Paper No. : 11 
       Paper Title: Food Analysis and Quality Control 
       Module-21: Analysis of anti-nutrients in foods 
        
       1.1 Methods for quantification of the antinutritional factors in foods 
       Antinutrients are found at some level in almost all foods for a variety of  reasons. They are 
       natural or synthetic compounds that interfere with the absorption of nutrients present in the food.  
       Although they not necessarily toxic per se, are plant compounds which decrease the nutritional 
       value of a plant food, usually by making an essential nutrient unavailable or indigestible when 
       consumed by humans/animals. Several methods are used for the quantitative determination of 
       anti-nutritional factors in foods based on reports by different authors. These are: trypsin inhibitor 
       activities are determined according to Liener (1979); haemagglutinatin, Jaffe (1979); cyanogenic 
       glucosides  (HCN),  Bradbury  et  al  (1999);  oxalates,  Fasset,  (1996);  phytates,  Maga  (1983); 
       tannin, Dawra et al. (1988); saponinn, Brunner (1984); and alkaloids, Henry (1973) etc. 
       There are other some new methods for quantification of antinutritional factors also due to recent 
       advances in the nutritional sciences. Principles of these methods are discussed as under. 
       1.1.1  Phytic acid 
       Several methods are available for determining phytic acid concentrations in products. There are 
       many papers that report different modifications to these methods, but the ideal methodology is yet 
       to be agreed upon. The existing methodology needs to be optimized and standardized. There are 
       many different techniques that can be used for the identification of phytic acid, but there are no 
       direct  methods.  There  are  no  specific reagents  that  detect  phytic  acid  or  its  various  forms. 
       Moreover, phytic acid does not have a characteristic absorption  spectrum in  the  UV  or  visible 
       light region. Most analytical methods are based on extraction or isolation of phytic acid. 
       Most conventional  quantitative methods  for phytate analysis  have  been  based  on  the 
       procedure of Heubner and Standler (1914). These  methods involve sample extraction with acid 
       and subsequent precipitation  of the   Fe (III)–phytate complex   following   addition    of 
       ferric  chloride.  Phytate   is estimated either  by  determining  the  phosphorus (McCance   and    
       Widdowson, 1935), Fe (Wheeler and  Ferrel, 1971) or inositol  (Oberleas, 1971) in the isolated 
       phytate  complex, or indirectly based on the determination of  the  residual  Fe  in  the  solution  
       after  precipitation  of  ferric  phytate    from  a  known  concentration  of  ferric  salt  in  an  acid 
       solution  (Young, 1936). Later,  it was established that  in addition  to inositol  hexaphosphate, 
       ferric  ion will also precipitate myoinositol  pentaphosphate  and tetraphosphate in a dilute acid 
        
        
       solution,  with the  amount  of IP5 and  IP4 of the  precipitate depending on  the  amount   and  
       composition of wash solution  (Oberleas, 1971; Frolich et al. 1986; Phillippy et al., 1986). Small 
       amounts  of  inorganic  phosphate  may  also  co-precipitate  (Ellis  et  al.,  1977).  As  the 
       stoichiometric  ratio  of  phosphorus  to  Fe  in  Fe  (III)–IP  precipitates  is  affected  by  several 
       variables, the  results are  unreliable. 
       Harland  and  Oberleas  (1977)  introduced  the  use  of  an  anion  exchange  resin  column.  
       Phytic  acid  was eluted from  the  column  separately from  the  lower  inositol phosphates  and   
       inorganic    phosphate   employing    a stepped gradient  system  and  quantified  by measuring 
       the   phosphate  released   after   acid   hydrolysis   of  the phytate  fractions.  Ellis and Morris 
       further modified the anion  exchange  column  stage of the method (Ellis and Morris, 1986) and 
       it was  accepted as  an official  method by the AOAC in1986 (Harland   and Oberleas, 1986). 
       The  method   of  Harland  and  Oberleas  (1977)  has  also  been  modified  by  other 
       workers.   Phytic  acid  content can  be  measured after  elution  from  the  anion  exchange 
       column   either   based   on   the   reaction  between   ferric chloride  and  sulfosalisylic  acid 
                          ¨ 
       (Wade  reagent) (Latta and Eskin, 1980 ; Fru Hbeck et al.,1995) or  formation of  the  phytate–o-
       hydroxyhydroquinone-  phtalein–Fe(III)  complex  (Fujita  et  al.,  1986).  In  the  Plaami  and 
       Kumpulainen‟s modification (1995) total phosphorus determination   of   phytic    acid,    after    
       either    anion exchange  column  or ferric precipitation, was performed by inductively  coupled 
       plasma  atomic  emission  spectrometry (ICP-AES). March  et al. (1995)  liberated  phosphorus  
       from  phytic  acid  after  anion  exchange  column by enzymatic  hydrolysis  and  measured it 
       spectrophotometrically,    according  to  the  method    of  Uppstro¨  m  and  Svensson    (1980).  
       However,  in the  method  of Uppstro¨ m and Svensson (1980), phytic acid was calculated  from 
       the difference   between   phosphorus  content   before   and after enzymatic  hydrolysis  of the 
       sample  without  using anion  exchange  separation. 
       The  AOAC  anion-exchange  method  is  one  that  has  been  used  to  estimate phytic  acid  content  in 
       products. The  results  of  the  AOAC method   and  the  method   of Latta  and  Eskin  (1980) 
       and  Fujita  et al. (1986)  agree  with those  of the  earlier  Fe  precipitation methods.  Later  it 
       was shown  that  the  concentration of  phytic  acid determined by  all  these  methods may  be 
       systematically overestimated   because    lower    inositol    phosphates (IP3-5)  and  adenosine 
       triphosphate (ATP),  if present, may be associated  with IP6 (Phillippy  et al., 1988; 36  Lehrfeld 
       and Morris, 1992). 
        
                  
                 Near-infrared spectroscopy methods for the determination of phytic acid have been developed 
                 by De  Boever et  al.  (De  Boever et al., 1994).  NMR  methods  are  capable   of  measuring 
                 phytic  acid  and  myoinositols  with  a  lower  number  of phosphate groups  (Frolich et al. 
                            ¨ et al., 1980
                 1986, Erso              ). 
                 Blatny  et  al.  (1995)  developed  a  method  in  which  myoinositol  hexaphosphate  was 
                 determined with iso- tacophoresis. De  Koning  (1994) determined phytic  acid in food  by gas 
                 liquid   chromatography.  The  early  HPLC  methods  were  capable  of  separation  and 
                 determination of IP6  only  (Camire  and Clydesdal, 1982, Lee and  Abendroth, 1983). Newer 
                 methods are  capable  of separating and  determining the  other IPs also.  HPLC and  detection 
                 methods are described. 
                 The high-performance liquid chromatography (HPLC) method is the primary means of separation and 
                 quantification. HPLC is capable of separating phytic acid and inositol phosphates as separate entities. It 
                 also has the sensitivity and reproducibility to measure low concentrations in products. However HPLC 
                 method is also not without its share of problems. The reagents used in this method must be pure and free 
                 from metals or it will cause distortion in the readings. There are many different  modifications to the 
                 HPLC  method.  The  most  common  are  the  use  of  different  columns,  mobile  phases,  flow  rates, 
                 extraction solvents, and preparation techniques. 
                 A  strong  anion  exchange  HPLC  column  has  been  used  by  Mathews  et  al.  (1988)  for 
                 separation  in  food analysis. Rounds  and   Nielsen   obtained  better  separation  and sharper   
                 peaks   in  plant,   food   and   soil  samples   by gradient  anion  exchange  HPLC  instead  of 
                 the  isocratic elution  used  by  Cilliers  and  Van  Niekerk   (1986).  The  use of  reverse phase 
                 columns in ion-pair chromatography has also been presented in several papers (Sandberg and 
                 Ahderinne, 1986; Sandberg et al., 1989; Lehrfeld, 1994; Rounds, and   Nielsen, 1993) with food, 
                 intestinal content and faeces samples. 
                 Methods for measuring  phytic  acid have  been  reviewed by  Oberleas  and  Harland  (1986),  
                 and  phytic  acid  and other  myoinositol  phosphates more  recently  by Xu  et al. (1992). For 
                 food  and  nutrition  studies,  methods  which  can  determine  different  IPs  separately  are  an 
                 appropriate choice. 
                 1.1.2  Analytical  techniques  used  in  the  determination  of  polyphenolic  compounds  from 
                 foods 
                 The most representative analytical methods mentioned in the literature for the separation and or 
                 quantification of polyphenolic compounds found in foods shall be discussed here. In the first 
                  
        
       place chromatographic techniques such as fine layer chromatography, gas, and in particular high-
       performance liquid chromatography used for the determination of polyphenolic compounds shall 
       be discussed. 
       1.1.2.1 Thin Layer Chromatography (TLC) 
       Before the onset of chromatography, the analysis of polyphenolic compounds was an extremely 
       tedious  task  and  perhaps  the  most  difficult  endeavor  for  those  responsible  for  analytical 
       determination.  The  birth  of  paper  chromatography  revolutionized  the  analysis  of  organic 
       substances, and during the 1950s and 1960s paper chromatography was widely used for the 
       determination of polyphenolic compounds, especially when applied for flavonoids determination 
       (Robards and  Antolovich, 1997).  
       In no time paper chromatography was substituted by thin layer chromatography (TLC). It was 
       considered  a  very  simple  and  cheap  technique  that  offered  great  versatility  with  respect  to 
       simultaneous qualitative analysis of polyphenolic compounds in distinct samples through the 
       employment of adequate absorbents and specific reagents. The choice of stationary phase as well 
       as an adequate solvent depends on the studied polyphenolic structures. Consequently, the most 
       hydrophilic  flavonoids  were  separated  with  TLC  by  employing  stationary  phases  such  as 
       polyamide and microcrystaline cellulose. On the other hand, a classical stationary phase made of 
       silicone  gel  has  been  used  widely  to  separate  more  apolar  flavonoids  such  as  flavons  and 
       isoflavonoids.  Likewise,  this  technique  has  numerous  applications  in  the  analysis  of 
       anthocyanins  as  confirmed  by  many  bibliographical  pilot  studies.  The  detection,  as  is  well 
       known,  is  carried  out  by  close  inspection  of  migratory  spot  under  the  ultraviolet  light. 
       Furthermore, in the current chemical arsenal we dispose of an array of specific reagents that can 
       be applied to each compound, previously separated on the plate. Therefore, for the sake of an 
       example we may cite aluminum chloride, boron hydride, sodium, 190 and vanillin193 as the 
       most common reagents employed in TLC. Inasmuch as that, based on the ensuing reaction and in 
       virtue  of  the  generated  color,  it  is  possible  to  accomplish  identification  of  determined 
       compounds, or at least the involved species of polyphenolic family. Thus, for example, while 
       flavonoles and flavanones do not react with vanillin and HCl in the methanol medium, these 
       reagents nonetheless are capable of reducing flavanones giving off a red or violet color that 
       intensifies throughout reaction, allowing the identification of individual species from a complex 
       polyphenolic environment.