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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 : Carot 02
#2
Title:
Determination of Lutein and β-Carotene in Infant Formula and Adult Nutritionals
Reversed-Phase High Performance Liquid Chromatography
Author:
Greg Hostetler, Perrigo Nutritionals, Georgia, VT USA
Summary of Method:
Powder samples are reconstituted in water, and liquid sample is first spiked with an internal standard and
saponified with potassium hydroxide. Samples are then extracted with Methyl tert butyl ether
(MTBE) and THF, followed by hexane. The supernatants from the liquid-liquid extraction are dried
under nitrogen and reconstituted in IPA. Separation is done by 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:
Method uses sound methodology
It is clearly written and should be easy to follow
Has description of spectrophotometric as well as chromatographic determination of standards purity
Uses internal standard (Apocarotenal)
Saponification step is short and done at room temperature
No data are presented for alpha-carotene and Lycopene – doesn’t meet the scope specified in SMPR
Not enough validation data:
-
Accuracy by spike recovery of SRM1849a at 2 levels of all-trans lutein and all-trans b-carotene –
meet SMPR for recoveries and repeatability (no reference or certified carotenoid values is
available from NIST)
Carot-02 Review Forms