Application of hydrazide compounds as derivatization reagents

Abstract

The present invention discloses the application of a hydrazide compound as a derivatization reagent, specifically discloses an isotope-labeled substance or a salt thereof of the compound shown in Formula I, a preparation method of the isotope-labeled substance of the compound shown in Formula I, a composition for detecting and / or separating Substance X, and the application of Substance A as a derivatization reagent. The compound synthesis method of the present invention is simple, the reagents are cheap and easily available, and it can simultaneously detect a variety of carbonyl-containing compounds (including compounds containing one or more of carboxyl, aldehyde carbonyl and ketone carbonyl). When used as a derivatization reagent, it has the advantages of high coverage, high sensitivity and low quantification limit. #imgabs0#

Description

Technical Field

[0001] The present invention relates to the field of quantitative and in vitro diagnostic analysis of compounds containing one or more of carboxyl, aldehyde carbonyl and ketone carbonyl in biological samples, and particularly relates to the application of hydrazide compounds as derivatization reagents. Background Art

[0002] As important small molecule metabolites, carboxyl metabolites participate in multiple metabolic processes in organisms and play a key role. The changes in their types and contents in organisms are closely related to the occurrence and development of various diseases. Therefore, highly covered and highly sensitive quantitative analysis of such metabolites is of great significance for the study of their physiological functions, in vitro diagnosis of diseases, etc. Ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) combines the high separation ability of chromatography with the high sensitive quantitative ability of mass spectrometry, and is currently an effective means widely used for the quantitative analysis of metabolites in complex biological matrices. However, since most carboxyl metabolites are hydrophilic metabolites and are difficult to retain on reverse-phase chromatography, the negative ion detection mode of mass spectrometry is mostly used at present, resulting in low quantitative sensitivity and being unable to meet the quantitative requirements for low-concentration carboxyl metabolites in biological samples. In recent years, the proposal of probe sensitization technology based on stable isotope labeling has provided a solution to the problems existing in the quantitative process of carboxyl metabolites. Currently, the commonly used derivatization reagents for carboxyl metabolites mainly include amino, hydrazino and active halogen reagents, which have made certain progress in improving the hydrophobicity of metabolites and enhancing the sensitivity, but there are still problems such as very limited coverage and most of them do not investigate the separation and quantification of chiral carboxylic acid enantiomers. Therefore, there is an urgent need to develop a quantitative analysis method for carboxyl metabolites with high coverage, high sensitivity and capable of separating chiral carboxylic acids. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide the application of hydrazide compounds as derivatization reagents in view of the defects of low detection coverage, low sensitivity and high quantitative limit of existing derivatization reagents for carboxyl metabolites. The synthesis method of the hydrazide compounds of the present invention is simple, the reagents are cheap and easily available, and it can detect a variety of carbonyl-containing compounds (including compounds containing one or more of carboxyl, aldehyde carbonyl and ketone carbonyl), and has high coverage, high sensitivity and low quantitative limit.

[0004] The present invention provides an isotope-labeled substance or a salt thereof (referring to a salt of an isotope-labeled substance of the compound shown in Formula I) of the compound shown in Formula I,

[0005]

[0006] wherein, the carbon atom with "*" represents a chiral carbon atom and exists in the form of R configuration, S configuration, or a mixture of R configuration and S configuration. For example, the compound shown in Formula I is

[0007] Among them, in the isotope-labeled compound of the compound represented by Formula I, the isotope abundances of one or more atoms are different from their natural abundances.

[0008] For example, at least one 1 H in the compound represented by Formula I is 2 replaced by

[0009] For example, at least one 12 C in the compound represented by Formula I is replaced by its heavier isotope 13 C.

[0010] For example, at least one 14 N in the compound represented by Formula I is replaced by its heavier isotope 15 N.

[0011] For example, at least one 16 O in the compound represented by Formula I is replaced by its heavier isotope 18 O.

[0012] Preferably, the isotope-labeled compound of the compound represented by Formula I is any one of the following compounds:

[0013]

[0014]

[0015]

[0016]

[0017]

[0018]

[0019]

[0020]

[0021] Among them, the salt of the isotope-labeled compound of the compound represented by Formula I is the salt formed by the isotope-labeled compound of the compound represented by Formula I and hydrochloric acid. Preferably, it is the salt formed by the isotope-labeled compound of the compound represented by Formula I and hydrochloric acid in a molar ratio of 1:1.

[0022] The present invention also provides a preparation method of an isotope-labeled compound of a compound represented by Formula I, which includes the following steps:

[0023] In a solvent and in the presence of a base, the "compound of formula II or a salt thereof" reacts with the compound of formula III and hydrazine hydrate in the coupling reaction shown below to form an isotope-labeled compound of formula I, and that's it.

[0024]

[0025] Among them, the carbon atom with "*" represents a chiral carbon atom and exists in the form of R configuration, S configuration, or a mixture of R configuration and S configuration.

[0026] Among the compound of formula II, the compound of formula III, or hydrazine hydrate, the isotope abundance of at least one atom is different from its natural abundance.

[0027] Among them, in the coupling reaction, the solvent can be selected from one or more of halogenated alkane solvents (such as dichloromethane and / or carbon tetrachloride), benzene solvents (such as toluene), and ether solvents (such as tetrahydrofuran and / or diethyl ether, more preferably anhydrous tetrahydrofuran with molecular sieve), and is preferably an ether solvent.

[0028] Among them, in the coupling reaction, the base can be an inorganic base and / or an organic base. The organic base can be triethylamine and / or pyridine. The inorganic base can be selected from one or more of carbonates (such as potassium carbonate and / or sodium carbonate, and again such as potassium carbonate), bicarbonates (such as sodium bicarbonate), and hydroxides (such as sodium hydroxide and / or potassium hydroxide), and is preferably a carbonate.

[0029] Among them, in the coupling reaction, the salt of the compound of formula II can be the hydrochloride salt of the compound of formula II.

[0030] Among them, in the coupling reaction, the molar ratio of the base to the "compound of formula II or a salt thereof" can be 2:1 - 10:1, such as 3:1.

[0031] Among them, in the coupling reaction, the molar ratio of the compound of formula III to the "compound of formula II or a salt thereof" can be 0.5:1 - 5:1, such as 0.9:1.

[0032] Among them, in the coupling reaction, the molar ratio of hydrazine hydrate to the "compound of formula II or a salt thereof" can be 10:1 - 500:1, such as 100:1.

[0033] Among them, in the coupling reaction, the molar concentration of the "compound of formula II or a salt thereof" in the solvent can be 0.001 - 10 mol / L, such as 0.06 mol / L.

[0034] Among them, in the coupling reaction, the feeding order of each material can be adding the compound shown in Formula III to the "compound shown in Formula II or its salt" and a base (preferably dropwise adding under an ice bath condition, and then reacting at room temperature for 30 minutes), and then adding hydrazine hydrate and an alcohol solvent (the alcohol solvent is preferably methanol, and preferably reacting at room temperature for 1 hour after adding).

[0035] Among them, the coupling reaction further includes the following post-treatment steps: removing the solvent (preferably by rotary evaporation), extraction (preferably extracting with dichloromethane), washing (preferably washing with saturated sodium carbonate solution), drying (preferably drying with anhydrous magnesium sulfate), filtering, removing the solvent (preferably by rotary evaporation), and freeze-drying.

[0036] The present invention also provides a composition for detecting and / or separating substance X, which includes:

[0037] Component A and a condensing agent;

[0038] The Component A is: (1) a compound shown in Formula I or its salt, and / or (2) an isotope-labeled substance of the compound shown in Formula I above or a salt of an isotope-labeled substance of the compound shown in Formula I;

[0039]

[0040] The carbon atom with "*" represents a chiral carbon atom, and exists in the form of R configuration, S configuration, or a mixture of R configuration and S configuration;

[0041] The substance X is a carbonyl-containing compound (referring to a compound containing one or more of a carboxyl group, an aldehyde carbonyl group, and a ketone carbonyl group).

[0042] Among them, the substance X is preferably a carboxyl-containing compound, and preferably, the carboxyl-containing compound is a small-molecule carboxyl compound, such as a carboxyl compound with a molecular weight less than 1000 Da; for example, a carboxyl metabolite, and more preferably, a carboxyl metabolite in human plasma, human urine, human feces, or human-derived cell A549.

[0043] Among them, the condensing agent is a condensing agent conventionally used in the art for the condensation of carboxyl groups and amino groups to undergo an acid amide reaction, such as selected from one or more of 2-(7-azabenzotriazol)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU), 2-(7-azabenzotriazol)-tetramethylurea hexafluorophosphate (HBTU), 6-chlorobenzotriazol-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU), O-benzotriazol-N,N,N',N'-tetramethylurea tetrafluoroborate (TBTU), 2-succinimidyloxy-1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU), 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (DMTMM), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), triphenylphosphine (TPP), and 1-hydroxy-7-azabenzotriazole (HOAT).

[0044] Among them, the molar ratio of the condensing agent to component A can be 1:10 - 10:1, such as 22:40.

[0045] Furthermore, the condensing agent is preferably EDC, HOAT, or "a combination of EDC and HOAT".

[0046] In the combination of EDC and HOAT, the molar ratio of EDC to component A can be 100:1 - 1:100, such as 20:40; the molar ratio of HOAT to component A can be 100:1 - 1:100, such as 2:40.

[0047] Among them, when component A is the compound shown in Formula I above and / or the isotope-labeled substance of the compound shown in Formula I above; the composition further comprises an acid, and the acid is preferably hydrochloric acid, more preferably 1M hydrochloric acid.

[0048] Preferably, the composition is any of the following compositions:

[0049] A) The composition consists of the following components: component A; and a condensing agent;

[0050] B) The composition consists of the following components: component A; a condensing agent; and an acid.

[0051] The present invention also provides the use of substance A as a derivatizing agent for detecting and / or separating substance X,

[0052] The substance A is (i) the compound shown in Formula I above or its salt; (ii) the composition above; or (iii) the isotope-labeled substance of the compound shown in Formula I above or its salt;

[0053] The substance X is a carbonyl-containing compound (referring to a compound containing one or more of carboxyl, aldehyde carbonyl, and ketone carbonyl).

[0054] Preferably, the application includes the following steps:

[0055] (1) The substance A reacts with the sample to obtain a derivatized product; the sample includes the substance X;

[0056] (2) Using liquid chromatography-mass spectrometry, the derivatized product is separated and / or detected, and that's it.

[0057] Among them, when the substance A is the "compound or its salt as shown in Formula I" or the "isotope-labeled compound or its salt as shown in Formula I", the above condensing agent can also be added in step (1).

[0058] Among them, the derivatization reaction can be carried out under acidic conditions, and the acidic condition is preferably hydrochloric acid, more preferably 1M hydrochloric acid. The volume ratio of the hydrochloric acid to the reaction system of the derivatization reaction can be 0.5 - 10%, for example, 4.5%.

[0059] Among them, the derivatization reaction is carried out in a solvent, and the solvent is preferably a nitrile solvent (such as acetonitrile) and water, and more preferably acetonitrile and water with a volume ratio of 1:1.

[0060] Among them, in the substance A, the molar ratio of the "compound or its salt as shown in Formula I" or the "isotope-labeled compound or its salt as shown in Formula I" to the reactive groups (one or more of ketone carbonyl, aldehyde carbonyl, and carboxyl) in the sample can be 10:1 - 500:1, for example, 200:1.

[0061] Among them, the reaction temperature of the derivatization reaction can be 20 - 80 °C, for example, 37 °C.

[0062] Among them, the reaction time of the derivatization reaction can be 10 - 120 minutes, for example, 60 minutes.

[0063] Among them, in the liquid chromatography-mass spectrometry, the chromatographic conditions of liquid chromatography are preferably: the chromatographic column is a reverse-phase chromatographic column (such as Agilent ZORBAX Eclipse Plus C18 chromatographic column); the column temperature is 40 to 55 degrees Celsius (for example, 50 degrees Celsius), the mobile phases A and B are water containing 0.005% - 0.5% formic acid and acetonitrile respectively, or water and methanol respectively (preferably water containing 0.1% formic acid and acetonitrile respectively), the flow rate is 0.2 to 0.5 milliliters per minute (for example, 0.5 milliliters per minute), the injection volume is 0.5 to 5 microliters (for example, 1 microliter), and gradient elution is carried out.

[0064] Preferably, the gradient of the gradient elution is expressed as a percentage of mobile phase B and may be: 0 - 4 minutes, 8%; 4 - 7 minutes, 8 - 20%; 7 - 11 minutes, 20 - 30%; 11 - 15 minutes, 30 - 50%; 15 - 18 minutes, 50 - 95%; 18 - 19 minutes, 95 - 100%.

[0065] Alternatively, the gradient of the gradient elution is expressed as a percentage of mobile phase B and may also be: 0 - 5 minutes, 2 - 7%; 5 - 6 minutes, 7%; 6 - 7 minutes, 7 - 8%; 7 - 12 minutes, 8 - 15%; 12 - 24 minutes, 15 - 21%; 24.1 - 27 minutes, 95%.

[0066] Among them, in the liquid chromatography - mass spectrometry technology, the analysis conditions of the mass spectrometry can adopt the multiple reaction monitoring mode in the positive ion mode. Further, it can also be: the ion source temperature is 350 to 550 degrees Celsius (for example, 450 degrees Celsius), the ionization voltage is 3500 to 5500 volts (for example, 3500 volts), the spray gas pressure is 40 to 55 psi (for example, 35 psi), and the pressure of the auxiliary heating gas is 40 to 60 psi (for example, 50 psi).

[0067] On the basis of conforming to the common knowledge in the art, the above - mentioned preferred conditions can be combined arbitrarily to obtain various preferred examples of the present invention.

[0068] The reagents and raw materials used in the present invention are all commercially available.

[0069] The positive and progressive effects of the present invention are as follows:

[0070] 1. The method for preparing the isotope - labeled compound of the hydrazide compound provided by the present application is simple, the reagents used are cheap and easily available, the reaction conditions are mild, and it can be obtained through simple extraction post - treatment, and it has excellent solubility in conventional solvents;

[0071] 2. The present application for the first time uses the compound of formula I, its isotope - labeled compound or their salts as derivatization reagents for separating and / or detecting carbonyl - containing compounds (referring to compounds containing one or more of carboxyl, aldehyde carbonyl and ketone carbonyl) in liquid chromatography - mass spectrometry. The reagent and the composition formed by it and the condensing agent have high derivatization reaction efficiency with carbonyl - containing compounds. Specifically, it has the same derivatization efficiency and mass spectrometry response for the R - / S - enantiomers of chiral carboxyl compounds. When two pure enantiomers cannot be obtained, one of the enantiomers or a mixed racemate can be used as an analytical reference for quantitative analysis; the chromatographic retention rules of chiral carboxyl compounds can help identify R - / S - enantiomers in the absence of optically pure reference substances.

[0072] 3. The isomers and enantiomers of chiral carboxyl compounds including chiral α-amino acids, chiral α-hydroxy acids, and chiral α-methyl acids can be separated under conventional reverse-phase chromatography conditions by the compound of formula I provided in this application, its isotope-labeled compound, or their salts and the compositions formed by them with condensing agents, so that quantitative analysis can be carried out on them, and the limit of quantification is 0.003 - 8.457 fmol (10 -15 mol).

[0073] 4. The quantitative analysis method established based on the compound of formula I provided in this application, its isotope-labeled compound, or their salts and the compositions formed by them with condensing agents is simple and rapid, and can complete the detection and quantitative analysis of 267 carboxyl compounds within 18 minutes, which is suitable for high-throughput analysis of large samples.

[0074] 5. Using the isotope-labeled form of the derivatizing reagent of this application, it can not only be used as an internal standard to assist in absolute quantification, but also be used to label different samples to achieve the detection of multiple samples in a single injection.

[0075] 6. The quantitative method established in this application can also achieve the quantitative detection of creatinine, and this analysis method can cover the high concentration of creatinine in human urine. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 It is the optimization of the SBPH derivatization conditions for 12 representative carboxyl compounds; among them; (a) screening of the amount of acid; (b - c) screening of the molar ratio of the condensing reagent to the reactive group of the analyte; (d) screening of the molar ratio of SBPH to the reactive group of the analyte; (e) reaction temperature; (f) reaction time.

[0077] Figure 2 It is to optimize the amount of the derivatizing reagent in urine samples.

[0078] Figure 3 It is the UPLC-MS / MS chromatogram of a single chiral enantiomer of a representative carboxyl compound labeled with SBPH.

[0079] Figures 4 - 7 They are respectively the UPLC-MS / MS chromatograms of some compounds among 267 carboxyl compounds.

[0080] Figures 8 - 11 They are respectively the UPLC-MS / MS chromatograms of carboxyl compounds with the same mass-to-charge ratio.

[0081] Among them, Figure 8The abbreviations in each represent: m / z 304 (A1: isobutyric acid; A2: butyric acid), m / z 318 (B1: acetoacetic acid; B2: R-2-methylbutyric acid; B3: S-2-methylbutyric acid; B4: isovaleric acid; B5: valeric acid), m / z 328 (C1: 2-furoic acid; C2: citraconic acid; C3: sorbic acid), m / z 358 (D1: 5-(hydroxymethyl)-2-furoic acid; D2: 2-octenoic acid), m / z 372 (E1: orotic acid; E2: 3-chlorobenzoic acid; E3: 3-nonenoic acid; E4: 2-nonenoic acid), m / z 381 (F1: R-phenylalanine; F2: S-phenylalanine), m / z 386 (G1: gallic acid; G2: 4-chlorophenylacetic acid), m / z 388 (H1: 4-ethyl octanoic acid; H2: 4-methyl nonanoic acid; H3: capric acid), m / z 397 (I1: R-tyrosine; I2: S-tyrosine), m / z 398 (J1: 3-(3-hydroxyphenyl)-3-hydroxypropionic acid; J2: p-hydroxyphenyllactic acid; J3: homovanillic acid; J4: isohomovanillic acid; J5: 2,6-dimethoxybenzoic acid), m / z 400 (K1: 3,4-dihydroxymandelic acid; K2: 10-undecenoic acid), m / z 404 (L1: 3-hydroxydecanoic acid; L2: S-2-hydroxydecanoic acid; L3: R-2-hydroxydecanoic acid), m / z 460 (M1: 3-hydroxytetradecanoic acid; M2: 2-hydroxytetradecanoic acid), m / z 535 (N1: malonic acid; N2: 3-hydroxypyruvic acid), m / z 547 (O1: fumaric acid; O2: maleic acid; O3: levulinic acid; O4: 2-pentanone acid; O5: 2-ketoisovaleric acid), m / z 577 (P1: adipic acid; P2: 3-methylglutaric acid; P3: 2-ketoglutaric acid), m / z 578 (Q1: R-glutamic acid; Q2: S-glutamic acid), m / z 591 (R1: pimelic acid; R2: 3-methyladipic acid);

[0082] Figure 9The abbreviations in each represent: m / z 339 (AA1: nicotinic acid; AA2: 2-pyridinecarboxylic acid), m / z 346 (AB1: 2-ethylvaleric acid; AB2: 3-ethylvaleric acid; AB3: R-2-methylhexanoic acid; AB4: S-2-methylhexanoic acid; AB5: 4-methylhexanoic acid; AB6: 5-methylhexanoic acid; AB7: heptanoic acid), m / z 347 (AC1: S-isoleucine; AC2: S-leucine), m / z 355 (AD1: 6-hydroxynicotinic acid; AD2: 3-hydroxy-2-pyridinecarboxylic acid), m / z 365 (AE1: R-methionine; AE2: S-methionine), m / z 368 (AF1: 4-hydroxyphenylacetic acid; AF2: 4-hydroxy-3-methylbenzoic acid; AF3: mandelic acid; AF4: 3-methoxybenzoic acid), m / z 369 (AG1: 3-aminosalicylic acid; AG2: 3-hydroxy-2-aminobenzoic acid), m / z 380 (AH1: 4-hydroxycinnamic acid; AH2: 3-hydroxycinnamic acid; AH3: 2-hydroxycinnamic acid; AH4: 3-phenylbutyric acid; AH5: phenylbutyric acid), m / z 384 (AI1: 4-hydroxymandelic acid; AI2: homogentisic acid; AI3: 3,4-dihydroxyphenylacetic acid; AI4: vanillic acid; AI5: 5-methoxysalicylic acid), m / z 369 (AG1: 3-aminosalicylic acid; AG2: 3-hydroxy-2-aminobenzoic acid), m / z 380 (AH1: 4-hydroxycinnamic acid; AH2: 3-hydroxycinnamic acid; AH3: 2-hydroxycinnamic acid; AH4: 3-phenylbutyric acid; AH5: phenylbutyric acid), m / z 384 (AI1: 4-hydroxymandelic acid; AI2: homogentisic acid; AI3: 3,4-dihydroxyphenylacetic acid; AI4: vanillic acid; AI5: 5-methoxysalicylic acid), m / z 410 (AJ1: p-aminohippuric acid; AJ2: trans-ferulic acid), m / z 420 (AK1: R-tryptophan; AK2: S-tryptophan), m / z 561 (AL1: pentenedioic acid; AL2: mesaconic acid; AL3: itaconic acid; AL4: 2-ketoisoleucine; AL5: 2-ketoisocaproic acid; AL6: 2-oxohexanoic acid), m / z 563 (AM1: glutaric acid; AM2: 2-methylsuccinic acid; AM3: ethylmalonic acid), m / z 581 (AN1: tartaric acid; AN2: thiodiacetic acid; AN3: benzoylformic acid), m / z 605 (AO1: isocitric acid; AO2: citric acid);

[0083] Figure 10The abbreviations in each represent: m / z 306 (BA1: 3-hydroxypropionic acid; BA2: lactic acid; BA3: methoxyacetic acid), m / z 316 (BB1: 4-pentenoic acid; BB2: 3,3-dimethylacrylic acid), m / z 330 (BC1: 2-methyl-4-pentenoic acid; BC2: 2-methyl-2-pentenoic acid), m / z 331 (BD1: R-proline; BD2: S-proline), m / z 333 (BE1: guanidine acetate; BE2: R-valine; BE3: S-valine; BE4: N-acetylglycine), m / z 350 (BF1: S-2,3-dihydroxyisovaleric acid; BF2: R-2,3-dihydroxyisovaleric acid), m / z 353 (BG1: 3-pyridineacetic acid; BG2: m-aminobenzoic acid; BG3: p-aminobenzoic acid), m / z 354 (BH1: 4-hydroxybenzoic acid; BH2: 3-hydroxybenzoic acid; BH3: 2-hydroxybenzoic acid), m / z 356 (BI1: imidazolepropionic acid; BI2: 3-(2-furan)propionic acid), m / z 360 (BJ1: 4-hydroxycyclohexanecarboxylic acid; BJ2: valproic acid; BJ3: R-2-methylheptanoic acid; BJ4: S-2-methylheptanoic acid; BJ5: octanoic acid), m / z 370 (BK1: 3,4-dihydroxybenzoic acid; BK2: 2,5-dihydroxybenzoic acid; BK3: 2,6-dihydroxybenzoic acid), m / z 396 (BL1: nicotinoylglycine; BL2: 3-(3-methoxyphenyl)propionic acid), m / z 405 (BM1: kynurenic acid; BM2: 3-indolepropionic acid; BM3: 1-methyl-3-indoleacetic acid), m / z 409 (BN1: 5,6-dihydroxy-2-indolecarboxylic acid; BN2: phenylacetylglycine; BN3: succinanilic acid), m / z 411 (BO1: p-hydroxymandelic acid; BO2: 3-hydroxymandelic acid; BO3: 5-acetylaminosalicylic acid; BO4: S-α-hydroxymandelic acid; BO5: R-α-hydroxymandelic acid; BO6: 2-hydroxymandelic acid), m / z 421 (BP1: xanthurenic acid; BP2: indole-3-lactic acid), m / z 549 (BQ1: succinic acid; BQ2: methylmalonic acid), m / z 587 (BR1: 2,5-furandicarboxylic acid; BR2: cis-aconitic acid), m / z 607 (BS1: 3-isopropylmalic acid; BS2: 2-isopropylmalic acid);

[0084] Figure 11The abbreviations in each represent: m / z 319 (CA1: R-2-aminobutyric acid; CA2: S-2-aminobutyric acid), m / z 320 (CB1: 3-hydroxybutyric acid; CB2: 3-hydroxyisobutyric acid; CB3: 2-hydroxyisobutyric acid; CB4: S-2-hydroxybutyric acid; CB5: R-2-hydroxybutyric acid), m / z 332 (CC1: 2-ethylbutyric acid; CC2: 2-methylvaleric acid; CC3: 3-methylvaleric acid; CC4: 4-methylvaleric acid; CC5: hexanoic acid), m / z 334 (CD1: 3-hydroxyisovaleric acid; CD2: 3-hydroxypentanoic acid; CD3: S-2-hydroxy-2-methylbutyric acid; CD4: R-2-hydroxy-2-methylbutyric acid; CD5: S-2-hydroxyisovaleric acid; CD6: R-2-hydroxyisovaleric acid; CD7: 2-hydroxypentanoic acid), m / z 335 (CE1: R-threonine; CE2: S-threonine), m / z 345 (CF1: R-pyroglutamic acid; CF2: S-pyroglutamic acid), m / z 348 (CG1: asparagine; CG2: 6-hydroxyhexanoic acid; CG3: 3-hydroxyhexanoic acid; CG4: 2-hydroxy-3-methylvaleric acid; CG5: S-2-hydroxyisohexanoic acid; CG6: R-2-hydroxyisohexanoic acid; CG7: S-2-hydroxyhexanoic acid; CG8: R-2-hydroxyhexanoic acid), m / z 362 (CH1: lysine; CH2: glutamine; CH3: ureidoisobutyric acid), m / z 366 (CI1: 3-phenylpropionic acid; CI2: p-methylphenylacetic acid), m / z 376 (CJ1: 3-hydroxyoctanoic acid; CJ2: S-2-hydroxyoctanoic acid; CJ3: R-2-hydroxyoctanoic acid), m / z 382 (CK1: p-hydroxycinnamic acid; CK2: (4-hydroxyphenyl)-2-propionic acid; CK3: 3-(3-hydroxyphenyl)propionic acid; CK4: 3-(2-hydroxyphenyl)propionic acid; CK5: phenyl lactic acid; CK6: 3-methoxyphenylacetic acid), m / z 564 (CL1: iminodiacetic acid; CL2: R-aspartic acid; CL3: S-aspartic acid), m / z 579 (CM1: 3-hydroxyglutaric acid; CM2: 2-hydroxyglutaric acid; CM3: 3-methylmalic acid; CM4: citramalic acid).

[0085] Figure 12 UPLC-MS / MS chromatograms of 30 pairs of enantiomers of chiral carboxyl compounds labeled with SBPH. Detailed implementation mode

[0086] The present invention will be further described below by way of examples, but the present invention is not limited to the scope of the described examples. For the experimental methods without specific conditions in the following examples, they are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0087] Experimental Materials: All carboxyl compound standard chemicals were purchased from commercial reagent companies, see Table 1 for details.

[0088] Anhydrous tetrahydrofuran used in the experiment was purchased from Adamas; methanol was purchased from Merck; and creatinine was purchased from Maclean.

[0089] Hydrazine hydrate, potassium carbonate, sodium carbonate, anhydrous magnesium sulfate, and dichloromethane were purchased from Aladdin Company.

[0090] 1-Hydroxy-7-azabenzotriazole (HOAT) was purchased from Sinopharm Chemical Reagent Company.

[0091] Mass spectrometry-grade formic acid, chromatography-grade acetonitrile, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), L-proline methyl ester, benzoyl chloride, and benzoyl chloride-d5 were purchased from Sigma.

[0092] Deuterated methanol (CD3OD) and creatinine-d3 were purchased from Cambridge Isotope Laboratories (USA).

[0093] Ultrapure water used for sample treatment and chromatographic mobile phase was purchased from Milli-Q system (Millipore, USA).

[0094] Biological samples: Human plasma, urine, and fecal samples were obtained from healthy Chinese adult volunteers recruited through the Human Phenotype Project and provided informed consent, as approved by the Fudan University Ethics Committee (PE21087). Once obtained using standard clinical sampling procedures, samples were immediately snap-frozen in liquid nitrogen and stored at −80°C until analysis. Human lung cancer A549 cells were obtained from Shanghai Pulmonary Hospital (Shanghai, China).

[0095] Experimental Instruments: One-dimensional proton spectra of SBPH and SBPH-d5 were performed on a Bruker 600 MHz superconducting nuclear magnetic resonance spectrometer. The data were acquired and processed using Bruker TopSpin 3.6.0 software. High-resolution mass spectrometry was performed on a Thermo OrbitrapExploris 240, Sciex X500R, or ZenoTOF 7600 mass spectrometer for SBPH, SBPH-d5, and their derivatives. Quantitative analysis was performed on a Sciex QTRAP 6500. + The quantification was performed on a mass spectrometer equipped with an electrospray ionization (ESI) source coupled to a Shimadzu LC-30AD UPLC system.

[0096] Pretreatment of biological samples: Mix 20 μL of human plasma or urine with 120 μL of pre-cooled acetonitrile (-20 °C), ultrasonicate in an ice bath for 5 min, and then centrifuge at 4 °C and 14,000 rpm for 10 min in a cryogenic centrifuge to obtain the supernatant.

[0097] Mix the cell sample (about 20 mg) with 100 μL of water and 600 μL of pre-cooled acetonitrile, freeze-thaw three times, ultrasonicate in an ice bath for 5 min, and then centrifuge at 4 °C and 14,000 rpm for 10 min in a cryogenic centrifuge to obtain the supernatant.

[0098] The fecal sample needs to be divided into two parallel samples. One is freeze-dried to measure its water content, and the other is used for detection and analysis. The pretreatment method of the latter (about 20 mg) is the same as that of the cell sample.

[0099] Preparation of standard stock solutions and mixed stock solutions: Dissolve the commercially purchased carboxyl compound standard chemicals in an acetonitrile-water (volume ratio 1:1) solution to obtain the standard stock solutions of each carboxyl compound with the concentrations shown in Table 1. Mix the standard stock solutions of each carboxyl compound according to the volume amounts shown in Table 1 to obtain a mixed stock solution of 268 carboxyl compounds with a total concentration of about 30 mM. Among them, 12 chiral carboxyl compound standard chemicals exist in the form of a mixture of R and S configurations, corresponding to 24 chiral carboxyl compounds of R or S configurations respectively. Therefore, the mixed stock solution of these 268 carboxyl compounds contains a total of 280 non-chiral and different chiral configuration carboxyl compounds.

[0100] Dissolve creatinine and creatinine-d3 in an acetonitrile-water (volume ratio 5:95) solution to make their concentrations reach 210 mM.

[0101] Preparation of mixed working solution L1 and internal standard working solution: Take 80 μL of the mixed stock solution, 20 μL of the creatinine solution, and 500 μL of acetonitrile-water (volume ratio 6:1) and mix them to obtain the mixed working solution L1. Take 80 μL of the mixed stock solution, 20 μL of the creatinine-d3 solution, and 500 μL of acetonitrile-water (volume ratio 6:1) solution and mix them to obtain the internal standard working solution.

[0102] Preparation of working solutions: Gradually dilute the mixed working solution L1 by dilution factors of 2, 5, 10, 25, 50, 125, 250, 625, 1250, 3125, 6250, 15625, 31250, 78125, 156250 to obtain 16 working solutions with concentration gradients of L1-L16 for drawing the standard curve, and select L7, L5, and L3 to represent the quality control products at low, medium, and high concentration levels respectively.

[0103] Table 1 Carboxyl compound standard chemicals, their sources, and the concentrations of the prepared stock solutions

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113] Example 1: Preparation of S-Benzoylprolyl Hydrazide and S-Benzoylprolyl Hydrazide-d5

[0114] S - Preparation of Benzoylprolyl Hydrazide (SBPH): Weigh S-proline methyl ester hydrochloride (331 mg, 2 mmol) and ground potassium carbonate powder (829 mg, 6 mmol) into a 100 mL round-bottom flask, and add anhydrous tetrahydrofuran with molecular sieve (20 mL) as the reaction solvent. Place the round-bottom flask in an ice bath, slowly add benzoyl chloride (207 μL, 1.8 mmol), and react at room temperature for 30 minutes after the addition is complete. Then add methanol (12 mL) and hydrazine hydrate (12.2 mL, 200 mmol), and react at room temperature for 1 hour. Rotavapor to remove the solvent, extract the product with dichloromethane (60 mL) three times, wash it with saturated sodium carbonate solution three times, combine the organic layers, dry over anhydrous magnesium sulfate, filter, rotavapor to remove the solvent, and lyophilize to obtain S-benzoylprolyl hydrazide (SBPH). Use 1H NMR and high-resolution mass spectrometry to identify its structure, and the results are as follows:

[0115] 1 H NMR(600MHz,CD3OD)δ / ppm 7.61-7.56(m,2H),7.51-7.38(m,3H),4.51(dd,J=8.2,6.0Hz,1H),3.66(ddd,J=10.5,7.1,4.5Hz,1H),3.52(ddd,J=10.3,7.3,4.7Hz,1H),2.33-2.26(m,1H),2.02-1.95(m,2H),1.91-1.83(m,1H).

[0116] HRMS-ESI(+),[M+H] +, m / z, calculated value: 234.1237, measured value: 234.1229.

[0117] S - Preparation of Benzoylprolyl Hydrazide - d5 (SBPH - d5): The synthesis method of S-benzoylprolyl hydrazide-d5 (SBPH-d5) is similar to that of SBPH, and only benzoyl chloride needs to be replaced with benzoyl chloride-d5. Its structure was identified by 1H NMR and high-resolution mass spectrometry, and the results are as follows:

[0118] 1 H NMR (600 MHz, CD3OD) δ / ppm 4.51 (dd, J = 8.2, 6.1 Hz, 1H), 3.66 (ddd, J = 10.5, 7.1, 4.5 Hz, 1H), 3.52 (ddd, J = 10.3, 7.3, 4.7 Hz, 1H), 2.34 - 2.27 (m, 1H), 2.08 - 1.93 (m, 2H), 1.91 - 1.81 (m, 1H).

[0119] HRMS-ESI(+), [M+H] + , m / z, calculated value: 239.1551, measured value: 239.1562.

[0120] Screening of derivatization conditions and verification of derivatization efficiency in Example 2

[0121] (1) Preparation of reagent solutions required for derivatization:

[0122] Preparation of EDC and HOAT solutions: Dissolve EDC and HOAT in acetonitrile-water (volume ratio 1:1) respectively to make their concentrations reach 1000 mM and 105 mM, and dilute with acetonitrile-water (volume ratio 1:1) solution to obtain the required concentrations below.

[0123] Preparation of hydrochloric acid solution: Slowly pour 10 mL of concentrated hydrochloric acid (12 M) into 110 mL of water to obtain 1 M hydrochloric acid.

[0124] Preparation of SBPH and SBPH-d5 solutions: Dissolve SBPH and SBPH-d5 prepared in Example 1 in acetonitrile-water (volume ratio 1:1) respectively to prepare 1500 mM stock solutions for standby, and dilute with acetonitrile-water (volume ratio 1:1) solution to obtain the required concentrations below.

[0125] (2) Screening of derivatization conditions:

[0126] The conditions of derivatization reactions, such as the amount of acid used, the ratio of the condensing agent and derivatization reagent to the analyte, reaction temperature and time, etc., were systematically optimized using representative carboxyl compounds (leucine and tryptophan representing amino acids, 2-(4-hydroxyphenyl)propionic acid and 2-methylvaleric acid representing methyl acids, 2-hydroxyisovaleric acid and mandelic acid representing hydroxy acids, citramalic acid and 2-methylsuccinic acid representing polycarboxylic acids, pyruvic acid and phenylpyruvic acid representing keto acids, vanillic acid and 3-hydroxybenzoic acid representing aromatic acids) (see Figure 1 ). Among them, hydrazide compounds can not only undergo acid amide condensation with carboxyl groups under the action of a condensing agent, but also directly undergo oximation reactions with carbonyl groups. The specific screening operations and results are as follows:

[0127] (a) Selection of the amount of hydrochloric acid used:

[0128] The standard stock solutions of the above representative carboxyl compounds were diluted to 0.10 mM with an acetonitrile-water (volume ratio 1:1) solution, and then 10 μL of each 0.10 mM solution of the above representative carboxyl compounds were taken and mixed to obtain a representative carboxyl compound mixed solution.

[0129] The optimization range of the hydrochloric acid amount is 0-10% of the total volume of the reaction system.

[0130] Accurately pipette 120 μL of the representative carboxyl compound mixed solution into an EP tube, add 10 μL of 140 mM EDC solution, 10 μL of 35 mM HOAT solution and 13 μL of 480 mM SBPH solution, and add 0, 1, 1.5, 3, 4.5, 6, 10% 1M hydrochloric acid to it respectively. There are five technical replicates for each hydrochloric acid amount. After vortex mixing, incubate at 37 °C for 1 hour (900 rpm). Transfer the above mixture to ice to cool and quench the reaction. The derivatized mixture was determined by liquid chromatography-high resolution mass spectrometry. An Acquity UPLC HSS T3 chromatographic column (2.1×100 mm, 1.8 μm) was used, the column temperature was set at 40 °C, the mobile phases A and B were water and acetonitrile containing 0.1% formic acid respectively, the flow rate was 0.4 mL per minute, and 1 μL was injected. The chromatographic elution gradient is expressed as the percentage of mobile phase B: 0-1 minute, 2%; 1-7 minutes, 2-95%; 7-8 minutes, 95%. The mass spectrometry was recorded in the positive ion mode using an electrospray ionization source, the ion source temperature was 550 °C, the ionization voltage was 5500 V, and the spray gas and auxiliary heating gas were set at 50 psi. Sciex OS software was used for data acquisition and processing. The optimal condition was determined when the average peak area of the carboxyl compound derivative (normalized with the maximum average peak area) reached the maximum. The results showed that when the hydrochloric acid amount was 4.5%, the peak area of the carboxyl compound derivative was the largest. Therefore, 4.5% hydrochloric acid amount was the optimal hydrochloric acid amount and was used for subsequent experiments.

[0131] (b) Selection of the ratio of EDC to the reactive group (carboxyl group) of the analyte:

[0132] The total concentration of carboxyl groups in the representative carboxyl compound mixed solution is approximately 0.117 mM.

[0133] The optimized range of the ratio of EDC to the reactive group of the analyte is 0 - 300 times its molar ratio.

[0134] Accurately pipette 120 μL of the representative carboxyl compound mixed solution into an EP tube, and add 10 μL of 0, 35, 70, 140, 210, 280, 420 mM EDC solutions to it respectively. Then add 10 μL of 35 mM HOAT solution, 13 μL of 480 mM SBPH solution, and 7 μL of hydrochloric acid. Each dosage has five technical replicates. After vortexing and mixing evenly, incubate at 37 °C for 1 hour (900 rpm). Transfer the above mixture to ice to cool and quench the reaction. The subsequent detection conditions are the same as above. The results show that when the ratio of EDC to the reactive group of the analyte is 100:1, the peak area of the carboxyl compound derivative is the largest. Therefore, the ratio of EDC to the reactive group of the analyte being 100:1 is the optimal EDC dosage and is used for subsequent experiments.

[0135] (c) Selection of the ratio of HOAT to the reactive group (carboxyl group) of the analyte:

[0136] The optimized range of the ratio of HOAT to the reactive group of the analyte is 0 - 75 times its molar ratio.

[0137] Accurately pipette 120 μL of the representative carboxyl compound mixed solution into an EP tube, add 10 μL of 140 mM EDC solution to it, and then add 10 μL of 0, 7, 14, 35, 70, 105 mM HOAT solutions, 13 μL of 480 mM SBPH solution, and 7 μL of hydrochloric acid. Each dosage has five technical replicates. After vortexing and mixing evenly, incubate at 37 °C for 1 hour (900 rpm). Transfer the above mixture to ice to cool and quench the reaction. The subsequent detection conditions are the same as above. The results show that when the ratio of HOAT to the reactive group of the analyte is 10:1, the peak area of the carboxyl compound derivative is the largest. Therefore, the ratio of HOAT to the reactive group of the analyte being 10:1 is the optimal HOAT dosage and is used for subsequent experiments.

[0138] (d) Selection of the ratio of SBPH to the reactive groups (carboxyl group and ketone carbonyl group) of the analyte:

[0139] The total concentration of the reactive groups (carboxyl group and ketone carbonyl group) in the representative carboxyl compound mixed solution is approximately 0.133 mM.

[0140] The optimized range of the ratio of SBPH to the reactive group of the analyte is 50 - 500 times of their molar ratio.

[0141] Accurately pipette 120 μL of the representative carboxyl compound mixed solution into an EP tube, add 10 μL of 140 mM EDC solution, 10 μL of 14 mM HOAT solution, 13 μL of 60, 120, 240, 360, 480, 600 mM SBPH solutions, and 7 μL of hydrochloric acid to it respectively. Each dosage has five technical replicates. After vortexing and mixing evenly, incubate at 37 °C for 1 hour (900 rpm). Transfer the above mixture to ice to cool and quench the reaction. The subsequent detection conditions are the same as above. The results show that when the ratio of SBPH to the reactive group of the analyte is 200:1, the peak area of the carboxyl compound derivative is the largest. Therefore, the ratio of SBPH to the reactive group of the analyte being 200:1 is the optimal dosage of SBPH and is used for subsequent experiments.

[0142] (e) Selection of reaction temperature:

[0143] The optimized range of temperature is 25 - 80 °C. Accurately pipette 120 μL of the representative carboxyl compound mixed solution into an EP tube, add 10 μL of 140 mM EDC solution, 10 μL of 14 mM HOAT solution, 13 μL of 240 mM SBPH solution, and 7 μL of hydrochloric acid to it respectively. Each dosage has five technical replicates. After vortexing and mixing evenly, incubate at 25, 37, 50, 60, 80 °C for 1 hour (900 rpm). Transfer the above mixture to ice to cool and quench the reaction. The subsequent detection conditions are the same as above. The results show that when the reaction temperature is 37 °C, the peak area of the carboxyl compound derivative is the largest. Therefore, 37 °C is the optimal reaction temperature and is used for subsequent experiments.

[0144] (f) Selection of reaction time:

[0145] The optimized range of reaction time is 10 - 120 minutes. Accurately pipette 120 μL of the representative carboxyl compound mixed solution into an EP tube, add 10 μL of 140 mM EDC solution, 10 μL of 14 mM HOAT solution, 13 μL of 240 mM SBPH solution, and 7 μL of hydrochloric acid to it respectively. Each dosage has five technical replicates. After vortexing and mixing evenly, incubate at 37 °C for 10, 20, 30, 60, 90, 120 minutes (900 rpm). Transfer the above mixture to ice to cool and quench the reaction. The subsequent detection conditions are the same as above. The results show that when the reaction time is 60 minutes, the peak area of the carboxyl compound derivative is the largest. Therefore, 60 minutes is the optimal reaction time and is used for subsequent experiments.

[0146] (g) Selection of the dosages of the condensing agent and derivatization reagent for biological samples:

[0147] Since the total amount of carboxyl analytes in biological samples is unknown, we selected urine samples with the most abundant types and contents of carboxyl analytes and optimized the specific dosages of condensing agents and derivatization reagents (see details in Figure 2 ). During this optimization process, we maintained the dosage ratio of EDC, HOAT, and SBPH at 100:10:200. By accurately pipetting 120 μL of the supernatant of urine pretreatment into an EP tube, we added 10 μL of EDC solutions with different concentrations (200, 400, 600, 800, 1000 mM), 10 μL of HOAT solutions (20, 40, 60, 80, 100 mM), 13 μL of SBPH solutions (300, 600, 900, 1200, 1500 mM), and 7 μL of hydrochloric acid to it. Each dosage had five technical replicates. After vortexing and mixing, the mixture was incubated at 37 °C for 1 hour (900 rpm). The above mixture was transferred to ice to cool and quench the reaction. The subsequent detection conditions were the same as above. The results showed that when the concentration of the SBPH solution was 600 mM, the peak area of the carboxyl compound derivative was the largest. Therefore, 400, 40, and 600 mM were the optimal dosages of EDC, HOAT, and SBPH and were used for subsequent experiments.

[0148] Through the above screening, the optimal derivatization reaction conditions were determined as follows: accurately pipette 120 μL of the working solution into an EP tube, add 10 μL of 400 mM EDC solution, 10 μL of 40 mM HOAT solution, 13 μL of 600 mM SBPH solution, and 7 μL of hydrochloric acid. After vortexing and mixing, the mixture was incubated at 37 °C for 1 hour (900 rpm). The above mixture was transferred to ice to cool and quench the reaction.

[0149] For biological samples, the above 120 μL of the working solution was replaced with 120 μL of the supernatant, and the other conditions remained unchanged.

[0150] (3) Verification of derivatization efficiency

[0151] The existing forms of carboxyl compounds in complex samples include fatty acids, amino acids, hydroxy acids, aromatic acids, keto acids, polycarboxylic acids, etc. and their possible chiral enantiomers.

[0152] To ensure that the above derivatization method has good derivatization efficiency for the above substances, the standard stock solutions of the representative carboxyl compounds in Table 2 were diluted to 0.10 mM with acetonitrile-water (volume ratio 1:1) solution respectively. Then, 10 μL of each 0.1 mM solution of the above carboxyl compounds was taken and mixed to obtain two parallel samples for determining the derivatization efficiency. One was the experimental group, which was operated according to the above optimal derivatization reaction conditions, and the other was the control group, which was operated in parallel, but the addition of SBPH was replaced with the same volume of acetonitrile-water (volume ratio 1:1). Liquid chromatography-high resolution mass spectrometry was used for detection, as follows:

[0153] Chromatographic analysis was performed using an Agilent ZORBAX Eclipse Plus C18 column (2.1×100 mm, 1.8 μm). The column temperature was set at 50 °C. Mobile phases A and B were water containing 0.1% formic acid and acetonitrile respectively, and the flow rate was 0.5 mL / min. The injection volume was 1 μL. The chromatographic elution gradient, expressed as the percentage of mobile phase B, was as follows: 0 - 5 minutes, 2 - 7%; 5 - 6 minutes, 7%; 6 - 7 minutes, 7 - 8%; 7 - 12 minutes, 8 - 15%; 12 - 24 minutes, 15 - 21%; 24.1 - 27 minutes, 95%. Mass spectrometry was recorded in the positive ion mode using an electrospray ionization source. The ion source temperature was 450 °C, the ionization voltage was 3500 V, and the spray gas and auxiliary heating gas were set at 50 psi. Sciex OS software was used for data acquisition and processing.

[0154] We calculated the difference between the mass spectrometry peak areas of the underivatized carboxyl compounds in the control group and those in the experimental group, and then calculated the ratio of this difference to the mass spectrometry peak area of the underivatized carboxyl compounds in the control group to obtain the derivatization efficiency of the carboxyl compounds.

[0155] It was found that the above method had a very high derivatization efficiency for the representative carboxyl compounds (92.8 - 100.0%), and the difference in the derivatization efficiency of all these R- / S-enantiomers was less than 3% (see Table 2 for details).

[0156] In addition, we also diluted the standard stock solutions of the R and S configurations of leucine, isoleucine, phenylalanine, tryptophan, phenyl lactic acid, 2-(4-hydroxyphenyl)propionic acid, mandelic acid, 2-hydroxyisocaproic acid, 2-hydroxyisovaleric acid, and 2-methylsuccinic acid to 0.10 mM respectively. Then, 10 μL of each 0.10 mM dilution of the R and S configurations of the above carboxyl compounds was taken and mixed to obtain mixed solutions of carboxyl compounds with R and S single configurations respectively. The above two mixed solutions of carboxyl compounds with single configurations were respectively operated according to the above optimal derivatization conditions, and then determined using the above liquid chromatography-high resolution mass spectrometry detection conditions.

[0157] It was found that no derivatives of chiral carboxyl compounds of the other configuration were seen in the chromatogram after derivatization of chiral carboxyl compounds in a single configuration. Therefore, it can be considered that no racemized derivatives caused by derivatization were observed during this derivatization process (see specifically Figure 3 ).

[0158] Table 2 Derivatization efficiency of representative carboxyl compounds

[0159]

[0160] a Derivatization efficiency ratio: The ratio of the derivatization efficiencies of the R- / S-carboxyl enantiomers labeled with SBPH.

[0161] Effect Example 1 S-Benzoylprolyl hydrazide (SBPH) is used for qualitative and quantitative analysis of highly covered carboxyl compounds

[0162] Test sample: The biological sample was derivatized with SBPH according to the optimal derivatization reaction conditions in Example 2. At the same time, the internal standard working solution was derivatized with SBPH-d5 under the same conditions for use as an internal standard. To achieve accurate quantification, the SBPH-derivatized biological sample and the SBPH-d5-derivatized internal standard working solution were mixed in a volume ratio of 10:1, dried with nitrogen and redissolved in acetonitrile-water (volume ratio 5:95) for LC-ESI-MS / MS detection and analysis.

[0163] Test method: Quantitative analysis was carried out on a Sciex QTRAP 6500 + mass spectrometer equipped with an electrospray ionization (ESI) source and coupled with a Shimadzu LC-30AD UPLC system. After parameter optimization, chromatographic analysis was performed using an Agilent ZORBAX Eclipse Plus C18 chromatographic column (2.1×100 mm, 1.8 μm), the column temperature was set at 50 °C, the mobile phases A and B were water and acetonitrile containing 0.1% formic acid respectively, the flow rate was 0.5 mL per minute, and the injection volume was 1 μL. The chromatographic elution gradient is expressed as the percentage of mobile phase B: 0 - 4 minutes, 8%; 4 - 7 minutes, 8 - 20%; 7 - 11 minutes, 20 - 30%; 11 - 15 minutes, 30 - 50%; 15 - 18 minutes, 50 - 95%; 18 - 19 minutes, 95 - 100%. Among them, 0 - 18 minutes is the mass spectrometry acquisition time. All derivatized carboxyl compounds were quantitatively analyzed in the positive ion mode using the multiple reaction monitoring mode (MRM), the ion source temperature was 450 °C, the ionization voltage was 3500 V, and the spray gas and auxiliary heating gas were set at 35 and 50 psi respectively. Sciex Analyst and OS (v1.7) software were used for data acquisition and processing respectively.

[0164] Test results:

[0165] (1) Qualitative analysis

[0166] After the carboxyl compounds (L3 high-concentration quality control products) are derivatized with SBPH, 267 carboxyl compounds can be detected by UPLC-MS / MS, including 49 saturated fatty acids, 18 unsaturated fatty acids, 44 amino acids, 50 hydroxy acids, 20 keto acids, 62 aromatic acids, and 24 heterocyclic acids (see Figures 4 - 7 , and the specific carboxyl metabolites corresponding to each serial number are shown in Table 3). For 65 groups of substances with the same mass-to-charge ratio (including isomers), they can all be separated by this method ( Figures 8 - 11 ). The above shows that this method can be effectively applied to the detection of most isomers.

[0167] (2) Establishment of the standard curve

[0168] Using the SBPH-d5 derivatized internal standard working solution as the internal standard, the above-mentioned stepwise diluted working solutions L1-L16 are derivatized with SBPH to obtain the standard curve, linear range, and correlation coefficient (R 2 ). Under the signal-to-noise ratios of 3 and 10, the detection limits (LOD) and quantification limits (LOQ) of each carboxyl compound are obtained respectively. The results show that the quantitative linear ranges of the 267 carboxyl compound derivatives can cover 2-4 orders of magnitude and have good linearity (R 2 > 0.99), and the detection limit (LOD, signal-to-noise ratio 3:1) and quantification limit (LLOQ, signal-to-noise ratio 10:1) are respectively lower than 5*10 -13 and 2*10 -12 mol, and the lowest values of LOD and LOQ can reach 3*10 -18 and 9*10 -18 mol (see Table 3 for details).

[0169] Table 3. Detection limits (LOD), lowest quantification limits (LOQ), linear ranges, and linear correlation coefficients (R 2 ) of 267 carboxyl compounds after SBPH derivatization

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178] (3) Intra-day and inter-day precision tests

[0179] The optimized derivatization reaction conditions were used to perform SBPH derivatization of the control samples at three concentration levels: low, medium, and high. Samples were collected five times daily, every four hours. Precision was assessed by analyzing the intra-day and inter-day variability of the samples collected over three consecutive days. Method validation results demonstrated that the intra-day and inter-day variability of all analytes at the low (L), medium (M), and high (H) concentration levels were less than 15% (Table 4).

[0180] Table 4. Intra-day (n=5) and inter-day (n=3) precision results for UPLC-MS / MS quantitative analysis of 267 carboxyl compounds

[0181]

[0182]

[0183]

[0184]

[0185]

[0186]

[0187]

[0188]

[0189] L, M, and H represent low, medium, and high concentration quality control working solutions, respectively; a : Intraday difference CV (%); b :Day difference CV (%)

[0190] (4) Recovery rate test

[0191] The accuracy of the method was evaluated by the recovery rates in classical biological fluids (human plasma and urine), fecal samples (human), and human-derived cells (A549). Quality control samples of carboxyl compounds at low, medium, and high concentration levels were added to the above biological samples respectively. The biological samples without added quality control were used as the control group. The difference in the measured carboxyl metabolite concentrations between the experimental group and the control group was calculated, and then the ratio of this difference to the actual added concentration of the carboxyl compound quality control was calculated. The results showed that the recovery rates of adding the above quality control samples at low, medium, and high concentration levels in human plasma, urine, fecal samples, and cell samples were all between 80 - 120% (Table 5).

[0192] Table 5. Recovery results of 267 carboxyl compounds in human plasma, urine, fecal samples, and A549 cell samples by UPLC-MS / MS quantitative analysis (n = 5)

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217] L, M, and H represent low, medium, and high concentration quality control working fluids, respectively; a : Human plasma; b : Human urine; c : Human fecal samples; d : Human A549 cells

[0218] (5) Applicability of the method to multiple biological samples

[0219] Derivatize the classical biological samples and mix them with the internal standard according to the optimal derivatization reaction conditions in Example 2, collect and process the data according to the above test method, and then calculate the quantitative results of the carboxyl metabolites in each biological sample through the established standard curve. To convert the results into the content corresponding to unit mass or volume, normalize each biological sample. Among them, the quantitative results of plasma samples are normalized by sample volume, the quantitative results of fecal samples are normalized by sample dry weight, the quantitative results of cell samples are normalized by sample wet weight, and the quantitative results of urine are normalized by sample creatinine. The results show that

[0220] This working method is applicable to the quantitative analysis of carboxyl metabolites in complex biological samples such as classical biological body fluids (human plasma and urine), fecal samples (human), and cells (A549) (Table 6).

[0221] Table 6. Quantitative results of the carboxyl metabolite composition in several typical complex biological samples (n = 5)

[0222]

[0223]

[0224]

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231] a Mean ± standard deviation (μmol / mmol Creatinine); b Mean ± standard deviation (μM); c Mean ± standard deviation (pmol / mg); N / A: Not detected.

[0232] (6) Quantitative detection of creatinine level in urine

[0233] Using creatinine-d3 as the internal standard, data acquisition and processing were carried out under the same chromatographic and mass spectrometric conditions as in Test Example 1 of the effect. Creatinine had a good linear relationship in the concentration range of 0.002 - 49.6 mM, with excellent precision (intra-day and inter-day CV < 10%) and recovery rate (94.8% - 113.5%), and was able to cover the high creatinine levels in human urine. The creatinine quantitative results in each urine sample could be calculated through the established standard curve.

[0234] From the above data, it can be seen that the measured quantitative values of the carboxyl metabolite composition in human fecal samples are similar to the reported results in Reference [1]. The quantitative data of the carboxyl metabolite composition in other samples are not commonly reported in the literature.

[0235] The quantitative analysis method established based on the probe of the present application is simple and rapid, and can complete the quantitative analysis of 267 carboxyl metabolites and creatinine within a detection time of 18 minutes, which is suitable for high-throughput analysis of large samples. Compared with the existing UPLC-MS / MS detection technology based on probe sensitization, although the detection time in Reference [1] is slightly shorter than that of the present application, its coverage is only short-chain fatty acids, which is much smaller than that of the present application. The carboxylic acid coverage of References [2] and [3] is slightly improved compared with Reference [1], but their analysis time is significantly increased, which is 2-3 times that of the present application. The carboxylic acid coverage of References [4] and [5] is relatively good among the carboxylic acids reported so far, but they did not perform absolute quantitative analysis of carboxylic acids. In addition, only the detection limits of eight carboxylic acids were evaluated in Reference [4], and its detection limits were 10-200 fmol, which were significantly higher than the detection limits of the present application, 0.003-51.9 fmol (except phthalic acid which was 408 fmol). References [6] and [7] are reports on the quantitative detection of carboxyl compounds based on carbohydrazide sensitized probes. Among them, the coverage of Reference [6] is only triterpenoid carboxyl compounds, which does not overlap with the carboxyl compounds detected in the present application and is not compared. The coverage of Reference [7] is only fatty acids and only relative quantification, and there are no data such as detection limits.

[0236] Considering the three aspects of detection coverage, sensitivity and efficiency, the present application is significantly superior to the traditional methods (Table 7).

[0237] Table 7. Detection coverage, sensitivity and detection efficiency of the probe of the present application and the probes in the existing literature

[0238]

[0239]

[0240] Effect Example 2: S-benzoylprolyl hydrazide is used for the quantitative analysis of chiral carboxylic acids

[0241] Detection samples: The working solution and biological samples were pretreated and derivatized with SBPH or SBPH-d5 according to the conditions in Effect Example 1.

[0242] Test method: The quantitative analysis conditions were the same as those in Effect Example 1 except for the chromatographic elution gradient. The chromatographic elution gradient is expressed as the percentage of mobile phase B: 0-5 minutes, 2-7%; 5-6 minutes, 7%; 6-7 minutes, 7-8%; 7-12 minutes, 8-15%; 12-24 minutes, 15-21%; 24.1-27 minutes, 95%.

[0243] Test results:

[0244] After the chiral carboxylic acids were derivatized with SBPH, diastereoisomers with different properties were formed, enabling the separation of 30 pairs of chiral carboxylic acid enantiomers on a common C18 reversed-phase chromatographic column ( Figure 12 , Table 8).

[0245] In this example of the effect, only the carboxyl compounds with numbers in the range of 216 - 268 in Table 1 were concerned. The test results are as follows:

[0246] Table 8. UPLC-MS / MS detection data of 30 pairs of chiral carboxylic acids

[0247]

[0248]

[0249] a Retention time (min); b Resolution; c Mass spectrometry response ratio.

[0250] As can be seen from the above table, this application shows the following obvious advantages: (1) Firstly, this application can simultaneously perform chiral separation and quantification on chiral α-amino acids (α-AA), chiral α-hydroxy acids (α-HA), and chiral α-methyl acids (α-MA), which has not been reported before; Amino acids with secondary amines or carboxylic acids without amino groups cannot be labeled by the aldehyde-based chiral recognition probes in References [8] and [9]. (2) This application can simultaneously separate the isomers and enantiomers of chiral carboxylic acids (such as R- / S-leucine and R- / S-isoleucine), and there is no racemization phenomenon caused by derivatization for these chiral carboxylic acids (such as Figure 2As shown, no chiral carboxylic acid of the other configuration was observed in the chromatogram after derivatization of the chiral carboxylic acid in a single configuration. It can be considered that no racemization phenomenon caused by derivatization was observed. (3) The R- / S-enantiomers have the same derivatization efficiency and similar mass spectrometry responses. When two pure enantiomers cannot be obtained, one of the enantiomers or a mixed racemate can be used as an analytical reference substance for quantitative analysis (for example, both RS-2-methylvaleric acid and S-2-methylvaleric acid can be used for the quantification of R- / S-2-methylvaleric acid). (4) The excess condensation reagent and its by-products are eluted at the front of all analytes to be measured, and the excess SBPH is also eluted at a time when no analyte to be measured is eluted (retention time = 7 minutes). Therefore, they can all be easily transferred to the waste liquid to avoid ionization suppression when entering the ion source. (5) The SBPH-labeled S-enantiomers of all chiral α-amino acids and chiral α-methyl acids tested in this application have better retention on a C18 reversed-phase chromatographic column than the corresponding R-enantiomers. On the contrary, the SBPH-labeled R-enantiomers of all chiral α-hydroxy acids have better retention on a C18 reversed-phase chromatographic column than the corresponding S-enantiomers. Therefore, when neither pure enantiomer can be obtained, this is very useful for identifying R- and S-enantiomers. For example, the two chromatographic peaks of SBPH-labeled RS-α-hydroxyhippuric acid can be easily identified because the retention of its R-enantiomer is weaker than that of the S-enantiomer.

[0251] In addition, for the vast majority of chiral carboxylic acids mentioned in the above table, their standard chemicals were used to determine which peak is the R or S configuration. It is also possible to assign the configuration of its chromatographic peak according to the type of carboxylic acid to which it belongs through the rule mentioned in point (5) above.

[0252] (2) Establishment of the standard curve

[0253] The standard curve was established using the same method as in Effect Example 1. The results of the methodology verification showed that the quantitative linear range of 60 chiral carboxylic acid enantiomers could cover 2 - 5 orders of magnitude and had good linearity (R 2 > 0.992), and the detection limit (LOD, signal-to-noise ratio 3:1) and the limit of quantitation (LOQ, signal-to-noise ratio 10:1) were respectively lower than 4×10 -15 and 9×10 -15 mol, and the lowest values of LOD and LOQ could reach 1×10 -18 and 3×10 -18 mol (Table 9).

[0254] Table 9. Detection limit (LOD), lowest limit of quantitation (LOQ), linear range and linear correlation coefficient (R 2 )

[0255]

[0256]

[0257]

[0258] (3) Intra-day and inter-day precision tests

[0259] The same method as in Effect Example 1 was used to derivatize the quality control products at three concentration levels of low, medium, and high for precision testing. The results showed that the CVs of intra-day differences and inter-day differences for all analytes were lower than 15% (Table 10).

[0260] Table 10. Intra-day (n = 5) and inter-day (n = 3) precision results of UPLC-MS / MS quantitative analysis of 30 pairs of chiral carboxylic acids

[0261]

[0262]

[0263] L, M, and H represent the quality control working solutions at low, medium, and high concentrations respectively; a : Intra-day difference CV (%); b : Inter-day difference CV (%)

[0264] (4) Recovery tests

[0265] The same method as in Effect Example 1 was used for the recovery test. The test results showed that the recoveries in human plasma, urine, feces, and A549 cell samples were all between 80 - 120% (Table 11).

[0266] Table 11. Recovery results of UPLC-MS / MS quantitative analysis of 30 pairs of chiral carboxylic acids in human plasma, urine, fecal samples, and cell samples (n = 5)

[0267]

[0268]

[0269]

[0270]

[0271]

[0272] L, M, and H represent the quality control working solutions at low, medium, and high concentrations respectively; a : Human plasma; b : Human urine; c : Human fecal sample;[[ID=6I]] d : Human A549 cells

[0273] As described above, this SBPH-based UPLC-MS / MS method is suitable for the accurate simultaneous quantitative analysis of these 60 chiral carboxylic acid enantiomers in four different biological matrices.

[0274] (5) Applicability of the method to multiple biological samples

[0275] Testing was carried out using the same method as in Effect Example 1. The results showed that this working method is applicable to the quantitative analysis of chiral carboxylic acids in complex biological samples such as classical biological fluids (human plasma and urine), fecal samples (human), and cells (A549) (Table 12). The measured quantitative values of chiral carboxylic acids in human urine were similar to the results reported in Reference [9]. Quantitative data on the composition of chiral carboxylic acids in other samples are not commonly reported in the literature.

[0276] Table 12. Quantitative results of the composition of carboxyl metabolites in several typical complex biological samples (n = 6)

[0277]

[0278]

[0279]

[0280] a Mean ± standard deviation (μmol / mmol Creatinine); b Mean ± standard deviation (μM); c Mean ± standard deviation (pmol / mg); N / A: not detected.

[0281] From the above data, it can be seen that the quantitative analysis method established based on the carboxyl compound-sensitized probe provided in this application can achieve the separation of isomers and enantiomers of chiral carboxylic acid metabolites including chiral α-amino acids, chiral α-hydroxy acids, and chiral α-methyl acids under conventional reversed-phase chromatography conditions, so that they can be quantitatively analyzed, and the limit of quantification is 0.003 - 8.457 fmol. Compared with the existing chiral carboxylic acid detection technologies based on probe sensitization, the detection coverage of Reference [8] is the best, but its detection limit is slightly higher. The detection sensitivity of Reference [9] is slightly improved compared with Reference [8], but its detection coverage is smaller. The detection time of References [10 - 12] is slightly shortened. Especially, Reference

[12] shows better detection sensitivity, but its detection coverage is less. Considering the three aspects of detection coverage, sensitivity, and efficiency at the same time, this application is significantly better than the traditional methods (Table 13).

[0282] Limit is also slightly higher. The detection sensitivity of Reference [9] is slightly improved compared with Reference [8], but its detection coverage is smaller. The detection time of References [10 - 12] is slightly shortened. Especially, Reference

[12] shows better detection sensitivity, but its detection coverage is less. Considering the three aspects of detection coverage, sensitivity, and efficiency at the same time, this application is significantly better than the traditional methods (Table 13).

[0283] Table 13. Detection Coverage, Sensitivity, and Detection Efficiency of the Probes in This Application and the Probes in the Existing Literature

[0284]

[0285]

[0286]

References

[0287] 1. Han, J.; Lin, K.; Sequeira, C.; Borchers, C. H. Anal. Chim. Acta 2015, 854, 86 - 94.

[0288] 2. Zheng, J.; Zheng, S. J.; Cai, W. J.; Yu, L.; Yuan, B. F.; Feng, Y. Q. Anal. Chim. Acta 2019, 1070, 51 - 59.

[0289] 3. Jiang, R. Q.; Jiao, Y.; Zhang, P.; Liu, Y.; Wang, X.; Huang, Y.; Zhang, Z. J.; Xu, F. G. Anal. Chem. 2017, 89, 12223 - 12230.

[0290] 4. Zheng, J.; Gong, G.-G.; Zheng, S.-J.; Zhang, Y.; Feng, Y.-Q. Anal. Chem. 2021, 93, 1604 - 1611.

[0291] 5. Zhao, S.; Li, L. Anal. Chem. 2018, 90, 13514 - 13522.

[0292] 6. Zheng, Y.; Liu, S.; Xing, J. P.; Zheng, Z.; Pi, Z. F.; Song, F. R.; Liu, Z. Q. Anal. Chem. 2018, 90, 13946 - 13952.

[0293] 7. Feng, Y.; Lv, Y. N.; Gu, T. J.; Chen, B. M.; Li, L. J. Anal. Chem. 2022, 94, 13036 - 13042.

[0294] 8. Ayon, N. J.; Sharma, A. D.; Gutheil, W. G. J. Am. Soc. Mass Spectrom. 2019, 30, 448 - 458.

[0295] 9. Zheng, J.Y.; Jiang, X.; Zhou, J.L.; Shi, Z.Q.; Liu, L.F.; Xin, G.Z. Anal. Chim. Acta 2019, 1077, 174 - 182.

[0296] 10. Shen, K.X.; Wang, L.; He, Q.; Jin, Z.; Chen, W.Y.; Sun, C.R.; Pan, Y.J. Anal. Chem. 2020, 92, 1763 - 1769.

[0297] 11. Huang, R.R.; Shen, K.X.; He, Q.; Hu, Y.Q.; Sun, C.R.; Guo, C.; Pan, Y.J. J. Proteome Res. 2021, 20, 3952 - 3962.

[0298] 12. Zheng, J.; Zheng, S.J.; Qi, C.B.; Wu, D.M.; Feng, Y.Q. Anal. Chem. 2019, 91, 11440 - 11446.

Claims

1. An isotopically labeled compound of the formula I or a salt thereof, Among them, The carbon atom with "*" represents a chiral carbon atom and exists in the form of R configuration, S configuration, or a mixture of R configuration and S configuration.

2. The isotope-labeled compound or its salt of the compound shown in Formula I as described in claim 1, characterized in that, It satisfies one or more of the following conditions: (1) At least one of the hydrogen atoms in the compound represented by Formula I is 1 substituted by 2 hydrogen; (2) At least one of the compounds represented by Formula I 12 C is replaced by its heavier isotope 13 C; (3) At least one of the compounds represented by Formula I 14 N is replaced by its heavier isotope 15 N; (4) At least one of the compounds represented by Formula I 16 O is replaced by its heavier isotope 18 O; and (5) The salt of the isotopically labeled compound of the formula I is a salt formed by the isotopically labeled compound of the formula I and hydrochloric acid.

3. The isotope-labeled compound or its salt of the compound shown by Formula I as described in Claim 1, characterized in that, The salt of the isotopically labeled compound of the formula I is a salt formed by the isotopically labeled compound of the formula I and hydrochloric acid in a molar ratio of 1:

1.

4. The isotope-labeled compound or its salt of the compound represented by Formula I as described in claim 1, characterized in that, The isotopically labeled compound of the formula I is any of the following compounds:

5. A method for preparing an isotopically labeled compound of the formula I according to any one of claims 1-4, which comprises the following steps: In a solvent and in the presence of a base, "the compound of the formula II or a salt thereof" undergoes the following coupling reaction with the compound of the formula III and hydrazine hydrate to form an isotopically labeled compound of the formula I, that's all; Wherein, the carbon atom with "*" represents a chiral carbon atom and exists in the form of R configuration, S configuration, or a mixture of R configuration and S configuration; In the compound of the formula II, the compound of the formula III, or hydrazine hydrate, the isotopic abundance of at least one atom is different from its natural abundance.

6. The method for preparing an isotope-labeled compound of the compound represented by Formula I as described in claim 5, characterized in that, It satisfies one or more of the following conditions: (1) In the coupling reaction, the solvent is selected from one or more of halogenated alkane solvents, benzene solvents, and ether solvents; (2) In the coupling reaction, the base is an inorganic base and / or an organic base; (3) In the coupling reaction, the salt of the compound of the formula II is the hydrochloride salt of the compound of the formula II; (4) In the coupling reaction, the molar ratio of the base to the "compound of the formula II or a salt thereof" is 2:1 - 10:1; (5) In the coupling reaction, the molar ratio of the compound of the formula III to the "compound of the formula II or a salt thereof" is 0.5:1 - 5:1; (6) In the coupling reaction, the molar ratio of hydrazine hydrate to the "compound of the formula II or a salt thereof" is 10:1 - 500:1; (7) In the coupling reaction, the molar concentration of the "compound of the formula II or a salt thereof" in the solvent is 0.001 - 10 mol / L; (8) In the coupling reaction, the feeding order of each material is that the compound of the formula III is added to the "compound of the formula II or a salt thereof" and the base, and then hydrazine hydrate and an alcohol solvent are added; and (9) The coupling reaction further includes the following post-treatment steps: removing the solvent, extraction, washing, drying, filtration, removing the solvent, and lyophilization.

7. The method for preparing an isotope-labeled compound of the compound represented by Formula I as described in claim 6, characterized in that, It satisfies one or more of the following conditions: (1) In the coupling reaction, the solvent is an ether solvent; (2) The organic base is triethylamine and / or pyridine; (3) The inorganic base is selected from one or more of carbonates, bicarbonates, and hydroxides; (4) In the coupling reaction, the molar ratio of the base to the "compound of the formula II or a salt thereof" is 3:1; (5) In the coupling reaction, the molar ratio of the compound represented by Formula III to the "compound represented by Formula II or its salt" is 0.9:1; (6) In the coupling reaction, the molar ratio of hydrazine hydrate to the "compound represented by Formula II or its salt" is 100:1; (7) In the coupling reaction, the molar concentration of the "compound represented by Formula II or its salt" in the solvent is 0.06 mol / L; (8) In the coupling reaction, the feeding order of each material is that the compound represented by Formula III is added dropwise to the "compound represented by Formula II or its salt" and a base under ice bath conditions, then reacted at room temperature for 30 minutes, and then hydrazine hydrate and an alcohol solvent are added; (9) The removal of the solvent is by rotary evaporation; (10) The extraction is by dichloromethane extraction; (11) The washing is with saturated sodium carbonate solution; (12) The drying is with anhydrous magnesium sulfate.

8. The method for preparing an isotope-labeled compound of the compound represented by Formula I as described in claim 6, characterized in that, It satisfies one or more of the following conditions: (1) In the coupling reaction, the feeding order of each material is that the compound represented by Formula III is added to the "compound represented by Formula II or its salt" and a base, then hydrazine hydrate and an alcohol solvent are added, and after addition, the reaction is carried out at room temperature for 1 hour; (2) In the coupling reaction, the feeding order of each material is that the compound represented by Formula III is added to the "compound represented by Formula II or its salt" and a base, then hydrazine hydrate and an alcohol solvent are added, and the alcohol solvent is methanol.

9. The method for preparing an isotope-labeled compound of the compound represented by Formula I as described in claim 6, characterized in that, The inorganic base is a carbonate.

10. The method for preparing an isotope-labeled compound of the compound represented by Formula I as described in claim 6, characterized in that, It satisfies one or more of the following conditions: (1) The halogenated alkane solvent is dichloromethane and / or carbon tetrachloride; (2) The benzene solvent is toluene; (3) The ether solvent is tetrahydrofuran and / or diethyl ether.

11. The method for preparing an isotope-labeled compound of the compound represented by Formula I as described in claim 6, characterized in that, The ether solvent is anhydrous tetrahydrofuran with molecular sieve.

12. The method for preparing an isotope-labeled compound of the compound represented by Formula I as claimed in claim 7, characterized in that, [[ID= / / 17]]It satisfies one or more of the following conditions: (1) The carbonate is potassium carbonate and / or sodium carbonate; (2) The bicarbonate is sodium bicarbonate; (3) The hydroxide is sodium hydroxide and / or potassium hydroxide.

13. A composition for detecting and / or separating substance X, which comprises: Component A and a condensing agent; Component A is: (1) a compound represented by Formula I or its salt, and / or (2) an isotope-labeled compound represented by Formula I as recited in any one of Claims 1-4 or its salt; The carbon atom with "*" is represented as a chiral carbon atom and exists in the form of R configuration, S configuration, or a mixture of R configuration and S configuration; The substance X is a carbonyl-containing compound, and the carbonyl-containing compound refers to a compound containing one or more of carboxyl, aldehyde carbonyl, and ketone carbonyl.

14. The composition for detecting and / or separating substance X according to claim 13, characterized in that, It satisfies one or more of the following conditions: (1) The substance X is a carboxyl-containing compound, and the carboxyl-containing compound is a small molecule carboxyl compound; (2) The condensing agent is selected from one or more of HATU, HBTU, HCTU, TBTU, TSTU, DMTMM, EDC, TPP, and HOAT; (3) The molar ratio of the condensing agent to Component A is 1:10 - 10:1; (4) When the component A is a compound represented by Formula I and / or an isotope-labeled substance of the compound represented by Formula I; the composition further comprises an acid; and (5) The composition is any one of the following compositions: A) The composition consists of the following components comprising: component A; and a condensing agent; B) The composition consists of the following components: component A; a condensing agent; and an acid.

15. The composition for detecting and / or separating substance X according to claim 14, characterized in that, It satisfies one or more of the following conditions: (1) The substance X is a carboxyl compound with a molecular weight less than 1000 Da; (2) The condensing agent is EDC, HOAT or "a combination of EDC and HOAT"; (3) The molar ratio of the condensing agent to the component A is 22:40; (4) When the component A is a compound represented by Formula I and / or an isotope-labeled substance of the compound represented by Formula I; the composition further comprises an acid, and the acid is hydrochloric acid.

16. The composition for detecting and / or separating substance X according to claim 15, characterized in that, It satisfies one or more of the following conditions: (1) The substance X is a carboxyl metabolite; (2) In the combination of EDC and HOAT, the molar ratio of EDC to the component A is 100:1 - 1:100; (3) In the combination of EDC and HOAT, the molar ratio of HOAT to the component A is 100:1 - 1:100; (4) When the component A is a compound represented by Formula I and / or an isotope-labeled substance of the compound represented by Formula I; the composition further comprises an acid, and the acid is 1M hydrochloric acid.

17. The composition for detecting and / or separating substance X according to claim 16, characterized in that, It satisfies one or more of the following conditions: (1) The substance X is a carboxyl metabolite in human plasma, human urine, human feces or human-derived cell A549; (2) In the combination of EDC and HOAT, the molar ratio of EDC to the component A is 20:40; (3) In the combination of EDC and HOAT, the molar ratio of HOAT to the component A is 2:

40.

18. Use of substance A as a derivatizing reagent for detecting and / or separating substance X, wherein the substance A is (i) a compound represented by Formula I or a salt thereof; (ii) the composition according to any one of claims 13 - 17; or (iii) an isotope-labeled substance of the compound represented by Formula I according to any one of claims 1 - 4 or a salt thereof; The carbon atom with "*" represents a chiral carbon atom and exists in the form of R configuration, S configuration, or a mixture of R configuration and S configuration; The substance X is a carbonyl-containing compound, and the carbonyl-containing compound refers to a compound containing one or more of carboxyl, aldehyde carbonyl and ketone carbonyl.

19. Use of substance A as a derivatization reagent according to claim 18, characterized in that, The use comprises the following steps: (1) Reacting the substance A with a sample to obtain a derivatized product; the sample includes substance X; (2) Separating and / or detecting the derivatized product by liquid chromatography - mass spectrometry.

20. Use of substance A as a derivatizing reagent according to claim 19, characterized in that, It satisfies one or more of the following conditions: (1) When the substance A is the "compound represented by Formula I or a salt thereof" or the "isotope-labeled substance of the compound represented by Formula I or a salt thereof", a condensing agent is further added in step (1); (2) The derivatization reaction is carried out under acidic conditions; (3) The derivatization reaction is carried out in a solvent; (4) The reaction temperature of the derivatization reaction is 20 - 80 °C; (5) The reaction time of the derivatization reaction is 10 - 120 minutes; (6) In the liquid chromatography - mass spectrometry technology, the chromatographic conditions of liquid chromatography are as follows: the chromatographic column is a reversed - phase chromatographic column; the column temperature is 40 to 55 °C, the mobile phases A and B are water containing 0.005% - 0.5% formic acid and acetonitrile respectively, or water and methanol respectively, the flow rate is 0.2 to 0.5 mL per minute, the injection volume is 0.5 to 5 μL, and gradient elution is performed; and (7) In the liquid chromatography - mass spectrometry technology, the analysis conditions of mass spectrometry adopt the multiple reaction monitoring mode in the positive ion mode.

21. Use of substance A as a derivatization reagent according to claim 19, characterized in that, In the substance A, the molar ratio of the "compound or its salt as shown in formula I" or the "isotope - labeled compound or its salt as shown in formula I" to the reactive group in the sample is 10:1 - 500:

1.

22. Use of substance A as a derivatization reagent according to claim 21, characterized in that, In the substance A, the molar ratio of the "compound or its salt as shown in formula I" or the "isotope - labeled compound or its salt as shown in formula I" to the reactive group in the sample is 200:

1.

23. Use of substance A as a derivatizing reagent according to claim 20, characterized in that, It satisfies one or more of the following conditions: (1) When the substance A is the "compound or its salt as shown in formula I" or the "isotope - labeled compound or its salt as shown in formula I", a condensing agent is further added in step (1), and the condensing agent is selected from one or more of HATU, HBTU, HCTU, TBTU, TSTU, DMTMM, EDC, TPP, and HOAT; (2) The derivatization reaction is carried out under acidic conditions, and the acidic condition is hydrochloric acid; (3) The derivatization reaction is carried out in a solvent, and the solvent is a nitrile solvent and water; (4) The reaction temperature of the derivatization reaction is 37 °C; (5) The reaction time of the derivatization reaction is 60 minutes; (6) The chromatographic column is an Agilent ZORBAX Eclipse Plus C18 chromatographic column; (7) The column temperature is 50 °C; (8) The mobile phases A and B are water containing 0.1% formic acid and acetonitrile respectively; (9) The flow rate is 0.5 mL per minute; (10) The injection volume is 1 μL; (11) In the liquid chromatography - mass spectrometry technology, the analysis conditions of mass spectrometry are that the ion source temperature is 350 to 550 °C, the ionization voltage is 3500 to 5500 V, the spray gas pressure is 40 to 55 psi, and the pressure of the auxiliary heating gas is 40 to 60 psi.

24. Use of substance A as a derivatization reagent according to claim 23, characterized in that, It satisfies one or more of the following conditions: (1) When the substance A is the "compound or its salt as shown in formula I" or the "isotope - labeled compound or its salt as shown in formula I", a condensing agent is further added in step (1), and the condensing agent is EDC, HOAT, or "a combination of EDC and HOAT"; (2) The derivatization reaction is carried out under acidic conditions, and the acidic condition is 1M hydrochloric acid; (3) The volume ratio of the hydrochloric acid to the reaction system of the derivatization reaction is 0.5 - 10%; (4) The nitrile solvent is acetonitrile; (5) The gradient of the gradient elution, expressed as the percentage of mobile phase B, is: 0 - 4 minutes, 8%; 4 - 7 minutes, 8 - 20%; 7 - 11 minutes, 20 - 30%; 11 - 15 minutes, 30 - 50%; 15 - 18 minutes, 50 - 95%; 18 - 19 minutes, 95 - 100%; Alternatively, the gradient of the gradient elution, expressed as the percentage of mobile phase B, is: 0 - 5 minutes, 2 - 7%; 5 - 6 minutes, 7%; 6 - 7 minutes, 7 - 8%; 7 - 12 minutes, 8 - 15%; 12 - 24 minutes, 15 - 21%; 24.1 - 27 minutes, 95%; (6) The ion source temperature is 450 degrees Celsius; (7) The ionization voltage is 3500 volts; (8) The spray gas pressure is 35 psi; (9) The pressure of the auxiliary heating gas is 50 psi.

25. Use of substance A as a derivatization reagent according to claim 24, characterized in that, It satisfies one or more of the following conditions: (1) In the combination of EDC and HOAT, the molar ratio of EDC to the "compound or its salt represented by formula I" or the "isotope-labeled compound or its salt represented by formula I" is 100:1 - 1:100; (2) In the combination of EDC and HOAT, the molar ratio of HOAT to the "compound or its salt represented by formula I" or the "isotope-labeled compound or its salt represented by formula I" is 100:1 - 1:100; (3) The volume ratio of hydrochloric acid to the reaction system of the derivatization reaction is 4.5%; (4) The derivatization reaction is carried out in a solvent, and the solvent is acetonitrile and water with a volume ratio of 1:

1.

26. Use of substance A as a derivatization reagent according to claim 25, characterized in that, It satisfies one or more of the following conditions: (1) In the combination of EDC and HOAT, the molar ratio of EDC to the "compound or its salt represented by formula I" or the "isotope-labeled compound or its salt represented by formula I" is 20:40; (2) In the combination of EDC and HOAT, the molar ratio of HOAT to the "compound or its salt represented by formula I" or the "isotope-labeled compound or its salt represented by formula I" is 2:40.

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