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A total on-line analysis of a target protein from a plasma sample was made using a selective immunoextraction step coupled on-line to an immobilized enzymatic reactor (IMER) for the protein digestion followed by LC-MS/MS analysis. For the development of this device, cytochrome c was chosen as model protein due to its well-known sequence. An immunosorbent (IS) based on the covalent immobilization of anti-cytochrome c antibodies on a solid support was made and an immunoextraction procedure was carefully developed to assess a selective extraction of the target protein from plasma. For the first time, IS was easily coupled on-line with a laboratory-made IMER based on pepsin. The whole on-line device (IS-IMER-LC-MS/MS) allowed the quantification of cytochrome c from 8.5pmol to 1.7nmol in buffer medium. Finally, this device was applied to the analysis of only 85pmol of cytochrome c from plasma with a RSD value lower than 10% (n=3)., (2009 Elsevier B.V. All rights reserved.)

A molecularly imprinted polymer (MIP) using zidovudine (AZT) as template and methacrylic acid as monomer was prepared. The synthesis of the MIP was performed in acetonitrile. The synthesized material was then tested for the solid-phase extraction of AZT from different media (pure organic solvents and hydro-organic mixtures). An optimised procedure was developed for the selective extraction of AZT with a recovery of 96% using the MIP and only 3% on a non-imprinted polymer used as control polymer. A specific capacity of 0.2 micromol g(-1) was determined. The specificity of the MIP was evaluated by studying the retention behaviour of two others nucleoside analogues. The feasibility of the MIP to selectively extract AZT and stavudine (d4T) from human serum was also demonstrated with recoveries of 80 and 85% respectively. The lower limit of quantification (LLOQ) and the lower limits of detection (LLOD) for AZT were 5.10(-7) and 10(-7) M respectively.

An in-line procedure for protein analysis using a trypsin-based immobilized enzymatic reactor (IMER) coupled to LC-MS/MS has been developed. Various IMERs were synthesized and characterized by estimating the digestion yield of a pattern peptide in UV detection. Laboratory-made IMERs were optimized by studying the effect of different parameters as the nature of the functionalized immobilization support (silica, agarose), the amount of immobilized trypsin and the binding density. The potential of the laboratory-made IMERs were compared with a batch digestion and with a commercial trypsin-based IMER. The laboratory-made IMER based on CNBr-activated Sepharose showed the best performances in terms of digestion yields, digestion time, price and repeatability (RSD<4%). Cytochrome c was then digested on this IMER and used in-line with LC-MS. The target protein was easily recognized by the Mascot database until 17pmol injected.

A molecularly imprinted polymer synthesized in dichloromethane, was evaluated for the selective extraction of a pharmaceutical compound from human plasma and integrated on-line with liquid chromatography. The target drug is an alpha-blocker called alfuzosin widely used for the treatment of benign prostatic hyperplasia. By a comprehensive approach of the retention mechanism, a selective extraction procedure was established by exploiting the development of electrostatic interactions between the target analyte and the selective support in LC compatible solvents. By applying this method to plasma, extraction recoveries close to 100% were obtained for alfuzosin while various pharmaceutical compounds currently found in biological fluids were not retained on the support. The high selectivity of the support coupled to the chromatographic system permitted an easy and fast analysis of the drug with a limit of quantification of 15 microg L(-1) by UV detection.