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Carotenoids quantification in Cassava

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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.

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Carotenoids quantification in Cassava

  1. 1. www.iita.orgA member of CGIAR consortium Carotenoids quantification in Cassava Food and Nutrition Sciences Laboratory Dr. Bussie Maziya-Dixon February, 2017
  2. 2. www.iita.orgA member of CGIAR consortium 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
  3. 3. www.iita.orgA member of CGIAR consortium  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
  4. 4. www.iita.orgA member of CGIAR consortium 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
  5. 5. www.iita.orgA member of CGIAR consortium 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
  6. 6. www.iita.orgA member of CGIAR consortium - 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
  7. 7. www.iita.orgA member of CGIAR consortium 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
  8. 8. www.iita.orgA member of CGIAR consortium 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
  9. 9. www.iita.orgA member of CGIAR consortium - 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.
  10. 10. www.iita.orgA member of CGIAR consortium 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
  11. 11. www.iita.orgA member of CGIAR consortium 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.
  12. 12. www.iita.orgA member of CGIAR consortium 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
  13. 13. www.iita.orgA member of CGIAR consortium 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
  14. 14. www.iita.orgA member of CGIAR consortium 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.
  15. 15. www.iita.orgA member of CGIAR consortium 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
  16. 16. www.iita.orgA member of CGIAR consortium 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).
  17. 17. www.iita.orgA member of CGIAR consortium 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
  18. 18. www.iita.orgA member of CGIAR consortium 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
  19. 19. www.iita.orgA member of CGIAR consortium 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
  20. 20. www.iita.orgA member of CGIAR consortium 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
  21. 21. www.iita.orgA member of CGIAR consortium 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
  22. 22. www.iita.orgA member of CGIAR consortium Literature consulted; Harvest Plus Handbook of Carotenoids Analysis. Delia B., Rodriguez-Amaya and Mieko Kimura 2004 Technical Monogram Series 2.
  23. 23. www.iita.orgA member of CGIAR consortium Thank you FoR LISTEnInG !.

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