Mobile phase, System suitability preparation, Standard preparation, and Chromatographic system
Proceed as directed in the Assay.
Use the Assay preparation.
Inject a volume (about 10 µL) of the Test preparation
into the chromatograph, record the chromatogram, and measure all the peak responses. Calculate the percentage of each impurity in the portion of Caffeine taken by the formula:
100(ri / rs),
in which ri
is the peak response for each impurity, and rs
is the sum of the responses of all the peaks: not more than 0.1% of any individual impurity is found; and not more than 0.1% of total impurities is found.
Transfer about 1.64 g of anhydrous sodium acetate, accurately weighed, to a 2-L volumetric flask, dissolve in and dilute with water to volume, and mix. Transfer 1910 mL of this solution to another 2-L volumetric flask, add 50 mL of acetonitrile and 40 mL of tetrahydrofuran, and mix. Adjust with glacial acetic acid to a pH of 4.5, mix, filter, and degas.
System suitability preparation
Transfer about 2 mg of theophylline, accurately weighed, to a 100-mL volumetric flask, add about 50 mL of Mobile phase, shake, and sonicate, if necessary, to dissolve. Dilute with Mobile phase to volume, and mix.
Transfer an acccurately weighed quantity of about 5.0 mg of USP Caffeine RS
to a 25-mL volumetric flask, add 5.0 mL of System suitability preparation
and 10 mL of Mobile phase,
shake, and sonicate, if necessary, to dissolve. Dilute with Mobile phase
to volume, mix, and filter.
Transfer about 10 mg of Caffeine, accurately weighed, to a 50-mL volumetric flask, add 10 mL of Mobile phase, shake, and sonicate, if necessary, to dissolve. Dilute with Mobile phase to volume, mix, and filter.
Chromatographic system (see Chromatography 621)
The liquid chromatograph is equipped with a 275-nm detector and a 4.6-mm × 15-cm column containing packing L1. The flow rate is about 1 mL per minute. Chromatograph the Standard preparation,
and record the peak responses as directed for Procedure:
the relative retention times for theophylline and caffeine are about 0.69 and 1.0, respectively; the resolution, R,
between theophylline and caffeine is not less than 6.0; the tailing factor for each of the peaks identified in the chromatogram is not more than 2.0; and the relative standard deviation is not more than 2.0%.
Separately inject equal volumes (about 10 µL) of the Standard preparation
and the Assay preparation
into the chromatograph, record the chromatograms, and measure the responses for the caffeine peaks. Calculate the quantity, in mg, of C8
in the portion of Caffeine taken by the formula:
50C(rU / rS),
in which C
is the concentration, in mg per mL, of USP Caffeine RS
in the Standard preparation;
are the peak responses for caffeine obtained from the Assay preparation
and the Standard preparation,