Development and validation of RP-HPLC method for the determination of doxycycline hyclate in spiked human urine and pharmaceuticals
An accurate, sensitive, precise, rapid, isocratic reversed phase HPLC (RP-HPLC) method for the determination of doxycycline hyclate in bulk drug and in tablets, and also in spiked human urine, was developed and validated. The best separation was achieved on a 250 mm × 4.0 mm i.d, 5.0 µm particle size C 8 reversed phase thermo column with acetonitrile-potassium dihydrogenorthophosphate buffer (pH 4.0), 40:60 (v/v) as mobile phase at a flow rate of 1.0 mL/min. UV detection was performed at 325 nm at ambient column temperature (25 °C). The method was linear over the concentration range of 30-300 µg/ mL (r=0.9994) with limits of detection and quantification of 0.02 and 0.1 µg/ mL, respectively. The drug was subjected to oxidation, acid and base hydrolysis, photolysis and heat as stress conditions. Degradation products were found interfering with the assay of doxycycline hyclate only in oxidation and base hydrolysis, therefore the method can be regarded as indicating stability in the case of acid hydrolysis, photolysis and heat stress conditions. The method was applied for determination of doxycycline hyclate in pharmaceuticals and spiked human urine with excellent recoveries. The method can be used for the quality control of doxycycline hyclate.