A quantitative method for dipeptides in liquor based on dansylation and liquid chromatography-mass spectrometry

Abstract

The present application relates to the technical field of liquor detection, and specifically to a method for quantitatively determining dipeptides in liquor based on dansylation and liquid chromatography-mass spectrometry, comprising the following steps: (1) taking a liquor sample and concentrating the liquor sample to obtain a concentrated sample; (2) measuring the concentrated sample to obtain a test sample, adding an internal standard to the test sample, adding a dansyl chloride acetonitrile solution after drying, and then adjusting the pH value to perform a derivatization reaction. After the reaction is completed, a mixed liquid is added, mixed evenly, and centrifuged, and the supernatant is obtained to obtain a test liquid; the mixed liquid includes a dansyl chloride acetonitrile solution, a pH buffer solution, and hydrochloric acid; (3) the test liquid is sent to a liquid chromatography-mass spectrometry instrument for detection, and the dipeptide is qualitatively and quantitatively analyzed based on the detection results. The present application can more accurately perform quantitative detection of dipeptides in liquor.

Description

Technical Field

[0001] The present application relates to the technical field of liquor detection, and in particular to a method for quantitatively determining dipeptides in liquor based on dansylation and liquid chromatography-mass spectrometry. Background Art

[0002] Dipeptides make a key contribution to the flavor of alcoholic beverages. Qualitative and quantitative analysis of dipeptide compounds helps to fully analyze the flavor of alcoholic products and further improve and regulate the flavor of alcoholic products.

[0003] Currently, existing methods utilize commercially available 6-aminoquinoline-N-hydroxysuccinimide carbamate derivatization kits to quantify dipeptides in sake. However, this method utilizes water to prepare the standard curve solution, ignoring the influence of matrix effects on analyte response, which in turn affects the authenticity and reliability of the quantitative results. Furthermore, due to the low levels of dipeptides in alcoholic beverages, the samples must be concentrated for testing. High concentration increases the concentration of carboxylic acids in alcoholic beverages, which affects the pH of the derivatization reaction, reducing derivatization efficiency and thus impacting the reliability of the results. Furthermore, the different matrices of the standard curve solution and the alcoholic beverage sample can also affect the authenticity and accuracy of the quantitative results.

[0004] Based on this, how to establish an accurate and reliable detection method with a low detection limit for detecting dipeptides in wine samples has become an urgent problem to be solved. Summary of the Invention

[0005] In order to solve the above problems, the present application provides a method for quantitative determination of dipeptides in liquor based on dansylation and liquid chromatography-mass spectrometry.

[0006] The present application provides a method for quantitative determination of dipeptides in liquor based on dansylation and liquid chromatography-mass spectrometry, which employs the following technical solutions:

[0007] A method for quantitatively determining dipeptides in liquor based on dansylation and liquid chromatography-mass spectrometry comprises the following steps:

[0008] (1) taking a liquor sample, and concentrating the liquor sample to obtain a concentrated sample;

[0009] (2) measuring the concentrated sample to obtain a sample to be tested, adding an internal standard to the sample to be tested, adding a dansyl chloride acetonitrile solution after drying, adjusting the pH value and performing a derivatization reaction, adding a mixed liquid after the reaction is completed, mixing uniformly and centrifuging, and taking the supernatant to obtain a liquid to be tested;

[0010] The mixed liquid includes dansyl chloride acetonitrile solution, pH buffer solution and hydrochloric acid;

[0011] (3) The liquid to be tested is sent to a liquid chromatography-mass spectrometer for detection, and the dipeptide is qualitatively and quantitatively analyzed based on the detection results.

[0012] Preferably, the step (1) comprises: taking 80 to 120 mL of the liquor sample, and concentrating the liquor sample 20 to 30 times to obtain a concentrated sample;

[0013] Preferably, the step (1) comprises: taking 100 mL of the liquor sample, and evaporating and concentrating the liquor sample 25 times to obtain a concentrated sample;

[0014] Preferably, the liquor sample is concentrated 25 times by rotary evaporation at 35-45° C. to obtain a concentrated sample;

[0015] More preferably, the liquor sample is concentrated 25 times by rotary evaporation at 40° C. to obtain a concentrated sample.

[0016] Preferably, the step (2) comprises: measuring 18 to 22 μL of the concentrated sample to obtain a sample to be tested, adding 9 to 11 μL of an internal standard to the sample to be tested, adding 8 to 12 μL of a dansyl chloride acetonitrile solution after freeze-drying, vortexing for 1 to 3 minutes, adding 8 to 12 μL of a buffer solution with a pH value of 9, vortexing for 1 to 3 minutes, adding an alkaline solution to adjust the pH value, and then performing a derivatization reaction, adding a mixed liquid after the reaction is completed, mixing evenly, and centrifuging, and taking the supernatant to obtain a liquid to be tested;

[0017] Preferably, the step (2) comprises: measuring 20 μL of the concentrated sample to obtain a sample to be tested, adding 10 μL of an internal standard to the sample to be tested, freeze-drying, adding 10 μL of a dansyl chloride acetonitrile solution, vortexing for 2 minutes, adding 10 μL of a buffer solution with a pH value of 9, vortexing for 2 minutes, adding an alkaline solution to adjust the pH value, and then performing a derivatization reaction, adding a mixed liquid after the reaction is completed, mixing evenly, and centrifuging, and taking the supernatant to obtain a liquid to be tested;

[0018] Preferably, the buffer solution is one of sodium carbonate / sodium bicarbonate buffer solution, potassium carbonate / potassium bicarbonate buffer solution, and sodium dihydrogen phosphate / disodium hydrogen phosphate buffer solution;

[0019] Preferably, the alkaline solution is a sodium hydroxide solution or a potassium hydroxide solution, or a mixed solution of a sodium hydroxide solution and a potassium hydroxide solution;

[0020] More preferably, the alkaline solution is a sodium hydroxide solution, and the concentration of the sodium hydroxide solution is 4 mol / L;

[0021] Preferably, the reaction temperature of the derivatization reaction is 55-65° C., and the reaction time is 1-1.5 h;

[0022] More preferably, the reaction temperature of the derivatization reaction is 60° C. and the reaction time is 1 h;

[0023] Preferably, the relative centrifugal force of the centrifugation is 14000-16000 g, and the centrifugation time is 8-12 min;

[0024] More preferably, the relative centrifugal force of the centrifugation is 15000 g, and the centrifugation time of the centrifugation is 10 min.

[0025] Preferably, the internal standard is L-phenylalanine- 13 C9- 15 N or L-serine- 13 C3- 15 N;

[0026] Preferably, the internal standard is L-phenylalanine- 13 C9- 15 N; the L-phenylalanine- 13 C9- 15 The concentration of N was 1 ng / mL.

[0027] Preferably, the concentration of the dansyl chloride acetonitrile solution is 3 to 5 mg / mL;

[0028] Preferably, the concentration of the dansyl chloride acetonitrile solution is 5 mg / mL.

[0029] Preferably, the step of vortexing for 2 minutes, adding an alkaline solution and adjusting the pH value before performing a derivatization reaction comprises: vortexing for 2 minutes, adding a 4 mol / L sodium hydroxide solution and adjusting the pH value to 6.7 for performing a derivatization reaction;

[0030] Preferably, the step of adding a 4 mol / L sodium hydroxide solution after vortexing for 2 minutes to adjust the pH value to 6.7 comprises: adding a 4 mol / L sodium hydroxide solution after vortexing for 2 minutes to obtain a reaction solution, detecting the reaction solution with pH test paper to obtain a colorimetric value corresponding to the pH value of the reaction solution; establishing a pH test paper standard colorimetric card colorimetric value-pH value curve with the pH value in the pH test paper standard colorimetric card as the horizontal coordinate and the colorimetric value of the pH test paper standard colorimetric card as the vertical coordinate; then establishing a volume-reaction solution pH value corresponding colorimetric value curve with the colorimetric value corresponding to the pH value of the reaction solution as the horizontal coordinate and the volume of the added 4 mol / L sodium hydroxide solution as the vertical coordinate; combining the pH test paper standard colorimetric card colorimetric value-pH value curve and the volume-reaction solution pH value corresponding colorimetric value curve; adjusting the pH value of the reaction solution to 6.7 by adjusting the volume of the added 4 mol / L sodium hydroxide solution;

[0031] The colorimetric value corresponding to the pH value of the reaction solution is obtained by taking a picture of the pH test paper used to detect the reaction solution and then taking the color from the picture;

[0032] The color value of the pH test paper standard color comparison card is obtained by taking a photo of the pH test paper standard color comparison card and then extracting the color from the photo.

[0033] Preferably, the mixed liquid comprises: 8 to 12 μL of dansyl chloride acetonitrile solution, 8 to 12 μL of a buffer solution with a pH value of 9, and hydrochloric acid;

[0034] Preferably, the mixed liquid comprises: 10 μL of dansyl chloride acetonitrile solution, 10 μL of a buffer solution with a pH value of 9, and 4 mol / L of hydrochloric acid, and the volume concentration of the hydrochloric acid in the entire system is 3%.

[0035] Preferably, the step (3) comprises: sending the liquid to be tested into a liquid chromatography-mass spectrometer for detection, and performing qualitative analysis based on the characteristic peaks of the spectrum; substituting the detected characteristic peak areas into a standard curve to perform quantitative analysis;

[0036] The standard curve is obtained by the following steps: weighing a dipeptide standard sample, preparing the dipeptide standard sample into standard solutions with different concentration gradients; taking 18 to 22 μL of the standard solutions with different concentration gradients, adding an internal standard, drying, and then adding a dansyl chloride acetonitrile solution to carry out a derivatization reaction; after the reaction is completed, adding a matrix solution, mixing evenly, and centrifuging, taking the supernatant and sending it to a liquid chromatography-mass spectrometer for detection, and plotting a standard curve with the obtained dipeptide chromatographic peak area as the ordinate and the dipeptide concentration in the standard solution as the abscissa;

[0037] The dipeptide standard sample comprises one of: Ala-Ile, Ala-Leu, Ile-Ala, Leu-Ala, Ile-Ser, Pro-Leu, Ile-Pro, Leu-Pro, Val-Leu, and Val-Ile;

[0038] The standard solutions with different concentration gradients include standard solutions with concentrations of 0.5pg / mL, 1pg / mL, 5pg / mL, 10pg / mL, 50pg / mL, 100pg / mL, 500pg / mL, 1ng / mL, 5ng / mL, 10ng / mL, 50ng / mL, 100ng / mL, 500ng / mL, and 1g / mL;

[0039] Preferably, the matrix solution is prepared by drying the sample to be tested and adding the mixed liquid and mixing them evenly;

[0040] Preferably, the relative centrifugal force of the centrifugation is 14000-16000 g, and the centrifugation time is 8-12 min;

[0041] More preferably, the relative centrifugal force of the centrifugation is 15000g, and the centrifugation time of the centrifugation is 10min;

[0042] Preferably, the dipeptides detected in the liquor include one or more of Ala-Ile, Ala-Leu, Ile-Ala, Leu-Ala, Ile-Ser, Pro-Leu, Ile-Pro, Leu-Pro, Val-Leu, and Val-Ile.

[0043] Preferably, the liquid chromatography parameters are set as follows: a BEH C8 (2.1 mm × 50 mm, 1.7 μm) chromatographic column is used; the column temperature is 48-52° C., the flow rate is 0.33-0.37 mL / min; the aqueous phase is an aqueous solution containing 0.1% by volume formic acid, the organic phase is an acetonitrile solution containing 0.1% by volume formic acid, and the chromatographic gradient is as follows: an initial gradient of 5% organic phase, maintained at 5% organic phase for 1 minute, the organic phase linearly increases to 100% from the 1st minute to the 24th minute, maintains 100% organic phase from the 24th minute to the 28th minute, reduces the organic phase to 5% from the 28th minute to the 28.1th minute, and equilibrates the gradient for 30 minutes;

[0044] Preferably, the column temperature is 50° C. and the flow rate is 0.35 mL / min.

[0045] Preferably, the mass spectrometry parameters are set as follows: using a Xevo TQXS series triple quadrupole mass spectrometry analysis system, the spray voltage positive ion is 2.8 to 3.2 kV; the desolvation gas flow rate is 640 to 660 L / h, the temperature is 340 to 360 ° C; the cone voltage is 28 to 32 V; the scanning mode is the multiple reaction monitoring mode; argon is the collision gas;

[0046] Preferably, the spray voltage for positive ions is 3kV; the desolvation gas flow rate is 650L / h, the temperature is 350°C; and the cone voltage is 30V.

[0047] This application has the following beneficial technical effects:

[0048] This application adjusts the pH value of the sample to be tested so that the dipeptide substance in the liquor can undergo a good derivatization reaction with dansyl chloride, thereby enabling the trace or trace amount of dipeptide substance in the liquor to be effectively detected, thereby improving the accuracy of detecting dipeptide substances in the liquor; at the same time, by adding a mixed liquid, the detection environment of the liquor sample is made the same as the standard solution environment, which can greatly reduce the interference of the liquor matrix and further improve the accuracy of detecting dipeptides in the liquor. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 is the colorimetric-pH fitting curve and equation;

[0050] Figure 2 The volume-color curve and equation of 4 mol / L NaOH added to wine sample A1;

[0051] Figure 3 The volume-color curve and equation of 4 mol / L NaOH added to wine sample A2;

[0052] Figure 4 The volume-color curve and equation of 4 mol / L NaOH added to wine sample A3;

[0053] Figure 5 The volume-color curve and equation of 4 mol / L NaOH added to wine sample A4;

[0054] Figure 6 The volume-color curve and equation of 4 mol / L NaOH added to A5 wine sample;

[0055] Figure 7 The volume-color curve and equation of 4 mol / L NaOH added to A6 wine sample;

[0056] Figure 8 The volume-color curve and equation of 4 mol / L NaOH added to A7 wine sample;

[0057] Figure 9 The volume-color curve and equation of 4 mol / L NaOH added to A8 wine sample;

[0058] Figure 10 The volume-color curve and equation of A9 wine sample with 4 mol / L NaOH added;

[0059] Figure 11 The volume-color curve and equation of 4 mol / L NaOH added to A10 wine sample;

[0060] Figure 12 is the relative intensity of 5 ng / mL dipeptide standard at different derivatization times;

[0061] Figure 13 is the relative intensity of 50 ng / mL dipeptide standard at different derivatization times;

[0062] Figure 14 is the relative intensity of 500 ng / mL dipeptide standard at different derivatization times;

[0063] Figure 15 is the relative intensity ratio of 50 ng / mL and 5 ng / mL dipeptide standards at different derivatization times;

[0064] Figure 16 is the relative intensity ratio of 500 ng / mL and 50 ng / mL dipeptide standards at different derivatization times;

[0065] Figure 17 is the ratio of the derivatization product to the prototype of Ser-13C3-15N at different sample / derivatization reagent ratios;

[0066] Figure 18 It is the ratio of the derivatized product to the prototype of Phe-13C9-15N at different sample / derivatization reagent ratios. DETAILED DESCRIPTION

[0067] The content of dipeptides in liquor is low and not easy to be detected. Therefore, when detecting dipeptides in liquor, the liquor sample needs to be concentrated and derivatized before detection. However, during the concentration process, the concentration of carboxyl compounds in the liquor increases, which reduces the pH value of the concentrated liquor sample, resulting in a decrease in the efficiency of the derivatization reaction between the dipeptides in the liquor sample and dansyl chloride, which will have a greater impact on the results of dipeptide detection in liquor. At the same time, when using a standard curve to quantitatively detect dipeptides in liquor, the preparation of standard solutions in the prior art often ignores the detection effect of the liquor matrix on the standard solution; resulting in a large error in the established standard curve, thereby reducing the accuracy of the quantitative detection results of dipeptides in liquor. Based on this, the present application provides a quantitative method for dipeptides in liquor based on dansylation and liquid chromatography-mass spectrometry to improve the accuracy of the quantitative detection results of dipeptides in liquor.

[0068] The present application is further described below with reference to the following embodiments.

[0069] Reagents: Alanyl-isoleucine (Ala-Ile), alanyl-leucine (Ala-Leu), leucyl-alanine (Leu-Ala), isoleucyl-serine (Ile-Ser), prolyl-leucine (Pro-Leu), isoleucyl-proline (Ile-Pro), leucyl-proline (Leu-Pro), valyl-leucine (Val-Leu), valyl-isoleucine (Val-Ile). The purity of all the above dipeptide standards is greater than 95%.

[0070] Example 1

[0071] The present application provides a method for quantitative determination of dipeptides in liquor based on dansylation and liquid chromatography-mass spectrometry, comprising the following steps:

[0072] (1) Take a liquor sample and concentrate the liquor sample to obtain a concentrated sample.

[0073] Specifically, 100 mL of liquor sample was taken, and the liquor sample was concentrated 25 times by rotary evaporation at 40° C. to obtain 4 mL of concentrated sample.

[0074] (2) measuring the concentrated sample to obtain a sample to be tested, adding an internal standard to the sample to be tested, adding a dansyl chloride acetonitrile solution after drying, adjusting the pH value and performing a derivatization reaction, adding a mixed liquid after the reaction is completed, mixing uniformly and centrifuging, and taking the supernatant to obtain a liquid to be tested;

[0075] The mixed liquid includes dansyl chloride acetonitrile solution, pH buffer solution and hydrochloric acid.

[0076] Specifically, 20 μL of concentrated sample was measured to obtain a test sample, and 10 μL of L-phenylalanine- 13 C9- 15 N was used as the internal standard. After freeze-drying at 0°C for 6 hours, 10 μL of 5 mg / mL dansyl chloride acetonitrile solution was added. After vortexing for 2 minutes, 10 μL of sodium carbonate / sodium bicarbonate buffer solution (0.4 mol / L, pH = 9.0) was added. After vortexing for 2 minutes, 4 mol / L sodium hydroxide solution was added to adjust the pH value to 6.7 for derivatization reaction. The reaction temperature of the derivatization reaction was 60°C, and the reaction time was 1 hour. After the reaction was completed, the mixed liquid was added, mixed evenly, and centrifuged at a relative centrifugal force of 15000g for 10 minutes. The supernatant was taken to obtain the liquid to be tested.

[0077] Further, vortexing for 2 minutes and then adding a 4 mol / L sodium hydroxide solution to adjust the pH value to 6.7 to perform a derivatization reaction, comprising: vortexing for 2 minutes and then adding a 4 mol / L sodium hydroxide solution to obtain a reaction solution, testing the reaction solution with a pH test paper to obtain a colorimetric value corresponding to the pH value of the reaction solution;

[0078] Establishing a pH test paper standard colorimetric card chromaticity value-pH value curve using the pH value of the pH test paper standard colorimetric card as the horizontal coordinate and the chromaticity value of the pH test paper standard colorimetric card as the vertical coordinate; establishing a volume-reaction solution pH value corresponding chromaticity value curve using the chromaticity value corresponding to the pH value of the reaction solution as the horizontal coordinate and the volume of the added 4 mol / L sodium hydroxide solution as the vertical coordinate; combining the pH test paper standard colorimetric card chromaticity value-pH value curve and the volume-reaction solution pH value corresponding chromaticity value curve; adjusting the pH value of the reaction solution to 6.7 by adjusting the volume of the added 4 mol / L sodium hydroxide solution;

[0079] The colorimetric value corresponding to the pH value of the reaction solution is obtained by taking a picture of the pH test paper used to detect the reaction solution and then taking the color from the picture;

[0080] The color value of the pH test paper standard color comparison card is obtained by taking a picture of the pH test paper standard color comparison card and then extracting the color from the picture.

[0081] It is understandable that due to the small volume of the reaction solution, the reaction solution cannot be adjusted by a pH meter. However, when pH test paper is used for testing and adjustment, only integer detection of the pH value can be performed, and the pH value of the solution cannot be accurately adjusted after the decimal point, and it is easy to over-adjust. The present application detects the chromaticity corresponding to the reaction solution by adjusting the volume of the added 4 mol / L sodium hydroxide solution, and then obtains the corresponding pH value according to the chromaticity, thereby achieving accurate adjustment of the pH value of the reaction solution.

[0082] Furthermore, the mixed liquid includes: 10 μL of dansyl chloride solution, 10 μL of a buffer solution with a pH value of 9, and 4 mol / L of hydrochloric acid, and the volume concentration of the hydrochloric acid in the entire system is 3%.

[0083] After the liquor sample is concentrated, the concentration of carboxyl substances increases, resulting in a decrease in the pH value of the reaction solution, which affects the derivatization reaction between the dipeptide in the liquor sample and dansyl chloride. The present application adjusts the pH value of the reaction solution to enable a better derivatization reaction between the dipeptide and dansyl chloride, making the derivatized dipeptide easier to detect, thereby improving the accuracy of detecting the dipeptide content in the liquor.

[0084] (3) The liquid to be tested is sent to a liquid chromatography-mass spectrometer for detection, and the dipeptide is qualitatively and quantitatively analyzed based on the detection results.

[0085] Specifically, the liquid to be tested is sent to a liquid chromatography-mass spectrometer for detection, and a qualitative analysis is performed based on the characteristic peaks of the spectrum; the detected characteristic peak areas are substituted into a standard curve for quantitative analysis.

[0086] Liquid chromatography was performed using a Waters Acquity series ultra-high performance liquid chromatograph (Waters, USA). The liquid chromatography parameters were as follows: a BEH C8 (2.1 mm × 50 mm, 1.7 μm) column; a column temperature of 50° C.; a flow rate of 0.35 mL / min; an aqueous phase containing 0.1% by volume formic acid in water; an organic phase containing 0.1% by volume formic acid in acetonitrile; and a chromatographic gradient as follows: an initial gradient of 5% organic phase, maintained at 5% organic phase for 1 minute, the organic phase linearly increased to 100% from the 1st minute to the 24th minute, maintained at 100% organic phase from the 24th minute to the 28th minute, reduced to 5% organic phase from the 28th minute to the 28.1st minute, and the gradient was equilibrated for 30 minutes.

[0087] The mass spectrometer used a XevoTQXS series triple quadrupole mass spectrometer analysis system (Waters, USA), and the mass spectrometer parameters were set as follows: a XevoTQXS series triple quadrupole mass spectrometer analysis system was used, the spray voltage was 3 kV for positive ions; the desolvation gas flow rate was 650 L / h, the temperature was 350°C; the cone voltage was 30 V; the scanning mode was the multiple reaction monitoring mode; and argon was the collision gas.

[0088] The standard curve was obtained by the following steps: Weigh the dipeptide standard samples separately. The dipeptide standard samples include: Ala-Ile, Ala-Leu, Ile-Ala, Leu-Ala, Ile-Ser, Pro-Leu, Ile-Pro, Leu-Pro, Val-Leu, and Val-Ile. Prepare each dipeptide standard sample into a standard solution with different concentration gradients using deionized water as the solvent. The standard solutions with different concentration gradients include: 0.5pg / mL, 1pg / mL, 5pg / mL, 10pg / mL, 50pg / mL, 100pg / mL, 500pg / mL, 1ng / mL, 5ng / mL, 10ng / mL, 50ng / mL, 100ng / mL, 500ng / mL, and 1g / mL standard solution. Take 20μL of each concentration gradient standard solution and add 10μL of 1ng / mL L-phenylalanine- 13 C9- 15 N was used as the internal standard, and after freeze-drying at 0°C for 6 hours, 10 μL of 5 mg / mL dansyl chloride acetonitrile solution was added, vortexed for 2 minutes, and then 10 μL of sodium carbonate / sodium bicarbonate buffer solution (0.4 mol / L, pH = 9.0) was added and vortexed for 2 minutes. According to the colorimetric value-pH value curve of the pH test paper standard colorimetric card, the pH value of the entire system was 6.7 at this time. The derivatization reaction was then carried out. The reaction temperature of the derivatization reaction was 60°C and the reaction time was 1 hour. After the reaction was completed, the matrix solution was added, mixed evenly, and centrifuged at a relative centrifugal force of 15,000 g for 10 minutes. The supernatant was sent to a liquid chromatography-mass spectrometer for detection. The obtained dipeptide chromatographic peak area was used as the ordinate, and the concentration of the dipeptide in the standard solution was used as the abscissa to draw a standard curve.

[0089] Furthermore, the matrix solution is prepared by freeze-drying the sample to be tested in step (2) at 0° C. for 6 h, adding the above mixed liquid and mixing evenly.

[0090] It is understood that the standard solution and the dansyl chloride acetonitrile solution can undergo a good derivatization reaction, and the pH value during the derivatization reaction is 6.7. Therefore, when testing liquor samples in this application, the pH value of the derivatization reaction solution is adjusted to 6.7, which also achieves the same reaction environment as the standard solution, making the test results more accurate.

[0091] The standard curve equations of the 10 dipeptides are shown in Table 1.

[0092] Table 1 Standard curve equation is as follows:

[0093] Dipeptide equation Ala-Ile y=136.92x-866638 Ala-Leu y=399.11x+24479 Ile-Ala y=563.87x-9816.4 Leu-Ala y=268.11x+189684 Ile-Ser y=261.82x-1E+06 Pro-Leu y=95.666x-832415 Ile-Pro y=2354.2x-379591 Leu-Pro y=199.79x-742635 Val-Leu y=194.33x-120133 Val-Ile y=279.45x-1E+06

[0094] y is the corrected peak area, x is the concentration fg / mL, and the concentration calculated by the above equation is reduced according to the concentration multiple to calculate the dipeptide concentration of the liquor sample.

[0095] The detection results of 10 dipeptides, including Ala-Ile, Ala-Leu, Ile-Ala, Leu-Ala, Ile-Ser, Pro-Leu, Ile-Pro, Leu-Pro, Val-Leu, and Val-Ile, are shown in Table 2. The parent ion in the table refers to the molecular ion, and product ion 1 and product ion 2 are the fragment ions after ionization.

[0096] Table 2 The detection results of 10 dipeptides are shown in Table 2

[0097]

[0098]

[0099] Method evaluation

[0100] The intraday precision of the test method was tested with the intraday relative standard deviation, and the interday precision of the test method was tested with the interday relative standard deviation. The intraday precision is to test the standard solution within one day, repeat the test 6 times and calculate its relative standard deviation. The interday precision is to test the standard solution after one day, repeat the test 6 times and calculate its relative standard deviation. According to the above method, the intraday precision and interday precision of standard solutions with concentrations of 5ng / mL (low), 50ng / mL (medium) and 500ng / mL (high) were calculated respectively. The detection limit is the lowest standard sample concentration with a signal-to-noise ratio close to 3, and the linear range is based on the R of the concentration-signal curve. 2 The minimum standard is 0.99, and the linear range is 2 to 4 orders of magnitude. The specific test data are shown in Table 3.

[0101] Table 3 Test data

[0102]

[0103]

[0104] As can be seen from the table, the quantification limit of the dipeptide quantification method in liquor in this application is at the ppb level, which is very low, indicating that this application is capable of detecting trace amounts of dipeptides in liquor. The intra-day standard deviation and inter-day standard deviation are also low, indicating that this application has high precision and small detection error.

[0105] Example 2

[0106] Ten liquor samples were taken separately and numbered A1 to A10. The samples were then concentrated according to the steps in Example 1 to obtain concentrated samples A1 to A10. 10 mL of each of the liquor samples A1 to A10 was then taken to obtain 100 mL of a mixed liquor sample. The mixed liquor sample was then concentrated according to the steps in Example 1 to obtain a mixed concentrated sample.

[0107] The concentrated samples of A1 to A10 were tested according to the test steps in Example 1. The color value-pH value curve of the pH test paper standard color card in this embodiment is as follows: Figure 1 The chromaticity curves corresponding to the volume of 4 mol / L sodium hydroxide solution added to A1 to A10 and the pH value of the reaction solution are shown as follows. Figures 2 to 11 shown.

[0108] Depend on Figures 2 to 11 It can be seen that the equations of the chromaticity curves corresponding to the volume-pH value of the reaction solution of liquor samples A1 to A10 are: A1 (y = 0.0002x 3 -0.216x 2 +0.998x-15.085), A2 (y=0.0132x 2 +0.9121x-13.881), A3 (y=0.0001x 3 -0.0162x 2 +0.6626x-7.5547), A4 (y=-0.0021x 2 +0.1796x-1.8136), A5 (y=0.0007x 3 -0.078x 2 +2.9059x-34.084), A6(y=-5E-05x 3 +0.0064x 2 -0.2437x+3.8104), A7 (y=0.0006x 2 -0.0225x+0.3899), A8 (y=3E-05x 3 -0.0045x 2 +0.2285x-2.1036), A9 (y=-0.0256x 2+1.5642x-21.983), A10 (y=-0.0002x 2 +0.0371x+0.5697). Based on the colorimetric value curve corresponding to the volume of 4 mol / L sodium hydroxide solution added to A1-A10 versus the pH value of the reaction solution and the colorimetric value-pH value curve of the pH test paper standard color chart, the volume of 4 mol / L NaOH added to adjust the pH of the reaction solution of A1-A10 to 6.7 was calculated. The results are shown in Table 4.

[0109] When preparing the standard solution, the sample to be tested in preparing the matrix solution in Example 1 was replaced with the mixed concentrated sample, and the remaining steps were the same as in Example 1.

[0110] Table 4 Volume of 4 mol / L NaOH added when the pH of the reaction solutions of A1 to A10 was adjusted to 6.7

[0111]

[0112]

[0113] The dipeptide contents in A1 to A10 detected in this example are shown in Table 5.

[0114] Table 5 Dipeptide content in A1 to A10

[0115]

[0116] It can be seen from Table 5 that the quantitative method in this application can be used to quantify the content of 10% in A1 to A10 liquor samples. -11 ~10 -8 mol / L dipeptides were accurately quantified with a very low limit of quantification. This indicates that the quantitative method for dipeptides in liquor of the present application is capable of accurately quantifying trace amounts of dipeptides in liquor.

[0117] Example 3 Investigating the Effect of Different Derivatization Reaction Times on Dipeptide Detection Results

[0118] This example is to explore the effect of derivatization reaction time on dipeptide detection results. The derivatization reaction time includes 5min, 10min, 20min, 40min, and 60min. The 10 dipeptide standard solutions prepared in Example 1 were subjected to derivatization reaction with dansyl chloride acetonitrile solution to explore the effect of different derivatization reaction times on dipeptide detection results. 10 dipeptide standard solutions with concentrations of 5ng / mL, 50ng / mL, and 500ng / mL were respectively subjected to derivatization reaction with dansyl chloride acetonitrile solution. The response intensity values ​​of different derivatization times are shown in FIG. Figures 12-14The ratio of the response intensity of 50 ng / mL dipeptide standard solution at different derivatization times to the response intensity of 5 ng / mL dipeptide standard solution at different derivatization times is shown in Figure 15 As shown; the ratio of the response intensity of 500 ng / mL dipeptide standard solution at different derivatization times to the response intensity of 50 ng / mL dipeptide standard solution at different derivatization times is shown as Figure 16 shown.

[0119] from Figures 12 to 14 It can be seen that after derivatization of the 10 dipeptide standard solutions at concentrations of 5 ng / mL, 50 ng / mL, and 500 ng / mL, the responses of most dipeptides gradually increased with increasing derivatization time, reaching a maximum at a derivatization time of 60 minutes. This indicates that changing the derivatization reaction time can increase the response value of dipeptide detection, thereby improving the accuracy of dipeptide detection.

[0120] from Figure 15 and Figure 16 As can be seen, as the derivatization time increases, the response ratios of dipeptide concentrations of 50 ng / mL to 5 ng / mL and 500 ng / mL to 50 ng / mL gradually approach the concentration ratios. This indicates that the dipeptide mass spectrometry response and derivatization efficiency are optimal when the derivatization time is 60 min.

[0121] Example 4 Investigating the Effect of the Ratio of Internal Standard Volume to Derivatization Reagent Volume on Detection Results

[0122] This example is to explore the effect of the volume ratio of the internal standard substance to the derivatization reagent on the detection results. The volume ratio of the internal standard substance to the derivatization reagent includes: 1:2, 1:1, 2.5:1, 5:1, 7.5:1, 10:1, and the internal standard of two amino acids, L-serine- 13 C3- 15 N(Ser- 13 C3- 15 N) and L-phenylalanine- 13 C9- 15 N(Phe- 13 C9- 15 N) Add two samples to be tested respectively, carry out derivatization reaction and detection according to the steps in Example 1. Internal standard Ser- 13 C3- 15 The relative intensity of the DNS derivatized product of N and the internal standard that did not undergo derivatization reaction is as follows Figure 17 Internal standard Phe- 13 C9- 15 The relative intensity of the DNS derivatized product of N and the internal standard that did not undergo derivatization reaction is as follows Figure 18 shown.

[0123] As can be seen from the figure, when the volume ratio of the internal standard to the derivatization reagent is 1:2 and 1:1, the relative intensity of the DNS derivatization products of the two internal standards far exceeds that of the underivatized internal standard. When the volume ratio of the internal standard to the derivatization reagent is greater than 2.5:1, the derivatization products are significantly reduced. The degree of derivatization of the internal standard can reflect the degree of derivatization of the dipeptide with dansyl chloride in the liquor sample. By controlling the volume ratio of the internal standard to the derivatization reagent, a more appropriate amount of dansyl chloride can be selected, promoting the derivatization reaction of the dipeptide in the liquor sample, thereby enabling more accurate detection of the dipeptide in the liquor.

[0124] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed, but as long as they are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A method for quantitative determination of dipeptides in liquor based on dansylation and liquid chromatography-mass spectrometry, characterized in that: The dipeptide comprises one or more of Ala-Ile, Ala-Leu, Ile-Ala, Leu-Ala, Ile-Ser, Pro-Leu, Ile-Pro, Leu-Pro, Val-Leu, and Val-Ile; and comprises the following steps: (1) taking a liquor sample, and concentrating the liquor sample to obtain a concentrated sample; (2) measuring the concentrated sample to obtain a sample to be tested, adding an internal standard to the sample to be tested, adding a dansyl chloride acetonitrile solution after drying, adjusting the pH value and then performing a derivatization reaction, adding a mixed liquid after the reaction is completed, mixing evenly and centrifuging, and taking the supernatant to obtain a liquid to be tested; The mixed liquid includes dansyl chloride acetonitrile solution, pH buffer solution and hydrochloric acid; (3) sending the liquid to be tested into a liquid chromatography-mass spectrometer for testing, and performing qualitative and quantitative analysis of the dipeptide based on the test results; The step (2) comprises: measuring 20 μL of the concentrated sample to obtain a sample to be tested, adding 10 μL of an internal standard to the sample to be tested, freeze-drying, adding 10 μL of a dansyl chloride acetonitrile solution, vortexing for 2 minutes, adding 10 μL of a buffer solution with a pH value of 9, vortexing for 2 minutes, adding an alkaline solution to adjust the pH value, and then performing a derivatization reaction. After the reaction is completed, a mixed liquid is added, mixed evenly, and then centrifuged, and the supernatant is taken to obtain a liquid to be tested; The reaction temperature of the derivatization reaction is 55-65°C, and the reaction time is 1-1.5h; The derivatization reaction comprises: adding a 4 mol / L sodium hydroxide solution after vortexing for 2 minutes to obtain a reaction solution, detecting the reaction solution with a pH test paper to obtain a colorimetric value corresponding to the pH value of the reaction solution; establishing a pH test paper standard colorimetric card colorimetric value-pH value curve with the pH value of the pH test paper standard colorimetric card as the horizontal coordinate and the colorimetric value of the pH test paper standard colorimetric card as the vertical coordinate; establishing a volume-reaction solution pH value corresponding colorimetric value curve with the colorimetric value corresponding to the pH value of the reaction solution as the horizontal coordinate and the volume of the added 4 mol / L sodium hydroxide solution as the vertical coordinate; combining the pH test paper standard colorimetric card colorimetric value-pH value curve and the volume-reaction solution pH value corresponding colorimetric value curve; and adjusting the pH value of the reaction solution to 6.7 by adjusting the volume of the added 4 mol / L sodium hydroxide solution. The liquid chromatography parameters were set as follows: a BEH C8 column was used as the chromatographic column, the aqueous phase was an aqueous solution containing 0.1% by volume formic acid, the organic phase was an acetonitrile solution containing 0.1% by volume formic acid, and the chromatographic gradient was as follows: an initial gradient of 5% organic phase, maintained at 5% organic phase for 1 minute, the organic phase linearly increased to 100% from the 1st minute to the 24th minute, maintained at 100% organic phase from the 24th minute to the 28th minute, and reduced to 5% organic phase from the 28th minute to the 28.1th minute, and the gradient was equilibrated for 30 minutes; The mass spectrometry parameters were set as follows: a Xevo TQXS series triple quadrupole mass spectrometry analysis system was used, the spray voltage for positive ions was 2.8 to 3.2 kV; the desolvation gas flow rate was 640 to 660 L / h, the temperature was 340 to 360° C.; the cone voltage was 28 to 32 V; the scanning mode was the multiple reaction monitoring mode; and argon was used as the collision gas.

2. The method according to claim 1, characterized in that The step (1) comprises: taking 80 to 120 mL of the liquor sample, and concentrating the liquor sample 20 to 30 times to obtain a concentrated sample.

3. The method according to claim 1 or 2, characterized in that The buffer solution is one of a sodium carbonate / sodium bicarbonate buffer solution, a potassium carbonate / potassium bicarbonate buffer solution, and a sodium dihydrogen phosphate / disodium hydrogen phosphate buffer solution.

4. The method according to claim 1, wherein The internal standard is L-phenylalanine- 13 C9- 15 N or L-serine- 13 C3- 15 NL.

5. The method according to claim 3, characterized in that The concentration of the dansyl chloride acetonitrile solution is 3-5 mg / mL.

6. The method according to claim 1, characterized in that ； The colorimetric value corresponding to the pH value of the reaction solution is obtained by taking a picture of the pH test paper used to detect the reaction solution and then taking the color from the picture; The color value of the pH test paper standard color comparison card is obtained by taking a photo of the pH test paper standard color comparison card and then extracting the color from the photo.

7. The method according to claim 3, characterized in that The mixed liquid includes: 8 to 12 μL of dansyl chloride acetonitrile solution, 8 to 12 μL of a buffer solution with a pH value of 9, and hydrochloric acid.

8. The method according to claim 1, characterized in that The step (3) comprises: sending the liquid to be tested into a liquid chromatography-mass spectrometer for testing, and performing qualitative analysis based on the characteristic peaks of the spectrum; substituting the detected characteristic peak areas into a standard curve to perform quantitative analysis; The standard curve is obtained by the following steps: weighing a dipeptide standard sample, preparing the dipeptide standard sample into standard solutions with different concentration gradients; taking 18 to 22 μL of the standard solutions with different concentration gradients, adding an internal standard, drying, and then adding a dansyl chloride acetonitrile solution to carry out a derivatization reaction; after the reaction is completed, adding a matrix solution, mixing evenly, and centrifuging, taking the supernatant and sending it to a liquid chromatography-mass spectrometer for detection, and plotting a standard curve with the obtained dipeptide chromatographic peak area as the ordinate and the dipeptide concentration in the standard solution as the abscissa; The dipeptide standard samples include: Ala-Ile, Ala-Leu, Ile-Ala, Leu-Ala, Ile-Ser, Pro-Leu, Ile-Pro, Leu-Pro, Val-Leu, Val-Ile; The standard solutions with different concentration gradients include standard solutions with concentrations of 0.5 pg / mL, 1 pg / mL, 5 pg / mL, 10 pg / mL, 50 pg / mL, 100 pg / mL, 500 pg / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, 500 ng / mL, and 1 g / mL.

9. The method according to claim 1, characterized in that The liquid chromatography parameters are set as follows: the chromatographic column has a specification of 2.1 mm×50 mm and a 1.7 μm; the column temperature is 48-52° C., and the flow rate is 0.33-0.37 mL / min.

10. The method according to claim 1, characterized in that The spray voltage for positive ions was 3 kV; the desolvation gas flow rate was 650 L / h, the temperature was 350° C.; and the cone voltage was 30 V.

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