Bioanalysis samples

Bioanalysis samples (saliva, urine, whole-blood, plasma, serum, cerebrospinal fluid, tissue, etc.) may contain water, ionic salts, proteins, lipids, anticoagulants, stabilizers, and other compounds that can interact on either a temporary or permanent basis with the stationary phases. These interactions decrease the column lifetime and interfere... 

Figure 11.16 Chromatograms of plasma samples obtained by using SPE-SFC with super-aitical desorption of the SPE cartridge (a) blank plasma (20 p.1), UV detection at 215 nm (b) blank plasma (20 p.1), UV detection at 360 nm (c) plasma (1 ml) containing 20 ng mitomycin C (MMC), UV detection at 360 nm. Reprinted from Journal of Chromatography, 454, W. M. A. Niessen et al., Phase-system switching as an on-line sample pretreatment in the bioanalysis of mitomycin C using supercritical fluid cliromatography, pp. 243-251, copyright 1988, with permission from Elsevier Science.

W. M. A. Niessen, R J. M. Bergers, U. R. Tjaden and J. van der Greef, Phase-system switclring as an on-line sample preti eatment in the bioanalysis of mitomycin C using supercritical fluid clrromatogi aphy , 7. Chromatogr. 454 243-251 (1988). 

Silica-based restricted access materials (RAM) have been developed for cleanup in bioanalysis, first for low molecular weight compounds in biofluids (Rbeida et al., 2005) and subsequently for biopolymers such as peptides (Wagner et al., 2002). A classification of different types of RAM has been given by Boos and Rudolphi (1997). Novel RAMs with strong cation-exchange functionality have been synthesized and implemented in the sample cleanup of biofluids. Racaityte et al. (2000) have shown that this type of RAM is highly suitable for the online extraction and analysis of... 

This chapter will review recent advances in mass spectrometry, liquid chromatography, and sample preparation techniques that aim at achieving high throughput. In particular, online solid phase extraction and multiplexed HPLC front ends for quantitative bioanalysis will be discussed in detail. 

Recent innovations in ionization techniques have allowed the development of ambient mass spectrometry. Mass spectra can be determined for samples in their native environment without sample preparation. Although the ambient mass spectrometry technique is still in its infancy, its potential for serving as a tool of choice for high-throughput bioanalysis is very encouraging. 

Another MS-based approach used in high-throughput bioanalysis utilizes a mass spectrometer equipped with several API spray probes. Each of the analytical columns in parallel is connected to a separate spray probe and each spray is sampled in rapid successions for data acquisition by the MS. A separate data hie for each spray is recorded. Several samples can be analyzed simultaneously on parallel columns5 6 in the course of a single chromatographic run. 

Chovan et al.30 described a system that integrates different components of bioanalysis including automatic in vitro incubation, automatic method development (mainly SRM transitions for LC/MS/ MS analysis), and a generic LC method for sample analysis to minimize human intervention and streamline information flow. Automaton software (Applied Biosystems) was used for automatic MS method development. Flow injection was used instead of a HPLC column to decrease run time to 0.8 min per injection. Two injections were performed. The first was performed to locate the precursor ion and optimal declustering potential (DP). The second injection was performed to locate the product ion and optimal collision energy (CE). 

Deng, Y. et al. 2002. High-speed gradient parallel liquid chromatography/tandem mass spectrometry with fully automated sample preparation for bioanalysis 30 seconds per sample from plasma. Rapid Commun. Mass Spectrom. 16 1116. 

A variety of online solid extraction devices and applications have been developed for bioanalysis. Many are easy to build in laboratories or commercially available. Unlike offline methods, minimal operator intervention is needed for daily sample analysis after online applications are set up, so the approach is both labor- and cost-effective. The technique can also minimize errors arising from manual operations, eliminate potential inconsistencies caused by different operators, and provide accessibility of LC/MS/MS applications to laboratories that have minimal analytical expertise. 

Because the instability of the N-oxide metabolite, which was subjected to decomposition during sample preparation (solvent evaporation during offline SPE), online SPE LC/MS became the method of choice for the application. Hsieh et al. (2004) built a system with two TFC cartridges and one analytical column, and another system with two TFC cartridges and two analytical columns for GLP quantitative bioanalysis of drug candidates. A Turbo C18 (50 x 1.0 mm, 5 /.mi, Cohesive Technologies), an Xterra MS C18 (30 x 2.0 mm, 2.5 /mi), and a guard column were used. Protein precipitation preceded injection. The cycle times for the two systems were 0.8 and 0.4 min. 

Jemal M., Xia Y., and Whigan D.B., 1998. The use of high-flow high performance liquid chromatography coupled with positive and negative ion electrospray tandem mass spectrometry for quantitative bioanalysis via direct injection of the plasma/serum samples. Rapid Commun Mass Spectrom 12 1389. 

Zhang N. et al., 2000. Integrated sample collection and handling for drug discovery bioanalysis. J Pharm BiomedAnal 23 551. 

The high-throughput concept for quantitative bioanalysis applies to steps such as assay development, sample collection and sorting, sample preparation, sample analysis, and data processing and reporting. Those processes are closely interlinked and improvement of process throughput is equally important. 

Due to its simplicity and wide applicability, PPT is important for sample pretreatment in early drug discovery when generic extraction of mixtures of candidates is more important than sensitivity. As a generic technique, PPT is attractive for high-throughput bioanalysis because it offers fast sample preparation and easy automation and requires minimal manual labor. 

Pre-concentration methods using online trace enrichment by applying chromatographic principles are also reported [66,69]. As described by Guzman and Meyers [71,72], this can be achieved by incorporating e.g. a solid-phase CE-concentrator tip at the inlet of the capillary. Undesired sample components can be flushed out prior to the CE separation run, providing faster and more specific analyses. Especially in the field of bioanalysis, where sample clean up and pre-concentration of analytes is usually critical, this approach may be preferred. 

The applicability of cinchonan carbamate CSPs for bioanalytical investigations using HPLC-ESI-MS/MS has been demonstrated by Fakt et al. [120]. The goal was the stereoselective bioanalysis of (R)-3-amino-2-fluoropropylphosphinic acid, a y-aminobutyric acid (GABA) receptor agonist, in blood plasma in order to determine whether this active enantiomer is in vivo converted to the 5-enantiomer. In this enantioselective HPLC-MS/MS bioassay, sample preparation consisted of... 

With the advent of API sources, LC/MS/MS allows the facile development of quantitative methods that are sensitive, selective, robust, and amenable to the rapid analysis of a majority of small molecules. In order to achieve high-throughput bioanalysis in support of pharmacokinetic studies, many approaches have been reported utilizing automated sample preparation and reducing analysis time by pooling samples, parallel analysis, and fast chromatography. 25,26,152,153... 

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