CN115015429A - Method for synchronously detecting middle-and-late-stage glycosylation end products of polygonatum sibiricum - Google Patents
Method for synchronously detecting middle-and-late-stage glycosylation end products of polygonatum sibiricum Download PDFInfo
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Abstract
A method for synchronously detecting advanced glycosylation end products in rhizoma Polygonati is provided. The invention belongs to the field of food detection. The invention aims to solve the problem that a method for detecting glycosylation end products in polygonatum is lacked at present, and a synchronous detection method of carboxymethyl lysine and carboxyethyl lysine suitable for representative products of the glycosylation end products in polygonatum products is defined. The method for synchronously detecting the advanced glycosylation end products in the sealwort comprises the following steps: pretreating a polygonatum sibiricum product, performing derivatization reaction, performing solid-phase extraction, drawing a standard curve and measuring the content, and synchronously detecting carboxymethyl lysine and carboxyethyl lysine generated in the polygonatum sibiricum processing process by using UPLC-MS/MS under a certain condition.
Description
Technical Field
The invention belongs to the field of food detection, and particularly relates to a method for synchronously detecting a middle-advanced glycosylation end product of rhizoma polygonati.
Background
Rhizoma Polygonati is not suitable for direct administration due to its strong irritation, and different processing methods are usually adopted to remove the irritation. Rhizoma Polygonati is rich in polysaccharide, and is very easy to produce glycosylation reaction in high temperature processing, and the generated glycosylation end product can induce diabetes and other diseases, and is contrary to the function of rhizoma Polygonati polysaccharide in treating type II diabetes.
At present, detection methods for glycosylation end products are mostly concentrated in the fields of dairy products, aquatic products and fried foods, and because pretreatment methods for different types of objects to be detected are different during detection, the pretreatment methods relate to retention rate of the content of the glycosylation end products in original objects to be detected, and further influence accuracy of the detection methods.
Disclosure of Invention
The invention aims to solve the technical problems that a method for detecting glycosylation end products in polygonatum is lacked at present and the existing means is inaccurate in detection and low in efficiency, and defines a synchronous detection method suitable for representative products of glycosylation end products in polygonatum products, namely carboxymethyl lysine and carboxyethyl lysine.
The method for synchronously detecting the middle-advanced glycosylation end products of rhizoma polygonati provided by the invention comprises the following steps:
step 1: pulverizing rhizoma Polygonati, sieving, mixing rhizoma Polygonati powder with n-hexane, eluting for 3-5 times, adding sodium tetraborate solution for reduction reaction, eluting with Sevag solution, adding hydrochloric acid into precipitate, incubating at 100-120 deg.C for 20-24 hr, and filtering to obtain hydrolysate;
step 2: adding a mixed isotope internal standard substance of CML-d4 and CEL-d4 into the hydrolysate, uniformly mixing, then adjusting the pH to 8.0, diluting with pure water by 5 times, adding an acetonitrile solution and a sodium tetraborate solution which is equal to the internal standard substance, uniformly mixing, adding the acetonitrile solution again in the mixing process, standing for 10min after uniformly mixing, and then adding formic acid to terminate the reaction to obtain a derivative solution;
and step 3: activating the derivative solution by methanol and balancing the derivative solution with a formic acid solution, and then performing solid-phase extraction by an SPE (solid phase extraction) column to obtain a solution to be detected;
and 4, step 4: drawing a standard curve;
and 5: detecting the advanced glycosylation end products in the solution to be detected by using UPLC-MS/MS, and calculating the actual content according to a standard curve.
Further limiting, in the step 1, the rhizoma polygonati is obtained by removing impurities from fresh rhizoma polygonati, cleaning, slicing, and then repeating steaming and drying for 9 times in a water-proof way, wherein the steaming is carried out for 2 hours and the drying is carried out for 3 hours at 100 ℃.
Further limiting, the volume ratio of the mass of the fine yellow powder to the volume of the normal hexane in the step 1 is 3 g: (20-30) mL.
Further limiting, the volume ratio of the mass of the rhizoma polygonati powder to the sodium tetraborate solution in the step 1 is 1 g: (10-20) mL, wherein the concentration of the sodium tetraborate solution is 0.1-0.3 mol/L.
Further limiting, in the step 1, the Sevag solution is a mixed solution of chloroform and n-butanol in a volume ratio of 4: 1.
Further limiting, the ratio of the mass of the precipitate to the volume of the hydrochloric acid in step 1 is 1 g: (15-25) mL, and the concentration of hydrochloric acid is 5-7 mol/L.
Further limiting, the volume ratio of the hydrolysis liquid to the internal standard substance in the step 2 is 1: (0.15-0.25), and the concentration of CEL-d4 in the internal standard substance is 0.7-0.9 mu g/mL.
Further limiting, the volume ratio of the acetonitrile solution and the hydrolysate added for the first time in the step 2 is (0.4-0.6): 1, the mass concentration of the sodium tetraborate solution is 4-6%, and the volume ratio of the acetonitrile solution to the hydrolysate added again is (0.1-0.3): 1.
further limiting, the volume ratio of the formic acid to the hydrolysate in the step 2 is (0.004-0.006): 1.
further limiting, the volume ratio of the methanol to the derivative liquid in the step 3 is 1: (2-4), wherein the volume ratio of the formic acid solution to the derivative solution is 1: (2-4), wherein the volume concentration of the formic acid solution is 0.4-0.6%.
Further limiting, the solid phase extraction process by SPE column in step 3 is: passing through an SPE column at the flow rate of 0.8-1.2mL/min, then leaching with 20% acetonitrile solution, leaching with dichloromethane after vacuum drying, vacuum drying again, then eluting with ammonia water/methanol solution and collecting eluent, blowing elution liquid nitrogen, concentrating and drying, finally re-dissolving with acetonitrile solution, vortex mixing uniformly, wherein the volume ratio of ammonia water to methanol in the ammonia water/methanol solution is 5: 95.
further limiting, the specific process of drawing the standard curve in the step 4 is as follows: (1) 10.0mg of each of carboxymethyl lysine and carboxyethyl lysine standard substance is prepared into mixed standard substance stock solution with the mass concentration of 100 mug/mL by using ultrapure water, and the mixed standard substance stock solution is stored at the temperature of-20 ℃; (2) diluting with 80% acetonitrile solution in gradient to obtain carboxymethyl lysine and carboxyethyl lysine standard solutions with mass concentrations of 2, 4, 6, 8 and 10 μ g/mL respectively, and performing sample injection according to liquid chromatography conditions, wherein each concentration is subjected to sample injection in parallel for three times; (3) and (4) drawing a standard curve by taking the concentration of the standard substance as a horizontal coordinate and the average value of the peak area as a vertical coordinate, and calculating a regression equation.
Further limiting, the detection conditions of the liquid chromatography-tandem mass spectrometry in the step 5 are as follows:
liquid chromatography conditions: a chromatographic column: waters cortecs HILIC (2.1X 100mm,1.6 μm), flow rate 0.4mL/min, column temperature 35 ℃, sample size 3 μ L, mobile phase A0.4% formic acid-acetonitrile, B50 mmol/L ammonium formate solution, gradient elution procedure: 0-3min, 60-80% A, 3-3.5min, 50-60% A, 3.5-4.5min, 50% A, 4.5-5min, 50-80% A, 5-7min, 80% A; mass spectrum conditions: the ionization being ESI + The scanning mode is MRM, the capillary voltage is 3.0kV, the ion source temperature is 150 ℃, the desolventizing temperature is 450 ℃, and the desolventizing airflowThe amount is 1000L/h, and the collision gas flow is 0.20 mL/min.
Mass spectrum conditions: the ionization being ESI + The scanning mode is MRM, the capillary voltage is 3.0kV, the ion source temperature is 150 ℃, the desolventizing temperature is 450 ℃, the desolventizing gas flow is 1000L/h, and the collision gas flow is 0.20 mL/min.
Compared with the prior art, the invention has the following remarkable effects:
1) the invention defines a rhizoma polygonati pretreatment means when detecting the content of carboxymethyl lysine and carboxyethyl lysine in the rhizoma polygonati product, and mainly aims to elute redundant protein, fat and impurities in the rhizoma polygonati, avoid excessive impurity peaks in the detection process and reduce the influence on the detection result.
2) The derivatization reaction aims at completing isotope internal standard, which is rare in the technical means of detecting the existing glycosylation end product, linear regression is carried out by utilizing the response abundance ratio of the object to be detected and the internal standard substance, and the quantitative determination of the object to be detected is realized by accurately measuring the isotope abundance.
Drawings
FIG. 1 is a graph of standard curve for carboxymethyllysine in example 1;
FIG. 2 is a graph showing a standard curve of carboxyethyl lysine in example 1;
FIG. 3 is a liquid chromatogram of a carboxymethyllysine standard (concentration of 2. mu.g/mL) in example 1;
FIG. 4 is a liquid chromatogram of a carboxyethyllysine standard (concentration of 2. mu.g/mL) in example 1.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not limit the invention.
The experimental procedures used in the following examples are conventional unless otherwise specified. The materials, reagents, methods and apparatus used, unless otherwise specified, are conventional in the art and are commercially available to those skilled in the art.
The terms "comprises," "comprising," "includes," "including," "has," "having," "contains," or any other variation thereof, as used in the following embodiments, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.
When an amount, concentration, or other value or parameter is expressed as a range, preferred range, or range defined by a list of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. For example, when a range of "1 to 5" is disclosed, the described range should be interpreted to include the ranges "1 to 4", "1 to 3", "1 to 2 and 4 to 5", "1 to 3 and 5", and the like. When a range of values is described herein, unless otherwise stated, the range is intended to include the endpoints thereof and all integers and fractions within the range. In the present description and claims, range limitations may be combined and/or interchanged, including all sub-ranges contained therein if not otherwise stated.
The indefinite articles "a" and "an" preceding an element or component of the invention are not intended to limit the number requirement (i.e., the number of occurrences) of the element or component. Thus, "a" or "an" should be read to include one or at least one, and the singular form of an element or component also includes the plural unless the number clearly indicates the singular.
The technical features of the embodiments of the present invention may be combined with each other as long as they do not conflict with each other.
The first embodiment is as follows: the method for synchronously detecting the middle-advanced glycosylation end products of rhizoma polygonati in the embodiment comprises the following steps:
step 1: pulverizing rhizoma Polygonati, sieving, mixing rhizoma Polygonati powder with n-hexane, eluting for 3-5 times, adding sodium tetraborate solution for reduction reaction, eluting with Sevag solution, adding hydrochloric acid into precipitate, incubating at 100-120 deg.C for 20-24 hr, and filtering to obtain hydrolysate;
step 2: adding a mixed isotope internal standard substance of CML-d4 and CEL-d4 into the hydrolysate, uniformly mixing, then adjusting the pH to 8.0, diluting with pure water by 5 times, adding an acetonitrile solution and a sodium tetraborate solution which is equal to the internal standard substance, uniformly mixing, adding the acetonitrile solution again in the mixing process, standing for 10min after uniformly mixing, and then adding formic acid to terminate the reaction to obtain a derivative solution;
and step 3: activating the derivative solution by methanol and balancing the derivative solution with a formic acid solution, and then performing solid-phase extraction by an SPE (solid phase extraction) column to obtain a solution to be detected;
and 4, step 4: drawing a standard curve;
and 5: detecting the advanced glycosylation end products in the solution to be detected by using UPLC-MS/MS, and calculating the actual content according to a standard curve.
The second embodiment is as follows: the first difference between the present embodiment and the specific embodiment is: in the step 1, the sealwort is obtained by removing impurities from fresh sealwort, cleaning, slicing, and then repeating steaming and drying for 9 times in a water-proof way, wherein each steaming time lasts for 2 hours, and the drying time lasts for 3 hours at 100 ℃. Other steps and parameters are the same as those in the first embodiment.
The third concrete implementation mode: the first difference between the present embodiment and the specific embodiment is: in the step 1, the volume ratio of the mass of the fine yellow powder to the volume of n-hexane is 3 g: (20-30) mL, wherein the volume ratio of the mass of the rhizoma polygonati powder to the sodium tetraborate solution is 1 g: (10-20) mL, wherein the concentration of a sodium tetraborate solution is 0.1-0.3mol/L, the pH is 9.2, a Sevag solution is a mixed solution of chloroform and n-butanol in a volume ratio of 4:1, and the volume ratio of the mass of a precipitate to the volume of hydrochloric acid is 1 g: (15-25) mL, and the concentration of hydrochloric acid is 5-7 mol/L. Other steps and parameters are the same as those in the first embodiment.
The fourth concrete implementation mode: the first difference between the present embodiment and the specific embodiment is: in the step 2, the volume ratio of the hydrolysate to the internal standard substance is 1: (0.15-0.25), and the concentration of CEL-d4 in the internal standard substance is 0.7-0.9 mu g/mL. Other steps and parameters are the same as those in the first embodiment.
The fifth concrete implementation mode: the first difference between the present embodiment and the specific embodiment is: the volume ratio of the acetonitrile solution and the hydrolysate added for the first time in the step 2 is (0.4-0.6): 1, the mass concentration of the sodium tetraborate solution is 4-6%, the pH value is 9.0, and the volume ratio of the acetonitrile solution to the hydrolysate added again is (0.1-0.3): 1. other steps and parameters are the same as those in the first embodiment.
The sixth specific implementation mode: the first difference between the present embodiment and the specific embodiment is: the volume ratio of the formic acid to the hydrolysate in the step 2 is (0.004-0.006): 1. other steps and parameters are the same as those in the first embodiment.
The seventh embodiment: the first difference between the present embodiment and the specific embodiment is: in the step 3, the volume ratio of the methanol to the derivative liquid is 1: (2-4), wherein the volume ratio of the formic acid solution to the derivative solution is 1: (2-4), wherein the volume concentration of the formic acid solution is 0.4-0.6%. Other steps and parameters are the same as those in the first embodiment.
The specific implementation mode eight: the first difference between the present embodiment and the specific embodiment is: the solid phase extraction process through the SPE column in the step 3 comprises the following steps: passing through an SPE column at the flow rate of 0.8-1.2mL/min, then leaching with 20% acetonitrile solution, leaching with dichloromethane after vacuum drying for 5min, then eluting with ammonia water/methanol solution and collecting eluent, blowing elution liquid nitrogen, concentrating and drying, finally re-dissolving with acetonitrile solution, and mixing by vortex, wherein the volume ratio of ammonia water to methanol in the ammonia water/methanol solution is 5: 95. other steps and parameters are the same as those in the first embodiment.
The specific implementation method nine: the first difference between the present embodiment and the specific embodiment is: the specific process of drawing the standard curve in the step 4 is as follows: (1) 10.0mg of each of carboxymethyl lysine and carboxyethyl lysine standard substance is prepared into mixed standard substance stock solution with the mass concentration of 100 mug/mL by using ultrapure water, and the mixed standard substance stock solution is stored at the temperature of-20 ℃; (2) diluting with 80% acetonitrile solution in gradient to obtain carboxymethyl lysine and carboxyethyl lysine standard solutions with mass concentrations of 2, 4, 6, 8 and 10 μ g/mL respectively, and performing sample injection according to liquid chromatography conditions, wherein each concentration is subjected to sample injection in parallel for three times; (3) and (4) drawing a standard curve by taking the concentration of the standard substance as a horizontal coordinate and the average value of the peak area as a vertical coordinate, and calculating a regression equation. Other steps and parameters are the same as those in the first embodiment.
The detailed implementation mode is ten: the first difference between the present embodiment and the specific embodiment is: the detection conditions of the liquid chromatogram-tandem mass spectrum in the step 5 are as follows: liquid chromatography conditions: a chromatographic column: waters cortecs HILIC chromatography column (2.1X 100mm,1.6 μm), flow rate 0.4mL/min, column temperature 35 ℃, sample size 3 μ L, mobile phase A0.4% formic acid-acetonitrile, B50 mmol/L ammonium formate solution, gradient elution procedure: 0-3min, 60-80% A, 3-3.5min, 50-60% A, 3.5-4.5min, 50% A, 4.5-5min, 50-80% A, 5-7min, 80% A; mass spectrum conditions: the ionization being ESI + The scanning mode is MRM, the capillary voltage is 3.0kV, the ion source temperature is 150 ℃, the desolventizing temperature is 450 ℃, the desolventizing gas flow is 1000L/h, and the collision gas flow is 0.20 mL/min. Mass spectrum conditions: the ionization being ESI + The scanning mode is MRM, the capillary voltage is 3.0kV, the ion source temperature is 150 ℃, the desolventizing temperature is 450 ℃, the desolventizing gas flow is 1000L/h, and the collision gas flow is 0.20 mL/min. Other steps and parameters are the same as those in the first embodiment.
Example 1, the method for synchronously detecting middle-and-late-stage glycosylation end products of polygonatum sibiricum of this example is performed according to the following steps:
the specific process of step 1 is as follows:
s1: removing impurities from fresh rhizoma Polygonati, cleaning, slicing, steaming over water for 2 hr, taking out, air drying, drying in oven at 100 deg.C for 3 hr, and repeating steaming over water and drying for 9 times to obtain rhizoma Polygonati product;
s2: pulverizing rhizoma Polygonati with a grinding machine, sieving with 60 mesh sieve, mixing 10g rhizoma Polygonati powder with 100mL n-hexane, and eluting for 3 times;
s3: taking 3g of the polygonatum sibiricum powder treated in the step S2, adding 45mL of sodium tetraborate solution with the concentration of 0.2mol/L for reduction reaction, and reducing for 12 h;
s4: eluting the rhizoma polygonati powder reduced by S3 by adopting Sevag solution (chloroform: n-butanol v: v ═ 4:1), discarding supernatant, collecting precipitate, repeatedly eluting for 3 times, then adding 15mL of hydrochloric acid with the concentration of 6mol/L into the precipitate, incubating for 24h at 110 ℃, and filtering to obtain hydrolysate;
step 2: adding 800 mu L of CML-d4 and CEL-d4 mixed isotope internal standard substance into 4mL of hydrolysate, uniformly mixing, wherein the concentration of CEL-d4 in the internal standard substance is 0.8 mu g/mL, then adjusting the pH value to 8.0 by using 50% potassium hydroxide solution, diluting by 5 times with pure water, adding 2mL of acetonitrile solution and 800 mu L of sodium tetraborate solution with the mass concentration of 5%, uniformly mixing, then standing for 10min, and then adding 20 mu L of formic acid to terminate the derivatization reaction to obtain a derivatization solution;
and step 3:
s1: 1mL of the derivative solution is activated by 3mL of methanol and is balanced by 3mL of formic acid solution with volume concentration of 0.5 percent;
s2: performing solid-phase extraction through an SPE column, passing the SPE column at the flow rate of 1mL/min, then leaching with 20% acetonitrile solution, leaching with dichloromethane after vacuum drying for 5min, then performing vacuum drying for 5min, then eluting with ammonia water/methanol (ammonia water: methanol v: v ═ 5: 95) solution, collecting eluent, blowing elution liquid nitrogen, concentrating and drying, finally re-dissolving with 50% acetonitrile solution according to the volume ratio of 1:2, and mixing uniformly by vortex to obtain a liquid to be detected;
and 4, step 4: the specific process of drawing the standard curve is as follows:
(1) 10.0mg of each of carboxymethyl lysine and carboxyethyl lysine standard substance is prepared into mixed standard substance stock solution with the mass concentration of 100 mug/mL by using ultrapure water, and the mixed standard substance stock solution is stored at the temperature of-20 ℃;
(2) performing gradient dilution by using an 80% acetonitrile solution to respectively obtain carboxymethyl lysine and carboxyethyl lysine standard substance solutions with mass concentrations of 2, 4, 6, 8 and 10 mu g/mL, detecting according to the liquid chromatography condition in the step 5, and performing parallel sample injection for three times at each concentration; liquid chromatography conditions: a chromatographic column: a Waters cortecs HILIC column (2.1X 100mm,1.6 μm) with a flow rate of 0.4mL/min, a column temperature of 35 deg.C, a sample volume of 3 μ L, a mobile phase A of 0.4% formic acid-acetonitrile, a mobile phase B of 50mmol/L ammonium formate solution, and a gradient elution procedure as shown in Table 1.
TABLE 1 gradient elution procedure
(3) Drawing a standard curve (shown in figure 1-2) by taking the concentration of the standard substance as an abscissa (X) and the average value of peak areas as an ordinate (Y), calculating a regression equation, and obtaining the regression equation of the standard curve of the carboxymethyl lysine, wherein the regression equation is that the concentration of the standard substance is 36.7327X-5.17226, R is 36.7327X-5.17226 2 0.997, the regression equation for carboxyethyllysine is Y-65.2657X-5.40813, R 2 At 0.995, the liquid chromatogram of the 2. mu.g/mL carboxymethyllysine standard is shown in FIG. 3, and the liquid chromatogram of the 2. mu.g/mL carboxyethyllysine standard is shown in FIG. 4.
And 5: detecting the advanced glycosylation end products in the liquid to be detected by using UPLC-MS/MS, and calculating the actual content according to a standard curve;
liquid chromatography conditions: a chromatographic column: a Waters cortecs HILIC column (2.1X 100mm,1.6 μm) with a flow rate of 0.4mL/min, a column temperature of 35 deg.C, a sample volume of 3 μ L, a mobile phase A of 0.4% formic acid-acetonitrile, a mobile phase B of 50mmol/L ammonium formate solution, and a gradient elution procedure as shown in Table 1.
Mass spectrum conditions: the ionization being ESI + The scanning mode is MRM, the capillary voltage is 3.0kV, the ion source temperature is 150 ℃, the desolvation temperature is 450 ℃, the desolvation airflow is 1000L/h, the collision airflow is 0.20mL/min, and the mass spectrum detection parameter setting table and the performance index of the object to be detected are shown in tables 2-3.
TABLE 2 Mass Spectrometry parameter setting Table
TABLE 3 Performance index Table
According to the detection, chromatographic peak areas of the carboxymethyl lysine and the carboxyethyl lysine which are respectively obtained are 12% and 0.2768%, a regression equation is substituted, and the content of the carboxymethyl lysine in the sealwort is 458.59 mu g/g and the content of the carboxyethyl lysine is 12.66 mu g/g through calculation.
The above embodiments are merely preferred embodiments of the present invention, and the present invention is not limited to the above embodiments, and modifications and changes thereof may be made by those skilled in the art within the scope of the claims of the present invention.
Claims (10)
1. A method for synchronously detecting advanced glycosylation end products in rhizoma polygonati is characterized by comprising the following steps:
step 1: pulverizing rhizoma Polygonati, sieving, mixing rhizoma Polygonati powder with n-hexane, eluting for 3-5 times, adding sodium tetraborate solution for reduction reaction, eluting with Sevag solution, adding hydrochloric acid into precipitate, incubating at 100-120 deg.C for 20-24 hr, and filtering to obtain hydrolysate;
step 2: adding a mixed isotope internal standard substance of CML-d4 and CEL-d4 into the hydrolysate, uniformly mixing, then adjusting the pH to 8.0, diluting with pure water by 5 times, adding an acetonitrile solution and a sodium tetraborate solution which is equal to the internal standard substance, uniformly mixing, adding the acetonitrile solution again in the mixing process, standing for 10min after uniformly mixing, and then adding formic acid to terminate the reaction to obtain a derivative solution;
and step 3: activating the derivative solution by methanol and balancing the derivative solution with a formic acid solution, and then performing solid-phase extraction by an SPE (solid phase extraction) column to obtain a solution to be detected;
and 4, step 4: drawing a standard curve;
and 5: detecting the advanced glycosylation end products in the solution to be detected by using UPLC-MS/MS, and calculating the actual content according to a standard curve.
2. The method according to claim 1, wherein in step 1, the rhizoma Polygonati is obtained by removing impurities from fresh rhizoma Polygonati, cleaning, slicing, and repeating steaming over water and drying for 9 times, wherein the drying is performed at 100 ℃ for 3 hours every 2 hours of steaming.
3. The method according to claim 1, wherein the volume ratio of the mass of the yellow refined powder to the volume of the normal hexane in the step 1 is 3 g: (20-30) mL, wherein the volume ratio of the mass of the rhizoma polygonati powder to the sodium tetraborate solution is 1 g: (10-20) mL, the concentration of the sodium tetraborate solution is 0.1-0.3mol/L, the Sevag solution is a mixed solution of chloroform and n-butanol according to the volume ratio of 4:1, and the volume ratio of the mass of the precipitate to the volume of hydrochloric acid is 1 g: (15-25) mL, and the concentration of hydrochloric acid is 5-7 mol/L.
4. The method according to claim 1, wherein the volume ratio of the hydrolysate to the internal standard substance in the step 2 is 1: (0.15-0.25), the concentration of CEL-d4 in the internal standard was 0.7-0.9. mu.g/mL.
5. The method according to claim 1, wherein the volume ratio of the acetonitrile solution to the hydrolysate added for the first time in the step 2 is (0.4-0.6): 1, the mass concentration of the sodium tetraborate solution is 4-6%, and the volume ratio of the acetonitrile solution to the hydrolysate added again is (0.1-0.3): 1.
6. the process according to claim 1, wherein the volume ratio of formic acid to hydrolysate in step 2 is (0.004-0.006): 1.
7. the method according to claim 1, wherein the volume ratio of the methanol to the derivative liquid in the step 3 is 1: (2-4), wherein the volume ratio of the formic acid solution to the derivative solution is 1: (2-4), wherein the volume concentration of the formic acid solution is 0.4-0.6%.
8. The method as claimed in claim 1, wherein the solid phase extraction process by SPE column in step 3 is: passing through an SPE column at the flow rate of 0.8-1.2mL/min, then leaching with 20% acetonitrile solution, leaching with dichloromethane after vacuum drying, vacuum drying again, then eluting with ammonia water/methanol solution and collecting eluent, blowing elution liquid nitrogen, concentrating and drying, finally re-dissolving with acetonitrile solution, vortex mixing uniformly, wherein the volume ratio of ammonia water to methanol in the ammonia water/methanol solution is 5: 95.
9. the method according to claim 1, wherein the specific process of drawing the standard curve in the step 4 is as follows: (1) 10.0mg of each of carboxymethyl lysine and carboxyethyl lysine standard substance is prepared into mixed standard substance stock solution with the mass concentration of 100 mug/mL by using ultrapure water, and the mixed standard substance stock solution is stored at the temperature of-20 ℃; (2) diluting with 80% acetonitrile solution in gradient to obtain carboxymethyl lysine and carboxyethyl lysine standard solutions with mass concentrations of 2, 4, 6, 8 and 10 μ g/mL respectively, and performing sample injection according to liquid chromatography conditions, wherein each concentration is subjected to sample injection in parallel for three times; (3) and (4) drawing a standard curve by taking the concentration of the standard substance as a horizontal coordinate and the average value of the peak area as a vertical coordinate, and calculating a regression equation.
10. The method according to claim 1, wherein the detection conditions of the liquid chromatography-tandem mass spectrometry in the step 5 are as follows: liquid chromatography conditions: a chromatographic column: waters cortecs HILIC, flow rate 0.4mL/min, column temperature 35 ℃, sample volume 3 uL, mobile phase A of 0.4% formic acid-acetonitrile, B of 50mmol/L ammonium formate solution, gradient elution program: 0-3min, 60-80% A, 3-3.5min, 50-60% A, 3.5-4.5min, 50% A, 4.5-5min, 50-80% A, 5-7min, 80% A; mass spectrum conditions: the ionization being ESI + The scanning mode is MRM, the capillary voltage is 3.0kV, the ion source temperature is 150 ℃, the desolventizing temperature is 450 ℃, the desolventizing gas flow is 1000L/h, and the collision gas flow is 0.20 mL/min.
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