Bioanalysis chromatographic separation

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 turbulent flow column switching (CS) approach has also been used for the bioanalysis of a single analyte, as well as for mixtures of multiple analytes. Jemal et al. used this scheme to effectively separate and quantify two positional isomers in plasma [114]. Multiple components in plasma were simultaneously quantified with good chromatographic separation and accuracy by Wu et al. [65]. Lim et al. used this approach to simultaneously screen for metabolic stability and profiling [115]. 

A major limitation of the DART/MS/MS method in bioanalysis is the potential metabolic interference resulting from ion source fragmentation. The helium atom at the excited state carries an internal energy of 19.8 eV, which is higher than the bond energy in many labile metabolites. Since no chromatographic separation is used in the DART/MS/MS method, metabolites with labile chemical bond, such as a glucuronides or A-oxides, may break down in the ion source and interfere with the assay of the parent compound [27], This limitation is believed to be more of a... 

The general process of bioanalysis is depicted in Figure 6.3. Following animal dosing and biological sample collection, the typical steps in the bioanalytical procedure are sample preparation, chromatographic separation, MS detection, and data analysis, aU of which are critical in determining assay quality [101-103]. 

MS-MS is currently very widely used in combination with chromatographic separation methods, especially LC. The obvious reason for this is the frequent use of soft ionization techniques in LC-MS interfacing, i.e. electrospray and atmospheric-pressure chemical ionization. MS-MS allows additional structural information as well as the molecular mass information to be obtained. On-line LC-MS-MS is currently the method-of-choice in quantitative bioanalysis in (pre-) clinical pharmacological studies during drug development in pharmaceutical industries. In these studies, the instrument is operated in... 

The on-line combination of a chromatographic separation technique (GC, LC, but also thin-layer chromatography (TLC), capillary zone electrophoresis (CZE) and supercritical fluid chromatography (SFC)) with MS enables the mass spectrometric characterization of components in complex mixtures after separation with minimal or no sample loss. It is especially useful in the identification of minor or trace components that are difficult to collect by fractionation of the column effluent or would be easily lost. Furthermore, as already indicated above, on-line GC-MS and LC-MS-MS are important tools in quantitative bioanalysis as well. Obviously, fractionation in large series of samples for routine quantitative applications would be extremely time-consuming and ineffective. 

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. 

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. 

Analytical separations, including bioanalysis, seek only information— what is in the sample and how much. An important clarification must be made here. Preparative separations also exist and are very common but seek to recover a product from the separative process for later use and not necessarily any information about the components being separated. These form the basis of vast industries such as ore refining and preparative scale chromatographic purification of important chemicals and pharmaceuticals. Of course the two often are operated concomitantly, yielding both valuable information and material. 

In addition to the MRM scan, full-scan data can be obtained using the EMS function on the QTRAP to provide valuable information for evaluation of the bioanalysis method. For example, dosing vehicle polyethylene glycol (PEG) has been found to cause ion suppression in electrospray ionization, and chromatographic resolution of PEG from the analytes of interest is crucial for reliable performance of a quantitative bioanalytical assay. The EMS scan of the incurred samples could provide valuable information on the presence and the retention time of PEG, which cannot be obtained by MRM (King et al., 2003). Phospholipids have been shown to cause ion suppression, especially when a generic extraction method such as protein precipitation is used. The EMS function on QTRAP can be used to elucidate the phospholipid profile and to monitor the separation of phospholipids from the analyte and the internal standard (King et al., 2003). 

---