ROUTINE CITRUS JUICE ANALYSES USING HPLC WITH AMPEROMETRIC DETECTION

Transcription

1 9. Gillspi, K Fungicid tsting. Taking it to th shds. Packingshd Nwslttr No. 41:3-4. Dpt. Agr. Waikri, So. Australia. 10. Holms, M. G Th dtrmination of thiabndazol (2-(4- thiazolyl)-bnzimidazol) in citrus fruit dips. Pstic. Sci. 3: Millr, V. L., C. J. Gould and E. Csonka Masurmnt of thiabndazol, bnomyl and folcidin fungicids in aquous suspn sions. J. AOAC 58: Millr, V. L., C. J. Gould and E. Csonka Analytical ractions for a fild tst for thiabndazol. Plant Dis. Rptr. 55: Sax. 1. L Dangrous proprtis of Industrial Matrials. Van Nostrand Rinhold Co. Nw York, N.Y. 14. Smoot, J. J. and C. F. Mlvin Dcay control of Florida citrus fruits with packinghous applications of thiabndazol. Proc. Fla. Stat Hort. Soc. 83: Tugwll, B. L Analysis of bnlat concntration in sampls takn from a commrcial citrus fungicid flood applicator. So. Au stralian Dpt. Agr. Fruit Packaging. Transport and Storag Rs. Rp. 1/73. Mimo, 3 pgs. 16. Wardowski, W. F. and Eldon Brown Stm-nd rot in th fall of Packinghous Nwslttr No. 138:1-2. Inst. Food Agr. Sci. Fla. Coop. Extnsion Srvic. 17. Wardowski, W. F., F. W. Hayward and J. D. Dnnis A floodrcovry TBZ fungicid tratmnt systm for citrus fruits. Proc. Fla. Stat Hort. Soc. 87: Windholz, M. (d.) Th Mrck Indx. Ninth Edition. Mrck & Co. Inc. Rahway, NJ. Proc. Fla. Stat Hort Soc. 103: ROUTINE CITRUS JUICE ANALYSES USING HPLC WITH AMPEROMETRIC DETECTION D. Richard Whit, jr. Stat of Florida Dpartmnt of Citrus Scintific Rsarch Dpartmnt Citrus Rsarch and Education Cntr 700 Exprimnt Station Rd. Lak Alfrd, FL Abstract. Analytical mthods usful for routin applications ar idally low-cost, simpl to prform, and yild rapid rsults which ar asy to intrprt. Chroma tog raphic mthods, par ticularly of complx sampls such as fruit juic, ar oftn difficult, can b xpnsiv and tim-consuming, and may yild rsults dvoid of intrprtation. With th propr choic of dtctor, th chromatographic analysis of vn th most difficult sampls can oftn b simplifid. During th past dc ad, thr has bn a rapid incras in th us of lc trochmical (EC) dtction mthods in high-prformanc liq uid chromatography (HPLC). Th suddn popularity is du to th dtctor's xtrm snsitivity, slctivity, and compatibil ity with rvrsd-phas and ion-xchang sparations. Thr HPLC/EC mthods wr dvlopd and/or adaptd for routin analysis of vitamin C, folic acid, sugars and sugar alcohols in citrus juic. Analysis tim for ach mthod was undr 10 mi nuts. Analytical rsults for som frsh citrus juics ar pr sntd, along with statistical considrations. Introduction Citrus juics ar an important ditary componnt for th maintnanc of good halth, and can b usful in th tratmnt and managmnt of a numbr of human dis ass (9). Frsh orang, grapfruit and thir juics, as wll as commrcially procssd products, typically rndr a palatabl blnd of svral ky nutrints. Th simpl sugars glucos, fructos, and sucros compris th major carbohydat fraction and mak up btwn 75 to 90% of th total solubl solids in juic; th rlativ amounts dpnd som what on sason and varity. Although its nutritional valu is not compltly undrstood, th sugar alcohol myo-inositol is prsnt at significant lvls. Probably th most im portant watr-solubl vitamins ar vitamin C (L-ascorbic Florida Agricultural Exprimnt Station Journal Sris No. N Th author's currnt addrss is: Th Proctr and Gambl Co., Winton Hill Tchnical Cntr, 6210 Cntr Hill Road, Cincinnati, OH acid) and folic acid. Othr nutrints includ thiamin (vita min B,), vitamin B6, niacin, riboflavin, pantothnic acid, bioflavonoids, potassium, othr minrals and trac l mnts (18). With th currnt mphasis on nutritional labling and an incrasd consumr intrst in nutrition, nw dmands ar bing placd on food and bvrag industris to spcify th quantity of nutritionally important componnts in foods. It is thrfor important to hav analytical mthods that ar accurat, ruggd, and of low-cost. Mthods that ar rapid, simpl to us or asily automatd ar prfrabl to mor costly, tim-consuming tchniqus. Citrus juic is a complx matrix and analysis oftn r quirs significant sampl clanup, som typ of sparation, followd by an accurat masurmnt of th analyt, which is oftn prsnt at vry low lvls. Th usfulnss of high prformanc liquid chromatography (HPLC) dpnds on th ability of th column to sparat, and th dtctor to sns th componnt of intrst. For complx mixturs, inadquat rsolution du to limitations in column pr formanc oftn rquirs that th dtctor b capabl of discriminating th analyt from othr coluting substancs. During th past dcad, thr has bn a significant incras in th popularity of lctrochmical (EC) dtc tion mthods. Primarily usd for th analysis of trac or ganic compounds in complx biological mdia, th usful nss of th EC dtctor stms from its slctivity, snsitiv ity, and rlativly low cost. Th thin-layr ampromtric cll (Figur 1) is widly usd bcaus of th simplicity of dsign, small cll volum, and compatibility with svral lctrod matrials. HPLC with ampromtric dtction prmits routin analysis of picomol amounts of lctroactiv substancs (8). Svral of th nutritional factors in cit rus juic ar lctroactiv and amnabl to ampromtric dtction. Th purpos of this work is to illustrat th utility of HPLC/EC for thr routin applications in citrus juic analysis: Vitamin C {L-ascorbic acid), folic acid, sugars and sugar alcohols. Vitamin C xists in citrus juic in th r ducd form and is asily oxidizd on th surfac of a glassy carbon lctrod (1). Folic acid xists in rducd form as 5-mthylttrahydrofolic acid (5-MTHF) or its polyglutamat drivativs (3). Sugars and sugar alcohols ar lc troactiv in alkalin solution and wr analyzd using high 247

2 To rfrnc and countr lctrods Machind Kl-F blocks From column Tflon Working lctrod Spacr Fig. 1. A thin-layr ampromtric dtctor cll dsign. (Adaptd from Kissingr, P. T., rf. 8). prformanc anion-xchang chromatography (HPAE) with pulsd ampromtric dtction (PAD) (7, 14). Ragnts Matrials and Mthods L-ascorbic acid was obtaind from Aldrich Chmical Co. Inc. (Milwauk, WI). 5-mthylttrahydrofolic acid (5- MTHF, barium salt) was obtaind from Sigma Chmical Co. (St. Louis, MO) and usd without furthr purification. An stimatd 80% purity was calculatd from th absorbanc at 290 nm of a frshly prpard standard solution using a publishd molar absorptivity of 2.9 x 104 (11). Ra gnt grad anhydrous dxtros (D-glucos), D-fructos, sucros (Fishr Scintific, Wst Havn, CT) and myo-inositol (Sigma) wr drid undr vacuum at 65 C for no lss than 3 hours. Othr ragnts includd ttrabutylammonium dihydrogn phosphat (TBAP, 1.0 M solution, Aldrich), glacial actic acid, concntratd sodium hydro xid, and mta-phosphoric acid (Fishr), sodium actat, concntratd phosphoric acid (Mallinckrodt, Paris, KY), concntratd prchloric acid (J. T. Bakr, Inc., Phillipsburg, NJ), and thylndiamin ttraactic acid, disodium salt (Na2EDTA, Sigma). Sampls Juics from tn arly sason citrus cultivars wr hand xprssd from fruit pickd at th Florida Citrus Ar bortum (Wintr Havn, FL). Tn ml portions of juic sampls wr clarifid by cntrifuging at 10,000 RPM and 2 C for 15 min. Prparation of Standards and Sampls Ascorbic acid assay. A stock solution was prpard by wighing 50.0 mg of L-ascorbic acid into a 100 ml vol umtric flask and diluting to mark with th HPLC mobil phas. A working standard was prpard by diluting th stock solution 100-fold with th mobil phas. Sampls wr prpard by piptting 1 ml of clarifid juic into a 100 ml flask and bringing to mark with 0.04 M prchloric acid. Sampls wr filtrd through 0.45 xm nylon syring-typ filtrs prior to injction. Folat assay. A primary stock solution of 5-MTHF was prpard by dissolving approximatly 12.5 mg of th salt in 100 ml of 0.05 M phosphat/actat buffr (adjustd to 248 ph 7.2 with concntratd NaOH) containing 4.0 g ascorbic acid and 5.0 g mta-phosphoric acid. This was kpt frozn at-20 C util ndd. Scondary stock solutions of approx imatly 1.25 jxg ml1 folat and 400 jig ml1 ascorbat wr prpard frsh daily by dilution of th primary stock with HPLC mobil phas A. On ml of ach clarifid juic was pipttd into sparat 1.5 ml microcntrifug tubs. Th ph was adjustd to about 5.0 using 1.0 M NaOH. This was followd by th addition of 0.2 ml of a conjugas nzym suspnsion, ncssary to dconjugat highr-ordr polyglutamat forms to 5-MTHF monoglutamat (3). Th sampls wr incubatd in a watr bath at 37 C for 90 min, coold rapidly on ic, thn cntrifugd at 10,000 RPM for 5 min. Th sampls wr finally filtrd through 0.45 xm nylon syring-typ filtrs and transfrrd to ambr autosamplr vials of 300 fil capacity for injction. Sampls wr kpt cold and shildd from light prior to injction. Sugar and sugar alcohol assay. A primary stock solution containing sucros, glucos, and fructos in a ratio 2:1:1 (approximatly 10% W/V total sugar) and myo-inositol (0.2% W/V) was prpard by wighing th drid solids into a 100 ml volumtric flask and diluting to volum with HPLC grad watr. This was kpt frozn until us. Juic sampls wr prpard by 100-fold dilution with HPLC grad watr. Th standard sugar mixtur was tratd in an idntical mannr. All sampls and standards wr fil trd through 0.2 xm nylon syring-typ filtrs prior to injction. HPLC Mthods Gnral Th HPLC systm consistd of Watrs (Milford, MA) Modl 510 and Modl 6000 LC pumps, a Modl 721 systm controllr, and Modl 710B WISP Autosam plr. Th EC dtctor was an E.G.&G. Princton Applid Rsarch Modl 400 (Princton, NJ). Data wr acquird with a 20-bit A/D convrtr (CSI Modl 160S, Autochrom Inc., Milford, MA) at a rat of no lss than 2 Hz. Pak hight or ara calculation was prformd using APEX Chromatography Softwar (Autochrom, Inc.) with th aid of a 286 AT-styl computr (CompuAdd Corp., Austin, TX). Ascorbic acid assay. Th mobil phas was prpard by dissolving 8.2 g of sodium actat and 150 mg of disodium EDTA with approximatly 500 ml of HPLC grad watr in a larg bakr. Th ph was adjustd to 5.0 with actic acid. This solution was thn transfrrd into a 1 L volumt ric flask and dilutd to mark with HPLC grad watr. Th solvnt was filtrd through a 0.45 xm filtr and dgassd by sparging with hlium. Th analytical column was a ZORBAX ODS (DuPont, Wilmington, DE), 4.6 mm x 25 cm, with 5 fjim packing. Th mobil phas flow rat was 0.8 ml min1. Injction volums wr 20 xl. Extrnal standard calibration was mad within vry four sampl injctions. Sparation was at ambint tmpratur. Th d tctor was quippd with a glassy carbon lctrod opr atd at mv (vs. Ag;AgCl, 3M NaCl) with th sn sitivity at 100 ia full scal. Folat assay. Mobil phas A was 10% mthanol in 0.05 M phosphat/actat buffr (ph 5.0) containing M TBAP. Mobil phas B was idntical xcpt that it con taind 30% mthanol. Both solvnts wr filtrd through a 0.45 xm filtr and dgassd by sparging with hlium. Sparations wr prformd at ambint tmpratur. A 10-port switching valv (Valco, Houston, TX) was confi-

3 gurd for sampl clan-up and backflush; its us is d scribd in dtail lswhr (20). A UV dtctor (Watrs Modl 441, 254 nm, snsitivity = 2.0 AUFS) was mployd to dtct unrtaind ascorbat as it lutd from th prcolumn. Th EC dtctor was quippd with a glassy car bon lctrod opratd at +200 mv (vs. Ag;AgCl) with th snsitivity at 10 na full scal. Th prcolumn was a Watrs Nova-Pak C18, 3.9 x 75 mm, with 4 xm packing. Th flow rat was 1.0 ml min1 and injction volums wr 20 (xl. Extrnal standard calibration was mad within vry four sampl injctions. Data wr corrctd for th purity of th standard and dilution which accompanid addition of nzym. Sugar and sugar alcohol assay. Sodium hydroxid (50% W/W) was dilutd to 0.14 M (ph 13) with filtrd (0.45 jxm) HPLC grad carbonat-fr watr and usd as th mobil phas. Car was takn to xclud carbon dioxid. Anion-xchang sparation was prformd with a Dionx (Sunnyval, CA) CarboPac PA1 column (4 x 250 mm) at ambint tmpraturs (23 ± 2 C). No guard column was usd in ordr to minimiz band sprading. Injction vol ums wr 20 xl. Extrnal standard calibration was mad within vry four sampl injctions. Tubing and fittings wr stainlss stl. Th pumping rat was 1.0 ml min-1. Th dtctor was quippd with a singl gold working lc trod and opratd in th puls mod at potntials (vs. Ag;AgCl): El = +50 mv (167 ms), E2 = mv (167 ms), and E3 = -950 mv (500 ms). Snsitivity was st at 100 xa full-scal and currnt was sampld during El. Rsults and Discussion Vitamin C is on of th principal nutritional compo nnts of citrus and many consumrs rly on citrus juic as a primary sourc of this vitamin. Thus, it is important that an ascorbic aid assay of citrus products b carrid out rg ularly. Figur 2 is an ovrlay of typical chromatograms obtaind using th dscribd mthod. Th analysis is com plt in undr four minuts. Som tailing is vidnt, how- Dtctor Rspons (MA) vr no attmpt was mad hr to improv th sparation; quantitation was satisfactorily prformd by by masuring pak hight. Analysis of svral frsh and procssd juics in our laboratory (2) gav a rang of ascorbic acid valus from 43.1 mg to 61.2 mg pr 100 ml of juic, agring wll with litratur valus. Rpatability is stimatd to b ±0.5 mg basd on rplicat injctions, corrsponding to a cofficint of variation of about 1%. Standard calibration curvs constructd by succssiv dilution of th standard wr linar (R2 = ) ovr th rang 12 to 100 mg pr 100 ml juic. Folats ar nutritionally ssntial du to thir rol as conzyms in th biosynthsis of nuclic acids, amino acids and protins. Citrus juic, in particular orang juic, has bn rportd to b a good sourc of ditary folat (6, 16). Microbiological assay using L. casi (5) has bn m ployd almost xclusivly for quantitating folat in citrus juics. Unfortunatly, th disparat growth rspons of this microorganism to polyglutamat forms (glun>3) (17) raiss doubts as to th rliability of th rsults. It was dtrmind arlir (19) that ascorbat was th main obstacl to slctiv dtction of 5-MTHF in citrus juic whn using HPLC with ampromtric dtction (E = mv vs. Ag;AgCl). It was thrfor ncssary to r duc th lvl of ascorbat in th sampls by using a clanup procdur. A 10-port switching valv nabld this xtraction to b carrid out automatically. At ph 5, 5- MTHF is ssntially anionic and th us of an ion-pairing ragnt (TBAP) in mobil phas A nhancs its rtntion on a C18 column (13). A small prcntag of mthanol (10%) facilitatd th washing of lss tightly bound, and possibly intrfring, compounds (such as ascorbat) through th prcolumn whil 5-MTHF was rtaind. In crasing th mthanol contnt to 30% in mobil phas B providd sufficint solvnt strngth to lut 5-MTHF. Ion-pairing ragnt (TBAP) was also addd to mobil phas B in ordr to prsrv th intgrity of th "ionpairs", othrwis pak shap dtrioration was notd. Fi gur 3 is a typical chromatogram obtaind by using th switching tchniqu. Th larg band which luts about 2.5 min aftr column switching is probably rsidual ascor bat, and prhaps othr unrtaind compounds lctroactiv at mv. Paks wr usually symmtrical in shap and pak hight was usd for th calculations. A sris of E C std L-ascorbic acid L-ascorbic acid in OJ 5-MTHF D t c t o r R Minuts Fig. 2. Rvrsd-phas HPLC/EC chromatogram of standard L-ascor bic acid and L-ascorbic acid in orang juic. Fig. 3. Rvrsd-phas HPLC/EC chromatogram of standard 5- MTHF in an ascorbat solution by dirct-injction with column-switch ing. Chromatographic conditions dscribd in txt. 249

4 standard dilutions ovr th rang 0.25 to xg ml' rsultd in a linar dtctor rspons, (R2 =.09992). In ordr to stimat th rproducibility of th HPLC mthod, rptitiv analyss (N = 14) of a singl unpasturizd juic sampl (12 Brix) wr carrid out. Rsults rangd from to xg ml1, with a man of fxg ml1 and a standard dviation of fxg ml1. This corrsponds to a cofficint of variation of about 2.8%. Ovr 100 commrcial juics hav bn analyzd for folat using th mthod (20). HPLC rsults wr gnrally highr than thos obtaind by microbiological assay. Thr ar two rasons why highr rsults ar to b xpctd whn using th HPLC mthod. First, no conjugas nzym trat mnt of juic sampls was mployd prior to th micro biological assay. Tamura (17) has shown that th rspons of L. casi to folats is dpndnt on th polyglutamyl chain lngth, with poorr rspons licitd for folat compounds with gratr than thr glutamic acid rsidus. Th highr lvls of 5-MTHF monoglutamat following th nzym tratmnt, confirmd by th HPLC analyss (19, 20), lnds support to arlir vidnc that polyglutamat forms ar prsnt in citrus juics (15). Scondly, calibration of th microassay mthod was prformd using th oxidizd folk acid monoglutamat (ptroylglutamic acid) rathr than th rducd 5-mthyl substitutd drivativ. Thr is vidnc that L. casi rsponds bttr to th formr, a factor which would undoubtdly lad to undrstimation of th lvl of 5-MTHF in th juic sampls (4). Th HPLC/EC mthod was usd hr to analyz th folat contnt of frshsquzd juic from tn arly-sason citrus cultivars. R sults ar givn in Tabl 1. Analysis of citrus fruits for carbohydrats is of spcial intrst sinc varital, gographic, sasonal, and maturity diffrncs can dramatically affct th composition. Myoinositol (i-inositol or hxahydroxycydohxan) is a com monly occurring sugar alcohol widly distributd in both plants and animals. Its discovry in citrus juic (12) and subsqunt quantitation (10, 22) showd that citrus was a rich sourc of this compound. In spit of its potntial im portanc to th citrus industry both from nutritional and conomic prspctivs, th dvlopmnt of suitabl mthodology for its routin analysis has largly bn ig nord. Sinc carbohydrats xhibit only wak absorbanc in th UV-Vis rgion, rfractiv indx (RI) has bn th con vntional mans of dtction aftr HPLC sparation. How vr, as a masur of a bulk proprty of solution, RI dos not offr slctivity for sugars ovr othr compounds. Th Dtctor Rspons (fj-a) myo-inositol glu ORANGE JUICE STANDARD su Minuts Fig. 4. HPAE/PAD chromatograms of sugars and sugar alcohols in orang juic and in standard solution. HPAE/PAD mthod is particularly wll-suitd for th analysis of complx sampls such as fruit juics which con tain componnts that may intrfr with RI dtction. Fi gur 4 illustrats th chromatogram obtaind undr th dscribd conditions. Analysis tim was undr 10 min. Multi-lvl calibration using fiv succssiv dilutions of th standard sugar mixtur xhibitd linar rsponss for th thr sugars and myo-inositol. Sucros data fit slightly bttr to a quadratic quation. Th standard dviations of th mthod wr calculatd from rplicat injctions (n > Tabl 1. D-glucos, D-fructos, sucros, myo-inositol, and folk acid in frsh-squzd juic from 10 arly sason citrus cultivars. Cultivar glucos (%W/V) fructos myo-inositol %W/V) 5-MTHF Barss lmon Prsian lim Pag orang Hamlin orang Orlando tanglo Minnola tanglo Robinson tangrin Star Ruby grapfruit Duncan grapfruit Marsh grapfruit 1.53 ±0.02* ± ± ± ± * rror stimats ar standard dviations basd on rplicat (n>5) analyss 250

5 5) of a standard sugar mix, similar in concntration to th sampls analyzd (21). Ths wr found to b 0.02, 0.04, 0.3, and %(W/V) for glucos, fructos, sucros, and myo-inositol rspctivly. Th HPAE/PAD mthod was usd to analyz th sugar contnt of frsh-squzd juic from tn arly-sason citrus cultivars. Rsults ar givn in Tabl 1. Conclusion HPLC with ampromtric dtction can b a usful tool with which to routinly analyz lctroactiv compounds in citrus juic. A simpl, rvrsd-phas, isocratic HPLC mthod was illustratd in which vitamin C analysis is com plt in lss than four minuts. An automatd columnswitching tchniqu allows th dtrmination of citrus juic folat by dirct injction of filtrd juic into th HPLC systm. Tratmnt of juics with a conjugas nzym prior to analysis is rcommndd for quantitating total folat. Analysis is complt within ight minuts. A high prform anc anion-xchang mthod with pulsd ampromtric dtction can b usd routinly to quantitat glucos, fruc tos, and sucros, as wll as th sugar alcohol, myo-inositol. Run tims ar lss than tn minuts. Th ampromtric dtction systm was found to b ruggd and xtrmly slctiv, providing suprior sn sitivity whr ndd. Th mthods illustratd ar simpl and rquir no sampl prparation othr than dilution and filtration. Litratur Citd 1. E.G.&G. Princton Applid Rsarch Dtrmination of ascor bic acid in fruit juic. Application Not: ECD FDOC Unpublishd data, Stat of Florida Dpartmnt of Citrus, Lak Alfrd, FL. 3. Grgory, J. F., Sartain, D. B., Day, B.P.F Fluoromtric dtr mination of folacin in biological matrials using high prformanc liquid chromatography. J. Nutr. 114: Hawks, J. G. and Villota, R Folats in foods: ractivity, stabil ity during procssing, and nutritional implications. Critical Rviws in Food Scinc and Nutrition. 28: Hrbrt, V Asptic addition mthod for Lactobacillus casi assay of folat activity in human srum. J. Clin. Pathol. 19: Hill, E. C. and Attaway, J. A Folic acid an ssntial vitamin prsnt in citrus fruit. Proc. Fla. Stat Hort. Soc. 84: Hughs, S. and Johnson, D. C High-prformanc liquid chromatographic sparation with tripl-puls ampromtric dtc tion of carbohydrats in bvrags. J. Agric. Food Chm. 30: Kissingr, P. T Ampromtric and coulomtric dtctors for high-prformanc liquid chromatography. Anal. Chm. 49:447A- 456A. 9. Krhl, W. A Th Rol of Citrus Fruits in Halth and Disas. Univrsity Prsss of Florida, Gainsvill, FL, 118 pp. 10. Krhl, W. A. and Cowgill, G. R Vitamin contnt of citrus products. Food Rs. 15: Larrab, A. R., Rosnthal, S., Cathou, R. E. Buchanan, J. M A mthylatd drivativ of ttrahydofolat as an intrmdiat of mthionin biosynthsis. J. Am. Chm. Soc. 83: Nlson, E. K. and Knan, G. L I-inositol in citrus fruits. Scinc 77: Rbllo, T Trac nrichmnt of biological folats on solidphas adsorption cartridgs and analysis by high-prssur liquid chromatography. Anal. Biochm. 166: Rocklin, R. D. and Pohl, C. A Dtrmination of carbohydrats by anion xchang chromatography with pulsd ampromtric d tction. J. Liquid Chromatogr. 6: Stokstad, E.L.R., Shin, Y. S., Tamura, T Distribution of folat forms in food and folat availability. In Folic acid: Biochmistry and Physiology in Rlation to th Human Nutrition Rquirmnt, Food and Nutrition Board, National Rsarch Council, NAS publication, Washington, D.C. 293 pp. 16. Striff, R Folat lvls in citrus and othr juics. Amr. J. Clin. Nutr. 24: Tamura, T., Shin, Y. S., Williams, M. A., Stokstad, E.L.R Lactobacillus casi rspons to ptroylpolyglutamats. Anal. Biochm. 49: Ting, S. V Nutrints and nutrition in citrus fruits. In Citrus Nutrition and Quality (Nagy, S., Attaway, J.A. ds.). ACS Sym posium Sris. Washington, DC. pp Whit, D. R., Dtrmination of 5-mthylttrahydrofolat in citrus juic by rvrsd-phas high-prformanc liquid chromatog raphy with lctrochmical dtction. J. Agric. Food. Chm.38: Whit, D. R., L, H. S., Krugr, R Rvrsd-phas HPLC/EC dtrmination of folat in citrus juic by dirct injction with col umn-switching. J. Agric. Food. Chm., (in prss). 21. Whit, D. R., Widmr, W. W Application of high-prform anc anion-xchang chromatography with pulsd ampromtric dtction to sugar analysis in citrus juics. J. Agric. Food Chm. 38: Wolford, R. W Inositol, a chmical constitunt of citrus fruits. Sunshin Stat Agricultural Rsarch Rport. (Octobr) pp Proc. Fla. Stat Hort. Soc. 103: MODELS FOR SEASONAL CHANGES IN BRIX AND RATIO OF CITRUS FRUIT JUICE Additional indx words. C. S. Chn Citrus Rsarch and Education Cntr Univrsity of Florida, IFAS 700 Exprimnt Station Road Lak Alfrd, FL Orangs. Forcasting mthod. Abstract. Sasonal changs in Brix and Brix/% acid ratio of Florida arly sason orangs and California 'Washington Navl' orangs wr fittd by linar modls. Th constant trm varid with diffrnt climatic conditions. Th rat of Florida Agricultural Exprimnt Station Journal Sris No. N Froc. Fla. Stat Hort. Soc. 103: chang was Brix/day and ratio/day for Florida arly sason orangs and ratio/day for California "Washington Navl" orangs, rspctivly. An application of th modls for arly prdiction of changs in Brix and Brix/% acid ratio throughout th harvsting priod was dvlopd. Producing high quality products is a common goal of both citrus growrs and procssors. High quality procssd citrus juics must com from high quality fruit. Tradition ally, producing high quality fruit is a rsponsibility of growrs. Fruit quality varis with varitis and stags of maturity as wll as othr factors throughout th sason. Th procssors rciv whatvr fruit is availabl as it ma turs throughout th sason and procss this into juic 251