361 BARBITURATE ASSAY
Internal Standard, Internal Standard Solution, Standard Preparation, and Assay Preparation Prepare as directed in the individual monograph.
Chromatographic System Under typical conditions, the gas chromatograph is equipped with a flame-ionization detector and contains a 4-mm × 0.9-m glass column packed with 3% liquid phase G10 on support 80- to 100-mesh S1A. The column is maintained at a temperature of 200 ± 10, and the injection port and detector are maintained at about 225, the column temperature being varied within the designated tolerance, as necessary, to meet System Suitability specifications and provide suitable retention times. Use a suitable carrier gas, such as dry nitrogen, at an appropriate flow rate, such as 60 to 80 mL per minute. Use on-column injection. [noteIf the instrument is not equipped for on-column injection, use an injection port lined with glass that has been washed successively with chromic acid cleansing solution, water, methanol, chloroform, a 1 in 10 solution of trimethylchlorosilane in chloroform, and chloroform.]
System Suitability (see Chromatography 621) Chromatograph five replicate injections of the Standard Preparation, and record peak responses as directed for Procedure: the relative standard deviation for the ratio RS is not more than 1.5%. In a suitable chromatogram, the resolution, R, between the barbituric acid and the Internal Standard is not less than the value given in the individual monograph, and the tailing factor, T, for each of the two peaks is not more than 2.0.
Procedure Inject a suitable portion (about 5 µL) of the Standard Preparation into a suitable gas chromatograph, and record the chromatogram. Similarly inject a suitable portion of the Assay Preparation, and record the chromatogram. Calculate the content of the barbiturate or barbituric acid in the assay specimen by the formula given in the individual monograph, in which RU is the ratio of the peak response of the barbituric acid to that of the Internal Standard obtained for the Assay Preparation; QS is the ratio of the weight of the barbituric acid to that of the Internal Standard in the Standard Preparation; Ci is the concentration, in mg per mL, of Internal Standard in the Internal Standard Solution; and RS is the ratio of the peak response of the barbituric acid to that of the Internal Standard in the Standard Preparation.