Preparation of Study Samples
The vancomycin study samples were thawed, mixed (by vortex for 10 s), and diluted to 2.5 mg/mL in HPLC-grade water (Fisher Scientific, Richmond, British Columbia; lot 092106). A 0.5-mL aliquot of each sample was then diluted with 0.5 mL of internal standard (metronidazole 1 mg/mL). The final theoretical concentrations were 1.25 mg/mL for vancomycin and 0.5 mg/mL for the internal standard. Each sample was passed through a 0.45-^m microfilter before withdrawal of a 10-^L sample for injection onto the column.
The HPLC instrumentation consisted of a delivery pump, an automatic injector system equipped with a 200-mL injector, a Symmetry RP18 3.0 x 100 mm column (Waters Ltd, Mississauga, Ontario; lot W13541F-011) with a Symmetry RP18 3.9 x 20 mm guard column (Waters Ltd; lot 239392051) and a UV detector set at 247 nm. The mobile phase consisted of a 26:74 (v/v) mixture of methanol (Fisher Scientific, Richmond, British Columbia; lot 092106) and a 0.01 mol/L solution of ammonium acetate (Fluka-Sigma- Aldrich, Oakville, Ontario; lot 1386428) at pH 3.0. All solvents were HPLC grade, and all were filtered before use. The flow rate was set at 1.0 mL/min. The assay was developed in the authors’ laboratory on the basis of previous published work. discount drugs canda
Accelerated Degradation of Vancomycin
A solution of vancomycin 25 mg/mL (in water) was prepared, mixed with 1N sodium hydroxide, adjusted to a final vancomycin concentration of 2.5 mg/mL (and 0.1N sodium hydroxide), and boiled for 10 min. Another vancomycin solution, in Ora-Sweet and water, was incubated at 100°C for 18 h. After the samples had cooled to room temperature, a 0.5 mL aliquot from each was combined with an equal volume of internal standard, and the combined solution was filtered and injected onto the column. The chromatograms obtained for the preparations of degraded vancomycin were compared with chromatograms obtained from the standard curve to determine any changes in concentration, retention time, and peak shape.
Statistical AnalysisThe means, SDs, and CVs were calculated for samples analyzed in triplicate or quadruplicate, as applicable. For each study day, the percentage of initial vancomycin concentration remaining was calculated for each sample. Stability was defined as maintenance of at least 90% of the initial vancomycin concentration. The percentage of vancomycin remaining at 75 days was calculated from the concentration at 75 days, as determined by linear regression, and the concentration observed on day 0, according to the following formula: concentration at 75 days ■ concentration on day 0 x 100%. The 95% confidence interval (CI) of the amount remaining on the last study day (day 75) was calculated from the lower limit of the 95% CI of the slope of the curve relating concentration to time, determined by linear regression, via computer analysis, according to the following formula: lower limit of the 95% CI of the concentration at 75 days ■ concentration on day 0 x 100%. If the percentage remaining at 75 days was not at least 90% of the initial concentration, then the time during which the solution would maintain at least 90% of its initial concentration was calculated according to the following formula: 10 ■ lower limit of the 95% CI of the slope of the curve relating concentration to time (determined by linear regression). kamagra soft tablets