Construction method of substance reference HPLC characteristic chromatogram of cape jasmine and fermented soybean soup

Abstract

The invention provides a construction method of a substance reference HPLC (High Performance Liquid Chromatography) characteristic chromatogram of cape jasmine and fermented soybean soup, which comprises the following steps: a, preparation of a test solution: taking a cape jasmine and fermented soybean soup sample, and dissolving and extracting by adopting a solvent to obtain a solution to be detected; and b, detecting the liquid to be detected by adopting a high performance liquid chromatography to obtain the HPLC characteristic chromatogram. The HPLC characteristic chromatogram which is simple, convenient and easy to implement, good in repeatability and capable of reflecting multiple components contained in the compound as much as possible is established, and the HPLC characteristic chromatogram is particularly important for quality control and evaluation on the substance standard of the cape jasmine and fermented soybean soup.

Description

Technical Field

[0001] This invention relates to a method for constructing a material reference HPLC characteristic spectrum of Gardenia and Fermented Soybean Decoction, belonging to the field of detection. Background Technology

[0002] Ancient classic prescriptions refer to formulas recorded in ancient Chinese medicine classics that are still widely used in clinical practice today, possessing definite efficacy and demonstrating the characteristics and advantages of traditional Chinese medicine treatment. Gardenia and Fermented Soybean Decoction is the 16th prescription in the "Catalogue of Ancient Classic Prescriptions (Second Batch)" (hereinafter referred to as the "Catalogue"). It originates from the "Treatise on Cold Damage" by Zhang Zhongjing, a physician of the Eastern Han Dynasty. Its composition is gardenia and fermented soybean. The prescription reads: Fourteen gardenias (broken), four he of fermented soybean (wrapped in cotton); boil the two ingredients in four liters of water, first boiling the gardenia until two and a half liters remain, then add the fermented soybean and boil until one and a half liters remain. Remove the dregs and divide into two doses. Take one dose warm. If vomiting occurs, stop taking the second dose. It has the effects of clearing stagnation and relieving irritability, and dispersing stagnant heat. It was first used to treat the syndrome of "heat stagnation in the chest and diaphragm," with symptoms such as restlessness, insomnia, vexation, and chest tightness. Modern applications mainly focus on the nervous system, primarily treating insomnia and depression. This prescription's research and development innovates upon the ancient foundation.

[0003] The quality standards for material references advocate for integrity and comprehensiveness. Gardenia and fermented soybean decoction is a compound preparation with complex components. The main components of gardenia include monoterpenes, iridoids, organic acid esters, and flavonoids; the main components of fermented soybean include flavonoids, amino acids, nucleosides, phenylpropanoids, and polysaccharides. This study, through qualitative analysis of the characteristic chromatograms of the Gardenia and fermented soybean decoction reference sample, can achieve simultaneous identification and control of both gardenia and fermented soybean, and is applicable to the identification and control of each individual herb, gardenia and fermented soybean.

[0004] Han Yan, Study on the Pharmacodynamic Material Basis of Gardenia and Fermented Soybean Decoction, Fujian University of Traditional Chinese Medicine, June 2015. Similarity evaluation was used to identify 23 common peaks in the chromatogram and 12 common peaks in fermented soybean. This method showed poor separation in the 0-20 min range; many peaks were not effectively separated at the beginning. Overall, the peaks were crowded at the beginning and sparse at the end. The interval between daidzein and genistein was approximately 15 min, and the inappropriate wavelength selection resulted in a large peak area for geniposide, making other peaks difficult to observe. The absence of ZZ (fermented soybean) and DDC (gardenia) chromatograms indicated limited information on the peaks of fermented soybean and gardenia. The absence of DDC did not reveal saffron glycosides, and the lack of peak information in ZZ resulted in insufficient peak quantity information. Therefore, this method cannot effectively characterize the chemical components contained in Gardenia and Fermented Soybean Decoction, nor is it suitable for the identification and control of individual herbs such as gardenia and fermented soybean. Summary of the Invention

[0005] This invention provides a method for constructing the HPLC characteristic chromatogram of Gardenia and Fermented Soybean Decoction as a material reference.

[0006] This invention provides a method for constructing the HPLC characteristic chromatogram of Gardenia and Fermented Soybean Decoction as a material reference, which includes the following steps: a. Preparation of the test solution: Take a sample of Gardenia and Fermented Soybean Soup, dissolve and extract it using a solvent to obtain the test solution; b. The test solution was detected by high performance liquid chromatography to obtain the HPLC characteristic chromatogram; The high-performance liquid chromatography conditions are as follows: The chromatographic column was a C18 column; acetonitrile was used as mobile phase A and 0.1% phosphoric acid as mobile phase B, with gradient elution performed under the following conditions:

[0007]

[0008] The preparation method of the test solution is as follows: take the Gardenia and Fermented Soybean Decoction sample, add methanol, extract by ultrasonication, filter, and take the filtrate to obtain the test solution.

[0009] The ultrasonic extraction conditions are as follows: power 600W, frequency 40KHz, and ultrasonic time 15min.

[0010] The chromatographic conditions are as follows: column length 250 mm, inner diameter 4.6 mm, particle size 5 μm; flow rate 1.0 ml / min; column temperature 30 °C; detection wavelength 260 nm; theoretical plate number calculated based on geniposide peak should not be less than 5000.

[0011] It also includes the preparation of gardenia reference solution and light fermented soybean reference solution; the preparation method is as follows: Take gardenia or fermented soybean as reference materials, decoct them in water, filter, evaporate the filtered liquid to dryness, add methanol, sonicate, filter, and use the filtrate as the reference solution.

[0012] The ultrasonic treatment conditions are: power 600W, frequency 40KHz; ultrasonic time 15min.

[0013] It also includes a reference solution, wherein the reference is geniposide, daidzein, or genistein; the preparation method is as follows: take the reference, dissolve it in methanol, and prepare a reference solution.

[0014] The HPLC characteristic chromatogram includes 20 characteristic peaks with relative retention times of: peak 1: 0.13, peak 2: 0.18, peak 3: 0.23, peak 4: 0.42, peak 5: 0.57, peak 6: 0.67, peak 7: 0.74, peak 8: 0.77, peak 9: 0.92, peak 11: 1.23, peak 12: 1.26, peak 13: 1.46, peak 14: 1.55, peak 15: 1.57, peak 16: 1.65, peak 17: 1.67, peak 18: 1.69, peak 19: 1.77, and peak 20: 1.80. The relative retention times should be within ±10% of the specified values.

[0015] The characteristic peaks are as follows: Peak 1: xanthine; Peak 2: hyperxanthine; Peak 9: genipin gentiopicroside; Peak 10: geniposide; Peak 12: rutin; Peak 14: crocin I; Peak 17: daidzein; Peak 18: daidzein; Peak 19: crocin III; Peak 20: genistein.

[0016] Multiple studies have reported the presence of xanthine and hypoxanthine in lightly fermented soybeans. However, there are currently no literature or patent reports on the identification and determination of these two components using high-performance liquid chromatography (HPLC). Nevertheless, there are reports of using HPLC-MS for their detection. For example, Wang Yihan et al. used ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS) to structurally identify the chemical components of black beans and lightly fermented soybeans. Their analysis revealed that these two components were only present in lightly fermented soybeans and not detected in black beans, which indirectly indicates that xanthine and hypoxanthine are... The substances are produced during fermentation, not inherent in black beans themselves. Literature by Lin Wangmin et al. also indicates that there are certain differences in nucleoside compounds between soybeans and fermented soybeans; xanthine and hypoxanthine are only present in fermented soybeans and not in soybeans. Studies by Gu Xiaoyu et al. have found that low-dose hypoxanthine has a significant anti-obesity effect on mice. Therefore, identifying xanthine and hypoxanthine as these two components can serve as a distinguishing point between fermented soybeans and black beans, and between fermented soybeans and soybeans. This also reflects more about the chemical components contained in Gardenia and Fermented Soybean Decoction, providing a theoretical basis for the research and further development of the pharmacodynamic material basis of Gardenia and Fermented Soybean Decoction.

[0017] This invention establishes a simple, easy-to-use, and highly reproducible HPLC characteristic chromatogram that can reflect as many components as possible in the compound formula, which is particularly important for the quality control and evaluation of the material reference of Gardenia and Fermented Soybean Decoction. Attached Figure Description

[0018] Figure 1HPLC characteristic chromatogram of Gardenia and Fermented Soybean Soup reference sample (peak 1: xanthine; peak 2: hyperxanthine; peak 9: genipin gentiopicroside; peak 10: geniposide; peak 12: rutin; peak 14: crocin I; peak 17: daidzein; peak 18: daidzein; peak 19: crocin III; peak 20: genistein). Figure 2 Different wavelengths in the standard decoction of Gardenia and Fermented Soybean Soup; Figure 3 Different mobile phases in the standard decoction of Gardenia and Fermented Soybean Soup; Figure 4 Mobile phase: acetonitrile-0.1% phosphoric acid; Figure 5 Mobile phase: acetonitrile-0.4% phosphoric acid; Figure 6 Mobile phase: acetonitrile-1% glacial acetic acid; Figure 7 Investigation of different column temperatures for standard Gardenia and Fermented Soybean Decoction; Figure 8 Investigation of different flow rates in the standard decoction of Gardenia and Fermented Soybean Soup; Figure 9 Delayed reaction of standard decoction of Gardenia and Fermented Soybean Decoction; Figure 10 An investigation into the standard extraction method of Gardenia and Fermented Soybean Decoction; Figure 11 Investigation on the extraction solvent of standard decoction of Gardenia and Fermented Soybean Soup; Figure 12 An investigation into the extraction time of the standard decoction of Gardenia and Fermented Soybean Soup; Figure 13 Investigation on the amount of solvent added to the standard decoction of Gardenia and Fermented Soybean Soup; Figure 14 Chromatographic peak identification of standard decoction of Gardenia and Fermented Soybean Soup; Figure 15 Chromatographic peak identification of standard decoction of Gardenia and Fermented Soybean Soup; Figure 16 Specificity assessment; Figure 17 Investigations using different chromatographic columns; Figure 18 Exploration using different instruments; Figure 19 Comparative chromatograms. Detailed Implementation

[0019] Example 1: Method for constructing HPLC characteristic chromatograms of Gardenia and Fermented Soybean Decoction reference sample [Characteristic chromatogram] Determined by high performance liquid chromatography (General Chapter 0512 of the 2020 edition of the Chinese Pharmacopoeia).

[0020] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile as mobile phase B and 0.1% phosphoric acid as mobile phase A, with gradient elution as specified in the table below; column temperature 30 °C; flow rate 1.0 mL / min; detection wavelength 260 nm; and theoretical plate number calculated based on the geniposide peak should be no less than 5000.

[0021]

[0022] Preparation of reference solutions: Accurately weigh 1g of Gardenia jasminoides reference material, add 50ml of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 25ml of methanol to the residue, sonicate (600W power, 40kHz frequency) for 15 minutes, shake well, filter, and collect the filtrate as the Gardenia jasminoides reference solution. Accurately weigh 0.5g of fermented soybean reference material, add 50ml of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 25ml of methanol to the residue, sonicate (600W power, 40kHz frequency) for 15 minutes, shake well, filter, and collect the filtrate as the fermented soybean reference solution. Accurately weigh an appropriate amount of geniposide reference standard, add methanol to prepare a solution containing approximately 60μg per ml, as the reference solution.

[0023] Preparation of the test solution: Take about 0.5g of this product (ZZCT-01), place it in an Erlenmeyer flask, accurately add 25ml of methanol, sonicate (power 600W, frequency 40kHz) for 15 minutes, shake well, filter, and take the filtrate to obtain the test solution.

[0024] The determination method involves precisely pipetting 5 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0025] The chromatogram of the test sample should show 20 characteristic peaks (see...). Figure 1The retention times should correspond to the 12 characteristic peaks in the chromatogram of Gardenia reference material and the 8 characteristic peaks in the chromatogram of fermented soybean reference material, with one peak corresponding to the retention time of the corresponding reference material peak. The peak corresponding to peak 10, geniposide reference material, is peak S. The relative retention times of each characteristic peak from peak 1 to peak 20 with peak S should be calculated and should be within ±10% of the specified value. The specified values ​​are: 0.13 (peak 1), 0.18 (peak 2), 0.23 (peak 3), 0.42 (peak 4), 0.57 (peak 5), 0.67 (peak 6), 0.74 (peak 7), 0.77 (peak 8), 0.92 (peak 9), 1.23 (peak 11), 1.26 (peak 12), 1.46 (peak 13), 1.55 (peak 14), 1.57 (peak 15), 1.65 (peak 16), 1.67 (peak 17), 1.69 (peak 18), 1.77 (peak 19), and 1.80 (peak 20).

[0026] Example 2: Screening test of the construction method of HPLC characteristic chromatogram of Gardenia and Fermented Soybean Decoction reference sample of the present invention. 1. Experimental instruments and materials High Performance Liquid Chromatograph (HPLC): Agilent 1260 HPLC, Shimadzu LC-20AD HPLC, Waters e2695 HPLC; Electronic balances: ME204E / 02, MS205DU, XP26 (Mettler-Toledo Instruments (Shanghai) Co., Ltd.); PH-20002 (Wuxin Weighing Instrument Co., Ltd.) Ultrapure water system: Cellular type 1810A (Chongqing Moore Water Treatment Equipment Co., Ltd.); Ultrasonic cleaner: KQ600DB model (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.); Chromatographic columns: Shim-pack GIST C18 AQ 250×4.6mm, 5μm; InertSustainAQ-C18 250×4.6mm, 5μm; Phenomenex Luna® C18(2) 100A 250×4.6mm, 5μm Acetonitrile, phosphoric acid, and glacial acetic acid were of chromatographic grade, water was ultrapure water, and all other reagents were of analytical grade.

[0027] Lightly fermented soybean (batch number: 121594-201804, China National Institutes for Food and Drug Control). Gardenia reference material (batch number: 120986-202111, China National Institutes for Food and Drug Control). Gardenoside (Jingpingniside) (Batch No.: 110749-202320, China National Institutes for Food and Drug Control, content calculated as 98.1%). Daidzein (batch number: 111502-202304, China National Institutes for Food and Drug Control, content calculated as 99.3%). Genistein (batch number: 111704-202104, China National Institutes for Food and Drug Control, content calculated as 98.8%). Rutin (batch number: 100080-202104012, China National Institutes for Food and Drug Control, content calculated as 92.2%). Geniposide (batch number: PS011212, Chengdu Pusi Biotechnology Co., Ltd., content >98.0%). Crocin I (batch number: 111588-202205, China National Institutes for Food and Drug Control, content calculated as 95.2%). Crocin III (batch number: PS013967, Chengdu Pusi Biotechnology Co., Ltd., content >98.0%). Soy flavonoids (batch number: wkq18040312, Victor Biotechnology Co., Ltd., content calculated as 99.1%). Xanthine (batch number: 140662-200802, China National Institutes for Food and Drug Control, content is calculated as 100%). Hypoxanthine (batch number: 140661-201704, China National Institutes for Food and Drug Control, content is calculated as 100%). Daidaidzein (batch number: 111738-202305, China National Institutes for Food and Drug Control, content calculated as 93.4%). Genistein (batch number: 111709-202303, China National Institutes for Food and Drug Control, content calculated as 99.6%). Two batches of negative samples (prepared by Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch numbers: ZZCT-YX-01 (ZZ missing), ZZCT-YX-01 (DDC missing)) Gardenia and fermented soybean decoction reference sample (prepared by Sichuan New Green Pharmaceutical Technology Development Co., Ltd., batch number: ZZCT-01).

[0028] 2. Determination of detection wavelength Based on the above-planned experimental conditions, a diode array detector was used to perform a full-band scan of the test solution, and chromatograms of the test solution were extracted at wavelengths of 220 nm, 240 nm, 250 nm, 260 nm, 270 nm, and 290 nm. For example... Figure 2 As shown.

[0029] The results showed that the peak of geniposide was much higher than that of daidzein and genistein. The wavelength of geniposide content was 238 nm, while that of daidzein and genistein was 260 nm. In addition, the detection wavelength of 260 nm had a large amount of peak information, and the chromatographic peaks were evenly distributed and of appropriate size. Therefore, the wavelength of the characteristic chromatographic method for the gardenia and fermented soybean soup reference sample was selected as 260 nm.

[0030] 3. Investigation of different mobile phases Based on the above-specified experimental conditions, the separation effects of four different mobile phases—acetonitrile-0.1% phosphoric acid, acetonitrile-0.4% phosphoric acid, acetonitrile-1% glacial acetic acid, and acetonitrile-water—were investigated. Figures 3-6 As shown.

[0031] The results showed that when the mobile phase was acetonitrile-0.1% phosphoric acid or acetonitrile-1% glacial acetic acid, the number of peaks in the reference sample was relatively large. When the mobile phase was acetonitrile-1% glacial acetic acid, the negative sample lacking gardenia had negative interference at the position of the peak after the target peaks of daidzein and genistein. Therefore, the mobile phase was finally selected as acetonitrile-0.1% phosphoric acid.

[0032] 4. Column temperature investigation Based on the above-planned experimental conditions, the column temperatures of 25℃, 30℃, and 35℃ were investigated respectively. Figure 7 As shown.

[0033] The results showed that, based on the above spectra, peaks 7 and 8 were not separated at a column temperature of 25℃; peaks 14 and 15 were not separated at a column temperature of 35℃, and peak 16 had poor separation. A column temperature of 30℃ was selected.

[0034] 5. Flow velocity assessment Based on the above-established experimental conditions, the flow rates of 0.8 mL / min, 1.0 mL / min, and 1.2 mL / min were investigated. Figure 8 As shown.

[0035] The results showed that, according to the above spectra, at a flow rate of 0.8 ml / min, peaks 14 and 15, and peaks 16 and 17 were not separated; at a flow rate of 1.2 ml / min, peaks 14 and 15 were not separated, and the positions of peaks 7 and 8 were swapped with poor separation. A flow rate of 1.0 ml / min was selected.

[0036] 6. Delayed testing Based on the above-planned experimental conditions, the chromatogram acquisition time will be doubled. For example... Figure 9 As shown.

[0037] The results showed that the sample had no obvious chromatographic peak after 100 minutes, so the sample detection time was set at 100 minutes.

[0038] In summary, the chromatographic conditions and system suitability test for the characteristic chromatogram of Gardenia and Fermented Soybean Soup reference sample were determined as follows: Octadecylsilane-bonded silica gel was used as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile was used as mobile phase A; 0.1% phosphoric acid solution was used as mobile phase B; gradient elution was performed according to the specifications in the table below; column temperature was 30℃; flow rate was 1.0 ml per minute; detection wavelength was 260 nm; and the theoretical plate number, calculated based on the geniposide peak, should not be less than 5000.

[0039]

[0040] 7. Examination of extraction methods Take 0.5g of this product (batch number: ZZCT-01), place it in a stoppered conical flask, add 25ml of methanol, heat under reflux and sonicate (600W power, 40kHz frequency) for 30 minutes respectively, cool, shake well, filter, and collect the filtrate. Results are as follows: Figure 10 .

[0041] The results showed that the change in extraction method did not significantly affect the number of sample peaks, and the ultrasonic method was fast and simple. Therefore, ultrasonic extraction was selected as the extraction method for the test sample.

[0042] 8. Investigation of extraction solvent Take 0.5g of this product (batch number: ZZCT-01) and place it in a stoppered conical flask. Add water, 30% methanol, 50% methanol, 70% methanol, and 25ml of methanol, respectively. Sonicate the mixture (600W power, 40kHz frequency) for 30 minutes, cool, shake well, filter, and collect the filtrate. Figure 11 As shown.

[0043] The results showed that peaks 17 and 20 were missing when the extraction solvent was water; peak 20 was missing when the extraction solvent was 30% methanol; all characteristic peaks were present when the extraction solvents were 50% methanol, 70% methanol, and methanol. Methanol was the simplest solvent to use and the sample solution was easier to filter, so methanol was chosen as the extraction solvent.

[0044] 9. Investigation of Solvent Addition Amount Take 0.5g of this product (batch number: ZZCT-01), place it in a stoppered conical flask, add 15ml, 25ml, and 50ml of methanol respectively, sonicate (600W power, 40kHz frequency) for 30 minutes, cool, shake well, filter, and collect the filtrate to obtain the product. Figure 12 As shown.

[0045] The results showed that when the solvent volume was 25 ml, the chromatographic peaks were of suitable size and the baseline was stable; therefore, the solvent volume of 25 ml was selected.

[0046] 10. Examination of extraction time Take 0.5g of this product (batch number: ZZCT-01), place it in a stoppered conical flask, add 25ml of methanol, sonicate (600W power, 40kHz frequency) for 15, 30, and 45 minutes, cool, shake well, filter, and collect the filtrate. Figure 13 As shown.

[0047] The results show that the sample was completely extracted at an extraction time of 15 minutes, therefore the extraction time was 15 minutes.

[0048] 11. Determine the method for preparing the test sample. The final test solution was prepared as follows: Take 0.5g of this product, place it in a stoppered conical flask, add 25ml of methanol, sonicate (power 600W, frequency 40kHz) for 15 minutes, cool, shake well, filter, and collect the filtrate to obtain the final product.

[0049] 12. Methodological Examination 12.1 Chromatographic peak identification and specificity assessment Preparation of the test solution: Prepare the test solution with characteristic chromatogram according to the experimental conditions proposed above.

[0050] Preparation of reference solution: Prepare the test solution with characteristic spectrum according to the experimental conditions proposed above.

[0051] Preparation of reference solutions: Accurately weigh appropriate amounts of daidzein, genistein, crocin I, crocin III, daidzein, rutin, genipin gentiopicroside, xanthine, hypoxanthine, and geniposide reference standards, and dissolve them separately in methanol to prepare solutions containing 50 μg per ml. Separately, accurately weigh appropriate amounts of daidzein and genistein reference standards, and dissolve them separately in methanol to prepare solutions containing 20 μg per ml.

[0052] Preparation of negative control solutions: Prepare negative sample solutions for soybeans lacking light color and gardenias lacking gardenias according to the experimental conditions proposed above.

[0053] The characteristic peaks of the standard decoction of Gardenia and Fermented Soybean Soup were located. For example... Figure 14 , 15 As shown in Figure 16.

[0054] The chromatographic peak identification results showed that peak 1 was xanthine, peak 2 was hypoxanthine, peak 9 was genipin gentiopicroside, peak 10 (S) was geniposide, peak 12 was rutin, peak 14 was crocin I, peak 17 was daidzein, peak 18 was genistein, peak 19 was crocin III, and peak 20 was genistein.

[0055] Specificity results show that this method has good specificity, identifying peaks 1, 2, 3, 4, 7, 17, 18, and 20 in the reference sample as originating from fermented soybean, and peaks 5, 6, 8, 9, 10 (S), 11, 12, 13, 14, 15, 16, and 19 as originating from gardenia. In the following methodological investigation, 20 characteristic peaks in the sample were examined.

[0056] 12.2 Precision Test Take the reference sample (batch number: ZZCT-01), prepare the test solution according to the proposed experimental method, inject the sample 6 times consecutively, and calculate the retention time and peak area of ​​each characteristic peak. As shown in Tables 1 and 2.

[0057] Table 1. Precision Study of Standard Gardenia and Fermented Soybean Decoction - Retention Time

[0058]

[0059] Table 2 Precision Study of Standard Gardenia and Fermented Soybean Decoction - Peak Area

[0060]

[0061] The results show that the RSD of the retention time of each characteristic peak is between 0.05% and 1.16%, and the RSD of the peak area is between 0.47% and 8.05%, indicating good instrument precision.

[0062] 12.3 Repeatability Test Six portions of the Gardenia and Fermented Soybean Decoction reference sample (batch number: ZZCT-01) were accurately weighed and prepared and measured according to the proposed experimental method. The relative retention time and relative peak area of ​​each characteristic peak were calculated. See Tables 3 and 4 for details.

[0063] Table 3. Repeatability Study of Standard Gardenia and Fermented Soybean Decoction - Relative Retention Time

[0064]

[0065] Table 4. Repeatability Study of Standard Gardenia and Fermented Soybean Decoction - Relative Peak Area

[0066]

[0067] The results show that the RSD of the relative retention time of each characteristic peak is between 0.00% and 3.11%, and the RSD of the peak area is between 0.26% and 5.83%, indicating that the method has good repeatability.

[0068] 12.4 Intermediate Precision Examination Based on the experimental conditions outlined above, two copies of the reference sample (batch number: ZZCT-01) were weighed by different personnel (A and B) at different times (T1 and T2) to prepare test samples. The samples were then measured on different instruments, and the relative retention time and relative peak area of ​​each characteristic peak were calculated. The results are shown in Tables 5 and 6.

[0069] Table 5. Personnel and Time Investigation of Standard Gardenia and Fermented Soybean Decoction - Relative Retention Time

[0070]

[0071] Table 6. Personnel and Time Survey of Standard Gardenia and Fermented Soybean Decoction - Relative Peak Area

[0072]

[0073] The results showed that when samples were prepared by different personnel at different times and measured on different instruments, the relative retention time RSD values ​​of each characteristic peak ranged from 0.00% to 3.78%, and the relative peak area RSD values ​​ranged from 1.99% to 28.31%. The smaller peak areas of peaks 7, 8, 14, and 16 may have caused the larger differences in relative peak area. The intermediate precision of the relative retention times of each characteristic peak was relatively good.

[0074] 12.5 Durability Test 12.5.1 Column robustness test Based on the above-planned experimental conditions, analysis was conducted using the following chromatographic columns: Shim-pack GIST C18 AQ 250×4.6mm, 5μm (column 1), InertSustainAQ-C18 250×4.6mm, 5μm (column 2), and Phenomenex Luna® C18(2) 100A 250×4.6mm, 5μm (column 3). The results are shown in the table below. Figure 17 As shown in Tables 7 and 8.

[0075] Table 7. Chromatographic column durability study of Gardenia and Fermented Soybean Decoction Standard Preparation - Relative Retention Time

[0076]

[0077] Continued from the table above

[0078]

[0079] Table 8. Chromatographic column durability study of Gardenia and Fermented Soybean Decoction Standard Preparation - Relative Peak Area

[0080]

[0081] Continued from the table above

[0082]

[0083] The results showed that the relative retention times (RSDs) of the characteristic peaks on the three columns ranged from 0.00% to 12.06%, and the relative peak areas ranged from 0.00% to 33.86%. The relative retention times of the different columns were relatively consistent. The data showed that the relative retention times and relative peak areas of the characteristic peaks on columns 1 and 2 were very similar. Therefore, the RSDs of the relative retention times and relative peak areas for columns 1 and 2 were calculated. The results are shown in Table 9-10. Table 9. Column Robustness Study - Relative Retention Time

[0084]

[0085] Continued from the table above

[0086]

[0087] Table 10 Column robustness study - relative peak area

[0088]

[0089] Continued from the table above

[0090]

[0091] The results showed that the relative retention time RSD of each characteristic peak on columns 1 and 2 ranged from 0.00% to 5.24%, and the relative peak area RSD ranged from 0.00% to 11.68%. Therefore, this method has a certain selectivity for chromatographic columns, and it is recommended to use column 1 Shimadzu GIST C18 AQ and column 2 Shimadzu InertSustain AQ-C18, or other columns with equivalent performance.

[0092] 12.5.2 Instrument Durability Test Based on the above-planned experimental conditions, analyses were conducted using Agilent 1260 (instrument 1), Waters e2695 (instrument 2), and Shimadzu LC-20AD (instrument 3), respectively. The results are shown in […]. Figure 18 Table 11-12.

[0093] Table 11. Durability Study of Instruments for Standard Gardenia and Fermented Soybean Decoction - Relative Retention Time

[0094]

[0095] Continued from the table above

[0096]

[0097] Table 12 Instrument Durability Test - Relative Peak Area

[0098]

[0099] Continued from the table above

[0100]

[0101] The results show that the relative retention time RSD values ​​of each characteristic peak of different instruments range from 0.00% to 4.23%; the relative peak area RSD values ​​range from 1.55% to 31.13%. The relative retention peak area varies greatly, and the relative retention time of different instruments is quite robust.

[0102] 12.5.3 Stability Assessment Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 4h, 8h, 12h, and 24h, respectively. As shown in Tables 13 and 14.

[0103] Table 13 Stability and Retention Time of Standard Gardenia and Fermented Soybean Decoction

[0104]

[0105] Continued from the table above

[0106]

[0107] Table 14 Stability of Gardenia and Fermented Soybean Decoction Standard Preparation - Peak Area

[0108]

[0109] Continued from the table above

[0110]

[0111] The results showed that the RSD of the retention time of each characteristic peak was 0.06%–3.01% and the RSD of the peak area was 1.03%–7.01% within 24 hours, indicating that the sample solution had good stability within 24 hours.

[0112] In summary, the relative retention times (RSDs) of the 20 peaks of Gardenia and Fermented Soybean Decoction using different chromatographic columns and instruments were all less than 6%, indicating good robustness of the relative retention times. However, the relative peak area (RSD) values ​​of each peak varied considerably. Therefore, this study will not include the relative peak area in the standard calculation.

[0113] Comparative Example 1 Referencing Han Yan's master's thesis, "A Study on the Pharmacological Material Basis of Gardenia and Fermented Soybean Decoction" Chromatographic conditions and system suitability test: C18 column (4.6*250mm, 5µm); mobile phase: 0.1% formic acid aqueous solution (A)-acetonitrile; gradient elution: 0-5 min, 5%-10% B; 5-15 min, 10% B; 15-25 min, 10%-20% B; 25-70 min, 20-40% B; 70-80 min, 40-95% B; 80-81 min, 95%-5% B; 81-90 min, 5% B. Flow rate: 1.0 mL / min; detection wavelength: 250 nm; column temperature: 25°C; injection volume: 20 µL. Chromatogram is shown below. Figure 19 .

[0114] The separation of this method from 0 to 20 min was poor. There were many peaks at the beginning, such as xanthine and hypoxanthine, which were not separated by this method, resulting in crowded peaks at the beginning and empty peaks at the end. The interval between daidzein and genistein was about 15 min, and the wavelength selection made the peak area of ​​geniposide very large, while other peaks were not very visible. The absence of ZZ and DDC chromatograms indicates that there is less chromatographic information of light fermented soybean and gardenia. No saffron glycosides were found in the missing DDC, and the number of peaks in the missing ZZ was too small to characterize the chemical components contained in gardenia fermented soybean soup well.

Claims

1. A method for constructing a material reference HPLC characteristic chromatogram of Gardenia and Fermented Soybean Decoction, characterized in that: It includes the following steps: a. Preparation of the test solution: Take a sample of Gardenia and Fermented Soybean Soup, dissolve and extract it using a solvent to obtain the test solution; b. The test solution was detected by high performance liquid chromatography to obtain the HPLC characteristic chromatogram; The high-performance liquid chromatography conditions are as follows: The chromatographic column was a C18 column; acetonitrile was used as mobile phase A and 0.1% phosphoric acid as mobile phase B, with gradient elution performed under the following conditions:

2. The method for constructing the material reference HPLC characteristic spectrum of Gardenia and Fermented Soybean Decoction according to claim 1, characterized in that: The preparation method of the test solution is as follows: take the Gardenia and Fermented Soybean Decoction sample, add methanol, extract by ultrasonication, filter, and take the filtrate to obtain the test solution.

3. The method for constructing the material reference HPLC characteristic spectrum of Gardenia and Fermented Soybean Decoction according to claim 2, characterized in that: The ultrasonic extraction conditions are: power 600W, frequency 40KHz; ultrasonic time 15min.

4. The method for constructing the material reference HPLC characteristic spectrum of Gardenia and Fermented Soybean Decoction according to claim 1, characterized in that: The chromatographic conditions are as follows: column length 250 mm, inner diameter 4.6 mm, particle size 5 μm; flow rate 1.0 ml / min; column temperature 30 °C; detection wavelength 260 nm; theoretical plate number calculated based on geniposide peak should not be less than 5000.

5. The method for constructing the material reference HPLC characteristic spectrum of Gardenia and Fermented Soybean Decoction according to claim 1, characterized in that: It also includes the preparation of gardenia reference solution and light fermented soybean reference solution; the preparation method is as follows: Take gardenia or fermented soybean as reference materials, decoct them in water, filter, evaporate the filtered liquid to dryness, add methanol, sonicate, filter, and use the filtrate as the reference solution.

6. The method for constructing the material reference HPLC characteristic spectrum of Gardenia and Fermented Soybean Decoction according to claim 5, characterized in that: The ultrasonic treatment conditions are: power 600W, frequency 40KHz; ultrasonic time 15min.

7. The method for constructing the material reference HPLC characteristic spectrum of Gardenia and Fermented Soybean Decoction according to claim 1, characterized in that: It also includes a reference solution, wherein the reference is geniposide, daidzein, or genistein; the preparation method is as follows: take the reference, dissolve it in methanol, and prepare a reference solution.

8. The method for constructing the material reference HPLC characteristic spectrum of Gardenia and Fermented Soybean Decoction according to any one of claims 1-7, characterized in that: The HPLC characteristic chromatogram includes 20 characteristic peaks with relative retention times of: peak 1: 0.13, peak 2: 0.18, peak 3: 0.23, peak 4: 0.23, peak 5: 0.23, peak 6: 0.23, peak 7: 0.23, peak 8: 0.23, peak 9: 0.23, peak 10: 0.23, peak 11: 0.23, peak 12: 0.23, peak 13: 0.23, peak 14: 0.23, peak 15: 0.23, peak 16: 0.23, peak 17: 0.23, peak 18: 0.23, peak 19: 0.23, peak 10: 0.23, peak 19: 0.23, peak 19: 0.23, peak 0.42, Peak 5: 0.57, Peak 6: 0.67, Peak 7: 0.74, Peak 8: 0.77, Peak 9: 0.92, Peak 11: 1.23, Peak 12: 1.26, Peak 13: 1.46, Peak 14: 1.55, Peak 15: 1.57, Peak 16: 1.65, Peak 17: 1.67, Peak 18: 1.69, Peak 19: 1.77, Peak 20: 1.80, the relative retention time should be within ±10% of the specified value.

9. The method for constructing the material reference HPLC characteristic spectrum of Gardenia and Fermented Soybean Decoction according to claim 8, characterized in that: The characteristic peaks are as follows: Peak 1: xanthine; Peak 2: hyperxanthine; Peak 9: genipin gentiopicroside; Peak 10: geniposide; Peak 12: rutin; Peak 14: crocin I; Peak 17: daidzein; Peak 18: daidzein; Peak 19: crocin III; Peak 20: genistein.

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