Medium: water; 1000 mL.

Apparatus 2: 50 rpm.

Time: 60 minutes.

Tolerances— Not less than 75% (Q) of the labeled amounts of butalbital (C11H16N2O3), caffeine (C8H10N4O2), and aspirin (C9H8O4) is dissolved in 60 minutes.

Solvent— Add 1 mL of phosphoric acid to 1000 mL of methanol, and mix.

Standard preparation— Prepare a solution of USP Salicylic Acid RS in Solvent having a known concentration of about 0.0012 mg per mL. Use this solution promptly.

Test preparation— [note—Perform this test on the same day the powder is removed from the Capsules.] Transfer an accurately weighed portion of the powder remaining from the preparation of the Assay preparation in the Assay, equivalent to about 65 mg of aspirin, to a 200-mL flask, add 100.0 mL of Solvent, and shake for about 1 minute. Promptly filter a portion of this solution, discarding the first 15 mL of the filtrate, and use the clear filtrate as the Test preparation. Use this solution within 20 minutes after the addition of the Solvent.

Procedure— Immediately place the cell containing the solution under test in the cell compartment of a spectrophotofluorimeter, and allow to equilibrate for 2 minutes. Concomitantly measure the intensities of the fluorescence of the Test preparation and the Standard preparation at 444 nm, using an excitation wavelength of 305 nm. Calculate the percentage of salicylic acid in the portion of Capsules taken by the formula: in which C is the concentration, in mg per mL, of USP Salicylic Acid RS in the Standard preparation; a is the quantity, in mg, of aspirin in the portion of Capsules taken to prepare the Test preparation, based on the labeled amount; and IU and IS are the fluorescence intensity readings obtained from the Test preparation and the Standard preparation, respectively. [note—If the intensity of the Test preparation greatly exceeds that of the Standard preparation, immediately transfer 5.0 mL of the Test preparation to a 50-mL volumetric flask, dilute with Solvent to volume, and mix. Immediately determine the intensity of this solution, and calculate the percentage of salicylic acid in the portion of Capsules taken by the formula: Not more than 2.5% is found.

0.01 M Phosphate buffer— Dissolve 1.361 g of monobasic potassium phosphate in 1000 mL of water, and mix.

Mobile phase— Prepare a suitable filtered and degassed mixture of 0.01 M Phosphate buffer and methanol (550:450), and adjust with phosphoric acid to a pH of 3.9. Make adjustments if necessary (see System Suitability under Chromatography 621).

pH 2.5 Buffer— Prepare a mixture of 0.01 M Phosphate buffer and methanol (550:450), and adjust with phosphoric acid to a pH of 2.5 ± 0.05.

Aspirin standard stock solution— Dissolve an accurately weighed quantity of USP Aspirin RS in pH 2.5 Buffer to obtain a solution having a known concentration of about 1.6 mg per mL, sonicating and shaking the solution, if necessary, to achieve complete dissolution. Use this solution within 24 hours.

Standard preparation— Dissolve accurately weighed quantities of USP Butalbital RS and USP Caffeine RS quantitatively in Aspirin standard stock solution to obtain a solution having known concentrations of about 1.6J mg of USP Butalbital RS and 1.6J¢ mg of USP Caffeine RS per mL, J and J¢ being the ratios of the respective labeled amounts, in mg, of butalbital and caffeine to the labeled amount, in mg, of aspirin per Capsule, sonicating and shaking the solution, if necessary, to achieve complete dissolution. Use this solution within 24 hours.

Salicylic acid solution— Prepare a solution of salicylic acid in pH 2.5 Buffer containing about 0.1 mg per mL. Pass this solution through a suitable filter of 0.5-µm or finer porosity.

Assay preparation— Remove, as completely as possible, the contents of not fewer than 20 Capsules. Transfer an accurately weighed portion of the powder, equivalent to about 325 mg of aspirin, to a 200-mL volumetric flask, dilute with pH 2.5 Buffer to volume, sonicate for about 30 minutes, and mix. Pass a portion of this solution through a suitable filter of 0.5-µm or finer porosity, and use the filtrate as the Assay preparation. Use this solution within 24 hours. [note—Reserve the remaining portion of the powder for the test for Limit of free salicylic acid.]

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a detector set at the wavelength of the isosbestic point of aspirin and salicylic acid at about 277 nm and a 210-nm detector, and a 3.9-mm × 30-cm column that contains packing L1 and is maintained at 35 ± 1. The flow rate is about 1 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the relative retention times are about 0.45 for caffeine, 0.6 for aspirin, and 1.0 for butalbital; the resolution, R, between the caffeine and aspirin peaks is not less than 2.0; the column efficiency determined from the butalbital peak is not less than 2000 theoretical plates; and the relative standard deviations of the caffeine, aspirin, and butalbital responses for replicate injections are not more than 2.0%. Inject 10 µL of the Salicylic acid solution, and record the peak response as directed for Procedure: the salicylic acid peak has the same retention time as that of the aspirin peak obtained in the chromatogram of the Standard preparation. [note—If the retention time of the salicylic acid peak differs from that of the aspirin peak, adjust the pH of the Mobile phase with 0.2 N potassium hydroxide or 1 M phosphoric acid so that the salicylic acid peak has the same retention time as that of the aspirin peak. The retention time of the salicylic acid peak decreases about 0.3 minute for each 0.1 pH increase. The retention time of the aspirin peak is essentially unaffected by such pH adjustments.]

Procedure— Separately inject equal volumes (about 10 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, using the 277-nm detector to record the caffeine and aspirin peak responses and the 210-nm detector to record the butalbital peak responses, and measure the areas for the major peaks. Calculate the quantities, in mg, of caffeine (C8H10N4O2), aspirin (C9H8O4), and butalbital (C11H16 N2O3) in the portion of Capsules taken by the same formula: in which C is the concentration, in mg per mL, of the appropriate USP Reference Standard in the Standard preparation; and rU and rS are the peak responses of the corresponding analyte obtained from the Assay preparation and the Standard preparation, respectively. Correct the amount of aspirin obtained for the amount of salicylic acid present by the formula: in which A is the quantity, in mg, of aspirin in the portion of Capsules taken to prepare the Assay preparation; and F is the percentage of salicylic acid obtained in the test for Limit of free salicylic acid.