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Table 3. b -Carotene precision data

cis - b -Carotene isomers, µg/100 g RTF

Total b -carotene, µg/100 g RTF

trans - b -Carotene, µg/100 g RTF

RSD r

RSD r

RSD r

,

RSD R

,

,

RSD R , %

,

RSD R

,

n

%

%

%

%

%

Product description/Batch No.

Infant GOS/FOS powder/50350017W1

1.01

5.92 6.38

3.19

7.20 7.09

4.20

5.65 4.90 6

Infant elemental powder/00795RF Child milk powder 1/00866RF00 Adult low-fat powder/00859RF00

6.04 0.11 NA a 0.45

5.57 6.91 9.21 84.2

14.9 0.35

6.80 10.0 6.00 8.73

21.0 0.46

6.77 9.07 6 4.75 26.7 6 NA NA 6 11.7 14.6 6

NA NA

NA

NA NA

NA

Infant hydrolyzed soy powder/410457651Z

23.4 20.2

0.36

8.57 9.02

0.82

Infant formula stage 1 milk powder/4044755861 SRM1849a powder/CLC10-B Infant milk powder/K16NTAV

3.67

4.77 2.31

9.40

5.52 5.19

13.1

4.69 4.25 6

0.07 5.28 0.90 3.14 0.06 0.95 5.88 7.64 0.54 219.7

12.4

2

0.13 12.9 1.85 6.60 0.20 11.0 11.6 33.8 0.72 593

5.87 54.4 6.53 3.51 5.89 7.84 2.01 8.63 3.80 7.33 10.1 34.5 3.28 6.75 2.35 4.67 4.46 6.01 4.02 12.0

0.20 18.2 2.75 9.74 0.26 11.9 17.4 41.4 1.27 812

6.82 88.6 6 5.48 3.07 6 5.86 10.1 12 2.06 9.51 12 3.78 7.31 12 12.1 60.0 12 3.24 5.79 12 2.31 4.81 12 4.32 5.78 12 3.86 16.2 12

3.32 3.18 6.57 17.2 2.87 12.1 3.87 7.38 3.57 22.9 2.46 5.72 3.85 7.43 4.61 23.3 20.5 1

Infant milk RTF 1/38300

SRM 1869 powder/CLC16-B

Adult high-protein RTF/00730RF00

Adult high-fat RTF/00729RF00

Adult high-fat RTF spiked/00729RF00

Infant soy powder/E10NWZC

Infant soy powder spiked/E10NWZC

Infant partially hydrolyzed milk powder/410057652Z

Infant partially hydrolyzed milk powder spiked/410057652Z

4.65

1.94 15.3

47.5

1.91 4.98

52.1

1.81 4.24 12

Infant milk RTF 2/EV4H2R

0.43 5.36

5.29 18.5 3.33 5.63

0.45 6.16

4.29 13.4 3.68 4.44

0.88 11.5

4.17 15.0 12 3.51 4.94 12

Child milk powder 2/4052755861

a NA = Not Applicable. No peaks detected.

14.9%. Lutein, b -carotene, and lycopene repeatability data are summarized in Tables 2 – 4, respectively, and all recovery data are summarized in Table 5. Excluding the child elemental powder, total lutein repeatability ranged from 1.89 to 5.58%. Total b -carotene repeatability ranged from 1.81 to 6.77%, and total lycopene repeatability ranged from 3.01 to 6.37%. Total lutein recoveries ranged from 90.3 to 95.3%. Total b -carotene recoveries ranged from 89.3 to 108%, and total lycopene recoveries ranged from 97.3 to 109%. The method demonstrated good linearity. For lutein, r averaged 0.99991 over a standard range of approximately 10 – 250 µg/L trans- lutein, with average calibration errors of <1%. For b -carotene and lycopene, r averaged 0.99993 and 0.9998 over standard ranges of approximately 25 – 500 and 5 – 100 µg/L, with average calibration errors of <1 and <1.5%, respectively. These data are summarized in Tables 6 – 8. LOQ requirements were met for all three carotenoids. LOQs were estimated to be 0.005, 0.002, and 0.005 µg/mL with standards and 0.4, 0.1, and 0.3 µg/100 g RTF with products for lutein, b -carotene, and lycopene, respectively, assuming 1 g sample was concentrated to 0.625 mL. Chromatograms from the analysis of several infant and adult nutritionals that have not been fortified with carotenoids help demonstrate the selectivity of this method for the determination of lutein and lycopene. Peaks are negligible or nonexistent in the lutein region of an infant partially hydrolyzed milk powder (Figure 8) and an adult nutritional RTF nutritional (Figure 10) and in the lycopene region of

several milk and soy infant nutritionals and an adult nutritional (Figures 6 – 10). AOAC Official Method 2017.04 cis- and trans- Lutein, cis- and trans - b -Carotene, and cis- and trans -Lycopene in Infant, Pediatric, and Adult Nutritionals Reversed-Phase HPLC with UV-Visible Detection First Action 2017 This reversed-phase HPLC method uses C30 chromatography and UV-Vis spectroscopy to determine cis and trans isomers of lutein, b -carotene, and lycopene in infant, pediatric, and adult nutritionals. Carotenoids are extracted from samples with 75 + 25 hexane – methyl tertiary butyl ether (MtBE) after saponification with a mixture of potassium hydroxide, tetrahydrofuran (THF), and methanol. After extraction, a portion of the organic layer is evaporated to dryness, and the residue is dissolved in 75 + 25 methanol with 10% butylated hydroxytoluene (BHT) – MtBE. Prepared samples are injected into a C30 HPLC column where cis and trans isomers of lutein, b -carotene, and lycopene are separated with a methanol – MtBE gradient and detected with UV-Vis spectroscopy at 445 nm. Refer to Material Safety Data Sheets (MSDS) of chemicals prior to use and follow the suggestions for personal protective equipment. A. Principle