MS-based methods

It is therefore not surprising that the interest in PyMS as a typing tool diminished at the turn of the twenty-first century and hence why taxonomists have turned to MS-based methods that use soft ionization methods such as electrospray ionization (ESI-MS) and matrix-assisted laser desorption ionization (MALDI MS). These methods generate information-rich spectra of metabolites and proteins, and because the molecular ion is seen, the potential for biomarker discovery is being realized. The analyses of ESI-MS and MALDI-MS data will still need chemometric methods, and it is hoped that researchers in these areas can look back and learn from the many PyMS studies where machine learning was absolutely necessary to turn the complex pyrolysis MS data into knowledge of bacterial identities. 

Other PTMs may involve changes in the chemical nature of amino acids (e.g., citrullination or deimination). Because many of these modifications result in mass changes that are measurable by MS, they are amenable to detection by MS-based approaches. A number of emerging MS-based strategies allow the identification of PTMs. Several MS-based methods to determine the types and sites of protein phosphorylation and ubiquitination have been developed. Phosphorylation occurs mainly on serine, threonine, and tyrosine residues at a frequency ratio of 1800 200 1 in vertebrates.70 Although the phosphorylation of tyrosine residues occurs less frequently in the proteome, it has been extensively studied. 

Phase Numbers of compounds Role or opportunity for MS-based methods... 

Table 4.2 MS-based methods proposed for use in lead discovery. Included from [4] with permission from Wiley Periodicals.

Recent trends in pesticide analysis in food aims for reduced sample pretreatments or simplified methodologies (as QuEChERS approaches), the use of online purification processes, the use of new adsorbents (such as molecular imprinted polymers (MIPs) and nanomaterials) for the extraction and clean-up processes, and focused on the development of large multiresidue methods, most of them based on LC-MS/ MS. In spite of the relevant role of LC-MS/MS, GC-MS-based methods still play an important role in pesticide analysis in food. Despite the development achieved in the immunochemical approaches, the need for multi-residue methods has supported the development and use of instrumental techniques. 

This screening system has also been applied successfully in the directed evolution of enantioselective EHs acting as catalysts in the kinetic resolution of chiral epoxides 95,96) (Section IV.A.4). Moreover, the firm Diversa has applied the MS-based method in the desymmetrization of a prochiral dinitrile (l,3-dicyano-2-hydroxypropane) catalyzed by mutant nitrilases 46). In this industrial application, one of the nitrile moieties was labeled selectively with as in N-17, which means that the two pseiido-eaaniiovaenc products (S)- N-18 and (J )-18 differ by one mass unit. This is sufficient for the MS system to distinguish between the two products quantitatively 46). 

Warren EN, Elms PJ, Parker CE, et al. Development of a protein chip a MS-based method for quantitation of protein expression and modification levels using an immunoaffmity approach. Anal. Chem. (2004) 76 4082-4092. 

Nine strategies consistently appear in LC/MS-based methods for accelerated development (Table 5.1). The nine strategies are standard methods, template structure identification, databases, screening, integration, miniaturization, parallel processing, visualization, and... 

TABLE 5.1 The nine strategies that consistently appear in LC/MS-based methods lor accelerated drug development... 

Interestingly, many of the current LC/MS approaches for pharmaceutical analysis are extensions of gas chromatography/mass spectrometry (GC/MS) (Foltz, 1978), mass spectrometry (Garland and Powell, 1981), and MS/MS (McLafferty, 1983)-based methods. With the introduction and widespread use of LC/MS-based methods, these fundamental approaches for quantitative and qualitative structure analysis became more routinely applicable to a wider scope of pharmaceuticals. 

Using this approach for natural products dereplication, data are routinely obtained from 40 gg of crude extract. Performance examples include the identification of 16 analogs of teicoplanin and 12 analogs of phenelfamycin from separate samples. The summary of results obtained for phenelfamycin is shown in Table 6.4. The correlation of fraction, retention time, and molecular weight provides the essential information for rapid dereplication and identification. The time required to dereplicate natural product samples is about 1 week with this LC/MS-based method compared to several weeks by previous methods that involve traditional isolation steps. The use of this LC/MS-based methodology results in greater clarity and confident decisions for proceeding with the full structural study of an active component derived from a culture. 

Combinatorial Mixture Screening The increased popularity of LC/MS-based methods combined with limited resources resulted in advances that effectively matched combinatorial chemistry samples (i.e., complexity) with instrument time. Richmond, Yates, and coworkers (Richmond et al, 1999 Yates et al.,2001) demonstrated the use of flow injection analysis (FIA)-LC/MS systems for rapid purity assessment and combinatorial mixture screening, respectively. These LC/MS-based applications addressed two critical bottlenecks HPLC... 

In the application described by Olah and workers, LC/MS-based methods are used to simultaneously assay plasma concentrations of up to 12 substances. The plasma is obtained from either single animals dosed with mixtures of lead compounds, or from multiple animals dosed with a single lead compound, after which aliquots of plasma from common time points are pooled. Essentially, simplified, less stringent versions of preclinical/clinical development procedures are used for sample preparation, assay validation, and analysis. A plasma concentration-time profile obtained from a dog administered simultaneously with 12 compounds is shown in Figure 6.21. Each... 

During the development of TAXOL , 90 taxane impurities were rapidly identified and added to the structure database. This MS/MS information is routinely obtained for impurities down to the 100 ng level (injected), and requires approximately 2-3 h for the analysis of each sample. The compounds are structurally categorized with profile group terminology. The LC/MS-based methods are significantly faster than previous methods based on scale-up, isolation, fractionation, and individual structural analysis. 

At the time, this application provided a powerful benchmark for the use of LC/MS-based methods in the pharmaceutical industry. This particular assay successfully supported several clinical studies with sensitive and reliable results. This performance was bench-marked on more than 4000 clinical samples and led to a wider scope of application and the development of routine, standard methods for quantitative bioanalysis. 

In this study, a LOQ of 20 pg/mL in cell lysates would be needed in order to quantify paclitaxel uptake by cells exposed to approx. 1 ng/mL drug, but the detection limits of published LC-MS/MS based methods were in the range of 0.1-0.25 ng/mL [10,15-21], Furthermore, concentrations of paclitaxel lower than 1 ng/mL are pharmacologically active, and therefore, a LOQ lower than 20 pg/mL is desirable. 

We refer to LC-MS-based methods for a PK study [61] as well as in vitro biotransformation studies with liver microsomes [23,62],... 

We herein refer to an in vitro biotransformation study in human liver microsomes [82] and a PK study in man [83] both analysed by LC-MS-based methods. 

Fig. 5 Statistical evaluation of LC-MS-based methods for tropane alkaloids referred in this chapter. (a) Relative frequency of ionization methods. +APCI positive atmospheric pressure chemical ionization, +ESI positive electrospray ionization, FAB fast atom bombardment, +TSP positive thermospray, (b) Relative frequency of scan modes used. MS full scan MS, MS/MS tandem mass spectrometry (product ion scan), MRM multiple reaction monitoring, SIM selected ion monitoring, (c) Relative frequency of mass analysers used. EBQtQ2 double focusing sector field mass spectrometer, IT ion trap, QqQ triple quadrupole, SQ single quadrupole. Considered publications were found by PubMed data-based search and references cited in these articles...

No further studies were found investigating stereoselective PK behaviour of any TA monitored by LC-MS-based methods. 

The three examples introduced above present applications of LC-MS-based methods to determine specific drug transfer into compartments relevant for systemic or local activity and thus providing basic pharmacological or toxicological data. 

Table 7 summarizes LC-MS-based methods that were applied to study metabolism of TA and Fig. 7 depicts some selected metabolites identified by mass spectrometry. 

A mandatory requirement when validating an LC-MS(-MS) based method according to the FDA guidelines is to evaluate and attempt to minimize the incidence of matrix effects [58], Matrix effects refer to the direct or indirect alteration or interference in response due to the presence of interfering substances in the sample [52], It can either reduce the analyte response (ion suppression) or enhance it (ion enhancement). Both can considerably compromise the accuracy of quantification and ion suppression may in the worst case even lead to decrease sensitivity and to false negative results. 

Fig. I The typical metabolomics workflow has three key steps the isolation of metabolites, detection of the metabolites, and data analysis. The isolation step is typically determined by the class of metabolite being measured because of the physicochemical properties of different metabolite classes (i.e., hydrophobic, hydrophilic), which require different enrichment protocols. Two principle methods for metabolite detection are NMR- and MS-based methods. Finally, the data analysis can be performed in a variety of ways depending on the problem...

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