Stability of Vancomycin 25 mg/mL in Ora-Sweet: METHODS

4 Jan

high-pressure liquid chromatography

Preparation of Vancomycin

Stock solutions of vancomycin 25 mg/mL were prepared by reconstituting commercially available vancomycin for injection (10 g/vial) (Pharmaceutical Partners of Canada, Richmond Hill, Ontario; lot 204369, expiry December 2009) with sterile water for injection (Baxter Corporation, Missis- sauga, Ontario; lot W9F15AO, expiry January 2011) and then diluting with a mixture of equal volumes of Ora-Sweet vehicle (Paddock Laboratories Inc, Minneapolis, Minnesota; lot 9085961, expiry February 2012) and distilled water (Ripple FX Water Inc, New Westminster, British Columbia; lot 9147102, expiry February 27, 2010). Portions of this preparation were transferred to fourteen 15-mL opaque blue polyethylene unit- dose cups with aluminum seal (Medical Packaging Inc, Ringoes, New Jersey; lot FD15MB0705) and six 50-mL amber polyvinyl chloride (recycle code 3) prescription bottles (Richards Packaging Inc, RIGO Products Division, Gloucester, Ontario). The volume dispensed was 5 mL into each unit-dose cup and 25 mL into each bottle. Half of the unit-dose cups and half of the plastic bottles were kept at room temperature (25°C) and the remainder of the containers were stored in the refrigerator (4°C).

The physical characteristics of the solutions were evaluated qualitatively at the time of preparation and at 15, 30, 40, 50, 63, and 75 days. As samples were collected throughout the study period, one individual (D.D.) measured the pH and visually examined the solutions for changes in colour against white and black backgrounds and to check for formation of precipitate. The same person (D.D.) also tasted the solutions. At each time point, 3 aliquots from one of the unit-dose cups at each temperature and 1 aliquot from each plastic bottle at each temperature were collected for determination of pH. The pH meter (model 8000, VWR, Mississauga, Ontario) was calibrated with commercially available standards at the begin­ning of each testing session. Immediately after the physical observations, three 1.5-mL samples from the selected unit-dose cup at each storage temperature were transferred into 2-mL polypropylene vials (Nalgene Company, Rochester, New York; lot 735276294). Similarly, one 1.5-mL sample from every bottle at both temperatures was transferred into a 2-mL polypropylene vial. As such, there were 3 samples for each combination of temperature and type of storage container on each study day. All vials were immediately stored at —85°C until analysis by a validated, stability-indicating HPLC method with ultraviolet (UV) detection.
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Preparation of Stocks, Standards, and Standard Curve

Standards were prepared as follows. Vancomycin (Sigma- Aldrich, Oakville, Ontario; lot 1418285) was reconstituted to a concentration of 10 mg/mL in water and was further diluted with water to concentrations of 8, 6, 4, 3, 2, 1, and 0.5 mg/mL to allow construction of the standard curve. The internal stan­dard metronidazole (Sigma-Aldrich, Oakville, Ontario; lot 095K0693) was prepared by dilution in water to a concentra­tion of 1 mg/mL. The standards were prepared by combining 0.5 mL of each stock solution and 0.5 mL of metronidazole 1 mg/mL. The final concentrations of vancomycin in the stan­dard samples injected onto the chromatograph were 4, 3, 2, 1.5, 1, 0.5, and 0.25 mg/mL. The final concentration of internal standard (metronidazole) was 0.5 mg/mL. To prevent injection of impurities onto the column, each standard was passed through a 0.45-^m microfilter before injection (Acrodisc GHP syringe filter, Gelman, Ann Arbor, Michigan; lot 21692572).

A 7-point calibration curve was prepared, with a blank (water) at the beginning of each run to prevent carry-over from one run to the next. The range of this calibration curve encompassed the diluted test concentration of vancomycin (1.25 mg/mL, as described in the next section). The calibration curve was generated by least-squares regression of the peak area ratio of vancomycin to internal standard and the concentration of each vancomycin standard. The precision of the assay was evaluated by intraday and interday validation methods. Intraday variability was determined by running stocks with concentrations of 2.5, 1.25, 0.62, and 0.31 mg/mL in quadru­plicate throughout a single day, whereas interday variability was determined by running stocks with the same concentrations (as in the testing for intraday variability) in quadruplicate daily for 4 days. The means, standard deviations (SDs), coefficients of variation (CVs), and accuracy were calculated. Acceptable limits were defined a priori as up to 10% (for coefficients of variation) and 90% or above (for accuracy). cialis professional