Method for measuring content of blood-entering chemical components of dripping pill with refreshing throat

Abstract

The invention relates to the technical field of pharmaceutical analysis, in particular to a method for determining the content of chemical components entering blood of dripping pills with a refreshing throat. According to the method, a gas chromatography-mass spectrometry (GC-MS) technology is adopted, and the chromatographic conditions are as follows: an SH-Stabilwax capillary column is 30 m * 0.32 mm in specification, and the film thickness is 0.25 mu m; the temperature of a sample inlet is 220 DEG C; the split-flow-free sample injection is realized; the sample size is 1 microliter; the flow of a chromatographic column is 2mL / min; the mass spectrum conditions are as follows: the ion source temperature is 200 DEG C; the interface temperature is 230 DEG C; the ionization mode is electron impact ion (EI); the detection voltage is 0.2 KV; the solvent is delayed for 2 minutes; and SIM mode. The method creatively realizes the simultaneous determination of the concentrations of 14 volatile components, namely beta-pinene, limonene, eucalyptol, 3-octanol, isomenthone, menthone, camphor, menthyl acetate, trans-caryophyllene, menthol, borneol, caryophylline, p-cymene and guaiacol, in the throat-refreshing dropping pill or a drug-containing serum sample after taking the throat-refreshing dropping pill. According to the research, accurate monitoring on the drug effect substance groups in the instant-swallowing and refreshing containing dropping pill is realized for the first time, so that not only is a key experimental basis provided for perfecting the quality control standard, but also a new view angle is provided for explaining the modern scientific connotation of multi-component synergistic effect of the instant-swallowing and refreshing containing dropping pill; more scientific data is provided for establishing comprehensive quality control and quantitative research of the medicine in the future.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis, in particular to a method for determining the content of chemical components of Yanlishuang oral dropping pills entering blood. Background Art

[0002] Yanli Shuang Oral Drops are a nationally exclusive Miao medicine product (National Medicine Standard Z20025286) produced by Guizhou Huangguoshu Shushuang Pharmaceutical Co., Ltd. This Miao medicine compound preparation, consisting of moxa slices, mugwort sesame oil, peppermint oil, menthol, and glycyrrhizic acid monoammonium salt, is said to dispel wind and heat, reduce swelling and pain, and clear the throat (Miao medicine: Song Gongzheng, Tai Kai Tai Meng). It is used to treat acute pharyngitis and acute exacerbations of chronic pharyngitis, characterized by sore throat, dry throat, and bad breath. Because Yanli Shuang Oral Drops are an early product upgraded from a local national standard, basic research is weak. The existing standard only controls menthol, borneol, and glycyrrhizic acid monoammonium salt. Patent CN116124922B measures the contents of camphor, menthone, borneol, and menthol, but does not clarify whether these ingredients are active ingredients. The pharmacological basis for the efficacy of Yanli Shuang Oral Drops remains unclear, and blood testing for its active ingredients is even more limited. The core difficulties are: ① Interference from complex biological matrices: High-abundance proteins, lipids, and endogenous metabolites in serum co-elute with trace target components, requiring complex pretreatment to improve selectivity; ② Metabolic transformation and coexistence of multiple components: Drugs may be metabolized into a variety of active or inactive products in the body, requiring simultaneous detection of the original drug and metabolites. Method development must cover the separation and quantification of multiple targets.

[0003] Traditional Chinese medicine preparations have complex ingredients, some of which may be difficult to be effectively absorbed by the body due to limited bioavailability and may not directly participate in the pharmacological effects. By systematically detecting the chemical components absorbed into the blood (blood-transiting components), candidate active substances that actually enter the systemic circulation can be effectively identified. This screening strategy based on in vivo exposure characteristics helps to focus on the research direction of key active components and avoid ineffective analysis of non-bioavailable components. In response to the needs of basic research on the active substances and clinical drug safety of Yanlishuang oral drops, the development of a quantitative analysis method for blood-transiting components with high sensitivity, good reproducibility and detection throughput is of great scientific value for clarifying the composition of its in vivo active substance group and revealing the synergistic mechanism of multiple components. It has become a key technical bottleneck in promoting the modernization of research on this preparation. Summary of the Invention

[0004] In order to solve the above technical problems existing in the prior art, the present invention provides a method for determining the content of chemical components of Yanlishuang oral dripping pills entering the blood, which is as follows:

[0005] A method for determining the content of chemical components of Yanlishuang oral dripping pills in blood, capable of simultaneously determining the concentrations of β-pinene, limonene, eucalyptol, 3-octanol, isomenthone, menthone, camphor, menthyl acetate, trans-caryophyllene, menthol, borneol, dianthinol, p-cymene, and guaiacol, using a GC-MS method for detection;

[0006] Chromatographic conditions: SH-Stabilwax capillary column, 30 m × 0.32 mm, 0.25 μm film thickness; injection port temperature 220°C; splitless injection; injection volume 1 μL; column flow rate: 2 mL / min;

[0007] Mass spectrometry conditions: ion source temperature 200°C; interface temperature 230°C; ionization mode: electron impact ionization (EI); detection voltage: 0.2 kV; solvent delay 2 min; SIM mode.

[0008] Furthermore, the chromatographic conditions and the specific temperature programming procedures are as follows:

[0009] Column oven temperature program

[0010]

[0011] Furthermore, the mass spectrometry conditions and the detected ion information of each component are as follows:

[0012] Detection ion information of 14 components including β-pinene

[0013]

[0014] Furthermore, in the GC-MS method, the test solution is prepared using Yanlishuang oral drops or a drug-containing serum sample after taking Yanlishuang oral drops.

[0015] Furthermore, the test solution is prepared by adding ethyl acetate to Yanli Shuang oral dripping pills. Specifically, the test solution is prepared by adding ethyl acetate to Yanli Shuang oral dripping pills to prepare a test solution containing 50 μg / mL of Yanli Shuang oral dripping pills and 50 ng / mL of naphthalene.

[0016] Furthermore, the drug-containing serum sample after taking Yanli Shuang oral drops is prepared by collecting the drug-containing serum of the individual after taking Yanli Shuang oral drops, then adding ethyl acetate to vortex and centrifuge, and taking the supernatant for sampling and analysis. Specifically, 100 μL of serum sample is placed in a 1.5 mL centrifuge tube, 100 μL of ethyl acetate is added to vortex and mix for 2 minutes, and the supernatant is centrifuged at 4°C and 12000 r·min. -1 Centrifuge for 10 min and take the supernatant for analysis.

[0017] Furthermore, the drug-containing serum of the individual after taking the Yanlishuang oral dropping pills is obtained by taking blood and placing it in a centrifuge tube containing an anticoagulant, centrifuging the whole blood, and taking the supernatant to obtain a serum sample.

[0018] Furthermore, the serum sample was stored at -80°C after preparation and thawed before use.

[0019] Compared with the prior art, the technical effects created by the present invention are embodied in:

[0020] (1) The present invention uses gas chromatography-mass spectrometry (GC-MS) technology. The chromatographic conditions are: SH-Stabilwax capillary column with specifications of 30m×0.32mm and film thickness of 0.25μm; injection port temperature of 220℃; splitless injection; injection volume of 1μL; column flow rate of 2mL / min; mass spectrometry conditions: ion source temperature of 200℃; interface temperature of 230℃; ionization mode of electron impact ionization (EI); detection voltage of 0.2KV; solvent delay of 2min; SIM mode. This method can simultaneously determine the concentrations of β-pinene, limonene, eucalyptol, 3-octanol, isomenthone, menthone, camphor, menthyl acetate, trans-caryophyllene, menthol, borneol, dianthinol, p-cymene, and guaiacol.

[0021] (2) The method of the present invention can be used to detect Yanli Shuang oral drops or drug-containing serum samples after taking Yanli Shuang oral drops, and has excellent specificity, repeatability, and stability. The ability to detect drug-containing serum samples fills the gap in blood detection methods for the active ingredients of Yanli Shuang oral drops. It has important scientific value in elucidating the composition of the active substance group in the body and revealing the mechanism of multi-component synergistic action.

[0022] (3) This application uses GC-MS to detect the blood-entering components of Yanli Shuang oral drops, and establishes a content determination method for Yanli Shuang oral drops using 14 components as indicators. This study has achieved the first precise monitoring of the active substance group in Yanli Shuang oral drops, which not only provides a key experimental basis for improving its quality control standards, but also provides a new perspective for explaining the modern scientific connotation of the "multi-component synergistic effect" of Yanli Shuang oral drops. It provides more scientific data for the establishment of comprehensive quality control and quantitative research on this drug in the future, which has far-reaching significance and value for Chinese medicine to better exert its clinical efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1The GC-MS SIM chromatograms of the serum of the treatment group (A), the control solution (B) and the serum of the blank group (C) are shown. 1. β-pinene; 2. limonene; 3. eucalyptol; 4. p-cymene; 5. 3-octanol; 6. menthone; 7. isomenthone; 8. camphor; 9. menthyl acetate; 10. trans-caryophyllene; 11. menthol; 12. borneol; IS. naphthalene; 13. dianthin; 14. guaiacol.

[0024] Figure 2 The GC-MS SIM chromatograms of the reference solution (A), the test solution (B), and the blank solution (C) are shown. 1. β-pinene; 2. limonene; 3. eucalyptol; 4. p-cymene; 5. 3-octanol; 6. menthone; 7. isomenthone; 8. camphor; 9. menthyl acetate; 10. trans-caryophyllene; 11. menthol; 12. borneol; IS. naphthalene; 13. dianthin; 14. guaiacol. DETAILED DESCRIPTION

[0025] The technical solution of the present invention is further defined below in conjunction with specific implementation methods, but the scope of protection required is not limited to the description.

[0026] Example:

[0027] 1. Materials

[0028] 1.1 Instrument

[0029] Gas chromatography-mass spectrometry (GC-MS-TQ8050NX, Shimadzu Enterprise Management (China) Co., Ltd.), ultra-performance liquid chromatography-triple quadrupole tandem mass spectrometer (UPLC-TQS, Waters, USA), low-temperature high-speed centrifuge (MICROFUGE64R, Beckman Coulter, USA), multi-tube vortex oscillator (Typ. VX-III, Beijing Tajin Technology Co., Ltd.), electronic balance (EL204, Mettler-Toledo Instrument Shanghai Co., Ltd.), ultrasonic cleaning machine (CQ250A-TS, Shanghai Yuejin Medical Optical Instrument Factory).

[0030] 1.2 Drug testing

[0031] Yanlishuang oral dripping pills (YLS) were provided by Guizhou Huangguoshu Shushuang Pharmaceutical Co., Ltd., with the batch numbers shown in Table 1. Ethyl acetate (chromatographic grade, Tianjin Kemeiou Chemical Reagent Co., Ltd.); β-pinene (batch number: WP24022005, purity 98%), limonene (batch number WP24012311, purity 98%), eucalyptol (batch number: WP24022210, purity 98%), 3-octanol (batch number WP24112209, purity 98%), isomenthone (batch number: WP24091304, purity 98%), trans-caryophyllene (batch number: WP23110105, purity 90%), and p-cymene (batch number: WP24060301, purity 98%) were all purchased from Sichuan Weikeqi Biotechnology Co., Ltd.; menthone (batch number: 1117 05-202106, purity 99%), menthyl acetate (batch number: 190044-202102, purity 98%), and menthol (batch number: 110728-202208, purity 99.6%) were purchased from the China Food and Drug Inspection Institute; camphor (batch number: PS020363, purity 99.30%) was purchased from Chengdu Pusi Biotechnology Co., Ltd.; borneol (batch number: MUST-22021501, purity 98%) was purchased from Chengdu Mansite Biotechnology Co., Ltd.; oleanolic acid (batch number: AB2637-0005, purity 98%) and guaiacol (batch number: AB3219-0005, purity 98%) were purchased from Chengdu Aifa Biotechnology Co., Ltd., and naphthalene (batch number: 21050063) was purchased from Tanmo Quality Inspection-Standard Material Center.

[0032] Table 117 Batch of Yanlishuang Oral Dropping Pills Sample Information

[0033]

[0034] 1.3 Animals

[0035] SD male rats, weighing 200 ± 20 g, were provided by Changsha Tianqin Biotechnology Co., Ltd., animal license number: SCXK (Xiang) 2019-0014.

[0036] 2. Methods and Results

[0037] 2.1 Identification of blood components of Yanlishuang oral drops

[0038] 2.1.1 Preparation of oral gavage solution

[0039] Grind the Yanlishuang oral dropping pills, weigh an appropriate amount of Yanlishuang oral dropping pill powder, dissolve it in 5% CMC-Na suspension to prepare an oral gavage drug dose of 840 mg / kg, and vortex mix for 1 minute to ensure that the drug is fully mixed.

[0040] 2.1.2 Preparation of reference solution

[0041] Accurately weigh appropriate amounts of β-pinene, limonene, eucalyptol, 3-octanol, isomenthone, menthone, camphor, menthyl acetate, trans-caryophyllene, menthol, borneol, dianthin, p-cymene, and guaiacol into a 10 mL volumetric flask. Add appropriate amount of ethyl acetate and ultrasonically dissolve. Dilute to volume to obtain a 100 μg / mL reference substance stock solution. Immediately before use, accurately measure an appropriate amount of the above stock solution (calculate the required volume based on the target concentration), dilute with ethyl acetate, and dilute to volume to obtain a mixed reference substance working solution of the desired concentration.

[0042] 2.1.3 Collection and preparation of serum samples

[0043] ① Collection of Drug-Containing Serum Samples: Twelve healthy male SD rats were fasted for 12 hours and then gavaged with 840 mg / kg of Yanlishuang Oral Dropping Pills. Before the first dose, 1 mL of blank blood was collected. Dosing was continued twice daily for 6 consecutive days. Blood was collected from the orbital cavity 30 minutes after the last dose and placed in a centrifuge tube containing anticoagulant. The whole blood was centrifuged and the supernatant was collected to obtain serum samples. The samples were then stored at -80°C for testing.

[0044] ② Preparation of serum samples: Thaw the serum sample on ice, take 100 μL and place it in a 1.5 mL centrifuge tube, add 100 μL of ethyl acetate and vortex for 2 min, then centrifuge at 4°C and 12000 r / min. -1 Centrifuge for 10 min and take the supernatant for analysis.

[0045] 2.1.4 GC-MS conditions

[0046] Chromatographic conditions: SH-Stabilwax (30 m × 0.32 mm, film thickness 0.25 μm) capillary column; injection port temperature 220°C; splitless injection; injection volume 1 μL; column flow rate: 2 mL / min; temperature program details are shown in Table 2

[0047] Table 2 Column oven heating program

[0048]

[0049] Mass spectrometry conditions: ion source temperature 200°C; interface temperature 230°C; electron impact ionization (EI) mode; detection voltage 0.2 kV; solvent delay 2 min; SIM mode. Detected ion information for each component is shown in Table 3.

[0050] Table 3 Detection ion information of 14 components including β-pinene

[0051]

[0052] 2.1.5 Original ingredients of Yanlishuang oral drops entering the blood

[0053] The serum of the drug administration group, mixed reference solution and blank group were accurately drawn and analyzed according to the given GC-MS conditions. The chromatogram is shown in Figure 1 Excluding the blank control, the chromatograms of the standards were compared and the blood-entering components of Yanlishuang oral drops were identified as follows: β-pinene, limonene, eucalyptol, p-cymene, 3-octanol, menthone, isomenthone, camphor, menthyl acetate, trans-caryophyllene, menthol, borneol, dianthin, and guaiacol.

[0054] 2.2 Determination of the content of components of Yanlishuang oral drops entering the blood.

[0055] 2.2.1 Preparation of reference solution

[0056] Same as “2.1.2”.

[0057] 2.2.2 Preparation of internal standard solution

[0058] Accurately weigh the naphthalene reference substance and prepare a 500 ng / mL internal standard stock solution in ethyl acetate.

[0059] 2.2.3 Preparation of test solution

[0060] Take an appropriate amount of Yanli Shuang oral dripping pills, grind them into powder, accurately weigh 0.1g, add ethyl acetate to dissolve, and prepare the naphthalene internal standard solution to prepare a test solution containing 50μg / mL of Yanli Shuang oral dripping pills and 50ng / mL of naphthalene.

[0061] 2.2.4 GC-MS conditions

[0062] GC-MS conditions are the same as those in “2.1.4”.

[0063] 2.2.5 Methodological Investigation

[0064] 2.2.5.1 Specificity Investigation

[0065] Specificity results are shown in Figure 2 , the blank solvent did not produce a peak at the corresponding position of the reference substance, and there was no obvious interference, indicating that the method had good specificity.

[0066] 2.2.5.2 Linear relationship investigation

[0067] Take an appropriate amount of the reference solution under "2.2.1" and inject it for detection according to the chromatographic conditions under "2.1.4". Perform linear regression with mass concentration as the abscissa (X) and peak area as the ordinate (Y). The linear regression equations for β-pinene, limonene, eucalyptol, p-cymene, 3-octanol, menthone, isomenthone, camphor, menthyl acetate, trans-caryophyllene, menthol, borneol, dianthin, and guaiacol are shown in Table 4. R 2 >0.998, indicating that each component has good linearity within its respective linear range.

[0068] Table 4 Results of linear relationship investigation of 14 components

[0069]

[0070] 2.2.5.3 Precision

[0071] Accurately pipette 1 μL of the mixed reference solution, and inject and test according to the chromatographic conditions under "2.1.4". Continuously inject and test 6 times, and the calculated RSD of the peak area of ​​14 components including β-pinene is between 0.54% and 2.56%, indicating that the instrument has good precision. The results are shown in Table 5.

[0072] Table 5 Precision inspection results

[0073]

[0074] 2.2.5.4 Repeatability

[0075] The same batch of YLS samples was prepared according to the method in "2.2.3" and 6 parallel preparations were made. The samples were injected and tested according to the chromatographic conditions in "2.1.4". The RSDs of the contents of 14 components including β-pinene were between 1.36% and 2.95%, indicating good repeatability of the method (see Table 6).

[0076] Table 6 Repeatability test results

[0077]

[0078]

[0079] 2.2.5.4 Stability investigation

[0080] The same batch of YLS samples were prepared according to the method in “2.2.3”. Samples were injected, tested and analyzed according to the chromatographic conditions in “2.1.4” at 0h, 2h, 4h, 8h, 12h and 24h after preparation. The RSDs of the contents of 14 components including β-pinene were between 0.98% and 2.97%, indicating that the YLS samples had good stability within 24h. The results are shown in Table 7.

[0081] Table 7 Stability test results

[0082]

[0083] 2.2.5.4 Sample recovery

[0084] Weigh 6 portions of Yanlishuang oral dropping pills with known component content, add the corresponding reference substances, prepare the test solution according to the method in "2.2.3", and perform sampling, detection and analysis according to the chromatographic conditions in "2.1.4". The average recovery of 14 components including β-pinene was calculated to be 96.77% to 101.82%, with RSD of 1.24% to 2.87%. The results are shown in Table 8.

[0085] Table 8 Recovery results of 14 ingredients in Yanlishuang oral dropping pills

[0086]

[0087]

[0088]

[0089] 2.2.5 Measurement results

[0090] 17 batches of YLS samples were prepared according to the test sample preparation method under "2.2.3". Samples were injected, tested and analyzed according to the chromatographic conditions under "2.1.4". The contents of 14 components in the YLS samples were calculated. The results are shown in Table 9. The results show that the contents of β-pinene, limonene, eucalyptol, p-cymene, 3-octanol, menthone, isomenthone, camphor, menthyl acetate, trans-caryophyllene, menthol, borneol, dianthinol and guaiacol in the 17 batches of YLS were 0.1867-0.4101, 0. 4486~1.084, 0.0540~0.3210, 0.0089~0.0161, 0.2571~0.4277, 1.635~2.277, 11.50~17.54, 5.180~7 .597, 2.005~2.586, 0.4214~0.8826, 38.93~43.99, 90.15~93.76, 0.7092~1.205, 0.3763~1.802mg / g.

[0091] 3. Discussion

[0092] This study used GC-MS to analyze the components of Yanlishuang oral drops that enter the bloodstream, and established a method for determining their content using 14 components as indicators. This study, for the first time, achieved precise monitoring of the active ingredient population in Yanlishuang oral drops. This not only provides key experimental evidence for improving quality control standards, but also offers a new perspective on the modern scientific implications of the "multi-component synergistic effect" of Yanlishuang oral drops. This provides more scientific data for comprehensive quality control and quantitative research on this drug, which has far-reaching significance and value for enhancing the clinical efficacy of Traditional Chinese Medicine.

[0093] Finally, it should be noted that the above embodiments are merely representative examples of the present invention. Obviously, the technical solutions of the present invention are not limited to the above embodiments and are subject to numerous variations. All variations that can be directly derived or conceived by a person of ordinary skill in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

[0094] Table 9 Determination of the contents of 14 ingredients in 17 batches of Yanlishuang oral drops (mg / g)

[0095]

[0096]

Claims

1. A method for determining the content of chemical components of Yanlishuang oral dripping pills in blood, which can simultaneously determine the concentrations of β-pinene, limonene, eucalyptol, 3-octanol, isomenthone, menthone, camphor, menthyl acetate, trans-caryophyllene, menthol, borneol, dianthinol, p-cymene, and guaiacol, characterized in that: GC-MS method was used for detection; Chromatographic conditions: SH-Stabilwax capillary column, 30 m × 0.32 mm, 0.25 μm film thickness; injection port temperature 220°C; splitless injection; Injection volume: 1 μL; Column flow rate: 2 mL / min; Mass spectrometry conditions: ion source temperature 200°C; interface temperature 230°C; ionization mode: electron impact ionization (EI); detection voltage: 0.2 kV; solvent delay 2 min; SIM mode.

2. The method for determining the content of chemical components of Yanlishuang oral dripping pills entering blood according to claim 1, characterized in that: The chromatographic conditions and the specific temperature program are as follows: Column oven temperature program 3. The method for determining the content of chemical components of Yanlishuang oral dripping pills entering blood according to claim 1, characterized in that: The mass spectrometry conditions and the detection ion information of each component are as follows: Detection ion information of 14 components including β-pinene 4. The method for determining the content of chemical components of Yanlishuang oral dripping pills entering blood according to claim 1, characterized in that: In the GC-MS method, the test solution is prepared using Yanlishuang oral dropping pills or a drug-containing serum sample after taking Yanlishuang oral dropping pills.

5. The method for determining the content of chemical components of Yanlishuang oral dropping pills entering blood according to claim 4, characterized in that: The test solution is prepared by adding ethyl acetate and internal standard stock solution to Yanlishuang oral dropping pills.

6. The method for determining the content of chemical components of Yanlishuang oral dripping pills entering blood according to claim 5, characterized in that: The test solution is specifically a solution containing 50 μg per 1 mL of Yanlishuang oral drops and ethyl acetate.

7. The method for determining the content of chemical components of Yanlishuang oral dripping pills entering blood according to claim 4, characterized in that: The drug-containing serum sample is prepared after taking Yanli Shuang oral dropping pills. The drug-containing serum of an individual is collected after taking Yanli Shuang oral dropping pills, and then ethyl acetate is added for vortex mixing and centrifugation, and the supernatant is taken for sampling and analysis.

8. The method for determining the content of chemical components of Yanlishuang oral dripping pills entering blood according to claim 7, characterized in that: The test solution was prepared by taking 100 μL of serum sample and placing it in a 1.5 mL centrifuge tube. 100 μL of ethyl acetate was added and vortexed for 2 min at 4 °C and 12000 r / min. -1 Centrifuge for 10 min and take the supernatant for analysis.

9. The method for determining the content of chemical components of Yanlishuang oral dripping pills entering blood according to claim 7, characterized in that: The drug-containing serum of the individual after taking the Yanlishuang oral dropping pills is obtained by taking blood and placing it in a centrifuge tube containing an anticoagulant, centrifuging the whole blood, and taking the supernatant to obtain a serum sample.

10. The method for determining the content of chemical components of Yanlishuang oral dripping pills entering blood according to claim 8, characterized in that: The serum samples were stored at -80°C after preparation and thawed before use.

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