A method for constructing almond HPLC fingerprint

Abstract

The application relates to the technical field of pharmaceutical analysis, and discloses a construction method of almond HPLC (High Performance Liquid Chromatography) fingerprint, which comprises the following steps: step 1, preparing a control reference solution by using tryptophan control; step 2, mixing almond powder and a methanol aqueous solution, and sequentially performing extraction, cooling and filtration treatment to obtain a test solution; and step 3, performing HPLC detection on the test solution and the control reference solution to obtain an almond HPLC fingerprint composed of sample common characteristic peaks. The construction method of the almond HPLC fingerprint provided by the application firstly constructs an almond HPLC fingerprint containing 11 common characteristic peaks of tryptophan, linoleic acid and oleic acid, the fingerprint chromatographic peak is good in shape, stable, excellent in repeatability and high in precision, and the method provides a new technical scheme and technical idea for identification, quality control and quality evaluation of almond and decoction pieces.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis technology, and in particular to a method for constructing an HPLC fingerprint of almond kernels. Background Technology

[0002] Almond kernels are the kernels of the sweet almond plant (Rosa aceae family). Amygdalus communis The dried, mature seeds of almonds (L.) are a classic medicinal material in Uyghur medicine and are included in the drug standards issued by the Ministry of Health (Uyghur Medicine Volume). Originally from West Asia, almonds are primarily produced in Xinjiang, my country, and are also distributed in Shaanxi and Gansu provinces. Almonds are not only a high-quality dried fruit with high nutritional value but also possess significant medicinal value. In Uyghur medical theory, almonds are believed to aid in nutrition, strengthen the body and brain, improve eyesight and complexion, and relieve coughs and intestinal discomfort. They are used for conditions such as weakness, cough with phlegm, chest tightness, constipation, and poor eyesight. Modern research has found that almonds mainly contain fats, proteins, vitamins, sugars, amino acids, and minerals such as calcium, copper, manganese, phosphorus, iron, magnesium, potassium, and sodium. They exhibit clear pharmacological activities in areas such as anti-oxidation, cardiovascular protection, immune regulation, respiratory system protection, intestinal health, and neurocognitive enhancement. In recent years, with the deepening of research on ethnic medicine and the development of the big health industry, the development and application of almonds have become increasingly widespread. However, there are still problems such as the imperfect standardized quality control system, which restricts the research on the material basis of almonds to mainly focus on the analysis of the nutritional components of almonds. Research on non-oil components is relatively scarce, and a standardized HPLC fingerprint construction method has not yet been established. As a result, the quality of almond medicinal materials and decoction pieces lacks a unified characterization standard, making it difficult to ensure their medicinal safety and efficacy.

[0003] High-performance liquid chromatography (HPLC) offers excellent selectivity, high separation efficiency, high sensitivity, and fast analysis speed. It can accurately analyze lipid-soluble components, water-soluble components, phenolic acids, flavonoids, and other substances in complex systems, and has become one of the most commonly used modern analytical techniques in pharmaceutical analysis. Establishing a fingerprint chromatogram of almond kernels using HPLC can reflect their overall component distribution, providing a scientific basis and technical support for the identification, quality evaluation, and improvement of quality standards for almond kernels. Summary of the Invention

[0004] The purpose of this invention is to provide a method for constructing an HPLC fingerprint of almond kernels. The constructed fingerprint has good peak shape, stability, excellent repeatability, and high precision, providing a new technical solution and approach for the identification, quality control, and quality evaluation of almond kernels and processed medicinal slices.

[0005] To achieve the above objectives, in a first aspect, the present invention provides a method for constructing an HPLC fingerprint of almond kernels, comprising the following steps: Step 1: Prepare a reference solution of tryptophan reference standard; Step 2: Mix almond kernel powder with methanol aqueous solution, and then perform extraction, cooling and filtration processes sequentially to obtain the test solution; Step 3: Perform high-performance liquid chromatography (HPLC) on the test solution and the reference solution to obtain the HPLC fingerprint of almond kernel composed of common characteristic peaks of the samples.

[0006] Furthermore, in step 1, the reference solution uses chromatographically pure methanol as the solvent, and the concentration of tryptophan is 2~10 μg / mL.

[0007] Furthermore, in the test solution of step 2, the particle size of the almond powder is such that it passes through a No. 2 to No. 3 sieve; the ratio of almond powder to methanol aqueous solution is (0.5~2.0g):(15~50mL); and the volume fraction of methanol in the methanol aqueous solution is 50%~100%.

[0008] Further, in step 2, the extraction method is ultrasonic or reflux, and the time is 20~60 min; the temperature after cooling is 15~25℃; filtration is carried out by filtering with a 0.22μm microporous membrane or high-speed centrifugation at 12000r / min for 5~15 min.

[0009] Furthermore, in step 3, the conditions for the high-performance liquid chromatography (HPLC) detection are as follows: acetonitrile is used as mobile phase A, and phosphoric acid aqueous solution is used as mobile phase B, with gradient elution. The specific steps are as follows: From 0 to 2 minutes, mobile phase A increased from 0% to 2%, while mobile phase B decreased from 100% to 98%. Over 2-25 minutes, mobile phase A increased from 2% to 20%, while mobile phase B decreased from 98% to 80%. Over 25-35 minutes, mobile phase A increased from 20% to 30%, while mobile phase B decreased from 80% to 70%. Over 35–45 minutes, mobile phase A increased from 30% to 65%, while mobile phase B decreased from 70% to 35%. Over 45-60 minutes, mobile phase A increased from 65% to 80%, while mobile phase B decreased from 35% to 20%.

[0010] Furthermore, the high-performance liquid chromatography (HPLC) detection satisfies at least one of the following conditions: 1) The detection wavelength is 260nm for 0~50 minutes and 210nm for 50~60 minutes; 2) The injection volume is 2~10 μL; 3) The flow rate is 0.8 mL per minute to 1.2 mL per minute; 4) Column temperature is 20~35℃; 5) An Agilent ZORBAX SB-Aq column was used, with dimensions of 4.6 × 250 mm and a particle size of 5 μm. 6) The concentration of the phosphoric acid aqueous solution is 0.1%~0.3%.

[0011] Furthermore, step 3 also includes: importing the high performance liquid chromatogram of the test sample solution into the "Traditional Chinese Medicine Fingerprint Similarity Evaluation System 2012 Edition" software for similarity calculation.

[0012] Furthermore, the HPLC fingerprint of almond kernels includes 11 characteristic peaks, of which peak 6 is the characteristic peak of tryptophan, peak 10 is the characteristic peak of linoleic acid, and peak 11 is the characteristic peak of oleic acid. Taking peak 6 as the reference peak, the relative retention times of peaks 1-5, peaks 7-11 and peak S are consistent as follows: peak 1 is 0.46, peak 2 is 0.52, peak 3 is 0.56, peak 4 is 0.64, peak 5 is 0.94, peak 7 is 1.23, peak 8 is 1.71, peak 9 is 1.98, peak 10 is 3.28, and peak 11 is 3.41, with a relative standard deviation of ±10%.

[0013] Secondly, this invention provides the application of the HPLC fingerprint of almond kernel constructed by the above-mentioned construction method in the identification, quality control and quality evaluation of almond kernel medicinal materials and / or processed slices.

[0014] Thirdly, this invention provides the application of the HPLC fingerprint of almond kernel constructed by the above-mentioned construction method in the chemical composition analysis of almond kernel.

[0015] The advantages and positive effects of the method for constructing an HPLC fingerprint of almond kernels described in this invention are as follows: 1. This invention is the first to construct an HPLC fingerprint of almond kernel containing 11 common characteristic peaks, including tryptophan, linoleic acid and oleic acid, providing a new technical solution and approach for the identification, quality control and quality evaluation of almond kernel and processed medicinal slices.

[0016] 2. The fingerprint chromatogram constructed in this invention has good peak shape, stable method, excellent repeatability and high precision, which makes up for the limitations of the study of the chemical composition of almond kernel and provides a basis for improving the quality standard of almond kernel, a traditional Uyghur medicine.

[0017] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0018] Figure 1 This is a chromatogram of the test solution in an embodiment of the present invention; Figure 2 These are chromatograms of the test samples at different detection wavelengths in embodiments of the present invention; Figure 3These are chromatograms of the test samples at different flow rates in embodiments of the present invention; Figure 4 These are chromatograms of the test samples at different column temperatures in the embodiments of the present invention; Figure 5 These are chromatograms of test samples treated with different extraction solvents in the embodiments of the present invention; Figure 6 These are chromatograms of test samples treated by different extraction methods in the embodiments of the present invention; Figure 7 These are chromatograms of test samples at different extraction times in embodiments of the present invention; Figure 8 This invention presents 33 batches of almond kernel medicinal materials with superimposed fingerprint spectra and a generated control spectra. Figure 9 This is a comparison fingerprint of almond kernels in an embodiment of the present invention; Figure 10 This is a comparison diagram of the reference solution and the almond test solution in the embodiments of the present invention. Detailed Implementation

[0019] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0020] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0021] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available materials.

[0022] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention.

[0023] This invention provides a method for constructing an HPLC fingerprint of almond kernels, which enables rapid and convenient monitoring of the overall chemical composition of almond kernels, providing a new technical solution for comprehensively evaluating the overall quality of almond kernels. This technical solution includes the following steps: Step 1: Prepare a reference solution of tryptophan reference standard; Step 2: Mix almond kernel powder with methanol aqueous solution, and then perform extraction, cooling and filtration processes sequentially to obtain the test solution; Step 3: Perform high-performance liquid chromatography (HPLC) on the test solution and the reference solution to obtain the HPLC fingerprint of almond kernel composed of common characteristic peaks of the samples.

[0024] In some embodiments, the reference solution in step 1 uses chromatographically pure methanol as the solvent, and the concentration of tryptophan is 2~10 μg / mL, specifically 2 μg / mL, 5 μg / mL, 8 μg / mL, or 10 μg / mL.

[0025] In some embodiments, the almond kernels in the test solution in step 2 are pulverized to a particle size that passes through a No. 2 or No. 3 sieve (specifically, a No. 2 or No. 3 sieve). In step 2, the sample weight of almond kernels is 0.5~2.0g, specifically 0.5g, 1.0g, 1.5g, or 2.0g, and the volume of methanol aqueous solution used is 15~50mL, specifically 15mL, 20mL, 25mL, or 50mL. In step 2, the volume fraction of methanol in the methanol-water solution is 50% to 100%, specifically 50%, 70%, 80%, or 100%, preferably 70%; In step 2, the extraction method is ultrasonic or reflux, preferably ultrasonic extraction; the extraction time is 20~60min, specifically 20min, 30min, 45min, 60min, preferably 30min. In step 2, the temperature after cooling is 15~25℃, specifically 15℃, 20℃, or 25℃; in step 2, the filtration includes filtration using a 0.22μm microporous membrane or high-speed centrifugation at 12000r / min for 10min.

[0026] The conditions for high-performance liquid chromatography detection include gradient elution using acetonitrile as mobile phase A and phosphoric acid aqueous solution as mobile phase B.

[0027] In some embodiments, the gradient elution is performed according to the following procedure: From 0 to 2 minutes, mobile phase A increased from 0% to 2%, while mobile phase B decreased from 100% to 98%. Over 2-25 minutes, mobile phase A increased from 2% to 20%, while mobile phase B decreased from 98% to 80%. Over 25-35 minutes, mobile phase A increased from 20% to 30%, while mobile phase B decreased from 80% to 70%. Over 35–45 minutes, mobile phase A increased from 30% to 65%, while mobile phase B decreased from 70% to 35%. Over 45-60 minutes, mobile phase A increased from 65% to 80%, while mobile phase B decreased from 35% to 20%.

[0028] In some embodiments, the high-performance liquid chromatography detection satisfies at least one of the following conditions: (1) The detection wavelength is 260nm for 0~50 minutes and 210nm for 50~60 minutes; (2) The injection volume is 2~10μL, specifically 2μL, 5μL, or 10μL; (3) The flow rate is 0.8 mL per minute to 1.2 mL per minute, specifically 0.8 mL per minute, 1.0 mL per minute, or 1.2 mL per minute; (4) The column temperature is 20~35℃, specifically 20℃, 25℃, 30℃, and 35℃; (5) An Agilent ZORBAX SB-Aq column was used, with a specification of 4.6×250 mm and a particle size of 5 μm; (6) The concentration of the phosphoric acid aqueous solution is 0.1%~0.3%, specifically 0.1%, 0.2% or 0.3%.

[0029] Furthermore, the construction method also includes: importing the high performance liquid chromatogram of the test sample solution into the "Chinese Herbal Medicine Fingerprint Similarity Evaluation System 2012 Edition" software to obtain the fingerprint spectrum of the almond kernel.

[0030] In some embodiments, the almond fingerprint spectrum includes 11 characteristic peaks, of which peak 6 is the characteristic peak of tryptophan, peak 10 is the characteristic peak of linoleic acid, and peak 11 is the characteristic peak of oleic acid. Taking peak 6 as the reference peak, the relative retention times of peaks 1-5, peaks 7-11 and peak S are as follows: peak 1 is 0.46, peak 2 is 0.52, peak 3 is 0.56, peak 4 is 0.64, peak 5 is 0.94, peak 7 is 1.23, peak 8 is 1.71, peak 9 is 1.98, peak 10 is 3.28, and peak 11 is 3.41, with a relative standard deviation within ±10%.

[0031] In summary, this invention establishes a method for constructing an HPLC fingerprint spectrum of almond kernels. The established fingerprint spectrum enables rapid and convenient monitoring of the overall chemical composition of almond kernels, providing a new technical solution for comprehensively evaluating the overall quality of almond kernels and offering new technical ideas for the quality control and quality evaluation of almond kernel medicinal materials and processed products.

[0032] The following examples further illustrate this point.

[0033] 1. Instruments and reagents: Instruments: High-performance liquid chromatograph (Agilent 1260Prime, Agilent Technologies); analytical balance 0.0001g (XSR105DU, Mettler Toledo); analytical balance 0.0001g (MS204TS, Mettler Toledo); ultrasonic cleaner (KQ-500DE, Kunshan Ultrasonic Instrument Co., Ltd.); Agilent ZORBAX SB-Aq column (4.6×250mm, 5μm).

[0034] Reagents: Acetonitrile (Fisher Chemical); phosphoric acid (Shanghai Maclean Biochemical Technology Co., Ltd.); methanol (Fisher Chemical); water used in the experiment was Wahaha purified drinking water. Acetonitrile, methanol, and phosphoric acid were all of chromatographic grade.

[0035] Reagents and medicinal materials: Tryptophan (batch number 140686-202205, 100% purity, China National Institutes for Food and Drug Control); Linoleic acid (batch number 111622-202105, 99.6% purity, China National Institutes for Food and Drug Control); Oleic acid (batch number 111621-202409, 99.3% purity, China National Institutes for Food and Drug Control); 33 batches of almond kernels (harvested in autumn 2024 from Kashgar region, Xinjiang) were identified as sweet almond (Rosa chinensis). Amygdalus communis Dried mature seeds of L.

[0036] 2. Chromatographic conditions and sample preparation: 2.1 Chromatographic conditions: An Agilent ZORBAX SB-Aq column (5 μm, 4.6 × 250 mm) was used; acetonitrile was used as mobile phase A and 0.2% phosphoric acid solution was used as mobile phase B, with gradient elution performed according to the specifications in Table 1; the column temperature was 25 °C; the flow rate was 1.0 mL / min; the detection wavelength was 260 nm from 0 to 50 min and 210 nm from 50 to 60 min; the injection volume was 5 μL.

[0037] Table 1 Gradient elution table

[0038] 2.2 Preparation of reference solution: Take an appropriate amount of tryptophan reference standard, accurately weigh it, dissolve it in methanol to prepare a solution containing 5 μg per 1 mL.

[0039] 2.3 Preparation of the test solution: Weigh 0.5-2.0g of the powder accurately, place it in a stoppered conical flask, add 25mL of 50%-100% methanol accurately, sonicate for 20-60 minutes, remove, cool to 15-25℃, filter through a 0.22μm filter membrane, and collect the filtrate to obtain the product.

[0040] 2.4 Determination Method: Accurately pipette 5 μL each of the reference solution and the test solution into the liquid chromatograph, perform the determination, and record the chromatogram. The result is as follows: Figure 1 The HPLC fingerprint of almond kernel is shown.

[0041] 3. Determination of chromatographic conditions: 3.1 Comparison of elution gradients: This invention screened chromatograms under different elution gradients through extensive experiments. Comparison revealed that when the gradient elution program is as shown in Table 1, the chromatogram baseline is more stable, the resolution is higher, and the analysis time is moderate. Therefore, the elution program in Table 1 was selected as the final elution gradient.

[0042] 3.2 Selection of detection wavelength: Following the elution procedure in Table 1, inject the test solution at a flow rate of 1.0 mL / min and a column temperature of 25℃. Perform a full wavelength scan from 190 to 400 nm. Based on the sample isoabsorption chromatogram, compare the chromatograms at wavelengths of 210 nm, 260 nm, and 280 nm. Figure 2 As shown in the figure. The results show that the chromatographic peaks responded most strongly at a detection wavelength of 210 nm. However, due to the proximity of the detection wavelength to the methanol terminal absorption, the baseline fluctuated significantly in the first 30 minutes. Peaks 10 and 11 were still detectable at this wavelength. Compared to 280 nm, the chromatographic peaks were more prominent in the first 10 minutes. Therefore, considering the overall number of chromatographic peaks, response, and baseline stability of the chromatogram, and to comprehensively represent the chemical composition of almond kernels, variable wavelength detection was chosen: 260 nm for 0–50 minutes and 210 nm for 50–60 minutes.

[0043] 3.3 Flow velocity assessment: This invention selected three different flow rates—0.8 mL / min, 1.0 mL / min, and 1.2 mL / min—for investigation, and the results are as follows: Figure 3 As shown, there are no significant differences in the fingerprint profiles of almonds at flow rates of 0.8–1.2 mL / min, and the separation of each characteristic peak is good.

[0044] 3.4 Column Temperature Investigation: This invention selected three different column temperatures—20℃, 25℃, and 30℃—for investigation, and the results are as follows: Figure 4As shown, there is no significant difference in the outline of almond fingerprint spectrum when the column temperature is between 20℃ and 30℃, and the separation of each characteristic peak is good.

[0045] 4. Preparation of the test solution: 4.1 Investigation of extraction solvent: One g of almond kernel sample was prepared in triplicate, using 50% methanol, 70% methanol, and methanol as extraction solvents, respectively. The samples were then analyzed. Results showed differences in the number and size of characteristic peaks extracted with the three solvents. For example, peaks 10 and 11 had very small areas in 50% methanol, but the largest areas in methanol, followed by 70% methanol. There were no significant differences in baseline stability and resolution among the different extraction solvents. Figure 5 As shown. Therefore, 70% methanol was chosen as the test solution for preparing the fingerprint spectrum of almond kernels, which can take into account both the number and area of ​​chromatographic peaks, and more comprehensively show the chemical composition characteristics of almond kernel medicinal materials.

[0046] 4.2 Examination of extraction methods: The test solution was prepared using 70% methanol as solvent by ultrasonic extraction and water bath reflux extraction, respectively, and then injected for analysis. The results showed no significant differences in the number of characteristic peaks, total peak area, baseline stability, and resolution under different extraction methods. Figure 6 As shown. Therefore, considering all factors, ultrasonic extraction, which is simpler and easier to perform, is the preferred extraction method for almond kernel samples.

[0047] 4.3 Extraction time: Using 70% methanol as solvent, 1g of almond kernels was weighed and prepared in quadruplicate for extraction times of 20min, 30min, 45min, and 60min, respectively. The samples were then injected for analysis. Results showed no significant differences in the number of characteristic peaks, total peak area, baseline stability, and resolution at different extraction times. Figure 7 As shown, ultrasound for 20–60 minutes has no significant effect on the fingerprint profile of almond kernels, and it can effectively characterize their chemical components. From an economic and efficiency perspective, 30 minutes is the preferred extraction time.

[0048] 5. Methodological Examination: 5.1 Precision: The same almond sample solution was injected six times consecutively, and the fingerprint spectrum was recorded. Using tryptophan as the reference peak S, the relative retention time and relative peak area of ​​the remaining characteristic peaks were calculated. The results are shown in Tables 2 and 3. The RSD of the relative retention time of each characteristic peak was 0.01%–0.43%, and the RSD of the relative peak area was 0.44%–1.78%, indicating good instrument precision.

[0049] Table 2. Relative retention time results from precision experiments

[0050] Table 3. Relative peak area results of precision experiments

[0051] 5.2 Repeatability: Take 1g of almond kernel material, prepare 6 parallel samples, and inject the samples for analysis. Record the fingerprint spectrum, using tryptophan as the reference peak S, and calculate the relative retention time and relative peak area of ​​the remaining characteristic peaks. The results are shown in Tables 4 and 5. The RSD of the relative retention time of each characteristic peak is 0.01%~0.03%, and the RSD of the relative peak area is 1.87%~3.86%, indicating that the method has good repeatability.

[0052] Table 4. Relative retention time results of repeatability experiments

[0053] Table 5. Relative peak area results of repeatability experiments

[0054] 5.3 Stability: The same almond kernel sample solution was injected and analyzed at 0, 4, 8, 12, 16, and 24 hours, and the chromatograms were recorded. Using tryptophan as the reference peak S, the relative retention times and relative peak areas of the other characteristic peaks were calculated. The results are shown in Tables 6 and 7. The relative retention time RSD of each characteristic peak was 0.02%–0.07%, and the relative peak area RSD was 0.23%–1.72%, indicating that the solution had good stability within 24 hours.

[0055] Table 6. Results of relative retention times in stability experiments

[0056] Table 7. Results of relative peak area in stability experiments

[0057] 6. Fingerprint analysis of multiple batches of almond kernels: Thirty-three batches of almond kernel medicinal materials were prepared into test solutions, and their chromatograms were recorded. The results were imported into the National Pharmacopoeia Commission's "Traditional Chinese Medicine Fingerprint Similarity Evaluation System 2012 Edition" software to match the chromatographic peaks and establish fingerprint spectra. The generated overlay spectra are shown below. Figure 8 As shown, the similarity of the 33 batches of almond kernels ranged from 0.795 to 0.998. The generated control fingerprint chromatograms are shown below. Figure 9 As shown, there are a total of 11 peaks, of which peak 6 is tryptophan, peak 10 is linoleic acid, and peak 11 is oleic acid.

[0058] Based on the determination results of 33 batches of almond kernels, it was determined that the fingerprint spectrum of almond kernels should show 11 characteristic peaks, among which peak 6 should correspond to the retention time of the reference peak. The peak corresponding to the tryptophan reference peak is peak S. The relative retention times of the remaining characteristic peaks were calculated, and their relative retention times should be within ±10% of the specified values, which are 0.46 (peak 1), 0.52 (peak 2), 0.56 (peak 3), 0.64 (peak 4), 0.94 (peak 5), 1.23 (peak 7), 1.71 (peak 8), 1.98 (peak 9), 3.28 (peak 10), and 3.41 (peak 11).

[0059] 7. Identification of characteristic peaks in almond fingerprint spectrum: By comparing the retention times of the test sample and the reference solution using ultraviolet 3D spectral analysis, three chemical components—tryptophan, linoleic acid, and oleic acid—were identified in the fingerprint chromatogram of almond kernels, corresponding to peaks 6, 10, and 11, respectively. The comparison graph between the test sample solution and the reference solution is shown below. Figure 10 As shown.

[0060] Using the technical solution of this invention, an HPLC fingerprint of almond kernel containing 11 common characteristic peaks, including tryptophan, linoleic acid and oleic acid, was constructed for the first time, realizing the monitoring of the overall chemical composition of almond kernel.

[0061] Therefore, the method for constructing an HPLC fingerprint of almond kernels described in this invention produces fingerprints with good peak shapes, stable methods, excellent repeatability, and high precision. This provides a new technical solution for comprehensively evaluating the overall quality of almond kernels and offers new technical ideas for the quality control and evaluation of almond kernel medicinal materials and processed products.

[0062] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for constructing an HPLC fingerprint of almond kernels, characterized in that, Includes the following steps: Step 1: Prepare a reference solution of tryptophan reference standard; Step 2: Mix almond kernel powder with methanol aqueous solution, and then perform extraction, cooling and filtration processes sequentially to obtain the test solution; Step 3: Perform high-performance liquid chromatography (HPLC) on the test solution and the reference solution to obtain the HPLC fingerprint of almond kernel composed of common characteristic peaks of the samples.

2. The construction method according to claim 1, characterized in that, In step 1, the reference solution uses chromatographically pure methanol as the solvent, and the concentration of tryptophan is 2~10 μg / mL.

3. The construction method according to claim 1, characterized in that, In the test solution of step 2, the particle size of the almond powder is such that it passes through a No. 2 to No. 3 sieve; the ratio of almond powder to methanol aqueous solution is (0.5~2.0g):(15~50mL); the volume fraction of methanol in the methanol aqueous solution is 50%~100%.

4. The construction method according to claim 1, characterized in that, In step 2, the extraction method is ultrasonic or reflux, and the time is 20~60 min; the temperature after cooling is 15~25℃; filtration is carried out by filtering with a 0.22μm microporous membrane or high-speed centrifugation at 12000r / min for 5~15 min.

5. The construction method according to claim 1, characterized in that, In step 3, the high-performance liquid chromatography (HPLC) detection conditions are as follows: acetonitrile is used as mobile phase A, and phosphoric acid aqueous solution is used as mobile phase B, with gradient elution. The specific steps are as follows: From 0 to 2 minutes, mobile phase A increased from 0% to 2%, while mobile phase B decreased from 100% to 98%. Over 2-25 minutes, mobile phase A increased from 2% to 20%, while mobile phase B decreased from 98% to 80%. Over 25-35 minutes, mobile phase A increased from 20% to 30%, while mobile phase B decreased from 80% to 70%. Over 35–45 minutes, mobile phase A increased from 30% to 65%, while mobile phase B decreased from 70% to 35%. Over 45-60 minutes, mobile phase A increased from 65% to 80%, while mobile phase B decreased from 35% to 20%.

6. The construction method according to claim 5, characterized in that, The high-performance liquid chromatography (HPLC) detection satisfies at least one of the following conditions: 1) The detection wavelength is 260nm for 0~50 minutes and 210nm for 50~60 minutes; 2) The injection volume is 2~10 μL; 3) The flow rate is 0.8 mL per minute to 1.2 mL per minute; 4) Column temperature is 20~35℃; 5) An Agilent ZORBAX SB-Aq column was used, with dimensions of 4.6 × 250 mm and a particle size of 5 μm. 6) The concentration of the phosphoric acid aqueous solution is 0.1%~0.3%.

7. The construction method according to claim 1, characterized in that, Step 3 also includes: importing the high performance liquid chromatogram of the test sample solution into the "Chinese Medicine Fingerprint Similarity Evaluation System 2012 Edition" software for similarity calculation.

8. The construction method according to claim 1, characterized in that, The HPLC fingerprint of almond kernels includes 11 characteristic peaks, of which peak 6 is the characteristic peak of tryptophan, peak 10 is the characteristic peak of linoleic acid, and peak 11 is the characteristic peak of oleic acid. Taking peak 6 as the reference peak, the relative retention times of peaks 1-5, peaks 7-11 and peak S are consistent as follows: peak 1 is 0.46, peak 2 is 0.52, peak 3 is 0.56, peak 4 is 0.64, peak 5 is 0.94, peak 7 is 1.23, peak 8 is 1.71, peak 9 is 1.98, peak 10 is 3.28, and peak 11 is 3.41, with a relative standard deviation of ±10%.

9. The application of the HPLC fingerprint of almond kernel constructed by the construction method according to any one of claims 1 to 8 in the identification, quality control and quality evaluation of almond kernel medicinal materials and / or processed slices.

10. The application of the HPLC fingerprint of almond kernel constructed by the construction method according to any one of claims 1 to 8 in the chemical composition analysis of almond kernel.

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