LC-MS-Based Approaches

The LC-MS approach is currently the most popular approach for lipid analysis [55-57], although it may not be the best choice for global lipid analysis (i.e., lipidomics). The rationale behind the LC-MS approaches is to maximally exploit the LC separation technology with the most sensitive detection power of MS currently available. Three major factors are generally considered for successful development of LC-MS methodology in lipidomics, as well as understanding the principles of those developments. 

The third factor is to set up the MS (or MS/MS) parameters to identify and quantify the eluted individual lipid species as many as possible. The particular features in LC-MS analysis are that the lipid concentrations in eluents are constantly changing, and identification and quantification of lipid species have to be done in a very limited time frame. These features are in contrast to shotgun lipidomics therefore, totally different settings and methodologies from those in shotgun lipidomics have to be employed. 

There are three different, but commonly used, methods in the analysis of lipids by LC-MS related to the MS settings as follows  

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LC/MS Contributions. The use of LC/MS-based approaches has expanded rapidly in drug discovery during the past several years. Applications that range from the quick molecular weight confirmation of synthetic lead compounds (Taylor et al., 1995 Pullen et al., 1995) to novel and highly selective methods for structure identification (Carr et al., 1993) have been demonstrated. Analysis strategies... 

The capillary LC/MS-based approach for peptide mapping performed by Arnott and colleagues features miniaturized sampleloading procedures, which are routinely amenable to small quantities of peptides. The reliable characterization of protein/peptide mixtures in conjunction with the widely used 2-DGE methods offers a powerful fingerprinting approach in the pharmaceutical industry. Low femtomole detection limits (typically <50 femtomole) with a mass accuracy of +0.5Da provide unique advantages for protein identification. Liberal parameters for mass range and unmatched masses are used for the initial protein search, whereas more conservative parameters are used to reduce the number of matches and to improve the confidence in the search. 

The ESI-LC/MS-based approaches that feature ion trap (Gatlin et al, 1998 Washburn et ah, 2001) and quadrupole time-of-flight (QTOF) (Blackburn and Moseley, 1999) mass spectrometers are routinely used for the identification and characterization of proteins. Nanoelectrospray LC/MS formats (Figure 6.3) are used to provide lower limits of detection and fully automated sample preconcentration and desalting. On-line LC/MS approaches for protein expression profiling are also used with ESI-TOF (Banks and Gulcicek, 1997 Chong et al., 2001) and ESI-Fourier transform (FT) (Kelleher... 

With an increasing number of novel lead candidates that enter into preclinical development, considerable resources are needed to identify impurities. LC/MS-based approaches provide integrated sample cleanup and structure analysis procedures for the rapid analysis of impurities. This advantage was demonstrated during the preclinical development of TAXOL . LC/MS played an important role for the identification of impurities contained in extracts and process intermediates from Taxus brevifolia and T. baccata biomass. Because drugs derived from natural sources often have a very diverse set of structural analogs, it is important to determine which... 

The determination of the concentration of a drug in plasma is an accepted surrogate marker for drug exposure. The use of LC-MS-based approaches for the quantitative analysis of a drug or its metabolite in a physiological sample such as plasma or serum is perhaps the hallmark of PK studies in drug discovery. The PK profiles derived from LC-MS data provide bioavailability and half-life information for drug candidates. These PK profiles are used to directly compare lead compounds and select specific compounds for further study. 

Hydrophilic Interaction LC-MS-Based Approaches Hydrophilic interaction LC (HILIC) is a variant of normal-phase LC. HILIC uses hydrophilic stationary phases, but employs reversed-phase type eluents. Any polar chromatographic surface can be used for HILIC separations, even nonpolar bonded silicas. A typical mobile phase for HILIC includes acetonitrile with a small amount of water. However, any aprotic solvent miscible with water (e.g., tetrahydrofunan or dioxane) can be used. Alcohols can also be used with a higher concentration. Ionic additives, such as ammonium acetate and ammonium formate, are usually used to serve as the modifiers for controlling the pH and ion strength of the mobile phase. 

Although many systematic indices (e.g.. Lipid MAPS, Chemical Entries of Biological Interest (ChEBI), lUPAC International Chemical Identifiers (InChl), simplified molecular-input line entry system (SMILES)) were developed to list the chemical compounds, these indices (identifiers) can only be meaningful if the compound is totally identified. However, in practice, lipidomics analysis in many cases can only provide partial identification of lipid molecular structures at the current development of technology. Moreover, different lipidomics approaches provide different levels of stmctural identification of lipid species. Therefore, how to clearly express and report the information about the levels of identification for the structures of lipid species (which can be derived fi om MS analysis) is not only helpful for the readers but also important for bioinformatics and data communication. To this end, the analysis by shotgun lipidomics could be used as a typical example to explain these levels. Similar phenomena also exist in the analysis of lipid species employing LC-MS-based approaches. 

As lipidomic analysis is classified into the LC-MS-based approach and shotgun lipidomics, the requirement for sample preparation is also different. The former is more tolerant with the presence of inorganic ions in lipid extracts than the latter since the pre-column or even the analytical column can get rid of these extraction contaminants. However, the presence of inorganic residues may affect substantially the ionization stability as well as ionization efficiencies of different lipid classes and individual species of a class in shotgun lipidomics. Therefore, minimizing the inorganic residues, particularly eliminating any aqueous phase contamination, becomes crucial. 

For both shotgun lipidomics and LC-MS-based approaches, the dynamic range should be examined in the presence of sample matrices instead of using a pure standard. Under such conditions, the matrix effects (e.g., ion suppression) that become more severe in analysis of minor species (or classes) in the presence of abundant species (or classes) can be eliminated for sample analysis. 

There have been numerous studies conducted using both endogenous substrates and fluorescent probe substrates for the purpose of evaluating CYP inhibition potential of test compounds. Cohen et al. demonstrated a lack of correlation between CYP inhibitors in vitro when using fluorescent versus conventional P450 probe substrates, arguing for LC/ MS-based approaches. 

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