TEST 1—
Buffer solution—
Prepare as directed in the Assay.
Mobile phase—
Prepare a filtered and degassed mixture of
Buffer solution and methanol (56:44). Make adjustments if necessary (see
System Suitability under
Chromatography 
621
).
Diluent—
Prepare a mixture of Buffer solution and methanol (50:50).
Standard solution—
Transfer about 30 mg of
USP Acebutolol Hydrochloride RS, accurately weighed, to a 50-mL volumetric flask. Add about 12 mL of methanol, swirl to dissolve, dilute with
Diluent to volume, and mix. Dilute an accurately measured volume of this solution quantitatively, and stepwise if necessary, with
Diluent to obtain a solution having a known concentration of about 1.4 µg of
USP Acebutolol Hydrochloride RS per mL.
Test solution—
Transfer an accurately weighed portion of the contents of 20 opened Capsules, equivalent to about 250 mg of acebutolol, to a 100-mL volumetric flask, add about 25 mL of methanol, and shake by mechanical means for about 15 minutes. Dilute with Diluent to volume, and mix. Centrifuge a portion of this solution, and transfer 10.0 mL of the clear supernatant to a 100-mL volumetric flask. Dilute with Diluent to volume, and mix.
Chromatographic system (see Chromatography 621)—
The liquid chromatograph is equipped with a 240-nm detector and a 3.9-mm × 15-cm column that contains 4-µm packing L1. The flow rate is about 1 mL per minute. Chromatograph the
Standard solution, and record the peak responses as directed for
Procedure: the relative standard deviation for replicate injections is not more than 6.0%.
Procedure—
Separately inject equal volumes (about 35 µL) of the
Standard solution, Test solution, and
Diluent into the chromatograph, record the chromatograms for about two times the retention time of acebutolol, and measure the responses for all the peaks, disregarding any peaks corresponding to those obtained from the
Diluent. Calculate the percentage of each impurity eluting prior to the acebutolol peak in the portion of Capsules taken by the formula:
(336.44/372.89)(0.4C)(ri / rS),
in which 336.44 and 372.89 are the molecular weights of acebutolol and acebutolol hydrochloride, respectively;
C is the concentration, in µg per mL, of
USP Acebutolol Hydrochloride RS in the
Standard solution; ri is the peak response of any individual impurity obtained from the
Test solution; and
rS is the peak response of acebutolol obtained from the
Standard solution: not more than 0.5% of any individual impurity is found.
TEST 2—
Buffer solution—
Prepare as directed in the Assay.
Mobile phase—
Prepare a filtered and degassed mixture of
Buffer solution and methanol (50:50). Make adjustments if necessary (see
System Suitability under
Chromatography 621).
Standard solution—
Transfer about 30 mg of
USP Acebutolol Hydrochloride RS, accurately weighed, to a 50-mL volumetric flask. Add about 12 mL of methanol, swirl to dissolve, dilute with
Mobile phase to volume, and mix. Dilute an accurately measured volume of this stock solution quantitatively, and stepwise if necessary, with
Mobile phase to obtain a solution having a known concentration of about 1.4 µg of
USP Acebutolol Hydrochloride RS per mL.
Test solution—
Transfer an accurately weighed portion of the contents of 20 opened Capsules, equivalent to about 250 mg of acebutolol, to a 100-mL volumetric flask, add about 25 mL of methanol, and shake by mechanical means for about 15 minutes. Dilute with Mobile phase to volume, and mix. Centrifuge a portion of this solution, and transfer 10.0 mL of the clear supernatant to a 100-mL volumetric flask. Dilute with Mobile phase to volume, and mix.
Chromatographic system (see Chromatography 621)—
The liquid chromatograph is equipped with a 240-nm detector and a 3.9-mm × 15-cm column that contains 4-µm packing L1. The flow rate is about 1 mL per minute. Chromatograph the
Standard solution, and record the peak responses as directed for
Procedure: the relative standard deviation for replicate injections is not more than 6.0%.
Procedure—
Separately inject equal volumes (about 70 µL) of the
Standard solution, Test solution, and
Mobile phase into the chromatograph, record the chromatograms for about three times the retention time of acebutolol, and measure the responses for all the peaks, disregarding any peaks corresponding to those obtained from the
Mobile phase. Calculate the percentage of each impurity eluting after the acebutolol peak in the portion of Capsules taken by the formula:
(336.44/372.89)(0.4C)(ri / rS),
in which 336.44 and 372.89 are the molecular weights of acebutolol and acebutolol hydrochloride, respectively;
C is the concentration, in µg per mL, of
USP Acebutolol Hydrochloride RS in the
Standard solution; ri is the peak response of any individual impurity obtained from the
Test solution; and
rS is the peak response of acebutolol obtained from the
Standard solution: not more than 0.5% of any individual impurity is found. The sum of all individual impurities found in
Test 1 and
Test 2 is not more than 1.0%.
