Comparison of High-Performance Ion Chromatography and absorptiometric methods for the determination of phytic acid in food samples

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1 Anlusis, 1998, 26, EDP Sciences, Wiley-VCH Comprison of High-Performnce Ion Chromtogrphy nd bsorptiometric methods for the determintion of phytic cid in food smples P. Tlmond 1, *, G. Gllon 2, J.P. Guyot 1, I. Mbome Lpe 3 nd S. Trèche 2 1 Lbortoire de Biotechnologie Microbienne Tropicle, Centre ORSTOM de Montpellier, BP. 5045, 911 venue d Agropolis, Montpellier Cedex, Frnce 2 Lbortoire de Nutrition Tropicle, Centre ORSTOM de Montpellier, BP. 5045, 911 venue d Agropolis, Montpellier Cedex, Frnce 3 Food nd Nutrition Reserch Centre, P.O. Box 6163, Youndé, Cmeroon Abstrct. The objective of this pper consists in defining the interest of new high-performnce ion chromtogrphy method (HPIC) with chemiclly suppressed conductivity detector for phytic cid determintion in food smples. Firsly, ccurcy nd precision of the HPIC method hve been mesured. Secondly, The HPIC method nd clssicl bsorptiometric method were compred. The HPIC method ws more sensitive nd selective thn the bsorptiometric method which leded to n 27% overestimtion of the phytic cid content in legume seeds. Becuse of it is rpid nd esy to perform, the HPIC method ppers to be prticulrly suitble for routine nlysis of food smples. Key words. Phytic cid liquid chromtogrphy bsorptiometry food nlysis. Introduction Phytic cid, myo-inositolhexphosphte (Fig. 1), is common constituent of mny plnt foods [1].This molecule is highly chrged with six phosphte groups extending from the centrl inositol ring structure [2] nd therefore is n excellent cheltor of minerl ions [3]. Since phytte cnnot be bsorbed nd humns hve limited cpcity for hydrolysing the phytte molecule, negtive effect of phytic cid on minerl biovilbility cn be expected [4]. There hve been numerous reports documenting negtive effect of phytic cid on the bsorption of zinc, iron, copper nd clcium [5,6]. Fig. 1. Structure of Inositol 1,2,3,4,5,6-hexphosphte (or Phytic Acid). The clssic methods for determintion of phytic cid derives from the method of Heubner nd Stdler [7]. These methods re bsed on the precipittion of ferric ion with phytte in dilute cid solution nd nlysis of phosphorus or iron in the precipitte [8,9]. One of the disdvntges of the iron precipittion methods is tht not only inositol hexphosphte is precipitted, but other phosphorus-contining compounds ble to precipitte with ferric ion (i.e. inositol pentphosphte nd inositol tetrphosphte) s well [10]. Becuse of these disdvntges, HPLC methods were developed to improve the determintion of phytic cid [11]. However, smple extrcts must be first prepurified by pssing through n nion-exchnge resin to remove inorgnic phosphte nd concentrte inositol [12]. The first systems incorported refrctive index detection coupled with reversed-phse seprtion [13-17]. Spectrophotometric detection with post-column derivtiztion ws lter introduced to improve the nlysis. The bsence of chromophoric functionl groups within inositol hs led to the development of methods bsed on post-column derivtiztion nd spectrophotometric detection. Despite high sensitivity of detection, the dditionl derivtiztion rection constitutes potentil source of error nd is lso time-consuming. Rounds nd Nielsen [18] hve improved the seprtion by replcing the reversed-phse column by n nion-exchnge one, thus suppressing the prepurifiction step. However, post-derivtiztion ws still needed. A new method hs been recently proposed by our lbortory bsed on high performnce ion chromtogrphy (HPIC) following chemiclly suppressed conductivity detection [19,20]. This method does not require ny prepurifiction nd derivtiztion steps. The objective of our study ws the comprison of this HPIC method to the clssicl bsorptiometric method. * Correspondence nd reprints. Received My 26, 1998; revised August 25, 1998; ccepted September 8, Article vilble t or 396

2 Mterils nd methods Anlyticl instruments Beckmn (Fullerton, USA) DU 70 spectrophotometer ws used for colorimetric determintion. High Performnce Ion Chromtogrphy (HPIC) nlyses were performed with 4500i Dionex (Sunnyvle, USA) liquid chromtogrph equipped with n eluent delivery pump, n utoinjector nd using chemiclly suppressed conductivity. A 200 µl constnt volume injection loop ws used throughout. A centrifugl evportor Joun (Frnce) RC10.10 fitted with refrigerted trp cooled t 60 C ws used for smple preprtion. Smple solutions were diluted with Gilson (Middleton, USA) semi-utomtic dilutor 401 prior to injection. Regents nd solutions All chemicls used were of nlyticl grde nd deionized wter ws used for prepring the regent solutions. Deionized wter ws purified by Millipore ultr pure system to specific resistnce of 18 mω cm or greter. Sodium phytte (Sigm Ref. 3168) ws used for the preprtion of stndrd phytic cid solutions. Iron solution (50 µg ml 1 ) 625 µl of concentrted HNO 3 ws dded to 25 ml of commercil stndrd iron solution (Fe 3+ 1 g L 1, Titrisol, Merck, Ref. 9972) nd completed to 500 ml deionized wter to give the finl solution (50 µg ml 1 ). HPIC regents The mobile phse ws mixture of three solutions A, B nd C. Solution A ws prepred by dding 10.4 ml NOH (commercil solution t 50% (w/v) in wter, Bker) in wter (finl volume: 1 L). Solution B ws deionized wter:isopropnol 50:50 (v/v). Solution C ws deionized wter. Regenerted solution of nion suppressor ws 50 mmol L 1 sulphuric cid solution. All the mobile phses were pssed through 0.45 µm filter nd degssed before use. Smple preprtion Two flours were prepred from seeds of cowpe (Vign unguicult spp.) nd millet (Pennisetum spp.) vrieties cultivted in Senegl. As germintion reduces phytte content of legume seeds nd is likely to increse inositol pent or tetrphosphte, flour from germinted cowpe seeds ws lso prepred by first soking them in wter for 24 h nd keeping them wet t 28 C during 48 h sprouting. Absorptiometric method Triplicte smples (0.5 g) of the freeze-dried, finely-ground products were extrcted with 20 ml 0.5 mol L 1 HNO 3 for 3 4 h with continuous shking. After filtering, phytte content determintion ws performed on the filtrte by modifiction of the Holt s method (1955) [9]. The modified Holt procedure (1955) [8] dopted routinely in our lbortories for phytte contents determintion ws s follows: ml of the filtrte or stndrd sodium phytte solution (0.2 mmol L 1 ) ws diluted with distilled wter to finl volume of 1.4 ml. Then, 1.0 ml of solution of ferric solution contining 50 µg ml 1 Fe 3+ ws dded. After mixing, the test-tubes were cpped, plced in boiling wter-bth for 20 min, nd cooled to room temperture. Five ml of myl lcohol ws dded to ech test-tube followed by 0.1 ml of solution of mmonium thiocynte (100 g L 1 ). The use of myl lcohol recommended by Dvid nd Reid [9] considerly improved the precision of the ssy due to n increse in slope of the ssy curve. The contents of the test-tubes were immeditely mixed nd centrifuged t 4000 g for 5 min. The intensity of the colour in the myl lyer ws determined t 465 nm using spectrophotometer ginst n myl lcohol blnk, exctly 15 min fter the ddition of NH 4 SCN. Under these conditions n inverse reltionship ws found over rnge of phytte concentrtions from mmol L 1 to mmol L 1. HPIC method Phytte extrction The flour smple (0.2 g) ws introduced in Pyrex vil fitted with PTFE screw-cp. Ten ml of 0.5 mol L 1 HCl ws dded nd the vil ws cpped. The mixture ws heted under stirring for 5 min by immersing the vil in boiling wter. It ws then centrifuged t 4000 g for 10 min. The superntnt ws recovered nd 1.5 ml of 12 mol L 1 HCl were dded to obtin 2 mol L 1 HCl concentrtion in order to ensure the decomplextion of phyttes. This procedure ws found to llow the best extrction conditions.the resulting solution ws then shken nd evported to dryness in centrifugl evportor. The vil ws finlly stored t 8 C. Ten min before the injection, the residue ws resuspended in 1 ml of deionized wter nd pssed through 0.2 µm disposble filter (Acrodisc) tip-syringe ssembly. The filtrte ws then diluted in deionized wter (1/50) nd injected into the liquid chromtogrph. Chromtogrphic conditions The seprtion ws crried out on n Omnipc Px-100 nion-exchnge column (25 cm 4 mm I.D., Dionex, Sunnyvle, C.A., USA) equipped with n Omnipc Px-100 (8 µm) precolumn nd n nion suppressor (ASRS-I 4mm). Ech eluent of the mobile phse ws previously degssed in n ultrsonic bth nd then introduced in the eluent delivery system under helium pressure. The seprtion ws performed by grdient elution using 3 eluents: solvent A = 200 mmol L 1 NOH solution; solvent B = deionized Wter-Isopropnol (1:1, v/v); solvent C = deionized wter. After severl ssys, the grdient elution progrmme, shown in tble I, ws selected with totl chromtogrphy run time of 15 min. The nion suppressor ws continully regenerted with 50 mmol L 1 sulphuric cid solution. Severl solutions of phytic cid with concentrtions from 0.01 mmol L 1 to 0.16 mmol L 1 were prepred from stndrd solution for determintion by clibrtion. 397

3 Tble I. Grdient elution progrm for the seprtion of phytic cid. Elution Flow rte %A %B %C time (min) (ml/min) Sttisticl nlysis The nlysis of the vrince (One wy ANOVA) ws crried out using the Sigmstt Softwre (Jndel) ccording to the conventionl procedures. Fig. 2. Elution profile of phytic cid stndrd. Seprtion of phytic cid on n Omnipc Px-100 column; eluents: 200 mm NOH, Wter-Isopropnol (1:1, v/v) nd Wter (18 mω); detection: chemiclly suppressed conductivity using nd ASRS-I 4 mm). Results nd discussion Firstly, we tested the linerity, ccurcy, precision nd reproducibility of the HPIC method. Secondly, we compred the results obtined on the smples by the bsorptiometric nd HPIC methods. Phytic cid with concentrtions rnging from 0.01 mmol L 1 to 0.16 mmol L 1 ws nlysed on n nion exchnge column with chemiclly suppressed conductivity detector using three solvents mixture for grdient elution. The more phosphte group were retined on the column. The elution of phytic cid ws ttined by 54 55% 200 mmol L 1 NOH solution. An nion micromembrne suppressor (AMMS) ws pplied with conductivity detection. This suppressor ws continully regenerted with 50 mmol L -1 sulfurid cid in order to neutrlize. The use of suppressor reduces the conductivity of eluent nd enhnces the conductivity of the nlytes. The retention time of phytte ws 6.0 ± 0.2 min with no dy-to-dy vrition over 5-month period (Fig. 2). The re under the conductivity pek is proportionl to the phytic cid concentrtion over the entire rnge nd correltion coefficients obtined re shown in tble II. In order to determine whether b (y = x + b) ws significntly different from 0 or not, the hypothesis b = 0 [21] ws tested. As the test ws ccepted, the clibrtion curves pssing through zero cn be used (y = x, Tb. II). The vrition coefficient of the instrument precision s clculted with the vlues of six injections of phytic cid ws 0.57%. To determine the method reproducibility, six different smples from the sme btch of ungerminted cowpe (flour) were repetedly nlysed dily for four dys. Precision for replicte injection (n = 24) ws 5% [reltive stndrd devition (R.S.D.) of repetbility] nd 7% (R.S.D. of reproducibility). The HPIC method llows the quntittion of phytic cid down to 0.1 µmol L 1. The signl-to-noise rtio ws higher thn 10: the limit of detection ws therefore less thn µmol L 1. Recoveries were determined by crrying out five extrctions of the smple with incresing stndrd dditions of phytic cid solution (33%, 50%, 66%, 100% of the initil Tble II. Sttisticl clcultions on the regression between detector response (pek re) nd phytte concentrtion. y = ( ± s)x+(b ± s) Result y = ( ± s)x R 2 1 ± s (13592 ± 157)x+( ± ) NS (13180 ± 106)x (14194 ± 164)x+( ± ) NS (14317 ± 109)x (13937 ± 127)x+( ± ) NS (13908 ± 78)x (14739 ± 109)x+(2.767 ± 6.193) NS (14525 ± 97)x m = ± 98 s: Stndrd devition. : Significnce of the difference between b nd the null hypothesis (b = 0). NS: non significnt. R 2 : Correltion coefficient. m: Averge vlue of four slopes. concentrtion) nd rnged between 100% nd 105%. Recoveries re independent of the mount of phytic cid pplied, s is demonstrted by the linerity of the curve (R 2 = 0.98). Mny workers [13-17] hve determined phytic cid by using the reversed phse procedure in ssocition with ionpir regents. Nevertheless, pure reversed phse procedures showed only poor seprtions becuse the compounds were hrdly retined on the sttionry phse. Moreover, prepurifiction step with polystyrene-bsed strong nion exchnge column prevents the potentil problem of pcking mteril deteriortion, which could be encountered during prolonged use of silic-bsed column with the mobilephse used, nd concentrted the inositol phosphtes. This step is time-consuming nd prevented its use in routine. In greement with Tngenjj et l. [11], Grf nd Dintzis [13] nd Rounds nd Nielsen [18], we observed with this HPLC methods two min disdvntges: poor seprtion nd low sensitivity in the detection system (refrctometric index). For these resons, we decided to compre the HPIC method with 398

4 Tble III. Accurcy nd precision of methods (bsorptiometric nd HPIC). Colorimetric Method HPIC Method Phytte D.O Precision b Phytte Pek re Precision b (mmol L 1 ) 465 nm (mmol L -1 ) (µs) ± % ± % ± % ± % ± % ± % ± % ± % ± % ± % Men nd Stndrd Devition of five stndrds. b Precision = ± (t sx)/x. x: men of 5 dt. sx: s/5_. t: the clssicl bsorptiometric method for use in our lbortory. We studied the two methods to determine their precision nd ccurcy for the determintion of phytic cid nd to select the most pproprite experimentl protocol. Ech smple of phytte stndrd solutions ws prepred nd nlyzed in five replicte with HPIC nd bsorptiometric methods. The dt of men, stndrd devition nd precision by ech method re shown in tble III. The HPIC method ws clerly more ccurte thn the bsorptiometric method nd could be directly pplied to smples without prepurifiction. Severl kinds of cerels were extrcted nd phytte ws quntified by the HPIC method nd by the conventionl bsorptiometric method. Dt obtined by the bsorptiometric method, regrdless of the cerel flour used, were systemticlly higher by bout 27% thn those of the HPIC method (Tb. IV). This result is in greement with those of other uthors [10,11,14-17] who obtined higher vlues with the bsorptiometric method thn reversed-phse method derives. This difference between the chromtogrphic nd bsorptiometric methods is not suprising in view of the fct tht tretment of the extrcts with the ferric solution: precipittes smll mounts of inorgnic phosphtes nd does not llow one to distinguish between different forms of phytte (prticulrly, pentphosphte (IP5), tetrphosphte (IP4)) [22]. The ferric precipittion method cnnot be used for the determintion of phytic cid in ll foods becuse the presence of interfering substnces, such s reducing compounds leds to overestimted results. Conclusion The use of nion exchnge chromtogrphy with conductivity detection ffords rpid seprtion of phytic cid without postcolumn derivtiztion. Moreover, this method does not require ny smple prepurifiction. Phytte contents cn be determined with more convenience, precision nd speed thn the bsorptiometric clssic method. With the HPIC method the number of pre-chromtogrphic smple tretment steps re decresed (cid extrction, centrifugtion nd evportion). This provides the Tble IV. Comprison of two methods for determining phytic cid in food smples. nlysis of phytic cid with n dditionl lterntive to the existing methods. References Phytic cid (% w/w) Smple Absorptiometric HPIC % of Method (A) Method (B) overestimtion b Millet soun NG ± ± Cowpe NG 1.32 ± ± Millet soun G 0.49 ± ± Men ± SD of three replicte smples. b % percent difference between the bsorptiometric nd the HPIC methods: 100 (B 100/A). 1. Posternk, S. C. R. Soc. Biol. 1903, 55, Johnson, L. F.; Tle, M. E. Cn. J. Chem. 1969, 47, Vohr, P.; Gry, G.; Krtzer, F. H. Proc. Soc. Expl. Biol. Med. 1965, 47, Besnçon, P. In: L limenttion de complément du jeune enfnt, Trèche, S.; de Benoist, B.; Benbouzid, D.; Verster, A.; Delpeuch, F. Eds., Orstom éditions, Pris, 1995; p Noln, B.; Duffin, P. A.; Mc Weeny, D. J. J. Agric. Food Chem. 1975, 23, Oberles, D. 2 nd Ed. Ntionl Acdemy of Science, Wshington DC, 1973; p Heubner, W.; Stdler, H. Biochem Z. 1914, 64, Holt, R. J. Sci. Food Agric. 1955, 6, Dvies, N. T.; Reid, H. Br. J. Nutr. 1979, 41, De Bolnd, A. R.; Grner, G. B.; O Dell, B. L. J. Agric. Food Chem. 1975, 23, Tngendjj, B.; Buckle, K. A.; Wootton, M. J. Chromtogr. 1980, 197, Cosgrove, D. J. Biochem J. 1963, 89, Grf, E.; Dintzis, F. R. J. Agric. Food Chem. 1982, 30,

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