Spectrometric Identification Procedure

Fragment B is obtained by release of the C-1 part of the sialic acid molecule. Eliminations of OCOCH3 in 0-acetylated neuraminic acid derivatives and of NH2COCH3 in Neu5Ac derivatives, which in principle give rise to the same mje value as fragment B in the case of = CH3, contribute little to the abundancy of this ion. 

Fragment D is formed from fragment C by consecutive eliminations of R2OH and R4OH. It is evident that the occurrence of this fragment ion is dependent on the presence of C. 

Fragment E is formed by elimination of the side-chain C-7,8,9 and the substituent at C-5. This fragment ion is not observed if an O-acetyl group is attached to C-4, illustrating that the transition state in the McLafferty rearrangement is disfavored when the substituent at C-4 is an ester group rather than an ether group. 

Fragment F contains the C-8,9 part. Based on the same fragmentation rules as mentioned above for fragments C and Z), this ion can only readily be formed if an ether group is connected at C-8. 

Fragment G consists of the C-4,5 part of the sialic acid molecule. 

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The most widely used mass spectrometric identification procedure is MALDI-Tof analysis of the entire peptide mixture. Gas-phase matrix interaction with peptide ions in MALDI-Tof results in singly charged ions, giving a mass profile that is highly characteristic of the protein from which the peptides are derived. These peptide masses (actually protonated peptide molecular ions, MH+) can be used to search databases (either protein or nucleic acid databases) to identify the proteins. The two most important factors in successfully identifying proteins by this approach are the number of matching peptide masses and the accuracy of the peptide mass determination. 

Latexes of synthetic resins are identified by ir spectrometry. Selective extraction with organic solvents is used to obtain purified fractions of the polymers for spectrometric identification. Polymeric films can be identified by the multiple internal reflectance ir technique, if the film is smooth enough to permit intimate contact with the reflectance plate. TAPPI and ASTM procedures have not been written for these instmmental methods, because the interpretation of spectra is not amenable to standardization. 

Application of modem analytical procedures and physical methods for structure determination. In studies of microbial metabolism, the advantages resulting from the requirement for only extremely small quantities of material needed for gas- and liquid-chromatographic quantification, coupled to mass spectrometric identification, can hardly be overestimated. 

A further common problem the analyst faces when integrating SPC into analytical procedures for the determination of LAS is the scarcity of available reference compounds, thereby complicating their determination. Therefore, the identification of the analytes has to be performed by comparison of retention time and absolute peak area ratio between the deprotonated molecular ion and the fragment ion, relative to the ratio obtained from the authentic standard ( 20%). Retention times of SPC, for which no standards were available, can be determined once by mass spectrometric identification in full-scan mode. 

A number of variations on the procedure exist and have been reviewed by Privett [737]. For example, the positions of double bonds in monoenes containing both cis- and fr"ans-isomers, can be determined by conversion to epoxides, of which the configurational isomers are readily separable, and cleavage of these with periodic acid [245]. Similarly, partial oxymercuration of the double bonds followed by mass spectrometric identification of the products has been applied to the analysis of polyunsaturated fatty acids [719],... 

Off-Line Mass Spectrometric Product Identification Procedures... 

With respect to sample preparation, it is necessary to develop effective and fast procedures involving only a few steps in order to avoid contamination, reduce analysis time and to improve the quality of analytical work. Microsampling and the use of smaller sample sizes is required and also the further development of analytical techniques. In particular, there is a need for the development of online and/or hyphenated techniques in ICP-MS. Microsampling combined with the separation of small amounts of analytes will be relevant for several chromatographic techniques (such as the development of micro- and nano-HPLC). There is a demand for further development of the combination of LA-ICP-MS as an element analytical technique with a biomolecular mass spectrometric technique such as MALDI- or ESI-MS for molecular identification and quantification of protein phosphorylation as well as of metal concentrations, this also enables the study of post-translational modifications of proteins, e.g. phosphorylation. 

In addition, analysis using the spectrometric techniques may reveal other chemicals relevant to the test. Unambiguous identification of a chemical is obtained if at least two analytical, preferably spectrometric, techniques give consistent results. Analyses are mostly qualitative (identification). Work is conducted according to the ROPs and other documented procedures of demonstrated performance. In the case of a PT, the testing report... 

The analysis report must include necessary analytical data (chromatographic and spectrometric) supporting the identifications made, describe the sample preparation and analytical methods in detail (or make reference to ROPs, SOPs, or other procedures), and give information on the identified chemicals (CAS registry number, structural formula, and IUPAC or CWC name). 

The question can be posed as to whether the stereochemical conformation of a phosphoglyceride can be determined by FAB-MS. While no rigorous examination of synthetic sn-3 or sn-1 compounds by this technique (or any other mass spectrometric procedure) has been reported, it seems highly unlikely that any detectable or significant differences would be noted. Nonetheless, FAB-MS and other forms of mass spectrometry are powerful tools useful in the structural identification of organic compounds, and this possibility may develop. 

Note 2. The identification techniques which comprise part of the original procedure may be supplemented or supplanted with NMR and mass spectrometric methods of analysis (Faix and Meier 1987). 

In drug preparations, sensitivity is not a particular problem as there is always sufficient of the active constituent present. Suitable extraction procedures should be carried out and each fraction should then be subjected to chromatographic analysis. The mass spectra, together with the appropriate retention parameters, may then be used for identification purposes. In many cases mass spectrometric analysis is undertaken as confirmation of an identification based on the physical characteristics such as size, colour, markings, etc., of the tablet or capsule. Mass spectrometry may be used not only to identify the major components but also the trace impurities. This information may be used for quality control purposes, and may enable the route of manufacture of certain drugs to be determined. 

Sherlock, I.R., James, K.J., Caudwell, EB., and MacKintosh, C. 1997. The first identification of microcystins in Irish lakes aided by a new derivatisation procedure for electrospray mass spectrometric analysis. Natural Toxins 5 247-254. 

The study of natural products in plant extracts is an interesting challenge to LC-MS. Generally, the relevant compounds must be detected as minor components in complex mixtures. A combination of LC separation, especially to resolve isomeric stractures, and MS detection is needed. Furthermore, structural information is needed for the identification and dereplication of the unknown plant constituents. Because of the complexity of the sample pretreatment procedures involved in the isolation, MS in most cases is the only applicable spectrometric technique too much of a purified component would be needed for IR and NMR analysis. On-line analysis in relatively erode samples is obligatory for the detection of minor constitnents. When electrospray ionization (ESI) or atmospheric-pressnre chemical ionization (APCl) are applied for analyte ionization, structural information mnst be obtained by application of colhsion-indneed dissociation (CID), either via in-sonree CID or preferably via MS-MS or MS". LC-MS and LC-MS-MS have proved to be extremely snccessful in this area. 

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