Chromatographic Determination of Doxycycline Using Three New Reversed Phase Columns

Abstract

In the present study, a rapid, simple and economical reversed phase HPLC method has been developed for separation of Doxycycline from its therapeutically active impurities (Oxytetracycline and Methacycline). The samples were separated isocratically on three different new reversed phase C18 columns: Ascentis Express C18 column 10 cm x 4.6 mm, 2.7µm (Sigma Aldrich), Luna 2.5 µm C18 column and Onxy Monolithic C18 column 50 mm x 4.6 mm (Phenomenex). Isocratic potassium dihydrogen phosphate (0.005 M) pH=5 containing 0.2 g/L of sodium edetate (solvent A) and acetonitrile (solvent B) 80:20, v/v was used as a mobile phase at a flow rate of 0.5 mL/min under ambient conditions using UV detection at 254 nm. The Doxycycline was separated from its impurities Oxycycline and Methacycline in less than 10 min using fused core column and in less than 5 min using monolithic and luna columns. The linearity of the method was calculated to R² = 0.9998, 0.9999 and 0.9999 for luna, monolithic and fused core columns respectively over a concentration range of 0.07- 0.3 mg/mL. The limit of detection (LOD) was found to be 0.041, 0.030 and 0.012 µg/mL for luna, monolithic and fused core columns respectively. The limit of quantitation (LOQ) was found to be 0.120, 0.080 and 0.040µg/mL for luna, monolithic and fused core columns respectively. Additionally, the developed HPLC method showed an acceptable value of repeatability and intermediate precision. Furthermore, the asymmetrical factor for Doxycycline peak, resolution and number of theoretical plate for the three different columns were calculated. The simplicity and validity of the method makes it highly reliable and suitable for analysis of Doxycycline