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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 glyoxylic acid 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 allantoin 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 urea. 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 peptide 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 FeCl3 in 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 urine 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