Chromatographic methods with multiple detection

The particle beam LC/FT-IR spectrometry interface can also be used for peptide and protein HPLC experiments to provide another degree of structural characterization that is not possible with other detection techniques. Infrared absorption is sensitive to both specific amino acid functionalities and secondary structure. (5, 6) Secondary structure information is contained in the amide I, II, and III absorption bands which arise from delocalized vibrations of the peptide backbone. (7) The amide I band is recognized as the most structurally sensitive of the amide bands. The amide I band in proteins is intrinsically broad as it is composed of multiple underlying absorption bands due to the presence of multiple secondary structure elements. Infrared analysis provides secondary structure details for proteins, while for peptides, residual secondary structure details and amino acid functionalities can be observed. The particle beam (PB) LC/FT-IR spectrometry interface is a low temperature and pressure solvent elimination apparatus which serves to restrict the conformational motions of a protein while in flight. (8,12) The desolvated protein is deposited on an infrared transparent substrate and analyzed with the use of an FT-IR microscope. The PB LC/FT-IR spectrometric technique is an off-line method in that the spectral analysis is conducted after chromatographic analysis. It has been demonstrated that desolvated proteins retain the conformation that they possessed prior to introduction into the PB interface. (8) The ability of the particle beam to determine the conformational state of chromatographically analyzed proteins has recently been demonstrated. (9, 10) As with the ESI interface, the low flow rates required with the use of narrow- or microbore HPLC columns are compatible with the PB interface. 

The detection of strongly acidic sulfonates is performed by ESI-MS in the negative ion mode. In SIR the molecular anions, or in the case of polysulfonated compounds the dianions, are selected for detection, while multiple reaction monitoring (MRM) uses a loss of 64 amu [M-SO2] or 80 amu [M-SO2] from the parent anions. It is still unclear which structural elements govern whether mainly SO2 or SO3 is eliminated. The selectivity of MS, especially MS-MS, allows ion-pair extraction to be used for the enrichment of aromatic sulfonates from aqueous samples without interference from humic material as in UV detection. " No reliable LC-MS method with volatile alkylamines has been developed yet, as problems occur in chromatographic and mass spectrometric separation. 

Very recently, Stobo et al. proposed a multiple LC-MS method that allows the detection of YTX together with most of the DSP toxins. The method was set up on a triple quadrupole spectrometer. Chromatographic separation of multiple lipophilic toxins was achieved by using a base deactivated silica C8 column eluted with a 5 mM ammonium acetate-acetonitrile mobile phase under gradient conditions. The method was applied to the analysis of sheUflsh and was validated for the quantitative detection of OA, YTX, PTX-2, and Azaspiracid-1 (AZA-1). 

LC coupled with detection by mass spectrometry (MS) offers the potential for excellent sensitivity and specificity. Sample preparations suitable for niacin analysis by LC-UV and other methods should also be suitable for LC-MS. However, chromatographic methods developed for LC-UV or LC-fluorescence methods would in most cases have to be modified due to issues such as ion suppression and problems created by non-volatile species in commonly used mobile phases. Niacin has a relatively low molecular mass (123 Da), and thus in selected ion recording (SIR) mode, there may still be significant interference problems. With instruments that allow a multiple reaction monitoring mode (MRM), however, interference problems can potentially be avoided even without optimized chromatography or sample clean-up. 

Detailed comparisons of TLC to other chromatographic methods, especially HPLC, and TLC to HPTLC are presented in Chapter 1 of Ref. 1. TLC involves the concurrent processing of multiple samples and standards on an open layer developed by a mobile phase. Development is performed, usually without pressure, in a variety of modes, including simple one-dimensional, multiple, circular, and multidimensional. The detection of zones is done statically with an assortment of diverse possibilities. Paper chromatography, which was invented by Consden, Gordon, and Martin in 1944, is fundamentally very similar to TLC, differing mainly in the nature of the stationary phase. Paper chromatography has lost favor compared to TLC because the latter is faster, more efficient, and allows more versatility in the choice of stationary and mobile phases. 

Mass spectrometry is a technique for separating ions by their mass-to-charge ratios. This technique is widely used to detect sulfur compounds, also coupled with other techniques (see Table 7.2). Socher et al. [26] presented MS like a tool to structure elucidation of sulfonated compounds. Ion-pair LC-ESI-MS-MS with TrBA as ion-pair agent has proven to be a powerful tool for the analysis of aromatic compounds with multiple acidic groups [25]. MS can not only be coupled with chromatographic methods, it can also be coupled with CE [2]. 

An LC-MS-MS method to monitor lonafarnib (a novel anticancer drug that inhibits farnesyl transferase) in human plasma. Deuterated internal standard is used proteins are precipitated by acetonitrile. Reverse-phase chromatographic separation is performed using acetonitrile/water/formic acid (50 50 0.05, v/v/v) mobile phase. Time of analysis 8 min. A triple quadrupole tandem mass spectrometer in the positive-ion mode with multiple reaction monitoring is used for detection. The cahbration curve has been established in the 2.5-2500 ng/ml concentration range. The validated method was successfully used in phase I trials of the drug. 

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