ABSTRACT
PURPOSE: To develop an assay to evaluate the bioequivalence of overcoated and marketed montelukast formulations, the former to be used for future blinded clinical studies. METHODS: The method used automated 96-well sample preparation and dual-column HPLC analysis for increased throughput. Regression analysis was performed using the total peak height of montelukast and its photodegradent, a cis-ethenyl geometric isomer. This approach successfully compensated for montelukast's light sensitivity, allowing both clinical specimen handling and bioanalytical laboratory analysis to be conducted without extensive precautions being taken to protect samples from UV light. To ensure a molar equivalent fluorescence response between the cis (Z) and trans (E) isomers, the emission wavelength and detector attenuation were both increased just prior to the elution of the montelukast peak (i.e., the trans isomer), effectively dampening the response of the stronger fluorophore. Plasma proteins were precipitated using acetonitrile, and 50 microl of supernatant was injected onto an HPLC system consisting of two C18 analytical columns connected to a 10-port switching valve. Injections were overlapped on alternating columns allowing twice as many samples to be processed during each analytical run. RESULTS: The calibration curve was linear from 5 to 2000 ng ml(-1). The inter-day and intra-day precision expressed as coefficient of variation (%CV), were 1.1-6.1% and 3.1-6.7%, respectively. The accuracy, reported as percentage bias, was less than or equal to +/-9.1%. The absolute recovery was determined to be 94.3% and 98.1% at 15 and 1500 ng ml(-1), respectively. CONCLUSIONS: This assay represents a rapid, accurate, and sensitive method for the determination of montelukast in human plasma. The method has been successfully used to demonstrate the bioequivalence of the overcoated montelukast formulations to their equivalent marketed tablets.
