Related compounds
Solvent
Dissolve 2.4 g of monobasic sodium phosphate in 1000 mL of water, and adjust with phosphoric acid to a pH of 2.5.
Blank solution
Use the Solvent.
Solution A
Dissolve 6.9 g of monobasic sodium phosphate in 1000 mL of water, and adjust with phosphoric acid to a pH of 4.0.
Solution B
Prepare a mixture of Solution A and acetonitrile (550:450), degassing for not more than 2 minutes.
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).
[NOTEReducing the acetonitrile content increases the retention time of cefaclor and increases the resolution between cefaclor, delta-3 isomer and cefaclor.
]
Standard solution
Dissolve an accurately weighed quantity of
USP Cefaclor RS in
Solvent to obtain a solution having a known concentration of about 0.05 mg per mL. Sonicate briefly, if necessary, to dissolve, and avoid heating.
[NOTEUse this solution on the day it is prepared.
]
System suitability solution
Dissolve a quantity of USP Cefaclor, Delta-3 Isomer RS in the Standard solution to obtain a solution having a concentration of about 0.05 mg per mL.
Test solutions
Transfer about 50 mg of Cefaclor, accurately weighed, to each of two 10-mL volumetric flasks, dilute each with Solvent to volume, and mix. Sonicate briefly, if necessary, to dissolve, and avoid heating. [NOTEUse these Test solutions within 2 hours when stored at room temperature or within 20 hours when stored under refrigeration.]
Chromatographic system (see Chromatography 621)
The liquid chromatograph is equipped with a 220-nm detector and a 4.6-mm × 25-cm column that contains packing L1. The flow rate is about 1 mL per minute. The chromatograph is programmed as follows.
Time (minutes) |
Solution A
(%) |
Solution B
(%) |
Elution |
0 |
95 |
5 |
equilibration |
030 |
95®75 |
5®25 |
linear gradient |
3045 |
75®0 |
25®100 |
linear gradient |
4555 |
0 |
100 |
isocratic |
5560 |
0®95 |
100®5 |
reset composition |
6070 |
95 |
5 |
re-equilibration |
Chromatograph the
System suitability solution, and record the peak responses as directed for
Procedure: the retention time for the cefaclor peak is between 23 and 29 minutes; the resolution,
R, between cefaclor, delta-3 isomer and cefaclor is not less than 2.0; and the tailing factor for the cefaclor peak is not more than 1.2. Chromatograph the
Blank solution as directed for
Procedure. Examine the chromatogram for any extraneous peaks, and disregard any corresponding peaks observed in the chromatogram of the
Test solutions. [NOTEEnsure that any extraneous peaks observed do not represent carryover from previous injections.
]
Procedure
Separately inject equal volumes (about 20 µL) of the
Standard solution and the
Test solutions into the chromatograph, record the chromatograms, and measure all of the peak areas. Calculate the percentage of each cefaclor related compound in the portion of Cefaclor taken by the formula:
(CP/W)(ri / rS),
in which
C is the concentration, in mg per mL, of
USP Cefaclor RS in the
Standard solution; P is the designated potency, in µg per mg, of
USP Cefaclor RS;
W is the weight, in mg, of the portion of Cefaclor taken to prepare the respective
Test solution; ri is the peak response of an individual related compound in the chromatogram obtained from the
Test solution; and
rS is the peak response for the cefaclor peak in the chromatogram of the
Standard solution. Determine the mean values for each cefaclor related compound: not more than 0.5% of any individual cefaclor related compound is found, and not more than 2.0% of total cefaclor related compounds is found. In an acceptable determination, the difference between duplicate determinations of total cefaclor related compounds is not more than 0.2% absolute, or the variation from the mean of the two values is not more than 10%, whichever is greater.
Assay
Mobile phase
Dissolve 1 g of sodium 1-pentanesulfonate in a mixture of 780 mL of water and 10 mL of triethylamine. Adjust with phosphoric acid to a pH of 2.5 ± 0.1, add 220 mL of methanol, and mix. Make adjustments if necessary (see
System Suitability under
Chromatography 621).
Standard preparation
Transfer about 15 mg of
USP Cefaclor RS, accurately weighed, to a 50-mL volumetric flask, dilute with
Mobile phase to volume, and mix. Sonicate briefly, if necessary, to achieve dissolution, and avoid heating the solution.
[NOTEUse this
Standard preparation within 8 hours if stored at room temperature, or within 20 hours if stored under refrigeration.
]
Assay preparation
Transfer about 15 mg of Cefaclor, accurately weighed, to a 50-mL volumetric flask, dilute with Mobile phase to volume, and mix. Sonicate briefly, if necessary, to achieve dissolution, and avoid heating the solution. [NOTEUse this Assay preparation within 8 hours if stored at room temperature, or within 20 hours if stored under refrigeration.]
Resolution solution
Prepare a solution in Mobile phase containing about 0.3 mg of cefaclor and 0.3 mg of USP Cefaclor, Delta-3 Isomer RS per mL.
Chromatographic system (see Chromatography 621)
The liquid chromatograph is equipped with a 265-nm detector and a 4.6-mm × 25-cm column containing 5-µm packing L1. The flow rate is about 1.5 mL per minute. Chromatograph the
Resolution solution, and record the responses as directed for
Procedure: the relative retention times for cefaclor and cefaclor, delta-3 isomer are about 0.8 and 1.0, the resolution,
R, between the cefaclor peak and the cefaclor, delta-3 isomer peak is not less than 2.5, the tailing factor is not more than 1.5, and the relative standard deviation for replicate injections is not more than 2%.
Procedure
[NOTEUse peak areas where peak responses are indicated.
] Separately inject equal volumes (about 20 µL) of the
Standard preparation and the
Assay into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the potency, in µg per mg, of cefaclor (C
15H
14ClN
3O
4S) in each mg of the Cefaclor taken by the formula:
(WS / WU)(P)(rU / rS),
in which
WS and
WU are the weights, in mg, of
USP Cefaclor RS and of Cefaclor taken to prepare the
Standard preparation and the
Assay preparation, respectively;
P is the designated potency, in µg of cefaclor (C
15H
14ClN
3O
4S) per mg, of
USP Cefaclor RS; and
rU and
rS are the peak responses of the cefaclor peaks obtained from the
Assay preparation and the
Standard preparation, respectively.