Method for detecting amino acids in fresh pears

Abstract

The invention relates to the technical field of amino acid detection, in particular to a method for detecting amino acid in fresh pears, an experimental instrument and a reagent, sample preparation and chromatographic and mass spectrum conditions. The method utilizes triple quadrupole mass spectrometry, is suitable for determination of low-concentration amino acid, has good selectivity, allows a target substance to have good chromatographic separation and mass spectrometry response, allows a standard curve to be good in a linear range, and can be applied to detection of low-concentration amino acid. The intra-day and inter-day precision, the accuracy, the quantitation limit, the recovery rate and the matrix effect all meet the verification requirements of related chemical methods, and the method is suitable for rapid and simple detection of the amino acids in the pear fruits.

Description

Technical Field

[0001] The present invention relates to the technical field of amino acid detection, in particular to a method for detecting amino acids in fresh pears. Background Art

[0002] Crown pears are primarily produced in Zhao County, Shijiazhuang, Hebei Province. They are rich in nutrients, including certain amounts of protein, fat, carotene, vitamin B1, vitamin B2, malic acid, and other essential substances for the human body. According to relevant research reports, the type and content of amino acids in pears is one of the indicators for evaluating pear quality. Fresh pears contain a relatively complete range of amino acids, at least 17 of which are essential amino acids (EAAs) and 10 non-essential amino acids (NEAAs). They also include some medicinal amino acids (MAAs), branched-chain amino acids (BCAAs), and aromatic amino acids (AAAs). The results identified in the first part indicate that temperature and time affect amino acid metabolism. According to relevant research, in addition to participating in the synthesis of proteins and peptides in the human body, some amino acids have been shown to have functions in maintaining growth, reproduction, and immunity. They are also involved in the formation of other fruit components and the synthesis of flavor compounds, such as arginine, glutamine, leucine, and proline.

[0003] At present, research on amino acids is mostly focused on biological samples, such as human blood, urine, and neonatal disease screening. There are fewer research mechanisms applied to fruit matrices. In existing studies, the main method for detecting amino acids in fruits is amino acid analyzers. Although there are many models of amino acid analyzers, their principles are basically cation exchange separation and post-column ninhydrin derivatization detection. However, the ion exchange resin is easily compressed, and the acceleration of the mobile phase flow rate is not proportional to the increase in pressure. The measurement time is relatively long. In addition, the amino acids are separated by ion exchange columns and mixed with ninhydrin derivatization agents for reaction, resulting in relatively large post-column diffusion and broadened peaks, resulting in poor practicality. Therefore, a method for detecting amino acids in fresh pears is urgently needed to improve the above problems. Summary of the Invention

[0004] To solve the above technical problems, the present invention provides a method using a triple quadrupole mass spectrometer, which is suitable for the determination of lower concentrations of amino acids. The method has good selectivity, the target substance has good chromatographic separation and mass spectrometric response, the standard curve is well within the linear range, and the intra-day and inter-day precision, accuracy, limit of quantification, recovery rate and matrix effect all meet the requirements for verification of relevant chemical methods. The method is suitable for the rapid and simple detection of amino acids in pear fruits and is a method for detecting amino acids in fresh pears.

[0005] A method for detecting amino acids in fresh pears of the present invention, step 1, experimental instruments and reagents:

[0006] Experimental instruments: TQ-S tandem quadrupole mass spectrometer, CAPCELL PAK ADME chromatographic column, centrifuge, vortex oscillator, electric thermostatic water bath, nitrogen blow dryer, ultrapure water system and electronic analytical balance;

[0007] Reagents: acetonitrile, methanol, n-butanol, acetyl chloride, amino acid standards, and deuterated isotope standards;

[0008] Step 2: Preparation of standard solution:

[0009] Prepare amino acid standard stock solutions, amino acid internal standard stock solutions, and derivatization reagents;

[0010] Step 3: Sample preparation:

[0011] Weigh 0.1 g of sample into a 1.5 mL plastic centrifuge tube with a stopper, add 20 μL of internal standard, then add 1 mL of 90% acetonitrile, vortex mix, perform low-temperature ultrasonic extraction and refrigerated centrifugation, transfer the supernatant to a clean nitrogen blowpipe, blow dry with nitrogen, add 100 μL of derivatization reagent to the nitrogen blowpipe, vortex and oscillate for 2 minutes, derivatize at 50°C for 15 minutes, blow dry with nitrogen, add 300 μL of water, reconstitute with acetonitrile, filter through a 0.22 μm filter, and wait for detection;

[0012] Step 4. Chromatographic and mass spectrometric conditions:

[0013] Liquid chromatography conditions:

[0014] a. Chromatographic column: CAPCELL PAK ADME chromatographic column, 150 mm × 2.1 mm, 5 μm;

[0015] b. Mobile phase: 2.5 mM ammonium acetate 0.1% formic acid in water (A) + 2.5 mM ammonium acetate 0.1% formic acid in acetonitrile (B); gradient elution;

[0016] c. Column temperature: 40°C;

[0017] d. Injection volume: 5 μL;

[0018] Mass spectrometry conditions:

[0019] Ion source: electrospray ion source;

[0020] Scanning mode: positive ion scanning;

[0021] Capillary voltage: 0.5kV;

[0022] Desolventization temperature: 1000℃;

[0023] Desolventizing gas flow rate: 500L / Hr;

[0024] Collision gas: argon, flow rate 1.7 mL / min;

[0025] Mass spectrometry scanning mode: segmented multiple reaction monitoring mode acquisition.

[0026] Preferably, the amino acid standard stock solution: accurately weigh 10 mg of the amino acid standard substance, dissolve it in water and dilute to the scale, and prepare a standard stock solution with a concentration of 100 μg / mL for storage. Accurately transfer a sufficient amount of the amino acid standard stock solution as needed, dilute it with water, and prepare a standard intermediate solution with a concentration of 1 μg / mL;

[0027] Preferably, the amino acid internal standard stock solution: accurately weigh 10 mg of the amino acid internal standard standard substance, dissolve it with water and dilute to the scale, and prepare a standard stock solution with a concentration of 100 μg / mL. Dilute the internal standard intermediate solution with water as needed to prepare a standard working solution with a concentration of 1 μg / mL. Before use, dilute the standard intermediate solution with the initial mobile phase to a working solution of appropriate concentration to ensure that the concentration of each internal standard is 200 ng / mL.

[0028] Preferably, the derivatization reagent is configured with n-butanol and acetyl chloride in a ratio of 9:1, and the reaction principle is R1OH+ClR2O=R2OOR1+HCl;

[0029] Reaction principle: After the derivatization reaction, the amino acid compound reacts with n-butanol hydrochloride to generate butylated amino acid.

[0030] Preferably, the amino acid standard stock solution and the amino acid internal standard stock solution in step 2 need to be stored at 4° C. in the dark.

[0031] Preferably, the purity of the amino acid standard and the deuterated isotope standard in step 1 is greater than 95%.

[0032] Preferably, after vortex mixing in step 3, low-temperature ultrasonic extraction is performed for 15 minutes, and refrigerated centrifugation is performed at 12000 r / min for 10 minutes.

[0033] Compared with the prior art, the present invention has the following beneficial effects: the method utilizes a triple quadrupole mass spectrometer, is suitable for the determination of lower concentrations of amino acids, has good selectivity, the target substances have good chromatographic separation and mass spectrometric response, the standard curve is well within the linear range, and the intra-day and inter-day precision, accuracy, limit of quantification, recovery rate and matrix effect all meet the requirements for validation of relevant chemical methods, and is suitable for the rapid and simple detection of amino acids in pear fruit. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 is a schematic diagram of the amino acid derivatization reaction of the present invention;

[0035] Figure 2 It is the TIC diagram of different chromatographic columns of the present invention;

[0036] Figure 3 1 is a TIC diagram of different mobile phases of the present invention;

[0037] Figure 4 is a comparison chart of different extraction solvents of the present invention;

[0038] Figure 5 is a comparison diagram of different extraction volumes of the present invention;

[0039] Figure 6 It is the derived temperature effect diagram of the present invention;

[0040] Figure 7 It is a time effect diagram derived from the present invention;

[0041] Figure 8 It is a derived volume rendering of the present invention;

[0042] Figure 9 It is a schematic diagram of the specificity investigation of the present invention;

[0043] Figure 10 It is a schematic diagram of screening differential compound contents of the present invention. DETAILED DESCRIPTION

[0044] To facilitate understanding of the present invention, the present invention will be described more fully below with reference to the accompanying drawings. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thorough and comprehensive.

[0045] Example: Figures 1 to 10 As shown, a method for detecting amino acids in fresh pears, step 1, experimental instruments and reagents:

[0046] Experimental instruments: WATERS ACQUITY UPLC-XEVO TQ-S tandem quadrupole mass spectrometer (WATERS, USA); CAPCELL PAKADME chromatographic column (150 mm × 2.1 mm, 5 μm); TDL-5-A centrifuge (Simga, Germany); vortex oscillator (Scientific, USA); DK-8D electric thermostatic water bath (Shanghai Yiheng Technology Co., Ltd.); nitrogen blow dryer (Shanghai Eurofins Technology Co., Ltd.); Milli-Q Element ultrapure water system (Millipore, USA); electronic analytical balance (METTLERTOLEDO, Switzerland); 0.22 μm filter membrane (Merck, Germany); acetonitrile, methanol, n-butanol, and acetyl chloride (chromatographically pure, purchased from CNW, Germany).

[0047] Reagents: acetonitrile, methanol, n-butanol, and acetyl chloride (chromatographically pure, purchased from CNW, Germany);

[0048] Amino acid standards: glycine, alanine, serine, proline, valine, threonine, leucine, isoleucine, asparagine, aspartic acid, glutamine, methionine, phenylalanine, arginine, tyrosine, glutamic acid, lysine, histidine, tryptophan, and cysteine ​​were purchased from Sigma and Dr. Ehrenstorfer Company with a purity greater than or equal to 95%.

[0049] Deuterated isotope standards: glycine-D5, alanine-D4, serine-D3, proline-D3, valine-D8, threonine-D2, leucine-13C, isoleucine-D10, asparagine-D3, aspartic acid-D3, glutamine-13C5, methionine-D3, phenylalanine-D8, arginine-N4, tyrosine-13C6, glutamate-15N, cysteine-D6, lysine-D4, histidine-C6, and tryptophan-D5 were purchased from Sigma and Dr. Ehrenstorfer Company with a purity greater than 95%;

[0050] The structural formulas of the above standard products are shown in Table 1.

[0051] Table 1 Standards information

[0052]

[0053]

[0054]

[0055] Step 2: Preparation of standard solution:

[0056] Amino acid standard stock solution: Accurately weigh 10 mg (accurate to 0.0001 g) of amino acid standard substance, dissolve in water and dilute to the mark to prepare a standard stock solution with a concentration of 100 μg / mL. Store at 4°C in the dark. Accurately pipette an appropriate amount of amino acid standard stock solution as needed, dilute with water, and prepare a standard intermediate solution with a concentration of 1 μg / mL.

[0057] Amino acid internal standard stock solution: Weigh 10 mg (accurate to 0.0001 g) of amino acid internal standard standard substance, dissolve it in water and dilute to the mark, prepare a standard stock solution with a concentration of 100 μg / mL, store it at 4°C in the dark, dilute the internal standard intermediate solution with water as needed to prepare a standard working solution with a concentration of 1 μg / mL, store it at 4°C in the dark, and dilute the standard intermediate solution with the initial mobile phase to a working solution of appropriate concentration before use to ensure that the concentration of each internal standard is 200 ng / mL;

[0058] Derivatization reagent: n-butanol and acetyl chloride are prepared in a ratio of 9:1. The reaction principle is R1OH+ClR2O=R2OOR1+HCl;

[0059] Reaction principle: After derivatization, the amino acid compound reacts with n-butanol hydrochloride to form butylated amino acid;

[0060] Step 3: Sample preparation:

[0061] Weigh 0.1 g of sample into a 1.5 mL plastic centrifuge tube with a stopper, add 20 μL of internal standard, then add 1 mL of 90% acetonitrile, vortex mix, perform low-temperature ultrasonic extraction and refrigerated centrifugation, transfer the supernatant to a clean nitrogen blowpipe, blow dry with nitrogen, add 100 μL of derivatization reagent to the nitrogen blowpipe, vortex and oscillate for 2 minutes, derivatize at 50°C for 15 minutes, blow dry with nitrogen, add 300 μL of water, reconstitute with acetonitrile, filter through a 0.22 μm filter, and wait for detection;

[0062] Step 4. Chromatographic and mass spectrometric conditions:

[0063] Liquid chromatography conditions:

[0064] a. Chromatographic column: CAPCELL PAK ADME column, 150 mm × 2.1 mm, 5 μm;

[0065] b. Mobile phase: 2.5 mM ammonium acetate 0.1% formic acid in water (A) + 2.5 mM ammonium acetate 0.1% formic acid in acetonitrile (B); gradient elution. Elution conditions are shown in Table 2.

[0066] c. Column temperature: 40°C;

[0067] d. Injection volume: 5 μL;

[0068] Table 2 Gradient elution conditions

[0069] Table 2 Gradient elution conditions

[0070]

[0071] Mass spectrometry conditions:

[0072] Ion source: electrospray ion source;

[0073] Scanning mode: positive ion scanning;

[0074] Capillary voltage: 0.5kV;

[0075] Desolventization temperature: 1000℃;

[0076] Desolventizing gas flow rate: 500L / Hr;

[0077] Collision gas: argon, flow rate 1.7 mL / min;

[0078] Mass spectrometry scanning mode: Segmented multiple reaction monitoring mode acquisition, conditions are shown in Table 3;

[0079] Table 3 Monitored ion pairs, retention times, and collision energies for each compound

[0080] compound

[0081]

[0082]

[0083]

[0084] Results and Verification:

[0085] 1. Selection of liquid phase conditions:

[0086] 1.1. Selection of chromatographic column:

[0087] There are many types of amino acid compounds with different properties. The currently reported detection methods all use reversed-phase C18 columns, HILIC columns, and ADME columns. Therefore, the experiment selected Waters Acquity BEH C18 (100mm×2.1mm, 1.7μm), Waters Acquity BEH HILIC (100mm×2.1mm, 1.7μm), and CAPCELL PAK ADME (150mm×2.1mm, 5μm) columns for comparison. The experiment found that 20 substances obtained relatively symmetrical chromatographic peak shapes on the CAPCELL PAK ADME (150mm×2.1mm, 5μm) column, with good reproducibility, appropriate retention for compounds of different polarities, and low background value for mass spectrometry detection. Therefore, the experiment selected the CAPCELL PAK ADME (150mm×2.1mm, 5μm) column. Taking proline and serine as examples, the results are as follows: Figure 2 As shown;

[0088] 1.2. Selection of mobile phase:

[0089] The experiment selected water-methanol and water-acetonitrile systems as mobile phases respectively, and examined the response values ​​of amino acids in the two groups. The comparison showed that compared with the water-methanol system, the peak response value was higher when water-acetonitrile was used as the mobile phase, and the water-methanol system would cause peak bifurcation. Adding formic acid to the mobile phase can improve the tailing of the sample, poor peak symmetry, and other conditions when the peak is eluted, and can also improve the sensitivity of the test. Therefore, adding 0.1% formic acid to the mobile phase and adding ammonium acetate solution at the same time can adjust the pH and reduce peak tailing. Finally, 2.5mM ammonium acetate 0.1% formic acid water A + 2.5mM ammonium acetate 0.1% formic acid acetonitrile solution B was selected as the mobile phase, and the effects of different gradient mobile phases on the peak shape and sensitivity of the target were examined. Finally, the proportion of organic phase in the mobile phase was changed by gradient elution (Table 1) to obtain a suitable retention time. The target can be effectively separated with good peak shape and symmetry and less interference. Taking leucine and isoleucine as examples, the results are as follows Figure 3 As shown;

[0090] 2. Establishment of mass spectrometry conditions:

[0091] Using water-acetonitrile as the base mobile phase and a "T" three-way connection, the mass spectrometry conditions of 20 target compounds were optimized. Full scans were performed in both positive and negative ion modes to select appropriate molecular ion peaks and ionization modes. Liquid chromatography was coupled with a triple quadrupole mass spectrometer to optimize the ionization efficiency of each target compound, including the ion source temperature, desolvation gas temperature and flow rate, and cone gas flow rate. Detailed information on the 20 amino acid compound standard solutions is shown in Table 2.

[0092] 3. Optimization of sample pretreatment conditions:

[0093] 3.1. Extraction solvent optimization:

[0094] A 0.1 g (accurate to 0.01 g) sample was added with 100 μL of a mixed standard solution with a mass concentration of 1 μg / mL. The extraction solvent selection test was conducted, and the extraction effects of acetonitrile, 90% acetonitrile, and water:methanol (7:3) were compared. The results showed that the response value of acetonitrile extraction was lower than that of 90% acetonitrile extraction, and the nitrogen blow-drying time of water:methanol (7:3) was longer. Therefore, 90% acetonitrile was finally selected as the extraction solvent.

[0095] 3.2 Extraction volume optimization:

[0096] Based on 90% acetonitrile as the extraction solvent, the extraction volume was optimized and the extraction effects of 500 μL, 1 mL and 500 μL (two extractions) were compared. The results showed that 1 mL had the best extraction effect and the highest recovery rate, so 1 mL was selected as the extraction volume.

[0097] 3.3、Derivative Condition Optimization:

[0098] Prepare the n-butanol hydrochloride (3 mol / L) reagent according to the ratio of n-butanol: acetyl chloride (9:1) to ensure the acidic environment for derivatization. At the same time, optimize the amount of derivatization reagent, time and temperature

[31] ;

[0099] 3.3.1. The influence of derivatization temperature on derivatization effect:

[0100] When the derivatization time was 10 min and the amount of derivatization agent added was 100 μL, each group was repeated 6 times, and the total area of ​​20 amino acids was taken as the response value. Figure 5 It can be seen that at 50°C, the total area of ​​amino acids reaches its maximum value. When the derivatization temperature continues to increase, the total area of ​​amino acids gradually decreases. This shows that a proper temperature increase will accelerate the reaction, but too high a temperature will affect the stability of n-butanol hydrochloride. However, due to the different thermal stabilities of different amino acids, the optimal derivatization temperature is not fixed for amino acids.

[0101] 3.3.2. The impact of derivation time on derivation effect:

[0102] When the derivatization temperature is 50℃ and the amount of derivatization agent added is 100μL, the total area of ​​20 amino acids is taken as the response value. Figure 6 It can be seen that when the derivatization time is between 5 and 15 minutes, the total area of ​​amino acids increases significantly with the extension of derivatization time, which indicates that the amino acids are not completely reacted within this time range. When the reaction time reaches 15 minutes, the total area of ​​amino acids reaches the maximum, and as the time continues to extend, the total area of ​​amino acids tends to be stable.

[0103] 3.3.3. The influence of the volume of the derivatizing agent on the derivatization effect:

[0104] When the derivatization temperature is 50℃ and the derivatization time is 10min, the total area of ​​20 amino acids is taken as the response value. Figure 8 It can be seen that when the amount of derivatization agent is less than 100 μL, the total area value of amino acids increases significantly (P<0.05) with the increase of the amount of derivatization agent. When the amount of derivatization agent is 100 μL, the total area value of amino acids increases significantly (P<0.01). When the amount of derivatization agent is further increased, the total area value of amino acids decreases first and then tends to be stable.

[0105] 4. Investigation of matrix effect:

[0106] Matrix effects (ME) refer to the signal suppression or enhancement caused by coeluting substances during chromatographic separation that do not alter the ionization of the analyte. This effect originates from the influence of substances coeluting with the analyte on the ionization process of the analyte during the chromatographic separation process. Coeluting interfering substances can be divided into endogenous and exogenous impurities, so matrix effects need to be evaluated. This experiment used an internal standard method to reduce or offset matrix effects. The responses of the analyte in the presence and absence of matrix components were compared. ME is equal to the response of the extracted blank matrix added to the analyte / the response of the analyte in pure solution. Because the chemical properties of the isotopic internal standard are the same as those of the analyte, the influence of matrix effects can be largely offset during the sample extraction and ionization process. Samples were selected, and blank substrate spiked solutions were prepared at concentrations of 1, 2, and 10 times the mass of the sample. Standard solutions of the same concentration were also prepared. The matrix effect was determined as the percentage of the response intensity of the two. Each sample was measured six times, and the matrix effect ranged from 70% to 101%. The results are shown in Table 6.

[0107] 5. Method validation:

[0108] 5.1 Specificity

[0109] The primary objective is to examine whether the method can accurately determine the characteristics of the analyte in the presence of other components (impurities, degradation products, excipients, etc.). Specificity reflects the method's ability to accurately and uniquely determine the analyte in the presence of coexisting substances and is a measure of the degree of mutual interference in complex sample analysis. To evaluate method specificity, solvent blanks (90% acetonitrile, n-butanol hydrochloride) and samples were spiked with and without spiked solutions.

[0110] 5.2 Linear Relationship, Detection Limit and Quantification Limit:

[0111] In the experiment, a certain amount of mixed standard solution and deuterated isotope internal standard mixed standard solution were added to the blank solution to prepare 8 standard series with concentrations of 1 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, 400 ng / mL and 1000 ng / mL. The response peak area of ​​the quantitative ion (Y axis) was plotted against the corresponding mass concentration (X axis, ng / mL)

[36] . The results are shown in Table 4. The results show that there is a good linear relationship between the response peak area of ​​the quantitative ion and the sample concentration in the range of 1 to 1000 ng / mL for the 20 amino acids, and the correlation coefficient R2 is greater than 0.99. The signal-to-noise ratio (S / N) is set as the detection limit of the method, and S / N=10 is set as the quantification limit. The detection limit of the 20 amino acids is 0.3 ng / mL, and the quantification limit is 1 ng / mL.

[0112] Table 4 Linear equations and correlation coefficients

[0113] Table 4 Linear equations and correlation coefficients

[0114]

[0115]

[0116] 5.3 Matrix Effect and Addition Recovery

[0117] Since there was no suitable blank substrate, the sample spike method was selected to investigate the matrix effect and the recovery test. The spike recovery test was performed at 1 times, 2 times, and 10 times the experimental lower limit. Each sample was repeated 6 times. The matrix effect ranged from 70.83% to 129%, the RSD ranged from 0.52% to 10.87%, the average recovery rate was 73.17% to 125.27%, and the precision ranged from 0.79% to 13.43%. The deviation of the characteristic ion abundance ratio of the spiked sample and the standard substance was within 15%. The results are shown in Table 5.

[0118] Table 5 Matrix effects of 20 amino acids and their addition

[0119]

[0120]

[0121] 5.4 Precision:

[0122] Precision is mainly used to measure the closeness between the results of multiple sampling and determination of the same homogeneous sample under the same conditions. We weighed 0.1g of pear fruit sample, and prepared low, medium and high concentration samples at 1 times, 2 times and 10 times the limit of quantification, respectively. Six samples of each concentration were prepared in parallel and measured continuously for 3 days to investigate the intra-day and inter-day precision. The results showed that the intra-day precision was 1.64%-11.63%, the inter-day precision was 5.04%-14.96%, and the relative standard deviation (RSD) of the intra-day and inter-day precision was ≤15%. The experimental results met the determination requirements of GB / T 27417-2017

[38] . The specific experimental data are shown in Table 6 below.

[0123] Table 6 Precision investigation

[0124] Table 6 Precision investigation

[0125]

[0126] 5.5 Stability

[0127] The main purpose was to investigate the magnitude of temporal changes in the standards under the influence of temperature, humidity, or light, which can provide a scientific basis for their production, packaging, and storage. The amino acid standards and internal standard stock solutions were stored at 4°C and tested at 1, 2, 4, and 8 weeks (n=3) and analyzed after three freeze-thaw cycles (freezing at -20°C and thawing at room temperature). According to GB5009.295-2023, if the analyte area changes within 15% compared to the freshly prepared analyte area, the analyte is considered stable in terms of storage conditions and time. The results are shown in Table 7.

[0128] Table 7 Stability of the method

[0129]

[0130]

[0131] 5.6. Durability inspection:

[0132] Durability mainly examines the ability of the measurement results to withstand small changes in the measurement conditions. According to the Guidelines for Validation of Analytical Methods in the 2020 edition of the Chinese Pharmacopoeia, typical UPLC-MS / MS variation factors include mobile phase composition, different brands or batches of the same type of chromatographic columns, column temperature, and flow rate. Therefore, this experiment mainly examined the concentration changes of amino acid standards under column temperature (20°C ± 0.05°C), flow rate (0.4 mL / min ± 0.05), and mobile phase concentration (2.5 mmol ± 0.05). The results showed that the RSD range was less than 2%, indicating good durability.

[0133] Table 8 Durability of the method

[0134]

[0135]

[0136] 6. Actual sample measurement:

[0137] The established amino acid determination method was used to determine actual samples at different times and temperatures. No cysteine, lysine, histidine, or tryptophan was detected. The results of the remaining 16 amino acids are as follows:

[0138] Table 9 Measured values ​​of temperature samples at different time

[0139]

[0140] 7. Comparison with other methods:

[0141] The established method was compared with existing amino acid detection methods (see Table 10). Currently, the LC-MS method detects more amino acids in blood samples and fewer in food, but it rarely involves pear fruit matrices. The existing methods have problems such as cumbersome and complex operations and a small number of detection types. The method established in this study can directly measure multiple amino acids in fruits, with a short detection cycle of only 9 minutes and a lower detection limit. The established method was compared with existing different amino acid detection methods (see Table 10);

[0142] Table 10 Comparison of amino acid detection methods

[0143]

[0144]

[0145] The main functions achieved by the present invention are: this method utilizes triple quadrupole mass spectrometry, is suitable for the determination of lower concentrations of amino acids, has good selectivity, the target substances have good chromatographic separation and mass spectrometric response, the standard curve is well within the linear range, and the intra-day and inter-day precision, accuracy, limit of quantification, recovery rate and matrix effect all meet the requirements for verification of relevant chemical methods, and is suitable for the rapid and simple detection of amino acids in pear fruit.

[0146] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for detecting amino acids in fresh pears, characterized in that: Step 1. Experimental instruments and reagents: Experimental instruments: TQ-S tandem quadrupole mass spectrometer, CAPCELL PAKADME chromatographic column, centrifuge, vortex oscillator, electric thermostatic water bath, nitrogen blow dryer, ultrapure water system and electronic analytical balance; Reagents: acetonitrile, methanol, n-butanol, acetyl chloride, amino acid standards, and deuterated isotope standards; Step 2: Preparation of standard solution: Prepare amino acid standard stock solutions, amino acid internal standard stock solutions, and derivatization reagents; Step 3: Sample preparation: Weigh 0.1 g of sample into a 1.5 mL plastic centrifuge tube with a stopper, add 20 μL of internal standard, then add 1 mL of 90% acetonitrile, vortex mix, perform low-temperature ultrasonic extraction and refrigerated centrifugation, transfer the supernatant to a clean nitrogen blowpipe, blow dry with nitrogen, add 100 μL of derivatization reagent to the nitrogen blowpipe, vortex and oscillate for 2 minutes, derivatize at 50°C for 15 minutes, blow dry with nitrogen, add 300 μL of water, reconstitute with acetonitrile, filter through a 0.22 μm filter, and wait for detection; Step 4. Chromatographic and mass spectrometric conditions: Liquid chromatography conditions: a. Chromatographic column: CAPCELL PAK ADME chromatographic column, 150 mm × 2.1 mm, 5 μm; b. Mobile phase: 2.5 mM ammonium acetate 0.1% formic acid in water (A) + 2.5 mM ammonium acetate 0.1% formic acid in acetonitrile (B); gradient elution; c. Column temperature: 40°C; d. Injection volume: 5 μL; Mass spectrometry conditions: Ion source: electrospray ion source; Scanning mode: positive ion scanning; Capillary voltage: 0.5kV; Desolventization temperature: 1000℃; Desolventizing gas flow rate: 500L / Hr; Collision gas: argon, flow rate 1.7 mL / min; Mass spectrometry scanning mode: segmented multiple reaction monitoring mode acquisition.

2. A method for detecting amino acids in fresh pears according to claim 1, characterized in that: Amino acid standard stock solution: Accurately weigh 10 mg of amino acid standard substance, dissolve it in water and dilute to the scale, prepare a standard stock solution with a concentration of 100 μg / mL for storage. Accurately pipette sufficient amount of amino acid standard stock solution as needed, dilute with water to prepare a standard intermediate solution with a concentration of 1 μg / mL.

3. A method for detecting amino acids in fresh pears according to claim 1, characterized in that: Amino acid internal standard stock solution: Weigh 10 mg of amino acid internal standard standard substance, dissolve it in water and dilute to the scale to prepare a standard stock solution with a concentration of 100 μg / mL. Dilute the internal standard intermediate solution with water as needed to prepare a standard working solution with a concentration of 1 μg / mL. Before use, dilute the standard intermediate solution with the initial mobile phase to a working solution of appropriate concentration to ensure that the concentration of each internal standard is 200 ng / mL.

4. A method for detecting amino acids in fresh pears according to claim 1, characterized in that: Derivatization reagent: n-butanol and acetyl chloride are prepared in a ratio of 9:

1. The reaction principle is R1OH+ClR2O=R2OOR1+HCl; Reaction principle: After the derivatization reaction, the amino acid compound reacts with n-butanol hydrochloride to generate butylated amino acid.

5. A method for detecting amino acids in fresh pears as claimed in claim 3, characterized in that: The amino acid standard stock solution and the amino acid internal standard stock solution in step 2 must be stored at 4° C. in the dark.

6. A method for detecting amino acids in fresh pears according to claim 1, characterized in that: The purity of the amino acid standard and the deuterated isotope standard in step 1 are both greater than 95%.

7. A method for detecting amino acids in fresh pears according to claim 1, characterized in that: After vortex mixing in step 3, low-temperature ultrasonic extraction was performed for 15 minutes, and refrigerated centrifugation was performed at 12000 r / min for 10 minutes.

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