A method for constructing a millet sprout characteristic map and application thereof

Abstract

The application belongs to the technical field of pharmaceutical analysis, and particularly relates to a construction method of a millet sprout characteristic spectrum and application. The construction method comprises preparation of a test sample solution, determination by high performance liquid chromatography, and use of methanol as a mobile phase A, water as a mobile phase B, and a specific gradient elution procedure. The characteristic spectrum of the millet sprout obtained by the construction method has a smooth baseline, good separation degree between characteristic peaks, can display more chemical components, and is helpful for comprehensive research on chemical components of the millet sprout. Further, the construction method is also helpful for identification of the millet sprout from easily confused products, such as differentiation of the millet sprout from malt, rice sprout and the like.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of pharmaceutical analysis, and particularly relates to a construction method of a millet sprout characteristic spectrum and application. BACKGROUND

[0002] Millet sprout is a processed product of mature fruits of Setaria italica (L.) Beauv. of the family Poaceae, which is soaked in water, dried under appropriate temperature and humidity, and dried when the radicles are about 6 mm long. Millet sprout is used for food digestion in clinic. There is little research on its chemical composition, and there is no characteristic spectrum to comprehensively display the chemical composition of millet sprout.

[0003] In addition, millet sprout is similar to malt and rice sprout, and it is difficult to distinguish millet sprout from malt and rice sprout. How to effectively distinguish millet sprout from malt and rice sprout is also of great significance for clinical application. SUMMARY

[0004] Therefore, the technical problem to be solved by the application is to overcome the defects in the prior art, such as little research on the chemical composition of millet sprout, so as to provide a construction method of a millet sprout characteristic spectrum and application.

[0005] To this end, the application provides the following technical solutions.

[0006] The application provides a construction method of a millet sprout characteristic spectrum, comprising the following steps:

[0007] Preparation of a test sample solution: the test sample is prepared into a test sample solution;

[0008] Determination: high performance liquid chromatography is used for determination.

[0009] The chromatographic conditions comprise: methanol as mobile phase A, water as mobile phase B, and gradient elution; wherein the gradient elution program is: 0-5 min, 0% mobile phase A, 100% mobile phase B; 5-30 min, 0→1% mobile phase A, 100→99% mobile phase B; 30-60 min, 1→15% mobile phase A, 99→85% mobile phase B.

[0010] The chromatographic conditions further comprise: octadecylsilane-bonded silica gel as a filler, column length 250 mm, inner diameter 4.6 mm, and particle size 5.0 μm; and / or,

[0011] The column temperature is 28-32℃; and / or,

[0012] The wavelength is 260-280 nm; and / or,

[0013] The flow rate is 0.9-1.1 ml / min; and / or,

[0014] The injection amount is 5-15 μL.

[0015] The chromatographic conditions include: column temperature of 30℃; and / or, wavelength of 260nm; and / or, flow rate of 1.0ml / min; and / or, injection volume of 10μL.

[0016] The test sample is at least one of the following: millet sprout decoction pieces, millet sprout formula granules, millet sprout standard decoction freeze-dried powder and millet sprout medicinal materials.

[0017] The preparation method of the test sample solution comprises: taking the test sample, adding a solvent, extracting, and filtering;

[0018] Preferably, the solvent for extraction is an organic solvent.

[0019] Preferably, the solvent for extraction is 10%-50% methanol.

[0020] Preferably, the ratio of the mass (g) of the test sample to the volume (ml) of the solvent is (0.01-0.02):1.

[0021] The construction method further comprises preparation of a reference solution.

[0022] Further, in the preparation of the reference solution, the ratio of the mass (g) of the reference to the volume (ml) of the extraction solvent is (0.04-0.08):1.

[0023] The characteristic spectrum obtained by the construction method comprises 8 characteristic peaks.

[0024] Taking peak No. 6 as the reference peak, the specified values of the relative retention times of peak No. 2, peak No. 3, peak No. 4 and peak No. 7 are 0.35, 0.45, 0.54 and 1.68 respectively.

[0025] Among them, the relative retention time of the characteristic peak is within ±10% of the specified value.

[0026] The ratio of the peak area of peak No. 1 to the peak area of peak No. 6 is not less than 0.44.

[0027] Preferably, peak No. 1 is uracil, peak No. 3 is hypoxanthine, peak No. 5 is uridine, peak No. 6 is adenine, peak No. 7 is tryptophan, and peak No. 8 is adenosine.

[0028] The construction method further comprises the step of preparing a control solution.

[0029] Preferably, the control is at least one of the following: uracil, uridine, adenine and adenosine.

[0030] The present application also provides a method for identifying millet sprout and confused products, comprising using the above construction method.

[0031] Preferably, the adulterants include at least one of rice sprout and wheat sprout.

[0032] Preferably, when the ratio of the peak area of peak No. 1 to the peak area of peak No. 6 in the characteristic spectrum obtained by the construction method is not less than 0.44, the sample to be tested is a millet sprout.

[0033] The technical scheme of the present application has the following advantages:

[0034] 1. The present application provides a construction method of a millet sprout characteristic spectrum, which comprises preparation of a test sample solution, determination by high performance liquid chromatography, using methanol as mobile phase A, water as mobile phase B, and a specific gradient elution program. The construction method can obtain a millet sprout characteristic spectrum with a smooth baseline and good separation degree between characteristic peaks, which can exhibit more chemical components and is helpful for comprehensive research on the chemical components of millet sprout. Further, the construction method is also helpful for distinguishing millet sprout from its easily confused products, such as wheat sprout and rice sprout. BRIEF DESCRIPTION OF DRAWINGS

[0035] In order to more clearly illustrate the specific embodiments of the present application or the technical scheme in the prior art, the following will briefly introduce the drawings needed to be used in the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0036] Figure 1 is the characteristic spectrum of 18 batches of millet sprout standard decoction freeze-dried powder; Figure 2 is the control spectrum obtained by fitting; Figure 3 is the spectrum of millet sprout control medicinal material reference; Figure 4 is the spectrum of the control solution; Figure 5 is the characteristic spectrum of 3 batches of millet sprout formula granules; Figure 6 is the characteristic spectrum of the control solution when verifying whether the chromatographic conditions and system suitability of the standard decoction of millet sprout decoction pieces are applicable to millet sprout formula granules; Figure 7 is the spectrum of millet sprout formula granules when verifying whether the chromatographic conditions and system suitability of the standard decoction of millet sprout decoction pieces are applicable to millet sprout formula granules; Figure 8 is the spectrum obtained by different gradient elution programs; Figure 9 is the investigation result at different wavelengths; Figure 10 is the spectrum obtained by investigation of different mobile phases; Figure 11 is the spectrum obtained by investigation of different flow rates; Figure 12 is the spectrum obtained by investigation of different column temperatures; Figure 13 is the spectrum obtained by investigation of different extraction solvents; Figure 14 is the spectrum for precision investigation in the methodological investigation;Figure 15 is a plot of the repeatability investigation in the methodological investigation; Figure 16 is a plot of the intermediate precision investigation in the methodological investigation; Figure 17 is a plot of the specificity investigation in the methodological investigation; Figure 18 is a plot of the stability investigation in the methodological investigation; Figure 19 is a plot of the column temperature robustness investigation in the methodological investigation; Figure 20 is a plot of the malt sprout standard infusion freeze-dried powder and the malt standard infusion freeze-dried powder; Figure 21 is a plot of the malt sprout standard infusion freeze-dried powder and the rice sprout standard infusion freeze-dried powder. DETAILED DESCRIPTION

[0037] The following examples are provided to better enable those skilled in the art to further understand and practice the application, and are not intended to limit the scope of the application or the content of the disclosure. Any product that is the same or similar to the present application that is obtained by the disclosure of the present application or by combining the present application with other prior art features falls within the scope of the present application. If the specific experimental steps or conditions are not mentioned in the examples, the operation or conditions can be carried out according to the conventional experimental steps described in the literature in the art. If the reagents or instruments used are not mentioned by the manufacturer, they are conventional reagent products that can be obtained by purchase.

[0038] Instrument

[0039] Waters e2695 chromatographic system includes a four-element solvent manager (Alliance), a sample manager (Alliance), a chromatographic column oven (Alliance), a diode array ultraviolet detector (2998-PDA Detector), and an Empower chromatographic management system. Electronic balance: METTLER TOLEDO (Switzerland Mettler-Toledo) XPE56 (one millionth); XS204, XSE205, XS205 (one hundred thousandth); Sartorius (Sartorius) ME36S (one millionth). Ultrasonic instrument: Kunshan Ultrasonic Instrument Co., Ltd. KQ-500DE digital ultrasonic instrument. Chromatographic column: Waters Atlantis T3 C18, 4.6x250mm, 5μm.

[0040] Reagents

[0041] Methanol: chromatographically pure, water is purified water, and other reagents are analytical pure; Uracil control batch number: 100469-201302, purchased from China Institute for Drug Control, purity 99.6%; Hypoxanthine control batch number: 140661-202005, purchased from China Institute for Drug Control, purity 99.4%; Uridine control batch number: 110887-202104, purchased from China Institute for Drug Control, purity 99.6%; Adenine control batch number: 110886-201102, purchased from China Institute for Drug Control, purity 99.4%; Tryptophan control batch number: 140686-201904, purchased from China Institute for Drug Control, purity 99.9%; Adenosine control batch number: 110879-201703, purchased from China Institute for Drug Control, purity 99.7%;

[0042] Sprout control drug material: batch number 270034-202109, purchased from Shanghai Hongyong Biological Technology Co., Ltd.; Sprout formula granules: 2208001Y, 2208002Y, 2208003Y; 18 batches of sprout decoction standard freeze-dried powder batch number: 2103001Y, 2103002Y, 2103003Y, 2103004Y, 2103006Y, 2103007Y, 2103010Y, 2103011Y, 2103012Y, 2103013Y, 2103014Y, 2103015Y, 211101Y, 211102Y, 211103Y, respectively, S1-S18.

[0043] Example 1

[0044] The present embodiment provides a method for constructing a characteristic map of sprout, comprising the following steps:

[0045] Preparation of test sample solution: Take a suitable amount of sprout decoction standard freeze-dried powder, grind it finely, take about 0.5g, accurately weigh and place it in a conical flask with a stopper, accurately add 25ml of 20% methanol, weigh the weight, ultrasonic treatment (power 250W, frequency 40kHz) for 30 minutes, cool down, weigh again, make up the weight loss with 20% methanol, shake well, filter, and obtain the solution.

[0046] Preparation of reference solution: Take 2g of sprout control drug material, place it in a conical flask with a stopper, add 50ml of water, heat reflux for 30 minutes, take it out, cool down, filter, evaporate the filtrate to dryness, add 25ml of 20% methanol, tightly stop, ultrasonic treatment (power 250W, frequency 40kHz) for 30 minutes, take it out, cool down, shake well, filter, take the filtrate, and use it as the reference solution of the control drug material.

[0047] Preparation of the control solution: Take the appropriate amount of uracil control, hypoxanthine control, uridine control, adenine control, tryptophan control, adenosine control, and accurately weigh, add 20% methanol solution to make a mixture solution containing 30 μg of uracil, 20 μg of hypoxanthine, 30 μg of uridine, 20 μg of adenine, 20 μg of tryptophan, and 20 μg of adenosine per 1 ml, as the control solution.

[0048] Determination: 10 μL of the test solution, the reference solution of the medicinal material, and the control solution were respectively injected into the liquid chromatograph for determination; wherein, the chromatographic conditions included: methanol as mobile phase A, water as mobile phase B, gradient elution; wherein, the gradient elution program: 0-5 min, 0% mobile phase A, 100% mobile phase B; 5-30 min, 0→1% mobile phase A, 100→99% mobile phase B; 30-60 min, 1→15% mobile phase A, 99→85% mobile phase B; the flow rate was 1.0 ml / min, the column temperature was 30°C, and the detection wavelength was 260 nm.

[0049] Example 2

[0050] The present embodiment provides a method for constructing the characteristic chromatogram of millet sprout formula granules, comprising the following steps:

[0051] Preparation of the test solution: Take the appropriate amount of millet sprout formula granules, grind finely, take about 0.5 g, accurately weigh, place in a conical flask with a plug, accurately add 25 ml of 20% methanol, weigh, ultrasonic treatment (power 250 W, frequency 40 kHz) for 30 minutes, cool, re-weigh, make up the weight loss with 20% methanol, shake well, and filter to obtain.

[0052] Preparation of the reference solution: Take 2 g of the control medicinal material of millet sprout, place in a conical flask with a plug, add 50 ml of water, heat reflux for 30 minutes, take out, cool, filter, evaporate the filtrate to dryness, add 25 ml of 20% methanol, tightly plug, ultrasonic treatment (power 250 W, frequency 40 kHz) for 30 minutes, take out, cool, shake well, filter, take the filtrate, as the reference solution of the control medicinal material.

[0053] Preparation of the control solution: Take the appropriate amount of uracil control, hypoxanthine control, uridine control, adenine control, tryptophan control, adenosine control, and accurately weigh, add 20% methanol solution to make a mixture solution containing 30 μg of uracil, 20 μg of hypoxanthine, 30 μg of uridine, 20 μg of adenine, 20 μg of tryptophan, and 20 μg of adenosine per 1 ml, as the control solution.

[0054] Determination: precisely pipette 10 μL of the test sample solution, the reference sample solution of the control medicinal material and the control substance solution respectively, inject into the liquid chromatograph, and determine; wherein, the chromatographic conditions include: using methanol as mobile phase A and water as mobile phase B for gradient elution; wherein, the gradient elution procedure is as follows: 0-5 min, 0% mobile phase A and 100% mobile phase B; 5-30 min, 0→1% mobile phase A and 100→99% mobile phase B; 30-60 min, 1→15% mobile phase A and 99→85% mobile phase B; the flow rate is 1.0 ml / min, the column temperature is 30°C, and the detection wavelength is 260 nm.

[0055] Determination of characteristic chromatogram

[0056] Take 18 batches of standard freeze-dried powder of millet sprout decoction, prepare the test sample solution according to the method of Example 1, and determine, the results are shown in the following table and Figure 1 . Among them, the control chromatogram is obtained by Mark fitting, see Figure 2 ; the reference sample chromatogram of the control medicinal material of millet sprout is shown in Figure 3 ; and the chromatogram of the control substance solution is shown in Figure 4 .

[0057] Table 1 Determination results of relative retention time of characteristic chromatogram of 18 batches of standard freeze-dried powder of millet sprout decoction

[0058]

[0059]

[0060] Table 2 Determination results of relative peak area of characteristic chromatogram of 18 batches of standard freeze-dried powder of millet sprout decoction

[0061]

[0062]

[0063] Table 3 Relative retention time of standard decoction control chromatogram of millet sprout decoction pieces

[0064] Number Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 (S) Peak 7 Peak 8 Retention time 7.185 9.949 12.746 15.424 17.897 28.505 47.769 57.012 Relative retention time 0.25 0.35 0.45 0.54 0.63 1.00 1.68 2.00

[0065] Table 4 Relative peak area of standard decoction control chromatogram of millet sprout decoction pieces

[0066] Number Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 (S) Peak 7 Peak 8 Peak area 154.55 34.669 210.289 58.016 205.521 99.357 103.055 165.881 Relative peak area 1.556 0.349 2.116 0.584 2.069 1.000 1.037 1.670

[0067] Eight common peaks in the characteristic chromatogram of 18 batches of standard decoction pieces of Gu Yaji were selected as characteristic peaks, and the retention time of the chromatographic peaks in the reference chromatogram of the control drug material was corresponded. By comparing the chromatogram of the control solution, peak 1, peak 3, peak 5, peak 6, peak 7 and peak 8 corresponded to the retention time of uracil, hypoxanthine, uridine, adenine, tryptophan and adenosine respectively, so it was confirmed that peak 1 was uracil, peak 3 was hypoxanthine, peak 5 was uridine, peak 6 was adenine, peak 7 was tryptophan and peak 8 was adenosine. The peak corresponding to peak 6 adenine was S peak, the relative retention time of each characteristic peak to S peak was calculated, and the relative retention time of each characteristic peak was within ± 10% of the specified value. The specified values of the relative retention time of peak 2, peak 3, peak 4 and peak 7 were 0.35, 0.45, 0.54 and 1.68 respectively.

[0068] Verification of the chromatographic conditions and system suitability of Gu Yaji formula granules

[0069] Three batches of Gu Yaji formula granules were taken, the test solution was prepared according to Example 2 and determined, and the results are shown in the following table and Figure 5 ; the results showed that the relative retention time and relative peak area of the characteristic chromatogram obtained from different batches of Gu Yaji formula granules were within the required range.

[0070] Table 5 Relative retention time results of three batches of Gu Yaji formula granules

[0071]

[0072] Table 6 Relative peak area results of three batches of Gu Yaji formula granules

[0073]

[0074]

[0075] Another Gu Yaji formula granules was prepared into test solution according to Example 2 and determined, to verify whether the chromatographic conditions and system suitability of Gu Yaji decoction pieces standard decoction were suitable for Gu Yaji formula granules. The results are shown in the following table and Figure 6-7 .

[0076] Table 7 Results of control experiment

[0077] Name Retention time Area Height Width Separation Symmetry factor Theoretical plates Uracil 7.252 1355904 158937 52 - 1.035 16950 Hypoxanthine 12.941 329043 24656 56.7 20.073 0.976 22072 Uridine 18.103 330585 18183 70.2 12.643 0.958 23287 Adenine 29.044 696601 24299 114.4 18.094 1.041 24462 Tryptophan 47.861 150021 7027 66.6 28.995 0.958 115248 Adenosine 57.238 294737 19743 58.2 19.824 0.931 346256

[0078] Table 8 Chromatogram of Gu Yaji formula granules

[0079] Peak number Name Retention time Area Height Width Separation Symmetry factor Theoretical plates 1 Uracil 7.248 49168 5485 36 1.01 15854 2 2 10.063 16371 1417 46.6 10.694 0.969 18634 3 Hypoxanthine 12.876 129635 9333 52.1 8.549 1.025 20171 4 4 15.667 45431 2898 47 7.262 1.004 22892 5 Uridine 18.063 166436 9239 63.8 5.451 0.967 23375 6 Adenine 29.122 57496 2142 77.7 18.929 1.099 26939 7 Tryptophan 47.806 32430 1574 50.8 29.941 1.022 120391 8 Adenosine 57.01 203783 13731 56.6 19.735 0.925 346861

[0080] The characteristic chromatogram of Gu Yaji formula granules has good system suitability, therefore, the construction method of Gu Yaji standard decoction is suitable for the characteristic chromatogram of Gu Yaji formula granules.

[0081] Investigation of gradient elution procedure in experimental example 1

[0082] This experimental example investigated different gradient elution procedures, specifically including: the same batch of millet sprout standard decoction freeze-dried powder was prepared into test sample solution according to Example 1, with gradient elution procedure as the variable, detection wavelength was 270 nm, column temperature was 40℃, and other methods were determined according to Example 1. The results are shown in Table Figure 8 , and the gradient elution procedures and flow rates are as follows:

[0083] (1) Gradient elution procedure 1

[0084] Time (min) Flow rate (ml / min) Mobile phase A (%) Mobile phase B (%) 0 1.0 0 100 5 1.0 0 100 25 1.0 1 99 40 1.0 15 85 50 1.0 0 100 60 1.0 0 100

[0085] (2) Gradient elution procedure 2:

[0086] Time (min) Flow rate (ml / min) Mobile phase A (%) Mobile phase B (%) 0 1.0 0 100 15 1.0 0 100 20 1.0 1 99 40 1.0 15 85 50 1.0 0 100 60 1.0 0 100

[0087] (3) Gradient elution procedure 3:

[0088] Time (min) Flow rate (ml / min) Mobile phase A (%) Mobile phase B (%) 0 1.0 0 100 15 1.0 0 100 40 1.0 5 95 50 1.0 5 95 55 1.0 0 100

[0089] (4) Gradient elution procedure 4:

[0090] Time (min) Mobile phase A (methanol) Mobile phase B (water) 0 0 100 5 0 100 30 1 99 31 5 95 55 15 85 60 90 10 61 0 100 70 0 100

[0091] (5) Gradient elution procedure 5:

[0092] Time (min) Mobile phase A (methanol) Mobile phase B (water) 0 0 100 5 0 100 60 15 85 70 90 10 71 0 100 80 0 100

[0093] (6) Gradient elution procedure 6:

[0094] Time (min) Mobile phase A (methanol) Mobile phase B (water) 0 0 100 5 0 100 30 1 99 60 15 85 70 90 10 71 0 100 80 0 100

[0095] The results show that gradient elution procedures 1-3 are compared, and the symmetry of the characteristic chromatographic peaks obtained by gradient elution procedure 3 is better and the baseline is more stable. The comparison results of gradient elution procedures 3, 4, 5 and 6 show that the separation degree of each chromatogram in the characteristic chromatogram obtained by gradient elution procedure 6 is higher, and the baseline is more stable, therefore, gradient elution procedure 6 is preferred. Since the chromatographic peaks in the chromatogram appear within 60 min, 60-80 min is to return to the original gradient change for the next needle determination, therefore, 60-80 min is omitted.

[0096] Investigation of wavelength in experimental example 2

[0097] This experimental example investigated the wavelength, specifically including: the millet sprout standard decoction freeze-dried powder test sample solution was prepared according to Example 1, 3D full wavelength scanning was used, and 4 wavelengths with more peak information were selected, as shown in Figure 9 , from top to bottom in the figure correspond to wavelengths of 300 nm, 280 nm, 260 nm and 230 nm respectively.

[0098] The optimal wavelength was determined according to the number of chromatographic peaks and the peak height. The results show that the peak information amount of the characteristic spectrum obtained at a wavelength of 260 nm is relatively large, and the response values of the various chromatographic peaks are relatively high, and thus the wavelength is preferably 260 nm.

[0099] Experimental Example 3 Investigation of Mobile Phase

[0100] In this experimental example, different mobile phases were investigated. Specifically, the same batch of standard decoction powder of millet sprouts was taken, and a test solution was prepared according to Example 1, and the mobile phase was taken as the variable. The mobile phase 1 was a 0.1% phosphoric acid aqueous solution-methanol system; the mobile phase 2 was a 0.07% triethylamine-methanol system; and the mobile phase 3 was a water-methanol system. Other conditions were determined according to the method of Example 1, and the results are shown in Table 1. Figure 10 .

[0101] The information amount of the detected chromatographic peaks and the system adaptability parameters were taken as the selection indexes. The results show that the baseline of the spectrum obtained by the mobile phase 3 is relatively stable, and the symmetry is relatively optimal, and thus the mobile phase 3 is preferably selected.

[0102] Experimental Example 4 Investigation of Flow Rate

[0103] In this experimental example, different flow rates were investigated. Specifically, the same batch of standard decoction powder of millet sprouts was taken, and a test solution was prepared according to Example 1, and the flow rate was taken as the variable. The flow rates were 0.9 ml / min, 1.0 ml / min and 1.1 ml / min, respectively. Other conditions were determined according to the method of Example 1, and the results are shown in Table 2, from top to bottom corresponding to 1.1 ml / min, 1.0 ml / min and 0.9 ml / min, respectively. From the above results, it can be seen that different flow rates have no effect on the retention time and peak area of the chromatographic peaks, and the separation degree between the various chromatographic peaks is good, and all are suitable for the present application. The commonly used 1.0 ml / min is preferably selected. Figure 11

[0104] Experimental Example 5 Investigation of Column Temperature

[0105] In this experimental example, different column temperatures were investigated. Specifically, the same batch of standard decoction powder of millet sprouts was taken, and a test solution was prepared according to Example 1, and the column temperature was taken as the variable. The column temperatures were 25°C, 30°C and 35°C, respectively. Other conditions were determined according to the method of Example 1, and the results are shown in Table 3. Figure 12 From the above results, it can be seen that when the column temperature is 30°C, the symmetry of the various chromatographic peaks in the spectrum is the best, and the interference from the surrounding small chromatographic peaks is relatively small. Therefore, the column temperature is preferably 30°C.

[0106] ​According to the above content, the preferred chromatographic condition is as follows: octadecylsilane-bonded silica gel is used as the filler (the column length is 250 mm, the inner diameter is 4.6 mm, and the particle size is 5.0 μm); methanol-water is used as the mobile phase, the column temperature is 30 °C, the detection wavelength is 260 nm, and the gradient elution procedure is as follows: 0-5 min, 0% mobile phase A and 100% mobile phase B; 5-30 min, 0→1% mobile phase A and 100→99% mobile phase B; and 30-60 min, 1→15% mobile phase A and 99→85% mobile phase B.

[0107] Experimental Example 6 Investigation of the Extraction Solvent of the Test Solution

[0108] In this experimental example, different extraction solvents of the test solution were investigated, specifically including: the same batch of the standard decoction powder of wheat germ was used to prepare the test solution according to Example 1, and the extraction solvent used to prepare the test solution was used as the variable, and the extraction solvent was 10% methanol, 20% methanol, 30% methanol, 50% methanol, 70% methanol, 10% ethanol, 20% ethanol, 30% ethanol, 50% ethanol, and 70% ethanol, respectively, and other operations were performed according to the method of Example 1, and the results are shown in the following table and Figure 1. Figure 13 .

[0109] Table 9 Comparison of the chromatographic peak system suitability parameters of different extraction solvents

[0110]

[0111]

[0112]

[0113] The experimental results show that a higher concentration of the extraction solution can affect the peak shape of the chromatographic peak, and in combination with the peak shape and the peak area, 20% methanol is preferably used as the extraction solvent of the test solution.

[0114] Experimental Example 7 Investigation of the Extraction Method of the Test Solution

[0115] In this experimental example, different extraction methods were investigated, specifically including: the same batch of the standard decoction powder of wheat germ was used to prepare the test solution according to Example 1, and the extraction method was used as the variable, and other operations were performed according to Example 1; the extraction methods included ultrasonic extraction and heating reflux extraction, and the details are as follows:

[0116] The ultrasonic extraction was the same as that in Example 1; the steps of the heating reflux extraction included: about 0.5 g of the standard decoction powder of wheat germ was accurately weighed, placed in a conical flask with a plug, 25 ml of 20% methanol was accurately added, the weight was determined, heated for reflux for 30 min, cooled, the weight was determined again, the lost weight was made up with 20% methanol, shaken uniformly, filtered, and the test solution was obtained.

[0117] Table 10 Comparison of chromatographic peak system suitability parameters of extraction methods

[0118]

[0119]

[0120] From the above experimental results, it can be seen that the ultrasonic extraction and heating reflux extraction have little effect on the system suitability parameters of the characteristic spectrum, and the ultrasonic extraction is preferred for the convenience of sample processing.

[0121] Experimental Example 8 Investigation of extraction time

[0122] In this experimental example, the extraction time of different test sample solutions was investigated. Specifically, the same batch of millet sprout standard decoction lyophilized powder was prepared into a test sample solution according to Example 1, and the extraction time (i.e. ultrasonic time) was used as a variable, with ultrasonic times of 15 min, 30 min, 45 min and 60 min, respectively. Other parameters were determined according to the method of Example 1, and the results are as follows:

[0123] Table 11 Comparison of chromatographic peak system suitability parameters of extraction time

[0124]

[0125]

[0126] The experimental results show that within the range of 15-60 min of extraction time, the differences in the total peak area of the characteristic peaks are small, and each characteristic component can be completely extracted. Considering the extraction efficiency, the extraction time of 30 min is preferred.

[0127] Experimental Example 9 Investigation of the amount of extraction solvent

[0128] In this experimental example, the different amounts of extraction solvent were investigated. Specifically, the same batch of millet sprout standard decoction lyophilized powder was prepared into a test sample solution according to Example 1, with an amount of test sample of about 0.5 g, and 20% methanol was used as a variable, with volumes of 25 ml and 50 ml, respectively. Other parameters were determined according to Example 1, and the results are as follows:

[0129] Table 12 Comparison of chromatographic peak system suitability parameters of solvent amount

[0130]

[0131] The experimental results show that within the range of 25-50 ml of extraction solvent, as the amount of solvent increases, the peak area of the characteristic peaks decreases proportionally, and each characteristic component can be completely extracted. Considering the response value of the characteristic peaks, 25 ml is preferred as the amount of extraction solvent.

[0132] Experimental Example 10 Methodology investigation

[0133] (1) Precision of the instrument

[0134] The same batch of Gu Yama formula granules (batch number 2208001Y) was taken, and the relative retention time and relative peak area of each common peak were determined according to Example 2, which was repeated 6 times. The results are shown in the following table and Figure 14 ; The results showed that the RSD of the relative retention time and relative peak area of each characteristic peak to the reference material S peak was small, indicating that the instrument precision was good.

[0135] Table 13 Instrument precision relative retention time test results

[0136]

[0137] Table 14 Instrument precision relative peak area test results

[0138]

[0139] (2) Reproducibility

[0140] The same batch of Gu Yama formula granules (batch number 2208001Y) was taken, and 6 test sample solutions were prepared according to Example 2, and the relative retention time and relative peak area of each common peak were determined. The results are shown in the following table and Figure 15 ; The results showed that the RSD of the relative retention time and relative peak area of each characteristic peak to the reference material S peak was small, indicating that the reproducibility was good.

[0141] Table 15 Reproducibility relative retention time test results

[0142]

[0143]

[0144] Table 16 Reproducibility relative peak area test results

[0145]

[0146] (3) Intermediate precision of different operators

[0147] The same batch of Gu Yama formula granules (batch number 2208001Y) was taken, and test sample solutions were prepared by 2 different inspectors at different times according to Example 2, and the relative retention time and relative peak area of each common peak were determined using the same equipment. Each inspector did the experiment 2 times, and there were 4 groups of results, which are shown in the following table and Figure 16 ; The results showed that the RSD of the relative retention time and relative peak area of each characteristic peak to the reference material S peak was small, indicating that the intermediate precision was good.

[0148] Table 17 Intermediate precision relative retention time test results

[0149]

[0150] Table 18 Intermediate precision relative peak area test results

[0151]

[0152]

[0153] (4) Specificity investigation

[0154] The millet formula granule test sample solution and negative granule solution (with maltodextrin as a negative control) were prepared according to Example 2 to investigate whether the negative granule would cause interference, as shown in Figure 17 The experimental results showed that the test sample blank control had no interference with the characteristic spectrum, and could be used as a detection method for the characteristic spectrum of the millet formula granule.

[0155] (5) Stability investigation

[0156] Several test sample solutions were prepared from the same batch of millet formula granules according to Example 2, and were injected at 0, 2, 4, 8, 16, 18, and 24 hours, respectively, to determine the relative retention time and relative peak area of the common peaks, as shown in the following table and Figure 18 The results showed that the relative retention time and relative peak area of each characteristic peak had small RSD, and the test sample solution was stable within 24 h, meeting the determination requirements.

[0157] Table 19 Stability relative retention time test results

[0158]

[0159] Table 20 Stability relative peak area test results

[0160]

[0161]

[0162] (6) Column temperature durability investigation

[0163] The same batch of millet formula granules was taken to prepare a test sample solution according to Example 2, which was determined at column temperatures of 28℃, 30℃, and 32℃, respectively, to investigate the influence of column temperature on the relative retention time and relative peak area of the characteristic peaks, as shown in the following table and Figure 19 , from top to bottom in the figure correspond to 32℃, 30℃, and 28℃, respectively. The results showed that when the column temperature changed, it had little effect on the separation effect of the characteristic peaks, and the present application had good durability at different column temperatures.

[0164] Table 21 Relative retention time results at different column temperatures

[0165]

[0166] Table 22 Relative peak area results at different column temperatures

[0167]

[0168] Example 3

[0169] The present example provides a method for identifying millet sprouts and adulterants, including rice sprouts and wheat sprouts, comprising the following steps:

[0170] Preparation method of millet sprout standard decoction lyophilized powder test sample solution: take an appropriate amount of millet sprout standard decoction lyophilized powder, finely grind, take about 0.5 g, accurately weigh and place in a conical flask with a stopper, accurately add 20% methanol 25 ml, weigh, ultrasonic treatment (power 250 W, frequency 40 kHz) for 30 minutes, cool, re-weigh, make up the weight loss with 20% methanol, shake well, filter, and obtain.

[0171] Preparation method of rice sprout standard decoction lyophilized powder test sample solution: take an appropriate amount of rice sprout standard decoction lyophilized powder, finely grind, take about 0.5 g, accurately weigh and place in a conical flask with a stopper, accurately add 20% methanol 25 ml, weigh, ultrasonic treatment (power 250 W, frequency 40 kHz) for 30 minutes, cool, re-weigh, make up the weight loss with 20% methanol, shake well, filter, and obtain. Three rice sprout standard decoction lyophilized powder test sample solutions are prepared according to this method.

[0172] Preparation method of wheat sprout standard decoction lyophilized powder test sample solution: take an appropriate amount of wheat sprout standard decoction lyophilized powder, finely grind, take about 0.5 g, accurately weigh and place in a conical flask with a stopper, accurately add 20% methanol 25 ml, weigh, ultrasonic treatment (power 250 W, frequency 40 kHz) for 30 minutes, cool, re-weigh, make up the weight loss with 20% methanol, shake well, filter, and obtain. Six wheat sprout standard decoction lyophilized powder test sample solutions are prepared according to this method.

[0173] Accurately pipette 10 μL of each of the above test sample solutions into a liquid chromatograph for determination; wherein the chromatographic conditions include: methanol as mobile phase A, water as mobile phase B, gradient elution; wherein the gradient elution program is: 0-5 min, 0% mobile phase A, 100% mobile phase B; 5-30 min, 0→1% mobile phase A, 100→99% mobile phase B; 30-60 min, 1→15% mobile phase A, 99→85% mobile phase B; flow rate is 1.0 ml / min, column temperature is 30°C, and detection wavelength is 260 nm. The results are shown in Figure 20-21 and the following table.

[0174] Table 23. Results of relative retention time determination of characteristic spectra of germinated rice and rice sprout standard decoctions (freeze-dried powder)

[0175]

[0176] Table 24 Characteristic Spectra of Germinated Rice and Rice Sprout Standard Decoction (Freeze-dried Powder) | Result of Relative Peak Area Measurement |

[0177]

[0178]

[0179] Figure 20 S1 in the figure is the characteristic spectrum of the freeze-dried powder of germinated rice decoction, and S2-S7 are the characteristic spectra of the freeze-dried powder of maltated rice decoction. Figure 21 S1 in the figure is the characteristic spectrum of the freeze-dried powder of germinated rice standard decoction, and S2-S4 are the characteristic spectra of the freeze-dried powder of germinated rice standard decoction. Figure 20-21 The ratio of the peak area of ​​peak 1 to peak 6 in the germinated rice spectrum is between 0.468 and 5.772; the ratio of the peak area of ​​peak 1 to peak 6 in the malted wheat spectrum is between 0.124 and 0.249; and the ratio of the peak area of ​​peak 1 to peak 6 in the germinated rice spectrum is between 0.339 and 0.423.

[0180] When distinguishing between germinated rice and malt, the ratio of the peak areas of peak 1 and peak 6 in the spectrum of germinated rice and malt can be differentiated. Germinated rice and malt can be distinguished by stipulating that the relative peak area of ​​peak 1 and peak 6 should not be less than 0.44.

[0181] When distinguishing between germinated rice and sprouted rice, the chromatogram of sprouted rice shows a higher chromatographic peak between peaks 4 and 5, while the chromatogram of germinated rice does not show this chromatographic peak between peaks 4 and 5. Furthermore, the ratio of the peak areas of peaks 1 and 6 in the chromatograms of germinated rice and sprouted rice are somewhat different. Germinated rice and sprouted rice can be distinguished by stipulating that the relative peak area of ​​peaks 1 and 6 should not be lower than 0.44.

[0182] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for constructing a characteristic map of a malt sprout, characterized by, The method comprises the following steps: Preparation of test sample solution: the test sample is prepared into a test sample solution; Determination: using high performance liquid chromatography to determine; The chromatographic conditions comprise: using octadecylsilane-bonded silica gel as the filler; using methanol as the mobile phase A and water as the mobile phase B for gradient elution; wherein, the gradient elution program is: 0-5 min, 0% mobile phase A, 100% mobile phase B; 5-30 min, 0→1% mobile phase A, 100→99% mobile phase B; 30-60 min, 1→15% mobile phase A, 99→85% mobile phase B; the wavelength is 260-280 nm; The test sample is at least one of the standard decoction of millet sprout slices, the formula granules of millet sprout, the freeze-dried powder of standard decoction of millet sprout and the medicinal material of millet sprout; When the test sample is the standard decoction of millet sprout slices, the formula granules of millet sprout or the freeze-dried powder of standard decoction of millet sprout, the extraction solvent used in the preparation of the test sample solution is 10%-50% methanol; When the test sample is the medicinal material of millet sprout, the preparation method of the test sample solution is: taking 2 g of the control medicinal material of millet sprout, placing it in a conical flask with a plug, adding 50 ml of water, heating for reflux for 30 minutes, taking it out, cooling, filtering, evaporating the filtrate to dryness, then adding 25 ml of 20% methanol into the residue, tightly plugging, performing ultrasonic treatment with the power of 250 W and the frequency of 40 kHz for 30 minutes, taking it out, cooling, shaking uniformly, filtering, and taking the filtrate; The control sample comprises: uracil, hypoxanthine, uridine, adenine, tryptophan and adenosine.

2. The construction method of claim 1, wherein, The chromatographic conditions further comprise: the specification of the chromatographic column is: column length 250 mm, inner diameter 4.6 mm, and particle size 5.0 μm; and / or, The column temperature is 28-32 ℃; and / or, The flow rate is 0.9-1.1 ml / min; and / or, The injection amount is 5-15 μL.

3. The construction method of claim 1, wherein, The chromatographic conditions comprise: the column temperature is 30 ℃; and / or, the wavelength is 260 nm; and / or, the flow rate is 1.0 ml / min; and / or, the injection amount is 10 μL.

4. The construction method according to any one of claims 1 to 3, characterized in that, When the test sample is the standard decoction of millet sprout slices, the formula granules of millet sprout or the freeze-dried powder of standard decoction of millet sprout, the preparation method of the test sample solution comprises: taking the test sample, adding a solvent, extracting, and filtering; The ratio of the mass of the test sample to the volume of the solvent is 0.01-0.02:1, the unit of the mass is g, and the unit of the volume of the solvent is ml.

5. The construction method according to any one of claims 1 to 3, characterized in that, The method further comprises the preparation of a reference solution; When the reference solution is prepared, the ratio of the mass of the reference to the volume of the extraction solvent is 0.04-0.08:1, the unit of the mass is g, and the unit of the volume of the solvent is ml.

6. The construction method according to any one of claims 1 to 3, characterized in that, The characteristic spectrum obtained by the construction method comprises eight characteristic peaks; Taking peak No. 6 as the reference peak, the specified values of the relative retention time of peak No. 2, peak No. 3, peak No. 4 and peak No. 7 are respectively: 0.35, 0.45, 0.54 and 1.68; The relative retention time of the characteristic peak is within ±10% of the specified value.

7. The construction method of claim 6, wherein, The ratio of the peak area of peak No. 1 to the peak area of peak No. 6 is not less than 0.44; Peak No. 1 is uracil, peak No. 3 is hypoxanthine, peak No. 5 is uridine, peak No. 6 is adenine, peak No. 7 is tryptophan, and peak No. 8 is adenosine.

8. The construction method of claim 7, wherein, The method further comprises the step of preparing a control sample solution.

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