'Ot) 'fB 'splce 'E66I 'SZ '9661 '7 le ueqJ ,(q umoqssB raque^op 1111dq patdeccv du4 paltaceX ola{ Jeqlo Jo prrourar ;oud lnoqll/t\ poqeur sq1 ,(q pauruuelep 'slcnpord paroloc fpul eq lourruc saldureseurseld'eJoJeJeqL '(9) ursloqqeur eutmd go emleeJ3 su s1ue1draq8lq ut lueserd -runs ecnpord ol HdNC rnp\ lcuoJII?c splc? op>l JeI[o asnuJJq '(9) spuryu uI sseruolq 'Je^e/t\oH osIBsr ulotuelry lIIsuIIuru I€IqoJcr-uIJo 'prcu c11,(xo,(1trog cgrceds lou sI poqlau s.JaqcJog plq ^\oU Ipunselul Jo xepq uu sepnold eJoJeJeI[puu sauFnd ',{ressesau 'snqJ 'ueturu s epruefcute; urnrss4od LDI^\ uoneplxo ou -oJcrruJo opdn qlIA\ peleleJJoJsI uoDeJcxeuloluufiv 'HOBN LnI^\ eu{erIIB eputu ueqa (utu OZSle umrulxutu ecueq eql ur pecnpoJdsseuotq plqoJJTtu uo4 seurmd snouatoxe 3o -rosqu) 'poo8 xaldtuoc 'daaqs Joloo peJ elqas e secnpord HdNC-€lel ,(1ddnsa8rul e Jo esruJeq (14ourur 0I Jo uoqs4uecuoc -,fuof1E tpIA eulzerp eq1'(1145q) euperp,(q1,{ueqdo4ruIp-t'Z roy ,{up4ourur 9I puP aF$c roJ ,{up4ourur 00€ ot dn) eurm -,{q1,(ueqd oI 'pe^\eu peculder(91) sJeqcJog'uoqslnu{ slql eluoJJe^o eql ur uIolIIelI€ Jo sennlmnb aErel eleJJxe seJo^IqJeH '3urury pclluc sermberempeco:d eql os ,ttryqsls aroqdouorqc -eJ uaeq seq sepn|s ptuFre uI uIoIrBIIs Jo luel'uemsBeluuI lse '(Iuu '(7) /r\ol sr spolDelu eseql Jo uopslFull eJe^eslsolu eql OlE -re1ul',{pueco6 aurorpufs s.a,(egut lue8e ctxol pnuepd e 'uolssrure) '*.u3;o 'JaAo 'uonslrcxe :uru gst eJuacserong fq peteatap su papclldun ueeq seq uIolIrBII? acueserdeql uI '(t 'g) s lcnpord Fug eql'epr-rmfcpel urnlss4od tpt^\ uonJrcJ Jo4iy -eJoIN olta ut suollJueJ[BcIpuJ ea4 elsJlpul(eur eruseld 'eulzvrp,(qlfueqd '(eusuld ;o pee6ur auuqdelfueqd S3snqcr-q^\'(tt )'p ur uoquruecuoc ulolltu11e1eql aoqs selpn1sluete1 'senbruqcol le otls) ,(q tuq $ poqtelu rBIIIulsy (€I-I I) cUuur ur l4ourrl 0S-S) sppg ,{poq ueunq ur luaserd eru uIolIIBIle 'saldurus '(Z) -o1nuro (g1-g) pnueru rprrrr pcrSolotq o1,(llnJssmJns Jo slrmotueecun'snq,L *rt auorqcol,b fq ro (1) sleJlpur pellddu ueJq seqpoqlau sql luu 0ZS l3 lururnx€Iu uoqd:osqu ee4 fq proe Jun Jo uorleplxo qEnorq swumq ,(q pecnpord 'epruefcrual ue qlr,u eroqdourorqcu ol urntsselod,{q uonBplxo eq uBJ ulolllslp eIIIos 13^e^\oH ,{poq uuumq eql uI es?cFmJo 'euozurpfqlfueqd JeUp 'peue^uoc sI qcr-q^\ e errr8 01 eulzeJp ecuesqueqlJo esneteqtusqoqnuceurmd;o lcnpord pug eqt se '(8unueq 'eurorpu,ft -,(q,(ueqd qlr/r\ slileJ uorl plru c11[xof19 ertnher uroluslle eleJcxelou op slluumH wqfp-qcsr1 eqt suoDJ?eJqloq) eem puu plJu c1,(xof1E 01 tunlpetu ,plcu ul puu lno8 sBqcns ses?eslpueumq uI pello^w esoqt ,(pelncnred 'tus11oqulaIu peplar pezflorpfq ueqt pue plcu croluelleol uoqnlos(gOeN) eul1u{Ie auFrnd3o sde,nrpudeW Jo uonsplcnle ol 'uloluelle uIolIrPII€ selpqs ,(1rea u1paz,(1orp,(q sr urotlrBllv'oEe srea{ 99 ueql erotu (r) femuo3 eJe^\ uoquurrue1ep Jo 1so61 qc.BeseJpcrEolorq;o seare ,{w;n1 pue Sunoa ,(q padolerreplul{t sI poqleuJ pesn ,{poururoJ lsou Jo luerrroJns?euarmbar 'peleJcxa prre JISSBIJeq1, 1cnpo:d peJoloce errlE ol splJe o1e{ tpr^\ sa^q 'sI 'uoll3€eJ $ pIrBEeJn puu prcu c1,{xo,{13ol JeL&nUpezloqul -?^uap eurzurp,(qJo uort€suspuoJ lstp reMrqc5 lou aroJeJelp -eru 'SIBLUIITB 'euun puPpoolq -rur-u4U aql uo peseq eJB spoqleu cqeuroloqdo4cedS sr uloluell€ eulmur uI uI poFJcxe u1luasard q U lusloqulec aurmd;o lcnpord IBug eql sI uloluel fularuoloqdo4cadS -p'sapda: eurospuu'spJlq'selelrrud uerp reqto sIEtrr\J -rrreru uon?pxo pezfpFc-esuJlJn ulol; uI'pIJB ctmJo V 'sourmd;o pnpord Jlloqu1ec3 sI uIoltrBII Y ',{qdurSopurorqc(31 PmbgPue peurroJst 11 'rolerlpul gql) ra,{eluFtl puu Hd tpl^\ uon€4p rllouilp{Ie ',fulauotoqdoaceds tJorp :uoncopp puu uoqeredes uo pesuq 'seuotepc 'uollBu!urel € otul IIBJ uoneuluuelap uIorrBIIs JoJ spoqler\l 'slcnpo:d obz-9'0Vm -op spoqleu Fqunl uo Prqnacuurruqdur (84otuti gzl-'(,il uloluEllProl lo lueudole^ap rcl uo ua^!6 spnpord crlerusocur (E4ourrl tl) obz'1enqolusll?Jo suorprl pue sedA eldues luala$lp Alllqeuns -uecuocpctd,(1'(epu1o1ue1p,{xorp,(qtp umururnp) uxoply se oresuatr|uoC'F!nb!l puelerteFulql) ^qdBrooleu -or qcns stnrp JeclnqIIBeluos uI osIB ptr? (apuloluelp umuttunp r,lcpuE' uollerl$ culoul! IBI le l{.4euolotldo4seds 'urceJt 'ood aql'pa/$e!A ,korpr(qorolqJ Jo uloluelp se) pue u14s :sauooapc uleu e re^oc spoqleu'l lcnsdq -oJ pclnaceuJeqd puP'qlarusoc -uruqs 'duos sB qcns spnpord JnelusoJ ,{uetu ut luerper8u ue ere seldues 'lecg6ogo;qu! ulouellE6u1u;urelep rol spoqlalt\l sr puu uorlJelo:d u11sro; ,(gecrlnedereqlposn sI ulolw11v

.^\usJud\.suuolqsf .uouunN puslod 0I I -s0 pue ,{Eo1ors,(r{dIsuJruv Jo et$qsq lls^\ouulal) eqJ xrz3Tv rox Nvf puB DrsalszsorYtrl HOsIJfoll )n' g56 ZgV ueepreqv'urnq$lJng'el$psul I{JJmseUDa/t\od NilrJ Mflsnx

saldruBslBcpnaJBrrrJBqd puu 6crleursoJ6lBJpolo;g u; uIoluBIMo uolleulrurele(I

s3cN3lCSCISN3UOI'SC|I3nSOC'SCnUO

"1oA 9661't'ON'61 TVNOIIVNIIIIINICVOV do'IvNllnof :''rv Ig NaHJ 829 CHENErAL.: JounrqelorAOAC INTERNATIoNALVoL.79, No. 3' 1996 529

Instead of using the Rimini-Schryver reaction, Sumi et al' the sensitivity is low (detection concentration ftrnge used by (17) converted enzymatically with glyoxylate Katz et al. was 1.34.3 mmol/L). are given in reductase in the presenceof NADH. The NAD* produced is Reagentsused for spectrophotometricmethods determined fluorometrically (emission, 460 nm; excitation, Table l. 340 nm). The method requires a small sample volume (50 pL)' and sample pretreafinent to convert to glyoxylic acid Titration in weak NaOH is similar to that in the method of Young and Conway (7). With this method,otherketo acidsdo notinterfere. Allantoin is also determined for pharmaceuticalpurposes by Other approachesalso have been used.Crokaert (18) used alkaline titration of the acidic succinimide proton. Generally, poor the reaction of allantoin with diacetylmonoxime (DAM) and titration is less sensitive and, because of its selectivity, Complex ma- thiosemicarbazide (TSC) based on the presence of a ureido requires removal of many interfering substances. physiological samples cannot be structure. The reaction is usually used for determining . trixes such as body fluids or When heated in an acidic (6N HCI) medium, allantoin con- tirated. (22) prepurified cream formulations denseswith ttre DAM-TSC reagentto form a colored complex Weber and Higgins pmol per aliquot of sample) (absorbancemaximum at 525 nm). However, the reaction is (containingat least 158 allantoin 545 column. They titrated the eluatewith 0.1M nonspecific and gives positive readings with urea and other with a Celite to a pH of ll.5-12, as monitored by a glass pH elec- compounds with a bond. Yuki et al. (19) thereforere- NaOH node. Billabert et al. (23) and Willemot and Parry Q4) de- moved urea by incubating sampleswith urease;adding FeCl3in scribed proceduresfor extracting allantoin with N,Ndimethyl- the allantoin--DAM-Tsc reaction systemincreases the color in- formamide (DMD and subsequent titration in nonaqueous tensity of tlre final product. solution (toluene-methanol) with tetrabutylammonium hy- Vrbaski et al. (20) used the Ehrlich reagent, which consists droxide solution. Use of sodium methoxide as titrant also was of I g p-dimethylaminobenzaldehyde (pDMAB) in a mixture testedin nonaqueousmedia, but it gave a lessprecise pH end- of 25 mL concentratedHCI and 75 mL methanol. They deter- point (24). The concentration of extracted allantoin had to be mined allantoin in Agrostemma gintlwge L. seed after exfrac- >63 mmol/L. tion with water. The allantoin-pDMAB complex (absorbance maximum at 440 nm) is stableat <40oC. However, the reaction is sufficiently sensitive only for samples with allantoin at Chromatography The presenceof urea leadsto >1.9 mmoVl and is nonspecific. Thin- Layer Ch roma@ raqhy overestimation of allantoin. Katz et al. QD used the reaction of allantoin with ammo- Before LC techniques became popular, TLC techniques nium copper tartrate and Folin acid molyMate reagent(mixture were usedas semiquantitative methodsfor fast and inexpensive of brominated sodium molydate, H2SOa, H3PO4, and acetic estimation of allantoin in natural samples.TLC still is very use- acid). Reductionofmolybdenum producesa color (absorbance ful as a rapid test of product quality in the pharmaceutical in- maximum at 685 nm). They applied the method to cosmetics dtstry (25-21). Cellulose or silica gel are the usual absorbens. and pharmaceuticalpreparations. They exftacted allantoin with TLC methods have been used to delermine allantoin in mam- hot ammonium sulfate solution and decolorized the extract by malian or serum after extraction with organic solvents passing it through an activated-carboncolumn. Becausethe re- (28,29). For body fluids, pDMAB and its derivatives are the action is nonselective, sample cleanup is important' Moreover, most frequently used color developers. Allantoin appearsas a

Table 1. Methodsfor spectrophotometricdetermination of allantoin

Sample Reagenta Detection SensitivityD Reference

Bloodserum, urine, plant and Phenylhydrazine 522nm 3'l pmol/L 7-13 animaltissues Urine DNPH 520nm 31 pmoVL 15 Plasma Glyorylatereductase, NADH Fluorescenceemission, 63 pmol/L 17 460nm (excilation,340 nm) Seedextract pDMAB(Ehrlich reagent) z[40nm 1.9mmoVl 20 '126 '18, Plasma,urine DAM+ TSC 525nm pmoVl 19 Cosmeticsand pharmaceuticals Ammoniumcopper tartrate 685nm 1.3mmouL 21 reagentand Folin acid molybdatereagent Pharmaceuticals Phenylephrinehydrochloride Fluorescenceemission, 31pmol/L 14 450nm (excitation,370 nm)

DNpH= 2,4-dinitrophenylhydrazine,pDMAB = pdimethylaminobenzaldehyde,DAM = diacetylmonoxime,TSC = thiosemicarbazide. Lowestconcentration of standardused by authors. 630 CnrN Er Al.: JounNaron AOAC lvrnnNenoNerY or. 79.No. 3. 1996 yellow spot, which can be measuredwith a densitometer with Moreover, because allantoin is poorly retained on a C1s col- a 445 run filter. This method can quantitate allantoin at urnn,some methods (33, 35, 36) requireeither a longercolumn >95 pmoUl, and it has been incorporated in the AOAC official (60 or 30 cm) or 2 columns (2 x 25 cm) connectedin series. method for detection of urine stains on food and packages(30). The modified columns increasethe retention time for allantoin. In the official method, the concenftation of allantoin in the ex- In some LC systems, however, they can produce excessively tract must be >3.2 mmol/L for detection. Ferrera et al. (3 1) used high pressures. p-dimethylaminocinnamaldehyde (pDMAC) as developer. To avoid problems due to short wavelengths and long col- pDMAC gives a 2-4 times better sensitivity for allantoin than umns, allantoin is converted,prior to LC separation,to a deriva- pDMAB, although it has a shorter shelf life. Details of TLC tive containing a chromophore with an aromatic ring absorbing methods are given in Table 2. in the near-UV range. Nakao et al. (41) derivatized allantoin LiquidChromatography with p-ninobenzaldehyde (pNBA) dissolved in DMF in the presenceof the basic catalyst 1,8-diazabicyclo(5.4.0)7-unde- Determination by LC methods usually is combined with cene. The allantoin-pNBA complex, which is stable for more analysis of other derivatives such as xanthine, hypoxan- than 6 h, is monitored at270nm. Hfuotaet al. (42) derivatized thine, and . The affinities of these compounds to the allantoin with xanthydrol in to form xanthylallan- stationary phaseof LC columns are similar to that of allantoin. toin, which is determinedat2l4wn. The methodis very sensi- Most methodsuse Cl8 reversed-phasehydrophobic columns to tive (detectionconcentration,0.6 pmoVl-) and specific. Deri- separateallantoin or its derivatives. With most LC methods, the vatization and sample preparation are, however, time generally lowest detectable concentration of allantoin is consuming, and the product is unstable at room temperature. 10 pmoL/L, although the concentration for accurate analysis Grootveld and Halliwell (3), Chen et al. (16), and Kock et al. needs to be higher. A summary of LC methods is given in Th- (43) converted allantoin to glyoxylic acid, which then forms a ble 3. hydrazone with DNPH. The colored product is monitored at Methods for reversed-phaseseparation of allantoin were re- 360 nm. The product is stable at room temperatureand readily ported by Duerre and Anderssen (32), Tiemeyer and Giesecke separatedwith a short C1scolumn. The derivatization chemis- (33), Lux et al. (34), Balcellset al. (35), Diezetal. (36), Resines try is the sameas that of Borchers' (15) colorimetric method, et al. (37, 38), Eitemanet al. (39), andTerzuoli et d. (a0). These but chromatographic separation avoids interference from keto methods were used for allantoin in urine and deproteinized plasma plasma. Allantoin is detected at short wavelengths (195 to acids, especially for samples.However, like the method 220 twt, becauseit has a very low molar absorption coefficient of Young and Conway (7) and others that determine allantoin at wavelengths >220 nm). At these wavelengths, however, as glyoxylic acid, this method does not distinguish allantoin, many compounds in urine and plasma also have high absor- allantoic acid, and glyoxylic acid. Thus, allantoin content can bances,leading to excessivebackground noise due !o peaks of be overestimated when glyoxylic acid is present, although the closely eluting compounds. Accuracy of quantitation is likely glyoxylic acid likely originates from allantoin decomposition affected when allantoin concentration is low (<60 pmol/L). during storage.Applications of reversed-phaseLC to allantoin

Table 2. Methodsfor thin-layerchromatographic determination of allantoin Sample Adso]tenl Visualizingreagenta SensitivityD Reference

Pharmaceuticals Silicagel Methanol or ethyl methyl pDMAB (Ehrlichreagent) 3.2mmoul ketone-acetone-{orm ic aci#water(,lO+2+1+6) Pharmaceuticals Silicagel Ethy' methyl pDMAB 27 ketone-acetone'{orm ic acid{Yater(4O+2+1+6) Pharmaceuticals(plant Cellulose Acetic aci(Fwater-rFbutyl UVzsl or FeClrl-tartaric acid tinctures) alcohol(28 + 61 + 11) Urine,serum, lymph Cellulose 1.0N acetic pDMAB 95 pmouL 29 acid+thanol--nbutyl afcohol(2 +2+7\ Urine Cellulose nbuVl alcohol-acetic pDMAB 126pmol/L 2gc acid-water(2+1+1) Urinestains Cellulose nbutyl alcohoF

pDMAC= pdimethylaminocinnamaldehyde,pDMAB = edimethylaminobenzaldehyde. Lowestconcentration of standardused by authors. Methodswere only for qualitativedetection, not for quantitativeanalysis. CueN Er Ar.: JOURNALOF AOAC INIERNAT'IONru-VOI' 79, NO. 3' 1996 63I

o (,) () s @ N o c) I S

= .J J J E * €E E E E Ei c E o 1 o,l c9 ro qRi q o o) U) o oJl

E s E E E E .9 (\l E EE E' E E E C) N o d is s R H R $ F o o R Es F o (\J ; 3= i €: 3 3 3 3 3 3 l tr

-! E o E tllllllll o) o (o o o

'il 6 E t- (! r )o dl a | --z z o o o E Fo .L x 6'^'o^g EEg ; o oI eI *P a =8 o $EEE i* g () E EqEESTEE Er e: ; o e e €6 EE i;E i: i -= o Ead ;E E^FEE5Na:; : E f:E-€-E E.; gggiEEItEE*;5l--e ET"TFEE EE o .> g u'igi U) Ei SfrEtEE E#E tE$g EIEE; iEE E ei c p i-e o E6=8=8EEEESENHEE$HH $E€H5a;"= €cF c so E E c E F Ee E 9et 5 c .9 o o : : € ; s! 6 : :i i 5 €F?E o .E o rn-E EE E E[*E$565EE5 E y iq* c, E '= o F tc AgH E5=5.5 It E ?sgs ?i;s fi Hs .9 6 E eCL }i;EgE€E CD EeE,E,g€EESF€E.E'65 EEE€ o (! ooo E o o ouro o o (uo (!(u(5 gEg 3 E o.oo- s s s s o Eoo oo(l) p .F o o u, 'lt) = (g o EEEE E E E (u o) ooo g g g o- ooo sEe I E o o (r E(r ,9E3 I & d o U) E o t, o G. E 6 oA Eee , Eg € t = e.Q e 5g o_ F €F e €€E -g *o E€E E € E E e >6 ll E = an8 E-' d €a d dd F s F s a @ F 'pupbar c)! d p.6 6 E sr prcs crolueF prrs urolrr8ll? qloq seuruualep rcql poqleu E ggx 3 o o+z= i)+ 0) 'snql 'prcs 'uoqerederd oL ?^' o crolrrullu o1 asodurocep,{utu utoluqp 3rr BU' P S.t olu uIolIIBIle O l\) gH eldures Jo ued s (7 eyqel) uoDnlos eullu:{p iA F o 'slue^los A Jo uoqJe.qxe uee\[ cnre8ro qll/t\ uonJurxe eAIoAuI {+ B* s sarnpecorduourruoJ 'sts,{puu eJoJeqpe^ouar oq NnIu qJIqA\ o< F'; cs. 'xlqsur - =l o (e1qn1os,(pood ro) elqnlosul-Jep^\3 uI e.IBfgensn ('c1e o!! @ g 'surseJc'srep,rod'soodureqs) o scnerusoJpIIe spJqnet€urJeltrd olS ooE 53 !t 'errrsuedxe 'pesn oo eJotu sI t1 qSnorpp oslE sr uone4FJsJlln,{q xx d ',{ressecau OJD oo seFlelour e8rel go IE^oIued eJoJeJeIDsI uolltu.qxe d. I. !ro peluedar (ob09 uIolLrBIIu rood selt8 5-di (o(o 3 Vlr-' Jo,{rerrocer loueqleu !lu ooo. 'p o qtr,n euseld Jo uoqceJlxee13urs pql punog (77) la slor .q@ 'uonJer1xe :> -rH aseqd-prtosqtrm peldnocst uorleurelordap',ftus c" 'lorruqleu 'pIcB '(y3;) 3! -sooeuJI Jo cnece plJe JlpceoJolqcul U,4 '(VCa) 'pezrurelordep 3q prcu cuolqcrad qtrm ,(gensn lsntu g 53kri o selduresenssq pue erus?ld'uollerlg ro uoge8nyruec sertnhar - N 3 'uoqnpp !, fgensn ss,{puu J-I aleudorddu qtm ,{prarp pe,(es dl q ',{ltun ol o -su oq rreo seldures euun ol esolJ eJeA\spoqleu eseql

uollPrPderdalduPs 6' CI'

'selduzs pcrSolorq qlrA\petsel ueeq lou a^?q spoqleu eseql o o 'E4ouri pelsal (gS) Euem q) 0S-OI Jo a8uur uoueruetuoc B o 6 ' o o lu steuufcoql umtss4od pue 4eadu se r-urJ 0S0Z vul ZLLIp + d o sreeddeutolu€11y :g; SuruTsluoc{slp B olut passarderu (epuu o -,(co1rtrt 'fletuopqd E unrssqod) pnpu?ls lulualut puu eldumg I :{ -o.qceds pale+ur ,(q (sumuc) saldurus cqetusoo ul ulol (tl -uelp parolruoru(€g) 3w16'14ouur 1;g-Zg1go e8uer uon 'p 'uIIu 1' -erlueJuocJueurl e pasn(ZE) le esleC 9 urnl^\ sgmod d o 'uuoJ =,o -uoJ JerDouror; peprudas ueql sI uloltrellv oIUoIIIBIrB I - 'poqeu 3 qt ur lueserd sr urorrpll? 1uqlos 6< ol pesrurs 11daldutus E: s1t1tq '(iZJ) srsetoqdo4raleeuoz frelpduc ,(q spc4neceur (o z = l, -ruqd 'p o o prmscqetusoc uI uIolIrBIlBpeuruuepp (29 le esleC o (oo o =r o o fo 9. o i 36 E spoqlew reqlo - o 3 ol 9c,c)J 'spunoduoc pezrseql o -i -s -u,(s ,{gecgeru,ftue uor; uloluslp;o 'elduruxo ro3 'uoquredee c gg q) errrlEredardJoJ InJesn st puu saldruzs xelduroc ssal ,{1enge1ar \ '(IS) o S8 roy fluo elqqrns s.meddupoqlatu reilBI aL[L sapour uoq ft 5 33 -cerelur-uor pue (uo4fuospu crqoqdorpfq) aseqd-pesJelal Jo 'slcu.uxa ut spunoduroc eurmd raqlo uonuurquoc e Sursn 1ue1d o U) CD N) o puu uroilre11eeprudas ol (02€H-SD {edgusV) uunlor 1eBreu at, 'IB 33 -,{1odocloqorlu 1,(urMyodludr-Wsv ue pesn (9) ta sruq.rqsv oo <: rr '@S-til sonerusoJo1 pelldde useq ssq tnunloJ (uuo;-*11cruot -pc) a8ueqcxa-uol IIe uo uoquredasJeU" eptpol urntss4od Puu eprolqcodfq turupos tDI^\ uonezqe^uep uunloqsod uo paseq t s o poqleu 'pasn ueoq osIB e^uq sepou uoperedas raql6 -@5 o V (.r'@ d 'gffiV 'It) paqurs o o -ep ueoq eABqsonelusoc puu slecqnaJ?urluqdut uogeuruuapp

'E 'ON '6' "IoA 9661 TVNOTTVNIMTNICVOV sO TvNuno[ :'1V rg NaHJ Z€9 'epueuljog{qgau4pi/ = ,ptce - ,ppe N Jl lO clareoroltlctrl VCI cuolqcred= VCd , eurzerplqp(ueqdqlm uorpear oJoJgqloqoolp ;{uleosrr.u.to;o.rolLlc r.llr,r\ ptce .uragord qlIxo{16psperue Ouruqeluoceldues sa;durespoolqenssu ilrouroloqdoJpedS olotl/r^rlum ppe q;Ixolp ol utoluEllppoua^uoc lPutluBJo ons€[ lueld gx-Mog0v xa^oo qyarrpelgnd ,alplacB E ctrauroloqdoJpadS ,vcJ_ cuncJouJLll!/r4 palBlldpald qu{ pappJxs onss!llo^!-l (aOeqlp punoq-eue;tsp{uaqd)uungoo paseqd-peuenar 5 c'l tlyl pqpnd ,y36 qlrm Uoqs pezrue6ou.ro;1 anssl ]ueld II culeurogoqdolpedg ptcBc[aoE qlt^ paztuteloJdao PuJSpld ,t pe6nluluecpue c1 (eerpguaguoo;tuy) polailllelyn Eulseld uomlosHOeN qI,$ pemlllsuocat cile pue'zN ,c-1 c-l uueru{q pogp {g peleuorpe.r3 Buseld .ulnlas zt peueep c'l rq requnJpue roueqqau, ^oo3tlr:$jno eulsBld & cll_ VCd Aq pezruraloldag qdull ,urueg 6t culeuoloqdolpeds VCI ^q pezrurelordeg €rusEld 9e'gt'9r c'l VCI lo VCd i(q pezurelo.rdeq eusEld '' 8e-S€'6€ c'l peJouulolpue pe6qu1ua3 ouun 6e c-l eoczl-.|ngz0.0 wA\ peperu3 leyl rt1no6 z9 1ZC Jele,u wt\ *oo: scrlorusocpue slBctmsoeulJeqd gt'9v cl lPcunocPur,leqd loueqloul Lr'9V c'l ro eluuuolsoptllt,\ pelnltp lale,$ qu,upelceJlrJ ctlotusoc w c'l loueqlau, %oL Uut polcPrlrJ lec[nacPurJeqd uoqJec peleillcB tz culeuolorldolpeds Iq peropoceprOSz(rHN) loq qI,$ popelyJ c|laursoc 09 3'l ullorroqe ptuordw %s )o )otet *" #5L c[auJsoc w ,pueqle ct Jl lO loqle;{q1e W,vrpslcprye,lpUas (uo;1o;.ro uee.rc) cneulsog ecuol0loH poqpn eoJnp@oJd epueg uolpulurapp uloluBlleJol salnpetoJduo;preda.rd adrues .? onpl

cFmruo{ paprudes eq lsnu uloluelle .saldurusqcns qlLl6 .(€) 'eSurots s4eed,neu aq1 3o rp8ua1qlrl\ ezrs u pasueJrur prl? .uounlos lsql [e^\[I?H ple^loorc,(q peqgcsapse eu[s{IB ur parruoJera^t (Cf ,{q paeredas ,0.g se) srpad meu Z puu 0.9 pepeq ueq^\ uloluell? ol prJu 'porrmco Hd rlm Jo uorsJe^uocpnredgo asnec 1ulnq uonrsodruocepou .0.€ gd .s{ep -eq ty gg o1dn rog ulolu?llB elEr,unseJe^orrEJ plJ? cq,(xo,{18 ol ulolrrellB parols prrp '0'g '0'€ Jo c.Os l3 0'g Hd lu urolrrBlluJo {tilqels arD srsflorp,(q uo peseqspoqleru .uuseld {reumq qJns .urolrrpllu 'p .prce su peurur"xe (SS) le olorrluuru1 c11,{xo,{1to1 fpug pue Jo ecu-Be ,{po 1nq lueluoc plcu JFm qt1q rnpr seldurusrog plce crolu?lle ol ulolupllg 'uoneuruuelep esoduocap rrgcrunrper[ eurle{Ie uV uloluellB r0r^\ ereJ.relu .arog 'uloluullsJo lou [Ln ,{arp uoqrsodurocepplqoJcr-ur llqr-qu ol unrperu JrprJg -eJer{L 'qJosq? prc? .plJB .uem lou op crolrrullg pup crlfxo,tp ur paJols {gensn e.lu uoqerrruuelep urolrrplle 'suonrpuoc .ulr rog seldureg qcns Jepun gZZ prru g.II_g.01 gd p pourul .apudorddu oq pFoc uoBezDuluep lno -rerap aq ulolusp prp pe1se33ns{aq1 .ZI -q1rn .suorlrsoduroc Hd lnrm lr.rslsuoJ uogaredespanp Sursn spoqleu xelduroc sutstuar pue q dn qlr^t sesuanrr uopnlos 0I Hd Hd snoanbuur ssel qlr^t Jo suonelueJuoJ rrolupllu qtlq saldures rog trrrr .Ip W$ SZZv sJrmqJosqus.urotrrellu luq pmoJ (9S) la 'eursuld su qcns 'urolrrefieJo 'InJesn {Erse{n I suouerluecuoc^\ol Burur4uoc spr aJBecrreqJosqe qSnor$ -ng An $l ulolrrplle Jolr-uotu1ur{l {poqgo srsfluuuanmcce roJelq?np^ eq ppom (97_17 ,91 spoqtau 'aloJareqJ .prce 'g) J.I cr1,txo.(18ot ulotuege go srsfyorp qlEuelele,t alqrsn ro e8rrun11reqErUu -fq rc Eurqrosqepunod uo pesuqsporpeul fq as red permruaep lornrec uloluellu -ruoJ u rrrroJ .31 'prcu 4 ol uoqyzq?^uep urolrrulle aseqd_pesraler c11fxo,fit puu uroluulle qloq urz1uooseldruzs ueq16 fq 'snoeS4uulpe ssfpuu rog eq feur (g 1) sJeqcrog poqeur 'sp1cu Jo tunoJ* olq ue{4 eq eq1 ole{ ureluoc lou op l?rp saldurus xaldruoc ssel Jod tou Ur^\ sepsuepuoJ eseql tuu eJrnqrosqp OEZ_ffiI te fq u1o1 ,(llrpcelos re11eqflezrqeleJ eql Jo esnuJaqsaldures pcrEolorq -Ir3lI? rolruolll spoqlau .uoqnlos lerD uJ to J.I QIla HouN roJ acrogc eql rrrBuar ppoc (1) i(e,ruo3 pue Bunoayo poqleur qll1l\ srs;(1orpr(qpepuelxe {€elh {q urotuelu se paJeAoJeJ pcrsselJ erp taFtuoloqdo4ceds B rltrJt\ .adfi eldrues pue eq wc sprmoduoc eserp .prcu Z cl1fxoflE yo srsuqeql uo urol luaudmha oIWIIE^? uo spuedeppoqtau elqqms -UBII? lsotu errl euuueleppql spoqteu J.I pu? crleuroloqdo4cods rpr11 'prcu c11fxof13puu m.olue1pJo sapsuepuoc sBpergpuepr eJe,r uolssncslo gt9 '€ 'oN'6t '1oA'rvNorrvNuirrNJ 966I JvovcoTVN[nOt :..rvJg MrHJ 'SS'utaq2 'reqsen{J '(ut"P Z8-O8 puy'[g'rossv'[ (ZL6D'I'f (82) Peqslqndun'ueq3'g'Y) 'eporc€le rznE-gtrE rcpercp *CIVN se (n Of'Or eruereJer IJEVFV '6t8osowott13'bn't '5i1'uo,{1euo3 "f @gOD ? tuueqs OZ) qlu\ eponcola Suqro,n U) apo.uoele ctrauroradruz Susuas 'lE 'S 's?tuef ''f 'plsruolnl ZEI-lEl alzoulolld (9t6I) ? gZ) -+CVN rrBIII!!\ sts,{1eueuopraf,t4 a\ou t$I/t\ pesn pIIE sllumloc gzl-t7t'n'32'turottd (1.-66}'-l'ezaet1 GZ) ssep arodloauoJ oluo pszl1lqoultut eJBesqJnpeJ ep1,{xo,{p 'W 'uw ',fire4 "1 'asuJrolrrelle 'esernollrulp 'qcuordde 'aseclol 06€-68€ U u)ryd Q,L6l\'9 ry louep.l1[ (iZ) prre JBIIuls E uI 99-99'W'4 -rrpllBprrB sserlloluPlle JoJ suoDJseJ ^\ols eAsI{SJosuesolq eseql 'wrotld 'uuv ',{rru4 ''f 'loluellll(\ "V 'ueqemg 'psz{Iqoulm OL6D'9 ? (€Z) 'Je^e^\oq JPJ oS eJB oseJloluelle puB esPulol IZ8I-6I 8I -wlle qqq.r\ uo aponJole cr$eurouuelod Sursuas-€HN ue pue '65 'pS 'rurDrld '16'1'sut33tg ''f'C (ZZ) 'ese3lolll?Ile 'essutol T (0t6I) ? tegel[ pJzlFqoululr arEes4JnPaJ 4el,{xor(13 pue 0I€-€0€'sl'waq) -uelle qJr.I/r\ uo aponJele crleuroradure Sursues*clvN rI€ :elq 'pusoJ'ros't 'g'S'Bcrel "U'esm1,"y'5'ae) (W) (W61) I d' -rssod o,t1'lsal e u pesneq,{eu sJosuesolg '16'uaq)otg are sadft lods g0€-t0€ 'paroldxe aq elqelnu?nb 'C'tl[ED "g 'ceful-ctfrug''I lsntu 1tuv QL60 ry I'W'p{seqr1 (02) 'f1r,rr1ra1es '87'nng poqlaur aql e{elu ot s,(u [ esPeJsul(uru lueqros 08Et-9LEt -pB qlrm 'uuoqA'tuaq) 'H 'l1ese.,rreX' ''H 'p{n ar$ ur saur,{zueEurzlpqouruu ,(q suogcuar crurfzue (OB6D ry 1 eu{e1 (0t ) 'ftrrlncales 'errqslqu€nbruras 'lV'lolg 'ut!q) '2oS'ilng 31 Euqdno3 rood e,ruq,(arp pue 9I0I-I00I (6961)'d'ua?{orJ (8I) 'alrsuedxeut '>l ro a,rqaqsnb .,(po eru ,(eq1 tnq pue eldruts Lg9-t99'SL'uaqxorglDuV (916) 'solletusot 'r-rtrwqe)lB'L'U 'olourxe) ' 'Hsl) 'I '"peuln ' 'IunS arp spoqatu J'IJ pw slecqnaJuluJeqd;o8uqse1 T ,\. I (U) 'pepaeu pnpord JoJ ,(pnbe sr enbr-uqrele qcns $ esn LVZ-WZ'719tSor lrgasn 'U'g "f'C'el,()'I"g'X'ueqJ pleg:og anbnrqcet e pIrB'slu?ururu w ,(1ddnsurelord -maorll)'t G66D ^otlsro? (St) ..1c4sdrp,, 'louv 'U'srequog xepul IrB s? pesn sl eu1muI uoluEIIB 'alduruxa ro; eg-Zlg'6L'uaqrorg QL6i (SI) I?lqonrju Jo 'uourrnu u1 'selduruspcrSolorq rog padolelep eq lsnu 0t I-99I' L6 ryssazn1o8n4otr (7261) 1Bumre cryceds puu spoqleur e^nqllu?nO 'sprnnecuuueqd I'oruuv d"'X'erruuoleN'I'oll''y'ereEu5'1'olTe) (lt) slssl lods ' 'uaqcotg'puv 'I'E 'aued pa1etuolnsIIB oltn eq p1noc ZVE-EE;LZ G96D (et ) ro; ssacord uoncnpord Pelu€au peregu q4tr EE-EZ'ES'c1.t7y'poog'tts7 (066I) dars pcu,(puu atp'(€S) f4eruoloqdo4cads 'fa'speag "61'Cd'lle^oH"f 'uoseqrel\l"g'X'ueqJ 'JeqlmJ ? ftl) paroldxa eq NnLu saldurus 'FSSselJns L9t-E9l pcrSolorq qlr^\ esn pnuetod sU sraaddeseldures 'J'uossrr?f ''g'1'Sreqpuq 'e,rr1eure1p '6l'say'ryBV't'paft,S (686D ry (tt) JuetusoJ rOI^\ esn sll pue In sJaJJo(ZS) poqeu 'uorleredes LEE_€,EE'IE'PAU UJ eq;- crgderSopuorqo Jo esuepue ,fir,rqcelas 4aouuuaA'dxg'r1cty (996l)'H'rallen4 4r''9',(lsmzurog (Ot) yo satelue.r'peeulqruoc ppo,n qceorddu s511 ,{qdur8opurorqc ' 'X "f LZZ-ArcLV wapoq puv (Z,L6]') turerB^Is? "IBuuI.N (6) asuqd-pes:ar'er,(q uo4urudes o1 .rogd xelduoc gyqqd-utol Lgl-ttr -uplle rre LruoJol lua8uer qcllJqg I0I^\ pozne^uop eq wc ulol 'Ee'uaqrolg'puv 'J'UIrq ''O'C's1e8on (g) '17) @L6D rapIre^ T -ue1e'aldurexarog 'paroldxe eq lsnu (gV'ZV g elqel ut 'uoneznu^uep 'uos 298-6€8 pelsq asottr1ot uoopP? ur Jo spoqleu eJolu 'Zil'uattJ'lolg't (2160 g'3',{e,nuo3 ''9'g'tunoa (D ry -sar slrlt roJ puu 'prcu c1,{xo,(13Stmnuluoc selduresro3 elqucrld spowa Kr1do1 g Eg-6S' lZ W's du aq tou feu g41qq-ep1,fuof13 truoJ ol urotuelleJo uoqszrl 'uaqrolg "N'Hnqe^ "H'?rPqIqsV (9) T (066I)'X'Instlw T -e^lJep 'sutunloJ 't 'rg uurnloeard uo paseqspoqlehl aseqd-pasrer'er zrt-ttt'89 unN (0661) 'paroldxa 'S'O'u^\org ''d'il'^o{sro "O'Oglla^oH "g'X'ueqJ ,(q paurnar ilern lou sI UIoIIIEIIBesneJoq 3q plnoJ T (9) "S'ueJ"V) suumloJ eseqd-pesrerrarJo asn IIBID Jerpo sepou uoDeredas gZEy'6't qXSUIG6f''D'Y'C's'$Bd ?8 :auuDls (t) 'J-I '(97-91) 'Wa''t 'g'IIo/I\[I"H "N 'ple^potD fq ulolu?fie Jo uoueuruueleplrerrp rog tueEu uapas 086I) ? (€) 80H08 -ar ro qrllrqg retpla qll^\ ulotuu11s uoq Ktldot g'utaq c JSI-6IVq lueEeer Jo 96-| 6' ZSZ's -JBoJ -o!g 'qilV 'ee'I 'I'S ',(erots"g'n 'sueu?t\l lcenp uo pesuq spoqleu ueql StmrmsuoJeurq eJolrr lnq (Lg6I) a'C ? Q) 'p a^utales erolu e.m(f le mms pue (t) fer'ruo3 puu Eunol lgt- LEE'81'unuuoJ'sav t) "S''I'3uo16''4's4ctg spoqlaur eq1 'pennber aJuelqellp^s ,{puxatd Ileqt sempac pxryDAaatg (96611'O'U',(eC ? (t) Jo -ord alrlceles eJolu puu ralduns 'spoqleu cuauolot{dolmds 'aperu secuaJalaH JoC oq uuc spoqlaru Surlsxe w $uaualordun requng 'Jerueepessncsp 'lducsnuerueql go uosRaleql roJ'luetuemseeuJelsmoJe epr,rordlou seop utunloc 'S'fa 'S'C uI ulollrull€ urscqFc ggdlaq ro; ,temeq pue u^\org {ueql eifi aseqd-pesrelere Eursn ,{q salduresuurseld IrBIunq ;o uoDeredeslJenCI'uonuJudes 3r1 Jq;? Jo aJoJeqpszne^uap '(It) sluaut6peuhou)lcv sr urolusllu qcrq^\ uI '(8U) q1qsoutt pue uurefolol ptre 'p 'p ta oerpN'(Zt) ta erorn+{q asoq are spoqlerua^qBIuolIB 'paz^{1euu '(slueururru ''t'a) ptcu Inqf qol aq o1seldums 3o ed,$ at11JoJ elqqms ?ursqd uerunq rog IIJoI Jlm 'poqleu -wlIB sr lr lelil aJnsueol lusuodur sI lI Suusua fue Sundop€ eJou ,{lpuoluodord Suruwluoc selduresuuseld ro; pesn 'snql 'JeI$o '(utsp 'g'X) eJoJeg 'palordun eq tsrur spoqleu eql ul )pe/t\ eq eroJarerTlrrur poqleu eql gaqsqqndun'ueqJ tnq pedser euo ur poo8 uego eJe pue ,$1c11dunspue ,(1urr;ea1 uon€uruuelap urolIrBII€ tpt,rr aleFslq lou seop TIourTi 00I> '(9I) 'p '(et'e) -as w frel urolu?llu Eururuuapp roJ elqulr-B^uspolpaur eql lB ptru rlrn p ueqJ Jo poqleu eql Wr,ry\ ptce

'€ 'oN '61 "1oA 966I TVNOLIVNIIIrNIJVOV JOTVNunOf :'1V rg NaHJ ]€9 '€ 966t q{'6{. ]q\ Fuo$rudul JVOVF F.D( T nll rugbU

aDouroqd l0z_ffi(,.z? Z Ll-ggl' g7't8 otouou13 (f tg.T2crmerfiorumf..f .{elsetF.I ..S.Fourqo ,.I ..) .qorrH 861 )'f,I'Dls^\olruzs (SS) T (€g6I)'I'ell|sl) ? I.esB^r?){ (ZV) mlo8qof (t .el{onst?W nZ9-519.9111r1ssry 66D.I4l 99eI-Z9El'Sg'uaqJ.puv .ouolqo ..S.oloulEtu?I '[6'cossy.t .epuoH.) ?' I'nsterue>IpJ''1'nsteuoy''W (ES) eg6l) 1.e{auo1?..) .o?TBN(tl) g-l'Llll .f 'U.r-ueted LVI-trl.ED rtopuatry G6) ?..g,ollaug o1cy'sKt1do1g'ultlcolg (ZOOD.g.olleuuphl ..U .1ueE -BW"W'nmlrrrzrrl''-I .rurzarv ..W .UIopuBd..T .IonzreJ "O'uourrs1'.U.rurcuo{l,.W.rur-qrrzzld..V,oueJqS? (Ol) -?d (tS) -uaqJ Ie €g9{gg,glt, .louv .z.snruas (.uaqJ puy) aqlong i;1661.).-I.Eue11 -aq 'T'lrI 9tZ-lt7'6t XuaJ (eS) O6D "rarqsf,?..n.U.olIpro0..V.W.u?tualg (Og) 'puv v6l-tgl.Lz'srauTl 6S8Z-t982'91t8o1mu 'Il{'U'olleruuEl ..f.u.asrec -orqJ 'bn 't .uqv .I.hl .zale ..V.f \n66D ?..I.N.Hr.ruqrehl (zs) (E6;6I).f.W T .seursey (Sg) SgI-S4'9gg t8otoutottqs.y .1a9 'N ''W'ps?) '1 Z0Z_661 t8ot (066I) "le4H ? "ruqr8euua"y 'rqcnEo11(tS) -Dworq) 't .ze!11 ..1.I .ulrv ..V.f .seurse1 Q66I) 1.h{ ? I ee) 'gg7 ztz-L€z 09El-LEEI'91'tSosout tSotowotq)'t (Zg6D .flnsJ ..H .ouo|I,.f .asp/l\e) (OE) -ott13'by1 't .seurse6 ,.f.I .uuv . .zerq ) ? (266)'V.f ? I I.W (Sg) t?otow 9lZ_@2,797 tS I-gSI' 911'tB otowotrq2 -orqJ 't 'llnsJ ,.V .e{BN ..H .ouen..f .esB/he) 't ..n.f .o{ed ..V.f Qg6D ) T (OV) Q66D'C :aved ? .Ep?nC..f .sllerl€g (SS) 'ggryo8oy t9E-6Vt 9LybLg'6Z.uaq) plasung(1661 turu,(o1oa (gt) -o!g 'un 'uuv .sqruops ..C .oopl?N,.O .xn-I I.qrqsoury 4r.1 (Z66I).f, ? $t) n6Z-n6Z'tL'uaq) .gO.rossy (886I).f.U .1pe^t{ IouV T eV) El-ll'&557luaqroq-WV fuB6D.C.opeserg?..11 tefaurera (gg) .1ouy 66-L66.tL AV-ZE'&I uaqroq (7g6i.U.t .uessrepuv .a .auen( '!rS'wrottd't .oruseg ..f T (Zt) Qg6D a'C "ues ,.\.Z,lplvz 'lL'uaqJ T J @V) 9S8-SS8 .puv go 'nx ..u'f,,n ..ff.t-I 'rossy 'f.f i.l.t .aseog..S.U LZt-ZTt'11ndagG,oelD's'z ? (st) T (gg6l) ireqsBr{t? .?rerreC (tg) nLz-LgT'L9'dunH Sl,.St6pus g7.0g6scos .yn .uofuq 'uroqd otrv .erulrg ..J -rV'JVOV''pA Qg6D.f T Tqtzaq.'U.sruueq (W) t1tgI(0661 ) s1s(.1ouyspoqtay,l (Oe) .1g .waqcorg lo Tmcgffg 0l €-€0€ .unJ .uaqJ 'ullJ't jqE-LLE.0L.uaqrotg nE (gOOl).g.tu1rerg 5lro) (eV) 'louv ,suqdog ..VC ..f ?,.g.xno^teq..U OL6l).4'g T euoerurs u?r{Brqv GZ) '€ 'ON '6' "ro^ .Tv s€9 966I TvNotrvNlrs.tNlJvov do TvNunof : Jg NaHJ