Immunoaffinity in-tube solid phase microextraction coupled with liquid chromatography with fluorescence detection for determination of interferon α in plasma samples

• A rare application of in-tube SPME for biopharmaceutical (protein) analyses was presented.
• A sensitive and selective immunoaffinity in-tube SPME/LC-FD method was established.
• This method has been applied to interferon alpha in plasma samples for therapeutic drug monitoring.
• The in-tube SPME variables was optimized and developed method has been validated.
• Low LOQ in a short time analysis was obtained.

The present work describes a sensitive and specific automated immunoaffinity in-tube solid-phase microextraction coupled with liquid chromatography with fluorescence detection (LC-FD) method for the determination of interferon α2a in plasma samples for therapeutic drug monitoring. To this end, the intrinsic selectivity of the monoclonal antibodies has been combined with in-tube solid phase microextraction by immobilization of these antibodies into the fused silica capillary. The in-tube SPME variables, such as plasma sample volume, draw/eject volume, number of draw-eject cycles, as well as desorption procedure have been optimized, in order to improve the sensitivity of the proposed method. Analyses of interferon α2a in plasma samples were carried out within 12 min (sample preparation and LC analyses). The response of the proposed method was linear over a dynamic range 0.006–3.0 MIU mL−1, with a correlation coefficient of 0.998. The inter-day precision of the method had a coefficient of variation lower than 6.2%. The proposed automated method has adequate analytical sensitivity and selectivity for the determination of interferon α2a in plasma samples for therapeutic drug monitoring. The proposed method was successfully applied to the plasmas samples analyses from patients in therapy with interferon α-2a, demonstrating a rare application of in-tube SPME for biopharmaceutical (protein) analyses.