Sodium hydroxide solution— Immediately before use, dissolve 4.3 g of sodium hydroxide in carbon dioxide-free water to make 1000 mL.

Procedure— To 2.00 g of Hypromellose Acetate Succinate, previously dried, add Sodium hydroxide solution to make 100.0 g, insert a stopper into the vessel, and dissolve by constant shaking for 30 minutes. Adjust the temperature of the solution to 20 ± 0.1, and determine the viscosity in a suitable viscosimeter, as directed for Procedure for Cellulose Derivatives under Viscosity 911. Its viscosity is not less than 80% and not more than 120% of that stated on the label.

Phosphoric acid solution— Transfer 1.0 mL of 1.25 M phosphoric acid into a 50-mL volumetric flask, and dilute with water to volume.

0.02 M Phosphate buffer— Dissolve 5.44 g of monobasic potassium phosphate in 2 L of water.

Diluent— Adjust 0.02 M Phosphate buffer with 1 N sodium hydroxide to a pH of 7.5.

Succinic acid stock solution— Accurately weigh about 130 mg of succinic acid into a 100-mL volumetric flask. Add about 50 mL of water, and swirl the contents until the succinic acid is fully dissolved. Fill the flask with water to volume.

Mobile phase— Adjust the 0.02 M Phosphate buffer to a pH of 2.8 by the dropwise addition of 6 M phosphoric acid. Pass through a 0.22-µm nylon filter.

Standard solution— Transfer 4.0 mL of the Acetic acid stock solution into a 25-mL volumetric flask. To the same flask, transfer 4.0 mL of the Succinic acid stock solution, dilute with Mobile phase to volume, and mix. Prepare this solution in duplicate.

Test solution— Accurately weigh about 102 mg of Hypromellose Acetate Succinate into a glass vial. Transfer 4.0 mL of Diluent to the vial, and stir the content for 2 hours. Then, transfer 4.0 mL of the Phosphoric acid solution to the same vial to bring the pH of the Test solution to 3 or less. Invert the vial several times to ensure complete mixing, centrifuge, and use the clear supernatant as the Test solution.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 215-nm detector and a 4.6-mm × 15-cm column that contains 5-µm packing L1. The column temperature is maintained between 20 and 30. The flow rate is about 1 mL per minute, and the run time is about 15 minutes. Chromatograph the first preparation of the Standard solution, and record the peak responses as directed for Procedure: the column efficiency, determined from the succinic acid peak, is not less than 8000 theoretical plates; the tailing factor of this peak is between 0.9 and 1.5; and the relative standard deviation for six replicate injections is not more than 2.0% for each peak. Chromatograph the second preparation of the Standard solution: the difference in peak areas between the two standard solutions for both acetic and succinic acid peaks does not exceed 2%. [NOTE—After each run sequence, the column should be flushed first by 50% water and 50% acetonitrile for 60 minutes and then by 100% methanol for 60 minutes. The column should be stored in 100% methanol.]

Procedure— Separately inject equal volumes (about 10 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the peak areas corresponding to acetic and succinic acids. Calculate the percentage of free acetic acid, Afree, in the portion of Hypromellose Acetate Succinate taken by the formula: in which WA is the weight of glacial acetic acid, in mg, used to prepare the Acetic acid stock solution; W is the weight of Hypromellose Acetate Succinate, in mg, used to prepare the Test solution; and rUA and rSA are the peak responses for acetic acid obtained from the Test solution and the Standard solution, respectively. Calculate the percentage of free succinic acid, Sfree, in the portion of Hypromellose Acetate Succinate taken by the formula: in which WS is the weight of succinic acid, in mg, used to prepare the Succinic acid stock solution; rUS and rSS are the peak responses for succinic acid obtained from the Test solution and the Standard solution, respectively; and W is as defined above: the sum of free acetic acid and free succinic acid found does not exceed 1.0%.

Phosphoric acid solution, Acetic acid stock solution, Succinic acid stock solution, Mobile phase, Standard solution, and Chromatographic system— Proceed as directed in the test for Limit of free acetic and succinic acids.

Test solution— Accurately weigh about 12.4 mg of Hypromellose Acetate Succinate into a glass vial. Transfer 4.0 mL of 1.0 N sodium hydroxide to the vial, and stir the solution for 4 hours. Then, add 4.0 mL of 1.25 M phosphoric acid to the same vial to bring the pH of the solution to 3 or less. Invert the test sample solution vial several times to ensure complete mixing, and pass through a 0.22-µm filter. Use the clear filtrate as the Test solution.

Procedure— Separately inject equal volumes (about 10 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the peak areas corresponding to acetic and succinic acids. Calculate the percentage of acetic acid, A, in the portion of Hypromellose Acetate Succinate taken by the formula: in which WA is the weight of acetic acid, in mg, used to prepare the Acetic acid stock solution; WU is the weight of Hypromellose Acetate Succinate, in mg, used to prepare the Test solution; and rUA and rSA are the peak responses for acetic acid obtained from the Test solution and the Standard solution, respectively. Calculate the percentage of acetyl groups (–COCH3) in the portion of Hydroxypropyl Methylcellulose Acetate Succinate taken by the formula: in which Afree is the percentage of free acetic acid, as determined in the test for Limit of free acetic and succinic acids; and A is as defined above. Calculate the percentage of succinic acid, S, in the portion of Hypromellose Acetate Succinate taken by the formula: in which WS is the weight of succinic acid, in mg, used to prepare the Succinic acid stock solution; WU is as defined above; and rUS and rSS are the peak responses for succinic acid obtained from the Test solution and the Standard solution, respectively. Calculate the percentage of succinoyl groups (–COC2H4COOH) in the portion of Hydroxypropyl Methylcellulose Acetate Succinate taken by the formula: in which S is as defined above; and Sfree is the percentage of free succinic acid, as determined in the test for Limit of free acetic and succinic acids.

Hydriodic acid— Use a reagent having a specific gravity of at least 1.69, equivalent to 55% hydrogen iodide.

Solution A— Prepare a mixture of water and methanol (90:10).

Solution B— Prepare a mixture of methanol and water (85:15).

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).

Standard stock solution— Transfer 2 mL of o-xylene into a stoppered, 10-mL volumetric flask, place the flask on a balance, and tare. Add about 200 µL of methyl iodide, insert the stopper into the flask, and accurately weigh: the weight of methyl iodide is about 350 mg. Tare the flask again, add about 34 µL of isopropyl iodide, and accurately weigh the flask: the recorded weight of isopropyl iodide is about 50 mg. Dilute with o-xylene to volume, and mix.

Standard solution— Transfer about 85 mg of adipic acid into an 8-mL vial (or other suitable container), add 2 mL of Hydriodic acid, and add 2.0 mL of the Standard stock solution. Shake and allow the phases to separate. Carefully transfer approximately 1.5 mL of the o-xylene (top) layer to a small vial, making sure that the bottom aqueous layer is not disturbed. Transfer 1.0 mL of this solution into a 10-mL volumetric flask, and dilute with methanol to volume. [NOTE—This solution is stable for 8 hours at 5.]

Test solution— Accurately weigh about 65 mg of Hypromellose Acetate Succinate into a 5-mL reaction vial, and add 2.0 mL of o-xylene and about 100 mg of adipic acid. Add 2.0 mL of Hydriodic acid, and close the vial tightly with a cap. [Caution—Use a cap that has a top safety relief valve, such as a Minniert valve, to prevent accidental explosion of the vial under high pressure when heated. ] Weigh the vial before heating, and place the vial into a heating block at 150. Shake the vial after 5 minutes and after 30 minutes of heating. Remove the vial from the heating block after 1 hour of heating, and cool. Weigh the vial. If the weight loss is greater than 10 mg, discard the mixture, and prepare another reaction solution. Carefully transfer approximately 1.5 mL of the top o-xylene layer into a small glass vial, making sure that the bottom aqueous layer is not disturbed. Transfer 1.0 mL of this solution into a 10-mL volumetric flask, and dilute with methanol to volume. [NOTE—This solution is stable for 8 hours at 5.]

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm × 15-cm column that contains 5-µm packing L1. The column temperature is maintained at 30. The flow rate is about 1 mL per minute. The chromatograph is programmed as follows. [NOTE—These gradient elution times are established on an HPLC system with a dwell volume of approximately 2.0 mL. The injection time can be adjusted relative to the start of a run to accommodate the change in dwell volume from one HPLC system to another to achieve the separation described.] Chromatograph the Standard solution, and record the peak responses as directed for Procedure: the column efficiency, determined from the methyl iodide peak, is not less than 10,000 theoretical plates; the tailing factor of this peak is between 0.9 and 1.5; and the relative standard deviation for replicate injections is not more than 2.0% for each peak.

Procedure— Separately inject equal volumes (about 10 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the peak areas corresponding to methyl iodide and isopropyl iodide. Calculate the percentage of methoxy groups (–OCH3) in the portion of Hypromellose Acetate Succinate taken by the formula: in which WM is the weight of methyl iodide, in mg, used to prepare the Standard stock solution; W is the weight of Hypromellose Acetate Succinate, in mg, used to prepare the Test solution; and rUM and rSM are the peak responses for methyl iodide obtained from the Test solution and the Standard solution, respectively. Calculate the percentage of 2-hydroxypropoxy groups (–OCH2CHOHCH3) in the portion of Hypromellose Acetate Succinate taken by the formula: in which WI is the weight of isopropyl iodide, in mg, used to prepare the Standard stock solution; rUI and rSI are the peak responses for isopropyl iodide obtained from the Test solution and the Standard solution, respectively; and W is as defined above.

(Official January 1, 2007)