A simple and rapid high-performance liquid chromatographic method for determining tobramycin in pharmaceutical formulations by direct UV detection

Abstract

Background: Tobramycin, an aminoglycoside antibiotic, is a polar pharmaceutical compound which lacks a UV absorbing chromophore. Due to the absence of a UV absorbing chromophore and high polar nature of this antibiotic, the analysis of such compounds becomes a major challenge.

Objective: To overcome these problems, a novel method for the determination aminoglycoside tobramycin was developed and validated based on reversed-phase high-performance liquid chromatography (RP-HPLC) with UV detector.

Materials and methods: An isocratic mobile phase consists of buffer 0.05 M diammonium hydrogen phosphate, pH adjusted to 10.0 using tetramethyl ammonium hydroxide. Chromatography was carried out at 25°C on a Purosphere RP-8e, 250 mm × 4.6 mm, 5mm. The detection was carried out using variable wavelength UV-Vis detector set at 210 nm. The compounds were eluted isocratically at a steady flow rate of 1.0 mL/min.

Result and discussion: Tobramycin retention time was about 9.0 min with an asymmetry factor of 1.4. A logarithmic calibration curve was obtained from 0.47 to 0.71 mg/mL (r > 0.9998). Within-day %RSD was 0.29 (n = 6, 0.60 mg/mL) and between-day %RSD was 0.54 Specificity/ selectivity experiments revealed the absence of interference from excipients, recovery from spiked samples was between 99.0-100.0 percent.

Conclusions: A HPLC method based on UV detection has been developed and validated for determination of tobramycin from ophthalmic solution. The method is simple, rapid, specific, accurate (error 0.80%), precise (RSD <2.0%) and linear (r2=0.9998). The described method is suitable for routine analysis and quality control of ophthalmic solution containing tobramycin.

Keywords: Amino glycoside; derivatization; isocratic; reverse phase chromatography; tobramycin.

Figure 1

(a) Chromatogram of tobramycin showing fi rst investigation. Chromatographic column: BioSep SEC-S2000, 300 mm × 7.8 mm, mobile phase: 0.05 M potassium dihydrogen phosphate, pH adjusted to 7.0 using potassium hydroxide, flow rate 1 ml/min. Detector wavelength: 205 nm. (b) Chromatogram of tobramycin showing second investigation. Chromatographic column: Purosphere RP-8e, 250 mm × 4.6 mm, 5μm, mobile phase: 0.05 M potassium dihydrogen phosphate, pH adjusted to 7.0 using potassium hydroxide ,flow rate 1 ml/min. Detector wavelength: 205 nm

Figure 2

Effect of pH on (peak area) and effi ciency (shown as plate number N/column) and capacity factor of tobramycin column: Purosphere RP-8e, 250 × 4.6 mm, 5μm, mobile phase: 0.05 M diammonium hydrogen phosphate, pH adjusted to 10 using tetramethyl ammonium hydroxide

Figure 3

A typical chromatogram of test sample by proposed methods column: Purosphere RP-8e, 250 × 4.6 mm, 5μm, mobile phase: 0.05 M diammonium hydrogen phosphate, pH adjusted to 10.0 using tetramethyl ammonium hydroxide, flow rate 1 ml/min, λ set at 210 nm