Lecture 14 - Vitamins Flashcards
define a vitamin
relatively low-molecular weight compounds which are required in small quantities for normal metabolism
why do we analyze for vitamins?
- nutritional value
- health benefits
- regulatory requirements
- shelf life
- affects packaging
how are vitamins classified?
based on their solubility
fat soluble: ADEK
water soluble: B and C
what’s an IU?
unit of measurement based on BIOLOGICAL ACTIVITY or effect
what units can vitamins be expressed in?
- mg
- ug
- IU
- US Pharmacopeia (USP)
- % DV
how do vitamin extractions usually go?
remove from its biological matrix prior to analysis
what methods of extraction are there for vitamins?
- heat
- acid
- alkali
- solvents
- enzymes
how to extract ascorbic acid?
cold extraction with metaphosphoric acid/acetic acid
how to extract vitamin B1 and B2?
boiling or autoclaving, plus enzyme treatment
how to extract niacin?
autoclaving acid (everything else) or alkali (cereal) products
how to extract folate?
enzyme extraction with a-amylase, protease and y-glutamyl hydrolase
how to extract vitamins A, E, or D?
organic solvent extraction, saponification, and re-extracctino with organic solvents
what challenges does milk present for vitamin D analysis?
- relatively low concentration
- poor stability in response to heat, light, oxidation
- possible interference from milk components
so we use solvent extraction; saponification; re-extraction
what are the classifications of vitamin assays?
- bioassays
- microbiological assays
- physiochemical assays
what’s a bioassay method?
- rats
- used to analyze vit B12 and D (line test)
principle of microbiological assays?
- growth of microorganisms is proportional to their requirement for a specific vitamin
- vitamin being tested acts as a growth limiting nutrient
how is microbial growth measured?
- turbidity
- acid production
- gravimetrically
- respiration
microbiological assay for niacin?
niacin:
- weight sample
- H2SO4
- autoclave
- filter mixture
- add Difco Niacin Assay Medium to filtrate
- autoclave again
- add lactobacillus plantarum
- incubate
- measure optical density
microbiological assay for folate?
- weigh sample
- add buffer
- add a-amylase, protease, and conjugate and allow for the digestion of the matrix components
- deactivate enzymes by boiling the mixture for 5 mins
- filter
- add bacterial growth medium to filtrate
- autoclave
- add lactobacillus case
- incubate
- measure optical density and compare with bacterial growth of medium containing known amount of vitamin
what method do you use to analyze vitamin A?
- vit A is sensitive to UV light, high temps, and moisture so need to avoid this
- HPLC is the only acceptable method to provide accurate method
steps for analyzing vitamin A:
- test sample saponified (with KOH)
- vit A extracted into organic solvent (diethyl ether) and concentrated
- vit A isomers are determined by HPLC on a silica column
what method do you use to analyze for vitamin C?
- vit C is oxidized to L-dehydro-ascorbic acid by redox indicator dye 2,6-dichloroindophenol (DCP)
- DCP is reduced to colorless form
- end point indicated by appearance of rose-pink color
true or false: vitamin C is not susceptible to oxidative deterioration
false, it is
what accelerates oxidative deterioration of vitamin C?
- high pH
- presence o f ferric and cupric ions
what do you do if your complex has a lot of Fe or Cu ions?
include a chelating agent (EDTA)
expand EDTA
ethylenediaminetetraacetic acid
what method is used to analyze for thiamine?
thiochrom fluorometric procedure
describe the steps of the thiochrom fluorometric procedure
- extraction of thiamine in H2SO4 with dilute acid
- enzymatic hydrolysis (takadiastase) of thiamine’s phosphate-esters at pH 4.0-4.5 in presence of sodium acetate
- chromatographic cleanup
- oxidation to thiochrome with alkaline potassium ferricyanide
- measurement using fluorescence intensity using a fluorometer
- compare value against a known concentration
what factors are considered in the selection of methods?
- method accuracy/precision
- need for bioavailability info
- time & instrumental requirements
- personnel
- matrix nature
- number of samples
- regulatory requirements
- suitability of method
microbiological assays are suitable for both fat and water soluble vitamins
false. mostly used for water-soluble vitamins (niacin, B12, and pantothenic acid)
