CN114509508A - Method for detecting tryptophan and metabolite and application - Google Patents
Method for detecting tryptophan and metabolite and application Download PDFInfo
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- CN114509508A CN114509508A CN202011288293.XA CN202011288293A CN114509508A CN 114509508 A CN114509508 A CN 114509508A CN 202011288293 A CN202011288293 A CN 202011288293A CN 114509508 A CN114509508 A CN 114509508A
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- alumina
- tryptophan
- acid
- solution
- metabolites
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- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 title claims abstract description 88
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 title claims abstract description 86
- 239000002207 metabolite Substances 0.000 title claims abstract description 72
- 238000000034 method Methods 0.000 title claims abstract description 52
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 120
- 239000012071 phase Substances 0.000 claims abstract description 31
- QZAYGJVTTNCVMB-UHFFFAOYSA-N serotonin Chemical compound C1=C(O)C=C2C(CCN)=CNC2=C1 QZAYGJVTTNCVMB-UHFFFAOYSA-N 0.000 claims abstract description 29
- YGPSJZOEDVAXAB-UHFFFAOYSA-N kynurenine Chemical compound OC(=O)C(N)CC(=O)C1=CC=CC=C1N YGPSJZOEDVAXAB-UHFFFAOYSA-N 0.000 claims abstract description 25
- 238000004458 analytical method Methods 0.000 claims abstract description 18
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 claims abstract description 17
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims abstract description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 16
- 229940076279 serotonin Drugs 0.000 claims abstract description 12
- YRKCREAYFQTBPV-UHFFFAOYSA-N acetylacetone Chemical compound CC(=O)CC(C)=O YRKCREAYFQTBPV-UHFFFAOYSA-N 0.000 claims abstract description 10
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims abstract description 8
- 235000019253 formic acid Nutrition 0.000 claims abstract description 8
- 238000000622 liquid--liquid extraction Methods 0.000 claims abstract description 8
- 238000000638 solvent extraction Methods 0.000 claims abstract description 8
- 239000012074 organic phase Substances 0.000 claims abstract description 7
- 238000000926 separation method Methods 0.000 claims abstract description 7
- 238000001819 mass spectrum Methods 0.000 claims abstract description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims abstract description 4
- VZTDIZULWFCMLS-UHFFFAOYSA-N ammonium formate Chemical compound [NH4+].[O-]C=O VZTDIZULWFCMLS-UHFFFAOYSA-N 0.000 claims abstract description 4
- 238000004811 liquid chromatography Methods 0.000 claims abstract description 4
- 229960004799 tryptophan Drugs 0.000 claims description 85
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 54
- 238000001514 detection method Methods 0.000 claims description 37
- 239000000243 solution Substances 0.000 claims description 27
- 239000012086 standard solution Substances 0.000 claims description 24
- 239000006228 supernatant Substances 0.000 claims description 24
- 150000002500 ions Chemical class 0.000 claims description 23
- 239000000126 substance Substances 0.000 claims description 23
- APJYDQYYACXCRM-UHFFFAOYSA-N tryptamine Chemical compound C1=CC=C2C(CCN)=CNC2=C1 APJYDQYYACXCRM-UHFFFAOYSA-N 0.000 claims description 21
- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical compound C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 claims description 20
- HCZHHEIFKROPDY-UHFFFAOYSA-N kynurenic acid Chemical compound C1=CC=C2NC(C(=O)O)=CC(=O)C2=C1 HCZHHEIFKROPDY-UHFFFAOYSA-N 0.000 claims description 18
- LDCYZAJDBXYCGN-VIFPVBQESA-N 5-hydroxy-L-tryptophan Chemical compound C1=C(O)C=C2C(C[C@H](N)C(O)=O)=CNC2=C1 LDCYZAJDBXYCGN-VIFPVBQESA-N 0.000 claims description 17
- 239000002253 acid Substances 0.000 claims description 16
- SIOXPEMLGUPBBT-UHFFFAOYSA-N picolinic acid Chemical compound OC(=O)C1=CC=CC=N1 SIOXPEMLGUPBBT-UHFFFAOYSA-N 0.000 claims description 16
- 101100348341 Caenorhabditis elegans gas-1 gene Proteins 0.000 claims description 12
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- RKJUIXBNRJVNHR-UHFFFAOYSA-N indolenine Natural products C1=CC=C2CC=NC2=C1 RKJUIXBNRJVNHR-UHFFFAOYSA-N 0.000 claims description 10
- 238000004949 mass spectrometry Methods 0.000 claims description 10
- YGPSJZOEDVAXAB-QMMMGPOBSA-N L-kynurenine Chemical compound OC(=O)[C@@H](N)CC(=O)C1=CC=CC=C1N YGPSJZOEDVAXAB-QMMMGPOBSA-N 0.000 claims description 9
- 239000011259 mixed solution Substances 0.000 claims description 9
- LDCYZAJDBXYCGN-UHFFFAOYSA-N oxitriptan Natural products C1=C(O)C=C2C(CC(N)C(O)=O)=CNC2=C1 LDCYZAJDBXYCGN-UHFFFAOYSA-N 0.000 claims description 9
- GJAWHXHKYYXBSV-UHFFFAOYSA-N pyridinedicarboxylic acid Natural products OC(=O)C1=CC=CN=C1C(O)=O GJAWHXHKYYXBSV-UHFFFAOYSA-N 0.000 claims description 9
- 210000002966 serum Anatomy 0.000 claims description 9
- FBZONXHGGPHHIY-UHFFFAOYSA-N xanthurenic acid Chemical compound C1=CC=C(O)C2=NC(C(=O)O)=CC(O)=C21 FBZONXHGGPHHIY-UHFFFAOYSA-N 0.000 claims description 9
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- SIOXPEMLGUPBBT-RHQRLBAQSA-N 3,4,5,6-tetradeuteriopyridine-2-carboxylic acid Chemical compound [2H]C1=NC(C(O)=O)=C([2H])C([2H])=C1[2H] SIOXPEMLGUPBBT-RHQRLBAQSA-N 0.000 claims description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N acetic acid Substances CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 3
- 150000001450 anions Chemical class 0.000 claims description 3
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- VCKPUUFAIGNJHC-LURJTMIESA-N 3-hydroxy-L-kynurenine Chemical compound NC1=C(O)C=CC=C1C(=O)C[C@H]([NH3+])C([O-])=O VCKPUUFAIGNJHC-LURJTMIESA-N 0.000 claims description 2
- 229910052708 sodium Inorganic materials 0.000 claims description 2
- 239000011734 sodium Substances 0.000 claims description 2
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 claims 13
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- 230000037361 pathway Effects 0.000 abstract description 5
- 150000004945 aromatic hydrocarbons Chemical class 0.000 abstract description 2
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- 239000000523 sample Substances 0.000 description 32
- OLNJUISKUQQNIM-UHFFFAOYSA-N indole-3-carbaldehyde Chemical compound C1=CC=C2C(C=O)=CNC2=C1 OLNJUISKUQQNIM-UHFFFAOYSA-N 0.000 description 16
- WJXSWCUQABXPFS-UHFFFAOYSA-N 3-hydroxyanthranilic acid Chemical compound NC1=C(O)C=CC=C1C(O)=O WJXSWCUQABXPFS-UHFFFAOYSA-N 0.000 description 14
- GOLXRNDWAUTYKT-UHFFFAOYSA-N 3-(1H-indol-3-yl)propanoic acid Chemical compound C1=CC=C2C(CCC(=O)O)=CNC2=C1 GOLXRNDWAUTYKT-UHFFFAOYSA-N 0.000 description 12
- DUUGKQCEGZLZNO-UHFFFAOYSA-N 5-hydroxyindoleacetic acid Chemical compound C1=C(O)C=C2C(CC(=O)O)=CNC2=C1 DUUGKQCEGZLZNO-UHFFFAOYSA-N 0.000 description 12
- WHOOUMGHGSPMGR-UHFFFAOYSA-N indol-3-ylacetaldehyde Chemical compound C1=CC=C2C(CC=O)=CNC2=C1 WHOOUMGHGSPMGR-UHFFFAOYSA-N 0.000 description 12
- ZFRKQXVRDFCRJG-UHFFFAOYSA-N skatole Chemical compound C1=CC=C2C(C)=CNC2=C1 ZFRKQXVRDFCRJG-UHFFFAOYSA-N 0.000 description 12
- BYHJHXPTQMMKCA-QMMMGPOBSA-N N-formyl-L-kynurenine Chemical compound [O-]C(=O)[C@@H]([NH3+])CC(=O)C1=CC=CC=C1NC=O BYHJHXPTQMMKCA-QMMMGPOBSA-N 0.000 description 10
- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 description 10
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- DRLFMBDRBRZALE-UHFFFAOYSA-N melatonin Chemical class COC1=CC=C2NC=C(CCNC(C)=O)C2=C1 DRLFMBDRBRZALE-UHFFFAOYSA-N 0.000 description 9
- 229950006238 nadide Drugs 0.000 description 9
- YJPIGAIKUZMOQA-UHFFFAOYSA-N Melatonin Natural products COC1=CC=C2N(C(C)=O)C=C(CCN)C2=C1 YJPIGAIKUZMOQA-UHFFFAOYSA-N 0.000 description 8
- BAWFJGJZGIEFAR-NNYOXOHSSA-N NAD zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-N 0.000 description 8
- BXFFHSIDQOFMLE-UHFFFAOYSA-N indoxyl sulfate Chemical compound C1=CC=C2C(OS(=O)(=O)O)=CNC2=C1 BXFFHSIDQOFMLE-UHFFFAOYSA-N 0.000 description 8
- 229960003987 melatonin Drugs 0.000 description 8
- 210000002700 urine Anatomy 0.000 description 7
- XGILAAMKEQUXLS-UHFFFAOYSA-N 3-(indol-3-yl)lactic acid Chemical compound C1=CC=C2C(CC(O)C(O)=O)=CNC2=C1 XGILAAMKEQUXLS-UHFFFAOYSA-N 0.000 description 6
- FSBKJYLVDRVPTK-UHFFFAOYSA-N Cinnavalininate Chemical compound C1=CC=C2OC3=CC(=O)C(N)=C(C(O)=O)C3=NC2=C1C(O)=O FSBKJYLVDRVPTK-UHFFFAOYSA-N 0.000 description 6
- 239000003290 indole 3-propionic acid Substances 0.000 description 6
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
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- RWZYAGGXGHYGMB-UHFFFAOYSA-N anthranilic acid Chemical compound NC1=CC=CC=C1C(O)=O RWZYAGGXGHYGMB-UHFFFAOYSA-N 0.000 description 4
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- VCKPUUFAIGNJHC-UHFFFAOYSA-N 3-hydroxykynurenine Chemical compound OC(=O)C(N)CC(=O)C1=CC=CC(O)=C1N VCKPUUFAIGNJHC-UHFFFAOYSA-N 0.000 description 2
- 102100038238 Aromatic-L-amino-acid decarboxylase Human genes 0.000 description 2
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 2
- WJXSWCUQABXPFS-VFHCMKGYSA-N OC1=C(C(C(=O)O)=CC=C1[2H])N([2H])[2H] Chemical compound OC1=C(C(C(=O)O)=CC=C1[2H])N([2H])[2H] WJXSWCUQABXPFS-VFHCMKGYSA-N 0.000 description 2
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- SIKJAQJRHWYJAI-HOSXNMPPSA-N 1,2,3,4,5,6,7-heptadeuterioindole Chemical compound [2H]C1=C([2H])C([2H])=C2N([2H])C([2H])=C([2H])C2=C1[2H] SIKJAQJRHWYJAI-HOSXNMPPSA-N 0.000 description 1
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- KUIYMLGDWSLTTD-UHFFFAOYSA-N 2-(5-hydroxy-1H-indol-3-yl)acetic acid Chemical compound OC=1C=C2C(=CNC2=CC1)CC(=O)O.OC=1C=C2C(=CNC2=CC1)CC(=O)O KUIYMLGDWSLTTD-UHFFFAOYSA-N 0.000 description 1
- LHZHXEJKKMIKME-UHFFFAOYSA-N 2-hydroxy-3-(1H-indol-3-yl)propanoic acid Chemical compound N1C=C(C2=CC=CC=C12)CC(C(=O)O)O.N1C=C(C2=CC=CC=C12)CC(C(=O)O)O LHZHXEJKKMIKME-UHFFFAOYSA-N 0.000 description 1
- KAWMNUUYCFHGKS-UHFFFAOYSA-N 3-(1H-indol-3-yl)propanoic acid Chemical compound C1=CC=C2C(CCC(=O)O)=CNC2=C1.C1=CC=C2C(CCC(=O)O)=CNC2=C1 KAWMNUUYCFHGKS-UHFFFAOYSA-N 0.000 description 1
- CGWWRMKUJFUTPT-UHFFFAOYSA-N 3-methyl-1h-indole Chemical compound C1=CC=C2C(C)=CNC2=C1.C1=CC=C2C(C)=CNC2=C1 CGWWRMKUJFUTPT-UHFFFAOYSA-N 0.000 description 1
- 229940000681 5-hydroxytryptophan Drugs 0.000 description 1
- 101710151768 Aromatic-L-amino-acid decarboxylase Proteins 0.000 description 1
- 108090000121 Aromatic-L-amino-acid decarboxylases Proteins 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 101001037256 Homo sapiens Indoleamine 2,3-dioxygenase 1 Proteins 0.000 description 1
- 101001037261 Homo sapiens Indoleamine 2,3-dioxygenase 2 Proteins 0.000 description 1
- 102100040061 Indoleamine 2,3-dioxygenase 1 Human genes 0.000 description 1
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- 108010068073 Kynurenine-oxoglutarate transaminase Proteins 0.000 description 1
- CZCIKBSVHDNIDH-NSHDSACASA-N N(alpha)-methyl-L-tryptophan Chemical compound C1=CC=C2C(C[C@H]([NH2+]C)C([O-])=O)=CNC2=C1 CZCIKBSVHDNIDH-NSHDSACASA-N 0.000 description 1
- 102100040653 Tryptophan 2,3-dioxygenase Human genes 0.000 description 1
- 101710136122 Tryptophan 2,3-dioxygenase Proteins 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
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- 208000015114 central nervous system disease Diseases 0.000 description 1
- 238000006114 decarboxylation reaction Methods 0.000 description 1
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- DRLFMBDRBRZALE-NZLXMSDQSA-N n-[1,1,2,2-tetradeuterio-2-(5-methoxy-1h-indol-3-yl)ethyl]acetamide Chemical compound C1=C(OC)C=C2C(C([2H])([2H])C([2H])(NC(C)=O)[2H])=CNC2=C1 DRLFMBDRBRZALE-NZLXMSDQSA-N 0.000 description 1
- 230000003955 neuronal function Effects 0.000 description 1
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- 238000005191 phase separation Methods 0.000 description 1
- MDAWATNFDJIBBD-UHFFFAOYSA-M potassium;1h-indol-3-yl sulfate Chemical compound [K+].C1=CC=C2C(OS(=O)(=O)[O-])=CNC2=C1 MDAWATNFDJIBBD-UHFFFAOYSA-M 0.000 description 1
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- AKMJJGSUTRBWGW-UHFFFAOYSA-N pyridine-2-carboxylic acid Chemical compound OC(=O)C1=CC=CC=N1.OC(=O)C1=CC=CC=N1 AKMJJGSUTRBWGW-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N2030/042—Standards
- G01N2030/045—Standards internal
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
Abstract
The invention discloses a method for detecting tryptophan and metabolites and application thereof, and relates to the technical field of metabolic component analysis, wherein the method comprises the step of detecting tryptophan and metabolites which are extracted by a liquid-liquid extraction method and separated by using a C18 chromatographic column by using LC-MS/MS. The mobile phase used for the liquid chromatography separation consisted of an aqueous phase consisting of 0.3% strength formic acid, 25mM ammonium formate and water and an organic phase consisting of 0.3% strength formic acid, 0.25% strength acetylacetone and methanol. The double solution is 40% methanol water solution. Mass spectrum scanning adopts MRM technology; wherein, the addition of acetylacetone solves the problem of peak tail removal which can not be solved by the conventional mobile phase. The existing method can only detect one of a serotonin metabolic pathway, a kynurenine metabolic pathway and an aromatic hydrocarbon metabolic pathway, and the invention can simultaneously detect 25 key metabolites of the three pathways, provides more clues for the diagnosis, treatment and prevention of diseases and has wider application range.
Description
Technical Field
The invention relates to the technical field of metabolome analysis, in particular to a method for detecting tryptophan and metabolites and application thereof.
Background
Tryptophan is used for the synthesis of various proteins, and is also involved in various diseases such as neuronal function, autoimmune diseases, CNS diseases, infectious diseases, and cancer. There are three pathways for tryptophan metabolism, one of which is the serotonin pathway leading to the formation of tryptamine by decarboxylation of aromatic-L-Amino Acid Decarboxylase (AADC); which is the aromatic hydrocarbon metabolic pathway leading to the formation of 5-hydroxytryptamine (5-HT) by tryptophan hydroxylase (TPH); and the third is a kynurenine pathway which is characterized in that three rate-limiting enzymes of IDO1, IDO2 and TDO are converted into N-formyl kynurenine (NFK), the NFK forms kynurenine (Kyn) under the action of kynurenine formamide enzyme (AFMID), the Kyn is converted into Anthranilic Acid (AA) through kynurenine enzyme (KYNU), and the Kyn generates Kynurenine (KA) under the action of kynurenine aminotransferase (KATI-KATIII). In addition, tryptophan metabolism is also directly or indirectly affected by intestinal microorganisms. Therefore, the purpose of treating diseases is achieved by using molecules targeting specific pathways or producing microorganisms for regulating tryptophan metabolism.
LC-MS/MS is a combination technology developed in recent years and has application in the fields of medicine and in-vivo medicine analysis, stimulant and drug detection, pesticide or veterinary drug residue analysis and the like. It is known that the chemical structure of substances in tryptophan metabolic pathways is greatly different, and the detection difficulty is increased along with the increase of detection substances. At present, although the LC-MS method for detecting tryptophan and metabolites is reported, only five or six substances in a certain path or only one path can be detected, and the detection of a plurality of substances covering a plurality of metabolic paths cannot be realized.
Disclosure of Invention
The invention provides a method for detecting tryptophan and metabolites and application thereof, which adopts a liquid-liquid extraction method to extract the tryptophan and the metabolites in a sample, detects the tryptophan and the metabolites after liquid phase separation, and adopts an isotope internal standard method to quantify, aiming at solving the problems that the prior art cannot detect the tryptophan and the metabolites in the whole channel and has peak tailing.
The technical scheme adopted by the invention is as follows:
a method for detecting tryptophan and metabolites comprises performing mass spectrum detection on tryptophan and metabolites extracted by liquid-liquid extraction and separated by C18 chromatographic column by LC-MS/MS; the mobile phase adopted by the liquid chromatography separation consists of a water phase and an organic phase, wherein the water phase consists of 0.3% formic acid, 25mM ammonium formate and water, the organic phase consists of 0.3% formic acid, 0.25% acetylacetone and methanol, and the redissolution is 40% methanol aqueous solution; mass spectrometry scans used MRM techniques.
Further, in the liquid-liquid extraction method, the treatment of precipitated protein extraction tryptophan and metabolite extraction is included, and the adopted extracting solution is a methanol/acetonitrile mixed solution, wherein the volume ratio of methanol to acetonitrile to water is 2: 2: 1; after the protein is precipitated, centrifugation is carried out and the supernatant containing tryptophan and metabolite is extracted.
Further, concentrating and drying the supernatant to obtain tryptophan and metabolites, and then re-dissolving by using the re-solution.
Further, before the extraction of tryptophan and metabolites from the solid sample to be detected, the method also comprises the step of homogenizing the solid sample to be detected.
Specifically, the method comprises the following steps:
and step S1, preparing internal standard solutions, respectively weighing each isotope internal standard, adding 40% methanol solution to dissolve the isotope internal standards to prepare a mixed internal standard solution with the concentration of 1 mu g/ml for later use.
And step S2, preparing standard substance solutions, weighing the standard substances respectively, adding 40% methanol solution, and dissolving to obtain a mixed standard substance solution of 25 μ g/ml for later use.
Step S3, constructing a standard curve, and diluting the standard solution obtained in the step S2 to 2.5ng/ml in a gradient manner; 100 μ L of each standard solution with gradient concentration is taken, and each diluted standard solution is treated as follows: adding 10 μ L of the mixed internal standard solution with the concentration of 1 μ g/ml obtained in the step S1, carrying out vortex mixing, adding 400 μ L of methanol/acetonitrile mixed solution, carrying out vortex for 30 seconds, centrifuging for 10min at 14000g, taking the supernatant, carrying out vacuum drying, finally re-dissolving with 100 μ L of 40% methanol solution, centrifuging for 15min at 14000g and 4 ℃, taking the supernatant, and carrying out LC-MS/MS analysis to obtain a standard curve.
Step S4, analyzing the sample, taking 100 mu L of serum sample, adding 10 mu L of mixed internal standard solution with the concentration of 1 mu g/ml obtained in the step S1, and mixing in a vortex manner; adding 400 mu L of the methanol/acetonitrile mixed solution, whirling for 30 seconds, and then centrifuging for 10min by 14000 g; collecting supernatant, vacuum drying, re-dissolving with 100 μ L40% methanol solution, centrifuging at 14000g and 4 deg.C for 15min, collecting supernatant, LC-MS/MS analyzing, and calculating the concentration of corresponding substance according to the standard curve constructed in step S3.
More specifically, in step S1, the internal isotope standards include Serotonin-d4, 2-Picolinic-d4 Acid, 5-Hydroxyindole-3-acetic-2 ¸ 2-d2 Acid, 3-Hydroxyanthracycline Acid-d3, Tryptomine-d 4, 3-Indole Sulfate sodium Salt-d 4, L-Kynurenine-d4, Indole-d7 and Meldonin-d 4.
More specifically, in step S2, the standard includes L-kynurenine, Tryptamine, Indole, Quinolic Acid, Indole-3-carboxaldehyde, 3-Methyl-indele, Indole-3-propinic Acid, Kynuric Acid, 3-hydroxy-L-kynurenine, Indoleactate (IAA), Serotonin, 5-hydroxy-L-tryptophan, Picolinic Acid, 5-hydroxy-3-acetic Acid, 3-hydroxy-anthracyanic Acid, N-Methyl-kynurenine, Xanthonic Acid, Cinnalitininic Acid, Indolyl-beta-D-glucuronide, 3-hydroxy-3-cyanohydrin, N-Methyl-tryptophane, Xantholytic Acid, and Metalacetic Acid, and P-Indole-3-beta-D-glucopyranoside.
More specifically, the liquid phase method in the LC-MS/MS analysis used in the step S3 and step S4 is as follows: separating by UPLC system, placing the sample in an automatic sample injector at 4 deg.C, at 45 deg.C, at 400 μ L/min, and at 5 μ L; the gradient of the related liquid phase is 0-2min, and the B phase is maintained at 15%; 2-9min, phase B changes linearly from 15% to 98%; 9-11min, maintaining phase B at 98%; 11-11.5min, phase B changes linearly from 98% to 15%; 11.5-14min, keeping phase B at 15%.
More specifically, the mass spectrometry conditions in the LC-MS/MS analysis used in step S3 and step S4 are set as follows:
mass spectrometry was performed in positive/negative ion mode using 5500 QTRAP mass Spectrometer (SCIEX); 5500 QTRAP ESI source positive ion conditions are as follows: source temperature of 450 deg.c; ion Source Gas1 (Gas1): 45; ion Source Gas2 (Gas2): 45; curtain gas (CUR) 40; ionSapary Voltage flowing (ISVF) 4500V; 5500 QTRAP ESI source anion conditions are as follows: source temperature of 450 deg.C; ion Source Gas1 (Gas1): 45; ion Source Gas2 (Gas2): 45; curtain gas (CUR) 40; and detecting the ion pair to be detected by adopting an MRM mode.
Specifically, the detection method is applied to a tryptophan and metabolite detection system, a detection system platform, a detection device or a kit.
Compared with the prior art, the invention has the advantages that:
the invention can lead tryptophan and metabolite to have good baseline separation through the specially developed re-solution and mobile phase, and can detect more tryptophan and metabolites. The method has good linearity, repeatability and accuracy. The method realizes the detection of tryptophan and metabolites by a sample-by-sample quantitative method, greatly improves the detection efficiency and reduces the detection cost.
In addition, the method for detecting tryptophan and metabolites disclosed by the invention needs a small amount of samples, has strong specificity, high sensitivity and good stability, can detect 25 kinds of tryptophan and metabolites at the same time within 15min of machine loading, and covers three metabolic pathways of tryptophan. The kit is suitable for common biological sample types such as animal tissues, feces, serum, plasma and the like, has wide application range, is even suitable for saliva, and realizes real non-invasion.
Detailed Description
The following describes specific embodiments of the present invention. Numerous details are set forth below in order to provide a thorough understanding of the present invention, but it will be apparent to those skilled in the art that the present invention may be practiced without these details.
A method for detecting tryptophan and metabolites comprises the following steps:
step S1, preparing internal standard solutions, respectively weighing isotope internal standards, adding 40% methanol solution to dissolve the isotope internal standards to prepare a mixed internal standard solution with the concentration of 1 mu g/ml for later use; the isotope internal standard comprises Serotonin-d4 (deuterated Serotonin), 2-Picolinic-d4 Acid (deuterated Picolinic Acid), 5-Hydroxyindole-3-acetic-2 ¸ 2-d2 Acid (deuterated-5-Hydroxyindole-3-acetic Acid), 3-Hydroxyanthranilic Acid-d3 (deuterated-3-Hydroxyanthranilic Acid), Tryptamine-d4 (deuterated Tryptamine), 3-Indoxyl Sulfate Potasium Salt-d 4 (deuterated Potassium Indole Sulfate), L-Kynurene-d 4 (deuterated Kynurenine), Indole-d7 (deuterated Indole), and Melanon-d 4 (deuterated Melatonin).
And step S2, preparing standard substance solutions, weighing the standard substances respectively, adding 40% methanol solution, and dissolving to obtain a mixed standard substance solution of 25 μ g/ml for later use. Wherein the standard substance comprises L-kynurenine, Tryptamine, Indole, Quinolinic Acid, Indole-3-carboxaldehyde, 3-Methyl-Indole (3-methylindole),
Indole-3-propionic Acid (3-indolepropionic Acid), Kynurenic Acid (Kynurenic Acid), 3-hydroxyyl-L-kynurenine (3-hydroxykynurenine), Indole Acid (IAA) (indoleacetic Acid), Serotonin (Serotonin), 5-hydroxyyl-L-tryptophane (5-hydroxytryptophan), Picolinic Acid (Picolinic Acid), 5-Hydroxyindole-3-acetic Acid (5-Hydroxyindole-3-acetic Acid), 3-hydroxyindolenine Acid (3-Hydroxyanthranilic Acid), N-formamyl-kynurenine (N-formylkynurenine), Xanthurenic Acid (xanthuric Acid), cinnalytic Acid (cinnamyl-beta-D-glucuronide), cinnamyl-beta-D-glucuronide (Indole-D-glucopyranoside), Indole-3-acetylaldehyde-3-acetic Acid (Indole-3-acetic Acid), and Indole-L-3-L-D-glucuronide (Indole-3-D-acetyl-acetic Acid) 3-Indoxyl Sulfate Potasssium Salt (Potassium Indoxyl Sulfate), Indole-3-lactic Acid (3-indolelactic Acid), Melatonin (Melatonin), beta-Nicotinamide adenine dinucletotide (NAD +, oxidative coenzyme I), and L-tryptophan (tryptophan).
Step S3, constructing a standard curve, and diluting the standard solution obtained in the step S2 to 2.5ng/ml in a gradient manner; 100 mu L of each standard solution with gradient concentration is taken, and each diluted standard solution is treated as follows: adding 10 μ L of the mixed internal standard solution with the concentration of 1 μ g/ml obtained in the step S1, carrying out vortex mixing, adding 400 μ L of methanol/acetonitrile mixed solution, carrying out vortex for 30 seconds, centrifuging for 10min at 14000g, taking the supernatant, carrying out vacuum drying, finally re-dissolving with 100 μ L of 40% methanol solution, centrifuging for 15min at 14000g and 4 ℃, taking the supernatant, and carrying out LC-MS/MS analysis to obtain a standard curve.
Step S4, analyzing a sample, namely adding 10 mu L of serum sample into the mixed internal standard solution with the concentration of 1 mu g/ml obtained in the step S1, carrying out vortex mixing, adding 400 mu L of methanol/acetonitrile mixed solution, carrying out vortex for 30 seconds, centrifuging for 10min by 14000g, taking supernatant, carrying out vacuum drying, finally re-dissolving by 100 mu L of 40% methanol solution, centrifuging for 15min at 14000g and 4 ℃, taking supernatant, carrying out LC-MS/MS analysis, and converting the analysis result into the corresponding substance concentration by using the standard curve constructed in the step S3;
the liquid phase method in the LC-MS/MS analysis used in step S3 and step S4 is as follows: separating by UPLC system, placing the sample in an autosampler at 4 deg.C, column temperature of 45 deg.C, flow rate of 400 μ L/min, and sample amount of 5 μ L. The gradient of the related liquid phase is 0-2min, and the B phase is maintained at 15%; 2-9min, phase B changes linearly from 15% to 98%; 9-11min, maintaining phase B at 98%; 11-11.5min, phase B changes linearly from 98% to 15%; 11.5-14min, keeping phase B at 15%.
The mass spectrum conditions in the LC-MS/MS analysis used in step S3 and step S4 are set as follows:
mass spectrometry was performed using a 5500 QTRAP mass Spectrometer (SCIEX) in positive/negative ion mode. 5500 QTRAP ESI source positive ion conditions are as follows: source temperature of 450 deg.C; ion Source Gas1 (Gas1): 45; ion Source Gas2 (Gas2): 45; curtain gas (CUR) 40; ionSapary Voltage flowing (ISVF) 4500V; 5500 QTRAP ESI source anion conditions are as follows: source temperature of 450 deg.C; ion Source Gas1 (Gas1): 45; ion Source Gas2 (Gas2): 45; curtain gas (CUR) 40; ionsapart volume flowing (ISVF): 4500V, detecting the ion pair to be detected by adopting an MRM mode, and detecting the ion pair to be detected by the object to be detected in table 1.
TABLE 1 Tryptophan MRM parameters
| Analyte | Q1(Da) | Q3(Da) | DP（V) | CE（V) | CXP（V) | EP（V) |
| L-kynurenine | 209.2 | 192 | 70 | 12 | 5 | 10 |
| Picolinic acid | 124.2 | 77.9 | 90 | 22 | 5 | 10 |
| 5-Hydroxyindole-3-acetic acid | 192.2 | 146.1 | 83 | 19 | 5 | 10 |
| Tryptamine | 161.2 | 144.1 | 60 | 15 | 5 | 10 |
| Melatonin | 233.2 | 174.1 | 80 | 22 | 5 | 10 |
| Xanthurenic acid | 206.1 | 159.9 | 100 | 25 | 5 | 10 |
| N-formyl-kynurenine | 237.2 | 192 | 67 | 18 | 5 | 10 |
| Cinnavalininic acid | 301.2 | 283 | 145 | 25 | 5 | 10 |
| 3-Methyl-indole | 132.2 | 117.1 | 144 | 29 | 5 | 10 |
| Kynurenic acid | 190.2 | 172 | 100 | 19 | 5 | 10 |
| 5-Hydroxy-L-Tryptophan | 221.2 | 204 | 80 | 15 | 5 | 10 |
| Serotonin | 177.2 | 160.1 | 70 | 16 | 5 | 10 |
| Indoleacetate(IAA) | 176.1 | 130.2 | 73 | 30 | 5 | 10 |
| Indole-3-acetaldehyde | 160.1 | 117.1 | 108 | 32 | 5 | 10 |
| Tryptophan | 205.2 | 188.1 | 72 | 15 | 5 | 10 |
| 3-Hydroxyanthranilic acid | 151.9 | 108 | -50 | -16 | -10 | -10 |
| Indole | 115.9 | 40.2 | -150 | -46 | -10 | -10 |
| 3-Indoxyl sulfate | 211.9 | 79.9 | -60 | -29 | -10 | -10 |
| Quinolinic acid | 165.8 | 78 | -40 | -18 | -10 | -10 |
| 3-Hydroxyl-L-kynurenine | 223 | 206 | -60 | -12 | -10 | -10 |
| Indole-3-carboxaldehyde | 143.9 | 116 | -110 | -33 | -10 | -10 |
| Indole-3-propionic acid | 187.9 | 59.1 | -60 | -20 | -10 | -10 |
| Indole-3-lactic acid | 204 | 158 | -56 | -20 | -10 | -10 |
| Indoxyl-b-D-glucuronide | 308 | 113 | -75 | -18 | -10 | -10 |
| NAD | 662.1 | 539.9 | -70 | -18 | -10 | -10 |
| L-kynurenine-d4 | 213.3 | 96.1 | 75 | 18 | 5 | 10 |
| Picolinic acid-d4 | 128 | 110.1 | 105 | 15 | 5 | 10 |
| 5-Hydroxyindole-3-acetic-2,2-acid-d2 | 194.2 | 148 | 77 | 23 | 5 | 10 |
| Tryptamine-d4 | 165.2 | 148.1 | 95 | 17 | 5 | 10 |
| Melatonin-d4 | 237.3 | 178.1 | 95 | 21 | 5 | 10 |
| Serotonin-d4 | 181.2 | 164.1 | 92 | 15 | 5 | 10 |
| 3-Hydroxyanthranilic acid-d3 | 154.9 | 111.1 | -65 | -17 | -10 | -10 |
| Indole-d6 | 122.1 | 42 | -61 | -50 | -10 | -10 |
| 3-Indoxyl sulfate-d4 | 215.7 | 79.9 | -60 | -35 | -10 | -10 |
The following further analysis of the test results of this example was performed to verify the feasibility of the present invention: and establishing a standard curve by adopting an isotope internal standard quantitative method and taking the concentration of the standard substance as an x axis and the ratio of the peak area of the standard substance to the peak area of the internal standard substance as a y axis. The concentrations of tryptophan and its metabolites in human serum were calculated from this curve. The signal-to-noise ratio of the characteristic ion MRM chromatographic peak is greater than 3 as the detection Limit (LOD), and the signal-to-noise ratio is greater than 10 as the quantification Limit (LOQ), and the results are as follows:
TABLE 2 concentration of Tryptophan and its metabolites in human serum
| Substance(s) | Retention time (min) | Linear range (ng/ml) | Linear equation of equations | Coefficient of correlation R | LOD（ng/ml） | LOQ（ng/ml） |
| L-kynurenine | 1.28 | 2.5-2500 | Y=0.03264X-0.16292 | 0.99919 | 0.5 | 2.5 |
| Picolinic acid | 0.80 | 5-2500 | Y=0.00122X-0.01604 | 0.99937 | 1 | 5 |
| 5-Hydroxyindole-3-acetic acid | 3.20 | 5-2500 | Y=0.00400X+0.05520 | 0.99985 | 2.5 | 5 |
| 3-Hydroxyanthranilic acid | 2.84 | 5-1250 | Y=0.16709X+3.84881 | 0.99766 | 1 | 5 |
| Tryptamine | 2.77 | 0.5-1250 | Y=0.01345X+0.03374 | 0.99922 | 0.1 | 0.5 |
| Indole | 5.63 | 125-2500 | Y=7.935e-6X-3.96528e-4 | 0.99915 | 0.5 | 12.5 |
| Melatonin | 5.77 | 0.5-50 | Y=0.08144X-0.03241 | 0.99826 | 0.5 | 0.5 |
| 3-Indoxyl sulfate | 3.28 | 5-1250 | Y=0.00311X+0.00160 | 0.99951 | 0.5 | 5 |
| Xanthurenic acid | 3.51 | 5-2500 | Y=2.00113e-4X-2.38663e-4 | 0.99733 | 2.5 | 5 |
| Quinolinic acid | 0.86 | 12.5-2500 | Y=4.21765e-5X-0.00847 | 0.99937 | 2.5 | 12.5 |
| N-formyl-kynurenine | 1.36 | 5-2500 | Y=0.01160X+0.02012 | 0.99786 | 1 | 5 |
| Cinnavalininic acid | 6.63 | 125-2500 | Y=2.34374e-4X-0.03133 | 0.9972 | 40 | 125 |
| 3-Hydroxyl-L-kynurenine | 0.91 | 2.5-2500 | Y=0.00105X-0.03766 | 0.99828 | 0.5 | 2.5 |
| Indole-3-carboxaldehyde | 5.62 | 2.5-50 | Y=4.83721e-4X+0.00141 | 0.99742 | 0.5 | 2.5 |
| 3-Methyl-indole | 7.47 | 50-2500 | Y=1.67625e-5X+0.00654 | 0.99899 | 10 | 50 |
| Indole-3-propionic acid | 6.58 | 12.5-500 | Y=3.45745e-5X-0.00608 | 0.99681 | 1 | 12.5 |
| Kynurenic acid | 5.62 | 1.25-50 | Y=0.01749X+0.03472 | 0.99922 | 0.25 | 1.25 |
| Indole-3-lactic acid | 5.41 | 5-2500 | Y=0.00101X-0.14566 | 0.99901 | 1 | 5 |
| Indoxyl-b-D-glucuronide | 2.79 | 12.5-1250 | Y=1.54470e-4X+0.00128 | 0.99653 | 2.5 | 12.5 |
| 5-Hydroxy-L-Tryptophan | 1.07 | 12.5-2500 | Y=0.00936X-0.12621 | 0.9964 | 2.5 | 12.5 |
| Serotonin | 1.03 | 2.5-500 | Y=0.01011X-0.02356 | 0.9994 | 0.5 | 2.5 |
| Indoleacetate(IAA) | 5.88 | 2.5-2500 | Y=0.00426X-0.00496 | 0.9985 | 0.5 | 2.5 |
| Indole-3-acetaldehyde | 5.91 | 50-2500 | Y=9.91965e-6X-6.94595e-4 | 0.99889 | 5 | 50 |
| Tryptophan | 2.30 | 5-500 | Y=0.00160X-0.27164 | 0.99957 | 0.5 | 5 |
| NAD | 0.72 | 1.25-500 | Y=6.04493e-5X+2.90352e-4 | 0.99967 | 0.1 | 1.25 |
Therefore, the 25 tryptophan and metabolites thereof have good linear relation in the respective concentration linear range, and meet the quantitative requirement.
The stability and recovery rate of the method for detecting tryptophan and metabolites thereof disclosed by the present invention were tested, and the results are shown in table 3, wherein the Average Value (AV), Coefficient of Variation (CV) and recovery Rate (RE) of the values were analyzed.
TABLE 3 stability and recovery test results for serum samples
| Component Name | AV(ng/ml) | CV(%) | RE(%) |
| L-kynurenine | 443.56 | 4.90 | 92.26 |
| Picolinic acid | 34.55 | 2.86 | 99.67 |
| 5-Hydroxyindole-3-acetic acid | 12.40 | 4.44 | 88.01 |
| 3-Hydroxyanthranilic acid | 7.60 | 8.95 | 113.49 |
| Tryptamine | 0.64 | 7.60 | 117.21 |
| Indole | 12.97 | 13.48 | 119.91 |
| Melatonin | 0.56 | 2.43 | 94.10 |
| 3-Indoxyl sulfate | 778.24 | 7.76 | 107.79 |
| Xanthurenic acid | 30.10 | 3.32 | 83.28 |
| Quinolinic acid | 275.63 | 2.38 | 116.96 |
| N-formyl-kynurenine | 123.20 | 4.10 | 101.80 |
| Cinnavalininic acid | 2110.91 | 14.53 | 107.34 |
| 3-Hydroxyl-L-kynurenine | 66.78 | 7.57 | 114.32 |
| Indole-3-carboxaldehyde | 3.33 | 10.76 | 94.04 |
| 3-Methyl-indole | 51.2 | 12.69 | 81.64 |
| Indole-3-propionic acid | 2086.06 | 7.59 | 106.46 |
| Kynurenic acid | 4.66 | 10.07 | 94.83 |
| Indole-3-lactic acid | 527.25 | 3.27 | 111.60 |
| Indoxyl-b-D-glucuronide | 10.47 | 9.89 | 102.55 |
| 5-Hydroxy-L-Tryptophan | 16.18 | 8.63 | 80.08 |
| Serotonin | 44.12 | 3.74 | 100.22 |
| Indoleacetate(IAA) | 351.09 | 1.75 | 92.15 |
| Indole-3-acetaldehyde | 51.75 | 9.76 | 90.81 |
| Tryptophan | 9524.63 | 4.54 | 118.06 |
| NAD | 1.36 | 12.6 | 89.16 |
The results in table 3 show that the method for detecting tryptophan and metabolites thereof disclosed by the invention can be used for detecting 25 tryptophan and metabolites thereof in a human serum sample, the stability of the detection result is good, and the recovery rate is 80-120%.
In short, the method for detecting the tryptophan and the metabolites uses LC-MS/MS to carry out mass spectrum detection on the tryptophan and the metabolites which are extracted by a liquid-liquid extraction method and separated by using a C18 chromatographic column; the mobile phase adopted by the liquid chromatography separation consists of a water phase and an organic phase, wherein the water phase consists of 0.3% formic acid, 25mM ammonium formate and water, the organic phase consists of 0.3% formic acid, 0.25% acetylacetone and methanol, and the redissolution is 40% methanol aqueous solution; mass spectrometry scans used MRM techniques. In the liquid-liquid extraction method, the method comprises the following steps: (1) treating the precipitated protein to extract tryptophan and metabolites, wherein the adopted extracting solution is a methanol/acetonitrile mixed solution, and the volume ratio of methanol to acetonitrile to water is 2: 2: 1; after the protein is precipitated, centrifugation is carried out and the supernatant containing tryptophan and metabolite is extracted. (2) Concentrating and drying the supernatant to obtain tryptophan and metabolite, and then re-dissolving by using the re-solution. (3) In addition, before extracting tryptophan and metabolites from the solid sample to be tested, the solid sample to be tested is required to be homogenized.
The detection method disclosed by the invention can be applied to a detection system, a detection system platform, a detection device or a kit of tryptophan and metabolites.
On the basis of the tests, the tryptophan and the metabolites thereof of three samples of urine, feces and animal tissues are respectively extracted and detected by adopting the tryptophan and the metabolite detection method disclosed by the invention. The extraction methods of tryptophan and its metabolites from the three samples are as follows.
Firstly, urine sample tryptophan and metabolite extraction and detection thereof, the urine sample that this embodiment adopted is normal human urine, and the concrete step is as follows:
1) extracting a human urine, and unfreezing a sample at the temperature of 4 ℃;
2) vortex for 30s, and suck 100. mu.L into a 1.5mL centrifuge tube;
3) adding 10 μ L of internal standard solution (1 μ g/ml), adding 400 μ L of precooled extract methanol/acetonitrile mixture (volume ratio of water, methanol and acetonitrile =1:2: 2);
4) centrifuging in a centrifuge for 15min (14000 rcf 4 deg.C), and vacuum drying 400 μ L of supernatant;
5) adding 100 mu L of 40% methanol aqueous solution for redissolving during mass spectrometry detection;
6) vortex and mix evenly for 30s, centrifuge for 15min at 14000 rcf 4 ℃;
7) taking the supernatant, and detecting on a machine.
The results are shown in Table 4.
TABLE 4 detection results of tryptophan and its metabolites in urine
| Component Name | CON(ng/ml) |
| L-kynurenine | 1776.16 |
| Picolinic acid | 202.10 |
| 5-Hydroxyindole-3-acetic acid | 4748.94 |
| 3-Hydroxyanthranilic acid | 1167.68 |
| Tryptamine | 166.15 |
| Indole | 167.37 |
| Melatonin | 1.09 |
| 3-Indoxyl sulfate | 58660.65 |
| Xanthurenic acid | 753.15 |
| Quinolinic acid | 16717.82 |
| N-formyl-kynurenine | 293.97 |
| Cinnavalininic acid | 3431.90 |
| 3-Hydroxyl-L-kynurenine | 899.63 |
| Indole-3-carboxaldehyde | 21.60 |
| 3-Methyl-indole | 350.51 |
| Indole-3-propionic acid | 12.64 |
| Kynurenic acid | 4.73 |
| Indole-3-lactic acid | 996.55 |
| Indoxyl-b-D-glucuronide | 1098.94 |
| 5-Hydroxy-L-Tryptophan | 342.52 |
| Serotonin | 77.07 |
| Indoleacetate(IAA) | 2386.32 |
| Indole-3-acetaldehyde | 2807.31 |
| Tryptophan | 27441.35 |
| NAD | 0.84 |
Therefore, the method for detecting tryptophan and the metabolite thereof disclosed by the invention can effectively detect the tryptophan and the metabolite thereof in the 25 th urine sample.
Secondly, extracting and detecting tryptophan and metabolites thereof in a stool sample, wherein the stool sample adopted in the embodiment is normal human stool, and the method comprises the following specific steps:
1) extracting human excrement, and unfreezing the sample at the temperature of 4 ℃;
2) weighing 100 +/-5 mg of a fecal sample in a homogenizing tube;
3) adding 10 μ L of internal standard solution (1 μ g/ml), adding 2 black ceramic beads of 4 mm and 5 zirconia beads of 2 mm, and screwing the cover;
4) adding 500 μ L of a pre-cooled extract methanol/acetonitrile mixture (water, methanol, acetonitrile in a volume ratio =1:2: 2), quenching with liquid nitrogen for 5s, and homogenizing 3 times (20 s each time) in an MP sample preparation apparatus;
5) centrifuging in a centrifuge for 15min (14000 rcf 4 deg.C), and vacuum drying 400 μ L of supernatant;
6) adding 100 mu L of 40% methanol aqueous solution for redissolving during mass spectrometry detection;
7) vortex and mix evenly for 30s, centrifuge for 15min at 14000 rcf 4 ℃;
8) taking the supernatant, and detecting on a machine.
The results are shown in Table 5.
TABLE 5 results of detection of tryptophan and its metabolites in feces
| Component Name | CON(ng/g) |
| L-kynurenine | 275.79 |
| Picolinic acid | 147.85 |
| 5-Hydroxyindole-3-acetic acid | 163.37 |
| 3-Hydroxyanthranilic acid | 123.82 |
| Tryptamine | 84.40 |
| Indole | 1.84 |
| Melatonin | 0.24 |
| 3-Indoxyl sulfate | 51.79 |
| Xanthurenic acid | 965.85 |
| Quinolinic acid | 8504.07 |
| N-formyl-kynurenine | 121.68 |
| Cinnavalininic acid | 93333.10 |
| 3-Hydroxyl-L-kynurenine | 15.50 |
| Indole-3-carboxaldehyde | 377.14 |
| 3-Methyl-indole | 11387.71 |
| Indole-3-propionic acid | 1.26 |
| Kynurenic acid | 1.65 |
| Indole-3-lactic acid | 57152.11 |
| Indoxyl-b-D-glucuronide | 7.54 |
| 5-Hydroxy-L-Tryptophan | 120.18 |
| Serotonin | 84.93 |
| Indoleacetate(IAA) | 6859.27 |
| Indole-3-acetaldehyde | 2.13 |
| Tryptophan | 163613.41 |
| NAD | 203.44 |
Therefore, the method for detecting tryptophan and the metabolite thereof disclosed by the invention can effectively detect the tryptophan and the metabolite thereof in the 25 th fecal sample.
Extracting and detecting tryptophan and metabolites thereof in a tissue sample, wherein the tissue sample adopted in the embodiment is a human tumor mass tissue, and the specific steps are as follows:
1) extracting a human tumor mass tissue, and unfreezing a sample at the temperature of 4 ℃;
2) weighing a tissue sample of 100 +/-5 mg in a homogenizing tube;
3) adding 10 μ L of internal standard solution (1 μ g/ml), adding 2 black ceramic beads of 4 mm and 5 zirconia beads of 2 mm, and screwing the cover;
4) adding 500 μ L of a pre-cooled extract methanol/acetonitrile mixture (water, methanol, acetonitrile in a volume ratio =1:2: 2), quenching with liquid nitrogen for 5s, and homogenizing 3 times (20 s each time) in an MP sample preparation apparatus;
5) centrifuging in a centrifuge for 15min (14000 rcf 4 deg.C), and vacuum drying 400 μ L of supernatant;
6) adding 100 mu L of 40% methanol aqueous solution for redissolving during mass spectrometry detection;
7) vortex and mix evenly for 30s, centrifuge for 15min at 14000 rcf 4 ℃;
8) taking the supernatant, and detecting on a machine.
The results are shown in Table 6.
TABLE 6 results of tryptophan and its metabolites in tissues
| Component Name | CON(ng/g) |
| L-kynurenine | 86.13 |
| Picolinic acid | 433.87 |
| 5-Hydroxyindole-3-acetic acid | 91.83 |
| 3-Hydroxyanthranilic acid | 1.07 |
| Tryptamine | 0.57 |
| Indole | 0.98 |
| Melatonin | 0.51 |
| 3-Indoxyl sulfate | 129.45 |
| Xanthurenic acid | 0.75 |
| Quinolinic acid | 302.12 |
| N-formyl-kynurenine | 28.62 |
| Cinnavalininic acid | 0.67 |
| 3-Hydroxyl-L-kynurenine | 1.24 |
| Indole-3-carboxaldehyde | 16.00 |
| 3-Methyl-indole | 0.19 |
| Indole-3-propionic acid | 10.15 |
| Kynurenic acid | 67.76 |
| Indole-3-lactic acid | 188.04 |
| Indoxyl-b-D-glucuronide | 0.96 |
| 5-Hydroxy-L-Tryptophan | 130.78 |
| Serotonin | 108.65 |
| Indoleacetate(IAA) | 77.70 |
| Indole-3-acetaldehyde | 12.67 |
| Tryptophan | 44445.45 |
| NAD | 2.43 |
Therefore, the method for detecting tryptophan and the metabolite thereof disclosed by the invention can effectively detect the tryptophan and the metabolite thereof in the tissue sample.
In conclusion, the specially-developed re-solution and the specially-developed mobile phase can enable tryptophan and metabolites to have good baseline separation, and can detect more tryptophan and metabolites. That is, the mobile phase developed by the present invention can improve the chromatographic behavior of tryptophan and its metabolites. The mobile phase enables the chromatographic behavior to have better separation and peak type, so that certain substances with peak type severe tailing can be accurately and quantitatively detected, for example, Cinnavillinic acid, and the traditional mobile phase is adopted, and the peak type tailing cannot obtain an effective detection result; by adopting the detection method, the peak type of the Cinnavillinic acid can be effectively improved, and an accurate detection result can be obtained. The method has good linearity, repeatability and accuracy. The method realizes the detection of tryptophan and metabolites by a sample-by-sample quantitative method, greatly improves the detection efficiency and reduces the detection cost.
In addition, the method for detecting tryptophan and metabolites disclosed by the invention needs a small amount of samples, has strong specificity, high sensitivity and good stability, can detect 25 kinds of tryptophan and metabolites at the same time within 15min of machine loading, and covers three metabolic pathways of tryptophan. The kit is suitable for common biological sample types such as animal tissues, feces, serum, plasma and the like, has wide application range, is even suitable for saliva, and realizes real non-invasion.
The above description is only an embodiment of the present invention, but the design concept of the present invention is not limited thereto, and any insubstantial modifications made by using the design concept should fall within the scope of infringing the present invention.
Claims (10)
1. A method for detecting tryptophan and metabolites, comprising: performing mass spectrometric detection on tryptophan and metabolites extracted by a liquid-liquid extraction method and separated by using a C18 chromatographic column by using LC-MS/MS; the mobile phase adopted by the liquid chromatography separation consists of a water phase and an organic phase, wherein the water phase consists of 0.3% formic acid, 25mM ammonium formate and water, the organic phase consists of 0.3% formic acid, 0.25% acetylacetone and methanol, and the redissolution is 40% methanol aqueous solution; mass spectrometry scans used MRM techniques.
2. The method for detecting tryptophan and metabolites according to claim 1, wherein: the liquid-liquid extraction method comprises the steps of extracting tryptophan and metabolite from precipitated protein, wherein the adopted extracting solution is a methanol/acetonitrile mixed solution, and the volume ratio of methanol to acetonitrile to water is 2: 2: 1; after the protein is precipitated, centrifugation is carried out and the supernatant containing tryptophan and metabolite is extracted.
3. The method for detecting tryptophan and metabolites according to claim 2, wherein: concentrating and drying the supernatant to obtain tryptophan and metabolite, and then re-dissolving the re-solution.
4. The method for detecting tryptophan and metabolites according to claim 2, wherein: before the extraction of tryptophan and metabolites from the solid sample to be detected, the method also comprises the step of homogenizing the solid sample to be detected.
5. The method for detecting tryptophan and metabolites according to claim 1, comprising the steps of:
step S1, preparing internal standard solutions, respectively weighing each isotope internal standard, adding 40% methanol solution to dissolve to prepare a mixed internal standard solution with the concentration of 1 mu g/ml for later use;
step S2, preparing standard substance solutions, respectively weighing each standard substance, adding 40% methanol solution to dissolve to prepare a mixed standard substance solution of 25 μ g/ml for later use;
step S3, constructing a standard curve, and carrying out gradient dilution on the standard solution obtained in the step S2 to 2.5 ng/ml; 100 μ L of each standard solution with gradient concentration is taken, and each diluted standard solution is treated as follows: adding 10 mu L of mixed internal standard solution with the concentration of 1 mu g/ml obtained in the step S1, carrying out vortex mixing, adding 400 mu L of methanol/acetonitrile mixed solution, carrying out vortex for 30 seconds, centrifuging for 10min at 14000g, taking supernate, carrying out vacuum drying, finally re-dissolving with 100 mu L of 40% methanol solution, centrifuging for 15min at 14000g and 4 ℃, taking supernate, and carrying out LC-MS/MS analysis to obtain a standard curve;
step S4, analyzing the sample, taking 100 mu L of serum sample, adding 10 mu L of mixed internal standard solution with the concentration of 1 mu g/ml obtained in the step S1, and mixing in a vortex manner; adding 400 mu L of the methanol/acetonitrile mixed solution, whirling for 30 seconds, and then centrifuging for 10min by 14000 g; and taking the supernatant, drying the supernatant by using vacuum, redissolving the supernatant by using 100 mu L of 40% methanol solution, centrifuging the redissolved solution at 14000g and 4 ℃ for 15min, taking the supernatant, performing LC-MS/MS analysis, and converting the analysis result into the concentration of a corresponding substance by using the standard curve constructed in the step S3.
6. The method according to claim 5, wherein the method comprises the steps of: in step S1, the isotope internal standards include Serotonin-d4, 2-Picolinic-d4 Acid, 5-Hydroxyindole-3-acetic-2 ¸ 2-d2 Acid, 3-Hydroxyanthracranic Acid-d3, Tryptomine-d 4, 3-Indole Sulfate sodium Salt-d 4, L-Kynurenine-d4, indium-d 7 and Meldonin-d 4.
7. The method for detecting tryptophan and metabolites according to claim 6, wherein: in step S2, the standard includes L-kynurenine, Tryptamine, Indole, Quinolic Acid, indium-3-carboxaldehyde, 3-Methyl-indele, Indole-3-propinic Acid, Kynurenic Acid, 3-hydroxy-L-kynurenine, induced title (IAA), Serotonin, 5-hydroxy-L-tryptophan, Picolinic Acid, 5-hydroxy-3-acetic Acid, 3-hydroxy-anthraninic Acid, N-formaldehyde-kynurenine, Xanthurenic Acid, cinnalinic Acid, Indole-beta-D-glucuronide, Indole-3-thionine, antibiotic-beta-3-alumina, and antibiotic-3-lactone, alumina-3-beta-cellulose, alumina-3-beta-lactone, alumina-3-alumina-beta-cellulose, alumina-3-alumina-lactone, alumina-3-cellulose, alumina-3-alumina-cellulose, alumina-3-cellulose, alumina-alumina, alumina-alumina, alumina-alumina, alumina-alumina, alumina-alumina, alumina-alumina, alumina-alumina, alumina-alumina, alumina-alumina, alumina-alumina, alumina-alumina, alumina-alumina.
8. The method for detecting tryptophan and metabolites according to claim 6, wherein: the liquid phase method in the LC-MS/MS analysis used in the step S3 and the step S4 is as follows: separating by UPLC system, placing the sample in an automatic sample injector at 4 deg.C, at 45 deg.C, at 400 μ L/min, and at 5 μ L; the gradient of the related liquid phase is 0-2min, and the B phase is maintained at 15%; 2-9min, phase B changes linearly from 15% to 98%; 9-11min, maintaining phase B at 98%; 11-11.5min, phase B changes linearly from 98% to 15%; 11.5-14min, keeping phase B at 15%.
9. The method according to claim 6, wherein the method comprises the steps of: the mass spectrum conditions in the LC-MS/MS analysis used in step S3 and step S4 are set as follows:
mass spectrometry was performed in positive/negative ion mode using 5500 QTRAP mass Spectrometer (SCIEX); 5500 QTRAP ESI source positive ion conditions are as follows: source temperature of 450 deg.c; ion Source Gas1 (Gas1): 45; ion Source Gas2 (Gas2): 45; curtain gas (CUR) 40; ionSapary Voltage flowing (ISVF) 4500V; 5500 QTRAP ESI source anion conditions are as follows: source temperature of 450 deg.C; ion Source Gas1 (Gas1): 45; ion Source Gas2 (Gas2): 45; curtain gas (CUR) 40; and detecting the ion pair to be detected by adopting an MRM mode.
10. The method for detecting tryptophan and metabolites according to any one of claims 1 to 4, wherein: the detection method is applied to a detection system, a detection system platform, a detection device or a kit of tryptophan and metabolites.
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