A method for constructing and identifying HPLC characteristic map of gum myrrh or preparation thereof

Abstract

The present application relates to a kind of gummy myrrh or the construction method of HPLC characteristic map of its preparation.The present application is directed to gummy myrrh characteristic and points out linalool, and determines six characteristic peaks, provides sufficient basis for the quality control of gummy myrrh, ensures that gummy myrrh medicinal material, decoction piece and its standard decoction quality are uniform, stable.

Description

Technical Field

[0001] This invention relates to a method for constructing HPLC characteristic chromatograms of myrrh or its preparations. Background Technology

[0002] Myrrh (gum myrrh) is the dried resin of *Commiphora myrrha* Engl. or *Commiphora molmol* Engl., plants of the Burseraceae family. It has the effects of dispersing blood stasis, relieving pain, reducing swelling, and promoting tissue regeneration. It is used to treat chest pain, stomach pain, dysmenorrhea, amenorrhea, postpartum blood stasis, abdominal masses, rheumatic pain, traumatic injuries, carbuncles, and sores. The standard decoction is a freeze-dried powder made from the medicinal materials according to a fixed preparation process.

[0003] Natural myrrh is the dried resin of the *Viola yedoensis* tree (family Burseraceae). Its main chemical components include furanocyanindiene, turmericene, bicyclosporine, β-juniperene, and β-oligone. Myrrh gum is the dried resin of the *Viola yedoensis* tree (family Burseraceae). Its main chemical components include α-trans-bergamotene, α-santalene-α-cis-bergamotene, trans-β-farnesene, trans-β-juniperene, α-bisabolene, β-bisabolene, trans-γ-bisabolene, cis-γ-bisabolene, and β-santalaldehyde. (Zhang Wenting, et al., HPLC Characteristic Chromatography Study of Natural and Myrrh Gum, *Chinese Journal of Modern Applied Pharmacy*, February 2019, Vol. 36, No. 3). Both natural and myrrh gum are irregular clumps or granules, with similar colors (brownish-yellow to brownish-red) and no significant difference in size. This literature studied natural myrrh and mucilage myrrh, but the method developed only confirmed two characteristic peaks for mucilage myrrh and no peak components were identified, which could not meet the detection requirements.

[0004] Regarding quality testing methods for myrrh, there are currently relevant literature reports. For example, Li Xia et al., "Study on Fingerprint Spectra of Myrrh from Different Origins," *Traditional Chinese Medicine Guide*, April 2019, Vol. 25, No. 7, established the HPLC characteristic chromatogram of myrrh. Studies on the fingerprint chromatogram of natural myrrh, although also detecting mucilage myrrh, show that their fingerprint chromatographic method for mucilage myrrh basically does not produce characteristic peaks (see this literature). Figure 3 ).

[0005] Yan Ganming, et al., Study on the analysis and HPLC content determination of sesquiterpenoid components in myrrh and its processed products based on UPLC-Q / TOF-MS technology, Chinese Pharmaceutical Journal, September 2023, Vol. 58, No. 17. This article only mentions that myrrh samples were used for testing, without indicating whether they were natural myrrh or mucilage myrrh.

[0006] Application No. 202210312708.5, Invention Title: A Quality Detection Method for Vinegar-Myrrh Standard Decoction, provides a method for quality detection of vinegar-myrrh standard decoction, including determination of the decoction's properties, dry extract yield, thin-layer chromatography identification, extracts, characteristic chromatograms, and curcumin content. This literature focuses on the study of processed myrrh products; although colloidal myrrh is used as a reference material, no characteristic peaks were observed. The Guangdong Provincial Standard for Quality of Traditional Chinese Medicine Formula Granules, Vinegar-Myrrh (Natural Myrrh) Formula Granules, Guangdong PFKL20230007, also discloses the characteristic chromatogram of processed natural myrrh product, vinegar-myrrh formula granules. Current HPLC characteristic chromatogram methods are mostly for natural myrrh and are not perfect for separating substances from colloidal myrrh. Bao Sheng, Research on Chemical Composition and Quality Evaluation Methods of Natural Myrrh, Master's Thesis, Nanjing University of Chinese Medicine, June 2021. This paper studies the fingerprint spectrum of natural myrrh, but the nine characteristic peaks identified by its fingerprint spectrum method for myrrh are not found in myrrh. This indicates that the method is only applicable to natural myrrh and not to myrrh. Yang Bao, Research on Chemical Composition of Myrrh, Jiangxi University of Chinese Medicine, June 2023. Although the study on the chemical composition of myrrh mentions the presence of camphor in the volatile oil of myrrh, existing HPLC / UPLC characteristic spectrum construction methods do not identify and separate it. In summary, the characteristic spectra of myrrh and natural myrrh differ significantly. Current HPLC / UPLC characteristic spectrum methods are mostly for natural myrrh and are not perfect for the separation of substances in myrrh. Summary of the Invention

[0007] This invention provides a method for constructing and identifying HPLC characteristic chromatograms of myrrh or its preparations.

[0008] This invention provides a method for constructing HPLC characteristic chromatograms of myrrh or its preparations, comprising the following steps:

[0009] a. Preparation of the test solution: Dissolve and extract the raw material of the test sample using a solvent to obtain the test solution;

[0010] b. The test solution is determined by high performance liquid chromatography to obtain a high performance liquid chromatogram of myrrh or its preparation;

[0011] The chromatographic conditions for the high-performance liquid chromatography (HPLC) method are as follows: a C18 column, methanol as mobile phase A, water as mobile phase B, gradient elution, and the elution conditions are as follows:

[0012]

[0013] The preparation method of the test sample solution is as follows: take the test sample, add methanol aqueous solution, sonicate, cool, shake well, filter, and take the filtrate to obtain the solution.

[0014] The methanol concentration is 70%, and the ultrasonic treatment conditions are: power 600W, frequency 40kHz, and treatment time 30 minutes.

[0015] It also includes the preparation of a reference solution, wherein the reference is myrrh reference material and linalool reference standard;

[0016] The preparation method of linalool reference standard is as follows:

[0017] Take an appropriate amount of linalool reference standard, accurately weigh it, and add methanol aqueous solution to prepare a reference standard solution;

[0018] The preparation method of the reference solution is as follows: take myrrh, add methanol aqueous solution, sonicate, cool, shake well, filter, and take the filtrate to obtain the reference solution.

[0019] The C18 column has a length of 150 mm, an inner diameter of 2.1 mm, and a particle size of 1.8 μm.

[0020] The high-performance liquid chromatography (HPLC) uses a flow rate of 0.2 ml / min, a column temperature of 30°C, and a detection wavelength of 300 nm. The theoretical plate number, calculated based on the linalool peak, should be no less than 10,000.

[0021] It uses the aforementioned quality detection method to establish a characteristic chromatogram, which contains 6 common chromatographic peaks with relative retention times of: peak 1: 0.60, peak 2: 0.81, peak 4: 1.13, peak 5: 1.35, and peak 6: 1.40, with relative retention times within ±10%.

[0022] Peak 3 is linalool.

[0023] The aforementioned gelatinous myrrh or its preparations refer to gelatinous myrrh medicinal materials, processed slices, standard decoctions, and their formulated granules.

[0024] This invention provides a method for identifying myrrh or its preparations, comprising the following steps:

[0025] a. Pre-treatment of the sample to be tested;

[0026] b. Construct the feature map according to the construction method described above;

[0027] c. Characteristic peak comparison: If identical characteristic peaks appear in peaks 1-6, then it is myrrh.

[0028] This invention identifies linalool based on the characteristics of myrrh and determines six characteristic peaks, providing sufficient basis for the quality control of myrrh and ensuring the uniformity and stability of the quality of myrrh raw materials, processed slices and standard decoctions. Attached Figure Description

[0029] Figure 1 Characteristic chromatogram of standard decoction (peak 3(S): linalool);

[0030] Figure 2 Characteristic spectrum of myrrh (gum myrrh) medicinal material (peak 3(S): linalool);

[0031] Figure 3 Characteristic spectrum of myrrh (colloidal myrrh) formulation granules (peak 3(S): linalool);

[0032] Figure 4 Ultraviolet absorption spectrum of myrrh (colloidal myrrh) formulation granules;

[0033] Figure 5 Column temperature investigation;

[0034] Figure 6 Flow velocity study;

[0035] Figure 7 Investigation of different injection volumes;

[0036] Figure 8 Delayed investigation;

[0037] Figure 9 Extraction methods were examined;

[0038] Figure 10 Extraction solvent investigation;

[0039] Figure 11 Extraction time consideration;

[0040] Figure 12 Spectral peak identification;

[0041] Figure 13 Linalool spectrum - reference standard;

[0042] Figure 14 Linalool spectrum - standard decoction;

[0043] Figure 15 Intermediate precision assessment;

[0044] Figure 16 Instrument durability test;

[0045] Figure 17 Durability study of chromatographic column for myrrh (collagenous myrrh) standard decoction;

[0046] Figure 18 Characteristic spectrum of standard decoction of myrrh (collagenous myrrh) (peak 3 (S): linalool, S1 to S16 are 16 batches of standard decoction respectively);

[0047] Figure 19Reference characteristic chromatogram (peak 3(S): linalool; column: ZORBAX SB C18, 2.1 mm × 150 mm, 1.8 μm);

[0048] Figure 20 Extraction methods were examined;

[0049] Figure 21 Extraction solvent investigation;

[0050] Figure 22 Extraction time consideration;

[0051] Figure 23 Chromatographic peak identification;

[0052] Figure 24 Linalool spectrum - reference standard;

[0053] Figure 25 Linalool spectrum - medicinal materials;

[0054] Figure 26 Intermediate precision assessment;

[0055] Figure 27 Instrument durability test;

[0056] Figure 28 Column durability study;

[0057] Figure 29 Characteristic spectrum of myrrh (gum myrrh) (peak 3 (S): linalool, S1: control material, S2 to S17: 16 batches of materials respectively);

[0058] Figure 30 Characteristic chromatogram of myrrh (gum myrrh) (peak 3(S): linalool, column: ZORBAX SBC18, 2.1mm×150mm, 1.8μm);

[0059] Figure 31 Chromatograms of myrrh (colloidal myrrh) formulation granules at different wavelengths;

[0060] Figure 32 Column temperature investigation;

[0061] Figure 33 Flow velocity study;

[0062] Figure 34 Investigation of different injection volumes;

[0063] Figure 35 Delayed investigation;

[0064] Figure 36 Extraction methods were examined;

[0065] Figure 37 Extraction solvent investigation;

[0066] Figure 38 Extraction time consideration;

[0067] Figure 39 Sample weighing and quantity determination;

[0068] Figure 40 Chromatographic peak identification;

[0069] Figure 41 Linalool spectrum - reference standard;

[0070] Figure 42 Linalool spectrum - formulation granules;

[0071] Figure 43 Intermediate precision assessment;

[0072] Figure 44 Column durability study;

[0073] Figure 45 Instrument durability test;

[0074] Figure 46 Characteristic spectrum of myrrh (colloidal myrrh) formulation granules (S1~S3: three batches of formulation granules);

[0075] Figure 47 Characteristic chromatogram of myrrh (colloidal myrrh) formulation granules (peak 3(S): linalool, column: ZORBAX SBC18, 2.1mm×150mm, 1.8μm);

[0076] Figure 48 Comparison of colloidal myrrh and natural myrrh (210 / 240nm);

[0077] Figure 49 Comparison of natural myrrh and gum myrrh;

[0078] Figure 50 Characteristic spectrum of quality standards for traditional Chinese medicine formula granules in Guangdong Province. Detailed Implementation

[0079] Example 1: HPLC characteristic chromatogram of myrrh or its preparations

[0080] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 150 mm, inner diameter 2.1 mm, particle size 1.8 μm); methanol as mobile phase A and water as mobile phase B, with gradient elution as specified in the table below; flow rate 0.2 mL / min; column temperature 30 °C; detection wavelength 300 nm. The theoretical plate number, calculated based on the linalool peak, should be no less than 10,000.

[0081]

[0082] Preparation of the reference solution: Take 1.0 g of myrrh (collagenous myrrh) reference material, add 25 ml of 70% methanol, sonicate (600 W power, 40 kHz frequency) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution. Separately, take an appropriate amount of linalool reference standard, accurately weigh it, and add 70% methanol to prepare a solution containing 2.5 mg per ml, as the reference solution.

[0083] Preparation of the test solution: Take 0.3g of myrrh (collagenous myrrh) standard decoction powder, add 25ml of 70% methanol, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0084] The determination method involves precisely pipetting 3 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0085] The chromatogram of the test sample should show six characteristic peaks, and the retention times should correspond to the six characteristic peaks in the chromatogram of the reference medicinal material. Peak 3 should correspond to the retention time of the reference material, and the peak corresponding to the linalool reference material is the S peak. Calculate the relative retention times of each characteristic peak and the S peak; the relative retention times should be within ±10% of the specified values. The specified values ​​are 0.60 (peak 1), 0.81 (peak 2), 1.13 (peak 4), 1.35 (peak 5), and 1.40 (peak 6). (See...) Figure 1 )

[0086] HPLC characteristic chromatographic methods for myrrh (gum myrrh) medicinal materials and processed slices:

[0087] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 150 mm, inner diameter 2.1 mm, particle size 1.8 μm); methanol as mobile phase A and water as mobile phase B, with gradient elution as specified in the table below; flow rate 0.2 mL / min; column temperature 30 °C; detection wavelength 300 nm. The theoretical plate number, calculated based on the linalool peak, should be no less than 10,000.

[0088]

[0089] Preparation of the reference solution: Take 1.0 g of myrrh (collagenous myrrh) reference material, add 25 ml of 70% methanol, sonicate (600 W power, 40 kHz frequency) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution. Separately, take an appropriate amount of linalool reference standard, accurately weigh it, and add 70% methanol to prepare a solution containing 2.5 mg per ml, as the reference solution.

[0090] Preparation of the test solution: Take 1g of myrrh (collagenous myrrh) powder (passed through a No. 3 sieve), add 25ml of 70% methanol, seal tightly, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0091] The determination method involves precisely pipetting 3 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0092] The chromatogram of the test sample should show six characteristic peaks, and the retention times should correspond to the six characteristic peaks in the chromatogram of the reference medicinal material. Peak 3 should correspond to the retention time of the reference material, and the peak corresponding to the linalool reference material is the S peak. Calculate the relative retention times of each characteristic peak and the S peak; the relative retention times should be within ±10% of the specified values. The specified values ​​are 0.60 (peak 1), 0.81 (peak 2), 1.13 (peak 4), 1.35 (peak 5), and 1.40 (peak 6). (See...) Figure 2 )

[0093] HPLC characteristic chromatographic method for myrrh (colloidal myrrh) formulation granules:

[0094] [Characteristic chromatogram] Determined by high performance liquid chromatography (General Chapter 0512, Chinese Pharmacopoeia 2020 Edition).

[0095] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 150 mm, inner diameter 2.1 mm, particle size 1.8 μm); methanol as mobile phase A and water as mobile phase B, with gradient elution as specified in the table below; flow rate 0.2 mL / min; column temperature 30 °C; detection wavelength 300 nm. The theoretical plate number, calculated based on the linalool peak, should be no less than 10,000.

[0096]

[0097] Preparation of the reference solution: Take 1.0 g of myrrh (collagenous myrrh) reference material, add 25 ml of 70% methanol, sonicate (600 W power, 40 kHz frequency) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution. Separately, take an appropriate amount of linalool reference standard, accurately weigh it, and add 70% methanol to prepare a solution containing 2.5 mg per ml, as the reference solution.

[0098] Preparation of the test solution: Take an appropriate amount of myrrh (colloidal myrrh) formulation granules, grind them into a fine powder, take 0.3g, add 25ml of 70% methanol, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0099] The determination method involves precisely pipetting 3 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0100] The chromatogram of the test sample should show six characteristic peaks, and the retention times should correspond to the six characteristic peaks in the chromatogram of the reference medicinal material. Peak 3 should correspond to the retention time of the reference material, and the peak corresponding to the linalool reference material is the S peak. Calculate the relative retention times of each characteristic peak and the S peak; the relative retention times should be within ±10% of the specified values. The specified values ​​are 0.60 (peak 1), 0.81 (peak 2), 1.13 (peak 4), 1.35 (peak 5), and 1.40 (peak 6). (See...) Figure 3 )

[0101] Example 2: Drafting Instructions for the HPLC Characteristic Chromatography of Myrrh (Colloidal Myrrh) Standard Decoction

[0102] 1. Experimental instruments and materials

[0103] Agilent 1290 UHPLC; Waters UHPLC; Thermo Fisher Vanquish F UHPLC;

[0104] Cellular 1810A Ultrapure Water System (Shanghai Moler Scientific Instruments Co., Ltd.);

[0105] KQ-600DB Ultrasonic Cleaner (Kunshan Ultrasonic Instrument Co., Ltd.);

[0106] Methanol was of chromatographic grade, water was ultrapure water, and all other reagents were of analytical grade.

[0107] Chromatographic columns: ZORBAX SB C18 2.1×150mm, 1.8μm; ZORBAX Eclipse Plus C18 2.1×150mm, 1.8μm; ACQUITY HSS T3 2.1×150mm, 1.8μm

[0108] Linalool reference standard (China National Institutes for Food and Drug Control, batch number: 111503-201603, purity: 97.8%);

[0109] Myrrh (gum myrrh) reference material (China National Institutes for Food and Drug Control, batch number: 121250-201806);

[0110] Preparation of myrrh (collagenous myrrh) standard decoction freeze-dried powder (Sichuan Xinlvse Pharmaceutical Technology Development Co., Ltd.): 16 batches of standard decoction freeze-dried powder.

[0111] 2. Proposed chromatographic conditions

[0112] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 150 mm, inner diameter 2.1 mm, particle size 1.8 μm); methanol as mobile phase A and water as mobile phase B, with gradient elution as specified in the table below; flow rate 0.2 mL / min; column temperature 30 °C; detection wavelength 300 nm. The theoretical plate number, calculated based on the linalool peak, should be no less than 10,000.

[0113]

[0114] Preparation of the reference solution: Take 1.0 g of myrrh (collagenous myrrh) reference material, add 25 ml of 70% methanol, sonicate (600 W power, 40 kHz frequency) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution. Separately, take an appropriate amount of linalool reference standard, accurately weigh it, and add 70% methanol to prepare a solution containing 2.5 mg per ml, as the reference solution.

[0115] Preparation of the test solution: Take 0.3g of myrrh (collagenous myrrh) standard decoction powder, add 25ml of 70% methanol, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0116] The determination method involves precisely pipetting 3 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0117] 3. System applicability assessment

[0118] Based on the above-specified experimental conditions, a full-band scan was performed using a diode array detector, and chromatograms of the test solution were extracted at wavelengths of 220 nm, 240 nm, 260 nm, 280 nm, 300 nm, and 320 nm. The results are shown below. Figure 4 .

[0119] The results showed that the chromatographic peak information was greater and the chromatographic baseline was more stable when the detection wavelength was 300 nm, so the detection wavelength was determined to be 300 nm.

[0120] 3.1 Column Temperature Investigation

[0121] Based on the above-planned experimental conditions, the effects were investigated at column temperatures of 25℃, 30℃, and 35℃. Figure 5 As shown in Table 1.

[0122] Table 1. Column Temperature Investigation—Relative Retention Time of Characteristic Peaks

[0123]

[0124] The results showed that the RSD of the relative retention times of each characteristic peak at different column temperatures ranged from 0.00% to 2.50%, indicating that the method is feasible. At a column temperature of 30℃, the chromatographic peaks were all symmetrical, and the resolution met the requirements. Therefore, a column temperature of 30℃ was chosen for further investigation.

[0125] 3.2 Flow velocity investigation

[0126] Based on the above-specified experimental conditions, the flow rates of 0.2 ml / min, 0.3 ml / min, and 0.4 ml / min were investigated respectively. (See attached figures.) Figure 6 Table 2.

[0127] Table 2. Flow velocity investigation—relative retention time of characteristic peaks

[0128]

[0129] The results showed that the RSD of the relative retention time of each characteristic peak at different flow rates ranged from 3.42% to 8.81%, indicating that the method is feasible. At a flow rate of 0.3 ml / min, the peak shapes of each characteristic peak were good; therefore, a flow rate of 0.2 ml / min was selected for further investigation.

[0130] 3.3 Investigation of different injection volumes

[0131] Based on the above-established experimental conditions, the results were investigated for injection volumes of 2 μL, 3 μL, and 4 μL. The results are shown below. Figure 7 .

[0132] The results showed that when the injection volume was 3 μL, the peak shape and separation of each characteristic peak were good. Therefore, according to convention, the injection volume of 3 μL was selected for further investigation.

[0133] 3.4 Delayed Examination

[0134] Based on the experimental conditions outlined above, the data acquisition time was extended to 60 minutes. The results are shown below. Figure 8 .

[0135] The results showed that there were basically no large chromatographic peaks after 30 minutes, and the analysis time for the characteristic chromatographic method of myrrh (colloidal myrrh) formulation particles was finally determined to be 30 minutes.

[0136] In summary, the chromatographic conditions for the characteristic chromatogram of the standard myrrh (collagenous myrrh) decoction were determined as follows: octadecylsilane-bonded silica gel as the packing material (column length 150 mm, inner diameter 2.1 mm, particle size 1.8 μm); methanol as mobile phase A, water as mobile phase B, gradient elution as specified in the table below; flow rate 0.2 ml / min; column temperature 30℃; detection wavelength 300 nm. The theoretical plate number, calculated based on the linalool peak, should be no less than 10,000.

[0137]

[0138] The determination method involves precisely pipetting 3 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0139] Subsequent formulations of granules and medicinal herbs were examined under these chromatographic conditions.

[0140] 4. Investigation into the preparation of the test solution.

[0141] 4.1 Examination of Extraction Methods

[0142] Take 0.3g of myrrh (collagenous myrrh) standard decoction, place it in a stoppered conical flask, add 25ml of 70% methanol, sonicate (600W, 40kHz) and reflux for 30 minutes, cool, shake well, filter, and collect the filtrate. Inject and determine the results according to the above method. See the results below. Figure 9 .

[0143] As can be seen from the figure above, there is no significant difference in the final results between reflux extraction and ultrasonic extraction. Therefore, ultrasonic extraction, which is simpler to operate, was chosen as the extraction method for preparing the test solution in the determination of the characteristic spectrum of myrrh (collagenous myrrh) standard decoction.

[0144] 4.2 Investigation of Extraction Solvents

[0145] Take 0.3g of myrrh (collagenous myrrh) standard decoction and place it in a stoppered conical flask. Add 25ml each of water, 30% methanol, 50% methanol, 80% methanol, methanol, and ethanol. Sonicate the solution (600W, 40kHz) for 30 minutes, cool, shake well, filter, and collect the filtrate. Inject and determine the chromatographic results according to the above conditions. The results are shown in the figure. Figure 10 .

[0146] The results showed that the chromatographic peak shape and resolution were better in the test solution extracted with 70% methanol. Therefore, 70% methanol was selected as the extraction solvent for the characteristic chromatogram of myrrh (collagenous myrrh) standard decoction.

[0147] 4.3 Examination of extraction time

[0148] Take approximately 0.3g of myrrh (collagenous myrrh) standard decoction and place it in a stoppered conical flask. Add 25ml of 70% methanol and sonicate (600W power, 40kHz frequency) for 20, 30, and 40 minutes respectively. Cool, shake well, filter, and collect the filtrate. Inject and determine the chromatographic results according to the above conditions. The results are shown in the figure. Figure 11

[0149] The results showed no significant difference between different extraction times. Taking all factors into consideration, the extraction time for the test sample was tentatively set at 30 minutes.

[0150] In summary, the preparation method of the test solution for the characteristic chromatogram of the standard decoction of myrrh (collagenous myrrh) is determined as follows: Take 0.3g of myrrh powder, add 25ml of 70% methanol, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the product.

[0151] 5. Methodological investigation

[0152] 5.1 Chromatographic Peak Identification

[0153] Preparation of test solution: Prepare the standard decoction test solution of myrrh (collagenous myrrh) according to the experimental conditions proposed above.

[0154] Preparation of the reference herb solution: Take 1.0 g of myrrh (collagenous myrrh) reference herb, add 25 ml of 70% methanol, sonicate (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference herb solution.

[0155] Preparation of reference solution: Weigh an appropriate amount of linalool reference standard accurately, add 70% methanol to prepare a solution containing 2.5 mg per ml.

[0156] Preparation of negative control solution: A negative control solution lacking myrrh (collagenous myrrh) was prepared according to the experimental conditions outlined above. Results are shown below. Figure 12-14 .

[0157] 5.2 Precision Test

[0158] Take the standard decoction of myrrh (collagenous myrrh) as the test sample solution, and inject it 6 times consecutively according to the proposed experimental method, 3 μl each time. Calculate the retention time and peak area. The results are shown in Table 3-4.

[0159] Table 3 Precision Examination—Retention Time of Characteristic Peaks

[0160]

[0161] Table 4 Precision Examination—Peak Area of ​​Characteristic Peaks

[0162]

[0163] The results show that the RSD values ​​of the retention times of each characteristic peak are between 0.04% and 0.10%, indicating that the instrument has good precision.

[0164] 5.3 Repeatability Test

[0165] Accurately weigh 6 portions of myrrh (collagenous myrrh) standard decoction lyophilized powder, and prepare and determine them according to the proposed experimental method. See Tables 5 and 6.

[0166] Table 5 Repeatability Tests—Relative Retention Times of Characteristic Peaks

[0167]

[0168] Table 6 Repeatability Tests - Relative Peak Areas of Characteristic Peaks

[0169]

[0170] The results show that the relative retention time RSD values ​​of each characteristic peak are in the range of 0.00-0.88%, indicating that the method has good repeatability.

[0171] 5.4 Intermediate Precision Examination

[0172] Myrrh (collagenous myrrh) standard decoction lyophilized powder was prepared and measured by different personnel (A, B) at different times (I, II) according to the proposed experimental method. The results were measured by injection on instruments a and b, respectively. The results are shown in Tables 7 and 8. Figure 15

[0173] Table 7 Intermediate Precision - Relative Retention Time

[0174]

[0175] Table 8 Intermediate Precision - Relative Peak Area

[0176]

[0177] As shown above, under different personnel preparing the test solution and different test solution preparation times, the RSD of the relative retention time of each characteristic peak is between 0.44% and 2.28%, indicating that the intermediate precision of this method is good.

[0178] 5.5 Stability Test

[0179] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 2h, 4h, 8h, 16h, and 24h. See Table 9-10.

[0180] Table 9. 24-hour stability study—retention time of characteristic peaks

[0181]

[0182]

[0183] Table 10 24-hour stability study—peak area of ​​characteristic peaks

[0184]

[0185] The results showed that the RSD of the retention time of the corresponding characteristic peak was between 0.05% and 0.09%, and the sample solution was relatively stable within 24 hours.

[0186] 5.6 Durability Test

[0187] Instrument durability test

[0188] Based on the above-established experimental conditions, ultra-high performance liquid chromatography (UHPLC) was conducted using Agilent 1290, Waters, and Thermo Fisher Vanquish F systems, respectively. The results are shown below. Figure 16 Table 11-12.

[0189] Table 11 Instrument Durability Test—Relative Retention Time of Characteristic Peaks

[0190]

[0191] Table 12 Instrument Durability Test—Relative Peak Area of ​​Characteristic Peaks

[0192]

[0193] The results show that when the samples are detected using the above three instruments, the RSD of the relative retention time of the characteristic peak is between 0.36% and 3.11%, which indicates that the instrument column of this method has good durability.

[0194] Column durability study

[0195] Based on the above-planned experimental conditions, the chromatographic columns ZORBAX SB C18 2.1×150mm, 1.8μm, ZORBAX Eclipse Plus C18 2.1×150mm, 1.8μm, and ACQUITY were tested respectively. HSS T3 2.1×150mm, 1.8μm was investigated. See Figure 17 Table 13-14.

[0196] Table 13 Column robustness study—relative retention times of characteristic peaks

[0197]

[0198] Table 14 Column Robustness Study—Relative Peak Area of ​​Characteristic Peaks

[0199]

[0200] The results showed that the RSD of the relative retention time of the characteristic peaks was between 0.63% and 1.53% when using the three chromatographic columns described above, indicating that the column robustness of this method is good. It is recommended to use a chromatographic column (2.1 mm × 150 mm, 1.8 μm) for further investigation.

[0201] 5.7 Establishment of Limits for Relative Retention Time

[0202] Table 15 summarizes the methodological examination items and validation results:

[0203] Table 15 Summary of RSD% for Methodological Results – Retention Time, Relative Retention Time

[0204]

[0205] As shown in the table above, different instruments have a significant impact on peak 1. In order to increase the reproducibility and applicability of the method, the relative retention time of each peak is tentatively set at 10%.

[0206] 6. Determination of characteristic peaks and establishment of reference spectra

[0207] The characteristic spectra of 16 batches of this product were determined using the proposed method, and the relative retention times and relative peak areas were calculated. See [link / reference]. Figure 18 Table 16-17.

[0208] Table 16. Relative retention times of 16 batches of standard decoctions of myrrh (collagenous myrrh)

[0209]

[0210]

[0211] Table 17 Relative Peak Areas of 16 Batches of Myrrh (Colloidal Myrrh) Standard Decoctions

[0212]

[0213] The results showed that the relative retention times of each characteristic peak were stable, with the RSD% within 10%. Therefore, the specified range for the relative retention time of each peak was tentatively set at ±10%. The relative peak areas varied too much between batches to be specified, and therefore were not included in the main text. Since the chromatographic conditions for the characteristic chromatograms of the standard decoction and the formulated granules were the same, the specified values ​​were consistent with those for the formulated granules.

[0214] The final specification stipulates that the chromatogram of the test sample should show six characteristic peaks, and these peaks should correspond to the retention times of the six characteristic peaks in the chromatogram of the reference medicinal material. Peak 3 should correspond to the retention time of the reference material, and the peak corresponding to the linalool reference material is designated as peak S. The relative retention times of each characteristic peak and peak S should be calculated, and these relative retention times should be within ±10% of the specified values. The specified values ​​are 0.60 (peak 1), 0.81 (peak 2), 1.13 (peak 4), 1.35 (peak 5), and 1.40 (peak 6).

[0215] The similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine (2012 version) was used to synthesize chromatographic fingerprints of 16 batches of myrrh (gelatinous myrrh) standard decoctions, and a reference chromatogram of the characteristic chromatograms of myrrh (gelatinous myrrh) standard decoctions was established. (See attached image.) Figure 19 .

[0216] 7. Method for determining the characteristic chromatograms of standard decoctions of myrrh (collagenous myrrh)

[0217] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 150 mm, inner diameter 2.1 mm, particle size 1.8 μm); methanol as mobile phase A and water as mobile phase B, with gradient elution as specified in the table below; flow rate 0.2 mL / min; column temperature 30 °C; detection wavelength 300 nm. The theoretical plate number, calculated based on the linalool peak, should be no less than 10,000.

[0218]

[0219] Preparation of the reference solution: Take 1.0 g of myrrh (collagenous myrrh) reference material, add 25 ml of 70% methanol, sonicate (600 W power, 40 kHz frequency) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution. Separately, take an appropriate amount of linalool reference standard, accurately weigh it, and add 70% methanol to prepare a solution containing 2.5 mg per ml, as the reference solution.

[0220] Preparation of the test solution: Take 0.3g of myrrh (collagenous myrrh) standard decoction powder, add 25ml of 70% methanol, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0221] The determination method involves precisely pipetting 3 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0222] Drafting Instructions for HPLC Characteristic Chromatography of Myrrh (Gel Myrrh) Medicinal Material and Slices:

[0223]

Feature Map

[0224] 1. Experimental Instruments and Materials

[0225] Agilent 1290 UHPLC; Waters UHPLC; Thermo Fisher Vanquish F UHPLC;

[0226] Cellular 1810A Ultrapure Water System (Shanghai Moler Scientific Instruments Co., Ltd.);

[0227] KQ-600DB Ultrasonic Cleaner (Kunshan Ultrasonic Instrument Co., Ltd.);

[0228] Methanol was of chromatographic grade, water was ultrapure water, and all other reagents were of analytical grade.

[0229] Chromatographic columns: ZORBAX SB C18 2.1×150mm, 1.8μm; ZORBAX Eclipse Plus C18 2.1×150mm, 1.8μm. HSS T3 2.1×150mm, 1.8μm

[0230] Linalool reference standard (China National Institutes for Food and Drug Control, batch number: 111503-201603, purity: 97.8%);

[0231] Myrrh (gum myrrh) reference material (China National Institutes for Food and Drug Control, batch number: 121250-201806);

[0232] Myrrh (gum myrrh): 16 batches of myrrh (gum myrrh) medicinal materials

[0233] 2. Proposed chromatographic conditions

[0234] The column was packed with octadecylsilane-bonded silica gel (150 mm column length, 2.1 mm inner diameter, 1.8 μm particle size); methanol was used as mobile phase A and water as mobile phase B, and gradient elution was performed according to the specifications in the table below; the flow rate was 0.2 mL / min; the column temperature was 30 °C; and the detection wavelength was 300 nm. The theoretical plate number, calculated based on the linalool peak, should be no less than 10,000.

[0235]

[0236]

[0237] Preparation of the reference solution: Take 1.0 g of myrrh (collagenous myrrh) reference material, add 25 ml of 70% methanol, sonicate (600 W power, 40 kHz frequency) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution. Separately, take an appropriate amount of linalool reference standard, accurately weigh it, and add 70% methanol to prepare a solution containing 2.5 mg per ml, as the reference solution.

[0238] The preparation of the test solution is the same as that of the control medicinal material solution.

[0239] The determination method involves precisely pipetting 3 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0240] 3. Preparation and investigation of the test solution

[0241] 3.1 Investigation of extraction solvent

[0242] Take two portions of myrrh (collagenous myrrh), 1g each, add 25ml of 70% methanol, and sonicate (600W power, 40kHz frequency) and reflux for 30 minutes respectively. Cool, shake well, filter, and collect the filtrate. Inject according to "Chromatographic Conditions and System Suitability Results". The results are shown in the table below. Figure 20 .

[0243] The results showed that there was no significant difference in the final effect between reflux extraction and ultrasonic extraction. Therefore, ultrasonic extraction, which is simpler to operate, was chosen as the extraction method for preparing the test solution in the determination of the characteristic spectrum of myrrh (collagenous myrrh).

[0244] 3.2 Investigation of Extraction Solvents

[0245] Take six portions of myrrh (collagenous myrrh), 1.0 g each, and add 25 ml each of water, ethanol, 30% methanol, 50% methanol, 70% methanol, and methanol to each portion. Sonicate the mixture (600 W, 40 kHz) for 30 minutes, cool, shake well, filter, and collect the filtrate. Inject the sample according to the "Chromatographic Conditions and System Suitability Results". See the results below. Figure 21 .

[0246] The results showed that the test sample with ethanol as solvent lost the corresponding peaks. The test sample solutions extracted with water, methanol, 30% methanol, 50% methanol and 80% methanol had better peak shapes and resolution. Taking all factors into consideration, 70% methanol was selected as the extraction solvent for the characteristic chromatogram of myrrh (collagenous myrrh).

[0247] 3.3 Examination of extraction time

[0248] Take three portions of myrrh (collagenous myrrh), 1g each, add 25ml of 70% methanol to each, and sonicate (600W power, 40kHz frequency) for 20 minutes, 30 minutes, and 40 minutes respectively. Cool, shake well, filter, and collect the filtrate. Inject and determine according to the results of the "Chromatographic Conditions and System Suitability Test". The results are shown in the figure. Figure 22 .

[0249] The results showed that an extraction time of 20 minutes was sufficient, but to ensure complete extraction of the solution, the extraction time for the test sample was determined to be 30 minutes.

[0250] In summary, the preparation method of the test solution for the characteristic spectrum of myrrh (collagenous myrrh) is determined as follows: Take 1g of the powder (passed through a No. 3 sieve), add 25ml of 70% methanol, seal tightly, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the product.

[0251] 4. Methodological Examination

[0252] 4.1 Chromatographic Peak Identification

[0253] Preparation of test solution: Prepare myrrh (collagenous myrrh) test solution according to the experimental conditions proposed above.

[0254] Preparation of reference solution: Take 1.0 g of myrrh (collagenous myrrh) reference material, add 25 ml of 70% methanol, sonicate (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution of the reference material.

[0255] Preparation of reference solution: Weigh an appropriate amount of linalool reference standard accurately, add 70% methanol to prepare a solution containing 2.5 mg per ml.

[0256] Preparation of negative control solution: Prepare negative control solution for myrrh (gelatinous myrrh) according to the experimental conditions proposed above.

[0257] The sample was injected and analyzed according to the results of the "Chromatographic Conditions and System Suitability Test". The results are shown in [Figure Number]. Figure 23-25 .

[0258] The results showed that peak 6 was the linalool peak. The following methodological investigation investigated six characteristic peaks in the sample.

[0259] 4.2 Precision Test

[0260] Take the myrrh (gelatinous myrrh) sample solution and inject it 6 times consecutively according to the proposed experimental method, 3 μl each time. Calculate the retention time and peak area. The results are shown in Table 18-19.

[0261] Table 18 Precision Examination—Retention Time of Characteristic Peaks

[0262]

[0263] Table 19 Precision Examination—Peak Area of ​​Characteristic Peaks

[0264]

[0265] The results show that the RSD values ​​of the retention times of each characteristic peak are between 0.03% and 0.11%, indicating that the instrument has good precision.

[0266] 3.3 Repeatability Test

[0267] Accurately weigh 6 portions of myrrh (gelatinous myrrh) and prepare and determine it according to the proposed experimental method. See Table 20-21.

[0268] Table 20 Repeatability Tests—Relative Retention Times of Characteristic Peaks

[0269]

[0270]

[0271] Table 21 Repeatability Tests—Relative Peak Area of ​​Characteristic Peaks

[0272]

[0273] The results show that the relative retention time (RSD%) of each characteristic peak is between 0.00% and 0.29%, indicating that the method has good repeatability.

[0274] 4.4 Stability

[0275] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 2h, 4h, 8h, 12h, and 24h. See Tables 22-23.

[0276] Table 22 24-hour stability study—retention time of characteristic peaks

[0277]

[0278] Table 23 24-hour stability study—relative peak area of ​​characteristic peaks

[0279]

[0280] The results showed that the RSD of the retention time of the corresponding characteristic peak was between 0.04% and 0.11%, and the RSD of the peak area was between 0.60% and 3.15%, indicating that the sample solution was relatively stable within 24 hours.

[0281] 4.5 Intermediate Precision Examination

[0282] Myrrh (gelatinous myrrh) was prepared and measured by different personnel at different times according to the prescribed experimental method. The results were measured on different instruments, and the results are shown in Tables 24-25. Figure 26 .

[0283] Table 24 Intermediate Precision - Relative Retention Time

[0284]

[0285] Table 25 Intermediate Precision - Relative Peak Area

[0286]

[0287] As shown above, the RSD of the relative retention times of each characteristic peak is between 0.00% and 1.89% under different test solution preparation personnel and different test solution preparation times, indicating that the intermediate precision of this method is good.

[0288] 4.6 Durability Test

[0289] Different instruments

[0290] Based on the above-established experimental conditions, the Agilent 1290, Waters, and Thermo Fisher ultra-high performance liquid chromatography (UHPLC) instruments were investigated respectively. See [link / reference]. Figure 27 Tables 26-27.

[0291] Table 26 Instrument Durability Test—Relative Retention Time of Characteristic Peaks

[0292]

[0293] Table 27 Instrument Durability Test—Relative Peak Area of ​​Characteristic Peaks

[0294]

[0295] The results show that when the samples are detected using the above three instruments, the RSD of the relative retention time of the characteristic peak is between 0.72% and 6.91%, indicating that the instrument column of this method has good robustness.

[0296] Investigation of different chromatographic columns

[0297] Based on the above-planned experimental conditions, the chromatographic columns ZORBAX SB C18 2.1×150mm, 1.8μm, ZORBAX Eclipse Plus C18 2.1×150mm, 1.8μm, and ACQUITY were tested respectively. The HSS T3 was investigated using a 2.1×150mm, 1.8μm aperture. See [link / reference]. Figure 28 Tables 28-29.

[0298] Table 28 Column Robustness Study—Relative Retention Times of Characteristic Peaks

[0299]

[0300] Table 29 Column Robustness Study—Relative Peak Area of ​​Characteristic Peaks

[0301]

[0302] The results showed that the RSD of the relative retention time of the characteristic peaks was between 1.02% and 5.59% when using the three chromatographic columns described above, indicating that the column robustness of this method is good. A 2.1 mm × 150 mm, 1.8 μm column is recommended for further investigation.

[0303] 4.7 Establishment of Limits for Relative Retention Time

[0304] Table 30 summarizes the methodological examination items and validation results:

[0305] Table 30 Summary of RSD% of Methodological Results – Retention Time, Relative Retention Time

[0306]

[0307] As shown in the table above, different instruments have a significant impact on peak 4. In order to increase the reproducibility and applicability of the method, the relative retention time of each peak is tentatively set at 10%.

[0308] 5. Verification of the characteristic spectrum of myrrh (gelatinous myrrh) medicinal material

[0309] According to the "characteristic spectrum determination method" obtained from the above investigation, 16 batches of myrrh (gum myrrh) medicinal materials were tested. The characteristic spectra of the 16 batches of myrrh (gum myrrh) medicinal materials are shown below. Figure 29 The relative retention time and relative peak area of ​​the characteristic peaks are shown in Tables 31-32.

[0310] Table 31 Relative Retention Time of Characteristic Atlases of Myrrh (Gel Myrrh)

[0311]

[0312]

[0313] Table 32 Relative Peak Area of ​​16 Batches of Myrrh (Gel Myrrh)

[0314]

[0315] The results showed that the relative retention times of each characteristic peak were stable, with the RSD% within 10%. Therefore, the specified range for the relative retention time of each peak was tentatively set at ±10%. The relative peak areas varied too much between batches to be specified, and therefore were not included in the main text. Since the chromatographic conditions for the characteristic chromatograms of the medicinal materials and the formulated granules were the same, the specified values ​​were consistent with those for the formulated granules.

[0316] The final specification stipulates that the chromatogram of the test sample should show six characteristic peaks, and these peaks should correspond to the retention times of the six characteristic peaks in the chromatogram of the reference medicinal material. Peak 3 should correspond to the retention time of the reference material, and the peak corresponding to the linalool reference material is designated as peak S. The relative retention times of each characteristic peak and peak S should be calculated, and these relative retention times should be within ±10% of the specified values. The specified values ​​are 0.60 (peak 1), 0.81 (peak 2), 1.13 (peak 4), 1.35 (peak 5), and 1.40 (peak 6).

[0317] The 2012 version of the Chinese herbal chromatographic fingerprint similarity evaluation system was used to synthesize 21 batches of myrrh (gum myrrh) medicinal materials, and a reference characteristic chromatogram of myrrh (gum myrrh) medicinal materials was established. See [link to relevant documentation]. Figure 30 .

[0318] 6. In summary, the method for determining the characteristic chromatogram of myrrh (gum myrrh) is as follows:

[0319] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 150 mm, inner diameter 2.1 mm, particle size 1.8 μm); methanol as mobile phase A and water as mobile phase B, with gradient elution as specified in the table below; flow rate 0.2 mL / min; column temperature 30 °C; detection wavelength 300 nm. The theoretical plate number, calculated based on the linalool peak, should be no less than 10,000.

[0320]

[0321] Preparation of the reference solution: Take 1.0 g of myrrh (collagenous myrrh) reference material, add 25 ml of 70% methanol, sonicate (600 W power, 40 kHz frequency) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution. Separately take the reference solution from the [Assay] section as the reference solution.

[0322] Preparation of the test solution: Take 1g of the powder (passed through a No. 3 sieve), add 25ml of 70% methanol, seal tightly, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0323] The determination method involves precisely pipetting 3 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0324] Drafting Instructions for the HPLC Characteristic Chromatography of Myrrh (Colloidal Myrrh) Formulation Granules:

[0325]

Feature Map

[0326] 1. Experimental Instruments and Materials

[0327] Agilent 1290 UHPLC; Waters UHPLC; Thermo Fisher Vanquish F UHPLC;

[0328] Cellular 1810A Ultrapure Water System (Shanghai Moler Scientific Instruments Co., Ltd.);

[0329] KQ-600DB Ultrasonic Cleaner (Kunshan Ultrasonic Instrument Co., Ltd.);

[0330] Chromatographic columns: ZORBAX SB C18 2.1×150mm, 1.8μm; ZORBAX Eclipse Plus C18 2.1×150mm, 1.8μm. HSS T3 2.1×150mm, 1.8μm

[0331] 2. Reagents and reagents

[0332] Methanol was of chromatographic grade, water was ultrapure water, and all other reagents were of analytical grade.

[0333] Linalool reference standard (China National Institutes for Food and Drug Control, batch number: 111503-201603, purity: 97.8%);

[0334] Myrrh (gum myrrh) reference material (China National Institutes for Food and Drug Control, batch number: 121250-201806);

[0335] Preparation of Myrrh (Colloidal Myrrh) Formula Granules (Sichuan New Green Pharmaceutical Technology Development Co., Ltd.): Three batches of formula granules.

[0336] 3. Proposed chromatographic conditions

[0337] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 150 mm, inner diameter 2.1 mm, particle size 1.8 μm); methanol as mobile phase A and water as mobile phase B, with gradient elution as specified in the table below; flow rate 0.2 mL / min; column temperature 30 °C; detection wavelength 300 nm. The theoretical plate number, calculated based on the linalool peak, should be no less than 10,000.

[0338]

[0339] Preparation of the reference solution: Take 1.0 g of myrrh (collagenous myrrh) reference material, add 25 ml of 70% methanol, sonicate (600 W power, 40 kHz frequency) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution. Separately, take an appropriate amount of linalool reference standard, accurately weigh it, and add 70% methanol to prepare a solution containing 2.5 mg per ml, as the reference solution.

[0340] Preparation of the test solution: Take an appropriate amount of myrrh (colloidal myrrh) formulation granules, grind them into a fine powder, take 0.3g, add 25ml of 70% methanol, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0341] The determination method involves precisely pipetting 3 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0342] Based on the above-specified experimental conditions, a full-band scan was performed using a diode array detector, and chromatograms of the test solution were extracted at wavelengths of 220 nm, 240 nm, 260 nm, 280 nm, 300 nm, and 320 nm. The results are shown below. Figure 31 .

[0343] The results showed that the chromatographic peak information was greater at a detection wavelength of 300 nm. Therefore, the detection wavelength of the myrrh (colloidal myrrh) formulation particle characteristic chromatographic method was finally determined to be 300 nm.

[0344] Column temperature investigation

[0345] Based on the above-planned experimental conditions, the effects were investigated at column temperatures of 25℃, 30℃, and 35℃. Figure 32 As shown in Table 33.

[0346] Table 33 Column Temperature Investigation—Relative Retention Time of Characteristic Peaks

[0347]

[0348] The results showed that the RSD of the relative retention times of each characteristic peak at different column temperatures ranged from 0.00% to 2.50%, indicating that the method is feasible. At a column temperature of 30℃, the chromatographic peaks were all symmetrical, and the resolution met the requirements. Therefore, a column temperature of 30℃ was chosen for further investigation.

[0349] Flow velocity investigation

[0350] Based on the above-specified experimental conditions, the flow rates of 0.2 ml / min, 0.3 ml / min, and 0.4 ml / min were investigated respectively. (See attached figures.) Figure 33 Table 34.

[0351] Table 34 Flow velocity investigation—Relative retention time of characteristic peaks

[0352]

[0353] The results showed that the RSD of the relative retention time of each characteristic peak at different flow rates ranged from 3.42% to 8.81%, indicating that the method is feasible. At a flow rate of 0.3 ml / min, the peak shapes of each characteristic peak were good; therefore, a flow rate of 0.2 ml / min was selected for further investigation.

[0354] Injection volume investigation

[0355] Based on the above-established experimental conditions, the results were investigated for injection volumes of 2 μL, 3 μL, and 4 μL. The results are shown below. Figure 34 .

[0356] The results showed that when the injection volume was 3 μL, the peak shape and separation of each characteristic peak were good. Therefore, according to convention, the injection volume of 3 μL was selected for further investigation.

[0357] Delayed examination

[0358] Based on the experimental conditions outlined above, the data acquisition time was extended to 60 minutes. The results are shown below. Figure 35 .

[0359] The results showed that there were basically no large chromatographic peaks after 30 minutes, and the analysis time for the characteristic chromatographic method of myrrh (colloidal myrrh) formulation particles was finally determined to be 30 minutes.

[0360] In summary, the chromatographic conditions for the characteristic chromatogram of myrrh (colloidal myrrh) formulation particles were determined as follows: Octadecylsilane-bonded silica gel as the packing material (column length 150 mm, inner diameter 2.1 mm, particle size 1.8 μm); methanol as mobile phase A, water as mobile phase B, gradient elution as specified in the table below; flow rate 0.2 ml / min; column temperature 30℃; detection wavelength 300 nm. The theoretical plate number, calculated based on the linalool peak, should be no less than 10,000.

[0361]

[0362] The determination method involves precisely pipetting 3 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0363] Subsequent standard decoctions and medicinal herb slices were examined under these chromatographic conditions.

[0364] 4. Preparation of the test solution

[0365] 4.1 Examination of Extraction Methods

[0366] Take an appropriate amount of myrrh (colloidal myrrh) granules, grind them finely, take 0.3g, place them in a stoppered conical flask, add 25ml of 70% methanol, seal tightly, and test the extraction methods for the sample: reflux and ultrasonic (600W power, 40kHz frequency). The extraction time is 30 minutes. Cool, shake well, filter, and collect the filtrate to obtain the sample. Figure 36 As shown.

[0367] As can be seen from the figure above, there is no significant difference in the final results between reflux extraction and ultrasonic extraction. Therefore, ultrasonic extraction, which is simpler to operate, was chosen as the extraction method for preparing the test solution in the determination of the characteristic spectrum of myrrh (colloidal myrrh) formulation particles.

[0368] 4.2 Investigation of Extraction Solvents

[0369] Take an appropriate amount of myrrh (colloidal myrrh) granules, grind them finely, take 0.3g, place them in a stoppered conical flask, add 25ml each of methanol, 30% methanol, 50% methanol, 70% ethanol, water, and ethanol, seal tightly, sonicate (600W power, 40kHz frequency) for 30 minutes, cool, shake well, filter, and collect the filtrate to obtain the product. Figure 37 As shown.

[0370] The results showed that using 70% methanol as the extraction solvent yielded chromatographic peaks with high information content and good separation, therefore 70% methanol was chosen as the extraction solvent.

[0371] 4.3 Examination of extraction time

[0372] Take an appropriate amount of myrrh (colloidal myrrh) granules, grind them finely, take 0.3g, place them in a stoppered conical flask, add 25ml of 70% methanol, seal tightly, and sonicate (600W power, 40kHz frequency) for 20 minutes, 30 minutes, and 40 minutes respectively. Cool, shake well, filter, and collect the filtrate to obtain the product. Figure 38 As shown.

[0373] The results showed that an extraction time of 20 minutes was sufficient, but to ensure complete extraction of the solution, the extraction time for the test sample was determined to be 30 minutes.

[0374] 4.4 Sample Quantity Examination

[0375] Take an appropriate amount of myrrh (colloidal myrrh) granules, grind them finely, and take 0.2g, 0.3g, and 0.4g respectively. Place them in stoppered conical flasks, add 25ml of 70% methanol to each, seal tightly, and sonicate (600W power, 40kHz frequency) for 30 minutes. Cool, shake well, filter, and collect the filtrate to obtain the product. Figure 39 As shown.

[0376] The results showed that when the solvent volume was 25 ml and the sample weight was 0.3 g, the peak sizes in the chromatogram were appropriate. Therefore, the sample weight was determined to be 0.3 g.

[0377] 4.5 Determine the method for preparing the test sample

[0378] In summary, the preparation method of the test solution for the characteristic chromatogram of myrrh (colloidal myrrh) formulation granules is determined as follows: Take an appropriate amount of myrrh (colloidal myrrh) formulation granules, grind them finely, take 0.3g, place them in a stoppered conical flask, add 25ml of 70% methanol, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the product.

[0379] 5. Feature mapping method is used to determine

[0380] Determined by high performance liquid chromatography (General Chapter 0512, Part IV, Chinese Pharmacopoeia 2020 Edition).

[0381] The column was packed with octadecylsilane-bonded silica gel (150 mm column length, 2.1 mm inner diameter, 1.8 μm particle size); methanol was used as mobile phase A and water as mobile phase B, and gradient elution was performed according to the specifications in the table below; the flow rate was 0.2 mL / min; the column temperature was 30 °C; and the detection wavelength was 300 nm. The theoretical plate number, calculated based on the linalool peak, should be no less than 10,000.

[0382]

[0383] Preparation of the reference solution: Take 1.0 g of myrrh (collagenous myrrh) reference material, add 25 ml of 70% methanol, sonicate (600 W power, 40 kHz frequency) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution. Separately, take an appropriate amount of linalool reference standard, accurately weigh it, and add 70% methanol to prepare a solution containing 2.5 mg per ml as the reference solution.

[0384] Preparation of the test solution: Take an appropriate amount of myrrh (colloidal myrrh) formulation granules, grind them into a fine powder, take 0.3g, add 25ml of 70% methanol, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0385] The determination method involves precisely pipetting 3 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0386] 5.1 Chromatographic Peak Identification

[0387] Preparation of test solution: Prepare a myrrh (colloidal myrrh) formulation granule test solution according to the experimental conditions proposed above.

[0388] Preparation of reference solution: Take 1.0 g of myrrh (collagenous myrrh) reference material, add 25 ml of 70% methanol, sonicate (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution of the reference material.

[0389] Preparation of reference solution: Weigh an appropriate amount of linalool reference standard accurately, add 70% methanol to prepare a solution containing 2.5 mg per ml.

[0390] Preparation of negative control solution: Prepare negative control solution of myrrh (colloidal myrrh) formulation granules according to the experimental conditions proposed above.

[0391] The characteristic spectral peaks of myrrh (colloidal myrrh) formulation granules were located. See [link / reference]. Figures 40-42 .

[0392] The results showed that peak 6 was the linalool peak. The following methodological investigation investigated six characteristic peaks in the sample.

[0393] 5.2 Precision Test

[0394] Take the test solution of myrrh formulation granules (batch number: 032520003), and inject it 6 times consecutively according to the proposed experimental method, 3 μl each time. Calculate the retention time and peak area of ​​each characteristic peak. As shown in Tables 35-36.

[0395] Table 35 Precision Examination—Retention Time of Characteristic Peaks

[0396]

[0397] Table 36 Precision Examination—Peak Area of ​​Characteristic Peaks

[0398]

[0399] The results show that the retention time RSD of each characteristic peak is between 0.06% and 0.14%, and the peak area RSD of each characteristic peak is between 0.09% and 2.00%, indicating that the instrument has good precision.

[0400] 5.3 Repeatability Test

[0401] Take 6 portions of myrrh (colloidal myrrh) formulation granules and prepare and determine them according to the proposed experimental method. See Tables 37-38.

[0402] Table 37 Repeatability Tests—Relative Retention Time of Characteristic Peaks

[0403]

[0404] Table 38 Repeatability Tests—Relative Peak Area of ​​Characteristic Peaks

[0405]

[0406] The results show that the relative retention time RSD% of each characteristic peak is between 0.00% and 0.64%, and the relative retention peak area RSD% is between 0.37% and 7.14%, indicating that the method has good repeatability.

[0407] 5.4 Intermediate Precision Examination

[0408] Myrrh (colloidal myrrh) granules (batch number: 032520003) were prepared and tested by different personnel at different times according to the proposed experimental method. The results were measured using different instruments, and the results are shown in Tables 39-40. Figure 43 .

[0409] Table 39 Intermediate Precision - Relative Retention Time

[0410]

[0411] Table 40 Intermediate Precision - Relative Peak Area

[0412]

[0413]

[0414] From Tables 39-40 and Figure 43It can be seen that the RSD of the relative retention time of each characteristic peak is between 0.00% and 1.30% under different test solution preparation personnel and different test solution preparation times, indicating that the intermediate precision of this method is good.

[0415] 5.5 Stability

[0416] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 2h, 4h, 8h, 16h, and 24h. See Tables 41-42.

[0417] Table 41 24-hour stability study—retention time of characteristic peaks

[0418]

[0419] Table 42 24-hour stability study—relative peak area of ​​characteristic peaks

[0420]

[0421] The results showed that the RSD of the retention time of the corresponding characteristic peak was between 0.13% and 0.28%, and the RSD of the peak area was between 0.08% and 7.17%, indicating that the sample solution was relatively stable within 24 hours.

[0422] 5.6 Durability Test

[0423] Column durability study

[0424] Based on the above-established experimental conditions, the chromatographic columns with different packing materials were tested: ZORBAX SB C18 2.1×150mm, 1.8μm; ZORBAX Eclipse Plus C18 2.1×150mm, 1.8μm; and ACQUITY. The HSS T3 was investigated using a 2.1×150mm, 1.8μm aperture. See [link / reference]. Figure 44 Tables 43-44.

[0425] Table 43 Column robustness study—Relative retention times of characteristic peaks

[0426]

[0427] Table 44 Column robustness test—Relative peak area of ​​characteristic peaks

[0428]

[0429] The results showed that the RSD of the relative retention time of the characteristic peaks was between 1.01% and 5.05% when using the three columns described above, indicating that the columns used in this method have good robustness. ZORBAX SB C18, 2.1 mm × 150 mm, 1.8 μm, is recommended for further investigation.

[0430] 5.7 Instrument Durability Test

[0431] Based on the above-established experimental conditions, the Agilent 1290, Waters, and Thermo Fisher ultra-high performance liquid chromatography (UHPLC) instruments were investigated respectively. See [link / reference]. Figure 45 Tables 45-46.

[0432] Table 45 Instrument Durability Test—Relative Retention Time of Characteristic Peaks

[0433]

[0434] Table 46 Instrument Durability Test—Relative Peak Area of ​​Characteristic Peaks

[0435]

[0436] The results show that when the samples are detected using the above three instruments, the RSD of the relative retention time of the characteristic peak is between 0.00% and 1.06%, indicating that the instrument column of this method has good robustness.

[0437] 6. Determination of characteristic peaks and establishment of reference spectra

[0438] Setting limits for relative retention time

[0439] Table 47 summarizes the methodology, the examination items, and the verification results.

[0440] Table 47 Summary of RSD% of Methodological Results — Retention Time — Relative Retention Time

[0441]

[0442]

[0443] As shown in the table above, different flow rates have a significant impact on peak 6. In order to increase the reproducibility and applicability of the method, the relative retention time of each peak is tentatively set at 10%.

[0444] 7. Validation results of 3 batches of myrrh (colloidal myrrh) formulation granules

[0445] The characteristic spectra of three batches of this product were determined using the proposed method, and the relative retention times and relative peak areas were calculated. (See attached image.) Figure 46 Tables 48-49.

[0446] Table 48 Relative retention times of three batches of myrrh (colloidal myrrh) formulation granules

[0447]

[0448] Table 49 Relative Peak Area of ​​Granules in Three Batches of Myrrh (Colloidal Myrrh) Formulation

[0449]

[0450] The relative retention times of each characteristic peak are stable, and the RSD% of the relative retention time is within 10%. Therefore, the specified range of the relative retention time of each peak is tentatively set at ±10%. The relative peak area of ​​each batch varies too much, so it cannot be specified. Therefore, the relative peak area is not included in the main text.

[0451] The final specification stipulates that the chromatogram of the test sample should show six characteristic peaks, and the retention times should correspond to the six characteristic peaks in the chromatogram of the reference medicinal material. The peak corresponding to the linalool reference is designated as the S peak. The relative retention times of each characteristic peak and the S peak should be calculated, and these relative retention times should be within ±10% of the specified values. The specified values ​​are 0.60 (peak 1), 0.81 (peak 2), 1.13 (peak 4), 1.35 (peak 5), and 1.40 (peak 6).

[0452] The 2012 version of the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System was used to synthesize three batches of myrrh (gelatinous myrrh) formulation granules, and a reference chromatogram of the characteristic chromatograms of myrrh (gelatinous myrrh) formulation granules was established. See [link / reference]. Figure 47 .

[0453] 7. Method for determining the characteristic spectral features of myrrh (colloidal myrrh) formulation granules

[0454] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 150 mm, inner diameter 2.1 mm, particle size 1.8 μm); methanol as mobile phase A and water as mobile phase B, with gradient elution as specified in the table below; flow rate 0.2 mL / min; column temperature 30 °C; detection wavelength 300 nm. The theoretical plate number, calculated based on the linalool peak, should be no less than 10,000.

[0455]

[0456] Preparation of the reference solution: Take 1.0 g of myrrh (collagenous myrrh) reference material, add 25 ml of 70% methanol, sonicate (600 W power, 40 kHz frequency) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution. Separately, take an appropriate amount of linalool reference standard, accurately weigh it, and add 70% methanol to prepare a solution containing 2.5 mg per ml, as the reference solution.

[0457] Preparation of the test solution: Take an appropriate amount of myrrh (colloidal myrrh) formulation granules, grind them into a fine powder, take 0.3g, add 25ml of 70% methanol, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0458] The determination method involves precisely pipetting 3 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0459] 2.3 Technical Effects

[0460] A high-performance liquid chromatography (HPLC) method was established for the identification of myrrh (gum myrrh), processed medicinal materials, standard decoctions, and formulated granules, providing a more scientific basis for their identification.

[0461] Comparative Example 1: Yan Ganming, et al., Study on the analysis and HPLC content determination of sesquiterpenoid components in myrrh and its processed products based on UPLC-Q / TOF-MS technology:

[0462] The results of testing using myrrh gum using the above methods are as follows:

[0463] Depend on Figure 48 It can be seen that the method in this literature is for the detection of natural myrrh, but it is not suitable for myrrh and its characteristic peaks cannot be detected.

[0464] Comparative Example 2: Application No. 202210312708.5, Invention Title: A Quality Testing Method for a Standard Vinegar and Myrrh Decoction:

[0465] The determination of natural myrrh and gum myrrh was obtained Figure 49 :

[0466] As shown in the figure, this method is applicable to natural myrrh, but it does not have characteristic peaks for mucilage myrrh and cannot represent it well, so it is not suitable for mucilage myrrh varieties.

[0467] Comparative Example 3: Guangdong Province's Quality Standard for Traditional Chinese Medicine Formula Granules, Vinegar-processed Myrrh (Natural Myrrh) Formula Granules, Guangdong

[0468] PFKL20230007

[0469] According to this standard, myrrh was tested, and the results are shown in the figure. Figure 50 :

[0470] As shown in the figure, this quality is for natural myrrh and is not applicable to mucilage myrrh, and it only has 4 characteristic peaks. However, the method of this patent is developed for mucilage myrrh and has 6 characteristic peaks.

Claims

1. A method for constructing an HPLC characteristic spectrum of myrrh or its preparations, characterized in that: It includes the following steps: a. Preparation of the test solution: Take the test sample, add methanol aqueous solution, sonicate, cool, shake well, filter, and take the filtrate to obtain the solution; b. The test solution is determined by high performance liquid chromatography to obtain a high performance liquid chromatogram of myrrh or its preparation; The chromatographic conditions for the high-performance liquid chromatography (HPLC) method are as follows: a C18 column, methanol as mobile phase A, water as mobile phase B, gradient elution, and the elution conditions are as follows: ； It also includes the preparation of a reference solution, wherein the reference is myrrh reference material and linalool reference standard; The preparation method of linalool reference standard is as follows: Take an appropriate amount of linalool reference standard, accurately weigh it, and add methanol aqueous solution to prepare a reference standard solution; The preparation method of the reference solution is as follows: take myrrh, add methanol aqueous solution, sonicate, cool, shake well, filter, and take the filtrate to obtain the solution; The C18 column has a length of 150 mm, an inner diameter of 2.1 mm, and a particle size of 1.8 μm.

2. The method for constructing the HPLC characteristic chromatogram of myrrh or its preparations according to claim 1, characterized in that: The concentration of the methanol aqueous solution is 70%, and the ultrasonic treatment conditions are: power 600W, frequency 40kHz, and treatment time 30 minutes.

3. The method for constructing the HPLC characteristic chromatogram of myrrh or its preparations according to claim 1 or 2, characterized in that: The high-performance liquid chromatography (HPLC) is performed at a flow rate of 0.2 ml per minute, a column temperature of 30°C, and a detection wavelength of 300 nm. The theoretical plate number, calculated based on the linalool peak, should be no less than 10,000.

4. The method for constructing the HPLC characteristic chromatogram of myrrh or its preparations according to claim 1 or 2, characterized in that: The constructed characteristic chromatogram contains 6 common chromatographic peaks with relative retention times of: peak 1: 0.60, peak 2: 0.81, Peak 4: 1.13, peak 5: 1.35, peak 6: 1.40, relative retention time within ±10%.

5. The method for constructing the HPLC characteristic chromatogram of myrrh or its preparations according to claim 4, characterized in that: Peak 3 is linalool.

6. The method for constructing the HPLC characteristic chromatogram of myrrh or its preparations according to claim 5, characterized in that: The aforementioned gelatinous myrrh or its preparations refer to gelatinous myrrh medicinal materials, processed slices, standard decoctions, and their formulated granules.

7. A method for identifying myrrh or its preparations, characterized in that: It includes the following steps: a. Pre-treatment of the sample to be tested; b. Construct a feature map according to the construction method of any one of claims 1-6; c. Characteristic peak comparison: If identical characteristic peaks appear in peaks 1-6, then it is myrrh.

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