Aztec Marigold Zeaxanthin Extract
C40H56O2 568.87
(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-
,-Carotene-3,3¢-diol
[148-68-3].
231
:
NMT 20 µg/g2S (USP38)
+'&img=../../images/v38332/cas-144-68-3.gif&casNumber=148-68-3&usp=38&nf=33&chemicalStructureImage=true&ID='+parent.myID,600,500);)
C40H56O2 568.87
(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-
,-Carotene-3,3¢-diol
[148-68-3].DEFINITION
Change to read:
Aztec Marigold Zeaxanthin Extract is a purified extract, derived from the flowers of
2S (USP38)
IDENTIFICATION
• A.
Sample solution:
Use the Sample solution from the test for Content of Total Carotenoids.
Analysis:
Record the UV-Vis spectrum from 300–600 nm.
Acceptance criteria:
The Sample solution shows a shoulder at about 428 nm, an absorption maximum at about 450 nm, and another maximum at about 478 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¢R-,-carotene-3,3¢-diol from the Standard solution, as obtained in the test for Stereoisomeric Composition.
,-carotene-3,3¢-diol from the Standard solution, as obtained in the test for Stereoisomeric Composition.
COMPOSITION
• Content of Total Carotenoids
[Note—Use low-actinic glassware. ]
Sample stock solution:
Use the Sample stock solution from the test for Content of Zeaxanthin.
Sample solution:
Transfer 2.0 mL of the Sample stock solution to a 100-mL volumetric flask, dilute with ethanol to volume, and mix well.
Instrumental conditions
Analytical wavelength:
450 nm
Cell path:
1 cm
Blank:
Ethanol
Analysis
Sample:
Sample solution
[Note—The absorbance reading should be between 0.2 AU to 1.0 AU. If not, readjust the dilution of the solution. ]
Calculate the percentage of the total carotenoids as zeaxanthin (C40H56O2):
Result = A/(C × F)
| A | = | = absorbance of the Sample solution |
| C | = | = concentration of the Sample solution (g/mL) |
| F | = | = coefficient of extinction (E1%) of zeaxanthin in ethanol (100 mL·g 1·cm1), 2480 |
Acceptance criteria:
NLT 36.0% of total carotenoids (T) as zeaxanthin (C40H56O2) on the dried basis
• Content of Zeaxanthin
[Note—Use low-actinic glassware. ]
Solution A:
Methyl tert-butyl ether
Solution B:
Water
Solution C:
Methanol
Diluent:
Mixture of hexane, ethanol, acetone, and toluene (10:6:7:7)
Mobile phase:
Gradient elution. See Table 1.
Table 1
| Time (min) |
Solution A (%) |
Solution B (%) |
Solution C (%) |
|---|---|---|---|
| 0.0 | 5 | 7 | 88 |
| 15 | 15 | 7 | 78 |
| 30 | 45 | 7 | 48 |
| 60 | 75 | 6.5 | 18.5 |
| 66 | 75 | 6 | 19 |
| 76 | 5 | 7 | 88 |
| 86 | 5 | 7 | 88 |
Standard stock solution:
Transfer 20.0 mg of USP Aztec Marigold Zeaxanthin Extract RS into a 100-mL volumetric flask, add 75 mL of Diluent to the flask to suspend the sample, and sonicate for 5 min. Dilute with Diluent to volume, and mix well. Allow the insolubles to settle for at least 10 min. Pass the supernatant through a membrane filter of 0.45-µm pore size.
Standard solution:
Transfer 0.5 mL of the Standard stock solution to an 8-mL vial, evaporated to dryness with an aid of a stream of nitrogen. Dissolve the residue in 4.0 mL of a mixture of methyl tert-butyl ether and methanol (5:95).
Sample stock solution:
Transfer 20.0 mg of Extract to a 100-mL volumetric flask, add 75 mL of Diluent to the flask to suspend the sample, and sonicate for 5 min. [Caution—Electrostatic charges may cause the sample to sputter and stick to the sides of the flask. Carefully wash down the product into the flask using Diluent.
] Dilute with Diluent to volume, and mix well. Allow the insolubles to settle for at least 10 min. Pass the supernatant through a membrane filter of 0.45-µm pore size.
Sample solution:
Transfer 0.5 mL of the Sample stock solution to an 8-mL vial, and evaporate to dryness with the aid of a stream of nitrogen. Dissolve the residue in a 4.0-mL mixture of methyl tert-butyl ether and methanol (5:95).
Chromatographic system
Mode:
HPLC
Detector:
450 nm
Column:
2.0-mm × 15-cm; 3-µm packing L62
Flow rate:
0.4 mL/min
Injection volume:
10 µL
System suitability
Sample:
Standard solution
[Note—The approximate relative retention times for lutein and zeaxanthin are 0.87 and 1.0, respectively. ]
Suitability requirements
Chromatographic similarity:
The chromatogram from the Standard solution is similar to the reference chromatogram provided with the USP Aztec Marigold Zeaxanthin Extract RS being used.
Resolution:
NLT 1.0 between zeaxanthin and lutein
Tailing factor:
NMT 2.0 for the zeaxanthin peak
Relative standard deviation:
NMT 2.0% for the zeaxanthin peak
Analysis
Sample:
Sample solution
Calculate the percentage of all-trans-zeaxanthin (C40H56O2) in the portion of the sample taken:
Result = (rU/rT) × T
| rU | = | = peak response of all-trans-zeaxanthin from the Sample solution |
| rT | = | = sum of the responses for all the peaks from the Sample solution |
| T | = | = percentage of total carotenoids as determined in the test for Content of Total Carotenoids |
Acceptance criteria:
NLT 30.0% of all-trans-zeaxanthin on the dried basis
Change to read:
• Lutein and Other Related Compounds
[Note—Use low-actinic glassware. ]
Mobile phase, Standard solution, Sample solution, Chromatographic system, and System suitability:
Proceed as directed in the test for Content of Zeaxanthin.
Analysis
Sample:
Sample solution
Calculate the percentage of lutein in the portion of the sample taken:
Result = (rU/rT) × T
| rU | = | = peak response of lutein from the Sample solution |
| rT | = | = sum of the responses for all the peaks from the Sample solution |
| T | = | = percentage of total carotenoids as determined in the test for Content of Total Carotenoids |
Calculate the percentage of other related compounds in the portion of the sample taken:
Result = (rU/rT) × 100
| rU | = | = peak response of individual related compounds peaks from the Sample solution |
| rT | = | = sum of the responses for all the peaks from the Sample solution |
Acceptance criteria
Lutein:
NMT 8.0% on the dried basis
Other related compounds:
NMT
2S (USP38)
• Stereoisomeric Composition
[Note—Use low-actinic glassware. ]
Mobile phase:
Gradient elution. See Table 2.
Table 2
| Time (min) |
n-Hexane (%) |
2-Propanol (%) |
|---|---|---|
| 0.0 | 95 | 5 |
| 50 | 95 | 5 |
| 55 | 50 | 50 |
| 63 | 50 | 50 |
| 65 | 95 | 5 |
| 75 | 95 | 5 |
Standard stock solution:
0.1 mg/mL of USP Aztec Marigold Zeaxanthin Extract RS in methylene chloride. Sonicate if necessary to dissolve the sample.
Standard solution:
Transfer 1.0 mL of the Standard stock solution into a 15-mL test tube, and evaporate to dryness with the aid of a stream of nitrogen. Dissolve the residue in a 10.0-mL mixture of 2-propanol and hexane (5:95). Pass through a membrane filter of 0.45-µm pore size.
Sample stock solution:
Weigh 10 mg of Extract into a 100-mL volumetric flask, add 85 mL of methylene chloride, sonicate, and swirl to dissolve. Dilute with methylene chloride to volume.
Sample solution:
Transfer 1.0 mL of the Sample stock solution into a 15-mL test tube, and evaporate to dryness with the aid of a nitrogen stream. Dissolve the residue in a 10.0-mL mixture of 2-propanol and hexane (5:95). Pass the solution through a membrane filter of 0.45-µm pore size.
Chromatographic system
Mode:
HPLC
Detector:
450 nm
Column:
4.6-mm × 25-cm; 5-µm packing L51
Flow rate:
0.8 mL/min
Injection volume:
20 µL
System suitability
Sample:
Standard solution
[Note—The approximate relative retention times for (3R,3¢S meso)-zeaxanthin, (3R,3¢R)-zeaxanthin, and (3R,3¢R,6¢R)-lutein are 0.92, 1.00, and 1.12, respectively. ]
Suitability requirements
Resolution:
The resolution between each pair of peaks due to (3R,3¢S meso)-zeaxanthin, (3R,3¢R)-zeaxanthin, and (3R,3¢R,6¢R)-lutein is NLT 1.0.
Chromatogram similarity:
The chromatogram from the Standard solution is similar to the reference chromatogram provided with the USP Aztec Marigold Zeaxanthin Extract RS being used.
Analysis
Samples:
Standard solution and Sample solution
Identify the peaks of the relevant analytes in the chromatogram of the Sample solution by comparing with those in the chromatogram of the Standard solution obtained from the System suitability.
Calculate the percentages of (3R,3¢S meso)-zeaxanthin and (3R,3¢R)-zeaxanthin in the portion of the sample taken:
Result = (rU/rT) × 100
| rU | = | = peak response of the corresponding analyte |
| rT | = | = sum of the responses for two peaks |
Acceptance criteria
(3R,3¢R)-Zeaxanthin:
NLT 99.0%
(3R,3¢S meso)-Zeaxanthin:
NMT 1.0%
CONTAMINANTS
Delete the following:
231:
NMT 20 µg/g2S (USP38)
Add the following:
233
Acceptance criteria
Arsenic:
NMT 0.5 µg/g
Cadmium:
NMT 1.0 µg/g
Lead:
NMT 5.0 µg/g
Mercury:
NMT 0.1 µg/g2S (USP38)
2S (USP38)
Delete the following:
281:
NMT 10.0%2S (USP38)
SPECIFIC TESTS
• Loss on Drying 731
731
Analysis:
Dry a sample under vacuum at 105
for 3 h.
for 3 h.
Acceptance criteria:
NMT 25.0%
ADDITIONAL REQUIREMENTS
• Packaging and Storage:
Preserve in tight, light-resistant containers.
• USP Reference Standards 11
11
USP Aztec Marigold Zeaxanthin Extract RS
Auxiliary Information—
Please check for your question in the FAQs before contacting USP.
| Topic/Question | Contact | Expert Committee |
|---|---|---|
| Monograph | Huy T. Dinh, M.S.
Senior Scientific Liaison (301) 816-8594 |
(DS2010) Monographs - Dietary Supplements and Herbal Medicines |
| Reference Standards | RS Technical Services 1-301-816-8129 rstech@usp.org |
USP38–NF33 Page 5894
USP38–NF33 Supplement : No. 2 Page 7782
Pharmacopeial Forum: Volume No. 40(5)