A: Infrared Absorption 197S—

B: Ultraviolet Absorption 197U—

C: The retention time of the major peak in the chromatogram of the Assay preparation corresponds to that in the chromatogram of the Standard preparation, as obtained in the Assay.

Mobile phase— Prepare as directed in the Assay.

Standard solution— 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 0.001 mg per mL.

Test solution— Transfer about 100 mg of Pentobarbital, accurately weighed, to a 100-mL volumetric flask, add about 80 mL of Mobile phase, and sonicate until dissolved. Dilute with Mobile phase to volume, and mix.

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 solution, 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 15.0%.

Procedure— Separately inject equal volumes (about 10 µL) of the Standard solution and Test solution into the chromatograph, record the chromatograms, and measure the areas for the major peaks. Calculate the percentage of any impurity in the portion of Pentobarbital taken by the formula: in which C is the concentration, in mg per mL, of USP Pentobarbital RS in the Standard solution; F is the relative response factor of the impurity according the table below; W is the weight, in mg, of pentobarbital, on the dried basis, used to prepare the Test solution; ri is the peak area for any impurity in the Test solution; and rS is the peak area for pentobarbital in the Standard solution: the impurities meet the requirements given in the table below:

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— Transfer about 100 mg of Pentobarbital, accurately weighed, to a 100-mL volumetric flask, add about 80 mL of Mobile phase, and sonicate until dissolved. Dilute with Mobile phase to volume, and mix. Transfer 10.0 mL of this solution to a 100-mL volumetric flask. Dilute with Mobile phase to volume, and mix.

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 quantity, in mg, of C11H18N2O3 in the portion of Pentobarbital taken by the formula: in which C is the concentration, in mg per mL, of USP Pentobarbital RS in the Standard preparation; and rU and rS are the peak areas obtained from the Assay preparation and the Standard preparation, respectively.