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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
010002000300040005000600070008000
0100200300400500600700
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: [email protected] 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