A: Infrared Absorption 197K.

B: The retention time of the major peak in the chromatogram of the Test preparation corresponds to that in the chromatogram of the Standard preparation, as obtained in the Assay.

Mobile phase and Diluent— Prepare as directed in the Assay.

Standard solution— Dilute a suitable volume of the Standard preparation, prepared as directed in the Assay, quantitatively, and stepwise if necessary, to obtain a solution having a known concentration of about 0.032 mg per mL of lorazepam.

Peak identification solution— Dissolve accurately weighed amounts of USP Lorazepam RS, USP Lorazepam Related Compound A RS, USP Lorazepam Related Compound B RS, USP Lorazepam Related Compound C RS, USP Lorazepam Related Compound D RS, and USP Lorazepam Related Compound E RS in Diluent to obtain a solution having a final concentration of about 3.2 mg per mL of lorazepam and 0.032 mg per mL each of lorazepam related compound A, lorazepam related compound B, lorazepam related compound C, lorazepam related compound D, and lorazepam related compound E.

Test solution— Dissolve an accurately weighed quantity of Lorazepam in Diluent, and dilute quantitatively, and stepwise if necessary, to obtain a solution having a known concentration of about 3.2 mg per mL of lorazepam.

Chromatographic system (see Chromatography 621)— Proceed as directed in the Assay. Chromatograph the Peak identification solution, record the peak responses as directed for Procedure, and identify the peaks, using the retention times given in Table 1. The resolution, R, between lorazepam related compound A and lorazepam related compound E is not less than 1.2. Chromatograph the Standard solution, and record the peak responses as directed for Procedure: the tailing factor for lorazepam is not more than 2.0 and the relative standard deviation for replicate injections is not more than 5% for lorazepam.

Procedure— Separately inject equal volumes (about 100 µL) of the Standard solution and the Test solution into the chromatograph, collect the data for at least 50 minutes, and measure the responses for all the peaks. Calculate the percentage of each impurity in the portion of Lorazepam taken by the formula: in which F is the relative response factor for any given impurity found in Table 1; CS and CU are the concentrations of lorazepam in the Standard solution and the Test solution, respectively; ri is the peak response for each impurity in the Test solution; and rS is the peak response for lorazepam obtained from the Standard solution. The limit for each related compound is given in Table 1.

Mobile phase— Prepare a mixture of water, acetonitrile, and glacial acetic acid (50:50:1.2). Make adjustments if necessary (see System Suitability under Chromatography 621).

Diluent— Prepare a mixture of methanol and water (75:25).

Standard preparation— Dissolve an accurately weighed quantity of USP Lorazepam RS in Diluent, and dilute quantitatively, and stepwise if necessary, to obtain a solution having a known concentration of about 0.1 mg of lorazepam per mL.

Assay preparation— Dissolve an accurately weighed quantity of Lorazepam in Diluent, and dilute quantitatively, and stepwise if necessary, to obtain a solution having a known concentration of about 0.1 mg per mL of lorazepam.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a sample compartment chiller maintained at 4, a UV detector set at 230 nm, and a 4.6-mm × 25-cm column that contains 5-µm packing L1. The column temperature is maintained at 5. The flow rate is about 1.0 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the tailing factor for lorazepam is not more than 2.0, and the relative standard deviation for replicate injections is not more than 2.0% for lorazepam.

Procedure— Separately inject equal volumes (about 20 µL) of the Standard preparation and the Assay preparation into the chromatograph, collect the data for at least 50 minutes, and measure the responses for all the peaks. Calculate the percentage of Lorazepam in the portion of sample taken by the formula: in which CS and CU are the concentrations of lorazepam in the Standard preparation and the Assay preparation, respectively; and rU and rS are the peak responses for lorazepam obtained from the Assay preparation and the Standard preparation, respectively.