Bioanalysis sample preparation

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. 

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. 

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. 

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. 

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... 

In bioanalysis, High-Performance Liquid Chromatography (HPLC) is the analytical technique most frequently used. Often, extended sample preparation is required to make a biological sample (the matrix) suitable for HPLC-analysis. The compound of interest, the analyte, has to be isolated from the matrix as selective and quantitative as possible. The quality of the sample preparation largely determines the quality of the total analysis procedure. In a survey Majors [2] showed that approximately 30% of an error generated during sample analysis was due to sample preparation, which indicates the need for error reduction and quality improvement in sample preparation. 

A systematical approach of sample preparation methods and optimisation of the quality aspects of sample preparation may enhance the efficiency of total analytical methods. This approach may also enhance the quality and knowledge of the methods developed, which actually enhances the quality of individual sample analyses. Unfortunately, in bioanalysis, systematical optimisation of sample preparation procedures is not common practice. Attention to systematical optimisation of assay methods has always been mainly on instrumental analyses problems, such as minimising detection limits and maximising resolution in HPLC. Optimisation of sample extraction has often been performed intuitively by trial and error. Only a few publications deal with systematical optimisation of liquid-liquid extraction of drugs from biological fluids [3,4,5]. 

Chang, M. S., Ji, Q., Zhang, J., and El-Shourbagy, T. A. (2007a). Historical review of sample preparation for chromatographic bioanalysis Pros and cons. Drug Dev. Res. 68 107-133. 

The work of Kaye and colleagues and Pleasance and co-workers provided the interest and motivation to extend sample preparation capabilities into an off-line batch mode process. This motivation was also stimulated by sample preparation bottlenecks, which typically occurred during on-line bioanalysis, where the limiting factor was associated with extraction and cleanup procedures. The rationale was to perform sample preparation tasks with an automated procedure followed by the transfer. 

Simpson, H. Berthemy, A. Buhrman, D. Burton, R. Newton, J. Kealy, M. Wells, D. Wu, D. 1998. High throughput liquid chromatography/mass spectrometry bioanalysis using 96-well disk solid phase extraction plate for the sample preparation. Rapid Commun. Mass Spectrom., 12,75-82. 

Abstract Internal standards play critical roles in ensuring the accuracy of reported concentrations in LC-MS bioanalysis. How do you find an appropriate internal standard so that analyte losses and experimental variations during sample preparation, chromatographic separation, and mass spectrometric detection could be corrected How is the concentration of an internal standard determined Should internal standard responses be monitored during the analysis of incurred samples What are the main causes for internal standard response variations How do they impact the quantitation Why are stable isotope labeled internal standards preferred And yet one should still have an open-mind in their usage for the analysis of incurred samples. All these questions are addressed in this chapter supported by theoretical considerations and practical examples. 

An internal standard is expected to track the analyte in all the three distinctive stages of LC-MS bioanalysis, i.e., sample preparation (extraction), chromatographic separation, and mass spectrometric detection. Though the emphasis should usually be... 

Internal standards play critical roles in ensuring the accuracy of final reported concentrations in quantitative LC-MS bioanalysis through the correction of variations during sample preparation, LC-separation, and MS detection. The physical-chemical properties of an internal standard, particularly hydrophobicity and ionization properties should be as close as possible to those of the corresponding analyte to better track the variations the analyte experiences. For this reason, stable isotope labeled internal standards should be used whenever possible. However, efforts should still be made to obtain clean extracts, adequate chromatographic separation, and optimized ionization mode and conditions. 

The use of MRM methods for quantitative bioanalysis often reduces sample preparation and analysis time. The MRM method that used LC/ESI-MS/MS for the quantitative analysis of an anticancer drug, Yondelis (Ecteinascidin 743, ET-743, trabectedin. Scheme 9), in human plasma was demonstrated by Rosing et al. [103]. The full-scan mass spectrum of ET-743 (MW 762) contained an abundant [MH+ - H2O] ion at m/z 744 as a result of loss of water molecules from in-source CID (spectrum not shown). The internal standard, ET-729 (Scheme 9, MW 747), exhibited similar performance in the full-scan mass spectrum an abundant [MH+ - H2O] ion at tn/z 730 was produced. The product ion spectra of ET-743 and ET-729 exhibited the most abundant fragment ions at m/z 495 and m/z 479, respectively (spectra not shown). The product ion at m/z 495 (C27H31N2O7) was formed in the collision cell after cleavage of the sulfur bond and ester binding at C-11 [103]. 

R. Dams, M. A. Huestis, W. E. Lambert, and C. M. Murphy, Matrix effect in bioanalysis of illicit drugs with LC-MS/MS Influence of ionization type, sample preparation, and biofiuid, /. Am. Soc. Mass Spectrom. 14 (2003), 1290-1294. 

Related to the volume processed, the detection limit of biosensors has to be considered. Take, for example, a hypothetical biosensor for HIV detection that has a detection limit of one virus per analysis. If only 100 nl of sample are analyzed, then the detection limit of biosensor cannot be below 10,000 virus particles per milliliter of sample, which is neither a stunning nor an acceptable limit of detection for rapid detection early in the disease. Thus, in this case, a sample preparation step that reduces a 1 ml blood sample to 100 nl is of imminent importance (i.e., now, reduced reagent costs make the miniaturized biosensor advantageous over a macroscopic counter part). The same problem is encountered in most environmental and food sample analyses. Thus, the incorporation of sample pretreatment steps into the bioanalysis is very important, and for miniaturized biosensors even more than for their macro cotmterparts. 

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