A: Thin-Layer Chromatographic Identification Test 201—

Test solution— Weigh the contents of not fewer than 20 Capsules, mix, and transfer an accurately weighed portion of the powder, equivalent to about 150 mg of arginine, to a 100-mL volumetric flask. Dissolve in water, and then sonicate for 15 minutes. Dilute with water to volume, mix, and filter.

Standard solution— Dissolve an accurately weighed quantity of USP l-Arginine RS. or USP Arginine Hydrochloride RS in water to obtain a solution having a known concentration of about 1.5 mg per mL.

Application volume: 5 µL.

Developing solvent system— Prepare a mixture of isopropyl alcohol and ammonium hydroxide (70:30).

Spray reagent— Dissolve 0.2 g of ninhydrin in 100 mL of a mixture of butyl alcohol and 2 N acetic acid (95:5).

Procedure— Proceed as directed for Thin-Layer Chromatography under Chromatography 621. Dry the plate between 100 and 105 until the ammonia disappears completely. Spray with Spray reagent, and heat between 100 and 105 for about 15 minutes. Examine the plate under white light. The principal spot from the Test solution corresponds in appearance and RF value to that of the Standard solution.

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.

Medium: 0.1 N hydrochloric acid; 900 mL.

Apparatus 2: 100 rpm.

Time: 60 minutes.

Procedure— Determine the amount of C6H14N4O2 dissolved by employing the procedure set forth in the Assay, making any necessary modifications.

Tolerances— Not less than 75% of the labeled amount of C6H14N4O2 is dissolved in 60 minutes.

Phosphate buffer— In a 1000-mL volumetric flask, dissolve 6.9 g of monobasic sodium phosphate in 800 mL of water, adjust with phosphoric acid to a pH of 3.5, dilute with water to volume, and mix.

2.3 mM 1-Octanesulfonic acid sodium salt solution— In a 1000-mL volumetric flask, dissolve 0.5 g of 1-octanesulfonic acid sodium salt in 800 mL of Phosphate buffer, dilute with Phosphate buffer to volume, and mix.

Mobile phase— Prepare a filtered and degassed mixture of 2.3 mM 1-Octanesulfonic acid sodium salt solution and acetonitrile (95:5). Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard preparation— Dissolve an accurately weighed quantity of USP l-Arginine RS or USP Arginine Hydrochloride RS in Phosphate buffer, and dilute quantitatively, and stepwise if necessary, with Phosphate buffer to obtain a solution having a known concentration of about 1.5 mg per mL.

Assay preparation— Weigh the contents of not fewer than 20 Capsules, mix, and transfer an accurately weighed portion of the powder, equivalent to about 150 mg of arginine, to a 100-mL volumetric flask. Dissolve in 80 mL of Phosphate buffer, and then sonicate for 15 minutes. Dilute with Phosphate buffer to volume, mix, and filter.

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 packing L7. The flow rate is about 0.8 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the column efficiency is not less than 1500 theoretical plates; and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 10 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 arginine (C6H14N4O2) in the portion of Capsules taken by the formula: in which C is the concentration, in mg per mL, of USP l-Arginine RS or USP Arginine Hydrochloride RS in the Standard preparation; and rU and rS are the peak responses obtained from the Assay preparation and the Standard preparation, respectively.USP32