Unknown analysis using mass spectrometry

A widely accepted quantification procedure for accurate elemental analysis and isotope ratio measurements is isotope dilution mass spectrometry. In this procedure the sample is spiked with a known amount of a less-abundant enriched isotope of the same element, and the ratio of the unknown number of analyte atoms to the known number of the spike atoms is measured using mass spectrometry. 

In many areas of chemistry, chemical methods of structure proof have given way to spectroscopic methods (Chapter 14) and analysis by mass spectrometry. In the field of carbohydrate chemistry, however, chemical reactions play an important part in the elucidation of the structures of unknown carbohydrates. The reason for this is that many proteins and lipids contain carbohydrates that have very complex structures, and these do not yield easily to spectroscopic methods. Also, since it is notoriously difficult to crystallize many carbohydrates, proof of structure by X-ray crystallography is often not possible. In this section we will discuss methods for determining determine the structure of a monosaccharide by chemical methods. Several synthetic reactions can be used to convert one monosaccharide into another. If the structure of a related monosaccharide is known, and the mechanism of the reaction that interconverts them is well understood, then the structure of the unknown monosaccharide can be established. 

This example can be used in reverse to show the usefulness of looking for such isotopes. Suppose there were an unknown sample that had two molecular ion peaks in the ratio of 3 1 that were two mass units apart then it could reasonably be deduced that it was highly likely the unknown contained chlorine. In this case, the isotope ratio has been used to identify a chlorine-containing compound. This use of mass spectrometry is widespread in general analysis of materials, and it... 

Tandem mass spectrometry or ms/ms was first introduced in the 1970s and gained rapid acceptance in the analytical community. The technique has been used for stmcture elucidation of unknowns (26) and has the abiUty to provide sensitive and selective analysis of complex mixtures with minimal sample clean-up (27). Developments in the mid-1980s advancing the popularity of ms/ms included the availabiUty of powerhil data systems capable of controlling the ms/ms experiment and the viabiUty of soft ionisation techniques which essentially yield only molecular ion species. 

Laser ionization mass spectrometry or laser microprobing (LIMS) is a microanalyt-ical technique used to rapidly characterize the elemental and, sometimes, molecular composition of materials. It is based on the ability of short high-power laser pulses (-10 ns) to produce ions from solids. The ions formed in these brief pulses are analyzed using a time-of-flight mass spectrometer. The quasi-simultaneous collection of all ion masses allows the survey analysis of unknown materials. The main applications of LIMS are in failure analysis, where chemical differences between a contaminated sample and a control need to be rapidly assessed. The ability to focus the laser beam to a diameter of approximately 1 mm permits the application of this technique to the characterization of small features, for example, in integrated circuits. The LIMS detection limits for many elements are close to 10 at/cm, which makes this technique considerably more sensitive than other survey microan-alytical techniques, such as Auger Electron Spectroscopy (AES) or Electron Probe Microanalysis (EPMA). Additionally, LIMS can be used to analyze insulating sam-... 

A modified nucleotide found in RNA sequencing could either be a new nucleotide of unknown chemical structure or it could correspond to an already known modified nucleotide (up to now about 90 different modified nucleotides have been identified in RNA). Keith [124] proposed preparative purifications of major and modified ribonucleotides on cellulose plates, allowing for their further analysis by UV or mass spectrometry. Separation was realized by two-dimensional elution using the following mobile phases (1) isobutyric acid-25% ammonia-water (50 1.1 28.9,... 

To assure consistency and speed in multidisciplinary structure analysis of low-MW compounds involving various techniques (IR, NMR, MS, etc.) most industrial laboratories use a Standard Operating Procedure (SOP). In such schemes IR analysis is frequently used as a cheap filter for a quick starting control and as a means for verification. As IR detects only structural units identification of an unknown compound on the basis of IR is difficult. Mass spectrometry is used as the prime identification tool and is especially important in the determination of the exact mass and gross formulae. While structural prognostication on the basis of MS is difficult for the non-expert, a posteriori interpretation is quite feasible. H NMR is both easy and cheap, however requires greater sample quantities than either... 

High resolution is used to determine the exact mass of an ion species in a mixture knowledge of the exact mass of an unknown substance allows its atomic composition to be established. Target analysis exact mass determination proves the presence of a particular ion species (compound) in a mixture. Mass spectrometry is perhaps the only method that can be used to find the empirical formulae of compounds that are not completely pure. 

Mass spectrometry can be specific in certain cases, and would even allow on-line QA in the isotope dilution mode. MS of molecular ions is seldom used in speciation analysis. API-MS allows compound-specific information to be obtained. APCI-MS offers the unique possibility of having an element- and compound-specific detector. A drawback is the limited sensitivity of APCI-MS in the element-specific detection mode. This can be overcome by use of on-line sample enrichment, e.g. SPE-HPLC-MS. The capabilities of ESI-MS for metal speciation have been critically assessed [546], Use of ESI-MS in metal speciation is growing. Houk [547] has emphasised that neither ICP-MS (elemental information) nor ESI-MS (molecular information) alone are adequate for identification of unknown elemental species at trace levels in complex mixtures. Consequently, a plea was made for simultaneous use of these two types of ion source on the same liquid chromatographic effluent. 

Visible and UV spectrometry are of secondary importance to other spectral methods for the identification and structural analysis of unknown compounds. This is a direct consequence of the broad bands and rather simple spectra which make differentiation between structurally related compounds difficult. As an adjunct to infrared, magnetic resonance and mass spectrometry, however, they can play a useful role. They can be particularly helpful in confirming the presence of acidic or basic groups in a molecule from the changes in band position and intensity associated with changes in pH (p. 369). 

PMR spectrometry is an extremely useful technique for the identification and structural analysis of organic compounds in solution, especially when used in conjunction with infrared, ultraviolet, visible and mass spectrometry. Interpretation of PMR spectra is accomplished by comparison with reference spectra and reference to chemical shift tables. In contrast to infrared spectra, it is usually possible to identify all the peaks in a PMR spectrum, although the complete identification of an unknown compound is often not possible without other data. Some examples of PMR spectra are discussed below. 

Hyphenated analytical techniques such as LC-MS, which combines liquid chromatography and mass spectrometry, are well-developed laboratory tools that are widely used in the pharmaceutical industry. Eor some compounds, mass spectrometry alone is insufficient for complete structural elucidation of unknown compounds nuclear magnetic resonance spectroscopy (NMR) can help elucidate the structure of these compounds (see Chapter 20). Traditionally, NMR experiments are performed on more or less pure samples, in which the signals of a single component dominate. Therefore, the structural analysis of individual components of complex mixtures is normally time-consuming and less cost-effective. The... 

Once an assessment on a particular impurity has been made all process-related compounds will be examined to confirm that the impurity of interest is indeed an unknown. An easy way of doing this is to compare the retention times of known process-related compounds to that in question. If this analysis confirms that the compound is an unknown, the next step would be to obtain an LC-MS on the compound. Mass spectrometry provides structural information which aids in determining structure. In some cases, mass spectrometry will be enough to identify the compound. In other cases, more complicated methods like LC-NMR are needed or the impurity will need to be isolated in order to obtain additional information. Compounds that are not purified often contain high levels of by-products and can be used for this purpose. Alternatively, mother liquors from crystallizations also contain levels of by-products. Other ways of obtaining larger quantities of impurities include flash chromatography which is typically used for normal phase separations or preparative HPLC which is more common for reversed phase methods. Once a suitable quantity of the compound in question has been obtained a full characterization can be carried out to identify it. 

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