A simplified method for the isolation of urinary ascorbic acid as the 2,4-dinitrophenylosazone

SHORT

A Simplified Ascorbic

537

COMWUNICATIONS

Method for the Isolation of Acid as the 2,4-Dinitrophenylosazone

Urinary

Isolation of radioactive urinary aticorbic acid as a 2,4-dinit,rophenylhydrazine (DNPHj derivative has ken useful in studies on ascorbic acid biosynthesis and metabolism (l-3 1. Either t’he ascorbic acid was isolated from the urine by ion-exchange cllrom:~togr:tplly and the dcrivative formed by reflusing bromine-oxidized ascorbic acid with DNPH (41, or the DNPH derivative was formctl in raw urine and separated from the various hydrazones and osazones formed by absorption chromatography (3). The latter technique is tedious and time-consuming and not suited for multiple determinations. The former method, in our hands, required some modification to reduce the impurities deriretl from the ion-exchange resin and to improve yieltls. The prcxnt report describes the integration of several existing ascorbic acid analytical procedures resulting in a simple technique for the isolation of urinary radioactive ascorbic acidDNPH derivatives in good yields. The improvements in this method result from: (al use of a mild oxidizing agent (p-benzoquinonej to oxidize t.he ascorbic acid prior to derivative formnt.ion; (Di substit.ution of Dowes 1 (acetate form) resin for Xmherlite IR.-4H 10H form) ; and (c) use of acetonitrile as the recrystallizing solvent for the DNPH derivative. Twenty-four-hour urine sampIt+ were collected from human subjects receiving an oral dose of ~~-aworlGc-l-l”C acid and acidified with conc’entrated hydrochloric acid (1 ml concentrated HCljlOOO ml urine). The initial concentration of as:corbic acid was detwmined in the urine by the calorimetric procedure of Phnffcrt i:i 1; carrier ascorbic acid was then added to a 50 ml urine aliquot to Iwing the aworbic acid conccnt.ration to 2 mg/mI. The aliquot of urine n-a~ poured direc+ly ont’o a column (2 X 15 cm) containing 20 gm of dry Dowcs I-S4 resin (J. T. Baker Chemical Co.) in the acetate form. After allowing tl~c urine t,o pass slowly through the column (flow rate = 0.5 ml/mini . t.he column was washed wit.11 200 ml of distilled Twter. Ascorbic acid was cluted from the column with 1 .j7 HCI, and the eluat,e collecte(l in 25 ml fractions. The concentration of ascorbic acid in the fractions was determined 1.1~titrating 1 ml aliquots with st,andard iodine solution, using starch as tlw intlicntor. Usually, TO-SO% of the initial ai;corhic acid was prewnt, in fractions 2 and 3, which mere pooled. To the pooled fraction!: was added a 0.95 molar equivalent (based on the iodine titration) of p-benzoquinone (Fkher Scientific Co.! in 5 ml

of methanol (6). After standing fnr 10 min, DKPH reagent (2% 2,4dinitrophenylhydrazinc in 3 LJ’ HCI, 4: 1 mole ratio of Dh’PH: ascorbic acid) was :~cltlcrl ~111 the samples nllowcd to stand at 5’C for 2-3 days. The derivntivc forme w:~:: filtered through Whatman Ko. 50 filter paper and imnicdiatcly (i.e.. without drying at the filkr pump) waxhcd into a beaker with ncctonitrilc. T11c couil)ound xws dissolved in boiling acetonitrilc, concentrated to a .sni:rll s-olunic (10-15 ml l, ant1 allowed to crystallize at 5°C. After three rccr~stnllizat,ionu, the specific activity of the derivative was constant, and any traces of p-bcnzoquinonc-DKPH had been rcinoved-as dctcctcd by cl~roni:~toglnl~h~- on thin-layer plates (8). Annl!/.sU: m.p. 291°C; fount1 C 39.95cjb, H 2.9370, N 19.60%; t)hcoretical (2,4-dinitrophcnylo+azone of nworbic acid) C 40..5670, H 2.62Cjo, N 20.97%. IR I~ontls wre found at 1753 and 1228 cm’, indicating an intnct y-lnctone. For ixdionctivity detcririinntions, tlic derivative w:w tli+olvcd in ncetonit,rile, :tppliccl to ncighctd planchete, clricd, and countrd n-ith a ;\‘uclc:w-Cliic:~go gas-flow counter. The ascorbic acid-I>NPH tlcrivntive proved difficult to recrystallize from solvents ot,lier than acetonitrile. In several solvents (isopropanol, ethanol i , or on drying wit,11 the use of a water vacuum pwip, the compound underwent a structural change resulting in a dark red crystalline modification that was difficult to recrystallize. The use of acctonitrile as the solvent resulted in a rc~pro~lucible derivative wit’h little loss on recl~~~t~alliznt,iol1. yields for the dcrivntive formation nftcr ouinonc oxidation ranged from 60 to 7070. I’re of ~~iuberlitc IR-413 resin. even when repeatedly washed with acid ant1 base (41, resulted in considerable release of formaldehyde from the resin which yielded a yellow DNPH derivative (1n.p. 166”C, IR pattern identical with t,hat of the DNPH derivative from authentic formnl~lchgde). Recovery of ascorbic acid from the Dower 1 column routinely ran from 85 to 95%’ of that piwent in the enriched 50 ml urine alicluot, without nccessitnting the prior rcmot-al of oxalic acid (4). REFEREh-CES 1. HOROWITZ,

H.

H.,

DOERSCHUK,

A.

P., AND

KING,

C. G.,

J. Biol.

Chem.

199,

193

(1952). E. M., SAUBERLICH, H. E., WOLFSKILL, S. J., WALLACE, W. T., AKD DEAN, E. E., PTOC. Sot. Exptl. Biol. Med. 109, 737 (1962). 3. ATKISS, G. L.. DEAN, B. M., GHIFFEN, W. J., AND WATTS, R. TV. E., J. Biol. Chem. 239, 2975 (1X4). 4. JACKEL, S. S., MOSB.KFI, E. H., ASD KING, C. G., Arch. Biochcm. BiophzJs. 31, 442 (1951). 2. BAKER,

SHORT

5. SHAFFTRT, R. R., .~ND 6. DAHN, H., AND MOLL,

KIXGSLET,

H., Hclzl.

539

COMMVSIChTIONS

G. R., J. Biol. Chew 212, 59 (1955) Chim. Acta 47, 1860 (1964).

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