A: Infrared Absorption 197K.

B: [note—Conduct this test promptly, with a minimum exposure to light.] Dissolve 25.0 mg of it, previously dried, in 0.25 mL of chloroform; mix with about 30 mL of methanol previously warmed to 50; transfer the mixture with the aid of warm methanol to a 250-mL volumetric flask; cool the solution to room temperature; dilute with methanol to volume; and mix. Pipet 10 mL of this solution into a 50-mL volumetric flask, add 36 mL of chloroform, dilute with methanol to volume, and mix: the UV absorption spectrum of a 1 in 50,000 solution so obtained exhibits the same maxima in the range of 255 nm to 350 nm as that of a similar solution of USP Reserpine RS, concomitantly measured; and the respective absorptivities, determined with reference to a mixture of 36 volumes of chloroform and 14 volumes of methanol as the blank, at the wavelength of maximum absorbance at about 268 nm, do not differ by more than 3.0%.

Mobile phase— Prepare a filtered and degassed 1:1 mixture of acetonitrile and ammonium chloride solution (1 in 100). Make adjustments if necessary (see System Suitability under Chromatography 621). The pH is about 5.6.

Standard preparation— Dissolve an accurately weighed quantity of USP Reserpine RS in Mobile phase, and dilute quantitatively, and stepwise if necessary, with Mobile phase to obtain a solution having a known concentration of about 10 µg per mL.

Assay preparation— Transfer about 10 mg of Reserpine, accurately weighed, to a 100-mL volumetric flask. Dilute with Mobile phase to volume, and mix. Dilute 1.0 mL of this solution with 9.0 mL of Mobile phase, and mix.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 268-nm detector and a 4.6-mm × 25-cm column that contains packing L1. The flow rate is about 1.5 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the column efficiency determined from the analyte peak is not less than 1500 theoretical plates; the tailing factor for the analyte peak is not more than 1.5; and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 20 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in mg, of C33H40N2O9 in the portion of Reserpine taken by the formula: in which C is the concentration, in µg per mL, of USP Reserpine RS in the Standard preparation; and rU and rS are the peak responses obtained from the Assay preparation and the Standard preparation, respectively.