stability

Assay Development and Validation

Figure 1 is a typical chromatogram obtained using the newly developed HPLC method to simultaneously analyse IR and LV. With this separation method, IR eluted at 14.7 min and the ring-opened carboxylate, produced by the addition of either sodium hydroxide or sodium hypochlorite (pH 10), eluted at 13.0 min. Sodium hypochlorite also produces other degradation products (Figure 1A). The addition of increasing amounts of sodium hydroxide to an IR sample increases the formation of the ring-opened carboxylate as the pH increases from 4 to 8 (Figure 2). Above a pH of 8, the ring-opened carboxylate product is the predominate species in solution. Addition of hydrochloric acid to such a sample (e.g., a sample at pH 10.14 through the addition of sodium hydroxide), results in the re-formation of IR within 30 min (when the pH dropped to less than 5). LV and its primary degradation product eluted at 5.7 min and 2.5 min, respectively. The separation of the degradation products from both IR and LV, as well as the similarity of the UV spectra for peaks associated with IR and LV to authentic standards of each, indicates that the method is specific for both IR and LV.

Duplicate analysis of a 0.05 mg/mL IR quality control sample containing LV demonstrated that concentrations were estimated with less than 3% deviation between the observed and known concentrations of IR. The coefficient of variation on duplicate analysis of standards averaged less than 1.5% within days and less than 3% between days. These analyses indicated that the IR concentrations were measured accurately and reproducibly and that differences of 10% or more could be confidently detected with acceptable error rates. Viagra Online Canadian Pharmacy

Figure 1. A Chromatogram of a mixture

Figure 1. A: Chromatogram of a mixture of expired 10 mg/mL leucovorin and a sample of 0.2 mg/mL irinotecan with 10 pL of 0.5% sodium hypochlorite added. B: Chromatogram of a sample from the stability study (3.60 mg/mL leucovorin and 0.32 mg/mL irinotecan) 24 h after mixing, showing only undegraded leucovorin and irinotecan with about 22% as the ring-opened carboxylate.

Duplicate analysis of a 0.20 mg/mL LV quality control sample containing IR demonstrated that concentrations were estimated with less than 0.7% deviation between the observed and known concentrations of LV. The coefficient of variation on duplicate analysis of standards averaged less than 1.6% within days and less than 0.9% between days.
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Figure 2. The pH dependency of irinotecan degradation

Figure 2. The pH dependency of irinotecan degradation. Eight samples ranging in pH from 4.12 to 10.14 (using oncentrations of sodium hydroxide) were prepared, and the percentage of irinotecan remaining after 30 min was measured. Above pH 7, irinotecan degrades quickly. At pH 6.27, only 3% of the initial irinotecan concentration was lost, and at pH 6.79 only 5% of the irinotecan was lost at 30 min. These results are similar to those of Fassberg and Stell2 and Dodds and others.

Based on the separation of IR and LV from each other and from their degradation products and thedemonstration that concentrations of both IR and LV could be measured accurately and reproducibly (as indicated by within-day and between-day coefficients of variation), this method can be considered stability-indicating for both IR and LV.
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