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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. Ste...
p-Anisidine Value Analysis is for Determination
of Oxidation Level of Oil Substance

www.ifosprogram.com
www.nutrasource.c...
p-Anisidine Value Analysis Procedure Has
Been Included in Many Official Methods
1. The United States Pharmacopeia and the
...
p-Anisidine Test is One of the
Three Common Analytical Tests
for Oil Oxidation
• Acid Value
• p-Anisidine Value
• Peroxide...
Acceptable Limits for Oxidative
Stability Parameters in Marine Oils
(Recommended Reporting Threshold)
____________________...
Oxidation Pathways of Oil
Autooxidation

• Free radical formation starts the chain
reaction of degradation to unsaturated ...
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 ...
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...
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.nutraso...
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
w...
New Ingredients Are Introduced
• Colour additives
• Flavour additives
• Vitamin additives
• Others

www.ifosprogram.com
ww...
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-anisi...
There should be a procedure
available to solve this issue.
1. A lot of research work on oil oxidation has
been nicely done...
There are Two Groups of Analytical Methods
Routine Test Methods

Research Methods

Advantages
• Fast
• Easy to be performe...
Strategies
Simple approach – new sample preparation to
separate degradation products

www.ifosprogram.com
www.nutrasource....
Simple Procedure
After several trials of different sample
preparations.
Procedures of extraction and separation are too
te...
Advanced Technologies
1. GC-Flame Ionization Detector. Too complicated to
identify and quantify the oxidized compounds.
2....
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 con...
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...
p-Anisidine HPLC-UV Method
Chromatographic Conditions
HPLC column, 4.6 mm × 250 mm
Column Temperature:
25˚C
Detection:
UV ...
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....
p-Anisidine HPLC-UV Method
Summary of Accuracy Results
Concentration Level

% Recovered

Average

99
50%

99

99%
RSD = 0%...
p-Anisidine HPLC-UV Method
Summary of System Suitability Study
Injection No.

Retention
Time
(Minutes)

Peak Area
(μV*sec)...
p-Anisidine HPLC-UV Method
Summary of System Suitability Study
Injection No.

Retention
Time
(Minutes)

Peak Area
(μV*sec)...
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 T...
Conclusion
1. The method is validated. It has
good specificity, good precision
and accuracy.
2. The most important feature...
Acknowledgements
Diteba Team
Lucy Zhao
Linda Chang

Nutrasource Diagnostics Inc.
Team
Kevin Yan
Martha Wlodek
William Rowe...
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
Vita...
Biomedical Research Findings Support Its
Usage with Important Indications
1. Cardiovascular diseases (CVDs) are the number...
p-Anisidine Value test is to determine the
secondary oxidation compounds in the oil
products. This spectrophotometric test...
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
Novel Approaches to Omega-3 Stability Testing
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Novel Approaches to Omega-3 Stability Testing

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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.

Published in: Technology
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Novel Approaches to Omega-3 Stability Testing

  1. 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. 2. p-Anisidine Value Analysis is for Determination of Oxidation Level of Oil Substance www.ifosprogram.com www.nutrasource.ca
  3. 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. 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. 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. 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. 7. Oxidation Pathways of Oil Autooxidation www.nutrasource.ca
  8. 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. 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
  10. 10. www.ifosprogram.com www.nutrasource.ca
  11. 11. Why Do We Have Problems with the pAnisidine Value Test? www.ifosprogram.com www.nutrasource.ca
  12. 12. The Test Has No Problem to Evaluate and Compare the Oxidation Level of Simple Marine Oil. www.ifosprogram.com www.nutrasource.ca
  13. 13. Marine Oil Products Changed When they Became Important Natural Health Products www.ifosprogram.com www.nutrasource.ca
  14. 14. The Challenge Comes From the Advanced Formulated Products Containing Marine Oil and Other Additives www.ifosprogram.com www.nutrasource.ca
  15. 15. New Ingredients Are Introduced • Colour additives • Flavour additives • Vitamin additives • Others www.ifosprogram.com www.nutrasource.ca
  16. 16. The New Formulations Interfere With p-Anisidine Tests www.ifosprogram.com www.nutrasource.ca
  17. 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. 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. 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. 20. Strategies Simple approach – new sample preparation to separate degradation products www.ifosprogram.com www.nutrasource.ca
  21. 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. 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. 23. IR Spectrum for Oil Degradation Samples www.ifosprogram.com www.nutrasource.ca
  24. 24. IR Spectrum for Oil Degradation Samples www.ifosprogram.com www.nutrasource.ca
  25. 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. 26. Chromatograms of p-Anisidine Value Test Preparations Blank, Oil Sample and Reacted www.ifosprogram.com www.nutrasource.ca
  27. 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. 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. 29. p-Anisidine HPLC-UV Method Specificity Chromatogram www.nutrasource.ca
  30. 30. p-Anisidine HPLC-UV Method Typical Chromatogram of QL (S/N=15, 3.162 µg/mL) www.nutrasource.ca
  31. 31. p-Anisidine HPLC-UV Method Linearity Study (Range: 0.1002 to 0.5008 mg/mL) www.nutrasource.ca
  32. 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. 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. 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. 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. 36. p-Anisidine HPLC-UV Method Cross Reference of Known AV values and Specification Set-up www.nutrasource.ca
  37. 37. p-Anisidine HPLC-UV Method Cross Reference of Known AV values and Specification Set-up www.nutrasource.ca
  38. 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. 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
  40. 40. 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
  41. 41. Thank you! © Nutrasource Diagnostics Inc. www.nutrasource.ca
  42. 42. 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
  43. 43. 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
  44. 44. 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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