Identification—
Dilute a volume of Elixir with alcohol to a concentration of about 1mg of pentobarbital per mL.Apply 50µLof this solution and 50µLof a Standard solution of
USP Pentobarbital RSin alcohol containing 1mg per mLas streaks about 1cm in length along the spotting line to a suitable thin layer chromatographic plate (see
Chromatography á621ñ)coated with a 0.25-mm layer of chromatographic silica gel mixture.Allow the streaks to dry,and develop the chromatogram in a solvent system consisting of a mixture of isopropyl alcohol,ammonium hydroxide,chloroform,and acetone (9:4:2:2)until the solvent front has moved about three-fourths of the length of the plate.Remove the plate from the developing chamber,mark the solvent front,and allow the solvent to evaporate.Locate the spots by viewing the plate under short-wavelength UVlight:the
RFvalue of the principal spot obtained from the test solution corresponds to that obtained from the Standard solution.
Assay—
Internal standard—
n-Tricosane.
Internal standard solution—
Dissolve an accurately weighed quantity of n-tricosane in chloroform,and quantitatively dilute with chloroform to obtain a solution having a known concentration of about 0.6mg per mL.
Standard preparation—
Dissolve accurately weighed quantities of
USP Pentobarbital RSand
n-tricosane in chloroform,and quantitatively dilute with chloroform to obtain a solution that contains,in each mL,known amounts of about 1mg of
USP Pentobarbital RSand about 0.4mg of
n-tricosane.
Assay preparation—
Transfer an accurately measured volume of Elixir,equivalent to about 20mg of pentobarbital,to a separator,add 1mLof dilute hydrochloric acid (1in 5),and extract with four 10-mLportions of chloroform.Filter the extracts through about 15g of anhydrous sodium sulfate that is supported on a funnel by a small pledget of glass wool.Collect the combined filtrate in a 50-mLvolumetric flask,wash the sodium sulfate with 5mLof chloroform,dilute with chloroform to volume,and mix.Combine 4.0mLof this solution with 1.0mLof Internal standard solutionin a suitable container,and reduce the volume to about 1.5mLby evaporation,with the aid of a stream of dry nitrogen,at room temperature.
Chromatographic system andSystem suitability—
Proceed as directed for
Chromatographic Systemand
System Suitabilityunder
Barbiturate Assay á361ñ,the resolution,
R,between pentobarbital and
n-tricosane being not less than 2.3.
[NOTE—Relative retention times are,approximately,0.5for
n-tricosane and 1.0for pentobarbital.
]
Procedure—
Proceed as directed for
Procedureunder
Barbiturate Assay á361ñ.Calculate the quantity,in mg,of C
11H
18N
2O
3in each mLof the Elixir taken by the formula:
12.5(RU)(QS)(Ci)/V(RS),
in which
Vis the volume,in mL,of Elixir taken;and the other terms are as defined therein.