Natural Antioxidant Measurement
KEMIN IS ASSURANCE
Zhuhai
Indaiatuba
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Lipid Oxidation
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Antioxidant
Unsaturated Lipids
Lubuza, T. P. Kinetics of Lipid Oxidation in Foods, CRC Crit. Rev.
Food Technol. 1971. 2, 355-405.
Initiation Propagation Termination
Multi-Analysis Approach
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Primary Oxidation
Antioxidant Residuals
Secondary Oxidation
Resistance to
Antioxidant Chemistry
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γ γ TocopherolTocopherolγ γ TocopherolTocopherol •
+ RH O
What To Measure?
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• Synthetic antioxidants are fairly easy to deal with. Synthetic
antioxidant formulations typically contain high concentrations of
high purity antioxidant molecules.
• BHA • BHT • Propyl Gallate • TBHQ • Ethoxyquin
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Green Tea
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• Green tea includes a large range of molecules with
activity.
J. Agric. Food Chem. 2013, 61, 11484-11493
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Natural Antioxidant Challenges
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• Natural antioxidants are complex mixtures of active molecules of
varying concentration and activity.
• Antioxidant Activity and reactivity is very complex. Depends
heavily on the matrix. Antioxidants need to be matched to the
situation. Reactivity (general)
Alpha Tocopherol
Ascorbic Acid
Carnosic Acid
Green Tea
Gamma Tcoopherol
Rosemary Extract
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• Rosemary extract is a mixture of phenolic diterpenes and
flavonoids of varying concentration.
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What to Measure Challenges
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Biological Antioxidants:
Shelf Life Antioxidants:
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ctivity In
cr ea
si ng
A nt
io xi
da nt
A ct
iv ity
O
OH
CH3
CH3
CH3
(CH2CH2CH2CH)3CH3
CH3
O
OH
O
OH
Delta homologues have the greatest antioxidant activity
Alpha homologues have the greatest Vitamin E activity
Chemical Structures of Natural Tocopherol Homologues
Typical Diet Stability by Tocopherol Homologue
Alpha Gamma Delta
Antioxidant Activity (alpha = 100%)
alpha tocopherol 100 beta tocopherol 130 gamma tocopherol 200 delta
tocopherol 300-500
*Riken Vitamin Co., Ltd. © Kemin Industries, Inc. and its group of
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Industries, Inc., U.S.A.
Chart1
control
7
12
15
18
Chart2
28
24
20
6
Sheet1
300-500
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Polar Paradox Do antioxidants partition into areas where lipid
oxidation is prevalent?
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Vegetable Oil with nonpolar antioxidant
Antioxidant Molecule
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2H
2H
•OH
Richheimer, et al, Recent Research and Developments in Oil
Chemistry 3:45-58, 1999 © Kemin Industries, Inc. and its group of
companies 2019. All rights reserved. ®™ Trademarks of Kemin
Industries, Inc., U.S.A.
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What To Measure Challenges
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• Final molecule to measure after process of elimination
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Natural Antioxidant Challenges
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• Natural antioxidants can have higher initial sacrifice rates than
synthetic antioxidants.
• Meal is stable with low residual rates but it introduces analysis
and/or application questions.
1200 ppm Naturox TX applied to a
protein meal
meal
• Questions: - Was the correct amount of antioxidant applied? - Are
there application issues?
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General Application Concerns: 1.Antioxidant dosage confirmation,
2.Equipment validation: Homogeneity, 3.Antioxidant sacrifice
evaluation.
Tracer properties : EU Authorized molecule for Pet Food
application. Heat stable and no pro-oxidant, Soluble in Antioxidant
solution, Easy to analyze by GC method.
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Indirect Measurement - Electrochemical • Cyclic Voltammetry •
Amperometry
Direct Measurement - Chromatography • Gas Chromatography (GC) •
High Performance Liquid Chromatography (HPLC)
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Indirect Measurement Positives • Equipment cost is relatively cheap
• Easy to implement. • Minimal sample preparation is often needed.
• Total Phenolic Kits available on the market.
• Inability to measure and isolate key compounds of interest.
• Total phenolics usually do not correlate with antioxidant
activity.
• Complex matrixes such as pet food diets have many compounds that
would cause interference (vitamins, carotenoids, and non
antioxidant phenolic compounds)
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Indirect Measurement Negatives
Antioxidant Measurement – Direct Measurements • Gas Chromatography
(GC)
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Chromatography
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Definition: The separation of compounds in a mixture.
Stationary Phase
Chromatography Column
min5 7.5 10 12.5 15 17.5 20 22.5 25 27.5
mAU
0
100
200
300
400
DAD1 A, Sig=280,4 Ref=off
(M:\BUTCH\2004FI~1\04APRI~1\040413\001-0601.D)
1 6.
70 4
e
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Rosemary GC Methods
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• No official methods available • Extraction using acetonitrile
followed by solvent extract. • Derivatization step. • Non polar
columns (0 to 5% phenyl) • Detector – FID • Method Positives –
Effective with low residual level carnosic
acid levels. Have had issues with low level carnosic analysis on
HPLC methods due to interferences in complex matrixes.
• Method Negatives – Derivatization reagent can cause increased GC
maintenance.
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Antioxidant Measurement – Direct Measurements • High Performance
Liquid Chromatography (HPLC)
Reference: KN CLS pictures approved © Kemin Industries, Inc. and
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Kemin Industries, Inc., U.S.A.
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Tocopherol HPLC Methods
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• Range of methods such as AOCS Ce 8-89 or EN 12822 • Both Reverse
Phase Method and Normal Phase Methods
exist. NH2 (normal) / C8 or C18 silica (reverse) • Detector – 280
nm UV detection • FLD Detection - 290 nm excitation and 330 nm
emission • Method Positives – Popular methodology • Method
Negatives – 280 nm UV detector methods have
broad interferences with a large range of compounds other than the
antioxidant compound of interest. Have seen range of false
positives for BHA and BHT.
• Method Negatives – Reverse phase less rugged than normal phase
methods. UV detection less sensitive and selective than the FLD
detection. KN uses FLD detectors.
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Carnosic Acid HPLC Method
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• No official methods • Acetone extraction. • Acidified
water/acetonitrile mobile phase. • Reverse Phase Method - C8 silica
column • Detector – 280 nm UV detection • Method Positives –
Popular methodology for high
concentration or simply matrix (fats and oils). No derivatization
step needed.
• Method Negatives – Interferences in complex matrixes such as pet
food diet due to 280 nm reactivity with other compounds.
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Green Tea HPLC Method
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• No official methods. Range of HPLC methods in literature. •
Acetonitrile extraction • Water, acetonitrile mobile phase. •
Reverse Phase Method - C8 silica column • Detector – 280 nm UV
detection • Method Positives – Efficient. Haven’t observed
interferences. • Method Negatives – 280 nm again. Chances of
interferences. Due to large number of green tea catechins, need
higher application rates to measure individual catechins on
finished pet food diet.
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Antioxidant Verification
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• GC-MS-QTOF • Same method as GC method • GC results can be
verified using
MS and MS/MS analysis and library matching
• Sensitivity – ppb
• Sensitivity – ppb
Mass Spectrometry Fingerprint
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Creative Commons BY-NC-SA 3.0
Natural Antioxidant Method Accuracy
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Method Sensitivity Precision(Typical) Accuracy Indirect Methods
(Catechin Kit) low ppm variable variable GC - Carnosic Acid 5 ppm
+/- 10% variable HPLC - DAD - Tocopherols 10 ppm +/- 10% variable
HPLC - FLD - Tocopherols 1 ppm +/- 5% variable HPLC - Green Tea
20-30+ ppm +/- 5% variable
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find high degree of variability with accuracy • Extraction and
Instrument validation is critical • Proficiency program
participation is critical
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.
Based in the US 1-2 Agricultural feed/ingredient samples monthly;
also 1-2 pet food/ingredient samples each quarter Moisture, Water
Activity
Based in the US Edible fat series, composed of vegetable shortening
and margarine; 2 samples each quarter Free Fatty Acid value,
Peroxide Value, OSI time
Based in the UK Synthetic Antioxidants in Vegetable Oil: BHA, BHT;
once per year
Based in Germany and conducted in German Edible Fat series,
composed of an unknown vegetable oil; once per year Tocopherols,
Peroxide Value, Free Fatty AcidNot an “official” proficiency
program, but important. Based
in the US, Belgium and Australia. Our CLS exchange pet food samples
between CLS laboratories and compare results for full range of
analysis such as synthetics, tocopherols, PV’s, FFA’s, moisture,
water activity, OSI,
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THANK YOU
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Ben Bowen photos KN
Natural Antioxidant Measurement
Slide Number 3
Slide Number 4
Polar Paradox
How to Measure Natural Antioxidants
Indirect Measurement Positives