AOAC Final Action Methods in 2019

Table 2016.13D. Interlaboratory study results for determination of total β-carotene a Sample No. of labs used No. of outlier labs b Mean s r c s R d RSD r RSD R HorRat NIST SRM 1869 9 1 10.9 0.3 1.2 2.3 11.3 0.51 Milk-based toddler formula powder 8 2 13.5 0.8 1.1 6.0 8.5 0.39 Milk-based infant formula powder 1 10 0 15.3 0.7 2.1 4.5 13.7 0.65 Soy-based infant formula powder 9 1 18.6 0.8 1.9 4.5 10.4 0.50 Milk-based infant formula powder 2 9 1 20.3 0.4 2.5 1.8 12.2 0.60 Infant formula RTF 8 2 25.1 0.3 2.2 1.1 8.8 0.45 a  Results expressed as µg/100 g reconstituted final product. b  Values from outlier laboratories were not included in the statistical analysis. c  s r = SD for repeatability. d  s R = SD for reproducibility.

standard stock solution, E ( a ), to a 100 mL volumetric flask and dilute to volume with cyclohexane. Immediately measure by UV- Vis spectroscopy and calculate purity according to section H ( a ). ( c )  Individual carotenoid working solutions for chromatographic purity check (200 μg/100 mL).— ( 1 ) For lutein and β-carotene .—With an adjustable pipet, transfer 100 μL of each standard stock solution, E(a) , to separate 10 mL volumetric flasks and dilute each to volume with sample solution. ( 2 )  For lycopene .—With an adjustable pipet, transfer 200 μL standard stock solution, E ( a ), to a 10 mL volumetric flask and dilute to volume with sample solution. ( 3 )  For apocarotenal .—Transfer 1.0 mL standard stock solution, E(a) , to a 100 mL volumetric flask and dilute to volume with sample solution. Store in glass vials at −20°C for up to 1 month and use for ISTD, E(i) . Analyze by UHPLC on the same day prepared, and calculate the chromatographic purity of each working solution according to section H ( a ). (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 solution. Store in glass vials at −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 solution. Store in glass vials 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 2016.13G and dilute to volume with sample solution. Store in glass vials at −20°C for up to 1 month. Solutions may be aliquoted to HPLC vials prior to storing in the freezer.

( e )  Lutein containing approximately 10% zeaxanthin .—USP Part No. 1370804. ( f )  β-Carotene system suitability reference standard .—USP Part No. 1065491. E. Standards Preparation Standard solution preparation is summarized in Table 2016.13F and detailed below. Notes : Use glass volumetric pipets and flasks for preparation of all standard solutions unless otherwise noted. Standard solution volumes may be scaled up or down provided good laboratory practices are followed. Note : Carotenoids can precipitate from solution when stored in the freezer. All standard solutions should be thawed, sonicated 2 min, and vortexed following removal from the freezer. ( a )  Carotenoid stock solutions (10000–20000 μg/100 mL).— Weigh (to 0.01 mg) approximately 5 mg each of lutein, D(a) ; β-carotene, D(b) ; and apocarotenal, D(c) , reference standards into separate 25 mL volumetric flasks. Add approximately 20 mL MTBE/vitamin E, C(o) , to each, sonicate for 2–3 min, and dilute to volume with MTBE/vitamin E solution. Weigh (to 0.01 mg) approximately 5 mg lycopene, D ( d ), reference standard into a 50 mL volumetric flask. Add approximately 40 mL THF/vitamin E solution, C ( p ), sonicate 2–3 min, and dilute to volume with THF/ vitamin E solution. Store stock solutions in glass vials 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 standard stock solution, E ( a ), to a 100 mL volumetric flask and dilute to volume with pure ethanol. Transfer 1.0 mL β-carotene standard stock solution, E ( a ), to a 100 mL volumetric flask and dilute to volume with cyclohexane. Transfer 2.0 mL lycopene

Table 2016.13E. Interlaboratory results for determination of total lycopene a Sample No. of labs used No. of outlier labs b Mean s r c

s

RSD r

RSD R

HorRat

d

R

NIST SRM 1869

8

2

30.1

0.5

2.2

1.6

7.4

0.39

a  Results expressed as µg/100 g reconstituted final product. b  Values from outlier laboratories were not included in the statistical analysis. c  s r = SD for repeatability. d  s R = SD for reproducibility.

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