Content of monensin A and B activity
Using the results of the calculations in the
Assay, calculate the percentage of monensin A activity in the Monensin under test by the formula:
100A / P,
in which
A is the potency, in µg per mg, of monensin A in the Monensin under test, as determined in the
Assay, and
P is the potency, in µg of monensin, in each mg of the Monensin under test, as determined in the
Assay: not less than 90% is found. Calculate the percentage of monensin A activity plus monensin B activity in the Monensin under test by the formula:
100(A + B) / P,
in which
B is the potency, in µg per mg, of monensin B in the Monensin under test, as determined in the
Assay, and the other terms are as defined above: not less than 95% is found.
Assay
Mobile phase
Prepare a filtered and 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 3 g of vanillin in a mixture of methanol and sulfuric acid (95:2). [CautionTo 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 vanillin.]
Standard preparation
Dissolve an accurately weighed quantity of
USP Monensin Sodium RS quantitatively in methanol to obtain a solution containing the equivalent of 1000 µg of monensin per mL. Dilute an accurately measured volume of this stock solution quantitatively with
Diluent to obtain a solution containing 20.0 µg of monensin per mL.
Assay preparation
Transfer about 500 mg of Monensin, accurately weighed, to a 250-mL flask, 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 monensin per mL.
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 0.9 for monensin B, 1.0 for monensin A, 1.3 for narasin A, and 1.5 for narasin 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 is not less than 3.5. Chromatograph the
Standard preparation, and record the peak responses as directed under
Procedure: the tailing factor is not more than 1.4, and the relative standard deviation for replicate injections is not more than 2.0%.
[NOTEAfter use, flush the system with methanol.
]
Procedure
[NOTEUse peak areas where peak responses are indicated.
] Separately inject equal volumes (about 200 µL) of the
Standard preparation and the
Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks, including a peak for monensin C/D, if present, at a retention time of about 1.1 relative to that of the main monensin A peak in the chromatogram obtained from the
Assay preparation. Calculate the quantity, in µg, of monensin A in each mg of the Monensin taken by the formula:
(CFD / 100,000W)(rU / rS),
in which
C is the concentration, in µg per mL, of monensin activity in the
Standard preparation, based on the quantity of
USP Monensin Sodium RS taken, its designated potency, in µg per mg, and the extent of dilution,
F is the designated percentage of monensin A in
USP Monensin Sodium RS,
D is the dilution factor used in preparing the
Assay preparation,
W is the quantity, in g, of Monensin taken to prepare the
Assay preparation, and
rU and
rS are the monensin A peak responses obtained from the
Assay preparation and the
Standard preparation, respectively. Calculate the quantity, in µg, of monensin B in each mg of the Monensin taken by the same formula, except that
rU is the monensin B peak response obtained from the
Assay preparation and
rS is the monensin A peak response obtained from the
Standard preparation. Calculate the quantity, in µg, of monensin C/D in each mg of the Monensin taken by the same formula, except that
rU is the monensin C/D peak response obtained from the
Assay preparation. Calculate the potency, in µg of monensin, in each mg of the Monensin taken by the formula:
A + 0.28B + 1.5C / D,
in which
A is the quantity, in µg, of monensin A in each mg of the Monensin taken, as calculated above, and
B is the quantity, in µg, of monensin B in each mg of the Monensin taken, and
C/D is the quantity, in µg, of monensin C/D in each mg of Monensin taken, as calculated above.