Carotenoids quantification in Cassava

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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 -800C 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 factorvolume = total volume of extract 25 mlA1%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.

Identification452

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= 0.999Y=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 !.

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Carotenoids quantification in Cassava