Assay
Mobile phase
Dissolve 5.75 g of monobasic ammonium phosphate in 700 mL of water, add 3.2 mL of a 40% solution of tetrabutylammonium hydroxide, 280 mL of methanol, and 25 mL of tetrahydrofuran, and mix. Adjust with phosphoric acid to a pH of 4.5 ± 0.1, pass through a filter having a 0.5-µm or finer porosity, and degas. Make adjustments if necessary (see
System Suitability under
Chromatography 621).
Standard preparation
Quantitatively dissolve an accurately weighed quantity of
USP Cefmetazole RS in
Mobile phase to obtain a solution having a known concentration of about 200 µg of cefmetazole (C
15H
17N
7O
5S
3) per mL.
[NOTEUse this solution within 10 minutes.
]
Resolution solution
Prepare a solution of
USP Cefmetazole RS in 0.01 N sodium hydroxide containing about 1 mg per mL. Heat at 95
for 10 minutes. To 1 mL of this solution add 2 mL of
Standard preparation, and dilute with
Mobile phase to obtain 20 mL of solution. This solution contains cefmetazole and cefmetazole lactone (resolution compound).
Assay preparation
Transfer about 20 mg of Cefmetazole, accurately weighed, to a 100-mL volumetric flask, dilute with Mobile phase to volume, and mix. [NOTEUse this solution within 10 minutes.]
Chromatographic system
(see
Chromatography 621)The liquid chromatograph is equipped with a 214-nm detector and a 4.6-mm × 25-cm column that contains packing L1. The flow rate is about 2 mL per minute. Chromatograph the
Resolution solution, and record the peak responses as directed for
Procedure: the resolution,
R, between cefmetazole and cefmetazole lactone is not less than 3.0. Chromatograph the
Standard preparation, and record the peak responses as directed for
Procedure: the column efficiency is not less than 1250 theoretical plates; the tailing factor is not less than 0.94 and not more than 1.6; 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 areas for the major peaks. Calculate the quantity, in µg, of cefmetazole (C
15H
17N
7O
5S
3) in each mg of Cefmetazole taken by the formula:
100(C / M)(rU / rS),
in which
C is the concentration, in µg per mL, of cefmetazole (C
15H
17N
7O
5S
3) in the
Standard preparation; M is the quantity, in mg, of Cefmetazole taken to prepare the
Assay preparation; and
rU and
rS are the cefmetazole peak responses obtained from the
Assay preparation and the
Standard preparation, respectively.