Establishment of a method for determining the fingerprint spectrum of a kind of Tongqiao Jiuxin oil and a method for detecting the content of its components

Abstract

The application discloses a method for establishing a fingerprint spectrum of Tongqiao Jiuxin oil and a method for detecting component content, and belongs to the technical field of traditional Chinese medicine quality detection. The technical problem to be solved is that the accuracy of the existing detection method of the Tongqiao Jiuxin oil needs to be improved. The technical solution is that a method for establishing a fingerprint spectrum of Tongqiao Jiuxin oil or a method for detecting component content is adopted, liquid chromatography is used, acetonitrile is used as a mobile phase A, a 0.1-0.2% mass concentration phosphoric acid solution is used as a mobile phase B, and a specific gradient elution program is used for elution.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine quality testing technology, specifically to a method for establishing a fingerprint spectrum of Tongqiao Jiuxin Oil and a method for detecting the content of its components. Background Technology

[0002] Tongqiao Jiuxin Oil originates from the Suhexiang Pill recorded in the Taiping Huimin Heji Jufang (Formulary of the Bureau of Imperial Medical Services). It is composed of sandalwood, costus root, agarwood, frankincense, styrax, borneol, menthol, camphor, and musk. It possesses aromatic and orifice-opening, qi-regulating, and pain-relieving effects, and is used to treat chest pain, phlegm-induced coma, sudden abdominal pain, and seasonal malaria. Studies have shown that Tongqiao Jiuxin Oil has significant anti-myocardial ischemia and analgesic effects, and also has a significant therapeutic effect on animal models of altitude sickness. Furthermore, Tongqiao Jiuxin Oil is effective and safe for unstable angina pectoris.

[0003] Tongqiao Jiuxin Oil is currently included in the "Drug Standards of the Ministry of Health of the People's Republic of China" (Volume 20 of Traditional Chinese Medicine Compound Preparations). The current quality standards only limit the content of borneol, menthol, and camphor, which are present in larger quantities. Each gram of Tongqiao Jiuxin Oil should contain 163.2-220.8 mg of borneol, 48.45-65.55 mg of menthol, and 5.1-6.9 mg of camphor. The standards also specify thin-layer chromatography for styrax and costus root, but lack qualitative and quantitative regulations for agarwood, sandalwood, and frankincense, which is detrimental to overall quality control. Therefore, it is essential to appropriately improve the quality standards for Tongqiao Jiuxin Oil.

[0004] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects:

[0005] The lack of qualitative and quantitative regulations for medicinal materials such as agarwood, sandalwood, and frankincense in Tongqiao Jiuxin Oil is detrimental to overall quality control.

[0006] In solving the above problems or overcoming the above defects, the present invention encountered the following difficulties and obstacles:

[0007] Currently, no other existing technologies related to the establishment of fingerprint chromatograms and component content detection methods for Tongqiao Jiuxin Oil have been found. Because the content of agarwood, sandalwood, frankincense, and other medicinal materials differs significantly from that of borneol, menthol, and camphor, it is not possible to simultaneously use gas chromatography for qualitative and quantitative studies. Summary of the Invention

[0008] The purpose of this invention is to provide:

[0009] A method for establishing a fingerprint spectrum of Tongqiao Jiuxin Oil and a method for detecting its component content, and related technologies, to solve technical problems such as improving the detection accuracy of Tongqiao Jiuxin Oil, or a combination thereof.

[0010] Terminology Explanation:

[0011] Unless otherwise defined, all technical terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this subject matter pertains. Unless otherwise stated, all patents, patent inventions, and disclosures cited throughout this document are incorporated herein by reference in their entirety. Where multiple definitions exist for terms herein, the definitions provided in this chapter shall prevail.

[0012] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0013] The definition of the standard chemical term can be found in the reference "Gao Wenyuan. Modern Chinese Medicine Quality Control and Technology [M]. Science Press, 2010."

[0014] Unless otherwise stated, conventional methods within the scope of the art, such as repeatability testing, stability testing, durability testing, and accuracy testing, shall be used.

[0015] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0016] The terms “optional / arbitrary” or “optionally / arbitrarily” mean that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation.

[0017] The term "Tongqiao Jiuxin Oil" used in this article refers to an oil made from sandalwood, costus root, agarwood, frankincense, styrax, borneol, menthol, camphor, and musk. The preparation method is as follows (according to the preparation method of Tongqiao Jiuxin Oil in the "Drug Standards of the Ministry of Health of the People's Republic of China" (Volume 20 of Traditional Chinese Medicine Compound Preparations):

[0018] The above nine ingredients are as follows: Agarwood, sandalwood, and costus root are crushed into coarse particles, extracted four times by reflux with 95% ethanol for 2 hours each time, filtered, the filtrates are combined, the ethanol is recovered, and the extract is concentrated to about half the amount of the medicinal materials in the prescription, and the extract is reserved for use; Frankincense is crushed into coarse particles, extracted four times by reflux with 95% ethanol for 2 hours each time, filtered, the filtrates are combined, the ethanol is recovered, and the extract is concentrated to about half the amount of the medicinal materials in the prescription, and the extract is reserved for use; Musk is extracted six times by reflux with 95% ethanol, 10 ml each time for 2 hours each time, the extract is filtered, combined, frozen at below 10℃ for more than 4 hours, filtered again, and reserved for use; an appropriate amount of liquid paraffin and vegetable oil are mixed, added to the above reserved extract, stirred, refrigerated for 24 hours, the supernatant is poured off, filtered by pressure, the remaining oil is dehydrated with anhydrous sodium sulfate, filtered by pressure, and the filtrates are combined. Separately, slowly add borneol, camphor, menthol, and styrax to the above oil solution, stir to dissolve, then add musk extract, mix well, weigh the total weight, add about 380g of vegetable oil, filter with dry filter paper (without filter material), make 1000g, and package to obtain the product.

[0019] The term "negative sample" as used in this article refers to a sample whose formulation and preparation process are identical to the test sample, except for the absence of the target medicinal ingredient. For example, a "negative sample lacking styrax" means that styrax was removed from the test sample formulation, while all other conditions remained unchanged.

[0020] All percentages of the mobile phase in this article are expressed as volume percentages.

[0021] In a first aspect, the present invention provides: a method for establishing a fingerprint spectrum of Tongqiao Jiuxin Oil, employing liquid chromatography with acetonitrile as mobile phase A and 0.1-0.2% (w / w) phosphoric acid solution as mobile phase B. The gradient elution program is as follows: 0-5 min, mobile phase A X1%→X1%, mobile phase B Y1%→Y1%; 5-16 min, mobile phase A X1%→X2%, mobile phase B Y1%→Y2%; 16-44 min, mobile phase A X2%→X3%, mobile phase B Y2%→Y3%; 44-52 min, mobile phase A X3%→X3%, mobile phase B Y3%→Y3%; 52-75 min, mobile phase A X3%→X4%, mobile phase B Y3%→Y4%; 75-88 min, mobile phase A X4%→X5%, mobile phase B Y4%→Y5%; 88-115 min, mobile phase A X5%→X5%, mobile phase B Y1%→Y1%; 52-75 min, mobile phase A X5%→X5%, mobile phase B Y1%→Y2%; 52-75 min, mobile phase A X5%→X4%, mobile phase B Y1%→Y2%; 52-75 min, mobile phase A X5%→X4%, mobile phase B Y1%→Y2%; 52-75 min, mobile phase A X5%→X5 ...5%, mobile phase B Y1%→Y2%; 52-75 min, mobile phase A X5%→X5%, mobile phase B Y1%→Y2%; 52-7 Y5%→Y5%; where X1=15-17%, X2=18-20%, X3=52-54%, X4=58-60%, X5=77-79%; Y1=83-85%, Y2=80-82%, Y3=46-48%, Y4=40-42%, Y5=21-23%.

[0022] This includes technical features such as chromatographic conditions for liquid chromatography and the method and steps for establishing the fingerprint spectrum of Tongqiao Jiuxin Oil.

[0023] Among them, the chromatographic conditions of the technical characteristic liquid chromatography method are selected from: gradient elution program, column packing material, detection wavelength, column temperature, theoretical plate number, and flow rate.

[0024] The preferred gradient elution program is as follows: 0-5 min, mobile phase A 16%→16%, mobile phase B 84%→84%; 5-16 min, mobile phase A 16%→19%, mobile phase B 84%→81%; 16-44 min, mobile phase A 19%→53%, mobile phase B 81%→47%; 44-52 min, mobile phase A 53%→53%, mobile phase B 47%→47%; 52-75 min, mobile phase A 53%→59%, mobile phase B 47%→41%; 75-88 min, mobile phase A 59%→78%, mobile phase B 41%→22%; 88-115 min, mobile phase A 78%→78%, mobile phase B 22%→22%.

[0025] The packing material for the chromatographic column is selected from octadecylsilane-bonded silica gel.

[0026] The detection wavelength for technical features is selected from 210-230nm.

[0027] The detection wavelength for the technical features is selected from 212nm.

[0028] The technical characteristic column temperature is selected from 30-40℃.

[0029] Among them, the theoretical plate number of the technical characteristics is selected from: based on the cinnamic acid-3-phenylpropyl ester peak, it should not be less than 5000.

[0030] The technical characteristic flow rate is selected from 0.9-1.1 ml / min.

[0031] The steps for establishing the fingerprint spectrum of the Tongqiao Jiuxin Oil are selected from the following steps: preparation of reference solution, preparation of test solution, preparation of negative sample solution, preparation of control solution and establishment of fingerprint spectrum.

[0032] The preparation of the technical feature reference solution is as follows: take cinnamic acid-3-phenylpropyl ester reference standard, accurately weigh it, add anhydrous ethanol to prepare a solution containing 50-100 μg of cinnamic acid-3-phenylpropyl ester reference standard per 1 ml.

[0033] The preparation of the test solution for the technical feature is as follows: take Tongqiao Jiuxin Oil, accurately weigh it, add anhydrous ethanol to prepare a solution containing 5-10 mg of Tongqiao Jiuxin Oil per 1 ml.

[0034] The preparation of the negative sample solution with technical characteristics is selected from: taking different negative samples and preparing different negative sample solutions according to the preparation method of the test sample solution; the negative samples are prepared according to the preparation method of Tongqiao Jiuxin Oil, respectively, for the absence of styrax, frankincense, sandalwood, agarwood and costus root.

[0035] The technical characteristic reference solution is prepared by taking agaric tetraol, dehydroauracene lactone, 11-carbonyl-β-acetylboswellic acid, 3-phenylpropyl cinnamic acid, and cinnamic acid ester, and adding anhydrous ethanol to prepare the reference solution. Alternatively, the six reference standards can be mixed first and then anhydrous ethanol can be added, or the six reference standards can be prepared separately with anhydrous ethanol and then mixed thoroughly.

[0036] The steps for establishing the technical fingerprint spectrum are selected from: using the liquid chromatography method to detect the reference solution, test solution, negative sample solution and control solution to obtain the fingerprint spectrum.

[0037] Secondly, this invention provides a method for detecting the content of components in a heart-clearing and mind-saving oil, employing liquid chromatography with acetonitrile as mobile phase A and 0.1-0.2% phosphoric acid solution as mobile phase B. The gradient elution program is as follows: 0-5 min, mobile phase A X1%→X1%, mobile phase B Y1%→Y1%; 5-16 min, mobile phase A X1%→X2%, mobile phase B Y1%→Y2%; 16-44 min, mobile phase A X2%→X3%, mobile phase B Y2%→Y3%; 44-52 min, mobile phase A X3%→X3%, mobile phase B Y3%→Y3%; 52-75 min, mobile phase A X3%→X4%, mobile phase B Y3%→Y4%; 75-88 min, mobile phase A X4%→X5%, mobile phase B Y4%→Y5%; 88-115 min, mobile phase A X5%→X5%, mobile phase B Y1%→Y1%; 52-75 min, mobile phase A X5%→X5%, mobile phase B Y1%→Y2%; 52-75 min, mobile phase A X5%→X4%, mobile phase B Y1%→Y2%; 52-75 min, mobile phase A X5%→X4%, mobile phase B Y1%→Y2%; 52-75 min, mobile phase A X5%→X5 ...5%, mobile phase B Y1%→Y2%; 52-75 min, mobile phase A X5%→X5%, mobile phase B Y1%→Y2%; 52-75 min, mobile Y5%→Y5%; wherein, X1=15-17%, X2=18-20%, X3=52-54%, X4=58-60%, X5=77-79%; Y1=83-85%, Y2=80-82%, Y3=46-48%, Y4=40-42%, Y5=21-23%; the components include at least one of agaric tetraol, dehydroauracene lactone, 3-phenylpropyl cinnamic acid and 11-carbonyl-β-acetylsalicylic acid.

[0038] This includes technical features such as chromatographic conditions for liquid chromatography and methods for detecting component content.

[0039] Among them, the chromatographic conditions of the technical characteristic liquid chromatography method are selected from: gradient elution program, column packing material, detection wavelength, column temperature, theoretical plate number, and flow rate.

[0040] The preferred gradient elution program is as follows: 0-5 min, mobile phase A 16%→16%, mobile phase B 84%→84%; 5-16 min, mobile phase A 16%→19%, mobile phase B 84%→81%; 16-44 min, mobile phase A 19%→53%, mobile phase B 81%→47%; 44-52 min, mobile phase A 53%→53%, mobile phase B 47%→47%; 52-75 min, mobile phase A 53%→59%, mobile phase B 47%→41%; 75-88 min, mobile phase A 59%→78%, mobile phase B 41%→22%; 88-115 min, mobile phase A 78%→78%, mobile phase B 22%→22%.

[0041] The packing material for the chromatographic column is selected from octadecylsilane-bonded silica gel.

[0042] The detection wavelength for technical features is selected from 210-230nm.

[0043] The preferred wavelength for detecting technical features is 225nm.

[0044] The technical characteristic column temperature is selected from 30-40℃.

[0045] Among them, the theoretical plate number of the technical characteristics is selected from: based on the cinnamic acid-3-phenylpropyl ester peak, it should not be less than 5000.

[0046] The technical characteristic flow rate is selected from 0.9-1.1 ml / min.

[0047] The detection method steps for the content of technical characteristic components are selected from: preparation of the test solution, preparation of the mixed reference solution, and detection steps.

[0048] The preparation of the test solution for the technical feature is as follows: take Tongqiao Jiuxin Oil, accurately weigh it, add anhydrous ethanol to prepare a solution containing 5-10 mg of Tongqiao Jiuxin Oil per 1 ml.

[0049] The preparation of the technical feature mixed reference solution is as follows: take agaric tetraol, dehydroauracene lactone, cinnamic acid-3-phenylpropyl ester, and 11-carbonyl-β-acetylbosuccinic acid reference standards, accurately weigh them, add anhydrous ethanol to prepare a mixed reference solution, and that is obtained.

[0050] The technical feature detection step is selected from: using the liquid chromatography method to detect the test solution and the mixed reference solution to obtain the content of at least one component among agaric tetraol, dehydroauracene lactone, cinnamic acid-3-phenylpropyl ester and 11-carbonyl-β-acetylbosuccinic acid.

[0051] The present invention has at least the following beneficial effects:

[0052] Compared with existing technologies, the present invention has better technical effects in terms of improving detection accuracy, repeatability, stability and durability.

[0053] This invention employs HPLC fingerprinting to establish characterization methods for styrax, costus root, agarwood, sandalwood, and frankincense in Tongqiao Jiuxin Oil. Through HPLC content determination, methods for determining the content of agaric tetraol, dehydrocostus lactone, 3-phenylpropyl cinnamic acid, and 11-carbonyl-β-acetylbosuccinic acid in Tongqiao Jiuxin Oil were established. These methods are simple, rapid, and highly specific, and can further improve the quality evaluation methods for Tongqiao Jiuxin Oil, thereby ensuring its stable and controllable quality. Attached Figure Description

[0054] Figure 1 The images show liquid chromatograms of different chromatographic columns used in Example 1.

[0055] Figure 2 These are liquid chromatograms of operations performed by different personnel in Example 1.

[0056] Figure 3 This is the confirmed spectrum of the common peak in Example 1.

[0057] Figure 4 This is a chromatogram confirming the attribution of the medicinal materials with common peaks in Example 1.

[0058] Figure 5 This is a spectrum confirming the chemical composition of the common peaks in Example 1.

[0059] Figure 6 This is the liquid chromatogram for the content determination in Example 2.

[0060] Figure 7 The results are from the linearity test of agaric tetraol.

[0061] Figure 8 The results are from a linearity test of dehydroauric acid lactone.

[0062] Figure 9 The results are from the linearity test of 3-phenylpropyl cinnamic acid.

[0063] Figure 10 The results are from a linear assay of 11-carbonyl-β-acetylboronic acid.

[0064] Figure 11 This is a liquid chromatogram at a wavelength of 254 nm.

[0065] Figure 12 This is a liquid chromatogram at a wavelength of 205 nm.

[0066] Figure 13 This is the liquid chromatogram of elution gradient A at 225 nm.

[0067] Figure 14 This is the liquid chromatogram of elution gradient B at 225 nm.

[0068] Figure 15 This is a liquid chromatogram of the elution gradient C at 225 nm.

[0069] Figure 16 The image shows the liquid chromatogram of methanol as mobile phase A at 225 nm.

[0070] Figure 17 Liquid chromatogram at 225 nm with 0.2% phosphoric acid as mobile phase B. Detailed Implementation

[0071] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0072] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0073] Example 1

[0074] 1. Experimental materials

[0075] 1.1 Instruments

[0076] Agilent 1260 high-performance liquid chromatograph; PX36 / A electronic balance (Mettler Toledo Technologies (China) Co., Ltd.); SECURA225D-1CN electronic balance (Sartorius Group); Zhongpu Red C18 (250mm×4.6mm×5μm) liquid chromatography column (Zhongpu Technology (Fuzhou) Co., Ltd.).

[0077] 1.2 Reagents

[0078] Anhydrous ethanol (chromatographic grade).

[0079] 1.3 Standard Products

[0080] Agaric tetraol (batch number: 11980-201904), dehydroauracene lactone (batch number: 11525-202313), and 11-carbonyl-β-acetylboswellic acid (batch number: 11760-202103) were all purchased from the China National Institutes for Food and Drug Control. 3-Phenylacetic acid ester (batch number: C16286696) and cinnamyl cinnamate (batch number: C16253723) were both purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0081] 1.4 Test Sample

[0082] Tongqiao Jiuxin Oil, batch number UT30002, was purchased from Guangzhou Baiyunshan Xingqun (Pharmaceutical) Co., Ltd.; the styrax, frankincense, sandalwood, costus root, and agarwood were qualified samples after purchase and testing according to the pharmacopoeia method.

[0083] 1.5 Negative samples

[0084] According to the preparation method of Tongqiao Jiuxin Oil in the "Drug Standards of the Ministry of Health of the People's Republic of China" (Volume 20 of Traditional Chinese Medicine Formulas), negative samples were prepared for those lacking styrax, frankincense, sandalwood, agarwood, and costus root.

[0085] Negative Sample 1 (lacking styrax): Agarwood, sandalwood, and costus root were coarsely pulverized and extracted four times by reflux with 95% ethanol for 2 hours each time. The extracts were filtered, the filtrates were combined, the ethanol was recovered, and the extract was concentrated to about half the amount of the medicinal materials in the prescription. The extract was prepared for use. Frankincense was coarsely pulverized and extracted four times by reflux with 95% ethanol for 2 hours each time. The extracts were filtered, the filtrates were combined, the ethanol was recovered, and the extract was concentrated to about half the amount of the medicinal materials in the prescription. The extract was prepared for use. Musk was extracted six times by reflux with 95% ethanol, 10 ml of ethanol each time for 2 hours each time. The extracts were filtered, combined, and then frozen at below 10°C for more than 4 hours before being filtered again. The extract was prepared for use. An appropriate amount of liquid paraffin and vegetable oil (tea oil) were mixed, and the above-prepared extract was added. The mixture was stirred, refrigerated for 24 hours, the supernatant was poured off, and the mixture was filtered. The remaining oil was dehydrated with anhydrous sodium sulfate, filtered, and the filtrates were combined. Add borneol, camphor, and menthol slowly to the above oil solution and stir until dissolved. Then add musk extract, mix well, weigh the total amount, add about 380g of vegetable oil, and filter with dry filter paper (without filter material) to make 1000g. Package the mixture.

[0086] Negative Sample 2 (lacking frankincense): Agarwood, sandalwood, and costus root were coarsely pulverized and extracted four times by reflux with 95% ethanol, 2 hours each time. The extracts were filtered, combined, and the ethanol was recovered and concentrated to about half the amount of the medicinal materials in the prescription. The extract was reserved for use. Musk was extracted six times by reflux with 95% ethanol, 10 ml of ethanol each time, 2 hours each time. The extracts were filtered, combined, and frozen at below 10℃ for more than 4 hours, then filtered again for use. An appropriate amount of liquid paraffin and vegetable oil (tea oil) were mixed, added to the above reserved extract, stirred, refrigerated for 24 hours, the supernatant was poured off, and the mixture was filtered. The remaining oil was dehydrated with anhydrous sodium sulfate, filtered, and the filtrates were combined. Borneol, camphor, menthol, and styrax were slowly added to the above oil, stirred until dissolved, and then the musk extract was added. The mixture was mixed well, the total weight was determined, and about 380g of vegetable oil was added. The mixture was filtered through dry filter paper (without filter material) to make 1000g, which was then packaged.

[0087] Negative Sample 3 (lacking sandalwood): Agarwood and costus root were crushed into coarse particles, extracted four times by reflux with 95% ethanol for 2 hours each time, filtered, the filtrates were combined, the ethanol was recovered and concentrated to about half the amount of medicinal materials in the prescription, and the extract was reserved for use; Frankincense was crushed into coarse particles, extracted four times by reflux with 95% ethanol for 2 hours each time, filtered, the filtrates were combined, the ethanol was recovered, the extract was concentrated to about half the amount of medicinal materials in the prescription, and the extract was reserved for use; Musk was extracted six times by reflux with 95% ethanol, 10 ml of ethanol each time for 2 hours each time, the extract was filtered, combined, frozen at below 10℃ for more than 4 hours, filtered again, and reserved for use; An appropriate amount of liquid paraffin and vegetable oil (tea oil) were mixed, the above reserved extract was added, stirred, refrigerated for 24 hours, the supernatant was poured off, filtered by pressure, the remaining oil was dehydrated with anhydrous sodium sulfate, filtered by pressure, and the filtrates were combined. Separately, slowly add borneol, camphor, menthol, and styrax to the above oil solution, stir to dissolve, then add musk extract, mix well, weigh the total weight, add about 380g of vegetable oil, filter with dry filter paper (without filter material), make 1000g, and package to obtain the product.

[0088] Negative Sample 4 (lacking agarwood): Sandalwood and costus root were crushed into coarse particles, extracted four times by reflux with 95% ethanol for 2 hours each time, filtered, the filtrates were combined, the ethanol was recovered and concentrated to about half the amount of medicinal materials in the prescription, and the extract was reserved for use; Frankincense was crushed into coarse particles, extracted four times by reflux with 95% ethanol for 2 hours each time, filtered, the filtrates were combined, the ethanol was recovered, the extract was concentrated to about half the amount of medicinal materials in the prescription, and the extract was reserved for use; Musk was extracted six times by reflux with 95% ethanol, 10 ml of ethanol each time for 2 hours each time, the extract was filtered, combined, frozen at below 10℃ for more than 4 hours, filtered again, and reserved for use; An appropriate amount of liquid paraffin and vegetable oil (tea oil) were mixed, the above reserved extract was added, stirred, refrigerated for 24 hours, the supernatant was poured off, the liquid was filtered by pressure, the remaining oil was dehydrated with anhydrous sodium sulfate, filtered by pressure, and the filtrates were combined. Separately, slowly add borneol, camphor, menthol, and styrax to the above oil solution, stir to dissolve, then add musk extract, mix well, weigh the total weight, add about 380g of vegetable oil, filter with dry filter paper (without filter material), make 1000g, and package to obtain the product.

[0089] Negative Sample 5 (lacking costus root): Agarwood and sandalwood were crushed into coarse particles, extracted four times by reflux with 95% ethanol for 2 hours each time, filtered, the filtrates were combined, the ethanol was recovered and concentrated to about half the amount of medicinal materials in the prescription, and the extract was reserved for use; Frankincense was crushed into coarse particles, extracted four times by reflux with 95% ethanol for 2 hours each time, filtered, the filtrates were combined, the ethanol was recovered, the extract was concentrated to about half the amount of medicinal materials in the prescription, and the extract was reserved for use; Musk was extracted six times by reflux with 95% ethanol, 10 ml of ethanol each time for 2 hours each time, the extract was filtered, combined, frozen at below 10℃ for more than 4 hours, filtered again, and reserved for use; An appropriate amount of liquid paraffin and vegetable oil (tea oil) were mixed, the above reserved extract was added, stirred, refrigerated for 24 hours, the supernatant was poured off, the liquid was filtered by pressure, the remaining oil was dehydrated with anhydrous sodium sulfate, filtered by pressure, and the filtrates were combined. Separately, slowly add borneol, camphor, menthol, and styrax to the above oil solution, stir to dissolve, then add musk extract, mix well, weigh the total weight, add about 380g of vegetable oil, filter with dry filter paper (without filter material), make 1000g, and package to obtain the product.

[0090] 2. Experimental methods and results

[0091] 2.1 Chromatographic conditions

[0092] HPLC fingerprint analysis: determined by high performance liquid chromatography (General Chapter 0512, Chinese Pharmacopoeia 2025).

[0093] Octadecylsilane-bonded silica gel was used as the packing material; acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B. Gradient elution was performed according to the specifications in Table 1; the detection wavelength was 212 nm; the column temperature was 30 °C; the theoretical plate number, calculated based on the cinnamic acid-3-phenylpropyl ester peak, should not be less than 5000; and the flow rate was 1.0 mL / min.

[0094] Table 1 Gradient elution program

[0095]

[0096] 2.2 Preparation of the reference solution

[0097] Accurately weigh 3-phenylpropyl cinnamic acid reference standard, add anhydrous ethanol to prepare a solution containing 100 μg per ml.

[0098] 2.3 Preparation of the test solution

[0099] Accurately weigh 50mg of Tongqiao Jiuxin Oil, place it in a 5ml volumetric flask, and add anhydrous ethanol to the mark.

[0100] 2.4 Preparation of negative sample solution

[0101] Take approximately 50 mg of each of the five negative samples and prepare different negative sample solutions according to the test sample solution preparation method.

[0102] 2.5 Preparation of mixed reference solution

[0103] Take appropriate amounts of agaritol, dehydroauracene lactone, 11-carbonyl-β-acetylboswellic acid, 3-phenylpropyl cinnamic acid, and cinnamate, and add anhydrous ethanol to prepare a mixed reference solution. The concentrations of agaritol, dehydroauracene lactone, 11-carbonyl-β-acetylboswellic acid, 3-phenylpropyl cinnamic acid, and cinnamate in the mixed reference solution are 2.59 μg / mL, 19.38 μg / mL, 12.62 μg / mL, 139.40 μg / mL, and 122.28 μg / mL, respectively.

[0104] 2.6 Determination Method

[0105] Accurately pipette 10 μl of the reference solution and the test solution into the liquid chromatograph, measure and record the chromatograms.

[0106] The fingerprint chromatogram of the test sample should show chromatographic peaks with the same retention time as the reference peak. According to the similarity evaluation system for chromatographic fingerprint chromatograms of traditional Chinese medicine, the similarity between the fingerprint chromatogram of the test sample and the fingerprint chromatogram of the reference should not be less than 0.90.

[0107] 3. Methodological Examination

[0108] 3.1 Repeatability Experiment

[0109] Six portions of approximately 50 mg each were accurately weighed from the same batch of sample and prepared in parallel according to the method described in section "2.3". The chromatographic conditions described in section "2.1" were then used for determination. Peak 7 (3-phenylpropyl cinnamic acid) was used as the reference peak (S), and the relative retention time and relative peak area of ​​each common peak were calculated. The results showed that the RSD of the relative retention time of each common chromatographic peak ranged from 0.0322% to 1.2756%, and the RSD of the relative peak area ranged from 0.0017% to 0.0469%. The results indicate that the method has good repeatability. The results are shown in Tables 2 and 3.

[0110] Table 2. Relative retention times of common peaks in repeatability experiments

[0111]

[0112] Table 3. Relative peak areas of common peaks in repeatability experiments

[0113]

[0114] 3.2 Stability Test

[0115] Take the same batch of test solution and inject it into the liquid chromatogram at 0, 4, 8, 12, 18, 24, 32, 40, and 48 hours according to the chromatographic conditions in section "2.1". Record the chromatograms. Using peak 7 (3-phenylpropyl cinnamic acid) as the reference peak (S), calculate the relative retention time and relative peak area of ​​each common peak. The results show that the relative retention time RSD of each common chromatographic peak is 0.0076%-0.2016%, and the relative peak area RSD is 0.1177%-1.8588%. The results indicate that the test solution is stable within 48 hours. The results are shown in Tables 4 and 5.

[0116] Table 4. Relative retention times of common peaks in stability experiments

[0117]

[0118] Table 5. Relative peak areas of common peaks in stability experiments

[0119]

[0120] 3.3 Durability Assessment

[0121] Evaluation of different chromatographic columns: Two chromatographic columns from different batches were tested in the experiment. Column 1: Mid-Spectrum Red C18 (250mm × 4.6mm × 5μm), serial number (SN): 018031691; Column 2: Mid-Spectrum Red C18 (250mm × 4.6mm × 5μm), serial number (SN): 018031690. The results showed that all columns had good column efficiency, symmetrical peak shapes of the target chromatographic peaks, and good separation effects. The results are shown in the figure. Figure 1 Where A represents SN: 018031691; B represents SN: 018031690.

[0122] 3.4 Intermediate Precision

[0123] 3.4.1 Assessment of different personnel

[0124] Two different individuals were examined. The results showed little difference in their test results. (See attached results.) Figure 2 .

[0125] 4. Confirmation and attribution of common peaks

[0126] 4.1 Confirmation of common peaks

[0127] For 10 batches of Tongqiao Jiuxin Oil samples, test solutions were prepared according to section "2.3", and the chromatographic conditions in section "2.1" were used for determination. Chromatograms were recorded, and 10 common peaks were identified by combining the characteristic peaks of the reference medicinal material. (See section "2.1"). Figure 3 There are 10 common peaks: peak 1 is agaritol, peak 4 is dehydroauric acid lactone, peak 6 is cinnamyl cinnamate, peak 7 is 3-phenylpropyl cinnamate, and peak 10 is 11-carbonyl-β-acetylmonic acid.

[0128] 4.2 Assignment of common peaks

[0129] 4.2.1 Classification of medicinal materials

[0130] Take the negative sample solutions from section "2.4" and the test solution from section "2.3", and inject them sequentially according to the chromatographic conditions in section "2.1", recording the chromatograms. Based on the retention time information of the chromatographic peaks, assign the common peaks in the chromatograms to the medicinal materials. Results: Of the 10 common peaks: peaks 1 and 3 belong to agarwood; peaks 2, 5, 6, and 7 belong to styrax; peak 4 belongs to costus root; peak 8 belongs to sandalwood; and peaks 9 and 10 belong to frankincense. See details... Figure 4 Among them, S1: Tongqiao Jiuxin Oil; S2: Styrax; S3: Costus Root; S4: Frankincense; S5: Sandalwood; S6: Agarwood.

[0131] 4.2.2 Classification of Chemical Components

[0132] Take the mixed reference solution from section "2.5" and the test solution from section "2.3" respectively, and inject them sequentially according to the chromatographic conditions from section "2.1," recording the chromatograms. Assign the chemical components to the common peaks in the chromatograms based on their retention times. Results: Peak 1 is linalool, Peak 4 is dehydroauracene lactone, Peak 6 is cinnamyl cinnamate, Peak 7 is 3-phenylpropyl cinnamate, and Peak 10 is 11-carbonyl-β-acetylbric acid. See details... Figure 5 Among them, S1: Tongqiao Jiuxin Oil; M2: 11-carbonyl-β-acetylbosuccinic acid; M3: agaric tetraol; M4: dehydroauracene lactone; M5: cinnamic acid cinnamate; M6: cinnamic acid-3-phenylpropyl ester.

[0133] Example 2

[0134] 1. Experimental materials

[0135] 1.1 Instruments

[0136] Agilent 1260 high-performance liquid chromatograph; PX36 / A electronic balance (Mettler Toledo Technologies (China) Co., Ltd.); SECURA225D-1CN electronic balance (Sartorius Group); Zhongpu Red C18 (250mm×4.6mm×5μm) liquid chromatography column (Zhongpu Technology (Fuzhou) Co., Ltd.).

[0137] 1.2 Drug Testing

[0138] Anhydrous ethanol (chromatographic grade).

[0139] 1.3 Standard Products

[0140] Agaric tetraol (batch number: 11980-201904), dehydroauracene lactone (batch number: 11525-202313), and 11-carbonyl-β-acetylbric acid (batch number: 11760-202103) were all purchased from the National Institutes for Food and Drug Control. 3-Phenylacetic acid ester (batch number: C16286696) was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0141] 1.4 Sample

[0142] Tongqiao Jiuxin Oil, batch number UT30002, was purchased from Guangzhou Baiyunshan Xingqun (Pharmaceutical) Co., Ltd.

[0143] 2. Experimental methods and results

[0144] 2.1 Chromatographic conditions

[0145] The content of components in Tongqiao Jiuxin Oil was determined by high performance liquid chromatography (General Chapter 0512, Chinese Pharmacopoeia 2025).

[0146] Octadecylsilane-bonded silica gel was used as the packing material; acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B. The flow rate was 1.0 mL / min, and gradient elution was performed according to the specifications in Table 1; the detection wavelength was 225 nm; the column temperature was 30 °C; and the theoretical plate number, calculated based on the cinnamic acid-3-phenylpropyl ester peak, should not be less than 5000.

[0147] 2.2 Preparation of mixed reference solution

[0148] Take appropriate amounts of agaric tetraol, dehydroauracene lactone, 3-phenylpropyl cinnamic acid, and 11-carbonyl-β-acetylbosuccinic acid reference standards, accurately weigh them, and add anhydrous ethanol to prepare solutions containing 50 μg, 100 μg, 1000 μg, and 100 μg per ml, respectively, as mother liquor. Further dilute the mother liquor to prepare mixed reference standard solutions with concentrations of 2 μg, 20 μg, 100 μg, and 10 μg per ml, respectively, to obtain the final product.

[0149] 2.3 Preparation of the test solution

[0150] Accurately weigh 50mg of Tongqiao Jiuxin Oil, place it in a 5ml volumetric flask, and add anhydrous ethanol to the mark.

[0151] 2.4 Determination Methods

[0152] Accurately pipette 10 μl of the mixed reference solution and the test solution separately, inject them into the liquid chromatograph, determine the concentration, and record the chromatogram. The results are as follows: Figure 6 As shown. Among them, peak 1: agaritol, peak 4: dehydroauric acid lactone, peak 7: cinnamic acid-3-phenylpropyl ester, peak 10: 11-carbonyl-β-acetylmethane.

[0153] 3. Methodological Examination

[0154] 3.1 Repeatability Experiment

[0155] Six portions of approximately 50 mg each from the same batch of samples were accurately weighed and prepared in parallel according to the method described in section "2.3". The chromatographic determination was performed under the conditions described in section "2.1", and the contents were calculated. The average content of agaric tetraol was 0.026%, with an RSD of 0.58% (n=6); the average content of dehydroauracene lactone was 0.147%, with an RSD of 0.45% (n=6); the average content of 3-phenylpropyl cinnamic acid was 0.961%, with an RSD of 0.36% (n=6); and the average content of 11-carbonyl-β-acetylbosuccinic acid was 0.122%, with an RSD of 0.62% (n=6), indicating that the method has good precision. Specific results are shown in Table 6.

[0156] Table 6 Repeatability Test Results

[0157]

[0158] 3.2 Accuracy Test

[0159] Mixed reference stock solution: Take 2 mL, 5 mL, 3 mL, and 3 mL of the reference stock solutions of agaritol, dehydroauracene lactone, 3-phenylpropyl cinnamic acid, and 11-carbonyl-β-acetylbosuccinic acid, respectively, and add them to a 25 mL volumetric flask to make up to volume to prepare a mixed reference stock solution. The final actual concentrations of agaritol, dehydroauracene lactone, 3-phenylpropyl cinnamic acid, and 11-carbonyl-β-acetylbosuccinic acid are 4.15 μg / mL, 19.91 μg / mL, 148.33 μg / mL, and 15.14 μg / mL, respectively.

[0160] Preparation of the sample recovery solution: Take 9 portions of the same batch of sample with known content, each approximately 25 mg, accurately weigh them, and accurately add 1 ml, 2 ml, and 3 ml of the mixed reference standard stock solution (repeat three times for each concentration), and determine the content according to the proposed content determination method.

[0161] Results of the spiking recovery test: As shown in Tables 7-10, the recovery rates of agaric tetraol, dehydroauracene lactone, cinnamic acid-3-phenylpropyl ester, cinnamic acid-3-phenylpropyl ester, and 11-carbonyl-β-acetylboronic acid were 98.09% and 2.33% (n=9), respectively. The recovery rates were 100.59% and 2.89% (n=9). The spiking recovery test results met the requirements.

[0162] Table 7. Recovery results of agaric tetraol.

[0163]

[0164] Table 8. Recovery results of dehydroaucklandia lactones.

[0165]

[0166] Table 9. Recovery results of 3-phenylpropyl cinnamic acid.

[0167]

[0168] Table 10 Recovery results of 11-carbonyl-β-acetylboronic acid

[0169]

[0170] 3.3 Examination of Linear Relationships

[0171] Mixed reference stock solution: Take 1 mL, 4 mL, 2 mL, and 2 mL of the reference stock solutions of agaritol, dehydroauracene lactone, 3-phenylpropyl cinnamic acid, and 11-carbonyl-β-acetylbosuccinic acid, respectively, and add them to a 10 mL volumetric flask to make up to volume to prepare a mixed reference stock solution. The final actual concentrations of agaritol, dehydroauracene lactone, 3-phenylpropyl cinnamic acid, and 11-carbonyl-β-acetylbosuccinic acid are 5.186 μg / mL, 38.768 μg / mL, 278.810 μg / mL, and 25.240 μg / mL, respectively.

[0172] Dilute with anhydrous ethanol to prepare reference solutions containing 0.519, 1.037, 2.074, 3.112, 3.889, and 5.186 μg of agaritol; 3.877, 7.754, 15.507, 23.261, 29.076, and 38.768 μg of dehydroauracene lactone; 27.881, 55.762, 111.524, 167.286, 209.107, and 278.810 μg of 3-phenylpropyl cinnamic acid; and 2.524, 5.048, 10.096, 15.144, 18.930, and 25.24 μg of 11-carbonyl-β-acetylbric acid per 1 ml. According to the proposed content determination method, inject 10 μl of each of the above reference solutions into the liquid chromatograph and record the chromatogram.

[0173] Standard curves were plotted with injection volume (ng) on ​​the x-axis and peak area on the y-axis. The regression equations for agaric tetraol, dehydroauracene lactone, and cinnamic acid-3-phenylpropyl ester were obtained: Y = 1589.7X - 742.74, with a correlation coefficient r = 0.9999; Y = 1301.2X + 280.75, with a correlation coefficient r = 1; Y = 1807.6X + 24408, with a correlation coefficient r = 1; and Y = 450.55X - 2775.3, with a correlation coefficient r = 1. The results are shown in Table 11. Figures 7-10 .

[0174] The results showed that agaric tetraol had a good linear relationship with the peak area ratio in the range of 5.19 ng to 51.86 ng, dehydroauracene lactone in the range of 38.77 ng to 387.68 ng, cinnamic acid-3-phenylpropyl ester in the range of 278.81 ng to 2788.10 ng, and 11-carbonyl-β-acetylbosuccinic acid in the range of 25.24 ng to 252.40 ng.

[0175] Table 11 Results of Linear Experiments

[0176]

[0177] 3.4 Precision Test

[0178] Mixed reference stock solution: Take 1 mL, 4 mL, 2 mL, and 2 mL of the reference stock solutions of agaritol, dehydroauracene lactone, 3-phenylpropyl cinnamic acid, and 11-carbonyl-β-acetylbosuccinic acid, respectively, and add them to a 10 mL volumetric flask to make up to volume to prepare a mixed reference stock solution. The final actual concentrations of agaritol, dehydroauracene lactone, 3-phenylpropyl cinnamic acid, and 11-carbonyl-β-acetylbosuccinic acid are 2.59 μg / mL, 19.38 μg / mL, 139.40 μg / mL, and 12.62 μg / mL, respectively.

[0179] The above-mentioned mixed reference standard stock solution was injected six times consecutively according to the proposed content determination method. The peak area of ​​each component was recorded, and the RSD value of the peak area was calculated. The results showed that the average peak area of ​​agaric tetraol was 4146.33, with an RSD value of 0.55%; the average peak area of ​​dehydroauracene lactone was 253262.33, with an RSD value of 0.09%; the average peak area of ​​cinnamic acid-3-phenylpropyl ester was 2433358.83, with an RSD value of 0.12%; and the average peak area of ​​11-carbonyl-β-acetylbosuccinic acid was 54027.83, with an RSD value of 0.72%. This indicates that the instrument has good precision. The results are shown in Table 12.

[0180] Table 12 Precision Test Results

[0181]

[0182] 3.5 Intermediate Precision

[0183] The same batch of test samples was taken and measured by two individuals, A and B, at different times using the same instrument according to the assay method. The peak areas of each group of chromatographic peaks were recorded, and the RSD values ​​of the four components were calculated. The results showed little difference between the measurements taken by different operators, indicating that the intermediate precision of this method is good. The results are shown in Table 13.

[0184] Table 13 Results of intermediate precision test

[0185]

[0186] 4. Durability test

[0187] 4.1 Stability Test

[0188] Accurately weigh the same batch of test solutions and inject them at 0h, 4h, 8h, 12h, 18h, 24h, 32h, 40h, and 48h after preparation. Record the peak areas of each group and calculate the RSD values ​​of the peak areas. The results show that the RSD value of the peak area of ​​agaric tetraol is 0.61%; the RSD value of the peak area of ​​dehydroauracene lactone is 1.15%; the RSD value of the peak area of ​​cinnamic acid-3-phenylpropyl ester is 0.14%; and the RSD value of the peak area of ​​11-carbonyl-β-acetylbosuccinic acid is 0.62%. This indicates that the test solutions have good stability after being stored for 48 hours after preparation. The results are shown in Table 14.

[0189] Table 14 Stability Test Results

[0190]

[0191] 4.2 Investigation at different flow velocities

[0192] The content determination method in the proposed standard was followed, and different flow rates were adjusted for detection. The results showed that the flow rate between 0.9 and 1.1 ml / min had little impact on the determination results, and the separation was good in all cases. The results are shown in Table 15.

[0193] Table 15 Test results at different flow rates

[0194]

[0195] 4.3 Chromatographic column investigation

[0196] According to the content determination method in the proposed standard, the content was detected using two chromatographic columns: Column 1: Mid-Spectrum Red C18 column (250mm×4.6mm×5μm), serial number (SN): 018031690; Column 2: Mid-Spectrum Red C18 column (250mm×4.6mm×5μm), serial number (SN): 018031691. The results showed no significant difference, as shown in Table 16.

[0197] Table 16 Results of experiments with different chromatographic columns

[0198]

[0199] Example 3

[0200] 1. Investigation of different detection wavelengths

[0201] Because the target components agaric tetraol, dehydroauracene lactone, 3-phenylpropyl cinnamic acid, and 11-carbonyl-β-acetylbosuccinic acid have different ultraviolet absorption characteristics, and there is no reference standard for sandalwood as specified in the pharmacopoeia, it is necessary to consider the response values ​​of chromatographic peaks at different wavelengths to reflect the characteristics of different medicinal materials as much as possible.

[0202] Preliminary experiments showed that agaric tetraol had strong absorption at the end and at a wavelength of 250-255 nm, dehydroaurolide had strong absorption at the end, cinnamic acid-3-phenylpropyl ester had strong absorption at a wavelength of 255-270 nm, and 11-carbonyl-β-acetylbosuccinic acid had strong absorption at a wavelength of 250-255 nm; the chromatographic peak of sandalwood had strong absorption at the end.

[0203] Taking all factors into consideration, a detection wavelength of 212 nm was selected for fingerprint spectroscopy research, which can characterize the characteristic peaks of agarwood, costus root, styrax, sandalwood, and frankincense. A detection wavelength of 225 nm was selected for content determination research, which can ensure that the peak shapes and resolution of the four components, agaritol, dehydrocostane lactone, cinnamic acid-3-phenylpropyl ester, and 11-carbonyl-β-acetylbosperidin, meet the quantitative requirements.

[0204] The results were compared at wavelengths of 254nm and 205nm, such as Figures 11-12 As shown. Figure 11 The chromatographic peaks are at a wavelength of 254 nm. Among them, peak 1 is agaritol, peak 7 is 3-phenylpropanoic acid, and peak 10 is 11-carbonyl-β-acetylbosuccinic acid. The chromatographic peaks corresponding to agaritol, 3-phenylpropanoic acid, and 11-carbonyl-β-acetylbosuccinic acid have better chromatographic effects, but the chromatographic peaks of dehydrocostane lactone and sandalwood have no response. Figure 12 The chromatographic peaks at a wavelength of 205 nm are: peak 1 is agaritol, peak 4 is dehydroauric acid lactone, peak 7 is cinnamic acid-3-phenylpropanoate, and peak 10 is 11-carbonyl-β-acetylmascarboxylic acid. The chromatographic peaks corresponding to dehydroauric acid lactone and cinnamic acid-3-phenylpropanoate show better chromatographic effects, while the separation of agaritol and 11-carbonyl-β-acetylmascarboxylic acid is poor.

[0205] 2. Investigation of different elution gradients

[0206] This study initially investigated various elution gradients. This section only presents a few representative gradients and summarizes their separation effects. In elution gradient A (Table 17), the separation of agaritol and dehydroausne lactone was poor, and 11-carbonyl-β-acetylbosinic acid eluted slowly. In elution gradient B (Table 18), the separation of agaritol and dehydroausne lactone was poor, with weak elution after 82 min, and 11-carbonyl-β-acetylbosinic acid did not elute. In elution gradient C (Table 19), the separation of dehydroausne lactone and 11-carbonyl-β-acetylbosinic acid was poor. Details are as follows... Figures 13-15 As shown in the figure. Among them, peak 1 is agaritol, peak 4 is dehydroauric acid lactone, peak 7 is 3-phenylpropyl cinnamic acid, and peak 10 is 11-carbonyl-β-acetylmethane.

[0207] Table 17 Elution Gradient A

[0208]

[0209] Table 18 Elution Gradient B

[0210]

[0211] Table 19 Elution gradient C

[0212]

[0213] 3. Examination of the elution system

[0214] This study further investigated the concentration of phosphoric acid and the choice of organic phase (acetonitrile and methanol). The results showed that, under the same elution gradient (Table 1), using methanol as the organic phase significantly reduced elution capacity and worsened separation efficiency. Figure 16 Therefore, acetonitrile is preferred as the organic phase; high concentration 0.2% phosphoric acid ( Figure 17 There is no significant difference between the commonly used concentration of 0.1% phosphoric acid and the 0.1% phosphoric acid. Considering column protection, 0.1% phosphoric acid is preferred.

[0215] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for establishing a fingerprint spectrum of a heart-clearing and heart-saving oil, characterized in that, Liquid chromatography was used with acetonitrile as mobile phase A and 0.1-0.2% phosphoric acid solution as mobile phase B. The gradient elution program was as follows: 0-5 min, mobile phase A X1%→X1%, mobile phase B Y1%→Y1%; 5-16 min, mobile phase A X1%→X2%, mobile phase B Y1%→Y2%; 16-44 min, mobile phase A X2%→X3%, mobile phase B Y2%→Y3%; 44-52 min, mobile phase A X3%→X3%, mobile phase B Y3%→Y3%; 52-75 min, mobile phase A X3%→X4%, mobile phase B Y3%→Y4%; 75-88 min, mobile phase A X4%→X5%, mobile phase BY4%→Y5%; 88-115 min, mobile phase A X5%→X5%, mobile phase B Y5%→Y5%. Among them, X1=15-17%, X2=18-20%, X3=52-54%, X4=58-60%, X5=77-79%; Y1=83-85%, Y2=80-82%, Y3=46-48%, Y4=40-42%, Y5=21-23%; Octadecylsilane-bonded silica gel is used as a filler; The preparation of the test solution includes: accurately weighing Tongqiao Jiuxin Oil, adding anhydrous ethanol to prepare a solution containing 5-10 mg of Tongqiao Jiuxin Oil per 1 ml; The preparation of the reference solution includes: taking agaric tetraol, dehydroauracene lactone, 11-carbonyl-β-acetylsalicylic acid, 3-phenylpropyl cinnamic acid and cinnamic acid ester, and adding anhydrous ethanol to prepare the reference solution.

2. The method for establishing according to claim 1, characterized in that, In the liquid chromatography method, and / or the detection wavelength is 210-230 nm; and / or column temperature of 30-40℃; And / or the theoretical plate number, calculated based on the 3-phenylpropyl cinnamic acid peak, should be no less than 5000; And / or the flow rate is 0.9-1.1 ml / min; The following gradient elution program can be used: 0–5 min, mobile phase A 16%→16%, mobile phase B 84%→84%; 5–16 min, mobile phase A 16%→19%, mobile phase B 84%→81%; 16–44 min, mobile phase A 19%→53%, mobile phase B 81%→47%; 44–52 min, mobile phase A 53%→53%, mobile phase B 47%→47%; 52–75 min, mobile phase A 53%→59%, mobile phase B 47%→41%; 75–88 min, mobile phase A 59%→78%, mobile phase B 41%→22%; 88–115 min, mobile phase A 78%→78%, mobile phase B 22%→22%.

3. The method for establishing according to claim 2, characterized in that, The detection wavelength is 212nm.

4. The method for establishing according to claim 1, characterized in that, The method also includes the preparation of a reference solution and a negative sample solution.

5. The method for establishing according to claim 4, characterized in that, The preparation of the reference solution includes: accurately weighing cinnamic acid-3-phenylpropyl ester reference standard, adding anhydrous ethanol to prepare a solution containing 50-100 μg of cinnamic acid-3-phenylpropyl ester reference standard per 1 ml; The preparation of the negative sample solution includes: taking different negative samples and preparing different negative sample solutions according to the preparation method of the test sample solution; the negative samples are prepared according to the preparation method of Tongqiao Jiuxin Oil, respectively, for the absence of styrax, frankincense, sandalwood, agarwood and costus root.

6. The method for establishing according to claim 4, characterized in that, The method further includes detecting the reference solution, test solution, negative sample solution and control solution using the liquid chromatography method to obtain a fingerprint spectrum.

7. A method for detecting the content of components in a heart-clearing and mind-saving oil, characterized in that, Liquid chromatography was used with acetonitrile as mobile phase A and 0.1-0.2% phosphoric acid solution as mobile phase B. The gradient elution program was as follows: 0-5 min, mobile phase A X1%→X1%, mobile phase B Y1%→Y1%; 5-16 min, mobile phase A X1%→X2%, mobile phase B Y1%→Y2%; 16-44 min, mobile phase A X2%→X3%, mobile phase B Y2%→Y3%; 44-52 min, mobile phase A X3%→X3%, mobile phase B Y3%→Y3%; 52-75 min, mobile phase A X3%→X4%, mobile phase B Y3%→Y4%; 75-88 min, mobile phase A X4%→X5%, mobile phase BY4%→Y5%; 88-115 min, mobile phase A X5%→X5%, mobile phase B Y5%→Y5%. Among them, X1=15-17%, X2=18-20%, X3=52-54%, X4=58-60%, X5=77-79%; Y1=83-85%, Y2=80-82%, Y3=46-48%, Y4=40-42%, Y5=21-23%; The ingredients include agaritol, dehydroaurate lactone, 3-phenylpropyl cinnamic acid and 11-carbonyl-β-acetylammonic acid; Octadecylsilane-bonded silica gel is used as a filler; The detection method also includes the preparation of a mixed reference solution: accurately weigh agaric tetraol, dehydroauracene lactone, 3-phenylpropyl cinnamic acid and 11-carbonyl-β-acetylbosuccinic acid reference standards, add anhydrous ethanol to prepare a mixed reference solution, and the solution is obtained. Preparation of the test solution: Take Tongqiao Jiuxin Oil, accurately weigh it, add anhydrous ethanol to prepare a solution containing 5-10 mg of Tongqiao Jiuxin Oil per 1 ml.

8. The detection method according to claim 7, characterized in that, In the liquid chromatography method, the detection wavelength is 210-230 nm; and / or column temperature of 30-40℃; And / or the theoretical plate number, calculated based on the 3-phenylpropyl cinnamic acid peak, should be no less than 5000; And / or the flow rate is 0.9-1.1 ml / min; The following gradient elution program can be used: 0–5 min, mobile phase A 16%→16%, mobile phase B 84%→84%; 5–16 min, mobile phase A 16%→19%, mobile phase B 84%→81%; 16–44 min, mobile phase A 19%→53%, mobile phase B 81%→47%; 44–52 min, mobile phase A 53%→53%, mobile phase B 47%→47%; 52–75 min, mobile phase A 53%→59%, mobile phase B 47%→41%; 75–88 min, mobile phase A 59%→78%, mobile phase B 41%→22%; 88–115 min, mobile phase A 78%→78%, mobile phase B 22%→22%.

9. The detection method according to claim 8, characterized in that, The detection wavelength is 225nm.

10. The detection method according to claim 9, characterized in that, The detection method further includes using the liquid chromatography method to detect the test solution and the mixed reference solution to obtain the contents of agaritol, dehydroauracene lactone, 3-phenylpropyl cinnamic acid and 11-carbonyl-β-acetylbosuccinic acid.

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