Oligosaccharide markers and their application in identification of bird's nest

Abstract

The present invention relates to quality control markers, and methods of using such markers to identify bird's nest. In particular, the present invention relates to oligosaccharide markers, and their use in rapid, specific, efficient, and low-cost identification of bird's nest raw materials and related products, and methods of identification.

Description

TECHNICAL FIELD

[0001] The present application relates to quality control markers, and methods of identifying bird's nest using such markers. In particular, the present application relates to oligosaccharide markers, and their use in and methods of identifying bird's nest raw materials and related products thereof, which are fast, specific, efficient, low cost, high sensitivity, high specificity, and highly reproducible. BACKGROUND

[0002] Bird's nest (edible bird's nest, EBN) is a nest built by the saliva and down of various species of swiftlets (Aerodramus or Collocalia) of the swiftlet family. The main component is glycoprotein, which contains 62-63% of protein and 25-27% of carbohydrate. Modern studies have shown that it has a wide range of pharmacological effects, such as immune promotion, antiviral, mitogenic, and cardiotonic. Therefore, as a functional food with high reputation in Asia, bird's nest is deeply loved by consumers and has high market value.

[0003] Currently, a series of methods have been developed for the identification of bird's nest, but all of the methods involved have the disadvantages of poor specificity, long time consumption, and complicated operation. For example, identification by determining the content of sialic acid in bird's nest, and identification by determining monosaccharides have been reported in the literature. However, the specificity of sialic acid, mannose, N-acetyl-galactosamine, and other monosaccharides is low, and they are widely present in animal products such as chicken eggs and milk, resulting in low accuracy of the identification results. For another example, identification by determining the amino acid composition and polypeptide spectrum using real-time fluorescent PCR (targeting the genes of fibrinogen and NADH dehydrogenase) and SDS-PAGE two-dimensional gel electrophoresis. However, these methods have low sensitivity and are not suitable for bird's nest samples with low protein content. At the same time, these methods involve multiple steps such as extraction, characterization, and enzymatic digestion, which are complicated and time-consuming, resulting in high cost in the detection of large quantities of samples. Similar limitations exist in other means such as infrared spectroscopy, LC-MS combined with statistics.

[0004] Therefore, there is an urgent need to develop the screening and identification of unique marker components in bird's nest, and to establish an identification method that is simple, fast, accurate, low cost, high sensitivity, high specificity, highly reproducible, and suitable for commercial application of large quantities of samples. SUMMARY

[0005] The present application relates to oligosaccharide markers for identifying bird's nest, a method for preparing oligosaccharide markers, a method for identifying bird's nest by oligosaccharide markers, and the use of oligosaccharide markers in the rapid, specific, efficient, and low-cost identification of bird's nest raw materials and related products thereof.

[0006] The oligosaccharide marker and the application thereof in rapid identification of bird's nest have the following characteristics: high specificity, high sensitivity, simple operation, 30 minutes for detection, high practicability, applicable to bird's nest raw materials and corresponding products, wide market prospect and great economic and social benefits.

[0007] In one aspect of the present application, the present application relates to a method for identifying bird's nest, comprising:

[0008] a. detecting carbohydrates in the bird's nest sample;

[0009] b. identifying one or more oligosaccharide markers of the carbohydrates in the bird's nest sample.

[0010] In one aspect of the present application, in the above-mentioned method for identifying bird's nest, the detection method is selected from chromatographic analysis, spectroscopic analysis, mass spectrometric analysis, chromatographic analysis, chromatographic-spectroscopic analysis, chromatographic-mass spectrometric analysis, capillary electrophoresis analysis, immunoassay, nucleic acid aptamer binding analysis and combinations thereof; preferably, the detection method is liquid chromatography-mass spectrometry, particularly preferably LC-DAD-Q-TOF-MS and liquid chromatography-triple quadrupole mass spectrometry (LC-QqQ-MS / MS).

[0011] In one aspect of the present application, in the above-mentioned method for identifying bird's nest, the chromatographic conditions of the liquid chromatography are that the chromatographic column of the liquid chromatography is selected from silica gel matrix chromatographic column, polymer matrix chromatographic column or other inorganic filler chromatographic column, preferably C18 octadecyl bonded silica gel liquid chromatographic column, C8 octyl bonded silica gel liquid chromatographic column, NH2 amino bonded silica gel chromatographic column, glycol bonded silica gel chromatographic column, phenyl bonded silica gel chromatographic column, ion exchange chromatographic column or amide bonded silica gel chromatographic column.

[0012] In one aspect of the present application, in the above-mentioned method for identifying bird's nest, the chromatographic conditions of the liquid chromatography are that the mobile phase of the liquid chromatography is selected from water, formic acid, acetic acid, acetonitrile, isooctane, n-hexane, n-heptane, cyclohexane, carbon disulfide, carbon tetrachloride, benzene, xylene, toluene, chlorobenzene, dichloromethane, tetrahydrofuran, ethyl acetate, chloroform, aniline, pyridine, acetone, methanol, ethanol, isopropanol, n-propanol or combinations thereof, preferably the mobile phase of the liquid chromatography is selected from water, formic acid, acetic acid, acetonitrile or combinations thereof.

[0013] In one aspect of the present application, in the above-mentioned method for identifying bird's nest, the mass spectrometry is Q-TOF mass spectrometry.

[0014] In one aspect of the present application, the method for identifying bird's nest further comprises a step of preparing the bird's nest sample into a sample solution before identifying the sample, preferably, the sample solution is prepared using an aqueous solvent, more preferably, the aqueous solvent is methanol or water, and most preferably, the aqueous solvent is methanol: water with a volume ratio of 0% to 70%.

[0015] In one aspect of the present application, the method for identifying bird's nest further comprises a step of pre-column derivatization of the bird's nest sample, preferably, the derivatization is performed using a derivatization reagent with a chromophore group, such as 2-AA, 2-AB, PMP, 2-AP, ABP, ABME, HOA, AMAC, 3-(acetylamino)-6-amino-acridine (AA-Ac), ANTS, phenylhydrazine, dansylhydrazine, etc., more preferably, the derivatization is performed using ABEE (4-aminobenzoic acid ethyl ester, Benzocaine).

[0016] In one aspect of the present application, the method for identifying bird's nest comprises the following steps:

[0017] (1) preparing a sample solution;

[0018] (2) derivatizing the sample solution in step (1) with ABEE;

[0019] (3) separating and detecting the derivatized sample using liquid chromatography-mass spectrometry to obtain the retention time and mass-to-charge ratio of the sample;

[0020] (4) comparing the retention time and mass-to-charge ratio of the sample with the markers to identify the sample.

[0021] In one aspect of the present application, in the method for identifying bird's nest, the oligosaccharide markers in the chromatogram of the liquid chromatography-mass spectrometry are: an oligosaccharide marker with a retention time of 4.26 min and a mass-to-charge ratio of 619.2403; an oligosaccharide marker with a retention time of 5.38 min and a mass-to-charge ratio of 822.3202; or an oligosaccharide marker with a retention time of 6.49 min and a mass-to-charge ratio of 660.2664.

[0022] In one aspect of the present application, in the method for identifying bird's nest, the bird's nest sample is a bird's nest raw material or a bird's nest product, wherein, preferably, the bird's nest raw material includes bird's nest cup, bird's nest horn, bird's nest silk, bird's nest cake, bird's nest shred, or bird's nest strip, and more preferably, the bird's nest raw material includes white bird's nest cup, yellow bird's nest cup, blood bird's nest cup, or hairy bird's nest cup; the bird's nest product includes instant bird's nest, thick bird's nest, or bird's nest drink.

[0023] In one aspect of the present application, the present application relates to a method for identifying bird's nest, comprising:

[0024] a. detecting carbohydrates in a bird's nest sample;

[0025] b. identifying one or more oligosaccharide markers of carbohydrates in the bird's nest sample;

[0026] wherein the detection method is liquid chromatography-mass spectrometry, and the chromatographic conditions are: using C18 octadecyl bonded silica liquid chromatography column; the mobile phase of the liquid chromatography is: 0.1% to 0.5% formic acid aqueous solution and 0.1% to 0.5% formic acid acetonitrile solution;

[0027] the mass spectrometry is Q-TOF mass spectrometry;

[0028] the oligosaccharide marker is one or more of the following: oligosaccharide marker with a retention time of 4.26 min and a mass-to-charge ratio of 619.2403; oligosaccharide marker with a retention time of 5.38 min and a mass-to-charge ratio of 822.3202; or oligosaccharide marker with a retention time of 6.49 min and a mass-to-charge ratio of 660.2664;

[0029] preferably, the oligosaccharide marker is the one with a retention time of 4.26 min and a mass-to-charge ratio of 619.2403;

[0030] Optionally, the bird's nest sample is pre-column derivatized with ABEE.

[0031] In another aspect of the present application, the present application relates to a method for preparing oligosaccharide markers for identifying bird's nest, comprising:

[0032] a. detecting carbohydrates in a bird's nest sample;

[0033] b. identifying one or more oligosaccharide markers of carbohydrates in the bird's nest sample;

[0034] c. isolating the one or more oligosaccharide markers;

[0035] d. analyzing the isolated one or more oligosaccharide markers.

[0036] In yet another aspect of the present application, the present application relates to the use of the above-mentioned oligosaccharide markers in the identification of bird's nest. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 . Flow chart of bird's nest identification.

[0038] Figure 2 . Different forms of bird's nest and commonly used substitutes in the market.

[0039] Figure 3ABEE (4-aminobenzoic acid ethyl ester, Benzocaine) labeled bird's nest and adulterants UPLC-UV chromatogram (λ = 305 nm). Chromatogram, which is detected in multiple batches of samples to overlap the graph (bird's nest n = 134; agar n = 8; egg white n = 5; fish gelatin powder n = 7; milk n = 6; pigskin n = 4; rice powder n = 4; starch n = 3; swim bladder n = 3; tremella n = 6).

[0040] Figure 4 The spectrum of the selected specific markers is shown in the following figures:

[0041] A: ABEE labeled sugar UPLC-UV chromatogram (λ = 305 nm) of typical bird's nest, in which the chromatographic peaks with obvious signals are marked with 1-9;

[0042] B: Comparison spectrum of 9 peaks of extracted ion chromatogram (EIC) in bird's nest and related adulterants, in which bird's nest is EBN; adulterants include: agar (A), egg white (B), fish gelatin powder (C), milk (D), pigskin (E), rice powder (F), starch (G), swim bladder (H) and tremella (I).

[0043] Figure 5 The negative ion mode extracted ion flow contrast chromatogram and the corresponding mass spectrum of the three specific ABEE labeled markers of bird's nest are shown in the following figures:

[0044] A: ABEE-BNM001: m / z = 619.2409 ± 0.050, Rt = 4.26 min;

[0045] B: ABEE-BNM002: m / z = 822.3216 ± 0.050, Rt = 5.38 min;

[0046] C: ABEE-BNM003: m / z = 660.2590 ± 0.050, Rt = 6.49 min;

[0047] D: Mass spectrum of ABEE-BNM001, ABEE-BNM002 and ABEE-BNM003.

[0048] Figure 6 The negative ion mode multiple reaction monitoring chromatogram of the representative marker BNM001 of bird's nest is shown in the following figure:

[0049] Figures 7A-7E The chemical structure of ABEE-BNM001 is identified as follows:

[0050] Figure 7A The chemical structure of ABEE-BNM001 is shown in the following figure:

[0051] Figure 7B . 1 HNMR, 13 C-NMR and DEPT 135 spectra;

[0052] Figure 7C .HSQC spectra;

[0053] Figure 7D . 1 H- 1 H COSY spectra; and

[0054] Figure 7E .HMBC spectra.

[0055] Figure 8 Sample preparation and results of ABEE derivatization condition optimization.

[0056] Figure 9 Specific ABEE labeled marker BNM001 in different morphological bird's nest in peak area comparison chart.

[0057] Figures 10A-10B Identification results of bird's nest products, wherein:

[0058] Figure 10A Specific ABEE labeled marker BNM001 in different product types in the number of batches detected and not detected comparison chart;

[0059] Figure 10B Specific ABEE labeled marker BNM001 in different product types in peak area comparison chart,

[0060] Data are expressed as mean ± SD. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001 are significantly different compared with another group. DETAILED DESCRIPTION

[0061] DEFINITIONS

[0062] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as those of a person of ordinary skill in the art to which the application belongs, but in the event of a conflict, the meaning in the present specification shall prevail.

[0063] As used in the specification and claims, the singular forms "a," "an" and "the" include plural references unless the context clearly dictates otherwise.

[0064] Unless otherwise specified, all percentages (%) in the present specification are percentages by weight (wt%).

[0065] All numerical values or expressions involving numbers of quantities of components, process conditions, and so forth as used in the specification and claims are to be understood as being modified in all instances by the term "about," unless expressly indicated otherwise. The term "about" when used in reference to a number or a numerical range means that the number or numerical range is an approximation within experimental variability (or within statistical experimental error), and thus the number or numerical range can vary by +5 between the stated number or numerical range.

[0066] All ranges involving same component or property include the endpoints, which can be combined independently. Because the ranges are continuous, they include every value between the minimum and maximum values. It is also understood that any numerical range recited in this application is intended to include all sub-ranges of the range.

[0067] When the application is defined as a range in connection with a physical property, such as molecular weight, or a chemical property, all combinations and subcombinations of the range, as well as specific embodiments therein, are intended to be included. The term "comprising" (and related terms such as "comprise" or "comprises" or "having" or "including") includes such embodiments as those "consisting essentially of" and those "consisting of."

[0068] "and / or", when used in the context of the specification and claims, should be understood to mean "one or both" of the associated components, i.e., the components are present either jointly or separately in some cases. Multiple components listed with "and / or" should be interpreted in the same manner, i.e., "one or more" of the associated components. Other components can optionally be present in addition to those specifically identified in the "and / or" clause, whether related to those specifically identified or not. Thus, as a non-limiting example, a reference to "A and / or B", when used in a conjunctive sense, such as "comprising", can refer solely to A (optionally including components other than B), solely to B (optionally including components other than A), or to both A and B (optionally including additional components). Likewise, as a non-limiting example, a reference to "A and / or B" when used in a disjunctive sense, such as "comprising", can refer solely to A (optionally including components other than B), solely to B (optionally including components other than A), or to both A and B (optionally including additional components).

[0069] It should be understood that, in any method claimed in the application, including more than one step or act, the steps and acts of the method can occur in any order, unless otherwise specifically noted herein.

[0070] Abbreviations used in the application have their usual meaning in the chemical, biological and pharmaceutical arts.

[0071] The term "chromatography" includes methods for separating chemical substances and generally involves the process of carrying a mixture of analytes through a moving liquid or gas stream ("mobile phase") and separating them into different components as they flow through or over a stationary liquid or solid phase ("stationary phase") due to differential partitioning of the analytes between the mobile phase and the stationary phase. The stationary phase can be, among other things, a finely divided solid, a sheet of filter material, or a thin film of liquid on a solid surface.

[0072] The chromatography can be column chromatography (i.e., where the stationary phase is deposited or packed into a column), such as liquid chromatography, high performance liquid chromatography (HPLC), or ultra-high performance liquid chromatography (UHPLC). Details of chromatography are well known in the art (Bidlingmeyer, Practical HPLC Methodology and Applications, John Wiley & Sons Inc., 1993). Exemplary types of chromatography include, without limitation, high performance liquid chromatography (HPLC), UHPLC, normal phase HPLC (NP-HPLC), reverse phase HPLC (RP-HPLC), ion exchange chromatography (IEC), such as cation or anion exchange chromatography, hydrophilic interaction chromatography (HILIC), hydrophobic interaction chromatography (HIC), size exclusion chromatography (SEC), including gel filtration chromatography or gel permeation chromatography, chromatofocusing, affinity chromatography, such as immunoaffinity, immobilized metal affinity chromatography, and the like. Chromatography, including one-, two-, or multi-dimensional chromatography, can be used with other analytical methods, such as mass spectrometry.

[0073] The term "mass spectrometry" (MS) refers to a technique for identifying and / or quantifying molecules in a sample. MS includes ionizing molecules in a sample, forming charged molecules; separating the charged molecules according to mass-to-charge ratio; and detecting the charged molecules. MS can provide qualitative and quantitative detection of molecules in a sample. Molecules can be ionized and detected by any suitable method known to one of skill in the art. Some examples of mass spectrometry are "tandem mass spectrometry" or "MS / MS," which is a technique in which multiple rounds of mass spectrometry occur, either simultaneously using more than one mass analyzer, or sequentially using a single mass analyzer.

[0074] The term "mass spectrometer" refers to a device capable of volatilizing / ionizing an analyte to form gas phase ions and determining their absolute or relative molecular weights. Suitable volatilization / ionization methods are matrix assisted laser desorption ionization (MALDI), electrospray, laser / light, heat, electricity, nebulization / spray, etc., or combinations thereof. Suitable mass spectrometry formats include, but are not limited to, ion trap instruments, quadrupole instruments, electrostatic and magnetic sector instruments, time-of-flight instruments, time-of-flight tandem mass spectrometers (TOF MS / MS), Fourier transform mass spectrometers, Orbitraps, and hybrid instruments composed of different combinations of these types of mass spectrometry instruments. In turn, these instruments can be coupled to a variety of other instruments that separate samples (e.g., liquid chromatography or solid phase adsorption techniques based on chemical or biological properties) and ionize the samples for introduction into the mass spectrometer, including matrix assisted laser desorption (MALDI), electrospray or nano-spray ionization (ESI), or combinations thereof.

[0075] The term "derivitization" is a method of converting a compound into a similar chemical structure using chemical transformation. The purpose of derivitization of a sample is mainly to convert a difficult-to-analyze substance into a similar chemical structure but easy-to-analyze substance, facilitating quantification and separation. When a substance needs to be detected but is not easy to be detected, such as no ultraviolet absorption, etc., it can be treated, such as adding a chromophore, etc., to generate a detectable substance. The derivitization method is widely used in instrumental analysis. The general chemical derivatization method mainly has the following purposes: improving the sensitivity of sample detection; improving the separation degree of sample mixture; suitable for further structure identification, such as mass spectrometry, infrared or nuclear magnetic resonance, etc.

[0076] It is to be understood that the terms used herein are for the purpose of describing specific embodiments and are not intended to be limiting. Furthermore, any method, apparatus, and material similar or equivalent to those described herein can be used in the practice or testing of the present application, with the preferred methods, apparatus, and materials described below.

[0077] In one embodiment of the present application, the present application relates to a method for identifying bird's nest based on oligosaccharide markers in a bird's nest sample, comprising identifying one or more of oligosaccharide marker BNM001 with a molecular weight of 471.4299, oligosaccharide marker BNM002 with a molecular weight of 674.3202, or oligosaccharide marker BNM003 with a molecular weight of 512.2664 in the bird's nest sample.

[0078] In one embodiment of the present application, the present application relates to a method for identifying bird's nest, comprising:

[0079] a. detecting carbohydrates in the bird's nest sample;

[0080] b. identifying one or more oligosaccharide markers of the carbohydrates in the bird's nest sample.

[0081] In one embodiment of the present application, the present application relates to a method for preparing oligosaccharide markers for identifying bird's nest, comprising:

[0082] a. detecting carbohydrates in a bird's nest sample;

[0083] b. identifying one or more oligosaccharide markers of the carbohydrates in the bird's nest sample;

[0084] c. isolating the one or more oligosaccharide markers;

[0085] d. analyzing the isolated one or more oligosaccharide markers.

[0086] Any detection method can be used in the above-mentioned method for identifying one or more oligosaccharide markers in a bird's nest sample or the method for preparation.

[0087] In one aspect of the present application, in the above-mentioned method for identifying bird's nest or the method for preparing oligosaccharide markers for identifying bird's nest, in some embodiments, at least one detection method is selected from the group consisting of microscopy, macroscopic examination, spectroscopy, spectrometry, mass spectrometry, chromatography, and combinations thereof. Preferably, the detection method is selected from the group consisting of chromatographic analysis, spectroscopic analysis, mass spectrometric analysis, chromatographic-spectrometric analysis, chromatographic-spectrometric analysis, chromatographic-mass spectrometric analysis, capillary electrophoresis analysis, immunoassay analysis, nucleic acid aptamer binding analysis, and combinations thereof. Exemplary identification methods include, but are not limited to, mass spectrometry, high-performance thin-layer chromatography, Fourier transform infrared spectroscopy, ultraviolet-visible spectroscopy, thin-layer chromatography, gas-liquid chromatography (GC), high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), nuclear magnetic resonance (NMR), antibody detection method, Raman spectroscopy, capillary electrophoresis, liquid chromatography, gel permeation chromatography, ion chromatography, and combinations thereof.

[0088] More preferably, the detection method is liquid chromatography-mass spectrometry, particularly preferably LC-DAD-Q-TOF-MS.

[0089] In some embodiments, the LC-MS method comprises ultra-performance liquid chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOF-MS), ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS / MS), reversed-phase liquid chromatography-mass spectrometry (RPLC-MS), hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS), liquid chromatography-triple quadrupole tandem mass spectrometry (LC-QqQ-MS / MS), hydrophilic interaction liquid chromatography-triple quadrupole tandem mass spectrometry (HILIC-QqQ-MS / MS), electrostatic repulsion-hydrophilic interaction liquid chromatography-mass spectrometry (ERLIC-MS), liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS), liquid chromatography-tandem mass spectrometry (LC-MS / MS), multidimensional liquid chromatography coupled with tandem mass spectrometry (LC / LC-MS / MS). In some preferred embodiments, the liquid chromatography-mass spectrometry method is liquid chromatography time-of-flight mass spectrometry (LC-QTOF-MS) or liquid chromatography-triple quadrupole tandem mass spectrometry (LC-QqQ-MS / MS). In some more preferred embodiments, the liquid chromatography-mass spectrometry method is liquid chromatography-diode array detector (DAD)-time-of-flight mass spectrometry (LC-DAD-QTOF-MS). In some embodiments, the LC-MS method in the present specification is performed by standard techniques well known in the art.

[0090] In one embodiment of the present application, in the above-mentioned method for identifying bird's nest or the method for preparing oligosaccharide markers for identifying bird's nest, the chromatographic conditions of the liquid chromatography are that the chromatographic column of the liquid chromatography is selected from a silica gel matrix chromatographic column, a polymer matrix chromatographic column, or other inorganic filler chromatographic column, preferably, a C18 octadecyl bonded silica liquid chromatographic column, a C8 octane bonded silica liquid chromatographic column, an NH2 amino bonded silica chromatographic column, a glycol group bonded silica chromatographic column, a phenyl bonded silica chromatographic column, an amide group bonded silica chromatographic column, or an ion exchange chromatographic column.

[0091] Exemplary chromatographic columns include, but are not limited to:

[0092] C18 octadecyl bonded spherical silica liquid chromatography column 250 x 4.6 mm, 5 μm; C18 octadecyl bonded spherical silica liquid chromatography column 200 x 4.6 mm, 5 μm; C18 octadecyl bonded spherical silica liquid chromatography column 150 x 4.6 mm, 5 μm; C18 octadecyl bonded spherical silica liquid chromatography column 250 x 10 mm, 5 μm; C18 octadecyl bonded spherical silica liquid chromatography column 250 x 22 mm, 5 μm; C18 octadecyl bonded spherical silica liquid chromatography column 250 x 4.6 mm, 10 μm; C18 octadecyl bonded spherical silica liquid chromatography column 200 x 4.6 mm, 10 μm; C18 octadecyl bonded spherical silica liquid chromatography column 150 x 4.6 mm, 10 μm; C18 octadecyl bonded spherical silica liquid chromatography column 250 x 10 mm, 10 μm; C18 octadecyl bonded spherical silica liquid chromatography column 250 x 22 mm, 10 μm; C8 octane bonded spherical silica; NH2 amino bonded spherical silica; Diol diol bonded spherical silica; Phenyl phenyl bonded spherical silica; spherical silica Kromasil™ packed column / packed packing liquid chromatography column; ion exchange chromatography column (strong cation exchange column, strong anion exchange column SCX strong cation exchange silica 100 x 4.6 mm, 5 μm; SAX strong anion exchange silica 150 x 4.6 mm, 5 μm; chiral column (ligand exchange, cyclodextrin, polysaccharide chiral column); LEC coated ligand exchange chiral column 50 x 4.6 mm, 5 μm; LE bonded ligand exchange chiral column; CAD starch tris(3,5-dimethylphenylcarbamate) coated silica column; COD cellulose tris(3,5-dimethylphenylcarbamate) coated silica column; COB cellulose tribenzoate coated silica column; CD beta-cyclodextrin bonded silica column; Melamine special column for triazine; "PS macroporous polystyrene microspheres 250 x 4.6 mm 8 μm"; "UF macroporous urea-formaldehyde resin microspheres 250 x 4.6 mm 8 μm"; spherical silica liquid chromatography packing (spherical 5 μm); C18 octadecyl bonded spherical silica 4-5 μm; C8 octane bonded spherical silica 4-5 μm; NH2 amino bonded spherical silica 4-5 μm; Diol diol bonded spherical silica 4-5 μm; Phenyl phenyl bonded spherical silica 4-5 μm; Sil spherical silica 4-5 μm; SH mercapto bonded spherical silica 4-5 μm; CHO aldehyde bonded spherical silica 4-5 μm; Prepline preparative chromatography packing (spherical 10 μm, amorphous); S Sil fully porous spherical silica 10 μm; C18 octadecyl bonded spherical silica 10 μm; C8 octane bonded spherical silica 10 μm; NH2 amino bonded spherical silica 10 μm; Diol diol bonded spherical silica 10 μm; Phenyl phenyl bonded spherical silica 10 μm.phenyl column, cyano column, amino column, amide column, glycol column, quaternary amino silica gel strong anion exchange column, propyl amino silica gel weak anion exchange column, sulfonic acid silica gel strong cation exchange column, carboxylic acid silica gel weak cation exchange column, silica gel column, polymer matrix solid phase extraction column, cross-linked polystyrene matrix strong anion exchange column, WAX cross-linked polystyrene matrix weak anion exchange column, SCX cross-linked polystyrene matrix strong cation exchange column, WCX cross-linked polystyrene matrix weak cation exchange column, MCX cross-linked polystyrene matrix mixed cation exchange column, MAX cross-linked polystyrene matrix mixed anion exchange column, PS non-polar cross-linked polystyrene column, HLB polar embedded cross-linked polystyrene column, non-silica matrix solid phase extraction column, MS magnesium silicate column, AL aluminum oxide column, GPC graphitized carbon column, special solid phase extraction column, Pd palladium ion adsorption column, IDA iminodiacetic acid column, PBA phenylboronic acid column, IDA-Ni histidine-tagged protein column, IDA-Fe phosphorylated protein column, PRS malachite green column, PSA propyl ethylene diamine, MEL melamine column.

[0093] Preferably, silica gel matrix solid phase extraction column, such as C18 carbon octadecyl column; C8 carbon octyl column; C4 carbon tetradecyl column; amide column.

[0094] More preferably, ACQUITY UPLC BEH C18 column or Waters UPLC XBridge BEH Amide column.

[0095] Any commonly used mobile phase can be used in the chromatographic method of the present application, in some embodiments, the mobile phase of the liquid chromatography is selected from water, formic acid, acetic acid, acetonitrile, isooctane, n-hexane, n-heptane, cyclohexane, carbon disulfide, carbon tetrachloride, benzene, xylene, toluene, chlorobenzene, dichloromethane, tetrahydrofuran, ethyl acetate, chloroform, aniline, pyridine, acetone, methanol, ethanol, isopropanol, n-propanol, and combinations thereof. Preferably, the mobile phase of the liquid chromatography is selected from water, formic acid, acetic acid, acetonitrile, or combinations thereof.

[0096] In some embodiments, the mobile phase (eluent) for liquid chromatography is aqueous formic acid and aqueous formic acid in acetonitrile. In some specific embodiments, the eluent is 0.1-0.5% aqueous formic acid A and 0.1-0.5% formic acid in acetonitrile B. In some specific embodiments, the eluent A is 0.1-0.4% aqueous formic acid. In some specific embodiments, the eluent A is 0.1-0.3% aqueous formic acid. In some specific embodiments, the eluent A is 0.1-0.2% aqueous formic acid. In some specific embodiments, the eluent A is 0.1-0.2% aqueous formic acid. In other specific embodiments, the eluent B is 0.1-0.4% formic acid in acetonitrile. In other specific embodiments, the eluent B is 0.1-0.3% formic acid in acetonitrile. In other specific embodiments, the eluent B is 0.1-0.2% formic acid in acetonitrile. In some specific embodiments, the eluent A is 0.5% aqueous formic acid. In some specific embodiments, the eluent A is 0.4% aqueous formic acid. In some specific embodiments, the eluent A is 0.3% aqueous formic acid. In some specific embodiments, the eluent A is 0.2% aqueous formic acid. In some specific embodiments, the eluent A is 0.1% aqueous formic acid. In other specific embodiments, the eluent B is 0.5% formic acid in acetonitrile. In other specific embodiments, the eluent B is 0.4% formic acid in acetonitrile. In other specific embodiments, the eluent B is 0.3% formic acid in acetonitrile. In other specific embodiments, the eluent B is 0.2% formic acid in acetonitrile. In other specific embodiments, the eluent B is 0.1% formic acid in acetonitrile.

[0097] In some embodiments, the eluent flow rate ranges from 0.3 ± 0.05 mL / min. In some specific embodiments, the eluent flow rate ranges from 0.3 ± 0.04 mL / min. In some specific embodiments, the eluent flow rate ranges from 0.3 ± 0.03 mL / min. In some specific embodiments, the eluent flow rate ranges from 0.3 ± 0.02 mL / min. In some specific embodiments, the eluent flow rate ranges from 0.3 ± 0.01 mL / min. In some preferred embodiments, the eluent flow rate is 0.3 mL / min.

[0098] In some embodiments, the column temperature of the chromatographic column of the liquid chromatography is 30-50 °C. In some specific embodiments, the column temperature of the chromatographic column is 30-45 °C. In some specific embodiments, the column temperature of the chromatographic column is 30-40 °C. In some specific embodiments, the column temperature of the chromatographic column is 30-35 °C. In some specific embodiments, the column temperature of the chromatographic column is 50 °C. In some specific embodiments, the column temperature of the chromatographic column is 45 °C. In some specific embodiments, the column temperature of the chromatographic column is 40 °C. In some specific embodiments, the column temperature of the chromatographic column is 35 °C. In some specific embodiments, the column temperature of the chromatographic column is 30 °C.

[0099] In some embodiments, the detection wavelength of the diode array detector (DAD) is 190-760 nm, preferably 305 nm.

[0100] In one aspect of the present application, in the above-mentioned method for identifying bird's nest or the method for preparing oligosaccharide markers for identifying bird's nest, the mass spectrometry is Q-TOF mass spectrometry.

[0101] In one aspect of the present application, in the above-mentioned method for identifying bird's nest or the method for preparing oligosaccharide markers for identifying bird's nest, the step of preparing the bird's nest sample into a sample solution before sample identification is further included, preferably, the sample solution is prepared using an aqueous solvent, more preferably, the aqueous solvent is methanol or water, and most preferably, the aqueous solvent is a methanol: water aqueous solvent with a volume ratio of 0-70%.

[0102] In one aspect of the present application, in the above-mentioned method for identifying bird's nest, the following steps are included:

[0103] (1) preparing a sample solution, wherein the sample solution is directly used in the subsequent steps without derivatization;

[0104] (2) separating and detecting the sample using liquid chromatography-mass spectrometry to obtain the retention time and mass-to-charge ratio of the sample;

[0105] (3) comparing the retention time and mass-to-charge ratio of the sample with the markers to identify the sample.

[0106] In one aspect of the present application, in the above-mentioned method for identifying bird's nest or the method for preparing oligosaccharide markers for identifying bird's nest, the step of pre-column derivatization of the bird's nest sample is further included, preferably, the derivatization is performed using a derivatization reagent with a chromophore, such as 2-AA, 2-AB, PMP, 2-AP, ABP, ABME, HOA, AMAC, 3-(acetylamino)-6-amino-acridine (AA-Ac), ANTS, phenylhydrazine, dansylhydrazine, etc., and more preferably, the step of derivatization is performed using ABEE.

[0107] In one aspect of the present application, in the method for identifying bird's nest as described above, it comprises the following steps:

[0108] (1) preparing a sample solution;

[0109] (2) derivatizing the sample solution in step (1) with ABEE;

[0110] (3) separating and detecting the derivatized sample using liquid chromatography-mass spectrometry to obtain the retention time and mass-to-charge ratio of the sample;

[0111] (4) comparing the retention time and mass-to-charge ratio of the sample with the markers to identify the sample.

[0112] In one aspect of the present application, in the method for identifying bird's nest as described above, in the chromatogram of the liquid chromatography-mass spectrometry, the ABEE derivatized oligosaccharide marker is one or more of the following: ABEE derivatized oligosaccharide marker with a retention time of 4.26 min and a mass-to-charge ratio of 619.2403; ABEE derivatized oligosaccharide marker with a retention time of 5.38 min and a mass-to-charge ratio of 822.3202; or ABEE derivatized oligosaccharide marker with a retention time of 6.49 min and a mass-to-charge ratio of 660.2664.

[0113] In one aspect of the present application, the present application relates to a method for identifying bird's nest, comprising:

[0114] a. detecting the carbohydrates in the bird's nest sample;

[0115] b. identifying one or more oligosaccharide markers of the carbohydrates in the bird's nest sample.

[0116] In the method for identifying bird's nest as described above, the one or more oligosaccharide markers are oligosaccharide marker BNM001 with a mass-to-charge ratio of 471.4299, oligosaccharide marker BNM002 with a molecular weight of 674.3202, and / or oligosaccharide marker BNM003 with a molecular weight of 512.2664.

[0117] In one aspect of the present application, in the method for identifying bird's nest or the method for preparing oligosaccharide markers for identifying bird's nest as described above, the bird's nest sample is a bird's nest raw material or a bird's nest product, wherein preferably the bird's nest raw material comprises bird's nest cups, bird's nest horns, bird's nest silk, bird's nest cakes, bird's nest pieces or bird's nest strips, more preferably the bird's nest raw material comprises white bird's nest cups, yellow bird's nest cups, blood bird's nest cups, and hair bird's nest cups; the bird's nest product comprises instant bird's nest, thick bird's nest or bird's nest drink.

[0118] In some embodiments, the method for identifying bird's nest or the method for preparing oligosaccharide markers for identifying bird's nest comprises extracting the markers before sample identification. The extraction of the markers comprises preparing the bird's nest sample into a sample solution with an aqueous solvent before sample identification, centrifugation, and taking the supernatant for use.

[0119] In some embodiments, if the bird's nest sample to be detected is a bird's nest raw material, the preparation of the sample solution comprises adding an aqueous solvent to the dry bird's nest sample powder, vortexing to mix, and then ultrasonication. In some specific embodiments, the ultrasonication condition is ultrasonication at room temperature for 1-15 minutes. In some more specific embodiments, the ultrasonication time is 1-10 minutes. In some more specific embodiments, the ultrasonication time is 1-5 minutes. In some more specific embodiments, the ultrasonication time is 5-15 minutes. In some more specific embodiments, the ultrasonication time is 5-10 minutes. In some more specific embodiments, the ultrasonication time is selected from 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, or 15 minutes. In some more specific embodiments, the ultrasonication time is 5 minutes. In other embodiments, if the bird's nest sample to be detected is a bird's nest product, the preparation of the sample solution comprises directly taking the solution in the bird's nest product or directly dissolving the bird's nest product in an aqueous solvent.

[0120] In some embodiments, the aqueous solvent is an aqueous solution of an alcohol. In some embodiments, the aqueous solvent is an aqueous solution of methanol. In some embodiments, the aqueous solvent is an aqueous solution of ethanol. In some embodiments, the aqueous solvent is an aqueous solution of propanol. In some embodiments, the aqueous solvent is an aqueous solution of butanol. In some embodiments, the aqueous solvent is an aqueous solution of pentanol. In some embodiments, the aqueous solvent is a solvent with a methanol:water volume ratio of 0% to 70%. In some embodiments, the aqueous solvent is a solvent with a methanol:water volume ratio of 0% to 60%. In some embodiments, the aqueous solvent is a solvent with a methanol:water volume ratio of 0% to 50%. In some embodiments, the aqueous solvent is a solvent with a methanol:water volume ratio of 0% to 40%. In some embodiments, the aqueous solvent is a solvent with a methanol:water volume ratio of 0% to 30%. In some embodiments, the aqueous solvent is a solvent with a methanol:water volume ratio of 0% to 20%. In some embodiments, the aqueous solvent is a solvent with a methanol:water volume ratio of 0% to 10%. In some embodiments, the aqueous solvent is a solvent with a methanol:water volume ratio of 70%. In some embodiments, the aqueous solvent is a solvent with a methanol:water volume ratio of 60%. In some embodiments, the aqueous solvent is a solvent with a methanol:water volume ratio of 50%. In some embodiments, the aqueous solvent is a solvent with a methanol:water volume ratio of 40%. In some embodiments, the aqueous solvent is a solvent with a methanol:water volume ratio of 30%. In some embodiments, the aqueous solvent is a solvent with a methanol:water volume ratio of 20%. In some embodiments, the aqueous solvent is a solvent with a methanol:water volume ratio of 10%. In some embodiments, the aqueous solvent is water.

[0121] In some embodiments, the method for identifying bird's nest comprises derivatizing the bird's nest sample before performing sample identification, and the derivatization method is selected from the group consisting of: derivatization using derivatization reagent with chromophore ABEE, 2-AA, 2-AB, PMP, 2-AP, ABP, ABME, HOA, AMAC, 3-(acetylamino)-6-amino-acridine (AA-Ac), ANTS, phenylhydrazine or dansylhydrazine, or methylation, acetylation or reduction method, etc. In some specific embodiments, the derivatization method is derivatizing the sample using derivatization reagent with chromophore ABEE, 2-AA, 2-AB, PMP or 2-AP. In some specific embodiments, the derivatization method is derivatizing the sample using derivatization reagent with chromophore ABEE.

[0122] In some embodiments, the derivatization reaction temperature is 60-90 °C. In some embodiments, the derivatization reaction temperature is 60-85 °C. In some embodiments, the derivatization reaction temperature is 60-80 °C. In some embodiments, the derivatization reaction temperature is 65-90 °C. In some embodiments, the derivatization reaction temperature is 65-85 °C. In some embodiments, the derivatization reaction temperature is 65-80 °C. In some embodiments, the derivatization reaction temperature is 70-90 °C. In some embodiments, the derivatization reaction temperature is 70-85 °C. In some embodiments, the derivatization reaction temperature is 70-80 °C. In some embodiments, the derivatization reaction temperature is 75-90 °C. In some embodiments, the derivatization reaction temperature is 75-85 °C. In some embodiments, the derivatization reaction temperature is 75-80 °C. In some embodiments, the derivatization reaction temperature is 60 °C. In some embodiments, the derivatization reaction temperature is 65 °C. In some embodiments, the derivatization reaction temperature is 70 °C. In some embodiments, the derivatization reaction temperature is 75 °C. In some embodiments, the derivatization reaction temperature is 80 °C. In some embodiments, the derivatization reaction temperature is 85 °C. In some embodiments, the derivatization reaction temperature is 90 °C.

[0123] In some embodiments, the derivatization reaction time is 1-30 minutes. In some embodiments, the derivatization reaction time is 1-25 minutes. In some embodiments, the derivatization reaction time is 1-20 minutes. In some embodiments, the derivatization reaction time is 1-15 minutes. In some embodiments, the derivatization reaction time is 1-10 minutes. In some embodiments, the derivatization reaction time is 5-30 minutes. In some embodiments, the derivatization reaction time is 5-25 minutes. In some embodiments, the derivatization reaction time is 5-20 minutes. In some embodiments, the derivatization reaction time is 5-15 minutes. In some embodiments, the derivatization reaction time is 5-10 minutes. In some embodiments, the derivatization reaction time is 30 minutes. In some embodiments, the derivatization reaction time is 25 minutes. In some embodiments, the derivatization reaction time is 20 minutes. In some embodiments, the derivatization reaction time is 15 minutes. In some embodiments, the derivatization reaction time is 5-10 minutes. In some embodiments, the derivatization reaction time is 5 minutes. In some embodiments, the derivatization reaction time is 1 minute.

[0124] In some embodiments, the method of identifying bird’s nest of the present application comprises the following steps:

[0125] (1) preparing a sample solution, wherein the sample solution is directly used for the subsequent steps without derivatization;

[0126] (2) Separating and detecting the sample by liquid chromatography-mass spectrometry to obtain the retention time and / or mass-to-charge ratio of the sample;

[0127] (3) Comparing the retention time and / or mass-to-charge ratio of the sample with the marker to identify the sample.

[0128] In some embodiments, the present application relates to a method for identifying a bird's nest sample based on oligosaccharide markers, comprising the following steps:

[0129] (1). Sample preparation

[0130] After the sample powder is treated with 50% methanol aqueous solution and ultrasonic treatment, the supernatant is taken out and directly used for further analysis without a derivatization step;

[0131] (2). LC-QqQ-MS / MS Analysis

[0132] Liquid chromatography conditions: using a chromatographic column Waters UPLC XBridge BEH Amide (2.1 mm x 100 mm, 3.5 μm, Waters, Milford, USA), and eluting with a linear gradient of 0.1-0.5%, preferably 0.1% formic acid aqueous solution A and 0.1-0.5%, preferably 0.1% formic acid acetonitrile solution B;

[0133] Preferably, the eluent flow rate is 0.4 mL / min, and the column temperature is 30°C;

[0134] Preferably, the solvent gradient is as follows: 0-4 minutes, 78-50% B; 4-5 minutes, 50-100% B; 5.1 minutes, 100-78% B; 5.1-7 minutes, 78% B. The injection volume is 6 μL;

[0135] Preferably, the mass spectrometry conditions are as follows: negative ion mode, optimal operation under multiple reaction monitoring (MRM);

[0136] More preferably, the mass spectrometry parameters are as follows: the qualitative ion pair is 470.1 / 87.0, the flow rate of the atomizing gas (N2) is 7.0 L / min, the atomizing gas temperature is 300°C; the sheath gas flow rate is 8.0 L / min; the sheath gas temperature is 350°C; the collision voltage is 10 V; and the fragment voltage is 220 V;

[0137] After the above analysis, the information of the chromatogram of the bird's nest sample is obtained;

[0138] (3). Confirmation of the marker in the bird's nest sample

[0139] The retention time of each substance in the information of the chromatogram of the bird's nest sample obtained in step 2 is compared with the retention time 2.143 min of the marker BNM001.

[0140] In some embodiments, the method for identifying bird's nest of the present application comprises the following steps:

[0141] (1) preparing a sample solution;

[0142] (2) derivatizing the sample solution in step (1) with ABEE;

[0143] (3) separating and detecting the derivatized sample using liquid chromatography-mass spectrometry to obtain the retention time and mass-to-charge ratio of the sample;

[0144] (4) comparing the retention time and mass-to-charge ratio of the sample with the markers to identify the sample.

[0145] In some embodiments of the present application, the present application provides one or more oligosaccharide markers that can be used for identifying bird's nest samples. In some specific embodiments, the oligosaccharide marker is one or more of oligosaccharide marker BNM001 with a molecular weight of 471.4299, oligosaccharide marker BNM002 with a molecular weight of 674.3202, or oligosaccharide marker BNM003 with a molecular weight of 512.2664.

[0146] In some embodiments, the present application relates to a method for identifying bird's nest samples based on oligosaccharide markers, which comprises the following steps:

[0147] (1). Sample preparation

[0148] After the sample powder is treated with distilled water and ultrasonic treatment, the supernatant is taken for further analysis;

[0149] (2). Sample derivatization

[0150] The supernatant solution or bird's nest product solution described above is added with ABEE, glacial acetic acid and NaBH3CN, mixed uniformly and reacted; after cooling, water and diethyl ether are added to the solution, the mixture is mixed thoroughly and centrifuged; the upper ABEE solution is removed, the lower aqueous phase is dried, redissolved in 70% volume ratio of methanol aqueous solution, and subjected to LC-DAD-qTOF-MS analysis;

[0151] (3). LC-DAD-qTOF-MS analysis

[0152] The liquid chromatography conditions are as follows: using a chromatographic column ACQUITY UPLC BEH C18, 1.7 μm, and using 0.1-0.5%, preferably 0.1% formic acid aqueous solution A and 0.1-0.5%, preferably 0.1% formic acid acetonitrile solution B linear gradient elution;

[0153] Preferably, the eluent flow rate is 0.3 mL / min, and the column temperature is 30°C;

[0154] Preferably, the solvent gradient is as follows: 0-7 min, 15% B; 7-9 min, 15-100% B; 9-9.1 min, 100-15% B; 9.1-12 min, 15% B; injection volume is 2 μL;

[0155] Preferably, the mass spectrometry conditions are as follows: the ESI source is used, and the best operation is in the negative ion full scan mode;

[0156] More preferably, the mass spectrometry parameters are as follows: the flow rate of the atomization gas N2 is 7.0 L / min, the atomization gas temperature is 300°C; the sheath gas flow rate is 8.0 L / min; the sheath gas temperature is 350°C; the atomizer is 40 psi; the capillary voltage is 3000 V; the cone voltage is 65 V; the multiplication voltage is 750 V; the fragment voltage is 150 V; and the mass scan range is set to 100-2000 charge mass;

[0157] After the above analysis, the spectrum information of the bird's nest sample is obtained;

[0158] (4). Confirmation of the marker in the bird's nest sample

[0159] The retention time and mass spectrum information of each substance in the spectrum information of the bird's nest sample obtained in step 3 are compared with the retention time 4.26 min and the mass-to-charge ratio 619.2403 of the ABEE-labeled marker BNM001.

[0160] In some embodiments of the present application, the present application provides an oligosaccharide marker BNM001 that can be used for the identification of a bird's nest sample, which has a chemical structure of formula (A), and the structure of ABEE-labeled (ABEE-BNM001) is B:

[0161]

[0162] In some embodiments of the present application, the present application provides one or more oligosaccharide markers for use in the identification of a bird's nest sample. In some specific embodiments, the present application provides the use of the oligosaccharide marker BNM001 in the identification of a bird's nest sample. In some specific embodiments, the present application provides the use of the oligosaccharide marker BNM002 in the identification of a bird's nest sample. In some specific embodiments, the present application provides the use of the oligosaccharide marker BNM003 in the identification of a bird's nest sample.

[0163] Examples

[0164] Other objects of the present application will be apparent to those skilled in the art from consideration of the description and examples that follow. The examples described below are illustrative only and are not intended to be limiting in any way upon the scope of the present application. Unless otherwise noted, technical or conditions described in the examples are in accordance with those described in the literature or in accordance with the product instructions. Unless otherwise noted, reagents or instruments used are conventional products available commercially. In the quantitative test in the following examples, three repeated experiments were set up and the results were averaged.

[0165] In the following examples, the materials, reagents and instruments involved are as follows:

[0166] Materials and reagents:

[0167] 134 kinds of raw materials of bird’s nest (RM001-RM134) (see Table 1A) were purchased from Hong Kong market. The sialic acid content was determined according to the industry standard SNT 3644-2013. The results showed that all of them contained sialic acid. Figure 2 A), including 90 kinds of Cup, 7 kinds of The horn, 8 kinds of Large strip, 8 kinds of Small strip, 6 kinds of Pieces and 15 kinds of The broken.

[0168] 83 kinds of bird’s nest products (PD01-PD83) were purchased from Thailand, Vietnam, mainland China and Hong Kong market according to the product instructions. They were classified into 44 kinds of instant bird’s nest, 18 kinds of thick bird’s nest and 21 kinds of bird’s nest drinks.

[0169] Common substitutes included agar (A1-A8), egg white (EW1-EW8), fish gelatin powder (GE1-GE7), milk (M1-M6), pigskin (P1-P4), rice flour (RF1-RF4), starch (ST1-ST3), swim bladder (SB1-SB3) and tremella fuciformis (SF1-SF6) (see Table 1B). Figure 2 B).

[0170] Acetonitrile (mass spectrometry), formic acid (mass spectrometry pure), methanol (analytical pure), glacial acetic acid (analytical pure) and diethyl ether (analytical pure) were purchased from Thermo Fisher. Sodium cyanoborohydride (NaBH3CN), 4-aminobenzoic acid ethyl ester (Benzocaine, ABEE) and sialic acid were purchased from Sigma.

[0171] Instruments: An Agilent 1290U HPLC system equipped with a binary pump, a temperature-controlled column, an autosampler, a degasser, and a diode array detector (DAD, λ = 305 nm); an Agilent 6540Q-TOF mass spectrometer with a quadrupole time-of-flight (Q-TOF) mass spectrometer, a triple quadrupole tandem mass spectrometer, and a JetStream electrospray ionization (ESI) source. The system was controlled by Mass Hunter B.06 software.

[0172] Example 1: Screening and Detection of Specific Biomarkers

[0173] in accordance with Figure 1 The detection procedure shown involves parallel ultrasonic treatment of the raw bird's nest and various substitute samples, followed by derivatization and full-scan analysis in negative ion mode using LC-DAD-qTOF-MS. The resulting chromatogram is shown in Figure 3. The specific experimental steps are as follows:

[0174] 1. Sample preparation

[0175] Weigh 25 mg of dried sample powder, add 0.5 mL of distilled water, vortex to mix, and sonicate at room temperature for 5 minutes. Centrifuge the sonicated sample at 15,000 rpm for 5 minutes. Collect the supernatant for further analysis.

[0176] 2. Sample derivatization

[0177] Take 100 μL of the supernatant or product solution above into a 2 mL centrifuge tube, add 400 μL of 0.6 mol / L ABEE, 80 μL of glacial acetic acid, and 80 μL of 1.4 mol / L NaBH3CN, mix thoroughly, and react at 80 °C for 5 minutes. After cooling, add water and diethyl ether to the solution, mix thoroughly, and centrifuge at 15,000 rpm for 5 minutes. Remove the upper layer (ABEE solution), dry the lower aqueous phase, redissolve in 200 μL of 70% (v / v) methanol aqueous solution, and perform LC-DAD-qTOF-MS analysis.

[0178] 3. LC-DAD-qTOF-MS analysis

[0179] Liquid chromatography conditions: An ACQUITY UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 μm, Waters, Milford, USA) was used, eluted with a linear gradient of 0.1% formic acid aqueous solution (A) and 0.1% formic acid acetonitrile solution (B), at a flow rate of 0.3 mL / min and a column temperature of 30 °C. The solvent gradient was as follows: 0–7 min, 15% B; 7–9 min, 15–100% B; 9–9.1 min, 100–15% B; 9.1–12 min, 15% B. The injection volume was 2 μL.

[0180] Mass spectrometry conditions: Using an ESI source, the optimal operating parameters in negative ion full scan mode are as follows: nebulizer gas (N2) flow rate 7.0 L / min, nebulizer gas temperature 300℃; sheath gas flow rate 8.0 L / min; sheath gas temperature 350℃; nebulizer 40 psi; capillary voltage 3000 V; cone voltage 65 V; multiplication voltage 750 V; fragment voltage 150 V. The mass scan range is set to 100-2000 charged mass (m / z).

[0181] Depend on Figure 3 It can be seen that, using this method, each sample exhibits rich carbohydrate fingerprint information, and the information consistency among various samples is good.

[0182] To further identify unique markers in bird's nest, the fingerprint information of bird's nest was compared in detail with that of substitutes. The results for the bird's nest samples were as follows: Figure 4 As shown in Figure A, nine distinct common peaks were found, with mass-to-charge ratios of 619.2403 (peak 1), 457.1882 (peak 2), 822.3202 (peak 3), 659.3143 (peak 4), 457.1882 (peak 5), 660.2664 (peak 6), 415.1768 (peak 7), 443.2092 (peak 8), and 415.1768 (peak 9). The ion chromatograms of the substitutes were extracted one by one according to their mass-to-charge ratios and compared with those of bird's nest to examine the specificity of each peak. The results are as follows. Figure 4 As shown in B and Table 1, peaks 2, 4, 5, 7, and 9 are non-specific peaks: isomer peaks 2 and 5 are present in gelatin powder, milk, and pig skin; peak 4 is present in egg white and tremella; isomer peaks 7 and 9 are present in egg white, milk, pig skin, fish swim bladder, and tremella. Therefore, these five peaks cannot be used as specific markers for bird's nest. While peak 8 has good specificity, it was not detected in some bird's nest samples, therefore, this peak is a non-common peak.

[0183] The other three peaks, peaks 1, 3, and 6, all exhibited good specificity and commonality, as shown in the extracted ion chromatography and mass spectrometry signals. Figure 5 As shown.

[0184] Furthermore, the detection of these three peaks showed good repeatability. Six parallel samples from the same batch were prepared and measured, and the peak areas of the three peaks were recorded. The results showed that the relative standard deviation (RSD%) of peak 1 was 4.13%, peak 2 was 5.04%, and peak 3 was 5.67%, making them potential biomarkers. Peak 1 had the largest response signal and a detection limit of 0.03 μg / ml, which can meet the identification and analysis requirements of different bird's nest products.

[0185] In summary, the substances represented by peaks No. 1, No. 3 and No. 6 can all be used as markers for identifying bird's nest. Preferably, the substance represented by peak No. 1, i.e. the substance with a retention time of 4.26 min and a mass-to-charge ratio of 619.2403, is a representative marker for ABEE-labeled ABEE, which is specific, highly sensitive and reproducible.

[0186] Table 1. Screening results of natural markers for bird's nest based on retention time, mass-to-charge ratio, specificity and commonality

[0187]

[0188] a Substitutes refer to agar, egg white, fish glue powder, milk, pigskin, rice powder, starch, swim bladder and tremella;

[0189] b Common peaks refer to peaks that exist in multiple batches (n≥100) of bird's nest raw material samples;

[0190] c “+” refers to a positive result, i.e. the peak can be detected or the conditions for specificity and commonality are met, and “-” refers to a negative result, i.e. the peak cannot be detected or the conditions for specificity and commonality are not met.

[0191] Example 2. Detection of representative markers without ABEE labeling

[0192] The bird's nest raw material and each substitute sample were treated with ultrasound in parallel, and the bird's nest product solution was taken for LC-QqQ-MS / MS negative ion mode MRM analysis. The obtained chromatogram is shown in Figure 6 The specific experimental steps are as follows:

[0193] 1. Sample preparation

[0194] 50 mg of dry sample powder was weighed, 0.4 mL of 50% methanol aqueous solution was added, vortexed and mixed, and treated with ultrasound at room temperature for 5 minutes. The sample treated with ultrasound was centrifuged at 15,000 rpm for 5 minutes. The supernatant was taken for sample analysis. In addition, 100 μL of bird's nest product solution was taken, 100 μL of methanol was added, vortexed and mixed, and the mixed sample was centrifuged at 15,000 rpm for 5 minutes. The supernatant was taken for sample analysis.

[0195] 2. LC-QqQ-MS / MS analysis

[0196] Liquid chromatography conditions: A Waters UPLC XBridge BEH Amide column (2.1 mm × 100 mm, 3.5 μm, Waters, Milford, USA) was used, eluted with a linear gradient of 0.1% formic acid aqueous solution (A) and 0.1% formic acid acetonitrile solution (B), at a flow rate of 0.4 mL / min and a column temperature of 30 °C. The solvent gradient was as follows: 0–4 min, 78–50% B; 4–5 min, 50–100% B; 5.1 min, 100–78% B; 5.1–7 min, 78% B. The injection volume was 6 μL.

[0197] Mass spectrometry conditions: The optimal operating parameters under ESI source, negative ion mode, and multiple reaction monitoring (MRM) are as follows: BNM001 qualitative ion pair 470.1 / 87.0, nebulizer gas (N2) flow rate 7.0 L / min, nebulizer gas temperature 300℃; sheath gas flow rate 8.0 L / min; sheath gas temperature 350℃; collision voltage 10V; fragment voltage 220V.

[0198] Depend on Figure 6 It is known that by using this method, the representative marker BNM001 can be detected in bird's nest raw materials and products, while it is not detected in common substitutes, indicating that identification can be completed even without ABEE labeling. This invention relates to markers with strong specificity.

[0199] Example 3: Separation and structural identification of representative markers

[0200] The results above indicate that BNM001 is the most abundant marker in EBN. ABEE-BNM001 was separated by semi-preparative liquid chromatography and high-performance liquid chromatography, and its precise chemical structure was characterized by one-dimensional (1D-NMR), two-dimensional (2D-NMR), and high-resolution mass spectrometry (MS). ABEE-BNM001 was dissolved in D2O, and TMS was used as an internal standard. 1 H and 13 C-NMR spectra were measured at 400 MHz and 100 MHz, respectively.

[0201] High-resolution mass spectrometry data ( Figure 7B The negative ion ESI was 619.2409, suggesting that ABEE-BNM001 is an ABEE-labeled sialylated disaccharide, and BNM001 is a sialylated disaccharide. NMR analysis further supports this finding. Figures 7B-7E To obtain more structural information, see the structural formula. Figure 7A .

[0202] Example 4: Optimization of Sample Preparation and Derivatization Methods

[0203] To establish a rapid identification method, the sample preparation process and the processing process are optimized.

[0204] For sample preparation, i.e. marker extraction process, according to the marker extraction step in Example 1, different solvents (0.5 mL distilled water, 50% methanol (V:V) aqueous solution, 70% methanol (V:V) aqueous solution) and different ultrasonic treatment time (1, 5, 10 minutes) were investigated to obtain the optimal conditions. As shown in Figure 8 With the increase of the proportion of organic solvent, the marker signal gradually decreased, indicating that the solubility of the marker in the aqueous solution was optimal, and the 50% methanol (V:V) solution was suboptimal. At the same time, the marker peak area did not increase significantly after 5 min of ultrasonic treatment, and the optimal ultrasonic treatment time was 5 min, and the suboptimal ultrasonic treatment time was 10 min.

[0205] For chromophore reagent derivatization process, according to the sample derivatization step in Example 1, different reaction temperatures (60℃, 70℃, 80℃, 90℃) and reaction times (1, 5, 10, 30 minutes) were investigated. The results are shown in Figure 8 With the increase of reaction temperature, the marker peak area showed an increasing trend, and decreased at 90℃, so the optimal reaction temperature was 80℃, and the suboptimal reaction temperature was 70℃. At the same time, we found that the marker peak area did not show significant difference after 5 min and 10 min of reaction, so the optimal reaction time was 5 min, and the suboptimal reaction time was 10 min. During the condition optimization process, it was found that too high temperature and / or too long reaction time might damage the stability of the derivative.

[0206] In summary, the optimal conditions are aqueous solution extraction, ultrasonic treatment for 5 min, and derivatization at 80℃ for 5 min.

[0207] Example 5 Marker BNM001 is contained in different forms of bird's nest

[0208] The collected 134 different forms of bird's nest raw materials were processed in parallel, and LC-DAD-qTOF-MS test was performed to record the specific marker peak area. After completing the sample weight correction, the data were statistically analyzed according to different forms of bird's nest classification. The results are shown in Figure 9 Although there are some differences between various bird's nests, they all contain the marker BNM001, which is consistent with the detection of sialic acid according to the industry standard SNT3644-2013.

[0209] Example 6 Application of oligosaccharide marker BNM001 in identification of bird's nest related products

[0210] 83 different types of bird's nest products collected from different markets were tested by LC-DAD-qTOF-MS in parallel, and ion flow chromatograms of specific markers were extracted, and the retention time and mass spectrum information were compared.

[0211] The determination results are shown in Figure 10A and Figure 10B Among the 83 products, 14 instant bird's nests and 5 bird's nest drinks were not detected for the oligosaccharide marker BNM001. Upon inspection of the original products, none of the products not detected for the oligosaccharide marker BNM001 had obvious bird's nest foam-like material, and some product samples presented a jelly-like turbidity sample, suspected to be agar or other unknown gel-like material. To further confirm the detection results, 100 mg of the product was freeze-dried and subjected to the same processing method for retesting, and the marker was still not detected, confirming that the 19 samples were counterfeit products.

[0212] At the same time, the sialic acid in the product was detected according to the industry export standard (GACS-000-1017-19(0)). The comparison of the detection results is shown in Table 3. The results show that sialic acid was not detected in 18 of the above-mentioned 19 counterfeit products, and the sialic acid content in one sample (PD-40) was higher than the detection limit, which may be related to the artificial addition of sialic acid. The overall trend of the marker BNM001 (represented by ABEE-BNM001) content in the remaining 64 samples was concentrated bird's nest > instant bird's nest > bird's nest drink. Among different manufacturers of instant bird's nests, there were large differences, which explained why there was no unified quality grade standard in the bird's nest product market, and therefore different manufacturers divided the products differently, i.e., instant bird's nests may be concentrated bird's nests or bird's nest drinks.

[0213] Table 3. Detection results of specific marker BNM001 and sialic acid in 83 batches of bird's nest products

[0214]

[0215]

[0216] Note: a Determination according to the marker BNM001 in the present application; b Determination of sialic acid according to the bird's nest industry export standard (GACS-000-1017-19(0)); and " - " means that the peak area of BNM001 in the bird's nest is less than the detection limit (0.03 μg / ml) or the peak area of sialic acid is less than the detection limit (7.50 μg / ml).

Claims

1. A method for identifying bird’s nest and bird’s nest substitutes, comprising: a. detecting carbohydrates in a bird’s nest sample; b. identifying an oligosaccharide marker of the carbohydrates in the bird’s nest sample wherein the oligosaccharide marker has a chemical structure of A ， the bird’s nest substitutes are selected from agar, egg white, fish glue powder, milk, pigskin, rice powder, starch, swim bladder or tremella.

2. The method of claim 1, wherein, the detection method is selected from chromatography, spectroscopy, mass spectrometry, chromatography-mass spectrometry, chromatography-spectrometry, chromatography-spectrometry-mass spectrometry, capillary electrophoresis, immunoassay, aptamer binding assay or a combination thereof.

3. The method of claim 2, wherein, the immunoassay is selected from specific antibody assay, ELISA assay or colloidal gold method; the spectroscopy is selected from nuclear magnetic resonance spectroscopy (NMR), ultraviolet spectrophotometry, near-infrared spectrophotometry, X-ray powder diffraction, Raman spectroscopy or Fourier transform infrared spectroscopy; the chromatography and combined analysis method is selected from thin layer chromatography, gas chromatography (GC), high performance liquid chromatography (HPLC), liquid chromatography, hydrophilic interaction chromatography, gel permeation chromatography, ion chromatography, chromatography-mass spectrometry such as liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), gas chromatography-infrared spectroscopy, gas chromatography-liquid chromatography or liquid chromatography-nuclear magnetic resonance.

4. The method of claim 1, wherein, the detection method is liquid chromatography-mass spectrometry.

5. The method of claim 1, wherein, the detection method is LC-DAD-Q-TOF-MS or liquid chromatography-triple quadrupole mass spectrometry (LC-QqQ-MS / MS).

6. The method of differentiating bird's nest from bird's nest substitute according to any one of claims 3-5, wherein, the chromatographic conditions of the liquid chromatography are selected from a chromatographic column of the liquid chromatography selected from silica gel matrix chromatographic column, polymer matrix chromatographic column or other inorganic filler chromatographic column.

7. The method of claim 6, wherein, the chromatographic column of the liquid chromatography is selected from C18 octadecyl bonded silica gel liquid chromatographic column, C8 octane bonded silica gel liquid chromatographic column, NH2 amino bonded silica gel chromatographic column, glycol bonded silica gel chromatographic column, phenyl bonded silica gel chromatographic column, ion exchange chromatographic column or amide bonded silica gel chromatographic column.

8. The method of claim 6, wherein, the chromatographic column of the liquid chromatography is selected from ACQUITY UPLC BEH C18 chromatographic column or Waters UPLC XBridge BEH Amide chromatographic column.

9. The method of claim 6, wherein, the chromatographic conditions of the liquid chromatography are selected from a mobile phase of the liquid chromatography selected from water, formic acid, acetic acid, acetonitrile, isooctane, n-hexane, n-heptane, cyclohexane, carbon disulfide, carbon tetrachloride, benzene, xylene, toluene, chlorobenzene, dichloromethane, tetrahydrofuran, ethyl acetate, chloroform, aniline, pyridine, acetone, methanol, ethanol, isopropanol, n-propanol or a combination thereof.

10. The method of claim 6, wherein, the mobile phase of the liquid chromatography is selected from water, formic acid, acetic acid, acetonitrile or a combination thereof.

11. The method of claim 6, wherein, the mobile phase of the liquid chromatography is 0.1% to 0.5% formic acid in water and 0.1% to 0.5% formic acid in acetonitrile.

12. The method of claim 1, wherein the method further comprises a step of preparing the sample solution from the sample of bird's nest before the sample is identified.

13. The method of claim 12, wherein the sample solution is prepared using an aqueous solvent.

14. The method of claim 13, wherein the aqueous solvent is methanol or water.

15. The method of claim 13, wherein the aqueous solvent is a mixture of methanol and water at a volume ratio of 0% to 70%.

16. The method of claim 1 or 12, wherein the method further comprises a step of pre-column derivatization of the sample of bird's nest.

17. The method of claim 16, wherein, The step of pre-column derivatization of the sample of bird's nest comprises derivatization using 2-AA, 2-AB, PMP, 2-AP, ABP, ABME, HOA, AMAC, 3-(acetylamino)-6-amino-acridine (AA-Ac), ANTS, phenylhydrazine or dansylhydrazine.

18. The method of claim 16, wherein, The step of pre-column derivatization of the sample of bird's nest comprises derivatization using ABEE.

19. The method of claim 1, wherein the method comprises the following steps: (1) preparing a sample solution; (2) derivatizing the sample solution of step (1) with ABEE; (3) separating and detecting the derivatized sample using liquid chromatography-mass spectrometry to obtain the retention time and mass-to-charge ratio of the sample; (4) comparing the retention time and mass-to-charge ratio of the sample with the markers to identify the sample.

20. The method of claim 1, comprising: a. detecting the carbohydrates in the sample of bird's nest; b. identifying the oligosaccharide markers of the carbohydrates in the sample of bird's nest; wherein the sample of bird's nest is pre-column derivatized with ABEE; the detection method is liquid chromatography-mass spectrometry, wherein the chromatographic conditions are: using a C18 octadecyl-bonded silica gel liquid chromatography column; the mobile phase of the liquid chromatography is: 0.1% to 0.5% formic acid aqueous solution and 0.1% to 0.5% formic acid acetonitrile solution; the mass spectrometry is Q-TOF mass spectrometry.

21. The method of claim 1, comprising the following steps: (1) preparing a sample solution, wherein the sample solution is used directly in the subsequent steps without derivatization; (2) separating and detecting the sample using liquid chromatography-mass spectrometry to obtain the retention time of the sample; (3) comparing the retention time and / or mass-to-charge ratio of the sample with the markers to identify the sample.

22. The method of claim 1, comprising: a. detecting the carbohydrates in the sample of bird's nest; b. identifying the oligosaccharide markers of the carbohydrates in the sample of bird's nest; wherein the sample of bird's nest is not derivatized; the detection method is liquid chromatography-mass spectrometry, wherein the chromatographic conditions are: using an amido-bonded silica gel chromatography column; the mobile phase of the liquid chromatography is: 0.1% to 0.5% formic acid aqueous solution and 0.1% to 0.5% formic acid acetonitrile solution; The mass spectrometer is a QqQ-MS / MS mass spectrometer.

23. The method of claim 17-20, wherein the structure of the ABEE-labeled oligosaccharide marker is B: 。 24. The method of claim 1, wherein the bird's nest sample is a bird's nest raw material or a bird's nest product.

25. The method of claim 24, wherein the bird's nest raw material is selected from the group consisting of bird's nest cup, bird's nest horn, bird's nest silk, bird's nest cake, bird's nest shred, or bird's nest strip.

26. The method of claim 24, wherein the bird's nest raw material is selected from the group consisting of white bird's nest cup, yellow bird's nest cup, blood bird's nest cup, or hair bird's nest cup.

27. The method of claim 24, wherein the bird's nest product is selected from the group consisting of instant bird's nest, thick bird's nest, or bird's nest drink.

28. Use of the oligosaccharide marker of any one of claims 1-27 in bird's nest identification.

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