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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