Stability of Trisodium Citrate and Gentamicin Solution: METHODS part 2

Validation of Gentamicin Assay

Three solutions of gentamicin standard (40 mg dissolved and made up to 100 mL in water) were prepared. These solutions were further diluted with water to prepare 3 sets of gentamicin solutions with concentrations of 80, 160, 240, 320, and 400 ^g/mL. Phenylisocyanate derivatives were prepared from samples of each of these solutions, and the resulting solutions were analyzed as described above. These data were used to calculate the linearity and range of the method, with linear regression of response or peak area as a function of concentration.

For gentamicin, 240 ^g/mL was selected as the target concentration, and the solutions at 160, 240, and 320 ^g/mL, representing about 66%, 100%, and 130% of the target con­centration, were used to determine accuracy and precision. The coefficients from the previous regression analysis were used to calculate a concentration value for each standard solution, which were expressed as a percentage (calculated concentration divided by theoretical concentration). Accuracy was assessed from the mean of the 9 values and precision from the relative standard deviation (SD) of this mean. On the following day, 3 fresh solutions were prepared and diluted to generate another 3 solutions with concentrations of 160, 240, and 320 ^g/mL. Phenylisocyanate derivatives were prepared and analyzed. The resulting data and the data from the previous day were used to calculate between-day precision.
Forced degradation was used to demonstrate the specifici­ty of the method. One-millilitre volumes of each of 6% H O , 0.1 mmol/L HCl, and 0.1 mmol/L NaOH were mixed individually with 1.0 mL of gentamicin 40 mg/mL in 100-mL flasks and left overnight. A control flask, containing only the gentamicin solution, was also prepared. The next morning, each flask was made up to volume with water, and four 0.5-mL samples were taken from each flask for preparation of phenylisocyanate derivatives and HPLC analysis.
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To determine whether the presence of citrate would interfere with analysis of gentamicin, samples containing gentamicin 2.5 mg/mL and sodium citrate 40 mg/mL were also prepared. For this part of the study, 4 solutions were prepared: gentamicin alone, citrate alone, gentamicin and citrate combined, and a control (distilled water). Phenylisocyanate derivatives were prepared from 4 samples from each flask, and these solutions were analyzed for gentamicin content.

Validation of Citrate Assay

Three solutions of sodium citrate standard (320 mg dissolved and made up to 100 mL in water) were prepared. These solutions were further diluted with water to prepare 3 sets of citrate solutions with concentrations of 400, 800, 1600, 2400, and 3200 ^g/mL. Samples from each of these solutions were analyzed using the HPLC assay described above. These data were used to calculate the linearity and range of the method, with linear regression of response or peak area as a function of concentration. kamagra soft tabs

For sodium citrate, 1600 ^g/mL was selected as the target concentration, and the solutions at 800, 1600, and 2400 pg/mL, representing about 50%, 100%, and 150% of the target concentration, were used to determine accuracy and precision. The coefficients from the previous regression analysis were used to calculate a concentration value for each standard solution, which were expressed as a percentage (calculated concentration divided by theoretical concentration). Accuracy was assessed from the mean of the 9 values and precision from the relative SD of this mean. On the following day, 3 fresh solutions were prepared and diluted to generate another 3 solutions with concentrations of 800, 1600, and 2400 ^g/mL. Samples of these solutions were analyzed. The resulting data and the data from the previous day were used to calculate between-day precision.

Forced degradation was used to demonstrate the specifici­ty of the method. One-millilitre volumes of each of 6% H O ,J 2 2′ 1 mmol/L HCl, and 1 mmol/L NaOH were mixed individual­ly with 0.342 mL of sodium citrate 46.7% in 100-mL flasks and left overnight. Two controls, one consisting of 0.342 mL of citrate and one containing only 1.0 mL of H2O2, were also prepared. The next morning, each flask was made up to volume with water, and four 0.1-mL samples were taken from each flask for HPLC analysis.

To determine whether the presence of gentamicin would interfere with analysis of citrate, samples containing sodium citrate 40 mg/mL and gentamicin 2.5 mg/mL were also prepared. For this part of the study, 4 samples were prepared; citrate alone, gentamicin alone, citrate and gentmicin combined, and a control (distilled water). Four samples from each flask were analyzed for citrate content. viagra jelly

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