A: Infrared Absorption 197K.

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: It meets the requirements of the tests for Chloride 191.

Diluted phosphoric acid, Buffer, Mobile phase, and Chromatographic system— Proceed as directed in the Assay.

Standard solution— Use the System suitability preparation, prepared as directed in the Assay.

Test solution— Use the Assay stock preparation.

Procedure— Inject a volume (about 20 µL) of the Test solution into the chromatograph, record the chromatogram, and measure the peak responses. Calculate the percentages of individual amitriptyline related compounds in the portion of Amitriptyline Hydrochloride taken by the formula: in which rU is the individual peak response for each amitriptyline related compound obtained from the Test solution; rS is the response of the corresponding peak in the Standard solution; CS is the concentration, in mg per mL, of each of the amitriptyline related compounds in the Standard solution; and CT is the concentration, in mg per mL, of amitriptyline hydrochloride in the Test solution. The related compound requirements are listed in Table 1. [note—Discard any peak with a relative retention time of less than 0.22. Use the response of the amitriptyline hydrochloride peak obtained from the Standard solution and the concentration of amitriptyline hydrochloride in the Standard solution to calculate the percentage of unknown individual impurities.]

Diluted phosphoric acid— Prepare a mixture of phosphoric acid and water (1:10), and mix well.

Buffer— Dissolve 1.42 g of dibasic sodium phosphate (Na2HPO4) in 1 L of water, and adjust with Diluted phosphoric acid to a pH of 7.7.

Mobile phase— Prepare a mixture of filtered and degassed methanol and Buffer (7:3).

Standard preparation— Dissolve an accurately weighed amount of USP Amitriptyline Hydrochloride RS in Mobile phase to obtain a solution having a known concentration of 0.2 mg per mL of amitriptyline hydrochloride.

Assay stock preparation— Dissolve an accurately weighed amount of Amitriptyline Hydrochloride in Mobile phase to obtain a solution having a known concentration of about 1 mg per mL of amitriptyline hydrochloride.

Assay preparation— Dilute the Assay stock preparation with Mobile phase (1:5).

Stock solution A— Dissolve an accurately weighed amount of USP Amitriptyline Related Compound A RS in methanol to obtain a solution having a known concentration of about 1 mg per mL of amitriptyline related compound A.

Stock solution B— Dissolve accurately weighed amounts of USP Amitriptyline Hydrochloride RS, USP Amitriptyline Related Compound B RS, USP Cyclobenzaprine Hydrochloride RS, and USP Nortriptyline Hydrochloride RS in Mobile phase to obtain a solution having a known concentration of about 0.4 mg per mL of amitriptyline hydrochloride and about 0.6 mg per mL each of amitriptyline related compound B, cyclobenzaprine hydrochloride, and nortriptyline hydrochloride.

System suitability preparation— Dilute suitable volumes of Stock solution A and Stock solution B with Mobile phase to obtain a solution having 1 µg per mL of amitriptyline hydrochloride, 0.5 µg per mL of amitriptyline related compound A, and 1.5 µg per mL each of amitriptyline related compound B, cyclobenzaprine hydrochloride, and nortriptyline hydrochloride.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 215-nm detector and a 4.6-mm × 25-cm column that contains 5-µm packing L7. The flow rate is about 1.5 mL per minute. The column temperature is maintained at 45. Chromatograph the System suitability preparation, and record the peak areas as directed for Procedure: the approximate relative retention times for related compounds are given in Table 1; and the resolution, R, between amitriptyline related compound B and nortriptyline is not less than 1.5. Chromatograph the Standard preparation, and record the peak areas as directed for Procedure: the relative standard deviation for amitriptyline replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 20 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms for about 40 minutes, and measure the responses for the major peaks. Calculate the percentage of C20H23N·HCl in the portion of Amitriptyline Hydrochloride taken by the formula: in which CS and CU are the concentrations, in mg per mL, of the Standard preparation and the Assay preparation; and rU and rS are the peak responses obtained from the Assay preparation and the Standard preparation, respectively.