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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-3 _____
Title:
Determination of beta-Carotene, Lutein and Lycopene in Infant and Adult/Pediatric Nutritional
Formula Using High Performance Liquid Chromatography With UV Detection.
Author:
Abbot Nutrition
Summary of Method:
After protected (sodium ascorbate and BHT) saponification using 3 % KOH in 10:4:1
MeOH:H2O:THF, lutein, beta-carotene, and lycopene are extracted from infant, pediatric, and adult
nutritionals using 75:25 hexane:MTBE. After evaporation to dryness, the carotenoids are dissolved 75:25
MeOH with 10% BHT:MTBE, then separated and quantitated by HPLC using a methanol:MTBE gradient
mobile phase through a C30 column terminated with UV detection at 445 nm.
Method Scope/Applicability: I
nfant, pediatric, and adult nutritionals in powder, ready to feed liquid, and
liquid concentrate forms.
General comments about the method:
Author(s) should consider rewriting the method summary to reflect actual conditions of saponification (see
above).
Is the methanol used in Sample Prep step d the methanol solution with BHT added? If so, that should be so
stated. If not, does the author think the BHT present in the THF is sufficient to protect the carotenoids
through the procedure up to step o?
Determination of Standard Concentrations step b may not be adequate for purpose. What if a standard of
any one of the analytes is purchased that has an impurity that absorbs @ 445 and is not in the respective
time range? Worse yet, what if that impurity falls in another analytes time range, thus overstating the
purity of the analyte in consideration, and understating the purity of the other analyte? I believe each of
the analytes should be injected independently, and data collected at its respective absorbance wavelength
shown in 6a.
I am assuming the data in columns 3 and 4 of the Method Performance vs SMPR requirements are
reversed, otherwise this method is clearly out to lunch and deserves no further consideration.