natural antioxidant measurement

Natural Antioxidant Measurement
KEMIN IS ASSURANCE
Zhuhai
Indaiatuba
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Lipid Oxidation
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
Primary Oxidation
Antioxidant Residuals
Secondary Oxidation
Resistance to
Antioxidant Chemistry
γ γ TocopherolTocopherolγ γ TocopherolTocopherol •
+ RH O
What To Measure?
• 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
Green Tea
• Green tea includes a large range of molecules with activity.
J. Agric. Food Chem. 2013, 61, 11484-11493
Natural Antioxidant Challenges
• 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
• Rosemary extract is a mixture of phenolic diterpenes and flavonoids of varying concentration.
What to Measure Challenges
Biological Antioxidants:
Shelf Life Antioxidants:
www.kemin.com/assurance
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 companies 2019. All rights reserved. ®™ Trademarks of Kemin Industries, Inc., U.S.A.
Chart1
control
7
12
15
18
Chart2
28
24
20
6
Sheet1
300-500
Polar Paradox Do antioxidants partition into areas where lipid oxidation is prevalent?
Vegetable Oil with nonpolar antioxidant
Antioxidant Molecule
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.
What To Measure Challenges
• Final molecule to measure after process of elimination
Natural Antioxidant Challenges
• 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?
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.
Indirect Measurement - Electrochemical • Cyclic Voltammetry • Amperometry
Direct Measurement - Chromatography • Gas Chromatography (GC) • High Performance Liquid Chromatography (HPLC)
Reference: KN CLS pictures approved © Kemin Industries, Inc. and its group of companies 2019. All rights reserved. ®™ Trademarks of Kemin Industries, Inc., U.S.A.
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)
Indirect Measurement Negatives
Antioxidant Measurement – Direct Measurements • Gas Chromatography (GC)
Chromatography
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
Rosemary GC Methods
• 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.
Antioxidant Measurement – Direct Measurements • High Performance Liquid Chromatography (HPLC)
Tocopherol HPLC Methods
• 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.
Carnosic Acid HPLC Method
• 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.
Green Tea HPLC Method
• 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.
Antioxidant Verification
• 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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Natural Antioxidant Method Accuracy
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
find high degree of variability with accuracy • Extraction and Instrument validation is critical • Proficiency program participation is critical
emin Industries, Inc. and its group of companies 2019. All rights reserved. ®™ Trademarks of Kemin Industries, Inc., U.S.A.
.
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,
THANK YOU
© Kemin Industries, Inc. and its group of companies 2019. All rights reserved. ®™ Trademarks of Kemin Industries, Inc., U.S.A. Ben Bowen photos KN
Natural Antioxidant Measurement
Slide Number 3
Slide Number 4
Polar Paradox
How to Measure Natural Antioxidants
Indirect Measurement Positives

natural antioxidant measurement

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