Mobile phase— Prepare a filtered and degassed mixture of hexanes and tetrahydrofuran (97.75:2.25). Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard preparation— Dissolve an accurately weighed quantity of USP Pyrethrum Extract RS in hexanes, and dilute stepwise with hexanes to obtain a solution having a known concentration of about 0.5 mg of pyrethrins per mL.

Assay preparation— Transfer an accurately weighed quantity of Extract, equivalent to about 50 mg of pyrethrins, to a 100-mL volumetric flask, dilute with hexanes to volume, and mix.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 240-nm detector and a 4.6-mm × 25-cm column that contains 5-µm packing L10. The flow rate is about 1.5 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the resolution, R, between the individual pyrethrin peaks is not less than 2.0; and the relative standard deviation for replicate injections is not more than 1.0%.

Procedure— 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 six major peaks. The relative retention times are about 0.78 to 0.80 for jasmolin I; 0.86 to 0.87 for cinerin I; 1.0 for pyrethrin I; 1.95 to 2.15 for jasmolin II; 2.15 to 2.4 for cinerin II; and 2.55 to 2.88 for pyrethrin II. Calculate the quantity, in mg, of pyrethrins in the portion of Extract taken by the formula: in which CSi is the concentration, in mg per mL, of the individual pyrethrin of interest in the Standard preparation; and rUi and rSi are the peak responses obtained from the Assay preparation and the Standard preparation, respectively. Calculate the ratio of Pyrethrins I to Pyrethrins II in the portion of the Extract taken by the formula: in which A and B are the sums of the quantities of Pyrethrins I and Pyrethrins II, respectively.