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Additive-493 Zeaxantin Trans JECFA

This document provides specifications for zeaxanthin-rich extract from Tagetes erecta, including: 1) It is obtained through hexane extraction and purification of the oleoresin from red flowers of T. erecta, yielding zeaxanthin as the main carotenoid along with other carotenoids, fats, oils, and waxes. 2) The extract must contain not less than 30% total carotenoids and 65% of that must be zeaxanthin. 3) Identification and purity tests are outlined to verify the extract meets the specifications.

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33 views

Additive-493 Zeaxantin Trans JECFA

This document provides specifications for zeaxanthin-rich extract from Tagetes erecta, including: 1) It is obtained through hexane extraction and purification of the oleoresin from red flowers of T. erecta, yielding zeaxanthin as the main carotenoid along with other carotenoids, fats, oils, and waxes. 2) The extract must contain not less than 30% total carotenoids and 65% of that must be zeaxanthin. 3) Identification and purity tests are outlined to verify the extract meets the specifications.

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ZEAXANTHIN-RICH EXTRACT from TAGETES ERECTA

(TENTATIVE)

Information is required on the non-zeaxanthin components in total


carotenoids and on the composition of the non-carotenoid components
by end of 2006.

New specifications prepared at the 63rd JECFA (2004) and published in


FNP 52 Add 12 (2004).

SYNONYMS INS No. 161h(ii)

DEFINITION These specifications apply to zeaxanthin rich extract from red flowers
of Tagetes erecta L. obtained with hexane extraction and subsequent
purification of oleoresin by saponification, crystallization and washing
of separated crystals alternatively in hexane and methanol to achieve
desired purity. Beside zeaxanthin which is the main colouring principle,
other carotenoids can be contained in various amounts, as well as
fats, oils and waxes originating from the plant material.

Chemical Names (all-E)-1,1'-(3,7,12,16-Tetramethyl-1,3,5,7,9,11,13,15,17-


octadecanonaene-1,18-diyl)bis[2,6,6-trimethylcyclohexene-3-ol];
3R,3'R-β,β-Carotene-3,3'-diol

C.A.S. number 144-68-3 (Zeaxanthin)


Chemical formula C40H56O2 (Zeaxanthin)
Structural formula
H3C OH
H3C CH3 CH3 CH3

CH3 CH3 H3C CH3


HO CH3

Formula weight 568.89 (Zeaxanthin)

Assay Not less than 30 % of total carotenoids (as zeaxanthin) and not less
than 65 % of zeaxanthin in total carotenoids

DESCRIPTION Orange-red crystalline powder, with little or no odour

FUNCTIONAL USES Colour, nutrient supplement

CHARACTERISTICS

IDENTIFICATION

Test for carotenoid The colour of the solution of the sample in acetone disappears after
successive additions of a 5 % solution of sodium nitrite and 1N sulfuric
acid.

Spectrophotometry A solution of the sample in chloroform/ethanol (1:9) shows maximum


(Vol. 4) absorption at ca. 453 nm

PURITY

Residual solvents (Vol. 4) Hexane and methanol, not more than 50 mg/kg, singly or in
combination

Lead (Vol. 4) Not more than 2 mg/kg


Determine using an atomic absorption technique appropriate to the
specified level. The selection of sample size and method of sample
preparation may be based on the principles of the methods described in
Volume 4, “Instrumental Methods”.

METHOD OF Total carotenoids (as zeaxanthin)


ASSAY Proceed as directed in FNP 5 under Colouring Matters, Total Content
by Spectrophotometry, Procedure 2, using the following conditions:
w = amount (g) to obtain adequate absorbance
V1 = V2 = V3 = 100 ml
v1 = v2 = 10 ml
A1%1cm = 2540
λ max = 453 nm

Zeaxanthin in total carotenoids


Determine by reverse phase HPLC using the following conditions:
Column: C18, 5 µm (4.6 x 250 mm)
Flow: 0.6 ml/min
Wave length: 453 nm
Injection volume: 10 µl
Column temperature: ambient
Injector temperature: 10o
Run time: 15 min
Mobile phase: acetonitrile/water/chloroform (82:2:15)

Sample preparations:
Weight a sample containing approximately 7 to 9 mg of zeaxanthin
(determined according to theoretical content) into a 100 ml volumetric
flask. Dissolve and bring to volume with chloroform. Place 1 ml of the
resultant solution into volumetric flask and dilute to 25 ml using the
mobile phase solution as a diluent.
Zeaxanthin as a % of total carotenoids equals:

A1 x 100

A1 + A2

where
A1 = area of the trans- zeaxanthin peak
A2 area of trans-lutein peak

Identification of the peak areas corresponding to trans-zeaxanthin and


trans-lutein is verified by comparison of the retention times with
standards.

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