A method for determining the content of blood chemical components of Yanyishuang buccal drop pills

Abstract

This invention relates to the field of pharmaceutical analysis technology, specifically a method for determining the content of chemical components in the blood of a throat lozenge. The invention employs gas chromatography-mass spectrometry (GC-MS) with the following chromatographic conditions: SH-Stabilwax capillary column (30m × 0.32mm, film thickness 0.25μm); injection port temperature 220℃; splitless injection; injection volume 1μL; column flow rate 2mL / min. Mass spectrometry conditions include: ion source temperature 200℃; interface temperature 230℃; ionization mode: electron impact ionization (EI); detection voltage: 0.2KV; solvent delay 2min; SIM mode. This innovative method allows for the simultaneous determination of the concentrations of 14 volatile components in Yanlishuang oral drops or in serum samples containing the drug after administration, including β-pinene, limonene, eucalyptol, 3-octanol, isomenthone, menthone, camphor, menthyl acetate, trans-caryophyllene, menthol, borneol, caryophyllene, p-cymene, and guaiacol. This study is the first to achieve precise monitoring of the pharmacodynamic components of Yanlishuang oral drops in vivo, providing crucial experimental evidence for improving its quality control standards. Furthermore, it offers a new perspective on the modern scientific implications of the "multi-component synergistic effect" of Yanlishuang oral drops, providing more scientific data for future comprehensive quality control and quantitative research of this drug.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis technology, specifically a method for determining the content of chemical components in the blood of a throat lozenge. Background Technology

[0002] Yanlishuang Oral Drops are a nationally exclusive Miao medicine product (National Medicine Approval Number Z20025286) developed by Guizhou Huangguoshu Shushuang Pharmaceutical Co., Ltd. It is a compound Miao medicine preparation composed of Artemisia argyi, Artemisia argyi oil, peppermint oil, menthol, and glycyrrhizic acid monoammonium salt. It has the effects of dispelling wind and heat, reducing swelling and relieving pain, and clearing the throat (Miao medicine: Song Gongzheng, Taikai Taimeng). It is used to treat acute pharyngitis and acute exacerbations of chronic pharyngitis, with symptoms such as sore throat, dry throat, and halitosis. Because Yanlishuang Oral Drops were an early product upgraded from a local standard to a national standard, basic research is weak. The existing standard only controls menthol, borneol, and glycyrrhizic acid monoammonium salt; patent CN116124922B has determined the content of camphor, menthone, borneol, and menthol, but does not clarify whether they are the active ingredients. The pharmacodynamic material basis for the efficacy of Yanlishuang Oral Drops is not yet clear, and the detection methods for its active ingredients in blood are even more lacking. The core challenges are: ① Interference from complex biological matrices: High abundance of proteins, lipids, and endogenous metabolites in serum co-elute with trace amounts of target components, requiring complex pretreatment to improve selectivity; ② Metabolic transformation and coexistence of multiple components: Drugs may be metabolized into multiple active or inactive products in vivo, requiring simultaneous detection of the original drug and metabolites, and the method development needs to cover the separation and quantification of multiple targets.

[0003] Traditional Chinese medicine (TCM) preparations have complex compositions, some of which may be difficult for the body to absorb effectively due to limited bioavailability and may not directly participate in pharmacological effects. By systematically detecting chemical components absorbed into the bloodstream (blood-transmitting 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 research on key active components and avoid ineffective analysis of non-bioavailable components. To address the needs of pharmacodynamic research and clinical safety of Yanlishuang oral drops, the development of a quantitative analytical method for blood-transmitting components with high sensitivity, good reproducibility, and high throughput is of significant scientific value for elucidating the composition of its in vivo pharmacodynamic material group and revealing the synergistic mechanism of multi-component action. This method has become a key technological bottleneck in advancing the modernization of research on this preparation. Summary of the Invention

[0004] To address the aforementioned technical problems in the existing technology, this invention provides a method for determining the content of chemical components in the blood of throat lozenges, as detailed below:

[0005] A method for determining the content of chemical components in the blood of a throat-soothing oral droplet can simultaneously determine the concentrations of β-pinene, limonene, eucalyptol, 3-octanol, isomenthone, menthone, camphor, menthyl acetate, trans-caryophyllene, menthol, borneol, caryophyllene, p-cymene, and guaiacol, using GC-MS for detection.

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

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

[0008] Furthermore, the specific temperature program for the chromatographic conditions is as follows:

[0009] Column oven heating program

[0010]

[0011] Furthermore, the specific ion information for each component under the mass spectrometry conditions is as follows:

[0012] Ion information of 14 components including β-pinene

[0013]

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

[0015] Furthermore, the test solution is prepared by adding ethyl acetate to the throat lozenges. Specifically, the throat lozenges are prepared by adding ethyl acetate to the throat lozenges to prepare a test solution containing 50 μg / mL of throat lozenges and 50 ng / mL of naphthalene.

[0016] Furthermore, the serum sample prepared using the method described above is obtained by collecting serum from individuals who have taken the Yanlishuang oral drops. Ethyl acetate is then added, vortexed, centrifuged, and the supernatant is used for analysis. Specifically, 100 μL of serum sample is placed in a 1.5 mL centrifuge tube, 100 μL of ethyl acetate is added, vortexed for 2 min, and incubated at 4°C and 12000 rpm. -1 Centrifuge for 10 minutes, then collect the supernatant for analysis.

[0017] Furthermore, the serum sample obtained from the individual who took the Yanli Shuang oral drops was prepared by collecting blood and placing it in a centrifuge tube containing an anticoagulant, centrifuging the whole blood, and collecting the supernatant.

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

[0019] Compared with the prior art, the technical effects of this invention are reflected in:

[0020] (1) This invention employs gas chromatography-mass spectrometry (GC-MS). Chromatographic conditions: SH-Stabilwax capillary column, 30m × 0.32mm, film thickness 0.25μm; injection port temperature 220℃; splitless injection; injection volume 1μL; column flow rate 2mL / min; mass spectrometry conditions: ion source temperature 200℃; interface temperature 230℃; ionization mode: electron impact ionization (EI); detection voltage: 0.2KV; solvent delay 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, caryophyllein, p-cymene, and guaiacol.

[0021] (2) The method of this invention can be used to detect the active ingredients of Yanlishuang oral drops or serum samples containing the drug after taking Yanlishuang oral drops, and has excellent specificity, repeatability, and stability. It fills the gap in the detection methods for the active ingredients of Yanlishuang oral drops in blood. It has significant scientific value for elucidating the composition of its pharmacodynamic components in vivo and revealing the synergistic mechanism of its multi-component action.

[0022] (3) This application uses GC-MS to detect the blood-entry components of Yanlishuang oral drops and establishes a method for content determination of Yanlishuang oral drops using 14 components as indicators. This study is the first to achieve precise monitoring of the pharmacodynamic substances in Yanlishuang oral drops, which not only provides key experimental evidence 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 Yanlishuang oral drops. It provides more scientific data for establishing comprehensive quality control and quantitative research of this drug in the future, which has profound significance and value for better exerting the clinical efficacy of traditional Chinese medicine. Attached Figure Description

[0023] Figure 1The GC-MS SIM chromatograms of serum from the treatment group (A), the control solution (B), and the blank group (C) are shown below; 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; 15. naphthalene; 13. caryophyllein; 14. guaiacol.

[0024] Figure 2 The GC-MS SIM chromatograms of the reference solution (A), the test solution (B), and the blank control solution (C) are shown below; 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; 15. naphthalene; 13. caryophyllein; 14. guaiacol. Detailed Implementation

[0025] The technical solution of the present invention will be further defined below with reference to specific embodiments, but the scope of protection is not limited to the description made.

[0026] Example:

[0027] 1. Materials

[0028] 1.1 Instruments

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

[0030] 1.2 Test Drugs

[0031] Yanshu Kou Shuang Kou Han Di Wan (YLS) was provided by Guizhou Huangguoshu Lishuang Pharmaceutical Co., Ltd., and the batch numbers are shown in Table 1 below. Ethyl acetate (chromatographic grade, Tianjin Kemiou 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%), p-cymene (batch number: WP24060301, purity 98%) were all purchased from Sichuan维克奇Biological Technology Co., Ltd.; menthone (batch number: 111705-202106, purity 99%), menthyl acetate (batch number: 190044-202102, purity 98%), menthol (batch number: 110728-202208, purity 99.6%) were all purchased from the National Institutes for Food and Drug Control; camphor (batch number: PS020363, purity 99.30%) was purchased from Chengdu Pusi Biological Technology Co., Ltd.; borneol (batch number: MUST-22021501, purity 98%) was purchased from Chengdu Mantest Biological Technology Co., Ltd.; caryophyllin (batch number: AB2637-0005, purity 98%), guaiol (batch number: AB3219-0005, purity 98%) were all purchased from Chengdu Aifa Biological Technology Co., Ltd., naphthalene (batch number: 21050063) was purchased from Tanmo Quality Inspection - Reference Material Center.

[0032] Table 1 Information of 17 batches of Yanshu Kou Shuang Kou Han Di Wan samples

[0033]

[0034] 1.3 Animals <000008!>

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

[0036] 2. Methods and Results

[0037] 2.1 Identification of blood components of Yanshu Kou Shuang Kou Han Di Wan <00!0087>

[0038] !>2.1.1 Preparation of intragastric administration solution <(

[0039] Take Yanshu Kou Shuang Kou Han Di Wan and grind it. Weigh an appropriate amount of Yanshu Kou Shuang Kou Han Di Wan powder and dissolve it in 5% CMC-Na suspension to make an intragastric administration drug dose of 840 mg / kg, and vortex for 1 min to ensure that the drug is fully mixed.

[0040] 2.1.2 Preparation of reference substance solution

[0041] Accurately weigh appropriate amounts of β-pinene, limonene, eucalyptol, 3-octanol, isomenthone, menthone, camphor, menthyl acetate, trans-caryophyllene, menthol, borneol, caryophyllene, p-cymene, and guaiacol, and place them in a 10 mL volumetric flask. Add an appropriate amount of ethyl acetate and sonicate to dissolve. Dilute to the mark to obtain a 100 μg / mL reference stock solution. 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 the mark to obtain the mixed reference working solution of the required 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 but allowed free access to water. They were then administered 840 mg / kg of throat lozenges via gavage. One mL of blank blood was collected before the first administration, followed by continuous administration for 6 days, twice daily. Thirty minutes after the last administration, blood was collected from the orbital rim and placed in a centrifuge tube containing anticoagulant. The whole blood was centrifuged, and the supernatant was collected as the serum sample, stored at -80°C for analysis.

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

[0045] 2.1.4 GC-MS Conditions

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

[0047] Table 2 Column Temperature Chamber Heating Program

[0048]

[0049] Mass spectrometry conditions: ion source temperature 200℃; interface temperature 230℃; ionization mode: electron impact ionization (EI); detection voltage: 0.2KV; solvent delay 2 min; SIM mode. See Table 3 for detailed ion information for each component.

[0050] Table 3 shows the ion detection information for 14 components including β-pinene.

[0051]

[0052] 2.1.5 Throat-Clearing Droplets Enter the Bloodstream as Original Formation Components

[0053] Serum from the drug administration group, the mixed reference solution, and the blank group were precisely pipetted and analyzed according to the given GC-MS conditions. The chromatograms are shown in the figure. Figure 1 Excluding blank controls, the chromatograms of the standard were compared to identify the blood-forming components of Yanlishuang oral drops: β-pinene, limonene, eucalyptol, p-cymene, 3-octanol, menthone, isomenthone, camphor, menthyl acetate, trans-caryophyllene, menthol, borneol, caryophyllein, and guaiacol.

[0054] 2.2 Determination of the content of components entering the bloodstream from the throat-soothing oral drops.

[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 naphthalene reference standard and prepare an internal standard stock solution of 500 ng / mL with ethyl acetate.

[0059] 2.2.3 Preparation of the test solution

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

[0061] 2.2.4 GC-MS Conditions

[0062] The GC-MS conditions are the same as in "2.1.4".

[0063] 2.2.5 Methodological Examination

[0064] 2.2.5.1 Specificity Examination

[0065] See the specificity results Figure 2 The blank solvent did not produce a peak at the corresponding position of the reference standard, indicating no significant interference, which shows that the method has good specificity.

[0066] 2.2.5.2 Examination of Linear Relationships

[0067] Take an appropriate amount of the reference solution from section "2.2.1" and inject it for detection according to the chromatographic conditions from section "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, caryophyllein, and guaiacol are shown in Table 4. R 2 A value greater than 0.998 indicates that each component exhibits good linearity within its respective linear range.

[0068] Table 4 shows the results of the linear relationship examination of the 14 components.

[0069]

[0070] 2.2.5.3 Precision

[0071] Accurately pipette 1 μL of the mixed reference solution and inject it for detection under the chromatographic conditions in section “2.1.4”. Perform 6 consecutive injections and measurements. The calculated peak areas RSD of 14 components, including β-pinene, were between 0.54% and 2.56%, indicating that the instrument has good precision. The results are shown in Table 5.

[0072] Table 5 Precision test results

[0073]

[0074] 2.2.5.4 Repeatability

[0075] Six parallel YLS samples were prepared using the method described in section “2.2.3” and analyzed under the chromatographic conditions described in section “2.1.4”. The RSDs of the contents of 14 components, including β-pinene, were found to be between 1.36% and 2.95%, indicating that the method has good repeatability (see Table 6).

[0076] Table 6 Results of Repeatability Testing

[0077]

[0078]

[0079] 2.2.5.4 Stability Assessment

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

[0081] Table 7. Results of Stability Study

[0082]

[0083] 2.2.5.4 Recovery rate

[0084] Six samples of the Yanli Shuang oral droplet with known component content were weighed, and corresponding reference standards were added to each sample. The test solutions were prepared according to the method in section "2.2.3", and the samples were injected and analyzed according to the chromatographic conditions in section "2.1.4". The average recovery rates of 14 components, including β-pinene, were calculated to be 96.77%–101.82%, and the RSDs were 1.24%–2.87%. The results are shown in Table 8.

[0085] Table 8. Recovery rates of 14 components in Yanli Shuang Kou Nao Drip Pills

[0086]

[0087]

[0088]

[0089] 2.2.5 Measurement Results

[0090] Seventeen batches of YLS samples were prepared according to the test sample preparation method in section "2.2.3" and analyzed by chromatographic conditions in section "2.1.4". The contents of 14 components in the YLS samples were calculated, and 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, caryophyllein, 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 detect the blood-entry components of Yanlishuang oral drops and established a method for content determination of Yanlishuang oral drops using 14 components as indicators. This study is the first to achieve precise monitoring of the pharmacodynamic components of Yanlishuang oral drops in vivo, providing crucial experimental evidence for improving its quality control standards. It also offers a new perspective on elucidating the modern scientific connotation of the "multi-component synergistic effect" of Yanlishuang oral drops, providing more scientific data for future comprehensive quality control and quantitative research of this drug. This has profound significance and value for better leveraging 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 solution of the present invention is not limited to the above embodiments, and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the content disclosed in this invention should be considered within the scope of protection of this invention.

[0094] Table 9. Results of content determination (mg / g) of 14 components in 17 batches of Yanli Shuang Kou Ling (a type of oral rehydration tablet).

[0095]

[0096]

Claims

1. A method for determining the content of chemical components entering the bloodstream in a throat lozenge, simultaneously determining the concentrations of fourteen chemical components entering the bloodstream, including β-pinene, limonene, eucalyptol, 3-octanol, isomenthone, menthone, camphor, menthyl acetate, trans-caryophyllene, menthol, borneol, caryophyllene, p-cymene, and guaiacol, characterized in that... Detection was performed using the GC-MS method; Chromatographic conditions: SH-Stabilwax capillary column, 30 m × 0.32 mm, film thickness 0.25 μm; injection port temperature 220℃; splitless injection; Injection volume: 1 μL; Column flow rate: 2 mL / min; Mass spectrometry conditions: ion source temperature 200 ℃; interface temperature 230 ℃; ionization mode: electron impact ionization (EI); detection voltage: 0.2 kV; solvent delay 2 min; SIM mode; The GC-MS method wherein the test solution is prepared by adding ethyl acetate and internal standard stock solution to the throat lozenges; The specific temperature program for the chromatographic conditions is as follows: Column oven heating program 2. The method for determining the content of chemical components in the blood of the throat-soothing oral drops according to claim 1, characterized in that, The specific mass spectrometry conditions and ion detection information for each component are as follows:

3. The method for determining the content of chemical components in the blood of the throat-soothing oral drops according to claim 1, characterized in that, The test solution is specifically a solution prepared by adding ethyl acetate to the throat lozenges, containing 50 μg per 1 mL.

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