Document B5Ex1BgxJr0Eo0kvavNRmLBmX

ti,n of 14c-N-Zthyl TOSZ in "to 13iotranSfOrmA lpeed for one week After Admin istration in innuary 28, 1983 Condur-ted At: During: ConduCtsd Byz 3teport BY: Reviewed BY: Riker Laboratoriest 3M Center St. p&ulf Kinnegota Inc. 55144 October 1980 to Jwe 198' S. J. Gibson D. johmon . D. Jo on, Ks ?RelRel-se@ejarch SPecial'-sSt Tz-a:@g@ R. E. Ober, PhD Managere Drug MetabOli- Date Date Table of contents page ry Introduction Materials and 140t'hods ... Carbon-14 Labeled M-Ethyl FOSE Animals ...... Dosing Preparation of DOge/Feed Mixture .... .......... ........................... Administration of Doe sample Collection Radiametric AnalYses 4 Sample Prp&ration for "dienattic )Ln&lyo" .................. knalynes of Samples .......*-*,* .......... Metabolite isolation and Identific&tion and Discussion Results .......................... References o ... 10 List List Of rigures ......o----o ...................... ....................14 Of Appendices ...***,**"**** page !oe-a-rx mg isolated frcm rat faces and A major m@t.blit- of '4C-N-thyl roSE SC*tiC &Cid (WSAA)- identifiedc,alsit2ea-Nc-ceotuhnYtls ppferrfaltuerlceOaCsttan5&0s%uloffOntBhxeidCO&Zbw -1t4ioenxcorfet&emdisitnure TfheeceMsOtbaebt- en 24-49 hours ahfitcehrrcaetseswaetrieontoofd affotrinIiwseterka. The mean of 14---"...OthPyOlSE/f*ed w total dose was 300 mg/kg/wG*k- Although collection of faces for source material for metabolite isolation was the primary purpose of this study* the rats were fad normal chow for three weeks foucwing the one weak of 14C-N-ethYlTC)szlteedadministra- tion andiplasma and select tissues were collected at sacrifice and analyzed for carbon-14 content. The mean cSrbcn-14 contents exPrOssL*das tigof N-.*thylTOSE equival*nts/9of wst tissue in Tarious tissues were: liver, 233; spleen, 201*and red blood cells, 30. Plasma contained 62 tig -.N--*thylIPOSZ/21 Of Plasm. The tissuee concentr&ti&A's equivalemnynt9s f1o4lClowing the last dose after I week of f eding 14C-N-.ethyl at 21 d consistent with Ahat would be gx"cted after multiple dosing IPOSE are with a couPOund that is slowly eliminated. page 2 introduction d by 314and is also a key intermediate ir, _qE is a product $01 it was shown Nt-hEethsyylntrhOesis of other products. in a previous studty i(n1)l,iver extracts Luorooctanesulfonate is & metabolite Pres*n a single dme of that perf:at 96 hours after administration in food Of from ratey, roSF.. From the same liv6r extracts* a second metabolite 1AC-N-ath a as perflucrOOctan0sulfonamide. was isolat d and tentatively identifie ant in the liver extract' 05 was Other -id:ntified tabo3-Lt--.re prom a nt experiment, three shown by thaidmni-nliasYtOerrerdad1i4oCc_hNr-lettohgyrlmsr.ose iInnfteheed Pfro*raI wTeheek afencdefsL'CCOtlsectiOns rats were .eke after the dosing period. were oo3.lected for two ' IAC-w-*thyl rose dose wore pooled and from 24-48 hours after the lost ,a identified. The results are extracts(I- A metaboliteinwatsheiosaonlatt*exdt of other fluorochosic&l metabolism reported and discussed studies. materials and methods ma!L@ Carbon-14 Labeled N-Eth 1 "0 C295 -CH2cB2oB C-7Fl5*CF2SG2- *Denotes position of carbon-14 label ido ethanol. , roSE is 2-N-ethyl perfluorooetanesulfonam N-EthY in this study was identical to the 14c-N-ethyl The 14C-N-ethyl "SE used 3dy reported *arlier; used in the single dooe 14C-N-ethYl ROSE at% ion repeated for rosz tails of the radi0chemical purity deteminatactivity of the lot of the de r.reported (I)- The specific these two studies i in these studies (i@ikorIsotope Inventory 14c-tii-ethylTOSE used of the specific 0.020 UCi/log. On the basis ical Numbs r 468) is o.483 haracterization, and radiochem activity determination@ chemical c e 14C-N-othY I roSE was judged purity reported selparaelY (2). th suitable for metabolism studies. A,nimals a were conditioned Prior to dosing, three male Charles River CD rat&- cages. Body weights t,i,less steel metabolism overnight in individual No. 3, 219 g. The 2, 210 g; and 15lat were, Rat NO- I- @269 9; fROaO6t7,-@oa.nd water before and during the rat had free sccoss to exp:riment. ies, Willmington, massachus*tts- a Charles River Breedirg Laborator uis, Kiesouri. Purina Lab Ch- , Rlaton Purina Company, St- 1,0 page 3 p_r_.L@@.tl-n o@' ID)Ooe@eed3 1"4'ix@ltull@ prior to this The i&c-li-ethyl rosz/feed aixture, PrOP-Rd just 1 . The details Of study dome study (1) was also used for the single the carbon-14 content are r*PO rted the Pr'eparation And the assay for was prepared by weighing 250 mg (1). @ 14c@N@ethyl TOSE solution I as A volumetric flask, adjusting 14c-ti@-ethyRlOSE into a I liter CIA arting. The absolute ethanol, and mixing by i- and t1o4Cv-oNl-uemtehywlitPhOSE solution was transferred to a 4 liter beaker Purins Ground Ch- was added The 14C-N-othyl i to FOSE/feed a glass tray and 500 g a then transferred mistur wa stirred for one hour t of the 6ose/feed the ethanol was evaporated. The carbon-14 conten mixture was detemined by combustion Administration of Dose Chad with wire to the inside of each cag@- A A feed cup -8 Att .53 mg 14C-N-ethYl 130 g portion of P:rina Ground Ch- containing 0 Posz/g of chow was weighed into a separate wide.@outh, brown glass jar for each rat And portions Of the niitturewere frequently trans- I cup*. Care was taken Dct to Place ferr,d to the corresponding fee@d that significant spilling by the so much of the mixture in the cui,s The jars containing the IAC-N-othyl 1poSE/feed rats could occur. BO&Ied at room temperature. it took mixture were kept tightly stork for the rats to eat 13()9 of t-he14C-N-etbyl TOSE/fe0dfor omniextuwereek. The total dome of 14C-41-othylrose administered was Rat No. 1, 256 mg/kg; Rat No. 2, 329 ag/kgl and Rat tio- 3, 315 mg/kg- After the 14C-N-othyl FOSE/feed mixture was consumed (168 hours), the rats were given free access to normal chow. Durirbg the dosing period, urine wO feces were collected and stored d at this time)- Urine and feces were collected at froz0e:n@ (not analyze of administration of intervals for I week following cessation 24 h feed mixture; thereafter, urine and feces were the 14C-N-ethyl IPOSE/ collected an P0019 for individual rats until sacrifice. At 21 days after the last ingestion of 14C-li-ethylIPOSE,the rats were anesthetized with diethyl ether; blood was drawn from the descending aorta and immediately transferred to a heparinized tube. Plasma was prepared promptly by centrifugation. Ali(riotswere taken for analysis and the samples were then frozen. The rats were sacrificed by exsanguination and liver, spleen, brain, kidneys, lungs, and testes were oollected as whole organs. The femurs and tibias were collected for bone sarrow of subcutaneous and abdominal fat# skeletal muscle, sampling. Samples and digestive tract (esophagus, stomach, and intestines), and the rem ining carcass were collected for each rat. page 4 .. t.Ic Analyses prepared for carbon-14 analysis by Faces liver# and spleen were sample f the h--g--to 'n"O em- ..,.,.g ,d aliq_oting a blenders by homog ' Ro"enizing we done in Waring The bustion cone 9-a-. to on: part of biological materialno, in duplicate' adding nine parts Of wewraeterweighed into combustion ' les Of hamOgenAktas ("0 g) te between care was taken to Six wethe homoignetno& combustion NaMP"ngs'All 5o-tPher 00 nes. red blood Cells were ighed t n&lyzed; these iection) were no ,amplas collected (see maxPlO C01 lysig or for metabolite able for further one samples remain &V&il isolation/identification studios' busted with a gmples weighed directly were c' ,as doterainod by somoger.ates and ,.ry of cagbon-14 Packard Model 306 oxidizer. "cl liver) spiked with ccebusting suitable blank hc"enateg (feces and es were cosibusted 14C-N-ethyl ROSE solution; these reference 58OP1 set. middle, and end Of the analytic&' sadoupplleico1t.e0 ml at the beginning, d counted directly) urine collections were GsxPled an into scintillation luots of each limple were pipett*d directly SaMled before alito and 15 m, Aquagolo was addl-d. Plasma was 5 ml vial iplicate.0.5 ml aliquots Plus () freezing and counted directlyl dt viis and 15 ml d directly into scintillation adapted before water were PiPett cold and dark Aquagole added. ;:I samples were countidg- done using a Packard Model 3380 All radioetric measurements were and urine Scintillation S;,ectrcoeter. For plea" Tri-Carb Liquid the counting efficiency for each sample was B&Zopjes counted dirnegctalyk#nown &Mount of internal standard to each determined by add . corrected for back- irlg. After each B=Pl* was Cal "ample and recount ting efficiency nd and for coun the carb--i4 content was ency for each samp le 9 rOU c@u nting effici zation) culated. For ccabusted 2-PleO, (Autmatic External Standardi was determined k*, use of the ABS external standard, a known amount Of ra tio method- To Calibrate the elected samples in the group (three internal standard was added to 5 1 and these SaMleg th low AW ratios r@d three with high ratio6l de wi - For ccmbusted samples, a correction was so were recounted xidizer. The percent recovery 95%) from the 0 r combusted with recovery (mean, 14C-N-ethyl FOS based on referenc1 e S..ples of for the as each sample set. Packard Downers company, inatr,@ent Grove, Illinois. Inc., 2200 Warronville Road, page 5 Isolation and Identification Metabolite rib,d. A short descrip . section, & ,,gthy procedur is de&C ,,n&tes were In thi ,f this length3r procedure fc)110": F*ces wahsolch@romatograph *d on tpiooonlaencdd extracted with other- The 03ctrecahcrtomatographed an a silicic acid -lusn and a fractieownawsaslorcated by .thirt-layerchtc"- second column. A ou,jorastabolit reparative TLC Cooparison Of tography (TLC) and isolated using P unds suggested system with standard goference cool- Rfis in varic's the structure to be CBF17S02NcR2CE3- I cB2COOR The data from go,& chromatography/mass spectroscopy (GCI"KS)confirm the identification. es collected 24-48 hours after The fecal homogOrIte pool from fOc laced into twelve the last administration of 14C.N.ethyl POSE was P To each tube we 45 ml es centrifuge tub (25 ml b-Ogenate/tube)Lixtures were sh aken for 15 mi:.tes addedme15 ml diethy other and t-he ed and the ether was ch&nical shlaker. The tubes wr. e caonnytroiffutghe interph-e meterial)- on :vd. (care -- taken not to tt-i0m0evs. The other vaka pooled and rem The extraction was arteipnegateevdapfoirvaetor so that what remained was mostly reduced with a rot been carried over with the ether. This water the water that had .1, V:V). The layers were layer was extractsd with chloroform tained 5-9 09 Of separated. RadicmetricallY, the chloroform con d 70% Of the carbo"-14 thy, MSE equivalents. The extraction remove tographed N-el chloroform layer I's chrome from the fecal homOgOftat*, The a was packed in chloroform into by column chrmatography (Unisila-- extract Of the 4 CM x 40 cm 91888 column). a2 The chloroform Ltract was place a on the COIL=N- aque'ous residue from the ether I i passed through the column and e hundred ml Of chloroform wal ,a, of chloroformr riv r&ction. pollowirg the 500 ically, collectea as one f was passed through the C01%mn. Radiometr Soo ml of methanol d 4.6 mg equivalents Of 14c-N-ethyl th chloroform fraction contains1.3 mc). Thus, the chloroform column po:E and the methanol contained erial in the fecal homoqenatle fraction contained 55% of the mat L4clanalysis Of carbon-14 The procedure for thin-layer chromatography (T (1). The chloroform labeled compounds has been descrTiLb@eCd anpdrevtihoeusrl&ydiocbroaatOgIMO is lumn fraction was analyzed comprised .90% of the co 1. A componenbty at Rf 0.43 shown in rigure column fraction or @50* Of the c.rb--14 in the rbon-14 in thi 1 nSE ingestion- Cfa....I-24-48 h:urs after the last 14c-N-ethY milicic acid 100/120 mesh, Clarkson Chemical Unisilr activated company, Inc., Mil liamsport, PA. page 6 rotating evaporator evaporated with The chloroform C01' fraction was column chromatography- UnLailO .w a, ,d chromatographed by en y. 4()CM glass to a f ck,d in bOnXOT,,chlorOfOrm , viv) (I" in a 2.4 on this a-nd was pa form column traction was placed column and the chlora ll.ctd a. 50C)-1 Of benze" column. rifty &I fractions were cO for, (lt2,v:v)r and (iti, viv), 500 al Of b*nzene@chlorc COJUmn. Aliquots chloroform sed through the then 500 mi of chloroform were pag So a, fractions were analysed r dicnetric&llyl (100 oll of the :a So =I fractions and most of the carbon-14 appeared to be in thr (lpractions 17, 18, collected from the 112 benzene-,chloroform Oluticng aphy and and ig). By inspection of the column durincj the chromatogr al tions, it was apparent that such of the fac of the varicus frac rbon_14 by this colim chrcmato- pigments were separated from the CA graplhy PrOcs&IrO- The pooled fraction, 17, ,a, and 19 contained 3.9 scj of lAc-N-*thyl IoSz equivalents. column were The pooled fractions 17, 18, and 19 from the second silica 11 volume and streaked on six 20 cm x 20 cm evaporated to a am loped to 15 cm above the Pre- ge plates-". The plates -re dev, are of loo ml chloroform, 35 ml ads1 orbent band in loo al of a sixt, nium hydroxide. The carbon-14 methanol, and 5 m@l concentrated m was located by scraping 0.5 cm segments laterally from a center 5 cm 1 20 cm section of the 20 co x 20 cP Pl&t*- (Only a narrow strip @l em was scraped.) The carbon-14 was located at Rf 0.33. A 1.0 Cm wide zone centered at Rf 0.33 was scraped fr- the six plates and the silica gel was eluted with methanol- The methanol from the elution was evaporated to a small volume and applied to a 2o cm x 20 cm Silica gel plate that had been preand air dried. The plat* was developed to 15 cm washed in methanol mixing loo al chloroform, 100 ml in loo ml of solvent prepared by methanol, and 2 mi concentrated acetic acid. The carbon-14 wa -etrically located radi (a narrowr band from the origin to the :5olcvment a of the plate laterally in 0* front was scraped from the cent segments and counted). The Rf Of the carbon-14 labeled material was 0.77. A 1.0 ca zone centered at Rf 0.77 was scraped and the si.lica gel was eluted with acetone and then chloroform-methanol (1:1, es were combined, evaporated to a =all volume, and v:v). The eluat ica gel plate and developed to is = in the same streaked On-mt&h-soill-&cetic chloroform as before. The acid solvent BYst g a segment ::a counting. The carbon-14 was located by scrapii 0 cm zone centered carbon-14 was again located at Rs0f 0.77 - A I- eluted with acetone. ilica gel was at Rf 0.77 was scraped and the. ag of W-ethyl 1posE equivalents. The acetone eluate contained 1. Iligure 2 is a radjochromatc)grm of the carbon-14 labeled Material eiuted from the second plate with acetone; the TLC PlAte w*l ]prepared from lo()mi chloroform, developed in 100 ul Of & mixture loo mi methanol, and 2 ml concentrated acetic acid- Analtech, 75 Blue Ron Drive, Newark, Delaware. page spotted Reference standards were prepared irkme-tihca-naollly)andlltbolite &liquots of the purified (radi-h wpiltahte&. The Structures of the reference standards werel along an TL4C 1) CeFl7SO2N"2 2) CBI'17SO2NW285 3) cs?17SO2NCB2CO2E C2H5 4) CSP17SO2NWB2CO21 loped in loo mi of a mixture prepared from The pi tes were do- methanoll and 5 al concentrated am=nium 100 mlachloro-c=. 35 ()a, of a mixture prepared from 100 ml hydroxide (system A) or in 10 chloroform, 100 al methanol, mr4 2 al concentr ted acetic acid (system 8). The spots an the plate were locat:d with acidified palladium chlorid or browhenol blue spray reagents. The Rf Of the metabolite wa: confirmed radimetrically bY scraping and counting- The Rf was 0.28 in system A and 0.77 in System B- The TLC of the metabolite and the reference standards showed that the Rf of standard 3 I[CSF17SO2NCB2CO2H) was the same in both systems I C2H5 and different than the Rf of standards metabolite was submitted to 3H Central 14c1, 2, and 4. The Research (i. N. Schroepfer) for GC/MS analysis (see Appendix 1). GC/KS data indicate identical retention times and an exact match of El spectrum with standard 3, and the Cl Spectrum of the metabolite matches exactly with the Cl spectrum of standard 3. in addition, it is possible to &Ssign the major fragments observed consistent with the structure of standard 3. rall, the identical thin-layer chrcoatogra;ihy Rf-s, identical gc retention times, identical El and Cl spectrum, and the interpretation oovfe structural data from mass spectra show that the metabolite is CeF17SD2NC82CO2H I C2R5 The mthanol fraction from the first silica gel column was evaporated and spotted on a silica gel plate and developed to 15 cm in 100 ml Of nixture of 100 mi chloroform, 100 al methanol, and 2 ml acetic cid. The radiochromatogrm : is shown in Figure 3. page 8 Itesults and DisCuOai*n "bolite Of 14LC_V_thy, roSE was isolated from faces Aidmeanjtoirfiesdo as 2-N-ethyl perfluorooetanesulfonamido &Cetic acid (PPSAA). The metabolite accounts for at least 50% of the carbon-14 excreted in a at 24-48 hours after the last dome (see Appendix 2) in a facer. Poon which rate were fed 14c-w-ethyl rosz for I week. Since the reexgtirmaecntioinI is from feces collected (24-48 hours after the last dose) following a week of dosing, the contents reflect what would k)e expected at various times after insingle dose. As reported previously (1) by one week following a single dose of Ik-M-othyl POSE, -%,50o%f the carbon-14 These data suggest that a large portion of the is eliminated via faces carbon-14 eliminated vi; fc.. J. ppSAA. 2. ddition, three other metabolites were extract,ed from feces. Together, these other unidentified 15%of the carbon-1i4n thispool. metabolites account for In a previousexperimentp,erfluorooetanesulfownaasteidentifieidn liverextractsfromratsat 48 hoursaftera singleoraldoseof i4C-N-ethyrlose (i). Perfluorooctanesufoancactoeuntefdorat least 22% of the carbon-1i4n theliverpool (4.4%of done.).From thesame , a secondmetabolitwehichaccountefdorat least32% of the ecxartbroanc-1t4 in the liver pool was tentatively identified an perfluoro- a octanesulfonamide (6.4% of dose) (1). identification of perfluorooctanesulfonate in liver as a metabolite at 4.4% of the done after a single dose and identification of FFSAA as a metabolite comprising 50% of the carbon-14 in feces during the second twerity-fourhour period after the last dome following a week of feeding 14C-N-ethyl ROSE establishes that both compounds are major metabolites r, these data do not lead to conclusions as to of N-ethyl ROSE. Howeve the relative proportion of the biotransformation to either coupound. Since it ran be assumed that the time course of elimination of PFSAA and perfluorooctanesulfon&te is vastly different (be ed on the amount of PTSAA in feces and on previous metabolism studie: on perfluorooctanesulfonate which is eliminated slowly via feces (<St of dose/week) (3)] " since the possibility that FFSAA is further biatransformed to per- exists, it would require a kinetic study to ascerfluorooctanesulfonate tain the rate and extent of biatransformation of N_ethyl ROSE to these compounds. For the sane reasons given for PFSAA, the relative extent of the biotransformation of N-ethyl POSE to the three metabolites tentatively identified as perfluorooctanesulfonamide (1) and the metabolites in the methanol column fraction (Figure 3) (which, since they have riotbeen identified, may include perfluorooctanesulfonate and perfluorooctane- oulfonamide) is not established with these data. The mean carbon-14 content expressed as jigof N-ethyl POSE equivalents/9 liver, 21131oVIeen, 201 and red of wet tissue in various tissues were- blood calls, 30. Plasma contained 62 iogequivalent@ 4C-N-ethyl ?OSE/ml of plasma. (individual tissue and plasma concentrations are .) The tissue concentrations at 21 days following listed6 in Appendix 3 week of feeding 14C-N-ethyl ROSE are consistent thiethIwnhtatdwoosueldaftbeerwcIpected from multiple dosing with a slowly eliminated compound. page 9 References and siotran8tormation Of N-Et@hyl Sj, Johnson jD: Absorption 1. GibgiOn -ribution and Zlwnation or Carbotl-i4After rose and Tissue Dist thy, IOSZ_14C in read to Rats (Report) AdnLinistration Of N-Z January 19, 1983- c yE, Synthesis and characterization of N-sthyl 2. JPOoShEn-s1o4nC, J(DReapnodrtB)ehD'etcember 11, 19eG, tion Extent and Route of Excretion and Distribu 3. John on JD- in Rats After a SinglO Intravenous Dose of of T:tai carbon-14 ; Decelnber 28, 1979- rc-95-14C (Report) page 10 List Of Figures Thir,-LayofR&di-hrla"rm of Chloroform Figure It Column praction vB-56531-19 Thin-Layer itadiochrcmatogran Of C01Figure 2% lpr&Ctions 17, 18, " 19 to-56S31-23 Thin-Layer Radiachrcmatograu of Methanol .Figure 3: Column P'raction NB-56531-26 page 11 so pigure 1 Thir@-Iayer RadjochromatOgr&m Of Chloroform COlwml Fraction Pre- adsorbent sc;F uniplate; IG3O5 eme-thhlaonroolform a=nonium hydroxide 5 Total CpM on plate 19,221 4D 63 co 30 C:) C) 20 10 0-1 0 234557 10 DISTfit4CE FROM ORIGIN tCM) page 12 70 so EL3 So Figure 2 Thin-Layer Radi(>chrc>lnatogrl Of column Fractions 17, le, and 19 Pre-a dsorbent SGF Uniplate: 100 Chlorofori@ loo methanol 2 acetic acid Total CPM on Plate 18,428 40 C-) CD L-- 30 LC:) F- 20 10 -:1 1 2 3 4 .5 0 a 9 10 1 12 13 14 15 DISTHNGE FROM ORIGIN (CM) page 13 40 Figure 3 Thin-Layer Radjochromatogram of Methanol C01- Fraction Pre-adsorbent sGF Uniplate: loo chloroform loo methanol 2 acetic acid Total CpM on Plate 1,742 30 cr_ z CL 20 0 C:) C3 mL'i 12 13 14 15 67 DISTRNCE FROM ORIGIN (CM) page 14 )Isms Spectral hnalYOLO Of IAC.N.Ethyl Appendix 1: rgchnioal RePOrt SuBmaryt NB-56531-2eu-2B3c 14 in races After an Oral DOse of Appendix 2: zxcretion of Total CarbonR-ats for I Week No-56531-13-14 14C-N-Zthvl rose Fed to Carbon-14 Content at 21 I)ays in Tissues After an Oral Doze for 1 Weak NB-56531-14-16 Appendix 3: of 14C-N-Ethyl ROSE Fsd to Rats ^ppencsix -L SUMMARY E.:@.h 13,@1981@ -A TECHNICAL RIEPORT TO: TECH@ICAL COMOUN,RATIONS CENTER - 2ol-2cN "Porw itsfOrCC m@ Pro ertics RESEAR'CliLABORATOltll!S Anal tic&l and c Esolation of Trace yluorochanicals Service to Riker - I mass szectral lysis of N-Ethyl A,RNo. 7454 - FOSE-1 C Labe ad - March 13. .1981 Research LaborAtO L 0502 AOOOO07 454 J. D. Johnson - 218-2-02 j. N- Schroe)fer sgcunlty 10 30"d ICOoRpWwO" rmkbnliall (S,," ORD$: "rtwtm from3M Tt...@ a n OOW CLMngprr OFACTIVS: Rquest No. C58236 svpl.mt"W=l poj,,t No. 91505026 ------------------- 076647 3 N@ Ch"WWg Repofftd 0 Va k. CRLAP Requestor J. D. Johnson Analytical Report Chemical Analysis CI/MS Nestative M$ M dmgr"" by dw Temn@l Comnwn@ti"sConurto T.t2o().w2w6d0s)TWOfib~ inionooon OTEPORTANMAC abwt3M,omtoCo po 0 "D .itiscono-v -Mk*-Iw ROWL"* f the results from the two sm3Wles using Based on the agreament 0 the major component in GC El ICS, and Cl MS, the 9 tructure of 1 to the structure the' .,Zple actabolite is probably ldentica of the major component in the standard-N-thYl FOSE- Cl mS of the -thylated gtilndardcan help detect this Negative but cannot confirm the serue ture by itselfcompound o.liw, C-4n CE14TP-&-L AiLkl@TTICAL IABOP-tTOP-y Report No ------t-k!L@---------- Date ------- Subject: mass spectra, Analysis of N-Fthyl FOSE-14C Labeled Dept. Name ...Riker ------ praj. No..t:L%CL59Zt Reque Reque:t No. -------- Dated ---ma-r-c-h--3,---i%t@ ------ Report: a &&spit metabolite of N-EthYl for r-C/MS&ualysis- Fthe The requestor submitteds,tandard of N-Fthll -nSE havin POSE-14C labeled and a. ,m2cR3 following structure: c@p,?SOzN 11 cu2cooH ich the roquestor separated The simple matabcl:Ltew a an extract from rat feceswh,,,,, ... contlininst to= zd dividedeq-117 into three on various TLC -'I-cor about 10oug. 100.ooo epm of Experimental the sample metabolite and standard; each Diazosethavic(CH2N,) reacted with to evaporate eXrON8 CR2N2. Flectron tra resulted from the sample Sample bad an air stre-emblown over it impact (EI) and Chamic'Liionization (CI) -me speciactrmeter (MS) via a 9&6 metabolite and standard introduced into the uses OV'ad the method of introdutrion, chrou&tOgr0afTMhs(oGpce:r).atTioanble-ld"t*hteo-at'h,es*paemcptlreum&Dn&ulmybjer * Table lalndlair$dtsmetthheylated the made pectr& resulted from the St caonnddituinomnsethyulseadt.ed iNnetgratoidvuecedCldiUr"eoctl"yIinto the MS. Di--uBs_iO" - and the standard. when I mention the sample metabolite . The major yor this discusgicm- forms unless specified Otherwise ad identical I will refer to their MethYlAted iplemetabolite and standard show component in the CC trace of the gas mass spectrum of this component in the time (8.6 minutes)- The El0 pectram Of the major component retenti,o,ntabolice compared ecxoancttaliyntthethgeoE=l* -88 base 0P'Oak-116 =/z--probably agmnt is the sample standard. in the TheyCOeOaCcHh,). The largest abundant high-aass fr@ Both mass dsuaemetion(e-Na(cChH2sCiHm3p)lCeH2-540 m/2 which is due to SO,N(CH2CRi)-CH2 Ceptparent ion of 599 , I. which 11 fragment at 600 =/Z-& in othrr gautples* The Cl spectra show a very msienelf cl phanom,non observed Both sample spcctro oho,, is probably due C* 6 ,uplas compare exactly- and 600 U/Z. The negative mass SPectra of the twsopectralft&glwntsl 116, 540, he parent IOV primarily three mass uragethyl&tcdstandard shows a 596 for t ci Sao. spectrum of the + 1. spectrum of the methylated standard shows a barely detectable The no.5,LtiveCl Mass a fragment at 527e*lp/z f 72 I/z from 59q M/Z)- ' isi m/z fragmLent at 600 u/z, (probably (losesondthe bass P*allat due to C 17SOT)l fragment at 483 S/Z , of 419 m/z.C6Fl7)- (probably due to 109 ,tRNo. 7454 'Mi,,-h13, 1981 'page 2 C ..lule-ioll the results froo the two 88=Plos using r'C,El MS' Based on the agrO*Iol2tof .Ompcmant in the sa"le Imtabolite is udard-N- and Cl MS, the structureof thetumramjoorfrthe -jor t cooWI'Our"in the eta prbably identicalto the a ruc ethyl FOSE. le3ple S,Lmple metab olit9 of N-Pthyl FOST "C sethylated standard of N-Zthyl ToSE nethylated Standard Of N-w-thylPOSP TABLE I Method of introduction cc cc GC GC DP D? Mode of olmati-OU El ci zi ci -Cl -Cl mass spectrum W-bet_ AA5624 AA5625 AA5622 A&5623 AA5639 AA5638 TABLE 11 Vari.- 2_140Gas CFh-r"ormatoRraph Col_,=n. 2. ,tar, glass, 5% OV on 80/100 mesh . loooc to 330*c at io*c/ain. r-"- crature: 270'C e-Ct0-r- t-8 260-C (Varian 'F'D) chrom C,-HP S!lEitrjlas- helium at 25 mls/min. dupont_zimi Sou_rce: Chemical Ionization sLiLas -. lsobut&ne h,PA_Xan&.. 15-617 u/z Single stage Jac tive CT) ltro,o (act to 420()vforNlga !UitElier: Bendix channe N. Schroapfer JNS/ra Appendix 2 Excretion of Total Carbon-14 in Paces After an Oral Dose of 14C-M-Ethil ROSE red to Rats for I Week= Time Period Of Collection (RrB) I ant vtmber z - --------x + SD 0-24 2" a 48-72 72-96 96-120 120-144 144-168 168-504 5.74b2.10 1.52 1.14 0.66 0.49 0.48 2.35 4.68 3.12 1.92 1.12 0.65 0.52 0.43 2.23 5.20 3.23 1.87 1.33 c.66 0.65 0.44 2.39 5.23 + 0.53 2.82 + 0.63 1.77 + 0.22 1.19 + 0.12 0.66 + 0.00 0.55 + 0.09 0.45 + 0.03 2.32 + 0.09 were: Rat No. il 256 mg/kg; Rst No. 2t Total doses ftd rats 329 mg/kgi and Rat No. 3, 315 mg/kg. Data art expressed as mg of N-ethyl ROSE *quivalents excreted during time Period- Appendix 3 Carbon-14 content at 21 Days in Tissues After an Oral Dose of 14C-N-Zthyl TOSE Ftd to Rats for I Wask@@ Rat Number Liver 1 220.gb- 2 211.3 3 266.6 Mean + SD 232.9 + 29.6 Spleen 21.6 17.5 21.8 20.3 + 2.4 Plasma Red Blood Cells 61.9 55.5 8.9 28.3 24.8 62.1 + 6.70 30.2 + 6.5 1, 256 mg/kgl Rat No. 2, Total doses fed rats were: Rat No. 329 wA;/kg; " Rat No. 3. 315 mg/kgData are expressed as jigof ?i--othyriosz equivaients/g of wet tissue or mi of fluid.