Analysis of pyrimidine bases in biological materials by gas chromatography-mass spectrometry

SUMBEARY

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Siiultaueous dctecth by’s annbinationof gas cbromatograp~ fragspazkomctry for total ion numitoring meatography and &asvphy+nass was developedfbr d&crmmatioa of ucacil,thymineand cyto&e presentia biological . materralsaspyrimidinekac#~thdr~~detivat~Thedeteccianlimitffor uradt,thymineand~inthe~~~O.oOl~~~forplesnrraand O.OOl-O.WS pg/gwetweightfor tissues. Thosefor uraciland tbyminein plasmaand tissrresandforcytosineinpIesmainth+~dmcthodwcnaZccg/mlorgwet weight.and thatfor cytosinein tissues pvas 2.5pg/gwet weigb~ An accurateand saAtive assay-fordemmiation of pyrimidine bases was establisbul.

IIWRODUCTION

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Tbeow~cotuma(~mx2lnmm_)oftEat~~was packed with 3% OV-17 QI~Clmxnosorb W AW (80-100 mesh) (Gashno Kogyo,

ofthcinjcctoraad 0saka#Japan)andamditi~at28(pfar24h.~~ tbcd&&orwassetat22OOaQdam&s?swerccauiedoutwith8ninitWc&lmxl tanpsaturcofl#pandatemperahae riscoflo”/min.Hdiumwasrwdaotbeauria gssata%ow-meof#)nll/Jnia. : Themessspctnrmeterwasopcratcdpndtrtbcfo%owiogeonditions:ianizationeneqy,7Oev;inozationamen& 300fl; aaxka&m voltage.3.0W; and ian multiplkrvoltagc,1.4kV.DaeiminationsbyGMS-~w~arrriedoutatb sametimeasthosebyGGMF.The~tiansBdeetcdametItcmo~ion peaks(M+)ofm/e256and27Ofortlaenspcctivesilylatcddaivativesofaracirand thymineaad tfscbase ion peaks(M-C&) of m/e 240 and 2’75for the s%ylated derivatives of cytosineand theinternalstandard5-&lorouracil.

-TSANDDISCUSSION

extra&on and sepmationof pyrimidine basesf’rombiologicalmaterials gave good rcproduciiity and remverywhencxeactionwas conductedas folIows: proteinwasdenatured by extraction withchloroformin HCI andthesnpematant was adjustcdtopH6.Owith OSM NaH#04andextracM withethylacetate.Therecoveries ofuracil,~~and~nemre~w.8oo~fromplasmaandaa’loo~fromvarioas ~.Extrsction~~datsobeachkvedwith~~ccbutirin~with subseqaintGlylation.S-Chlmmmcilwas chosenas an intanal stqndardfar muUipk ion detection by GGrMF. The fkagmcnt ionsdetected wcre.themokcukr ion peaks intbe~spctraofttae~k~dnivatmesof~andtbymine~~2),andthe baseiau peaksfor M-_CAI of theGXyIatecl derivatives of cytosiueand 3-chlomrracil @Sg. 2). Cmsqedy, goad sepaaiion and sati&aory sensitivity were achieved. Fig.3shows~~~by~~~of~c~~ofurecit,thymiaeand cytosinc,~d~inanextract~m~liverofK-~nademia.~~ti~ timw wme 1.7,Z&3.7 and 25 mill-forthe saylatedd&vat&s of Ilmcigthymin~ cytosineand Ichtomrusacil, rcspaSveIy,and theircomqondiug p&s were condlrmea-bym speetrometiy and mass chromatography. The detectionlimitsfor ~~~and~inthisG
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FWy, we studied the influences of mwIeosidesand nuckotides of pyrimidine on~determinatioaofuracil,tbymineandcgtosinebythepresentmethod:for ~inationofthe~~of~m~ti~of~~nudeosidesandnu~~~, au~~sampEesofthesc~m~~~werrd~Xv~ipp~andtissaehom~ate ofliva,andthenapalypdfortheconamtrationofpyrimidine~detected,Tbe resultsshowed that the decompositionof nuduxkk and nuckotides had no appreciable it&cncc on the inemmmntofpyrimi~baseswiththcprcsentmctb~. Cbqgcs in the concentrationof pyrimidinc bases were exam&d by incubation of pkmaandtissuehomogcnateoflivcrwithsbakingat Pfor 1%Mb-b&orcmcasu+g

tszr g&_ spl-

28lio.78nmol

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AadB42(1911)549.

5V.hf3llu.V.hdkodmdESmoltovb,I.~., 119 (1976) 355. 6P.RBcow4s.EbHdcamdF.L.mnky.A w.9 99