A fast on-line micro-column sample pretreatment with a HPLC column switching technique and large volume sample injection for determination of non-steroidal anti-inflammatory drugs in human serum

Abstract

A fully automated HPLC column-switching method with fast sample pretreatment on a short micro-column was developed for sensitive and quantitative determination of non-steroidal anti-inflammatory drugs (ketoprofen, ibuprofen, sodium diclofenac and flurbiprofen) in human serum with UV detection. The serum samples were diluted 3 : 1, with 2% acetic acid in acetonitrile, and centrifuged at 4200g for 10 min. 200 μL of supernatant was injected directly into the HPLC system. An on-line sample clean-up was achieved on a pretreatment micro-column LiChrospher® RP-18 ADS 25 mm × 2.0 mm, 25 μm (Merck) with a washing mobile phase (acetonitrile : 0.085% phosphoric acid; 10 : 90) at a flow rate of 2.5 mL min⁻¹. The valve switch to an analytical column was set at the 7^th min in a back-flush mode. Separation of all antiflogistics and internal standard butylparaben was performed using a Chromolith Performance RP 18e 100 × 4.6 mm column (Merck) and a mobile phase consisting of 0.085% phosphoric acid and acetonitrile in gradient mode. The column temperature was set at 45 °C and a flow rate of 1.0 mL min⁻¹ and a UV detector at 210 nm. Standard and matrix calibrations were carried out under the optimal chromatographic conditions in the linearity range 7–2000 ng mL⁻¹ with good linearity – correlation coefficients for all compounds are in the range 0.9956–0.9999, n ≥ 6. Limits of quantification were in the range 7–56 ng mL⁻¹. The accuracy of the method defined as a mean recovery was in the range 81.97–118.40%. Intra-day method precision was measured at three concentration levels and relative standard deviations were in the range 0.07–1.92%. The developed method showed high sample throughput, fully automated sample clean-up and analysis in time less than 17 min.