Chromatographic purity—
Examine the chromatogram of the
Assay preparation, as obtained in the
Assay, for peaks that elute at the following retention times in relation to ethopabate: 0.33,
p-aminosalicylic acid (ethopabate related compound B); 0.64, methyl 2-ethoxy-4-aminobenzoate (ethopabate related compound C); 0.68, methyl 2-hydroxy-4-aminobenzoate (ethopabate related compound D); 0.9, methyl 4-acetamido-2-hydroxybenzoate (ethopabate related compound A); and 1.6, ethyl 4-acetamido-2-ethoxybenzoate (ethopabate related compound E). Calculate the percentage of diazotizable substances, represented by peaks for ethopabate related compounds B, C, and D, if present, by the formula:
(0.72rB + 0.68rC + 0.74rD) / 0.01rU,
in which 0.72, 0.68, and 0.74 are the response factors of ethopabate related compounds B, C, and D, respectively, relative to that of ethopabate,
rB,
rC, and
rD are the responses of the peaks observed for ethopabate related compounds B, C, and D, respectively, and
rU is the ethopabate peak response obtained from the
Assay preparation: not more than 0.5% of diazotizable substances is found. Calculate the percentage of any other impurities by the formula:
100
AE As,
in which
AE is the percentage of total peak area represented by the main ethopabate peak in the chromatogram obtained from the
Assay preparation, and
As is the percentage of peak area represented by the sum of the peaks for ethopabate related compounds B, C, and D: not more than 2.0% of other impurities is found.
[NOTE—Exclude from the total peak area the responses of any minor peaks that are 0.01% or less than that of the main ethopabate peak.
]
Assay—
Mobile phase—
Dissolve 3 g of sodium 1-hexanesulfonate in 1 L of water, and adjust with phosphoric acid to a pH of 2.5. Prepare a filtered and degassed mixture of this solution, methanol, and acetonitrile (450:150:30). Make adjustments if necessary (see
System Suitability under
Chromatography 
621
).
Diluent—
Prepare a mixture of methanol and water (50:50).
Standard preparation—
Prepare a solution of
USP Ethopabate RS in
Diluent having a known concentration of about 0.4 mg per mL. If necessary, filter this solution through a filter having a porosity of 0.5 µm or finer, and use the filtrate as the
Standard preparation. Use this solution on the day prepared.
Assay preparation—
Transfer about 40 mg of Ethopabate, accurately weighed, to a 100-mL volumetric flask, add about 80 mL of Diluent, and dissolve with the aid of sonication. If necessary, filter this solution through a filter having a porosity of 0.5 µm or finer, and use the filtrate as the Assay preparation. Use this solution on the day prepared.
Chromatographic system
(see
Chromatography 621)—The liquid chromatograph is equipped with a 268-nm detector and a 3.9-mm × 30-cm column that contains packing L11 and is maintained at about 40
. The flow rate is about 1 mL per minute. Chromatograph the
Resolution solution, and record the peak responses as directed under
Procedure: the relative retention times are about 0.9 for methyl 4 acetamido-2-hydroxybenzoate (ethopabate related compound A) and 1.0 for ethopabate, the column efficiency is not less than 4000 theoretical plates, the resolution,
R, between the ethopabate related compound A peak and the ethopabate peak is not less than 1.2, and the tailing factor is not more than 1.5. Chromatograph the
Standard preparation, and record the peak responses as directed under
Procedure: the relative standard deviation for replicate injections is not more than 1.0%.
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 of the responses for the major peaks. Calculate the quantity, in mg, of C
12H
15NO
4 in the portion of Ethopabate taken by the formula:
100C(rU / rS),
in which
C is the concentration, in mg per mL, of
USP Ethopabate RS in the
Standard preparation, and
rU and
rS are the ethopabate peak responses obtained from the
Assay preparation and the
Standard preparation, respectively.
;)
