NL2029775B1

NL2029775B1 - Method for detecting multiple advanced glycation end products in dairy products

Info

Publication number: NL2029775B1
Application number: NL2029775A
Authority: NL
Inventors: Fu Linglin; Zhu Wenxiu; Wang Shunyu; Wang Yanbo; Zhang Qiaozhi; Wang Chong; Li Linfang; Chen Jian
Original assignee: Univ Zhejiang Gongshang; Zhe Jiang Li Zi Yuan Food Ltd
Priority date: 2021-09-09
Filing date: 2021-11-16
Publication date: 2023-01-03
Also published as: CN113702546A

Abstract

The present invention discloses a method for detecting multiple advanced glycation end products in dairy products. Fig. l

Description

METHOD FOR DETECTING MULTIPLE ADVANCED GLYCATION END

PRODUCTS IN DAIRY PRODUCTS

TECHNICAL FIELD

The present invention relates to the technology field of food detection, specifically to a method for detecting multiple advanced glycation end products in dairy products.

BACKGROUND

Advanced glycation end products (AGEs) are a group of stable end-products generated by a series of reactions between free amino groups of proteins, amino acids, lipids or nucleic acids and the carbonyl group of reducing sugars through condensation, rearrangement, cleavage and oxidative modification. At present, more than 40 kinds of AGEs have been characterized and identified, wherein about 15 AGEs are found in foods, comprising free-type and peptide/protein-bound AGEs. Some AGEs such as carboxymethyl lysine (CML) are derived from lysine, while some AGEs are produced by modification of arginine, or by cross-linking these two amino acids at the same time.

Milk and dairy products with high nutritional value and unique flavor have become one of the main sources of nutrition in people's daily life. Milk products are rich in components such as lactose, whey protein, and caseins, which are prone to Maillard reaction to form various AGEs during food processing, storage and transportation.

Evidence keeps showing that the intake of food-borne AGEs has adverse effects on the human health and is closely related to many chronic diseases such as diabetes and chronic kidney disease. Due to the complexity and intractability of food substrates, as well as the wide variety of AGEs and complex structures, there has been a lack of universal, accurate and efficient methods for the detection of AGEs in food. At present, the detection methods of AGEs in food mainly comprise enzyme-linked immunosorbent assay, gas chromatography-mass spectrometry, high performance liquid chromatography, liquid chromatography tandem mass spectrometry and so on. Among them, the enzyme-linked immunoassay has limited application due to the unsatisfied accuracy and antibody specificity, while the gas chromatography-mass spectrometry method needs to derivatize the target, thereof the method is relatively cumbersome, and the recovery rate of the target is not high. Although liquid chromatography has a relatively high instrument popularity and simple operation, its detection sensitivity is not satisfied.

When detecting AGEs, using a liquid chromatography coupled with tandem mass spectrometer would greatly improve the sensitivity, selectivity and analysis speed of the method. However, the liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the related field can only detect one to three AGEs products, and the efficiency is low, where it is difficult to objectively reflect the content and distribution of different AGEs in the food. In addition, the mass spectrometry technique is susceptible to errors caused by the matrix effect of samples, therefore it is necessary to fully consider the matrix effect when detecting samples with different matrices to improve the accuracy of the results.

BRIEF SUMMARY OF THE INVENTION

Aiming at the above problems, the present invention provides a method for detecting multiple advanced glycation end products in dairy products. The method can detect 11

AGEs in dairy products at one time, comprising carboxymethyl lysine (CML), carboxyethyl lysine (CEL), pyrraline, pentosidine, methylacetaldehyde-hydroxyimidazolone isomer (MG-H1/MG-H2/MG-H3), argpyrimidine, glyoxal-hydroimidazolone (G-HI), glyoxal-lysine dimer (GOLD) and methylglyoxal-lysine dimer (MOLD), in order to achieve a comprehensive and accurate analysis of the content of AGEs in food.

In order to achieve the above objective, the embodiment of the present invention, on the one hand, provides a method for detecting multiple advanced glycation end products in dairy products, including the following steps: (1) adding the isotope internal standards into the dairy products after centrifugal degreasing, then adding borate buffer solution and sodium borohydride solution into the dairy products to obtain a mixed solution; (2) carrying out acid hydrolysis on the mixture, concentrating the mixture by vacuum centrifugation to near dryness, redissolving it with water, filtering to remove impurities thereof to obtain treated dairy products; (3) adding AGEs standards into the treated dairy products to make various AGEs standard solution, multiplying integral chromatographic area ratio of the isotope internal standard corresponding to each standard by the concentration of the corresponding isotope internal standard as a function of concentration of each standard to draw a standard curve;

(4) carrying out UPLC-MS/MS detection on the treated dairy products, obtaining the area of AGEs peak in the dairy products, and the concentration of each AGEs is calculated by substituting it into the standard curve.

According to the method of detecting multiple advanced glycation end products in dairy products in an embodiment of the present invention, the method calculates the correction factor by the isotope internal standard and the peak height/area of the corresponding standard, then uses the correction factor to correct the error caused by the pre-treatment of the sample. Pre-treatment steps thereof are simple, easy to operate, less impurity, and the standard curve is based on milk matrix, therefore greatly reduces the interference of matrix effect on the detection. The method can detect 11 AGEs of CML, CEL, pyrraline, pentosidine, MG-H1/3, MG-H2, argpyrimidine, G-H1, GOLD and MOLD at one time, and 11 AGEs have ideal separation effect, therefore achieve comprehensive and accurate analysis of the content of each AGE in the sample.

In addition, the method of detecting multiple advanced glycation end products in dairy products provided by the previously described embodiment of the present invention also includes the following additional technical characteristics:

Optionally, in step (1), the isotope internal standards include CML-d4, CEL-d4,

GOLD-15N2, MG-H1-d3 and G-H1-13C2.

Optionally, in step (1), 10 mL - 15 mL of 0.2 mol/L pH 9.2 borate buffer and 5 mL - 8 mL of 1 mol/L sodium borohydride solution prepared by 0.1 mol/L sodium hydroxide solution are added into each milliliter of dairy products, incubating for 2 to 4 h at room temperature to obtain a mixed solution.

Optionally, in step (2), the acid hydrolysis conditions are: adding 20 mL - 25 mL of 6 mol/L HCI into each milliliter of dairy products, and acid hydrolysis at 110 °C for 24 to 25 h.

Optionally, in step (1), the concentration of the isotope internal standard is 100 ng/mL ~200 ng/mL

Optionally, in step (1), the centrifugal degreasing conditions are: rotating speed is 4500-6000 g, temperature is 0-5 °C, time is 10-15 min.

Optionally, in step (2), the vacuum centrifugal concentration conditions are: vacuum degree is 0-1 mbar, rotation speed is 200-300 g, temperature is not higher than -20 °C.

Optionally, in step (3), the standard curve of CML is Y=0.01143X+0.000163, the standard curve of CEL is Y=0.02832X-0.01327, the standard curve of pyrraline is

Y=0.13448X-0.02140, and the standard curve of pentosidine is Y=0.00876 X+0.01663, the standard curve of MG-H1/3 is Y=0.00878X-0.00238, the standard curve of MG-H2 is

Y=0.00519X-0.00110, the standard curve of argpyrimidine is Y=0.05723X+0.05402, the standard curve of G-H1 is Y=0.00913X-0.00137, the standard curve of GOLD is

Y=0.01109X-0.00384, and the standard curve of MOLD is Y=0.04551X-0.0044.

Optionally, in step (4), the UPLC conditions are: column: Waters ACQUITY HSS T3 100 mmx2.1 mm 18 um; mobile phase A: aqueous solution containing 0.1% v/v formic acid; mobile phase B: acetonitrile; column temperature: 30 °C; flow rate: 0.25 mL/min; sample introduction volume: 5 pL; The sample is eluted by gradient elution, and the gradient is set to 98% A: 2% B, 95% A: 5% B, 90% A: 10% B, 92%A: 8%B, 98%A: 2%B. Therefore, by optimizing the liquid phase conditions such as the chromatographic column, mobile phase, and elution gradient, the detection time is shortened, the detection of 11 AGEs can be completed within 10 minutes, which greatly improves the detection efficiency.

Optionally, in step (4), the triple quadrupole mass spectrometry conditions are: ion source: electro-ion spray; scanning mode: positive ion mode; detection mode: MRM; desolventizing gas temperature: 600 °C; ion source temperature: 120 °C; desolventizing gas flow rate: 1000 L/h; gas flow rate of sampling cone: 200 L/h; collision gas flow rate: 0.20 mL/min; capillary voltage: 600 V; fragmentation voltage: 40 V.

The additional characteristics and advantages of the present invention will be partly described below, and some will become apparent from the following description or be known through implementation of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. I illustrates the schematic diagram of the chemical formula of 11 AGEs quantifiable according to an embodiment of the present invention;

FIG. 2 illustrates the ion chromatogram of CML quantified in ultra-high-temperature sterilized milk according to an embodiment of the present invention;

FIG. 3 illustrates the ion chromatogram of CEL quantified in ultra-high-temperature sterilized milk according to an embodiment of the present invent;

FIG. 4 illustrates the 10n chromatogram of MG-H1/3 quantified in ultra-high-temperature sterilized milk according to an embodiment of the present invention;

FIG. 5 illustrates the ion chromatogram of MG-H2 quantified in ultra-high-temperature sterilized milk according to an embodiment of the present invent;

FIG. 6 illustrates the ion chromatogram of GH-1 quantified in ultra-high-temperature 5 sterilized milk according to an embodiment of the present invent;

FIG. 7 illustrates the ion chromatogram of GOLD quantified in ultra-high-temperature sterilized milk according to an embodiment of the present invent;

FIG. 8 illustrates standard curve of CML in milk matrix according to an embodiment of the present invent;

FIG. 9 illustrates standard curve of CEL in milk matrix according to an embodiment of the present invent;

FIG. 10 illustrates standard curve of MG-H1/3 in milk matrix according to an embodiment of the present invent;

FIG. 11 illustrates standard curve of MG-H2 in milk matrix according to an embodiment of the present invent;

FIG. 12 illustrates standard curve of GH-1 in milk matrix according to an embodiment of the present invent; and

FIG. 13 illustrates standard curve of GOLD in milk matrix according to an embodiment of the present invent.

DETAILED DESCRIPTION OF THE INVENTION

The following describes the technical solutions of the present invention through specific embodiments. It should be understood that, one or more steps mentioned in the present invention do not exclude other steps prior to or behind the sequenced steps or other steps inserted among those clearly mentioned steps. It should also be understood that, those embodiments are used to merely describe the present invention, not to limit the scope of the present invention. Moreover, unless otherwise specified, the serial numbers of steps are only convenient tools for distinguishing the steps instead of limiting the sequence of the steps or limiting the implementable scope of the present invention. Change or adjustment of the relative relationship of the serial numbers should also be deemed to fall within the implementable scope of the present invention if there is no substantial change to the technical contents.

To facilitate a better understanding of the previously described technical solution, exemplary embodiments of the present invention will be described in further detail.

Although exemplary embodiments of the present invention are illustrated, it should be understood that the present invention can be implemented in various forms and should not be limited by embodiments described herein. On the contrary, those embodiments are provided to help more thoroughly understand the present invention and entirely pass on the scope of the present invention to those skilled in the art.

The instruments and reagents used in the present invention were:

Agilent 1290 ultra performance liquid chromatograph, Agilent Technologies, USA;

Agilent 6470 Triple quadrupole mass spectrometer, Agilent Technologies, USA;

AUW220D electronic balance, Shimadzu Corporation, Japan;

Allegra X-30R multifunctional tabletop high-speed centrifuge, Beckman Coulter,

Germany;

ED56 natural convection oven, Binder GmbH, Germany;

Milli-Q Advantage A 10 ultra-pure water meter, Bedford, USA;

ZLS-2 vacuum centrifugal concentrator, Hunan Hexi Instrument Equipment Co., Ltd;

CML, CEL standard products, Cayman Chemical, USA;

Pyrraline standard, pentosidine standard, MG-H1/3 standard, MG-H2 standard, argpyrimidine standard, G-H1 standard, GOLD standard, MOLD standard, CML-d4 standard, CEL-d4 standard, GOLD-15N2 standard, MG-H1-d3 standard, G-H1-13C2 standard, Iris Biotech GmbH, Germany;

Sodium tetraborate, sodium borohydride, sodium hydroxide, hydrochloric acid, formic acid, acetonitrile, Shanghai Aladdin Biochemical Technology Co., Ltd;

Storage bottle, Agilent Technologies, USA; 0.22 um filter membrane, 2.5 mL syringe, threaded tube, sample bottle, centrifuge tube,

Shenggong Bioengineering (Shanghai) Co., Ltd;

The following describes the present invention by reference to specific embodiments. It should be noted that those embodiments are merely descriptive and do not limit the present invention in any way.

Embodiment

Extraction of AGEs

Taking 1 mL of ultra-high-temperature sterilized milk into a 1.5 mL centrifuge tube, degreasing at 4500g for 15 minutes, taking 100 uL sample from the bottom of the centrifuge tube into a threaded tube, adding 10 pL of 10 pg/mL CML-d4, 10 pL of 10 ng/mL CEL-d4, 10 uL of 10 pg/mL GOLD-15N2, 10 pL of 10 pg/mL MG-H1-d3, and 10 uL of 10 pg/mL G-H1-13C2 isotope internal standards to correct for errors, adding 1 mL of 0.2 mol/L pH 9.2 borate buffer and 0.5 mL of sodium borohydride solution prepared by 1 mol/L sodium hydroxide solution, incubating for 2 h at room temperature to obtain a mixed solution;

Adding 1.5 mL of 6 mol/L HCI to the mixture, acid hydrolyzing at 110 °C for 24 h to dissociate the bound AGEs, therefore turn them into free AGEs, thereafter, concentrating the mixture by vacuum centrifugation to near dryness at a vacuum of 0.1 mbar, rotating speed 200 g, -20 °C, adding 5 mL of ultra-pure water to redissolve the residue, then obtaining the redissolution;

Passing the redissolution through a 0.22 um filter membrane, obtaining the filtrate;

The use of ion source as electrospray mass spectrometry to analyze target analytes in food samples may cause ionization inhibition or ionization enhancement due to the chromatographic elution of matrix components, thereby affecting the quantitative results.

Therefore, the matrix effect level of the established method was evaluated by measuring the slope change (%) of the matrix standard curve and the solvent standard curve, the matrix effect level was calculated by the following formula:

Matrix effect (%) = (Slope of the matrix standard curve - slope of the solvent standard curve) / slope of the matrix standard curve

A negative level (<10%) of the matrix effect indicated that the matrix may suppress the mass spectrometry signal, while a positive level (>10%) indicated that the matrix may enhance the mass spectrometry signal. When the matrix effect level remained in the range of -10-10%, it could be considered that there is no matrix effect during the analysis of the target analyte.

Making the standard curve:

Using 5 pg/mL CML, 5 pg/mL CEL, 5 pg/mL pyrraline, 5 pg/mL pentosidine, 5 pg/mL

MG-H1, 5 pg/mL MG-H2, 5 pg/mL MG-H3, 5 pg /mL argpyrimidine, 5 pg/mL G-HI, 5 ug/mL GOLD, 5 pg/mL MOLD to prepare a mixed standard solution with a final concentration of 1 pg/mL for each standard, and then using 10 pg/mL CML- d4, 10 pg/mL

CEL-d4, 10 pg/mL GOLD-15N2, 10 pg/mL MG-H1-d3, 10 pg/mL G-H1-13C2 to prepare a mixed internal standard solution with a final concentration of 1 ug/ mL for each internal standard, and finally prepared 1 mL mixed standard solution containing 0.1 pg/mL mixed internal standard solution of 5 ng/mL, 25 ng/mL, 50 ng/mL, 100 ng/mL, 250 ng/mL and 500 ng /mL, the matrix was the filtrate obtained in the above steps, and the standard solution preparation conditions were shown in Table 1;

Table 1 Standard solution preparation conditions ngmL ng/mL ng/mL ng/mL ng/mL ng/mL

Mixed standard solution ~~ 5uL ~~ 25ul 30pL 100uL 2504 S00 ul

Mixed internal standard lution 100 ul 100pL 100pL 100uL 100uL 100 pL

Matrix [06 uL 100 uL 100 uL 100 uL 100 pL 100 pL

Ultra-pure water 795uL 775uL 750uL 700uL 550uL 300 pL

The preparation condition of the standard solution of the solvent standard curve was to replace the same amount of the matrix added in Table 1 with ultra-pure water;

Multiplying the integral chromatographic peak area ratio of the isotope internal standard corresponding to each standard by the concentration (Y) of each corresponding isotope internal standard as a function of the standard concentration (X) to draw a standard curve to obtain the equation. As shown in FIG. 8-FIG. 13. The standard curve of CML was

Y=0.01143X+0.000163, the standard curve of CEL was Y=0.02832X-0.01327, the standard curve of pyrraline was Y=0.13448X-0.02140, the standard curve of pentosidine was Y=0.00876X+0.01663, the standard curve of MG-H1/3 was Y=0.00878X-0.00238, the standard curve of MG-H2 was Y=0.00519X-0.00110, the standard curve of argpyrimidine was Y=0.05723X+0.05402, the standard curve of G-Hl was Y=0.00913

X-0.00137, the standard curve of GOLD was Y=0.01109X-0.00384, and the standard curve of MOLD was Y=0.04551X-0.0044.

The matrix effect level result was calculated according to the matrix effect formula. The matrix effect of CML was -3.2%, the matrix effect of CEL was -2.6%, the matrix effect of pyrraline was -1.7%, the matrix effect of pentosidine was -5.4%, the matrix effect of

MG-H1/3 was -4.8%, the matrix effect of MG-H2 was -2.9%, the matrix effect of argpyrimidine was -3.5%, the matrix effect of G-H1 was -2.8%, the matrix effect of

GOLD was -1.7%, and the matrix effect of MOLD was -4.1%.

UPLC-MS/MS detection

The conditions of UPLC: column: Waters ACQUITY HSS T3 (100 mmx2.1 mm 18 um); mobile phase A: an aqueous solution containing 0.1% (v/v) formic acid; mobile phase B: acetonitrile; column temperature: 30°C; flow rate: 0.25 mL/min; sample instruction volume: 5 pL; the gradient elution is shown in Table 2;

Table 2 Gradient elution program

Time (min) 0 2 6 9 10

A 98% 95% 90% 92% 98%

B 2% 5% 10% 8% 2%

Using the above gradient elution, CML, CEL, pyrraline, pentosidine, MG-H1/3, MG-H2, argpyrimidine, G-HI, GOLD, MOLD had extremely short detection time and excellent separation effect, the retention times were 4.32 min, 4.92 min, 5.67 min, 8.64 min, 6.22 min, 7.29 min, 5.61 min, 8.12 min, 8.33 min, which was beneficial to improve the accuracy and precision of analyzing and quantifying; and under this elution gradient , the analyte retention value was appropriate and the peak shape was good.

The conditions of triple quadrupole mass spectrometry: ion source: electro-ion spray; scanning mode: positive ion mode; detection mode: MRM; desolventizing gas temperature: 600 °C; ion source temperature: 120 °C; desolventizing gas flow rate: 1000 L/h; gas flow rate of sampling cone: 200 L/h; collision gas flow rate: 0.20 mL/min; capillary voltage: 600 V;

fragmentation voltage: 40 V,

MRM mass spectrometer parameters were shown in Table 3:

Table 3 MRM mass spectrum parameters

Analyte Prerequisite ion Quantitative ion (m/z) Collision energy (eV) (m/z)

CML 2050 116 84 27

CEL 219.0 130* 17 84 26

Pyrraline 255.1 130% 18 174.9 17

Pentosidine 380.2 251.0% 32 317.1 35

MG-H1/3 229.1 114.1% 20 210.8 12

MG-H2 229.1 116.0% 19 211.0 16

Argpyrimidine 255.1 139.7* 22 116.0 21

G-H1 215.2 116.1% 19 152.2 20

GOLD 328.2 130.1* 31 283.1 30

MOLD 342.3 297.1% 30 252.2 40

CML-d4 209.0 134.0% 16

CEL-d4 223.0 134.0% 18

GOLD-15N2 330.0 130.8* 28

MG-H1-d3 231.0 116.0% 21

G-H1-13C2 217.1 153.9% 18

TT

The quantitative ion pair in the table was marked with "*".

Carrying out UPLC-MS/MS detection on the treated samples, the detection mode was

MRM, obtaining the area of AGEs peak in the sample, calculating the concentration of

AGEs in the sample.

The results were shown in FIG. 2 to FIG. 7, the ion chromatographic peaks of CML, CEL,

MG-H1/3, MG-H2, G-H1, GOLD in ultra-high-temperature sterilized milk, according to the standard curve, the concentration of CML was 0.555 pg/mL, the concentration of CEL was 0.240 pg/mL, the concentration of MG-H1/3 was 19.917 ng/mL, the concentration of

MG-H2 was154.239 pg/mL, the concentration of G-HI was 8.708 pg/mL, the concentration of GOLD was 1.346 pg/mL. The concentrations of the other AGEs were all lower than the quantitative limitation of the method.

In summary, according to the embodiment of the present invention, the content of each

AGEs in the food can be comprehensively and accurately analyzed.

In the description of the present invention, reference terms including one embodiment, some embodiments, example, specific example or some examples refer to that specific characteristics, structures, materials or features which are described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In the present description, the schematic expression of the above terms should not be construed as always aiming at identical embodiments or examples. Besides, the described specific characteristics, structures, materials or features may be combined in an appropriate way in any one of or more embodiments. In addition, those skilled in the art can blend and combine different embodiments or examples described in the present invention.

Although the embodiments of the present invention have been illustrated and described above, it can be understood that the previously described embodiments are exemplary, and cannot be construed as limiting the present invention. Those ordinarily skilled in the art can change, modify, replace and transform the previously described embodiments within the scope of the present invention.

Claims

CONCLUSIONS I. A method for detecting multiple advanced glycation end products in dairy products, involving the following steps:

1. adding internal isotope standards to the dairy products after centrifugal degreasing and then adding borate buffer solution and sodium borohydride solution to the dairy products to obtain a mixed solution;

ii. carrying out acid hydrolysis of the mixture, concentration of the mixture by vacuum centrifugation until almost dry, redissolving it in water, filtering it to remove the impurities, thus obtaining treated dairy products;

iii. adding advanced glycation end products (AGEs) to the treated dairy products with the aim of obtaining different AGE standards, multiplying the integral ratio of the chromatographic surface of the isotope internal standard corresponding to each standard by concentration of the corresponding isotope internal standard as a function of the concentration of each standard for the purpose of drawing a standard curve; and iv. performing UPLC-MS/MS detection on the treated dairy products and obtaining the area of the AGE peak in the dairy products, calculating the concentration of each AGE by substituting it into the standard curve.

The method of claim 1, wherein the method comprises: in step i, the internal standards of the isotope are CML-d4, CEL-d4, GOLD-15N2, MG-H1-d3 and G-H1-13C2.

The method according to claim 1, wherein the method comprises: in the step i, prepare 10 mL-15 mL of 0.2 mol/L pH 9.2 borate buffer and 5 mL-8 mL of 1 mol/L sodium borohydride solution by adding 0.1 mol/l sodium hydroxide solution to each milliliter of dairy products, with incubation for 2 to 4 hours at room temperature to obtain a mixed solution.

The method of claim 1, wherein the method comprises: in step ii, the acid hydrolysis conditions are: add 20 ml-25 ml of 6 mol/L HCl to each milliliter of dairy products, and perform acid hydrolysis at 110°C for 24 to 25 o'clock.

The method of claim 1, wherein the method comprises: in step i, the concentration of the internal standards of the isotope are 100 ng/ml - 200 ng/ml.

The method according to claim 1, wherein the method comprises: in step i, the centrifugal degreasing conditions are the following: rotation speed 4500 - 6000 g, the temperature 0 - 5 °C and the time 1015 min.

The method according to claim 1, wherein the method comprises: in step ii, the centrifugal vacuum conditions are: vacuum ratio 0 - 1 mbar, rotation speed 200 - 300 g and the temperature is not higher than -20°C.

The method of claim 1, wherein the method comprises: in step iii, the standard curve of carboxymethyl lysine (CML) is:

Y=0.01143X+0.000163, the standard curve of carboxyethyl lysine (CEL) is the following

Y=0.02832X-0.01327, the standard curve of pyrraline is the following

Y=0.13448X-0.02140, and the standard curve of pentosidine is Y=0.00876

X+0.01667, the standard curve of methyl acetaldehyde-hydroxyimidazolone (MG-H)1/3 the following is Y=0. 00878X-0.00238, the standard curve of MG-H2 is the following is Y=0.00519X-0.00110, the standard curve of argpyrimidine is the following Y=0.05723X+0.05402, the standard curve of glyoxal-hydroimidazolone ( G-H)l next is Y=0. 00913

The method of claim 1, wherein the method comprises: in step iv, the UPLC conditions are: column: Waters ACQUITY HSS T3 100mm ~ 2.1mm Jun 18; mobile phase A: aqueous solution containing 0.1% v/v formic acid; mobile phase B: acetonitrile, column temperature: 30 °C; flow rate: 0.25 ml/min/; sample introduction volume: 5 pL; the sample is diluted by gradient dilution and the gradient is set to 98% A: 2% B, 95% A: 5% B, 90% A: 10% B, 92% A: 8% B, 98%A:2% b.

The method of claim 1, wherein the method comprises: in step iv, the conditions regarding the triple quadrupole mass spectrometry are:

ion source: electro-ion spray; scan mode: positive ion mode; detection mode: MRM; temperature of the desolvent gas: 600 °C, source temperature ions: 120 °C, flow rate desolvent gas: 1000 l/hour; flow rate gas steel cone: 200 l/hour; impact gas flow rate 0.20 ml/min; capillary voltage: 600 V; fragmentation voltage: 40

V.