A: Infrared Absorption 197K—Do not dry specimens.

B: Acid solvent—The UV absorption spectrum of a solution in 0.1 N hydrochloric acid (1 in 3000) exhibits maxima and minima at the same wavelengths as that of a similar solution of USP Ketamine Hydrochloride RS, concomitantly measured, and the respective absorptivities, at the wavelengths of maximum absorbance at about 269 and 276 nm, do not differ by more than 3.0%.

Mobile phase— Dissolve 0.95 g of sodium 1-hexanesulfonate in 1 L of a solution consisting of a mixture of water and acetonitrile (3:1). Add 4 mL of acetic acid, and mix.

Standard solution— Dissolve accurately weighed quantities of USP Ketamine Hydrochloride RS and USP Ketamine Related Compound A RS in Mobile phase (sonicate if necessary) to prepare a solution containing about 0.005 mg per mL of each compound. Prepare immediately before use.

Test solution— Transfer an accurately weighed quantity of about 50.0 mg of Ketamine Hydrochloride to a 50-mL volumetric flask. Dissolve in and dilute with Mobile phase to volume, sonicating if necessary.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 215-nm detector and a 4.0-mm × 4.0-mm guard column with a 4.0-mm × 12.5-cm analytical column that contains 5-µm packing L1. The flow rate is about 1.0 mL per minute. Chromatograph the Standard solution, and record the peak responses as directed for Procedure: the order of elution is ketamine hydrochloride followed by ketamine related compound A; the resolution, R, between these two peaks is not less than 2.0; the retention time of ketamine hydrochloride is between 3.0 and 4.5 minutes (if necessary, adjust the concentration of water and acetonitrile); and the tailing factor is not greater than 1.5.

Procedure— Separately inject equal volumes (about 20 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, identify the ketamine hydrochloride and ketamine related compound A peaks, and measure the areas of the major peaks. Calculate the area percentage of each impurity, relative to ketamine hydrochloride, in the portion of Ketamine Hydrochloride taken by the formula: in which C is the concentration, in mg per mL, of USP Ketamine Hydrochloride RS in the Standard solution; W is the weight, in mg, of Ketamine Hydrochloride taken to prepare the Test solution; ri is the peak area of each individual impurity peak in the Test solution; and rS is the response of the ketamine hydrochloride peak obtained from the Standard solution. Not more than 0.1% of ketamine related compound A is found; the response of no other unknown impurity is greater than 0.3% of the ketamine peak area; and the sum of the responses of all unknown impurity peaks is not greater than 1.0% of the ketamine peak response.

Buffer— Dissolve 5.75 g of monobasic ammonium phosphate in 1000 mL of water. Add 6 mL of triethylamine, and adjust with phosphoric acid to a pH of 3.0.

Mobile phase— Prepare a filtered and degassed mixture of Buffer and methanol (65:35). Make adjustments if necessary (see System Suitability under Chromatography 621).

System suitability solution— Transfer about 12.5 mg each, of USP Ketamine Hydrochloride RS and USP Ketamine Related Compound A RS, both accurately weighed, to a 50-mL volumetric flask, dissolve in Mobile phase with the aid of sonification if necessary, dilute with Mobile phase to volume, and mix. Transfer 10.0 mL of the solution so obtained to a 100-mL volumetric flask, dilute with Mobile phase to volume, and mix.

Standard preparation— Transfer about 10 mg of USP Ketamine Hydrochloride RS, accurately weighed, to a 50-mL volumetric flask, add about 20 mL of Mobile phase, and sonicate to dissolve. Dilute with Mobile phase to volume, and mix.

Assay preparation— Transfer about 20 mg of Ketamine Hydrochloride, accurately weighed, to a 100-mL volumetric flask, add about 35 mL of Mobile phase, and sonicate to dissolve. Dilute with Mobile phase to volume, and mix.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 220-nm detector and a 4.6-mm × 25-cm column that contains 5-µm packing L1. The flow rate is about 1.0 mL per minute. Chromatograph the System suitability solution, and record the peak responses as directed for Procedure: the order of elution is ketamine followed by ketamine related compound A; the resolution, R, between ketamine and ketamine related compound A is not less than 2.0; the column efficiency determined from the ketamine peak is not less than 9400 theoretical plates; and the tailing factor determined from the ketamine peak is not more than 1.6. Chromatograph the Standard preparation, and record the ketamine peak response as directed for Procedure: the relative standard deviation for replicate injections is not more than 0.6%.

Procedure— Separately inject equal volumes (about 20 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 C13H16ClNO· HCl in the portion of Ketamine Hydrochloride taken by the formula: in which C is the concentration, in mg per mL, of USP Ketamine Hydrochloride RS in the Standard preparation; and rU and rS are the ketamine peak responses obtained from the Assay preparation and the Standard preparation, respectively.