High Performance Liquid Chromatographic Method for Determination of Doxycycline in Human Plasma and its Application in Pharmacokinetic Studies

An accurate, sensitive and reproducible high performance liquid chromatographic (HPLC) method for the quantitation of doxycycline in plasma has been developed and validated. The drug and the internal standard were eluted from a (X-Bondapak C18 column (3.9 mm x 150 mm, I.D., 5 jxm particle size) at room temperature with a mobile phase consisting of acetonitrile and water (28:72, % v/v). The flow rate was 0.8 ml/min. A UV detector set at 346 nm was used to monitor the effluent. Each analysis required no longer than 6 min. Quantitation was achieved by measurement of the peak area ratio of the drug to the internal standard. The limit of detection was 10.0 ng/ml and the limit of quantification of doxycycline in plasma was 0.10 (ig/ml. The standard curve ranged from 0.1 to 2.5 |ig/ml. The intraday coefficient of variation (C.V., %) ranged from 1.444 to 3.016%, and interday (C.V., %) from 1.572 to 2.705% at four different concentrations. The relative recoveries ranged from 98.43 to 105.13% and the absolute recoveries ranged from 54.08 to 62.56% at four different concentrations. Stability studies showed that doxycycline is stable for at least 4 weeks in plasma after freezing at - 20 °C. The method was applied for the determination of the pharmacokinetic parameters of doxycycline after oral administration of 100 mg capsules of two commercially available formulations to 6 human volunteers