Lycopene

C40H56 536.88 [502-65-8].
DEFINITION
Lycopene is a mixture of geometrical isomers of lycopene. It contains NLT 96.0% and NMT 101.0% of lycopene (C40H56), calculated on the dried basis.
IDENTIFICATION
•  A. Ultraviolet-Visible Absorption 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 of A476/A508 is 1.10–1.14.
•  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 all-E-Lycopene, 5Z-Lycopene, and Related Compounds.
COMPOSITION
•  Content of Lycopene
Sample stock solution:  Transfer 25 mg of Lycopene and 25 mg of butylated hydroxytoluene into a 100-mL volumetric flask. Add 60 mL of methylene chloride, and sonicate to dissolve. Dilute with methylene chloride to volume.
Sample solution:  Transfer 2.0 mL of the Sample stock solution into a 200-mL volumetric flask, and dilute with cyclohexane to volume.
Instrumental conditions 
Mode:  UV-Vis
Analytical wavelength:  476 nm
Blank:  Cyclohexane
Analysis 
Sample:  Sample solution
Calculate the percentage of lycopene (C40H56) in the portion of Lycopene 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== concentration of the Sample solution (mg/mL)
Acceptance criteria:  96.0%–101.0% on the dried 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.
Sample stock solution:  Transfer 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.
Sample solution:  Pipet 2 mL of the Sample stock solution into a 50-mL volumetric flask, and add 8 mL of tetrahydrofuran. Dilute with tert-butyl methyl ether to volume.
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 all-E-lycopene and 5Z-lycopene peaks are 1.0 and about 1.07, respectively. ]
Suitability requirements 
Resolution:  NLT 1.0 between 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 Lycopene taken:
Result = (rE/rT) × T
rE== peak response of the all-E-lycopene isomer
rT== sum of the responses of all the peaks
T== percentage of total lycopene isomers obtained in the test for Content of Lycopene
Acceptance criteria:  NLT 70.0% of all-E-lycopene
Calculate the percentage of the 5Z-lycopene isomer in the portion of Lycopene taken:
Result = (r5Z/rT) × T
r5Z== peak response for the 5Z-lycopene isomer
rT== sum of the responses of all the peaks
T== percentage of total lycopene isomers obtained in the test for Content of Lycopene
Acceptance criteria:  NMT 23.0% of the 5Z-lycopene isomer
Calculate the percentage of related compounds in the portion of Lycopene taken:
Result = (rS/rT) × T
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
T== percentage of total lycopene isomers obtained in the test for Content of Lycopene
Acceptance criteria:  NMT 9.0% of other related compounds calculated as lycopene
IMPURITIES
•  Residue on Ignition 281: NMT 0.2%
•  Heavy Metals, Method II 231: NMT 10 ppm
SPECIFIC TESTS
•  Loss on Drying 731: Dry a sample in vacuum over phosphorus pentoxide at 40 for 4 h: it loses NMT 0.2% of its weight.
ADDITIONAL REQUIREMENTS
•  Packaging and Storage: Preserve in tight, light-resistant containers, under inert gas, and store in a cool place.
•  Labeling: Label it to indicate whether the article is obtained from natural sources or is prepared synthetically. If obtained from natural sources, label it to indicate the natural source, including its Latin binomial.
•  USP Reference Standards 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.
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USP35–NF30 Page 1370
Pharmacopeial Forum: Volume No. 30(6) Page 2073