Identification, Ultraviolet Absorption 
197U
—
Solution:
10 µg per mL, prepared as follows. Transfer about 50 mg to a glass-stoppered, 100-mL volumetric flask, add 25 mL of methanol and 5 mL of glacial acetic acid to dissolve the specimen, dilute with methanol to volume, and mix. Pipet 2 mL of this solution into a 100-mL volumetric flask, dilute with methanol to volume, and mix.
Ratio:
the ratio (A230/A279(sh)) is between 0.90 and 1.25.
Composition—
Solution A—
Prepare a mixture of 0.05 M ammonium acetate and acetonitrile (71:29).
Solution B—
Prepare a mixture of acetonitrile and 0.05 M ammonium acetate (60:40).
Mobile phase—
Use variable mixtures of
Solution A and
Solution B as directed for
Chromatographic system. Make adjustments if necessary (see
System Suitability under
Chromatography 621).
Standard solution—
Quantitatively dissolve an accurately weighed quantity of
USP Nystatin RS in dimethyl sulfoxide to obtain a solution having a known concentration of about 0.4 mg per mL.
[NOTE—Protect this solution from light and use within 24 hours, if stored in the refrigerator.
]
Test solution—
Transfer about 20 mg of Nystatin, accurately weighed, to a low-actinic 50-mL volumetric flask, dissolve in and dilute with dimethyl sulfoxide to volume, and mix. [NOTE—Use this solution within 24 hours, if stored in the refrigerator.]
System suitability solution—
Dissolve about 20 mg of Nystatin in 25 mL of methanol, dilute with water to 50 mL, and mix. To 10.0 mL of this solution add 2.0 mL of diluted hydrochloric acid, and allow to stand at room temperature for 1 hour.
Chromatographic system (see Chromatography 621)—
The liquid chromatograph is equipped with a 304-nm detector and a 4.6-mm × 15-cm column that contains base-deactivated end-capped 5-µm packing L1. The column is maintained at a constant temperature of about 30
. The flow rate is about 1 mL per minute. The chromatograph is programmed as follows.
Time
(minutes) |
Solution A
(%) |
Solution B
(%) |
Elution |
| 0–25 |
100 |
0 |
isocratic |
| 25–35 |
100®0 |
0®100 |
gradient |
| 35–40 |
0 |
100 |
isocratic |
| 40–45 |
0®100 |
100®0 |
gradient |
| 45–50 |
100 |
0 |
equilibrium |
Chromatograph the
System suitability solution, and record the peak responses as directed for
Procedure: the resolution,
R, between the two major peaks is not less than 3.5. Chromatograph the
Standard solution, and record the peak responses as directed for
Procedure: the main nystatin A1 peak elutes at about 14 minutes.
Procedure—
Inject about 20 µL of the
Test solution into the chromatograph, record the chromatogram, and measure the peak area responses for all of the peaks, disregarding any peaks eluting in less than 2 minutes. Calculate the percentage of each peak by the formula:
100ri / rT,
in which
ri is the response of an individual peak; and
rT is the total of all of the responses, any peaks eluting in less than 2 minutes being disregarded. Not less than 85.0% of nystatin A1 is found; and not more than 4.0% of any other individual component is found.
Suspendibility (where packaged for use in the extemporaneous preparation of oral suspensions)—
Transfer about 200 mg, accurately weighed, to a 250-mL beaker containing 200.0 mL of water, and disperse by stirring gently with a stirring rod. Allow to stand for 2 minutes, and observe the suspension: the material is in suspension, and little or no sediment is present on the bottom of the beaker. If there is any sediment, assay the undisturbed suspension as directed for nystatin under
Antibiotics—Microbial Assays 81, using an accurately measured volume of it blended in a high-speed blender for 3 to 5 minutes with a sufficient accurately measured volume of dimethylformamide to give a concentration of about 400 USP Nystatin Units per mL. Dilute this stock solution quantitatively with
Buffer No. 6 to obtain a
Test Dilution having a concentration assumed to be equal to the median dose level of the Standard: not less than 90.0% of the expected number of USP Nystatin Units is found, based on the potency obtained in the
Assay.
Loss on drying 731—
Dry about 100 mg, accurately weighed, in a capillary-stoppered bottle in vacuum at a pressure not exceeding 5 mm of mercury at 60
for 3 hours: it loses not more than 5.0% of its weight.
