Miniaturization of the bioanalytical process

This work presents a systematic study of macroporous polymer monoliths and their use as stationary phases for microscale separation and as supports for immobilized trypsin digestion in pipette tip format for bioanalysis. Reversed-phase/cation-exchange mixed-mode polymer monolithic columns were prepared in situ within fused-silica capillaries via UV-initiated free-radical polymerization reaction. Control of the porous properties was achieved by varying the ratio of porogenic solvents (e.g. 1,4-butanediol and 1-propanol), the type of porogenic solvents used and the relative amounts of functional monomers and cross-linker. These columns were successfully used for the separation of a mixture of selected acidic drugs and beta-blockers, namely ketoprofen, ibuprofen, diclofenac, arterenol and propranolol. Monolithic columns with higher amounts of cross-linker were shown to give better repeatability. Furthermore, the separation mechanism of the investigated compounds was shown to be dual mode hydrophobic/ion-exchange interaction of the analytes with the hydrophobic and cation-exchange monolith. All these properties demonstrated the potential of these devices for solid-phase extraction and sample enrichment purposes in miniaturized formats. Based on the success of the previous work a trypsin-immobilized monolithic polymer with pipette-tip format was also investigated to explore the utility of using a tip-based protein digestion methodology in a bioanalytical setting. The excellent performance of immobilized enzymatic polypropylene pipette (IMEPP) tips was characterized using MicrOTOF-Q quadrupole time-of-flight MS and triple quadrupole LC-MS/MS systems. Very high sequence coverages of over 90% were achieved for the digestion of proteins ranging from low molecular weight to high molecular weight proteins with short contact times prior to MS analysis, which is comparable to 24 h digestion in solution. In addition, quantitative analysis of target proteins spiked in rat plasma was demonstrated for the first time with relatively good linearity over a wide range from 40-1000 ng/mL. The developed IMEPP tips exhibit high plasma loading capacity up to 40 uL of plasma that can be used to yield the highest efficiency for digestion. The IMEPP tip approach is thus rapid, reliable, and robust suggesting the potential of this approach to improve sample throughput for pharmaceutical and pharmacokinetic studies, leading to faster and safer discovery of new drugs to treat diseases. The trypsin immobilized polymer monolith was prepared in situ in syringe-compatible glass tube and evaluated for the digestion of protein. Preliminary results showed that the enzyme on the immobilized bed exhibited high proteolytic performance in this special format. The present glass tube bioreactor provides a promising platform for the full automation, on-line coupling to detection systems, short sample preparation times and high-throughput protein digestion.