ABSTRACT
A liquid chromatography (HPLC) method with UV detection was developed for determination of sodium hyaluronate in pharmaceutical formulation. Sodium hyaluronate is a polymer of disaccharides, composed of d-glucuronic acid and d-N-acetylglucosamine, linked via alternating ß-1, 4 and ß-1, 3 glycosidic bonds. Being a polymer compound it lacks a UV absorbing chromophore. In the absence of a UV absorbing chromophore and highly polar nature of compound, the analysis becomes a major challenge. To overcome these problems a novel method for the determination of sodium hyaluronate was developed and validated based on size exclusion liquid chromatography (SEC) with UV detection. An isocratic mobile phase consisting of buffer 0.05 M potassium dihydrogen phosphate, pH adjusted to 7.0 using potassium hydroxide (10%) was used. Chromatography was carried out at 25 °C on a BioSep SEC S2000, 300 mm×7.8 mm column. The detection was carried out using variable wavelength UV-vis detector set at 205 nm. The compounds were eluted isocratically at a steady flow rate of 1.0 mL/min. Sodium hyaluronate retention time was about 4.9 min with an asymmetry factor of 1.93. A calibration curve was obtained from 1 to 38 g/mL (r>0.9998). Within-day % RSD was 1.0 and between-day % RSD was 1.10. Specificity/selectivity experiments revealed the absence of interference from excipients, recovery from spiked samples for sodium hyaluronate was 99-102. The developed method was applied to the determination of sodium hyaluronate in pharmaceutical drug substance and product.
ABSTRACT
A liquid chromatography (HPLC) method with UV detection was developed for determination of sodium hyaluronate in pharmaceutical formulation. Sodium hyaluronate is a polymer of disaccharides, composed of D-glucuronic acid and D-N-acetylglucosamine, linked via alternating β-1, 4 and β-1, 3 glycosidic bonds. Being a polymer compound it lacks a UV absorbing chromophore. In the absence of a UV absorbing chromophore and highly polar nature of compound, the analysis becomes a major challenge. To overcome these problems a novel method for the determination of sodium hyaluronate was developed and validated based on size exclusion liquid chromatography (SEC) with UV detection. An isocratic mobile phase consisting of buffer 0.05 M potassium dihydrogen phosphate, pH adjusted to 7.0 using potassium hydroxide (10%) was used. Chromatography was carried out at 25 1C on a BioSep SEC S2000, 300 mm ? 7.8 mm column. The detection was carried out using variable wavelength UV-vis detector set at 205 nm. The compounds were eluted isocratically at a steady flow rate of 1.0 mL/min. Sodium hyaluronate retention time was about 4.9 min with an asymmetry factor of 1.93. A calibration curve was obtained from 1 to 38 g/mL (r40.9998). Within-day%RSD was 1.0 and between-day%RSD was 1.10. Specificity/selectivity experiments revealed the absence of interference from excipients, recovery from spiked samples for sodium hyaluronate was 99-102. The developed method was applied to the determination of sodium hyaluronate in pharmaceutical drug substance and product.
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.
