A: It responds to the Thin-layer Chromatographic Identification Test 201, a test solution of it diluted, if necessary, with water, to obtain a concentration of 0.5 mg of alfentanil per mL. Prepare a Standard solution in water to obtain a concentration of 0.54 mg per mL of USP Alfentanil Hydrochloride RS. Apply 200 µL each of the Standard solution and the test solution, develop the plate using a solvent system consisting of a mixture of chloroform, methanol, and formic acid (85:10:5), and visualize the spots using Dragendorff's reagent.

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 Chromatographic system— Proceed as directed in the test for Chromatographic purity under Alfentanil Hydrochloride.

Standard solution— Use the Standard preparation as obtained in the Assay.

Test solution— Use the Assay preparation.

Procedure— Inject a volume (about 25 µL) of the Test solution into the chromatograph, record the chromatogram, and measure the responses for all of the peaks. Calculate the percentage of each impurity in the portion of Injection taken by the formula: in which ri is the response of each impurity peak, and rs is the sum of all of the peaks: the sum of all the impurities is not more than 2.0%.

(Official January 1, 2007)

Mobile phase and Chromatographic system— Proceed as directed in the test for Chromatographic purity under Alfentanil Hydrochloride.

Standard preparation— Dissolve an accurately weighed quantity of USP Alfentanil Hydrochloride RS in saline TS, and dilute quantitatively, and stepwise if necessary, with saline TS to obtain a solution having a known concentration of about 0.54 mg per mL.

Assay preparation— Transfer an accurately measured volume of Injection to a suitable container, and dilute with saline TS, if necessary, to obtain a concentration of about 0.50 mg per mL of alfentanil.

Procedure— Separately inject equal volumes (about 25 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 C21H32N6O3 in each mL of the Injection taken by the formula: in which 416.52 and 452.98 are the molecular weights of alfentanil and alfentanil hydrochloride, respectively; C is the concentration, in mg per mL, of USP Alfentanil Hydrochloride RS in the Standard preparation; and rU and rS are the peak responses obtained from the Assay preparation and the Standard preparation, respectively.