Novel Approaches to Omega-3 Stability Testing

Novel Approaches toOmega-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. [email protected]

www.nutrasource.ca

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

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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)

p-Anisidine Test is One of the Three Common Analytical Tests

for Oil Oxidation


• Acid Value• p-Anisidine Value

• Peroxide Value

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 )

______________________________________________________

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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Oxidation Pathways of Oil Autooxidation

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Traditional p-Anisidine Value Test


1. 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.

2. 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.

3. 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.

.

Traditional P-Anisidine Value Test


4. 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.

5. 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

Why Do We Have Problems with the p-Anisidine Value Test?


The Test Has No Problem to Evaluate and Compare the Oxidation Level of Simple

Marine Oil.


Marine Oil Products Changed When they Became Important Natural Health Products


The Challenge Comes From the Advanced Formulated Products Containing Marine Oil

and Other Additives


New Ingredients Are Introduced


• Colour additives• Flavour additives• Vitamin additives

• Others

The New Formulations Interfere

With p-Anisidine Tests


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.

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??

There are Two Groups of Analytical Methods


Routine Test Methods

Advantages• Fast• Easy to be performed• Used in industry for century?• Cost effective• Common Apparatus or

InstrumentDisadvantages:• Not specific• Not directly quantitative• Easily Interfered

Research Methods

Advantages• Relatively Specific• Can be quantitativeDisadvantages:• Time consuming• Cost• Require instruments• Difficult to be used in QC

environment

Strategies


Simple approach – new sample preparation to separate degradation products

Simple Procedure


After several trials of different sample preparations.

Procedures of extraction and separation are too tedious and not feasible for QC methods.

Oops!

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.

IR Spectrum for Oil Degradation Samples


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.

Chromatograms of p-Anisidine Value Test Preparations

Blank, Oil Sample and Reacted


Channel @ PDA 245.0 nm; Acquired By yingluz

AU

0.00

0.02

0.04

0.06

0.08

Minutes

0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00

Channel @ PDA 245.0 nm; Acquired By yingluzAU

0.00

0.02

0.04

0.06

0.08

0.10

Minutes

0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00

Channel @ PDA 245.0 nm; Acquired By yingluz

isoo

ctan

e -

3.15

6

AU

0.00

0.02

0.04

Minutes0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00

What do we need for this method?


1. Simple routine method2. Economical and practical3. Relatively specific4. Relatively precise and accurate

All approaches explored are Not doable and Not All approaches explored are Not doable and Not affordable!affordable!

What else? Nothing!!

p-Anisidine HPLC-UV Method


Chromatographic Conditions

HPLC column, 4.6 mm × 250 mmColumn Temperature: 25˚CDetection: UV at 254nm

Injection Volume: 10µL Flow Rate: 1.0 mL/minute

Run Time: 10 minutes Mobile Phase: Hexane: 2-Propanol (50:50)


Specificity Chromatogram

Channel @ W2996 PDA 254.0 nm at 1.2

AU

-0.05

0.00

0.05

0.10

0.15

0.20

Minutes

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

Injection Peaks

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Typical Chromatogram of QL(S/N=15, 3.162 µg/mL)

Channel @ W2996 PDA 254.0 nm at 1.2

3.30

5

p-A

nisi

dine

- 4.

855

AU

0.000

0.001

0.002

0.003

0.004

0.005

Minutes

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

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p-Anisidine HPLC-UV MethodLinearity Study

(Range: 0.1002 to 0.5008 mg/mL)Equation Y = 8.49e+006 X - 7.06e+003R 2̂ 0.999889

R 0.999944 Cal Curve Id 5424

Norm Intercept/Slope -0.002770 Residual Sum of Squares 8.044077e+008

Calibration Plot

Are

a

0

1x106

2x106

3x106

4x106

5x106

Am ount

0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50

1

2

3

4

5

Mean

% RSD

Nam e X Value Response Calc. Value Manual Ignore RF

p-Anis idine

p-Anis idine

p-Anis idine

p-Anis idine

p-Anis idine

0.100200

0.200300

0.300500

0.400600

0.500800

857435.001071

1686026.997872

2530386.492799

3381242.004378

4259300.996942

2542878.298613

52.85

0.101871

0.199510

0.299008

0.399271

0.502740

No

No

No

No

No

No

No

No

No

No

8557235.539636

8417508.726270

8420587.330447

8440444.344429

8504994.003479

8468154.0

0.7

Peak: p-Anis idine

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p-Anisidine HPLC-UV MethodRepeatability Results

SampleNo.

Assay (%)

RepeatabilityIntermediate

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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p-Anisidine HPLC-UV Method Summary of Accuracy Results

Concentration Level % Recovered Average

50%

99

99%RSD = 0%

99

99

100%

100

100%RSD =0 %

100

100

150%

101

101%RSD =1 %

101

102

Average of 9 samples = 100% RSD = 1%

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p-Anisidine HPLC-UV MethodSummary of System Suitability Study

Injection No.Retention

Time(Minutes)

Peak Area (μV*sec)

USP Tailing Factor

Theoretical 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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p-Anisidine HPLC-UV MethodCross Reference of Known AV values

and Specification Set-up

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Specification Set-up

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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.

Conclusion1. 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 p-Anisidine value test.

3. The method shows good characteristics of a quality control method.

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© Nutrasource Diagnostics Inc.

Acknowledgements

Diteba Team Lucy Zhao

Linda ChangNutrasource Diagnostics Inc.

TeamKevin Yan

Martha Wlodek William RoweMike Wlodek

www.nutrasource.ca


Thank you!Thank you!

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p-Anisidine HPLC-UV MethodRecommended Test Criteria

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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

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 preventable4. Omega-3s EPA and DHA can help by lowering blood

triglycerides , preventing fatty deposits and reducing blood pressure

p-Anisidine Value test is to determine the secondary oxidation compounds in the oil products. This spectrophotometric test is

easy to be interfered.


Novel Approaches to Omega-3 Stability Testing

Technology

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