2.4. Forced Degradation Test

Forced degradation tests using various decomposition pathways were carried out as follows: Mannitol was dissolved in water for injection (WFI) at a concentration of 100 mg/mL. The drug stock solution was prepared by dissolving pemetrexed disodium hemipentahydrate in mannitol solution at a concentration of 100 mg/mL. Each test solution was prepared by following the method described below for each degradation condition and being filtered immediately through 0.2 µm Nalgene filter units (Nalgene, Rochester, NY, USA) for sterilization, followed by filling 20 mL of test solution in a glass vial and sealing with a cap. The vials containing test solution were stored at specific conditions, considering various decomposition mechanisms, and the samples were withdrawn at predetermined time intervals, and then they were diluted suitably and analyzed by HPLC. Each experiment was performed in quadruplicates.

The pH of the stock solution was adjusted to 7.0 ± 0.1 by the addition of 0.1 N of NaOH, and the stock solution was then diluted to a final drug concentration of 25 mg/mL, using WFI. The prepared test sample vials were secondary packed, using a box to block the light, and then stored at 60 °C for 4 weeks.

The test solution was prepared by dilution of the stock solution to a final drug concentration of 25 mg/mL, using 0.01 N of HCl. The prepared test sample vials were secondary packed, using a box to block the light, and then stored at 25 °C for a day.

The test solution was prepared by dilution of the stock solution to a final drug concentration of 25 mg/mL, using 1 N of NaOH. The prepared test sample vials were secondary packed, using a box to block the light, and then stored at 25 °C for a day.

A 6% solution of hydrogen peroxide, a strong oxidizing agent, in WFI, was added to the two times diluted stock solution, at a volume/volume ratio of 1:1. The final concentration of hydrogen peroxide and pemetrexed was 3% and 25 mg/mL, respectively. The prepared test sample vials were secondary packed, using a box to block the light, and then stored at 25 °C for a day.

The test solution was prepared by dilution of the stock solution to a final drug concentration of 25 mg/mL, using WFI, and then the vials containing the test solution samples were stored in a photostability chamber equipped with one ultraviolet lamp with 22 W UV power, at 25 °C, for 1 day, in accordance with the Guidance for Industry Q1B Photostability Testing of New Drug Substances and Products by ICH [28].