Stability of Trisodium Citrate and Gentamicin Solution: RESULTS part 2

Validation of Citrate Assay

Regression analysis of peak area as a function of concen­tration for standard solutions of sodium citrate with concentra­tions ranging from 400 to 3200 ^g/mL demonstrated linearity, and the coefficient of determination (r2) was 0.9996 (n = 15). The 3 sets of solutions at concentrations 800, 1600, and 2400 ^g/mL had an accuracy of 99.61% (n = 9) and a within- day precision, expressed as relative SD, of 1.06% (n = 9). The between-day precision was 0.88% (n = 18). Chromatograms of citrate showed one peak eluting at 11.0 min.

Forced degradation was used to demonstrate the specificity of the method. The samples mixed with either 1 mmol/L HCl or 1 mmol/L NaOH showed no indication of degradation, and the citrate concentration remained essentially unchanged, with 99.92% ± 0.08% remaining for the HCl-treated samples, 99.97% ± 0.14% remaining for the NaOH-treated samples, and 100.0% ± 0.39% remaining for the control. The sample mixed with H2O2 did show signs of degradation. The content remaining dropped to 91.98% ± 0.21% and a new peak appeared at 14.5 min, but it was too small to be integrated. The peak for citrate had a retention time of 11.1 min, and the peak for H2O2 had a retention time of 15.7 min. Chromatograms of the control and peroxide-treated samples are presented in Figure 2.
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Figure 2. Chromatograms from forced degradation

Figure 2. Chromatograms from forced degradation of citrate after overnight exposure to 6% hydrogen peroxide. A: Untreated control with citrate peak at about 11.1 min. B: Treated sample with new peak at about 14.5 min. The hydrogen peroxide eluted at about 15.7 min. The sample is about 10% degraded. AU = absorbance units.

Samples containing citrate alone, gentamicin alone, both citrate and gentamicin, and a control (distilled water) were analyzed to determine whether the presence of gentamicin in samples would interfere with analysis of citrate. The 2 solutions containing citrate yielded similar chromatograms, with no statistically significant difference between them in terms of peak area (p = 0.44). The sample containing only gentamicin generated no peaks under the conditions of the assay. These results indicated that the presence of gentamicin in the sample did not affect the analysis of citrate. Cialis Jelly

Stability Study

Four samples of each formulation were withdrawn at intervals over 112 days, stored frozen at -20°C, and then analyzed for concentration of gentamicin and citrate using the validated HPLC methods described above. Analysis of all samples was complete by study day 126, so the longest storage time in the freezer was 126 days (for the samples obtained on day 0); the median storage time for the frozen samples was 98 days. All of the solutions retained at least 99% of the labelled concentration over time (Table 1). None of the chromatograms for either gentamicin or citrate showed the presence of unexpected peaks or peaks associated with the breakdown products seen in the forced degradation studies.

Table 1. Concentrations of Gentamicin and Citrate for the 3 Study Formulations as a Function of Time, Expressed as Percentage of Label Content

Formulation C

Formulation A

Formulation B

Study Day

(Gentamicin)

(Citrate)

Gentamicin

Citrate


0


101.3±
2.27


103.3±
0.64


102.0±
0.75


102.8±
2.15


7


102.8±
0.87


104.3±
0.29


99.9±
1.64


101.5±
1.55


14


101.4±
3.19


104.6±
2.61


100.6±
0.13


102.2±
1.03


28


101.1±
1.94


103.9±
1.54


99.6 ± 0.60


101.8± 0.56


42


100.9±
1.30


104.2±
0.94


100.9±
1.73


102.6±
1.92


56


101.8± 2.30


104.4±
0.84


100.4±
2.08


100.8±
1.54


112


99.9±
1.13


103.4±
0.59


100.1±
1.86


101.6± 0.90


*Values are means± standard deviation(n= 4).

For each formulation, the concentration of drug present in the 4 samples obtained at each time point were compared by ANOVA. For formulation A (gentamicin 2.5 mg/mL), there was no significant change in gentamicin concentration over the 112-day study period (p = 0.60). Similarly, for formulation B (sodium citrate 40 mg/mL), there was no significant change in citrate concentration over the same period (p = 0.57). For for­mulation C (gentamicin and citrate combined), there was no change in the concentration of either gentamicin (p = 0.34) or citrate (p = 0.055) over the study period.

None of the formulations showed a significant change in either gentamicin or citrate concentration over the 112-day period (Table 1). This result was confirmed with regression analysis of concentration as a function of time: in all cases, the slope of the regression line was not significantly different from zero (p > 0.05). From the linear regression analysis of the concentration—time data of formulation C, we calculated that 99.97% of the labelled amount of gentamicin and 101.3% of the labelled amount of citrate remained at day 112. The lower limit of the 95% CI also indicated that more than 98.17% of the gentamicin and more than 99.57% of the citrate remained on day 112. canada viagra online

The pH of each formulation did not change significantly from baseline to the end of the observation period (p > 0.05 for all comparisons). The initial and final pH values were 4.86 ± 0.06 and 4.97 ± 0.10, respectively, for formulation A; 6.56 ± 0.05 and 6.60 ± 0.02, respectively, for formulation B; and 6.47 ± 0.03 and 6.50 ± 0.02, respectively, for formulation C. At the end of the study period, samples of each formulation remained clear and colourless by visual inspection. The storage temperature was 23.9 ± 0.9°C (n = 10) over the course of the study.

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