Table 1. Data for LOD and LOQ of Anthocyanins

Analyte Calibration Range (μg/mL)

LOD (μg/mL)

LOQ (μg/mL)

Dp3Glu 0.39–50 1.56 6.25Cy3Gal 0.39–25 0.78 3.13Cy3Glu 0.39–25 0.78 3.13Pet3Glu 0.39–25 0.20 0.78Peo3Gal 0.39–10 0.56 2.34Peo3Ara 0.39–25 0.78 3.13Mal3Gal 0.39–25 0.20 0.78

Table 2. Determination of Anthocyanins in NIST and USP Samples

Analyte NIST Sample (μg/mL) USP Sample (μg/mL)Dp3Glu 14.9 12.2Cy3Gal 8.01 7.00 Cy3Glu 7.59 6.61 Pet3Glu 6.08 5.14 Peo3Ara 5.76 5.22 Peo3Gal 1.53 1.45 Mal3Gal 2.58 2.24

Table 3. Recovery of Anthocyanins in Different Bilberry-Based Products

Sample Analytes Amount Spiked (µg/mL) % Recovery

Eye Antioxidant

Dp3Glu 10 89.3Cy3Gal 3.0 94.8Cy3Glu 3.0 81.1

Peo3Gal 0.5 96.2Pet3Glu 3.0 92.6Mal3Gal 0.5 94.2Peo3Ara 3.0 91.2

Brand A Bilberry-Based Nutritional

Supplement

Dp3Glu 25 90.2Cy3Gal 10 82.8Cy3Glu 10 78.7

Peo3Gal 4.0 98.1Pet3Glu 10 78.4Mal3Gal 10 107Peo3Ara 10 104

Brand B Bilberry-Based Nutritional

Supplement

Dp3Glu 20 97.6Cy3Gal 10 84.0Cy3Glu 10 93.6

Peo3Gal 2.0 105Pet3Glu 10 83.7Mal3Gal 3.0 101Peo3Ara 5.0 108

28820

R3 R5 R3 Anthocyanin

H H Glucose/Arabinose/Galactose Pelargonidin 3-Glucose/Arabinose/Galactose

OH H Glucose/Arabinose/Galactose Cyanidin 3-Glucose/Arabinose/Galactose

OH OH Glucose/Arabinose/Galactose Delphinidin 3-Glucose/Arabinose/Galactose

OCH3 OH Glucose/Arabinose/Galactose Petunidin 3-Glucose/Arabinose/Galactose

OCH3 H Glucose/Arabinose/Galactose Peonidin 3-Glucose/Arabinose/Galactose

OCH3 OCH3 Glucose/Arabinose/Galactose Malvidin 3-Glucose/Arabinose/Galactose

OH

HO

0-R3

OH

R3'

R5'

O+

Peaks: Conc. (μg/mL) 1. Dp3Gal — 2. Dp3Glu 20.1 3. Cy3Gal 11.0 4. Dp3Ara — 5. Cy3Glu 12.3 6. Pet3Gal — 7. Cy3Ara — 8. Dp — 9. Pet3Glu 9.91 10. Peo3Gal 3.91 11. Pet3Ara — 12. Peo3Glu — 13. Mal3Gal 8.76 14. Peo3Ara 12.7 15. Cy — 16. Mal3Glu — 17. Mal3Ara — 18. Pet — 19. Peo — 20. Mal —

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

20

19

18

1716

15

1413

12

11

9

876

5

4

31

2

-0.50

8

Minutes

mAU

Column: Acclaim 120, C18, 2.2 µm, analytical (2.1 × 150 mm)Eluent: A: 10% Formic acid B: 10% Formic acid, 22.5% methanol, 22.5% acetonitrileGradient: 0.0–12.0 min 9% B 12.0–25.0 min 35% B 25.0–30.0 min 50% B Hold for 5 min at 9% BTemp.: 35 °CFlow: 0.475 mL/minDetection: Absorbance, Vis 520 nmInj. Volume: 2.0 µLSample: 125 µg/mL15 Anthocyanin standard

29008

10

29009

Peaks: Conc. (μg/mL) 1. Dp3Gal — 2. Dp3Glu 5.15 3. Cy3Gal 2.23 4. Dp3Ara — 5. Cy3Glu 2.02 6. Pet3Gal — 7. Cy3Ara — 8. Pet3Glu 1.89 9. Peo3Gal 0.28 10. Pet3Ara — 11. Mal3Gal 4.27 12. Peo3Ara 1.49 13. Cy — 14. Mal3Glu —

Column: Acclaim 120, C18, 2.2 µm, analytical (2.1 × 150 mm)Eluent: A: 10% Formic acid B: 10% Formic acid, 22.5% methanol, 22.5% acetonitrileGradient: 0.0–12.0 min 9% B 12.0–25.0 min 35% B 25.0–30.0 min 50% B Hold for 5 min at 9% BTemp.: 35 °CFlow: 0.475 mL/minDetection: Absorbance, Vis 520 nmInj. Volume: 2.0 µLSample: Eye antioxidant

0 2 4

1

2

34

5 67

8

9

10

1112

13

14

6 8 10 12 14 16 18 20 22 24 26 28 30-0.1

0

1.1

Minutes

mAU

29010

Peaks: Conc. (μg/mL) 1. Dp3Gal — 2. Dp3Glu 21.7 3. Cy3Gal 11.9 4. Dp3Ara — 5. Cy3Glu 10.6 6. Pet3Gal — 7. Cy3Ara — 8. Dp — 9. Pet3Glu 8.76 10. Peo3Gal 3.31 11. Pet3Ara — 12. Peo3Glu — 13. Mal3Gal 6.26 14. Peo3Ara 8.88 15. Cy — 16. Mal3Glu — 17. Mal3Ara — 18. Pet — 19. Peo — 20. Mal —

Column: Acclaim 120, C18, 2.2 µm, analytical (2.1 × 150 mm)Eluent: A: 10% Formic acid B: 10% Formic acid, 22.5% methanol, 22.5% acetonitrileGradient: 0.0–12.0 min 9% B 12.0–25.0 min 35% B 25.0–30.0 min 50% B Hold for 5 min at 9% BTemp.: 35 °CFlow: 0.475 mL/minDetection: Absorbance, Vis 520 nmInj. Volume: 2.0 µLSample: Nutritional supplement A

0

1

2 3

4

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30-1

0

12

5 6

7

810

11

1213

15

14

16

17 1819

9

20

Minutes

mAU

29011

Peaks: Conc. (μg/mL) 1. Dp3Gal — 2. Dp3Glu 20.8 3. Cy3Gal 10.2 4. Dp3Ara — 5. Cy3Glu 9.99 6. Pet3Gal — 7. Cy3Ara — 8. Delphinidin — 9. Pet3Glu 8.40 10. Peo3Gal 1.12 11. Pet3Ara — 12. Peo3Glu — 13. Mal3Gal 3.27 14. Peo3Ara 6.96 15. Cyanidin — 16. Mal3Glu — 17. Mal3Ara — 18. Petunidin — 19. Peonidin — 20. Malvidin —

Column: Acclaim 120, C18, 2.2 µm, analytical (2.1 × 150 mm)Eluent: A: 10% Formic acid B: 10% Formic acid, 22.5% methanol, 22.5% acetonitrileGradient: 0.0–12.0 min 9% B 12.0–25.0 min 35% B 25.0–30.0 min 50% B 30.0–35.0 min 9% BTemp.: 35 °CFlow: 0.475 mL/minDetection: Absorbance, Vis 520 nmInj. Volume: 2.0 µLSample: Nutritional supplement B

0 2

1

2

3

45

6

7

8 10

11

12

13

14 16

1718

1920

15

9

4 6 8 10 12 14 16 18 20 22 24 26 28 30-0.5

0

7

Minutes

mAU