Bioanalysis procedures

Automation using a robotic liquid handling system eliminated most of the tedious steps encountered with traditional manual extraction procedures. Automated 96-well SPE and LLE techniques using robotic liquid handlers have been successfully implemented to support high-throughput bioanalysis.5... 

For those readers who are not yet familiar with mass spectrometry, the introduction provides an explanation of the basics of mass spectrometry and its instrumentation as well as practical aspects and applications in bioanalysis. Next, a block of three chapters shows different affinity selection procedures suitable to identify hits from combinatorial compound libraries. This subject, being metaphorically speaking a search for a needle in a haystack, is of outstanding relevance for big pharma . The techniques described here offer real high throughput capabilities and are implemented already in the routine industrial screening... 

A typical application of GC to the determination of a drug in plasma is in the determination of the anti-epileptic drug valproic acid after solid phase extraction (see Ch. 15) by GC with flame ionisation detection. In this procedure, caprylic acid, which is isomeric with valproic acid, was used as an internal standard. The limit of detection for the drug was 1 pg/ml of plasma. The trace shown in Figure 11.25 indicates the more extensive interference from background peaks extracted from the biological matrix which occurs in bioanalysis compared to the quality control of bulk materials. 

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]. 

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. 

Another popular and selective extraction technique widely used in bioanalysis is solid phase extraction (SPE). SPE is a separation process utilizing the affinity of the analytes to a solid stationary phase. By manipulating the polarity and pH of the mobile phase, the analytes of interest or undesired impurities pass through stationary phase sequentially according to their physical and chemical properties. For a SPE procedure, a wash step refers to the elution of the unwanted impurities which are discarded and the elution step refers to the elution of the analytes of interest which are collected. While the fundamental remains the same in decades, the continuing invention and introduction of new commercial stationary phases and accessory devices have boosted the application of SPE in bioanalysis and many other fields. 

The typical column-switching setup for on-Une SPE-LC-MS is shown in Figure 1.3. In a typical application, the sample is loaded by the autosampler onto a precolunm. The sample volume can be larger than the typical injection volume of an analytical column. Analytes are adsorbed onto the chosen stationary phase under weak solvent conditions, while more hydrophilic sample constituents are flushed through. A washing step of the SPE column may be included in the procedure. Next, the valves are switched from the load to the inject position. The SPE coluum is eluted, in most cases in backflush mode, and the analytes are transferred to the LC coluum for separation and subsequent LC-MS detection. Examples of on-line SPE-LC-MS are discussed in Ch. 7.3.2 in enviroiunental analysis, in Ch. 11.6.4 for quantitative bioanalysis, and in Ch. 17.5.2 for peptide analysis. 

In quantitative bioanalysis, the application of SRM in a triple-quadrupole instrument is the method-of-choice. SRM provides ultimate selectivity, because only compounds are detected, which after sample pretreatment of the matrix are detected within a selected retention time window in the chromatogram as the stracture-specific fragment at a particular m/z in MSj, generated by collision-induced dissociation (CID) from a precursor ion with a particular m/z of the compound of interest, selected in MS . The ultimate selectivity results in good sensitivity and low detection limits. Software procedures for the automatic optimization of instrament... 

The SRM transitions for the analyte and its IS are preferentially selected as a common neutral loss. In many automatic optimization procedures for SRM, the selection of the SRM transition is based on the maximum response. However, from selectivity point of view, additional criteria based on structure specificity, selectivity, and enhanced S/N in the analysis of real samples are important. The selection of an SRM transition by evaluating the background noise and the absence of interferences was reported by Woolf et al. [33] for the bioanalysis of an indinavir metabolite. 

Quantitative bioanalysis has been reported for a wide variety of drugs and their metabolites for various stages of drag development. In this section, procedures and results for five compounds (classes) are reviewed. The five compounds were selected, because a number of studies were reported for each compound. This allows to illustrate the diversity in strategies in quantitative bioanalysis. 

Until recently, on-line SPE via valve-switching techniques was not veiy popular in bioanalytical LC-MS. With the introduction of the Spark Holland Prospekt II, online SPE-LC-MS is promoted for quantitative bioanalysis. The typical procedure and column-switehing setup for on-line SPE-LC-MS is discussed Ch. 1.5.4. 

A closely related variation of on-line SPE involves off-line removal of proteins prior to sample injection. Usually this procedure is accomplished by the addition of an organic solvent (e.g., acetonitrile) that contains an internal standard [63]. Variations of this strategy have been applied in our own laboratory for plasma bioanalysis [49] as well as in vitro screens [60]. A number of variations on this theme have often appeared in the literature for the simultaneous analysis of multiple analytes. An example of this type of analysis is described by Zell and co-workers, who used perchloric acid pretreatment prior to on-line SPE for the bioanalysis of a platelet inhibitor, its ester prodrug, and an active metaboUte [64]. Analytical ranges from 1 ng/mL to 1000 ng/mL are fairly typical for this technology, where most appUcations involve narrow-bore columns and begin with 50-100 juL plasma. Despite the need for a deliberate precipitation step, indirect SPE is generally more robust and less expensive than the other alternatives presented here. 

Because of the serial nature of LC-MS, much of the current discussion has centered on ways to reduce LC-MS/MS cycle time. Often the rate-limiting step for drug-discovery bioanalysis lies in the speed with which methods (sample preparation and chromatography) can be prepared for NCEs. One of the frequently overlooked steps is the need to tune and optimize MS/MS transitions for the various analytes studied. Fortunately, most MS vendors now offer semi- or fully automated procedures to perform this task. The origin for these procedures can be traced to the seminal work of Whalen et al., who published an automated procedure known as AUTOSCAN [106]. Itis possible to establish experimental conditions with this procedure in the flow-injection analysis (FIA) mode for 96 analytes in less than one hour. 

In addition to some early applications in bioanalysis, ambient ionization mass spectrometry has been used as an imaging tool to study drug distribution in tissue sections. Most of the work reported so far involved the use of DESI as the ambient ionization method. Compared to other mass spectrometry-based tissue imaging techniques such as MALDI and SIMS, DESI allows tissue samples to be analyzed under ambient conditions without sample preparation, which simplifies the procedure and prevents the redistribution of analytes during matrix deposition. A major drawback of DESI as an imaging tool is its relatively low spatial resolution (typically 250 pm) and therefore cannot be used for cellular or subcellular imaging. 

It was found that EVLS is capable to detect (i) minor signals hidden in major ones, (ii) small changes in ODN structure and the interaction between ODN and electrode surface, and (iii) potentially closed signals (resolution of overlapped peaks). On the basis of the above-mentioned advantages, EVLS in connection with the adsorption procedure fulfills the requirements for a perspective and promising tool for qualitative and quantitative studies in bioanalysis in bio- and nanotechnologies. Therefore, the implementation of EVLS in electrochemical analyzers should be of great interest. 

Serious matrix problems may be experienced in quantitative bioanalysis, especially in ESI. Signal suppression due to unknown matrix interferences is often observed. Changes in the sample pretreatment procedures may be successful in solving the problem, but in some cases changing over to APCI, when applicable, appears to be the only feasible solution. 

The sensitivity of the method is evaluated by the limit of detection and quantification. In general, there are no specific criteria for the limit of detection, but for bioanalysis purposes, the limit of quantification is established for the concentration with precision and accuracy lower than 20%. Limits of detection and quantification obtained in CE with UV detection are generally higher than in HPLC, owing to the small optical path in the detection window and injected volumes. As discussed previously, by using preconcentration procedures (off-line sample preparation and/or stacking sample injection techniques) and a more sensitive detection system, particularly LIE and MS detection, suitable detection or quantification limits could be obtained for the apphcation of the methods to the analysis of real samples. 

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