AOAC SPIFAN Nutrients ERP Review (March 14, 2019)

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

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

UV-Vis solution (200 μg/100 mL)

Intermediate solution in sample solvent

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

10 10

50 50 50 50

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

400 400 200

β-Carotene Lycopene Apocarotenal

100

5

10

—

—

100

50

1200

−20°C for up to 1 month. (e)  Mixed carotenoid intermediate solution (400 μg/100 mL).— Combine 2.0 mL each of lutein, β-carotene, and lycopene 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 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 h. Cool to room temperature, add 250 μL apocarotenal stock solution, E(a) , and dilute to volume with IPA. Store in refrigerator for up to 3 months. (i)  Internal standard (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(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) .

E. Standards Preparation Standard solution preparation is summarized in Table 2016.13A and detailed below. ( a )  Carotenoid stock solutions (20 000 μg/100 mL).— Weigh (to 0.01 mg) approximately 10 mg each of lutein, D(a) ; β-carotene, D(b) ; and apocarotenal, D(c) , reference standards into separate 50 mL volumetric flasks. Add approximately 40 mL MTBE/ vitamin E, C(m) , to each, sonicate for 2 to 3 min, and dilute to volume with MTBE/vitamin E. Weigh (to 0.01 mg) approximately 5 mg each of lycopene, D ( d ), reference standard into a 50 mL volumetric flask. Add approximately 40 mL THF/vitamin E, C ( n ), to each, sonicate 2–3 min, and dilute to volume with THF/ vitamin E. Store stock solutions at −20°C for up to 6 months and check their purity each time new standard solutions are made from them. ( b )  Standard measuring solutions for UV-Vis spectroscopy potency check (200 μg/100 mL).— Transfer 1.0 mL lutein and β-carotene standard stock solutions, E(a) , to separate 100 mL volumetric flasks and dilute each to volume with MTBE. Transfer 2.0 mL lycopene standard stock solution, E ( a ), to a 100 mL volumetric flask and dilute to volume with hexane. ( c )  Individual carotenoid working solutions for chromatographic purity check (200 μg/100 mL).— ( 1 ) For lutein and β-carotene .— Transfer 100 μL of each standard stock solution, E(a) , to separate 10 mL volumetric flasks and dilute each to volume with sample solvent. ( 2 )  For lycopene .—Transfer 200 μL standard stock solution, E ( a ), to a 10 mL volumetric flask and dilute to volume with sample solvent. ( 3 )  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

Table 2016.13B. 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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