AOAC
INFANT FORMULA
TASK FORCE(s)
SME Working Group
Saturday, September 25th, 2010
Orlando, FL
Vitamin A
Jonathan W. DeVries
Medallion Laboratories
Minneapolis, MN
September 25, 2010
[email protected]
Background and History
• 1915-McCollum and Davis isolate “fat soluble
factor A”, a growth promoting factor in animal
fat.
• 1920-Term “vitamin A” was suggested.
• 1919-22-”Growth promoting factor” isolated in
plants (carotene)
Background and History
• 1925-Xerophthalmia (dry eyes, no tears,
eventual blindness)
• 1925-Night blindness
• 1926-Discovery of intense color formation with
SbCl3
• 1928-UV absorption at 328 nm max reported
Background and History
• 1930- β-Carotene shown to have provitamin A
activity
• 1930-31-Structures for retinol and β-Carotene
• 1937-First synthesis of retinol.
• 1950-First synthesis of β-Carotene
Background and History
• Definition: “Vitamin A” is used when
reference is made to the biological activity,
and applies to more than one vitamin A
active substance.
Background and History
Primary sources in diet
• Retinol (and dehydroretinol and retinol esters),
• Retinal (and dehyroretinal),
• Retinoic acid,
• Carotenoids (primarily α, β, and γ carotene)
Primary Vitamin A Forms Available
for Fortification
(4 forms)
• Retinol (animal sources or synthetic)
• Retinyl Acetate
• Retinyl Palmitate
• Carotenoids (plant sources or synthetic)
Retinol
Retinyl Acetate
Retinyl Palmitate
CAROTENOIDS
Beta Carotene
Activity
• International Units
• 0.3 µg retinol,
• 0.334 µg retinol acetate
• 0.55 µg retinol palmitate
• 0.6 µg beta carotene
• 1.2 µg alpha carotene
Activity
• Retinol Equivalents (RE) = 1 ug retinol
• 1.0 µg retinol
• 1.15 µg retinyl acetate
• 1.83 µg retinyl palmitate
• 12 µg all-trans-beta carotene in foods
• 24 µg other provitamin A carotenoids
Biologic Activity
• Essential for vision-(aldehyde form)
• Essential for bone growth
• Reproductive functions in adults
Biologic Activity (Signs of deficiency)
• Loss of appetite
• Retarded growth
• Kerinization of membranes
• Infection
• Death
Biologic Activity (Signs of deficiency)
• Increase cerebrospinal fluid pressure
• Rough scaly skin, rough hair
• Muscular weakness
• Nephritis (kidney inflammation)
• Fetal death
• Night blindness, xerophthalmia
Minimum Dosages
• Combes - 20 ug/kg/day
• National Academies 1958 – 6 ug/kg/day
Symptoms of Overdose
(Combes)
• Eczema and Erythema (skin redness)
• Alopecia (hair loss)
• Bone exostoses (growth on bones) and Fractures
• Hemorrhage
• Hepatotoxicity
• Death
Upper Limits (Institute of Medicine)
(preformed vitamin A)
Gender
Age
Condition
UL μg/day
Male
> 19 yr
3000
Male
14 to 18 yr
2800
Female/Male
9 to 13 yr
1700
Female/Male
4 to 8 yr
900
Female/Male
0 to 3 yr
600
Female
14 to18yr
Pregnant/Lactating
/Other
2800
Female
19 to 50 yr
Pregnant/Lactating
/Other
3000
ADEQUATE INTAKES
Institute of Medicine (2001)
• Infants (0-6 months) 400 ug RAE/day
(400 ug/day preformed vitamin A)
• Infants (7-12 months) 600 ug RAE/day
(600 ug/day preformed vitamin A)
Regulations (Vitamin A content)
Regulation
Regulating Entity
Units
Minimum
Maximum
Australia/
New Zealand
μg RE/100 kJ
(μg RE/100 kcal)
14
(58.6)
43
(180.0)
China
μg RE/100 kJ
μg RE/100 kcal
14
59
43
180
CODEX
μg RE/100 kJ
μg RE/100 kcal
14
60
43
180
Regulations (Vitamin A content)
Regulation
Regulating Entity
Units
Minimum
Maximum
European Union
μg RE/100 kJ
μg RE/100 kcal
14
60
43
180
Indonesia
μg RE/100 kcal
(μg RE/100 kJ)
75
(17.9)
150
(35.8)
United States
IU/100 kcal
(μg RE/100 kJ)
(μg RE/100 kcal)
250
(17.9)
(75)
750
(53.7)
(225)
Regulations (CODEX Energy)
Regulation
Regulating Entity
Units
Minimum
Maximum
Australia/
New Zealand
kJ/L
(kcal/100 mL)
2500
(59.7)
3150
(75.2)
CODEX
And China
kJ/100 mL
kcal/100 mL
250
60
295
70
Calculated Content Range for IF
(based on regulations)
• Minimum = 35.0 μg RE/100 mL
(0.597 x 58.6)
• Maximum = 169.2 μg RE/100 mL
(0.752 x 225)
OFFICIAL/APPROVED METHODS
(Spectophotometric)
• AOAC 960.45 Vitamin A in Margarine
• AACC 86-01-02 Vitamin A -- Ultraviolet
Absorption Method
• Retinol, Carotenoids
• Saponify, extract, alumina/magnesia
cleanup, evaporate, UV @ 310, 325, 450 nm
OFFICIAL/APPROVED METHODS
(Colorimetric (Carr-Price)
• AOAC 974.29 Vitamin A in Mixed Feeds,
Premixes, and Human and Pet Foods
• AACC 86-05.01Vitamin A and Carotene
• AACC 86-02.01Vitamin A -- Carr-Price
Method
• AACC 86-03.01Vitamin A in Enriched
Flour
OFFICIAL/APPROVED METHODS
(Colorimetric (Carr-Price)
• AOAC 974.29
• AACC 86-02.01, 86-03.01, 86-05.01
• Saponify, extract, alumina
chromatography, carotene @ 440 nm,
react retinol with SbCl3, read at 620 nm.
OFFICIAL/APPROVED METHODS
(Mechanized Chromatography-Retinol)
• AOAC 992.04 Vi ta min A (Retinol
Isomers) in Milk and Milk-Based Infant
Formula-LC
• Saponify, extract in C6H14, (C16H34
protectant), normal phase LC @ 340nm
with retinol standards.
OFFICIAL/APPROVED METHODS
(Mechanized Chromatography-Retinol)
• AOAC 992.06 Vitamin A (Retinol) in
Milk-Based Infant Formula-LC
• Saponify (pyrogallol protectant), extract
in CH2Cl2:C6H14, normal phase LC @ 336
nm with retinyl palmitate standards.
OFFICIAL/APPROVED METHODS
(Mechanized Chromatography-Retinol)
• AOAC 2001.13-Vitamin A (Retinol) in
Foods (JAOAC Int’l, 85 pp 424-434 (2002).
• AACC 86-06.01 Analysis of Vitamins A and
E by High-Performance Liquid
Chromatography
• Saponify (pyrogallol protectant), dilute,
reverse phase LC @ 313 or 328 nm with
retinol or retinyl palmitate standards.
OFFICIAL/APPROVED METHODS
(Mechanized Chromatography-Retinol)
• Horrat 0.51 to 3.75
(0.51 to 1.49 without dried egg powder)
• Recovery 100 +/- 13%
OFFICIAL/APPROVED METHODS
(Mechanized Chromatography-Other)
• AOAC 2002.06 Retinyl Palmitate
(Vitamin A) in Fortified Fluid Milk (LC)
• Extract with hexane, LC on normal phase
• Horrat-0.4 to 1.3
• Recovery study not done
OFFICIAL/APPROVED METHODS
(Carotenes-Spectrophotometric)
• AOAC 970.64-Carotenes and Xanthophylls
in Dried Plant Materials and Mixed Feeds
• Saponify, extract, manual chromatography,
Absorbance @ 436 and 474 nm
OFFICIAL/APPROVED METHODS
(Carotenes-Spectrophotometric)
• AOAC 938.04-Carotenoids in Macaroni
Products
• AOAC 975.23-Carotenoids in Eggs
• Saponify, extract, manual chromatography,
Absorbance @ 436 nm
OFFICIAL/APPROVED METHODS
(Carotenes-Mechanized Chromatography)
• AOAC 2005.07-beta Carotene in
Supplements and Raw Materials
• (Digest with protease) extract
CH2Cl2:alcohol, reversed phase
chromatography, detection @ 445, 448
nm
Cautions
• Retinol and carotenes very sensitive to
oxidation
• Light
• Heat/Oxygen
• Some isomerization occurs during analysis
• Retinyl esters reasonably stable
CAUTIONS (cont)
• Light
• Yellow lab lighting
• Low Actinic Glassware
• Oxygen
• Add antioxidants (pyrogallic acid)
• Nitrogen atmosphere during saponifications
Analytical Questions
• Total Vitamin A activity (include carotenes)
or just Fortificants.
• Encapsulations (Alginates, gelatins,
starches, waxes)
Analytical Challenges
• Standards are rarely pure
• Spectrophotometric measurement
• Chromatography measurement
• Multiply
• Key issue not chromatography
• Key issue is in sample preparation
• Column temperature control
Corn-Soy Blend Study
JAOAC Int’l, 93 pp 638-649 (2010)
• Direct extraction (of esters) had poorer
precision than saponification/extraction
• Reference material analyzed accurately but
with 7.1% CV
• Laboratories following Official Methods
equivocally produced more accurate and
precise results than those using “other
methods”
Fitness for Purpose Statement
• An Analytical method(s) to determine
Retinol, Retinyl Esters and Carotenes in the
Concentration Ranges (as a minimum
requirement) from 35 ug RE/100 mL to 169
ug RE/100 mL of liquid and reconstituted
solid infant formula, milk or soy based and
adult nutritionals. Time to signal not
necessarily significant.
Fitness for Purpose Statement (cont)
• Vitamin A levels meeting regulatory
requirements will be covered by the
concentration ranges of this quantitative
reference assay conducted in a laboratory
by a trained chemist.