Sensitivity analysis mass spectrometry

Coupling chromatographic procedures with immunochemical techniques can also provide a very sensitive and specific analytical system for either determinative or confirmatory analysis. If the antibody used is very specific for the analyte of interest and the antibody reactivity is known to be sensitive to small variations in the structure of the analyte tested, positive reactions with the method are strongly indicative that an analyte of defined structural characteristics is present in the sample. Full rigorous confirmation, however, would depend on further analysis by mass spectrometry, which is the method of choice in confirmatory analysis. Mass spectrometry gives specific information on the identity and structure of the compound of interest. Coupled with chromatographic techniques it becomes a very powerful confirmatory tool for both quantitative and qualitative assessment of drug residues in foods. 

Because the characterization of support-bound intermediates is difficult (see below), solid-phase reactions are most conveniently monitored by cleaving the intermediates from the support and analyzing them in solution. Depending on the loading, 5-20 mg of support will usually deliver sufficient material for analysis by HPLC, LC-MS, and NMR, and enable assessment of the outcome of a reaction. Analytical tools that are particularly well suited for the rapid analysis of small samples resulting from solid-phase synthesis include MALDI-TOF MS [3-5], ion-spray MS [6-8], and tandem MS [9]. MALDI-TOF MS can even be used to analyze the product cleaved from a single bead [5], and is therefore well suited to the identification of products synthesized by the mix-and-split method (Section 1.2). The analysis and quantification of small amounts of product can be further facilitated by using supports with two linkers, which enable either release of the desired product or release of the product covalently bound to a dye [10-13], to an isotopic label [11], or to a sensitizer for mass spectrometry [6,14,15] (e.g., product-linker-dye- analytical linker -Pol). 

Mass spectrometry has become an essential analytical tool for a wide variety of biomedical applications such as food chemistry and food analysis. Mass spectrometry is highly sensitive, fast, and selective. By combining mass spectrometry with HPLC, GC, or an additional stage of mass spectrometry (MS/MS), the selectivity increases considerably. As a result, mass spectrometry may be used for quantitative as well as qualitative analyses. In this manual, mass spectrometry is mentioned frequendy, and extensive discussions of mass spectrometry appear, for example, in units describing the analyses of carotenoids (unitfia) and chlorophylls (unit F4.5). In particular, these units include examples of LC/MS and MS/MS and the use of various ionization methods. 

Reversed-phase HPLC is widely utilized to generate a peptide map from digested protein, and the MS online method provides rapid identification of the molecular mass of peptides. The HPLC-MS-FAB online system is a sensitive and precise method for low-MW peptides (<3000 Da) even picomol quantities can be detected. However, as the MW of the analytes increases, the ionization of peptides becomes more difficult and decreases the sensibility of the FAB-MS (112). Electrospray ionization (ESI-MS) was found to be an efficient method for the determination of molecular masses up to 200,000 Da of labile biomolecules, with a precision of better than 0.1%. Molecular weights of peptide standards and an extensive hydrolysate of whey protein were determined by the HPLC-MS-FAB online system and supported by MALDI-TOF (112). Furthermore, HPLC-MS-FAB results were compared with those of Fast Performance Liquid Chro-motography (FPLC) analysis. Mass spectrometry coupled with multidimensional automated chromatography for peptide mapping has also been developed (9f,l 12a). 

Also HPLC with tandem mass spectrometric detection (HPLC-MS/MS) provides a suitable method of analysis. We have found that a single column is sufficient [3,5], however, we must emphasise that unknown substances similar in mass to 8-oxodG needs to be separated from 8-oxodG. For high sensitivity in mass spectrometry the peak height in HPLC is very important. The amount detected is proportional both to the peak height and to the area under the curve. 

In inorganic chemistry, mixtures of metal ions in solution can be analyzed by electron-impact mass spectrometry. First the metal ions are complexed with an organic ligand (usually various substituted acetylacetonates) to form volatile metal chelates. If many metal ions are anticipated, the mixture is separated by GC and the separated fractions identified by mass spectrometry. Simple mixtures can be analyzed directly using the mass spectrometer. Because of the high sensitivity of mass spectrometry, trace analysis is possible. 

All above mentioned criteria are fulfilled by electrochemical methods. Electrochemical methods are attractive for ecological research also because they enable immediate measurement of changes in the component concentrations and because they can be frequently used for continuous monitoring and are suitable for field work, where the systematic error caused by transport and storage of the sample is avoided. It is true that there are a number of much more sensitive physical methods (X-ray electron difraction, neutron activation analysis, mass spectrometry, etc.) which, however, cannot be used in the field and are very expensive. 

Mass Spectrometry. Different varieties of mass spectrometry have for several years been used to identify the chemical nature of cobalamins produced from microorganisms (Kumudha et al. 2010). The sensitivity of mass spectrometry is still insufficient for measurement of cobalamins in the picomolar range and this method is therefore of limited interest for analysis of human serum. If the sensitivity is improved, mass spectrometry is predicted to be a very strong tool for identification of the varieties of cobalamins and cobalamin analogues present in human serum. 

Improvements in the instrumentation, ionization sources, high-resolution mass analyzers, and detectors [67-69], in recent years have taken mass spectrometry to a different level of HPLC-MS for natural product analysis. Mass spectrometry detection offers excellent sensitivity and selectivity, combined with the ability to elucidate or confirm chemical structures of flavonoids [70-72]. Both atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) are most commonly used as ionization sources for flavonoid detection [73-76]. Both negative and positive ionization sources are applied. These sources do not produce many fragments, and the subsequent collision-induced dissociation energy can be applied to detect more fragments. Tandem mass spectrometry (MS , n> 2) provides information about the relationship of parent and daughter ions, which enables the confirmation of proposed reaction pathways for firagment ions and is key to identify types of flavonoids (e.g., flavones, flavonols, flavanones, or chalcones) [77-80]. 

The high separation power of HPLC, together with excellent selectivity and sensitivity of mass spectrometry (HPLC-MS), overcame the above problem in the analysis of triterpenoid saponins. HPLC-MS [1, 5, 7, 10, 17, 19, 28-30, 37, 39-42] plays an important role in the analysis of triterpenoid saponins. 

The commonly used methods for the determination of these drugs are immunoassays and chromatography. Most immunoassays for immunosuppressants are semiautomated since extraction of drugs from the whole blood is needed before analysis. Immunoassays are convenient due to automation, but have problems with cross-reactivity with drug metabolites (3, 6). Both polyclonal and monoclonal antibody-based assays are available. Monoclonal antibody-based immunoassays are more specific. HPLC with ultraviolet detection and tandem mass spectrometry are commonly used chromatographic methods for the assay of immunosuppressants. Due to their specificity and sensitivity, tandem mass spectrometry assays are preferred and are now in wide use (7, 8). The other major advantage of tandem mass spectrometry assays is their ability to simultaneously measure several immunosuppressants (7-10). Pharmacokinetic properties of CSA, sirolimus, and tacrolimus are shown in Table 1 (3, 6, 11). 

Takemori, N. Komori, N. Matsumoto, H. Highly sensitive multistage mass spectrometry enables small-scale analysis of protein glycosylation from two-dimensional polyacrylamide gels. Electrophoresis 2006, 27, 1394-1406. 

Ions are also used to initiate secondary ion mass spectrometry (SIMS) [ ], as described in section BI.25.3. In SIMS, the ions sputtered from the surface are measured with a mass spectrometer. SIMS provides an accurate measure of the surface composition with extremely good sensitivity. SIMS can be collected in the static mode in which the surface is only minimally disrupted, or in the dynamic mode in which material is removed so that the composition can be detemiined as a fiinction of depth below the surface. SIMS has also been used along with a shadow and blocking cone analysis as a probe of surface structure [70]. 

Section 13 22 Mass spectrometry exploits the information obtained when a molecule is ionized by electron impact and then dissociates to smaller fragments Pos itive ions are separated and detected according to their mass to charge (m/z) ratio By examining the fragments and by knowing how classes of molecules dissociate on electron impact one can deduce the structure of a compound Mass spectrometry is quite sensitive as little as 10 g of compound is sufficient for analysis... 

The techniques described thus far cope well with samples up to 10 kDa. Molecular mass determinations on peptides can be used to identify modifications occurring after the protein has been assembled according to its DNA code (post-translation), to map a protein structure, or simply to confirm the composition of a peptide. For samples with molecular masses in excess of 10 kDa, the sensitivity of FAB is quite low, and such analyses are far from routine. Two new developments have extended the scope of mass spectrometry even further to the analysis of peptides and proteins of high mass. 

Few of the naturally occurring elements have significant amounts of radioactive isotopes, but there are many artificially produced radioactive species. Mass spectrometry can measure both radioactive and nonradioactive isotope ratios, but there are health and safety issues for the radioactive ones. However, modem isotope instmments are becoming so sensitive that only very small amounts of sample are needed. Where radioactive isotopes are a serious issue, the radioactive hazards can be minimized by using special inlet systems and ion pumps in place of rotary pumps for maintaining a vacuum. For example, mass spectrometry is now used in the analysis of Pu/ Pu ratios. 

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