Validation of Gentamicin AssayRegression analysis of peak area as a function of concentration for standard solutions of gentamicin with concentrations ranging from 80 to 400 ^g/mL demonstrated linearity, and the coefficient of determination (r2) was 0.9924 (n = 15). The 3 sets of solutions at concentrations of 160, 240, and 320 ^g/mL had an accuracy of 101.0% (n = 9) and a within- day precision, expressed as relative SD, of 2.21% (n = 9). The between-day precision was 2.52% (n = 18). Chromatograms of the phenylisocyanate derivatives prepared from the gentamicin standard showed 3 peaks eluting at 5.3, 5.6, and 6.0 min, which represented gentamicin C^ C , and C2.To quantify the gentamicin, the areas of these 3 peaks were summed. Gentamicin C2 and C are isomers that this assay was unable to resolve; they co-eluted as one peak, so the chromatograms showed 3 rather than 4 peaks. Other authors have quantified gentamicin on the basis of the sum of 3 peaks.
The specificity of the method was established using forced degradation. The samples mixed with 0.1 mmol/L HCl showed no indication of degradation, and the gentamicin concentration remained essentially unchanged, with 99.63% ± 0.29% remaining for the HCl-treated samples and 99.10% ± 0.57% remaining for the control. The samples treated with 0.1 mmol/L NaOH showed some degradation, with 98.21% ± 0.29% remaining and the presence of a new peak at 4.4 min. The samples mixed with H2O2 also showed signs of degradation, with 4 new peaks appearing on the chromatogram. Quantification of the gentamicin in these samples indicated that only 18.11% ± 0.83% of the drug originally present remained. Three of the new peaks eluted early, with retention times of 2.4, 3.7, and 4.4 min, respectively, and the fourth eluted late, at about 8.4 min. The peaks associated with gentamicin eluted between 5 and 6 min. Chromatograms of the control and peroxide-treated samples are presented in Figure 1.
Figure 1. Chromatograms from forced degradation of gentamicin after overnight exposure to 6% hydrogen peroxide. A: Untreated control, with gentamicin peaks at about 5.3, 5.5, and 6.0 min. B: Treated sample with new peaks at about 2.4, 3.7, 4.4, and 8.4 min. The sample is about 80% degraded. AU = absorbance units.
Samples containing gentamicin alone, citrate alone, gentamicin and citrate combined, and a control (distilled water) were analyzed to determine whether the presence of citrate in samples would interfere with analysis of gentamicin. The 2 solutions containing gentamicin yielded similar chro- matograms, with no statistically significant differences between them in terms of peak area (p = 0.92). The sample containing only citrate generated no peaks and was identical with the chro- matogram of the control preparation. These results indicated that the presence of citrate in the sample did not affect the analysis of gentamicin.