HPLC determination of bioactive components in oil …...HPLC determination of bioactive components...

HPLC determination of bioactive components in oil for varietal screening Clare Flakelar BSc(Hons) PhD Student Charles Sturt University, Wagga Wagga, Australia

Saskatoon Canada July 2015

Valuable Minor Components

Carotenoids 70-130 ppm

Tocopherols 700-1200 ppm

• Vitamin E • Natural Antioxidants • Disease fighting

• Vitamin A activity • Skin and eye health • Lutein and β-Carotene

Sterols 7000 - 10000 ppm

• Lowers LDL cholesterol • Cardiovascular benefits

Introduction and

background

Research focus and

aims

Method development Results

Conclusion and future research

Introduction and

background

Research focus and

aims

Method development Results

Conclusion and future research

Current commercial processing losses

- 40% of sterols

- 50% of tocopherols

- 90% of carotenoids

CRUDE REFINED

Introduction and

background

Research focus and

aims

Method development Results

Conclusion and future research

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Ster

ol C

once

ntra

tion

(ppm

)

ppm

Effect of processing on bioactive components

Tocopherols Carotenoids Sterols

Where is research and industry up to? • Methods published for simultaneous determination of

compounds but few on three different classes

• NP methods for sterols -> require MS for quantification of individual sterols due to co-elution

• Currently sample pre-treatment techniques and analysis times are lengthy

• Very few reports on simultaneous quantification of free and

esterified sterols (none for oils)

Introduction and

background

Research focus and

aims

Method development Results

Conclusion and future research

• Publication

Introduction and

background

Research focus and

aims

Method development Results

Conclusion and future research

Introduction and

background

Research focus and

aims

Method development Results

Conclusion and future research

• To develop a method to simultaneously measure the main bioactive compounds (tocopherols, carotenoids and sterols) in canola oil

• Method requirements: 1. Minimal sample preparation 2. Short, reproducible run time 3. Satisfactory elution of compounds

SOLVENTS • Hexane used as the solvent for

extraction, sample preparation, dilutions and main HPLC solvent

• Ethyl acetate used with hexane

in a gradient elution

Introduction and

background

Research focus and

aims

Method development Results

Conclusion and future research

INSTRUMENTATION • A Varian Star 9010 binary

pump • Agilent 1200 series

autosampler • Phenomenex luna silica

column (150 mm x 4.6 mm, 3 µm)

Introduction and

background

Research focus and

aims

Method development Results

Conclusion and future research

DETECTORS • Agilent diode array detector

(DAD) • Agilent 6410 Triple quadrupole

tandem mass spectrometer (MS/MS) detector.

Introduction and

background

Research focus and

aims

Method development Results

Conclusion and future research

Introduction and

background

Research focus and

aims

Method development Results

Conclusion and future research

Method validation • Co-elution influence – very small • Verification of free sterol and sterol esters

Unsaponified oil Saponified oil

Introduction and

background

Research focus and

aims

Method development Results

Conclusion and future research

MS chromatogram of standard

TIC

Introduction and

background

Research focus and

aims

Method development Results

Conclusion and future research

Introduction and

background

Research focus and

aims

Method development Results

Conclusion and future research

MS chromatogram of oil sample

TIC

Introduction and

background

Research focus and

aims

Method development Results

Conclusion and future research

MS chromatogram of oil sample

Introduction and

background

Research focus and

aims

Method development Results

Conclusion and future research

Introduction and

background

Research focus and

aims

Method development Results

Conclusion and future research

Introduction and

background

Research focus and

aims

Method development Results

Conclusion and future research

Tocopherols (mg/kg oil) Carotenoids (mg/kg oil)

α-tocopherol γ-tocopherol δ-tocopherol β-carotene Lutein

crude canola 387.5 717.9 14.0 4.09 31.6

RBD canola 475.5 663.4 15.4 n.d. n.d.

Carotino blend 319.6 443.7 28.9 255 n.d.

sunflower 714.0 72.4 16.7 1.38 n.d.

olive 261.2 n.d. n.d. 3.08 9.76

*Average values, For crude canola oil n=9, commercial oils n = 2

Edible oil samples

Introduction and

background

Research focus and

aims

Method development Results

Conclusion and future research

Free sterols (mg/kg oil)* Esterified sterols (mg/kg oil)*

β-sitosterol Campesterol Brassicasterol β-sitosterol Campesterol Brassicasterol

crude canola 1979 1628 1093 761.9 1383 554.8

RBD canola 2122 2025 854.6 811.9 1712 422.8

Carotino blend 1469 1321 549.2 721.8 1423 386.9

sunflower 1481 391.4 21.85 403.2 162.1 39.66

olive 1176 89.78 n.d. 61.48 15.76 n.d.

*Average values, For crude canola oil n=9, commercial oils n = 2

• New method developed for simultaneous analysis of bioactive compounds

• To be used for further studies for larger data sets • Has now been applied to a number of vegetable oils and

further applications

Conclusions

Introduction and

background

Research focus and

aims

Method development Results

Conclusion and future research

ACKNOWLEDGEMENTS Contact Email: cflakelar@csu.edu.au Twitter: @clflake Supervisors Assoc. Prof. Paul Prenzler (Principal Supervisor) Dr Greg Doran Dr Julia Howitt Assoc. Prof. David Luckett

THANK YOU – ANY QUESTIONS?

Affiliates / Funding

Introduction and

background

Research focus and

aims

Method development Results

Conclusion and future research