Development and validation of stability indicating HPLC–PDA method for the simultaneous determination of benzoyl peroxide and tretinoin in topical dosage form

Materials and chemicals

Benzoyl peroxide (BPO) was obtained from Cambrex Karlskoga, Sweden while Tretinoin (TRN) was obtained from Chongqing huapont shengchem pharmaceuticals, China. The Twyneo (Benzoyl peroxide 3% and Tretinoin 0.1%) cream was purchased online. All the commercial grade excipients used for the method validation study were kindly gifted by Horizon Healthcare, Pakistan. We used Honeywell-made HPLC-grade acetonitrile, methanol, and water. Potassium dihydrogen phosphate, orthophosphoric acid, and all other chemicals were used of analytical grade, manufactured by Sigma Aldrich. The mobile phase, standards, and samples were filtered through 0.22 µm polyamide membrane filters.

Chromatographic system

Shimadzu HPLC quaternary gradient LC-10AD equipped with coolant autosampler, column oven, and photodiode array multi-channel detector was used for the whole study. Chromatographic separation was accomplished by the reverse phase isocratic method using SHIMPAC® octadecyl silane C18 column, 4.6 mm internal diameter, 15 cm length, and 5 µm particle size. The final mobile phase contains a 25% volume of 0.01 M solution of potassium phosphate buffer in distilled water adjusted pH 2.5 with orthophosphoric acid and 75% volume of acetonitrile. The flow rate of the mobile phase was 1.5 mL/min, injection volume 20 µL, autosampler coolant temperature 4 °C, and the column temperature was set at 30 °C.

Preparation of standard solution

The stock standard solution of TRN drug substance was prepared by transferring 50.0 mg in a 100 mL amber-colored or low actinic volumetric flask, dissolved in methanol by using ultra sonicator, and dilute to volume. The final standard solution was prepared by transferring 30.0 mg of BPO in another 100 mL amber-colored volumetric flask, dissolved in 40 mL methanol, adding 2 mL of TRN stock solution, and mix well then diluted to volume with methanol. Hence the target concentrations were for BPO (0.3 mg/mL) and TRN (0.01 mg/mL).

Preparation of sample solution

Mix the contents of three creams, and transfer 1.0 g accurately weighed quantity of cream (nominally equivalent to 30 mg of BPO and 1 mg of TRN) into 100 mL amber-colored volumetric flask, dissolve the active ingredients by using ultra sonicator and dilute to volume. Finally, filter this solution through 0.22 µm polyamide membrane filter before injection.

The stability of the solutions was evaluated for 24 h at intervals of 0, 6, 12, and 24 h while the standard and sample solutions were both maintained in the refrigerator at 2 °C to 8 °C, protected from light.

Preparation of placebo solution

The placebo solution was prepared by transferring the excipients (anhydrous citric acid; 2.5 mg, butylated hydroxytoluene; 1 mg, carbomer homopolymer type C; 1 mg, cetrimonium chloride; 0.5 mg, cetyl alcohol; 70 mg, cyclomethicone; 10 mg, edetate disodium; 1 mg, glycerin; 20 mg, imidurea; 1 mg, (S)-lactic acid; 1 mg, macrogol stearate; 25 mg, mono and di-glycerides; 10 mg, polyquaternium-7; 10 mg, silicon dioxide; 1 mg, squalane; 20 mg, tetraethyl orthosilicate; 1.5 mg, white wax; 20 mg, and purified water; 765 mg) into 100 mL beaker. Add methanol, stir by magnetic stirrer, and heat at 50 °C for 15 min then transferred to 100 mL volumetric flask make up the volume to mark with methanol. This solution was sonicated for 20 min with intermittent shaking then filtered through 0.22 µm polyamide membrane filter before injection.

Preparation of solutions for stress studies

Stress study or Forced degradation involved the submission of samples under various environmental conditions like acid, alkali, oxidative, photolytic, and thermal with humidity to verify what are the degradation products. The control standard solution was prepared as described above. 5 mL of 1 N solution of hydrochloric acid for acidic stress, 5 mL of 1 N solution of sodium hydroxide for alkali stress, and 5 mL of 3% v/v solution of hydrogen peroxide for oxidative stress was separately added in separate samples and separate placebo solutions, then diluted with methanol to volume. All samples were kept at room temperature for 24 h. Photolytic stress was applied by keeping the sample in Laminar flow hood under UV light for 24 h. Thermal and humidity stress was applied at two different conditions for 24 h, one is at room temperature and other is 40 °C temperature with 75% relative humidity in climatic chamber.

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