title: Critical development by design of a rugged HPLC-MS/MS method for direct determination of ibuprofen enantiomers in human plasma.
abstract: 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 3(2) design (LLE) and D-Optimal Experimental Design (SPE). Excellent recovery values, 80% (mean) and 95% (mean) for LLE and SPE respectively, were obtained using 50μL 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 0.1-50mg/L with a lower limit of detection of 0.02mg/L. The applicability of the method was demonstrated through determination of R-Ibu and S-Ibu in human plasma after oral administration of 400mg rac-Ibu.
authors.affiliation: Faculty of Pharmacy, University Ss Cyril and Methodius, 1000 Skopje, Macedonia. Electronic address: natalijan@ff.ukim.edu.mk.
authors.forename: Natalija
authors.initials: N
authors.lastname: Nakov
authors.affiliation: Faculty of Pharmacy, University Ss Cyril and Methodius, 1000 Skopje, Macedonia.
authors.forename: Rumenka
authors.initials: R
authors.lastname: Petkovska
authors.affiliation: Faculty of Pharmacy, University Ss Cyril and Methodius, 1000 Skopje, Macedonia.
authors.forename: Liljana
authors.initials: L
authors.lastname: Ugrinova
authors.affiliation: Faculty of Pharmacy, University Ss Cyril and Methodius, 1000 Skopje, Macedonia.
authors.forename: Zoran
authors.initials: Z
authors.lastname: Kavrakovski
authors.affiliation: Faculty of Pharmacy, University Ss Cyril and Methodius, 1000 Skopje, Macedonia.
authors.forename: Aneta
authors.initials: A
authors.lastname: Dimitrovska
authors.affiliation: Alexander University Hospital, Faculty of Medicine, Medical University, 1431 Sofia, Bulgaria.
authors.forename: Dobrin
authors.initials: D
authors.lastname: Svinarov
mesh_terms: D000328:Adult; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D007052:Ibuprofen / Q000097:blood* / Q000737:chemistry* / Q000493:pharmacokinetics; D057230:Limit of Detection; D016014:Linear Models; D059625:Liquid-Liquid Extraction; D015203:Reproducibility of Results; D052616:Solid Phase Extraction; D013237:Stereoisomerism; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D007052:Ibuprofen
keywords: Experimental design; Ibuprofen enantiomers; LC-MS/MS; Matrix effect; Sample preparation; in vitro Chiral interconversion