Chromatographic purity
[NOTEProtect all solutions from light, and use amber autosampler vials and low-actinic glassware.
]
Solution A, Solution B, and Mobile phase
Proceed as directed in the Assay.
System suitability stock solution
Prepare a solution in a mixture of methanol and acetonitrile (3:2) containing about 0.1 mg of
USP Fluvastatin Related Compound A RS and about 0.1 mg of USP Fluvastatin Related Compound B RS per mL.
System suitability solution
Transfer about 50 mg of
USP Fluvastatin for System Suitability RS, accurately weighed, to a 100-mL volumetric flask, and dissolve in 35 mL of
Solution B. Add 5.0 mL of
System suitability stock solution into the flask, dilute with
Solution A to volume, and mix.
[NoteThe
System suitability stock solution and the
System suitability solution are stable for up to 6 months if stored in a refrigerator.
]
Standard solution
Use the Standard preparation, prepared as directed in the Assay.
Test solution
Use the Assay preparation, prepared as directed in the Assay.
Chromatographic system
Proceed as directed in the Assay, except use the liquid chromatograph equipped with either a programmable variable wavelength detector or two separate detectors capable of monitoring at 305 nm and at 365 nm. Chromatograph the System suitability solution, and record the peak responses at 305 nm as directed for Procedure. Identify the peaks corresponding to fluvastatin, fluvastatin anti-isomer, fluvastatin hydroxydiene, and fluvastatin t-butyl ester. The resolution, R, between fluvastatin anti-isomer and fluvastatin is not less than 1.6; the column efficiency is not less than 700 theoretical plates for the fluvastatin peak; and the tailing factor is not more than 3.0. Chromatograph the Standard solution, and record the peak responses at 305 nm as directed for Procedure: the relative standard deviation for replicate injections is not more than 1.0%.
Procedure
Separately inject equal volumes (about 20 µL) of the
Standard solution and the
Test solution into the chromatograph, record the chromatograms at 305 nm and 365 nm, identify the impurities listed in
Table 1, and measure the peak responses.
[Note3-Hydroxy-5-keto fluvastatin is monitored using a wavelength of 365 nm, and all other compounds are monitored at 305 nm.
] Calculate the percentage of each impurity, except for 3-hydroxy-5-keto fluvastatin, in the portion of Fluvastatin Sodium taken by the formula:
100F(CS / CT)(ri (305) / rS (305)),
in which
F is the relative response factor as listed in
Table 1 [NoteUse
F equal to 1.0 for unknown impurities
];
CS is the concentration, in mg per mL, of
USP Fluvastatin Sodium RS in the
Standard solution; CT is the concentration, in mg per mL, of Fluvastatin Sodium in the
Test solution; ri(305) is the peak response at 305 nm for each impurity obtained from the
Test solution; and
rS(305) is the peak response at 305 nm for the fluvastatin peak obtained from the
Standard solution.
Calculate the percentage of 3-hydroxy-5-keto fluvastatin in the portion of Fluvastatin Sodium taken by the formula:
100F(CS / CT)(ri(365) / rS (365)),
in which F, CS, and CT are as defined above; ri (365) is the peak response at 365 nm for 3-hydroxy-5-keto fluvastatin obtained from the Test solution; and rS(365) is the peak response at 365 nm for the fluvastatin peak obtained from the Standard solution. In addition to not exceeding the limits for each impurity in Table 1, not more than 0.1% of any other individual impurity is found; and not more than 1.0% of total impurities is found.
Table 1
Name |
Relative Retention Time |
Relative Response Factor (F) |
Limit (%) |
Fluvastatin N-ethyl
analog |
0.7 |
0.9 |
0.1 |
Fluvastatin anti-
isomer |
1.2 |
1.0 |
0.8 |
3-Hydroxy-5-keto
fluvastatin |
1.5 |
0.0371 |
0.1 |
3-Keto-5-hydroxy
fluvastatin |
1.6 |
1.6 |
0.1 |
Fluvastatin hydroxydiene2 |
2.0 |
1.1 |
0.1 |
Fluvastatin short
chain aldehyde |
3.0 |
0.7 |
0.1 |
Fluvastatin t-butyl
ester3 |
3.4 |
1.1 |
0.2 |
1
At 365 nm
|
2
Fluvastatin related compound A
|
3
Fluvastatin related compound B
|
Assay
Solution A
Add 20 mL of 25% aqueous tetramethylammonium hydroxide solution to 880 mL of water. Adjust with about 2.3 mL of phosphoric acid to a pH of 7.2 ± 0.2. Add 100 mL of a mixture of methanol and acetonitrile (3:2), mix, and filter.
Solution B
Add 20 mL of 25% aqueous tetramethylammonium hydroxide solution and 80 mL of water to 900 mL of a mixture of methanol and acetonitrile (3:2). Adjust with about 2.3 mL of phosphoric acid to a pH of 7.2 ± 0.2, mix, and filter. Make adjustments if necessary (see
System Suitability under
Chromatography 621).
Mobile phase
Use variable mixtures of Solution A and Solution B as directed for Chromatographic system.
System suitability preparation
Transfer an accurately weighed quantity of
USP Fluvastatin for System Suitability RS to a suitable volumetric flask, dissolve first in
Solution B, using 40% of the final volume, then dilute with
Solution A to volume, and mix to obtain a solution having a known concentration of about 0.5 mg of fluvastatin sodium per mL.
Standard preparation
Transfer an accurately weighed quantity of
USP Fluvastatin Sodium RS to a suitable volumetric flask, dissolve first in
Solution B, using 40% of the final volume, then dilute with
Solution A to volume, and mix to obtain a solution having a known concentration of about 0.5 mg of fluvastatin sodium per mL.
Assay preparation
Transfer about 25 mg of Fluvastatin Sodium, accurately weighed, to a 50-mL volumetric flask. Dissolve in 20 mL of Solution B, dilute with Solution A to volume, and mix.
Chromatographic system (see Chromatography 621)
The liquid chromatograph is equipped with a 305-nm detector and a 4.6-mm × 5-cm column that contains 5-µm packing L1. The flow rate is about 3.0 mL per minute, and the column temperature is maintained at 35
. The chromatograph is programmed as follows.
[NoteAdjust the start time of the gradient step and the equilibration time for each instrument.
]
Time (minutes) |
Solution A
(%) |
Solution B
(%) |
Elution |
06 |
60 |
40 |
isocratic |
620 |
60®18 |
40®82 |
linear gradient |
2020.1 |
18®60 |
82®40 |
linear gradient |
20.125.1 |
60 |
40 |
equilibration |
Chromatograph the
System suitability preparation, and record the peak responses as directed for
Procedure: the relative retention times are about 1.0 for fluvastatin and 1.2 for fluvastatin anti-isomer; the resolution,
R, between fluvastatin anti-isomer and fluvastatin is not less than 1.6; the column efficiency is not less than 700 theoretical plates for the fluvastatin peak; and the tailing factor is not more than 3.0. Chromatograph the
Standard preparation, and record the peak responses as directed for
Procedure: the relative standard deviation for replicate injections is not more than 1.0%.
Procedure
Separately inject equal volumes (about 20 µL) of the
Standard preparation and the
Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the fluvastatin peaks. Calculate the quantity, in mg, of C
24H
25FNNaO
4 in the portion of Fluvastatin Sodium taken by the formula:
50C(rU / rS),
in which
C is the concentration, in mg per mL, of
USP Fluvastatin Sodium RS in the
Standard preparation; and
rU and
rS are the fluvastatin peak responses obtained from the
Assay preparation and the
Standard preparation, respectively.