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Specificity
The SMPR calls for the determination of all-
trans
and
cis
isomers of lutein and β-carotene, as
well as separation of lutein from zeaxanthin. Specificity was evaluated with visual inspection
of chromatograms and by measuring resolution of system suitability standard mixtures.
Because apocarotenal is used as an internal standard, samples were prepared without internal
standard to ensure there were no interfering peaks. Standard mixtures were isomerized with
heat to identify major cis isomers.
Peak assignments were based on relative retention times from previous studies using C30
columns and methanol/MTBE as the mobile phase (Aman et al., 2005; Marx et al, 2000;
Ping and Lian, 2006; Puspitasari-Nienaber, 2002). Samples spiked with all-
trans
-lutein and
β-carotene showed no isomerization during sample preparation. All experiments showed
separation of all-
trans
-lutein and β-carotene from their major
cis
isomers, apocarotenal, α-
carotene, lycopene, and zeaxanthin.
Figure 4. Lutein system suitability solution.
Figure 5. Beta-carotene system suitability solution.
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