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FOR EXPERT REVIEW PANEL USE ONLY
NOT FOR DISTRIBUTION
1
Expert Review Panel for Infant Formula and Adult Nutrition
Evaluation of Method
Caro-02
Title:
Determination of Lutein and β-Carotene in Infant Formula and Adult Nutritionals
Author:
Greg Hostetler Perrigo
Summary of Method:
ISTD added to recon. or liquid samples prior to saponification with potassium
hydroxide. Carotenoids extracted with MTBE and THF, followed by hexane. The supernatants from the
liquid-liquid extraction are dried under nitrogen and reconstituted in IPA. Reversed-phase chromatography
on a C30 column. All-trans lutein and β-carotene are separated from their major cis isomers, as well as
zeaxanthin and α-carotene.
Method Scope/Applicability:
Applicable to the determination of all-
trans
-lutein,
cis
isomers of lutein, all-
trans-β-carotene, and
cis
isomers of β-carotene in infant formula and adult nutritionals.
General comments about the method:
Lutein has a variety of cis- and trans-isomers; the most important source of lutein in nature is extracted
from marigold, its major form is trans-isomer, and the biological activity of trans-isomer is much higher
than that of cis-isomer, causing a significant difference between its biological potency. However, under the
influence of light, heat and air oxidation etc., the transformation between cis- and trans-isomers of lutein
can occur, resulting in cis- lutein.
ISTD is recommended as recovery of lutein is very variable and degradation caused by light heat and
sensitivity to oxygen. Apocartenal is appropriate ISTD since there is already some innate zeaxanthin from
lutein raw ingredient – also cheaper. Brief cold saponification is recommended with addition of an anti-
oxidant, (Vitamin E)
Method Clarity:
To comply with SPIFAN guidelines, add sample preparation for homogeneous vs non-homogeneous
samples – 25g plus 200 mL water etc. Sample weight is very small if product is non-homogeneous.
Should the intercept be forced through zero – what is the reasoning for this?
Add chromatograms to the method - (in the SLV report).
Method Safety Concerns:
General safety section needs to be added to the method
Pros/Strengths: