Chromatographic purity—
Solution A—
Prepare a filtered and degassed mixture of water and acetonitrile (77:23).
Solution B—
Prepare a filtered and degassed mixture of water and acetonitrile (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
).
Diluent:
a mixture of water and acetonitrile (1:1).
Standard solution—
Dissolve an accurately weighed quantity of
USP Prednisolone RS in
Diluent, and dilute quantitatively, and stepwise if necessary, with
Diluent to obtain a solution having a known concentration of about 0.01 mg per mL.
System suitability solution—
Dissolve an accurately weighed quantity of
USP Prednisolone RS and hydrocortisone in
Diluent to obtain a solution having a known concentration of about 1 mg per mL and 0.06 mg per mL, respectively.
Test solution—
Transfer about 25 mg of Prednisolone, accurately weighed, to a 25-mL volumetric flask, dissolve in and dilute with Diluent to volume, and mix.
Chromatographic system (see Chromatography 621)—
The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm × 30-cm column that contains 5-µm packing L1. The flow rate is about 1.5 mL per minute. The column temperature is maintained at 40
. The chromatograph is programmed as follows.
Time
(minutes) |
Solution A
(%) |
Solution B
(%) |
Elution |
| 0 |
100 |
0 |
equilibration |
| 0–25 |
100 |
0 |
isocratic |
| 25–45 |
100®0 |
0®100 |
linear gradient |
| 45–60 |
0 |
100 |
isocratic |
| 60–61 |
0®100 |
100®0 |
linear gradient |
| 61–100 |
100 |
0 |
re-equilibration |
Chromatograph the
System suitability solution, and record the peak responses as directed for
Procedure: the relative retention times for prednisolone and hydrocortisone are about 1.0 and 1.06, respectively; and the height of the smallest peak is not less than 2 times the height of the valley between the prednisolone and hydrocortisone peaks. Chromatograph the
Standard solution, and record the peak responses as directed for
Procedure: the relative standard deviation for replicate injections is not more than 5.0% for the prednisolone peak.
Procedure—
Separately inject equal volumes (about 20 µL) of the
Standard solution and
Test solution into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the percentage of each impurity in the portion of Prednisolone taken by the formula:
2500(C / W)(ri / rS),
in which
C is the concentration, in mg per mL, of
USP Prednisolone RS in the
Standard solution; W is the weight, in mg, of prednisolone used to prepare the
Test solution; ri is the peak response for each impurity in the
Test solution; and
rS is the peak response obtained from the
Standard solution: no impurity greater than 1.0% and only one peak greater than 0.5% is found; and not more than 2.0% of total impurities is found.
Assay—
Mobile phase—
Prepare a solution containing a mixture of butyl chloride, water-saturated butyl chloride, tetrahydrofuran, methanol, and glacial acetic acid (95:95:14:7:6). Make adjustments if necessary (see
System Suitability under
Chromatography 621).
Internal standard solution—
Prepare a solution of betamethasone in tetrahydrofuran containing 5 mg per mL. Dilute this solution with water-saturated chloroform, and mix to obtain a solution having a final concentration of 0.5 mg per mL.
Standard preparation—
Transfer about 10 mg of
USP Prednisolone RS, accurately weighed, to a 100-mL volumetric flask, and dissolve in 5.0 mL of methanol. Add 20.0 mL of
Internal standard solution, and mix. Dilute with water-saturated chloroform to 100.0 mL, and mix.
Assay preparation—
Transfer about 10 mg of Prednisolone, accurately weighed, to a 100-mL volumetric flask, and dissolve in 5.0 mL of methanol. Add 20.0 mL of Internal standard solution, and mix. Dilute with water-saturated chloroform to 100.0 mL, and mix.
Chromatographic system (see Chromatography 621)—
The liquid chromatograph is equipped with a 254-nm detector and a 4-mm × 30-cm column that contains packing L3. The flow rate is about 1 mL per minute. Chromatograph four replicate injections of the
Standard preparation, and record the peak responses as directed for
Procedure: the relative retention times are about 0.7 for betamethasone and 1.0 for prednisolone; the resolution,
R, between prednisolone and betamethasone is not less than 3.5; 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 C
21H
28O
5 in the portion of Prednisolone taken by the formula:
0.1C(RU / RS),
in which
C is the concentration, in µg per mL, of
USP Prednisolone RS in the
Standard preparation; and
RU and
RS are the peak response ratios of prednisolone to the internal standard obtained from the
Assay preparation and the
Standard preparation, respectively.
;)
)-.