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					Purification, Analysis, and Structures
  of Avenanthramides from Oats:
 Unique Components for Targeted
      Nutraceutical Applications
               F. William Collins
   Eastern Cereals and Oilseeds Research Centre
                    Ottawa, ON.
       8th IOC Minneapolis, MN, June 2008
2   Purification, Analysis, and Structures
       of Avenanthramides from Oats
                             OVERVIEW
      Need for improved methods of analysis
      Group separation for reproducibility
      Co-extracted compounds and
          their effects on quantitation
      UV Spectral properties of avenanthramides
      2-D HPLC-MSMS techniques to map total
            avenanthramide diversity
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3
    NEED FOR IMPROVED METHODS

 Avenanthramide components are complex
         mixture of over 30 individual members
 Several other groups of compounds in oat
         extracts can interfere with UV-based HPLC
         estimation of avenanthramides
 Bioactivity and bioavailability studies leading to
         possible health claims for avenanthramides
         in future oat products will require more
         accurate/robust analytical techniques
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4   Purification, Analysis, and Structures
       of Avenanthramides from Oats
            SUMMARY OF PROCEDURE
1. Stabilization and extraction with aqueous EtOH
2. Removal of lipophilic phenolics and prolamines
  using hydrophobic binding to Octyl Sepharose
3. Removal of flavonoids glycosides and avenacins
   using aromatic absorption on Sephadex LH-20
4. Concentration of avenanthramides

5. HPLC-PDA UV spectrometric standardization
    and quantization using avenanthramide A
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5            THERE ARE TWO TYPES OF
             AVENANTHRAMIDES IN OATS…

             4                                       1.    Substitution with
                                                           hydroxyl and or
     5             3       O        7'                     methoxy functions
                                          2'
                                                           at C-4 and/or C-5
     6                                         3'
                       N                                   positions have been
                       H       8'                          reported
                                     6'
    HO            O                             OH
                                          5'
                                                      2.   Substitution with
         Anthranilic acid (2-aminobenzoic)                 hydroxyl and or
                                                           methoxy functions
                               +
                                                           at C-3’ have also
     p-Coumaric acid (4-hydroxycinnamic)
                                                           been reported
         =       AVENANTHRAMIDE: TYPE I


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6      …..BASED ON THE CHAIN LENGTH
          OF THE CONJUGATING ACID

                                                      1.   Substitution with
                       O                                   hydroxyl and or
                                  9'        7'
                                                           methoxy functions
                   N                                       at C-4 and/or C-5
                   H        10'        8'
                                                           positions have been
    HO         O                                 OH        reported

                                                      2.   Substitution with
       Anthranilic acid (2-aminobenzoic)                   hydroxyl and or
                                                           methoxy functions
                            +                              at C-3’ have also
                Avenalumic acid                            been reported
    5-(4-hydroxyphenyl)-2,4-pentadienoic acid

           =   AVENANTHRAMIDE: TYPE 2

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EXTRACTION AND GROUP SEPARATION
 7
     OF AVENANTHRAMIDES...
                10 g ground oats               Procedure is scaleable

                                               Denatures enzymes (e.g.
    1. 75 mL boiling, stirred, acidified
                                               peroxidases, esterases, etc)
         (0.1% HOAc) 80% EtOH

  Stir for 20 min with ambient cooling and     Cooling and sedimenting
    decant into graduated glass column         aids coalescing of insoluble
                                               and oil phases and forms
    Allow to settle and pack by gravity to     bed with good flow-through
    form a percolation extraction bed, Vb      properties

  Drain extract and percolate a further 3xVb   Percolation is an extremely
     fresh solvent to give a “percolate”       efficient extraction method

                                               HPLC evaluation at this
  Add ~ 3 mL of Octyl-Sepharose beads to
                                               point, detection at 330nm
     percolate and evaporate solvent

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8       HPLC Profiles During Group
      Separation Of Avenanthramides...
120                                                                                     VAO 48
           OD 330nm
                                                      Avenacosylate peaks (in part)    (hulless)
            (mAU)
100                                                                                   Crude EtOH
                                          “Prolamine” peaks (in part)                   extract
                                          “Glycolipid” peaks (in part)
 80                                       “Phosphatide” peaks (in part)


 60                             Avenanthramide zone


 40

 20

 0

           5     10 15 20 25 30 35 40 45 50 55 60 65
                             Time (min)
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9           AVENACOSYLATES: UV Interfering
                Components from Oats
          There are 3 major classes of avenacosylates in oats
    HO
                     O
    RO                                                                                Hydroxy-cinnamoyl
                O
     R = CH3; 1-O-feruloyl-n-hexacosan-1-ol         (C26 alcohol)                        esters of n-
     R = H;   1-O-caffeoyl-n-hexacosan-1-ol         (C26 alcohol)                          alkanols

     HO                                                                  O
                     O                                                         OR2
    R1O                                                              O
                 O                                                             OH
    R1 = R2 = CH3; 1,26-di-O-feruloyl-n-                            (C26  -diol)
                        hexacosan-1,26-diol                                           Hydroxy-cinnamoyl
    R1 = H;    1-O caffeoyl-26-O-feruloyl-n-
                                                                    (C26  -diol)
                                                                                       esters of n-alkan-
         R2 = CH3        hexacosan-1,26-diol
                                                                                           α,ω-diols
    R1 = R2 = CH3; 1,26-di-O-caffeoyl-n-                            (C26  -diol)
                        hexacosan-1,26-diol
    HO
                                                                O
    R2O             O
                                                                    O
                                                                                        Mixed hydroxy-
                O                             HO
                                                                                      cinnamoyl esters of
                                                                O
                                              R1O
                                                            O
                                                                               OH
                                                                                         ω-hydroxy-n-
                                                                    O
R1 = CH3; 1-O caffeoyl-2-O-feruloyl-3-O-(26'-O-                                OH
                                                                                      alkanoyl glycerides
R2 = OH     caffeoyl)-n-hexacosanoyl-D-glycerol                         O

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EXTRACTION AND GROUP SEPARATION
10
     OF AVENANTHRAMIDES...
                10 g ground oats               Procedure is scaleable

                                               Denatures enzymes (e.g.
    1. 75 mL boiling, stirred, acidified
                                               peroxidases, esterases, etc)
         (0.1% HOAc) 80% EtOH

  Stir for 20 min with ambient cooling and     Cooling and sedimenting
    decant into graduated glass column         aids coalescing of insoluble
                                               and oil phases and forms
   Allow to settle and pack by gravity to      bed with good flow-through
   form a percolation extraction bed, Vb       properties

  Drain extract and percolate a further 3xVb   Percolation is an extremely
     fresh solvent to give a “percolate”       efficient extraction method


  Add ~ 3 mL of Octyl-Sepharose beads to       Collins et al, US Patent
     percolate and evaporate solvent           #6,495,140 (2002)

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EXTRACTION AND GROUP SEPARATION
11
     OF AVENANTHRAMIDES...
   2.    Re-suspend in acidified 50% EtOH     Prolamines and lipophilic
          and transfer with washings to top   phenolics are bound to the
         of ~ 30 mL Octyl-Sepharose column    beads in 50% EtOH
        (pre-equil. in acidified 50% EtOH)
                                              All avenanthramides and
                                              hydrophilic components are
    Elute with 3xVb acidified 50% EtOH
                                              not bound to Octyl
                                              Sepharose in 50% EtOH
    Evaporate eluate to dryness and re-
   suspend in ~ 2mL acidified 40% EtOH        HPLC evaluation at this
                                              point, detection at 330nm
    3. Transfer with washings to top of a ~
     30 mL Sephadex LH-20 column pre-         All avenanthramides are
       equilibrated in acidified 40% EtOH     bound to Sephadex LH-20
                                              in 40% EtOH: saponins,
   Elute with 2xVb acidified 40% EtOH to      sugars, amino acids,
         remove polar components              flavone-glycosides etc are
                                              not bound
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12      HPLC Profiles During Group
      Separation Of Avenanthramides...
120                                                                                   VAO 48
           OD 330nm
                                                                                     (hulless)
            (mAU)
100                                                                                 Crude EtOH
       “flavone glycosides”                           “Prolamine” peaks (in part)   extract after
                                                                                     removal of
 80
                       “Avenacins”                                                   lipophilics

 60                               Avenanthramide zone



 40                   luteolin aglycone apigenin aglycone

                                           tricin aglycone
 20

 0

           5     10 15 20 25 30 35 40 45 50 55 60 65
                             Time (min)
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13          Other UV Absorbing Components
               from Oats: The Avenacins
                                                    O
                                                                                              Triterpene
                                                             R   R= O                      saponins with a
                                                H
                                            O                             O   HN            chromophoric
                                                                                   CH3
                                                                                            group with UV
                                                                  Avenacin A-1           absorption max at
   HO                                                   OH
           O                                                                             ~ 325 nm (A-1, B-1)
 HO               O
  HO                        O                                    R=   O
           OH          O
                                        H
             HO                                                           O
                                   OH
            HO                                                   Avenacin A-2
                      O O                           O
           HO
            HO
                      OH                                 O
                                            O   H            R
                                                                 R=   O


                                                    OH                    O   HN
   HO                                                                              CH3
           O
 HO              O
  HO                        O                                    Avenacin B-1
           OH          O
                                H 3C    H
             HO
            HO                                                   R=   O
                      O O
          HO                                                              O
           HO
                      OH                                         Avenacin B-2

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14          Other UV Absorbing Components
                  from Oats: Flavones
                                                           OH
                    HO
                  H 3C
                                                                    OH   • UV absorption spectral
                                      HO           O                     maxima similar to those
                  O         OH                                           of avenanthramides
      HO      O       OH
     HO                          OH
                                              OH O
      HO     O
        H3CO        O                      3',4',5,7-(OH)4-flavone
                                                  (Luteolin)             • Chromatographic
                OH O
                                                               OH        properties overlap with
                                      HO             O                   avenanthramides
  Isoswertisin-2"-O-rhamnoside
 (Apigenin-8-C-(2"-O--L-rhamno-
   pyranosyl)--D-glucoside-7-O-
           methyl ether)                      OH O                       • However, flavone
                                           4',5,7-(OH)4-flavone          derivatives appear to be
                                 OH             (Apigenin)
                                                         OCH3            minor components in oat
          HO            O
  HO
          O
                                                            OH           groat tissues (~5%?)
 HO
  HO                                  HO         O
           OH                                                 OCH3
                  OH O

          Isovitexin                        OH O
          Apigenin-6-C-
                                             4',5,7'-(OH)3-3',5'-
          -D-glucoside                       (OMe)2-flavone
                                                   (Tricin)

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EXTRACTION AND GROUP SEPARATION
15
     OF AVENANTHRAMIDES...
   2.    Re-suspend in acidified 50% EtOH     Prolamines and lipophilic
          and transfer with washings to top   phenolics are bound to the
         of ~ 30 mL Octyl-Sepharose column    beads in 50% EtOH
        (pre-equil. in acidified 50% EtOH)
                                              All avenanthramides and
                                              hydrophilic components are
    Elute with 3xVb acidified 50% EtOH
                                              not bound to Octyl
                                              Sepharose in 50% EtOH
    Evaporate eluate to dryness and re-
   suspend in ~ 2mL acidified 40% EtOH
                                              HPLC analysis to look at
                                              profile
   3. Transfer with washings to top of a ~
    30 mL Sephadex LH-20 column pre-
      equilibrated in acidified 40% EtOH

    Elute with 2xVb acidified 40% EtOH to
          remove polar components
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EXTRACTION AND GROUP SEPARATION
16
     OF AVENANTHRAMIDES...
   2.    Re-suspend in acidified 50% EtOH     Prolamines and lipophilic
          and transfer with washings to top   phenolics are bound to the
         of ~ 30 mL Octyl-Sepharose column    beads in 50% EtOH
        (pre-equil. in acidified 50% EtOH)
                                              All avenanthramides and
                                              hydrophilic components are
     Elute with 3xVb acidified 50% EtOH
                                              not bound to Octyl
                                              Sepharose in 50% EtOH
    Evaporate eluate to dryness and re-
   suspend in ~ 2mL acidified 40% EtOH
                                              HPLC analysis to look at
                                              profile
   3. Transfer with washings to top of a ~
    30 mL Sephadex LH-20 column pre-          All avenanthramides are
      equilibrated in acidified 40% EtOH      bound to Sephadex LH-20
                                              in 40% EtOH: saponins,
   Elute with 2xVb acidified 40% EtOH to      sugars, amino acids,
         remove polar components              flavone-glycosides etc are
                                              not bound
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EXTRACTION AND GROUP SEPARATION
17
     OF AVENANTHRAMIDES...

                                               All avenanthramides are not
    Recover avenanthramides by eluting
                                               bound to Sephadex LH-20
       column with 3xVb 95% EtOH
                                               in 95% EtOH

  4. Evaporate eluate to dryness, take up in   Procedure results in base-
     known volume of 50% EtOH (~ 2-5 mL),      line resolution of all the
     pass sub-sample through 0.45μ filter      avenanthramides and can
                                               be easily scaled up to gram
                                               quantities

    HPLC-rapid scan               HPLC-diode   Molecular Weight Range
     (“diode array”)              array-MSMS   scanned between 100-700
                                               daltons
         Can also use different HPLC
        column:solvent configurations


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18      HPLC Profiles During Group
      Separation Of Avenanthramides...
120                                                       VAO 48
           OD 330nm
                                                         (hulless)
            (mAU)
100                                                     Final purified
                                                       avenanthramide
 80                                                        extract


 60
                                 Avenanthramide zone   Flat base line for
                                                            ease of
                                                          integration
 40

 20

 0

           5     10 15 20 25 30 35 40 45 50 55 60 65
                             Time (min)
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19    Comparison of HPLC Profiles During
       Separation Of Avenanthramides...
                                 Avenanthramide zone
120

100                                                    Crude EtOH
                                                         extract

 80

 60
                                                       After Octyl
                                                       Sepharose
 40

 20
                                                       After LH-20

 0

           5     10 15 20 25 30 35 40 45 50 55 60 65
                             Time (min)
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20    Spectral analyses of Avenanthramides

120                                                        VAO 48
           OD 330nm
                                                          (hulless)
            (mAU)
100

 80                                                     High degree
                                                           of peak
                                                         resolution
 60                                                      facilitates
                                                          spectral
 40                                                    analysis and
                                                       identification
 20

 0

           5     10 15 20 25 30 35 40 45 50 55 60 65
                             Time (min)
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21     Spectral analyses of avenanthramides

                                                                    (mAU)
    OD                               HO
                                                     O
  330nm                                                      OCH3
  (mAU)                            B             N
                                                 H
                                       HO    O               OH
                                                 m/z = 329




                                                                    400
       250
 λ
                                                                    (mAU)

       275                                                          300
(nm)
       300
                                                                    200

       325
                                                                    100
       350

       375                                                           0

                               Retention Time (min)
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22     Spectral analyses of avenanthramides

                                  HO                                (mAU)
                                                 O
 OD
330nm                             P          N
                                                             OCH3
                                             H
(mAU)                               HO   O       m/z = 355   OH




                                                                    400
       250
 λ
                                                                    (mAU)

       275                                                          300
(nm)
       300
                                                                    200

       325
                                                                    100
       350

       375                                                           0

                               Retention Time (min)
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23   Spectral analyses of avenanthramides

                   300s
                                                    HO
                                                                          O
                                                                                  OCH3
                             335
                                   350s           B                   N
                                                                      H
                                                         HO       O               OH
                                                                      m/z = 329


                                                 All Type I avenanthramides
                                                show a shoulder between 290
                                                         and 310nm

                                                  HO
                                                                      O

                                                  P               N
                                                                                  OCH3
                                      360                         H
                               340s                 HO        O                   OH
                                                                      m/z = 355



                                               All Type II avenanthramides lack
                                                          this shoulder

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24         HPLC DIODE ARRAY-MSMS OF
               AVENANTHRAMIDES
                        Hypersil® C18 5μ column:
                                          A: Methanol
                        Solvents          B: H2O
                                          C: 5% Acetic acid
                                   Gradient
         Time           A(%)         B(%)        C(%)     Flow rate

          0               40          55          5       0.8 mL/min
         80              60           35          5       0.8 mL/min
         85              80           15          5       0.8 mL/min
         88             100            0          0       0.8 mL/min
         91             100            0          0       0.8 mL/min
         94              40           55          5       0.8 mL/min
         105             40           55          5       0.8 mL/min

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25         HPLC DIODE ARRAY-MSMS OF
               AVENANTHRAMIDES
                        Polaris® Amide C18 5μ column:
                                          A: Methanol
                        Solvents          B: H2O
                                          C: 5% Formic acid
                                    Gradient
         Time           A(%)          B(%)       C(%)    Flow rate

          0              55            40        5       0.8 mL/min
          96             95            0         5       0.8 mL/min
         100             95            0         5       0.8 mL/min
         105             55            40        5       0.8 mL/min
         115             55            40        5       0.8 mL/min



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26        HPLC DIODE ARRAY-MSMS OF
              AVENANTHRAMIDES
                         INSTRUMENTATION
                           HPLC:
      Thermo Separation Products® (Thermo Finnigan)
                        P4000 pump
                        SN 4000 Controller
                        SpectraSystem UV3000 detector UV 250-400nm;
                        Chromquest software
                          MASS SPEC-MASS SPEC:
                                 (MS-MS)
     Thermo Finnigan LCQ Advantage® with DA detector
     Mode:               electrospray ionization neg. mode
     Source voltage:     4.5 Kvolts
     Capillary voltage:  -10v
     Capillary temp:     300C Sheath gas flow = 80% full
     Aux gas flow:       20% max. (no stream splitting)
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27           HPLC DIODE ARRAY-MSMS OF
                 AVENANTHRAMIDES
     2200 OD 330nm                                          AC Baton
     2000  (mAU)                                            (hulless)
     1800
     1600                                              More than 30
                                                          peaks
     1400
                                                       detectected
     1200
     1000
      800
      600
      400
      200
         0
             0      10        20    30     40     50   60       70      80
                               Retention Time (min)
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28           HPLC DIODE ARRAY-MSMS OF
                 AVENANTHRAMIDES
     1000
             OD 330nm
     900      (mAU)                                        AC Baton
                                                           (hulless)
     800
     700
     600                                                   More than 20
                                                              peaks
     500                                                   detectected
     400
     300
     200
      100
         0
             0     10     20     30   40    50   60   70    80   90    100
                               Retention Time (min)
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29           KEY TO AVENANTHRAMIDE ID IS
              THE MS-MS IONS m1 AND m2
                                                                                MS parameters
         R                                         R                            optimized for
R                                         R                                     generation of [M-1] -
                     O                                          O
                                 n                                  n
                 N                   R                      N              R    Parent ion M-1 –
                 H                                          H                   (always even
    -O       O                       OH       -O        O                  OH
                                                                                number) gives
                                                                                molecular weight of
         Parent ion [M-1]-                             Parent ion [M-1]-
                                                                                the avenanthramide
             R                                     R
                                                                                Daughter m1 - gives
     R                                    R                                     molecular weight of
                         O                                      O   n
                                                                           R    the anthranilic moiety
                     N       -                     -        N
                                                            H
             O
                     O                                                     OH   Daughter m2 - gives
                                                   Daughter ion m2 -            molecular weight of
     Daughter ion m1 -
                                                                                both ring moieties


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30                       STRUCTURES OF TYPE I
                          AVENANTHRAMIDES
                                               R=H   R = OCH3   R = OH
                           O
                                   R
                       N

             HO    O
                       H
                                   OH
                                                D       E        F
        HO
                           O

                       N
                       H
                               R
                                                A       B        C
         HO        O           OH

                  OH
                           O
                               R
                       N
                       H                        G        H        K
             HO    O           OH


                  OH
         HO
                           O
                               R
                       N
                       H                       AA       BB       CC
             HO    O           OH


                  OCH3
         HO
                           O
                               R
                       N
                       H
                                                X        Y        Z
             HO    O           OH

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31    STRUCTURES AND CHROMATOGRAPHIC
       PROPERTIES OF AVENANTHRAMIDES

                   TYPE 1 AVENANTHRAMIDES
 100                                         anthranilic             SUMMARY
                 Rt (min)
                 Amide
     80                    5-OH                                      5 Different
                        anthranilic                 D                anthranilic
                                         F                 E         acid amides
     60                                   4-OH                       with either
                              A        anthranilic
                                                                     p-coumaric,
                     C   B                                           ferulic, or
     40                      G
                     AA BB K                                         caffeic acids
                             XY H
     20           CC      Z
                4,5-di(OH)          4-OH,5-OMe
                anthranilic          anthranilic
     0
            0               20            40               60   80
                                      Rt (min) C18
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32                      STRUCTURES OF TYPE II
                          AVENANTHRAMIDES
                            O
                                               R=H   R = OCH3   R = OH
                        N        R

          HO        O
                        H
                                 OH            L        M          N
        HO
                            O
                                  R
                        N

           HO       O
                        H
                                  OH
                                               O        P          Q
               OH

                            O
                                 R

         HO      O
                        N
                        H
                                 OH
                                                R       S          T
                OH
        HO
                            O
                                     R
                        N

          HO        O
                        H
                                     OH
                                               OO       PP        QQ
                OCH3
         HO
                            O
                                 R

          HO        O
                        N
                        H
                                 OH
                                                U        V         W
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33   STRUCTURES AND CHROMATOGRAPHIC
      PROPERTIES OF AVENANTHRAMIDES
               TYPE 2 AVENANTHRAMIDES
                                                              anthranilic
     100                              5-OH                                     SUMMARY
               Rt (min)            anthranilic
               Amide                                                 L
     80                                                  N            M        5 Different
                                          O                                    anthranilic
     60                                       P                                acid amides
                               Q                     R       S                 with either
                                               T                    4-OH
                                                                               avenalumic, 3’-
                                   OO                            anthranilic
     40                                                                        OMe-
                          QQ        PP               U                         avenalumic, or
                                                 W               V             3’-OH-
     20
                               4,5-di(OH)                                      avenalumic
                               anthranilic 4-OH,5-OMe                          acids
                                            anthranilic
      0
           0              20            40               60               80
                                   Rt (min) C18
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34        2-D HPLC MS-MS “MAPPING”OF
               AVENANTHRAMIDES
                                                                                    Based on:
 100                                                                                1) Rt in 2 solvents
              Rt (min)                                                 L            2) UV spectra
              Amide                                       N                         3) MS pattern
     80
                                                    O D                    M
                                           F                                        There are about
                                     Q                    E                         30 different
     60                      A                      P
                                                              R                     avenanthramides
                    C                    OO                                         not including
                               B        G PP                      S
     40                                       T                                     dimeric forms
                             QQ        X                      U
                        AA                  H
                CC                   K   Y                                          Each one
     20                  BB                  W                    V
                                 Z                                                  identified by an
                                                                                    alphabetic
     0                                                                              designation
          0              20                    40                 60           80
                                         Rt (min) C18
      Agriculture and  Agriculture et
      Agri-Food Canada Agroalimentaire Canada
35              RELATIVE AMOUNTS OF EACH
                   AVENANTHRAMIDE…..
Rt (min) OD (AU)                                            AC Baton        Relative
Amide 2.5
                                                            (hulless)     quantitative
     100   2.0                                                            estimations:

     80        1.5                                                           Major
                                                                        Avenanthramides
               1.0
     60                                                                     A, B, C
                0.5
                     0                                                     O, P, Q
     40
                                                                          OO, PP, QQ
     20
                                                                          AA, BB, CC

      0
           0             20             40             60          80
                                        Rt (min) C18
      Agriculture and  Agriculture et
      Agri-Food Canada Agroalimentaire Canada
36                                        GENOTYPIC VARIATION IN
                                         AVENANTHRAMIDE LEVELS
CONCENTRATION IN PPM




                       500


                       400


                       300


                       200


                       100
                        50
                        0

                                                           VARIETIES / LINES   AV A
                                                                               AV B
                                                                               AV C
                                                                               TOTAL

                               Strong genotype x environment interaction as well

                             Agriculture and  Agriculture et
                             Agri-Food Canada Agroalimentaire Canada
37             TEAM CONTRIBUTORS AND
                ACKNOWLEDGEMENTS….

          AAFC                            USDA TUFTS UNIVERSITY


     Synthesis ,                                              Antioxidant
                                             Vascular
     GMP Purification,
     Analytical,                           Biology Lab:      Research Lab:
     Germplasm                            In vitro studies   In vivo studies

     F. William Collins                  Mohsen Medani        Jeffrey Blumberg
     Vern Burrows                          Liping Liu         Paul Milbury
     Winson Orr                           Ligia Zubik         Chung-Yen Chen
     Dwight Colley                       Melissa Marko        Ting Li
     Nicole Fillion                       Weimin Guo          Jennifer O’Leary


     Agriculture and  Agriculture et
     Agri-Food Canada Agroalimentaire Canada

				
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