A method for constructing an HPLC characteristic map of watermelon cream, application and quality detection method

Abstract

The application discloses a kind of construction methods, applications and quality detection methods of watermelon frost HPLC characteristic atlas, comprising the following steps: (1) amino acid is selected as reference, and reference solution is prepared;(2) watermelon frost test sample solution is prepared;(3) chromatographic conditions: with amino acid analysis special column as chromatographic column, with acetonitrile-0.1mol / L sodium acetate solution as mobile phase A, with acetonitrile-water as mobile phase B, the volume ratio of acetonitrile and 0.1mol / L sodium acetate solution in the mobile phase A is 7:93, the volume ratio of acetonitrile and water in the mobile phase B is 80:20;Gradient elution is carried out, and the theoretical plate number is not less than 150000 according to valine peak calculation;(4) reference solution and test sample solution are respectively taken, and injected into liquid chromatograph, and determined, and watermelon frost HPLC characteristic atlas is obtained.The method is stable and reliable, can control the quality of watermelon frost as a whole, guarantees product batch stability and consistency.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of pharmaceutical quality control, and particularly relates to a construction method, application and quality detection method of HPLC characteristic spectrum of watermelon frost. BACKGROUND

[0002] Watermelon frost is a kind of white to yellowish white crystalline powder prepared from the mature fresh fruit of Citrullus lanatus (Thunb.) Matsumu.et Nakai and nitre, and has the effects of clearing heat and reducing fire, eliminating swelling and relieving pain, and is used for treating sore throat, laryngeal swelling and pain, and aphtha.

[0003] As a raw medicinal material, watermelon frost is often combined with other effective medicinal ingredients or excipients to prepare medicinal compositions for treating or preventing oral and throat diseases, or oral health food, cleaning products, etc. For example, watermelon frost-containing tablets, watermelon frost throat moistening tablets, watermelon frost sprays, watermelon frost mouthwashes, watermelon frost throat moistening candies, etc.

[0004] The quality standard of watermelon frost (raw frost) generally refers to the detection method of watermelon frost in the first part of Chinese Pharmacopoeia 2020 edition. At present, the detection methods include thin layer chromatography identification method, content determination method for sodium sulfate, and heavy metal inspection.

[0005] The application number 201010300225.0 of the patent application filed by the applicant discloses a quality control method of watermelon frost, which comprises an identification method, an inspection method and a content determination method of watermelon frost. The identification method is one or more of amino acid, sodium salt and sulfate identification; the inspection is one or more of heavy metal inspection and arsenic salt inspection; and the content determination comprises total nitrogen content and sodium sulfate content.

[0006] However, the precision of the detection method in the existing standard needs to be improved, and there is no high performance liquid chromatography detection method for watermelon frost at present, and there is a lack of characteristic spectrum of watermelon frost. If a high performance liquid chromatography detection method for watermelon frost can be provided to construct a characteristic spectrum, it will be beneficial to improve the quality control of watermelon frost.

[0007] Therefore, the present application is proposed. SUMMARY

[0008] The present application aims to overcome the deficiencies of the prior art, and provides a construction method, application and quality detection method of HPLC characteristic spectrum of watermelon frost. The present application provides a high performance liquid chromatography detection method for watermelon frost, and for the first time establishes a characteristic spectrum of watermelon frost, which is beneficial to further improve the quality control standard of watermelon frost and lays a foundation for ensuring the quality and clinical efficacy of watermelon frost related products.

[0009] In order to solve the above technical problems, the basic concept of the technical solution adopted by the present invention is:

[0010] The first object of the present invention is to provide a method for constructing a watermelon frost HPLC characteristic spectrum, comprising the following steps:

[0011] (1) Select amino acids as controls and prepare reference solutions;

[0012] (2) preparing watermelon frost test solution;

[0013] (3) Chromatographic conditions: using a column specifically designed for amino acid analysis as the chromatographic column, acetonitrile-0.1 mol / L sodium acetate solution as the mobile phase A, and acetonitrile-water as the mobile phase B, wherein the volume ratio of acetonitrile to 0.1 mol / L sodium acetate solution in the mobile phase A is 7:93, and the volume ratio of acetonitrile to water in the mobile phase B is 80:20; performing gradient elution, the theoretical plate number calculated based on the valine peak is not less than 150,000; preferably, the pH value of mobile phases A and B is 6.40-6.60;

[0014] (4) The reference solution and the test solution were respectively aspirated and injected into a liquid chromatograph for determination to obtain the HPLC characteristic spectrum of watermelon frost.

[0015] In a further embodiment, the conditions for gradient elution of the mobile phase include:

[0016]

[0017] Alternatively, from 15 min to 76 min, the proportion of mobile phase A is overall increased or decreased by ≤2%, and the proportion of mobile phase B is overall decreased or increased by ≤2% accordingly.

[0018] The present applicant has found in experiments that when the proportion of the mobile phase in gradient elution is adjusted within a range of ≤2%, the results can meet the analytical requirements and there is no significant difference. When the proportion of the mobile phase is as shown in the above table, the separation effect is the best.

[0019] In a further embodiment, the chromatographic conditions also include: a detection flow rate of 0.78 mL / min to 0.82 mL / min; a detection wavelength of 254 nm; and a column temperature of 38 to 42°C.

[0020] In a further embodiment, the chromatographic column is selected from Welch Amino Acid columns, AgelaTechnologies Vensil AA columns, or Sepax AAA columns.

[0021] In a further embodiment, when injected into a liquid chromatograph, the injection volume is 10 μl.

[0022] In a further aspect, the amino acid control substances selected in step (1) include glutamic acid control substance, glycine control substance, arginine control substance, alanine control substance, valine control substance, isoleucine control substance, leucine control substance, phenylalanine control substance, and lysine hydrochloride control substance.

[0023] In a further aspect, the method for preparing the reference solution in step (1) includes: separately taking each amino acid control substance, and adding 0.1 mol / L hydrochloric acid solution to prepare a solution containing each amino acid; then taking a certain volume of the solution, adding 0.1 mol / L phenyl isothiocyanate acetonitrile solution and 1 mol / L triethylamine acetonitrile solution, shaking, reacting in a 50°C water bath, taking out and cooling, adding 50% acetonitrile, shaking, adding an equal volume of n-hexane, shaking, standing, taking the lower layer solution, filtering, and taking the subsequent filtrate to obtain the reference solution.

[0024] Preferably, each amino acid control substance is taken, and 0.1 mol / L hydrochloric acid solution is added to prepare a solution containing 10 μg / ml glutamic acid and alanine, 12 μg / ml arginine, 8 μg / ml glycine, valine, isoleucine, leucine, phenylalanine, and lysine hydrochloride; then a certain volume of the solution is taken, 0.5 times the solution volume of 0.1 mol / L phenyl isothiocyanate acetonitrile solution and 0.5 times the solution volume of 1 mol / L triethylamine acetonitrile solution are added, shaking, reacting in a 50°C water bath, taking out and cooling, adding 50% acetonitrile to a total volume, the total volume being 5 times the solution volume, shaking, adding an equal volume of n-hexane, shaking, standing, taking the lower layer solution, filtering, and taking the subsequent filtrate to obtain the reference solution.

[0025] As a specific embodiment, the method for preparing the reference solution includes:

[0026] The glutamic acid control substance, glycine control substance, arginine control substance, alanine control substance, valine control substance, isoleucine control substance, leucine control substance, phenylalanine control substance, and lysine hydrochloride control substance are separately taken, accurately weighed, and 0.1 mol / L hydrochloric acid solution is added to prepare a solution containing 10 μg / ml glutamic acid and alanine, 12 μg / ml arginine, 8 μg / ml glycine, valine, isoleucine, leucine, phenylalanine, and lysine hydrochloride per 1 ml. 5 ml of the above reference solution is accurately measured and placed in a 25 ml volumetric flask, 2.5 ml of 0.1 mol / L phenyl isothiocyanate acetonitrile solution and 2.5 ml of 1 mol / L triethylamine acetonitrile solution are added, shaking, reacting in a 50°C water bath for 1 hour, then taking out, cooling, adding 50% acetonitrile to the calibration mark, shaking, taking 10 ml, adding 10 ml of n-hexane, shaking, standing for 30 minutes, taking the lower layer solution, filtering, and taking the subsequent filtrate to obtain the reference solution.

[0027] Further, in step (2), the preparation method of the watermelon frost test sample solution comprises the following steps:

[0028] The watermelon frost sample is added with 6 mol / L hydrochloric acid solution, shaken uniformly, sealed with nitrogen, hydrolyzed at 110℃ for 12-48 hours, cooled, mixed uniformly, and filtered.

[0029] Preferably, the watermelon frost sample is added with 6 mol / L hydrochloric acid solution, shaken uniformly, sealed with nitrogen, hydrolyzed at 110℃ for 24 hours, cooled, mixed uniformly, and filtered.

[0030] Preferably, the watermelon frost sample is added with 6 mol / L hydrochloric acid solution, shaken uniformly, sealed with nitrogen, hydrolyzed at 110℃ for 24 hours, cooled, mixed uniformly, and filtered.

[0031] As a specific embodiment, the preparation method of the watermelon frost test sample solution comprises the following steps:

[0032] The second object of the present application is to provide an application of the construction method of the HPLC characteristic chromatogram of watermelon frost as described in the above scheme in the quality detection of watermelon frost raw materials.

[0033] The third object of the present application is to provide a quality detection method of watermelon frost raw materials, which comprises the following steps:

[0034] The HPLC chromatogram of the watermelon frost raw material to be detected is obtained by using the construction method as described in the above scheme, and is compared with the HPLC characteristic chromatogram of watermelon frost constructed by the construction method as described above.

[0035] Further, the quality detection method of watermelon frost raw materials comprises the following steps:

[0036] 1) The HPLC chromatogram of the watermelon frost raw material to be detected is obtained by using the construction method as described above, and the peak corresponding to the valine reference peak is taken as the S peak, and the relative retention time of various characteristic peaks and the S peak is calculated.

[0037] 2) comparing the relative retention time obtained in step 1) with the relative retention time of characteristic peaks in the HPLC characteristic spectrum of watermelon frost constructed by the construction method described above;

[0038] 3) judging whether the quality of the watermelon frost to be tested meets the standard according to the comparison result of step 2).

[0039] Preferably, in 3), it is determined based on the comparison result of step 2) whether the watermelon frost to be detected simultaneously contains the 9 characteristic peaks in the HPLC characteristic spectrum of watermelon frost constructed by the construction method described above. If so, it meets the standard; if not, it does not meet the standard.

[0040] After adopting the above technical solution, the present invention has the following beneficial effects compared with the prior art.

[0041] 1. The present invention establishes for the first time an HPLC detection method for watermelon frost (raw material frost) and constructs a characteristic spectrum of watermelon frost, which is conducive to further improving the quality control standard of watermelon frost, and is conducive to comprehensively evaluating the quality of watermelon frost drugs, laying the foundation for ensuring the quality and clinical efficacy of watermelon frost-related products.

[0042] 2. The present invention adopts high-performance liquid phase gradient elution method to study watermelon frost, establishes a characteristic spectrum, calibrates 9 common peaks by retention time for qualitative identification, and carries out methodological validation of the characteristic spectrum, proving that the method is stable and reliable, thereby controlling the quality of watermelon frost as a whole and ensuring the stability and consistency of product batches.

[0043] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The accompanying drawings are part of this invention and are used to provide a further understanding of the invention. The exemplary embodiments of the invention and their descriptions are used to explain the invention, but do not constitute an undue limitation of the invention. Obviously, the drawings described below are only some embodiments. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without inventive effort. In the accompanying drawings:

[0045] Figure 1 This is the reference characteristic spectrum of the present invention; wherein, peak 1 is glutamic acid; peak 2 is glycine; peak 3 is arginine; peak 4 is alanine; peak 5 (S) is valine; peak 6 is isoleucine; peak 7 is leucine; peak 8 is phenylalanine; and peak 9 is lysine hydrochloride.

[0046] Figure 2 This is the HPLC chromatogram of the acetonitrile-0.1 mol / L sodium acetate (7:93)-acetonitrile-water (80:20) solution (mobile phase) in 1.1.1;

[0047] Figure 3 is the high performance liquid chromatogram of the negative control sample solution in 1.1.1 without watermelon frost;

[0048] Figure 4 is the high performance liquid chromatogram of the 9 amino acid mixed standard solution in 1.1.1; wherein, peak 1: glutamic acid t R = 6.903 R = 2.675 T = 0.885 n = 6.160 x 10 3 ; peak 2: glycine t R = 13.993 R = 18.636 T = 0.928 n = 2.032 x 10 4 ; peak 3: arginine t R = 16.887 R = 7.145 T = 0.967 n = 2.742 x 10 4 ; peak 4: alanine t R = 19.387 R = 3.470 T = 0.962 n = 3.796 x 10 4 ; peak 5: valine t R = 28.490 R = 5.381 T = 0.957 n = 2.423 x 10 5 ; peak 6: isoleucine t R = 32.503 R = 3.961 T = 0.971 n = 3.041 x 10 5 ; peak 7: leucine t R = 32.961 R = 1.910 T = 0.948 n = 3.091 x 10 5 ; peak 8: phenylalanine t R = 35.188 R = 4.083 T = 0.944 n = 3.442 x 10 5 ; peak 9: lysine hydrochloride t R = 38.019 R = 1.500 T = 0.939 n = 2.619 x 10 5 ;

[0049] Figure 5 is the high performance liquid chromatogram of the 200723 batch solution of the test sample in 1.1.1; wherein, peak 1: glutamic acid t R = 6.964 T = 0.800 n = 5.764 x 10 3 ; peak 2: glycine t R = 14.021 R = 2.663 n = 1.888 x 10 4 ; peak 3: arginine t R = 16.911 R = 1.289 T = 0.951 n = 2.685 x 10 4Peak 4: Alanine t R =19.413 R=2.062 T=0.961 n=4.091×10 4 Peak 5: Valine t R =28.496 R=5.317 T=0.995 n=2.373×10 5 Peak 6: Isoleucine t R =32.493 R=1.559 n=2.208×10 5 Peak 7: Leucine t R =32.956 R=1.753 n=3.052×10 5 Peak 8: Phenylalanine t R =35.196 R=1.204 T=0.938 n=3.461×10 5 Peak 9: Lysine hydrochloride R =38.009 R=1.516 n=2.591×10 5 ;

[0050] Figure 6 This is the HPLC chromatogram of the negative control sample solution lacking watermelon frost in 1.1.2 using the mobile phase of acetonitrile-0.1 mol / L sodium acetate (7:93)-acetonitrile-methanol-water (60:20:20);

[0051] Figure 7 This is the HPLC chromatogram of the mixed standard solution of 9 amino acids in 1.1.2; Peak 1: Glutamic acid R =7.112T=0.904 n=4.446×10 3 Peak 2: glycine t R =14.603 R=15.266 T=0.859 n=1.184×10 4 Peak 3: Arginine t R =18.052 R=5.706 T=0.790 n=1.112×10 4 Peak 4: Alanine t R =20.647 R=2.973 T=0.880 n=2.631×10 4 Peak 5: Valine t R =30.748 R=4.929 T=0.862 n=1.701×10 5 Peak 6: Isoleucine t R =35.884 R=3.229 T=0.945 n=1.914×10 5 Peak 7: Leucine t R=36.497 R=1.787 T=0.777 n=1.821×10 5 Peak 8: Phenylalanine t R =39.827 R=1.510 T=0.867 n=1.373×10 5 Peak 9: Lysine hydrochloride R =44.241 R=6.965 T=0.875 n=1.088×10 5 ;

[0052] Figure 8 This is the HPLC chromatogram of the test sample 200723 batch solution in 1.1.2, where Peak 1: Glutamic acid t R =7.153 R=2.684 T=0.833 n=5.136×10 3 Peak 2: glycine t R =14.617 R=2.178 T=0.850n=1.347×10 4 Peak 3: Arginine t R =18.066 n=7.816×10 3 Peak 4: Alanine t R =20.657 R=2.387 T=0.936 n=2.986×10 4 Peak 5: Valine t R =30.743 R=1.906 T=1.324 n=1.571×10 5 Peak 6: Isoleucine t R =35.879 R=1.049 n=1.824×10 5 Peak 7: Leucine t R =36.485 R=1.665 n=1.625×10 5 Peak 8: Phenylalanine t R =39.595 R=0.729 n=2.390×10 4 Peak 9: Lysine hydrochloride R =44.240 R=7.002 T=1.073 n=1.396×10 5 ;

[0053] Figure 9 This is the HPLC chromatogram of the negative control sample solution lacking watermelon frost in 1.1.3 using the mobile phase of acetonitrile-0.05 mol / L sodium acetate (7:93)-acetonitrile-methanol-water (60:20:20);

[0054] Figure 10This is the HPLC chromatogram of the mixed standard solution of 9 amino acids in 1.1.3; Peak 1: Glutamic acid R =6.753 T=0.824 n=6.978×10 3 Peak 2: glycine t R =14.618 R=18.319 T=0.890 n=1.284×10 4 Peak 3: Arginine t R =18.469 R=6.676 T=0.811 n=1.340×10 4 Peak 4: Alanine t R =20.692 R=3.891 T=0.890 n=2.741×10 4 Peak 5: Valine t R =30.769 R=4.851 T=0.871n=1.726×10 5 Peak 6: Isoleucine t R =35.917 R=3.200 n=1.920×10 5 Peak 7: Leucine t R =36.532 R=1.799 n=1.849×10 5 Peak 8: Phenylalanine t R =39.886 R=1.347 n=1.420×10 5 Peak 9: Lysine hydrochloride R =44.273 R=7.633 T=0.902 n=1.086×10 5 ;

[0055] Figure 11 This is the HPLC chromatogram of the test sample 200723 batch solution in 1.1.3, where Peak 1: Glutamic acid t R =6.834 R=2.055 T=0.757 n=3.912×10 3 Peak 2: glycine t R =14.645 R=2.402 T=0.851n=1.514×10 4 Peak 3: Arginine t R =18.539 R=1.451 T=0.782 n=1.322×10 4 Peak 4: Alanine t R =20.724 R=2.270 T=0.968 n=3.195×10 4 Peak 5: Valine t R=30.780 R=4.243 T=1.479 n=1.313×10 5 Peak 6: Isoleucine t R =35.840 R=2.879 T=0.943 n=8.969×10 4 Peak 7: Leucine t R =36.539 R=1.657 n=1.747×10 5 Peak 8: Phenylalanine t R =39.617; Peak 9: Lysine hydrochloride t R =44.280 R=6.177 T=0.904 n=1.166×10 5 ;

[0056] Figure 12 This is the HPLC chromatogram of the negative control sample solution lacking watermelon frost in 1.1.4 using the mobile phase of acetonitrile-0.05 mol / L sodium acetate (7:93)-acetonitrile-water (80:20);

[0057] Figure 13 This is the HPLC chromatogram of the mixed standard solution of 9 amino acids in 1.1.4; Peak 1: Glutamic acid R =6.643 R=1.758 T=0.829 n=6.792×10 3 Peak 2: glycine t R =14.193 R=19.020 T=0.877 n=1.405×10 4 Peak 3: Arginine t R =17.609 R=6.469 T=0.820 n=1.533×10 4 Peak 4: Alanine t R =19.766 R=3.295 T=0.891 n=3.015×10 4 Peak 5: Valine t R =28.901 R=5.448 T=0.877n=2.128×10 5 Peak 6: Isoleucine t R =33.029 R=3.786 T=1.042 n=2.599×10 5 Peak 7: Leucine t R =33.496 R=1.841 T=0.859 n=2.726×10 5 Peak 8: Phenylalanine t R =35.804 R=3.643T=0.782 n=2.184×10 5Peak 9: Lysine hydrochloride R =38.875 R=1.854 T=0.854 n=2.015×10 5 ;

[0058] Figure 14 This is the HPLC chromatogram of the test sample 200723 batch solution in 1.1.4, Peak 1: Glutamic acid t R =6.602 R=2.233 T=1.107 n=3.631×10 3 Peak 2: glycine t R =14.222 R=2.323 T=0.880 n=1.479×10 4 Peak 3: Arginine t R =17.694 R=1.309 T=0.867 n=6.279×10 3 Peak 4: Alanine t R =19.795 R=2.343 T=0.919 n=3.119×10 4 Peak 5: Valine t R =28.898 R=4.688 T=0.871 n=2.061×10 5 Peak 6: Isoleucine t R =33.025 n=1.933×10 5 Peak 7: Leucine t R =33.487 R=1.652 n=2.453×10 5 Peak 8: Phenylalanine t R =35.798 n=2.051×10 5 Peak 9: Lysine hydrochloride R =38.865 R=1.781 T=0.856n=1.888×10 5 ;

[0059] Figure 15 This is the HPLC chromatogram of the sample 200723 batch solution in 1.2; Peak 1: Glutamic acid t R =7.082 R=3.267 T=0.894 n=1.055×10 4 Peak 2: glycine t R =14.370 R=3.243 T=0.966 n=2.909×10 4 Peak 3: Arginine t R =17.441 R=1.449 T=0.950 n=3.155×10 4 Peak 4: Alanine tR =19.974 R=2.884 T=0.989 n=4.723×10 4 Peak 5: Valine t R =28.798 R=4.540 T=1.003n=3.443×10 5 Peak 6: Isoleucine t R =32.813 R=2.098 T=0.755 n=3.384×10 5 Peak 7: Leucine t R =33.281 R=2.063 T=0.990 n=4.476×10 5 Peak 8: Phenylalanine t R =35.479 R=1.246T=0.876 n=2.788×10 5 Peak 9: Lysine hydrochloride R =38.416 R=1.745 T=0.946 n=3.341×10 5 ;

[0060] Figure 16 This is the maximum absorption spectrum of each characteristic peak of the test product 200723 batch watermelon frost in 1.2;

[0061] Figure 17 This is the purity chart of each characteristic peak of the test sample 200723 batch solution in 1.2;

[0062] Figure 18 It is Welch in 1.3 HPLC chromatogram of the solution of the test sample batch 200723 for Amino Acid detection;

[0063] Figure 19 This is the HPLC chromatogram of the sample batch 200723 solution tested by Agela Technologies Vensil AA in 1.3;

[0064] Figure 20 This is the HPLC chromatogram of the solution of sample batch 200723 tested by Sepax AAA in 1.3;

[0065] In the figures, the names of peaks 1 to 9 are the same as those in the Figure 1 .

[0066] It should be noted that these drawings and textual descriptions are not intended to limit the conceptual scope of the present invention in any way, but rather to illustrate the concept of the present invention for those skilled in the art by referring to specific embodiments. DETAILED DESCRIPTION

[0067] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. The following embodiments are used to illustrate the present invention but are not used to limit the scope of the present invention.

[0068] Example 1

[0069] 1. Instruments and reagents

[0070] WATERS e2695 high performance liquid chromatograph; WATERS 2489; WATERS 2998 detector.

[0071] Hydrochloric acid, triethylamine, phenyl isothiocyanate, n-hexane, and acetonitrile were of chromatographic grade, water was ultrapurified water, and other reagents were of analytical grade.

[0072] Glutamic acid reference substance (lot number: 140690-201604), arginine reference substance (lot number: 140685-201707, content 99.9%), alanine reference substance (lot number: 140680-201604), valine reference substance (lot number: 140681-201703, content 99.5%), isoleucine reference substance (lot number: 140683-201302, content 99.9%) , leucine reference substance (batch number: 140687-201905, content 99.9%), glycine reference substance (batch number: 140689-202006), phenylalanine reference substance (batch number: 140676-201706), lysine hydrochloride reference substance (batch number: 140673-201509, content 99.8%); all provided by the China Food and Drug Inspection Institute for amino acid identification and content determination.

[0073] 2. Solution preparation

[0074] Preparation of Reference Solution: Accurately weigh appropriate amounts of glutamic acid reference substance, glycine reference substance, arginine reference substance, alanine reference substance, valine reference substance, isoleucine reference substance, leucine reference substance, phenylalanine reference substance, and lysine hydrochloride reference substance. Add 0.1 mol / L hydrochloric acid solution to prepare a solution containing 10 μg each of glutamic acid and alanine, 12 μg of arginine, and 8 μg each of glycine, valine, isoleucine, leucine, phenylalanine, and lysine hydrochloride per 1 ml. Accurately measure 5 ml of the above reference solution and place it in a 25 ml volumetric flask. Add 2.5 ml of a 0.1 mol / L solution of phenyl isothiocyanate in acetonitrile and 2.5 ml of a 1 mol / L solution of triethylamine in acetonitrile. Shake well. Incubate in a 50°C water bath for 1 hour. Remove from heat, cool, and add 50% acetonitrile to the mark. Shake well. Take 10 ml, add 10 ml of n-hexane, shake, let stand for 30 minutes, take the lower layer solution, filter, and take the filtrate to obtain the product.

[0075] Preparation of test solution: Take 500 mg of watermelon frost in a 20 ml headspace bottle, accurately weigh it, accurately add 10 ml of 6 mol / L hydrochloric acid solution, shake evenly, blow nitrogen above the liquid surface of the headspace bottle for 120 seconds and then seal it. Hydrolyze at 110 ° C for 24 hours, cool, mix, open the bottle, filter, accurately measure 5 ml of the filtrate, evaporate to dryness below 60 ° C, add a little water if necessary, repeat the evaporation 1 to 2 times, dissolve the residue in 0.1 mol / L hydrochloric acid solution, transfer it to a 5 ml volumetric flask, add 0.1 mol / L hydrochloric acid solution to the scale, shake well, place it in a 25 ml volumetric flask, add 2.5 ml of 0.1 mol / L phenyl isothiocyanate in acetonitrile solution and 2.5 ml of 1 mol / L triethylamine in acetonitrile solution, shake well, react in a 50 ° C water bath for 1 hour, take out, cool, add 50% acetonitrile to the scale, and shake well. Take 10 ml, add 10 ml of n-hexane, shake, let stand for 30 minutes, take the lower layer solution, filter, and take the filtrate to obtain the product.

[0076] 3. Chromatographic conditions and system suitability test

[0077] The amino acid analysis column was used as the chromatographic column (Welch Amino Acid chromatographic column (column length, 250 mm, inner diameter, 4.6 mm, particle size, 5 μm); mobile phase A: acetonitrile-0.1 mol / L sodium acetate solution (dissolve 13.6 g of sodium acetate in 900 ml of water, adjust the pH to 6.5 with acetic acid, and add water to 1000 ml) (7:93); mobile phase B: acetonitrile-water (80:20); gradient elution as specified in Table 1; flow rate, 0.8 ml / min; detection wavelength, 254 nm; column temperature, 40°C. The number of theoretical plates, calculated based on the valine peak, should be no less than 150,000.

[0078] Table 1

[0079]

[0080] Determination method: Accurately aspirate 10μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0081] The control characteristic spectrum of the present invention is as follows Figure 1 The characteristic spectrum of the test sample should also show 9 characteristic peaks, and the retention time should be consistent with that of the reference substance.

[0082] The following is the methodological investigation and results

[0083] 1. Chromatographic conditions

[0084] 1.1 Selection of mobile phase:

[0085] 1.1.1 Use acetonitrile-0.1 mol / L sodium acetate solution (adjust pH to 6.5 with acetic acid) (7:93) as mobile phase A, and acetonitrile-water (80:20) as mobile phase B. The gradient elution conditions for the mobile phases are the same as those in Table 1.

[0086] Column: Welch Amino Acid 5μm(4.6×250mm)

[0087] Flow rate: 0.8 mL / min Column temperature: 40°C

[0088] Accurately weigh appropriate amounts of glutamate reference substance, glycine reference substance, arginine reference substance, alanine reference substance, valine reference substance, isoleucine reference substance, leucine reference substance, phenylalanine reference substance, and lysine hydrochloride reference substance, and prepare reference solution according to the preparation method of Example 1. Then accurately weigh 0 mg of watermelon frost test sample and 500 mg of test sample, and prepare sample solution according to the preparation method of test solution in Example 1 for determination.

[0089] Results: The negative control sample lacking watermelon frost had no absorption peaks at the corresponding positions of the glutamic acid, glycine, arginine, alanine, valine, isoleucine, leucine, and lysine hydrochloride reference peaks, indicating that the negative sample had no interference and the chromatographic peak separation effect of the test sample was good. The HPLC chromatograms of the mobile phase, the negative control sample lacking watermelon frost, the 9 amino acid mixed standards, and the test sample are shown in Figure 2. Figures 2 to 5 .

[0090] 1.1.2 Use acetonitrile-0.1 mol / L sodium acetate solution (adjust pH to 6.5 with acetic acid) (7:93) as mobile phase A and acetonitrile-methanol-water (60:20:20) as mobile phase B. Use the gradient elution conditions in Table 1. Inject the reference substance, negative control sample, and test solution prepared in 1.1.1 into the liquid chromatograph for determination.

[0091] Results: The negative control sample lacking watermelon frost had no absorption peaks at the corresponding positions of the glutamic acid, glycine, arginine, alanine, valine, isoleucine, leucine, phenylalanine, and lysine hydrochloride reference peaks, indicating that the negative sample had no interference. However, under this condition, the peak width of each amino acid chromatographic peak of the 9 amino acid mixed standard became larger, the peak shape of each amino acid chromatographic peak in the test sample became worse, and the separation was poor. The HPLC chromatograms of the negative control sample lacking watermelon frost, the 9 amino acid mixed standard, and the test sample are shown in Figure 2. Figures 6-8 .

[0092] 1.1.3 Use acetonitrile-0.05 mol / L sodium acetate solution (adjust pH to 6.5 with acetic acid) (7:93) as mobile phase A and acetonitrile-methanol-water (60:20:20) as mobile phase B. Use the gradient elution conditions in Table 1. Inject the reference solution, negative control sample, and test solution prepared in 1.1.1 into the liquid chromatograph for determination.

[0093] Results: The negative control sample lacking watermelon frost had no absorption peaks at the corresponding positions of the glutamic acid, glycine, arginine, alanine, valine, isoleucine, leucine, phenylalanine, and lysine hydrochloride reference peaks, indicating that the negative sample had no interference. However, under this condition, the peak width of each amino acid chromatographic peak of the 9 amino acid mixed standard became larger, the peak shape of each amino acid chromatographic peak in the test sample became worse, and the separation was poor. The HPLC chromatograms of the negative control sample lacking watermelon frost, the 9 amino acid mixed standard, and the test sample are shown in Figure 2. Figures 9-11 .

[0094] 1.1.4 Use acetonitrile-0.05 mol / L sodium acetate solution (adjust pH to 6.5 with acetic acid) (7:93) as mobile phase A and acetonitrile-water (80:20) as mobile phase B. Use the gradient elution conditions in Table 1. Inject the reference substance, negative control sample, and test solution prepared in 1.1.1 into the liquid chromatograph for determination.

[0095] Results: The negative control sample lacking watermelon frost had no absorption peaks at the corresponding positions of the glutamic acid, glycine, arginine, alanine, valine, isoleucine, leucine, and lysine hydrochloride reference peaks, indicating that the negative sample had no interference. However, under this condition, the peak width of each amino acid chromatographic peak of the 9 amino acid mixed standard became slightly larger, the peak shape of each amino acid chromatographic peak in the test sample became worse, and the separation of the phenylalanine chromatographic peak was poor. The HPLC chromatograms of the negative control sample lacking watermelon frost, the 9 amino acid mixed standard, and the test sample are shown in Figure 2. Figures 12-14 .

[0096] 1.2 Selection of detection wavelength

[0097] Use acetonitrile-0.1mol / L sodium acetate solution (adjust pH to 6.5 with acetic acid) (7:93) as mobile phase A and acetonitrile-water (80:20) as mobile phase B. Use the gradient elution conditions in Table 1 for the mobile phase. Inject the reference substance and test solution prepared in 1.1.1 into the liquid chromatograph for determination. Detection is performed using a PDA detector (210-400nm). The maximum absorption spectra of each characteristic peak are shown in the attached figure. Figure 15-16The maximum absorption wavelength of each amino acid characteristic peak is around 247nm, among which the maximum absorption wavelength of the main characteristic peak of valine is 248.6nm. Since the valine peak is an S peak, and the detection wavelength of the chromatographic conditions of the pre-column PITC derivatization amino acid determination method is 254nm, there is no significant difference with the maximum absorption wavelength of each amino acid determined. Comprehensive analysis shows that 254nm is used as the inspection wavelength of the characteristic spectrum. The absorption values ​​of each characteristic peak are appropriate, the baseline is relatively stable, and the separation is good.

[0098] In summary, the mobile phase was determined to be acetonitrile-0.1 mol / L sodium acetate solution (pH adjusted to 6.5 with acetic acid) (7:93) as mobile phase A, acetonitrile-water (80:20) as mobile phase B, the flow rate was 0.8 ml / min, the detection wavelength was 254 nm, and the column temperature was 40°C.

[0099] 1.3 Determination of the number of theoretical plates

[0100] Column 1: Welch Three brands of chromatographic columns were tested, including Amino Acid 5μm 4.6×250mm, Agela Technologies Vensil AA 5μm 4.6×250mm, and Column 3: Sepax AAA 5μm 4.6×250mm. The separation results were all good. The theoretical plate numbers were calculated based on the valine peak of the measured component. The results are shown in Table 2, and the chromatograms are shown in Table 2. Figure 18 、 19 , 20.

[0101] Table 2 Determination of the number of theoretical plates

[0102]

[0103] According to the test results, when the theoretical plate number N is lower than 150,000, the separation degree between the valine peak and its adjacent chromatographic peak is less than 1.5, which does not meet the analysis requirements. Therefore, it is stipulated that the theoretical plate number calculated based on the valine peak should not be lower than 150,000.

[0104] 2. Specificity test

[0105] 2.1 Negative interference test: Preparation of negative control sample solution lacking watermelon frost: Take 0 mg of watermelon frost and prepare a sample solution according to the preparation method of the test solution in Example 1 to obtain a negative control sample solution lacking watermelon frost.

[0106] Use acetonitrile-0.1mol / L sodium acetate solution (adjust pH to 6.5 with acetic acid) (7:93) as mobile phase A, acetonitrile-water (80:20) as mobile phase B, and the gradient elution conditions in Table 1. Inject the reference substance, negative control sample, and test solution prepared in 1.1.1 into the liquid chromatograph for determination.

[0107] Results: The negative control sample lacking watermelon frost had no absorption peaks at the corresponding positions of the glutamic acid, glycine, arginine, alanine, valine, isoleucine, leucine, and lysine hydrochloride reference peaks, indicating that the negative sample had no interference. The chromatograms are shown in Figure 2. Figures 3-5 .

[0108] 2.2 Peak purity test: acetonitrile-0.1mol / L sodium acetate solution (adjust pH to 6.5 with acetic acid) (7:93) was used as mobile phase A, acetonitrile-water (80:20) was used as mobile phase B, and the mobile phase adopted the gradient elution conditions in Table 1. The test solution prepared in 1.1.1 was injected into the liquid chromatograph for determination. The results showed that the purity values ​​of the characteristic peaks of glutamic acid, glycine, arginine, alanine, valine, isoleucine, leucine, phenylalanine and lysine hydrochloride were all lower than the automatic threshold value, indicating that there were no overlapping peaks. The chromatograms are shown in Table 1. Figure 15 、 17 .

[0109] 3. Preparation of test solution

[0110] Take 500mg of watermelon frost in a 20ml headspace bottle, accurately weigh it, accurately add 10ml of 6mol / L hydrochloric acid solution, shake evenly, blow nitrogen above the liquid surface of the headspace bottle for 120 seconds and then seal it. Hydrolyze at 110℃ for 24 hours, cool, mix, open the bottle, filter, accurately measure 5ml of the filtrate, evaporate to dryness below 60℃, add a little water if necessary, repeat the evaporation 1 to 2 times, dissolve the residue in 0.1mol / L hydrochloric acid solution, transfer it to a 5ml volumetric flask, add 0.1mol / L hydrochloric acid solution to the scale, shake well, place it in a 25ml volumetric flask, add 2.5ml of 0.1mol / L phenyl isothiocyanate in acetonitrile solution and 2.5ml of 1mol / L triethylamine in acetonitrile solution, shake well, react in a 50℃ water bath for 1 hour, take it out, cool, add 50% acetonitrile to the scale, and shake well. Take 10 ml, add 10 ml of n-hexane, shake, let stand for 30 minutes, take the lower layer solution, filter, and take the filtrate to obtain the product.

[0111] Hydrolysis time of the test sample: Watermelon Frost (Batch No. 200723) was hydrolyzed for 12, 24, 36, and 48 hours. Test solutions were prepared according to the above-described test solution preparation method. The prepared test solutions were injected into a liquid chromatograph and analyzed. The results are shown in Table 3. The sum of the peak areas of the nine characteristic amino acid peaks of the test solutions prepared under different hydrolysis time conditions was calculated. As shown in the table, there was no significant difference in the sum of the peak areas of the nine characteristic amino acid peaks among the test solutions prepared under different hydrolysis time conditions. The ranking from highest to lowest was: 36 hours of hydrolysis, 48 ​​hours of hydrolysis, 24 hours of hydrolysis, and 12 hours of hydrolysis. Considering the simplicity of the experimental operation and cost-effectiveness, the hydrolysis time for this product was determined to be 24 hours.

[0112] Table 3 Test results of different hydrolysis times

[0113]

[0114] 4. Precision test

[0115] This product (batch number: 200723) was used to prepare a test solution according to the method of Example 1. The injection volume was 10 μl and the samples were injected six times continuously. The results are shown in Table 4. The peak corresponding to the valine reference peak was designated as the S peak, and the relative retention time of each characteristic peak was calculated. As can be seen from the table, the relative retention time RSD of each characteristic peak was less than 3% when the test solution was measured on the same instrument, indicating that the precision of the HPLC instrument used can meet the test detection requirements.

[0116] Table 4 Precision test results

[0117]

[0118]

[0119] 5. Repeatability test

[0120] Watermelon frost samples from the same batch (Batch No. 200723) were mixed uniformly and six aliquots were accurately weighed to prepare test solutions. The samples were injected separately. The results are shown in Table 5. The peak corresponding to the valine reference peak was designated the S peak, and the relative retention time of each characteristic peak was calculated. As shown in the table, the RSDs for the relative retention times of each characteristic peak were less than 3%, demonstrating good reproducibility.

[0121] Table 5 Repeatability test results

[0122]

[0123]

[0124] 6. Durability test

[0125] 6.1 Stability test:

[0126] Take watermelon frost test solution (batch number: 200723), according to the chromatographic conditions of Example 1, respectively, at time intervals of 0, 4, 8, 12, 16, 24, 30, and 36 hours, respectively, 10 μl, the results are shown in Table 6. The peak corresponding to the valine reference substance peak is the S peak, and the relative retention time of each characteristic peak is calculated. As can be seen from the table, the relative retention time RSD of each characteristic peak is less than 3% when the test solution is measured within 36 hours. The test results show that the test solution is relatively stable when measured within 36 hours.

[0127] Table 6 Stability test results

[0128]

[0129] 6.2 Investigation of different chromatographic columns:

[0130] Take three batches of watermelon frost samples (batch numbers: 200706, 200723, 200807), and prepare the test solution according to the determination method of Example 1. The test was conducted using three brands of chromatographic columns: Amino Acid 5μm 4.6×250mm, Column 2: Agela Technologies Vensil AA 5μm 4.6×250mm, and Column 3: Sepax AAA 5μm 4.6×250mm. The peak corresponding to the valine reference peak was designated as the S peak, and the relative retention time of each characteristic peak was calculated. The results are shown in Table 7. The results demonstrate that all three selected columns meet the system suitability requirements of the method.

[0131] Table 7 Investigation results of chromatographic columns from different manufacturers / brands

[0132]

[0133] 6.3 Investigation of different mobile phase ratios:

[0134] The same batch number sample (Batch No. 200723) was taken and prepared according to the determination method of Example 1. The reference solution and the test solution were prepared. The mobile phase ratio was adjusted downward by 2%. The mobile phase gradient elution conditions are shown in Table 8. The samples were injected into the liquid chromatograph respectively. The peak corresponding to the valine reference peak was taken as the S peak. The relative retention time of each characteristic peak was calculated. The determination results are shown in Table 9. The test results show that the overall mobile phase ratio was reduced by 2%, and the determination results met the analysis requirements and there was no significant difference.

[0135] Table 8 Mobile phase gradient elution conditions

[0136]

[0137]

[0138] Column: Welch Amino Acid 5μm(4.6×250mm)

[0139] Flow rate: 0.8 mL / min Column temperature: 40°C

[0140] Table 9 Investigation results of different mobile phase ratios

[0141]

[0142] 6.4 Investigation of mobile phase pH value:

[0143] Take the same batch number sample (batch number: 200723), according to the determination method of Example 1, prepare the test solution, respectively with acetonitrile-0.1mol / L sodium acetate solution (with acetic acid to adjust the pH value to 6.40) (7:93), acetonitrile-0.1mol / L sodium acetate solution (with acetic acid to adjust the pH value to 6.50) (7:93), acetonitrile-0.1mol / L sodium acetate solution (with acetic acid to adjust the pH value to 6.60) (7:93) as mobile phase A, with acetonitrile-water (80:20) as mobile phase B, respectively injected into the liquid chromatograph. The peak corresponding to the valine reference substance peak is the S peak, and the relative retention time of each characteristic peak is calculated respectively. The determination results are shown in Table 10. The test results show that the mobile phase pH value fluctuates within the range of 6.40 to 6.60, and the determination results can meet the analysis requirements and there is no significant difference.

[0144] Table 10 Comparative test results of different mobile phase pH values

[0145]

[0146] 6.5 Investigation of different column temperatures:

[0147] Samples from the same batch (Batch No. 200723) were prepared using the assay method described in Example 1. The column temperatures were set at 38°C, 40°C, and 42°C, respectively, and the samples were injected into the liquid chromatograph. The peak corresponding to the valine reference peak was designated the S peak, and the relative retention time of each characteristic peak was calculated. The results are shown in Table 11. The results showed that the results met the analytical requirements within the column temperature range of 38-42°C, with no significant differences.

[0148] Table 11 Comparative test results at different column temperatures

[0149]

[0150] 6.6 Investigation of different flow rates:

[0151] Take the same batch number sample (batch number: 200723), according to the determination method of Example 1, prepare the test solution, set the flow rate to 0.78mL / min, 0.80mL / min, and 0.82mL / min, respectively, and inject it into the liquid chromatograph respectively. The peak corresponding to the valine reference substance peak is the S peak, and the relative retention time of each characteristic peak is calculated respectively. The determination results are shown in Table 12. The test results show that the flow rate fluctuates within the range of 0.78mL / min to 0.82mL / min, and the determination results can meet the analysis requirements and there is no significant difference.

[0152] Table 12 Comparative test results at different flow rates

[0153]

[0154] 7. Sample determination

[0155] According to the characteristic spectrum determination method of Example 1, Welch Twenty samples were measured using an Amino Acid column, and 15 samples were measured using an Agela Technologies Vensil AA column. The peak corresponding to the valine reference peak was designated as the S peak. The relative retention time of each characteristic peak was calculated. The results are shown in Tables 13 and 14.

[0156] The results further illustrate that the HPLC determination method of Example 1 of the present application is stable and reliable, which is beneficial for controlling the quality of watermelon frost as a whole and ensuring the stability and consistency of product batches.

[0157] Table 13 Test results of 20 batches of samples

[0158]

[0159] Table 14 Agela column 15 batches of sample determination results

[0160]

[0161] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as above in terms of a preferred embodiment, it is not intended to limit the present invention. Any technician familiar with this patent can make slight changes or modifications to equivalent embodiments using the above technical content without departing from the scope of the technical solution of the present invention. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the solution of the present invention.

Claims

1. A method for constructing an HPLC characteristic spectrum of watermelon frost, characterized in that: The steps include: (1) Select amino acids as reference substances and prepare reference substance solutions; amino acids selected as reference substances include glutamic acid reference substance, glycine reference substance, arginine reference substance, alanine reference substance, valine reference substance, isoleucine reference substance, leucine reference substance, phenylalanine reference substance, and lysine hydrochloride reference substance; (2) Prepare watermelon frost test solution, including: Take a watermelon frost sample, add 6mol / L hydrochloric acid solution, shake evenly, seal with nitrogen, hydrolyze at 110℃ for 12-48 hours, cool, mix, and filter; take the filtrate, evaporate to dryness below 60℃, dissolve the residue in 0.1mol / L hydrochloric acid solution, shake well, add 0.1mol / L phenyl isothiocyanate acetonitrile solution and 1mol / L triethylamine acetonitrile solution, shake well, react in a 50℃ water bath, take out and cool, add 50% acetonitrile, shake well, add an equal volume of n-hexane, shake, let stand, take the lower layer solution, filter, take the filtrate, and obtain the test solution; (3) Chromatographic conditions: The chromatographic column was selected from Welch Ultimate ® Amino Acid chromatographic column, Agela Technologies Vensil AA column or Sepax AAA column; acetonitrile-0.1 mol / L sodium acetate solution as mobile phase A, acetonitrile-water as mobile phase B, the volume ratio of acetonitrile to 0.1 mol / L sodium acetate solution in mobile phase A being 7:93, and the volume ratio of acetonitrile to water in mobile phase B being 80:20; gradient elution with a theoretical plate number of not less than 150,000 calculated based on the valine peak; the pH value of mobile phase A being 6.40-6.60; detection wavelength being 254 nm; The conditions for gradient elution with mobile phase include: Alternatively, from 15 min to 76 min, the proportion of mobile phase A is increased by ≤2% overall, and the proportion of mobile phase B is correspondingly decreased by ≤2% overall; Alternatively, from 15 min to 76 min, the proportion of mobile phase A is decreased by ≤2% overall, and the proportion of mobile phase B is correspondingly increased by ≤2% overall; (4) Aspirate the reference solution and the test solution separately, inject them into the liquid chromatograph, and measure them to obtain the HPLC characteristic spectrum of watermelon frost.

2. The construction method according to claim 1, characterized in that The chromatographic conditions also include: a detection flow rate of 0.78 mL / min to 0.82 mL / min; and a column temperature of 38 to 42°C.

3. The construction method according to claim 1 or 2, characterized in that In step (1), the preparation method of the reference solution includes: taking each amino acid reference substance separately, adding 0.1 mol / L hydrochloric acid solution to prepare a solution containing each amino acid; then taking a certain volume of the solution, adding 0.1 mol / L phenyl isothiocyanate acetonitrile solution and 1 mol / L triethylamine acetonitrile solution, shaking, reacting in a 50°C water bath, taking out and cooling, adding 50% acetonitrile, shaking, adding an equal volume of n-hexane, shaking, allowing to stand, taking the lower layer of solution, filtering, and taking the filtrate to obtain the reference solution.

4. The construction method according to claim 1 or 2, characterized in that In step (1), each amino acid reference substance is taken separately, and 0.1 mol / L hydrochloric acid solution is added to prepare a solution containing 10 μg / ml glutamic acid and alanine, 12 μg / ml arginine, 8 μg / ml glycine, valine, isoleucine, leucine, phenylalanine and lysine hydrochloride; then a certain volume of the solution is taken, 0.5 times the volume of the solution is added with 0.1 mol / L phenyl isothiocyanate in acetonitrile solution and 0.5 times the volume of the solution is added with 1 mol / L triethylamine in acetonitrile solution, shaken, reacted at 50°C, taken out and cooled, 50% acetonitrile is added to the total volume, the total volume is 5 times that of the solution, after shaking, an equal volume of n-hexane is added, shaken, allowed to stand, the lower layer of solution is taken, filtered, and the filtrate is taken to obtain the reference solution.

5. The construction method according to claim 1 or 2, characterized in that: In step (2), Take a watermelon frost sample, add 6mol / L hydrochloric acid solution, shake evenly, seal with nitrogen, hydrolyze at 110℃ for 24 hours, cool, mix, and filter; take the filtrate, evaporate to dryness below 60℃, dissolve the residue in 0.1mol / L hydrochloric acid solution, and shake to obtain a solution; add 0.5 times the volume of the solution of 0.1mol / L phenyl isothiocyanate in acetonitrile solution and 0.5 times the volume of the solution of 1mol / L triethylamine in acetonitrile solution, shake well, react at 50℃, take out and cool, add 50% acetonitrile to the total volume, the total volume is 5 times that of the solution, shake evenly, add an equal volume of n-hexane, shake, let stand, take the lower layer of solution, filter, and take the filtrate to obtain the test solution.

6. Use of the method for constructing the HPLC characteristic spectrum of watermelon frost according to any one of claims 1 to 5 in quality detection of watermelon frost raw materials.

7. A method for detecting watermelon frost raw materials, characterized in that: include: The HPLC spectrum of the watermelon frost raw material to be detected is obtained by the construction method according to any one of claims 1-5, and the HPLC spectrum is compared with the HPLC characteristic spectrum of the watermelon frost constructed by the construction method according to any one of claims 1-5.

8. The detection method according to claim 7, characterized in that The steps include: 1) Obtaining an HPLC spectrum of the watermelon frost raw material to be tested using the construction method described in any one of claims 1 to 5, and using the peak corresponding to the valine reference peak as the S peak, calculating the relative retention time of various characteristic peaks and the S peak; 2) comparing the relative retention time obtained in step 1) with the relative retention time of characteristic peaks in the HPLC characteristic spectrum of watermelon frost constructed by the construction method according to any one of claims 1 to 5; 3) judging whether the quality of the watermelon frost raw material to be tested meets the standard according to the comparison result of step 2).

9. The detection method according to claim 8, characterized in that In step 3), it is determined based on the comparison result of step 2) whether the watermelon frost raw material to be tested also contains the characteristic peaks in the watermelon frost HPLC characteristic spectrum constructed by the construction method according to any one of claims 1 to 5. If so, it meets the standard; if not, it does not meet the standard.

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