Method for constructing characteristic maps of malt and its preparations and their uses

Abstract

The invention belongs to the technical field of traditional Chinese medicine detection, and specifically provides a method for constructing a characteristic spectrum of malt and a preparation thereof and a use thereof. In the method for constructing the characteristic spectrum of malt and the preparation thereof, octadecylsilane bonded silica gel is used as a filler, a mobile phase is a potassium dihydrogen phosphate aqueous solution-methanol with a pH value of 3.4-3.6 and a concentration of 0.02-0.08 mol / L, 218-222 nm is selected as a detection wavelength, 0.15-0.22 ml / min is selected as a flow rate, 33-37 DEG C is selected as a column temperature, and a specific gradient elution program is adopted to obtain multiple common characteristic peaks, and achieve good separation of these common characteristic peaks. The obtained characteristic spectrum has a stable baseline, good characteristic peak shape, high separation, and short detection time, providing a basis for quality detection and control of malt and the preparation thereof, achieving characterization of the overall components of malt and the preparation thereof, and having stable reproducibility.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a method for constructing a characteristic spectrum of malt and a preparation thereof and uses thereof. Background Art

[0002] Malt is obtained by germinating and drying the mature fruit of Hordeum vulgare L., a grass plant. Malt is spindle-shaped, 8-12 mm long and 3-4 mm in diameter. It has a pale yellow surface, with a five-veined lemma on the dorsal surface and palea on the ventral surface. After removing the palea and palea, a longitudinal groove is observed on the ventral surface. At the base of the radicle, sprouts and fibrous roots emerge. The sprouts are lanceolate and approximately 0.5 cm long. The fibrous roots are numerous, slender, and curved. The texture is hard, white in cross section, and powdery. It is odorless and has a slightly sweet taste. After soaking the grains in water and maintaining a suitable temperature and humidity, sun-dry or low-temperature dry them when the sprouts reach approximately 0.5 cm. Malt's main functions and indications include: digestion and elimination of food stagnation; and lactation. This includes indigestion of staple food; abdominal distension and diarrhea; nausea and vomiting; loss of appetite; milk stagnation; and breast tenderness.

[0003] The 2020 edition of the Chinese Pharmacopoeia only includes properties, microscopic identification and thin-layer chromatography identification. So far, there is no clear chemical component index to evaluate malt medicinal materials. Simply measuring the amount of one or several active ingredients can no longer fully reflect the quality of the medicinal materials. Chinese herbal formula granules are extracted from Chinese herbal medicine slices with water and produced by extraction, vacuum concentration, spray drying, granulation and other processes. Their clinical efficacy should be consistent with that of decoctions. Standard decoctions are the material benchmark for measuring whether Chinese herbal formula granules are basically consistent with decoctions. However, whether it is a standard decoction or a Chinese herbal granule, the microscopic and property identification characteristics of the original medicinal materials and decoction slices have been lost, that is, it is impossible to inspect and identify the shape, size, color, surface, texture, etc. of the medicinal materials. Therefore, it is necessary to establish a method for rapid identification of the characteristic spectrum of malt and its preparations to provide a basis for effective control and scientific evaluation of the quality of malt and its preparations. Summary of the Invention

[0004] Therefore, the object of the present invention is to provide a method for constructing a characteristic map of malt and its preparations and its use, which provides a relatively comprehensive, systematic and effective rapid evaluation method for the quality evaluation and control of malt and its preparations.

[0005] Specifically, the present invention discloses a method for constructing a characteristic spectrum of malt and its preparation, comprising the following steps:

[0006] (1) Preparation of malt test solution: The extraction solvent for preparing the malt test solution is selected from hydrochloric acid aqueous solution, water or methanol aqueous solution, wherein the volume fraction of methanol in the methanol aqueous solution is not greater than 30%, and the concentration of hydrochloric acid in the hydrochloric acid aqueous solution is not greater than 0.2 mol / L;

[0007] (2) The malt test sample solution was detected by ultra-high performance liquid chromatography to obtain a characteristic spectrum of the test sample. Octadecylsilane bonded silica gel was used as a filler, and a potassium dihydrogen phosphate aqueous solution-methanol with a pH value of 3.4-3.6 and a concentration of 0.02-0.08 mol / L was used as a mobile phase for gradient elution. The detection wavelength was 218-222 nm, the flow rate was 0.15-0.22 ml / min, the column temperature was 33-37°C, and the gradient elution program included: 0→5 minutes→10 minutes→20 minutes→30 minutes→35 minutes. The volume percentage of methanol in the mobile phase was 0%→0-3%→3%→5%→22-25%→0%.

[0008] According to any one of the construction methods of the present invention, step (1) comprises:

[0009] 1) Weigh the malt sample and extract it with a solvent to obtain an extract;

[0010] 2) Separate the solid and liquid of the extract and take the liquid, which is the test solution.

[0011] According to any one of the construction methods of the present invention, step (1) satisfies any one or more of AC:

[0012] A. In step 1), the amount of extraction solvent is 25-100 times the amount of the test sample;

[0013] B. In step 1), the extraction method is ultrasonic extraction, shaking extraction or reflux extraction, and the extraction time is 15 min-2 h;

[0014] C. In step 2), the solid-liquid separation is independently selected from centrifugation or filtration.

[0015] According to the construction method described in any one of the present invention, the step (1) comprises: taking 0.2-1.0 g of the test sample, adding 25 ml of solvent, ultrasonically extracting for 15-60 min, taking it out, cooling it, weighing it again, making up the lost weight with solvent, filtering it, and taking the filtrate to obtain it.

[0016] The concentration of potassium dihydrogen phosphate in the aqueous solution containing potassium dihydrogen phosphate is 0.08 mol / L; the pH value of the aqueous solution containing potassium dihydrogen phosphate is 3.5.

[0017] In certain preferred embodiments, the construction method further includes the step of preparing a reference solution using at least one of adenine, tyrosine, uridine, N-methyltyramine, phenylalanine, hordenine, adenosine and tryptophan, and the step of detecting the reference solution by ultra-high performance liquid chromatography according to any of the above construction methods to obtain a reference spectrum of the reference substance.

[0018] In certain preferred embodiments, the method for preparing the reference solution comprises the steps of dissolving N-methyltyramine and hordenine reference substances in a methanol aqueous solution having a volume fraction of no more than 30%; or, the method comprises the steps of dissolving adenine, tyrosine, uridine, N-methyltyramine, phenylalanine, hordenine, adenosine and tryptophan in a methanol aqueous solution having a volume fraction of no more than 30%.

[0019] In certain preferred embodiments, the method for preparing the reference solution comprises the following steps: respectively dissolving adenine, tyrosine, uridine, N-methyltyramine, phenylalanine, hordenine, adenosine and tryptophan in a methanol aqueous solution having a volume fraction of not more than 30% to prepare a reference solution containing 10-100 μg of adenine, 10-100 μg of tyrosine, 10-100 μg of uridine, 10-100 μg of N-methyltyramine, 10-100 μg of phenylalanine, 10-100 μg of hordenine, 10-100 μg of adenosine and 10-100 μg of tryptophan per 1 ml.

[0020] According to any one of the construction methods of the present invention, the malt and its preparation (or test sample) is selected from at least one of malt medicinal materials, malt decoction pieces and malt preparations; wherein the malt preparation is a traditional Chinese medicine preparation prepared by using an extract obtained by malt extraction (such as a decoction obtained by decoction) in accordance with conventional pharmaceutical processes, with or without the addition of conventional pharmaceutical excipients.

[0021] Preferably, the malt preparation can be in the form of, but not limited to, dry powder, tablets, granules, capsules, ointments, solutions, and the like.

[0022] More preferably, the malt product provided by the present invention is at least one selected from malt medicinal materials, malt decoction pieces, malt standard decoction freeze-dried powder and malt formula granules.

[0023] In certain preferred embodiments, the characteristic spectrum of the malt and its preparation has 8 characteristic peaks, peak 4 and peak 6 should correspond to the retention times of the reference peaks of N-methyltyramine and hordenine, respectively; the peak corresponding to the hordenine reference peak is the S peak, and the relative retention times of the remaining characteristic peaks and the S peak are within ±10% of the specified value, and the specified values ​​of peaks 1-peak 3, peak 5, and peak 7-peak 8 are 0.36, 0.38, 0.57, 0.95, 1.45, and 1.61, respectively.

[0024] In the present invention, corresponding means that the RSD of the retention times of the two peaks is less than 5%, less than 3% or 1%, and / or corresponding means that the overlap of the two peaks is not less than 50%.

[0025] In the present invention, the control characteristic spectrum of malt and its preparations can also use the characteristic spectrum obtained by a single batch or multiple batches of malt medicinal materials, malt pieces or malt preparations according to any construction method described in the present invention; optionally, the control characteristic spectrum of malt and its preparations can also use the characteristic spectrum obtained by multiple batches of malt, malt pieces or malt preparations according to any construction method described in the present invention to prepare the control characteristic spectrum through the average value or median method.

[0026] Optionally, at least 2 batches of malt medicinal materials, malt decoction pieces or malt preparations are used to obtain a control characteristic spectrum, for example, 3 batches, 5 batches, 11 batches, 15 batches of malt medicinal materials, 18 batches of malt standard decoction freeze-dried powder, and 3 batches of malt formula granules.

[0027] The present invention also provides the use of the method for constructing the characteristic map of the malt and its preparation in the quality detection of malt products.

[0028] The present invention also provides a method for detecting the quality of malt and its preparations, comprising the step of comparing a characteristic spectrum of a malt product to be tested with a reference characteristic spectrum of malt and its preparations; the characteristic spectrum of the malt product to be tested is obtained using the malt product to be tested according to any one of the construction methods described in the present invention, and the reference characteristic spectrum of the malt and its preparations is selected from any one of the following (1) to (4):

[0029] (1) It has 8 common characteristic peaks, the retention time of each common characteristic peak has an RSD of less than 5.0% compared with the specified value of the retention time of the following common characteristic peaks, and the specified values ​​of the retention time of each common characteristic peak are 5.975 min, 6.398 min, 9.456 min, 15.115 min, 15.897 min, 16.653 min, 24.09 min and 26.807 min respectively;

[0030] (2) It has eight common characteristic peaks. Peak 4 and Peak 6 should correspond to the retention times of the reference peaks of N-methyltyramine and hordenine, respectively. The peak corresponding to the hordenine reference peak is the S peak. The relative retention times of each characteristic peak and the S peak are within ±10% of the specified value. The specified values ​​of Peaks 1-3, Peak 5, and Peaks 7-8 are 0.36, 0.38, 0.57, 0.95, 1.45, and 1.61, respectively.

[0031] (3) A malt fingerprint obtained by any of the above-mentioned construction methods using a single batch or multiple batches of malt test samples;

[0032] (4) Using multiple batches of malt test samples and the fingerprints obtained according to any of the construction methods described above, a control fingerprint is prepared by the average or median method.

[0033] The malt product is selected from at least one of malt medicinal materials, malt decoction pieces and malt preparations; wherein the malt preparation is a traditional Chinese medicine preparation prepared from the extract obtained by malt extraction (such as a decoction obtained by decoction) according to conventional pharmaceutical processes, with or without conventional pharmaceutical excipients.

[0034] Preferably, the malt preparation can be in the form of, but not limited to, dry powder, tablets, granules, capsules, ointments, solutions, and the like.

[0035] More preferably, the malt product provided by the present invention is at least one selected from malt medicinal materials, malt decoction pieces, malt standard decoction freeze-dried powder and malt formula granules.

[0036] In certain embodiments, a traditional Chinese medicine chromatographic fingerprint similarity evaluation software is used to generate a reference characteristic spectrum of malt and its preparations.

[0037] In certain preferred embodiments, after generating the control characteristic spectrum of malt and its preparations using the Chinese medicine chromatographic fingerprint similarity evaluation software, the step of marking the common characteristic peaks is also included.

[0038] The technical solution of the present invention has the following advantages:

[0039] 1. The method for constructing a characteristic spectrum of malt and its preparation provided by the present invention uses octadecylsilane bonded silica gel as a filler, a potassium dihydrogen phosphate aqueous solution-methanol with a pH value of 3.4-3.6 and a concentration of 0.02-0.08 mol / L, a detection wavelength of 218-222 nm, a flow rate of 0.15-0.22 ml / min, a column temperature of 33-37° C., and a specific gradient elution program. Multiple common characteristic peaks can be obtained, and these common characteristic peaks are well separated. The obtained characteristic spectrum has a stable baseline, good characteristic peak shape, high resolution, and short detection time, providing a basis for quality detection and control of malt and its preparation, achieving overall component characterization of malt and its preparation, and stable reproducibility.

[0040] By optimizing the extraction conditions such as chromatographic conditions, extraction solvent, and sampling volume, the optimal extraction process and chromatographic conditions were determined, which resulted in a higher peak area and better separation effect, enabling more comprehensive quality monitoring of malt formula granules.

[0041] 2. The method for constructing characteristic spectra of malt and its preparations provided by the present invention has good peak shape and separation of characteristic peaks and good repeatability of characteristic peaks; this method has high working efficiency and has the advantages of simplicity, stability, high precision and good repeatability. It can be applied to different types of malt preparations, can control the consistency of the quality of malt preparations, and quickly achieve quality control of different types of malt preparations.

[0042] The malt preparation detection method provided by the present invention can better reflect the characteristic peaks that characterize the types and numbers of the overall chemical components in the malt preparation by controlling the chromatographic conditions, thereby achieving quality control of the malt preparation based on the relative retention time of each characteristic peak.

[0043] 3. The method for constructing a characteristic spectrum of malt and its preparation provided by the present invention, after identification by a reference substance, shows that Peak 1 is adenine; Peak 2 is tyrosine; Peak 3 is uridine; Peak 4 is N-methyltyramine; Peak 5 is phenylalanine; Peak 6 is hordenine; Peak 7 is adenosine; and Peak 8 is tryptophan. These eight characteristic peaks identified in the characteristic spectrum are stable common peaks of 15 batches of malt preparations from different production areas, which can well ensure the accuracy of the method for malt quality control. In addition, these eight characteristic peaks have good peak shape, peak separation effect, and peak symmetry, which can well control the retention time and relative retention time of the characteristic peaks, greatly improving the reproducibility and applicability of the method. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0045] Figure 1 is the chromatogram of N-methyltyramine reference solution; Figure 2 This is the chromatogram of hordenine reference solution; Figure 3 This is the chromatogram of the sample of standard decoction of malt slices (lyophilized powder); Figure 4 1 chromatogram for method optimization; Figure 5 Chromatogram for method optimization 2 flow rate 0.20 ml / min; Figure 6 Chromatogram for method optimization 2 flow rate 0.15 ml / min; Figure 7 This is the chromatogram under 0.02mol / L-KH2PO4 (PH=3.5); Figure 8 This is the chromatogram under 0.05mol / L-KH2PO4 (PH=3.5); Figure 9 This is the chromatogram under 0.08mol / L-KH2PO4 (PH=3.5); Figure 10 This is the chromatogram under the conditions of 0.08mol / L-KH2PO4 (PH=2.5); Figure 11 This is the chromatogram under 0.08mol / L-KH2PO4 (PH=2.8); Figure 12 This is the chromatogram under the conditions of 0.08mol / L-KH2PO4 (PH=3.2); Figure 13 This is the chromatogram under 0.08mol / L-KH2PO4 (PH=3.5); Figure 14 This is the chromatogram under 0.08mol / LKH2PO4 (PH=3.8); Figure 15 This is the chromatogram under the conditions of 0.08mol / L-KH2PO4 (PH=4.0); Figure 16 This is the chromatogram under the conditions of 0.08mol / L-KH2PO4 (pH not adjusted, pH=4.58); Figure 17 Chromatogram for method optimization 3 flow rate 0.15 ml / min; Figure 18 Chromatogram for method optimization 3 flow rate 0.20 ml / min; Figure 19 The chromatogram is at a wavelength of 210 nm; Figure 20 The chromatogram is at a wavelength of 220 nm; Figure 21 The chromatogram is at a wavelength of 230 nm; Figure 22 The chromatogram is at a wavelength of 250 nm; Figure 23 The chromatogram is at a wavelength of 280 nm; Figure 24 The chromatogram is obtained with water as the extraction solvent; Figure 25 This is the chromatogram using 0.1 mol / L hydrochloric acid as the extraction solvent; Figure 26 This is the chromatogram using 10% methanol as the extraction solvent; Figure 27 This is the chromatogram using 30% methanol as the extraction solvent; Figure 28 This is the chromatogram using 50% methanol as the extraction solvent; Figure 29 The chromatogram is obtained using methanol as the extraction solvent; Figure 30 This is the characteristic spectrum of the malt control medicinal material; Figure 31 Characteristic spectra of 15 batches of standard decoctions of malt slices (lyophilized powder); Figure 32 Characteristic spectra of three batches of malt formula granules; Figure 33 This is the control characteristic spectrum of malt formula granules; Peak 1: adenine; Peak 2: tyrosine; Peak 3: uridine; Peak 4: N-methyltyramine; Peak 5: phenylalanine; Peak 6: hordenine; Peak 7: adenosine; Peak 8: tryptophan; Figure 34 Positioning of reference substances for different characteristic peaks (from bottom to top: S1: chromatogram of malt formula particles; S2: adenine; S3: tyrosine; S4: uridine; S5: N-methyltyramine; S6: phenylalanine; S7: hordenine; S8: adenosine; S9: tryptophan); Figure 35 This is the chromatogram of negative formula particles; Figure 36 Chromatography of the test sample for malt formula granules; Figure 37 This is a comparison chart of the characteristic spectrum of malt formula particles and the characteristic spectrum of rice sprouts; Figure 38 This is a comparison chart of the characteristic spectrum of malt formula particles and the characteristic spectrum of barley sprouts. DETAILED DESCRIPTION

[0046] The following examples are provided for a better understanding of the present invention and are not intended to limit the best mode of implementation. They do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the scope of protection of the present invention.

[0047] If specific experimental procedures or conditions are not specified in the examples, the procedures or conditions described in the literature in the field can be followed. Reagents or instruments used without manufacturer's information are commercially available. % Methanol refers to the volume percentage of methanol in the methanol-water solution.

[0048] Example 1 Malt slices standard decoction freeze-dried powder

[0049] This embodiment provides a method for preparing a freeze-dried powder of a standard decoction of malt slices, comprising the following steps: taking malt medicinal materials, removing impurities as specified in the Chinese Pharmacopoeia, decocting the malt slices twice, adding 8 times the weight of water for the first time, soaking the malt slices for 30 minutes, boiling the malt slices over high heat, then decocting the malt slices over low heat for 30 minutes, filtering the malt slices, adding 6 times the weight of water for the second time, boiling the malt slices over high heat, then decocting the malt slices over low heat for 25 minutes, filtering the malt slices, combining the filtrates, concentrating the filtrates (at 65° C.) to a concentrated extract with a relative density of approximately 1.00 g / ml, freeze-drying the malt slices in a freeze dryer, and pulverizing the malt slices into a powder.

[0050] Example 2 Malt Formula Granules

[0051] This embodiment provides a method for preparing malt formula granules, comprising the following steps: taking malt medicinal materials, removing impurities according to the Chinese Pharmacopoeia, heating and refluxing and extracting them twice, adding 8 times the weight of water for the first time, soaking for 30 minutes, heating and refluxing and extracting for 30 minutes, filtering, adding 6 times the weight of water for the second time, extracting for 20 minutes, filtering, combining the filtrates, concentrating the filtrates to a relative density of 1.02 to 1.10 g / ml at 60° C., adding an appropriate amount of auxiliary material (maltodextrin), drying, and granulating to obtain the malt formula granules.

[0052] Example 3

[0053] This embodiment provides a method for constructing a malt fingerprint, comprising the following steps:

[0054] (1) Preparation of test solution: 0.5 g of the test sample (the freeze-dried powder of the standard decoction of malt slices prepared in Example 1 is used in this example) is accurately weighed and placed in a stoppered conical flask. 25 ml of 10% methanol is accurately added and weighed. The solution is ultrasonically treated (power 300 W, frequency 40 kHz) for 30 minutes. The solution is taken out, cooled, and weighed again. The lost weight is supplemented with 10% methanol. The solution is shaken and filtered. The filtrate is obtained.

[0055] (2) Preparation of reference solution: Take about 5 g of malt control medicinal material, place it in a stoppered conical flask, add 50 ml of water, heat and reflux for 30 minutes, remove, filter, evaporate the filtrate to dryness, add 25 ml of 10% methanol to the residue, ultrasonically treat (power 300 W, frequency 40 kHz) for 30 minutes, remove, cool, filter, and take the filtrate as the reference solution of the control medicinal material. Take appropriate amounts of N-methyltyramine and hordenine reference substances, accurately weigh them, add 10% methanol to make a solution containing 15 μg of each per 1 ml, and use it as the reference solution of the reference substance;

[0056] (3) Ultra-high performance liquid chromatography detection: 2 μl of the test solution and reference solution were accurately aspirated and detected by ultra-high performance liquid chromatography; octadecylsilane bonded silica gel was used as the filler (chromatographic column: Waters ACQUITY HSS T3, 2.1×150 mm, 1.8 μm); use methanol as mobile phase A and 0.08 mol / L potassium dihydrogen phosphate solution (adjusted to pH 3.50 with 10% phosphoric acid) as mobile phase B, using gradient elution as specified in the table below; column temperature 35°C, flow rate 0.2 ml / min, detection wavelength 220 nm. The number of theoretical plates calculated based on the hordenine peak should be no less than 5000.

[0057] Table 1 Gradient elution conditions

[0058]

[0059]

[0060] See the results Figure 1-3 As shown in the table below, the characteristic spectrum of the freeze-dried powder of the standard decoction of malt slices has 8 characteristic peaks, among which the retention times of peak 4 and peak 6 should correspond to the retention times of the reference peaks of N-methyltyramine and hordenine, respectively; the peak corresponding to the hordenine reference peak is the S peak, and the relative retention times of each characteristic peak and the S peak are 0.36 (peak 1), 0.38 (peak 2), 0.57 (peak 3), 0.96 (peak 5), 1.44 (peak 7), and 1.61 (peak 8). The system adaptability parameters of the freeze-dried powder of the standard decoction of malt slices and the reference solution are shown in Table 2 below. The results show that the baseline of the characteristic spectrum is stable, the characteristic peaks have good peak shape, and the separation is high.

[0061] Table 2 System adaptability parameters of lyophilized powder and reference solution of malt slice standard decoction

[0062]

[0063] Example 4

[0064] This embodiment provides a method for constructing a malt fingerprint, comprising the following steps:

[0065] (1) Preparation of test solution: 0.5 g of the malt formula granules prepared in Example 2 was accurately weighed and placed in a stoppered conical flask. 25 ml of 10% methanol was accurately added and weighed. The solution was ultrasonically treated (power 300 W, frequency 40 kHz) for 30 minutes. The solution was removed, cooled, and weighed again. The lost weight was supplemented with 10% methanol. The solution was shaken and filtered. The filtrate was obtained.

[0066] (2) Preparation of reference solution: Take appropriate amounts of adenine, tyrosine, uridine, N-methyltyramine, phenylalanine, hordenine, adenosine, and tryptophan, accurately weigh them, and add 10% methanol to make a solution containing 15 μg of each per ml to obtain the reference solution.

[0067] (3) Ultra-high performance liquid chromatography detection: same as in Example 3.

[0068] Experimental Example 1 Optimization of chromatographic conditions

[0069] 1. Instruments:

[0070] Chromatograph 1: Waters ACQUITY UPLC H-Class PLUS chromatography system, including quaternary solvent manager (ACQ-QSM), autosampler (ACQ-FTN), imported column oven (ACQ-CM), diode array UV detector (ACQ-PDA), and Empower chromatography management system;

[0071] Chromatograph 2: Thermo Vanquish Flex UHPLC chromatography system, including a quaternary solvent manager (Vanquish Quaternary Pump F VF-P20-A), an autosampler (Vanquish Split Sampler FTVF-A10-A-02), an imported column oven (Vanquish Column Compartment H VH-C10-A-02), and a DAD detector (Vanquish VF-D40-A).

[0072] Chromatograph 3: The chromatograph used was a Thermo UltiMate 3000 chromatography system, including an LPG-3400SDN quaternary pump, a WPS-3000SL autosampler, a DAD diode array detector, and a Chromeleon chromatography workstation.

[0073] Electronic balance: METTLER TOLEDO (Swiss Mettler) ME36S, XS204, XSE205, XS205

[0074] Ultrasonic instrument: KQ-500DE CNC ultrasonic instrument produced by Kunshan Ultrasonic Instrument Co., Ltd.

[0075] Column: Waters ACQUITY HSS T3, 2.1×150mm, 1.8μm; JADE-RAK ODS-AQ (250mm×4.6mm, 5μm), Waters Atlantis T3 (250mm×4.6mm, 5μm).

[0076] 2. Drug testing:

[0077] Adenine reference substance (batch number: 110866-202203, purchased from China Food and Drug Administration, purity 99.8%);

[0078] N-methyltyramine reference substance (batch number: 21101, purchased from the Hong Kong Institute of Standards Materials, purity 99.33%)

[0079] Hordenine reference substance (batch number: 21060704, purchased from Chengdu Gelipu Biotechnology Co., Ltd., purity 99.84%)

[0080] Adenosine reference substance (batch number: 110879-201703, purchased from the China Food and Drug Administration, purity 99.7%);

[0081] Tyrosine reference substance (batch number: 140609-201914, purchased from China Food and Drug Administration, purity 99.9%);

[0082] Tryptophan reference substance (batch number: 140686-201904, purchased from China Food and Drug Administration, purity 99.9%);

[0083] Uridine reference substance (batch number: 110887-202104, purchased from China Food and Drug Administration, purity 99.6%);

[0084] Phenylalanine reference substance (batch number: 020079-202111, purchased from Shanghai Hongyong Biotechnology Co., Ltd., purity ≥98%)

[0085] Reagents: Methanol was of chromatographic grade, water was ultrapure water; other reagents were of analytical grade.

[0086] 3. UPLC method optimization

[0087] Since the characteristic peak separation of the characteristic spectrum of the standard decoction of malt pieces was poor on HPLC, the UPLC chromatographic conditions of the characteristic spectrum method of the standard decoction of malt pieces were optimized based on the HPLC investigation results. The UPLC chromatographic conditions of the freeze-dried powder test solution of the same batch of malt standard decoction prepared according to the method of Example 3 were optimized using chromatograph 1 according to the following method.

[0088] (1) Selection of mobile phase gradient and flow rate

[0089] The effects of different gradient programs on the separation effect of this product were investigated. The elution programs of method optimization 1 and method optimization 2 are shown in the table below, respectively. Both methods used a Waters ACQUITY HSS T3 (150 mm × 2.1 mm, 1.8 μm) column, the mobile phase was methanol (A)-0.05 mol / L potassium dihydrogen phosphate aqueous solution (pH = 3.0 adjusted with phosphoric acid) (B), the flow rate was 0.2 ml / min, the gradient elution was according to the table below, the detection wavelength was 220 nm, and the column temperature was 35°C.

[0090] Table 3 Method Optimization 1 Elution Program (Gradient 1)

[0091]

[0092] Table 4 Method Optimization 2 Elution Program (Gradient 2)

[0093]

[0094] like Figure 4-5 As shown in Figure 2, the separation of characteristic peaks in the 10-15 minute segment is improved compared with gradient 1. Based on the optimization of method 2 above, the flow rate was adjusted to 0.15 ml / min. The other conditions were the same and the same malt standard decoction freeze-dried powder test solution was tested. The results are shown in Figure 2. Figure 6 As shown in the figure, it can be seen that when the flow rate is 0.15 ml / min, the overall separation between chromatographic peaks has been greatly improved. However, in the characteristic spectrum of the standard decoction of malt slices, there are many impurities in the characteristic peaks at 0 to 10 minutes, and the separation is poor. Therefore, the chromatographic conditions were further optimized.

[0095] (2) Investigation of mobile phase buffer salt concentration, aqueous phase pH value, elution procedure and flow rate

[0096] The effects of different potassium dihydrogen phosphate buffer concentrations (0.02 mol / L, 0.05 mol / L, 0.08 mol / L) on the separation of this product were investigated. Except for the concentration of potassium dihydrogen phosphate buffer, the other conditions were as follows: a Waters ACQUITY HSS T3 (150 mm × 2.1 mm, 1.8 μm) column was used, the mobile phase was methanol (A)-potassium dihydrogen phosphate aqueous solution (pH adjusted to 3.0 with phosphoric acid) (B), the flow rate was 0.15 ml / min, and gradient elution was performed according to the elution program optimized in step 2 above. The detection wavelength was 220 nm and the column temperature was 35°C. Figure 7-9 .

[0097] The same sample solution of lyophilized powder of standard malt decoction was taken to investigate the effects of adjusting 0.08 mol / L potassium dihydrogen phosphate buffer with phosphoric acid to different pH values ​​(pH values ​​were 2.5, 2.8, 3.2, 3.5, 3.8, and 4.0) and not adjusting (pH value was 4.58) on the separation effect of this product. Except for the pH value of potassium dihydrogen phosphate buffer, the other conditions were as follows: a Waters ACQUITY HSS T3 (150 mm × 2.1 mm, 1.8 μm) column was used, the mobile phase was methanol (A)-0.05 mol / L potassium dihydrogen phosphate aqueous solution (pH value adjusted with phosphoric acid) (B), the flow rate was 0.15 ml / min, and gradient elution was performed according to the elution program optimized in 2 according to the above method. The detection wavelength was 220 nm and the column temperature was 35°C.

[0098] See the results Figure 10-16 As shown in the figure, it can be seen that the concentration and pH value of potassium dihydrogen phosphate in the mobile phase have a great influence on the separation of chromatographic peaks. When the pH value is less than or equal to 3.2, the separation effect of the chromatographic peaks at 5-10 minutes and 15-20 minutes is poor. When the pH value is greater than or equal to 3.8, the separation effect of the chromatographic peaks at 5-10 minutes is poor. The concentration of potassium dihydrogen phosphate can be 0.02-0.08 mol / L and the pH value can be 3.4-3.6. In particular, when the concentration of potassium dihydrogen phosphate is 0.08 mol / L and the pH value is 3.50, the separation effect of the chromatographic peaks at 5-10 minutes and 15-20 minutes is the best. Therefore, the concentration of potassium dihydrogen phosphate in the mobile phase is tentatively set to 0.08 mol / L and the pH value is 3.50, and the elution gradient is further optimized.

[0099] The elution schedule is shown in the table. The remaining conditions are as follows: a Waters ACQUITY HSS T3 column (150 mm × 2.1 mm, 1.8 μm) was used, with a mobile phase consisting of methanol (A) and a 0.08 mol / L aqueous potassium dihydrogen phosphate solution (pH adjusted to 3.5 with phosphoric acid) (B), a flow rate of 0.15 ml / min, a detection wavelength of 220 nm, and a column temperature of 35°C. (This is referred to as method optimization 3.) The results are shown in the table. Figure 17 .

[0100] After gradient optimization, the separation of the chromatographic peaks at 30 minutes increased, but the separation at 15-20 minutes was poor. Therefore, based on the optimization of the above method 3, the flow rate was adjusted to 0.20 ml / min. The other conditions were the same and the same malt standard decoction freeze-dried powder test solution was tested. The results are shown in Figure 2. Figure 18 .

[0101] Table 5 Method Optimization 3 Elution Program

[0102]

[0103] From the results in the attached figure, it can be seen that when the concentration of potassium dihydrogen phosphate is 0.08 mol / L, the pH is 3.50, and the flow rate is 0.2 ml / min, the chromatographic peak separation of the characteristic spectrum 15-20 minutes is significantly improved, and the chromatographic peak separation is better.

[0104] (3) Wavelength selection

[0105] The same sample solution of lyophilized powder of standard malt decoction was taken to investigate the effect of different wavelengths (210 nm, 220 nm, 230 nm, 250 nm, 280 nm) on the separation effect of this product. Except for the wavelength, the other conditions were as follows: a Waters ACQUITY HSS T3 (150 mm × 2.1 mm, 1.8 μm) column was used, the mobile phase was methanol (A)-0.08 mol / L potassium dihydrogen phosphate aqueous solution (pH = 3.5 adjusted with phosphoric acid) (B), the flow rate was 0.2 ml / min, and gradient elution was performed according to the 3 elution programs optimized according to the above method. The column temperature was 35 ° C.

[0106] See the results Figure 19-23 As shown in the figure, when the wavelength is 230nm and above, the response values ​​of the characteristic peaks are low and the characteristic is poor. At 210nm, the baseline is not flat. Therefore, it can be seen that the wavelength can be 218-222nm. In particular, when the detection wavelength is 220nm, the response values ​​of the characteristic peaks in the chromatogram are good and the baseline is flat.

[0107] (4) Investigation of different flow rates

[0108] The same batch of malt formula granule test solution prepared according to the method of Example 4 was taken and the flow rates were set to 0.18 ml / min, 0.2 ml / min, and 0.22 ml / min, respectively, according to the determination method of Example 4. Peak 6 was used as the S peak, and the relative retention times of the eight common characteristic peaks were calculated. The separation effect of each characteristic peak when the flow rate was changed was examined. The RSD value of the relative retention time of the eight characteristic peaks was less than 5%, indicating that the flow rate can be selected between 0.18 and 0.22 ml / min. The peak area of ​​Peak 4 will be affected when the flow rate changes. Preferably, a fixed flow rate (0.2 ml / min) is recommended for determination.

[0109] Table 6 Comparison of measurement results at different flow rates

[0110]

[0111]

[0112] Table 7 Relative peak area results at different flow rates

[0113]

[0114] (5) Investigation of different column temperatures

[0115] The same batch of malt formula granule test solution prepared according to the method of Example 4 was taken and the column temperature was set to 33°C, 35°C, and 37°C, respectively, according to the determination method of Example 4. Peak 6 was used as the S peak, and the relative retention time of the eight common characteristic peaks was calculated. The relative retention time of each characteristic peak was examined when the column temperature changed. The results showed that the relative retention time RSD of the eight characteristic peaks in the characteristic spectrum at the three column temperatures was less than 5%. Therefore, the column temperature can be selected to be 33-37°C. When the column temperature changes, the peak area of ​​peaks 1, 2, and 6 will be affected. Preferably, this method recommends a fixed column temperature (35°C) for measurement.

[0116] Table 8 Relative retention time results at different column temperatures

[0117]

[0118] Table 9 Relative peak area results at different column temperatures

[0119]

[0120] (6) Investigation of different chromatographic columns

[0121] The same batch of malt formula granule test solution prepared according to the method of Example 4 was used to investigate the separation of the characteristic spectrum of the malt formula granule using a Waters ACQUITY HSS T3 (2.1 mm × 150 mm, 1.8 μm) and a SHIMADZU Shim-pack GIST-HPAQ-C18 (2.1 mm × 150 mm, 1.9 μm) chromatographic column. The results showed that under these chromatographic conditions, the elution times of the chromatographic peaks of the malt formula granule varied significantly between the columns, which had a certain impact on the separation of the characteristic peaks. The relative retention times of the eight common characteristic peaks, with peak 6 as the S peak, were calculated. The results showed that the RSD values ​​of the relative retention times of the eight characteristic peaks were less than 10%, and the retention times relative to the peak area were greater than 10%. The Waters ACQUITY HSS T3 column is recommended for the determination of the characteristic spectrum of the malt formula granule.

[0122] Table 10 Comparison of the results of different chromatographic columns

[0123]

[0124]

[0125] Table 11 Relative peak area results of different chromatographic columns

[0126]

[0127] Experimental Example 2 Preparation of test solution

[0128] (1) Selection of extraction solvent

[0129] The effects of 0.1 mol / L hydrochloric acid solution, water, 10% methanol, 30% methanol, 50% methanol and methanol on the content of the standard decoction of malt slices were investigated. 0.5 g of the standard decoction of malt slices (lyophilized powder) was accurately weighed and placed in a stoppered conical flask. 25 ml of 0.1 mol / L hydrochloric acid aqueous solution, water, 10% methanol, 30% methanol, 50% methanol and methanol were accurately added respectively, weighed, ultrasonically extracted (power 300 W, frequency 40 kHz) for 30 minutes, cooled, weighed again, and the weight was supplemented with the corresponding solvent to make up the lost weight. The mixture was shaken, filtered and the filtrate was taken to obtain the test solution, which was tested according to the chromatographic conditions under Example 3.

[0130] Table 12 Chromatogram parameters of malt slices standard decoction (lyophilized powder) under different extraction solvents

[0131]

[0132]

[0133] The results are shown in the table above and Figure 24-29 As shown, the results show that when the methanol concentration is relatively high, the peak shape deteriorates and the extraction effect is relatively poor. The characteristic peak areas of the standard decoction of malt slices extracted by 0.1mol / L hydrochloric acid aqueous solution, water, 10% methanol, and 30% methanol are basically the same. Therefore, hydrochloric acid aqueous solution, water or methanol aqueous solution can be selected as the extraction solvent, wherein the volume fraction of methanol in the methanol aqueous solution is not greater than 30%, and the concentration of hydrochloric acid in the hydrochloric acid aqueous solution is not higher than 0.2mol / L. Taking into account the peak shape of each chromatographic peak and the stability of the sample solvent, 10% methanol is preferably used as the extraction solvent for the characteristic spectrum of the standard decoction of malt slices.

[0134] (2) Investigation of extraction methods

[0135] Take 0.5 g of the standard decoction of malt slices (lyophilized powder), accurately weigh it, put it in a stoppered conical flask, accurately add 25 ml of 10% methanol, weigh it, shake it for extraction, ultrasonic extraction (power 400 W, frequency 40 kHz), and reflux extraction for 30 minutes each, let it cool, weigh it again, make up the lost weight with 10% methanol, shake it well, filter it, and take the filtrate to obtain the test solution, which was detected according to the chromatographic conditions of Example 3.

[0136] Table 13 Results of the investigation of extraction methods

[0137]

[0138]

[0139] The experimental results show that the characteristic peak areas of the standard decoction of malt slices measured by the three extraction methods are basically the same, indicating that the extraction method has little effect on the determination of the characteristic spectrum of the standard decoction of malt slices. Considering the convenience of operation, ultrasonic extraction is preferred as the extraction method for the determination of the characteristic spectrum of the standard decoction of malt slices.

[0140] (3) Investigation of extraction time

[0141] Take 0.5 g of the standard decoction of malt slices (lyophilized powder), accurately weigh it, put it in a stoppered conical flask, accurately add 25 ml of 10% methanol, weigh it, and ultrasonically extract it (power 400 W, frequency 40 kHz) for 15, 30, 45, and 60 minutes, respectively. Cool it, weigh it again, make up the lost weight with 10% methanol, shake it well, filter it, and take the filtrate to obtain the test solution, which was detected according to the chromatographic conditions of Example 3.

[0142] Table 14 Results of the investigation on extraction time

[0143]

[0144]

[0145] The experimental results show that the peak areas of the characteristic spectra of the standard decoction of malt slices are basically the same at different extraction times, and all can be extracted completely. Therefore, 15-60 minutes (for example, 30 minutes) can be selected as the extraction time for the determination of the characteristic spectra of the standard decoction of malt slices.

[0146] (4) Investigation of sampling volume

[0147] Take 0.2, 0.5, 0.8, and 1.0 g of the standard decoction of malt slices (lyophilized powder) respectively, accurately weigh them, place them in a stoppered conical flask, accurately add 25 ml of 10% methanol, weigh the weight, and ultrasonically extract (power 400 W, frequency 40 kHz) for 30 minutes. Let cool, weigh the weight again, make up the lost weight with 10% methanol, shake well, filter, and take the filtrate to obtain the test solution, which was detected according to the chromatographic conditions of Example 3.

[0148] Table 15 Investigation results of sampling volume

[0149]

[0150]

[0151] The experimental results show that, under the sampling volume of 0.2g to 1.0g, as the sampling volume increases, the characteristic peak area of ​​the characteristic spectrum of the standard decoction of malt pieces basically increases proportionally, indicating that under the sampling volume of 0.2g to 1.0g, the standard decoction of malt pieces can be completely extracted. Considering the comprehensive peak area, 0.5g is preferred as the sampling volume for the determination of the characteristic spectrum of the standard decoction of malt pieces.

[0152] (5) Investigation of ultrasonic power

[0153] Take 0.5 g of the standard decoction of malt slices (lyophilized powder), accurately weigh it, put it in a stoppered conical flask, accurately add 25 ml of 10% methanol, weigh it, and ultrasonically extract it at 200 W, 250 W, and 300 W powers for 30 minutes, let it cool, weigh it again, make up the lost weight with 10% methanol, shake it well, filter it, and take the filtrate to obtain the test solution, which was detected according to the chromatographic conditions of Example 3.

[0154] Table 16 Results of investigation on extraction power

[0155]

[0156]

[0157] The experimental results show that under different ultrasonic powers, the characteristic peaks of the characteristic spectrum of the standard decoction of malt slices are basically the same, indicating that the ultrasonic power has no effect on the determination of the characteristic spectrum of the standard decoction of malt slices. Therefore, 300W min is selected as the ultrasonic power for the determination of the characteristic spectrum of the standard decoction of malt slices.

[0158] (6) Investigation of injection volume

[0159] Take 0.5g of the standard decoction of malt slices (lyophilized powder), accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of 10% methanol, weigh it, ultrasonicate it (power 300W, frequency 40kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with 10% methanol, shake it well, filter it, and take the filtrate. According to the chromatographic conditions of Example 3, inject 1, 2, 3, and 5μl of it respectively.

[0160] Table 17 Investigation results of injection volume

[0161]

[0162]

[0163] The experimental results showed that as the injection volume increased, the characteristic peak area of ​​the standard decoction of malt slices increased proportionally, and no obvious overload phenomenon occurred at each injection volume. Considering the comprehensive peak shape, 2μl was selected as the injection volume for the characteristic spectrum determination of the standard decoction of malt slices.

[0164] Experimental Example 3 Establishment of characteristic spectrum of malt formula particles

[0165] 1. Construction of multi-batch feature maps

[0166] Take 15 batches of malt slices standard decoction freeze-dried powder samples (batch number: 2103001Y, 2103002Y, 2103003Y, 2103004Y, 2103006Y, 2103007Y, 2103010Y, 2103011Y, 2103012Y, 2103013Y, 2103014Y, 2103015Y, 211101Y, 211102Y, 211103Y) and malt control medicinal materials, and obtain 15 batches of malt slices standard decoction freeze-dried powder characteristic maps according to the ultra-performance liquid chromatography of Example 3, see Figure 31 and 30 Three batches of malt formula granule samples (batch numbers: 2205001Y, 2205002Y, 2205003Y) were taken and tested by ultra-high performance liquid chromatography according to Example 4 to obtain characteristic spectra of the three batches of malt formula granules, as shown in FIG. Figure 32 shown.

[0167] The fingerprint similarity evaluation software "Chinese Herbal Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" compiled by the Pharmacopoeia Committee was used to generate the reference characteristic spectrum. Figure 33 As shown. By identifying and specifying the characteristic peaks with the reference substance, the characteristic spectrum of the standard decoction of malt slices was confirmed. Combining the characteristic peaks of malt medicinal materials and reference medicinal materials, 8 characteristic common peaks were selected as the characteristic peaks of the standard decoction of malt slices, and the chromatographic peak numbers 1 to 8 were rearranged according to the order of the chromatographic peaks.

[0168] The determination results show that there are 8 characteristic peaks in the characteristic spectra of 15 batches of standard decoctions of malt slices (lyophilized powder) and 3 batches of malt formula granules, among which the retention times of peak 4 and peak 6 should correspond to the retention times of the reference peaks of N-methyltyramine and hordenine, respectively; the peak corresponding to the hordenine reference peak is the S peak, and the relative retention time of each characteristic peak and the S peak is calculated. The relative retention time of each characteristic peak is within ±10% of the specified value.

[0169] Table 18 Relative retention time determination results of characteristic spectra of 15 batches of malt decoction standard decoction (lyophilized powder) and 3 batches of formula granules

[0170]

[0171]

[0172] Table 19 Determination results of relative peak areas of characteristic spectra of 15 batches of standard decoctions of malt slices (lyophilized powder) and 3 batches of formula granules

[0173]

[0174]

[0175] 2. Specified value

[0176] The reference spectrum was fitted using Mark peak fitting, and the eight chromatographic peaks with good separation in the obtained malt UPLC characteristic spectrum were confirmed, and they should correspond to the eight characteristic peaks in the reference medicinal material chromatogram, among which the retention times of peak 4 and peak 6 should correspond to the retention times of the N-methyltyramine and hordenine reference substance peaks, respectively; the peak corresponding to the hordenine reference substance peak was the S peak, and the relative retention time of each characteristic peak and the S peak was calculated. The relative retention time should be within the range of ±10% of the specified value, and the specified value is 0.36 (peak 1), 0.38 (peak 2), 0.57 (peak 3), 0.95 (peak 5), 1.45 (peak 7), and 1.61 (peak 8).

[0177] The relative retention times and relative peak areas of the obtained control characteristic spectra, as well as the control herbal material characteristic spectra, are shown in the corresponding tables below. The control herbal material characteristic spectra of malt, the characteristic spectra of 15 batches of malt decoction standard decoctions (lyophilized powder) and 3 batches of formula granules, and the control characteristic spectra of malt formula granules are shown in the corresponding spectra below.

[0178] The reference spectrum was fitted using Mark peak fitting.

[0179] Table 20 Relative retention time of malt formula granule comparison chart

[0180]

[0181] Table 21 Relative peak areas of malt formula granule comparison chart

[0182]

[0183] Table 22 Relative retention time of characteristic spectrum of malt control medicinal materials

[0184]

[0185] Table 23 Relative peak areas of characteristic spectrum of malt control medicinal materials

[0186]

[0187] 3. Identification of characteristic peaks

[0188] 2 μL of the reference solution and the test solution prepared in Example 4 were respectively injected into the liquid chromatograph, and the sample was injected and measured according to the chromatographic conditions of Example 4 to obtain a characteristic spectrum.

[0189] Feature maps such as Figure 34 As shown, by comparing with the reference solution spectrum, it was determined that peak 1 was adenine; peak 2 was tyrosine; peak 3 was uridine; peak 4 was N-methyltyramine; peak 5 was phenylalanine; peak 6 was hordenine; peak 7 was adenosine; and peak 8 was tryptophan. According to the peak identification and peak selection results of the characteristic peaks in the standard decoction of malt slices, the chromatographic peaks that are easily obtained by the reference sample are N-methyltyramine and hordenine. In the characteristic spectrum of the standard decoction of malt slices, hordenine has a relatively moderate response and is a content indicator component of the standard decoction of malt slices, which is relatively stable. In summary, hordenine is taken as the S peak of the characteristic spectrum.

[0190] 4. Similarity Analysis

[0191] The fingerprint similarity evaluation software "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" compiled by the Pharmacopoeia Committee was used to calculate the similarity between the characteristic spectra of 15 batches of freeze-dried powder samples of standard decoction of malt slices and 3 batches of formula granules and the control characteristic spectra. The results are shown in the table below, and the similarities are all greater than 0.95.

[0192] Table 24 Similarity results

[0193] batch number Similarity 2103001Y 0.998 2103002Y 0.992 2103003Y 0.999 2103004Y 0.978 2103006Y 0.991 2103007Y 0.984 2103010Y 1 2103011Y 0.997 2103012Y 0.994 2103013Y 0.995 2103014Y 0.999 2103015Y 0.999 211101Y 0.981 211102Y 0.981 211103Y 0.981 Granules 2205001Y 0.991 Granules 2205002Y 0.991 Granules 2205003Y 0.991 Comparison chart 1

[0194] Experimental Example 4 Methodology Verification

[0195] 1. Precision

[0196] 1.1 Instrument precision: Take the same malt formula granule test sample (2205002Y), prepare one sample according to the test sample of Experimental Example 1, No. 3 (2), repeat the injection 6 times, and determine the relative retention time and relative peak area of ​​each common peak according to the chromatographic conditions of Experimental Example 1, No. 3 (3). Take peak No. 6 as the S peak, calculate its relative peak area and relative retention time. And calculate RSD. The results show that the relative retention time RSD range of the 8 characteristic peaks of the six precision experimental samples is 0% to 0.2%, and the relative peak area RSD range is 0% to 0.9%, indicating that the characteristic spectrum instrument precision is good.

[0197] 1.2 Repeatability test: Take the same malt formula granule test sample (2205002Y) and repeat the preparation of 6 samples according to the test sample preparation method of Experimental Example 1, No. 3 (2), and determine the relative retention time and relative peak area of ​​each common peak according to the chromatographic conditions of Experimental Example 1, No. 3 (3). Take peak No. 6 as the S peak, calculate its relative peak area and relative retention time. And calculate RSD. The results show that the relative retention time RSD range of the 8 characteristic peaks of the six repeatability experimental samples is 0% to 0.2%, and the relative peak area RSD range is 0% to 0.6%, indicating that the characteristic spectrum has good repeatability.

[0198] 1.3 Intermediate precision (different operators)

[0199] Three inspectors took the same portion of malt formula granules at different times, prepared samples according to the test sample preparation method of Experimental Example 1, Section 3(2), and used the same equipment to determine the relative retention time and relative peak area of ​​each common peak according to the chromatographic conditions of Experimental Example 1, Section 3(3). Peak No. 6 was taken as the S peak, and its relative peak area and relative retention time were calculated. RSD was also calculated. The results showed that the RSD range of the relative retention time of the eight characteristic peaks was 0% to 0.2%, and the RSD range of the relative peak area was 0% to 0.7%, indicating that the characteristic spectrum instrument has good precision.

[0200] 2. Exclusivity

[0201] Take malt formula granules and negative granules (obtained by granulating with maltodextrin as an auxiliary material without adding malt extract) respectively, and prepare the test sample solution and negative control solution according to the test sample preparation method of Experimental Example 1, Section 3 (2). Figure 35 and 36 The blank control of the test sample has no interference with the characteristic spectrum and can be used as a detection method for the characteristic spectrum of malt formula particles.

[0202] 3. Stability

[0203] Take an appropriate amount of the same malt formula granule sample and prepare the test solution according to the method of Experimental Example 1, Section 3 (2). The solution was measured according to the chromatographic conditions of Experimental Example 1, Section 3 (3) at 0, 2, 4, 8, 16, and 24 hours to obtain its characteristic spectrum. With Peak 9 as the reference peak, the relative peak area and relative retention time of the 8 characteristic peaks were calculated. The RSD was also calculated. The experimental results showed that after examining the stability of the solution for 24 hours, the RSD of the relative retention time of each characteristic peak was in the range of 0% to 0.3%, and the RSD of the area of ​​each characteristic peak within 24 hours was in the range of 0% to 1.1%. The stability was good.

[0204] 4. Durability inspection of different instruments

[0205] The same batch of malt formula granule test solution prepared according to the method of Example 4 was taken and tested on UPLC instruments of different brands according to the determination method of Example 4. The relative retention time of each characteristic peak on the UPLC instruments of different brands was investigated. The results showed that the RSD range of the relative retention time of the eight characteristic peaks in the characteristic spectra of the three different brands of UPLC instruments was 0% to 3.8%, the relative retention time variation was small, the chromatographic peak reproducibility was good, the difference was small, and the durability of the different instruments was good.

[0206] Table 25 Relative retention time results of different instruments

[0207]

[0208] Table 26 Relative peak area results of different instruments

[0209]

[0210] Experimental Example 5 Application of the method for constructing characteristic maps of malt and its preparations in identifying malt and its preparations.

[0211] The main counterfeit malt products are barley sprouts and rice sprouts. These three varieties all have the effect of helping digestion, but in addition to the digestion effect, they also have other different functions. Therefore, they should not be mixed in clinical practice.

[0212] This embodiment provides a method for identifying malt and its preparation, comprising the following steps: using lyophilized powder of a standard decoction of malt and lyophilized powder of a standard decoction of rice sprout as test samples, respectively, to be identified, preparing test sample solutions according to the method of Example 3, and detecting under the chromatographic conditions of Example 3. The results are shown in the following table and accompanying drawings. The lyophilized powder of a standard decoction of malt and the lyophilized powder of a standard decoction of rice sprout are test samples prepared according to the preparation method of Example 1, with the only difference being that 6 batches of malt and rice sprout medicinal materials are used instead of the malt medicinal materials in Example 1.

[0213] Table 27 Retention time results of characteristic spectrum determination of standard decoction of barley sprout and rice sprout

[0214]

[0215]

[0216] Table 28 Peak area determination results of characteristic spectrum of standard decoction of barley sprout and rice sprout

[0217]

[0218] As shown in the table above and Figure 37 and 38 As shown, the constructed characteristic spectra of freeze-dried powder of standard decoction of barley malt slices and freeze-dried powder of standard decoction of rice bud slices were compared with the control characteristic spectra constructed in Experimental Example 3. The standard decoction of barley malt slices lacked peak 6 (hordenine), and the standard decoction of rice bud slices lacked peak 4 (N-methyltyramine) and peak 6 (hordenine), which formed a clear distinction with the characteristic spectra of malt.

[0219] This confirms that the method can be used to identify malt and its counterfeits (barley sprouts, rice sprouts).

[0220] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A method for constructing a characteristic profile of malt and its preparation, characterized in that: The following steps are included: (1) Preparation of malt test solution: The extraction solvent for preparing the malt test solution is selected from hydrochloric acid aqueous solution, water or methanol aqueous solution, wherein the volume fraction of methanol in the methanol aqueous solution is not greater than 30%, and the concentration of hydrochloric acid in the hydrochloric acid aqueous solution is not greater than 0.2 mol / L; (2) The malt test sample solution was detected by ultra-high performance liquid chromatography to obtain a characteristic spectrum of the test sample. Octadecylsilane bonded silica gel was used as a filler, and a potassium dihydrogen phosphate aqueous solution-methanol with a pH value of 3.4-3.6 and a concentration of 0.02-0.08 mol / L was used as the mobile phase for gradient elution. The detection wavelength was 218-222 nm, the flow rate was 0.15-0.22 ml / min, the column temperature was 33-37 °C, and the gradient elution program included: 0→5 min→10 min→20 min→30 min→35 min. The volume percentage of methanol in the mobile phase was 0%→0-3%→3%→5%→22-25%→0%; The malt and its preparation are selected from at least one of malt medicinal materials, malt decoction pieces and malt preparations; the malt preparation is selected from at least one of malt formula granules and malt standard decoction freeze-dried powder.

2. The construction method according to claim 1, characterized in that Step (1) includes: 1) Take the malt sample, add solvent to extract, and obtain the extract; 2) Separate the solid and liquid of the extract and take the liquid, which is the test solution.

3. The construction method according to claim 2, characterized in that Step (1) satisfies any one or more of the following AC: A. In step 1), the amount of extraction solvent used is 25-100 times the amount of the test sample; B. In step 1), the extraction method is ultrasonic extraction, shaking extraction or reflux extraction, and the extraction time is 15 minutes to 2 hours; C. In step 2), the solid-liquid separation is independently selected from centrifugation or filtration.

4. The construction method according to any one of claims 1 to 3, characterized in that The concentration of potassium dihydrogen phosphate in the aqueous solution of potassium dihydrogen phosphate is 0.08 mol / L; the pH value of the aqueous solution containing potassium dihydrogen phosphate is 3.

5.

5. The construction method according to claim 4, characterized in that The construction method further includes the steps of preparing a reference solution using at least one of adenine, tyrosine, uridine, N-methyltyramine, phenylalanine, hordenine, adenosine and tryptophan, and obtaining a reference spectrum of the reference solution by detecting the reference solution using ultra-high performance liquid chromatography according to the construction method of claim 4.

6. The construction method according to claim 5, characterized in that: The preparation method of the reference solution comprises the following steps: dissolving N-methyltyramine and hordenine reference substances in a methanol aqueous solution with a volume fraction of no more than 30%; or dissolving adenine, tyrosine, uridine, N-methyltyramine, phenylalanine, hordenine, adenosine and tryptophan in a methanol aqueous solution with a volume fraction of no more than 30%.

7. The construction method according to claim 6, characterized in that: The characteristic spectrum of the malt and its preparation has 8 characteristic peaks, peak 4 and peak 6 should correspond to the retention times of the reference peaks of N-methyltyramine and hordenine, respectively; the peak corresponding to the hordenine reference peak is the S peak, and the relative retention times of the remaining characteristic peaks and the S peak are within ±10% of the specified value. The specified values ​​of peak 1-peak 3, peak 5, and peak 7-peak 8 are 0.36, 0.38, 0.57, 0.95, 1.45, and 1.61, respectively.

8. Use of the method for constructing a characteristic profile of malt and its preparation according to any one of claims 1 to 7 in the identification or quality inspection of malt products.

9. A method for identifying or testing the quality of malt and its preparations, characterized in that: The method comprises the steps of comparing a characteristic spectrum of the product to be identified or the malt product to be tested with a reference characteristic spectrum of malt and its preparation; the characteristic spectrum of the product to be identified or the malt product to be tested is obtained by using the product to be identified or the malt product to be tested according to the construction method according to any one of claims 1 to 6, and the reference characteristic spectrum of malt and its preparation is selected from any one of the following (1) to (4): (1) It has 8 common characteristic peaks, and the RSD of the retention time of each common characteristic peak is less than 5.0% compared with the specified value of the retention time of the following common characteristic peaks. The specified values ​​of the retention time of each common characteristic peak are 5.975min, 6.398min, 9.456min, 15.115min, 15.897min, 16.653min, 24.09min and 26.807min respectively; (2) It has 8 common characteristic peaks. Peak 4 and Peak 6 should correspond to the retention time of the reference peaks of N-methyltyramine and hordenine, respectively. The peak corresponding to the hordenine reference peak is the S peak. The relative retention time of each characteristic peak and the S peak is within the range of ±10% of the specified value. The specified values ​​of Peak 1-Peak 3, Peak 5, and Peak 7-Peak 8 are 0.36, 0.38, 0.57, 0.95, 1.45, and 1.61, respectively. (3) A malt fingerprint obtained by the construction method according to any one of claims 1 to 6 using a single batch or multiple batches of malt test samples; (4) Using multiple batches of malt test samples and the fingerprints obtained according to the construction method described in any one of claims 1 to 6, a control fingerprint is prepared by the average or median method.

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