Lycopene Preparation
DEFINITION
Lycopene Preparation is a combination of Lycopene with one or more inert substances and suitable antioxidants. It may be in a solid or oily liquid form. It contains NLT 95.0% and NMT 120.0% of the labeled amount of lycopene (C40H56), calculated on the anhydrous basis.
IDENTIFICATION
• A. Ultraviolet-Visible Absorption 
197U
197U
Wavelength range:
300–700 nm
Test solution:
Prepare as directed for the Sample solution in the test for Content of Lycopene.
Acceptance criteria:
Meets the requirements in the chapter. The ratio A476/A508 is 1.10–1.14 in cyclohexane. The ratio A472/A504 is 1.09–1.13 in isopropyl alcohol.
COMPOSITION
• Content of Lycopene
Procedure for oily preparations
Sample stock solution:
Transfer a weighed quantity of oily Preparation containing 25 mg of lycopene to a 100-mL volumetric flask. Add 25 mg of butylated hydroxytoluene and 60 mL of methylene chloride, and sonicate to dissolve. Dilute with methylene chloride to volume.
Sample solution:
2.0 mL of the Sample stock solution diluted with cyclohexane to 200.0 mL
Instrumental conditions
Mode:
UV-Vis
Analytical wavelength:
476 nm
Blank:
Cyclohexane
Analysis
Sample:
Sample solution
Calculate the percentage of the labeled amount of lycopene (C40H56) in the portion of Preparation taken:
Result = AU/[(a × CU)] × 100
| AU | = | = absorbance of the Sample solution |
| a | = | = absorptivity of the pure lycopene in cyclohexane, 331 (mL·mg-1·cm-1) |
| CU | = | = nominal concentration of lycopene in the Sample solution (mg/mL) |
Acceptance criteria:
95.0%–120.0% on the anhydrous basis
Procedure for solid preparations
Sample stock solution:
Transfer a weighed quantity of solid Preparation, equivalent to approximately 5 mg of lycopene, into a 200-mL volumetric flask. Add about 60 units of bacterial alkaline protease preparation, or another suitable enzyme, and about 25 mg of butylated hydroxytoluene. Add 2.5 mL of dilute ammonium hydroxide (2 in 100) in water, and mix. Place in an ultrasonic bath at 50
for 10 min, rotate the flask occasionally to avoid having the material stick to the glass surface, and continue until the material is dispersed with no lumps. Add 5 mL of tetrahydrofuran, 40 mL of dehydrated alcohol, and mix. Place in an ultrasonic bath for about 1 min. Cool to room temperature, and dilute with tert-butyl methyl ether to volume. Shake vigorously, then allow to stand until the solid has settled.
for 10 min, rotate the flask occasionally to avoid having the material stick to the glass surface, and continue until the material is dispersed with no lumps. Add 5 mL of tetrahydrofuran, 40 mL of dehydrated alcohol, and mix. Place in an ultrasonic bath for about 1 min. Cool to room temperature, and dilute with tert-butyl methyl ether to volume. Shake vigorously, then allow to stand until the solid has settled.
Sample solution:
2.0 mL of the Sample stock solution diluted with isopropyl alcohol to 25.0 mL
Instrumental conditions
Mode:
UV-Vis
Analytical wavelength:
472 nm
Blank:
Isopropyl alcohol
Analysis
Sample:
Sample solution
Calculate the percentage of the labeled amount of lycopene in the portion of Preparation taken:
Result = AU/[(a × CU)] × 100
| AU | = | = absorbance of the Sample solution |
| a | = | = absorptivity of the pure lycopene in isopropyl alcohol, 320 (mL·mg-1·cm-1) |
| CU | = | = nominal concentration of lycopene in the Sample solution (mg/mL) |
Acceptance criteria:
95.0%–120.0% on the anhydrous basis
• Content of All-E-Lycopene, 5Z-Lycopene, and Related Compounds
Mobile phase:
tert-Butyl methyl ether, methanol, and tetrahydrofuran (784:665:74)
Standard solution:
Transfer a weighed quantity of USP Lycopene RS, equivalent to approximately 5 mg of lycopene, into a 250-mL volumetric flask. Add about 60 units of bacterial alkaline protease preparation, or another suitable enzyme, and about 25 mg of butylated hydroxytoluene. Add 2.5 mL of dilute ammonium hydroxide (2 in 100) in water, and mix. Place in an ultrasonic bath at 50 for 10 min, rotate the flask occasionally to avoid having the material stick to the glass surface, and continue until the material is dispersed with no lumps. Add 5 mL of tetrahydrofuran, 40 mL of dehydrated alcohol, and mix. Place in an ultrasonic bath for about 1 min. Cool to room temperature, and dilute with tert-butyl methyl ether to volume. Shake vigorously, then allow the precipitate to settle. Filter the supernatant.
for 10 min, rotate the flask occasionally to avoid having the material stick to the glass surface, and continue until the material is dispersed with no lumps. Add 5 mL of tetrahydrofuran, 40 mL of dehydrated alcohol, and mix. Place in an ultrasonic bath for about 1 min. Cool to room temperature, and dilute with tert-butyl methyl ether to volume. Shake vigorously, then allow the precipitate to settle. Filter the supernatant.
Sample solution for oily preparations:
Transfer a quantity of oily Preparation, equivalent to 15 mg of lycopene, to a 25-mL volumetric flask, and dissolve in tetrahydrofuran containing 50 mg/L of butylated hydroxytoluene. Dilute with the same solvent to volume. Pipet 2 mL of this solution into a 50-mL volumetric flask, and add 8 mL of tetrahydrofuran. Dilute with tert-butyl methyl ether to volume.
Sample solution for solid preparations:
Transfer a weighed quantity of solid Preparation, equivalent to approximately 5 mg of lycopene, into a 250-mL volumetric flask. Add about 60 units of bacterial alkaline protease preparation, or another suitable enzyme, and about 25 mg of butylated hydroxytoluene. Add 2.5 mL of dilute ammonium hydroxide (2 in 100) in water, and mix. Place in an ultrasonic bath at 50 for 10 min, rotate the flask occasionally to avoid having the material stick to the glass surface, and continue until the material is dispersed with no lumps. Add 5 mL of tetrahydrofuran, 40 mL of dehydrated alcohol, and mix. Place in an ultrasonic bath for about 1 min. Cool to room temperature, and dilute with tert-butyl methyl ether to volume. Shake vigorously, then allow the precipitate to settle. Filter the supernatant.
for 10 min, rotate the flask occasionally to avoid having the material stick to the glass surface, and continue until the material is dispersed with no lumps. Add 5 mL of tetrahydrofuran, 40 mL of dehydrated alcohol, and mix. Place in an ultrasonic bath for about 1 min. Cool to room temperature, and dilute with tert-butyl methyl ether to volume. Shake vigorously, then allow the precipitate to settle. Filter the supernatant.
Chromatographic system
Mode:
LC
Detector:
UV 472 nm
Column:
4.6-mm × 25-cm, 5-µm packing L62; second column connected in series, 4.6-mm × 25-cm, 3-µm packing L62. [Note—New columns may require conditioning. ]
Flow rate:
1 mL/min
Injection size:
10 µL
System suitability
Sample:
Standard solution
[Note—The relative retention times for the all-E-lycopene and 5Z-lycopene peaks are 1.0 and about 1.07, respectively. ]
Suitability requirements
Resolution:
NLT 1.0 between the all-E-lycopene and 5Z-lycopene peaks
Tailing factor:
0.8–2.0 for the all-E-lycopene peak
Relative standard deviation:
NMT 2.0% for the all-E-lycopene peak
Analysis
Sample:
Sample solution
Calculate the percentage of all-E-lycopene in the portion of Preparation taken:
Result = (rE/rT) × 100
| rE | = | = peak response of the all-E-lycopene isomer |
| rT | = | = sum of the responses of all the peaks |
Acceptance criteria:
NLT 65.0% of all-E-lycopene
Calculate the percentage of the 5Z-lycopene isomer in the portion of Preparation taken:
Result = (r5Z/rT) × 100
| r5Z | = | = peak response for the 5Z-lycopene isomer |
| rT | = | = sum of the responses of all the peaks |
Acceptance criteria:
NMT 23.0% of the 5Z-lycopene isomer
Calculate the percentage of related compounds in the portion of Preparation taken:
Result = (rS/rT) × 100
| rS | = | = sum of the responses of all peaks except the peak for all-E-lycopene and the peak for 5Z-lycopene |
| rT | = | = sum of the responses of all the peaks |
Acceptance criteria:
NMT 14% of other related compounds calculated as lycopene
SPECIFIC TESTS
• Water Determination, Method I 921:
NMT 8.0%
921:
NMT 8.0%
ADDITIONAL REQUIREMENTS
• Packaging and Storage:
Preserve in tight, light-resistant containers under inert gas. Store the oily Preparation in a cool place and the solid Preparation at controlled room temperature.
• Labeling:
Label it to state the name and content of added antioxidants and inert substances. Label it to indicate whether the article is prepared with lycopene from natural sources or with synthetic lycopene. If prepared with lycopene from natural sources, label it to indicate the natural source, including its Latin binomial.
• USP Reference Standards 11
11
USP Lycopene 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.
Scientific Liaison 1-301-816-8594 |
(DS2010) Monographs - Dietary Supplements |
| Reference Standards | RS Technical Services 1-301-816-8129 rstech@usp.org |
USP35–NF30 Page 1371
Pharmacopeial Forum: Volume No. 30(6) Page 2075