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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-03

Title:

Determination of β-Carotene, Lutein and Lycopene in Infant and Adult/Paediatric Nutritionals

Formula using HPLC with UV Detection

Author:

Abbott Nutrition

Summary of Method:

Carotenoids are extracted from samples with 75:25 hexane: MTBE following

saponification with a mix of KOH, THF and methanol. Following extraction a portion of the organic layer is

evaporated to dryness and the residue dissolved in 75:25 10% BHT:MTBE. Analysis is by HPLC with

separation of the cis and trans isomers, beta carotene and lycopene on a C30 carotenoid column with a

methanol: MTBE gradient and detection by UV spec at 445nm.

Method Scope/Applicability:

Applicable to the determination of

cis

and

trans

-lutein, cis and

trans

-β-

carotene, and

cis

and

trans

lycopene in infant formula and adult nutritionals.

General comments about the method:

Need to include scope/applicability – can use sentence from introduction in SLV report

Requires more detail on sample weigh – sample weight for liquid products included but no detail on

powder or reconstituted products. Assume 1 g is used for all but need to be stated.

To comply with SPIFAN guidelines, add sample preparation for homogeneous vs non-homogeneous

samples – 25g plus 200 mL water etc.

Add note indicating that liquid and reconstituted samples should be prepared fresh prior to analysis.

Add chromatograms to the method - (in the SLV report).

Suggest laying out standard and sample concentration calculations in a clearer format with glossary.

Consider two curves – one for low concentration and one for high carotenoid concentrations or allow for

optimizing curve to suit concentration of samples being analyzed.

Method Safety Concerns:

General safety section needs to be added to the method.

Pros/Strengths:

Good detail on materials, chemicals, reference standard and sample preparation.

Both UV Spectrophotometric and Chromatographic purity performed.