Chapter 11 Vitamins Analysis

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Chapter 11 Vitamins Analysis
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Introduction

• 1. What is vitamins?
• VITAMIN VITA (LIFE)-AMINE
• 2. Vitamins classification
• Hydrosoluble vitamins B, C,
• Liposoluble Vitamins A, D, E, K
• 3. Vitamins and health

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Vitamins determination

• The general techniques for determining vitamins
  can be used for biological liquids and plant or
  animal tissues. The general principles are the
  same, but the extraction methods differ depending
  on the matter being analyzed. For many decades,
  the determination of vitamins has been based on
  the evolution of diverse technologies from
  microbiological methods to more sophisticated
  techniques like liquid or gaseous phase
  chromatography coupled with specific detection
  systems(ultraviolet or fluorimetric).
• The real problem at present is no longer the
  methods of determination rather, it is
  extraction procedures. The current change in
  legislation that permits vitamin supplement food
  substances necessitates constant analytical
  control, and underlines the importance of the
  methods for determining these vitamins.

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VA2
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Global Extraction Tech for Liposoluble Vitamins
Add 10 ml of 33 KOH and 40 ml ethanol
Weigh proper amount of the food substance and
transfer to a 150 ml round flask
Cool very rapidly, then add 17 ml of 25 HCl, and
cool again
Boil at reflux for 30 mins
Add 50 ml of petroleum ether and shake vigorously
for 3 mins
Wait to obtain complete separation of the two
phases
Evaporate to dry in a vacuum at a temperature of
35 degree C
Remove and filter the organic phase through
anhydrous sodium phases
Redissolve the residue with a know volume of
hexane for HPLC analysis
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Extraction of Vitamin A

• 1. Weigh 5-10 g of the previously crushed food
  substance into a 1 L round flask.
• 2. Add 20 ml of a 50 NaOH solution and warm the
  mixture in a water bath.
• 3. Then, add 100 ml of diethyl alcohol and 2 ml
  of a hydroquinone solution that was obtained by
  dissolving 20 g in 100 ml of pure alcohol.
• 4. Maintain the water bath at 90? for 30 minutes.
• 5. Pour the contents of the round flask into a
  decanting vial and add 100 ml of water.
• 6. Add 50 ml of ethylic ether and shake.
• 7. Add 50 ml of petroleum ether. Shake and allow
  it to decant.
• 8. Extract once or twice with 50 ml of petrol
  ether.
• 9. Wash the ether phase three times with 100 ml
  of water.
• 10. Filter, evaporate, and concentrate until 1 ml
  is obtained.
• It must be noted that all of these steps are
  conducted away from light.
• Moreover, saponification with NaOH is not useful
  with nonfatty products.

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Extraction of Vitamin D2 or D3
1. Weigh between 5 and 10 g of the sample food
substance. 2. Add 1 g of pyropanol, 90 ml of a
mixture of 60 ml absolute ethanol, and 30 ml of a
50 potash solution. 3. Extract three times, each
time with 50 ml of petroleum ether. 4. Wash the
ether extract and material three times with
water. 5. Filter, evaporate, and concentrate
until 1 ml is obtained. Saponification with the
alcoholic potash mixture is not necessary if the
products to be analyzed do not contain fats.
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Extract of Vitamin E

• 1. Weigh between 5 and 10 g of the food substance
  that you crush.
• 2. Add 100 ml of ascorbic acid methanol solution
  obtained by mixing 0.5 g of ascorbic acid, 4 ml
  of water, and 20 ml of ethanol brought to 100 ml
  with methanol.
• 3. Keep in boiling water for 15-20 minutes.
• Add 15 ml of a 70 KOH solution Place
  again in the water bath for 40 minutes.
• 4. Decant the contents of the flask into a
  separation flask vial, washing the flask with 50
  ml of water.
• 5. Add 120 ml of ethylic ether and stir the
  mixture. Decant and filter on Na2SO4.
• 6. Extract again with 120 ml of ethyl ether.
• 7. Filter, evaporate, and concentrate to 1 ml.
• Saponification with potash is not
  necessary for nonfat products.

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Other Extraction Techniques

• Other Extraction Techniques Specific to Each
  Liposoluble Vitamin and the Product Being
  Analyzed
• These techniques are detailed in the official
  analysis methods proposed by the Official
  Association of Analytical Chemists (OAAC).

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Determination Methods

• Numerous determination techniques can be
  proposed
• ? determination by fluorimetry and by
  colorimetry
• ? determination by liquid phase chromatography,
  with ultraviolet or fluorimetric detection
• ? determination by gas phase chromatography
  (vitamin E)
• ? microbiological determination.

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Determination of Vitamin A

• After extraction, the determination is carried
  out on the solvent of the liquid extraction.
• Colorimetric Determination
• Through this method, carotenoids and vitamin A
  are determined.
• Determination of carotenoids.
• Carotenoids are determined at 450 nm. After
  evaporating the ether phase of the extracted
  solution, dissolved the extraction with 1 ml of
  hexane. Determine the O.D. of this phase at 450
  nm.
• Determination of vitamin A.
• The hexane phase obtained earlier is taken
  again and concentrated in a vacuum. Redissolve
  the extract in a chloroform. Then, to the volume
  of chloroform, add four volumes of the
  trifluoroacetic acid reagent prepared by mixing 1
  v of trifluoroacetic acid with three volumes of
  chloroform. Then, observe the DO at 620 nm

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Determination of Vitamins D

• Determination of Vitamins D2,D3,and Their
  Metabolites
• If the sample contains all the vitamian D
  metabolites, then you can carry out a liquid
  chromatography under the following conditions
• Column fatty acid
  analysis column
• Solvent MeCN
  (acetonitrile), 55
• Mixture of
  water/acetic acid
• (4 ml of
  acetic per liter) 45
• Flow rate 1 ml/min
• Wavelength 265 nm
• Solvent temperature 25?
• Temperature of oven 40?

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Determination of Vitamin E

• A number of methods can be used to determine this
  vitamin.
• 1. Colorimetric Determination
• After extraction and evaporation,
  re-dissolve the residue using n-heptane. Add 1 ml
  of dipyridil solution, then determine the
  absorbance at 460 nm. Methods derived from this
  one have been recommended for use with ferric
  chloride with a reading at 510 nm.
• 2. Determination by Liquid Chromatography
• Using the extract prepared as described
  earlier, proceed with an HPLC determination under
  the following conditions
• Column Lichrosorb R P
  8,25 cm , 4.6 mm, 5 µm
• Solvent methanol/water
  (928)
• Flow rate 1.5 ml/min
• Wavelength 288 nm
• Solvent temperature 25?
• Temperature of oven 40?
• For some foods, all three vitamins (A, D,
  E) can be determined, or only vitamins A and
  E simultaneously.

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Extraction of Hydrosoluble Vitamins

• 1. General principle is that the sample must be
  crashed as finely as possible.
• 2. Enzymatic extraction is conducted with
  amylolytic or proteolytic enzymes.

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Tech of extracting the ensemble of hydrosoluble
vitamins
Weigh 5-10 g sample into a 250 ml flask and add
65 ml 0.1 M HCl
homogenize the sample after crushing it finely
and rapidly (if necessary)
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Heat at 100 deg C for 30 mins in a water bath
Cool and adjust to pH4.5 with 2.5 M NaOAc
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Add 50mg ß-amylase and 50 mg takadiastase,
incubate at 37 oven for overnight
Decant quantitatively into a 100 ml volumetric
flask and adjust volume with water
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?
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Filter the supernatant and do further treatment
to the filtrate if necessary
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Ascorbic Acid Extraction

• 1. In a breaker weigh to about 0.1 mg of a
  certain product quantity as a function of the
  assumed vitamin C content of the sample food
  substance.
• 2. Decant into a 50 ml volumetric flask using
  0.4 metaphosphoric acid.
• 3. Bring the volume to 50 ml with this solution.
  
• . For the analysis of a liquid, pippet the
  sample directly into the flask and adjust the
  volume to 50 ml with the 0.4 metaphosphoric acid
  solution.
• 4. Filter the solution an a cellulose acetate
  membrane (0.2 µg), then pass the filtered
  substance through a SEP-PAK C18 cartridge
  (supplied by Waters, a division of Millipore).
  Eliminate the first 2 ml, then collect 5 ml for
  analysis by RP-HPLC.
• (Note It must be pointed out that some
  authors may propose a determination of ascorbic
  and dehydro-L-sacorbic acids for which the
  extraction technique is different from the
  preceding one).

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Determination of Water Soluble Vitamins

• 1. Determination of Vitamin B1 Thiamine
• 1.1 Fluorimetric Determination
• After the action of potassium
  ferricyanide in the presence of potash, determine
  fluorescence using a 360-365 nm primary filter
  and a 460-480 nm secondary filter.
• 1.2 Microbiological Determination
• A number of lactobacilli can be used,
  such as Lactobacillus fermentum and Lactobacillus
  viridiceus ATCC 1270 C, depending on the chemical
  methods of determination. The statistical
  calculation of activity is based on the six-point
  method.
• 1.3 Determination by Liquid Chromatography
• Chromatographic conditions
• Column C 18, 25 cm 4.6
  mm, 5µm
• Solvent methanol
• 0.005 M sodium
  acetate
• adjusted to 4.5 pH
  (30-70)
• Flow rate 1 ml/min
• Detection excitation 366
  nm
• 
  emission 435 nm

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Principle of NP-C and RP-C
Reverse phase chromatography
Normal phase chromatography
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• So much for this lesson
• and see you next time !