HPLC method development for determination of doxycycline in human seminal fluid

• We report development and validation of HPLC-UV method for doxycycline quantification in human seminal fluid.
• Tetracycline was used as an internal standard.
• Sample preparation was based on precipitation of the proteins using perchloric acid.
• The method showed good precision (RSD < 7.0%), accuracy (recovery > 80%) and correlation coefficient (r = 0.998).
• The method was applied to doxycycline determination in seminal fluids of male subjects treated with doxycycline-hyclate.

The present paper reports the development and validation of an analytical method for doxycycline quantification in human seminal fluid by HPLC with UV detection. The separation of doxycycline was achieved at 40 °C on a reversed-phase C18 column using isocratic elution. The mobile phase consisted of acetonitrile (A) and water buffered at pH 2.5 with a concentrated orthophosphoric acid (B) in the volume ratio of 20:80 (v/v), respectively. The detection was performed at 350 nm. As an internal standard (IS), tetracycline was used. The proposed method involves the extraction of doxycycline from seminal fluid based on acidic precipitation of the proteins using perchloric acid. The method showed good intra- and inter-day precisions (RSD < 7.0%), good accuracy (recovery for doxycycline > 80%), and high correlation coefficient (r = 0.998) for standards subjected to the entire procedure. The detection and quantification limits were 0.087 μg/ml and 0.264 μg/ml. The developed method was used to analyze doxycycline in the seminal fluids obtained from male subjects who were treated with doxycycline-hyclate. The mean doxycycline concentrations of 0.89 ± 0.07 μg/ml and 0.45 ± 0.26 μg/ml were detected in seminal fluid after 6 h and 12 h, respectively. This is the first study reporting extraction and HPLC determination of doxycycline in this complex sample and can be very useful in support of clinical and pharmacokinetic studies on this antibiotic.