Compliance assay—
pH 3.6 Buffer—
Transfer about 0.900 g of anhydrous dibasic sodium phosphate and about 1.298 g of citric acid monohydrate to a 1-liter volumetric flask, dissolve in and dilute with water to volume, and mix.
pH 7.0 Buffer—
Transfer about 5.68 g of anhydrous dibasic sodium phosphate and about 3.63 g of monobasic potassium phosphate to a 1-liter volumetric flask, dissolve in and dilute with water to volume, and mix.
Solution A—
Combine 900 mL of pH 3.6 Buffer and 100 mL of acetonitrile in a suitable container. Pass the resulting solution through a filter having a 5-µm or finer porosity, and degas.
Solution B—
Combine 200 mL of pH 3.6 Buffer and 800 mL of acetonitrile in a suitable container. Pass the resulting solution through a filter having a 5-µm or finer porosity, and degas.
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
).
Standard preparation—
Using low-actinic volumetric glassware, dissolve an accurately weighed quantity of
USP Cefazolin RS in
pH 7.0 Buffer, and dilute quantitatively, and stepwise if necessary, with
pH 7.0 Buffer to obtain a solution having a known concentration of about 0.32 mg per mL. Maintain at 4
prior to injection.
Assay preparation—
Transfer 1.0 mL of Ophthalmic Solution to a 10-mL low-actinic volumetric flask, dilute with
pH 7.0 Buffer to volume, and mix. Maintain at 4
prior to injection.
Chromatographic system (see Chromatography 621)—
The liquid chromatograph is equipped with a 273-nm detector and a 3.9-mm × 30-cm column that contains 10-µm packing L1. The flow rate is about 2 mL per minute and the column temperature is maintained at 25
. The chromatograph is programmed as follows.
Time
(minutes) |
Solution A
(%) |
Solution B
(%) |
Elution |
| 0 |
100 |
0 |
equilibration |
| 0–15 |
100®0 |
0®100 |
linear gradient |
| 15–25 |
100 |
0 |
isocratic |
Chromatograph the
Standard preparation, and record the peak responses as directed for
Procedure: the column efficiency is not less than 1500 theoretical plates; the tailing factor is not more than 1.5; 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 cefazolin (C
14H
14N
8O
4S
3) in 10 mL of Ophthalmic Solution taken by the formula:
100C(rU / rS),
in which
C is the concentration, in mg per mL, of
USP Cefazolin RS in the
Standard preparation; and
rU and
rS are the peak responses obtained from the
Assay preparation and the
Standard preparation, respectively.
