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Related compounds—
[NOTE—On the basis of information regarding the manufacturing process, perform either
Test 1 or
Test 2 and
Test 3.
]
TEST 1—USP29
Mobile phase—
Prepare a mixture of water and acetonitrile (80:20).
System suitability solution—
Use the Standard solution.
Standard solution—
Transfer accurately weighed quantities of USP Fluconazole RS, USP Fluconazole Related Compound A RS, USP Fluconazole Related Compound B RS, and USP Fluconazole Related Compound C RS to a suitable volumetric flask, dissolve in acetonitrile, dilute quantitatively, and stepwise if necessary, with Mobile phase to volume, and mix to obtain a solution having known concentrations of 10 µg of each per mL.
Test solution—
Transfer about 30 mg of Fluconazole, accurately weighed, to a 10-mL volumetric flask, dissolve in and dilute with Mobile phase to volume, and mix.
Chromatographic system (see Chromatography 
621
)—
The liquid chromatograph is equipped with a 260-nm detector and a 4.6-mm × 15-cm column that contains 3.5-µm packing L1. The flow rate is about 0.5 mL per minute. The column temperature is maintained at 40
. Chromatograph the
Standard solution, and record the peak responses as directed for
Procedure: typical retention times are about 4.9 minutes for fluconazole related compound A, 8.0 minutes for fluconazole related compound B, 8.5 minutes for fluconazole related compound C, and 9.9 minutes for fluconazole; the resolution,
R, between fluconazole related compound B and fluconazole related compound C is not less than 1.5; and the relative standard deviation of each peak for replicate injections is not more than 5.0%.
Procedure—
Separately inject equal volumes (about 20 µL) of the
Standard solution and the
Test solution into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the percentage of fluconazole related compound A, fuconazole related compound B, fluconazole related compound C, and any other impurities in the portion of Fluconazole taken by the formula:
1000(C/W)(rU / rS)
in which
C is the concentration, in mg per mL, of USP Fluconazole Related Compound A RS, USP Fluconazole Related Compound B RS, USP Fluconazole Related Compound C RS, or USP Fluconazole RS, respectively, in the
Standard solution; W is the weight, in mg, of Fluconazole taken to prepare the
Test solution; rU is the peak response obtained from the
Test solution; and
rS is the average peak response of fluconazole related compound A, fluconazole related compound B, fluconazole related compound C, or fluconazole obtained from replicate injections of the
Standard solution: not more than 1.0% of any impurity with a relative retention time (RRT) of about 0.6 is found; not more than 0.2% of any impurity with an RRT of 0.5 or an RRT of about 0.9 is found; not more than 0.1% of any other individual impurity is found; not more than 0.2% of total other impurities is found; and not more than 1.2% of total impurities is found.
TEST 2—
Acetate buffer—
Prepare a 0.04 M anhydrous sodium acetate solution, adjust with 1 N acetic acid to a pH of 5.0, and mix.
Solution A:
filtered and degassed Acetate buffer.
Solution B:
acetonitrile.
Solution C:
methanol.
Mobile phase—
Use variable mixtures of
Solution A, Solution B, and
Solution C as directed for
Chromatographic system. Make adjustments if necessary (see
System Suitability under
Chromatography 621).
Diluent—
Prepare a mixture of Acetate buffer and methanol (84:16).
Standard solution—
Dissolve an accurately weighed quantity of USP Fluconazole 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 suitable quantities of USP Fluconazole RS and
USP Desacetyl Diltiazem Hydrochloride RS in
Diluent. Dilute quantitatively, and stepwise if necessary, with
Diluent to obtain a solution containing about 0.02 mg per mL and 0.006 mg per mL, respectively.
Test solution—
Transfer about 200 mg of Fluconazole, accurately weighed, to a 100-mL volumetric flask, and dissolve in and dilute with Diluent to volume.
Chromatographic system (see Chromatography 621)—
The liquid chromatograph is equipped with a 261-nm detector and a 4.0-mm × 10-cm column that contains packing L1. The flow rate is 1 mL per minute. The chromatograph is programmed as follows.
Time
(minutes) |
Solution
A (%) |
Solution
B (%) |
Solution
C (%) |
Elution |
| 0–10 |
80 |
5 |
15 |
isocratic |
| 10–20 |
80®30 |
5®55 |
15 |
linear gradient
(A and B) |
| 20–23 |
30 |
55 |
15 |
isocratic |
| 23–25 |
30®80 |
55®5 |
15 |
reset composition |
| 25–30 |
80 |
5 |
15 |
re-equilibration |
Chromatograph the
System suitability solution, and record the peak responses as directed for
Procedure: the relative retention times are 1.0 for fluconazole and about 1.2 for desacetyl hydrochloride; the resolution,
R, between fluconazole and desacetyl diltiazem hydrochloride is not less than 10.0; the column efficiency for fluconazole is not less than 30,000 theoretical plates; and the tailing factor,
T, is not more than 1.4. Chromatograph the
Standard solution, and record the peak responses as directed for
Procedure: the relative standard deviation for replicate injections is less than 5.0%.
Procedure—
Separately inject equal volumes (about 20 µL) of the
Standard solution and the
Test solution into the chromatograph, record the chromatograms, and measure the peak responses. Calculate the percentage of each impurity in the portion of Fluconazole taken by the formula:
10,000(ri / rS)(C/W)(1/F)
in which
ri is the peak response of each impurity obtained from the
Test solution; rS is the peak response of fluconazole obtained from the
Standard solution; C is the concentration, in mg per mL, of USP Fluconazole RS in the
Standard solution; W is the weight, in mg, of Fluconazole taken to prepare the
Test solution; and
F is the relative response factor as determined from the following table.
| Relative Response Factor (F) |
Relative Retention Time (RRT) |
| 0.72 |
0.17–0.37 |
| 0.85 |
1.20–1.32 |
| 1.21 |
0.48–0.60 |
| 0.96 |
1.14–1.18 |
| 0.97 |
0.67–0.79 |
| 1.0 |
all other peaks |
Not more than 0.1% of any individual impurity is found; and not more than 0.5% of total impurities is found.
TEST 3—
Adsorbent:
0.25-mm layer of chromatographic silica gel mixture.
Test solution—
Dissolve an accurately weighed quantity of Fluconazole in methanol to obtain a solution containing approximately 50 mg per mL.
Standard solutions—
Dissolve an accurately weighed quantity of USP Fluconazole RS in methanol to obtain Standard solution A having a known concentration of about 1 mg per mL (2.0%). Quantitatively dilute portions of this solution with methanol to obtain Standard solution B and Standard solution C having known concentrations of about 0.1 mg per mL (0.2%) and 0.05 mg per mL (0.1%), respectively.
Developing solvent system—
Prepare a mixture of chloroform, methanol, and ammonium hydroxide (80:20:1).
Application volume:
10 µL.
Spray reagent A—
Dissolve about 170 mg of silver nitrate in 100 mL of water.
Spray reagent B (Potassium iodoplatinate solution)—
Dissolve about 375 mg of chloroplatinic acid in 5 mL of 1 N hydrochloric acid. Dissolve about 5 g of potassium iodide in 50 mL of water, and store in a light-resistant container. Prepare a mixture of water, the potassium iodide solution, and the chloroplatinic acid solution (20:9:1).
Procedure—
Proceed as directed for
Thin-Layer Chromatography under
Chromatography 621. Spray the dry plate with
Spray reagent A, and expose the plate to 365-nm UV light for 10 to 20 minutes. Dry the plate for 20 minutes between 80
and 90
, then spray the plate with
Spray reagent B. Allow the plate to dry. Examine the plate and compare the intensities of any secondary spots observed in the chromatogram of the
Test solution with those of the principal spots in the chromatograms of the
Standard solutions: no spot from the chromatogram of the
Test solution with an
RF value of between 0.10 to 0.25 and 0.27 to 0.41 is larger or more intense than that obtained from
Standard solution B (0.2%).
USP29
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