves, crush them and soak them in water at a ratio of 1g:15ml, then steam distill them for 2.5h, collect the volatile oil and store it in another container; take Artemisia annua, Nepeta tenuifolia, Mentha, Forsythia suspensa, Patchouli, Artemisia argyi and Tangerine peel, crush them and soak them in water at a ratio of 1g:15ml, then steam distill them for 3h, collect the volatile oil, mix the collected volatile oils evenly, take an appropriate amount of the mixed volatile oil, dilute it 20,000 times with N,N-dimethylformyl, and perform GC-MS analysis after passing through a 0.22μm organic phase filter.

[0013] Preferably, the materials of various medicinal materials are 6818g of Pinus koraiensis, 6818g of Eucalyptus grandiflora, 45g each of Artemisia annua, Nepeta tenuifolia, Mentha, Forsythia suspensa, Patchouli, and 23g each of Artemisia argyi and dried tangerine peel.

[0014] Preferably, the n-hexane extract of the Yinhu Ganmao Powder is prepared by weighing 1.0 g of the powder in the Yinhu Ganmao Powder into a conical flask, adding 10 ml of n-hexane, weighing, ultrasonic extraction for 30 minutes, cooling to make up the weight, centrifuging at 8000 rpm for 20 minutes, and taking the supernatant and filtering it through a 0.22 μm filter membrane for GC-MS analysis.

[0015] The present invention also provides a method for simultaneously determining the contents of multiple components in Yinhu Ganmao Powder volatile oil based on GC-MS technology combined with MRM, and the quality evaluation method comprises the following steps:

[0016] (1) Preparation of Yinhu Ganmao Powder Volatile Oil: 6818g of Pinus lanceolata and 6818g of Eucalyptus globulus were crushed and soaked in water at a ratio of 1g:15ml, then steam distilled for 2.5h, and the volatile oil was collected and stored in a separate container; 45g each of Artemisia annua, Nepeta tenuifolia, Mentha, Forsythia suspensa, and Patchouli, 23g each of Artemisia argyi and Tangerine peel were crushed and soaked in water at a ratio of 1g:15ml, then steam distilled for 3h, and the volatile oil was collected. The collected volatile oils were mixed evenly, and an appropriate amount of the mixed volatile oil was diluted 20,000 times with N,N-dimethylformamide, and then filtered through a 0.22μm organic phase filter for GC-MS analysis;

[0017] (2) The GC analysis procedure of Yinhu Ganmao Powder volatile oil was as follows: column temperature was maintained at 80°C for 10 min, increased to 150°C at a rate of 10°C / min and maintained for 30 min, increased to 200°C at a rate of 25°C / min and maintained for 15 min, the carrier gas was nitrogen (99.99%), the split ratio was 10:1, the injection volume was 1 μL, the injection port temperature was 200°C, and the detector temperature was 250°C;

[0018] (3) Mass spectrometry identification was performed using a mass spectrometry library (NIST20-1, NIST20-2, NIST20s). The mass spectrometry conditions were as follows: the ion source was electron bombardment ionization (EI), the energy was 70 eV, the ion source temperature was 200 °C, the interface temperature was 250 °C, the full scan mode was m / z 45-500, the electron multiplier voltage was 1.5 kV, and the collision gas was argon (99.99%);

[0019] (4) The mass spectrometry identification results are combined with multiple reaction monitoring (MRM) to perform detection analysis and component content determination.

[0020] Compared with the prior art, the method of the present invention has the following advantages:

[0021] 1. The present invention establishes a method for simultaneously determining the contents of multiple components in Yinhu Ganmao Powder or volatile oil using GC-MS technology, wherein more than 50 compounds were identified in the volatile oil of Yinhu Ganmao Powder, and more than 25 compounds were identified in the n-hexane extract of the powder.

[0022] 2. The present invention establishes a method for MRM quantitative analysis of 19 compounds in the volatile oil of Yinhu Ganmao Powder. The quantitative results of the 19 components in the volatile oil of Yinhu Ganmao Powder are reliable, and the specific contents of the 19 compounds with relatively high contents in the volatile oil of Yinhu Ganmao Powder are further determined. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1This is the GC-MS total ion current graph of the volatile oil of Yinhu Ganmao Powder.

[0024] Figure 2 This is the GC-MS total ion current of the n-hexane extract of Yinhu Ganmao San powder.

[0025] Figure 3 This is the GC-MS chromatogram of the MRM extraction of 19 components of Yinhu Ganmao Powder volatile oil. (A. α-pinene; B. camphene; C. sabinene; D. β-pinene; E. α-terpinene; F. limonene; GP-cymene; H. eucalyptol; I. linalool; J. (-)-menthone; K. 4-terpene alcohol; L. menthol; M. α-terpineol; N. tridecane; O. pulegone; P. trans-caryophyllene; Q. α-caryophyllene; R. hexadecane; S. patchouli alcohol). DETAILED DESCRIPTION

[0026] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0027] Example 1

[0028] 1. Experimental Materials

[0029] 1.1 Instruments and Equipment

[0030] Nexis TQ2030 GC (Shimadzu Corporation, Japan); TQ8040 Nexis MS (Shimadzu Corporation, Japan); DB-624UI gas column (30m×0.25mm×1.40μm, Agilent Technologies, USA); high-speed low-temperature centrifuge (SIGMA, Germany); carbon dioxide incubator (Taicang Yisigao Medical Technology Co., Ltd.); microplate reader (TECAN, Switzerland); analytical balance (Mettler Toledo Technology Co., Ltd.).

[0031] 1.2 Reagents and test drugs

[0032] Medicinal materials and reagents Yinhu Ganmao San (202106291, 202101233, 2021103082) were all provided by Guangxi Yuanantang Pharmaceutical Co., Ltd.; N,N-dimethylformamide (DMF, HPLC ≥ 99.9, C14428283) and dimethyl sulfoxide (DMSO, HPLC ≥ 99.9, C14585519) were purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; α-terpinene (98%, CFN70238), β-pinene (98%, CF N93287), α-pinene (98%, CFN93285), trans-caryophyllene (98%, CFN90502), 4-terpene alcohol (98%, CFN94877), α-terpinene (98%, CFN70238) were purchased from Chemfaces; tridecane (98.5%, 190191-201701), linalool (98.8%, 111503-202004), pulegone (99.8%, 111706-20190 7), (-)-menthone (99.9%, 111705-202106), menthol (99.8%, 110728-201707), limonene (96%, 100470-201302), dexamethasone sodium phosphate (100016-202117) were purchased from China Food and Drug Administration; α-terpineol (98.7%, 0913-RC-OO42) and camphene (99.4%, D6-12-5-1) were purchased from CATO; eucalyptol (98%, 221212), hexadecane (99.5%, 20230320), p-cymene (98%, 230505) were all obtained from Beijing North Weiye Institute of Metrology and Technology; patchouli alcohol (97%, Shanghai Acemec Biochamical, 95523846C); sabinene (98%, Shanghai Yuanye Biotechnology Co., Ltd., M23HB179089); α-caryophyllene (95%, Hebei Bailingwei Ultrafine Materials Co., Ltd., L310X43).

[0033] 2. Experimental Methods

[0034] 2.1 Preparation of Yinhu Ganmao Powder Volatile Oil

[0035] Take 6818g of Pinus koraiensis and 6818g of Eucalyptus globulus leaves, grind them into powder and soak them in water at a ratio of 1g:15ml, then steam distill them for 2.5h, collect the volatile oil and store it in another container; take 45g each of Artemisia annua, Nepeta tenuifolia, Mentha, Forsythia suspensa, and Patchouli, and 23g each of Artemisia argyi and dried tangerine peel, grind them into powder and soak them in water at a ratio of 1g:15ml, then steam distill them for 3h, collect the volatile oil, mix the collected volatile oils evenly, take an appropriate amount of the mixed volatile oil, dilute it 20,000 times with N,N-dimethylformyl, and pass it through a 0.22μm organic phase filter membrane for GC-MS analysis.

[0036] 2.2 Preparation of n-hexane extract of Yinhu Ganmao powder

[0037] Weigh 1.0 g of the powder from Yinhu Ganmao Powder into a conical flask, add 10 ml of n-hexane, weigh, and perform ultrasonic extraction for 30 min. Cool to make up the weight, centrifuge at 8000 rpm for 20 min, and take the supernatant and filter it through a 0.22 μm filter membrane for GC-MS analysis.

[0038] 2.3 GC-MS conditions

[0039] The GC analysis program of Yinhu Ganmao Powder volatile oil was 80℃ for 10min, then increased to 150℃ at a rate of 10℃ / min for 30min, then increased to 200℃ at a rate of 25℃ / min for 15min, the carrier gas was nitrogen (99.99%), the split ratio was 10:1, the injection volume was 1μL, the injection port temperature was 200℃, and the detector temperature was 250℃;

[0040] The GC analysis program of the n-hexane extract of Yinhu Ganmao Powder was as follows: maintain at 50°C for 1 min, increase at 10°C / min to 180°C for 30 min, increase at 10°C / min to 240°C for 30 min, the carrier gas was nitrogen (99.99%), the split ratio was 10:1, the injection volume was 1 μL, the injection port temperature was 200°C, and the detector temperature was 250°C.

[0041] Mass spectrometry conditions: the ion source was electron bombardment ionization (EI), the energy was 70 eV, the ion source temperature was 200°C, the interface temperature was 250°C, the full scan mode was m / z 45-500, the electron multiplier voltage was 1.5 kV, and the collision gas was argon (99.99%).

[0042] 2.4 Mass spectrometry identification of volatile oil and powder components

[0043] The compounds were identified by literature review and NIST20-1, NIST20-2, and NIST20s databases using Shimadzu reanalysis software. We also checked the mass spectrum data of the base peak mass-to-charge ratio and relative abundance conditions as a direct comparison.

[0044] 2.5 MRM quantitative analysis of target compounds

[0045] After comparing the precursor ion data with the standard databases NIST20-1, NIST20-2, NIST20s, and literature review, this study conducted a quantitative analysis of 19 compounds with relatively high contents in volatile oils. First, product ion scanning of the target compound was used to optimize the optimal fragmentation voltage of its product ion fragments and determine the Q3 ion of MRM. Based on the MRM scan of the mixed standard solution, the ion ratio of the MRM method was reset to establish a method for MRM quantitative analysis of these 19 compounds.

[0046] 3. Experimental Results

[0047] 3.1 GC-MS Identification Results of Yinhu Ganmao Powder Volatile Oil

[0048] The Q3Scan full scanning method was used to analyze the components of volatile oils. Figure 1 The total ion current (TIC) of the volatile oil of Yinhu Ganmao Powder was used. According to the literature review and database data as well as the mass spectrum data of the base peak mass-to-charge ratio and relative abundance conditions, a total of 50 compounds were identified (Table 1), among which eucalyptol had the highest relative content of 28.25%.

[0049] 3.2 GC-MS Identification Results of Hexane Extract of Yinhu Ganmao Powder

[0050] Q3Scan was used to analyze the components in the n-hexane extract of the powder ( Figure 2 ), according to the literature review and database data as well as the mass-to-charge ratio of the base peak and the mass spectrum data of the relative abundance conditions, a total of 25 compounds were identified (Table 2), among which eucalyptol had the highest content of 34.83%. The identification results of the top components with the highest relative content were the same as those in the volatile oil, so the 50 compounds to be identified in the medicinal oil were used for subsequent network pharmacology analysis.

[0051] 3.3 MRM quantitative analysis of 19 compounds in Yinhu Ganmao Powder volatile oil

[0052] In order to clarify the content of relatively high components in the volatile oil of Yinhu Ganmao Powder, 19 components were selected for quantitative analysis according to the relative percentage content and the conditions of commercially available standards. The standard products were compared with the components in the volatile oil. The results are as follows Figure 3As shown, the retention time of the 19 compounds is consistent with that of the volatile oil, and the solvent DMF has no interference with the detection. The optimal CE voltage and ion abundance of each target component were obtained by optimizing the product ion scanning conditions of the 19 target compounds. The results are shown in Table 3. The MRM method was established based on the optimal CE voltage, quantitative ion pair and qualitative ion pair, and the standard solution was prepared. The methodology was verified using N,N-dimethylformamide as the solvent blank. The results showed that the solvent had no interference with the detection, and the MRM extraction mass chromatographic peak of the Yinhu Ganmao powder volatile oil was consistent with the RT of the standard solution.

[0053] 3.4 Methodological Review

[0054] Standard curves of different concentrations of 19 mixed compounds were prepared, and the GC-MS quantitative method established for the 19 compounds was methodologically investigated. The results (Table 4) show that the linearity, stability, repeatability, precision and accuracy of the established method are good, indicating that the quantitative results of the 19 components in the volatile oil of Yinhu Ganmao Powder are reliable, and the specific contents of these 19 relatively high compounds in the volatile oil of Yinhu Ganmao Powder are further determined.

[0055] Table 1 GC-MS identification results of Yinhu Ganmao Powder volatile oil

[0056]

[0057]

[0058]

[0059] Table 2 GC-MS identification results of n-hexane extract of Yinhu Ganmao San medicinal powder

[0060]

[0061]

[0062] Table 3 Quantitative and qualitative ion pair information of 19 compounds in Yinhu Ganmao Powder volatile oil

[0063]

[0064]

[0065] Table 4 Determination of the contents of 19 ingredients in Yinhu Ganmao Powder

[0066]

[0067]

[0068] Although the present invention has been described in detail above by means of general description, specific implementation methods and tests, it is obvious to those skilled in the art that some modifications or improvements may be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A method for simultaneously determining the contents of multiple components in Yinhu Ganmao Powder based on GC-MS technology, the method comprising a method for simultaneously determining the contents of multiple components in Yinhu Ganmao Powder powder or volatile oil, characterized in that: The method comprises the following steps: (1) subjecting the extract or volatile oil of Yinhu Ganmao San medicinal powder to a GC analysis procedure; (2) Mass spectrometry identification was performed using a mass spectrometry library. The mass spectrometry conditions were as follows: the ion source was electron impact ionization (EI), the energy was 70 eV, the ion source temperature was 200°C, the interface temperature was 250°C, the full scan mode was m / z 45-500, the electron multiplier voltage was 1.5 kV, and the collision gas was argon with a purity of 99.99%.

2. The method according to claim 1, characterized in that The mass spectrum library includes one of NIST20-1, NIST20-2, and NIST20s, or a combination of two or more thereof.

3. The method according to claim 2, characterized in that The GC analysis procedure of the volatile oil of Yinhu Ganmao Powder is as follows: the column temperature is maintained at 80°C for 10 minutes, increased to 150°C at a rate of 10°C / min and maintained for 30 minutes, increased to 200°C at a rate of 25°C / min and maintained for 15 minutes, the carrier gas is nitrogen with a purity of 99.99%, the split ratio is 10:1, the injection volume is 1 μL, the injection port temperature is 200°C, and the detector temperature is 250°C.

4. The method according to claim 3, characterized in that: The preparation method of the volatile oil of Yinhu Ganmao Powder is as follows: take Pinus kirilowii and Eucalyptus globulus leaves, grind them into powder and soak them in water at a ratio of 1g:15ml, then steam distill them for 2.5h, collect the volatile oil and store them in another container; take Artemisia annua, Nepeta tenuifolia, Mentha, Forsythia suspensa, Patchouli, Artemisia argyi and Tangerine peel, grind them into powder and soak them in water at a ratio of 1g:15ml, then steam distill them for 3h, collect the volatile oil, mix the collected volatile oils evenly, take an appropriate amount of the mixed volatile oils, dilute them 20,000 times with N,N-dimethylformyl, and perform GC-MS analysis after filtering them through a 0.22μm organic phase filter membrane.

5. The method according to claim 4, characterized in that The materials of various medicinal materials are 6818g of Pinus tabebuia, 6818g of Eucalyptus globulus, 45g each of Artemisia annua, Nepeta tenuifolia, Mentha, Forsythia suspensa, Patchouli, 23g each of Artemisia argyi and dried tangerine peel.

6. The method according to claim 2, characterized in that The Yinhu Ganmao Powder extract is a n-hexane extract of Yinhu Ganmao Powder, and the GC of the n-hexane extract of Yinhu Ganmao Powder is shown.

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