Tiletamine and Zolazepam for Injection
»Tiletamine and Zolazepam for Injection is a sterile dry mixture of Tiletamine Hydrochloride and Zolazepam Hydrochloride.It contains the equivalent of not less than 90.0percent and not more than 110.0percent of the labeled amounts of tiletamine (C12H17NOS)and zolazepam (C15H15FN4O).
Packaging and storage— Preserve in Containers for Sterile Solidsas described under Injections á1ñ.
Labeling— Label it to indicate that it is for veterinary use only.
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 10mg of tiletamine and 10mg of zolazepam per mL.Prepare two Standard solutions containing in each mL10mg of USP Tiletamine Hydrochloride RSand 10mg of USP Zolazepam Hydrochloride RS,respectively.Separately apply 2µLof the test solution and the Standard solutions to a thin-layer chromatographic plate (see Chromatography á621ñ)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 UVlight:the RFvalues of the principal spots obtained from the test solution correspond to those obtained from the Standard solutions.
Bacterial endotoxins á85ñ— It contains not more than 0.07USP Endotoxin Unit per mg of combined tiletamine and zolazepam equivalents.
Sterility á71ñ— It meets the requirements when tested as directed for Membrane Filtrationunder Test for Sterility of the Product to be Examined.
pHá791ñ: between 2.0and 3.5,when constituted as directed in the labeling.
Water,Method Iá921ñ: not more than 20mg in a container containing the equivalent of 250mg of tiletamine and 250mg of zolazepam.
Other requirements— It meets the requirements under Injections á1ñand for Uniformity of Dosage Units á905ñ.
Assay—
Internal standard solution— Prepare a solution of tetraphenylethylene in chloroform containing 10mg per mL.
Standard preparation— Transfer accurately weighed quantities of about 116mg of USP Tiletamine Hydrochloride RSand 113mg of USP Zolazepam Hydrochloride RSto a 250-mLflask,add 2mLof water,and swirl to dissolve.Add 30mLof Alkaline borate buffer,pH10.0(see Buffer Solutionsin the section Reagents,Indicators,and Solutions),and swirl.Add 5.0mLof Internal standard solutionand 95.0mLof chloroform,and shake by mechanical means for 30minutes.Allow the phases to separate,and use the chloroform layer as the Standard preparation.
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 100mg of tiletamine and 100mg of zolazepam,to a 250-mLflask.Add 30.0mLof Alkaline borate buffer,pH10.0(see Buffer Solutionsin the section Reagents,Indicators,and Solutions),and swirl.Add 5.0mLof Internal standard solutionand 95.0mLof chloroform,and shake by mechanical means for 30minutes.Allow the phases to separate,and use the chloroform layer as the Assay preparation.
Chromatographic system(see Chromatography á621ñ)— The gas chromatograph is equipped with a flame-ionization detector and a 2-mm ×1.24-m column that contains 3%phase G2on 100-to 120-mesh support S1AB.Helium is used as the carrier gas flowing at a rate of about 40mLper minute.The column temperature is maintained at about 150for 0.5minute after injection and is programmed to rise to 230at a rate of 10per minute.The injector port is maintained at 160and the detector at 250.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the relative retention times are about 0.4for tiletamine,0.8for zolazepam,and 1.0for tetraphenylethylene.
Procedure— Separately inject equal volumes (about 2µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the area responses for the major peaks.Calculate the quantity,in mg,of tiletamine (C12H17NOS)in each mLof the constituted solution taken by the formula:
(223.33/259.79)(W/V)(RU/RS),
in which 223.33and 259.79are the molecular weights of tiletamine base and tiletamine hydrochloride,respectively;Wis the weight,in mg,of USP Tiletamine Hydrochloride RStaken to prepare the Standard preparation;Vis the volume,in mL,of the constituted solution taken to prepare the Assay preparation;and RUand RSare the peak area response ratios of the tiletamine peak to the tetraphenylethylene peak obtained from the Assay preparationand the Standard preparation,respectively.Calculate the quantity,in mg,of zolazepam (C15H15FN4O)in each mLof the constituted solution taken by the formula:
(286.31/322.77)(W/V)(RU/RS),
in which 286.31and 322.77are the molecular weights of zolazepam base and zolazepam hydrochloride,respectively;Wis the weight,in mg,of USP Zolazepam Hydrochloride RStaken to prepare the Standard preparation;Vis the volume,in mL,of the constituted solution taken to prepare the Assay preparation;and RUand RSare the peak area response ratios of the zolazepam peak to the tetraphenylethylene peak obtained from the Assay preparationand the Standard preparation,respectively.
Auxiliary Information— Staff Liaison:Ian DeVeau,Ph.D.,Senior Scientist
Expert Committee:(VET)Veterinary Drugs
USP28–NF23Page 1930
Phone Number:1-301-816-8178