Five implants were kept at 4°C and five were kept at 37°C. Every 7 days, the solution in each container was mixed to ensure homogeneity and 350μL was then removed and placed into a cryo-tube for analysis. Following sample removal, isotonic saline (350μL) was added to the container so the volume was kept consistent. U0126 MEK Samples were collected for a total of 28 weeks and were kept in a −80°C freezer until analysis. Saline samples were analyzed using HPLC with ultraviolet absorption. The system consisted
of a 2695 separations module, a 2487 absorbance selleck chemicals Paclitaxel detector (Waters, Milford, MA, USA). Terbinafine was extracted from saline samples using a hexane extraction and was separated Inhibitors,research,lifescience,medical on a Symmetry Shield C18 (4.6 × 100mm, 5μm) column with a guard column. The mobile phase was a mixture of (A) 20mM phosphoric acid with 0.1% triethylamine adjusted to pH 3.0 and (B) acetonitrile (65:35). The flow rate was 1.1mL/min and the column temperature ambient. Absorbance was measured at 224nm. Standard curves for analysis were
prepared Inhibitors,research,lifescience,medical by fortifying saline with terbinafine to produce a linear concentration range of 5–1500ng/mL. Average recovery for terbinafine was 95% while intra- and interassay variability were less than 10%. The lower limit Inhibitors,research,lifescience,medical of quantification was 5ng/mL. Following HPLC analysis, the amount of terbinafine released by each implant during each interval was calculated. The mean release of terbinafine with standard deviations was calculated for the different temperatures at each time point. Data was tested for normalcy with a Bartlett’s test for inequality of variances. If the values were normally distributed, a t-test was performed to determine if a significant difference in amount of terbinafine released was present at the Inhibitors,research,lifescience,medical two temperatures. If the data was not normally
distributed, a Mann-Whitney/Wilcoxon Inhibitors,research,lifescience,medical two-sample test was used to determine if differences existed. Significance was set at P < 0.05 and analysis was performed with EpiInfo (CDC, Atlanta, GA, USA). 3. Results Samples were collected and analyzed with HPLC for a total of 28 weeks after initial placement into isotonic saline. A sample was not collected during week 23. The mean amount released from the implants at the two different temperatures during the 28 weeks is shown in Table 1/Figure 2. The amount released from the Carfilzomib implants at 37°C was significantly greater than 4°C at the 1 (P < 0.01), 17 (P < 0.01), 26 (P = 0.03), and 28 (P = 0.04) week time points; the amount released from implants at 4°C was greater than 37°C at the 2 (P = 0.04) and 3 (P = 0.02) week time points. The mean amount of terbinafine released weekly across the 28 weeks was approximately 1.7μg at 4°C and 4.3μg at 37°C. Figure 2 Terbinafine impregnated implants were placed into isotonic saline at 4°C (n = 5) and 37°C (n = 5). Samples were collected every 7 days and terbinafine concentrations were determined with HPLC.