Identification—
B:
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.
C:
To about 20 mg add 5 mL of hydrochloric acid and 10 mL of water, and heat to boiling to effect hydrolysis. To the cooled solution add 2 mL of sodium nitrite solution (1 in 1000), shake, add 1 mL of ammonium sulfamate solution (1 in 200), then shake for 2 minutes, and add 1 mL of N-(1-naphthyl)ethylenediamine dihydrochloride solution (1 in 1000): a reddish violet color is produced.
Related compounds—
Transfer 50.0 mg to a 10-mL conical flask, add 2.5 mL of acetone, and shake. Allow any undissolved particles to settle, and apply 50 µL of the supernatant to a suitable thin-layer chromatographic plate (see
Chromatography 
621
) coated with a 0.25-mm layer of chromatographic silica gel. Apply to the same plate 10 µL of an acetone solution containing 100 µg per mL of
USP Chlordiazepoxide Related Compound A RS and 10 µL of an acetone solution containing 10 µg per mL of USP 2-Amino-5-chlorbenzophenone RS. Develop the chromatogram in a chromatographic chamber (not previously saturated with the developing solvent) in ethyl acetate 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 on the plate by lightly spraying with 2 N sulfuric acid, drying at 105
for 15 minutes, and then spraying in succession with sodium nitrite solution (1 in 1000), ammonium sulfamate solution (1 in 200), and N-(1-naphthyl)ethylenediamine dihydrochloride solution (1 in 1000). Any spots from the test solution are not greater in size or intensity than the spots at the respective
RF values produced by the Standard solutions, corresponding to not more than 0.1% of chlordiazepoxide related compound A, and to not more than 0.01% of 2-amino-5-chlorobenzophenone.
Assay—
[NOTE—Use low-actinic glassware in this procedure.
]
Mobile phase—
Prepare a filtered and degassed mixture of methanol and water (60:40). Make adjustments if necessary (see
System Suitability under
Chromatography 621).
Standard preparation—
Dissolve an accurately weighed quantity of
USP Chlordiazepoxide RS in
Mobile phase, and dilute quantitatively, and stepwise if necessary, with
Mobile phase to obtain a solution having a known concentration of about 200 µg per mL.
Assay preparation—
Transfer about 100 mg of Chlordiazepoxide, accurately weighed, to a 50-mL volumetric flask, dissolve in
Mobile phase, sonicate for 5 minutes, dilute with
Mobile phase to volume, and mix. Pass this solution through a membrane filter having a 0.5-µm or finer porosity. Transfer 10 mL of this solution to a 100-mL volumetric flask, dilute with
Mobile phase, and mix.
Chromatographic system
(see
Chromatography 621)—The liquid chromatograph is equipped with a 254-nm detector and a 3.9-mm × 30-cm column that contains packing L1. The flow rate is about 1 mL per minute. Chromatograph the
Standard preparation, and record the peak responses as directed for
Procedure: the column efficiency is not less than 3600 theoretical plates: the tailing factor is not more than 2.0; and the relative standard deviation for replicate injections is not more than 2.0%.
Procedure—
Separately inject equal volumes (about 5 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 C
16H
14ClN
3O in the portion of Chlordiazepoxide taken by the formula:
0.5C(rU / rS),
in which
C is the concentration, in µg per mL, of the USP Reference Standard in the
Standard preparation; and
rU and
rS are the peak responses obtained from the
Assay preparation and the
Standard preparation, respectively.
;)
