A quality detection method for Nuan Gan Jian

Abstract

The present invention discloses a quality detection method for Nuan Gan Jian, belonging to the technical field of quality detection of traditional Chinese medicine. The method includes characteristic spectrum identification, and specifically comprises the following steps: (1) preparing a reference solution of syringin, a reference solution of norisoboldine, a reference solution of agarwood tetraol, a reference solution of cinnamaldehyde, a reference solution of cinnamic acid, and a reference solution of 6-gingerol; (2) preparing a test solution from Nuan Gan Jian granules; and (3) performing liquid chromatography detection on the six reference solutions and the test solution. The method has good comparability of detection results, reliable quality control, high detection efficiency, adapts to modern production needs, and realizes effective control of the quality of Nuan Gan Jian granules.

Description

Technical Field

[0001] The present invention relates to the technical field of quality detection of traditional Chinese medicines, and more particularly to a quality detection method for Nuan Gan Jian. Background Art

[0002] The Nuan Gan Jian formula uses cinnamon and fennel as the main ingredients to warm and nourish the liver and kidneys, while angelica and wolfberry replenish the liver and blood. Linderae scutellariae and agarwood promote qi circulation and dispel cold. It is primarily used to treat hernia pain caused by liver and kidney deficiency and cold-cold syndrome, lower abdominal pain, and menstrual irregularities due to uterine cold. As a classic formula, Nuan Gan Jian boasts rigorous formulation and proven efficacy, supported by both ancient theoretical foundations and modern clinical validation. It is currently a representative formula being promoted for translational applications. However, current testing of Nuan Gan Jian only targets one or two specific components, failing to fully reflect its overall quality. This results in some active ingredients being unmonitored, impacting the quality control of Nuan Gan Jian granules. Summary of the Invention

[0003] The purpose of the present invention is to overcome the deficiencies of the above-mentioned prior art and provide a quality detection method for Nuan Gan Jian, which has good comparability of detection results, reliable quality control and high detection efficiency.

[0004] In order to achieve the above object, the present invention adopts the following technical solutions:

[0005] A quality detection method for Nuan Gan Jian, comprising characteristic spectrum identification, specifically comprising the following steps:

[0006] (1) Prepare syringin reference solution, norisoboldine reference solution, agarwood tetraol reference solution, cinnamaldehyde reference solution, cinnamic acid reference solution, and 6-gingerol reference solution respectively;

[0007] (2) Prepare the test solution using Nuan Gan Jian granules;

[0008] (3) Perform liquid chromatography on the above six reference substance solutions and test sample solutions respectively.

[0009] In summary, the present invention has the following beneficial effects:

[0010] The quality of Nuan Gan Jian granules was tested through characteristic spectrum identification, thin layer identification, syringin content determination, norisobolidine content determination, agarwood tetraol content determination, and cinnamic acid content determination. The test results are comparable, the quality control is reliable, the test efficiency is high, and it meets the needs of modern production, realizing effective control of the quality of Nuan Gan Jian granules. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1Chromatogram of the syringin reference solution obtained to verify the positioning and system adaptability of the Nuan Gan Jian granule characteristic spectrum determination method (peak 1 in the figure is the syringin peak);

[0012] Figure 2 Chromatogram of the reference solution of norisoboldine obtained to verify the positioning and system adaptability of the Nuan Gan Jian granule characteristic spectrum determination method (peak 1 in the figure is norisoboldine);

[0013] Figure 3 Chromatogram of the agarwood tetraol reference solution obtained to verify the positioning and system adaptability of the Nuan Gan Jian granule characteristic spectrum determination method (peak 1 in the figure is the agarwood tetraol peak);

[0014] Figure 4 Chromatogram of the cinnamic aldehyde reference solution obtained to verify the positioning and system adaptability of the Nuan Gan Jian granule characteristic spectrum determination method (peak 1 in the figure is the cinnamic aldehyde peak);

[0015] Figure 5 Chromatogram of a cinnamic acid reference solution obtained to verify the positioning and system adaptability of the Nuan Gan Jian granule characteristic spectrum determination method (peak 1 in the figure is the cinnamic acid peak);

[0016] Figure 6 Chromatogram of the 6-gingerol reference solution obtained to verify the positioning and system adaptability of the Nuan Gan Jian granule characteristic spectrum determination method (peak 1 in the figure is the 6-gingerol peak);

[0017] Figure 7 The chromatogram of the test sample solution was obtained to verify the positioning of the Nuan Gan Jian granule characteristic spectrum determination method and the system adaptability;

[0018] Figure 8 The chromatogram obtained to verify the column temperature durability of the Nuan Gan Jian granule characteristic spectrum determination method at 25°C;

[0019] Figure 9 The chromatogram obtained to verify the column temperature durability of the Nuan Gan Jian granule characteristic spectrum determination method at 35°C;

[0020] Figure 10 The chromatogram was obtained to verify the flow rate durability of the Nuan Gan Jian granule characteristic spectrum determination method at a flow rate of 0.18 mL / min;

[0021] Figure 11 The chromatogram was obtained to verify the flow rate durability of the Nuan Gan Jian granule characteristic spectrum determination method at a flow rate of 0.22 mL / min;

[0022] Figure 12 The chromatogram obtained to verify the durability of the characteristic spectrum determination method of Nuan Gan Jian granules at a wavelength of 260 nm;

[0023] Figure 13 The chromatogram obtained to verify the durability of the characteristic spectrum determination method of Nuan Gan Jian granules at a wavelength of 270 nm;

[0024] Figure 14 This is the chromatogram obtained by determining the characteristic spectrum of Nuan Gan Jian granules of batch number NGJ-K-25041603;

[0025] Figure 15 The chromatogram was obtained to verify the column temperature durability at 25℃ for the determination of syringin, norisoboldine and agarwood tetraol in Nuan Gan Jian granules;

[0026] Figure 16 The chromatogram was obtained to verify the column temperature durability of the determination method of syringin, norisoboldine and agarwood tetraol in Nuan Gan Jian granules at 35℃;

[0027] Figure 17 The chromatogram was obtained to verify the flow rate robustness of the method for determining the contents of syringin, norisoboldine and agarwood tetraol in Nuan Gan Jian granules at a flow rate of 0.18 mL / min;

[0028] Figure 18 The chromatograms were obtained to verify the robustness of the determination method of syringin, norisoboldine and agarwood tetraol in Nuan Gan Jian granules at a flow rate of 0.22 mL / min.

[0029] Figure 19 The chromatogram was obtained to verify the wavelength durability of the determination method of syringin, norisoboldine and agarwood tetraol in Nuan Gan Jian granules at 205 nm;

[0030] Figure 20 The chromatogram was obtained to verify the wavelength durability of the determination method of syringin, norisoboldine and agarwood tetraol in Nuan Gan Jian granules at 215 nm;

[0031] Figure 21 Chromatogram of the reference solution obtained to verify the positioning and system suitability of the determination method of cinnamic acid content in Nuan Gan Jian granules;

[0032] Figure 22 The chromatogram of the test sample solution was obtained to verify the positioning and system suitability of the determination method of cinnamic acid content in Nuan Gan Jian granules;

[0033] Figure 23 The chromatogram was obtained to verify the column temperature durability of the method for determining the cinnamic acid content in Nuan Gan Jian granules at 25°C;

[0034] Figure 24 The chromatogram was obtained to verify the column temperature durability of the method for determining the cinnamic acid content in Nuan Gan Jian granules at 35°C;

[0035] Figure 25The chromatogram was obtained to verify the robustness of the method for determining the cinnamic acid content in Nuan Gan Jian granules at a flow rate of 0.18 mL / min;

[0036] Figure 26 Chromatogram obtained to verify the robustness of the method for determining cinnamic acid content in Nuan Gan Jian granules at a flow rate of 0.22 mL / min;

[0037] Figure 27 The chromatogram was obtained to verify the wavelength durability of the determination method of cinnamic acid content in Nuan Gan Jian granules at 275 nm;

[0038] Figure 28 The chromatogram was obtained to verify the wavelength durability of the determination method of cinnamic acid content in Nuan Gan Jian granules at 285 nm;

[0039] Figure 29 This is the chromatogram obtained from the specificity test of the thin-layer identification method of Nuan Gan Jian granules under ultraviolet light at 254nm;

[0040] Figure 30 This is the chromatogram obtained from the specificity test of the thin-layer identification method of Nuan Gan Jian granules under ultraviolet light at 365nm;

[0041] Figure 31 This is the chromatogram obtained from the 4°C durability test of the thin-layer identification method of Nuan Gan Jian granules under ultraviolet light at 254nm;

[0042] Figure 32 This is the chromatogram obtained from the 4°C durability test of the thin-layer identification method of Nuan Gan Jian granules under ultraviolet light at 365nm;

[0043] Figure 33 This is the chromatogram obtained from the durability test of the thin-layer identification method of Nuan Gan Jian granules at 30°C under ultraviolet light at 254nm;

[0044] Figure 34 This is the chromatogram obtained from the durability test of the thin-layer identification method of Nuan Gan Jian granules at 30°C under ultraviolet light at 365nm;

[0045] Figure 35 This is the chromatogram obtained from the durability test of the thin-layer identification method of Nuan Gan Jian granules under 30% humidity under ultraviolet light at 254nm;

[0046] Figure 36 This is the chromatogram obtained from the durability test of the thin layer identification method of Nuan Gan Jian granules under 30% humidity under ultraviolet 365nm;

[0047] Figure 37 This is the chromatogram obtained from the durability test of the thin-layer identification method of Nuan Gan Jian granules under 70% humidity under ultraviolet light at 254nm;

[0048] Figure 38 This is the chromatogram obtained from the durability test of the thin layer identification method of Nuan Gan Jian granules under 70% humidity under ultraviolet 365nm;

[0049] Figure 39 This is the chromatogram obtained by the thin layer identification test of Nuan Gan Jian granules using a thin layer plate purchased from Qingdao Ocean Chemical Co., Ltd. under ultraviolet light at 254nm;

[0050] Figure 40 This is the chromatogram obtained by performing a thin layer identification test of Nuan Gan Jian granules using a thin layer plate purchased from Qingdao Ocean Chemical Co., Ltd. under ultraviolet light at 365nm;

[0051] Figure 41 This is the chromatogram obtained by the thin layer identification test of Nuan Gan Jian granules using a thin layer plate purchased from Qingdao Shuoyuan Silica Gel Technology Co., Ltd. under ultraviolet light at 254nm;

[0052] Figure 42 This is the chromatogram obtained by the thin layer identification test of Nuan Gan Jian granules using a thin layer plate purchased from Qingdao Shuoyuan Silica Gel Technology Co., Ltd. under ultraviolet light at 365nm;

[0053] Figure 43 This is the chromatogram obtained by the thin layer identification test of Nuan Gan Jian granules using a thin layer plate purchased from Qingdao Xinchanglai Silica Gel Co., Ltd. under ultraviolet 254nm;

[0054] Figure 44 The chromatogram is obtained by performing a thin layer identification test of Nuan Gan Jian granules using a thin layer plate purchased from Qingdao Xinchanglai Silica Gel Co., Ltd. under ultraviolet light at 365nm.

[0055] Note: Figures 7 to 14 Peak 3 is the peak of syringin, Peak 5 is the peak of norisoboldine, Peak 6 is the peak of agarwood tetraol, Peak 9 is the peak of cinnamaldehyde, Peak 12 is the peak of cinnamic acid, and Peak 13 is the peak of 6-gingerol. Figures 15 to 20 Peak 1 in the middle is the syringin peak, peak 2 is the norisoboldine peak, and peak 3 is the agarwood tetraol peak; Figures 21 to 28 The middle peak 1 is all cinnamic acid peak; Figure 29 、 Figure 31 、 Figure 33 、 Figure 35 、 Figure 37 、 Figure 39 、 Figure 41 、 Figure 43 From left to right in the middle are negative sample solution, test solution, wolfberry control medicinal material solution, angelica control medicinal material solution, Poria control medicinal material solution, ferulic acid reference solution, and Pachycorylic acid B reference solution; Figure 30 、 Figure 32 、 Figure 34 、 Figure 36 、 Figure 38 、 Figure 40 、 Figure 42 、 Figure 44From left to right in the middle are negative sample solution, test solution, wolfberry control medicinal material solution, angelica control medicinal material solution, Poria control medicinal material solution, ferulic acid reference solution, and ligustilide reference solution. DETAILED DESCRIPTION

[0056] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0057] Example 1 Preparation of Nuan Gan Jian Granules

[0058] The preparation method of Nuan Gan Jian granules is as follows: taking the decoction pieces constituting Nuan Gan Jian granules, adding water to decoct, filtering, concentrating the filtrate to a clear paste, drying the clear paste to a dry paste, adding or not adding maltodextrin, mixing, and granulating;

[0059] The decoction pieces that make up Nuan Gan Jian granules (Nuan Gan Jian granules are composed of the following decoction pieces: 166 g of angelica sinensis, 199 g of wolfberry, 133 g of Poria cocos, 133 g of fennel, 100 g of cinnamon bark, 133 g of Linderae serrata, 66 g of agarwood, and 71 g of ginger) were taken and three batches were prepared according to the above method. The specific production data are shown in Table 1.

[0060] Table 1

[0061]

[0062] Example 2 Quality Inspection of Nuan Gan Jian Granules

[0063] 1. Characteristic spectrum identification

[0064] 1.1 Detection method

[0065] 1.1.1 Preparation of reference substance solutions: Using methanol as solvent, prepare 10 μg / mL syringin reference substance solution, 50 μg / mL norisoboldine reference substance solution, 20 μg / mL agarwood tetraol reference substance solution, 5 μg / mL cinnamaldehyde reference substance solution, 5 μg / mL cinnamic acid reference substance solution, and 40 μg / mL 6-gingerol reference substance solution.

[0066] 1.1.2 Preparation of test solution: Take an appropriate amount of Nuan Gan Jian granules, grind them into fine powder, take 0.5 g, weigh accurately, place in a stoppered conical flask, add 25 mL of 50 wt% methanol accurately, weigh the weight, ultrasonically treat (power 250 W, frequency 50 kHz) for 30 minutes, let cool, weigh again, make up the lost weight with 50 wt% methanol, shake well, filter, and take the filtrate.

[0067] 1.1.3 Determination: Accurately pipette 2 μL of the reference substance solution and the test solution into a high performance liquid chromatograph, measure, record the chromatogram, and calculate the relative retention time.

[0068] Liquid chromatography conditions were as follows: column: ChromCore AQ C18, 2.1×100 mm, 1.8 μm; detection wavelength: 265 nm; flow rate: 0.2 mL / min; column temperature: 30°C; injection volume: 2 μL; mobile phase: methanol as mobile phase A, 0.1 wt% phosphoric acid as mobile phase B, gradient elution as shown in Table 2:

[0069] Table 2

[0070]

[0071] 1.2 Validation of characteristic pattern detection methodology

[0072] 1.2.1 Positioning and system suitability: Take appropriate amounts of syringin, norisoboldine, aganetetrol, cinnamic aldehyde, cinnamic acid, and 6-gingerol reference substances, accurately weigh them, and add methanol to prepare 10 μg / mL syringin reference substance reference solution, 50 μg / mL norisoboldine reference substance reference solution, 20 μg / mL aganetetrol reference substance reference solution, 5 μg / mL cinnamic aldehyde reference substance reference solution, 5 μg / mL cinnamic acid reference substance reference solution, and 40 μg / mL 6-gingerol reference substance reference solution. Take an appropriate amount of Nuan Gan Jian granules (batch number: NGJ-K-25041603), grind them into powder, take 0.5 g, place it in a stoppered conical flask, accurately add 25 mL of 50 wt% methanol, weigh the weight, and ultrasonically treat (power 250 W, frequency 50 kHz) for 30 minutes. Let it cool, weigh it again, make up the lost weight with 50 wt% methanol, shake it well, filter it, and take the filtrate as the test solution.

[0073] Accurately pipette 2 μL of reference solution and test solution respectively, inject into high performance liquid chromatography, measure, and record the chromatogram. Figures 1 to 7As can be seen from the figure, in the chromatogram of the reference solution, the retention time of syringin is 16.607 min, the theoretical plate number is 42317, and the tailing factor is 2.02; the retention time of norisoboldine is 19.660 min, the theoretical plate number is 24518, and the tailing factor is 0.94; the retention time of agarwood tetraol is 26.837 min, the theoretical plate number is 31956, and the tailing factor is 0.95; the retention time of cinnamaldehyde is 46.307 min, the theoretical plate number is 41762, and the tailing factor is 0.83; the retention time of cinnamic acid is 53.642 min, the theoretical plate number is 173234, and the tailing factor is 0.91; the retention time of 6-gingerol is 70.464 min, the theoretical plate number is 1403320, and the tailing factor is 0.85. In the chromatogram of the test solution, the retention time of syringin was 16.608 min, the number of theoretical plates was 66446, and the tailing factor was 0.72; the retention time of norisoboldine was 19.848 min, the number of theoretical plates was 31479, and the tailing factor was 0.55; the retention time of agallotetraol was 26.720 min, the number of theoretical plates was 38636, and the tailing factor was 0.73; the retention time of cinnamaldehyde was 46.154 min, the number of theoretical plates was 76288, and the tailing factor was 0.85; the retention time of cinnamic acid was 53.690 min, the number of theoretical plates was 162661, and the tailing factor was 1.02; and the retention time of 6-gingerol was 70.309 min, the number of theoretical plates was 1217199, and the tailing factor was 0.61. This indicates that the system suitability is good.

[0074] 1.2.2 Durability test

[0075] 1.2.2.1 Column temperature durability: According to the specified chromatographic conditions, change the column temperature by ±5°C, take Nuan Gan Jian granules (batch number: NGJ-K-25041603), prepare the test solution according to the method under "1.1.2", accurately pipette 2μL of the test solution, inject it into the high performance liquid chromatograph, record the retention time of the common peak, take the peak corresponding to the syringin reference substance peak as the S1 peak, calculate the relative retention time of the characteristic peaks of Peak 1, Peak 2, Peak 4 and the S1 peak and RSD value, the peak corresponding to the agarwood tetraol reference peak is the S2 peak, and the relative retention time and RSD value of peak 7 and peak 8 characteristic peaks and S2 peak are calculated. The peak corresponding to the cinnamaldehyde reference peak is the S3 peak, and the relative retention time and RSD value of peak 10 and peak 11 characteristic peaks and S3 peak are calculated. The peak corresponding to the 6-gingerol reference peak is the S4 peak, and the relative retention time and RSD value of peak 14 characteristic peak and S4 peak are calculated. The results are shown in Table 3. Related chromatograms are shown in Figures 8 and 9 .

[0076] Table 3

[0077]

[0078] The relative retention time RSD of peak 1 is greater than 5%, so the column temperature is specified to be 30℃.

[0079] 1.2.2.2 Flow rate durability

[0080] According to the specified chromatographic conditions, the flow rate was changed by ±0.02 mL / min, and a sample (batch number: NGJ-K-25041603) was taken. The test solution was prepared according to the method under "1.1.2". 2 μL of the test solution was accurately aspirated and injected into the high performance liquid chromatograph. The retention time of the common peak was recorded. The peak corresponding to the syringin reference peak was designated as the S1 peak. The relative retention time and RSD value of the characteristic peaks of peak 1, peak 2, and peak 4 were calculated with respect to the S1 peak. The peak corresponding to the agallochetol reference peak was designated as the S2 peak. The relative retention time and RSD value of the characteristic peaks of peak 7 and peak 8 were calculated with respect to the S2 peak. The peak corresponding to the cinnamaldehyde reference peak was designated as the S3 peak. The relative retention time and RSD value of the characteristic peaks of peak 10 and peak 11 were calculated with respect to the S3 peak. The peak corresponding to the 6-gingerol reference peak was designated as the S4 peak. The relative retention time and RSD value of the characteristic peak of peak 14 were calculated with respect to the S4 peak. The results are shown in Table 4. Figure 10-11 .

[0081] Table 4

[0082]

[0083] 1.2.2.3 Wavelength durability

[0084] According to the specified chromatographic conditions, the wavelength was changed by ±5nm, and Nuan Gan Jian granules (batch number: NGJ-K-25041603) were taken. The test solution was prepared according to the method under "1.1.2". 2μL of the test solution was accurately aspirated and injected into the high performance liquid chromatograph. The retention time of the common peak was recorded. The peak corresponding to the syringin reference peak was taken as the S1 peak. The relative retention time and RSD value of the characteristic peaks of peak 1, peak 2, and peak 4 were calculated with respect to the S1 peak. The peak corresponding to the agarwood tetraol reference peak was taken as the S2 peak. The relative retention time and RSD value of the characteristic peaks of peak 7 and peak 8 were calculated with respect to the S2 peak. The peak corresponding to the cinnamaldehyde reference peak was taken as the S3 peak. The relative retention time and RSD value of the characteristic peaks of peak 10 and peak 11 were calculated with respect to the S3 peak. The peak corresponding to the 6-gingerol reference peak was taken as the S4 peak. The relative retention time and RSD value of the characteristic peak of peak 14 were calculated with respect to the S4 peak. The results are shown in Table 5. Figure 12-13 .

[0085] Table 5

[0086]

[0087] The results of the above durability tests showed that the flow rate and wavelength durability of this method met the requirements for content determination, and the column temperature was specified to be 30°C.

[0088] 1.3 Sample testing

[0089] Three batches of Nuan Gan Jian granules were tested according to "1.1". The relative retention time results of the characteristic spectrum of Nuan Gan Jian granules are shown in Table 6. Figure 14 This is the characteristic spectrum of Nuan Gan Jian granules with batch number NGJ-K-25041603 (tested at column temperature of 30°C, flow rate of 0.2 mL / min, and wavelength of 265 nm).

[0090] Table 6

[0091]

[0092] 2. Determination of Syringin, Norisoboldine, and Agarwood Tetraol Content

[0093] 2.1 Determination method

[0094] 2.1.1 Preparation of reference solution: Take appropriate amount of syringin, norisoboldine, and agallochetol reference substances, accurately weigh them, and add methanol to prepare 10 μg / mL syringin reference solution, 40 μg / mL norisoboldine reference solution, and 20 μg / mL agallochetol reference solution, respectively.

[0095] 2.1.2 Preparation of test solution: Take an appropriate amount of Nuan Gan Jian granules (batch number: NGJ-K-25041603), grind it into powder, take 0.5 g, accurately weigh it, put it into a stoppered conical flask, accurately add 25 mL of 50 wt% methanol, weigh it, and treat it with ultrasound (power 250 W, frequency 50 kHz) for 30 minutes. Let it cool, weigh it again, make up the lost weight with 50 wt% methanol, shake it well, filter it, and take the filtrate to obtain the product.

[0096] 2.1.3 Determination: Accurately pipette 2 μL of each of the test solution and reference solution into a high performance liquid chromatograph, record the peak area, and calculate the content;

[0097] Chromatographic conditions were as follows: column: ChromCore AQ C18, 2.1×100 mm, 1.8 μm; detection wavelength: 210 nm; flow rate: 0.2 mL / min; column temperature: 30°C; injection volume: 2 μL; mobile phase: methanol as mobile phase A, 0.1 wt% phosphoric acid as mobile phase B, gradient elution as shown in Table 7:

[0098] Table 7

[0099]

[0100] 2.2 Methodological validation

[0101] 2.2.1 Positioning and system applicability

[0102] Accurately weigh appropriate amounts of syringin, norisoboldine, and aloeswood tetraol reference standards. Add methanol to prepare 10 μg / mL syringin, 40 μg / mL norisoboldine, and 20 μg / mL aloeswood tetraol reference solutions, respectively. Grind an appropriate amount of Nuan Gan Jian Granules (Batch No. NGJ-K-25041603) and take 0.5 g. Place in a stoppered conical flask and accurately add 25 mL of 50 wt% methanol. Weigh the weight and sonicate (power 250 W, frequency 50 kHz) for 30 minutes. Allow to cool, weigh again, and make up the loss with 50 wt% methanol. Shake well, filter, and collect the filtrate as the test solution. Accurately pipette 2 μL each of the reference solution and test solution into a high-performance liquid chromatograph for measurement and record the chromatogram. Analysis of the chromatograms demonstrates good system suitability.

[0103] 2.2.2 Durability test

[0104] 2.2.2.1 Column temperature durability

[0105] According to the specified chromatographic conditions, the column temperature was changed by ±5°C, and Nuan Gan Jian Granules (Batch No.: NGJ-K-25041603) were prepared. The test solution was prepared according to the method under "2.1.2" and the contents of syringin, norisoboldine, and agarwood tetraol were determined. The results are shown in Table 8. The relevant chromatograms are shown in Figure 15-16 .

[0106] Table 8

[0107]

[0108] The RSDs of the contents of syringin, norisoboldine and aganetetrol were all greater than 5%, so the column temperature was set at 30°C.

[0109] 2.2.2.2 Flow rate durability:

[0110] According to the specified chromatographic conditions, the flow rate was changed by ±0.02 mL / min, and Nuan Gan Jian Granules (Batch No.: NGJ-K-25041603) were prepared. The test solution was prepared according to the method under "2.1.2" and the contents of syringin, norisoboldine, and agarwood tetraol were determined. The results are shown in Table 9. The relevant chromatograms are shown in Figure 17-18 .

[0111] Table 9

[0112]

[0113] 2.2.2.3 Wavelength Durability: Following the specified chromatographic conditions, vary the wavelength by ±5 nm, prepare Nuan Gan Jian Granules (Batch No.: NGJ-K-25041603), and prepare the test solution according to the method in 2.1.2. Determine the contents of syringin, norisoboldine, and agarwood tetraol. The results are shown in Table 10. The relevant chromatograms are shown in Figures 19 and 20 .

[0114] Table 10

[0115]

[0116] The results of the above durability tests showed that the flow rate and wavelength durability of this method met the requirements for content determination, and the column temperature was specified to be 30°C.

[0117] 2.3 Sample determination: Three batches of Nuan Gan Jian granules were taken and the content was determined according to the method, see Table 11.

[0118] Table 11

[0119]

[0120] 3. Determination of silicic acid content in meat

[0121] 3.1 Determination method

[0122] 3.1.1 Preparation of reference solution: Take an appropriate amount of cinnamic acid reference substance, accurately weigh it, and add methanol to make a 5 μg / mL reference solution.

[0123] 3.1.2 Preparation of test solution: Take an appropriate amount of Nuan Gan Jian granules, grind them into powder, take 0.5 g, weigh accurately, place in a stoppered conical flask, add 25 mL of 50 wt% methanol solution accurately, weigh the weight, ultrasonically treat (power 250 W, frequency 50 kHz) for 30 minutes, let cool, weigh again, make up the lost weight with 50 wt% methanol, shake well, filter, and take the filtrate.

[0124] 3.1.3 Determination: Accurately pipette 2 μL of each reference solution and test solution, inject into high performance liquid chromatography, record the peak area, and calculate the content;

[0125] The chromatographic conditions were as follows: chromatographic column: ChromCore AQ C18, 2.1×100 mm, 1.8 μm; mobile phase: same as the mobile phase and gradient elution procedure in “2.1.3”; detection wavelength: 280 nm; flow rate: 0.20 mL / min; column temperature: 30°C; injection volume: 2 μL.

[0126] 3.2 Methodological validation

[0127] 3.2.1 Positioning and system adaptability

[0128] Accurately weigh an appropriate amount of cinnamic acid reference standard and add methanol to prepare a solution containing 5 μg per 1 mL. This serves as the reference solution. Grind an appropriate amount of Nuan Gan Jian granules (batch number: NGJ-K-25041603) and take 0.5 g. Place in a stoppered conical flask and accurately add 25 mL of a 50 wt% methanol solution. Weigh the weight and sonicate (power 250 W, frequency 50 kHz) for 30 minutes. Allow to cool and weigh again. Make up the lost weight with 50 wt% methanol, shake well, filter, and collect the filtrate as the test solution. Accurately pipette 2 μL each of the reference solution and the test solution into a high-performance liquid chromatograph for measurement and record the chromatogram. The chromatogram of the reference solution shows a retention time of 53.642 min, a theoretical plate number of 173,234, and a tailing factor of 0.91. The retention time of cinnamic acid in the chromatogram of the test solution is 53.691 min, the theoretical plate number is 155954, and the tailing factor is 1.04. This shows that the system applicability is good. The relevant chromatogram is shown in Figure 21-22 .

[0129] 3.2.2 Durability test

[0130] 3.2.2.1 Column temperature durability

[0131] According to the specified chromatographic conditions, the column temperature was changed by ±5°C, and Nuan Gan Jian Granules (Batch No.: NGJ-K-25041603) were taken. The test solution was prepared according to the method under "3.1.2" and the content of cinnamic acid was determined. The results are shown in Table 12. The relevant chromatograms are shown in Figure 23-24 .

[0132] Table 12

[0133]

[0134] The RSD of the content results was greater than 10%, so the column temperature was set to 30°C.

[0135] 3.2.2.2 Flow rate durability

[0136] According to the specified chromatographic conditions, the flow rate was changed by ±0.02 mL / min, and Nuan Gan Jian Granules (Batch No.: NGJ-K-25041603) were taken. The test solution was prepared according to the method under "3.1.2" and the content of cinnamic acid was determined. The results are shown in Table 13. The relevant chromatograms are shown in Figure 25-26 .

[0137] Table 13

[0138]

[0139] 3.2.2.3 Wavelength durability

[0140] According to the specified chromatographic conditions, change the wavelength ± 5nm, take a sample (batch number: NGJ-K-25041603), prepare the test solution according to the method under "3.1.2", and determine the content of cinnamic acid. The results are shown in Table 14. The relevant chromatogram is shown in Figure 27-28 .

[0141] Table 14

[0142]

[0143] The results of the above durability tests showed that the flow rate and wavelength durability of this method met the requirements for content determination, and the column temperature was specified to be 30°C.

[0144] 3.3 Sample determination

[0145] Three batches of Nuan Gan Jian granules were taken and the content was determined according to the method. The results are shown in Table 15.

[0146] Table 15

[0147]

[0148] 4. Thin layer identification

[0149] 4.1 Detection method

[0150] 4.1.1 Preparation of test solution: Take 5 g of Nuan Gan Jian granule powder, add 50 mL of 80 wt% methanol, heat to reflux, filter, concentrate the filtrate to 20 mL, and extract with ethyl acetate twice by shaking, adding 20 mL each time. Combine the ethyl acetate extracts, evaporate to dryness, and dissolve the residue in 1 mL of acetone to obtain the test solution.

[0151] 4.1.2 Preparation of Reference Solution and Reference Herb Solution: 1.0 g of ligustilide reference, pachymic acid B reference, and ferulic acid reference were added to methanol to prepare reference solution at a concentration of 1 mg / mL. 1.0 g of wolfberry reference herb was added to 50 mL of water and boiled for 30 min. The mixture was cooled, filtered, and the filtrate was evaporated to dryness. 50 mL of 80 wt% methanol was added, and the mixture was heated under reflux for 1 h. The filtrate was concentrated to 20 mL and extracted with ethyl acetate twice by shaking, adding 20 mL each time. The ethyl acetate extracts were combined and evaporated to dryness. The residue was dissolved in 1 mL of acetone to prepare the wolfberry reference herb solution. 1.0 g of angelica reference herb was added to 10 mL of methanol and sonicated for 30 min. The mixture was filtered, and the filtrate was concentrated to 2 mL to prepare the angelica reference herb solution. 1.0 g of Poria reference herb was added to 10 mL of methanol and sonicated for 30 min. The mixture was filtered, and the filtrate was concentrated to 2 mL to prepare the Poria reference herb solution.

[0152] 4.1.3 Determination: (1) Take 10 μL each of the pachymic acid B reference solution, ferulic acid reference solution, wolfberry control medicinal material solution, angelica control medicinal material solution, poria control medicinal material solution, and test solution, and spot them on a silica gel G thin layer plate, use cyclohexane-ethyl acetate-formic acid with a volume ratio of 6:4:0.1 as the developing solvent, develop, remove, dry, and place under a 254 nm ultraviolet lamp for inspection; (2) Take 10 μL each of the ligustilide reference solution, ferulic acid reference solution, wolfberry control medicinal material solution, angelica control medicinal material solution, poria control medicinal material solution, and test solution, and spot them on a silica gel G thin layer plate, use cyclohexane-ethyl acetate-formic acid with a volume ratio of 6:4:0.1 as the developing solvent, develop, remove, dry, and place under a 365 nm ultraviolet lamp for inspection.

[0153] 4.2 Methodological validation

[0154] 4.2.1 Specificity test

[0155] Prepare Nuan Gan Jian Granules (Batch No. NGJ-K-25041601) as described in Section 4.1.1 for the Nuan Gan Jian Granules test solution. Prepare the negative sample solution using maltodextrin as described in Section 4.1.1. Prepare the ligustilide reference solution, pachymic acid B reference solution, ferulic acid reference solution, wolfberry fruit reference solution, angelica root reference solution, and poria reference solution as described in Section 4.1.2. 10 μL each of negative sample solution, pachymic acid B reference solution, ferulic acid reference solution, wolfberry fruit reference medicinal material solution, angelica control medicinal material solution, poria control medicinal material solution, and test solution were aspirated and spotted on silica gel G thin layer plate, and cyclohexane-ethyl acetate-formic acid with a volume ratio of 6:4:0.1 was used as the developing solvent. The plate was developed, removed, air-dried, and examined under a 254 nm ultraviolet lamp. 10 μL each of negative sample solution, ligustilide reference solution, ferulic acid reference solution, wolfberry fruit reference medicinal material solution, angelica control medicinal material solution, poria control medicinal material solution, and test solution were aspirated and spotted on silica gel G thin layer plate, and cyclohexane-ethyl acetate-formic acid with a volume ratio of 6:4:0.1 was used as the developing solvent. The plate was developed, removed, air-dried, and examined under a 365 nm ultraviolet lamp. The thin layer chromatogram at 254 nm is shown in FIG. Figure 29 , 365nm thin layer chromatogram see Figure 30 The results showed that the test sample chromatogram showed a fluorescent spot of the same color at the corresponding position in the reference sample and reference medicinal material chromatograms; the negative sample chromatogram showed no fluorescent spot at the corresponding position in the reference sample and reference medicinal material chromatograms. Therefore, this thin-layer chromatography identification method is specific.

[0156] 4.2.2 Durability test

[0157] Prepare Nuan Gan Jian Granules (Batch No. NGJ-K-25041601) as described in Section 4.1.1 for the Nuan Gan Jian Granules test solution. Prepare the negative sample solution using maltodextrin as described in Section 4.1.1. Prepare the ligustilide reference solution, pachymic acid B reference solution, ferulic acid reference solution, wolfberry fruit reference solution, angelica root reference solution, and poria reference solution as described in Section 4.1.2.

[0158] 4.2.2.1 Temperature durability

[0159] 10 μL each of the negative sample solution, pachymic acid B reference solution, ferulic acid reference solution, wolfberry fruit control medicinal material solution, angelica control medicinal material solution, Poria control medicinal material solution, and test sample solution were aspirated and spotted on silica gel G thin layer plates, respectively. The plates were developed with cyclohexane-ethyl acetate-formic acid in a volume ratio of 6:4:0.1, developed, removed, air-dried, and examined under a 254 nm ultraviolet lamp at 4°C and 30°C, respectively. 10 μL each of the negative sample solution, ligustilide reference solution, ferulic acid reference solution, wolfberry fruit control medicinal material solution, angelica control medicinal material solution, Poria control medicinal material solution, and test sample solution were aspirated and spotted on silica gel G thin layer plates, respectively. The plates were developed with cyclohexane-ethyl acetate-formic acid in a volume ratio of 6:4:0.1, developed at 4°C and 30°C, respectively. The plates were removed, air-dried, and examined under a 365 nm ultraviolet lamp. The thin layer chromatogram at 4℃ and 254nm is shown in Figure 31 , the thin layer chromatogram at 30℃ and 254nm is shown in Figure 33 , the thin layer chromatogram at 4℃ and 365nm is shown in Figure 32 , the thin layer chromatogram at 30℃ and 365nm is shown in Figure 34 The results showed that in the chromatogram of the test sample, fluorescent spots of the same color appeared at the corresponding positions in the chromatogram of the reference sample and the chromatogram of the reference medicinal material.

[0160] 4.2.2.2 Humidity durability

[0161] 10 μL each of the negative sample solution, pachymic acid B reference solution, ferulic acid reference solution, wolfberry control medicinal material solution, angelica control medicinal material solution, Poria control medicinal material solution, and test sample solution were aspirated and spotted on silica gel G thin layer plates, respectively. The plates were developed with cyclohexane-ethyl acetate-formic acid in a volume ratio of 6:4:0.1, respectively, under a humidity of 30% and 70%, respectively. The plates were taken out, air-dried, and examined under a 254 nm ultraviolet lamp. 10 μL each of the negative sample solution, ligustilide reference solution, ferulic acid reference solution, wolfberry control medicinal material solution, angelica control medicinal material solution, Poria control medicinal material solution, and test sample solution were aspirated and spotted on silica gel G thin layer plates, respectively. The plates were developed with cyclohexane-ethyl acetate-formic acid in a volume ratio of 6:4:0.1, respectively, under a humidity of 30% and 70%, respectively, respectively, the plates were taken out, air-dried, and examined under a 365 nm ultraviolet lamp. The thin layer chromatogram at 30% humidity and 254 nm is shown in Figure 35 , the thin layer chromatogram at 70% humidity and 254nm is shown in Figure 37 , the thin layer chromatogram at 30% humidity and 365nm is shown in Figure 36 , the thin layer chromatogram at humidity 70% and 365nm is shown in Figure 38 The results showed that in the chromatogram of the test sample, fluorescent spots of the same color appeared at the corresponding positions in the chromatogram of the reference sample and the chromatogram of the reference medicinal material.

[0162] 4.2.2.3 Thin layer boards from different manufacturers

[0163] 10 μL each of negative sample solution, pachymic acid B reference solution, ferulic acid reference solution, wolfberry fruit reference medicinal material solution, angelica root reference medicinal material solution, tuckahoe reference medicinal material solution, and test solution were taken and spotted on silica gel G thin layer plates, respectively, with cyclohexane-ethyl acetate-formic acid with a volume ratio of 6:4:0.1 as the developing solvent, developed, taken out, dried, and placed on three thin layer plates from different manufacturers (Qingdao Ocean Chemical Co., Ltd., Qingdao Shuoyuan Silica Gel Technology Co., Ltd., Qingdao Xinchanglai Silica Gel Co., Ltd.), respectively. Inspect under 254nm ultraviolet light; aspirate 10μL each of negative sample solution, ligustilide reference solution, ferulic acid reference solution, wolfberry control medicinal material solution, angelica control medicinal material solution, Poria control medicinal material solution, and test solution, and spot them on silica gel G thin layer plate, using cyclohexane-ethyl acetate-formic acid with a volume ratio of 6:4:0.1 as the developing agent, develop, take out, dry, and inspect under 365nm ultraviolet light on three different manufacturers' thin layer plates (same as the above manufacturers). The thin layer chromatogram at 254nm using thin layer plates purchased from Qingdao Ocean Chemical Co., Ltd., Qingdao Shuoyuan Silica Gel Technology Co., Ltd., and Qingdao Xinchanglai Silica Gel Co., Ltd. is shown in the figure. Figure 39 、 Figure 41 、 Figure 43The thin layer chromatogram at 365 nm was obtained using thin layer plates purchased from Qingdao Ocean Chemical Co., Ltd., Qingdao Shuoyuan Silica Gel Technology Co., Ltd., and Qingdao Xinchanglai Silica Gel Co., Ltd. Figure 40 、 Figure 42 、 Figure 44 The results showed that in the chromatogram of the test sample, fluorescent spots of the same color appeared at the corresponding positions in the chromatogram of the reference sample and the chromatogram of the reference medicinal material.

[0164] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiment. All technical solutions based on the concept of the present invention are within the scope of protection of the present invention. It should be noted that for those skilled in the art, various improvements and modifications that do not depart from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A quality detection method for Nuan Gan Jian, characterized in that: Including characteristic spectrum identification, the specific steps are: (1) Prepare syringin reference solution, norisoboldine reference solution, agarwood tetraol reference solution, cinnamaldehyde reference solution, cinnamic acid reference solution, and 6-gingerol reference solution respectively; (2) Prepare the test solution using Nuan Gan Jian granules; (3) Perform liquid chromatography on the six reference substance solutions and the test solution, record the chromatograms, and calculate the relative retention times; The chromatographic conditions for characteristic spectrum determination are: Column: ChromCore AQ C18, 2.1 × 100 mm, 1.8 μm; Mobile phase: methanol as mobile phase A, 0.1 wt% phosphoric acid as mobile phase B; gradient elution; detection wavelength: 265 nm; flow rate: 0.2 mL / min; column temperature: 30°C; injection volume: 2 μL; The gradient elution program was: 。 2. The method for detecting the quality of Nuan Gan Jian according to claim 1, wherein: The quality inspection method also includes the determination of syringin, norisoboldine, and agarwood tetraol content, and the specific steps are as follows: (1) Prepare syringin reference solution, norisoboldine reference solution, and agarwood tetraol reference solution respectively; (2) Prepare the test solution using Nuan Gan Jian granules; (3) The three reference solutions and the test solution were subjected to liquid chromatography detection, and the contents of syringin, norisoboldine, and agarwood tetraol were calculated based on the obtained chromatograms.

3. The quality detection method of Nuan Gan Jian according to claim 2, characterized in that: The chromatographic conditions for the determination of syringin, norisoboldine and agarwood tetraol are as follows: Column: ChromCore AQ C18, 2.1 × 100 mm, 1.8 μm; Mobile phase: methanol as mobile phase A, 0.1wt% phosphoric acid as mobile phase B; gradient elution; detection wavelength: 210nm; flow rate: 0.2mL / min; column temperature: 30℃; injection volume: 2μL.

4. The quality detection method of Nuan Gan Jian according to claim 1, characterized in that: The quality testing method also includes the determination of cinnamic acid content, and the specific steps are as follows: (1) Prepare cinnamic acid reference solution; (2) Prepare the test solution using Nuan Gan Jian granules; (3) Perform liquid chromatography on the cinnamic acid reference solution and the test solution, and calculate the cinnamic acid content based on the obtained chromatogram.

5. The quality detection method of Nuan Gan Jian according to claim 4, characterized in that: The chromatographic conditions for the determination of cinnamic acid content are: Column: ChromCore AQ C18, 2.1 × 100 mm, 1.8 μm; Mobile phase: methanol as mobile phase A, 0.1wt% phosphoric acid as mobile phase B; gradient elution; detection wavelength: 280nm; flow rate: 0.20mL / min; column temperature: 30℃; injection volume: 2μL.

6. The method for quality inspection of Nuan Gan Jian according to claim 1, characterized in that: The quality inspection method also includes thin layer identification, and the specific steps are: (1) Prepare the test solution using Nuan Gan Jian granules; (2) Prepare ligustilide reference solution, pachymic acid B reference solution, and ferulic acid reference solution; Prepare wolfberry fruit control medicinal material solution, angelica sinensis control medicinal material solution, and Poria cocos control medicinal material solution; (3) Spot the ligustilide reference solution, pachymic acid B reference solution, ferulic acid reference solution, wolfberry reference medicinal material solution, angelica reference medicinal material solution, poria reference medicinal material solution, and test solution on a silica gel G thin layer plate and place it under ultraviolet light for inspection.

7. The method for detecting the quality of Nuan Gan Jian according to claim 6, wherein: Cyclohexane-ethyl acetate-formic acid was used as the developing solvent in thin layer identification.

8. The method for detecting the quality of Nuan Gan Jian according to any one of claims 1 to 7, wherein: The Nuan Gan Jian granules are prepared by the following method: taking the decoction pieces constituting the Nuan Gan Jian granules, decocting with water, filtering, concentrating the filtrate to a clear paste, drying the clear paste to a dry paste, adding or not adding auxiliary materials, mixing, and granulating.

Images