LC-MS studies

TABLE 12.1 Chromatographic Modes and Conditions Used for LC-MS Study of Tryptic Peptides Separation Selectivity... 

FIGURE 13.4 Total ion chromatograms from the ID LC/MS analysis of a yeast ribosomal protein fraction separated using 0.1% TFA (Panel a) and 0.1% formic acid (Panel b) as mobile phase modifiers. TFA produced narrower, more concentrated, peaks for mass analysis that did not overcome the significant electrospray ionization suppression associated with using this modifier for LC/MS studies, resulting in an overall reduction in component intensities. 

In LC-MS, specific ionisation conditions can be required for different types of species. This means that in LC-MS studies on extractable additives, it is necessary to use a range of experimental conditions to cover detection of all types of potential species. Depending on instrument type, it is also possible to isolate ions in complex matrices and obtain positive identifications by further unique fragmentation of these ions (by MS-MS or MSn). Quantitative methods based on this secondary ionisation can be employed. The mass accuracy of LC-MS detection systems continues to improve. Accurate mass measurement improves the certainty of identification. Advanced systems are typically offering 1-2 ppm (mass dependent) mass accuracy. 

Phase 1 clinical trials of the natural polyphenol quercetin (33) against several types of cancer have shown that this compound is well tolerated when administered at a dose of 70 mg/kg by i.v. bolus [63,64]. Further studies revealed that 33 inhibits GST Pl-1 completely after 2 h at a concentration of 25 pM however, addition of GSH partially restores activity. HPLC and LC-MS studies indicate that 33 inhibits GST Pl-1 through the formation of a covalent yet reversible bond with GST Pl-1 cysteine residue at position 47. 

For comparison of time necessary to quantify the compounds examined by FIA—MS and MS—MS, LC was also examined. API— LC—MS studies with RP-Cis (AE, betaine, FADA) or PLRP (quat) column applied for separation were performed. Quantitative results could be obtained with SDs < 5%, while the invested time in parallel increased by a factor > 12 compared with FIA-MS. 

Fulcrand, H. et al., LC-MS study of acetaldehyde induced polymerisation of flavan-3-ols. In 18th International Conference on Polyphenols (eds J. Vercauteren, C. Cheze, M. Dumon, and J. Weber), Bordeaux, France, 1996, p. 203. 

LC-MS/MS is recognized as the technique of choice for analysing many environmental pollutants, for example fluorinated alkyl substances (FASs). These organic compounds contain carbon and fluorine, and arise as a result of biological and geochemical processes, and may be produced synthetically for heat-, oil- and water-resistant products. FAS contaminants have been identihed in environmental and biological samples, and can bioaccumulate to toxic levels. LC-MS/MS has been widely used to study FAS contaminants in water, sediment and biological samples, and may confirm and extend more limited LC-MS studies. 

Two approaches are often used to improve the detection limit, including selected ion monitoring (SIM) and multiple reaction monitoring (MRM). In LC/MS studies, it is often desirable to increase detection sensitivity by hmit-ing the mass analyzer scan to just one ion— that is, SIM. In this mode, a single ion of interest is monitored continuously by a mass spectrometer and no other ions are detected. This results in signihcant improvement of signal-to-noise ratio. SIM trades specihcity for sensitivity. In general, the sensitivity in SIM is increased by a factor of 100 to 1000 over full-scan mass spectra. This can be quite useful in detection and quantihcation of specihc compounds at low levels. 

Using this optimized method shown in Figure 8-49 that starts at 25v/v% acetonitrile, LC-MS studies were performed to determine the [M -i- H]+ ion of the impurity that has been resolved from the main peak. The mass spectrum of Product M was taken and was shown to be spectrally homogeneous. The mass spectrum of the impurity (RRT 1.04) that has now been resolved from the main peak was also taken. The UV and the total ion chromatograms are shown in Figure 8-50. This impurity, RRT 1.04, has the same [M -i- H]+ ion that was co-eluting with the main component in the initial separation on the C8... 

The anionie surfactants of major importance are the linear alkylbenzene sulfonates (LAS). Other important elasses are alkyl sulfates, sulfonates, and ethoxy-sulfates (AES). Few LC-MS studies have been published. 

Both signal -to-noise and absolute signal intensity were the criteria used to select the mode of ionization for LC-MS (Table IV). Except for the sulfates and 4-nitrophenol in PCI, there is a decrease in S/N when the solvent is changed from acetonitrile to acetonitrile-buffer. The opposite is observed for the sulfates and 4-nitrophenol. In most cases, there is a parallel loss of signal intensity when going from acetonitrile to acetonitrile-buffer. The deleterious effects of the buffer on S/N is least pronounced in PCI. With acetonitrile-ammonium formate mobile phase, Isobutane PCI provides optimum sensitivity when compared to methane PCI or El. In both methane and isobutane NCI, peak broadening was unacceptably large, and NCI spectra were difficult to interpret. Based on these considerations, isobutane PCI was chosen for LC-MS studies. 

Biomarker identification represents a major bottleneck in nontargeted MS-based metabolomics and in particular in LC-MS studies. This happens... 

Liu et al. (2003) focused their method paper on the instrumental set-up for on-line capillary LC coupled with MS-MS via a low flow-rate interface, hoping that miniaturization would improve sensitivity for many biomedical applications. The system design and perfonnance were tested on DHEA sulfate and pregnenolone sulfate quantitation from 5 pL of plasma from one male volunteer. Sample preparation was simple - addition of aqueous methanol and of the deuterated analog internal standards, protein precipitation, supernatant filtration through a Cig bed, evaporation and reconstitution in 100 pL of mobile phase, of which 20 pL were injected for LC-MS-MS analysis. The DHEA sulfate concentration found was in agreement with literature values based on GC-MS and LC-MS studies. 

For dereplication and structure determination purposes, one of the most useful results from an LC-MS study is the identification of a compound s molecular ion. From the molecular ion, it is usually possible to determine the compound s molecular weight to the nearest atomic mass unit. This can be carried out by experimentally changing the mobile phase buffer and observing the resulting adduct ions in order to determine the adduct composition of the molecular ion e.g., M +H, M" +Li, M +Na, M —H. The unit-molecular-weight... 

LC-MS studies indicate that tyrosinase oxidation of 4-fluorocatechol gives several products including 2,3-dihydroxy-6-fluorodioxin, formed via fluoride displacement from 4-fluoro-l,2-benzoquinone and cyclisa-tion of the resulting 4-(2-hydroxyaryloxy)-l,2-benzoquinone (08MI1). 

In a LC-MS study [175] applying FAB besides TSP, APCl, ESI, plasma desorption (PD) ionisation a series of N- and P-containing pesticides were studied. Collision-induced dissociation (CID) spectra were recorded. Pesticide residues could readily be identified, confirmed and quantified. The results for several different polar pesticides were compared with APCI and ESI and were presented [176]. 

APCI-LC-MS [325] was applied to determine phenylurea herbicides in water samples with detection limits all in the low pg range, whereas ESI-LC-MS was used to analyse the acidic polar pesticides. Under APCI conditions the specific fragment ion of phenylureas mjz 72 corresponding to [0=C=N (CH3)2] could be observed [325]. Fragmentation pathways were presented. In a TSP-LC-MS study covering 15 phenylurea and thiourea pesticides APCI-LC-MS/MS was used to elucidate fragmentation behavior observed under TSP conditions [175]. 

Hiller and coworkers reported an NMR and LC-MS study on the structure and stability of l-iodosyl-4-methoxybenzene and 1-iodosyl-4-nitrobenzene in methanol solution [195]. Interestingly, LC-MS analyzes provided evidence that unlike the parent iodosylbenzene, which has a polymeric structure (Section 2.1.4), the 4-substituted iodosylarenes exist in the monomeric form. Both iodosylarenes are soluble in methanol and provide acceptable and NMR spectra however, gradual oxidation of the solvent was observed after several hours. Unlike iodosylbenzene, the two compounds did not react with methanol to give the dimethoxy derivative ArI(OMe)2 [195]. 

Zhang, J.-R., Gazers, A. R., Lutzke, B. S. and Hall, E. D. (1995) HPLC-chemiluminescence and thermospray LC/MS study of hydroperoxides generated from phosphatidylcholine. Free Rad. Biol Med., 18, 1-10. 

Magnetic analyzers are often used in various combinations with electrostatic sectors. Sector instruments are characterized by good ion transmission. Depending on the order of these analyzers, very high resolution and wide mass ranges can be achieved. Sector instruments suffer from a relatively low scan rate, which makes their application in GC-MS and LC-MS studies somewhat limited. At the same time, some types of tandem mass spectrometry experiments with mass-selected ions can be performed only on this type of equipment. 

Mobile-phase additives are known to play an important role in the efficient separation of chromatographic peaks in LC-MS. Mobile-phase additives used for LC-MS studies are most often volatile molecules that can be easily evaporated in the LC-MS interface to prevent ionization suppression and/or detection contamination. The most commonly used mobile-phase additives in LC-MS analysis are the small organic acids, acetic and formic acids, together with their ammonium salts. Trifluoroacetic acid (TFA) is also widely used in LC-MS analysis despite reports of ionization suppression in the ESI mode. Some studies suggest that the drawback of TFA use can be overcome for some neutral analytes by using minute concentrations of the acid in the mobile phase. A number of studies presented here used TFA as the mobile-phase additive, and in all those instances, ESI was the ionization interface. However, formic acid was the additive most commonly used, followed closely by TFA and then by the ammonium salts of both formic and acetic acid. The type of glucuronide conjugate (O vs. N) did not appear to influence the type of mobile-phase additive used. 

ESI (LC-MS) Study of protein folding (cytochrome c), hydrogen/deuterium exchange Yang and Smith [231 ]... 

Recently Achour et al. [55] used an LC-MS proteomic approach with human liver micro-somes and reported a mean value of 39 pmol/ mg protein. This value was -one half of that found for P450 3A4 in the same set of samples (Fig. 9.2d). The concentration was much less in the other samples (Fig. 9.2a, b, c), with another LC-MS study reporting only 0.5 and 7 pmol P450 2B6/mg protein [54]. 

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