100A / [A + (D + I)]

Diluent— Prepare a mixture of methanol and water (9:1).

Mobile phase— Prepare a degassed mixture of methanol, water, and glacial acetic acid (94:6:0.1). Make adjustments if necessary (see System Suitability under Chromatography 621).

Neutralized methanol— Add 1 g of sodium bicarbonate to 4 L of methanol, mix, and filter.

Derivatizing reagent— Dissolve 30 g of vanillin in a mixture of methanol and sulfuric acid (950:20) in a container protected from light. [Caution—To avoid splattering, add the sulfuric acid carefully and slowly with a pipet; do not pour. Allow the mixture of methanol and sulfuric acid to cool before adding the vanillin. ] Do not filter.

Resolution solution— Prepare a solution in Neutralized methanol containing about 3 mg of USP Narasin RS and 1 mg of USP Monensin Sodium RS per mL. Transfer 2 mL of this solution to a 200-mL volumetric flask, dilute with Diluent to volume, and mix.

Standard preparations— Dissolve an accurately weighed quantity of USP Narasin RS in Neutralized methanol to obtain a solution having a known concentration of about 1 mg per mL. Transfer 1.0 mL of this stock solution to a 200-mL volumetric flask, and transfer 2.0 mL and 4.0 mL of the stock solution to two separate 100-mL volumetric flasks, dilute each with Diluent to volume, and mix. These solutions contain about 5, 20, and 40 µg of USP Narasin RS per mL. Using the designated percentage of narasin A in the USP Narasin RS, calculate the exact narasin A concentration, in µg per mL, in each of the Standard preparations.

Assay preparation— Transfer about 5 g of Narasin Granular, accurately weighed, to a suitable container, add 200.0 mL of Diluent, and shake by mechanical means for 1 hour. Allow the solids to settle, and quantitatively dilute an accurately measured volume of the supernatant with Diluent to obtain a solution containing about 20 µg of narasin per mL. Pass a portion of this solution through a filter having a 0.5-µm or finer porosity, and use the filtrate as the Assay preparation.

Chromatographic system (see Chromatography 621)—The liquid chromatograph is equipped with a 4.6-mm × 25-cm column that contains packing L1. The column outlet is attached to a tee, the opposing arm is attached to a tube from which is pumped the Derivatizing reagent, and the outlet is connected to a 2-mL postcolumn reaction coil maintained at 98. The outlet of the reaction coil is connected to a detector set at 520 nm. The Mobile phase and the Derivatizing reagent flow at the rate of about 0.7 mL per minute. Chromatograph the Resolution solution, and record the peak responses as directed for Procedure: the relative retention times are about 0.7 for monensin B, 0.75 for monensin A, 1.0 for narasin A, and 1.1 for narasin D + I; and the resolution, R, between the monensin B peak and the monensin A peak is not less than 1.25, and between the monensin A peak and the narasin A peak not less than 3.5. Chromatograph the Standard preparations, and record the peak responses as directed for Procedure: the tailing factor for the narasin A peak is not more than 1.4 when calculated by the formula: in which W0.1 is the width of the peak at 10% of peak height; and f is the distance from the peak maximum to the leading edge of the peak, the distance being measured at a point on the baseline at which 10% peak height is reached. The relative standard deviation for replicate injections is not more than 10%. [note—After use, flush the system with methanol.]

Procedure— Separately inject equal volumes (about 200 µL) of the Standard preparations and the Assay preparation into the chromatograph, and measure the areas of the peak responses for the narasin A and narasin D + I peaks [note—Narasin D and narasin I will co-elute under this chromatographic system.]

(0.001)(CA FA + CD +I FD +I)(VE / M)

(1.510D + 0.012I) / (D + I)