Chemicals, Reagents, and Materials
The study was performed between November 2007 and May 2008. Commercially prepared gentamicin 40 mg/mL for injection (Sandoz Canada Inc, Boucherville, Quebec; lot 139819, expiry May 2009), sodium citrate 46.7% for injection (TriCitrasol, Cytasol Laboratories Inc, Braintree, Massachusetts; lot B82T, expiry January 2011), and USP-grade sterile water for injection (Hospira Health Care Corp, Montreal, Quebec; lot 74-337-DK, expiry January 2011) were used to prepare the study solutions, which were stored in polyethylene syringes with luer-lock caps (MedXL Inc, Montreal, Quebec; lot F86331). Gentamicin sulphate USP (PCCA Canada, London, Ontario; lot C104339) and sodium citrate USP (Spectrum, Gardenia, California; lot KC066) were used as reference standards. High-performance liquid chromatography (HPLC)-grade acetonitrile (lot 891499) and phosphoric acid (lot 082037) and analytical-grade hydrochloric acid (lot 4100030) and sodium hydroxide (lot 016045) were purchased from Fisher Scientific (Nepean, Ontario). Hydrogen peroxide USP (6%) was purchased from Pure Standard Products (Edmonton, Alberta; lot 189). Phenylisocyanate (Fluka; lot 1378398), triethylamine (lot 047K1041), and trifluoroacetic acid (Fluka; lot 046710) were purchased from Sigma-Aldrich (St Louis, Missouri). Samples for analysis were stored frozen in cryovials (Nunc, Rochester, New York; lot 089675), and the mobile phase was filtered through Supor-200 polyethersulfone 0.2-^m membranes (Pall, Gelman Laboratories, East Hills, New York; lot 51321).
The chromatographic system was manufactured by Knauer (Berlin, Germany) and consisted of a Wellchrom K-501 pump, a Wellchrom K-2501 variable-wavelength ultraviolet (UV) detector, a Basic-Marathon autosampler, and a Wellchrom HPLC interface box. Eurochrome 2000 operating and data-acquisition software (Knauer, Berlin, Germany) was used. All chromatography was performed at room temperature, and the temperature of the experimental environment was recorded periodically over the study period.
For the gentamicin analysis, an isocratic reversed-phase HPLC method based on that of Kim and others20 was followed, except for use of a 150 x 4.6 mm cyano column with a particle size of 4 ^m (Jones, Hengoed, UK; lot 9650001) instead of a C18 column. The mobile phase consisted of acetonitrile— water-trifluoroacetic acid (400:600:1 v/v/v) at a flow rate of 1.0 mL/min. The injection volume was 20 ^L, detection was by UV absorbance at 240 nm, and the concentration of gentamicin was quantified by means of external standards and peak areas. A stock solution containing gentamicin 400 ^g/mL in water was made fresh daily and was used to prepare the standard solutions, also in water. Because gentamicin has no inherent UV-absorbing properties, phenylisocyanate derivatives of the drug, which absorb strongly at 240 nm, were prepared according to the method described by Kim and others.20,21 Briefly, each sample was diluted with water to a gentamicin concentration of about 250 ^g/mL. A 0.5-mL volume of this solution was then mixed with 0.25 mL of a 0.5% v/v solution of triethylamine in acetonitrile, and 0.25 mL of a 0.5% v/v solution of phenylisocyanate in acetonitrile was added. Kim and others20 reported that the reaction is very rapid at room temperature and the derivatives are stable for at least 24 h at room temperature. The phenylisocyanate and triethylamine reagents are also stable at room temperature for at least 24 h.
For citrate analysis, an isocratic ion-exchange HPLC method, as described in a Supelco application note, was used. The mobile phase consisted of 0.1 % w/v phosphoric acid at a flow rate of 0.5 mL/min. A 300 x 7.8 mm polystyrene divinyl- benzene resin column (Supelcogel C-610H, Supelco Canada, Mississauga, Ontario; lot 59320U) was used. The injection volume was 20 ^L, detection was by UV absorbance at 210 nm, and the concentration of citrate was quantified with external standards. A stock solution containing sodium citrate 3200 ^g/mL in water was made fresh daily and was used to prepare the standard solutions. Samples were prepared by diluting the test solution with water to a concentration of sodium citrate of about 1600 ^g/mL. Viagra Super Active
A typical run for analysis of the stored samples was 14 samples, 1 blank, and 6 standards. For gentamicin, the standards consisted of 2 each at 160, 240, and 320 ^g/mL, representing about 65%, 100%, and 130% of the target concentration; for citrate the standards consisted of 2 each at 800, 1600, and 2400 ^g/mL, representing about 50%, 100%, and 150% of the target concentration. The samples were diluted in water to 250 ^g/mL for gentamicin (1:10) and to 1600 ^g/mL for citrate (1:25). The resulting 21 vials were coded and randomized, phenylisocyanate derivatives were prepared in the case of the gentamicin samples, and the samples were run. Quantitation was achieved by regression analysis of the standards, with the slope and intercept coefficients being used to calculate the concentrations of the samples from their respective peak areas.