chapter 8: Analysis of selected Vitamins, Minerals

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chapter 8 Analysis of selected Vitamins,
Minerals Non-nutritional additives
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Vitamin analysis

• Generally difficult to measure vitamin content of
  food
• chemically heterogeneous group of essential
  micronutrients
• present in very small amounts
• often unstable during analysis
• Bioassays using humans or animals
• Microbiological assays
• Physico-chemical assays
• titrimetric / spectrophotometric
• fluorimetry
• chromatographic
• enzymatic

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Example of a bioassay for the analysis of vitamins

• Vitamin D
• rats fed vit D depleted diet for 18-25 days
• groups of rats fed (8 to 11 days) known amounts
  of vitamin D
• other groups of rats fed food sample (8 to 11
  days) being analysed
• vitamin D potency of the sample is measured as
  bone calcification level at end of tibia bone
  compared to calcification in rats fed known
  amount of vit D

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Example of a microbiological assay for vitamins

• Limited to water soluble vitamins
• Growth of specific microorganism in extract of
  vitamin containing sample is compared with growth
  of the microorganism in presence of known amounts
  of the vitamin
• Growth measure in terms of
• turbidity
• acid production
• gravimetry
• respiration

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Niacin microbiological assay procedure

• Sample dissolved autoclaved in 1N H2SO4
• Range of dilutions of sample extract made in
  assay broth autoclave (1hr at 121C)
• Range of niacin standards made in same way as
  sample autoclave (10 min. at 121C)
• Inoculated with Lactobacillus plantarum,
  incubated 37oC for 16-18 hr
• Measure turbidity by absorbance at any wavelength
  between 540nm and 660nm.
• Compare absorbance of sample incubations with
  standards to calculate niacin content of
  original sample

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HPLC analysis of vitamins- vitamin E (Tocopherols
Tocotrienols)

• Vitamin E is present in food as eight compounds
  (?-?-?- and ?-) tocopherols and tocotrienols
• In order to estimate the Vit E activity ?
  tocopherol equivalent
• quantification of each Vit E form is required

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Structure of vitamin E Tocopherols Tocotrienols
RI
RII
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• Method
• saponify
• mix with 6 (wt/vol) pyrogallol in ethanol, heat
  at 70C sonicate
• add 60 KOH solution digest at 70C sonicate
• extract with hexane
• filter inject into normal phase HPLC
• use florescence detector at 290 and 330 nm
• quantify by external standard from peak area by
  linear regression

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Example of a titrimetric analysis of vitamins

• 2, 6-Dichloroindophenol (DCPIP or DPIP)
  titrimetric method for ascorbic acid
• Sample homogenised in metaphosphoric acetic acid
  solution
• Filtered and diluted
• Standard solutions of ascorbic acid prepared
• Standards and samples titrated to pink endpoint
  with dichloroindophenol dye
• mg ascorbic acid/ml sample C x V x (DF / WT)
• C mg ascorbic acid/mL dye
• V ml of dye used for titration of diluted
  sample
• DF dilution factor
• WT sample wgt, g

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Example of fluorimetric analysis of vitamins

• Fluorometric thiamin (vitamin B1) determination
• sample and standards dissolved in HCl
• enzymatic hydrolysis of phosphate esters of
  thiamin
• sample clean up using ion exchange cartridge
• converte thiamin to thiachrome using potassium
  ferrocyanide
• immiscible isobutyl alcohol added, shaken and
  left to separate into two layers
• isobutyl alcohol upper layer now containing
  thiochrome decanted off into fluorescence tube
• fluorescence measured (at 365-435nm) compared
  to standard solutions

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Comparison of methods used for vitamin analysis

• Bioassays
• extremely time consuming
• dont necessarily require preparation of an
  extract
• limited to animals rather than humans
• Microbiological
• limited to water extractable vitamins
• require vitamin extraction but less sample
  preparation than physicochemical assays
• not necessarily measure of bioavailability in
  humans
• Physicochemical (require vit. Extraction)
• relatively simple, accurate precise
• can involve high capital outlay (ie HPLC)
• establishing the peak identity is essential

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ASH ANALYSIS

• Ash inorganic residue remaining after either
  ignition or complete oxidation of organic matter
  in a foodstuff
• dry ashing (proximate analysis)
• whole grain, cereals and dried vegetables
• wet ashing (oxidation, preparation for elemental
  analysis)
• meat and meat products
• microwave (low temperature ashing)
• volatile elements
• Ash content of fresh food is rarely gt5

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Dry ashing

• Sample (5-10 g) weighed into
  crucible and pre-prepared
• Ignited in muffle furnace 12-18 hr, 550oC
• water volatiles vapourised
• organic substances oxidised to water vapour,
  carbon dioxide, and oxides of nitrogen
• minerals converted to oxides, sulphates,
  phosphates, chlorides and silicates
• some elements may be partially lost through
  volatilisation eg. Fe, Se, Pb, Hg
• Cool furnace and open door carefully as ash may
  be fluffy
• Cool in desiccator and calculate ash weight as
  percentage of original sample

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Wet ashing

• Wet oxidising of organic substances
• Place 1g dried sample of food in H2SO4 HNO3
• Heated to 200?C on hot plate in fume-hood
  brown-yellow fume will evolve
• sample should become colourless
• Cool and transfer oxidised food solution to 50 mL
  volumetric flask
• Make to volume with ultra pure water
• Follow wash down procedure for fume-hood

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Microwave ashing

• Totally automated
• May carry out dry or wet ashing
• Wet ashing is preformed in open or closed vessels
  (Teflon, quartz or Pyrex) which withstand
  pressures of gt1500psi
• acids may be heated past their boiling point
• ensures complete digestion in 30 min.
• permits use of nitric acid when normally we would
  require sulfuric acid

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• Time, temperature, pressure microwave power
  parameters are adjustable
• may ramp the temperature according to
  preprogramming
• Dry ashing may reach up to 1200C
• uses the same protocol and crucibles as muffle
  furnace ashing
• generally 20 min. in a microwave oven is equal to
  4hr in a muffle furnace

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Analysis of specific minerals

• Flame photometry and atomic absorbtion
  spectroscopy
• EDTA complexation titration
• Redox reactions
• Precipitation titration
• Colorimetric methods
• Ion selective electrodes

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Contamination

• Milling and grinding with steel grinders
• Old glassware can contaminate samples for
  micro-elemental analysis
• glass is acid washed triple rinsed with ultra
  pure water
• Solvents including water may contain high amounts
  of minerals
• need pure reagents high in cost
• run reagent blank

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EDTA complexometric titration

• Formation of stable complexes of metal ions with
  ethylenediaminetetraacetic acid (EDTA)
• except alkali metals (Na)
• Via the presence of donor oxygen and nitrogen
  atoms EDTA is able to form six, five member
  chelate rings

• Fennema, 1996 p.625

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Water hardness - EDTA titration

• Adjust water sample pH to 10 by adding buffer
  solution (NH4OH Na2EDTA MgCl2) and Calmagite
  indicator solution
• Titrate with 0.01 EDTA to a blue endpoint
• This method is suitable to assess Ca in ashed
  fruits and vegetables

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• Add magnesium salt and enough EDTA to bind all
  magnesium.
• In buffer solution the Ca replaces the Mg bound
  to the EDTA.
• The free magnesium binds to Calmagite,
• pink magnesium Calmagite complex persists until
  all Ca in the sample has been titrated with the
  EDTA.
• Excess EDTA removes Mg from Clamagite and
  produces a blue endpoint

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Redox reactions for mineral measurement

• Revision!
• oxidation
• addition of oxygen OR removal of hydrogen
• removal of electrons increase in positive
  charge
• reduction
• removal of oxygen OR addition of hydrogen
• addition of electrons reduction in positive
  charge

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Iron determination using redox reaction and
colorimetry

• Food sample dried and ashed
• Dissolve ash in HCl and filter with rinsing
• Add aliquot of filtrate containing iron to
  solution of hydroxylamine hydrochloride
• Add acetate buffer and colour developing reagent
  such as 0.1 orthophenanthroline
• Dilute and read absorbance of colour at 510 nm
• To calculate iron content compare absorbance of
  sample to standard curve generated by known
  concentrations of iron chloride
• prepared by dissolving analytical grade iron
  wire in HCl

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Precipitation (Mohr) titration analysis of salt
in butter

• Mohr titration Based on formation of an orange
  coloured solid, silver chromate after silver from
  silver nitrate has complexed with all available
  chloride
• AgNo3 NaCl ? AgCl (Cl- is complexed) NaNo3
• 2AgCl K2CrO4 ? Ag2CrO4 (orange all Cl- is
  complexed) 2KCl
• butter is melted in boiling water in a conical
  flask
• potassium chromate solution is added
• titrated with silver nitrate until an orange
  brown colour persists for 30 sec
• exact normality of silver nitrate solution
  standardised against known amount of potassium
  chloride
• salt in food calculated from concentration
  titration volume of standardised silver nitrate

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Colorimetric assay for phosphorous

• Sample of food is ashed
• Add HCl and nitric acid
• Heat to dissolved ash, cool and make up to volume
  in water
• Add 20 ml of molybdovanadate reagent to aliquot
  of ash solution
• After colour development (10 min.) due to
  formation of phosphomolybdovanadate
• read absorbance at 400 nm
• To calculate phosphorus content compare
  absorbance of sample to standard curve generated
  by known concentrations of potassium dihydrogen
  phosphate

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Ion-selective electrodes

• Similar concept to pH electrodes that measure H
  ions can be applied to other ions and even
  dissolved gases
• Chemical composition of glass in electrode is one
  means of making electrodes sensitive to specific
  ions
• 71 SiO2, 11 Na2O and 18 Al2O3 for K
• A typical glass membrane sodium indicating
  electrode operates in the range of 1 - 10-6 M
• Usually develop a calibration curve of electrode
  potential (millivolts) developed in standard
  solutions
• plot on semilog paper electrode potential vs
  logarithms of the standard concentrations

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Preservatives - Sulphur dioxide

• When sulphur dioxide or sulphur salts are
  dissolved in water an equilibrium between sulphur
  dioxide and a range of ions is established as
  follows
• SO2 H20 ? H2SO3 ? H HSO3- ?
  H SO32-
• sulphur sulphurous acid
  bisulphite sulphite
• dioxide

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Sulphur dioxide determination

• Pale coloured liquid foods or foods that can be
  dispersed in water
• digest food with cold alkali (pH 13),
• acidify to produce un-dissociated sulphurous acid
• sulphur dioxide reacts with standard iodine
  solution
• SO2 I2 2H2O 2I- 2H H2SO4
  I 2
• excess iodine reacts with starch to give a dark
  blue endpoint
• Foods not easily dispersed in water or intensely
  coloured
• distilling of sulphur dioxide from the acidified
  food
• titrating the colourless sulphur dioxide directly
  with standard iodine solution as it distills over

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Nitrates and nitrites

• Levels of nitrites (NO2) nitrates (NO3) in
  foods are controlled by international regulation
• have the potential to form toxic nitrosamines and
  to interfere with infant metabolism
• Aqueous extraction of the food
• to reduce nitrate loss, pH of extract is gt5
• De-proteinisation of the extract at the
  isoelectric point of the contaminating proteins
  followed by filtration

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• Extracted nitrates reduced to nitrites by
  nicotinamide-adenine dinucleotide phosphate
  (NADPH) in the presence of the enzyme nitrate
  reductase
• Nitrate NADPH ? nitrite NADP H2O
• The nitrites originally present in the sample
  plus those converted from nitrates converted to a
  diazo dye
• Nitrite sulphanilamide NED diazo dye
• Level of dye can then be determined
  colorimetrically with reference to a standard
  curve
• Nitrite and nitrates in food calculated as nitrite

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Hunter L, a, b system for quantifying colour of
foods

• Concept of a 3D colour space or colour sphere
• Vertical coordinate (L)
• L 0 represents black
• L 100 represents white
• Horizontal coordinate (a)
• the more negative value the more green
• zero grey
• the more positive value the more red
• Horizontal coordinate (b)
• the more negative value the more blue
• zero grey
• the more positive value the more yellow