Determination of sodium hyaluronate in pharmaceutical formulations by HPLC-UV

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 °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.

Keywords: Chromophore; Derivatization; Hyaluronic acid; Size exclusion chromatography; Sodium hyaluronate.

Fig. 1

(A) Mobile phase: 0.01 M phosphoric acid, pH adjusted to 3.0 using 10% potassium hydroxide, flow rate 0.50 mL/min, column, ultrahydrogel, 300 mm×7.8 mm, 1000 Å and detector wavelength set at 205 nm. (B) Mobile phase: 0.1 M ammonium dihyrogen phosphate, flow rate 0.50 mL/min, column, ultrahydrogel, 300 mm×7.8 mm, 1000 Å and detector wavelength set at 205 nm.

Fig. 2

Effect of pH on retention time, detection response (peak area) and efficiency (as shown as plate number N/column) of sodium hyaluronate. Column, BioSep SEC S2000, 300 mm×7.8 mm, mobile phase: 0.05 M potassium hydrogen phosphate, pH adjusted to shown below using potassium hydroxide (10% solution), flow rate of l.0 mL/min, wavelength set at 205 nm and injection volume 10 μL.

Fig. 3

Effect of buffer on detection response (peak area) and capacity factor of sodium hyaluronate. Column, BioSep SEC S2000, 300 mm×7.8 mm, mobile phase: buffer (potassium hydrogen phosphate) at varied concentration as shown in below, pH adjusted to 7.0 using potassium hydroxide (10% solution), flow rate of l.0 mL/min,wavelength set at 205 nm and injection volume 10 μL.

Fig. 4

A typical HPLC chromatogram of sodium hyaluronate. Mobile phase: 0.05 M potassium dihydrogen phosphate, pH adjusted to 7.0 using potassium hydroxide (10% solution). Column, BioSep SEC S2000, 300 mm×7.8 mm, flow rate of l.0 mL/min, wavelength was set at 205 nm and injection volume 10 μL.