Method for detecting multiple dipeptides in ruminant rumen fluid

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to a method for detecting multiple dipeptides in ruminant rumen fluid. The detection method provided by the invention is simple, has strong operability, and can detect 28 dipeptides. As the content of the dipeptide in the rumen fluid is relatively low, the acetonitrile is utilized to precipitate the protein of the sample to be determined, and then the dansyl chloride is utilized to perform derivation and determination, so that not only can the impurities such as the protein of the sample be removed to the greatest extent and the matrix effect be reduced, but also the detection amount of the dipeptide in the sample can be ensured; further, the accuracy of detecting 28 dipeptides in rumen fluid is improved. The pretreatment method adopting acetonitrile precipitation and dansyl chloride derivation is simple and easy to operate. Meanwhile, the target object detected by using the ultra-high performance liquid chromatography-mass spectrometry is good in peak shape, high in separation degree and good in retention on a chromatographic column, and the detection accuracy is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and in particular relates to a method for detecting multiple dipeptides in ruminant rumen fluid. Background Art

[0002] Nutrients in the diet are an important guarantee for maintaining high production of livestock and poultry, among which dietary protein is a key nutrient that determines the nutritional performance of livestock and poultry. The classical protein and amino acid theory has formed a variety of nutritional systems, but protein and amino acids still face many challenges in further improving animal performance, such as the differences in the requirements of different animals for the nutritional value and digestibility of feed protein, and the degree of utilization of various amino acids in feed by livestock and poultry is not completely affected by the level of a single limiting amino acid, that is, it does not completely follow the balanced amino acid theory. Small peptides are an important class of protein metabolites in the body, which play an important nutritional function in the body and also show great potential in regulating the production performance of livestock and poultry. However, the current research on peptides basically starts from the perspective of amino acid supplementation, and has not really opened up the road to new theoretical research on livestock and poultry peptide nutrition. Limited by the precision detection technology, the main existence form, distribution and metabolic circulation rules of small peptides in livestock and poultry are still unclear.

[0003] At present, the research on small peptides is mainly focused on the human body, and small peptides are mainly dipeptides and tripeptides. Dipeptides are oligopeptides composed of two amino acid residues. They are diverse in types and functions, but the large number of isomers and wide concentration range also bring huge challenges to the qualitative and quantitative analysis of dipeptide metabolites and the study of biological functions. Liquid chromatography-mass spectrometry (LC-MS / MS) stands out from many analytical instruments due to its high sensitivity and high selectivity and has become the main instrument for analyzing dipeptide metabolites. However, the detection of dipeptides is currently mainly concentrated in human blood, tissues and red wine, and there are almost no reports on the detection of dipeptides in ruminant rumen fluid. Therefore, there is an urgent need for a method for detecting multiple dipeptides in ruminant rumen fluid. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the purpose of the present invention is to provide a method for detecting multiple dipeptides in ruminant rumen fluid, which has good accuracy and high sensitivity in detecting dipeptides in rumen fluid.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for detecting multiple dipeptides in ruminant rumen fluid, comprising the following steps:

[0007] The sample to be tested is mixed with acetonitrile, and after protein precipitation, solid-liquid separation is performed to obtain a supernatant;

[0008] The supernatant, the sodium bicarbonate buffer solution and the dansyl chloride acetonitrile solution are mixed to carry out a derivatization reaction to obtain a dansyl chloride-dipeptide derivative system;

[0009] Using sodium hydroxide solution to remove hydrochloric acid in the dansyl chloride-dipeptide derivative system to obtain a dansyl chloride-dipeptide derivative system after impurities are removed;

[0010] The excess sodium hydroxide in the decontaminated dansyl chloride-dipeptide derivative system is removed by using a formic acid-acetonitrile aqueous solution system, and the obtained solution is frozen and layered to obtain a supernatant;

[0011] The supernatant is subjected to ultra-high performance liquid chromatography-tandem mass spectrometry detection to obtain the peak area of ​​each dipeptide; the content of each dipeptide in the sample to be tested is calculated by external standard method according to the peak area of ​​each dipeptide and the corresponding predetermined standard curve;

[0012] The conditions for the ultra-high performance liquid chromatography detection include: the mobile phase system is a mobile phase A and a mobile phase B, the mobile phase A is a formic acid aqueous solution, and the mobile phase B is a formic acid acetonitrile solution;

[0013] The flow rate of the mobile phase system is 0.3 mL / min;

[0014] The elution mode is gradient elution;

[0015] The procedure of the gradient elution is:

[0016] 0.0min: the volume percentage content of the mobile phase A is 90%;

[0017] 0-1min: the volume percentage of the mobile phase A is uniformly reduced from 90% to 80%;

[0018] 1-12 min: the volume percentage of the mobile phase A is uniformly reduced from 80% to 0%;

[0019] 12-16 min: the volume percentage content of the mobile phase A is 0%;

[0020] 16-17min: the volume percentage of the mobile phase A increases uniformly from 0% to 90%;

[0021] 17-20min: the volume percentage of the mobile phase A is 90%;

[0022] The mass spectrometry detection conditions include: the scanning mode is the positive ion mode; the positive ion voltage is 5500V; the ion source temperature is 550°C, and the scanning mode is the multiple reaction monitoring mode.

[0023] Preferably, the concentration of the sodium bicarbonate buffer solution is 0.08-0.12 mol / L; the concentration of the dansyl chloride acetonitrile solution is 35-45 mmol / L; and the concentration of the sodium hydroxide solution is 0.20-0.30 mmol / L.

[0024] Preferably, the concentration of formic acid in the formic acid-acetonitrile aqueous solution system is 0.38-0.45 mol / L; the formic acid-acetonitrile aqueous solution system is obtained by dissolving formic acid in an acetonitrile aqueous solution; and the volume ratio of acetonitrile to water in the acetonitrile aqueous solution is 1:1.

[0025] Preferably, the volume ratio of the supernatant, the sodium bicarbonate buffer solution and the dansyl chloride acetonitrile solution is 15:9:5.

[0026] Preferably, the volume ratio of the dansyl chloride acetonitrile solution to the sodium hydroxide solution is 5:1;

[0027] The volume ratio of the sodium hydroxide solution to the formic acid-acetonitrile aqueous solution system is 1:3.

[0028] Preferably, the temperature of the derivatization reaction is 35-45° C., and the time of the derivatization reaction is 40-50 min.

[0029] Preferably, the temperature of removing hydrochloric acid in the dansyl chloride-dipeptide derivative system by using sodium hydroxide solution is 40° C. and the time is 5 to 15 minutes.

[0030] Preferably, the ruminant comprises cattle and sheep; the dipeptide comprises diGly, L-alanyl-glycine, glycyl-DL-alanine, L-alanyl-L-alanine, glycyl-L-proline, alanyl-proline, prolyl-alanine, glycyl-L-valine, phenylalanyl-glycine, DL-alanyl-DL-methionine, DL-alanyl-DL-valine, phenylalanyl-alanine, glycine-L-leucine, L-alanyl -L-leucine, L-valyl-L-methionine, valyl-valine, L-alanyl-L-glutamate, DL-alanyl-DL-leucine, phenylalanyl-proline, valyl-tyrosine, DL-leucyl-DL-valine, prolyl-phenylalanine, L-phenylalanyl-L-tryptophan, leucyl-leucine, alanyl-histidine, tyrosyl-leucine, L-tyrosyl-L-phenylalanine, L-leucyl-L-tyrosine.

[0031] Preferably, the volume ratio of formic acid to water in the formic acid aqueous solution is 1:1000; the volume ratio of formic acid to acetonitrile in the formic acid acetonitrile solution is 1:1000.

[0032] Preferably, the chromatographic column for ultra-high performance liquid chromatography detection is kinetic XB-C18 Chromatographic column; column temperature is 40℃; injection volume is 3μL.

[0033] The invention provides a method for detecting multiple dipeptides in ruminant rumen fluid, comprising the following steps: mixing a sample to be tested with acetonitrile, performing protein precipitation, and performing solid-liquid separation to obtain a supernatant; mixing the supernatant, a sodium bicarbonate buffer solution, and a dansyl chloride acetonitrile solution, and performing a derivatization reaction to obtain a dansyl chloride-dipeptide derivative system; using a sodium hydroxide solution to remove hydrochloric acid in the dansyl chloride-dipeptide derivative system to obtain a dansyl chloride-dipeptide derivative system after impurities are removed; using a formic acid-acetonitrile aqueous solution system to remove excess sodium hydroxide in the dansyl chloride-dipeptide derivative system after impurities are removed, and freezing and stratifying the obtained solution to obtain a supernatant; performing ultra-high performance liquid chromatography-tandem mass spectrometry detection on the supernatant to obtain a peak area of ​​each dipeptide; and obtaining the content of each dipeptide in the sample to be tested according to the peak area of ​​each dipeptide and a predetermined standard curve corresponding thereto; the conditions for ultra-high performance liquid chromatography detection include: a mobile phase system of mobile phase A and Mobile phase B, the mobile phase A is a formic acid aqueous solution, and the mobile phase B is a formic acid acetonitrile solution; the flow rate of the mobile phase system is 0.3 mL / min; the elution mode is gradient elution; the program of the gradient elution is: 0.0 min: the volume percentage content of the mobile phase A is 90%; 0-1 min: the volume percentage content of the mobile phase A is uniformly reduced from 90% to 80%; 1-12 min: the volume percentage content of the mobile phase A is uniformly reduced from 80% to 0%; 12-16 min: the volume percentage content of the mobile phase A is 0%; 16-17 min: the volume percentage content of the mobile phase A is uniformly increased from 0% to 90%; 17-20 min: the volume percentage content of the mobile phase A is 90%; the conditions for the mass spectrometry detection include: the scanning mode is the positive ion mode; the positive ion voltage is 5500 V; the ion source temperature is 550° C., and the scanning mode is the multiple reaction monitoring mode.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] (1) The detection method of the present invention is simple and highly operable, and can detect 28 dipeptides. (2) Because the content of dipeptides in rumen fluid is relatively low, the present invention uses acetonitrile to precipitate the sample protein to be determined, and then uses dansyl chloride to derivatize and then determine. This can not only remove the protein and other impurities in the sample to the greatest extent and reduce the matrix effect, but also ensure the detection amount of dipeptides in the sample, thereby improving the accuracy of detecting 28 dipeptides in rumen fluid. The pretreatment method of acetonitrile precipitation and dansyl chloride derivatization used in the present invention is simple and easy to operate. At the same time, the target object detected by the present invention using the above-mentioned ultra-high performance liquid chromatography-mass spectrometry has good peak shape, high separation, and good retention on the chromatographic column, thereby improving the detection accuracy.

[0036] The data of the embodiment show that the detection limit of the dipeptide is 0.13-3.78 ng / mL, the quantification limit is 0.43-12.5 ng / mL, the repeatability relative standard deviation of the retention time and the peak area is less than 15.0%, and the average recovery rate is between 60.0 and 129.0%, which can achieve accurate quantitative determination of 28 dipeptides in ruminant rumen fluid. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The total ion chromatogram of 28 dansyl chloride-dipeptide derivatives. DETAILED DESCRIPTION

[0038] The present invention provides a method for detecting multiple dipeptides in ruminant rumen fluid, comprising the following steps:

[0039] The sample to be tested is mixed with acetonitrile, and after protein precipitation, solid-liquid separation is performed to obtain a supernatant;

[0040] The supernatant, the sodium bicarbonate buffer solution and the dansyl chloride acetonitrile solution are mixed to carry out a derivatization reaction to obtain a dansyl chloride-dipeptide derivative system;

[0041] Using sodium hydroxide solution to remove hydrochloric acid in the dansyl chloride-dipeptide derivative system to obtain a dansyl chloride-dipeptide derivative system after impurities are removed;

[0042] The excess sodium hydroxide in the decontaminated dansyl chloride-dipeptide derivative system is removed by using a formic acid-acetonitrile aqueous solution system, and the obtained solution is frozen and layered to obtain a supernatant;

[0043] The supernatant is subjected to ultra-high performance liquid chromatography-tandem mass spectrometry detection to obtain the peak area of ​​each dipeptide; the content of each dipeptide in the sample to be tested is calculated by external standard method according to the peak area of ​​each dipeptide and the corresponding predetermined standard curve;

[0044] The conditions for the ultra-high performance liquid chromatography detection include: the mobile phase system is a mobile phase A and a mobile phase B, the mobile phase A is a formic acid aqueous solution, and the mobile phase B is a formic acid acetonitrile solution;

[0045] The flow rate of the mobile phase system is 0.3 mL / min;

[0046] The elution mode is gradient elution;

[0047] The procedure of the gradient elution is:

[0048] 0.0min: the volume percentage content of the mobile phase A is 90%;

[0049] 0-1min: the volume percentage of the mobile phase A is uniformly reduced from 90% to 80%;

[0050] 1-12 min: the volume percentage of the mobile phase A is uniformly reduced from 80% to 0%;

[0051] 12-16 min: the volume percentage content of the mobile phase A is 0%;

[0052] 16-17min: the volume percentage of the mobile phase A increases uniformly from 0% to 90%;

[0053] 17-20min: the volume percentage of the mobile phase A is 90%;

[0054] The mass spectrometry detection conditions include: the scanning mode is the positive ion mode; the positive ion voltage is 5500V; the ion source temperature is 550°C, and the scanning mode is the multiple reaction monitoring mode.

[0055] The method comprises mixing a sample to be tested with acetonitrile, performing protein precipitation, and then performing solid-liquid separation to obtain a supernatant.

[0056] As an embodiment, the acquisition of the sample to be tested includes: filtering fresh rumen fluid of ruminants through four layers of gauze, centrifuging, and taking the supernatant as the sample to be tested.

[0057] As one embodiment, the ruminant includes cattle or sheep.

[0058] As an embodiment, the centrifugal speed is 11000-13000 rpm, the centrifugal temperature is 2-6°C, and the centrifugal time is 5-15 min. In a specific embodiment, the centrifugal speed is 12000 rpm, the centrifugal temperature is 4°C, and the centrifugal time is 10 min.

[0059] As an embodiment, the volume ratio of the sample to be tested and acetonitrile is 1:3-6. As another embodiment, the volume ratio of the sample to be tested and acetonitrile is 1:4-5. In a specific embodiment, the volume ratio of the sample to be tested and acetonitrile can be 1:4.

[0060] As an embodiment, the solid-liquid separation method is refrigerated centrifugation.

[0061] After obtaining the supernatant, the present invention mixes the supernatant, a sodium bicarbonate buffer solution and a dansyl chloride acetonitrile solution, and performs a derivatization reaction to obtain a dansyl chloride-dipeptide derivative system.

[0062] As an embodiment, the concentration of the sodium bicarbonate buffer solution is 0.08-0.12 mol / L. As another embodiment, the concentration of the sodium bicarbonate buffer solution is 0.09-0.11 mol / L. In a specific embodiment, the concentration of the sodium bicarbonate buffer solution can be 0.10 mol / L.

[0063] As an embodiment, the concentration of the dansyl chloride acetonitrile solution is 35-45 mmol / L. As another embodiment, the concentration of the dansyl chloride acetonitrile solution is 38-42 mmol / L. In a specific embodiment, the concentration of the dansyl chloride acetonitrile solution is 40 mmol / L.

[0064] As an embodiment, the volume ratio of the supernatant, the sodium bicarbonate buffer solution and the dansyl chloride acetonitrile solution is 15:9:5; the volume ratio of the dansyl chloride acetonitrile solution and the sodium hydroxide solution is 5:1.

[0065] As one embodiment, the temperature of the derivatization reaction is 35-45°C, and the time of the derivatization reaction is 40-50 min; as another embodiment, the temperature of the derivatization reaction is 38-42°C, and the time of the derivatization reaction is 43-48 min; in a specific embodiment, the temperature of the derivatization reaction can be 40°C, and the time of the derivatization reaction can be 45 min.

[0066] After the dansyl chloride-dipeptide derivative system is obtained, the present invention uses a sodium hydroxide solution to remove hydrochloric acid in the dansyl chloride-dipeptide derivative system to obtain the dansyl chloride-dipeptide derivative system after impurities are removed.

[0067] As an implementation manner, the concentration of the sodium hydroxide solution is 0.20-0.30 mmol / L. In a specific embodiment, the concentration of the sodium hydroxide solution can be 0.25 mmol / L.

[0068] As an embodiment, the temperature for removing hydrochloric acid in the dansyl chloride-dipeptide derivative system using sodium hydroxide solution is 40°C and the time is 5 to 15 minutes. As another embodiment, the time is 8 to 12 minutes; in a specific embodiment, the time can be 10 minutes.

[0069] After obtaining the impurity-removed dansyl chloride-dipeptide derivative system, the present invention uses a formic acid-acetonitrile aqueous solution system to remove excess sodium hydroxide in the impurity-removed dansyl chloride-dipeptide derivative system, and the resulting solution is frozen and layered to obtain a supernatant. As an embodiment, the formic acid-acetonitrile aqueous solution system is a mixed solution of formic acid, water and acetonitrile; the volume ratio of water to acetonitrile in the formic acid-acetonitrile aqueous solution system is 1:1, and the concentration of formic acid in the formic acid-acetonitrile aqueous solution system is 0.38-0.45 mol / L. In a specific embodiment, the concentration of formic acid in the formic acid-acetonitrile aqueous solution system can be 0.42 mol / L.

[0070] As an embodiment, the volume ratio of the sodium hydroxide solution to the formic acid-acetonitrile aqueous solution system is 1:3.

[0071] As an embodiment, during the freezing and stratification, the temperature is -20°C and the freezing and stratification time is ≥30 minutes.

[0072] After obtaining the supernatant, the present invention performs ultra-high performance liquid chromatography-tandem mass spectrometry detection on the supernatant to obtain the peak area of ​​each dipeptide; according to the peak area of ​​each dipeptide and the corresponding predetermined standard curve, the content of each dipeptide in the sample to be tested is calculated by the external standard method.

[0073] As an embodiment, the dipeptide includes diglycine, L-alanyl-glycine, glycyl-DL-alanine, L-alanyl-L-alanine, glycyl-L-proline, alanyl-proline, prolyl-alanine, glycyl-L-valine, phenylalanyl-glycine, DL-alanyl-DL-methionine, DL-alanyl-DL-valine, phenylalanyl-alanine, glycine-L-leucine, L-alanyl-L- Leucine, L-valyl-L-methionine, valyl-valine, L-alanyl-L-glutamate, DL-alanyl-DL-leucine, phenylalanyl-proline, valyl-tyrosine, DL-leucyl-DL-valine, prolyl-phenylalanine, L-phenylalanyl-L-tryptophan, leucyl-leucine, alanyl-histidine, tyrosyl-leucine, L-tyrosyl-L-phenylalanine, L-leucyl-L-tyrosine.

[0074] As an embodiment, the conditions for the ultra-high performance liquid chromatography detection include: the mobile phase system is mobile phase A and mobile phase B, the mobile phase A is a formic acid aqueous solution, and the mobile phase B is a formic acid acetonitrile solution; the volume ratio of formic acid to water in the formic acid aqueous solution is 1:1000; the volume ratio of formic acid to acetonitrile in the formic acid acetonitrile solution is 1:1000. The flow rate of the mobile phase system is 0.3 mL / min.

[0075] As an embodiment, the elution method is gradient elution;

[0076] As an embodiment, the procedure of the gradient elution is:

[0077] 0.0min: the volume percentage content of the mobile phase A is 90%;

[0078] 0-1min: the volume percentage of the mobile phase A is uniformly reduced from 90% to 80%;

[0079] 1-12 min: the volume percentage of the mobile phase A is uniformly reduced from 80% to 0%;

[0080] 12-16 min: the volume percentage content of the mobile phase A is 0%;

[0081] 16-17min: the volume percentage of the mobile phase A increases uniformly from 0% to 90%;

[0082] 17-20min: the volume percentage of the mobile phase A is 90%.

[0083] As an implementation manner, the conditions for mass spectrometry detection include: the scanning mode is a positive ion mode; the positive ion voltage is 5500V; the ion source temperature is 550°C, and the scanning mode is a multiple reaction monitoring mode.

[0084] As an implementation method, the curtain gas is high-purity nitrogen with a pressure of 35 psi; Gas1 and Gas2 are zero-grade air with a pressure of 55 psi.

[0085] As an embodiment, the preparation of the predetermined standard curve comprises the following steps:

[0086] Prepare mixed standard working solutions of various dipeptide standards at a series of concentrations;

[0087] The mixed standard working solution of the series of concentrations, the sodium bicarbonate buffer solution and the dansyl chloride acetonitrile solution are mixed to carry out a derivatization reaction to obtain a dansyl chloride-dipeptide derivative system;

[0088] Using sodium hydroxide solution to remove hydrochloric acid in the dansyl chloride-dipeptide derivative system to obtain a dansyl chloride-dipeptide derivative system after impurities are removed;

[0089] The excess sodium hydroxide in the decontaminated dansyl chloride-dipeptide derivative system is removed by using a formic acid-acetonitrile aqueous solution system, and the obtained solution is frozen and layered to obtain a supernatant;

[0090] The supernatant was subjected to ultra-performance liquid chromatography-tandem mass spectrometry detection to obtain the peak area of ​​each dipeptide standard, and then, the standard curve of each dipeptide standard was obtained with the peak area of ​​each dipeptide standard as the ordinate and the respective concentration as the abscissa.

[0091] In the present invention, the information of dipeptide standards is shown in Table 1.

[0092] Table 1 Dipeptide standard information

[0093]

[0094]

[0095] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0096] Example 1

[0097] Take the standard stock solution of various dipeptides with a concentration of 1.00 mg / mL, and prepare a mixed standard solution using acetonitrile-water system (the volume ratio of acetonitrile and water is 80:20) as the diluent; then use this as the mother solution to prepare a series of mixed standard working solutions, and their concentrations are shown in Table 2.

[0098] Take 300 μL of the mixed standard working solution of each concentration, add 180 μL of 0.10 mol / L sodium bicarbonate-sodium carbonate buffer solution, and mix evenly; then add 100 μL of 40 mmol / L dansyl chloride acetonitrile solution, mix evenly; then place it on a 40°C metal plate to react for 45 minutes; after the reaction is completed, add 20 μL of 0.25 mol / L sodium hydroxide solution, mix evenly, and continue to react for 10 minutes, then add 60 μL of 0.425 mol / L formic acid-acetonitrile aqueous solution (the volume ratio of acetonitrile to water in the acetonitrile aqueous solution is 1:1) solution, mix evenly, and place it at -20°C for freezing and stratification; take about 100 μL of the upper clear liquid in an inner tube, and perform ultra-high performance liquid chromatography-mass spectrometry analysis, wherein the ultra-high performance liquid chromatography-mass spectrometry conditions are as follows:

[0099] The chromatographic column is kinetic XB-C 18Chromatographic column, column temperature of 40°C, injection volume of 3 μL; mobile phase A: formic acid aqueous solution (the volume ratio of formic acid to water is 1:1000), mobile phase B: formic acid acetonitrile solution (the volume ratio of formic acid to acetonitrile is 1:1000), mobile phase flow rate of 0.3 mL / min; gradient elution program: 0.0 min: the volume percentage content of the mobile phase A is 90%; 0-1 min: the volume percentage content of the mobile phase A is uniformly reduced from 90% to 80%; 1-12 min: the mobile phase The volume percentage of A is uniformly reduced from 80% to 0%; 12-16min: the volume percentage of mobile phase A is 0%; 16-17min: the volume percentage of mobile phase A is uniformly increased from 0% to 90%; 17-20min: the volume percentage of mobile phase A is 90%; the mass spectrometry conditions are positive ion mode, positive ion voltage: 5500V, ion source temperature 550°C, scanning mode is multiple reaction monitoring (MRM) mode, and the target MRM ion pair information is shown in Table 3. The gas curtain gas pressure is 35psi; Gas1 and Gas2 pressures are both 55psi. The concentration of the target is used as the horizontal coordinate and the peak area of ​​the target after derivatization is used as the vertical coordinate to obtain a linear regression equation; the linear range, linear equation and linear correlation coefficient of various dipeptides are shown in Table 4.

[0100] Table 2 Concentration of mixed standard working solution (ng / mL)

[0101]

[0102]

[0103] Table 3 MRM parameters of dansyl chloride-derivatized dipeptides

[0104]

[0105]

[0106] Note: 1 is the quantitative ion pair; 2 is the qualitative ion pair.

[0107] Table 4 Linear range, linear equation and linear correlation coefficient of dipeptides

[0108] Target Linear equation Linear correlation coefficient Linear range (ng / mL) Dns-Gly-Gly Y = 5.7049E4X + 8.57659E4 0.997 1.88-30.0 Dns-Ala-Gly Y = 4.33E4X + 1.026E5 0.992 1.88-45.0 Dns-Gly-Ala Y = 29291.42X + 22588.99 0.999 0.94-25.0 Dns-Ala-Ala Y = 7480.993X + 10149.9 0.997 1.41-30.0 Dns-Gly-Pro Y = 26928.83X + 22745.26 0.994 2.50-60.0 Dns-Ala-Pro Y = 4.24938E4X + 6533.7 0.994 0.94-45.0 Dns-Pro-Ala Y = 1.08756E5X + 8.34011E4 0.996 1.88-30.0 Dns-Gly-Val Y = 4.04215E4X + 7272.11 0.996 0.94-45.0 Dns-Phe-Gly Y = 3.97672E4X + 6.84103E4 0.994 3.75-45.0 Dns-DL-Ala-DL-Met Y = 3.11459E4X + 2099.74 0.993 0.94-45.0 Dns-DL-Ala-DL-Val Y = 4.3514E4X + 1641.2 0.997 0.94-45.0 Dns-Phe-Ala Y = 22307.81X + 8343.1 0.992 12.5-150 Dns-Gly-Leu Y = 6.59820E4X + 4216.86 0.997 0.94-45.0 Dns-L-Ala-L-Leu Y = 8.47433E4X + 131778 0.994 0.63-60.0 Dns-L-Val-L-Met Y = 28236.27X + 3.9197E4 0.996 0.56-30.0 Dns-H-Val-Val-OH Y = 5.84528E4X - 10829.2 0.997 1.09-105 Dns-Ala-Gln Y = 20556.117X + 2309.95 0.999 1.88-45.0 Dns-DL-Ala-DL-Leu Y = 1.9078e4X + 606.9 0.997 0.47-45.0 Dns-Phe-Pro Y = 7.33885E4X + 14857.34 0.999 0.94-30.0 Dns-Val-Tyr Y = 29146.7X + 4.17904E4 0.999 1.88-30.0 Dns-DL-Leu-DL-Val Y = 6.34319E4X + 18395.73 0.998 0.94-30.0 Dns-H-Pro-Phe-OH Y = 6.27435E4X - 12881.22 0.998 0.94-45.0 Dns-L-Phe-Trp Y = 7751.928X + 2874.69 0.997 2.50-60.0 Dns-Leu-Leu Y = 6.91356E4X - 3812.3 0.999 0.13-45.0 Dns-Ala-His Y = 18331.01X + 10185.54 0.999 0.63-60.0 Dns-H-Tyr-Leu Y = 4394.832X + 4585.87 0.996 1.88-30.0 Dns-Tyr-Phe Y = 708.325X - 1272.16 0.995 12.5-130 Dns-L-Leu-L-Tyr Y = 7031.41X - 986.70 0.996 1.88-45.0

[0109] Example 2

[0110] A method for detecting multiple dipeptides in sheep rumen fluid, comprising the following steps:

[0111] (1) Sample acquisition: After collecting fresh sheep rumen contents through four layers of gauze, centrifuge at a speed of 12000 rpm and a temperature of 4°C for 10 min, and take the supernatant to obtain the sample to be measured;

[0112] (2) Sample protein precipitation: Take 100 μL of the sample to be determined in step (1), add 400 μL of cold acetonitrile (stored at 4°C), vortex to mix evenly, let stand at -20°C for 10 min, then centrifuge at 12000 rpm and 4°C, take the supernatant, and obtain the protein precipitation supernatant, i.e., the sample to be derivatized;

[0113] (3) Sample derivatization: 300 μL of the sample to be derivatized obtained in step (2) was added with 180 μL of 0.10 mol / L sodium bicarbonate-sodium carbonate buffer solution and mixed evenly; 100 μL of 40 mmol / L dansyl chloride acetonitrile solution was added thereto and mixed evenly; then the sample was placed on a metal plate at 40°C for reaction for 45 min; after the reaction was completed, 20 μL of 0.25 mol / L sodium hydroxide solution was added thereto, mixed evenly and continued to react for 10 min, then 60 μL of 0.425 mol / L formic acid-acetonitrile aqueous solution (the volume ratio of acetonitrile to water in the acetonitrile aqueous solution was 1:1) was added thereto, mixed evenly and placed at -20°C for freezing and stratification, and about 100 μL of the upper clear liquid was taken into an inner tube for ultra-high performance liquid chromatography-mass spectrometry analysis, wherein the ultra-high performance liquid chromatography-mass spectrometry conditions are as follows:

[0114] The chromatographic column is kinetic XB-C 18 Chromatographic column, column temperature of 40°C, injection volume of 3 μL; mobile phase A: formic acid aqueous solution (the volume ratio of formic acid to water is 1:1000), mobile phase B: formic acid acetonitrile solution (the volume ratio of formic acid to acetonitrile is 1:1000), mobile phase flow rate of 0.3 mL / min; gradient elution program: 0.0 min: the volume percentage content of the mobile phase A is 90%; 0-1 min: the volume percentage content of the mobile phase A is uniformly reduced from 90% to 80%; 1-12 min: the mobile phase The volume percentage of A decreases from 80% to 0% at a uniform rate; 12-16min: the volume percentage of the mobile phase A is 0%; 16-17min: the volume percentage of the mobile phase A increases from 0% to 90% at a uniform rate; 17-20min: the volume percentage of the mobile phase A is 90%; the mass spectrometry conditions are positive ion mode, positive ion voltage: 5500V, ion source temperature 550°C, scanning mode is multiple reaction monitoring (MRM) mode, and the target MRM ion pair information is shown in Table 3. The pressure of the curtain gas (high-purity nitrogen) is 35psi; Gas1 and Gas2 are zero-grade air, and the pressure is 55psi.

[0115] Example 3

[0116] A method for detecting multiple dipeptides in cattle rumen fluid, comprising the following steps:

[0117] (1) Sample acquisition: After the collected fresh bovine rumen contents were passed through four layers of gauze, they were centrifuged at a speed of 12000 rpm and a temperature of 4°C for 10 min to obtain the supernatant to obtain the sample to be measured;

[0118] (2) Sample protein precipitation: Take 100 μL of the sample to be determined in step (1), add 400 μL of cold acetonitrile (stored at 4°C), vortex to mix evenly, let stand at -20°C for 10 min, then centrifuge at 12000 rpm and 4°C, take the supernatant, and obtain the protein precipitation supernatant, i.e., the sample to be derivatized;

[0119] (3) Sample derivatization: 300 μL of the sample to be derivatized obtained in step (2) was added with 180 μL of 0.10 mol / L sodium bicarbonate-sodium carbonate buffer solution and mixed evenly; 100 μL of 40 mmol / L dansyl chloride acetonitrile solution was added thereto and mixed evenly; then the sample was placed in a 40°C water bath to react for 45 min; after the reaction was completed, 20 μL of 0.25 mol / L sodium hydroxide solution was added thereto, mixed evenly and continued to react for 10 min, then 60 μL of 0.425 mol / L formic acid-acetonitrile aqueous solution (the volume ratio of acetonitrile to water in the acetonitrile aqueous solution was 1:1) was added thereto, mixed evenly and placed in a -20°C frozen stratification, and about 100 μL of the upper clear liquid was taken into an inner tube for ultra-high performance liquid chromatography-mass spectrometry analysis, wherein the ultra-high performance liquid chromatography-mass spectrometry conditions are as follows:

[0120] The chromatographic column is kinetic XB-C 18 Chromatographic column, column temperature of 40 DEG C, injection volume of 3 μL; mobile phase A: formic acid aqueous solution (the volume ratio of formic acid to water is 1:1000), mobile phase B: formic acid acetonitrile solution (the volume ratio of formic acid to acetonitrile is 1:1000), flow rate of 0.3 mL / min; gradient elution program: 0.0 min: the volume percentage content of the mobile phase A is 90%; 0-1 min: the volume percentage content of the mobile phase A is uniformly reduced from 90% to 80%; 1-12 min: the volume percentage content of the mobile phase A is uniformly reduced from 80% to 0%; 12-16 min: the volume percentage content of the mobile phase A is 0%; 16-17 min: the volume percentage content of the mobile phase A is uniformly increased from 0% to 90%; 17-20 min: the volume percentage content of the mobile phase A is 90%. The mass spectrometry conditions are positive ion mode, positive ion voltage: 5500V, ion source temperature 550°C, and scanning mode is multiple reaction monitoring (MRM) mode. The specific parameters are shown in Table 3. The curtain gas pressure is 35psi; the pressures of Gas1 and Gas2 are both 55psi. The test results of Example 2 and Example 3 are shown in Table 5.

[0121] Table 5 Dipeptide content in cattle and sheep rumen fluid samples

[0122]

[0123]

[0124] Example 4

[0125] Determination of detection limit and quantification limit

[0126] Take the standard stock solution of various dipeptides with a concentration of 1.00 mg / mL, and prepare a mixed standard solution with acetonitrile-water system (the volume ratio of acetonitrile and water is 80:20) as the diluent; then use this as the mother solution to prepare a series of standard working solutions; after the sample is derivatized according to step (3) of Example 1, it is tested, and the target compound is further diluted and derivatized according to the peak response intensity to determine its quantitative limit and detection limit. The concentration of the target object 3 times the signal-to-noise ratio is its detection limit, and the concentration of 10 times the signal-to-noise ratio is its quantitative limit. The results are shown in Table 6.

[0127] Table 6 Quantification limit and detection limit of dipeptides

[0128]

[0129]

[0130] Example 5

[0131] Determination of precision and repeatability

[0132] After the standard working solutions std1, std3 and std5 were derivatized according to step (3) of Example 2, the standard samples were measured in parallel 6 times on the same day (n=6), and the average concentration (Mean) and standard deviation (SD) of the measurement results were calculated, and the precision (CV%) was calculated as SD÷Mean×100, and the coefficient of variation was calculated; the measurement was continued for three consecutive days (n=18), and the average concentration (Mean) and standard deviation (SD) of the measurement results were calculated, and the repeatability (CV%) was calculated as SD÷Mean×100. The results are shown in Table 7 below.

[0133] Table 7 Precision and reproducibility of standards

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141] The results in Table 7 show that the sample derivatization method of the present invention has good precision and repeatability in determining 28 dipeptides.

[0142] Example 6

[0143] Determination of the average recovery rate of the detection method of Example 2

[0144] The sheep rumen contents after passing through four layers of gauze obtained in step (1) of Example 2 were used as test samples, and three levels of mixed standard solutions were added to the test samples, with 6 replicates for each treatment, and the sample spike recovery was determined according to the detection method of Example 2. The results are shown in Table 8.

[0145] Spiked recovery (%) = (measured value - initial value) / added value × 100.

[0146] Table 8 Spiked recovery results of rumen fluid samples

[0147]

[0148]

[0149]

[0150] The results in Table 8 show that the average recovery rates of 28 dipeptides detected in rumen fluid samples by the detection method of the present invention are between 60.0% and 129.0%.

[0151] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. Other embodiments can be obtained based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for detecting multiple dipeptides in ruminant rumen fluid, characterized in that: The following steps are involved: The sample to be tested is mixed with acetonitrile, and after protein precipitation, solid-liquid separation is performed to obtain a supernatant; The supernatant, the sodium bicarbonate buffer solution and the dansyl chloride acetonitrile solution are mixed to carry out a derivatization reaction to obtain a dansyl chloride-dipeptide derivative system; Using sodium hydroxide solution to remove hydrochloric acid in the dansyl chloride-dipeptide derivative system to obtain a dansyl chloride-dipeptide derivative system after impurities are removed; The excess sodium hydroxide in the decontaminated dansyl chloride-dipeptide derivative system is removed by using a formic acid-acetonitrile aqueous solution system, and the obtained solution is frozen and layered to obtain a supernatant; The supernatant is subjected to ultra-high performance liquid chromatography-tandem mass spectrometry detection to obtain the peak area of ​​each dipeptide; the content of each dipeptide in the sample to be tested is calculated by external standard method according to the peak area of ​​each dipeptide and the corresponding predetermined standard curve; The conditions for the ultra-high performance liquid chromatography detection include: the mobile phase system is a mobile phase A and a mobile phase B, the mobile phase A is a formic acid aqueous solution, and the mobile phase B is a formic acid acetonitrile solution; The flow rate of the mobile phase system is 0.3 mL / min; The elution mode is gradient elution; The procedure of the gradient elution is: 0.0min: the volume percentage content of the mobile phase A is 90%; 0-1min: the volume percentage of the mobile phase A is uniformly reduced from 90% to 80%; 1-12 min: the volume percentage of the mobile phase A is uniformly reduced from 80% to 0%; 12-16 min: the volume percentage content of the mobile phase A is 0%; 16-17min: the volume percentage of the mobile phase A increases uniformly from 0% to 90%; 17-20min: the volume percentage of the mobile phase A is 90%; The mass spectrometry detection conditions include: the scanning mode is the positive ion mode; the positive ion voltage is 5500V; the ion source temperature is 550°C, and the scanning mode is the multiple reaction monitoring mode.

2. The detection method according to claim 1, characterized in that The concentration of the sodium bicarbonate buffer solution is 0.08-0.12 mol / L; the concentration of the dansyl chloride acetonitrile solution is 35-45 mmol / L; and the concentration of the sodium hydroxide solution is 0.20-0.30 mmol / L.

3. The detection method according to claim 1, characterized in that The concentration of formic acid in the formic acid-acetonitrile aqueous solution system is 0.38-0.45 mol / L; the formic acid-acetonitrile aqueous solution system is obtained by dissolving formic acid in an acetonitrile aqueous solution; and the volume ratio of acetonitrile to water in the acetonitrile aqueous solution is 1:

1.

4. The detection method according to claim 2 or 3, characterized in that: The volume ratio of the supernatant, the sodium bicarbonate buffer solution and the dansyl chloride acetonitrile solution is 15:9:

5.

5. The detection method according to claim 1, characterized in that The volume ratio of the dansyl chloride acetonitrile solution to the sodium hydroxide solution is 5:1; The volume ratio of the sodium hydroxide solution to the formic acid-acetonitrile aqueous solution system is 1:

3.

6. The detection method according to claim 1, characterized in that The temperature of the derivatization reaction is 35-45° C., and the time of the derivatization reaction is 40-50 min.

7. The detection method according to claim 1, characterized in that The temperature of removing hydrochloric acid in the dansyl chloride-dipeptide derivative system by using sodium hydroxide solution is 40° C. and the time is 5 to 15 minutes.

8. The detection method according to claim 1, characterized in that The ruminant includes cattle or sheep; the dipeptide includes diGly, L-alanyl-glycine, glycyl-DL-alanine, L-alanyl-L-alanine, glycyl-L-proline, alanyl-proline, prolyl-alanine, glycyl-L-valine, phenylalanyl-glycine, DL-alanyl-DL-methionine, DL-alanyl-DL-valine, phenylalanyl-alanine, glycine-L-leucine, L-alanyl- L-Leucine, L-Valyl-L-Methionine, Valyl-Valine, L-Alanyl-L-Glutamate, DL-Alanyl-DL-Leucine, Phenylalanyl-Proline, Valyl-Tyrosine, DL-Leucyl-DL-Valine, Prolyl-Phenylalanine, L-Phenylalanyl-L-Tryptophan Leucyl-Leucine, Alanyl-Histidine, Tyrosyl-Leucine, L-Tyrosyl-L-Phenylalanine, L-Leucyl-L-Tyrosine.

9. The detection method according to claim 1, characterized in that: The volume ratio of formic acid to water in the formic acid aqueous solution is 1:1000; the volume ratio of formic acid to acetonitrile in the formic acid acetonitrile solution is 1:1000.

10. The detection method according to claim 1, characterized in that: The chromatographic column for the ultra-high performance liquid chromatography detection is kinetic XB-C 18 Chromatographic column; column temperature is 40℃; injection volume is 3μL.

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