Assay Development and Validation

HPLC Method for Simultaneous Analysis of Irinotecan and Leucovorin A high-performance liquid chromatography (HPLC) method was developed that allowed simultaneous ana lyses of IR and LV and ensured separation of the 2 drugs from each other and their degradation products. The mobile phase consisted of a mixture of 10% acetonitrile and 90% 0.05 mol/L potassium phosphate monobasic (pH adjusted to 4.1), which was gradually changed to 35% acetonitrile and 65% 0.05 mol/L potassium phosphate monobasic (pH adjusted to 4.1) over 10 min. The pH of the mixed solution was adjusted to 4.1 with 1 mol/L phosphoric acid. Each sample was analyzed for 30 min. The mobile phase was pumped at 1 mL/min through a 15 cm x 4.6 mm reverse-phase C18, 3-pm column (Supelcosil ABZ Plus, Supelco, Mississauga, Ontario) using a 600E system controller and pump (Waters Corp, Mississauga, Ontario). IR and LV were detected at 244 nm and 240 nm, respectively, using a scanning variable-wavelength detector (Spectra System UV6000LP, Thermo Separation Products, Freemont, California); the chromatograms were recorded directly into a computer database using ChromQuest software (Thermo Separation Products). The assay was developed using samples of IR, LV, and their respective degradation products to ensure accurate measurement of IR and LV stability. The UV spectral purity of the IR and LV peaks was compared between degraded and undegraded samples to demonstrate the specificity of the method. This method is unique and differs from previously published methods of IR analysis because of differences in type of column, mobile-phase constituents, temperature, method or wavelength of detection, and ability to separate IR from LV.

Accelerated Degradation of Irinotecan

During development of the HPLC method to separate IR and LV from their degradation products, the pH dependency of IR degradation and formation of the ring-opened carboxylate was evaluated, as was the formation of other irreversible degradation products. The ring-opened carboxylate product of IR was generated by the addition of sodium hydroxide to solutions of IR. A stock solution of IR was prepared by dissolving an accurately weighed quantity of approximately 10 mg of IR hydrochloride trihydrate powder (CPT-11, class A primary standard, Pharmacia Corporation, Mississauga, Ontario; lot C, expiry September 1, 2004) in 25 mL of distilled water to achieve a final IR concentration of 0.4 mg/mL. Eight separate samples with pH ranging from 4.12 to 10.14 were prepared by mixing 1 mL of the 0.4 mg/mL IR solution and 1 mL of sodium hydroxide ranging from 0.002 mol/L to 0.016 mol/L. The pH was determined immediately after mixing, and the concentration of IR was determined 30 min after addition of the sodium hydroxide. HPLC was used to monitor separation of IR and the primary degradation product, the ring-opened carboxylate. To the sample of pH 10.14, 1 mL of 0.016 mol/L hydrochloric acid was added. The pH was determined immediately after mixing and the concentration of IR was determined 30 min later.
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Given that sodium hydroxide produces only the ring-opened product, a second set of experiments was performed, in which 10-pL aliquots of various sodium hypochlorite solutions ranging in concentration from 0.25% to 1% were added to 1-mL samples of 0.2 mg/mL IR (prepared by further dilution of the 0.4 mg/mL stock solution). These samples were immediately chromatographed, and the concentration of IR was determined.

Chromatograms from all solutions prepared above were inspected for the appearance of additional peaks, changes in retention time, and changes in peak shape. The ultraviolet (UV) spectral purity (200-365 nm, 6-nm bandwidth, determined with a UV3000LP deuterium lamp, Thermo Separation Products) of the leading edge, middle, and tail of the IR peak in a chromatogram of an authentic undegraded sample and the sample taken at time 0 were also compared. Samples of degraded IR were used to develop an HPLC method for simultaneous analysis of IR, LV, and their degradation products. canadian cialis