Carotenoids quantification in Cassava

www.iita.orgA member of CGIAR consortium
Carotenoids quantification in Cassava
Food and Nutrition Sciences Laboratory
Dr. Bussie Maziya-Dixon
February, 2017

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

 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

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

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

- 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

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

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

- 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 -800
C storage condition.

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

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.

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

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

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.

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

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 factor
volume = total volume of extract 25 ml
A1%1cm = absorption coefficient of carotene in PE (2592).

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

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.
Identification
452
478

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= 0.999
Y=964.1x + 101.45

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

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

Literature consulted;
Harvest Plus Handbook of Carotenoids Analysis.
Delia B., Rodriguez-Amaya and Mieko Kimura 2004
Technical Monogram Series 2.

Thank you FoR LISTEnInG !.

Carotenoids quantification in Cassava

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