14-vitamine Flashcards
define vitamin
Vitamins are defined as relatively low-molecular weight compounds which are required in small quantities for normal metabolism.
Health benefits Regulatory requirement Addition of ____ to fluid milk is a legal requirement under Health Canada’s Food and Drugs Act
Vitamin A, Vitamin C and Vitamin D
how is vitamin calssified?
water soluble BC
fat soluble ADEK
why analysis vitamin?
Nutritional quality of food because of the health benefits Humans do not synthesize vitamin and therefore need to them from food
obtain
Regulatory requirement
Addition of Vitamin A, Vitamin C and Vitamin D to fluid milk
is a legal requirement under Health Canada’s Food and Drugs Act
One of the determinants of shelf-life of food
Vitamins are relatively labile compounds that are affected by factors such as heat, light, air, pH, other food components
Indicate effect of food processing/packaging/and storage on the nutritional quality of food
what is the vitamin unit?
Usually expressed in units of mg or μ g per tablet or food serving
how can vitamin expressed?
international units (IU), US Pharmacopeia (USP) units , and % Daily Value (DV).
what is IU? what does it based on? what does it used for?
The IU is a unit of measurement for the amount of a substance, based on measured biological activity or effect. It is used for vitamins, hormones, vaccines, and similar biologically active substances.
The precise definition of 1 IU ______ substance to substance.
differs from
3 ex of IU
1 IU of vitamin A is the biological equivalent of 0.3 μg retinol, 0.6 μg β -carotene, and 1.2 μg of other pro-vitamin A active carotenoids (e.g., α -carotene and β -cryptoxanthin).
1 IU of vitamin C is the biological equivalent of 50 μg L -ascorbic acid.
1 IU of vitamin D is the biological equivalent of 0.025 μg cholecalciferol/ergocalciferol.
the pathway of sample processing
sample extraction get extract
- clean up get purified extract
- quantitative and qualitative analysis
why analysis of vitamins is performed by different chemical, physical, and biological methods.
Vitamins belong to different classes of organic substances
vitamin assays in most situation involve the extraction of a vitamin from_____ to analysis.
biological matrix prior to analysis.
which treatment used to extract vitamin
heat, acid, alkali, solvent, enzyme
what is the function of extraction procedure?
is it specific/non specific for vitamin?
extraction procedures are specific for each vitamin and designed to stabilize the vitamin.
In some case, some procedures are applicable to the ______ of more than one vitamin.
combined extraction
Typical extraction procedures :
Ascorbic acid?
Cold extraction with metaphosphoric acid/acetic acid
Typical extraction procedures :B1,B2
Boiling or autoclaving in acid plus enzyme
treatment.
Typical extraction procedures :B3
Autoclaving in acid (non-cereal products) or alkali (cereal products).
Typical extraction procedures :B9
Enzyme extraction with α –amylase, protease and γ -glutamyl hydrolase.
Typical extraction procedures :AED
Organic solvent extraction, saponification, and re- extraction with organic solvents.
what is the chanllenge of determine VD in milk?
Relatively low concentration
Poor stability in response to heat, light, oxidation,
Possible interference from milk components (e.g., fat, emulsifiers, amino acids,
proteins, sterols, and minerals)
what is the key in VD determination
sample preparation
procedure of extract VD
solvent extraction
saponification
re-extraction
3 type of vitamin assay
bioassay
microbiological assay
physicochemical assay
physicochemical contain?
spectrophotometric methods.
- 2- fluorometric methods.
- 3- chromatographic methods.
- 4- enzymatic methods.
- 5- immunological methods. 3.6- radiometric methods.
bioassay involve?
animal
micorbiological assay use?
protozoan, bacteria yeast, organism
Bioassays at the present are used only for the analysis of ?
vitamins B12 and D.
Vitamin D assay using__models
rat
Line test for Vit D is based upon?
the cure of experimentally induced rickets in rats during a specific period, as measured by the calcification of bones, the extent of calcification being determined by the “line test’’
Bone sections (logitudinal) are stained with___
silver for observing calcification
how to define amount of VD?
Compare the extent of this line formation with that of reference group receiving oil containing
Microbiological assays are limited to the analysis of __. The methods are very sensitive and specific for __.
water-soluble vitamins.
each vitamin.
The growth of microorganisms is ____to their requirement for a specific vitamin.
proportional
The vitamin tested will act as the__?
growth limiting nutrient.
how to test growth limiting nutrient?
The growth of a certain microorganism in an extract of a vitamin-containing sample is compared against the growth of this microorganism in the presence of known quantities of that vitamin.
Bacteria, yeast, or protozoans are used as test ___?
organisms
how can growth be measured?
turbidity , acid production, gravimetric, or
by respiration .
With bacteria and yeast, _____ is the most commonly
employed system
turbidimetry
procedure of analysis Niacin
1- Weigh the sample.
2- Add 1N H2SO4.
3-Autoclave 30min at 121-123 0C.
4-Filter the mixture, discard and residue.
5- To the filtrate add Difco Niacin Assay Medium (assay medium is devoid of Niacin).
6-Autoclave 10min at 121-123 0C.
7- Add Lactobacillus plantarum ATCC 8014. 8-Incubate at 37 0C for 16-24hr.
9- Measure the optical density and compare with that of bacteria grown in medium containing known amount of the vitamin (Niacin)
procedure of analysis folate
1- Weigh the sample (2g).
2- Add 50ml buffer
3-Add α-amylase, protease, and conjugate and allow for the digestion of the matrix components
4-Deactivate enzymes by boiling the mixture for 5min.
5-Filter the mixture and discard the residue
6- Add the filtrate with bacterial growth medium
7-Autoclave for 10min at 121-123 0C.
8- Add Lactobacillus casei (spp. rhamnosus) ATCCTM 7469 .
9-Incubate at 37 0C for 20-24hr.
10-Measure the optical density and compare with that of bacteria grown in medium containing known amount of the vitamin (folate)
VA is sensitive to?
UV air high temp, moisture
what method is used to measure VA activity
only HPLC
Principle of VA activity measurement
The test sample is saponified with ethanolic KOH, vitamin A (retinol) is extracted into organic solvent and then concentrated. Vitamin A isomers – all-trans- retinol and 13-cis-retinol – levels are determined by HPLC on a silica column.
steps of VA
1- Transfer 40 ml of ready-to-use formula or fluid milk to a 100-ml digestion
flask containing a stirring bar.
2- Add 10 ml of ethanolic pyrogallol (absorb oxygen) solution and 40-ml ethanolic KOH
(for saponification of lipids—>Potassium salts of fatty acids and glycerol).
3-stir at room temperature for 18 hr.
4-Extract vitamin A with hexane:diethyl ether mixture
5- Analyze the organic layer using HPLC for Vit A.
what is the principle of analysis VC?
L-ascorbic acid is oxidized to L-dehydro-ascorbic acid by the 2,6-dichloroindophenol (DCP).
what is 2,6 DCP
oxidation– reduction indicator dye,
Presence of ascorbic acid in solution/juice keep the pH ___
acidic
DCP is___ colored dye in acidic solution.
red
DCP specifically/nonspecifically oxidize ascorbic acid
finally turn ___ color?
specifically
colorless
the end point of DCP is ___ color?
rose-pink
VC is susceptible to?
oxidative deterioration that is accelerated by high pH and the presence of ferric and cupric ions.
If significant amounts of ferric Fe, cupric Cu ions are present in the sample to be analyzed,___ need to add with the extraction
EDTA
what is EDTA
chelating agent?
titrant DCP will ____ from ___ to ___ color. as L-ascorbic acid which is ___ color will change to L-dehydroascrobic acid which is ___ color
reduce
red to colorless
colorless
brown
Thiamine use which method?
Thiochrome fluorometric
write down B1 procedure
- Extraction of thiamine in 0.1N H2SO4with dilute acid
- Enzymatic hydrolysis of thiamine’s phosphate-esters (using diesterase) at pH 4.0-4.5 in presence of 1.2 M sodium acetate
- Chromatographic cleanup (i.e., purification),
- Oxidation to thiochrome
- Measurement using fluorescence intensity
what does enzymatic do?
hydrolysis of thiamine’s phosphate-esters
what does the pH need for thiochrome fluorometric?
4-4.5
what solution need to hydrolysis thiamine’s phosphate-ester?
sodium acetate
how to oxidation to thiochrome?
using alkaline potassium ferricyanide
what need to be considered as selection of method?
- Method accuracy and precision (Consider the use of official methods)
- The need for bioavailability information
- Time and instrumentation requirements
- Personnel requirements
- The type of biological matrix to be analyzed
- The number of samples to be analyzed
- Regulatory requirements – Must official AOAC International methods be used?
- Suitability of the method (Applicability of microbiological assays is limited to water-soluble vitamins (most commonly niacin, B12, and pantothenic acidB5)).
Cold extraction with metaphosphoric acid/acetic acid. extract?
ascorbic acid
Autoclaving in acid (non-cereal products) or alkali (cereal products).extract?
B3 Niacin
Boiling or autoclaving in acid plus enzyme
treatment. extract?
B1 or B2
Organic solvent extraction, saponification, and re- extraction with organic solvents.extract?
A.E.D
Enzyme extraction with α –amylase, protease and γ -glutamyl hydrolase. extract?
folate
B12, D using ___ assay?
bioassay
B3,B9 using___ assay?
microbiological
VA using___ method?
physicochemical– chromatographic
only HPLC
water soluble vitamin using __ assay?
microbiological
B1 using___ method?
physicochemical– thiochrome fluorometric
VC using ___ indicator?
DCP
