A method for constructing HPLC characteristic spectrum of linseed medicinal materials, decoction pieces, extracts and preparations thereof and its application

Abstract

The present invention belongs to the field of detection technology, and in particular relates to a method for constructing an HPLC characteristic spectrum of flaxseed medicinal materials, decoction pieces, extracts, and preparations thereof, and its application. The HPLC characteristic spectrum construction method provided by the present invention comprises the following steps: preparing a test sample, wherein the test sample is flaxseed medicinal materials, flaxseed decoction pieces, extracts, and preparations thereof; dissolving the test sample to obtain a test sample solution; measuring the test sample solution by high performance liquid chromatography to obtain an HPLC characteristic spectrum of the corresponding test sample; the chromatographic conditions of the high performance liquid chromatography are: a C18 column; mobile phase A is acetonitrile, mobile phase B is a 0.2 vol% acetic acid solution, and gradient elution. The characteristic spectrum construction method provided by the present invention can provide a more scientific basis for the identification of flaxseed medicinal materials, decoction pieces, extracts, and preparations thereof.

Description

Technical Field

[0001] The present invention belongs to the field of detection technology, and in particular relates to a method for constructing an HPLC characteristic spectrum of flaxseed medicinal materials, decoction pieces, extracts, and preparations thereof, and applications thereof. Background Art

[0002] Flaxseed, the dried, mature seeds of Linum usitatissimum L. (Linaceae), has moisturizing and wind-relieving properties. To ensure the uniformity and stability of flaxseed medicinal materials, decoction pieces, extracts, and formulated granules, it is essential to develop a new characteristic mapping method to control their quality. Summary of the Invention

[0003] In view of this, the object of the present invention is to provide a method for constructing an HPLC characteristic spectrum of linseed medicinal materials, decoction pieces, extracts and preparations thereof and its application. The construction method provided by the present invention can provide a more scientific basis for the identification of linseed medicinal materials, decoction pieces, extracts and preparations thereof.

[0004] The present invention provides a method for constructing an HPLC characteristic spectrum of linseed medicinal materials, decoction pieces, extracts, and preparations, comprising the following steps:

[0005] preparing test samples, wherein the test samples are flaxseed medicinal materials, flaxseed decoction pieces, flaxseed extracts and preparations thereof;

[0006] Dissolving the test sample to obtain a test sample solution;

[0007] The test solution is measured by high performance liquid chromatography to obtain a HPLC characteristic spectrum of the corresponding test sample;

[0008] The chromatographic conditions of the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column; the mobile phase A is acetonitrile, the mobile phase B is a 0.2 vol% acetic acid solution, and the elution is performed in a gradient manner.

[0009] In the construction method provided by the present invention, the flaxseed slices are prepared by processing flaxseed medicinal materials, that is, removing impurities to obtain the flaxseed slices; the flaxseed extract is a freeze-dried powder prepared by extracting the flaxseed slices; and the flaxseed preparation is a granule prepared by concentrating the flaxseed extract.

[0010] In the construction method provided by the present invention, the dissolving solvent is preferably a 30 vol% methanol solution, which has a large amount of information on the chromatographic peaks and a good separation degree.

[0011] In the construction method provided by the present invention, the dissolution is preferably carried out under ultrasonic assistance; the power of the ultrasonic assistance is preferably 580-620 W, more preferably 600 W; the frequency of the ultrasonic assistance is preferably 35-45 kHz, more preferably 40 kHz; the time of the ultrasonic assistance is preferably 25-35 min, more preferably 30 min.

[0012] In the construction method provided by the present invention, when the test sample is linseed medicinal material or linseed decoction pieces, the specific process of dissolving and preparing the test sample solution preferably includes: decocting the test sample with water, filtering, evaporating the filtrate to dryness, mixing the evaporation residue with a solvent, ultrasonic-assisted dissolution, cooling, shaking, and filtering. The resulting filtrate is the test sample solution; wherein the dosage ratio of the test sample to water is preferably (0.8-1.2) g:50 mL, more preferably 1 g:50 mL; the decoction time is preferably 25-35 min, more preferably 28-32 min, and even more preferably 30 min; and the dosage ratio of the solvent to the test sample is preferably 25 mL:(0.8-1.2) g, more preferably 25 mL:1 g.

[0013] In the construction method provided by the present invention, when the test sample is linseed extract, the specific process of dissolving and preparing the test sample solution preferably includes: mixing the test sample with a solvent, ultrasonic-assisted dissolution, cooling, shaking, and filtering. The resulting filtrate is the test sample solution; wherein the dosage ratio of the test sample to the solvent is preferably (0.3-0.7) g:25 mL, more preferably 0.5 g:25 mL.

[0014] In the construction method provided by the present invention, when the test sample is a linseed preparation, the specific process of dissolving and preparing the test sample solution preferably includes: grinding the test sample and mixing it with a solvent, ultrasonic-assisted dissolution, cooling, shaking, filtering, and the resulting filtrate is the test sample solution; wherein the dosage ratio of the test sample to the solvent is preferably (0.3-0.7) g:25 mL, more preferably 0.5 g:25 mL.

[0015] In the construction method provided by the present invention, when performing the high performance liquid chromatography determination, the specific process of the gradient elution is preferably:

[0016] 0-7 min, phase A: 1 vol%, phase B: 99 vol%;

[0017] 7-20 min, phase A: 1-4 vol%, phase B: 99-96 vol%;

[0018] 20-28 min, phase A: 4 vol%, phase B: 96 vol%;

[0019] 28-36 min, phase A: 4-10 vol%, phase B: 96-90 vol%;

[0020] 36-48 min, phase A: 10-20 vol%, phase B: 90-80 vol%.

[0021] In the construction method provided by the present invention, when performing the high performance liquid chromatography determination, the flow rate of the mobile phase is preferably 0.8 mL / min. Under this flow rate condition, the chromatogram peak shape obtained is better and the baseline is more stable.

[0022] In the construction method provided by the present invention, when performing the high performance liquid chromatography determination, the detection wavelength is preferably 278 nm. Under this detection wavelength condition, the chromatogram obtained has a larger amount of information and a more stable baseline.

[0023] In the construction method provided by the present invention, when performing the high performance liquid chromatography determination, the injection volume is preferably 10 μL.

[0024] In the construction method provided by the present invention, when the high performance liquid chromatography determination is performed, the filler in the chromatographic column is octadecylsilane bonded silica gel; the column length of the chromatographic column is preferably 200 to 300 mm, more preferably 250 mm; the inner diameter of the chromatographic column is preferably 4 to 5 mm, more preferably 4.6 mm; the particle size of the filler in the chromatographic column is preferably 3 to 7 μm, more preferably 5 μm.

[0025] In the construction method provided by the present invention, when performing the high performance liquid chromatography determination, the column temperature of the chromatographic column is preferably 30° C. Under this column temperature condition, the peak shape of the chromatogram obtained is relatively symmetrical and the retention time is relatively suitable.

[0026] In the construction method provided by the present invention, when performing the high performance liquid chromatography determination, the theoretical plate number calculated based on the tryptophan peak should be no less than 3000.

[0027] In the construction method provided by the present invention, it is preferred that the following steps are further included:

[0028] Dissolve the linseed control medicinal material to obtain a control medicinal material reference solution; dissolve tryptophan to obtain a reference substance reference solution;

[0029] The control medicinal material reference solution and the reference substance reference solution are measured by high performance liquid chromatography to obtain a chromatogram of the reference substance; and the components of the HPLC characteristic spectrum of the test sample are qualitatively analyzed based on the chromatogram of the reference substance.

[0030] In the construction method provided by the present invention, the specific process of dissolving and preparing the control medicinal material reference solution preferably includes: decocting linseed control medicinal material with water, filtering, evaporating the filtrate to dryness, mixing the evaporation residue with a solvent, dissolving with ultrasound-assisted method, cooling, shaking, and filtering. The obtained filtrate is the control medicinal material reference solution. The ratio of the linseed control medicinal material to water is preferably (0.8-1.2) g:50 mL, more preferably 1 g:50 mL; the decocting time is preferably 25-35 min, more preferably 28-32 min, and even more preferably 30 min; the solvent is preferably a 30 vol% methanol solution; the ratio of the solvent to the linseed control medicinal material is preferably 25 mL:(0.8-1.2) g, more preferably 25 mL:1 g; the ultrasonic-assisted power is preferably 580-620 W, more preferably 600 W; the ultrasonic-assisted frequency is preferably 35-45 kHz, more preferably 40 kHz; and the ultrasonic-assisted time is preferably 25-35 min, more preferably 30 min.

[0031] In the construction method provided by the present invention, the specific process of dissolving and preparing the reference substance solution preferably includes: mixing tryptophan with a solvent to obtain a reference substance solution; wherein the solvent is preferably a 30 vol% methanol solution; the dosage ratio of the tryptophan to the solvent is preferably (8 to 12) μg:1 mL, more preferably 10 μg:1 mL.

[0032] In the construction method provided by the present invention, it is preferred that the following steps are further included:

[0033] The similarity of an HPLC characteristic spectrum of linseed medicinal material is evaluated using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and an HPLC standard characteristic spectrum of linseed medicinal material consisting of six characteristic peaks is obtained. In the HPLC standard characteristic spectrum, peak 6 corresponding to the peak of a tryptophan reference substance is the S peak. The relative retention times of the characteristic peaks and the S peak are calculated, and the relative retention times are within ±10% of the specified values, which are: 0.35 (peak 1), 0.55 (peak 2), 0.65 (peak 3), 0.69 (peak 4), 0.73 (peak 5), and 1.00 (peak 6).

[0034] In the construction method provided by the present invention, it is preferred that the following steps are further included:

[0035] The similarity of HPLC characteristic spectra of linseed slices is evaluated by using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and an HPLC standard characteristic spectra of linseed slices consisting of 6 characteristic peaks is obtained; in the HPLC standard characteristic spectra, peak 6 corresponding to the peak of a tryptophan reference substance is the S peak, and the relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time is within the range of ±10% of the specified value, and the specified value is: 0.35 (peak 1), 0.55 (peak 2), 0.65 (peak 3), 0.69 (peak 4), 0.73 (peak 5), and 1.00 (peak 6).

[0036] In the construction method provided by the present invention, it is preferred that the following steps are further included:

[0037] The similarity of the HPLC characteristic spectrum of the linseed extract is evaluated using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and an HPLC standard characteristic spectrum of the linseed extract consisting of six characteristic peaks is obtained. In the HPLC standard characteristic spectrum, peak 6 corresponding to the peak of a tryptophan reference substance is the S peak. The relative retention times of the characteristic peaks and the S peak are calculated, and the relative retention times are within ±10% of the specified values, which are: 0.35 (peak 1), 0.55 (peak 2), 0.65 (peak 3), 0.69 (peak 4), 0.73 (peak 5), and 1.00 (peak 6).

[0038] In the construction method provided by the present invention, it is preferred that the following steps are further included:

[0039] The similarity of the HPLC characteristic spectrum of the flaxseed preparation was evaluated using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and an HPLC standard characteristic spectrum of the flaxseed preparation consisting of six characteristic peaks was obtained. In the HPLC standard characteristic spectrum, peak 6 corresponding to the peak of a tryptophan reference substance was the S peak. The relative retention times of the characteristic peaks and the S peak were calculated, and the relative retention times were within ±10% of the specified values, which were: 0.35 (peak 1), 0.55 (peak 2), 0.65 (peak 3), 0.69 (peak 4), 0.73 (peak 5), and 1.00 (peak 6).

[0040] The present invention also provides a method for identifying flaxseed medicinal materials, decoction pieces, extracts, and preparations, which uses the HPLC characteristic spectrum obtained by the construction method described in the above technical solution as a basis for identifying flaxseed medicinal materials, decoction pieces, extracts, and preparations.

[0041] Compared with the existing technology, the present invention provides a method for constructing HPLC characteristic spectra of linseed medicinal materials, decoction pieces, extracts, and preparations and its application. The method provided by the present invention can be used to establish HPLC characteristic spectra of linseed medicinal materials, decoction pieces, extracts, and preparations, has good repeatability, precision, and stability, and can provide a more scientific basis for the identification of linseed medicinal materials, decoction pieces, extracts, and preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0043] Figure 1 This is a chromatographic peak identification diagram of the characteristic spectrum of flaxseed extract provided in Example 1 of the present invention;

[0044] Figure 2 is a spectrum of the tryptophan reference substance provided in Example 1 of the present invention;

[0045] Figure 3 This is a spectrum of tryptophan in the test sample provided in Example 1 of the present invention;

[0046] Figure 4 This is a graph showing the experimental results of different instruments used to examine the flaxseed extract provided in Example 1 of the present invention;

[0047] Figure 5 This is a graph showing the experimental results of different chromatographic columns for the flaxseed extract provided in Example 1 of the present invention;

[0048] Figure 6 This is a characteristic spectrum verification diagram of 16 batches of linseed extract samples provided in Example 1 of the present invention;

[0049] Figure 7 This is a comparison graph of the characteristic graph of the linseed extract provided in Example 1 of the present invention;

[0050] Figure 8 This is a graph showing the experimental results of investigating the dissolution mode during the preparation of the flaxseed medicinal material test solution provided in Example 2 of the present invention;

[0051] Figure 9 This is a chromatographic peak identification diagram of the characteristic spectrum of flaxseed medicinal material provided in Example 2 of the present invention;

[0052] Figure 10 This is a graph showing the experimental results of flaxseed medicinal materials investigated using different instruments, as provided in Example 2 of the present invention;

[0053] Figure 11 This is a graph showing the experimental results of different chromatographic columns for investigating flaxseed medicinal materials provided in Example 2 of the present invention;

[0054] Figure 12 This is a characteristic spectrum verification diagram of 16 batches of flaxseed medicinal material samples provided in Example 2 of the present invention;

[0055] Figure 13 This is a comparison of the characteristic spectrum of flaxseed medicinal materials provided in Example 2 of the present invention;

[0056] Figure 14 This is a characteristic spectrum verification diagram of 16 batches of linseed decoction pieces samples provided in Example 2 of the present invention;

[0057] Figure 15 This is a comparison graph of the characteristic graph of the linseed decoction pieces provided in Example 2 of the present invention;

[0058] Figure 16 This is a chromatogram of different wavelengths of the flaxseed formula granules provided in Example 3 of the present invention;

[0059] Figure 17 This is a column temperature chromatogram provided in Example 3 of the present invention;

[0060] Figure 18 This is a flow rate investigation chromatogram provided in Example 3 of the present invention;

[0061] Figure 19 This is a graph showing the experimental results of investigating the dissolution mode during the preparation of the test solution provided in Example 3 of the present invention;

[0062] Figure 20 This is a graph showing the experimental results of investigating the dissolving solvent during the preparation of the test solution provided in Example 3 of the present invention;

[0063] Figure 21 This is a graph showing the experimental results of investigating the dissolution time during the preparation of the test solution provided in Example 3 of the present invention;

[0064] Figure 22 This is a graph showing the experimental results of investigating the amount of solvent added during the preparation of the test solution provided in Example 3 of the present invention;

[0065] Figure 23 This is a chromatographic peak identification diagram of the characteristic spectrum of the linseed formula granules provided in Example 3 of the present invention;

[0066] Figure 24 This is a graph showing the experimental results of different instruments used to examine the flaxseed formula particles provided in Example 3 of the present invention;

[0067] Figure 25This is a graph showing the experimental results of different chromatographic columns used to examine the flaxseed formula particles provided in Example 3 of the present invention;

[0068] Figure 26 This is a verification diagram of the characteristic spectra of three batches of flaxseed formula granules provided in Example 3 of the present invention;

[0069] Figure 27 This is a comparison chart of the characteristic chart of the flaxseed formula granules provided in Example 3 of the present invention. DETAILED DESCRIPTION

[0070] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0071] Table 1 Summary of origin information of flaxseed medicinal materials

[0072]

[0073] Preparation of flaxseed slices

[0074] Take linseed medicinal materials and remove impurities to obtain linseed slices. 16 batches of linseed medicinal materials were processed into 16 batches of linseed slices. The specific corresponding information is shown in Table 2.

[0075] Table 216 Batch of Flaxseed Processing Correspondence

[0076]

[0077] Preparation of linseed extract

[0078] Take 100g of flaxseed slices, add water and decoct twice, add 10 times the water for the first decoction, soak for 30 minutes, boil, keep boiling for 30 minutes, filter through a 200-mesh sieve, and immediately cool to room temperature; add 8 times the water for the second decoction, boil, keep boiling for 20 minutes, filter through a 200-mesh sieve, combine the decoctions, immediately cool to room temperature, concentrate, freeze-dry, and package to obtain the product.

[0079] 16 batches of linseed extracts were prepared from 16 batches of linseed slices. The corresponding information is shown in Table 3 below.

[0080] Table 3 Corresponding table of linseed extract preparation

[0081]

[0082] Example 1

[0083] Construction of HPLC characteristic spectrum of flaxseed extract:

[0084] 1) Experimental instruments and materials

[0085] Instrument: Waters, Agilent high performance liquid chromatograph. Unless otherwise specified, Waters high performance liquid chromatograph is used by default.

[0086] Electronic balances: ME204E / 02, MS205DU, XP26 (Mettler-Toledo Instruments Co., Ltd.);

[0087] Ultrapure water machine: Cell type 1810A (Shanghai Moller Scientific Instrument Co., Ltd.);

[0088] Ultrasonic cleaner: KQ-600DB (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0089] Chromatographic column: AtlantisTM T34.6×250mm (filler particle size 5μm), HSS T34.6×250mm (filler particle size 5μm), Shim-pack Scepter C18-1204.6×250mm (filler particle size 5μm), unless otherwise specified, AtlantisTM T34.6×250mm (filler particle size 5μm) chromatographic columns are used by default.

[0090] 2) Reagents and test drugs

[0091] Acetic acid and acetonitrile were of chromatographic grade, water was ultrapure water, tryptophan (China Food and Drug Inspection Institute, batch number: 140686-202205, content is 100.0%), and the remaining reagents were of analytical grade;

[0092] Linseed as a control medicinal material (Chengdu Pusi Biotechnology Co., Ltd., batch number: PS030308);

[0093] Linseed extract freeze-dried powder (prepared by Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch numbers: YMZ-BT-230601, YMZ-BT-230701, YMZ-BT-230702, YMZ-BT-230703, YMZ-BT-230704, YMZ-BT-230705, YMZ-BT-230706, YMZ-BT-230707, YMZ-BT-230708, YMZ-BT-230709, YMZ-BT-230710, YMZ-BT-230711, YMZ-BT-230712, YMZ-BT-230713, YMZ-BT-230714, YMZ-BT-230715, YMZ-BT-230716).

[0094] 3) Feature spectrum detection method

[0095] Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel was used as the filler; acetonitrile was used as mobile phase A, and 0.2 vol% acetic acid solution was used as mobile phase B, with gradient elution performed as specified in Table 4; the flow rate was 0.8 mL / min; the column temperature was 30°C; the detection wavelength was 278 nm; and the number of theoretical plates calculated based on the tryptophan peak should be no less than 3000.

[0096] Table 4 Gradient elution program

[0097] Time (minutes) Mobile phase A (vol%) Mobile phase B (vol%) 0～7 1 99 7～20 1→4 99→96 20～28 4 96 28～36 4→10 96→90 36～48 10→20 90→80

[0098] Preparation of reference solution: Take 1 g of linseed control medicinal material, place it in a stoppered conical flask, add 50 mL of water, boil for 30 minutes, filter, evaporate the filtrate to dryness, add 25 mL of 30 vol% methanol to the residue, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the control medicinal material reference solution; take an appropriate amount of tryptophan reference substance, accurately weigh it, and add 30 vol% methanol to make a solution containing 10 μg of tryptophan per 1 mL, which is used as the reference substance solution.

[0099] Preparation of test solution: Take an appropriate amount of the product, grind it into powder, take about 0.5 g, accurately weigh it, place it in a stoppered conical flask, accurately add 25 mL of 30 vol% methanol, stopper it tightly, weigh it, and treat it ultrasonically (power 600 W, frequency 40 kHz) for 30 minutes. Let it cool, weigh it again, make up the lost weight with 30 vol% methanol, shake it well, filter it, and take the filtrate.

[0100] Determination method: Accurately aspirate 10 μL of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0101] 4) Methodological investigation

[0102] 4.1) Chromatographic peak identification

[0103] According to the experimental conditions proposed above, a linseed extract test solution, a tryptophan reference solution, and a linseed reference medicinal material reference solution were prepared. At the same time, a negative control solution lacking linseed extract (i.e., a blank solution) was prepared according to the experimental conditions proposed above for chromatographic detection.

[0104] The characteristic peaks of flaxseed extract were located, and the results are shown in Figures 1 to 3 , Figure 1 This is the chromatographic peak identification diagram of the characteristic spectrum of flaxseed extract provided in Example 1 of the present invention. Figure 2 is a spectrum of the tryptophan reference substance provided in Example 1 of the present invention, Figure 3This is a spectrum of tryptophan in the sample provided in Example 1 of the present invention. Figures 1 to 3 It can be seen that the retention time and spectrum of the tryptophan reference substance can match the retention time and spectrum of the target peak in the flaxseed extract. Figure 1 There is one-to-one correspondence and no interference from the negative solution, so the method has good specificity.

[0105] 4.2) Repeatability study

[0106] Six portions of linseed extract (batch number: YMZ-BT-230601) were accurately weighed and prepared and measured according to the proposed experimental method. The results are shown in Table 5.

[0107] Table 5 Repeatability study - relative retention time of characteristic peaks

[0108]

[0109] As shown in Table 5, the relative retention times of the characteristic peaks are consistent, and the RSD of the relative retention times is between 0.00% and 0.40%. This method has good repeatability.

[0110] 4.3) Intermediate precision study

[0111] Based on the experimental conditions proposed above, 12 portions of linseed extract were accurately weighed to prepare test solutions, which were then measured on Waters (No. 1-6) and Agilent (No. 7-12) high performance liquid chromatographs. The results are shown in Table 1. Figure 4 and Table 6, Figure 4 This is a graph showing the experimental results of different instruments for investigating the flaxseed extract provided in Example 1 of the present invention.

[0112] Table 6 Intermediate precision - relative retention time of characteristic peaks

[0113]

[0114] pass Figure 4 As shown in Table 6, this method has good intermediate precision.

[0115] 4.4) Durability test

[0116] On the basis of the experimental conditions proposed above, the chromatographic columns were: AtlantisTM T34.6×250mm / 5μm (chromatographic column 1), HSS T34.6×250mm / 5μm (chromatographic column 2) and Shim-pack ScepterC18-1204.6×250mm / 5μm (chromatographic column 3) were investigated. Figure 5 and Table 7, Figure 5 This is a graph showing the experimental results of different chromatographic columns for the flaxseed extract provided in Example 1 of the present invention.

[0117] Table 7 Column durability investigation - relative retention time of characteristic peaks

[0118]

[0119]

[0120] pass Figure 5 As shown in Table 7, the RSDs of the relative retention times of the characteristic peaks of different brands of chromatographic columns range from 0.00 to 10.69%.

[0121] 4.5) Stability study

[0122] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 4h, 8h, 12h, 16h and 24h respectively. The results are shown in Table 8.

[0123] Table 8 Stability Study—Characteristic Peak Retention Time

[0124]

[0125] As shown in Table 8, the RSD of the corresponding characteristic peak retention time is between 0.08% and 0.22%, and the sample solution is relatively stable within 24 hours.

[0126] In summary, the RSDs of the retention time / relative retention time of each characteristic peak met the requirements in all the above investigations, and this method is good.

[0127] 5) Determination of characteristic peaks and establishment of reference maps

[0128] 5.1) Verification results of 16 batches of flaxseed extract

[0129] The proposed method was used to determine the characteristic spectra of 16 samples of this product and calculate the relative retention time. The results are shown in Figure 6 and Table 9, Figure 6 This is a characteristic spectrum verification diagram of 16 batches of linseed extract samples provided in Example 1 of the present invention. The batch numbers from bottom to top are: YMZ-BT-230701, YMZ-BT-230702, YMZ-BT-230703, YMZ-BT-230704, YMZ-BT-230705, YMZ-BT-230706, YMZ-BT-230707, YMZ-BT-230708, YMZ-BT-230709, YMZ-BT-230710, YMZ-BT-230711, WFG-BT-210912, YMZ-BT-230713, YMZ-BT-230714, YMZ-BT-230715, and YMZ-BT-230716.

[0130] Table 9 Relative retention times of characteristic peaks of 16 batches of linseed extract samples

[0131]

[0132]

[0133] Six peaks with good reproducibility were selected as characteristic peaks based on the principles of stable relative retention times, consistent detection across all batches of samples, and relatively high peak heights. Table 9 shows that when Peak 6 was used as the S peak, the RSDs of the relative retention times of the characteristic peaks of Peaks 1 through 5 ranged from 0.00 to 1.15%.

[0134] 5.2) Establishment of relative retention time limit values

[0135] The summary of the methodology inspection items and verification results is shown in Table 10:

[0136] Table 10 Summary of RSD (%) results of each item of the methodology - relative retention time (retention time)

[0137]

[0138] Table 10 shows that, with the exception of column durability, the RSD values of the retention times or relative retention times of each characteristic peak met the requirements in all of the above considerations, indicating that this method is good. The above six characteristic peaks will be included in subsequent investigations.

[0139] Final regulations: It is tentatively stipulated that 6 characteristic peaks should appear in the chromatogram of the test sample, and the retention times should correspond to the 6 characteristic peaks in the chromatogram of the control medicinal material reference substance, among which Peak 6 should correspond to the retention time of the tryptophan reference substance peak; the peak corresponding to the tryptophan reference substance peak is the S peak, and the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time should be within the range of ±10% of the specified value, and the specified values are: 0.35 (peak 1), 0.55 (peak 2), 0.65 (peak 3), 0.69 (peak 4), 0.73 (peak 5), and 1.00 (peak 6).

[0140] Sixteen batches of flaxseed extracts were synthesized using the Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version), and a comparison of the characteristic spectra of flaxseed extracts was established, such as Figure 7 As shown, Figure 7 This is a comparison graph of the characteristic graph of the flaxseed extract provided in Example 1 of the present invention.

[0141] 6) Determination of the characteristic spectrum method of flaxseed extract

[0142] Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel was used as the filler; acetonitrile was used as mobile phase A, and 0.2 vol% acetic acid solution was used as mobile phase B, with gradient elution performed as specified in Table 4; the flow rate was 0.8 mL / min; the column temperature was 30°C; the detection wavelength was 278 nm; and the number of theoretical plates calculated based on the tryptophan peak should be no less than 3000.

[0143] Preparation of reference solution: Take 1 g of linseed control medicinal material, place it in a stoppered conical flask, add 50 mL of water, boil for 30 minutes, filter, evaporate the filtrate to dryness, add 25 mL of 30 vol% methanol to the residue, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the control medicinal material reference solution; take an appropriate amount of tryptophan reference substance, accurately weigh it, and add 30 vol% methanol to make a solution containing 10 μg of tryptophan per 1 mL, which is used as the reference substance solution.

[0144] Preparation of test solution: Take an appropriate amount of the product, grind it into powder, take about 0.5 g, accurately weigh it, place it in a stoppered conical flask, accurately add 25 mL of 30 vol% methanol, stopper it tightly, weigh it, and treat it ultrasonically (power 600 W, frequency 40 kHz) for 30 minutes. Let it cool, weigh it again, make up the lost weight with 30 vol% methanol, shake it well, filter it, and take the filtrate.

[0145] Determination method: Accurately aspirate 10 μL of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0146] Example 2

[0147] Construction of HPLC characteristic spectrum of flaxseed medicinal materials and decoction pieces:

[0148] 1) Experimental instruments and materials

[0149] Same as Example 1, no further details will be given.

[0150] 2) Reagents and test drugs

[0151] Linseed medicinal material (prepared by Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch number: YMZ01

[0152] , YMZ02, YMZ03, YMZ04, YMZ05, YMZ06, YMZ07, YMZ08, YMZ09, YMZ10, YMZ11, YMZ12, YMZ13, YMZ14, YMZ15, YMZ16);

[0153] Linseed slices (prepared by Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch numbers: YMZ-YP-01, YMZ-YP-02, YMZ-YP-03, YMZ-YP-04, YMZ-YP-05, YMZ-YP-06, YMZ-YP-07, YMZ-YP-08, YMZ-YP-09, YMZ-YP-10, YMZ-YP-11, YMZ-YP-12, YMZ-YP-13, YMZ-YP-14, YMZ-YP-15, YMZ-YP-16);

[0154] Other reagents and drugs are the same as those in Example 1 and will not be described in detail.

[0155] 3) Feature spectrum detection method

[0156] Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel was used as the filler; acetonitrile was used as mobile phase A, and 0.2 vol% acetic acid solution was used as mobile phase B, with gradient elution performed as specified in Table 4; the flow rate was 0.8 mL / min; the column temperature was 30°C; the detection wavelength was 278 nm; and the number of theoretical plates calculated based on the tryptophan peak should be no less than 3000.

[0157] Preparation of reference solution: Take 1 g of linseed control medicinal material, place it in a stoppered conical flask, add 50 mL of water, boil for 30 minutes, filter, evaporate the filtrate to dryness, add 25 mL of 30 vol% methanol to the residue, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the control medicinal material reference solution; take an appropriate amount of tryptophan reference substance, accurately weigh it, and add 30 vol% methanol to make a solution containing 10 μg of tryptophan per 1 mL, which is used as the reference substance solution.

[0158] Preparation of test solution: Take an appropriate amount of the product powder (passed through No. 3 sieve), take about 1.0 g, place it in a stoppered conical flask, add 50 mL of water, boil for 30 minutes, filter, evaporate the filtrate to dryness, add 25 mL of 30 vol% methanol to the residue, stopper it, and ultrasonically treat it (power 600 W, frequency 40 kHz) for 30 minutes. Let it cool, shake well, filter, and take the filtrate.

[0159] Determination method: Accurately aspirate 10 μL of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0160] 4) Preparation of test solution (dissolution method)

[0161] Test solution 1 (ultrasonic sample): Take about 1.0 g of the product powder (passed through No. 3 sieve) (batch number: YMZ05), place it in a stoppered conical flask, add 25 mL of 30 vol% methanol, stopper it tightly, and ultrasonically treat it (power 600 W, frequency 40 kHz) for 30 minutes. Cool, shake well, filter, and take the filtrate to obtain the product.

[0162] Test solution 2 (reflux sample): Take about 1.0 g of the product powder (passed through No. 3 sieve) (batch number: YMZ05), place it in a stoppered conical flask, add 25 mL of 30 vol% methanol, stopper it tightly, reflux for 30 minutes, cool, shake well, filter, and take the filtrate.

[0163] Test solution 3 (water extraction): Take about 1.0 g of the product powder (passed through No. 3 sieve) (batch number: YMZ05), add 50 mL of water, boil for half an hour, filter, evaporate to dryness, add 25 mL of 30 vol% methanol to the residue, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate.

[0164] According to the experimental conditions proposed above, chromatographic determination was performed on ultrasonic sample, reflux sample and water extraction sample respectively. The results are shown in Figure 8 , Figure 8 This is a graph showing the experimental results of the dissolution mode of the flaxseed medicinal material sample solution prepared in Example 2 of the present invention. Figure 8 It can be seen that the chromatographic peak information of the water extraction sample is larger, which is better than that of the ultrasonic sample and reflux sample. Therefore, this experiment chose water decoction followed by ultrasonication as the dissolution method of the test sample.

[0165] 5) Methodological investigation

[0166] 5.1) Chromatographic peak identification

[0167] According to the experimental conditions proposed above, linseed medicinal material test solution, tryptophan reference solution, and linseed control medicinal material reference solution were prepared. At the same time, according to the experimental conditions proposed above, a negative control solution lacking linseed medicinal material (i.e., blank solution) was prepared for chromatographic detection.

[0168] The characteristic spectrum peaks of flaxseed medicinal materials were identified, and the results are shown in Figure 9 , Figure 9 This is the chromatographic peak identification diagram of the characteristic spectrum of flaxseed medicinal material provided in Example 2 of the present invention. Figure 9 , the six characteristic peaks in the medicinal materials were included in the subsequent investigation and methodological research was carried out.

[0169] 5.2) Repeatability study

[0170] Six portions of linseed (batch number: YMZ05) were precisely weighed and prepared and tested according to the proposed experimental method. The results are shown in Table 11.

[0171] Table 11 Repeatability study - relative retention time of characteristic peaks

[0172]

[0173] As shown in Table 11, the relative retention RSD of each characteristic peak of repeatability is 0.00% to 0.75%, and the method has good repeatability.

[0174] 5.3) Intermediate precision study

[0175] Based on the experimental conditions proposed above, 12 portions of linseed were accurately weighed to prepare test solutions, which were then analyzed on Waters (No. 1-6) and Agilent (No. 7-12) high performance liquid chromatographs. The results are shown in Table 1. Figure 10 and Table 12, Figure 10 This is a graph showing the experimental results of different instruments used to examine the flaxseed medicinal material provided in Example 2 of the present invention.

[0176] Table 12 Intermediate precision - relative retention time of characteristic peaks

[0177]

[0178] pass Figure 10 As shown in Table 12, this method has good intermediate precision.

[0179] 5.4) Durability test

[0180] On the basis of the experimental conditions proposed above, the chromatographic columns were: AtlantisTM T34.6×250mm / 5μm (chromatographic column 1), HSS T34.6×250mm / 5μm (chromatographic column 2) and Shim-pack ScepterC18-1204.6×250mm / 5μm (chromatographic column 3) were investigated. Figure 11 and Table 13, Figure 11 This is a graph showing the experimental results of different chromatographic columns for the flaxseed medicinal material provided in Example 2 of the present invention.

[0181] Table 13 Column durability investigation - relative retention time of characteristic peaks

[0182]

[0183] pass Figure 11 As shown in Table 13, the RSD values of the relative retention times vary greatly when using different chromatographic columns. Column 1 is recommended.

[0184] 5.5) Stability study

[0185] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 4h, 8h, 12h, 16h and 24h respectively. The results are shown in Table 14.

[0186] Table 14 Stability Study—Characteristic Peak Retention Time

[0187]

[0188]

[0189] As shown in Table 14, the RSD of the corresponding characteristic peak retention time is between 0.17% and 1.15%, and the sample solution is relatively stable within 24 hours.

[0190] In summary, the RSDs of the retention time / relative retention time of each characteristic peak met the requirements in all the above investigations, and this method is good.

[0191] 6) Determination of characteristic peaks and establishment of reference maps

[0192] 6.1) Verification results of 16 batches of flaxseed medicinal materials

[0193] The proposed method was used to determine the characteristic spectra of 16 samples of this product and calculate the relative retention time. The results are shown in Figure 12 and Table 15, Figure 12 This is a characteristic spectrum verification diagram of 16 batches of flaxseed medicinal material samples provided in Example 2 of the present invention. Figure 12 In the figure, the batch number represented by each spectrum is S1:YMZ01, S2:YMZ02, S3:YMZ03, S4:YMZ04, S5:YMZ05, S6:YMZ06, S7:YMZ07, S8:YMZ08, S9:YMZ09, S10:YMZ10, S11:YMZ11, S12:YMZ12, S13:YMZ13, S14:YMZ14, S15:YMZ15, S16:YMZ16.

[0194] Table 1 Relative retention time of characteristic peaks of 1516 batches of flaxseed medicinal materials

[0195]

[0196] Based on the principle that the relative retention time is stable and all batches of samples can be detected with relatively high peaks, a total of 6 peaks with good reproducibility were selected as characteristic peaks.

[0197] 6.2) Establishment of relative retention time limit values

[0198] The summary of the methodology inspection items and verification results is shown in Table 16:

[0199] Table 16 Summary of RSD (%) results of each item of the methodology - relative retention time (retention time)

[0200]

[0201]

[0202] As shown in Table 16, the RSD values of the retention times or relative retention times of the characteristic peaks met the requirements in all the above investigations, indicating that the method was good. The above six characteristic peaks were included in the subsequent investigation.

[0203] Final regulations: It is tentatively stipulated that 6 characteristic peaks should appear in the chromatogram of the test sample, and the retention times should correspond to the 6 characteristic peaks in the chromatogram of the control medicinal material reference substance, among which Peak 6 should correspond to the retention time of the tryptophan reference substance peak; the peak corresponding to the tryptophan reference substance peak is the S peak, and the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time should be within the range of ±10% of the specified value, and the specified values are: 0.35 (peak 1), 0.55 (peak 2), 0.65 (peak 3), 0.69 (peak 4), 0.73 (peak 5), and 1.00 (peak 6).

[0204] The Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version) was used to synthesize 16 batches of flaxseed medicinal materials, and a comparison map of the characteristic spectrum of flaxseed medicinal materials was established, such as Figure 13 As shown, Figure 13 This is a comparison graph of the characteristic graph of flaxseed medicinal material provided in Example 2 of the present invention.

[0205] 6.3) Verification of characteristic spectra of 16 batches of flaxseed slices

[0206] The proposed method was used to determine the characteristic spectra of 16 samples of this product and calculate the relative retention time. The results are shown in Figure 14 and Table 17, Figure 14 This is a characteristic spectrum verification diagram of 16 batches of linseed decoction pieces samples provided in Example 2 of the present invention. Figure 14 , the representative batch numbers of each spectrum are S1: YMZ-YP-01, S2: YMZ-YP-02, S3: YMZ-YP-03, S4: YMZ-YP-04, S5: YMZ-YP-05, S6: YMZ-YP-06, S7: YMZ-YP-07, S8: YMZ-YP-0 8, S9: YMZ-YP-09, S10: YMZ-YP-10, S11: YMZ-YP-11, S12: YMZ-YP-12, S13: YMZ-YP-13, S14: YMZ-YP-14, S15: YMZ-YP-15, S16: YMZ-YP-16.

[0207] Table 1 Relative retention time of characteristic peaks of 1716 batches of flaxseed slices

[0208]

[0209] Based on the principle that the relative retention time is stable and all batches of samples can be detected with relatively high peaks, a total of 6 peaks with good reproducibility were selected as characteristic peaks.

[0210] Final regulations: It is tentatively stipulated that 6 characteristic peaks should appear in the chromatogram of the test sample, and the retention times should correspond to the 6 characteristic peaks in the chromatogram of the control medicinal material reference substance, among which Peak 6 should correspond to the retention time of the tryptophan reference substance peak; the peak corresponding to the tryptophan reference substance peak is the S peak, and the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time should be within the range of ±10% of the specified value, and the specified values are: 0.35 (peak 1), 0.55 (peak 2), 0.65 (peak 3), 0.69 (peak 4), 0.73 (peak 5), and 1.00 (peak 6).

[0211] The Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version) was used to synthesize 16 batches of flaxseed slices, and a comparison map of the characteristic spectrum of flaxseed slices was established. Figure 15 As shown, Figure 15 This is a comparison graph of the characteristic graph of the linseed decoction pieces provided in Example 2 of the present invention.

[0212] 7) Determination of characteristic spectrum method for flaxseed medicinal materials and decoction pieces

[0213] Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel was used as the filler; acetonitrile was used as mobile phase A, and 0.2 vol% acetic acid solution was used as mobile phase B, with gradient elution performed as specified in Table 4; the flow rate was 0.8 mL / min; the column temperature was 30°C; the detection wavelength was 278 nm; and the number of theoretical plates calculated based on the tryptophan peak should be no less than 3000.

[0214] Preparation of reference solution: Take 1 g of linseed control medicinal material, place it in a stoppered conical flask, add 50 mL of water, boil for 30 minutes, filter, evaporate the filtrate to dryness, add 25 mL of 30 vol% methanol to the residue, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the control medicinal material reference solution; take an appropriate amount of tryptophan reference substance, accurately weigh it, and add 30 vol% methanol to make a solution containing 10 μg of tryptophan per 1 mL, which is used as the reference substance solution.

[0215] Preparation of test solution: Take an appropriate amount of the product powder (passed through No. 3 sieve), take about 1.0 g, place it in a stoppered conical flask, add 50 mL of water, boil for 30 minutes, filter, evaporate the filtrate to dryness, add 25 mL of 30 vol% methanol to the residue, stopper it, and ultrasonically treat it (power 600 W, frequency 40 kHz) for 30 minutes. Let it cool, shake well, filter, and take the filtrate.

[0216] Determination method: Accurately aspirate 10 μL of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0217] Example 3

[0218] Construction of HPLC characteristic spectrum of flaxseed formula granules:

[0219] 1) Experimental instruments and materials

[0220] Same as Example 1, no further details will be given.

[0221] 2) Reagents and test drugs

[0222] Flaxseed formula granules (prepared by Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch numbers: 18060089, B01, B02, B03);

[0223] Other reagents and drugs are the same as those in Example 1 and will not be described in detail.

[0224] 3) Feature spectrum detection method

[0225] Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel was used as the filler; acetonitrile was used as mobile phase A, and 0.2 vol% acetic acid solution was used as mobile phase B, with gradient elution performed as specified in Table 4; the flow rate was 0.8 mL / min; the column temperature was 30°C; the detection wavelength was 278 nm; and the number of theoretical plates calculated based on the tryptophan peak should be no less than 3000.

[0226] Preparation of reference solution: Take 1 g of linseed control medicinal material, place it in a stoppered conical flask, add 50 mL of water, boil for 30 minutes, filter, evaporate the filtrate to dryness, add 25 mL of 30 vol% methanol to the residue, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the control medicinal material reference solution; take an appropriate amount of tryptophan reference substance, accurately weigh it, and add 30 vol% methanol to make a solution containing 10 μg of tryptophan per 1 mL, which is used as the reference substance solution.

[0227] Preparation of test solution: Take an appropriate amount of the product, grind it into powder, take about 0.5 g, accurately weigh it, place it in a stoppered conical flask, accurately add 25 mL of 30 vol% methanol, stopper it tightly, weigh it, and treat it ultrasonically (power 600 W, frequency 40 kHz) for 30 minutes. Let it cool, weigh it again, make up the lost weight with 30 vol% methanol, shake it well, filter it, and take the filtrate.

[0228] Determination method: Accurately aspirate 10 μL of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0229] 4) Investigation of chromatographic conditions

[0230] 4.1) Wavelength selection

[0231] Based on the experimental conditions proposed above, the chromatograms of the test solution at wavelengths of 260nm, 270nm, 278nm, 290nm, and 300nm were extracted respectively. The results are shown in Figure 16 , Figure 16 The chromatograms at different wavelengths of the flaxseed formula granules provided in Example 3 of the present invention are shown. It can be seen that when the detection wavelength is 278 nm, the chromatographic peak information volume is larger and the chromatogram baseline is more stable, so the detection wavelength is determined to be 278 nm.

[0232] 4.2) Column temperature investigation

[0233] Based on the experimental conditions proposed above, the test solution was chromatographed at column temperatures of 25°C, 30°C, and 35°C. The results are shown in Figure 17 , Figure 17 This is the column temperature chromatogram provided in Example 3 of the present invention. It can be seen that at 30°C, the chromatogram peak shape is relatively symmetrical and the retention time is relatively suitable, so the column temperature is selected to be 30°C.

[0234] 4.3) Flow rate investigation

[0235] Based on the experimental conditions proposed above, the test solution was chromatographed at flow rates of 0.8 mL / min, 1.0 mL / min, and 1.2 mL / min, respectively. The results are shown in Figure 18 , Figure 18 This is the flow rate investigation chromatogram provided in Example 3 of the present invention. It can be seen that when the flow rate is 0.8 mL / min, the chromatogram peak shape is relatively symmetrical and the retention time is appropriate, so the flow rate is selected to be 0.8 mL / min.

[0236] 5) Preparation of test solution

[0237] 5.1) Investigation of dissolution mode

[0238] Take an appropriate amount of this product (batch number: 18060089), grind it into powder, take about 0.5 g, accurately weigh it, place it in a stoppered conical flask, accurately add 25 mL of 30 vol% methanol, stopper it, weigh it, reflux the sample or ultrasonically treat it (power 600 W, frequency 40 kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with 30 vol% methanol, shake it well, filter it, and take the filtrate to obtain the test solution.

[0239] According to the experimental conditions proposed above, the reflux treatment and ultrasonic treatment of the test solution were chromatographically determined, and the results are shown in Figure 19 , Figure 19 This is the experimental result diagram of the dissolution mode during the preparation of the test solution provided in Example 3 of the present invention. Figure 19 It can be seen that the chromatographic effects of ultrasound and reflux treatment are basically the same. In this experiment, ultrasound was selected as the dissolution method for the test sample.

[0240] 5.2) Investigation of dissolving solvent

[0241] Take the granules of this product (batch number: 18060089) and grind them into powder. Take about 0.5 g, weigh accurately, place in a stoppered conical flask, dissolve with water, 30 vol% methanol, 70 vol% methanol, methanol, and 50 vol% ethanol, respectively, add 25 mL of each, stopper tightly, weigh, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, weigh again, make up the lost weight with the corresponding solvent, shake well, filter, and take the filtrate to obtain the test solution.

[0242] According to the experimental conditions proposed above, the test solutions prepared with different solvents were chromatographically determined. The results are shown in Figure 20 , Figure 20 This is a graph showing the experimental results of the investigation of the dissolving solvent during the preparation of the test solution provided in Example 3 of the present invention. Figure 20 It can be seen that when the solvent is 30 vol% methanol, the chromatographic peak information volume is larger and the peak shape is better, so the solvent of the test sample is determined to be 30 vol% methanol.

[0243] 5.3) Investigation of dissolution time

[0244] Take an appropriate amount of this product (batch number: 18060089), grind it into powder, take about 0.5 g, place it in a stoppered conical flask, add 25 mL of 30 vol% methanol, stopper it tightly, and treat the sample with ultrasound (power 600 W, frequency 40 kHz) for 20 minutes, 30 minutes, and 40 minutes respectively. Cool, shake well, filter, and take the filtrate to obtain the test solution.

[0245] According to the experimental conditions proposed above, the test solution treated with ultrasound for different times was subjected to chromatographic determination. The results are shown in Figure 21, Figure 21 This is a graph showing the experimental results of the dissolution time during the preparation of the test solution provided in Example 3 of the present invention. Figure 21 It can be seen that under different ultrasonic time conditions, the chromatographic effects are basically the same, but in order to ensure that the test sample can be fully dissolved and extracted, the ultrasonic treatment time of the test sample is determined to be 30 minutes.

[0246] 5.4) Investigation of solvent addition amount

[0247] Take an appropriate amount of this product (batch number: 18060089), grind it into powder, take about 0.5 g, place it in a stoppered conical flask, add 10 mL, 25 mL, and 50 mL of 30 vol% methanol respectively, stopper it, and ultrasonically treat it (power 600 W, frequency 40 kHz) for 30 minutes. Cool it, shake it well, filter it, and take the filtrate to obtain the test solution.

[0248] According to the experimental conditions proposed above, the test solution with different solvent addition amounts was chromatographically determined. The results are shown in Figure 22 , Figure 22 This is the experimental result diagram of the amount of solvent added during the preparation of the test solution provided in Example 3 of the present invention. Figure 22 It can be seen that when the amount of solvent added is 25 mL, the chromatographic peak area of the characteristic spectrum is moderate, so the amount of solvent added for the test sample is determined to be 25 mL.

[0249] 6) Methodological investigation

[0250] 6.1) Chromatographic peak identification

[0251] According to the experimental conditions proposed above, a flaxseed formula granule test solution, a tryptophan reference substance solution, and a flaxseed control medicinal material reference substance solution were prepared. At the same time, a negative control solution lacking the flaxseed formula granule (i.e., a blank solution) was prepared according to the experimental conditions proposed above for chromatographic detection.

[0252] The characteristic spectrum peaks of flaxseed medicinal materials were identified, and the results are shown in Figure 23 , Figure 23 This is a chromatographic peak identification diagram of the characteristic spectrum of linseed formula granules provided in Example 3 of the present invention. Figure 23 , the six characteristic peaks in the medicinal materials were included in the subsequent investigation and methodological research was carried out.

[0253] 6.2) Repeatability study

[0254] Six portions of flaxseed formula granules were accurately weighed and prepared and tested according to the proposed experimental method. The results are shown in Table 18.

[0255] Table 18 Repeatability study - relative retention time of characteristic peaks

[0256]

[0257] As shown in Table 18, the relative retention times of the characteristic peaks are consistent, and the method has good repeatability.

[0258] 6.3) Intermediate precision study

[0259] Based on the experimental conditions proposed above, 12 portions of flaxseed granules were accurately weighed to prepare test solutions, which were then measured on Waters (No. 1-6) and Agilent (No. 7-12) high performance liquid chromatographs, respectively. Figure 24 and Table 19, Figure 24 1 is a graph showing the experimental results of different instruments used to examine the flaxseed formula particles provided in Example 3 of the present invention.

[0260] Table 19 Intermediate precision - relative retention time of characteristic peaks

[0261]

[0262] pass Figure 24 As can be seen from Table 19, this method has good intermediate precision.

[0263] 6.4) Durability assessment

[0264] On the basis of the experimental conditions proposed above, the chromatographic columns were: AtlantisTM T34.6×250mm / 5μm (chromatographic column 1), HSS T34.6×250mm / 5μm (chromatographic column 2) and Shim-pack ScepterC18-1204.6×250mm / 5μm (chromatographic column 3) were investigated. Figure 25 and Table 20, Figure 25 This is a graph showing the experimental results of different chromatographic columns for the flaxseed formula granules provided in Example 3 of the present invention.

[0265] Table 20 Column durability inspection - relative retention time of characteristic peaks

[0266]

[0267] pass Figure 25 As shown in Table 20, the relative peak area RSD values vary greatly when using different chromatographic columns. Column 1 is recommended.

[0268] 6.5) Stability study

[0269] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 4h, 8h, 12h, 16h and 24h respectively. The results are shown in Table 21.

[0270] Table 21 Stability Study—Characteristic Peak Retention Time

[0271]

[0272] As shown in Table 21, the RSD values of the corresponding characteristic peak retention times are 0.06% to 0.21%, and the sample solution is relatively stable within 24 hours.

[0273] In summary, the RSDs of the retention time / relative retention time of each characteristic peak met the requirements in all the above investigations, and this method is good.

[0274] 7) Determination of characteristic peaks and establishment of reference maps

[0275] 7.1) Verification results of three batches of flaxseed formula granules

[0276] The proposed method was used to determine the characteristic spectra of three batches of samples of this product and calculate the relative retention time. The results are shown in Figure 26 and Table 22, Figure 26 This is a verification diagram of the characteristic spectra of three batches of flaxseed formula granules provided in Example 3 of the present invention.

[0277] Table 2 Relative retention times of characteristic peaks of 23 batches of flaxseed formula granules

[0278]

[0279] Based on the principle that the relative retention time is stable and all batches of samples can be detected with relatively high peaks, a total of 6 peaks with good reproducibility were selected as characteristic peaks.

[0280] 7.2) Establishment of relative retention time limit values

[0281] The summary of the methodology inspection items and verification results is shown in Table 23:

[0282] Table 23 Summary of RSD (%) results of each item of the methodology - relative retention time (retention time)

[0283]

[0284] Table 23 shows that, with the exception of column durability, the RSD values of the retention times or relative retention times of each characteristic peak met the requirements in all of the above considerations, indicating that this method is good. These six characteristic peaks will be included in subsequent investigations.

[0285] Final regulations: It is tentatively stipulated that 6 characteristic peaks should appear in the chromatogram of the test sample, and the retention times should correspond to the 6 characteristic peaks in the chromatogram of the control medicinal material reference substance, among which Peak 6 should correspond to the retention time of the tryptophan reference substance peak; the peak corresponding to the tryptophan reference substance peak is the S peak, and the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time should be within the range of ±10% of the specified value, and the specified values are: 0.35 (peak 1), 0.55 (peak 2), 0.65 (peak 3), 0.69 (peak 4), 0.73 (peak 5), and 1.00 (peak 6).

[0286] Three batches of flaxseed formula granules were synthesized using the Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version), and a comparison map of the characteristic spectrum of flaxseed formula granules was established, such as Figure 27 As shown, Figure 27 This is a comparison chart of the characteristic chart of the flaxseed formula granules provided in Example 3 of the present invention.

[0287] 8) Determination of characteristic spectrum of flaxseed formula particles

[0288] Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel was used as the filler; acetonitrile was used as mobile phase A, and 0.2 vol% acetic acid solution was used as mobile phase B, and gradient elution was performed according to the requirements in Table 1; the flow rate was 0.8 mL / min; the column temperature was 30°C; the detection wavelength was 278 nm; and the number of theoretical plates calculated based on the tryptophan peak should be no less than 3000.

[0289] Preparation of reference solution: Take 1 g of linseed control medicinal material, place it in a stoppered conical flask, add 50 mL of water, boil for 30 minutes, filter, evaporate the filtrate to dryness, add 25 mL of 30 vol% methanol to the residue, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the control medicinal material reference solution; take an appropriate amount of tryptophan reference substance, accurately weigh it, and add 30 vol% methanol to make a solution containing 10 μg of tryptophan per 1 mL, which is used as the reference substance solution.

[0290] Preparation of test solution: Take an appropriate amount of the product, grind it into powder, take about 0.5g, weigh it accurately, put it into a stoppered conical flask, accurately add 25mL of 30vol% methanol, stopper it tightly, weigh it, ultrasonicate it (power 600W, frequency 40kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with 30vol% methanol, shake it well, filter it, and take the filtrate to obtain

[0291] Determination method: Accurately aspirate 10 μL of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0292] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for constructing HPLC characteristic patterns of linseed medicinal materials, decoction pieces, extracts, and preparations, characterized in that: The following steps are involved: preparing test samples, wherein the test samples are flaxseed medicinal materials, flaxseed decoction pieces, flaxseed extracts and flaxseed preparations; Dissolve the test sample to obtain a test sample solution; the dissolving solvent is a 30 vol% methanol solution; The test solution is measured by high performance liquid chromatography to obtain a HPLC characteristic spectrum of the corresponding test sample; The chromatographic conditions of the high performance liquid chromatography method are as follows: a C18 column; a detection wavelength of 278 nm; acetonitrile as mobile phase A, a 0.2 vol% acetic acid solution as mobile phase B, and gradient elution; The gradient elution is specifically as follows: 0~7min, phase A: 1vol%, phase B: 99vol%; 7~20min, phase A: 1~4vol%, phase B: 99~96vol%; 20~28min, phase A: 4vol%, phase B: 96vol%; 28~36min, phase A: 4~10vol%, phase B: 96~90vol%; 36~48min, phase A: 10~20vol%, phase B: 90~80vol%.

2. The construction method according to claim 1, characterized in that The dissolution is carried out under the assistance of ultrasound, wherein the power of the ultrasound assistance is 580-620 W, the frequency is 35-45 kHz, and the time is 25-35 min.

3. The construction method according to claim 1, characterized in that The chromatographic conditions of the high performance liquid chromatography method also include: a mobile phase flow rate of 0.8 mL / min; an injection volume of 10 μL; a column temperature of 30° C.; and a theoretical plate number calculated based on the tryptophan peak of not less than 3000.

4. The construction method according to claim 1, wherein: The following steps are also included: Dissolve the linseed control medicinal material to obtain a control medicinal material reference solution; dissolve tryptophan to obtain a reference substance reference solution; The control medicinal material reference solution and the reference substance reference solution are measured by high performance liquid chromatography to obtain a chromatogram of the reference substance; and the components of the HPLC characteristic spectrum of the test sample are qualitatively analyzed based on the chromatogram of the reference substance.

5. The construction method according to claim 4, characterized in that The following steps are also included: The similarity of the HPLC characteristic spectrum of linseed medicinal material was evaluated using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and an HPLC standard characteristic spectrum of linseed medicinal material consisting of 6 characteristic peaks was obtained; in the HPLC standard characteristic spectrum, peak 6 corresponding to the peak of the tryptophan reference substance was the S peak, and the relative retention time of each characteristic peak and the S peak was calculated. The relative retention time was within the range of ±10% of the specified value, and the specified value was: 0.35 (peak 1), 0.55 (peak 2), 0.65 (peak 3), 0.69 (peak 4), 0.73 (peak 5), 1.00 (peak 6).

6. The construction method according to claim 4, characterized in that The following steps are also included: The similarity of the HPLC characteristic spectrum of the linseed slices was evaluated using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and an HPLC standard characteristic spectrum of the linseed slices consisting of 6 characteristic peaks was obtained; in the HPLC standard characteristic spectrum, peak 6 corresponding to the peak of the tryptophan reference substance was the S peak, and the relative retention time of each characteristic peak and the S peak was calculated. The relative retention time was within the range of ±10% of the specified value, and the specified value was: 0.35 (peak 1), 0.55 (peak 2), 0.65 (peak 3), 0.69 (peak 4), 0.73 (peak 5), 1.00 (peak 6).

7. The construction method according to claim 4, characterized in that: The following steps are also included: The similarity of the HPLC characteristic spectrum of the linseed extract was evaluated using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and an HPLC standard characteristic spectrum of the linseed extract consisting of 6 characteristic peaks was obtained; in the HPLC standard characteristic spectrum, peak 6 corresponding to the peak of the tryptophan reference substance was the S peak, and the relative retention time of each characteristic peak and the S peak was calculated. The relative retention time was within ±10% of the specified value, and the specified value was: 0.35 (peak 1), 0.55 (peak 2), 0.65 (peak 3), 0.69 (peak 4), 0.73 (peak 5), 1.00 (peak 6).

8. The construction method according to claim 4, characterized in that: The following steps are also included: The similarity of the HPLC characteristic spectrum of the flaxseed preparation was evaluated using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and an HPLC standard characteristic spectrum of the flaxseed preparation consisting of 6 characteristic peaks was obtained; in the HPLC standard characteristic spectrum, peak 6 corresponding to the peak of the tryptophan reference substance was the S peak, and the relative retention time of each characteristic peak and the S peak was calculated. The relative retention time was within ±10% of the specified value, and the specified value was: 0.35 (peak 1), 0.55 (peak 2), 0.65 (peak 3), 0.69 (peak 4), 0.73 (peak 5), 1.00 (peak 6).

9. A method for identifying linseed medicinal materials, decoction pieces, extracts, and preparations, characterized in that: The HPLC characteristic spectrum obtained by the construction method according to any one of claims 1 to 8 is used as the basis for identifying flaxseed medicinal materials, decoction pieces, extracts and preparations.

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