Tomato Extract Containing Lycopene
DEFINITION
Tomato Extract Containing Lycopene is an ethyl acetate extract of the natural tomato lipids. It is produced from the pulp of ripe fruits of Lycopersicon esculentum Mill. (Fam. Solanaceae), after removing the tomato water-soluble fraction. It contains NLT 95.0% and NMT 105.0% of the labeled amount of lycopene (C40H56). It contains NLT 4.7% and NMT 12.0% of lycopene (C40H56), NLT 0.8% of the combined amount of phytofluene (C40H68) and phytoene (C40H64), NLT 0.2% of beta carotene (C40H56), and NLT 1.0% of tocopherols (C28H48O2) on the anhydrous basis. Tocopherols may be added as antioxidants.
IDENTIFICATION
•  A. Presence of Lycopene, Phytofluene, and Phytoene
Analysis:  Proceed as directed in the test for Content of Other Carotenoids and Tocopherols.
Acceptance criteria:  The retention times of the peaks for lycopene, phytofluene, and phytoene from the Sample solution correspond to those from Standard solution C.
•  B. Ratio of all-E-Lycopene and 5Z-Lycopene
Butylated hydroxytoluene stock solution:  Proceed as directed in the test for Content of Lycopene.
Mobile phase:  0.05% diisopropylethylamine in n-hexane; sonicate for 3–4 min
Sample solution:  Proceed as directed in the test for Content of Lycopene, except dilute 5 to 100 with n-hexane
Chromatographic system 
Mode:  LC
Detector:  UV-Vis 472 nm
Column:  Two 4.0-mm × 25-cm columns; 5-µm packing L3 (300 pore size), connected in a series
Column temperature:  22
Flow rate:  0.5 mL/min
Injection size:  10 µL
[Note—The peak for all-E-lycopene elutes in 30–45 min. ]
[Note—The relative retention times for all-E-lycopene and 5Z-lycopene are 1.00 in the range and 1.04–1.10, respectively. ]
Analysis 
Sample:  Sample solution
Measure the areas of the two major peaks, and calculate their area ratio:
Result = rU1/rU2
rU1== peak area of 5Z-lycopene
rU2== peak area of all-E-lycopene
Acceptance criteria:  NMT 0.10 for the area ratio
COMPOSITION
•  Content of Lycopene
Butylated hydroxytoluene stock solution:  5 mg/mL of butylated hydroxytoluene in methylene chloride. [Note—This solution can be stored protected from light for up to 3 months. ]
Mobile phase:  Acetonitrile, methylene chloride, n-hexane, and methanol (850:25:25:100). Add 0.05% of diisopropylethylamine, mix, and sonicate for 3–4 min.
Diluent:  Acetonitrile, methylene chloride, n-hexane, butylated hydroxytoluene, and methanol (600: 150: 100: 0.5: 150). Add 0.05% of diisopropylethylamine, mix, and sonicate for 3–4 min.
Standard solution A:  Transfer a weighed quantity of USP Lycopene RS, equivalent to approximately 5 mg of lycopene, to a 100-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, mix, and place in an ultrasonic bath at 50 for 10 min, rotating the flask occasionally to avoid having the material stick to the glass surface. Continue until the material is dispersed with no lumps. Add 5 mL of tetrahydrofuran, and shake until no colored precipitate remains. Add another portion of 2 mL of tetrahydrofuran and 40 mL of Diluent, and shake until the mixture is homogeneous. Dilute with Diluent to volume, shake vigorously, and allow to stand, if necessary, until the solid has settled.
Calculate the exact concentration of this solution by the following method.
Standard solution B:  To 2.0 mL of Standard solution A add 10 mL of alcohol and 10 mL of Butylated hydroxytoluene stock solution, and dilute with n-hexane to 100 mL. Prepare in triplicate.
Determine the absorbance of Standard solution B at the maximum absorbance at about 472 nm, using a mixture of alcohol, Butylated hydroxytoluene stock solution, and n-hexane (10:10:80) as the blank.
Calculate the concentration of Standard solution A, in µg/mL, of lycopene:
Result = (AX/F) × 50,000
AX== average of the absorbance of the three preparations of Standard solution B
F== absorptivity of pure lycopene in n-hexane at 472 nm, 345
Standard solution C:  Transfer a weighed quantity of USP Tomato Extract Containing Lycopene RS, equivalent to about 6 mg of lycopene, to a 100-mL volumetric flask, and dissolve in 1 mL of Butylated hydroxytoluene stock solution and 9 mL of methylene chloride, using a sonicator. Dilute with Diluent to volume to obtain a solution having a known concentration of about 0.06 mg/mL of lycopene.
Sample stock solution:  Warm the Tomato Extract Containing Lycopene to 50 in a water bath. Mix well with a glass rod or a spatula. Weigh and dissolve a quantity of 1–1.2 g of the sample in 10 mL of Butylated hydroxytoluene stock solution and 30 mL of methylene chloride, and sonicate the solution for 1 min to dissolve the sample completely. Cool to room temperature, and dilute with methylene chloride to 100 mL.
Sample solution:  Dilute the Sample stock solution with Diluent (1 in 10).
Chromatographic system 
Mode:  LC
Detector:  UV-Vis 472 nm
Column:  4.6-mm × 25-cm; 5-µm packing L7
Column temperature:  39 ± 1
Flow rate:  0.7 mL/min
Injection size:  10 µL
System suitability 
Sample:  Standard solution A
[Note—The retention time for lycopene is about 6 min. ]
Suitability requirements 
Relative standard deviation:  NMT 1.5% for the lycopene peak area
Analysis 
Samples:  Standard solution A or Standard solution C, and Sample solution
Measure the responses of the major lycopene peaks.
Calculate the percentage of lycopene in the portion of Tomato Extract Containing Lycopene taken:
Result = (rU/rS) × CS × (V/W) × D × 100
rU== peak area for lycopene from the Sample solution
rS== peak area for lycopene from Standard solution A or Standard solution C
CS== concentration of lycopene in Standard solution A or Standard solution C (µg/mL)
V== volume of the Sample stock solution (mL)
W== weight of Tomato Extract Containing Lycopene taken to prepare the Sample stock solution (mg)
D== dilution factor used to prepare the Sample solution from Sample stock solution
Acceptance criteria:  NLT 95.0%–NMT 105.0% of the labeled amount of lycopene; NLT 4.7%–NMT 12.0% of lycopene (C40H56)
•  Content of Other Carotenoids and Tocopherols (Phytofluene, Phytoene, Beta Carotene, and Tocopherols)
Butylated hydroxytoluene stock solution, Diluent, Standard solution A, Standard solution B, Standard solution C, and Sample solution:  Proceed as directed in the test for Content of Lycopene.
Mobile phase:  Acetonitrile, methylene chloride, n-hexane, and methanol (19:1:1:19). Add 0.05% of diisopropylethylamine, mix, and sonicate for 3–4 min.
Chromatographic system 
Mode:  LC
Detector:  UV-Vis; 472 nm for lycopene; 450 nm for beta carotene; 350 nm for phytofluene; and 288 nm for phytoene and tocopherol
Column:  4.6-mm × 25-cm; 5-µm packing L1
Column temperature:  39 ± 1
Flow rate:  0.6 mL/min
Injection size:  10 µL
System suitability 
Sample:  Standard solution C
[Note—The relative retention times are about 0.6 for the tocopherol isomers, 1.0 for all-E-lycopene, 1.5–1.7 for the beta carotene isomers, 1.6–1.8 for the phytofluene isomers, and 1.8–2.2 for phytoene. ]
Suitability requirements 
Chromatogram similarity:  The chromatogram of Standard solution C is similar to the reference chromatogram provided with the lot of USP Tomato Extract Containing Lycopene RS being used.
Relative standard deviation:  NMT 2% for all-E-lycopene
Analysis 
Samples:  Standard solution A and Sample solution
Identify the locus of the peaks for the lycopene isomers, the beta carotene isomers, the phytofluene isomers, and phytoene by comparison with the reference chromatogram provided with the corresponding lot of USP Tomato Extract Containing Lycopene RS. Measure the sum of the peak responses of the lycopene isomers at 472 nm in Standard solution A. [note—The lycopene isomers may be resolved in more than one peak in this chromatographic system. ] In the Sample solution, measure the sum of the peak responses of the beta carotene isomers at 450 nm, the sum of the peak responses of the phytofluene isomers at 350 nm, the peak response of phytoene at 288 nm, and the sum of the peak responses of all tocopherols at 288 nm.
Determine the concentration of Standard solution A as directed in the test for Content of Lycopene.
Calculate the percentage of beta carotene in the portion of Tomato Extract Containing Lycopene taken:
Result = (rU/rS) × CS × (V/W) × D × F × 100
rU== sum of the peak responses for the beta carotene isomers at 450 nm from the Sample solution
rS== peak area for lycopene at 472 nm from Standard solution A
CS== concentration of lycopene in Standard solution A (µg/mL)
V== volume of the Sample stock solution (mL)
W== weight of Tomato Extract Containing Lycopene taken to prepare the Sample stock solution (mg)
D== dilution factor used to prepare the Sample solution from Sample stock solution
F== absorptivity ratio of pure lycopene to pure beta carotene, 345/259.2
Calculate the percentage of phytofluene in the portion of Tomato Extract Containing Lycopene taken:
Result = (rU/rS) × CS × (V/W) × D × F × 100
rU== sum of the peak responses for phytofluene isomers at 350 nm from the Sample solution
rS== sum of the peak responses for the lycopene isomers at 472 nm from Standard solution A
CS== concentration of lycopene in Standard solution A (µg/mL)
V== volume of the Sample stock solution (mL)
W== weight of Tomato Extract Containing Lycopene taken to prepare the Sample stock solution (mg)
D== dilution factor used to prepare the Sample solution from Sample stock solution
F== absorptivity ratio of pure lycopene to pure phytofluene, 345/135
Calculate the percentage of phytoene in the portion of Tomato Extract Containing Lycopene taken:
Result = (rU/rS) × CS × (V/W) × D × F × 100
rU== area of the phytoene peak response at 288 nm from the Sample solution
rS== sum of the peak responses for the lycopene isomers at 472 nm from Standard solution A
CS== concentration of lycopene in Standard solution A (µg/mL)
V== volume of the Sample stock solution (mL)
W== weight of Tomato Extract Containing Lycopene taken to prepare the Sample stock solution (mg)
D== dilution factor used to prepare the Sample solution from Sample stock solution
F== absorptivity ratio of pure lycopene to pure phytoene, 345/125
Calculate the percentage of tocopherols in the portion of Tomato Extract Containing Lycopene taken to prepare the Sample solution:
Result = (rU/rS) × CS × (V/W) × D × F × 100
rU== sum of the peak responses for all the tocopherol peaks at 288 nm from the Sample solution
rS== sum of the peak responses for the lycopene isomers at 472 nm from Standard solution A
CS== concentration of lycopene in Standard solution A (µg/mL)
V== volume of the Sample stock solution (mL)
W== weight of Tomato Extract Containing Lycopene taken to prepare the Sample stock solution (mg)
D== dilution factor used to prepare the Sample solution from Sample stock solution
F== absorptivity ratio of pure lycopene to the average absorptivity for tocopherols, 345/8.5
Acceptance criteria:  NLT 0.8% of the combined amount of phytofluene (C40H68) and phytoene (C40H64), NLT 0.2% of beta carotene (C40H56), and NLT 1.0% of tocopherols (C28H48O2), on the anhydrous basis
CONTAMINANTS
•  Heavy Metals, Method II 231: NMT 10 µg/g
•  Articles of Botanical Origin, Test for Aflatoxins 561: NMT 4 ng/g of total aflatoxins B1, B2, G1, and G2; NMT 2 ng/g of aflatoxin B1
•  Microbial Enumeration Tests 2021: The total aerobic microbial count does not exceed 103 cfu/g, and the total combined molds and yeasts count does not exceed 2 × 102 cfu/g.
•  Absence of Specified Microorganisms 2022: It meets the requirements of the tests for absence of Salmonella species, Escherichia coli, and Pseudomonas aeruginosa.
SPECIFIC TESTS
•  Clarity of Solution
Analysis:  Warm the sample to 50 in a water bath. Mix well with a glass rod or a spatula, and transfer 1 g of the Extract directly into a 100-mL volumetric flask. Add 50 mL of methylene chloride, and sonicate the solution for 1 min to completely dissolve the sample. Bring to room temperature, and dilute with methylene chloride to volume.
Acceptance criteria:  The solution is clear: no deposit or turbidity is formed.
•  Viscosity 911
Analysis:  Equilibrate the Tomato Extract Containing Lycopene at 37 in a 30-mL glass vial. Determine the viscosity using a rotational viscosimeter equipped with a spindle (No. 6) having a cylinder 1.47 cm in diameter and 0.16 cm high attached to a shaft 0.32 cm in diameter, with a distance of 3.02 cm from the top of the cylinder to the lower tip of the shaft. The spindle is rotating at the appropriate speed and immersion depth to obtain a scale reading of 10%–90% of full scale. Calculate the viscosity, in centipoises, by multiplying the scale reading by the constant for the spindle and speed used.
Acceptance criteria:  NMT 5000 centipoises
•  Particle Size Distribution
Analysis:  Transfer 1 drop to a microscope slide, and spread evenly. Isopropanol may be used as a diluent, if necessary. Examine the slide under a microscope equipped with a calibrated ocular micrometer, using 450× magnification. Scan the slide, and note the size of the individual particles.
Acceptance criteria:  NLT 98% of the particles are less than 20 µm in length when measured along the longest axis, NLT 60% of the particles are less than 5 µm, and NLT 40% of the particles are less than 2 µm.
ADDITIONAL REQUIREMENTS
•  Packaging and Storage: Preserve in tight, light-resistant containers, and store in a cool place.
•  Labeling: Label it to state the content of lycopene as a percentage and to state that the material should be heated to 50 and mixed before use. Label it to indicate the Latin binomial and the part of the plant from which the article is derived.
•  USP Reference Standards 11
USP Lycopene RS
USP Tomato Extract Containing Lycopene RS
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