Direct Injection Microemulsion HPLC Method for Simultaneous Determination of Morphine, Tramadol and Lornoxicam in Biological Fluids Using Monolithic Column

Objective: A rapid and environmental friendly microemulsion liquid chromatographic method was developed for simultaneous quantification of morphine (MOR), tramadol (TRA) and lornoxicam (LOR) in biological fluids. Methods: Microemulsion used in this study was an aqueous solution containing sodium dodecyl sulfate (0.12 M), n-propanol (10%), ethyl acetate (0.75%), tri-ethyl amine (0.3%), orthophosphoric acid (0.15 %) and the pH was adjusted to 3.0 with orthophosphoric acid. Chromatographic separation was carried out on a monolithic C18 column and a mobile phase flow rate of 1.0 mL min−1 was applied throughout the analysis. The data was monitored using UV-detection mode at a wavelength of 220 nm. Results: Under the optimized conditions, all the studied drugs were well resolved and completely eluted within 6 min. The proposed method was linear over the concentration ranges of 0.5−100, 0.75−125 and 0.25−50 μg mL−1; limits of detection of 0.074, 0.086 and 0.056 μgmL−1 and limits of quantification of 0.21, 0.28 and 0.12 were recorded for TRA, LOR, and MOR, respectively. The developed method was fully validated according to the ICH guidelines. The method was successfully utilized to estimate the tested analytes in plasma and urine samples, which were directly injected into the chromatographic system after suitable dilution with the microemulsion. Conclusion: The developed method is considered to be very efficient to analyze the cited drugs in different biological fluids with low running costs and short analysis time.