Chromatographic purity
Solution A
Proceed as directed for Mobile phase in the Assay.
Solution B
Prepare filtered and degassed methanol.
Mobile phase
Use variable mixtures of
Solution A and
Solution B as directed under
Chromatographic system. Make adjustments, if necessary (see
System Suitability under
Chromatography 621).
System suitability solution
Proceed as directed in the Assay.
Standard solution
Dissolve an accurately weighed quantity of
USP Gemcitabine Hydrochloride RS and USP Cytosine RS in water, and dilute quantitatively, and stepwise if necessary, to obtain a solution having a known concentration of about 2 µg per mL of each.
Test solution
Transfer about 50 mg of Gemcitabine Hydrochloride, accurately weighed, to a 25-mL volumetric flask, dissolve in and dilute with water to volume, and mix.
Chromatographic system (see Chromatography 621)
Proceed as directed under
Assay. The chromatograph is programmed as follows.
Time (minutes) |
Solution A
(%) |
Solution B
(%) |
Elution |
08 |
97 |
3 |
isocratic |
813 |
97®50 |
3®50 |
linear gradient |
1320 |
50 |
50 |
isocratic |
2025 |
50®97 |
50®3 |
re-equilibration |
Chromatograph the
System suitability solution, and record the peak responses as directed for
Procedure: the relative retention times are about 0.5 for gemcitabine
-anomer and 1.0 for gemcitabine; the resolution,
R, between gemcitabine
-anomer and gemcitabine is not less than 8.0; and the tailing factor for gemcitabine is not more than 1.5. Chromatograph the standard solution, and record the peak responses as directed for
Procedure: the relative retention times are about 0.1 for cytosine and 1.0 for gemcitabine; the relative standard deviation for replicate injections is not more than 2.0%.
Procedure
Separately inject a volume (about 20 µL) of the
Standard solution and
Test solution into the chromatograph, record the chromatogram, and measure all of the peak responses. Calculate the percentage of cytosine in the portion of Gemcitabine taken by the formula:
2.5(Cc / W)(rt / rs)
in which
Cc is the concentration of USP Cytosine RS in the
Standard solution, in µg per mL;
W is the weight, in mg, of Gemcitabine taken;
rt is the peak response for cytosine in the
Test solution; and
rs is the response for cytosine in the
Standard solution: not more than 0.1% of cytosine is found. Calculate the percentage of each impurity other than cytosine in the portion of Gemcitabine taken by the formula:
2.5(Cs / W)(ri / rs)
in which
Cs is the concentration of
USP Gemcitabine Hydrochloride RS in the
Standard solution, in µg per mL;
W is the weight, in mg, of Gemcitabine taken;
ri is the peak response for each impurity in the
Test solution; and
rs is the response due to gemcitabine in the
Standard solution: not more than 0.1% of gemcitabine
-anomer or any other individual impurity is found; and the sum of all impurities is not more than 0.2%. Exclude from the sum of all impurities any peaks that are below the limit of quantitation (0.02%).
Assay
Mobile phase
Prepare a filtered and degassed solution containing 13.8 g of monobasic sodium phosphate and 2.5 mL of phosphoric acid in 1000 mL of water. [NOTEThe pH of this solution is between 2.4 and 2.6.]
System suitability solution
Transfer about 10 mg of Gemcitabine Hydrochloride to a small vial, add 4 mL of a solution containing 168 mg of potassium hydroxide per mL of methanol, cap tightly, and sonicate. Heat at 55
for 6 to 16 hours, allow to cool, and transfer the contents to a 100-mL volumetric flask with successive washes of 1% (v/v) phosphoric acid. Dilute with 1% phosphoric acid to volume, and mix.
[NOTEThis solution contains about 0.02 mg per mL of gemcitabine
-anomer.
]
Standard preparation
Dissolve an accurately weighed quantity of
USP Gemcitabine Hydrochloride RS in water, and dilute quantitatively, and stepwise if necessary, with water to obtain a solution having a known concentration of about 0.1 mg per mL.
Assay preparation
Transfer about 20 mg of Gemcitabine Hydrochloride, accurately weighed, to a 200-mL volumetric flask, dissolve in and dilute with water to volume, and mix.
Chromatographic system (see Chromatography 621)
The liquid chromatograph is equipped with a 275-nm detector and a 4.6-mm × 25-cm column that contains 5-µm packing L7. The flow rate is about 1.2 mL per minute. Chromatograph the
System suitability solution, and record the peak responses as directed for
Procedure: the resolution,
R, between the gemcitabine
-anomer and gemcitabine is not less than 8.0; and the tailing factor determined from gemcitabine is not more than 1.5. 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 major peaks. Calculate the quantity, in mg, of C
9H
11F
2N
3O
4·HCl in the portion of Gemcitabine Hydrochloride taken by the formula:
200C(rU / rS),
in which
C is the concentration, in mg per mL, of
USP Gemcitabine Hydrochloride RS in the
Standard preparation; and
rU and
rS are the peak responses obtained from the
Assay preparation and the
Standard preparation, respectively.