NAPHTHALENESULFONYL COMPOUNDS, AND PREPARATION METHODS AND APPLICATIONS

Abstract

The present disclosure provides naphthalenesulfonyl compounds, preparation methods and application thereof. Specifically disclosed is a compound of formula (I) or a salt thereof, which serves as a specific derivatization reagent capable of reacting with hydroxyl and amino groups. The compound features simple synthesis, high reactivity, and ready availability at low cost, and is capable of improving chromatographic separation behaviors of target compounds and enhancing the detection sensitivities of these compounds.

##STR00001##

Claims

1. A compound of formula (I) or a salt thereof: ##STR00052## wherein R.sub.1 and R.sub.1 are independently selected from C.sub.1-7 alkyl; R.sub.2 is selected from H, C.sub.1-7 alkyl, or benzyl; and X is selected from OH or halogen.

2. The compound of formula (I) or the salt thereof according to claim 1, wherein R.sub.1 and R.sub.1 are the same.

3. The compound of formula (I) or the salt thereof according to claim 1, wherein, in R.sub.1 or R.sub.1, the C.sub.1-7 alkyl is C.sub.1-4 alkyl; and/or in R.sub.2, the C.sub.1-7 alkyl is C.sub.1-4 alkyl; and/or in X, the halogen is Cl.

4. The compound of formula (I) or the salt thereof according to claim 3, wherein the C.sub.1-7 alkyl in R.sub.1 or R.sub.1 is C.sub.2-4 alkyl or ethyl.

5. The compound of formula (I) or the salt thereof according to claim 3, wherein the C.sub.1-7 alkyl in R.sub.2 is isobutyl.

6. The compound of formula (I) or the salt thereof according to claim 1, wherein the compound of the formula (I) is ##STR00053##

7. The compound of formula (I) or the salt thereof according to claim 1, wherein the salt of the compound of formula (I) is a salt obtained from the compound of formula (I) and an acid, and the acid is an inorganic acid or an organic acid.

8. A preparation method for a compound of formula (I), comprising: subjecting a compound of formula (III) to a condensation reaction with a compound of formula (IV) in the presence of an activating agent and a base in a solvent to obtain the compound of formula (I): ##STR00054## wherein, X is OH, R.sub.1 and R.sub.1 are independently selected from C.sub.1-7 alkyl, and R.sub.2 is selected from H, C.sub.1-7 alkyl, or benzyl.

9. The preparation method for the compound of formula (I) according to claim 8, wherein, in the condensation reaction, the solvent is N,N-dimethylformamide (DMF); and/or in the condensation reaction, the activating agent comprises one or more of 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole; and/or in the condensation reaction, the base is an organic base.

10. The preparation method for the compound of formula (I) according to claim 9, wherein the organic base comprises N-methylmorpholine and/or pyridine.

11. The preparation method for the compound of formula (I) according to claim 8, further comprising a process (1-1) or a process (1-2); wherein the process (1-1) comprises: subjecting a compound of formula (VI) to a reductive amination reaction with a compound of formula (A-1) and a compound of formula (A-2) in the presence of a reducing agent in a solvent to obtain the compound of formula (III): ##STR00055## the process (1-2) comprises: subjecting a compound of formula (VI) to an alkylation reaction with a compound of formula (B-1) and a compound of formula (B-2) in the presence of a base in a solvent to obtain the compound of formula (III): ##STR00056## wherein X.sub.1 and X.sub.2 are independently halogen.

12. The preparation method for the compound of formula (I) according to claim 11, wherein, in the process (1-2), the halogen is I.

13. The preparation method for the compound of formula (I) according to claim 11, wherein, in the reductive amination reaction, the solvent is methanol, acetonitrile, or a buffer of sodium acetate or phosphate with a pH of 2-12; and/or in the reductive amination reaction, the reducing agent is sodium cyanoborohydride and/or 2-methylpyridine borane; and/or in the alkylation reaction, the solvent is acetonitrile; and/or in the alkylation reaction, the base is carbonate or bicarbonate.

14. The preparation method for the compound of formula (I) according to claim 13, wherein the carbonate is a potassium carbonate.

15. A preparation method for a compound of formula (I), comprising: subjecting a compound of formula (III) to a condensation reaction with a compound of formula (IV) in the presence of an activating agent and a base in a solvent to obtain a compound of formula (V): ##STR00057## and subjecting the compound of formula (V) to an acylation reaction with a chlorinating agent to obtain the compound of formula (I): ##STR00058## wherein, X is Cl, R.sub.1 and R.sub.1 are independently selected from C.sub.1-7 alkyl, and R.sub.2 is selected from H, C.sub.1-7 alkyl, or benzyl.

16. The preparation method for the compound of formula (I) according to claim 15, wherein, in the condensation reaction, the solvent is N,N-dimethylformamide (DMF); and/or in the condensation reaction, the activating agent comprises one or more of 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole; and/or in the condensation reaction, the base is an organic base; and/or in the acylation reaction, the solvent comprises tetrahydrofuran and/or toluene; and/or in the acylation reaction, the chlorinating agent comprises phosphorus pentachloride and/or acetyl chloride.

17. The preparation method for the compound of formula (I) according to claim 15, further comprising a process (1-1) or a process (1-2); wherein, the process (1-1) comprises: subjecting a compound of formula (VI) to a reductive amination reaction with a compound of formula (A-1) and a compound of formula (A-2) in the presence of a reducing agent in a solvent to obtain the compound of formula (III): ##STR00059## the process (1-2) comprises: subjecting a compound of formula (VI) to an alkylation reaction with a compound of formula (B-1) and a compound of formula (B-2) in the presence of a base in a solvent to obtain the compound of formula (III): ##STR00060## wherein X.sub.1 and X.sub.2 are independently halogen.

18. The preparation method for the compound of formula (I) according to claim 17, wherein, in the reductive amination, the solvent reaction is methanol, acetonitrile, or a buffer of sodium acetate or phosphate with a pH of 2-12; and/or in the reductive amination reaction, the reducing agent comprises sodium cyanoborohydride and/or 2-methylpyridine borane; and/or in the alkylation reaction, the solvent is acetonitrile; and/or in the alkylation reaction, the base is carbonate or bicarbonate.

19. The preparation method for the compound of formula (I) according to claim 18, wherein the carbonate is a potassium carbonate.

20. An isotope-labeled compound of formula (II) or a salt thereof: ##STR00061## wherein Y is ##STR00062## R.sub.1 and R.sub.1 are independently selected from C.sub.1-7 alkyl; R.sub.2 is selected from H, C.sub.1-7 alkyl, or benzyl; X is selected from OH or halogen; and at least one atom in Y is substituted by a heavier isotope thereof.

21. The isotope-labeled compound of formula (II) or the salt thereof according to claim 20, wherein, at least one .sup.1H in Y is substituted by a heavier isotope .sup.2H thereof, and/or at least one .sup.12C in Y is substituted by a heavier isotope .sup.13C thereof; and/or at least one .sup.14N in Y is substituted by a heavier isotope .sup.15N thereof, and/or at least one .sup.16O in Y is substituted by a heavier isotope .sup.18O thereof.

22. The isotope-labeled compound of formula (II) or the salt thereof according to claim 21, wherein the isotope-labeled compound of formula (II) is any one of the following compounds: ##STR00063## ##STR00064## ##STR00065## ##STR00066## ##STR00067## ##STR00068## ##STR00069## ##STR00070## ##STR00071## ##STR00072## ##STR00073## ##STR00074## ##STR00075## ##STR00076## ##STR00077## ##STR00078## ##STR00079## ##STR00080## ##STR00081## ##STR00082## ##STR00083## ##STR00084## ##STR00085## ##STR00086## ##STR00087## ##STR00088## wherein R.sub.0 is ##STR00089## and X is OH or Cl.

23. Application of the compound of formula (I) or the salt thereof according to claim 1 as a derivatization reagent for detecting and/or separating compounds containing hydroxyl and/or amino groups.

24. Application of the isotope-labeled compound of formula (II) or the salt thereof according to claim 20 as a derivatization reagent for detecting and/or separating compounds containing hydroxyl and/or amino groups.

Description

DETAILED DESCRIPTION

[0070] Hereinafter the present invention is further described with reference to the embodiments. However, the present disclosure is not limited to the scope as defined by the embodiments described. Experimental methods with specific conditions unstated in the following embodiments are routine methods with customary conditions that are readily known by a person skilled in the art, or selected in accordance with the specifications of relevant products.

[0071] The sources of experimental reagents in the following examples are as listed in Table 1.

TABLE-US-00001 TABLE 1 Experimental reagents and sources Reagent Source L-leucine Aladdin Acetaldehyde Aladdin 4-(4,6-dimethoxytriazin-2-yl)-4- Aladdin methylmorpholine hydrochloride (DMTMM) N,N-dimethylformamide (DMF) Aladdin Dichloromethane Aladdin Tetrahydrofuran (THF) Aladdin Methanol Sigma Acetonitrile (ACN) Sigma Deuterated methanol (MeOD) Sigma Deuterated acetonitrile (CD.sub.3CN) Sigma Deuterium oxide (D.sub.2O) Sigma 4-methylmorpholine (NMM) Sigma 5-aminonaphthalene sulfonic acid (ANSA) TCI Sodium bicarbonate, petroleum ether, ethyl China National Pharmaceutical Group acetate Double-distilled water Prepared by a double-distillation apparatus using purified water Reverse-phase filler DAISOCEL SP-120-50-ODS-RPC

[0072] Qualitative analysis of each starting material and product was performed by an AB Sciex ExionLC UHPLC system, which included a PDA detector, an auto-sampler, a binary gradient pump, a temperature control unit, and the like modules, and was equipped with an ACQUITY UPLC HSS T3 C18 reverse-phase chromatographic column (1.8 am, 2.1 mm?100 mm). Experiments such as molecular weight and mass spectrometric cleavage of each starting material and product were performed on an AB Sciex X 500R TOE. Fine purification of N,N-diethyl leucyl amido naphthalene sulfonic acid was achieved by an Agilent 1100 series LC system, which included a VWD detector, an auto-sampler, a binary gradient pump, and a temperature control unit, and was equipped with a YMC Pack GDS-A C18 chromatographic was column (5 am, 10 mm?25 mm). Structure and purity information for the starting materials and products in synthetic steps was provided by Bruker Ascend 600 MHz NMR. Anke N-1001D-OSB2100 rotary evaporator was used to remove organic solvent, Christ ALPHA 1-2 LD plus freeze dryer was used to remove water, and glass instruments (Beijing Xinweier Instrument Co.) such as the chromatography column were used to complete the synthesis reaction at each step.

Example 1 Synthesis of N,N-diethyl-L-leucine

[0073] ##STR00038##

[0074] L-leucine powder (800 mg, 6 mmol) was first weighed into a 100 mL round-bottomed flask, 40 mL of sodium acetate or phosphate buffer (0.2 M, pH=2-12) was added and then stirred at 37? C. to dissolve, then sodium cyanoborohydride powder (1.6 g, 24 mmol) was added, and then a acetaldehyde solution (3.4 mL, 60 mmol) was dropwise added. The reaction mixture was stirred at 30-40? C. for 20-28 hours, and finally a 6 mol/L HCl solution (4 mL, 24 mmol) was added and stirred for 10 min to stop the reaction. The organic reagent was removed using a rotary evaporator, and the reactant was lyophilized using a lyophilizer, and then purified by reverse-phase column chromatography to obtain pure N,N-diethylleucine.

[0075] The structure of the pure N,N-diethyl L-leucine was confirmed using NMR and mass spectrometry.

[0076] .sup.1H NMR (600 MHz, D.sub.2O buffer, pH 7.4): ? 0.971 (dd, 6H), 1.298 (t, 6H), 1.650 (m, 2H), 1.760 (m, 1H), 3.247 (m, 4H), 3.668 (dd, 1H); MS+(TOF) m/z 188.1645.

Example 2 Synthesis of N,N-diethyl-L-leucine

[0077] ##STR00039##

[0078] L-leucine powder (800 mg, 6 mmol) was first weighed into a 100 mL round-bottomed flask, 40 mL of sodium acetate or phosphate buffer (0.2 M, pH=2-12) was added and then stirred at 37? C. to dissolve, then 2-methylpyridine borane (1.3 g, 12 mmol) was added, and then a acetaldehyde solution (3.4 mL, 60 mmol) was dropwise added. The reaction mixture was stirred at 30-40? C. for 20-28 hours, and finally a 6 mol/L HCl solution (4 mL, 24 mmol) was added and stirred for 10 min to stop the reaction. The organic reagent was removed using a rotary evaporator, the reactant was lyophilized using a lyophilizer, and then purified by reverse-phase column chromatography to obtain pure N,N-diethylleucine.

Example 3 Synthesis of N,N-diethyl-L-leucine

[0079] ##STR00040##

[0080] Leucine powder (800 mg, 6 mmol) was first weighed into a 100 mL round-bottomed flask, ground potassium carbonate powder (4.8 g, 6 mmol) and 40 mL of acetonitrile were added, and an iodoethane solution (9.6 mL, 120 mmol) was dropwise added under stirring. The reaction mixture was reacted under reflux at 90? C. for 20-28 hours. Excess potassium carbonate was removed by filtration and the solvent was evaporated. The crude product is filtered by addition of diethyl ether, and the precipitate was washed several times with diethyl ether and finally recrystallized from acetonitrile to obtain a purified product.

Example 4 Synthesis of N,N-diethyl leucylamino naphthalene sulfonic acid

[0081] ##STR00041##

[0082] N,N-diethylleucine (800 ?L, 16 mmol) was dissolved in DMF, DMTMM (6.9 mg, 24 mmol) was added, NMM (43.3 ?L, 320 mmol) was dropwise added and vortexed for a moment, and 5-aminonaphthalene sulfonic acid powder (71.6 mg, 640 mmol) was added but not vortex. The reaction mixture was gently placed on a metal shaker, reacted for 8-24 hours at room temperature in 12 groups. The product was purified by extraction, and 192 mL of dichloromethane and 19.2 mL of double distilled water were added to extract impurities to obtain a supernatant.

Example 5 Synthesis of N,N-diethyl leucylamino naphthalene sulfonic acid

[0083] ##STR00042##

[0084] N,N-diethylleucine (35.7 mg, 192 mmol) was dissolved in DMF and activated by the addition of 1.2 equivalents of EDC (44.2 mg, 230 mmol) and HOBt (31.1 mg, 230 mmol). 1.5 equivalents of 5-aminonaphthalene sulfonic acid (64.4 mg, 287.5 mmol) were added thereto, and 2 mL of pyridine was dropwise added thereto, and the reaction was allowed to proceed at normal temperature overnight with stirring.

[0085] The organic reagent was removed using a rotary evaporator and the crude product was purified by reverse-phase column chromatography using a pad of ODS C18 to obtain about 26 mg of yellow-brown powder. Fine purification was carried out using semi-preparative liquid chromatography Agilent 1100 LC-VWD coupled to a rotary evaporator to remove the solvent to obtain a pure product.

[0086] The structure of the pure product was confirmed using NMR and mass spectrometry.

[0087] .sup.1H NMR (600 MHz, meOD): ? 1.232, 1.070 (dd, 6H), 1.435 (t, 6H), 1.832 (m, 2H), 2.050 (m, 1H), 3.411, 3.502 (q, 4H), 4.359 (dd, 1H), 7.213 (m, 1H), 7.402 (m, 1H), 7.457 (m, 1H), 7.692 (m, 1H), 8.037 (m, 1H), 8.722 (m, 1H); MS+(TOF) m/z 393.1845.

Example 6 Synthesis of N,N-diethyl leucyl amido naphthalene sulfonyl chloride

[0088] ##STR00043##

[0089] N,N-diethyl leucylaminonaphthalenesulfonic acid (26.1 mg, 0.07 mmol) was weighed and dissolved in 5 mL of toluene as a solvent under sonication at a molar ratio of 1:50, and excess phosphorus pentachloride (0.7 g, 3.33 mmol) was weighed into a reaction flask. The reaction mixture was allowed to react at room temperature for 1-3 hours. Ice ethyl acetate was added for extraction, an ice saturated sodium bicarbonate solution was gradually dropwise added to quench the reaction, pH was adjusted to 7, and the supernatant is taken and evaporated to dryness to obtain an N,N-diethyl leucyl amido naphthalene sulfonyl chloride crude product.

[0090] The crude product was purified using normal phase column chromatography packed with silica gel, petroleum ether, ethyl acetate, and acetonitrile as eluents, 1:1 (acetonitrile/ethyl acetate) and 1:2 (acetonitrile/ethyl acetate) elution fractions were combined and the solvent was dried up by rotary evaporation to obtain pure N,N-diethyl leucyl amido naphthalenesulfonyl chloride.

[0091] The structure of the pure product was confirmed using NMR.

[0092] .sup.1H NMR (600 MHz, CD.sub.3CN): ? 1.203 (dd, 6H), 1.558 (t, 6H), 1.958 (m, 2H), 7.892 (m, 1H), 8.026 (m, 1H), 8.135 (m, 1H), 8.593 (m, 1H), 8.816 (m, 1H), 8.964 (m, 1H); MS+(TOF) m/z 411.1495.

Example 7 Synthesis of N,N-diethyl leucyl amido naphthalene sulfonyl chloride (DELANS-Cl)

[0093] ##STR00044##

[0094] N,N-diethyl leucylaminonaphthalenesulfonic acid (26.1 mg, 0.067 mmol) was weighed and dissolved in 4 mL THF. 20 equivalents of oxalyl chloride (112 ?L, 1.33 mmol) were diluted to 500 ?L of THF in an ice bath and added into a reaction flask. With 3 drops of DMF added, the reaction mixture was stirred at room temperature and reacted for 10 minutes to 30 minutes. The THF and oxalyl chloride were removed by rotary evaporation.

[0095] The crude product was purified using normal phase column chromatography packed with silica gel, petroleum ether, ethyl acetate, and acetonitrile as eluents, 1:1 (acetonitrile/ethyl acetate) and 1:2 (acetonitrile/ethyl acetate) elution fractions were combined and the solvent was dried up by rotary evaporation to obtain pure N,N-diethyl leucyl amido naphthalenesulfonyl chloride.

Example 8 Synthesis of d.SUB.2.-N,N-diethyl L-leucine

[0096] ##STR00045##

d.SUB.2.-N,N-diethyl L-leucine

[0097] The synthesis steps for d.sub.2-N,N-diethyl L-leucine were the same as those for d.sub.2-N,N-diethyl L-leucine, except that deuterated sodium cyanoborohydride was used as a starting material for the synthesis. (M+H].sup.+=190.1771)

Example 9 Synthesis of d.SUB.2.-N,N-diethyl leucylamino naphthalene sulfonic acid (d2-DELANS-Cl)

[0098] ##STR00046##

d.SUB.2.-N,N-diethyl leucylamino naphthalene sulfonic acid

[0099] The synthesis steps for d.sub.2-N,N-diethyl leucyl amido naphthalene sulfonic acid were the same as those for N,N-diethyl leucyl amido naphthalene sulfonic acid, except that d.sub.2-N,N-diethyl L-leucine was used as a starting material for the synthesis. (M+H].sup.+=395.1968)

Example 10 Synthesis of d.SUB.2.-N,N-diethyl leucyl amido naphthalene sulfonyl chloride

[0100] ##STR00047##

d.SUB.2.-N,N-diethyl leucyl amido naphthalene sulfonyl chloride

[0101] The synthesis steps for d.sub.2-N,N-diethyl leucyl amido naphthalenesulfonyl chloride were the same as those for N,N-diethyl leucyl amido naphthalenesulfonyl chloride, except that d.sub.2-N,N-diethyl leucylamino naphthalene sulfonic acid was used as a starting material for the synthesis.

[0102] The structure of the pure product was confirmed using NMR and mass spectrometry.

[0103] .sup.1H NMR (600 MHz, CD.sub.3CN): ? 1.203 (dd, 6H), 1.518 (d, 3H), 1.592 (d, 3H), 1.959 (m, 2H), 3.479 (m, 2H), 4.388 (m, 1H), 7.875 (m, 1H), 8.038 (m, 1H), 8.132 (m, 1H), 8.584 (m, 1H), 8.831 (m, 1H), 8.959 (m, 1H); MS+(TOF) m/z 413.1602.

Effects Example 1 Derivatization Reaction of the Derivatization Reagent DELANS-Cl on Nucleoside Metabolites

N,N-diethyl leucyl amido naphthalene sulfonyl chloride (DELANS-Cl

[0104] ##STR00048##

d.SUB.2.-N,N-diethyl leucylamino naphthalene sulfonic acid (d.SUB.2.-DELANS-Cl

[0105] ##STR00049##

[0106] The derivatization reagents described above can be used to derivatize amino-, hydroxyl-containing compounds like the classical dansyl chloride, and can also be used to derivatize nucleosides and are described below.

Preparation of Working Solution:

[0107] Powder of nucleoside metabolites listed in Table 2 was accurately weighed separately, and dissolved in a sodium carbonate/sodium bicarbonate buffer solution with a concentration of 250 mM and pH of 9.4 to obtain a standard stock solution of each metabolite at a concentration listed in Table 2. 50 ?L of each of the single standard solutions was mixed to obtain a mixed stock solution of 65 nucleoside metabolites, which was an initial mixed stock solution. The initial mixed stock solution was diluted to obtain a working solution S.sub.1 with a total concentration of about 4 mM, and then the solution was diluted step by step according to a dilution ratio of 1:2:4:10:20:40:100:200:400:1000:2000:4000 to obtain a working solution of totally 12 concentration gradients of S.sub.1, S.sub.2, S.sub.3, S.sub.4, S.sub.5, S.sub.6, S.sub.7, S.sub.8, S.sub.9, S.sub.10, S.sub.11 and S.sub.12.

TABLE-US-00002 TABLE 2 Concentrations of substances in standard stock solution, initial mixed stock solution, working solution S.sub.1, and S.sub.1 derivative reaction solution Concentration Concentration of each Concentration Concentration of each substance in of standard of initial substance in derivative stock mixed stock working reaction solution solution solution S.sub.1 solution S.sub.1 No. Metabolite (mM) (mM) (mM) (mM) 1 Adenine 10.00 0.15 0.0410 911.68 2 Guanine 10.00 0.15 0.0410 911.68 3 Hypoxanthine 10.00 0.15 0.0410 911.68 4 Xanthine 10.00 0.15 0.0410 911.68 5 Uracil 15.00 0.23 0.0615 1367.52 6 Cytosine 10.00 0.15 0.0410 911.68 7 Thymine 10.00 0.15 0.0410 911.68 8 Adenosine 10.00 0.15 0.0410 911.68 9 Guanosine 6.91 0.11 0.0284 630.15 10 Uridine 50.00 0.77 0.2051 4558.40 11 Cytidine 10.00 0.15 0.0410 911.68 12 Thymidine 10.00 0.15 0.0410 911.68 13 Inosine 10.00 0.15 0.0410 911.68 14 Xanthosine 10.00 0.15 0.0410 911.68 15 Deoxyadenosine 10.00 0.15 0.0410 911.68 16 Deoxyguanosine 10.00 0.15 0.0410 911.68 17 Deoxyuridine 10.00 0.15 0.0410 911.68 18 Deoxycytidine 10.00 0.15 0.0410 911.68 19 Deoxyinosine 10.00 0.15 0.0410 911.68 20 1-methyladenosine 10.00 0.15 0.0410 911.68 21 2-methyladenosine 10.00 0.15 0.0410 911.68 22 N.sup.6-methyladenosine 10.00 0.15 0.0410 911.68 23 2-O-methyladenosine 15.00 0.23 0.0615 1367.52 24 N.sup.6,2-O-dimethyladenosine 10.00 0.15 0.0410 911.68 25 N.sup.1,2-O-dimethyladenosine 10.00 0.15 0.0410 911.68 26 3-O-methyladenosine 10.00 0.15 0.0410 911.68 27 8-methyladenosine 10.00 0.15 0.0410 911.68 28 3-methylcytidine 10.00 0.15 0.0410 911.68 29 N.sup.4-methylcytidine 10.00 0.15 0.0410 911.68 30 5-methylcytidine 10.00 0.15 0.0410 911.68 31 5-hydroxymethylcytidine 10.00 0.15 0.0410 911.68 32 2-O-methylcytidine 50.00 0.77 0.2051 4558.40 33 N.sup.4,2-O-dimethylcytidine 10.00 0.15 0.0410 911.68 34 2-C-methylcytidine 10.00 0.15 0.0410 911.68 35 5-methyl-2-O-methylcytidine 50.00 0.77 0.2051 4558.40 36 1-methylguanosine 10.00 0.15 0.0410 911.68 37 2-methylguanosine 4.00 0.06 0.0164 364.67 38 N.sup.2,N.sup.2-dimethylguanosine 10.00 0.15 0.0410 911.68 39 7-methylguanosine 10.00 0.15 0.0410 911.68 40 2-O-methylguanosine 10.00 0.15 0.0410 911.68 41 5-methyluridine 10.00 0.15 0.0410 911.68 42 2-O-methyluridine 10.00 0.15 0.0410 911.68 43 5-methyl-2-O-methyluridine 10.00 0.15 0.0410 911.68 44 2-O-methoxyinosine 10.00 0.15 0.0410 911.68 45 5-methyldeoxycytidine 10.00 0.15 0.0410 911.68 46 5-hydroxymethyldeoxycytidine 50.00 0.77 0.2051 4558.40 47 N.sup.6-methyldeoxyadenosine 100.00 1.54 0.4103 9116.81 48 3-methyladenine 11.05 0.17 0.0453 1007.41 49 5-methylcytosine 10.00 0.15 0.0410 911.68 50 5-hydroxymethylcytosine 15.00 0.23 0.0615 1367.52 51 5-formylcytosine 10.00 0.15 0.0410 911.68 52 5-carboxylcytosine 15.00 0.23 0.0615 1367.52 53 7-methylguanine 50.00 0.77 0.2051 4558.40 54 5,6-dihydrouracil 10.00 0.15 0.0410 911.68 55 2-aminoadenosine 10.00 0.15 0.0410 911.68 56 8-aminoadenosine 10.00 0.15 0.0410 911.68 57 8-oxoadenosine 10.00 0.15 0.0410 911.68 58 Dihydrouridine 10.00 0.15 0.0410 911.68 59 Pseudouridine 10.00 0.15 0.0410 911.68 60 Orotidine 10.00 0.15 0.0410 911.68 61 5-methoxycarbonylmethyluridine 10.00 0.15 0.0410 911.68 62 5-aminomethylcarbamoylmethyluridine 4.00 0.06 0.0164 364.67 63 5-methoxycarbonylmethyl-2- 10.00 0.15 0.0410 911.68 thiouridine 64 5-methoxycarbonylmethyl-2-O- 10.00 0.15 0.0410 911.68 methyluridine 65 N.sup.4-acetylcytidine 8.40 0.13 0.0345 765.81

Preparation of Internal Standard Solution:

[0108] A d.sub.2-DELANS-Cl solution with a concentration of 5 mmol/L was prepared with the solvent of the isotope-labeled derivatization reagent d.sub.2-DELANS-Cl as a dry acetonitrile solution. 50 ?L of a working solution S.sub.2 was transferred into a 500 ?L EP tube using a pipette, 400 ?L of a d2-DELANS-Cl solution (5 mM, dissolved in acetonitrile) was sucked and added thereto, the reaction EP tube was placed on a metal shaker, the reaction temperature was set to 37? C., and the reaction was carried out at a shaking frequency of 900 rpm for 5 hours. The reaction mixture was immediately cooled on ice to quench the reaction. Upon completion of the derivatization reaction, 100 ?L of the reaction solution was taken out, diluted 5-fold with an acetonitrile solution, and mixed well to obtain the internal standard solution. The obtained solution was sealed and stored at low temperature of ?20? C. or ?80? C.

Derivatization Reaction of Derivatization Reagent DELANS-Cl with Standard Solution and Establishment of Linear Curve:

[0109] A DELANS-Cl solution with a concentration of 5 mmol/L was prepared with a dry acetonitrile solution as the solvent of the derivatization reagent. First, 5 ?L of a standard solution (namely, working solution with various concentration gradients) (dissolved in a sodium bicarbonate buffer solution with a concentration of 250 mM and pH of 9.4) was transferred using a pipette, and placed into a 500 ?L EP tube, then 40 ?L of a DELANS-Cl solution (5 mM, acetonitrile solution) was sucked and added thereto, the reaction EP tube was placed into a metal shaker, the reaction temperature was set to 37? C., and the reaction was carried out at a shaking frequency of 900 rpm for 5 hours. The reaction mixture solution was then immediately cooled on ice to quench the reaction. Upon completion of the derivatization reaction, 8 ?L of the reaction solution was taken out, and diluted 5-fold with the acetonitrile solution, and then 10 ?L of an internal standard solution (volume ratio: 4:1) was added and mixed well. Treated samples were sealed and stored at low temperature of ?20? C. or ?80? C. before being subjected to analysis by the UHPLC-MS system.

Derivatization Reaction of Derivatization Reagent DELANS-Cl with Sample:

[0110] The derivatization reaction of nucleoside metabolites in the sample was as follows. 5 L of a sample solution (urine sample, serum sample, tissue sample, and lung cancer cell sample respectively) was taken out into a 1.5 mL EP tube, 40 ?L of a DELANS-Cl solution was transferred (5 mM, dissolved in an acetonitrile solution) thereto, the reaction EP tube was placed on a metal shaker, the reaction temperature is set to 37? C., and the reaction was carried out at a shaking frequency of 900 rpm for 5 hours. The reaction mixture solution was immediately cooled on ice to quench the reaction. Finally, upon completion of the derivatization reaction, 8 ?L of the reaction solution was taken out, diluted 5-fold with the acetonitrile solution, and 10 ?L of internal standard solution (volume ratio: 4:1) was added and mixed well. UHPLC-MS system analysis was prepared.

[0111] Urine samples were collected from adult male morning urine. Serum samples were collected from healthy adults in accordance with the relevant requirements of scientific research ethics of Fudan University and national laws. Tissue samples were taken from rabbit liver. Lung cancer cell samples: the cell sample selected in the experiment was non-small cell lung adenocarcinoma cell line A549.

[0112] For the derivatization reaction of nucleoside metabolites with derivatization reagents N,N-dimethylamino naphthalene sulfonyl chloride (DNS-Cl) and N,N-diethylamino naphthalene sulfonyl chloride (DENS-Cl), reference is made to DELANS-Cl.

N,N-dimethylamino naphthalene sulfonyl chloride (DNS-Cl

[0113] ##STR00050##

N,N-diethylamino naphthalene sulfonyl chloride (DENS-Cl

[0114] ##STR00051##

Test Method:

[0115] The liquid phase was equipped with a Waters ACQUITY UPLC HSST3 C18 reverse-phase chromatographic column (Waters, Technologies, Milford. USA). The column temperature was 40? C., and the autosampler temperature was 4? C. Mobile phase A was 0.1% formic acid in water (MilliQ ultrapure water) and mobile phase B was 0.1% formic acid in acetonitrile. The elution gradient (B %) was as follows: 0-0.5 min: 2-25%, 0.5-3. 6 min: 25%, 3.6-3.7 min: 25-30%, 3.7-4.5 min: 30%, 4.5-6 min: 30-40%, 6-7 min: 40-90%, 7-8 min: 95%. The flow rate was 0.5 mL/min and the injection volume was 1 ?L.

[0116] Mass spectrum AB Sciex 6500 plus QTRAP (ESI-MS/MS) employed a positive ion mode with ion source (chamber) conditions as follows: air curtain gas pressure 35 psi, collision cell gas flow selection medium, ion spray voltage 4500 V, spray gas pressure 55 psi, spray gas temperature 400? C., and auxiliary heater gas pressure 50 psi. The scanning mode was scheduled multiple reaction monitoring (sMRM) mode. The common daughter ion of light-labeled derivatization product was m/z 142. 2, and that of heavy-labeled derivatization product is m/z 144. 2. The collision energy (CE) of each derivatization product was respectively optimized upon derivatization under each derivatization standard.

Test Results 1 Linear Range, Correlation Coefficient, and Limit of Quantification of 65 Nucleoside Metabolites

[0117] DELANS-Cl was reacted with the working solutions of various concentration gradients to obtain the linear ranges, linear correlation coefficients, and minimum quantitation limits of 65 nucleoside metabolites.

TABLE-US-00003 TABLE 3 Linear range, linear correlation coefficient, and limit of quantification of 65 nucleoside metabolites Limit of Linear range (nM) Linear quantitation Lower Upper correlation No. Metabolite Linear equation LOQ (fmol) limit limit coefficient (R.sup.2 1 Adenine y = 0.0091x + 0.0116 0.198 0.182 729.345 0.998 2 Guanine y = 0.0118x + 0.0129 0.203 0.182 729.345 0.997 3 Hypoxanthine y = 0.0122x + 0.0049 0.246 0.182 729.345 0.996 4 Xanthine y = 0.0078x + 0.0131 0.045 0.182 729.345 0.998 5 Uracil y = 0.0078x + 0.0138 0.032 0.274 1094.017 0.998 6 Cytosine y = 0.0226x + 0.5196 0.517 1.823 729.345 0.997 7 Thymine y = 0.0185x + 0.0060 0.079 0.182 729.345 0.997 8 Adenosine y = 0.0202x + 0.0164 0.521 0.182 729.345 0.997 9 Guanosine y = 0.0174x + 0.0247 0.286 0.252 504.123 0.998 10 Uridine y = 0.0033x + 0.0084 1.216 0.912 3646.724 0.997 11 Cytidine y = 0.0175x + 0.2168 0.368 0.365 729.345 0.997 12 Thymidine y = 0.0228x + 0.1164 1.351 0.729 729.345 0.995 13 Inosine y = 0.0151x + 0.0418 0.351 0.182 729.345 0.996 14 Xanthosine y = 0.0165x + 0.0743 0.445 0.365 729.345 0.998 15 Deoxyadenosine y = 0.0260x + 0.7010 1.057 0.729 729.345 0.997 16 Deoxyguanosine y = 0.0290x + 0.0036 0.217 0.182 729.345 0.995 17 Deoxyuridine y = 0.0278x + 0.0619 2.224 1.823 729.345 0.997 18 Deoxycytidine y = 0.0711x + 0.1102 3.039 1.823 729.345 0.997 19 Deoxyinosine y = 0.0130x + 0.0007 0.090 0.182 729.345 0.997 20 1-methyladenosine y = 0.0154x + 0.0056 0.109 0.182 729.345 0.999 21 2-methyladenosine y = 0.0220x + 0.1307 0.038 0.182 729.345 0.997 22 N.sup.6-methyladenosine y = 0.0206x + 0.0041 0.200 0.182 729.345 0.997 23 2-O-methyladenosine y = 0.0248x + 0.3439 7.597 2.735 729.345 0.995 24 N.sup.6,2-O-dimethyladenosine y = 0.0075x + 0.0117 1.552 0.729 729.345 0.998 25 N.sup.1,2-O-dimethyladenosine y = 0.0267x + 2.6341 1.614 1.823 729.345 0.997 26 3-O-methyladenosine y = 0.0431x + 0.0202 6.078 1.823 729.345 0.997 27 8-methyladenosine y = 0.0222x + 0.0046 0.450 0.365 729.345 0.998 28 3-methylcytidine y = 0.0133x + 0.0019 0.314 0.182 729.345 0.995 29 N.sup.4-methylcytidine y = 0.0220x + 0.0898 3.090 1.823 729.345 0.999 30 5-methylcytidine y = 0.0246x + 0.0033 3.171 0.729 729.345 0.997 31 5-hydroxymethylcytidine y = 0.0217x + 0.3079 2.279 1.823 729.345 0.997 32 2-O-methylcytidine y = 0.0013x + 0.0091 4.144 1.823 729.345 0.998 33 N.sup.4,2-O-dimethylcytidine y = 0.0460x + 0.0256 7.597 1.823 729.345 0.999 34 2-C-methylcytidine y = 0.0219x + 0.0017 8.481 3.647 729.345 0.998 35 5-methyl-2-O-methylcytidine y = 0.0076x + 0.2137 9.910 9.117 729.345 0.996 36 1-methylguanosine y = 0.0215x + 0.0022 0.145 0.182 729.345 0.997 37 2-methylguanosine y = 0.0249x + 0.0083 0.374 0.292 729.345 0.995 38 N.sup.2,N.sup.2-dimethylguanosine y = 0.0133x + 0.0208 7.013 1.823 729.345 0.996 39 7-methylguanosine y = 0.0107x + 0.0044 0.414 0.182 729.345 0.998 40 2-O-methylguanosine y = 0.0310x + 0.0078 0.424 0.182 729.345 0.996 41 5-methyluridine y = 0.0184x + 0.0042 0.264 0.182 729.345 0.997 42 2-O-methyluridine y = 0.0323x + 0.0051 1.140 0.365 729.345 0.998 43 5-methyl-2-O-methyluridine y = 0.0358x + 0.0886 0.986 0.729 729.345 0.996 44 2-O-methoxyinosine y = 0.0136x + 0.0115 0.038 0.182 729.345 0.997 45 5-methyldeoxycytidine y = 0.0077x + 0.1500 0.506 0.365 729.345 0.997 46 5-hydroxymethyldeoxycytidine y = 0.0039x + 0.0874 17.532 9.117 729.345 0.998 47 N.sup.6-methyldeoxyadenosine y = 0.0012x + 0.0048 40.519 18.234 729.345 0.997 48 3-methyladenine y = 0.0306x + 0.0612 0.611 0.201 729.345 0.999 49 5-methylcytosine y = 0.0207x + 0.1625 0.157 0.365 729.345 0.996 50 5-hydroxymethylcytosine y = 0.0164x + 0.0390 1.403 0.547 729.345 0.998 51 5-formylcytosine y = 0.0089x + 0.0314 0.744 0.365 729.345 0.999 52 5-carboxylcytosine y = 0.0135x + 0.0632 0.257 1.094 729.345 0.996 53 7-methylguanine y = 0.0042x + 0.0397 3.721 1.823 729.345 0.997 54 5,6-dihydrouracil y = 0.0130x + 0.0192 0.276 0.182 729.345 0.998 55 2-aminoadenosine y = 0.0229x + 0.0007 0.829 0.365 729.345 0.998 56 8-aminoadenosine y = 0.0140x + 0.0005 0.445 0.182 729.345 0.999 57 8-oxoadenosine y = 0.0126x + 0.0105 0.536 0.182 729.345 0.999 58 Dihydrouridine y = 0.0237x + 0.0040 0.368 0.365 729.345 0.998 59 Pseudouridine y = 0.0105x + 0.0009 0.068 0.182 729.345 0.997 60 Orotidine y = 0.0023x + 0.0055 6.181 3.647 729.345 0.996 61 5-methoxycarbonylmethyluridine y = 0.0214x + 0.0003 0.376 0.365 729.345 0.998 62 5-aminomethylcarbamoylmethyluridine y = 0.0359x + 0.0285 1.488 0.729 729.345 0.998 63 5-methoxycarbonylmethyl-2-thiouridine y = 0.0026x + 0.0303 52.096 18.234 729.345 0.995 64 5-methoxycarbonylmethyl-2-O- y = 0.0260x + 0.0376 0.285 0.182 729.345 0.997 methyluridine 65 N.sup.4-acetylcytidine y = 0.0295x + 0.0021 0.557 0.306 729.345 0.997 indicates data missing or illegible when filed

Test Results 2

[0118] The results of sensitivity comparison of derivatization reaction of DELANS-Cl with DNS-Cl and DENS-Cl are listed in Table 4.

TABLE-US-00004 TABLE 4 DELANS-Cl vs DNS-Cl and DENS-Cl derivatization sensitivity comparison DELANS-Cl DELANS-Cl LOQ for LOQ for LOQ for vs DNS-Cl vs DENS-Cl DELANS-Cl DNS-Cl DENS-Cl sensitivity sensitivity method method method improvement enhancement No. Metabolite (fmol) (fmol) (fmol) factor factor 1 Adenine 0.198 0.148 0.289 0.75 1.46 2 Guanine 0.203 12.662 6.119 62.50 30.20 3 Hypoxanthine 0.246 6.907 3.453 28.03 14.02 4 Xanthine 0.045 9.117 5.180 203.00 115.34 5 Uracil 0.032 0.810 0.595 25.03 18.37 6 Cytosine 0.517 2.650 13.898 5.13 26.91 7 Thymine 0.079 0.556 0.289 7.01 3.64 8 Adenosine 0.521 15.195 2.849 29.17 5.47 9 Guanosine 0.286 1.575 2.864 5.50 10.00 10 Uridine 1.216 11.748 1.521 9.66 1.25 11 Cytidine 0.368 3.506 5.698 9.52 15.47 12 Thymidine 1.351 42.207 4.604 31.25 3.41 13 Inosine 0.351 189.938 22.237 541.68 63.42 14 Xanthosine 0.445 3.506 5.698 7.88 12.81 15 Deoxyadenosine 1.057 10.130 25.682 9.58 24.30 16 Deoxyguanosine 0.217 1.302 2.192 6.00 10.10 17 Deoxyuridine 2.224 15.452 7.473 6.95 3.36 18 Deoxycytidine 3.039 25.324 112.000 8.33 36.86 19 Deoxyinosine 0.090 2.374 2.590 26.43 28.84 20 1-methyladenosine 0.109 0.127 0.375 1.17 3.46 21 2-methyladenosine 0.038 2.730 8.082 72.75 215.43 22 N.sup.6-methyladenosine 0.200 1.266 1.190 6.32 5.94 23 2-O-methyladenosine 7.597 14.245 43.831 1.88 5.77 24 N.sup.6,2-O-dimethyladenosine 1.552 6.512 21.104 4.20 13.60 25 N.sup.1,2-O-dimethyladenosine 1.614 7.597 22.792 4.71 14.13 26 3-O-methyladenosine 6.078 15.195 7.597 2.50 1.25 27 8-methyladenosine 0.450 0.684 0.829 1.52 1.84 28 3-methylcytidine 0.314 0.712 1.952 2.27 6.21 29 N.sup.4-methylcytidine 3.090 3.618 7.236 1.17 2.34 30 5-methylcytidine 3.171 3.506 35.753 1.11 11.27 31 5-hydroxymethylcytidine 2.279 4.341 10.130 1.90 4.44 32 2-O-methylcytidine 4.144 18.993 33.518 4.58 8.09 33 N.sup.4,2-O-dimethylcytidine 7.597 1.302 172.340 0.17 22.68 34 2-C-methylcytidine 8.481 2.279 10.244 0.27 1.21 35 5-methyl-2-O-methylcytidine 9.910 45.584 223.451 4.60 22.55 36 1-methylguanosine 0.145 0.190 0.570 1.31 3.94 37 2-methylguanosine 0.374 18.990 10.483 50.77 28.03 38 N.sup.2,N.sup.2-dimethylguanosine 7.013 4.798 20.720 0.68 2.95 39 7-methylguanosine 0.414 0.190 1.140 0.46 2.75 40 2-O-methylguanosine 0.424 1.302 0.912 3.07 2.15 41 5-methyluridine 0.264 4.144 1.216 15.68 4.60 42 2-O-methyluridine 1.140 14.245 13.024 12.50 11.43 43 5-methyl-2-O-methyluridine 0.986 8.140 1.349 8.26 1.37 44 2-O-methoxyinosine 0.038 2.171 1.212 57.62 32.18 45 5-methyldeoxycytidine 0.506 45.584 113.963 90.00 225.00 46 5-hydroxymethyldeoxycytidine 17.532 63.311 284.900 3.61 16.25 47 N.sup.6-methyldeoxyadenosine 40.519 5.534 11.395 0.14 0.28 48 3-methyladenine 0.611 4.131 97.620 6.77 159.89 49 5-methylcytosine 0.157 1.727 9.027 10.98 57.43 50 5-hydroxymethylcytosine 1.403 5.698 8.339 4.06 5.95 51 5-formylcytosine 0.744 12.662 7.597 17.01 10.21 52 5-carboxylcytosine 0.257 19.877 37.500 77.40 146.02 53 7-methylguanine 3.721 21.104 2.952 5.67 0.79 54 5,6-dihydrouracil 0.276 21.707 19.648 78.57 71.12 55 2-aminoadenosine 0.829 1.530 4.240 1.85 5.12 56 8-aminoadenosine 0.445 1.838 2.903 4.13 6.53 57 8-oxoadenosine 0.536 18.381 27.460 34.27 51.20 58 Dihydrouridine 0.368 2.399 11.396 6.51 30.94 59 Pseudouridine 0.068 0.414 1.249 6.09 18.36 60 Orotidine 6.181 1.599 9.117 0.26 1.48 61 5-methoxycarbonylmethyluridine 0.376 5.698 0.166 15.16 0.44 62 5-aminomethylcarbamoylmethyluridine 1.488 9.402 2.600 6.32 1.75 63 5-methoxycarbonylmethyl-2-thiouridine 52.096 2170.667 75.973 41.67 1.46 64 5-methoxycarbonylmethyl-2-O-methyluridine 0.285 24.508 7.859 86.02 27.59 65 N.sup.4-acetylcytidine 0.557 9.190 5.698 16.50 10.23

[0119] By comparison between the improved sensitivities of the derivatization reagent DELANS-Cl of the present disclosure and the commercial derivatization reagent DNS-Cl, it can be seen that the sensitivities of more than 58 metabolites in the nucleoside metabolite library (65) are improved. Compared with DNS-Cl, the derivatization reagent DELANS-Cl of the present disclosure improves the sensitivity up to 541 times. Compared with DENS-Cl, the derivatization reagent DELANS-Cl of the present disclosure increases the sensitivity up to 225 times.

Test Results 3

[0120] The derivatization reagent DELANS-Cl can be used to derivatize amino- and hydroxyl-containing metabolites (i.e. nucleoside metabolites) in urine, serum, tissue and cell samples. Upon derivatization, the derivatized samples can be quantitatively detected by ultra performance liquid chromatography-mass spectrometry (UHPLC-MS/MS) to detect 58, 55, 59 and 60 nucleoside metabolites, respectively. The analysis results are listed in Table 5.

TABLE-US-00005 TABLE 5 Detection results of nucleoside metabolites in urine, serum, tissue, and cell samples Urine Serum Tissue Cell No. Metabolite (10.sup.?1 ?M) (10.sup.?1 ?M) (10.sup.?2 nmol/mg) (10.sup.?2 nmol/mg) 1 Adenine 122.0 ? 5.7 104.4 ? 4.1 52.8 ? 5.8 147.4 ? 8.3 2 Guanine 89.4 ? 7.9 ND .sup.197 ? 10.2 133.7 ? 8.8 3 Hypoxanthine 207.5 ? 17.8 13.9 ? 1.4 875.8 ? 80.3 91.9 ? 10.6 4 Xanthine 414.2 ? 16.8 8.4 ? 0.9 1774.2 ? 87.4 145.1 ? 9.4 5 Uracil 386.8 ? 15.4 0.8 ? 0.1 324.1 ? 10.6 149.0 ? 5.9 6 Cytosine 0.1 ? 0.0 0.1 ? 0.0 237.9 ? 25.4 11.0 ? 1.0 7 Thymine 10.8 ? 0.5 0.7 ? 0.2 3.2 ? 0.5 2.4 ? 0.3 8 Adenosine 23.1 ? 3.2 1.4 ? 0.4 2269.4 ? 175.1 2231.5 ? 187.0 9 Guanosine 3.1 ? 1.1 0.1 ? 0.0 3261.8 ? 294.3 .sup.2121 ? 216.6 10 Uridine 11.6 ? 2.0 16.6 ? 1.6 2551.2 ? 103.7 1995.9 ? 100.0 11 Cytidine 15.1 ? 2.6 ND .sup.2498 ? 302.3 2017.9 ? 201.6 12 Thymidine 15.6 ? 4.4 63.1 ? 12.4 18979.1 ? 1308.5 13179.1 ? 1847.5 13 Inosine 19.4 ? 4.6 2.8 ? 1.4 2801.6 ? 241.1 1248.2 ? 117.6 14 Xanthosine 34.6 ? 7.7 5.0 ? 1.7 40.6 ? 9.5 ND 15 Deoxyadenosine ND 1292.8 ? 247.1 8450.8 ? 580.0 4144.2 ? 329.0 16 Deoxyguanosine 121.4 ? 26.2 14.1 ? 2.3 844.1 ? 91.5 1069.5 ? 104.1 17 Deoxyuridine 14.4 ? 5.3 11.0 ? 3.4 687.7 ? 92.0 99.7 ? 10.5 18 Deoxycytidine ND 27.6 ? 8.4 571.5 ? 114.5 363.7 ? 47.3 19 Deoxyinosine 0.3 ? 0.1 0.1 ? 0.1 168.8 ? 18.4 3.4 ? 0.4 20 1-methyladenosine 110.4 ? 7.7 0.2 ? 0.1 24.4 ? 2.2 15.2 ? 0.9 21 2-methyladenosine 4.9 ? 3.7 2.5 ? 1.4 11.4 ? 1.7 1.1 ? 0.1 22 N.sup.6-methyladenosine 1.0 ? 0.2 0.4 ? 0.2 5.8 ? 1.5 5.9 ? 1 23 2-O-methyladenosine 113.7 ? 17.7 8.1 ? 3.6 6650.6 ? 926.3 1953.2 ? 177.9 24 N.sup.6,2-O-dimethyladenosine 25.5 ? 10.6 7.3 ? 1.7 616.5 ? 51.2 118.9 ? 8.1 25 N.sup.1,2-O-dimethyladenosine 129.6 ? 47.8 1.4 ? 0.0 1638.1 ? 185.9 496.4 ? 65.1 26 3-O-methyladenosine 4.7 ? 0.9 3.1 ? 2.1 176.5 ? 18.3 31.6 ? 5.5 27 8-methyladenosine 12.7 ? 7.2 4.2 ? 2.2 42.1 ? 6.0 27.9 ? 4.7 28 3-methylcytidine 34.8 ? 4.0 0.4 ? 0.2 36.4 ? 6.1 5.4 ? 1.0 29 N.sup.4-methylcytidine ND ND ND 16.1 ? 8.1 30 5-methylcytidine 9.3 ? 3.7 3.2 ? 1.4 21.3 ? 4.1 13.6 ? 3.1 31 5-hydroxymethylcytidine 14.1 ? 4.7 26.916.9 .sup.1154 ? 108.8 404.4 ? 39.0 32 2-O-methylcytidine 632.7 ? 73.9 9.4 ? 5.7 20587.7 ? 2113.8 7091.8 ? 685.8 33 N.sup.4,2-O-dimethylcytidine 18.6 ? 11.3 7.8 ? 2.0 ND ND 34 2-C-methylcytidine 13.7 ? 2.2 4.4 ? 1.7 ND 12.4 ? 2.7 35 5-methyl-2-O-methylcytidine ND ND 311.5 ? 92.7 54.2 ? 17.4 36 1-methylguanosine 33.7 ? 4.5 0.3 ? 0.1 14.3 ? 2.9 7.5 ? 1.1 37 2-methylguanosine 8.2 ? 1.0 ND 1.8 ? 1.4 0.8 ? 0.2 38 N.sup.2,N.sup.2-dimethylguanosine 217.5 ? 28.5 2.2 ? 0.6 26.4 ? 5.3 10.6 ? 1.9 39 7-methylguanosine 0.9 ? 0.3 0.2 ? 0.1 22.6 ? 2.1 12.4 ? 1.2 40 2-O-methylguanosine 5.8 ? 1.0 0.9 ? 0.2 38.4 ? 12.3 25.3 ? 5.1 41 5-methyluridine 1.1 ? 0.8 1.9 ? 1.1 73.4 ? 6.1 35.1 ? 5.3 42 2-O-methyluridine 6 ? 2.8 1.8 ? 0.5 110.3 ? 19.9 76.6 ? 16 43 5-methyl-2-O-methyluridine 12.1 ? 2.5 5.4 ? 1.4 357.6 ? 101.3 32.1 ? 8.2 44 2-O-methoxyinosine 4.5 ? 0.5 0.7 ? 0.2 11.2 ? 1.8 3.4 ? 0.6 45 5-methyldeoxycytidine 13.3 ? 4.sup. 1.2 ? 0.0 63.3 ? 9.3 10.4 ? 1.5 46 5-hydroxymethyldeoxycytidine 11.8 ? 0.0 ND ND ND 47 N.sup.6-methyldeoxyadenosine ND ND 5938.6 ? 866.8 2490.8 ? 520.0 48 3-methyladenine 30.6 ? 13.0 8.6 ? 1.0 330.0 ? 35.0 85.6 ? 9.8 49 5-methylcytosine ND ND 454.5 ? 43.3 346.8 ? 33.1 50 5-hydroxymethylcytosine ND 6.6 ? 3.5 328.0 ? 43.6 355.4 ? 51.5 51 5-formylcytosine 323.3 ? 25.6 27.7 ? 3.7 192.8 ? 23.8 ND 52 5-carboxylcytosine 284.2 ? 53.6 82.8 ? 15.8 1522.8 ? 299.6 293.3 ? 55.5 53 7-methylguanine 5895.5 ? 737.6 20.9 ? 4.8 497.9 ? 109.9 276.3 ? 91.0 54 5,6-dihydrouracil 254.7 ? 9.0 0.1 ? 0.1 243.3 ? 37.1 107.7 ? 7.9 55 2-aminoadenosine 8.3 ? 2.9 1.9 ? 1.3 ND 2.3 ? 0.5 56 8-aminoadenosine 2.3 ? 0.5 0.5 ? 0.3 9.5 ? 1.9 5.8 ? 1.4 57 8-oxoadenosine 7.2 ? 2.1 1.8 ? 0.6 4867.6 ? 491.2 3239.8 ? 331.7 58 Dihydrouridine 284.6 ? 30.7 5.1 ? 1.7 171.6 ? 28.1 46.3 ? 7.3 59 Pseudouridine 1369.9 ? 59.8 15 ? 1.7 260.3 ? 15.5 154.4 ? 9.3 60 Orotidine 30 ? 12.5 ND ND ND 61 5-methoxycarbonylmethyluridine 3.2 ? 1.3 0.4 ? 0.2 34.7 ? 9.0 23.2 ? 5.9 62 5-aminomethylcarbamoylmethyluridine 21.1 ? 3.6 2.2 ? 0.8 339.0 ? 55.1 181.1 ? 38.8 63 5-methoxycarbonylmethyl-2-thiouridine 51.8 ? 11.0 ND 63.7 ? 50.0 41.2 ? 6.1 64 5-methoxycarbonylmethyl-2-O- 10.4 ? 1.3 1.5 ? 0.8 999.2 ? 193.5 121.3 ? 25.8 methyluridine 65 N.sup.4-acetylcytidine 24.2 ? 6.2 2.5 ? 1.0 146.0 ? 24.2 97.1 ? 17.9

[0121] Comments: nd means not detected or below the limit of quantitation.

Test Result 4

[0122]

TABLE-US-00006 TABLE 6 Retention time of working solution S.sub.2 and metabolites in working solution S.sub.2 upon derivatization with DELANS-Cl Retention time without Retention time upon derivatization (min) derivatization (min) Adenine 1.021 4.402 Guanine 1.052 3.298 Hypoxanthine 1.023 4.149 Xanthine 1.040 2.610 Uracil 1.047 4.631 Cytosine 1.047 3.084 Thymine 0.544 5.646 Adenosine 0.761 1.970 Guanosine 2.099 1.921 Uridine 2.495 3.004 Cytidine 1.080 1.835 Thymidine 1.083 4.979 Inosine 2.250 1.981 Xanthosine 2.001 2.053 Deoxyadenosine 2.866 4.065 Deoxyguanosine 2.474 3.835 Deoxyuridine 2.623 4.534 Deoxycytidine 2.388 3.791 Deoxyinosine 1.408 4.179 1-methyladenosine 1.493 1.491 2-methyladenosine 2.014 2.001 N.sup.6-methyladenosine 1.991 2.441 2-O-methyladenosine 3.206 4.790 N.sup.6,2-O-dimethyladenosine 2.961 5.615 N.sup.1,2-O-dimethyladenosine 2.961 2.484 3-O-methyladenosine 2.432 4.407 8-methyladenosine 2.011 2.275 3-methylcytidine 1.621 1.574 N.sup.4-methylcytidine 1.418 1.982 5-methylcytidine 1.440 1.881 5-hydroxymethylcytidine 1.437 1.840 2-O-methylcytidine 2.663 2.681 N.sup.4,2-O-dimethylcytidine 2.323 3.036 2-C-methylcytidine 2.477 1.911 5-methyl-2-O-methylcytidine 1.173 2.861 1-methylguanosine 2.039 2.041 2-methylguanosine 2.061 2.752 N.sup.2,N.sup.2-dimethylguanosine 2.201 2.371 7-methylguanosine 2.173 1.441 2-O-methylguanosine 3.117 4.511 5-methyluridine 3.197 3.480 2-O-methyluridine 2.718 4.831 5-methyl-2-O-methyluridine 2.949 5.270 2-O-methoxyinosine 3.820 4.851 5-methyldeoxycytidine 1.081 2.441 5-hydroxymethyldeoxycytidine 1.086 1.795 N.sup.6-methyldeoxyadenosine 1.170 3.181 3-methyladenine 3.303 5.558 5-methylcytosine 0.756 5.274 5-hydroxymethylcytosine 0.526 2.596 5-formylcytosine 0.408 5.533 5-carboxylcytosine 1.442 4.632 7-methylguanine 1.727 4.841 5,6-dihydrouracil 1.075 4.690 2-aminoadenosine 1.318 1.636 8-aminoadenosine 1.318 1.551 8-oxoadenosine 1.457 1.844 Dihydrouridine 1.089 2.631 Pseudouridine 1.111 2.132 Orotidine 1.894 2.515 5-methoxycarbonylmethyluridine 3.346 3.852 5-aminomethylcarbamoylmethyluridine 1.925 2.556 5-methoxycarbonylmethyl-2- 4.499 5.248 thiouridine 5-methoxycarbonylmethyl-2-O- 4.433 5.318 methyluridine N.sup.4-acetylcytidine 3.375 3.533

[0123] By comparison, partially underivatized metabolites have poor retention on the chromatographic column (retention time close to the dead volume of the column, about 0.5 min), and metabolites with poor retention on the chromatographic column were improved after derivatization, with retention time within 3-6 min.