Internal standard— Butabarbital.

Internal standard solution— Dissolve an accurately weighed quantity of Butabarbital in chloroform, and quantitatively dilute with chloroform to obtain a solution having a known concentration of about 0.7 mg per mL.

Standard preparation— Dissolve accurately weighed quantities of USP Secobarbital RS and Butabarbital in chloroform, and quantitatively dilute with chloroform to obtain a solution that contains, in each mL, known amounts of about 1.2 mg of USP Secobarbital RS and about 0.9 mg of Butabarbital.

Assay preparation— Transfer an accurately measured volume of Oral Solution, equivalent to about 22 mg of secobarbital, to a separator, add 1 mL of dilute hydrochloric acid (1 in 5), and extract with four 10-mL portions of chloroform. Filter the extracts through about 15 g of anhydrous sodium sulfate that is supported on a funnel by a small pledget of glass wool. Collect the combined filtrate in a 50-mL volumetric flask, wash the sodium sulfate with 5 mL of chloroform, dilute with chloroform to volume, and mix. Combine 4.0 mL of this solution with 2.0 mL of Internal standard solution in a suitable container, and reduce the volume to about 1.5 mL by evaporation, with the aid of a stream of dry nitrogen, at room temperature.

Chromatographic system and System suitability— Proceed as directed for Chromatographic System and System Suitability under Barbiturate Assay 361, the resolution, R, between secobarbital and butabarbital being not less than 3.0. [note—Relative retention times are, approximately, 0.6 for butabarbital and 1.0 for secobarbital.]

Procedure— Proceed as directed for Procedure under Barbiturate Assay 361. Calculate the quantity, in mg, of secobarbital (C12H18N2O3) in each mL of the Oral Solution taken by the the formula: in which V is the volume, in mL, of Oral Solution taken; and the other terms are as defined therein.