Content of narasin A—
Using the chromatogram of the
Assay preparation obtained as directed in the
Assay, calculate the percentage of narasin A by the formula:
100A / [A + (D + I)],
in which
A is the narasin A biopotency and
D +
I is the narasin
D +
I biopotency. Not less than 85% of narasin A is found.
Assay—
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 liters of methanol, mix, and filter.
Diluent—
Prepare a mixture of methanol and water (9:1).
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.
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 dilute an accurately measured volume of the supernatant quantitatively 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 porosity of 0.5 µm or less, and use the filtrate as the Assay preparation.
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.
Chromatographic system
(see
Chromatography 621)—The liquid chromatograph is equipped with a 4.6-mm × 25-cm column that contains packing L1, and the outlet of which is attached to a tee, the opposing arm of which is attached to a tube from which is pumped the
Derivatizing reagent, and the outlet of which 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 under
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; 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 under
Procedure: the tailing factor for the narasin A peak is not more than 1.4 when calculated by the formula:
W0.1 / 2f,
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.
Plot the three narasin A peak responses in the chromatograms obtained from the Standard preparations versus the concentration, in µg per mL, of narasin A, and draw the straight line best fitting the three plotted points. From the graph so obtained, and the narasin A peak response in the chromatogram obtained from the Assay preparation, determine the concentration, CA, in µg per mL, of narasin A in the Assay preparation. From the same graph and the narasin D + I peak response in the chromatogram obtained from the Assay preparation, determine the concentration, CD+I, in µg per mL, of narasin D + I in the Assay preparation. Calculate the biopotency, in mg per g, of the Narasin Granular taken to prepare the Assay preparation by the formula:
(CA + CD+IF)(VE / M),
in which F is the biopotency conversion factor for narasin D + I; V is the extraction volume, in mL; E is the dilution factor used in diluting the extract to the final estimated concentration of 20 µg per mL; and M is the weight, in g, of Narasin Granular taken to prepare the Assay preparation. Calculate the bioconversion factor, F, for narasin D + I by the formula:
(1.402D + 0.0111I) / (D + I),
in which
D and
I are the specified percentages of narasin D and narasin I in
USP Narasin RS, 1.402 is the factor for converting narasin D to narasin D biopotency, and 0.0111 is the factor for converting narasin I to narasin I biopotency.
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-ethyl-6-[5-[2-(5-ethyltetrahydro-5-hydroxy-6-methyl-2H-pyran-2-yl)-15-hydroxy-2,10,12-trimethyl-1, 6,8-trioxadispiro[4.1.5.3]pentadec-13-en-9-yl]-2-hydroxy-1,3-dimethyl-4-oxoheptyl]tetrahydro-3,5-dimethyl-.