Simultaneous Determination of Urinary

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1 Simultaneus Determinatin f Urinary Ascrbic Acid and Creatinine by Highperfrmance Liquid Chrmatgraphy (Accepted n Mar. 26, 1996) Yshihir Shimada Sanae K Key wrds : Urinary Ascrbic Acid, Creatinine, High-perfrmance Liquid Chrmatgraphy Abstract A simple high-perfrmance liquid chrmatgraphic methd is described fr the determinatin f ascrbic acid and dehydrascrbic acid. Use f tetra-n-butylammnium brmide as an in-pairing regent in the mbile phase yielded cmplete separatin f ascrbic acid. Ttal ascrbic acid was determined by reducing the dehydrascrbic acid t ascrbic acid thrugh treatment with DL-hmcysteine. The high-perfrrnance liquid chrmatct graphic methd was then applied t the analysis f human urine. This methd can als be available fr the simultaneus determinatin f urinary ascrbic acid and creatinine. Intrductin Ascrbic acid () and dehydrascrbic acid (DH) are equally bilgically active frms f vitamin C in human and guinea pig (1). Therefre, and DH shuld be determined t knw the ttal amunt f vitamin C in vegetables. has been determined by the dye-titratin methd using 2,6-dichlrphenlindphenl. Ttal ascrbic acid (T) cmprises.the sum f and DH is determined by the clrimetric methd using 2,6-dichlrphenlindphenl by xidizing t DH and using 2,4-dinitrphenylhydrazine (DNPH) by subsequent frmatin sazne (2). Hwever, these methds are time-cnsuming and may verestimate and DH wing t the presence f xidizable cmpunds ther than vitamin C in urine samples. In additin, it is difficult t visually determine the titratin endpint. -89-

2 Yshihir Shimada Sanae K Recently, several high-perfrmance liquid chrmatgraphic (HPLC) methds have been develped fr analysis f and Dum. These methds adpt varius clumn materials, mbile phases and ultravilet (UV) r electrchemical detectrs(3--6). was well separated frm ther cmpunds in range juice by use f an in-pairing regent in the mbile phase (7,8). Determinatin f T by HPLC requires a rapid methd f cnverting necessary fr detectin. Hughes (9) described a rapid and cmplete reductin f D t by DL-hmcysteine. In this study we reprt relatively simple HPLC prcedure fr estimatin f DH t by DL-hmcysteine and uses tetra-n-butylammnium brmide as an in-pairing regent in the mbile phase. This methd was used fr simultaneus determinatin f urinary and creatinine (Cr). T cntents in vegetables were determinatin in rder t cmpare ur HPLC methd with the DNPH methd. Materials and methds Regents. DL-hmcysteine hydrate was btained frm Aldrich Chemical Cmpany, Inc., Milwaukee, Wi, USA. L(+)-ascrbic acid, creatinine, tetra-n-butylammnium brmide and ther chemicals were btained frm the Wak Pure Chemical Ind., Ltd., Osaka, Japan. All the regents used were f reagent grade. Highmaperfrmance liquid chrmatgraphy. Separatin f was achieved with a Yanac L-5000 liquid chrmatgraphic apparatus equipped with a Rhedyne Mdel 7125 injectr. Clumn effiuents were mnitred at 265 nm with a Yanac M-515 variable-wavelength detectr. Peak areas were determined using an SIC Chrmatcrder 12. A Shdex RSpak DET613 clumn (150 mm Å~ 6 mm inside diameter) was used. The mbile phase was cmpsed f 8 mm phsphate buffer, ph 6.8, cntaining 3 mm tetra-n-butylammnium brmide. The flw rate was 1.0 ml/min. Sample preparatin. Urine; A vlunteer (37 year-ld, male) tk a single ral dse f 225 mg, 150 mg and 75 mg f separately. Urine was cllected every ne hur. Tw ml f urine were received 2 ml f metaphsphric acid. The mixture was centrifuged fr 20 min at 3000 rpm. The supernatant was diluted 40-flds with distilled water. Vegetable; Ten g f Pakuchi and Tahtuai which are Chinese vegetable were hmgenized with 10 ml f 109(r metaphsphric acid in a mrtar with sea sand. The slurry btained was transferred t a centrifuge tube with 20 ml f 5P06 metaphsphric acid and centrifuged fr 20 min at 3000 rpm. The supernatant was diluted 20-flds with distilled water. assay. One ml f O.08 M K2HP04 was added t 4 ml f diluted urine sample t give a -90-

3 Determinatin f Urinary and Cr by HPLC final ph f 7.0. Fr vegetables, a 10-ml f diluted supernatant was diluted with O.13 ml f 2.5 M K2HP04 t give a final ph f 7.0. A 20-pl aliqut f this slutin was injected int the HPLC system. ' T assay. T was assayed by adding O.O15 g f hmcysteine t 5 ml f neutralized sample fr assay. After 30 min at 25Åé, a 20- pt1 aliqut f this slutin was injected int the HPLC system. The cncentratin f DH was calculated by subtracting the amunt f frm that f T. Results Separatin f and Cr. Typical chrmatgrams are shwn in Fig.1 fr the elutin f standard mixed slutins f and Cr (A) and thse in urine administered rally (B). Retentin times were 4.1 min fr standard Cr and 4.6 min fr standard. The peaks f the same retentin time as standards were seen in human urine. and Cr were cmpletely separated frm ther unknwn peaks. Fig.2 shws chrmatgrams f in vegetables befre and after incubatin with hmcysteine. The hmcysteine treatment resulted in the appearance f extra peak at 3.5 min due t hmcysteine, but it culd be readily separated frm. Cr L Cr O246 O Time (m i n.) Fig.1 Chrmatgrams Fr (A) standard (1.6ptglml) and Cr (24ptglml),(B) and Cr in human urine. L"J.- t 4J- ua" O7 i, 6OZL6 6O 2L68 Ti me (m i n.) Fig.2 Chrmatgrams fr (A) standard (2pglml),(B) in Chinese vegetable,(c) in Chinese vegetable incubatin with hmcysteine. -91-

4 Yshihir Shimada Sanae K Tablel Recveries f ascrbic acid() and creatinine(cr) frm "-li urine. ' IIE 20 0 Added Fund Recvery 3 (pt g/ml) (p g/ml) (P06) v O 18 I ` E a 99.0 J = > A. < O 1325 S O l 20 Cr 500 l T by bnph methd<pglml) Fig.3 Crrelatinbetweenquantities f T estimated by DNPH methd and by this HPLC methd. Recvery. Recveries f added t urine ranged frm 96.0 t (r and thse f Cr frm 96.2 t (Table 1). Crrelatin. A cmparisn was made f the cncentratins f T fund in varius vegetables by DNPH methd and thse by ur HPLC rnethd (Fig. 3). The relatinship between the values btained by the tw methds was described by a regressin line y=o.9997x - O.3657, r==o.9979 (p<o.ooi), where x represents the values btained by DNPH methd and y represents thse btained by ur H[PLC methd. Analysis f in urine administered rally. Urinary T, and DH were analyzed in rder t cmpare the DH in T ranged frm 2. Time curses f urinary relatin t the Cr excretin administratin f 225 mg, 150 mg and 75 mg f /va separately are shwn in Fig.4. Urinary excretin f related t the Cr excretin reached its maximum values in 3 hurs in each dse f. A small peak was als bserved in 8 hurs after administratin f all kinds f dse. urinary cncentratins (Table 2). The urinary 5 t 3.9 P06. excretin in after ral excretln Table2 Cmparisn f the unna ry cncentratin f DH with that f T. Cncentratin ln unne T (mg/ml) O.435 O.418 O17(3 O.345 O.360 O.212 O.333 O.35I O.205 Numbers in parentheses indicate centages(906) f DH in TA-A. T : ttal ascrbic acid A-A: ascrbic acid DH : dehydrascrbic acid DH 9) O12(3 5) O09(2 5) O07(3 5) f per- -92-

5 Determinatin f Urinary and Cr by HPLC L'- nu" [ b 30 N- '- E +J v : > 100 L aj [ : DO -7 ' iti,a N,x ZN t ! x NxN 'N. XS, Å~.XtL v.lr/-A't>szÅ~.ss O Time after administratin(hrs.) -' --L ;=X sxnr:=--'-' Fig.4 Urinary excretin f related t the Cr excretin as a functin f timeafter ral administratin f (225mg, 150mg -----, 75mg ). Discussin Determinatin T f by HPLC requires a rapid cnversin f DH t, since DHA-A des absrb nt UV ray which is necessary fr detectin. Hughes (9) described a simple and cnvenient prcedure fr the reductin f DH t with DL-hmcysteine. This methd been has applied t the analysis f T by HPLC (10,11). In the present assay, use f reducing this regent als prduced excellent agreement between the clrimetric d meth using DNPH and ur HPLC methd as shwn by crrelatin cefficient (r= O.998). The retentin time f was delayed by the increase in amunt f tetra-n-butylammnium brmide as an in-pairing regent in the mbile phase. Use f tetra-n-butylammnium brmide in cnditin f this study gave cmplete separatin f, Cr and ther unknwn peaks in urine. In analysis f in vegetables, a clear peak f was als btained, and the separatin was nt interfered by peak f DL-hmcysteine added in sample. ' Our HIPLC methd appears t have a higher specificity than the clrimetric methd, because the btained values by the clrimetric methd were higher than the HPLC values m many f the samples. We determined the amunt f urinary excretin f after ral administratin f. was majr the cmpund excreted in urine in cmparisn with DH. The purpse f -93-

6 Yshihir Shimada Sanae K this investigatin was t examine a pssibility f mnitring vitamin C intake using the simple assay methd f urinary. A special merit f this HPLC methd is that urinary Cr can be determined simultaneusly. Cr adjustment has been thugh t be an effective measure in case f the spt urine specimen which is very cncentrated r diluted. several studies have been reprted fr the examinatin f the urinary excretin f after administratin f a large dse f (12,13). In the present paper, we examined the excretin when a small dsage f was taken. Urinary excretin f crrected by Cr excretin reached its maximum value in 3 hurs after administratin f A-A. This seems t be related with the fact that the peak plasma vitamin C cncentratin ccurred in 3 hurs after ral adrninistratin f vitamin C (250 mg) as reprted by Murata et al (14). Therefre, we prpse that the intake f vitamin C can be mnitred by measurement f urinary 3 hurs after meal. References dehydrascrbic acid in fruit and vegetables by high-perfrmance liquid chrmatgraphy. JChrmatgr (1983) 256, Augustin J, Beck C and Marusek GI : Quantitative determinatin f ascrbic acid in ptates and ptat prducts by high perfrmance liquid chrmatgraphy. J Fd Sci (1981) 46, Rse RC and Nahrwld DL : Quantitative analysis f ascrbic acid and dehydrascbic acid by high-perfrmance liquid chrmatgraphy. Anal Bichem (1981) 114, Sabry JM, Fisher KH and Ddds }vfi.: Human utilizatin f dehydrascrbic acid. J Nutr (1956) 64, Re JH, Mills MB, Oesterling MJ and Damrn CM : The determinatin f diketc"-1-gulnic acid, dehydr-1-ascrbic acid, and 1-ascrbic acid in the same tissue extract by the 2,4-dinitrphenylhydrazine methd. J Bil Chem (1948) 174, Wimalasiri P and Wills RBH : Simultaneus analysis f ascrbic acid and.pachla LA and Kissinger PT : Determinatin f ascrbic acid in fdstuffs, pharmaceuticals, and bdy fluids by liquid chrmatgraphy with electrchemical detectin. Anal Chem (1976) 48, Sd SP, Sartri LE, Wittmer DP and Haney WG : High-pressure liquid chrmatgraphic determinatin f ascrbic acid in selected fds and multivitamin prduets. Anal Chem (}976) 48, Keating RW and Haddad PR : Simultaneus determinatin f ascrbic acid and -94-

7 Determinatin f Urinary and Cr by I-IPLC dehydrascrbic acid by reversed-phase in-pair high-perfrmance Iiquid chrmatgraphy with pre-clumn derivatisatin. J Chrmatgr (1982) 245, Hughes RE : The use f hmcysteine in the estimatin f dehydrascrbic acid. Bichem J (1956) 64, Dennisn DB, Brawley TG and Hunter GLK : Rapid high-perfrmance liquid chrmatgraphic determinatin f ascrbic acid and cmbined ascrbic acid-dehydrascrbic acid in beverages. JAgric Fd Chem (1981) 29, Behrens WA and Mad re R : A highly sensitive high-perfrmance liquid chrmatgraphy methd fr the estimatin f ascrbic and dehydrascrbic acid in tissues, bilgical fluids, and fds. Anal Bichem (1987) 165, Yung S, Mayershn and Rbinsn JB : Ascrbic acid absrptin in man: Influence f divided dse and fd. Life Sci (1981) 28, Yung S, Mayershn M and Rbinsn JB : Ascrbic acid absrptin in humans : A crnparisn amng several dsage frms. JPharm Sci (1982) 71, Murata A, Baba R, Matsuka M, Otsuka H, Takauchi T and Itkawa Y : Plasma levels and urinary excretin f vitamin C after ral administratin f vitamin C preparatins in healthy male adult. Vitamins (1989) 63, (in Japanese). -95-

METHOD 8032 ACRYLAMIDE BY GAS CHROMATOGRAPHY

METHOD 8032 ACRYLAMIDE BY GAS CHROMATOGRAPHY 1.0 SCOPE AND APPLICATION 1.1 Methd 8032 is used t determine trace amunts f acrylamide mnmer in aqueus matrices. This methd may be applicable t ther matrices.