Method for detecting various amino acids in fish meat

Abstract

The invention relates to the technical field of biochemical detection, and discloses a method for detecting various amino acids in fish meat, which comprises the following steps: mixing a fish meat sample with a formic acid aqueous solution, grinding and homogenizing at low temperature, then centrifuging, and taking supernate after centrifuging; adding a protein precipitant into the supernate, carrying out vortex uniform mixing, centrifuging, taking the supernate, carrying out low-temperature refrigeration on the obtained supernate, centrifuging again, and taking the final supernate as a to-be-detected solution; and injecting the to-be-detected liquid into an LC-Q-TOF system for analysis and detection to obtain a detection result. The method provided by the invention can simultaneously detect 17 amino acids in fish meat, can ensure separation of leucine and isoleucine isomeride, and is high in recovery rate, low in detection limit, high in sensitivity and good in stability.

Description

Technical Field

[0001] This invention relates to the field of biochemical detection technology, and more specifically, to a method for detecting multiple amino acids in fish meat. Background Technology

[0002] Amino acids are the basic building blocks of protein molecules in living organisms and are closely related to life activities. Metabolic activities in organisms cannot proceed without amino acids, and fish, as an important food source, are a crucial pathway for humans to obtain amino acids. Furthermore, amino acids are one of the main sources of umami flavor in fish. Glutamic acid, aspartic acid, glycine, and alanine, among others, have a significant impact on fish flavor. Measuring the content of free amino acids in fish helps control fish quality in aquaculture and is of great significance to the aquaculture industry.

[0003] Existing methods for amino acid detection mainly include pre-column derivatization-ion exchange chromatography, gas chromatography, liquid chromatography, capillary electrophoresis, and liquid chromatography-tandem mass spectrometry (LC-MS / MS). Among these, the most commonly used are amino acid analyzers, high-performance liquid chromatography (HPLC), and LC-MS / MS. Due to the inherent properties of amino acids, such as low volatility and lack of UV absorption, derivatization is often required during chromatographic detection. This process is complex, involves expensive derivatization reagents, has poor reproducibility, and cannot be applied to all amino acids with a single derivatization reagent. Furthermore, chromatographic detection requires absolute resolution in the chromatogram, thus increasing the analysis time as the number of amino acids detected increases. These shortcomings lead to prolonged detection cycles, low experimental efficiency, and high costs. In contrast, liquid chromatography-tandem mass spectrometry (LC-MS / MS) offers significant advantages. As a highly selective and sensitive detection tool, it quantifies amino acids based on their mass-to-charge ratio (m / z), eliminating the need for derivatization, shortening detection time, improving efficiency, saving costs, and enabling isotope-labeled quantification. However, the detection method of liquid chromatography-tandem mass spectrometry (LC-MS / MS) has high requirements for pretreatment and is easily affected by matrix interference. Due to the structural characteristics of amino acids, ion fragments with similar or even the same mass number may appear during the detection process of liquid chromatography-tandem mass spectrometry (LC-MS / MS), which may interfere with the acquisition.

[0004] Currently, amino acid mass spectrometry analysis is mainly achieved through liquid chromatography-tandem mass spectrometry (LC-MS / MS), primarily used to detect free amino acids in plants or plasma. Complete protocols for detecting free amino acids in muscle are rare. This is mainly because, firstly, muscle samples have a richer matrix and higher protein content compared to plasma or plant tissues, making them more complex. LC-MS / MS is sensitive to matrix effects, and pretreatment of complex samples for LC-MS / MS detection presents significant challenges. Secondly, due to the high polarity of amino acids, they are difficult to retain on ordinary C18 columns. Some detection methods still require derivatization or ion-pairing reagents to ensure retention time on C18 columns. Both of these methods have their drawbacks. Derivatization, by reacting derivatizing reagents with amino acids to generate less polar compounds and thus achieve retention, involves cumbersome pretreatment procedures, is time-consuming, and the derivatizing reagents are expensive and not universally applicable. Using ion-pairing reagents as the mobile phase will increase the matrix effect of mass spectrometry. Moreover, mass spectrometry has limited tolerance to ion-pairing reagents. Ion-pairing reagents can cause enhanced matrix interference, decreased mass spectrometry signal, increased mass spectrometry maintenance costs, and even affect detection results.

[0005] In the prior art, patent publication number CN117030878A, entitled "A Stable Method for the Detection of Underrivatized Free Amino Acids and Its Application," discloses a method of precipitating proteins from samples using deoxycholate and trichloroacetic acid, followed by detection by liquid chromatography-mass spectrometry (LC-MS). To reduce matrix interference and sample complexity, trichloroacetic acid precipitation significantly reduces the sample matrix. However, trichloroacetic acid has a significant impact on the uniformity of the C18 column and the ESI (extra-spray ionization) source in the mass spectrometer, leading to decreased column efficiency and increased background noise from the mass spectrometer ion source with long-term use. Patent publication number CN112730723A, entitled "A Method for the Detection of 22 Free Amino Acids in Plasma by Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry," discloses extraction with formic acid, methanol, and ethyl acetate, followed by nitrogen drying and reconstitution for detection. The addition of heptafluorobutyrate ion-pairing reagent to the mobile phase can contaminate the mass spectrometer and increase maintenance costs. The above-mentioned liquid chromatography-tandem mass spectrometry (LC-MS / MS) detection methods all have the problems of high requirements for pretreatment, severe matrix interference when detecting complex samples, and the addition of fluorine or chlorine-containing compounds in the pretreatment or mobile phase, which greatly damages the mass spectrometry.

[0006] Therefore, providing a method for detecting multiple amino acids in fish meat based on liquid chromatography-quadrupole time-of-flight mass spectrometry that can solve the above-mentioned technical problems has important practical significance. Summary of the Invention

[0007] In view of this, the present invention proposes a method for detecting multiple amino acids in fish meat, aiming to solve at least one of the aforementioned background technical problems.

[0008] This invention proposes a method for detecting multiple amino acids in fish meat, comprising the following steps: Step 1: Mix the fish meat sample with formic acid aqueous solution, grind and homogenize at low temperature, then centrifuge and take the supernatant after centrifugation; Step 2: Add protein precipitant to the supernatant, vortex to mix, centrifuge to collect the supernatant, refrigerate the obtained supernatant at low temperature, centrifuge again, and take the final supernatant as the test solution. Step 3: Inject the test solution obtained in Step 2 into the LC-Q-TOF system for analysis and detection, and obtain the detection results; The protein precipitant is a mixed solution of methanol, acetonitrile, and formic acid. The LC-Q-TOF system uses an amide column for chromatographic separation, and the mobile phase does not contain ion-pairing reagents.

[0009] Preferably, the volume fraction of formic acid in the formic acid aqueous solution is 0.1%; the ratio of the fish meat sample to the formic acid aqueous solution is 0.1g:50μL.

[0010] Preferably, the volume ratio of methanol, acetonitrile, and formic acid in the protein precipitant is 1.5~2.5:1.5~2.5:0.001~0.005.

[0011] Preferably, the temperature in the low-temperature grinding homogenate is 4°C; the low-temperature refrigeration temperature is -15~-25°C, and the time is 15-30 min.

[0012] Preferably, the amide column has dimensions of 2.1mm × 100mm and a diameter of 1.7μm; the column temperature is 30-40℃.

[0013] Preferably, the mobile phase comprises: mobile phase A and mobile phase B, wherein mobile phase A is an aqueous solution containing 5-20 mmol / L ammonium acetate and 0.01%-0.05% formic acid; Mobile phase B is acetonitrile; gradient elution is used in the analysis and detection, with a flow rate of 0.3-0.5 mL / min.

[0014] Preferably, the gradient elution procedure is as follows: 0 min: Mobile phase A 10%, Mobile phase B 90%; 4 min: Mobile phase A 20%, Mobile phase B 80%; 8 min: Mobile phase A 60%, Mobile phase B 40%; 10.0 min: Mobile phase A 60%, Mobile phase B 40%; 11 min: Mobile phase A 10%, Mobile phase B 90%; 13 min: Mobile phase A 10%, Mobile phase B 90%.

[0015] Preferably, the mass spectrometry detection conditions for the LC-Q-TOF are: electrospray ionization source, positive ion mode; scanning mass range 50-500 m / z; spray voltage 3000-4000 V; ion source temperature 300-400 °C; and transfer tube temperature 300-350 °C.

[0016] Preferably, the amino acids that the method can simultaneously detect include glycine, alanine, serine, proline, valine, threonine, leucine, isoleucine, aspartic acid, lysine, glutamic acid, methionine, histidine, phenylalanine, arginine, tyrosine, and cystine.

[0017] This invention also provides the application of the method described above in the simultaneous detection of multiple amino acids.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The method provided by this invention can simultaneously detect 17 amino acids in fish meat without derivatization or the use of ion-pairing reagents. It can ensure the separation of leucine and isoleucine isomers, with high recovery, low detection limit, high sensitivity, and good stability. Based on the advantages of ultra-high resolution and ultra-high quality precision of LC-Q-TOF, its resistance to matrix interference is also higher than that of liquid chromatography-tandem mass spectrometry (LC-MS / MS). It can meet the requirements for analyzing complex samples and can distinguish compounds with very similar mass numbers, including isomers and compounds with similar elemental compositions. LC-Q-TOF has stronger resistance to interference and higher selectivity for complex biological samples (plasma, tissue), environmental samples, food and other samples with severe matrix interference. By obtaining the precise mass number of the compound, accurate to 4 decimal places or more, it can clearly characterize isotope pattern recognition and use the isotope peak clusters of the compound for confirmation. It can resist interference and reduce complex pretreatment, and avoid the interference problem of ion fragments with the same mass number on liquid chromatography-tandem mass spectrometry (LC-MS / MS). In addition, liquid chromatography-tandem time-of-flight mass spectrometry (LC-Q-TOF) can more clearly characterize isotope patterns and more easily identify and confirm compounds using isotope peak clusters.

[0019] 2. The protein pretreatment precipitation method of this invention is simple to operate, causes little damage to the sample, has a high recovery rate of the target analyte, and the protein is well precipitated, with minimal impact on the chromatographic column and mass spectrometer. Furthermore, this invention requires a small sample volume, with an injection volume of only 1 μL to simultaneously detect 17 amino acids. For precious samples with complex matrices, no complicated pretreatment is required, ensuring sample recovery and detection results.

[0020] 3. This invention uses an Amide 2.1X100mm 1.7um chromatographic column, eliminating the need for ion-pairing reagents as the mobile phase. The conventional mass spectrometry mobile phase is sufficient to ensure compound retention time and separate isomers, reducing column and mass spectrometry losses. This invention also features a short analysis time, completing the acquisition and analysis of a single sample in 13 minutes, enabling rapid analysis and detection of large quantities of complex matrix samples.

[0021] 4. The reagents used in the pretreatment and sample analysis of this invention are all conventional chemical reagents, which are simple to operate. Even for samples with complex technical matrices, there is no need for expensive pretreatment purification consumables, and the cost of the entire detection process is low. This invention has good detection repeatability, high sensitivity, strong specificity, and good anti-interference performance. Attached Figure Description

[0022] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Furthermore, the same reference numerals denote the same parts throughout the drawings. In the drawings: Figure 1 This is the mass spectrometry characteristic spectrum of glycine (Gly); Figure 2 This is the mass spectrometry characteristic spectrum of alanine (Ala); Figure 3 Mass spectrometry characteristic spectrum of serine (Ser); Figure 4 The mass spectrometry characteristic spectrum of proline (Pro); Figure 5 Mass spectrometry characteristic spectrum of valine (Val); Figure 6 The mass spectrometry characteristic spectrum of threonine (Thr); Figure 7 The mass spectrometry characteristic spectrum of leucine (Leu); Figure 8 The mass spectrometry characteristic spectrum of isoleucine (Ile); Figure 9 The mass spectrometry characteristic spectrum of aspartic acid (Asp); Figure 10 The mass spectrometry characteristic spectrum of lysine (Lys); Figure 11 Mass spectrometry characteristic spectrum of glutamic acid (Glu); Figure 12 This is a mass spectrometry characteristic spectrum of methionine (Met); Figure 13 This is a mass spectrometry profile of histidine (His). Figure 14This is the mass spectrometry characteristic spectrum of phenylalanine (Phe); Figure 15 Mass spectrometry characteristic spectrum of arginine (Arg); Figure 16 The mass spectrometry characteristic spectrum of tyrosine (Tyr); Figure 17 Mass spectrometry characteristic spectrum of cystine (Cys)2; Figure 18 The total ion current chromatogram (TIC chromatogram) of 17 amino acids is shown. Detailed Implementation

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0024] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included within this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0025] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0026] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0027] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0028] This invention provides a method for detecting multiple amino acids in fish meat. Specifically, this method is based on liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QFS) and includes the following steps: Step 1: Mix the fish meat sample with formic acid aqueous solution, grind and homogenize at low temperature, then centrifuge and take the supernatant after centrifugation; Step 2: Add protein precipitant to the supernatant, vortex to mix, centrifuge to collect the supernatant, refrigerate the obtained supernatant at low temperature, centrifuge again, and take the final supernatant as the test solution. Step 3: Inject the test solution obtained in Step 2 into the LC-Q-TOF system for analysis and detection, and obtain the detection results; The protein precipitant is a mixed solution of methanol, acetonitrile, and formic acid. The LC-Q-TOF system uses an amide column for chromatographic separation, and the mobile phase does not contain ion-pairing reagents.

[0029] Step 1: Mix the fish meat sample with formic acid aqueous solution, grind and homogenize at low temperature, then centrifuge and take the supernatant after centrifugation; In this invention, the volume fraction of formic acid in the formic acid aqueous solution is preferably 0.1%; the ratio of the amount of fish meat sample to the formic acid aqueous solution is preferably 0.1g:50μL.

[0030] This invention first mixes fish meat samples with formic acid aqueous solution in a certain proportion, and grinds them at 4°C with a grinding homogenizer at 6000 Hz for 2 minutes. Following this, the mixture is rapidly centrifuged for 30 seconds to obtain the supernatant. This invention extracts amino acids from fish meat by adding a certain proportion of formic acid aqueous solution and grinding at 4°C. This method is simple, rapid, and requires no derivatization; the addition of formic acid facilitates amino acid dissolution and results in a high recovery rate.

[0031] Step 2: Add protein precipitant to the supernatant, vortex to mix, centrifuge to collect the supernatant, refrigerate the obtained supernatant at low temperature, centrifuge again, and take the final supernatant as the test solution. The protein precipitant is preferably a mixed solution of methanol, acetonitrile, and formic acid; In this invention, the preferred volume ratio of methanol, acetonitrile, and formic acid in the protein precipitant is 1.5~2.5:1.5~2.5:0.001~0.005.

[0032] Specifically, in this invention, the protein precipitant is mixed with the supernatant obtained in step 1 by vortexing for 30 seconds each time, repeated 3 times. Then, centrifugation is performed at 4°C for 15 minutes. After centrifugation, the sample is refrigerated at -15 to -25°C for 15-30 minutes, followed by a second centrifugation at 4°C for 15 minutes. Freezing and centrifuging the intermediate sample and then refrigerating it for 15-30 minutes more effectively removes proteins and reduces the impact on the chromatographic column. The entire pretreatment process can be completed within 1 hour.

[0033] In this invention, the temperature in the low-temperature grinding homogenate is preferably 4°C; the temperature of the low-temperature refrigeration is preferably -15~-25°C, and the time is preferably 15-30 min.

[0034] Step 3: Inject the test solution obtained in Step 2 into the LC-Q-TOF system for analysis and detection, and obtain the detection results; In this invention, the liquid chromatography-tandem time-of-flight mass spectrometry (LC-Q-TOF) instrument is from Agilent Technologies, USA. This invention uses LC-Q-TOF as the detection instrument, whose ultra-high resolution is far superior to that of liquid chromatography-tandem mass spectrometry (LC-MS / MS), offering greater advantages for the detection of isomers and samples with complex matrices.

[0035] The LC-Q-TOF system described in this invention uses an amide column for chromatographic separation, and the mobile phase does not contain ion-pairing reagents. More specifically: In this invention, the amide column has the following specifications: Amide 2.1mm×100mm, 1.7μm; and the column temperature is 30-40℃.

[0036] In this invention, the mobile phase comprises: mobile phase A and mobile phase B, wherein mobile phase A is an aqueous solution containing 5-20 mmol / L ammonium acetate and 0.01%-0.05% formic acid; Mobile phase B is acetonitrile; gradient elution is used in the analysis and detection, and the flow rate is preferably 0.3-0.5 mL / min, more preferably 0.4 mL / min.

[0037] In this invention, the gradient elution procedure is as follows: 0 min: Mobile phase A 10%, Mobile phase B 90%; 4 min: Mobile phase A 20%, Mobile phase B 80%; 8 min: Mobile phase A 60%, Mobile phase B 40%; 10 min: Mobile phase A 60%, Mobile phase B 40%; 11 min: Mobile phase A 10%, Mobile phase B 90%; 13 min: Mobile phase A 10%, Mobile phase B 90%.

[0038] In this invention, the mass spectrometry detection conditions of the LC-Q-TOF are as follows: electrospray ionization source, positive ion mode; scanning mass range 50-500 m / z; spray voltage 3000-4000 V; ion source temperature 300-400 °C; transmission tube temperature 300-350 °C.

[0039] In this invention, the amino acids that the method can simultaneously detect include glycine, alanine, serine, proline, valine, threonine, leucine, isoleucine, aspartic acid, lysine, glutamic acid, methionine, histidine, phenylalanine, arginine, tyrosine, and cystine.

[0040] This invention utilizes an Amide 2.1 x 100 mm 1.7 μm column, effectively separating 17 amino acids without adding ion-pairing reagents to the mobile phase or derivatizing the sample. This effectively avoids the problem of amino acids lacking retention time in C18 columns due to polarity issues, requiring the use of ion-pairing reagents or sample derivatization to enhance retention time. Adding 20 μM ammonium acetate and 0.1% formic acid to the mobile phase, gradient elution, and signal acquisition of all 17 amino acids is completed within 13 minutes. Each amino acid exhibits high signal intensity, effectively separating isomers and improving analytical efficiency. This provides a simple, rapid, and efficient method for amino acid detection in complex matrix samples such as muscle.

[0041] Example 1 1. Reagent preparation Formic acid aqueous solution for homogenization: Prepare a 0.1% formic acid aqueous solution for later use; Reagent for protein precipitation: Prepare by mixing methanol, acetonitrile, and formic acid in a volume ratio of 2:2:0.002, and set aside for later use; Standard: Purchase 17 amino acid mixed standards from Anpu, with a concentration of 2500 nmol / ml, and use 1% hydrochloric acid aqueous solution as solvent for later use.

[0042] 2. Sample pretreatment Weigh 0.3g of fish meat sample, put it into a 1.5mL EP tube, and add 2-3 magnetic beads into the tube; Add 1.5 mL of the prepared 0.1% formic acid aqueous solution to the EP tube; The EP tube was placed in an environment of 4℃ and ground into a homogenate at a power of 6000Hz for 3 minutes to obtain a homogenate solution. Measure 200 μL of homogenate and transfer it to a new centrifuge tube. Add 800 μL of protein precipitation reagent to the centrifuge tube. Mix using a vortex method, each vortex lasting 30 seconds, repeating the vortexing process 3 times; Place the centrifuge tubes at 4°C and centrifuge at 12000g for 15 minutes. Collect the supernatant after centrifugation. Place the supernatant at -20℃ for 20 minutes; The supernatant after refrigeration was placed at 4°C and centrifuged at 12000g for 15 minutes. The supernatant was then collected to complete the sample pretreatment.

[0043] 3. Detection by liquid chromatography-quadrupole time-of-flight mass spectrometry Instrumentation: Liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-Q-TOF) system from Agilent Technologies, USA. Chromatographic conditions: Column: Amide 2.1×100mm 1.7μm; Column temperature: 35℃; Injection volume: 1 μL; Mobile phase: Mobile phase A is an aqueous solution containing 20 mmol ammonium acetate and 0.1% formic acid, and mobile phase B is pure acetonitrile; Gradient elution program: 0 min (A: 10%, B: 90%, flow rate 0.4 mL / min) → 4 min (A: 20%, B: 80%, flow rate 0.4 mL / min) → 8 min (A: 60%, B: 40%, flow rate 0.4 mL / min) → 10 min (A: 60%, B: 40%, flow rate 0.4 mL / min) → 11 min (A: 10%, B: 90%, flow rate 0.4 mL / min) → 13 min (A: 10%, B: 90%). Mass spectrometry conditions: Ion source: Electrospray ionization (ESI), positive ion mode; Spray voltage: 3500V; Transmission tube temperature: 320℃; Ion source temperature: 350℃; Auxiliary air flow rate: 3L / min; Full scan mass range: 50-500 m / z; Detection: Inject the pretreated supernatant into the sample vial, perform detection according to the above chromatographic and mass spectrometric conditions, and record the detection data.

[0044] Example 2 1. Reagent preparation Formic acid aqueous solution for homogenization: Prepare a 0.1% formic acid aqueous solution for later use; Reagent for protein precipitation: Prepared by mixing methanol, acetonitrile, and formic acid in a volume ratio of 1.5:1.5:0.001, and set aside for later use; Standard: Purchase 17 amino acid mixed standards from Anpu, with a concentration of 2500 nmol / ml, and use 1% hydrochloric acid aqueous solution as solvent for later use.

[0045] 2. Sample pretreatment Weigh 0.3g of fish meat sample, put it into a 1.5mL EP tube, and add 2-3 magnetic beads into the tube; Add 1.5 mL of the prepared 0.1% formic acid aqueous solution to the EP tube; The EP tube was placed in an environment of 4℃ and ground into a homogenate at a power of 6000Hz for 3 minutes to obtain a homogenate solution. Measure 200 μL of homogenate and transfer it to a new centrifuge tube. Add 800 μL of protein precipitation reagent to the centrifuge tube. Mix using a vortex method, each vortex lasting 30 seconds, repeating the vortexing process 3 times; Place the centrifuge tubes at 4°C and centrifuge at 12000g for 15 minutes. Collect the supernatant after centrifugation. Place the supernatant at -15℃ for 15 minutes; The supernatant after refrigeration was placed at 4°C and centrifuged at 12000g for 15 minutes. The supernatant was then collected to complete the sample pretreatment.

[0046] 3. Detection by liquid chromatography-quadrupole time-of-flight mass spectrometry Instrumentation: Liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-Q-TOF) system from Agilent Technologies, USA. Chromatographic conditions: Column: Amide 2.1×100mm 1.7μm; Column temperature: 30℃; Injection volume: 1 μL; Mobile phase: Mobile phase A is an aqueous solution containing 5 mmol ammonium acetate and 0.1% formic acid, and mobile phase B is pure acetonitrile; Gradient elution program: 0 min (A: 10%, B: 90%, flow rate 0.4 mL / min) → 4 min (A: 20%, B: 80%, flow rate 0.4 mL / min) → 8 min (A: 60%, B: 40%, flow rate 0.4 mL / min) → 10 min (A: 60%, B: 40%, flow rate 0.4 mL / min) → 11 min (A: 10%, B: 90%, flow rate 0.4 mL / min) → 13 min (A: 10%, B: 90%). Mass spectrometry conditions: Ion source: Electrospray ionization (ESI), positive ion mode; Spray voltage: 3000V; Transmission tube temperature: 300℃; Ion source temperature: 300℃; Auxiliary air flow rate: 3L / min; Full scan mass range: 50-500 m / z; Detection: Inject the pretreated supernatant into the sample vial, perform detection according to the above chromatographic and mass spectrometric conditions, and record the detection data.

[0047] Example 3 1. Reagent preparation Formic acid aqueous solution for homogenization: Prepare a 0.1% formic acid aqueous solution for later use; Reagent for protein precipitation: Prepared by mixing methanol, acetonitrile, and formic acid in a volume ratio of 2.5:2.5:0.005, and set aside for later use; Standard: Purchase 17 amino acid mixed standards from Anpu, with a concentration of 2500 nmol / ml, and use 1% hydrochloric acid aqueous solution as solvent for later use.

[0048] 2. Sample pretreatment Weigh 0.3g of fish meat sample, put it into a 1.5mL EP tube, and add 2-3 magnetic beads into the tube; Add 1.5 mL of the prepared 0.1% formic acid aqueous solution to the EP tube; The EP tube was placed in an environment of 4℃ and ground into a homogenate at a power of 6000Hz for 3 minutes to obtain a homogenate solution. Measure 200 μL of homogenate and transfer it to a new centrifuge tube. Add 800 μL of protein precipitation reagent to the centrifuge tube. Mix using a vortex method, each vortex lasting 30 seconds, repeating the vortexing process 3 times; Place the centrifuge tubes at 4°C and centrifuge at 12000g for 15 minutes. Collect the supernatant after centrifugation. Place the supernatant at -25°C for 30 minutes; The supernatant after refrigeration was placed at 4°C and centrifuged at 12000g for 15 minutes. The supernatant was then collected to complete the sample pretreatment.

[0049] 3. Detection by liquid chromatography-quadrupole time-of-flight mass spectrometry Instrumentation: Liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-Q-TOF) system from Agilent Technologies, USA. Chromatographic conditions: Column: Amide 2.1×100mm 1.7μm; Column temperature: 40℃; Injection volume: 1 μL; Mobile phase: Mobile phase A is an aqueous solution containing 20 mmol ammonium acetate and 0.1% formic acid, and mobile phase B is pure acetonitrile; Gradient elution program: 0 min (A: 10%, B: 90%, flow rate 0.4 mL / min) → 4 min (A: 20%, B: 80%, flow rate 0.4 mL / min) → 8 min (A: 60%, B: 40%, flow rate 0.4 mL / min) → 10 min (A: 60%, B: 40%, flow rate 0.4 mL / min) → 11 min (A: 10%, B: 90%, flow rate 0.4 mL / min) → 13 min (A: 10%, B: 90%). Mass spectrometry conditions: Ion source: Electrospray ionization (ESI), positive ion mode; Spray voltage: 4000V; Transfer tube temperature: 350℃; Ion source temperature: 400℃; Auxiliary air flow rate: 3L / min; Full scan mass range: 50-500 m / z; Detection: Inject the pretreated supernatant into the sample vial, perform detection according to the above chromatographic and mass spectrometric conditions, and record the detection data.

[0050] Comparative Example 1 1. Sample pretreatment Weigh 0.1g of homogenized fish meat sample into a centrifuge tube, add 1mL of 0.1mol / L hydrochloric acid solution, vortex to mix, extract by sonication at 4℃ for 30 minutes, centrifuge at 4℃ and 12000 rpm for 15 minutes, and take the supernatant.

[0051] Pre-column derivatization: Take 100 μL of supernatant, add 20 μL of 0.1 mol / L borate buffer (pH 9.0) and 50 μL of 6-aminoquinoline-N-hydroxysuccinimide carbamate (AQC) derivatizing reagent (3 mg / mL acetonitrile solution), and vortex to mix. Incubate the mixture in a 55°C water bath for 10 minutes to complete derivatization. Extract the derivatized solution twice with 0.5 mL of ethyl acetate to remove excess reagent, and collect the lower aqueous phase. Filter the aqueous phase through a 0.22 μm microporous membrane to obtain the test solution.

[0052] 2. Detection by liquid chromatography-tandem mass spectrometry (LC-MS / MS) Instrument: Liquid chromatography-tandem triple quadrupole mass spectrometry system.

[0053] Chromatographic conditions: Chromatographic column: C18 column (2.1 mm × 100 mm, 1.7 μm).

[0054] Column temperature: 40℃.

[0055] Injection volume: 5 μL.

[0056] Mobile phase: Mobile phase A: An aqueous solution containing 0.1% heptafluorobutyric acid (HFBA) and 5 mmol / L ammonium formate. (Note: Use a fluoride ion-pairing reagent.) Mobile phase B: An acetonitrile solution containing 0.1% formic acid.

[0057] Gradient elution program: 0 min (A: 95%, B: 5%) → 10 min (A: 60%, B: 40%) → 12 min (A: 5%, B: 95%) → 15 min (A: 5%, B: 95%) → 16 min (A: 95%, B: 5%), equilibration for 5 minutes. Total run time: 21 minutes.

[0058] Flow rate: 0.3 mL / min.

[0059] 3. Mass spectrometry conditions: Ion source: Electrospray ionization (ESI) source, positive ion mode.

[0060] Scanning method: Multiple response monitoring (MRM).

[0061] Ion source temperature: 300℃.

[0062] Spray voltage: 3000V.

[0063] Test data results The following analysis was performed on the detection process and data of Example 1. (1) The mass spectrometry characteristic spectra of glycine, alanine, serine, proline, valine, threonine, leucine, isoleucine, aspartic acid, lysine, glutamic acid, methionine, histidine, phenylalanine, arginine, tyrosine, and cystine obtained from the sample obtained in Example 1 are as follows: Figures 1-17 As shown.

[0064] (2) Linear range and sensitivity verification Since fish tissue free of amino acids could not be obtained, a blank spiked method was used for verification. Three different concentrations of amino acids were added to prepare three different spiked sample series. The concentration range of cystine (Cys)2 was 0-50 nmol / ml, and the concentration range of the other amino acids was 0-100 nmol / ml. After processing the prepared samples according to the aforementioned sample pretreatment method, they were analyzed by mass spectrometry. The detection data were linearly fitted by mass spectrometry software to obtain the linear range, standard curve equation, linear correlation coefficient, and detection limit. The amino acid data are shown in Table 1. Table 1 Amino Acid Data

[0065] As shown in Table 1, the linear correlation of each amino acid is good, and the correlation coefficients are all greater than 0.995.

[0066] (3) Precision and accuracy testing Using a blank spiking method, different volumes of amino acid standard solution were added to the blank system to obtain low, medium, and high concentration spiked samples. Three samples were prepared for each concentration. After processing according to the aforementioned pretreatment steps, the samples were analyzed. Some amino acid detection data are shown in Table 2. Table 2. Amino Acid Detection Data

[0067] As shown in the table above, the precision of the sample testing is good, with RSD% all less than 10%.

[0068] (4) Actual sample testing Five fish meat samples were selected and processed according to the aforementioned pretreatment steps before being analyzed. Three parallel samples were prepared for each group. The amino acid detection data of some fish meat samples are shown in Table 3. Table 3. Detection data of various amino acids in fish meat samples.

[0069] As can be seen from the table above, the results of parallel samples are stable when using this method to detect various amino acids in fish meat, with RSD% less than 10%.

[0070] The following analysis is conducted on the detection process and results of Comparative Example 1. Taking three representative amino acids—aspartic acid (Asp), leucine (Leu), and isoleucine (Ile)—as examples, the relevant key performance data are explained in detail: Within the linear range of 0-100 nmol / ml for aspartic acid, leucine, and isoleucine, their linear correlation coefficient (R0) was... 2 The values ​​were 0.9989, 0.9921, and 0.9905, respectively. The calculated limits of detection (LODs) were 0.045 nmol / ml, 0.038 nmol / ml, and 0.041 nmol / ml, respectively. Based on this, it can be seen that the LODs of the comparative examples are generally higher than those of the method of this invention, and the method described in this invention is more sensitive.

[0071] Samples were spiked at three concentration levels: low (5 nmol / ml), medium (25 nmol / ml), and high (50 nmol / ml). The recoveries of aspartic acid were 92.5%, 105.8%, and 97.3%, with relative standard deviations (RSDs) of 1.5%, 2.1%, and 1.8%, respectively. The recoveries of leucine and isoleucine ranged from 88% to 107%, but due to poor isomer separation, their RSDs at low concentrations reached 3.2% and 4.5%, respectively, and were generally higher than 2.0% at medium and high concentrations. Under optimized gradient elution, complete baseline separation of the chromatographic peaks of leucine and isoleucine was not achieved, with peak heights exceeding 10%, making accurate quantification impossible, especially at low concentrations where the error was more pronounced.

[0072] In summary, the present invention adopts a derivatization-free pretreatment scheme, which only requires low-temperature grinding and extraction and two-step low-temperature centrifugation to precipitate proteins. The process is simple and can be completed within 1 hour. It is easy to operate, has good reproducibility, and the analysis cycle of a single sample in the example is 13 minutes, which is much shorter than the 21 minutes (including equilibration) of the comparative example. Combined with faster sample pretreatment, the overall analytical throughput is greatly improved.

[0073] Furthermore, this embodiment of the invention utilizes the separation advantages of amide columns to clearly achieve baseline separation of leucine and isoleucine, solving the problem of inaccurate quantification caused by insufficient separation in comparative methods.

[0074] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A method for detecting multiple amino acids in fish meat, characterized in that, Includes the following steps: Step 1: Mix the fish meat sample with formic acid aqueous solution, grind and homogenize at low temperature, then centrifuge and take the supernatant after centrifugation; Step 2: Add protein precipitant to the supernatant, vortex to mix, centrifuge to collect the supernatant, refrigerate the obtained supernatant at low temperature, centrifuge again, and take the final supernatant as the test solution. Step 3: Inject the test solution obtained in Step 2 into the LC-Q-TOF system for analysis and detection, and obtain the detection results; The protein precipitant is a mixed solution of methanol, acetonitrile, and formic acid. The LC-Q-TOF system uses an amide column for chromatographic separation, and the mobile phase does not contain ion-pairing reagents.

2. The method for detecting multiple amino acids in fish meat according to claim 1, characterized in that, The volume fraction of formic acid in the formic acid aqueous solution is 0.1%; the ratio of the fish meat sample to the formic acid aqueous solution is 0.1g:50μL.

3. The method for detecting multiple amino acids in fish meat according to claim 1, characterized in that, The volume ratio of methanol, acetonitrile, and formic acid in the protein precipitant is 1.5~2.5:1.5~2.5:0.001~0.

005.

4. The method for detecting multiple amino acids in fish meat according to claim 1, characterized in that, The temperature during the low-temperature grinding homogenization is 4℃; the temperature during the low-temperature refrigeration is -15~-25℃, and the time is 15-30min.

5. The method for detecting multiple amino acids in fish meat according to claim 1, characterized in that, The amide column has dimensions of 2.1mm × 100mm and a diameter of 1.7μm; the column temperature is 30-40℃.

6. The method for detecting multiple amino acids in fish meat according to claim 1, characterized in that, The mobile phase comprises mobile phase A and mobile phase B, wherein mobile phase A comprises an aqueous solution of 5-20 mmol / L ammonium acetate and 0.01%-0.05% formic acid; mobile phase B is acetonitrile; gradient elution is used in the analysis and detection, and the flow rate is 0.3-0.5 mL / min.

7. The method for detecting multiple amino acids in fish meat according to claim 6, characterized in that, The gradient elution procedure is as follows: 0 min: Mobile phase A 10%, Mobile phase B 90%; 4 min: Mobile phase A 20%, Mobile phase B 80%; 8 min: Mobile phase A 60%, Mobile phase B 40%; 10 min: Mobile phase A 60%, Mobile phase B 40%; 11 min: Mobile phase A 10%, Mobile phase B 90%; 13 min: Mobile phase A 10%, Mobile phase B 90%.

8. The method for detecting multiple amino acids in fish meat according to claim 1, characterized in that, The mass spectrometry detection conditions for the LC-Q-TOF are as follows: electrospray ionization source, positive ion mode; scanning mass range 50-500 m / z; spray voltage 3000-4000 V; ion source temperature 300-400 °C; transmission tube temperature 300-350 °C.

9. The method for detecting multiple amino acids in fish meat according to any one of claims 1-8, characterized in that, The amino acids that the method can simultaneously detect include glycine, alanine, serine, proline, valine, threonine, leucine, isoleucine, aspartic acid, lysine, glutamic acid, methionine, histidine, phenylalanine, arginine, tyrosine, and cystine.

10. The application of the method according to any one of claims 1 to 9 in the simultaneous detection of multiple amino acids.

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