Validation of HPLC Method for Determination of Doxycycline hyclate in turkey serum

Abstract

An accurate, sensitive and reproducible high performance liquid chromatographic (HPLC) method for the quantitation of doxycycline in turkey serum has beendeveloped and validated. The doxycycline standard was eluted from a C18 column (4.6 mm i.d., 250 mm, 5 µm particle size) at 15 ⁰C with a mobile phase consisting of 5% acetic acid : methanol : acetonitrile (55:20:25, v/v/v). A UV detector set at 347 nm was used to monitor the effluent. Retention time of doxycycline was approximately at 4 min and the run time was 10 min. The column temperature adjusted at 15 ⁰C with a flow rate 1ml/min and injection volume 25 µl. Doxycycline was extracted from serum by ACN. Linearity was confirmed in the concentration range 0.1-2 µg/ml, had regression coefficients (R2) 0.9999 for doxycycline quantification in turkey serum. The analytical conditions gave a recoveries in a range of 97.57% to 99.83%. The limit of detection (LOD) was 0.035 µg/ mL and limit of quantification (LOQ) was 0.108 µg/ml. The relative standard deviation (RSD %) was less than 2% in all cases. The developed method was accurate, precise and rapid for simultaneous estimation of doxycycline in turkey serum samples.