6. AOACSPIFANMethods-2018Awards

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H ostetler : J ournal of AOAC I nternational V ol . 100, N o . 3, 2017  3

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

Stock solution (20000 μg/100 mL) in MTBE/vitamin E

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

UV-Vis solution (200 μg/100 mL) in MTBE

Intermediate solution in sample solvent

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

10 10 10

50 50 50

1.0 1.0

100 100

0.1 0.1 1.0

10 10

2.0 2.0 3.0

100

400 400

β-Carotene Apocarotenal

—

—

100

50

1200

250 μL apocarotenal stock solution, E(a) , and dilute to volume with IPA. Store in the refrigerator for up to 3 months. (i)  Internal standard solution (ISTD).— 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(n) . 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(n) . Note on range: Although this 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 nine 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-mix until dissolved. Let sit for up to 15 min at room temperature. Note : For nonhomogeneous powder samples, first dissolve 25 g powder sample with 200 mL water (record weights of both powder sample and water) and then transfer approximately 5 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 approximately 5 g sample into a 50 mL centrifuge tube. F. Sample Preparation

separate 100 mL volumetric flasks and dilute each to volume with MTBE. Prepare fresh when needed. (c)  Individual carotenoid working solutions for chromatographic purity check (200 μg/100 mL).— ( 1 ) For lutein and β-carotene .—Transfer 100 μL aliquots of each standard stock solution, E(a) , to separate 10 mL volumetric flasks and dilute each to volume with sample solvent. Prepare fresh when needed. ( 2 ) For apocarotenal .—Transfer 1.0 mL standard stock solution, E(a) , to a 100 mL volumetric flask and dilute to volume with sample solvent. Store at −20°C for up to 1 month and use for internal standard, E(i) . (d) Apocarotenal intermediate solution (1200 μg/100 mL).— Transfer 3.0 mL apocarotenal stock solution, E(a) , to a 50 mL volumetric flask and dilute to volume with sample solvent. Store at −20°C for up to 1 month. (e)  Mixed carotenoid intermediate solution (400 μg/100 mL).— Combine 2.0 mL each of lutein and β-carotene standard stock solutions, E(a) , in a 100 mL volumetric flask and dilute to volume with sample solvent. Store at −20°C for up to 1 month. (f)  Calibration solutions.— Transfer apocarotenal intermediate solution, E(d) , and mixed carotenoid intermediate solution, E(e) , to volumetric flasks according to Table 2016.13B and dilute to volume with sample solvent. Store at −20°C for up to 1 month. (g) β-Carotene system suitability solution.— Transfer 20 mg β-carotene system suitability reference standard, D(e) , 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, filter the cloudy suspension through 0.2 μm PTFE, and dilute the clear filtrate 1:4 with IPA. Store in the refrigerator for up to 3 months. (h) Lutein system suitability solution.— Transfer 10 mg USP lutein, D(d) , to a 100 mL volumetric flask, dilute to volume with ROH, and mix. Transfer 1 mL to a 50 mL volumetric flask. Add approximately 35 mL IPA, stopper loosely, and heat in a water bath at 80°C for 2 hours. Cool to room temperature, add

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

Apocarotenal intermediate solution, mL

Mixed carotenoid intermediate solution, mL

Lutein concn, μg/100 mL

Apocarotenal concn, μg/100 mL

β-Carotene concn, μg/100 mL

Calibration solution

Total volume, mL

1 2 3 4 5

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

8.0 5.0 2.0 1.0

80 32

80 32

100

4

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