Specific chromatogram construction method, quality detection method and content determination method of black sesame formula granule preparation

Abstract

The invention relates to the technical field of traditional Chinese medicine analysis and detection, and relates to a specific chromatogram construction method, a quality detection method and a content determination method of a black sesame formula granule preparation. According to the method for constructing the characteristic chromatogram of the black sesame preparation, ultra-high performance liquid chromatography is adopted for detection, and the chromatographic conditions include that octadecylsilane chemically bonded silica serves as a filler, methyl alcohol serves as a mobile phase A, water serves as a mobile phase B, and gradient elution is carried out. The method can comprehensively reflect the characteristic peak information of the sample, has the advantages of good peak pattern, good separation degree, high precision, good reproducibility and strong specificity, can comprehensively detect the quality of the black sesame preparation, and is short in detection time.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine analysis and detection, and relates to a characteristic spectrum construction method, a quality detection method and a content determination method of a black sesame formula granule preparation. Background Art

[0002] Black sesame is the dried mature seeds of Sesamumindicum L., a plant of the Sesamaceae family. This product is mild in nature, sweet in taste, and enters the liver, kidney, and large intestine meridians. It is a tonic Chinese herbal medicine with the effects of nourishing the liver and kidney, benefiting the essence and blood, and moisturizing the intestines. It is used for deficiency of essence and blood, dizziness, tinnitus and deafness, premature graying of hair, hair loss after illness, and constipation due to dry intestines. The quality standard of this medicinal material is included in the 2020 edition of the "Chinese Pharmacopoeia", in which there is no characteristic spectrum and content determination detection method under the black sesame item. At present, there are few literatures on the quality research of black sesame, most of which are studies on the chemical composition and pharmacological effects of black sesame, and there are even fewer studies on black sesame preparations. Therefore, the characteristic spectrum and content determination detection method of black sesame preparations are established to provide a scientific basis for the comprehensive establishment of quality control standards for black sesame preparations.

[0003] At present, in the research on the characteristic spectrum of black sesame formula granules, there are defects such as long detection time, fewer chromatographic peaks, poor separation of characteristic peaks, and relatively complicated detection methods in content determination, which makes it difficult to conduct comprehensive quality control and effective ingredient content determination of 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 a black sesame preparation, which can perform overall quality control and identification of the black sesame preparation, and provide a scientific basis for comprehensively establishing quality control standards for black sesame formula 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, which not only establishes content determination indicators for lignan components in black sesame preparations, but also establishes detection methods for the content of oleic acid and linoleic acid, which are oil components with relatively high content. This method can simply, quickly and efficiently determine the content of effective ingredients in black sesame.

[0006] The embodiment disclosed in the present invention relates to a method for constructing a characteristic spectrum of a black sesame preparation, including detection by ultra-high performance liquid chromatography, wherein the chromatographic conditions include:

[0007] Octadecylsilane bonded silica gel was used as filler, methanol was used as mobile phase A, and water was used as mobile phase B. Gradient elution was performed. The elution procedure was 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) The column temperature is 33-37°C; optionally, the column temperature is 35°C;

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

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

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

[0015] In some embodiments, the method further includes preparing a test sample solution, including: taking a test sample, adding an extraction solvent, extracting, separating a solid from a liquid, and taking a 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, 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 volume ratio of the test sample and the extraction solvent is 0.5g:15~50ml.

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

[0022] Preparation of control medicinal material solution: taking control medicinal material, adding extraction solvent, extracting, solid-liquid separation, evaporating the filtrate to dryness, adding resolvent, and taking liquid;

[0023] And / or, preparation of reference solution: take sesamin reference, add solvent to make 1 ml of solution containing 20-40 μg; optionally, 1 ml of solution containing 30 μg; optionally, the solvent is 50-100 v / v% methanol or its aqueous solution; optionally, 70 v / v% methanol aqueous solution is selected.

[0024] In some embodiments, the preparation of the control medicinal material 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 number of extractions in the extraction step is 1 to 2 times;

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

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

[0030] 5) The re-dissolving solvent is 50-100 v / v% methanol or its aqueous solution; optionally, 70 v / v% methanol aqueous solution is selected;

[0031] 6) The mass volume ratio of the control medicinal material and the reconstitution solvent is 2g:5~10ml.

[0032] The embodiment disclosed in the present invention relates to a method for detecting the content of a black sesame preparation, wherein a test solution and a reference solution are taken and respectively detected by ultra-high performance liquid chromatography in the method for constructing a characteristic spectrum of the black sesame preparation;

[0033] Control substances included sesamin.

[0034] In some embodiments, a method for detecting the content of oleic acid and / or linoleic acid is also included, including detection by ultra-high performance liquid chromatography, and the chromatographic conditions include:

[0035] Octadecylsilane bonded silica gel is used as a filler, acetonitrile is used as a mobile phase A, 0.08-0.12 v / v% acetic acid solution is used as a mobile phase B, and the volume ratio of mobile phase A to mobile phase B is 79-81:21-19 for elution;

[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) The column temperature is 38-42°C; optionally, the column temperature is 40°C;

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

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

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

[0042] Optionally, it also includes preparing a test sample solution: taking a test sample, adding an extraction solvent, extracting, separating the solid from the liquid, and taking a 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 volume ratio of the test sample and the extraction solvent is 0.2g:10~20ml;

[0048] Optionally, the method further includes preparing a reference solution: taking an oleic acid reference substance and a linoleic acid reference substance, adding a solvent to prepare a mixed solution containing 65 to 75 μg of oleic acid and 75 to 85 μg of linoleic acid per 1 ml;

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

[0050] The embodiment disclosed in the present invention relates to a method for quality detection of a black sesame preparation, comprising the steps of comparing a characteristic spectrum of a sample to be tested with a control characteristic spectrum of a black sesame preparation;

[0051] The characteristic spectrum of the sample to be tested is constructed according to the construction method;

[0052] The control characteristic spectrum 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 corresponding to the peak of the sesamin reference substance is the S peak. The relative retention times of peaks 1-5, peak 7 and S peak are calculated. The relative retention times should be within the range of ±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 product to obtain the characteristic spectrum according to the construction method, a control characteristic spectrum is prepared by the average value or median method.

[0055] In some embodiments, in (1), peak 6 corresponds to sesamin; and peak 7 corresponds to sesamelin.

[0056] In some embodiments, the black sesame preparation includes black sesame formula granules, water extract, etc.

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

[0058] 1. A method for constructing a characteristic spectrum of a black sesame preparation provided by the present invention comprises detecting by ultra-high performance liquid chromatography, wherein the chromatographic conditions comprise: using octadecylsilane bonded silica gel as a filler, using methanol as a mobile phase A, and using water as a mobile phase B, and performing gradient elution, wherein the elution procedure is as follows: 0 to 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 to 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 to 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, The method comprises the following steps: the first step is to prepare a mobile phase A and a second step is to prepare a mobile phase B. The first step is to prepare a mobile phase B and a second step is to prepare a mobile phase A. The second step is to prepare a mobile phase B. The second step is to prepare a mobile phase B. The third step is to prepare a mobile phase A. The fourth step is to prepare a mobile phase B. The fourth step is to prepare a mobile phase B. The fifth step is to prepare a mobile phase B. The fifth step is to prepare a mobile phase A. The fifth step is to prepare a mobile phase B. The fifth step is to prepare a mobile phase B. The fifth step is to prepare a mobile phase B. The fifth step is to prepare a mobile phase A. The fifth step is to prepare a mobile phase B. The fifth step is to prepare a mobile phase B. The fifth step is to prepare a mobile phase B. The fifth step is to prepare a mobile phase A.

[0059] 2. The present invention provides a method for detecting the content of a black sesame preparation, comprising taking a test solution and a reference solution, and respectively adopting the ultra-high performance liquid chromatography method in the method for constructing a characteristic spectrum of the black sesame preparation for detection; the reference substance includes sesamin; the above method detects the content of sesamin, an effective ingredient in the black sesame preparation, with high accuracy, high precision, strong specificity, wide linear range, high correlation and strong durability.

[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 an ultra-high performance liquid chromatography method. The detection method is simple and easy to operate. It can also detect the content of the effective ingredients oleic acid and linoleic acid with high accuracy, high precision, strong specificity, wide linear range, high correlation and strong durability. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0062] Figure 1 Chromatogram of the test sample in Example 1 of the present invention;

[0063] Figure 2 Chromatogram of the reference substance in Example 1 of the present invention;

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

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

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

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

[0068] Figure 7 Chromatograms of 15 batches of black sesame formula granules (K467CP01-15) in Example 3 of the present invention; the chromatograms correspond to K467CP01-15 from bottom to top;

[0069] Figure 8 The reference characteristic spectrum in Example 3 of the present invention;

[0070] Fig. 9 Characteristic peak identification results in Example 3 of the present invention;

[0071] Fig.10 Chromatogram of reference medicinal material in Example 3 of the present invention;

[0072] Fig.11 Specific chromatogram in Example 6 of the present invention;

[0073] Fig.12 Sesamin linear graph in Example 6 of the present invention;

[0074] Fig.13 The ultraviolet absorption spectrum of oleic acid in Example 8 of the present invention;

[0075] Fig.14 The ultraviolet absorption spectrum of linoleic acid in Example 8 of the present invention;

[0076] Fig.15 The chromatogram of method 1 in Example 8 of the present invention;

[0077] Fig.16 Chromatogram of method 2 in Example 8 of the present invention;

[0078] Fig.17 Chromatogram of the specificity experiment in Example 9 of the present invention;

[0079] Fig.18 Linear graph of oleic acid in Example 9 of the present invention;

[0080] Fig.19 The linear graph of linoleic acid in Example 9 of the present invention;

[0081] Fig. 20 Characteristic spectrum of black sesame formula particles in Comparative Example 1 of the present invention. DETAILED DESCRIPTION

[0082] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0083] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0084] The following instruments, reagents and reagents are:

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

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

[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 testing:

[0091] Sesamin reference substance (batch number: 110836-201706, purity: 99.9%, China Food and Drug Inspection Institutes);

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

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

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

[0095] Black sesame reference medicinal material (batch number: 121339-201803, China Food and Drug Inspection Institute);

[0096] The black sesame square granules can be prepared by conventional methods in the art, for example, in the present invention, they are prepared according to the following steps: 80 kg of black sesame slices are taken, and the mixture is extracted twice by boiling (100° C.), the amount of water added in the first decoction is 10 times the amount of the slices (10 ml of water is added per gram of the slices), and the extraction is performed for 1 hour, the amount of water added in the second decoction is 8 times the amount of the slices (8 ml of water is added per gram of the slices), and the extraction is performed for 1 hour, the mixture is filtered while hot through a 150-mesh filter cloth, and the filtrate is concentrated under reduced pressure at 80° C. to a relative density of 1.05 to 1.10 (60° C.), spray-dried, the air inlet temperature is set to 180° C.±5, dry granulated, and the packaging specifications are 1 g / bag, 100 g / bottle, and 250 g / bottle, and the mixture is sealed and stored. Black sesame formula granules with batch numbers K467CP01, K467CP02, K467CP03, K467CP04, K467CP05, K467CP06, K467CP07, K467CP08, K467CP09, K467CP10, K467CP11, K467CP12, K467CP13, K467CP14 and K467CP15 were prepared using black sesame slices from different batches.

[0097] Reagents:

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

[0099] Example 1

[0100] This embodiment provides a method for constructing a characteristic spectrum of a black sesame preparation, including detection by ultra-high performance liquid chromatography, wherein the chromatographic conditions include:

[0101] Octadecylsilane bonded silica gel was used as the filler (column length: 100 mm, inner diameter: 2.1 mm, particle size: 1.7 μm, Waters ACQUITY UPLC BEH Shield RP18); methanol was used as mobile phase A, water was used as mobile phase B, and gradient elution was performed according to the provisions in the following table; the flow rate was 0.3 ml per minute, the column temperature was 35°C, and the detection wavelength was 287 nm. The number of theoretical plates 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 reference solution: Take 2g of black sesame reference medicinal material, place in a stoppered conical flask, add 25ml of water, heat and reflux for 40 minutes, cool, shake, filter, evaporate the filtrate to dryness, add 70v / v% methanol to dissolve the residue, place in a 10ml volumetric flask, add 70v / v% methanol to the scale, filter, and take the filtrate as the reference medicinal material reference solution. Take an appropriate amount of sesamin reference substance, accurately weigh, add 70v / v% methanol to make a solution containing 30μg per 1ml, and obtain it as the reference substance solution.

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

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

[0107] The test sample should show 7 common characteristic peaks, and the retention time should correspond to that of the reference medicinal material. Peak 6 and Peak 7 should correspond to the retention time of sesamin and sesamolin reference substances, respectively. Peak 6 corresponding to the sesamin reference substance peak is the S peak. Calculate the relative retention time of peaks 1-5, peak 7 and S peak. The relative retention time should be within ±10% of the specified value. The specified values ​​of peaks 1-5 and peak 7 are: 0.42, 0.57, 0.66, 0.71, 0.75, 1.05, respectively; Peak 6 corresponds to sesamin; Peak 7 corresponds to sesamolin. The results are as follows Figures 1 to 3 shown.

[0108] Example 2 Optimization of Chromatographic Conditions

[0109] 1. Gradient elution program optimization

[0110] The method of Example 1 was followed, except that the gradient elution procedure was in accordance with Method 1 in the following table.

[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 The characteristic peaks of the characteristic spectrum of black sesame formula granules obtained by this method are concentrated between 6 and 9 minutes, and the separation of each characteristic peak is poor, which needs further optimization. Therefore, the method 2 in the table below is used.

[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 The characteristic spectrum of black sesame formula granules obtained by this method has a large interval between 10 and 16 minutes, and the reference peak is later than that of method 1. The elution gradient is further optimized, and method 3 in the table below is 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 the above method optimization, method three has more chromatographic information and the system suitability experimental results are good. Therefore, method three is used as the gradient elution method for the characteristic spectrum of black sesame particles.

[0120] Table 5 Peak information in the chromatogram of the test solution of method 3

[0121] Peak Retention time (minutes) Separation Tailing Factor Theoretical 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 test solution

[0123] 2.1 Selection of extraction solvent:

[0124] The same method as in Example 1 was used, except that the extraction solvents were 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. The results are shown in the following table:

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

[0126] Solvent type 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] It can be seen from the above table that the peak areas of the characteristic peaks are smaller when ethanol is used as the solvent. Taking into account that the peak areas of the characteristic peaks are higher and the peak shapes are better when 70% methanol is used as the extraction solvent, 70% v / v methanol is selected as the extraction solvent.

[0128] 2.2. Selection of extraction solvent dosage

[0129] According to Example 1, the extraction solvent was used in an amount of 15 ml, 25 ml, and 50 ml, respectively. The peak area of ​​each characteristic peak was measured. In order to avoid the influence of the test sample concentration on the peak area, the peak area was converted by ratio with the test sample concentration (i.e., the corrected data), and the results are shown in the table below.

[0130] Table 7 Peak area table of characteristic spectra of different extraction solvent amounts

[0131]

[0132] It can be seen from the above table that the peak areas of each characteristic peak at each extraction solvent dosage are not much different. In order to fully extract the sample, 25 ml is selected as the extraction solvent dosage.

[0133] 2.3. Extraction method selection

[0134] The extraction was carried out according to Example 1, with ultrasonic treatment (power 250W, frequency 40kHz) for 30 minutes and reflux extraction for 30 minutes. The peak area of ​​each characteristic peak was measured. The results are shown in the following table.

[0135] Table 8 Peak area table of characteristic spectra of different extraction 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 Reflow 446215 32755 75943 10603 16380 297250 76713

[0137] It can be seen from the above table that 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 is determined as the extraction method for preparing the test solution.

[0138] 2.4. Selection of extraction time

[0139] The extraction time was 20, 30 and 40 minutes respectively according to Example 1. The results are shown in the following table.

[0140] Table 9 Peak area table of characteristic spectra 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 slightly different under different extraction times. Considering the principle of saving time, the ultrasonic time is selected as 20 minutes.

[0143] Example 3 Establishment of the control characteristic spectrum

[0144] Take 15 batches of black sesame formula granules (K467CP01-15) as test samples and implement according to the method of Example 1. The results are as follows Figure 7 The Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version) was used to synthesize 15 batches of samples, and a reference spectrum of the characteristic spectrum of black sesame formula granules (i.e., the reference characteristic spectrum) was established. Figure 8 As shown in the figure, according to the principle of stable relative retention time and the fact that each batch of samples can be detected and the peak is relatively high, a total of 7 peaks with good repeatability were selected as characteristic peaks. Peak 6 is the sesamin reference peak (S peak), and the relative retention time of peaks 1 to 5, peak 7 and S peak is calculated. The relative retention time of each characteristic peak is 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 medicine chromatographic fingerprint similarity evaluation system (2012 edition). 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 Formula Granules-K467CP02 0.995 3 Black Sesame Formula Granules-K467CP03 0.997 4 Black Sesame Formula Granules-K467CP04 0.995 5 Black Sesame Formula 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 Formula 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 formula granules is greater than 0.99.

[0150] Characteristic peak identification

[0151] Sesamin and sesamolin reference substances were taken in appropriate amounts, accurately weighed, and 70% methanol was added to prepare solutions containing 30 μg of sesamin and 30 μg of sesamolin per 1 ml to obtain sesamin reference substance solutions and sesamolin reference substance solutions. The black sesame formula granule test solution was prepared according to the test solution preparation method of Example 1. The sesamin reference substance solution, sesamolin reference substance solution and black sesame formula granule test solution were tested and compared according to the chromatographic conditions of Example 1. The results are as follows: Fig. 9 .

[0152] The retention times of Peak 6 and Peak 7 in the chromatogram of the black sesame formula granules test sample are consistent with those of the chromatogram peaks of sesamin and sesamolin reference substances, respectively, and it can be confirmed that Peak 6 is sesamin and Peak 7 is sesamolin.

[0153] Comparison with black sesame control medicinal material

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

[0155] Therefore, it is stipulated that 7 characteristic peaks should be presented in the chromatogram of the test sample, and should correspond to the retention time of the reference medicinal materials. Among them, peak 6 and peak 7 should correspond to the retention time of sesamin and sesamolin reference substances respectively. The peak corresponding to the sesamin reference substance is the S peak. The relative retention times of peaks 1 to 5, peak 7 and S peak are calculated. Their relative retention times should 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 Methodology Verification

[0157] 1. Precision experiment

[0158] 1.1. Repeatability

[0159] Take 6 portions of black sesame formula granules (batch number: K467CP01), measure according to Example 1, obtain its characteristic spectrum, calculate the relative retention time and relative peak area of ​​peaks 1 to 5, peak 7 and S peak with peak 6 as reference peak S, and calculate the RSD value. The results are shown in the following table.

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

[0161]

[0162] Table 12 Peak area and relative peak area of ​​black sesame repeatability test

[0163]

[0164] The results of the repeatability experiment showed that the relative retention time RSD of the seven identification peaks of the six repeatability experimental 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 formula granules (batch number: K467CP01) were taken, and intermediate precision tests were performed on different instruments by different analysts at different times, and the characteristic spectrum was obtained according to Example 1. Peak 6 was used as the reference peak S, and the relative retention time and relative peak area of ​​peaks 1 to 5, peak 7 and S were calculated, and the RSD value was calculated. The results are shown in the following table.

[0167] Table 13 Retention time and relative retention time of intermediate precision test of black sesame

[0168]

[0169]

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

[0171]

[0172] The relative retention time RSD of each characteristic peak of intermediate precision is between 0.0% and 0.0%, and the relative peak area RSD is between 0.0% and 2.0%, indicating that the intermediate precision of the characteristic spectrum is good.

[0173] 5.2 Solution stability study

[0174] Take black sesame formula granules (batch number: K467CP01) test solution, prepare black sesame formula granules test solution according to the method of Example 1, respectively at 0h, 2h, 4h, 6h, 8h, 10h, 12h, 24h, measure according to the method of Example 1, analyze the characteristic peaks therein, and the results show that the test solution is stable within 24h. The results are shown in the table below.

[0175] Table 15 Retention time and relative retention time of black sesame durability test

[0176]

[0177]

[0178] Table 16 Peak area and relative peak area of ​​black sesame durability test

[0179]

[0180] After examining the stability of the solution for 24 hours, the relative retention time RSD of the seven identified peaks was in the range of 0.0% to 0.1%, and the relative peak area RSD was in the range of 0.0% to 1.6%, indicating that the characteristic spectrum analysis method of black sesame particles used in this experiment is stable, reliable, and has good reproducibility.

[0181] 3 Durability test

[0182] 3.1 Investigation of different column temperatures

[0183] To investigate the effect of slight changes in column temperature on the detection method, an appropriate amount of black sesame formula granules were taken, and a test solution was prepared according to Example 1. The column temperatures were 33°C, 35°C and 37°C, respectively. The rest of the characteristic spectrum of the black sesame formula granule test solution was detected according to the method of Example 1. The results showed that the method had good durability for different column temperatures.

[0184] Table 17 Retention time and relative retention time of black sesame at different column temperatures

[0185]

[0186]

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

[0188]

[0189] The RSDs of the relative retention times of the seven identified peaks in the samples were all within the range of ±10% of the specified value, and this method had good durability for different column temperatures.

[0190] 3.2 Investigation of different flow rates

[0191] Take an appropriate amount of black sesame formula granules, prepare a test solution and measure it according to Example 1. Use different flow rates (0.28 ml / min, 0.30 ml / min and 0.32 ml / min) to investigate the durability of the chromatographic method in Example 1 with respect to flow rate.

[0192] Table 19 Retention time and relative retention time of black sesame at different flow rates

[0193]

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

[0195]

[0196] The RSDs of the relative retention times of the seven identified peaks in the samples were all within the range of ±10% of the specified value, and the method had good durability for different flow rates.

[0197] Example 4: Determination of Sesamin Content in Black Sesame Formula Granules

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

[0199] Octadecylsilane bonded silica gel was used as the filler (column length: 100 mm, inner diameter: 2.1 mm, particle size: 1.7 μm, Waters ACQUITY UPLC BEH Shield RP18); methanol was used as mobile phase A, water was used as mobile phase B, and gradient elution was performed according to the provisions in the following table; the flow rate was 0.3 ml per minute, the column temperature was 35°C, and the detection wavelength was 287 nm. The number of theoretical plates 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 reference solution: Take 2g of black sesame reference medicinal material, place in a stoppered conical flask, add 25ml of water, heat and reflux for 40 minutes, cool, shake, 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 take the filtrate as the reference medicinal material reference solution. Take an appropriate amount of sesamin reference substance, accurately weigh, add 70% methanol to make a solution containing 30μg per 1ml, and obtain it as the reference substance solution.

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

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

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

[0206] Table 22 Content determination results of 15 batches of black sesame formula granules

[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 - Preparation of Test Solution

[0209] 1. Investigation of extraction solvent

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

[0211] Table 23 Comparison of different extraction solvents

[0212]

[0213] The above results show that the sesamin content is higher when methanol, 70% methanol and ethanol are used as extraction solvents. Combined with the poor peak shapes of the characteristic peaks of methanol and ethanol in the characteristic spectrum, 70% methanol is selected as the extraction solvent based on comprehensive considerations.

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

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

[0216] Table 24 Extraction solvent dosage investigation results

[0217]

[0218] It can be seen from the above table that there is no significant difference in the sesamin content of each extraction solvent dosage. In order to fully extract the sample, 25 ml is selected as the extraction solvent dosage.

[0219] 3. Extraction time investigation

[0220] The preparation of the test solution was carried out according to Example 4, and the extraction of the reagent was carried out for 20, 30, and 40 minutes respectively. The content of sesamin was calculated, and the results are shown in the table below.

[0221] Table 25 Results of different extraction time contents

[0222]

[0223]

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

[0225] 4. Investigation of extraction methods

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

[0227] Table 26 Content results of different extraction methods

[0228]

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

[0230] Example 6 Methodological Verification of the Detection Method for Determining Sesame Content in Black Sesame Formula Granules

[0231] 1. Accuracy

[0232] Take an appropriate amount of sesamin reference substance, accurately weigh it, and add 70% methanol to make a sesamin reference substance solution containing 2.3132 mg per 1 ml. Take 9 portions of black sesame formula granules with known content (sesamin content is 2.3 mg / g), each portion is about 0.25 g, and each 3 portions are 1 group, accurately weigh it, and accurately add 25 ml of reference substance solution containing 0.5783 mg, 1.1566 mg, and 1.7349 mg of sesamin according to low, medium, and high concentrations, respectively, and the remaining operating example 4 method is used to 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 by the recovery test ranged from 94.54% to 100.72%, with an average recovery rate of 98.9% and an RSD value of 2.0%, which met the recovery rate requirements of the methodology validation, indicating that the results measured by this method are accurate.

[0237] 2Precision

[0238] 2.1 Repeatability: 6 portions of black sesame granules (batch number: K467CP01) were taken and measured according to the method of Example 4. The results are shown in the table below.

[0239] Table 28 Repeatability test

[0240]

[0241] The average sesamin content in black sesame granules measured in the repeatability test was 2.3 mg / g, and the RSD value was 0.8%, which met the repeatability requirements of the methodology validation.

[0242] 2.2 Intermediate precision: Different analysts conducted intermediate precision tests at different times 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 of Example 4. The results are shown in the table below.

[0243] Table 29 Intermediate precision results

[0244]

[0245] The content of sesamin in black sesame granules measured by the intermediate precision test was 2.3 mg / g, with an RSD value of 0.7%, which was consistent with the RSD of 1.0% in the repeatability test, meeting the requirements for intermediate precision of methodological 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 a chromatogram according to the method of Example 4, and the results are shown in Fig.11 .

[0248] By examining the specificity, it was found that there was no interference at the corresponding position of the peak in the chromatogram of the test sample.

[0249] 4. Linear

[0250] Take an appropriate amount of sesamin reference substance, add 70% methanol to make a solution containing 0.1512 mg per 1 ml, as the mother solution of sesamin reference substance, recorded as ①; accurately draw 5 ml, 1 ml, and 1 ml of the mother solution of sesamin reference substance respectively, and place them in 10 ml, 10 ml, and 20 ml volumetric flasks, add 70% methanol to the scale, shake well, and filter, respectively, and record them as ② to ④; accurately draw 45 ml and place it in a 10 ml volumetric flask, add 70% methanol to the scale, shake well, and filter, and record them as ⑤. Accurately draw 2 μl of the subsequent filtrate from ① to ⑤ and inject it into the liquid chromatograph, and measure 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, and the regression equation is y=8817324.0455x+1727.3639, R 2 =1.0000, and its linear range is 0.0038mg / ml~0.1512mg / ml. The results are shown in the table below. Fig.12 .

[0251] Table 30 Sesamin linear relationship investigation

[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] According to the precision, accuracy and linearity test results and the results of sesamin content in 15 batches of black sesame granules, the range is 0.0038 mg / ml to 0.1512 mg / ml.

[0255] 6. Durability inspection

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

[0257] Table 31 Sesamin stability results

[0258]

[0259] Summary: According to the above measurement results, the RSD of the sesamin peak area within 24 hours was 0.5%, indicating that the sample had good stability within 24 hours.

[0260] 6.2 Investigation of 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 of Example 4. The test was carried out at different column temperatures (33°C, 35°C and 37°C) according to the method of Example 4 to investigate the durability of the experimental method for column temperature. The results are shown in the table below.

[0262] Table 32 Sesame content determination results at different column temperatures

[0263]

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

[0265] 6.3 Investigation of different flow rates:

[0266] 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 4. Different flow rates (0.28 ml / min, 0.30 ml / min and 0.32 ml / min) were used to measure according to the method of Example 4 to investigate the durability of the experimental method for flow rate. The results are shown in the table below.

[0267] Table 33 Sesamin content determination results at different flow rates

[0268]

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

[0270] 6.4 Investigation of different chromatographic columns:

[0271] Take black sesame granules (batch number: K467CP01), prepare the test solution according to the method of Example 4, use different types of chromatographic columns to measure according to the method of Example 4, and investigate the durability of the experimental method for different chromatographic columns. 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 Sesamin content determination results of different chromatographic columns

[0276]

[0277] The RSD value of sesamin content measured by different chromatographic columns was 0.7%, which met the system suitability requirements, indicating that the method had good durability for different chromatographic columns.

[0278] 6.5 Investigation of different wavelengths:

[0279] Take black sesame granules (batch number: K467CP01), prepare the test solution according to the method of Example 4, use different wavelengths (285nm, 287nm, 289nm) to measure according to the method of Example 4, and investigate the durability of the experimental method for different wavelengths. The results are shown in the table below.

[0280] Table 35 Sesamine content determination results at different wavelengths

[0281]

[0282] The RSD value of sesamin content measured at different wavelengths was 0.5%, indicating that the method had good durability for different wavelengths.

[0283] Example 7 Method for detecting the content of oleic acid and linoleic acid in black sesame preparation

[0284] This embodiment provides a method for detecting the content of oleic acid and linoleic acid in a black sesame preparation, comprising detecting by high performance liquid chromatography, wherein the chromatographic conditions include:

[0285] Octadecylsilane bonded silica gel was used as filler (column length 100 mm, inner diameter 2.1 mm, particle size 1.7 μm, Waters ACQUITY UPLC BEH C18); acetonitrile-0.1 v / v% acetic acid solution (80:20) was used as mobile phase; flow rate was 0.3 ml per minute; column temperature was 40°C; detection wavelength was 205 nm. The theoretical plate number calculated based on the linoleic acid peak should not be less than 10,000.

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

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

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

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

[0290] Table 36 Content determination results of 15 batches of black sesame formula granules

[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] Through 3D chromatographic scanning, the maximum absorption wavelength of oleic acid and linoleic acid is around 200nm. Considering the reduction of interference from the end absorption of the solvent, the detection wavelength of oleic acid and linoleic acid content was selected as 205nm. Figure 13-14 .

[0296] 2. Optimization of chromatographic conditions

[0297] Method 1 was used for detection, which differed from Example 7 in that the mobile phase was acetonitrile-0.1% acetic acid solution (90:10). The detection results were as follows: Fig.15 . As can be seen from the figure, when the black sesame granule test solution was tested under the chromatographic conditions of method 1, the linoleic acid peak time was too early, resulting in a distance too close to the peak of the component eluted earlier, which easily led to poor separation. Therefore, the method of reducing the proportion of organic phase in the mobile phase was considered. Method 2 was used for detection. The difference from method 1 is that the mobile phase is acetonitrile-0.1% acetic acid solution (80:20). The test results are as follows Fig.16 As can be seen from the figure, when the black sesame granule test solution was determined using the chromatographic conditions of method 2, the theoretical plate number, tailing factor, and separation of the linoleic acid peak and oleic acid peak all met the system suitability requirements, and the retention time was moderate. Finally, method 2 was determined as the chromatographic condition for the determination of oleic acid and linoleic acid content in black sesame formula granules.

[0298] 3. Preparation of test solution

[0299] 3.1. Investigation of extraction solvent

[0300] The method was implemented according to Example 7, except that the extraction solvents were water, 50% methanol, methanol, 50% ethanol, and anhydrous ethanol. The test results are shown in the following table.

[0301] Table 37 Extraction solvent investigation table

[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 show that the total amount of oleic acid and linoleic acid is the highest when methanol is used as the extraction solvent, so methanol is selected as the extraction solvent.

[0304] 3.2. Investigation of the amount of extraction solvent

[0305] The same method as in Example 7 was used, except that the amount of the extraction solvent was 10 ml, 15 ml, and 20 ml, respectively. The results are shown in the table below.

[0306] Table 38 Extraction solvent dosage investigation results

[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] From the above test results, it can be seen that there is no obvious 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 amount of extraction solvent added is selected as 10 ml.

[0309] 3.3. Extraction time investigation

[0310] The same method as in Example 7 was used, except that the extraction time was 20, 30, and 40 minutes, respectively. The results are shown in the following table:

[0311] Table 39 Content results of different extraction time

[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 show that there is no significant difference in the total amount of oleic acid and linoleic acid in the test solution obtained at different ultrasonic times. In order to ensure sufficient extraction, the extraction time was determined to be 30 minutes.

[0314] 3.4. Investigation of extraction methods

[0315] The same method as in Example 7 was used, except that the extraction methods were ultrasonic treatment (power 250W, frequency 40kHz) for 30 minutes and reflux extraction for 30 minutes. The results are shown in the following table:

[0316] Table 40 Content results of 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 Reflow 6.5 7.5 14.0

[0318] The above results show 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, so ultrasonic extraction method was selected to save time.

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

[0320] 1. Accuracy

[0321] Accurately weigh 17.053 mg of oleic acid reference substance (purity 99.1%) and 18.825 mg of linoleic acid reference substance (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, and use them as reference substance mother solution. Accurately measure 5 ml, 10 ml, and 15 ml of reference substance mother solution and place them in 50 ml volumetric flasks, add methanol to dilute to the mark, shake well, and use them as reference substance solution 1, reference substance solution 2, and reference substance solution 3, respectively.

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

[0323]

[0324] Table 41 Oleic acid recovery test results

[0325]

[0326] Table 42 Linoleic acid recovery test results

[0327]

[0328] The recovery rate of oleic acid measured by the recovery test ranged from 97.7% to 102.8%, and the recovery rate of linoleic acid ranged from 97.5% to 102.9%. According to regulations, when the content of the component to be tested is 0.1%, the recovery rate of the sample should be between 90% and 108%, so it meets the recovery rate requirements of the method validation, indicating that the total amount of oleic acid and linoleic acid measured by this method is accurate.

[0329] 2Precision

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

[0331] Table 43 Repeatability test

[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 average content of oleic acid measured in the repeatability test was 6.6 mg / g, with an RSD value of 1.0%; the average content of linoleic acid was 7.5 mg / g, with an RSD value of 0.5%. The average sum of the two components was 14.1 mg / g, with an RSD value of 0.5%, which met the repeatability requirements of the methodology validation.

[0334] 2.2 Intermediate precision: Different analysts conducted intermediate precision tests at different times 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 of 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 of different instruments (%) 0.3 1.1 0.6

[0337] The average content of oleic acid measured in the intermediate precision test was 6.6 mg / g, with an RSD value of 0.0%, the average content of linoleic acid was 7.6 mg / g, with an RSD value of 0.5%, and the average value of the sum of the two components was 14.2 mg / g, with an RSD value of 0.3%. Compared with the test results of the repeatability test, the RSD value of oleic acid was 0.3%, the RSD value of linoleic acid was 1.1%, and the RSD value of the total amount of the two components was 0.6%, which met the precision requirements of the method validation.

[0338] 3. Exclusiveness

[0339] Take black sesame granules test solution, oleic acid and linoleic acid mixed reference solution, blank solution (methanol) and obtain chromatogram according to the method of Example 7. The results are shown in Fig.17 By investigating the specificity, it was found that there was no interference at the corresponding position of the peak in the chromatogram of the test product.

[0340] 4 Linear

[0341] Take appropriate amount of oleic acid and linoleic acid reference substances, weigh accurately, add methanol to prepare a mixed reference substance solution containing 0.18290 mg of oleic acid and 0.20713 mg of linoleic acid per 1 ml, as linear 5 solution; take 10 ml of linear 5 solution and place it in a 25 ml volumetric flask, add methanol to the scale, as linear 4; take 15 ml of linear 5 solution and place it in a 50 ml volumetric flask, add methanol to the scale, as linear 3; take 10 ml of linear 5 solution and place it in a 50 ml volumetric flask, add methanol to the scale, as linear 2; take 5 ml of linear 5 solution and place it in a 50 ml volumetric flask, add methanol to the scale, as linear 1. Accurately take 3 μl of the filtrate of each of the above solutions and inject it into a high performance liquid chromatograph. Determine the peak area of ​​the oleic acid and linoleic acid chromatographic peaks according to the method in Example 7. Use the peak area of ​​the chromatographic peak of each component as the abscissa and the concentration of each component as the ordinate to draw a standard curve, and the regression equation of oleic acid is y=4×10-7 x+0.0004, R 2 =1.0000, its linear range is 0.01829mg / ml~0.18290mg / ml; the regression equation of linoleic acid is y=7×10 -8 x-0.0003, R 2 =1.0000, the linear range is 0.02071mg / ml~0.20713mg / ml, the results are shown in Table 45 and Table 46, Fig.18 , Fig.19 .

[0342] Table 45 Oleic acid linear relationship investigation

[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 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] According to the precision, accuracy and linearity experimental results and the results of multiple batches of black sesame granules, the range of oleic acid is 0.01829mg / ml~0.18290mg / ml, and the range of linoleic acid is 0.02071mg / ml~0.20713mg / ml.

[0348] 6. Durability

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

[0350] Table 47 Oleic acid stability results

[0351]

[0352] Table 48 Linoleic acid stability results

[0353]

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

[0355] 6.2 Investigation of 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 of Example 7. The test was carried out at different column temperatures (38°C, 40°C and 42°C) according to the method of Example 7 to investigate 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 chromatograms obtained at different column temperatures showed that the theoretical plate number, tailing factor and separation degree of each peak of oleic acid and linoleic acid met the system suitability requirements. The RSD value of oleic acid content was 0.9%, the RSD value of linoleic acid content was 0.8%, and the RSD value of the total content of the two components was 0.7%, indicating that the method has good durability for different column temperatures.

[0360] 6.3 Investigation of different flow rates:

[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. Different flow rates (0.28 ml / min, 0.30 ml / min and 0.32 ml / min) were used to measure according to the method of Example 7 to investigate the durability of the experimental method for flow rate. The results are shown in the table below.

[0362] Table 50 Different flow rate content determination results

[0363]

[0364] The chromatograms obtained at different flow rates showed that the theoretical plate number, tailing factor and separation degree of each peak of oleic acid and linoleic acid met the system suitability requirements. The RSD value of the oleic acid content was 0.9%, the RSD value of the linoleic acid content was 0.8%, and the RSD value of the total content of the two components was 0.4%, indicating that the method has good durability for different flow rates.

[0365] 6.4 Investigation of different wavelengths:

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

[0367] Table 51 Content determination results at different detection wavelengths

[0368]

[0369] The chromatograms obtained at different wavelengths, the theoretical plate number, tailing factor and separation degree of each peak of oleic acid and linoleic acid all met the system suitability requirements. The RSD value of oleic acid content was 0.9%, the RSD value of linoleic acid content was 0.8%, and the RSD value of the total content of the two components was 0.7%, indicating that the method has good durability for 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, use different types of chromatographic columns to measure according to the method of Example 7, and investigate the durability of the experimental method for different chromatographic columns. The results are shown in the table below.

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

[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 of different chromatographic columns

[0376]

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

[0378] 6.6 Investigation of different acid concentrations:

[0379] Take black sesame granules (batch number: K467CP01), prepare the test solution according to the method of Example 7, use different acid concentrations (0.08% acetic acid, 0.10% acetic acid, 0.12% acetic acid) to measure according to the method of Example 7, and investigate the durability of the experimental method for different mobile phase acid concentrations. The results are shown in the table below.

[0380] Table 53 Results of determination of different acid concentrations

[0381]

[0382] The chromatograms obtained under different acetic acid concentrations showed that the theoretical plate number, tailing factor and separation degree of each peak of oleic acid and linoleic acid met the system suitability requirements. The RSD value of oleic acid content was 0.9%, the RSD value of linoleic acid content was 0.8%, and the RSD value of the total content of the two components was 0.4%, indicating that the method has good durability for different acetic acid concentrations.

[0383] Comparative Example 1

[0384] This comparative example adopts the method for constructing a map of fried black sesame medicinal materials, fried black sesame decoction pieces, standard decoctions and their formula particles disclosed in CN117330671A and its application, and detects the black sesame formula particles in Example 1 according to the method in Example 6 thereof. The results are as follows Fig. 20 As shown, in Comparative Example 1, the elution time is 65 minutes, and 6 characteristic peaks are identified. In the first 20 minutes, the effective characteristic peaks are only Peak 1, Peak 2 and Peak 3. The detection time is long and the separation degree of each chromatographic peak is low.

[0385] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for constructing a characteristic spectrum of a black sesame preparation, characterized in that: The method includes ultra-high performance liquid chromatography detection, and the chromatographic conditions include: Octadecylsilane bonded silica gel was used as filler, methanol was used as mobile phase A, and water was used as mobile phase B. Gradient elution was performed. The elution procedure was as follows: 。 2. The method for constructing a characteristic spectrum of a black sesame preparation 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; optionally, the flow rate is 0.3 ml per minute; 2) The column temperature is 33-37°C; optionally, the column temperature is 35°C; 3) The detection wavelength is 285nm to 289nm; optionally, the detection wavelength is 287nm; 4) The injection volume is 1 to 3 μl; optionally, the injection volume is 2 μl; 5) The specifications of the chromatographic column are: column length 100 mm, inner diameter 2.1 mm, and particle size 1.7 μm.

3. The method for constructing a characteristic spectrum of a black sesame preparation according to claim 1 or 2, characterized in that: The method also includes the preparation of a test sample solution, including: taking the test sample, adding an extraction solvent, extracting, separating the solid from the liquid, and taking the liquid.

4. The method for constructing a characteristic spectrum of a black sesame preparation according to claim 3, characterized in that: The preparation of the test solution includes at least one of the following conditions: 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, 70 v / v% methanol aqueous solution is selected; 2) The extraction method used in the extraction step is ultrasonic extraction or reflux extraction; 3) The extraction time in the extraction step is 20-40 minutes; 4) The mass volume ratio of the test sample and the extraction solvent is 0.5g:15~50ml.

5. The method for constructing a characteristic spectrum of a black sesame preparation according to any one of claims 1 to 4, characterized in that: Also included is the preparation of reference solutions, including: Preparation of control medicinal material solution: taking control medicinal material, adding extraction solvent, extracting, solid-liquid separation, evaporating the filtrate to dryness, adding resolvent, and taking liquid; And / or, preparation of reference solution: take sesamin reference, add solvent to make 1 ml of solution containing 20-40 μg; optionally, 1 ml of solution containing 30 μg; optionally, the solvent is 50-100 v / v% methanol or its aqueous solution; optionally, 70 v / v% methanol aqueous solution is selected.

6. The method for constructing a characteristic spectrum of a black sesame preparation according to claim 5, characterized in that: The preparation of the control medicinal material solution includes at least one of the following conditions: 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; 2) The extraction method used in the extraction step is ultrasonic extraction or reflux extraction; 3) The number of extractions in the extraction step is 1 to 2 times; 3) The extraction time in the extraction step is 20-40 minutes; 4) The mass volume ratio of the reference medicinal material and the extraction solvent is 2g:25-50ml; 5) The re-dissolving solvent is 50-100 v / v% methanol or its aqueous solution; optionally, 70 v / v% methanol aqueous solution is selected; 6) The mass volume ratio of the control medicinal material and the reconstitution solvent is 2g:5~10ml.

7. A method for detecting the content of a black sesame preparation, characterized in that: Taking the test solution and the reference solution, respectively, and detecting them by the ultra-high performance liquid chromatography method in the method for constructing the characteristic spectrum of the black sesame preparation according to any one of claims 1 to 6; Control substances included sesamin.

8. The method for detecting the content of black sesame preparation according to claim 7, characterized in that: It also includes a method for detecting the content of oleic acid and / or linoleic acid, including detection by ultra-high performance liquid chromatography, and the chromatographic conditions include: Octadecylsilane bonded silica gel is used as a filler, acetonitrile is used as a mobile phase A, 0.08-0.12 v / v% acetic acid solution is used as a mobile phase B, and the volume ratio of mobile phase A to mobile phase B is 79-81:21-19 for elution; Optionally, the chromatographic conditions further include at least one of the following conditions: 1) The flow rate is 0.28-0.32 ml per minute; optionally, the flow rate is 0.3 ml per minute; 2) The column temperature is 38-42°C; optionally, the column temperature is 40°C; 3) The detection wavelength is 203nm to 207nm; optionally, the detection wavelength is 205nm; 4) The injection volume is 1 to 5 μl; optionally, the injection volume is 3 μl; 5) The specifications of the chromatographic column are: column length 100 mm, inner diameter 2.1 mm, particle size 1.7 μm; Optionally, it also includes preparing a test sample solution: taking a test sample, adding an extraction solvent, extracting, separating the solid from the liquid, and taking a liquid; Optionally, the preparation of the test solution includes at least one of the following conditions: 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; 2) The extraction method used in the extraction step is ultrasonic extraction or reflux extraction; 3) The extraction time in the extraction step is 20-40 minutes; 4) The mass volume ratio of the test sample and the extraction solvent is 0.2g:10~20ml; Optionally, the method further includes preparing a reference solution: taking an oleic acid reference substance and a linoleic acid reference substance, adding a solvent to prepare a mixed solution containing 65 to 75 μg of oleic acid and 75 to 85 μg of linoleic acid per 1 ml; Optionally, the solvent is water, 30-100 v / v% methanol or its aqueous solution, or 30-100 v / v% ethanol or its aqueous solution.

9. A method for quality inspection of black sesame preparations, characterized in that: The method comprises the steps of comparing the characteristic spectrum of the sample to be tested with the control characteristic spectrum of the black sesame preparation; The characteristic spectrum of the sample to be tested is constructed according to the construction method described in any one of claims 1 to 6; The control characteristic spectrum of the black sesame preparation is selected from any one of the following (1)-(2): (1) It has 7 common characteristic peaks. Peak 6 corresponding to the peak of the sesamin reference substance is the S peak. The relative retention times of peaks 1-5, peak 7 and S peak are calculated. The relative retention times should be within the range of ±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. (2) Using at least one batch of black sesame preparation standard product and obtaining the characteristic spectrum according to the construction method described in any one of claims 1 to 6, a control characteristic spectrum is prepared by using the average value or median method.

10. The quality detection method of black sesame preparation according to claim 8, characterized in that: In (1), peak 6 corresponds to sesamin; peak 7 corresponds to sesamolin.

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