A method for detecting bornyl acetate in a traditional Chinese medicine compound preparation

Abstract

The present invention discloses a method for detecting bornyl acetate in a traditional Chinese medicine compound preparation, comprising the following steps: (1) preparing a reference solution and a test solution; (2) setting chromatographic conditions of a gas chromatograph, taking the reference solution and the test solution, injecting them into the gas chromatograph for detection, and recording chromatograms; the detection method selects bornyl acetate as a detection indicator, optimizes the chromatographic conditions and the test sample preparation method, overcomes the matrix effects of other medicinal decoction pieces and auxiliary materials during the preparation of the test sample, improves the detection sensitivity by distillation enrichment, and finally confirms the detection method of bornyl acetate, a volatile component in Wuda granules, thereby achieving quality control and standardization. The method ensures batch consistency, eliminates the complex ingredients of the traditional Chinese medicine compound preparation, and eliminates the susceptibility of bornyl acetate to factors such as the origin of the raw materials and the extraction process, thereby ensuring the efficacy and clinical efficacy and guaranteeing the stability of the preparation quality.

Description

Technical Field

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

[0002] The invention patent with 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 ingredients such as Linderae umbellatae, Amomum villosum, ginseng, peach kernel and betel nut. The granules made from the traditional Chinese medicine composition are called "Wuda Granules". Among them, Amomum villosum is the dried mature fruit of the Zingiberaceae plant Amomum villosum, and Linderae umbellatae is the dried tuberous root of the Lauraceae plant Linderae umbellatae. Both Amomum villosum and Linderae umbellatae are the main ingredients of Wuda Granules. The volatile oil of Amomum villosum contains volatile components such as L-camphor and bornyl acetate, while the volatile oil of Linderae umbellatae contains volatile components such as bornyl acetate and camphor. Bornyl acetate has antibacterial, anti-inflammatory, sedative and anthelmintic effects and has been widely used in medicine, cosmetics and daily chemical products.

[0003] The preparation method of Wuda Granules involves the operations of extracting and adding volatile oils. Since borneol acetate comes from both the monarch medicine Amomum villosum and the monarch medicine Linderae odoratum, and borneol acetate is an indicator component for the content determination item of Amomum villosum medicinal materials and decoction pieces in the 2020 edition of the "Chinese Pharmacopoeia", controlling borneol acetate can achieve the purpose of controlling the two monarch medicines. Bornyl acetate is selected as the gas phase qualitative indicator, which is conducive to detecting the quality of Wuda Granules.

[0004] However, the detection of bornyl acetate has the following difficulties:

[0005] (1) Due to the poor physical stability of volatile oils, they are easily volatilized, oxidized and photolyzed. Especially during the preparation and storage of granules, volatile oils may be destroyed, resulting in a decrease in their content and a weakening of their odor, and even the production of oxidation products (such as peroxides). In addition, there are many uncontrollable factors in the scale-up production of granules, and the volatile oil extraction rate is low. Therefore, it is difficult to establish a unified quality control indicator using quantitative detection methods.

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

[0007] (3) Volatile oils are highly sensitive to process parameters. When adding volatile oils, the timing of addition before / after drying, atomization pressure, and the selection of carrier excipients will affect the retention rate of volatile oils. Slight changes in the process may cause the content of volatile oils to fluctuate beyond 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 traditional TLC (thin layer chromatography) may not be able to specifically distinguish the target volatile components.

[0009] (5) Storage stability is low. When the granules absorb moisture or the packaging is not sealed enough, the volatile oil will gradually dissipate or deteriorate. It is difficult to establish a unified quality control index using quantitative detection methods, and it is difficult to reasonably evaluate product stability.

[0010] The document "Study on the Identification Method of Bornyl Acetate in Eryi Pills by GC" 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 standard for Chinese medicine formula preparations, it can be seen that the Amomum villosum therein is finely powdered for medicinal use, and its volatile component (bornyl acetate) usually exists in its native form, with a high and stable content. Its detection matrix is ​​relatively simple, mainly plant tissue, with fewer interfering substances. Therefore, the volatile components can be directly extracted and measured by ultrasonic treatment.

[0011] The invention patent application with application number CN202311190353.8 discloses a method for determining the ingredients 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", it can be seen that the Amomum villosum therein is a fine powder used as medicine and has not been extracted with water. Therefore, its volatile component content is high and the detection difficulty is low. The test sample can be prepared by dissolving in an organic solvent and ultrasonic treatment.

[0012] The invention patent application with application number CN202410447887.2 discloses a method for determining the quality of volatile drugs in bronchitis rubber paste and its application, but does not disclose a detection method for borneol acetate. Toluene is used as a solvent in the preparation of the test sample, but toluene has a relatively low polarity and is not suitable for the detection of borneol acetate; and the volatile components are of various types and complex properties, so it is necessary to develop and select specific suitable solvents and methods for component detection based on the specific components.

[0013] After extensive literature and patent research and retrieval, there is currently no method for qualitative detection of borneol acetate. The borneol acetate content in Wuda Granules is significantly reduced due to processes such as water extraction, which greatly increases the difficulty of detection and requires a higher sensitivity of the method. In addition, after extraction, excipients are added to prepare 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 method for detecting bornyl acetate in a traditional Chinese medicine compound preparation that can achieve quality control and standardization and ensure batch consistency.

[0015] In order 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 comprises the following steps:

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

[0018] Reference solution: Take an appropriate amount of borneol acetate reference substance, accurately weigh it, and add ethyl acetate to make a solution containing 40µg borneol acetate per 1ml;

[0019] Test solution: Take 20g of the Chinese herbal compound preparation, place it in a flask, add an appropriate amount of water and a few zeolite particles, and operate according to the volatile oil determination method (2020 edition of the "Chinese Pharmacopoeia" Part IV General Rules 2204), add water from the upper end of the analyzer until it fills the scale and overflows into the flask, then add 2ml of ethyl acetate, connect a reflux condenser, heat to boiling and keep it boiling for 5 hours, let cool, separate the ethyl acetate layer, place it in a 2ml measuring flask, add ethyl acetate to the scale, then add 0.5g of anhydrous sodium sulfate, shake well, and take the supernatant as the test solution;

[0020] (2) Setting the chromatographic conditions of the gas chromatograph, taking the reference solution and the test solution, injecting them into the gas chromatograph for detection, and recording the chromatogram;

[0021] The prescription of the Chinese medicine compound preparation includes Linderae serrata, Amomum villosum, Ginseng, Prunus persica and Areca catechu, and the Chinese medicine compound preparation is a granule.

[0022] As a further improvement of the above technical solution: the chromatographic conditions are: the chromatographic column is DB-1, 30mm×0.25mm×0.25μm; N2 is the carrier gas; the flow rate is 2ml / min; FID detector; the detector temperature is 320℃; the injection port temperature is 250℃; and the injection volume is 1μL.

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

[0024] As a further improvement of the above technical solution: in the chromatographic conditions, the temperature rising program is: the starting temperature is 60°C and maintained for 1 minute, then raised to 120°C at a rate of 6°C / min and maintained for 3 minutes, and then raised to 260°C at a rate of 20°C / min and maintained for 5 minutes.

[0025] As a further improvement of the above technical solution: N2 is the tail gas with a flow rate of 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 present invention has the following beneficial effects:

[0027] The present invention discloses a method for detecting bornyl acetate in a traditional Chinese medicine compound preparation, which selects bornyl acetate as a detection indicator. By optimizing the chromatographic conditions and the method for preparing the test sample solution, the matrix effects of other medicinal decoction pieces and excipients are overcome during the preparation of the test sample, and the detection sensitivity is improved by distillation enrichment. The method for detecting bornyl acetate, a volatile component in Wuda granules, is finally confirmed, thereby achieving quality control and standardization. Batch consistency is ensured, the complex ingredients of the traditional Chinese medicine compound preparation are eliminated, and bornyl acetate is easily affected by factors such as the origin of the raw materials and the extraction process, thereby ensuring the efficacy and clinical efficacy and guaranteeing the stability of the preparation quality. It is convenient to design long-term / accelerated tests according to the ICH guidelines, thereby formulating the shelf life of the preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The chromatogram is the chromatogram of Example 1 in the method for detecting 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 The chromatogram of the test solution of Example 3 of the present invention is shown in FIG.

[0031] Figure 4 This is an enlarged superimposed chromatogram of the peach kernel decoction volatile oil test solution and the particle test solution in Example 3 of the present invention.

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

[0033] Figure 6 This is a chromatogram of the ultrasonic extraction (anhydrous ethanol) test solution of Example 5 of the present invention.

[0034] Figure 7 This is an enlarged chromatogram of the ultrasonic extraction (anhydrous ethanol) test solution of Example 5 of the present invention.

[0035] Figure 8 This is a chromatogram of the ultrasonic extraction (ethyl acetate) test solution of Example 5 of the present invention.

[0036] Figure 9 This is a chromatogram of the extraction (toluene) test solution of Example 5 of the present invention.

[0037] Figure 10 This is a chromatogram of the extraction (n-butanol) test solution of Example 5 of the present invention.

[0038] Figure 11 This is a chromatogram of the extraction (ethyl acetate) test solution of Example 5 of the present invention.

[0039] Figure 12 This is a chromatogram of the test sample solution after evaporation and redissolution in Example 6 of the present invention.

[0040] Figure 13 The chromatogram of the extraction (ethyl acetate) test solution of Example 6 of the present invention.

[0041] Figure 14 This is a chromatogram of the distilled (ethyl acetate enriched) test sample solution of Example 6 of the present invention.

[0042] Figure 15 This is a chromatogram of the ground test solution of Example 7 of the present invention.

[0043] Figure 16 This is a chromatogram of the unground test solution of Example 7 of the present invention.

[0044] Figure 17 This is an enlarged chromatogram of the unground test solution of Example 7 of the present invention.

[0045] Figure 18 The chromatograms are superimposed of the Yin-deficient solution and the finished preparation solution of Example 8 of the present invention. DETAILED DESCRIPTION

[0046] The present invention will be further described in detail below with reference to the accompanying drawings and specific examples, but the scope of protection 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 (China) Technology Co., Ltd.), Agilent 7000D triple quadrupole gas chromatograph / gas spectrometer (Agilent (China) Technology Co., Ltd.), KQ500DE digitally controlled ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.), ME204E electronic balance (Mettler-Toledo International Trading (Shanghai) Co., Ltd.).

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

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

[0050] Test samples: Wuda Granules (Guangdong Provincial Hospital of Traditional Chinese Medicine), concentrate lacking Amomum villosum and Linderae serrata (homemade).

[0051] Reference substance: Bornyl acetate (China Food and Drug Inspection Institute).

[0052] Example 1:

[0053] With reference to the 2020 edition of the Chinese Pharmacopoeia, under the item of content determination of Amomum villosum, the gas chromatography preliminary experimental conditions were formulated. The experimental contents are as follows:

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

[0055] Reference solution: Accurately weigh 60 mg of bornyl acetate and place it in a 30 ml volumetric flask. Add anhydrous ethanol to dilute to the constant volume. Accurately pipette 1 ml and add anhydrous ethanol to 2 ml. Shake well to obtain the solution (concentration is approximately 1 mg / ml).

[0056] Wuda granules test solution: Take an appropriate amount of Wuda granules, grind them into powder, weigh 5g, place them in a conical flask, accurately add 15ml of methanol, ultrasonicate for 30 minutes, filter, and take the filtrate to obtain the solution.

[0057] (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, and record the chromatogram;

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

[0059] Table 1: Gas phase heating program of Example 1

[0060]

[0061] Experimental results: The chromatogram of Example 1 is as shown in the attached Figure 1 As shown, borneol acetate was not detected in the Wuda granules test solution, and the analysis time was relatively long.

[0062] Example 2:

[0063] (1) Preparation of test solution:

[0064] Wuda granules test solution: Take an appropriate amount of Wuda granules, grind them into powder, take about 1g, weigh accurately, place in a stoppered conical flask, add 10ml of water to dissolve, transfer to a separatory funnel, rinse the conical flask with a small amount of water, combine the washing liquid into the same separatory funnel, extract with 10ml of ethyl acetate, let it stand to separate the layers, separate the ethyl acetate layer into a centrifuge tube covered with 0.5g of anhydrous sodium sulfate, let it stand, separate the ethyl acetate solution into a 10ml volumetric flask, shake well, filter, and take the filtrate to obtain the product.

[0065] (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, and record the chromatogram;

[0066] The chromatographic conditions were basically the same as those in Example 1, except that the split ratio was 5:1 and the temperature program was as shown in Table 2:

[0067] Table 2: Gas phase heating program of Example 2

[0068]

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

[0070] Example 3:

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

[0072] Peach kernel single decoction volatile oil test solution: weigh 200g of peach kernel slices, crush them, add 12 times water to extract for 2.5 hours, and collect the volatile oil at the same time. Take the collected volatile oil, place it in a 20ml volumetric flask, dissolve it with anhydrous ethanol to the volume, then transfer 1ml, place it in a 50ml volumetric flask, dilute it with water to the volume, take 19ml of aromatic water, place it in a 50ml volumetric flask, dilute it with water to the volume, transfer it to a separatory funnel, rinse it with a small amount of purified water, combine the liquids into a separatory funnel, add ethyl acetate, shake and extract twice, 25ml each time, combine the two upper extracts, remove water with an appropriate amount of anhydrous sodium sulfate, and take the supernatant to obtain the solution.

[0073] (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, and record the chromatogram;

[0074] The chromatographic conditions were substantially the same as those in Example 2, except that a splitless mode was used.

[0075] Experimental results: The chromatogram of the test solution of Example 3 is shown in the attached figure. Figure 3 The superimposed chromatograms of the test solution of the volatile oil of the single decoction of peach kernel and the test solution of the granules are shown in the attached figure. Figure 4The comparison of target peak areas of Example 2 and Example 3 with different split ratios is shown in Table 3. The results show that Example 3 adopts a splitless mode, which shortens the analysis time, and the peak time is located in the middle of the analysis time, and the total analysis time is more appropriate. Figure 4 It can be seen that peach kernel will interfere with the detection of bornyl acetate.

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

[0077]

[0078] Example 4:

[0079] (1) Prepare the test solution: same as in 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, and record the chromatogram;

[0081] The chromatographic conditions were the same as those in Example 3, except that the temperature program was as shown in Table 4:

[0082] Table 4: Gas Phase Temperature Rising Program for Example 4

[0083]

[0084] Experimental results: The chromatogram of Example 4 is shown in the attached diagram. Figure 5 As shown in the figure, the results show that the peach kernel decoction solution has no interference with the determination of bornyl acetate. Under this chromatographic condition, the specificity is good and the heating program is qualified.

[0085] In summary, the chromatographic conditions can be determined as the chromatographic conditions of Example 4, that is, the chromatographic conditions are: the chromatographic column is DB-1, 30mm×0.25mm×0.25μm; N2 is the carrier gas; the flow rate is 2ml / min; the non-split mode is adopted; FID detector; the detector temperature is 320℃; the injection port temperature is 250℃; the injection volume is 1μL; N2 is the tail gas, the flow rate is 25ml / min, the air flow rate is 300ml / min, and the hydrogen flow rate is 30ml / min; the heating program: the starting temperature is 60℃ and maintained for 1min, then increased to 120℃ at 6℃ / min and maintained for 3min, and then increased to 260℃ at a rate of 20℃ / min and maintained for 5min.

[0086] Example 5:

[0087] (1) Preparation of test solution:

[0088] Preparation of ultrasonic extraction (anhydrous ethanol) test solution: Take an appropriate amount of Wuda granules, grind it into powder, take about 5g, accurately weigh it, put it into a stoppered conical flask, accurately add 15ml of anhydrous ethanol, stopper it, weigh it, and ultrasonically treat it (power 300W, frequency 40kHz) for 30 minutes. Shake well, filter, and take the filtrate.

[0089] Preparation of ultrasonic extraction (ethyl acetate) test solution: Take an appropriate amount of Wuda granules, grind it into powder, take about 1g, accurately weigh it, place it in a stoppered conical flask, accurately add 10ml of ethyl acetate, stopper it, weigh it, and treat it ultrasonically (power 300W, frequency 40kHz) for 30 minutes. Let it cool, make up the lost weight with ethyl acetate, shake it well, filter it, and take the filtrate.

[0090] Preparation of extraction (toluene) test solution: Take an appropriate amount of Wuda granules, grind them into powder, take about 1g, weigh accurately, place in a stoppered conical flask, add 10ml of water to dissolve, transfer to a separatory funnel, rinse the conical flask with a small amount of water, combine the washing liquid into the same separatory funnel, add 10ml of toluene to extract, let it stand to separate, take the toluene layer into a centrifuge tube covered with 0.5g of anhydrous sodium sulfate, remove water, filter, and take the filtrate.

[0091] Preparation of extraction (n-butanol) test solution: Take an appropriate amount of Wuda granules, grind them into powder, take about 1g, weigh accurately, place in a stoppered conical flask, add 10ml of water to dissolve, transfer to a separatory funnel, rinse the conical flask with a small amount of water, combine the washing liquid into the same separatory funnel, add 5ml of n-butanol to extract, let it stand to separate, take the n-butanol layer and transfer it to a centrifuge tube covered with 0.5g of anhydrous sodium sulfate to remove water, filter, and take the filtrate.

[0092] Preparation of extraction (ethyl acetate) test solution: Take an appropriate amount of Wuda granules, grind them finely, take about 1g, weigh accurately, place in a stoppered conical flask, add 10ml of water to dissolve, transfer to a separatory funnel, rinse the conical flask with a small amount of water, combine the washing liquid into the same separatory funnel, add 10ml of ethyl acetate to extract, let it stand to separate, take the ethyl acetate layer into a centrifuge tube covered with 0.5g of anhydrous sodium sulfate, remove water, filter, and take the filtrate.

[0093] (2) Setting the chromatographic conditions of the gas chromatograph, the chromatographic conditions are the same as those in Example 4, taking the reference solution and the test solution, injecting them into the gas chromatograph for detection, and recording the chromatogram;

[0094] Experimental results: The chromatogram of the test solution after ultrasonic extraction (anhydrous ethanol) is shown in the attached figure. Figure 6 As shown, the enlarged chromatogram of the ultrasonic extraction (anhydrous ethanol) test solution is shown in the attached Figure 7 As shown, the chromatogram of the test solution after ultrasonic extraction (ethyl acetate) is shown in the attached Figure 8 The chromatogram of the extracted (toluene) test solution is shown in the attached Figure 9 The chromatogram of the extracted (n-butanol) test solution is shown in the attached Figure 10 The chromatogram of the extracted (ethyl acetate) test solution is shown in the attached Figure 11 The results of the analysis of the differences in component content of the test samples extracted with different solvents are shown in Table 5. The results indicate that the baseline of the chromatogram of the test sample prepared using ultrasonic extraction (anhydrous ethanol) was unstable. The RSD% of the bornyl acetate peak area was large when the test sample was prepared using different solvents, indicating that the extraction solvent affected the dissolution of the component. The bornyl acetate peak area was larger in the chromatogram of the sample extracted with ethyl acetate, indicating that extraction with ethyl acetate was preferred for the test sample preparation.

[0095] Table 5: Analysis of the differences in the content of components in the test samples extracted with different solvents

[0096]

[0097] Example 6:

[0098] (1) Preparation of test solution:

[0099] Preparation of the test solution after extraction (ethyl acetate): Take an appropriate amount of the finished product of Wuda Granules, grind it finely, take about 2g, accurately weigh it, place it in a stoppered conical flask, add 50ml of water to dissolve it, transfer it to a separatory funnel, rinse the conical flask with a small amount of water, combine the washings into the same separatory funnel, extract twice with ethyl acetate, 25ml each time, let it stand and separate, combine the extracts, place it in an evaporating dish and evaporate it to almost dryness, dissolve it with ethyl acetate to 2ml, pass through 0.5g of anhydrous sodium sulfate, shake well, filter, and take the filtrate.

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

[0101] Preparation of distillation (ethyl acetate enrichment) test solution: Take an appropriate amount of Wuda granules finished product, grind it finely, take about 20g, place it in a 500ml round-bottom flask, add 200ml of water and a few zeolites, and operate according to the volatile oil determination method (2020 edition of the "Chinese Pharmacopoeia" Part IV General Rules 2204), add water from the upper end of the measuring instrument to fill the scale and overflow into the flask, then add 2ml of ethyl acetate, connect a reflux condenser, heat to boiling and keep boiling for 5 hours, cool, separate the ethyl acetate layer, place it in a 2ml volumetric flask, add ethyl acetate to the scale, then add 0.5g of anhydrous sodium sulfate, shake well, and take the supernatant as the test solution.

[0102] (2) Setting the chromatographic conditions of the gas chromatograph, the chromatographic conditions are the same as those in Example 4, taking the reference solution and the test solution, injecting them into the gas chromatograph for detection, and recording the chromatogram;

[0103] Experimental results: After extraction (ethyl acetate), the chromatogram of the test solution after evaporation and redissolution is as shown in the attached figure. Figure 12 The chromatogram of the extracted (ethyl acetate) test solution is shown in the attached Figure 13 The chromatogram of the distilled (ethyl acetate enriched) test solution is shown in the attached Figure 14 The results of the peak area difference analysis of the test sample components using different extraction methods are shown in Table 6. The results show that the peak area RD% of bornyl acetate is larger when the test samples are prepared using different methods, indicating that there are significant differences in extraction efficiency. The volatile oil in Wuda granules is extracted and then sprayed, and the transfer rate and content of bornyl acetate are both low. Therefore, the components need to be enriched during the test sample preparation process to improve the response. When the sample concentration is extracted, the bornyl acetate evaporates with the solvent during the solvent extraction process, and it cannot be effectively enriched. Distillation (ethyl acetate enrichment) is more conducive to enriching the target component, and the peak area of ​​the target peak is larger. In summary, distillation (ethyl acetate enrichment) is a better method for preparing the test sample solution.

[0104] Table 6: Results of peak area difference analysis of test sample components using different extraction methods

[0105]

[0106] Example 7:

[0107] (1) Preparation of test solution:

[0108] Preparation of grinding test solution: Take an appropriate amount of Wuda granules finished product, grind it finely, take about 20g, place it in a 500ml round-bottom flask, add 200ml of water and a few zeolites, and operate according to the volatile oil determination method (2020 edition of the "Chinese Pharmacopoeia" Part IV General Rules 2204), add water from the upper end of the measuring instrument until it fills the scale and overflows into the flask, then add 2ml of ethyl acetate, connect a reflux condenser, heat to boiling and keep boiling for 5 hours, cool, separate the ethyl acetate layer, place it in a 2ml measuring flask, add ethyl acetate to the scale, then add 0.5g of anhydrous sodium sulfate, shake well, and take the supernatant as the test solution.

[0109] Preparation of the test solution without grinding: Take about 20g of the finished product of Wuda Granules, place it in a 500ml round-bottom flask, add 200ml of water and a few zeolites, and operate according to the volatile oil determination method (2020 edition of the "Chinese Pharmacopoeia" Part IV General Rules 2204), add water from the upper end of the measuring instrument until it fills the scale and overflows into the flask, then add 2ml of ethyl acetate, connect a reflux condenser, heat to boiling and keep boiling for 5 hours, cool, separate the ethyl acetate layer, place it in a 2ml measuring flask, add ethyl acetate to the scale, then add 0.5g of anhydrous sodium sulfate, shake well, and take the supernatant as the test solution.

[0110] (2) Setting the chromatographic conditions of the gas chromatograph, the chromatographic conditions are the same as those in Example 4, taking the reference solution and the test solution, injecting them into the gas chromatograph for detection, and recording the chromatogram;

[0111] Experimental results: The chromatogram of the ground test solution is shown in the attached Figure 15 The chromatogram of the unground test solution is shown in the attached Figure 16 The enlarged chromatogram of the unground test solution is shown in the attached Figure 17 The results of the analysis of the differences in peak area of ​​the test sample components after grinding are shown in Table 7. The results show that the peak area RD of the bornyl acetate solution in the ground and unground test solution is 23.99%, which is a significant difference. This may be due to the volatilization of volatile components during the grinding process. In summary, in order to reduce the loss of volatile components, the test sample was prepared without grinding.

[0112] Table 7: Analysis results of differences in peak area of ​​test sample components due to grinding

[0113]

[0114] In summary, by investigating the effects of extraction solvent, extraction method and grinding, it was found that the test solution obtained using the test sample preparation method of Example 7 (i.e., distillation (ethyl acetate enrichment)) was chromatographically purer and had less interference than the test sample solutions obtained by the ultrasonic method (ethanol and ethyl acetate as solvents) and the extraction method (toluene, n-butanol and ethyl acetate as extractants), and the borneol acetate extraction rate was higher, which greatly improved the sensitivity and applicability of the method detection. Therefore, the final preparation method of the test solution is the same as that in Example 7, that is, 20 g of the finished product of Wuda Granules is taken, placed in a 500 ml round-bottom flask, 200 ml of water and a few zeolites are added, and the volatile oil determination method (2020 edition of the "Chinese Pharmacopoeia" Part IV General Rules 2204) is followed. Water is added from the upper end of the measuring instrument until the scale is filled and overflows into the flask, then 2 ml of ethyl acetate is added, a reflux condenser is connected, heated to boiling and kept at a slight boiling for 5 hours, cooled, the ethyl acetate layer is separated, placed in a 2 ml measuring flask, ethyl acetate is added to the scale, and 0.5 g of anhydrous sodium sulfate is added, shaken, and the supernatant is taken as the test solution.

[0115] Example 8:

[0116] (1) Prepare blank solution and test solution:

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

[0118] Reference solution: same as in Example 1.

[0119] Negative test solution: Take about 20g of the concentrated solution without Amomum villosum and Linderae scorzonerifolia, place it in a 500ml round-bottom flask, add 200ml of water and a few zeolites, and operate according to the volatile oil determination method (2020 edition of the "Chinese Pharmacopoeia" Part IV General Chapter 2204), add water from the upper end of the determiner until it fills the scale and overflows into the flask, then add 2ml of ethyl acetate, connect a reflux condenser, heat to boiling and keep boiling for 5 hours, cool, separate the ethyl acetate layer, place it in a 25ml volumetric flask, add ethyl acetate to the scale, then add 0.5g of anhydrous sodium sulfate, shake well, and take the supernatant as the test solution.

[0120] Particle test solution: same as Example 7.

[0121] Experimental results: The superimposed chromatograms of the yin-deficient solution and the finished product solution are shown in the attached figure. Figure 18 As shown in the figure, 1 is the blank solution; 2 is the bornyl acetate reference solution; 3 is the particle test solution; 4 is the negative test solution; the results show that the blank solution and the negative test solution have no interference with the detection of the target peak, and the specificity meets the requirements.

[0122] The present invention discloses a method for detecting bornyl acetate in a traditional Chinese medicine compound preparation, which selects bornyl acetate as a detection indicator. By optimizing the chromatographic conditions and the method for preparing the test sample solution, the matrix effects of other medicinal decoction pieces and excipients are overcome during the preparation of the test sample, and the detection sensitivity is improved by distillation enrichment. The method for detecting bornyl acetate, a volatile component in Wuda granules, is finally confirmed, thereby achieving quality control and standardization. Batch consistency is ensured, the complex ingredients of the traditional Chinese medicine compound preparation are eliminated, and bornyl acetate is easily affected by factors such as the origin of the raw materials and the extraction process, thereby ensuring the efficacy and clinical efficacy and guaranteeing the stability of the preparation quality. It is convenient to design long-term / accelerated tests according to the ICH guidelines, thereby formulating the shelf life of the preparation.

[0123] Although the present invention has been disclosed above with reference to preferred embodiments, this is not intended to limit the present invention. Any person skilled in the art can, without departing from the scope of the technical solution of the present invention, utilize the technical content disclosed above to make many possible changes and modifications to the technical solution of the present invention, or modify it into an equivalent embodiment with equivalent changes. Therefore, any simple modification, equivalent change, and modification made to the above embodiments based on 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 Chinese medicine compound preparation, characterized in that: The following steps are involved: (1) Preparation of reference solution and test solution: Reference solution: Take an appropriate amount of bornyl acetate reference substance, accurately weigh it, and add ethyl acetate to make a solution containing 40µg bornyl acetate per 1ml; Test solution: Take 20g of the traditional Chinese medicine compound preparation, place it in a flask, add an appropriate amount of water and a few zeolite grains, and operate according to the volatile oil determination method. Add water from the upper end of the analyzer until it fills the scale and overflows into the flask, then add 2ml of ethyl acetate, connect a reflux condenser, heat to boiling and maintain a slight boiling for 5 hours, let cool, separate the ethyl acetate layer, place it in a 2ml measuring flask, add ethyl acetate to the scale, then add 0.5g of anhydrous sodium sulfate, shake well, and take the supernatant as the test solution; (2) Setting the chromatographic conditions of the gas chromatograph, taking the reference solution and the test solution, injecting them into the gas chromatograph for detection, and recording the chromatogram; The prescription of the Chinese medicine compound preparation includes Linderae Root, Amomum Villosum, Ginseng, Peach Kernel and Areca Nut, and the Chinese medicine compound preparation is Wuda Granules from Guangdong Provincial Hospital of Traditional Chinese Medicine; The chromatographic conditions are as follows: a DB-1 chromatographic column, 30 mm × 0.25 mm × 0.25 μm; an FID detector; a splitless flow mode; and a heating program: a starting temperature of 60°C, maintained for 1 minute, then increased to 120°C at a rate of 6°C / min, maintained for 3 minutes, and then increased to 260°C at a rate of 20°C / min and maintained for 5 minutes.

2. The method for detecting bornyl acetate in the Chinese medicinal compound preparation according to claim 1, wherein: The chromatographic conditions are as follows: N2 is the carrier gas; the flow rate is 2 ml / min; the detector temperature is 320°C; the injection port temperature is 250°C; and the injection volume is 1 μL.

3. The method for detecting bornyl acetate in the Chinese medicine compound preparation according to claim 2, wherein: The chromatographic conditions were as follows: N2 was the tail gas with a flow rate of 25 ml / min, air flow rate of 300 ml / min, and hydrogen flow rate of 30 ml / min.

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