Stability of Norepinephrine Solutions: METHODS part 2
On study day 0, 4 mL of a 1 mg/mL solution of norepinephrine (norepinephrine bitartrate injection USP, Sandoz Canada Inc; lot 149812, expiry April 2010) was withdrawn from a vial and further diluted in a 50-mL polyvinyl chloride (PVC) minibag of 5% D5W (Baxter Corporation, Mississauga, Ontario; lot P230821, expiry June 2010) to prepare 16 minibags with nominal concentrations of 64.5 mg/L (after consideration of 4 mL of drug volume and assuming 8 mL of overfill). This procedure was repeated to prepare 16 samples containing 64.5 mg/L of norepinephrine in 50-mL PVC minibags of 0.9% NS (Hospira, Saint-Laurent, Quebec; lot 70 011 JT, expiry April 1, 2010).
For each diluent (D5W and NS), 8 minibags were stored at 4°C and 8 were stored at room temperature (23°C). For the bags stored at each temperature, half (n = 4) were protected from ambient fluorescent room light (using Amber Zip Bags, 127 mm x 203 mm, 3 mil; thebagcompany Inc, West Springfield, Massachusetts; obtained through PharmaSystems Inc, Markham, Ontario), and the other 4 were exposed to ambient fluorescent room light. On study days 0, 1, 2, 3, 4, 8, 9, 10, 11, 14, 18, 21, 23, 25, 28, 30, 36, 42, and 61, the concentration of nor- epinephrine was determined in duplicate, as described below, and visual inspection was completed.
On each analysis day, 1-mL samples were drawn from each PVC minibag, and 10-pL samples were injected directly into the chromatographic system, in duplicate, without further dilution or preparation.
Data Reduction and Statistical Analysis
After determination of the coefficient of variation for replicate determinations of concentration for an assay, a power calculation demonstrated that 2 replicates were required to ensure that the analytical method could distinguish between concentrations that differed by at least 10%. Mean results from different days for each test were compared statistically to determine if there was an association between the observed result and time (linear regression). Analysis of variance (ANOVA) was used to test differences in degradation rate between combinations of temperature, diluent, and concentration. The 5% level was used as the a priori cutoff for significance. Concentrations were considered “acceptable” or “within acceptable limits” if the concentration was greater than 90% of the initial (day 0) concentration and the amount found on that day, with 95% confidence, exceeded 90% of the initial concentration.