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Sample Preparation
Note on range: While this method can quantify carotenoids in the range of 1-1300 μg/100 g, it is
recommended to only quantify a 100-fold difference with a single preparation. For example, the
range of 1-100 μg/100 g works well for infant formula, but the range of 15-1500 μg/100 g would
work best for samples with the highest carotenoid concentrations.
(a)
Prepare up to 9 samples at a time.
(b)
Weights – Weigh all samples (powders and liquids) to 0.1 mg.
(1)
Powders – Weigh approximately 625 mg powder sample into a 50 mL centrifuge tube. Add 5
mL water, cap, and vortex until dissolved. Let sit up to 15 min at room temperature.
(2)
Liquid ready-to-feed (RTF) with individual carotenoid concentrations ≤200 μg/100 g – Shake
bottle or can on a reciprocating shaker 10 min before opening. Pipette 5 mL sample into a
50 mL centrifuge tube.
(3)
Infant formula concentrate – Shake bottle or can on a reciprocating shaker 10 min before
opening. Pipette 2.5 mL sample into a 50 mL centrifuge tube. Add 2.5 mL water, cap, and
vortex 10 sec. Let sit up to 15 min at room temperature.
(4)
RTF sample with individual carotenoid concentrations >200 μg/100 g – Shake bottle or can
on a reciprocating shaker 10 min before opening. Pipette 2 mL sample into a 50 mL
centrifuge tube. Add 3 mL water, cap, and vortex 10 sec. Let sit up to 15 min at room
temperature.
(c)
Pipette 5.0 mL ISTD solution to each tube.
(d)
Add 1.5 mL KOH solution to each tube with a repeater pipette.
(e)
Shake on reciprocating shaker 5 min.
(f)
Add 8 mL extraction solution to each tube with a repeater pipette.
(g)
Shake 10 min.
(h)
With a repeater pipette or dispenser, add 10 mL water and 10 mL hexane to each tube.
(i)
Shake 1 min.
(j)
Centrifuge at 1000 rpm for 5 min.
(k)
Transfer a portion of the supernatant to a 12 mL scintillation vial.
(1)
Samples with individual carotenoid concentrations ≤ 50 μg/100 g – use 10 mL supernatant
(2)
Samples with individual carotenoid concentrations > 50 μg/100 g – use 3 mL supernatant
(l)
Dry under nitrogen at ≤40 °C.
(m) Reconstitute dried extract in sample solvent and vortex to mix.
(1)
Samples with individual carotenoid concentrations ≤ 100 μg/100 g – add 0.5 mL
(2)
Samples with individual carotenoid concentrations 100-500 μg/100 g – add 1 mL
(3)
Samples with individual carotenoid concentrations 500-1000 μg/100 g – add 2 mL
(4)
Samples with individual carotenoid concentrations 1000-1500 μg/100 g – add 3 mL
(n)
Filter through 0.2 μm PTFE syringe filter prior to injection.
METHOD
FOR ERP USE ONLY
DO NOT DISTRIBUTE