Sialic acid quantitative analysis method

Abstract

The invention belongs to the technical field of biomolecule detection, and particularly relates to a quantitative analysis method for sialic acid. According to the method, a basic type sialic acid standard substance is used as an internal standard substance, dimethylamine and deuterated dimethylamine are used as labeling reagents, amido bonds are generated between the labeling reagents and carboxyl groups in the internal standard substance and a biological sample, sialic acid is labeled at the same time, the samples with light and heavy labels are mixed, and the mixture passes through a reduction end of 3-nitrophenylhydrazine derivative sialic acid. After detection is completed through a liquid chromatography-mass spectrometry method, absolute quantitative analysis can be performed on sialic acid in a biological sample on the basis of combined use of an internal standard method and an external standard method. As O-acetylated sialic acid and non-O-acetylated sialic acid of the same type can generate similar secondary mass spectrums and same quantitative ions, absolute quantitative analysis can be approximately performed on O-acetylated sialic acid by using the method and the device.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomolecule detection, and in particular relates to a sialic acid quantitative analysis method. Background Art

[0002] Sialic acid is a family of monosaccharides with a nine-carbon core structure. It often serves as the terminal component of glycoproteins and glycolipids in organisms. It includes three basic types: N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc), and deaminoneuraminic acid (KDN). These basic sialic acids can be further modified with acetyl, lactyl, methyl, and phosphate groups to form even more diverse structures. To date, over 70 types of sialic acid have been discovered in nature.

[0003] Sialic acid with modified groups, particularly O-acetylated sialic acid, has been found to be closely associated with numerous biological functions, such as pathogen binding and infection of hosts, embryonic development and immune processes, cancer development and progression, drug targets, and antibody activity. Quantitative analysis of sialic acid with modified groups would facilitate monitoring its dynamic changes within organisms and provide a deeper understanding of its biological functions.

[0004] Chemical derivatization of sialic acid followed by detection and analysis by liquid chromatography-mass spectrometry (LC-MS) is a commonly used technique for studying sialic acid in biological samples. Depending on the target, the methods for deriving sialic acid can be divided into two categories. The basic operation of the first method is to first dissociate sialic acid from the glycoconjugate and then derive the free sialic acid, which is mainly used for the quantitative analysis of various sialic acids and isomers. The basic operation of the second method is to directly derivatize sialic acid on the glycoconjugate, which is often used to analyze the type and connection mode of sialic acid on the glycoconjugate. Although the second method is the main development direction, this method is currently unable to resolve the isomers of sialic acid and is difficult to use for absolute quantitative analysis, so the first method is still widely used.

[0005] There have been many reports on the first type of methods. However, in practical applications, these methods have shown some limitations, mainly involving the following aspects: ① Some methods face the problem of no internal standard, which affects the accuracy of the analysis results; ② The use of 13③ The internal standard can be introduced by using an isotope-labeled derivatization reagent, but such derivatization reagents usually need to be customized and are expensive; ④ The lack of O-acetylated sialic acid standards on the market seriously affects the accuracy of existing methods in the quantitative analysis of such sialic acids. In short, the existing first type of method has two major limitations: the analysis cost based on the internal standard method is high; and it is not very suitable for the absolute quantitative analysis of O-acetylated sialic acid.

[0006] Regarding the second type of method, there have been reports using dimethylamine as a derivatization reagent (literature: A linkage-specific sialic acid labeling strategy reveals different site-specific glycosylation patterns in SARS-CoV-2 spike protein produced in CHO and HEK cell substrates, Frontiers in Chemistry, 2021), and there have also been reports using 3-nitrophenylhydrazine as a derivatization reagent (literature: Analysis of O-glycans by oxidative release combined with 3-nitrophenylhydrazine derivatization, ACS Omega, 2025). The present invention combines these two derivatization reagents for dual labeling of free sialic acid to suppress the defects of the first type of method. Summary of the Invention

[0007] This invention establishes a novel sialic acid quantitative analysis method that, to a certain extent, mitigates the shortcomings of existing analytical methods for free sialic acid. Firstly, it uses an inexpensive, readily available labeling reagent instead of expensive internal standards or derivatization reagents. Secondly, a secondary labeling reagent modifies the fragmentation pattern of sialic acid molecules during mass spectrometry analysis, resulting in similar secondary mass spectra and identical quantitative ions for sialic acids of the same type. This allows working curves established based on Neu5Ac, Neu5Gc, and KDN standards to be largely applicable to their respective O-acetylated forms.

[0008] The technical concept of this method is based on stable isotope labeling of sialic acid and LC-MS technology. Dimethylamine (d0-DMA) is used to lightly label the carboxyl groups of sialic acid in one sample; deuterated DMA (d6-DMA) is used to heavily label the carboxyl groups of sialic acid in another sample. After mixing the light and heavy labeled products, the reducing ends of the sialic acids are derivatized with 3-nitrophenylhydrazine (3-NPH). After desalting, the product ions are detected by LC-MS. The content of one product ion (i.e., the quantification ion) is derived from the common portion of various sialic acid molecules. This significantly reduces the correlation between the quantification ion and the sialic acid modification group, expanding the applicability of the working curve.

[0009] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0010] A method for quantitative analysis of sialic acid, comprising:

[0011] Method 1: When performing absolute quantitative analysis of sialic acid in a sample

[0012] (1) According to the type of sialic acid to be detected, the corresponding sialic acid standard is selected as the internal standard. When the Neu5Ac standard is used as the internal standard, a heavy labeling reagent (CD3)2NH or (CD3)2NH·HCl is added to a certain concentration of the Neu5Ac standard solution for heavy labeling; a light labeling reagent (CH3)2NH or (CH3)2NH·HCl is added to a series of concentrations of the Neu5Ac standard solution for light labeling; or the aforementioned light and heavy labeling reagents are swapped for use; the light and heavy labeled products are mixed, and 3-nitrophenylhydrazine (3-NPH) or its hydrochloride is added to derivatize the reducing end of the sialic acid for double labeling, and the mixed sample is detected by liquid chromatography-mass spectrometry (LC-MS). The extracted ion chromatographic peak area corresponding to the two specific product ions (i.e., C1-C3 of the standard) generated by the dissociation of the standard doubly labeled with d0-DMA / d6-DMA and 3-NPH is used as the quantitative basis, and a working curve of the extracted ion chromatographic peak area-concentration corrected by the internal standard based on the Neu5Ac standard is established;

[0013] (2) After acid hydrolyzing the biological sample to be tested to obtain a sample containing free sialic acid, the acid-hydrolyzed sample and the internal standard are subjected to light and heavy labeling, wherein the light and heavy labeling reagents of the internal standard are the same as those used to establish the working curve, the light and heavy labeled products are mixed, and 3-nitrophenylhydrazine or its hydrochloride is added for double labeling. The mixed sample is detected by LC-MS to obtain the extracted ion chromatogram peaks of Neu5Ac-like sialic acids in the acid-hydrolyzed sample and Neu5Ac in the internal standard. The peak area of ​​the extracted ion chromatogram peak generated by Neu5Ac-like sialic acids in the acid-hydrolyzed sample and corrected by the internal standard is substituted into the above working curve to obtain the content of Neu5Ac-like sialic acids in the sample to be tested.

[0014] The Neu5Ac-like sialic acid includes Neu5Ac, Neu4,5Ac2, Neu5,7Ac2, Neu5,8Ac2, and Neu5,9Ac2.

[0015] When the internal standard is Neu5Gc standard, a working curve based on the Neu5Gc standard is established to quantify Neu5Gc-like sialic acids (including Neu5Gc, Neu4Ac5Gc, Neu5Gc7Ac, Neu5Gc8Ac, and Neu5Gc9Ac).

[0016] When the internal standard is a KDN standard, a working curve based on the KDN standard is established to quantify KDN-like sialic acids (including KDN and KDNAc).

[0017] Method 2: When comparing the relative content differences of the same sialic acid (including Neu5Ac, Neu4,5Ac2, Neu5,7Ac2, Neu5,8Ac2, Neu5,9Ac2, Neu5Gc, Neu4Ac5Gc, Neu5Gc7Ac, Neu5Gc8Ac, Neu5Gc9Ac, KDN, KDNAc) between different samples

[0018] After acid hydrolysis of the biological sample to be tested to obtain a sample containing free sialic acid, any one of them is taken as an internal standard or an appropriate amount is taken from each acid-hydrolyzed sample and mixed, and the mixed sample is used as the internal standard or a control group (which also needs acid hydrolysis) is used as the internal standard, and a heavy labeling reagent (CD3)2NH or (CD3)2NH·HCl is added for heavy labeling; a light labeling reagent (CH3)2NH or (CH3)2NH·HCl is added to each acid-hydrolyzed sample for light labeling; or the aforementioned light and heavy labeling reagents are added to the sample. The labeled acid-hydrolyzed samples are then mixed with an equal amount of labeled internal standard, and 3-nitrophenylhydrazine or its hydrochloride is added to derivatize the reducing end of sialic acid for double labeling. The mixed samples are detected by liquid chromatography-mass spectrometry to obtain extracted ion chromatographic peaks of the same sialic acid in each acid-hydrolyzed sample and the internal standard. The peak areas of the extracted ion chromatographic peaks of the same sialic acid in each acid-hydrolyzed sample, corrected for the internal standard, are compared with the internal standard, thereby comparing the relative amount differences of the same sialic acid among different samples.

[0019] Method 1 differs from Method 2 primarily in the use of internal standards. Sialic acid (Neu5Ac, Neu5Gc, KDN) standards are used as internal standards. This combined use of internal and external standards allows for absolute quantitative analysis of sialic acid in biological samples. Because modified (O-acetylated) sialic acids produce similar MS / MS spectra and identical quantification ions as unmodified (basic) sialic acids of the same type, absolute quantitative analysis of modified (O-acetylated) sialic acids can be similarly performed using the test sample (either one or a mixture) as the internal standard.

[0020] Furthermore, the biological sample is a tissue, body fluid, protein sample, etc.; acid hydrolysis of the biological sample to be tested includes: acid hydrolyzing the biological sample to be tested in a 1.5-2.0 mol / L acetic acid solution, preferably, placing it in a 70-80°C water bath for 2.5-3.0 hours; and recovering the supernatant after high-speed centrifugation, which contains the sialic acid to be quantified.

[0021] Furthermore, the heavy labeling comprises: mixing the sample with a 1.0-2.0 mol / L deuterated dimethylamine hydrochloride solution containing 10 v / v% N-methylmorpholine and a 0.2-0.4 mol / L 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) solution, wherein the solvent is dimethyl sulfoxide; and placing the sample in the dark at 30-40° C. for 1.0-1.5 hours;

[0022] Furthermore, the light labeling comprises: mixing the sample with a 1.0-2.0 mol / L dimethylamine hydrochloride solution containing 10 v / v% N-methylmorpholine and a 0.2-0.4 mol / L 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) solution, wherein the solvent is dimethyl sulfoxide; and placing the sample in the dark at 30-40° C. for 1.0-1.5 hours;

[0023] Furthermore, the double labeling comprises: adding 0.10-0.14 mol / L 3-nitrophenylhydrazine hydrochloride solution to the sample, wherein the solvent is 70 v / v % acetonitrile; placing in the dark at 20-30° C. for 1.0-1.5 hours;

[0024] Furthermore, when the total mass of sialic acid in the sample (including the internal standard) does not exceed 2.0 μg, the amount of d0-DMA or d6-DMA added is 0.02-0.04 mmol, the amount of HATU added is 0.004-0.008 mmol, and the amount of 3-NPH added is 0.010-0.014 mmol; when the total mass of sialic acid in the sample (including the internal standard) exceeds 2.0 μg, the amount of the added reagents is increased proportionally.

[0025] Furthermore, the concentrations of the standard solution in the series include 0.05, 0.1, 0.5, 1.0, 5.0, 10.0, and 50.0 μmol / L.

[0026] Furthermore, the sample needs to be desalted before liquid chromatography-mass spectrometry detection.

[0027] Furthermore, the liquid chromatography conditions include: a C18 chromatographic column; mobile phase A: 0.1 v / v% formic acid solution; mobile phase B: acetonitrile; and a gradient elution program as follows: 0 to 35 minutes, mobile phase B from 10% to 90%.

[0028] Furthermore, detection is performed by secondary mass spectrometry of LC-MS; preferably, data acquisition and detection of product ions are performed in parallel reaction monitoring (PRM) or multiple reaction monitoring (MRM) mode of liquid chromatography-mass spectrometry.

[0029] In this method, even after single-labeling the carboxyl group (C1) of sialic acid with d0-DMA / d6-DMA, its hydrophilicity remains strong and is washed away by water after direct desalting, resulting in essentially no ion chromatography peak signal. Following single-labeling of the carboxyl group of sialic acid with d0-DMA / d6-DMA, the carbonyl group (C2) of sialic acid is subsequently labeled with 3-NPH. This not only adjusts the fragmentation pattern of sialic acid molecules during mass spectrometry analysis, resulting in similar secondary mass spectra and identical quantitative ions for similar sialic acids, but also enhances the hydrophobicity of sialic acid molecules, allowing the removal of most water-soluble impurities during subsequent desalting and reducing the need for additional sialic acid enrichment.

[0030] Compared with the existing technology, the advantages and beneficial effects of the present invention are as follows: ① This method does not require the use of isotope-labeled sialic acid standards or the customization of isotope-labeled derivatization reagents, and only inexpensive and readily available derivatization reagents are used, which has a significant cost advantage; ② In response to the problem that O-acetylated sialic acid standards are unavailable or difficult to obtain, this method can approximately absolutely quantitatively analyze such sialic acids in the absence of O-acetylated sialic acid standards; ③ When using this method for relative quantitative analysis of O-acetylated sialic acid, differences in ionization efficiency and matrix effects between samples are taken into account, thereby improving the accuracy of the analysis results. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Flowchart of the sialic acid quantitative analysis method established in the present invention.

[0032] Figure 2 (AC) shows the mass spectra of double-labeled Neu5Ac, derived from a standard with a concentration of 5.0 μmol / L. Figure 2A The primary mass spectrum of Neu5Ac double labeled with d0-DMA / d6-DMA and 3-NPH ([MH] - ); Figure 2B is the secondary mass spectrum of Neu5Ac doubly labeled with d0-DMA and 3-NPH; Figure 2C This is the MS / MS spectrum of Neu5Ac doubly labeled with d6-DMA and 3-NPH.

[0033] Figure 3 This is a working curve drawn based on the data of Neu5Ac standard.

[0034] Figure 4 (AC) is a chromatogram of double-labeled sialic acid in mouse serum, from sample A3 in Example 2. Figure 4A The extracted ion chromatograms of Neu5Ac in 0.5 μL mouse serum and Neu5Ac in the internal standard (5.0 μmol / L) are shown; Figure 4BThe extracted ion chromatograms of Neu5,9Ac2 in 0.5 μL mouse serum and Neu5Ac (5.0 μmol / L) in the internal standard are shown; Figure 4C This is a composite image of the extracted ion chromatograms of Neu5Ac, Neu5Gc, Neu5,9Ac2, and Neu5Gc9Ac in 0.5 μL mouse serum.

[0035] Figure 5 (AD) is the mass spectra of Neu5Ac and Neu5,9Ac2 in double-labeled mouse serum, which are from sample A3 in Example 2. Figure 5A-5B The primary mass spectrum of Neu5Ac double-labeled with d0-DMA and 3-NPH in mouse serum (m / z=470.1917, [MH] - ) and secondary mass spectra; Figure 5C-5D The primary mass spectrum of Neu5,9Ac2 double-labeled with d0-DMA and 3-NPH in mouse serum (m / z = 512.2041, [MH] - ) and secondary mass spectra.

[0036] Figure 6 (AD) shows the extracted ion chromatogram data of Neu5,9Ac2 in the test mouse serum with different volumes and Neu5,9Ac2 in the internal standard. Figures 6A-6C Extracted ion chromatograms of Neu5,9Ac2 in the double-labeled serum S1 sample (0.5 μL serum), S2 sample (1.0 μL serum), S3 sample (2.0 μL serum), and internal standard (0.2 μL serum); Figure 6D is the difference between the relative quantitative analysis result and the theoretical value. DETAILED DESCRIPTION

[0037] The applicant will further describe the technical solution of the present invention below with reference to specific embodiments and drawings.

[0038] In the examples, the light labeling reagent is dimethylamine hydrochloride, (CH3)2NH·HCl, denoted as d0-DMA; the heavy labeling reagent is deuterated dimethylamine hydrochloride, (CD3)2NH·HCl, denoted as d6-DMA;

[0039] 2-(7-Azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, denoted as HATU.

[0040] Example 1: Drawing a working curve

[0041] (1) Re-labeling of Neu5Ac standard as internal standard: Prepare a Neu5Ac standard solution with a concentration of 100.0 μmol / L, take 50 μL and dry it in vacuum; dissolve deuterated dimethylamine hydrochloride in dimethyl sulfoxide (containing 10 v / v% N-methylmorpholine) to a concentration of 2.0 mol / L; dissolve HATU in dimethyl sulfoxide to a concentration of 0.4 mol / L; mix the dried Neu5Ac with 20 μL of d6-DMA solution and 20 μL of HATU solution, and then place it at 40°C in the dark for 1 h; dilute the reaction solution with 960 μL of water to obtain an internal standard solution (5.0 μmol / L) for later use.

[0042] (2) Light labeling of Neu5Ac standard for working curve: prepare a series of Neu5Ac standard solutions (purity 98%, Aladdin Reagent (Shanghai) Co., Ltd.) with a concentration of 0.05, 0.1, 0.5, 1.0, 5.0, 10.0, 50.0 μmol / L, take 50 μL of each solution and vacuum dry it; dissolve dimethylamine hydrochloride in dimethyl sulfoxide (containing 10 v / v% N-methylmorpholine) to a concentration of 2.0 mol / L; dissolve HATU in dimethyl sulfoxide to a concentration of 0.4 mol / L; mix the dried Neu5Ac with 20 μL of d0-DMA solution and 20 μL of HATU solution, and then place it at 40°C in the dark for 1 hour to serve as a series of standard solutions.

[0043] (3) Double labeling: Dissolve 3-nitrophenylhydrazine hydrochloride in 70 v / v% acetonitrile to a concentration of 19 mg / mL; mix each series of standard solutions in (2) with 50 μL of the internal standard solution in (1), 10 μL of water, and 100 μL of 3-NPH solution, and then place in the dark at room temperature (25°C) for 1 hour; add 1 mL of water to dilute the sample solution to obtain a series of double-labeled standard solutions.

[0044] (4) Desalting: Wash a C18 solid phase extraction cartridge (specification: containing 100 mg of filler) with 2 mL of water and 2 mL of 80 v / v% acetonitrile (containing 0.1 v / v% trifluoroacetic acid) solution in sequence; equilibrate the C18 cartridge with 2 mL of water; load the double-labeled series of standard solutions in (3) into the cartridge respectively; wash the cartridge with 1 mL of water; elute sialic acid from the C18 cartridge with 1 mL of 50 v / v% ethanol solution; recover the eluate and vacuum dry it, then store it in a -20°C refrigerator for later use.

[0045] (5) Liquid Chromatography-Mass Spectrometry Analysis: The sample treated in (4) was dissolved in 50 μL of 20 v / v% acetonitrile solution and analyzed by LC-MS. The liquid chromatography model used in this example was M5-MicroLC (SCIEX, USA); the mass spectrometry model was TripleTOF5600+ (SCIEX, USA).

[0046] Liquid chromatography conditions: The sample solution was introduced into the liquid phase via an injection system and then onto the analytical column (C18, 3.0 μm, 0.3 mm × 150 mm, Phenomenex, USA). Elution was completed using a gradient elution program (mobile phase A: 0.1 v / v% formic acid solution; mobile phase B: acetonitrile) at a flow rate of 7 μL / min and a column temperature of 45°C. The mobile phase gradient elution program was as follows: 0 min, 10% B; 2 min, 10% B; 25 min, 45% B; 35 min, 90% B; 39 min, 90% B; 40 min, 10% B.

[0047] Mass spectrometry conditions: negative ion mode; ion source temperature 250°C; spray voltage -4500 V; ion source gas GS1 is 17 psi; ion source gas GS2 is 18 psi; curtain gas CUR is 35 psi; the primary mass spectrometry MS scanning range is m / z = 300 to 900 Da; the secondary mass spectrometry MS / MS adopts PRM mode, the scanning range is m / z = 100 to 800 Da, and the collision energy CE is -25±3 V.

[0048] In PRM mode, the mass spectrometer's first-stage mass analyzer selects a specific precursor ion (for example, m / z = 470.2 for Neu5Ac labeled with d0-DMA; m / z = 512.2 for O-acetylated Neu5Ac labeled with d0-DMA). The precursor ion is dissociated in the second-stage mass analyzer to produce product ions, which are then separated and detected by the third-stage mass analyzer. In this mode, the product ions correspond to specific precursor ions, and the content of a particular product ion (i.e., quantification ion) can be used to analyze the corresponding precursor ion (i.e., sialic acid). Even if different substances produce the same product ion, the source of the product ion can be distinguished based on the precursor ion selected by the first-stage mass analyzer.

[0049] Figure 1 Flowchart of the sialic acid quantitative analysis method established in the present invention.

[0050] Figure 2 (AC) shows the mass spectra of double-labeled Neu5Ac, derived from a standard with a concentration of 5.0 μmol / L. Figure 2A is the primary mass spectrum of Neu5Ac doubly labeled with d0-DMA / d6-DMA and 3-NPH; Figure 2Bis the secondary mass spectrum of Neu5Ac doubly labeled with d0-DMA and 3-NPH; Figure 2C Figure 2 (AC) shows the complete mass-to-charge ratio of Neu5Ac doubly labeled with d6-DMA and 3-NPH is 470.1904 ([MH] - ), the complete mass-to-charge ratio of Neu5Ac double-labeled with d6-DMA and 3-NPH was 476.2260 ([MH] - ); All standards (Neu5Ac) doubly labeled with d0-DMA and 3-NPH can produce a product ion with m / z = 249.10Da, and all internal standards (Neu5Ac) doubly labeled with d6-DMA and 3-NPH can produce a product ion with m / z = 255.14Da. ​​The extracted ion chromatographic peak areas corresponding to these two product ions can be used as the basis for quantification.

[0051] (6) Draw the working curve:

[0052] A working curve was drawn based on the data generated from a series of concentrations of Neu5Ac standard, such as Figure 3 Wherein, x is the concentration of the sialic acid standard in μmol / L; y is the internal standard-corrected extracted ion chromatogram peak area, i.e., the extracted ion chromatogram peak area of ​​the Neu5Ac standard labeled with d0-DMA divided by the extracted ion chromatogram peak area of ​​the internal standard labeled with d6-DMA, multiplied by the internal standard concentration (in this example, the internal standard concentration is 5.0 μmol / L).

[0053] The working curve is y=0.20+1.85x, and the linear correlation coefficient R 2 =0.9988; the lower and upper limits of quantification were 0.05 and 50.0 μmol / L, respectively, which are slightly better than the relevant research results in recent years (the lower and upper limits of quantification were 0.16 and 33.0 μmol / L, respectively, see the reference: Changes in the serum and tissue levels of free and conjugated sialic acids, Neu5Ac, Neu5Gc, and KDN in mice after the oral administration of edible bird's nests: an LC-MS / MS quantitative analysis, Separations, 2024, 11); the detection limit was 0.02 μmol / L (signal-to-noise ratio ≥5).

[0054] It is known that mouse serum contains various types of sialic acid, including Neu5Ac, Neu5Gc, KDN, Neu5,9Ac2, and Neu5Gc9Ac, with a total concentration generally ranging from 2.0 to 8.0 mmol / L.

[0055] Example 2: Absolute quantitative analysis of Neu5Ac and Neu5,9Ac2 in mouse serum

[0056] (1) Re-labeling of Neu5Ac standard as internal standard: Prepare a Neu5Ac standard solution with a concentration of 100.0 μmol / L, take 50 μL and dry it in vacuum; dissolve deuterated dimethylamine hydrochloride in dimethyl sulfoxide (containing 10 v / v% N-methylmorpholine) to a concentration of 2.0 mol / L; dissolve HATU in dimethyl sulfoxide to a concentration of 0.4 mol / L; mix the dried Neu5Ac with 20 μL of d6-DMA solution and 20 μL of HATU solution, and then place it at 40°C in the dark for 1 h; dilute the reaction solution with 960 μL of water to obtain an internal standard solution (5.0 μmol / L) for later use.

[0057] (2) Preparation of serum samples to be tested:

[0058] Dissociation of sialic acid in S1 serum sample: 10 μL of mouse serum was mixed with 100 μL of acetic acid solution (4 mol / L) and 90 μL of water, and then placed in an 80°C water bath for 3 h. The sample solution was centrifuged in a refrigerated centrifuge at 12,000 g for 30 min. Three 10 μL aliquots of supernatant (each equivalent to 0.5 μL of serum) were recovered and vacuum dried, respectively designated A1, A2, and A3.

[0059] Light labeling of sialic acid in S2 serum sample: Dimethylamine hydrochloride was dissolved in dimethyl sulfoxide (containing 10 v / v% N-methylmorpholine) to a concentration of 2.0 mol / L; HATU was dissolved in dimethyl sulfoxide to a concentration of 0.4 mol / L; dried A1, A2, and A3 samples were mixed with 20 μL of d0-DMA solution and 20 μL of HATU solution, and then placed in the dark at 40°C for 1 h to obtain A1, A2, and A3 sample solutions.

[0060] (3) Double labeling: 3-Nitrophenylhydrazine hydrochloride was dissolved in 70 v / v% acetonitrile to a concentration of 19 mg / mL; the A1, A2, and A3 sample solutions in (2) S2 were mixed with 50 μL of the internal standard solution in (1), 10 μL of water, and 100 μL of 3-NPH solution, and then placed in the dark at room temperature (25°C) for 1 hour; 1 mL of water was added to dilute the sample solution; the sample solution was centrifuged in a refrigerated centrifuge at a speed of 12,000 g for 10 minutes, and then the supernatant was recovered to obtain double-labeled serum A1, A2, and A3 sample solutions to be tested.

[0061] (4) Desalting: Wash a C18 solid phase extraction cartridge (specification: containing 100 mg of filler) with 2 mL of water and 2 mL of 80 v / v% acetonitrile (containing 0.1 v / v% trifluoroacetic acid) solution in sequence; equilibrate the C18 cartridge with 2 mL of water; load the double-labeled serum A1, A2, and A3 sample solutions in (3) into the cartridge, respectively; wash the cartridge with 1 mL of water; elute sialic acid from the C18 cartridge with 1 mL of 50 v / v% ethanol solution; recover the eluate, vacuum dry it, and then store it in a -20°C refrigerator for later use.

[0062] (5) Liquid chromatography-mass spectrometry analysis: The sample treated in (4) was dissolved in 50 μL of 20 v / v% acetonitrile solution and detected by LC-MS. The liquid chromatography and mass spectrometry conditions were exactly the same as those in Example 1.

[0063] Figure 4 (AC) is a chromatogram of sialic acid in the double-labeled mouse serum A3 sample solution. Figure 4A Extracted ion chromatograms of Neu5Ac in 0.5 μL (1 / 20 of a 10 μL serum sample was taken for subsequent processing after acid hydrolysis with acetic acid) mouse serum and Neu5Ac (5.0 μmol / L) in the internal standard;

[0064] Figure 4B The extracted ion chromatograms of Neu5,9Ac2 in 0.5 μL mouse serum and Neu5Ac (5.0 μmol / L) in the internal standard are shown; Figure 4C The combined extracted ion chromatograms of Neu5Ac, Neu5Gc, Neu5,9Ac2, and Neu5Gc9Ac in 0.5 μL of mouse serum are shown. Figures 4 (A-C) demonstrate that the LC-MS analysis simultaneously measures the levels of various sialic acids, including Neu5Ac, Neu5Gc, Neu5,9Ac2, and Neu5Gc9Ac, in mouse serum.

[0065] FIG5 (AD) is the mass spectra of the double-labeled Neu5Ac and Neu5,9Ac2 in the mouse serum A3 sample solution to be tested. Figure 5A-5B The primary mass spectrum of Neu5Ac double-labeled with d0-DMA and 3-NPH in mouse serum (m / z=470.1917, [MH] - ) and secondary mass spectra; Figure 5C-5D The primary mass spectrum of Neu5,9Ac2 double-labeled with d0-DMA and 3-NPH in mouse serum (m / z = 512.2041, [MH] - ) and secondary mass spectra.

[0066] d0-DMA / d6-DMA labels the carboxyl group (C1) of sialic acid with light and heavy labels, and 3-NPH labels the carbonyl group (C2) of sialic acid. Different sialic acids present in serum samples will be labeled together. Moreover, for the same type of sialic acid (such as Neu5Ac and Neu5,9Ac2), similar secondary mass spectra and the same quantitative ions can be generated after dual labeling with DMA and 3-NPH. For example, all Neu5Ac-type sialic acids labeled with d0-DMA can produce product ions with m / z = 249.10Da (corresponding to C1-C3 of sialic acid, see Figure 2B 、 Figure 5D All Neu5Ac-type sialic acids labeled with d6-DMA can generate product ions with m / z = 255.14 Da (corresponding to C1-C3 of sialic acid, see Figure 2C The working curves obtained in Example 1 are therefore quite general for the same type of sialic acid and have little to do with whether the sialic acid contains a modification group (e.g., O-acetylation) (O-acetylation primarily occurs at C4, C7, C8, and C9 of sialic acid). Therefore, the working curves established based on Neu5Gc and KDN standards are also largely applicable to their respective O-acetylated forms.

[0067] The peak area of ​​the extracted ion chromatogram (IIC) peak produced by Neu5Ac in serum samples was corrected by the internal standard (i.e. Figure 4A The peak area of ​​the plasma sample divided by the peak area of ​​the internal standard, and then multiplied by the concentration of the internal standard) was substituted into the working curve to obtain the Neu5Ac content in the test serum sample. Combined with the dilution factor of the serum sample (in this embodiment, after the sample was acid-hydrolyzed with acetic acid, a supernatant equivalent to 0.5 μL of serum was taken for subsequent processing. Then, after a series of treatments, the sample was finally dissolved in 50 μL of 20 v / v% acetonitrile solution. Therefore, it is equivalent to diluting the serum sample 100 times before LC-MS analysis), the Neu5Ac content in the serum sample to be tested can be obtained.

[0068] As shown in Table 1, the concentration of Neu5Ac in mouse serum was calculated to be 0.175±0.007 mmol / L based on the working curve and sample dilution multiple. This test result (LC-MS negative ion mode, double labeling method) is comparable to the results of a previously reported related study (LC-MS negative ion mode, label-free method) (Reference: Changes in the serum and tissue levels of free and conjugated sialic acids, Neu5Ac, Neu5Gc, and KDN in mice after the oral administration of edible bird's nests: an LC-MS / MS quantitative analysis, Separations, 2024, 11).

[0069] Table 1 Neu5Ac content in mouse serum (mmol / L)

[0070]

[0071] Since Neu5Ac and Neu5,9Ac2 can produce similar secondary mass spectra, the working curve obtained based on the Neu5Ac standard can be approximately applicable to the analysis of Neu5,9Ac2. Figure 4B The peak area of ​​the plasma sample divided by the peak area of ​​the internal standard, and then multiplied by the concentration of the internal standard) is substituted into the working curve to obtain the Neu5,9Ac2 content in the test serum sample. Combined with the dilution factor of the serum sample (in this embodiment, the serum sample is equivalent to being diluted 100 times), the Neu5,9Ac2 content in the serum sample to be tested can be obtained.

[0072] As shown in Table 2, the concentration of Neu5,9Ac2 in mouse serum was calculated to be 0.143±0.019 mmol / L based on the working curve and sample dilution factor. This result (LC-MS negative ion mode, double labeling) is comparable to that of a previously reported study (high performance liquid chromatography, fluorescence labeling) (Serum contents of sialic acids in mice bearing different tumors, Chinese Science Bulletin, 1994, 39).

[0073] Table 2 Neu5,9Ac2 content in mouse serum (mmol / L)

[0074]

[0075]

[0076] Example 3: Relative quantitative analysis of Neu5,9Ac2 in mouse serum

[0077] (1) Preparation of samples as internal standards:

[0078] S11 dissociation: 40 μL of mouse serum was mixed with 100 μL of acetic acid solution (4 mol / L) and 60 μL of water, and then placed in an 80°C water bath for 3 h. The sample solution was centrifuged in a refrigerated centrifuge at 12,000 g for 30 min. 20 μL of the supernatant (equivalent to 4.0 μL of serum) was recovered and vacuum-dried to serve as the internal standard.

[0079] S12 relabeling: Dissolve deuterated dimethylamine hydrochloride in dimethyl sulfoxide (containing 10 v / v% N-methylmorpholine) to a concentration of 2.0 mol / L; dissolve HATU in dimethyl sulfoxide to a concentration of 0.4 mol / L; mix the dried internal standard with 20 μL of d6-DMA solution and 20 μL of HATU solution, then incubate at 40°C in the dark for 1 h; dilute the reaction solution with 960 μL of water and set aside as the internal standard solution.

[0080] (2) Preparation of serum samples to be tested:

[0081] Dissociation of sialic acid in S21 serum samples: 5 μL, 10 μL, and 20 μL of mouse serum were mixed with 100 μL of acetic acid solution (4 mol / L), respectively, and an appropriate amount of water was added to bring the total volume to 200 μL. The sample solution was placed in an 80°C water bath for 3 h and then centrifuged in a refrigerated centrifuge at 12,000 g for 30 min. 20 μL of supernatant was recovered from each of the three samples, designated S1, S2, and S3 (equivalent to 0.5 μL, 1.0 μL, and 2.0 μL of serum, respectively), and vacuum dried.

[0082] Light labeling of sialic acid in the S22 serum sample: dimethylamine hydrochloride was dissolved in dimethyl sulfoxide (containing 10 v / v% N-methylmorpholine) to a concentration of 2.0 mol / L; HATU was dissolved in dimethyl sulfoxide to a concentration of 0.4 mol / L; S1, S2, and S3 samples were mixed with 20 μL of d0-DMA solution and 20 μL of HATU solution, and then placed in the dark at 40°C for 1 h to obtain S1, S2, and S3 sample solutions.

[0083] (3) Double labeling: 3-Nitrophenylhydrazine hydrochloride was dissolved in 70 v / v% acetonitrile to a concentration of 19 mg / mL; the S1, S2, and S3 sample solutions in (2) were mixed with 50 μL of the internal standard solution in (1) (equivalent to 0.2 μL of serum), 10 μL of water, and 100 μL of 3-NPH solution, and then placed in the dark at room temperature (25°C) for 1 hour; 1 mL of water was added to dilute the sample solution; the S1, S2, and S3 sample solutions were centrifuged in a refrigerated centrifuge at a speed of 12,000 g for 10 minutes, and then the supernatant was recovered to obtain the double-labeled serum S1, S2, and S3 sample solutions to be tested.

[0084] (4) Desalting: Wash a C18 solid phase extraction cartridge (specification: 100 mg filler) with 2 mL of water and 2 mL of 80% v / v acetonitrile (containing 0.1 v / v% trifluoroacetic acid) solution, respectively; equilibrate the C18 cartridge with 2 mL of water; load the double-labeled serum S1, S2, and S3 sample solutions in (3) into the cartridge, respectively; wash the cartridge with 1 mL of water; elute sialic acid from the C18 cartridge with 1 mL of 50% v / v ethanol solution; recover the eluate, vacuum dry it, and then store it in a -20°C refrigerator for later use.

[0085] (5) Liquid chromatography-mass spectrometry analysis: The sample treated in (4) was dissolved in 50 μL of 20 v / v% acetonitrile solution and detected by LC-MS. The liquid chromatography and mass spectrometry conditions were exactly the same as those in Example 1.

[0086] Mouse serum contains several O-acetylated sialic acids. Taking the most common Neu5,9Ac2 as an example, the extracted ion chromatogram data of Neu5,9Ac2 in double-labeled mouse serum with different volumes and Neu5,9Ac2 in internal standard were obtained, as shown in Figure 6 (AD). Figure 6A The extracted ion chromatograms of Neu5,9Ac2 in the double-labeled serum S1 sample (0.5 μL serum) and the internal standard (0.2 μL serum);

[0087] Figure 6B The extracted ion chromatograms of Neu5,9Ac2 in the double-labeled serum S2 sample (1.0 μL serum) and the internal standard (0.2 μL serum); Figure 6C The extracted ion chromatograms for Neu5,9Ac2 in the dual-labeled serum S3 sample (2.0 μL serum) and the internal standard (0.2 μL serum). The three internal standard data were flattened using their average, and the individual serum sample data were corrected using the multiple of the average value and each internal standard data.

[0088] The extracted ion chromatographic peak areas of S1, S2, and S3 samples corrected by internal standards (i.e. Figure 6A、 Figure 6B 、 Figure 6C The peak area ratio of S1, S2, and S3 samples (multiplied by the above-mentioned multiple) is 1.00:1.89:3.76, which is less than 10% different from the theoretical value of relative quantity (1:2:4). The difference between the relative quantitative analysis results and the theoretical value is shown in Figure 6D Compared with the method using similar sialic acid as the internal standard, this method uses the same sialic acid as the internal standard, which suppresses the adverse effects on analytical accuracy caused by differences in ionization efficiency and matrix effects.

[0089] The above embodiments should not limit the present invention in any way, and any technical solutions obtained by equivalent replacement or equivalent conversion fall within the protection scope of the present invention.

Claims

1. A method for quantitative analysis of sialic acid, characterized in that: When performing absolute quantitative analysis of sialic acid in a sample, the following method is used: (1) According to the type of sialic acid to be detected, the corresponding sialic acid standard is selected as the internal standard. When the Neu5Ac standard is used as the internal standard, a heavy labeling reagent (CD3)2NH or (CD3)2NH·HCl is added to a certain concentration of the Neu5Ac standard solution for heavy labeling; a light labeling reagent (CH3)2NH or (CH3)2NH·HCl is added to a series of concentrations of the Neu5Ac standard solution for light labeling; or the aforementioned light and heavy labeling reagents are swapped for use; the light and heavy labeled products are mixed, and 3-nitrophenylhydrazine or its hydrochloride is added to derivatize the reducing end of the sialic acid for double labeling, and the mixed sample is detected by liquid chromatography-mass spectrometry. The extracted ion chromatographic peak area corresponding to the two specific product ions generated by the dissociation of the standard doubly labeled with d0-DMA / d6-DMA and 3-NPH is used as the quantitative basis to establish a working curve of the extracted ion chromatographic peak area-concentration corrected by the internal standard based on the Neu5Ac standard; (2) After acid hydrolyzing the biological sample to be tested to obtain a sample containing free sialic acid, the acid-hydrolyzed sample and the internal standard are subjected to light and heavy labeling, wherein the light and heavy labeling reagents of the internal standard are the same as those used to establish the working curve, the light and heavy labeled products are mixed, and 3-nitrophenylhydrazine or its hydrochloride is added for double labeling, and the mixed sample is detected by LC-MS to obtain the extracted ion chromatogram peaks of Neu5Ac-like sialic acids in the acid-hydrolyzed sample and Neu5Ac in the internal standard, and the peak area of ​​the extracted ion chromatogram peak generated by Neu5Ac-like sialic acids in the acid-hydrolyzed sample and corrected by the internal standard is substituted into the above working curve to obtain the content of Neu5Ac-like sialic acids in the sample to be tested; the Neu5Ac-like sialic acids include Neu5Ac, Neu4,5Ac2, Neu5,7Ac2, Neu5,8Ac2, and Neu5,9Ac2; When the internal standard is a Neu5Gc standard, a working curve based on the Neu5Gc standard is established to quantify Neu5Gc-like sialic acids; the Neu5Gc-like sialic acids include Neu5Gc, Neu4Ac5Gc, Neu5Gc7Ac, Neu5Gc8Ac, and Neu5Gc9Ac; When the internal standard is a KDN standard, a working curve based on the KDN standard is established to quantify KDN-like sialic acids; the KDN-like sialic acids include KDN and KDNAc.

2. The sialic acid quantitative analysis method according to claim 1, characterized in that When comparing the relative content of the same sialic acid between different samples, the following method is used: After acid hydrolyzing the biological sample to be tested to obtain a sample containing free sialic acid, any one of the samples is taken as an internal standard, or an appropriate amount of each acid-hydrolyzed sample is taken and mixed, and the mixed sample is used as the internal standard, or a control group is used as the internal standard, and a heavy labeling reagent (CD3)2NH or (CD3)2NH·HCl is added for heavy labeling; a light labeling reagent (CH3)2NH or (CH3)2NH·HCl is added to each acid-hydrolyzed sample for light labeling; Alternatively, the light and heavy labeling reagents are swapped; each labeled acid-hydrolyzed sample is then mixed with an equal amount of a labeled internal standard, and 3-nitrophenylhydrazine or its hydrochloride is added to derivatize the reducing end of the sialic acid for double labeling. The mixed sample is detected by LC-MS coupling to obtain the extracted ion chromatographic peak of the same sialic acid in each acid-hydrolyzed sample and the internal standard. The peak area of ​​the extracted ion chromatographic peak of the same sialic acid in each acid-hydrolyzed sample, corrected for the internal standard, is compared with the internal standard, thereby comparing the relative amount difference of the same sialic acid in different samples.

3. The sialic acid quantitative analysis method according to claim 1 or 2, characterized in that: The biological sample is a tissue, body fluid, or protein sample. The acid hydrolysis of the biological sample includes: acid hydrolyzing the biological sample in a 1.5-2.0 mol / L acetic acid solution.

4. The sialic acid quantitative analysis method according to claim 1 or 2, characterized in that: The heavy / light labeling comprises: mixing the sample with a 1.0-2.0 mol / L deuterated dimethylamine hydrochloride solution or a dimethylamine hydrochloride solution containing 10 v / v% N-methylmorpholine, and a 0.2-0.4 mol / L 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate solution, wherein the solvent is dimethyl sulfoxide; and placing the sample in the dark at 30-40° C. for 1.0-1.5 hours.

5. The sialic acid quantitative analysis method according to claim 1 or 2, characterized in that: The double labeling comprises: adding 0.10-0.14 mol / L 3-nitrophenylhydrazine hydrochloride solution to the sample, wherein the solvent is 70 v / v % acetonitrile; and placing the sample in the dark at 20-30° C. for 1.0-1.5 hours.

6. The sialic acid quantitative analysis method according to claim 1 or 2, characterized in that: When the total mass of sialic acid in the sample does not exceed 2.0 μg, the amount of d0-DMA or d6-DMA added is 0.02-0.04 mmol, the amount of HATU added is 0.004-0.008 mmol, and the amount of 3-NPH added is 0.010-0.014 mmol. When the total mass of sialic acid in the sample exceeds 2.0 μg, the amount of the added reagents is increased proportionally.

7. The sialic acid quantitative analysis method according to claim 1 or 2, characterized in that: The product ions were detected by liquid chromatography-mass spectrometry in parallel reaction monitoring mode or multiple reaction monitoring mode.

8. The sialic acid quantitative analysis method according to claim 1 or 2, characterized in that: Liquid chromatography conditions include: C18 chromatographic column; mobile phase A: 0.1 v / v% formic acid solution; mobile phase B: acetonitrile; gradient elution program is as follows: 0-35 min, mobile phase B from 10% to 90%.

9. The sialic acid quantitative analysis method according to claim 1 or 2, characterized in that: Before liquid chromatography-mass spectrometry detection, the sample needs to be desalted.

10. The method for quantitative analysis of sialic acid according to claim 1, wherein The concentrations of the standard solution series include 0.05, 0.1, 0.5, 1.0, 5.0, 10.0, and 50.0 μmol / L.

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