The chromatographic purity of pegaptanib sodium was determined by HPLC using both anion exchange and reversed-phase methods. For anion exchange analysis, an HPLC system (A1100; Agilent, Palo Alto, CA) was used, operated at 80°C with an analytical column (DNA Pac PA-100 column; 4.6 × 250 mm; Dionex Corp., Sunnyvale, CA) and a variable-wavelength UV detector with collection at 260 nm. Mobile phase A consisted of 1% acetonitrile and 10 mM Tris (pH 8), and mobile phase B consisted of 1% acetonitrile, 10 mM Tris, 1 M ammonium chloride (pH 8). A multistep gradient (0%–40% B in 21 minutes, 54% B at 30 minutes, and 80% B at 43 minutes) and a flow rate of 1.5 mL/min were used. For reversed-phase analysis, a separation module (Alliance 2695; Waters, Milford, MA) was used, operated at 30°C with a PRP-1 column (4.6 × 250 mm, 10 μm; Hamilton Co., Reno, NV), and a photodiode array detector (2996; Waters) with data extracted at 260 nm. Mobile phase A was an aqueous solution of triethylammonium acetate, whereas mobile phase B was 100% acetonitrile. Gradient conditions (5%–70% B in 40 minutes) and a flow rate of 1 mL/min were used in the analysis. All chromatograms were acquired and processed by the system software (Empower; Waters). The concentration of pegaptanib sodium was determined based on a calibration curve from 25 to 500 μg/mL (R 2 = 0.9974).