Lutein in food supplementsLutein in food supplements

Irena Vovk1, Breda Simonovska1, Danica Agbaba2

and Gordana Popović2

1 National Institute of Chemistry Ljubljana Slovenia1 National Institute of Chemistry, Ljubljana, Slovenia2 University of Belgrade, Faculty of Pharmacy

• Lutein and its structural isomer zeaxanthin were found asWhy lutein?

• Lutein and its structural isomer zeaxanthin were found as exclusive carotenoids in macula and retina of human eyesE i l f h i i• Essential for human vision

• As strong antioxidants they might also have a beneficial effect on health, although the underlying mechanisms areeffect on health, although the underlying mechanisms are mostly unknown and object of intensive investigations

• Higher intake of both micronutrients, resulting in higher plasma and tissues concentrations was recommended forplasma and tissues concentrations, was recommended for prevention of the age-related macular degeneration and cataract

• Numerous different formulations of food supplements containing lutein recently appeared on the market

Marigold

• Calendula officinalis L.(lutein – about 10% of the total (carotenoids)

• Tagetes erecta L.(lutein – about 90% of the total carotenoids)

• China - the most important supplier of lutein from Tagetes erecta Llutein from Tagetes erecta L.

Lutein and zeaxanthin in the diet• Lutein (spinach, kale, orange, mango, egg yolk, E161b)• Zeaxanthin (corn, E161h)• Estimation of the average daily intake of lutein with the

diet: 2 mg (usually less!) and about 10 times less zeaxanthin

• Recommended daily dose: ?• Toxicity: 20 mg is GRAS (Generally recognized as

f )safe)• High intakes can result in carotenodermia, a harmless

and reversible hyperpigmentationyp p g

Analytical problems• Different contents per unit (e.g. 0.25 – 20 mg) • Lutein present: as a mixture of lutein esters with p

saturated high fatty acids or free (not always declared)• Difficult extraction of lutein or lutein esters from

different formulationsdifferent formulations• Stability• Separation and identificationp

USP PHARMACOPEIA 2007, 2009

LUTEIN• As purified fraction from saponified oleoresin of p p

Tagetes erecta L.LUTEIN PREPARATION• A combination of lutein with one or more inert

substances in solid or liquid form

PRINCIPLE• A combination of spectrophotometric determination• A combination of spectrophotometric determination

of total carotenoids and HPLC determination based on peak area % belonging to all-trans-luteinp g g

USP PHARMACOPEIA 2007, 2009LIMITATIONS• Only free all-trans-lutein (geometric isomers not even

i d)mentioned)• Analysis of lutein esters impossible

Oth t id i t f• Other carotenoids interfere• Yellow flavonoids interfere• High consumption of organic solvents for a questionable• High consumption of organic solvents for a questionable

extraction• Error in volume definition for calculation of luteinError in volume definition for calculation of lutein

content in solid lutein preparations without proteins

Not useful for determination of luteinNot useful for determination of lutein in most of the food supplements.

Food supplements with lutein

Appearance of units (tablets, capsules)pp ( , p )

• Tablet (lutein not evenly distributed in placebo)• Tablet (evenly colored)• Tablet (evenly colored)• Capsule with pastous content

C l ith il t t• Capsule with oily content• Two-pieces capsule with dry content

Additional bioactive ingredients in food

Vitamin C (30 180 mg)

supplements with lutein

• Vitamin C (30-180 mg)• Vitamin E (4-30 mg) • Flavonoids (as “bioflavonoids” extracts of blue• Flavonoids (as bioflavonoids , extracts of blue

berry, grape skins, pycnogenol etc) • Zn (2.5-10 mg)( . g)• Fish oil, soy oil• Some preparations contain additional bioactive p p

compounds (mainly vitamins and minerals)

•Additional substances can be present as usual carriers dd t o a substa ces ca be p ese t as usua ca e sin pharmaceuticals.

Development of analytical method for the determination of lutein

PRINCIPLES• Grinding and homogenization of samples which

determination of lutein

• Grinding and homogenization of samples which enable weighing of small aliquots

• Optimized saponification (concentration of base, l i )solvent, time, temperature)

• Optimized extraction from basic solution• Preparation of standard solutions for calibration and• Preparation of standard solutions for calibration and

samples in the same way• HPLC determination of all-trans-lutein,

all-trans-zeaxanthin, cis-isomers of lutein

0 50,6

P bli h d b ti iti

0 20,30,40,5

APublished absorptivitiesenable determination of lutein concentration

00,10,2

350 400 450 500 550

of lutein concentration

OH

wavelength (nm)

HO luteinOH

lutein

HO zeaxanthin

60

Standard after saponificationm

AU

20

40

ein

ein

n zeax

anth

in

n n

0

20

13-c

is-lu

t e13

'-cis

-lut

all-t

rans

-lute

i n

all-t

rans

-z 9-

cis-

lute

in 9'

-cis

-lute

in

Minutes0 2 4 6 8 10 12 14 16 18 20

60

AU

40an

thin

Sample M8

mA

20

13'-c

is-lu

tein

13'-c

is-lu

tein

ans-

lute

in

all-t

rans

-zea

xa

-cis

-lute

in

'-cis

-lute

in

Minutes0 2 4 6 8 10 12 14 16 18 20

0

all-t

ra 9 9 '

Validation of the developed analytical methodPARAMETERS• Selectivity

p y

y• Range• Linearity• System precision• Intra-day and inter-days precision• Accuracy• Accuracy• LOD, LOQ• StabilityStability

Accuracy

• Spiking of placebo with free lutein or lutein esters at 3 levels, 3 replicates for each level

• Criteria: recovery 95-105%, RSD 5%

Free lutein Lutein esters

Level Mean recovery RSD (n=3) Mean recovery RSD (n=3)(%) (%) (%) (%) (%)

50 103.28 1.63 101.00 0.91100 100 27 4 86 96 10 2 36100 100.27 4.86 96.10 2.36140 98.24 1.49 98.43 2.96

Additional characteristics of the developed th d

• Usable for all kinds of samples (tablets, l ith d t il t t)

method

capsules with dry, pastous or oily content)• Selective for lutein and zeaxanthin• Determination of free lutein and zeaxanthin or a

mixture of lutein esters• Determination of 4 cis isomers of lutein• Used organic solvents with low toxicity• Low consumption of organic solvents• Small mass of homogenised sample neededg p

160Results

140

160te

in

100

120

lue

of lu

t

USP 2009 limits

60

80

clar

ed v

a

20

40

% o

f dec

0

20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

Food supplementsOne sample without declared content: 2.31 mg/unit

Results 25

20

declared determined

140

160

15it (m

g)

80

100

120

140

ed v

alue

of l

utei

n GRAS

Average daily

10

15

n pe

r un

i

0

20

40

60

% o

f dec

lare Average daily

intake from food

5

10

lute

in 01 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

Food supplements

5

01 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

Food supplements

Conclusion• The results of 31 analysed samples showed the

importance of control of lutein in food supplements% of declared content of lutein Number of samples

Without lutein 5

1-50 4

51-89 5

90-130 14

131-145 2

• The consumer should consider the content of lutein in mg per unit (0.25 – 20 mg !) and expired datein mg per unit (0.25 20 mg !) and expired date (stability !)

Acknowledgements

Grant from the:

Acknowledgements

• Bilateral project (Slovenia-Serbia; BI- RS/08-09-025): Determination of active ingredients in dietary supplementsdietary supplements