Chemical Stability of Irinotecan and Leucovorin

To specifically evaluate the stability of IR and LV, HPLC was used to determine the concentrations in each experimental solution at time 0 and at 0.5 h, 1 h, and 24 h after mixing. Mean concentrations (3 determinations at each observation time) of IR and LV for solutions stored in glass, PVC, and PABs are reported in Tables 2, 3, and 4, respectively. These data show that the concentration of IR declined in all 18 experiments over the 24-h experimental period. In 5 of the 6 treatment scenarios (1a, 1b, 2a, 2b, and 3b), the mean concentration of IR was greater than 98% of the original concentration at 0.5 h and was greater than 91% of the original concentration at 24 h. The sixth treatment scenario (3a), with initial projected concentrations of IR 0.32 mg/mL and LV 3.60 mg/mL, had a more rapid degradation of IR. At 0.5 h, the mean concentration of IR was between 91.57% and 95.09%, but continued to decline (to 76.30% to 78.34% of the initial concentration) until 24 h. In these solutions, the only degradation product that was evident was the ring-opened carboxylate (Figure 3).

Analyses of these data by multiple linear regression demonstrated that there was no effect of container type (p = 0.62) or initial IR concentration (p = 0.80) on the rate of IR degradation, but there was a significant effect of ini­tial LV concentration (p < 0.0001) and time (p < 0.001) on the rate of IR degradation. The significant effects of both time and initial LV concentration were entirely due to the loss of IR in the experimental solution containing 0.32 mg/mL IR and 3.60 mg/mL LV, in which the IR concentration degraded to 76.30% to 78.34% of the initial concentration at 24 h. It is noteworthy that the degradation of IR was not linear; IR concentration declined by approximately 12% on average in the first hour of incubation and then by a further approximately 12% over the next 23 h.
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Table 3. Observed Mean Concentration (± Standard Deviation) of Irinotecan and Leucovorin in 5% Dextrose in Water Solutions stored in Polyvinyl Chloride


Scenario 1a


Scenario 1b


Scenario 2a


Scenario 2b


Scenario 3a


Scenario 3b


Characteristic


(IR 0.56,


(IR 0.53,


(IR 0.59,


(IR 0.56,


(IR 0.32,


(IR 0.30,


LV 0.94)


LV 0.74)


LV 0.66)


LV 0.27)


LV 3.60)


LV 0.68)


Irinotecan


Initial concentration (mg/mL)


0.65±0.03


0.55±0.01


0.47±0.12


0.54±0.00


0.31±0.00


0.34±0.00


%
remaining


At 0.5
h


99.08±0.41


98.70±0.18


99.15±0.78


99.40±0.25


91.76±0.28


99.45±1.26


At 1
h


97.50±0.27


98.10±0.19


99.42±0.89


98.82±0.04


88.69±0.67


97.03±0.34


At 24
h


92.00±0.50


92.58±0.32


96.17±0.23


97.33±0.26


78.34±0.81


95.08±0.32


Leucovorin


Initial concentration (mg/mL)


0.87±0.03


0.70±0.02


0.68±0.02


0.26±0.00


3.55±0.01


0.66±0.01


% remaining


At 0.5
h


101.02±1.79


99.67±2.25


99.65±1.21


99.77±0.10


103.39±2.87


98.98±1.32


At 1 h


100.52±0.86


100.94±0.69


100.62±0.79


100.05±0.19


101.83±0.39


99.78±0.58


At 24 h


100.36±1.32


100.03±1.49


99.42±1.06


99.14±0.26


101.57±0.51


100.69±5.20

Analysis of LV concentrations by multiple linear regression demonstrated that there was no trend for a decrease in concentration and so there was no significant effect of container type (p = 0.12), initial IR concentration (p = 0.80), initial LV concentration (p = 0.54), or time (p = 0.34) over the 24-h period. The mean concentration of LV at 24 h was greater than 96% of the original concentration in all treatment scenarios.
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Table 4. Observed Mean Concentration (± Standard Deviation) of Irinotecan and Leucovorin in 5% Dextrose in Water Solutions Stored in Polypropylene-Polyethylene Copolymer Bags


Scenario 1a


Scenario 1b


Scenario 2a


Scenario 2b


Scenario 3a


Scenario 3b


Characteristic


(IR 0.56,


(IR 0.53,


(IR 0.59,


(IR 0.56,


(IR 0.32,


(IR 0.30,


LV 0.94)


LV 0.74)


LV 0.66)


LV 0.27)


LV 3.60)


LV 0.68)


Irinotecan


Initial concentration (mg/mL)


0.65±0.03


0.55±0.01


0.47±0.12


0.54±0.00


0.31±0.00


0.34±0.00


% remaining


At 0.5
h


99.16±0.56


99.08±0.13


99.74±0.78


99.52±0.15


91.57±1.22


98.92±0.12


At 1 h


97.18±0.56


98.15±0.47


99.09±0.31


98.63±0.54


88.56±0.79


97.10±0.71


At 24 h


91.88±0.41


92.28±0.42


96.61±1.14


97.21±0.60


76.30±0.69


94.55±0.59


Leucovorin


Initial concentration (mg/mL)


0.95±0.01


0.72±0.01


0.65±0.02


0.23±0.01


3.67±0.03


0.67±0.01


% remaining


At 0.5
h


99.81±0.37


99.56±0.86


100.16±2.46


106.81±5.77


96.82±2.80


100.50±1.37


At 1 h


100.27±0.24


101.90±0.69


100.45±2.06


107.08±7.19


99.67±2.34


100.12±0.56


At 24 h


101.17±1.28


100.30±0.88


102.38±2.00


97.66±1.66


99.86±0.62


100.24±0.03

The rapid degradation of IR observed in treatment scenario 3a was likely due to the higher pH of the solution created by high concentration of LV (Table 2). The pH dependency of IR stability was evaluated in a separate experiment undertaken as part of developing the new HPLC method. These data (Figure 2) indicate that at a pH above 7.82, IR converts rapidly to the ring- opened carboxylate. At pH 7.82, 14.9% of the initial IR concentration was lost in 30 min, whereas at pH 8.31, 74.9% of the initial IR concentration was lost in 30 min. The highest pH for any of the experimental solutions was observed for treatment scenario 3a (pH 6.50). In the pH dependency study, 3.0% of the initial IR concentration was lost in 30 min at pH 6.27 and 5.2% of the initial IR concentration was lost at pH 6.79. These results are in agreement with the percent remaining in all experiments conducted with the mixture of 0.32 mg/mL IR and 3.60 mg/L LV, in which between 4.9% and 8.4% of the initial IR concentration was lost in 0.5 h at pH 6.50. viagra jelly

Figure 3. A Chromatogram of a freshly prepared

Figure 3. A: Chromatogram of a freshly prepared mixture of leucovorin (3.60 mg/mL) and irinotecan (0.32 mg/mL). B: Chromatogram of the same sample, 24 h later. The retention times were 5.7 min for leucovorin, 14.7 min for irinotecan, and 13 min for the irinotecan degradation product.