solvelit was allowed to run a distalice of 1.; cni. -4fter air drying, the finished chromatogram was heated in an oveii a t SOo for ap~)roximately5 min to remove niojt, of the acetic acid. C. Spray Reagents.-For the detection of compounds, the following reagents were used to apray the developed thin layer c+hroniatograms: !i) iodine reagent,6 iodine-15; in CC14; (ii) Pauly iiaed to detect possible phenolic metabolites which yield strong reddish to brow-n colors when sprayed a d h t,his reagent,: (iii) diazotized o-dianiaidine reagent, similar to the l'auly reageiit except that sulfanilic acid is replaced by o-di:iiiisidine dihydrochloride (12 g ) in reagent (a); used to detect possible phenolic metabolites; niethoxamine yields a moderat'e \)rowii color aiid IIlh a faint yellow color when sprayed xith this reagent ; phenolic compounds yield stronger reddish to brown colors: (iv) diazotized benzidine reagent, similar to the I'auly reagent except that sulfanilic acid is replaced by benzidine dihj-drochluride (9 g ) ; used to locate methoxamine which yields a stroily yellm color when sprayed with diazotized benzidine; pheiiolic cumpounds yield reddish to brown colors when sprayed with this reagent. The Rf valnes of all reference compounds in the ten thin layer solvelit systems used in this investigation arc: shown in Table I. Analysis of Drugs by Thin Layer Chromatography. A. Analysis of Phenolic Metabolites in Urine.-Chromatography of the pheiiolic metabolites in the urine of drug-treated animals was done after acid hydrolysis of conjugates in the urine. For each 1.0 nil of rat urine or standard solution 0.3 ml of concentrated idded, and the solutioii was heated for 1 hr o n a boilingwater Iiath. After allowing the solution to cool in an ice bath, the p1-I was adjusted to 10 bj- the addition of 1.2 ml of concentrated NII3 and 1 nil of p H 10 1 AI ammonia bufier.5 A 3-ml portion of aqueous phase was extracted with 30 nil of CHE12, arid a 20-nil aliquot of the organic phase 11-as evaporated to dryness. T h e residue was dissolved in 1 ml of methanol and a 15-pl portioii of the solution was applied to a thin layer plate. Uiitie obt,ained from a dog \vas treated similarly except that the iiriiiury re4due was dismlved in a few drops of methanol, and the entire extract was applied to the thin layer plate. The conditions iised f ( ~ the r confirmation and estimation of the 0-dealkylated metabolites of IIlh and methoxamine in dog are shon-vn in Table
11. B. Analysis of Methoxamine in Urine of Drug-Treated Dogs.-To each milliliter of standard solution, control urine, or urine obtained from a methoxamine or IX4-treated animal, 2 nil of 0 . 5 AVS a O I I was added and the alkaline solution was extracted with Y O nil of a 1:1 mixture of CCII-CHCIZ. A 20-in1 portion of the organic phase was washed with 0.1 vol. of 0.5 ATNaOH; a 15-in1 portion of the washed solvelit \vas evaporated. The resiclue was chromatographed with sj-stem 3 (see Table I ) and the thin 1:tyer plate was sprayed with diazotized benzidine.s To 3 ml of urine obtained from a TX4-treated dog, 1 nil of 2 -\-? ; a 0 5 was added and the alkaline urine was extracted with 60 nil of the CC14-CHCL mixture. A 30-ml portion of the organic phase \vas washed TT-ith 0.1 vol. of 0.5 S KaOH and a 40-ml portion of the Lvashed solvent was treated as described above. Spectrophotometric Analysis of Metabolites of IMA. A. The Isolation of 2-Hydroxy-IMA for Spectrophotometric Analysis.-The CE12C12 extract of 2 ml of hydrolyzed rat urine was applied along with referelice 2-hydroxy-111.4 to three doubly lq-ered thin layer plates9 prepared as described in the chromatogi,aphic bectioii. After the chromatograms were developed with .\olvetit system 3, the referelice 2-hydroxj--IlI.A o n each plate was loc,;ited by spraying with the iodine reagent. 'The adjaceint a . i w ( I . the chromatographed iirine extract was then eluted. The cl(~5iJl'~)ellt was removed from the plate. packed in a t h i d e tube (ti..j-niin diameter, supplied by Emil Greiner), and washed with 30 nil of distilled methanol. The solvent was removed by evaporatioii aiid the residue was dissolved in 3.0 ml of 0.1 LV HCI. A i3.2-ni! pwtioii of the acidic aolution was diluted with 3.8 ml of (i;) Reference 1,p 2 1 i .
( 7 ) "Hiochemists Handbook,'' Van Piostrand Co., Inc., Princeton, X. ,J., 1961, I, 3.5. ( 8 ) 'l'iie plirnolic compounds present in the urine from drhlg-treated nniinals tliat reset \!itli the benzidine suray are n u t e x t r a c t e d h y the above iirocrdnre. I n f d c t . .5-Iiydroxy-IhI.i (200 gg;inl added to oontrul i:rine) \\-as nut d e t c ~ r t r r lon the t l c Dlates t h a t x e r e processed as described f u r metliorilinini'. !!)I i i l o ~ i l i l ct t i i i , 01 itlashing t a i ~ and t n i w i l l c a l n o u n ~u f nbaurbeni IISIVIi u I,rei>ure a 1)iilte.
TABLE I
h?f X 100 ------siyaielll"-----AYEILIGE
1
ColIl~1)ulld
2
3
4
~
5
6
7
8
!I
10
AIetlioxamiiie 71 17 28 53 0 1: 12 10 37 38 2-Hydroxymethoxamine 33 0 6 17 0 0 0 0 20 34 1x4 89 5 2 7 3 85 29 47 34 31) 70 Ti 2-Hydroxy-IMA 84 38 51 77 19 37 22 16 70 70 5-Hydroxy-II\IA 76 23 28 57 7 17 12 9 j l 63 a 1: CHC13-EtOH-H20-AcOH (20: 10: 15: l), lower phase; 2 : CHC13-EtOH-H20-AcOII (20: 10:35:1), lower phase; 3 : CHC13-EtOH-H20-AcOH (20: 10: 35: 2 ) , l o m r phase; 4: CIIC13-E;tOH-HaO-rZcOH ( 2 0 :10: 35:4),lower phase; 5: CJIC1,l\IeOH-HzO-AcOH (20: 10:25: l), lower phase; 6: CIIC1,~leOH-H20-dcOH (20: 10:25: Z), lou-er phase; 7 : ethyleiie dichloride-PlleOH-H20-ilcOH (20: 15: 10: I), lower phase; S: benzene-EtOH-H20-.4cOH (20: 10: 10: 1), upper phaae; I): beiizeiie-PrOH-H,O-BcOII (20: 10: 20: l), upper phase; 10: benzeiie-PrOH-H?O-AcOI1 (20: 10: 10: 1 ), upper phase.
TABLE I1 Drily gir en
Ilethoxamine
1x4
Compd eatd
2-Hydroxy metho-iamiiie 2-Hydroxy-
IlIA 11214
Cliroma-
Vol of urine for anal
tographic qolx ent sg stein"
1 ml
1
2 ml
8
Sura? reavent
Diazotized 0dianisidine Diazotized odianisidine Pauly reagenth
2 ;-Hydro\y1 ml 1x4 ILIA 2-IIydl OXY3 ml Diazotized o1 methoxamine dianisidine a See Table I. b l\lethoxamine, \\ith mobility 4milar to that of 5-hydroxy-IMA, does not react with the Pauly reagent.
1.0 S HCI, another 0.2-ml portion of the acidic solution was made alkaline x i t h 3.8 ml of 1.0 KaOH, and a 0.1-ml portion of the acidic solution was made alkaline with 3.9 nil of 1.0 *Y SaOII. The acidic and alkaline soliiticiiis were measured spectrophotometrically in a Cary 3Iodel 1.5 recording spectrophotometer. The absorbance was determined from :340 to 220 mh againht the appropriate refeiwice soliitioris. 'i'he spec*triimof the metabolite which had ,,,A, 290 and 221 nip in ac,id and, , ,A 306 and 236.5 mp in base as ideiitical with that of referelice 2-hydroxy-1M.A. B. Measurement of 0-Dealkylated Metabolites in Rat Urine.-The amount of pheiiolic mei:rt)olites i i i rate urii:e m-:t* determined spectrophotometrically. '9 ;LmI portioii of the hydrolyzed, buffered ( p H 10) uriiie !vas extrwted with 30 ml of ethylene dichloride and a 20-ml portioii I:f the organic phase was reextracted with 10 ml of 0.5 S NaOH. The ac sorbaiice of the alkaline solution a t 306 m p was nieasiired i i i a Beckman Model DU spectrophotometer aiid the total yhi.iiulic metabolites were calculated ii.4ng the extinction coeficiei;t of 3-hydroxyI3lA. Analysis of Methoxamine in Dog Urine.--To 5 ml of uriiie from a dog dosed with 1A1a4J1 ml of 2 .\' XaOH the alkalilie urine was extracted wiih 00 nil 01 the CCl4-CHCla mixture (1 :I ). The aqueous layel, was disc:ti,ded, atid the oi.gxiiic layer was u-ashed with 6 nil of 0 . 5 .\- S : i O I l . The alkaline ~vaslt was discarded arid the organic la-.ei, \v:L.. ev:~l~c~rated. The rehidue was dissolved i i i a small vohime of niethaiiol aild applied to a doubly layered thin layer pIa1eg and chcomatographed aloiig v-ith a sample of ailthentic methoxaniiiie with system 3. -Ailthentic methoxamine was located O I I the thiii layer plate with the iodine reagent and the adjarelit metabolite area \vas eliited as described for the elutioii of 2-li~-droxy-IlI~A except that 20 ml of methanol was used tu elrite the iiriir:iry extract. After the solvent \vas removed by evrtpr~ratii~n. t h e reqidlie wa:, dissolved i i i 1.5 nil of 0 . 1 .Y TICI. .A 0..5-nil purtioii ( ~ fthe acidic. S U ~ l l t i i J l l \\-a< diliited with 1 nil of 1 .\ IICl and another 0.5-niI portioii of [he acidic wliition was made alkalilie with 1 In1 of 1 -I S'a O H . Thc acidic aiid allralilie soliitioiis were nien-iu,cd spectrophotonieti,ic,all>. i i i ii Ci:ti,). 3lotiel 1.3 rec.ordiiig ~ l , c c ~ t l o I ) ~ i ~ ) i ~ ~ i i i c l c l . . 1lie o b r u i ~ l ~ a i i cwas e detcrniiiied Il,oni 340 tu 230 nip :igaiii.\( ttic r 7
Results and Discussion L'riiies for drug-treated aninialn were :wid Ii>.drolj.ztvl chromatographed aloiig with hjdrolyzed cmitrol
:iiid
uriiies and standards (which consisted of the Lippropriwt,e reference compound dissolved in L: control uriiie sample). The standards in the control uriiie were similar twiicentrat,ion t o that of the urinary metabolite so that after the thin layer chromatogram was sprayed with a dye reagent! the phenolic metabolite in urine c d d be estimated by :L visual (.omparison with the st aiidartl::. Thc identit!. of srver:il metabolites iv:r,< hlished t)!. c*lironiato uriiie aloiig with t,hc i i i several thiii layer solve Metabolism of IMA in Rats. ' l ' l i t h rat \v:w ;;clec*tctl for the iiiitial st'udies because this spcries lind bccii sho\rii to metabolize IlIA at :I niurh more rapid rate t hsii t tic dog.lrJ ,Iiialy of the r:it urine by tht, niet,hyl or:iiige proredure of Hiirii5, cf a / . , 1 1showed t h t 0111y :tbont, 2.094 of the tiosc w:is escwtetl uiic~hniiged. I"or the initial c,hromatiigrapliic. studies, the rat i i r i i i c \ W:LS 1iydrolj.zetl with H?SO,. Alethyleiic dirlilo18ide extracats of t h c hydrol~zcd uriiies were rhroniatogrwphed on paper. Two spots were located (IZi 0.25, 0.75) when the developed rhron~at~ograni\vas spra!,cd with the Paul?- rexgciit . These results suggested the preseiivc of two phenolic metabolites of IlIA. The intensit,?- of the cdor of the spot ivith Rf 0.25 \vas much strotigcr t1i:tu tlic otlier spot ( R i 0.7,;). .Iiitheiitir >-liydrox>.-IA\I.\ Iiatl Ii'f 0.25 : i i i c l referrlic*u2-h>~tlros~~-I~\I,i 11:rtl /if 0.T.i \vIiett c*hroliii-Ltograplic~lwith t lio p:ipw 1~1iroii~;itogi~:i~~liic~ s\.stem. 111 ordei* t o :ircwnulatc suffi(~ioiit ni:itwi:d so tlxit tliv ideiitit?. of thc c~hrcini:itogt~:c~~lii~~:~lly slowor iiioviiig nictabolitt! (with /if0.25) cwultl t w defitiitelj- e :I 1:wgct volume of uriiie fro 1 clrug-trentctl rat \v:1s hydrol\rzed n-ith acid. L4 ( t:illiiie niet:ibolitc w:is isolated from the h>drol\.z 1i.i t i :ui(1 ( Y ) I 1rlunivel!. identified ;IS .i-tijdrosy-I.\I.I :rs tlesc*rik)ed i i i the Experinieiitd Sectioii. Identification of 2-Hydroxy-IMA as a -Metabolite of IMA.-I)uring the iiiore recent studies it was tletermiiieti by the sl)cctroi)hotonietric. methods described earlier that corijugates 1)reserit in the rat' urine were completely with HC1. The Iiyacts were cliroiii:ttogra~)hecloii tliiii layer 1)l:Ltes with solvent system 3 , :md two spot's were' locatecl (I& 0.28, 0.51) after s1)r:yiiig the l)littcs nit11 tlie 1':tuly rc:igoiit or tliazotizotl ~)-~li~i,tiisi(liiie. Tlie iii(11 :iI)iilit c tvit 11 Ii'f 0.2s c.oi,i,c,sl)ciiidetl t ( 1 .j-lr>drox~.1 ALI ivliose isohtioii u x s clesc~ibed,: ~ i i c l ~ v i t l /if i 0.51 c*orrcs:poiidedt o the 2-Iij.dro nictabolite and the reference ?-hydroxy simil:tr mobilities when chromatographed in three other tenis ( 2 . 5 , mid 6). To measure the ultra(L
\ 10) Internal stamlards \\-err also ctironiatopraiJlIrd in order to eliminate d o u b t caused b y variation in lii i a l n e s of a particular substance chromatograplieii not only a t diffi-rent tinrt'r, I I I I I r v t ' i i i\ Iit'ii c.liromatographed XI different locations o n the same plate. Tliis in\ d v e d tile chroniatograplrs of an e s ~ r a cof~ drng urine t o nliicli referencr
