Method for simultaneously determining contents of 16 amino acids in asparagus cochinchinensis

Abstract

The invention discloses a method for simultaneously determining the contents of 16 amino acids in radix asparagi, and belongs to the technical field of traditional Chinese medicinal material detection. The detected 16 amino acids in the radix asparagi are tyrosine, arginine, phenylalanine, histidine, methionine, glutamic acid, lysine hydrochloride, aspartic acid, leucine, isoleucine, threonine, valine, proline, serine, alanine and glycine respectively. The method for simultaneously determining 16 amino acids in radix asparagi through the ultra-high performance liquid chromatography-tandem mass spectrometry is established by determining parameters such as an extraction method, chromatographic conditions and the like of a radix asparagi sample, and compared with a traditional amino acid determination method, the method has the advantages of being easy and convenient to operate, rapid, sensitive, accurate and specific in selectivity, and has wide application prospects. Meanwhile, the method fills up the research blank of the amino acid content in asparagus fern from different producing areas.

Description

Technical Field

[0001] The invention relates to the technical field of detection of traditional Chinese medicinal materials, and in particular to a method for simultaneously determining the contents of 16 kinds of amino acids in asparagus cochinchinensis. Background Art

[0002] Asparagus cochinchinensis (Lour.) Merr, a plant of the Liliaceae family, is a dried root tuber. It has the effects of nourishing yin and moistening dryness, clearing the lungs and promoting fluid production. It is often used for dry coughs caused by dry lungs, sticky sputum caused by coughing, sore waist and knees, hot flashes caused by bone steaming, internal heat and thirst, damage to body fluids caused by heat diseases, dry throat and thirst, dry intestines and constipation, etc. It is one of the traditional Chinese medicinal materials in my country. The main pharmacological components of asparagus cochinchinensis are amino acids (mainly glutamic acid, valine, phenylalanine, serine, threonine, proline, etc.), sugars, spirosteroids, etc. Asparagus cochinchinensis has been used as a wild product for medicinal purposes or candied fruit for consumption in the past dynasties. Many Chinese medicine factories in China are using asparagus cochinchinensis raw materials to produce Chinese patent medicines, and its demand is showing a substantial growth trend, making the raw materials extremely scarce. Asparagus cochinchinensis extracts also have anti-tumor, anti-inflammatory, anti-aging, and hypoglycemic effects. At the same time, modern pharmacological studies have found that amino acids are one of the material bases for exerting anti-tumor, anti-viral, and anti-aging effects, among which amino acids are the effective ingredients for anti-virus and anti-tumor. Therefore, the efficacy of Asparagus cochinchinensis can be inferred by measuring the content of various amino acids in Asparagus cochinchinensis, but the determination of amino acid content in Asparagus cochinchinensis is still a research gap.

[0003] Amino acids are the basic building blocks of proteins and nitrogen metabolites of traditional Chinese medicine. Amino acids in traditional Chinese medicine are the main or auxiliary ingredients for treating diseases. Amino acid analysis is an important part of the analysis of effective ingredients in traditional Chinese medicine. For a long time, scholars have conducted a lot of research on the amino acid content in botanical medicines. Such as the identification of counterfeit Chinese herbal medicines, the study of the relationship between the medicinal properties of medicinal plants and species, the study of the distinction between authentic medicinal materials, and the research and development of new medicinal materials. In terms of the identification of botanical medicines, since there are many kinds of amino acids in medicinal materials, and there are obvious differences between different families and genera, the relationship between some Chinese herbal medicines and counterfeit drugs that are difficult to distinguish by morphological classification can be explored through the amino acid content. Moreover, this distinction method is relatively simple and has a wider range of applicability, uniformity and scientificity.

[0004] In the early days, amino acid content analysis was performed by separation with ion exchange resins, followed by post-column derivatization reactions, and the derivatives were detected by ultraviolet detectors. However, this method has long analysis time, complex operation, low sensitivity, and expensive instruments, which seriously limits its promotion and application. In recent years, the detection methods of amino acids in Chinese medicinal materials have been improved, mainly including automatic amino acid analysis technology, high performance liquid chromatography, and gas chromatography-mass spectrometry technology. Among them, the automatic amino acid analysis technology has the defects of being greatly affected by the pH of the buffer solution and the differences in the detection results of different instruments; the high performance liquid chromatography technology has the defects of complicated and cumbersome pre-column derivatization operation and long analysis time; the gas chromatography-mass spectrometry technology has the defect of incomplete reaction with hydroxyl and indole groups. Summary of the invention

[0005] The present invention is to solve the above technical problems existing in the prior art and provides a method for simultaneously determining the contents of 16 kinds of amino acids in Asparagus cochinchinensis. The determination method of the present invention has the characteristics of simple operation, rapidity, sensitivity, accuracy, and selectivity.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] A method for simultaneously determining the contents of 16 kinds of amino acids in asparagus, wherein the 16 kinds of amino acids are tyrosine, arginine, phenylalanine, histidine, methionine, glutamic acid, lysine hydrochloride, aspartic acid, leucine, isoleucine, threonine, valine, proline, serine, alanine, and glycine; the method comprises the following steps:

[0008] (1) Preparation of reference solution: Dissolve the 16 amino acid standards with hydrochloric acid solution to prepare a mixed amino acid standard stock solution, dilute the mixed amino acid standard stock solution with hydrochloric acid solution, mix well, and prepare a mixed amino acid standard intermediate solution with a mass concentration of 100 μg / mL, and dilute the mixed amino acid standard intermediate solution stepwise with 50% acetonitrile solution to prepare a series of mixed standard working solutions of different concentrations;

[0009] (2) Preparation of the sample solution to be tested: crush the asparagus cochinchinensis, place it in a hydrolysis tube, add hydrochloric acid, place it in a drying oven for hydrolysis, take out the hydrolysis tube, cool it to room temperature, mix it, centrifuge it, draw the supernatant into a test tube, place it in a nitrogen blower, evaporate it to dryness, add 50% acetonitrile to dissolve the residue, filter it through a microporous membrane after it is fully dissolved, and prepare the sample solution to be tested for analysis;

[0010] (3) analyzing the contents of the 16 amino acids in the sample solution by ultra-high performance liquid chromatography-tandem mass spectrometry;

[0011] Chromatographic conditions are as follows: chromatographic column: HILIC-Z chromatographic column, 2.7 μm, 2.1 mm×100 mm; guard column: BEHAmide, 2.1×5 mm, 1.7 μm; mobile phase: 20 mmol / L ammonium formate-water solution containing formic acid as mobile phase A, 20 mmol / L ammonium formate-acetonitrile solution containing formic acid as mobile phase B; gradient elution: 0-12 min, 100% B-70% B; 12-13 min, 70% B-100% B; 13-20 min, 100% B; column temperature: 30°C; flow rate: 0.3 mL / min; injection volume: 1 μL;

[0012] The mass spectrometry conditions were as follows: ionization mode: ESI positive ion mode; drying gas temperature: 300°C; drying gas flow rate: 5.0 L / min; nebulizer pressure: 45 psi; sheath gas temperature: 250°C; sheath gas flow rate: 11 L / min; capillary voltage: 3500 V; and nozzle voltage: 500 V.

[0013] Furthermore, the specific operation steps of step (1) are as follows: take the 16 amino acid standards respectively, dissolve them with 0.1mol / L hydrochloric acid solution and prepare a mixed amino acid standard stock solution with a mass concentration of 1 mg / mL for each amino acid; accurately pipette 1.0mL of the mixed amino acid standard stock solution into a 10mL volumetric flask, dilute it with 0.1mol / L hydrochloric acid solution and make it to the scale, mix it, and prepare a mixed amino acid standard intermediate solution with a mass concentration of 100μg / mL; dilute the mixed amino acid standard intermediate solution step by step with 50% acetonitrile solution to prepare a total of 9 series of mixed standard working solutions with different concentrations; the mass concentrations of the 16 amino acids in the 9 concentrations of the mixed standard working solutions are 0.3μg / mL, 0.625μg / mL, 1.25μg / mL, 2.5μg / mL, 5μg / mL, 10μg / mL, 20μg / mL, 40μg / mL, and 60μg / mL.

[0014] Furthermore, the specific operation steps of step (2) are as follows: crush the asparagus sample, accurately weigh 0.2g and place it in a hydrolysis tube, add 10mL of 6mol / L hydrochloric acid, tighten the screw cover, place it in a 150±2℃ electric heating blast drying oven, and hydrolyze for 3h; take out the hydrolysis tube and cool it to room temperature; mix well, open the hydrolysis tube, transfer it to a 50mL volumetric flask, rinse the hydrolysis tube with water, transfer it to the volumetric flask, and adjust the volume to the scale; centrifuge, accurately pipette 2mL of supernatant into a test tube, place it in a nitrogen blower, and evaporate to dryness at 50℃; accurately add 2mL of 50% acetonitrile to dissolve the residue, and after it is fully dissolved, filter it through a 0.22μm microporous membrane to obtain the sample solution for instrumental analysis.

[0015] Furthermore, in step (3), the mass spectrometry parameters of the 16 amino acids are as follows:

[0016]

[0017] The present invention also provides a quality control method for asparagus, comprising the following steps:

[0018] (1) The 16 amino acid content data measured above are associated with the batch, origin, and harvesting time of the asparagus root to achieve quality traceability;

[0019] (2) In batch management, by comparing the 16 amino acid content data of different batches of medicinal materials, their differences and consistency are evaluated to provide a basis for the storage, transportation and use of medicinal materials;

[0020] (3) Establishing a dynamic monitoring mechanism for the contents of the 16 amino acids, regularly or irregularly testing the amino acids of Asparagus cochinchinensis, and timely discovering abnormal changes in the contents of the 16 amino acids by comparing historical data with current data, and issuing early warning signals. Based on the early warning signals, taking corresponding intervention measures, the intervention measures include adjusting planting conditions and improving processing technology.

[0021] Beneficial effects:

[0022] The invention establishes a method for simultaneously determining 16 kinds of amino acids in asparagus by ultra-high performance liquid chromatography-tandem mass spectrometry by determining parameters such as an extraction method and chromatographic conditions of an asparagus sample. Compared with a traditional amino acid determination method, the method has the characteristics of simple operation, rapidity, sensitivity, accuracy, and selectivity. At the same time, the method supplements the research gap of the amino acid content in asparagus from different origins, provides theoretical support for its quality control, and provides a reference for the research on the determination of the amino acid content of traditional Chinese medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a comparison chart of the effects of different hydrolysis conditions on the total amino acid content of asparagus extract;

[0024] Figure 2 The total ion current of the amino acid reference substance;

[0025] Figure 3 It is the total ion current of the detected sample;

[0026] Figure 4 is the MRM diagram of Phe;

[0027] Figure 5 is the MRM diagram of Leu;

[0028] Figure 6 is the MRM map of Ile;

[0029] Figure 7 is the MRM diagram of Met;

[0030] Figure 8 is the MRM diagram of Tyr;

[0031] Fig. 9 is the MRM diagram of Val;

[0032] Fig.10 MRM diagram for Pro;

[0033] Fig.11 is the MRM diagram of Ala;

[0034] Fig.12 is the MRM diagram of Thr;

[0035] Fig.13 is the MRM diagram of Gly;

[0036] Fig.14 is the MRM map of Ser;

[0037] Fig.15 is the MRM diagram of Glu;

[0038] Fig.16 is the MRM diagram of Asp;

[0039] Fig.17 is the MRM map of His;

[0040] Fig.18 is the MRM diagram of Arg;

[0041] Fig.19 This is the MRM diagram of Lys. DETAILED DESCRIPTION

[0042] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0043] Example

[0044] A method for simultaneously determining the contents of 16 kinds of amino acids in Asparagus cochinchinensis comprises the following steps:

[0045] 1.1 Instrument

[0046] Ultra-high performance liquid chromatography-tandem mass spectrometer (Agilent 1290 / 6460A, Agilent Technologies Co., Ltd.); electric blast drying oven (WFLL-1258E, Tianjin Test Instrument Co., Ltd.); analytical balance (XS205DU, Mettler-Toledo, sensitivity 0.01 mg); analytical balance (AE200, Mettler-Toledo, sensitivity 0.0001 g); constant temperature CNC ultrasonic cleaner (KQ-500GDV, Kunshan Ultrasonic Instrument Co., Ltd.); ultrapure water machine (H20pro-UV-T, Sartorius, Germany); centrifuge (ROTANTA460R, Degent Technology Co., Ltd.); nitrogen blowdown instrument (TTL-DCⅢ, Beijing Tongtai Lian Technology Development Co., Ltd.); nitrogen generator (LCMS301-E, Degent Technology Co., Ltd.).

[0047] 1.2 Materials

[0048] Amino acid standards (China Food and Drug Inspection Institute, batch number: 140624-201508): tyrosine (purity ≥99.8%), arginine (purity ≥99.9%), phenylalanine (purity ≥100.0%), histidine (purity ≥99.9%), methionine (purity ≥99.9%), glutamic acid (purity ≥99.9%), lysine hydrochloride (purity ≥100.0%), aspartic acid (purity ≥99.9%), leucine (purity ≥99.9%), isoleucine (purity ≥99.9%), threonine (purity ≥99.9%), valine (purity ≥99.9%), proline (purity ≥99.9%), serine (purity ≥99.9%), alanine (purity ≥99.9%), glycine (purity ≥99.9%). Water is ultrapure water, prepared by H20pro-UV-T ultrapure water machine; ammonium formate (analytical grade, Shandong Xiya Chemical Co., Ltd., batch number: P22456); formic acid (chromatographic grade, Tianjin Komiou Chemical Reagent Co., Ltd., batch number: 20200110); acetonitrile (chromatographic grade, Merck KGaA, batch number: JB129230); hydrochloric acid (superior grade, Sinopharm Chemical Reagent Co., Ltd., batch number: 20221111). Asparagus cochinchinensis was purchased from Yulin Chinese herbal medicine market and identified as authentic by Qiu Yixian, deputy director pharmacist of Yulin Food and Drug Inspection and Testing Center. They are all tuberous roots of Asparagus cochinchinensis (Lour.) Merr. of the Liliaceae plant.

[0049] 2 Experimental methods

[0050] 2.1 Solution preparation

[0051] 2.1.1 Preparation of 0.1 mol / L hydrochloric acid solution

[0052] Take 9 mL of concentrated hydrochloric acid, pour it into a reagent bottle pre-filled with an appropriate amount of water, dilute it to 1000 mL with water, and mix well.

[0053] 2.1.2 Preparation of 6 mol / L hydrochloric acid solution

[0054] Take 500mL of concentrated hydrochloric acid, pour it into a reagent bottle pre-filled with an appropriate amount of water, dilute it to 1000mL with water, and mix well.

[0055] 2.2 Preparation of mobile phase

[0056] 2.2.1 Preparation of 200mmol / L ammonium formate solution

[0057] Weigh 12.61 g of ammonium formate, dissolve it in 1000 mL of water, and adjust the pH to 3.0 with formic acid.

[0058] 2.2.2 Preparation of mobile phase A (20 mmol / L ammonium formate-water solution)

[0059] Accurately pipette 100 mL of 200 mmol / L ammonium formate solution, add 900 mL of water, and mix well.

[0060] 2.2.3 Preparation of mobile phase B (20 mmol / L ammonium formate-acetonitrile solution)

[0061] Accurately pipette 100 mL of 200 mmol / L ammonium formate solution, add 900 mL of acetonitrile and mix well.

[0062] 2.3 Preparation of reference solution

[0063] Weigh an appropriate amount of each amino acid standard (accurate to 0.01 mg), dissolve it with 0.1 mol / L hydrochloric acid solution and prepare a mixed amino acid standard stock solution with a mass concentration of 1 mg / mL for each amino acid. Accurately pipette 1.0 mL of the mixed amino acid standard stock solution into a 10 mL volumetric flask, dilute it with 0.1 mol / L hydrochloric acid solution and make up to the mark, mix well, and prepare a mixed amino acid standard intermediate solution with a mass concentration of 100 μg / mL. Dilute the mixed amino acid standard intermediate solution stepwise with 50% acetonitrile solution to prepare a total of 9 mixed standard working solutions of different concentrations. The mass concentrations of the 16 amino acids in the 9 concentrations of the mixed standard working solutions are 0.3 μg / mL, 0.625 μg / mL, 1.25 μg / mL, 2.5 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, and 60 μg / mL.

[0064] 2.4 Preparation of test solution

[0065] The present invention determines the sample pretreatment conditions when preparing the solution to be tested: the hydrolysis interference conditions of the sample pretreatment are investigated, a group of hydrolysis tubes are filled with high-purity nitrogen before sealing, and phenol is added as a protective agent, the purpose of which is to prevent oxidation, and another group of untreated hydrolysis tubes are taken for comparison, and the results show that there is no obvious difference between the two; the hydrolysis temperature and time are then investigated, and the results show that as the temperature increases, the hydrolysis efficiency is also correspondingly improved, and the hydrolysis time is also correspondingly shortened; when the hydrolysis temperature is 140°C and the hydrolysis time is 4h, this condition is equivalent to the hydrolysis temperature of 110°C and the hydrolysis time of 22h, and the results of the two are equivalent; when the hydrolysis temperature is increased to 150°C and 160°C, the hydrolysis efficiency is improved; when the hydrolysis time is extended to 5h at 150°C, the measured amino acid content decreases; after comprehensive consideration, the hydrolysis temperature of 150°C and the hydrolysis time of 3h are finally selected, and the influence of different hydrolysis conditions on the total amino acid content extracted from asparagus is detailed in Figure 1 .

[0066] The preparation steps of the test solution are as follows: crush the asparagus sample, accurately weigh 0.2g and place it in a hydrolysis tube, add 10mL of 6mol / L hydrochloric acid, tighten the screw cap, place it in a (150±2)℃ electric heating blast drying oven, and hydrolyze for 3h. Take out the hydrolysis tube and cool it to room temperature. Mix well, open the hydrolysis tube, transfer it to a 50mL volumetric flask, rinse the hydrolysis tube with water several times, transfer it to a volumetric flask, and adjust the volume to the scale. Centrifuge, accurately pipette 2mL of supernatant into a test tube, place it in a nitrogen blower, and evaporate it to dryness at 50℃. Accurately add 2mL of 50% acetonitrile to dissolve the residue, and after it is fully dissolved, filter it through a 0.22μm microporous membrane and perform instrumental analysis.

[0067] During sample testing, it was found that if the sample hydrolyzate is not completely deacidified during the nitrogen blowdown process, the retention time of leucine will drift or even become undetectable; in subsequent sample processing, the hydrolyzate is evaporated to dryness, and normal detection can be restored.

[0068] 2.5 Instrument working conditions

[0069] 2.5.1 Chromatographic conditions

[0070] Selection of chromatographic columns: Hilic, Amide and PFP columns were used to detect the amino acid mixed standard solution. The retention effects of Hilic and Amide columns were comparable, and both achieved good separation effects. However, the retention of PFP columns was poor, and it was difficult to achieve good separation. After comprehensive consideration, Hilic columns were selected.

[0071] Selection of mobile phase: In Hilic chromatographic columns, the solvent elution ability from weak to strong is: acetonitrile < methanol < water. Water is the strongest elution solvent in the mobile phase. The effect of methanol and acetonitrile as organic phases on the retention time of amino acid compounds showed that when methanol was used as the organic phase, the analyte quickly flowed out with the mobile phase and failed to be separated. However, when acetonitrile was used as the organic phase, better separation could be achieved. When pure organic solvents were used in the organic phase, it was found that the chromatographic peaks would have a more serious tailing phenomenon. If formic acid or ammonium acetate was added to both the aqueous phase and the organic phase, both of them could significantly improve the peak shape of the chromatographic peaks. Increasing the buffer salt concentration can improve the peak shape, but high buffer salt concentration will have a negative impact on the sensitivity of mass spectrometry detection. Adjusting the pH of the mobile phase will also affect the detection sensitivity. Experiments show that when formic acid is used to adjust the pH, the higher the acidity, the higher the sensitivity. Considering the impact on the instrument and the chromatographic column, the pH was finally adjusted to 3, about 0.25% formic acid (volume fraction). Through the above experiments, the mobile phase was determined to be 20 mmol / L ammonium formate-water solution (containing 0.25% formic acid): 20 mmol / L ammonium formate-acetonitrile solution (containing 0.25% formic acid).

[0072] Chromatographic parameters determination: Run a gradient from 100% acetonitrile to 50% acetonitrile. The results show that all elution and separation of amino acid standards have been completed at 70% acetonitrile. According to the characteristics of Hilic chromatographic columns, Hilic chromatographic columns are not suitable for gradient elution with too fast changes and too short column equilibration time. Finally, the elution time of 100% acetonitrile to 70% acetonitrile is determined to be 12 minutes, and the column equilibration time is determined to be 8 minutes. The flow rate and gradient changes of chromatographic conditions were investigated. The results showed that when the flow rate was 0.8mL / min, the retention time would be advanced and concentrated, and the separation effect was poor. The flow rates of 0.3mL / min and 0.5mL / min can achieve good separation. Considering the influence of column pressure caused by flow rate on the chromatographic column, the flow rate of 0.3mL / min was finally selected.

[0073] In summary, the chromatographic conditions finally determined by the present invention are as follows:

[0074] Chromatographic column: HILIC-Z chromatographic column (2.7 μm, 2.1 mm×100 mm, Agilent Technologies Inc.); guard column: BEHAmide (2.1×5 mm, 1.7 μm, Waters Technologies Inc.); mobile phase: 20 mmol / L ammonium formate-water solution (containing formic acid) as mobile phase A, 20 mmol / L ammonium formate-acetonitrile solution (containing formic acid) as mobile phase B; gradient elution: 0-12 min (100% B-70% B), 12-13 min (70% B-100% B), 13-20 min (100% B); column temperature: 30°C; flow rate: 0.3 mL / min; injection volume: 1 μL.

[0075] 2.5.2 Mass spectrometry conditions

[0076] Ionization mode: ESI positive ion mode; drying gas temperature: 300℃; drying gas flow rate: 5.0L / min; nebulizer pressure: 45psi; sheath gas temperature: 250℃; sheath gas flow rate: 11L / min; capillary voltage: 3500V; nozzle voltage: 500V; drying gas, nebulizer gas, collision gas and sheath gas are all high-purity nitrogen; details of quantitative ion pairs, qualitative ion pairs, fragmentation voltage and collision gas energy are shown in Table 1.

[0077] Table 1 Detection parameters of mass spectrometry

[0078]

[0079] 3 Experimental Results

[0080] 3.1 Methodological considerations

[0081] 3.1.1 Establishment of standard curve

[0082] Accurately measure 1 ml of each concentration of the mixed standard working solution under item "2.3", detect according to the instrument conditions of item "2.5", and draw a standard curve with the peak area of ​​each reference substance as the ordinate and the mass concentration as the abscissa. The results show that the correlation coefficient (r) of the 16 amino acids within their respective mass concentration ranges is greater than 0.99, and the linear relationship is good. The detection limit and quantification limit are based on the signal intensity equal to three times (S / N=3) and ten times (S / N=10) of the baseline noise, respectively. The results show that both the detection limit and the quantification limit can meet the requirements for the determination of amino acids in asparagus samples. Relevant data are shown in Table 2. The total ion current of the amino acid reference substance is shown in Figure 2 The total ion current of the sample is shown in Figure 3 , the MRM chromatograms of 16 amino acids are shown in Figure 4-19 .

[0083] Table 2 Linear parameters and lower limits of determination

[0084]

[0085]

[0086] 3.1.2 Precision

[0087] Take 1 mL of the mixed standard working solution with a concentration of 10 μg / ml under item "2.3" and perform continuous sampling and measurement 6 times according to the instrument conditions of item "2.5". Calculate the response value of each reference substance based on the peak area. The results are shown in Table 3, indicating that the precision of this method is good.

[0088] Table 3 Precision test results

[0089]

[0090] 3.1.3 Repeatability

[0091] Take 6 samples from the same batch and prepare the test solution according to the method under "2.4". Determine according to the instrument conditions under "2.5" and calculate the content by the regression curve. The results are shown in Table 4, indicating that the method has good repeatability.

[0092] Table 4 Repeatability test results

[0093]

[0094] 3.1.4 Stability

[0095] Take a sample and prepare the test solution according to the method under "2.4". Determine the content according to the instrument conditions under "2.5" at 0, 4, 8, 12, 16, 20 and 24 hours respectively. Calculate the content by regression curve. The results are shown in Table 5, indicating that the test solution has good stability within 24 hours.

[0096] Table 5 Stability test results

[0097]

[0098]

[0099] 3.1.5 Spike recovery

[0100] Mixed standard solutions of three concentration levels, low, medium and high, were added to the sample, processed according to the method in "2.4", and measured according to the instrument conditions in "2.5". Each concentration level was measured three times in parallel, and the recovery rate and relative standard deviation (RSD) of the measured value were calculated. The results are shown in Table 6. As can be seen from Table 6, the results meet the requirements of mass spectrometry.

[0101] Table 6 Experimental results of sample recovery of 16 amino acids

[0102]

[0103]

[0104]

[0105] 3.2 Sample determination

[0106] According to the above method, 21 batches of asparagus samples (S1-S21) purchased were measured, and the 21 asparagus samples were respectively from Guangxi Zhuang Autonomous Region (15), Anhui (1), Hebei (2), Sichuan (1), and Yunnan (2). The results of the detection and analysis are shown in Table 7. The results show that among the samples tested, Phe, Leu, Ile, Tyr, Gly, Lys, Ala, and Val all have the highest content in S13, Met, Thr, His, Asp, Arg, all have the highest content in S17, Pro has the highest content in S8, Ser has the highest content in S6, and Glu has the highest content in S18, as shown in Table 7 for details.

[0107] Table 7 Analysis results of samples

[0108]

[0109]

[0110] The method of the present invention is used to simultaneously determine the contents of 16 kinds of amino acids in Asparagus cochinchinensis. After obtaining these data, its application is far more than a simple numerical record, but can be deeply applied to multiple aspects, especially in quality control and evaluation. The following are some possible application scenarios:

[0111] 1. Quality Control

[0112] 1. Establish personalized quality standards:

[0113] (1) Based on the data measured by UHPLC-MS / MS, personalized amino acid content standards for Asparagus cochinchinensis can be established. These standards can be more detailed and accurate than existing standards, taking into account the effects of different origins, years, processing methods, and other factors on amino acid content.

[0114] (2) Monitor the changes in amino acid content in Asparagus cochinchinensis through regular testing to ensure the stability and consistency of the quality of the medicinal material.

[0115] 2. Quality traceability and batch management:

[0116] (1) The UHPLC-MS / MS data can be linked to the batch, origin, harvesting time and other information of the medicinal materials to achieve quality traceability. This helps to quickly locate the problem batch, analyze the cause, and take corresponding improvement measures.

[0117] (2) In batch management, by comparing the amino acid content data of different batches of medicinal materials, their differences and consistency can be evaluated to provide a scientific basis for the storage, transportation and use of medicinal materials.

[0118] 2. Innovation in Evaluation Methods

[0119] 1. Comprehensive evaluation from multiple dimensions:

[0120] (1) In addition to the single amino acid content value, other indicators (such as total amino acid content, essential amino acid ratio, correlation between specific amino acids and active ingredients, etc.) can also be combined for multi-dimensional comprehensive evaluation. This evaluation method can more comprehensively reflect the quality characteristics of Asparagus cochinchinensis.

[0121] (2) Utilize statistical analysis and data mining techniques to establish a mathematical model or scoring system between amino acid content and medicinal material quality, and realize quantitative and standardized evaluation of medicinal material quality.

[0122] 2. Dynamic monitoring and early warning:

[0123] (1) Establish a dynamic monitoring mechanism for amino acid content and test Asparagus cochinchinensis regularly or irregularly. By comparing historical data with current data, abnormal changes in amino acid content can be detected in a timely manner and early warning signals can be issued.

[0124] (2) Based on the early warning signals, take appropriate intervention measures (such as adjusting planting conditions, improving processing technology, etc.) to ensure the stability and safety of the quality of the medicinal materials.

[0125] 3. Study on the correlation with medicinal ingredients:

[0126] (1) To study the correlation between the amino acid content and the active ingredients (such as saponins, polysaccharides, etc.) in Asparagus cochinchinensis. Through correlation analysis or regression analysis, the potential mechanism of amino acids in the formation of active ingredients and pharmacological effects was revealed.

[0127] (2) This correlation study helps to deeply understand the material basis of the efficacy and quality formation of Asparagus cochinchinensis, and provides a scientific basis for the quality control and clinical application of the medicinal material.

[0128] 3. Practical Application Scenarios

[0129] 1. Pharmaceutical company quality control: In pharmaceutical companies, the data measured by UHPLC-MS / MS are used to conduct strict quality control on Asparagus cochinchinensis to ensure the quality and efficacy of the drugs.

[0130] 2. Research by scientific research institutions: Scientific research institutions can use these data to conduct in-depth pharmacological and mechanism studies, and provide a theoretical basis and experimental basis for the development of new drugs.

[0131] 3. Market supervision: Market supervision departments can use these data to conduct random inspections and supervision on Asparagus cochinchinensis in the market, crack down on counterfeit and shoddy products, and protect the rights and interests of consumers.

[0132] Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art may make some modifications and improvements without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be based on the definition of the claims.

Claims

1. A method for simultaneously determining the contents of 16 kinds of amino acids in Asparagus cochinchinensis, characterized in that: The 16 amino acids are tyrosine, arginine, phenylalanine, histidine, methionine, glutamic acid, lysine hydrochloride, aspartic acid, leucine, isoleucine, threonine, valine, proline, serine, alanine, and glycine; the method comprises the following steps: (1) Preparation of reference solution: Dissolve the 16 amino acid standards with hydrochloric acid solution to prepare a mixed amino acid standard stock solution, dilute the mixed amino acid standard stock solution with hydrochloric acid solution, mix well, and prepare a mixed amino acid standard intermediate solution with a mass concentration of 100 μg / mL, and dilute the mixed amino acid standard intermediate solution stepwise with 50% acetonitrile solution to prepare a series of mixed standard working solutions of different concentrations; (2) Preparation of the sample solution to be tested: crush the asparagus cochinchinensis, place it in a hydrolysis tube, add hydrochloric acid, place it in a drying oven for hydrolysis, take out the hydrolysis tube, cool it to room temperature, mix it, centrifuge it, draw the supernatant into a test tube, place it in a nitrogen blower, evaporate it to dryness, add 50% acetonitrile to dissolve the residue, filter it through a microporous membrane after it is fully dissolved, and prepare the sample solution to be tested for analysis; (3) analyzing the contents of the 16 amino acids in the sample solution by ultra-high performance liquid chromatography-tandem mass spectrometry; The chromatographic conditions were as follows: column: HILIC-Z column, 2.7 μm, 2.1 mm × 100 mm; guard column: BEHAmide, 2.1 × 5 mm, 1.7 μm; Mobile phase: 20mmol / L ammonium formate-water solution containing formic acid as mobile phase A, 20mmol / L ammonium formate-acetonitrile solution containing formic acid as mobile phase B; gradient elution: 0-12min, 100%B-70%B; 12-13min, 70%B-100%B; 13-20min, 100%B; column temperature: 30℃; flow rate: 0.3mL / min; injection volume: 1μL; The mass spectrometry conditions were as follows: ionization mode ESI positive ion mode; drying gas temperature 300°C; drying gas flow rate 5.0 L / min; nebulizer pressure 45 psi; Sheath gas temperature 250°C; sheath gas flow rate 11 L / min; capillary voltage 3500 V; nozzle voltage 500 V.

2. The method for simultaneously determining the contents of 16 kinds of amino acids in Asparagus cochinchinensis according to claim 1, characterized in that: The specific operation steps of step (1) are as follows: take the 16 amino acid standards respectively, dissolve them with 0.1mol / L hydrochloric acid solution and prepare a mixed amino acid standard stock solution with a mass concentration of 1 mg / mL for each amino acid; accurately pipette 1.0mL of the mixed amino acid standard stock solution into a 10mL volumetric flask, dilute it with 0.1mol / L hydrochloric acid solution and make it to the scale, mix it, and prepare a mixed amino acid standard intermediate solution with a mass concentration of 100μg / mL; dilute the mixed amino acid standard intermediate solution step by step with 50% acetonitrile solution to prepare a series of mixed standard working solutions of a total of 9 different concentrations; the mass concentrations of the 16 amino acids in the mixed standard working solutions of the 9 concentrations are 0.3μg / mL, 0.625μg / mL, 1.25μg / mL, 2.5μg / mL, 5μg / mL, 10μg / mL, 20μg / mL, 40μg / mL, and 60μg / mL.

3. The method for simultaneously determining the contents of 16 kinds of amino acids in Asparagus cochinchinensis according to claim 1, characterized in that: The specific operation steps of step (2) are as follows: crush the asparagus sample, accurately weigh 0.2 g and place it in a hydrolysis tube, add 10 mL of 6 mol / L hydrochloric acid, tighten the screw cover, place it in a 150±2°C electric heating blast drying oven, and hydrolyze for 3 hours; take out the hydrolysis tube and cool it to room temperature; mix well, open the hydrolysis tube, transfer it to a 50 mL volumetric flask, rinse the hydrolysis tube with water, transfer it to the volumetric flask, and dilute to the scale; After centrifugation, accurately pipette 2 mL of supernatant into a test tube, place in a nitrogen blower, and evaporate to dryness at 50°C; accurately add 2 mL of 50% acetonitrile to dissolve the residue, filter through a 0.22 μm microporous membrane after sufficient dissolution, and obtain the sample solution for instrumental analysis.

4. The method for simultaneously determining the contents of 16 kinds of amino acids in Asparagus cochinchinensis according to claim 1, characterized in that: In step (3), the mass spectrometry parameters of the 16 amino acids are as follows:

5. A method for controlling the quality of Asparagus cochinchinensis, characterized in that: The steps include: (1) associating the 16 amino acid content data determined in any one of claims 1 to 4 with the batch, origin, and harvesting time of the Asparagus cochinchinensis to achieve quality traceability; (2) In batch management, by comparing the 16 amino acid content data of different batches of medicinal materials, their differences and consistency are evaluated to provide a basis for the storage, transportation and use of medicinal materials; (3) Establishing a dynamic monitoring mechanism for the contents of the 16 amino acids, regularly or irregularly performing amino acid detection on the Asparagus cochinchinensis using the method of any one of claims 1 to 4, timely discovering abnormal changes in the contents of the 16 amino acids by comparing historical data with current data, and issuing an early warning signal. Based on the early warning signal, taking corresponding intervention measures, the intervention measures include adjusting planting conditions and improving processing technology.

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