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Carotenoids quantification in Cassava
Food and Nutrition Sciences Laboratory
Dr. Bussie Maziya-Dixon
February, 2017
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Outline
Introduction Carotenoids of cassava Field sampling Sample preparations Extraction Quantification of Total
Carotenoids using UV-Vis Spectrophotometer
Separation of carotenoids - High Performance Liquid Chromatography (HPLC)
Identification & quantification Special precautions during
carotenoids analysis Quality Control Near Infrared Reflectance
Spectroscopy
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Carotenoids are one of the most essential groups of natural pigments, with wide distribution in food crops, structural diversity and numerous functions in the biological systems.
They are a class of over 750 pigment synthesized by plants, algae and photosynthetic bacterial.
Carotenoids are the precursor of vitamin A & are powerful antioxidants that help in preventing some form of cancer and other degenerative diseases. Carotenoids cannot be produced by human and therefore needs to be obtained from the diet.
1.0 Introduction
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Classification of Carotenoids
Carotene• Oxygen free Carotenoids which
contains only carbon & Hydrogen.
• Readily soluble in petroleum Ether & hexane.
• Found in yellow roots cassava and carrots and gives bright orange color.
• E.g. Lycopene, β- Carotene
Xanthophyl• Contains 1 or more Oxygen atoms
and other groups such as hydroxy, epoxy, keto, carboxy and methoxy groups.
• Dissolve best in Methanol & Ethanol.
• Generally yellow in color.
• E.g. Lutein, Zeaxanthin
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Structurally, an important characteristic of carotenoids are the
extended conjugated double-bonds system, which constitutes
the light-absorbing chromophore that gives carotenoids their
unique color. This also provides the visible absorption
spectrum that serves as a basis for their identification and
quantification.
Chemical Structure
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- Hydrocarbon carotenoids (e.g.,
β-carotene, α-carotene, lycopene)
are known as carotenes, and
oxygenated derivatives are called
xanthophylls.
- Oxygen substituted carotenoids
are β-cryptoxanthin, Lutein,
Zeaxanthin, canthaxanthin and
violaxanthin amongst others.
β-carotene
β-cryptoxanthin
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Provitamin A carotenoids are β-carotene, α-carotene, and
β-cryptoxanthin. β−carotene which is the most potent Provitamin A
is predominant in cassava; studies have shown that cassava contains
about 90% of β−carotene.
Carotenoids must have an unsubstituted β-ring with an 11
conjugated-carbon polyene chain to have vitamin A activity.
Therefore α-carotene and β-cryptoxanthin exhibits about 50% of
vitamin A activity of β-carotene in cassava.
2. 0 Carotenoids of cassava
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Sampling is one of the fundamental factors that determines the reliability
of analytical data obtained from sample analysis. The aim of sampling is
to secure a portion of the material that is representative of the entire lot
under investigation.
However, the predisposition of carotenoids to certain environmental
factors such as heat and sunlight requires the selection of healthy samples
and careful handling during harvesting and transport to the laboratory for
subsequent analysis.
3.0 Field Sampling
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- Maturity
- Post harvest physiological degradation
- Sample quantity
- Storage and analysis time
• Matured and healthy tubers with no traces of cuts should be selected during field sampling
• Harvesting should be before sun rises and sent to the laboratory as soon as possible.
• 3 yellow flesh big, medium and small sized tubers should be provided for each cultivar under investigation.
• Samples should be processed within 24 hours after harvest, analyzed immediately or stored under -20 or -800C storage condition.
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Sample preparation is done to homogenize the large sample brought into
the laboratory and subsequently reduce to the sample size needed for
analysis, while at the same time maintaining its integrity and
representativity
Three storage roots of different sizes (large, medium and small) for each
variety under study is washed thoroughly with potable water to remove
dirt and adhering sand particles and air-dried on a clean concrete surface.
4.0 Sample Preparation
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The storage roots are then peeled manually using a stainless steel knife,
rinsed with de-ionized water, and cut longitudinally (from the proximal end
to the distal end) into four equal parts. Two opposite sections from each root
is combined, manually chopped into small pieces and mixed thoroughly.
This is wrapped in an aluminum foil and then transferred into a properly
labeled whirl pak for subsequent analysis. All sample preparations are done
under subdued light.
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A portion of about 10g of homogenous sample is weighed into a mortar
and about 3g of hyflosupercel (celite) is added. The mixture is ground
with 50 ml of cold acetone. After proper maceration in the mortal, the
mixture is filtered with suction using a buchner funnel with filter paper.
The mortar, pestle, funnel, and residue are washed with small amounts of
acetone, receiving the washings in the suction flask through the funnel.
Extraction is repeated 3-4 times until the final residue washed with
acetone is devoid of color.
5.0 Extraction with cold acetone
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Partitioning to petroleum ether
About 20ml of petroleum ether (PE) is
transfered into a 500 ml separatory funnel
with teflon stop-cock and the acetone
extract is added. 300mls of distilled water
is slowly added, allowing to flow along
the walls of the funnel without shaking to
avoid formation of an emulsion. The two
phases are allowed to separate and the
aqueous lower phase is discarded.
PE phase
Aqueous phase
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For about 3-4 times, 200ml of distilled water is used to wash to remove
residual acetone. The PE phase is collected in a 25ml volumetric flask
making the solution pass through a small funnel containing anhydrous
sodium sulfate (about 15 g) to remove residual water. Volume is made
up to mark using PE and proceed to spectrophotometric measurements.
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7.0 Quantification of Total carotenoids
Carotenoids in solution obey the Beer–Lambert law, that is, their absorbance is directly proportional to the concentration. Therefore total carotenoids are quantified using a UV/Vis spectrophotometer.
Final extract from the partitioning step is made up to mark in the volumetric flask and read on the spectrophotometer using PE as a blank.
UV-Vis Spectrophotometer
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The Total carotenoids (TC) content is calculated using the formula:
TC (µg/g) = A x volume (ml) x DF x 104
A 1%1cm x sample weight (g)
where A= absorbance; DF= Dilution factorvolume = total volume of extract 25 mlA1%1cm = absorption coefficient of carotene in PE (2592).
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8.0 Separation of carotenoids using High Performance Liquid Chromatography (HPLC)
High Performance Liquid Chromatography system (HPLC)
Separation of carotenoids in cassava is carried out using Waters e2695 HPLC systems equipped with a Photodiode Array (PDA) Detector. The PE extract is concentrated and dried down under nitrogen gas and reconstituted in 1 ml of dichloromethane: methanol (50:50), this is filtered through 0.22mm PTFE syringe filter (Millipore) directly into injection vials and 10µL is injected into the system.
Chromatographic conditions:
Mobile Phase: 50% MTBE : 50 % MeOH
Polymeric Column: YMC C30, 5µm, 4.6 x 250 mm
Isocratic elution for 10min
Flow rate: 1ml/min
Equilibration: 10min
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The chromatographic behaviour and the
UV/visible absorption spectrum provide the
basis for the identification of carotenoids.
Both the wavelengths of maximum
absorption (λ max) and the shape of the
spectrum (spectral fine structure) are
characteristic of the chromophore.
Identification452
478
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Quantification
Carotenoids are quantified using a
calibration plot of peak area vs conc.
obtained from the injection of
commercial standard solutions for each
carotenoids prepared at varying
concentrations. Carotenoids
concentrations in the samples are
extrapolated from the calibration graph.
The samples and standards are subjected
to the same chromatographic conditions.
R2= 0.999Y=964.1x + 101.45
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The major challenge in carotenoids analysis is their instability; therefore the following special precautionary measures are ensured to avoid quantitative losses of carotenoids during sample preparation and laboratory analysis. • completion of the analysis within the shortest possible time;
• protection from light, thus carotenoids analysis are done under subdued light
• avoiding high temperatures and contact with acids and other oxidizing agents, thus antioxidants such as Butylated Hydroxyl Toluene (BHT) is often added to solvents and standards.
• Use of high purity solvents which are free from impurities.
10.0 Special precautions in carotenoids analysis
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Quality Control
• For preparation and validation of calibration curves
• To check the suitability of the chromatographic systems as well as a test for accuracy of analytical data
• These are used regularly as checks during each batches of sample run as accuracy.
• Duplicate runs of each sample is done to test for precision
• Annual PM is done to ensure optimum system performance
• Only HPLC grade solvents are used for analysis
Commercial Standards
Standard Reference Materials(SRM)
Selected Cassava varieties with high β-carotene
Replicate analysis
Performance Maintenance (PM) of HPLC systems
Purity of solvents
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Literature consulted;
Harvest Plus Handbook of Carotenoids Analysis.
Delia B., Rodriguez-Amaya and Mieko Kimura 2004
Technical Monogram Series 2.
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Thank you FoR LISTEnInG !.