AOAC Final Action Methods in 2019

Table 2016.13F. Composition and nominal concentrations of carotenoid standard solutions

Working solution (200 μg/100 mL) in sample solution

UV-Vis solution (200 μg/100 mL)

Intermediate solution in sample solution

Stock solution

Standard, mg

Total volume, mL

Stock solution, mL

Total volume, mL

Stock solution, mL

Total volume, mL

Stock solution, mL

Total volume, mL

Concn, μg/100 mL

Analyte

Lutein

5 5 5 5

25 25 50 25

1.0 1.0 2.0

100 100 100

0.1 0.1 0.2 1.0

10 10 10

2.0 2.0 2.0 3.0

100 100 100

400 400 200

β-Carotene Lycopene Apocarotenal

—

—

100

50

1200

(g)  β-Carotene system suitability solutions.— To make the stock solution, transfer 20 mg β-carotene system suitability reference standard, D(f) , to a 50 mL volumetric flask. Add 1 mL water and 4 mL THF and sonicate for 5 min. Dilute to volume with IPA and sonicate for 5 min. Cool to room temperature and filter the cloudy suspension through a 0.2 μm PTFE syringe filter. To prepare the working solution, dilute 5 mL of the filtered stock solution to 25 mL with IPA. Store in glass vials in the refrigerator up to 3 months or at –20°C for up to 6 months. (h)  Lutein system suitability solutions.— To make the stock solution, transfer 10 mg USP lutein, D(e) , to a 100 mL volumetric flask, dilute to volume with ROH, and mix. Store at –20°C for up to 6 months. To prepare the working solution, transfer 1 mL stock solution to a 50 mL volumetric flask. Add approximately 35 mL IPA to the 50 mL flask, stopper loosely, and heat in a water bath at 80°C for 120 min. Cool to room temperature, add 250 μL apocarotenal stock solution, E ( a ), with an adjustable pipet, and dilute to volume with IPA. Store in glass vials in the refrigerator up to 3 months or at –20°C for up to 6 months. (i)  ISTD solution.— Prepare immediately before use. ( 1 ) For infant formula and samples with low carotenoid concentrations (up to 100 μg individual carotenoid per 100 g) .— Transfer 4.0 mL apocarotenal working solution, E(c) , to a 50 mL volumetric flask and dilute to volume with pyrogallol solution, C(q) . This is enough solution for nine samples. ( 2 )  For samples with individual carotenoid concentrations >100 μg/100 g .—Transfer 4.0 mL apocarotenal intermediate solution, E(d) , to a 50 mL volumetric flask and dilute to volume with pyrogallol solution, C(q) . Note : The apocarotenal solutions used to make the ISTD should be made from the same apocarotenal stock solution, E ( a ), as that used to make the calibration solutions, E ( f ).

F. Sample Preparation Note on range: While the method can quantify carotenoids in the range of 1–1300 μg/100 g, it is recommended to quantify only 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 12 samples at a time. ( b )  Weights .—Weigh all reconstituted and liquid samples to 0.1 mg. ( 1 ) Powders .—Record weights of both powder sample and water to 4 significant figures. Reconstitute 25 g powder sample with 200 g of 35°C water in a reagent bottle and shake well. Mix on a spin plate 1–5 min until completely dispersed and no solids are visible. To ensure homogeneity, shake again immediately before transferring 5.25 ± 0.25 g reconstituted sample into a 50 mL centrifuge tube. ( 2 )  Liquid RTF with individual carotenoid concentrations ≤200 μg/100 g. —Shake bottle or can on a reciprocating shaker 10 min before opening. Transfer 5.25 ± 0.25 g sample into a 50 mL centrifuge tube. ( 3 )  RTF sample with individual carotenoid concentrations >200 μg/100 g .—Shake bottle or can on a reciprocating shaker 10 min before opening. Transfer approximately 2 g sample into a 50 mL centrifuge tube. Add 3 mL water, cap, and vortex 10 s. Let sit up to 15 min at room temperature. ( 4 )  Infant formula concentrate .—Shake bottle or can on a reciprocating shaker 10min before opening. Transfer approximately 2.5 g sample into a 50 mL centrifuge tube. Add 2.5 mL water, cap, and vortex 10 s. Let sit up to 15 min at room temperature. ( c ) Volumetrically pipet 5.0 mL of the appropriate ISTD solution from E(i) to each tube. ( d ) Add 1.5 mL KOH solution, C(n) , to each tube with a repeater pipet. ( e ) Shake on reciprocating shaker for 5 min.

Table 2016.13G. Composition and nominal concentrations of carotenoid calibration solutions

Intermediate solution, mL

Concn, μg/100 mL

Calibration solution

Apocarotenal

Mixed carotenoid

Total volume, mL

Lutein

Apocarotenal

β-Carotene

Lycopene

C1 C2 C3 C4 C5

2.0 2.0 2.0 2.0 8.0

15.0

25 25 25 25

240 128

96 96 96 96 96

240 128

120

8.0 5.0 2.0 1.0

64 40 16

80 32

80 32

100

4

4

2

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