Mobile phase— Prepare a filtered and degassed pH 3.5 mixture of 0.01 M monobasic potassium phosphate and acetonitrile (65:35). Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard preparation— Dissolve an accurately weighed quantity of USP Pentobarbital RS in Mobile phase, and dilute quantitatively, and stepwise if necessary, with Mobile phase to obtain a solution having a known concentration of about 0.1 mg per mL.

Assay preparation— Quantitatively dilute a suitable volume of Injection with Mobile phase to obtain a solution having a known concentration of about 0.1 mg per mL.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 214-nm detector and a 4.6-mm × 25-cm column that contains 5-µm packing L1. The flow rate is about 1.0 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the capacity factor, k¢, is not less than 2.5; the column efficiency is not less than 15,000 theoretical plates; the tailing factor 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 10 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 percentage of C11H17N2NaO3 in the portion of Injection taken by the formula: in which 248.25 and 226.27 are the molecular weights of pentobarbital sodium and pentobarbital, respectively; CS is the concentration, in mg per mL, of USP Pentobarbital RS in the Standard preparation; CU is the final concentration, in mg per mL, of the Assay preparation; and rU and rS are the peak areas obtained from the Assay preparation and the Standard preparation, respectively.