A method for constructing characteristic spectra, a quality detection method, and a content determination method for a black sesame formula granule preparation.

Abstract

This invention relates to the field of traditional Chinese medicine analysis and detection technology, specifically to a method for constructing a characteristic spectrum of a black sesame granule preparation, a quality detection method, and a content determination method. The method for constructing a characteristic spectrum of a black sesame preparation provided by this invention includes detection using ultra-high performance liquid chromatography (UHPLC). The chromatographic conditions include: using octadecylsilane-bonded silica gel as the packing material, methanol as mobile phase A, and water as mobile phase B, with gradient elution. The characteristic spectrum constructed by this method contains numerous characteristic peaks and identifying components, comprehensively reflecting the characteristic peak information of the sample. It exhibits good peak shape, good resolution, high precision, good reproducibility, and strong specificity, enabling comprehensive quality detection of black sesame preparations, and the detection time is short.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine analysis and testing technology, and to a method for constructing characteristic spectra, a quality testing method, and a content determination method for a black sesame formula granule preparation. Background Technology

[0002] Black sesame is the dried, mature seed of *Sesamumindicum* L., a plant in the family Elymusaceae. It is neutral in nature, sweet in taste, and enters the liver, kidney, and large intestine meridians. Belonging to the category of tonic Chinese herbal medicines, it has the effects of tonifying the liver and kidneys, replenishing essence and blood, and moistening the intestines. It is used for deficiency of essence and blood, dizziness, tinnitus, deafness, premature graying of hair, hair loss after illness, and constipation due to intestinal dryness. The quality standard for this medicinal material is included in Part I of the 2020 edition of the *Chinese Pharmacopoeia*, but there is no characteristic chromatogram or content determination method under the entry for black sesame. Currently, there is limited literature on the quality of black sesame, mostly focusing on its chemical composition and pharmacological effects, with even less research on black sesame preparations. Therefore, establishing a characteristic chromatogram and content determination method for black sesame preparations will provide a scientific basis for comprehensively establishing quality control standards for black sesame preparations.

[0003] Currently, research on the characteristic chromatograms of black sesame granules suffers from drawbacks such as long detection time, few chromatographic peaks, poor separation of characteristic peaks, and complex detection methods in content determination, making it difficult to conduct comprehensive quality control and determine the content of effective components in black sesame preparations. Summary of the Invention

[0004] Therefore, the first technical problem to be solved by the present invention is to provide a method for constructing a characteristic spectrum of black sesame preparations. The characteristic spectrum method can perform overall quality control and identification of black sesame preparations, and provide a scientific basis for comprehensively establishing quality control standards for black sesame granules.

[0005] The second technical problem to be solved by the present invention is to provide a method for detecting the content of black sesame preparations. This method not only establishes content determination indicators for lignans in black sesame preparations, but also establishes a detection method for the content of oleic acid and linoleic acid, which are oily components with high content. This method can determine the content of effective components in black sesame in a simple, rapid and efficient manner.

[0006] The embodiments disclosed in this invention relate to a method for constructing characteristic chromatograms of a black sesame preparation, including detection using ultra-high performance liquid chromatography (UHPLC), with chromatographic conditions including:

[0007] Using octadecylsilane-bonded silica gel as the packing material, methanol as mobile phase A, and water as mobile phase B, gradient elution was performed. The elution procedure is as follows:

[0008]

[0009] In some embodiments, the chromatographic conditions further include at least one of the following conditions:

[0010] 1) The flow rate is 0.28–0.32 ml per minute; optionally, the flow rate is 0.3 ml per minute.

[0011] 2) Column temperature is 33-37℃; optionally, the column temperature is 35℃.

[0012] 3) The detection wavelength is 285nm~289nm; optionally, the detection wavelength is 287nm;

[0013] 4) The injection volume is 1–3 μl; optionally, the injection volume is 2 μl.

[0014] 5) The chromatographic column specifications are: column length 100mm, inner diameter 2.1mm, and particle size 1.7μm.

[0015] In some embodiments, the preparation of the test solution is also included, comprising: taking the test sample, adding an extraction solvent, extracting, separating the solid and liquid, and taking the liquid.

[0016] In some embodiments, the preparation of the test solution includes at least one of the following conditions:

[0017] 1) The extraction solvent is water, 30-100 v / v% methanol or its aqueous solution, or 30-100 v / v% ethanol or its aqueous solution; optionally, a 70 v / v% methanol aqueous solution is selected.

[0018] 2) The extraction method used in the extraction step is ultrasonic extraction or reflux extraction;

[0019] 3) The extraction time in the extraction step is 20-40 minutes;

[0020] 4) The mass-to-volume ratio of the test sample to the extraction solvent is 0.5g:15-50ml.

[0021] In some embodiments, the preparation of a reference solution is also included, including:

[0022] Preparation of reference herb solution: Take the reference herb, add the extraction solvent, extract, separate the solid and liquid, evaporate the filtrate to dryness, add the redissolved solvent, and collect the liquid;

[0023] And / or, preparation of reference solution: Take sesamin reference standard and add solvent to prepare a solution containing 20-40 μg per ml; optionally, a solution containing 30 μg per ml; optionally, the solvent is 50-100 v / v% methanol or its aqueous solution; optionally, a 70 v / v% methanol aqueous solution is selected.

[0024] In some embodiments, the preparation of the control herbal solution includes at least one of the following conditions:

[0025] 1) The extraction solvent is water, 30-100 v / v% methanol or its aqueous solution, or 30-100 v / v% ethanol or its aqueous solution;

[0026] 2) The extraction method used in the extraction step is ultrasonic extraction or reflux extraction;

[0027] 3) The extraction process is repeated 1 to 2 times;

[0028] 3) The extraction time in the extraction step is 20-40 minutes;

[0029] 4) The mass-to-volume ratio of the reference medicinal material to the extraction solvent is 2g:25-50ml;

[0030] 5) The resolvent is 50-100 v / v methanol or its aqueous solution; optionally, a 70 v / v methanol aqueous solution is selected.

[0031] 6) The mass-volume ratio of the reference medicinal material to the reconstitution solvent is 2g:5-10ml.

[0032] The embodiments disclosed in this invention relate to a method for detecting the content of black sesame preparations, wherein a test sample solution and a reference solution are taken and detected by ultra-high performance liquid chromatography in the method for constructing the characteristic spectrum of the black sesame preparations.

[0033] The reference standard includes sesamin.

[0034] In some embodiments, the method for detecting the content of oleic acid and / or linoleic acid also includes ultra-high performance liquid chromatography (UHPLC), with chromatographic conditions including:

[0035] Elution was performed using octadecylsilane-bonded silica gel as the packing material, acetonitrile as mobile phase A, and 0.08–0.12 v / v% acetic acid solution as mobile phase B, with a volume ratio of mobile phase A to mobile phase B of 79–81:21–19.

[0036] Optionally, the chromatographic conditions further include at least one of the following conditions:

[0037] 1) The flow rate is 0.28–0.32 ml per minute; optionally, the flow rate is 0.3 ml per minute.

[0038] 2) Column temperature is 38–42℃; optionally, the column temperature is 40℃.

[0039] 3) The detection wavelength is 203nm to 207nm; optionally, the detection wavelength is 205nm.

[0040] 4) The injection volume is 1–5 μl; optionally, the injection volume is 3 μl.

[0041] 5) The chromatographic column specifications are: column length 100mm, inner diameter 2.1mm, and particle size 1.7μm;

[0042] Optionally, it also includes the preparation of the test solution: take the test sample, add the extraction solvent, extract, separate the solid and liquid, and take the liquid;

[0043] Optionally, the preparation of the test solution includes at least one of the following conditions:

[0044] 1) The extraction solvent is water, 50-100 v / v% methanol or its aqueous solution, or 50-100 v / v% ethanol or its aqueous solution; optionally, methanol is selected.

[0045] 2) The extraction method used in the extraction step is ultrasonic extraction or reflux extraction;

[0046] 3) The extraction time in the extraction step is 20-40 minutes;

[0047] 4) The mass-to-volume ratio of the test sample to the extraction solvent is 0.2g:10-20ml;

[0048] Optionally, the preparation of reference solution is also included: take oleic acid reference standard and linoleic acid reference standard, add solvent to prepare a mixed solution containing 65-75 μg of oleic acid and 75-85 μg of linoleic acid per 1 ml;

[0049] Optionally, the solvent is water, 30-100 v / v% methanol or an aqueous solution thereof, or 30-100 v / v% ethanol or an aqueous solution thereof.

[0050] The embodiments disclosed in this invention relate to a quality detection method for black sesame preparations, including the step of comparing the characteristic spectrum of the sample to be tested with the control characteristic spectrum of the black sesame preparation;

[0051] The feature spectrum of the sample to be tested was constructed according to the construction method described above.

[0052] The control characteristic chromatogram of the black sesame preparation is selected from any one of the following (1)-(2):

[0053] (1) It has 7 common characteristic peaks. Peak 6, which corresponds to the peak of the sesamin reference standard, is the S peak. Calculate the relative retention times of peaks 1-5, peak 7 and S peak. The relative retention times should be within ±10% of the specified values. The specified values ​​of peaks 1-5 and peak 7 are 0.42, 0.57, 0.66, 0.71, 0.75 and 1.05, respectively.

[0054] (2) Using at least one batch of black sesame preparation standard, the characteristic spectrum obtained according to the construction method described above is used to prepare a control characteristic spectrum by the average value or median method.

[0055] In some implementations, in (1), peak 6 corresponds to sesamin; peak 7 corresponds to sesamolin.

[0056] In some embodiments, black sesame preparations include black sesame granules, water extracts, etc.

[0057] The technical solution of this invention has the following advantages:

[0058] 1. The present invention provides a method for constructing a characteristic chromatogram of a black sesame preparation, comprising detection by ultra-high performance liquid chromatography (UHPLC). The chromatographic conditions include: using octadecylsilane-bonded silica gel as the packing material, methanol as mobile phase A, and water as mobile phase B, performing gradient elution. The elution program is as follows: 0–3 minutes, the proportion of mobile phase A is 30% v / v → 35% v / v, and the proportion of mobile phase B is 70% v / v → 65% v / v; 3–7 minutes, the proportion of mobile phase A is 35% v / v → 40% v / v, and the proportion of mobile phase B is 65% v / v → 60% v / v; 7–9 minutes, the proportion of mobile phase A is 40% v / v → 52% v / v, and the proportion of mobile phase B is 60% v / v → 48% v / v. From 9 to 13 minutes, the proportion of mobile phase A changes from 52% v / v to 70% v / v, and the proportion of mobile phase B changes from 48% v / v to 30% v / v. From 13 to 18 minutes, the proportion of mobile phase A changes from 70% v / v to 90% v / v, and the proportion of mobile phase B changes from 30% v / v to 10% v / v. From 18 to 19 minutes, the proportion of mobile phase A changes from 90% v / v to 30% v / v, and the proportion of mobile phase B changes from 10% v / v to 70% v / v. The characteristic spectrum constructed by the above method has a large number of characteristic peaks and identifying components, which can comprehensively reflect the characteristic peak information of the sample. It has good peak shape, good separation, high precision, good reproducibility, and strong specificity, and can comprehensively detect the quality of black sesame preparations with a short detection time.

[0059] 2. The present invention provides a method for detecting the content of black sesame preparations, wherein the test sample solution and the reference solution are taken and detected by ultra-high performance liquid chromatography in the method for constructing the characteristic spectrum of the black sesame preparations; the reference standard includes sesamin; the above method for detecting the content of the active ingredient sesamin in black sesame preparations has high accuracy, high precision, strong specificity, wide linear range, high correlation and strong robustness.

[0060] Furthermore, it also includes a method for determining the content of oleic acid and linoleic acid in black sesame preparations. The detection method adopts ultra-high performance liquid chromatography, which is simple and easy to operate. Moreover, it has high accuracy, high precision, strong specificity, wide linear range, high correlation, and strong durability for detecting the content of the effective components oleic acid and linoleic acid. Attached Figure Description

[0061] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0062] Figure 1 The chromatogram of the test sample in Example 1 of this invention;

[0063] Figure 2 Chromatogram of the reference standard in Example 1 of this invention;

[0064] Figure 3 Chromatogram of the reference medicinal material in Example 1 of this invention;

[0065] Figure 4 Chromatogram of method 1 in embodiment 2 of the present invention;

[0066] Figure 5 Chromatogram of method 2 in embodiment 2 of the present invention;

[0067] Figure 6 Chromatogram of method 3 in embodiment 2 of the present invention;

[0068] Figure 7 Chromatograms of 15 batches of black sesame formula granules (K467CP01~15) in Example 3 of this invention; K467CP01~15 are listed from bottom to top in the figure;

[0069] Figure 8 The comparative feature map in Example 3 of this invention;

[0070] Figure 9 Feature peak identification results in Embodiment 3 of the present invention;

[0071] Figure 10 Chromatogram of the reference medicinal material in Example 3 of this invention;

[0072] Figure 11 The specific chromatogram in Embodiment 6 of this invention;

[0073] Figure 12 Linear graph of sesamin in Example 6 of this invention;

[0074] Figure 13 The ultraviolet absorption spectrum of oleic acid in Example 8 of this invention;

[0075] Figure 14 The ultraviolet absorption spectrum of linoleic acid in Example 8 of this invention;

[0076] Figure 15 Chromatogram of Method 1 in Embodiment 8 of the present invention;

[0077] Figure 16 Chromatogram of method 2 in embodiment 8 of the present invention;

[0078] Figure 17 Chromatogram of the specificity experiment in Example 9 of this invention;

[0079] Figure 18 The linear graph of oleic acid in Example 9 of this invention;

[0080] Figure 19 Linear graph of linoleic acid in Example 9 of this invention;

[0081] Figure 20 Characteristic spectrum of black sesame granules in Comparative Example 1 of this invention. Detailed Implementation

[0082] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0083] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0084] The instruments, reagents, and reagents used in the following examples are:

[0085] Instruments: Waters ACQUITY UPLC H-Class ultra-high performance liquid chromatograph (TUV Detector); ML204T electronic balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.); JA1002 electronic balance (Changzhou Lucky Electronic Equipment Co., Ltd.); MSA6.6S-0CE-DM electronic balance (Sartorius Scientific Instruments (Beijing) Co., Ltd.); KQ-100DE CNC ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.); DK-S26 electric thermostatic water bath (Beijing Zhongyi Guoke Technology Co., Ltd.);

[0086] Chromatographic column: Waters ACQUITY UPLC BEH Shield RP18 (2.1mm × 100mm, 1.7μm);

[0087] Waters ACQUITY UPLC HSS T3 (2.1mm×100mm, 1.8μm);

[0088] Waters CORTECS UPLC T3 (2.1mm×100mm, 1.6μm);

[0089] Waters ACQUITY BEH Shield RP18 (2.1×100mm, 1.7μm)

[0090] Drug trials:

[0091] Sesamin reference standard (batch number: 110836-201706, purity: 99.9%, China National Institutes for Food and Drug Control);

[0092] Sesamin reference standard (batch number: AY260041-202306, purity: ≥98%, Foshan Aoyu Biotechnology Co., Ltd.);

[0093] Linoleic acid reference standard (batch number: 111622-202105, purity: 99.6%, China National Institutes for Food and Drug Control);

[0094] Oleic acid reference standard (batch number: 111621-202108, purity: 99.1%, China National Institutes for Food and Drug Control);

[0095] Black sesame reference material (batch number: 121339-201803, China National Institutes for Food and Drug Control);

[0096] Black sesame granules can be prepared using conventional methods in the field. For example, in this invention, they are prepared according to the following steps: Take 80 kg of black sesame slices, boil (100°C) twice, add water to the first decoction at 10 times the amount of slices (10 ml of water per gram of slices), and extract for 1 hour. Add water to the second decoction at 8 times the amount of slices (8 ml of water per gram of slices), and extract for 1 hour. Filter while hot using a 150-mesh filter cloth. Concentrate the filtrate under reduced pressure at 80°C until the relative density is 1.05–1.10 (60°C). Spray dry the granules, setting the inlet air temperature to 180°C ± 5°C. Granulate by dry method. Packaging specifications are 1 g / bag, 100 g / bottle, and 250 g / bottle. Store in a sealed container. Black sesame granules with batch numbers K467CP01, K467CP02, K467CP03, K467CP04, K467CP05, K467CP06, K467CP07, K467CP08, K467CP09, K467CP10, K467CP11, K467CP12, K467CP13, K467CP14, and K467CP15 were prepared using different batches of black sesame slices.

[0097] Reagents:

[0098] Methanol was of chromatographic grade; water was distilled water (Watsons); ethanol was of analytical grade.

[0099] Example 1

[0100] This embodiment provides a method for constructing characteristic chromatograms of black sesame preparations, including detection using ultra-high performance liquid chromatography (UHPLC), with chromatographic conditions including:

[0101] The column was packed with octadecylsilane-bonded silica gel (100 mm column length, 2.1 mm inner diameter, 1.7 μm particle size, Waters ACQUITY UPLC BEH Shield RP18); methanol was used as mobile phase A and water as mobile phase B, and gradient elution was performed according to the specifications in the table below; the flow rate was 0.3 mL / min, the column temperature was 35 °C, and the detection wavelength was 287 nm. The theoretical plate number, calculated based on the sesamin peak, should not be less than 10,000.

[0102] Table 1. Gradient elution conditions

[0103] Time (minutes) Mobile phase A (% v / v) Mobile phase B (% v / v) 0～3 30→35 70→65 3～7 35→40 65→60 7～9 40→52 60→48 9～13 52→70 48→30 13～18 70→90 30→10 18～19 90→30 10→70

[0104] Preparation of the reference solution: Take 2g of black sesame reference material, place it in a stoppered conical flask, add 25ml of water, heat under reflux for 40 minutes, cool, shake well, filter, evaporate the filtrate to dryness, dissolve the residue in 70v / v% methanol, place in a 10ml volumetric flask, add 70v / v% methanol to the mark, filter, and use the filtrate as the reference solution. Separately, take an appropriate amount of sesamin reference standard, accurately weigh it, and add 70v / v% methanol to prepare a solution containing 30μg per 1ml, which is the reference solution.

[0105] Preparation of the test solution: Take an appropriate amount of black sesame formula granules (batch number K467CP01), grind them into a fine powder, take about 0.5g, weigh accurately, place in a stoppered conical flask, accurately add 25ml of 70% v / v methanol, weigh, sonicate (power 250W, frequency 40kHz) for 20 minutes, remove, cool, weigh again, replenish the lost weight with 70% methanol, shake well, filter, and take the filtrate to obtain the test solution.

[0106] Determination method: Accurately pipette 2 μl of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0107] The test sample should exhibit 7 common characteristic peaks, corresponding to the retention times of the reference herb. Peaks 6 and 7 should correspond to the retention times of the sesamin and sesamolinin reference standards, respectively. Peak 6, corresponding to the sesamin reference standard peak, is designated as peak S. The relative retention times of peaks 1-5, peak 7, and peak S should be calculated and should be within ±10% of the specified values. The specified values ​​for peaks 1-5 and peak 7 are 0.42, 0.57, 0.66, 0.71, 0.75, and 1.05, respectively. Peak 6 corresponds to sesamin; peak 7 corresponds to sesamolinin. Results are as follows... Figures 1-3 As shown.

[0108] Example 2: Optimization of Chromatographic Conditions

[0109] 1. Gradient elution program optimization

[0110] The method of Example 1 is implemented, except that the gradient elution procedure is performed according to Method 1 in Table 1 below.

[0111] Table 3 Gradient Elution Table

[0112] Time (min) Mobile phase A (% v / v) Mobile phase B (% v / v) 0～3 30→33 70→67 3～12 33→72 67→28 12～18 72→90 28→10 18～19 90→30 10→70

[0113] The results are as follows Figure 4 As shown in the figure. The characteristic peaks of the black sesame formula granules obtained by this method are concentrated between 6 and 9 min, and the separation of each characteristic peak is not good, which requires further optimization. Therefore, method 2 in the table below is followed.

[0114] Table 3 Gradient Elution Table

[0115] Time (min) Mobile phase A (% v / v) Mobile phase B (% v / v) 0～3 30→35 70→65 3～8 35→40 65→60 8～12 40→51 60→49 12～14 51→70 49→30 14～18 70→90 30→10 18～19 90→30 10→70

[0116] The results are as follows Figure 5 As shown. The characteristic spectrum of black sesame granules obtained by this method has a large interval between 10 and 16 min, and the reference peak is further down than that of Method 1. Therefore, to further optimize the elution gradient, Method 3 in the table below was used.

[0117] Table 4 Gradient Elution Table

[0118] Time (minutes) Mobile phase A (% v / v) Mobile phase B (% v / v) 0～3 30→35 70→65 3～7 35→40 65→60 7～9 40→52 60→48 9～13 52→70 48→30 13～18 70→90 30→10 18～19 90→30 10→70

[0119] The results are as follows Figure 6 As shown in the table below, after optimization using the above methods, Method 3 provides more spectral information and demonstrates good system applicability in experiments. Therefore, Method 3 is selected as the gradient elution method for the characteristic spectral data of black sesame seeds.

[0120] Table 5. Peak information in the chromatogram of the test sample solution for Method 3.

[0121] Peak Retention time (minutes) Resolution Tail factor Theoretical number of plates 1 6.113 3.4 0.9 28828 2 8.159 3.8 1.1 37294 3 9.438 4.2 0.8 127638 4 10.234 2.4 1.0 223760 5 10.744 2.3 1.0 210648 6 14.215 2.9 1.1 352835 7 14.972 1.9 1.1 403879

[0122] 2. Preparation of the test solution

[0123] 2.1 Selection of extraction solvent:

[0124] The procedure was carried out according to Example 1, except that the extraction solvents were selected as 30% v / v methanol, 50% v / v methanol, 70% v / v methanol, methanol, 30% v / v ethanol, 50% v / v ethanol, 70% v / v ethanol, ethanol, and water, respectively. The results are shown in the table below:

[0125] Table 6. Peak area of ​​characteristic spectra of different extraction solvents

[0126] Solvent types Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 (S) Peak 7 30% methanol 420868 32350 71490 11305 16048 222578 72985 50% methanol 427358 32595 73138 11403 16490 257323 74993 70% methanol 430825 33188 74560 11538 17230 293203 77503 methanol 418525 31195 73898 11485 11740 285735 76613 30% ethanol 422770 32958 73800 11860 16115 276493 74375 50% ethanol 428288 31883 74500 11223 13843 256835 73735 70% ethanol 431868 32253 74623 11898 15685 258113 60983 ethanol 127530 28333 31843 10310 13663 183825 43065 water 412750 30483 70628 10918 15068 134583 47773

[0127] As shown in the table above, the peak areas of each characteristic peak are relatively small when ethanol is used as the solvent. Considering all factors, the peak areas of each characteristic peak are relatively large and the peak shapes are better when 70% methanol is used as the extraction solvent. Therefore, 70% v / v methanol is selected as the extraction solvent.

[0128] 2.2 Selection of Extraction Solvent Amount

[0129] The extraction solvent was used in accordance with Example 1, with volumes of 15 ml, 25 ml, and 50 ml, respectively. The peak areas of each characteristic peak were measured. To avoid the influence of the sample concentration on the peak area, the peak areas were converted to the sample concentration by ratio (i.e., corrected data), and the results are shown in the table below.

[0130] Table 7. Peak area of ​​characteristic spectra for different extraction solvent amounts.

[0131]

[0132] As shown in the table above, the peak areas of each characteristic peak are not significantly different depending on the amount of extraction solvent used. Therefore, 25 ml was selected as the extraction solvent amount to ensure sufficient extraction of the sample.

[0133] 2.3 Extraction Method Selection

[0134] The extraction was performed according to Example 1, using ultrasonic treatment (250W power, 40kHz frequency) for 30 minutes and reflux extraction for 30 minutes. The peak areas of each characteristic peak were measured. The results are shown in the table below.

[0135] Table 8 Peak area of ​​feature maps extracted using different methods

[0136] Extraction method Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 (S) Peak 7 ultrasound 430825 33188 74560 11538 17230 293203 77503 reflux 446215 32755 75943 10603 16380 297250 76713

[0137] As shown in the table above, there is no significant difference in the peak area of ​​each characteristic peak in the test solution obtained by different extraction methods. Considering the convenience of experimental operation, ultrasonic treatment was selected as the extraction method for preparing the test solution.

[0138] 2.4 Selection of extraction time

[0139] The extraction was performed according to Example 1, with extraction times of 20, 30, and 40 minutes, respectively. The results are shown in the table below.

[0140] Table 9 Peak area of ​​feature maps at different extraction times

[0141] Extraction time Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 (S) Peak 7 20min 428843 33028 72353 11410 17943 295435 76028 30min 430825 33188 74560 11538 17230 293203 77503 40min 433353 33385 74603 11573 18663 297263 77233

[0142] The analysis results show that the number of characteristic peaks and response values ​​are relatively similar under different extraction times. Considering the principle of saving time, the ultrasound time was selected as 20 minutes.

[0143] Example 3: Establishment of Comparative Feature Maps

[0144] Fifteen batches of black sesame granules (K467CP01-15) were used as test samples, and the method of Example 1 was followed. The results are as follows: Figure 7 As shown. The chromatographic fingerprint similarity evaluation system for traditional Chinese medicine (2012 version) was used to synthesize a reference chromatogram (i.e., a reference characteristic chromatogram) for 15 batches of samples, establishing a characteristic chromatogram for black sesame formula granules. See [link to reference chromatogram]. Figure 8 As shown, based on the principles of stable relative retention times, detectability in all batches of samples, and relatively high peak values, seven peaks with good repeatability were selected as characteristic peaks. Peak 6 is the sesamin reference peak (S peak). The relative retention times of peaks 1-5, peak 7, and peak S peak were calculated. The relative retention times of each characteristic peak were 0.42 (peak 1), 0.57 (peak 2), 0.66 (peak 3), 0.71 (peak 4), 0.75 (peak 5), and 1.05 (peak 7).

[0145] Similarity calculation

[0146] The similarity of 15 batches of black sesame formula granules was calculated using the Chinese herbal chromatographic fingerprint similarity evaluation system (2012 version), and the results are shown in the table.

[0147] Table 10 Similarity Results

[0148] serial number sample Similarity 1 Black Sesame Formula Granules - K467CP01 0.992 2 Black sesame granules - K467CP02 0.995 3 Black sesame granules - K467CP03 0.997 4 Black sesame granules - K467CP04 0.995 5 Black sesame granules - K467CP05 0.998 6 Black sesame formula granules - K467CP06 0.994 7 Black sesame formula granules - K467CP07 0.996 8 Black sesame formula granules - K467CP08 0.999 9 Black Sesame Formula Granules - K467CP09 0.993 10 Black Sesame Formula Granules - K467CP10 0.997 11 Black Sesame Formula Granules - K467CP11 0.999 12 Black Sesame Formula Granules - K467CP12 0.996 13 Black sesame granules - K467CP13 0.997 14 Black Sesame Formula Granules - K467CP14 0.999 15 Black Sesame Formula Granules - K467CP15 0.995

[0149] The similarity of each batch of black sesame granules was greater than 0.99.

[0150] Identification of characteristic peaks

[0151] Accurately weigh appropriate amounts of sesamin and sesamolin reference standards, respectively, and add 70% methanol to prepare solutions containing 30 μg sesamin and 30 μg sesamolin per ml, thus obtaining sesamin reference standard solutions and sesamolin reference standard solutions. Prepare black sesame granule test solutions according to the test solution preparation method in Example 1. Analyze and compare the sesamin reference standard solution, sesamolin reference solution, and black sesame granule test solutions under the chromatographic conditions of Example 1. The results are as follows: Figure 9 .

[0152] In the chromatogram of the black sesame formula granules test sample, peaks 6 and 7 have the same retention times as the chromatograms of sesamin and sesamolin, respectively, confirming that peak 6 is sesamin and peak 7 is sesamolin.

[0153] Comparison with black sesame as a reference medicinal material

[0154] Take 2g of black sesame reference material, place it in a stoppered conical flask, add 25ml of water, heat under reflux for 30 minutes, remove, cool, shake well, filter, evaporate the filtrate to dryness, dissolve the residue in 70% methanol and transfer to a 10ml volumetric flask, shake well, filter, and collect the filtrate to obtain the reference material solution. Prepare the black sesame granule test solution according to the test solution preparation method in Example 1. Compare the black sesame reference material solution and the black sesame granule test solution under the chromatographic conditions of Example 1. The results are as follows: Figure 10 .

[0155] Therefore, it is stipulated that the chromatogram of the test sample should show 7 characteristic peaks, which should correspond to the retention times of the reference medicinal material. Among them, peaks 6 and 7 should correspond to the retention times of the sesamin and sesamolin reference standards, respectively. The peak corresponding to the sesamin reference standard is the S peak. The relative retention times of peaks 1 to 5, peak 7 and S peak should be calculated. The relative retention times should all be within ±10% of the specified values. The specified values ​​are: 0.42 (peak 1), 0.57 (peak 2), 0.66 (peak 3), 0.71 (peak 4), 0.75 (peak 5), and 1.05 (peak 7).

[0156] Example 4 Methodological Validation

[0157] 1. Precision Experiment

[0158] 1.1 Repeatability

[0159] Six portions of black sesame granules (batch number: K467CP01) were taken and their characteristic spectra were obtained according to Example 1. The relative retention times and relative peak areas of peaks 1-5, peak 7 and peak S were calculated using peak 6 as reference peak S, and the RSD values ​​were calculated. The results are shown in the table below.

[0160] Table 11 Retention time and relative retention time of black sesame seeds in repeatability test

[0161]

[0162] Table 12 Peak area and relative peak area of ​​black sesame seeds in repeatability studies

[0163]

[0164] The repeatability test results showed that the relative retention time RSD of the seven identifier peaks of the six repeatability test samples was in the range of 0.0% to 0.1%, and the relative peak area RSD was in the range of 0.0% to 4.0%, indicating that the repeatability of the characteristic spectrum was good.

[0165] 1.2 Intermediate Precision

[0166] Black sesame granules (batch number: K467CP01) were used. Intermediate precision tests were conducted by different analysts at different times on different instruments, according to Example 1. Characteristic spectra were obtained. Peak 6 was used as the reference peak S. The relative retention times and relative peak areas of peaks 1-5, peak 7, and peak S were calculated, and the RSD values ​​were calculated. The results are shown in the table below.

[0167] Table 13 Retention time and relative retention time of black sesame seeds for intermediate precision study

[0168]

[0169]

[0170] Table 14 Peak area and relative peak area of ​​black sesame intermediate precision test

[0171]

[0172] The relative retention time (RSD) of each characteristic peak in the intermediate precision ranges from 0.0% to 0.0%, and the relative peak area (RSD) ranges from 0.0% to 2.0%. This indicates that the intermediate precision of the characteristic spectrum is good.

[0173] 5.2 Solution stability study

[0174] A test solution of black sesame granules (batch number: K467CP01) was prepared according to the method in Example 1. The solution was then analyzed at 0h, 2h, 4h, 6h, 8h, 10h, 12h, and 24h, following the method in Example 1. The characteristic peaks were determined, and the results showed that the test solution was stable within 24h. The results are shown in the table below.

[0175] Table 15. Durability Study of Black Sesame Seeds: Retention Time and Relative Retention Time

[0176]

[0177]

[0178] Table 16 Peak area and relative peak area in the durability test of black sesame seeds

[0179]

[0180] After examining the solution stability over 24 hours, the relative retention time (RSD) of the seven marker peaks was within the range of 0.0% to 0.1%, and the relative peak area (RSD) was within the range of 0.0% to 1.6%. This indicates that the black sesame granule characteristic spectral analysis method used in this experiment is stable, reliable, and has good reproducibility.

[0181] 3. Durability Test

[0182] 3.1 Investigation at different column temperatures

[0183] The influence of minute changes in column temperature on the detection method was investigated. An appropriate amount of black sesame formula granules was taken and a test solution was prepared according to Example 1. The column temperatures were 33℃, 35℃ and 37℃, respectively. The characteristic spectrum detection of the black sesame formula granule test solution was performed according to the method in Example 1. The results showed that the method has good robustness to different column temperatures.

[0184] Table 17 Retention Time and Relative Retention Time of Black Sesame Seeds at Different Column Temperatures

[0185]

[0186]

[0187] Table 18 Peak area and relative peak area of ​​black sesame seeds at different column temperatures

[0188]

[0189] The relative retention times (RSDs) of the seven marked peaks in the sample were all within ±10% of the specified value, indicating that this method is robust to different column temperatures.

[0190] 3.2 Investigation of different flow velocities

[0191] Take an appropriate amount of black sesame granules, prepare a test solution according to Example 1, and determine the concentration. The robustness of the chromatographic method in Example 1 to different flow rates (0.28 ml / min, 0.30 ml / min, and 0.32 ml / min) was investigated.

[0192] Table 19 Retention Time and Relative Retention Time of Black Sesame Seeds at Different Flow Rates

[0193]

[0194] Table 20 Peak area and relative peak area of ​​black sesame seeds at different flow velocities

[0195]

[0196] The relative retention times (RSDs) of the seven marked peaks in the sample were all within ±10% of the specified value, indicating that this method is robust to different flow rates.

[0197] Example 4: Determination method for sesamin content in black sesame granules

[0198] This embodiment provides a method for determining the content of black sesame granules in a formula, including detection using ultra-high performance liquid chromatography (UHPLC), with chromatographic conditions including:

[0199] The column was packed with octadecylsilane-bonded silica gel (100 mm column length, 2.1 mm inner diameter, 1.7 μm particle size, Waters ACQUITY UPLC BEH Shield RP18); methanol was used as mobile phase A and water as mobile phase B, and gradient elution was performed according to the specifications in the table below; the flow rate was 0.3 mL / min, the column temperature was 35 °C, and the detection wavelength was 287 nm. The theoretical plate number, calculated based on the sesamin peak, should not be less than 10,000.

[0200] Table 21. Gradient elution conditions

[0201] Time (minutes) Mobile phase A (% v / v) Mobile phase B (% v / v) 0～3 30→35 70→65 3～7 35→40 65→60 7～9 40→52 60→48 9～13 52→70 48→30 13～18 70→90 30→10 18～19 90→30 10→70

[0202] Preparation of the reference solution: Take 2g of black sesame reference material, place it in a stoppered conical flask, add 25ml of water, heat under reflux for 40 minutes, cool, shake well, filter, evaporate the filtrate to dryness, dissolve the residue in 70% methanol, place in a 10ml volumetric flask, add 70% methanol to the mark, filter, and use the filtrate as the reference solution. Separately, take an appropriate amount of sesamin reference standard, accurately weigh it, and add 70% methanol to prepare a solution containing 30μg per ml, which is the reference solution.

[0203] Preparation of the test solution: Take an appropriate amount of black sesame formula granules (batch number: K467CP01), grind them into a fine powder, take about 0.5g, weigh accurately, place in a stoppered conical flask, accurately add 25ml of 70% v / v methanol, weigh, sonicate (power 250W, frequency 40kHz) for 20 minutes, remove, cool, weigh again, replenish the lost weight with 70% methanol, shake well, filter, and take the filtrate to obtain the test solution.

[0204] Determination method: Accurately pipette 2 μl of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0205] Fifteen batches of black sesame granules were taken and their content was determined according to Example 4.

[0206] Table 22 Results of content determination of black sesame formula granules in 15 batches

[0207] batch number Sesamin content (mg / g) K467CP01 2.3 K467CP02 3.2 K467CP03 3.8 K467CP04 4.3 K467CP05 2.8 K467CP06 2.7 K467CP07 2.8 K467CP08 2.9 K467CP09 3.6 K467CP10 4.5 K467CP11 3.9 K467CP12 2.5 K467CP13 2.3 K467CP14 3.9 K467CP15 2.7

[0208] Example 5: Determination of Sesamin Content in Black Sesame Formula Granules - Study on Preparation of Test Solution

[0209] 1. Investigation of extraction solvents

[0210] Following Example 4, the extraction reagents used in the preparation of the test solution were 30% methanol, 50% methanol, 70% methanol, methanol, 30% ethanol, 50% ethanol, 70% ethanol, ethanol, and water, respectively. The sesamin content was calculated, and the results are shown in the table below.

[0211] Table 23 Comparison of different extraction solvents

[0212]

[0213] The above results indicate that the sesamin content is higher when methanol, 70% methanol, and ethanol are used as extraction solvents. Considering that the peak shapes of methanol and ethanol in the characteristic spectrum are poor, 70% methanol was chosen as the extraction solvent after comprehensive consideration.

[0214] 2. Investigation on the amount of extraction solvent used

[0215] The extraction reagents used in the preparation of the test solution were 15 ml, 25 ml, and 50 ml, respectively, as per Example 4. The sesamin content was calculated, and the results are shown in the table below.

[0216] Table 24 Results of the analysis of extraction solvent dosage and content

[0217]

[0218] As shown in the table above, there is no significant difference in the sesamin content among different amounts of extraction solvents. In order to fully extract the sample, 25 ml was selected as the amount of extraction solvent.

[0219] 3. Examination of extraction time

[0220] Following Example 4, the extraction time for the sample solution preparation was 20, 30, and 40 minutes, respectively. The sesamin content was calculated, and the results are shown in the table below.

[0221] Table 25 Results of content at different extraction times

[0222]

[0223]

[0224] The above results indicate that the sesamin content in the test solution obtained with different ultrasonic times is similar. Considering the rapid and effective sample processing, the extraction time was determined to be 20 minutes.

[0225] 4. Examination of extraction methods

[0226] As per Example 4, the extraction methods for preparing the test sample solution were ultrasonic treatment (power 250W, frequency 40kHz) for 20 minutes and reflux extraction for 20 minutes. The sesamin content was calculated, and the results are shown in the table below.

[0227] Table 26 Content results for different extraction methods

[0228]

[0229] The above results indicate that there is no significant difference in the sesamin content in the test solution obtained by different extraction methods. Considering the convenience of experimental operation, ultrasonic treatment was selected as the extraction method for preparing the test solution.

[0230] Example 6: Methodological Validation of the Sesamin Content Determination Method for Black Sesame Formula Granules

[0231] 1. Accuracy

[0232] Accurately weigh an appropriate amount of sesamin reference standard and add 70% methanol to prepare a sesamin reference standard solution containing 2.3132 mg / ml. Take 9 portions of black sesame formula granules with known content (sesamin content of 2.3 mg / g), each approximately 0.25 g, and divide into groups of 3 portions. Accurately weigh each portion and add 25 ml of the reference standard solution containing 0.5783 mg, 1.1566 mg, and 1.7349 mg of sesamin, respectively, according to the low, medium, and high concentrations. Perform the remaining operations as described in Example 4, and calculate the recovery rate according to the following formula. The results are shown in the table below.

[0233]

[0234] Table 27 Accuracy Test Results

[0235]

[0236] The recovery rate of sesamin measured in the recovery test ranged from 94.54% to 100.72%, with an average recovery rate of 98.9% and an RSD of 2.0%, which met the recovery rate requirements for method validation, indicating that the results obtained using this method are accurate.

[0237] 2 Precision

[0238] 2.1 Repeatability: Six portions of black sesame granules (batch number: K467CP01) were tested according to the method in Example 4. The results are shown in the table below.

[0239] Table 28 Repeatability Tests

[0240]

[0241] The average sesamin content in black sesame granules measured in the repeatability test was 2.3 mg / g, with an RSD of 0.8%, which meets the repeatability requirements for method validation.

[0242] 2.2 Intermediate Precision: Intermediate precision tests were conducted at different times by different analysts using another Agilent 1290 ultra-high performance liquid chromatograph. Six portions of black sesame granules (batch number: K467CP01) were taken and measured according to the method in Example 4. The results are shown in the table below.

[0243] Table 29 Intermediate Precision Results

[0244]

[0245] The intermediate precision test showed that the sesamin content in the black sesame granules was 2.3 mg / g, with an RSD of 0.7%, which is consistent with the RSD of 1.0% in the repeatability test, meeting the requirements for intermediate precision in method validation.

[0246] 3. Exclusivity

[0247] Take an appropriate amount of the extraction reagent used for the sesamin granule test solution and prepare a blank control solution according to the preparation method of the test solution in Example 4; take the black sesame granule test solution, sesamin reference solution, and blank control solution to obtain chromatograms according to the method in Example 4, and the results are shown in [Figure 4]. Figure 11 .

[0248] The specificity was examined and it was found that there was no interference at the corresponding positions of the peaks in the chromatogram of the test sample.

[0249] 4. Linear

[0250] Take an appropriate amount of sesamin reference standard and add 70% methanol to prepare a solution containing 0.1512 mg per ml, which is used as the sesamin reference standard stock solution, denoted as ①. Accurately pipette 5 ml, 1 ml, and 1 ml of the sesamin reference standard stock solution into 10 ml, 10 ml, and 20 ml volumetric flasks respectively, add 70% methanol to the mark, shake well, and filter, denoted as ② to ④ respectively. Accurately pipette 5 ml of ④ into a 10 ml volumetric flask, add 70% methanol to the mark, shake well, and filter, denoted as ⑤. Accurately pipette 2 μl of the filtrates from ① to ⑤ into the liquid chromatograph, and determine the peak area of ​​the sesamin chromatographic peak according to the method in Example 4. With the peak area of ​​the sesamin chromatographic peak as the ordinate and the concentration of sesamin as the abscissa, the regression equation is obtained as y = 8817324.0455x + 1727.3639, R. 2 =1.0000, and its linear range is 0.0038 mg / ml to 0.1512 mg / ml. The results are shown in the table below. Figure 12 .

[0251] Table 30. Examination of the linear relationship of sesamin

[0252] serial number ① ② ③ ④ ⑤ Sesamin concentration (mg / ml) 0.1512 0.0756 0.0151 0.0076 0.0038 Sesamin peak area 1334171 669779 135337 68264 34514

[0253] 5. Scope

[0254] Based on the results of precision, accuracy, and linearity experiments, as well as the results of sesamin content in 15 batches of black sesame granules, the range was 0.0038 mg / ml to 0.1512 mg / ml.

[0255] 6. Durability Test

[0256] 6.1 Stability test: Take the same batch of black sesame granules (batch number: K467CP01), prepare the test solution according to the test solution preparation method in Example 4, and measure the stability at 0h, 2h, 4h, 6h, 8h, 10h, 12h and 24h according to the method in Example 4. Record the peak area of ​​sesamin. The results are shown in the table below.

[0257] Table 31 Results of Sesamin Stability

[0258]

[0259] Summary: Based on the above measurement results, the RSD of the sesamin peak area within 24 hours is 0.5%, indicating that the sample has good stability within 24 hours.

[0260] 6.2 Investigation at different column temperatures:

[0261] Black sesame granules (batch number: K467CP01) were taken and prepared into a test solution according to the test solution preparation method in Example 4. The solution was then measured at different column temperatures (33℃, 35℃ and 37℃) according to the method in Example 4 to examine the durability of the experimental method for column temperature. The results are shown in the table below.

[0262] Table 32 Results of Sesamin Content Determination at Different Column Temperatures

[0263]

[0264] The RSD value of sesamin content measured at different column temperatures was 2.5%, which meets the system suitability requirements, indicating that the method has good column temperature durability.

[0265] 6.3 Investigation of different flow velocities:

[0266] Black sesame granules (batch number: K467CP01) were used to prepare a test solution according to the preparation method of the test solution in Example 4. The solution was tested at different flow rates (0.28 ml / min, 0.30 ml / min and 0.32 ml / min) according to the method in Example 4 to examine the robustness of the experimental method to the flow rate. The results are shown in the table below.

[0267] Table 33 Results of Sesamin Content Determination at Different Flow Rates

[0268]

[0269] The RSD value of sesamin content measured at different flow rates was 2.5%, which meets the system suitability requirements, indicating that the method has good flow rate durability.

[0270] 6.4 Investigation of different chromatographic columns:

[0271] Black sesame granules (batch number: K467CP01) were taken and a test solution was prepared according to the method in Example 4. Different types of chromatographic columns were used to determine the test solution according to the method in Example 4. The robustness of the experimental method to different chromatographic columns was investigated. The results are shown in the table below.

[0272] Column 1: Waters ACQUITY UPLC BEH Shield RP18 (2.1 mm × 100 mm, 1.7 μm)

[0273] Column 2: Waters ACQUITY UPLC HSS T3 (2.1 mm × 100 mm, 1.8 μm)

[0274] Column 3: Waters CORTECS UPLC T3 (2.1 mm × 100 mm, 1.6 μm)

[0275] Table 34 Results of Sesamin Content Determination Using Different Chromatographic Columns

[0276]

[0277] The RSD value of sesamin content measured by different chromatographic columns was 0.7%, which meets the system suitability requirements, indicating that the method has good robustness to different chromatographic columns.

[0278] 6.5 Examination of different wavelengths:

[0279] Black sesame granules (batch number: K467CP01) were taken and a test solution was prepared according to the method in Example 4. The test solution was measured at different wavelengths (285nm, 287nm, 289nm) according to the method in Example 4. The robustness of the experimental method to different wavelengths was investigated. The results are shown in the table below.

[0280] Table 35 Results of Sesamin Content Determination at Different Wavelengths

[0281]

[0282] The RSD value of sesamin content measured at different wavelengths was 0.5%, indicating that the method is robust to different wavelengths.

[0283] Example 7: Method for detecting oleic acid and linoleic acid content in black sesame preparations

[0284] This embodiment provides a method for detecting the content of oleic acid and linoleic acid in black sesame preparations, including detection by high performance liquid chromatography (HPLC), with chromatographic conditions including:

[0285] The column was filled with octadecylsilane-bonded silica gel (100 mm column length, 2.1 mm inner diameter, 1.7 μm particle size, Waters ACQUITY UPLC BEH C18); the mobile phase was acetonitrile-0.1 v / v acetic acid solution (80:20); the flow rate was 0.3 mL / min; the column temperature was 40 °C; and the detection wavelength was 205 nm. The theoretical plate number, calculated based on the linoleic acid peak, should be no less than 10,000.

[0286] Preparation of reference solution: Take appropriate amounts of oleic acid reference standard and linoleic acid reference standard, accurately weigh them, and add methanol to prepare a mixed solution containing 70 μg of oleic acid and 80 μg of linoleic acid per 1 ml.

[0287] Preparation of the test solution: Take an appropriate amount of black sesame formula granules (batch number: K467CP01), grind them into a fine powder, take about 0.2g, weigh it accurately, place it in a stoppered conical flask, accurately add 10ml of methanol, weigh it, sonicate (power 250W, frequency 40kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with methanol, shake it well, filter it, and take the filtrate to obtain the test solution.

[0288] Determination method: Accurately pipette 3 μl of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0289] Fifteen batches of black sesame granules were taken and their content was determined according to Example 7.

[0290] Table 36 Results of content determination of black sesame formula granules in 15 batches

[0291]

[0292]

[0293] Example 8: Investigation of the detection method for oleic acid and linoleic acid content in black sesame preparations

[0294] 1. Selection of detection wavelength

[0295] 3D chromatographic scanning revealed that the maximum absorption wavelengths of oleic acid and linoleic acid were around 200 nm. Considering the need to reduce interference from solvent end absorption, a detection wavelength of 205 nm was selected for the determination of oleic acid and linoleic acid content. Results are shown below. Figures 13-14 .

[0296] 2. Optimization of chromatographic conditions

[0297] The detection was performed using Method 1, the difference from Example 7 being that the mobile phase was acetonitrile-0.1% acetic acid solution (90:10). The detection results are as follows. Figure 15 As shown in the figure, when the black sesame granule sample solution was analyzed using Method 1 chromatographic conditions, the linoleic acid peak eluted too early, resulting in a close proximity to the previously eluted component peaks and potentially poor separation. Therefore, reducing the proportion of organic phase in the mobile phase was considered to adjust the method. Method 2 was used, differing from Method 1 in that the mobile phase was acetonitrile-0.1% acetic acid solution (80:20). The detection results are as follows. Figure 16 As shown in the figure, when the content of black sesame granules was determined using the chromatographic conditions of Method 2, the theoretical plate number, tailing factor, and resolution of the linoleic acid and oleic acid peaks all met the system suitability requirements, and the retention time was moderate. Therefore, Method 2 was ultimately determined as the chromatographic conditions for the determination of oleic acid and linoleic acid content in black sesame granules.

[0298] 3. Preparation of the test solution

[0299] 3.1 Investigation of extraction solvent

[0300] The procedure was carried out according to Example 7, except that the extraction solvents were water, 50% methanol, methanol, 50% ethanol, and anhydrous ethanol, respectively. The test results are shown in the table below.

[0301] Table 37 Analysis of Extraction Solvents

[0302] Extraction solvent Oleic acid (mg / g) Linoleic acid (mg / g) Total amount (mg / g) water 1.8 2.1 3.9 50% methanol 3.2 4.8 8.0 methanol 6.6 7.5 14.1 50% ethanol 5.7 7.0 12.7 Anhydrous ethanol 3.7 7.7 11.4

[0303] The above results indicate that the total amount of oleic acid and linoleic acid is highest when methanol is used as the extraction solvent, therefore methanol was chosen as the extraction solvent.

[0304] 3.2 Investigation on the amount of extraction solvent used

[0305] The procedure was carried out according to Example 7, except that the extraction solvent volume was 10 ml, 15 ml, and 20 ml, respectively. The results are shown in the table below.

[0306] Table 38 Results of content analysis based on extraction solvent dosage

[0307] Extraction solvent dosage Oleic acid (mg / g) Linoleic acid (mg / g) Total amount (mg / g) 10ml 6.6 7.5 14.1 15ml 6.5 7.5 14.0 20ml 6.6 7.5 14.1

[0308] The above test results show that there is no significant difference in the total amount of oleic acid and linoleic acid after extraction with different solvent dosages. Therefore, considering the principle of saving solvent, the extraction solvent dosage was selected as 10 ml.

[0309] 3.3. Examination of extraction time

[0310] The procedure was performed according to Example 7, with the difference being that the extraction times were 20, 30, and 40 minutes, respectively. The results are shown in the table below:

[0311] Table 39 Results of content at different extraction times

[0312] Extraction time Oleic acid (mg / g) Linoleic acid (mg / g) Total amount (mg / g) 20min 6.5 7.5 14.0 30min 6.6 7.5 14.1 40min 6.5 7.5 14.0

[0313] The above results indicate that there is no significant difference in the total amount of oleic acid and linoleic acid in the test solution obtained from different ultrasonic times. To ensure sufficient extraction, the extraction time was determined to be 30 min.

[0314] 3.4. Examination of Extraction Methods

[0315] The procedure was carried out according to Example 7, the difference being that the extraction methods were ultrasonic treatment (power 250W, frequency 40kHz) for 30 minutes and reflux extraction for 30 minutes, respectively. The results are shown in the table below:

[0316] Table 40 Content results for different extraction methods

[0317] Extraction method Oleic acid (mg / g) Linoleic acid (mg / g) Total amount (mg / g) ultrasound 6.6 7.5 14.1 reflux 6.5 7.5 14.0

[0318] The above results indicate that there is no significant difference in the total amount of oleic acid and linoleic acid in the test solution obtained by different extraction methods. Therefore, ultrasonic extraction was chosen to save time.

[0319] Example 9: Methodological validation of the method for detecting oleic acid and linoleic acid content in black sesame preparations.

[0320] 1. Accuracy

[0321] Accurately weigh 17.053 mg of oleic acid reference standard (purity 99.1%) and 18.825 mg of linoleic acid reference standard (purity 99.6%) and place them in a 50 ml volumetric flask. Add an appropriate amount of methanol to dissolve and dilute to the mark. Shake well to obtain the reference standard stock solution. Accurately measure 5 ml, 10 ml, and 15 ml of the reference standard stock solution and place them in separate 50 ml volumetric flasks. Add methanol to the mark and shake well to obtain reference solution 1, reference solution 2, and reference solution 3, respectively.

[0322] Take black sesame granules with known content (oleic acid 6.6 mg / g, linoleic acid 7.5 mg / g), grind them finely, take 9 portions, each 0.1 g, accurately weigh them, and number them 1 to 9. For portions 1 to 3, accurately add 10 ml of the above-mentioned reference solution 1; for portions 4 to 6, accurately add 10 ml of the above-mentioned reference solution 2; for portions 7 to 9, accurately add 10 ml of the above-mentioned reference solution 3. The remaining operations are the same as the preparation method of the test solution in Example 7. Detect the solution according to the method in Example 7, and calculate the recovery rate according to the following formula. The results are shown in Tables 41 and 42.

[0323]

[0324] Table 41 Results of Oleic Acid Recovery Rate Test

[0325]

[0326] Table 42 Results of Linoleic Acid Recovery Rate Test

[0327]

[0328] The recovery rates of oleic acid and linoleic acid, as determined in the recovery tests, ranged from 97.7% to 102.8% and from 97.5% to 102.9%, respectively. According to regulations, the recovery rate should be between 90% and 108% when the content of the analyte is 0.1%. Therefore, this meets the recovery rate requirements for method validation, indicating that the total oleic acid and linoleic acid results obtained using this method are accurate.

[0329] 2 Precision

[0330] 2.1 Repeatability: Six samples of black sesame granules (batch number: K467CP01) were tested according to the method in Example 7. The results are shown in the table below.

[0331] Table 43 Repeatability Tests

[0332] serial number Oleic acid (mg / g) Linoleic acid (mg / g) Total amount (mg / g) 1 6.6 7.5 14.1 2 6.5 7.5 14.0 3 6.7 7.5 14.2 4 6.6 7.4 14.0 5 6.6 7.5 14.1 6 6.6 7.5 14.1 average value 6.6 7.5 14.1 RSD (%) 1.0 0.5 0.5

[0333] The repeatability test showed that the average content of oleic acid was 6.6 mg / g with an RSD of 1.0%; the average content of linoleic acid was 7.5 mg / g with an RSD of 0.5%. The total average content of the two components was 14.1 mg / g with an RSD of 0.5%, which meets the repeatability requirements for method validation.

[0334] 2.2 Intermediate Precision: Intermediate precision tests were conducted at different times by different analysts using another Agilent 1290 ultra-high performance liquid chromatograph. Six samples of black sesame granules (batch number: K467CP01) were taken and measured according to the method in Example 7. The results are shown in the table below.

[0335] Table 44 Intermediate Precision Results

[0336] serial number Oleic acid (mg / g) Linoleic acid (mg / g) Total amount (mg / g) 1 6.6 7.7 14.3 2 6.6 7.6 14.2 3 6.6 7.6 14.2 4 6.6 7.6 14.2 5 6.6 7.6 14.2 6 6.6 7.6 14.2 average value 6.6 7.6 14.2 RSD (%) 0.0 0.5 0.3 RSD (%) for different instruments 0.3 1.1 0.6

[0337] The intermediate precision test showed that the average content of oleic acid was 6.6 mg / g with an RSD of 0.0%, the average content of linoleic acid was 7.6 mg / g with an RSD of 0.5%, and the total average content of the two components was 14.2 mg / g with an RSD of 0.3%. Compared with the repeatability test results, the RSD values ​​for oleic acid and linoleic acid were 0.3% and 1.1%, respectively, and the total RSD value for the two components was 0.6%, which meets the precision requirements for method validation.

[0338] 3 Exclusivity

[0339] Chromatograms were obtained by taking the black sesame granule test solution, the mixed oleic acid and linoleic acid reference solution, and the blank solution (methanol) according to the method in Example 7. The results are shown in [Figure 7]. Figure 17 The specificity was examined, and it was found that there was no interference at the corresponding positions of the peaks in the chromatogram of the test sample.

[0340] 4 linear

[0341] Accurately weigh appropriate amounts of oleic acid and linoleic acid reference standards, and prepare a mixed reference solution containing 0.18290 mg of oleic acid and 0.20713 mg of linoleic acid per 1 ml with methanol, as the linearity 5 solution; transfer 10 ml of the linearity 5 solution to a 25 ml volumetric flask and add methanol to the mark, as the linearity 4 solution; transfer 15 ml of the linearity 5 solution to a 50 ml volumetric flask and add methanol to the mark, as the linearity 3 solution; transfer 10 ml of the linearity 5 solution to a 50 ml volumetric flask and add methanol to the mark, as the linearity 2 solution; transfer 5 ml of the linearity 5 solution to a 50 ml volumetric flask and add methanol to the mark, as the linearity 1 solution. Accurately pipette 3 μl of the filtrate from each of the above solutions and inject it into a high-performance liquid chromatograph. Determine the peak area of ​​oleic acid and linoleic acid according to the method in Example 7. Plot a standard curve with the peak area of ​​each component as the abscissa and the concentration of each component as the ordinate. The regression equation for oleic acid is obtained as y = 4 × 10⁻⁶.-7 x+0.0004, R 2 =1.0000, with a linear range of 0.01829 mg / ml to 0.18290 mg / ml; the regression equation for linoleic acid is y = 7 × 10 -8 x-0.0003, R 2 =1.0000, and its linear range is 0.02071 mg / ml to 0.20713 mg / ml. The results are shown in Tables 45 and 46. Figure 18 , Figure 19 .

[0342] Table 45. Examination of the linear relationship of oleic acid.

[0343] serial number 1 2 3 4 5 Oleic acid concentration (mg / ml) 0.01829 0.03658 0.05487 0.07316 0.18290 oleic acid peak area 46844 93031 140356 189480 472828

[0344] Table 46. Investigation of the linear relationship of linoleic acid

[0345] serial number 1 2 3 4 5 Linoleic acid concentration (mg / ml) 0.02071 0.04143 0.06214 0.08285 0.20713 oleic acid peak area 302212 605475 903048 1207370 3002115

[0346] 5. Scope

[0347] Based on the results of precision, accuracy, and linearity experiments, as well as the results of multiple batches of black sesame granules, the oleic acid range is 0.01829 mg / ml to 0.18290 mg / ml, and the linoleic acid range is 0.02071 mg / ml to 0.20713 mg / ml.

[0348] 6. Durability

[0349] 6.1 Stability test: Black sesame granules (batch number: K467CP01) were taken and the test solution was prepared according to the method of Example 7. The stability was measured at 0h, 2h, 4h, 6h, 8h, 10h, 12h and 24h respectively according to the method of Example 7. The peak areas of oleic acid and linoleic acid were recorded. The results are shown in Tables 47 and 48.

[0350] Table 47 Oleic Acid Stability Results

[0351]

[0352] Table 48 Stability Results of Linoleic Acid

[0353]

[0354] Based on the above measurement results, the RSD of the oleic acid peak area was 0.6% and the RSD of the linoleic acid peak area was 0.5% within 24 hours, indicating that the sample had good stability within 24 hours.

[0355] 6.2 Investigation at different column temperatures:

[0356] Black sesame granules (batch number: K467CP01) were taken and prepared into a test solution according to the test solution preparation method in Example 7. The solution was then measured at different column temperatures (38℃, 40℃ and 42℃) according to the method in Example 7 to examine the durability of the experimental method for column temperature. The results are shown in the table below.

[0357] Table 49 Results of content determination at different column temperatures

[0358]

[0359] The theoretical plate number, tailing factor, and resolution of each peak of oleic acid and linoleic acid obtained at different column temperatures all met the system suitability requirements. The RSD values ​​of oleic acid content were 0.9%, linoleic acid content were 0.8%, and the total RSD value of the two components was 0.7%, indicating that the method has good robustness to different column temperatures.

[0360] 6.3 Investigation of different flow velocities:

[0361] Black sesame granules (batch number: K467CP01) were taken and prepared into a test solution according to the preparation method of the test solution in Example 7. The test solution was measured at different flow rates (0.28 ml / min, 0.30 ml / min and 0.32 ml / min) according to the method in Example 7 to investigate the robustness of the experimental method to the flow rate. The results are shown in the table below.

[0362] Table 50 Results of content determination at different flow rates

[0363]

[0364] The theoretical plate number, tailing factor, and resolution of each peak of oleic acid and linoleic acid obtained at different flow rates all met the system suitability requirements. The RSD values ​​of oleic acid content were 0.9%, linoleic acid content were 0.8%, and the total RSD value of the two components was 0.4%, indicating that the method has good robustness to different flow rates.

[0365] 6.4 Examination of different wavelengths:

[0366] Take black sesame granules (batch number: K467CP01), prepare the test solution according to the method of Example 7, and measure the wavelengths at different wavelengths (203nm, 205nm, 207nm) according to the method of Example 7 to investigate the robustness of the experimental method to different wavelengths. The results are shown in the table below.

[0367] Table 51 Results of content determination at different detection wavelengths

[0368]

[0369] The theoretical plate number, tailing factor, and resolution of each peak of oleic acid and linoleic acid obtained at different wavelengths all meet the system suitability requirements. The RSD values ​​of oleic acid content and linoleic acid content were 0.9% and 0.8%, respectively, and the RSD value of the total content of the two components was 0.7%, indicating that the method has good robustness to different wavelengths.

[0370] 6.5 Investigation of different chromatographic columns:

[0371] Take black sesame granules (batch number: K467CP01), prepare the test solution according to the method of Example 7, and use different types of chromatographic columns to determine the test solution according to the method of Example 7. The robustness of the experimental method to different chromatographic columns is investigated. The results are shown in the table below.

[0372] Column 1: Waters ACQUITY UPLC BEH C18 (2.1×100mm, 1.7μm)

[0373] Column 2: Waters ACQUITY UPLC BEH Shield RP18 (2.1 mm × 100 mm, 1.7 μm)

[0374] Column 3: Waters ACQUITY UPLC HSS T3 (2.1 mm × 100 mm, 1.8 μm)

[0375] Table 52 Results of content determination using different chromatographic columns

[0376]

[0377] The chromatograms obtained using different chromatographic columns showed that the theoretical plate number, tailing factor, and resolution of the oleic acid and linoleic acid peaks all met the system suitability requirements. The RSD values ​​for oleic acid content were 0.9%, linoleic acid content was 0.8%, and total component content was 0.4%, indicating that this method has good robustness for content determination using different chromatographic columns.

[0378] 6.6 Investigation of different acid concentrations:

[0379] Black sesame granules (batch number: K467CP01) were taken and a test solution was prepared according to the method in Example 7. Different acid concentrations (0.08% acetic acid, 0.10% acetic acid, 0.12% acetic acid) were used to determine the test solution according to the method in Example 7. The robustness of the experimental method to different mobile phase acid concentrations was investigated. The results are shown in the table below.

[0380] Table 53 Results of Determination of Different Acid Concentrations

[0381]

[0382] The chromatograms obtained at different acetic acid concentrations showed that the theoretical plate number, tailing factor, and resolution of each peak of oleic acid and linoleic acid all met the system suitability requirements. The RSD values ​​for oleic acid content were 0.9% and linoleic acid content were 0.8%, with a total RSD value of 0.4%, indicating that the method has good robustness to different acetic acid concentrations.

[0383] Comparative Example 1

[0384] This comparative example uses the method disclosed in CN117330671A for constructing and applying the chromatograms of roasted black sesame medicinal material, roasted black sesame slices, standard decoction, and their formulation granules. The black sesame formulation granules in Example 1 were tested according to the method in Example 6. The results are as follows: Figure 20 As shown, in Comparative Example 1, the elution time was 65 minutes, and 6 characteristic peaks were identified. In the first 20 minutes, only peaks 1, 2, and 3 were effective characteristic peaks. The detection time was relatively long, and the resolution of each chromatographic peak was low.

[0385] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for constructing a characteristic map of black sesame formula granules, characterized in that, This includes detection using ultra-high performance liquid chromatography (UHPLC), with chromatographic conditions including: A Waters ACQUITY UPLC BEH Shield RP18 column with a length of 100 mm, an inner diameter of 2.1 mm, and a particle size of 1.7 µm was used. Methanol was used as mobile phase A, and water was used as mobile phase B. Gradient elution was performed, and the detection wavelength was 285 nm to 289 nm. The elution program is as follows: The preparation of the test solution includes: taking the test sample, adding the extraction solvent, extracting, separating the solid and liquid, and taking the liquid; the extraction solvent is water, a 30~70 v / v% methanol aqueous solution or a 30~70 v / v% ethanol aqueous solution; The characteristic spectrum has 7 common characteristic peaks, including characteristic peaks of sesamin and sesamolin.

2. The method for constructing the characteristic map of black sesame formula granules according to claim 1, characterized in that, The chromatographic conditions also include at least one of the following conditions: 1) The flow rate is 0.28~0.32 ml per minute; 2) Column temperature is 33~37℃; 3) The detection wavelength is 287nm; 4) The injection volume is 1~3 μl.

3. The method for constructing the characteristic spectrum of black sesame formula granules according to claim 2, characterized in that, The chromatographic conditions also include at least one of the following conditions: 1) The flow rate is 0.3 ml per minute; 2) Column temperature is 35℃; 3) The injection volume is 2 μl.

4. The method for constructing the characteristic spectrum of black sesame formula granules according to claim 3, characterized in that, The preparation of the test solution includes at least one of the following conditions: 1) The extraction method used in the extraction step is ultrasonic extraction or reflux extraction; 2) The extraction time in the extraction step is 20-40 minutes; 3) The mass-volume ratio of the test sample to the extraction solvent is 0.5g:15~50ml.

5. The method for constructing the characteristic spectrum of black sesame formula granules according to any one of claims 1-4, characterized in that, It also includes the preparation of the reference solution, including: Preparation of reference herb solution: Take the reference herb, add the extraction solvent, extract, separate the solid and liquid, evaporate the filtrate to dryness, add the reconstitution solvent, and take the liquid; Preparation of reference solution: Take sesamin reference standard and add solvent to prepare a solution containing 20~40µg per ml.

6. The method for constructing the characteristic spectrum of black sesame formula granules according to claim 5, characterized in that, The reference solution is a solution containing 30 µg of sesamin reference standard per ml; And / or, the solvent in the reference solution is a 50-70 v / v% aqueous methanol solution.

7. The method for constructing the characteristic spectrum of black sesame formula granules according to claim 6, characterized in that, The solvent in the reference solution is a 70 v / v methanol aqueous solution.

8. The method for constructing the characteristic spectrum of black sesame formula granules according to claim 5, characterized in that, The preparation of the control herbal solution includes at least one of the following conditions: 1) The extraction solvent is water, a 30~70 v / v% methanol aqueous solution, or a 30~70 v / v% ethanol aqueous solution; 2) The extraction method used in the extraction step is ultrasonic extraction or reflux extraction; 3) The extraction process is repeated 1-2 times. 4) The extraction time in the extraction step is 20-40 minutes; 5) The mass-to-volume ratio of the reference medicinal material to the extraction solvent is 2g:25~50ml; 6) The resolvent is a 50~70 v / v% methanol aqueous solution; 7) The mass-volume ratio of the reference medicinal material to the reconstitution solvent is 2g:5~10ml.

9. The method for constructing the characteristic spectrum of black sesame formula granules according to claim 8, characterized in that, 6) The resolvent should be a 70 v / v% methanol aqueous solution.

10. A method for quality testing of black sesame granules, characterized in that, This includes the step of comparing the characteristic spectrum of the sample to be tested with the control characteristic spectrum of black sesame formula granules; The feature spectrum of the sample to be tested is constructed according to the construction method of any one of claims 1-6; The control feature spectrum of the black sesame formula granules was prepared by using the feature spectrum obtained from 15 batches of black sesame formula granule standards according to the construction method described in any one of claims 1-9, and then using the average value or median method.

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