HPLC method for determining cyclic adenosine monophosphate content in jujube

Abstract

The invention discloses an HPLC (High Performance Liquid Chromatography) method for determining the content of cyclic adenosine monophosphate in jujubes. The HPLC method comprises the following steps: (1) preparing a reference solution; (2) preparing a test solution; and (3) respectively injecting the reference substance solution and the test solution for HPLC determination, recording a chromatogram, and calculating the adenosine cyclophosphate content by a peak area method under the chromatographic conditions that a C18 bonded silica gel chromatographic column is adopted, 0.2% phosphoric acid aqueous solution-acetonitrile is used as a mobile phase, the volume flow rate is 1.0 mL / min, the detection wavelength is 256 nm, the column temperature is 30 DEG C, and the injection volume is 5 L. Scientific basis is provided for formulating the Chinese Pharmacopoeia Chinese date content limit standard and selecting the producing area of Chinese dates in classic famous prescription preparations, and quality standardization and product industrialization of traditional Chinese medicines are promoted.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine quality control and analysis technology, specifically relating to an HPLC method for determining the content of cyclic adenosine monophosphate in jujube. Background Technology

[0002] Jujube, the dried, ripe fruit of the jujube plant (Ziziphus jujuba Mill.), belonging to the Rhamnaceae family, is a traditional Chinese medicinal herb. It is sweet and warm in nature, and enters the spleen, stomach, and heart meridians. It has the effects of tonifying the middle energizer and replenishing qi, nourishing blood and calming the mind, and is widely used to treat symptoms such as spleen deficiency with poor appetite, fatigue, loose stools, and hysteria in women. According to classic medical texts such as the *Shang Han Lun*, jujube was used in 58 out of 113 prescriptions, demonstrating its important role in traditional Chinese medicine preparations. Modern pharmacological studies have shown that jujube contains various active ingredients, including polysaccharides, flavonoids, phenols, cyclic nucleotides, triterpenes, alkaloids, amino acids, fatty acids, and dietary fiber, which have antioxidant, anti-fatigue, anti-inflammatory, antibacterial, antitumor, anti-aging, immune-regulating, blood sugar-lowering, and blood lipid-lowering effects. Cyclic adenosine monophosphate (cAMP) is one of the main water-soluble components of jujube. As an intracellular "second messenger," it is stable to acid and heat, and can regulate gene expression, relax smooth muscle, dilate blood vessels, promote nerve regeneration, and enhance immune function. It also plays a positive role in the prevention and treatment of cardiovascular diseases, diabetes, and neurodegenerative diseases. Studies have shown that the level of cAMP is closely related to the quality and efficacy of jujube, and it is expected to serve as a quality marker for jujube.

[0003] my country boasts the richest jujube resources in the country, with 704 existing varieties. Major producing areas include Hebei, Henan, Shandong, and Shaanxi provinces, with Xinjiang accounting for over 30% of the national planting area and over 50% of the output. Jujubes from Xinjiang often exhibit higher levels of chemical components compared to those from other regions. However, the cAMP content of jujubes is significantly affected by factors such as variety, origin, harvest time, and drying method, resulting in a wide range of reported levels in the literature, ranging from 0.0114 to 1.0762 mg / g. For example, Hou Guangyue et al. (“Determination of Oleanolic Acid, Ursolic Acid, CAMP and CAMP in Jujube by High Performance Liquid Chromatography”, *Chinese Journal of Health Laboratory Technology*, July 2018, Vol. 28, No. 13) used HPLC to simultaneously determine oleanolic acid, ursolic acid, cAMP and cyclic guanosine in jujube. The mobile phase was acetonitrile-0.1% formic acid aqueous solution (10:90, V / V). The linear range was 1.0-100.0 µg / mL, the recovery rate was 85%-115%, and the cAMP content of jujube from different origins was 0.083-0.372 mg / g. Li Hui et al. (“Determination of CAMP Content in Jujube from Northern my country by High Performance Liquid Chromatography”, *Analytical Testing Technology and Instruments*, March 2020, Vol. 26, No. 1) used HPLC to determine the cAMP content of 16 kinds of jujube from northern China. The mobile phase was methanol-2% acetic acid (5:95, V / V), and the linear range was 0.1-1.0 µg / mL. The recovery rate was 102.1%, with the highest concentration of 0.372 mg / g found in Xinjiang. Other studies have determined concentrations ranging from 0.24 to 0.56 mg / g, 0.0366 to 0.2434 mg / g, 0.0128 to 0.7082 mg / g, 0.1106 to 0.1925 mg / g, 0.0329 to 0.1975 mg / g, 0.0352 to 0.4421 mg / g, 0.0114 to 0.2854 mg / g, and 0.10 to 0.44 mg / g. Even the concentration of 0.1914 to 2.0632 mg / g found in grafted one-year-old fresh jujubes at the Jujube Resource Nursery of Tarim University was found in fresh jujubes. The national drug standard for "Jujube Formula Granules" stipulates that each 1 g of granules (equivalent to 1.2 g of medicinal slices) contains 0.08-0.35 mg / g of cAMP. Based on a 100% extraction rate, the cAMP content of jujube is 0.0667-0.2917 mg / g. Currently, neither the 2020 nor the 2025 editions of the Chinese Pharmacopoeia (Part I) specify a limit for cAMP content under the jujube section. The lack of systematic comparative data on production areas restricts the quality control of jujube medicinal materials and the selection of jujube production areas in classic prescriptions (such as Huangqi Guizhi Wuwu Tang).

[0004] The determination of cAMP content in jujube mainly employs high-performance liquid chromatography (HPLC). The mobile phase is often methanol-potassium dihydrogen phosphate buffer (e.g., ratio 30:70 or 80:2, detection wavelength 260 nm, column temperature 30 ℃), or methanol-2% acetic acid, acetonitrile-0.1% formic acid, etc. Some studies use liquid chromatography-mass spectrometry (LC-MS / MS) or ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS / MS) to improve sensitivity, but the equipment is expensive and the operation is complex, making it unsuitable for routine application. Fluorescence resonance energy transfer (FRET) and isophase non-radioactive isotope methods are suitable for high-throughput detection, but not for the quantitative analysis of traditional Chinese medicinal materials. However, existing HPLC methods have significant drawbacks: the use of potassium dihydrogen phosphate buffer salts easily leads to increased column pressure, decreased column efficiency, and unstable baseline, affecting resolution and repeatability; for example, while Hou Guangyue et al.'s method can detect multiple components, the 0.1% formic acid in the mobile phase is volatile and has poor pH stability; Li Hui et al.'s methanol-2% acetic acid system is simple, but acetic acid is highly corrosive and easily affects column life; extraction conditions often involve heating and boiling methanol or ethanol (e.g., 80 °C, 2 h), resulting in significant interference from impurities and cumbersome operation; although acetonitrile-phosphoric acid aqueous solution mobile phase systems have been reported, they are mostly gradient elution with non-specifically optimized ratios (e.g., 0.1% phosphoric acid for whole-herb detection of traditional Chinese medicine), making it difficult to achieve baseline separation of the cAMP peak (resolution > 1.5) and low detection limits; sample preparation is not standardized (e.g., freezing followed by pulverization, particle size), resulting in poor comparability of results. Furthermore, existing literature reports on cAMP content from different origins are scattered and lack large-scale, multi-batch systematic determination, failing to provide a reliable basis for the formulation of standards in the Chinese Pharmacopoeia. These problems lead to inaccurate quality evaluation of jujubes, affecting the quality stability and efficacy consistency of traditional Chinese medicine preparations. There is an urgent need to develop a stable, reliable, and green HPLC method, and to combine it with origin analysis to lay the foundation for quality control. Summary of the Invention

[0005] The purpose of this invention is to overcome the defects of the prior art and provide an HPLC method for determining the content of cyclic adenosine monophosphate in jujube.

[0006] The technical solution of the present invention is as follows:

[0007] An HPLC method for determining the content of cyclic adenosine monophosphate in jujube includes the following steps:

[0008] (1) Preparation of reference solution: Accurately weigh cyclic adenosine monophosphate reference standard, dissolve and dilute it with water to a stock solution with a concentration of 400-410 µg / mL, and prepare reference solutions of different concentrations based on the stock solution;

[0009] (2) Preparation of test solution: After freezing the jujube sample, crush it through a No. 2 sieve, accurately weigh 0.5 g of powder, add 25 mL of water, sonicate, add weight and shake well, filter to obtain the filtrate;

[0010] (3) Inject the above reference solution and test solution separately for HPLC determination, record the chromatograms, and calculate the cyclic adenosine monophosphate (cAMP) content by peak area method. The cAMP content in the jujube sample is calculated based on the dried jujube sample. The chromatographic conditions for this step are as follows: use a C18 bonded silica gel column, use 0.2% phosphoric acid aqueous solution-acetonitrile as the mobile phase, flow rate 1.0 mL / min, detection wavelength 256 nm, column temperature 30 ℃, injection volume 5 µL, and the volume ratio of phosphoric acid aqueous solution to acetonitrile in the mobile phase is 97.2:2.8.

[0011] In a preferred embodiment of the present invention, the C18 bonded silica column is an Agilent 5TC-C18 (2) with dimensions of 4.6 mm × 250 mm and 5 µm.

[0012] In a preferred embodiment of the present invention, the elution method of the mobile phase is as follows: 0-9 min, 2.8% acetonitrile; 9-10 min, 2.8%-100% acetonitrile; 10-14 min, 100% acetonitrile; 14-14.1 min, 100%-2.8% acetonitrile; 14.1-21 min, 2.8% acetonitrile.

[0013] In a preferred embodiment of the present invention, the parameters of the ultrasonic treatment are 500 W power and 80 kHz frequency.

[0014] More preferably, the ultrasonic treatment time is 10 minutes.

[0015] In a preferred embodiment of the invention, the filtration is performed using a 0.45 µm filter membrane.

[0016] In a preferred embodiment of the present invention, the linear range of the reference solution is 1.00-60.01 µg / mL, and the linear regression equation is y=12.1850x+0.4465.

[0017] In a preferred embodiment of the present invention, the moisture content of the dried product is determined by drying method according to General Chapter 0832 of the 2020 edition of the Chinese Pharmacopoeia.

[0018] In a preferred embodiment of the present invention, the jujube sample is the dried, mature fruit of Ziziphus jujuba Mill., a plant of the Rhamnaceae family.

[0019] More preferably, the jujube samples are sourced from Xinjiang, Shanxi, and Hebei.

[0020] The beneficial effects of this invention are:

[0021] 1. This invention uses a 0.2% phosphoric acid aqueous solution-acetonitrile (97.2:2.8), which is superior to the existing acetonitrile-0.1% formic acid or methanol-2% acetic acid system. It avoids the column pressure increase and column efficiency decrease caused by buffer salts, achieves baseline separation of cAMP peak and impurity peak (resolution >1.5), theoretical plate number ≥8000, moderate retention time, and high sensitivity at a detection wavelength of 256 nm.

[0022] 2. The sample preparation of the present invention adopts freezing, pulverizing and passing through a No. 2 sieve, and ultrasonic extraction with water. It has a high extraction rate, less interference from impurities, simple operation, and is green and environmentally friendly, which is superior to the existing water heating and boiling or ethanol extraction.

[0023] 3. The linear range of this invention is 1.00-60.01 µg / mL (r=0.9964), the instrument precision RSD is 0.47%, the injection precision RSD is 2.28%, the repeatability RSD is 1.31%, the intermediate precision RSD is 3.78%, the stability (within 35 h) RSD is 2.11%, the average recovery rate is 88.97% (RSD 3.83%), and the robustness study (different columns, flow rates, column temperatures, pH, ratios) has an RSD of <2.01%, which meets the requirement of no more than 4% in the 2020 edition of the Chinese Pharmacopoeia. The stability is better than that of existing methanol-acetic acid or formic acid systems.

[0024] 4. In practical applications, this invention determined the cAMP content of 18 batches of jujubes from 12 origins to be 0.0461-0.4098 mg / g (based on dried product), revealing the pattern that Xinjiang-origin jujubes have higher cAMP content (0.1409-0.3633 mg / g) and Shanxi-origin jujubes have lower cAMP content (0.0461-0.1939 mg / g), which is included in the overall range of existing technologies (0.0114-1.0762 mg / g). This provides a scientific basis for the formulation of the cAMP content limit standard for jujubes in the Chinese Pharmacopoeia and the selection of the origin of jujubes in classic prescriptions, thus promoting the standardization of traditional Chinese medicine quality and the industrialization of products. Attached Figure Description

[0025] Figure 1 This is the HPLC chromatogram of the reference solution in Example 1 of the present invention.

[0026] Figure 2 This is the HPLC chromatogram of the test solution in Example 1 of the present invention.

[0027] Figure 3 This is the HPLC chromatogram of the blank solvent in Example 1 of the present invention. Detailed Implementation

[0028] The technical solution of the present invention will be further explained and described below with reference to specific embodiments and accompanying drawings.

[0029] Example 1

[0030] 1. Instruments and Materials

[0031] 1.1 Instruments: Agilent 1260 high performance liquid chromatograph, including a low-pressure gradient quaternary pump, autosampler, incubator, DAD UV detector, Chem Station chromatography workstation (Agilent Technologies, USA); XS205 electronic analytical balance (Mettler-Toledo, Switzerland, d=0.01 mg); KQ-500VDB three-frequency digitally controlled ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.).

[0032] 1.2 Materials: Acetonitrile (batch number: 1023QH067, chromatographic grade, Tianjin Biaoshiqi Technology Development Co., Ltd.), phosphoric acid (batch number: 20210530, analytical grade, Xilong Scientific Co., Ltd.), ultrapure water. cAMP (batch number 140709-202306, purity 99.9%), purchased from the China National Institutes for Food and Drug Control, chemical structural formula:

[0033] The 18 batches of jujubes tested in this embodiment were all produced in the authentic producing areas or main producing areas, and were identified as dried mature fruits of the jujube (Ziziphus jujuba Mill.) plant of the Rhamnaceae family. They all met the requirements of genuine products in the 2020 edition of the Chinese Pharmacopoeia.

[0034] 2. Methods and Results

[0035] 2.1 Chromatographic conditions: Agilent 5 TC-C 18 (2) Chromatographic column (4.6 mm × 250 mm, 5 µm); mobile phase: 0.2% phosphoric acid aqueous solution (A) - acetonitrile (B) (97.2:2.8), isocratic elution (0~9 min, 2.8%B; 9~10 min, 2.8%~100%B; 10~14 min, 100%B; 14~14.1 min, 100%~2.8%B; 14.1~21 min, 2.8%B); detection wavelength: 256 nm; flow rate: 1 mL / min; column temperature: 30 ℃; injection volume: 5 µL; theoretical plate number calculated based on cAMP peak should not be less than 8000.

[0036] 2.2 Preparation of reference solution: Accurately weigh 10.23 mg of cAMP reference standard and place it in a 25 mL volumetric flask. Dissolve it in water, add water to the mark, and shake well to obtain a reference stock solution with a concentration of 408.79 µg / mL. Store at 4 ℃ for later use.

[0037] 2.3 Preparation of the test solution: Jujubes were frozen at -20 ℃ for 24 h, then quickly pulverized and passed through a No. 2 sieve to obtain jujube powder, which was then sealed for later use. Approximately 0.5 g of the jujube powder (batch number: DZ2023120402) was accurately weighed into a 100 mL stoppered conical flask. 25 mL of water was accurately added, the flask was sealed tightly, and the weight was verified. The flask was then sonicated (500 W, 80 kHz) for 10 min, cooled, and weighed again. The lost weight was replenished with water, and the solution was shaken well. The solution was filtered through a 0.45 µm filter membrane, and the filtrate was collected as the test solution.

[0038] 2.4 Specificity Assessment: Under the conditions described in section 2.1, the cAMP peak achieved baseline separation from other chromatographic peaks (resolution greater than 1.5), and there was no interference from the blank solvent (water). The HPLC chromatograms of the reference solution, test solution, and blank solvent are shown below. Figures 1 to 3 As shown.

[0039] 2.5 Linearity Study: Accurately pipette 12.20, 30.60, 61.20, 91.70, 153.00, 245.00, 367.00, 550.00, and 734.00 µL of the cAMP reference stock solution from section “2.2” into separate 5 mL volumetric flasks. Add water to the mark and mix well to obtain a series of reference solutions with concentrations of 1.00, 2.50, 5.00, 7.50, 12.51, 20.03, 30.01, 44.97, and 60.01 µg / mL, respectively. Record the chromatogram and peak area according to the chromatographic conditions in section “2.1”. Plot the peak area y as the ordinate and the mass concentration x (µg / mL) as the abscissa to obtain the linear regression equation: y = 12.1850x + 0.4465, r = 0.9964. This indicates that cAMP exhibits good linearity in the range of 1.00–60.01 µg / mL.

[0040] 2.6 Instrument precision test: Take the same cAMP reference solution and inject it 6 times consecutively under the chromatographic conditions in section "2.1". The RSD of the cAMP peak area should be 0.47% instead of 2.28%, indicating that the instrument precision is good.

[0041] 2.7 Injection Precision Test: Take the same sample solution and inject it 6 times consecutively under the chromatographic conditions in section "2.1". The RSD of the cAMP peak area should be 2.28% instead of 3.94%. This indicates that the injection precision of this method is good.

[0042] 2.8 Determination of Limits of Detection and Quantitation: The cAMP reference stock solution from section “2.2” was serially diluted according to the specified ratio, and then injected under the chromatographic conditions described in section “2.1”. The limits of detection and quantitation for cAMP were calculated using signal-to-noise ratios of 3:1 and 10:1, respectively. The limits of detection and quantitation for cAMP were found to be 0.2055 µg / mL and 0.6849 µg / mL, respectively.

[0043] 2.9 Repeatability test: Six test solutions were prepared in parallel using jujube powder (batch number: DZ2023120402) according to the method in section "2.3", and the cAMP content was determined according to the chromatographic conditions in section "2.1". The RSD of cAMP content was 1.31%, indicating that the method has good repeatability.

[0044] 2.10 Stability Study: Test solutions from the same batch (batch number: DZ2023120402) were analyzed at 0, 7, 12, 16, 18, 21, 24, 31, and 35 h according to the chromatographic conditions described in section “2.1”. The RSD of cAMP content was 2.11%, indicating that the test solution was stable within 35 h.

[0045] 2.11 Intermediate precision assessment: Unlike the repeatability test, the experimenters prepared 6 parallel solutions of jujube (batch number: DZ2023120402) according to the method in section “2.3”, and injected them 6 times consecutively under the chromatographic conditions in section “2.1”. The RSD of cAMP content was calculated to be 3.78%, indicating that the intermediate precision of the method is good.

[0046] 2.12 Recovery Test: Accurately weigh 0.20 g, 0.20 g, 0.20 g, 0.25 g, 0.25 g, 0.25 g, 0.30 g, 0.30 g, and 0.30 g of jujube powder with known cAMP content (batch number: DZ2023120402, cAMP content 357.04 µg / g), respectively, and place them in 100 mL stoppered conical flasks. Accurately add 178, 178, 178, 222, 222, 222, 266, 266, and 266 µL of cAMP reference stock solution (cAMP concentration 408.79 µg / mL), respectively. Accurately add 25 mL of water, and then adjust the volume difference of the added reference stock solution to 266 µL for each of the first six test samples with water. Seal tightly, weigh, and sonicate (power 500W, frequency 80 kHz) for 10 minutes. After cooling, weigh the sample again, make up the lost weight with water, shake well, filter through a 0.45 µm filter membrane, and collect the filtrate to obtain the test solution for recovery determination. Then determine the recovery rate according to the chromatographic conditions under section "2.1", calculate the recovery rate, and the results are shown in Table 1.

[0047]

[0048] Table 1 Results of the recovery test

[0049] As shown in Table 1, the cAMP recovery rate in this example was 84.60%-95.80%, with an average recovery rate of 88.97% and an RSD of 3.83%, which meets the requirements of the 2020 edition of the Chinese Pharmacopoeia for a recovery rate limit of 85%-110% and an RSD limit of 4%.

[0050] 2.13 Durability Test:

[0051] 2.13.1 Detection using different chromatographic columns: Take the test solution (batch number: DZ2023120402) from section "2.3", and use Welch AQ-C columns. 18 Agilent 5TC-C 18 (2) Agilent ZORBAX SB-C 18 All samples were 250 mm × 4.6 mm and 5 µm in size. They were measured under the chromatographic conditions described in section “2.1”. The results are shown in Table 2.

[0052] 2.13.2 Investigation of different flow rates: Take the test solution (batch number: DZ2023120402) under section “2.3”, and the flow rates are 0.99 mL / min, 1.00 mL / min and 1.01 mL / min respectively. The rest are determined according to the chromatographic conditions under section “2.1”. The results are shown in Table 2.

[0053] 2.13.3 Observation at different column temperatures: Take the test solution (batch number: DZ2023120402) under section “2.3”, and the column temperatures are 29 ℃, 30 ℃ and 31 ℃ respectively. The rest are determined according to the chromatographic conditions under section “2.1”. The results are shown in Table 2.

[0054] 2.13.4 Investigation of different pH values ​​(different concentrations of phosphoric acid aqueous solution): Take the test solution (batch number: DZ2023120402) under section “2.3”, with phosphoric acid aqueous solution concentrations of 0.19% phosphoric acid aqueous solution, 0.20% phosphoric acid aqueous solution and 0.21% phosphoric acid aqueous solution, respectively. The rest are determined according to the chromatographic conditions under section “2.1”. The results are shown in Table 2.

[0055] 2.13.5 Investigation of different mobile phase ratios: Take the test solution (batch number: DZ2023120402) under section “2.3”, and the ratio of 0.2% phosphoric acid aqueous solution to acetonitrile in the mobile phase is 97.3:2.7, 97.2:2.8, and 97.1:2.9, respectively. The rest are determined according to the chromatographic conditions under section “2.1”. The results are shown in Table 2.

[0056] Table 2 Durability Test Results

[0057]

[0058] As shown in Table 2, the RSD% of this embodiment for three different types and manufacturers of chromatographic columns, flow rates, column temperatures, mobile phase pH values, and mobile phase ratios were all less than 4%, indicating that the method has good robustness and meets the requirements of the 2020 edition of the Chinese Pharmacopoeia.

[0059] 2.14 Determination of cAMP content in samples: The moisture content of 18 batches of jujubes was determined by drying method according to General Chapter 0832 of the 2020 edition of the Chinese Pharmacopoeia. The results are shown in Table 3. Two test solutions were prepared for each batch according to the method in section "2.3". The cAMP content was determined under the chromatographic conditions in section "2.1". The details of the origin of the 18 batches of jujubes and the cAMP content calculated on a dried basis are shown in Table 3.

[0060] Table 3. Results of cAMP content determination in jujube production areas (n=2)

[0061]

[0062] As shown in Table 3, the moisture content of the 18 batches of jujubes ranged from 6.34% to 12.82%, all meeting the requirement of "not exceeding 13%" in the General Rules for the Inspection of Medicinal Materials and Processed Pieces of the 2020 Edition of the Chinese Pharmacopoeia (0212). In this example, the cAMP content of 18 batches of jujubes from 12 producing areas, calculated on a dried basis, ranged from 0.0461 to 0.4098 mg / g. Among them, the cAMP content of 7 batches of jujubes from Xinjiang was relatively high, ranging from 0.1409 to 0.3633 mg / g, including those from Alar City, Ruoqiang County, Hotan City, Aksu City, Kashgar City, Turpan City, and Hami City; the cAMP content of 4 batches of jujubes from Shanxi was relatively low, ranging from 0.0461 to 0.1939 mg / g, including those from Yuncheng City, Jiaocheng County, Linfen City, and Liulin County; and the cAMP content of 1 batch of jujubes from Shijiazhuang City, Hebei Province, was 0.1829 mg / g.

[0063] 3. Discussion

[0064] This embodiment employed a single-factor method to investigate the extraction solvents (water, 20% methanol, 30% methanol, 40% methanol, 50% methanol, methanol, 50% ethanol, anhydrous ethanol), extraction methods (ultrasonic extraction, reflux extraction), ultrasonic frequencies (45 kHz, 80 kHz, 100 kHz), and extraction times (10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 70 min). The results showed that water was the most environmentally friendly extraction solvent, resulting in a higher extraction rate. Ultrasonic extraction resulted in less interference from impurities than reflux extraction and was simpler to operate. The highest extraction rate was achieved at an ultrasonic frequency of 80 kHz. The RSD of cAMP content at different ultrasonic times at 80 kHz was 1.93%, with little difference. To save time, the optimal conditions for preparing the jujube sample solution were finally determined.

[0065] In this embodiment, various mobile phases were investigated, including methanol-0.1% phosphoric acid aqueous solution, methanol-0.1% phosphoric acid aqueous solution-0.1% triethylamine aqueous solution, acetonitrile-0.02 mol / L potassium dihydrogen phosphate aqueous solution, acetonitrile-0.1% phosphoric acid aqueous solution-0.1% triethylamine aqueous solution, acetonitrile-0.1% phosphoric acid aqueous solution, and acetonitrile-0.2% phosphoric acid aqueous solution. Ultimately, acetonitrile-0.2% phosphoric acid aqueous solution was selected as the mobile phase. With isocratic elution, the cAMP chromatographic peaks were well separated from the baseline, and the retention time was moderate.

[0066] This embodiment avoids the weaknesses of using phosphate buffer systems and establishes an HPLC method using an acetonitrile-0.2% phosphoric acid aqueous solution system. The cAMP content of 18 batches of jujubes from 12 origins was determined, and the content ranged from 0.0461 to 0.4098 mg / g (calculated on a dried basis), which is within the total range of 0.0114 to 1.0762 mg / g reported in existing technologies. Analysis of the reasons for the large variations in jujube cAMP content revealed that it is related to factors such as jujube variety, planting location, harvest time, drying method, sample preparation method (whether pitted, whether whole jujubes were crushed together, particle size, moisture content, extraction solvent, solvent ratio, extraction method, extraction time, and number of extractions), determination method, and whether the results were calculated on a dried basis.

[0067] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. An HPLC method for determining the content of cyclic adenosine monophosphate in jujube, characterized in that: Includes the following steps: (1) Preparation of reference solution: Accurately weigh cyclic adenosine monophosphate reference standard, dissolve and dilute it with water to a stock solution with a concentration of 400-410 µg / mL, and prepare reference solutions of different concentrations based on the stock solution; (2) Preparation of test solution: After freezing the jujube sample, crush it through a No. 2 sieve, accurately weigh 0.5 g of powder, add 25 mL of water, sonicate, add weight and shake well, filter to obtain the filtrate; (3) Inject the above reference solution and test solution separately for HPLC determination, record the chromatograms, and calculate the cyclic adenosine monophosphate (cAMP) content by peak area method. The cAMP content in the jujube sample is calculated based on the dried jujube sample. The chromatographic conditions for this step are as follows: use a C18 bonded silica gel column, use 0.2% phosphoric acid aqueous solution-acetonitrile as the mobile phase, flow rate 1.0 mL / min, detection wavelength 256 nm, column temperature 30 ℃, injection volume 5 µL, and the volume ratio of phosphoric acid aqueous solution to acetonitrile in the mobile phase is 97.2:2.

8.

2. The HPLC method according to claim 1, characterized in that: The C18 bonded silica column is an Agilent 5 TC-C18 (2) with dimensions of 4.6 mm × 250 mm and 5 µm.

3. The HPLC method according to claim 1, characterized in that: The elution method of the mobile phase is as follows: 0-9 min, 2.8% acetonitrile; 9-10 min, 2.8%-100% acetonitrile; 10-14 min, 100% acetonitrile; 14-14.1 min, 100%-2.8% acetonitrile; 14.1-21 min, 2.8% acetonitrile.

4. The HPLC method according to claim 1, characterized in that: The parameters for the ultrasonic treatment are 500 W power and 80 kHz frequency.

5. The HPLC method as described in claim 4, characterized in that: The ultrasonic treatment time is 10 minutes.

6. The HPLC method according to claim 1, characterized in that: The filtration was performed using a 0.45 µm filter membrane.

7. The HPLC method according to claim 1, characterized in that: The linear range of the reference solution was 1.00-60.01 µg / mL, and the linear regression equation was y=12.1850x+0.4465.

8. The HPLC method according to claim 1, characterized in that: The moisture content of the dried product was determined by drying method according to General Chapter 0832 of the 2020 edition of the Chinese Pharmacopoeia.

9. The HPLC method according to any one of claims 1 to 8, characterized in that: The jujube sample was the dried, ripe fruit of Ziziphus jujuba Mill., a plant in the Rhamnaceae family.

10. The HPLC method according to claim 9, characterized in that: The jujube samples were sourced from Xinjiang, Shanxi, and Hebei.

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