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.

Mobile phase and Standard stock preparation— Prepare as directed in the Assay.

Impurity stock solution— Dissolve accurately weighed quantities of USP Imipramine Hydrochloride RS and USP Iminodibenzyl RS in a suitable volume of Mobile phase to obtain a solution having a known concentration of about 50 µg per mL of iminodibenzyl and 56.5 µg per mL of imipramine hydrochloride.

Trimipramine related compound A solution— Dissolve a suitable quantity of USP Trimipramine Related Compound A RS in Mobile phase to obtain a solution having a concentration of about 50 µg per mL.

Trimipramine stock solution— Quantitatively dilute the Standard stock preparation with Mobile phase to obtain a solution having a known concentration of about 70 µg per mL of trimipramine maleate.

System suitability solution— Transfer about 7 mg of USP Trimipramine Maleate RS to a 10-mL volumetric flask, dissolve in a small amount of Mobile phase, add 0.1 mL each of the Impurity stock solution and the Trimipramine related compound A solution, and dilute with Mobile phase to volume.

Standard solution— Transfer 5.0 mL each of the Impurity stock solution and the Trimipramine stock solution with Mobile phase to a 50-mL volumetric flask, and dilute with Mobile phase to volume. Dilute the resulting solution quantitatively, and stepwise if necessary, with Mobile phase to obtain a final solution having a known concentration of about 0.5 µg per mL each of iminodibenzyl, imipramine (free base), and trimipramine (free base). [note—This solution is stable for one day at room temperature. The concentration of imipramine (free base), in µg per mL, can be calculated using the molecular weights of imipramine (282.41) and imipramine hydrochloride (318.88). The concentration of trimipramine (free base), in µg per mL, can be calculated using the molecular weights of trimipramine (294.43) and trimipramine maleate (410.51).]

Test solution— Use the Assay stock preparation.

Chromatographic system— Prepare as directed in the Assay. Chromatograph about 10 µL of the System suitability solution, and record the peak responses as directed for Procedure: the resolution, R, between imipramine and trimipramine related compound A is not less than 1.5. [note—For identification purposes, the approximate relative retention times of the specified impurities are given in Table 1.]

Procedure— Separately inject about 10 µL of the Standard solution and the Test solution into the chromatograph, and record the chromatogram for three times the retention time for trimipramine. Identify the components based on their relative retention times in Table 1. Measure the peak areas of all the peaks in the Test solution. Calculate the percentage of imipramine and iminodibenzyl in the portion of Trimipramine Maleate taken by the formula: in which CS is the concentration, in mg per mL, of any given impurity (free base) in the Standard solution; CT is the concentration of Trimipramine Maleate, in mg per mL, in the Test solution; rU is the individual peak response of the given impurity obtained from the Test solution; and rS is the corresponding response for the same impurity obtained from the Standard solution. Calculate the percentage of trimipramine related compound A in the portion of Trimipramine Maleate taken by the formula: in which 3.6 is the relative response factor for trimipramine related compound A; CS is the concentration, in mg per mL, of trimipramine (free base) in the Standard solution; CT is the concentration of Trimipramine Maleate, in mg per mL, in the Test solution; rU is the peak response for trimipramine related compound A obtained from the Test solution; and rS is the peak response of trimipramine obtained from the Standard solution. Calculate the percentage of each unknown impurity in the portion of Trimipramine Maleate taken by the formula: in which CS is the concentration, in mg per mL, of trimipramine (free base) in the Standard solution; CT is the concentration of Trimipramine Maleate, in mg per mL, in the Test solution; ri is the individual peak response of the given impurity obtained from the Test solution; and rS is the response for trimipramine obtained from the Standard solution: the limits of the related compounds are given in Table 1. [note—Disregard any peak due to the maleate counterion eluting at a relative retention time of about 0.13.]

Buffer solution— Dissolve about 1.4 g of anhydrous dibasic sodium phosphate in 1 L of water. Adjust with phosphoric acid to a pH of 7.7.

Mobile phase— Prepare a filtered and degassed mixture of acetonitrile, methanol, and Buffer solution (18:12:10). Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard stock preparation— Dissolve an accurately weighed quantity of USP Trimipramine Maleate RS in a suitable volume of Mobile phase to obtain a solution having a known concentration of about 0.7 mg per mL.

Standard preparation— Transfer 3 mL of the Standard stock preparation to a 10-mL volumetric flask, and dilute with Mobile phase to volume to obtain a final solution having a known concentration of about 0.21 mg per mL of trimipramine maleate.

Assay stock preparation— Dissolve an accurately weighed quantity of Trimipramine Maleate in a suitable volume of Mobile phase to obtain a solution having a known concentration of about 0.7 mg per mL.

Assay preparation— Transfer 3 mL of the Assay stock preparation to a 10-mL volumetric flask, and dilute with Mobile phase to volume to obtain a final solution having a known concentration of about 0.21 mg per mL of trimipramine maleate.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm × 25-cm column that contains 5-µm packing L7. The flow rate is about 1.0 mL per minute. The column temperature is maintained at 30. Chromatograph about 20 µL of the Standard preparation, and record the peak responses as directed for Procedure: the tailing factor for the trimipramine maleate peak is not more than 2.0; and the relative standard deviation for replicate injections of the Standard preparation 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 up to 1.5 times the retention time of trimipramine maleate, and measure the responses for the trimipramine maleate peak. Calculate the percentage of C20H26N2·C4H4O4, in the portion of Trimipramine Maleate taken by the formula: in which CS is the concentration, in mg per mL, of USP Trimipramine Maleate RS in the Standard preparation; CU is the concentration, in mg per mL, of Trimipramine Maleate in the Assay preparation; and rU and rS are the trimipramine peak responses obtained from the Assay preparation and the Standard preparation, respectively.