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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

www.nutrasource.ca
p-Anisidine Value Analysis is for Determination
of Oxidation Level of Oil Substance

www.ifosprogram.com
www.nutrasource.ca
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)
www.ifosprogram.com
www.nutrasource.ca
p-Anisidine Test is One of the
Three Common Analytical Tests
for Oil Oxidation
• Acid Value
• p-Anisidine Value
• Peroxide Value

www.ifosprogram.com
www.nutrasource.ca
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 )

______________________________________________________

www.ifosprogram.com
www.nutrasource.ca
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.

www.nutrasource.ca
Oxidation Pathways of Oil
Autooxidation

www.nutrasource.ca
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.

www.ifosprogram.com
www.nutrasource.ca
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

www.ifosprogram.com
www.nutrasource.ca
www.ifosprogram.com
www.nutrasource.ca
Why Do We Have Problems with the pAnisidine Value Test?

www.ifosprogram.com
www.nutrasource.ca
The Test Has No Problem to Evaluate and
Compare the Oxidation Level of Simple
Marine Oil.

www.ifosprogram.com
www.nutrasource.ca
Marine Oil Products Changed When they
Became Important Natural Health Products

www.ifosprogram.com
www.nutrasource.ca
The Challenge Comes From the Advanced
Formulated Products Containing Marine Oil
and Other Additives

www.ifosprogram.com
www.nutrasource.ca
New Ingredients Are Introduced
• Colour additives
• Flavour additives
• Vitamin additives
• Others

www.ifosprogram.com
www.nutrasource.ca
The New Formulations Interfere
With p-Anisidine Tests

www.ifosprogram.com
www.nutrasource.ca
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.

www.ifosprogram.com
www.nutrasource.ca
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??
www.ifosprogram.com
www.nutrasource.ca
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

www.ifosprogram.com
www.nutrasource.ca
Strategies
Simple approach – new sample preparation to
separate degradation products

www.ifosprogram.com
www.nutrasource.ca
Simple Procedure
After several trials of different sample
preparations.
Procedures of extraction and separation are too
tedious and not feasible for QC methods.
Oops!

www.ifosprogram.com
www.nutrasource.ca
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.

www.ifosprogram.com
www.nutrasource.ca
IR Spectrum for Oil Degradation Samples

www.ifosprogram.com
www.nutrasource.ca
IR Spectrum for Oil Degradation Samples

www.ifosprogram.com
www.nutrasource.ca
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.
www.ifosprogram.com
www.nutrasource.ca
Chromatograms of
p-Anisidine Value Test Preparations
Blank, Oil Sample and Reacted

www.ifosprogram.com
www.nutrasource.ca
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!!
www.ifosprogram.com
www.nutrasource.ca
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)

www.ifosprogram.com
www.nutrasource.ca
p-Anisidine HPLC-UV Method
Specificity Chromatogram

www.nutrasource.ca
p-Anisidine HPLC-UV Method
Typical Chromatogram of QL
(S/N=15, 3.162 µg/mL)

www.nutrasource.ca
p-Anisidine HPLC-UV Method
Linearity Study
(Range: 0.1002 to 0.5008 mg/mL)

www.nutrasource.ca
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%

www.nutrasource.ca
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%

www.nutrasource.ca
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

www.nutrasource.ca
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

www.nutrasource.ca
p-Anisidine HPLC-UV Method
Cross Reference of Known AV values
and Specification Set-up

www.nutrasource.ca
p-Anisidine HPLC-UV Method
Cross Reference of Known AV values
and Specification Set-up

www.nutrasource.ca
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.

www.nutrasource.ca
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.
© Nutrasource Diagnostics Inc.

www.nutrasource.ca
Novel Approaches to Omega-3 Stability Testing
Novel Approaches to Omega-3 Stability Testing
Novel Approaches to Omega-3 Stability Testing
Novel Approaches to Omega-3 Stability Testing
Novel Approaches to Omega-3 Stability Testing
Novel Approaches to Omega-3 Stability Testing
Novel Approaches to Omega-3 Stability Testing
Novel Approaches to Omega-3 Stability Testing
Acknowledgements
Diteba Team
Lucy Zhao
Linda Chang

Nutrasource Diagnostics Inc.
Team
Kevin Yan
Martha Wlodek
William Rowe
Mike Wlodek
© Nutrasource Diagnostics Inc.

www.nutrasource.ca
Thank you!

© Nutrasource Diagnostics Inc.

www.nutrasource.ca
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

www.nutrasource.ca
Novel Approaches to Omega-3 Stability Testing
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
www.ifosprogram.com
www.nutrasource.ca
p-Anisidine Value test is to determine the
secondary oxidation compounds in the oil
products. This spectrophotometric test is
easy to be interfered.

www.ifosprogram.com
www.nutrasource.ca

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Novel Approaches to Omega-3 Stability Testing

  • 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 www.nutrasource.ca
  • 2. p-Anisidine Value Analysis is for Determination of Oxidation Level of Oil Substance www.ifosprogram.com www.nutrasource.ca
  • 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) www.ifosprogram.com www.nutrasource.ca
  • 4. p-Anisidine Test is One of the Three Common Analytical Tests for Oil Oxidation • Acid Value • p-Anisidine Value • Peroxide Value www.ifosprogram.com www.nutrasource.ca
  • 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 ) ______________________________________________________ www.ifosprogram.com www.nutrasource.ca
  • 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. www.nutrasource.ca
  • 7. Oxidation Pathways of Oil Autooxidation www.nutrasource.ca
  • 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. www.ifosprogram.com www.nutrasource.ca
  • 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 www.ifosprogram.com www.nutrasource.ca
  • 11. Why Do We Have Problems with the pAnisidine Value Test? www.ifosprogram.com www.nutrasource.ca
  • 12. The Test Has No Problem to Evaluate and Compare the Oxidation Level of Simple Marine Oil. www.ifosprogram.com www.nutrasource.ca
  • 13. Marine Oil Products Changed When they Became Important Natural Health Products www.ifosprogram.com www.nutrasource.ca
  • 14. The Challenge Comes From the Advanced Formulated Products Containing Marine Oil and Other Additives www.ifosprogram.com www.nutrasource.ca
  • 15. New Ingredients Are Introduced • Colour additives • Flavour additives • Vitamin additives • Others www.ifosprogram.com www.nutrasource.ca
  • 16. The New Formulations Interfere With p-Anisidine Tests www.ifosprogram.com www.nutrasource.ca
  • 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. www.ifosprogram.com www.nutrasource.ca
  • 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?? www.ifosprogram.com www.nutrasource.ca
  • 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 www.ifosprogram.com www.nutrasource.ca
  • 20. Strategies Simple approach – new sample preparation to separate degradation products www.ifosprogram.com www.nutrasource.ca
  • 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! www.ifosprogram.com www.nutrasource.ca
  • 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. www.ifosprogram.com www.nutrasource.ca
  • 23. IR Spectrum for Oil Degradation Samples www.ifosprogram.com www.nutrasource.ca
  • 24. IR Spectrum for Oil Degradation Samples www.ifosprogram.com www.nutrasource.ca
  • 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. www.ifosprogram.com www.nutrasource.ca
  • 26. Chromatograms of p-Anisidine Value Test Preparations Blank, Oil Sample and Reacted www.ifosprogram.com www.nutrasource.ca
  • 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!! www.ifosprogram.com www.nutrasource.ca
  • 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) www.ifosprogram.com www.nutrasource.ca
  • 29. p-Anisidine HPLC-UV Method Specificity Chromatogram www.nutrasource.ca
  • 30. p-Anisidine HPLC-UV Method Typical Chromatogram of QL (S/N=15, 3.162 µg/mL) www.nutrasource.ca
  • 31. p-Anisidine HPLC-UV Method Linearity Study (Range: 0.1002 to 0.5008 mg/mL) www.nutrasource.ca
  • 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% www.nutrasource.ca
  • 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% www.nutrasource.ca
  • 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 www.nutrasource.ca
  • 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 www.nutrasource.ca
  • 36. p-Anisidine HPLC-UV Method Cross Reference of Known AV values and Specification Set-up www.nutrasource.ca
  • 37. p-Anisidine HPLC-UV Method Cross Reference of Known AV values and Specification Set-up www.nutrasource.ca
  • 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. www.nutrasource.ca
  • 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. © Nutrasource Diagnostics Inc. www.nutrasource.ca
  • 48. Acknowledgements Diteba Team Lucy Zhao Linda Chang Nutrasource Diagnostics Inc. Team Kevin Yan Martha Wlodek William Rowe Mike Wlodek © Nutrasource Diagnostics Inc. www.nutrasource.ca
  • 49. Thank you! © Nutrasource Diagnostics Inc. www.nutrasource.ca
  • 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 www.nutrasource.ca
  • 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 www.ifosprogram.com www.nutrasource.ca
  • 53. p-Anisidine Value test is to determine the secondary oxidation compounds in the oil products. This spectrophotometric test is easy to be interfered. www.ifosprogram.com www.nutrasource.ca