Identification—
Constitute a container of Tiletamine and Zolazepam for Injection with a volume of water sufficient to yield a test solution containing the equivalent of about 10 mg of tiletamine and 10 mg of zolazepam per mL. Prepare two Standard solutions containing in each mL 10 mg of
USP Tiletamine Hydrochloride RS and 10 mg of
USP Zolazepam Hydrochloride RS, respectively. Separately apply 2 µL of the test solution and the Standard solutions to a thin-layer chromatographic plate (see
Chromatography 
621
) coated with a 0.25-mm layer of chromatographic silica gel mixture, and allow the spots to dry. Place the plate in a saturated chamber containing ethyl acetate as the solvent system and lined with filter paper. Develop the chromatogram until the solvent front has moved about three-fourths of the length of the plate. Remove the plate from the chamber, mark the solvent front, allow the plate to air-dry, and examine under short-wavelength UV light: the
RF values of the principal spots obtained from the test solution correspond to those obtained from the Standard solutions.
Assay—
Internal standard solution—
Prepare a solution of tetraphenylethylene in chloroform containing 10 mg per mL.
Assay preparation—
Constitute a container of Tiletamine and Zolazepam for Injection with the volume of water specified in the labeling. Transfer an accurately measured volume of the resultant solution, equivalent to about 100 mg of tiletamine and 100 mg of zolazepam, to a 250-mL flask. Add 30.0 mL of
Alkaline borate buffer,
pH 10.0 (see
Buffer Solutions in the section
Reagents, Indicators, and Solutions), and swirl. Add 5.0 mL of
Internal standard solution and 95.0 mL of chloroform, and shake by mechanical means for 30 minutes. Allow the phases to separate, and use the chloroform layer as the
Assay preparation.
Chromatographic system (see Chromatography 621)—
The gas chromatograph is equipped with a flame-ionization detector and a 2-mm × 1.24-m column that contains 3% phase G2 on 100- to 120-mesh support S1AB. Helium is used as the carrier gas flowing at a rate of about 40 mL per minute. The column temperature is maintained at about 150
for 0.5 minute after injection and is programmed to rise to 230
at a rate of 10
per minute. The injector port is maintained at 160
and the detector at 250
. Chromatograph the
Standard preparation, and record the peak responses as directed for
Procedure: the relative retention times are about 0.4 for tiletamine, 0.8 for zolazepam, and 1.0 for tetraphenylethylene.
Procedure—
Separately inject equal volumes (about 2 µL) of the
Standard preparation and the
Assay preparation into the chromatograph, record the chromatograms, and measure the area responses for the major peaks. Calculate the quantity, in mg, of tiletamine (C
12H
17NOS) in each mL of the constituted solution taken by the formula:
(223.33/259.79)(W/V)(RU / RS),
in which 223.33 and 259.79 are the molecular weights of tiletamine base and tiletamine hydrochloride, respectively
; W is the weight, in mg, of
USP Tiletamine Hydrochloride RS taken to prepare the
Standard preparation; V is the volume, in mL, of the constituted solution taken to prepare the
Assay preparation; and
RU and
RS are the peak area response ratios of the tiletamine peak to the tetraphenylethylene peak obtained from the
Assay preparation and the
Standard preparation, respectively. Calculate the quantity, in mg, of zolazepam (C
15H
15FN
4O) in each mL of the constituted solution taken by the formula:
(286.31/322.77)(W/V)(RU / RS),
in which 286.31 and 322.77 are the molecular weights of zolazepam base and zolazepam hydrochloride, respectively
; W is the weight, in mg, of
USP Zolazepam Hydrochloride RS taken to prepare the
Standard preparation; V is the volume, in mL, of the constituted solution taken to prepare the
Assay preparation; and
RU and
RS are the peak area response ratios of the zolazepam peak to the tetraphenylethylene peak obtained from the
Assay preparation and the
Standard preparation, respectively.
