Rapid and sensitive UPLC-MS/MS method for the determination of etodolac in small-volume rat plasma: Application to rat real samples

A rapid, simple, and sensitive UPLC-MS/MS method was developed and validated for the determination of etodolac in rat plasma using flurbiprofen as an internal standard (IS). Etodolac and IS were detected using electrospray ionization in negative ion multiple reaction monitoring mode by monitoring the transitions (precursor to product) m/z 286.2212.1 and 243.2199.2, respectively. The developed new extraction procedure for etodolac in rat plasma does not contain more than one stage. Chromatographic separation was performed on reverse phase C-18 column with a gradient mobile phase, which consisted of methanol for solvent A and 5mM ammonium formate in water solvent B percentages varying at a flow rate of 0.4mL/min. The LLOQ for etodolac was determined as 1ng/mL. The calibration curve for etodolac was linear (r>0.996 for plasma) well within the range of 1-5000ng/mL. Quantification of etodolac in rat plasma is possible in a short chromatographic run time (1.91min) with this method. The proposed method was fully validated by determining specificity, linearity, LLOQ, precision and accuracy, recovery, matrix effect, and stability. The validated method was successfully applied to plasma samples obtained from rats.