CN1532544A - Method for detecting organic acid/amino acid metabolite in urine by gas chromatography-mass spectrometry of filter paper sheet - Google Patents
Method for detecting organic acid/amino acid metabolite in urine by gas chromatography-mass spectrometry of filter paper sheet Download PDFInfo
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- CN1532544A CN1532544A CNA031187978A CN03118797A CN1532544A CN 1532544 A CN1532544 A CN 1532544A CN A031187978 A CNA031187978 A CN A031187978A CN 03118797 A CN03118797 A CN 03118797A CN 1532544 A CN1532544 A CN 1532544A
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- 210000002700 urine Anatomy 0.000 title claims abstract description 37
- 238000000034 method Methods 0.000 title claims abstract description 25
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 title claims abstract description 22
- 150000001413 amino acids Chemical class 0.000 title claims abstract description 21
- 150000007524 organic acids Chemical class 0.000 title claims abstract description 19
- 239000002207 metabolite Substances 0.000 title claims abstract description 15
- 238000004458 analytical method Methods 0.000 claims abstract description 18
- 238000000605 extraction Methods 0.000 claims abstract description 11
- 238000006146 oximation reaction Methods 0.000 claims abstract description 7
- 230000002503 metabolic effect Effects 0.000 claims abstract description 6
- UIUXUFNYAYAMOE-UHFFFAOYSA-N methylsilane Chemical compound [SiH3]C UIUXUFNYAYAMOE-UHFFFAOYSA-N 0.000 claims abstract description 3
- DDRJAANPRJIHGJ-UHFFFAOYSA-N creatinine Chemical compound CN1CC(=O)NC1=N DDRJAANPRJIHGJ-UHFFFAOYSA-N 0.000 claims description 10
- 230000002485 urinary effect Effects 0.000 claims description 10
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 9
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 9
- RTZKZFJDLAIYFH-UHFFFAOYSA-N ether Substances CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 6
- 229940109239 creatinine Drugs 0.000 claims description 5
- 239000007789 gas Substances 0.000 claims description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 4
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 4
- FUZZWVXGSFPDMH-UHFFFAOYSA-N hexanoic acid Chemical compound CCCCCC(O)=O FUZZWVXGSFPDMH-UHFFFAOYSA-N 0.000 claims description 4
- 239000003960 organic solvent Substances 0.000 claims description 4
- 239000002253 acid Substances 0.000 claims description 3
- HIXDQWDOVZUNNA-UHFFFAOYSA-N 2-(3,4-dimethoxyphenyl)-5-hydroxy-7-methoxychromen-4-one Chemical compound C=1C(OC)=CC(O)=C(C(C=2)=O)C=1OC=2C1=CC=C(OC)C(OC)=C1 HIXDQWDOVZUNNA-UHFFFAOYSA-N 0.000 claims description 2
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical class [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 2
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 2
- 238000007664 blowing Methods 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims description 2
- 230000009514 concussion Effects 0.000 claims description 2
- 238000001816 cooling Methods 0.000 claims description 2
- 238000009795 derivation Methods 0.000 claims description 2
- 238000001035 drying Methods 0.000 claims description 2
- YAQXGBBDJYBXKL-UHFFFAOYSA-N iron(2+);1,10-phenanthroline;dicyanide Chemical compound [Fe+2].N#[C-].N#[C-].C1=CN=C2C3=NC=CC=C3C=CC2=C1.C1=CN=C2C3=NC=CC=C3C=CC2=C1 YAQXGBBDJYBXKL-UHFFFAOYSA-N 0.000 claims description 2
- WQEPLUUGTLDZJY-UHFFFAOYSA-N n-Pentadecanoic acid Natural products CCCCCCCCCCCCCCC(O)=O WQEPLUUGTLDZJY-UHFFFAOYSA-N 0.000 claims description 2
- 229910052757 nitrogen Inorganic materials 0.000 claims description 2
- 238000012545 processing Methods 0.000 claims description 2
- WTDHULULXKLSOZ-UHFFFAOYSA-N Hydroxylamine hydrochloride Chemical compound Cl.ON WTDHULULXKLSOZ-UHFFFAOYSA-N 0.000 claims 1
- 238000012512 characterization method Methods 0.000 claims 1
- 238000003745 diagnosis Methods 0.000 abstract description 4
- 150000004715 keto acids Chemical class 0.000 abstract description 4
- 230000007547 defect Effects 0.000 abstract description 3
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- 238000011160 research Methods 0.000 abstract description 3
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- 238000012216 screening Methods 0.000 abstract description 2
- 238000000926 separation method Methods 0.000 abstract description 2
- 150000004716 alpha keto acids Chemical class 0.000 abstract 1
- 238000001212 derivatisation Methods 0.000 abstract 1
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- 238000012106 screening analysis Methods 0.000 abstract 1
- QRMZSPFSDQBLIX-UHFFFAOYSA-N homovanillic acid Chemical compound COC1=CC(CC(O)=O)=CC=C1O QRMZSPFSDQBLIX-UHFFFAOYSA-N 0.000 description 6
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 6
- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 6
- QIAFMBKCNZACKA-UHFFFAOYSA-N N-benzoylglycine Chemical compound OC(=O)CNC(=O)C1=CC=CC=C1 QIAFMBKCNZACKA-UHFFFAOYSA-N 0.000 description 5
- WHBMMWSBFZVSSR-UHFFFAOYSA-N 3-hydroxybutyric acid Chemical compound CC(O)CC(O)=O WHBMMWSBFZVSSR-UHFFFAOYSA-N 0.000 description 4
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 description 4
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 4
- JFCQEDHGNNZCLN-UHFFFAOYSA-N anhydrous glutaric acid Natural products OC(=O)CCCC(O)=O JFCQEDHGNNZCLN-UHFFFAOYSA-N 0.000 description 3
- WLJVXDMOQOGPHL-UHFFFAOYSA-N benzyl-alpha-carboxylic acid Natural products OC(=O)CC1=CC=CC=C1 WLJVXDMOQOGPHL-UHFFFAOYSA-N 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 150000002500 ions Chemical class 0.000 description 3
- 239000004310 lactic acid Substances 0.000 description 3
- 235000014655 lactic acid Nutrition 0.000 description 3
- 229960003424 phenylacetic acid Drugs 0.000 description 3
- 239000003279 phenylacetic acid Substances 0.000 description 3
- HKZLPVFGJNLROG-UHFFFAOYSA-M silver monochloride Chemical compound [Cl-].[Ag+] HKZLPVFGJNLROG-UHFFFAOYSA-M 0.000 description 3
- 239000001384 succinic acid Substances 0.000 description 3
- 239000001124 (E)-prop-1-ene-1,2,3-tricarboxylic acid Substances 0.000 description 2
- RBQYTVXHDIGUCS-UHFFFAOYSA-N 2-hydroxy-2-(4-hydroxy-3-methylphenyl)acetic acid Chemical compound CC1=CC(C(O)C(O)=O)=CC=C1O RBQYTVXHDIGUCS-UHFFFAOYSA-N 0.000 description 2
- VZCYOOQTPOCHFL-OWOJBTEDSA-N Fumaric acid Chemical compound OC(=O)\C=C\C(O)=O VZCYOOQTPOCHFL-OWOJBTEDSA-N 0.000 description 2
- AEMRFAOFKBGASW-UHFFFAOYSA-N Glycolic acid Chemical compound OCC(O)=O AEMRFAOFKBGASW-UHFFFAOYSA-N 0.000 description 2
- OFOBLEOULBTSOW-UHFFFAOYSA-N Malonic acid Chemical compound OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 description 2
- 229940091181 aconitic acid Drugs 0.000 description 2
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- GTZCVFVGUGFEME-IWQZZHSRSA-N cis-aconitic acid Chemical compound OC(=O)C\C(C(O)=O)=C\C(O)=O GTZCVFVGUGFEME-IWQZZHSRSA-N 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000016245 inborn errors of metabolism Diseases 0.000 description 2
- -1 oxammonium hydrochlorides Chemical class 0.000 description 2
- 229940107700 pyruvic acid Drugs 0.000 description 2
- 238000012113 quantitative test Methods 0.000 description 2
- TYFQFVWCELRYAO-UHFFFAOYSA-N suberic acid Chemical compound OC(=O)CCCCCCC(O)=O TYFQFVWCELRYAO-UHFFFAOYSA-N 0.000 description 2
- GTZCVFVGUGFEME-UHFFFAOYSA-N trans-aconitic acid Natural products OC(=O)CC(C(O)=O)=CC(O)=O GTZCVFVGUGFEME-UHFFFAOYSA-N 0.000 description 2
- 238000010792 warming Methods 0.000 description 2
- QBYIENPQHBMVBV-HFEGYEGKSA-N (2R)-2-hydroxy-2-phenylacetic acid Chemical compound O[C@@H](C(O)=O)c1ccccc1.O[C@@H](C(O)=O)c1ccccc1 QBYIENPQHBMVBV-HFEGYEGKSA-N 0.000 description 1
- RTBFRGCFXZNCOE-UHFFFAOYSA-N 1-methylsulfonylpiperidin-4-one Chemical compound CS(=O)(=O)N1CCC(=O)CC1 RTBFRGCFXZNCOE-UHFFFAOYSA-N 0.000 description 1
- YURNCBVQZBJDAJ-UHFFFAOYSA-N 2-heptenoic acid Chemical compound CCCCC=CC(O)=O YURNCBVQZBJDAJ-UHFFFAOYSA-N 0.000 description 1
- JVGVDSSUAVXRDY-UHFFFAOYSA-N 3-(4-hydroxyphenyl)lactic acid Chemical compound OC(=O)C(O)CC1=CC=C(O)C=C1 JVGVDSSUAVXRDY-UHFFFAOYSA-N 0.000 description 1
- SJZRECIVHVDYJC-UHFFFAOYSA-N 4-hydroxybutyric acid Chemical compound OCCCC(O)=O SJZRECIVHVDYJC-UHFFFAOYSA-N 0.000 description 1
- 229940006015 4-hydroxybutyric acid Drugs 0.000 description 1
- 239000005711 Benzoic acid Substances 0.000 description 1
- 239000004215 Carbon black (E152) Substances 0.000 description 1
- 206010059521 Methylmalonic aciduria Diseases 0.000 description 1
- 208000003943 Multiple carboxylase deficiency Diseases 0.000 description 1
- XCOBLONWWXQEBS-KPKJPENVSA-N N,O-bis(trimethylsilyl)trifluoroacetamide Chemical compound C[Si](C)(C)O\C(C(F)(F)F)=N\[Si](C)(C)C XCOBLONWWXQEBS-KPKJPENVSA-N 0.000 description 1
- 229920000305 Nylon 6,10 Polymers 0.000 description 1
- IWYDHOAUDWTVEP-UHFFFAOYSA-N R-2-phenyl-2-hydroxyacetic acid Natural products OC(=O)C(O)C1=CC=CC=C1 IWYDHOAUDWTVEP-UHFFFAOYSA-N 0.000 description 1
- 108010046334 Urease Proteins 0.000 description 1
- WDJHALXBUFZDSR-UHFFFAOYSA-M acetoacetate Chemical compound CC(=O)CC([O-])=O WDJHALXBUFZDSR-UHFFFAOYSA-M 0.000 description 1
- 208000037863 aminoacidopathy Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 235000010233 benzoic acid Nutrition 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 239000012159 carrier gas Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000002242 deionisation method Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- UKFXDFUAPNAMPJ-UHFFFAOYSA-N ethylmalonic acid Chemical compound CCC(C(O)=O)C(O)=O UKFXDFUAPNAMPJ-UHFFFAOYSA-N 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- CTIKAHQFRQTTAY-UHFFFAOYSA-N fluoro(trimethyl)silane Chemical compound C[Si](C)(C)F CTIKAHQFRQTTAY-UHFFFAOYSA-N 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 239000001530 fumaric acid Substances 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 235000013905 glycine and its sodium salt Nutrition 0.000 description 1
- 239000001307 helium Substances 0.000 description 1
- 229910052734 helium Inorganic materials 0.000 description 1
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
- 150000002430 hydrocarbons Chemical class 0.000 description 1
- 150000002596 lactones Chemical class 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 229960002510 mandelic acid Drugs 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- ZIYVHBGGAOATLY-UHFFFAOYSA-N methylmalonic acid Chemical compound OC(=O)C(C)C(O)=O ZIYVHBGGAOATLY-UHFFFAOYSA-N 0.000 description 1
- 201000003694 methylmalonic acidemia Diseases 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- CXMXRPHRNRROMY-UHFFFAOYSA-N n-Decanedioic acid Natural products OC(=O)CCCCCCCCC(O)=O CXMXRPHRNRROMY-UHFFFAOYSA-N 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- WOMAZEJKVZLLFE-UHFFFAOYSA-N propionylglycine Chemical compound CCC(=O)NCC(O)=O WOMAZEJKVZLLFE-UHFFFAOYSA-N 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 150000003442 suberic acids Chemical class 0.000 description 1
- 150000005846 sugar alcohols Chemical class 0.000 description 1
- WRUSVQOKJIDBLP-HWKANZROSA-N tiglylglycine Chemical compound C\C=C(/C)C(=O)NCC(O)=O WRUSVQOKJIDBLP-HWKANZROSA-N 0.000 description 1
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention discloses a method for detecting organic acid/amino acid metabolites in urine by gas chromatography-mass spectrometry of filter paper sheets. The method comprises the steps of carrying out elution, oximation reaction, extraction and methylsilane derivatization on urine collected by filter paper, and detecting the urine sample by adopting a gas chromatograph-mass spectrometer combination. The method of the invention has the following advantages: 1. the urine specimen is collected by adopting the filter paper, so that the urine specimen is suitable for the characteristics of numerous population, broad range of personnel and unbalanced regional development in China, is favorable for large-scale high-risk screening, research and remote analysis of the genetic metabolic defect, and accords with the national conditions of China; 2. the oximation treatment process is adopted, the detection of the alpha-ketoacid is facilitated, and important branched-chain ketoacid metabolic disorder cannot be missed to diagnosis; 3. through extraction, the recovery rate of the organic acid/amino acid metabolite is high, and the repeatability is good. The impurity peaks of the chromatogram are few, so that the chromatographic capillary column and the mass spectrum ion source are not polluted; 4. the qualitative/quantitative analysis conditions of the gas chromatography-mass spectrometry are stable, the separation effect of the chromatographic peaks of the sample is good, the analysis time of the sample is short, and the method is suitable for high-throughput sample screening analysis.
Description
Technical field
The invention belongs to the detection method of organic acid or amino acid metabolites, be specifically related to adopt urine filter paper to detect the method for urinary organic acid/amino acid metabolites by gas chromatography-mass spectrometry analysis.
Background technology
Owing to contain the whole end or the mesostate of amino acid, carbohydrate, lipid and many other endogenous compound in the urine, these organic compounds have higher relatively water-soluble and renal clearance, its concentration also is higher than serum and other body fluid usually, and sample is easy to collect most.Previously China only can carry out Preliminary detection to hereditary metabolic defect; Dependence is delivered to external cooperation unit with sample and is determined that carrying out of the examination of the hereditary metabolic disease that seriously lags behind, treatment and pathogenesis research work influences the integral level of this medical domain of China.
Gas chromatography-mass spectrography (the gas chromatography-massspectrometry that North America and Europe are adopted, GC-MS) method is directly analyzed for utilizing the crude urine sample, or express delivery is not suitable for China's national situation to inspection center after freezing, easily fails to pinpoint a disease in diagnosis the branched-chain keto acids dysbolism.In recent years Japanese scholar uses filter paper and collects the urine sample, directly carrying out GC-MS without organic extraction after urease is handled analyzes, in order to identify organic acid and partial amino-acid and sugar alcohol metabolic product, attempt to carry out carrying out hereditary metabolic disease neonate population screening with the GC-MS method.But assorted peak, gas chromatography peak is a lot of, unfavorable interpretation of result, and very easily pollute gas chromatographic column and mass ion source, its simple urine amino acid analysis result also is not enough to diagnosis and studies complicated aminoacidopathy.
Summary of the invention
After the objective of the invention is to urine filter paper carried out oximate, extraction, utilize gas chromatography-mass spectrometry analysis to detect the method for urinary organic acid/amino acid metabolites.
The present invention is achieved in that 1) adopt filter paper to collect the urine sample; 2) filter paper urine sample is carried out wash-out and handle, measure creatinine content and determine urine concentration, obtain urine specimen; 3) urine specimen is finished the oximation reaction of a-ketone acid; 4) adopt organic solvent to extract; 5) sample of above-mentioned processing being carried out methyl-monosilaneization derives; 6) adopting gas chromatograph-mass spectrometer to unite detects urine sample.Set up organic acid, amino acid and other metabolic product mass-spectrogram storehouses according to the material that will detect.
Adopt the method that the present invention set up to have following advantage: 1, to adopt filter paper to collect the urine sample, be fit to populous, vast in territory, the regional uneven in development characteristics of China, help carrying out high-risk examination, research and the remote analysis of large-scale hereditary metabolic defect, tally with the national condition; 2, routine is carried out the oximate handling procedure before the extraction, is beneficial to detecting of 2-ketoacid, unlikelyly fails to pinpoint a disease in diagnosis important branched-chain keto acids dysbolism; 3, use ethyl acetate and twice organic extraction of ether, press the creatinine content quantitative test.Organic acid/amino acid metabolites recovery height, good reproducibility.The assorted peak of chromatogram is few, unlikely pollution chromatogram capillary column and mass ion source; 4, selected chromatography-mass spectroscopy qualitative/quantitative test conditional stability, sample chromatographic peak good separation, the mass spectrogram information completely is easy to computing machine spectrum storehouse and identifies.The sample analysis time is short, is fit to the examination of high flux sample and analyzes.
Description of drawings
Normal urine organic acid/amino acid GC-MS that Fig. 1 this method detects analyzes chromatogram;
The methylmalonic aciduria urine GC-MS that Fig. 2 this method detects analyzes chromatogram
The multiple carboxylase deficiency disease urine GC-MS that Fig. 3 this method detects analyzes chromatogram
Embodiment
1, urine specimen collection: adopt No. 3 filter paper of homemade Xinhua, be cut into 5cm * 8cm size, collect urina sanguinis or inferior arbitrarily urine, dry slightly in the rearmounted special filter paper dry bag.
2, filter paper urine sample disposal: the dry filter paper urine sample is with 2ml deionization distilled water gradation wash-out, and is centrifugal, collects eluent.
3, determine urine sample concentration: measure creatinine content and determine urine concentration, get suitable 2.5 μ mol creatinine urine samples.
4, add mark pentadecanoic acid and 2-ketone caproic acid in the 50 μ g in the sample, with 100 μ l oxammonium hydrochlorides, 200 μ l0.25M sulfuric acid, the saturated ammonium chloride mixing of 500 μ l 45min to finish the oximation reaction of 2-ketoacid, improve the recovery of branched-chain keto acids.
5, under sour environment, carry out twice extraction with 3ml ethyl acetate and ether respectively, carefully separate and merge the organic solvent phase, remove a little remaining moisture content with 1g anhydrous sodium sulfate (concussion, centrifugal), add saturated 24 hydrocarbon of external standard (C24), do with the purified nitrogen air-blowing.
6, add 100 μ l methyl-monosilane derivating agent N in the sample after the extraction, O-pair-(TMS)-trifluoroacetamide and 1% trimethyl silicon fluoride (BSTFA+1%TMS), put 60 ℃ of derivation 30 ~ 60min on the drying heater, the cooling back is diluted to 500 μ l with normal hexane.Getting on the 1 μ l sample carries out GC-MS and analyzes.
7, gas chromatograph-mass spectrometer Conjoint Analysis: use Agilent 5890/5973N type GC-MS and carry out sample analysis, be equipped with 7673 type automatic samplers, HP-5 capillary chromatographic column, column length 30m, diameter 0.32mm, thickness 0.25 μ m.Chromatogram and mass spectrophotometry condition are as follows:
Adopt no shunting sample introduction, injector temperature is set at 250 ℃.Helium is carrier gas (1ml/min), 250 ℃ of interface temperatures.Mass Spectrometer Method adopts the total ion scan mode of electronics dissociative pattern (EI 70eV), mass-to-charge ratio (m/z) scope 50 ~ and to 600, scan period 0.4s.The chromatograph box temperature programme is since 60 ℃, and 20 ℃/min stops 4min after being warming up to 90 ℃, is warming up to 300 ℃ with 8 ℃/min then, about 25min of sample analysis time.Computer system record chromatogram and mass spectrum figure are used the abundance of the reference evaluation of mass-spectrogram storehouse and each chromatographic peak and are carried out qualitative and quantitative analysis.
According to above-mentioned multiple urine sample is detected:
As shown in Figure 1, each chromatographic peak is accredited as according to this among the figure: 1. lactic acid; 2. glycollic acid; 3.3-hydroxybutyric acid; 4. urea; 5. acetoacetate; 6. ethyl malonic acid; 7. succinic acid; 8. interior mark-I; 9.3-methylpentene diacid; 10.3-hydroxyl hexane diacid lactone; 11. hexane diacid; 12. heptenoic acid; 13.4-hydroxyl phenylacetic acid; 14. unsaturated suberic acid; 15. suberic acid; 16. aconitic acid; 17. homovanillic acid; 18. citric acid; 19. hippuric acid; 20.3-methyl-4-hydroxymandelic acid; 21. interior mark-II; 22.3-hydroxyl decanedioic acid; 23.4-hydroxyl hippuric acid; 24. external standard
As shown in Figure 2, each chromatographic peak is accredited as according to this among the figure: 1. lactic acid; 2. glycollic acid; 3.3-hydracrylic acid; 4. pyruvic acid; 5.3-hydroxybutyric acid; 6. methylmalonic acid; 7. malonic acid; 8. benzoic acid; 9. succinic acid; 10. interior mark-I; 11. fumaric acid; 12. glutaric acid; 13. mandelic acid; 14.2-hydroxyl glutaric acid; 15.3-hydroxyl hexane diacid; 16.3-hydroxyl-3-methylglutaric acid; 17.4-hydroxyl phenylacetic acid; 18. homovanillic acid; 19.4-hydroxyphenyl lactic acid; 20. interior mark-II; 213-methyl-4-hydroxyphenyl-lactic acid; 22. external standard
As shown in Figure 3, each chromatographic peak is accredited as according to this among the figure: 1. lactic acid; 2.3-hydracrylic acid; 3. pyruvic acid; 4.4-hydroxybutyric acid; 5.3-hydroxyl glutaric acid; 6. urea; 7. succinic acid; 8. interior mark-I; 9. Propionylglycine; 10. isobutyryl glycocoll; 11.3-hydroxyl-3-methylglutaric acid; 12.3-Tiglylglycine; 13. Tiglylglycine; 14.4-hydroxyl phenylacetic acid; 15. aconitic acid; 16. homovanillic acid; 17. hippuric acid; 18.3-methyl-4-hydroxymandelic acid; 19. interior mark-II; 20. external standard.
Claims (7)
1, the filter paper gas chromatography-mass spectrometry analysis detects the method for urinary organic acid/amino acid metabolites, and its characterization step is: 1) adopt filter paper to collect the urine sample; 2) filter paper urine sample is carried out wash-out and handle, measure creatinine content and determine urine concentration, obtain urine specimen; 3) urine specimen is finished the oximation reaction of a-ketone acid; 4) adopt organic solvent to extract; 5) sample of above-mentioned processing being carried out methyl-monosilaneization derives; 6) adopting gas chromatograph-mass spectrometer to unite detects urine sample.
2, the filter paper gas chromatography-mass spectrometry analysis detects the method for urinary organic acid/amino acid metabolites according to claim 1, and it is characterized in that: the urine sample of described collection is a urina sanguinis.
3, the filter paper gas chromatography-mass spectrometry analysis detects the method for urinary organic acid/amino acid metabolites according to claim 1, it is characterized in that: the oximation reaction of described a-ketone acid is mark pentadecanoic acid and a 2-ketone caproic acid in adding in sample, mix with oxammonium hydrochloride, sulfuric acid, saturated ammonium chloride, finish the oximation reaction of 2-ketoacid.
4, the filter paper gas chromatography-mass spectrometry analysis detects the method for urinary organic acid/amino acid metabolites according to claim 1, and it is characterized in that: described extraction is to adopt ethyl acetate and ether to carry out twice extraction, carefully separates and merge the organic solvent phase.
5,, detect the method for urinary organic acid/amino acid metabolites as filter paper gas chromatography-mass spectrometry analysis as described in claim 1 or 4, it is characterized in that: after extraction, sample is carried out remaining moisture content and remove, its method is to add anhydrous sodium sulfate by concussion, centrifugal, remove remaining moisture content, add external standard C24 again, do with the purified nitrogen air-blowing.
6, the filter paper gas chromatography-mass spectrometry analysis detects the method for urinary organic acid/amino acid metabolites according to claim 1, it is characterized in that: it is to add the methyl-monosilane derivating agent in sample that described methyl-monosilaneization is derived, put 60 ℃～90 ℃ derivation 30～60min on the drying heater, dilute with normal hexane the cooling back.
7, the filter paper gas chromatography-mass spectrometry analysis detects the method for urinary organic acid/amino acid metabolites according to claim 1, it is characterized in that: set up organic acid, amino acid and other metabolic product mass-spectrogram storehouses.
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