Method for identifying homalomena occulta component in sample

Abstract

The invention discloses a method for identifying a homalomena occulta component in a sample, which is used for separating and identifying the homalomena occulta characteristic component in the sample by using an ultra performance liquid chromatograph and taking acetonitrile and water as mobile phases. According to the method, an ultra-high performance liquid chromatography-evaporative light scattering detection method taking 1, 4, 7-cineotriol as an index is established for the first time to identify whether a sample contains the obscured homalomena rhizome or not and determine the content of the obscured homalomena rhizome; wherein 1, 4, 7-cineotriol is a water-soluble specific component, and the method is particularly suitable for detection of a homalomena occulta water extraction process preparation sample; the method is simple to operate, methodology verifies that the reproducibility and the stability are good, the recovery rate is reliable, the detection cost is low, the detection efficiency is high, the method has good guiding significance in the aspects of process control of homalomena occulta medicinal material selection and preparation processing and product quality stability, and a powerful guarantee is provided for industrialization and quality control of the homalomena occulta medicinal material selection and preparation processing.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of traditional Chinese medicine, and particularly relates to a method for identifying components of Homalomena occulta in a sample. BACKGROUND

[0002] Homalomena occulta is the dried rhizome of Homalomena occulta (Lour.) Schott in Araceae, with bitter and pungent tastes and warm nature, and is attributed to the liver and kidney channels. It has the effects of expelling wind and dampness and strengthening bones and joints, and is clinically used for treating wind-damp arthralgia, cold pain in the waist and knees, and the like. The chemical components of Homalomena occulta mainly include volatile oils (such as linalool) and sesquiterpenes, and also include phenolic acids, alkaloids, polysaccharides and the like. Modern pharmacological studies show that Homalomena occulta has the activities of anti-inflammatory analgesia, anti-osteoporosis, antioxidant and anti-tumor, and has a significant potential in the treatment of rheumatic diseases and degenerative bone diseases. With the development of modernization of traditional Chinese medicine, Homalomena occulta formula granules have gradually been popularized, but the quality control needs to take into account the dual needs of traditional decoction pieces and modern preparations.

[0003] Potential counterfeits of Homalomena occulta are mainly derived from closely related plants in the same family, such as the rhizomes of Alocasia macrorrhizos or Amorphophallus konjac. Such counterfeits are similar in shape to Homalomena occulta, and currently mainly rely on the standards in the Chinese Pharmacopoeia (2020 edition) for identification of medicinal materials, including observation of characteristics, microscopic identification, thin-layer chromatography comparison and determination of volatile oil components by gas chromatography. In addition, existing studies attempt to expand the methods: Jiang Li-juan et al. simultaneously determine linalool and protocatechuic acid by quantitative nuclear magnetic resonance hydrogen spectrum, and He Dan et al. evaluate the batch consistency of medicinal materials by high-performance liquid chromatography fingerprint combined with similarity.

[0004] However, the existing methods mainly focus on non-water-soluble components such as volatile oils and phenolic acids, and although they have a certain specificity, the detection of non-water-soluble components is difficult to adapt to the quality control of samples (such as formula granules) after water extraction process due to changes in components or other factors: for example, linalool is significantly lost during the water extraction process due to its low polarity, and the effective component content of the preparation cannot be truly reflected; protocatechuic acid has pharmacological activity, but it is widely present in many plants and lacks specificity. This easily leads to the fact that adulteration or the use of substitute products are not discovered in time, affecting the efficacy and safety. SUMMARY

[0005] The purpose of the present application is to provide a method for identifying whether Homalomena occulta components are contained in a sample based on the characteristic component 1,4,7-cadinatriol of Homalomena occulta.

[0006] Technical scheme: The method for identifying Homalomena occulta components in a sample provided by the present application comprises the following steps:

[0007] (1) preparing a sample solution to be tested;

[0008] (2) Set the parameters of the chromatographic conditions on the ultra-high performance liquid chromatograph:

[0009] (21) Chromatographic column, chromatographic column with octadecylsilane-bonded silica gel filler as stationary phase;

[0010] (22) Eluent, including mobile phase A and mobile phase B, wherein mobile phase A is acetonitrile, and mobile phase B is water;

[0011] (23) Elution mode, flow rate of mobile phase is 0.2-0.4 mL / min, gradient elution:

[0012] 0 min, volume ratio of mobile phase A is 6-10%, volume ratio of mobile phase B is 90-94%,

[0013] 5 min, volume ratio of mobile phase A is 13-17%, volume ratio of mobile phase B is 83-87%,

[0014] 12 min, volume ratio of mobile phase A is 26-30%, volume ratio of mobile phase B is 70-74%;

[0015] (24) Column temperature for elution, 20-40℃;

[0016] (3) Inject the sample solution to be tested into the ultra-high performance liquid chromatograph, analyze and record the chromatogram, compare with the chromatogram of 1,4,7-cembratrienol standard product, and identify.

[0017] Preferably, the preparation of the sample solution to be tested in step 1 comprises: adding methanol-water solution to the sample, extracting, 0.22 μm filtering, and obtaining the sample solution to be tested.

[0018] Preferably, the concentration of methanol in the methanol-water solution is not higher than 70%, and the amount is 15-50 mL per gram of sample; the extraction is any one of ultrasonic treatment, heating reflux, and shaking extraction, and the extraction time is 15-60 min.

[0019] Preferably, the extraction is ultrasonic extraction at a power of 200-300 W and a frequency of 35-45 kHz for 15-60 min.

[0020] Preferably, the chromatographic column with octadecylsilane-bonded silica gel filler as stationary phase in step 21 is a chromatographic column with a column length of 100 mm, an inner diameter of 2.1 mm, and a particle size of 1.6 μm, and the chromatographic column is selected from any one of Agilent ZORBAX SB Aq RRHD, specification 2.1×100 mm, 1.8 μm; Waters CORTECS C18, specification 2.1 mm×100 mm, 1.6 μm; Thermo HypersilGLOD aQ, specification 2.1×100 mm, 1.9 μm.

[0021] Preferably, the flow rate of the mobile phase in step 23 is 0.25-0.35 mL / min.

[0022] Preferably, the elution column temperature in step 24 is 25-35°C.

[0023] Preferably, in step 3, the volume of sample injected into the high-performance liquid chromatograph is 2-8 μL.

[0024] Preferably, the ultra-high performance liquid chromatograph is an ultra-high performance liquid chromatograph equipped with an evaporative light scattering detector, wherein the ultra-high performance liquid chromatograph is selected from either Thermo VANQUISH or Waters H-Class ultra-high performance liquid chromatograph systems, and the evaporative light scattering detector is Alltech ELSD6000.

[0025] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: 1. This invention establishes for the first time an ultra-high performance liquid chromatography-evaporative light scattering detection method using 1,4,7-cineole as an indicator, which can accurately identify whether a sample contains *Homalomena occulta* and can be used for content determination; 2. 1,4,7-cineole is a water-soluble specific component of *Homalomena occulta*, and this method is particularly suitable for the detection of *Homalomena occulta* water-extracted formulation samples; 3. This method is simple to operate, has been validated, and exhibits good reproducibility, stability, and reliable recovery. It also has low detection cost and high detection efficiency, providing good guidance for the selection of *Homalomena occulta* medicinal materials, process control of formulation processing, and product quality stability, and providing strong support for its industrialization and quality control. Attached Figure Description

[0026] Figure 1 The figure shows the results of liquid chromatography-mass spectrometry analysis of 1,4,7-eucalyptol reference standard and *Homalomena occulta* medicinal material.

[0027] Figure 2 This is a representative ultra-high performance liquid chromatography (UHPLC) analysis spectrum of the Qiannianjian formula granules;

[0028] Figure 3 This is a representative ultra-high performance liquid chromatography (UHPLC) chromatogram of Qiannianjian herbal tablets;

[0029] Figure 4 This is a representative ultra-high performance liquid chromatography (UHPLC) analysis spectrum of the Qiannianjian standard decoction;

[0030] Figure 5 Ultra-high performance liquid chromatography (UHPLC) spectra of *Homalomena occulta* formulation granules under different extraction solvents;

[0031] Figure 6 Ultra-high performance liquid chromatography (UHPLC) spectra of *Henochia glutinosa* formula granules under different extraction methods;

[0032] Figure 7 Ultra-high performance liquid chromatography (UHPLC) spectra of *Henochia glutenin* formula granules extracted at different times;

[0033] Figure 8 Ultra-high performance liquid chromatography (UHPLC) spectra of *Homalomena occulta* formula granules with different extraction volumes;

[0034] Figure 9 The graph shows the linear relationship between the logarithm of the injection amount (μg) and the logarithm of the peak area for ultra-high performance liquid chromatography analysis of 1,4,7-cineoletriol.

[0035] Figure 10 The ultra-high performance liquid chromatography (UHPLC) chromatogram of the Qiannianjian formula granules in the specificity test;

[0036] Figure 11 The ultra-high performance liquid chromatography (UHPLC) chromatograms of the Qiannianjian formula granules in the overall test are shown.

[0037] Figure 12 Ultra-high performance liquid chromatography (UHPLC) spectra of Qiannianjian formula granules at different flow rates;

[0038] Figure 13 Ultra-high performance liquid chromatography (UHPLC) spectra of the Qiannianjian formula granules at different column temperatures;

[0039] Figure 14 The images show the ultra-high performance liquid chromatography (UHPLC) spectra of Qiannianjian formula granules under different chromatographic columns. Detailed Implementation

[0040] The technical solution of the present invention will be further described below.

[0041] Example 1: Sample and Standard Preparation

[0042] The specific species and sources of *Henochia lingua* medicinal materials and counterfeit medicinal materials are shown in Table 1 below:

[0043] Table 1. Specific species and sources of *Hymenoplastics* medicinal materials and counterfeit medicinal materials.

[0044]

[0045]

[0046] Qiannianjian formula granules, batch numbers: 2023080019, 2023080029, 2023080039; Qiannianjian standard decoction, batch numbers: 2019120101DG, 2019120102DG, 2019120103DG, 2019120104DG, 2019120105DG, 2019120106DG, 2019120107DG, 2019120108DG, 2019120109DG, 2019120110DG; Qiannianjian medicinal slices, batch number: 2019120101Y Products P, 2019120102YP, 2019120103YP, 2019120104YP, 2019120105YP, 2019120106YP, 2019120107YP, 2019120108YP, 2019120109YP, 2019120110YP; counterfeit taro slices, batch numbers: 2023080101YP, 2023080201YP; counterfeit taro standard decoction, batch numbers: 2023080101DG, 2023080201DG, were all prepared and supplied by Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0047] The medicinal slices are prepared by removing impurities, washing, moistening, slicing and drying the medicinal material or counterfeit products such as *Houttuynia cordata* and *Symplocos edulis*, according to the description in the current edition of the Chinese Pharmacopoeia.

[0048] The standard decoction is prepared by decocting *Hylocereus undatus*, *Alocasia macrorrhiza*, or *Spatholobus suberectus* slices twice for 30 minutes each time, in accordance with the requirements of the "Management Standards for Traditional Chinese Medicine Decoction Rooms in Medical Institutions". The decoction is then concentrated under reduced pressure and freeze-dried.

[0049] The formula granules are obtained by extracting the slices of *Homalomena occulta*, *Alocasia macrorrhiza*, or *Spatholobus suberectus* twice with water for 30 minutes each time. After the extract is concentrated under reduced pressure, maltodextrin is added and spray-dried. Then, magnesium stearate and silica are added and granulated by dry method.

[0050] 1,4,7-Cephalocarbazone triol reference standard was purchased from Yunnan Xili Biotechnology Co., Ltd., batch number BBP01070, with a purity of 98.0%, and no pretreatment is required before use.

[0051] Example 2: Identification of water-soluble characteristic components of *Hymenochaete medica*

[0052] (1) Take the *Hypericum perforatum* obtained in Example 1, grind it into powder and pass it through a No. 2 sieve. Weigh 0.7g of each powder, add 10mL of 10% methanol, seal tightly, weigh, and sonicate at 250W power and 40kHz frequency for 30min. After cooling to room temperature, weigh again and replenish the lost weight with 10% methanol. After shaking well, filter through 0.22μm and take the filtrate to obtain the test solution.

[0053] Weigh out 1,4,7-cineoletriol and prepare a 180 μg / mL solution of 1,4,7-cineoletriol using 50% methanol-water solution as a reference solution.

[0054] (2) Analysis was performed using a liquid chromatography-mass spectrometry system, with the following liquid chromatography conditions:

[0055] Setting chromatographic parameters on a Waters H-Class ultra-high performance liquid chromatograph equipped with an Alltech ELSD6000 evaporative light scattering detector:

[0056] The chromatographic column was a Waters CORTECS C18, 2.1 mm × 100 mm, 1.6 μm;

[0057] The eluent consists of mobile phase A and mobile phase B, where mobile phase A is acetonitrile and mobile phase B is water;

[0058] The elution method is gradient elution, with a mobile phase flow rate of 0.3 mL / min. The specific elution gradient is as follows or as shown for counterfeit products:

[0059] Elution time (min) Phase A (%) Phase B (%) 0 8 92 5 15 85 12 28 72 13 90 10 20 90 10

[0060] The elution column temperature was 30℃. 5 μL of the test solution and 2 μL of the reference solution were injected into the liquid chromatography system, and the chromatograms were analyzed and recorded. The separated substances were then analyzed by mass spectrometry under the following conditions: dual-jet ESI ion source, drying gas temperature 300℃, flow rate 8 L / min, nebulizer pressure 35 psi; positive ion mode acquisition, capillary voltage 4000 V, capillary outlet voltage 175 V, cone voltage 65 V; high-resolution mode data acquisition, mass-to-charge ratio acquisition range m / z 100–2000, sampling rate 1 spectra / s, sampling time 1000 ms / spectra; nebulizer pressure 5 psi.

[0061] The results are as follows Figure 1 As shown, the mass spectrometry fragments of the sample and the reference are consistent, indicating that the sample of *Hymenoplastics* contains the target compound 1,4,7-eucalyptanetriol.

[0062] Example 3: Identification of water-soluble characteristic components in the water-extracted preparation of *Hypericum perforatum*

[0063] (1) Take the granules of the formula of Qiannianjian, the slices of Qiannianjian or its counterfeit products, and the standard decoction of Qiannianjian or its counterfeit products obtained in Example 1 to prepare a test solution; use 1,4,7-cineoletriol as a reference standard to prepare a reference standard solution;

[0064] Preparation of the test solution of the formula granules: After grinding into powder and passing through a No. 2 sieve, weigh 0.3g of the Qiannianjian formula granules, add 10mL of 10% methanol, seal tightly, weigh, sonicate at 250W power and 40kHz frequency for 30min, cool to room temperature and weigh again, replenish the lost weight with 10% methanol, shake well and filter at 0.22μm, take the filtrate to obtain the Qiannianjian formula granule test solution;

[0065] Preparation of the test solution of the medicinal slices: After grinding into powder and passing through a No. 2 sieve, weigh 0.7g of either *Homalomena occulta* or its counterfeit medicinal slices, add 10mL of 10% methanol, seal tightly, weigh, and sonicate at 250W power and 40kHz frequency for 30min. After cooling to room temperature, weigh again, replenish the lost weight with 10% methanol, shake well, filter through 0.22μm, and collect the filtrate to obtain the test solution of either *Homalomena occulta* or its counterfeit medicinal slices.

[0066] Preparation of standard decoction test solution: Weigh 0.3g of either Qiannianjian or the counterfeit decoction powder, add 10mL of 10% methanol, seal tightly, weigh, sonicate at 250W power and 40kHz frequency for 30min, cool to room temperature and weigh again, replenish the lost weight with 10% methanol, shake well and filter at 0.22μm, take the filtrate to obtain the standard decoction test solution of Qiannianjian or the counterfeit.

[0067] Weigh 1,4,7-Cephalocarbazone triol and prepare a 180 μg / mL 1,4,7-Cephalocarbazone triol solution using 50% methanol-water solution as a reference solution;

[0068] (2) Set the chromatographic parameters on a Waters H-Class ultra-high performance liquid chromatograph equipped with an Alltech ELSD6000 evaporative light scattering detector:

[0069] The chromatographic column was a Waters CORTECS C18, 2.1 mm × 100 mm, 1.6 μm;

[0070] The eluent consists of mobile phase A and mobile phase B, where mobile phase A is acetonitrile and mobile phase B is water;

[0071] The elution method is gradient elution, with a mobile phase flow rate of 0.3 mL / min. The specific elution gradient is as follows or as shown for counterfeit products:

[0072] Elution time (min) Phase A (%) Phase B (%) 0 8 92 5 15 85 12 28 72 13 90 10 20 90 10

[0073] The elution column temperature was 30℃. 5 μL of the test solution and 2 μL of the reference solution were injected into the ultra-high performance liquid chromatography system, and the chromatograms were analyzed and recorded. Using 1,4,7-cineoletriol as the content determination index, the external standard two-point logarithmic equation y = 1.8253x + 12.4902 was obtained, where x is the logarithmic value of the 1,4,7-cineoletriol content (μg) and y is the logarithmic value of the peak area. Based on this calculation, the quantitative analysis of the sample was performed.

[0074] The results are shown in Table 3-5. Figures 2-4 As shown,

[0075] Table 3. Content of 1,4,7-Cephalocarbazone Triol in Qiannianjian Formula Granules

[0076] Batch Content (mg / g) 2023080019 2.2 2023080029 2.2 2023080039 2.2

[0077] Table 4. Content of 1,4,7-Cineoletriol in Qiannianjian or Counterfeit Herbal Slices

[0078]

[0079]

[0080] Table 5. Content of 1,4,7-Cephalocarbazone triol in standard decoctions of Qiannianjian or counterfeit products.

[0081] Serial number Content (%) 2019120101DG 0.25 2019120102DG 0.24 2019120103DG 0.27 2019120104DG 0.27 2019120105DG 0.29 2019120106DG 0.31 2019120107DG 0.36 2019120108DG 0.23 2019120109DG 0.32 2019120110DG 0.30 2023080101DG -- 2023080201DG --

[0082] The test results above show that Qiannianjian formula granules, Qiannianjian decoction pieces, and Qiannianjian standard decoction all contain 1,4,7-cineoletriol, while it was not detected in counterfeit products. 1,4,7-cineoletriol can be used to distinguish genuine Qiannianjian products from counterfeit products.

[0083] Example 4: Establishment and Methodological Study of the Identification Method for Homalomena occulta

[0084] 1. Preparation method of test solution

[0085] 1.1 Selection of extraction solvents at different concentrations

[0086] After grinding the Qiannianjian formula granules (2023080019) into powder and passing them through a No. 2 sieve, take 0.3g and divide into 5 parallel groups, with 2 portions per group. Add 10% methanol, 30% methanol, 50% methanol, 70% methanol, or 10mL of methanol, seal tightly, weigh, and sonicate at 250W power and 40kHz frequency for 30min. After cooling to room temperature, weigh again and replenish the lost weight with methanol of the corresponding concentration. Shake well and filter through 0.22μm. Take the filtrate to obtain the test solution of Qiannianjian formula granules extracted with methanol of different concentrations.

[0087] 3 μL of each test solution was injected into the liquid chromatograph, and the chromatographic conditions of Example 2 were used for determination. The content of 1,4,7-cineoletriol was calculated, and the results are as follows: Figure 5 As shown in Table 5, the content of 1,4,7-cineoletriol was relatively high when 10% methanol or 30% methanol was used as the extraction solvent. Subsequently, 10% methanol was selected as the extraction solvent for subsequent experiments.

[0088] Table 5. Detection results of extraction solvents at different concentrations

[0089]

[0090] 1.2 Selection of Different Extraction Methods

[0091] After grinding the Qiannianjian formula granules 2023080019 into powder and passing them through a No. 2 sieve, take 0.3g and divide into 3 parallel groups, 2 portions per group. Add 10mL of 10% methanol, seal tightly, weigh, and perform the following treatments respectively: a) ultrasonic treatment at 250W power and 40kHz frequency for 30min, b) heating and reflux for 30min, or c) shaking for 30min. After all treatments are cooled to room temperature, weigh again, replenish the lost weight with 10% methanol, shake well, filter through 0.22μm, and collect the filtrate to obtain the test solutions of Qiannianjian formula granules extracted by different methods.

[0092] 3 μL of each test solution was injected into the liquid chromatograph, and the chromatographic conditions of Example 2 were used for determination. The content of 1,4,7-cineoletriol was calculated, and the results are as follows: Figure 6 As shown in Table 6, there was no significant difference in the content of 1,4,7-cineoletriol obtained by the three extraction methods, and ultrasonic extraction was subsequently selected.

[0093] Table 6. Detection results of different extraction methods

[0094]

[0095] 1.3 Selection of different extraction durations

[0096] After grinding the Qiannianjian formula granules 2023080019 into powder and passing them through a No. 2 sieve, take 0.3g and divide into 4 parallel groups, 2 portions per group. Add 10mL of 10% methanol, seal tightly, weigh, and treat with ultrasound at 250W power and 40kHz frequency for 15, 30, 45 or 60min respectively. After all treatments are cooled to room temperature, weigh again, and make up the weight loss with 10% methanol. After shaking well, filter through 0.22μm and collect the filtrate to obtain the test solution of Qiannianjian formula granules with different extraction methods.

[0097] 3 μL of each test solution was injected into the liquid chromatograph, and the chromatographic conditions of Example 2 were used for determination. The content of 1,4,7-cineoletriol was calculated, and the results are as follows:Figure 7 As shown in Table 7, there was no significant difference in the content of 1,4,7-cineoletriol obtained from different extraction times, and the subsequent extraction time was selected as 30 min.

[0098] Table 7. Detection results for different extraction times

[0099]

[0100] 1.4 Selection of different extraction volumes

[0101] After grinding the Qiannianjian formula granules 2023080019 into powder and passing them through a No. 2 sieve, take 0.3g and divide into 3 parallel groups, 2 portions per group. Add 5, 10 or 15 mL of 10% methanol, seal tightly, weigh, and treat with ultrasound at 250W power and 40kHz frequency for 30min. After all treatments are cooled to room temperature, weigh again, and replenish the lost weight with 10% methanol. Shake well and filter through 0.22μm. Take the filtrate to obtain the test solution of Qiannianjian formula granules with different extraction methods.

[0102] 3 μL of the test solution with extraction solvent volumes of 5 mL and 10 mL, and 6 μL of the test solution with extraction solvent volume of 15 mL, were injected into the liquid chromatograph. The chromatographic conditions of Example 2 were followed, and the content of 1,4,7-cineoletriol was calculated. The results are as follows: Figure 8 As shown in Table 8, there was no significant difference in the content of 1,4,7-cineoletriol obtained from different extraction volumes. To ensure sufficient extraction and save solvent, an extraction volume of 10 mL was selected for subsequent extractions.

[0103] Table 8. Detection results for different extraction volumes

[0104]

[0105]

[0106] Based on the above results, the final method for preparing the test sample for determining the content of Qiannianjian granules is as follows:

[0107] Add 10 mL of 10% methanol to 0.3 g of the sample to be tested, weigh it, and then sonicate it for 30 min at a power of 250 W and a frequency of 40 kHz. After cooling to room temperature, weigh it again, replenish the lost weight with 10% methanol, shake well, filter it through 0.22 μm, and take the filtrate to obtain the test sample.

[0108] 2. Methodological Validation

[0109] 2.1 Linearity Validation

[0110] 0.6 μL, 0.8 μL, 1.0 μL, 1.2 μL, 1.5 μL, 2.0 μL, or 2.5 μL of a 209.92 μg / mL 1,4,7-cineole triol reference solution were injected into the liquid chromatograph, and the chromatographic conditions of Example 1 were followed. A standard curve was plotted with the logarithm of peak area on the ordinate and the logarithm of injection volume (μg) on ​​the abscissa. The regression equation for 1,4,7-cineole triol was obtained, and the results are as follows. Figure 9 As shown in Table 9, the injection amount of 1,4,7-cineoletriol showed a good linear relationship with the peak area in the range of 0.1259–0.5248 μg, with a regression equation of y = 1.7832x + 12.4283, R0. 2 =0.9997, where x is the logarithm of the injection amount (μg) of 1,4,7-cineoletriol, and y is the logarithm of the peak area.

[0111] Table 9. Relationship between the logarithmic values ​​of the injection volume and the logarithmic values ​​of the peak area of ​​1,4,7-Cephalocarbazonetriol reference standard.

[0112]

[0113]

[0114] 2.2 Precision Verification

[0115] 2.2.1 Instrument precision test

[0116] After grinding the Qiannianjian formula granules 2023080019 into powder and passing them through a No. 2 sieve, take 0.3g of the powder, add 10mL of 10% methanol, weigh the powder, and then use an ultrasonic treatment with a power of 250W and a frequency of 40kHz for 30min. After cooling to room temperature, weigh the powder again, and use 10% methanol to make up the lost weight. Shake well, filter through a 0.22μm filter, and take the filtrate to obtain the test sample.

[0117] 3 μL of the test solution was injected into the liquid chromatograph, and the determination was carried out under the chromatographic conditions of Example 2. The determination was repeated 6 times, and the peak area of ​​1,4,7-cineole was recorded. The RSD was calculated, and the results are shown in Table 10 below. The peak area RSD was 3.15%, which indicates that the instrument precision test was good.

[0118] Table 10 Instrument Precision Test

[0119] Serial number Peak area LN (peak area) 1 12588 9.4405 2 12646 9.4451 3 12689 9.4485 4 12879 9.4634 5 13589 9.5170 6 13310 9.4963 Mean 12950 9.4685 RSD % 3.15 0.33

[0120] 2.2.2 Repeatability Test

[0121] After grinding the Qiannianjian formula granules 2023080019 into powder and passing them through a No. 2 sieve, take 0.3g of the powder, add 10mL of 10% methanol, weigh the powder, and then use an ultrasonic treatment with a power of 250W and a frequency of 40kHz for 30min. After cooling to room temperature, weigh the powder again, and use 10% methanol to make up the lost weight. Shake well, filter through a 0.22μm filter, and take the filtrate. Repeat this process 6 times to obtain test samples 1-6.

[0122] Inject 3 μL of each of the test solutions 1-6 into the liquid chromatograph and perform the determination under the chromatographic conditions of Example 2. Record the peak area of ​​1,4,7-cineoletriol and calculate its RSD. The results are shown in Table 11 below. The RSD of the repeatability test of the samples is 1.89%, indicating good repeatability.

[0123] Table 11 Repeatability test results of the samples

[0124]

[0125] 2.2.3 Intermediate Precision Test

[0126] Experimenter A took the formula granules of Qiannianjian (2023080019), ground them into powder, passed them through a No. 2 sieve, took 0.3g, added 10mL of 10% methanol, weighed it, and then used ultrasonic treatment with a power of 250W and a frequency of 40kHz for 30min. After cooling to room temperature, weighed it again, and the weight loss was made up with 10% methanol. The mixture was shaken well, filtered through 0.22μm, and the filtrate was collected to obtain test sample A.

[0127] Test sample B was prepared by experimenter B using the same method.

[0128] At different times, experimenter A injected 3 μL of sample A into a Waters H-Class ultra-high performance liquid chromatograph; experimenter B injected 3 μL of sample B into a Thermo Fisher VANQUISH ultra-high performance liquid chromatograph. The chromatographic conditions of Example 2 were followed, and the results were repeated 3 times. The peak area of ​​1,4,7-cineole triol was recorded, and its RSD was calculated. The results are shown in Table 12 below. The RSD of the intermediate precision of the sample was 1.89%, indicating that the intermediate precision was good.

[0129] Table 12 Intermediate Precision Test

[0130]

[0131]

[0132] 2.3 Accuracy Test

[0133] Take 0.15g of a sample with a 1,4,7-cineole triol content of 2.1mg / g, divide it into 3 groups, 3 portions per group, for a total of 9 portions, and add 1, 2 or 3 mL of 1,4,7-cineole triol solution with a concentration of 157μg / mL to each group respectively;

[0134] Then add 10 mL of 10% methanol, weigh it, and sonicate it for 30 min using a power of 250 W and a frequency of 40 kHz. After cooling to room temperature, weigh it again, replenish the lost weight with 10% methanol, shake well, filter through 0.22 μm, and take the filtrate to obtain 3 groups of test solutions: 50%, 100%, and 150%, with 3 portions of each group.

[0135] Inject 3 μL of each sample into the liquid chromatograph and determine the chromatographic conditions as described in Example 2. Calculate the recovery rate using the formula shown below and calculate the RSD:

[0136]

[0137] Table 13 Accuracy Results

[0138]

[0139]

[0140] The results are shown in Table 13. The recovery rate of 1,4,7-cineole triol was between 96.32% and 107.77%, and the accuracy test was good.

[0141] 2.4 Specificity Test

[0142] The excipients added to the Qiannianjian formula granules are maltodextrin, silicon dioxide, and magnesium stearate. This experiment investigated the effect of negative excipient samples lacking Qiannianjian on the characteristic chromatogram of Qiannianjian formula granules.

[0143] Take a negative excipient sample lacking Homalomena occulta, grind it into powder and pass it through a No. 2 sieve. Take 0.3g of the powder, add 10mL of 10% methanol, weigh it, and then use an ultrasonic treatment with a power of 250W and a frequency of 40kHz for 30min. After cooling to room temperature, weigh it again and make up the lost weight with 10% methanol. Shake well, filter it through a 0.22μm filter, and take the filtrate to obtain the negative test sample.

[0144] After grinding the Qiannianjian formula granules 2023080019 into powder and passing them through a No. 2 sieve, take 0.3g of the powder, add 10mL of 10% methanol, weigh the powder, and then use an ultrasonic treatment with a power of 250W and a frequency of 40kHz for 30min. After cooling to room temperature, weigh the powder again, and use 10% methanol to make up the lost weight. Shake well, filter through a 0.22μm filter, and take the filtrate to obtain the test sample.

[0145] Take 3 μL of the negative test solution, the test solution, and 0.18 mg / mL 1,4,7-cineole triol reference solution and inject them into the liquid chromatograph, respectively, and determine them according to the chromatographic conditions of Example 2.

[0146] The results are as follows Figure 10 As shown, solvents and excipients do not interfere with the determination of the characteristic chromatogram of Qiannianjian formula granules, indicating that this method has good specificity.

[0147] 2.5 Integral Test

[0148] After grinding the Qiannianjian formula granules 2023080019 into powder and passing them through a No. 2 sieve, take 0.3g of the powder, add 10mL of 10% methanol, weigh the powder, and then use an ultrasonic treatment with a power of 250W and a frequency of 40kHz for 30min. After cooling to room temperature, weigh the powder again, and use 10% methanol to make up the lost weight. Shake well, filter through a 0.22μm filter, and take the filtrate to obtain the test sample.

[0149] Inject 3 μL of the test solution into the liquid chromatograph and perform the determination under the chromatographic conditions of Example 2, but with the elution gradient having the highest acetonitrile ratio, the elution time is doubled. Record the chromatogram, and the results are as follows. Figure 11 As shown, doubling the analysis time did not interfere with the determination of 1,4,7-cineoletriol content.

[0150] 2.6 Durability Test

[0151] 2.6.1 Stability Test

[0152] After grinding the Qiannianjian formula granules 2023080019 into powder and passing them through a No. 2 sieve, take 0.3g of the powder, add 10mL of 10% methanol, weigh the powder, and then use an ultrasonic treatment with a power of 250W and a frequency of 40kHz for 30min. After cooling to room temperature, weigh the powder again, and use 10% methanol to make up the lost weight. Shake well, filter through a 0.22μm filter, and take the filtrate to obtain the test sample.

[0153] 3 μL of the sample solution was injected at 0, 2, 4, 8, 12, 18, and 24 hours after preparation, and the chromatographic conditions of Example 2 were used for determination. The peak area values ​​were recorded and the RSD was calculated. The results are shown in Table 14 below. The logarithmic value of the peak area of ​​1,4,7-cineoletriol was 0.36%, indicating that the sample solution had good stability within 24 hours.

[0154] Table 14 Results of Stability Test

[0155] Time (h) Peak area LN (peak area) 0 12588 9.4405 2 13310 9.4963 4 13001 9.4728 8 12978 9.4710 12 13354 9.4996 18 13321 9.4971 24 12185 9.4080 Mean 12962 9.4693 RSD % 3.38 0.36

[0156] 2.6.2 Testing at different flow rates

[0157] After grinding the Qiannianjian formula granules 2023080019 into powder and passing them through a No. 2 sieve, take 0.3g of the powder, add 10mL of 10% methanol, weigh the powder, and then use an ultrasonic treatment with a power of 250W and a frequency of 40kHz for 30min. After cooling to room temperature, weigh the powder again, and use 10% methanol to make up the lost weight. Shake well, filter through a 0.22μm filter, and take the filtrate to obtain the test sample.

[0158] Inject 3 μL of the test sample. Set the mobile phase flow rate to 0.25, 0.30, or 0.35 mL / min. Perform the determination under the same chromatographic conditions as in Example 2. Record the peak area values ​​and calculate the RSD. The results are as follows: Figure 12 As shown in Table 15 below, within the range of 0.25-0.35 mL / min, the flow rate variation had no significant effect on the content determination of the sample, and the flow rate durability was good.

[0159] Table 15 Investigation of different flow velocities

[0160]

[0161] 2.6.3 Testing at different column temperatures

[0162] After grinding the Qiannianjian formula granules 2023080019 into powder and passing them through a No. 2 sieve, take 0.3g of the powder, add 10mL of 10% methanol, weigh the powder, and then use an ultrasonic treatment with a power of 250W and a frequency of 40kHz for 30min. After cooling to room temperature, weigh the powder again, and use 10% methanol to make up the lost weight. Shake well, filter through a 0.22μm filter, and take the filtrate to obtain the test sample.

[0163] Inject 3 μL of the test sample, set the column temperature to 25, 30, or 35 °C, and perform the determination under the same chromatographic conditions as in Example 2. Record the peak area values ​​and calculate the RSD. The results are as follows: Figure 13 As shown in Table 16 below, within the range of 25-35℃, column temperature variation has no significant effect on the content determination of the sample, and the column temperature durability is good.

[0164] Table 16 Investigation at different column temperatures

[0165]

[0166] 2.6.4 Testing with different chromatographic columns

[0167] After grinding the Qiannianjian formula granules 2023080019 into powder and passing them through a No. 2 sieve, take 0.3g of the powder, add 10mL of 10% methanol, weigh the powder, and then use an ultrasonic treatment with a power of 250W and a frequency of 40kHz for 30min. After cooling to room temperature, weigh the powder again, and use 10% methanol to make up the lost weight. Shake well, filter through a 0.22μm filter, and take the filtrate to obtain the test sample.

[0168] Inject 3 μL of the test sample. Use either an Agilent ZORBAX SB Aq RRHD column (2.1 × 100 mm, 1.8 μm), a Waters CORTECS C18 column (2.1 mm × 100 mm, 1.6 μm), or a Thermo Hypersil GLOD aQ column (2.1 × 100 mm, 1.9 μm) for the chromatographic determination. All other chromatographic conditions are the same as in Example 2. Record the peak area values ​​and calculate the RSD. The results are as follows: Figure 14 As shown, different chromatographic columns have no significant effect on the content determination of the sample, indicating that the chromatographic columns have good applicability.

Claims

1. A method for identifying the component of *Homalomena occulta* in a sample, characterized by the following steps: include: (1) Prepare the sample solution to be tested; (2) Setting chromatographic parameters on the ultra-high performance liquid chromatograph: (21) Chromatographic column, a chromatographic column with octadecylsilane bonded silica gel as the stationary phase; (22) The eluent includes mobile phase A and mobile phase B, wherein mobile phase A is acetonitrile and mobile phase B is water; (23) Elution method: The flow rate of the mobile phase is 0.2-0.4 mL / min, and the gradient elution is as follows: 0 min, the volume ratio of mobile phase A is 6-10% and the volume ratio of mobile phase B is 90-94%; 5 min, the volume ratio of mobile phase A is 13-17% and the volume ratio of mobile phase B is 83-87%; 12 min, the volume ratio of mobile phase A is 26-30% and the volume ratio of mobile phase B is 70-74%. (24) The elution column temperature is 20-40℃; (3) Inject the sample solution to be tested into an ultra-high performance liquid chromatograph, analyze and record the chromatogram, and compare it with the chromatogram of 1,4,7-cineole triol standard for identification.

2. The method for identifying the Homalomena component in a sample according to claim 1, characterized in that, Step 1, the preparation of the sample solution to be tested, includes: adding methanol-water solution to the sample, extracting, filtering, and obtaining the sample solution to be tested.

3. The method for identifying the Homalomena component in a sample according to claim 2, characterized in that, The methanol concentration in the methanol-water solution is not higher than 70%, and the amount used is 15-50 mL per gram of sample.

4. The method for identifying the Homalomena component in a sample according to claim 2, characterized in that, The extraction is performed by any one of ultrasonic treatment, heating and reflux, or shaking extraction, with an extraction time of 15-60 minutes.

5. The method for identifying the Homalomena component in a sample according to claim 4, characterized in that, The extraction is performed using ultrasound at 200-300W power and 35-45kHz frequency for 15-60 minutes.

6. The method for identifying the Homalomena component in a sample according to claim 1, characterized in that, The chromatographic column with octadecylsilane-bonded silica gel as the stationary phase in step 21 has a column length of 100 mm, an inner diameter of 2.1 mm, and a particle size of 1.6-1.9 μm.

7. The method for identifying the Homalomena component in a sample according to claim 1, characterized in that, The flow rate of the mobile phase in step 23 is 0.25-0.35 mL / min.

8. The method for identifying the Homalomena component in a sample according to claim 1, characterized in that, The elution column temperature in step 24 is 25-35℃.

9. The method for identifying the Homalomena component in a sample according to claim 1, characterized in that, In step 3, the volume of sample injected into the high-performance liquid chromatograph is 2-8 μL.

10. The method for identifying the Homalomena component in a sample according to claim 1, characterized in that, Its features are, The ultra-high performance liquid chromatograph is an ultra-high performance liquid chromatograph equipped with an evaporative light scattering detector.

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