Lutein Preparation
DEFINITION
Lutein Preparation is a combination of Lutein 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 lutein, calculated as C40H56O2 on the anhydrous basis. It contains NLT 85.0% of lutein and NMT 9.0% of zeaxanthin of the total carotenoid content.
IDENTIFICATION
• A. Ultraviolet Absorption 
197U
197U
Analytical wavelength:
300–700 nm
Sample solution:
Prepare as directed for the Sample solution in the test for Content of Total Carotenoids.
Ratio:
A446/A474, 1.09–1.14
• 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 Lutein.
COMPOSITION
• Content of Total Carotenoids
Diluent:
Hexanes, acetone, toluene, and dehydrated alcohol (10:7:7:6)
Sample stock solution A (for solid lutein preparations labeled as containing gelatin):
Transfer an amount of Preparation, equivalent to 3.5 mg of lutein, to a 50-mL centrifuge tube. Add 15 mL of warm water, 60 units of bacterial alkaline protease preparation, and 1 mg of bromelain. Cap and sonicate for 20 min with occasional swirling. Cool to room temperature, and add 20.0 mL of methylene chloride. Shake for 1 min, and centrifuge for 5 min at 2000 rpm. Remove the upper aqueous phase, and add 2–3 g of anhydrous sodium sulfate to the remaining red layer.
Sample stock solution B (for other solid lutein preparations):
Transfer an amount of Preparation, equivalent to 1.5 mg of lutein, to a 50-mL centrifuge tube. Add 15 mL of warm water, cap, and sonicate for 30 min with occasional swirling. Cool to room temperature, and add 30.0 mL of ethyl acetate and 2–3 g of sodium chloride. Shake for 1 min, and centrifuge for 5 min at 2000 rpm. Use the upper orange-red layer.
Sample stock solution C (for liquid lutein suspensions in oil):
Transfer a weighed amount of Preparation equivalent to 20 mg of lutein to a 100-mL volumetric flask, and dilute with Diluent to volume. Add a magnetic bar, and stir for 30 min.
Sample solution:
Transfer 1.0 mL of Sample stock solution A, or 1.0 mL of Sample stock solution B, or 1.0 mL of Sample stock solution C into a 100-mL volumetric flask, and dilute with dehydrated alcohol to volume.
Spectrometric conditions
Analytical wavelength:
446 nm
Cell path:
1 cm
Blank:
Dehydrated alcohol
Analysis
Sample:
Sample solution
Calculate the percentage of total carotenoids (T) as lutein (C40H56O2) in the Preparation:
Result = (A × V × D × 100)/(F × W)
| A | = | = absorbance of the Sample solution |
| F | = | = absorptivity of the lutein in alcohol, 255.0 (mL/mg·cm) |
| V | = | = volume of organic solvent (20.0 mL for Sample stock solution A, 30.0 mL for Sample stock solution B, and 100.0 mL for Sample stock solution C) used in preparing the Sample stock solutions |
| D | = | = dilution factor used to prepare the Sample solution from Sample stock solutions |
| W | = | = weight of Preparation taken to prepare the Sample stock solutions (mg) |
• Content of Lutein
Diluent:
Hexanes, acetone, toluene, and dehydrated alcohol (10:7:7:6)
Mobile phase:
Hexane and ethyl acetate (75:25)
Standard solution:
150 µg/mL of USP Lutein RS in Mobile phase
Sample solution:
Transfer 1.0 mL of Sample stock solution A, or 1.0 mL of Sample stock solution B, or 2.0 mL of Sample stock solution C from the test for Content of Total Carotenoids into a suitable vial. Evaporate the solvent to dryness under a stream of nitrogen. Add 1.0 mL of Mobile phase, and sonicate to dissolve.
Chromatographic system
Mode:
LC
Detector:
UV 446 nm
Column:
4.6-mm × 25-cm; 5-µm packing L3
Flow rate:
1.5 mL/min
Injection size:
10 µL
System suitability
Sample:
Standard solution
[Note—The relative retention times for lutein and zeaxanthin are about 1.0 and 1.05, respectively. ]
Suitability requirements
Resolution:
NLT 1.0 between lutein and zeaxanthin
Tailing factor:
NMT 2
Relative standard deviation:
NMT 2.0%
Analysis
Sample:
Sample solution
Calculate the percentage of lutein relative to total carotenoids in the Preparation taken:
Result = (rU/rT) × 100
| rU | = | = individual peak response of lutein |
| rT | = | = sum of the responses of all the peaks |
Calculate the percentage of lutein in the Preparation taken:
Result = (rU/rT) × T
| rU | = | = individual peak response of lutein in the Sample solution |
| rT | = | = sum of the responses of all the peaks |
| T | = | = percentage of total carotenoids as determined in the test for Content of Total Carotenoids |
Acceptance criteria:
NLT 85.0% of lutein in the total carotenoid content, and the Preparation contains 95.0%–130.0% of the labeled amount of lutein, calculated as C40H56O2, on the anhydrous basis.
• Zeaxanthin and Other Related Compounds
Solvent, Mobile phase, Standard solution, Sample solution, and Chromatographic system:
Proceed as directed in the test for Content of Lutein.
Analysis
Sample:
Sample solution
Injection size:
10 µL
Calculate the percentage of zeaxanthin relative to total carotenoids in the Preparation taken:
Result = (rU/rT) × 100
| rU | = | = individual peak response of zeaxanthin |
| rT | = | = sum of the responses of all the peaks |
Acceptance criteria
Zeaxanthin:
NMT 9.0%
Any other single related compound:
NMT 1.0%
Total related compounds (including zeaxanthin):
NMT 15.0%
IMPURITIES
Inorganic Impurities
• Residue on Ignition 281:
NMT 2.0%
281:
NMT 2.0%
• Lead 251:
NMT 1 ppm
251:
NMT 1 ppm
• Heavy Metals 231:
NMT 10 ppm
231:
NMT 10 ppm
SPECIFIC TESTS
• Water Determination, Method I 921:
NMT 10.0%
921:
NMT 10.0%
ADDITIONAL REQUIREMENTS
• Packaging and Storage:
Preserve in tightly sealed, light- and oxygen-resistant containers. Store in a cool place.
• Labeling:
The label states that this article is not intended for direct administration to humans or to animals.
• USP Reference Standards 11
11
USP Lutein RS
Auxiliary Information—
Please check for your question in the FAQs before contacting USP.
| Topic/Question | Contact | Expert Committee |
|---|---|---|
| Monograph | Natalia Davydova
Scientific Liaison 1-301-816-8328 |
(DS2010) Monographs - Dietary Supplements |
| Reference Standards | RS Technical Services 1-301-816-8129 rstech@usp.org |
USP35–NF30 Page 1369
Pharmacopeial Forum: Volume No. 33(2) Page 255