Assay—
Solution A—
Transfer about 26.2 mL of acetic acid and about 99.12 g of potassium acetate to a 4-L volumetric flask. Add 2000 mL of water, and mix to dissolve. Dilute with water to volume, and pass through a 0.45-µm nylon filter.
Solution B—
Use acetonitrile.
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
).
Extraction solution:
a mixture of 400 mL of acetic acid and 600 mL of water.
Dilution buffer—
Dissolve about 205 g of potassium acetate in about 800 mL of water. Adjust with acetic acid to a pH of 7.5 to 8.2. Dilute with water to 1000 mL, and pass through a 0.45-µm nylon filter.
10% Acetic acid solution—
Add about 10.0 mL of acetic acid to a 100-mL volumetric flask. Mix, and dilute with water to volume.
System suitability solution—
Dissolve an accurately weighed quantity of
USP Cephapirin Sodium RS in
10% Acetic acid solution to prepare a solution containing a known concentration of about 2.0 mg per mL. Heat the solution at 50
for 12 to 18 hours.
Standard preparation—
In duplicate, accurately weigh about 50 mg of
USP Cephapirin Sodium RS, and transfer into a 25-mL volumetric flask. Add about 2.5 mL of
Extraction solution and about 15.0 mL of
Dilution buffer, and agitate to dissolve. Add 7.0 mL of acetonitrile, and mix well. Allow the solution to return to room temperature, and dilute with water to volume.
Assay preparation—
In duplicate, weigh about 60 mg of Cephapirin Benzathine, and transfer into a 25-mL volumetric flask. Add about 2.5 mL of Extraction solution and 15.0 mL of Dilution buffer, and mix to dissolve. Add 7.0 mL of acetonitrile, and mix. Allow the flask to return to room temperature, and dilute with water to volume.
Chromatographic system (see Chromatography 621)—
The liquid chromatograph is equipped with a 260-nm detector, a 3.2-mm × 15-mm guard column that contains 7-µm packing L1 and a 3.9-mm × 15-cm analytical column that contains 4-µm packing L1. The flow rate is about 2.0 mL per minute, and the columns are heated to 40
. The chromatograph is programmed as follows.
Time
(minutes) |
Solution A
(%) |
Solution B
(%) |
Elution |
| 0–6 |
91.5 |
8.5 |
isocratic |
| 6–10 |
91.5®80.0 |
8.5®20.0 |
linear |
| 10–12 |
80.0 |
20.0 |
isocratic |
| 12 |
80.0®91.5 |
20.0®8.5 |
return to initial |
| 12–21 |
91.5 |
8.5 |
re-equilibration |
Chromatograph the
System suitability solution and the
Standard preparation, and record the peak heights and valleys as directed for
Procedure. Using the results from the
System suitability solution, calculate the percentage of the height of the valley taken by the formula:
100(rV / ri),
in which
rV is the height of the valley between cephapirin and any impurity; and
ri is the impurity peak height. The percentage of the height of the valley is not more than 25% for the impurity peaks adjacent to the cephapirin peak.
[note—The
System suitability solution is acceptable as long as the cephapirin peak is larger than the two peaks on either side of the cephapirin peak.
] The relative standard deviation for replicate injections of the
Standard preparation is not more than 3.0%.
Procedure—
Separately inject equal volumes (about 2 µL) of the duplicate
Standard preparation and the duplicate
Assay preparation into the chromatograph, record the chromatograms, and measure the areas for the major peaks. Calculate the quantity, in µg, of C
17H
17N
3O
6S
2 in each mg of Cephapirin Benzathine taken by the formula:
P(WS / WU)(VU / VS)(rU / rS),
in which
P is the assigned potency, in µg of cephapirin per mg, of
USP Cephapirin Sodium RS;
WS and
WU are the quantities of
USP Cephapirin Sodium RS and Cephapirin Benzathine, in mg, used to prepare the
Standard preparation and the
Assay preparation, respectively;
VS and
VU are the final volumes, in mL, of the
Standard preparation and the
Assay preparation, respectively; and
rU and
rS are the average peak areas of the cephapirin peaks obtained from the
Assay preparation and the
Standard preparation, respectively.
;)
