In the last decade, nutritional oil products such as omega-3s have grown to a multi-billion dollar industry globally. As a result, new analytical testing challenges have arisen from the current trend toward producing more attractive and more appetizing products created for a wider consumer base. In formulating these "new and improved," better looking and better tasting products, different color and flavor additives are used, which can interfere with the most popular analytical procedure for determining the secondary oxidation of nutritional oil products, the p-anisidine value test. In order to overcome these analytical challenges, a new alternative method for testing these additive-laden products has been established and is ready for use in this fast growing marketing segment. Dr. Steven Li provides details about the latest innovation in omega-3 testing and its application in the omega-3 industry, at GOED Exchange 2014.

1. Novel Approaches to
Omega-3 Stability Testing
Performing the p-Anisidine Value (TAV)
Test by a New HPLC-UV Method
Dr. Steve Li, MD, M.Sc. (Clin.Pharm)
Director of Laboratory Operations
Diteba, a Nutrasource Diagnostics Inc. company
info@nutrasource.ca
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2. p-Anisidine Value Analysis is for Determination
of Oxidation Level of Oil Substance
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3. p-Anisidine Value Analysis Procedure Has
Been Included in Many Official Methods
1. The United States Pharmacopeia and the
National Formulary (USP–NF)
2. European Pharmacopeia (EP)
3. American Oil Chemists' Society (AOCS)
4. Food Chemicals Codex (FCC)
5. Association of Official Analytical Chemists
(AOAC)
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4. p-Anisidine Test is One of the
Three Common Analytical Tests
for Oil Oxidation
• Acid Value
• p-Anisidine Value
• Peroxide Value
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5. Acceptable Limits for Oxidative
Stability Parameters in Marine Oils
(Recommended Reporting Threshold)
________________________________________
Acid Value
NMT 3 mg KOH/Kg
Peroxide value (PV)
NMT 5 mEq/Kg
Anisidine value (AV)
NMT 20
TOTOX value
NMT 26 (2 x PV + AV )
______________________________________________________
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6. Oxidation Pathways of Oil
Autooxidation
• Free radical formation starts the chain
reaction of degradation to unsaturated fatty
acid.
• The major initial oxidation products, ROOH
hydroperoxide, form compounds responsible
for off-flavors and odors.
Such secondary products include
aldehyde and ketones, such as
hexanal, pentanal, and malonaldehyde.
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7. Oxidation Pathways of Oil
Autooxidation
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8. Traditional p-Anisidine Value Test
1.
2.
3.
.
The anisidine value is defined as 100 times the optical density
measured in a 1 cm cell of a solution containing 1 g of the
substance to be examined in 100 ml of a mixture of solvents and
reagents.
Test solution (a). Dissolve 0.500 g of the substance to be
examined in iso-octane and dilute to 25.0 ml with the same
solvent.
Test solution (b). To 5.0 ml of test solution (a) add 1.0 ml of a 2.5
g/l solution of p-anisidine R in glacial acetic acid R, shake and
store protected from light.
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9. Traditional P-Anisidine Value Test
4.
5.
Reference solution. To 5.0 ml of isooctane add 1.0 ml of a 2.5 g/l
solution of p-anisidine in glacial acetic acid, shake and store
protected from light.
Measure the absorbance of test solution (a) at the maximum at
350 nm using isooctane as the compensation liquid. Measure
the absorbance of test solution (b) at 350 nm exactly 10 min
after its preparation using the reference solution as the
compensation liquid.
The p-anisidine value (p-A.V.) is given by the formula:
25 (1.2 As – Ab)
pAV = ------------------------m
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11. Why Do We Have Problems with the pAnisidine Value Test?
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12. The Test Has No Problem to Evaluate and
Compare the Oxidation Level of Simple
Marine Oil.
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13. Marine Oil Products Changed When they
Became Important Natural Health Products
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14. The Challenge Comes From the Advanced
Formulated Products Containing Marine Oil
and Other Additives
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15. New Ingredients Are Introduced
• Colour additives
• Flavour additives
• Vitamin additives
• Others
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16. The New Formulations Interfere
With p-Anisidine Tests
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17. Advanced Formulations Bring Big Challenges to
the Traditional p-Anisidine Value Test
1. Interference creates wrong p-anisidine
results and generates misleading certificates;
2. Interference causes failed performance of
the test method;
3. No Practical Alternative Test Available to
replace p-Anisidine method for the combined
oil formulations.
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18. There should be a procedure
available to solve this issue.
1. A lot of research work on oil oxidation has
been nicely done and available; True!
2. It should not be too difficult to develop an
alternative test method; True!
How??
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19. There are Two Groups of Analytical Methods
Routine Test Methods
Research Methods
Advantages
• Fast
• Easy to be performed
• Used in industry for century?
• Cost effective
• Common Apparatus or
Instrument
Disadvantages:
• Not specific
• Not directly quantitative
• Easily Interfered
Advantages
• Relatively Specific
• Can be quantitative
Disadvantages:
• Time consuming
• Cost
• Require instruments
• Difficult to be used in QC
environment
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20. Strategies
Simple approach – new sample preparation to
separate degradation products
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21. Simple Procedure
After several trials of different sample
preparations.
Procedures of extraction and separation are too
tedious and not feasible for QC methods.
Oops!
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22. Advanced Technologies
1. GC-Flame Ionization Detector. Too complicated to
identify and quantify the oxidized compounds.
2. GC-MS. Features good identification. Too complicated
to identify and quantify the oxidized compounds.
3. LC-UV. Too difficult to identify the oxidized
compounds.
4. LC-MS. Too difficult of sample preparation and too
expensive to quantify the oxidized compounds.
5. IR or FTIR (Fourier Transform Infrared spectroscopy).
Very good test mechanism but very difficult to be a
routine analytical method.
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23. IR Spectrum for Oil Degradation Samples
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24. IR Spectrum for Oil Degradation Samples
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25. IR method seems working but...
The method can not be used as a quality
control method because there are no
appropriate control samples and the peak
areas are not very reproducible, meaning
poor precision.
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26. Chromatograms of
p-Anisidine Value Test Preparations
Blank, Oil Sample and Reacted
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27. What do we need for this method?
1. Simple routine method
2. Economical and practical
3. Relatively specific
4. Relatively precise and accurate
All approaches explored are Not doable and Not
affordable!
What else? Nothing!!
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28. p-Anisidine HPLC-UV Method
Chromatographic Conditions
HPLC column, 4.6 mm × 250 mm
Column Temperature:
25˚C
Detection:
UV at 254nm
Injection Volume:
10µL
Flow Rate:
1.0 mL/minute
Run Time:
10 minutes
Mobile Phase: Hexane: 2-Propanol (50:50)
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29. p-Anisidine HPLC-UV Method
Specificity Chromatogram
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30. p-Anisidine HPLC-UV Method
Typical Chromatogram of QL
(S/N=15, 3.162 µg/mL)
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31. p-Anisidine HPLC-UV Method
Linearity Study
(Range: 0.1002 to 0.5008 mg/mL)
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32. p-Anisidine HPLC-UV Method
Repeatability Results
Sample
No.
Assay (%)
Repeatability
Intermediate
Precision
1
86.0
84.1
2
85.6
83.4
3
85.0
85.8
4
83.6
84.9
5
85.1
86.6
6
84.4
83.7
Mean
85.0
84.8
%RSD
1.0
1.5
Mean of 12
Samples
84.9%
RSD
1.2%
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33. p-Anisidine HPLC-UV Method
Summary of Accuracy Results
Concentration Level
% Recovered
Average
99
50%
99
99%
RSD = 0%
99
100
100%
100
100%
RSD =0 %
100
101
150%
101
101%
RSD =1 %
102
Average of 9 samples = 100%
RSD = 1%
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34. p-Anisidine HPLC-UV Method
Summary of System Suitability Study
Injection No.
Retention
Time
(Minutes)
Peak Area
(μV*sec)
USP Tailing
Factor
Theoretica
l Plate
Number
1
4.85
2111474
2.3
2858
2
4.85
2125521
2.3
2843
3
4.85
2127289
2.3
2831
4
4.86
2139357
2.3
2808
5
4.85
2131861
2.3
2842
6
4.85
2146601
2.3
2836
Mean
4.85
2130350
2.3
2836
%RSD
0.03
0.6
0.9
0.6
Overall %RSD
N/A
0.6%
N/A
N/A
Check Std
Recovery (%)
N/A
101.0%
N/A
N/A
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35. p-Anisidine HPLC-UV Method
Summary of System Suitability Study
Injection No.
Retention
Time
(Minutes)
Peak Area
(μV*sec)
USP Tailing
Factor
Theoretica
l Plate
Number
1
4.85
2111474
2.3
2858
2
4.85
2125521
2.3
2843
3
4.85
2127289
2.3
2831
4
4.86
2139357
2.3
2808
5
4.85
2131861
2.3
2842
6
4.85
2146601
2.3
2836
Mean
4.85
2130350
2.3
2836
%RSD
0.03
0.6
0.9
0.6
Overall %RSD
N/A
0.6%
N/A
N/A
Check Std
Recovery (%)
N/A
101.0%
N/A
N/A
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36. p-Anisidine HPLC-UV Method
Cross Reference of Known AV values
and Specification Set-up
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37. p-Anisidine HPLC-UV Method
Cross Reference of Known AV values
and Specification Set-up
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38. p-Anisidine HPLC-UV Method
Specification Set-up
From the data collected from our current tests
and stability studies the TAV acceptance
criteria has been recommended. Most of the
oil samples with over anisidine values >20
give the TAV results around 69% to 72%
anisidine % remaining . The range is very
small and well correlated with p-Anisidine
results.
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39. Conclusion
1. The method is validated. It has
good specificity, good precision
and accuracy.
2. The most important feature is that
the method demonstrates very
good correlation with traditional pAnisidine value test.
3. The method shows good
characteristics of a quality control
method.
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48. Acknowledgements
Diteba Team
Lucy Zhao
Linda Chang
Nutrasource Diagnostics Inc.
Team
Kevin Yan
Martha Wlodek
William Rowe
Mike Wlodek
© Nutrasource Diagnostics Inc.
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49. Thank you!
© Nutrasource Diagnostics Inc.
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50. p-Anisidine HPLC-UV Method
Recommended Test Criteria
Flavoured Marine oil
TAV
p-anisidine
1. Marine oil containing
Vitamin A
yes
no
2. Marine oil containing
Vitamin A + D
yes
no
3. Marine oil containing
orange Flavour
yes
no
4. Marine oil containing low
level of orange flavour
yes
no
5. Marine oil containing
Tangerine lime
(concentration unknown)
yes
no
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52. Biomedical Research Findings Support Its
Usage with Important Indications
1. Cardiovascular diseases (CVDs) are the number one cause of
death globally
2. The most common disorders: hypertension (arteries), heart
attacks (coronary arteries) and strokes (brain arteries). The
changes are all related to blood vessels, a build-up of fatty
deposits on the inner walls of the blood vessels, blood clots
and bleeding
3. Importantly, these conditions are reversible and preventable
4. Omega-3s EPA and DHA can help by lowering blood
triglycerides , preventing fatty deposits and reducing blood
pressure
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53. p-Anisidine Value test is to determine the
secondary oxidation compounds in the oil
products. This spectrophotometric test is
easy to be interfered.
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