Critical development by design of a rugged HPLC-MS/MS method for direct determination of ibuprofen enantiomers in human plasma

• Fully validated LC-MS/MS method for analysis of ibuprofen enantiomers in plasma.
• Enantiomers were chromatographically separated without derivatization.
• Matrix effect investigation showed that LLE yields cleaner extracts than the SPE.
• Confirmation of the absence of R/S interconversion during sample processing.
• The method was successfully applied on volunteers using only 50 μL sample volume.

Development and validation of a HPLC-MS/MS method for direct determination of R- and S-ibuprofen (Ibu) in human plasma without a need of derivatization or other complexities such as postcolumn infusion of solvents or reagents was performed. Critical steps were investigated during method development using experimental design to achieve a reliable and rugged assay. The LC-MS/MS separation of R-Ibu and S-Ibu was obtained on Lux Cellulose chiral column utilizing 0.1% (v/v) acetic acid in mixture of methanol and water (90:10%, v/v) as a mobile phase. Two types of extraction procedure for Ibu and Ketoprofen (internal standard, IS) were optimized using Full factorial 32 design (LLE) and D-Optimal Experimental Design (SPE). Excellent recovery values, 80% (mean) and 95% (mean) for LLE and SPE respectively, were obtained using 50 μL plasma. The matrix effect was assessed for both of the extraction procedures, including hyperlipidaemic and haemolyzed plasma. The extensive investigation of matrix effect showed that LLE yields cleaner extracts than the SPE. The result of the investigation of in vitro interconversion of R-Ibu and S-Ibu showed that it does not occur under the influence of pH, temperature, and in the overall analytical procedure. The validation data, adhered to EMA guideline for validation of bioanalytical methods, showed that the proposed method provides accurate and reproducible results in range of 0.1–50 mg/L with a lower limit of detection of 0.02 mg/L. The applicability of the method was demonstrated through determination of R-Ibu and S-Ibu in human plasma after oral administration of 400 mg rac-Ibu.