Particle size distribution—
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 about 450× magnification (see
Optical Microscopy 
776
). Scan the slide, and note the size of the individual particles: not fewer than 98% of the particles are less than 20 µm in length when measured along the longest axis, not fewer than 60% of the particles are less than 5 µm, and not fewer than 40% of the particles are less than 2 µm.
Content of lycopene—
Butylated hydroxytoluene stock solution—
Dissolve 2.5 g of butylated hydroxytoluene in methylene chloride to make 500 mL. [NOTE—This solution can be stored protected from light for up to 3 months.]
Mobile phase—
Prepare a filtered and degassed mixture of acetonitrile, methanol, methylene chloride, and n-hexane (850:100:25:25). Add 0.05% of diisopropylethylamine, mix well, and sonicate for 3 to 4 minutes.
Diluting solution—
Prepare a mixture of acetonitrile, methylene chloride, methanol, n-hexane, and butylated hydroxytoluene (600:150:150:100:0.5). Add 0.05% of diisopropylethylamine, mix well, and sonicate for 3 to 4 minutes.
Standard solution A—
Transfer an accurately weighed quantity of
USP Lycopene RS, equivalent to 6 to 8 mg of lycopene, to a 100-mL volumetric flask, and dissolve in 10 mL of methylene chloride, using a sonicator. Dilute with
Diluting solution to volume to obtain a solution having a known concentration of about 0.07 mg of lycopene per mL.
Determine the exact concentration of Standard solution A by employing the following method.
Standard solution B—
Transfer 2.0 mL of Standard solution A to a 100-mL volumetric flask, add 10 mL of alcohol and 10 mL of Butylated hydroxytoluene stock solution, dilute with n-hexane to volume, and mix.
Procedure—
Determine the absorbance of this solution at the maximum absorbance at about 472 nm. Calculate the concentration of
Standard solution A, in ppm of lycopene, by the formula:
50,000Ax /345,
in which
Ax is the absorbance of
Standard solution B, and 345 is the absorptivity for pure lycopene in
n-hexane at 472 nm.
Standard solution C—
Transfer an accurately weighed quantity of
USP Tomato Extract Containing Lycopene RS, equivalent to 6 to 8 mg of lycopene, to a 100-mL volumetric flask, and dissolve in 10 mL of methylene chloride, using a sonicator. Dilute with
Diluting solution to volume to obtain a solution having a known concentration of about 0.07 mg of lycopene per mL.
Test solution—
Warm the sample of the Extract to 50
in a water bath. Mix well with a glass rod or a spatula. Accurately weigh a quantity of 1 to 1.2 g of the sample into a 100-mL volumetric flask. Add 10 mL of
Butylated hydroxytoluene stock solution and 30 mL of methylene chloride, and sonicate the solution for 1 minute in order to dissolve the sample completely. Cool to room temperature, dilute with methylene chloride to volume, and mix well. Transfer 5.0 mL to a 50-mL volumetric flask, and dilute with
Diluting solution to volume.
Chromatographic system (see Chromatography 621)—
The liquid chromatograph is equipped with a 472-nm detector and a 4.6-mm × 25-cm column that contains 5-µm packing L7 maintained at a temperature of 39 ± 1
. The flow rate is about 0.7 mL per minute. Chromatograph
Standard solution A, record the chromatograms, and measure the peak responses as directed for
Procedure: the retention time for lycopene is about 6 minutes; and the relative standard deviation of the lycopene peak area for replicate injections is not more than 1.5%.
Procedure—
Separately inject equal volumes (10 µL) of
Standard solution A or
Standard solution C, and the
Test solution into the chromatograph, record the chromatograms, and measure the responses of the major lycopene peaks. Calculate the percentage of lycopene in the portion of Extract taken by the formula:
100(C/W)(rU / rS),
in which
C is the concentration, in ppm, of
Standard solution A or
Standard solution C; W is the weight, in mg, of Extract taken to prepare the
Test solution; and
rU and
rS are the areas of the lycopene peak responses obtained from the
Test solution and
Standard solution A or
Standard solution C, respectively.
Content of other carotenoids and tocopherols (phytofluene, phytoene, beta carotene, and tocopherols)—
Butylated hydroxytoluene stock solution, Diluting solution, Standard solution A, Standard solution B, Standard solution C, and Test solution—
Proceed as directed in the test for Content of lycopene.
Mobile phase—
Prepare a filtered and degassed mixture of acetonitrile, methanol, methylene chloride, and n-hexane (475:475:25:25). Add 0.05% of diisopropylethylamine, mix well, and sonicate for 3 to 4 minutes.
Chromatographic system (see Chromatography 621)—
The liquid chromatograph is equipped with a 4.6-mm × 25-cm column that contains 5-µm packing L1 maintained at a temperature of 39 ± 1º and a detector set at 472 nm for lycopene, at 450 nm for beta carotene, at 350 nm for phytofluene, and at 288 nm for phytoene and tocopherol. The flow rate is about 0.6 mL per minute.
Chromatograph
Standard solution C, record the chromatograms, and measure the peak responses and the peak retention times as directed for
Procedure: the chromatogram of
Standard solution C is similar to the Reference Chromatogram provided with the
USP Tomato Extract Containing Lycopene RS; the relative retention times in the chromatogram of
Standard solution C are about 0.6 for the peaks of the tocopherol isomers, 1.0 for the peaks of the lycopene isomers, 1.5 to 1.7 for the peaks of the beta carotene isomers, 1.6 to 1.8 for the peaks of the phytofluene isomers, and 1.8 to 2.2 for the phytoene peak; and the relative standard deviation for replicate injections for the peak of the lycopene isomers is not more than 2%.
Procedure—
Separately inject equal volumes (10 µL) of
Standard solution A and the
Test solution into the chromatograph, and record the chromatograms. Identify the locus of the peaks for the lycopene isomers, beta carotene isomers, 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. In the
Test 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 response of the 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 Extract taken by the formula:
100(C/W)(rU1 / rS)(345/259.2),
in which C is the concentration, in ppm, of Standard solution A or Standard solution C; W is the weight, in mg, of Extract taken to prepare the Test solution; rU1 is the sum of the peak responses for the beta carotene isomers at 450 nm obtained from the Test solution; rS is the sum of the peak responses for the lycopene isomers at 472 nm obtained from Standard solution A; and 345 and 259.2 are the absorptivities for pure lycopene and for pure beta carotene, respectively.
Calculate the percentage of phytofluene in the portion of Extract taken by the formula:
100(C/W)(rU2 / rS)(345/135),
in which C is the concentration, in ppm, of Standard solution A or Standard solution C; W is the weight, in mg, of Extract taken to prepare the Test solution; rU2 is the sum of the peak responses for phytofluene isomers at 350 nm obtained from the Test solution; rS is the sum of the peak responses for the lycopene isomers at 472 nm obtained from Standard solution A or Standard solution C; and 345 and 135 are the absorptivities for pure lycopene and for pure phytofluene, respectively.
Calculate the percentage of phytoene in the portion of Extract taken by the formula:
100(C/W)(rU3 / rS)(345/125),
in which C is the concentration, in ppm, of Standard solution A or Standard solution C; W is the weight, in mg, of Extract taken to prepare the Test solution; rU3 is the area of the phytoene peak response at 288 nm obtained from the Test solution; rS is the sum of the peak responses for the lycopene isomers at 472 nm obtained from Standard solution A; and 345 and 125 are the absorptivities for pure lycopene and for pure phytoene, respectively.
Calculate the percentage of tocopherols in the portion of Extract taken to prepare the Test solution by the formula:
100(C/W)(rU4 / rS)(345/8.5),
in which C is the concentration, in ppm, of Standard solution A or Standard solution C; W is the weight, in mg, of Extract taken to prepare the Test solution; rU4 is the sum of the peak responses for all the tocopherol peaks at 288 nm obtained from the Test solution; rS is sum of the peak responses for the lycopene isomers at 472 nm obtained from Standard solution A; 345 is the absorptivity for pure lycopene; and 8.5 is the average absorptivity for tocopherols.
pore size), connected in a series and kept at 22