11/9/2020 USP-NF meso-Zeaxanthin Preparation

Printed on: Mon Nov 09 2020, 17:43:55 pm

Printed by: Shruti Kharidia
O cial Status: Currently O cial on 09-Nov-2020
O cial Date: O cial as of 1-Jan-2018
Document Type: DIETARY SUPPLEMENTS
DocId: 1_GUID-4CFC1971-CC7B-43E3-BA35-91195D5ABA85_3_en-US
Printed from: https://online.uspnf.com/uspnf/document/1_GUID-4CFC1971-CC7B-43E3-BA35-91195D5ABA85_3_en-US
© 2020 USPC

meso-Zeaxanthin Preparation
DEFINITION
meso-Zeaxanthin Preparation is a combination of meso-Zeaxanthin with one or more inert substances. It may be in a solid or a liquid
form. It contains NLT 95.0% and NMT 130.0% of the labeled amount of total carotenoids, calculated as zeaxanthin (C40H56O2) on the
anhydrous basis. It contains NLT 85.0% of zeaxanthin and NMT 9.0% of lutein of the total carotenoid content.

IDENTIFICATION
• A.
Sample solution: Prepare as directed for Sample solution A or Sample solution B in the test for Content of Total Carotenoids.
Analysis: Record the UV-Vis spectrum from 300 to 600 nm.
Acceptance criteria: The Sample solution shows a shoulder at about 427 nm, an absorption maximum at about 453 nm, and another
maximum at about 480 nm.
• B. The retention time of the major peak of the Sample solution corresponds to that of the Standard solution, as obtained in the test for
Content of Zeaxanthin.
• C. The retention time of the major peak of the Sample solution corresponds to that of (3R,3′S-meso)-zeaxanthin from the Standard
solution, as obtained in the test for Stereoisomeric Composition.

COMPOSITION
• CONTENT OF TOTAL CAROTENOIDS
Sample stock solution A (for solid Preparations): Transfer an amount of Preparation equivalent to 7.5 mg of zeaxanthin to a 100-

L
mL low-actinic volumetric ask, add 5 mL of water, and sonicate at 60° for 5 min. Add 50 mL of alcohol, and dilute with methylene
chloride to volume. Shake thoroughly to obtain a ne dispersion, transfer 10 mL of this dispersion to a centrifuge tube, and
IA
centrifuge. Discard the foremost 2 mL of the supernatant. Use the clear solution.
Sample solution A: Transfer 1.0 mL of Sample stock solution A to a 50-mL low-actinic ask, and dilute with a mixture of cyclohexane
and alcohol (9:1) to volume.
Sample stock solution B (for liquid Preparations): Transfer an amount of Preparation equivalent to 7.5 mg of meso-zeaxanthin to a
100-mL low-actinic volumetric ask, add 20 mL of chloroform, and sonicate for 5 min. Cool the solution to room temperature, and
IC

dilute with cyclohexane to volume. Shake thoroughly to obtain a ne dispersion, transfer 10 mL of this dispersion to a centrifuge
tube, and centrifuge.
Sample solution B: Transfer 1.0 mL of Sample stock solution B to a 50-mL low-actinic ask, and dilute with cyclohexane to volume.
Instrumental conditions
FF

(See Ultraviolet-Visible Spectroscopy 〈857〉.)

Analytical wavelength: 453 nm
Cell: 1 cm
Blank: Cyclohexane
Analysis
O

Sample: Sample solution

Calculate the percentage of total carotenoids (T) as zeaxanthin (C40H56O2) in the portion of Preparation taken:

Result = A/(C × F)

A = absorbance of the Sample solution

C = concentration of Preparation in Sample solution A or Sample solution B (g/mL)

F = coe cient of extinction (E1%) of zeaxanthin in cyclohexane (100 mL · g-1 · cm-1), 2540

Calculate the percentage of the labeled amount of total carotenoids:

Result = (T/L) × 100

T = percentage of total carotenoids found as calculated above

L = labeled amount of total carotenoids (%)

Acceptance criteria: 95.0%–130.0% of the labeled amount of total carotenoids as zeaxanthin (C40H56O2) on the anhydrous basis
• CONTENT OF ZEAXANTHIN
[NOTE—Use low-actinic glassware.]
Mobile phase: Hexane and ethyl acetate (75:25)

https://online.uspnf.com/uspnf/document/1_GUID-4CFC1971-CC7B-43E3-BA35-91195D5ABA85_3_en-US?source=TOC 1/4
11/9/2020 USP-NF meso-Zeaxanthin Preparation

Standard solution: 75 µg/mL of USP meso-Zeaxanthin RS in Mobile phase

Sample solution: Proceed as directed in Sample stock solution A or Sample stock solution B in the test for Content of Total
Carotenoids. Pass through a membrane lter of 0.45-µm pore size.
Chromatographic system
(See Chromatography 〈621〉, System Suitability.)
Mode: LC
Detector: UV 453 nm
Column: 4.6-mm × 25-cm; 3-µm packing L3
Flow rate: 1.5 mL/min
Injection volume: 10 µL
System suitability
Sample: Standard solution
[NOTE—The relative retention times for zeaxanthin and lutein are about 1.0 and 0.95, respectively.]
Suitability requirements
Resolution: NLT 1.0 between zeaxanthin and lutein
Tailing factor: NMT 2.0
Relative standard deviation: NMT 2.0%
Analysis
Sample: Sample solution
Calculate the percentage of zeaxanthin relative to total carotenoids in the portion of Preparation taken:

Result = (rU/rT) × 100

rU = individual peak response of zeaxanthin

rT = sum of all the peak responses

Calculate the percentage of zeaxanthin in the portion of Preparation taken:

L
Result = (rU/rT) × T
IA
rU = individual peak response of meso-zeaxanthin in the Sample solution

rT = sum of all the peak responses

T = percentage of total carotenoids as determined in the test for Content of Total Carotenoids
IC

Acceptance criteria: NLT 85.0% of zeaxanthin in the total carotenoid content

• LUTEIN AND OTHER RELATED COMPOUNDS
Mobile phase, Standard solution, Sample solution, and Chromatographic system: Proceed as directed in the test for Content of
Zeaxanthin.
FF

Injection volume: 10 µL
Analysis
Sample: Sample solution
Calculate the percentage of lutein and other related compounds relative to total carotenoids in the portion of Preparation taken:

Result = (rU/rT) × 100

O

rU = peak response of lutein or other individual related compounds

rT = sum of all the peak areas

Acceptance criteria
Lutein: NMT 9.0%
Any other individual related compound: NMT 1.0%
Total related compounds (including lutein): NMT 15.0%
• STEREOISOMERIC COMPOSITION
[NOTE—Use low-actinic glassware.]
Mobile phase: Gradient elution (see Table 1)

Table 1

Time (min) n-Hexane (%) 2-Propanol (%)

0.0 95 5

https://online.uspnf.com/uspnf/document/1_GUID-4CFC1971-CC7B-43E3-BA35-91195D5ABA85_3_en-US?source=TOC 2/4
11/9/2020 USP-NF meso-Zeaxanthin Preparation

Time (min) n-Hexane (%) 2-Propanol (%)

50 95 5

55 50 50

63 50 50

65 95 5

75 95 5

Standard solution: Transfer 2.5 mg of USP meso-Zeaxanthin RS to a 50-mL volumetric ask, add 25 mL of dehydrated alcohol, and
sonicate at 60° for 2–5 min to dissolve the substance. Cool the ask to room temperature and dilute with n-hexane to volume.
Transfer 1.0 mL of the resultant solution to a 15-mL test tube, and evaporate with a stream of nitrogen to dryness. Dissolve the
residue in a 10.0-mL mixture of 2-propanol and n-hexane (5:95). Pass through a membrane lter of 0.45-µm pore size.
Sample solution: Proceed as directed for Sample stock solution A or Sample stock solution B in the test for Content of Total
Carotenoids. Transfer 1.0 mL of Sample stock solution A or Sample stock solution B to a 15-mL test tube, and evaporate with a
stream of nitrogen to dryness. Dissolve the residue in a 10.0-mL mixture of 2-propanol and n-hexane (5:95). Pass through a
membrane lter of 0.45-µm pore size.
Chromatographic system
(See Chromatography 〈621〉, System Suitability.)
Mode: HPLC
Detector: 453 nm
Column: 4.6-mm × 25-cm; 5-µm packing L51
Column temperature: 30°
Flow rate: 0.8 mL/min
Injection volume: 20 µL
System suitability
L
IA
Sample: Standard solution
[NOTE—The approximate relative retention times for (3S,3′S)-zeaxanthin, (3R,3′S-meso)-zeaxanthin, (3R,3′R)-zeaxanthin, and
(3R,3′R,6′R)-lutein are 0.94, 1.00, 1.06, and 1.11, respectively.]
Suitability requirements
IC

Resolution: NLT 1.0 between each pair of peaks due to (3S,3′S)-zeaxanthin, (3R,3′S-meso)-zeaxanthin, (3R,3′R)-zeaxanthin, and
(3R,3′R,6′R)-lutein
Chromatogram similarity: The chromatogram from the Standard solution is similar to the reference chromatogram provided with
the lot of USP meso-Zeaxanthin RS being used.
Analysis
FF

Samples: Standard solution and Sample solution

Identify the peaks of the relevant analytes in the chromatogram of the Standard solution by comparison with the reference
chromatogram provided with the USP Reference Standard being used.
Calculate the percentage of (3S,3′S)-zeaxanthin, (3R,3′S-meso)-zeaxanthin, and (3R,3′R)-zeaxanthin:
O

Result = (rU/rT) × 100

rU = peak response of the corresponding analyte

rT = sum of all the peak responses

Acceptance criteria
(3R,3′S-meso)-Zeaxanthin: NLT 85.0%
(3R,3′R)-Zeaxanthin: NMT 15.0%
(3S,3′S)-Zeaxanthin: NMT 1.0%

IMPURITIES
• RESIDUE ON IGNITION 〈281〉: NMT 2.0%

SPECIFIC TESTS
• WATER DETERMINATION, Method I〈921〉: NMT 8.0% for solid Preparations; NMT 1.0% for liquid Preparations

ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Preserve in tightly sealed, light- and oxygen-resistant containers. Store in a cool place.
• LABELING: The label states the name and content of any carriers and antioxidants added to the formulation, and the content of total
carotenoids as zeaxanthin.

https://online.uspnf.com/uspnf/document/1_GUID-4CFC1971-CC7B-43E3-BA35-91195D5ABA85_3_en-US?source=TOC 3/4
11/9/2020 USP-NF meso-Zeaxanthin Preparation

• USP REFERENCE STANDARDS 〈11〉

USP meso-Zeaxanthin RS
β,β-Carotene-3,3′-diol (3R,3′S)-;
(3R,3′S-meso)-Zeaxanthin.
C40H56O2 568.88

Auxiliary Information- Please check for your question in the FAQs before contacting USP.

Topic/Question Contact Expert Committee

MESO-ZEAXANTHIN PREPARATION Maria Monagas NBDS2020 Non-botanical Dietary

Scienti c Liaison Supplements

Chromatographic Database Information: Chromatographic Database

Most Recently Appeared In:

Pharmacopeial Forum: Volume No. 41(2)

Page Information:

USP43-NF38 - 5572
USP42-NF37 - 5528
USP41-NF36 - 5157

Current DocID: GUID-4CFC1971-CC7B-43E3-BA35-91195D5ABA85_3_en-US

Previous DocID: GUID-4CFC1971-CC7B-43E3-BA35-91195D5ABA85_1_en-US

L
IA
IC
FF
O

https://online.uspnf.com/uspnf/document/1_GUID-4CFC1971-CC7B-43E3-BA35-91195D5ABA85_3_en-US?source=TOC 4/4