.Ben-Amotz and Fishler (10) Method. The maize sample (0.6 g)
was extracted twice with tetrahydrofuran/methanol (5 mL, 50:50 v/v)
by mixing and centrifugation. Hexanes (10 mL) and sodium chloride
(2 mL, 10% w/v) were added to the combined organic layers. Rather
than using a separatory funnel, layers were separated in a large glass
test tube (50 mL). The organic layer was transferred to a new tube and
dried under argon.
Kurilich and Juvik (21) Method. Carotenoids were released from
dried maize (0.6 g) by adding ethanol (6 mL) containing 0.1% BHT
(w/v), mixing by vortex for 20 s, and placing in an 85 °C water bath
for 5 min. Potassium hydroxide (120 íL, 80% w/v) was added to the
heated ethanol-maize mixture, to saponify potentially interfering oils.
Samples were mixed by vortex and returned to the 85 °C water bath
for 10 min with an additional mixing at 5 min. After saponification,
samples were immediately placed in ice, and cold deionized water (3
mL) was added. Carotenoids were extracted 3 times with hexanes (3
mL) using centrifugation (1200g) to separate the layers. Combined
organic layers were washed with deionized water (3 mL), and the
organic layer was removed to a new test tube. The remaining aqueous
layer was extracted twice more with hexanes. The combined organic
layers were dried under argon.
Several modifications were performed to improve chromatography
and verify importance of saponification and heating steps. Modification
of the saponification process included addition of 500 íL of potassium
hydroxide (80% w/v) instead of 120 íL. Modifications of the heating
steps included changing the extraction and saponification temperature
from 85 °C to 23 or 60 °C. All temperature changes were performed
with the additional base modification described previously.