Determination of Doxycycline in Human Plasma by Liquid Chromatography-Mass Spectrometry after Liquid-Liquid Extraction and its Application in Human Pharmacokinetics Studies

A sensitive and reproducible liquid chromatography-mass spectrometric (LC-MS) method has been developed and validated for the quantifi cation of doxycycline in human plasma, after liquid-liquid extraction (LLE). Best chromatographic resolution was achieved on a reverse-phase Phenomenex C 18 column using the mobile phase acetonitrile-5mM ammonium acetate (80:20) in isocratic elution with a total run time of 3.5 min. The analyte, doxycycline was detected by using an electrospray ionization mass spectrometer in the selected ion monitoring (SIM) mode. Linear plot was obtained in the concentration range of 0.5-5.0 g/ml (r 2 = 0.998). Lower limit of quantifi cation (LLOQ) was found to be 100 ng/ml in 500l of plasma. Average recovery of the analyte was found to range from 96.08 to 104.60% in plasma at the concentrations of 1.0, 3.0 and 5.0 g/ml. Intra-and inter-day relative standard deviation was found to be 0.13-0.74 and 0.11-0.34 respectively. The present method was successfully applied in the pharmacokinetic study of doxycycline in human plasma.