Method for detecting bornyl acetate in traditional Chinese medicine compound preparation

Abstract

The invention discloses a method for detecting bornyl acetate in a traditional Chinese medicine compound preparation. The method comprises the following steps: (1) preparing a reference solution and a test solution; (2) setting chromatographic conditions of a gas chromatograph, respectively injecting the reference solution and the test solution into the gas chromatograph for detection, and recording chromatograms; according to the detection method, bornyl acetate is selected as a detection index, and chromatographic conditions and a test sample preparation method are optimized, so that when a test sample is prepared, the matrix effects of other medicinal decoction pieces, auxiliary materials and the like are broken through, and the detection sensitivity is improved in a distillation enrichment manner; and finally, the detection method of the volatile component bornyl acetate in the Wudao granules is confirmed, so that the quality control and standardization are realized. The batch consistency is ensured, the complex components of the traditional Chinese medicine compound preparation are eliminated, the influence of factors such as raw material production places and extraction processes on the bornyl acetate is avoided, the drug effect and the clinical curative effect are ensured, and the quality stability of the preparation is ensured.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical analysis, and particularly relates to a method for detecting bornyl acetate in a traditional Chinese medicine compound preparation. Background Art

[0002] The invention patent with the application number CN201410778959.8 discloses a traditional Chinese medicine composition for promoting gastrointestinal function recovery after abdominal surgery and its preparation method. The traditional Chinese medicine composition includes medicinal flavors such as Lindera aggregata (Sims) Kosterm., Amomum villosum Lour., Panax ginseng C. A. Mey., Prunus persica (L.) Batsch, and Areca catechu L., and the granule prepared from the traditional Chinese medicine composition is called "Wuda Granules". Among them, Amomum villosum Lour. is the dried ripe fruit of Amomum villosum Lour. of the Zingiberaceae family, and Lindera aggregata (Sims) Kosterm. is the dried tuberous root of Lindera aggregata (Sims) Kosterm. of the Lauraceae family. Both Amomum villosum Lour. and Lindera aggregata (Sims) Kosterm. are the monarch drugs of Wuda Granules. The volatile oil of Amomum villosum Lour. contains volatile components such as (-)-camphor and bornyl acetate, and the volatile oil of Lindera aggregata (Sims) Kosterm. contains volatile components such as bornyl acetate and camphor. Among them, bornyl acetate has functions such as antibacterial, anti-inflammatory, sedative, and anthelmintic, and has been widely used in medicine, cosmetics, and daily chemical products.

[0003] The preparation method of Wuda Granules involves operations of extracting volatile oil and adding volatile oil. Since bornyl acetate comes from both the monarch drug Amomum villosum Lour. and the monarch drug Lindera aggregata (Sims) Kosterm., and bornyl acetate is the index component for the content determination item of Amomum villosum Lour. medicinal materials and cut pieces in the 2020 edition of the Chinese Pharmacopoeia, controlling bornyl acetate can achieve the purpose of controlling the two monarch drugs. Selecting bornyl acetate as the gas-phase qualitative index is conducive to detecting the quality of Wuda Granules.

[0004] However, there are the following difficulties in the detection of bornyl acetate:

[0005] (1) Due to the poor physical stability of volatile oil, it is easy to volatilize, oxidize, and photolyze. Especially during the preparation and storage of granules, the volatile oil may be damaged, resulting in a decrease in its content and a weakening of the odor, and even the generation of oxidation products (such as peroxides). Moreover, there are many uncontrollable factors in the large-scale production of granules, and the extraction rate of volatile oil is low. Therefore, it is difficult to establish a unified quality control index using quantitative detection methods.

[0006] (2) When adding volatile oil, it is necessary to evenly disperse the volatile oil in the granules. However, the surface properties (hydrophilicity) of the granules after water extraction are poorly compatible with the volatile oil (hydrophobicity), and local enrichment or migration is likely to occur, resulting in large differences in the content between granules of the same batch, affecting the dosage accuracy, and it is difficult to ensure uniformity.

[0007] (3) The volatile oil is highly sensitive to process parameters. When adding volatile oil, the addition timing before / after drying, atomization pressure, and the selection of carrier excipients will all affect the retention rate of the volatile oil. Minor changes in the process may cause the content fluctuation of the volatile oil to exceed the standard range.

[0008] (4) Due to the complex composition of the compound, other fat-soluble components will interfere with the detection of volatile oils, and the traditional TLC method (thin-layer chromatography analysis) may not be able to specifically distinguish the target volatile components.

[0009] (5) Low storage stability. When the granule absorbs moisture or the packaging sealing is insufficient, the volatile oil will gradually escape or deteriorate. It is difficult to establish a unified quality control index for quantitative detection methods, and it is difficult to reasonably evaluate the product stability.

[0010] The literature "Study on the Identification Method of Bornyl Acetate in Eryi Pills by GC Method" discloses a method for detecting bornyl acetate in traditional Chinese medicine pills. However, combined with the preparation method of "Eryi Pills" disclosed in the first volume of the Ministry of Health's Drug Standards for Traditional Chinese Medicine Formulas, the amomum fruit in it is used as fine powder, and its volatile components (bornyl acetate) usually exist in the original form, with high and stable content. Its detection matrix is relatively simple, mainly plant tissues, and there are fewer interfering substances. Therefore, the volatile components can be directly extracted and determined by ultrasonic treatment.

[0011] The patent application with the application number CN202311190353.8 discloses a method for determining the components in Gushen Dingchuan Pills and its application. However, combined with the preparation method of Gushen Dingchuan Pills in the 2020 edition of the Chinese Pharmacopoeia, the amomum fruit in it is used as fine powder and has not undergone water extraction. Therefore, its volatile component content is high and the detection difficulty is low. The preparation of the test sample can be achieved through organic solvent dissolution and ultrasonic treatment.

[0012] The patent application with the application number CN202410447887.2 discloses a method for determining the quality of volatile drugs in tracheitis rubber plaster and its application, but does not disclose the detection method of bornyl acetate. Toluene is used as a solvent during the preparation of the test sample, but toluene has a relatively small polarity and is not suitable for the detection of bornyl acetate; moreover, there are many types of volatile components with complex properties, and specific appropriate solvents and methods need to be developed and selected for component detection according to the specific components.

[0013] Currently, after a large number of literature and patent research and retrieval, there is no method for qualitatively detecting bornyl acetate. The content of bornyl acetate in Wuda granules is significantly reduced due to processes such as water extraction, greatly increasing the detection difficulty and requiring higher sensitivity for the method; moreover, after extraction, the addition of excipients results in granules, and the excipients may also interfere with the extraction or detection of volatile components. Summary of the Invention

[0014] The technical problem to be solved by the present invention is to overcome the deficiencies of the prior art and provide a detection method for bornyl acetate in traditional Chinese medicine compound preparations that can achieve quality control and standardization and ensure batch consistency.

[0015] To solve the above technical problems, the present invention adopts the following technical solutions:

[0016] A method for detecting bornyl acetate in a traditional Chinese medicine compound preparation, comprising the following steps:

[0017] (1) Preparation of reference solution and test solution:

[0018] Reference solution: Weigh an appropriate amount of bornyl acetate reference substance accurately, and dissolve it in ethyl acetate to prepare a solution containing 40 μg of bornyl acetate per 1 ml;

[0019] Test solution: Take 20 g of the traditional Chinese medicine compound preparation, place it in a flask, add an appropriate amount of water and several grains of zeolite, and operate according to the volatile oil determination method (General Rule 2204, Volume IV of the Chinese Pharmacopoeia 2020 Edition). Add water from the upper end of the measuring device to fill the graduated part and overflow into the flask, then add 2 ml of ethyl acetate, connect the reflux condenser, heat to boiling and keep slightly boiling for 5 hours, cool, separate the ethyl acetate layer, place it in a 2 ml volumetric flask, add ethyl acetate to the scale, add another 0.5 g of anhydrous sodium sulfate, shake well, and take the supernatant as the test solution;

[0020] (2) Set the chromatographic conditions of the gas chromatograph, take the reference solution and the test solution, inject them into the gas chromatograph for detection respectively, and record the chromatogram;

[0021] Among them, the prescription of the traditional Chinese medicine compound preparation includes Lindera aggregata (Sims) Kosterm., Amomum villosum Lour., Panax ginseng C. A. Mey., Prunus persica (L.) Batsch, and Areca catechu L., and the traditional Chinese medicine compound preparation is a granule.

[0022] As a further improvement of the above technical solution: The chromatographic conditions are as follows: The chromatographic column is DB-1, 30 mm × 0.^{25} mm × 0.25 μm; N2 is the carrier gas; the flow rate is 2 ml / min; FID detector; the detector temperature is 320 °C; the injection port temperature is 250 °C; the injection volume is 1 μL.

[0023] As a further improvement of the above technical solution: In the chromatographic conditions, the splitless mode is adopted.

[0024] As a further improvement of the above technical solution: In the chromatographic conditions, the temperature programming is as follows: The initial temperature is maintained at 60 °C for 1 min, then it is raised to 120 °C at a rate of 6 °C / min and maintained for 3 min, and then it is raised to 260 °C at a rate of 20 °C / min and maintained for 5 min.

[0025] As a further improvement of the above technical solution: N2 is the make-up gas, the flow rate is 25 ml / min, the air flow rate is 300 ml / min, and the hydrogen flow rate is 30 ml / min.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] The detection method of bornyl acetate in the traditional Chinese medicine compound preparation disclosed by the present invention selects bornyl acetate as the detection index. Through the optimization of chromatographic conditions and the preparation method of test solution, when preparing the test solution, the matrix effects of other medicinal herb slices and excipients are overcome, and the detection sensitivity is improved by means of distillation enrichment. Finally, the detection method of the volatile component bornyl acetate in Wuda granules is confirmed, thus realizing quality control and standardization. The batch consistency is ensured, and the complex components of the traditional Chinese medicine compound preparation and the influence of factors such as the raw material origin and extraction process on bornyl acetate are excluded, ensuring the medicinal effect and clinical efficacy, and guaranteeing the quality stability of the preparation. It is convenient to design long-term / accelerated tests according to the ICH guidelines, so as to formulate the expiration date of the preparation. Description of the Drawings

[0028] Figure 1 It is the chromatogram of Example 1 in the detection method of bornyl acetate in the traditional Chinese medicine compound preparation of the present invention.

[0029] Figure 2 It is the chromatogram of Example 2 in the present invention.

[0030] Figure 3 It is the chromatogram of the test solution of Example 3 in the present invention.

[0031] Figure 4 It is the enlarged superimposed chromatogram of the test solution of the volatile oil obtained by single decoction of peach kernels and the test solution of the granules in Example 3 of the present invention.

[0032] Figure 5 It is the chromatogram of Example 4 in the present invention.

[0033] Figure 6 It is the chromatogram of the test solution obtained by ultrasonic extraction (absolute ethanol) in Example 5 of the present invention.

[0034] Figure 7 It is the enlarged chromatogram of the test solution obtained by ultrasonic extraction (absolute ethanol) in Example 5 of the present invention.

[0035] Figure 8 It is the chromatogram of the test solution obtained by ultrasonic extraction (ethyl acetate) in Example 5 of the present invention.

[0036] Figure 9 It is the chromatogram of the test solution obtained by extraction (toluene) in Example 5 of the present invention.

[0037] Figure 10 It is the chromatogram of the test solution obtained by extraction (n-butanol) in Example 5 of the present invention.

[0038] Figure 11 It is the chromatogram of the test solution obtained by extraction (ethyl acetate) in Example 5 of the present invention.

[0039] Figure 12 Chromatogram of the sample solution after evaporation to dryness and reconstitution by extraction (ethyl acetate) in Example 6 of the present invention.

[0040] Figure 13 Chromatogram of the sample solution by extraction (ethyl acetate) in Example 6 of the present invention.

[0041] Figure 14 Chromatogram of the sample solution by distillation (ethyl acetate enrichment) in Example 6 of the present invention.

[0042] Figure 15 Chromatogram of the ground sample solution in Example 7 of the present invention.

[0043] Figure 16 Chromatogram of the unground sample solution in Example 7 of the present invention.

[0044] Figure 17 Enlarged chromatogram of the unground sample solution in Example 7 of the present invention.

[0045] Figure 18 Superimposed chromatogram of the solution lacking yin and the finished preparation solution in Example 8 of the present invention. Detailed implementation manners

[0046] The present invention will be further described in detail below in conjunction with the accompanying drawings of the specification and specific embodiments, but the protection scope of the present invention is not limited thereby. The materials and instruments used in the following examples are all commercially available products.

[0047] Instruments: Agilent 7890B + 7694E gas chromatograph (Agilent Technologies (China) Co., Ltd.), Agilent 7000D triple quadrupole gas chromatography-mass spectrometry instrument (Agilent Technologies (China) Co., Ltd.), KQ500DE numerically controlled ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.), ME204E electronic balance (Mettler-Toledo International Trade (Shanghai) Co., Ltd.).

[0048] Chromatographic column: DB-1 (30m × 0.25mm × 0.25 µm) (Agilent Technologies (China) Co., Ltd.).

[0049] Reagents: absolute ethanol (Sinopharm Chemical Reagent Co., Ltd.), ethyl acetate (Shanghai Aladdin Biochemical Technology Co., Ltd.), toluene (Sinopharm Chemical Reagent Co., Ltd.), n-butanol (Sinopharm Chemical Reagent Co., Ltd.), anhydrous sodium sulfate (Shanghai Aladdin Biochemical Technology Co., Ltd.).

[0050] Test samples: Wuda Granules (Guangdong Provincial Hospital of Traditional Chinese Medicine), concentrated solution lacking fructus amomi and lindera aggregata (self-made).

[0051] Reference substance: Bornyl acetate (National Institutes for Food and Drug Control).

[0052] Example 1:

[0053] Referring to the content determination item of "Amomum villosum" in Part I of the Chinese Pharmacopoeia (2020 Edition), the preliminary gas chromatography experimental conditions were determined, and the experimental content is as follows:

[0054] (1) Preparation of reference substance solution and test sample solution:

[0055] Reference substance solution: Weigh accurately 60 mg of bornyl acetate, place it in a 30 ml volumetric flask, dilute it to the mark with anhydrous ethanol, accurately pipette 1 ml, add anhydrous ethanol to 2 ml, and shake well to obtain. (Concentration is about 1 mg / ml)

[0056] Test sample solution of Wuda Granules: Take an appropriate amount of Wuda Granules, grind them finely, weigh 5 g, place it in a conical flask, accurately add 15 ml of methanol, ultrasonicate for 30 minutes, filter, and take the subsequent filtrate to obtain.

[0057] (2) Set the chromatographic conditions of the gas chromatograph, take the reference substance solution and the test sample solution, inject them into the gas chromatograph for detection respectively, and record the chromatogram;

[0058] Chromatographic conditions are as follows: DB-1 capillary column (100% dimethyl polysiloxane as the stationary phase) (column length is 30 m, inner diameter is 0.25 mm, film thickness is 0.25 μm); N2 is the carrier gas; flow rate is 2 ml / min; FID detector, detector temperature is 320 °C, injection port temperature is 250 °C; split ratio is 20:1, injection volume is 1 μL; N2 is the make-up gas, flow rate is 25 ml / min, air flow rate is 300 ml / min, hydrogen flow rate is 30 ml / min; the temperature programming is shown in Table 1:

[0059] Table 1: Gas chromatographic temperature programming of Example 1

[0060]

[0061] Experimental result: The chromatogram of Example 1 is as attached Figure 1 shown, bornyl acetate was not detected in the test sample solution of Wuda Granules, and the analysis time was relatively long.

[0062] Example 2:

[0063] (1) Preparation of test sample solution:

[0064] Wuda Granules test solution: Take an appropriate amount of Wuda Granules, grind them finely, take about 1 g, weigh accurately, place in a stoppered conical flask, add 10 ml of water to dissolve, transfer to a separating funnel, rinse the conical flask with a small amount of water, and combine the rinsing liquid into the same separating funnel. Extract with 10 ml of ethyl acetate, let stand for layer separation, separate the ethyl acetate layer into a centrifuge tube containing 0.5 g of anhydrous sodium sulfate, let stand, transfer the ethyl acetate solution to a 10-ml volumetric flask, shake well, filter, and take the continuous filtrate, that is obtained.

[0065] (2) Set the chromatographic conditions of the gas chromatograph. Take the reference solution and the test solution, and inject them into the gas chromatograph for detection respectively, and record the chromatogram.

[0066] The chromatographic conditions are basically the same as those in Example 1, and the difference is that: the split ratio is 5:1, and the temperature programming is shown in Table 2:

[0067] Table 2: Gas phase temperature programming of Example 2

[0068]

[0069] Experimental results: The chromatogram of Example 2 is as attached Figure 2 shown. The peak area of bornyl acetate is small, the sensitivity is low, and the effect is not ideal.

[0070] Example 3:

[0071] (1) Prepare the test solution: the same as in Example 2.

[0072] Taoren single decoction volatile oil test solution: Weigh 200 g of Taoren decoction pieces, crush them, add 12 times the amount of water and extract for 2.5 hours, and collect the volatile oil at the same time. Take the collected volatile oil, place it in a 20-ml volumetric flask, dissolve and make up the volume with absolute ethanol, then transfer 1 ml, place it in a 50-ml volumetric flask, dilute and make up the volume with water. Take 19 ml of aromatic water, place it in a 50-ml volumetric flask, dilute and make up the volume with water, transfer to a separating funnel, rinse with a small amount of purified water, combine the liquid into the separating funnel, add ethyl acetate, shake and extract 2 times, 25 ml each time, combine the upper layer extraction liquids of the two times, remove water with an appropriate amount of anhydrous sodium sulfate, and take the supernatant, that is obtained.

[0073] (2) Set the chromatographic conditions of the gas chromatograph. Take the reference solution and the test solution, and inject them into the gas chromatograph for detection respectively, and record the chromatogram.

[0074] The chromatographic conditions are basically the same as those in Example 2, and the difference is that: the splitless mode is adopted.

[0075] Experimental results: The chromatogram of the test solution of Example 3 is as attached Figure 3 shown; The enlarged superimposed chromatogram of the Taoren single decoction volatile oil test solution and the granule test solution is as attached Figure 4As shown, the comparison of the peak areas of the target peaks with different split ratios in Example 2 and Example 3 is shown in Table 3. The results show that Example 3 adopts a splitless mode, which shortens the analysis time, and the peak emergence time is in the middle part of the analysis time. The total analysis time is relatively appropriate, but as can be seen from the appendix Figure 4 it can be seen that peach kernels will interfere with the detection of bornyl acetate.

[0076] Table 3: Comparison of the peak areas of the target peaks with different split ratios in Example 2 and Example 3

[0077]

[0078] Example 4:

[0079] (1) Preparation of the test solution: The same as Example 2.

[0080] (2) Set the chromatographic conditions of the gas chromatograph. Take the reference solution and the test solution, inject them into the gas chromatograph for detection respectively, and record the chromatogram;

[0081] The chromatographic conditions are the same as those in Example 3, and the difference is that the temperature programming is shown in Table 4:

[0082] Table 4: Gas chromatographic temperature programming of Example 4

[0083]

[0084] Experimental results: The chromatogram of Example 4 is as shown in the appendix Figure 5 As shown, the results show that the single decoction solution of peach kernels has no interference on the determination of bornyl acetate. Under these chromatographic conditions, the specificity is good and the temperature programming inspection is qualified.

[0085] In summary, the chromatographic conditions can be determined as those of Example 4, that is, the chromatographic conditions are: the chromatographic column is DB-1, 30 mm×0.25 mm×0.25 μm; N2 is used as the carrier gas; the flow rate is 2 ml / min; the splitless mode is adopted; the FID detector; the detector temperature is 320 °C; the injection port temperature is 250 °C; the injection volume is 1 μL; N2 is used as the make-up gas, the flow rate is 25 ml / min, the air flow rate is 300 ml / min, and the hydrogen flow rate is 30 ml / min; the temperature programming: the initial temperature is 60 °C and is held for 1 min, then it is raised to 120 °C at a rate of 6 °C / min and held for 3 min, and then it is raised to 260 °C at a rate of 20 °C / min and held for 5 min.

[0086] Example 5:

[0087] (1) Preparation of the test solution:

[0088] Preparation of the test solution by ultrasonic extraction (anhydrous ethanol): Take an appropriate amount of Wuda Granules finished product, grind it finely, take about 5 g, weigh accurately, place it in a stoppered conical flask, accurately add 15 ml of anhydrous ethanol, stopper tightly, weigh, perform ultrasonic treatment (power 300 W, frequency 40 kHz) for 30 minutes, shake well, filter, and take the subsequent filtrate to obtain the solution.

[0089] Preparation of the test solution by ultrasonic extraction (ethyl acetate): Take an appropriate amount of Wuda Granules finished product, grind it finely, take about 1 g, weigh accurately, place it in a stoppered conical flask, accurately add 10 ml of ethyl acetate, stopper tightly, weigh, perform ultrasonic treatment (power 300 W, frequency 40 kHz) for 30 minutes, let it cool, make up the lost weight with ethyl acetate, shake well, filter, and take the subsequent filtrate to obtain the solution.

[0090] Preparation of the test solution by extraction (toluene): Take an appropriate amount of Wuda Granules, grind it finely, take about 1 g, weigh accurately, place it in a stoppered conical flask, add 10 ml of water to dissolve it, transfer it to a separating funnel, rinse the conical flask with a small amount of water, and combine the rinsing solution into the same separating funnel. Add 10 ml of toluene for extraction, let it stand for layer separation, take the toluene layer and transfer it to a centrifuge tube containing 0.5 g of anhydrous sodium sulfate for water removal, filter, and take the subsequent filtrate to obtain the solution.

[0091] Preparation of the test solution by extraction (n-butanol): Take an appropriate amount of Wuda Granules, grind it finely, take about 1 g, weigh accurately, place it in a stoppered conical flask, add 10 ml of water to dissolve it, transfer it to a separating funnel, rinse the conical flask with a small amount of water, and combine the rinsing solution into the same separating funnel. Add 5 ml of n-butanol for extraction, let it stand for layer separation, take the n-butanol layer and transfer it to a centrifuge tube containing 0.5 g of anhydrous sodium sulfate for water removal, filter, and take the subsequent filtrate to obtain the solution.

[0092] Preparation of the test solution by extraction (ethyl acetate): Take an appropriate amount of Wuda Granules, grind it finely, take about 1 g, weigh accurately, place it in a stoppered conical flask, add 10 ml of water to dissolve it, transfer it to a separating funnel, rinse the conical flask with a small amount of water, and combine the rinsing solution into the same separating funnel. Add 10 ml of ethyl acetate for extraction, let it stand for layer separation, take the ethyl acetate layer and transfer it to a centrifuge tube containing 0.5 g of anhydrous sodium sulfate for water removal, filter, and take the subsequent filtrate to obtain the solution.

[0093] (2) Set the chromatographic conditions of the gas chromatograph. The chromatographic conditions are the same as those in Example 4. Take the reference solution and the test solution, inject them into the gas chromatograph for detection respectively, and record the chromatogram.

[0094] Experimental results: The chromatogram of the test solution by ultrasonic extraction (anhydrous ethanol) is as shown in the appendix Figure 6 The enlarged chromatogram of the test solution by ultrasonic extraction (anhydrous ethanol) is as shown in the appendix Figure 7 The chromatogram of the test solution by ultrasonic extraction (ethyl acetate) is as shown in the appendix Figure 8 The chromatogram of the test solution by extraction (toluene) is as shown in the appendixFigure 9 As shown, the chromatogram of the test sample solution extracted with (n-butanol) is as attached Figure 10 As shown, the chromatogram of the test sample solution extracted with (ethyl acetate) is as attached Figure 11 As shown, the results of the differential analysis of the component contents of the test samples extracted with different solvents are shown in Table 5. The results show that when the test sample solution is prepared by ultrasonic extraction (anhydrous ethanol), the chromatogram baseline is unstable. When the test samples are prepared by extraction with different solvents, the RSD% of the peak area of bornyl acetate is relatively large, indicating that the extraction solvent has an impact on the dissolution of the components. The peak area of bornyl acetate in the chromatogram of the test sample extracted with (ethyl acetate) is relatively large. Therefore, it is better to prepare the test sample by extraction with (ethyl acetate).

[0095] Table 5: Results of the differential analysis of the component contents of the test samples extracted with different solvents

[0096]

[0097] Example 6:

[0098] (1) Preparation of the test sample solution:

[0099] Preparation of the test sample solution after evaporation to dryness and re-dissolution after extraction with (ethyl acetate): Take an appropriate amount of the finished product of Wuda granules, grind it finely, take about 2 g, weigh it accurately, place it in a stoppered conical flask, add 50 ml of water to dissolve it, transfer it to a separating funnel, wash the conical flask with a small amount of water, and combine the washing liquid into the same separating funnel. Extract twice with 25 ml of ethyl acetate each time, let it stand for layering, combine the extraction liquid, place it in an evaporating dish and evaporate it to near dryness, dissolve it to 2 ml with ethyl acetate, pass through 0.5 g of anhydrous sodium sulfate, shake well, filter, and take the continued filtrate to obtain the solution.

[0100] Preparation of the test sample solution extracted with (ethyl acetate): Take an appropriate amount of the finished product of Wuda granules, grind it finely, take about 2 g, weigh it accurately, place it in a stoppered conical flask, add 10 ml of water to disperse it evenly, transfer it to a separating funnel, add 5 ml of ethyl acetate for extraction, take the upper layer liquid, and remove water through about 0.5 g of anhydrous sodium sulfate to obtain the solution.

[0101] Preparation of the test sample solution by distillation (ethyl acetate enrichment): Take an appropriate amount of the finished product of Wuda granules, grind it finely, take about 20 g, place it in a 500 ml round-bottomed flask, add 200 ml of water and several boiling chips, operate according to the volatile oil determination method (General Chapter 2204, Volume IV of the Chinese Pharmacopoeia 2020 Edition), add water from the upper end of the measuring device to fill the graduated part and overflow into the flask, then add 2 ml of ethyl acetate, connect the reflux condensation device, heat to boiling and keep slightly boiling for 5 hours, let it cool, separate the ethyl acetate layer, place it in a 2 ml volumetric flask, add ethyl acetate to the scale, add another 0.5 g of anhydrous sodium sulfate, shake well, and take the supernatant as the test sample solution.

[0102] (2) Set the chromatographic conditions of the gas chromatograph. The chromatographic conditions are the same as those in Example 4. Take the reference solution and the test solution, inject them into the gas chromatograph for detection respectively, and record the chromatogram.

[0103] Experimental results: The chromatogram of the test solution after extraction (ethyl acetate) and evaporation to dryness and re-dissolution is as shown in the appendix Figure 12 as shown, the chromatogram of the test solution after extraction (ethyl acetate) is as shown in the appendix Figure 13 as shown, the chromatogram of the test solution after distillation (ethyl acetate enrichment) is as shown in the appendix Figure 14 as shown. The analysis results of the peak area differences of the components in the test solutions prepared by different extraction methods are shown in Table 6. The results show that when the test samples are prepared in different ways, the relative deviation (RD%) of the peak area of bornyl acetate is relatively large, indicating that there are significant differences in extraction efficiency. For Wuda granules, the volatile oil is sprayed in after extraction, and the transfer rate and content of bornyl acetate are both low. Therefore, during the preparation of the test sample, it is necessary to enrich the components to improve the response. When using the extraction method, during the process of evaporating the solvent to enrich the sample concentration, bornyl acetate evaporates with the solvent and cannot be effectively enriched. When using the distillation (ethyl acetate enrichment) method, it is more conducive to enriching the target components, and the peak area of the target peak is larger. In summary, it is better to prepare the test solution by the distillation (ethyl acetate enrichment) method.

[0104] Table 6: Analysis results of the peak area differences of the components in the test solutions prepared by different extraction methods

[0105]

[0106] Example 7:

[0107] (1) Preparation of the test solution:

[0108] Preparation of the ground test solution: Take an appropriate amount of the finished Wuda granules, grind them finely, take about 20 g, place them in a 500-ml round-bottom flask, add 200 ml of water and several boiling chips, and operate according to the volatile oil determination method (General Chapter 2204, Volume IV of the Chinese Pharmacopoeia 2020 Edition). Add water from the upper end of the measuring device to fill the graduated part and overflow into the flask, then add 2 ml of ethyl acetate, connect the reflux condenser, heat to boiling and keep slightly boiling for 5 hours, cool, separate the ethyl acetate layer, place it in a 2-ml volumetric flask, add ethyl acetate to the scale, and then add 0.5 g of anhydrous sodium sulfate, shake well, and take the supernatant as the test solution.

[0109] Preparation of the non-ground test solution: Take about 20 g of the finished product of Wuda Granules, place it in a 500-ml round-bottom flask, add 200 ml of water and several boiling chips, and operate according to the volatile oil determination method (General Rule 2204, Part IV of the Chinese Pharmacopoeia 2020 Edition). Add water from the upper end of the measuring device until the graduated part is filled and overflows into the flask. Then add 2 ml of ethyl acetate, connect the reflux condenser, heat to boiling and maintain gentle boiling for 5 hours. Let it cool, separate the ethyl acetate layer, place it in a 2-ml volumetric flask, add ethyl acetate to the scale, add another 0.5 g of anhydrous sodium sulfate, shake well, and take the supernatant as the test solution.

[0110] (2) Set the chromatographic conditions of the gas chromatograph. The chromatographic conditions are the same as those in Example 4. Take the reference solution and the test solution, inject them into the gas chromatograph for detection respectively, and record the chromatogram.

[0111] Experimental results: The chromatogram of the ground test solution is as shown in the appendix Figure 15 as shown, and the chromatogram of the non-ground test solution is as shown in the appendix Figure 16 as shown, and the enlarged chromatogram of the non-ground test solution is as shown in the appendix Figure 17 as shown. The analysis results of the peak area difference of the components in the test solution with and without grinding are shown in Table 7. The results show that the relative difference (RD) of the peak area of bornyl acetate in the ground and non-ground test solutions is 23.99%, which is quite different, probably due to the volatilization of volatile components during the grinding process. In summary, in order to reduce the loss of volatile components, the test solution is prepared without grinding.

[0112] Table 7: Analysis results of the peak area difference of the components in the test solution with grinding

[0113]

[0114] In summary, by investigating the extraction solvent, extraction method, and the influence of grinding, it is found that the test solution obtained by using the test solution preparation method in Example 7 (i.e., distillation (ethyl acetate enrichment)) has a purer chromatogram, less interference, and a higher extraction rate of bornyl acetate compared to the test solutions obtained by the ultrasonic method (using ethanol and ethyl acetate as solvents) and the extraction method (using toluene, n-butanol, and ethyl acetate as extractants), greatly improving the sensitivity and applicability of the method for detection. Therefore, the final determined preparation method of the test solution is the same as that in Example 7, that is: Take 20 g of the finished product of Wuda Granules, place it in a 500-ml round-bottom flask, add 200 ml of water and several boiling chips, and operate according to the volatile oil determination method (General Rule 2204, Part IV of the Chinese Pharmacopoeia 2020 Edition). Add water from the upper end of the measuring device until the graduated part is filled and overflows into the flask. Then add 2 ml of ethyl acetate, connect the reflux condenser, heat to boiling and maintain gentle boiling for 5 hours. Let it cool, separate the ethyl acetate layer, place it in a 2-ml volumetric flask, add ethyl acetate to the scale, add another 0.5 g of anhydrous sodium sulfate, shake well, and take the supernatant as the test solution.

[0115] Example 8:

[0116] (1) Preparation of blank solution and test solution:

[0117] Blank solution: Take an appropriate amount of ethyl acetate to obtain.

[0118] Reference solution: Same as Example 1.

[0119] Negative test solution: Take about 20 g of the concentrated solution without fructus amomi and radix linderae, place it in a 500 ml round-bottom flask, add 200 ml of water and several grains of zeolite, operate according to the volatile oil determination method (General Chapter 2204, Volume IV of the Chinese Pharmacopoeia 2020 Edition), add water from the upper end of the measuring device to fill the graduated part and overflow into the flask, then add 2 ml of ethyl acetate, connect the reflux condenser, heat to boiling and keep slightly boiling for 5 hours, cool, separate the ethyl acetate layer, place it in a 25 ml volumetric flask, add ethyl acetate to the scale, add another 0.5 g of anhydrous sodium sulfate, shake well, and take the supernatant as the test solution.

[0120] Granular test solution: Same as Example 7.

[0121] Experimental results: The superimposed chromatograms of the negative solution and the finished preparation solution are as shown in the appendix Figure 18 As shown. In the figure, 1 is the blank solution; 2 is the reference solution of bornyl acetate; 3 is the granular test solution; 4 is the negative test solution; the results show that the blank solution and the negative test solution have no interference on the detection of the target peak, and the specificity meets the requirements.

[0122] The detection method of bornyl acetate in the traditional Chinese medicine compound preparation disclosed in the present invention selects bornyl acetate as the detection index. Through the optimization of chromatographic conditions and the preparation method of the test solution, when preparing the test solution, it breaks through the matrix effects of other medicinal herb slices and excipients, etc., and improves the detection sensitivity by means of distillation enrichment. Finally, the detection method of the volatile component bornyl acetate in Wuda granules is confirmed, thus realizing quality control and standardization. It ensures batch consistency, excludes the influence of complex components of traditional Chinese medicine compound preparations and the susceptibility of bornyl acetate to factors such as raw material origin and extraction process, guarantees the medicinal effect and clinical efficacy, and ensures the quality stability of the preparation. It is convenient to design long-term / accelerated tests according to the ICH guidelines, so as to formulate the expiration date of the preparation.

[0123] Although the present invention has been disclosed above in preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make many possible changes and modifications to the technical solution of the present invention by using the technical content disclosed above without departing from the scope of the technical solution of the present invention, or modify it into equivalent embodiments with equivalent changes. Therefore, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention shall fall within the scope of protection of the technical solution of the present invention.

Claims

1. A method for detecting bornyl acetate in a traditional Chinese medicine compound preparation, characterized in that: It includes the following steps: (1) Prepare the reference solution and the test solution: Reference solution: Weigh an appropriate amount of bornyl acetate reference substance accurately, and dissolve it in ethyl acetate to prepare a solution containing 40 μg of bornyl acetate per 1 ml; Test solution: Take 20 g of the traditional Chinese medicine compound preparation, place it in a flask, add an appropriate amount of water and several zeolite grains, operate according to the volatile oil determination method. Add water from the upper end of the measuring device to fill the graduated part and overflow into the flask. Then add 2 ml of ethyl acetate, connect the reflux condenser, heat to boiling and keep slightly boiling for 5 hours. Let it cool, separate the ethyl acetate layer, place it in a 2-ml volumetric flask, add ethyl acetate to the scale, add 0.5 g of anhydrous sodium sulfate, shake well, and take the supernatant as the test solution; (2) Set the chromatographic conditions of the gas chromatograph. Take the reference solution and the test solution, inject them into the gas chromatograph for detection respectively, and record the chromatogram; Among them, the prescription of the traditional Chinese medicine compound preparation includes Lindera aggregata (Sims) Kosterm., Amomum villosum Lour., Panax ginseng C. A. Mey., Prunus persica (L.) Batsch, and Areca catechu L., and the traditional Chinese medicine compound preparation is a granule.

2. The detection method of bornyl acetate in the traditional Chinese medicine compound preparation according to claim 1, wherein: The chromatographic conditions are as follows: The chromatographic column is DB-1, 30 mm×0.25 mm×0.25 μm; N2 is used as the carrier gas; the flow rate is 2 ml / min; FID detector; the detector temperature is 320 °C; the injection port temperature is 250 °C; the injection volume is 1 μL.

3. The detection method of bornyl acetate in the traditional Chinese medicine compound preparation according to claim 2, characterized in that: In the chromatographic conditions, the splitless mode is adopted.

4. The detection method of bornyl acetate in the traditional Chinese medicine compound preparation according to claim 3, characterized in that: In the chromatographic conditions, the temperature programming is as follows: The initial temperature is maintained at 60 °C for 1 min, then it is raised to 120 °C at a rate of 6 °C / min and maintained for 3 min, and then it is raised to 260 °C at a rate of 20 °C / min and maintained for 5 min.

5. The detection method of bornyl acetate in the traditional Chinese medicine compound preparation according to claim 4, characterized in that: N2 is used as the make-up gas, the flow rate is 25 ml / min, the air flow rate is 300 ml / min, and the hydrogen flow rate is 30 ml / min.

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